Bioelectrochemically Functional Nanohybrids through Co-Assembling

Publication Date (Web): June 3, 2005. Copyright .... Ling Xiang , Zhinan Zhang , Ping Yu , Jun Zhang , Lei Su , Takeo Ohsaka and Lanqun Mao. Analytica...
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Bioelectrochemically Functional Nanohybrids through Co-Assembling of Proteins and Surfactants onto Carbon Nanotubes: Facilitated Electron Transfer of Assembled Proteins with Enhanced Faradic Response Yiming Yan,† Wei Zheng, Meining Zhang,† Li Wang, Lei Su, and Lanqun Mao* Center for Molecular Science, Institute of Chemistry, the Chinese Academy of Sciences, Beijing 100080, People’s Republic of China Received January 6, 2005. In Final Form: May 2, 2005 Preparation and bioelectrochemical properties of functional nanohybrids through co-assembling of hemeproteins (i.e., horseradish peroxidase, hemoglobin, myoglobin and cytochrome c) and surfactants onto carbon nanotubes (CNTs) are described. The prepared protein-surfactant-CNT nanohybrids are found to possess facilitated interfacial electron transfer of the proteins with enhanced faradic responses. The enhancements are ascribed for the first time to the properties of the surfactants for facilitation of protein electrochemistry and the improved portion of electroactive proteins assembled, of which the latter assignment is closely associated with the electrochemical and structural properties of the nanotubes and the threedimensional architecture of the CNT film confined onto the glassy carbon electrode. It is proposed that the single and/or small bundles of the nanotubes in the CNT film electrode can be rationally functionalized with surfactants to be functional nanoelectrodes capable of facilitating electron transfer of proteins. The three-dimensional confinement of these functional nanowires onto the GC electrode essentially increases the portion of electroactive proteins assembled in the nanohybrids. These properties of the proteinsurfactant-CNT nanohybrids, combined with the bioelectrochemical catalytic activity, could make them useful for development of bioelectronic devices and investigation of protein electrochemistry at functional interfaces.

Introduction Carbon nanotubes (CNTs) emerged as a new kind of carbon material with unique electronic, structural, and mechanical properties.1 Extensive efforts over last several years have witnessed a marked progress in synthesis, manipulation, functionalization, and application of CNTs.2 Besides their high impact in other fields, the special electronic and structural properties of CNTs essentially make them both fundamentally interesting in electrochemistry and attractive for practical applications.3 For example, CNTs have been demonstrated to possess distinct electrochemical properties from other kinds of carbonbased materials, for example, glassy carbon (GC) and graphite, which substantially make them useful for electrochemical applications.3 Our recent interests in electrochemistry of CNTs have been focused on development of CNT-based electrochemical devices such as biosensors and biofuel cells.4 Direct electron transfer of biomacromolecules (i.e., enzymes and proteins) with enhanced faradic responses * Corresponding author. Fax: +86-10-62559373. E-mail: lqmao@ iccas.ac.cn. † Also in Graduate School of the CAS. (1) (a) Iijima, S. Nature (London) 1991, 354, 56. (b) Ajayan, P. M. Chem. Rev. 1999, 99, 1787-1799. (2) (a) Thess, A.; Lee, R.; Nikolaev, P.; Dai, H.; Petit, P.; Robert, J.; Xu, C.; Lee, Y. H.; Kim, S. G.; Rinzler, A. G.; Colbert, D. T.; Scuseria, G. E.; Tomanek, D.; Ficher, J. E.; Smalley, R. E. Science 1996, 273, 483. (b) Haddon, R. C. Acc. Chem. Res. 2002, 35, 997-997. (c) Baughman, R. H.; Zakhidov, A. A.; Heer, W. A. Science 2002, 297, 787. (3) (a) Wang, J. Electroanalysis 2005, 17, 7. (b) Zhao, Q.; Gan, Z.; Zhuang, Q. Electroanalysis 2002, 14, 1609. (c) Dai, L.; Soundarrajan, P.; Kim, T. Pure Appl. Chem. 2002, 74, 1753. (d) Wang, J.; Musameh, M.; Lin, Y. J. Am. Chem. Soc. 2003, 125, 2408. (e) Wang, J.; Musameh, M. Anal. Chem. 2003, 75, 2075-2079. (f) Luo, H.; Shi, Z.; Li, N.; Gu, Z.; Zhuang, Q. Anal. Chem. 2001, 73, 915-920. (g) Moore, R. R.; Banks, C. E.; Compton, R. G. Anal. Chem. 2004, 76, 2677-2682. (h) Wang, J.; Li, M.; Shi, Z.; Li, N.; Gu, Z. Anal. Chem. 2002, 74, 1993-1997.

at CNTs is highly desired for development of CNT-based enzymatic biosensors and biofuel cells since this property is envisaged to make it possible to develop so-called thirdgeneration biosensors with a high sensitivity and novel biofuel cells with a high performance.5 It is known that the structural properties inherent in the biomacromolecules substantially make it difficult to achieve their direct electrochemistry, for example, at conventional electrodes such as GC, Au, and Pt electrodes, and to date, some elegant strategies based on rational surface modification, for example, with self-assembled monolayers (SAMs), surfactants, biocompatible polymers, and metal (oxide) nanoparticles, have been demonstrated to circumvent such a problem.6 Superior to other kinds of carbon-based materials, CNTs have been demonstrated to be able to promote direct electron transfer of proteins.7 Despite that the use of CNTs (4) (a) Gong, K.; Dong, Y.; Xiong, S.; Chen, Y.; Mao, L. Biosens. Bioelectron. 2004, 20, 253-259. (b) Zhang, M.; Gong, K.; Zhang, H.; Mao, L. Biosens. Bioelectron. 2005, 20, 1270-1276. (c) Zhang, M.; Yan, Y.; Gong, K.; Mao, L.; Guo, Z.; Chen, Y. Langmuir 2004, 20, 8781-8785. (d) Gong, K.; Zhang, M.; Yan, Y.; Su, L.; Mao, L.; Xiong, S.; Chen, Y. Anal. Chem. 2004, 76, 6500-6505. (e) Zheng, W.; Li, Q.; Yan, Y.; Su, L.; Mao, L. Indian J. Chem., Sect. A: Inorg., Bio-inorg., Phys., Theor. Anal. Chem., in press. (5) (a) Kano, K.; Ikeda, T. Electrochemistry 2003, 71, 86-99. (b) Tian, Y.; Mao, L.; Okajima, T.; Ohsaka, T. Anal. Chem. 2004, 76, 4162-4168. (c) Ohsaka, T.; Tian, Y.; Shioda, M.; Kasahara S.; Okajima, T. Chem. Commun. 2002, 990-991. (6) (a) Jeuken, L. J. C. Biochim. Biophys. Acta 2003, 1604, 67-76. (b) Chattopadhyay, K.; Mazumdar, S. Bioelectrochemistry 2000, 53, 17-24. (c) Rusling, J. F. Acc. Chem. Res. 1998, 31, 363-369. (d) Huang, R.; Hu, N. Biophys. Chem. 2004, 104, 199-208. (e) Liu, H.; Tan. Z.; Lu, Z.; Zhang, Z.; Zhang, M.; Pang, D. Biosens. Bioelectron. 2004, 20, 294304. (f) Jia, J.; Wang, B.; Wu, A.; Cheng, G.; Li, Z.; Dong, S. Anal. Chem. 2002, 74, 2217-2223. (g) Zhou, Y.; Hu, N.; Zeng, Y.; Rusling, J. F. Langmuir 2002, 18, 211-219. (h) Nadzhafova, O. Y.; Zaitsev, V. N.; Drozdova, M. V.; Vaze, A.; Rusling, J. F. Electrochem. Commun. 2004, 6, 205-209. (i) Liu, H.; Wang, L.; Hu, N. Electrochim. Acta 2002, 47, 2515-2523.

10.1021/la050043z CCC: $30.25 © 2005 American Chemical Society Published on Web 06/03/2005

Functional Nanohybrids by Co-Assembling onto CNTs

would largely facilitate the investigations on protein electrochemistry, the faradic responses achieved hitherto for the biomacromolecules at CNTs are relatively weak. As a consequence, a strategy for efficiently enhancing the faradic responses of the biomacromolecules at CNTs is highly desired, for development of the CNT-based bioelectronic devices, in particular. Moreover, the enhanced faradic responses of the biomacromolecules are also of great importance in voltammetric investigations of interfacial electron transfer of the biomacromolecules at the CNT electrode since the CNT electrode generally has a large surface area and, hence, shows a great charge current in the voltammograms, rendering difficulties in differentiating the faradic signal of biomacromolecules. CNTs have several features such as a large and functionalizable surface and high conductivity that can be exploited to accomplish the purposes mentioned above. More importantly, the CNT film electrode prepared by confining CNTs onto the substrate has been demonstrated to possess a three-dimension architecture, in which each separated single bundle and/or small bundles of the nanotubes essentially behave like individual nanoelectrodes.8 These electrodes, once being rationally designed, could be used to facilitate direct electron transfer of the enzymes and proteins, even of those far from the substrate electrode (e.g., GC electrode). Such a property can be envisaged to improve the portion of electroactive enzymes and proteins and thereby the enhancement in the faradic responses. Despite these striking properties, to the best of our knowledge, the rational uses of CNTs to prepare functional nanohybrids with excellent bioelectrochemical properties have been less explored so far. In the present study, we use surfactants to decorate CNTs for achieving the direct electron transfer of heme proteins [i.e., horseradish peroxidase (HRP), hemoglobin (Hb), myoglobin (Mb), and cytochrome c (Cyt-c)] coassembled onto the CNTs. The prepared protein-surfactant-CNT nanohybrids are found to possess good electron-transfer properties for the proteins with enhanced faradic responses and bioelectrochemical catalytic activities. This demonstration could pave a new way to CNTbased bioelectrochemical devices and protein electrochemistry at functional interfaces. Experimental Section Reagents and Materials. Multiwalled carbon nanotubes (MWNTs, diameter, 30-40 nm; purity > 95%; length, 0.5-40 µm) and single-walled carbon nanotubes (SWNTs, diameter < 2 nm, purity > 90%, length, 0.5-50 µm) were obtained from Shenzhen Nanotech Port Co., Ltd. (Shenzhen, China). The CNTs were purified by refluxing the as-received CNTs in 2.6 M HNO3 for 36 h prior to use. HRP, Mb, Hb, and Cyt-c were all purchased from Sigma and used without further purification. Sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), Triton X-100, N,N-dimethylformamide (DMF), and hydrogen peroxide (H2O2) were purchased from Beijing Chemical Co. (7) (a) Davis, J. J.; Coles, R. J.; Hill, H. A. O. J. Electroanal. Chem. 1997, 440, 279-292. (b) Gooding, J. J.; Wibowo, R.; Liu, J.; Yang, W.; Losic, D.; Orbons, S.; Mearns, F. J.; Shapter, J. G.; Hibbert, D. B. J. Am. Chem. Soc. 2003, 125, 9006-9007. (c) Yu, X.; Chattopadhyay, D.; Galeska, I.; Papadimitrakopoulos, F.; Rusling, J. F. Electrochem. Commun. 2003, 5, 408-411. (d) Zhao, G.; Zhang, L.; Wei, X.; Yang, Z. Electrochem. Commun. 2003, 5, 825-829. (e) Wang, L.; Wang, J.; Zhou, F. Electroanalysis 2004, 16, 627-632. (f) Cai, C.; Chen, J. Anal. Biochem. 2004, 332, 75-83. (g) Zhao, Y.; Zhang. W.; Chen, H.; Luo, Q.; Li, S. F. Y. Sens. Actuators, B 2002, 87, 168-172. (h) Yang, P.; Zhao, Q.; Gu, Z.; Zhuang, Q. Electroanalysis 2004, 16, 97-100. (i) Zhao, Y.; Bi, Y.; Zhang, W.; Luo, Q. Talanta 2005, 65, 489-494. (8) (a)Li, J.; Cassell, A.; Delzeit, L.; Han, J.; Meyyappan, M. J. Phys. Chem. B 2002, 106, 9299-9305. (b) Davis, J. J.; Coleman, K. S.; Azamian, B. R.; Bagshaw, C. B.; Green, M. L. H. Chem.sEur. J. 2003, 9, 37323739.

Langmuir, Vol. 21, No. 14, 2005 6561 (Beijing, China). Other chemicals were of analytical grade or higher and used as received. Aqueous solutions were prepared with doubly distilled water. Preparation of Protein-Surfactant-CNT Hybrids on GC Electrodes. GC (3-mm diameter, Bioanalytical Systems, Inc.) electrodes were used as the substrate for preparation of the nanohybrids. The electrodes were polished first with emery paper and then with aqueous slurries of fine alumina powders (1 and 0.05 µm) on a polishing microcloth and were finally rinsed with doubly distilled water in an ultrasonic bath for 10 min. Proteinsurfactant-CNT nanohybrids confined on GC electrodes were prepared as follows. In a typical experiment, 2 mg/mL MWNTs were dispersed into DMF to obtain a homogeneous dispersion under sonication. A total of 2 µL of the resulting dispersion was coated onto GC electrodes, and the electrodes (MWNT-modified GC) were dried to evaporate the solvent. The proteins and surfactants were co-assembled onto the MWNTs by immersing the MWNT-modified GC electrodes into 0.10 M phosphate buffer containing 10 mg/mL protein (i.e., HRP, Hb, Mb, or Cyt-c) and surfactant (5.8 mg/mL SDS, 0.85 mg/mL CTAB, or 0.50 mg/mL Triton X-100) for 8 h. The electrodes (denoted as protein/ surfactant/MWNT-modified GC electrodes, hereafter) were rinsed with distilled water prior to electrochemical measurements. Apparatus and Measurements. Electrochemical measurements were carried out with a computer-controlled CHI660A electrochemical analyzer (CHI, Austin, U.S.A.) in a conventional and two-compartment cell. Protein/surfactant/MWNT-modified GC electrodes were used as the working electrode, and a platinum spiral wire was used as the counter electrode. All potentials were referred to a Ag/AgCl electrode (KCl-saturated). A 0.10 M phosphate buffer (pH 7.0) was used as the supporting electrolyte. The electrolyte was deoxygenated by purging pure N2 into the solution for about 30 min, and N2 gas was kept flowing over the solution during electrochemical measurements. Quartz crystal microbalance (QCM) measurements used for monitoring the mass of protein (i.e., HRP), surfactant (i.e., SDS), and MWNTs in the nanohybrids were performed with a QCM analyzer (CHI440, CHI, Inc.) with 8 MHz of Au resonators (geometric area, 0.50 cm2). The Au resonators were first sonicated in ethanol and distilled water each for 3 min and then pretreated by dipping one droplet of piranha solution (a 1:3 mixture of 30% H2O2 and concentrated H2SO4; note that this mixture violently reacts with organics) onto the resonators for 10 min. The resonators were finally rinsed with distilled water and ethanol and dried with N2 blowing. The HRP-SDS-MWNT hybrid was confined onto the Au resonator, and the mass of each constituent was measured with the following procedures. A total of 7 µL of the MWNT suspension in DMF was dip-coated onto the Au resonator. After drying in air, the MWNT-modified Au resonator was rinsed with distilled water and dried with N2 blowing. The mass of the MWNTs confined onto the resonator was measured by a QCM frequency shift and calculated with the Sauerbery equation shown below.9

M/A (g cm-2) ) -∆F (Hz)/(1.45 × 108) After that, the MWNT-modified resonator was immersed into 5.8 mg/mL SDS solution for 1 h. The SDS/MWNT-modified resonator was rinsed with distilled water, dried with N2 blowing, and subjected to the QCM measurement for the mass of SDS assembled onto the MWNTs. Further assembling of HRP onto the SDS/MWNT-modified resonator was conducted by immersing the resonator into 0.10 M phosphate buffer containing 10 mg/ mL HRP and 5.8 mg/mL SDS for 8 h. The Au resonator was then rinsed with distilled water and dried with N2 blowing, and the mass of HRP co-assembled was measured by QCM as mentioned above. The HRP/MWNT nanohybrid was prepared with the same procedures as those for the HRP-SDS-MWNT hybrid, with an exception that no SDS was assembled onto the MWNTs or added in the HRP solution. The masses of the MWNTs confined onto the Au resonator and HRP adsorbed onto the MWNTs were also measured with QCM. For calculation of the amount of HRP capable of conducting direct electron transfer (denoted as electroactive HRP) assembled (9) Sauerbrey, G. Z. Phys. 1959, 155, 206.

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Figure 1. Typical cyclic voltammograms of MWNT- (dotted curve), HRP/MWNT- (dashed curve), and HRP/SDS/MWNTmodified (solid curves) GC electrodes in 0.10 M N2-saturated phosphate buffer (pH 7.0). Scan rate, 100 mV s-1. The HRP/ SDS/MWNT-modified GC electrodes were prepared by immersing the MWNT-modified GC electrodes into 0.10 M phosphate buffer containing 10 mg/mL HRP and 5.8 mg/mL SDS for different times of (from inner to outer) 0.5, 1, 2, and 5 h. in both kinds of the nanohybrids (i.e., HRP-SDS-MWNT and HRP-MWNT), the HRP-SDS-MWNT hybrid was prepared on the GC electrode with the same procedures as those for the Au resonator. That is, the GC electrode used for voltammetric measurements was first coated with the MWNTs with the same surface coverage as that at the Au resonator used for QCM measurements. Then, SDS and HRP were stepwise assembled onto the confined MWNTs with the same conditions as those for the Au resonators. The electrodes prepared are denoted as protein/ MWNT-surfactant-modified GC electrodes, hereafter. The amount of electroactive HRP in the nanohybrids was calculated by integrating the cathodic/anodic peak in the voltammograms of the HRP/MWNT-SDS-modified and HRP/MWNT-modified GC electrodes at a scan rate of 25 mV s-1 in 0.10 M N2-saturated phosphate buffer. All experiments were carried out at room temperature.

Results and Discussion Cyclic Voltammetry of Protein-Surfactant-CNT Nanohybrids. Figure 1 depicts typical cyclic voltammograms obtained with the HRP/SDS/MWNT-modified GC electrodes prepared by immersing the MWNT/GC electrodes into an aqueous solution of HRP and SDS for different times (i.e., 0.5, 1, 2, and 5 h, solid curves). For comparison, voltammograms of the MWNT-modified (dotted curve) and HRP/MWNT-modified (no SDS assembly, dashed curve) GC electrodes were also given. As shown, the HRP/MWNT-modified GC electrode prepared by immersing the MWNT-modified GC electrode in the aqueous solution of HRP containing no SDS shows a pair of slight redox peaks (dashed curve) with a peak-to-peak separation of about 75 mV and a near unity of the ratio of cathodic-to-anodic peak current (at 100 mV s-1). The formal potential (E°′) calculated by averaging the cathodic and anodic peak potentials was found to be -0.35 V (vs Ag/AgCl). These features are characteristic of the reversible electrode process of the heme FeIII/FeII redox couple in HRP molecules,10 suggesting that the direct electron transfer of HRP adsorbed onto the MWNTs can be achieved. The observed weak response is in agreement with earlier reports on the direct electron transfer of the heme proteins, for example, HRP, Mb, Hb, and catalase at the CNT electrodes,7d-i and may also coincide with the almost unobservable responses of Cyt-c and microperoxidase 11 onto CNTs randomly confined onto the (10) Ferapontova, E. E. Electroanalysis 2004, 16, 1101-1112.

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Figure 2. Cyclic voltammograms of the MWNT/SDS-modified GC electrode in 0.10 M N2-saturated phosphate buffer in the absence (dotted curve) and presence (solid curve) of 6.0 mg/mL Cyt-c. Inset, cyclic voltammograms of the MWNT/SDS-modified GC electrode in the same solution in a narrow potential range to enlarge the redox peaks of Cyt-c. Scan rate, 100 mV s-1.

substrate, which could be remarkably improved through pretreatment or alignment of the CNTs on the substrate.3h,7b,c As can be also seen from Figure 1 (solid curves), the HRP/SDS/MWNT-modified GC electrodes exhibit a pair of well-defined redox peaks, which could be ascribed to the reversible redox process of the heme FeIII/FeII couple in HRP co-assembled with SDS onto the MWNTs. We reasoned this from the small peak-to-peak separation (65 mV) and the near unity of the ratio of cathodic-to-anodic peak current (both at 100 mV s-1) as well as the formal potential (-0.35 V vs Ag/AgCl) of the redox peaks. Almost the same electrochemical character was obtained for other kinds of protein/SDS/MWNT-modified electrodes, suggesting that the direct electronic communication between heme proteins confined within the surfactant matrix and the MWNTs could be facilitated. The E°′ values obtained for Hb, Mb, and Cyt-c in the nanohybrids were -0.30, -0.32, and -0.34 V (vs Ag/AgCl), respectively. These values are much more negative than those for the heme proteins in their native state [e.g., ca. 0.06 V vs saturated calomel electrode, SCE (i.e., 0.10 V vs Ag/AgCl) as reported for Cyt-c].11 To understand the essence for the big difference in the formal potentials of the heme proteins obtained in this study from the reported values, we used Cyt-c as a typical example to study the electrochemical process of heme proteins at the GC electrode modified with SDS-functionalized MWNTs (SDS/MWNT-modified electrode). Figure 2 depicts cyclic voltammograms obtained with the SDS/MWNT-modified GC electrode in 0.10 M phosphate buffer containing Cyt-c. As shown, the electrode exhibits three couples of redox peaks at E°′ of -0.35 (peak I), -0.05 (peak II), and +0.05 V (peak III). Peak I was ascribed to the redox process of Cyt-c adsorbed onto the SDS/MWNTmodified electrode since the redox peaks were still recorded after the electrode was taken out from the solution, rinsed with distilled water, and then recycled in N2-saturated phosphate buffer (data not shown). Peak II was ascribed to the redox process of oxygen-containing moieties at the MWNTs since it was recorded in the solution containing no Cyt-c (dotted line). Peak III was attributed to the redox process of Cyt-c in the solution phase since the E°′ value obtained here was very close to that reported for Cyt-c in the solution phase.3h It has been addressed that electrochemical behavior (e.g., E°′) of Cyt-c is closely relevant to its state.12 For instance, Cyt-c in its native state (e.g., those in solution phase or in adsorbed form without a conformational change) shows a redox process with E°′ of 0.06

Functional Nanohybrids by Co-Assembling onto CNTs

V versus SCE (i.e., 0.10 V vs Ag/AgCl),11 while the denatured Cyt-c exhibits a redox process at -0.44 V versus SCE (i.e., -0.40 V vs Ag/AgCl), for example, on a bare Au electrode.12a For the case of Cyt-c adsorbed onto electrodes modified with functional films, for example, SAMs and surfactants, Cyt-c can essentially interact with the films, avoid its direct adsorption onto the bare electrode, and, hence, prevent it from being denatured onto the modified electrode surface.12a,13 The interactions between Cyt-c and the films confined onto electrodes (e.g., electrostatic and/ or hydrophobic interactions) essentially lead to structural alternations restricted to the heme crevice, resulting in an open conformation (state II) compared to the closed structure in the native state (state I) of Cyt-c and thereby a decrease in the redox potential.12b,d,14 The adsorbed form of Cyt-c is likely to be the state II conformer that is active and not denatured as reported by Hildebrandt et al.12b,c Since our main aim is to develop CNT-based bioelectronic devices in which an efficient integration of the proteins and enzymes into the functional hybrids is essentially required, this study primarily focuses on the electrochemical response of the heme proteins adsorbed into the surfactant matrix confined onto GC electrodes (i.e., peak I). Earlier reports have suggested that supramolecular surfactants could interact with CNTs with hydrophobic interactions, leading to the adsorption of surfactant molecules onto CNTs and solubilization of CNTs in aqueous solution.15 On the other hand, heme proteins (e.g., HRP, Mb, Hb, and Cyt-c) could be confined within or coassembled with surfactants onto various solid substrates, for example, pyrolytic graphite (PG), GC, Au, and Pt electrodes, and studies concerning the as-formed protein film voltammetry have been widely conducted previously.6b,c,16 In terms of these facts, it is very likely that the proteins and surfactants demonstrated here could also be co-assembled onto CNTs in probably the same way as those onto, for example, PG and GC electrodes. Upon revisiting Figure 1, we can easily find that the peak currents obtained at the HRP/SDS/MWNT-modified GC electrodes clearly increase with increasing the time for immersing the MWNT-modified electrodes into the aqueous solution of HRP and SDS and level off when the immersion time reached 5 h (solid curves, from inner to outer). Such phenomena could be reflective of the coassembling process of both species onto MWNTs, forming bioelectrochemically functional nanohybrids. Interestingly, a comparison of the voltammograms obtained at the HRP/MWNT-modified GC electrode (dashed curve, Figure 1) with that at the HRP/SDS/ MWNT-modified GC electrode (solid curves) reveals that the peak currents obtained at the latter electrode are (11) (a) Henderson, R. W.; Rawlinson, W. A. Biochem. J. 1956, 62, 21. (b) Hawkridge, F. M.; Kuwana, T. Anal. Chem. 1973, 45, 7021. (12) (a) Sagara, T.; Niwa, K.; Sone, A.; Hinnen, C.; Niki, K. Langmuir 1990, 6, 254. (b) Hildebrandt, P.; Stockburger, M. Biochemistry 1989, 28, 6710. (c) Rivas, L.; Murgida, D. H.; Hildebrandt, P. J. Phys. Chem. B 2002, 106, 4823. (d) Rikhie, J.; Sampath, S. Electroanalysis 2005, 17, 762-768. (13) (a) Lojou, E Ä .; Bianco, P. J. Electroanal. Chem. 2000, 485, 71. (b) Imabayashi, S.; Mita, T.; Kakiuchi, T. Langmuir 2005, 21, 2474. (14) Abass, A. K.; Hart, J. P. Electrochim. Acta 2001, 46, 829. (15) (a) Richard, C.; Balavoine, F.; Schultz, P.; Ebbesen, T. W.; Mioskowski, C. Science 2003, 300, 775-778. (b) Yurekli, K.; Mitchell, C. A.; Krishnamoorti, R. J. Am. Chem. Soc. 2004, 126, 9902-9903. (c) Moore, V. C.; Strano, M. S.; Haroz, E. H.; Hauge, R. H.; Smalley, R. E.; Schmidt, J.; Talmon, Y. Nano Lett. 2003, 3, 1379-1382. (d) Islam, M. F.; Rojas, E.; Bergey, D. M.; Johnson, A. T.; Yodh, A. G. Nano Lett. 2003, 3, 269-273. (16) (a) Shumyantseva, V. V.; Ivanov, Y. D.; Bistolas, N.; Scheller, F. W.; Archakov, A.; Wollenberger, U. Anal. Chem. 2004, 76, 60466052. (b) Armstrong, F. A.; Wilson, G. S. Electrochim. Acta 2000, 45, 2623-2645. (c) Le´ger, C.; Elliot, S. J.; Hoke, K. R.; Jeuken, L. J. C.; Jones, A. K.; Armostrong, F. A. Biochemistry 2003, 42, 8653-8662.

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remarkably larger than that at the former one. In addition, such an enhancement in the redox peak currents of the proteins was found to be general for other kinds of heme proteins, for example, Hb, Mb, and Cyt-c, and with other kinds of surfactants, for example, Triton X-100 and CTAB, as well as onto SWNTs (not shown). Despite the facts that the surfactants have been used for solubilization, thereby avoiding the aggregation of CNTs, and that the surfactantdispersed CNTs have been used for facilitating electron transfer of the enzymes and proteins,7f,17,19 the enhancements in the faradic responses observed here have been less demonstrated so far. Elucidation on the Enhanced Faradic Responses. As detailed in the experimental section, the protein/ surfactant/MWNT- and protein/MWNT-modified GC electrodes were prepared almost under identical conditions, for example, the same loading of MWNTs onto GC electrodes and the same aqueous solution of protein used. Moreover, the time used for the adsorption of the protein from the aqueous solution containing no surfactant was intentionally prolonged to 12 h so that the adsorption of the protein was saturated. The only difference in the conditions employed was the addition of the surfactant in the aqueous solution of the protein for the preparation of the protein/surfactant/MWNT-modified GC electrodes, suggesting that the observed remarkably enhanced faradic current responses at this kind of electrode are possibly associated with the co-assembly of surfactant onto the MWNTs. Indeed, previous efforts have elegantly demonstrated that supramolecular surfactants could be used for facilitating interfacial electron transfer of enzymes and proteins. This property of the surfactants is essentially based on their capability for deterging the electrode surface to remove the adsorbed pollutant which may block the electron transfer and/or formation of a biomimic membrane to effectively maintain a proper orientation of these biomacromolecules favorable for their direct electron transfer.6c,18 We believe that such properties of the surfactants may also be suited for the present case and are partially responsible for the observed enhanced faradic responses of the proteins assembled in the protein/ surfactant/CNT nanohybrids (Figure 1). Beside this, as will be demonstrated below, we found that, compared with those achieved at PG, GC, and MWNT-modified GC electrodes, a larger portion of the enzymes and proteinassembled protein/surfactant/CNT nanohybrids could be capable of conducting direct electron transfer. Such an advantage, which is closely associated with the electrochemical and structural properties of the nanotubes and the three-dimensional architecture of the CNT film functionalized with protein and surfactant, essentially constitutes another essence for the observed enhanced responses mentioned above. We used QCM to measure the masses of the MWNTs confined onto the substrate and the surfactant and protein (taking SDS and HRP as an example) assembled onto the MWNTs. Meanwhile, the amount of electroactive HRP was calculated from the voltammograms of the HRP/SDS/ MWNT-modified GC electrode as detailed in the experimental section. These results are displayed in Table 1. As (17) Patosky, F; Weizmann, Y; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 2113-2117. (18) (a) Huang, R.; Hu, N. Bioelectrochemistry 2001, 54, 75-81. (b) Liu, H.; Hu, N. Anal. Chim. Acta 2003, 481, 91-99. (19) (a) Zhou, Y.; Li, Z.; Hu, N.; Zeng, Y.; Rusling, J. F. Langmuir 2002, 18, 8572-8579. (b) Li, Z.; Hu, N. J. Electroanal. Chem. 2003, 558, 155-165. (c) Wang, Q.; Lu, G.; Yang, B. Langmuir 2004, 20, 13421347. (d) Yu, X.; Sotzing, G. A.; Papadimitrakopoulos, F.; Rusling, J. F. Anal. Chem. 2003, 75, 4565-4571.

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Table 1. Mass of MWNT, HRP, and Electroactive HRP Assembled in the Nanohybrids

HRP/MWNT HRP/SDS/MWNT

MWNT, µg

SDS, µg

HRPads, µg

HRPactive, µg

HRPactive/ HRPads

2.17 2.35

0 0.44

0.65 0.60

0.11 0.51

16.70 % 84.90 %

Scheme 1. Carton Diagrams of (A) Bioelectrochemically Functional Individual through Co-Assembling of Protein and Surfactant onto a Single MWNT and (B) Nanohybrids Consisting of Single Biofunctional Nanotubes onto the GC Electrode

Table 2. Ratio of Electroactive Protein to Total Protein Immobilized in Various Matrixes matrix

ratio of proteinelectroactive (%)

ref

MWNT/SDS/HRP MWNT/HRP polyacrylamide hydrogel/HRP agarose hydrogel/HRP clay/HRP eastman AQ/HRP gluten/HRP sol-gel/Mb active carbon/HRP sol-gel/CTAB/Mb DHP-PDDA/Hb

84.90 16.70 0.32 10.00 2.00 0.70 1.4-7.3 13.95 13.70 17.00 5.00

this work this work 6d 6e 6g 18a 18b 19c 20 6h 6i

shown, both kinds of nanohybrids (protein/surfactant/ MWNT and protein/MWNT) almost have the same loading of HRP. However, the amount of electroactive HRP and, thus, the ratio of electroactive HRP to total HRP assembled in the HRP/SDS/MWNT are greater than those in the HRP/MWNT nanohybrid (almost 5-fold, Table 1). This striking property of the protein-surfactant-CNT nanohybrids is remarkable since the portion of the electroactive proteins is greatly improved compared with those in the other matrix, for example, surfactants, biocompatible polymers, and metal (oxide) nanoparticles (Table 2). Additionally, while some techniques such as layer-by-layer and sol-gel could be used for increasing the loading of the proteins, the portion of the electroactive proteins did not increase accordingly as demonstrated previously.19 The remarkably improved portion of electroactive protein-assembled protein-surfactant-CNT nanohybrids, which may not be solely related with the co-assembly of surfactant onto the MWNTs, is considered to be closely associated with the electrochemical and structural properties of the nanotubes and the architecture of the CNT film confined protein-surfactant-CNT nanohybrids. As reported previously, very different from bare PG and GC electrodes, the MWNT film confined onto the GC electrode essentially behaves like three-dimensional ensembles consisting of separated single nanotubes and/or small bundles of the MWNTs.8a Similar to PG and GC electrodes, the separated single nanotubes and/or small bundles of the MWNTs could be used to facilitate the direct electrochemistry of enzymes and proteins upon modification with surfactants (Scheme 1), although such a prediction could not be verified in this case because of the difficulties in manipulation of the separated single nanotubes and/or small bundles of the MWNTs. Upon three-dimensional confinement of these protein/surfactant/MWNT units onto the GC electrode, the total faradic responses of the asformed protein/surfactant/MWNT nanohybrids are the sum of those obtained at biofunctional individuals (Scheme 1). In such a case, the most distinct feature of the protein/ surfactant/MWNT nanohybrids from those with the other kinds of electrodes, for example, PG, GC, and active carbon, is that each separated nanotube (single nanotubes and/or small bundles of the MWNTs) can be essentially used as an individual nanoelectrode. These individual electrodes can be rationally functionalized to facilitate electron transfer of the biomacromolecules, even of those far from the GC substrate. As a consequence, this intrinsic feature essentially enables a large portion of the enzymes and

proteins assembled to be capable of conducting direct electron transfer, resulting in a remarkably improved ratio of electroactive molecules to total molecules assembled in the hybrids. These properties, which are superior to those of other kinds of carbon-based electrodes, for example, PG, GC, and active carbon,20 combined with the unique properties of the surfactant in facilitation of protein electrochemistry, are most likely responsible for the observed enhanced faradic responses (Figure 1). It has been reported that the presence of SDS essentially solubilizes the MWNTs in solution, yielding a homogeneous dispersion of the MWNTs in which the MWNTs mainly present in a form of single nanotubes and/or small bundles of the MWNTs.15 The better separation of the MWNTs in the presence of the surfactant relative to its absence may also constitute an essence for the enhanced portion of electroactive proteins assembled in the nanohybrids. However, such a possibility was excluded. We reasoned this by comparing the voltammograms of the nanohybrids prepared with different procedures; the electrodes prepared by stepwise assembling the surfactant and the protein onto the MWNTs (i.e., protein/MWNTsurfactant-modified GC electrodes) shown in Figure 3 exhibit almost the same redox peak currents as those at the electrodes prepared by co-assembling of the surfactant and the protein onto the MWNTs (i.e., protein/surfactant/ MWNT-modified GC electrodes) shown in Figure 1. As further evidence, we found that the MWNTs used here were well-dispersed into DMF upon sonication, and the as-formed MWNT film consisting of separated single and small bundles of nanotubes were readily obtained without a large aggregation (scanning electron microscopy image not shown here) provided the prepared MWNT dispersion was immediately confined onto the GC electrode. The observed enhanced faradic responses of the proteins in the protein-surfactant-MWNT nanohybrids were not due to the accelerated rate of electron transfer of the proteins with the surfactants co-assembled onto the (20) Sun, D.; Cai, C.; Li, X.; Xing, W.; Lu, T. J. Electroanal. Chem. 2004, 566, 415-421.

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Figure 3. Typical cyclic voltammograms of HRP/MWNT-SDS(solid curve), HRP/MWNT- (dashed curve), and MWNTmodified (dotted curve) GC electrodes in 0.10 M N2-saturated phosphate buffer. Scan rate, 100 mV s-1.

Figure 5. Cyclic voltammograms of the HRP/SDS/MWNTmodified GC electrode in 0.10 M N2-saturated phosphate buffer at various potential scan rates of (from inner to outer) 25, 100, 300, 500, 700, and 900 mV s-1. The inset shows the plot of the peak currents versus potential scan rate.

Figure 4. Cyclic voltammograms of Cyt-c/SDS/MWNT- (solid curve), Cyt-c/MWNT- (dashed curve), and MWNT/SDS-modified (dotted curve) GC electrodes in 0.10 M N2-saturated phosphate buffer. Scan rate, 100 mV s-1. Note that the loading of MWNTs onto the GC electrode in this experiment was 2 µg, which was half of those used in Figures 1-3.

Figure 6. Typical cyclic voltammograms of MWNT-modified (curves 2 and 4) and HRP/SDS/MWNT-modified (curves 1 and 3) GC electrodes in 0.10 M N2-saturated phosphate buffer in the absence (curves 1 and 2) and presence (3 and 4) of 2.0 mM H2O2. Scan rate, 100 mV s-1.

MWNTs, since the reversibility of the redox processes of both kinds of the nanohybrids (i.e., protein-surfactantMWNT and protein-MWNT) was found to be essentially identical at a given scan rate (Figure 1). Moreover, the electron-transfer rates of the proteins in both nanohybrids (e.g., HRP-SDS-MWNT and HRP-MWNT), calculated with the Laviron equation,21 were found to be 3.5 and 4.6 s-1, respectively. These values are in agreement with those reported previously and essentially in the same level for both nanohybrids.7b,f Furthermore, the faradic responses in the proteinsurfactant-MWNT nanohybrids could not be due to the redox process of the heme groups dissociated from the proteins even though the use of the protein-surfactant mixture to prepare protein-surfactant-CNT nanohybrids confined onto GC electrodes in this study may cause partial denaturation of the proteins and, thus, loss bioactivity in the case of enzymes. We reasoned this from the experiments obtained with Cyt-c as depicted in Figure 4. Unlike those in HRP, Hb, and Mb, the heme group in Cyt-c is covalently linked to the protein moiety through strong thioether bonds and, thus, is unable to be readily dissociated from the protein.22 As shown in Figure 4, an enhancement in the faradaic response of Cyt-c in the Cytc/SDS/MWNT-modified electrode was clearly achieved. (21) Laviron, E. J. Electroanal. Chem. 1979, 101, 19-28. (22) (a) Peterson, J.; Saleem, M. M. M.; Silver, J.; Wilson, M. T. J. Inorg. Biochem. 1983, 19, 165. (b) Bonanni, B.; Alliata, D.; Bizzarri, A. R.; Cannistraro, S. ChemPhysChem 2003, 4, 1183.

This demonstration may conclude that the enhanced faradaic current responses observed for HRP, Hb, and Mb could not be due to the redox process of heme groups dissociated from these proteins even though whether the proteins are in their bioactive states still needs more experimental evidence. Electrochemistry and Bioelectrocatalysis of the Nanohybrids. Figure 5 shows cyclic voltammograms of the HRP/SDS/MWNT-modified GC electrode at various scan rates. The small peak-to-peak separation and the linear relationship between the peak currents and potential scan rate in the range of 25-900 mV s-1 indicate that the redox process of the prepared nanohybrid is a reversible and surface-confined process. Moreover, the E°′ value of the HRP/SDS/MWNT-modified GC electrode changes linearly with solution pH with a slope of about -50 mV/pH (not shown), indicating that the redox process involves one electron and one proton. Having demonstrated the redox properties of the nanohybrids, we next investigated their bioelectrochemically catalytic activity toward, for exmaple, H2O2, as shown in Figure 6. The presence of H2O2 in solution clearly increased the cathodic peak current, while it decreased the reversed oxidation peak current (curve 3) compared with its absence (curve 1). This observation, along with the large positive shift of the potential for H2O2 reduction at the HRP/SDS/MWNT-modified GC electrode compared with that at the MWNT-modified GC electrode (curve 4), indicates that the nanohybrids possess a good catalytic activity toward the reduction of H2O2. The catalytic

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activities of the nanohybrids and their enhanced faradic responses as well as the high conductivity would enable them to be relatively useful for the development of new bioelectronic nanodevices, for example, enzyme-based biosensors and biofuel cells. Conclusions We have demonstrated here that the bioelectrochemically functional nanohybrids prepared by co-assembling of heme proteins and surfactants onto CNTs possess facilitated direct electron transfer with enhanced faradic responses for the proteins. The enhancement has been for the first time ascribed to the properties of the surfactants for facilitating of protein electrochemistry and the electrochemical and structural properties of CNTs as well as the three-dimensional architecture of the CNT film electrode. It is demonstrated that each separated sur-

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factant-functionalized nanotube in the CNT film threedimensionally confined on GC electrode can be essentially used as an individual functional nanoelectrode for facilitating the direct electron transfer of the proteins, enabling the enhancement in the portion of electroactive proteins assembled in the protein/surfactant/MWNT nanohybrids. This demonstration may pave a facile route to CNT-based bioelectronic nanodevices and would be useful for investigation of the electron-transfer properties of the proteins at functional interfaces. Acknowledgment. We gratefully acknowledge the financial support from National Natural Science Foundation of China (Grants 20375043 and 20435030) and Chinese Academy of Sciences (Grant KJCX2-SW-H06). LA050043Z