Anal. Chem.
1994,66, 1520-1529
Prediction of Pocket-Portable and Implantable Glucose
Enzyme Electrode Performance from Combined Species
Permeability and Digital Simulation Analysis
Rathbun K. Rhodes,'ptp$ Mark C. Shults,tp* and Stuart J. Updiket
Department of Medicine, University of Wisconsin Medical Center, 600 Hbhland Avenue,
Madison, Wisconsin 53792, and Markwell Medical Institute, 1202 Ann Street, Madison, Wisconsin 537 13
Development of enzyme electrode sensors can be facilitated by
the use of computer simulation modeling techniques. In this
work, the model accounts for contacting solution effects,
changes in species diffusion and partition coefficients across
multiple membrane layer interfaces,cofactor limitationeffects,
enzyme loading,enzymekineticsand decay, and other variatiolrs
in electrode layer geometry. Experimentally determined single
species membrane permeabilities are included in the digital
simulations to predict the performance characteristics of a
multilayered glucose oxidase based enzyme electrode. Infor-
mation obtained includes range of sensor linearity under
different substrate and cofactor concentration combinations,
sensor time response and output as permeability parameters
and enzymeloading are varied, sensorwashout and interference
effects, and predictionof decay of sensorenzyme concentration
and resultant estimated sensor lifetime as a function of
contacting solution conditions and sensor permeability.
In the development of amperometric enzyme electrodes,
the need arises to quickly identify optimized sensor membrane
materials and geometries with a minimumof experimentation.
These sensor materials can serve one or more purposes,
including the following: (1) tailoring of electroactive species
flux and thus device output and time response, (2) preferential
extracting of either substrate or cofactor to linearize signal
in the desired measurement regime, (3) providing an immo-
bilized enzyme reaction layer for conversion of substrate and
cofactor to products (any of which may be monitored
electrochemically), (4) discriminating against either electro-
active interferences or other chemically reactive constituents
which would degrade underlying materials, and ( 5 ) providing
the requisite hydration and osmotic properties to maintain
electrode stability. This process can be expedited by the
development of a sensor simulation model which, with the
inclusion of a few known or experimentally determined
membrane material permeability parameters, can be iterated
upon in different final geometries to predict transient and
steady-state response curves. Thus, the experimenter can
specify the type of material and appropriate dimensions
required to fit the desired application.
Previous digital simulation models of enzyme electrodes
have been used to explain observed electrode transient behavior
and to predict steady-state outputs for a variety of membrane
parameters.1-8These papers explain extension of substrate
University of Wisconsin Medical Center.
* Markwell Medical Institute.
1520 Analytical Chemistry, Vol. 66,No. 9, May 1, 1994
measurement linearity through membrane diffusion control,'
the importance of the relative permeabilities of substrate and
cofactor in defining the usable sensor measurement range,"
the idea of simulating speciesflux through multiple membrane
layer^,^ transient matching of experiment to theory when
enzyme activity is varied and simultaneous step changes occur
for substrate and c0factor,6.~and decay of enzyme concen-
tration over time as a function of contacting solution
composition.8 In most of these simulations, the calculations
were performed in dimensionlessunits and in all cases assumed
no perturbation of concentrations in external contacting
solutions.
The sensor simulation model used in this paper solves the
problem of multiple species diffusion through a diffusion-
controlling resistance layer with reaction in an immobilized
enzyme layer. The mathematics to describe the simulation
can be reproduced easily by reviewing the diffusional portion
of solutions to prior simulations9and then adding in a reaction
term to account for enzyme kinetics. The species concentration
profiles and sensor outputs are solved by the Newton-Raphson
method carried through iteratively to a desired degree of
convergence.lo
This model expands upon the previous enzyme electrode
simulations by the inclusion of an unlimited number of
interfaces, thus allowing for effects due to both membrane/
solution partition and partition from one membrane material
to another. This permits changes in species diffusion coef-
ficient as an interface is crossed and allows different element
thickness from layer to layer. The model makes provisions
for calculating complete transients where any relevant species
is the limiting reactant and allows the typical calculations for
response variations due to membrane thickness, enzyme
activity, enzyme decay factors and kinetics, and species
diffusioncharacteristics. It allows concentration profile carry-
over and parameter modification from one simulation to the
next, thus making possible the description of significant but
previously unreported phenomena, such as sensor washout
Mell, L. D.; Maloy, J. T. Anal. Chem. 1975, 47, 299-307.
Leypoldt, J. K.; Gough, D.A. Anal. Chem. 19&4,56, 2896-2904.
Leypoldt, J. K.; Gough, D.A. Biotechnol. Bioeng. 1982, 24, 2705-2719.
Leypoldt, J. K.; Gough, D. A. Applied Biochemistry and Bioengineering;
Academic Press: New York, 1981; Vol. 3, pp 175-206.
( 5 )' Bergel, A.; Comtat, M. Anal. Chem. 1984, Sa, 29W2909.
(61' Tse, P. H. S.; Gough D.A. Anal. Chem. 1987, 59, 2239-2344.
(7j Lucisano, J.; Gough, D. A. A M I . them. I.WI,~O, 1272-1281.
( 8 ) Tse, P. H. S.; Gough, D.A. Biotechnol. Bioeng. 1987, 29, 705-713.
(9) Brumleve, T. R.; Buck, R. P. J. Electroanal. Chem. 1978,90, 1-31.
(10) Camahan B.; Luther H. A.: Wilkcs, J. B. AppliedNumerical Methods; John
Wiley; New York, 1969; pp 274-321, 436540.
0003-27OOf 94/0366-1520%04.50/0
0 1994 Amerlcan Chemlcal Socletv
Tabk 1
paramete9 solutionb resistance
D (GLU) 3.6 X 10-6 2.2 x 10-7
K 1 0.075
D (02) 2.1 x 10-4 6.0 x 10-7
K 1 3.5
D (HzOz) 1.0 x 10-4 5.8 x 10-7
K 1 1.41
D (ASC) (2-4) X 10-6 1.8x 10-7
K 1 0.15
D (ACETA) (2-4) X 106 5.2 X 10-8
K 1 8.8
Thickness$ cm 0.089 0.0040
Q Units for D are centimeters squared per seconds and X is dimensionless. K is the ratio of the equilibrium membrane layer concentration
relative to the solution layer concentration. b Note that literature solution layer diffusion coefficientshave been multiplied by 10 to account
for mixed diffusion/convection effects. Not measurable. d Thicknesses are known to be within &20%for the thin interferencelayer and within
+5% for all other layers.
transients and interference effects. We have chosen to carry
out these simulations without normalizing to dimensionless
units since we are working in a relatively small subset of
parameter space, and in our experience, the results were easier
to explain to nonspecialists who wished to evaluate our work.
Several experimental methods exist for obtaining the
necessary single-species diffusion and partition coefficients
to include in the simulation model. One can assume that the
concentration step technique commonly used with membranes
contacting gas is valid and that only minimal perturbation of
external concentration occurs.1 However, when membrane
permeability approaches that of the contacting solution, this
assumption is badly in error as speciesconcentration gradients
extend well into solution. Even rapid stirring cannot com-
pletely alleviate this problem.12 A better approach to separate
the membrane from solution permeabilities has been dem-
onstrated by Gough and Leypoldt with a rotating disk
technique.13J4Variations of limiting current by adjusting the
electrode rotation rate allow extrapolation to the membrane
permeability. Further combination of this technique with
analysis of transients due to voltage steps15 or concentration
steps16 allows an elegant separation of the membrane per-
meability into its diffusion and partition components.
In the absence of rotating disk instrumentation, a further
technique comes to mind and in fact is used in obtaining the
permeation parameters for our model. A concentration step
experiment is run and analyzed as for a gas contacting
membrane to obtain initial diffusion and partition coefficients.
These values, with the appropriate distance grid describing
membrane and solution regions, are then included in the
simulation model and iterated through several cycles providing
updated values for these parameters until the experimental vs
calculated transient is matched with respect to both output
and time response. This resultant information for single species
is then included as part of the complete enzyme electrode
model. Since the sensor’s solution boundary layer is not as
well-defined as in the RDE method, this technique does not
provide the same degree of accuracy in calculating diffusion
(11) Manoy, K. H.; Okun, D. A.; Reilley, C. N. J. Elecrroanal. Chem. 1962, 4,
65-92.
(12) Malone, D. M.; Anderson, J. L. AIChE J. 1977, 23, 177.
(13) Gough, D. A.; Leypldt, J. K. Anal. Chem. 1979,51,439-444.
(14) Gough, D. A.; Leypoldt, J. K. Anal. Chem. 1980,52, 1126-1130.
(15) Gough, D. A.; Leypoldt, J. K. AIChE J. 1980, 26, 1013-1019.
(16) Gough, D. A.; Leypldt, J. K. J. Electrochem. Soc. 1980,127, 1278-1286.
enzyme interference hydration
1.0 x 10-8 nmc 1.0 x 10-8
0.15 0 0.15
1.0 x 1od 1.0 x 10-7 1.0 x 10-6
1 1 1
5.0 X 10-8 3.3 x 10-8 5.0 X le
1 1 1
1.0 x 10-8 nmc 1.0 x 10-8
0.25 0 0.25
5.0 x 10-7 1.0 x 10-8 5.0 X 10-’
1.5 1 1.5
O.OOO64 o.OOO1 O.OOO64
and partition coefficients. However, for our purposes in
describing an overall electrode system and in making relative
comparisons of parameter variation on electrode performance,
this technique proved useful.
EXPERIMENTAL SECTION
The permeability parameters of the complete system are
listed in Table 1 under generic headings which describe the
contacting solution and membrane layer functions in sequence
(Le., solution, resistance, enzyme, interference, and hydration).
Four replicate samples of well-defined thickness for each
membrane layer type were used to verify the permeability
parameters obtained. Thicknesses for all layers were directly
measurable by micrometer to within f5%. Membranes were
given 24 h to hydrate fully prior to parameter measurement.
Resistance and interference layer permeability parameters
were obtained directly from single layer measurements.
Enzyme and hydration layer permeability parameters were
obtained indirectly from multilayer measurements where all
other layer parameters had already been determined. In the
complete four-layer membrane, the interference layer is 10
times thinner than the samples used for initially obtaining
-
permeability parameters and thickness is calculated to within
f20% from preparation volume, solution percent solids, and
resultant membrane square footage.
Enzyme layers of constant thickness, but with concentration
of glucose oxidase (Sigma Type VII) varied from 0.78 pM to
0.2 mM, were prepared to study effects of enzyme loading.
The0.2 mM concentration resulted in -4% enzyme by weight
within the enzyme layer. This by weight percentage scaled
proportionately with lowering of enzyme concentration.
Glutaraldehyde (Sigma Grade I, diluted to 0.1%) served as
the cross-linker. All four membrane layers were polyurethane
in nature and prepared by standard coating techniques.
Membrane samples for study are available from the authors.
The complete four-layer membranes define a portion of the
system used in the Direct 30/30, a pocket-portable electro-
chemical glucose sensing unit for diabetic patients.”
Diffusion and partition coefficients for the species hydrogen
peroxide, oxygen, glucose, ascorbic acid, and acetaminophen
~ ~~~~
(17) Updike, S.J.; Shults, M. C.; Capelli, C. C.; von Heimburg, D.; Rhodes, R.
K.; Tipton-Joseph, N.; Anderson, B.; Koch, D. D. Diobetes Care 1988, 11,
801-807.
Ana&ticaI Chemistry, Voi. 66,No. 9, May 1, 1994 IS21

Figure 1. Cross-sectional view of three-electrode cell for permeability
measurements: (1) bottom case, (2) top case, (3) electrode insert, (4)
electrodes, (5) snapon membrane holder, (6) membrane, (7) sample
well, and (8) space for electronics.
in the polymer materials of interest were determined by the
combination of permeability measurements with iterative
digital simulations. All solutions used were prepared with
distilled, deionized water (McGaw), reagent grade chemicals
(J. T. Baker, Aldrich, Amend), and research grade gases
(Matheson). Direct 30/30 top cases in standard three-
electrode c~nfigurationl~ were used as the electrode test
platform (Figure 1). The working and counter electrodes
were 0.020-in.-diameter Pt wire while the reference electrode
was a 0.020-in.-diameter silver wire. All electrodes were potted
in epoxy, and the final assembly cut to a 0.200 in.-radius
dome, which was then polished at the surface. The silver wire
reference electrode was then chemically chloridized.18 An
eight-channel, three-electrode potentiostat (Markwell Medi-
cal) with switching box was used to direct sensor outputs for
computer acquisition. Potentials were set to -0.6 V (vs AgC1)
for oxygen reduction and +0.6 V for hydrogen peroxide,
ascorbic acid, and acetaminophen oxidations. Glucose pa-
rameters were determined indirectly by monitoring the
peroxide formed in a thin layer of laminated polyurethane/
glucose oxidase on the electrode side of the membranes.
The transient responses for solution concentration steps
were acquired with a Macintosh Plus using a Remote
Measurement Systems A/D board and Quickbasic software.
Since the three electrodes were situated directly beneath the
membrane, concentration steps were made by blotting dry
the top side of the membrane and then pipeting the solution
of interest into a 50-pL well. Data acquisition synchronization
with sample application was assured by applying the sample
at the sound of a computer-generated tone. In cases where
oxygen exclusion was required for extended times, a capped
polycarbonate cylinder was positioned above the electrode to
increase the solution volume to 0.5 mL. This allowed easy
maintenance of a constant pOz. The concentration steps used
to determine the permeability parameters included 0-5 mM
for hydrogen peroxide, 0-1 1 mM for glucose, 0-160 mmHg
for oxygen, 0-6 mM for ascorbic acid, and 0-0.8 mM for
acetaminophen. A trace of catalase was included in the glucose
solutions to scavenge any enzyme-produced hydrogen peroxide
which diffused back through the membranes to the contacting
solution.
Additional concentration steps were performed on the
complete membrane system to provide the data required for
model verification. Linearity was checked by varying the
glucose level from 0 to 55 mM in air-saturated solution. Further
variation was made by checking the higher glucose level steps
(22-55 mM) as a function of solution oxygen concentration
(30-160 mmHg, verified by either Radiometer BMS3 Mk2
Blood MicroSystemor ColeParmer 551360oxygen monitor).
(18) Sandifer, J. R.And. Chem. 1981, 53,312-316.
1522 AnalyNcal Chemlsiw, Vol. 88, No. 9, M y 1, 1994
Exposure times for these specific glucose and oxygen con-
centration permutations were varied and then the electrode
assembly was reexposed to storage solution (air saturated with
no glucose). This allowed following of sensor washout curves.
Interference transients were also checked by performing
concentration steps using freshly prepared solutions of ascorbic
acid or acetaminophen.
Enzyme deactivation rates were measured using our lower
loading enzyme recipes in a manner equivalent to that described
by Gough.8 This information was then entered into our
simulation model, the simulated steady-state species concen-
trations and enzyme fractions were calculated as a function
of contacting solution exposure and membrane permeabilities,
and sensor lifetime was simulated.
To look at the kinetics of the possible decay mechanisms,
contacting solutions were used which would leave glucose
oxidase in either its resting state or with known fractions of
the reduced forms. The starting enzyme concentration was
*/@ofthe standard recipe or lower so that the electrode output
to a 11 mM glucose concentration step was approximately
linear with enzymeconcentration. Theconcentration of active
enzyme was then calculated from the ratio of sensor output
after a defined decay period to the starting sensor output. The
averages from four membrane samples were used to calculate
remaining active enzyme concentration.
To look at the deactivation of the resting-state enzyme,
membranes were suspended in a phosphate-buffered saline
(pH 7.05) contacting solution at a p02 of 150. Two sets of
samples were tested at temperatures of 22 and 37 OC. Fresh
samples were selected periodically over 4 months from these
storage solutions and tested after mounting on Direct 30/30
cases.
To study peroxide-induced decay pathways of the reduced
enzyme fractions, the phosphate-buffered saline solution also
included 27.5 mM glucose and 5 mM hydrogen peroxide.
Decay measurements were made using membrane samples
exposed to this solution for varying lengths of time. Solution
p0z was set to either 0 or 30 mmHg by continuous degassing.
This provided a high level of convection and allowed accurate
calculation of oxygen and glucose concentration within the
enzyme layer. These oxygen levels produced preferential decay
pathways for the enzyme. At 30 min to 4-h intervals,
membrane samples were removed, rinsed in phosphate-
buffered saline to quench the decay, and tested as before for
remaining active enzyme concentration. This process was
repeated over four to six time intervals until at least a 60%
decay in active enzyme concentration occurred.
Simulations of electrode output and membrane layer
concentration profiles were run on a Macintosh SE/30
computer using True Basic as the programming language.
The time required to run any simulation is dependent on the
number of species, elements, and time steps desired. The
typical transients shown here used eight or less species, 10
elements per layer, and 120-480 time steps. The species
included glucose, oxygen, hydrogen peroxide, interferent,
oxidized enzyme, glucose-complexed enzyme, reduced enzyme,
peroxide-complexed enzyme, and deactivated enzyme. An
eight-species, 50-element, 120-time step simulation required
-20 min to complete. In addition, the algorithm is designed
in such a way that calculation times will scale directly with
changes in any of these threevariables. To verify the accuracy
of the simulation, the time step size was varied from 0.05 to
5 s to find the times which showed no appreciable distortion
of the simulated current curves due to diffusional/kinetic
mismatches. In addition, mass balance checks were made on
the resultant concentration profiles to ascertain that no
distortion occurred.
The simulation of enzyme decay as a function of contacting
solution required an important approximation. This was
related to the simulation time step that can be used without
-
distortion of the concentration profiles. For sensor transient
development, it took 10 min of computer time to simulate
1 min of real time. However, simulating months to years of
enzyme decay with these small time steps presented a
computational bottleneck. This was addressed by realizing
that enzyme decay occurred slowly. To start the simulation,
initial steady-state concentration profiles for specific contacting
solution conditions were first established using a time step of
0.25-1 s. However, from this point on we scaled up the decay
kinetics from seconds to days as long as two conditions were
met. First, the product of the enzyme decay kinetics and the
time step was sufficiently small so that no more than 1% of
the hydrogen peroxide steady-state concentration was removed
during any time step by reaction with enzyme subpopulations.
Second, after each half-loading of simulated enzyme decay,
the simulation was put into a "zero decay" mode until it was
verified that glucose, oxygen, and hydrogen peroxide were
indeed at the correct steady-state concentrations for the
remaining active enzyme. These considerations guaranteed
that enzyme subpopulations were correct and minimized the
underestimation of decay that would occur at highest enzyme
loading when the simulation was being based on hydrogen
peroxide concentration that was a few percent low. Worst-
caseerrors with this approximation resulted in underestimated
decay times of 10-15%. Thus, as long as the concentration
estimates of the enzyme subpopulation fractions are correct
and hydrogen peroxide is close to the correct steady state,
propagation of error in enzyme decay during the course of the
simulation is reasonable.
RESULTS
Determination of Model Parameters. The gas contacting
model serves as the starting point and predicts steady-state
and transient output currents of the form
is, = (nFAD,K,C,)/X (1)
and
it = i,(l + 2~(-l)"e-"") where z = (?r2Dmt)/X2 (2)
and C, equals the bulk solution concentration (mol/cm3), X
themembrane thickness (cm), t the time (s), D, the membrane
diffusion coefficient (cm2/s), and Km the dimensionless
membrane partition coefficient and n, F, A , and ?r have their
usual meaning.
When expanded, eq 2 leads to
it/iss= 1 - 2e-' + 2e4' -2 +
~ ~2e-16'
' ...
(3)
Thus, by determining the factor z at different fractional cur-
rents, it becomes possible to solve for D, using the equation
D, = (zX2)/(?r2t) (4)
where for example z = 1.37 at it/iss= 0.5. The partition
coefficient then is calculated from
Km = (issu)/ (nFADmCg) (5)
We typically find the averages for D , and Kmby determining
the times to fractional currents of 0.1, 0.3, 0.5, 0.7, and 0.9
using the corresponding z factors of 0.66,l .OO,1.37,1.89, and
3.00.
These D / K pairs serve as the starting point in the iterative
process to match experimental data to theoretical transients.
After these values are plugged into the relevant distance grid
which defines our experimental conditions, the concentration
step is then simulated with literature values for solution
diffusion coefficients in all solution-layer elements. Curve
matching of simulated to experimental data for a more
permeable species, such as hydrogen peroxide, is performed
first to check for mismatches in transient curve shape due to
solution permeability effects. In this experimental situation,
where sample application is less well defined than with a RDE,
the classical approximation of D,ff = D a t a sensor boundary
layer of defined thickness and D,ff >> D in bulk solution is
not likely to apply. We have used the approximation of D,ff
= 1OD throughout the entire solution layer. This approxima-
tion is suggested through the high-quality curve shape match
of experiment to simulation, but it should be recognized that
this factor would be different if applied to a different system
with different permeability parameters and geometry. Once
this modeling of the solution layer is defined, then iterations
in the membrane resistance layer D / K pairs with further
matching of real to simulated data completes the parameter
determination. This process is repeated for glucose, hydrogen
peroxide, and oxygen as each successive membrane layer is
added.
The uncertainty introduced by this model of solution
permeability has little effect on the reliability of experimentally
determined membrane resistance layer permeability param-
eters for the less permeable species, which include glucose,
ascorbic acid, and acetaminophen. However, for more
permeable species such as oxygen and hydrogen peroxide, the
tabulated values of the resistance layer permeability com-
ponents are probably not better than 45-896 for D and f 1 0 -
12% for K . This does not imply equivalent errors in the total
layer permeability so much as a lack of resolution between its
D and K components.
Representative fitted curves are shown for two- and four-
layer membranes for hydrogen peroxide and glucose in Figures
2 and 3, respectively. All subsequent simulation predictions
are made for the full sensor system where four of the layers
are membrane materials and the fifth layer is the contacting
solution. The full permeability parameter sets for these layers
are included in Table 1.
Oxygen Cofactor ConcentrationLimitationEffects. Figure
4 shows the simulated oxygen concentration profiles obtained
through 120 s for the glucose concentration step of Figure 3
(0-1 1 mM glucose, p02 = 160 mmHg = 0.000 25M). Notice
that oxygen reservoirs are present in the membrane layers
prior to the concentration step. Once the step occurs, new
profiles are generated with the glucose profiles reaching a
Analytical Chemism, Vol. 66, No. 9, May 1, 1994 1523
 40 r

0
0 30 60
0 30 60 TIME (SECONDS)
TIME (SECONDS)
Flgwe 2. Slmulated(solid1ines)vsexperimental sensor outputtransients
for 0.5 mM hydrogen peroxide (filled squares) and 11 mM glucose
(open squares) concentratlon steps through two-layer membrane.
Electrode area is calculated to be 2.0 X cm2.

20 r

0 11 22 33 44 55
mM GLUCOSE

Flgure 5. (a) Simulated output transients for (1) 5.5, (2) 11.O, (3)27.5,
and (4) 55 mM glucose concentration steps with a contactlng blood
pO2 of 30 mmHg. (b) Simulated outputs at (1) 10, (2) 15, and (3) 20
0 60 120 s for the four blood glucose concentration steps at a pOzof 30 mmHg.
Note that best linearity Is obtained at shorter measurement times.
TIME (SECONDS)
Flgure 3. Shnulated (solkl1lnes)vsexperknentalsensor output transients
for 0.5 mM hydrogen peroxlde (filled squares) and 11 mM glucose
(open squares) concentration steps through four-layer membrane. When dissolved oxygen concentration decreases in the
Electrode area is calculated to be 1.8 X lo3 cm2. contacting solution, as is the case for many relevant matrices
Membrane Layers - Function and Thicknesses
such as venous blood samples (0.1-0.025 mM), oxygen flux
into the membrane very badly lags glucose flux. As Figure
hydration interference enzyme resistance solution
0.00064 cm 0.0001 cm 0.00064 cm 0.0040 cm 0.089 cm 5a shows for simulations of 5.5-55 mM glucose with 0.05
mM dissolved oxygen, membrane oxygen quickly becomes
Oxygen Concentration Profiles exhausted and the sensor converts from a glucose-dependent
to an oxygen-dependent sensor. At the higher glucose levels,
the current begins to decay as early as 15 s after sample
application and current is now controlled by oxygen rather
than glucose flux. Thus, steady-state current is no longer
useful for analytical purposes. However, by simulating a
family of output transients as a function of glucose concen-
0
D. trations at the lowest concentration of oxygen to be encoun-
I L
E tered, it is possible to identify a usable time window over
which glucose linearity is maintained. This time window can
then be incorporated into the analysis software, as was done
for the Direct 30/30, to provide a high degree of measurement
linearity. This is demonstrated in Figure 5b.
Discrimination Against Interference Effects. The ability
to discriminate against electroactive interferences is usually
assumed to be due to the complete rejection (i.e., partition
coefficient of 0) of the species at one of the membrane
interfaces. However, as the species partition and diffusion
coefficients of Table 1 show, there must be an additional mode
of interference discrimination since the K for ascorbic acid is
vanishingly small while that for acetaminophen is large. This
second mode can be explained on the basis of the much slower
diffusion coefficient of acetaminophen relative to the other
species. As Figure 6 suggests, we can use the kinetic mode
to measure substrate in the first 30 s before development of
the interference signal at greater times. An analogous type
of time domain discrimination has been demonstrated pre-

1524 Analytical Chemistry, Vol. 66, No. 9, May 1, 1994

viously by Sandifer18 for chloride potentiometric sensors in
the presence of bromide.
Sensor Washout Effects. One phenomenon of significance
which has not been reported in previous enzyme electrode
simulation studies is that of sensor washout. This relaxation
in sensor output occurs when the contacting sample solution
is replaced with one containing no substrate and a normal
level of cofactor. The speed of this relaxation or recovery
process determines the fastest possible sensor cycle time, which
is of great practical importance to the analyst trying to run
a series of samples. Three classes of relaxation curves can
occur during sensor washout. In the simplest case, when
cofactor remains in excess during the entire measurement,
the curve relaxes toward its zero level in a time comparable
to the initial transient development time.
In situations where the enzyme layer cofactor has been
depleted due to extended exposure at high substrate concen-
tration, the relaxation curve to wash solution is nonmonotonic
(Figure 7a). When the contacting cofactor concentration is
low to start with and then is brought back to normal levels,
this transient can be quite striking. This occurs because
increased cofactor flux reaches and reacts with the ac-
cumulated glucose in the enzyme layer. The resultant output
moves to its highest level with cofactor flux maximization and
holds this level until the residual substrate has been completely
scavenged from the enzyme layer. At this point, the cofactor
begins to replenish itself in the enzyme layer and a region of
relatively fast decay occurs with removal of the last residual
substrate and reaction product.
The last class of relaxation curve occurs for a slowly
diffusing interferent which has not achieved its maximum
inward flux. Upon return to wash solution, the output transient
appears unaffected and continues to grow in magnitude.
However, at some point the bidirectional flux of species will
begin to diminish the concentration profile near the electrode.
At this point the output transient reverses and begins its slow
decay back toward zero (Figure 7b). Samples of this type
give an extremely slow decay and can increase the cycle time
and apparent baseline of the sensor when the interferent
concentration is high.
Enzyme Loading Effects. In setting up enzyme loading
permutations, enzyme was lowered by factors of 2 from the
standard recipe to a ' 1 2 5 6 recipe. Assuming homogeneous
distribution in the enzyme layer, no enzyme decay during
preparation, and concentrations calculable from normal
geometric factors, enzyme concentrations ranging from 2.00
X 10-4to 7.81 X M can be predicted. For the standard
glucose oxidase mechanism,19
E
ki
+ G -,RL -..R + L
ki
(6)
R + 0, -
k,
E*-H202 E -+
k4
H202 (7)
the room-temperature rate constants given in the literature20
are kl = 12 200 M d , k2 = 1220 s-l, ks = 2 000 000 Mss-',
and kq = 660 s-l. Combining this information with the
previously determined species permeability data gives the curve
(19)Gibson. Q.H.; Swoboda, B. E. P.; Massey, V. J. Biol. Chem. 1964, 239,
3927-3934.
(20) Duke, F. R.; Weibel, M.; Page, D. S.; Bulgrin, V. G.; Luthy, J. J. Am.Chem.
Soc. 1969, 91, 3904-3909
'Or 2
0
0 30 60
TIME (SECONDS)
Figure6. Simulated output response fora 1 1 mM glucose concentratlon
step in the absence (1) and presence (2) of 0.4 mM acetaminophen
interference.
fits shown in Figure 8a for transient current as a function of
enzyme loading. The match is quite good at highest loading
but appears to lag in time and underestimate current magnitude
as loading is decreased.
To try to obtain a better match, the different k values are
individually increased. Increases in k2, k3, and k4 by up to
a factor of 2 leave essentially the same transient as before.
Increasing kl produces a faster development of the transient
as well as an increase in magnitude. When kl is increased by
a factor of 2, fairly good matches result for ail enzyme recipes,
as shown in Figure 8b.
Enzyme Decay Measurements. Prior work has demon-
strated that glucose oxidase can decay through three primary
pathways.8 An apparent "autoinactivation" can occur when
the enzyme layer is submersed in fluid containing no glucose
and no hydrogen peroxide. This resting-state enzyme shows
a simple first-order decay of the form
dxldt = k,,(a - x) where a = total and
x = inactive enzyme (8)
with the solution
x = a(1 - with a half-life of r1,2 = 0.693/k,,
(9)
In this work deactivation of the resting-state enzyme at 37
OC, as shown in Figure 9a, was fit to project a half-life for
this pathway of 105 days. From this information, klt is
-
calculated to be 7.64 X l e 8 s-l. This is 15% slower than
that measured in previous work.8 This rate of decay did not
change when externally added peroxide but no glucose was
added to the contacting solution.
The other two pathways of enzyme deactivation occur in
the presence of hydrogen peroxide when contacting solution
conditions force enzyme to the reduced or peroxide-complexed
forms. The reduced form is exclusively formed in glucose
solutions with zero oxygen. Both forms are present in
significant fractions when glucose solutions are at low PO*.
These decay pathways are "psuedo" first order and of the
form
dx/dt = (kit + k2&)(~ - X) + k&)(c - x)) ( a - X) (10)
where c = peroxide, fR = fraction of reduced enzyme, and fc
= fraction of peroxide-complexed enzyme, thus leading to the
solution
= a( 1 - e-(ki,)(t)-(kz,)(/~)(c)(f)-(kjr)(/~)(c)(r))
(1 1)
Analytical Chemistry, Vol. 66, No. 9,May 1, 1994 1525
 1.0 s

,0
K
K
3
u ,6
n
W
N
i ,4
a
E
K
0 ,2
z
0 90 180
TINE (SECONDS) .o
0 120 240 360 480 600
TIME (DAYS)
1.0

ii
w -8
K
K
I)
u ,6
n
W
N
.4
E
K
0 90 180 p #2
TINE (SECONDS)
Flgure 7. (a) Experimental (squares) vs simulated (sotid line) output .o
transient for the case where the inittal contacting sdutlon ia 27.5 mM 0 3 6 9 12 15
Inglucose and at a p02of 30 mmHg, while thewash solutionintroduced TINE (HOURS)
at 90 s contains 0 mM glucose and Is at a pO2 of 160 mmHg. (b)
Simulated output transient for the slowly diffusing Interference
acetaminophen Introduced at 0.4 mM concentration at t = 0 s with
a return to an lnterferencafree wash solution at t = 60 s.

10
..................................................................................................
I i\
h

2
v

I-
22OC
E 5
K
K
3
u
0 5 10 15 20 25
0 TINE (HOURS)
0 60 120 Flgure 9. (a) Enzyme decay at 37 O C from restingstate enzyme.
TINE (SECONDS) Exponential fit projects an enzyme half-life of 105 days. Points are the
average value of four sample membranes. (b) Enzyme decay at 22
and 37 O C for a contacting solution of 0 mmHg pop, 27.5 mM glucose,
and 5 mM hydrogen peroxide. The simulation predicts an equilibrium
.I enzyme layer glucose concentration of 4.1 mM. (c) Enzyme decay at

t _I
. / - - 22 and 37 O C for a contacting solution of 30 mmHg p02, 27.5 mM
glucose, and 5 mM hydrogenperoxide. The sknulatkm predictsa steady-
state enzyme layer glucose Concentration of 2.9 mM and an enzyme
layer pop of 18 mmHg.

low oxygen, and externally added hydrogen peroxide are shown

in Figure 9b,c. To determine rate constants from these decay
0 60 120 curves, the enzyme fractions are first calculated. At a
TIME (SECONDS) contacting solution p02 of 0, the enzyme is fully converted to
Flgure 8. (a) Experimental vs simulated transients for 1 1 mM glucose the reduced form by excess glucose. At a contacting solution
step as a function of enzyme concentration. (1)2.0 X lo4, (2)3.13
X lO-O,and (3)7.81 X lo-' M. All kinetic parameters are at literature pOz of 30 mmHg (0.05 mM) with oxygen flux to the enzyme
values. (b) Experimental vs simulated transients for 1 1 mM glucose layer from both sides of the membrane, simulations using the
step as a function of enzyme concentration: (1)2.0 X lo4, (2)1.25 previously defined permeability parameters and modified
X (3)3.13X and (4)7.81 X le7 M. k, istwkethe Herature
value. enzyme kinetics predict enzyme layer glucose and oxygen
concentrations of 2.9 and 0.028 mM. This results in predicted
and enzyme fractions of 0.522 for the reduced form and 0.044 for
the complexed form. Rate constants at 22 and 37 O C are then
fl,2 = 0.693/(k1,+ kZ,CfR)c+ k&&) (12) calculated to be 0.013 and 0.029 (M.s)-' for the reduced
The results for enzyme concentration decay in excess glucose, enzyme and 0.075 (Mas)-' and 0.132 (M.s)-' for thecomplexed

1526 AnalyticalChemlstty, Vol. 66,No. 9, M y 7, 1994


0 180 360
TIME (SECONDS)
Flgure 10. Simulated output transients for 22 mM glucose (filled
symbols)vs11 mM glucose (opensymbols)for Direct 30130 membrane
sensor covered with an additional 0.0025-cm-thick resistance layer.
The glucose diffusion coefflcient for the extra layer is either (1) 5.50
X (2) 2.75 X or (3) 1.84 X cm2/s. Oxygen diffusion
coefficient did not change from that for the Direct 30130 membrane.
enzyme. These experiments compare favorably with the 37
OC decay constants of 0.02 and 0.76 (M.s)-' measured
previously.8
Simulationof Permeability Parameters for an Implantable
Glucose Sensor. This model was used in predicting the required
membrane permeabilities for an in vivo subcutaneous glucose
sensor. The challenge here was to continuously maintain linear
response to glucose in the useful clinical range (1 5 - 2 2 mM)
with tissue oxygen tension as low as 30 mmHg. Thus, at
equilibrium the flux of oxygen from contacting environment
to the enzyme layer must always be greater than that for
glucose. Simply increasing membrane oxygen permeability
does minimal good since this will result in a depletion layer
into the contacting solution and only a small increase in oxygen
flux to the sensor area. The alternative is to lower glucose
flux through the outermost resistance membrane. This can
be done by lowering the partition or diffusion coefficients.
The former would be preferred since this would not decrease
the sensor response time. For us, finding materials with low
partition coefficients that maintain reasonable diffusion
coefficients has proved illusive. Thus, theoretical and ex-
perimental work has focused on materials with comparable
partition coefficients and decreased diffusion coefficients.
Membrane and solution permeabilities listed in Table 1
serve as the simulation starting point. The contacting solution
p02 is set to 30 mmHg. Now a fifth membrane layer is
interposed between this solution and the previous membranes.
As the glucose diffusion coefficient is lowered by up to a factor
of 12 in this fifth layer (with no change in glucose partition
coefficient and oxygen permeability), the family of simulated
curves shown in Figure 10 is generated. This desired set of
parameters predicts the development of a linear signal from
0 to 22 mM. This strategy allowed us to develop membrane
resistance layers which, upon continuous aqueous solution
exposure, could provide in vitro glucose linearity through
22 mM when pOz is as low as 20 mmHg. In addition, these
membranes have now been used in subcutaneous glucose
sensors for periods of up to three months in a dog model.21
Estimation of Implantable Sensor Lifetime Limit Due to
Enzyme Decay. A useful presentation of remaining active
(21) Updike, S.J.; Shults, M. C.; Gilligan, B. J.; Rhodes, R . K.;Luebow, J. 0.;
vonHeimburg, D. ASAIO Trans., in press.
0
5
6
7
a
9
10
11
0 240 480
TIME (SECONDS)
Figure 11. Simulated sensor output vs time for 0-1 1 mM glucose
concentrationsteps using membranesof implantablepermeabiilty with
varied enzyme loading. ID numbers represent the number of half-
loadings below the standard recipe of 2.00 X lo4 M: (0) 2.00 X lo4,
(5) 6.25 X (6) 3.12 X lo", (7) 1.56 X lo4, (8) 7.83 X lo-', (9)
3.92 X lo-', (10) 1.96 X lo-', and (11) 9.80 X M.
-3
- -4
W
I
w
2 -5
t;
Q -6
0
0
-I -7
-0
0 225 450 675 900
TIME (DAYS)
Figure 12. Simulated active enzyme Concentration decay curves vs
time for Implantable membranes continuously exposed to different
glucose concentrations of 0.0, 11.0, 22.0, 24.8, and 27.5 mM at a
contacting solution pOn of 30 mmHg. Starting active enzyme
-
concentrationis 2 X lo4 M; (.I and (- -1 respectively represent the
enzyme concentrations where 90 and 50% of the sensor output
remains.
enzyme concentration and its correspondence with sensor
output is given in Figures 11 and 12. Figure 11 shows the
simulated output transients to a 1 1 mM glucose concentration
step for sensors with implantable membrane permeabilities
and enzyme concentration lowered in 50% increments from
0.2 mM to 0.098 pM. Note that approximately six half-
loadings of enzyme must be removed before signal drops more
than 10% and that nearly nine half-loadings must be removed
to drop the signal more than 50%. These serve as standard
curves for predicted sensor output.
In Figure 12, the active enzyme concentration vs time has
been simulated for membranes with implantable sensor
permeabilities. The contacting solution p02 was 30 mmHg
and glucose was varied from 0 to 27.5 mM. Note that these
plotted enzyme concentrations are the lumped sum average
of remaining active enzyme throughout all enzyme elements.
These remaining active enzyme concentrations can be com-
pared to Figure 11 to predict the remaining sensor output to
a standard glucose challenge. Two lines are drawn at the
enzyme concentration levels where 90 and 50% of the standard
loading sensor output remains. At 90% sensor output, only
minimal changes in overall sensor performance will have
occurred. However, at the 50% sensor output level, the sensor
time response will be so slow (2-4 times slower) and the output
so enzyme concentration dependent that the sensor is no longer
useful.
Analytical Chemistty, Vol. 66,No. 9, May 1, 1994 1527
 There are several points to note from this plot. The first
is that, even with no glucose exposure, the enzyme concentra-
tion will decrease from resting-state enzyme decay. This
represents the best-case limit of electrode lifetime. The second
is that as long as the glucose flux is lower than the oxygen flux
into the enzyme layer, the enzyme decay will be only slightly
faster than this slowest decay pathway. Finally, once glucose
flux is greater than oxygen flux, glucose will accumulate in
the enzyme layer, the fractions of reduced and complexed
enzyme concentrations will increase, and enzyme concentration
decay will accelerate. The contacting solution concentrations
where this occurs will be very sensitiveto both initial membrane
permeabilities and changes in permeability that may occur in
the sensor membranes due to aging, foreign body encapsula-
tion, or membrane pore occlusion. However, if as a starting
point we are willing to believe this simulated time to 50%
output, then we can speculate that glucose concentrations in
-
the typical diabetic patient range can be in continuous contact
with the sensor for 2 years without appreciably accelerating
the speed of enzyme decay. Thus, enzyme lifetime is not
likely to be the primary limiting factor in implantable sensor
development.
DISCUSSI ON
In our system, there are several experimental situations
which we need to be able to predict accurately before we can
consider our model complete. These include the following:
(1) single species permeability measurements corrected for
contacting solution effects, (2) electrode output magnitude
and time responses as a function of membrane thickness (not
cofactor limited), (3) range of substrate linearity as a function
of cofactor concentration, (4)output transients vs time for
cofactor-limited cases, (5) output transients vs time for
different enzyme loadings (at constant membrane thickness
and solution conditions), and ( 6 ) washout transients vs time
for a variety of solution cofactor/substrate concentration
permutations and exposure times.
We start first with the simplest case, that of determining
single species diffusion and partition coefficients. For the
directly electroactive species this is easy. However, when we
get to the enzyme converted species measurements, a degree
of uncertainty must initially be accepted in making the
measurement. This uncertainty occurs since we are using a
single output transient to try to explain several possibly
counteracting effects including (1) the bidirectional flux of
peroxide generated in the enzyme layer, (2) enzyme activity
and kinetics, (3) cofactor concentration, and (4) the substrate
and cofactor partition coefficients in resistance and enzyme
layers. In fact, no single transient of this type is uniquely
determined as tradeoffs in species concentrations, species DIK
pair, and enzyme kinetics and loading can provide essentially
indistinguishable curves.
To assess our initial glucose permeability estimate we
provide an experimental situation that will force a nonmono-
tonic output transient. For our system this is provided simply
by setting up solution conditions where cofactor will quickly
be depleted. Thus our simulation substrate/cofactor fluxes
will have to be matched correctly to reproduce the experimental
sensor output magnitude and more importantly the time when
cofactor becomes the limiting reagent and sensor output decays
1528 AnalyticalChemistry, Vol. 66,No. 9, May 1, 1994
with loss of cofactor. The subset of parameter tradeoffs which
can match this curve type is much smaller than that for the
simple glucose transient with adequate cofactor, and thus we
approach convergence on the final parameter set.
A more stringent test is the prediction of the washout curve
after cofactor-limited situations. In these situations, substrate
will have accumulated in the enzyme layer after cofactor was
depleted. Once the contacting solution is replaced with another
containing no substrate and normal levels of cofactor,
accumulated substrate will begin to either react or diffuse
away. The shape and magnitude of the output washout curve
and the time required for removal of residual substrate can
be used to verify enzyme kinetic parameters and partition of
substrate from the resistance into the enzyme layer. This is
checked at several widely spaced permutations of substrate
and cofactor concentrations to verify that some unknown
artifact has not assisted in this match.
The final test for the model is its ability to predict transient
response curves as a function of enzyme loading. That we
could simply take literature values for the enzyme kinetics as
determined in solution and have them transfer so well to the
immobilized membrane case is quite remarkable. The small
increase in kl to match a 250-fold swing in enzyme loading
implies that only minimal deactivation occurred in the
production of these membranes.
The question arises as to what is considered a good fit of
simulation to experimental data. It is not possible to simply
do a least-squares fit and project from the residuals which of
the simulation parameters should be changed to get a better
fit. For this work, the answer has been empirical in that if
all relevant situations are matched without experiencing large
errors in transient development time response or output, then
we will be satisfied with the model. This seems reasonable
since there are unquestionably certain assumptions in the model
which are oversimplified, such as layer homogeneity, unidi-
mensional diffusion, uniform convection in the contacting
solution resulting in an average “effective” diffusion coefficient,
no temperature or membrane stretching effects on DIKpairs,
no time lags on solution exchanges, etc. Thus, the ability to
predict all major trends as opposed to an exact fit for one
transient is our criterion for “goodness of fit”.
We have found many applications for this methodology in
our work. This model serves us well in quality control of
resistance membrane polymer permeability and subsequent
calculation of required thickness prior to production. It has
been used to follow enzyme decay over the shelf life of
membranes. It proved valuable in defining the measurement
time window in the software for the Direct 30130 and provided
guidance in establishing the required permeability range for
our implantable glucose sensor development. Finally, it was
used in combination with enzyme decay measurements to
estimate the possible lifetime theoretically obtainable for
sensors in an in vivo environment.
CONCLUSIONS
A simulation strategy has been developed for modeling the
glucose oxidase enzyme electrode. It provides for the
calculation of sensor performance information, such as range
of linearity, time response, output magnitudes, cofactor
limitation regimes, and enzyme loading and decay effects. It
also allows one to establish different sets of contacting solution
conditions (pure diffusion, RDE boundaries, convection, etc.)
which can produce strikingly different performance patterns
for the identical set of membrane parameters. Concentration
profiles can be simulated under one set of solution boundary
conditions and the decay then followed for a different set of
conditions. This allows for visualization of sensor washout
transients, which provides information related to sensor cycle
time and answers questions about enzyme loading and reaction
mechanisms. Any number of layers can be handled, providing
single-layer parameters can be adequately measured. There
is great flexibility in handling of input parameters, and in
fact, if one wishes, these can be changed during the course of
the simulation (Le., enzyme activity decay, growth of stagnant
layer thicknesses, etc.). Finally, while the experimental
verification of this simulation model was performed with the
glucoseoxidase system, this modeling strategy should be easily
extendible to other enzyme electrode systems and has the
potential to be used with minimal modification to describe the
detection of neutral species by spectroscopic as well as
electrochemical means.
ACKNOWLEDGMENT
Markwell Medical Institute thanks the SBIR program
within NIH for grant support (DK 4065743) pertaining to
simulated and experimental implantable glucose sensors during
the course of this work.
Recehred for revlew October 1, 1993. Accepted February 8,
1994.'
Abstract published in Advance ACS Absrracrs, March 15, 1994.
Anelytical Chemisby, Vol. 66,No. 9, May 1, 1994 1529