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Application of Thiolated Gold Nanoparticles for the Enhancement of Glucose Oxidase Activity Pratibha Pandey,†,§ Surinder P. Singh,*,† Sunil K. Arya,†,§ Vinay Gupta,‡ Monika Datta,§ Sukhvir Singh,† and Bansi D. Malhotra*,† Biomolecular Electronics & Conducting Polymer Research Group, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi - 110012, India, Department of Physics and Astrophysics, UniVersity of Delhi, Delhi - 110007, India, and Department of Chemistry, UniVersity of Delhi, Delhi - 110007, India ReceiVed October 3, 2006. In Final Form: December 1, 2006 Glucose oxidase (GOx) has been covalently immobilized onto chemically synthesized thiolated gold nanoparticles (5-8 nm) via N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The lower value of the Michaelis-Menton constant obtained for the immobilized (3.74 mM) GOx compared with that for the free (5.85 mM) GOx suggests significant enhancement in the activity of GOx attached to thiolated gold nanoparticles. The covalently immobilized GOx thiolated nanoparticles exhibit a response time of 30 s, a shelf life of more than 6 months, and improved tolerance to both pH and temperature.
Introduction Biosensors have attracted much attention in recent times because of the potential applications of these devices in the clinical diagnostics, environmental monitoring, pharmaceuticals, and food processing industries due to their fast response and ease of operation.1-3 The stability of enzymes is crucial for the fabrication of biosensors. A number of techniques have been used for the immobilization of enzymes on different substrates to improve the enzymatic activity and stability.4-10 Nanostructure materials exhibit interesting properties such as a large surface-to-volume ratio, high surface reaction activity, high catalytic efficiency, and strong adsorption ability11-16 that make them potential candidate materials to play a catalytic role in the fabrication of a biosensor. The large surface area of nanomaterials is likely to provide a better matrix for the immobilization of enzymes, leading to increased enzyme loading per unit mass of particles. The * Corresponding author. Phone: +91-11-25734273. Fax: 91-1125726938. E-mail:
[email protected] (B.D.M.); singhsp@ mail.nplindia.ernet.in (S.P.S.). † National Physical Laboratory. ‡ Department of Physics and Astrophysics, University of Delhi. § Department of Chemistry, University of Delhi. (1) Luo, X.; Morrin, A.; Killard, J. A.; Smyth, R. M. Electroanalysis 2006, 18, 319. (2) Malhotra, B. D.; Chaubey, A.; Singh, S. P. Anal. Chim. Acta 2006, 578, 59. (3) Gerard, M.; Chaubey, A.; Malhotra, B. D. Biosens. Bioelectron. 2002, 17, 345. (4) Dai, Z.; Liu, S.; Ju, H.-X.; Chen, H.-Y. Biosens. Bioelectron. 2004, 19, 861. (5) Liu, H. H.; Tian, Z. Q.; Lu, Z. X. Biosens. Bioelectron. 2004, 20, 294. (6) Zhong, X.; Yuan, R.; Chai, Y.; Liu, Y.; Dai, J.; Tang, D. Sens. Actuators, B 2004, 104, 191. (7) Xian, Y.; Hu, Y.; Liu, F.; Xian, Y.; Wang, H.; Jin, L. Biosens. Bioelectron. 2006, 21, 1996. (8) Ren, X.; Meng, X.; Tang, F. Sens. Actuators, B 2005, 110, 358. (9) Rossi, M. L.; Quach, A. D.; Rosenzweig, Z. Anal. Bioanal. Chem. 2004, 380, 606. (10) Zhang, S.; Wang, N.; Niu, Y.; Sun, C. Sens. Actuators, B 2005, 109, 367. (11) Pendry, J. Science 1999, 285, 1687. (12) Hudson, S. D.; Jung, H.-T.; Percec, V.; Johansson, W.-D.; Cho, G.; Balagurusamy, K. Science 1997, 278, 449. (13) Svoboda, K.; Block, S. M. Opt. Lett. 1994, 19, 930. (14) Feldstein, M. J.; Keating, C. D.; Liau, Y.-H.; Natan, M. J.; Scherer, N. F. J. Am. Chem. Soc. 1997, 119, 6638. (15) Fukumi, K.; Chayahara, A.; Kadono, K.; Sakaguchi, T.; Horino, Y.; Miya, M.; Fujii, K.; Hayakawa, J.; Satou, M. J. Appl. Phys. 1994, 75, 3075. (16) Hagland, R. F.; Yang, L.; Magruder, R. H.; Wittig, J. E.; Becker, K.; Zuhr, R. A. Opt. Lett. 1993, 18, 373.
multipoint attachment of enzyme molecules to nanomaterial surfaces reduces protein unfolding, resulting in the enhanced stability of the enzyme attached to the nanoparticle surfaces.17 The enzyme-attached nanoparticles facilitate enzymes to act as free enzymes in solution and in turn improve the enzymesubstrate interaction by avoiding the potential aggregation of the free enzyme.18 Jia et al. studied the activity of an enzyme loaded onto nanoparticles in the framework of collision theory, relating mobility, particle size, and viscosity through the Stokes-Einstein equation, suggesting that the particle size and viscosity of the reaction media affect the mobility of the enzyme catalyst, which in turn alters the activity of the enzyme attached to the particles.18 The metallic nanomaterials are known for their ubiquitous optical plasmon resonance, which allows these materials to be used in the optoelectronic and biological fields in areas such as ultrafast optical switching, optical stability, chemical and biological sensing, surface-enhanced spectroscopy, biotagging, and drug delivery.14-16 For the development of biosensors, particularly glucose biosensors, different matrices, such as titania sol-gel membranes,19 titania nanoporous films,20 polypyrrole/ carbon nanotubes,21 carbon nanotube/nanoelectrodes,22 poly(ortho-amino-benzoic acid),23 Langmuir-Blodgett films of poly3-hexylthiophene,24 silane/Au electrode/Au colloids,25 ZrO2/ chitosan composite films,26 and magnetite nanoparticles,9 have been used to immobilize enzymes. Recently, gold nanoparticles have been identified as a biocompatible material exhibiting fascinating optical and electronic properties and providing a compatible environment for the enzymes.27-29 Gold nano(17) Mozhaev, V. V.; Melik-Nubarov, N. S.; Sergeeva, M. V.; Sikns, V.; Martinek, K. Biocatalysis 1990, 3, 179. (18) Jia, H.; Zhu, G.; Wang, P. Biotechnol. Bioeng. 2003, 84, 406. (19) Rajgopalan, T. J.; Heller, R. A. Anal. Chem. 1994, 66, 2451. (20) Fiorito, P. A; Torresi, S. J. Braz. Chem. Soc. 2001, 12, 729. (21) Tsai, C. Y; Li, C. S; Liao, W. S. Biosens. Bioelectron. 2006, 22, 495. (22) Lin, Y.; Lu, F.; Tu, Y.; Ren, Z. Nano Lett. 2004, 4, 191. (23) Cai, H.; Xu, C.; He, P.; Fnag, Y. J. Electroanal. Chem. 2001, 510, 78. (24) Ramnathan, K.; Pandey, S. S.; Kumar, R.; Gulati, A.; Surya, A.; Murthy, N.; Malhotra, B. D. J. Appl. Polym. Sci. 2000, 78, 662. (25) Singhal, R.; Chaubey, A.; Srikhirin, T.; Aphiantrakul, S.; Pandey, S. S.; Malhotra, B. D. Curr. Appl. Phys. 2003, 3, 275. (26) Yang, Y. H.; Yang, H. F.; Yang, M. H.; Liu, Y. L.; Shen, G. L.; Yu, R. Q. Anal. Chim. Acta 2004, 525, 213. (27) Daniel, M.-C.; Astruc, D. Chem. ReV. 2004, 104, 293. (28) Crumbliss, A. L.; Perine, S. C.; Stonehuerner, J.; Tubergen, K. R.; Zhao, J.; Henkens, R. W.; O’ Daly, J. P. Biotechnol. Bioeng. 1992, 40, 483.
10.1021/la062901c CCC: $37.00 © 2007 American Chemical Society Published on Web 01/30/2007
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Figure 1. Enzyme functionalization on thiolated gold nanoparticles via EDC and NHS chemistry.
particles have also been used as electron-transfer mediators and electric wires for enhancing the electron-transfer rate between the active centers of enzymes and electrodes. The gold nanoparticles, therefore, can act as tiny conduction centers to facilitate electron transfer to realize sensitive biosensors.30-36 Studies for the immobilization of cholesterol oxidase, lipase, and glucose oxidase (GOx) onto iron oxide nanoparticle surfaces have recently been demonstrated with enhanced activity.37,38 Not much effort has been made to covalently immobilize GOx on biocompatible gold nanoparticles. In the present work, we have immobilized GOx onto thiol-modified gold nanoparticles. Experimental Section Materials. Hydrogen tetrachloroaurate(III) (HAuCl4‚3H2O), Nhydroxysuccinimide (NHS), N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC), 11-mercaptoundecanoic acid, GOx (200 U/mg), and horseradish peroxidase (HRP) (E.C. 1.11.1.7, 200 U/mg) were purchased from Sigma (Aldrich). All chemicals were used without further purification. Deionized water was used in the preparation of aqueous solutions. Preparation of Thiol-Modified Gold Nanoparticles. Thiolmodified gold nanoparticles were synthesized at room temperature using the procedure of Shi et al.39 The methanolic solution of 11mercaptoundecanoic acid was added to a 10 mM methanolic solution of HAuCl4 (10 mL) along with vigorous stirring. Subsequently, 5 mL of freshly prepared 0.3 M aq NaBH4 was added to the mixture dropwise with stirring. The reaction mixture turned deep brown, indicating the formation of gold nanoparticles. The stirring was continued for about 2 h, and the functionalized gold nanoparticles were retrieved from methanol using ultracentrifugation. The excess of thiol was removed by successive washing with methanol. Covalent Immobilization of GOx on Synthesized Thiolated Gold Nanoparticles. The GOx was covalently attached to thiolfunctionalized nanoparticles by activation of the -COOH group using EDC and NHS. Thiol-functionalized gold nanoparticles (25 (29) Crumbliss, A. L.; Stonehuerner, J.; Henkens, R. W.; Zhao, J.; O’Daly, J. P. Biosens. Bioelectron. 1993, 8, 331. (30) Shipway, A. N.; Lahav, M.; Willner, I. AdV. Mater. 2000, 12, 993. (31) Ferapontova, E. E.; Grigorenko, V. G.; Egorovo, A. M.; Borchers, T.; Ruzgas, T.; Gorton, L. Biosens. Bioelectron. 2001, 16, 147. (32) Bharathi, S.; Nogmi, M.; Ikeda, S. Langmuir 2001, 17, 1. (33) Lei, C. X.; Hua, S. Q.; Shen, G. L.; Yu, R. Q. Talanta 2003, 59, 981. (34) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. Science 2003, 299, 1877. (35) Jia, J.; Wang, B.; Wu, A.; Cheng, G.; Li, Z.; Dong, S. Anal. Chem. 2002, 74, 2217. (36) Xu, Q.; Mao, C.; Liu, N.-N.; Zhu, J.-J.; Sheng, J. Biosens. Bioelectron. 2006, 22, 768. (37) Kouassi, G. K.; Irudayaraj, J.; McCarty, G. J. Nanobiotechnol. 2005, 3, 1. (38) Huang, S. H.; Liao, M. H.; Chen, D. H. Biotechnol. Prog. 2003, 19 (3), 1095. (39) Shi, W.; Sahoo, Y.; Swihart, M. T. Colloids Surf., A: Physicochem. Eng. Aspects 2004, 246, 109.
mg) were dispersed in 5 mL of phosphate buffer solution (50 mM, pH 7.0) containing 0.02 M EDC and 0.01 M NHS for about 2 h with continuous stirring for activation of the -COOH group. The GOx solution (5 mg mL-1) was then added to the -COOH-activated gold nanoparticle solution. The EDC-NHS-activated -COOH group bound with the -NH2 group of GOx, resulting in the formation of a covalent CO-NH amide bond. The GOx-bound gold nanoparticles were collected by centrifugation and washed with phosphate buffer (pH 7.0) several times (until no enzyme was found in the supernatant solution). After washing, the gold nanoparticle solution was redispersed in the phosphate buffer solution of pH 7.0. A 90% loading of enzymes onto the thiolated gold nanoparticles was estimated by UV-visible measurements. The stability of the colloidal solution was found to be retained after the attachment of enzymes onto the thiolated gold nanoparticles, which could be attributed to the fact that the isoelectric point of GOx is 4.2, indicating that the GOx will be negatively charged at pH 7.0,40 and hence the negatively charged enzyme would act as a stabilizing agent for the colloidal solution to prevent aggregation. The GOx enzyme-bound gold nanoparticles were used for the measurement of activity and stability. A schematic of the covalently linked enzyme is shown in Figure 1.
Results and Discussion The UV-visible spectrum of gold nanoparticles is known to exhibit a strong surface plasmon absorbance band at about 520 nm. The resonance frequency of nanoparticles has been found to depend upon its size, shape, material properties, surrounding media, and proximity to other nanoparticles.41 Thiol-functionalized gold nanoparticles show the characteristic surface plasmon peak at about 525 nm (Figure 2), confirming the formation of gold nanoparticles, and the broadening of the peak could be attributed to an indirect indication of the presence of thiol on the surface of the gold nanoparticles. The binding of GOx to the gold nanoparticles results in a shift of the absorption band toward a higher wavelength (533 nm) along with further broadening of the absorption band, suggesting the immobilization of the GOx enzyme on the thiolated gold nanoparticles. The transmission electron microscopy (TEM) images of thiolfunctionalized gold nanoparticles and GOx immobilized on gold nanoparticles further support the attachment of GOx. The microstructural investigation of the thiolated and GOx-attached gold nanoparticles was carried out using TEM to reveal the size, shape, and distribution/separation of the particles. All TEM micrographs were recorded with a transmission electron microscope (Jeol, model JEM 200 CX) operated at 160 kV. (40) Wei, A.; Sun, X. W.; Wang, J. X.; Lei, Y.; Cai, X. P.; Li, C. M.; Dong, Z. L.; Huang, W. Appl. Phys. Lett. 2006, 89, 123902-1. (41) Aslan, K.; Lakowicz, J. R.; Geddes, C. D. Anal. Biochem. 2004, 330, 145.
Enhancing GOx ActiVity Via Thiolated Au Nanoparticles
Figure 2. Thiol-modified gold nanoparticles (---) and enzymefunctionalized thiolated gold nanoparticles (‚-‚-‚-).
Figure 3. TEM images of thiolated and enzyme-bound gold nanoparticles.
Figure 3a shows a bright-field electron micrograph of the thiolated gold nanoparticles recorded at a magnification of 300 000×, and it is revealed that the nearly spherical thiolated gold nanoparticles are distributed uniformly with a low tendency of agglomeration among the particles. The size of the spherical thiolated gold particles is found to vary from 3 to 8 nm with an average size of about 5 nm. The TEM micrograph of the thiolated gold nanoparticles recorded at 375 000× magnification attached with a GOx enzyme is shown in Figure 3b. From the micrographs, it is observed that the size of the particles has increased, with a slightly higher tendency of agglomeration, which may be due to the presence of the enzyme. The size of the particles varies from 6 to 11 nm with an average size of 9 nm. The absence of the characteristic peak at 2500 cm-1 for S-H stretching in the Fourier transform infrared (FT-IR) spectra of
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thiol- and GOx-functionalized gold nanoparticles (Figure 4a,b) indicates the formation of a Au-S bond on the surface of the gold nanoparticles. The observed slight shift (2-4 cm-1) in the peak of the -CH2 group toward a lower frequency along with significant narrowing confirms the thiol functionalization of the gold nanoparticles and is in agreement with the literature reported for the attachment of thiol on gold nanoparticles.42,43 The narrowing of the stretching bands is attributed to the rigid chain conformation on the gold nanoparticle surface, which generates a crystalline-like state of the chains and restricts their mobility. The peaks observed at 2916 and 2848 cm-1 are due to C-H (CH3) and C-H (CH2) stretching frequencies, respectively. The additional peaks seen at 1655 cm-1 and 3369 cm-1 (Figure 4b) confirm the binding of GOx. The sharp peak at 1655 cm-1 attributed to CdO stretching is known as the amide I band, and the peak at 3369 cm-1 is assigned to NH deformation (amide II band). The results are in agreement with the existing literature where cholesterol oxidase was covalently bound on the surface of magnetic nanoparticles.37 Activity and Binding Kinetics of Covalently Immobilized GOx on Thiolated Gold Nanoparticles. The ability of amino acids present at the active sites of the enzyme to interact with the substrate depends on their electrostatic state, which in turn depends on the pH of the solution.44 To find the optimum pH, the activity of free and gold nanoparticle-bound GOx has been investigated in the pH range of 6.0-8.0 at 30 °C. The optimum catalytic activity for both bound and free GOx enzymes was observed at pH 7.0. Gold nanoparticle-bound GOx enzyme shows enhanced activity at pH 7.0 relative to that of the free GOx enzyme. The activity of free and immobilized GOx enzymes on thiolated gold nanoparticle surfaces was monitored using UV-visible spectroscopy. To carry out the enzyme assay, free and gold nanoparticle-bound GOx enzymes (100 µL) were mixed in 3 mL of phosphate-buffered saline (PBS) containing 50 µL of HRP (1 mg/mL), 20 µL of o-dianisidine dye (1%), and 100 µL of glucose as a substrate separately. The absorbance recorded at 405 nm (Figure 5) indicates enhanced activity of immobilized GOx on the gold nanoparticles. Additionally, GOx activity increases with the increase in concentration of glucose up to 300 mg/dL, whereafter it reaches saturation. The value of the MichaelisMenton kinetic parameter (Km and Vmax), which gives an indication of the enzyme-substrate kinetics, was determined by the analysis of the slope of enzymatic reactions. The Km value for an enzymatic reaction determines the affinity of the enzyme for the substrate, whereas the value of Vmax provides the maximum rate of enzyme reaction when the enzyme is saturated by the substrate. These parameters have been estimated using the Lineweaver-Burke plot, that is, the graph of the inverse of absorption versus the inverse of glucose concentration (Figure 6), where the inverse of the intercept at the x-axis and y-axis gives the value of Km and Vmax, respectively. The smaller value of Km indicates the increased affinity of enzyme for substrate. The values of Km in the present enzymatic assay were found to be 3.74 and 5.85 mM/L for the immobilized GOx and the free GOx, respectively. The value of Vmax (1.42 µM min-1mg-1) obtained for the covalently immobilized GOx is one order higher in magnitude than that of free GOx (0.25 µM min-1mg-1). However, the value of Km for the covalently immobilized GOx is 1.56-fold lower in magnitude in comparison with the value obtained for free GOx. The secondary and tertiary structure of the enzyme is known to play an important (42) Hostetler, M. J.; Stokes, J. J.; Murray, R. W. Langmuir 1996, 12, 3604. (43) Sahoo, Y.; Pizem, H.; Fried, T. D.; Golodnitsky, L.; Burstein, C. N.; Sukenik, G. Markovich, Langmuir 2001, 17, 7907. (44) Kang, Y.; Marangoni, A. G.; Yada, R. Y. J. Food Biochem. 1994, 17, 389.
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Figure 4. FT-IR spectra of (a) thiol-functionalized gold nanoparticles and (b) enzyme-thiol-modified gold nanoparticles.
Figure 6. Lineweaver-Burke plots of (a) free GOx and (b) GOx bound to thiolated gold nanoparticles at pH 7.0.
Figure 5. Enzyme activity of (a) free GOx enzymes and (b) GOx covalently immobilized onto thiol-functionalized gold nanoparticles.
role in the enzyme activity,45 as the rearrangement and conformational changes in these structures may result in the enhancement or suppression of enzyme activity. To investigate the conformational changes, circular dichroism (CD) studies were performed. The CD peak at 222 nm (Figure 7a) is a characteristic peak of the secondary structure of enzymes. The shift in the 222 nm CD peak toward a higher wavelength with a decrease in intensity (Figure 7b) reveals the conformational change in the secondary structure of the enzyme.46 The observed low value of Km in the present study reflects the high affinity and the enhanced activity of the immobilized GOx on thiolated gold nanoparticles.
Figure 7. CD spectra of (a) free GOx and (b) GOx attached to gold nanoparticles.
(45) Pollegioni, L.; Wels, G.; Pilone, M. S.; Ghisla, S. J. Biochem. 1999, 264, 140. (46) Verma, A.; Simard, J. M.; Rotello, V. M. Langmuir 2004, 20, 4178.
The high affinity of the GOx for the substrate can be attributed to the favorable conformational changes in GOx upon binding
Enhancing GOx ActiVity Via Thiolated Au Nanoparticles
Langmuir, Vol. 23, No. 6, 2007 3337 Table 1.
serial no. 1 2 3 4 5 6 7 8
immobilization matrix
sensing element
method of immobilization
linearity (mM)
Km (mM)
titania sol-gel membrane TiO2 nanoporous film polypyrrole/carbon nanotubes carbon nanotube/ nanoelectrode Au NP/ Cyst/ AuE ZrO2/ chitosan film magnetite nanoparticles thiolated gold nanoparticles
GOx GOx GOx GOx GOx GOx GOx GOx
entrapment entrapment entrapment covalent covalent entrapment covalent covalent
0.07-15 6-30 4 30 0.01-10 0.125-9.5 20 15
6.38 6.08
with the thiolated gold nanoparticles, resulting in the enhanced availability of active sites of enzyme to glucose. These favorable conformational changes in GOx after the immobilization result in a reduction in the value of Km (3.74 mM/L). To the best of our knowledge, the Km value, which is related to the activity and stability of the enzyme, is the lowest for the GOx enzyme immobilized onto the gold nanoparticles in comparison with the studies reported on different nanoparticles.9 The lowest value of Km clearly indicates the usefulness of thiolated gold nanoparticles for glucose sensing application. To confirm the reproducibility of the system, each set of experiment was carried out in triplicate, and similar results within the maximum error of 2-3% were obtained. This reproducibility in the results indicates the promising application of gold nanoparticles in biosensors. Thermal Stability and Shelf Life. The thermal stability of free and covalently immobilized GOx has been investigated in the range of 20-50 °C. The GOx covalently immobilized onto thiolated gold nanoparticles shows higher GOx activity in comparison with that of free GOx. The higher thermal stability of the covalently bound GOx on thiolated nanoparticles suggests an improved resistance of the GOx to temperature. The observed increased thermal stability of GOx may be attributed to the fact that covalent immobilization of GOx prevents unfolding of the structure, leading to reduced dissociation of oligomeric proteins to subunits.47
16 3.45 6.8 3.74
shelf life 80 days
30 days 90 days >180 days
ref 19 20 21 22 25 26 9 present work
Figure 8 shows the Arrhenius plot of ln (absorbance) as a function of reciprocal temperature in the range of 20-50 °C. The activation energies in the lower and higher temperature range were found to be 5.9 and 19.2 kJ/mol, respectively, suggesting better activity of the enzyme at lower temperatures. The shelf life of the enzyme bound to gold nanoparticles was investigated using UV-visible studies for a given concentration of glucose at regular intervals of 15 days for 6 months. It was found that GOx-bound particles retain almost 97% of their initial activity at zero days, even after a duration of 6 months (data not shown), suggesting the stability of bound enzymes for more than 6 months. A comparison of the present investigation has been summarized in Table 1 in which it is clear that thiolated gold nanoparticles are a promising matrix for the immobilization of enzyme and the realization of fast and sensitive biosensors.
Conclusions We have chemically synthesized thiolated gold nanoparticles (3-8 nm) and demonstrated that GOx enzyme can be covalently immobilized onto the surface of these nanoparticles. GOx binding has been confirmed by UV-visible, FT-IR, and TEM studies. CD studies indicate a conformational change in the GOx structure. The lower Km value (3.74 mg/dL) of GOx shows enhanced activity, indicating a favorable conformational change in the GOx structure upon attachment to thiolated gold nanoparticles. These GOxthiolated gold nanoparticles can be used for the estimation of glucose up to 300 mg/dL and are stable for more than 6 months when stored at 4 °C. This result suggests that these biocompatible thiol-modified gold nanoparticles may prove to be a promising matrix for the immobilization of other enzymes and proteins with enhanced stability and activity for biosensor applications. Work is in progress to directly adsorb these GOx-modified nanoparticles onto a desired electrode in order to realize a glucose biosensor. Acknowledgment. We are grateful to Dr. Vikram Kumar, Director NPL, for his interest in this work. We thank all the members of BECPRG for interesting discussions. P.P. is grateful to the University Grant Commission (UGC), India, for the award of a Junior Research Fellowship. Financial support received under the DST sponsored project DST/TSG/ME/2002/19 is gratefully acknowledged. B.D.M. thanks the Japan Society for the Promotion of Science for the award of an Invitation Fellowship during September-October 2006.
Figure 8. Arrhenius plot of ln absorbance versus the reciprocal of absolute temperature.
LA062901C (47) Mozhaev, V. V. Trends Biotechnol. 1993, 11, 88.
