ACTIVITY MEASUREMENTS OF ENZYMES CONJUGATED TO...
Transcript of ACTIVITY MEASUREMENTS OF ENZYMES CONJUGATED TO...
ACTIVITY MEASUREMENTS OF ENZYMES CONJUGATED TO SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES UNDER AN
ALTERNATING MAGNETIC FIELD
By
TAPOMOY BHATTACHARJEE
A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2014
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ACKNOWLEDGMENTS
I’d like to thank my advisor, Dr. Carlos Rinaldi, for granting me the freedom to
pursue my academic and professional interests over the past one and a half years and
for guiding me in my first steps toward a career in research. I’m also indebted to Dr. Jon
Dobson of the Department of Biomedical Engineering and Material Science Engineering
at University of Florida for engaging discussions and suggestions. I should also take this
opportunity to thank my fellow colleagues Ana C. Bohorquez and Adam Monsalve for
their continuous help and support.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 7
LIST OF FIGURES .......................................................................................................... 9
LIST OF ABBREVIATIONS ........................................................................................... 11
ABSTRACT ................................................................................................................... 12
CHAPTER
1 BACKGROUND....................................................................................................... 14
1.1 Superparamagnetic Iron Oxide Nanoparticle .................................................... 14
1.1.1 Néel Relaxation ....................................................................................... 15 1.1.2 Brownian Relaxation................................................................................ 16
1.2 Local Heating Phenomena ................................................................................ 17 1.3 Objective ........................................................................................................... 18
1.4 Bio-Conjugation Chemistry ............................................................................... 19 1.4.1 EDC/ NHS Chemistry .............................................................................. 19
1.4.2 Sulfo SMCC Chemistry ............................................................................ 20
1.5 -Amylase ......................................................................................................... 21
1.6 β-Galactosidase ................................................................................................ 22
2 MATERIALS AND METHODS................................................................................. 23
2.1 Materials ........................................................................................................... 23 2.2 Nanoparticle Characterization ........................................................................... 23
2.2.1 PEG550 Batch-1...................................................................................... 23 2.2.2 PEG550 Batch-2...................................................................................... 25
2.2.3 IO-CMDX particles:.................................................................................. 28 2.3 Free Enzyme Activity ........................................................................................ 30
2.3.1 -Amylase activity ................................................................................... 30 2.3.2 β-Galactosidase activity........................................................................... 32
2.4 Conjugated Enzyme activity .............................................................................. 33
2.4.1 -Amylase activity ................................................................................... 33 2.4.2 β-Galactosidase activity........................................................................... 34
2.5 Bio-Conjugation ................................................................................................ 34
2.5.1 For -Amylase ......................................................................................... 34 2.5.2 For β- Galactosidase ............................................................................... 36
2.5.2.1 Scheme 1 ....................................................................................... 36 2.5.2.2 Scheme 2 ....................................................................................... 37
2.6 Wash Procedure of Conjugated Enzyme .......................................................... 38 2.6.1 Amicon Ultra-15 Centrifugal Filter Units .................................................. 38
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2.6.2 Sephacryl S400 HR Columns .................................................................. 39
2.6.2.1 -Amylase separation .................................................................... 39
2.6.2.2 β-Galactosidase separation ........................................................... 41 2.7 Inactivation Study.............................................................................................. 44
2.7.1 For -amylase ................................................................................... 44 2.7.2 For β-galactosidase .......................................................................... 46
3 RESULTS ................................................................................................................ 48
3.1 Selectivity of Sulfo-SMCC over EDC ................................................................ 48 3.2 Activity of free enzyme ...................................................................................... 50
3.2.1 For -Amylase ......................................................................................... 50 3.2.2 For β-Galactosidase ................................................................................ 51
3.3 Activity of Enzyme Conjugated to Iron Oxide nanoparticle ............................... 52
3.3.1 For -Amylase ......................................................................................... 52 3.3.2 For β-Galactosidase ................................................................................ 53
3.4 Activity under AMF ............................................................................................ 54
3.4.1 For -Amylase: ........................................................................................ 54
3.4.1.1 Study 1: .......................................................................................... 54 3.4.1.2 Study 2: .......................................................................................... 55
3.4.1.3 Study 3: .......................................................................................... 57 3.4.2 For β-galactosidase: ................................................................................ 59
3.4.2.1 IO-CMDX conjugate Study 1: ......................................................... 59 3.4.2.2 IO-CMDX conjugate Study 2: ......................................................... 60
3.4.2.3 PEG550 Conjugate Study 1: .......................................................... 61 3.4.2.4 PEG550 Conjugate Study 2: .......................................................... 62
3.4.2.5 PEG550 Conjugate Study 3: .......................................................... 62 3.4.2.6 PEG550 Conjugate Study 4: .......................................................... 63
4 CONCLUSION ........................................................................................................ 64
APPENDIX
-AMYLASE CONJUGATION TRIALS USING EDC/NHS CHEMISTRY ...................... 65
Scheme 1 ................................................................................................................ 65 Scheme 2 ................................................................................................................ 66
Scheme 3 ................................................................................................................ 67 Scheme 4 ................................................................................................................ 68
Scheme 5 ................................................................................................................ 69 Scheme 6 ................................................................................................................ 70
Scheme 7 ................................................................................................................ 71
LIST OF REFERENCES ............................................................................................... 72
BIOGRAPHICAL SKETCH ............................................................................................ 76
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LIST OF TABLES
Table page 2-1 beta- gal conjugation scheme 1: Absorbance of sample and control after
incubation ........................................................................................................... 36
2-2 beta- gal conjugation scheme 2: Absorbance of sample and control after incubation ........................................................................................................... 37
2-3 Absorbance of the particles; A1-12 refers to drop 1-12, B1-12 refers to drop 13-24 and so on, H 12 is the control ................................................................... 40
2-4 Absorbance of the starch iodine assay; A1-12 refers to drop 1-12, B1-12 refers to drop 13-24 and so on, H 12 is the control ............................................. 41
2-5 Difference of absorbance normalized by control; A1-12 refers to drop 1-12, B1-12 refers to drop 13-24 and so on, H 12 is the control .................................. 41
2-6 Absorbance of the ONPG assay from column effluent; A1-12 refers to drop 1-12, B1-12 refers to drop 13-24 and so on ........................................................... 43
3-1 (𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒) for Starch Iodine assay of free enzyme after 45 min
incubation at different temperature ..................................................................... 50
3-2 (𝐴𝑠𝑎𝑚𝑝𝑙𝑒 − 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙) for ONPG assay of free beta-Galactosidase after 60 min incubation at different temperature .............................................................. 51
3-3 (𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒) for Starch Iodine assay of conjugated enzyme after 45 min incubation at different temperature ......................................................... 52
3-4 (𝐴𝑠𝑎𝑚𝑝𝑙𝑒 − 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 ) for ONPG assay of conjugated beta-Galactosidase after 60 min incubation at different temperature.......................... 53
3-5 Activity under AMF: Study 1: Absorbance after application of AMF.................... 54
3-6 Activity under AMF: Study 1: Absorbance of Control .......................................... 54
3-7 Activity under AMF: Study 2: Absorbance of after application of AMF ................ 55
3-8 Activity under AMF: Study 2: Absorbance of Control .......................................... 56
3-9 Activity under AMF: Study 3: Absorbance after application of AMF.................... 57
3-10 Activity under AMF: Study 3: Absorbance of Control .......................................... 57
3-11 IO-CMDX Beta gal conjugate Study 1: Absorbance of sample and control ........ 59
3-12 IO-CMDX Beta gal conjugate Study 2: Absorbance of sample and control ........ 60
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3-13 PEG550 (batch2) Beta gal conjugate Study 1: Absorbance of sample and control ................................................................................................................. 61
3-14 PEG550 (batch2) Beta gal conjugate Study 2: Absorbance of sample and control ................................................................................................................. 62
3-15 PEG550 (batch2) Beta gal conjugate Study 3: Absorbance of sample and control ................................................................................................................. 62
3-16 PEG550 (batch2) Beta gal conjugate Study 4: Absorbance of sample and control ................................................................................................................. 63
A-1 Scheme 2 reaction mixture preparation. ............................................................. 66
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LIST OF FIGURES
Figure page 1-1 Energy of a single domain particle under applied field ....................................... 15
1-2 EDC Chemistry; R is IO nanoparticle and R' is the enzyme here ....................... 20
1-3 Sulfo- SMCC Chemistry; R is the IO nanoparticle and R' is the enzyme ............ 21
2-1 DLS of PEG550-Batch 1 ..................................................................................... 23
2-2 Zeta potential of PEG550-Batch 1 particles ........................................................ 24
2-3 SAR measurement of PEG550-Batch 1 particles ............................................... 25
2-4 DLS of PEG550-Batch 2 ..................................................................................... 25
2-5 Zeta potential of PEG550-Batch 2 particles ........................................................ 26
2-6 M vs. H Plot for PEG550 batch 2 particles.......................................................... 27
2-7 SAR measurement of PEG550-Batch 2 particles ............................................... 28
2-8 DLS of IO-CMDX particles .................................................................................. 28
2-9 Zeta potential of IO-CMDX particles ................................................................... 29
2-10 M vs. H Plot for IO-CMDX particles .................................................................... 29
2-11 SAR measurement of PEG550-Batch 2 particles ............................................... 30
2-12 Absorbane of starch iodine complex at different wavelength .............................. 31
2-13 beta-Galactosidase assay .................................................................................. 32
2-14 Scheme 8: Starch Iodine assay with Washed particles; ‘S1’ and ‘S2’ refers to Sample and contains washed particles after reaction; 'C2' refers to Negative Control and contains pure starch solution only; 'C1' refers to positive control and contains 2.5 ug of Amylase.......................................................................... 35
2-15 Wash using Amicon Ultra-15 Centrifugal Filter Units .......................................... 38
2-16 . Starch-Iodine assay of column effluent ............................................................. 40
2-17 ONPG assay of column effluent ........................................................................ 42
2-18 Particle effluent from the column ....................................................................... 44
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2-19 Coil for applying AMF ........................................................................................ 45
2-20 Inactivation study set up .................................................................................... 46
3-1 alpha-Amylase (PDB ID: 6TAA) with Lysine residues marked in Yellow ............ 48
3-2 Terminal amine of alpha-Amylase (PDB ID: 6TAA) ............................................ 49
3-3 Free alpha-Amylase activity normalized by the maximum .................................. 50
3-4 Free beta-Galactosidase activity normalized by the maximum ........................... 51
3-5 Normalized activity of alpha-Amylase conjugated to Iron Oxide Nanoparticle .... 52
3-7 Activity under AMF: Study 1: Temperature throughout the process ................... 55
3-8 Activity under AMF: Study 2: Temperature throughout the process ................... 56
3-9 Activity under AMF: Study 3: Temperature throughout the process ................... 58
3-10 Activity of conjugated alpha-Amylase under AMF .............................................. 59
3-11 IO-CMDX Beta gal conjugate Study 1: Temperature throughout the process .... 60
3-12 IO-CMDX Beta gal conjugate Study 2: Temperature throughout the process .... 61
3-13 Activity of conjugated beta-Gal under AMF ........................................................ 63
A-1 Scheme 1: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure starch solution only ...................................................................... 66
A-2 Scheme 2: Starch Iodine assay with Washed particles; '1'-'6' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure starch solution only ............................................................... 67
A-3 Scheme 3: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure starch solution only ...................................................................... 68
A-4 Scheme 5: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure starch solution only ...................................................................... 69
A-5 Scheme 6: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C1' refers to Negative Control and contains pure starch solution only; 'C2' refers to positive control and contains 2.5 ug of Amylase with Starch solution................................................. 70
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LIST OF ABBREVIATIONS
AMF Alternating Magnetic Field
DLS Dynamic Light Scattering
IO Iron Oxide
ILP Intrinsic Loss Power
ONPG o-nitrophenyl-β-D-galactoside
SAR Specific Absorption Rate
SQUID Superconducting Quantum Interference Device
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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science
ACTIVITY MEASUREMENTS OF ENZYMES CONJUGATED TO SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES UNDER AN
ALTERNATING MAGNETIC FIELD
By
Tapomoy Bhattacharjee
May 2014
Chair: Carlos Rinaldi Major: Chemical Engineering
Iron oxide magnetic nanoparticles have the capacity to dissipate the energy of an
alternating magnetic field (AMF) in the form of heat. Heat is generated either due to
Brownian relaxation, i.e., due to physical rotation of particles under AMF or due to Néel
Relaxation, i.e., rotation of magnetic dipole only. This physical property can be used
effectively for inducing local heating phenomena. Enzymes can be inactivated by
inducing heat energy. Here the objective was to inactivate an enzyme using the benefit
of local heating phenomena. Iron oxide nanoparticles synthesized by the co-
precipitation of Ferric Chloride and Ferrous Chloride were used. Particle sizes were
characterized using Dynamic Light Scattering (DLS). Relaxation properties of the
nanoparticles were determined by AC susceptibility measurements. -Amylase and β-
Galactosidase from Aspergillus Oryzae were used for heat inactivation study. Both -
Amylase and β-Galactosidase were covalently conjugated to Iron Oxide nanoparticles.
Conjugates were separated and washed. Remote inactivation studies were conducted
under a magnetic field of 33.7 kA/m at a frequency of 255 kHz. Activity of -Amylase
was measured using starch iodine assay at 618 nm. Activity of β-Galactosidase was
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measured using ONPG assay at 420 nm. Experiments done throughout this study
confirms that both the enzymes are bound to the surface of Iron Oxide nanoparticles.
Iron Oxide nanoparticles have also shown heat dissipation in presence of an AMF.
Conjugated particles were washed properly and no presence of free enzyme was
ensured. Activity of the conjugated particles, in case of both the enzymes, were
measure at different temperatures with external heating. It was observed that enzyme
bound to the IO nanoparticle can be inactivated by external heating. However, when
placed under AMF, both the enzymes conjugated to IO nanoparticles, did not show any
significant inactivation. Inference that can be drawn that local heating due to IO
nanoparticles under AMF was not sufficient to inactivate the enzymes.
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CHAPTER 1 BACKGROUND
1.1 Superparamagnetic Iron Oxide Nanoparticle
Superparamagnetism is magnetic phenomena demonstrated by nanoscale
ferromagnetic and ferrimagnetic materials. In this case the nanoparticles demonstrate
magnetic properties in presence of a magnetic field only.
Superparamagnetism occurs in case of Iron Oxide (IO) nanoparticles when the
material is composed of very small crystallites (< 30 nm). Superparamagnetic Iron
Oxide nanoparticles are generally made from ferrimagnetic iron oxides, such as
magnetite (Fe3O4) and maghemite (γ-Fe2O3).1 Superparamagnetic Iron Oxide
nanoparticles are widely used for numerous biomedical and bioengineering applications
such as MRI contrast enhancement 2, tissue repair, immunoassay, gene transfection 3,
4, tumor targeting, detoxification of biological fluids, hyperthermia 5, drug delivery and
cell separation, etc.6, 7 Superparamagnetic Iron Oxide nanoparticles are also useful as
probes for measurement of physical properties such as viscosity of a fluid 8. In a recent
study, Klyachko et. al.9, have shown that low-frequency alternating magnetic field
changes the reaction kinetics of an enzyme when covalently bound to the surface of
Superparamagnetic Iron Oxide nanoparticle. Iron oxide magnetic nanoparticles have the
capacity to dissipate the energy of an alternating magnetic field (AMF) in the form of
heat 10, 11. Heat is generated either due to Brownian relaxation, i.e., due to physical
rotation of particles under AMF or due to Néel Relaxation, i.e., rotation of magnetic
dipole only 12.
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1.1.1 Néel Relaxation
Single domain magnetic nanoparticles may respond to a time-varying magnetic
field by the rotation of its magnetic moment through realignment of the magnetic spin
without particle rotation. This phenomena is known as Néel Relaxation. Relaxation time
is defined as the amount of time the magnetization of the material remains in one stable
direction after removal of an external field.
Realignment of the magnetic spin of these particles requires an activation energy
EB [Fig 1-1]. IO nanoparticles usually relax by the Néel mechanism when kT is higher
than this EB.
Figure 1-1. Energy of a single domain particle under applied field 13
For Superparamagnetic Iron Oxide nanoparticles relaxing by Néel mechanism,
Néel Relaxation time is given by: 14
𝜏𝑁 = 𝜏0 exp (𝜋𝐾𝐷𝑚
3
6𝑘𝑇) … … … … … 1
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Where K the particle magnetocrystalline anisotropy constant, Dm is the diameter
of the magnetic core, k is Boltzmann’s constant, T is the absolute temperature, 𝜏0 is a
characteristic time.
1.1.2 Brownian Relaxation
Single domain magnetic nanoparticles may also respond to a time-varying
magnetic field by the rotation of the particle-locked magnetic moment by physical
particle reorientation through rotational Brownian motion. This phenomena is known as
Brownian Relaxation. In this case, realignment of the magnetic spin is not possible as
EB is higher than kT at normal temperature.
For Superparamagnetic Iron Oxide nanoparticles relaxing by Brownian
mechanism in a Newtonian fluid, Brownian Relaxation time is given by: 14
𝜏𝐵 =𝜋𝐷ℎ
3𝜂0
2𝑘𝑇(1 + 3𝜆)… … … … 2
Where 𝜏𝐵 Brownian Relaxation time, Dh is the particle hydrodynamic diameter,
𝜂0 is the suspending medium viscosity, k is Boltzmann’s constant, T is the absolute
temperature and λ is a non-dimensional slip coefficient characterizing the amount of
hydrodynamic slip at the nanoparticle surface.
Under an AMF, these particles dissipate heat due to the viscous interaction of the
particle surface with the surrounding fluid.
Relaxation occurs by the faster of these two mechanisms. An effective relaxation
time is given by:
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𝜏 =𝜏𝐵𝜏𝑁
𝜏𝐵 + 𝜏𝑁… … … … 3
1.2 Local Heating Phenomena
Local energy delivery using magnetite particulate matter was first introduced by
Gilchrist et al. in 1957 10. Local heating phenomena is widely used in case of magnetic
fluid hyperthermia 15. In 2011, Creixell et. al. reported that epidermal growth factor-
conjugated iron oxide nanoparticles targeting the epidermal growth factor receptor
(EGFR) result in a 99% reduction in cell viability and clonogenic survival when exposed
to an AMF, without a perceptible rise in medium temperature 5. In a more recent study,
Polo-Corrales et. al. have demonstrated that the surface temperature of iron oxide
nanoparticles in an alternating magnetic field (AMF) remains higher than the
temperature of the surrounding medium through the temperature induced change in
fluorescence of a thermoresponsive/fluorescent polymer consisting nanoparticle 16.
Under the influence of an AMF single domain magnetic nanoparticles can
dissipate heat by both relaxation mechanisms; both Néel and Brownian 1. This heat
generation is dependent on the amplitude and frequency of the AMF applied. According
to Rosensweig’s model17, volumetric energy dissipation rate from a superparamagnetic
nanoparticle (P) is given by:
𝑃 = 𝜋 𝜇𝑜𝑥0𝐻2𝑓2𝜋𝑓𝜏
[1 + (2𝜋𝑓𝜏)2]… … … … 4
Where, 𝑥0 is initial susceptibility of the nanoparticles, H is magnetic field amplitude,
𝜇𝑜 is permeability of free space, 𝑓 is frequency of AMF in Hz and 𝜏 is effective
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relaxation time. Increasing the field and frequency thus increases the amount of heat
generated by these magnetic nanoparticles.
The amount of heat generated by superparamagnetic nanoparticles can be
quantified by a parameter, SAR (Specific Absorption Rate). Unit of SAR is W/gm of
nanoparticle.
𝑆𝐴𝑅 = 𝐶𝑃
𝑚𝑠𝑎𝑚𝑝𝑙𝑒
𝑚𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒
∆𝑇
∆𝑡|
𝑡→0… … … … 5
Here, 𝐶𝑃 is heat capacity of the nanoparticle suspension medium, 𝑚𝑠𝑎𝑚𝑝𝑙𝑒 is
mass of the sample, 𝑚𝑛𝑎𝑛𝑜𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒is mass of the nanoparticles suspended and
∆𝑇
∆𝑡|
𝑡→0is the initial slope of the time vs. temperature curve for the suspension under
AMF 18. Now, as SAR is field and frequency dependent, Kallumadil et. al. introduced a
new parameter, Intrinsic Loss Power (ILP) 11. Unit of ILP is nHm2/kg of nanoparticle.
𝐼𝐿𝑃 =𝑆𝐴𝑅
𝐻2𝑓… … … … 6
1.3 Objective
Enzyme stability is related to enzyme structure and to factors in the
microenvironment. Temperature dependence of enzyme activity is related to the
structure, thermodynamics and kinetics of the enzymatic reaction19. Different enzymes
have previously been immobilized on magnetic nanoparticles for several application
such as enzyme recovery20, 21, bio sensing22, 23 etc. The objective of this study was to
use Superparamagnetic Iron Oxide nanoparticles with surface modification, to control
activity of an enzyme remotely. Previous attempts by Klyachko et. al. 9 incorporates a
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non-heating magnetic field to change the reaction kinetics of an enzyme immobilized on
the surface of IO nanoparticles predominantly by deformation of the enzyme by
mechanical stress. As IO nanoparticles dissipate heat under an AMF, enzymes
covalently conjugated on to the surface of these nanoparticles also receive the energy.
Here, the idea was to change the activity of an enzyme using this heat energy. In that
way, by controlling amplitude, frequency and application time of AMF, activity of the
enzyme can be controlled.
1.4 Bio-Conjugation Chemistry
1.4.1 EDC/ NHS Chemistry
EDC (or EDAC; 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) is
used for conjugating biological substances containing carboxylates and amines. Both
EDC itself and the isourea formed as the by-product of the crosslinking reaction are
water-soluble and may be removed easily by dialysis or gel filtration. Here the Iron oxide
nanoparticles were coated with dextran and thus contains carboxylic groups. -Amylase
enzyme contains amine groups. EDC reacts with carboxylic acids to create an active-
ester intermediate. In the presence of an amine nucleophile, an amide bond is formed
with release of an isourea by-product. EDC may be used to form active ester
functionalities with carboxylate groups using the water-soluble compound NHS that
couple rapidly with amines on target molecules.
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Figure 1-2. EDC Chemistry; R is IO nanoparticle and R' is the enzyme here 24
1.4.2 Sulfo SMCC Chemistry
Sulfo-SMCC, sulfosuccinimidyl-4-( N-maleimidomethyl)cyclohexane-1-
carboxylate, is a watersoluble analog of SMCC that possesses a negatively charged
sulfonate group on it NHS ring. Sulfo-SMCC reacts with amine-containing molecules to
form stable amide bonds. Its maleimide end then reacts to a sulfhydryl-containing
compound to create a thioether linkage. Here the Iron oxide nanoparticles were coated
with Poly Ethylene Glycol and contains amine groups. -Amylase enzyme contains
sulfhydryl groups.
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Figure 1-3. Sulfo- SMCC Chemistry; R is the IO nanoparticle and R' is the enzyme 24
1.5 -Amylase
-Amylase is a protein enzyme (EC 3.2.1.1) that catalyzes the hydrolysis of
internal -1, 4-glucan links in polysaccharides containing 3 or more -1, 4-linked D-
glucose units, such as starch, yielding smaller units like glucose and maltose 25. It was
named -Amylase as the hydrolysis products are in the alpha configuration 26. -
Amylase has a molecular weight ranging from 51 kDa to 54 kDa depending on its
source. -Amylase form Aspergillus Oryzae (PDB ID: 6TAA) consists of 499 amino
acids27.
The main substrate of -Amylase is starch. Starch is a polymer of glucose linked
to one another through the C1 oxygen, known as the glycosidic bond. Two types of
glucose polymers are present in starch: amylose and amylopectin. Amylose is a linear
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polymer consisting of up to 6000 glucose units with -1-4 glycosidic bonds 28. -
amylase is widely used in the starch industry to hydrolyze starch into fructose and
glucose syrups 29. -amylase has its application in detergent30, 31, textile31, food, biofuel
and paper industry as well 32.
Activity of -Amylase on a starch substrate can be monitored by a
spectrophotometric method. Amylose of starch forms a blue colored complex with
Iodine which can be quantified using a spectrophotometer 33. This color of starch iodine
complex is a result of iodine entrapped inside a helical chain of amylose 34.
1.6 β-Galactosidase
β-Galactosidase (β-gal; EC 3.2.1.23), is a hydrolase enzyme that catalyzes the
hydrolysis of D-galactosyl residues from polymers, oligosaccharides or secondary
metabolites 35. It hydrolyses β(1–3) and β(1–4) galactosyl bonds in oligo- and
disaccharides and also catalyzes the reverse reaction of thehydrolysis, often called
transglycosylation 36. β-Galactosidase from Aspergillus Oryzae (PDB ID: 4IUG) has 985
residues 36.This enzyme is mainly used for lactose removal from milk products and for
the production of galactosylated products 37, 38. This enzyme’s function in the cell is to
cleave lactose to glucose and galactose so that they can be used as carbon/energy
sources. The synthetic compound o-nitrophenyl-β-D-galactoside (ONPG) is also
recognized as a substrate and cleaved to yield galactose and o-nitrophenol which has a
yellow color. β-Galactosidase activity can be measured by this method 39.
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CHAPTER 2 MATERIALS AND METHODS
2.1 Materials
-Amylase, Potassium Iodide, Iodine and starch from Potato was bought from
Sigma. EDC, NHS and Sulfo-SMCC were bought from Thermo Scientific™ Pierce™.
IO-CMDx and PEG-550 Iron oxide nanoparticles were produced in the laboratory and
provided by A C Bohorquez. Sephacryl S400 HR gel filtration medium was bought from
GE Healthcare Life Sciences. Ambrell® EasyHeat LI 8310 induction heater was used to
apply the alternating magnetic field. An AccuBlock™ Digital dry bath is used for heating.
Spectrophotometric analysis was done using a Shimadzu UV-2600 spectrophotometer.
2.2 Nanoparticle Characterization
2.2.1 PEG550 Batch-1
Hydrodynamic diameter of PEG550-Batch 1 particles were measured by dynamic
light scattering (DLS). Hydrodynamic diameter of these particles was 64 nm [Fig: 2-1]
Figure 2-1. DLS of PEG550-Batch 1
Zeta potential of these nanoparticles were 15.3 +/- 0.72 mV [Fig: 2-2]
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Figure 2-2. Zeta potential of PEG550-Batch 1 particles
SAR of PEG550-Batch 1 particles were measured at 37.5 kA/m field and 340
kHz frequency. To calculate the time dependent temperature rise 200 µL of PEG550
suspension in water (0.36 mgFe/mL) was subjected to the AMF for 60 s and first 20 s of
temperature data was used to calculate the slope [Fig: 2-3]. ILP was calculated from
SAR.
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Figure 2-3. SAR measurement of PEG550-Batch 1 particles
SAR of PEG550 Batch 1 was 1099 W/g of Fe in water and 947 W/g of Fe in 1.5%
Agar. ILP of PEG550 in water was 2.3 nHm2/kg of Fe. ILP of PEG550 particles
immobilized in a 1.5% agar matrix was 1.98 nHm2/kg of Fe. 14% of particles relax by
Brownian mechanism.
2.2.2 PEG550 Batch-2
Hydrodynamic diameter of PEG550-Batch 2 particles was 53 nm.
Figure 2-4. DLS of PEG550-Batch 2
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Zeta potential of these nanoparticles were 11.62 +/- 1.89 mV [Fig 2-5]
Figure 2-5. Zeta potential of PEG550-Batch 2 particles
Saturation magnetization is measured with a SQUID and is found to be 26 kA/m.
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Figure 2-6. M vs. H Plot for PEG550 batch 2 particles
SAR of PEG550-Batch 2 particles were measured at 37.5 kA/m field and 340
kHz frequency. To calculate the time dependent temperature rise 200 µL of PEG550
suspension in water (2.8 mgFe/mL) was subjected to the AMF for 60 s and first 20 s of
temperature data was used to calculate the slope [Fig: 2-7]. ILP was calculated from
SAR. SAR of PEG550 Batch 2 in water was 540 W/g of Fe. ILP of PEG550 Batch 2 in
water was 1.13 nHm2/kg of Fe. SAR of these particles at 50°C (255 kHz and 33.7 kA/m)
is 57 W/g of Fe.
28
Figure 2-7. SAR measurement of PEG550-Batch 2 particles
2.2.3 IO-CMDX particles:
Hydrodynamic diameter of IO-CMDX particles was 49 nm.
Figure 2-8. DLS of IO-CMDX particles
Zeta potential of IO-CMDX particles was -5.9 +/- 1.83 mV.
29
Figure 2-9. Zeta potential of IO-CMDX particles
Saturation magnetization is measured with a SQUID and is found to be 200
kA/m.
Figure 2-10. M vs. H Plot for IO-CMDX particles
SAR of IO-CMDX particles were measured at 37.5 kA/m field and 340 kHz
frequency. 200 µL of IO-CMDX suspension (10 mg particle/mL) in water was subjected
30
to the AMF for 60 s and first 20 s of temperature data was used to calculate the slope
[Fig: 2-11]. SAR of IO-CMDX in water was 598 W/g of Fe. ILP of IO-CMDX in water was
1.251 nHm2/kg of Fe. SAR of these particles at 50°C (255 kHz and 33.7 kA/m) is 30
W/g of Fe.
Figure 2-11. SAR measurement of PEG550-Batch 2 particles
2.3 Free Enzyme Activity
2.3.1 -Amylase activity
To know the absorption wavelength of the Starch-Iodide complex formed, a 100
µg/mL starch solution was prepared. Potassium Iodide and Iodine was added to water
to a final concentration of 2.5mM to form the Iodine reagent. 20 µL of 2.5mM Iodine
reagent is added to the starch solution. A full range (300 -700 nm) absorption scan was
then made which gave the absorption wavelength as 618 nm.
31
Figure 2-12. Absorbane of starch iodine complex at different wavelength
Activity of -Amylase was measured by a method described by Xiao et. al. 40. To
quantify the activity of free enzyme (pH 7.4) at different temperatures, 200 µL of 2
mg/mL starch solution was incubated with 10 µL of 24 µg /mL free -Amylase solution
for 45 min using a dry bath at 5 different temperatures (20°C, 30°C, 45°C, 60°C and
80°C). After 45 min the reaction was quenched with 20 µL of 1M HCl and 20 µL of
Iodine reagent was added to that. Absorbance of these solutions were measured at 618
nm. These are Asample.
Again, 200 µL of 2 mg/mL starch solution was diluted with 10 uL PBS 1X and
incubated for 45 min using a dry bath at 5 different temperatures (20°C, 30°C, 45°C,
60°C and 80°C). After 45 min the reaction was quenched with 20 uL of 1M HCl and 20
uL of Iodine reagent was added to that. Absorbance of these solutions were measured
at 618 nm. These are Acontrol.
Activity was defined as U (µg of starch degraded per minute per µg of enzyme
used).
32
𝑈 = (𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒)
(𝐴𝑢𝑔 × 0.1) × 45 × 0.24… … … … 7
Where, Aug is the absorbance for 1 µg/mL solution.
Normalized activity was used which was defined as:
𝑈𝑁 = (𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒)
(𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒)| 𝑚𝑎𝑥
… … … … … 8
2.3.2 β-Galactosidase activity
β-Galactosidase activity was measure by the method described by Miller 39. Here
β-galactosidase was assayed by measuring hydrolysis of the chromogenic substrate, o-
nitrophenyl-β-Dgalactoside (ONPG) as shown in Fig 2-13. The reaction was stopped by
adding Na2CO3 which shifts the reaction mixture to pH 11. At this pH most of the o-
nitrophenol gets converted to the yellow colored anionic form and β-galactosidase is
inactivated. The amount of o-nitrophenol formed was measured by determining the
absorbance at 420 nm.
Figure 2-13. beta-Galactosidase assay
To measure β-Galactosidase activity, a 5 U/mL solution of β-Galactosidase in
PBS 1X was prepared. 250 µL of 10 mg/ mL ONPG solution in PBS 1X was incubated
with 20 µL of β-Galactosidase solution for 60 min. 20 µL of 0.5M Na2CO3 solution was
33
added to the reaction mixture to quench the reaction. Absorbance of the reaction
mixture was measured at 420 nm. These are Asample.
Again, 250 µL of 10 mg/ mL ONPG solution was incubated with 20 µL of PBS 1X
for 60 min and 20 µL of 0.5M Na2CO3 solution was added to it. Absorbance of this
solution was measured at 420 nm. These are Acontrol.
Activity was defined as UG (µg of o-Nitrophenol produced per minute per U of
enzyme used).
𝑈𝐺 = (𝐴𝑆𝑎𝑚𝑝𝑙𝑒 − 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙)
(𝐴𝑢𝑔 × 0.1) × 60 × 0.01… … … … 9
Where, Aug is the absorbance for 1 µg/mL o-Nitrophenol solution.
Normalized activity was used which was defined as:
𝑈𝐺𝑁 = (𝐴𝑆𝑎𝑚𝑝𝑙𝑒 − 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙)
(𝐴𝑆𝑎𝑚𝑝𝑙𝑒 − 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙)| 𝑚𝑎𝑥
… … … … 10
2.4 Conjugated Enzyme activity
2.4.1 -Amylase activity
To quantify the activity of conjugated enzyme (pH 7.4) at different temperatures,
200 µL of 2 mg/mL starch solution was incubated with 10 uL of PEG550 conjugated -
Amylase solution for 45 min using a dry bath. After 45 min the reaction is quenched with
20 uL of 1M HCl and 20 uL of Iodine reagent is added to that. Absorbance of these
solutions were measured at 618 nm. These are Asample here. Activity normalized by
maximum were measured at 5 different temperatures (20°C, 30°C, 45°C, 60°C and
80°C).
34
2.4.2 β-Galactosidase activity
To measure conjugated β-Galactosidase activity, 200 µL of 10 mg/ mL ONPG
solution in PBS 1X was incubated with 20 µL of conjugated β-Galactosidase solution for
60 min. 20 µL of 0.5M Na2CO3 solution was added to the reaction mixture to quench the
reaction. Absorbance of the reaction mixture was measured at 420 nm. These are
Asample.
Again, 200 µL of 10 mg/ mL ONPG solution was incubated with 20 µL of PBS 1X
for 60 min and 20 µL of 0.5M Na2CO3 solution was added to it. Absorbance of this
solution was measured at 420 nm. These are Acontrol.
Now, 20 µL of conjugated β-Galactosidase was diluted using 200 µL PBS. 20 µL
of 0.5M Na2CO3 solution was added to it. These are Aparticle.
Activity was defined as UG (µg of o-Nitrophenol produced per minute per U of
enzyme used).
𝑈𝐺 = (𝐴𝑆𝑎𝑚𝑝𝑙𝑒 − 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒)
(𝐴𝑢𝑔 × 0.1) × 60 × 0.01… … … … 11
Where, Aug is the absorbance for 1 µg/mL o-Nitrophenol solution.
Normalized activity was used which was defined as:
𝑈𝐺𝑁 = (𝐴𝑆𝑎𝑚𝑝𝑙𝑒 − 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒)
(𝐴𝑆𝑎𝑚𝑝𝑙𝑒 − 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒)| 𝑚𝑎𝑥
… … … … 12
2.5 Bio-Conjugation
2.5.1 For -Amylase
Different schemes were attempted to conjugate Iron Oxide nanoparticles to -
Amylase using EDC and NHS as cross-linker. -Amylase could not be conjugated to IO-
35
CMDX nanoparticles using EDC and NHS as cross-linker. Details of these procedures
are described in Appendix A.
-Amylase was conjugated to PEG550 (batch 1) nanoparticles using Sulfo-
SMCC as cross-linker. To do so, 5 mL of PEG550 particle suspension in water was
taken (pH=5.81). The buffer was changed to PBS 1x using a 30 kDa membrane under
centrifugation (pH=7.2); final volume was 6mL. 2.2 mg of dry Sulfo-SMCC was taken in
two vials. 2.5 mL of PEG550 suspension was added to each vial. Reaction was carried
on for 1 hour under room temperature. These reaction mixtures were washed with PBS
1x two times using 30kDa membrane using centrifuge. 2 mL of 45 mg/mL -Amylase
solution in PBS 1x was added to each vial.all vial is shakekeat another is shaken at 25
°C for 24 hours. After reaction these particles were washed using a Sephacryl S 400
HR column. Washed particles confirm presence of -amylase on incubation with
starch followed by the addition of iodine reagent. [Figure 2-14]. For further conjugations
9 mg of -Amylase was used and reaction was carried out at 25 °C.
Figure 2-14. Tapomoy Bhattacharjee. Scheme 8: Starch Iodine assay with Washed particles; ‘S1’ and ‘S2’ refers to Sample and contains washed particles after reaction; 'C2' refers to Negative Control and contains pure starch solution only; 'C1' refers to positive control and contains 2.5 ug of Amylase. 10th November, 2013.
36
2.5.2 For β- Galactosidase
Two different conjugation schemes were attempted to conjugate β-Galactosidase
to Iron Oxide nanoparticles.
2.5.2.1 Scheme 1
20 mg (2 mg core) IO-CMDx particles were suspended in 2 mL DI water
(pH=5.4). 10 mg of dry EDC and 6 mg of Sulfo-NHS was added to the particle
suspension. This was shaken for 15 min. 22.4 mg (~ 300 U) of β-galactosidase was
added to the reaction mixture. pH was adjusted to 7.5 using 1M NaOH. This reaction is
carried for 3 hours under room temperature. These particles were washed using two
Sephacryl S 400 HR column collecting first 16 particle drops each time. To see if
there is any conjugated β-galactosidase bound to the particle, ONPG assay was used.
200 uL ONPG incubated with 30 uL particle solution for 3 hour. For control, 200 uL PBS
incubated with 30 uL particle solution for 3 hour. The reaction was stopped by 30 µL of
0.5M Na2CO3 solution. Absorbance was measured at 420 nm.
Table 2-1. beta- gal conjugation scheme 1: Absorbance of sample and control after incubation
Run Absorbance at 420 nm
Sample control
Run 1 0.477 0.336
Run 2 0.487 0.315
Run 3 0.509 0.332
Run 4 0.497 0.348
Average 0.493 0.333
SD 0.014 0.014
Higher absorbance in sample solution indicates the presence of β-galactosidase
on the surface of the particle.
37
2.5.2.2 Scheme 2
1 mL of PEG550 particle suspension (2.8 mg Fe/mL) in water was taken. The
buffer was changed to PBS 1x using a 30 kDa membrane under centrifugation. 2.2 mg
of dry Sulfo-SMCC was added to the particle suspension. Reaction was carried on for 1
hour under room temperature. This reaction mixtures were washed with PBS 1x two
times using 30kDa membrane under centrifugation. 22.4 mg (~ 300 U) of β-
galactosidase was added to the vial. Reaction was carried on for 24 hour under 25 °C
temperature. After reaction these particles were washed using two Sephacryl S 400
HR column collecting first 16 particle drops each time. To see if there is any
conjugated β-galactosidase bound to the particle, ONPG assay was used. 200 uL
ONPG incubated with 20 uL particle solution for 3 hour. For control, 200 uL PBS
incubated with 20 uL particle solution for 3 hour. The reaction was stopped by 20 µL of
0.5M Na2CO3 solution. Absorbance was measured at 420 nm.
Table 2-2. beta- gal conjugation scheme 2: Absorbance of sample and control after
incubation
Run Absorbance at 420 nm
sample control
Run 1 0.826 0.397
Run 2 0.878 0.402
Run 3 1.048 0.439
Run 4 0.973 0.424
Average 0.931 0.416
SD 0.099 0.020
Higher absorbance in sample solution indicates the presence of β-galactosidase
on the surface of the particle.
38
2.6 Wash Procedure of Conjugated Enzyme
2.6.1 Amicon Ultra-15 Centrifugal Filter Units
At the initial stage Amicon Ultra-15 Centrifugal Filter Units with 100 kDa
molecular weight cutoff were used to separate conjugated particles from free enzyme. 2
mL of enzyme conjugated particles were washed 11 times for 10 min each at 2500 rpm.
Each time the washed residue is re-suspended in 2mL of PBS 1X. After each wash
effluent were monitored using a UV spectrophotometer. This type of wash was
discarded as traces of free enzyme were found even after 11 washes [Fig 2-15]
Figure 2-15. Wash using Amicon Ultra-15 Centrifugal Filter Units
39
2.6.2 Sephacryl S400 HR Columns
To avoid any free enzyme a gel filtration step was introduced using Sephacryl
S400 HR as separating medium. This separating medium has a MW cutoff of 9000kDa
and nanoparticle cutoff of 31nm (Hydrodynamic diameter).
To load these columns 12 mL of raw media was diluted with 5 mL PBS 1X to
form a slurry. Blank PD-10 columns were washed with 20% ethanol and filled with PBS
1X to remove any air bubble. The slurry was then added slowly and continuously while
the solid media settles down along the column. The column was then washed
thoroughly using PBS 1X.
2.6.2.1 -Amylase separation
To confirm the separation efficiency, 2mL of PEG550 (0.36 mg Fe/mL) particles
were mixed with 9mg of -Amylase and concentrated to 0.5 mL using a 30 kDa
membrane under centrifugation. This mixture was then run through Sephacryl S400 HR
columns. 95 droplets coming out of the column (after the first drop from the particle
band) were collected in a 96 well plate. 100 uL of starch solution was added to each
well. 96th well is a control and pure starch solution is added to it. Absorbance of each
well was measured using a plate reader at 618 nm. This is absorbance of the particles
[Table 2-3]. This plate was shaken at 25C for 90 min. 20uL of 1M HCl was added to
each well to quench the reaction. 20uL of Iodine reagent is added to each well.
Absorbance was measured using a plate reader at 618 nm. This is absorbance of the
assay [Table 2-4]. The difference of Table 2-3 and Table 2-4 is normalized by the
difference of H12 of both table and plotted in Table 2-5. It shows that the free enzyme
starts to elute after 21 drops. A10 and A11, i.e., drop 10 and 11 has experimental error.
40
Figure 2-16. Tapomoy Bhattacharjee. Starch-Iodine assay of column effluent. 6th December, 2013.
Table 2-3. Absorbance of the particles; A1-12 refers to drop 1-12, B1-12 refers to drop 13-24 and so on, H 12 is the control
1 2 3 4 5 6 7 8 9 10 11 12
A 0.058 0.068 0.093 0.161 0.139 0.118 0.083 0.14 0.202 0.336 0.209 0.138
B 0.213 0.218 0.213 0.192 0.164 0.134 0.099 0.105 0.163 0.242 0.198 0.154
C 0.141 0.125 0.11 0.103 0.096 0.09 0.086 0.083 0.081 0.077 0.076 0.072
D 0.086 0.073 0.073 0.069 0.069 0.066 0.066 0.062 0.062 0.061 0.062 0.058
E 0.057 0.06 0.058 0.067 0.058 0.056 0.056 0.056 0.057 0.055 0.054 0.051
F 0.054 0.052 0.056 0.053 0.054 0.053 0.051 0.051 0.054 0.051 0.054 0.048
G 0.045 0.049 0.049 0.052 0.054 0.052 0.049 0.049 0.05 0.049 0.048 0.048
H 0.05 0.048 0.048 0.049 0.048 0.048 0.049 0.06 0.047 0.049 0.049 0.048
41
Table 2-4. Absorbance of the starch iodine assay; A1-12 refers to drop 1-12, B1-12 refers to drop 13-24 and so on, H 12 is the control
1 2 3 4 5 6 7 8 9 10 11 12
A 0.828 0.764 0.783 0.866 0.827 0.799 0.719 0.806 0.852 0.832 0.47 0.849
B 0.908 0.903 0.917 0.882 0.858 0.818 0.773 0.787 0.814 0.757 0.712 0.678
C 0.658 0.638 0.579 0.512 0.479 0.408 0.32 0.271 0.215 0.15 0.126 0.113
D 0.104 0.122 0.092 0.086 0.082 0.08 0.075 0.072 0.072 0.071 0.068 0.069
E 0.064 0.065 0.067 0.064 0.063 0.061 0.06 0.081 0.059 0.058 0.057 0.056
F 0.058 0.057 0.056 0.056 0.055 0.053 0.054 0.054 0.055 0.053 0.052 0.051
G 0.055 0.052 0.051 0.052 0.052 0.052 0.051 0.058 0.05 0.05 0.05 0.057
H 0.051 0.05 0.05 0.049 0.05 0.05 0.049 0.049 0.049 0.051 0.05 0.812
Table 2-5. Difference of absorbance normalized by control; A1-12 refers to drop 1-12, B1-12 refers to drop 13-24 and so on, H 12 is the control
1 2 3 4 5 6 7 8 9 10 11 12
A 1.01 0.91 0.90 0.92 0.90 0.89 0.83 0.87 0.85 0.65 0.34 0.93
B 0.91 0.90 0.92 0.90 0.91 0.90 0.88 0.89 0.85 0.67 0.67 0.69
C 0.68 0.67 0.61 0.54 0.50 0.42 0.31 0.25 0.18 0.10 0.07 0.05
D 0.02 0.06 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01
E 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.03 0.00 0.00 0.00 0.01
F 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
G 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.00 0.00 0.00 0.01
H 0.00 0.00 0.00 0.00 0.00 0.00 0.00 -0.01 0.00 0.00 0.00 1.00
To overcome this, enzyme conjugated Iron Oxide nanoparticles were separated
from free enzyme using two freshly prepared columns. Only 16 drops were collected
each time after the first particle drop.
2.6.2.2 β-Galactosidase separation
To confirm the separation efficiency, 20 mg of beta-gal was dissolved in 0.5 mL
PBS 1X. This mixture was then run through Sephacryl S400 HR column. 192 droplets
coming out of the column (after the introduction of protein solution) were collected in two
96 well plate. 200 uL of ONPG solution was added to each well. This plate was shaken
at 25C for 120 min. 20uL of 0.5M Na2CO3 was added to each well to quench the
reaction [Fig 2-16]
42
Figure 2-17. Tapomoy Bhattacharjee. ONPG assay of column effluent. 25th February, 2014
Absorbance was measured using a plate reader at 420 nm. This is absorbance
of the assay. It shows that the free enzyme starts to elute after 92 drops (H8) [Table 2-
6]
43
Table 2-6. Absorbance of the ONPG assay from column effluent; A1-12 refers to drop 1-12, B1-12 refers to drop 13-24 and so on
1 2 3 4 5 6 7 8 9 10 11 12
A 0.049 0.046 0.048 0.048 0.05 0.048 0.05 0.049 0.047 0.049 0.048 0.048
B 0.046 0.047 0.046 0.047 0.049 0.049 0.047 0.048 0.048 0.051 0.052 0.048
C 0.055 0.05 0.05 0.05 0.05 0.049 0.047 0.046 0.047 0.049 0.047 0.047
D 0.05 0.045 0.045 0.047 0.044 0.046 0.045 0.046 0.049 0.05 0.054 0.05
E 0.049 0.053 0.049 0.051 0.051 0.052 0.047 0.048 0.048 0.047 0.051 0.053
F 0.047 0.053 0.047 0.045 0.046 0.046 0.045 0.052 0.064 0.056 0.059 0.056
G 0.055 0.051 0.053 0.054 0.054 0.069 0.062 0.075 0.076 0.098 0.117 0.102
H 0.061 0.064 0.067 0.069 0.083 0.091 0.082 0.117 0.109 0.124 0.159 0.2
I 0.761 1.142 1.584 1.799 1.895 1.931 1.929 1.933 1.88 1.937 1.939 1.901
J 1.585 1.699 1.778 1.808 1.835 1.88 1.879 1.896 1.867 1.933 1.938 1.93
K 1.488 1.666 1.789 1.815 1.868 1.878 1.902 1.91 1.895 1.947 1.943 1.907
L 1.508 1.683 1.747 1.787 1.843 1.867 1.872 2.175 1.882 1.903 1.924 1.891
M 1.49 1.678 1.803 1.816 1.893 1.893 1.896 1.908 1.901 1.914 1.915 1.871
N 1.546 1.722 1.806 1.841 1.875 1.892 1.896 1.9 1.876 1.887 1.868 1.815
O 1.581 1.707 1.825 1.778 1.815 1.816 1.826 1.795 1.743 1.755 1.717 1.651
P 1.498 1.602 1.63 1.645 1.663 1.644 1.652 1.647 1.592 1.569 1.524 1.484
Again, 20 mg of IO-CMDX was dissolved in 0.5 mL PBS 1X. This suspension
was then run through Sephacryl S400 HR column. 96 droplets coming out of the column
(after the introduction of particle suspension) were collected in a 96 well plate.
It shows that the free particle starts to elute after 80 drops (G8) [Fig 2-17]. If 16
drops collected from the first column, there will be some free enzyme in the effluent.
But, amount of this free enzyme is small and gets removed at the second column.
44
Figure 2-18. Tapomoy Bhattacharjee. Particle effluent from the column. 25th February, 2014.
2.7 Inactivation Study
2.7.1 For -amylase
To remotely inactivate the enzyme bound to the surface of Superparamagnetic
Iron Oxide Nanoparticles, an Alternating Magnetic Field (AMF) of 33.7 kA/m and 255
kHz is applied. In an 8 well strip 200 µL of starch solution was taken in every well. The
well strip was covered with a paraffin film and placed inside the coil [Fig 2-18]. The coil
was placed inside an incubator of constant temperature [Fig 2-19]. Starch stock solution
was preheated inside the coil to a certain temperature. The well strip was then taken out
and 10 µL of conjugated particle solution was added in every well. The well strip was
again covered with a paraffin film and place inside the coil. AMF was applied for 90 min
(33.7 kA/m, 255 kHz). The well strip was then taken out of the coil and 20 µL of 1M HCl
45
was added to each well to quench the reaction. 20 µL of iodine reagent was added to
each well. Absorbance of each well was then measured at 618 nm.
For control the above mentioned procedure was followed without application of
AMF.
Figure 2-19. Tapomoy Bhattacharjee. Coil for applying AMF. 20th September, 2013.
46
Figure 2-20. Tapomoy Bhattacharjee. Inactivation study set up. 20th September, 2013.
2.7.2 For β-galactosidase
To remotely inactivate the enzyme bound to the surface of Superparamagnetic
Iron Oxide Nanoparticles, an Alternating Magnetic Field (AMF) of 33.7 kA/m and 255
kHz is applied. In an 8 well strip 200 µL of ONPG solution was taken in 3 wells in such a
way that the samples goes at the center of the coil. Other wells were filled with DI water.
For PEG550 conjugates 10 µL and for IO-CMDX conjugates 30 µL of conjugated
particle solution was added in every well. The well strip was sealed with a film and
placed inside the coil [Fig 2-18]. The coil was placed inside an incubator of constant
temperature [Fig 2-19]. AMF was applied for 2hr for PEG550 conjugates and 3hr for IO-
CMDX conjugates. The reaction was quenched after application of AMF using 0.5 M
Na2CO3. Absorbance of each well was then measured at 420 nm.
48
CHAPTER 3 RESULTS
3.1 Selectivity of Sulfo-SMCC over EDC
EDC reacts with carboxylic acids to create an active-ester intermediate. This
ester intermediate is stabilized by NHS. In the presence of an amine nucleophile, an
amide bond is formed with release of an isourea by-product. In case of a protein, this
amine nucleophile is generated by the terminal amine group and Lysine residue. To
have this primary amine groups in a nucleophilic state pH of the reaction medium
should be higher than the pKa of the individual residue.
pKa of individual residues of -Amylase (PDB ID: 6TAA) is obtained from The
PROPKA Web Interface. -Amylase has 20 Lysine residues and only 3 of them has no
columbic interaction with other residues [Fig 3-1]. Average pKa of these Lysine residues
is approximately 10. -Amylase has only one terminal amine group with a pKa of 7.27.
Figure 3-1. alpha-Amylase (PDB ID: 6TAA) with Lysine residues marked in Yellow
49
The terminal amine works as a nucleophile at pH of 7.4 (PBS). From The
PROPKA Web Interface it is found that this terminal amine is 36% buried and is in
columbic interaction with three Aspartic acid residues. So, it is inferred that this terminal
amine nucleophile does not interact with the ester intermediate due to steric hindrance
and columbic interaction with other residues.
Figure 3-2. Terminal amine of alpha-Amylase (PDB ID: 6TAA)
In case of Lysine residues, pH of the reaction mixture has to be increased
beyond 10 in order to produce amine nucleophile. Now, Half-life of the intermediate
ester is in between 4-5 hours at pH 7 and 0°C. This half-life decreases to 10 min at pH
8.6 and 4°C. 24 So, it is inferred here that increasing the pH of the reaction medium
beyond 10, force the intermediate ester to degrade immediately.
Sulfo-SMCC reacts with amine-containing molecules to form stable amide bonds.
Its maleimide end then reacts to a sulfhydryl-containing compound to create a thioether
linkage.
50
3.2 Activity of free enzyme
3.2.1 For -Amylase
Difference of absorbance of Control and Sample after following the procedure
described in Chapter 2.3, is listed in Table 3-1. Normalized activity is plotted in Fig 3-3.
Table 3-1. (𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒) for Starch Iodine assay of free enzyme after 45 min
incubation at different temperature
10 µL of 24 µg / mL free enzyme incubated with starch solution
(𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒)
Temperature Run 1 Run 2 Run 3 Average SD
20 C 1.155 1.127 1.144 1.142 0.014
30 C 1.169 1.146 1.148 1.154 0.013
45 C 1.044 1.063 1.09 1.066 0.023
60 C 0.86 0.784 0.841 0.828 0.040
80 C 0.188 0.055 0.044 0.096 0.080
Figure 3-3. Free alpha-Amylase activity normalized by the maximum
51
3.2.2 For β-Galactosidase
Difference of absorbance of Sample and Control after following the procedure
described in chapter 2.3, is listed in Table 3-2. Normalized activity is plotted in Fig 3-4.
Table 3-2. (𝐴𝑠𝑎𝑚𝑝𝑙𝑒 − 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙) for ONPG assay of free beta-Galactosidase after 60
min incubation at different temperature
20 µL of 5U / mL free enzyme incubated with 250 µL of ONPG solution
(𝐴𝑠𝑎𝑚𝑝𝑙𝑒−𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 )
Temperature, C Run 1 Run 2 Run 3 Average SD
20 0.522 0.317 0.424 0.421 0.103
30 0.589 0.601 0.636 0.609 0.024
45 0.885 0.886 0.882 0.884 0.002
60 0.721 0.750 0.749 0.740 0.016
70 0.169 0.173 0.161 0.168 0.006
80 0.010 0.012 0.017 0.013 0.004
Figure 3-4. Free beta-Galactosidase activity normalized by the maximum
52
3.3 Activity of Enzyme Conjugated to Iron Oxide nanoparticle
3.3.1 For -Amylase
Difference of absorbance of Control and Sample after following the procedure
described in chapter 2.4, is listed in Table 3-3. Normalized activity is plotted in Fig 3-5.
Table 3-3. (𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒) for Starch Iodine assay of conjugated enzyme after
45 min incubation at different temperature
10 µL of Conjugated particle incubated with starch solution
(𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 − 𝐴𝑠𝑎𝑚𝑝𝑙𝑒)
Temperature Run 1 Run 2 Run 3 Average SD
20 C 0.67 0.642 0.629 0.647 0.021
30 C 0.808 0.788 0.778 0.791 0.015
45 C 0.781 0.794 0.781 0.785 0.008
60 C 0.478 0.418 0.398 0.431 0.042
80 C 0.027 0.03 0.032 0.030 0.003
Figure 3-5. Normalized activity of alpha-Amylase conjugated to Iron Oxide Nanoparticle
53
3.3.2 For β-Galactosidase
Difference of absorbance of Sample and Control after following the procedure
described in chapter 2.4, is listed in Table 3-4. Normalized activity is plotted in Fig 3-7.
Table 3-4. (𝐴𝑠𝑎𝑚𝑝𝑙𝑒 − 𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙−𝐴𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 ) for ONPG assay of conjugated beta-
Galactosidase after 60 min incubation at different temperature
20 µL of PEG550 conjugated beta galactosidase incubated with 200 µL of ONPG solution
(𝐴𝑠𝑎𝑚𝑝𝑙𝑒−𝐴𝐶𝑜𝑛𝑡𝑟𝑜𝑙 −𝐴𝑝𝑎𝑟𝑡𝑖𝑐𝑙𝑒 )
Temperature, C Run 1 Run 2 Run 3 Average SD
20 0.168 0.18 0.156 0.168 0.012
30 0.261 0.233 0.243 0.246 0.014
45 0.397 0.431 0.411 0.413 0.017
50 0.406 0.45 0.447 0.434 0.025
60 0.389 0.412 0.439 0.413 0.025
70 0.003 0.025 0.023 0.017 0.012
Figure 3-6. Conjugated beta-Galactosidase activity normalized by the maximum
54
3.4 Activity under AMF
Activity of enzyme covalently bound to the surface of the superparamagnetic Iron
Oxide nanoparticles was measured using the protocol mentioned in chapter 2.7.
3.4.1 For -Amylase:
3.4.1.1 Study 1:
Incubator temperature: 45°c
Absorbance after Application of AMF:
Table 3-5. Activity under AMF: Study 1: Absorbance after application of AMF
Sample ID Absorbance at 618nm
Absorbance
Average SD
1 0.685 0.738 0.042
2 0.771
3 0.747
4 0.71
5 0.715
6 0.744
7 0.711
8 0.819
Absorbance of Control:
Table 3-6. Activity under AMF: Study 1: Absorbance of Control
Sample ID Absorbance at 618nm
Absorbance
Average SD
1 0.656 0.728 0.046
2 0.716
3 0.712
4 0.706
5 0.705
6 0.799
7 0.776
8 0.754
55
Temperature profile throughout the process:
Figure 3-7. Activity under AMF: Study 1: Temperature throughout the process
3.4.1.2 Study 2:
Incubator temperature: 45°c
Absorbance after Application of AMF:
Table 3-7. Activity under AMF: Study 2: Absorbance of after application of AMF
Sample ID Absorbance at 618nm
Absorbance
Average SD
1 0.591 0.593 0.043
2 0.582
3 0.563
4 0.558
5 0.576
6 0.553
7 0.646
8 0.672
56
Absorbance of Control:
Table 3-8. Activity under AMF: Study 2: Absorbance of Control
Sample ID Absorbance at 618nm
Absorbance
Average SD
1 0.512 0.554 0.031
2 0.527
3 0.534
4 0.542
5 0.561
6 0.579
7 0.571
8 0.606
Temperature profile throughout the process:
Figure 3-8. Activity under AMF: Study 2: Temperature throughout the process
57
3.4.1.3 Study 3:
Incubator temperature: 50°c
Absorbance after Application of AMF:
Table 3-9. Activity under AMF: Study 3: Absorbance after application of AMF
Sample ID Absorbance at 618nm
Absorbance
Average SD
1 0.942 0.899 0.027
2 0.939
3 0.891
4 0.869
5 0.885
6 0.893
7 0.896
8 0.875
Absorbance of Control:
Table 3-10. Activity under AMF: Study 3: Absorbance of Control
Sample ID Absorbance at 618nm
Absorbance
Average SD
1 0.995 0.874 0.088
2 0.946
3 0.895
4 0.802
5 0.899
6 0.82
7 0.719
8 0.915
58
Temperature profile throughout the process:
Figure 3-9. Activity under AMF: Study 3: Temperature throughout the process
Now, from study 1, 2 and 3 it is observed that the absorbance of sample placed
under AMF not significantly different from the absorbance of the control [Fig: 3-11]. As
there is no statistical difference between these two absorbance, it can be concluded that
the AMF does not have any significant effect on the activity.
59
Figure 3-10. Activity of conjugated alpha-Amylase under AMF
3.4.2 For β-galactosidase:
3.4.2.1 IO-CMDX conjugate Study 1:
Absorbance of Sample and control:
Table 3-11. IO-CMDX Beta gal conjugate Study 1: Absorbance of sample and control
Sample ID Absorbance at 420 nm
sample control
1 0.610 0.635 2 0.620 0.657 3 0.629 0.674
Average 0.620 0.655 SD 0.010 0.020
60
Temperature throughout the process:
Figure 3-11. IO-CMDX Beta gal conjugate Study 1: Temperature throughout the process
3.4.2.2 IO-CMDX conjugate Study 2:
Absorbance of Sample and control:
Table 3-12. IO-CMDX Beta gal conjugate Study 2: Absorbance of sample and control
Sample ID Absorbance at 420 nm
sample control
1 0.531 0.555 2 0.653 0.678 3 0.689 0.688
Average 0.624 0.640 SD 0.083 0.074
61
Temperature throughout the process:
Figure 3-12. IO-CMDX Beta gal conjugate Study 2: Temperature throughout the
process
3.4.2.3 PEG550 Conjugate Study 1:
Maintained temperature during the process: 50°C
Absorbance of Sample and control:
Table 3-13. PEG550 (batch2) Beta gal conjugate Study 1: Absorbance of sample and control
Sample ID Absorbance at 420 nm
sample control
1 0.685 0.799 2 0.718 0.717 3 0.766 0.658
Average 0.723 0.725 SD 0.041 0.071
62
3.4.2.4 PEG550 Conjugate Study 2:
Maintained temperature during the process: 50°C
Absorbance of Sample and control:
Table 3-13. PEG550 (batch2) Beta gal conjugate Study 2: Absorbance of sample and
control
Sample ID Absorbance at 420 nm
sample control
1 0.402 0.456 2 0.403 0.419 3 0.389 0.427
Average 0.398 0.434 SD 0.008 0.019
3.4.2.5 PEG550 Conjugate Study 3:
Maintained temperature during the process: 50°C
Absorbance of Sample and control:
Table 3-15. PEG550 (batch2) Beta gal conjugate Study 3: Absorbance of sample and control
Sample ID Absorbance at 420 nm
sample control
1 0.708 0.715 2 0.715 0.742 3 0.709 0.731
Average 0.711 0.729 SD 0.004 0.014
63
3.4.2.6 PEG550 Conjugate Study 4:
Maintained temperature during the process: 50°C
Absorbance of Sample and control:
Table 3-16. PEG550 (batch2) Beta gal conjugate Study 4: Absorbance of sample and
control
Sample ID Absorbance at 420 nm
sample control
1 0.638 0.616 2 0.629 0.601 3 0.618 0.618
Average 0.628 0.612 SD 0.010 0.009
Now, from the above studies it is observed that the absorbance of sample placed
under AMF not significantly different from the absorbance of the control [Fig: 3-14]. As
there is no statistical difference between these two absorbance, it can be concluded that
the AMF does not have any significant effect on the activity.
Figure 3-13. Activity of conjugated beta-Gal under AMF
64
CHAPTER 4 CONCLUSION
Experiments done throughout this study confirms that both the enzymes are
bound to the surface of Iron Oxide nanoparticles. Iron Oxide nanoparticles have also
shown heat dissipation in presence of an AMF. Conjugated particles were washed
properly and no presence of free enzyme was ensured. Activity of the conjugated
particles, in case of both the enzymes, were measure at different temperatures with
external heating. It was observed that enzyme bound to the IO nanoparticle can be
inactivated by external heating. However, when placed under AMF, both the enzymes
conjugated to IO nanoparticles, did not show any significant inactivation. Inference that
can be drawn that local heating due to IO nanoparticles under AMF was not sufficient to
inactivate the enzymes.
65
APPENDIX
-AMYLASE CONJUGATION TRIALS USING EDC/NHS CHEMISTRY
Scheme 1
100mg of IO-CMDx particles were dispersed in 10 mL PBS 1x (1 mg core/mL). 6
mL of particle suspension was taken in 15mL centrifuge tube and pH is taken down to
5.17 with 5 µL 1M HCl. 59 uL of EDC (10 mg/mL in PBS 1x) added to it followed by the
addition of 35 uL of NHS (10 mg/mL in PBS 1x). This sample was mixed in a shaker at
4°C at 600 rpm for 15 min. The reaction mixture was washed with 30 kDa membrane
under centrifugation (3200 rpm; 3 times, 5 min each) to remove excess EDC and NHS.
Then 471 uL of 20mg/mL -amylase solution in PBS 1x was added to it. This reaction
mixture was shaken for 4 hours at 4°C at 600 rpm. After the reaction was complete, the
reaction mixture was washed with 100 kDa membrane under centrifuge. These washed
particles were incubated with starch solution for 2 hours. On addition of iodine reagent,
dark blue color formation was seen which refers to the absence of -amylase. [Figure
A-1]
Conclusion: -amylase was not conjugated to IO-CMDx nanoparticles.
66
Figure A-1. Tapomoy Bhattacharjee. Scheme 1: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure starch solution only. 1st. October, 2013.
Scheme 2
420 mg IO-CMDx particles were dispersed in 17.5 mL PBS 1x (pH: 7.4). 30 mg
of -amylase was dissolved in 5 mL PBS 1x. 360 mg EDC was dissolved in 3 mL PBS
1x. Six set of reaction mixture were prepared following Table A-1.
Table A-1. Scheme 2 reaction mixture preparation.
Sample -amylase solution Particle suspension
EDC solution
1 10uL 2.5mL 0.5mL 2 50uL 2.5mL 0.5mL 3 0.1mL 2.5mL 0.5mL 4 0.2mL 2.5mL 0.5mL 5 0.5mL 2.5mL 0.5mL 6 1mL 2.5mL 0.5mL
These reaction mixtures were shaken at room temperature at 600 rpm for 2
hours. All of these reaction mixtures were then washed with a 100 kDa membrane
under centrifuge to remove unreacted -amylase and reaction by products. These
washed particles were incubated with starch solution for 2 hours. On addition of iodine
67
reagent, dark blue color formation was seen which refers to the absence of -amylase.
[Figure A-2]
Conclusion: -amylase was not conjugated to IO-CMDx nanoparticles.
Figure A-2. Tapomoy Bhattacharjee. Scheme 2: Starch Iodine assay with Washed particles; '1'-'6' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure starch solution only. 25th October, 2013.
Scheme 3
24mg IO-CMDx particles in PBS 1x were washed and suspended in phosphate
buffer. 10 mg EDC and 10 mg NHS each dissolved in 0.5 mL DI water was added to
particle suspension. This mixture was shaken moderately at room temperature for
15min. Excess EDC and NHS was washed times using 100 kDa membrane. Washed
particles were suspended in phosphate buffer. Resultant pH was 7.7. Now 0.5 mL of 6
mg/mL -amylase solution in PBS 1x was added to it. The reaction was conducted for 2
hours at room temperature. After the reaction was completed, the particle suspension
was washed with 100 kDa membrane under centrifuge. Washed particles doesn’t
confirm any presence of -amylase with starch iodine assay.
68
Conclusion: -amylase was not conjugated to IO-CMDx nanoparticles.
Figure A-3. Tapomoy Bhattacharjee. Scheme 3: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure starch solution only. 28th October, 2013.
Scheme 4
20mg of IO-CMDx particle suspension (2 mg core) in PBS 1x was taken in 5mL
vial and pH is taken down to 4.97 with 2 µL 1M HCl. 118 µL of EDC (0.1 mg/mL in
PBS 1x) was added to it (EDC: COOH of particle=1:1). Then, 35 µL of NHS (0.2
mg/mL in PBS 1x) was added to it (NHS: COOH =1:1). This reaction mixture was mixed
in a shaker at room temperature at 300 rpm for 5 min. 523 µL of 6mg/mL -amylase
solution in PBS 1x (Enzyme: COOH =1:1) was added to the reaction vial. The reaction
is carried on for 2 hours at room temperature at 300 rpm. After the completion of the
reaction, Particle suspension was washed with 100 kDa membrane under centrifuge to
remove unreacted -amylase. Washed particles doesn’t confirm any presence of -
amylase with starch iodine assay.
69
Conclusion: -amylase was not conjugated to IO-CMDx nanoparticles.
Scheme 5
20 mg (2 mg core) IO-CMDx particles were suspended in 2 mL MES buffer
(pH=4.75). Particle suspension was filtered using 0.2µ syringe filter. 1mL of 50 mg/mL
EDC solution in MES was added to the particle suspension (10 times Stoichiometric
amount). 1mL of 30 mg/mL of NHS solution in MES was added to the particle
suspension (10 times Stoichiometric amount). This reaction mixture was shaken for 30
min. pH was adjusted to 7.1 using 1M NaOH. 1mL of 90 mg/mL alpha amylase solution
in PBS (7.4 pH) added to this particle suspension. (10 times Stoichiometric amount).
This reaction is carried for 2 hours under room temperature. After reaction these
particles were washed using a 100 kDa membrane. Washed particles were incubated
with starch solution for 2 hours. On addition of iodine reagent, dark blue color formation
was seen which refers to the absence of -amylase. [Figure A-4]
Conclusion: -amylase was not conjugated to IO-CMDx nanoparticles.
Figure A-4. Tapomoy Bhattacharjee. Scheme 5: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C' refers to Control and contains pure starch solution only. 5th November, 2013.
70
Scheme 6
60 mg cIO-CMDx particles were suspended in 6 mL MES buffer (pH=4.75).
Particle solution was filtered using 0.2u syringe filter. 1ml of 5 mg/mL EDC solution in
MES was added to 2mL particle suspension (Stoichiometric amount). 1mL of 3 mg/mL
of NHS solution in MES was added to the particle suspension (Stoichiometric amount).
pH after addition of EDC and NHS is 4.9. This mixture is shaken for 30 min. 1mL of 90
mg/mL alpha amylase solution in PBS (7.4 pH) was then added to this particle
suspension. (10X Stoichiometric amount). At this point, pH was adjusted to 9.9 using
0.1 M NaOH. This reaction was carried on for 12 hours under room temperature. After
reaction these particle solution was washed using a 100 kDa membrane under
centrifugation. Washed particles doesn’t confirm any presence of -amylase with starch
iodine assay. [Figure A-5]
Conclusion: -amylase was not conjugated to IO-CMDx nanoparticles.
Figure A-5. Tapomoy Bhattacharjee. Scheme 6: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C1' refers to Negative Control and contains pure starch solution only; 'C2'
71
refers to positive control and contains 2.5 ug of Amylase with Starch solution. 7th November, 2013.
Scheme 7
60 mg of cIO-CMDx particles were suspended in 6 mL MES buffer (pH=4.78).
Particle solution is filtered using 0.2u syringe filter. 1ml of 5 mg/mL EDC solution in MES
was added to 2mL particle suspension (1X Stoichiometric amount). 1mL of 3 mg/mL of
NHS solution in MES is added to the particle suspension (1X Stoichiometric amount).
pH after addition of EDC and NHS was 4.9. The reaction mixture is shaken for 30
min.1mL of 90 mg/mL alpha amylase solution in PBS (7.4 pH) added to this particle
suspension. (10X Stoichiometric amount). pH at this point was 4.97. pH was adjusted to
7.04 using NaOH. This reaction is carried on for 13 hours under room temperature.
Washed particles doesn’t confirm any presence of -amylase with starch iodine assay.
[Figure A-6]
Conclusion: -amylase was not conjugated to IO-CMDx nanoparticles.
Figure A-6. Tapomoy Bhattacharjee. Scheme 7: Starch Iodine assay with Washed particles; 'S' refers to Sample and contains washed particles after reaction; 'C2' refers to Negative Control and contains pure starch solution only; 'C1' refers to positive control and contains 2.5 ug of Amylase with Starch solution. 8th November, 2013.
72
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76
BIOGRAPHICAL SKETCH
Tapomoy Bhattacharjee had his schooling from Ramkrishna Vivekananda
Mission Vidyabhaban, Barrackpore. In 2008, He received a National Merit Scholarship
Award from Govt. of India for the period of 2008 to 2012. In 2012, he graduated with a
Bachelor of Chemical Engineering degree from Jadavpur University, India.