Amperometric Response Enhancement of the Immobilized Glucose Oxidase Enzyme Electrode Leroy D. Mell' and J. T. Maloy* Department of Chemistry, West Virginia University, Morganto wn, W. Va. 26506
Digltal simulation has been used to model the amperometrlc response of an immobilized enzyme electrode followlng periods of electrode inactivity. Average currents greater (by a factor of 10) than the steady-state current response are observed experlmentally and predlcted by the simulatlon. Callbration curves have been constructed for glucose concentrations from 0.1 to 1.5 mM where the steady-state current response is significantly below observed background currents (ca. 0.15 FA). The extent of enhancement is predicted by the simulation model to be related to 7, the time allowed for buildup of electroactive product within the membrane, and lndependent of the velocity of the enzyme reactlon and substrate concentration. Agreement between simulation theory and experimental behavior is observed using a glucose oxldase electrode meeting diffusion-control crlterla established by the simulation model.
Enzyme electrodes are widely used in analytical chemistry at this time. In combination with ion selective electrodes, enzyme electrodes allow for the potentiometric determination of a wide variety of substrates ( I , 2). Amperometric methods have also been proposed (3-5). In either case, attempts have been made to increase the sensitivity of the method for use in cases where small sample volumes containing low concentrations of substrate must be analyzed. Various methods of enhancement have found application in analytical chemistry. In the analysis of water samples, membrane concentrating techniques for the analysis of pollutants and trace metals have been employed (6, 7). Blaedel and Kissel (8) have shown that ion exchange membranes can be used for the concentration enrichment of ionic species and, in particular, to increase the sensitivity of ion selective electrodes. Lowry (9, IO) has reported a novel microanalytical system which utilizes enzymatic cycling to chemically amplify the concentration of substrates important in clinical and biological analysis. A model for an amperometric enzyme electrode obtained through digital simulation and applied to the immobilized glucose oxidase enzyme electrode system has been reported previously ( 5 ) .In this treatment, digital simulation was used to model the amperometric response of the glucose-sensitive electrode. From the results of the simulation, optimum operational parameters for a given enzyme electrode system were predicted and pertinent kinetic and diffusion properties of the system were evaluated. The amperometric glucose oxidase electrode has been employed by monitoring the steady-state current response due to reduction of iodine produced in the following enzymesubstrate reaction sequence
. Glucose + O2
Glucose oxidase
--+
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(1)
Present address, Navy Medical Research Institute, Bethesda, Md.
20014.
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Of particular interest in this case is the apparent buildup of iodine within the membrane thin layer due to the ongoing enzyme catalysis during periods of electrode inactivity. This increase in iodine concentration throughout the membrane when electrolysis is not occurring, is observed experimentally as an increase in the average current (relative to steady-state current) passed in short time intervals following the onset of electrolysis. The enhancement of amperometric response increases the sensitivity of the enzyme electrode system and thereby lowers the limits of detection placed on the system due to relatively high background currents. In this work, the effect of measured periods of electrode inactivity upon the average current passed during short time intervals following the onset of electrolysis is treated experimentally and theoretically. As in the previous effort ( 5 ) ,the prediction of the amperometric and coulometric electrode response is effected through digital simulation techniques.
EXPERIMENTAL The instrumentation, the procedure for enzyme immobilization, and a description of the solutions and reagents used have been given previously (5).The procedure for measuring the current-time and charge-time curves was altered slightly to allow for periods of electrode inactivity between successive chronoamperometric and chronocoulometric experiments. Figure 1 shows the simultaneous current-time and charge-time curves obtained for a 12 mM glucose solution. After the addition of glucose, the enzyme electrode potential was stepped to -0.2 V vs. SCE and the electrode was allowed to reach steady-state operation. In Figure 1,this steady-state current was recorded between points a and b. Point b marks the initiation of a period of electrode inactivity during which no potential was applied so that no current passed; this and subsequent periods of electrode inactivity were initiated after steady-state conditions had been obtained. The time interval T , measured between points band c, represents the time between successive simultaneous chronoamperometric-chronocoulometric experiments during which the electrode operated a t open circuit potential. At point c, the electrolysis was resumed by again stepping the electrode potential to -0.2 V vs. SCE. The corresponding amperometric-coulometric response was recorded for the duration t between points c and d. At point d, steady-state current conditions had been reached and the electrolysis experiment was repeated in the sequence specified. Experimentally, the magnitude of the charge which accumulates to time t during the electrolysis experiment varies with T . For identical electrolysis times an increase in Q ( t )can be brought about by increasing T provided that T is less than that time required for a maximum amount of product to accumulate within the membrane. This effect may be observed by comparing the values of Q ( t )obtained for identical values o f t with the duration of the previous period of electrode inactivity ( T ) . The overall effect of variations in T is such that the accumulated charge must be referenced with respect to T as well as t . This relationship is investigated in the digital simulation reported below. For the experimental current-time and charge-time curves shown in Figure 1the steady-state current response, i,, is approximately 0.3 PA with a corresponding background current, ib, of 0.15 FA. The relative thickness of the recorder response for the current-time curve may be due to pen fluctuations that occur as the sample solution is stirred. Previous studies (5) have demonstrated that this stirring is necessary to maintain reproducible boundary conditions a t the solution-membrane interface. These pen fluctuations may also be due
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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Figure 1. Simultaneous current-time
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These experimental curves were obtained with a typical enzyme electrode for a 12 mM glucose solution. The time between successive chronoamperometric and chronocoulometric experlments is represented by T ; tis the time duration of the electrolysis experiment
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to increased noise pickup at the high impedancemembrane covered electrode.
DIGITAL SIMULATION The appearance of product at the electrode surface, and the subsequent current response is dependent upon several factors including the bulk glucose concentration, the rate of enzyme catalysis, and the rate of diffusion of substrate and product within the membrane thin layer. The digital simulation model for the amperometric glucose oxidase electrode discussed previously accounts for these factors with two dimensionless parameters, CIK, and V. The former compares the bulk substrate concentration to the Michaelis constant, K,, and may be varied to show the substrate concentration dependence of the current. The latter is defined as
V = k 3 C ~ ‘d/ O K ,
(3)
where h3 is the rate constant governing the formation of product from enzyme-substrate complex in the catalysis sequence; CE represents the total enzyme concentration in the membrane layer; d is the thickness of the membrane layer; and D is the diffusion coefficient of substrate within the membrane thin layer. The parameter V compares the maximum rate of the enzyme reaction, k & ~ with , a known time associated with diffusion through the membrane thin layer, d 2/D. Because the enzyme-substrate reaction proceeds even when no electrolysis occurs a t the electrode surface, product buildup occurs across the membrane thin layer. In the absence of electrolysis, the extent of product buildup a t the electrode surface will depend on the length of time between amperometric experiments as well as those factors that determine conventional steady-state response. Generally, the longer the enzyme electrode is allowed to stand in substrate solution, the higher the distribution of product concentration across the membrane thin layer. This increase in product concentration will be observed as long as 7 does not exceed the time necessary to establish a steady-state distribution of product across the membrane thin layer in the absence of any electrode reaction. Once this steady-state distribution is obtained (when the rate of product transport into the bulk solution equals the rate of product generation in the membrane thin layer), no further increase in 7 will increase the amount of product in the membrane. Thus, for a given short period of time into the 1598
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Simulated concentration profiles for product, P, at various electrode inactivity
Values of DT/d2 are indicated on each concentration’profile and range from 0.001 to 3.0. In each case V = 100. Panels b and c represent moderate and low substrate concentration; panel a, high substrate concentration. Note that all concentrations are rendered dimensionless with K,. The dashed line represents the steady-state glucose concentration profile
amperometric experiment, the average current passed due to electrolysis of product generated during periods of electrode inactivity will be higher than the steady-state current approached a t longer electrolysis times. The digital simulation program previously described which provided the model for the amperometric enzyme electrode needed only to be altered slightly to account for the current enhancement due to variations in the time between successive experiments. In that treatment, the diffusion-limited reaction of the product at the electrode was simulated by first establishing the proper boundary values (zero product concentration) at both the electrode-membrane interface and the membrane-solution interface. The model catalysis-electrolysis was then allowed to proceed for a pre-determined number of iterations (each of duration d 2 / D L ,where L is a known number of iterations, typically 1000) until a dimensionless steady-state current had been achieved. The effect of electrode inactivity, then, may be simulated by altering the boundary condition a t the electrode-membrane interface to account for the buildup of product in the vicinity of the electrode. (The other boundary condition, maintained by solution stirring, remains unchanged.) If the product accumulation is allowed to proceed for a known number of time iterations, M, before electrolysis is continued, a time parameter involving 7 results
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
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