Electrochemical studies of glucose oxidase immobilized on glutathione coated gold nanoparticles

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1 Indian Journal of Biochemistry & Biophysics Vol. 44, April 2007, pp Electrochemical studies of glucose oxidase immobilized on glutathione coated gold nanoparticles Sridevi Akella 1 and Chanchal K Mitra 2 * School of Life Sciences, University of Hyderabad, Hyderabad , India Received 06 November 2006 ; revised 15 March 2007 Glutathione (L-γ -glutamyl-l-cysteinyl-l-glycine; GSH) forms a surface monolayer on gold nanoparticles by tethering via sulfur bonds (Au:GSH). In the present study, glucose oxidase (GOx; EC ) was immobilized by covalent chemical coupling reactions on to Au:GSH nanoparticles and the enzyme coupled nanoparticles formed a stable colloid (stable for several weeks) in water. The immobilized enzyme was investigated for electrochemical characteristics to monitor the FAD (prosthetic group of the GOx) redox potentials. Various concentrations of substrate (glucose) were added to check the oxidation characteristics. It was observed that with increase in substrate concentrations, the oxidation rate increased proportionally with the current. The present study demonstrated that GOx was effectively coupled to the gold nanoparticle (Au:GSH). The coupled nanoparticle system could be used in a potential biosensor application. Similarly, other enzymes (e.g., horseradish peroxidase) could be immobilized to the Au:GSH nanoparticles via the peptide arm (GSH) to achieve the desired characteristics needed for a specific application in biosensor. Keywords: Glutathione, Glucose oxidase, Gold nanoparticle, Biosensor The gold colloid can be easily prepared by treating a solution of gold chloride (HAuCl 4 ) with a suitable reducing agent. The gold nanoparticles are produced as a colloidal solution having the particle size in the range of Å (1-10 nm); the particle size may vary depending on the nature of preparation. The nanoparticles exhibit various colors according to the structure and size. When a strong reducing agent like sodium borohydride (NaBH 4 ) is used to reduce tetrachloroauric (III) acid (HAuCl 4 ), a ruby red colored colloid is formed. The Au nanoparticle colloidal solution is usually unstable and thus a suitable protecting agent is required to protect it from coagulation. Gold, due to its affinity towards sulfur, binds strongly with many proteins, which adsorb on to the Au surface via their thiol ( SH) groups, producing a clear colloidal solution and thus stable Au nanoparticle colloid can be obtained 1. *Author for correspondence Fax: neuron555@yahoo.co.in; 2 c_mitra@yahoo.com Abbreviations: GOx, glucose oxidase; CV, cyclic voltammetry (voltammogram), NHS, N-hydroxy succinimide; EDC, 1-ethyl-3- (3-dimethylaminopropyl) carbodimide hydrochloride; FAD, flavin adenine dinucleotide; HRP, horseradish peroxidase. Glutathione (GSH) protects the cell membrane against oxidative and other types of stress. It is transported as monoethyl esters 2. Thus, GSH esters may be of practical importance in protection against radiation and various types of chemical toxicity produced during oxygen reduction. GSH provides a reservoir of reducing equivalents, capable of preventing the effects of oxidants on sensitive -SH groups. It is a co-factor in several enzymatic reactions and plays an important role in intracellular cysteine storage, intra-organ cysteine transport, amino acid transport, disulfide bond reduction and detoxification of reactive electrophiles, free radicals and hydrogen peroxide. GSH contains three functional groups (two COOH and a SH groups), of which SH group is involved in forming the surface monolayers with Au nanoparticles 3. The GSH coats the Au nanoparticle with a monolayer in a stereo-specific manner. The free COOH groups available on the GSH are useful in covalent immobilization of the enzymes. Each GSH molecule though can in principle bind to two proteins via the two COOH groups, but steric hindrance prevent attachment of more than ~0.5 molecules of a protein of moderate size.

2 AKELLA & MITRA: GLUCOSE OXIDASE COATED GOLD NANOPARTICLES 83 Glucose oxidase (GOx), a flavoenzyme is widely used in biosensors to detect glucose, which is its substrate. In the present study, GOx has been covalently coupled to the free COOH groups of GSH present on the Au surface (Fig. 1) using carbodiimide and N-hydroxysuccinimide (NHS) as the coupling agents. The GOx-coupled nanoparticles have been painted on the surface of an Au or Pt electrode to study their electrochemical behavior. The redox behavior has been studied using cyclic voltammetry (CV). The electrochemistry revealed characteristic peaks in CV plots, suggesting successful electron transfer. This integrated moiety (Au:GSH:GOx) can be used to detect glucose in nano to micro scale and can be used in clinical diagnostics. It should be recalled that the present sensor work is based on the covalent coupling of the enzyme to gold nanoparticles. Materials and Methods Chemicals Gold and platinum working electrodes were from CH Instruments, Texas, USA. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were from Sigma- Aldrich, St Louis, USA. Tetrachloroauric (III) acid, glutathione (GSH) and carbodiimide [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride] (EDC) were obtained from Himedia (Bombay, India). N-hydroxysuccinimide (NHS) was from a local supplier and D-glucose, KCl and all other chemicals are of AR grade and from Qualigens India. Procedure The reactions leading to the formation of Au colloid and the subsequent reaction with GSH can be shown as and detailed in the next section. The size distribution of the nanoparticles produced by this chemical reaction depends on the concentrations of the reactants, time of reactions and other factors. HAuCl + NaBH Au + H + B H + NaCl Au + GSH Au:GS + H + Glucose oxidase (GOx) was coupled to the Au:GSH nanoparticles by carbodiimide (EDC) which activates the COOH functional group present in GSH of the Au:GSH nanoparticles. Thus, activated COOH groups of GSH couples to the NH 2 group of the GOx by activating the carbonyl group to form amine derivative of O-acyl intermediate, which reacts with the nucleophile and forms an amide link between GSH and GOx. The intermediate is soluble in water and gets rapidly hydrolyzed, but the addition of NHS can prevent the hydrolysis 4. NHS converts unstable derivative to an amine-reactive ester and thus accelerate the coupling reaction. Preparation of gold colloid by reduction with NaBH 4 75 µmoles of HAuCl 4 (15 ml of 5 mm HAuCl 4 ; 25 mg) and 75 µmoles of NaBH 4 (15 ml of 5 mm of NaBH 4 ; 3 mg) were both separately dissolved in 15 ml of distilled water and freshly prepared, and (b) 15 ml of gold chloride (HAuCl 4 ) solution (yellow colored) was reduced, when 15 ml NaBH 4 solution was added dropwise and mixed thoroughly. A ruby red colored colloid was formed. Fig. 1 Diagrammatic representation of Au nanoparticle covered with a GSH monolayer (Au:GSH), attached to the Au atoms by sulfur bonds [The number of GSH molecules per nanoparticle depends on the size of the central core of Au particle. The Au:GSH has a large negative charge under normal experimental conditions as the COOH groups will be mostly ionized except at low ph] Preparation of Au nanoparticles with GSH as surface coating (Au:GSH). 25 µmoles of GSH (5 mm GSH; 10 mg in 5 ml of double-distilled water) was added to the ruby red colored colloid prepared above. Addition of GSH turned the colloid ink-blue in color and the Au nanoparticles slowly precipitated out (0.5-1 h). The precipitate was collected and washed with methanol (about 5 ml) twice and dissolved in 10 ml of 50 mm

3 84 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, APRIL 2007 phosphate buffer, ph 9 (GSH is a tripeptide and its solubility in water is ph-dependent) 5. This solution could be directly used for the enzyme coupling. The purple solid was stored at 4ºC, easily dissolved back in solution (sometimes a gentle sonication may be needed), giving a ruby red solution. Immobilization of glucose oxidase (GOx) on GSH-coated Au nanoparticles (Au:GSH:GOx) To 5 ml of (Au:GSH) colloid, 20 mg of EDC (dissolved in 1 ml of 50 mm phosphate buffer, ph 9) was added and allowed to react for 1 h. Thereafter, 20 mg of NHS (dissolved in 1 ml of 50 mm phosphate buffer, ph 9) was added and the reaction mixture was placed in an ice bath and maintained at 0-4ºC. After 1 h, 2 mg of GOx (dissolved in 1 ml of 50 mm buffer, ph 9) was added and allowed to react at room temperature for 7-8 h. A dark-purple colored precipitate was obtained which was centrifuged at 5,000 rpm for 10 min and washed with methanol to remove excess unreacted reagents. The precipitate consisting of Au nanoparticles tethered with GSH and immobilized enzyme was dissolved in same phosphate buffer and stored at 4ºC and was the starting material for electrochemical studies. Optical and spectral studies of Au colloid The ruby-red color of Au colloid was due to the surface plasmon resonance (SPR) lying in the visible range 6 ( nm). When GSH was added to the colloidal solution, there was a shift in the band from 520 nm to about 560 nm (ink-blue color), indicating that GSH had formed a coating on nanoparticles. The color change was probably because of the increase in size, due to the layering of the surface of Au nanoparticles with an insulating molecular layer of GSH. Laser scattering studies suggested some aggregations as only the smaller nanoparticles could contribute to the color. GOx activity assay Activity assay of the coupled enzyme was carried out using o-dianisidine dye. The dye is colourless in reduced form and reacts in presence of horseradish peroxidase (HRP) and an oxidizing agent to produce a brown colored solution, measurable at 436 nm 7. GOx catalyzed the oxidation of glucose to δ-gluconolactone and H 2 O 2 was produced. The generation of H 2 O 2 was indirectly measured by the oxidation of the dye in presence of HRP. Both GOx and HRP reactions were coupled set of reactions. The hydrogen peroxide produced by GOx in the reduction of O 2 was used by HRP to oxidize the leuco dye as shown in the reaction below. About 100 µl (750 nmoles) of Au nanoparticles was present in the sample and it was estimated that 0.5 U/mg of was coupled to the 75 µg Au colloidal solution i.e, about U/mg of GOx was coupled to 1 µmole of gold nanoparticle colloid, which was optimum. This implied that GOx was bound in the covalent coupling reactions. As a control experiment, the same experiment was carried out without the coupling agent to see the effects of adsorption, but no detectable enzyme was found bound to the nanoparticles. The number of enzyme molecules attached per nanoparticles was not determined, as the size distribution was not quite uniform. GOx β-d + O + H O H2O2 2 2 HRP + o-dianisidine (red) δ-gluconolactone + H O 2 2 o -Dianisidine (ox) + H O The enzyme activity of the bound GOx was determined using the following equation: Corrected A/min Total volume Dilution Activity = 8.3 Sample volume Results Cyclic voltammograms of immobilized GOx to the nanoparticles on Au and Pt electrode surface Au and Pt electrodes were cleaned using a rouge polishing cloth, followed by electrochemical cleaning. A potential scan of 800 mv to +100 mv was taken to fit the window. The 10 mm KCl solution was used as the supporting electrolyte in all our experiments. Blank electrode scans were taken to ensure that electrodes were clean, before actual scans were obtained. ~1 µl of Au:GSH or Au:GSH:GOx colloidal solution samples of the Au nanoparticles were gently placed on to the Au or Pt electrodes and allowed to spread on the surface. The electrodes were dried in air approximately for 1 day. The electrodes were placed in the electrochemical chamber (a 25 ml capacity wide and short test-tube, fitted with a teflon lid through which three electrodes were inserted) and were scanned for further analysis. The CV results for the Au electrode are presented in Fig. 2A. The blank scan, plot (I) referred about the initial readings of the electrode. A hump implied 2

4 AKELLA & MITRA: GLUCOSE OXIDASE COATED GOLD NANOPARTICLES 85 Fig. 2 (A): Cyclic voltammograms (CVs) of (I) blank, (II) Au: GSH nanoparticle, and (III) immobilized GOx to the Au:GSH nanoparticle on Au electrode [The electrode surface was painted on using a colloidal solution of gold nanoparticles. The electrochemical parameters were initial E (V) =100 mv; high E (V) =100 mv; low E (V) = -800 mv; scan polarity = negative; and scan rate = 10 mv/s. Reference electrode was Ag/AgCl; (B): The graph has three scans taken on Pt electrode, similar to the scans of Au electrode] clearly that the oxygen decomposition was at about 600 mv 8. At this potential, only dissolved oxygen could be reduced, which was the only oxidant present. Plot (II) shows the peak, due to the presence of GSH at a potential of 327 mv. The standard redox potential of GSH/GSSG couple was around 310 mv, which could be correlated to our present experimental data 9. This suggested that the redox characteristics of GSH had not changed significantly upon attachment with the Au particles. Plot (III) shows the CV of GOx a flavoenzyme having FAD as its prosthetic group. The FAD redox potentials in various enzymes ranged from 190 to 490 mv 5,10. In plot (III), the FAD peak was seen at 480 mv, which was acceptable, suggesting that GOx bound to the Au nanoparticles was active in the electron transfer. It was also noted that the GSH peak disappeared after coupling of the GOx (and the FAD peak appeared at a lower potential). The CVs did not show reversibility, suggesting that the electron transfer was still slow. However, useful conclusions such as oxidation and reduction potentials could still be made as such CVs were well studied and documented in literature 11. Fig. 3 CV Plots for various concentrations of glucose on Au: GSH:GOx nanoparticle [(A), Au electrode, and (B), Pt electrode. All other details in the graph are given in Fig. 1 (Experimental conditions)] Fig. 4 A current vs glucose concentration graph showing that with increase in the substrate concentration the current was decreased [The current values were taken at the fixed potential of 300 mv from Fig. 3] The CV plots with different concentrations of glucose (substrate for the GOx) are shown in Fig. 3. The current or rather the peak height in the CV depended directly on the substrate concentration in solution (assuming a diffusion-controlled process). It could be seen that with increase in the substrate concentrations, the peak heights were increased (ignoring the sign of the current) and the increase in peak height was almost linear with increased concentration of glucose (Fig. 4). The range of peak positions for Au electrode was around 490 mv to

5 86 INDIAN J. BIOCHEM. BIOPHYS., VOL. 44, APRIL mv and for Pt electrode was around 400 mv to 380 mv (at which the current values were recorded). For effective comparison, one constant potential had been taken, at which all the current values were compared. Fig. 2B shows results of CV of the Pt electrode treated exactly similarly as the Au electrode. Plot (I) refers to the blank Pt electrode with a hump seen at 400 mv to 600 mv, presumably due to the presence of dissolved oxygen. Results for the electrode coated with Au:GSH nanoparticles plot (II) showed a peak at 211 mv, which might be compared with the potential (-310 mv) obtained with gold electrode as the base. A small peak in plot (III) at 400 mv (cf. 480 mv for Au) indicated the presence of FAD. Although the results were very similar for both the electrodes, they were not identical, indicating that the base metal played some role. This was not surprising as the jumping of electrons from the active site of the enzyme to the conduction band of the electrode may need some activation energy. We noted that for Pt electrode the FAD potential decreased (in magnitude), suggesting a reduction in the activation energy for the electron transfer process. Although the solution contained dissolved oxygen, the peaks were still clearly seen, suggesting that the effect of dissolved oxygen was relatively minor. It was, however, entirely possible in a sufficiently well deaerated sample, the peak heights could have been larger. It may be stated here that as the effective concentrations were very low, it was extremely difficult to completely remove all the dissolved oxygen in our experimental set-up. The dissolved oxygen was not expected to affect the potentials significantly 8. Fig. 4 depicts the substrate concentration vs. current plots for Au and Pt electrodes at 300 mv potential (vs Ag/AgCl reference). The current values were determined manually from the CV plots at the same potential. We noted that the Pt electrode showed more sensitivity (larger current/concentration slope) towards the substrate. Discussion The change in color of the Au nanoparticles upon reaction with GSH clearly suggested an increase in size. However, we considered both the plasmon absorption and conventional light scattering, as these phenomena are common for Au colloids only. We carried out laser light scattering of the colloids to estimate the size distributions in the Au colloids at different stages of the experiment. The median size of the Au nanoparticles increased from 140 to 170 nm (size distribution was quite broad and 30% of nanoparticles had size <40 nm, making application of theoretical analysis very difficult). Enzyme immobilization on an electrode (for a biosensor application) was common in electrochemical techniques 11. Direct adsorption of an enzyme on to an electrode often gives rise to a slow electron transfer. The use of Au nanoparticles coupled to an enzyme may be advantageous, as well as a novel protocol, since more surface area can be allowed for an enzyme to bind. Coupling reaction could be done on to a nanoparticle and then applied to the electrode surface. The plots in Fig. 4 indicated that the GOx was able to exchange electrons to the base electrode. Some improvements were needed, as the concentration range for the substrate (glucose) was still inadequate and the linearity was far from excellent. The CV plots were far from reversible and only the reduction peaks were clearly seen. However, nanoparticles as electrode modifiers were relatively new and need more detailed study. We also noted that the nature of the base electrode also played some important role as the electrochemistry on Au and Pt electrodes was similar, but there were also small differences in details (mainly in the redox potentials). This also needs to be explored in detail. Earlier, we suggested that covalent coupling of the enzymes with a reasonably long spacer arm could preserve the enzyme activity to a significant extent. When coupled to the active site of a redox enzyme (e.g., GOx), the electron transfer rates increased dramatically 10. In case of simple adsorption, when the adsorbate was expected to be randomly oriented, ~50% of the activity was lost (50% of the molecules will be having their active site facing the wrong way) and the access to the active site was severely restricted. In the present case, the Au nanoparticles acted merely as a carrier (possibly) and electrons transfer took place from the enzyme active site to the electrode. In essence, the nanoparticles offered a large surface area on the electrode. Nanoparticles could be purified by ultracentrifugation or by electrophoresis on purified agarose, but the yields were quite small. A suitable modification of the chemical reaction that produces smaller and more uniform particle size is currently under investigation. In this paper, we

6 AKELLA & MITRA: GLUCOSE OXIDASE COATED GOLD NANOPARTICLES 87 demonstrated that nanoparticles could be successfully used in the covalent coupling of redox enzymes and they could be functionally detected using direct electron transfer to a base electrode. Acknowledgements The work was supported by Vetenskapsradet (The Swedish Research Council). References 1 Salata O V (2004) Nanobiotechnol 2:3 doi.10/11, 1186/ Meister A (1989) On the Biochemistry of Glutathione: In Glutathione Centennial, Molecular Perspectives and Clinical Implications (Tanigiuchi N, Higashi T, Sakamoto Y & Meister A, eds.), pp 3-17, Academic Press, New York 3 Schaff T G, Knight G, SchaFigullin M N, Borkman M N & Whetten R L (1998) J Phys Chem B 102, Katz E, Carbodiimide coupling using EDC, 5 Calvin M (1954) Mercaptans and Disulfides: Some Physics, Chemistry, and Speculation. Proceedings of the Symposium on Glutathione (Colowick S, Schwarz D R, Lazarow A, Racker E, Stadman E and Waelsch H, eds), pp 3-26, Academic Press Inc, New York 6 Surface plasmon resonance, Surface_plasmon_resonance 7 Assay procedure for glucose oxidase, 8 Akella S & Mitra C K (2003) IANCAS Bull II (1), Scheller F, Strand G, Neumann B, Kuhn M & Ostrowski W (1979) Bioelectrochem. Bioenerg 6, Savitri D & Mitra C K (1998) Bioelectrochem Bioenerg 47, Bard A J & Faulkner L R (1980) Introduction and overview of electrode processes in Electrochemical methods Fundamentals and applications, pp 1-43, John Wiley & Sons

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