Electrochemical Biosensor Based on Integrated Assembly of

The AuNPs on the silicate network efficiently catalyze the oxidation of NADH ..... Metal Nanoparticles in Nanomedicine: Advantages and Scope ..... Ele...
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Anal. Chem. 2006, 78, 6332-6339

Electrochemical Biosensor Based on Integrated Assembly of Dehydrogenase Enzymes and Gold Nanoparticles Bikash Kumar Jena and C. Retna Raj*

Department of Chemistry, Indian Institute of Technology, Kharagpur, 721 302, India

Development of a highly sensitive nanostructured electrochemical biosensor based on the integrated assembly of dehydrogenase enzymes and gold (Au) nanoparticle is described. The Au nanoparticles (AuNPs) have been selfassembled on a thiol-terminated, sol-gel-derived, 3-D, silicate network and enlarged by hydroxylamine seeding. The AuNPs on the silicate network efficiently catalyze the oxidation of NADH with a decrease in overpotential of ∼915 mV in the absence of any redox mediator. The surface oxides of AuNP function as an excellent mediator, and a special inverted “V” shape voltammogram at less positive potential was observed for the oxidation of NADH. The AuNP self-assembled sol-gel network behaves like a nanoelectrode ensemble. The nanostructured electrode shows high sensitivity (0.056 ( 0.001 nA/nM) toward NADH with an amperometric detection limit of 5 nM. The electrode displays excellent operational and storage stability. A novel methodology for the fabrication of a NADHdependent dehydrogenase biosensor based on the integration of dehydrogenase enzyme and AuNPs with the silicate network is developed. The enzymatically generated NADH is, in turn, electrocatalytically detected by the AuNPs on the silicate network. The integrated assembly has been successfully used for the amperometric biosensing of lactate and ethanol at a potential of -5 mV. The biosensor is very stable and highly sensitive, and it has a fast response time. The excellent performance validates the integrated assembly as an attractive sensing element for the development of new dehydrogenase biosensors. Nanostructured metal and semiconductor particles have been extensively studied in the past decade because of their fascinating chemical, optical, and electronic properties.1-11 The size-depend* Corresponding author. Phone: +91-3222-283348. Fax: +91-3222-282252. E-mail: [email protected]. (1) Moffitt, M.; Eisenberg, A. Chem. Mater. 1995, 7, 1178-1184. (2) Feldheim, D. L.; Grabar, K. C.; Natan, M. J.; Mallouk, T. E. J. Am. Chem. Soc. 1996, 118, 7640-7641. (3) Valden, M.; Lai, X.; Goodman, D. W. Science 1998, 281, 1647-1650. (4) Sarathy, K. V.; Thomas, P. J.; Kulkurni, G. U.; Rao, C. N. R. J. Phys. Chem. B 1999, 103, 399-401. (5) Crooks, R. M.; Zhao, M.; Sun, L.; Chechik, V.; Yeung, L. K. Acc. Chem. Res. 2001, 34, 181-190. (6) Jia, J.; Wang, B.; Wu, A.; Cheng, G.; Li, Z.; Dong, S. Anal. Chem. 2002, 74, 2217-2223. (7) Wang, Y.; Tang, Z.; Correa-Duarte, M. A.; Liz-Marzan, L. M.; Kotov, N. A. J. Am. Chem. Soc. 2003, 125, 2830-2831.

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ent, unique characteristics of these particles are very promising for practical application in such diverse areas as catalysis, optoelectronics, etc.11 Particularly, the AuNPs has received considerable attention in analytical electrochemistry.12-20 For instance, Willner’s group has extensively studied the electrochemical and optical applications of AuNPs.11-15 Although Au is known to be a poor catalyst in the bulk form, recent studies show that the nanosized Au particles exhibit excellent catalytic activity.21 Haruta and co-workers have shown that highly dispersed, nanometer-sized Au particles are really very active in many important reactions.22,23 Zhong et al. investigated the electrocatalytic effect of core-shell AuNPs toward the oxidation of methanol and CO.16,19,24 Recently, Ohsaka et al. demonstrated the electrocatalytic activity of electrodeposited AuNPs toward O2 reduction.17 The nanoparticles are very different from their bulk counterparts, and it is believed that the catalytic activity originates from the quantum scale dimension.25 The large surface-to-volume ratio and the presence of active sites on the fine particle are the driving force in the nanoparticle-assisted catalysis.16,26 Because nanometer-sized Au particles show extraordinary catalytic activity, the deliberate tailoring of transducers with AuNPs is a promising approach in the development of sensing devices. The sol-gel technology provides a versatile way to prepare a 3-D silicate network through the hydrolysis and condensation of (8) Daniel, M.-C.; Astruc, D. Chem. Rev. 2004, 104, 293-346. (9) Lu, Q.; Hu, S.; Pang, D.; He, Z. Chem. Commun. 2005, 2584-2585. (10) Tang, Z.; Wang, Y.; Sun, K.; Kotov, N. A. Adv. Mater. 2005, 17, 358-363. (11) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042-6108. (12) Xiao, Y.; Pavlov, V.; Shlyahovsky, B.; Willner, I. Chem.sEur. J. 2005, 11, 2698-2704. (13) Zayats, M.; Baron, R.; Popov, I.; Willner, I. Nano Lett. 2005, 5, 21-25. (14) Shipway, A. N.; Katz, E.; Willner, I. ChemPhysChem 2000, 1, 18-52. (15) Xiao, Y.; Pavlov, V.; Levine, S.; Niazov, T.; Markovitch, G.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 4519-4522. (16) Maye, M. M.; Lou, Y.; Zhong, C.-J. Langmuir 2000, 16, 7520-7523. (17) El-Deab, M. S.; Okajima, T.; Ohsaka, T. J. Electrochem. Soc. 2003, 150, A851-A857. (18) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27-36. (19) Zhong, C.-J.; Maye, M. M. Adv. Mater. 2001, 13, 1507-1511. (20) Raj, C. R.; Jena, B. K. Chem. Commun. 2005, 2005-2007. (21) Biswas, P. C.; Nodasaka, Y.; Enyo, M.; Haruta, M. J. Electroanal. Chem. 1995, 381, 167-177. (22) Haruta, M.; Yamada, N.; Kobayashi, T.; Iijima, S. J. Catal. 1989, 115, 301309. (23) Haruta, M. Catal. Today 1997, 36, 153-166. (24) Lou, Y.; Maye, M. M.; Han, L.; Luo, J.; Zhong, C.-J. Chem. Commun. 2001, 473-474. (25) Alivisatos, A. P. Science 1996, 271, 933-937. (26) Haruta, M.; Date, M. Appl. Catal. A 2001, 222, 427-437. 10.1021/ac052143f CCC: $33.50

© 2006 American Chemical Society Published on Web 08/17/2006

silicon alkoxide precursors.27 The sol-gel-derived three-dimensional (3-D) network is particularly attractive in the development of sensing devices, because the network exhibits tunable porosity, high thermal stability, and chemical inertness. Enzymes and proteins encapsulated into the sol-gel derived silicate network have been extensively studied for different applications.28 Interestingly, the encapsulated biomolecules retain their native functions and properties.29 This biocomposite material is permeable to a wide variety of molecules, but it retains the encapsulated biocomponent inside the network, and hence, quickly responding biosensing devices can be developed. (3Mercaptopropyl)trimethoxysilane (MPTS) is known to form a 3-D structure that is full of thiol tail groups by the hydrolysis and condensation process.30 The thiol group has a strong affinity to Au and is able to chemisorb onto the surface of Au through the cleavage of the S-H bond.31 Because the thiol groups are distributed throughout the network, the nanosized Au particle can be conveniently self-assembled on the thiol groups present both inside and on the surface of the network. The electrochemical oxidation of NADH to enzymatically active NAD+ has attracted much interest because over 300 dehydrogenases require NADH as a cofactor.32 This pyridine coenzyme oxidation can be used as an electrochemical transduction reaction in developing dehydrogenase-based amperometric biosensors. It is generally observed that the direct electrochemical oxidation of the coenzyme is electrochemically irreversible and requires high overpotential as large as 1 V on solid electrodes, although the formal potential of the NADH/NAD+ redox couple at pH 7 is 0.32 (NHE).33 The large overpotential required for the oxidation invites interference from other easily oxidizable species present in the real sample. The fouling of the electrode surface by the adsorption of reaction intermediates on the electrode is another major concern in achieving fast electron transfer.34 A convenient way to decrease the high overpotential and avoid the fouling effect is to use suitable mediators that can facilitate the electron transfer kinetics. In this way, much effort has been made to identify/ develop new materials that can effectively overcome the kinetic (27) (a) Brinker, C. J.; Scherer, G. W. Sol-Gel Science; Academic Press: New York, 1990. (b) Hench, L. L.; West, J. K. Chem. Rev. 1990, 90, 33-72. (c) Avnir, D. Acc. Chem. Res. 1995, 28, 328-334. (d) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 66, 1120A-1127A. (28) (a) Dave, B. C.; Dunn, B.; Valentine, J. S.; Zink, J. I. Anal. Chem. 1994, 66, 1120A-1127A. (b) Collinson, M. M.; Howells, A. R. Anal. Chem. 2000, 72, 702A-709A. (c) Avnir, D.; Coradin, T.; Lev, O.; Livage, J. J. Mater. Chem. 2006, 16, 1013-1030. (d) Wang, J.; Pamidi, P. V. A. Anal. Chem. 1997, 69, 4490-4494. (29) (a) Guilbault, G. G. Analytical Uses of Immobilized Enzymes; Marcel Dekker: New York, 1984. (b) Gill, I.; Ballesteros, A. Trends Biotechnol. 2000, 18, 282-296. (30) (a) Wang, J.; Pamidi, P. V. A.; Zanette, D. R. J. Am. Chem. Soc. 1998, 120, 5852-5853. (b) Chen, X.; Wilson, G. S. Langmuir 2004, 20, 8762-8767. (c) Jia, J.; Wang, B.; Wu, A.; Cheng, G.; Li, Z.; Dong, S. Anal. Chem. 2002, 74, 2217-2223. (d) Bharathi, S.; Nogami, M.; Ikeda, S. Langmuir 2001, 17, 1-4. (31) Finklea, H. O. In Bard, A. J., Rubinstein, I., Eds.; Electroanalytical Chemistry, Marcel Dekker: New York, 1996; Vol 19, p 109-335. (32) (a) Gorton, L.; Dominguez, E. In Electrochemistry of NAD(P)+/NAD(P)H, Encyclopedia; Wilson, G. S., Ed.; Wiley-VCH: Weinheim, 2002, Vol 9, pp 67-143. (b) Gorton, L.; Dominguez, E. Rev. Mol. Biotech. 2002, 82, 371392. (33) (a) Moiroux, J.; Elving, P. J. Anal. Chem. 1978, 50, 1056-1062. (b) Clarke, W. M. Oxidation-Reduction Potential of Organic System; Williams & Williams Co.: Baltimore, MD, 1960. (34) Jaegfeldt, H. Bioelectrochem. Bioenerg. 1981, 8, 355-370.

barriers for the electrochemical regeneration of NAD+.35-43 Nevertheless, such electrodes possess certain drawbacks, such as leaching of mediator and lack of long-term stability, which may limit their analytical application.44 Recently, we have shown the use of nanosized, citrate-stabilized Au particles as an electrocatalyst for the oxidation NADH.20 Although we have observed the oxidation of NADH at less positive potential, the sensitivity of the electrode was low. The hydroxylamine seeding of surface-confined AuNPs has been proven to be a valuable tool in the controlled growth of Au nanostructures.45 The hydroxylamine-mediated growth of a Au nanostructure would result in the formation of nanoelectrode ensembles with a high area fraction.46 Significant improvement in the sensitivity is expected at such nanoelectrode ensembles.47 In an effort to develop a highly sensitive and selective dehydrogenase-based biosensor without any redox mediator, in the present investigation, we have exploited the hydroxylamineenlarged AuNPs assembled on a sol-gel-derived silicate network as an electrocatalyst. The biosensor is developed by integrating the enlarged AuNPs and dehydrogenase enzyme with the solgel network. The electrocatalytic activity of the enlarged AuNPs is combined with the enzymatic activity of dehydrogenase enzymes for the sensing of lactate/ethanol. The biosensor described herein is based on the electrocatalytic sensing of enzymatically generated NADH by the integrated assembly without any redox mediator. MATERIALS AND METHODS Materials.(3-Mercaptopropyl)trimethoxysilane(MPTS),HAuCl4, lactate dehydrogenase (LDH), and alcohol dehydrogenase (ADH) were obtained from Sigma-Aldrich and used as received. LDH (400-600 units/mg) and ADH (50 units/mg) were obtained as lyophilized powder. All other chemicals used in this investigation were of analar grade unless mentioned otherwise. All the solutions were prepared with Millipore water. Instrumentation. Electrochemical measurements were performed using two-compartment, three-electrode cell with a polycrystalline Au working electrode, a Pt wire auxiliary electrode, and a Ag/AgCl (3M NaCl) reference electrode. Cyclic voltammograms were recorded using a computer-controlled CHI643B (35) Alvarez-Gonzalez, M. I.; Saidman, S. B.; Lobo-Castanon, M. J.; MirandaOrdieres, A. J.; Tunon-Blanco. P. Anal. Chem. 2000, 72, 520-527. (36) Ramesh, P.; Sampath, S. Anal. Chem. 2000, 72, 3369-3373. (37) Rao, T. N.; Yagi, I.; Miwa, T.; Tryk, D. A.; Fujishima, A. Anal. Chem. 1999, 71, 2506-2511. (38) Pariente, F.; Tobalina, F.; Moreno, G.; Hernandez, L.; Lorenzo, E.; Abruna, H. D. Anal. Chem. 1997, 69, 4065-4075. (39) Katz, E.; Lotzbeyer, T.; Schlereth, D. D.; Schuhmann, W.; Schmidt, H.-L. J. Electroanal. Chem. 1994, 373, 189-200. (40) De-los-Santos-Alvarez, N.; Lobo-Castanon, M. J.; Miranda-Ordieres, A. J.; Tunon-Blanco, P.; Abruna, H. D. Anal. Chem. 2005, 77, 2624-2631. (41) Zhang, M.; Gorski, W. J. Am. Chem. Soc. 2005, 127, 2058-2059. (42) Wang, J.; Musameh, M. Anal. Chem. 2003, 75, 2075-2079. (43) Tian, S.; Liu, J.; Zhu, T.; Knoll, W. Chem. Commun. 2003, 2738-2739. (44) (a) Gilmartin, M. A. T.; Hart, J. P. Analyst 1995, 120, 1029-1045. (b) Mano, N.; Thienpont, A.; Kuhn, A. Electrochem. Commun. 2001, 3, 585-589. (45) Brown, K. R.; Natan, M. J. Langmuir 1998, 14, 726-728. (46) (a) Cheng, W.; Dong, S.; Wang, E. Langmuir 2002, 18, 9947-9952. (b) Jin, Y.; Kang, X.; Song, Y.; Zhang, B.; Cheng, G.; Dong, S. Anal. Chem. 2001, 73, 2843-2849. (47) (a) Menon, V. P.; Martin, C. R. Anal. Chem. 1995, 67, 1920-1928. (b) Weber, S. G. Anal. Chem. 1989, 61, 295-302. (c) Liu, Z.; Niwa, O.; Kurita, R.; Horiuchi, T. Anal. Chem. 2000, 72, 1315-1321. (d) Reller, H.; KirowaEisner, E.; Gileadi, E. J. Electroanal. Chem. 1984, 161, 247-268.

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electrochemical analyzer. X-ray diffraction analysis of self-assembled AuNPs before and after seeding was carried out with Phillips X′pert PRO X-ray diffraction unit using Ni-filtered CuKR (λ ) 1.54 Å) radiation. Scanning electron microscopic measurements were performed with a JEOL JEM 6700F field emission scanning electron microscope (FESEM). FTIR measurements were performed with a Nexus 870 (Thermonicolet). UV-visible absorption spectral measurements were carried out using a Shimadzu UV-1601 double-beam spectrophotometer. The UVvisible diffuse reflectance spectra (DRS) of AuNPs on the goldcoated glass slides were measured with a Shimadzu UV-2401 PC spectrophotometer. Preparation of Colloidal AuNPs. All glassware used in the following preparation of colloidal nanoparticles were cleaned with freshly prepared aqua regia and rinsed thoroughly with water. (Caution: aqua regia is a powerful oxidizing agent and it should be handled with extreme care). Citrate-stabilized AuNPs of ∼2.6-nm diameter were prepared by adding 0.64 mL of 1.15% trisodium citrate and freshly prepared 0.08% NaBH4 (0.32 mL) in 1% trisodium citrate to 30 mL of water containing 1% HAuCl4 (0.32 mL) and stirring the solution for 10 min at room temperature. Preparation of MPTS Sol. The MPTS sol was prepared by dissolving MPTS, methanol, and water (as 0.1 M HCl) in a molar ratio of 1:3:3 and stirring the mixture vigorously for 30 min. For the fabrication of the biosensor, the MPTS sol-enzyme biocomposite was prepared by the following procedure: the MPTS sol was prepared by taking 24 µL of MPTS and 10 µL (0.1 M) of HCl in 2 mL of water and stirring the mixture vigorously for 30 min. Then 0.5 mL of MPTS sol was mixed with 0.5 mL of LDH or ADH solution (8 mg/mL in 5 mM PBS of pH 8) and stirred for 2-3 min for the encapsulation of enzymes into the network. The resulting sol-gel biocomposite was stored at 4° C. Self-Assembling of AuNPs on Sol-Gel Network. The polycrystalline Au electrode of geometrical surface area 0.031 cm2 was polished repeatedly with alumina (0.06 µm) and sonicated in water for 15 min. The well-polished electrode was then subjected to electrochemical pretreatment by cycling the potential between - 0.2 and 1.5 V in 0.25 M H2SO4 at a scan rate of 10 V/s for ∼10 min or until a voltammogram characteristic of a clean polycrystalline Au electrode was obtained. The cleaned Au electrode was thoroughly rinsed with water and ethanol and was soaked in 0.5 mL of MPTS sol for 10 min. MPTS sol chemisorbs on the polycrystalline Au electrode and exists as a 3-D silicate network.48 The resulting MPTS sol-modified electrode was thoroughly rinsed with water to remove the physically adsorbed MPTS sol and immersed into colloidal AuNP for 18 h. The nanosized Au particles chemisorb on the thiol groups of the silicate network. For the FESEM, FTIR, and DRS measurements, the goldcoated cover slip was first modified with the MPTS sol to get the sol-gel network, and the AuNPs were self-assembled as described earlier. For XRD and UV-visible absorption spectral measurements, the microscopic glass slide or cover slip was functionalized with MPTS by soaking the cleaned glass slides or cover slip in a methanolic solution of MPTS for 18 h. This MPTS-functionalized (48) The existence of a 3-D silicate network was confirmed by FT-IR spectral measurements. The characteristic asymmetric vibrational stretching was observed at 1000-1200 cm-1. The FTIR spectra are given in the Supporting Information.

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glass slide or coverslip was washed with a copious amount of water, and then AuNPs were self-assembled as before. Hydroxylamine Enlargement of AuNPs on the Sol-Gel Network. The enlargement of AuNPs on the sol-gel network was carried out by using hydroxylamine seeding.45 Hydroxylamine is capable of reducing Au3+ to bulk metal, and it has been shown that this reaction is accelerated by Au surfaces.45,49 The surfacecatalyzed reduction of Au3+ by hydroxylamine leads to the enlargement of the small particles. The AuNP self-assembled electrode/cover slip was soaked in a solution containing 0.3 mM hydroxylamine and 0.3 mM HAuCl4, and the solution was shaken constantly for 20 min. The resulting electrodes were rinsed repeatedly with water and kept in phosphate buffered solution (PBS) before being subjected to electrochemical experiments. Hereafter, the AuNP self-assembled electrodes before and after hydroxylamine seeding will be referred to as nAu and nAuS, respectively. Fabrication of Biosensor. The amperometric biosensor for the sensing of lactate and ethanol was fabricated as illustrated in Scheme 1. The cleaned polycrystalline Au electrode was soaked in the MPTS sol-enzyme biocomposite for 20 min for the spontaneous adsorption of the enzyme-encapsulated sol-gel network onto the electrode surface. The AuNPs were then selfassembled on this enzyme-encapsulated sol-gel network at 4° C. The hydroxylamine enlargement of the AuNPs on the network was carried out as described earlier (Scheme 1). To obtain the SEM image of the integrated assembly, a gold-coated cover slip was used instead of a polycrystalline Au electrode. All electrochemical experiments were performed in an Ar atmosphere. PBS (0.1 M, pH 7.2) was used as a supporting electrolyte in all experiments. All the experiments were repeated at least three times, and reproducible results were obtained. The calibration plots for the sensing of NADH, lactate, and alcohol were made from three independent measurements. RESULT AND DISCUSSION Characterization of AuNPs on the Sol-Gel Network. The binding of AuNPs with the thiol groups was investigated by UVvisible spectral measurements. The AuNP self-assembled solgel network shows a characteristic surface plasmon band at 522 nm, whereas the sol-gel network does not show any band (Supporting Information), indicating that AuNPs chemisorbed onto the thiol groups of the silicate network. The hydroxylamineenlarged AuNP on the network exhibits the red-shifted plasmon band at 536 nm due to the enlargement in the size. The binding of AuNPs on the thiol groups of the sol-gel network was further confirmed by FT-IR spectral measurements. The characteristic band at 2560 cm-1 for the -SH group50 disappears upon the chemisorption of AuNP onto the thiol groups (Supporting Information). The size of the AuNPs on the sol-gel network and the surface morphology of the integrated assembly of the enzyme (ADH) and AuNPs have been examined by FESEM. Figure 1 shows the FESEM image obtained for the hydroxylamine enlarged AuNPs and the integrated assembly. The AuNPs on the network have a size distribution between 70 and 100 nm and are almost spherical (49) Stremsdoerfer, G.; Perrot, H.; Martin, J. R.; Clechet, P. J. Electrochem. Soc. 1988, 135, 2881-2885. (50) Kim, C. H.; Han, S. W.; Ha, T. H.; Kim, K. Langmuir 1999, 15, 8399-8404.

Scheme 1. Scheme Illustrating the Integration of Dehydrogenase Enzyme (LDH or ADH) and AuNPs with the Sol-Gel-Derived 3D Network

in shape. The nanoparticles are uniformly distributed throughout the network; they can be considered ensembles of nanoelectrodes. The aggregates of encapsulated enzyme exist throughout the network and have a bright, islandlike structure (Figure 1b). The distribution of both nanoparticles and the enzyme in the network facilitate the access of the substrate and result in a fast amperometric response. The XRD measurement after hydroxylamine seeding showed four peaks corresponding to (111), (200), (220), and (311) planes (Supporting Information). The peak for the Au(111) plane is more intense than the others, indicating that the (111) is the predominant orientation. The surface area of the AuNPs has been determined by chronoamperometry using K3Fe(CN)6 as the redox probe. The surface area of hydroxylamineenlarged AuNPs on the sol-gel network was 0.076 cm2. Cyclic voltammetry and impedance techniques are powerful tools to probe the nature of the modified electrodes and the influence of nanoparticles on electron-transfer kinetics. The MPTS sol-modified electrode shows sluggish electron-transfer kinetics for the Fe(CN)64-/3- redox couple, with a peak-to-peak separation (∆Ep) of ∼200 mV (Supporting Information); the silicate network on the electrode surface impedes the electron-transfer process. On the other hand, the nAu electrode exhibits reversible redox response, which is very similar to that of the unmodified polycrystalline Au electrode. The AuNPs on the silicate network tune the electrochemical characteristics of the MPTS sol-modified electrode and favor the electron-transfer reaction. In the case of nAuS electrode, significant enhancement in the voltammetric peak current for the Fe(CN)64-/3- redox couple was observed. This is ascribed to the increase in the surface area. The Rct values calculated from the impedance measurement (Supporting Information) for the MPTS modified Au, nAu, and nAuS electrodes

confirm the facilitated electron transfer on the nanoparticlemodified electrodes. Figure 2 shows the cyclic voltammograms obtained for the nAu and nAuS electrodes in the absence of any redox probes. The nAu electrode does not show any characteristic peak in the potential range used, whereas the nAuS electrode shows two pairs of peaks at +0.2 ( 0.01 and -0.14 ( 0.01 V. The appearance of two voltammetric peaks for the nAuS electrode suggests that hydroxylamine seeding influenced the surface morphology of the AuNP. The voltammetric features observed for the nAuS electrodes are similar to those of the Au(111) single-crystal electrode, indicating that AuNPs on the silicate network have a Au(111) face (vide supra). The Au(111) electrode shows two pairs of voltammetric peaks at +0.2 and -0.2 V (SCE) in phosphate buffer solution and were ascribed to the formation of surface oxides.51 In the present investigation, the voltammetric peaks observed for nAuS electrodes are ascribed to the formation of incipient hydrous oxides (AuOH or Au(OH)3) by the process of premonolayer oxidation. Such voltammetric response has not been observed either at the MPTS sol-modified electrode or at unmodified polycrystalline Au electrode, revealing that the voltammetric features of the nAuS electrode are due to the presence of AuNPs on the sol-gel network. Electrochemical Oxidation of NADH. The main objective of the present investigation is to examine the electrocatalytic activity of the AuNPs toward the oxidation of NADH and the development of dehydrogenase biosensors. First, we investigated the electrocatalytic property of the AuNPs on the sol-gel network. The oxidation of NADH on the unmodified polycrystalline Au (51) Adzic, R. R.; Hsiao, M. W.; Yeager, E. B. J. Electroanal. Chem. 1989, 260, 475-485.

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Figure 3. Cyclic voltammograms for the oxidation of NADH (0.5 mM) on (a) MPTS sol-modified polycrystalline Au and (b) nAuS electrodes in 0.1 M PBS. Scan rate, 10 mV/s.

Figure 1. FESEM images obtained for (a) hydroxylamine enlarged AuNPs and (b) integrated assembly of hydroxylamine enlarged AuNPs and enzyme ADH on MPTS sol-gel network.

Figure 2. Cyclic voltammograms of (a) nAu and (b) nAuS electrodes in 0.1 M PBS (7.2). Scan rate, 100 mV/s.

electrode requires high overpotential, and it occurs at >0.85 V. The voltammogram is ill-defined due to the fouling of the electrode surface by the adsorption of intermediates formed during oxidation (data not shown). The MPTS sol-modified electrode does not show any characteristic voltammogram for NADH (Figure 3 a). However, as can be readily seen from Figure 3b, the nAuS electrode displays a unique inverted “V” shape voltammogram, and the 6336 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006

oxidation occurs at -0.065 V, indicating that the AuNPs efficiently catalyze the oxidation of NADH. In the case of the nAu electrode, the oxidation peak appears at 0.07 V.20 Comparison of the voltammetric features obtained for the oxidation of NADH at the nAu and nAuS electrodes reveals that the hydroxylamine seeding of the AuNPs results in a 4.3-fold enhancement in the catalytic current associated with a 135-mV negative shift in the peak potential. Here, it should be mentioned that the catalytic current and the oxidation potential depend on the hydroxylamine seeding time; a gradual increase in the oxidation current and a negative shift in the peak potential was noticed while increasing the seeding time up to 20 min (Supporting Information). As the hydroxylamine seeding increases the surface area of the AuNP, high catalytic effect has been observed. The AuNPs on the network facilitate the electron transfer and decrease the overpotential of ∼915 mV for the oxidation of NADH. The onset potential for the oxidation of NADH on the nAuS electrode is much more negative (less than - 0.4 V), implying that the electrode has excellent electrocatalytic activity. The most significant observation with the nanostructured electrode is that NADH can be oxidized at less positive potential without the need for redox mediator to shuttle the electrons from NADH to the electrode surface. It is quite surprising to observe such a very large decrease in the overpotential in the absence of any redox mediator. AuNPs on the electrode surface function as an efficient mediator for the oxidation of NADH. The peak current scales approximately linearly (Supporting Information) with ν1/2 at the lower scan rates, suggesting that the oxidation process is diffusion-controlled. The special inverted “V” shape of the voltammogram implies that the oxidation process involves surfacebound species. The electrocatalytic features obtained at the nAuS electrode resemble those observed at a gold single-crystal electrode, Au(111), for the oxidation of NADH.52 The electrocatalytic effect of the Au(111) electrode was explained by considering the surface-bound oxide species, which function like a mediator. In the present investigation, the electrocatalytic activity of AuNPs can be rationalized by considering the incipient hydrous oxide/ adatom mediator model.53 The AuNP surface undergoes oxidation (52) Xing, X.; Shao, M.; Liu, C.-C. J. Electroanal. Chem. 1996, 406, 83-90. (53) (a) Burke, L. D. Gold Bull. 2004, 37, 125-135. (b) Burke, L. D.; Nugent, P. F. Gold Bull. 1998, 31, 39-50.

Figure 4. (A) Cyclic voltammograms for the oxidation of NADH on nAuS electrode at different concentrations in 0.1 M PBS. Each addition increased the concentration of NADH by 0.1 mM. Scan rate, 10 mV/s. (B) Corresponding calibration plot.

at unusually low potential (premonolayer oxidation), resulting in the formation of incipient oxides. The surface oxide apparently exists at the interface as either AuOH or Au(OH)3, and these oxide species are considered the mediators for the oxidation of NADH. Since the nAuS transducer is highly sensitive toward NADH and the oxidation takes place at much less positive potential, it has been used for the sensing of NADH at low concentration. Figure 4 depicts the voltammogram of NADH at various concentrations on the nAuS electrode. A gradual increase in the peak current during each addition of NADH was noticed. The peak currents linearly increase with wide concentrations of NADH (up to 0.5 mM). The cathodic peak for the surface oxides completely disappeared upon the addition of NADH, indicating the involvement of surface oxides in the catalytic process. At lower concentration of NADH, the oxidation peak appeared at less positive potential, and it slightly shifted to the positive side when the concentration was increased. The potential utility of the present nanostructured electrode was further examined by recording the amperometric response at different concentrations of NADH. Figure 5 displays the amperometric traces obtained for the oxidation of NADH on the nAuS electrode. The electrode was polarized at -5 mV, and aliquots of NADH were injected into a stirred supporting electrolyte solution. A fast and stable response was obtained within 3 s upon every injection. The sensitivity and limit of detection (S/N ) 12) were 0.056 ( 0.001 nA/nM and 5 nM, respectively. It is

Figure 5. Amperometric i-t curve for the detection of NADH on nAuS electrode. (A) 1 µM and (B) 5 nM NADH was injected into the stirred 0.1 M PBS at regular intervals; arrows indicate the NADH injection. The electrode was polarized at -5 mV. Inset shows the calibration plot. The noise is due to stirring of the supporting electrolyte.

surprising to observe such a very low detection limit in the absence of a redox mediator. The AuNPs on the sol-gel network play two important roles in the oxidation process: (i) the surfacebound incipient hydrous oxides of the AuNPs function as an efficient mediator (vide supra), and (ii) the AuNP self-assembled sol-gel network behaves like a nanoelectrode ensemble. As shown in Figure 1a, the hydroxylamine-enlarged AuNPs on the sol-gel network exist as ensembles of nanoelectrodes. In such nanoelectrode ensembles, the kinetics of the electrochemical reactions would be almost free of ohmic-drop effect and masstransport limitation.54 It has been demonstrated47 that micro-/ nanoelectrode ensembles exhibit a very low detection limit due to (i) a better signal/noise ratio and (ii) enhancement in the mass transport. In the present investigation, the observed very low detection limit can be ascribed to the existence of nanoelectrode ensembles. Stability of the electrode is very important for practical biosensing applications. The stability of the present electrode was tested by using the same transducer for 25 repeated voltammetric measurements of PBS containing 100 µM NADH. The coefficient (54) Scharifker, B. R. In Modern Aspects of Electrochemistry; Bockris, J. O. M., Conway, B. E., White, R. E., Eds.; Plenum Press: New York, 1992; Vol. 22, p 467.

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of variation in the peak current was calculated to be 0.42%. To further ascertain the operational stability of the present electrode, voltammetric measurement in a supporting electrolyte solution containing 100 µM NADH was performed with the nAuS electrode, and the peak current for the oxidation of NADH was measured at regular intervals (5 h) over a period of 30 h. The magnitude of peak current has not changed appreciably (Supporting Information) during the whole set of experiments (30 h), demonstrating that the nAuS electrode is very stable, and it retains its sensitivity toward the oxidation of NADH. The long-time storage stability of the electrode was tested by using the same electrode for 8 days. The electrode was used at regular intervals (every 24 h) for the voltammetric oxidation of NADH (100 µM) and stored in PBS after every measurement. No observable change in the peak current and peak potential for the oxidation of NADH was noticed up to 4 days; only an 11% decrease in the peak current was noticed after 8 days (Supporting Information). It is very interesting to note that the peak potential remained the same, even after 8 days. All of these results reveal that the present electrode is stable and can be used for a long time. The long-term stability of the electrode can be explained by considering the improved electron-transfer kinetics. The facilitated electron-transfer kinetics for the oxidation of NADH limit the amount of radical intermediates and their dimerization, which is the usual cause for the fouling of an electrode surface during the oxidation process. To check the reproducibility of the results, four different nAuS electrodes have been used for the voltammetric measurement of a solution containing NADH (100 µM). All four electrodes exhibit a similar voltammetric response for the oxidation of NADH. The standard deviation in the peak current obtained for the four different electrodes was calculated to be 0.07 µA, showing that the results are reproducible. It is worth comparing the present electrode with the other electrodes based on exfoliated graphite,36 carbon nanotubes,40,41 redox mediators,31,38,55 and redox polymers37,42 used for the detection of NADH. Recently, Alvarez and co-workers reported the excellent detection limit of 3 nM, with a graphite electrode modified with redox mediator derived from coenzyme A at 0.1 V.55 However, a rapid loss of the sensitivity due to the leaching of the redox mediator and fouling of the electrode surface55 was noticed. These data indicate that the analytical performance of the nAuS electrode is superior to the existing NADH electrodes. Amperometric Biosensor for Lactate and Ethanol. The excellent sensitivity and high stability of the present electrode toward the detection of NADH at low potential makes it attractive for the development of dehydrogenase-based amperometric biosensors. The dehydrogenase enzymes catalyze the oxidation of variety of substrate in the presence of cofactor NAD+. This is useful in biosensors and biotransformation applications. During the enzymatic reaction, the cofactor gets reduced to NADH. Since the amount of NADH generated during the enzymatic reaction is proportional to the substrate concentration of sample, the substrates can be conveniently quantified by the amperometric sensing of enzymatically generated NADH. In the present investigation, the nanostructured NADH electrode was employed for the amperometric sensing of lactate and ethanol using the (55) de-los-Santos-Alvarez, N.; Lobo-Castanon, M. J.; Miranda-Ordieres, A. J.; Tunon-Blanco, P. Electroanalysis 2005, 17, 445-451.

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Scheme 2. Schematic Representation for the Bioelectrocatalytic Sensing of Lactate and Ethanol Using the Integrated Assembly

dehydrogenase enzymes. The lactate and ethanol biosensor was fabricated according to Scheme 1 (vide supra). The enzyme encapsulated into the network efficiently catalyzes the oxidation of lactate or ethanol in the presence of cofactor NAD+ (Scheme 2). Figures 6 and 7 display the amperometric response of the biosensor toward lactate and ethanol, respectively. No current was observed in the absence of either NAD+ or the substrate (lactate or ethanol), confirming that the observed response is due to the oxidation of enzymatically generated NADH. For the practical application of any biosensor, a fast response time is essential. Interestingly, the present transducer shows a very fast response (2-3 s) toward the substrate. The electrode linearly responded to lactate or ethanol at lower concentration, and saturation response was reached at higher concentration, as expected for a Michaelis-Menten-type process. The linear Lineweaver-Burk plot of 1/icat vs 1/Cs will be a diagnostic of kinetic control of the amperometric response. The apparent Michaelis-Menten constant, KMapp for lactate and ethanol was calculated to be 0.91 and 2 mM, respectively. These values are in close agreement with the values reported in the literature.56 The integrated nanostructured biosensor could detect lactate and ethanol as low as 0.1 and 20 µM, respectively. The sensitivity of the biosensor toward lactate (56) (a) Kwan, R. C. H.; Hon, P. Y. T.; Mak, K. K. W.; Renneberg, R. Biosens. Bioelectron. 2004, 19, 1745-1752. (b) Ciolkosz, M. K.; Jordan, J. Anal. Chem. 1993, 65, 164-168.

Figure 8. Effect of NAD+ concentration on the amperometric response of lactate biosensor. LDH loading, 8 mg/mL; [lactate], 0.1 mM. Electrode potential, -5 mV.

Figure 6. Amperometric trace obtained for the biosensing of lactate at the integrated biosensor. 0.1 mM lactate was injected into the stirred 5 mM PBS at regular intervals. Arrows indicate the lactate injection. Electrode potential, -5 mV. Inset shows the calibration plot.

loading and substrate concentration (0.1 mM). As shown in Figure 8, the catalytic current increases with increasing concentration of NAD+ and reaches a maximum value at around 4 mM for both ethanol and lactate. A further increase in the concentration of NAD+ results in a decrease in the catalytic current, probably due to the inhibitory effect of NAD+, as observed previously.57 CONCLUDING REMARKS We have described the electrocatalytic activity of AuNPs toward the oxidation of NADH and the development of amperometric dehydrogenase biosensor for the sensing of lactate and ethanol. The AuNPs efficiently catalyze the oxidation of NADH in the absence of any redox mediator. The nAuS electrode is very stable and highly sensitive, and it can detect concentrations as low as 5 nM of NADH at -5 mV in neutral pH. The biosensor was developed by integrating the dehydrogenase enzyme and AuNPs with the sol-gel-derived silicate network. The amperometric signal of the biosensor is based on the electrocatalytic detection of enzymatically generated NADH. The integrated biosensor is highly sensitive, and it shows a stable and fast response. The major advantage of the present biosensor is that it does not require any redox mediator. Because L-lactate is a metabolite that reflects oxygen supply to body organs, the present biosensor could be used for practical applications.

Figure 7. Amperometric trace obtained for the biosensing of ethanol at the integrated biosensor. Each addition increased the concentration of ethanol by 0.5 mM. Other conditions are the same as in Figure 6.

and ethanol was obtained from the slope of the linear plots obtained at lower concentrations and was found to be 446.8 and 47 nA/mM, respectively. Note that the difference in the sensitivity of the electrode toward lactate and ethanol is due to the difference in the amount of enzyme loaded into the sol-gel network. The effect of the concentration of NAD+ on the biosensor response was also investigated with a constant amount of enzyme (57) Pariente, F.; Lorenzo, E.; Tobalina, F.; Abruna, H. D. Anal. Chem. 1995, 67, 3936-3944.

ACKNOWLEDGMENT This work was supported by grants from CSIR (01/1895/03/ EMR/-11) and DST (SR/FTP/CSA-15/2002). The authors are grateful to Dr. Asim Bhowmik, Indian Association for the Cultivation of Sciences, Kolkata, for DRS measurement. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review December 6, 2005. Accepted July 18, 2006. AC052143F

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