Electrochemical Biosensing Platforms Using Platinum Nanoparticles

nanoparticles on carbon nanotubes whereas cyclic volta- mmetry confirmed an electrical contact through SWCNTs between Pt nanoparticles and the glassy ...
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Anal. Chem. 2004, 76, 1083-1088

Electrochemical Biosensing Platforms Using Platinum Nanoparticles and Carbon Nanotubes Sabahudin Hrapovic, Yali Liu, Keith B. Male, and John H. T. Luong*

Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada H4P 2R2

Platinum nanoparticles with a diameter of 2-3 nm were prepared and used in combination with single-wall carbon nanotubes (SWCNTs) for fabricating electrochemical sensors with remarkably improved sensitivity toward hydrogen peroxide. Nafion, a perfluorosulfonated polymer, was used to solubilize SWCNTs and also displayed strong interactions with Pt nanoparticles to form a network that connected Pt nanoparticles to the electrode surface. TEM and AFM micrographs illustrated the deposition of Pt nanoparticles on carbon nanotubes whereas cyclic voltammetry confirmed an electrical contact through SWCNTs between Pt nanoparticles and the glassy carbon (GC) or carbon fiber backing. With glucose oxidase (GOx) as an enzyme model, we constructed a GC or carbon fiber microelectrode-based biosensor that responds even more sensitively to glucose than the GC/GOx electrode modified by Pt nanoparticles or CNTs alone. The response time and detection limit (S/N ) 3) of this biosensor was determined to be 3 s and 0.5 µM, respectively. Nanoparticles frequently display unusual physical and chemical properties, depending upon their size, shape, and stabilizing agents. Catalysis can be considered as one of the most popular applications of transition metals, especially noble metals, because of their high catalytic activities for many chemical reactions. Nanoparticles also facilitate the electron transfer and can be easily modified with a wide range of biomolecules and chemical ligands. Such characteristics together with an ease of miniaturization of sensing devices to nanoscale dimensions make nanoparticles suitable for important applications in chemical/biochemical sensing. Electrochemical behavior and applications of nanoparticles have witnessed a significant growth in the past few years.1 The modification of electrode surfaces with redox-active metal nanoparticles has led to the development of various electrochemical sensors. In particular, platinum nanoparticles have been an intensive research subject for the design of electrodes.2 Nanostructured Pt films were electrodeposited onto microelectrodes to increase the surface areas with enhanced mass transport characteristics3 and shown to be excellent amperometric sensors for H2O2 over a wide range of concentrations.4 The detection of this target analyte is of interest to many fields but particularly in biosensing because H2O2 is released during the oxidation of the substrate by a pertinent oxidoreductase in the presence of oxygen.5 * Corresponding author. Tel: 514-496-6175. Fax: 514-496-6265. E-mail: [email protected]. 10.1021/ac035143t CCC: $27.50 Published 2004 Am. Chem. Soc. Published on Web 12/18/2003

Since the discovery of carbon nanotubes (CNTs) by highresolution transmission electron microscopy (TEM),6 intensive research has been conducted and revealed several unique properties of these exciting materials.7 Among a plethora of diversified applications, there has been growing interest to use CNTs in biosensor platforms8 and nanoscale electronic devices owing to the ability of CNTs to promote electron-transfer reactions with enzymes and other biomolecules.9 The potential application of CNTs to fabricate electrochemical sensors was also reviewed.8f The immobilization of biomolecules with CNTs resulted in a new class of biosensors with improved performances.10 CNTs have been effectively suspended and solubilized in Nafion, a perfluorosulfonated polymer, to facilitate the modification of electrode surfaces toward the development of an amperometric biosensor for glucose.8b γ-(Aminopropyl)triethoxysilane was used as a (1) (a) Chiou, C.-Y.; Chou, T.-C. Electroanalysis 1996, 8, 1179. (b) Casella, I. G.; Destradis, A.; Desimori, E. Analyst 1996, 121, 249. (c) Casella, I. G.; Gatta, M.; Guascito, M. M. R.; Cataldi, T. R. I. Anal. Chim. Acta 1997, 357, 63. (d) Brown, K. R.; Fox, A. P.; Natan, M. J. J. Am. Chem. Soc. 1996, 118, 1154. (e) Lahav, M.; Shipway, A. N.; Willner, I.; Nielsen, M. B.; Stoddart, J. F. J. Chem. Soc., Perkin. Trans. 1999, 11, 1925. (f) Shipway, A. N.; Lahav, M.; Blonder, R.; Willner, I. Chem. Mater. 1999, 11, 13. (g) Lahav, M.; Shipway, A. N.; Willner, I.; Nielsen, M. B.; Stoddart, J. F. J. Electroanal. Chem. 2000, 482, 217. (h) Lahav, M.; Gabai, R.; Shipway, A. N.; Willner, I. Chem. Commun. 1999, 1937. (i) Patolsky, F.; Gabriel, T.; Willner, I. J. Electroanal. Chem. 1999, 479, 69. (j) Zheng, W.; Maye, M. M.; Leibowitz, F. L.; Zhong, C.-J. Anal. Chem. 2000, 32, 2190. (k) Labande, A.; Astruc, D. Chem. Commun. 2000, 1007. (l) Boal, A. K.; Rotello, V. M. J. Am. Chem. Soc. 1999, 121, 4914. (m) Zhao, J.; Henkens, R. W.; Stonehuerner, J.; O’Daly, J. P.; Crumbliss, A. L. J. Electroanal. Chem. 1992, 32, 109. (n) Crumbliss, A. L.; O’Daly, J. P.; Perine, S. C.; Stonehuerner, J.; Tubergen, K. R.; Zhao, J.; Henkens, R. W. Biotechnol. Bioeng. 1992, 40, 483. (o) Zhao, J.; O’Daly, J. P.; Henkens, R. W.; Perine, S. C.; Stonehuerner, J.; Crumbliss, A. L. Biosens. Bioelectron. 1996, 11, 493. (p) Crumbliss, A. L.; Stonehuerner, J.; Henkens, R. W.; Zhao, J.; O’Daly, J. P. Biosens. Bioelectron. 1993, 8, 331. (q) Hu, X.-Y.; Xiao, Y.; Chen, H.-Y. J. Electroanal. Chem. 1999, 466, 26. (r) Xiao, Y.; Hu, X.-Y.; Chen, H.-Y. Anal. Chim. Acta 1999, 391, 73. (s) Celej, M. S.; Rivas, G. Electroanal. 1998, 10, 771. (2) (a) Gamez, A.; Richard, D.; Gallezot, P.; Gloguen, F.; Faure, R.; Durand, R. Electrochim. Acta 1996, 41, 307. (b) Antoine, O.; Bultel, Y.; Durand, R. J. Electroanal. Chem. 2001, 499, 85. (c) Genies, L.; Faure, R.; Durand, R. Electrochim. Acta 1998, 44, 1317. (d) Gloaguen, F.; Leger, J. M.; Lamy, C. J. Appl. Electrochem. 1997, 27, 1052. (e) Pron’kin, S. N.; Tsirlina, G. A.; Petrii, O. A.; Vassiliev, S. Yu. Electrochim. Acta 2001, 46, 2343. (f) SollaGullon, J.; Montiel, V.; Aldaz, A.; Clavilier, J. J. Electroanal. Chem. 2000, 491, 69. (3) (a) Attand, G. S.; Barlett, P. N.; Coleman, N. R. B.; Elliot, J. M.; Owen, J. R.; Wang, J. H. Science 1997, 278, 838. (b) Birkin, P. R.; Elliot; J. M.; Watson, Y. E. Chem. Com. 2000, 1693. (c) Elliot, J. M.; Birkin, P. R.; Barlett, P. N.; Attard, G. S. Langmuir 1999, 15, 7411. (4) Evans, S. A. G.; Elloit, J. M.; Andrews, L. M.; Barlett, P. N.; Doyle, P. J.; Denuault, G. Anal. Chem. 2002, 74, 1322. (5) Clark, L. C., Jr. Membrane polarographic electrode system and method with electrochemical compensation. U.S. Patent 3539455, 1965. (6) Iijima, S. Nature 1991, 354, 56.

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solubilizing agent for CNTs as well as an immobilization matrix for glucose oxidase (GOx) to construct a mediatorless biosensor for efficiently monitoring direct electroactivity of GOx at the electrode surface.8g The CNT/Nafion-modified glassy carbon electrode offers a significant decrease in the overvoltage for the hydrogen peroxide reaction. Although a bare glassy carbon (GC) electrode exhibits no redox activity for H2O2 over most of the potential range, the CNT-coated electrode displays significant oxidation and reduction currents starting around +0.20 V.8b The current response to H2O2 by Pt electrodes is under mixed kinetic and diffusion control and further complicated by competitive adsorption of dioxygen onto Pt surface sites and the protonation of the adsorbed H2O2 complex.11 A lack of Pt surface sites limits the reaction and results in a depression of response for higher concentrations of H2O2.11 The key idea of this paper is to combine Pt nanoparticles and CNTs to modify a GC electrode and a carbon fiber microelectrode (CFM) in order to improve their electroactivity for H2O2. We demonstrate that platinum nanoparticles are in electrical contact, through the single-wall carbon nanotube (SWCNT), with the glassy carbon or carbon fiber backing, enabling the composite structure to be used as an electrode. With GOx as an enzyme model, we construct a biosensor, which responds even more sensitively to glucose than those modified by platinum nanoparticles or CNTs alone. The performance of the GC- and CFMbased biosensors with respect to sensitivity, linear range, and response time is presented and discussed. EXPERIMENTAL SECTION Materials. Single-wall carbon nanotubes were obtained from Carbon Nanotechnology Inc. (Houston, TX). Nafion-perfluorinated ion-exchange resin (5 wt %), glutaraldehyde (25%), and GOx (7) (a) Buongo-Nardelli, M.; Fatterbert, J.-L.; Orlikowski, D.; Roland, C.; Zhao, Q.; Bernholc, J. Carbon 2000, 38, 1703. (b) Wong, E. W.; Sheehan, P. E.; Lieber, C. M. Science 1997, 277, 1971. (c) Yakobson, B. I.; Smalley, R. E. Am. Sci. 1997, 85, 324. (d) Mintmire, J. W.; Dunlap, B. I.; White, C. T. Phys. Rev. Lett. 1992, 68, 631. (e) Chopra, N.; Benedict, L.; Crespi, V.; Cohen, M. L.; Louie, S. G.; Zettl, A. Nature 1995, 377, 135. (f) Thess, A.; Lee, R.; Nikolaev, P.; Dai, H.; Petit, P.; Robert, J.; Xu, C.; Hee-Lee, Y.; Kim, S. G.; Rinzler, A. G.; Colbert, D. T.; Scuseria, G. E.; Tomanek, D.; Fischer, J. E.; Smalley, R. E. Science 1996, 273, 483. (g) Liu, J.; Rinzler, A. G.; Dai, H.; Hafuer, J. H.; Bradley, R. K.; Boul, P. J.; Lu, A.; Iverson, T.; Shelimov, K.; Huffman, C. B.; Rodriguez-Macias, F.; Shon, Y.-S.; Lee, T. R.; Colbert, D. T.; Smalley, R. E. Science 1998, 280, 1253. (h) Baughman, R. H.; Zakhidov, A. A.; Iqbal, Z.; Barisci, J. N.; Spinks, G. M.; Wallace, G. G.; Mazzoldi, A.; De Rossi, D.; Rinzler, A. G.; Jaschinski, O.; Roth, S.; Kertesz, M. Science 1999, 284, 1340. (8) (a) Wang, J.; Li, M.; Shi, Z.; Li, N.; Gu, Z. Anal. Chem. 2002, 74, 1993. (b) Wang, J.; Musameh, M.; Lin, Y. J. Am. Chem. Soc. 2003, 125, 2408. (c) Gooding, J. J.; Wibowo, R.; Liu, J. Q.; Yang, W.; Losic, D.; Orbons, S.; Mearns, F. J.; Shapter, J. G.; Hibbert, D. B. J. Am. Chem. Soc. 2003, 125, 9006. (d) Azamian, B. R.; Davis, J. J.; Coleman, K. S.; Bagshaw, C. B.; Green, M. L. H. J. Am. Chem. Soc. 2002, 124, 12664. (e) Besterman, K. B.; Lee, J.-O.; Wiertz, F. G. M.; Heering, A.; Dekker, C. Nano Lett. 2003, 3/6, 727. (f) Zhao, Q.; Gan, Z.; Zhuang, Q. Electroanal. 2002, 14/23, 1609. (g) Luong, J. H. T.; Hrapovic, S.; Wang, D.; Bensebaa, F.; Simard, B. Electroanalysis, in press. (9) Guiseppi-Elie, A.; Lei, C.; Baughman, R. H. Nanotechnology 2002, 13, 559. (10) (a) Davis, J. J.; Green, M. L. H.; Hill, H. A. O.; Leung, Y. C.; Sadler, P. J.; Slaon, J.; Xavier, A. V.; Tsang, S. C. Inorg. Chim. Acta 1998, 272, 261. (b) Matyshevska, P.; Karlash, A. Y.; Shtogun, Y. V.; Benilov, A.; Kirgizov, Y.; Gorchinskyy, K. O.; Buzaneva, E. V.; Prylutskyy, Y. I.; Scharff, P. Mater. Sci. Eng. 2001, C15, 249. (c) Chen, R. J.; Zhang, Y.; Wang, D.; Dai, H. J. Am. Chem. Soc. 2001, 123, 3838. (d) Balavoine, F.; Schultz, P.; Richard, C.; Mallouh, V.; Ebbesen, T. W.; Mioskowski, C. Angew. Chem., Int. Ed. 1999, 38, 1912. (11) Hall, S. B.; Khudaish, E. A.; Hart, A. L. Electrochim. Acta 1998, 43, 579.

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(type: X-S, Aspergillus niger, 128 000 units/g) were obtained from Aldrich-Sigma (St. Louis, MO). Preparation of Pt Nanoparticles. Pt nanoparticles (diameter 2-3 nm) were prepared as described by Harriman et al.12 In brief, H2PtCl6‚6H2O (4 mL of 5% aqueous solution) was added to distilled water (340 mL) and heated to 80 °C with stirring in a 500-mL round-bottom flask. After adding 60 mL of sodium citrate (1% aqueous solution), the resulting solution was maintained at 80 ( 0.5 °C. The course of the reduction was followed by absorption spectroscopy, and the end of the reaction was marked by the disappearance of the absorption bands of PtCl62-. Instrumentation. TEM data were recorded using a Philips CM20 200-kV electron microscope equipped with an Oxford Instruments energy-dispersive X-ray diffraction spectrometer (Link exl II) and an UltraScan 1000 CCD camera. To obtain TEM images, a drop of the Pt nanoparticle suspension, SWCNTs dissolved in 5% Nafion, or SWCNTs in the presence of Pt nanoparticles were dropped on a standard TEM sample holder containing holey carbon film, 300-mesh copper grid and allowed to dry in air. AFM micrographs were obtained using a Nanoscope IV (Digital Instruments, Veeco, Santa Barbara, CA) with a silicon tip operated in tapping mode. For imaging, SWCNTs (1 g/L) were dissolved in dichloromethane and immoblized on a piece of silicon wafer (Virginia Semiconductor, Fredericksburg, VA), while platinum nanoparticles (diluted 50-fold) were immoblized on poly(diallyldimethylammonium) chloride (PDDA) activated glass using the procedure described by Hrapovic et al.13 In addition, SWCNTs (1 mg) were dissolved in a 1-mL mixture of platinum nanoparticle solution and 5% Nafion (9:1 ratio). After 20 min of sonication, the sample was diluted 10-fold with distilled water and immoblized on PDDA activated glass before imaging. Cyclic voltammetry (CV), linear sweep voltammetry, and amperometric measurements were performed using an electrochemical analyzer coupled with a picoampere booster and Faraday cage (CHI 601A, CH Instruments, Austin, TX). A Pt wire (Aldrich, 99.9% purity, 1 mm diameter) and an Ag/AgCl, 3 M NaCl (BAS, West Lafayette, IN) electrode were used as counter and reference electrodes, respectively. Electrode Preparation. Glassy carbon electrodes (3 mm in diameter, BAS) and carbon fiber microelectrodes (BAS, 11 ( 2 µm diameter) were carefully polished with polishing paper (grid 2000) and subsequently with alumina until a mirror finish was obtained. After 5 min of sonication to remove the alumina residues, the electrodes were immersed in concentrated H2SO4 for 3 min followed by thorough rinsing with water and ethanol. The electrode was then transferred to the electrochemical cell for cleaning by cyclic voltammetry between -0.5 and +1.2 V versus Ag/AgCl at 100 mV s-1 in 50 mM phosphate buffer, pH 7.2, until a stable CV profile was obtained. For the activation of the electrode’s surface as well as the improvement of CNT and Pt nanoparticle adhesion on the GC electrode surface, the cycling was terminated by stepping the potential to +1.2 V for 2 min. The prepared electrodes were dried under a nitrogen stream and used immediately for modification. (12) Harriman, A.; Millward, G. R.; Neta, P.; Richoux, M. C.; Thomas, J. M. J. Phys. Chem. 1998, 92, 1286. (13) Hrapovic, S.; Liu, Y.; Enright, G.; Bensabaa, F.; Luong, J. H. T. Langmuir 2003, 19, 3958.

SWCNTs were dissolved in a mixture of 100 µL of Nafion perfluorinated ion-exchange resin and 900 µL of Pt nanoparticle solution, designated as the stock solution. The volume of the Pt nanoparticle stock solution was varied to optimize the Pt nanoparticle concentration. For electrode preparation, a concentration of 2 mg of SWCNT in 1 mL was usually used; however, for optimization of the SWCNT concentration, experiments were also performed at 0.5, 1.0, and 1.5 g/L. About 20-40 min of sonication was necessary to get uniformly dispersed SWCNTs depending on their concentration. Glassy carbon electrodes were then modified by a 3-µL drop of SWCNT or Ptnano solution and dried in air. Uniform films containing a network of SWCNT and Pt nanoparticles were formed. The enzyme solution was prepared by dissolving 20 mg of GOx in 1 mL of 50 mM phosphate buffer, pH 7.2. All aqueous solutions were prepared using Milli Q (Millipore) A-10 gradient (18 MΩ‚cm) deionized water. A 3-µL drop of the enzyme solution was dried on the SWCNT/Pt-modified GC electrode. Glutaraldehyde (3.0 µL, 2.5%) was applied on the resulting electrode to cross-link the enzyme. The enzyme-modified electrodes were stored in 50 mM phosphate buffer, pH 7.2, at 4 °C. Cyclic voltammetry and amperometry were carried out in 50 mM phosphate buffer, pH 7.2. Different stock concentrations of anhydrous β-D-glucose (BDH, Toronto, ON, Canada) were prepared in 50 mM phosphate buffer, pH 7.2, and stored at 4 °C (mutarotation was allowed for at least 24 h before use). The active surface area of the electrodes prior to enzyme immobilization was determined by steady-state voltammetry in a solution of 20 mM K4Fe(CN)6 (99% purity, A&C Ltd., Montreal, PQ, Canada) with 0.2 M potassium chloride as the supporting electrolyte. RESULT AND DISCUSSION Deposition of Pt Nanoparticles on SWCNTs. Carbon nanotubes are very hydrophobic and cannot be wet by liquids with surface tensions higher than 100-200 mN/m.14a Therefore, most metals would not adhere to carbon nanotubes and physisorbed Pt particles can be easily removed by agitating the material in liquid followed by agglomeration.14b Surface modification and sensitization activation have been attempted to improve metal deposition onto carbon nanotubes. The first approach is associated with the oxidation of the carbon nanotube surface to create functional groups and increase metal nucleation.14c-d The second procedure involves the generation of small nuclei in order to promote metal deposits on carbon nanotubes.14e-f Deposition of metal onto SWCNTs is also difficult because of the greater inertness, smaller size, and higher curvature of these materials compared to multiwall carbon nanotubes.14d In general, physical adsorption of Pt onto SWCNTs is not effective.14e Our strategy for the deposition of Pt nanoparticles on SWCNTs is simple, effective, and different. First, Nafion, a perfluorosulfonated and negatively charged polymer, was used to solubilize and disperse SWCNTs. It was then possible to deposit Pt nanoparticles on Nafion-modified carbon nanotubes, mainly due to charged interactions. As shown from the TEM micrograph in Figure 1 (top, right (14) (a) Dujardin, E.; Ebbesen, T. W.; Hiura, H.; Tanigaki, K. Science 1994, 265, 1850. (b) Lordi, V.; Yao, N.; Wei, J. Chem. Mater. 2001, 13, 733. (c) Sun, Y. P.; Fu, K.; Lin, Y.; Huang, W. Acc. Chem. Res. 2002, 35, 1096. (d) Ebbesen, T. W.; Hiura, H.; Bischer, M. E. Adv. Mater. 1996, 8, 155. (e) Ang, L. M.; Hor, T. S. A.; Xu, G. Q.; Yung, C. H.; Zhao, S. P.; Wang, J. L. S. Carbon 2003, 38, 363. (f) Liu, Z.; Lin, X.; Zhang, W.; Han, M.; Gan, L. M. Langmuir 2002, 18, 4054.

Figure 1. Upper image: TEM micrograph of untreated SWCNTs dissolved in 5% Nafion (diluted 10-fold with water), Left inset: AFM tapping-mode height image (size, 10 µm × 10 µm; data scale, 100 nm) of SWCNTs. Right inset: TEM image of Pt nanoparticles in 5% Nafion (diluted 10-fold with water). Lower image: TEM micrograph of SWCNT in the presence of Pt nanoparticles. Inset: AFM tappingmode phase image (size, 1 µm × 1 µm; data scale, 20 nm) of one SWCNT in the presence of Pt nanoparticles.

inset), a high and homogeneous dispersion of spherical Pt metal clusters was prepared. AFM analysis (figure not shown) indicated that the Pt nanoparticle size was 2.7 ( 0.7 nm (n ) 4737 particles, image size 5 µm × 5 µm; the sample was diluted 50-fold), which is in good agreement with Harriman et al.12 No change in the particle size was noticed after 6 months when the Pt colloid was stored in the dark at 4 °C. The addition of 0.5% Nafion to the Pt nanoparticle solution also did not interfere with imaging or affect the particle size (2.5 ( 0.7, n ) 3027 particles, image size 5 µm Analytical Chemistry, Vol. 76, No. 4, February 15, 2004

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Figure 2. (A) Polarization curves for unmodified and modified GC electrodes: (a) GC, (b) GC/CNT, (c) GC/Ptnano, and (d) GC/CNT + Ptnano in 0.5 M H2SO4 at 1 mV s-1 with nitrogen bubbling. Inset: Cyclic voltammetry (second cycle) in 0.5 M H2SO4 at 10 mV s-1: (b) GC/ CNT, (c) GC/Ptnano, and (d) GC/CNT + Ptnano. (B) Estimation of electroactive surface area by cyclic voltametry in 20 mM Fe(CN)64and 0.2 M KCl at 20 mV s-1 vs Ag/AgCl (3 M NaCl) reference electrode: (a) GC, (b) GC/CNT, (c) GC/Ptnano, and (d) GC/CNT + Ptnano.

× 5 µm; the sample was diluted 100-fold). Using SWCNTs with high purity provides a well-defined support material to allow more direct study of metal-carbon nanotube interactions. Nafionsolubilized SWCNTs were entangled into micrometer-sized clusters with individual bundles protruding from the edges as seen in Figure 1 (top). Both TEM and AFM were used to show a very clean surface for carbon nanotubes. Platinum clusters agglomerated to some extent and dispersed on the surface of SWCNTs with a dense distribution (Figure 1, bottom). The inset of Figure 1 (bottom) shows a high magnification view of a single bundle with the typical deposition of Pt nanoparticles. In the case of both TEM and AFM images, Pt nanoparticles were not observed in the background of the images, indicating the absence of free Pt nanoparticles. Electrochemical Characterization. Linear sweep voltammetry was conducted for four different types of electrodes: bare GC, GC modified by CNT, GC modified by Pt nanoparticles, and GC modified by CNT + Ptnano. The GC/CNT + Ptnano electrode exhibited the highest activity toward hydrogen and oxygen evolution reactions as promising electrocatalytic materials. The CNT-modified GC electrode provoked a negative shift of corrosion potential Ecorr toward less noble values from -0.275 V for the bare GC electrode to -0.42 V versus Ag/AgCl (Figure 2A) for the CNTmodified GC electrode due to the specific electrochemical properties of CNTs. The presence of Pt nanoparticles in the GC/CNT (Figure 2A, curve c) electrode redirected Ecorr in the opposite direction (from -0.42 to -0.31 V) due to the more noble electrochemical characteristics of platinum. The shift toward less 1086

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negative (more noble) values of potentials clearly shows the presence of Pt nanoparticles in the matrix revealing higher stability of the GC/CNT + Ptnano electrode. CV further revealed that the CNT/Ptnano-modified electrode exhibited a significant electrocatalytic activity for the oxygen reduction reaction, in agreement with the literature data.14f With the CNT/Ptnano-modified GC electrode, the largest oxygen reduction wave between -0.15 and +0.45 V was observed. Although this wave, a typical feature for Pt, was also observed for the GC electrode modified by Ptnano, the peak area was much smaller. For the CNT/GC or GC electrode, the activity toward O2 reduction was practically negligible. CV experiments were used to record oxidation and reduction currents toward H2O2 (5 mM in 50 mM phosphate buffer, pH 7.2 at 50 mV s-1). Again, the same trend was observed with the CNT/Ptnanomodified electrode exhibiting the highest current values. No significant activity was observed for the other three electrodes over most of the potential range (figure not shown). Estimation of the Active Surface Area of Electrodes. Figure 2B represents steady-state CVs (second cycle recorded) for the bare and the three modified GC electrodes in 20 mM Fe(CN)64and 0.2 M KCl at 20 mV s-1 versus Ag/AgCl (3 M NaCl) reference electrode. The well-defined oxidation and reduction peaks due to the Fe3+/Fe2+ redox couple were noticeable at +0.30 and +0.17 V versus Ag/AgCl in forward and reverse scans, respectively. The GC/CNT + Ptnano electrode exhibited the highest electroactive surface area according to the Randles-Sevcik equation.15

Ip ) 2.69 × 105AD1/2n 3/2γ 1/2C where n is the number of electrons participating in the redox reaction, A is the area of the electrode (cm2), D is the diffusion coefficient of the molecule in solution (cm2 s-1),16 C is the concentration of the probe molecule in the bulk solution (mol cm-3), and γ is the scan rate of the potential perturbation (V s-1). The Fe(CN)64-/3- redox system used in this study is one of the most extensively studied redox couples in electrochemistry and exhibits a heterogeneous one-electron transfer (n ) 1). C is equal to 20 mM, and the diffusion coefficient (D) is (6.70 ( 0.02) × 10-6 cm2 s-1. For the GC/Ptnano-modified electrode, the peaks were still pronounced, but their shift and a lower Ipeak value illustrated a decrease in the electroactive surface area, mainly due to the lack of CNTs that act as nanoconnectors between Pt nanoparticles and the electrode. The average value of the electroactive surface area for optimized GC/CNT + Ptnano electrodes (2.0 g/L SWCNT in 100% Ptnano solution) was (2.8 ( 0.3) × 10-2 cm2 (n ) 6) compared to (8.6 ( 0.1) × 10-3 cm2 (n ) 6) for the GC/Ptnano electrode. In contrast, the CNT-modified GC electrode displayed no redox waves, likely due to the semiconducting properties of CNTs (Figure 2B, curve b). Nevertheless, the resultant current was higher than that of the bare GC electrode (Figure 2B, curve a), indicating an electrical contact between SWCNTs and the GC backing. The bare carbon fiber microlectrode in the Fe(CN)64-/3- redox system shows a typical CV for microelectrodes with a sigmoidal shape and diffusion-limited current plateau (Figure 3A inset). The (15) Bard, A. J.; Faulkner, L. R. Electrochemical Methods-Fundamentals and Applications; John Wiley and Sons: New York, 2000. (16) Hrapovic, S.; Luong, J. H. T. Anal. Chem. 2003, 75, 3308.

Figure 3. Cyclic voltammograms (second run recorded) for unmodified and modified carbon fiber microelectrodes (d ) 11 ( 0.5 µm) in 20 mM Fe(CN)64- and 0.2 M KCl at 20 mV s-1 vs Ag/AgCl (3 M NaCl) reference electrode. (A) Carbon fiber microelectrode modified by Nafion. Inset: bare carbon fiber microelectrode before modification, (B) carbon fiber electrode modified by SWCNT/Nafion, (C) Ptnano/Nafion-modified C fiber electrode, and (D) Ptnano/SWCNT/Nafion-modified microelectrode.

relationship between the steady-state diffusion-limited current and the effective radius of the microelectrode can be described as17

Ilim ) KnFDCrapp

The K factor is geometry-dependent and the size of a particular microelectrode can only be estimated if the K value is known (K ) 4 was used in this study considering the disk electrode’s geometry). According to these calculations, the average diameter of the carbon fiber electrode (four electrodes evaluated) used in this study prior to any modification was 11 ( 0.5 µm. The resulting surface areas of SWCNT- or Pt nanoparticle-modified microelectrodes were estimated in the same way. Similarly to GC electrodes, after coating the carbon fiber microelectrode with only Nafion (3µL aliquots for all modification procedures), the current was substantially reduced and still displayed a steady-state diffusion plateau (Figure 3A). In contrast to GC electrodes, a SWCNTNafion-modified electrode exhibited a constant current increase, typically for unsteady-state behavior caused probably by high film resistance due to electronic properties of CNTs (Figure 3B). Microelectrodes modified with Pt nanoparticles (Figure 3C) exhibited a well-defined sigmoidal shape, typically for diffusionlimiting processes obtained with microelectrodes. There was also an increase in the electroactive surface area compared to Nafion microelectrodes. In the case of surface modification with SWCNT/ Ptnano, a drastic increase of the surface area was observed (Figure 3D). The resultant current was in the range of microamperes with a virtually peak-shaped profile. The estimated electroactive surface area was close to that of a standard GC disk electrode with a diameter of 3 mm (in these two cases, 3-µL aliquots of SWCNT/ (17) (a) Schulte, A.; Chow, R. H. Anal. Chem. 1996, 68, 3054. (b) Strein, T. G.; Ewing, A. G. Anal. Chem. 1992, 64, 1368.

Ptnano solutions were used). A significant increase of the estimated active surface area clearly shows that the network of Pt nanoparticles and SWCNT is electronically conductive. In fact, different amounts of nanocomposite material added on carbon fiber electrodes could control the electrode area. In addition, such modified microelectrodes (or macroelectrodes upon modification) exhibited practically the same electrocatalytic behavior toward hydrogen peroxide oxidation as standard GC electrodes. Detection of Hydrogen Peroxide. In addition to high electrochemical behavior for Fe(CN)64-, the combined CNT + Ptnano-modified GC electrode exhibited the highest electrocatalytic activity toward hydrogen peroxide. As shown in Figure 4 (inset, left), the detection limit of 25 nM is noticeably better than that obtained using mesoporous Pt electrodes (4.5 µM).4 For comparison, the detection limit was 1.5 and 150 µM for the GC/Ptnanoand GC/CNT-modified electrodes, respectively. The GC/CNT + Ptnano electrode exhibited a linear range from 25 nM to 10 µM (R2 ) 0.997, sensitivity of 3.57 A M-1 cm-2), and also from 100 µM to 2 mM (R2 ) 0.996, sensitivity of 1.85 A M-1 cm-2). In the concentration range between 2 and 10 mM, the linearity rapidly decreased, and a H2O2 concentration of >10 mM was considerd too high and fouled the electrode’s surface. Repeated use of electrodes did not affect the long-term stability as long as the measurement was not performed at high concentrations of H2O2 (>10 mM). A few CV runs with a scan rate of 100 mV s-1 in phosphate buffer effectively released the blocked active Ptnano sites. However, only low detection limit, high sensitivity, and reproducibility toward the detection of hydrogen peroxide were feasible when the measurement was conducted at low concentrations of hydrogen peroxide (