Biocatalytic Carbon Paste Sensors Based on a Mediator Pasting

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Anal. Chem. 2004, 76, 3735-3739

Biocatalytic Carbon Paste Sensors Based on a Mediator Pasting Liquid Nathan S. Lawrence, Randhir P. Deo, and Joseph Wang*

Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico 88003

The preparation and advantages of a new generation of carbon paste enzyme electrodes where the redox mediator acts also as the pasting liquid are described. The mediator pasting liquid concept is illustrated for amperometric biosensing of glucose in connection with either the tertpentylferrocene or n-butylferrocene mediator/binder along with the glucose oxidase enzyme. The attractive performance and advantages of the new device is indicated from comparison to a conventional carbon paste biosensor using a mineral oil binder and the dimethylferrocene electron acceptor. The simplified preparation of the biosensor is coupled with a greatly improved sensitivity and an extended linear range. The mediator pasting liquid imparts high thermal stability onto the embedded enzyme and leads to good resistance to oxygen effects. Owing to the huge mediator reservoir, stability problems associated with the leaching of the mediator are greatly reduced. The fundamental aspects of the electrode behavior have been examined first in the absence of the enzyme. Variables affecting the performance of the new carbon paste biosensor have been investigated and optimized. Such use of the electron acceptor as a binder as well as the mediator offers considerable promise for the biosensing of numerous analytes of clinical and environmental significance. Amperometric biosensors based on the incorporation of an enzyme within a carbon paste matrix have gained considerable attention.1-3 Such devices offer several important advantages, including renewability, versatility, controlled bulk modification, low background current, internal oxygen supply, and high stability.2,3 Carbon pastes have thus been widely used as hosts for a wide range of oxidase and dehydrogenase enzymes. These bioelectrodes are commonly prepared by dispersing graphite powder (along with the enzyme and mediator) within a waterimmiscible, nonconducting pasting liquid. The mediator, added at 1-5 wt % level, acts as a nonphysiological electron acceptor that shuttles electrons between the redox center of the enzyme and the electrode surface.4-6 * To whom correspondence should be addressed. Tel: 1-505-646-2140. E-mail: [email protected]. (1) Gorton, L. Electroanalysis 1995, 7, 23. (2) Wang, J.; Fang, L. J. Am. Chem. Soc. 1998, 120, 1049. (3) Kulys, J. Biosens. Bioelectron. 1999, 14, 473. (4) Frew, J.; Hill, H. A. Anal. Chem. 1987, 59, 933A. (5) Wang, J. Electroanalysis 2001, 13, 983. (6) Ghindilis, A.; Atanasov, P.; Wilkins, E. Electroanalysis 1997, 9, 661. 10.1021/ac049943v CCC: $27.50 Published on Web 05/13/2004

© 2004 American Chemical Society

This article reports on a new strategy for preparing carbon paste enzyme electrodes in which the redox mediator acts also as the pasting liquid. Our findings indicate that the elimination of the traditional oil binder actually leads to several distinct advantages, including a substantially higher sensitivity, a significantly wider linear dynamic range, higher stability, and a greatly simplified preparation. Since the mediator serves also as the binder, the resulting bioelectrodes are fundamentally different from commonly used carbon paste biosensors. The new concept is illustrated for amperometric biosensing of glucose in connection with the tert-pentylferrocene (t-PFc) and n-butylferrocene (n-BuFc) mediators/binders. Ferrocene derivatives represent one of the major groups of mediators owing to their attractive redox properties and fast reaction with the reduced enzyme.5 A variety of ferrocene-based carbon paste enzyme electrodes have thus been developed.7-10 The preparation, characterization, and attractive performance of the new alkylferrocene-based biocomposites are illustrated in the following sections. EXPERIMENTAL SECTION Apparatus. Cyclic voltammetric measurements were conducted using a PGSTAT12 computer-controlled potentiostat (EcoChemie BV, Utrecht, The Netherlands), and a Bioanalytical Systems (BAS) CV-27 voltammograph, in connection with a BAS X-Y recorder, were used for the amperometric experiments. A standard three-electrode configuration was employed along with a typical 15-mL cell. A carbon paste disk (see below) served as the working electrode with platinum wire as the counter electrode and a Ag/AgCl (3 M KCl, model CHI111, CH Instruments, Austin, TX) reference electrode. Reagents. Glucose oxidase (EC 1.1.3.4, Type X-S from Aspergillus niger, 157 500 units/g solid), β-D(+)-glucose, tertpentylferrocene, n-butylferrocene, 1,1-dimethylferrocene (DMFc), KH2PO4, K2HPO4, mineral oil (all from Aldrich), uric acid, acetaminophen, ascorbic acid, and stearic acid (Sigma) were used without further purification. The glucose stock solution (2 M in water) was used at least 24 h after its preparation. The graphite powder (Grade 38, 1-2 µm) was purchased from Fisher Scientific (7) Wang, J.; Wu, L. H.; Lu, Z.; Li, R.; Sanchez, J. Anal. Chim. Acta 1990, 228, 251. (8) Szuwarski, N.; Gueguen, S.; Boujtita, M.; Murr, N. E. Electroanalysis 2001, 13, 1237. (9) Smolander, M.; Gorton, L.; Lee, H. S.; Skotheim, T.; Lan, H. L. Electroanalysis 1995, 7, 941. (10) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. O.; Aston, W. J.; Higgins; I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56, 667.

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(Fair Lawn, NJ). Deoxygenation of the cell chamber was carried out using helium (Air Liquide America Corp., Houston, TX). Preparation of the Paste Electrodes. The conventional carbon paste electrodes were constructed by using a 60:40 graphite powder to mineral oil weight-to-weight ratio. Once weighed, the constituents were thoroughly hand-mixed (∼30 min) to produce a paste. This paste was then packed into the cavity (2-mm diameter, 2 mm deep) of a 1-mL plastic syringe holder (Becton Dickinson Co., Franklin Lakes, NJ); a bare copper wire had been inserted through the opposite end to produce electrical contact. The t-PFc paste electrodes were constructed in a similar manner, with the t-PFc mediator replacing the mineral oil. Various t-PFc-to-graphite weight-to-weight ratios were examined to ascertain the best mixing ratio. The glucose biosensors were constructed in a manner analogous to the bare paste electrodes with the addition of glucose oxidase (GOx) to the mixture. For this purpose, GOx was mixed first with the ferrocene compound, followed by addition of graphite and a thorough mixing. Most sensing work employed a graphite/ ferrocene/GOx weight ratio of 50:40:10. The traditional carbon paste glucose biosensors, based on the DMFc mediator and mineral oil binder, were constructed using a graphite/mineral oil/ DMFc/GOx weight ratio of 39:48:3:10. The composite surface was smoothed on a weighing paper (VWR Scientific products, West Chester, PA) and rinsed carefully with double-distilled water prior to each experiment. Procedure. Measurements were carried out in a phosphate buffer (0.05 M, pH 7.4) supporting electrolyte medium. Amperometric batch measurements were conducted under forced convection (stirring) by applying a potential of +0.40 V and allowing the transient currents to decay to a steady-state value (usually within 15 min). Cyclic voltammograms were recorded following an initial 90-min stabilization at +0.40 V. All measurements were performed at room temperature. RESULTS AND DISCUSSION The mixed enzyme/carbon paste strategy has received considerable attention as it results in versatile and renewable reagentless biosensors.1,2 Such devices commonly rely on dissolving the mediator in the pasting liquid. The new carbon paste enzyme electrode concept, in which the redox mediator acts as the binder and as the electron acceptor, is illustrated for amperometric biosensing of glucose in connection with the t-PFc and n-BuFc mediators/binders. Since the replacement of the oil binder with the mediator pasting liquid results in a fundamentally different electrode, the new ferrocene-carbon paste electrodes were first characterized in the absence of the enzyme. Figure 1 displays the influence of the t-PFc loading upon the resistance (b) and capacitance (×) of the composite electrode. The electrode resistance is not affected by the t-PFc content between 10 and 20 wt %, then increases slowly (from 18 to 30 Ω) between 20 and 40 wt %, and more rapidly thereafter. The capacitance [estimated from background cyclic voltammograms at different scan rates (υ) and monitoring the i versus υ dependence at 0.0 V] decreases rapidly upon raising the t-PFc loading from 10 to 20 wt % and more slowly up to 50 wt %. While the resistance is affected primarily by the corresponding changes in the graphite content, the capacitance profile appears to reflect changes in the coverage of the graphite particles (by 3736 Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

Figure 1. Effect of the t-PFc loading (wt %) on the resistance (b) and capacitance (×) of the carbon paste electrode.

Figure 2. (A) Cyclic voltammetric response of the t-PFc-based paste electrode in a blank phosphate buffer solution (0.05 M, pH 7.4). Scan rate, 100 mV s-1. (B) Plot of log(ip,a) vs log(ν). (C) Effect of the pH on the oxidation peak potential of the t-PFc paste electrode.

the binder/mediator) at the surface/solution interface. The mediator content of traditional carbon paste bioelectrodes has little effect on the electrode resistance or capacitance. Electrodes containing more than 30 wt % t-PFc appeared pasty, while a lower ferrocene content led to a dry (yet still analytically useful) formulation. Figure 2A displays a cyclic voltammogram of the t-PFc carbon paste electrode in the blank phosphate buffer solution. A welldefined response, with Ep,a and Ep,c of 0.465 and 0.402 V, respectively, a midpoint potential of 0.432 V, and an ip,c/ip,a value of 1.00 is observed. The peak separation of 63 mV indicates a one-electron diffusion-controlled redox process. However, cyclic voltammetric experiments at different scan rates (υ) resulted in a linear log(ip,a) versus log(υ) plot over the 50-1000 mV/s range (B). The slope of the log(ip,a) versus log(υ) plot gave a value of 0.67, suggesting a mixture of diffusion- and surface-controlled processes. (Slopes of 0.50 and 1.00 are expected for ideal reactions of solutions and surface processes, respectively.) Analysis of the voltammetric peak shape further supports this theory; the cathodic peak appears sharper than the anodic one (with broadening of the latter at lower scan rates). We also examined the scan rate dependence of Qp,a/Qp,c. The results revealed that, for scan rates below 50 mV/s, the Qp,a/Qp,c ratio steadily decreases after which it levels off (not shown). This behavior can be rationalized as follows; when the mediator pasting liquid t-PFc(l) is oxidized, it forms the soluble t-PFc+(aq) species, at the three-phase graphite/ t-PFc/electrolyte boundary, consistent with previous studies of n-BuFc.11,12 It can be envisaged that t-PFc+(aq) will first dissolve (11) Wadhawan, J. D.; Evans, R. G.; Compton, R. G. J. Electroanal. Chem. 2002, 533, 71. (12) Wittstock, G.; Emons, H.; Heineman, W. R. Electroanalysis 1996, 8, 143.

Figure 3. Hydrodynamic voltammograms for 2 mM glucose at the DMFc- (A) and t-PFc- (B) based biosensors. Electrode composition, 60:20:20 graphite/t-PFc/GOx (A) and 30:50:10:10 mineral oil/graphite/ DMFc/GOx (B). Electrolyte, 0.05 M phosphate buffer (pH 7.4); stirring rate, ∼300 rpm.

into the diffusion layer (eq 1), followed by diffusion into the bulk solution (eq 2). Upon reversal of the scan direction, the t-PFc+(aq,TL)

t-PFc(l) a t-PFc+(aq,TL) + e-

(1)

t-PFc+(aq,TL) a t-PFc+(aq,bulk)

(2)

within the diffusion layer is reduced back to the insoluble t-PFc(l), which precipitates back onto the electrode surface.13 This mechanism is qualitatively consistent with the data detailed in Figure 2. At fast scan rates (>50 mV s-1), only the thin layer of t-PFc+(aq,TL) is formed, with a minimal loss to the bulk solution, possibly leading to a mixed control. In contrast, at slower scan rates, the newly formed t-PFc+(aq) has more time to diffuse from the bulk electrode into the diffusion layer (t-PFc+(aq,TL)) and then into the bulk electrolyte solution (t-PFc+(aq,bulk)), leading to a significant increase in the charge ratios. Finally, it was found that the peak potentials were independent of the pH over a wide range (from 2.5 to 10; Figure 2C), while the peak currents were nearly independent of the pH (not shown). These findings are consistent with eqs 1 and 2. Figure 3 displays typical hydrodynamic voltammograms for glucose at the conventional (DMFc-based) biosensor (A) and using the new t-PFc-enzyme paste electrode (B). The curves were developed pointwise over the range -0.20 to +0.70 V, in the presence of 2 mM glucose, and display a nearly sigmoidal response. The signal of the DMFc biosensor starts above 0.00 V, increases slowly up to +0.30 V, and levels off thereafter. The new t-PFc-based biosensor displays a significantly (∼15-fold) larger response, starting above +0.10 V, rising sharply up to +0.40 V, and more slowly thereafter. The half-wave potentials [+0.10 (A) and +0.26 (B) V] reflect the standard potentials of the corresponding ferrocene derivatives. The use of the mediator pasting liquid leads to higher sensitivity and a wider dynamic range compared to conventional carbon paste biosensors. Figure 4 compares the response to (13) Dietz, S. D.; Bell, W. L.; Cook, R. L. J. Organomet. Chem. 1997, 545-546, 67.

Figure 4. Current-time recordings to successive 4 mM glucose additions at the 50:40:10 graphite/t-PFc/GOx (A), the 50:40:10 graphite/n-BuFc/GOx (B), and the 39:48:3:10 mineral oil/graphite/ DMFc/GOx (C) electrodes. Potential, +0.4V; other conditions, as in Figure 3.

increasing levels of glucose in 4 mM steps at the new carbon paste bioelectrodes (A, B) and at the conventional (DMFc) biosensor (C). The response of the DMFc-based biosensor increases rapidly at first and nearly levels off above 16 mM. In contrast, the new t-PFc and n-BuFc mediator/binder enzyme electrodes respond favorably to these glucose additions, yielding large signals over the entire 4-60 mM range. As expected from the voltammetric profiles, the DMFc biosensor displays significantly smaller glucose signals (note the different current scales). Note that the three biosensors have similar graphite and enzyme “loadings”, indicating that the substantially higher sensitivity and wider linear range reflects the higher mediator/binder content and not the influence of the graphite composition on the electron-transfer rate.14 The three biosensors display similar response times of 17 (A), 18 (B), and 22 (C) s. A similar noise level was indicated by recording the baseline current at +0.40 V using an amplified scale. The resulting calibration plots (shown in Figure 5A) illustrate that the new mediator-based biosensors offer a substantially higher sensitivity and a greatly extended linear dynamic range compared to the mineral oil/DMFc bioelectrode (a, b vs c). While the n-BuFc biosensor displays a lower sensitivity (0.200 µA cm-2 mM-1) than the t-PFc based device (sensitivity, 0.304 µA cm-2 mM-1), it offers a wider linear range (up to 24 vs 16 mM). Using the conventional DMFc biosensor, linearity prevails up to 8 mM (sensitivity, 0.033 µA cm-2 mM-1). Apparently, the use of the alkylferrocene pasting liquid results in a larger resistance to the substrate mass transport compared to that observed at conventional carbon paste electrodes based on the mineral oil binder (see discussion of the mechanism below). It has been shown previously that increasing the mediator loading within the conventional carbon paste enzyme electrodes extends the linear range in accordance with the enzymatic pingpong mechanism.15,16 However, the quantity of mediator that can (14) Rice, M. E.; Galus, Z.; Adams, R. N. J. Electroanal. Chem. 1983, 143, 89. (15) Saby, C.; Mizutani; F.; Yabuki, S. Anal. Chim. Acta 1995, 304, 33. (16) Amine, A.; Kauffmann, J. M.; Guilbault, G. G. Anal. Lett. 1993, 26, 1281.

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Figure 6. Effect of varying (A) the quantity of t-PFc (b ) 60:30:10, + ) 70:20:10, 0 ) 80:10:10) and (B) the quantity of GOx (b ) 60: 20:20, + ) 70:20:10, 0 ) 75:20:5) on the amperometric response to glucose. Conditions as in Figure 4.

see above).16 Similarly, and in accordance to the mechanism proposed by Kulys et al.,17 glucose diffuses from the bulk solution and reduces the enzyme. The oxidized t-PFc mediator, dissolved in the diffusion layer, then interacts with the reduced form of GOx:

GOx(red) + t-PFc(aq)+ f GOx(ox) + t-PFc(l)

Figure 5. (A) Calibration plots for glucose based on the data of Figure 4. (B) The corresponding Lineweaver-Burk plots. Electrodes: (a) 50:40:10 graphite/t-PFc/GOx, (b) 50:40:10 graphite/nBuFc/GOx, and (c) 39:48:3:10 mineral oil/graphite/DMFc/GOx.

be incorporated into these electrodes is limited ( ∼3-10 wt %) by their large oil binder content. The new electrode configuration obviates the need for an oil binder and hence overcomes this limitation. The sensitivity and dynamic range of the new biosensor compares favorably with those reported for conventional carbon paste enzyme electrodes.3,7,15 For example, an early DMFc-based glucose sensor displayed a sensitivity of 0.023 µA cm-2 mM-1 and a linearity up to 8 mM.7 Figure 5B displays Lineweaver-Burk plots for the three biosensors (based on the data of Figure 4). Such plots are linear for the new biosensors and yield Km values of 59 (n-BuFc) and 107 (t-PFc) mM. The corresponding Lineweaver-Burk plot for the conventional DMFc biosensor is linear only at low glucose concentrations and yields an apparent Km value of 20 mM. Such a Km is in good agreement with those (19-33 mM) reported for other mediator-based glucose carbon paste biosensors (including DMFc ones).15,16 A similar value (20 mM) was obtained from the corresponding linear Eadie-Hoftsee plot (not shown), reflecting the nondiffusional limitations. Both the n-BuFc and t-PFc bioelectrodes yielded nonlinear Eadie-Hoftsee plots, characteristic of hindered diffusion, with a large curvature at low glucose concentrations. In view of the complex mechanism of carbon paste biosensors, it is not clear at this stage whether the catalytic processes take place in the electrode interior or at the surface/solution boundary. Increased Km values with higher mediator loadings have been reported in connection with conventional carbon paste biosensors15,16 and were attributed to increased external mass transport resistance (by the ferrocenium ion dissolved in the diffusion layer; 3738

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(3)

This leads to precipitation of t-PFc and its reoxidization back to t-PFc+ at the three-phase (graphite/t-PFc/electrolyte) boundary (eq 1). One advantage of carbon paste biosensors is the ability to vary the loading of the individual components and hence tailor the response characteristics. Figure 6 displays the influence of the t-PFc (A) and GOx (B) loadings upon the response to glucose. Increasing the t-PFc content from 10 to 30 wt % resulted in a 1520% higher sensitivity as well as in a larger deviation from linearity. Increasing the enzyme loading from 5 to 20 wt % enhanced the linearity and, hence, the sensitivity at higher glucose concentrations (Figure 6B). A nearly identical response is observed up to 8 mM glucose using the different enzyme loadings. The mediator pasting liquid appears to impart high thermal stability onto the resulting biosensor, in a manner analogous to mineral oil binders of conventional carbon pastes.18 For example, full activity of glucose oxidase was retained over a 48-h period of thermal stress at 60 °C. The leaching of the oxidized form of mediators (to the contacting solution) commonly affects the stability of the corresponding oxidase carbon paste biosensors. While a similar problem exists using the new mediator pasting liquid configuration, it is not as severe in view of the huge mediator reservoir. The t-PFc and mineral oil/DMFc biosensors displayed 30 and 65% decrease of the response, respectively, following a 4-h stirring at +0.40 V; only 5 (t-PFc) and 30% (DMFc) current diminutions were observed following a similar period at open circuit. These data emphasize the stability advantage of the mediator pasting liquid approach compared to the conventional carbon paste designs. The replacement of oxygen by artificial electron acceptors, such as ferrocene derivatives, is commonly used for addressing the oxygen demand of oxidase-based enzyme electrodes.4,5,10 Accordingly, it is important to examine whether the new ferrocene pasting liquids can address the oxygen limitation of glucose (17) Kulys, J.; Schuhman, W.; Schmidt, H. Anal. Lett. 1992, 25, 1011. (18) Wang, J.; Liu, J.; Cepra, G. Anal. Chem. 1997, 69, 3124.

uric acid, and acetaminophen. These interferences were nearly eliminated by using an operating potential of +0.25 V and incorporating stearic acid (at 10 wt %) into the paste (not shown). Such bulk modification has been used in conventional carbon paste designs and provides the necessary charge repulsion of anionic interferences.7

Figure 7. Calibration plots for glucose at the t-PFc- (A, B) and DMFc-based (C, D) carbon paste biosensors in the absence (A, C) and presence (B, D) of oxygen. Conditions as in Figure 4.

biosensors. Figure 7 compares calibration plots for glucose at the t-PFc (A, B) and DMFc/mineral oil (C, D) carbon paste biosensors in the presence (B, D) and absence (A, C) of oxygen. Both devices respond favorably to these glucose additions under oxygen-free conditions. A slight oxygen interference is observed in both cases, with the response decreasing in the presence of oxygen (due to the competing oxygen reaction). For example, current changes of 7 and 15% are observed at the t-PFc and DMFc biosensors, respectively, at the 2 × 10-2 M glucose level. Notice again the significantly higher sensitivity of the t-PFc biosensor and its wider linear range (in the presence of oxygen). A greater deviation from linearity is observed in the absence of oxygen (A, C). We also examined potential interferences from electroactive compounds commonly present in physiological fluids.5 As expected from the absence of a permselective membrane barrier, the response of the t-PFc-based glucose biosensor was affected by the presence of physiological levels (0.1 mM) of ascorbic acid,

CONCLUSIONS We have demonstrated a new generation of carbon paste biosensors based on the use of the redox mediator as the pasting liquid. Since the mediator serves also as the binder, the resulting devices are fundamentally different from those based on conventional carbon paste bioelectrodes. The elimination of the common mineral oil binder not only simplifies the preparation but also led to several distinct analytical advantages, including a substantially higher sensitivity and a wider linear range. Bulk modification of the entire electrode material is still possible by dispersing the necessary additives within the mediator pasting liquid. Further work is required for obtaining better understanding of the response mechanism of the new carbon paste bioelectrode. While the new concept has been illustrated in connection with ferrocene mediators and glucose oxidase, it could be readily expanded to other artificial electron acceptors and enzymes. By providing a simplified avenue for preparing renewable biosensors, the mediator-pasting liquid fabrication scheme holds great promise for routine biosensing applications. ACKNOWLEDGMENT Financial support from the National Science Foundation (Award CHE 02009707) is gratefully acknowledged.

Received for review January 9, 2004. Accepted April 12, 2004. AC049943V

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