Studies on Differential Behavior of Silver Nanoparticles Towards Thiol ...

Current Nanoscience, 2012, 8, 000-000

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Studies on Differential Behavior of Silver Nanoparticles Towards Thiol Containing Amino Acids Aswathy Ravindran, N. Chandrasekaran and Amitava Mukherjee* Centre for Nanobiotechnology, School of Bio Sciences & Technology, VIT University, Vellore, India Abstract: Silver nanoparticles offer a broad range of applications in biomedical and bioanalytical areas. In lieu of inadequate prior reports the present study aims to investigate the differential interaction of silver nanoparticles with cysteine and glutathione. Cysteine and glutathione were chosen as the model biomolecules owing to its strong affinity towards silver nanoparticles and potential applications in the field of biomedicine. Cysteine induced aggregation of particles but under similar experimental conditions glutathione failed to cause aggregation. The interaction of silver nanoparticles with cysteine resulted in a shift in the plasmon bands to higher wavelengths, which was further confirmed by microscopic studies which showed randomly arranged aggregates of the particles. The disappearance of the – SH band in the FT-IR spectra and the shift in the peaks of COO- and NH3+ groups indicated the S-Ag interaction and the aggregation of the particles. Further DLS and zeta potential measurements showed relatively high degree of polydispersity confirming the aggregation of the particles. The positive amine group of cysteine formed salt bridges with carboxylate groups which result in the aggregation of the particles. Unlike in the cysteine treated samples the FT-IR spectra of glutathione treated samples did not show any shift in the peaks of COOand NH3+ stretching, confirming that these groups have not taken part in the interaction in order to cause the aggregation of the particles. We anticipate that further improvements on this approach will enable the exploitation of the nanoparticles functionalized with amino acids containing thiol group towards bio-sensing applications.

Keywords: Atomic force microscopy (AFM), cysteine, glutathione, silver nanoparticles, thiol, transmission electron microscopy (TEM). 1. INTRODUCTION The study of the interactions between biologically pertinent molecules and nanoparticles has attracted increasing interest because of the potential applications in sensors, biosensors, and biomedical diagnostics [1-3]. New discoveries with the potential applications of silver and gold nanoparticles especially biofunctionalized nanoparticles have become the focus of research in biomedical and bioanalytical areas in recent years [4-8]. The interaction between the organic ligands and the surface of inorganic nanoparticles would pave the way for the coupling of biomolecular recognition systems to generate novel materials. The interaction of gold and silver (Ag) nanoparticles with biomolecules has been studied intensively [9, 10]. The ideal agents for the functionalization of nanoparticles are amino acids; this is due to the presence of different functional groups in the amino acids [11]. Since plasmonic nanoparticles exhibit surface plasmon oscillations in the visible wavelength range, their optical properties attract intense scientific and technological interest. Zhong et al., illustrated that gold nanoparticles reduced and stabilized by citrate have a negative surface charge and preferentially bind to thiol, amine, cyanide or diphenylphosphine functional groups [12]. Thiol containing amino acids such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play an important role in many biochemical pathways [13, 14]. Their level in biological fluids like human plasma and urine are important for clinical diagnostics of a variety of diseases [15]. Glutathione (GSH) is a tripeptide (-GluCys-Gly) that contains an -SH group. It is known to protect the red cells from oxidative damage when it is present in adequately high quantities (~5 mM) and to maintain the normal reduced state of the cell because of its antioxidant nature. It also plays an important role in the detoxification of the cell and is responsible for eliminating harmful organic peroxides and free radicals. It binds to toxins, such as heavy metals, solvents, and pesticides, and converts them into a form that can be excreted in urine or bile [16]. Many studies have *Address correspondence to this author at the School of Bio Sciences and Technology, VIT University, Vellore-632014; Tel: 91 416 2202620; Fax: 91-416-2243092; E-mail: [email protected]

1573-4137/11 $58.00+.00

recently been performed to understand the reactivity of this biologically relevant molecule in the presence of nanoparticles. As an important biologically active and thiol-containing amino acid, cysteine is actively involved in a number of important cellular functions and its level in biological fluids such as human plasma and urine is significant for clinical diagnostics of a variety of diseases. For example, L-cysteine on a gold surface was used to immobilize protein molecules. Cysteine on a solid surface is an important issue in protein study as well as in differentiating amino acid molecules [17]. Recently a few studies [18, 19] reported the interfacial interaction of biomolecules with gold nanoparticles, but the interaction mechanisms and especially the nanoscale effects about silver nanoparticles and bio-macromolecules has received less attention. Our group working for last few years on interface of plasmonic nanoparticles with biomolecules reported recently on the interaction of colloidal silver nanoparticles with a model protein Bovine serum albumin [20]. Still the nano-bio interface of silver nanoparticles with amino acids, especially thiol containing ones, is an area that remains relatively unexplored. The unique optical properties and surface binding affinity of silver nanoparticles to thiol-containing amino acids or peptide at the physiological pH provide an intriguing opportunity to develop nanoprobes to address some of the primary questions related to the role of thiols in the biological systems. The primary objective of the current investigation was to study and compare the behavior of different thiol containing amino acids like cysteine and glutathione towards silver nanoparticles in the physiological conditions. 2. MATERIALS AND METHODS 2.1. Materials Silver nitrate and trisodium citrate dihydrate (Na3C6H5O7. 2H2O) were purchased from SD fine Chemicals (high purity, above 99.5%), Mumbai. L-cysteine was obtained from Sigma Aldrich, USA (high purity) and glutathione was procured from Sisco Research Laboratories, Mumbai. Deionized and distilled water was used in all experiments.

© 2012 Bentham Science Publishers Ltd.

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2.2. Preparation of Silver Nanoparticles The silver colloid was prepared according to Lee and Meisel’s method [21], by the reduction of silver nitrate with trisodium citrate. In a typical experiment, to 100 ml of redistilled deionized water, 18 mg of silver nitrate was dissolved and the solution was heated to boiling. 2 ml of 1 % trisodium citrate aqueous solution was added into the boiling silver nitrate drop wise, accompanied with vigorous stirring. The mixed solution was kept for boiling for further 20 min. Finally, a green gray silver colloid was obtained. The formation of silver nanoparticles was confirmed using UVvisible spectroscopy by a prominent peak in the range of 400-500 nm [22]. In order to calculate the concentration of Ag NPs, the synthesized colloid was centrifuged at 12,000 rpm for 10 min. Clear supernatant was collected and filtered through a 0.22 mm sterilized filter. The final concentrations of silver in the synthesized colloid were measured by Atomic Absorption Spectrophotometer (Spectra Varian AA 240) after acidification by 1 % nitric acid. 2.3. Characterization Morphological and structural features of silver nanoparticles before and after interaction were studied using Transmission electron microscopy (TEM) (Technai 10 Philips) operated at an accelerated voltage of 80 kV. The silver nanoparticle colloids and their mixtures with the amino acid solutions were adsorbed for 2 min on the copper grids followed by drying of the sample. Atomic force microscopy (AFM) investigations were executed from colloidal solutions containing silver nanoparticles both in the absence and presence of cysteine and glutathione respectively at ambient conditions. AFM studies were carried out by drop coating the dispersion containing the particles onto a glass slide after required reaction time and scanning at a rate of 100 mV/s in the range 50m50m using Nanosurf easysurf 2 (Switzerland).The 90Plus Particle Size Analyzer (Brookhaven Instruments Corporation Holtsville, NY, USA) was used to analyze the particle size (hydrodynamic diameter) by dynamic light scattering and for the zeta potential measurements by laser Doppler electrophoresis. The measurements were executed on to silver colloidal solutions and on the mixtures in the 2:1 volume ratio of this sample with (10-3 M) cysteine and glutathione solutions. Fourier transform infrared spectroscopic (FT-IR) studies of the silver colloid and the Ag-cysteine and Ag-glutathione mixtures were carried out by mixing the sample with KBr and was scanned from 400 to 1400 cm1 using Perkin Elmer Spectrum One FT-IR spectrometer operated at a resolution of 4 cm1. 2.4. Interaction of Silver Nanoparticles with Glutathione For the interaction of silver nanoparticles with glutathione 0.1M stock solution of glutathione was prepared and the necessary dilutions (0.01M and 0.001M) were made from this stock using nano pure water (high concentrations of amino acid solutions have been previously applied in similar studies with gold nanoparticles [23]). The experiment was conducted using various concentrations of glutathione and a fixed concentration (10-3 M) of silver nanoparticles. A solution containing 10-3 M of silver nanoparticles was interacted with 10-1 M, 10-2 M and 10-3 M of glutathione for 10 minutes. For 10-3 M silver nanoparticles and 10-1 M glutathione, the vol: vol ratios taken were 5:1, 2.5:1 and 2:1 respectively. For 10-3 M silver nanoparticles and 10-2 M glutathione, the ratios taken were 5:1, 3:1, 2.5:1, and 2:1 respectively. For 10-3 M silver nanoparticles and 10-3 M glutathione, the ratios taken were 5:1, 3:1, 2.5:1, and 2:1 respectively. The mixtures were then kept on the rotary shaker for 10 min at 120 rpm at room temperature for interaction. After interaction, the absorbance spectra of the various samples are recorded using the UV – vis Spectrophotometer over a range of 200 – 600 nm.

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2.5. Interaction of Silver Nanoparticles with Cysteine The interactions of silver nanoparticles with different concentrations of cysteine were studied. The concentrations and the ratios used for the interaction studies were similar to that as discussed in section 2.4. 3. RESULTS The localized surface plasmon resonances are collective oscillations of the conduction electrons confined to metallic nanoparticles. For silver nanoparticles max values were reported in the visible range 400-500 nm [22]. In the present study the extinction spectrum of the synthesized silver nanoparticles in the colloidal solution exhibited a narrow and apparent peak located at 443 nm reflecting the spherical shape uniformity of the silver nanoparticles. The shape, size distributions and concentration of the nanoparticles determine the characteristics of the surface plasmon bands [24]. The optical absorption spectrum of metal nanoparticles is dominated by the SPR and an increase of particle size always results in the red-shift of plasmon band to higher wavelength ranges [25, 26]. As shown in Fig. (1a, b and c), when 10-1, 10-2 and 10-3 M solutions of cysteine were interacted with silver nanoparticles for 10 minutes at a pH of 6.4, the particles formed aggregates and a noticeable red shift in max by 10-40 nm was noted. Beyond ten minutes, the particles formed visible clumps which settled down in time. In all the three cases, a distinct shift in the plasmon peak towards the right for a decrease in ratios from 5:1 to 2:1 (Ag: Cys) was observed with 2:1 ratio of Ag: Cys exhibiting maximum red shift of 30-40 nm. This red shifted band can be ascribed due to the aggregation of the silver nanoparticles in the presence of the adsorbed molecule, cysteine in this case. Our results were in substantial agreement with the already reported data on gold nanoparticles and cysteine [27]. The UV-visible spectral data clearly indicated cysteine mediated aggregation of the particles. A representative TEM image of the synthesized silver nanoparticles is shown in Fig. (2a). The synthesized particles were mostly spherical in shape with an effective diameter of 10 nm. The Transmission Electron Micrograph of the silver nanoparticles functionalized with 0.001M cysteine solution showed aggregation of the nanoparticles Fig. (2b). The average size of the interacted particles in the cluster was found to be 24 nm. The primary size data from TEM was commensurate with the aggregation behavior as observed in UV-vis spectral data. However, under the similar experimental conditions with a pH of 6.4 and an interaction time of 10 minutes glutathione exhibited a different mode of interaction. When silver nanoparticles were interacted with a 10-3 M solution of glutathione, there was no significant spectral shift in the Ag-GSH samples when compared to the initial absorption spectrum of silver nanoparticles. It was also observed that the particles did not aggregate even after ten minutes of interaction with silver. Similar results were found in 10-2 and 10-1 M glutathione solutions too Fig. (3a, b and c). Fig. (4a) represents the AFM images of the synthesized silver nanoparticles. The AFM images showed mostly spherical particles with approximately 20 nm in diameter. Ag/Cys interacted samples showed aggregated nanoparticles as shown in Figs. (4b) where as the particles on interaction with glutathione showed dispersed particles which were mostly spherical in shape Fig. (4c). Dynamic Light scattering [28, 29] was also used to characterize the colloidal silver solution. The hydrodynamic diameter of the prepared silver nanoparticles was found to be 55.1 nm and its zeta potential was -30.66 mV. Ag-GSH interacted samples gave a hydrodynamic diameter 56.6 nm and a zeta potential value of -29.83 mV which was similar to that of silver particles. The interaction with cysteine caused an increase in average hydrodynamic diameter

Studies on Differential Behavior of Silver Nanoparticles

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Fig. (1). Optical spectrum of silver colloidal solutions with different concentrations of cysteine solution in different ratios, expressed in terms of CAg / Ccysteine ratio. (A) Optical spectrum of silver colloidal solutions with 0.1M cysteine solution. (B) Optical spectrum of silver colloidal solutions with 0.01M cysteine solution in different ratios. (C) Optical spectrum of silver colloidal solutions with 0.001M cysteine solution in different ratios.

Fig. (2). (A) Transmission electron micrograph of as synthesized silver nanoparticles and (B) Transmission electron micrograph of silver nanoparticles with 0.001M cysteine solution.


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Fig. (3). Optical spectrum of silver colloidal solutions with different concentrations of glutathione solution in different ratios, expressed in terms of CAg / Cglutathione ratio. (A) Optical spectrum of silver colloidal solutions with 0.1M glutathione solution. (B) Optical spectrum of silver colloidal solutions with 0.01M glutathione solution. (C) Optical spectrum of silver colloidal solutions with 0.001M glutathione solution.

to 152 nm and a decrease in zeta potential value of -21.43 mV. Therefore, the addition of cysteine reduces the (negative) surface charge of the silver nanoparticles, thus, increasing their tendency for aggregation. Therefore DLS and the zeta potential observations were also in substantial agreement with the spectroscopic data and the microscopic observations confirming the differential interaction. FT-IR spectroscopy was performed to identify the characteristic bands in cysteine and glutathione after conjugation with silver nanoparticles. Generally, amino acids exist as zwitterions (internal salts) and exhibit spectra characteristic of both carboxylate and primary amine salts [30]. The spectra obtained for cysteine and glutathione were in good agreement with the FT-IR spectra of a typical thiol containing amino acid. A band at 2597 cm-1 (2600 2550 cm-1 –SH stretch) or a band at 715 cm-1 (710-685 cm-1 C-S stretch) virtually confirmed the presence of thiol groups. The bands at 1614 and 1398 cm-1 corresponded to the asymmetric and symmetric stretches of COO-. A prominent peak at 3208 cm-1 (30003500 cm-1) signified NH3+ stretches. However slight changes were observed in the spectra of Ag/ Cys samples. For example, the very broad band at 3454 cm1 corresponded to –NH3 + stretching in the cysteine - silver nanoparticle aggregates. The asymmetric stretching of COO shifted from 1614 to 1633.68 and the band due to sym-

metric stretching of COO - was absent in the interacted sample. A shift in the positions of COO- and NH3+ stretching can possibly be due to the change in dipole moments when cysteine binds to the metal surface with a high electron density [31]. Significantly, the band belonging to S–H (2597 cm-1) and C-S (715 cm-1) disappeared in the spectra of cysteine–silver nanoparticle aggregates. The band shift of –NH3+ and COO stretching may be induced by the binding of cysteine to silver nanoparticles, indicating that S–Ag interaction has really occurred Fig. (5a and 5b). Unlike in the cysteine treated samples the FT-IR spectra of glutathione treated samples did not show any shift in the peaks of COO- and NH3+ stretching, confirming that these groups have not taken part in the interaction in order to cause the aggregation of the particles. However the spectra revealed the absence of –SH bands Fig. (6a and 6b) predicting the possible attachment of the thiol group on to surface of the nanoparticles through the cysteine moiety of glutathione molecule. 4. DISCUSSION The main objectives of our studies were to compare the nature of interaction between silver and thiol containing biomolecules like cysteine and glutathione. We note that both cysteine and glutathione in spite of being thiol containing amino acids differed in



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Fig. (4). (A) AFM image of as synthesized silver nanoparticles, (B) AFM image of silver nanoparticles with 0.001M cysteine and (C) AFM image of silver nanoparticles with 0.001M glutathione.

their behavior towards silver nanoparticles. A possible model illustrating both the cysteine bindings to silver nanoparticles and the formation of particle aggregates is schematized in Fig. (7). Cysteine is a thiol [SH] containing polar amino acid with limited hydrophilic properties [32]. The isoelectric pH of cysteine is 5.07 with its pKa values being 1.96 (for the carboxylic group), 8.18 (for the amine group) and 10.28 (for the thiol group). Under acidic conditions, the functional groups of cysteine are protonated thus the thiol groups of cysteine bind to silver surface through ligand exchange reactions [33, 34] and the cysteine molecule has still two functional groups free to form bonds between particles. The positive aminno group of cysteine formed salt bridges with carboxylate groups which result in the aggregation of the particles. Besides the electrostatic interac-

tion mechanism by salt bridges, a possible binding of biothiols on the surface of the nanoparticles through covalent bonding has also been reported [35]. Glutathione (GSH) is a tripeptide that contains an –SH group which can be easily adsorbed onto the surface of metallic nanoparticles. Glutathione consists of three amino acids glutamic acid, cysteine, and glycine therefore its binding modes are pH sensitive. There are several probable binding or anchoring points in glutathione which makes different binding modes possible. The binding points in glutathione are the two carboxylic groups in the glutamic acid and glycine residues, three –NH2 group in the three amino acids and the sulfur atom in the cysteine residue. In glutathione the pKa values corresponding to the two carboxylic groups


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Fig. (5). (A) FTIR spectra of cysteine and (B) cysteine interacted silver nanoparticles.

on the glutamate and glycine moieties are 2.56 and 3.50, and the cysteine residue has a pKa of 9.42 [36]. According to Lim et al., Binding via -amino groups is preferential at low pH and is suppressed at neutral and high pH, due to electrostatic repulsion between the particle surface and the charged carboxyl groups. Thus at intermediate or higher pH, dissociation of the carboxyl group would be expected to hinder the binding of Ag colloids via -amine group, resulting in little or no cross-linking [36]. The UV-vis spec-

trum of the glutathione interacted with silver did not exhibit any peak shift as in the case of cysteine. It exhibited a low reactivity possibly because of the steric hindrance of glutathione [37]. 5. CONCLUSION Thiol containing amino acids like cysteine and glutathione differed in their behavior towards silver nanoparticles. Cysteine induced aggregation of particles but under similar experimental


Fig. (6). (A) FTIR spectra of glutathione and (B) glutathione interacted silver nanoparticles.


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Fig. (7). Schema of cysteine binding to citrate capped Ag NPs (b) bonds formation between Ag NPs.

conditions glutathione failed to cause aggregation. UV–vis spectroscopy, DLS, zeta potential, TEM and AFM were used to confirm the differential nature of interaction of the biomolecules towards silver nanoparticles. Our data indicate that the aggregation of silver nanoparticles can be induced by cysteine, an amino acid possessing an additional thiol functional group besides the alpha-amine. The aggregation of particles induced by cysteine could be explained primarily through the zwitterionic-type electrostatic interactions between the charged amine and acid groups of cysteine molecules, bound to the silver nanoparticles by their –SH groups. The new insight into the affinity of silver nanoparticles towards -SH containing amino acid can lead to the development of various bioanalytical techniques and may pave the way for controlled drug delivery applications [38-40]. ACKNOWLEDGMENT The authors are greatly acknowledged to Mr. Lakshmipathy and Mr. Prabhu of VIT- TBI for helping us to carry out UV-vis spectroscopic and FT-IR studies.

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Received: January 11, 2011

Revised: July 4, 2011

Accepted: October 13, 2011

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