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Materials Chemistry and Physics xxx (2014) 1e9

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Materials Chemistry and Physics

journal homepage: www.elsevier .com/locate/matchemphys

Water-soluble acetylated chitosan-stabilized gold nanospherebioprobes

Lanh T. Le a, Hai-Phong Nguyen b, Quang-Khieu Dinh b, Thai-Long Hoang b,Quoc-Hien Nguyen c, Thai-Hoa Tran b, *, Thanh-Dinh Nguyen d, *

a Technical and Economic College of Quang Nam, Quang Nam, Viet Namb Department of Chemistry, College of Sciences, Hue University, Hue, Viet Namc Research and Development Center for Radiation Technology, Vietnam Atomic Energy Institute, HoChiMinh, Viet Namd Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada

h i g h l i g h t s

* Corresponding authors.E-mail addresses: tthoa@hueuni.edu.vn (T.-H.

(T.-D. Nguyen).

http://dx.doi.org/10.1016/j.matchemphys.2014.10.0240254-0584/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: L.T. Le, etand Physics (2014), http://dx.doi.org/10.1016

g r a p h i c a l a b s t r a c t

� Water-soluble chitosan was derivedfrom surface acetylation.

� Uniform Au nanospheres were syn-thesized in the presence of acetylatedchitosan.

� Au nanocolloids were efficient bio-probes for detecting melamine, bac-teria, and uric acid.

a r t i c l e i n f o

Article history:Received 21 June 2014Received in revised form14 September 2014Accepted 15 October 2014Available online xxx

Keywords:GoldNanostructureSurfactant-assisted synthesisElectrochemical measurementsOptical properties

a b s t r a c t

Sustainable biopolymers are intriguing motifs for transferring the distinct biocompatibility and recog-nition into inorganic nanoparticles, which have a potential to be used in biomedicine applications. Herewe report scalable production of water-soluble chitosan polymers from N-acetylation. One-pot aqueoussynthesis of water-dispersible gold nanospheres is developed using the acetylated chitosan as a stabilizerand a reducing agent. The inherent aqueous solubility of the acetylated chitosan renders the goldnanospheres dispersible in water and enables for controlling the uniform size and monodispersity. Theacetylated chitosan-stabilized gold nanospheres with plasmonic and biocompatible properties are usedas efficient bioprobes for the selective detection of various biochemical agents of melamine, bacteria, anduric acid.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Gold (Au) materials play an essential role in the life since the useof Au solids as pigments for producing rudy-colored stained glasses

Tran), ntdinhc@chem.ubc.ca

al., Water-soluble acetylated/j.matchemphys.2014.10.024

in the ancient age. Metallic colloid science begunwith experimentsof Michael Faraday on Au sols in the mid-nineteenth century [1]. Asreducing the particle size of a material to several nanoscale meters,Au nanoparticles arise unique optoelectronic properties differentfrom bulk counterparts [2]. The outstanding feature of the Aunanoparticles is surface enhance Raman scattering (SERS) phe-nomenon derived from collective oscillation of conduction elec-trons upon interaction with electromagnetic radiation [3,4]. Theplasmonic property makes the Au nanoparticles of considerable

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interest for biosensing [5e7]. Recent progress in the preparation ofthe Au bioprobes has dealt with the possibility of the selectivedetection towards chemical stimuli such asmelamine, bacteria, uricacid, etc [8e12].

The scientific community has devoted for many syntheticmethods to monodisperse, uniform Au nanoparticles withcontrolled geometries [13e16]. Chemical strategies for growing theAu nanocrystals in solution phase in the presence of organic sta-bilizers have significant advances in engineering the structuralgeometries [17]. The capped Au nanoparticles can be obtained withthe surfaces capturing from hydrophobicity to hydrophilicity,depending on the surfactants employed in the preparation. In 1994,Brust et al. [18] first reported biphasic method for the growth ofhydrophobic Au nanoparticles in interfaces by transferring Auprecursors from water to toluene followed by capping thiol-alkylsurfactants. Solvothermal treatment of Au precursors in oleyl-amine/organic solvent can produce hydrophobic oleylamine-capped Au nanoparticles [19]. The hydrophilic Au nanoparticlesare commonly synthesized by using aqueous routes of polyolreduction [20,21] and seed-mediated growth [22].

The intriguing feature of the Au nanoparticles is able to tunetheir plasmonic properties through molecular level design byvarying the particle size of the core and by surface modificationwith suitable functional molecules [23,24]. A major thrust has beenthe use of the Au nanoparticles for biomedicine in biosensing ofemerging biochemical agents (e.g., melamine, bacteria, and uricacid) [25]. For example, Ai et al. [26] proposed ligandeexchangereaction of citrate-stabilized Au nanoparticles with 1-(2-mercaptoethyl)-1,3,5-triazinane-2,4,6-trione (MTT) to generatewater-soluble MTT-stabilized Au colloidal dispersion. The MTT-stabilized Au nanoparticles tuned the color from wine red to blueupon exposure to melamine, resulting from hydrogen-bondingrecognition between melamine and MTT. Chang et al. [27] re-ported ultrasensitive method for the detection of Staphylococcusaureus cells by aptamer-conjugated Au nanoparticles based on thesimple measurement of the resonance light-scattering signal.Sharma et al. [28] performed the hierarchical assembly of Aunanostructures on indium tin oxide substrates to design electro-chemical biosensors that show good reproducibility, stability, andselectivity for the detection of uric acid, ascorbic acid, and glucosein the blood samples. The Au nanoparticles are more compatiblewith biomedical applications in terms of the hydrophilic surfaceand good stability. It is highly desirable to synthesize the hydro-philic Au nanoparticles in water instead of using the non-aqueousmethods. The hydrophilic particles can be used directly inbiomedicine, whereas the hydrophobic particles require furthersurface modification to hydrophobicity. Various stabilizing re-agents, such as thiol ligands [18], polyethylene glycol [29], polyvinylalcohol [30], polyvinyl pyrollidine [31], and cetyl-trimethylammonium bromide [22], are popularly used to preventhydrophilic Au nanoparticles from aggregation. Among them, bio-molecules represent an attractive choice for the hydrophilic Auparticle synthesis [32,33]. The biomolecules adsorbed on the Auparticles act as stabilizers and biocompatible agents, thus second-ary surface modifications may not require for selective biotargeting[34e36].

Chitin is a natural polymer consisting of N-acetyl-D-glucos-amine units, which is the main structural component in the shellsof crabs, shrimps, insects [37] and the cell walls of fungi [38].Similarly the principle derivative of chitin, chitosan can be obtainedby treating chitin with concentrated base for deacetylation of N-acetyl groups. Chitosan has been widely exploited for biomedicineand pharmaceutical applications in drug, gene delivery, and stabi-lizer owing to its biocompatible, biodegradable, bioactive proper-ties [39]. These preeminent properties greatly improve along with

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an increase in the aqueous solubility of the chitosan [40,41]. Thereis a growth demand for new methods to prepare chitosan bio-polymers with a surface modification to improve the aqueous sol-ubility. Once chitosan is soluble in water, it can be ideal for using asan effective stabilizer for the aqueous synthesis of monodisperse Auparticles. Chitosan isolated from high-volume food wastes ofshrimp and crab shells could be a viable renewable resource forpreparing biomolecule stabilizers in the particle synthesis. It isworth noting that there are a few reports on chitosan-stabilized Auparticles, but appear the drastic aggregation resulting from thepoor aqueous solubility of the prepared chitosan. Liu et al. [42]represented the conjugation of dithiolane lipoic acid and acryl-oyloxyethyl phosphorylcholine to chitosan backbones to formwater-soluble polymers that stabilized Au nanoparticles in water.This molecule modification however involves a few synthetic stepsto obtain the aqueous solubility.

Herein, we reported simple acetylation of chitosan to water-soluble polymers that acted simultaneously as a stabilizer and areducing agent for the size-controlled synthesis of Au nanospheresin water. The acetylated chitosan-assisted Au nanospheres well-dispersed and stabilized in water with size uniformity and mono-dispersity. The Au nanocolloids were used to design effective bio-probes for the selective detection of appropriate biomolecules.

2. Experimental section

2.1. Preparation of water-soluble acetylated chitosan

2.1.1. Chitosan purificationDried shrimp shells (25 g) were soaked in a NaOH aqueous so-

lution (500 mL, 5 wt%) at 80 �C for 6 h to remove protein. Theproducts were cooled to room temperature and washed thoroughlywith water. Next, removal of calcium carbonate minerals in theshells was accomplished by treating the shells with a HCl aqueoussolution (500 mL, 7 vol%) for 4 d at room temperature to producecrude chitin. The deacetylation and degradation of the crude chitin(12.5 g) were carried out by sequentially treating with a HClaqueous solution (50 mL, 4 M) and a NaOH aqueous solution(25mL, 33%) to form chitosanwith degree of deacetylation (DDA) of~91% and average weight of ~1.7 � 105 g mol�1. White chitosanflakes obtained were used as starting material for preparing water-soluble chitosan.

2.1.2. Acetylation of chitosanDried chitosan (25 g) was immersed in lactic acid (500 mL, 3%)

to form 5 wt% chitosan dispersion within 24 h. In addition of H2O2(10mL, 0.5%) to chitosan/lactic acid dispersion under stirring for 3 hfollowed by (CH3COO)2O (15 mL) under stirring for 2 h were per-formed for acetylation of chitosan at room temperature. The reac-tion mixturewas then adjusted pH up to 7 by NH4OH (15mL, 5%) toform an acetylated chitosan dispersion. This dispersion wasprecipitated by ethanol to collect acetylated chitosan and this pu-rification was repeated several times to remove impurities. Theproducts were dried at 40 �C under air to obtain brown acetylatedchitosan powders with DAA of ~50% and average weight of~1.5 � 105 g mol�1. The powders dispersible in water to form ahomogeneous acetylated chitosan dispersion that was used forpreparing water-dispersible Au nanospheres. The acetylation yieldof chitosan was about 87 wt% following the above procedure.

2.1.3. Synthesis of acetylated chitosan-stabilized Au nanospheresA reaction dispersion (50 mL) was prepared by mixing the

aqueous acetylated chitosan dispersion (25 mL, 1 wt%) with HAuCl4(2.5 mL, 10 mM) and distilled water. The yellow mixed dispersionwas heated to 85 �C under stirring for desired times to obtain a

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purple homogeneous dispersion of acetylated chitosan-stabilizedAu nanospheres. The particle size of the Au nanospheres wastuned by varying the reaction duration within 2e31 h and theacetylated chitosan concentration.

2.2. Biosensing study: melamine, bacteria, uric acid detections

Melamine detection was accomplished by preparing the water-dispersible Au nanosphere dispersion (0.5 mL, 0.5 mM) uponaddition of different melamine concentrations(0.05e10.00 mg L�1). Visible changes in colors and aggregation ofthe mixed dispersion were observed and characterized by UVevisspectroscopy to evaluate melamine detection. Calibration curve foranalyte quantification was made based on transmission differencebetween l520 nm and l650 nm (Figure S7). Different melamine con-centrations (0.05e1.00 mg L�1) were added to raw milks (5.0 mL)obtained from local supermarket and then mixed with CCl3COOH(1.5 mL) under vigorous stirring for 5 min. Protein-contained ag-gregates from the mixtures were removed by centrifugation andcollected an aqueous solution. A transparent solution was obtainedby adjusting pH of the sample up to 7 by 1 M NaOH followed byfiltering [43]. The water-dispersible Au nanosphere dispersion(2.5 mL, 0.5 mM) was added to these milk-induced solutions for theanalyte quantification.

Antibacterial resistance of Escherichia. Coli (E.C), Salmonellatyphimurium (S.T), Listeria monocytogenes (L.M), and S. aureus (S.A)bacteria over the acetylated chitosan-stabilized Au nanosphereswas investigated by institute of veterinary research and develop-ment of central Vietnam (http://phanvienthuy.com.vn/en/). Thesespecific bacteria were incubated in sterile test tubes containingbrain heart infusion (BHI) agar under vibration and then stored in achamber at 37 �C for 8 h. Bacteria grew upwithin this durationwerequantitated by using UVevis spectroscopy and the resulting solu-tion was diluted to the final concentration of the growing bacteriaat 105 cfu mL�1 for antibacterial resistant test. The Au nanospheredispersionwas diluted to 0.05e50.00 mgmL�1 by BHI agar medium.The bacteria species (100 mL, 105 cfu mL�1) were added to 100 mLthe Au colloidal dispersions with different particle concentrationsin the sterile test tubes. The changes in transparency and aggre-gation of the test solutions were evaluated by antibacterial resis-tance of the Au particles. Kanamycin antibiotics were thenincubated in these tubes for comparative purpose. Alamar blueindicators were ultimately added to these tubes to tune yellow toblue in order to a visible enhancement.

The detection of uric acid was performed on Au nanoelectrodesusing differential pulse voltammetry (DPV) technique. Au nano-electrodes were designed by binding the acetylated chitosan-stabilized Au nanospheres on the surface of glassy carbon elec-trodes (CE) through L-cysteine cross-linkers. The CE electrode'ssurface was mechanically polished with 0.05 mm-sized aluminaslurry and then immersed in 2 M KOH and sonicated in 2 M H2SO4to completely remove trace adsorbates. The cleaned CE electrodeswere washed with ethanol, distilled water and dried at ambientconditions. These electrodes were further cleaned by running acyclic voltammetry (CV) from �1.5 V to 2.5 V in 0.1 M phosphatebuffered saline (PBS) and thenwashed with distilled water. Bindingthe Au nanospheres on the CE electrode surface is composed by twosteps. The DPV system equipped with the CE electrode was firstscanned in the electrolyte aqueous solution of 0.1 M PBS and10�3 M L-cysteine (pH~7) at the scan rate of 100 mV s�1 with po-tential varying from�1.5 V to 2.5 V to form a L-cysteine layer on theCE surface. L-cysteine-coated CE electrodes were then immersed inthe acetylated chitosan-stabilized Au nanosphere dispersion at 4 �Cfor 12 h and dried at ambient conditions to obtain Au particle/cys/CE electrodes. The designed Au nanoelectrodes were used to detect

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uric acid in the aqueous solution and human urine solution (10 mL)containing different uric acid concentrations (6.0e30.0 mM) in thepresence of 0.1 M PBS.

The analyte quantifications of melamine and uric acid weredone using a standard addition method (Figure S8). The limit ofdetection (LOD) and the limit of quantification (LOQ) for melamineand uric acid were obtained by using equations LOD ¼ 3sx/y/b andLOQ ¼ 10sx/y/b where sy/x and b are the estimated standard devia-tion and the slope of the analytical calibration function of eachsubstance, respectively, with a 95% (degrees of freedom ¼ 3) con-fidence level [8].

3. Results and discussion

Crude chitinwas prepared by sequentially treating shrimp shellswith dilute base and acid at room temperature for removal ofprotein and minerals, respectively. Chitin was retreated withconcentrated acid and base at 90 �C to form chitosan (Fig. 1a).Chitosan was immersed in dilute lactic acid within 24 h to obtain a5 wt% chitosan dispersion. This dispersion was then mixed withdilute H2O2 and (CH3COO)2O to yield an acetylated chitosandispersion. Acetylated chitosan powders were collected byprecipitating the dispersion with ethanol followed by drying at40 �C under air (Fig. 1b). The acetylated chitosan products weredispersed in water to form a homogeneous aqueous dispersion(Fig. 1c).

Chitosan structure before and after acetylation was confirmedby Fourier transform infrared (FTIR) and proton nuclear magneticresource (1H NMR) spectroscopy. IR spectra (Fig. 1d) show absor-bance peaks at 1655 cm�1 for NeH bending vibrations of amide Iband, at 1570 cm�1 for NeH bending vibrations of nonacetylated 2-aminoglucose primary amines, and at 1555 cm�1 for stretching ofamide II band. Pristine chitosan with DDA of ~91% shows thepresence of 2-aminoglucose and 2-acetamidoglucose repeat unitsconfirmed by bands at 1655, 1570, and 1555 cm�1. After N-acety-lation, the vibrational band corresponding to the primary aminogroups at 1570 cm�1 disappeared, while prominent bands at 1655and 1555 cm�1 were observed. The intensities of the carbonyl andamide II bands of the acetylated chitosan were proportional to theextended acetylation to 5 h. This analysis assumes that the acety-lation reaction predominantly occurred on the free primary aminegroups of the pristine chitosan. This prediction further confirmedby 1H NMR spectra (Fig. 1e) shows that for the pristine chitosan,peaks at 2.6 ppm are ascribed to three N-acetyl protons (HeAc) ofN-acetylglucosamine and at 3.6 ppm for a H2 proton (H-2D) ofglucosamine. The ring protons (H2eH6) are considered to resonateat 4.1e4.3 ppm. Peaks at 5.1 and 5.31 ppm were assigned to H1protons of glucosamine (H-1A) and N-acetylglucosamine (H-1D)residues, respectively. The acetylated chitosan shows new peaks at1.2 and 1.9 ppm assigned respectively to CH3 and (eCO)eCH2e ofanhydride acetic residues appeared with enhanced intensitiesalong with the extended acetylation. Like IR spectroscopy, weobserved no peak shift and impurity peaks, except an enhancedpeak intensity in the acetylated chitosan. This indicates that theadditional acetyl groups were bound to chitosan upon acetylationleading to an increased hydrogen components in the acetylatedproduct relative to the pristine chitosan, likely resulting in theenhanced peak intensity. These analyses clearly demonstrate thateNH2 groups of the chitosan molecules were partially substitutedby eNHCOCH3 to recover N-acetylated chitosan.

Crystalline structure of chitosan before and after acetylationwascharacterized by powder X-ray diffraction (PXRD) (Fig. 1f). Thepristine chitosan shows a broad diffraction peak at 19� 2q and othertwo small sharp peaks at 12 and 21� 2q were ascribed to thediffraction of the plane of the crystal region in chitosan. After N-

chitosan-stabilized gold nanosphere bioprobes, Materials Chemistry

Fig. 1. Water-soluble acetylated chitosan. (a) Chitosan flakes from purified shrimp shells. (b) Acetylated chitosan from acetylation. (c) Homogeneous dispersion of the acetylatedchitosan in water. (d) FTIR spectra of acetylated chitosan prepared at extended reaction duration. (e) 1H NMR spectra of chitosan before (bottom) and after (top) acetylation. (f) PXRDpatterns of chitosan before and after acetylation.

L.T. Le et al. / Materials Chemistry and Physics xxx (2014) 1e94

acetylation, the peak at 19� 2q remains intact, but the peaks at 12and 21� 2q disappear in the acetylated chitosan, indicating adecrease in the crystallinity of the acetylated chitosan. We sur-mised that regeneration of chitin polymers from soluble-wateracetylated chitosan monomers by precipitating in ethanol rapidlyleads to a reduction in hydrogen bonding between chitosan fibrils,presumably due to the presence of water molecules in the polymercrystallites. This suggests that the reacetylation is responsible forthe strong decrease of crystallinity of the chitosan fibrils, thusfacilitating high aqueous solubility of the acetylated products.

The acetylated chitosan is not only a polymeric stabilizer butalso a reducing agent in the Au nanoparticle synthesis. Note thataldehyde groups of glucose make these molecules reducing sugarsfor the metal preparation. This reducing property resembles that ofchitosan polysaccharide, thus the reduction of Au3þ to Au0 by theacetylated chitosan may occur the same mechanism. In this work,we synthesized Au particles in the acetylated chitosan dispersionwithout using any toxic reducing additives (e.g., hydrazine or so-dium borohydride). In a typical procedure, the acetylated chitosandispersion was mixed with a HAuCl4 precursor and heated to 85 �Cto form a purple homogeneous solution (in web version) indicativeof the generation of Au nanocolloids (inset of Fig. 2a). The reactionduration and stabilizer concentration were found to be crucialfactors for precise control over the size of monodisperse Au parti-cles in the bulk solution. The stabilized Au nanoparticles werecharacterized with respect to their structures and surfaceproperties.

UVevis spectra (Fig. 2a) of the Au colloidal solution prepared atdifferent reaction times show that a peak at 520 nm appears after2 h reaction and whose intensity increases with aging to 31 h.Comparative adsorption edges indicate that HAuCl4 solution andacetylated chitosan dispersion show a peak at 310 and 296 nm,respectively (Figure S3) and whose positions shift to 520 nm after

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heating. This indicates the reduction of Au3þ to acetylated chitosan-stabilized Au nanoparticles occurring along with reaction time.Transmission electron microscopy (TEM) images (Fig. 2b, Fig. S1) ofthe reaction solution prepared at 8 h show uniformity and mono-dispersity of spherical Au nanoparticles with sizes of 8e10 nm andnarrow size distribution of 8.5 ± 0.95 nm (Figure S2).

The stabilized Au nanospheres were characterized by FTIR and1H NMR spectroscopies to prove the acetylated chitosan adsorbedon the particle surfaces. IR bands characteristic of the acetylatedchitosan molecules were observed for the sample before and aftercapping the Au particles (Fig. 2c). Unlike the acetylated chitosan,new IR bands at 1020 cm�1 and 1680 cm�1 were observed for thestabilized Au particles. The origin of these vibrations is thought toresult from the formation of carboxyl groups in the acetylatedchitosan upon the surface passivation. This suggests that thealdehyde groups of the stabilizers reduce Au3þ to Au0 and thenconvert to carboxylic acid ones. These analyzed results demon-strate the adsorption of the acetylated chitosan on the Au particlesurfaces in the assistance of the reduction of the aldehyde groups ofthe stabilizers. PXRD patterns (Fig. 2d) of the acetylated chitosan-stabilized Au nanospheres show broad diffraction peaks of (111),(200), (220), (311) lattice planes characteristic of a cubic phase andcrystallinity of the Au nanostructure [44]. The average particle sizeestimated to be ~8.0 nm from the diffraction peak of the (111) planein accordance with the TEM observation.

The major challenge in using biomacromolecules to control theparticle growth often gives rise to random aggregation owing totheir complex functional networks [42]. Our approach overcomesthis obstacle in terms of the successful preparation of the water-soluble acetylated chitosan. Since chitosan dissolves as a poly-cation in dilute acid, the water-dispersible Au nanospheres stabi-lized by the acetylated chitosan obtained might possess positivelycharge surface to establish repulsive forces between particles,

chitosan-stabilized gold nanosphere bioprobes, Materials Chemistry

Fig. 2. Structural features of water-soluble acetylated chitosan-stabilized Au nanospheres. (a) UVevis spectra of the formation of the Au particles from a HAuCl4 0.50 mM/acetylatedchitosan 0.50 wt% solution upon aging; inset showing a homogeneous aqueous dispersion of the Au nanocolloids. (b) TEM images of the Au nanoparticles prepared at 8 h (c) FTIRspectra of acetylated chitosan-stabilized Au nanospheres (top) and acetylated chitosan (bottom). (d) PXRD pattern of acetylated chitosan-stabilized Au nanospheres. Water-solublechitosan is denoted as WSC.

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leading to high stability of the aqueous dispersion of the mono-disperse nanospheres.

The homogeneity of the reaction solution based on water-soluble chitosan stabilizers can allow to perform the synthesis inresponse to the control of the particle sizes by changing recipes. Thesize variation of the Au nanospheres from 16 to 9, 7 nm can beconducted by increasing the acetylated chitosan concentrationfrom 0.25 to 0.5, 1.0 wt%, presumably due to sufficient surfacepassivation at higher stabilizer loading (Fig. 3). Changing the con-centration of the Au3þ precursor in a limited range of0.25e1.00 mM can linearly tune the size of Au nanospheres from 6to 15 nm (Figure S4). Otherwise, we observed that the chitosan-stabilized Au nanospheres after synthesis disperse in water but

Fig. 3. Tunable sizes of monodisperse stabilized Au nanospheres. TEM images of the Au nan(b) 0.50, (c) 1.00 wt%.

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aggregate lightly after 6 months. It is acceptable that thebiomolecule-conjugated Au nanocolloids with long-term stabili-zation in water provide great benefit for practical applications inbiomedicine. Thus, we addressed this obstacle by adding a smallamount of free acetylated chitosan (0.30wt%) to the as-prepared Aunanosphere dispersion. This extended experiment allowed forfurther coating of stabilizers on the particles, thus improving thegood stabilization of the dispersion beyond 6 months (Figure S5).

The SERS signal of the Au nanoparticles is sensitive to chemicalstimuli capable of functioning as biochemical responses. Biocom-patibility, biodegradability, and non-toxicity of the plasmonic Aunanomaterials have inspired materials chemists to seek out theirnew applications in biomedicine, particularly bioimaging

oparticles prepared at 8 h using variable acetylated chitosan concentrations at (a) 0.25,

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phototherapy for cancer tumors [21,45]. Ideally, the integration ofthese unique properties of the Au particles into biological systemsenables to interact specifically with target molecules. Here we usedthe water-dispersible chitosan-stabilized Au nanospheres as bio-probes to trace various emerging agents including melamine, bac-teria, and uric acid.

Melamine can cause serious damage to organs of animal orhuman beings because of hydrolysis to toxic cyanuric acid in body[46]. Contamination of milk products with melamine over a limiteddose has been an important worldwide concern for recent years.Significant endeavors in the use of the Au nanoparticles have beenstudied for the analyte quantification of melamine residues[47e49]. In the continuation of developing this technique, we candetermine melamine in aqueous solutions and raw milks over thestabilized Au nanospheres.

The Au nanosphere dispersion loaded with melamine exhibits avisible change in color from purple through violet to deep bluewithincreasing the melamine concentration from 0.05 to 10.00 mg L�1

within 2 min (Fig. 4a). UVevis spectra (Fig. 4c) of melamine-loadedAu dispersion show that the position of the plasmonic peakmaintains alongside the appearance of a new adsorption peak at650 nm characteristic of melamine. The absorbance at 520 nmdeclined and enhanced at 650 nm simultaneously according to theincrease of the melamine concentration. The plasmonic peaknoticeably blue-shifted probably due to the aggregation of the Au

Fig. 4. Melamine detection over water-dispersible Au nanospheres. (a) Visual color changetrations of melamine. (c) UVevis spectra of the melamine-loaded Au particle dispersion withmilks by Au nanospheres. (d) UVevis spectra of a mixture of the Au particles and milk-inducreferences to color in this figure legend, the reader is referred to the web version of this a

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nanospheres in the presence of melamine. Intensity ratio of thesepeaks (A650/A520) is proportional to the melamine concentrationvarying from 0.05 to 1.00mg L�1 (correlation coefficient, r, is 0.999).LOD, LOQ, and analyte sensitivity for melamine was found to be0.049 mg L�1 and 0.117e0.224 mg L�1, and 2.141 L mg�1, respec-tively (Figure S7, Table S1). These analyzed results are comparableto the values reported by Guan et al. [43] using chitosan-capped Aunanoparticles.

The applicability of this analysis to raw milk samples wasexamined with the standard addition method. No melamine wasdetected in the pretreated milk sample. We then added certainamounts of melamine varying from 0.05 to 1.00 mg L�1 to thissample. It is apparent that the resulting mixture exhibits the similarchanges in the color and blue-shift and decline of the plasmonicsignal as the prepared melamine-loaded aqueous solution(Fig. 4b,d). This method gave an acceptable recovery value of94e111% in a good agreement with our high performance liquidchromatography analysis on the same sample. This further con-firms that our nanomaterials are highly sensitive to the tracemelamine detection even in the raw milks. These demonstrate thatthese Au nanomaterials not only detectmelamine, but also offer theeffectively analyte quantification. The presence of melamine in thecolloidal dispersion led to the formation of larger aggregates of theAu nanospheres as observed by electron microscopy (Figure S6).The role of the acetylated chitosan stabilizer is to place the analyte

s from purple to blue of the Au particle dispersion upon addition of variable concen-different melamine concentrations (mg L�1). (b) Visible detection of melamine in raw

ed solution with different melamine concentrations (mg L�1). (For interpretation of therticle.)

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molecules in order to obtain interactions between the adsorbedfunctional groups (e.g., eNH2) with the melamine molecules. Theaggregation into larger particles caused by these interactions,facilitating selective decline of the plasmonic resource, while pre-venting other amplification signals of the analyzed solution[47e49].

Despite the possibility of good bacteria detection of antibioticagents is known but cause of other auxiliary symptoms [11,12]. Herewe used the water-dispersible Au nanospheres to recognize thepresence of specific toxic bacteria. Different bacteria speciesincluding E.C, S.T, L.M, and S.A were used in this comparative studywithout and with using Kanamycin antibiotic and Alamar blue in-dicator. Towards yellow blank samples containing bacteria alone,the bacteria grew up along time to aggregate the Au particles intoturbid mixtures. In addition of the Au particle dispersion(0.1e50.0 mg mL�1) to the bacteria-contaminated samples whichremain the obvious yellow homogeneity without any aggregation.These mixed solutions transformed into transparent at a higher Auparticle loading (Fig. 5a). The minimum inhibitory concentration(MIC) of the Au nanosphere antibacterial agent to kill a particularbacteriumwas found to be 0.1e0.2 mg mL�1 in this study, since fewhave been investigated for the Au-based antibiotics for these bac-teria [12,50,51]. Kanamycin antibiotics were then incubated in thebacteria solution loaded with the Au particles. The yellow color ofthe resulting samples remains intact with more transparent indi-cating further antibacterial resistance of the antibiotics. Alamarblue indicators were ultimately added to these dispersions tochange the color from yellow to blue in order to be more visible forthe bacteria detection (Fig. 5b). This observation assumes that theAu nanospheres worked effectively to restrict the growth of thebacteria and these four bacteria species have the similar antibac-terial resistance over the Au nanospheres. So far, it should beconsidered these water-soluble acetylated chitosan-stabilized Aunanospheres with the most promising potential uses for real bac-terial species.

Analysis of trace amounts of uric acid, particularly in urine andblood, is commonly implemented in biomedicine [52,53]. Here weused the acetylated chitosan-stabilized Au nanospheres as brickunits to design nanoelectrodes for selectively detecting uric acid byusing DPV technique. The chitosan-stabilized Au particles werebound on the CE electrode's surface by using L-cysteine cross-linkers (Fig. 6a). Analyzed aqueous solutions were prepared withvarying the uric acid concentration from 6 to 30 mMat pH~4.0 in thepresence of 0.1 M PBS for experimental test.

Fig. 5. Antibacterial resistance of water-soluble acetylated chitosan-stabilized Au nanospheconcentration varying from 0.05 to 50.00 mg mL�1 without (a) and with (b) using Alamarreader is referred to the web version of this article.)

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Cyclic voltammetry (CV) profiles of the electrodes in prepareduric acid solutions show that the electrodes before and afterfunctionalizing with Au particles have only a stable anodic signalwithout cathodic peak, suggesting that irreversible electrochemicalreactions of uric acid occurred on the electrodes (Fig. 6b,c). Incomparison, the Au/cys/CE electrodes show a stable anodic peakwith intensity 12-fold higher than that of the Au/CE electrodes,arising from good contacts and binding of the particles to the CEsurface in the assistance of L-cysteine molecules. Also, wedemonstrated the extreme sensitivity of these nanoelectrodes withanonic peak intensity tuned disproportionally with the uric acidconcentration in a range of 6e30 mM with the correlation coeffi-cient of 0.9983 (Fig. 6d). The analyte quantification of the nano-electrodes of uric acid shows LOD, LOQ, highly analyte sensitivity,and RSD of 2.66 ± 0.29 mM, 7.5e12.0 mM, and 1.386e1.591 mA mM�1,and 0.938e3.609%, respectively (Figure S8, Table S2). We exten-sively used the designed Au nanoelectrodes for the analyte quan-tification of uric acid in human urine using the standard additionmethod. Urine samples after addition of 0.1 M PBS and certainvolumes of uric acid were characterized by DPV to obtain CV curves.We observed that the urine sample appears an anonic peak withtunable intensity with adding uric acid varying from 6 to 18 mM(Fig. 6e). This analysis allowed to calculate an acceptable recoveryvalue of 94.72e126.40%. This feature confirms the selective adap-tation of the designed Au nanoelectrodes for the detection of uricacid even in human urine. We speculate that the presence of thefree amino groups in the acetylated chitosan layer may beresponsible for electrostatic interactions with acidic groups of uricacid. This makes the acetylated chitosan a scavenger towards targetmolecules, which once close to Au particles can easily react andpromote the detection of uric acid. Au-Prussian blue nano-electrodes were recently designed to detect uric acid with effi-ciency slightly higher than our result, but no conclusion ofanalyzing haman urine showed [54].

Biomolecules bring great benefits to biomedical nanotech-nology. DNA and peptide are intriguing hierarchical platforms forthis purpose but face cost-effectiveness. Seeking out the sustain-able water-soluble chitosan biopolymers is a prospective strategyfor developing affordable sensing devices. The water-soluble acet-ylated chitosan may be beneficial for the extended synthesis ofother metal particles, a variety of oxide nanostructures with hy-drophilic surfaces and their assemblies. Furthermore, the aqueouschitosan dispersion may co-assemble into organized bioplasticmembranes embedded with metallic particles that offer access tonew materials for antibacterial resistance detection. One elegant

res. Bacteria-incubated solutions loaded with water-dispersible Au nanospheres withblue indicators. (For interpretation of the references to color in this figure legend, the

chitosan-stabilized gold nanosphere bioprobes, Materials Chemistry

Fig. 6. Design of Au nanosphere-based electrodes for uric acid detection. (a) Binding of Au nanospheres on CE electrode using L-cysteine cross-linkers. CV curves of CE electrodesbefore (b) and after (c) binding Au nanospheres by L-cysteine. CV curves of (d) aqueous solution and (e) human urine with adding different uric acid concentrations recorded in0.1 M PBS buffer, pH~4.0 at the scan rate of 100 mV s�1 on the Au nanoelectrodes.

L.T. Le et al. / Materials Chemistry and Physics xxx (2014) 1e98

example recently reported by Bae et al. [41] was the use of chitosanoligosaccharide for stabilizing ferrimagnetic iron oxide nanocubesfollowed by conjugating with L-3,4-dihydroxyphenylalanine todesign effective heat nanotherapeutic agents for hyperthermiccancer treatment. As well, but not less important, the water-solubleacetylated chitosan is a useful alternative to expensive DNA andpeptide in bioconjugation of the functional nanoparticles for bio-imaging medicine.

4. Conclusions

We have investigated the acetylation of chitosan prepared frompurified shrimp shells to yield acetylated chitosan polymers withwater solubility. This is the simple and affordable method forscalable production of the homogeneous chitosan dispersion in thechemical laboratory. The water-solution chitosan was used simul-taneously as a sustainable stabilizer and a reducing agent in thesynthesis of Au nanoparticles inwater. AuCl4�/chitosan mixtures area stable, dispersible system for the controlled growth of water-dispersible acetylated chitosan-stabilized Au nanospheres withtunable size from 6 to 15 nm. Taking the biocompatibility into ac-count, the stabilized Au nanospheres acted as efficient bioprobes.We used the Au nanocolloids to detect melamine and toxic bacteria

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in the solution, otherwise analyzed uric acid by designing selectiveAu-based nanoelectrodes. These Au nanospheres recognized theappropriate biochemical agents at trace quantity even in severalreal samples of raw milk and human urine. These novel acetylatedchitosan-stabilized Au nanospheres may detect the presence ofother biochemical species and be potentially used in biomedicine,particularly in cancer phototherapy.

Acknowledgments

We are grateful to the National Foundation for Science andTechnology Development of Vietnam � No. 104.03-2012.54. T.D.Nthanks to the Natural Sciences and Engineering Research Council(NSERC) of Canada for a Postdoctoral Fellowship.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.matchemphys.2014.10.024.

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