Anal. Chem. 1994,66, 1988-1992
Ultrathin Porous Carbon Films as Amperometric Transducers for Biocatalytic Sensors Joseph Wang' and Qlang Chen
Department of Chemistry and Biochemistry, New Mexico State UniversnY, Las Cruces, New Mexico 88003 Clifford L. Renschler and Chrstlne WhAe
Sandia National Laboratories, Division 18 12, Mail Stop 0367, Albuquerque, New Mexico 87185-0367 Novel ultrathin (0.4 pm) porous carbon fdms are employed as transducers for amperometric biosensors. Such foamlike nanoscopicfilmscouple the advantagesof high enzymeloadings (within the micropore hosts) and large microscopic area with a small geometric area. Both electropolymerization and metalization are used to entrap the enzyme within the micropores. Scanningelectronmicroscopy shedsusefulinsights into the unique morphology of the growing enzyme layer. The greatly enhanced sensitivity is coupled with a fast and stable response. Factors influencing the performanceof porous-filmbased biosensors are examined and discussed. The improved performance is illustrated in connectionwith glucoseand p b e d sensors. The latter offers a remarkably low detection limit of 2.5 X 10-8 M. The new nanoscopic foams should prove useful for many other electroanalytical applications. The application of amperometric enzyme electrodes in chemical analysis has greatly increased during the past Such sensing devices rely on the intimate coupling of an enzyme layer and a solid-electrode transducer. Most often the transducer is a metallic (Pt, Au) or carbon disk, which is covered with an appropriate enzyme membrane. While significant effort has been devoted to the immobilization of the biological layer and to the mediation of electrons between this layer and the transducer, little attention has been given to the explorationof new (more efficient)transducer materials. This article describes the utility of novel ultrathin porous carbon films as transducers for glucose and phenol biosensors. Porous electrodes are not commonly used as transducers in connection with enzyme-catalyzed reactions. Only flowthrough reticulated vitreous carbon (RVC) electrodes have been widely used for on-line b i o m o n i t ~ r i n g .While ~ ~ ~ offering large surface areas (and hence enhanced signals), porous bioelectrodes cannot be miniaturized, as often desired in clinical diagnostics. Porous carbon films, developed recently at Sandia ~~
( 1 ) Turner, A. P., Karube, I., Wilson, G., Eds. Eiosensors: Fundamentals and Applications; Oxford Scientific: Oxford, England, 1987. (2) Wan& J. Electroanalytical Techniques in Clinical Chemistry and Loboratory Medicine; VCH Publishers: New York, 1988. (3) Kobos, R. Trends Anal. Chem. 1987, 6 , 6. (4) Blaedel, W. J.; Wang, J. Anal. Chem. 1980, 52, 1426. ( 5 ) Sasso, S.; Pierce, R.; Walla, R.; Yacynych, A. AMI. Chem. 1990, 62, 11 11.
1988 AnalytlcalChemistty, Vd. 88, No. 13, Ju& 1, 1994
National Laboratories? overcome this limitation as they combine the high surface area of porous electrodes with a 0.1-1.0pmfilm thickness. Inde4,onecanviewthesefoamlike films as macroscopic foams whose thickness dimension collapsed. Such ultrathin porous films are produced by pyrolytic decompositionof spin cast polyacrylonitrile (PAN). Fully dense (solid) carbonized PAN films, prepared by film casting under low humidity, have been used recently for preparing enzyme nanael~ctrodes.~ The thin porous films, used in the present work, are produced by casting PAN solutions in a humid environment. Theuptakeof water induces phase separation, resulting in a foamlike morphology with unique cell structure (see Figure 1A). In addition to their attractive morphology,such porous films possess high electrical conductivity and controlled thickness. The resulting submicrometer pores can thus be used for entrapping high levels of enzymes. The biosensing opportunities and advantages accrued from these developmentsare explored in the following sections.
EXPERIMENTAL SECTION Apparatus. Amperometric and chronoamperometric experiments were performed with the Bioanalytical Systems (BAS) Model CV-27 voltammetric analyzer, in connection with a BAS X-Y-t recorder. The enzyme electrode, the reference electrode (Ag/AgCl, Model RE-1 (BAS)), and the platinum-wire auxiliary electrode were inserted into the 10mL cell (Model VC-2, BAS) through holes in its Teflon cover. The electrode surface was characterized by using a Phillips Model 501B scanning electron microscope (operated at a 7.2 kV accelerating voltage). Reagents. All solutionswere prepared with deionized water and reagent-grade chemicals. Glucose oxidase (GOx from Aspergillus niger, EC 1.1.3.4, 166100 U/g), was received from Sigma (catalog no. G-7141), while tyrosinase (from mushrooms, EC 1.14.18.1, 4200 U/mg) was obtained from United States Biochemicals Corp. (catalog no. 22885). Phenol, glucose, catechol, and o-phenylenediamine were also (6) Renschler. C. L.; Sylwater, A. P.; Salgado, L. V. J. Mater. Res. 1989,4,452. ( 7 ) Wang, J.; Nasw, N.; Rewhler, C. L.AMI. Lrrt. 1993, 26, 1333.
0003-2700184103661988504.50lo 1994 American chemical Socle@
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distilled water and stored in phosphate buffer at 4 OC (when not in use). The tyrosinaseelectrodewas prepared in a similar manner in the presence of 16,800 U (4 mg) of tyrosinase. Glucose oxidase/polymer-platinum and glassy-carbon electrodes, used for comparison purposes, were prepared as describedabove using commercial(BAS) 2-mm diameter disk electrodes. Porous film enzyme electrodes were also prepared by codeposition of glucose oxidase and rhodium. A stirred 500pL 0.03 M NaCl solution, containing 100 ppm rhodium and 830 U GOx (adjusted to pH 5.2) was used. The codeposition proceeded at -0.8 V for 15 min. Procedure. Amperometric and chronoamperometric experiments were performed at room temperature under batch conditions. The former were carried out using a stirred (400 rpm) solution and after the transient background current decayed to a negligible value.
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Figure 1. Scanning electron micrographs of the thin porous carbon film electrode before (A) and after (B) depositing the PPD/GOx. Magnification: 13000X.
purchased from Sigma and used without further purification. The rhodium solution ( 1000 ppm, atomic absorptionstandard) was received from Aldrich. The supporting electrolyte was a 0.05 M phosphate buffer (pH 7.4) solution. Electrode Assembly and Enzyme Immobilization. PANbased porous carbon films (400 nm thickness) were prepared at Sandia National Labs on one side of a quartz plate (25 X 25 mm) by spin casting (2000 rpm) a 6% PAN solution in humid (80%) air and carbonizing at 1100 OC. Dense (solid) carbon films were prepared in a similar fashion, but under 0% humidity. The plate was cut in half to give two 12.5 X 25 mm slides. The (solid) carbon film was subsequently covered almost entirely with a nonconducting epoxy (Cole Parmer Co.), leaving only two 2 X 3 mm exposed areas (on both ends) for use as the working electrode and for electrical contact. The latter was established to a copper wire via a silver-filled epoxy. Glucose oxidase/poly(o-phenylenediamine) films were electrochemicallygrown on the carbon film electrodes from an unstirred 500-pL acetate buffer (pH 5.2) solution containing 830 U (5 mg) GOx and 5 X 10-3M o-phenylenediamine.The electropolymerizationproceeded for 20 min at +0.65 V. The resulting enzyme electrode was thoroughly rinsed with double-
RESULTS AND DISCUSSION Scanning Electron Microscopy. Electropolymerization has been used for localizing the enzyme within the submicron pores of the porous carbon film transducer. Poly(pheny1enediamine) (PPD) films, known for their permselective and fast respon~e,~.~ have been used throughout most of this work. Figure 1 displays scanning electron micrographs (SEMs) of the porous carbon film, obtained with 13000X magnification, before (A) and after (B) the growth of the poly(pheny1enediamine)/glucose oxidase (PPD/GOx) layer. The naked carbon film has a foam-like morphology, with connected carbon "lines" of 0.1 pm average width and a pore size of around 0.8 pm. The PPD/GOx film is deposited on the struts of the entire carbon network, thus reducing the average pore size to around 0.5 pm. Yet, the enzyme-coated electrode maintains the three-dimensional open-cell morphology and large surface area. A smaller-magnification (3250X) image of the PPD/GOx/carbon film (Figure 2B) reveals a relatively uniform sponge-like structure of the enzyme-carbon network, as compared to a reticular microstructure characterizing the naked porous film (Figure 2A). Such enzyme microstructures result in an attractive biosensor performance, as discussed in the following sections. Characterization of PPD/GOx Porous-Film Sensors. Figure 3 displays the chronoamperometric (A) and amperometric (B) response of the PPD/GOx/dense- (solid-) film (a) and PPD/GOx/porous-film (b) electrodes to successive increments (5 X 10" M) of the glucose concentration. While both carbon films have the same geometric area, the porous one offers a significant enhancement of the response. Coupled with its low noise level, such an electrode results in a detection limit around 5 X M (based on S/N = 3), as compared to 5 X lo4 M for the dense-film based sensor. The porous electrode offers also attractive dynamic properties, with attainment of steady-state currents within 15 s. Figure 4 compares calibration plots for glucose (at +0.8 V) at the PPD/GOx/dense-film (A), PPD/GOx/platinum (B) and PPD/GOx/porous-film (C) electrodes over the 5 X lo4 to 5 X M range. Significantly higher signals are observed at the porous film electrode (e.g. 5- and ll-fold (8) Malitesta, C.; Palmisano, F.; Torsi, L.; Zambonin, P. Anal. Chem. 1990,62, 2735.
AnaIytimlChemisfry, Vol. 66, No. 13, Juv 1, 1994
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CONCENTRATION (mM) Figure 4. Calibration plots for glucose (5 X lo4 to 5 X lo3 M) at the PPD/GOx/dense-carbon(A), PPD/GOx/Pt (B),and PpD/GOx/porous carbon (C)electrodes. Other conditions are as in Figure 3B.
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Figure 2. Scanning electron micrographs of the thin porous carbon film electrode before (A) and after (B)depositing the PPD/GOx. Magnification: 3250X. ..
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TIME Figure 3. Chronoamperometric (A) and amperometric (B) response of the PPD/GOx/dense-carbon (a) and PPD/GOx/porous-carbon (b) sensors to successive increments of 5 X 1O4 M glucose. Key: operating potential, +0.80 V; electrolyte, 0.05 M phosphate buffer (pH 7.4); stirring rate (B), 300 rpm.
enhancements over the platinum and dense-film electrodes, respectively, at 1 X 10-3M). Notice also that while the porous electrode displays a curvature in the response (characteristic of biocatalytic control), the platinum- and dense-film-based sensors yield highly linear plots. Apparently, the electropolymerization onto these solid transducers results in a buildup of a thicker PPD layer, and yet mass transport limited reactions. No response was observed for analogous measure1990 Analytical Chemistry, Vol. 66, No. 13, July 1, 1994
ments at a glassy-carbon based sensor (not shown). An apparent Km value of 4.69 mM was calculated from the correspondingLineweaver-Burk plot of the porous-film data. Extended linearity over the entire range was observed at the porous-film sensor upon doubling the biocatalytic activity in the polymerization solution (not shown), indicating a change to mass transport limitation. This was accompanied by a decreased sensitivity (slope of 0.47 vs 0.60 pA/mM). The greatly enhanced biosensor response accrued from the porous film transducer is attributed not only to a larger microscopic area (vs that of the dense film), but also to its morphology that results in higher enzyme loadings within the micropores. Conventional (nonenzymatic) amperometric measurements of ferrocyanide or dopamine at the porous film yielded only 3-4-fold signal enhancements (vs the dense film), as compared to a ll-fold magnification of the biosensor response. The relatively small signal enhancement for the nonenzymatic reactions reflects the changes in the surface area, and is in agreement with the 4-fold increase in the chargingcurrent (indicated from blank cyclic voltammograms; not shown). This small change is expected from the nanoscopic film thickness. We estimate that the films used here consist of three to four overlaid rings of carbon. Hence, the significant enhancement of the biosensor response is attributed primarily to the large void volume of the porous films that results in high loading of the bioactive materials. The porous carbon film represents an active form of carbon, which offers a facile anodic detection of relevant oxidizable compounds (e.g. hydrogen peroxide). Figure 5 displays a M glucose over hydrodynamic voltammogram for 2 X the 0.2-0.9-V potential range. Apparently, the oxidation of the liberated peroxide species starts at 0.4 V, with the response rising sharply up to +0.8 V, and levels off at higher values. Common glassy-carbon or carbon-paste transducers usually require higher operating potentials (with theoxidation starting around 0.6 and 0.8 V, respectively). The drawn out wave suggests that the oxidation process is not reversible. All subsequent glucose sensing was thus carried out at +0.8 V. Potential interferences, from coexisting electroactive species can be addressed by the discriminative (permselective) properties of the PPD Metal-doped carbon films (currently being developed at Sandia NL) should further lower the operating potential.
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I I I POTENTIAL IVI 5 10 Flgunl. Effectoftheoperatingpotentialuponthechronoampermtric TIME CONCENTRATION(mM) responsefor2 X l~Mglu~~~~atthePPDlGOxlporowcarbonsensor. Figure 7. Chronoampermtrlc responseof the Rh/GOx/porouscarbon Other conditions are as in Figure 3A. sensor to successive increments of 5 X lo4 M glucose wlth a potential step to +0.3 V. Other conditlons are as in Figure 3A.
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TIME Flgure 8. Amperometric response of the PPDltyroslnaselporous carbon sensor to successive Increments of 2 X lo-' M catechol. Operating potential: -0.2 V. Other conditions are as in Figure 38.
The response of the PPD/GOx/porous carbon film sensor is reproducible and stable. The short-term stability was evaluated in a series of 80 repetitive chronoamperometric measurements of 2 X 10-3 M glucose (not shown; conditions, as in Figure 3A). Such a prolonged experiment yielded a mean peak current of 756.1 nA, a range of 700-786 nA, and a relative standard deviation of 2.7%. The sensor displayed a highly stable response for about 30 days (with intermittent usage and storage at 4 OC in phosphate buffer; not shown). A slow decrease in the sensitivity (of up to 25%) was observed during the 40-50-day period, with a faster decrease at longer periods. A similar behavior was reported for the PPD/GOx/ reticulated-vitreous carbon sensor, and attributed to the stabilization action of PPD.5
PPD/Tyrosinase Porous-Film Electrodes. Other biocatalytic sensors can benefit from the attractive properties of the porous-carbon film transducer. For example, the entrapment of tyrosinase on the walls of the micropores results in effective detection of the corresponding phenolic and catechol substrates. Figure 6 displays calibration data for catechol at the PPD/tyrosinase/porous-carbon sensor. A well-defined and fast amperometria response is observed (at -0.2 V) for these 2 X lV7 M increments in the substrate concentration. An extremely low detection limit of 2.5 X le8M catechol can be estimated based on the signal-to-noisecharacteristics (S/N = 3) of these data. A slight curvature was indicated from the resulting calibration plot (not shown). A highly linear plot resulted from another calibration experiment involving ten increments of the phenol concentration (over the 1 X 106 to
1 X l e 5 M range; not shown). The response of the PPD/ tyrosinase/porous film sensor is also highly reproducible; a relative standard deviation of 2.0% characterized 20 repetitive chronoamperometric measurements of 5 X 10-6 M catechol (not shown). Structural differencesand/or the lower operating potential (Le. lower noise level) may account for the lower detection limit of the tyrosinasesensor (compared to theglucose one). Metalized Carbon Film Enzyme Electrodes. Another attractive route for localizing enzymes within the submicrometer pores of the porous film transducer is electrochemical codeposition with an appropriate metal. Such one-step metalization/immobilizationscheme has been used successfully for the preparation of carbon-fiber enzyme microelect r o d e ~ . ~InJ ~particular, the codeposition of glucose oxidase with platinum, rhodium, or palladium couples an efficient enzyme entrapment with a low-potential electrocatalytic detection of the peroxide product. Figure 7 demonstrtates the utility of the rhodinized porous carbon film enzyme (GOx) electrode for the detection of low glucose concentrations. Potential stepsto +0.3 V offer a favorablechronoamperometric response to the 5 X 1 V M changes in the glucose concentration. The response increases linearly with the substrate concentration up to about 5 X le3M; a slight curvature is observed at upper levels. Nevertheless, a well-defined concentration dependence is observed over the entire (5 X lo"' to 1.2 X le2M) range examined. The corresponding Lineweaver-Burk plot yields an apparent Km value of 34.2 mM. Conclusions. Ultrathin porous carbon films have been shown to be attractive materials for the preparation of amperometric enzyme electrodes. Such nanoscopic foams impart high sensitivity to biocatalytic sensors due to high enzymeloadings and large microscopic area. Yet, their small geometric area should be attractive for fabricating enzymebased diagnostic strips. The coupling of carbon films with microfabrication techniques' should be extremely useful for obtaining reproducible diagnostic strips. While the concept is illustrated in connection with tyrosinase and glucose oxidase, (9) Ikariyama,Y.; Yamanchi, S.; Yukiashi, T.;Ushioda, H. 1.Electrochem. Soc. 1989, 136, 702.
(10) Wang, J.; Angnts, L. And. Chem. 1992,64,456. ( 1 1 ) Niwa, 0.;Tabei, H. Anal. Chem. 1994.66, 285.
it could be extended to other enzyme/substrate systems, and to other bioactive materials, in general. Such porous films may prove useful in many other electroanalyticalapplications, including flow detection, stripping analysis or as twodimensional composite electrodes (by filling their pores with an insulator). The film morphology can be tailored, by manipulating the preparation conditions, to further improve the performance, as needed for meeting future analytical challenges.
ACKNOWLEDGMENT This work was supported through Sandia National Laboratories under U S . Department of Energy Contract DEAC04-94AL85000. The authors acknowledge G. Zender for taking the SEMs. Received for review January 3, 1994.
Accepted March 23,
1994.6 Abstract published in Advance ACS Abstracts, May 15, 1994.
