Amperometric Biosensors Based on Redox Polymer− Carbon

Anal. Chem. 2005, 77, 3183-3188

Amperometric Biosensors Based on Redox Polymer-Carbon Nanotube-Enzyme Composites Pratixa P. Joshi, Stephen A. Merchant, Youdan Wang, and David W. Schmidtke*

University of Oklahoma Bioengineering Center, School of Chemical, Biological and Materials Engineering, University of Oklahoma, 100 East Boyd, Norman, Oklahoma 73019

Based on their size and unique electrical properties, carbon nanotubes offer the exciting possibility of developing ultrasensitive, electrochemical biosensors. In this study, we describe the construction of amperometric biosensors based on the incorporation of single-walled carbon nanotubes modified with enzyme into redox polymer hydrogels. The composite films were constructed by first incubating an enzyme in a single-walled carbon nanotube (SWNTs) solution and then cross-linking within a poly[(vinylpyridine)Os(bipyridyl)2Cl2+/3+] polymer film. Incorporation of SWNTs, modified with glucose oxidase, into the redox polymer films resulted in a 2-10-fold increase in the oxidation and reduction peak currents during cyclic voltammetry, while the glucose electrooxidation current was increased 3-fold to ∼1 mA/cm2 for glucose sensors. Similar effects were also observed when SWNTs were modified with horseradish peroxidase prior to incorporation into redox hydrogels.

Initial studies with SWNTs10 and MWNTs11-13 as electrochemical sensors demonstrated that they have fast electron-transfer kinetics suggesting that they may serve as excellent transducers for biosensors. This has led more recently to an upsurge of research on incorporating carbon nanotubes into biosensing platforms. Some of the electrode configurations employing carbon nanotubes that have been developed include the following: (a) mixing nanotubes with a binder and packing them as a paste electrode,11,14 (b) solution casting nanotubes onto glassy carbon electrodes,10,15-18 (c) microfabrication of nanoelectrode ensembles19,20 and arrays,20-22 and (d) attaching individual12 or microbundles13 of MWNTs to the end of wires. In a majority of the recent papers on enzyme-based biosensors utilizing carbon nanotubes, the enzyme is either physically adsorbed onto the nanotube16-19 or entrapped by electropolymerization.23,24 The detection scheme in most of these sensors is to measure a byproduct of the enzyme-catalyzed reaction such as H2O2.14,16,19,23 Recently a few studies have reported direct electron

As the need and desire to monitor our health and environment in real time increases, correspondingly the demand for reliable miniature sensors that can measure a wide range of molecules also increases. Carbon nanotubes, which are 10 000 times thinner than a human hair and 100 times stronger than steel,1 exhibit several unique electrical, geometrical, and mechanical properties that make them attractive materials for the construction of ultrasensitive electrochemical biosensors. They are found in two distinct types of structures: single-wall carbon nanotubes (SWNTs)2,3 and multiwall carbon nanotubes (MWNTs).4 Depending on their diameter and helicity, carbon nanotubes can exhibit metallic or semiconducting electrical properties.2 Based on these unique properties, several different sensing applications have been proposed, such as probe tips in atomic force microscopy,5,6 chemical sensors,7 and nanomechamical sensors.8,9

(7) Kong, J.; Franklin, N. R.; Zhou, C. W.; Chapline, M. G.; Peng, S.; Cho, K. J.; Dai, H. J. Science 2000, 287 (5453), 622-625. (8) Poncharal, P.; Wang, Z. L.; Ugarte, D.; de Heer, W. A. Science 1999, 283 (5407), 1513-1516. (9) Li, C. Y.; Chou, T. W. Appl. Phys. Lett. 2004, 84 (25), 5246-5248. (10) Luo, H. X.; Shi, Z. J.; Li, N. Q.; Gu, Z. N.; Zhuang, Q. K. Anal. Chem. 2001, 73 (5), 915-920. (11) Britto, P. J.; Santhanam, K. S. V.; Ajayan, P. M. Bioelectrochem. Bioenerg. 1996, 41 (1), 121-125. (12) Campbell, J. K.; Sun, L.; Crooks, R. M. J. Am. Chem. Soc. 1999, 121 (15), 3779-3780. (13) Nugent, J. M.; Santhanam, K. S. V.; Rubio, A.; Ajayan, P. M. Nano Lett. 2001, 1 (2), 87-91. (14) Wang, J.; Musameh, M. Analyst 2003, 128 (11), 1382-1385. (15) Wu, K. B.; Fei, J. J.; Hu, S. S. Anal. Biochem. 2003, 318 (1), 100-106. (16) Gan, Z. H.; Zhao, Q.; Gu, Z. N.; Zhuang, Q. K. Anal. Chim Acta 2004, 511 (2), 239-247. (17) Guiseppi-Elie, A.; Lei, C. H.; Baughman, R. H. Nanotechnology 2002, 13 (5), 559-564. (18) Wang, J. X.; Li, M. X.; Shi, Z. J.; Li, N. Q.; Gu, Z. N. Anal. Chem. 2002, 74 (9), 1993-1997. (19) Lin, Y. H.; Lu, F.; Tu, Y.; Ren, Z. F. Nano Lett. 2004, 4 (2), 191-195. (20) Li, J.; Ng, H. T.; Cassell, A.; Fan, W.; Chen, H.; Ye, Q.; Koehne, J.; Han, J.; Meyyappan, M. Nano Lett. 2003, 3 (5), 597-602. (21) Gooding, J. J.; Wibowo, R.; Liu, J. Q.; Yang, W. R.; Losic, D.; Orbons, S.; Mearns, F. J.; Shapter, J. G.; Hibbert, D. B. J. Am. Chem. Soc. 2003, 125 (30), 9006-9007. (22) Chen, R. J.; Bangsaruntip, S.; Drouvalakis, K. A.; Kam, N. W. S.; Shim, M.; Li, Y. M.; Kim, W.; Utz, P. J.; Dai, H. J. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (9), 4984-4989. (23) Gao, M.; Dai, L.; Wallace, G. G. Synth. Met. 2003, 137 (1-3), 1393-1394. (24) Callegari, A.; Cosnier, S.; Marcaccio, M.; Paolucci, D.; Paolucci, F.; Georgakilas, V.; Tagmatarchis, N.; Vazquez, E.; Prato, M. J. Mater. Chem. 2004, 14 (5), 807-810.

* To whom correspondence should be addressed. Tel: (405) 325-7944. Fax: (405) 325-5813. E-mail: [email protected]. (1) Yu, M. F.; Files, B. S.; Arepalli, S.; Ruoff, R. S. Phys. Rev. Lett. 2000, 84 (24), 5552-5. (2) Ebbesen, T. W.; Lezec, H. J.; Hiura, H.; Bennett, J. W.; Ghaemi, H. F.; Thio, T. Nature 1996, 382 (6586), 54-56. (3) Iijima, S.; Ichihashi, T. Nature 1993, 363 (6430), 603-605. (4) Iijima, S. Nature 1991, 354 (6348), 56-58. (5) Dai, H. J.; Hafner, J. H.; Rinzler, A. G.; Colbert, D. T.; Smalley, R. E. Nature 1996, 384 (6605), 147-150. (6) Wong, S. S.; Joselevich, E.; Woolley, A. T.; Cheung, C. L.; Lieber, C. M. Nature 1998, 394 (6688), 52-55. 10.1021/ac0484169 CCC: $30.25 Published on Web 04/12/2005

© 2005 American Chemical Society

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transfer for glucose oxidase by either (a) adsorbing the enzyme directly onto carbon nanotubes,17,21,25 (b) first immobilizing its FAD center onto SWNTs and then reconstituting the enzyme,26,27 or (c) using a diffusional mediator.25 With this in mind we have decided to investigate a more generalized approach by immobilizing enzymes in redox hydrogels. In biosensors based on redox hydrogels, electron transfer occurs when the tethered redox centers of the redox polymer collide with the redox centers of the immobilized enzymes.28 Redox polymer-based biosensors have been constructed with a variety of enzymes: amine oxidase,29 glucose oxidase,30-32 glucose dehydrogenase,33 glycerol phosphate oxidase,34 horseradish peroxidase,35 lactate oxidase,34,36 and pyruvate oxidase.37 In general, these sensors exhibit high current densities and have been reported to be greater than 1 mA/cm2 in some cases.33,38 Furthermore, a recent study has reported that the incorporation of 10-µm graphite particles into a redox polymer-enzyme hydrogel led to a dramatic increase in the sensor output.39 In this paper, we test the hypothesis that, due to their excellent electrical properties, the incorporation of SWNTs into redox polymer hydrogels will result in highly sensitive biosensors. To our knowledge, this is the first report of the incorporation of SWNTs into redox polymer-enzyme films. EXPERIMENTAL SECTION Chemicals and Solutions. Glucose oxidase (GOX) from Aspergillus niger (EC 1.1.3.4, type X-S, 245.9 units/mg of solid, 75% protein), horseradish peroxidase (HRP; EC 1.11.1.7, type VI, 250-300 units/mg), and D-glucose were all purchased from Sigma Chemical Co. (St. Louis, MO), while poly(ethylene glycol) diglycidyl ether 400 (PEGDGE) was obtained from Polysciences (Warrington, PA). All chemicals were used as received. The redox polymer, designated as PVP-Os, was synthesized by partially complexing the pyridine nitrogens of poly(4-vinylpyridine) with Os(bpy)2Cl+/2+ and then partially quaternizing the resulting polymer with 2-bromoethylamine according to a previously published protocol.30 AP-grade SWNTs (diameter 15-17 Å) were purchased from Carbolex (Lexington, KY). Phosphate-buffered (25) Azamian, B. R.; Davis, J. J.; Coleman, K. S.; Bagshaw, C. B.; Green, M. L. H. J. Am. Chem. Soc. 2002, 124 (43), 12664-12665. (26) Patolsky, F.; Weizmann, Y.; Willner, I. Angew. Chem., Int. Ed. 2004, 43 (16), 2113-2117. (27) Liu, J.; Chou, A.; Rahmat, W.; Paddon-Row, M. N.; Gooding, J. J. Electroanal 2005, 17 (1), 38-46. (28) Heller, A. Acc. Chem. Res. 1990, 23 (5), 128-134. (29) Niculescu, M.; Frebort, I.; Pec, P.; Galuszka, P.; Mattiasson, B.; Csoregi, E. Electroanalysis 2000, 12 (5), 369-375. (30) Gregg, B. A.; Heller, A. Anal. Chem. 1990, 62 (3), 258-263. (31) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1993, 65 (23), 35123517. (32) Taylor, C.; Kenausis, G.; Katakis, I.; Heller, A. J. Electroanal. Chem. 1995, 396 (1-2), 511-515. (33) Ye, L.; Heller, A.; Hammerle, M.; Schuhmann, W.; Schmidt, H. L.; Olsthoorn, A. J. J.; Duine, J. A. Abstr. Pap. Am. Chem. Soc. 1993, 205, 34-Iec. (34) Katakis, I.; Heller, A. Anal. Chem. 1992, 64 (9), 1008-1013. (35) Vreeke, M.; Maidan, R.; Heller, A. Anal. Chem. 1992, 64 (24), 3084-3090. (36) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1994, 66 (15), 24512457. (37) Revzin, A. F.; Sirkar, K.; Simonian, A.; Pishko, M. V. Sens. Actuators, B 2002, 81 (2-3), 359-368. (38) Mao, F.; Mano, N.; Heller, A. J. Am. Chem. Soc. 2003, 125 (16), 49514957. (39) Binyamin, G.; Cole, J.; Heller, A. J. Electrochem. Soc. 2000, 147 (7), 27802783.

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saline solution (PBS), pH 7.4, was prepared from 8 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH2PO4, and 1.15 g/L Na2HPO4 in Nanopure deionized water. Stock solutions of 2 M glucose were allowed to mutarotate for 24 h before use and subsequently kept refrigerated at 4 °C, while solutions of hydrogen peroxide were prepared by diluting a 50% solution. Solutions of PVP-Os polymer (10 mg/ mL in water), PEGDGE (2.5 mg/mL in water), GOX (13.9 mg/ mL in 20 mM MES buffer, pH 6), and HRP (10 mg/mL in PBS) were used for sensor construction. The PEGDGE solution was always freshly prepared and used within 15 min. Electrodes and Electrochemical Instrument. Cyclic voltammetry (CV) and constant potential measurements were performed with a CH Instruments bipotentiostat (CHI832, Austin, TX) in a three-electrode cell configuration with a saturated calomel reference electrode (SCE) and a platinum wire counter electrode. Constant temperature (25 ( 1 °C) was maintained during the experiments by using a water-jacketed electrochemical cell connected to a circulating water bath. Glucose Oxidase Sensors. Glucose-sensitive sensors were constructed by two methods (Figure 1). For type A sensors, 10 µL of a SWNT dispersion in water (0.4 mg/mL) was solution cast on top of a polished glassy carbon electrode and allowed to dry overnight at room temperature to form a SWNT film on top of the electrode. A redox hydrogel was made from solutions of PVPOs (10 mg/mL in water), GOX (13.9 mg/mL in 20 mM MES, pH 6), and freshly dissolved PEGDGE (2.5 mg/mL in water) mixed in a 59:36:5 vol % ratio. Next a 3.3-µL aliquot of the redox hydrogel was deposited on top of the glassy carbon electrode that had been modified with SWNTs and dried under vacuum for at least 24 h. For type B sensors, 13.9 mg of GOX was dissolved in a dispersion of SWNTs (6.9 mg/mL) and incubated for 18 h at 4 °C before mixing with PVP-Os and PEGDGE. After mixing, a 3.3-µL aliquot of the solution was then deposited on top of a polished 3-mmdiameter glassy carbon electrode and allowed to dry under vacuum for at least 24 h. For type B sensors, the composition of the redox hydrogel was 59 vol % PVP-Os polymer, 36 vol % GOX/ SWNT dispersion, and 5 vol % PEGDGE unless otherwise stated. Horseradish Peroxidase Sensors. Hydrogen peroxide-sensitive sensors were constructed by incubating a dispersion of SWNTs with HRP (5 mg of HRP/mL of NT dispersion) overnight before mixing with PVP-Os (10 mg/mL in water) and PEGDGE (2.5 mg/mL in water). After mixing, a 3.3-µL aliquot of the solution was then deposited on top of polished 3-mm-diameter glassy carbon electrode and allowed to dry for at least 24 h. The composition of the layer was 45 vol % PVP-Os polymer, 33 vol % HRP/SWNTs dispersion, and 22 vol % PEGDGE. Calculations and Statistics. Current densities were calculated using the geometric surface area of a 3-mm-diameter electrode. Values are presented as mean ( standard error of the mean (SEM), and statistical significance was assessed when appropriate by a Student t-test for paired data, with P < 0.05 considered as statistically significant. RESULTS AND DISCUSSION Electrochemical Characterization of Type A Glucose Oxidase Sensors. To investigate whether a film of SWNTs on a bare GCE could enhance the electron-transfer process and the response to glucose, CVs and constant potential experiments were performed with type A glucose oxidase electrodes. Figure 2A shows

Figure 1. Schematic of the construction of type A and type B sensors. (A) Fabrication of type A sensors in which a film of SWNTs was first cast onto a bare glassy carbon electrode and allowed to dry, before an aliquot of the redox hydrogel was cast on top of the SWNT-coated electrode. (B) Fabrication of type B sensors in which SWNTs were first incubated with an enzyme solution, before they were incorporated into the redox hydrogel. An aliquot of the redox hydrogel solution containing the enzyme-modified SWNTs was then cast on top of a bare glassy carbon electrode.

Figure 2. Electrochemical characterization of glucose oxidase sensors. (A) Cyclic voltammograms of a GCE modified with the redox hydrogel alone (-); a GCE modified first with a film of SWNT and then coated with the redox hydrogel (- - -) (type A sensor); (iii) a GCE modified with a redox hydrogel containing GOX-treated SWNTs (s) (type B sensor). Scan rate 50 mV/s. (B) Glucose calibration curves for the three types of sensors described in (A). T ) 25 °C; E ) 0.5 V vs SCE. Values are mean ( SEM.

the CVs at a GCE and a SWNT-coated GCE modified with a glucose-sensitive redox hydrogel film. At the GCE modified with the redox hydrogel film alone, a pair of well-defined redox waves corresponding to the oxidation and reduction of the redox polymer’s Os(bpy) complexes are seen at 336 and 281 mV versus SCE, respectively (∆Ep ) 55 mV). For the case of the SWNTcoated GCE modified with the same redox hydrogel film, the

oxidation and reduction peak potentials broaden slightly (Epox )344 mV, Epred ) 276 mV, ∆Ep ) 68 mV), and there is an increase in both the background current and the peak currents. The fact that the background voltammetric response increased for the SWNT-coated GCE is not surprising since similar effects of SWNT films cast on GCEs have been reported by others10,18 and is most likely due to a larger electrode area undergoing double layer charging and background reactions. What is rather surprising is the fact that the oxidation and reduction peak currents increase by a factor of 2 despite the fact that the same amount of redox polymer (i.e., osmium centers) were deposited in both cases. This suggests that, in the case of the electrode with the redox polymer-enzyme film alone, not all of the redox centers were in electrical communication with the electrode surface and that the increased working electrode area provided by the SWNT film allowed for more of the redox polymer’s osmium centers to be accessible. The fact that there was virtually no change in the shape or peak potentials of the CV suggests that the SWNT film did not affect the electron-transfer process between the redox polymer film and the GCE support. The response of the type A electrodes to glucose at 25 °C was investigated by poising the electrodes at 0.5 V versus SCE and measuring the output current as aliquots of a stock 2 M glucose were added to a well-stirred PBS solution (Figure 2B). Both the GCE and the SWNT-coated GCE displayed Michaelis-Mententype behavior with apparent Km’s of 8.3 ( 0.4 (n ) 29 electrodes) and 7.8 ( 0.6 mM (n ) 14 electrodes), respectively. Although there appears to be a slight increase in the glucose oxidation current of the SWNT-coated GCE at higher glucose concentrations, statistically this was insignificant (P ) 0.42). This result is somewhat surprising given the fact that the CV data showed an increased electrode surface area and more osmium centers available. These results suggest that the current is not limited by the transfer of electrons through the redox polymer matrix to the Analytical Chemistry, Vol. 77, No. 10, May 15, 2005

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electrode surface but rather electron transfer from the FAD redox center of the enzyme to either the SWNTs or the redox polymer. Electrochemical Characterization of Type B Glucose Oxidase Sensors. Recently it has been reported that SWNTs improve the activity and stability of enzyme-containing polymer films40 and that the incorporation of 10-µm graphite particles into redox polymer films results in a dramatic increase in the current sensitivity.39 With this in mind, we investigated whether incorporating SWNTs modified with adsorbed enzyme into the redox hydrogels before coating onto an electrode surface would result in an enhanced sensitivity. To test this, SWNTs were incubated with GOX for 18 h at 4 °C (referred to as GOX-treated SWNTs) prior to cross-linking with the redox polymer. Figure 2A shows the steady-state cyclic voltammogram for a type B sensor. Similar to type A sensors, the redox peak potentials changed slightly (Epox )332 mV, Epred) 264 mV, ∆Ep ) 68 mV). However for type B sensors, the oxidation and reduction peak currents increased ∼6fold despite the fact that the same amount of redox polymer was used in coating the bare GCEs. Figure 2B shows the response of type B sensors to glucose and demonstrates that the incorporation of the GOX-treated SWNTs into the redox hydrogel results in a 2.5-fold increase in the steady-state current response. In addition, the Km for type B sensors was increased to 13.2 ( 0.7 mM (n ) 7 electrodes). We obtained similar results with SWNTs that were first purified by nitric acid treatment and then annealed.41 In some aspects, our results agree with those recently reported by Binyamin et al.,39 where the incorporation of graphite particles into redox hydrogels also led to an enhanced current density (1.6 mA/cm2). However, in their study, only hydrophilic graphite particles with a specific diameter (5-45-µm diameter) led to an increased current density. If the particles were too small (0.4 µm), too big (75 µm), or hydrophobic, the increase was not observed. In addition, the incorporation of graphite particles increased the peak separation from reversible behavior (∆Ep ) 66 mV for no particles) to irreversible behavior (∆Ep ) 180 mV) for 45-µm graphite particles. In our study, the SWNTs had diameters of 1517 Å and were not hydrophilic. In comparing the results of the type A and type B sensors, it becomes evident that the SWNTs are not just increasing the electroactive surface area. If this were the case, we would have expected that type A sensors would also exhibit an increased response to glucose. The exact mechanism for this increased response to glucose in type B sensors is unknown at this time. Previous studies with glucose oxidase adsorbed onto carbon nanotubes have reported direct electron transfer with its FAD center.17,21,25-27 It has been suggested that the distance between the FAD center and the electrode surface is reduced below the normal distance of 13 Å due to either (a) partial unfolding of glucose oxdiase as it adsorbs onto the nanotube42,43 or (b) the nanotube being able to penetrate closer to the FAD center due to its small diameter.17,27 Although we cannot rule out either of these possibilities at this time, we hypothesize that in our sensors the enzyme becomes partially (40) Rege, K.; Raravikar, N. R.; Kim, D. Y.; Schadler, L. S.; Ajayan, P. M.; Dordick, J. S. Nano Lett. 2003, 3 (6), 829-832. (41) Joshi, P. P.; Schmidtke, D. W. Unpublished results. (42) Liu, J. Q.; Paddon-Row, M. N.; Gooding, J. J. J. Phys. Chem. B 2004, 108 (24), 8460-8466. (43) Zhao, Y. D.; Zhang, W. D.; Chen, H.; Luo, Q. M. Anal. Sci. 2002, 18 (8), 939-941.

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Figure 3. Effect of the SWNT weight fraction (A) Representative CVs of type B sensors modified with redox hydrogels containing different weight fractions of GOX-modified-SWNTs. Scan rate 50 mV/ s. (B) Glucose calibration curves for type B sensors modified with redox hydrogels containing different weight fractions of GOX-modifiedSWNTs. T ) 25 °C; E ) 0.5 V vs SCE (mean ( SEM).

unfolded when incubated with nanotubes, which allows for improved access to the FAD centers by the osmium redox centers. Effect of Varying the Amount of GOX and SWNTs in the Film. Having determined that SWNTs increase the sensor response, we then studied the electrode response as a function of SWNT concentration at a fixed loading of enzyme. Figure 3A demonstrates the effect of varying the amount of GOX-treated SWNTs from 0 to 31.0 wt % in the redox hydrogel on the cyclic voltammetry. There appears to be an almost linear increase in the oxidation and reduction peak currents with increasing amounts of GOX-treated SWNTs, suggesting that more and more of the redox centers of the polymer are becoming accessible. In contrast to this, Figure 3B shows that the response to glucose does not continually increase with the amount of GOX-treated SWNTs incorporated. Rather, the current output plateaus at ∼70 µA between 17.5 and 31.0 wt %. If one calculates the corresponding current densities for these sensors using the geometric surface area of the 3-mm-diameter electrode, these sensor outputs are ∼1 mA/cm2. Why the glucose response plateaus while the CV

Figure 4. Effect of the GOX weight fraction. (A) Oxidation peak currents during cyclic voltammetry (scan rate 50 mV/s) as a function of the GOX weight fraction for type B sensors. (B). Dependence of the limiting catalytic current on the GOX weight fraction for type B sensors. T ) 25 °C, 100 mM glucose; E ) 0.5 V vs SCE. The data points are the average of 3-4 electrodes (mean ( SEM).

response increases is unknown at this time and under investigation. We hypothesize that initially increasing the amount of SWNTs in the films allows for more of the total enzyme in the film to be electrically accessible, but at some point, either all the enzyme becomes accessible or the response is limited by the electron transfer from the enzyme to the polymer’s redox center. The fact that the CV response continually increases suggests that the current response is not limited by electron transfer through the redox polymer film. We then performed the complementary experiment in which the SWNT concentration was fixed and the loading of GOX in the film was varied from 20 to 60 wt %. Figure 4A demonstrates the effect of varying the amount of GOX from 20 to 60.0 wt % in the redox polymer film on the oxidation peak during cyclic voltammetry. The maximal oxidation peak current was observed at 30 wt %, and increasing the amount of GOX in the film above this led to a decrease in the response. In addition, the separation between the oxidation and reduction peaks also increased with increasing amounts of GOX in the films (data not shown). These observations are most likely due to either (a) dilution of the redox centers in the film, (b) reduction in the bulk conductivity of the film due to the increased concentration of the insulating enzyme, or (c) a decreased segmental mobility of the polymer due to electrostatic complexation between the positively charged polymer and negatively charged enzyme.44,45 Figure 4B shows the effect of varying the amount of GOX-treated SWNTs from 20 to 60.0 wt % in the redox hydrogel on the glucose response. Similarly, the (44) Rajagopalan, R.; Aoki, A.; Heller, A. J. Phys. Chem. B 1996, 100 (9), 37193727. (45) Surridge, N. A.; Diebold, E. R.; Chang, J.; Neudeck, G. W. In Diagnostic Biosensor Polymers; Usmani, A. M., Akmal, N., Eds.; ACS Symposium Series 556; American Chemical Society: Washington, DC, 1994; p 355. (46) Gregg, B. A.; Heller, A. J. Phys. Chem. B 1991, 95 (15), 5976-5980.

Figure 5. Effect of film thickness. (A) Dependence of the oxidation peak currents during cyclic voltammetry (scan rate 5 mV/s) on the total amount of material coated for type B sensors prepared with a fixed composition. (B) Dependence of the limiting catalytic current on the on the total amount of material coated for type B sensors prepared with a fixed composition. T ) 25 °C, 100 mM glucose; E ) 0.5 V vs SCE. The data points are the average of 3-4 electrodes (mean ( SEM).

limiting current initially increases, plateaus, and then decreases with increasing amounts of GOX in the films. These results are consistent with previous observations31,32,45 and suggest that at low enzyme concentrations the response to glucose is controlled by the amount of enzyme in the film, while at higher concentrations, it is controlled by electron transfer from the enzyme to the polymer redox centers or slow electron transfer through the polymer film.31,44 Effect of Film Thickness. The effect of film thickness on the cyclic voltammetry and glucose response was studied by fixing the film composition and varying the amount of material (301250 µg/cm2) that was applied to the electrode surface. Figure 5A is a plot of the oxidation peak current measured for sensors with and without GOX-treated SWNTs. For sensors made without GOX-treated SWNTs, the oxidation peak current initially increases with film thickness and then plateaus at ∼7 µA for thicknesses of 400 µg/cm2 and above. In contrast, when GOX-treated SWNTs were incorporated into the films, the current increased up to ∼33 µA and plateaus at 950 µg/cm2. When very thin films (∼40 µg/ cm2) are made, there is essentially no difference in the oxidation peak currents whether SWNTs are present, which suggests that all of the osmium redox centers are accessible. However, as the films become thicker it clearly becomes evident that the incorporation of SWNTs into the films allows for more of the osmium redox centers to become accessible. Figure 5B is a plot of the limiting current measured for sensors with and without GOX-treated SWNTs in the presence of 100 mM glucose. Once again, for very thin films there was essentially no difference between the current response of the films. However, as the films became thicker the presence of GOX-treated SWNTs Analytical Chemistry, Vol. 77, No. 10, May 15, 2005

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sensor’s response to hydrogen peroxide. Increasing the amount of HRP-modified SWNTs initially led to higher currents with a maximum occurring at 10 wt %; however, when 20 wt % was used, the response was reduced. At this time it is not clear why the response to hydrogen peroxide was reduced and did not plateau as in the case with glucose oxidase. The fact that the oxidation peaks increased with increasing amounts of SWNTs suggests that more of the osmium sites became accessible, which should lead to better electron transfer. However, the fact that the peak separation distance increased from 65 to 91 mV may suggest that the rate of electron transfer through the film may have become limiting. Alternatively, the reduced response to hydrogen peroxide may be a result of the enzyme becoming irreversibly denatured at higher concentrations of SWNTs. The source of this reduction is currently under investigation.

Figure 6. Electrochemical characterization of type B peroxidasebased sensors (A) Representative CVs of electrodes modified with redox hydrogels containing different wt fractions of HRP-modifiedSWNTs. Scan rate 50 mV/s. (B) Glucose calibration curves for electrodes modified with redox hydrogels containing different weight fractions of HRP-modified SWNTs.. T ) 25 °C; E ) 0.0 V vs SCE (mean ( SEM).

allowed for more of the FAD centers in the film to be electrochemically accessible. Electrochemical Characterization of Peroxidase-Based Sensors. We next investigated whether the enhanced sensitivity produced by incorporating GOX-treated SWNTs into redox hydrogel films was unique to glucose oxidase or applicable to other redox enzymes. To test this, we incubated varying amounts of SWNTs with HRP for 18 h before incorporation into a redox hydrogel and deposition on a bare GCE. Figure 6A compares the CVs of electrodes with varying amounts of HRP-modified SWNTs incorporated into redox hydrogels. As the amount of HRP-modified SWNTs was increased, the peak currents increased up to 6-fold (20 wt %), while there was a gradual broadening of the redox peak potentials (65 ( 4 mV at 0 wt % to 91 ( 8 mV at 20 wt %). Figure 6B shows the response of these sensors to hydrogen peroxide. Similar to the results with glucose oxidase, the incorporation of HRP-modified SWNTs into the redox hydrogels increased the

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CONCLUSIONS We have shown that the incorporation of SWNTs modified with enzymes into redox hydrogels results in a 2-3-fold increase in the sensor’s current output while the amount of electrochemically accessible osmium redox centers increases up to 10-fold. It appears that this increased output is not just due to the increased electrochemical surface area that SWNTs may provide, because casting the redox hydrogel on top of a SWNT film did not lead to substantial increases. Rather, this dramatic increase occurs when SWNTs are first incubated with the enzyme before incorporation into the redox hydrogel. We hypothesize that this increased response is due to partial unfolding of the enzyme which reduces the electron-transfer distance and makes the enzyme’s redox centers more accessible. The fact that increased sensitivities were observed with two different enzymes suggests that this may be a generalized phenomenon and applicable to a variety of redox enzyme-based sensors. Finally, the high current densities (>1 mA/cm2) exhibited by the glucose oxidase-based sensors should allow for the construction of sensors with reduced dimensions needed for their use in everyday applications. ACKNOWLEDGMENT This work was supported in part by the Oklahoma NSF EPSCoR Grant (EPS 0132534). We thank Paul Coleman for his help in carrying out some of the experiments. Received for review October 26, 2004. Accepted February 24, 2005. AC0484169