In Situ Cationic Ring-Opening Polymerization and Quaternization

Laccase (EC 1.10.3.2, from Trametes versicolor) was obtained from Sigma ... for the development of electrochemical biosensors with the Fe(CN)63−-con...
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Anal. Chem. 2008, 80, 6587–6593

In Situ Cationic Ring-Opening Polymerization and Quaternization Reactions To Confine Ferricyanide onto Carbon Nanotubes: A General Approach to Development of Integrative Nanostructured Electrochemical Biosensors Ling Xiang,† Zhinan Zhang,† Ping Yu,† Jun Zhang,*,† Lei Su,† Takeo Ohsaka,‡ and Lanqun Mao*,† Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, the Chinese Academy of Sciences (CAS), Beijing 100080, China, and Department of Electronic Chemistry, Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan This study demonstrates a new and relatively general route to the development of multiwalled carbon nanotube (MWNT)-based integrative electrochemical biosensors by confining ferricyanide redox mediator onto MWNTs. The ferricyanide-confined MWNTs are synthesized first through grafting of epoxy chloropropane onto MWNTs with in situ cationic ring-opening polymerization and then introducing the positively charged methylimidazolium moieties into the grafted polymer with a quaternization reaction. The grafted polymers with positively charged methylimidazolium moieties tethered onto MWNTs can essentially be used to confine redox-active ferricyanide onto MWNTs to form a redox mediator-confined nanocomposite with a good stability and excellent electrochemical property. The synthetic nanocomposite with surface-confined ferricyanide is demonstrated to be well-competent as the efficient electronic transducers for the general development of electrochemical biosensors upon combination with biorecognition units, which is illustrated by using glucose oxidase and laccase as two model biorecognition units. This study essentially paves a facile and general approach to the development of integrative nanostructured electrochemical biosensors. As one kind of one-dimensional carbon nanostructure, carbon nanotubes (CNTs) represent one of the members in the carbon family and possess very distinct structural and electronic properties from other kinds of carbon materials frequently used in electrochemistry, such as glassy carbon (GC), graphite, and diamond.1 For instance, CNTs are rich in surface chemistry and are very beneficial to electrochemical studies in terms of their unique structural and electronic properties and of rationally surface-functionalizable capabilities. So far, the structural and electronic differences of CNTs from other kinds of carbon materials mentioned above have largely stimulated the everincreasing research interests both in the fundamental understand* To whom correspondence should be addressed. Fax: +86-10-62559373 (L.M.). E-mail: [email protected] (L.M.); [email protected] (J.Z.). † Institute of Chemistry, CAS. ‡ Tokyo Institute of Technology. 10.1021/ac800733t CCC: $40.75  2008 American Chemical Society Published on Web 08/01/2008

ing of the structure-property relationship associated with the CNTs and in the practical development of new types of CNT-based electrochemical sensors and biosensors.2 To this end, the past decade has witnessed substantial progress on the studies of CNTbased electrochemistry and on the uses of CNTs to develop electrochemical sensors, as summarized in previous excellent reviews.1c-f,2a,b Although recent attempts have suggested that the combination of the unique electronic and structural properties of CNTs with the specific recognition ability of the biomacromolecules, such as redox enzymes and proteins, could potentially offer a facile but effective approach to the nanostructured electrochemical biosensors with improved performance, several key factors remain yet to be addressed in such a pursuit,1c-f,2a,b of which the facilitation of electronic communication between the biorecognition units and CNT electronic transducers represents one of the most important issues, and such a factor remains a challenge in the development of CNT-based electrochemical biosensors. The distinct structural and electronic properties of CNTs have enabled them to be advantageous over other kinds of electrode materials in facilitating direct electron transfer of the redox enzymes and proteins,3 and this advantage essentially make CNTs very useful for the development of new types of the so-called thirdgeneration electrochemical biosensors without the use of any redox mediators. However, such a direct electron-transfer-based strategy is yet limited for the general development of nanostructured electrochemical biosensors because only several kinds of (1) (a) Baughman, R. H.; Zakhidov, A. A.; de Heer, W. A. Science 2002, 297, 787. (b) Tasis, D.; Tagmatarchis, N.; Bianco, A.; Prato, M. Chem. Rev. 2006, 106, 1105. (c) Dai, L.; Soundarrajan, P.; Kim, T. Pure Appl. Chem. 2002, 74, 1753. (d) Gong, K.; Yan, Y.; Zhang, M.; Su, L.; Xiong, S.; Mao, L. Anal. Sci. 2005, 21, 1383. (e) Gooding, J. J. Electrochim. Acta 2005, 50, 3049. (f) Zhao, Q.; Gan, Z.; Zhuang, Q. Electroanalysis 2002, 14, 1609. (g) Luo, H.; Shi, Z.; Li, N.; Gu, Z.; Zhuang, Q. Anal. Chem. 2001, 73, 915. (h) Su, L.; Gao, F.; Mao, L. Anal. Chem. 2006, 78, 2651. (i) Gong, K.; Zhu, X.; Zhao, R.; Xiong, S.; Mao, L.; Chen, C. Anal. Chem. 2005, 77, 8158–8165. (2) (a) Katz, E.; Willner, I. ChemPhysChem 2004, 5, 1084. (b) Wang, J. Electroanalysis 2005, 17, 7. (c) Yan, Y.; Zheng, W.; Su, L.; Mao, L. Adv. Mater. 2006, 18, 2639. (d) Zhang, M.; Smith, A.; Gorski, W. Anal. Chem. 2004, 76, 5045. (e) Wang, J.; Musameh, M.; Lin, Y. J. Am. Chem. Soc. 2003, 125, 2408. (f) Wang, J.; Musameh, M. Anal. Chem. 2003, 75, 2075. (g) Chakraborty, S.; Raj, C. R. J. Electroanal. Chem. 2007, 609, 155. (h) Xiang, L.; Lin, Y.; Yu, P.; Su, L.; Mao, L. Electrochim. Acta 2007, 52, 4144.

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redox enzymes and proteins, typically as heme proteins, can conduct their direct electron transfers onto CNTs. This limitation substantially constitutes a straightforward consequence for the wide use of electron-transfer mediators to shuttle the electron transfer between the biorecognition units and CNT electronic transducers in the recent development of nanostructured electrochemical biosensors.4 As a redox species with excellent electrochemical properties, ferricyanide (Fe(CN)63-) has been widely used both in fundamental electrochemical studies and in the electrochemical applications.5 More importantly, the good biochemical reaction properties of this redox species with many kinds of enzymes and proteins, such as flavine adenine dinucleotide-containing oxidases and cytochrome c,6 substantially enabled it to be one of the most widely used electron-transfer mediators for the redox enzymes and proteins in the development of electrochemical biosensors, among all kinds of the redox mediators reported so far.7 Nevertheless, the confinement of such kind of widely used and water-soluble redox mediator onto CNTs remains a challenge, rendering difficulties in developing integrative electrochemical biosensors with surface-confined Fe(CN)63-, although several methods have been previously reported to confine Fe(CN)63- onto an electrode surface.8 Actually, such a difficulty has been encountered at other kinds of the electrodes frequently used in electrochemistry and has constituted the main consequence for the wide uses of solution-phased, rather than surface-confined, Fe(CN)63- as the redox mediator for the development of most kinds of mediated electron-transfer-based electrochemical biosensors.4b,6b,7a,b In comparison with those with the redox mediatorintegrated electrochemical biosensors, the electroanalytical methods with the electrochemical biosensors with solution-phased redox mediators may suffer from the limitations from higher costs, lower efficiency, and higher environmental burden. (3) (a) Yan, Y.; Zheng, W.; Zhang, M.; Wang, L.; Su, L.; Mao, L. Langmuir 2005, 21, 6560. (b) Gao, F.; Yan, Y.; Su, L.; Wang, L.; Mao, L. Electrochem. Commun. 2007, 9, 989. (c) Zheng, W.; Li, Q.; Su, L.; Yan, Y.; Zhang, J.; Mao, L. Electroanalysis 2006, 18, 587. (d) 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. (e) Wang, J.; Li, M.; Shi, Z.; Li, N.; Gu, Z. Anal. Chem. 2002, 74, 1993. (f) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2000, 39, 1180. (4) (a) Yan, Y.; Su, L.; Mao, L. J. Nanosci. Nanotechnol. 2007, 7, 1625. (b) Liu, Y.; Dong, S. Biosens. Bioelectron. 2007, 23, 593. (c) Liu, Y.; Huang, L.; Dong, S. Biosens. Bioelectron. 2007, 23, 35. (5) (a) Holt, K. B. Langmuir 2006, 22, 4298. (b) Moore, R. R.; Banks, C. E.; Compton, R. G. Anal. Chem. 2004, 76, 2677. (c) Conyers, J. L.; White, H. S., Jr Anal. Chem. 2000, 72, 4441. (6) (a) May, J. M.; Qu, Z.; Cobb, C. E. J. Biol. Chem. 2004, 279, 14975. (b) Shul’ga, A. A.; Koudelka-Hep, M.; de Roolj, N. F. Anal. Chem. 1994, 66, 205. (c) Ahmed, A. J.; Millett, F. J. Biol. Chem. 1981, 256, 1611. (7) (a) For a review: Chaubey, A.; Malhotra, B. D. Biosens. Bioelectron 2002, 17, 441. (b) Smit, M. H.; Rechnitz, G. A. Anal. Chem. 1993, 65, 380. (c) Le´veˆque, V. J.-P.; Vance, C. K.; Nick, H. S.; Silverman, D. N. Biochemistry 2001, 40, 10586. (d) Baker, M. A.; Lane, D. J. R.; Ly, J. D.; Pinto, V. D.; Lawen, A. J. Biol. Chem. 2004, 279, 4811. (e) Kurita, R.; Yabumoto, N.; Niwa, O. Biosens. Bioelectron. 2006, 21, 1649. (f) Liu, S.; Lin, B.; Yang, X.; Zhang, Q. J. Phys. Chem. B 2007, 111, 1182. (g) Weigel, M. C.; Tritscher, E.; Lisdat, F. Electrochem. Commun. 2007, 9, 689. (h) Razola, S. S.; Aktas, E.; Vire, J. C.; Kauffmann, J.-M. Analyst 2000, 125, 79. (8) (a) Gros, P.; Comtat, M. Biosens. Bioelectron. 2004, 20, 204. (b) Nakagawa, T.; Tsujimura, S.; Kano, K.; Ikeda, T. Chem. Lett. 2003, 32, 54. (c) Vasantha, V. S.; Chen, S. Electrochim. Acta 2005, 51, 347. (d) Raoof, J.-B.; Ojani, R.; Rashid-Nadimi, S. Electrochim. Acta 2005, 50, 4694. (e) Kumar, A.; Rajesh; Chaubey, A.; Grover, S. K.; Malhotra, B. D. J. Appl. Polym. Sci. 2001, 82, 3486.

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Scheme 1. Synthetic Route of the Poly(ECH)-Grafted and MIM-Tethered MWNTs

In this study, we wish to report a new method for stably confining Fe(CN)63- electron-transfer mediator onto CNTs to form efficient electronic transducers for the general development of the integrative electrochemical biosensors. The strategy demonstrated here is essentially based on the creation of positively charged moieties onto CNTs by first grafting CNTs with polyether via an in situ cationic ring-opening polymerization of epoxy chloropropane9 and then introducing positively charged methylimidazolium (MIM) moieties through a quaternization reaction between methylimidazole and polyether grafted onto CNTs. The positively charged MIM moieties introduced onto the CNTs can essentially interact with Fe(CN)63- through an electrostatic interaction and can consequently confine such kind of redox mediators onto CNTs (Scheme 1). The prepared nanocomposite with surface-confined Fe(CN)63- redox mediator can be used as efficient electronic transducers for the general development of redox enzyme-based electrochemical biosensors in terms of the good electrochemical property of Fe(CN)63- for shuttling the electron transfer of many kinds of redox enzymes and proteins. This study could pave a platform for the development of the integrative nanostructured electrochemical biosensors. EXPERIMENTAL SECTION Chemical and Materials. Multiwalled carbon nanotubes (MWNTs, diameter 20 to ∼30 nm, length >1 µm) were purchased from Shenzhen Nanoport Co. Ltd. (Shenzhen, China). Glucose oxidase (GOx, EC 1.1.3.4, from Aspergillus niger), poly(diallyldimethylammonium chloride) (PDDA, Mw ) 200 000-350 000, 20% aqueous solution), and methylimidazole were obtained from Sigma and used as supplied. Laccase (EC 1.10.3.2, from Trametes versicolor) was obtained from Sigma and purified with a method described in our early work.3c Bovine serum albumin (BSA), glutaraldehyde (50% aqueous solution), and glucose were all purchased from Beijing Chemical Reagent Co. Ltd. (Beijing, China). Other chemicals were of analytical grade or higher and used as received. Aqueous solutions were prepared with doubly distilled water. Synthesis of Polyether-Grafted and MethylimidazoliumTethered MWNTs. For grafting of the polyether onto MWNTs, MWNTs were first treated in a mixture of concentrated sulfuric acid and nitric acid (volume ratio 3:1) under sonication to introduce oxygen-containing moieties on the open ends and (9) (a) Tsubokawa, N.; Yoshihara, T. Colloids Surf., A 1993, 81, 195. (b) Tsubokawa, N. Polym. J. 2005, 37, 637.

defects on the sidewall of the nanotubes to produce MWNTs functionalized with carboxylic groups. The resulting MWNTs were allowed to react with thionyl chloride to produce carbonyl chloride (-COCl) moieties onto MWNTs, according to a method reported previously.10 Polyether grafting onto MWNTs was accomplished by the in situ cationic ring-opening polymerization of epoxy chloropropane (ECH) initiated by the -COCl moieties onto MWNTs under the catalysis of FeCl3. In a typical experiment, 20.0 mL of ECH, 0.20 g of MWNTs with -COCl moieties, and 0.20 g of FeCl3 were mixed in a 50 mL flask under N2 atmosphere. The polymerization was carried out at room temperature (25 °C) under magnetic stirring for 12 h. The resulting mixture was dispersed into a large excess of methanol to precipitate the polyether-grafted MWNTs. The precipitated polyether-grafted MWNTs were washed with tetrahydrofuran to remove the ungrafted polymer, and the obtained polyether-grafted MWNTs were filtered and dried at 70 °C overnight under vacuum. Positively charged MIM moieties were introduced onto the polyether-grafted MWNTs by mixing 0.20 g of polyether-grafted MWNTs and 20 mL of methylimidazole into 40.0 mL of ethanol and refluxing the mixture under stirring for 12 h. The product was first washed with excess ethanol, then filtered with 220 nm PTFE membrane, and finally dried at 70 °C under vacuum overnight to give the MIM-tethered MWNTs. Preparation of Electrochemical Biosensors. Glassy carbon electrodes (GC, 3 mm diameter, Bioanalytical Systems, Inc.) were used as the substrate for the development of electrochemical biosensors with the Fe(CN)63--confined MWNTs as the electronic transducers. The electrodes were polished first with emery paper, and then with aqueous slurries of fine alumina powders (0.3 and 0.05 µm) on a polishing microcloth, and were finally rinsed with doubly distilled water in an ultrasonic bath for 10 min. The synthetic MIM-tethered MWNTs (2 mg/mL) were dispersed into doubly distilled water to obtain a homogeneous dispersion under a bath sonication, and 1 µL of the resulting dispersion was coated onto GC electrodes. After solvent evaporation, the electrodes (denoted as MIM/MWNT-modified GC) were first immersed into the aqueous solution of 1.0 mM K3Fe(CN)6 for 10 min, then taken out from the solution, and finally rinsed with distilled water to obtain the Fe(CN)63-/MIM/MWNT-modified GC electrodes. We found that the adsorption of Fe(CN)63- onto the MIM/MWNTmodified GC electrodes was a quick process and was saturated after immersing the electrodes into 1.0 mM aqueous solution of K3Fe(CN)63- for 10 min. The amount of Fe(CN)63- confined onto the MIM-tethered MWNTs was determined by integrating the voltammetric peak obtained at the Fe(CN)63-/MIM/MWNTmodified GC electrode and was estimated to be 5.24 × 10-3 mg Fe(CN)63- per milligram of MWNTs. For comparative study, the MWNTs were noncovalently functionalized with positively charged PDDA to create a positively charged layer onto MWNTs, which was expected to confine K3Fe(CN)6 onto MWNTs via the electrostatic interaction. For such a purpose, 2 mg MWNTs were mixed into 1 mL 0.2% PDDA aqueous solution to give a uniform dispersion under a bath sonication, and 1 µL of the resulting dispersion was coated onto GC electrodes to form the PDDA/ (10) (a) Coleman, J. N.; Khan, U.; Gun’ko, Y. K. Adv. Mater. 2006, 18, 689. (b) Grossiord, N.; Loos, J.; Regev, O.; Koning, C. E. Chem. Mater. 2006, 18, 1089. (c) Moniruzzaman, M.; Winey, K. I. Macromolecules 2006, 39, 5194. (d) Mylvaganam, K.; Zhang, L. C. J. Phys. Chem. B 2004, 108, 15009.

MWNT-modified electrodes. After being air-dried, the PDDA/ MWNT-modified electrodes were immersed into the aqueous solution of 1.0 mM K3Fe(CN)6 for 10 min, then taken out from the solution, and finally rinsed with distilled water to obtain the Fe(CN)63-/PDDA/MWNT-modified GC electrodes. GOx and laccase were used as the model biorecognition units to demonstrate the generality of the development of the electrochemical biosensors with the Fe(CN)63--confined MWNTs as the electronic transducers. A 1.5 µL volume of laccase solution in 0.10 M phosphate buffer (pH 6.0) was mixed with 0.5 µL of BSA (1%) aqueous solution to give a laccase-BSA mixture that was totally coated onto the Fe(CN)63-/MIM/MWNT-modified GC electrodes. After that, 1.0 µL of glutaraldehyde aqueous solution (40 mM) was pipetted to the electrode surface to cross-link laccase onto MWNTs. Similarly, a mixture of 6.0 µL of GOx solution (10 mg/mL) and 2.0 µL of BSA (1%) were totally coated onto the Fe(CN)63-/MIM/MWNT-modified GC electrodes, and 2.0 µL of glutaraldehyde aqueous solution (1%) was applied onto the electrode surface to cross-link GOx onto MWNTs. The resulting electrodes (denoted as laccase/Fe(CN)63-/MIM/MWNT-modified and GOx/Fe(CN)63-/MIM/MWNT-modified GC) were rinsed with distilled water, dried at ambient temperature, and stored in a refrigerator at 4 °C while not in use. For comparison, the electrodes without confinement of Fe(CN)63- onto MWNTs (i.e., GOx/MIM/MWNT-modified GC electrodes) were also prepared by cross-linking the same amount of GOx onto the MIM/MWNTmodified GC electrodes, and the electrodes were rinsed with distilled water and dried at ambient temperature before use. Apparatus and Measurements. Electrochemical measurements were carried out on a computer-controlled electrochemical analyzer (CHI660B, CHI, Austin, TX) in a two-compartment electrochemical cell with the modified GC electrodes as working electrode, a platinum spiral wire as counter electrode, and a Ag/ AgCl electrode (saturated with KCl) as reference electrode. A 0.10 M HAc-NaAc buffer (pH 5.0) and 0.10 M phosphate buffer solution (pH 7.0) were used as supporting electrolyte for the electrochemical studies of the electrochemical biosensors based on laccase and GOx, respectively. Thermogravimetric analysis (TGA) was carried out on PerkinElmer TGA-7 instrument with a heating rate of 20 °C/min in the nitrogen flow (20 mL/min). X-ray photoelectron spectroscopy (XPS) was performed on an ESCALab220i-XL electron spectrometer from VG Scientific using 300 W Al KR radiation. RESULTS AND DISCUSSION Characterization of Poly(ECH)-Grafted and MIM-Tethered MWNTs. Both grafting of the poly(ECH) onto MWNTs to form the poly(ECH)-grafted MWNTs and introducing of the MIM moieties onto the poly(ECH)-grafted MWNTs to form the MIMtethered MWNTs through the quaternization reaction, shown in Scheme 1, were characterized with XPS, as displayed in Figure 1A. The peaks at 283.3 and 532.2 eV in the spectra of the treated (curve 1), poly(ECH)-grafted MWNTs (curve 2), and MIMtethered (curve 3) MWNTs could be ascribed to carbon atoms (C 1s) of the MWNTs and/or of the grafted polymer and to oxygen atoms (O 1s) of the oxygenated groups and/or the ester bond between the grafted polymer and MWNTs and the ether bonds in each repeated union of the grafted polymer, respecAnalytical Chemistry, Vol. 80, No. 17, September 1, 2008

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Figure 1. (A) XPS spectra of the treated (1), poly(ECH)-grafted (2), and MIM-tethered (3) MWNTs. (B) Digital photos of treated (1), poly(ECH)-grafted (2), and MIM-tethered (3) MWNTs dispersed into a binary mixture of CH2Cl2/H2O at room temperature. The upper solvent was H2O, and the lower one was CH2Cl2. The photo of the treated MWNTs dispersed in the binary mixture was taken 5 h after the dispersion was prepared, and the photos for the other two kinds of dispersions were taken 2 weeks after the dispersions were prepared.

tively.11 The presence of the peak at 199.7 eV in the spectrum of the poly(ECH)-grafted MWNTs (curve 2), which was ascribed to the chloride atoms (Cl 2p),11a,12 substantially suggests successful grafting of poly(ECH) onto MWNTs. When compared with the spectrum of the poly(ECH)-grafted MWNTs (curve 2), the spectrum of the MIM-tethered MWNTs (curve 3) exhibits a new peak at 399.5 eV, which was assigned to the nitrogen atoms (N 1s) in the MIM ring,11a,13 demonstrating the introduction of the MIM moieties onto the poly(ECH)-grafted MWNTs through the quaternization reaction to form the positively charged MIMtethered MWNTs. The formation of poly(ECH)-grafted and MIM-tethered MWNTs through the grafting and quaternizing protocols described above was verified from the different dispersibility of the as-formed nanocomposites in a binary mixture of CH2Cl2/H2O, as displayed in Figure 1B. Due to the initial introduction of carboxylic acid groups on MWNTs through the acidic treatment, the prepared MWNTs exhibit improved solubility in water but tend to agglomerate on the interface of CH2Cl2/H2O after several hours (1). After being grafted with the polyether, the formed poly(ECH)grafted MWNTs become highly hydrophobic and could thus be stably dispersed into CH2Cl2 for at least 2 weeks (2). The subsequent introduction of MIM moieties onto the poly(ECH)grafted MWNTs essentially leads to solubilization of the resulting (11) (a) Wanger, C. D.; Riggs, W. M.; Davis, L. E.; Moulder, J. F.; Muilenberg, G. E. Handbook of X-ray Photoelectron Spectroscopy; Perkin-Elmer: Eden Prairie, MN, 1978. (b) Yu, P.; Lin, Y.; Xiang, L.; Su, L.; Zhang, J.; Mao, L. Langmuir 2005, 21, 9000. (12) Huezo, L.; Crumer, C.; Soto, C.; Tysoe, W. T. Langmuir 1994, 10, 3571. (13) (a) Ho ¨fft, O.; Bahr, S.; Himmerlich, M.; Krischok, S.; Schaefer, J. A.; Kempter, V. Langmuir 2006, 22, 7120. (b) Park, M.; Lee, J.; Lee, B.; Lee, Y.; Choi, I. S.; Lee, S. Chem. Mater. 2006, 18, 1546.

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Figure 2. High-resolution XPS spectra of Cl 2p at the MIM-tethered MWNTs. Upper panel, poly(ECH)-grafted MWNTs with parts of the poly(ECH) being involved into the quaternization reaction with methylimidazole.

MIM-tethered MWNTs into water (3). This obvious change in the dispersibility into the binary mixture of CH2Cl2/H2O among the treated MWNTs, poly(ECH)-grafted MWNTs, and MIMtethered MWNTs again suggests the successful polyether grafting and thus MIM tethering onto MWNTs. In addition, TGA results of the treated MWNTs and poly(ECH)grafted MWNTs (data not shown) suggest that the pretreated MWNTs decompose slightly with a weight loss of about 5% with the increasing of temperature up to 600 °C, possibly due to the loss of carboxylic groups on the surface of MWNTs, but the poly(ECH)-grafted MWNTs show a poor thermostability and decompose greatly with a weight loss of about 40% when the temperature was increased up to 450 °C, which was due to decomposition of the poly(ECH) grafted onto MWNTs. The percentage of the poly(ECH) grafted onto MWNTs involved in the quaternization reaction with methylimidazole was quantified through the high-resolution XPS spectrum of Cl 2p at the MIMtethered MWNTs, as shown in Figure 2. The spectrum of Cl 2p could be split into two peaks at 197.6 and 200.9 eV. The former was attributed to chloride ions adsorbed onto the polymer chain through the electrostatic interaction with the positively charged MIM ring,11a whereas the latter was ascribed to the chloride atoms in each unit of poly(ECH) that does not react with methylimidazole (Figure 2, upper panel). According to the integral area of the each normalized peak of Cl 2p, the percentage of the poly(ECH) grafted onto MWNTs involved in the quaternization reaction with methylimidazole was evaluated to be about 40%. The high contents of both the poly(ECH) grafted and the MIM tethered onto MWNTs essentially provides plenty of positively charged moieties onto MWNTs for efficiently confining Fe(CN)63- electron-transfer mediator for the general development of the integrative electrochemical biosensors, as described below. Confinement of Fe(CN)63- onto the MIM-Tethered MWNTs. The adsorption of Fe(CN)63- onto the MIM-tethered MWNTs was characterized by cyclic voltammetry (CV) with the MIM/MWNT-modified GC electrode in the aqueous solution of

Figure 3. (A) CVs obtained at the MIM/MWNT-modified GC electrode in phosphate buffer (pH 7.0) containing 1.0 × 10-4 M K3Fe(CN)6 when the electrode was immersed into the solution for different times of (from inner to outer) 10 and 30 s and 1, 2, 3, 4, 5, 6, 7, 10, and 30 min. The dotted curve represents a typical CV obtained with the same electrode in phosphate buffer containing no K3Fe(CN)6. Scan rate, 100 mV s-1. (B) Kinetic plot of ln(1 - θ) vs immersion time.

K3Fe(CN)6, as displayed in Figure 3A. The peak currents of the redox wave at +0.14 V clearly increase with increasing the immersing time, suggesting the adsorption and the continuous growth of Fe(CN)63- onto MIM-tethered MWNTs. The adsorption kinetics was calculated from the kinetic plot shown in Figure 3B, according to the equation14 ln(1 - θ) ) -kct where k is the adsorption rate constant, t is the time for immersing the electrode in the solution of K3Fe(CN)6, and c is the concentration of K3Fe(CN)6. θ is the fractional coverage of Fe(CN)63- and is equal to Γ0′/Γ0, where Γ0′ and Γ0 are the surface coverage of Fe(CN)63- at any time and at the saturation plateau, respectively, which were determined by integrating the peaks displayed in Figure 3A. The k value was calculated from the slope of the kinetic plot and was estimated to be 9.42 × 104 cm3 mol-1 s-1. Figure 4A depicts typical CVs obtained at the MIM-tethered MWNTs confined onto GC electrodes in pure phosphate buffer containing no K3Fe(CN)6 after the electrodes were first immersed into the aqueous solution of K3Fe(CN)6, then taken out from the solution, and finally rinsed with distilled water. After being first immersed into the K3Fe(CN)6 solution and then taken out from such solution and rinsed with distilled water, the electrodes exhibit (14) (a) Yan, J.; Zhou, Y.; Yu, P.; Su, L.; Mao, L.; Zhang, D.; Zhu, D. Chem. Commun., in press. (b) Stiles, R. L.; Balasubramanian, R.; Feldberg, S. W.; Murray, R. W. J. Am. Chem. Soc. 2008, 130, 1856.

Figure 4. (A) CVs obtained at the MIM-tethered MWNTs confined onto a GC electrode in 0.10 M phosphate buffer (pH 7.0) before (dotted line) and after (solid line) the electrode was immersed into 1 mM K3Fe(CN)6 for 10 min and rinsed with distilled water. Scan rate, 100 mV s-1. (B) XPS spectrum of the Fe(CN)63-/MIM-tethered MWNTs. Inset, high-resolution XPS spectrum of Fe 2p at the Fe(CN)63-/MIM-tethered MWNTs.

one pair of well-defined redox waves, which were ascribed to the redox process of Fe(CN)63- adsorbed onto MWNTs, revealing that Fe(CN)63- was confined onto MWNTs. The confinement of Fe(CN)63- onto MWNTs through the in situ cationic ring-opening polymerization and the quaternization reactions was also verified with the XPS spectrum obtained with the Fe(CN)63--confined MWNTs, as displayed in Figure 4B. The peaks at 710.1 and 723.6 eV in the spectrum of the Fe(CN)63-confined MWNTs (inset) were ascribed to Fe atoms (Fe 2p) onto the MWNTs.11a These peaks were not recorded at the pretreated MWNTs (Figure 1, curve 1), revealing the successful confinement of Fe(CN)63- onto the MWNTs through in situ cationic ringopening polymerization and quaternization reactions described above. Figure 5 displays the consecutive CVs obtained at the Fe(CN)63-/ MIM/MWNT-modified electrode in 0.10 M phosphate buffer under magnetic stirring. For comparison, the consecutive CVs obtained at the Fe(CN)63-/PDDA/MWNT-modified electrode under the same conditions are also given. The confinement of Fe(CN)63- onto MWNTs through the present strategies with the interaction between MIM moieties and Fe(CN)63- anion was stable: the peak currents recorded for the confined Fe(CN)63remain almost unchanged upon consecutive potential cycling the electrode for at least 50 cycles under magnetic stirring of the solution, as depicted in Figure 5A. Although the method for a creating positively charged layer onto MWNTs through the noncovalent functionalization of MWNTs with positively charged Analytical Chemistry, Vol. 80, No. 17, September 1, 2008

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Figure 5. Consecutive CVs for 50 cycles obtained at the Fe(CN)63-/ MIM/MWNT-modified (A) and the Fe(CN)63-/PDDA/MWNT-modified (B) GC electrodes in 0.10 M phosphate buffer (pH 7.0) under magnetic stirring of the solution. Scan rate, 100 mV/s. Stirring rate, 200 rpm.

PDDA polyelectrolyte was more facile than the approach demonstrated here, our control experiments with the as-prepared Fe(CN)63-/PDDA/MWNT-modified electrode revealed that the confinement of Fe(CN)63- onto the MWNTs with such kind of polyelectrolyte was less stable: the current recorded at the Fe(CN)63-/PDDA/MWNT-modified electrode gradually decreased with consecutive potential cycling, and only half of the original current was preserved after 50 cycles under magnetic stirring of the solution (Figure 5B). The high stability of Fe(CN)63- confined onto the MWNTs through our strategy described above could be due to the strong interaction between MIM moieties and Fe(CN)63- anion, as reported previously.15 In addition to the good stability, the prepared Fe(CN)63-confined MWNTs exhibit excellent electrochemical properties. In 0.10 M phosphate buffer (pH 7.0), the peak currents obtained at the Fe(CN)63-/MIM/MWNT-modified electrode are linear with potential scan rate within the potential range from 10 to 400 mV s-1 (data not shown). This, together with the small peak-to-peak potential separation and the slight dependence of the peak potential on potential scan rate, reveals that the redox process of Fe(CN)63- confined onto MWNTs is a fast and surface-confined process. Such a property, along with the good stability demonstrated above, essentially enables the prepared Fe(CN)63-confined MWNTs to be well-competent as the efficient electronic transducers for the general development of integrative electrochemical biosensors with surface-confined, rather than solution(15) Chi, Y. S.; Hwang, S.; Lee, B. S.; Kwak, J.; Choi, I. S.; Lee, S. Langmuir 2005, 21, 4268.

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Figure 6. (A) Amperometric I-t response obtained at the GOx/ Fe(CN)63-/MIM/MWNT-modified GC electrode in 0.10 M phosphate buffer solution (pH 7.0) to successive addition of glucose with the concentrations indicated in the figure. The electrode was polarized at +0.20 V. Inset (left), amperometric I-t response obtained at the GOx/MIM/MWNT-modified GC electrode (i.e., without confining Fe(CN)63- redox mediator onto MWNTs) in 0.10 M phosphate buffer solution (pH 7.0) to the successive addition of 0.1 mM glucose with a time interval of 100 s from t ) 500 to 2400 s. Inset (right), plot of current response vs the concentration of glucose. (B) CVs obtained at the laccase/Fe(CN)63-/MIM/MWNT-modified GC electrode in 0.10 M HAc-NaAc buffer (pH 5.0) saturated with N2 (dotted curve), ambient air (dashed curve), or O2 (solid curve). Scan rate, 10 mV s-1.

phased, redox mediators for shuttling the electron transfer of the biorecognition units, as described below. Toward Electrochemical Biosensing. By using GOx and laccase as the model biorecognition units, we demonstrated the uses of the MWNT nanocomposite with surface-confined Fe(CN)63- redox mediator synthesized by the in situ cationic ringopening polymerization and quaternization reactions as the electronic transducers to develop integrative electrochemical biosensors. Figure 6A displays typical amperometric response at the GOx/Fe(CN)63-/MIM/MWNT-modified GC electrode toward glucose. The addition of glucose in solution clearly results in the increase in the current response at the electrode, whereas the same addition did not result in a recordable current response at the electrode without confinement of Fe(CN)63- onto MWNTs (i.e., GOx/MIM/MWNT-modified GC electrode) (left inset). This demonstrates that the as-prepared electrochemical biosensor with the Fe(CN)63--confined MWNTs as the electronic transducer and GOx as the biorecognition unit is very responsive to glucose. The current was linear with the concentration of glucose within the concentration from 0.05 to 0.5 mM (I (nA) ) 39.19Cglucose (mM) + 1.78, γ ) 0.982) (right inset). The detection limit of the biosensor for glucose was 0.01 mM (S/N ) 3).

Figure 6B shows CVs obtained at the laccase/Fe(CN)63-/ MWNT-modified GC electrode for O2 reduction in 0.10 M HAc-NaAc buffer (pH 5.0). At the MIM-tethered MWNTmodified GC electrode without immobilization of the biorecognition unit of laccase, O2 reduction occurs at -0.40 V (data not shown). At the laccase/Fe(CN)63-/MIM/MWNT-modified GC electrode, the presence of O2 in solution clearly increases the reduction peak current and decreases the reversed oxidation peak current of Fe(CN)63- confined onto MWNTs, as shown in Figure 6B, suggesting that the O2 reduction occurs under the bioelectrocatalysis of laccase with surface-confined Fe(CN)63- as the redox mediator. These demonstrations with GOx and laccase as the model biorecognition units substantially suggest that the Fe(CN)63--confined MWNTs prepared by polyether-grafting and MIM-tethering approaches could be used as the efficient electronic transducers for the general development of integrative nanostructured electrochemical biosensors. CONCLUSIONS In summary, we have demonstrated a new and relatively general approach to the development of integrative electrochemical biosensors with ferricyanide-confined MWNT nanocomposite as the efficient electronic transducers synthesized by first grafting

of ECH onto MWNTs through in situ cationic ring-opening polymerization, then introducing the MIM moieties through the quaternization reaction, and finally confining ferricyanide anion onto the MIM-tethered MWNTs. The good stability and the excellent electrochemical properties of the as-prepared ferricyanide-confined MWNT nanocomposite substantially enable its applications as the efficient electronic transducers for the general development of the integrative electrochemical biosensors upon combination with biorecognition units. This study essentially paves a facile and general approach to the development of integrative nanostructured bioelectrochemical devices, such as biosensors and biofuel cells. ACKNOWLEDGMENT We gratefully acknowledge the financial support from the NSF of China (Grant Nos. 20435030, 20575071, 20625515, and 20721140650 for L.M. and 20705034 for L.S.), the National Basic Research Program of China (2007CB935603), the Chinese Academy of Sciences (KJCX2-YW-H11), and the Center for Molecular Science, Institute of Chemistry. Received for review April 14, 2008. Accepted July 1, 2008. AC800733T

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