690
Anal. Chem. 9002, 6 4 , 630-635
because an enzyme is generally deactivated to some extent by immobilization onto a solid support. In Figure 8 the simulated and experimental calibration curves show fairly good agreement with each other. A larger discrepancy between the two for the cross-linked membrane electrode may reflect the situation that the present model does not completely accommodate enzyme cross-linked membrane electrodes even though the membrane is much thinner than the diffusion layer. A cause for this discrepancy may be that the ec value is smaller in the crw-linked membrane electrode than in the monolayer electrode, though the value of ec is common between the two cases in the simulation. The response saturation for the cross-linkedmembrane electrode beyond Cs = lo+ mol at the mediator (disaolved oxygen) concentration of 2.5 X mol is a real phenomenon because the response is increased by oxygen bubbling into the electrolyte so1ution.l
NOMENCLATURE a b
C
c, co
D e F
> Jpot.
kl,
kE n
kz
r
total surface density of the enzyme (mol cm-2) Subscripts C charge M mediator N charged mediator S substrate
ACKNOWLEDGMENT We are grateful to Dr.Y. Okawa for useful diecussions. This work was supported in part by the Chemical Materials Research and Development Foundation. Registry No. Glucose oxidase, 9001-37-0. REFERENCES (1) Okawa, Y.; Tsuzukl, H.; Yoshida, S.; Watanabe, T. Anal. Sc/. 1989, 5 , 507-512. (2) Tatsuma, T.; Okawa, Y.; Watanabe, T. Anal. Chem. 1989, 67, 2352-2355. (3) Miyasaka, T.; Koyama, K.; Watanabe, T. Chem. Len. 1990,627-630. (4) Tateuma, T.; Watanabe, T. Anal. Chim. Acta 1991, 242, 85-89. (5) Tatsuma, T.; Okawa, Y.; Tsuuki, H.; Yoshlda, S.; Watanabe, T. TMn w m F Y ~ 1991,202, S 145-150. (6) Tatsuma, T.; Watanabe, T. J . Ektraenal. Chem. Interfac&lE&ctro&em. 1991, 370, 149-157. (7) Tatsuma, T.; Watanabe, T. Anal. Chem. 1991, 63. 1580-1565. (8) Tatsuma, T.; Watanabe, T. Anal. Chem. 1992, 64, 143-147. (9) Mell, L. D.; MaiOy, J. T. Anal. Chem. 1976, 47, 299-307. (10) Gough, D. A.; LeypoMt, J. K. Appl. Bbchem. Bloeng. 1981, 3, 175-206. (11) LeypoMt, J. K.; Gough, D. A. Anal. Chem. 1984, 56, 2896-2904. (12) Tatsuma, T.; Watanabe, T. Anal. Chem., following article in this issue. (13) BOWWS, L. D. Anal. Chem. 1988, 56, 513A-530A. (14) FTabhu, V. G.; Zarapkar. L. R.; Dhaneshwar. R. G. .€kf".Acta 1981, 26. 725-729. (15) Glbson. Q. H.; Swoboda, B. E. P.; Massey, V. J . Bbl. Chem. 1984, 239, 3927-3934.
distance between the electrode surface and the plane of the enzyme active center (cm) thickness of the diffusion layer (cm) concentration in the bulk (mol ~ m - ~ ) concentration on the plane of enzyme active center (mol ~ m - ~ ) concentration on the electrode surface (mol ~ m - ~ ) diffusion coefficient (cm2s-l) transfer efficiency of char e or mass Faraday constant (C mol- f) output current density (A cm-2 s-l) flux (mol cm-2 s-l) potential rate (flux) of the enzymatic reaction (mol cm-2 s-l) enzymatic reaction rate constant (cm3 mol-' s-l) RECEIVED for review July 1, 1991. Accepted December 20, electrode reaction rate constant (cm s-l) charge number 1991.
Model Analysis of Enzyme Monolayer= and Bilayer-Modified Electrodes: The Transient Response Tetsu Tatsuma* and Tadashi Watanabe* Institute of Industrial Science, University of Tokyo, Roppongi, Minato-ku, Tokyo 106, Japan Yusuke Okawa Department of Image Science, Faculty of Engineering, Chiba University, Yayoi-cho, Chiba 260, Japan
The translent responses of enzyme monolayer- and bllayermodlfled electrodes were thwretlcally analyzed. The response was numerically dmulatod wlth parameters wed In the foreggolng paper. The tradent proteas condsto of three elementary steps: enzymatlc reactlon step, substrate and medlator consumptlon step, and charge-carrylng medlator accumulation step. Contrlbutlonr of the paranntors to them elementary steps and the overall response were examined. Experlmental observations are succeu(ully Interpreted In terms of the thwretlcal results.
INTRODUCTION Analysis of enzyme electrode responses is of much importance from the viewpoint of sensor design and optimization.
Steady-state response analysis gives valuable information for designing more sensitive sensors. In addition to the sensitivity, the response time is also a crucial factor in sensors. However, studies on transient analysis are scarce14 compared to those on steady-state analysis, due mainly to the complexity in the analytical procedure. We have been developing amperometric biosensors with monolayers or heterobilayers of such functional molecules as enzymes, immobilized on a solid electrode.+12 In the foregoing paper13 we analyzed the steady-state response of enzyme monolayer- and bilayer-modified electrodes on the basis of their simple models and theoretically proved the high sensitivity per enzyme molecule of the sensor. The procedure of the steady-state analysis was much simpler than that for enzyme multilayer-carrying electrodes fabricated by enzyme cross-linking or polymer entrapment. The simple equations
0003-2700/92/0364-0630$03.00/00 1992 Amerlcan Chemlcal Soclety
ANALYTICAL CHEMISTRY, VOL. 64, NO. 6, MARCH 15, 1992
to
and mediator concentrations in the vicinity of electrode surface decrease and then reach a steady state. Hence this step contributes to the response decay. In step 3, the charged mediator is accumulated in the vicinity of the electrode surface and then reaches a steady state. The catalytic current of a sensor is dictated by three factors: (1)the efficiencies of the substrate and mediator supply, es (=C,/Cs, see Figure 1) and eM(=Cd/C,), (2) the flux of chargecarrying mediator generation, J (function of es and eM), and (3) the efficiency of the enzyme/electrode charge transfer, eC.lo The catalytic current density i of the sensor is given by
Bilayer Monolayer
h
i = nFecJ “ O a
631
b
Distance ( c d Fl~uro1. Schematic drawing of concentratlon profiles of S, M, and N in the mode4 of enzyme monolayer-modifled electrode.
derived from the analysis predict the steady-state currents and are successfully used to interpret our own experimental observations. In the present paper, we analyze the transient responses of enzyme monolayer- and bilayer-modified electrodes based on the same interfacial models as before.” Three elementary steps, namely the (1)enzymatic reaction step, (2) substrate and mediator consumption step, and (3) charge-carrying mediator accumulation step, were first treated separately and then were combined to examine the overall transient process. The results were used to interpret our own experimental observations on enzyme monolayer- and bilayer-modified electrodes. In this paper, we introduce the 80% response time, which is time elapsed until the signal reaches 80% of the steady-state value, which is derived from a simple formula by the foregoing anal~si5.l~This significantly reduces the computation time. MODEL The model of an enzyme monolayer-modified electrode employed here is the same as the one in our preceding paper on steady-state response analy~i5.l~ The electrode senses a substrate on the basis of charge transfer from the substrate to electrode via an enzyme and a dissolved electron mediator. The reactions can be formulated as follows:
(4)
where n is the charge number and F is the Faraday constant. Steps 1-3 are the processes in which J, es and eM,and ec approach their steady state, respectively. SIMULATION Mass-Transfer Step. The mass-transfer step, namely the substrate and mediator consumption step or the charge-carrying mediator accumulation step, was numerically treated by dividing the diffusion layer into N layers with thickness Ax ( = b / N ) and concomitantly defining a unit time At. The concentration in the Xth layer at time t (=TAt), C(X,Tj,is calculated from the following equation derived from the initial concentration profiles at t = 0 and Fick’s second law:
C(X,T) = C(X,T - 1) + (DAt/k2)[C(X + l,T- 1)-
2C(X,T - 1) + C ( X - l,T - l ) ] ( 5 )
where D is a &ion coefficient. Here the value of DAt/Ax2 should be equal to or smaller than 0.5. The number of layers N was generally set at 100 in this work. Choice of a larger N value yields a more precise response time but requires a longer calculation time. Except for a few cases where a larger N was needed, it was ensured under different conditions that N = 100 is sufficient to yield the 80% response times within an error of several percent. Enzymatic Reaction Step. The enzymatic step consisting of eqs 1and 2 was also simulated numerically. The surface densities of E and E’ ( r E and rE*) are calculated from the following equations: rE(T)
= (1- k,C,s&)rE(T- 1)+ k&&Atr~,(T-1) (6)
rEu? = r - r,m
(7) The flux of chargecarrying mediator generation on the plane of the enzyme active center is given by J(T) =
where E, E’, S,P, M, and N denote the enzyme, the chargecanying enzyme, the substrate, the product, the mediator, and the charge-& mediator, respectively,and kl, kz, and kE are reaction rate The assumptions 1-9 made in the steady-state analysis” hold ale0 in the preaent analysis,where a, b, Cs, and CM denote the distance between the enzyme active center plane and the electrode surface, the stationary diffusion layer thickness, the concentration of S,and that of M, respectively. Figure 1 schematically illustrates the concentration profiles for S,M, and N in the vicinity of the electrode surface. Here we assume that the overall process consists of the following three elementary steps: (1)the enzymatic reaction step, (2) the substrate and mediator consumption step, and (3) the chargecarrying mediator (N)accumulation step. The step which reaches a steady state most sluggishly determines the response time. In step 1, the charged mediator (N)generation rate approaches a steady state. In step 2, the subetrate
~&.M~E’(T)
(8)
where r is the total surface density of E and E’. A preliminary examination showed that the 80% response times are obtained within an error of several percent when the unit time At is smaller than l/klCs X lo-’ and l/k2CMX lo-’. RESULTS AND DISCUSSION
I. Theoretical Evaluation. Chargecarrying Mediator Accumulation Step. Here the enzyme/electrode chargetransfer step alone is considered, and the other steps are assumed to be at their steady state. Further, C d = C M is assumed. As mentioned above, this is the step in which the enzyme/el&ode charge-transfer efficiency ec approaches ita steady-state value. Figure 2 shows simulated time courses of ec, which are normalized against the steady state, for a series of kE. The value of ec at the steady state, e=, is given by the following equation:” b-a en: = (9)
D N / ~+ Eb
832 ANALYTICAL CHEMISTRY, VOL. 64, NO. 6, MARCH 15, 1992
I
0.0 4
0
8
12
16
20
Time ( s ) Flgure 2. Simulated time courses of eC(normalized against the steady state) in the chargacarrying mediator accumulation step. Values of parameters: b = cm, a = 0 cm, DN= 10-~ cm2s-l,and k , = (I), io-' (21, (3), or (4) cm s-l. io3
0
0.2
0.4
0.6
0.8
1.0
a/b Flguro 5. Correlations between the simulated 80% response time of the charge-carrying mediator accumulation step and a . Values of parameters: b = lo-' cm, DN = cm2 s-', and k , = (0), lo-' (A), (0). or lo-' (V)cm s-'.
1.0 102
8
h
v
-
a 0
W
3
2
10'
s .*
PI
8 a
8
CT
0.5
w
E
100
z
8
0 m
0.0
10-1
0
2
4
6
8
10
Time (s) 10-2
10.~
k E (cm
io-2
5-l)
Dependencies of the simulated 80% response time of the charge-carrylng mediator accumulation step on k E . Values of pa- ~ rameters: a = 0 cm, DN= 10-~cm2 s-l,and b = lo3 (01, l ~ (A), or 10-l (0)cm. Flgure 3.
10-11
'
"
" "
' "'
10.~
J
"
10P
k E (cm s-')
Dependencies of the simulated 80% response time of the charge-carrying mediator accumulation step on k E . Values of pacm, a = o cm, and DN = 5 X 10" (01, 1 0 - ~(A), rameters: b = or 2 x (0) cm2 s-l. Flgure 4.
where DN is the diffusion coefficient of the charge-carrying mediator, N. The response time strongly depends on kE in the high kEregion. Figure 3 depicts the correlations between the 80% response time of ec and kE for various b. A thinner diffusion layer makes the response faster. Correlations between the 80% response time of ec and kE for a series of DN are shown in Figure 4. With DN increasing, the response time is decreased in the lower kE region, while increased in the higher kE region. Dependencies of the 80% response time of ec on u me shown in Figure 5 for various k p A shorter u makes
Figure 8. Simulated time courses of e, (nonnaiized against the Initial state) in the substrate consumption step. Values of parameters: b = lO-'cm, a = 0 cm, D , = cm2s-l, and k , r = lo4 (1). (3), or lo-' (4) cm s-l. (2),
the response faster. The response time of ec was found to be independent of the N generation rate; kl, r, and Ds do not affect the response time. A larger kE/DNmakes e= and thereby the sensitivity higher; hence a larger kE should be selected. A larger b leads to a faster response, but it generally drops the sensitivity of an enzyme monolayer-modified e1ectr0de.l~ Both steady-state and transient kinetics should be considered to optimize the b value. The distance a should be shortened to improve both sensor sensitivity and response time. Substrate and Mediator Consumption Step. Next we examine the substrate consumption step, though this is not significant for most of enzyme monolayer-modified electrodes in which substrate and mediator are not depleted. Here we assume that the other steps are much faster and that the enzymatic reaction step is in the linear region; namely reaction 1is much slower than reaction 2 and determines the enzymatic reaction rate. In this case, the mediator consumption rate need not be considered because the mediator supply to the enzyme is faster than the enzymatic reaction so that C , N C M holds even at the steady state.13 Under these conditions, the flux of substrate consumption at the active center plane is klI'Cd, where r = rE because reaction 2 is much faster than reaction 1.
As mentioned above, the substrate consumption step is the process in which the substrate supply efficiency es approaches its steady-state value. Figure 6 shows the simulated time courses of es for several values of k l r . The response time was found to be independent of Cs. With klF decreasing, the response time increases and then saturates, while the
ANALYTICAL CHEMISTRY, VOL. 64, NO. 6, MARCH 15, 1992
893
Table 1. Response Time of the Enzymatic Step kiCs, s-'
kpCM = 1S-'
100 10' 102
8.04 X lo-' 1.46 X lo-' 1.59 X 1.61 x 10-3 1.61 X lo-'
103 104
0
1
2
k&M
3
IO'S-'
1.46 X lo-' 8.04 X lo-* 1.46 X 1.59 x 10-3 1.61 X lo4
4
80% response time, s k2Cw = 102 8-1
1.59 X 1.46 X 8.04 x 10-3 1.46 x 10-3 1.59 X lo4
= lo3 8-'
k& 1.61 1.59 1.46 8.04 1.46
k&M
x 10-3 x 10-3 x 10-3 X
lo'
S-'
1.61 X lo4 1.61 X lo4 1.59 X lo4 1.46 X lo-' 8.04 x 10-5
lo4
x 104
5
Tine (s) -0 7. Simulated time courses of J (normalized against the steady state) In the enzymatic step. Values of parameters: k l C s = k,C, = 1 s-I and = 10-'* mol om-,.
r
steady-state es value rises. This step does not affect the total transient reaponse if the steady-state es value is close to unity. The dependence of the reaction time on b is similar to that in the charge-carrying mediator accumulation step; namely a thinner diffusion layer makes the response faster. The influence of Dson the reaction time of es is governed by the magnitude of klI' as in the charge-carrying mediator accumulation step; with Dsincreasing, the response time is decreased in the lower klF region, while increased in the higher klI' region. The control of r, which has become easier by the use of electropolymerized film recently, is important in this step. Larger I' shortens the response time, while smaller r makes this step negligible. For the steady-state kinetics,13larger r raises the sensor sensitivity but depresses Cs. In the saturated region, where reaction 2 determines the enzymatic reaction rate, C, is close to CSl3and the substrate consumption step need not be considered. In the nonlinear region bridging the linear and saturated regions, the flux of substrate consumption is expressed by k1kJC,C&/(klC, k2C&).13Therefore, the mediator consumption step must be considered simultaneously. On simulation of the mediator consumption step, the chargecarrying mediator accumulation step should also be considered concomitantly because the mediator regeneration on the electrode surface affects the consumption step. Such combined steps will be examined later in the section of the overall process. Enzymatic Reaction Step. The enzymatic step was simulated on an assumption that the system is kinetic controlled; Cas= Cs and C& = C M Figure 7 shows an example of the simulated time course of the charged mediator (N) generation rate, normalized against the steady state. The steady-state value of the generation rate, Jf,is as f01lows:'~
+
(10) The simulated response time was found to be independent of r. The 80% response times of the N generation rate are listed in Table I as a function of k,Cs and kzCM The response time is inversely proportional to both klCsand kzCMat a constant k,C,/k,C*
10
0
20
30
Time (mid F M 8. Simulated time causes of i(l),e, (2), and ec (3)(normallred against the steady state). Values of parameters: k , = k , = loe cm3 mol-' s-l,k , = 1 x 10-5 cm s-1, I' = 10-12 mol cm-2, b = 0.1 cm, cm2s-l, C , = mol cm-3, cM B = 0 cm, D , = D M= DN = = lo-' mol om3, and n = 2.
1.5
P
8?
1.0
-s
8
j
0.5
z
0.0
1
1
0
10
20
30
40
Time (mid Fbura 8. Simulated lraansient responses (normalized against the steady state). Values of parameters: k , = k , = 10' cm3 mol-' s-I, k , = lo-' cm s-', b = 0.1 cm, a = 0 cm, D , = 5 X lo* cm2s-', D, = DN = lo-' cm2s-', Cs5= C, = lo-' mol cm4, n = 2, and I? = 2 (3), or 2 x 10-l~(4) mol cm-,. x 10-l~(I), 5 x 10- (21,
Thus, C M alone is a controllable parameter which affects the response time of the enzymatic reaction step. Larger CM is preferable for the faster response as well as for the higher upper limit of the measurable Cs range. The rate constants kland k2 are generally lowered by enzyme immobilization onto an electrode surface. Efforts should be paid to retain the values of these constants, since smaller kl and k2 lead to delayed and lowered response. However, this step generally takes a shorter time than the charge-carrying mediator accumulation step and than the process of a stepwise change in substrate concentration from zero to Cson sample injection (>1s), so that this step is negligible before the overall response time in most cases. Overall Process. The above three steps cannot be treated independently when two or three of them are of comparable rates. Several examples of transient responses are shown in Figures 8 and 9. Tke currents are normalized against the steady-state values. In cases where es is not close to unity,
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namely where the substrate depletion step is not negligible, the overall process may be faster than the accumulation step. Figure 8 illustrates such an example. Curves 2 and 3 are for the elementary steps of substrate consumption and charged mediator accumulation, respectively. Curve 1, representing the overall process, shows an 80% response about 2-fold faster than curve 3. The transient responses depicted in Figure 9 exhibit current peaking and decaying. The charge-carrying mediator accumulates and the current increases firstly, then the accumulation is decelerated while approaching ita steady state, and fiially the accumulation becomes slower than the depletion of substrate or mediator to reault in a current decrease. Figure 9 indicates that the larger r becomes, namely the more significant the depletion becomes, the more conspicuous the peaking becomes. The peak current, as well as the steady-state current, depends linearly on the substrate concentration. In view of this, and also for the higher sensitivity and shorter response time, it is preferable to take the peak current rather than the steady-state current as the sensor output. Electrode Reaction Process. The electrode reaction process should also be considered in the case of enzyme heterobilayer-modified electrodes. This process is viewed as the transient process of underlayer enzyme system and can be simulated by a procedure similar to that for the monolayer system. The substrate for the underlayer system is the charged mediator (N) for the overlayer system. Hence the substrate concentration on the underlayer enzyme active center plane is regarded as the charged mediator (N) concentration on the electrode surface for the overlayer system, CON. The concentration profiles for a bilayer electrode are depicted in Figure 1. h mentioned above, a larger kE makes the response faster. From the viewpoint of response time, therefore, introduction of the underlying enzyme should improve kW Further, the transient process of the underlayer enzyme system should be faster than that for the overlayer system. 11. Comparison with Experimental Observations. Glucose Oxidase Monolayer-Modified Electrode. The present model is in line with the mechanism on a glucose oxidase monolayer-modified Sn02electrode, where S, M, and N are glucose, dissolved oxygen, and hydrogen peroxide, respectively. The 80% response time of this electrode was about 7 min at 5 mM glucose and at +900 mV vs Ag/AgC1.5 The transient response of this electrode was simulated with parameters evaluated as in the steady-state analysis:13 kl = 8 X 105cm3mol-' s-l, k2 = 3.2 X 108 cm3mol-' s-l, kE = 2 X lo4 cm s-l, r = 2 X 10-l2mol cm-2, b = 0.1 cm, a = 0 cm, Ds = cm2s-l, DN = 1.5 X cm2 7 X lo4 cm2s-l, D M = 2.5 X s-l, C M = 2.5 X lo-' mol cmT3,and n = 2. Under these conditions we obtain ec = 0.013, es = 1.00, and eM = 1.00. The substrate and mediator depletion steps can be neglected because es and eM are unity. The 80% response time of the respective steps for this electrode was calculated to be 0.3 s for the enzymatic step and 6.4 min for the hydrogen peroxide accumulation step. Therefore, the accumulation step determines the total response time here. A fairly good agreement between the simulated and experimental response time, as was also the case for the steady-state response,13supports the validity of the present analysis. The response time of the glucose oxidase electrode was examined at several electrode potentials, namely for a series of the kE value. However, no significant change in the response time was observed. This was rationalized by the response simulation; the 80% response time was calculated to be 6.5 and 6.1 m h for k E of 5 X lo-' and 1X lod cm s-l, respectively. Peroxidase Monolayer-Modified Electrode. Next a peroxidase monolayer-modified electrode, in which S, M, and
N are hydrogen peroxide, a ferrocene derivative, and its oxidized form, respectively, was examined. The electrode exhibited a response time of about 0.5-1 The electrode potential was +150 mV vs Ag/AgCl, and the mediator concentration was 0.2 mM. However, this response time may be determined by none of the above-mentioned factors but by the stirring effect. When the electrolyte solution was stirred, an anodic current probably reflecting electrooxidation of the ferrocene derivative was actually observed even in the absence of hydrogen peroxide. Though almost all the ferrocene exists in solution as the reduced form, a small fraction of it must exist as the oxidized form on the electrode surface. The size of this fraction can be determined by the Nemtian equation. After temporary stirring, the oxidized form was accumulated in the vicinity of the electrode surface and the anodic current decayed down to the background level in 0.5-1 min. Hence the true response time of the peroxidase electrode should be comparable to or shorter than 0.5-1 min. This stirring effect was also observed on a diaphorase monolayer-modified electrode employing ferricyanide or 2,6-dichloroindophenol as mediator.1° The enzymatic step and the oxidized ferrocene accumulation step were simulated by neglecting the stirring effect. The reaction of peroxidase occurs in three stages? (1) reaction of peroxidase and peroxide to give compound I, (2) reaction of compound I and mediator to give compound 11, and (3) reaction of compound I1 and mediator to give native peroxidase. Hence the simulation procedure for the enzymatic step was slightly modified here. Generally, enzymes are deactivated to some extent during immobilization onto an electrode surface. Even though the rate constants of immobilized peroxidase were assumed to be 2 orders-of-magnitude smaller than those in the native state, the simulated enzymatic step reached a steady state in 0.5 s. For the accumulation step, values of the parameters were assumed as follows: kE 1 cm s-l, b = 0.1 cm, a = 0 cm, and D M = lod cm2s-'. The 80% response times of this step were 40 and 0.7 s for kE = and cm s-', respectively. These results do not contradict the experimental observations. Glucose Oxidase/Peroxidase Bilayer-ModifiedElectrode. A glucose oxidase/peroxidase heterobilayer-modified electrode is a glucose sensor. In the electrode, hydrogen peroxide produced by the overlying glucose oxidase becomes the substrate for the underlying peroxidase. This electrode exhibited the 80% response time of about 0.5-2 min in the linear region, +150 mV vs Ag/AgCl (concentration of mediator, a ferrocene derivative, was 0.2 mM)? This response time is several times shorter than that for the glucose oxidase monolayer-modified electrode (see above). This can be caused by the accelerated hydrogen peroxide/electrode chargetransfer rate resulting from the introduction of peroxidase or, in other words, caused by the enhanced apparent kE for the peroxide. On the peroxidase monolayer-modified electrode, kE for hydrogen peroxide is about 2 orders-of-magnitude larger than that on a bare SnOz electrode. The response of the bilayer electrode was simulated with the same parametera as for the glucose oxidase monolayer electrode mentioned above, except for k E which was assumed to be 2 X lo4 cm here. The simulated 80% response time was 2.8 min, which is broadly in line with the experimental value. Peroxidase Model Electrode. We have devised a peroxiidase model electrode,llon which a peroxidase model (heme peptide) mimicking the vicinity of the peroxidase active center is immobilized on an Sn02electrode surface. The peroxidase model is small enough to communicate directly with the electrode, and hence the electrode functioned as a reagentless H202sensor. This means that the response process does not involve the charge-carrying mediator accumulation step.
696
Anal. Chem. 1902, 64, 635-041
Further, this electrode functions under kinetic-controlled conditions, hence the substrate depletion step is also negligible. These account for the short response time (
