Anal. Chem. 1986, 58, 2872-2874
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on reservoir size. Figure 5 shows a comparison of the fluorescence spectrum of pyrene in a cuvette to that of the same solution when flowed through a thin film. This suggests that molecular fluorescence in thin liquid f i i s may be a useful as a detection technique for liquid chromatography or flow injection analysis. Current research is aimed in this direction.
Registry No. CTAB, 57-09-0; pyrene, 129-00-0; quinine, 130-95-0;p-aminobenzoic acid, 150-13-0. LITERATURE CITED (1) Mysels, K. J. J . Phys. Chem. 1964, 68, 3441-3448. (2) Radoev, B. D.; Dimitrov, D. S.;Ivanov, I.B. Colloid Polym. Sci. 1974, 252,50-55.
(3) Ivanov, I. B.; Dirnitrov, D. S. Colloid Polym. Sci. 1974, 252, 982-990. (4) Manev, E. D.: Vassilieff, Chr. St.; Ivanov, I. 6.Colloid Polym. Sci. 1976, 254, 99-102. (5) Parker, C. A. Photoluminescence of Solutions: Eslevier: New York, 1968; Chapter 5.
Bradley T. Jones James D. Winefordner* Department of Chemistry University of Florida Gainesville, Florida 32611
RECEIVED for review March 25,1986. Accepted June 25,1986. This research was supported by NIH-5R01-GM11373-22.
Polyacrylamide/Graphite and Polyacrylamide/Titanium Dioxide Gel Electrodes Sir: Polyacrylamide (pacr) gels have attractive properties that are the basis for several important applications in bioanalytical chemistry. For example, they provide a hydrophilic environment with a controllable pore size that allows for the entrapment and immobilization of bioplymers in their active forms. Pacr gels have long been used as matrices for gel electrophoresis and gel permeation chromatography. Recently, we described the construction of an enzyme electrode, based on the ideas of Hill and co-workers ( I ) , that consisted of an enzyme/mediator system entrapped in a pacr interface between a carbon support bed and the solution ( 2 ) . These pacr-modified electrodes mediated the direct and specific amperometric oxidation of glucose. Others ( 3 )have studied pacr-modified electrodes, and a variety of applications based on the electrochemically driven swelling of polyelectrolyte gels can be envisioned (4). Thus these electrodes show promise as permeable, multicomponent interfaces between an electrode substrate and solution. The purpose of the work described here was 3-fold: first, to study charge transport through the ferrocene/graphite/pacr gel composite; second, to document preliminary investigations of the photoactivation process occurring in the ferrocene/Ti02/pacr gel; and third, to examine the analytical utility of the pacr electrode. EXPERIMENTAL SECTION Reagents and Apparatus. All experiments (unless otherwise noted) were carried out in pH 7 Sorenson phosphate buffer solutions (0.0667 M) and potentials are referred to the Ag/AgCl reference electrode. Filtered distilled water (Milli-Q System, Millipore) was used to prepare solutions. Ferrocene was used as received from ROC/RIC. Acrylamide (Aldrich gold label) and riboflavin (Merck & Co., Inc.) were used to prepare the gels. Linear sweep and cyclic voltammetry and differential pulse voltammetry were done on a BAS-100 electrochemical analyzer. Chronocoulometry was performed with a Princeton Applied Research Model 173/ 175 potentiostat/programmer combination. Charge data were taken in digital form using a Nicolet Model 200 oscilloscope. Electrode Fabrication. Photopolymerization of acrylamide has been described previously ( 2 , 5 ) . The photopolymerization is terminated before the gel becomes firm, and then the gel is mixed with powdered graphite (Union Carbide Corp., Grade 38) to yield a mixture which is 40% by weight graphite with the consistency of chewing gum. This composite is then packed into a strip of reticulated vitreous carbon (RVC) and mounted into a Teflon sleeve to give the gel/graphite interface a geometrical surface area of ca. 0.28 cm2. Colloidal Ti02 was prepared in a manner similar to that described by Duonghong et al. (6). One milliliter of Ti(OCH(CH&),
Table I. Chronocoulometry of Ferrocene/Graphite/Pacr Gels" w t % Fc
104(Q/t1'2)/~o~1 s-lI2
10.5 14.2 18.5 22.7
3.61b
25.7
109D1~2c/mo1 cm-2 s-l/2 10.lb
3.04
8.5
2.31 2.60
6.4 7.2
2.77
7.7
a Cottrell slopes for applied potential steps from 0.0 to 0.5 V in pH 7 Dhowhate buffer. bRelativestandard deviation = 10%.
was added to 20 mL of 2-propanol. A 0.2-mL aliquot of this solution was slowly injected into 20 mL of pH 1 HCl solution. The resulting suspension showed an absorption band at 340 nm in agreement with the literature (6). The precipitate was collected, dried at 120 OC, pulverized to a fine powder, and used as above, in place of the graphite, to make the Ti02 gel electrodes. An Aminco-Bowman spectrophotofluorometer, Model No. 4-8202, was used to measure the wavelength response of these electrodes. A spectroline, Model llSC-2, Hg lamp was used to activate the electrodes. For the studies described below, the electrodes were baked in a drying oven at 125 "C until they appeared to be completely dehydrated. Of course this step was omitted in the previous study of pacr/enzyme electrodes (2). After dehydration, the electrodes were loaded by application of an aliquot of solution containing the analyte, which was rapidly imbibed by the interface. The loaded electrodes were then immersed in the pH 7 buffer and subjected to electrochemical analysis.
RESULTS AND DISCUSSION Chronocoulometry of Ferrocene/Graphite/Pacr Gels. The mechanism of charge transport through a gel matrix containing mediator sites or redox couples is a complex process. Electron exchange reactions along with incorporation of aqueous electrolyte and motion of the pacr matrix must occur. Electrolysis results in huge volume changes, which must be considered in a practical or theoretical analysis. Accordingly, it is difficult to predict a priori the expected time dependence of the chronoamperometric current or the possible role of finite diffusion limitations of the gel matrix. Table I gives chronocoulometric Cottrell slope data for electrodes in which solid ferrocene is dispersed in the gel matrix. The composition range encompasses that employed in the enzyme electrodes previously described (2). The chronocoulometric data displayed linear Anson slopes of Q
C 1986 American Chemical Society 0003-2700/86/0358-2872$01,50/0
ANALYTICAL CHEMISTRY, VOL. 58, NO. 13, NOVEMBER 1986
0
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Scheme I
JFC
40
t
LIGHT OFF-LIGHT
ON-LG IM
I IO
i0
30
O F -
Qb
8b
60
7b
eb
CYCLE
Figure 1. Variation of the cathodic peak current at 20 mV/s for the reduction of ferricinium ion in a polyacrylamide/Ti02gel electrode upon illumination. I .ti
vs. t1I2 over an extremely wide time range. Even a t times as long as lo3 s apparent linear diffusion behavior is seen and there is no evidence of finite volume limitations on the charge transport process. During the course of the constant potential electrolyses, the pacr electrodes absorb solvent/electrolyte and swell to several times their initial size. Others ( 4 ) have observed similar phenomena for electrolysis in polyelectrolyte media. As a result the mass transport process operative is clearly complex, and it is surprising that simple Cottrell behavior is observed. Similar apparent diffusion control is also seen for the cyclic voltammetry of the ferrocene/graphite/pacr gels where the peak currents have classical Randles-Sevcik shapes and are proportional to u0,41 for sweep rates less than ca. 1 V/s. The data of Table I indicate that the charge transport rate constant is not strongly dependent on the ferrocene composition. There is a slight decrease in the Cottrell slopes as the percent ferrocene is increased. This may be associated with some kind of restriction of the film swelling process a t the higher percent ferrocene compositions. Finite volume limitations are not apparent since the ferrocene concentration is maintained approximately constant by dissolution from the entrapped particles. When the graphite is omitted from the electrode formulation, very small peak currents and Cottrell slopes are observed, presumably due simply to diffusion to the RVC support bed. Thus the role of the graphite is viewed as a way of extending the electrode area in a manner similar to the approach of Burgmayer and Murray (7) for redox polymer films. An effective electrode area of ca. 50 cm2 is calculated form the data of Table I and the solubility of ferrocene in water, 1.7 X M (B), assuming a diffusion coefficient of 1X cm2/s. Ferrocene/Ti02/Pacr Gels. In order to demonstrate the versatility of parc gel electrodes, experiments were carried out in which T i 0 2 particles were used in place of graphite in the electrode formulation. Upon exposure to UV/visible light these electrodes were “activated” with the Ti02 semiconductor particles effectively extending the electrode area. This behavior is shown in Figure l, which displays a plot of the peak current for the oxidation of ferrocene trapped in the gel upon successive cycles in the dark and upon illumination with a 254-nm Hg-vapor lamp (2000 pW/cm2). When the light is turned on, the peak currents gradually increase upon successive cycles (sweep rate, 20 mV/s) and reach a maximum after ca. 30 cycles. Temperature effects can be ruled out as a cause of this behavior since control experiments using a pacr composite coated thermocouple indicated that the temperature change was ca. 1 O C for a 10-min exposure to the light source. When the light source is turned off, the peak currents decay back to their original values only after long periods of time. This decay, which was not dependent on whether or not the electrode potential was cycled, is consistent with
1.7
I .E
1.5 c (
\
5
1.4
b W
2
1.3
8a
a
2
I.,
I. I .o
1
2
400
500
600
700
800
h(nrn)
Figure 2. Variation of the activation factor, 120/10 (see text), and the absorbance (arbitrary units) of TiO, colloidal solution with wavelength of excitation. The experlmental values of 120/10are given by the circles and the absorption spectrum (dashed line) is taken from ref 6.
surface charge build-up seen with slurry electrodes of T i 0 2 (9). The activation of these semiconductor/gel composites is understandable by a mechanism in which charge transport between Ti02 particles is mediated by the ferrocene+I0couple as shown in Scheme I. Further evidence that promotion of electrons from the valence to the conduction band of the semiconductor particles is involved in the activation process is afforded by the photoresponse experiment shown in Figure 2. In this experiment a series of electrodes made from a single batch of ferrocene/ Ti02/pacr was illuminated and cycled in a spectrofluorometer. The “activation factor”, defined by the ratio of the peak current on cycle number 20 in light to the peak current on the last cycle in the dark, tracks the absorption spectrum of a colloidal TiO, dispersion (IO). Voltammetry of Analyte Solutions Absorbed in Graphite/Pacr Gel Electrodes. The possible use of these electrodes as working electrodes for electroanalysis was investigated by using Fe(CN)63-/4-,benzoquinone (Q)/hydroquinone (H2Q),and methyl viologen dication (MV2+)/radical cation (MV+) redox couples. The procedure involves absorption and entrapping of a small volume of an analyte solution in a dehydrated gel electrode followed by voltammetric quantitation after contact is made to an external electrolyte solution. Advantages for this procedure over the usual practice of immersing the working electrode directly in the test solution include (i) the use of small volumes of analyte solution, (ii) minimizing adsorption of impurities on the active electrode
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Anal. Chem. 1986, 58, 2874-2876
surface due the large area/volume ratio in the gel electrode, and (iii) possible advantages in automation and the exclusion of macromolecules. For the relatively large geometrical area electrodes used in this study (0.28 cm2),aliquots of ca. 20-40 pL were applied to fresh electrode surfaces for ca. 5 min. The electrodes swell somewhat as the solution is absorbed by the gel. Application of additional aliquots to the surface did not result in absorption of more solution or increased peak currents. For Fe(CN)t- solutions differential pulse voltammograms exhibited a single wave a t 0.196 V after subtraction of the background current which contained peaks attributed to oxidation of the surface of the graphite particles. A calibration curve of peak current vs. Fe(CN)64-concentration was constructed by using data collected from three batches of graphite/pacr gel, each having slightly different characteristics (i.e., degree of polymerization and cross-linking). Although electrochemicalresponse varied somewhat because of variation of the polymer gel morphology, the calibration curve was linear over the concentration range 0.2-100 mM with a correlation coefficient of 0.9907 and an intercept of -80 PA. For the individual data sets obtained with the same gel, the correlation coefficients were 0.9938, 0.9960, and 0.9875. The significant feature of these data is the wide dynamic range for the solution entrapped in the gel electrode. It is anticipated that detection limits can be significantly lowered by minimization of background currents. Also by use of smaller electrodes, it should be feasible to analyze volumes of 1 FL or less. Thus this approach has promise for the analysis of small volumes containing amounts in the 100 nmol range or less. The technique was also applied to p-benzoquinone solutions (10% in ethanol), which gave a linear calibration curve over the concentration range 1-50 mM. For HzQ, however, very large oxidation currents were observed that did not give well-defined peaks. This phenomonen is under further study. Solutions of MV2+absorbed in the gel electrodes gave welldefined peak currents, but the dication rapidly diffused out of the swollen electrode limiting the analytical utility of the method. In this situation it will be necessary to carry out the voltammetric quantitation with the loaded gel electrode contacting the electrolyte solution in a thin-layer configuration.
CONCLUSIONS Polyacrylamide gel electrodes have been fabricated by use of relatively simple procedures that require only readily available, inexpensive materials. It has been demonstrated
that complex, multicomponent, electroactive interfaces can be constructed using ordinary pacr gels. Specifically, composite electrodes containing graphite particles, solid mediator (ferrocene), semiconductor particles (TiOz), and enzymes (glucose oxidase (2))have been shown to function as catalytic and photoresponsive gel electrodes. The possible analytical utility of these electrodes was demonstrated for the differential pulse voltammetric analysis of small volumes of ferrocyanide and p-benzoquinone solutions. Charge transport through the ferrocene-loaded pacr gels obeys the laws of simple linear diffusion over a wide time scale. The Cottrell slopes were not strongly dependent on the percent ferrocene in the electrode formulation a t loadings above ca. 10%. The absence of a simple finite volume limitation of the mass transport process is probably related by the large volume changes that take place upon electrolysis with these electrodes, but a complete understanding of the change transport process awaits further work. Registry No. pacr, 9003-05-8;TiOz, 13463-67-7;Fe(CN):-, 13408-63-4;graphite, 7782-42-5;p-benzoquinone, 106-51-4;ferrocene, 102-54-5.
LITERATURE CITED (1) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hili, H. A. 0.; Aston, W. J. HiQQinS, I. J.; Piotkin, E. V.; Sott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 5 6 , 667. (2) Lange, M. A.; Chambers, J. Q. Anal. Chim. Acta 1985, 175, 89. (3) Van Koppenhagen, J. E.;Majda, M. J. Nectroanal. Chem. 1985, 189, 379. (4) Osada, Y.; Hasebe, M. Chem. Lett. 1985, 1285. (5) Oster, G. K.; Oster, G.; Prati, G. J . Am. Chem. SOC. 1957, 79, 595. (6) Duonghong, D.; Borgarello, F.; Gratzel, M. J. Am. Chem. SOC.1981, 103, 4685. (7) Burgmayer, P.; Murray, R. W. J. Hectroanal. Chem. 1902, 135, 335. (8) Kolthoff, ,I. M.; Thomas, F. G. J. Phys. Chem. 1965, 6 9 , 3045. (9) Dunn, W.; Yosihiro. A.; Bard, A. J. J. Nectrochem. SOC.1981, 128,
222.
(IO) Duonghong, D.; Ramsden, J.; Gratzel, M. J. Am. Chem. SOC.1982, 104, 2977.
Mark A. Lange James Q. Chambers* Department of Chemistry University of Tennessee Knoxville, Tennessee 37996
RECEIVED for review March 18, 1986. Accepted July 1,1986. This research was supported by grants from Martin Marrietta Corp. and the National Science Foundation (Grant No. CHE-8219210).
Fiber-optic Probe for Kinetic Determination of Enzyme Activities Sir: The knowledge of enzyme concentration of a sample solution is of considerable practical interest in clinical analysis, and numerous methods have therefore been proposed for assays ( I , 2). Most of these are kinetic, based on the measurement of changes in the concentration of a natural or synthetic substrate. Assays have mainly been performed in solution or on “dry reagent” phases (3). None of them, however, is suitable for in vivo application. Our interest in photometric and fluorometric enzyme activity determination ( 4 , 5 )along with that in fiber-optic sensors (6) resulted in the idea to determine enzyme activities via fiber optics in very small sample volumes with the aim to develop a method for an in vivo assay in blood and related biomatter. Small optical fibers have already been used for in vivo sensing
of pH (3,oxygen (8),or pH, oxygen, and C 0 2simultaneously (9) because fiber sensors have certain advantages over electrochemical sensors. Biocompatibility, small size, and the lack of reference cells appear to be the most attractive features. Arnold (IO) has recently described the immobilization of the enzyme alkaline phosphatase (together with light-scattering material) a t the end of a fiber-optic probe. When immersed into a substrate solution such as p-nitrophenyl phosphate, the second fiber sees the formation of yellow pnitrophenolate. This communication describes our experiments directed toward a continuous kinetic enzyme assay via optical fibers. In contrast to the method of Arnold (IO), the substrate is immobilized in this case, and the formation of the hydrolysis
0003-2700/86/0358-2874$01.50/0 0 1986 American Chemical Society
