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O2 biofuel cell: Preparation, characterization and performance in serum
Electrochemistry Communications 9 (2007) 989–996 www.elsevier.com/locate/elecom
An enzymatic glucose/O2 biofuel cell: Preparation, characterization and performance in serum Feng Gao a, Yiming Yan b, Lei Su b, Lun Wang a, Lanqun Mao a
b,*
Anhui Key Laboratory of Functional Molecular Solids, College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, China b Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100080, China Received 22 November 2006; received in revised form 5 December 2006; accepted 5 December 2006 Available online 11 January 2007
Abstract This study demonstrates a new kind of single-walled carbon nanotubes (SWNT)-based compartment-less glucose/O2 biofuel cell (BFC) with glucose dehydrogenase (GDH) and bilirubin oxidase (BOD) as the anodic and cathodic biocatalysts, respectively, and with poly(brilliant creysl blue) (BCB) adsorbed onto SWNT nanocomposite as the electrocatalyst for the oxidation of NADH. The prepared GDH-polyBCB-SWNT bioanode exhibits an excellent electrocatalytic activity toward the oxidation of glucose biofuel; in 0.10 M phosphate buffer containing 20 mM NAD+ and 100 mM glucose, the oxidation of glucose commences at 0.25 V and the current reaches its maximum of 310 lA/cm2 at 0.05 V vs. Ag/AgCl. At the BOD-SWNT biocathode, a high potential output is achieved for the reduction of O2 due to the direct electron transfer property of BOD at the SWNTs. In 0.10 M phosphate buffer, the electrocatalytic reduction of O2 is observed at a high potential of 0.53 V vs. Ag/AgCl with an electrocatalytic current plateau of ca. 28 lA/cm2 at 0.45 V under ambient air and ca. 102 lA/cm2 under O2-saturated atmosphere. In 0.10 M phosphate buffer containing 10 mM NAD+ and 40 mM glucose under O2-saturated atmosphere, the power density of the assembled SWNT-based glucose/O2 BFC reaches 53.9 lW/cm2 at 0.50 V. The performance and the stability of the glucose/O2 BFC are also evaluated in serum. This study could offer a new route to the development of new kinds of enzymatic BFCs with a high performance and provide useful information on future studies on the enzymatic BFCs as in vivo power sources. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Enzymatic biofuel cells; Bioelectrocatalysis; Glucose oxidation; O2 reduction
1. Introduction Considerable attention has been recently paid on enzymatic biofuel cells (BFCs) because they have been recognized as a new kind of energy conversion technologies that possess striking properties, such as operation in mild conditions, i.e., ambient temperature and neutral pH, and potentiality to be used as in vivo power sources for bioelectronics including micropumps, pacemakers, neuromorphic circuits and so forth [1–5]. To date, much effort has been devoted to the key issues that have to be properly addressed in the transition of the enzymatic BFCs from *
Corresponding author. Tel.: +86 10 62646525; fax: +86 10 62559373. E-mail address: [email protected] (L. Mao).
1388-2481/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2006.12.008
theoretical concepts to practical devices [6–11]. These include establishment of new bioelectrocatalytic systems, stable immobilization of the electrocatalysts and biocatalysts into a three-dimensional conducting matrix, improvement of the power output and stability, and examination of the cell performance in real biological samples to evaluate the potentiality of the BFCs as in vivo power sources [12,13]. To this end, several new kinds of electrochemical concepts and assembled enzymatic BFCs have been demonstrated in recent a few years [14–24]. Our approaches to development of new kinds of enzymatic BFCs are essentially based on the uses of carbon nanotubes (CNTs) because, as a new kind of carbon-based nanostructures, CNTs have been proved to possess unique electronic and structural properties [25–29], which substan-
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tially enable them to be well competent both as electrode materials and as conducting matrix to accommodate the electrocatalysts (e.g., redox dyes) and biocatalysts (i.e., enzymes and proteins) used in the enzymatic BFCs [30– 40]. More remarkably, we have recently found that the uses of CNTs could largely facilitate direct electron transfer of blue multi-copper oxidases (e.g., laccase and bilirubin oxidase) that have been frequently used as the biocatalysts of the biocathodes in the enzymatic BFCs [41–43]. Such a property of CNTs essentially makes it possible for the catalytic reduction of O2 to occur at a more positive potential than those with electron transfer of blue multi-copper oxidases being mediated by redox mediators, resulting in a low potential loss in the as-prepared biocathodes. On the other hand, the uses of CNTs have been found to be also useful for the development of the dehydrogenase-based bioanodes in the enzymatic BFCs in terms of their capability to be used to accommodate the electrocatalysts for the oxidation of NADH [37,42,44]. It is known that at the dehydrogenase-based bioanodes the potentials for the oxidation of biofuels are essentially determined by the kinetics of the oxidation of NADH, in which the oxidized form of NADH, i.e., NAD+, is generally used as the cofactor for the dehydrogenases in the bioelectrocatalytic oxidation of biofuels [15,19,42,45,46]. As demonstrated previously, some kinds of organic compounds with p-conjugative structures, such as redox dyes, can interact with CNTs with p-stacking interactions to form a three-dimensional and redox-active CNT nanocomposite [32,44]. Such a property of the redox dyes and CNTs essentially makes it possible to prepare a conducting and stable matrix containing redox-active dyes that could be used as
the electrocatalysts for the oxidation of NADH and thereby for the development of new kinds of nanostructured dehydrogenase-based BFCs. In our previous attempts [32,42], we have prepared a methylene blue-single walled carbon nanotubes (MB-SWNT) nanocomposite through the noncovalent adsorption of MB onto SWNTs and found that, after being electrochemically polymerized, the nanocomposite (i.e., polyMB-SWNT) exhibits an excellent electrocatalytic activity toward the oxidation of NADH. Such a property has been used for developing a new kind of glucose/O2 BFC. As a continuation of our interests in the dehydrogenasebased enzymatic BFCs, we are currently searching for a new kind of redox dyes which processes a better electrocatalytic activity toward NADH oxidation than poly(methylene blue) used in our earlier attempt [42]. In the present work, we demonstrate a new kind of glucose/O2 BFC with glucose dehydrogenase (GDH) and bilirubin oxidase (BOD) as the anodic and cathodic biocatalysts, respectively, as schematically illustrated in Scheme 1. Instead of poly(methylene blue), poly(brilliant creysl blue) (BCB) is used here as the electrocatalyst for the oxidation of NADH because, BCB and MB both belong to the family of azine dyes with a p-conjugative structure. They can readily adsorb onto SWNTs to form stable dye-SWNT nanocomposites, of which the dye molecules can be electrochemically polymerized to give new kinds of polymer-SWNT nanocomposites. More importantly, as will be demonstrated later, the polyBCB exhibits a better electrocatalytic activity toward the oxidation of NADH than the polyMB and, as a consequence, the as-prepared GDH-based bioanode with the polyBCB as the electrocatalyst is envisaged to
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Scheme 1. (Upper panel) Schematic illustration of the SWNT-based glucose/O2 biofuel cell with polyBCB as the electrocatalyst for the oxidation of NADH. (Lower panel) Chemical structures of brilliant creysl blue (BCB) used in this study and methylene blue (MB) used previously.
F. Gao et al. / Electrochemistry Communications 9 (2007) 989–996
exhibit a higher potential output than that with the polyMB used previously as the electrocatalyst for NADH oxidation. Moreover, as one of the most important aspects of the enzymatic BFCs, the as-prepared glucose/O2 BFC is studied with respect to its performance in serum to further evaluate its potentiality as an in vivo power for bioelectronics in real biological systems. The study undertaken here has not been reported so far and could offer a new route to the development of new kinds of enzymatic BFCs with a high performance. 2. Experimental 2.1. Chemicals and reagents Single-walled carbon nanotubes (SWNTs, with an average diameter less than 2 nm and the length of about 50 lm) were purchased from Shenzhen Nanoport Co. Ltd. (Shenzhen, China). The SWNTs were purified by refluxing the as-received SWNTs in 2.6 M HNO3 for 10 h. Glucose dehydrogenase (GDH, E.C.1.1.1.47, from Aspergillus niger), bilirubin oxidase (BOD, E.C.1.10.3.2, from Myrothecium verrucaria), bovine serum albumin (BSA), D-(+)-glucose, brilliant creysl blue (BCB), and methylene blue (MB, structures shown in Scheme 1) were all obtained from Sigma. Reduced and oxidized forms of nicotinamide adenine dinucleotide (NADH and NAD+) were purchased from Aldrich. Other chemicals were at least analytical grade and used as received. All aqueous solutions were prepared with doubly distilled water. 2.2. Preparation of the bioanode and biocathode Glassy carbon electrodes (GC, 3-mm diameter, Bioanalytical Systems Inc.) were used as the substrate for fabricating the enzymatic glucose/O2 BFC. The electrodes were first polished with 0.3 and 0.05 lm alumina slurry on a polishing cloth and then sonicated in the acetone and distilled water each for 10 min. SWNTs were dispersed into N,Ndimethylformamide (DMF) to give a homogeneous suspension (1 mg/mL) under sonication. A 4 lL of the prepared suspension was dipped onto GC electrodes to obtain the SWNT-modified electrodes. After being air-dried, the SWNT-modified electrodes were immersed into the aqueous solution of BCB (0.4 mM) for 3 h. The electrodes (denoted as BCB/SWNT-modified electrodes) were then thoroughly rinsed with distilled water to remove the nonadsorbed BCB. The BCB/SWNT-modified electrodes were polarized at +0.80 V vs. Ag/AgCl in 0.10 M phosphate solution (pH 7.0) for 50 min for the polymerization of BCB to form a polyBCB-SWNT nanocomposite onto the electrodes. After that, the electrodes were taken out of the solution, thoroughly rinsed with distilled water, and air-dried. A 1% (wt.%) aqueous solution of BSA was mixed with the aqueous solution of GDH (15 mg/mL) with a volume ratio of 1:2 to give a GDH-BSA mixture and 6 lL of the resulting mixture was coated onto the polyBCB/
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SWNT-modified electrodes. A 2 lL of aqueous solution of glutaraldehyde (40 mM) was further coated onto the electrodes to cross-link the enzyme onto polyBCB-SWNT nanocomposite confined onto GC electrodes. The prepared electrodes were rinsed with distilled water, air-dried, and used as the GDH-polyBCB-SWNT bioanode for the oxidation of glucose in the glucose/O2 BFC. For comparison, a GDH-polyMB-SWNT bioanode was prepared with the procedures reported previously [42]. The biocathode of the glucose/O2 BFC was prepared by cross-linking the BOD-BSA (volume ratio, 1:2) onto the SWNT-modified electrodes with the same amount of glutaraldehyde as used for the GDH-BSA described above. After being rinsed with distilled water and air-dried, the electrodes were used as the BOD-SWNT biocathode for the reduction of O2 in the glucose/O2 BFC. 2.3. Apparatus and measurements Electrochemical measurements were performed on a computer-controlled electrochemical analyzer (CHI 660 A, Austin, USA). The prepared biocathode and bioanode were used as working electrode and platinum spiral wire was used as counter electrode. All potentials were referred to a KClsaturated Ag/AgCl electrode. A 0.10 M phosphate buffer (pH 7.0) was used as the supporting electrolyte. The glucose/O2 BFC was assembled by immersing the prepared bioanode and the biocathode into a 10-mL vessel (Scheme 1) and the cell performance was studied in 0.10 M phosphate solution (pH 7.0) containing 40 mM glucose and 10 mM NAD+ under ambient air. The performance of the assembled glucose/O2 biofuel cell was also evaluated in serum containing 10 mM NAD+. The serum was separated from the blood collected from a healthy person by centrifugating the blood at a speed of 4500 rpm for 20 min. The serum was diluted with the same volume of 0.10 M phosphate buffer before measurements. All electrochemical experiments were conducted at ambient temperature. 3. Results and discussion 3.1. GDH-polyBCB-SWNT bioanode for the oxidation of glucose Similar to methylene blue reported in our previous work [32], BCB was found also to be able to be stably adsorbed onto SWNTs to form a BCB-SWNT nanocomposite, possibly through the charge-transfer interaction between both components. However, a subsequent study on the oxidation of NADH at the BCB-SWNT nanocomposite reveals that the potential for the oxidation of NADH at the BCBSWNT nanocomposite (i.e., ca. 0.0 V) is more positive than the formal potential of BCB in the nanocomposite (i.e., 0.27 V) although this potential value is more negative compared with those both at bare GC electrode (i.e., +0.70 V) and pristine SWNTs confined onto GC electrode (i.e., +0.30 V) (data not shown). This comparison shows
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that the prepared BCB-SWNT nanocomposite does not exhibit an excellent electrocatalytic activity toward the oxidation of NADH. To improve the electrocatalytic activity of the BCB-SWNT nanocomposite, the BCB monomers entrapped into the SWNT nanocomposite were consequently electrochemically polymerized by polarizing the nanocomposite confined GC electrode at +0.80 V, since the polymers of the azine dyes have been reported to possess a better electrocatalytic activity toward the oxidation of NADH than the corresponding monomers [47,48]. Fig. 1 displays cyclic voltammograms (CVs) recorded at the BCB-SWNT nanocomposite confined onto GC electrode in 0.10 M phosphate buffer after the electrode was polarized for different time. As shown, the BCB-SWNT nanocomposite exhibits one pair of well-defined mirror-like redox waves at 0.27 V, which could be ascribed to the reversible redox process of BCB at SWNTs. Upon being poised at +0.80 V in the phosphate buffer, the as-formed nanocomposite shows a remarkable change in the voltammetric responses as shown in Fig. 1; the currents of the redox wave of the monomer were clearly decreased and, in the meantime, a pair of new redox wave at 0.11 V appeared with a very small peak-to-peak separation and with the peak currents increasing with prolonging the time employed for the electropolymerization. This redox wave could be attributed to the reversible redox process of the as-formed polyBCB at SWNTs. After the nanocomposite confined onto electrode was polarized for 50 min, the redox wave of the monomer was completely disappeared, suggesting a complete polymerization of BCB molecules in the nanocomposite to form a new kind of polyBCB-SWNT nanocomposite. Fig. 2a shows CVs for the oxidation of NADH at the formed polyBCB-SWNT nanocomposite confined onto GC electrode in 0.10 phosphate buffer. The presence of NADH into the solution clearly results in an increase in
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E / V vs. Ag/AgCl Fig. 1. CVs recorded at BCB-SWNT nanocomposite confined onto GC electrode in 0.10 M phosphate buffer (pH 7.0) after the nanocomposite was polarized at +0.80 V in 0.10 M phosphate buffer solution (pH 7.0) for different time of 0 (curve 1), 5 (curve 2), 15 (curve 3), 30 (curve 4), and 50 min (curve 5). Scan rate, 20 mV s1.
Fig. 2. (a) CVs obtained at the polyBCB-SWNT nanocomposite confined onto GC electrode in 0.10 M phosphate buffer in the absence (dotted line) and presence (solid line) of 2.5 mM NADH. Scan rate, 20 mV s1. (b) CVs obtained at the GDH-polyBCB-SWNT nanocomposite confined onto GC electrode in 0.10 M phosphate buffer containing 20 mM NAD+in the absence (dotted line) and presence (solid line) of 20 mM glucose. Scan rate, 20 mV s1. (c) Polarization curves at the GDHpolyBCB-SWNT bioanode in 0.10 M phosphate buffer (pH 7.0) containing 20 mM NAD+ and glucose with different concentrations of 20 (curve 1), 60 (curve 2), and 100 mM (curve 3). Dotted curve (4) represents the polarization curve at the GDH-polyMB-SWNT bioanode in 0.10 M phosphate buffer (pH 7.0) containing 20 mM NAD+ and 45 mM glucose. Scan rate, 1 mV s1.
the oxidation peak current and a decrease in the reversed reduction peak current of the polyBCB-SWNT nanocomposite. The potential for the oxidation of NADH at the pre-
F. Gao et al. / Electrochemistry Communications 9 (2007) 989–996
3.2. BOD-SWNT biocathode for the reduction of O2 in neutral pH Fig. 3a depicts CVs for O2 reduction at the BOD-SWNT biocathode prepared by cross-linking BOD onto pristine SWNT matrix confined onto GC electrode in 0.10 M phosphate buffer solution (pH 7.0). The O2 reduction at the BOD-SWNT biocathode commences at +0.50 V in the phosphate solution (pH 7.0), which is close to the redox potential of BOD at carbon nanotubes as reported previously [41]. This potential value is more positive than those obtained with the electron transfer of BOD being shuttled by redox mediators, revealing that a low overpotential is involved in O2 reduction at the prepared BOD-SWNT biocathode. As can be seen from the polarization curve displayed in Fig. 3b, the electrocatalytic reduction of O2 was observed at 0.53 V vs. Ag/AgCl (0.73 V vs. NHE), which is close to the thermodynamic equilibrium potential of E0O2 =H2 O (0.83 V vs. NHE at pH 7.0). The electrocatalytic current plateau reached ca. 28 lA/cm2 at 0.45 V under ambient air and ca. 102 lA/cm2 under O2-saturated atmosphere. As mentioned above, this direct electron communication between BOD and the SWNTs is highly expected for the development of the biocathodes with a high potential output because of a low overpotential involved in O2 reduction. 3.3. Cell performance Before assembling the as-prepared GDH-polyBCBSWNT bioanode and the BOD-SWNT biocathode to form
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pared polyBCB-SWNT nanocomposite (i.e., ca. 0.11 V) remains to be more negative than that at the BCB-SWNT nanocomposite, demonstrating that the polyBCB-SWNT nanocomposite show a better electrocatalytic activity toward the oxidation of NADH than the BCB-SWNT nanocomposite. Such an excellent electrocatalytic activity of the as-prepared polyBCB-SWNT nanocomposite toward the oxidation of NADH into NAD+ was subsequently exploited to develop a glucose dehydrogenase (GDH)-based bioanode for the oxidation of glucose biofuel with the mechanism shown in Scheme 1. As shown in Fig. 2b, the oxidation of glucose into gluconolacton occurs at a low potential of 0.11 V, which is close to that for the oxidation of NADH (Fig. 2a). At the prepared GDH-polyBCB-SWNT bioanode, the onset potential for the oxidation of glucose was observed at 0.25 V vs. Ag/AgCl and the current reached its maximum (i.e., 310 lA/cm2) at 0.05 V for 100 mM glucose, as shown in Fig. 2c. The onset potential achieved here is more negative than that at the GDH-polyMBSWNT bioanode demonstrated in our earlier work [42], as shown in curve 4, suggesting that the present uses of polyBCB instead of polyMB as the electrocatalyst for the oxidation of NADH endows the as-prepared GDH-polyBCB-SWNT bioanode with a high potential output.
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E / V vs. Ag/AgCl Fig. 3. (a) CVs obtained at BOD-SWNT biocathode in 0.10 M phosphate buffer (pH 7.0) saturated with N2(dotted line) or O2(solid line), scan rate, 10 mV s1. (b) Polarization curves of the BOD-SWNT biocathode for O2 reduction 0.10 M phosphate solution (pH 7.0) under ambient air (curve 1) and O2-saturated atmosphere (curve 2). Scan rate, 1 mV s1.
a glucose/O2 BFC, we studied the possible crossover between the prepared bioanode and biocathode, as shown in Fig. 4. The presence of O2 in solution did not result in an observable voltammetric response at the GDH-polyBCBSWNT bioanode (Fig. 4a). While the addition of glucose in solution did not produce a recordable voltammetric response at the BOD-SWNT biocathode (Fig. 4b). These demonstrate that the assembled SWNT-based glucose/O2 BFC suffers from little crossover between the prepared bioanode and biocathode. This property substantially makes it possible to fabricate a compartment-less glucose/O2 BFC in the present study. Fig. 5 displays the polarization curve and the relationship between the power density (P) with the current density (j) of the assembled SWNT-based glucose/O2 BFC in phosphate solution at pH 7.0. The open circuit voltage (OCV) of the BFC is ca. 0.73 V and the power density reaches 53.9 l W/cm2 at 0.50 V. When the cell operated continuously with an external loading of 1 MX resistance in a quiescent phosphate buffer (0.10 M, pH 7.0) containing 10 mM NAD+ and 40 mM glucose under ambient air, it lost 5% of its original power in the first 24 h and the power output dropped by 46% after a 7-day continuous work, as displayed in Fig. 6. The performance of the assembled
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Fig. 6. Stability of the assembled glucose/O2 biofuel cell as a function of time. The external load in the test was 1 MX. Other conditions are the same as those in Fig. 5. P0 represents the original power density of the glucose/O2 BFC and P represents the power density recorded in different time shown in the figure.
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Fig. 4. (a) CVs recorded at the GDH-polyBCB-SWNT bioanode in 0.10 M phosphate buffer saturated by N2 (solid line) or under ambient air (dotted line) containing 20 mM NAD+. Scan rate, 50 mV s1. (b) CVs recorded at the BOD-SWNT biocathode in 0.10 M N2-saturated phosphate buffer in the absence (dotted line) and presence (solid line) of 10 mM glucose. Scan rate, 10 mV s1.
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j Cell / A cm-2 Fig. 5. Polarization curve (d) of the assembled SWNT-based compartment-less glucose/O2 biofuel cell (Scheme 1) and the dependence of the power output (s) on the current density in the quiescent phosphate buffer (0.10 M, pH 7.0) containing 10 mM NAD+ and 40 mM glucose under O2saturated atmosphere.
SWNT-based glucose/O2 BFC is restricted to the current density of the BOD-SWNT biocathode. We are currently working on a new strategy to improve the performance of the biocathode for O2 reduction. Fig. 7 displays the performance of the assembled SWNT-based glucose/O2 BFC in serum containing 10 mM NAD+ under O2-saturated atmosphere. As shown in Fig. 7a, the open circuit voltage of the glucose/O2 BFC was 0.63 V and the power density was 5 l W/cm2 at 0.45 V. Compared with those of the BFC in the phosphate buffer containing 10 mM NAD+ and 40 mM glucose under O2-saturated atmosphere (Fig. 5), both the open circuit voltage and the power density of the BFC were decreased in serum, possibly due to the low concentration of glucose and the presence of chemical species, such as urate and ascorbic acid in the serum. It has been reported that urate could remarkably deactivate BOD, resulting in the decrease the enzyme activity [13,49]. While, as an independent experiment in 0.10 M phosphate buffer containing 10 mM NAD+ and 40 mM glucose under O2-saturated atmosphere, we have found the addition of ascorbic acid into the buffer leads to a clear decrease in the open circuit voltage of the glucose/O2 BFC (data not shown), indicating that ascorbic acid coexisting in the serum could lower the potential output of the BFC. These effects are essentially responsible for the poor stability and the low open circuit voltage of the assembled glucose/O2 BFC in serum as shown in Fig. 7. As can be seen from Fig. 7b and c, both the open circuit voltage (OCV) and the performance of the BFC were decreased as a function of time in the serum. Upon continuous operation for 13 h in the serum with an external load of 1 MX resistance, the glucose/O2 BFC lost its 35% power. These demonstrations imply that the maintenance of the enzyme activity and the avoidance of the potential crosstalk from the chemical species coexisting in the serum may constitute two of the key points to extend the enzymatic BFCs in real biological systems.
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electrocatalyst for the oxidation of NADH, the bioanode demonstrated here exhibits a more negative potential for the catalytic oxidation of NADH and thus for that of glucose biofuel. As a result, the prepared GDH-polyBCBSWNT bioanode has a high potential output. This study may offer a new strategy for the development of new kinds of enzymatic BFCs with a high performance and offer useful information for future studies on the enzymatic BFCs as in vivo power sources. Acknowledgements
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The present research was financially supported by NSF of China (Grant Nos. 20375043, 20435030 and 20575071), Chinese Academy of Sciences and Centre for Molecular Sciences, Institute of Chemistry. F. Gao thanks the financial support from the Key Project of Education Committee of Anhui Province (Grant No. 2007kj006A). L. Mao thanks NSF of China for Distinguished Young Scholars (Grant No. 20625515).
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Fig. 7. (a) Polarization curve (d) of the assembled SWNT-based compartment-less glucose/O2 biofuel cell (Scheme 1) and the dependence of the power output (s) on the current density in serum containing 10 mM NAD+ under O2-saturated atmosphere. (b) Open circuit voltage of the assembled glucose/O2 BFC in serum as a function of time. (c) Stability of the biofuel cell output as a function of time upon operation in serum with an external load of 1 MX resistance.
4. Conclusions By using poly(brilliant creysl blue) as the electrocatalyst for the catalytic oxidation of NADH, we have successfully developed a new kind of SWNT-based compartment-less glucose/O2 biofuel cell with GDH and BOD as the anodic and cathodic biocatalysts, respectively. Compared with the GDH-based bioanode with poly(methylene blue) as the
[1] S.C. Barton, J. Gallaway, P. Atanassov, Chem. Rev. 104 (2004) 4867. [2] R.A. Bullen, T.C. Arnot, J.B. Lakeman, F.C. Walsh, Biosens. Bioelectron. 21 (2006) 2015. [3] J. Kim, H.F. Jia, P. Wang, Biotechnol. Adv. 24 (2006) 296. [4] T. Ikeda, K. Kano, Biochim. Biophys. Acta 1647 (2003) 121. [5] F. Davis, S.P.J. Higson, Biosens. Bioelectron., in press. [6] N. Mano, F. Mao, A. Heller, J. Am. Chem. Soc. 124 (2002) 12962. [7] T. Chen, S.C. Barton, G. Binyamin, Z.Q. Gao, Y.C. Zhang, H.H. Kim, A. Heller, J. Am. Chem. Soc. 123 (2001) 8630. [8] I. Willner, Science 298 (2002) 2407. [9] R.F. Service, Science 296 (2002) 1223. [10] E. Katz, O. Lioubashevski, I. Willner, J. Am. Chem. Soc. 127 (2005) 3979. [11] E. Katz, I. Willner, J. Am. Chem. Soc. 125 (2003) 6803. [12] N. Mano, F. Mao, A. Heller, J. Am. Chem. Soc. 125 (2003) 6588. [13] A. Heller, Phys. Chem. Chem. Phys. 6 (2004) 209. [14] V. Soukharev, N. Mano, A. Heller, J. Am. Chem. Soc. 126 (2004) 8368. [15] G.T.R. Palmore, H. Bertschy, S.H. Bergens, G.M. Whitesides, J. Electroanal. Chem. 443 (1998) 155. [16] G.T.R. Palmore, H.H. Kim, J. Electroanal. Chem. 464 (1999) 110. [17] K.G. Lim, G.T.R. Palmore, Biosens. Bioelectron. 22 (2007) 941. [18] C.M. Moore, S.D. Minteer, R.S. Martin, Lab Chip 5 (2005) 218. [19] S. Topcagic, S.D. Minteer, Electrochim. Acta 51 (2006) 2168. [20] A. Ramanaviciusa, A. Kausaitea, A. Ramanavicienec, Biosens. Bioelectron. 20 (2005) 1962. [21] F. Sato, M. Togo, M.K. Islam, T. Matsue, J. Kosuge, N. Fukasaku, S. Kurosawa, M. Nishizawa, Electrochem. Commun. 7 (2005) 643. [22] D. Ivnitski, B. Branch, P. Atanassov, C. Apblett, Electrochem. Commun. 8 (2006) 1204. [23] F. Barrie`re, Y. Ferry, D. Rochefort, D. Leech, Electrochem. Commun. 6 (2004) 237. [24] Y. Liu, M.K. Wang, F. Zhao, B.F. Liu, S.J. Dong, Chem. Eur. J. 11 (2005) 4970. [25] P.M. Ajayan, Chem. Rev. 99 (1999) 1787. [26] D.M. Guldi, G.M. Rahman, F. Zerbetto, M. Parto, Accounts Chem. Res. 38 (2005) 871. [27] A. Hirsch, Angew. Chem. Int. Edit. 41 (2002) 1853. [28] S. Banerjee, T.H. Benny, S.S. Wong, Adv. Mater. 17 (2005) 17. [29] R.R. Moore, C.E. Banks, R.G. Compton, Anal. Chem. 76 (2004) 2677.
996
F. Gao et al. / Electrochemistry Communications 9 (2007) 989–996
[30] L. Qu, L. Dai, J. Am. Chem. Soc. 127 (2005) 10806. [31] C. Wei, L. Dai, A. Roy, T.B. Tolle, J. Am. Chem. Soc. 128 (2006) 1412. [32] Y. Yan, M. Zhang, K. Gong, L. Su, Z. Guo, L. Mao, Chem. Mater. 17 (2005) 3457. [33] K. Gong, X. Zhu, R. Zhao, S. Xiong, L. Mao, C. Chen, Anal. Chem. 77 (2005) 8158. [34] M. Zhang, K. Liu, K. Gong, L. Su, Y. Chen, L. Mao, Anal. Chem. 77 (2005) 6234. [35] D.M. Guldi, G.M.A. Rahman, N. Jux, D. Balbinot, U. Hartnagel, N. Tagmatarchis, M. Prato, J. Am. Chem. Soc. 127 (2005) 9830. [36] P.P. Joshi, S.A. Merchant, Y. Wang, D.W. Schmidtke, Anal. Chem. 77 (2005) 3183. [37] M. Zhang, A. Smith, W. Gorski, Anal. Chem. 76 (2004) 5045. [38] K. Besteman, J.O. Lee, F.G.M. Wiertz, H.A. Heering, C. Dekker, Nano Lett. 3 (2003) 727. [39] S. Hrapovic, Y. Liu, K.B. Male, J.H.T. Luong, Anal. Chem. 76 (2004) 1083.
[40] J.J. Davis, K.S. Coleman, B.R. Azamian, C.B. Bagshaw, M.L.H. Green, Chem. Eur. J. 9 (2003) 3732. [41] W. Zheng, Q. Li, L. Su, Y. Yan, J. Zhang, L. Mao, Electroanalysis 18 (2006) 587. [42] Y. Yan, W. Zheng, L. Su, L. Mao, Adv. Mater. 18 (2006) 2639. [43] Y. Yan, L. Su, L. Mao, J. Nanosci. Nanotechnol., in press. [44] M. Zhang, W. Gorski, J. Am. Chem. Soc. 127 (2005) 2058. [45] N. Yuhashia, M. Tomiyamab, J. Okudaa, S. Igarashia, K. Ikebukuroa, K. Sodea, Biosens. Bioelectron. 20 (2005) 2145. [46] N.L. Akers, C.M. Moore, S.D. Minteer, Electrochim. Acta 50 (2005) 2521. [47] A.A. Karyahin, E.E. Karyakina, W. Schuhmann, H.-L. Schmidt, Electroanalysis 11 (1999) 553. [48] A.A. Karyahin, E.E. Karyakina, H.-L. Schmidt, Electroanalysis 11 (1999) 149. [49] C. Kang, H. Shin, Y. Zhang, A. Heller, Bioelectrochemistry 68 (2006) 22.
An enzymatic glucose/O2 biofuel cell: Preparation, characterization and performance in serum Feng Gao a, Yiming Yan b, Lei Su b, Lun Wang a, Lanqun Mao a
b,*
Anhui Key Laboratory of Functional Molecular Solids, College of Chemistry and Materials Science, Anhui Normal University, Wuhu 241000, China b Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, The Chinese Academy of Sciences, Beijing 100080, China Received 22 November 2006; received in revised form 5 December 2006; accepted 5 December 2006 Available online 11 January 2007
Abstract This study demonstrates a new kind of single-walled carbon nanotubes (SWNT)-based compartment-less glucose/O2 biofuel cell (BFC) with glucose dehydrogenase (GDH) and bilirubin oxidase (BOD) as the anodic and cathodic biocatalysts, respectively, and with poly(brilliant creysl blue) (BCB) adsorbed onto SWNT nanocomposite as the electrocatalyst for the oxidation of NADH. The prepared GDH-polyBCB-SWNT bioanode exhibits an excellent electrocatalytic activity toward the oxidation of glucose biofuel; in 0.10 M phosphate buffer containing 20 mM NAD+ and 100 mM glucose, the oxidation of glucose commences at 0.25 V and the current reaches its maximum of 310 lA/cm2 at 0.05 V vs. Ag/AgCl. At the BOD-SWNT biocathode, a high potential output is achieved for the reduction of O2 due to the direct electron transfer property of BOD at the SWNTs. In 0.10 M phosphate buffer, the electrocatalytic reduction of O2 is observed at a high potential of 0.53 V vs. Ag/AgCl with an electrocatalytic current plateau of ca. 28 lA/cm2 at 0.45 V under ambient air and ca. 102 lA/cm2 under O2-saturated atmosphere. In 0.10 M phosphate buffer containing 10 mM NAD+ and 40 mM glucose under O2-saturated atmosphere, the power density of the assembled SWNT-based glucose/O2 BFC reaches 53.9 lW/cm2 at 0.50 V. The performance and the stability of the glucose/O2 BFC are also evaluated in serum. This study could offer a new route to the development of new kinds of enzymatic BFCs with a high performance and provide useful information on future studies on the enzymatic BFCs as in vivo power sources. Ó 2006 Elsevier B.V. All rights reserved. Keywords: Enzymatic biofuel cells; Bioelectrocatalysis; Glucose oxidation; O2 reduction
1. Introduction Considerable attention has been recently paid on enzymatic biofuel cells (BFCs) because they have been recognized as a new kind of energy conversion technologies that possess striking properties, such as operation in mild conditions, i.e., ambient temperature and neutral pH, and potentiality to be used as in vivo power sources for bioelectronics including micropumps, pacemakers, neuromorphic circuits and so forth [1–5]. To date, much effort has been devoted to the key issues that have to be properly addressed in the transition of the enzymatic BFCs from *
Corresponding author. Tel.: +86 10 62646525; fax: +86 10 62559373. E-mail address: [email protected] (L. Mao).
1388-2481/$ - see front matter Ó 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2006.12.008
theoretical concepts to practical devices [6–11]. These include establishment of new bioelectrocatalytic systems, stable immobilization of the electrocatalysts and biocatalysts into a three-dimensional conducting matrix, improvement of the power output and stability, and examination of the cell performance in real biological samples to evaluate the potentiality of the BFCs as in vivo power sources [12,13]. To this end, several new kinds of electrochemical concepts and assembled enzymatic BFCs have been demonstrated in recent a few years [14–24]. Our approaches to development of new kinds of enzymatic BFCs are essentially based on the uses of carbon nanotubes (CNTs) because, as a new kind of carbon-based nanostructures, CNTs have been proved to possess unique electronic and structural properties [25–29], which substan-
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tially enable them to be well competent both as electrode materials and as conducting matrix to accommodate the electrocatalysts (e.g., redox dyes) and biocatalysts (i.e., enzymes and proteins) used in the enzymatic BFCs [30– 40]. More remarkably, we have recently found that the uses of CNTs could largely facilitate direct electron transfer of blue multi-copper oxidases (e.g., laccase and bilirubin oxidase) that have been frequently used as the biocatalysts of the biocathodes in the enzymatic BFCs [41–43]. Such a property of CNTs essentially makes it possible for the catalytic reduction of O2 to occur at a more positive potential than those with electron transfer of blue multi-copper oxidases being mediated by redox mediators, resulting in a low potential loss in the as-prepared biocathodes. On the other hand, the uses of CNTs have been found to be also useful for the development of the dehydrogenase-based bioanodes in the enzymatic BFCs in terms of their capability to be used to accommodate the electrocatalysts for the oxidation of NADH [37,42,44]. It is known that at the dehydrogenase-based bioanodes the potentials for the oxidation of biofuels are essentially determined by the kinetics of the oxidation of NADH, in which the oxidized form of NADH, i.e., NAD+, is generally used as the cofactor for the dehydrogenases in the bioelectrocatalytic oxidation of biofuels [15,19,42,45,46]. As demonstrated previously, some kinds of organic compounds with p-conjugative structures, such as redox dyes, can interact with CNTs with p-stacking interactions to form a three-dimensional and redox-active CNT nanocomposite [32,44]. Such a property of the redox dyes and CNTs essentially makes it possible to prepare a conducting and stable matrix containing redox-active dyes that could be used as
the electrocatalysts for the oxidation of NADH and thereby for the development of new kinds of nanostructured dehydrogenase-based BFCs. In our previous attempts [32,42], we have prepared a methylene blue-single walled carbon nanotubes (MB-SWNT) nanocomposite through the noncovalent adsorption of MB onto SWNTs and found that, after being electrochemically polymerized, the nanocomposite (i.e., polyMB-SWNT) exhibits an excellent electrocatalytic activity toward the oxidation of NADH. Such a property has been used for developing a new kind of glucose/O2 BFC. As a continuation of our interests in the dehydrogenasebased enzymatic BFCs, we are currently searching for a new kind of redox dyes which processes a better electrocatalytic activity toward NADH oxidation than poly(methylene blue) used in our earlier attempt [42]. In the present work, we demonstrate a new kind of glucose/O2 BFC with glucose dehydrogenase (GDH) and bilirubin oxidase (BOD) as the anodic and cathodic biocatalysts, respectively, as schematically illustrated in Scheme 1. Instead of poly(methylene blue), poly(brilliant creysl blue) (BCB) is used here as the electrocatalyst for the oxidation of NADH because, BCB and MB both belong to the family of azine dyes with a p-conjugative structure. They can readily adsorb onto SWNTs to form stable dye-SWNT nanocomposites, of which the dye molecules can be electrochemically polymerized to give new kinds of polymer-SWNT nanocomposites. More importantly, as will be demonstrated later, the polyBCB exhibits a better electrocatalytic activity toward the oxidation of NADH than the polyMB and, as a consequence, the as-prepared GDH-based bioanode with the polyBCB as the electrocatalyst is envisaged to
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Scheme 1. (Upper panel) Schematic illustration of the SWNT-based glucose/O2 biofuel cell with polyBCB as the electrocatalyst for the oxidation of NADH. (Lower panel) Chemical structures of brilliant creysl blue (BCB) used in this study and methylene blue (MB) used previously.
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exhibit a higher potential output than that with the polyMB used previously as the electrocatalyst for NADH oxidation. Moreover, as one of the most important aspects of the enzymatic BFCs, the as-prepared glucose/O2 BFC is studied with respect to its performance in serum to further evaluate its potentiality as an in vivo power for bioelectronics in real biological systems. The study undertaken here has not been reported so far and could offer a new route to the development of new kinds of enzymatic BFCs with a high performance. 2. Experimental 2.1. Chemicals and reagents Single-walled carbon nanotubes (SWNTs, with an average diameter less than 2 nm and the length of about 50 lm) were purchased from Shenzhen Nanoport Co. Ltd. (Shenzhen, China). The SWNTs were purified by refluxing the as-received SWNTs in 2.6 M HNO3 for 10 h. Glucose dehydrogenase (GDH, E.C.1.1.1.47, from Aspergillus niger), bilirubin oxidase (BOD, E.C.1.10.3.2, from Myrothecium verrucaria), bovine serum albumin (BSA), D-(+)-glucose, brilliant creysl blue (BCB), and methylene blue (MB, structures shown in Scheme 1) were all obtained from Sigma. Reduced and oxidized forms of nicotinamide adenine dinucleotide (NADH and NAD+) were purchased from Aldrich. Other chemicals were at least analytical grade and used as received. All aqueous solutions were prepared with doubly distilled water. 2.2. Preparation of the bioanode and biocathode Glassy carbon electrodes (GC, 3-mm diameter, Bioanalytical Systems Inc.) were used as the substrate for fabricating the enzymatic glucose/O2 BFC. The electrodes were first polished with 0.3 and 0.05 lm alumina slurry on a polishing cloth and then sonicated in the acetone and distilled water each for 10 min. SWNTs were dispersed into N,Ndimethylformamide (DMF) to give a homogeneous suspension (1 mg/mL) under sonication. A 4 lL of the prepared suspension was dipped onto GC electrodes to obtain the SWNT-modified electrodes. After being air-dried, the SWNT-modified electrodes were immersed into the aqueous solution of BCB (0.4 mM) for 3 h. The electrodes (denoted as BCB/SWNT-modified electrodes) were then thoroughly rinsed with distilled water to remove the nonadsorbed BCB. The BCB/SWNT-modified electrodes were polarized at +0.80 V vs. Ag/AgCl in 0.10 M phosphate solution (pH 7.0) for 50 min for the polymerization of BCB to form a polyBCB-SWNT nanocomposite onto the electrodes. After that, the electrodes were taken out of the solution, thoroughly rinsed with distilled water, and air-dried. A 1% (wt.%) aqueous solution of BSA was mixed with the aqueous solution of GDH (15 mg/mL) with a volume ratio of 1:2 to give a GDH-BSA mixture and 6 lL of the resulting mixture was coated onto the polyBCB/
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SWNT-modified electrodes. A 2 lL of aqueous solution of glutaraldehyde (40 mM) was further coated onto the electrodes to cross-link the enzyme onto polyBCB-SWNT nanocomposite confined onto GC electrodes. The prepared electrodes were rinsed with distilled water, air-dried, and used as the GDH-polyBCB-SWNT bioanode for the oxidation of glucose in the glucose/O2 BFC. For comparison, a GDH-polyMB-SWNT bioanode was prepared with the procedures reported previously [42]. The biocathode of the glucose/O2 BFC was prepared by cross-linking the BOD-BSA (volume ratio, 1:2) onto the SWNT-modified electrodes with the same amount of glutaraldehyde as used for the GDH-BSA described above. After being rinsed with distilled water and air-dried, the electrodes were used as the BOD-SWNT biocathode for the reduction of O2 in the glucose/O2 BFC. 2.3. Apparatus and measurements Electrochemical measurements were performed on a computer-controlled electrochemical analyzer (CHI 660 A, Austin, USA). The prepared biocathode and bioanode were used as working electrode and platinum spiral wire was used as counter electrode. All potentials were referred to a KClsaturated Ag/AgCl electrode. A 0.10 M phosphate buffer (pH 7.0) was used as the supporting electrolyte. The glucose/O2 BFC was assembled by immersing the prepared bioanode and the biocathode into a 10-mL vessel (Scheme 1) and the cell performance was studied in 0.10 M phosphate solution (pH 7.0) containing 40 mM glucose and 10 mM NAD+ under ambient air. The performance of the assembled glucose/O2 biofuel cell was also evaluated in serum containing 10 mM NAD+. The serum was separated from the blood collected from a healthy person by centrifugating the blood at a speed of 4500 rpm for 20 min. The serum was diluted with the same volume of 0.10 M phosphate buffer before measurements. All electrochemical experiments were conducted at ambient temperature. 3. Results and discussion 3.1. GDH-polyBCB-SWNT bioanode for the oxidation of glucose Similar to methylene blue reported in our previous work [32], BCB was found also to be able to be stably adsorbed onto SWNTs to form a BCB-SWNT nanocomposite, possibly through the charge-transfer interaction between both components. However, a subsequent study on the oxidation of NADH at the BCB-SWNT nanocomposite reveals that the potential for the oxidation of NADH at the BCBSWNT nanocomposite (i.e., ca. 0.0 V) is more positive than the formal potential of BCB in the nanocomposite (i.e., 0.27 V) although this potential value is more negative compared with those both at bare GC electrode (i.e., +0.70 V) and pristine SWNTs confined onto GC electrode (i.e., +0.30 V) (data not shown). This comparison shows
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that the prepared BCB-SWNT nanocomposite does not exhibit an excellent electrocatalytic activity toward the oxidation of NADH. To improve the electrocatalytic activity of the BCB-SWNT nanocomposite, the BCB monomers entrapped into the SWNT nanocomposite were consequently electrochemically polymerized by polarizing the nanocomposite confined GC electrode at +0.80 V, since the polymers of the azine dyes have been reported to possess a better electrocatalytic activity toward the oxidation of NADH than the corresponding monomers [47,48]. Fig. 1 displays cyclic voltammograms (CVs) recorded at the BCB-SWNT nanocomposite confined onto GC electrode in 0.10 M phosphate buffer after the electrode was polarized for different time. As shown, the BCB-SWNT nanocomposite exhibits one pair of well-defined mirror-like redox waves at 0.27 V, which could be ascribed to the reversible redox process of BCB at SWNTs. Upon being poised at +0.80 V in the phosphate buffer, the as-formed nanocomposite shows a remarkable change in the voltammetric responses as shown in Fig. 1; the currents of the redox wave of the monomer were clearly decreased and, in the meantime, a pair of new redox wave at 0.11 V appeared with a very small peak-to-peak separation and with the peak currents increasing with prolonging the time employed for the electropolymerization. This redox wave could be attributed to the reversible redox process of the as-formed polyBCB at SWNTs. After the nanocomposite confined onto electrode was polarized for 50 min, the redox wave of the monomer was completely disappeared, suggesting a complete polymerization of BCB molecules in the nanocomposite to form a new kind of polyBCB-SWNT nanocomposite. Fig. 2a shows CVs for the oxidation of NADH at the formed polyBCB-SWNT nanocomposite confined onto GC electrode in 0.10 phosphate buffer. The presence of NADH into the solution clearly results in an increase in
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Fig. 2. (a) CVs obtained at the polyBCB-SWNT nanocomposite confined onto GC electrode in 0.10 M phosphate buffer in the absence (dotted line) and presence (solid line) of 2.5 mM NADH. Scan rate, 20 mV s1. (b) CVs obtained at the GDH-polyBCB-SWNT nanocomposite confined onto GC electrode in 0.10 M phosphate buffer containing 20 mM NAD+in the absence (dotted line) and presence (solid line) of 20 mM glucose. Scan rate, 20 mV s1. (c) Polarization curves at the GDHpolyBCB-SWNT bioanode in 0.10 M phosphate buffer (pH 7.0) containing 20 mM NAD+ and glucose with different concentrations of 20 (curve 1), 60 (curve 2), and 100 mM (curve 3). Dotted curve (4) represents the polarization curve at the GDH-polyMB-SWNT bioanode in 0.10 M phosphate buffer (pH 7.0) containing 20 mM NAD+ and 45 mM glucose. Scan rate, 1 mV s1.
the oxidation peak current and a decrease in the reversed reduction peak current of the polyBCB-SWNT nanocomposite. The potential for the oxidation of NADH at the pre-
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3.2. BOD-SWNT biocathode for the reduction of O2 in neutral pH Fig. 3a depicts CVs for O2 reduction at the BOD-SWNT biocathode prepared by cross-linking BOD onto pristine SWNT matrix confined onto GC electrode in 0.10 M phosphate buffer solution (pH 7.0). The O2 reduction at the BOD-SWNT biocathode commences at +0.50 V in the phosphate solution (pH 7.0), which is close to the redox potential of BOD at carbon nanotubes as reported previously [41]. This potential value is more positive than those obtained with the electron transfer of BOD being shuttled by redox mediators, revealing that a low overpotential is involved in O2 reduction at the prepared BOD-SWNT biocathode. As can be seen from the polarization curve displayed in Fig. 3b, the electrocatalytic reduction of O2 was observed at 0.53 V vs. Ag/AgCl (0.73 V vs. NHE), which is close to the thermodynamic equilibrium potential of E0O2 =H2 O (0.83 V vs. NHE at pH 7.0). The electrocatalytic current plateau reached ca. 28 lA/cm2 at 0.45 V under ambient air and ca. 102 lA/cm2 under O2-saturated atmosphere. As mentioned above, this direct electron communication between BOD and the SWNTs is highly expected for the development of the biocathodes with a high potential output because of a low overpotential involved in O2 reduction. 3.3. Cell performance Before assembling the as-prepared GDH-polyBCBSWNT bioanode and the BOD-SWNT biocathode to form
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pared polyBCB-SWNT nanocomposite (i.e., ca. 0.11 V) remains to be more negative than that at the BCB-SWNT nanocomposite, demonstrating that the polyBCB-SWNT nanocomposite show a better electrocatalytic activity toward the oxidation of NADH than the BCB-SWNT nanocomposite. Such an excellent electrocatalytic activity of the as-prepared polyBCB-SWNT nanocomposite toward the oxidation of NADH into NAD+ was subsequently exploited to develop a glucose dehydrogenase (GDH)-based bioanode for the oxidation of glucose biofuel with the mechanism shown in Scheme 1. As shown in Fig. 2b, the oxidation of glucose into gluconolacton occurs at a low potential of 0.11 V, which is close to that for the oxidation of NADH (Fig. 2a). At the prepared GDH-polyBCB-SWNT bioanode, the onset potential for the oxidation of glucose was observed at 0.25 V vs. Ag/AgCl and the current reached its maximum (i.e., 310 lA/cm2) at 0.05 V for 100 mM glucose, as shown in Fig. 2c. The onset potential achieved here is more negative than that at the GDH-polyMBSWNT bioanode demonstrated in our earlier work [42], as shown in curve 4, suggesting that the present uses of polyBCB instead of polyMB as the electrocatalyst for the oxidation of NADH endows the as-prepared GDH-polyBCB-SWNT bioanode with a high potential output.
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a glucose/O2 BFC, we studied the possible crossover between the prepared bioanode and biocathode, as shown in Fig. 4. The presence of O2 in solution did not result in an observable voltammetric response at the GDH-polyBCBSWNT bioanode (Fig. 4a). While the addition of glucose in solution did not produce a recordable voltammetric response at the BOD-SWNT biocathode (Fig. 4b). These demonstrate that the assembled SWNT-based glucose/O2 BFC suffers from little crossover between the prepared bioanode and biocathode. This property substantially makes it possible to fabricate a compartment-less glucose/O2 BFC in the present study. Fig. 5 displays the polarization curve and the relationship between the power density (P) with the current density (j) of the assembled SWNT-based glucose/O2 BFC in phosphate solution at pH 7.0. The open circuit voltage (OCV) of the BFC is ca. 0.73 V and the power density reaches 53.9 l W/cm2 at 0.50 V. When the cell operated continuously with an external loading of 1 MX resistance in a quiescent phosphate buffer (0.10 M, pH 7.0) containing 10 mM NAD+ and 40 mM glucose under ambient air, it lost 5% of its original power in the first 24 h and the power output dropped by 46% after a 7-day continuous work, as displayed in Fig. 6. The performance of the assembled
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Fig. 6. Stability of the assembled glucose/O2 biofuel cell as a function of time. The external load in the test was 1 MX. Other conditions are the same as those in Fig. 5. P0 represents the original power density of the glucose/O2 BFC and P represents the power density recorded in different time shown in the figure.
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Fig. 4. (a) CVs recorded at the GDH-polyBCB-SWNT bioanode in 0.10 M phosphate buffer saturated by N2 (solid line) or under ambient air (dotted line) containing 20 mM NAD+. Scan rate, 50 mV s1. (b) CVs recorded at the BOD-SWNT biocathode in 0.10 M N2-saturated phosphate buffer in the absence (dotted line) and presence (solid line) of 10 mM glucose. Scan rate, 10 mV s1.
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j Cell / A cm-2 Fig. 5. Polarization curve (d) of the assembled SWNT-based compartment-less glucose/O2 biofuel cell (Scheme 1) and the dependence of the power output (s) on the current density in the quiescent phosphate buffer (0.10 M, pH 7.0) containing 10 mM NAD+ and 40 mM glucose under O2saturated atmosphere.
SWNT-based glucose/O2 BFC is restricted to the current density of the BOD-SWNT biocathode. We are currently working on a new strategy to improve the performance of the biocathode for O2 reduction. Fig. 7 displays the performance of the assembled SWNT-based glucose/O2 BFC in serum containing 10 mM NAD+ under O2-saturated atmosphere. As shown in Fig. 7a, the open circuit voltage of the glucose/O2 BFC was 0.63 V and the power density was 5 l W/cm2 at 0.45 V. Compared with those of the BFC in the phosphate buffer containing 10 mM NAD+ and 40 mM glucose under O2-saturated atmosphere (Fig. 5), both the open circuit voltage and the power density of the BFC were decreased in serum, possibly due to the low concentration of glucose and the presence of chemical species, such as urate and ascorbic acid in the serum. It has been reported that urate could remarkably deactivate BOD, resulting in the decrease the enzyme activity [13,49]. While, as an independent experiment in 0.10 M phosphate buffer containing 10 mM NAD+ and 40 mM glucose under O2-saturated atmosphere, we have found the addition of ascorbic acid into the buffer leads to a clear decrease in the open circuit voltage of the glucose/O2 BFC (data not shown), indicating that ascorbic acid coexisting in the serum could lower the potential output of the BFC. These effects are essentially responsible for the poor stability and the low open circuit voltage of the assembled glucose/O2 BFC in serum as shown in Fig. 7. As can be seen from Fig. 7b and c, both the open circuit voltage (OCV) and the performance of the BFC were decreased as a function of time in the serum. Upon continuous operation for 13 h in the serum with an external load of 1 MX resistance, the glucose/O2 BFC lost its 35% power. These demonstrations imply that the maintenance of the enzyme activity and the avoidance of the potential crosstalk from the chemical species coexisting in the serum may constitute two of the key points to extend the enzymatic BFCs in real biological systems.
F. Gao et al. / Electrochemistry Communications 9 (2007) 989–996
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electrocatalyst for the oxidation of NADH, the bioanode demonstrated here exhibits a more negative potential for the catalytic oxidation of NADH and thus for that of glucose biofuel. As a result, the prepared GDH-polyBCBSWNT bioanode has a high potential output. This study may offer a new strategy for the development of new kinds of enzymatic BFCs with a high performance and offer useful information for future studies on the enzymatic BFCs as in vivo power sources. Acknowledgements
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The present research was financially supported by NSF of China (Grant Nos. 20375043, 20435030 and 20575071), Chinese Academy of Sciences and Centre for Molecular Sciences, Institute of Chemistry. F. Gao thanks the financial support from the Key Project of Education Committee of Anhui Province (Grant No. 2007kj006A). L. Mao thanks NSF of China for Distinguished Young Scholars (Grant No. 20625515).
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Fig. 7. (a) Polarization curve (d) of the assembled SWNT-based compartment-less glucose/O2 biofuel cell (Scheme 1) and the dependence of the power output (s) on the current density in serum containing 10 mM NAD+ under O2-saturated atmosphere. (b) Open circuit voltage of the assembled glucose/O2 BFC in serum as a function of time. (c) Stability of the biofuel cell output as a function of time upon operation in serum with an external load of 1 MX resistance.
4. Conclusions By using poly(brilliant creysl blue) as the electrocatalyst for the catalytic oxidation of NADH, we have successfully developed a new kind of SWNT-based compartment-less glucose/O2 biofuel cell with GDH and BOD as the anodic and cathodic biocatalysts, respectively. Compared with the GDH-based bioanode with poly(methylene blue) as the
[1] S.C. Barton, J. Gallaway, P. Atanassov, Chem. Rev. 104 (2004) 4867. [2] R.A. Bullen, T.C. Arnot, J.B. Lakeman, F.C. Walsh, Biosens. Bioelectron. 21 (2006) 2015. [3] J. Kim, H.F. Jia, P. Wang, Biotechnol. Adv. 24 (2006) 296. [4] T. Ikeda, K. Kano, Biochim. Biophys. Acta 1647 (2003) 121. [5] F. Davis, S.P.J. Higson, Biosens. Bioelectron., in press. [6] N. Mano, F. Mao, A. Heller, J. Am. Chem. Soc. 124 (2002) 12962. [7] T. Chen, S.C. Barton, G. Binyamin, Z.Q. Gao, Y.C. Zhang, H.H. Kim, A. Heller, J. Am. Chem. Soc. 123 (2001) 8630. [8] I. Willner, Science 298 (2002) 2407. [9] R.F. Service, Science 296 (2002) 1223. [10] E. Katz, O. Lioubashevski, I. Willner, J. Am. Chem. Soc. 127 (2005) 3979. [11] E. Katz, I. Willner, J. Am. Chem. Soc. 125 (2003) 6803. [12] N. Mano, F. Mao, A. Heller, J. Am. Chem. Soc. 125 (2003) 6588. [13] A. Heller, Phys. Chem. Chem. Phys. 6 (2004) 209. [14] V. Soukharev, N. Mano, A. Heller, J. Am. Chem. Soc. 126 (2004) 8368. [15] G.T.R. Palmore, H. Bertschy, S.H. Bergens, G.M. Whitesides, J. Electroanal. Chem. 443 (1998) 155. [16] G.T.R. Palmore, H.H. Kim, J. Electroanal. Chem. 464 (1999) 110. [17] K.G. Lim, G.T.R. Palmore, Biosens. Bioelectron. 22 (2007) 941. [18] C.M. Moore, S.D. Minteer, R.S. Martin, Lab Chip 5 (2005) 218. [19] S. Topcagic, S.D. Minteer, Electrochim. Acta 51 (2006) 2168. [20] A. Ramanaviciusa, A. Kausaitea, A. Ramanavicienec, Biosens. Bioelectron. 20 (2005) 1962. [21] F. Sato, M. Togo, M.K. Islam, T. Matsue, J. Kosuge, N. Fukasaku, S. Kurosawa, M. Nishizawa, Electrochem. Commun. 7 (2005) 643. [22] D. Ivnitski, B. Branch, P. Atanassov, C. Apblett, Electrochem. Commun. 8 (2006) 1204. [23] F. Barrie`re, Y. Ferry, D. Rochefort, D. Leech, Electrochem. Commun. 6 (2004) 237. [24] Y. Liu, M.K. Wang, F. Zhao, B.F. Liu, S.J. Dong, Chem. Eur. J. 11 (2005) 4970. [25] P.M. Ajayan, Chem. Rev. 99 (1999) 1787. [26] D.M. Guldi, G.M. Rahman, F. Zerbetto, M. Parto, Accounts Chem. Res. 38 (2005) 871. [27] A. Hirsch, Angew. Chem. Int. Edit. 41 (2002) 1853. [28] S. Banerjee, T.H. Benny, S.S. Wong, Adv. Mater. 17 (2005) 17. [29] R.R. Moore, C.E. Banks, R.G. Compton, Anal. Chem. 76 (2004) 2677.
996
F. Gao et al. / Electrochemistry Communications 9 (2007) 989–996
[30] L. Qu, L. Dai, J. Am. Chem. Soc. 127 (2005) 10806. [31] C. Wei, L. Dai, A. Roy, T.B. Tolle, J. Am. Chem. Soc. 128 (2006) 1412. [32] Y. Yan, M. Zhang, K. Gong, L. Su, Z. Guo, L. Mao, Chem. Mater. 17 (2005) 3457. [33] K. Gong, X. Zhu, R. Zhao, S. Xiong, L. Mao, C. Chen, Anal. Chem. 77 (2005) 8158. [34] M. Zhang, K. Liu, K. Gong, L. Su, Y. Chen, L. Mao, Anal. Chem. 77 (2005) 6234. [35] D.M. Guldi, G.M.A. Rahman, N. Jux, D. Balbinot, U. Hartnagel, N. Tagmatarchis, M. Prato, J. Am. Chem. Soc. 127 (2005) 9830. [36] P.P. Joshi, S.A. Merchant, Y. Wang, D.W. Schmidtke, Anal. Chem. 77 (2005) 3183. [37] M. Zhang, A. Smith, W. Gorski, Anal. Chem. 76 (2004) 5045. [38] K. Besteman, J.O. Lee, F.G.M. Wiertz, H.A. Heering, C. Dekker, Nano Lett. 3 (2003) 727. [39] S. Hrapovic, Y. Liu, K.B. Male, J.H.T. Luong, Anal. Chem. 76 (2004) 1083.
[40] J.J. Davis, K.S. Coleman, B.R. Azamian, C.B. Bagshaw, M.L.H. Green, Chem. Eur. J. 9 (2003) 3732. [41] W. Zheng, Q. Li, L. Su, Y. Yan, J. Zhang, L. Mao, Electroanalysis 18 (2006) 587. [42] Y. Yan, W. Zheng, L. Su, L. Mao, Adv. Mater. 18 (2006) 2639. [43] Y. Yan, L. Su, L. Mao, J. Nanosci. Nanotechnol., in press. [44] M. Zhang, W. Gorski, J. Am. Chem. Soc. 127 (2005) 2058. [45] N. Yuhashia, M. Tomiyamab, J. Okudaa, S. Igarashia, K. Ikebukuroa, K. Sodea, Biosens. Bioelectron. 20 (2005) 2145. [46] N.L. Akers, C.M. Moore, S.D. Minteer, Electrochim. Acta 50 (2005) 2521. [47] A.A. Karyahin, E.E. Karyakina, W. Schuhmann, H.-L. Schmidt, Electroanalysis 11 (1999) 553. [48] A.A. Karyahin, E.E. Karyakina, H.-L. Schmidt, Electroanalysis 11 (1999) 149. [49] C. Kang, H. Shin, Y. Zhang, A. Heller, Bioelectrochemistry 68 (2006) 22.