Anal. Chem. 2006, 78, 7044-7047
Acid Stability of Carbon Paste Enzyme Electrodes Joseph Wang,* Mustafa Musameh, and Jian-Wei Mo
The Biodesign Institute, Departments of Chemical & Materials Engineering and Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-5801
This note reports on the unusual protection of several enzymes against harsh pH conditions provided by carbon paste electrodes. Both glucose oxidase and polyphenol oxidase carbon paste amperometric biosensors display a remarkable resistance to acid deactivation compared to conventional biosensors prepared by electropolymeric entrapment of enzymes. For example, the carbon paste enzyme electrodes fully retain their activity upon stressing in strongly acidic conditions (pH ∼2.0-2.5) for prolonged periods, where conventional (polymer-based) biosensors rapidly lose most of their response. Such unusual acid stability of carbon paste enzyme electrodes is attributed to the “pH memory” of enzymes in the hydrophobic paste environment, to the barrier to hydronium ions provided by the pasting liquid and to decreased conformational mobility. Enzymes are effective biocatalysts that are widely used in a variety of bioassays, bioprocesses, bioremediation, and biofuel cells. A major drawback that hampers many of these biotechnological applications of enzymes is their limited stability. In particular, various important applications of enzymes involve extreme environments, such as high temperatures or harsh acidic conditions, which are outside the “stability window” of common biocatalysts.1,2 Methods for improving the stability of biosensors in extreme environments have focused primarily on the thermal stability of the corresponding enzymes.2 Few studies have been devoted to imparting high resistance to acid deactivation effects. The solution pH is one of the most influential parameters affecting the stability and activity of enzymes. Acid stability is a measure of the enzyme’s survival during stress in a harsh acidic medium. Dong’s group3 reported on an acid-stable hydrogen peroxide biosensor based on the immobilization of soybean peroxidase within a self-gelatinizable hydrogel. Convenient operation down to pH 2.5 was thus accomplished. Sol-gel encapsulation has also shown useful for extending the stability of enzymes in harsh acid conditions.4 In particular, entrapping enzymes in (surfactant-modified) silica sol-gel matrixes resulted in a high degree of protection in strongly acidic conditions.4 * To whom correspondence should be addressed. Tel: 1-480-727-0399. E-mail:
[email protected]. (1) Burton, S. G.; Cowan, D. A.; Woodley, J. M. Nature Biotechnol. 2002, 20, 37. (2) Dong, S.; Wang, B. Electroanalysis 2002, 14, 7. (3) (a) Wang B. Q.; Li, B.; Wang, Z. X.; Xu, G. B.; Wang, Q.; Dong, S. J.Anal. Chem. 1999, 71, 1935. (b)Wang, B.; Li, B.; Cheng, G.; Dong, S. Electroanalysis 2001, 13, 555. (4) Frenkel, H.; Avnir, M.; Avnir, D. J. Am. Chem. Soc. 2005, 127, 8077.
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Here we report on the effective pH protectability of enzymes provided by carbon paste electrode matrixes. Carbon paste electrodes (CPEs), which use graphite powder mixed with a waterimmiscible pasting liquid, represent a convenient matrix for the incorporation of biocatalysts.5 Earlier we described the unusual resistance of enzymes to heat-induced denaturation upon entrapment in carbon paste microenvironments.6 Dramatic improvement in the thermal stability was thus documented for six enzymes upon stressing the corresponding carbon paste biosensors at elevated temperatures (60-80 °C) for prolonged periods of several months. Kulys and Schmid7 reported on the high stability of a glucose oxidase carbon paste biosensor over 3 months at 35 °C. Amine and Kauffmann8 demonstrated the extended room-temperature stability of the “fragile” enzyme glutamate dehydrogenase within the paste matrix. The resistance of carbon paste enzyme electrodes to heat-induced denaturation has been attributed to the conformational (structural) rigidity of the biocatalyst within the highly hydrophobic pasting liquid, in a manner observed in organic-phase environments.9 However, there are no reports on the effective resistance of carbon paste biosensors to another common cause of enzyme deactivation, acid stress. In the following sections, we will demonstrate the dramatic protection of polyphenol oxidase (PPO) and glucose oxidase (GOx) against harsh pH conditions upon confinement in CPEs. Both GOx- and PPO-carbon paste biosensors retain their complete activity following prolonged immersions in highly acidic bulk solutions. Such protectability to acid deactivation effects provided by CPEs is attributed primarily to the “pH memory” of enzymes in organic media9 and to the barrier to hydronium ions provided by the hydrophobic pasting liquid. The unique pH stability of carbon paste biosensors indicates great promise for sensing applications as well as for biotechnological and industrial operations involving harsh acidic conditions. EXPERIMENTAL SECTION Apparatus. Amperometric experiments were performed with a Bioanalytical Systems (BAS) CV-27 voltammograph, in connection with a BAS X-Y recorder. The enzyme working electrodes, the reference electrode (Ag/AgCl, model CHI111, CH Instruments, Austin, TX), and platinum wire counter electrode were inserted into the 20-mL cell (BAS, model VC-2) through holes in its Teflon cover. A magnetic stirrer provided the convective (5) (6) (7) (8) (9)
Gorton, L. Electroanalysis 1995, 7, 23. Wang, J.; Liu, J.; Cepra, G. Anal. Chem. 1997, 69, 3124. Kulys, J. J.; Schmid, R. D. Bioelectrochem. Bioenerg. 1990, 24, 305. Amine, A.; Kauffmann, J. M. Bioelectrochem. Bioenerg. 1992, 28, 117. Klibanov, A. M. Nature 2001, 409, 241. 10.1021/ac060986g CCC: $33.50
© 2006 American Chemical Society Published on Web 09/06/2006
transport during the amperometric measurement. pH measurements were carried out using an Accumet Research AR50 pH meter equipped with a combination pH electrode (0-14 pH range) from Fisher Scientific (Pittsburgh, PA). Reagents. All solutions were prepared from double-distilled water. GOx (EC 1.1.3.4, Type X-S, Aspergillus niger, 234 900 units/g), tyrosinase [polyphenol oxidase (PPO), EC 1.14.18.1, mushroom, 6050 units/mg], D-(+)-glucose (97%), and potassium chloride (99%) were obtained from Sigma. Phenol (99%), 1,2-phenylenediamine dihydrochloride (99%), rhodium-on-carbon (5% Rh) (Rh-C), and mineral oil were received from Aldrich, while graphite powder (grade no. 38) and hydrochloric acid (37%) were obtained from Fisher and Spectrum Quality Products (Gardena, CA), respectively Preparation of Biosensors. Different biosensors, based on various enzymes and immobilization schemes, were prepared for comparing the pH stability. The glucose and phenol carbon paste biosensors were prepared by hand-mixing thoroughly 10 mg of GOx, 45 mg of Rh-C powder, and 55 mg of mineral oil, or 2 mg of PPO, 60 mg of graphite powder, and 40 mg of mineral oil, respectively. A portion of the resulting paste was then packed firmly into the electrode cavity (2-mm diameter, 2-mm depth) of a glass sleeve. Electrical contact was established via a copper wire. The paste surface was smoothed on a weighing paper and rinsed carefully with double-distilled water prior to each measurement. A glassy carbon (GC) electrode (3-mm diameter, model CHI104, CH Instruments) was used in connection with the electropolymeric entrapment of GOx or PPO. Prior to the enzyme immobilization, the glassy carbon substrate was polished with 3and 0.05-µm alumina slurries and then washed with doubledistilled water. The electropolymerization of polyphenylenediamine (PPD) was performed by applying a constant potential of +0.65 V for 20 min in a 0.05 M phosphate buffer solution (pH 6.0 for GOx and pH 7.0 for PPO), containing 5 mM 1,2-phenylenediamine and 1000 units/mL GOx or 6050 units/mL PPO. Subsequently, the PPD/enzyme layer was washed thoroughly with double-distilled water. Procedure. The pH stability of the biosensors was evaluated by immersing the different bioelectrodes in vials containing HCl solutions of different pH values (1.0-3.0) for a predetermined period (ranging from minutes to hours). The appropriate pH was adjusted by using 0.2 M hydrochloric acid and 0.2 M KOH. Subsequent amperometric measurements were performed in a phosphate buffer solution (0.05 M pH 7.4), by applying an operational potential of +0.60, +0.85, or -0.10 V onto the Rh-C/GOx/mineral oil, GC/GOx/PPD, and PPO biosensors, respectively. The solution was stirred at 300 rpm; the transient background current was allowed to decay to a steady-state value (in the absence of substrate). The substrate was then added, and the current-time tracing was recorded. Direct chronoamperometric measurements of glucose in a Pepsi soft beverage (Young America, MN; pH 2.5) were carried out using the Rh-C/GOx/mineral oil and Rh-C/mineral oil paste electrodes by stepping the operational potential to +0.60 V. All experiments were carried out at room temperature. RESULTS AND DISCUSSION Acid stability is a measure of the enzyme’s survival under conditions of extreme pH. The dramatic improvement in the acid
Figure 1. Amperometric response to 6 mM glucose (A, B) and to 0.1 mM phenol (C, D) at GC/GOx/PPD (A), Rh-C/GOx/mineral oil (B), GC/PPO/PPD (C), and graphite/PPO/mineral oil (D) biosensors, before (a) and after (b) immersing in a pH 2.2 (A, B) and pH 2.5 (C, D) medium for 60 min (A, B, D) and for 10 min (C). Detection potential, +0.85 (A), +0.60 (B), and -0.10 V (C,D); electrolyte, phosphate buffer (pH 7.4; 0.05 M), stirred at 300 rpm.
stability of carbon paste enzyme electrodes has been assessed by comparing the performance of these biosensors before and after immersion in a highly acidic medium for selected times. Figure 1 displays a comparative study of acid deactivation of GOx (A, B) and PPO (C, D) entrapped in a carbon paste matrix (B, D) or within an electropolymerized PPD film (A, C). The polymerbased biosensors display a rapid loss of the biocatalytic activity following immersion in acidic media [pH 2.2 (A), and pH 2.5 (C)] for 1 h (A) and for 10 min (C). For example, an 85% activity loss is observed for the PPD-entrapped GOx after a 60-min acid immersion (A; b vs a). The PPO-PPD bioelectrode displays an even faster and sharper activity loss, with a complete disappearance of its response after a 10-min immersion in a solution of pH 2.5. In contrast, both enzymes remain highly active upon confinement within the carbon paste matrix. No apparent change in the current signal or in the response time of both paste-based biosensors is observed after similar immersions in the acidic solutions (B, D; a vs b). Figure 2 examines the influence of the immersion time (in acidic media) upon the response of the GOx (A) and PPO (B) enzyme electrodes. The carbon paste biosensors display high stability in connection with the different immersion times, retaining their entire original response after a 60-min acid incubation (a, c). In contrast, both polymer-based enzyme electrodes display a rapid loss of the response (b, d). A complete decay of the response of the PPO-PPD device is observed following a 10-min immersion in the pH 2.5 solution (d). A slower decrease of 60 and 85% is observed after dipping the GOx-PPD electrode in the acidic (pH 2.2) medium for 10 and 60 min, respectively. Figure 3 assesses the influence of the pH of the incubation solution upon the stability of the GOx (a) and PPO (b) carbon paste enzyme electrodes. The GOx-carbon paste biosensor retains its complete response after dipping in acidic solutions of pH higher than 2.2 (a). A rapid decay of its signal (to 36% of its original value) is observed between pH 2.2 and 1.7, with a slower decrease at lower pH values. The PPO CPE offers higher stability (than the GOx sensor) in strongly acidic solutions (below pH 1.9; b vs a) and retains full activity above pH 2.5 (b). Note that the PPO- and GOx-carbon paste biosensors retain 42 and 15% of their original activity, respectively, even after dipping in an Analytical Chemistry, Vol. 78, No. 19, October 1, 2006
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Figure 2. Acid deactivation kinetics at pH 2.2 (A) and pH 2.5 (B) of GOx (a, b) and PPO (c, d) within a carbon paste matrix (a, c), and upon entrapment in PPD (b, d). The activity was recorded at the times indicated using amperometric monitoring of 6 mM glucose and 0.1 mM phenol at potentials of +0.60 (a), +0.85 (b), and -0.10 V (c, d). Other conditions, as in Figure 1. Figure 4. Amperometric response of the Rh-C/GOx/mineral oil biosensor to successive 2 mM additions of glucose in a phosphate buffer (pH 7.4) before (a) and after (b) immersing the biosensor for 60 min in a pH 2.2 (HCl) solution. Detection potential, +0.60 V, with a 300 rpm stirring.
extremely harsh acidic medium of pH 1.0. While the exact reason for the higher acid stability of PPO (versus GOx) is not fully understood, PPO is known to operate efficiently in extreme environments such as nonpolar organic media;10 this may explain, in part, its higher acid stability (vs GOx). Overall, the data of Figure 3 indicate that both enzyme electrodes display considerable insensitivity to the pH of the bulk solution above 2.2. While the exact reason for the resistance to acid-induced denaturation provided by the carbon paste matrix is not fully understood, we will offer some interpretations of this unusual observation. It has been proposed that enzymes hydrophobic media have a pH memory.9,11 Similarly, the pH protectability exhibited by carbon paste biosensors can be attributed to the retention of the ionization state of the protein ionogenic groups in a manner analogous to the pH memory of enzymes in organic solvents. Such pH memory disappears in aqueous bulk environ-
ments where (in contrast to hydrophobic media) the enzyme’s ionogenic groups rapidly change their ionization state. We suggest consideration also of the role of the pasting liquid as a barrier to hydronium ions as another possible protection mechanism. Owing to the difficulty of hydronium ions in penetrating the hydrophobic pasting-liquid “blanket” surrounding the enzyme, the carbon paste microenvironment is insensitive to variations in the bulk solution pH. Conformational rigidity, responsible for the enhanced thermal stability of carbon paste enzyme electrodes,6,9 may also contribute to some of the observed acid protection. Similar acid protection mechanisms, coupling the protein rigidity/“locking” and barrier to hydronium ions (matrix isolation), were suggested recently in connection with the entrapment of enzymes within surfactantmodified sol-gels4 or a lipid matrix.12 Figure 4 displays the amperometric response of the GOx CPE to successive 2 mM increments in the glucose concentration, as recorded before (a) and after (b) a 1-h incubation in an HCl (pH 2.2) solution. Completely identical current-time profiles are observed before and after such prolonged acid stress, with no apparent change in the sensitivity or dynamic properties. In both cases, the response increases linearly and rapidly over the entire 2-20 mM glucose range. Such wide dynamic range (in the absence of an external mass transport limiting coating) reflects the “built-in” mass transport limitation associated with the thin pasting-liquid layer covering the enzyme. Finally, we exploited the protectability of the enzyme against harsh pH imparted by CPEs for conducting assays of a real-life (Pepsi beverage) sample, which has a pH of 2.5. Chronoamperometric measurements of glucose in this soft drink were conducted by stepping the potential of the Rh-C/GOx/mineral oil CPE from an open circuit to +0.60 V. No apparent change in the glucose
(10) Kazandjian, R. Z.; Klibanov, A. M. J. Am. Chem. Soc. 1985, 107, 5448. (11) Zaks, A.; Klibanov, A. M. J. Biol. Chem. 1988, 263, 3194.
(12) Gole, A.; Vyas, S.; Sainkar, S. R.; Lachke, A.; Sasty, M. Langmuir 2001, 17, 5964.
Figure 3. Influence of the pH upon the enzymatic activity of Rh-C/GOx/mineral oil (a) and graphite/PPO/mineral oil (b) carbon paste bioelectrodes. The activity was examined after a 5-min dipping in the indicated pH medium, followed by measurements in a phosphate buffer. Operating potential, +0.60 (a) and -0.10 V (b); other conditions, as in Figure 1.
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signal was observed after dipping this biosensor in the acidic sample solution for a 16-h period (before stepping the potential), in comparison to the initial potential-step current response obtained after a very short 3-min immersion (not shown). A similar potential-step control experiment, conducted without the immobilized enzyme, yielded a substantially smaller current signal (of electroactive sample constituents), indicating that most of the response observed with the biosensor is due to the enzymatic reaction. In conclusion, we have demonstrated an unusually high level of protection of enzymes against harsh pH conditions upon confinement within a carbon paste matrix. The resulting GOxand PPO-carbon paste biosensors display an unusual resistance to acid deactivation effects, retaining their complete activity following prolonged immersions in highly acidic solutions. While the exact reason for the high acid stability of carbon paste enzyme electrodes is not fully understood, it is probably related to the pH memory of enzymes in organic media, to the barrier to hydronium ions provided by the hydrophopic pasting liquid, and to increased enzyme rigidity. More studies are needed to understand better the pH protectability offered by CPEs and the
role of the pasting liquid in the acid stabilization. Besides assessing different pasting liquids, we plan to explore various surfactant and lipid additives (within the pasting liquid) to further improve the acid stability. The new understanding will allow tailoring of the carbon paste composition to meet the demands of harsh pH conditions. The pH memory of enzymes in CPEs could also be exploited to tailor the biocatalytic activity of such biosensors by preexposing their enzymes to an aqueous solution of optimal pH. The unique pH stability of carbon paste biosensors indicates great promise for sensing applications as well as for numerous industrial and biotechnological operations involving harsh acidic conditions. ACKNOWLEDGMENT This research was supported by grants from the NIH (Awards R01A 1056047-01 and R01 EP 0002189) and NSF (CHE 0506529). M.M. acknowledges a fellowship from the Islamic Development Bank (Jeddah, Saudi Arabia). Received for review May 29, 2006. Accepted August 7, 2006. AC060986G
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