Preparation and Characterization of Carbon Nanotube Paste Electrodes

Sep 9, 2003 - Paste electrodes have been constructed using single-wall carbon nanotubes mixed with mineral oil. The electro- chemical behavior of such...
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Anal. Chem. 2003, 75, 5413-5421

Carbon Nanotube Purification: Preparation and Characterization of Carbon Nanotube Paste Electrodes Federica Valentini,† Aziz Amine,‡ Silvia Orlanducci,† Maria Letizia Terranova,† and Giuseppe Palleschi*,†

Dipartimento di Scienze e Tecnologie Chimiche, Universita` degli Studi di Roma Tor Vergata, via Della Ricerca Scientifica, 00133 Roma, Italy, and Faculte` des Sciences et Techniques, Universite` Hassan II Mohammedia, Morocco

Paste electrodes have been constructed using single-wall carbon nanotubes mixed with mineral oil. The electrochemical behavior of such electrodes prepared with different percentages of carbon nanotubes has been compared with that of graphite paste electrodes and evaluated with respect to the electrochemistry of ferricyanide with cyclic voltammetry. Carbon nanotubes were purified by a treatment with concentrated nitric acid, then oxidized in air. In addition, electrochemical pretreatments were carried out to increase the selectivity of carbon nanotube electrodes. Performances of carbon nanotube paste and carbon paste electrodes were evaluated by studying such parameters as current peak, ∆Ep, anodic and cathodic current ratio, and charge density toward several different electroactive molecules. Data interpretation based on the carbon nanotubes and carbon surface area is presented. Carbon nanotube paste and carbon paste electrodes were tested as H2O2 and NADH probes, and several analytical parameters were evaluated. The oxidative behavior of dopamine was examined at these electrodes. The two-electron oxidation of dopamine to dopaminequinone showed an excellent reversibility in cyclic voltammetry that was significantly better than that observed at carbon paste electrodes. Carbon electrodes are widely used in electroanalysis because of their low background current, wide potential window, chemical inertness, low cost, and suitability for various sensing and detection.1,2 Several forms of carbon that are suitable for electroanalytical applications are available. Among these, glassy carbon (GC) and carbon paste (CP) are the most popular carbon electrode materials. The recent discovery of carbon nanotubes,3,4 has attracted much attention because of their dimensions and structuresensitive properties.5,6 In particular, single-walled carbon nano* Corresponding author. Fax: +39-06-72594328. E-mail: giuseppe.palleschi@ uniroma2.it. † Universita ` degli Studi di Roma Tor Vergata. ‡ Universite ` Hassan II Mohammedia. (1) Kissinger, P. T.; Heineman, W. R. Laboratory Techniques in Electroanalytical Chemistry, 2nd ed.; Dekker: New York, 1996. (2) Wang, J. Electroanalytical Chemistry, 2nd ed.; Wiley: New York, 2000. (3) Iijima, S. Nature 1991, 56, 354. (4) Ebbesen, T. W.; Ajayan, P. M. Nature 1992, 349, 315. (5) Ajayan, P. M.; Iijima, S. Nature 1993, 361, 333. 10.1021/ac0300237 CCC: $25.00 Published on Web 09/09/2003

© 2003 American Chemical Society

tubes, consisting of hollow cylindrically wound graphene sheets, are fascinating systems for fundamental science as well as for technological applications.7 An interesting property is that this material can behave as either a metal or a semiconductor8,9 depending on size (typically 1-2 nm in diameter) and lattice helicity. The electronic properties of carbon nanotubes suggest that they might have the ability to mediate electron-transfer reactions with an electroactive species in solution10,11 when used as electrodes. Since the discovery of single-wall carbon nanotubes in 1993, only Liu et al., have reported the electrochemistry of cast films of single-wall carbon nanotubes on Pt and Au electrodes, but the films do not show well-resolved voltammograms.12 A very stable electrochemical behavior was only obtained casting a nitric acid-purified single-wall carbon nanotube suspension on a glassy carbon electrode. The resulting carbon nanotube films were used to catalyze the electrochemical reaction of some biomolecules, such as dopamine, epinephrine and ascorbic acid.13,14 In addition, the cytochrome c, which plays an important role in the biological respiratory chain, exhibited a stable electrochemical response at single-wall carbon nanotube film-modified electrodes, using only purified carbon nanotube suspensions on glassy carbon electrodes.15 In 1996, the carbon nanotubes were also used to fabricate carbon nanotube electrodes, which were successfully used only in the oxidation of dopamine.16 In that work,16 a composite electrode material was fabricated mixing multiwall carbon nanotubes and bromoform as an oil binder. Although direct electrochemistry of dopamine and protein17 at multiwall carbon nanotube (6) Hamada, N.; Sawada, S.; Oshiyama, A. Phys. Rev. Lett. 1992, 68, 1579. (7) Iijima S.; Ichihashi, T. Nature 1993, 363, 603. (8) Aihara, J.; Yamabe, T.; Hosoya, H. Synth. Met. 1994, 641, 309. (9) Saito, R.; Dresselhaus, G.; Dresselhaus, M. S. Physical Properties of Carbon Nanotubes; Imperial College Press: 1998. (10) Hiura, H.; Ebbesen, T. W.; Tanigaki, K. Adv. Mater. 1995, 7, 275. (11) Li, Q. W.; Luo, G. A. Sens. Actuators, B 1999, 59, 42-49. (12) Liu, C. Y.; Bard, A. J.; Wudl, F. Electrochem. Solid State Lett. 1999, 2, 577578. (13) Luo, H.; Shi, Z.; Li, N.; Gu, Z.; Zhuang, Q. Chem. J. Chin. Univ. 2000, 21, 1372-1374. (14) Luo, H.; Shi, Z.; Li, N.; Gu, Z.; Zhuang, Q. Anal. Chem. 2001, 73, 915-920. (15) Wang, J.; Li, M.; Shi, Z.; Li, N.; Gu, Z. Anal. Chem. 2002, 74, 1993-1997. (16) Britto, P. J.; Santhanam, K. S. V.; Ajayan, P. M. Bioelectrochem. Bioenerg. 1996, 41, 121-125. (17) Davis, J. J.; Coles, R. J.; Allen, A. O. H. J. Electroanal. Chem. 1997, 440, 279-282.

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electrodes has been reported, the electrochemical response is weak. Recently, a new carbon paste electrode material based on mixing glassy carbon microparticles with an organic pasting liquid was described.18,19 The resulting glassy carbon paste electrode combines the electrochemical properties of glassy carbon with the various advantages of paste electrodes.20 Glassy carbon pastes offer high electrochemical reactivity, a wide accessible potential window, and a low background current. In addition, they are inexpensive and easy to prepare, modify, and renew. In this paper, we describe for the first time the electrochemical behavior and the electroanalytical performance of a new carbon paste electrode obtained by mixing carbon nanotubes (CNT) with a mineral oil binder. The use of nanomaterials to modify electrodes, in addition to the common effects of their inherent physical and chemical properties, such as the large specific surface area and the abundant functional groups, makes them suitable for specific catalytic actions in the electrochemical reactions of certain substances.21,22 The effect of the surface state upon the electrode kinetics of carbon nanotube paste (CNTP) and carbon paste (CP) electrodes has been the subject of numerous investigations.23-25 Various activation procedures, particularly electrochemical ones, have thus been shown to influence the electrochemical reactivity of these electrodes. The electrochemical pretreatments of carbon nanotubes and graphite are able to change the electronic properties of the carbon material producing different electrocatalytic behaviors, as reported in this work. The exact species responsible for the redox peaks and the activation mechanism are uncertain so far, but they are thought to involve the formation of oxygen functional groups (i.e., carbonyl, hydroxyl, quinone, lactone, phenol, etc.)26-28 during the electroactivation. Many studies have reported that the electrode surface structure became very porous by such treatment,29,30 and the effective electrode surface area, as also described in this paper, increased.31,32 In this work, the carbon nanotube paste electrodes showed a very stable electrochemical behavior, so they can be used to study the electrochemistry of a wide range of molecules for promising biosensor applications. (18) Wang, J.; Kirgoz, U. A.; Mo, J.-W.; Lu, J.; Kawde, A. N.; Muck, A. Electrochem. Comun. 2001, 3, 203-208. (19) Ricci, F.; Gonc¸ alves, C.; Amine, A.; Gorton, L.; Palleschi, G.; Moscone, D. Electroanalysis, accepted. (20) Voorhies, J. D.; Adams, R. N. Anal. Chem. 1958, 30, 346. (21) Li, Q. W.; Wang, Y. M.; Luo, G. A. Mater. Sci. Eng. C 2000, 11, 71-74. (22) Li, Q. W.; Luo, G. A. Anal. Chim. Acta 2000, 379, 134-137. (23) McCreery, R. L. Carbon Electrodes: structural effects on electron-transfer kinetics. In Electroanalytical Chemistry; Bard, A. J., Ed.; Dekker: New York, 1991. (24) Engstrom, R. C. Anal. Chem. 1982, 54, 2310. (25) DuVall, S. H.; McCreery, R. L. Anal. Chem. 1999, 71, 4594. (26) Bowers, M. L.; Yenser, B. A. Anal. Chim. Acta 1991, 243, 43. (27) Cabannis, G. E.; Diamantis, A. A.; Murphy, W. R., Jr.; Linton, T. W.; Meyer, T. J. J. Am. Chem. Soc. 1985, 107, 1845. (28) Nagaoka, T.; Yoshino, T. Anal. Chem. 1986, 58, 1459. (29) Nagaoka, T.; Fukunaga, T.; Yoshino, T.; Watanabe, I.; Nakayama, T.; Okazaki, S. Anal. Chem. 1988, 60, 2766. (30) Kepley, L. J.; Bard, A. J. Anal. Chem. 1998, 60, 1459. (31) Eswaramoorthy, M.; Sen, R.; Rao, C. N. R. Chem. Phys. Lett. 1999, 304, 207-210. (32) Murata, K.; Kaneko, K.; Kokai, F.; Takahashi, K.; Yudasaka, M.; Iijima, S. Chem. Phys. Lett. 2000, 331, 14-20.

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In particular, different routes to improve the selectivity of carbon electrodes, such as electrochemical preanodization33 and cathodization and physical34 and chemical35 modifications, were reported. EXPERIMENTAL SECTION Apparatus and Procedure. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and chronoamperometry experiments were performed using an Autolab electrochemical system (Eco Chemie, Utrecht, The Netherlands) equipped with PGSTAT-12 and GPES software (Eco Chemie, Utrecht, The Netherlands). The electrochemical cell was assembled with a conventional three-electrode system: a carbon nanotube paste (CNTP) or a carbon paste (CP) working electrode (1-mm diameter), a Ag|AgCl/KCl (3M) reference electrode, and a Pt counter electrode. All experiments were carried out at room temperature. Cyclic voltammetry experiments were carried out at a scan rate of 100 mV/s over the relevant potential range using 0.2 M phosphate buffer (pH 7.0). DPV were performed with a pulse amplitude of 50 mV, a pulse width of 60 ms, a scan rate of 10 mV/s, a pulse interval of 200 ms, and a sampling time of 20 ms; Ei ) 0.2 V and Ef ) 0.7 V for NADH calibration curves. The dopamine calibration curves with DPV analysis were performed with a pulse amplitude of 50 mV, a pulse width of 60 ms, a scan rate of 50 mV/s, a pulse interval of 200 ms, and a sampling time of 20 ms; Ei ) -0.2 V and Ef ) 0.4 V. Micrographs of the electrode surfaces were obtained by scanning electron microscopy (SEM) using a Hitachi model S-3200N microscope. Raman spectra were collected using the 514.5-nm excitation from an Ar ion laser beam in the backscattering geometry. Reagents. Single-wall carbon nanotubes (CarboLex AP-Grade) and graphite particles (powder, 1-2 µm) were obtained from Aldrich, (Steinheim, Germany). Mineral oil was obtained from Fluka (Buchs, Switzerland). Potassium ferricyanide, catechol, hexaammineruthenium(III) chloride, sodium hexachloroiridate(III) hydrate, ferrocene monocarboxylic acid, dopamine, epinephrine, L-tyrosine, 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbic acid, uric acid, 4-acetamidophenol (acetaminophen), NADH, H2O2, caffeic acid, guanine, and serotonin (5-HT) hydrochloride were obtained from Sigma (St. Louis, MO). All chemicals from commercial sources were of analytical grade. All solutions were prepared with 0.2 M phosphate buffer, pH 7.0. Standard solutions were daily prepared in same buffer. For pH effect studies, acetate buffer (0.1 M, pH 5.0) and N-[2-hydroxyethyl]piperazine-N′[2ethanesulfonic acid] (HEPES, pH 7.0) were also used. Pretreatment of Carbon Nanotube Materials. Before use, the CNT material was submitted to various chemical-physical procedures to remove graphitic nanoparticles, amorphous carbon, and catalyst impurities.36-38 Following one of the purification methodologies, 100 mg of carbon nanotubes was oxidized at 400 °C using an air flow of 12 (33) Falat, L.; Cheng, H. Y. Anal. Chem. 1982, 54, 2108. (34) Fagan, D. T.; Hu, I. F.; Kuwana, T. Anal. Chem. 1985, 57, 2759. (35) Hernandi, K.; Siska, A.; Thien-Nga, L.; Forro`, L.; Kiricsi, I. Solid State Ionics 2001, 141-142, 203-209. (36) Colomer, J.-F.; Piedigrosso, P.; Fonseca, A.; Nagy, J. B. Synth. Met. 1999, 103, 2482-2483. (37) Bandow, S.; Asaka, S.; Zhao, X.; Ando, Y. Appl. Phys. A 1998, 67, 23-27. (38) Ajayan, P. M.; Ebbesen, T. W.; Ichihashi, T.; Iijima, S.; Tanigaki, K.; Hiura, H. Nature 1993, 362, 522.

mL/min (quartz tubular reactor of 14-mm diameter), for 1 h. To eliminate metal oxide catalysts, the oxidized amount of carbon nanotubes was dispersed in 60 mL of 6.0 M HCl for 4 h under ultrasonic agitation,; washed until the pH of the solution was neutral; and finally, dried. A different pretreatment used 0.05 g of carbon nanotubes dispersed in 60 mL of 2.2 M HNO3 for 20 h at room temperature with the aid of ultrasonic agitation (for 30 min), then washed with distilled water to neutrality and dried in an oven at 37 °C. Preanodization and precathodization treatments of the CNTPand CP-based electrodes were performed at 1.70 V for 3 min and -1.50 V for 3 min in phosphate buffer solution 0.2 M pH 7.0 by chronoamperometry. CNTP and CP Electrodes Preparation. Carbon nanotube paste electrodes were prepared by mixing carbon nanotubes and mineral oil at different ratios. The paste was carefully hand-mixed in a mortar and then packed into a cavity (1-mm diameter; 1-mm depth) at the end of a Teflon tube. The electrical contact was provided by a copper wire connected to the paste in the inner hole of the tube. The paste was kept at room temperature in a desiccator until used. Carbon paste electrodes were prepared in a similar way using graphite powder and mineral oil at different ratios. The surface of the resulting paste electrodes was smoothed and rinsed carefully with double-distilled water prior to each measurement. RESULTS AND DISCUSSION Characterization of the CNTP and CP Electrodes. Structural and Morphological Characterizations. Raman spectroscopy and scanning electron microscopy were routinely used to characterize the as-received nanotube samples and the samples submitted to the various processing steps. Figure 1a reports a typical Raman spectrum of purified material performed in the 140-250 cm-1 spectral region. This region, associated with the radial breathing mode (RBM) of carbon atoms, is the fingerprint of single-walled carbon nanotubes. The Raman signal with components between 150 and 200 cm-1 indicates the presence of single-walled carbon nanotubes with diameters ranging between 1.2 and 1.6 nm.39 The analysis of the Raman signals in the 1300-1600 cm-1 region, which is associated with the tangential modes of the graphite lattice, allowed information to be obtained about the phase purity of the treated material. As an example, we report in Figure 1b the spectra of a nanotube sample before (a) and after (b) purification by air oxidation. The decrease of the signal at ∼1590 cm-1, attributed to the E2g stretching vibration of graphite, is due to a decrease in the graphitic content of the sample. The residual peak is produced by the vibrational frequencies of the nanotubes themselves, and for single-walled nanotubes it is always found coupled with the peaks in the RBM region.39 The broad peak at ∼1350 cm-1 is found to be rather unaffected by the purification treatments. As has been suggested,37 this signal is likely produced by defective nanotubes. Figure 2 compares the typical morphological features of a CP and of a CNTP before use. The carbon paste (Figure 2a) is characterized by a surface formed by irregularly shaped micrometer(39) Rao, A. M.; Richter, E.; Bandow, S.; Chase, B.; Eklund, P. C.; Williams, K. A.; Fang, S.; Subbasawamy, K. R.; Melon, M.; Thess, A.; Smalley, R. E.; Dresselhaus, G.; Dresselhaus, M. Science 1997, 275, 187.

Figure 1. (a) Raman spectrum performed in the 140-250 cm-1 spectral region of a sample of carbon nanotubes (deposited on a Si substrate) after the purification by air oxidation; (b) Raman spectrum of a nanotube sample before (a) and after (b) purification by air oxidation, performed in the 1300-1600 cm-1 spectral region.

sized flakes of graphite. The nanotube paste shows a more uniform surface topography, formed by the assembling of rather large (2030 µm) smooth regions (Figure 2b). In this case, the carbon nanostructures are embedded inside the oil binder. Choice of the Paste Composition. The carbon nanotube paste electrodes were prepared by mixing different percentages of CNT (i.e., 33, 50, 60, 67, and 75%) and mineral oil. For all these electrodes, cyclic voltammetry was run in 1 mM K3Fe(CN)6 in phopshate buffer 0.2 M pH 7.04, between 0.6 and -0.3 V and with a scan rate of 100 mV s-1. The influence of the paste composition upon the separation of the cyclic voltammetric peak potentials (∆Ep) and peak current (Ip) for K3Fe(CN)6 is represented in Figure 3. Results obtained with CNT/oil ratios of 75/25 (w/w) and 33/67 (w/w) confirmed that the best reversibility in terms of ∆Ep and Ipa was obtained for 75/25 (w/w) CNT/oil composition. In fact, when the composition of the paste is similar to the dry carbon powder (which means that the mineral oil percentage is diminished in the CNT/oil composition) the electroanalytical response improves (lower ∆Ep and higher current Ip). On the contrary, the electroactivity of the 33/67 (w/w) paste electrode resulted in a higher resistance and, thus, a higher ∆Ep for Fe(CN)63-/ Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

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paste that leaked in a stirred solution, causing electrode instability. This behavior was not observed when using 60% of graphite and 40% of mineral oil, tested under the same conditions, confirming that the best paste composition was the 60/40 (w/w) CNT/ graphite/oil one. The reason is probably related to the different surface areas of the two materials (i.e., graphite and carbon nanotubes). The experimental value of carbon nanotube surface area was ∼300 m2/g, obtained using BET method.41,42 The value of the surface area of the graphite obtained from the manufacturer was between 6.5 and 10.5 m2/g.43 The graphite particles, having a lower surface area, are probably completely covered by a high amount of mineral oil (i.e., 50%). This hampers the electrical communications between graphite particles, resulting in a higher resistance and, thus, a higher ∆Ep for Fe(CN)63-/Fe(CN)64- redox couple. Resistance values were measured for four different singlewalled carbon nanotube paste compositions, used as working electrodes, and Hg as the reference electrode. Results showed that resistance values increased when the mineral oil percentage was increased. In fact, for 75/25 (w/w) CNT/mineral oil paste composition, the resistance was 160 Ω; for 60/40 (w/w), it was 195 Ω; for 50/50 (w/w), it was 279 Ω; and for 33/67 (w/w), it was 325 Ω. According to these results, the best paste composition, resulting in sharper voltammograms (lower ∆Ep and higher Ip) was the one prepared with 60% carbon nanotubes/graphite particles and 40% mineral oil. This composition also assured the construction of stable and robust graphite paste electrodes, which are essential to compare the electrochemical responses of new carbon nanotube-modified electrodes.

Figure 2. SEM images of the CP (a) and CNTP (b) electrodes. CNT/ and graphite/oil ratio, 60/40 (w/w) %; accelerating voltage, 10 KV.

Fe(CN)64- redox couple. This electrochemical behavior was explained because the amount of carbon nanotubes powder in the composition of the CNTP electrodes decreases while the mineral oil percentage increases, producing a very high resistance at carbon paste electrode surfaces. Results confirmed that the best reversibility in terms of ∆Ep and Ipa was obtained for a high ratio (w/w) CNT/mineral oil paste composition of 75/25 (w/w), as shown in Figure 3 and in agreement with the previous work based on graphite/oil paste composition reported by Adams et al.40 Since the high surface area of carbon nanotubes makes possible the construction of stable and robust paste electrodes with high amounts of mineral oil, we investigated 50/50 (w/w) CNT/oil composition, which resulted in a consistent paste useful to prepare the electrodes. In contrast, the same relative amount of mineral oil when mixed with graphite formed a less consistent (40) Rice, M. E.; Galus, Z.; Adams, R. N. J. Electroanal. Chem. 1983, 143, 89.

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ELECTROCHEMICAL REACTIVITY The paste composition prepared with 60% carbon nanotubes/ graphite particles and 40% mineral oil was then used to investigate the electrochemistry of a wide range of chemical species. Analysis of the ferricyanide faradaic current as a function of the scan rate resulted in a linear Ip vs v1/2 relationship over the 5-500 mV/s range (not shown; conditions as in Figure 4), indicating that the current is controlled by a semi-infinite linear diffusion. The electrochemical area of CNTP electrodes was evaluated using chronoamperometry in a 1 mM ferrocene monocarboxylic acid solution. The slope of the linear region of the I - t-1/2 plot in the short time region provides the product nFAC0D1/2π-1/2 using the Cottrell equation

id ) nFAC0D1/2(πτ)-1/2 where C0 ) 1 mM and D ) 7.96 × 10-10 cm2 s-1 44 are the concentration and diffusion coefficient, respectively, of ferrocene monocarboxylic acid, and the other parameters have their usual meanings. For CNTP electrodes with 60% of CNT powder and 40% of mineral oil, an electrochemical area of 0.040 ( 0.003 cm2 (41) Wong, S. S.; Joselevich, E.; Woolley, A. T.; Cheung, C. Li; Lieber, C. M. Nature 1998, 394, 52-55. (42) Peigney, C. H.; Laurent, E.; Bacsa Flahaut, R. R.; Rousset A. Carbon 2001, 39, 507-514. (43) Firm documentation, Aldrich (U.S.A.). (44) Handbook of Chemistry and Physics, 62nd ed.; CRC Press: Boca Raton, FL, 1981-1982.

Figure 3. The effect of the paste composition on the (A) peak potential separation (∆Ep) and (B) peak current (Ip) of 1 × 10-3 M ferricyanide. Compositions: 33/67, 50/50, 60/40, 67/33, and 75/25 (w/w)% (CNT/mineral oil) 1.2-1.4-nm particles; electrolyte and scan rate as in Figure 4.

was obtained, which is slightly different from the apparent geometric area of the electrode (0.031 cm2). For CNTP electrodes with 50% of CNT powder and 50% of mineral oil, the same value of 0.040 ( 0.003 cm2 was obtained for the electrochemical area. CP electrodes with 60% of graphite powder and 40% of mineral oil showed an electrochemical area of 0.035 ( 0.003 cm2. The relative difference between the electrochemical area and the geometric area was estimated to be 30 and 13% for CNTP and CP electrodes, respectively. This is probably due to the roughness factor of the carbon nanotube particles, which is bigger than graphite particles. The electrochemical characterization of CNTP electrodes was performed by cyclic voltammetry in 0.2 M phosphate buffer solution pH 7.04 at a scan rate of 100 mV/s. The advantages of these new modified electrodes were compared with conventional CPEs. The experimental conditions were changed only when ascorbic acid, NADH, and H2O2 were measured. For ascorbic acid, the cyclic voltammetry was run in 0.1 M acetate buffer solution pH 5.0; for NADH, the scan rate was 2 mV/s at carbon paste electrodes; and for hydrogen peroxide (5 mM), the scan rate was 10 mV/s at carbon nanotubes and graphite electrodes. Several different molecules were used to study the electroanalytical response at carbon nanotube paste electrodes and conventional carbon paste electrodes: potassium ferricyanide, sodium hexachloroiridate (III) hydrate, hexaammineruthenium (III) chloride, ferrocene monocarboxylic acid, catechol, dopamine, epinephrine, tyrosine, DOPAC, ascorbic acid, uric acid, acetaminophen, NADH, H2O2, caffeic acid, serotonin, and guanine. There are many biological species, such as dopamine, DOPAC, serotonin, epinephrine, ascorbic acid, which represent very important chemical neurotransmitters, and their investigation should find broad utility in electroanalysis. For all of these molecules, the voltammetric parameters are summarized in Table 1. Figure 4 shows cyclic voltammograms obtained at 100 mV/s for ferricyanide, dopamine, and NADH at CNTs (A) and carbon (B) paste electrodes without pretreatments. Ferricyanide (a)

displays a reversible response on the CNTP, as reported in Figure 4 and in Table 1. Broader peaks, with higher values of ∆Ep (data show in Table 1) are observed at the CP electrodes. For dopamine oxidation, the highest degree of reversibility was observed at the conventional CP electrodes (Figure 4b and Table 1), and the carbon nanotube electrochemistry improved following pretreatments of carbon nanomaterials (as reported in Table 1). Cyclic voltammetric experiments for NADH (Figure 4c) indicated an improved electrochemical reactivity toward the oxidation of this molecule, as compared to common CP electrodes. In fact, the electrochemical oxidation of NADH was investigated at different scan rates, ranging from 2 to 200 mV/s, at both CNTP and CP electrodes. Results showed that the NADH oxidation occurred at 2 mV/s at CP electrodes, and the oxidation peak was observed also at 200 mV/s at CNTP electrodes. Other molecules were also investigated, and the electrochemical reactivity is reported in Figure 1 of the Supporting Information. PRETREATMENT EFFECTS ON VOLTAMMETRY Figure 5 compares the effect of chemical and physical pretreatments on the potassium ferricyanide and caffeic acid, electrontransfer reaction at CNTP without treatments (a, c), and at CNTP electrodes treated by (b) HNO3 oxidation and (d) oxidation by air. The performances of CNTP electrodes improved when chemical-physical pretreatments of carbon nanotubes (HNO3 oxidation and oxidation by air) were used. In particular, we observed that the electroactivity of carbon nanotube paste electrodes was enhanced by HNO3 and by air oxidation treatments, resulting in sharper voltammograms (lower ∆Ep and higher Ip), as shown in Figure 5b, d and in Table 1 for potassium ferricyanide and caffeic acid, respectively. Other molecules were studied at CNTP electrodes after chemical and physical treatments, and the relative voltammograms are reported in Figure 2 of the Supporting Information. These results seemed to confirm several studies reported in the literature in which the air oxidation of carbon Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

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Figure 4. (A) Cyclic voltammograms for 1 × 10-3 M potassium ferricyanide (a), 0.1 × 10-3 M dopamine (b), and 0.1 × 10-3 M NADH (c) CNTP electrodes (CNT/oil ratio of 60/40 (w/w)); (B) CP electrodes (graphite/oil ratio of 60/40 (w/w)). Electrolyte, 0.2 M phosphate buffer (pH 7.0); scan rate, 100 mV/s. The diameter of electrodes was 1.0 mm.

nanotubes represents the best postsynthesis purification process able to generate a higher percentage of purified carbon nanotubes. The air oxidation combined with the elimination of catalyst by HCl treatment allowed production of nanotube materials with ∼90% purity45 in which the amorphous carbon and the graphite particles were destroyed faster than the single-wall carbon nanotubes (which are more stable toward the oxidation process). The effect of concentrated nitric acid on carbon nanotubes seems conversely to influence the surface structure and to generate new oxygenic groups.45 In fact, after the chemical oxidation by HNO3, the carbon nanotube paste composition changed and appeared more hydrophilic, less dry, and easier to handle. In addition, it seems that such a treatment results in a more efficient oxidation for dopamine and biomolecules, as reported in ref 16. It has to be noted that during the experiments, caffeic acid showed an electroactivity improvement with respect to conventional carbon paste electrodes independently of the pretreatment procedure of carbon nanotube materials. The high electrochemical reactivity and the selectivity improvement of the carbon nanotube electrodes by pretreatments and modifications of the electrode (45) Wang, Z.; Liu, J.; Liang, Q.; Wang, Y.; Luo, G. Analyst 2002, 127 (5), 653665.

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surface had a profound effect upon their analytical performances. The implications of these improvements are illustrated in connection to the biosensor applications or differential pulse voltammetry determination of dopamine and other significant catecholamines that belong to the family of chemical neurotransmitters. Comparable background currents were registered with CP and CNTP (nontreated under our experimental conditions, 0.2 M phosphate buffer solution, scan rate 100 mV/s, and potential ranging from 0.0 to 1.2 V). However, the background current increased up to a factor of 100 when carbon nanotubes were treated by oxidation by air or by nitric acid or were electrochemically treated by preanodization or precathodization. This was probably due to the increased porosity and surface area of carbon nanotubes. TOWARD BIOSENSOR APPLICATIONS It has been shown that carbon tends to be more compatible with biological tissues than other commonly used electrode materials. Conventional carbon paste composites have been used for the design of amperometric enzyme electrodes. As a new type of carbon nanomaterial, CNT with porous structure and large specific surface area combines the attractive advantages of biocomposite46 electrodes with an enhancement of the selectivity using different pretreatments of the electrode surface.

Table 1. Summary of the Cyclic Voltammetric Data for Several Redox Systems at CNTP and CPa,b Electrodes no pretreatment molecules Fe(CN)63-, 1 mM Na3IrCl6‚H2O, 1 mM Ru(NH3)6, 1 mM ferrocene Mc acid, 1 mM catechol, 1 mM dopamine, 0.1 mM epinephrine, 0.1 mM L-tyrosine,

0.1 mM

DOPAC, 0.1 mM ascorbic acid, 0.1 mM uric acid, 0.1 mM acetaminophen, 0.1 mM serotonin 5-HT, 0.1 mM guanine, 0.1 mM NADH, 0.1 mM H2O2, 5 mM caffeic acid, 1 mM

+1.70 V 3′

CP

NTP

CP

209a 1.5b 90 8.0 92 2.8 60 0.6 344 15.0 149 0.5 474 1.4 763 0.5 437 0.7 389 0.8 379 2.0 314 2.0 483 1.4 808 2.0 466 0.03 1.33 9.0 299 12.5

90 5 120 5.0 94 2.4 60 0.3 239 10.3 164 0.4 489 1.4 810 0.2 393 0.2 404 0.2 423 0.3 389 1.4 460 0.7 823 0.5 546 0.2 1.2 6.0 422 6.6

101 3.1

105 15.0

76 61.0

NTP

86 10.5

56 17.0

-1.50 V 3′

ox HNO3

ox air

CP

NTP

CP

NTP

120 4.7 90 7.0

78 8 70 60.0 62 18.0

239 3.7 90 8.0

74 5.5 80 17.0

153 3.2 140 4.16

344 13.0 149 0.5 400 1.7 772 2.0 420 0.7

165 10.0 70 2.0

314 10.0 194 1.0 362 0.5 776 1.1 433 0.7

285 11.0

1.3 2.5 209 11.0

118 26.0

450 1.6

439 2.4

1.4 19.0 239 15.0

1.2 16.0 246 16.0

CP

238 1.0

548 0.7 554 1.2 561 0.9 948 0.3 548 0.6 617 6.5

NTP

374 1.0 458 0.4 447 0.8 798 1.4 424 0.2 1.2 8.0 324 23.0

a ∆E (mV) for all the reversible electrochemical species, such as potassium ferricyanide, catechol, hexaammineruthenium (III) chloride, sodium p hexachloroiridate (III) hydrate, ferrocene monocarboxilic acid, dopamine, and caffeic acid. Epa (mV) for all the irreversible electrochemical species, such as epinephrine, L-tyrosine, 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbic acid, uric acid, 4-acetamidophenol (acetaminophen), NADH, H2O2, guanine, andserotonin (5-HT) hydrochloride. b Ipa (µA) is the current value of the anodic peak for all the electroactive species investigated. The experimental conditions were electrolyte, 0.2 M phosphate buffer (pH ) 7.0) and scan rate, 100 mV/s. Different conditions were investigated only for ascorbic acid: electrolyte, 0.1 M acetate buffer (pH ) 5.4), scan rate 100 mV/s; NADH: electrolyte, 0.2 M phosphate buffer (pH ) 7.0), scan rate 2 mV/s at carbon paste electrode (CP). H2O2: 0.2 M phosphate buffer (pH ) 7.0). Scan rate 10 mV/s for both CNTP and CP electrodes.

We reported the electronanalytical characterization at CNTP electrodes of hydrogen peroxide and NADH being substrates, products, and cofactors for more than 800 enzymes. Results indicated that cyclic voltammetric experiments improved the electrochemical reactivity toward the oxidation of hydrogen peroxide at CNTP electrodes, as compared to common CP electrodes. The peroxide oxidation at the CNTP electrodes started at 1.15 V, as compared to 1.33 V at the CP electrode surface, and this cyclic voltammetric experiment indicated an improved electrochemical reactivity for the hydrogen peroxide oxidation. This represents a great advantage in electrochemistry, because using CNTP electrodes, it is possible to determine the hydrogen peroxide at very low potential to obtain a sensitivity enhancement. The potential value of 0.950 V was the best to measure the hydrogen peroxide with CNTP electrodes. At this potential, we obtained the lowest background current and the shortest time to reach a stable analytical signal, which was far away from the water signal.

For this reason, we used a chronoamperometric method to measure the H2O2 to compare the sensitivity, the concentration range, and the detection limit for CNTP to conventional CP electrodes. In Table 2, all the analytical parameters are reported for hydrogen peroxide calibration curves. Such a facile oxidation of H2O2 at the CNTP electrodes greatly enhances the performances of oxidase-based composite biosensors. NADH was also measured by differential pulse voltammetry, and the resulting calibration plots reflect the same sensitivity for CNTP electrodes and the conventional CP electrodes for the range of concentration explored. The calculated detection limit was 2 µM. In Table 2, all of the analytical parameters are reported for NADH calibration curves. These results agreed with the literature47 for the enhancement of sensitivity and the stable electrochemistry of NADH obtained at carbon paste electrodes. CNTP electrodes showing the same sensitivity of CP electrodes for the NADH oxidation could represent an interesting way to solve electroanalytical problems of NADH oxidation, observed at

(46) Chen, R. J.; Zhang, Y.; Wang, D.; Dai, H. J. Am. Chem. Soc. 2001, 123, 3838-3839.

(47) Lobo, M. J.; Miranda, A. J.; Lopez-Fonseca, J. M.; Tunon, P. Anal. Chim. Acta 1996, 325, 33-42.

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Figure 5. Cyclic voltammograms for 1 × 10-3 M potassium ferricyanide at CNTP electrodes without treatment (a), 1 × 10-3 M potassium ferricyanide at CNTP electrodes treated with oxidation by HNO3 (b), 0,1 × 10-3 M caffeic acid at CNTP electrodes without treatment (c), and 0,1 × 10-3 M caffeic acid at CNTP electrodes treated with oxidation by air (d). Electrolyte and scan rate as in Figure 4. Pretreatment was carried out in a 0.2 M phosphate buffer solution (pH 7.0).

Table 2. Analytical Parameters for H2O2 Calibration Curves at CNTP and CP Electrodes Obtained by Chronoamperometry for 300 s, Dopamine Calibration Curves at CNTP and CP Electrodes Obtained by Differential Pulse Voltammetry (DPV), and β-NADH Calibration Curves at CNTP and CP Electrodes Obtained by DPV CPE 60/40

NTPE 60/40

slope nA/µM LOD, µM

H2O2 0.56 50

0.80 20

slope µA/µM LOD, µM

Dopamine 0.26 0.5

0.34 0.5

slope nA/µM LOD, µM

β-NADH 2.03 2

1.40 2

conventional solid electrode surfaces,48 in which the electrochemical reaction is highly irreversible and takes place at considerable overpotentials. Differential Pulse Voltammetry for the Determination of Dopamine. Dopamine is one of the most significant catecholamines and belongs to the family of the excitatory chemical (48) Jaegfeldt, H. J. Electroanal. Chem. 1980, 110, 295.

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neurotransmitters. The electrochemical determination of DA, however, has AA as a potential interfering agent, which is in excess in the biological fluids and has an oxidation potential similar to that of DA. All analytical parameters studied by the DPV method are reported in Table 2, where the calculated detection limit was 0.5 µM for CNTP and CP electrodes. According to these results, the detection limit was very low for dopamine oxidation at CNTP electrodes without treatment and CNTP electrodes after air oxidation, although the background current signal was very high. The detection limit calculated for dopamine improved significantly, as compared to other carbon nanotube-modified electrodes described in the literature.16 CONCLUSIONS A new kind of carbon nanotube paste-based modified electrodes has been prepared and characterized. Studies on the composition of the single-wall carbon nanotube paste electrodes showed a different behavior, as compared to graphite paste electrodes. In fact, some molecules showed a similar electrochemistry at CP electrodes without treatments and at CP electrodes after HNO3 oxidation, while for CNTP electrodes after HNO3 oxidation, the electrochemistry of ferricyanide, sodium hexachloroiridate(III) hydrate, catechol, dopamine, serotonin5HT, and caffeic acid improved significantly. The CNTP electrodes resulted in an effective probe for H2O2 detection and showed useful characteristics for the assembling of a biosensor based on hydrogen peroxide-producing oxidases.

The sensitivity enhancement toward dopamine oxidation indicated a promising application of CNT-modified electrodes for the investigation of many important neurotransmitters. ACKNOWLEDGMENT This work has been supported by the national P.O.N. (no. 12796) Project. F.V. and S.O. gratefully acknowledge the financial support of the Giovani Ricercatori Research Project supported by grants from Tor Vergata University.

SUPPORTING INFORMATION AVAILABLE Cyclic voltammograms for various molecules at CP and CNTP electrodes. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review January 9, 2003. Accepted July 11, 2003 AC0300237

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