BETA-CYCLODEXTRIN CONJUGATED MAGNETIC NANOPARTICLES FOR … · 2018. 1. 10. · beta-cyclodextrin...
Transcript of BETA-CYCLODEXTRIN CONJUGATED MAGNETIC NANOPARTICLES FOR … · 2018. 1. 10. · beta-cyclodextrin...
BETA-CYCLODEXTRIN CONJUGATED MAGNETIC
NANOPARTICLES FOR BIO- AND ENVIRONMENTAL
APPLICATIONS
ABU ZAYED MD. BADRUDDOZA
NATIONAL UNIVERISTY OF SINGAPORE 2011
BETA-CYCLODEXTRIN CONJUGATED MAGNETIC
NANOPARTICLES FOR BIO-AND ENVIRONMENTAL
APPLICATIONS
ABU ZAYED MD. BADRUDDOZA (B.Sc., Bangladesh University of Engineering & Technology)
A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF CHEMICAL & BIOMOLECULAR ENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE
2011
To My Parents
With Love
Acknowledgement
i
Acknowledgements I would like to take this opportunity to express my sincere thanks and grateful
acknowledgement to my supervisors, Associate Professorial Fellow Mohammad
Shahab Uddin and Associate Professor Kus Hidajat, for their valuable guidance,
support and encouragement throughout the research program. Their meticulous
attentions to details, incisive but constructive criticisms and insightful comments have
helped me shape the direction of this thesis research to the form it is presented here.
I would also like to give my deepest appreciation to all the staff members in the
Department of Chemical and Biomolecular Engineering and all my colleagues in the
lab, who have given me great help in my research work.
I would also like to extend my eternal gratitude to my parents, wife and my baby
daughter for their love, support and dedication.
Finally, my thanks to National University of Singapore for providing the Research
Scholarship and to the Department of Chemical and Biomolecular Engineering for
providing the facilities to take this project through in its entirety.
Table of contents
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Table of contents Acknowledgements ........................................................................................................... i Table of contents .............................................................................................................. ii Summary .......................................................................................................................... v Nomenclature ................................................................................................................ viii List of figures ................................................................................................................... x List of tables .................................................................................................................. xvi Chapter 1 Introduction ................................................................................................ 1
1.1 General background ............................................................................................... 1 1.2 Research objective and significance ...................................................................... 3 1.3 Organization of the thesis ....................................................................................... 6
Chapter 2 Literature Review ....................................................................................... 7
2.1 Magnetic nanoparticles .......................................................................................... 7 2.1.1 Synthesis of superparamagnetic iron oxide nanoparticles .............................. 7 2.1.2 Properties of magnetic particles ...................................................................... 9 2.1.3 Surface modification of magnetic particles ................................................... 11
2.2 Magnetic Separation ............................................................................................. 15 2.2.1 Parameters of magnetic separator ................................................................. 17 2.2.1 Applications of magnetic separation ............................................................. 18
2.3 Cyclodextrins ....................................................................................................... 18 2.3.1 Basic properties of cyclodextrins .................................................................. 19 2.3.2 Cyclodextrin inclusion complexes ................................................................ 21 2.3.3 Characterization of cyclodextrin inclusion complexes ................................. 23 2.3.4 Applications of cyclodextrin ......................................................................... 25
2.4 Protein refolding ................................................................................................... 26 2.5 Pollutants removal from wastewater .................................................................... 31 2.6 Adsorption and Desorption .................................................................................. 39
2.6.1 Adsorption equilibrium ................................................................................. 39 2.6.2 Adsorption kinetics ....................................................................................... 40 2.6.3 Desorption study ........................................................................................... 42
2.7 Scope of the thesis ................................................................................................ 43 Chapter 3 Characterization Techniques .................................................................... 46
3.1 Transmission Electron Microscopy (TEM) Measurement ................................... 46 3.2 X-ray Diffraction (XRD) ...................................................................................... 46 3.3 Vibrating Sample Magnetometer (VSM) ............................................................. 47 3.4 Thermogravimetric Analysis (TGA) .................................................................... 48 3.5 Differential Scanning Calorimetry (DSC) ............................................................ 48 3.6 Fourier Transform Infrared (FTIR) ...................................................................... 48 3.7 Brunauer-Emmett-Teller (BET) Method ............................................................. 49 3.8 Zeta Potential Analyzer ........................................................................................ 49 3.9 X-ray Photoelectron Spectroscopy (XPS) ............................................................ 51 3.10 Circular Dichroism (CD) .................................................................................... 51 3.11 Fluorescence ....................................................................................................... 53
Table of contents
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Chapter 4 β–cyclodextrin bonded magnetic nanoparticles as solid-phase artificial chaperone in refolding of proteins1 ................................................................................ 54
4.1 Introduction .......................................................................................................... 54 4.2 Experimental ........................................................................................................ 56
4.2.1 Materials ........................................................................................................ 56 4.2.2 Preparation of mono-tosyl-β-cyclodextrin (Ts-β-CD) .................................. 57 4.2.3 Preparation of Fe3O4 magnetic nanoparticles ................................................ 57 4.2.4 Preparation of 3-aminopropyltriethoxy silane (APES) modified magnetic nanoparticles (APES-MNPs) .................................................................................. 58 4.2.5 Fabrication of Ts-β-CD modified Fe3O4 nanoparticles (CD-APES-MNPs) . 58 4.2.6 Protein refolding experiments ....................................................................... 59
4.3 Results and Discussion ......................................................................................... 61 4.3.1 Synthesis of β-CD bonded magnetic nanoparticles ....................................... 61 4.3.2 Characterization of magnetic nanoparticles .................................................. 63 4.3.3 CD-APES-MNPs assisted CA Refolding ...................................................... 68 4.3.4 Structural analyses of the refolded products by intrinsic fluorescence and far-UV circular dichroism ............................................................................................ 76
4.4 Conclusions .......................................................................................................... 80 Chapter 5 Selective recognition and separation of nucleosides using carboxymethyl-β-cyclodextrin functionalized hybrid magnetic nanoparticles2 ...................................... 82
5.1 Introduction .......................................................................................................... 82 5.2 Experimental ........................................................................................................ 85
5.2.1 Materials ........................................................................................................ 85 5.2.2 Preparation of carboxymethyl-β-cyclodextrin (CM-β-CD) .......................... 85 5.2.3 Fabrication of CM-β-CD modified APES-MNPs (CMCD-APES-MNPs) ... 86 5.2.4 Adsorption of nucleosides ............................................................................. 86
5.3 Results and discussions ........................................................................................ 87 5.3.1 Synthesis and characterization of magnetic nanoparticles ............................ 87 5.3.2 Adsorption of nucleosides onto CMCD-APES-MNPs ................................. 93 5.3.3 Selective adsorption of A and G ................................................................... 98 5.3.4 UV-vis spectra analysis and determination of binding constants ............... 100 5.3.5 Interactions of the nucleosides with cyclodextrins ..................................... 103
5.4 Conclusions ........................................................................................................ 106 Chapter 6 Adsorptive removal of dyes from aqueous water using carboxymethyl-β-cyclodextrin conjugated magnetic nano-adsorbent3 ..................................................... 108
6.1 Introduction ........................................................................................................ 108 6.2 Experimental ...................................................................................................... 111
6.2.1 Materials ...................................................................................................... 111 6.2.2 Fabrication of CM-β-CD modified Fe3O4 nanoparticles [CMCD-MNP(C) & CMCD-MNP(P)] .................................................................................................. 111 6.2.3 Adsorption/desorption of methylene blue ................................................... 112
6.3. Results and Discussion ...................................................................................... 113 6.3.1 Synthesis and characterization of magnetic nanoparticles .......................... 113 6.3.2 Adsorption of MB onto CM-β-CD modified MNPs ................................... 122 6.3.3 Adsorption interactions ............................................................................... 135 6.3.4 Desorption and regeneration experiments ................................................... 137
6.4 Cost analysis ....................................................................................................... 139 6.5 Conclusions ........................................................................................................ 140
Table of contents
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Chapter 7 Carboxymethyl-β-cyclodextrin conjugated magnetic nano-adsorbents for removal of copper ions from aqueous water4 ............................................................... 142
7.1 Introduction ........................................................................................................ 142 7.2 Experimental ...................................................................................................... 145
7.2.1 Materials ...................................................................................................... 145 7.2.2 Adsorption/ desorption of Cu2+ ions ........................................................... 145
7.3 Results and Discussion ....................................................................................... 146 7.3.1 Synthesis and characterization of magnetic nanoparticles .......................... 146 7.3.2 Adsorption of Cu2+ ions on CMCD-MNP(C) ............................................. 149 7.3.3 Adsorption interactions ............................................................................... 159 7.3.4 Desorption and regeneration studies ........................................................... 163
7.4 Conclusions ........................................................................................................ 164 Chapter 8 Multifunctional core-shell silica nanoparticles with magnetic, fluorescence, specific cell targeting and drug-inclusion functionalities ...................... 166
8.1 Introduction ........................................................................................................ 166 8.2 Experimental ...................................................................................................... 169
8.2.1 Materials ...................................................................................................... 169 8.2.2 Synthesis of multifunctional core-shell silica magnetic nanoparticles ....... 169 8.2.3 Cell culture and intracellular uptake study .................................................. 172 8.2.4 Cytotoxicity assay ....................................................................................... 172 8.2.5 Inclusion/adsorption of retinoic acid ........................................................... 173 8.2.6 Release study ............................................................................................... 173
8.3 Results and discussions ...................................................................................... 174 8.3.1 Synthesis and characterization of as-synthesized magnetic nanoparticles .. 174 8.3.2 Cytotoxicity assay ....................................................................................... 182 8.3.3 Confocal microscopy observations ............................................................. 182 8.3.4 Drug inclusion/adsorption and release studies ............................................ 184
8.4 Conclusions ........................................................................................................ 189 Chapter 9 Summary and Recommendations ........................................................... 190
9.1 Summary of findings .......................................................................................... 190 9.2 Recommendations for future work ..................................................................... 192 9.3 Research opportunities ....................................................................................... 199
References .................................................................................................................... 201 Appendix A: Supporting information for Chapter 5 .................................................... 229 Appendix B: Supporting information for Chapter 8 .................................................... 237 Appendix C .................................................................................................................. 241 List of publications ....................................................................................................... 250
Summary
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Summary
Magnetic nanoparticles (MNPs) due to their high specific surface area,
biocompatibility, low toxicity and strong magnetic responsivity, have emerged as
excellent materials in many fields, such as biology, medicine, environment and material
science. In this work, we designed and fabricated β-cyclodextrin (β-CD) conjugated
MNPs which could be used in various bio- and environmental applications.
Cyclodextrins are natural oligosaccharides which have the molecular
inclusion/complexation capabilities through host-guest interactions with a wide variety
of organic and inorganic molecules. Tagging cyclodextrins with magnetic, stable
nanoparticles makes them magneto-responsive and may lead to a new generation of
adsorbents which will provide good opportunities for applications in the fields of bio-
separation/purification, contaminants removal from wastewater in environment
pollution cleanup and hydrophobic drug delivery. In this thesis work, tosyl-β-
cyclodextrin (Ts-β-CD) and carboxymethyl-β-cyclodextrin (CM-β-CD) conjugated
Fe3O4 MNPs were fabricated using different synthetic routes. Functionalized
nanoparticles were characterized with FTIR, TEM, XPS, XRD and TGA etc.
The use of Ts-β-CD grafted 3-aminopropyltriethoxysilane (APES) modified MNPs
(CD-APES-MNPs) as a solid-phase artificial chaperone to assist protein refolding in
vitro was demonstrated using carbonic anhydrase bovine (CA) as model protein. Our
refolding results show that a maximum of 85% CA refolding yield could be achieved
using these β-CD-conjugated magnetic nanoparticles which was at the same level as
that using liquid-phase artificial chaperone-assisted refolding. In addition, the
secondary and tertiary structures of the refolded CA were the same as those of native
Summary
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protein under optimal conditions. These results indicate that CD-APES-MNPs are
suitable and efficient materials (stripping agents) for solid-phase artificial chaperone-
assisted refolding due to easier and faster separation of these nanoparticles from the
refolded samples and also due to recycling of the stripping agents.
Selective recognition and separation of nucleosides (guanosine and adenosine) were
studied using CM-β-CD grafted APES modified MNPs (CMCD-APES-MNPs).
CMCD-APES-MNPs showed a higher adsorption ability and selectivity for guanosine
than adenosine under identical conditions. For better understanding to gain insights into
the molecular recognition mechanism of nucleosides and CM-β-CD, the inclusion
relation between the immobilized CM-β-CD and the guest substrates were investigated
through FTIR, UV-vis spectrophotometer and circular dichroism. Our results indicate
that this adsorbent would be a promising tool for easy and selective adsorption and
separation, analysis of nucleosides and nucleotides in biological samples. In fact, this
study provides a practical way to separate organic molecules based on the difference of
binding property by forming host–guest inclusion.
Adsorption behaviors of dyes (methylene blue) and heavy metals (Cu2+ ions) onto CM-
β-CD grafted magnetic nanocomposites were studied from equilibrium and kinetic
viewpoints. The grafted CM-β-CD on the iron oxides nanoparticles contributed to an
enhancement of the adsorption capacities because of the strong abilities of the multiple
hydroxyl/carboxyl groups and the inner cores of the hydrophobic cavity in CM-β-CD to
form complexes with metal ions and organic pollutants, respectively. The adsorption of
both pollutants onto CM-β-CD modified MNPs was found to be dependent on pH and
temperature. To gain insight into adsorption interactions, FTIR and XPS data were
introduced for better understanding. The regeneration and reusability studies suggest
Summary
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that CM-β-CD conjugated MNPs could be used as easily separable, recyclable and
effective adsorbents for the removal of organic/inorganic pollutants from aqueous
solution in environment pollution cleanup.
Highly uniform magnetic nanocomposite materials [Fe3O4@SiO2(FITC)-FA/CMCD
NPs] possessing an assortment of functionalities: superparamagnetism, luminescence,
cell-targeting, hydrophobic drug storage and delivery were also fabricated in this work.
Magnetic particle Fe3O4 is encapsulated within a shell of SiO2 that ensures
biocompatibility of the nanocomposite as well as act as a host for fluorescent dye
(FITC), cancer-targeting ligand (folic acid), and a hydrophobic drug storage-delivering
vehicle (β-CD). Inclusion/release of hydrophobic drug by these multifunctional
nanoparticles was studied using all-trans-retinoic acid (RA) as a model drug. Our
preliminary results suggest that such core-shell nanocomposite can be a smart
theranostic candidate for simultaneous bioimaging, magnetic control, cancer cell-
targeting and drug delivery.
Nomenclature
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Nomenclature Greek letters θ Half diffraction angle, (deg)
λ Wavelength of X-ray, (nm)
β Half width of XRD diffraction line (rad)
Hc Coercive field
Ms Saturation magnetization
Mr Remanent magnetization
d Particle size
ξ Zeta potential
TB Blocking temperature
Co Initial concentration (mg/ml)
Ce Equilibrium concentration (mg/ml)
qm Maximum adsorbed quantity (mg/g solid)
qe Adsorbed quantity at equilibrium (mg/g solid)
qt Adsorbed quantity at any time t (mg/g solid)
k1 Pseudo-first-order rate constant
k2 Pseudo-second-order rate constant
RL Dimensionless separation factor
Abbreviations CD Cyclodextrin
β-CD β-cyclodextrin
Ts-β-CD Mono-tosyl-β-cyclodextrin
Nomenclature
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CM-β-CD or CMCD Carboxymethyl-β-cyclodextrin
MNPs Magnetic nanoparticles
NPs Nanoparticles
SPIONs Superparamagnetic iron oxide nanoparticles
CA Carbonic anhydrase from bovine
G Guanosine
A Adenosine
MB Methylene blue
APES 3-aminopropyltriethoxy silane
CTAB Cetyltrimethylammonium bromide
TTAB Tetradecyltrimethylammonium bromide
SDS Sodium dodecyl sulfate
FITC Fluorescein isothiocyanate
FA Folic acid
RA All-trans-retinoic acid or vitamin A
BET Brunauer- Emmett-Teller method
CD Circular Dichroism
FTIR Fourier transform infrared spectroscopy
TEM Transmission electron microscopy
FESEM Field emission scanning electron microscopy
TGA Thermogravimetric analysis
VSM Vibrating sample magnetometer
XPS X-ray photoelectron spectroscopy
XRD X-ray diffraction
HPLC High performance liquid chromatography
List of Figures
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List of figures Figure 2-1 Theoretical magnetization versus magnetic field curve for
superparamagnetic (SPM) and ferri- or ferromagnetic nanoparticles (FM) where the coercive field (HC), the saturation magnetization (MS) and the remanent magnetization (MR) parameters are indicated [27]. ............................................... 10
Figure 2-2 Variation of the coercivity (HC) of magnetic nanoparticles with size [27]. . 11 Figure 2-3 (a) Ligand exchange; (b) ligand addition; and (c) inorganic coating. F
represents functional chemical group that can be used for further conjugation [29]. ................................................................................................................................ 12
Figure 2-4 Schematic diagram of the magnetic separation of non magnetic targets ..... 16 Figure 2-5 Structures and molecular dimensions of α, β, γ- cyclodextrins. ................... 19 Figure 2-6 Functional structure of cyclodextrin ............................................................. 20 Figure 2-7 Schematic representation of cyclodextrin inclusion complex formation: p-
Xylene is the guest molecules; the small circles represent the water molecules. .. 22 Figure 2-8 Processes for the recovery of inclusion body proteins [61]. ........................ 26 Figure 2-9 Different approaches of protein refolding; (A) dilution mode (B) dilution-
additive assisted protein refolding (C) artificial chaperone assisted protein refolding. ................................................................................................................ 28
Figure 2-10 A typical high-gradient magnetic separation facility [121]. ....................... 34 Figure 2-11 Schematic diagram of the superconducting HGMS system [123]. ............ 35 Figure 4-1 Preparation steps for fabricating β-CD bonded magnetic nanoparticles. ..... 63 Figure 4-2 FTIR spectra of (a) Uncoated Fe3O4 MNPs, (b) APES-MNPs, (c) CD-APES-
MNPs, and (d) β-CD. ............................................................................................. 64 Figure 4-3 Typical TEM images and hydrodynamic size distribution of APES-MNPs
(a) &(c) and CD-APES-MNPs (b) & (d), respectively. ......................................... 65 Figure 4-4 (i) XRD patterns of (a) uncoated magnetic nanoparticles (MNPs), (b) APES
modified magnetic nanoparticles (APES-MNPs), and (c) β-CD modified magnetic nanoparticles (CD-APES-MNPs).(ii) Magnetization curves for uncoated and β-CD coated Fe3O4 magnetic nanoparticles measured by VSM at room temperature. .... 67
Figure 4-5 Schematic diagram of CD-APES-MNPs assisted protein refolding in-vitro.
................................................................................................................................ 69
List of Figures
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Figure 4-6 Time course of refolding yield of CA (■) and residual CTAB concentration (●) in refolded samples in presence of 40 mg/ml CD-APES-MNPs. The CTAB concentration in the capturing step was set at 2mM and final CA concentration was 0.029 mg/ml. .......................................................................................................... 70
Figure 4-7 Relative changes in the recovered enzymatic activity of the assisted
renatured CA as a function of detergent type and the solid phase quantity. Protein-detergent (CTAB, □: TTAB, Δ and SDS, ○) complex solution (2.7 ml, with a detergent concentration of 2mM in Tris–Sulfate buffer, pH 7.75) was added to 1.3 ml suspension containing various amounts of β-CD bonded magnetic nanoparticles. The final protein concentration was 0.029 mg/ml. The residual CTAB concentration (●) in the supernatant of the suspension was also measured. ................................................................................................................................ 72
Figure 4-8 Proposed mechanism by Hanson and Gellman for cyclodextrin-induced
folding from a protein–detergent complex [187]. U–dn, protein–detergent complex generated during the capture step (contains only one protein molecule); U, unfolded protein molecule; d, one detergent molecule; U–dn–m, protein–detergent complex from which detergent has been partially stripped away; (U–dn–m)p, partially stripped protein–detergent complex that has self-associated to form a species containing multiple protein molecules; F, first detergent-free form of the protein (extent of folding unspecified); N, native state of the protein. .................. 73
Figure 4-9 Refolding yield of thermally denatured CA in the presence of different
concentrations of β-CD in liquid phase artificial chaperone assisted refolding approach. The denatured CA (0.043 mg/ml) was diluted with water containing different concentration of β-CD (0-16 mM). The error bars represent standard error of mean for three independent measurements. .............................................. 74
Figure 4-10 The effect of CA concentration and the amount of CD-APES-MNPs on CA
renaturation yield. The CTAB concentration in the capturing step was set at 2 mM. The protein concentration in the captured state were 0.043 (●), 0.075 (■) and 0.15 (▲) mg/ml. ............................................................................................................. 75
Figure 4-11 Effect of various concentrations of CD-APES-MNPs on the intrinsic
fluorescence emission of refolded CA samples. (a) Intrinsic fluorescence spectra of refolded samples in the presence of different amounts of CD-APES-MNPs (0 mg/ml, curve 1; 10 mg/ ml, curve 2; 20 mg/ml, curve 3; 30 mg/ml, curve 4; 40 mg/ml, curve 5 and 50 mg/ml, curve 7). Curve 6 represents the native CA. (b) Maximum emission wavelength peak point of the refolded CA in presence of various concentrations of CD-APES-MNPs. ......................................................... 77
Figure 4-12 ANS fluorescence spectra of native (6) CA and refolded CA in the presence
of 0 (curve 1), 10 (curve 2), 20 (curve 3), 30 (curve 4), 40 (curve 5), 50 (curve 7) and 60 (curve 8) mg/ml of CD-APES-MNPs added in the solution. The final protein concentration was 0.029 mg/ml for all samples. Samples were excited at 360 nm. ................................................................................................................... 78
Figure 4-13 Far-UV circular dichroism spectra of native (curve 1) and the refolded CA
samples in the presence of 10 (curve 4), 30 (curve 3) and 40 (curve 2) mg/ml of the
List of Figures
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CD-APES-MNPs added in the solution. Each spectrum was obtained at 25C with a 1mm path length cell and is the average of two scans. All necessary corrections were made for background absorption. .................................................................. 80
Figure 5-1 Chemical structure of adenosine (A) and guanosine (G) ............................. 84 Figure 5-2 Schematic illustration of the fabrication of the carboxymethyl-β-cyclodextrin
modified magnetic nanoadsorbents and mechanism for selective separation of nucleosides. ............................................................................................................ 88
Figure 5-3 FTIR spectra of as-synthesized magnetic nanoparticles-(a) uncoated MNPs,
(b) APES-MNPs, (c) CMCD-APES-MNPs and (d) CM-β-CD. ............................ 90 Figure 5-4 (a) TEM micrograph and size distribution of CMCD-MNPs measured by
TEM and (b) hydrodynamic size distribution by DLS. .......................................... 91 Figure 5-5 TGA curves for (a) uncoated MNPs, (b) APES-MNPs and (c) CMCD-
APES-MNPs. .......................................................................................................... 92 Figure 5-6 Zeta potentials of uncoated, APES and CM-β-CD modified magnetic
nanoparticles at different pH. ................................................................................. 93 Figure 5-7 Effect of pH on the adsorption of A and G by CMCD-APES-MNPs:
Conditions: Initial A and G concentration: 1.0 g/l, temperature: 25oC. ............... 94 Figure 5-8 Effect of contact time on nucleoside adsorption by CMCD-APES-MNPs.
Conditions: Initial A or G concentration: 1.0 g/l, pH: 5, temperature: 25oC, adsorbent: 110 mg. ................................................................................................. 95
Figure 5-9 Effects of initial concentrations on the adsorption of A and G onto CMCD-
APES-MNPs. Conditions: Initial A and G concentration: 0.1,0.2,0.4,0.6,0.8,1.0g/l, pH: 5, temperature: 25oC, adsorbent: 110 mg. ....................................................... 96
Figure 5-10 Langmuir plot illustrating the linear dependence of Ce/qe on qe for A and G.
................................................................................................................................ 97 Figure 5-11 Selective adsorption of A and G mixture solutions using (a) CMCD-APES-
MNPs and (b) APES-MNPs. Conditions: pH: 5, temperature: 25oC, adsorbent: 110mg. .................................................................................................................. 100
Figure 5-12 Absorption spectra of (a) adenosine and (b) guanosine (1x10–5 mol l–1) in
pH 5.0 buffers containing various concentrations of CM-β-CD. Concentration of CM-β-CD: (1) 0, (2) 1.0x10–3, (3) 2.0x10–3, (4) 2.5x10–3 and (5) 3.5x10–3 mol l–1. Insets: Double reciprocal plot for nucleoside complexes with CM-β-CD. .......... 103
Figure 5-13 FTIR spectra of adenosine and guanosine and their inclusion complexes
with β-CD. ............................................................................................................ 106 Figure 6-1 An illustration for the carboxymethylation and binding of cyclodextrin onto
Fe3O4 nanoparticles by two different methods. .................................................... 115
List of Figures
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Figure 6-2 FTIR spectra of (a) uncoated MNPs, (b) CMCD-MNP(P), (c) CMCD-MNP(C) and (d) CM-β-CD. ................................................................................. 116
Figure 6-3 TEM micrographs and hydrodynamic diameter distributions for CMCD-
MNP(C) (a) & (b); and CMCD-MNP(P) (c) & (d) respectively. ......................... 118 Figure 6-4 TGA curves for (a) bare Fe3O4 MNP, (b) CMCD-MNP(C) and (c) CMCD-
MNP(P). ............................................................................................................... 119 Figure 6-5 (i) XRD patterns and (ii) magnetization curves (a) uncoated Fe3O4 MNPs,
(b) CMCD-MNP(C) and (c) CMCD-MNP(P). .................................................... 120 Figure 6-6 Zeta potentials of bare Fe3O4 MNP, CMCD-MNP(C) and CMCD-MNP(P).
.............................................................................................................................. 121 Figure 6-7 The schematic representation of the selective adsorption and separation of
MB by CM-β-CD modified magnetic nanoparticles. ........................................... 123 Figure 6-8 UV-vis spectra of methylene blue (0.5 mg/ml) at pH~12 upon addition of (a)
0 mg MNPs; (b) 50 mg uncoated MNPs; (c) 50 mg CMCD-MNP(C); (d) 50 mg CMCD-MNP(P). .................................................................................................. 124
Figure 6-9 The molecular structure of MB. ................................................................. 125 Figure 6-10 Effect of pH on the adsorption of MB by CM-β-CD modified magnetic
nanoparticles. Conditions: (Initial MB concentration: 2 mg/ml, temperature: 25ºC, adsorbents: 120 mg). ............................................................................................ 125
Figure 6-11 Effect of contact time on the adsorption of MB onto CMCD-MNP(P) (a)
and CMCD-MNP(C) (b) and pseudo-second order kinetics of MB adsorption onto CMCD-MNP(P) (c) and CMCD-MNP(C) (d). Conditions: (Initial MB concentration: 0.25, 0.50 and 2 mg/ml, pH: 8, temperature: 25ºC). .................... 127
Figure 6-12 Equilibrium isotherm for the adsorption of MB on CMCD-MNP(P) and
CMCD-MNP(C) at 25ºC. ..................................................................................... 131 Figure 6-13 Langmuir plot illustrating the linear dependences of Ce/qe on Ce (a) and
Freundlich plot illustrating the linear dependences of lnqe on lnCe (b). .............. 132 Figure 6-14 Separation factor for MB onto CM-β-CD modified magnetic nanoparticles
at pH 8 and 25ºC. ................................................................................................. 134 Figure 6-15 FTIR spectra of CMCD-MNP(P) of before (a) and after (b) adsorption of
MB, and only MB (c). .......................................................................................... 136 Figure 6-16 (a) Desorption of MB from CMCD-MNP(P) as a function of acetic acid
concentration in methanol, and (b) performance of CMCD-MNP(P) by multiple cycles of regenaration. Adsorption conditions: (CMCD-MNP(P): 120 mg; MB concentration: 2 mg/ml; temperature: 25ºC; pH: 8; contact time: 2 hr). Desorption
List of Figures
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conditions: (CMCD-MNP(P): 120 mg; temperature: 25ºC; contact time: 2 hr; desorption eluent: methanol with 5 % acetic acid) .............................................. 138
Figure 7-1 (a) TGA curves for uncoated Fe3O4 MNPs and CMCD-MNP(C); (b) Effect
of the amount of CM-β-CD in solution on the grafted CM-β-CD contents onto magnetic nanoparticles. ........................................................................................ 147
Figure 7-2 Zeta potentials of uncoated and CM-β-CD coated magnetic nanoparticles
and copper(II) nitrate solution at different pH. .................................................... 148 Figure 7-3 Effect of pH on the adsorption of Cu2+ ions by CMCD-MNP(C) at 25ºC.
Open points: qe vs. initial pH values; solid points: equilibrium pH values vs. initial pH values. ............................................................................................................. 150
Figure 7-4 (a) Effect of contact time on Cu2+ adsorption by CMCD-MNP(C); (b)
pseudo-second-order kinetics for adsorption of Cu2+. (Initial pH: 6, temperature: 25ºC). .................................................................................................................... 153
Figure 7-5 Equilibrium isotherms for Cu2+ adsorption by CMCD-MNP(C) at different
temperature and by uncoated Fe3O4 nanoparticles (inset) at 25ºC and initial pH 6. .............................................................................................................................. 155
Figure 7-6 The Langmuir (a) and Freundlich (b) isotherm plots for Cu2+ ions adsorption
by CMCD-MNP(C) at pH 6. ................................................................................ 157 Figure 7-7 van’t Hoff plot for the adsorption of Cu2+ by CMCD-MNP(C). ................ 159 Figure 7-8 FTIR spectra of CMCD-MNP(C) before (a) and after (b) adsorption of Cu2+
ions. ...................................................................................................................... 160 Figure 7-9 XPS spectra of CMCD-MNP(C): (a) C1s before adsorption; (b) C1s after
adsorption; (c) O1s before adsorption; and (d) O1s after adsorption; (e) Cu(2p3/2) core-level spectrum of Cu-loaded CMCD-MNPs. ............................................... 162
Figure 7-10 (a) Kinetics of desorption of Cu2+ from Cu-loaded CMCD-MNP(C) in
0.1M citric acid solution; (b) performance of CMCD-MNPs by multiple cycles of regeneration. ......................................................................................................... 164
Figure 8-1 Synthesis of CM-β-CD conjugated fluorescein-doped magnetic mesoporous
silica nanoparticles [Fe3O4@SiO2(FITC)-FA/CMCD NPs]. ............................. 175 Figure 8-2 UV–vis spectra of (a) pure FITC, (b) pure folic acid, (c) Fe3O4@SiO2, (d)
Fe3O4@SiO2(FITC), and (e) Fe3O4@SiO2(FITC)–FA/NH2 NPs in water. ......... 176 Figure 8-3 Excitation (dashed line, recorded at 515 nm emission) and emission (solid
line, recorded at 480 nm excitation) spectra of Fe3O4@SiO2(FITC)-FA/CMCD NPs dispersed in water. Insets: visual photographs of (a) Fe3O4@SiO2(FITC) NPs dispersion in water under room light; (b) the green emission of these MNPs in water and (c) in response to a NdFeB magnet under the 365 nm excitation portable UV lamp. .............................................................................................................. 178
List of Figures
xv
Figure 8-4 TEM mages of (a) OA-MNPs, (b) Fe3O4@SiO2(FITC) NPs and (c) Fe3O4@SiO2(FITC)-FA/CMCD NPs. .................................................................. 179
Figure 8-5 (A) Zeta potentials of as-synthesized nanoparticles at pH 7.4; (B) TGA
curves of (a) Fe3O4@SiO2(FITC)-FA/NH2 and (b) Fe3O4@SiO2(FITC)-FA/CMCD MNPs. ................................................................................................................... 180
Figure 8-6 In vitro cell viability of HeLa cells incubated with Fe3O4@SiO2(FITC)-
FA/CMCD MNPs at different concentrations for 6 and 24 h at 37oC. ................ 182 Figure 8-7 CLSM images of cells incubated with Fe3O4@SiO2(FITC)-FA/CMCD NPs
at 37oC: (A) HeLa cells (FA-positive) and (C) MCF-7 cells (FA-negative). Bright field images of HeLa cells (B) and MCF-7 cells (D) were also supplied. The nanoparticle concentration is 50 µg/ml and the incubation time is 1 h. ............... 183
Figure 8-8 (a) Absorption spectra of RA (7.3x10–6 mol l–1) in pH 7.4 buffers containing
various concentrations of CM-β-CD. Concentration of CM-β-CD: (1) 0, (2) 7.5x10–5, (3) 1.1x10–4, (4) 1.6x10–4, (5) 2.3x10–4, (6) 5.6x10–4 and (7) 3.75x10–3 mol l–1. Inset: Double reciprocal plot for RA complexes with CM-β-CD. .......... 185
Figure 8-9 Effects of nanoparticle dosage on adsorption/inclusion of retinoic acid. ... 187 Figure 8-10 Release profile of retinoic acid from Fe3O4@SiO2(FITC)-FA/CMCD NPs.
.............................................................................................................................. 188 Figure 9-1 Schematic presentation of complexation of CMCD-MNPs magnetic
nanoparticles with both organic and inorganic pollutants. ................................... 196 Figure 9-2 Schematic of the proposed multistage countercurrent continuous contact
[313]. .................................................................................................................... 197 Figure 9-3 Schematic description of the overall process: in the first part (left), the target
solute is extracted from a large volume using modified magnetic nanoparticles. In the second part (right), the loaded magnetic nanoparticles are washed (enrichment) and release the solute into a small volume. The moving solids (the true moving bed, i.e. the nanoparticles) links the two tank systems and transports the nanoparticles-bound solute against the concentration gradient between the large tank (left, low concentration) and the small tank (right, high concentration) [314]. .............................................................................................................................. 198
List of Tables
xvi
List of tables Table 2-1 Materials used for coating or encapsulating iron oxide magnetic nanoparticles
and their bio- and environmental applications. ...................................................... 14 Table 2-2 List of magnetic separation applications [12]. ............................................... 18 Table 2-3 Properties of α, β, γ- cyclodextrin .................................................................. 21 Table 2-4 Protein refolding-dilution additive mode ....................................................... 29 Table 2-5 Liquid phase artificial chaperone-assisted protein refolding ......................... 30 Table 2-6 Solid-phase artificial chaperone-assisted protein refolding ........................... 31 Table 2-7 Adsorption capacities and experimental conditions of functionalized
magnetic nanoparticles for various heavy metals and dye removal from wastewater ................................................................................................................................ 36
Table 2-8 Adsorption capacities and experimental conditions of cyclodextrin based
materials for various dye and heavy metals removal from wastewater ................. 38 Table 4-1 Data on chemical analysis of magnetic nanoparticles modified with APES
and β-CD. ............................................................................................................... 66 Table 5-1 Adsorption isotherm parameters for A and G at pH 5 and 25oC ................... 98 Table 6-1 Characteristics of Methylene Blue (MB) dye .............................................. 111 Table 6-2 Adsorption kinetic parameters of MB onto the CM-β-CD modified magnetic
nano-adsorbents at pH 8 and 25ºC ....................................................................... 129 Table 6-3 Adsorption isotherm parameters for MB adsorption onto CM-β-CD modified
magnetic nanoparticles at 25ºC ............................................................................ 133 Table 6-4 Maximum adsorption capacities (qm in mg/g) for MB by some other
adsorbents reported in literature ........................................................................... 133 Table 7-1 Adsorption kinetic parameters of Cu2+ onto CMCD-MNP(C). ................... 154 Table 7-2 Adsorption isotherm parameters for Cu2+ ions adsorption on uncoated and
CM-β-CD coated magnetic nanoparticles at pH 6. .............................................. 156 Table 7-3 Comparison of maximum adsorption capacity of CMCD-MNP(C) with those
of some other magnetic adsorbents reported in literature for Cu2+ ions adsorption. .............................................................................................................................. 157
List of Tables
xvii
Table 7-4 Thermodynamic parameters for the adsorption of Cu2+ ions onto CMCD-MNP(C). ............................................................................................................... 159
Table 7-5 FTIR absorption frequencies (cm-1) of CMCD-MNP(C) before and after
adsorption and their possible assignments ........................................................... 160 Table 8-1 Surface composition of Fe3O4@SiO2(FITC), Fe3O4@SiO2(FITC)-FA/NH2
and Fe3O4@SiO2(FITC)-FA/CMCD nanoparticles obtained from XPS spectra. 181
Chapter: 1 Introduction
1
Chapter 1 Introduction
1.1 General background
Recently nano-sized materials, due to their unique size as well as physico-chemical
properties have attracted significant interest and have now become one of the most
important research and development frontiers in modern science. Among them,
magnetite (Fe3O4) nanoparticles due to their good biocompatibility, superior
superparamagnetic property, nontoxicity and easy preparation process, are becoming
very popular and promising materials now-a-days [1]. They have attracted increasing
attention and enormous interest in various fields, such as environmental and biomedical
applications including enzyme immobilization, protein separation, magnetic resonance
imaging (MRI), hyperthermia, targeting drug delivery system [2,3].
Magnetite nanoparticles with superparamagnetism can be easily magnetized with an
external magnetic field and demagnetized immediately once the external magnetic field
is removed, not retaining any magnetism [4]. However, due to high specific surface
energy and anisotropic dipolar attraction, magnetite nanoparticles tend to aggregate
together into larger clusters which lead to a possible loss of superparamagnetism and
limit their applications. Therefore, the surface modification of nanoparticles is
indispensable and the particle surface can be modified by inorganic or organic coating.
Surface functionalization of MNPs endows the particles some important properties that
the bare particles lack. Modification of the surface of MNPs not only prevents
aggregation/agglomeration of the particles, leading to colloidal stability, but also
renders them with water-solubility, biocompatibility, non-toxicity, nonspecific
adsorption to cells, and bioconjugation. Considerable efforts have been made to modify
the surface of magnetic nanoparticles and the preparation of organic–inorganic
Chapter: 1 Introduction
2
nanocomposites. The combination of inorganic and organic components in a single
particle at the nano-sized level has made accessible an immense area of new functional
materials [5]. Inorganic materials (silica, gold etc.) [2], natural [6,7] or synthetic
polymers [5,8] are frequently employed as coating materials to impart surface
reactivity. Natural polymers include chitosan, dextran, gelatin, starch, cyclodextrin etc.
and synthetic polymers are polyacrylic acid, polyvinyl chloride, poly vinyl alcohol etc.
Cyclodextrins (CDs) are a group of naturally cyclic oligosaccharides, with six, seven,
or eight glucose subunits linked by α-(1, 4) glycosidic bonds in a torus shaped structure
and are denominated as α-, β-, and γ-CD, respectively. Attention has recently been
focused on cyclodextrin based polymeric materials in a wide variety of applications due
to their unique sorption properties [9]. The sustained interest in the research and
application of cyclodextrin-based copolymer materials is attributed to the ability of
forming inclusion compounds with a wide range of organic molecules through host-
guest interactions: the interior cavity of the molecule provides a relatively hydrophobic
environment into which an apolar organic compound can be trapped. Recently, a
number of insoluble cyclodextrin polymer or co-polymers have been widely used for
various application such as contaminants removal from wastewater, protein refolding,
drug delivery etc. [10,11]. However, the difficulty in separating those powdery
cyclodextrin-based adsorbents, except high speed centrifugation, from treated effluent
limits their practical applications. Magnetic assisted adsorption separation technology
provides an alternative method to separate powdery adsorbents from solution
effectively.
Magnetic separation has been considered as an effective method for solid–liquid phase
separation techniques [12]. It has drawn more and more attention due to its merits of
Chapter: 1 Introduction
3
speed, accuracy, simplicity and effectiveness. Magnetic separation has numerous
applications in biotechnology and biomedical diagnostics such as cell sorting, nucleic
acid detachment, enzyme immobilization, drug delivery and protein adsorption and
purification [2,5,13-16]. Magnetic separation involves magnetic particles, carrier
liquids, complexes and target molecules. Magnetic separation has been applied in many
areas to remove, isolate and/or concentrate the desired components from a sample
solution. The principle of the magnetic separation process is, at first the magnetic
particles are combined with the intermediates (polymers, ligands, surfactant etc.) to
form a complex. These complex particles can interact with the target molecules and
then by using gradient magnetic field the target is separated from the mixture. The
basic concept is to utilize the physical interactions between magnetic complex particles
and target molecules and as well as the specific chemical interactions between the
particles and target molecules.
1.2 Research objective and significance
Many researchers focused on synthesis of nano-sized magnetic particles with various
surface reactivity such as natural or synthetic polymers (i.e. chitosan, polyacrylic acid,
poly-N-isopropylacrylamide etc.). However, no work so far has been carried out on the
preparation of organic-inorganic nanocomposite materials through covalent binding
between MNPs and CDs and their use as a tool for bioseparation/purification and in the
cleanup of environmental pollutants. The incorporation of inclusion/complexation
property of CDs and the superparamagnetic property of magnetite (Fe3O4) in one nano-
system is considered to be a promising tool for wide variety of applications such as bio-
separation, drug delivery, molecular recognition, environmental pollution control etc.
Chapter: 1 Introduction
4
In bio-applications, particularly in artificial chaperone-assisted protein refolding
approach [see Section 2-4], soluble β-CD or some insoluble β-CD polymers have been
used as stripping agents for detergent molecules. However, excessive soluble β-CD and
β-CD/detergent complexes are to be removed from the refolded products for
downstream purification. The insoluble stripping agents also suffer lower protein
refolding yield than the corresponding values obtained from the liquid-phase artificial
chaperone assisted method and separation inconvenience. Therefore, efforts are still
required to carry out investigation for new promising striping agent which can act as a
solid-phase artificial chaperone.
The use of β-CD as an adsorbent with molecular recognition function for the selective
removal of biomolecules from the aqueous stream is limited due to its inherent water
solubility. We presume that the adsorbent formed by anchoring β-CD to magnetite
would improve its molecular recognition ability and additionally, the reaction converts
β-CD into an insoluble matrix which in turn facilitate the separation. As far as we
know, cyclodextrin bonded magnetic nanoparticles for selective recognition and
separation of biomolecules has not been reported yet. Furthermore, functionalization of
the magnetic nano-surface by β-CD in well-defined host-guest interactions may also
lead to a generation of nanocarriers for hydrophobic drug delivery applications.
β-CD has potentials applications in pollutants removal from industrial wastewater as it
can facilitate the incorporation of various organic compounds such as dyes,
pharmaceutical waste chemicals, phenolic compounds etc. into its hydrophobic cavity
through host-guest interaction. It is also reported that β-CD has the ability to complex
heavy metals such as cadmium, lead, mercury etc. through the interactions between the
metal ions and multiple hydroxyl groups on it. Hence, β-CD is a procedure of choice
Chapter: 1 Introduction
5
for decontaminating technique. Considering the complexation property of β-CD and the
separation convenience of magnetic Fe3O4 nanoparticles, grafting of β-CD on the
magnetic nanoparticles is expected to be a promising alternative to develop magnetic
nano-adsorbent for the efficient removal of both organic and inorganic pollutants from
wastewater. To evaluate their effectiveness in clean up of environmental pollutants
adsorption equilibrium, adsorption kinetics and effects of various parameters (such as
pH, temperature etc.) on adsorption need to be studied in details.
The desired objectives of this work are summarized as follows:
1. Grafting of β-CD derivatives on magnetic nanoparticle surface using different
synthetic approaches and characterization of as-synthesized magnetic particles using
different analytical tools i.e. FTIR, TEM, XPS, TGA and elemental analysis.
2. Evaluation of the effectiveness of β-CD conjugated magnetic nanoparticles as solid-
phase artificial chaperone in refolding of protein in-vitro.
3. Individual and selective adsorption study of nucleosides (guanosine and adenosine)
onto CM-β-CD conjugated magnetic nanoparticles having molecular recognition and
magnetic properties.
4. Adsorption behaviors of organic dye (methylene blue) using CM-β-CD conjugated
magnetic nanoparticles as nano-adsorbents.
5. Adsorption of copper ions onto CM-β-CD conjugated magnetic nano-adsorbents and
study of adsorption interactions.
6. Synthesis and characterization of CM-β-CD grafted multifunctional silica core-shell
nanoparticles with the magnetic, fluorescent, specific cell targeting and inclusion
Chapter: 1 Introduction
6
functionalities and investigation of the possibility of the applications of these
nanoparticles in biomedical research fields, particularly for imaging, specific cell
targeting and hydrophobic drug inclusion/delivery etc.
1.3 Organization of the thesis
The present thesis is organized into eight chapters. Chapter 1 gives a brief introduction
of magnetic nanoparticles with magnetic separation technique and β-CD coated
magnetic nanoparticles, the objectives of the thesis and a structural organization of the
whole thesis. A review of related literature is presented in chapter 2. Chapter 3 presents
various characterization techniques that were used throughout my thesis work. Chapter
4 presents the characterization results of magnetic nanoparticles and also the results for
refolding of carbonic anhydrase (CA) using β-CD bonded magnetic nanoparticles as
solid-phase artificial chaperone. Chapter 5 shows the characterization results of as-
synthesized magnetic nano-adsorbents, adsorption behaviors of nucleosides (guanosine
and adenosine) at different experimental conditions and the adsorption mechanism. In
Chapter 6, the results of adsorption, desorption and adsorption interactions of organic
pollutant (methylene blue) with the nano-adsorbents are described where two types of
CD-conjugated magnetic nanoparticles were used as nano-adsorbents. Experimental
results on adsorptive removal of heavy metals (Cu2+ adsorption) from aqueous solution
using the same nanoadsorbents have been detailed in Chapter 7. Chapter 8 represents
the synthesis and characterization results of CM-β-CD grafted multifunctional silica
core-shell nanoparticles that combine the magnetic, fluorescent, specific cell targeting
and hydrophobic drug-inclusion functionalities. Overall conclusions together with
recommendations are given in Chapter 9.
Chapter: 2 Literature Review
7
Chapter 2 Literature Review
In Chapter 2, literature reviews on magnetic nanoparticles, their properties and surface
functionalization, cyclodextrin and its structural properties and applications, protein
refolding, pollutants removal from wastewater, adsorption and desorption processes are
presented.
2.1 Magnetic nanoparticles
The special and superior properties of nanomaterials have attracted much attention in
the past two decades. Particularly, magnetic nanoparticles (MNPs) with inherent
magnetic properties and high surface-to-volume ratio have continued to draw
considerable interest because of their diverse potential applications in biological,
environmental and medical diagnostic fields. One of the rapidly emergent research
subjects involving MNPs is their application in biological systems, including their
application in magnetic resonance imaging (MRI), targeted drug delivery, rapid
biological separation, biosensors, and magnetic hyperthermia therapy [2,3]. The most
commonly used magnetic particles are magnetite (Fe3O4) and maghemite (γ- Fe2O3).
Other types of magnetic particles are pure metal (Fe and Co) and spinel type
ferromagnets (MeO.Fe2O3, where M = Ni, Co, Mg, Zn, Mn).
2.1.1 Synthesis of superparamagnetic iron oxide nanoparticles
Due to unique size, biocompatibility, low toxicity and superparamagnetic properties,
magnetic nanoparticles are emerging as promising tools in various fields such as
physics, medicine, biology, environment and material science [1]. Several types of iron
oxides have been investigated recently in the field of nano-sized magnetic particles
(mostly maghemite, γ-Fe2O3, or magnetite, Fe3O4, single domains of about 5–20 nm in
Chapter: 2 Literature Review
8
diameter), among which Fe3O4 is a very promising and popular candidate since its
biocompatibility have already proven [1]. In last decades, numerous synthetic methods
have been developed to synthesize iron oxide nanoparticles including coprecipitation
[17], sol-gel synthesis [18], microemulsion synthesis [19], sonochemical reaction [20],
hydrothermal reaction [21], thermal decomposition [22], laser pyrolysis [23] etc.
Among these reported methods, the chemical co-precipitation may be the most
promising one because of its simplicity and productivity. In a chemical co-precipitation
method, aqueous FeCl3 and FeCl2 solutions are mixed at a concentration ratio of Fe3+
:
Fe2+
=2:1 in an aqueous ammonia solution, yielding Fe3O4 NPs with d = 3–15 nm. The
control of size, shape and composition of nanoparticles depends on the type of salts
used (chlorides, sulfates, nitrates, perchlorates), Fe2+
and Fe3+
ratio, pH, temperature and
ionic strength of the media [24,25]. In general, the overall reaction may be written as
follows [26]:
Fe2+ + 2Fe3+ + 8OH- Fe3O4 + 4H2O (2-1)
This method would critically affect the physical and chemical properties of the
nanosized magnetic particles. In addition, Fe3O4 NPs are not very stable under ambient
conditions and are easily oxidized to Fe2O3 or dissolved in an acidic medium. In order
to avoid the possible oxidation in the air, the synthesis of Fe3O4 NPs must be done in an
anaerobic condition. In this method, the reaction temperature is limited by the boiling
point of water, and iron oxide nanoparticles synthesized under these conditions exhibit
usually a low degree of crystallinity and large polydispersity
Compared to the co-precipitation method, the organic phase synthesis which relies on
pyrolysis of iron precursors with the presence of surfactants, allows better control over
Chapter: 2 Literature Review
9
the size and monodispersity of Fe3O4 nanoparticles. Two of the most widely used iron
precursors in organic phase iron oxide nanoparticle synthesis are Fe(acac)3 and
Fe(CO)5. Sun et al. showed that high-temperature reaction of Fe(acac)3 in the presence
of alcohol, oleic acid, and oleylamine can be used to produce size-controlled
monodisperse Fe3O4 nanoparticles [22]. The advantage of this method is that the
synthesis is not limited to simple iron oxide nanoparticles but can be extended to
various types of ferrite nanoparticles as well.
The microemulsion (water in oil: W/O) method using water droplets as nanoreactors in
a continuous phase (oil) in the presence of surfactant molecules is reported to be an
alternative and more controlled method [19]. The size of the NPs can be controlled by
controlling the size of the water droplets. Surfactants, which are responsible for
micellisation, can be utilised for the dispersion of iron oxide NPs.
2.1.2 Properties of magnetic particles
In contrast to bulk iron oxide, which is a multi-domain ferromagnetic material (exhibits
a permanent magnetization in the absence of a magnetic field), iron oxide magnetic
nanoparticles smaller than approximately 20–30 nm in size contain a single magnetic
domain with a single magnetic moment and exhibit superparamagnetism [4]. A material
in a paramagnetic phase is characterized by randomly oriented (or uncoupled) magnetic
dipoles, which can be aligned only in the presence of an external magnetic field and
along its direction. This type of material has no coercivity nor remanence, which means
that when the external magnetic field is switched off, the internal magnetic dipoles
randomize again, no extra energy is required to demagnetize the material and hence the
initial zero net magnetic moment is spontaneously recovered, see Figure 2-1. A
nanoparticle with such magnetic behavior is superparamagnetic (SPM).
Chapter: 2 Literature Review
10
Figure 2-1 Theoretical magnetization versus magnetic field curve for superparamagnetic (SPM) and ferri- or ferromagnetic nanoparticles (FM) where the coercive field (HC), the saturation magnetization (MS) and the remanent magnetization (MR) parameters are indicated [27].
The size reduction of magnetic materials shows interesting advantages that make them
more suitable for therapeutic and diagnostic techniques compared to their bulk
counterparts. Magnetic parameters such as the coercivity of the nanoparticles can be
finely tuned by decreasing their size. Moreover, a further reduction of the size below a
certain value of the radius, the so-called superparamagnetic radius (rSP), induces a
magnetic transition in particles where both ferro- and ferrimagnetic nanoparticles (FM)
become superparamagnetic and, as previously said, high magnetic moments are
observed under the effect of a magnetic field, but no remanent magnetic moment will
be present when the external magnetic field is removed. Superparamagetism is a
property strictly associated to nanostructured magnetic materials and arises when the
thermal energy is sufficiently high to overcome the magnetic stabilization energy of the
particle. Figure 2-2 explains how the coercivity of the nanoparticles varies (at a certain
temperature) when their size is decreased, until the superparamagnetic state is reached.
Chapter: 2 Literature Review
11
With increasing size, the particles will become blocked as thermal energy becomes
insufficient to allow the free rotation of spins. Superparamagnetic particles are usually
ordered below a blocking temperature, TB (blocking temperature is the transition
temperature between the ferrimagnetic and superparamagnetic state and is directly
proportional to the size of the particles).
Figure 2-2 Variation of the coercivity (HC) of magnetic nanoparticles with size [27].
2.1.3 Surface modification of magnetic particles
Control of the surface chemistry of superparamagnetic iron oxide nanoparticles
(SIONPs) is essential to develop SIONPs for bio-related applications. The stability of
SPIONs in suspension is controlled by three principal forces: (a) hydrophobic–
hydrophilic, (b) magnetic and (c) van der Waals [28]. Pristine SIONPs tend to
aggregate into large clusters, in suspension due to the attractive van der Waals forces in
order to minimize the total surface or interfacial energy. The resulting large
agglomerates reduce intrinsic superparamagnetic properties. Modification of the surface
of SIONPs not only prevents aggregation/agglomeration of the particles, leading to
colloidal stability, but also renders them with water-solubility, biocompatibility,
Chapter: 2 Literature Review
12
nonspecific adsorption to cells, and bioconjugation. Several approaches to modify the
surfaces of SIONPs have been investigated for the preparation of water-soluble SIONPs
which can be roughly divided into three categories: ligand exchange, ligand addition,
and inorganic coating [29] as shown as Figure 2-3.
Figure 2-3 (a) Ligand exchange; (b) ligand addition; and (c) inorganic coating. F represents functional chemical group that can be used for further conjugation [29].
In the first approach, ligand exchange, the native monolayer of hydrophobic surface
ligands is exchanged with ligands containing head groups that bind the magnetic
nanoparticle surface and hydrophilic tails that interact with aqueous solvent [3,13,30].
The most common molecules used are ligands such as oleic acid, lauric acid, alkane
sulphonic acids, and alkane phosphonic acids. Silanes were employed to exchange the
hydrophobic ligands on ferrite magnetic nanostructures [30]. The end group of silanes,
Chapter: 2 Literature Review
13
including isocyanine, acrylate, thiol, amino, and carboxylic groups, offer extensive
chemistry for the modification of nanostructures. Surfactant addition is achieved
through the adsorption of amphiphilic molecules that contain both a hydrophobic
segment and a hydrophilic component. The hydrophobic segment forms a double layer
structure with the original hydrocarbon chain, while hydrophilic groups are exposed to
the outside of the NPs, rendering them water soluble [31,32].
The third route for magnetic NP surface modification is the fabrication of an inorganic
shell, typically consisting of silica. Silica coatings are formed either via the Stöber
process [33] or through a microemulsion synthesis [34]. Silica has been widely used to
protect the core nanoparticles from the external environment, thereby improving the
stability of the NPs. In addition, silica is highly biocompatible and its surface can be
easily modified with amines, thiols, and carboxyl groups, which enables covalent
modification of the particle surfaces with biological molecules.
There are various kinds of materials that can be chosen for coating nanoparticles. Table
2-1 presents a list of materials which have been used as stabilizing agents during the
synthesis of iron oxide nanoparticles. These materials can be chemically anchored or
physically adsorbed on magnetic nanoparticles to form a single or double layer, which
creates repulsive (steric repulsion) forces to balance the van der Waals attractive forces
acting on the nanoparticles, and the magnetic particles are stabilized in the process.
Chapter: 2 Literature Review
14
Table 2-1 Materials used for coating or encapsulating iron oxide magnetic nanoparticles and their bio- and environmental applications.
Materials used
Size and size distribution
Applications Advantages Reference
Silica 10-200 nm, narrow
Cellular MRI, heavy metal removal from wastewater, drug delivery, fluorescence
Improves biocompatibility, hydrophilicity and chemical stability. Prevents aggregation of particles in liquid. Provides further functionalization
[35,36]
Polyethylene glycol (PEG)
20-40 nm, narrow
MRI contrasting
Improves the biocompatibility, blood circulation time and easy to functionalize
[3]
Chitosan 10-200 nm, broad
Heavy metals separation, drug delivery
Natural polysaccharide with hydrophilicity, biocompatibility, biodegradability, antibacterial properties and remarkable affinity for many biomacromolecules. Used in food, biotechnology, biomedicine, food ingredients, cosmetics, water treatment
[37,38]
Dextran 10-50 nm, narrow
Biomedical applications
Enhances the blood circulation time, stabilizes the colloidal solution
[6,39]
Polyacrylic acid
8-20 nm, broad
Heavy metals removal, dye removal, enzyme recovery
Increases the stability and biocompatibility of the particles and also helps in bioadhesion
[8,40]
Gum arabic 13-67 nm, narrow
Heavy metals removal
Natural, harmless and environment friendly polymer, wide applications as a stabilizer, thickening agent and hydrocolloid emulsifier, mostly used in food and pharmaceutical industries
[41,42]
Chapter: 2 Literature Review
15
Table 2-1 (Continued) Poly(3,4-ethylenedioxythiophene) (PEDOT)
11 nm, narrow
Heavy metals removal
Promising conducting polymers because of its high conductivity, excellent environmental stability, and simple acid/base doping/ dedoping chemistry
[43]
Humic acid 10-190 nm, broad
Heavy metals removal
Enhances the stability of nanodispersions by preventing their aggregation
[44]
Poly-N-isopropyl acrylamide (PNIPAAm)
10-25 nm, broad
Hyperthermia, protein separation, controlled drug release
Thermoresponsive polymer, surface hydrophilicity or hydrophobicity can be varied easily
[15,45,46]
Dimercapto- succinic acid (DMSA)
~6 nm Heavy metals removal
Excellent chelating agent for heavy metals
[47]
2.2 Magnetic Separation
Chromatography is a powerful technology for the purification of biological substances
in both analytical and preparative scales. However, packed bed chromatographic
column is prone to clogging so that the method is unable to process particulate feed
stocks such as whole fermentation broth, cell disruptions and unclarified biological
extracts. To overcome this drawback, various alternative separation techniques have
been developed, including fluidizing bed adsorption, expanded bed adsorption and
magnetic separations [12]. These techniques offer great opportunities for process
integration by achieving particulate removal and desired product capture in a single
operation.
Over the last three decades, magnetic separation technology has emerged as one of the
most promising separation technologies [12]. In this method, colloidal particles are
manipulated by mismatches in their magnetization. Magnetic separation has been
developed as a technique to separate mainly magnetic materials, but recent development
Chapter: 2 Literature Review
16
has made possible the separation of non magnetic materials also. Magnetic separation
involves the transportation of magnetic or magnetically susceptible particles in a
gradient magnetic field. Generally, magnetic separation could be divided into two parts:
1. Separation of magnetic materials and 2. Separation of non-magnetic materials. In the
first method, magnetic separation of the target molecule could be achieved without
further modification of magnetic materials. For example separation of colored
magnetic impurities from kaolin clay, magnetic particulates from stack gases, magnetic
impurities from wastewater treatment, magnetic materials in mineral beneficiations, and
magnetotactic bacteria containing magnetic particles inside their cells. Most of the
application of magnetic fractionation can be classified in the second type. The principle
of this separation process is to use magnetic particles modified with some
intermediates, such as surfactant, polymer and ligand to adsorb the target molecule,
which can be separated by applying magnetic field gradient. The whole process of
separation of non magnetic target by magnetic separation method is illustrated in Figure
2-4. The interaction mechanisms between the non-magnetic targets and the
intermediates, coated in advance on magnetic particles, could be either electrostatic
interaction, hydrophobic interaction, and/or ligand-specific interaction.
Figure 2-4 Schematic diagram of the magnetic separation of non magnetic targets
P
I
Magnetic particle
Intermediate
Preparation
P I P I: : T P I T
Surface Preparation Coupling Release
TargetMolecule
Magnetic field gradient
Chapter: 2 Literature Review
17
2.2.1 Parameters of magnetic separator
The magnetic force (Fm) acting on a magnetizable particle of volume Vp and particle
magnetization Mp, placed in a magnetic field H can be expressed as,
Fm = µ0VpMpgrad H (2-2)
where µ0 denotes permeability constant of the vacuum.
The particle magnetization (Mp) may be expressed by the magnetic volume
susceptibility χ and the magnetic field strength H where the volume susceptibility is a
constant for diamagnetic and paramagnetic substances and a function among others of
particle shape and size as well as field strength for the ferromagnetic or ferrimagnetic
substances.
Mp = χH (2-3)
The most significant external forces that compete with the magnetic force in a magnetic
separator are the force of gravity, centrifugal force, and the hydrodynamic drag. For a
spherical particle of density ρp the gravitational force (Fg) is given by
Fg = (ρp – ρf)Vpg (2-4)
where ρf and g are the density of the fluid medium and the acceleration due to gravity,
respectively.
The centrifugal force (Fc) can be expressed as
Fc = (ρp – ρf)ωVpr (2-5)
Chapter: 2 Literature Review
18
where r is the radial position of the particle and ω is the angular velocity.
The hydrodynamic drag force (Fd) can be obtained from Stokes’ equation
Fd = 6πηb(vf – vp) (2-6)
where η is the dynamic viscosity of the fluid and vf and vp are velocities of the fluid and
particle respectively.
2.2.1 Applications of magnetic separation
In the past decades, magnetic separation has shown to be useful in many promising
applications in various areas of chemical and biotechnological processes. Table 2-2
summarizes the applications of magnetic separations.
Table 2-2 List of magnetic separation applications [12].
Areas Application of magnetic separation Chemical processes Ores–mineral beneficiation Kaolin (clay) decolorization Water treatment and metal removal Food Industries Biotechnological processes Enzyme immobilization Cell sorting DNA and protein separation and purification Nucleic acid extraction Drug delivery Biocatalysis and diagnostics
2.3 Cyclodextrins
Cyclodextrins (CDs) are cyclic torus-shaped oligosaccharides consisting of six (α-
cyclodextrin), seven (β-cyclodextrin), eight (γ-cyclodextrin) or more glucopyranose
units linked by α-(1,4) bonds [9,48,49] (Figure 2-5). They are also known as
cycloamyloses, cyclomaltoses and Schardinger dextrins. They are produced as a result
Chapter: 2 Literature Review
19
of intramolecular transglycosylation reaction from degradation of starch by
cyclodextrin glucanotransferase (CGTase) enzyme [9].
Figure 2-5 Structures and molecular dimensions of α, β, γ- cyclodextrins.
2.3.1 Basic properties of cyclodextrins
The three major cyclodextrins are crystalline, homogeneous and non-hygroscopic
substances, which are of a torus-like macro ring shape, built up from glucopyranose
units. Based on the number of glucopyranose units, cyclodextrins are commonly
classified as α (6), β (7), γ (8)- cyclodextrins. β-CD is the most accessible, the lowest-
priced and generally the most useful. Cyclodextrins contain a relatively hydrophobic
internal cavity, which can include various inorganic and organic molecules and exhibits
regio- and stereoselectivity, and a hydrophilic surface, which has primary and
1.69nm 1.53nm
0.78nm
1.37nm
0.95nm 0.57nm
0.79nm
Chapter: 2 Literature Review
20
secondary hydroxyl groups. The main properties of those cyclodextrins are given in
Table 2-3.
In cyclodextrin molecules glucose units situated in the classical 4C1 conformation of
chains are linked through α-1,4 bonds. The interior of the cavity, which contains two
rings of C-H groups with a ring of glycosidic oxygen in between, is relatively,
hydrophobic, while the external faces with hydroxyl groups are hydrophilic. All
secondary hydroxyl groups (C2-OH and C3-OH) are situated on the wider end of the
cavity, whereas all primary hydroxyls (C6-OH) are situated on the narrower end, as
shown in Figure 2-6. The internal hydrophobic cavity is the key structural feature of the
cyclodextrins. It provides their ability to complex and holds a wide variety of inclusion
molecules. To bind with cyclodextrins, the inclusion molecule must have a size that fits, at
least partially, into the cavity, creating the complex. The inclusion compound, however,
does not have to be completely contained in the cavity. Complexes can be formed by the
insertion of some specific functional groups or part of the molecule to bind in the
hydrophobic cavity.
Figure 2-6 Functional structure of cyclodextrin
Chapter: 2 Literature Review
21
In cyclodextrins, the non-bonding electron pairs of the glycosidic oxygen bridges are
directed towards the inside of the cavity, giving it high electron density and some
Lewis-based characters. The C2-OH group of one glucopyranose unit can form a
hydrogen bond with the C3-OH group of the adjacent glucopyranose unit
intramolecularly. A complete secondary belt is formed by these H-bonds to give β-CD
molecules a rigid structure. Cyclodextrins are readily soluble in water, because all the
free hydroxyl groups are located on the outer surface of the ring [9]. Among them, β-
CD is observed to have the lowest solubility. The hydrogen belt in α-cyclodextrins is
incomplete because one glucopyranose unit is in a distorted position and only four out
of six possible H-bonds can be formed. γ-CDs have a non-coplanar and more flexible
structure so it is the most soluble among the three common cyclodextrins.
Table 2-3 Properties of α, β, γ- cyclodextrin
Properties α β γ
Number of glucose units 6 7 8
Molecular weight (g/mol) 972 1135 1297
Solubility in water at 25oC (g/100ml) 14.5 1.85 23.2
Volume of cavity (Å3)
Cavity diameter ( Å)
174
4.7-5.3
262
6-6.5
427
7.5-8.3
Outer diameter ( Å) 14.6 15.4 17.5
Height of the torus ( Å) 7.9 7.9 7.9
2.3.2 Cyclodextrin inclusion complexes
The most important property of cyclodextrin for both practical application and scientific
research is the ability to selectively form inclusion complexes with other molecules,
ions, and radicals [49]. Inclusion complexes are molecular compounds in which one
Chapter: 2 Literature Review
22
compound (host molecule) spatially encloses another (guest). The driving forces of the
formation of the inclusion complexes are: (1) van der Waals (or hydrophobic)
interactions between hydrophobic “guest” molecules and the CD cavity; (2) hydrogen
bonds between polar functional groups of “guest” molecules and CD hydroxyl groups.
The role of hydrogen bonds is not universal, because “guest” molecules such as
benzene, which cannot form hydrogen bonds, also form stable inclusion complexes; (3)
substitution of high energy (high enthalpy) water molecules in the CD cavities in the
complexation process. In aqueous solutions nonpolar CD cavities are occupied by water
molecules, which is energetically unfavorable (polar–nonpolar interactions), and,
therefore, they can be easily replaced by suitable molecules of the “guests” less polar
than water (Figure 2-7); (4) the release of the strain energy in the ring structure of CD
molecules; (5) charge-transfer interactions [49,50].
Figure 2-7 Schematic representation of cyclodextrin inclusion complex formation: p-Xylene is the guest molecules; the small circles represent the water molecules.
Complex formation is a dimensional fit between host cavity and guest molecule. The
ability of a cyclodextrin to form an inclusion complex with a guest molecule is a
function of two key factors [51]. The first is steric and depends on the relative size of
the cyclodextrin to the size of the guest molecule or certain key functional groups
within the guest. If the guest is of wrong size, it will not fit properly into the
Chapter: 2 Literature Review
23
cyclodextrin cavity. For example, the bulky anthracene can only fit into γ-CD.
Propionic acid is relatively small so it is compatible with α-CD but its fitting with large
cavity of γ-CD is unsatisfactory. The second critical factor is the thermodynamic
interactions between the different components of the system (cyclodextrin, guest,
solvent). For a complex to form there must be a favorable net energetic driving force
that pulls the guest into the cyclodextrin.
The stability of inclusion complexes also depends on the polarity of the “guest”
molecules. Highly hydrophilic molecules and strongly hydrated and ionized groups are
not, or weakly complexable with cyclodextrin. Only compounds less polar than water
can form inclusion complexes with cyclodextrin. The hydrophobic character of the
substituent determines the stability of the complexes. The stability of inclusion complex
increases with an increase in the electron donor nature of the “guest”.
2.3.3 Characterization of cyclodextrin inclusion complexes
Inclusion complexes can be characterized by a variety of spectrometric methods. UV
spectroscopy has been used to determine the stability constant and thermodynamic
parameters for interaction of guests with CD in aqueous solution [52]. The
complexation causes a change in the absorption spectrum of a guest molecule. During
the complexation, the chromophore of the guest molecule is transferred from an
aqueous medium to the non-polar cyclodextrin. These changes must be due to a
perturbation of the electronic energy levels of the guest caused either by direct
interaction with the CD, by the exclusion of solvating water molecules or by a
combination of these two effects [53]. Hypsochromic or bathochromic shift or increase
in the absorption intensity without change in the λmax has been considered as evidence
of the formation of the complex. Hydrogen bonding can be considered as the main force
Chapter: 2 Literature Review
24
behind the inclusion complex formation. As hydrogen bonding lowers the energy of ‘n’
orbitals, a hypsochromic shift (blue shift) is observed. Cleavage of the existing
hydrogen bonds in the compound can lead to a bathochromic shift due to complexation
[54,55]. An increase or decrease in the absorption intensity of UV band without change
in its λmax is also reported in certain inclusion complexes. For example, the absorption
intensity was increased when benzene was complexed with β-CD [52].
Circular Dichroism is a useful method to detect cyclodextrin inclusion complexes in
aqueous solution [56]. When an achiral guest molecule is included within the
asymmetric locus of the cyclodextrin cavity, new circular dichroism bands can be
induced in the absorption bands of the optically inactive guest. Not only achiral guest
molecules but also chiral guest molecules may show changes in circular dichroism
spectra upon the formation of inclusion complexes with cyclodextrin [56].
Infra-Red spectroscopy is also used to estimate the interaction between CD and the
guest molecules in the solid state. CD bands often change only slightly upon complex
formation and if a part of guest molecules is encapsulated in CD cavity, bands which
could be assigned to the included part of the guest molecules are easily masked by the
bands of the spectrum of cyclodextrin. Generally IR techniques are not suitable for the
detection of inclusion compounds because the resultant spectra have a superposition of
host and guest bands [55].
Recently, thermal analysis has also been used to detect the inclusion compounds. In
some cases no melting or decomposition peaks of the guests are observed on the
thermogram obtained by DTA or DSC after the formation of complexes [55,57]. The
Chapter: 2 Literature Review
25
thermal decomposition of inclusion compounds was studied only hardly. It was
observed that the decomposition of the complexed guest occurs above the
decomposition temperature of the uncomplexed guest molecule [53]. Apart from the
techniques described above, there are methods used to characterize or detect the
inclusion complexes such as, powder X-day diffraction [54,55,57], nuclear magnetic
resonance (1H-NMR) [54-57], fluorescence spectroscopy [53] etc.
2.3.4 Applications of cyclodextrin
The unique structure and properties of cyclodextrins have made them a popular choice
for a wide range of applications over the past few decades [51,58]. There are numerous
applications for cyclodextrins in the pharmaceuticals field [59,60]. Cyclodextrins can be
used as drugs either for complexation or as auxiliary additives such as carriers, diluents,
solubilisers or tablet gradients. The physical and chemical properties of the drugs can be
modified through the inclusion complex formation. These modifications are desirable in
the formation of oral drugs and have the potential to improve their physical and
chemical stability, as well as enhance the bioavailability of poorly soluble drugs. In
food and cosmetics industries, cyclodextrins can be used for molecular encapsulation of
flavors and fragrances based on their water retention properties. The major advantages
of cyclodextrins in this application include the protection of active ingredient against
various chemical reactions and elimination or reduction of undesirable tastes/odors and
microbiological contamination. Furthermore, cyclodextrins are widely used in chemical
technology [51]. For example, cyclodextrins can modify the reactivity of the guest
molecules through the alteration of the reaction pathways, enhancement of the
hydrophilicity and reactivity in aqueous systems, chiral recognition and reduction of the
free, uncomplexed guest substance. In the chemical industry, cyclodextrins are widely
Chapter: 2 Literature Review
26
used to separate isomers and enantiomers, to catalyse reactions, to aid in various
processes and to remove or detoxify waste materials.
2.4 Protein refolding
Protein refolding is an important downstream process in the field of biotechnology.
Many of these proteins are currently produced by Escherichia coli in the form of
insoluble, inactive aggregates often termed as inclusion bodies. The general strategy
used to recover active protein from inclusion bodies involves three steps: inclusion
body isolation and washing; solubilization of the aggregated protein; and refolding of
the solubilized protein (Figure 2-8). Although the efficiency of the first two steps can be
relatively high, renaturation yields may be limited by the accumulation of inactive
misfolded species as well as aggregates. Such aggregates must be separated from cell
debris and solubilized by exposing them to a strong denaturant, such as urea or
guanidine (GdmCl) or detergent sodium dodecyl sulphate (SDS) and n-cetyl
trimethylammonium bromide (CTAB).
Figure 2-8 Processes for the recovery of inclusion body proteins [61].
Chapter: 2 Literature Review
27
In conventional methods, refolding of unfolded polypeptide chains to their native state
is initiated through decreasing the denaturant concentration by direct dilution or dialysis
[62]. The main disadvantages of dilution refolding for commercial applications are the
need for larger refolding vessels and additional concentration steps after renaturation. In
addition, protein aggregates formed due to hydrophobic interactions between partially
folded polypeptide chains leads to low yields of biologically functional protein [63]. On
the other hand, renaturation yields using these membrane-based dialysis methods can be
significantly affected by protein binding to the membranes. The difference between
protein folding and aggregation is described as kinetic competition between
intramolecular reaction (folding) or inter molecular reaction (aggregation) by the
following reactions:
U → N (2-7)
U + U → A2 (2-8)
where U, N, and A2 represent the unfolded protein, native protein, and a dimer,
respectively [64].
Therefore, many strategies for in vitro protein refolding have focused on suppression of
aggregation along with improved renaturation yields. An efficient strategy to suppress
aggregation is the inhibition of the intermolecular interactions leading to aggregation by
the use of low molecular weight additives. This strategy is often termed as ‘dilution
additive approach’ (Figure 2-9). However, scaling up dilution processes leads to large
volumes and low protein concentration. A wide variety of additives has been used in
protein refolding (Table 2-4).
Chapter: 2 Literature Review
28
Recently, Rozema and Gellman developed a two-step protein refolding procedure using
CTAB as capturer to bind denatured protein, and β-CD as stripper to dissociate the
CTAB–protein complex so that the individual protein may fold into the native form
[65-68]. This method is also referred to as artificial chaperone-assisted protein refolding
which presents a useful way to inhibit the formation of protein aggregates. In addition
to applying this method to different proteins research into new “capturers” and
“strippers” has attracted growing attention and efforts in recent years (Table 2-5 and
Table 2-6).
Figure 2-9 Different approaches of protein refolding; (A) dilution mode (B) dilution-additive assisted protein refolding (C) artificial chaperone assisted protein refolding.
Denatured protein
Protein-detergent complex
Native protein (+some aggregates)
Capture step Stripping Step
Aggregate (inactive)
Dilution
Native protein (+some aggregates)
Dilute +additive
A
B
C
Chapter: 2 Literature Review
29
Table 2-4 Protein refolding-dilution additive mode
Model proteins (denaturant used)
Additives References
Glycosylated recombinant human deoxyribonuclease (rhDNAse) (Urea, GdmCl)
Polyethylene glycol (0.1-0.4 g/l)
[69]
Bovine Carbonic anhydrase (GdmCl)
α-CD (20-100 mM) [70]
Bovine Carbonic anhydrase II (GdmCl)
Detergents (CHAPS, Triton X-100, n-hexanol, n-pentanol, cyclohexanol)
[71]
β–lactamase (GdmCl)
Temperature-sensitive polymer (PNIPAAm)
[72]
Sulfertranferase enzyme rhodanese (Urea)
Detergent-phospholipids mixtures [73]
Rhodanese (GdmCl) Wide range of detergents used: ionic, zwitterionic, nonionic etc.
[74,75]
Calf intestinal alkaline phosphatase (CIP) (GdmCl)
Magnesium ions (<3mM) [76,77]
Rabbit muscle creatine kinase (GdmCl)
Proline, glycerol and heparin sodium [78]
Rabbit muscle creatine kinase (GdmCl)
Osmolytes, including dimethysulfoxide, glycine, proline and sucrose
[79]
Hen egg-white lysozyme(GdmCl) Proline (>1.5 M)
[80]
Hen egg-white lysozyme (GdmCl)
Acetone, acetoamide, or urea derivatives (1.4M)
[81]
Hen egg-white lysozyme(GdmCl denatured), bovine carbonic anhydrase (urea )
A varieties of cyclodextrins [82,83]
hen egg-white lysozyme(GdmCl) L-Arginine (0.4–1 M)
[84,85]
Yeast inorganic pyrophosphatase
Sugars (Trehalose, glycerol) [86]
α-amylase(GdmCl)
α-CD (100 mM)
[87,88]
Hen egg-white lysozyme (GdmCl)
Acetamide (1-3M), acetone (0.5-1.5M), thiourea (0.2-0.6M), l-arginine (0.4-1M) or glycerol (3.2-6.5M)
[89]
Hen egg white lysozyme, recombinant human lysozyme from E. coli BL21 (Urea)
CTAB [90]
Hen egg white lysozyme (GdmCl)
GdmCl (0.5-1.25M), Arginine (0.4-1M) [91]
Chapter: 2 Literature Review
30
Table 2-5 Liquid phase artificial chaperone-assisted protein refolding
Model proteins(denatured state)
Capturers Striping agents Refolding yields
References
Lysozyme (GdmCl)
CTAB β-CD, methyl-β-CD
[67]
Bovine carbonic anhydrase (CAB) (GdmCl/thermally)
Several types of detergents used: CTAB, SDS, STS, POE(10)L etc.
α-CD, β-CD, and methyl-β-CD
~80% (max.) [65]
Porcine heart citrate synthase (CS) (GdmCl/urea )
Several types of detergents used: CTAB, SDS, Triton X-100, ZW 3–14, POE(10)L etc.
β-CD ~65% (max.) [66]
Rabbit muscle creatine kinase (MM-CK) (Thermally)
SDS Hydroxypropyl-β-CD ~75% [92]
ALP (Urea) α –amylase (urea) CAB (thermally)
CTAB, DTAB, TTAB, SDS, STS, SHS
Alginate ALP~ 85% α –amylase-78% CAB-89%
[93]
CAB (GdmCl) Porcine heart citrate synthase ((GdmCl)
cholesterol group-bearing pullulan (CHP) nanogels
α-CD, β-CD, and methyl-β-CD, HP- β-CD
CAB~65% CS~76%
[94]
CAB (Thermally)
cholesterol group-bearing pullulan (CHP) nanogels
α-CD, β-CD, and methyl-β-CD, HP-β-CD
~90% [95]
Xylanase (GdmCl & urea)
CTAB, TTAB etc.
Methyl-β-CD
50~60% [96]
HCAB (GdmCl) Lysozyme (GdmCl)
CTAB Linear dextrins
HCAB-77% Lys-60%
[97]
α–amylase (GdmCl) CTAB α-CD ~75% [98]
Porcine heart citrate synthase (GdmCl) CAB(GdmCl) Lysozyme(GdmCl)
A wide variety of detergents
Cycloamylose ~90% [99]
Lysozyme CTAB β-CD-grafted-PNIPAAm(β-CD-g-PNIPAAm)
~70% [100]
ALP from calf intestine (Urea) CAB (thermally) α –amylase (Urea)
Detergents Detergents ~78-88% [101]
CAB (GdmCl) Lysozyme(urea)
Dextran-grafted-PNIPAAm
[102]
Chapter: 2 Literature Review
31
Table 2-6 Solid-phase artificial chaperone-assisted protein refolding Model proteins(denatured state)
Capturing agents
Solid-phase stripping agents
Refolding yield References
CAB (SDS denatured)
SDS β -Cyclodextrin-Polyurethane Polymer
~75% [103]
α-glucosidase (urea) SDS POLY RING-DEX B (β-cyclodextrin-epichlorohydrin copolymer)
50-60% [104]
Lysozyme (GdmCl) CAB ( GdmCl)
CTAB β -cyclodextrin-acrylamide copolymer beads
Lysozyme~65% CAB~80%
[105]
CAB (thermally denatured)
CTAB, TTAB, SDS
β-cyclodextrin-epichlorohydrin copolymer
~75% [106]
2.5 Pollutants removal from wastewater The removal of heavy metal pollutants and dyes from industrial wastewater has become
an important issue because of their adverse affects on human health and environment
[107,108]. Dyes are widely used in many industries in order to color their products,
such as cosmetics, leather, paper, printing textile finishing industries. The presence of
these dyes in water, even at very low concentrations, is highly visible and undesirable.
Most of the used dyes are stable to photodegradation, bio-degradation and oxidizing
agents [109]. However, heavy metals, unlike the organic contaminants, are not
biodegradable and tend to accumulate in living organisms. Due to the toxic and
carcinogenic properties of both inorganic and organic pollutants, it is necessary to treat
the pollutant containing water before being released into the environment.
Numerous approaches have been developed for the removal of these contaminants from
wastewater. Traditional heavy metal and dye removal methods include chemical
precipitation, ion exchange, liquid-liquid extraction, membrane filtration, adsorption
Chapter: 2 Literature Review
32
and biosorption [110,111]. Among these methods, adsorption has increasingly received
more attention due to its merits of effectiveness, efficiency and economy [41,112-115].
The common adsorbents primarily include activated carbons, zeolites, clays, industrial
by-products, agricultural wastes, biomass and polymeric materials [110,116]. However,
these adsorbents described above suffer from low adsorption capacities and separation
inconvenience. Therefore, efforts are still required to carry out investigation for new
promising adsorbents.
Adsorption using nano-sized adsorbents is becoming more popular as it possesses better
performance when compared to sub-micron to micron sized particles which need
internal porosities to ensure adequate surface area for adsorption. However, the
existence of intraparticle diffusion may lead to the decreases in the adsorption rate and
available capacity, particularly for macromolecules [40]. Therefore, to develop an
adsorbent with large surface area and small diffusion resistance has significant
importance in practical use [117]. Now-a-days, magnetic nanoparticles with tailored
surface chemistry have been widely used in various fields of applications. The
functional groups on the surface play an important role in determining the effectiveness,
capacity, selectivity, and reusability of these adsorbents. The basic properties of
nanoparticles- extremely small size, the absence of internal diffusion resistance and
high surface area to volume ratio, provide better kinetics for the adsorption of
contaminants from aqueous solutions [40]. Magnetic nano-adsorbents have the
advantages of both magnetic separation techniques and nano-sized materials, which can
be easily recovered or manipulated with an external magnetic field. The main advantage
of this technology consists in its capability of treating large amount of wastewater
within a short time and producing no contaminants. Magnetic materials, particularly
superparamagnetic Fe3O4 nanoparticles, provide a novel biomedical and environmental
Chapter: 2 Literature Review
33
purification technique because of their specific magnetic separation characteristics. A
list of magnetic materials that have been used as adsorbents so far for the removal of
heavy metals and dyes from wastewater is summarized in Table 2-7.
Attention has recently been focused on cyclodextrin based non-magnetic materials as
adsorbents. The most characteristic feature of CDs is the ability to form inclusion
compounds with various organic molecules through host-guest interactions: the interior
cavity of the molecule provides a relatively hydrophobic environment into which an
apolar pollutant can be trapped. The insoluble cyclodextrin polymer or immobilization
of derivatives of cyclodextrin onto inert support materials such as silica, have been
widely used for contaminants removal from wastewater [10,118]. It has been shown
that these materials could be effectively used for the removal of phenolic compounds
and dyes from wastewater. The list of cyclodextrin based materials is summarized in
Table 2-8. However, less work has been done for heavy metal removal from wastewater
using cyclodextrin based magnetic adsorbents. Combining the advantages of both
materials, a superior adsorbent could be developed for wastewater treatment.
When the magnetic particles containing pollutants move through a magnetic field, they
are rapidly and efficiently separated or captured. The capture of particles depends
strongly on the creation of these large magnetic field gradients, as well as on the
magnetic properties. There are three categories of separators based on magnet type:
permanent magnet-based (field of less than 1 T), electromagnet-based (max. 2.4 T) and
superconducting magnet-based (2 to 10 T). The high gradient magnetic separation
(HGMS), commonly used term in magnetic separations has been of scientific and
technological interest over the past decades [12,119]. The technique has shown great
advantages in ferromagnetic material separation and it has been industrialized, such as
Chapter: 2 Literature Review
34
magnetic separation in steel industry, steel making, steel rolling, and circulating cooling
water in thermal power plant and so on. An HGMS device comprises of a bed of
magnetically susceptible wires placed between two poles of an electromagnet, as shown
in Figure 2-10. When a magnetic field is on, the wires dehomogenize the magnetic field
in the column, producing large field gradients around the wires that attract magnetic
particles to their surfaces and trap them there. Recent research showed that the capture
of particles depends on particle sizes and aggregates of nanoparticles larger than 50 nm
showed significantly high capture efficiency in HGMS operations [120].
Figure 2-10 A typical high-gradient magnetic separation facility [121].
The superconducting HGMS is a new kind of separation technology was also used
many researchers. Instead of copper coil, the high gradient magnetic field of more than
10 T can be generated using superconducting coil in this separator [122]. Moreover, this
technology has many advantages such as low investment, energy saving, low operating
cost, easy handling, automatic control. Recently Kondo and Miura used magnetic
mesoporous carbon to remove the dissolved organic matter from wastewater efficiently
with the help of superconducting HGMS technique as shown in Figure 2-11[123].
Chapter: 2 Literature Review
35
Figure 2-11 Schematic diagram of the superconducting HGMS system [123].
Chapter: 2 Literature Review
36
Table 2-7 Adsorption capacities and experimental conditions of functionalized magnetic nanoparticles for various heavy metals and dye removal from wastewater
Adsorbent Adsorbate Adsorption capacity (mg/g)
pH Temperature (oC)
Kinetic model Isotherm References
Chitosan-coated Fe3O4 NPs modified with alpha-ketoglutaric acid
Cu(II) 96.15 6 22 Langmuir, Freundlich
[108]
Humic coated Fe3O4 NPs Hg(II) Pb(II) Cd(II) Cu(II)
97.70 92.40 50.40 46.30
6 20 - Langmuir [44]
Glutaraldehyde modified Fe3O4 NPs (GA-APTES-NPs)
Cu(II) 61.07 4 20 - Langmuir, Freundlich
[124]
Calcium alginate modified Fe3O4 NPs Cu(II) 60.00 5 20 Pore diffusion model
Langmuir [125]
Gum Arabic modified Fe3O4 NPs Cu(II) 38.50 5.1 27 - Langmuir, Freundlich
[41]
Amino functionalized Fe3O4@SiO2 core-shell NPs
Cu(II) Pb(II) Cd(II)
29.85 76.66 22.48
6 25 - Langmuir [126]
Chitosan bound MNPs Cu(II) 21.50 5 27 - Langmuir [37] Amino-functionalized MNPs Cu(II)
Cr(VI)12.43 11.24
5 2
25 Langmuir [8]
Carbon encapsulated MNPs Cu(II) Cd(II) Co(II)
3.21 1.77 1.23
9 r.t. - - [127]
Dimercaptosuccinic acid (DMSA) coated Fe3O4 NPs
Hg(II) 227.00 8.1 r.t. - Langmuir [47]
Magnetic (γ-Fe2O3) hydrogel Cr(VI) 205.00 4-6 23 - Langmuir [128] Carbon encapsulated MNPs (CEMNPs)
Cu(II) 3.21 (at initial conc. 20 mg/l)
8 20 Freundlich [129]
Cross-linked magnetic chitosan–diacetylmonoxime Schiff’s base resin (CSMO)
Cu(II) Co(II) Ni(II)
95 ± 4 60 ± 2
47 ± 1.5
5 28 Pseudo-first order model,
pseudo-second-order model
Langmuir, Freundlich and
Tempkin
[130]
Chitosan-magnetite composite Pb(II) Ni(II)
63.33 52.55
6 r.t. - Langmuir [38]
Chapter: 2 Literature Review
37
Table 2-7 (Continued)
Adsorbent Adsorbate Adsorption capacity (mg/g)
pH Temperature (oC)
Kinetic model Isotherm References
Dendrimer conjugated MNPs Zn(II) 24.30 7 25 Langmuir, Freundlich
[131]
Fe3O4 hollow spheres Neutral red 105.00 6 25 Pseudo-second-order model
Langmuir, Freundlich
[132]
Cross-linked magnetic chitosan–diacetylmonoxime Schiff’s base resin (CSMO)
Cu(II) Co(II) Ni(II)
95 ± 4 60 ± 2
47 ± 1.5
5 28 Pseudo-first order model,
pseudo-second-order model
Langmuir, Freundlich and
Temkin
[130]
γ-Fe2O3 Acridine orange (AO)
59.00 4.5 24 Pseudo-second-order model
Langmuir, Freundlich
[133]
Magnetic silica modified with amino groups
Acid Orange 10 (AO10)
61.33 3 25 Pseudo-first order model,
pseudo-second-order model
Langmuir, Freundlich
[134]
Carboxymethyl chitosan-conjugated Fe3O4 NPs
Crocein orange G (AO12)
Acid green 25 (AG25)
94.16
73.16
3
25 Pseudo-first order, pseudo-
second-order and intraparticle
diffusion model
Langmuir, Freundlich
[135]
Polyacrylic acid-bound Fe3O4 MNPs Methylene blue (MB)
199.00 9 25 - Langmuir [40]
MWCNTs filled with γ-Fe2O3 NPS Methylene blue Neutral red
42.30 77.50
6 25 - Freundlich
[136]
Fe3O4-activated carbon nanocomposite
Methylene blue 321.00 - 25 - Langmuir [137]
SDS coated Fe3O4 NPs Safranin O 769.23 3 r.t. Pseudo-second-order model
Langmuir, Freundlich
[138]
Carboxylic functionalized superparamagnetic mesoporous silica microspheres
Acridine orange Methylene blue
112.70 109.80
10 25 - Langmuir, Freundlich
[139]
Amine-functionalized silica magnetite (NH2/SiO2/Fe3O4 adsorbents)
Cu(II) Reactive black 5
10.41 217.00
5.5 3
25 Pseudo-first order model,
pseudo-second-order model
Langmuir, Freundlich
[140]
Chapter: 2 Literature Review
38
Table 2-8 Adsorption capacities and experimental conditions of cyclodextrin based materials for various dye and heavy metals removal from wastewater
Adsorbent Adsorbate Adsorption capacity (mg/g)
pH Temperature (oC)
Kinetic model Isotherm References
Cyclodextrin/carboxymethyl cellulose crosslinked polymers
C.I. Basic Green 4
(Malachite Green)
91.9 8 25 Pseudo-first order, pseudo-second-order and intraparticle diffusion model
Langmuir, Freundlich
[10]
β-CD polymer crosslinked by citric acid
Methylene blue 105 Deionized water
30 - Langmuir [11]
Cyclodextrin/carboxymethyl cellulose crosslinked polymer
Methylene blue 56.4 8 25 Pseudo-first order model, pseudo-second-order
model
Freundlich, Langmuir,
Temkin and generalized
[141]
Cyclodextrin/carboxymethyl cellulose crosslinked polymer
Astrazone blue Crystal violet Rhodamine B
53.2 42.4 35.8
8 25 pseudo-second-order and
intraparticle diffusion model
Langmuir, Freundlich
[113]
Cyclodextrin-starch based polymer
Congo red 36.2 5.8 25 - Langmuir, Freundlich
[142]
β-CD/chitosan copolymer Pb(II) 434.78 4,5 30 Pseudo-first order model, pseudo-second-order
model
Langmuir, Freundlich
[143]
Pyromellitic dianhydride (PMDA) grafted β-CD
Methylene blue Neutral red
497.5 555.6
- 25 Pseudo-first order model, pseudo-second-order
model
Langmuir, Freundlich
[144]
β-CD polymer (HMDI as crosslinking agent)
Evans Blue Chicago Sky Blue
10.59 12.09
2 25 - Langmuir, Freundlich
[145]
β-CD/citric acid copolymer Pb(II) Cd(II) Zn(II)
263.2 107.5 82.6
5 20 - Langmuir [146]
Chapter: 2 Literature Review
39
2.6 Adsorption and Desorption
Adsorption is one of the most important parts of separation. It has been widely used in
the chemical, biological, analytical, and environmental fields. In most cases, the
adsorbents have diameters in the range of sub-micron to micron and have large internal
porosities to ensure adequate surface area for adsorption. However, the diffusion
limitations within the particles lead to decrease in the adsorption rate and the available
capacity, particularly for macromolecules such as proteins and DNA. To understanding
adsorption more precisely we investigated the adsorption equilibrium and the
parameters affecting adsorption. However, desorption is also important for the recovery
of target molecules from the surface of the particles. Therefore, suitable adsorption
desorption parameters should be developed.
2.6.1 Adsorption equilibrium a) Langmuir Model
The Langmuir model assumes that the adsorption process takes place on a surface
composed of a fixed number of adsorption sites of equal energy, with one molecule
adsorbed per adsorption site until a monolayer coverage is obtained. The Langmuir
model is described by the equation [147]:
m ee
L e
q Cq
K C
(2-9)
The linear form of this equation can be represented as follow:
1e e
e m m L
C C
q q q K (2-10)
Chapter: 2 Literature Review
40
where Ce is the adsorbate liquid-phase equilibrium concentration, qe is the adsorbate
surface concentration, qm is the maximum adsorbate binding capacity and KL is
Langmuir constant, which represents the affinity between adsorbate and adsorbent. The
values of qm and KL can be obtained from the slope and intercept of the linear plots of
Ce/qe versus Ce.
b) Freundlich Model
This isotherm is an empirical equation to describe multilayer adsorption with
interaction between adsorbed molecules [148]. This model predicts that the solute
concentrations on the adsorbent will increase as long as there is an increase of the solute
concentration in the solution. Usually it applies to adsorption onto heterogeneous
surfaces with a uniform energy distribution and reversible adsorption. The Freundlich
equation suggests that the adsorption energy decreases exponentially on the completion
of the adsorption centers of an adsorbent. This equation is given as follows:
qe=KFCe1/n (2-11)
The commonly used linear form of the equation is:
lnqe = (1/n) lnCe + lnKF (2-12)
where KF and n are the Freundlich equilibrium constant and the Freundlich isotherm
power term (also called the heterogeneity factor), respectively. The values of KF and 1/n
can be determined from the slope and intercept of the linear plot of lnqe versus lnCe.
2.6.2 Adsorption kinetics In order to investigate the mechanism of adsorption and potential rate controlling steps
such as mass transport and chemical reaction processes, kinetic models have been used
Chapter: 2 Literature Review
41
to test experimental data. The adsorption of pollutants from aqueous solution plays an
important role in wastewater treatment; hence it is important to know the sorption rate
at which the pollutants are removed from aqueous solutions in order to design the
appropriate sorption treatment plants [149]. Several kinetic models have been reported
to describe the adsorption of pollutants (dyes, heavy metals, phenolic compounds etc.)
[149-152]. These kinetic models included the pseudo-first order equation, the pseudo-
second order equation and intraparticle diffusion model.
The pseudo-first-order equation of Lagergren is generally expressed as follows [150]:
1( )te t
dqk q q
dt (2-13)
Integrating this equation for the boundary conditions t = 0 to t = t and q = 0 to q = qt,
gives:
ln(qe – qt) = lnqe – k1t (2-14)
where qe and qt refer to the adsorption capacity at equilibrium and at any time, t (min),
respectively, (mg/ml) and k1 is the equilibrium rate constant of pseudo-first-order
sorption (min-1). The slope of the linear plot of ln(qe - qt) versus t is used to determine
the first-order rate constant, k1.
The pseudo-second order kinetic rate equation is expressed as follows [149]:
22 ( )t
e t
dqk q q
dt (2-15)
Integrating this equation for the boundary conditions t = 0 to t = t and q = 0 to q = qt,
and rearranging the variables, gives:
22
1 1
t e e
tt
q k q q (2-16)
Chapter: 2 Literature Review
42
where k2 is the equilibrium rate constant of pseudo-second-order adsorption (g mg-1
min-1). If pseudo-second order kinetics is applicable, the plot of t /qt against t of
equation [2-16] should give a linear relationship, from which qe and k2 can be
determined from the slope and intercept of the plot.
The dye sorption is governed usually by either the liquid phase mass transport rate or
the intraparticle mass transport rate. Hence diffusive mass transfer is incorporated into
the adsorption process. In diffusion studies, the rate can be expressed in terms of the
square root time. The mathematical dependence of qt versus t1/2 (known as the Weber-
Morris plot) is obtained if the process is considered to be influenced by diffusion in the
particles and convective diffusion in the solution. According to this model proposed by
Weber and Morris [153], the root time dependence may be expressed by Equation [2-
17]:
qt = kidt1/2 + C (2-17)
where qt is the amount of solute on the surface of the sorbent at time t (mg/g), kid the
intraparticle diffusion rate constant (mg/g min1/2), t the time (min), and C is the
intercept (mg/g). The kid values are found from the slopes of qt versus t1/2 plots.
2.6.3 Desorption study
Desorption study is important to explore the possibility of recyclability of the magnetic
nano-adsorbent. Desorption of solutes from interfaces depends essentially on the
conditions under which they have been adsorbed. Therefore, suitable desorbing agents
should be selected to release the adsorbed pollutants from magnetic particles. For heavy
metal desorption from magnetic nanoparticles, acidic solution (HCl or HNO3) could be
used as eluant [154-156]. EDTA which is a strong hexadentate chelating agent has also
Chapter: 2 Literature Review
43
been used as eluant for metals desorption [151,154]. For desorption of dye molecules,
methanol or ethanol solution has been shown to be effective eluant [40,157].
2.7 Scope of the thesis
The aim of this project is to synthesize and characterize β-CD coated MNPs (either bi-
functional or multifunctional) and their applications in biomolecule separation,
biomolecule refolding, drug delivery, organic/inorganic pollutants removal from
wastewater. Although nanoparticles have diverse applications in various fields of
bioscience, biotechnology and environment, only limited work has been published on
the preparation of organic-inorganic nanocomposite materials using β-CD coated
nanoparticles and their use as a tool for bio/chemical separation, drug delivery etc.
Based on the literature review the scopes of the present project are:
(a) Synthesis and characterization of β-CD coated magnetic nanoparticles
MNPs are synthesized by chemical precipitation method using FeCl3 and FeCl2 with
ammonia under nitrogen environment in a ratio of 2:1. These nanomagnetic particles
are then functionalized with β-CD derivatives e.g. mono-tosyl-β-CD (Ts-β-CD) and
carboxymethyl-β-CD (CM-β-CD) using different synthetic routes. Characterization of
the chemical, physical and magnetic properties of the as-synthesized nanomagnetic
particles are carried out using FTIR, XPS, BET, TEM, XRD and VSM.
(b) Application of Ts-β-CD bonded MNPs in biomolecule refolding
Ts-β-CD conjugated MNPs are used as a solid-phase artificial chaperone in refolding of
bovine carbonic anhydrase (CA) as model protein. CA is a Zn containing
(metalloenzyme). It is expected that with cumulative effects of chaperoning function of
Ts-β-CD and superparamagnetism of iron oxide, Ts-β-CD conjugated MNPs would
Chapter: 2 Literature Review
44
have potential applications in biomolecule i.e. protein refolding. To evaluate the
efficiency of TS-β-CD conjugated MNPs as solid-phase artificial chaperone in protein
refolding, results are compared with that using only β-CD as liquid-phase chaperone.
Also the structural changes of the refolded protein are studied. In the refolding study,
only Ts-β-CD grafted on magnetic particles is used which acts as protein refolding aid
whereas other CD derivative for example, CM-β-CD induces serious protein
aggregation [83].
(c) Application of CM-β-CD bonded MNPs in biomolecule recognition and separation
CM-β-CD conjugated core-shell APES-MNPs are used for individual and selective
adsorption of nucleosides. Adenosine (A) and guanosine (G) were selected as model
target guest molecules. For better understanding to gain insights into the molecular
recognition mechanism of nucleosides and β-CD, the characteristics of inclusion
complexes prepared are also investigated through UV-vis spectrophotometer, FTIR,
circular dichroism, DSC etc.
(d) Application of CM-β-CD bonded MNPs in wastewater treatment
CM-β-CD conjugated MNPs are used as nano-adsorbent for the removal of organic and
inorganic pollutants from wastewater. In organic pollutant removal study, methylene
blue (MB) is used as model contaminant because of their extensive use in various
industries. Methylene blue is a cationic dye which is toxic and carcinogenic. On the
other hand, the adsorption behavior of these adsorbents is investigated using Cu2+
(inorganic pollutant) as the target metal contaminant because of its extensive
environmental impacts. The CM-β-CD grafted onto Fe3O4 nanoparticles contributes to
an enhancement of the adsorption capacity of Fe3O4 nanoparticles because of the strong
Chapter: 2 Literature Review
45
complexation abilities of the multiple hydroxyl/carboxyl groups in CM-β-CD with
metal ions and of the hydrophobic cavity with organic contaminants. The adsorption
interactions are also investigated towards the removal of these pollutants.
(e) CM-β-CD grafted multifunctional core-shell silica MNPs for biomedical applications In this study, fluorescein dye-doped silica coated magnetic nanoparticles with core-shell
architecture is synthesized and then the silica surface is further functionalized with folic
acid and CM-β-CD. These as-synthesized nanoparticles has multiple functional
moieties- (i) silica shell which increases the stability and biocompatibility of these
nanoparticles, (ii) incorporated dye conjugate, (iii) folic acid which is a common anti-
cancer targeting ligand, and (iv) CM-β-CD which has encapsulation ability for
hydrophobic drugs. The possibility of the applications of these multifunctional
nanoparticles in biomedical fields, particularly for imaging, specific cell targeting and
anti-cancer drug inclusion/delivery is investigated.
It is believed that the present work on simple fabrication and applications of β-CD
conjugated magnetic nanoparticles for biomolecule refolding/separation, drug
inclusion/delivery, contaminants removal from wastewater can provide useful
information on the extension of these as-synthesized bifunctional or multifunctional
magnetic nanoparticles with combined properties of superparamagnetism and
complexation/ molecular recognition functions to multiple potential applications.
Chapter: 3 Characterization Techniques
46
Chapter 3 Characterization Techniques
3.1 Transmission Electron Microscopy (TEM) Measurement
A bright field transmission electron microscopy (TEM) was used for the size
measurement and size distribution of the magnetic nanoparticles. TEM images were
recorded on a JEOL 2010 transmission electron microscope at an accelerating voltage
of 200 kV. The TEM specimens were prepared by placing a drop of the nanoparticle
suspension on a carbon-coated copper grid (200 mesh and cover with formvar/carbon).
The copper film was then dried at room temperature for 24 h before the measurement.
3.2 X-ray Diffraction (XRD)
The structural properties and crystallographic structure of Fe304 powders were analyzed
by X-ray diffraction with shimadzu XRD-6000 diffractometer using a
monochromatized X-ray beam from nickel-filtered CuKα radiation (40 KV/ 30 mA,
wavelength λ=0.1504 nm) at a scanning rate of 2o min-1. The XRD patterns change was
plotted by intensity vs. 2θ in the range of 20o -70o. The average size of the crystals was
estimated using Debye-Scherrer’s formula:
cosCDB
(3-1)
where DC is the average crystalline diameter, Κ = particle shape factor (for spherical
particles, Κ = 0.9), λ is the X-ray wavelength, B is the angular line width of half-
maximum intensity and θ is the Bragg’s angle in degree.
Chapter: 3 Characterization Techniques
47
3.3 Vibrating Sample Magnetometer (VSM)
The VSM is the basic instrument for characterization of magnetic materials as a
function of magnetic field and temperature. It is based on vibrating a sample in a
magnetic field to produce an alternating electromagnetic field in the pickup coils.
Sample is placed between two electromagnet poles which are attached to pickup coils.
The electromagnets produce a high magnetic field (~90 kOe) to saturate the sample.
The sample vibrates vertically and the oscillation induces an AC signal (i.e. e.m.f.
according to the Faraday’s law) in the pickup coils. The amplitude of this signal is
proportional to the magnetic moment of the sample. A plastic cylinder cell containing
the sample was attached on a rod in the applied magnetic field from -15,000 to 15,000
Oe, in which the rod was vibrating in a certain rate. The magnetization curve of the
magnetic particles at room temperature was then plotted with the changes of magnetic
field strength and its direction. The magnetization curve (M-H hysteresis loop) of the
magnetic particles at room temperature is obtained by slowly sweeping the applied field
from a maximum positive field, through zero, to a maximum negative field and
reversing to the maximum positive field.
Wet magnetic particles were freeze-dried (Edwards freeze-dryer, ESM 1342) for 24 h
before VSM measurement. In this work, VSM (Lakeshore, Model 665) was used to
determine the saturation magnetization (MS) of the nanoparticles. VSM experiments
were carried by placing 10-20 mg dry powder samples (wrapped with aluminum foil) in
a VSM sample holder and then scanned from 2T to -2T (Tesla, 1 T = 104 Gauss) at the
room temperature. A small piece of nickel was used as a standard sample to calibrate
the VSM before all the VSM measurements.
Chapter: 3 Characterization Techniques
48
3.4 Thermogravimetric Analysis (TGA)
To measure the changes in the weight loss of the sample as a function of temperature
and time thermal analysis technique is used. The thermogravimetric analysis (TGA)
was performed on a thermal analysis system (Model: TA 2050). For TGA
measurements, 7-15 mg of the dried sample was loaded into the system and the mass
loss of dried sample was monitored under N2 at temperatures ranging from room
temperature to 800oC at a rate of 10oC/ min.
3.5 Differential Scanning Calorimetry (DSC)
DSC is mainly concerned with the amount of heat absorbed or released by a sample as
it is heated or cooled or kept at constant temperature (isothermal). In actual practice the
sample and reference materials are simultaneously heated or cooled at a constant rate.
The thermogram is plotted as mW vs T. DSC is a fast and relatively inexpensive
technique to characterize cyclodextrin inclusion complexes. The DSC records were
obtained with a Mettler Toledo DSC 882 apparatus. Between 4 and 10 mg of sample
was crimped in a sealed aluminium pan and heated at 10oC/ min in the range of room
temperature to 300oC using an empty sealed pan as a reference. Dry nitrogen was used
as purge gas and the N2 flow rate was 80 ml/min.
3.6 Fourier Transform Infrared (FTIR)
FTIR is an analytical technique for the identification of organic (and in some case for
inorganic) materials. It measures the absorption of various infrared light wavelengths by
the target sample, which can identify specific molecular components and structures.
Absorption bands in the range of 4000-1500 wavenumbers are typically due to
functional groups (e.g. –OH, C=O, N-H, and CH3, et al.). The region between 1500-400
wavelength numbers is considered as the fingerprint region, which is highly specific for
Chapter: 3 Characterization Techniques
49
each molecule and no two molecules have the same spectrum at this region. FTIR
measurements were performed in a transmission mode using a Shimadzu infrared
spectrometer (Model 8400) with KBr as background over the range of 4000-400 cm-1.
Samples were prepared by following steps: (1) First 1-2 mg particles/samples were
mixed with 150 mg KBr powder (highly transparent substance in IR range) using
spatula. (2) Then the resulting mixture was pounded with a pestle. (3) Finally a thin
pallet was formed under a press.
3.7 Brunauer-Emmett-Teller (BET) Method
The specific surface area was measured by BET analyzer (Model: Nova 3000). Surface
cleaning (degassing) of the dried solid magnetic particles were carried out by placing
the sample in a glass cell and heating under vacuum before the measurements. Once
clean, small amounts of gas molecules were admitted in steps into the sample chamber
and then stick to the surface of solid particles, which was carrying out in an external
bath maintained by liquid nitrogen. As the equilibrium adsorbate pressures approach
saturation, the surfaces of magnetic particles become completely occupied by adsorbate.
Knowing the density of the adsorbate, it is easy to calculate the specific area of the
magnetic particles.
3.8 Zeta Potential Analyzer
Zeta potential measurement is an easy and powerful way to investigate the charge
properties of particles and colloidal suspensions. According to the electrical double
layer (EDL) theory, the zeta potential represents the surface charge at the shear plane
which separates the Stern layer, attached to the particle, from the diffusive layer. It is
the most relevant parameter
Chapter: 3 Characterization Techniques
50
regarding electrostatic and electrokinetic properties of the particles, because the ions in
the Stern layer merely effect a static charge compensation. The zeta potential ζ is
usually deducted from the measurement of the electrophoretic mobility μe, based on the
Smoluchowski equation:
μe = ε. (ζ/η) (3-2)
where ε is the permittivity constant of the liquid involved and η is the viscosity of this
liquid. In experiments ζ is investigated as a function of the solution pH and the ionic
strength. If all the particles in suspension have a large negative or positive zeta potential
then they will tend to repel each other and there will be no tendency for the particles to
come together (flocculate). However, if the particles have low zeta potential values then
there will be no force to prevent the particles coming together and flocculating. The
general dividing line between stable and unstable suspensions is generally taken at
either +30 or -30 mV. Particles with zeta potentials more positive than +30 mV or more
negative than -30 mV are normally considered stable. The most important factor that
affects zeta potential is pH. In this thesis work, the zeta potentials of as-synthesized
uncoated or coated Fe3O4 magnetic nanoparticles (0.1 mg/ml) were measured in 10−3M
NaCl aqueous solution at different pH using a Malvern Zetasizer Nano-ZS model
ZEN3600 equipped with a standard 633 laser. The pH was adjusted with diluted HNO3
or NaOH solution. Hydrodynamic size and size distribution of the nanoparticles was
also measured using this same equipment by the dynamic light scattering (DLS)
technique. Hydrodynamic size (dH) is estimated from the following equation:
(3-3)
where k is the Boltzmann constant, T is the absolute temperature, D is the translational
diffusion coefficient, and η is the viscosity of the liquid. This method is based on the
Chapter: 3 Characterization Techniques
51
measurement of the speed at which particles are diffusing because of Brownian motion.
A light source, usually a laser, illuminates a sample cell containing the particles, and
fluctuations in the scattered intensity provide information on the particles’ dynamics.
3.9 X-ray Photoelectron Spectroscopy (XPS)
XPS is well established technique for surface analysis of particles, in which surfaces are
irradiated with soft X-ray and the emitted photoelectrons energy are analyzed. The
difference between X-ray energy and the photoelectron energies give the binding
energies (BEs) of the core level electrons, an atomic characteristic. XPS measurements
were made on an AXIS HSi spectrometer (Kratos Analytical Ltd.) using a
monochromatized Al Kα X-ray source (1486.6 eV photons) at a constant dwell time of
100 ms and a pass energy of 40 eV. The sample was mounted on the standard sample
studs by means of double sided adhesive tape. The core-level signals were obtained at a
photoelectron take-off angle 90° (with respect to the sample surface). The anode
voltage was 15 kV, and the anode current was 10 mA. The pressure in the analysis
chamber was maintained at 6.7 x10-6 Pa or lower during each measurement. All binding
energies (BEs) were referenced to the C1s neutral carbon peak at 284.6 eV. The spectral
deconvolution was performed using the curve-fitting program with the subtraction of
Shirley background; the line-width (full width at half-maximum) of the Gaussian peaks
was maintained constant for all components in a particular spectrum. Surface elemental
compositions were determined from XPS peak area ratios, after correction with the
experimentally determined sensitivity factors.
3.10 Circular Dichroism (CD)
Circular dichroism (CD) is the difference in absorption between left and right handed
circularly polarized light. Proteins contain elements of asymmetry and thus exhibit
Chapter: 3 Characterization Techniques
52
distinct circular dichroism signals. CD spectroscopy was used to measure the
conformational change of the refolded protein with respect to the native one. Solutions
of the native protein and the refolded protein were scanned over the wavelength range
200-250 nm by Jacob J810 spectropolarimeter, using 0.1 cm quartz cylindrical cell. All
circular dichroism measurements were carried out at 25C with the help of a
thermostatically controlled cell holder. Spectra were recorded at 1 nm intervals, with a
scan speed of 20 nm/min. Each spectrum was the average of two scans and noise in the
data was smoothed using the software. In all tests, circular dichroism spectra were
corrected for the background absorbance. The CD spectra measurements were
processed by Jasco’s spectra manager in millidegrees and were smoothed using the in-
built binomial smoothing algorithm. The collected data expressed in millidegrees
(mdeg) could be converted to mean molar ellipticity (deg cm2/dmol) using the relation:
θ mrd = θd (M/10CLNr)
(3-4)
where θmrd is the mean molar ellipticity per residue at 208 nm (deg cm2/dmol), θd is the
ellipticity in units of mdeg which is the experimentally measured value, M is the
molecular weight of the protein, C is the concentration of the sample in mg/ml, d is the
optical path length of the cuvette in centimeters and Nr is the number of amino residues.
In our refolding experiment, the secondary structures of native protein and refolded
protein were evaluated by comparing the α-helix contents which may be estimated
according to the following equation [158]:
% 100% (3-5)
Chapter: 3 Characterization Techniques
53
3.11 Fluorescence
Fluorescence is the phenomenon in which absorption of light of a given wavelength by
a fluorescent molecule is followed by the emission of light at longer wavelengths. The
distribution of wavelength-dependent intensity that causes fluorescence is known as the
excitation spectrum, and the distribution of wavelength-dependent intensity of emitted
energy is known as emission spectrum. The fluorescence measurements of the native
protein and the refolded protein by different desorption agent were carried out with
fluorescence spectrometer (Quantamaster, GL-3300 & GL-302 laser systems).
Tryptophan (Trp) is one of the three naturally occurring aromatic amino acid residues
that fluoresce when excited with UV light. When Trp residue is located in a
hydrophobic environment, the fluorescence is blue shifted relative to the emission when
it is located in a hydrophilic environment. When protein changes its conformation, the
exposure of Trp residues to the solvent may change and thereby affect fluorescence.
That’s why Trp fluorescence can be used for monitoring structural changes of proteins.
For the intrinsic protein fluorescence spectra of native and refolded CA samples, the
excitation wavelength was set at 280 nm and the emission was scanned from 290 to 400
nm for all samples. The scan rate was 1.0 nm/min-1 with a 10 nm bandpass for both
excitation and emission.
Chapter: 4
54
Chapter 4 β–cyclodextrin bonded magnetic nanoparticles as solid-phase artificial chaperone in refolding of proteins1
4.1 Introduction
The production of biologically active recombinant proteins in heterologous systems
often encounters the formation of inactive aggregates known as inclusion bodies [61].
Protein aggregation, which is a kinetically competitive process with protein folding
leads to low yields of biologically functional protein. Such aggregates must be
separated from cell debris and solubilized by exposing them to a strong denaturant, such
as urea or guanidine (GdmCl). Refolding of unfolded polypeptide chains to their native
state is initiated through decreasing the denaturant concentration by dialysis or direct
dilution. Several approaches have been developed to prevent protein aggregation and
also to facilitate protein refolding. One of the extensively used approaches is dilution
method, in which small molecules are added to mask or to eliminate the intermolecular
hydrophobic interactions among denatured proteins, thus prevent misfolding and/or
aggregation and promote refolding of proteins. Examples of such additives include
sugars [159,160], amino acids [159,161], acetoamide [162], alcohols [160,161],
polyethylene glycol [163], metals [164,165], detergents [166,167], and cyclodextrins
[168]. Cyclodextrins (CDs) are non-toxic, stable, cyclic oligosaccharides consisting of α
(1→ 4) linked D-glucose units which can be visualized as toroidal, hollow truncated
1. A.Z.M. Badruddoza, K. Hidajat, M.S. Uddin, Synthesis and characterization of beta-cyclodextrin-
conjugated magnetic nanoparticles and their uses as solid-phase artificial chaperones in refolding of
carbonic anhydrase bovine, J. Colloid Interface Sci. 346(2) (2010) 337-346.
Chapter: 4
55
cones with hydrophilic rims, and hydrophobic internal cavity. The ability of
cyclodextrin to sequester hydrophobic moieties makes them interesting and valuable in
many areas of research such as enantiomeric separation [169], targeted drug delivery
[60], removal of contaminants from wastewater [170] etc.
More recently, a systematic approach referred to as “artificial chaperone-assisted
refolding” has been developed by Rozema and Gellman [65-68] that effectively
promotes the renaturation yield of denatured proteins by suppressing the aggregate
formation. This method consists of two steps: in the first (capturing) step, the protein is
captured by a detergent and a stable protein–detergent complex is formed. The
detergent molecules shield the hydrophobic regions of denatured protein, thus prevent
aggregation of protein. In the second (stripping) step, β-CD is added which strips the
detergent molecules from the complexes with concomitant refolding of the protein.
Several other molecules like linear dextrin [171], cycloamylose [172] or alginate [173]
have also been reported which could replace cyclodextrin to act as the stripping agents.
However, with such systems, it is necessary to remove the detergent-stripping agent
complex and excessive stripping agents from the refolded products for downstream
purification which in turn leads to process complexity and increased production cost.
Now-a-days, magnetic nanoparticles with tailored surface chemistry have been widely
used in various fields of biotechnology. Due to unique size, biocompatibility and
superparamagnetic properties, magnetic nanoparticles are emerging as promising tools
for various fields of applications [174]. In the present work, we use β-CD conjugated
magnetic nanoparticles (CD-APES-MNPs) as stripping agents for protein refolding.
With cumulative effects of chaperoning behavior of β-CD and superparamagnetism of
iron oxide, CD-APES-MNPs is expected to have potential applications in protein
Chapter: 4
56
refolding. Because, the use of solid phase CD-APES-MNPs would offer the following
potential improvements over the soluble artificial chaperone assisted refolding: (1) steps
to remove the detergent-cyclodextrin complex and excess cyclodextrin can be omitted
and hence minimizes the cost; and (2) the magnetic nanoparticles bonded with β-CD
can be handled from the suspension easily by using low gradient magnetic field and
recycled after simple washing. Taking into the consideration of the recycling potential
of the solid phase, CD-APES-MNPs might be a suitable and efficient candidate for
protein refolding system.
A few insoluble solid adsorbents, such as β-CD-epichlorohydrin copolymer [175-177],
β-CD–acrylamide copolymer beads [177], β-CD-polyurethane polymer [103], β-CD
bonded silica particles [178] were previously used as host molecules for detergents in
artificial chaperone-assisted (ACA) refolding of various denatured proteins. Although
there were some previous work carried out on solid phase assisted protein refolding, no
or limited work on nanosized magnetic particles assisted protein refolding has been
published to the best of our knowledge. In the present study, β-CD conjugated magnetic
nanoparticles (CD-APES-MNPs) are synthesized and characterized. These synthesized
CD-APES-MNPs are then used as solid phase artificial chaperone in refolding of
bovine carbonic anhydrase (CA) as model protein.
4.2 Experimental
4.2.1 Materials
The following chemicals were used for synthesizing β-CD conjugated magnetic
nanoparticles: iron (II) chloride tetrahydrate (99%) [Alfa Aesar], iron (III) chloride
hexahydrate (98%) [Alfa Aesar], 3-aminopropyltriethoxysilane (APES, 98%) [Alfa
Aesar], ammonium hydroxide (25%) [Merck], β-cyclodextrin (99%) [Tokyo Kasie
Chapter: 4
57
Kogyo (Japan)], p-toluenesulfonyl chloride (98%) [Lancaster (UK)], carbonic
anhydrase (CA) from bovine erythrocyte [Sigma], 4-nitrophenyl acetate (pNPAc, 99%)
[Fluka], cetyltrimethylammonium bromide (CTAB, 99%) [Sigma],
tetradecyltrimethylammonium bromide (TTAB, 99%) [Sigma], sodium dodecyl sulfate
(SDS, 99%) [Sigma], cetyltrimethylammonium monohydrogen sulfate (CTAHS, 99%)
[Sigma], 1-anilinonaphtalene-8-sulfonate (ANS) [Sigma]. All other chemicals were of
analytical grade and were used as received without further purification. All protein
refolding experiments were generally performed in 20 mM Tris sulfate buffer, pH 7.75,
prepared using double distilled water.
4.2.2 Preparation of mono-tosyl-β-cyclodextrin (Ts-β-CD)
Mono-tosyl-β-cyclodextrin (Ts-β-CD) was synthesized according to the following
method. A solution of β-CD (18g, 16mmol) in 100 ml dry pyridine was prepared and
2.5g of p-toluenesulfonyl chloride (12 mmol) was added to this solution. The reaction
was carried out at 2-4C for a period of 8 h, and then 2 days at room temperature [179].
After concentrating the solution under vacuum, the mixture was poured into diethyl
ether. A white precipitate was collected and purified by repeated crystallization from
water. This product was finally washed by acetone and dried under vacuum at 60ºC.
Chemical composition for Ts-β-CD (C49H75O37S): C, 45.68%; H, 5.83%; S, 2.49%.
Found from elemantal analysis: C, 45.60%; H, 5.71%; S, 2.42%. IR (KBr): 3387 (ν,
OH), 2927 (ν, C-H), 1636 (δ, OH), 1368 (ν, SO2), 1157 (ν, C-O-C), 1029 (ν, C-O).
4.2.3 Preparation of Fe3O4 magnetic nanoparticles
Fe3O4 magnetite nanoparticles were prepared by chemical co-precipitation method
maintaining a molar ratio of Fe2+: Fe3+ = 1: 2 under a non-oxidizing environment and
alkaline condition [14]. To obtain 1g Fe3O4 precipitate, 0.86 g of FeCl2.4H2O and 2.36 g
Chapter: 4
58
of FeCl3.6H2O salts were reacted under a nitrogen atmosphere in 40 ml of de-aerated
Milli-Q water with vigorous stirring. 5 ml of NH4OH (25%) was added after the
solution was heated to 80ºC. The reaction was carried out further for another 30 min at
80ºC under constant stirring to ensure the complete growth of the nanoparticle crystals.
The resulting particles were then washed with Milli-Q water to remove any unreacted
chemicals and dried by freeze-drying for 24 h.
4.2.4 Preparation of 3-aminopropyltriethoxy silane (APES) modified magnetic nanoparticles (APES-MNPs)
To synthesize APES-MNPs, 3 g of Fe3O4 nanoparticles was suspended in 90 ml ethanol
and 1 ml H2O and sonicated for 30 min in the ultrasonic bath. The mixture was then
vigorously stirred in a 250 ml three neck flask equipped with a reflux condenser for 40
min. 10 ml of APES was then added slowly to the flask. The reaction was carried out
for 3 h at the refluxing temperature under nitrogen atmosphere and mechanical stirring.
The resulting nanoparticles were washed with water, then acetone, and these washings
were repeated for one more time. The resulting APES-MNPs were finally dried in the
air.
4.2.5 Fabrication of Ts-β-CD modified Fe3O4 nanoparticles (CD-APES-MNPs)
50 ml of DMF containing 2 g of APTS-MNPs was stirred in a three neck flask equipped
with a reflux condenser at room temperature. 0.25 g of Ts-β-CD dissolved in 10 ml of
DMF was added to this reaction mixture (pH 7~8). The temperature was increased to
60ºC and the reaction was carried out for 7 h under nitrogen atmosphere and
mechanical stirring. The reaction mixture was left overnight at room temperature. The
resulting particles were then collected using a permanent magnet and washed with DMF
and acetone to remove any unreacted chemicals and dried in the air.
Chapter: 4
59
4.2.6 Protein refolding experiments
4.2.6.1 Determination of protein concentration
The protein concentration of the native carbonic anhydrase solution was determined by
measuring the absorbance at 280 nm with an extinction coefficient (ε280) of 5.7×104
mol−1 cm−1 [180].
4.2.6.2 CA denaturation/ renaturation and assay
In a typical thermal denaturation of carbonic anhydrase (CA), 0.043 mg/ml of CA in 20
mM Tris sulfate (pH 7.75) containing 2 mM of each of detergents (CTAB, TTAB,
SDS) were heated to 70°C for 10 min in thermostatically controlled water bath
according to Rozema and Gellman [65]. Protein inactivation was confirmed by activity
determination as well as by fluorescence analysis. After cooling for 10 min, the CA-
detergent complex solutions were diluted with addition of certain amount of stock
solutions of suspended CD-APES-MNPs or β-CD (for liquid phase artificial chaperone
assisted refolding) to make solutions of 0.029 mg/ml CA, 1.35 mM detergent, 13.5 mM
Tris sulfate, pH 7.75. After incubation for overnight, the supernatant was collected by
applying magnetic field and assayed for enzymatic activity. In each CA refolding study,
3 ml of aliquots was used. For refolding kinetic analysis, the enzymatic activities were
monitored immediately after addition of CD-APES-MNPs to the CA solutions.
The enzymatic activities of renatured CA solutions were determined based on pNPAc
esterase activity of the enzyme according to Pocker and Stone [181]. Briefly, 45 μl of
52 mM pNPAc in dry acetonitrile was added to 0.45 ml of CA solution to make a
solution of 0.0264 mg/ml CA, 12.3 mM Tris-sulfate, pH 7.75, and 4.7 mM pNPAc.
After 10 s of mixing, the increase in the hydrolysis product of p-nitrophenolate was
Chapter: 4
60
monitored by measuring the increase in absorbance at 400 nm as a function of time. The
absorbance of the solution was measured every second for 30–60 s using a Model UV-
1601 Shimadzu spectrophotometer. The yield of the refolded CA was determined by
comparing the resulting initial rate with the initial rate of pNPAc hydrolysis by the
native enzyme under identical conditions and expressed as percentage relative to the
activity exhibited by the native enzyme.
4.2.6.3 Structure analyses using Fluorescence Spectra and Far-UV circular dichroism
Fluorescence experiments to observe the tertiary structural changes of refolded CA
were performed using a fluorescence spectrometer (Quantamaster, GL-3300 & GL-302
laser systems) at 25C, with a 10 mm path length sample cuvette. For ANS
fluorescence studies, to the refolded CA solutions, a concentrated stock of ANS (10
mM) was added to get a final concentration of 250 μM. The dye 1-anilinonaphthalene-
8-sulfonate (ANS) which shows enhanced fluorescence in hydrophobic environments
has been used as hydrophobic reporter probe to monitor protein conformational changes
by binding to the hydrophobic regions of a protein. The fluorescence intensity of ANS
increases when the dye binds to the hydrophobic regions of a protein. All samples were
incubated with this hydrophobic reporter probe for 30 min at 25C and the changes of
ANS fluorescence spectra were recorded between 400 nm and 600 nm using excitation
wavelength of 360 nm. For the intrinsic protein fluorescence spectra of native and
refolded CA samples, the excitation and emission wavelengths were set at 280 and 290–
400 nm range, respectively. The secondary structural changes of the CA samples were
evaluated by far-UV circular dichroism spectroscopy. Measurements were recorded
over wavelength range of 200–250 nm using a Jacob J810 spectropolarimeter with a 0.1
cm path length sample cell. All circular dichroism measurements were carried out at
Chapter: 4
61
25C with the help of a thermostatically controlled cell holder. Spectra were recorded at
1 nm intervals, with a scan speed of 20 nm/min. Each spectrum was the average of two
scans and noise in the data was smoothed using the software. The final CA
concentration after renaturation was set to 0.15 mg/ml. In all tests, circular dichroism
spectra were corrected for the background absorbance.
4.2.6.4 Detergent (CTAB) quantification
After completion of the refolding process, the residual CTAB concentration in the
renaturation buffer was measured based on ANS fluorescence according to Mannen et
al. [175]. The refolded samples in solid-phase artificial chaperone assisted refolding
system were incubated with 100 μM ANS (final) and the fluorescence emission was
measured at 480 nm after 30 min incubation at room temperature using a 360 nm
excitation wavelength with 1 cm path length quartz cell. Fluorescence intensity was
plotted against the final detergent concentration and the percent of residual detergent
measured according to fluorescence intensity of each of the refolded samples.
4.3 Results and Discussion
4.3.1 Synthesis of β-CD bonded magnetic nanoparticles
Fe3O4 magnetic nanoparticles with a mean diameter of about 9.5 nm were synthesized
by co-precipitation method from ferrous and ferric ion solutions with a molar ratio of
1:2 [14,15]. The magnetic nanoparticles prepared by this method had significant
numbers of hydroxyl groups on the surface from contacting with the aqueous phase. For
grafting Ts-β-CD, the amino groups were preliminarily bonded onto the surface of the
magnetic nanoparticles by the reaction with 3–aminopropyltriethoxysilane (APES)
[182]. The APES hydrolyzes in the presence of water to form silanol groups which
coupled with the surface metal hydroxyl groups, forming Si-O-Fe bonds upon
Chapter: 4
62
dehydration. Thus –NH2 groups remain reactive on the surface through direct silanation
of APES on magnetic particles. It is known that the homogeneous reaction between
amino chemicals and tosyl-containing compounds occurs at 60−70ºC in an aqueous
medium with pH 11.5 [183]. However, in our experiment, the grafting of β-CD on the
surface of APES-MNPs was carried out at 60ºC between aminopropyl iron and Ts-β-
CD for 7h in DMF at pH 7-8 because of the poor solubility of Ts-β-CD in water. At
higher pH (pH>11) aminopropyl iron considerably collapsed and no attachment of β-
CD was observed. Similar phenomenon was observed on the synthesis of β-CD bonded
silica particles prepared by Belyakova et al. [28]. The lower reaction temperature was
chosen in order to avoid the side reactions that may occur due to the interactions of
surface amino groups with DMF [28]. The concentration of β-CD in reaction medium
was varied to see its effect on grafting on the surface of magnetic nanoparticles and it
was observed that the amount of grafted β-CD was 0.042, 0.038 and 0.036 mmolg-1
when 0.25, 0.50 and 0.75 g of β-CD was used, respectively. The more β-CD added in
the reaction, the less β-CD was immobilized on the surface of magnetic nanoparticles.
The reaction procedures for fabricating CD-APES-MNPs are shown step by step in
Figure 4-1.
Chapter: 4
63
Figure 4-1 Preparation steps for fabricating β-CD bonded magnetic nanoparticles.
4.3.2 Characterization of magnetic nanoparticles
The reaction pathway for grafting β-CD on surfaces of magnetic nanoparticles was
verified by FTIR studies. Figure 4-2 shows the FTIR spectra of MNPs, APES-MNPs,
CD-APES-MNPs and β-CD in the 400-2000 cm-1 wave number range. It shows the
characteristic absorption band of Fe–O bonds in the tetrahedral sites is 586 cm-1. The
spectrum of APES-MNPs shows peak at 997 and 1110 cm-1 which can be attributed to
siloxane bonds (Si-O-H) and Si-O-Si groups, respectively due to APES deposition on
the MNPs’ surface. The broad band at 1626 cm-1 is due to –NH stretching vibrations.
The spectrum of β-CD shows the characteristic peaks at 947, 1028, and 1159 cm−1. The
APES-MNPs
Chapter: 4
64
peak at 947 cm−1 is due to the R-1,4-bond skeleton vibration of β-CD, and the peaks at
1028 and 1159 cm−1 corresponded to the antisymmetric glycosidic νa (C-O-C)
vibrations and coupled ν (C-C/C-O) stretch vibration [16,184]. All the significant peaks
of β-CD in the range of 900–1200 cm−1 are present in the spectrum of CD-APES-MNPs
with a small shift. Thus, all the above results indicate that β-CD has been grafted
successfully on APES-MNPs.
Figure 4-2 FTIR spectra of (a) Uncoated Fe3O4 MNPs, (b) APES-MNPs, (c) CD-APES-MNPs, and (d) β-CD.
Typical TEM images and size distribution of APES-MNPs and CD-APES-MNPs are
shown in Figure 4-3. Spherical or ellipsoidal magnetic nanoparticles are observed. TEM
image of β-CD bonded magnetic nanoparticles is almost the same as that of magnetic
nanoparticles coated with APES. The mean diameter of both APES-coated and β-CD
400 800 1200 1600 2000
% T
rans
mis
sion
(a.u
.)
Wavenumber, cm-1
947
10281159
9481001 1029
997
1110
(a)
(c)
(b)
(d)
586
586
586
1626
1626
Chapter: 4
65
functionalized nanoparticles is about 11.5 nm. No detectable layer of β-CD can be
observed on CD-APES-MNPs due to the small amount of β-CD attached on the surface
(0.042 mmolg-1 solid from elemental analysis). It is known that magnetic particles with
size less than about 30 nm with zero coercivity and zero remanence exhibits
superparamagnetism [185]. Therefore, both the magnetic particles coated with APES
and β-CD show superparamagnetic properties which are further evidenced by VSM
studies. Figure 4-3(c) and 4-3(d) also show the hydrodynamic diameter distribution of
APES-MNPs and CD-APES-MNPs in water determined by the DLS technique. The
mean hydrodynamic diameters were found to be 38.6 and 51.8 nm, respectively.
Figure 4-3 Typical TEM images and hydrodynamic size distribution of APES-MNPs (a) &(c) and CD-APES-MNPs (b) & (d), respectively.
0
5
10
15
20
8 9 10 11 12 13 14 15 16 17
Particle diameter (nm)
Fre
quen
cy (
%)
(a) (b)
(c) (d)
0
5
10
15
20
25
30
7 8 9 10 11 12 13 14 15 16 17
Particle diameter (nm)
Fre
quen
cy (
%)
Chapter: 4
66
Elemental analyses shown in Table 4-1 provide further evidence of the successful
immobilization of β-CD onto magnetic nanoparticle surface. The grafted amounts of
aminopropyl groups and β-CD moieties on the surface of magnetic nanoparticles (BET
area~109 m2/gm) calculated from elemental analyses are as 0.812 mmol/g and 0.042
mmol/g of solid, respectively. The concentration of surface amino groups (-NH2) is
0.812 mmol/gm (7.44 µmol/m2). It can be concluded that not all –NH2 groups on the
surface of APES-MNPs are bonded with tosyl groups of Ts-β-CD as the tosylation
reaction occurs 1:1 stoichiometic ratio. Taking into account the molecular area of Ts-β-
CD (=3.41 nm2) [28], the maximum quantity of Ts-β-CD grafted on the surface of
aminopropyl iron cannot exceed 0.054 mmol/g. Thus, the surface β-CD coverage ratio
of Fe3O4 magnetic nanoparticles was about 79%. Probably, the incompleteness (on
monolayer adsorption basis) of the surface coating is owing to the incompleteness of
surface tosylation and also due to existence of spatial resistance or the steric hindrance
of the β-CD moieties.
Table 4-1 Data on chemical analysis of magnetic nanoparticles modified with APES
and β-CD.
As-synthesized MNP samples %C %N %H
APES-MNPs 3.12 1.12 0.47
CD-APES-MNPs 4.96 1.09 0.73
The XRD patterns of MNPs, APES-MNPs and CD-APES-MNPs indicate five
characteristic peaks at 2θ = 30.1, 35.5, 43.1, 53.4, 57, and 63.1 as shown in Figure
4-4(i). These are related to their corresponding indices (220), (311), (400), (422), (511),
and (440), respectively. This reveals that the resultant nanoparticles are pure Fe3O4 with
a spinel structure and the modification of magnetic nanoparticles surface with APES or
β-CD does not result in the phase change of Fe3O4. The crystal size of CD-APES-MNPs
Chapter: 4
67
-80
-60
-40
-20
0
20
40
60
80
-15 -10 -5 0 5 10 15
M, e
mu/
g F
e 3O
4
H, kOe
Fe3O4 MNPs
CD-APES-MNPs
determined from the XRD pattern by using Scherrer’s equation is found to be 12.8 nm,
consistent with that observed from the TEM image.
Figure 4-4 (i) XRD patterns of (a) uncoated magnetic nanoparticles (MNPs), (b) APES modified magnetic nanoparticles (APES-MNPs), and (c) β-CD modified magnetic nanoparticles (CD-APES-MNPs).(ii) Magnetization curves for uncoated and β-CD coated Fe3O4 magnetic nanoparticles measured by VSM at room temperature.
(i)
(ii)
Chapter: 4
68
The magnetic properties of the uncoated and β-CD coated Fe3O4 nanoparticles were
measured by VSM at room temperature. The VSM technique is used to measure the
magnetization value of magnetic particles under applied magnetic field. For particles
with superparamagnetic property, when the applied magnetic field is removed, they
should exhibit no coercivity and remanence. In Figure 4-4(ii), the hysteresis loops that
are characteristic of superparamagnetic behavior can be clearly observed for both
MNPs and CD-APES-MNPs. Superparamagnetism is the responsiveness to an applied
magnetic field without retaining any magnetism after removal of the applied magnetic
field. The properties of superparamagnetism and high saturation magnetization value of
magnetic nanoparticles are the two important parameters in biomedical applications.
From M vs. H curve, the saturation magnetization value (Ms) of uncoated MNPs was
found to be 75 emu g−1 which is much less than its bulk counterpart (92 emu g−1) [186].
For CD-APES-MNPs, the magnetization obtained at same field was 64 emu g-1, lower
than that of uncoated Fe3O4. This is mainly attributed to the existence of non-magnetic
materials (APES and β-CD layer) on the surface of nanoparticles. This feature allows
the as-synthesized nanoparticles to be used even under relatively low external magnetic
field. The combination of magnetic property of iron oxide with chaperoning behavior of
β-CD suggests that these materials have great a potential to be used in protein refolding.
4.3.3 CD-APES-MNPs assisted CA Refolding As mentioned earlier, Rozema and Gellman [65,68] had developed an approach for
protein renaturation in which the aggregation of protein was prevented by detergents
which could shield the hydrophobic regions of denatured proteins. The detergent
molecules were then slowly stripped off the protein surface by cyclodextrins, allowing
the protein to refold. This approach requires an efficient detergent stripping agent and
further steps needed to remove detergent-CD complex and excess β-CD are necessary
Chapter: 4
69
in downstream purification process. But using solid phase CD-APES-MNPs as
stripping agent, further purification steps can be omitted. In addition, this solid phase
can be easily separated by using low gradient magnetic field and re-used it after simple
washing. A schematic diagram of the protein refolding process using β-CD conjugated
magnetic nanoparticles is shown in Figure 4-5.
Figure 4-5 Schematic diagram of CD-APES-MNPs assisted protein refolding in-vitro.
Figure 4-6 shows the kinetic properties of CA refolding at concentration of 0.029
mg/ml CA using 40 mg/ml of CD-APES-MNPs. We also measured the residual CTAB
concentration in refolded solution using ANS fluorescence probe at different time
intervals to check whether the refolding occurred concomitantly with the detergent
stripping steps. The CTAB concentration in the capturing step was set at 2mM which
Chapter: 4
70
was higher than the CMC of CTAB. The CMC of CTAB in the refolding buffer was
measured to be 0.63 mM, similar value was reported in literature [65]. As shown in
Figure 4-6 that only about 30-40 mins was required for the maximum refolding yield to
be reached in CD-APES-MNPs assisted CA refolding. Within this time, most of CTAB
molecules were stripped away by the CD-APES-MNPs and the residual CTAB
concentration also reached a plateau (~20%). Here, the stripping of CTAB form the
protein-detergent complexes was greatly limited by the amount of CD immobilized on
the solid surface. More CTAB could be stripped away using higher amount of CD-
bonded magnetic nanoparticles (Figure 4-7), but the refolding yield would decrease. To
solve this problem, one approach would be to speed up the stripping of detergent
molecule from the protein-detergent complexes by increasing the number of CD
molecules per unit volume or unit mass of solid matrix. However, this result indicates
that both detergent stripping process and refolding of CA are very fast processes and
occur concomitantly.
Figure 4-6 Time course of refolding yield of CA (■) and residual CTAB concentration (●) in refolded samples in presence of 40 mg/ml CD-APES-MNPs. The CTAB concentration in the capturing step was set at 2mM and final CA concentration was 0.029 mg/ml.
10
20
30
40
50
60
70
80
90
100
0
20
40
60
80
100
0 40 80 120 160
% R
esid
ual
CT
AB
Ref
old
ing
yiel
d,
%
Time, min
Chapter: 4
71
It was previously reported that the minimum detergent concentration i.e. the minimum
structural arrangement between CA and detergent is required to prevent CA aggregation
at the capture step [187,188]. This minimum structural arrangement is apparently
achieved at a detergent (CTAB, TTAB and SDS) concentration of 2 mM. Under these
conditions, the competent mixed micelles are formed [65]. Formation of competent or
incompetent protein-detergent complex strongly depends on the final concentration of
detergent used in the system. For example, CA–CTAB complexes generated with 0.2–
0.4 mM final CTAB concentration are ‘incompetent’: they do not lead efficiently to
refolded protein upon stripping, even though the amount of detergent is sufficient to
prevent detectable protein aggregation at the point of denaturant dilution [187]. It was
predicted that competent complexes contain only one protein molecule, whereas
incompetent protein–detergent complexes contain, on average, more than one protein
molecule per complex. So, in stripping step, the rate of detergent stripping which is
determined by the amount of β-CD (or β-CD : detergent ratio) is a key factor in
artificial chaperone assisted refolding approach [187]. Regarding this criteria, the
effects of CD-APES-MNPs on refolding yield of thermally denatured CA were
investigated. Figure 4-7 shows the refolding yield of CA upon addition of CD-APES-
MNPs (10–60 mg/ml) and as a function of detergent type. The maximum refolding
yield of CA of 85%, 74% and 48% were obtained in CD-APES-MNPs assisted
refolding for CTAB, TTAB and SDS, respectively. The order of refolding yields
(CTAB> TTAB> SDS) could be explained by the hydrocarbon chain length of the
detergent which influences the rate of detergent stripping from the detergent-protein
complexes [176,189]. It was reported that the detergent stripping, in the liquid-phase
ACA technique, is the rate-determining step of the whole process and the rate of this
step are controlled by β-CD/detergent binding constants [176,188,189].
Chapter: 4
72
Figure 4-7 Relative changes in the recovered enzymatic activity of the assisted renatured CA as a function of detergent type and the solid phase quantity. Protein-detergent (CTAB, □: TTAB, Δ and SDS, ○) complex solution (2.7 ml, with a detergent concentration of 2mM in Tris–Sulfate buffer, pH 7.75) was added to 1.3 ml suspension containing various amounts of β-CD bonded magnetic nanoparticles. The final protein concentration was 0.029 mg/ml. The residual CTAB concentration (●) in the supernatant of the suspension was also measured.
From Figure 4-7, it is also shown that the optimal refolding yield is achieved at
concentration of 40, 50 and 50 mg/ml of CD-APES-MNPs for CTAB, TTAB and SDS,
respectively which we refer to it as the optimal value and the refolding yield decreases
beyond these values for each of the three detergents. The reason for the decrease in
refolding efficiency at higher than optimal concentrations of the CD-APES-MNPs is
not clear. Yazdanparast et al. claimed that it was possibly due to the fact that solid-
phase ACA approach faces difficulties due to mass transfer limitation and/or the low
substitution content of β-CD in the CD-bearing solid phase [176]. Alternatively, there is
the possibility of ‘‘inappropriate’’ removal rate of the detergent molecules from the
detergent–protein complexes [103]. It could be better explained by the mechanism
proposed by Hanson and Gellman [187] where product-determining step occurs after a
0
20
40
60
80
100
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70
% R
esid
ual
CT
AB
Ref
old
ing
yiel
d,%
Conc. of CD bonded magnetic nanoparticles, mg/ml
Chapter: 4
73
portion of the detergent has been stripped from the protein complex U–dn–m as shown
in Figure 4-8. Under such a condition, the stripped and unfolded protein molecules will
form intermolecular hydrophobic interactions which will finally lead to aggregation
and/or misfolding. This view could be evaluated through analyzing the
microenvironment of the refolded and the partially refolded intermediates by using the
ANS fluorescence probe [190]. Use of too much striping agent might affect the
detergent stripping rates which in turn will lead to the exposure of hydrophobic surfaces
of the stripped and the unfolded intermediates which are quite suited for intermolecular
interactions and aggregate formations [103]. The lower yield obtained using higher
amount of magnetic nanoparticles values might also possibly be due to non-specific
binding of the folded or refolded enzymes to these nanoparticles. Non-specific
adsorption might occur due to residual amine (NH2) groups on the surface of magnetic
nanoparticles due to electrostatic interactions [191].
Figure 4-8 Proposed mechanism by Hanson and Gellman for cyclodextrin-induced folding from a protein–detergent complex [187]. U–dn, protein–detergent complex generated during the capture step (contains only one protein molecule); U, unfolded protein molecule; d, one detergent molecule; U–dn–m, protein–detergent complex from which detergent has been partially stripped away; (U–dn–m)p, partially stripped protein–detergent complex that has self-associated to form a species containing multiple protein molecules; F, first detergent-free form of the protein (extent of folding unspecified); N, native state of the protein.
Chapter: 4
74
In order to compare the solid-phase ACA refolding results with that of using liquid-
phase ACA refolding, renaturation of denatured CA was carried out in presence of
soluble β-CD (0-16 mM). The maximum refolding yield (85%) obtained using solid-
phase CD-APES-MNPs was almost in the same level as that for the liquid-phase ACA
refolding method using soluble β-CD (90%) as shown in Figure 4-9. Moreover, our
refolding results could be compared when using same protein concentration level with
those using other solid-phase ACA method. For example, Esmaeili et al. obtained CA
refolding yields of 80% and 54% using at 0.030 mg/ml β-CD-polyurethane polymer and
β-CD bonded silica particles at same CA concentration (0.030 mg/ml) in two different
studies [103,178]. Yamaguchi et al. also obtained same refolding yield (80%) using β-
CD–acrylamide copolymer hydrogel beads [177]. Our result indicates that CD-APES-
MNPs are suitable, effective and recyclable stripping agents for solid-phase ACA
technique. Moreover, use of solid phase CD-APES-MNPs in ACA method would
eliminate further tedious purification steps associated with liquid-phase ACA method.
Figure 4-9 Refolding yield of thermally denatured CA in the presence of different concentrations of β-CD in liquid phase artificial chaperone assisted refolding approach. The denatured CA (0.043 mg/ml) was diluted with water containing
0
20
40
60
80
100
0 4 8 12 16
Ref
old
ing
yiel
d,
%
Cyclodextrin, mM
Chapter: 4
75
different concentration of β-CD (0-16 mM). The error bars represent standard error of mean for three independent measurements.
It was previously observed that the refolding yield decreases with an increase in the
concentration of the protein to be refolded [63,103,176]. When refolding was performed
at high protein concentrations, inactive aggregates formed by intermolecular interaction
were the predominant products. Our data presented in Figure 4-10, clearly indicate that
a similar pattern also exist in solid-phase artificial chaperone assisted technique. As
shown in Figure 4-10, the maximum refolding yield for CA (85%) was obtained using
40 mg/ml CD-APES-MNPs at CA concentration of 0.043mg/ml in the captured step
(final CA concentration was 0.029 mg/ml). At higher protein concentrations (0.15
mg/ml in the captured step), the renaturation yields of CA dropped to 44.8%.
Figure 4-10 The effect of CA concentration and the amount of CD-APES-MNPs on CA renaturation yield. The CTAB concentration in the capturing step was set at 2 mM. The protein concentration in the captured state were 0.043 (●), 0.075 (■) and 0.15 (▲) mg/ml.
0
20
40
60
80
100
0 10 20 30 40 50 60
Ref
oldi
ng y
ield
, %
Conc. of CD bonded magnetic nanoparticles, mg/ml
0.043 mg/ml
0.075 mg/ml
0.15 mg/ml
Chapter: 4
76
4.3.4 Structural analyses of the refolded products by intrinsic fluorescence and far-UV circular dichroism
Fluorescence spectroscopy is a useful tool to study the structural and conformational
changes of protein molecules in solution. Intrinsic fluorescence analysis can perform
this kind of studies by differentiating the micro-environmental changes of tryptophan
residues upon excitation at 280 nm. Tertiary structure of the refolded (CA) products
was assessed by recording the fluorescence emission spectra as shown in Figure 4-
11(a). The sub-optimal concentration of CD-APES-MNPs causes variation of
fluorescence intensity of refolded samples implying that the tertiary structures are
different from those of native sample. However, the addition of optimal quantity of CD-
APES-MNPs (40 mg/ml, curve 5) to the captured solution caused a remarkable change
in the tertiary structure of the refolded sample in such a way that the pattern of its
tertiary structure became very similar to the native conformation. Figure 4-11(b) shows
the maximum emission peak point of the refolded CA upon addition of various
concentrations of CD-APES-MNPs. The fluorescence emission maximum for native
CA is at 340 nm, while the fluorescence emission maximum for refolded CA in
unassisted refolding method is at 346 nm, which is typical of water-exposed tryptophan
[67]. This implies that the unassisted refolded product is still in inactive CA-CTAB
complex form where the tertiary structure is lost. Detergent-protein complex shows
much higher fluorescence intensity than native CA, suggesting that the fluorescing side
chains (presumably tryptophan) are less subject to internal quenching in the detergent
complex than in the native folded state. However, with addition of various
concentrations of CD-APES-MNPs from 10 to 40 mg/ml, the fluorescence emission
maximum blue shifted from 346 to 340 nm. This indicates that CD-APES-MNPs at 40
mg/ml concentration are maximally assisting the captured CA to refold to its native
conformation.
Chapter: 4
77
Figure 4-11 Effect of various concentrations of CD-APES-MNPs on the intrinsic fluorescence emission of refolded CA samples. (a) Intrinsic fluorescence spectra of refolded samples in the presence of different amounts of CD-APES-MNPs (0 mg/ml, curve 1; 10 mg/ ml, curve 2; 20 mg/ml, curve 3; 30 mg/ml, curve 4; 40 mg/ml, curve 5 and 50 mg/ml, curve 7). Curve 6 represents the native CA. (b) Maximum emission wavelength peak point of the refolded CA in presence of various concentrations of CD-APES-MNPs.
The ANS fluorescence spectra of the native and the refolded CA in the presence of
various amounts of CD-APES-MNPs were also recorded in the wavelength range of
400-600 nm. All refolded samples were incubated at room temperature for 30 min in
0
20
40
60
80
100
120
140
160
180
305 320 335 350 365 380 395
Rel
ativ
e fl
uor
esce
nce
inte
nsi
ty
Wavelength, nm
1
2
3
4
7
5,6
(a)
338
340
342
344
346
348
0 20 40 60
Em
ax(n
m)
CD bonded magnetic nanopaticles, mg/ml
(b)
Chapter: 4
78
the presence of ANS solution (100 μM). The excitation wavelength was 360 nm. As
shown in Figure 4-12, at low CD-APES-MNPs concentrations, most of the released
protein molecules remained in their non-native states with exposed hydrophobic areas
attracting the ANS molecules leading to high fluorescence emission intensities [103].
However, at higher appropriate concentration of CD-APES-MNPs (40 mg/ml), more
efficient refolding has taken place which is evident by decrease in ANS fluorescence. It
is surprising to find that the ANS intensities of the samples with higher than 40 mg/ml
of CD-APES-MNPs were even smaller than that of the native CA. This might be caused
by various types of interactions among β-CD and the protein molecules [103] which
have limited the accessibility of ANS molecules to the proteins. Also, it could be
explained by the inappropriate CTAB stripping rate caused by using too-much stripping
agents which would lead to the exposure of hydrophobic surfaces of the stripped and
the unfolded intermediates suited for intermolecular interactions and aggregate
formations [103].
Figure 4-12 ANS fluorescence spectra of native (6) CA and refolded CA in the presence of 0 (curve 1), 10 (curve 2), 20 (curve 3), 30 (curve 4), 40 (curve 5), 50 (curve 7) and 60 (curve 8) mg/ml of CD-APES-MNPs added in the solution. The final
0
5
10
15
20
25
30
35
400 440 480 520 560 600
Wavelength, nm
Rel
ativ
e A
NS
flu
ores
cnce
inte
nsity
1
8
7
23
45
66
Chapter: 4
79
protein concentration was 0.029 mg/ml for all samples. Samples were excited at 360 nm.
Finally, to investigate whether the secondary structure of CA was successfully
recovered in solid phase CD-APES-MNPs assisted refolding method, the far-UV
circular dichroism spectra of refolded CA using different amounts of CD-APES-MNPs
were measured and compared with that of native CA. Circular dichroism measurements
are difficult with CTAB containing solutions, because of absorption by bromide ion;
therefore, cetyltrimethylammonium hydrogen sulfate (CTAHS) was used in these
studies. Control studies indicated very similar behavior for CTAB and CTAHS as
artificial chaperones [65]. Figure 4-13 shows the far-UV circular dichroism spectra in
200-250 nm range for native CA (curve 1) and for the refolded samples (curves 2, 3 and
4). From the figure, it can be seen that at lower concentration of CD-APES-MNPs (10
mg/ml), the far-UV circular dichroism spectra of the refolded CA have more negative
ellipticity which are attributed to higher α-helix content [65]. However, as the amount
of CD-APES-MNPs increases (curves 2 and 3), the secondary structural features of the
refolded CA become closer to that of the native CA (curve 1). The ellipticity values at
208 nm are different for curves 2, 3 and 4. These values are used to calculate the α-helix
content of proteins [158]. Based on the value of ellipticity obtained at different amount
of solid phase, this result also indicates that optimal refolding assistance was achieved
by using 40 mg/ml of CD-APES-MNPs. Only partial stripping of the detergent
molecules occurs in case of using too little CD-APES-MNPs which in turn leads to
partially stripped inactive intermediates. Under these circumstances, these partially
captured CA remain in the inactive detergent-protein complex states [187]. Sub-optimal
concentration of CD-APES-MNPs may allow the refolding intermediate complexes to
Chapter: 4
80
live long enough to form intermolecular interactions with each other which lead to
declined refolding yields [176].
Figure 4-13 Far-UV circular dichroism spectra of native (curve 1) and the refolded CA samples in the presence of 10 (curve 4), 30 (curve 3) and 40 (curve 2) mg/ml of the CD-APES-MNPs added in the solution. Each spectrum was obtained at 25C with a 1mm path length cell and is the average of two scans. All necessary corrections were made for background absorption.
4.4 Conclusions
In this work, we have described a new method to synthesize β-CD bonded magnetic
nanoparticles (CD-APES-MNPs) having combined effects of chaperoning behavior of CDs
and magnetic properties of iron oxide. The results from FTIR, XRD, TEM and VSM have
confirmed the formation of nanosized superparamagnetic particles functionalized with
β-CD. From the results of elemental analyses, the amount of grafted β-CD on the
surface of magnetic nanoparticles was calculated as 0.042 mmolg-1 solid. These as-
synthesized CD-APES-MNPs were effectively used in as solid phase artificial
chaperone in carbonic anhydrase (CA) refolding in vitro. Our result indicates that the
maximum refolding yields obtained were 85% for thermally denatured CA which was
-20
-16
-12
-8
-4
0
4
200 210 220 230 240 250
Elli
pti
city
, m
deg
Wavelength, nm
4
1
3
2
Chapter: 4
81
as the same level as that using liquid-phase artificial chaperone-assisted refolding and
the structures of the refolded CA were found as same as those of native protein. These
results suggest that CD-APES-MNPs could be used as a suitable and efficient stripping
agent in solid-phase artificial chaperone-assisted refolding due to easier and faster
separation of these stripping agents from the refolded samples and the recycling
potentials of these nanoparticles. This new approach will have a considerable impact on
reducing the production cost for the respective refolding processes by eliminating
further purification steps associated with the liquid-phase artificial chaperone assisted
refolding.
Chapter: 5
82
Chapter 5 Selective recognition and separation of nucleosides using carboxymethyl-β-cyclodextrin functionalized hybrid magnetic nanoparticles2
5.1 Introduction
As the constructing units of nucleic acids, nucleosides are involved in performing
significant functions in various biological processes. Analysis of the nucleosides
provides important information for the diagnosis of certain severe diseases and
metabolic disorders [192]. However, mixing of different nucleosides occurs frequently
when they perform various biological and chemical functions in biological raw
materials [193]. Extensive studies have been carried out to separate and quantify
nucleosides due to their important biomedical and pharmaceutical applications. The
traditional way to quantify nucleosides is ion-exchange method but unfortunately this
technique is unable to separate the nucleosides from their mixtures [194]. In the recent
years, chromatographic methods have been widely used to quantify and separate the
nucleosides in the mixing conditions, among which most of them are reverse phase high
performance liquid chromatography (RP-HPLC) with gradient elution or ion-pair
[192,195]. RP-HPLC requires organic solvents such as acetonitrile or methanol as
mobile phase to achieve satisfactory separation of the nucleosides within the reasonable
retention time. Umemura et al. utilized amphoteric surfactant to modify the
octadecylsilica (ODS) column of RH-HPLC so that a non-organic aqueous solvent can
be used as mobile phase instead [196]. However, the intrinsic disadvantages of HPLC
2. A.Z.M. Badruddoza, L. Junwen, K. Hidajat, M.S. Uddin, Selective recognition and separation of
nucleosides using carboxymethyl-β-cyclodextrin functionalized hybrid magnetic nanoparticles,
Colloids and Surfaces B: Biointerfaces, 92 (2012) 223-231.
Chapter: 5
83
are its cost and stringent requirements for sample purity. The nucleosides mixtures
usually contain small amounts of other large molecules and impurities from cell broth,
which may clog the column, bind to the mobile phase or precipitate out during the
separation process [197]. Another powerful alternative to determine nucleosides is
electrochemical detection. Recent literature reveals that several studies have been
carried out to use different electrodes such as mercury electrode [198], boron doped
diamond electrode [199] and fullerene-C60-modified glassy carbon electrode [198] to
improve the sensitivity and selectivity in the quantification and separation of adenosine
and guanosine. However, tedious sample preparation and high cost are the most
significant limitations of this method. In a recent study by Lan Jin et al, β-
cyclodextrin/layered double hydroxide intercalation compound was used for selective
adsorption of adenosine and guanosine [193]. The method is very effective and simple
but the subsequent removal of clay compound from nucleoside solution requires a very
long period of centrifugation and complete removal is difficult. Thus, development of a
simple, fast and accurate method to separate and detect nucleoside derivatives is
essential for accurate biomedical analysis. Although various magnetic beads or
magnetic latex particles were reported for molecular diagnosis consisting in the
extraction, purification, concentration, and amplification of nucleic acids [191,200],
cyclodextrin modified MNPs combining magnetic and molecular binding/recognition
properties would be useful as separators and receptors in biological research.
Recently, the functionalization of magnetic nanoparticle surfaces with macrocyclic host
molecules in well-defined host–guest interactions has gained significant attention
[201,202]. The combination of the physical properties of magnetic materials and the
molecular recognition ability of CD molecules adsorbed on the nanoparticles surface
makes them to exhibit promising applications in different fields such as catalysis [201],
Chapter: 5
84
drug delivery [16,202,203], environment [201,204,205], chemical sensing [206], and so
on. Recent work showed that based on differences of intermolecular weak interactions
i.g., inner interactions (van der waals force and hydrophobic interactions) and external
interactions (hydrogen bonding), β-CD could selectively separate several alcohols from
aldehyde solutions [207]. However, no work has been done so far utilizing the
combined properties of MNPs and CDs for the selective capture of biomolecules like
nucleosides.
Adenosine (A) Guanosine (G)
Figure 5-1 Chemical structure of adenosine (A) and guanosine (G)
Here, we developed a novel magnetic nanoadsorbent (CMCD-APTS-MNPs) by grafting
CM-β-CD onto the surface of APTS modified Fe3O4 MNPs and evaluated their
capacities to adsorb nucleosides. Adenosine (A) and guanosine (G) were selected as
model target guest molecules as shown in Figure 5-1. The individual and selective
adsorption behaviors of guanosine and adenosine with CMCD-APTS-MNPs were
studied. Effects of pH on adsorption were investigated. For better understanding to gain
insights into the molecular recognition mechanism of nucleosides and β-CD, the
inclusion relation between the immobilized β-CD and the guest substrates were also
investigated.
NH
N
N
O
NH2N
O
HOH
HH
HH
HO
1
2
3
4
567
8
9
1
24
567
8
9
N
NN
N
NH2
O
HOH
HH
HH
HO 3
Chapter: 5
85
5.2 Experimental
5.2.1 Materials
Iron (II) chloride tetrahydrate (99%), Iron (III) chloride hexahydrate (98%), N-
hydroxysuccinimide (NHS), 3-aminopropyltriethoxysilane (APTS, 98%), guanosine
(99%), adenosine (99%), chloroacetic acid (98+%) and N-(3-dimethylaminopropyl)-N’-
ethylcarbodiimide hydrochloride (EDC) was purchased from Alfa Aesar. Ammonium
hydroxide (25%) and ammonium phosphate, monobasic (99.99%) was purchased from
Merck (USA). β-CD (99%) was obtained from Tokyo Kasie Kogyo (Japan). All other
chemicals were used as received without further purification. The water in this work
was Milli-Q ultrapure water.
5.2.2 Preparation of carboxymethyl-β-cyclodextrin (CM-β-CD)
CM-β-CD was prepared following the procedure of literature [208], and the detail
synthesis of CM-β-CD was as following: a mixture of β-CD (10 g) and NaOH (9.3 g) in
water (37 ml) was treated with a 16.3% monochloroacetic acid solution (27ml) at 50ºC
for 5 h. Then the reaction mixture was cooled to room temperature, and pH was
adjusted (6~7) using HCl. The obtained neutral solution was then poured to superfluous
methanol solvent and white precipitates were produced. The solid precipitation was
then filtered and dried under vacuum to give carboxymethylated β-CD (CM-β-CD, 6 g).
1H NMR (300 MHz, D2O), δ (TMS, ppm): 5.08, 5.30 (s, 7H, H-1), 4.39 (s, 2.5H, H-7),
3.30-4.20 (m, 45.7H, H-2, H-3, H-4, H-5, H-6, H-7’). (The number of C is shown in
Figure 5-2). IR (KBr): ν (cm−1): 3140–3680 (–OH), 2923 (–CH), 1741 (C=O), 960–
1200 (C–O–C, C-C/C-O) (Figure 5-3d). The average number of carboxylate groups
(3.1) per CM-β-CD was calculated using 1H NMR [209].
Chapter: 5
86
5.2.3 Fabrication of CM-β-CD modified APES-MNPs (CMCD-APES-MNPs)
CM-β-CD was grafted onto APES-MNPs in the presence of EDC and NHS. Detailed
synthetic procedure for APES modified magnetic nanoparticles (APES-MNPs) are
given in Chapter 4 (Section 4.2.4). A solution of CM-β-CD (1.3 g, 1 mmol) in 40 ml of
distilled water was first activated with 0.192 g (1 mmol) of EDC and 0.115 g (1 mmol)
of NHS for 30cmin. With this mixture, 0.7 g of APES-MNPs dispersed in 10 ml of
distilled water was added and the pH of the reaction mixture was adjusted to 7.0. The
reaction was carried out at room temperature under constant stirring. After 24 h, the
product formed was separated by magnetic decantation and dried by freeze-drying.
5.2.4 Adsorption of nucleosides
Nucleosides adsorption experiments were carried out using batch equilibrium technique
in aqueous solutions at pH 4–9 at 25oC. In general, 110 mg of wet magnetic nano-
adsorbent was added to 5 ml of either guanosine or adenosine solution of various
concentrations and shaken in a thermostatic water-bath shaker operated at 230 rpm for a
specific period of time to reach adsorption equilibrium. After equilibrium was reached,
the magnetic nano-adsorbents were removed using a strong permanent magnet made of
Nd-Fe-B and the supernatant was collected. The amount of nucleosides in the
supernatant was then quantified by UV-vis spectrophotometer. The solution pH was
adjusted by NaOH or HCl.
To measure the adsorption selectivity of CMCD-APES-MNPS, adsorption experiments
were also carried out by putting same amount of adsorbent in a mixture of A and G
(containing equal amounts) with different concentrations. After adsorption equilibrium,
the supernatant were separated by magnetic decantation and the concentrations of A and
G were analyzed by HPLC.
Chapter: 5
87
For single nucleoside samples, UV-Vis spectrophotometer (Model 1601) was used to
determine the concentration of nucleosides. The detection wavelength of both
guanosine and adenosine was set at 260 nm. For mixed nucleoside samples, the amount
of residual nucleosides in solution was quantified by HPLC. HPLC experiments were
carried out with Agilent Technologies 1200 Series chromatography system equipped
with a 4.6 mm × 250 mm C18 column (Agilent). The mobile phase used was 0.05 M
(NH4)H2PO4 in methanol-water (10:90) at pH 5.0. The flow rate was 1.0 ml/min and the
UV detector was set 254 nm at 30oC.
5.3 Results and discussions
5.3.1 Synthesis and characterization of magnetic nanoparticles The reaction scheme for grafting CM-β-CD onto Fe3O4 nanoparticles is shown in
Figure 5-2. First, 3–aminopropyltriethoxysilane (APES) modified MNPs were
synthesized which provides free –NH2 groups on the surface. Then CM-β-CD moiety
was coupled on the magnetic surface through amide bond (–NH–CO–) formation.
When CMCD-APTS-MNPs were added into solution containing either A or G or both,
immobilized CM-β-CD adsorbed the nucleoside through inclusion and hydrophobic
interactions. The nucleoside adsorbed particles were then separated from solution by
applying an external magnetic field (Figure 5-2).
Chapter: 5
88
Figure 5-2 Schematic illustration of the fabrication of the carboxymethyl-β-cyclodextrin modified magnetic nanoadsorbents and mechanism for selective separation of nucleosides.
The successful grafting of carboxymethylated β-CD on the surface of APES-MNPs was
verified by FT-IR studies. Figure 5-3 shows IR spectra of bare MNPs, APES-MNPs,
CMCD-APES-MNPs, and CM-β-CD in the range of 400-4000 cm-1 wave number. It is
shown that the characteristics adsorption band of Fe–O bonds in the tetrahedral sites of
bare MNPs is 586 cm-1. Strong absorption band at 3422 cm-1 is due to O-H stretching
vibration. The spectrum of APES-MNPs shows peak at 997 and 1110 cm-1 which can be
Chapter: 5
89
attributed to siloxane bonds (Si-O-H) and Si-O-Si groups, respectively. The peaks at
2854 and 2953 cm-1 are assigned to C-H stretching vibration in aminopropyl groups.
This suggests that Fe3O4 surface is successfully coated with APES. In spectra for
CMCD-APES-MNPs, the characteristics peaks at 941, 1030 and 1153 cm-1 can be
observed due to immobilized CM-β-CD on the surface. The peak at 941 cm−1 was due
to the R-1,4-bond skeleton vibration of β-CD, and the peaks at 1030 and 1153 cm−1
corresponded to the antisymmetric glycosidic νas(C–O–C) vibrations and the coupled
ν(C–C/C–O) stretch vibration, respectively [16]. Thus, the comparison between FTIR
spectra of APES and CM-β-CD coated magnetite nanoparticles gives evidence for
successful grafting of CM-β-CD on the magnetite surface which is further supported by
XPS (Figure A-2 in Appendix A).
Chapter: 5
90
Figure 5-3 FTIR spectra of as-synthesized magnetic nanoparticles-(a) uncoated MNPs, (b) APES-MNPs, (c) CMCD-APES-MNPs and (d) CM-β-CD.
High resolution TEM was applied to characterize the size and morphology of the nano-
magnetic particles. Typical TEM image of CMCD–APES–MNPs is shown in Figure 5-
4(a). No significant layer due to CM-β-CD grafting can be observed on the surface of
magnetic nanoparticles. It shows that magnetic particles bonded with CM-β-CD were
spherical or ellipsoidal particles with nano-size range. The mean diameter calculated
from the TEM image was estimated to be 11.3 nm. These as-synthesized CM-β-CD
modified magnetic nanoparticles exhibit superparamagnetism as evidenced by VSM
(Figure A-1 in Appendix A). Figure 5-4(b) also shows the hydrodynamic diameter
distribution of CMCD-APES-MNPs in water determined by the dynamic light
scattering (DLS) technique. The mean hydrodynamic diameter was found to be 56.5
nm. The particle size from TEM measurements in a dried state cannot be compared
400 1400 2400 3400
% T
ran
smis
sion
Wavenumber, cm-1
1030
1155
997
997
948
1080
582
1741
1030
1155
9452923
1624
10803396
2924
2854
586
3398
3404
586
2923
1325
1217
1110
(a)
(b)
(c)
(d)
Chapter: 5
91
with the values from DLS measurements which are water-swollen particles and DLS
gives higher values compared to TEM.
Figure 5-4 (a) TEM micrograph and size distribution of CMCD-MNPs measured by TEM and (b) hydrodynamic size distribution by DLS.
The amount of CM-β-CD grafted on the surface of APES-MNPs was estimated from
the thermogravimetric analyses of uncoated, APES coated and CM-β-CD coated
magnetic nanoparticles. As shown in Figure 5-5, the TGA curve of uncoated MNPs
shows first weight loss of 1.6 % below 130oC which might be due to the loss of
adsorbed water in the sample. After this brief event of weight loss, an unexpected
weight gain takes place within the temperature range from 130 to 205oC. This weight
gain can be attributed to the oxidation of Fe3O4 to Fe2O3 [210]. The total weight loss
over the full temperature range is estimated to be 2.3% due to the loss of the adsorbed
water as well as dehydration of the surface –OH groups. The TGA curve for APES-
MNPs exhibits two steps of weight loss, contributed from the loss of residual water in
the sample in 30–200oC and the loss of APES (~2.7%) in the range of 200–450 oC.
From the TGA curve for CMCD-APES-MNPs, a drastic drop of 8.5% can be seen in
(a) (b)
Chapter: 5
92
the range 190–430oC and it is contributed from the thermal decomposition of APES and
CM-β-CD moieties. The TGA curves also confirm the successful grafting of CD
molecules onto the magnetic surface. From these data, the amount of CM-β-CD grafted
onto the surface of APES-MNPs could be estimated to be 5.8 wt% or 0.044 mmol/g.
Figure 5-5 TGA curves for (a) uncoated MNPs, (b) APES-MNPs and (c) CMCD-APES-MNPs.
Zeta potentials of uncoated and CM-β-CD coated magnetic nanoparticles (0.1 mg/ml)
were measured in 10-3 M NaCl aqueous solution at different pH. The solution pH was
adjusted by NaOH or HCl. As shown in Figure 5-6, the pH values of point of zero
charge (pHpzc) of uncoated Fe3O4 nanoparticles and APES coated magnetic particles
(APES-MNPs) are about 6.8 and 7.7, respectively. After being grafted with CM-β-CD,
the pHpzc has been shifted to 4.25, indicating that the grafting of CM-β-CD onto
uncoated MNPs is successful. Moreover, magnetic nanoparticles modified with CM-β-
CD yields acidic surface since pHpzc is lower than that of uncoated MNPs and this
85
88
91
94
97
100
0 200 400 600 800
%w
eigh
t los
s
Temperature, oC
(a)
(b)
(c)
Chapter: 5
93
surface acidity is due to the introduction of several oxygen-containing functional groups
[211].
Figure 5-6 Zeta potentials of uncoated, APES and CM-β-CD modified magnetic
nanoparticles at different pH.
5.3.2 Adsorption of nucleosides onto CMCD-APES-MNPs 5.3.2.1. Effects of pH It has been reported that the solution pH values are important controlling parameters on
the analysis and separation of nucleosides [192,212]. The effects of initial solution pH
on the adsorption of nucleosides onto CMCD-APES-MNPs were investigated from pH
4 to 9 with an initial A and G concentration of 1.0 g/l. The solution pH after adsorption
didn’t change significantly in the experiments. As shown in Figure 5-7, sorption
capacity increases from pH 4 to pH 5 for guanosine and pH 4 to pH 6 for adenosine.
Comparatively less adsorption takes place for both A and G at low pH (pH 4). At low
pH, A and G are positively charged to some extent [see Figure A-3 in Appendix A
where the distribution coefficient (δ) of A and G are calculated from the pH and pKa
-60
-40
-20
0
20
40
60
2 3 4 5 6 7 8 9 10 11 12
Zet
a p
oten
tial
, m
V
pH
Uncoated MNPs
APES-MNPS
CMCD-APES-MNPs
Chapter: 5
94
values and the dissociation equilibrium process of A and G are also shown]. The zeta-
potential analysis of the CMCD-APES-MNPs (Figure 5-6) shows that the magnetic
nanoparticles are also positively charged at pH 4. Therefore, the interactions between
the adsorption sites on CMCD-APES-MNPs and the G or A cations are electrostatically
repulsive. The optimum pH appears to be in the range of 5~7 and 7~9 for guanosine and
adenosine, respectively. Above pH 7, the sorption capacity of magnetic nanoparticles
decreases slightly for G. However, the largest difference in the adsorption capacity
between G and A is obtained at pH 5. Therefore, the selective adsorption of nucleosides
can be achieved at pH 5 when they are in a competitive environment. At pH 5, 3.1% of
adenosine molecule is positively charged and the mean charge is approximately +0.03V
[193]. Guanosine is almost a neutral molecule (δ= 100%) at this pH. It is well known
that CDs show stronger inclusion with neutral guest molecules than cations, accounting
for a larger adsorption capacity for G than that of A at pH 5.
Figure 5-7 Effect of pH on the adsorption of A and G by CMCD-APES-MNPs: Conditions: Initial A and G concentration: 1.0 g/l, temperature: 25oC.
Chapter: 5
95
5.3.2.2 Kinetic studies Figure 5-8 shows the effects of contact time on adsorption of nucleosides onto CMCD-
APES-MNPs under the standard conditions. The initial nucleoside concentration
provides the necessary driving force to overcome the mass transfer resistance A and G
between aqueous and solid phases. It can be seen that the most rapid adsorption of A
and G occurred at initial few minutes and then occurred slowly. The time taken to reach
adsorption equilibrium for both nucleosides adsorption is 80 min.
Figure 5-8 Effect of contact time on nucleoside adsorption by CMCD-APES-MNPs. Conditions: Initial A or G concentration: 1.0 g/l, pH: 5, temperature: 25oC, adsorbent: 110 mg.
5.3.2.3 Equilibrium Studies The adsorption isotherm for A and G at pH 5 and 25oC are shown in Figure 5-9. It can
be seen that the adsorption of A and G increased with increasing initial concentration.
The adsorption of G was consistently higher than that of A and the difference in
adsorption between A and G increased clearly at higher initial concentration. The
higher adsorption of G onto CMCD-APES-MNPs can be attributed to its stronger
0
4
8
12
16
20
0 20 40 60 80 100 120 140 160 180 200
q e(m
g/g)
Time (min)
G
A
Chapter: 5
96
affinity towards CM-β-CD. The evidence of adsorption of guanosine and adenosine
onto the magnetic surface is shown in Figure A-4 (Appendix A).
Figure 5-9 Effects of initial concentrations on the adsorption of A and G onto CMCD-APES-MNPs. Conditions: Initial A and G concentration: 0.1,0.2,0.4,0.6,0.8,1.0g/l, pH: 5, temperature: 25oC, adsorbent: 110 mg.
The adsorption equilibrium data were fitted by Langmuir isotherm. The Langmuir
equation can be expressed as:
1e e
e m m L
C C
q q q K (5-1)
where qe is the equilibrium adsorption capacity of A and G onto the adsorbents (mg/g),
Ce is the equilibrium concentration of A and G (mg/ml), qm is the maximum capacity of
adsorbent (mg/g) and KL is the Langmuir adsorption constant (ml/mg). The values of qm
and KL can be determined from the slope and y-intercept of the linear plot of Ce/qe
against Ce as shown in Figure 5-10. For predicting the favorability of an adsorption
0
4
8
12
16
20
0 0.2 0.4 0.6 0.8 1
q e(m
g/g)
Final concentration (g/l)
AGLangmuir fit
Chapter: 5
97
system, the Langmuir equation can also be expressed in terms of a dimensionless
separation factor (RL) defined as follows [213]:
0
1
1LL
RK C
(5-2)
where Co is the initial concentration of adsorbate. The RL indicates the favorability and
the capacity of adsorption system. When 0 < RL < 1.0, it represents good adsorption.
The adsorption parameters are shown in Table 5-1. The adsorption isotherms of A and
G are both fitted well by Langmuir isotherm with R2 values of 0.983 and 0.982,
respectively. The maximum capacity of adsorbent on A and G are 12.5 mg/g and 31.3
mg/g respectively, indicating that the adsorption of G is more than 2 times of that of A
under the same conditions. The higher values of RL indicate the high affinity of these
nano-adsorbents to the nucleosides.
Figure 5-10 Langmuir plot illustrating the linear dependence of Ce/qe on qe for A and G.
y = 0.0807x + 0.0255R² = 0.9832
y = 0.0335x + 0.0195R² = 0.9874
0.02
0.04
0.06
0.08
0.1
0.12
0 0.2 0.4 0.6 0.8 1
Ce/
q e
Ce
A
G
Chapter: 5
98
Table 5-1 Adsorption isotherm parameters for A and G at pH 5 and 25oC
Isotherm Model Parameters A G Langmuir qm (mg/g) 12.5 31.3
KL (ml/mg) 3.2 1.68 R2 0.983 0.982 RL 0.238-0.758 0.373-0.856
5.3.3 Selective adsorption of A and G
The mixture nucleoside solution was prepared by dissolving equal amount of A and G
in the same buffer (pH 5). CMCD-APTS-MNPs were added to the mixture solution and
subject to mechanical shaking for 12 h. HPLC was used to quantify the concentration of
remaining A and G in the supernatant. The adsorption capacities of the CMCD-APTS-
MNPs at different total concentration of nucleosides were plotted in Figure 5-11(a). The
adsorption of both A and G increased proportionally with the initial total concentration.
It can be seen that G was adsorbed preferentially over A, especially at higher initial
concentration. The selective adsorption of nucleoside molecules onto the CMCD-
APTS-MNPs could be attributed to a stronger recognition capability of CM-β-CD
toward G, while the recognition for A is relatively weak. Binding constant or inclusion
formation constant (K) which represents the inclusion interactions is measured between
nucleoside and CM-β-CD (see Section 5.2.3.5). It can be seen that the binding constant
of CM-β-CD towards G is larger than that towards A. Moreover, the shifting of
maximum absorption peak for G/CM-β-CD system is more obvious in compared to that
for A/CM-β-CD system indicating a stronger interaction between G and CM-β-CD.
From Figure 5-1, it can be seen, A and G have similar structures except for an extra
C=O present in G. Hence, the results for the selective adsorption of G over A can be
attributed to the stronger hydrophobic interactions and additional weak intermolecular
hydrogen bonding contributed by C=O group in G which is further evidenced through
Chapter: 5
99
the studies of interactions between nucleosides and CM-β-CD by UV-vis spectra and
FTIR analysis, respectively.
Thus, the structure and charge of the guest plays important role in host-guest
interactions. In order to further verify that the selective adsorption properties of the
adsorbents were due to the hydrophobic cavity of CM-β-CD, a similar experiment was
conducted using APTS-MNPs instead of CMCD-APTS-MNPs. The adsorption of A or
G onto APTS-MNPs is mainly governed by electrostatic attractive interactions since
APTS-MNPs are positively charged at pH 5 whereas G or A is almost neutral at the
same condition (Figure A-2, Appendix A). Previous studies on adsorption behavior of
oligonucleotides on cationic or anionic latex particles also showed that electrostatic
forces play a major role in the adsorption process [214]. The result in Figure 5-11(b)
indicated that the adsorption of A and G was approximately the same and hence it
proved that the selective adsorption was indeed directly related to the hydrophobic
cavity of CM-β-CD. However, nonspecific adsorption might occur due to residual
amine (NH2) groups on the surface of magnetic nanoparticles due to electrostatic
interactions [191]. But this nonspecific adsorption may be negligible in compared to the
inclusive adsorption due to CM-β-CD molecules which has more than 80% of the
surface coverage and act as effective binding sites for nucleosides adsorption.
Chapter: 5
100
Figure 5-11 Selective adsorption of A and G mixture solutions using (a) CMCD-APES-MNPs and (b) APES-MNPs. Conditions: pH: 5, temperature: 25oC, adsorbent: 110mg.
5.3.4 UV-vis spectra analysis and determination of binding constants The inclusion capability of CM-β-CD towards nucleosides was investigated by UV-vis
absorption measurements. In this study, the guest molecules G or A is assumed to be
included by CM-β-CD moiety grafted on CMCD-APTS-MNPs with the same
mechanism as parent CM-β-CD; while Fe3O4 or other molecule do not affect the
inclusion complexation. As the binding sites in the CMCD-APTS-MNPs are essentially
0
2
4
6
8
10
12
14
16
0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
q e(m
g/g)
Initial concentration (g/l)
G
A
(a)
1
2
3
4
5
6
0.1 0.3 0.5 0.7 0.9 1.1
q e(m
g/g)
Initial concentration (g/l)
G
A
(b)
Chapter: 5
101
the CM-β-CD apolar cavities and that the global properties of these sites are not
significantly different from the properties of the isolated CM-β-CD molecule, the
measurement of binding constant between CM-β-CD host and nucleoside guest
molecules could explain the reasons for selective inclusion of G to some extent. Figure
5-12 shows the UV-vis spectra in the absorption of nucleosides (1x10–5 mol l–1) in
phosphate buffer solution (pH 5) containing various concentrations of CM-β-CD. It is
clear that absorption peaks of A or G were enhanced, with the addition of CM-β-CD.
This increase is not just from additional CM-β-CD hosts added, but from the inclusion
of G or A within the relatively non-polar cyclodextrin cavity. A significant shift in the
absorption maximum (a blue shift from 253 nm to 248.3 nm) was observed in the case
of G upon addition of CM-β-CD (Figure 5-12a), whereas the absorption peak was
slightly shifted (from 259.9 nm to 259.1 nm) for A (Figure 5-12b). Compared with the
pristine G, the absorption bands of the products CMCD–APTS-MNPs/G (see Fig. S5)
shifted to a lower wavelength for about 4 nm. The reason may be that the strong
interaction due to additional hydrogen bonding between the host and the G was formed,
thus including the G guest much more tightly. This increase in absorption maxima and
shift in peak position upon CM-β-CD addition could be attributed to the fact that G or
A was becoming included in the hydrophobic cavity of CM-β-CD.
The inclusion equilibrium constant or binding constant (K) is an important parameter,
which represents the strength of the inclusion interaction. In other words, the stability
mainly relies on the host–guest interactions. The stability of the inclusion complex is
mainly determined by the geometric capability and the polarity of guest molecules
[215]. Geometric factors are crucial in determining the guest molecules that can
penetrate into the relatively hydrophobic cavity. On the other hand, only those
substrates with less polarity than water can form inclusion complexes with cyclodextrin
Chapter: 5
102
moieties. If CMCD: guest complex was formed with 1:1 stoichiometry, the inclusion of
CMCD and guest could be expressed as:
(5-3)
The inclusion constants of complexes are estimated according to the equation of Benesi
and Hildebrand (double-reciprocal method) [216]:
∆ (5-4)
where ∆A denotes the difference of absorption of guest molecule in the presence and
absence of CDs. α is a constant, and [CMCD]o denotes the concentration of CM-β-CD.
K can be calculated from a plot of 1/∆A vs. 1/[CMCD]o. Insets of Figure 5-12 shows the
double reciprocal plots of 1/∆A versus 1/[CMCD]0 for A and G with CM-β-CD at pH 5.
The plot exhibits good linearity (the linear regression coefficient R2 ~ 0.99). This
verifies the formation of inclusion complexes with a stoichiometry of 1:1 between
nucleosides and CM-β-CD. The relative inclusion constant of CM-β-CD with G and A
is estimated to be 47 and 18.6 M-1, respectively. From this result, the complex of G with
CM-β-CD should be more stable than that of A, and this is the driving force for the
inclusive separation. The 1:1 stoichiometry of complexes of parent β-CD with
nucleosides (both A and G) was also determined by the circular dichroism spectral
changes (Figure A-5, Appendix A). It can be seen that G has higher binding capacities
with β-CD in compared to A as the binding constant of β-CD with G (30.3 M-1) was
higher than that with A (17.9 M-1). This result also indicates that incorporation of
carboxylic groups in β-CD moieties can enhance the strength of host-guest interactions
with G molecules.
Chapter: 5
103
Figure 5-12 Absorption spectra of (a) adenosine and (b) guanosine (1x10–5 mol l–1) in pH 5.0 buffers containing various concentrations of CM-β-CD. Concentration of CM-β-CD: (1) 0, (2) 1.0x10–3, (3) 2.0x10–3, (4) 2.5x10–3 and (5) 3.5x10–3 mol l–1. Insets: Double reciprocal plot for nucleoside complexes with CM-β-CD.
5.3.5 Interactions of the nucleosides with cyclodextrins
In order to investigate the vibrational changes upon host-guest interaction between
nucleosides and CM-β-CD, FTIR spectroscopy was used. FTIR spectroscopy is a useful
technique used to confirm the formation of an inclusion complex. Figure 5-13 shows
the infrared spectra of adenosine, guanosine, β-CD and their inclusion complexes. The
Chapter: 5
104
inclusion complexes were prepared following the procedure described in the Section A-
6 (Appendix A) and the formation of inclusion complexes are also confirmed by
Differential Scanning Calorimetry (DSC) analysis (Figure A-6, Appendix A). In the IR
spectrum of adenosine, the bands at 1666, 1605 and 1572 cm-1 are mainly assigned to
the bending mode of NH2, C=N stretching and the conjugated vinylic group (C=C) in
the aromatic ring, respectively. The two strong bands at 1734 and 1695 cm-1 in the
spectra of free guanosine are assigned to the cyclic carbonyl (C6=O) stretching [217].
The bands at 1624 and 1609 cm-1 corresponds to NH2 bending mode and the aromatic
C=C vibrations, respectively. The other bands in the region of 1600-1400 cm-1 in
guanosine are assigned to C-C, C=N and C-N vibrations.
In the IR spectra of inclusion complexes, there is a significant change in the
characteristic bands absorption of adenosine and guanosine, particularly in the range of
1400-1800 cm-1. Some bands disappeared and some bands became less intense after
forming the inclusion complexes. The strong peaks of adenosine at 1572 and 1605 cm-1
reduced its intensity significantly and shifted to 1574 and 1609 cm-1, respectively in its
inclusion complexes, The aromatic C=C strong band of guanosine at 1609 cm-1
disappeared in the spectrum of its inclusion complex. These results indicate that the
aromatic rings of adenosine and guanosine might be included into the cavity of β-CD.
The vibrational band for the C=O groups (1742 cm-1) of CM-β-CD showed a strong
band at 1634 cm-1 upon complexation with G. Moreover, the cyclic carbonyl (C=O)
stretching band of guanosine at 1695 cm-1 reduced its intensity and shifted to 1693 cm-1
in its inclusion complex suggesting the strong interactions through intermolecular
hydrogen bonds between guanosine and CM-β-CD moiety [215]. The peak at 1539 cm-1
of guanosine is also shifted to 1537 cm-1 and its intensity is reduced. The band located
at 1643 cm-1 in the IR spectra of CM-β-CD which is correlated to δ-HOH bending of
Chapter: 5
105
water molecules attached to cyclodextrins shifted to 1637 cm-1 in that of the inclusion
complex. σC–O–H peaks at 1336 cm-1 and 1250 cm-1 in the G/ CM-β-CD inclusion
complexes have much higher intensity compared to that of CM-β-CD. These are the
evidences of formation of hydrogen bonds between the hydroxyl groups of CM-β-CD
and the carbonyl groups of G [215]. The electron cloud density of the oxygen atom of
carbonyl in G might lead to the formation of the hydrogen bond between CM-β-CD and
G. Recent study showed that hydrogen bond interactions (e.g., O-H…O and C-H…O
etc.) play an important role in the complexes of alcohol/ β-CD and aldehyde/ β-CD and
based on the strength of the interactions, β-CD could selectively separate the alcohols
from the mixed solution [207]. Yang et al. [218] utilized insoluble β-CD polymer for
selective separation of cinnamaldehyde and benzaldehyde based on hydrogen bond
interactions between carbonyl (aldehyde) and secondary hydroxyl groups present in β-
CD moieties. It can also be concluded preliminarily from the FTIR results that the
adenine and guanine part of the nucleosides was probably enclosed in the β-CD
hydrophobic cavity. Several studies were done using different techniques to investigate
the interactions between the base (purine) moieties and CDs. For example, Seno et al.
proposed that the adenine group of nicotinamide adenine dinucleotide phosphate
(NADP) is incorporated into the CD cavity from the measurements of high performance
liquid chromatography [219]. Hao et al. also reported the inclusion of adenine molecule
in the more apolar environment inside the β-CD cavity based on enhancement of the
fluorescence intensity of adenine [220]. Kondo and Nishikawa suggested that adenine
and β-CD forms an inclusion complex based on the results of an ultrasonic relaxation
study [221]. However, from our studies it can be seen that the CMCD-APTS-MNPs
could selectively recognize and adsorb G over A as β-CD cavity primarily provides
Chapter: 5
106
hydrogen bonds between β-CD and polar groups of G in addition to hydrophobic
interactions.
Figure 5-13 FTIR spectra of adenosine and guanosine and their inclusion complexes with β-CD.
5.4 Conclusions
In summary, a simple and straightforward synthetic method was developed for the
preparation CM-β-CD modified Fe3O4 nanoparticles that combine magnetic and
molecular recognition properties. The properties of this novel nano-adsorbent were
Chapter: 5
107
evaluated for selective adsorption of guanosine and adenosine. The grafting of CM-β-
CD onto the surface of the magnetic particles was confirmed by FTIR, TGA and XPS.
The adsorption of nucleosides was found to be pH dependent and the adsorption of A
and G onto CMCD-APES-MNPs were well-described by Langmuir isotherm model.
Under the competitive environment, selective adsorption of G over A occurred at pH 5
onto these nano-adsorbents. This selectivity was due to the multiple recognition of the
CM-β-CD which might give strong hydrophobic and extra interaction due to hydrogen
bonding towards G. The stronger interactions between G and CM-β-CD were confirmed
by measuring the binding constants which represent the inclusion interactions and also
by FTIR studies. The results showed that this adsorbent would have wide applications
in selective separation, concentration, and analysis of nucleotides/nucleosides in
biological sample.
Indeed, this study provides a practical and promising technique to separate organic
compounds with similar structures based on the difference of inclusional binding
property by forming host–guest interactions. Combining the unique properties of MNPs
(superparamagnetism and electrocatalytic) and cyclodextrins (excellent supramolecular
recognition and host–guest inclusion capability), it is also possible to obtain the new
materials which will provide good opportunities for applications in the fields of sensors,
electrocatalysis etc.
Chapter: 6
108
Chapter 6 Adsorptive removal of dyes from aqueous water using carboxymethyl-β-cyclodextrin conjugated magnetic nano-adsorbent3
6.1 Introduction
This chapter reports on synthesis and characterization of novel nanoadsorbents-
carboxymethyl-β-cyclodextrin conjugated magnetic nanocomposites and their use in the
removal of organic dyes from aqueous streams. The adsorption behaviors of a cationic
dye with theses nano-adsorbents have been discussed from the equilibrium and kinetic
viewpoints. Methylene blue (MB) was used in this study as the model dye compound
because of their extensive use in various industries.
Dye removal from industrial wastewater has become an important issue because of
environmental concern. Dye and dye stuffs are extensively used in many industries,
such as cosmetics, leather, paper, printing textile finishing industries. It has been
estimated that 10-15% of the dyes was lost during the dyeing process and released as
effluent. Since most of the used dyes are stable, recalcitrant, colorant, and even
potentially carcinogenic and toxic, their discharge into the environment without proper
treatment poses serious environmental, aesthetical and health problems [109]. The
removal of dyes from aqueous environment has been widely studied and used including
biological processes, coagulation, membrane filtration, adsorption, photocatalysis,
advance oxidation etc. Among these methods, adsorption has increasingly received
more attention because of its high efficiency, simplicity of design, and ease of operation
[114,115,141].
3. A.Z.M. Badruddoza, Goh Si Si Hazel, K. Hidajat, M.S. Uddin, Synthesis of carboxymethyl-β-cyclodextrin conjugated magnetic nano-adsorbent for removal of methylene blue, Colloids Surf. A: Physicochem Eng. Aspects 367 (2010) 85-95.
Chapter: 6
109
One of the most widely used method for the removal of organic pollutants is their
adsorption on activated carbon, arranged in different arrays such as filters, dispersed
powder or fluidized beds. Activated carbon has been efficiently used to adsorb many
kinds of pollutants as it is characterized by a high porosity and a very large surface area.
In spite of its high adsorption capacity, the use of activated carbon on a large scale is
limited by process engineering difficulties such as the dispersion of the activated carbon
powder and the cost of its regeneration. Even if activated carbon can be used in granular
form, a lack of selectivity in adsorption is observed with activated carbon. Thus,
continuous and great efforts are being made by many researchers on optimizing
adsorption and developing novel alternative adsorbents with high adsorptive capacity
and low cost.
Recently, natural polymers such as polysaccharide and their derivatives have gained
significant interest as low-cost adsorbents of choice for wastewater treatment because
of their particular structure, physico-chemical characteristics, chemical stability, high
reactivity and excellent selectivity towards organic compounds and metals [141]. An
extensive list of low-cost polysaccharide based adsorbents for dye removal has been
reported by Crini [107]. An important class of polysaccharide derivatives are
cyclodextrins (CD) which can form inclusion complexes with a wide variety of organic
compounds in its hydrophobic cavity through host-guest interactions and also can
complex various metal ions [9]. Therefore, CD complexation is a procedure of choice
for decontaminating techniques. Several types of water insoluble β-CD-based polymers
which have been used for removal of contaminants from wastewaters are summarized
in Table 2-7. This kind of polymer is usually synthesized by reacting β-CD with
crosslinking agents such as epichlorohydrin, diisocyanates, polycarboxylic acids and
anhydrides. In particular, the β-CD-based polymers are very efficient in removing acid,
Chapter: 6
110
direct, disperse and reactive dyes from aqueous solutions [114]. However, they have
low affinity for basic (cationic) dyes. Recently it has been reported that incorporation of
suitable groups e.g. carboxyl groups onto β-CD can enhance the capability to adsorb
basic dyes on the polymer beads [11,113,141]. However, the application of these
polymeric materials for treating wastewater requires an additional separation step to
remove the adsorbent from the aqueous system. A step in developing adsorbents with
superior properties would be the inclusion of magnetic particles into natural polymers,
thus combining the advantages of both materials.
This work aims at exploring the possibility to fabricate carboxymethyl-β-cyclodextrin
(CM-β-CD) conjugated Fe3O4 nanoparticles with exploitable characteristics as nano-
adsorbent for removal of dyes from aqueous solution. Here, two adsorbents CMCD-
MNP(C) and CMCD-MNP(P) were synthesized via carbodiimide and precipitation
method, respectively. The size, structure and surface properties of the CM-β-CD
modified magnetic nanoparticles were characterized using different analytical tools.
The adsorption capability was demonstrated using methylene blue (MB) as model dye.
MB is a cationic dye which is toxic and carcinogenic. Its characteristics are given in
Table 6-1. The adsorption behaviors of CMCD-MNP(C) and CMCD-MNP(P) towards
MB adsorption were studied using both equilibrium and kinetic viewpoints. The effects
of several parameters such as effect of pH, initial MB concentration etc. and mechanism
of MB adsorption onto CM-β-CD modified magnetic nanoparticles were investigated in
this report.
Chapter: 6
111
6.2 Experimental
6.2.1 Materials
In this work, the following chemicals were used for fabrication of CM-β-CD modified
magnetic nanoparticles: Iron (II) chloride tetrahydrate (99%) [Alfa Aesar], iron (III)
chloride hexahydrate (98%) [Alfa Aesar], ammonium hydroxide (25%) [Merck (USA)],
β-cyclodextrin (β-CD, 99%) [Kasie Kogyo Co. (Tokyo)], chloroacetic acid (99%) [Alfa
Aesar], carbodiimide (cyanamide, CH2N2, 98%) [Alfa Aesar], methylene blue (Basic
blue 9: CI-52015) [Sigma]. All other chemicals were of analytical grade and were used
as received without further purification. Milli-Q water was used throughout this study.
Table 6-1 Characteristics of Methylene Blue (MB) dye
C.I. name Methylene Blue
C.I. number 52015
Abbreviation MB
Dye Type Basic
Dye content (%) 99
Molecular Weight (gmol-1) 319.86
Molecular formulae C16H18ClN3S
Wavelength (nm) 664
Chemical structure
6.2.2 Fabrication of CM-β-CD modified Fe3O4 nanoparticles [CMCD-MNP(C) & CMCD-MNP(P)]
To synthesize CMCD-MNP(C), the binding of CM-β-CD onto the Fe3O4 magnetite
surface was conducted following the work of Liao and Chen [17] on the preparation of
polyacrylic acid (PAA) bound magnetic nanoparticles with slight modification. Detailed
Chapter: 6
112
procedures to synthesize CM-β-CD are given in Chapter 5 (Section 5.2.5). For the
binding of CM-β-CD, 100 mg of magnetic nanoparticles were first added to 2 ml of
buffer A (0.003M phosphate, pH 6, 0.1 M NaCl) and sonicated for 5 min. Then, the
reaction mixture was further sonicated for 15 min after adding 0.5 ml of carbodiimide
solution (0.025 g/ml in buffer A). Finally, 2.5 ml of CM-β-CD (50 mg/ml in buffer A)
was added and the reaction mixture was sonicated for another 90 min. The CM-β-CD
bound magnetic nanoparticles were recovered from the reaction mixture using a
permanent magnet and washed 2-3 times with buffer A and then dried in a vacuum
oven.
CMCD-MNP(P) was fabricated by one step co-precipitation method. Briefly, 0.86 g of
FeCl2.4H2O, 2.36 g FeCl3.6H2O and 1.5 g CM-β-CD were dissolved in 40 ml of de-
aerated Milli-Q water with vigorous stirring at a speed of 1,200 rpm. 5 ml of NH4OH
(25%) was added after the solution was heated to 90ºC. The reaction was continued for
1 hr at 90ºC under constant stirring and nitrogen environment. The resulting
nanoparticles were then washed with Milli-Q water few times to remove any unreacted
chemicals and dried in a vacuum oven.
6.2.3 Adsorption/desorption of methylene blue
The adsorption of methylene blue (MB) onto the CM-β-CD-bound iron oxide magnetic
nanoparticles was carried out in 0.03 M phosphate buffer at desired MB concentrations,
pH and temperatures. In general, 120-130 mg wet nanoparticles were added to 5 ml of
MB solution. The initial concentrations of MB solution were 0.1 to 3 mg/ml. The effect
of pH on the amount of dye removal was analyzed in the pH range from 2 to 12. The
pH was adjusted using HCl or NaOH solutions. After equilibrium was reached,
nanoparticles were removed magnetically from MB solution. The adsorption amounts
Chapter: 6
113
of MB were estimated from the concentration change of MB in solution after adsorption
by the spectrophotometric method at 664 nm (Shimadzu UV-1601 UV/vis
spectrophotometer). Calibration curves were plotted between absorbance and
concentration of the MB solution. The amount of MB adsorbed onto CMCD-MNP(C)
and CMCD-MNP(P) was calculated by a mass balance relationship,
( )e t e
Vq C C
w (6-1)
where V (ml) is the volume, Ct and Ce (mg/ml) are the initial and final solution
concentration of MB, respectively, and w (mg) is the dry mass of the solid.
For desorption experiment, the MB-sorbed nano-adsorbents were washed thoroughly
with deionized water. Desorption of MB was then performed by putting the MB-sorbed
nanoparticles into methanol solution containing 0–5% acetic acid [40]. After
equilibrium was reached, nanoparticles were removed by magnetic decantation from
MB solution and the concentration of MB in liquid solution was measured to estimate
the amount of MB desorbed.
6.3. Results and Discussion
6.3.1 Synthesis and characterization of magnetic nanoparticles
CMCD-MNP(C) (BET surface area~110.9 m2/g) was synthesized using two-steps
(carbodiimide method). The first step involves the formation of carboxyl group onto β-
CD by reacting chloroacetic acid with β-CD in the alkaline condition. The average
number of carboxylate groups (3.1) per molecule of CM-β-CD is determined using 1H
NMR [209] as well as by titrating the carboxylate groups with NaOH solution [222].
Before titrating CM-β-CD with NaOH, the CM-β-CD sodium salt was converted to its
acidic form by passing 50 ml of a 100 g/l CM-β-CD solution twice through a column
Chapter: 6
114
containing an ion exchange resin (H+). Using the degree of substitution of 3.1
carboxymethyl substituents (each with a molecular weight of 58 amu) per CM-β-CD
molecule, we calculated an average atomic mass for CM-β-CD of 1312 amu. This
molecular weight was used to calculate the grafted amount of CM-β-CD on the surface
of Fe3O4 nanoparticles. In the second step, CM-β-CD was covalently bonded onto the
surface of magnetic nanoparticles via carbodiimide activation. Here, carbodiimide
(cyanamide) was added in CM-β-CD solution to pre-activate the carboxyl (-COOH)
groups which in turn form covalent bonding with the surface OH groups on magnetite.
On the other hand, CMCD-MNP(P) (BET surface area~77 m2/g) was synthesized by
simple one-step co-precipitation where iron precursors (Fe2+ and Fe3+) and CM-β-CD
were mixed together in the reaction medium. In one-step method, the carboxyl groups
of CM-β-CD directly react with the surface OH groups on the magnetite to form Fe-
carboxylate. The step-by-step reaction procedures to synthesize CM-β-CD modified
magnetic nanoparticles are shown in Figure 6-1.
Chapter: 6
115
Figure 6-1 An illustration for the carboxymethylation and binding of cyclodextrin onto Fe3O4 nanoparticles by two different methods.
The binding of CM-β-CD on surfaces of magnetic nanoparticles was confirmed by
FTIR spectroscopy. Figure 6-2 shows the FTIR spectra of bare MNP, CMCD-MNP(C)
and CMCD-MNP(P) and CM-β-CD in the 400-4000 cm-1 wave number range. The
broad band at 330-3500 cm-1 is due to –OH stretching vibrations. The spectrum of CM-
β-CD shows the characteristic peaks at 947, 1030, 1157 and 1741 cm−1. The peak at 947
cm−1 is due to the R-1,4-bond skeleton vibration of β-CD, and the peaks at 1030 and
1157 cm−1 corresponded to the antisymmetric glycosidic νa(C-O-C) vibrations and
coupled ν(C-C/C-O) stretch vibration [11,16]. The peak at 1741 cm-1 corresponds to
carbonyl group (=CO) stretching which confirms the incorporation of the
carboxymethyl group (-COOCH3) into β-CD molecule. All the significant peaks of
Chapter: 6
116
CM-β-CD in the range of 900–1200 cm−1 are present in the spectrum of CMCD-
MNP(C) and CMCD-MNP(P) with a small shift. Moreover, as shown in Figure 6-2(b),
two main characteristic peaks appeared at 1623 and 1401 cm−1 due to bands of COOM
(M represents metal ions) groups, which indicates that the COOH groups of CM-β-CD
reacted with the surface OH groups of Fe3O4 particles resulting in the formation of the
iron carboxylate [223,224]. So it can be concluded that CM-β-CD has been grafted
successfully on the surface of both magnetic nano-adsorbents.
Figure 6-2 FTIR spectra of (a) uncoated MNPs, (b) CMCD-MNP(P), (c) CMCD-MNP(C) and (d) CM-β-CD.
Typical TEM images and particle size distributions of CMCD-MNP(P) and CMCD-
MNP(C) are shown in Figure 6-3(a) and 6-3(c) and their insets. Well-shaped spherical
or ellipsoidal magnetic nanoparticles are observed. The mean diameter of both CMCD-
MNP(P) and CMCD-MNP(C) is about 12 nm. This indicates that binding process did
400 800 1200 1600 2000 2400 2800 3200 3600 4000
% T
Wavenumber, cm-1
945
582
3396
3386
29242854
582
58614001028
947
1030
3394
1030
947
10781153
1155
1080
1618
1635
(a)
(b)
(c)
(d)
17412923
10801157
2924
Chapter: 6
117
not significantly result in the agglomeration and more changes in their sizes. Similar
results have been observed for the magnetic nanoparticles binding with carboxymethyl-
chitosan by one-step and two-steps method [224]. No significant layer of CM-β-CD can
be observed on these nano-adsorbents. Figure 6-3(b) and 6-3(d) also show the
hydrodynamic diameter distribution of CMCD-MNP(C) and CMCD-MNP(P) in water
determined by the DLS technique. The mean hydrodynamic diameters were found to be
28.2 and 43.6 nm, respectively. The particle size from TEM measurements in a dried
state cannot be compared with the values from DLS measurements which are water-
swollen particles and DLS gives higher values compared to TEM. This can be attributed
to the reason that polymer coating increases the hydrodynamic diameter and modified
iron oxide particles are slightly aggregated in water. The difference in sizes obtained
from DLS and TEM has also been observed with other nano-materials [16,202].
Chapter: 6
118
Figure 6-3 TEM micrographs and hydrodynamic diameter distributions for CMCD-MNP(C) (a) & (b); and CMCD-MNP(P) (c) & (d) respectively.
The amount of CM-β-CD grafted on the surface of MNP was estimated from the
thermogravimetric analyses of uncoated and CM-β-CD coated magnetic nanoparticles.
As shown in Figure 6-4, the TGA curves for CMCD-MNP(C) and CMCD-MNP(P)
exhibits two steps of weight loss, contributed from the loss of residual water in the
sample in 30–200oC and the loss of CM-β-CD in the range of 200–400oC. From the
TGA curve for CMCD-MNP(C) and CMCD-MNP(P), a drastic drop of 4.7% and 12%,
respectively can be seen in the range 190–400oC and it is contributed from the thermal
decomposition of CM-β-CD moieties. Below 200oC, the rate of weight loss is relatively
slow owing to the loss of residual water adhering to the sample surface and adsorbed in
the CD cavities. Thus, the TGA curves also confirm the successful grafting of CM-β-
CD molecules onto the magnetic surface.
0
2
4
6
8
10
12
14
16
18
7 8 9 10 11 12 13 14 15 16 17 18
Particle diameter (nm)
Fre
quen
cy (
%)
(a)
(b)
(d)
(c)
Chapter: 6
119
Figure 6-4 TGA curves for (a) bare Fe3O4 MNP, (b) CMCD-MNP(C) and (c) CMCD-MNP(P).
The XRD patterns of bare MNP, CMCD-MNP(C) and CMCD-MNP(P) indicates six
characteristic peaks at 2θ = 30.2o, 35.6o, 43.2o, 53.7o, 57.2o, and 62.9o as shown in
Figure 6-5(i). They relate to their corresponding indices (2 2 0), (3 1 1), (4 0 0), (4 2 2),
(5 1 1), and (4 4 0), respectively. This reveals that the resultant nanoparticles are pure
Fe3O4 with a spinel structure and the modification of the surface of magnetic
nanoparticles with CM-β-CD did not result in the phase change of Fe3O4. The crystal
sizes of CMCD-MNP(C) and CMCD-MNP(P) determined from the XRD pattern by
using Scherrer’s equation [Dhkl = 0.9λ/(βcos θ), where Dhkl is the average crystalline
diameter, λ is X-ray wavelength, β is the half width of XRD diffraction lines and θ is
Bragg’s angle in degree] are found to be 10.2 and 10.7 nm, respectively, slightly less
than that observed from the TEM image. Here, the (3 1 1) peak of the highest intensity
was picked out to evaluate the particle diameter of the MNPs.
Chapter: 6
120
Figure 6-5 (i) XRD patterns and (ii) magnetization curves (a) uncoated Fe3O4 MNPs, (b) CMCD-MNP(C) and (c) CMCD-MNP(P).
The magnetic properties of the bare and CM-β-CD coated Fe3O4 nanoparticles were
measured by VSM at room temperature. In Figure 6-5(ii), the hysteresis loops that are
characteristic of superparamagnetic behavior can be clearly observed for both bare and
CM-β-CD coated magnetic nanoparticles. Superparamagnetism is the responsiveness to
an applied magnetic field without retaining any magnetism after removal of the applied
magnetic field. From M vs. H curve, the saturation magnetization values (Ms) obtained
at same field for CMCD-MNP(C) and CMCD-MNP(P) were 68 and 54 emu g-1,
respectively, lower than that of bare Fe3O4 (75 emu g-1). This is mainly attributed to the
existence of non-magnetic materials (CM-β-CD layer) on the surface of nanoparticles.
However, the magnetization magnitude of sample synthesized by one-steps method is
lower than that by two-steps method [224]. This is because the adsorption quantity of
CM-β-CD onto Fe3O4 nanoparticles is higher; the magnetic particles amount per unit
quantity is lower. As a result, the saturation magnetization of the prepared by one-step
Chapter: 6
121
method is lower. These values of saturation magnetization are comparable to those of
other functionalized nanoadsorbents [108,225]. This feature allows the as-synthesized
nanoparticles for highly efficient magnetic manipulation when used as nano-adsorbents
for removal of dyes from aqueous solution under relatively low external magnetic field.
Zeta potentials of CMCD-MNP(P) and CMCD-MNP(C) (0.1 mg/ml of each adsorbent)
were measured in 10-3 M NaCl aqueous solution at different pH. The solution pH was
adjusted by NaOH or HCl. As shown in Figure 6-6, the isoelectric point (pI) of bare
Fe3O4 nanoparticles was about 6.8, consistent with the values reported in literature [40].
After being grafted with CM-β-CD, the pI for CMCD-MNP(C) and CMCD-MNP(P)
were shifted to 4.7 and 4.2, respectively indicating that the grafting of CM-β-CD onto
both adsorbents was successful. Thus, the CMCD-MNP(C) was positively charged at
pH<4.7 whereas CMCD-MNP(P) was positively charged at pH < 4.7.
Figure 6-6 Zeta potentials of bare Fe3O4 MNP, CMCD-MNP(C) and CMCD-MNP(P).
-60
-40
-20
0
20
40
60
1 2 3 4 5 6 7 8 9 10 11 12
Zet
a p
oten
tial
, m
V
pH
Bare Fe3O4 MNPCMCD-MNP(C)CMCD-MNP(P)
Chapter: 6
122
6.3.2 Adsorption of MB onto CM-β-CD modified MNPs
The CD molecules with proper size, shape, and polarity could fit the cavity to form
inclusion complex through host–guest interactions, which contribute to the application
of CDs in environmental pollution cleanup. CDs can remove typical organic pollutants,
such as dyes and phenolic compounds, from large volumes of water. In our work,
methylene blue (MB) was used as a model compound representing organic
contaminants. The use of MB as a model compound has been reported in earlier studies
[11,114,118,141,226,227]. Qualitative adsorption behavior of the MB on CM-β-CD-
functionalized nanoadsorbent is shown in Figure 6-7. Nanoadsorbents were shaken for
15-50 min in an alkaline aqueous solution of MB (blue colored). During the adsorption
process, the decrease in free MB could be qualitatively shown by the clear change of
the color in the aqueous phase, which indeed turned lighter and lighter with time. After
adsorption, the supernatant MB solution was separated by using strong magnet and
quantified by UV-vis spectrophotometer.
Chapter: 6
123
Figure 6-7 The schematic representation of the selective adsorption and separation of MB by CM-β-CD modified magnetic nanoparticles.
Figure 6-8 is the UV-vis spectra of MB before and after adsorption with different
nanoadsorbents at pH~12. It was found that CM-β-CD modified magnetic nanoparticles
had higher adsorption capabilities than uncoated magnetic particles, indicating surface
modification with CM-β-CD could enhance the adsorption capacities. Moreover,
CMCD-MNP(P) adsorbed more MB than CMCD-MNP(C) due to higher amount of
grafted CM-β-CD on the surface acting as the adsorption sites.
Chapter: 6
124
Figure 6-8 UV-vis spectra of methylene blue (0.5 mg/ml) at pH~12 upon addition of (a) 0 mg MNPs; (b) 50 mg uncoated MNPs; (c) 50 mg CMCD-MNP(C); (d) 50 mg CMCD-MNP(P).
6.3.2.1 Effect of pH
The effects of initial solution pH on the adsorption of methylene blue (MB) onto both
CMCD-MNP(P) and CMCD-MNP(C) were investigated at pH 2–12, 25ºC, and an
initial MB concentration of 2 mg/ml. As shown in Figure 6-10, very less adsorption of
MB took place on both adsorbents, when pH was less than 4. At lower pH values, the
dimethylamine group in MB molecule is changed, as shown in Figure 6-9 and the
strong hydrophilic MB may have weak interaction with hydrophobic cavity of CM-β-
CD. Weaker interaction of CM-β-CD with MB molecules was reported in acetic media
(pH 2) [228]. Moreover, at low pH the surface of adsorbents would be surrounded by
the hydrogen ions which compete with MB ions binding the sites of the sorbent.
Between the initial pH values of 4 and 10 (equilibrium pH 5.4 to 9.65), the solution pH
hardly affects the adsorption of MB onto both CMCD-MNP(P) and CMCD-MNP(C).
Here, the hydrophilic and hydrophobic groups in each MB molecule simultaneously
exist, which have the same interaction with the CMCD-MNPs. At pH > 10, the
0
0.2
0.4
0.6
0.8
1
1.2
400 450 500 550 600 650 700 750 800
Ab
sorb
ance
Wavenumber (nm)
(a)
(b)
(c)
(d)
(a)
(b)
(c)
(d)
(a)
(b)
(c)
(d)
(a)
(b)
(c)
(d)
Chapter: 6
125
adsorption capacities increase with increase of pH since MB molecules with ammonium
salt are neutralized and the interaction between neutral MB and the cavity of CM-β-CD
is strengthened. Similar observation was found on the binding of MB with self-
assembled multilayer thin-film fabricated with CM-β-CD/diazoresin [229]. As the
adsorption amount of MB remained almost same at pH 4–10, the following studies were
conducted at a fixed pH of 8 and also at optimum pH 12.
Figure 6-9 The molecular structure of MB.
Figure 6-10 Effect of pH on the adsorption of MB by CM-β-CD modified magnetic nanoparticles. Conditions: (Initial MB concentration: 2 mg/ml, temperature: 25ºC, adsorbents: 120 mg).
0
50
100
150
200
250
300
1 3 5 7 9 11 13
Ad
sorb
ed a
mou
nt
(mg/
g)
Equilibrium pH
CMCD-MNP(P)
CMCD-MNP(C)
Chapter: 6
126
6.3.2.2 Adsorption kinetics
Figure 6-11 shows the effects of contact time on adsorption of MB onto CM-β-CD
modified magnetic nanoparticles under various initial concentrations of MB and at
25ºC. The initial dye concentration provides the necessary driving force to overcome
the mass transfer resistance of MB between the aqueous and solid phases [230]. It can
be seen that a rapid adsorption of MB by both adsorbents occurred, with equilibrium
reached in approximately 20 and 50 min by CMCD-MNP(C) and CMCD-MNP(P),
respectively. Also the amount of dye adsorbed at equilibrium increased with the
increase in initial MB concentrations. Within almost 5-10 minutes, 85-90% MB of that
at equilibrium was achieved using both CMCD-MNP(P) and CMCD-MNP(C). Such a
fast adsorption rate could be attributed to the absence of internal diffusion resistance.
This suggests that both adsorbents could rapidly and effectively adsorb MB from
aqueous solution.
Chapter: 6
127
Figure 6-11 Effect of contact time on the adsorption of MB onto CMCD-MNP(P) (a) and CMCD-MNP(C) (b) and pseudo-second order kinetics of MB adsorption onto CMCD-MNP(P) (c) and CMCD-MNP(C) (d). Conditions: (Initial MB concentration: 0.25, 0.50 and 2 mg/ml, pH: 8, temperature: 25ºC).
The adsorption kinetics of MB onto CM-β-CD modified magnetic nanoparticles was
investigated with the help two kinetic models, namely the Lagergren pseudo-first order
and pseudo-second-order model. The Lagergren rate equation is one of the most widely
used adsorption rate equations for the adsorption of solute from a liquid solution. The
pseudo-first-order kinetic model is expressed by the following equation [150]:
1( )te t
dqk q q
dt (6-2)
Chapter: 6
128
Integrating this equation for the boundary conditions t = 0 to t = t and q = 0 to q = qt,
gives:
ln(qe - qt) = lnqe – k1t (6-3)
where qe and qt refer to the amount of dye adsorbed (mg/g) at equilibrium and at any
time, t (min), respectively, and k1 is the equilibrium rate constant of pseudo-first-order
sorption (1/min). The slope and intercept of the plot of ln(qe - qt) versus t are used to
determine the first-order rate constant, k1. The corresponding kinetic parameters are
shown in Table 6-1. It was found that the correlation coefficient (R2) has low value
(<89%) for both adsorbents for all initial MB concentrations studied and a very large
difference exists between qe,cal and qe,exp, indicating a poor pseudo-first-order fit to the
experimental data. The inapplicability of the pseudo-first-order model to describe the
kinetics of basic blue 3 and Malachite Green (a basic dye) by adsorption using
cyclodextrin based polymer was also observed in some previous work [10,113].
Another kinetic model is pseudo-second order model, which is expressed by [149]:
21( )t
e t
dqk q q
dt (6-4)
Rearranging the variables in Eq. 6-4 gives,
12( )t
e t
dqk dt
q q
(6-5)
Integrating this equation for the boundary conditions t = 0 to t = t and q = 0 to q = qt,
gives:
22
1 1
t e e
tt
q k q q (6-6)
Chapter: 6
129
where k2 is the equilibrium rate constant of pseudo second- order adsorption (g/mg
min). The slope and intercept of the plot of t/qt versus t were used to calculate the
second-order rate constant, k2 (Figures 6-11c and 6-11d). The corresponding kinetic
parameters from this model are listed in Table 6-2. The correlation coefficient (R2) for
the pseudo-second-order adsorption model has high value (> 99%) for both adsorbents.
The calculated equilibrium adsorption capacities, (qe,cal) by CMCD-MNP(P) and
CMCD-MNP(C) were 129.87 and 69.44 mg/g, respectively which were consistent with
the experimental data. These facts suggest that the pseudo-second-order adsorption
mechanism is predominant, and that the overall rate of the dye adsorption process
appears to be controlled by the chemisorption process [115]. Similar phenomena have
been observed for MB adsorption onto many adsorbents, for example- titanate
nanotubes [226], tea waste [231], β-CD/carboxymethyl cellulose polymer [141] etc.
Table 6-2 Adsorption kinetic parameters of MB onto the CM-β-CD modified magnetic nano-adsorbents at pH 8 and 25ºC
Adsorbent Initial conc. Co (mg/ml)
pseudo-first order pseudo-second order
k1, min-1 qeb, mg/g R2 qe
a, mg/g k2,g/(mg min) qeb, mg/g R2
CMCD-MNP(P)
0.25 0.005 5.93 0.719 47.68 0.032 44.70 0.999
0.50 0.015 7.77 0.798 74.60 0.020 73.53 0.999
2.00 0.023 25.20 0.785 130.96 0.004 129.87 0.999
CMCD-MNP(C)
0.25 0.013 3.84 0.662 27.23 0.026 26.46 0.999
0.50 0.016 5.00 0.658 42.53 0.020 41.33 0.998
2.00 0.016 4.10 0.883 68.75 0.016 69.44 0.999 a experimental b calculated
6.3.2.3 Adsorption isotherms of MB
The equilibrium isotherms for the adsorption of MB by CMCD-MNP(P) and CMCD-
MNP(C) at pH 8 and 12 and 25ºC are shown in Figure 6-12. The equilibrium data were
fitted by Langmuir and Freundlich isotherm equations. The Langmuir equation can be
expressed as:
Chapter: 6
130
1e e
e m m L
C C
q q q K (6-7)
where qe is the equilibrium adsorption capacity of MB on the adsorbent (mg/g), Ce the
equilibrium MB concentration in solution (mg/ml), qm the maximum capacity of
adsorbent (mg/g), and KL is the Langmuir adsorption constant (ml/mg). The linear form
of Freundlich equation, which is an empirical equation used to describe heterogeneous
adsorption systems, can be represented as follows:
lnqe = (1/n) lnCe + lnKF (6-8)
where qe and Ce are defined as above, KF is Freundlich constant (ml/mg), and n is the
heterogeneity factor. The values of qm and KL were determined from the slope and
intercept of the linear plots of Ce/qe versus Ce (Figure 6-13a) and of values of KF and
1/n were determined from the slope and intercept of the linear plot of lnqe versus lnCe
(Figure 6-13b). The isotherm parameters are shown in Table 6-3. The high values of
correlation coefficients, R2 (> 0.99) for both CMCD-MNP(P) and CMCD-MNP(C),
indicated that the equilibrium data were fitted better by Langmuir equation than by
Freundlich equation. Thus, the adsorption of MB on both adsorbents obeyed the
Langmuir adsorption isotherm. From Table 6-3, the qm values at optimum pH (~12) for
CMCD-MNP(P) and CMCD-MNP(C) were estimated to be 277.8 and 140.8 mg/g,
respectively, and the KL values for MNPs and CMCD-MNPs were calculated as 9.0 and
4.44 ml/mg, respectively. CMCD-MNP(P) could adsorb MB molecule almost twice
than that by CMCD-MNP(C) at both pH 8 and 12. This could be explained by the
amount of CM-β-CD grafted on the surface of both adsorbents. CMCD-MNP(P)
adsorbent contained more CM-β-CD (12 wt%) than CMCD-MNP(C) (4.7 wt%)
providing more adsorption sites for adsorption. The maximum amount of MB adsorbed
by both adsorbents in this work is compared to other adsorbents recently reported in
Chapter: 6
131
literature shown in Table 6-4. The adsorption capacities of these as-synthesized nano-
adsorbents for MB are comparatively higher than some of other cyclodextrin polymer
based adsorbents. For example, the adsorption capacities of MB on β-CD polymer (β-
CDP) crosslinked by citric acid and β-CD/carboxymethyl cellulose polymer were 105
mg/g [11] and 56.5 mg/g [141], respectively. Our results reveal the potential of these
materials to be an effective nano-adsorbent for removing basic dyes.
Figure 6-12 Equilibrium isotherm for the adsorption of MB on CMCD-MNP(P) and CMCD-MNP(C) at 25ºC.
0
50
100
150
200
250
300
0 0.5 1 1.5 2 2.5 3
q e(m
g/g)
Ce (mg/ml)
CMCD-MNP(P) at pH12CMCD-MNP(P) at pH 8CMCD-MNP(C) at pH12CMCD-MNP(C) at pH 8Langmuir fitting
Chapter: 6
132
Figure 6-13 Langmuir plot illustrating the linear dependences of Ce/qe on Ce (a) and Freundlich plot illustrating the linear dependences of lnqe on lnCe (b).
(a)
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0 0.5 1 1.5 2 2.5 3
Ce/
q e
Ce
CMCD-MNP(P) at pH 12CMCD-MNP(P) at pH 8CMCD-MNP(C) at pH12CMCD-MNP(C) at pH 8
(b)
2
2.5
3
3.5
4
4.5
5
5.5
6
-5 -3 -1 1
lnq e
lnCe
CMCD-MNP(P) at pH 12
CMCD-MNP(P) at pH 8
CMCD-MNP(C) at pH 12
CMCD-MNP(C) at pH 8
Chapter: 6
133
Table 6-3 Adsorption isotherm parameters for MB adsorption onto CM-β-CD modified magnetic nanoparticles at 25ºC
Isotherm Models
Parameters CMCD-MNP(P) CMCD-MNP(C)
pH 12 pH 8 pH 12 pH 8
Langmuir
qm (mg/g) 277.8 138.9 140.8 77.5
KL (ml/mg) 9.0 10.28 4.44 8.60
R2 0.999 0.998 0.993 0.996
RL 0.31-0.036 0.49-0.031 0.69-0.07 0.54-0.037
Freundlich n 2.913 2.97 2.20 2.84
KF (ml/g) 256.1 120.60 112.26 66.14
R2 0.921 0.957 0.975 0.945
Table 6-4 Maximum adsorption capacities (qm in mg/g) for MB by some other adsorbents reported in literature
Adsorbents Maximum adsorbed amount, qm (mg/g)
pH References
Activated carbon from steam activated bituminous coal
580.0 11 [232]
Fe3O4-activated carbon nanocomposite
321.0 NA* [137]
M-β-CD modified magnetic nanoparticles [CMCD-MNP(P)]
277.8 12 This study
Polyacrylic acid-bound iron oxide magnetic nanoparticles
199.0 9 [40]
Activated carbon from groundnut shell (GNSC)
164.9 7.4 [233]
CM-β-CD modified magnetic nanoparticles [CMCD-MNP(C)]
140.8 12 This study
β -CD polymer (β-CDP) crosslinked by citric acid
105.0 NA* [11]
Spent activated clay 91.23 5.5 [234] Tea waste 85.16 8 [231] β -CD/carboxymethyl cellulose polymer
56.5 8 [141]
Silica 56.2 11 [235] Yellow passion fruit waste 44.7 8 [227] Fe(III)/Cr(III) hydroxide 22.8 NA* [236] Activated carbon from waste biomass
16.43 6 [230]
*NA: Not available
Chapter: 6
134
The essential features of the Langmuir isotherm can be expressed in terms of a
dimensionless constant called separation factor or equilibrium parameter (RL) which is
given by the following equation [213]:
0
1
1LL
RK C
(6-9)
where C0 is the initial MB concentration (mg/ml) and KL is the Langmuir constant
related to the energy of adsorption (ml/mg). The value of RL indicates the shape of the
isotherms to be either unfavorable (RL > 1), linear (RL = 1), favorable (0 < RL < 1) or
irreversible (RL = 0). It was observed that the value of RL in the range of 0-1 (Table 6-2)
confirmed the favorable uptake of MB by both adsorbents. The calculated RL values at
different initial MB concentration are presented in Figure 6-14. Also higher RL values at
lower MB concentrations indicate that adsorption was more favorable at lower
concentration. In addition, the low RL values (< 0.05) implied that the interaction of MB
molecules with CM-β-CD modified magnetic nanoparticles might be relatively strong
[226].
Figure 6-14 Separation factor for MB onto CM-β-CD modified magnetic nanoparticles at pH 8 and 25ºC.
0
0.1
0.2
0.3
0.4
0.5
0.6
0 1 2 3
RL
Co (mg/ml)
CMCD-MNP(P)
CMCD-MNP(C)
Chapter: 6
135
The free energy change (∆G) for adsorption at 25oC was calculated using the following
equation:
∆G = −RT lnKa = −RT lnKc = −RT ln(Cads/Ce ) (6-10)
where T is the temperature (K), R the gas constant (Jmol-1K-1), Ce the dye concentration
in solution at equilibrium and Cads the concentration of dye adsorbed at equilibrium.
The above equation implies that only at very low concentration of solute, the
distribution constant (Kc) is equal to the thermodynamic equilibrium constant (Ka) and
only in this case it can be used for calculation of ∆G [237]. Kc can be estimated from the
slope of the linear plot of Cads vs. Ce and the linear relationship holds only for dilute
solutions. The calculated ∆G value was found to be −3.61 kJ/mol and -3.21 kJ/mol at
pH~8 and -4.62 KJ/mol and -2.98 KJ/mol at pH~12 for CMCD-MNP(P) and CMCD-
MNP(C), respectively. The negative values of free energy change indicate the
spontaneous nature of sorption and confirm affinity of sorbents for the dye.
6.3.3 Adsorption interactions
For understanding of the MB/CMCD-MNP(P) interaction, FTIR data were introduced
to gain insight into the adsorption mechanism. Figure 6-15 shows the spectra of
methylene blue (MB) and CMCD-MNP(P) nano-adsorbent before and after MB
adsorption. Figure 6-15(a) shows the broad peak at 3386 cm−1 corresponded to the
stretching vibration of highly -OH group on the CMCD-MNP(P). The other peaks at
1028 and 1153 cm−1 are due to C-OH and O-C-O groups of cyclodextrin moieties,
respectively. After being adsorbed by methylene blue, the peaks at 900-1100 cm-1 in the
spectrum for CMCD-MNP(P) have shifted which indicates that these functional groups
may be involved into interaction with MB. The absorption band at 3383 cm−1 also
shifts to higher wave number (3389 cm−1) which can be explained by the disappearance
Chapter: 6
136
of water molecules from the cyclodextrin cavity, similarly to the case of true inclusion
complexes [238]. In the spectrum for MB shown in Figure 6-15(c), the peak
corresponding to the vibration of the aromatic ring at 1600 cm−1 was very prominent.
Two peaks at 1490 and 1396 cm−1 are assigned to the C–N stretching vibrations [226].
The vibrations of the CH3 group are found at 1357 and 1342 cm−1. Several weak peaks
between 1252 and 669 cm−1 were ascribed to the C–H in plane and out of plane bending
vibrations [232]. In the spectrum of CMCD-MNP(P) after adsorption of MB (Figure 6-
15b), several peaks corresponding to MB with the aromatic ring vibration at 1606 cm−1,
the C–N stretching vibrations at 1492 and 1394 cm−1 and the CH3 group vibrations at
1354 and 1337 cm−1, can be observed which reflects the evidence for the strong
interaction between MB and CMCD-MNP(P). Similar MB-adsorbent interaction was
observed after adsorption of MB onto titanate nanotube [226].
Figure 6-15 FTIR spectra of CMCD-MNP(P) of before (a) and after (b) adsorption of MB, and only MB (c).
400 900 1400 1900 2400 2900 3400 3900
%T
Wavenumber, cm-1
10281623
3386
1606
10321337
1600
1396
1357
3389
1083
2923
586
586
13421028
1081
(b)
(a)
(c)
1490
1492
Chapter: 6
137
It is reported that due to its specific characteristics (the presence of cyclodextrin
moieties and complexing chemical groups), the sorption mechanism of cyclodextrin
based materials is different from those of other materials [10]. The main interactions in
the complexation process between cyclodextrin and other organic molecule are dipole-
dipole, hydrogen bonding, electrostatic, van der Waals, hydrophobic and charge transfer
interaction [9]. Among these forces, hydrophobic interactions have been mostly
recognized as the main factors involved in the inclusion complex with cyclodextrin. In
this study, the adsorption of MB is found to be pH dependent and the
hydrophilic/hydrophobic nature of MB depending on various pHs plays an important
role in the formation of inclusion complex with CM-β-CD (Figure 6-10). It has been
found that the adsorption process follows the pseudo-second-order kinetics and this
suggests that chemical sorption might be the rate-limiting step in controlling the
adsorption process. The negative values of free energy change (∆G) confirmed the
affinity of adsorbents for the MB dye.
6.3.4 Desorption and regeneration experiments
Desorption study was conducted to explore the possibility of recyclability of the
magnetic nano-adsorbent. Desorption of MB from MB loaded-CMCD-MNP(P) was
demonstrated using a methanol solution containing acetic acid (0-5%) as eluent [40]. It
can be seen from Figure 6-16(a) that pure methanol solution could desorb 57.2% of MB
from the nano-adsorbent surface. The percentage of MB desorbed could be increased by
using acetic acid in methanol solution. About 99.0% of desorption efficiency was
achieved when 5% (v/v) of acetic acid was used in methanol solution, respectively.
Desorption of MB from MB loaded-CMCD-MNP(P) was also carried out using acidic
buffer (pH< 3). Since adsorption of MB was very less at low pH as shown in Figure 6-
Chapter: 6
138
10, it was hypothesized that acidic buffer might aid in desorption of MB from the CM-
β-CD modified magnetic nanoparticles. The desorption efficiency was achieved also as
high as 79.7% when acidic buffer (pH = 2) was used as desorption eluent.
Figure 6-16 (a) Desorption of MB from CMCD-MNP(P) as a function of acetic acid concentration in methanol, and (b) performance of CMCD-MNP(P) by multiple cycles of regenaration. Adsorption conditions: (CMCD-MNP(P): 120 mg; MB concentration: 2 mg/ml; temperature: 25ºC; pH: 8; contact time: 2 hr). Desorption conditions: (CMCD-MNP(P): 120 mg; temperature: 25ºC; contact time: 2 hr; desorption eluent: methanol with 5 % acetic acid)
0
20
40
60
80
100
0 1 2.5 5
% R
emov
al
Acetic acid in methanol (vol%)
(a)
0
20
40
60
80
100
120
140
1 2 3 4
q e(m
g/g)
No. of cycles
(b)
Chapter: 6
139
As shown as Figure 6-16(b), CMCD-MNP(P) could be recycled four times for MB
adsorption with more than 90% of adsorption capacity being retained. This result
indicates the stability and reusability of the adsorbents in wastewater treatment.
6.4 Cost analysis
Since its first introduction for heavy metals removal, activated carbon has undoubtedly
been the most popular and widely used adsorbent for environmental pollution control.
Activated carbons are the most effective adsorbents as they can efficiently adsorb many
pollutants. Although the commercial activated carbon is relatively cheap, but further
modification of these adsorbents for some specific applications could be quite
expensive. The regeneration of saturated carbon by thermal and chemical procedure is
also expensive, and results in loss of the adsorbent. This had led many researchers to
search for more efficient adsorbents with high adsorption capacities and low cost.
Cost is actually an important parameter for comparing the adsorbent materials. But,
which low-cost adsorbent is better? There is no direct answer to this question because
each low-cost adsorbent has its specific physical and chemical characteristics such as
porosity, surface area, physical strength and chemical stability, as well as inherent
advantages and disadvantages in wastewater treatment. A sorbent can be considered
low-cost if it requires little processing, is abundant in nature, or is a by-product or waste
material from another industry [107,116]. This adsorbent should also possess high
capacity and adsorption rate, high selectivity for different concentrations and rapid
kinetics. β-CDs are synthetic polysaccharides, formed by the action of an enzyme on
starch. Starches are unique raw materials in that they are very abundant natural
polymers, inexpensive and widely available in many countries. β-CD molecules are
available commercially at a low-cost. Both soluble and insoluble CD-based materials
Chapter: 6
140
are also easy to prepare with relatively inexpensive chemical reagents and are available
in a variety of structures with a variety of properties. Anchoring the β-CD derivatives
on the surfaces of Fe3O4 nanoparticles in one step precipitation method is also a simple
and inexpensive method. The particles synthesized in this way, shows high adsorption
capacities and fast kinetics and they could be regenerated and recycled using simple
magnetic separation technology which is obviously a great advantage. Due to scarcity
of consistent cost information in the literature data, cost comparisons are difficult to
make. However, the relative cost of the material used in the present study can be
comparable with modified activated carbons.
6.5 Conclusions
In this work, CM-β-CD modified Fe3O4 nanoparticles with combined properties of
superparamagnetism and inclusion functionalities were successfully synthesized. The
binding of CM-β-CD on the magnetic surface was confirmed by FTIR, TGA and zeta
potential analysis. MB was recovered fast and efficiently from the aqueous solution
using the as-synthesized magnetic nanoadsorbents. The adsorption of MB on these
adsorbents was found to be pH dependant. The kinetics of MB adsorption onto both
adsorbents followed the pseudo-second-order model. The adsorption of MB reached
equilibrium within 50 min and 85-90% of MB being adsorbed within 20 min. The
equilibrium data for both CMCD-MNP(P) and CMCD-MNP(C) were fitted well by the
Langmuir isotherm model of adsorption, showing monolayer coverage of MB
molecules at the outer surface of these nanoparticles. The maximum sorption capacities
of MB for CMCD-MNP(P) and CMCD-MNP(C) were 277.8 and 140.8 mg/g,
respectively at pH~12 and 25ºC and the negative values of free energy change indicated
the spontaneity of adsorption process. The magnetic nano-adsorbents (CMCD-MNP(P))
Chapter: 6
141
prepared by one-step method showed better adsorption performance than CMCD-
MNP(C) prepared by two-steps method. This is because, CMCD-MNP(P) has higher
content of CM-β-CD which in turn provides more available active sites for adsorption.
The adsorbed MB could be desorbed from the surface by using methanol solution
containing acetic acid and these nano-adsorbents could be recycled and handled easily
by using magnetic field gradient. The experimental results suggest that the CM-β-CD
modified magnetic nanoparticles could be employed as efficient and suitable adsorbents
for the removal of methylene blue dye from wastewater.
Chapter: 7
142
Chapter 7 Carboxymethyl-β-cyclodextrin conjugated magnetic nano-adsorbents for removal of copper ions from aqueous water4
7.1 Introduction
This Chapter presents adsorption studies of heavy metal [Cu2+] ions onto CM-β-CD
bonded magnetic nanoparticles. Considered the special advantages of the adsorption
capacity of β-CD through complexation with multiple functional groups and the
separation convenience of magnetic Fe3O4 nanoparticles, grafting of β-CD on the
magnetic nanoparticles is considered as a promising alternative to develop magnetic
materials for the efficient removal of heavy metal pollutants.
Heavy metal pollution in industrial wastewater has attracted global attention because of
its adverse effects on the environment and human health [108,239]. The effluents of
industrial or municipal wastewaters contain considerable amount of toxic and polluting
heavy metals. One of those metals in focus is copper (Cu2+) which is an abundant and
naturally occurring element present in the wastewaters [108]. Copper ingestion might
cause vomit, cramps, liver damage, kidney damage and even death. It is also toxic to
aquatic organisms even at very small concentrations in natural water [152]. It is,
therefore, crucial to remove these heavy metals from wastewaters effectively before
their discharge into the environment.
4. A.Z.M. Badruddoza, A.S.H. Tay, P.Y. Tan, K. Hidajat, M.S. Uddin, Carboxymethyl-β-cyclodextrin
conjugated magnetic nanoparticles as nano-adsorbents for removal of copper ions: Synthesis and
adsorption studies, J. Hazard. Mater. 185(2-3) (2011) 1177-1186.
Chapter: 7
143
Currently heavy metal removal methods include chemical precipitation, ion exchange,
liquid-liquid extraction, membrane filtration, adsorption and biosorption [111]. Among
these methods, adsorption has increasingly received more attention as it is simple and
cost effective [41,112]. Recently, adsorption using magnetic nano-sized adsorbents has
attracted significant attentions in environmental applications due to the specific
characteristics [8,37,41,108,125,240]. Their properties- extremely small size, high
surface-area-to volume ratio and the absence of internal diffusion resistance provide
better kinetics for adsorption of metal ions from aqueous solution. Magnetic nano-
adsorbents have the advantages of both magnetic separation techniques and nano-sized
materials, which can be easily recovered or manipulated with an external magnetic
field. This technique overcomes many issues present in other physicochemical process
such as fouling problems and operation cost, and requires much less energy to achieve a
given level of separation [128]. The incorporation of magnetic particles with other
functionalized materials such as multiwalled carbon nanotubes (MWCNT)
[155,241,242], zeolites [243], activated carbon [244] etc. which are effective for the
removal of both organic and inorganic pollutants is potentially a promising method to
facilitate the separation and recovery of the adsorbents. Various types of magnetic
nano-adsorbents with tailored surface reactivity by using natural or synthetic polymers
such as, chitosan, gum arabic, alginate, poly(acrylic acid) etc. have been used recently
for heavy metal removal from wastewater [8,37,41,44,108,124,125,241,245]. However,
the effectiveness of magnetic nano-adsorbents modified with cyclodextrin–an important
class of polysaccharides has not been attempted.
Although there is less report on environmental applications of cyclodextrins for heavy
metal removal, there is evidence that metal can be complexed by cyclodextrins through
hydroxyl groups. Metal ion complexes with cyclodextrins could have a wide range of
Chapter: 7
144
applications in catalysis and molecular recognition [246]. Some properties such as
aqueous solubility and metal complexation potential can be altered by substituting
functional groups to the outside of the cyclodextrin [222]. For example, carboxymethyl-
β-cyclodextrin (CM-β-CD) has the ability to complex heavy metals such as cadmium,
nickel, strontium and mercury through the interactions between the metal ions and -
COOH functional groups [247,248]. Insoluble cyclodextrin polymers and cyclodextrin
immobilized on various supports including inorganic carriers are currently being
investigated for adsorptive specificity towards heavy metal ions [179,249]. Very
recently, Hu et al. showed that the grafted β-CD on the MWCNT/iron oxides
contributes to an enhancement of the adsorption capacity because of the strong
complexation abilities of the multiple hydroxyl groups in β-CD with the lead ions
[242]. However, to our knowledge, no adsorptive study of heavy metals on magnetic
nanoparticles covalently bonded with CM-β-CD has been reported so far.
In this study, a novel magnetic nano-adsorbent was synthesized for the adsorption of
metal ions by surface modification of Fe3O4 nanoparticles with CM-β-CD. The
adsorption behavior of these sorbents was investigated using Cu2+ ions as the target
metal contaminant because of its extensive environmental impacts. The effects of
several factors such as, pH, initial Cu2+ concentration and temperature were studied.
The adsorption interactions of Cu2+ onto CMCD-MNP(C) were investigated through
FTIR and XPS analyses. In addition, the regeneration and reusability of the adsorbents
were also evaluated.
Chapter: 7
145
7.2 Experimental
7.2.1 Materials
In this work, the following chemicals were used for fabricating CMCD-MNP(C): Iron
(II) chloride tetrahydrate (99%) [Alfa Aesar], iron (III) chloride hexahydrate (98%)
[Alfa Aesar], ammonium hydroxide (25%) [Merck], β-cyclodextrin (β-CD, 99%)
[Tokyo Kasie Kogyo (Japan)], carbodiimide (cyanamide, CH2N2, 98%) [Sigma],
copper(II) nitrate [Sigma], chloroacetic acid (99%) [Alfa Aesar]. All other chemicals
were of analytical grade and used as received without further purification.
7.2.2 Adsorption/ desorption of Cu2+ ions
The adsorption of Cu2+ by the CMCD-MNP(C) was investigated using batch
equilibrium technique in aqueous solutions at pH 2–6 at 25–55 ◦C. In general, 120 mg
of wet magnetic nano-adsorbent was added to 10 ml of copper nitrate solution of
various concentrations and shaken in a thermostatic water-bath shaker operated at 230
rpm. After equilibrium was reached, the magnetic nano-adsorbents were removed using
a strong permanent magnet made of Nd–Fe–B and the supernatant was collected. The
solution pH was adjusted by NaOH or HCl. The concentrations of copper ions were
measured using Inductive Couple Plasma Mass Spectrometry (Agilent ICP-MS 7700
series).
For the kinetic experiments, the initial copper ion concentrations were 50, 100 and 200
mg/l and the initial solution pH value was 6.0 (the solution pH was not controlled
during the experiments). These copper solutions containing the magnetic particles were
agitated for a contact time varied in the range of 0–240 min at a speed of 230 rpm at
25◦C. At various time intervals, samples were collected after separation of magnetic
adsorbents by magnetic decantation process and the copper concentration was
Chapter: 7
146
determined as mentioned previously. In the investigation of the effect of solution pH on
equilibrium copper adsorption, the initial Cu2+ concentrations in the solution were 50
and 200 mg/l, but the solution pH values were changed from 2 to 6. In the adsorption
isotherm experiments, the initial solution pH was 6.0 while the initial Cu2+
concentrations in the solutions were changed from 50 to 400 mg/l and temperatures
were varied from 25◦C to 55◦C. The samples were shaken for 4 h (which was enough to
achieve equilibrium) at 230 rpm. Duplicate experiments were carried out in each case
and the reproducibility was found to be fairly good. The error percentage was within
5%. Desorption study was conducted using 0.1 M acetic acid, 0.1 M citric acid and 0.1
M Na2EDTA as desorption eluents. Adsorption was first conducted using 120 mg of
wet CMCD-MNPs in 10 ml of 200 mg/l Cu2+ solution for 4 h at pH 6. Desorption was
then studied by adding 10 ml of either acid solution to the Cu2+-sorbed CMCD-
MNP(C). After shaking at 200 rpm for 3 h, the CMCD-MNPs were removed and the
concentration of copper ions was measured.
7.3 Results and Discussion
7.3.1 Synthesis and characterization of magnetic nanoparticles
The detailed synthesis procedures of CMCD-MNP(C) are shown step-by-step in
Chapter 6 (section 6.2.1). In Chapter 6, the characterization results of these nano-sized
magnetic particles performed by FTIR, TEM and VSM are also discussed. In this
section, only the results of characterization by TGA and zeta potential measurement
have been discussed.
The amount of CM-β-CD grafted on Fe3O4 MNPs is estimated from TGA analyses of
uncoated and CM-β-CD coated magnetic nanoparticles. As shown in Figure 7-1(a), the
TGA curve for CMCD-MNPs exhibits two steps of weight loss, contributed from the
Chapter: 7
147
loss of residual water in the sample in 30–190oC and the loss of CM-β-CD in the range
of 200–450oC. A drastic drop of weight loss can be seen in the range 190–400oC with
the occurrence of a weak heat absorption at 282.5oC and it is contributed from the
thermal decomposition of CM-β-CD. Thus, the TGA curves also confirm the successful
grafting of CM-β-CD onto the magnetic surface.
Figure 7-1 (a) TGA curves for uncoated Fe3O4 MNPs and CMCD-MNP(C); (b) Effect of the amount of CM-β-CD in solution on the grafted CM-β-CD contents onto magnetic nanoparticles.
-0.01
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
92
93
94
95
96
97
98
99
100
101
0 200 400 600
% w
eigh
t lo
ss
Temperature, oC
Fe3O4 MNPs
CMCD-
Der
ivat
ive
of w
eigh
t lo
ss
121.9 oC
282.5 oC
(a)
0
1
2
3
4
5
6
0 50 100 150 200 250 300 350
% C
M-β
-CD
gra
fted
on
to M
NP
s
CM-β-CD added in solution (mg)
(b)
Chapter: 7
148
The effect of the amount of CM-β-CD used in solution on the amount of grafted CM-β-
CD onto the surface of magnetic nanoparticles was also demonstrated in this study. The
experiment was performed in 5 ml of CM-β-CD (5-60 mg/ml) solution containing a
constant Fe3O4 amount of 100 mg. The percentage amount of CM-β-CD grafted on
Fe3O4 MNPs was estimated from the weight loss measured by TGA analysis. As shown
in Figure 7-1(b), with increasing amounts of CM-β-CD in solution, the amount of
grafted CM-β-CD on the nanoparticles increases first and then remains almost constant
when the amount of CM-β-CD in solution is above 150 mg. This may be due to the fact
that as the amount of Fe3O4 is fixed which means the number of surface hydroxyl (-OH)
groups are fixed, no more -OH groups are available which can react with the –COOH
groups on CM-β-CD to form metal carboxylate. Similar finding was reported in
covalent binding of poly(acrylic acid) (PAA) to Fe3O4 nanoparticles via carbodiimide
activation [5]. However, the maximum amount of CM-β-CD bound to Fe3O4
nanoparticles could be determined to be 4.8 wt% i.e. 0.0366 mmol/g.
Figure 7-2 Zeta potentials of uncoated and CM-β-CD coated magnetic nanoparticles and copper(II) nitrate solution at different pH.
-60
-40
-20
0
20
40
60
2 4 6 8 10 12
Zet
a p
oten
tial
, m
V
pH
Uncoated Fe3O4 MNPs
CMCD-MNPs
copper(II) nitrate
Chapter: 7
149
Zeta potentials of uncoated and CM-β-CD coated magnetic nanoparticles (0.1 mg/ml)
were measured in 10-3 M NaCl aqueous solution at different pH. The solution pH was
adjusted by NaOH or HCl. As shown in Figure 7-2, the pH value of point of zero
charge (pHpzc) of uncoated Fe3O4 nanoparticles is about 6.8, consistent with the values
reported in literature. After being grafted with CM-β-CD, the pHpzc has been shifted to
4.7, indicating that the grafting of CM-β-CD onto uncoated MNPs is successful.
Moreover, magnetic nanoparticles modified with CM-β-CD yields acidic surface since
pHpzc is lower than that of uncoated MNPs and this surface acidity is due to the
introduction of several oxygen-containing functional groups [211].
7.3.2 Adsorption of Cu2+ ions on CMCD-MNP(C) 7.3.2.1 Effect of pH
The pH of the aqueous solution is an important controlling parameter in the adsorption
process [108,112,250]. The effects of initial solution pH on Cu2+ adsorption onto
CMCD-MNP(C) were investigated at pH 2–6, 25ºC, and an initial Cu2+ ion
concentration of 50 and 200 mg/l. As shown in Figure 7-3, it is noteworthy that the
adsorption capacity of Cu2+ ions increases with the solution pH at an initial Cu2+
concentration of 200 mg/l. This might be due to the less insignificant competitive
adsorption of hydrogen ions [8,37,251]. At a lower initial Cu2+ concentration (50 mg/l),
the adsorption capacity increases with increasing the solution pH and then remains
almost unchanged at pH 4–6. This might be due to the fact that almost all Cu2+ ions are
adsorbed. It is well known that Cu(II) species can be present in aqueous solution in the
forms of Cu2+, Cu(OH)+, Cu(OH)2, Cu(OH)3− and Cu(OH)4
2− and the predominant
copper species at pH < 6.0 is Cu2+ [250,252]. Therefore, less adsorption of Cu2+ that
takes place on CMCD-MNP(C) at lower pH, can be explained by the fact that at these
Chapter: 7
150
pH values the H+ concentration is high, which can compete with copper cations for the
same adsorption sites [250]. The adsorption studies at pH> 6 were not conducted
because of the precipitation of Cu(OH)2 from the solution.
Figure 7-3 Effect of pH on the adsorption of Cu2+ ions by CMCD-MNP(C) at 25ºC. Open points: qe vs. initial pH values; solid points: equilibrium pH values vs. initial pH values.
The effect of pH can be also explained by considering the surface charge on the
adsorbent material. The zeta potential of Cu2+ solution is presented in Figure 7-2 and it
shows that copper solution has positive zeta potential at pH< 7. Since the zeta potentials
of the CMCD-MNP(C) are positive at pH< 4.7 (point of zero charge, pHpzc), the
interactions between the adsorption sites on CMCD-MNP(C) and the copper ions are
electrostatically repulsive. As the solution pH increases, the repulsion between
adsorbent surface and metal ions becomes weaker, thus enhancing the adsorption
capacity [253]. On contrary, at pH above pHpzc, the negatively charged CMCD-
MNP(C) nano-adsorbents attract the positively charged metal ions. Moreover, the
1
2
3
4
5
6
0
10
20
30
40
1 2 3 4 5 6
q e(m
g/g)
Initial pH
Eq
uili
bri
um
pH
Chapter: 7
151
adsorption of Cu2+ ions depends on the adsorption sites available for copper attachment
on the surface of CMCD-MNP(C), possibly through the complexation with mainly –OH
and –COOH functional groups (see the discussions in the section 7.2.2.5). The
equilibrium pH values are also plotted as a function of initial pH values for each
experimental data (Figure 7-3). One can see that the final pH value is higher than the
initial pH value for lower pH range. When the initial solution pH exceeds to 4.0, the
final pH value is lower than the initial one and this difference becomes higher for
higher adsorption capacity of CMCD-MNP(C). The pH decreasing after Cu2+ ions
adsorption suggests that part of H+ is released from the CMCD-MNP(C) surface to
solution. Due to the increase of pH, more surface functional groups are deprotonated to
provide more metal binding sites, which results in a high metal ion adsorption and more
H+ are released into the solution resulting in the decrease in the pH values after metal
adsorption [250].
7.3.2.2 Adsorption kinetics
Figure 7-4(a) shows the effects of contact time on adsorption of Cu2+ onto CMCD-
MNP(C) at different Cu2+ concentrations ranging from 50 to 200 mg/l and at 25ºC. It
can be seen that a rapid adsorption of Cu2+ by CMCD-MNP(C) occurs, with equilibrium
reached in approximately 30 min. The adsorption capacity increases as initial metal ion
concentration increases. Within 15 min, 90% of Cu2+ adsorption equilibrium is
achieved using CMCD-MNP(C). This suggests that CMCD-MNP(C) could easily and
rapidly adsorb Cu2+ from aqueous solution due to high specific surface area and the
absence of internal diffusion resistance. Rapid adsorption rate for copper removal has
also been observed using several magnetic nanoparticles based nano-adsorbents
[37,41,124]. However, a shaking time of 4 h was used in all further adsorption
experiments to ensure equilibrium.
Chapter: 7
152
The adsorption kinetics of Cu2+ ions onto CMCD-MNP(C) is investigated with the help
two kinetic models, namely the Lagergren pseudo-first-order and pseudo-second-order
model. The pseudo-first-order kinetic model is expressed by the following equation
[150]:
ln(qe – qt) = lnqe – k1t (7-1)
where qe and qt refer to the adsorption capacity of Cu2+ (mg/g) at equilibrium and at any
time, t (min), respectively, and k1 is the rate constant of pseudo-first-order adsorption
(min-1). The slope of the linear plot of ln(qe – qt) versus t is used to determine k1 (figure
not shown).
The pseudo-second-order kinetic rate equation is expressed as follows [149]:
22
1 1
t e e
tt
q k q q (7-2)
where k2 is the rate constant of pseudo-second-order adsorption (g mg-1min-1). The
slope and intercept of the plot of t/qt versus t are used to calculate k2 and qe,cal (Figure 7-
4b). The corresponding kinetic parameters from both models are listed in Table 7-1. It
is found that the correlation coefficients (R2) for the pseudo-first-order kinetic model
are less than 90% indicating a poor pseudo-first-order fit to the experimental data. On
the other hand, the correlation coefficient (R2) for the pseudo-second-order adsorption
model has high value (> 99%), and the q values (qe,cal) calculated from pseudo-second-
order model are more consistent with the experimental q values (qe,exp) than those
calculated from pseudo-first-order model. These facts suggest that the adsorption data
are well represented by pseudo-second-order kinetic model. The pseudo-second-order
adsorption has also been reported for many adsorbents such as natural kaolinite clay
Chapter: 7
153
[254], H2SO4 modified chitosan [154], chitosan-coated MNPs modified with alpha-
ketoglutaric acid [245].
Figure 7-4 (a) Effect of contact time on Cu2+ adsorption by CMCD-MNP(C); (b) pseudo-second-order kinetics for adsorption of Cu2+. (Initial pH: 6, temperature: 25ºC).
0
10
20
30
40
0 50 100 150 200 250
q t(m
g/g)
t (min)
200 mg/mL100 mg/mL50 mg/mLpseudo-second-order kinetics
(a)
0
4
8
12
16
0 40 80 120 160 200 240 280
t/qt
t (min)
200 mg/mL
100 mg/mL
50 mg/mL
(b)
Chapter: 7
154
Table 7-1 Adsorption kinetic parameters of Cu2+ onto CMCD-MNP(C).
Initial conc. Co(mg/ml)
pseudo-first-order pseudo-second-order k1 (min-1) qe,cal
(mg/g)R2 qe,exp
(mg/g)k2
g/(mg min)qe,cal (mg/g) R2
50 0.0053 10.92 0.688 14.88 0.038 14.47 0.999 100 0.0082 20.45 0.641 23.48 0.027 23.25 0.999 200 0.0116 34.75 0.896 36.77 0.009 36.90 0.999
7.3.2.3 Adsorption isotherm of Cu2+ ions The equilibrium isotherms for the adsorption of copper ions by uncoated MNPs at 25oC
and by CMCD-MNP(C) at different temperatures (25oC, 40oC and 55oC) in pH 6 are
shown in Figure 7-5. The equilibrium data are fitted by Langmuir and Freundlich
isotherm equations [255]. The Langmuir equation can be expressed as:
1e e
e m m L
C C
q q q K (7-3)
where qe is the amount of adsorbed material at equilibrium (mg/g), Ce the equilibrium
concentration in solution (mg/l), qm the maximum capacity of adsorbent (mg/g), and KL
is the “affinity parameter” or Langmuir constant (l/mg). The linear form of Freundlich
equation, which is an empirical equation derived to model the multilayer adsorption,
can be represented as follows:
lnqe = (1/n) lnCe + lnKF (7-4)
where qe and Ce are defined as above, KF is Freundlich constant (l/mg), and n is the
heterogeneity factor.
Chapter: 7
155
Figure 7-5 Equilibrium isotherms for Cu2+ adsorption by CMCD-MNP(C) at different temperature and by uncoated Fe3O4 nanoparticles (inset) at 25ºC and initial pH 6.
The values of qm and KL are determined from the slope and intercept of the linear plots
of Ce/qe versus Ce (Figure 7-6a) and the values of KF and 1/n are determined from the
slope and intercept of the linear plot of lnqe versus lnCe (Figure 7-6b). The isotherm
parameters are shown in Table 7-2. Namely, in all temperature studied, the Langmuir
isotherm shows a better fit to experimental data in compared to the Freundlich isotherm.
Moreover, Table 7-2 shows that the Langmuir constant, KL, related to the affinity of
binding sites decreases with increasing temperature indicating that the adsorption is
favorable at lower temperatures. The maximum adsorption capacities (qm) for uncoated
MNPs and CMCD-MNP(C) are found to be 20.8 and 47.2 mg/g, respectively, at 25oC.
The qm for uncoated MNPs is found quite similar as done by Banerjee and Chen (19.3
mg/g) [41]. CMCD-MNP(C), on the other hand, could adsorb copper ions almost or
more than twice that by uncoated MNPs indicating that the modification of magnetite
surface by CM-β-CD which has multiple hydroxyl and carboxyl functional groups
could enhance the adsorption capabilities. Hu et al. [242] also found that introducing
0
5
10
15
20
25
30
35
40
45
0 100 200 300 400
q e (
mg/
g)
Ce (mg/ml)
25oC40oC55oCLangmuir fitting
0
5
10
15
20
25
0 100 200 300 400 500
q e
Ce
Chapter: 7
156
cyclodextrin onto the surface of MWCNT/iron oxide composites enhances the
adsorption capacity with Pb(II). Chen et al. [241] studied the adsorption of Eu(III) onto
CNT-iron oxide composite in presence of poly(acrylic acid) (PAA) as a surrogate for
natural organic matter and found that the presence of PAA could strongly enhance the
Eu(III) adsorption and adsorbed PAA anions can be considered as a ‘bridge’’ between
the magnetic composite and Eu(III). A positive effect of PAA on Ni2+ adsorption at
pH< 8 using MWCNT is also reported by Yang et al. [256]. The high complexation
ability of pre-adsorbed PAA onto oxidized MWCNT with Ni2+ contributes to enhanced
adsorption on the surfaces. The maximum uptake of Cu2+ by CMCD-MNP(C) obtained
in this work is compared with other results reported in literature shown in Table 7-3.
However, the adsorption capacity of CMCD-MNP(C) for copper ions is comparatively
higher than some of other magnetic particles based adsorbents.
Table 7-2 Adsorption isotherm parameters for Cu2+ ions adsorption on uncoated and CM-β-CD coated magnetic nanoparticles at pH 6.
Adsorbents T (oC)
Langmuir isotherm constants Freundlich isotherm constants
KL (l/mg)
qm (mg/g)
R2 KF (mg/g)(l/mg)
n R2
CMCD-MNP(C)
25 0.0237 47.20 0.995 7.064 3.15 0.973 40 0.0221 42.01 0.993 5.044 2.78 0.961 55 0.0202 38.75 0.992 4.002 2.57 0.993
Uncoated MNPs
25 0.0514 20.80 0.984 4.635 3.86 0.853
Chapter: 7
157
Table 7-3 Comparison of maximum adsorption capacity of CMCD-MNP(C) with those of some other magnetic adsorbents reported in literature for Cu2+ ions adsorption.
Adsorbents Maximum adsorbed amount, qm (mg/g)
References
Chitosan-coated Fe3O4 NPs modified with alpha-ketoglutaric acid
96.15 [108]
Glutaraldehyde modified Fe3O4 NPs (GA-APTES-NPs)
61.07 [124]
Calcium alginate modified Fe3O4 NPs 60.00 [125] CM-β-CD modified MNPs 47.20 This study Gum Arabic modified MNPs 38.50 [41] Amino functionalized Fe3O4@SiO2 core-shell NPs
29.85 [126]
Chitosan bound MNPs 21.50 [37] Uncoated Fe3O4 nanoparticles 19.30 [41] Amino-functionalized MNPs 12.43 [8] Carbon encapsulated MNPs 3.21 [127]
Figure 7-6 The Langmuir (a) and Freundlich (b) isotherm plots for Cu2+ ions adsorption by CMCD-MNP(C) at pH 6.
7.3.2.4 Effect of temperature and thermodynamic parameters
The effect of temperature on the adsorption isotherm was investigated under isothermal
conditions in the temperature range of 25-55ºC and at pH 6. As shown in Figure 7-5,
the adsorption capacities for CMCD-MNP(C) decrease with the increase in temperature
Chapter: 7
158
from 25oC to 55oC. Thermodynamic parameters such as change in free energy (∆Go),
enthalpy (∆Ho), and entropy (∆So) are calculated using the following thermodynamic
functions:
∆Go = -RT lnKp (7-5)
∆ ° ∆ ° (7-6)
KP is the thermodynamic equilibrium constant, i.e., the ratio of the equilibrium
concentration of Cu2+ on CMCD-MNP(C) to that in solution. This constant KP for the
sorption process is determined by plotting ln(qe/Ce) versus qe and extrapolating to zero
qe [257,258]. Figure 7-7 shows the van’t Hoff plots of lnKP versus 1/T (R2=0.99) and
the values of ∆Ho and ∆So for CMCD-MNP(C) are determined from the slopes and
intercepts, respectively. The calculated thermodynamic parameters are listed in Table 7-
4. The negative value of all ∆Go indicates the feasibility of the process and spontaneous
nature of metal ion adsorption onto magnetic nano-adsorbents. Moreover, the
magnitude of ∆G◦ decreases with increasing temperature indicating that adsorption is
not favorable at higher temperatures. The changes of enthalpy (∆Ho) and entropy (∆So)
at 25-55ºC could be determined to be -19.26 kJmol-1 and -0.0546 kJmol-1K-1,
respectively for CMCD-MNP(C). The negative value of ∆Ho confirms the exothermic
nature of adsorption which is also supported by the decrease in value of Cu2+ uptake of
the adsorbent with the rise in temperature. Similar results have been obtained for Cu2+
adsorption by cellulosic fibers modified by β-CD [259]. The negative entropy change
(∆So) for the process is caused by the decrease in degree of freedom by the adsorbed
species and indicates the stability of adsorption process with no structural change at
solid–liquid interface [154,260].
Chapter: 7
159
Figure 7-7 van’t Hoff plot for the adsorption of Cu2+ by CMCD-MNP(C).
Table 7-4 Thermodynamic parameters for the adsorption of Cu2+ ions onto CMCD-MNP(C).
∆Go (kJmol-1) ∆Ho (kJmol-1) ∆So (kJmol-1K-1)
298.15K 313.15K 328.15K -2.93 -1.99 -1.29 -19.26 -0.0546
7.3.3 Adsorption interactions
For understanding of the copper/ CMCD-MNP(C) interactions, FTIR data are analyzed.
Figure 7-8 shows the spectra of CMCD-MNP(C) nano-adsorbent before and after Cu2+
adsorption and the possible IR frequencies of CMCD-MNP(C) are listed in Table 7-5.
FTIR spectrum of metal (Cu2+)-sorbed CMCD-MNP(C) shows that the peaks expected
at 1030, 1155, 1635 and 3423 cm-1 have shifted, respectively to 1028, 1153, 1628 and
3406cm-1. The shifting of the peaks indicates that their corresponding functional groups
(mainly hydroxyl and carbonyl groups) may directly be involved in interaction with
metal to form surface complexes. Since Cu2+ adsorption on the magnetic nano-
adsorbent affected all the chemical bonds with oxygen atoms, it is reasonable to assume
that the oxygen atoms would be the main adsorption sites for Cu attachment. After Cu2+
0.2
0.4
0.6
0.8
1
1.2
1.4
0.003 0.0031 0.0032 0.0033 0.0034
lnK
p
1/T (K-1)
Chapter: 7
160
adsorption, a new sharp adsorption band at 1385cm−1 can also be observed which is
assigned to nitrate (NO3-) anion [261,262]. Hence, NO3
- ions are on the surface of
CMCD-MNP(C) to balance the electrical charges of the adsorbed copper ions [262]. In
addition, the peak of Fe-O has a slight shift from 582 to 586 cm-1 as some of metal ions
are adsorbed onto the iron oxide [263].
Figure 7-8 FTIR spectra of CMCD-MNP(C) before (a) and after (b) adsorption of Cu2+ ions.
Table 7-5 FTIR absorption frequencies (cm-1) of CMCD-MNP(C) before and after adsorption and their possible assignments
CMCD-MNP(C) (before adsorption)
CMCD-MNP(C) (after adsorption)
Assignment
582 586 υFe–O 947 946 Skeletal vibration involving α-1,4 linkage
1030 1028 υasC-O-C α-glycoside bridge 1080 1080 υC-C 1155 1153 υC-OH
- 1385 NO3-
1635 1628 υC=O (COO-) 2924 2924 υasC-H 3423 3406 υO-H
υ: stretching vibration, υas: asymmetric stretching vibration
400 800 1200 1600 2000 2400 2800 3200 3600 4000
% T
ran
smis
sion
Wavenumber, cm-1
10301080
1155947
1385582 10801028
11532924
2924
586
3423
3406
946
(a)
(b)
1635
1628
Chapter: 7
161
To further verify the findings from the FTIR results, XPS studies of the CMCD-
MNP(C) before and after Cu2+ adsorption are conducted. Figure 7-9 shows the high
resolution spectra of the C1s and O1s regions. Deconvolution of the C1s peak produces
four peaks of binding energies 284.6, 286.2, 287.9 and 288.7eV, respectively (before
and after Cu2+ adsorption) (Figure 7-9a and 7-9b). These peaks can be assigned to the
carbon atoms in the forms of C–C (aromatic), C–O (alcoholic hydroxyl and ether), C=O
(carbonyl) and COO- (carboxyl and ester) [264]. However, C1s XPS spectra do not
clearly show any significant changes of the CMCD-MNP(C) before and after Cu2+
adsorption which indicates that C atoms are not involved in chemical adsorption of
Cu2+ ions.
Deconvolution of the O1s peak yields three individual component peaks which come
from different groups and overlap on each other (Figure 7-9c and 7-9d). Peaks at 529.8
and 530.6eV (before and after Cu2+ adsorption) are attributable to the C=O functional
groups [264]. Peaks at 530.7 and 532.5eV (observed before Cu2+ adsorption) and at
532.0 and 533.7eV (observed after Cu2+ adsorption) are attributable to the C-O groups
(alcoholic hydroxyl and ether) and COO- groups, respectively. The shift in the binding
energies after Cu2+ adsorption is likely to be caused by the binding of copper ions onto
the oxygen atoms and thus reduces its electron density. Similar observation has been
reported by Lim et al. [263] and Zheng et al. [264]. The relative content of oxygen in
C–O (alcoholic hydroxyl and ether) is reduced (from 39.2% to 33.3%) and that in form
of C=O increases (from 32.7% to 46.5%) after copper adsorption. These changes,
together with binding energy shift, suggest that oxygen atoms are the main adsorption
sites and the Cu2+–O coordinate bonds are formed in Cu2+ adsorption by CMCD-
MNP(C), which is in good agreement with FTIR results.
Chapter: 7
162
Figure 7-9 XPS spectra of CMCD-MNP(C): (a) C1s before adsorption; (b) C1s after adsorption; (c) O1s before adsorption; and (d) O1s after adsorption; (e) Cu(2p3/2) core-level spectrum of Cu-loaded CMCD-MNPs.
Figure 7-9e shows the Cu(2p3/2) core-level spectrum of the CMCD-MNP(C) with
adsorbed copper ions at pH 6.0. The binding energy of the copper at 935.1eV and the
presence of a satellite band nearby is representative of the oxidation state (+2) of copper
Chapter: 7
163
for the Cu(2p3/2) orbital [265,266]. This indicates the existence of Cu (i.e., Cu2+) being
adsorbed on the surface of the magnetic nano-adsorbents.
7.3.4 Desorption and regeneration studies
From practical point of view, repeated availability is a crucial factor for an advanced
adsorbent [155,255,267]. Such adsorbent has higher adsorption capability as well as
better desorption property which will reduce the overall cost for the adsorbent. To
evaluate the possibility of regeneration and reusability of CMCD-MNP(C) as an
adsorbent, we performed the desorption experiments. Desorption of Cu2+ from CMCD-
MNP(C) were demonstrated using three different eluent buffers, namely 0.1M citric
acid, 0.1M Na2EDTA and 0.1M acetic acid. Below pH 2, CMCD-MNP(C) tends to be
unstable and have a tendency to disintegrate. It is found that the quantitative desorption
efficiencies using citric acid, Na2EDTA and acetic acid were 96.2, 89.4 and 66.8%,
respectively. It is also noted that no obvious Fe ions were observed in the desorbent
solutions after desorption, an indication that there was no dissolution of Fe3O4
nanoadsorbents during the desorption process.
The reusability was checked by following the adsorption–desorption process for three
cycles and the adsorption efficiency in each cycle was analyzed. In each cycle, 120 mg
of CMCD-MNP(C) and 10 ml of an initial Cu2+ concentration of 200 mg/l was used for
the adsorption process, and 10 ml of 0.1M citric acid was used as desorption eluent. The
desorption kinetics of Cu2+ from Cu-loaded nanoparticles and the adsorption and
desorption capacity of CMCD-MNP(C) is presented in Figure 7-10. It is observed that
the desorption reaches equilibrium within 1 h using 0.1 M citric acid solution as
desorption eluent. Cu2+ adsorption capacity of CMCD-MNP(C) remains almost
constant for the three cycles, indicating that there are no irreversible sites on the surface
of CMCD-MNP(C). Recently Hu et al. [242] reported that β-cyclodextrin grafted
Chapter: 7
164
multiwalled carbon nanotubes/iron oxides (MWCNTs/iron oxides/CD) can be recycled
five times for Pb(II) adsorption with less than 5% of decline in efficiency, indicating
good reusability of the adsorbents. Our recyclability studies suggest that theses nano-
adsorbents can be repeatedly used as efficient adsorbents in wastewater treatment.
Figure 7-10 (a) Kinetics of desorption of Cu2+ from Cu-loaded CMCD-MNP(C) in 0.1M citric acid solution; (b) performance of CMCD-MNPs by multiple cycles of regeneration.
7.4 Conclusions
In this study, we have successfully synthesized a magnetic novel nano-adsorbent
comprising Fe3O4 nanoparticles modified with CM-β-CD having magnetic properties
0
20
40
60
80
100
0 20 40 60 80 100 120 140 160
% d
esor
pti
on
Time (min)
(a)
0
10
20
30
40
1 2 3
q e(m
g/g)
No. of Cycles
Adsorption Desorption(b)
Chapter: 7
165
exhibited by magnetite and adsorption properties of CM-β-CD. Grafting of CM-β-CD
onto the magnetic nano-adsorbents is confirmed by FTIR, TGA, and XPS analyses.
These magnetic nano-adsorbents were used to effectively remove Cu2+ from aqueous
solution. The solution pH greatly affects the adsorption of Cu2+ onto CMCD-MNP(C).
Adsorption of Cu2+ reaches equilibrium within 30 min and 90% of Cu2+ is being
adsorbed at 15 min. The kinetics of Cu2+ adsorption follows the pseudo-second-order
model. The equilibrium data are fitted well by the Langmuir model of adsorption. The
maximum adsorption capacity of CMCD-MNP(C), determined to be 47.2 mg/g, is
considerably higher than that of some other reported magnetic adsorbents. Furthermore,
the adsorption process is spontaneous and exothermic in nature. Both FTIR and XPS
analyses clearly reveal that the oxygen atoms on CMCD-MNP(C) are the main binding
sites for Cu2+ to form surface-complexes. In addition, the copper ions were desorbed
effectively from CMCD-MNP(C) by treatment with citric acid and Na2EDTA solution.
Results of this work suggest that the CMCD-MNP(C) may be promising adsorbents for
the removal of heavy metal ions from wastewater using the technology of magnetic
separation.
Chapter: 8
166
Chapter 8 Multifunctional core-shell silica nanoparticles with magnetic, fluorescence, specific cell targeting and drug-inclusion functionalities
8.1 Introduction
In recent years, the advent of multifunctional nanomaterials attracted enormous interest
of biomedical research community [268]. Rational combination of diverse building
blocks such as metallic or semiconductor nanomotifs, organic molecules/polymers,
biomolecules give rise to composite nanomaterials which exhibit a wide range of
diagnostic and therapeutic capabilities; bioimaging, specific cell targeting, drug storage
and controlled drug release etc [269,270]. Magnetic nanoparticles evidently are
becoming prominent inorganic building blocks of such multifunctional nanocomposites.
Because, colloidal magnetic nanoparticles (MNPs), typically iron oxide (Fe3O4), exhibit
superparamagnetism which can respond to external magnetic field. The fact that these
superparamagnetic materials do not retain any magnetization after removal of the
external magnetic field, and thus avoid aggregation, is a clear advantage for in vivo
applications [271]. MNPs can be magnetically guided inside a biological system, e.g,
artery or tumor by an external magnetic field [272] and tracked by magnetic resonance
imaging (MRI) [273]. Moreover, a metal/semiconductor/polymer coating on the
magnetic particles enhances their stability, biocompatibility, as well as expands
applicability by integrating more functionalities via physical and/or chemical methods.
A shell of silica around MNPs, known as Fe3O4@SiO2 core-shell, constitute the basic
architecture of most multifunctional nanocomposites. Because silica has low-toxicity,
less prone to degradation in a biological environment, plus numerous chemical methods
exist to functionalize silica shell with molecular, biomolecular and polymeric ligands
for biomedical purposes [274,275]. To date most of the magnetic core-shell
Chapter: 8
167
nanocomposites exhibit typically two or a maximum of three functionalities [275,276]
by combining the magnetic core a fluorescent label [277,278] or quantum dot
[279,280], cell targeting ligand [278,280,281] or drugs [282], however little attempt has
been made to incorporate four functionalities: magnetism, fluorescence, selective cell-
targeting and drug storage-delivery in one single platform was made [283,284]. We
present a novel core-shell nanocomposite which has such quadruple of functionalities
[36].
We rationally design the bottom-up synthesis of a core-shell nanocomposite by
stepwise coating of Fe3O4 magnetic crystal (~10 nm) with layers of silica shells for
colloidal protection, dye-doping and molecular fictionalization (Figure 8-1).
Microemulsion sol-gel chemistry was opted to construct the first layer of silica gel to
obtain Fe3O4@SiO2 core-shell structure. We then overlay a Fluorescent dye-conjugated
(FITC) silica layer to optically track the nanocomposite for any cellular uptake by
fluorescence imaging. FITC is chemically encapsulated into the silica shell to enhance
photochemical stability [270] and fluorescence intensity due to “caging effects” [285].
Being both magnetic and fluorescently labeled these materials have the potential for
bimodal imaging by optical and magnetic resonance means [284,286,287].
For a nanoparticulate delivery device for selective drug delivery to specific sites, the
challenge lies in successfully combining therapeutic and targeting functionalities along
with imaging capability into one single nano-system. To this end, the outermost layer of
silica is furnished with a cancer cell targeting ligand and an interesting ‘molecular cage’
for drug storage. We covalently conjugated folic acid to the amino-termini on the outer
silica shell. Folic acid or folate (FA) has emerged as an attractive cancer-targeting
ligand because cancerous cells overexpress high-affinity FA receptors (FARs) [288].
Chapter: 8
168
Thus FA-conjugated nanomaterials show preference towards malignant cells than the
healthy ones.
In order to armor our nanocomposite with a drug storage and delivery vehicle we finally
conjugated β-cyclodextrin molecules onto the shell. In the magnetic core-shell
literature, mesopores in the silica shell are used as the physical reservoir for storing
drug molecules [36,284]. However, in most cases these magnetic-mesoporous silica
nanocomposites can carry only hydrophilic drugs, because the mesopores of silica can
accommodate only water-soluble drug molecules [289-291]. Since the majority of
potent anticancer drugs are water-insoluble, applications of silica nanoparticles for
anticancer drugs delivery have been largely restricted. Unlike physically entrapping
drug molecules into the mesopores of silica, we relied on the supramolecular inclusion
of drug molecules into the doughnut-shaped β-cyclodextrin (β-CD) cavity. β-CD
molecule has a hydrophilic exterior while the internal cavity is sufficiently hydrophobic
to host water-insoluble drug molecules [292,293]. As a drug carrier, β-CD not only
facilitates solubilization, stabilization, and transport of hydrophobic drugs but also
minimizes unwanted side effects associated with the corresponding drugs
[60,117,294,295]. Several studies have been directed towards constructing MNP-
cyclodextrin nanocomposites as magnetically guided drug carrier [202,296]. However,
the agglomeration of these as-synthesized magnetic nanocarriers as evidenced by TEM
may limit their applications in biomedical research fields.
The feasibility of our magnetic nanocomposite as an optically trackable, cancer-cell
targeting and delivery vehicle for anticancer drugs is evaluated by investigating their
capability in discriminating between cancerous and healthy cells, cellular uptake,
fluorescent imaging, formation of hydrophobic drug-inclusion complexes and the in
vitro release profile using all-trans-retinoic acid (RA) as a model hydrophobic drug.
Chapter: 8
169
After combining superparamagnetic, luminescent, specific cell-targeting and
hydrophobic drug-encapsulation properties, these multi-functional nanoparticles pose
great potentials as a smart nano ‘Swiss army knife’ in diverse theranostic applications
[297,298].
8.2 Experimental 8.2.1 Materials
Iron (II) chloride tetrahydrate (99%), Iron (III) chloride hexahydrate (98%), 3-
aminopropyltriethoxysilane (APTS) (98+%), all-trans-retinoic acid (RA) (98%),
chloroacetic acid (99%) and N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide
hydrochloride (EDC) was purchased from Alfa Aesar. Triton X-100 and N-
hydroxysuccinimide (NHS) (98%) were purchased from Sigma-Aldrich. Ammonium
hydroxide (25%) was purchased from Merck (USA). 1-hexanol (>98%), oleic acid
(99%) and tetraethyl orthosilicate (TEOS) (99%) was purchased from Fluka. β-
cyclodextrin hydrate (99%) was obtained from Sinopharm Chemicals. Folate free
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and 3-[4,5-
dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide were purchased from Sigma-
Aldrich. All other chemicals were used purchased from either Sigma-Aldrich or Fisher
Scientific and received without further purification. All the experiments were
performed in deionized Milli-Q water.
8.2.2 Synthesis of multifunctional core-shell silica magnetic nanoparticles
8.2.2.1 Preparation of oleic acid coated Fe3O4 MNPs (OA-MNPs)
Oleic acid stabilized Fe3O4 MNPs were prepared according to the method described by
Lopez-Lopez et al. [299]. Briefly, 3.04 g FeCl3.6H2O and 1.26 g FeCl2.4H2O were
dissolved in 50 ml DI water under nitrogen and vigorous stirring with ultrasonification
Chapter: 8
170
for 10 min. 0.8 ml of oleic acid was then added immediately after 8 ml ammonium
hydroxide (NH4OH, 25 wt%) had been added to the solution, and the resulting emulsion
was stirred vigorously for 1 h at 25ºC. Then the solution was heated up to 95 ºC to
convert iron hydroxides into magnetite. As soon as that temperature was reached, the
suspension was cooled to room temperature. The mixture was then acidified to pH 5–6
with HNO3. The resulting particles were collected by magnetic decantation, washed 4-5
times with DI water to remove the unreacted reactants, then washed with ethanol 3
times to remove water and residual oleic acid. Finally the particles were dried in
vacuum oven for 3 h.
8.2.2.2 Synthesis of Fe3O4@SiO2(FTIC) NPs
Typically, 12 mg of dried magnetic Fe3O4 powder was dissolved first in 77 ml
cyclohexane and then 20 g triton X-100, 16 ml hexanol, and 3.4 mL H2O were added
with intense stirring to generate the microemulsion system [34]. 0.4 ml of tetraethyl
orthosilicate (TEOS) was added to this mixture. After 6 h of stirring, 1.2 ml aqueous
ammonia (25 wt %) was introduced drop wisely to initiate the TEOS hydrolysis. The
microemulsion was kept stirring for another 24 h, and then N-1-(3-
triethoxysilylpropyl)-N’-fluoresceyl thiourea (FITC-APTS) ethanolic solution and
TEOS (0.3 ml) was added to the microemulsion. FITC-APTS conjugate was prepared
by adding 1.25 mg FITC with 0.15 ml APTS in 0.2 ml absolute alcohol and stirring in
dark for 24 h in room temperature. For FITC-APTS conjugate synthesis, an excess
amount of APTS (molar ratio of APTS to FITC =20) was used to ensure complete
conversion of FITC to FITC-APTS conjugates. After stirring in dark for 24 h at room
temperature, ethanol was added to destabilize the microemulsion system. The resulting
MNPs were collected by magnetic separation and washed 5 times by anhydrous ethanol
Chapter: 8
171
and then by deionized water to remove surfactant and unreacted reactants and dried by
air.
8.2.2.3 Synthesis of Fe3O4@SiO2(FTIC)-FA/NH2 NPs
To attach folic acid (FA) to the Fe3O4@SiO2(FTIC) MNPs surface, FA-APTS
conjugate was prepared first. In a flask, 2 mg of folic acid and 1 µL of APTS were
mixed in 20 ml of DMSO. Next, 0.53 mg of N-hydroxysuccinimide (NHS) and 2.35 mg
of N,N'-dicyclohexylcarbodiimide (DDC) (FA: NHS: DCC = 1: 1: 2.5) were added into
the mixture and stirred for 2 h. In a separate flask containing 80 ml of toluene and the
Fe3O4@SiO2(FTIC) MNPs (50 mg) in DMSO suspension, the FA-APTS conjugate and
free APTS (7 µL) (total APTS/FA molar ratio = 8) solution was added, and the mixture
was stirred for 20 h at room temperature. The materials were recovered by magnetic
separation, washed twice with toluene, then ethanol and dried under vacuum.
8.2.2.4 Synthesis of Fe3O4@SiO2(FTIC)-FA/CMCD NPs
CM-β-CD was grafted onto Fe3O4@SiO2(FITC)-FA/NH2 NPs in the presence of EDC
and NHS. Detailed synthetic procedure for CM-β-CD is given in Chapter 5 (Section
5.2.2). A solution of CM-β-CD (0.65 g, 0.5 mmol) in 20 ml of distilled water was first
activated with 0.96 g (0.5 mmol) of EDC and 0.575 g (0.5 mmol) of NHS for 30 min.
With this mixture, 100 mg of dried Fe3O4@SiO2(FITC)-FA/NH2 was added and the pH
of the reaction mixture was adjusted to 6~7. The reaction was carried out at room
temperature under constant stirring. After 24 h, the product formed was separated by
magnetic decantation and dried by vacuum oven.
Chapter: 8
172
8.2.3 Cell culture and intracellular uptake study The HeLa cells were grown in DMEM (Dulbecco’s modified Eagle’s medium)
supplemented with 10% FBS (fetal bovine serum) at 37oC and 5% CO2. The MCF-7
cells were grown in DMEM supplemented with 10% FBS and 1% insulin (10 ml: 400
U) at 37oC and 5% CO2. Cells (5 x108/ L) were plated on 14 mm glass cover slips and
allowed to adhere for 24 h. Subsequently, after washing with phosphate buffer solution
(PBS), the cells were incubated in PBS buffer containing 50 µg/ml Fe3O4@SiO2(FTIC)-
FA/CMCD MNPs at 37oC for 1 h under 5% CO2, and then washed with PBS
sufficiently to remove excess nanoparticles. Nanoparticle uptake by HeLa and MCF-7
cells was studied by confocal microscopy methods. Confocal microscopy images of the
cells were acquired with a 488 nm laser on a Leica SP2 confocal laser scanning
microscope.
8.2.4 Cytotoxicity assay
In vitro cytotoxicity was evaluated by performing methyl thiazolyl tetrazolium (MTT)
assays on the HeLa cells. Cells were seeded into a 96-well cell culture plate at 5x104
per well in DMEM with 10% FBS at 37oC and 5% CO2 for 24 h; then the cells were
incubated with Fe3O4@SiO2(FTIC)-FA/CMCD NPs with different concentrations (0, 1,
10, 20, 30, 50 µg/ml diluted in DMEM) for 6 h or 24 h at 37oC under 5% CO2.
Untreated wells were used as control. Thereafter, MTT (10 µl, 5 mg/ml) was added to
each well, and the plate was incubated for 4 h at 37oC. The assays were carried out
according to the manufacturer’s instructions. The OD570 value (Abs.) of each well,
with background subtraction at 690 nm was measured by microplate reader (GENios,
Tecan, Switzerland).
Chapter: 8
173
8.2.5 Inclusion/adsorption of retinoic acid The inclusion of retinoic acid was performed by immersing precisely weighed amount
of Fe3O4@SiO2(FTIC)-FA/CMCD MNPs (about 0.12 g) in 10 ml methanol solution of
retinoic acid (0.2 mg/ml concentrations), and stirred with a thermostatic water-bath
shaker (200 rpm) for 12 h at room temperature. The magnetic nanoparticles were then
removed magnetically from the solution. The concentration of retinoic acid solution
was determined by using a spectrophotometer at 341 nm. The amount of included
retinoic acid (A) by Fe3O4@SiO2(FTIC)-FA/CMCD MNPs was calculated as follows:
A = V (C0–C1) (8-1)
where V is the volume of RA solution (ml), C0 is the initial concentration of RA
(mg/ml), C1 is the concentration of RA solution after inclusion or absorption (mg/ml).
8.2.6 Release study The drug release experiment was performed in an Erlenmeyer flask at 37oC in PBS
buffer (pH 7.4). 120 mg of Fe3O4@SiO2(FITC)-FA/CMCD NPs containing a known
amount of retinoic acid were suspended in 250 ml of release medium, stirred at 150 rpm
in a thermostatic water-bath heater maintained at 37oC. 5 ml of samples were
periodically removed and assayed. The volume of each sample withdrawn was replaced
by the same volume of fresh medium. The amount of released retinoic acid was
determined at 341 nm using a UV-Visible spectrophotometer. The drug release study
was performed in duplicate for each of the samples. To prevent decomposition of RA
during the release experiment, the samples were kept in dark.
Chapter: 8
174
8.3 Results and discussions
8.3.1 Synthesis and characterization of as-synthesized magnetic nanoparticles
The synthetic route for multifunctional core-shell magnetic nanoparticles
[Fe3O4@SiO2(FITC)-FA/CMCD NPs] is outlined in Figure 8-1. Fluorescently-doped
core-shell structure Fe3O4@SiO2(FITC) was obtained by a one-pot two-step method. In
the first step, oleic-acid-stabilized Fe3O4 MNPs was coated with a layer of silica to
make the basis of the core-shell structure. Fe3O4 MNPs were dispersed in a water-in-oil
(cyclohexane-hexanol) microemulsion prior to the sol-gel formation of the silica layer
onto the magnetite surface. In the second step, this emulsion was further treated with a
mixture of FITC-APTS conjugate and free APTS overlay thin FITC-doped silica on the
primary shell. The purpose of the primary silica shell is to protect the core Fe3O4 MNP.
As the dye molecules are incorporated into the second silica layer, any possibility of
FITC degradation or fluorescence quenching by Fe3O4 is minimized. Secondly,
encasing FITC within a thin layer of condensed APTS-layer would provide
photostability and additional chemical protection in the cellular environment. The
outermost layer of Fe3O4@SiO2(FITC) was then decorated with a cancer targeting
ligand, folate by silanization with folic acid-APTS conjugate. A fair excess of APTS
(molar ratio of APTS to FA = 10) was used to ensures sufficient free amino groups
dangling out from the outer surface for further functionalization. CM-β-CD, the
molecular host from hydrophobic drugs was attached onto the outermost surface via
amide bond formation between the carboxylic groups of CM-β-CD with free amino
groups.
Chapter: 8
175
Figure 8-1 Synthesis of CM-β-CD conjugated fluorescein-doped magnetic mesoporous silica nanoparticles [Fe3O4@SiO2(FITC)-FA/CMCD NPs].
Physical and chemical features of the core-shell multifunctional particles are
investigated by several analytical methods. The XRD pattern (Figure B-1 in Appendix
B) confirmed the existence of Fe3O4, and a broad peak ranging from 20˚ to 30˚ was
assigned to the amorphous silica. This indicates that silica is successfully coated onto
the Fe3O4 MNP surfaces. However, silica shell did not result in the phase change of
Fe3O4 nanoparticles. Further investigation with the FTIR spectra confirms the presence
of the silica shell and also the incorporation of APES-FITC conjugate in the silica
matrix (Figure B-2 in Appendix B). The sample exhibits a strong broad band at around
1090 cm-1 corresponding to the Si-O-Si asymmetric stretching vibration and a weak
band at 956 cm-1 associated to Si–OH vibration [288]. The bands at 1630 cm-1 and 1560
cm-1 are the bendings of N–H, characteristic of the presence of NH2 group. The peak at
Chapter: 8
176
2938 cm-1 due to the C–H stretching vibrations of alkyl groups. The magnetization
curve of Fe3O4@SiO2(FITC)-FA/CMCD NPs measured at 300 K exhibits no hysteresis
loop, which means that these multifunctional nanocomposites retain the
superparamagnetic property (Figure B-3 in Appendix B).
The successful attachment of fluorescent probe molecule (FITC) and cell-targeting
ligand (folate) into/onto the silica shell was investigated by UV-Vis spectroscopic
analyses (Figure 8-2). UV-vis absorption spectra of all the samples were measured in
aqueous medium. Fe3O4@SiO2 nanocomposites do not show any prominent absorption
peaks. Characteristic absorption bands for FITC and folate, at 488 and 280 nm,
respectively, are clearly visible in the case of Fe3O4@SiO2(FITC)-FA/NH2 NPs and are
not blue/red shifted due to the integration inside/on a silica matrix (when compared
with UV absorptions for free FITC and FA). Since FITC-APES conjugate was
covalently linked to the silica matrix, no dye leaking was observed in an aqueous
suspension.
Figure 8-2 UV–vis spectra of (a) pure FITC, (b) pure folic acid, (c) Fe3O4@SiO2, (d) Fe3O4@SiO2(FITC), and (e) Fe3O4@SiO2(FITC)–FA/NH2 NPs in water.
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The incorporation of FITC dye in Fe3O4@SiO2(FITC)-FA/CMCD NPs enabled the
nanoprobes with excellent fluorescent property in water (Figure 8-3). The emission
spectrum of the dye-doped multifunctional NPs shows a broad band ranging from 490
to 580 nm with a maxima at 515 nm. Fe3O4@SiO2(FITC)-FA/CMCD nanoprobes and
pure FITC dye in water have similar excitation and emission spectra (not shown here),
so the fluorescent characteristics of FITC dye did not alter after incorporation into a
silica-shell. The spectral properties (excitation and emission spectra) of
Fe3O4@SiO2(FITC)-FA/CMCD NPs were comparable with FITC dye-doped magnetic
silica nanoparticles in the literature [270,284]. Inset picture in Figure 8-3 shows
dispersions of these nanoparticles under room light and under the 365 nm UV
excitation. Clearly, these core-shell MNPs are highly dispersible in water and emit
strong green fluorescence under 365 nm UV excitation. Facile magnetic separation is
evident from its magnetic responsivity towards a permanent NdFeB magnet. Moreover,
after being harvested by an external magnet, absence of any fluorescence in the aqueous
part confirms that FITC is robustly embedded into the silica shell by chemical bonding
and does not leach out.
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Figure 8-3 Excitation (dashed line, recorded at 515 nm emission) and emission (solid line, recorded at 480 nm excitation) spectra of Fe3O4@SiO2(FITC)-FA/CMCD NPs dispersed in water. Insets: visual photographs of (a) Fe3O4@SiO2(FITC) NPs dispersion in water under room light; (b) the green emission of these MNPs in water and (c) in response to a NdFeB magnet under the 365 nm excitation portable UV lamp.
High resolution TEM images of as-prepared multifunctional Fe3O4@SiO2(FITC)-
FA/CMCD NPs show that highly monodispersed particles with mean diameter 70 nm
(silica shell thickness = 30 nm) are formed (Figure 8-4). Moreover, a thin ring on the
outer surface can be observed which may be due to the surface organic group [34]. For
each core-shell particle a singular oleic acid coated Fe3O4 particle, rather than a cluster,
is encased within a thick layer of silica shell. Oleic acid modified bare Fe3O4 MNP
nanoparticles have an average diameter of 9.5 nm. The synthesized highly
monodispersed core-shell silica nanoparticles is achieved by microemulsion-based sol-
gel growth of silica in limited domain of water in the water-in-oil microemulsion. It can
be assumed that aqueous microdroplets in the microemulsion are monodisperse in size
and contain a single Fe3O4 MNP particle around which a well-defined shell of silica is
grown through hydrolysis of tetraethyl orthosilicate (TEOS) and subsequent
condensation of silica onto the surface of Fe3O4 cores. The silica layer can be tuned by
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varying the ratio of water to surfactant (Figure B-4 in Appendix B). A thick silica shell
was preferred in this case since a thicker silica shell allowed high payloads of dye
molecules to be achieved. The uniformity of as-synthesized particles could be attributed
to the microemulsion method, which allows better control over the particle size and size
distribution.
Figure 8-4 TEM mages of (a) OA-MNPs, (b) Fe3O4@SiO2(FITC) NPs and (c) Fe3O4@SiO2(FITC)-FA/CMCD NPs.
The stepwise conjugation of functional groups on the silica surface was monitored by
measuring the surface charges and surface chemistry at different stages of synthesis
(Figure 8-1). As shown in Figure 8-5A, a negative zeta potential (ξ) value of -27 mV (at
pH 7.4) of dye-doped silica particles [Fe3O4@SiO2(FITC) NPs], can be attributed to the
presence of ionizable silanol groups of surface silica and acid functionalities (COOH
and phenolic-OH) of FITC molecules [300]. After modification with APTS/FA
conjugate, the ξ value of Fe3O4@SiO2(FITC)-FA/NH2 nanoparticles reverts to positive
value (ξ ~ +4.0 mV at pH 7.4) due to the presence of excess amount of amino groups
which exist mainly as NH3+. Folic acid (FA) is expected to furnish silica surface with
negative charge, however excess amount of NH2 groups presumable nullify it, making
the overall surface charge slightly positive. Further modification of the surface by
conjugating cyclodextrin groups is manifested in the reversal of surface charge back to
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a negative value -20 mV attributable to the high ionizability of CM-β-CD (pKa< 4)
groups. As a result, Fe3O4@SiO2(FITC)-FA/CMCD NPs were observed to be well
dispersed in aqueous medium without any visible aggregation (insets of Figure 8-3).
Good aqueous dispersibility and colloidal stability of the nanoparticles are essential for
their biological applications. Dynamic light scattering (DLS) analyses suggest long-
term colloidal stability over a few day time and narrow size distribution (hydrodynamic
diameter = ca. 125 nm and polydispersity index = 0.20) (Figure B-5 in Appendix B).
Figure 8-5 (A) Zeta potentials of as-synthesized nanoparticles at pH 7.4; (B) TGA curves of (a) Fe3O4@SiO2(FITC)-FA/NH2 and (b) Fe3O4@SiO2(FITC)-FA/CMCD MNPs.
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High density grafting of cyclodextrin molecules onto the outermost silica surface is
desirable as these are the host for hydrophobic drug molecules. TGA curves (25–800oC)
in Figure 8-5B shows the weight loss due to organics decomposition in both
Fe3O4@SiO2(FITC)-FA/NH2 and Fe3O4@SiO2(FITC)-FA/CMCD NPs; a comparison
of two TGA curves revealed an increased weight loss for Fe3O4@SiO2(FITC)-
FA/CMCD NPs, presumably due to the presence of substantial amount of CM-β-CD.
The amount of CM-β-CD grafted on the surface was calculated to be 0.034 mmol/g.
The average values of surface concentration obtained from XPS spectra of
Fe3O4@SiO2(FITC), Fe3O4@SiO2(FITC)-FA/NH2 and Fe3O4@SiO2(FITC)-FA/CMCD
are presented in Table 8-1. The elemental composition percentage of Si and N atom was
34.95% & 2.48% on the surface of Fe3O4@SiO2(FITC)-FA/NH2 and was 29.57% &
2.03% on the surface of Fe3O4@SiO2(FITC)-CMCD, respectively. The decreased
percentages may be attributed to the lack of Si and N in CM-β-CD moiety. In addition,
percentage of C atom was observed to increase from 16.8% to 23.3% after conjugation
of CM-β-CD. Therefore, XPS analysis together with the results of TGA confirmed the
successful grafting of CM-β-CD on the multifunctional magnetic surface.
Table 8-1 Surface composition of Fe3O4@SiO2(FITC), Fe3O4@SiO2(FITC)-FA/NH2 and Fe3O4@SiO2(FITC)-FA/CMCD nanoparticles obtained from XPS spectra.
Samples Surface elemental composition percentage (%)
C 1s O 1s Si 2p N 1s Fe3O4@SiO2(FITC) 15.62 46.90 36.46 1.02 Fe3O4@SiO2(FITC)-FA/NH2 16.80 45.76 34.95 2.48 Fe3O4@SiO2(FITC)-FA/CMCD 23.23 45.17 29.57 2.03
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8.3.2 Cytotoxicity assay After successful synthesis and full physical and chemical characterizations biomedical
relevance of these multifunctional core-shell particles was investigated. The
cytotoxicity of the Fe3O4@SiO2(FITC)-FA/CMCD MNPs was evaluated using an MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay with HeLa cells,
a human cervical carcinoma cell line. The concentration-dependent effect of
Fe3O4@SiO2(FITC)-FA/CMCD MNPs on the cell viability at 6 and 24 h was
determined (Figure 8-6). It is clear that the high cell viability can still be attained even
at a MNP concentration of 50 µg/ml. This indicates that the Fe3O4@SiO2(FITC)-
FA/CMCD MNPs is biocompatible at MNP concentrations up to 50 µg/ml.
Figure 8-6 In vitro cell viability of HeLa cells incubated with Fe3O4@SiO2(FITC)-FA/CMCD MNPs at different concentrations for 6 and 24 h at 37oC.
8.3.3 Confocal microscopy observations We carried out fluorescence imaging of Fe3O4@SiO2(FITC)-FA/CMCD NPs by for
their ability to discriminate between healthy and cancerous cells and cellular uptake.
After 1 hr incubation of HeLa and MCF-7 cells with MNPs at 37oC, cells were
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observed using confocal laser scanning microscope (CLSM) (Figure 8-7).
Corresponding bright field images of the cells were also recorded. A clear preference
for the cancerous cells (HeLa) by these MNPs over the healthy cells (MCF-7) is
demonstrated in Figure 8-7. Cytoplasm of all the HeLa cells (Figure 8-7A) is highly
fluorescent due to the presence of FITC-tagged MNPs, while most of MCF-7 cells are
either less or non-invaded by MNPs (by comparing dark and bright field images) under
similar conditions. This suggests that the Fe3O4@SiO2(FITC)-FA/CMCD NPs were
preferentially targeted to the cancer cells and were internalized, presumably by folate
receptor mediated endocytosis [281,288]. These results suggest that
Fe3O4@SiO2(FITC)-FA/CMCD NPs could be used for simultaneous targeting and
imaging of cancerous cells.
Figure 8-7 CLSM images of cells incubated with Fe3O4@SiO2(FITC)-FA/CMCD NPs at 37oC: (A) HeLa cells (FA-positive) and (C) MCF-7 cells (FA-negative). Bright field images of HeLa cells (B) and MCF-7 cells (D) were also supplied. The nanoparticle concentration is 50 µg/ml and the incubation time is 1 h.
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8.3.4 Drug inclusion/adsorption and release studies
Since Fe3O4@SiO2(FITC)-FA/CMCD NPs are capable of preferentially target and
invade cancerous cells, use of these particles as an anti-cancer drug carrier would be
ideal for tumor-specific drug delivery. In this direction, we investigated drug loading
capacity of these MNPs which are already decorated with well-known molecular drug
carrier: β-cyclodextrin. We chose Retinoic acid (RA) as a model hydrophobic anti-
cancer drug. In order to get an idea about supramolecular interactions between RA and
free CM-β-CD molecules in solution, host-guest type complex formation was
investigated by UV-vis spectroscopy. Figure 8-8 shows the UV-vis spectra of RA
(7.3x10–6 mol l–1) in 10 vol% methanol solutions (pH 7.4) containing various
concentrations of CM-β-CD. Previous studies showed that methanol even up to 20
vol% does not influence the inclusion of cyclodextrin with other guest molecules [301].
The UV absorption peak is shifted to shorter wavelengths with an increase in the CM-β-
CD concentrations, accompanied by the increase in absorbance. This indicates the
formation of the RA/CM-β-CD inclusion complexes. Similar inclusion behaviors of
cyclodextrin with various drugs by UV-Vis spectroscopy have been reported in
literature [302,303].
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Figure 8-8 (a) Absorption spectra of RA (7.3x10–6 mol l–1) in pH 7.4 buffers containing various concentrations of CM-β-CD. Concentration of CM-β-CD: (1) 0, (2) 7.5x10–5, (3) 1.1x10–4, (4) 1.6x10–4, (5) 2.3x10–4, (6) 5.6x10–4 and (7) 3.75x10–3 mol l–1. Inset: Double reciprocal plot for RA complexes with CM-β-CD.
The inclusion constants of complexes are estimated according to the double-reciprocal
method as in Eq 8-2.
0][
111
CDKA
(8-2)
where ∆A denotes the difference of absorption of guest molecule in the presence and
absence of CDs. α is a constant, and [CD]0 denotes the concentration of CDs. K can be
calculated from a plot of 1/∆A vs. 1/[CD]. Inset of Figure 8-8 shows the double
reciprocal plots of 1/∆A versus 1/[CD] for RA with CM-β-CD at pH 7.4. The plot
exhibits good linearity (the linear regression coefficient R2=0.998). This verifies the
formation of inclusion complexes with a stoichiometry of 1:1 between RA and CM-β-
CD. The relative inclusion constant of CM-β-CD with RA is estimated to be 779 M-1.
The lipophilic character of RA provides the main driving force for their inclusion into
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the apolar CDs cavities. Literature values for CD-drug inclusion constant varies from
100 to 10,000 M−1 [304]. For therapeutic applications, a moderate value for K is
desirable that ensures thermodynamic feasibility for drug inclusion in the drug loading
stage, while at the same time does not impede drug release when needed [305].
Figure 8-9 shows the capabilities of Fe3O4@SiO2(FITC)-FA/CMCD and
Fe3O4@SiO2(FITC)-FA/NH2 NPs for RA loading in methanol solution. The effects of
adsorbent dosage on RA adsorption/inclusion were studied. It was obvious that
Fe3O4@SiO2(FITC)-FA/CMCD NPs exhibited a considerable adsorption capability for
RA, but very less retinoic acid loading by Fe3O4@SiO2(FITC)-FA/NH2 MNPs was
observed. This revealed that the CM-β-CD grafted on the Fe3O4@SiO2(FITC)-FA/NH2
NPs could indeed enhance the adsorption of retinoic acid effectively due to its special
hydrophobic cavity structure which acts as a host–guest complex with RA. Maximum
adsorption capacity of these multifunctional nanoparticles with RA was estimated to be
3.0 mg/g which is comparable with previous studies. For example, Banerjee et al. found
in their magnetic nanoparticle assisted drug delivery experiments that the maximum
uptakes were 2.72 mg/g of ketoprofen using CD-citrate-GAMNPs [202], 7.53 mg/g of
RA using CD-GAMNPs [16], 1.68 mg/g of ketoprofen using HCD-GAMNP [203] and
7.53 mg/g of RA using HCD-HMDI-GAMNP [306].
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Figure 8-9 Effects of nanoparticle dosage on adsorption/inclusion of retinoic acid.
We then focused on the drug release profile from RA complexed-Fe3O4@SiO2(FITC)-
FA/CMCD NPs in phosphate buffer saline (pH 7.4) at 37oC. Figure 8-10 shows the
release profiles of RA-loaded Fe3O4@SiO2(FITC)-FA/CMCD NPs (RA loading
capacity of 3.0 µg/mg) as a function of time. The typical release profile was
characterized by an initial rapid release phase in the first 60 min when almost 23% of
the total drug was released. These results suggest that a portion of the total drug–CD
complexes in the nanocomposite are quite labile, whereas the rest apparently more
stable. Apart from Van der Walls interaction between hydrophobic part of RA and CD
cavity, at certain conformational arrangements hydrogen bonding between the polar
functional groups’ of RA and CD and the release of high energy water molecules from
the cavity during the complex formation may contribute to further stabilization for some
RA-CD complexes, contributing to slow down the release process [50]. Thus the
reversible and predictive nature of complex formation with hydrophobic molecules
makes CD to serve as a dual agent of drug loading and unloading in our
nanocomposite. Apart from relying on passive drug release (vide supra), the magnetic
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core can be exploited for magneto-responsive release of the drug from the MNP surface
[290,307] which we envisage as a potential research direction towards a remotely
controllable, sustained-release of drugs from CD-decorated nanocomposites.
Figure 8-10 Release profile of retinoic acid from Fe3O4@SiO2(FITC)-FA/CMCD NPs.
In the present work the release data were fitted to the Ritger–Peppas equation given as
follows [308]:
Mt/M∞ = ktn (8-3)
where Mt/M∞ is the amount of drug released, t is the time of release, k is a kinetic
constant, and n is a diffusion exponent which describes the drug release mechanism.
For spherical metrics, when n ≤0.43, diffusion is the major driving force, normally
termed ‘‘Fickian diffusion”. When n > 0.85 drug release is mainly controlled by
degradation, termed case II transport. When the value of n is between 0.43 and 0.85, the
drug release mechanism is anomalous. Our experimental data showed a fairly good fit
to the Ritger–Peppas equation for RA release as the correlation coefficient (R2) is 0.972.
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The value of the exponent n is 0.26, indicating a Fickian diffusion controlled
mechanism.
8.4 Conclusions
We synthesized highly uniform and monodispersed multifunctional magnetic
nanocomposite that is capable of performing a quadruple of activities: magnetic
manipulation, bioimaging, cell-targeting, drug storing-and-releasing. The synthesis
involves several stages of covalent attachments of functional molecules and ligands
orchestrated in rational way to ensure the chemical robustness. All functional groups
were capable of performing independently being within one single nanosystem. Our
magnetically maneuverable nanocomposite shows to be highly dispersible in aqueous
medium and benign towards healthy cells. Folate ligand leads to preferential
internalization into the target cancerous cells. Fluorescence tag acts as a beacon to know
the whereabouts of the particles within a cell and the cyclodextrin moiety can carry and
release drug pay-load in a biologically relevant environment. All these features are ideal
for a nano Trozan horse for targeted drug delivery and imaging. Magnetic core material
has two other features which could further expand the potency of our nanocomposite:
magnetic resonance would facilitate in vivo imaging (MRI) and magneto-responsive
drug release should enable a remotely controllable and sustained drug-delivery system.
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Chapter 9 Summary and Recommendations
9.1 Summary of findings
The main aim of this thesis work is to present a systematic and comprehensive study on
design, synthesis and characterization of β-cyclodextrin conjugated magnetic
nanoparticles and also their potential applications in the field of bio-/biomedical
applications as well as inorganic/organic pollutants removal from wastewater. To each
technique whether protein refolding, adsorption, it has been demonstrated that addition
of magnetic materials aides to improvisation in efficiency of separations.
Chapter 4 describes the method to synthesize β-CD bonded magnetic nanoparticles
(CD-APES-MNPs) having combined effects of chaperoning function of β-CD and
magnetic properties of iron oxide. These as-synthesized CD-APES-MNPs were
effectively used as solid phase artificial chaperone in carbonic anhydrase (CA)
refolding in vitro. The maximum refolding yields obtained were 85% for thermally
denatured CA which was as the same level as that using liquid-phase artificial
chaperone-assisted refolding and the structures of the refolded CA were obtained as
same as those of native protein. Moreover, these magnetic stripping agents could be
easily separated from the refolded samples using the technology of magnetic separation.
Taking into account the recycling potentials of these magnetic based stripping agents,
with significant cost-cutting capabilities by eliminating further purification steps
associated with the liquid-phase artificial chaperone assisted refolding, these stripping
agents would be very suitable materials for protein refolding.
In Chapter 5, a simple and straightforward synthetic method was developed for the
preparation of CM-β-CD modified APES-Fe3O4 nanoparticles that combine magnetic
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and molecular recognition properties. The adsorption of both adenosine (A) and
guanosine (G) onto CMCD-APES-MNPs was found to be pH dependent and the
adsorption equilibrium data were well-described by Langmuir isotherm. Under the
competitive environment, selective adsorption of G over A occurred at pH 5 onto these
nano-adsorbents. This selectivity was due to the multiple recognition sites of the CM-β-
CD which might give strong hydrophobic interaction and hydrogen bonding towards G.
The results showed that this adsorbent would have wide applications in selective
separation, concentration, and analysis of nucleotides/nucleosides in biological samples.
In Chapter 6 and 7, two novel nanoadsorbents, CMCD-MNP(C) and CMCD-MNP(P)
were synthesized via carbodiimide and precipitation method, respectively. These
adsorbents could effectively be used in methylene blue (MB) and Cu2+ ions removal
from aqueous solution. Surface modification of Fe3O4 nanoparticles by CM-β-CD could
greatly enhance the adsorption capacity of MB and Cu2+ ions due to inclusion behavior
and surface complexation with multiple hydroxyl and carboxyl groups of CM-β-CD,
respectively. The adsorption of MB and Cu2+ ions on these adsorbents was found to be
dependent on pH dependant and initial concentration of these pollutants. Moreover,
these pollutants could be recovered fast and efficiently from the aqueous solution. The
adsorption kinetics and isotherms studies demonstrated that the adsorption process
obeyed the pseudo-second-order kinetics and Langmuir isotherm model, respectively.
The maximum uptakes for MB and Cu2+ ions were 277.8 and 47.2 mg/g, respectively
which are considerably higher than that of some other reported magnetic adsorbents.
The negative values of free energy change indicated the spontaneity of adsorption
process. Moreover, the Cu2+ adsorption process was exothermic in nature. In addition,
desorption and regeneration studies suggest that theses nano-adsorbents could be
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repeatedly used as efficient and suitable adsorbents for the removal of both organic and
inorganic pollutants from wastewater using the technology of magnetic separation.
In Chapter 8, highly uniform and monodispersed multifunctional magnetic
nanocomposites were synthesized that are capable of performing a quadruple of
activities: magnetic manipulation, bioimaging, cell-targeting, drug storing and
releasing. The results of retinoic acid inclusion and release experiments indicate that
this system seems to be a very promising vehicle for the administration of hydrophobic
anticancer drugs. Moreover, these prepared nanoparticles possessing assortment of
functionalities would serve simultaneously in efficient bioimaging, specific cell
targeting, hydrophobic drug storage and controlled release systems.
This study has a lot of implications in the field of bio-/biomedical or environmental
applications. All the properties such as superparamagnetism, inclusion/ molecular
recognition properties found in one nano-system imply a wide variety of applications.
The bifunctional or multifunctional magnetic nanomaterials synthesized in this thesis
work can act as a promising candidate for bio-separation, environmental pollutants
cleanup and controlled drug delivery for biomedical applications.
9.2 Recommendations for future work
This thesis has successfully introduced methods for the development and synthesis of
magnetite/β-CD nanocomposites. Several bio- and environmental applications have
been demonstrated using these as-prepared nanoparticles combining magnetic
responsiveness and supramolecular host-guest behaviors. This thesis work is only a
preliminary study. There are several interesting directions for future work. Based on the
current study and above discussion further work should be carried out in the following
aspects:
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9.2.1 Protein refolding using cyclodextrin conjugated magnetic nanoparticles
(a) In Chapter 4, β-CD conjugated magnetic nanoparticles as stripping agents was used
in only CA refolding. Further work might focus on refolding of other structurally
diverse non-homologous proteins such as, hen’s egg white lysozyme (containing
disulfide links in the native state), citrate synthase (a dimer with almost exclusively α-
helical secondary structure) etc. using β-CD conjugated magnetic nanoparticles to
evaluate their efficiencies as stripping agents.
(b) Though refolding experiment was carried out at low protein concentrations (0.029
mg/ml CA) in this study, for many applications, it is advantageous to conduct refolding
at higher protein concentrations. CA refolding at higher protein concentrations can be
made more efficient by increasing the concentrations of the artificial chaperones and by
adding Na2SO4 as suggested by Rozema and Gellman [65]. However, in our study using
solid phase chaperones, CD-APES-MNPs at above optimal concentration (40 mg/ml)
would lead to lower refolding yields. To solve this problem, one approach would be to
synthesize stripping agents with increased number of cyclodextrin moieties per volume
or per mass of solid matrix (magnetic nanoparticles).
(c) In this study, CD-APES-MNPs were used as stripping agents to strip away the
detergent molecules from protein-detergent complex. However, the use of cyclodextrin
as protein refolding aids was reported previously where it could prevent the formation
of protein aggregates during renaturation [70,82,83]. Natural CDs may interact with
proteins via hydrophobic forces, hydrogen bonding and Van der Waals interactions.
Charged chemically modified CDs can also bind to the folding polypeptide chain via
electrostatic interactions. Our as-prepared CD-APES-MNPs could be used as refolding
aids in ‘dilution additive approach’ described in Section 2.4. Moreover, it could be used
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as a separation tool for separating the inclusion bodies (protein aggregates) from cell
debris.
(d) To evaluate the possibility of repeated use of CD-APES-MNPs as a solid-phase
artificial chaperone, regeneration of these stripping agents could be investigated after
recovery of refolded proteins. It could be regenerated by washing out adsorbed
detergent from solid phase with pure water [104].
9.2.2 Pollutants removal from aqueous water using cyclodextrin bonded magnetic
nanoparticles
(a) CM-β-CD conjugated magnetic nano-adsorbents were used as novel adsorbent for
the removal of methylene blue from aqueous streams. MB is a basic or cationic dye.
Adsorption behaviors of other dyes like acid, direct, disperse or reactive dye could be
studied with these magnetic nano-adsorbents to gain better understanding of adsorption
mechanism with different dyes. Similarly, these nano-adsorbents could be used for
adsorption of other heavy metals such as lead, cadmium etc. in non-competitive and
competitive adsorption environment. As CM-β-CD can complex various metals, its
complexation ability could be improved by increasing the number of carboxyl groups
i.g. the degree of carboxymethylation. Indeed, we have synthesized CM-β-CD polymer
(Mp = 13,000, 40wt% COOH groups) and grafted it on the magnetic nanoparticles.
These as-synthesized magnetic nanocomposites can be used for selective adsorption of
lead (II) from contaminated water. Currently, this work is in progress and some of
preliminary results are shown in Appendix C.
(b) Various ions exist in wastewater. The presence of other solutes may influence the
adsorption of any given adsorbate. Thus, K+, Na+, and Ca2+, as well as the anion of Cl−
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and NO3−, which are the commonly coexisting ions with metal ions could be chosen for
further adsorption study. Natural organic matter (NOM) also exists ubiquitously in
natural aquatic environments, with two of its main fractions as humic acid (HA) and
fulvic acid (FA). The presence of natural organic matter (NOM) may also influence the
adsorption of heavy metal ions or organic pollutants on adsorbents in natural
environment. Therefore, competitive adsorption of metal ions and methylene blue could
be investigated in presence of natural organic matters.
(c) In this thesis, a small and limited recyclability study was done for the removal of
methylene blue and Cu2+ ions from aqueous water. For repeated use of these magnetic
nanoadsorbents, their chemical stability in various chemical and physical environments
including acid or base media needs to be investigated. The stability could be tested
through treatment of the particles with acids and several washing steps described in
literature [309]. Furthermore, the stability of the functional moieties (CM-β-CD) on the
adsorbent surface could be examined by FTIR after desorption. At very low pH (pH
<1), the magnetic phase was observed to be unstable and underwent leaching which
might limit the use of these nanoadsorbent in combination with complexing agents in
acidic media. However, magnetic nanoparticles can be coated with graphitic carbon
which can protect and isolate the encapsulated magnetic core and provide them
excellent chemical and thermal stability [309-311] .
(d) Organic and inorganic contaminant mixtures present at contaminated sites is one of
the major challenges in remediating water. Due to the fundamentally different
physiochemical properties of these contaminants, research has to be extended focusing
on remediating organic and inorganic contaminants separately rather than with a single
treatment. Some previous studies on soil remediation purposes showed that CM- β-CD
Chapter: 9
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has the ability to simultaneously complex low-polarity organic molecules and heavy
metals. It was also found that the presence of organic molecules had a negligible effect
on the complexation of metal cations by CM-β-CD or vice versa [247,248]. Therefore,
CMCD-MNPs magnetic nanoparticles could be used for simultaneous removal of
organic and inorganic pollutants from wastewater as they can complex with both
pollutants as shown in Figure 9-1.
Figure 9-1 Schematic presentation of complexation of CMCD-MNPs magnetic nanoparticles with both organic and inorganic pollutants.
(e) The present work was conducted in batch lab-scale operation. A continuous
adsorption/desorption process more relevant for industrial processes could be developed
and studied. For this purposes, superconducting high gradient magnetic separator
(HGMS) suitable for a large-scale wastewater treatment in wastewater treatment plants
could be used. The availability of reasonably priced and efficient super-conducting
magnets and new methods for generating high magnetic field gradients enabled the use
of magnetic extractions on a large scale. Several research groups used HGMS for
contaminants removal from wastewater [123,312]. A multistage countercurrent
continuous contact process (Figure 9‐2) was proposed by Egashira and Hatton to
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improve the treatment capacity [313]. Very recently, Rossier et al. demonstrated the use
of a true moving bed of chemically modified, carbon-coated metal magnetic
nanoparticles to adsorb small amounts of a solute (arsenate) out of a large volume, and
the desorption of the solute into a smaller volume, thereby enriching the solution [314].
The schematic diagram of their overall process is shown in Figure 9-3. However, for
large-scale wastewater treatment, a large quantity of magnetic nanoadsorbents would be
required unless they have extremely high adsorption capacity, which might limit their
practical applicability in treatment plants. Also for large-scale applications, the need of
producing high magnetic field generators and developing materials with high saturated
magnetization is a reality.
Figure 9-2 Schematic of the proposed multistage countercurrent continuous contact [313].
Chapter: 9
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Figure 9-3 Schematic description of the overall process: in the first part (left), the target solute is extracted from a large volume using modified magnetic nanoparticles. In the second part (right), the loaded magnetic nanoparticles are washed (enrichment) and release the solute into a small volume. The moving solids (the true moving bed, i.e. the nanoparticles) links the two tank systems and transports the nanoparticles-bound solute against the concentration gradient between the large tank (left, low concentration) and the small tank (right, high concentration) [314].
9.2.3 Multifunctional silica core-shell magnetic nanoparticles for biomedical
applications
- Cellular Uptake of multifunctional nanoparticle in cancer cells could be evaluated and
measured quantitatively. Recent study by Yang et al. showed that the uptake of silica-
coated manganese oxide nanoparticles in targeted cells was quantitatively analyzed
through flow cytometry.
- Further experiments could be carried out on the specific delivery of hydrophobic drug
to the targeted cancer cells to assess their cytotoxicity effects on cells.
Chapter: 9
199
9.3 Research opportunities
In this thesis work, though various β-CD modified Fe3O4 nanoparticles has been
successfully synthesized and used in several potential applications, these
nanocomposites will also be useful in some other aspects as below:
Enantioseparation of amino acid enantiomers: The importance of amino acids in a
variety of bioscience, pharmaceutical areas and food analysis is indispensible. Amino
acids can be present as L- or D-isomers. Though they have identical physical and
chemical properties except for optical rotation, they can have very different
pharmacological properties and bioactive effects in stereo environments, specifically in
biological environments. Because of these similarities, chiral discrimination of
enantiomers is a very challenging task. Therefore chiral analysis of D/L-amino acids is
of increasing importance in pharmaceutical and biological fields. Though a number of
complicated approaches have been used for the chiral separation of organic compounds,
including gas chromatography (GC), high-performance liquid chromatography (HPLC),
capillary electrophoresis (CE) and the use of chiral reagents in chromatography [315],
most of these techniques are usually limited to the analytical scale, and only allow a
small amount of materials to be separated per run. However, β-CD modified Fe3O4
nanospheres synthesized in this thesis work as a chiral selecting system could be used in
efficient and economical enantioseparation of amino acid enantiomers [316]. The
combination of chiral recognition ability of amino acids enantiomers provided by β-CD
[169], and the magnetic separation ability of Fe3O4 nanoparticles, would provide an
effective tool to separate amino acid enantiomers.
Removal of pharmaceuticals, personal care products and endocrine disruptors
from wastewater: Over the last decade, environmental pollution by “emerging
Chapter: 9
200
contaminants” namely pharmaceuticals and personal care products (PPCPs) and also by
endocrine disrupting chemicals (EDCs) in different water sources and industrial
effluents has aroused the public concern [317,318]. Their occurrence is most often a
result of municipal wastewater discharge, as these compounds are not completely
removed in sewage treatment plants [319]. Wide range of PPCPs (naproxen, ibuprofen,
salicylic acid, carbamazepine, clofibric acid, diclofenac etc.) and EDCs (biphenol A, β-
Estradiol etc.) were found ubiquitously up to µg/l level in aqueous environment, soil,
wastewater, surface waters, sediments, groundwater, and even drinking water
[320,321]. Due to the adverse effects on living creatures and persistency of these PPCPs
and EDCs in environment, removal of these contaminants is of great importance. Based
on same principle used in Chapter 6 for the effective removal of dyes and in Chapter 8
for inclusion of hydrophobic drug (retinoic acid), β-CD modified Fe3O4
nanocomposites could also be used to remove PPCPs and EDCs from wastewater.
Chemical sensing. Selective recognition and separation of nucleosides using β-CD
bonded Fe3O4 nanocomposites has been demonstrated in Chapter 5. This method
provides a practical way to separate substrates based on the difference of binding
property by forming host–guest inclusion. Combining the unique properties of MNPs
(superparamagnetism and electrocatalytic) and CDs (excellent supramolecular
recognition and host–guest inclusion capability), it is possible to obtain the new
materials which will provide good opportunities for applications in the fields of sensors,
electrocatalysis etc. For detecting and monitoring chemicals, new electrodes modified
with β-CD bonded Fe3O4 nanocomposites could be designed. Recently, different β-CD
functionalized magnetic nanoparticles have been synthesized and electrochemical
response to dopamine, uric acid and tryptophan with the assistance of a magnet was
observed [206,322,323].
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Appendix
229
Appendix A: Supporting information for Chapter 5 A-1 Magnetic properties of CMCD-APES-MNPs
The magnetic properties of the uncoated and CM-β-CD coated Fe3O4 nanoparticles
were measured by vibrating sample magnetometer (VSM) at room temperature. From
M vs. H curves (Figure A-1), the saturation magnetization value (Ms) of uncoated
MNPs was found to be 75 emu g-1 which is much less than its bulk counterpart (92 emu
g-1) [186]. For CD-APES-MNPs, the magnetization obtained at the same field was 61.5
emu g-1, lower than that of uncoated Fe3O4. This is mainly attributed to the presence of
nonmagnetic materials (APES and CM-β-CD layer) on the surface of nanoparticles,
which might have quenched the magnetic moment. The high magnetic response is
further demonstrated by applying a magnet to the outside of a vial containing the
CMCD-APES-MNPs aqueous solution (the inset of Figure A-1). This feature allows the
as-synthesized nanoparticles for highly efficient magnetic manipulation when used as
nano-adsorbents even under a relatively low external magnetic field. The magnetic
separation was accomplished efficiently within few minutes in our experiments. The
saturated magnetization value of the magnetic particles could be compared with other
magnetic adsorbents reported in literature [108,225]. In our lab-scale batch experiment
we used permanent magnet which could induce fields less than 1 T. Though this
magnetic field is suitable in small volume applications, a high gradient magnetic field
separator, however, would be required in a large scale operation. A properly designed
magnet in a HGMS separator can greatly improves the performance. Different types of
separator based on magnet type could be used as discussed in Section 2.5. However, the
separation efficiency largely depends on the magnetic field applied, magnetization of
materials, volume of liquid handled etc. For large-scale operation, the need of
Appendix
230
producing high filed generators and developing materials with high saturation
magnetization is a reality.
Figure A-1 Magnetization curves for uncoated MNPs and CMCD-APES-MNPs. A-2 XPS analysis of as-synthesized magnetic nanoparticles
XPS analysis was applied to find the chemical binding in the as-synthesized CMCD-
APES-MNPs. The C1s deconvoluted spectrum is shown in Figure A-2. The C1s
spectrum can be curve-fitted into five peak components with binding energy of about
283.1, 284.6, 286.0, 287.3 and 288.7 eV, attributable to the carbon atoms in the forms
of Si–C, C–C (aromatic), C–O (alcoholic hydroxyl and ether), O=C–N and O=C–O
species, respectively [324]. The C–O/C–O–C peak is characteristic of β-CD, confirming
that the modification of magnetic nanoparticles with β-CD derivative was
successful.The broad peak for N1s could be fitted into two peaks at 398.8 and 400.4 eV
corresponding to –NHCO and –NH2 [281]. The appearance of –NHCO peak suggests
-80
-60
-40
-20
0
20
40
60
80
-15 -10 -5 0 5 10 15
M, e
mu
/g F
e 3O
4
H, kOe
CMCD-APES-MNPs
Fe3O4 MNPs
Appendix
231
394 396 398 400 402 404 406
Cou
nts (
a.u)
Binding energy (eV)
-NH-CO
-NH2
N 1s
that CM-β-CD is grafted on the surface of magnetic nanoparticles by amide bond
formation.
Figure A-2 XPS C1s and N1s spectra of CMCD-APES-MNPs. A-3 Effects of pH The surface charge of the adsorbent and the ionization degree of adsorbate are generally
affected by solution pH values. When pH< pKa, the non-dissociated species and the
dissociated species (bases form cations by protonation at pH) are dominated for organic
acids and organic bases, respectively; whereas when pH> pKa, the dissociated species
(anions) is dominant for organic acids and the non-dissociated species is dominant for
organic bases [325]. The fraction of non-dissociated species for organic acids (fAN) and
bases (fBN) can be respectively estimated by fA
N = (1 + 10pH−pKa)−1 and fBN = (1 +
10pKa−pH)−1, and the fraction of dissociated species for organic acids (fAD) and bases
(fBD) can be respectively estimated by fA
D = (1 + 10pKa−pH)−1 and fBD = (1 + 10pH−pKa)−1,
respectively. In order to properly interpret the nucleosides adsorption behavior, the
distribution of non-dissociated and dissociated species of guanosine and adenosine as a
function of pH is shown in Figure A-3.
279 281 283 285 287 289 291
Cou
nts (
a.u)
Binding enegry (eV)
C 1s
Si-C
C-C/C-H
C-O/C-O-
O=C-NO=C-O
Appendix
232
(A)
(B)
Figure A-3 (A) Distribution of guanosine (G) and adenosine (A) species as a function of
pH; (B) Ionization mechanism of A and G.
A-4 Spectral evidence of nucleoside adsorption onto CMCD-APES-MNPs
High resolution XPS spectra provide information on the chemical environment of each
of the detected atoms on the surface. In our study, the N 1s high-resolution spectra were
studied to examine the possible existence of chemical interaction in the adsorption of
nucleosides onto magnetic nanoparticles. Three chemical environments were
distinguishable in the N 1s high resolution spectrum after nucleoside adsorption (Figure
A-4). Deconvolution of the N 1s peak of CMCD-APES-MNPs before nucleoside
Appendix
233
adsorption produces two peaks of binding energies 398.8 and 400.4 eV corresponding
to –NHCO and –NH2 [281]. As guanine and adenine residues both contain 5 nitrogen
atoms but under different forms (3 –N=, 1 –NH– & 1 –NH2 for adenine residues and 2
–N=, 2 –NH– & 1 –NH2 for guanine residues), it was expected to find all the
corresponding N 1s deconvoluted peaks for CMCD-APES-MNPs after adsorption with
A or G. The N 1s spectrum for CMCD-APES-MNPs after A or G adsorption was fit
with three peaks at BEs of 398.2 eV, 399.5 eV and 400.5 eV corresponding to
conjugated nitrogen (–N=) in the nucleic bases, non-conjugated nitrogen (–NH– and N
with three single bonds) and the (–NH2), respectively, [326,327]. The presence of –N=
peaks on A or G-loaded CMCD-APES-MNPs confirms the adsorption of nucleosides
onto magnetic surfaces.
Figure A-4 N 1s high resolution peaks and peak fitting: (a) CMCD-APES-MNPs before
adsorption; (b) after guanosine adsorption; (c) after adenosine adsorption. A-5 Determination of binding constants between nucleoside and CDs
The inclusion capability of β-CD towards nucleosides was investigated by circular
dichroism spectra, respectively. Circular dichroism (CD) spectra were obtained on a
Jacob J810 spectropolarimeter at 25oC. The concentrations of adenosine (A) and
guanosine (G) were controlled at 0.075 mM and concentration of β-CD was varied from
2 mM to 18 mM. The molar ellipticities of β-CD:A and β-CD:G solutions at
Appendix
234
wavelength from 220 nm to 320 nm were measured separately by circular dichroism at
25oC with a 5-mm path length sample cell (data not shown here). Molar ellipticity
differences, ∆θ, were recorded at the wavelength at which the differences between pure
and complexed nucleosides were maximized (approx. 260 nm) as a function of β-CD
concentration. These spectral variations induced by β-CD and its derivatives would be
caused by the insertion of the nucleosides into optically active β-CD cavity and by the
conformational changes of the purine derivatives. The binding constants were obtained
from double reciprocal plots of 1/∆θ vs 1/[β-CD] according to the following equation:
∆ ∆ AB ‐ ∆ AB
(A-1)
where K is the binding constant and ∆θAB is the coefficient for right and left circularly
polarized light between the free and complexed nucleosides. From the slope and
intercept in Figure A-5 the binding constants of β-CD for A and G are calculated to be
17.9 and 30.3 M-1 respectively. The 1:1 stoichiometry of complexes of β-CD with A or
G is also suggested by UV-circular dichroism spectra. The formation constant of A is
very close to the literature value (16 M-1) reported by Suzuki et al. using the same
method [328]. The other study by Xiang and Anderson using solubility method also
yielded a similar value of 13 M-1 for adenosine complex [56].
Appendix
235
Figure A-5 Double reciprocal plots for A and G complexes to β-CD measured from
circular dichroism spectral data.
A-6 Preparation of inclusion complexes and DSC study
The required amount of A or G and aqueous solution of CM-β-CD (2.0 mM) were
mixed (1:1 molar ratio) and stirred at 25oC for 2 days with a thermostatic water-bath
shaker. After this period the solid residue was separated by centrifugation at 12,000 rpm
for 15 min and the upper liquid layer was filtered over 0.45 µm membrane. The
resulting solution was frozen at -18 oC for 24 h and then dried 48 h by lyophilization for
the solid inclusion complex to be collected.
Figure A-6 illustrates the DSC thermograms of β-CD, adenosine, guanosine and their
physical mixtures and inclusion complexes. The DSC curve of β-CD showed a broad
endothermic peak in the range of 130-170oC, which could be attributed to the release of
water molecule from the cavity (desolvation). The thermograms of adenosine and
guanosine were typical of highly crystalline compounds, characterized by a sharp
endothermic peak at 237.2 and 249.9oC corresponding to their melting point,
Appendix
236
respectively. In their physical mixtures, the DSC pattern revealed the presence of peaks
of both pure compounds except that the melting endotherm of adenosine and guanosine
slightly shifted to 234.2 and 254oC, respectively with a decrease in the peak intensities.
However, the absence of the characteristic peaks of adenosine and guanosine in the
adenosine:β-CD and guanosine:β-CD complexes is a strong evidence of the inclusion of
nucleosides inside the cyclodextrin cavities.
Figure A-6 DSC curves of (a) adenosine and (b) guanosine and their physical mixtures and inclusion complexes with CM-β-CD.
25 75 125 175 225 275
Hea
t flo
w (m
W)
Temperature (oC)
Inclusion complex
(237.2oC)
CM-β-CD
Adenosine
Physical mixture
(a)
25 75 125 175 225 275
Hea
t fl
ow (m
W)
Temperature (oC)
Inclusion complex
Physical mixture
CM-β-CD
Guanosine
249.9oC
(b)
Appendix
237
Appendix B: Supporting information for Chapter 8
Figure B-1 XRD patterns of a) Fe3O4 nanoparticles and b) Fe3O4@SiO2(FITC)
nanoparticles. The symbol of * indicates the broad peak of amorphous silica
Figure B-2 FTIR spectra of oleic acid capped-Fe3O4 (OA-MNPs) and fluorescent dye-
doped magnetic silica nanoparticles [Fe3O4@SiO2(FITC) NPs].
Appendix
238
Figure B-3 Field-dependent magnetization curve of Fe3O4@SiO2(FITC)]-FA/CMCD NPs at 300K. Effects of water-surfactant molar ratio on sizes of core-shell silica magnetic nanoparticles Bagwe et al. found that the microemulsion parameters such as reactant concentrations
(ammonium hydroxide), nature of surfactant molecules, and molar ratios of water to
surfactant and cosurfactant to surfactant influenced the fluorescence spectra, particle
size, and size distribution of Ru(bpy) dye-doped silica nanoparticles [329]. In this
study, in order to optimize the properties of the as-synthesized nanoprobes, a series of
nanoprobes were prepared with different thickness of the silica shell just by changing
the ratio of water to surfactant (triton X-100) in the synthetic process. When increasing
the ratio from 6.2 to 15, the average size of the as-synthesized MNPs decreases (Figure
B-4, TEM images). A thick silica shell allows high payloads of dye molecules and
therefore improving the fluorescent property of the nanoparticles, which is confirmed
by fluorescence emission spectra (Figure B-4). In case of smaller particles, the outer
silica shells in which the dye molecules are incorporated come nearer to the magnetic
Appendix
239
core resulting in fluorescence quenching. Also the outer layer contains low payloads of
dye molecules because of thin silica layer.
Figure B-4 TEM images Fe3O4@SiO2(FITC) nanoparticles (a-c) and their fluorescence emission spectra (recorded at 480 nm excitation).
Appendix
240
Figure B-5 (a) Hydrodynamic size distribution of Fe3O4@SiO2(FITC)]-FA/CMCD
NPs (b) Hydrodynamic diameter of Fe3O4@SiO2(FITC)]-FA/CMCD NPs measured by dynamic light scattering technique in deionized water.
Appendix
241
Appendix C
This section reports the synthesis of a novel nanoadsorbent- carboxymethyl-β-
cyclodextrin polymer grafted magnetic nanoparticles (CDpoly-MNPs). This polymer
having numerous carboxyl groups (Mp= 13,000, 40% COOH groups) has the ability to
adsorb heavy metal ions through complexation reaction with surface groups, mainly
carboxyl groups in the polymer matrix (Figure C-1). Competitive adsorption
experiments were conducted to elucidate the adsorption selectivity of CDpoly-MNPs on
lead ions.
C-1 Experimental
Synthesis of CM-β-CD polymer
CM-β-CD polymer was prepared following the procedure of literature [330], with the
detailed description as follows: β-cyclodextrin (5 g) was dissolved in 50 ml of 10%
(w/v) NaOH and 10 ml of epichlorohydrin was added. The system was vigorously
stirred for 8 h before another 5 ml of epichlorohydrin was added with stirring and the
mixture kept overnight at room temperature. The solution was concentrated to about 15
ml and precipitated by addition of cold ethanol (500 ml). The gummy precipitate was
crushed several times with ethanol in a mortar until a fine precipitate was obtained. The
precipitate was then washed again with ethanol and acetone and dried under high
vacuum overnight. The yield of β-CD/epichlorohydrin co-polymer was 80%. Two
grams of the above polymer was further dissolved in 50 ml 5% (w/v) NaOH and 2 g of
monochloroacetic acid was added. The system was vigorously stirred for 24 h,
neutralized with 2 M HCl, concentrated to about 15 ml and cooled to 4 oC. The
precipitated NaCl was filtered off and the supernatant was precipitated by addition of
Appendix
242
cold ethanol (500 ml). The gummy precipitate was crushed several times with ethanol
in a mortar until a fine precipitate was obtained. The precipitate was then washed two
more times with ethanol and acetone and dried under high vacuum overnight. The yield
of CM-β-CD polymer was 60%. The molecular weight of this polymer (Mp = 13000)
was determined by gel permeation chromatography on Fractogel EMD BioSEC (S)
(1.6×100 cm) calibrated with dextran standards as previously described [330].
Potentiometric titration of the –OCH2COOH groups with alkali gave a degree of
substitution of 40% (mol/mol D-glucose) for this polymer.
Synthesis of CM-β-CD polymer coated magnetic nanoparticles (CDpoly-MNPs)
CDpoly-MNPs were fabricated by one step co-precipitation method. Briefly, 0.86 g of
FeCl2.4H2O, 2.36 g FeCl3.6H2O and 1.5 g CM-β-CD polymer were dissolved in 40 ml
of de-aerated Milli-Q water with vigorous stirring at a speed of 1,200 rpm. 5 ml of
NH4OH (25%) was added after the solution was heated to 90 ºC. The reaction was
continued for 1 h at 90 ºC under constant stirring and nitrogen environment. The
resulting nanoparticles were then washed with Milli-Q water 5 to 6 times to remove any
unreacted chemicals and dried in a vacuum oven.
The step-by-step reaction procedures to synthesize CM-β-CD polymer modified
magnetic nanoparticles are shown in Figure C-1.
Appendix
243
Figure C-1 (A) Schematic presentation of CM-β-CD polymer grafting on Fe3O4 nanoparticles and (B) possible mechanism for adsorption of metal ions by CDpoly-MNPs.
Competitive Adsorption of heavy metal ions
Metal ions (Pb2+, Cd2+ and Ni2+) adsorption experiments were carried out using batch
equilibrium technique in aqueous solutions at pH 5.5 and at 25 oC. In general, an
average of 120 mg of wet magnetic nanoadsorbents (20% dry particle content) was
added to 10 ml of single, binary and ternary metals solution with each metal
concentration of 10 mg/ml and shaken in a thermostatic water-bath shaker operated at
230 rpm. After equilibrium was reached (~2 h), magnetic nanoadsorbents were
removed using a permanent Nd-Fe-B magnet and the supernatant was collected. The
Appendix
244
concentrations of Pb2+, Cd2+ and Ni2+ ions were measured using Inductive Couple
Plasma Mass Spectrometry (Agilent ICP-MS 7700 series).
C-2 Results and discussion
Characterization of magnetic nanoparticles
The grafting of CM-β-CD polymer on magnetic nanoparticles was confirmed by FTIR
and XPS analysis. Figure C-2 shows the FTIR spectra of CM-β-CD polymer, bare and
polymer coated Fe3O4 nanoparticles in the 4000-400 cm-1 wavenumber range. The
spectrum of CM-β-CD polymer shows the characteristic peaks at 1028, 1155 and 1710
cm−1. The peaks at 1028 and 1155 cm−1 correspond to the antisymmetric glycosidic
νa(C-O-C) vibrations and coupled ν (C-C/C-O) stretch vibration. The peak at 1710 cm-1
corresponds to carbonyl group (= CO) stretching which confirms the incorporation of
the carboxymethyl group (-COOCH3) into CM-β-CD polymer. The characteristic
adsorption band of magnetic nanoparticles is 586 cm-1 which is due to Fe–O bonds in
the tetrahedral sites. All the significant peaks of CM-β-CD polymer in the range of
900–1200 cm−1 are present in the spectrum of CDpoly-MNPs with a small shift.
Moreover, as shown in Fig. 1c, two main characteristic peaks appeared at 1628 and
1400 cm−1 due to bands of COOM (M represents metal ions) groups, which indicates
that the COOH groups of CM-β-CD polymer reacted with the surface OH groups of
Fe3O4 particles resulting in the formation of the iron carboxylate [205].
Appendix
245
Figure C-2 FTIR spectra of (a) CM-β-CD polymer, (b) uncoated MNPs and (b) CDpoly-MNPs.
Figure C-3 XPS C 1s spectrum of CDpoly-MNPs.
280 282 284 286 288 290 292 294
Cou
nts
(a.u
.)
Binding energy (eV)
C 1s
C-C/C-H
C-O/C-O-C
C=O
COO-
Appendix
246
The C 1s deconvoluted spectrum is shown in Figure C-3. The C 1s spectrum can be
curve-fitted into four peak components with binding energy of about 284.6, 286.1,
287.9 and 288.7 eV, attributable to the carbon atoms in the forms of C–C (aromatic),
C–O (alcoholic hydroxyl and ether), C=O (carbonyl) and COO- (carboxyl and ester)
species, respectively [264]. The C–O/C–O–C and C=O peaks are the characteristic
peaks of CM-β-CD polymer. Moreover, the presence of COO- peak at 288.7 eV
indicates that the COOH functional groups on CM-β-CD polymer reacted with surface
OH groups to form metal carboxylate (COOM). Thus, the modification of magnetic
particle surface with CM-β-CD polymer was confirmed.
Competitive adsorption of metal ions
To examine the competitive effects the metals exert on each other in multi-metal
solutions, the removal efficiencies of CDpoly-MNPs for each metal in single, binary
and ternary solutions were compared and are shown in Figure C-4. In can be seen that
the percentage removal of Pb2+, Cd2+ and Ni2+ ions in single-metal system (non-
competitive) were 99.5%, 55.9% and 24.3%, respectively. In binary metal solution
(Pb2+ - Cd2+ and Pb2+ - Ni2+), Pb2+ metal adsorption was slightly reduced (to 95.6% and
96.4% respectively) by the presence of Cd2+ or Ni2+. However, the percentage removal
of Cd2+ and Ni2+ were reduced significantly to 12.7% and 9.4% respectively in the same
binary mixtures. This indicates that Pb2+ ions were preferentially adsorbed on the
surface of CDpoly-MNPS as compared to Cd2+ or Ni2+ ions. In the binary Cd2+ - Ni2+
mixture, the removal of Cd2+ and Ni2+ were also decreased to 30.1% and 14.4%
respectively, showing the competitiveness of the two metals. In the ternary system, the
percentage removal of Pb2+ was slightly reduced (94.9%), whereas the percentage
Appendix
247
removal of Cd2+ and Ni2+ were lower than that of single or binary metal mixtures
(10.3% and 7.3% respectively).
Figure C-4 Percentage removal of Ni2+, Cd2+ and Pb2+ from single, binary and ternary mixtures (Each metal concentration: 100 mg/l, adsorbents: 120 mg, temperature: 25 °C, pH: 5.5 and contact time: 2 h).
The decrease in sorption capacity of same adsorbent in multi-metal solution than that of
single metal ion may be ascribed to the less availability of binding sites. The results
from the binary and ternary metal mixtures show that the presence of Cd2+ and/or Ni2+
has little influence on the adsorption of Pb2+ onto the CDpoly-MNPs, whereas the
adsorption of Cd2+ and Ni2+ are significantly reduced when in a competitive metal ion
environment. Hence, the order of removal efficiency for the three metal ions was Pb2+
>>Cd2+ > Ni2+, implying the stronger affinity of the adsorbent for Pb2+ than Cd2+ and
Ni2+. This tendency of higher adsorption of Pb2+ on different adsorbents containing –
COOH and –OH functional groups in multi-metal solutions was reported by other
studies [146,331].
Appendix
248
Recyclability study
The reusability was checked by following the adsorption–desorption process for four
cycles for Pb2+ ions and the adsorption efficiency in each cycle was analyzed and
presented in Figure C-5. In each cycle, 120 mg of CDpoly-MNPs and 10 ml of an initial
lead concentration of 300 ppm was used for the adsorption process, and 10 ml of 0.01
M nitric acid was used as desorption eluent. 0.01 M HNO3 could desorb around 96.0%
of Pb2+ ions from metal-loaded nanoparticles. The CDpoly-MNPs adsorbent kept its
adsorption capability after repeated adsorption–regeneration cycles with negligible
changes, indicating that there are almost no irreversible sites on the surface of CDpoly-
MNPs. Our recyclability studies suggest that these nanoadsorbents can be repeatedly
used as efficient adsorbents in wastewater treatment.
Figure C-5 Four consecutive adsorption–desorption cycles of CDpoly-MNPs adsorbent for Pb2+ (initial concentration: 300 mg/l, pH: 5.5, desorption agent: 10 ml of 0.01 mol/l HNO3).
0
20
40
60
1 2 3 4
q e(m
g/g)
No. of Cycles
Lead Adsorption Lead Desorption
Appendix
249
C-3 Conclusions
In this work, carboxymethyl-β-cyclodextrin polymer grafted magnetic nanoparticles
(CDpoly-MNPs) were synthesized successfully and used as easily separable, recyclable
and highly selective nanoadsorbent for the removal of Pb2+ ions from contaminated
water.
List of Publications
250
List of publications
Journal Articles
A.Z.M. Badruddoza, K. Hidajat, M.S. Uddin, Synthesis and characterization of beta-
cyclodextrin-conjugated magnetic nanoparticles and their uses as solid-phase artificial
chaperones in refolding of carbonic anhydrase bovine, J. Colloid Interface Sci. 346(2) (2010)
337-346.
Sudipa Ghosh, A.Z.M. Badruddoza, M.S. Uddin, K. Hidajat, Adsorption of chiral aromatic
amino acids onto carboxymethyl-β-cyclodextrin bonded Fe3O4/SiO2 core-shell nanoparticles, J.
Colloid Interface Sci. 354(2) (2011) 483-492.
A.Z.M. Badruddoza, Goh Si Si Hazel, K. Hidajat, M.S. Uddin, Synthesis of carboxymethyl-β-
cyclodextrin conjugated magnetic nano-adsorbent for removal of methylene blue, Colloids Surf.
A: Physicochem Eng. Aspects 367 (2010) 85-95.
A.Z.M. Badruddoza, A.S.H. Tay, P.Y. Tan, K. Hidajat, M.S. Uddin, Carboxymethyl-β-
cyclodextrin conjugated magnetic nanoparticles as nano-adsorbents for removal of copper ions:
Synthesis and adsorption studies, J. Hazard. Mater. 185(2-3) (2011) 1177-1186.
A.Z.M. Badruddoza, L. Junwen, K. Hidajat, M.S. Uddin, Selective recognition and separation
of nucleosides using carboxymethyl-β-cyclodextrin functionalized hybrid magnetic
nanoparticles, Colloids and Surfaces B: Biointerfaces, 92 (2012) 223-231.
A.Z.M. Badruddoza, Sudipa Ghosh, K. Hidajat, M.S. Uddin, Multifunctional core-shell SiO2
nanoparticles with magnetic, fluorescent, specific cell targeting and inclusion properties for
biomedical applications, Submitted.
Abu Zayed M. Badruddoza, Zayed Bin Zakir Shawon, Tay Wei Jin Daniel, Kus Hidajat,
Mohammad Shahab Uddin, Single and multi-component adsorption of lead, cadmium and
nickel onto cyclodextrin polymer modified magnetic nanoparticles, Manuscript in Preparation.
List of Publications
251
Conference Proceedings
Abu Zayed M. Badruddoza, Zayed Bin Zakir Shawon, Tay Wei Jin Daniel, Kus Hidajat and
Mohammad Shahab Uddin, Carboxymethyl-β-cyclodextrin polymer modified magnetic
nanoadsorbents for removal of endocrine disrupter and toxic metal ions, Proceedings of the
International Conference on Chemical Engineering 2011, ICChE2011, 29-30 December,
Dhaka, Bangladesh.
Zayed Bin Zakir Shawon, Abu Zayed M. Badruddoza, Soh Wei Min Louis, Kus Hidajat,
Mohammad Shahab Uddin, Accelerated procedure for synthesizing Janus magnetic
nanoparticles, Proceedings of the International Conference on Chemical Engineering 2011,
ICChE2011, 29-30 December, Dhaka, Bangladesh.
Conference Presentations Liang Hong, Abu Zayed Md. Badruddoza , Mohammad S Uddin and Kus Hidajat, Synthesis
and characterization of magnetic nano-particles functionalized with β-cyclodextrin for
biomolecule refolding, Particle, Florida, USA, May 10-13, 2008.
K Hidajat, A Z M Badruddoza, M S Uddin, L Hong, N T K Thuyen, Magnetic nano-sized
particles functionalized with beta-cyclodextrin and its use in lysozyme refolding process,
CHISA, Prague, Czech Republic, August 24-28, 2008.
A Z M Badruddoza, K Hidajat and M S Uddin, Solid-phase artificial chaperone assisted
refolding of carbonic anhydrase using β-cyclodextrin conjugated magnetic nanoparticles, VIII
European Symposium of the Protein Society, Zurich, Switzerland, June 14-18, 2009.
A Z M Badruddoza, K Hidajat and M S Uddin, Beta-cyclodextrin bonded magnetic
nanoparticles as solid-phase artificial chaperone for protein refolding, ICBN 2010, Biopolis,
Singapore, August 1-4, 2010.