Design variations of a polymerenzyme composite biosensor for glucose: Enhanced analyte sensitivity without increased oxygen dependence
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Available online 3 May 2005
redox center of the enzyme and the electrode surface
[7,8]. Using the avoenzyme glucose oxidase (GOx), the
GOx/FADH2 +O2!GOx/FAD+H2O2 2
H2O2!O2 +2H +2e 3where FAD is the oxidized form of the prosthetic group,avin adenine dinucleotide. Reaction (3) represents the
* Corresponding author. Tel.: +353 1 7162314; fax: +353 1 7162127.
E-mail address: Robert.ONeill@UCD.ie (R.D. ONeill).
Journal of Electroanalytical Chemist
Journal ofElectroanalytical0022-0728/$ - see front matter 2005 Elsevier B.V. All rights reserved1. Introduction
The incorporation of biological molecules into sensing
devices is an important strategy in the development of
fast, ecient and inexpensive assays for a wide range of
analytes in clinical, industrial and environmental applica-
tions . In particular, amperometric biosensors fabri-cated using immobilized enzymes can be categorized
broadly as rst, second or third generation devices
depending on the mode of electron transfer between the
majority of these sensors have focused on the detection
of glucose, both as a model system and because of the
importance of glucose as a target analyte [1,911].
The overall enzymatic process for GOx, that is simi-
lar for many oxidases, may be written as Reactions (1)
The glucose sensitivity and oxygen dependence of a variety of implantable biosensors based on glucose oxidase (GOx), incorpo-
rating an electrosynthesized poly-o-phenylenediamine (PPD) permselective barrier on 125-lm diameter Pt disks (PtD) and cylinders(PtC, 1-mm length), were measured and compared. Full glucose calibrations and experimental monitoring of solution oxygen con-
centration allowed us to determine apparent MichaelisMenten parameters for glucose and oxygen. In the linear region of glucose
response, the most sensitive biosensor design studied was PtD/PPD/GOx (enzyme deposited over polymer) that was 20 times moresensitive than the more widely used PtC/GOx/PPD (enzyme immobilized before polymer deposition) conguration. The oxygen
dependence, quantied as KM(O2), of both active and less active designs was surprisingly similar, a nding that could be rationalized
in terms of an increase in KM(G) with increased enzyme loading. The PtD/PPD/GOx design will now enable us to explore glucose
concentration dynamics in smaller and layered brain regions with good sensitivity and minimal interference from uctuations in
2005 Elsevier B.V. All rights reserved.
Keywords: Poly(o-phenylenediamine); Enzyme modied electrode; Amperometry; Glucose oxidase; Electropolymerization; Hydrogen peroxide;
Brain monitoringDesign variations of a polymfor glucose: Enhanced analy
Colm P. McMahon, Sarah J
Chemistry Department, University C
Received 20 January 2005; received in revisedoi:10.1016/j.jelechem.2005.03.026enzyme composite biosensorsensitivity without increasedendence
illoran, Robert D. ONeill *
e Dublin, Beleld, Dublin 4, Ireland
m 22 March 2005; accepted 24 March 2005
ry 580 (2005) 193202
194 C.P. McMahon et al. / Journal of Electroanalytical Chemistry 580 (2005) 193202electrochemical step and in rst generation devices is
generally carried out amperometrically, either directly
on the substrate surface at relatively high applied poten-
tials [12,13], or catalytically at lower potentials [14,15].
Thus, a two-substrate model is necessary to describe
the kinetics of GOx under conditions of varying concen-tration of both glucose and O2 . When the concen-
tration of the co-substrate is constant, however, the
two-substrate equation simplies to the one-substrate
MichaelisMenten form (Eq. (4)), where the current
density for the biosensor glucose response, Jgluc, is a
measure of the overall rate of the enzyme reaction,
and Jmax is the Jgluc value when the surface enzyme is
saturated with glucose (G). Dierent values of Jmax,determined under the same conditions, reect dierences
in the amount of active enzyme on the surface provided
the sensitivity of the electrode to H2O2 (Reaction (3))
does not vary, as is the case for the PPD-modied Pt cyl-
inders and disks used here [17,18]
J gluc Jmax1 KMGG
The true Michaelis constant for the one-substrate
case, KM, is dened (Eq. (5)) in terms of the rate con-
stants for the generalized reactions describing the overall
conversion of substrate to product, catalyzed by the en-zyme: substrate binding to enzyme (k1) and its reversal
(k1), and the conversion step itself (k2 or kcat) [19,20]
KM k1 k2k1 : 5
However, when Eq. (4) is used to approximate the
two-substrate case (e.g., glucose and oxygen), the
KM(G) is more complex, containing co-substrate terms.
KM(G) is then the apparent Michaelis constant and phe-
nomenologically denes the concentration of substrate
that gives half the Jmax response. If the concentrationof co-substrate is high and constant (as was the case in
the rst part of this study), the main factors aecting
changes in KM(G), for glucose biosensors of similar de-
sign, are those given in Eq. (5). Thus, the value of
KM(G) is sensitive to the binding constant, k1, and has
often been interpreted in terms of barriers to substrate/
enzyme binding [21,22].
Alternatively, if the concentration of glucose is xedand O2 levels are changed, then Eq. (6) can be used to
analyze the oxygen dependence of the glucose signal
, where J 0max is the maximum (plateau) response fora particular concentration of glucose, and KM(O2) is
the apparent Michaelis constant for oxygen. The option
of using a single two-substrate equation, such as Eq. (32)
in  or Eq. (1) in  that expresses explicitly the true
Michaelis constants for both substrate and co-substrate,was not used in this analysis because the apparentMichaelis constants dened separately are analyticallymore straightforward, are useful for dening the linear
range for glucose (1/2KM(G), Eq. (4)) and oxygen(1/2KM(O2), Eq. (6)) responses, and inuence the glu-cose slope in the linear region, i.e., Jmax/KM(G). Inaddition, the concentration of surface enzyme determin-
able in studies involving drop evaporation onto macro-disk electrodes  is not available for enzyme deposited
by dip evaporation, as used here for microdisks and cyl-
inders; this also limits the scope of the kinetic analysis of
the individual reaction steps
J gluc J0max
1 KMO2O2: 6
The three main problems associated with reliable glu-
cose detection using rst generation amperometric bio-
sensors are: analyte sensitivity (Reactions (1)(3)); the
inuence of changes in pO2 on the biosensor signal(Reaction (2)); and interference by electroactive compo-
nents present in the target medium (Reaction (3)). A
variety of strategies have been developed to optimize
the sensitivity, selectivity and stability of glucose biosen-
sors . Recent novel approaches to the ecient detec-
tion of GOx-generated H2O2 include the use of
platinized platinum cylinders , platinized boron-
doped diamond microbers , iron-loaded carbonnanotubes , and horseradish peroxidase coupled to
redox polymers . Targeted synthesis of enzyme
immobilization agents , incorporation of oxygen
storage media into the biosensor design , and the
use of inventive enzyme entrapment procedures 
have also led to advances in achieving these goals. The
choice of design for a given application, however, should
depend on the conditions, such as levels of glucose pres-ent, properties of specic electroactive interference, and
the relevant range of pO2.
A number of laboratories have described the develop-
ment and characterization of a rst generation glucose
sensor based on aGOx immobilized in poly(o-phenylene-
diamine) on Pt electrodes (PtGOxPPD) that possesses
a variety of properties indicating suitability for neuro-
chemical applications [22,3237]. These attributes in-clude fast response time, linearity over the relevant
concentration range, eective elimination of interference
by endogenous reducing agents such as ascorbic acid,
freedom from protein and lipid fouling, stability in vivo,
and ease of miniaturization . More recently, we have
demonstrated that a particular conguration of the Pt
GOxPPD design also displays minimal O2 interference
in applications both in vitro and in vivo over limited glu-cose and O2 concentration ranges . In this paper we
systematically explore variations in the PtGOxPPD
design, in attempts to increase glucose sensitivity with-
out compromising the range of pO2 over which these
biosensors can operate reliability. Designs involvingthe incorporation of enzyme in the monomer solution,
environments to perform all amperometric experiments
troanaand to collect, plot, and do a preliminary analysis ofthe data.
2.3. Working electrode preparation
The basic protocol for fabrication of the working
electrode biosensors has been described in detail recently
[47,48], and a number of variations are reported here.
Cylinders (1 mm long) of freshly cut Teon-insulatedPt wire (125 lm diameter, Advent Research Materials,a strategy that has been used frequently for PPD-based
biosensors [22,25,33,34,3946], were avoided here be-
cause dip-evaporation deposition of the enzyme prior
to electropolymerization has been shown to give good
eciency of enzyme loading compared to the co-
deposition of enzyme and polymer , and to enableus to develop protocols that could be used formore expen-
sive enzymes, such as glutamate oxidase, where co-immo-
bilization would not be a cost-eective option .
2. Materials and methods
2.1. Chemicals and solutions
The enzymes glucose oxidase (GOx) from Aspergillus
niger (EC 18.104.22.168, type VII-S), was obtained from Sigma
Chemical Co, as was o-phenylenediamine (1,2-diamino-
benzene), a-D-(+)-glucose and bovine serum albumin(BSA, fraction V). The L-ascorbic acid (AA, BDH Bio-
chemical grade) and all other reagents were used as sup-
plied. A stock 1 M solution of glucose was prepared inwater, left for 24 h at room temperature to allow equil-
ibration of the anomers, and then stored at 4 C. Allexperiments were carried out in a phosphate-buered
saline (PBS) solution, pH 7.4; NaCl (BDH, AnalaR
grade, 150 mM), NaH2PO4 (BDH, AnalaR grade, 40
mM) and NaOH (Sigma, 40 mM). A 200 U/mL solution
of GOx was prepared by dissolving 15 mg in 10 mL of
PBS. The 300 mM o-phenylenediamine (o-PD) mono-mer solution was prepared using 0.81 g of o-PD and
125 mg of BSA in 25 mL of PBS and sonicating at 25
C until dissolved.
2.2. Instrumentation and software
Experiments were computer controlled with data col-
lection accomplished using either a Biodata Microlinkinterface or a National Instruments (NI, Austin, TX)
AT-MIO-16 data acquisition board linked to a low-
noise, low-damping potentiostat (Biostat II, Electro-
chemical and Medical Systems, Newbury, UK).
In-house software was written in QuickBASIC (version
4.0) and NI LabWindows (version 2.1) QuickBASIC
C.P. McMahon et al. / Journal of ElecSuolk, UK), or disks made from the same wire, weredipped into a 200 U/mL solution of GOx to allow
adsorption of the enzyme (5 min), and then removed
to dry. This procedure was repeated four more times, ex-
cept that the electrodes were removed immediately fol-
lowing immersion. This dip-evaporation procedure
optimizes the loading of GOx on the Pt wire . Someof these Pt/GOx electrodes were exposed to glutaralde-
hyde (GA) vapor for 20 min to cross-link the enzyme;
these enzyme-modied cylinders and disks are termed
PtC/GOxGA (geometric area, 4.05 103 cm2) andPtD/GOxGA (1.23 104 cm2) biosensors, respec-tively. Other samples of the enzyme-coated wire were
immersed in PBS containing the o-PD monomer and
BSA, and electropolymerization carried out at +700mV vs. SCE for 15 min to produce PtC/GOx/PPD
BSA and PtD/GOx/PPDBSA sensors. Finally, a set of
biosensors were fabricated by dip-evaporation of GOx
onto polymer-modied electrodes followed by cross-
linking with GA, i.e., PtC/PPDBSA/GOxGA and
PtD/PPDBSA/GOxGA. The BSA was included in
the polymerization medium because this, and other pro-
teins , have been shown to increase the interferencerejecting properties of the PPD polymer [49,50].
A self-calibrating commercial membrane-covered
amperometric oxygen sensor (1 cm diameter) was usedto quantify solution oxygen concentration. The model
used was a CellOx 325 connected to an Oxi 340A meter
(Wissenschaftlich-Technische Werkstatten GmbH from
Carl Stuart Ltd., Dublin, Ireland), incorporating a tem-
perature probe for automatic compensation. Reliablequantication of O2 using this device required constant
stirring of the solution at a rate of 5 Hz using a glasspropeller mounted parallel to the shaft of the CellOx
325. The sensor range was 0.0199.9% O2 (100% corre-
sponding to air saturation) with a resolution of 0.1%.
This percentage was converted to an estimated concen-
tration of O2, taking 200 lM to correspond to 100%[51,52].
2.4. Amperometric experiments
All calibrations were performed in a standard three-
electrode glass electrochemical cell containing 20 mL
PBS at room temperature (22 C). A saturated calomelelectrode (SCE) and a large stainless steel needle served
as the reference and auxiliary electrodes, respectively.The applied potential for amperometric studies was
+700 mV vs. SCE. Glucose calibrations were performed
in quiescent air-saturated PBS by adding aliquots of glu-
cose stock solution to the electrochemical cell, and stir-
ring for 5 s. In oxygen dependence experiments, the
electrochemical cell was contained within an Atmos-
bage (Sigma) to avoid contamination by air . Theaddition of glucose was not carried out until the elec-trodes were well settled giving a steady background in
lytical Chemistry 580 (2005) 193202 195air-saturated PBS. After the addition of a single aliquot
of dierences observed between the glucose and oxy-
been used in the past to probe barriers to substrate/en-
zyme binding [21,22]. Finally, the value of KM(O2),
which is the concentration of oxygen needed to give half
the maximum biosensor response for a given xed con-
centration of glucose (Eq. (6)), provides an index of the
oxygen dependence of the immobilized enzyme as afunction of glucose concentration for each design of
0 20 40 60 80 1000
0 20 40 60 80 1000
[glucose] / mM
[glucose] / mM
0 1 2 3 4 50.0
Fig. 1. The eects of electrode geometry and enzyme distribution on
the glucose response of GOx-based biosensors. Points are means
SEM of current densities; curves were generated using non-linear
regression and Eq. (4) (R2 > 0.95). (Top) The geometric eect is
illustrated by glucose calibrations performed using cylinder- and disk-
based biosensors of the type where the GOx was adsorbed on the Pt
before electropolymerization of the PPDBSA composite. Although
the Jmax values were greater for the smaller electrodes (see Table 1), the
KM for glucose, KM(G), was not signicantly dierent for these two
designs: 15 2 mM (, n = 21); 15 1 mM (, n = 4); p > 0.94. (Inset)Part of the linear region glucose response for the PtC/GOx/PPDBSA
conguration, showing the regression line with R2 = 0.995 and using
the same axes as the main graph. (Bottom) The eect of enzyme
distribution was demonstrated on cylinder electrodes by sequentially
depositing GOx, and calibrating at each stage: rst on the bare Pt (1.
PtC/GOxGA); the PPDBSA matrix was then generated (2. PtC/GOx/
PPDBSA); nally, GOx was again deposited over the polymer layer
(3. PtC/GOx/PPDBSA/GOxGA). MichaelisMenten parameters for
larger populations of biosensors are given in Table 1.
roanagen responses for the various designs was calculated
using Students two-tailed unpaired t tests on theabsolute current densities, slopes or MichaelisMenten
3. Results and discussion
A number of sophisticated mathematical models of
the behavior of enzymes immobilized in surface layers
have been described [20,5356]. These complex analy-
ses are often needed to understand and optimize the
behavior of thick and/or conducting lms , andhave been used, for example, to determine the site
of H2O2 oxidation in polypyrrole-based biosensors
. However, a recent study has shown that substrate
diusion is not limiting for PPD layers incorporating
GOx, due to their small thickness . Therefore,
the basic MichaelisMenten parameters used here pro-
vide more readily accessible insights into factors aect-
ing the responsiveness of biosensors fabricated fromultra-thin (1030 nm [34,49,58,59]) insulating poly-
mers, such as PPD.
Thus, dierences in Jmax values (Eq. (4)), determined
under the same conditions, reect dierences in the
amount of active enzyme on the surface, provided the
sensitivity of the electrode to H2O2 (Reaction (3)) does
not vary. Changes in the apparent Michaelis constant
for glucose (KM(G), Eq. (4)), in the presence of a highand constant concentration of oxygen, are sensitive toof glucose, the bag was sealed and high-grade N2(
3.1. Glucose response of a cylinder- and disk-based
The use of PPD as a permselective polymer in biosen-
sor applications is well documented (see Section 1). Two
signicant advantages of PPD are its high permeabilityto the oxidase transduction molecule, H2O2 [17,49,60],
and its ultra-thin nature when formed in neutral media
that allows it to immobilize enzymes eciently
[22,33,34,61]. Moreover, for neurochemical monitoring
in vivo, the PPDenzyme matrix is stable over weeks
Fig. 1 (top). The sensitivity of PtC and PtD electrodes
to H2O2 was therefore measured as the slope of the cal-
ibration plot (0100 lM) that was linear in both cases.
en parameters, Jmax and KM(G), determined using non-linear regression and
calibration slopes in the linear region, for biosensors of dierent designs
cm2) KM(G) (mM) Slope (lA cm2 mM1)
.8 14.7 2.0 0.42 0.09
.3 6.4 0.3 3.0 0.3
.0 5.0 1.4 6.6 1.9
4 9.0 1.4 20.5 1.6
.1 15.0 1.0 0.82 0.18
Fig. 2. Schematics representing the biosensor design themes explored
in this study. Immobilization of enzyme before (top left) or after
(bottom left) deposition of the polymer (PPD) by electrosynthesis on
either 125-lm diameter Pt cylinder (PtC, 1-mm length, top right) ordisk (PtD, bottom right) electrodes. Enzyme was immobilized on top of
the polymer using GA vapor. Polymer thickness and enzyme diameter
are roughly to scale. For clarity, the polymer modier (BSA) has been
omitted from the scheme. Glucose oxidase (GOx) biosensors based on
all four permutations of these two themes were characterized: PtC/
GOx/PPDBSA; PtC/PPDBSA/GOx; PtD/GOx/PPDBSA; and PtD/
C.P. McMahon et al. / Journal of Electroanalytical Chemistry 580 (2005) 193202 197of continuous recording , and its excellent ascor-
bate-rejecting properties are a feature of PPD that has
allowed unambiguous detection of glucose under a vari-ety of physiological and pharmacological conditions in
relatively large brain structures, such as the corpus stri-
atum dorsalis [23,48,62]. For studies of smaller brain
regions, and in other applications where miniaturization
is desirable, the cylinder geometry is not suitable .
We have therefore investigated the properties of a range
of PPD-based glucose biosensors, specically the eects
of decreasing electrode size and increasing GOx loadingon analyte sensitivity and oxygen dependence.
The behavior of each biosensor design was character-
ized by a full calibration in quiescent air-saturated buf-
fer, and analyzed in terms the MichaelisMenten
constants (Jmax and KM(G), Eq. (4)) and the slope of
Jgluc in the linear region (LR) of glucose response. The
rst factor examined was geometry. Fig. 1 (top) shows
averaged calibrations for one cylinder and one diskdesign, both with the same enzyme conguration (poly-
merization following enzyme immobilization): PtC/GOx/
PPDBSA and PtD/GOx/PPDBSA. Clearly, the Jmaxfor the disk-based electrode was signicantly greater
than that for the cylinder with the same polymer
enzyme arrangement (see Table 1 for calibration
Before using Jmax as an index of active enzymeloading when dierent geometries and scales were in-
volved, it was necessary to determine whether subtle dif-
ferences in mass transport to the PtC and PtD electrodes
(see Fig. 2), or a disparity between the condition of the
cylinder and freshly cut disk surfaces, might also con-
tribute to the variations in Jmax values observed in
Means SEM (n = number of biosensors) of apparent MichaelisMent
Eq. (4) for glucose calibrations (see Fig. 1), together with the glucose
calibrated at +700 mV vs. SCE in air-saturated buer (pH 7.4)
Design n-value Jmax (lA
PtC/GOx/PPDBSA 21 6.0 0
PtC/PPDBSA/GOxGA 10 29.0 3
PtC/GOxGA 6 35.8 7
PtD/GOxGA 28 249 3
PtD/GOx/PPDBSA 4 15.8 3PtD/PPDBSA/GOxGA 4 128 33 7.1 1.1 10.5 1.7
roanaThere was no dierence between the two slopes (PtC,
225 15 nA cm2 lM1, n = 23; PtD, 220 10 nAcm2 lM1, n = 51), and both were signicantly higherthan that observed for H2O2 at Pt macrodisk electrodes
, indicating that radial diusion is equally ecient
for PtC and PtD electrodes of these dimensions overthe long time scales pertinent to these measurements.
Thus, given the similarity of H2O2 sensitivity for the
two geometries, the Jmax values in Fig. 1 (top) and Table
1 indicate that there was three times more active GOx
per unit area for PtD/GOx/PPDBSA compared with
PtC/GOx/PPDBSA biosensors. This is consistent with
retention of a dome of enzyme solution around the disk
tip (see Fig. 2) as it is removed vertically from the liquid,as expected from surface tension considerations. Upon
evaporation, the density of GOx on the disk surface
was therefore higher than that achieved when the corre-
sponding cylinder geometry (see Fig. 2) was prepared.
(The eect of the drop that formed on the bottom of
the PtC electrodes had a negligible contribution to the
overall response of the PtC-based biosensors because
the disk is 30 times smaller than the cylinder.)
3.2. Glucose response of cylinder-based biosensors with
dierent enzyme congurations
Biosensors fabricated from PtC wires (Fig. 2) were
used for an initial determination of the eects of enzyme
distribution with respect to the polymer matrix. Starting
by calibrating biosensors where the GOx was immobi-lized with GA on bare Pt (i.e., containing no polymer,
PtC/GOxGA), and then re-calibrating following elec-
tropolymerization, Fig. 1 (bottom) shows that the
PPD matrix caused a signicant lowering the glucose
sensitivity, and that re-loading GOx over the polymer
restored the response.
This nding was explored further using a larger pop-
ulation of similar biosensors (Table 1). There was no sig-nicant dierence between the Jmax values for biosensors
where the GOx was on the surface, either as PtC/GOx
GA (36 7 lA cm2, n = 6) or PtC/PPDBSA/GOxGA (29 4 lA cm2, n = 10, p > 0.35). The KM(G)values were also indistinguishable (5.0 1.4 mM, n = 6
vs. 6.4 0.3, n = 10, p > 0.23) and close to the value of
5 mM cited for unhindered GOx . In contrast, when
polymer was formed after enzyme immobilization, as inPtC/GOx/PPDBSA, the Jmax was lower (6 1
lA cm2, n = 21, p < 0.001 compared with no PPD)and the KM(G) was higher (15 2 mM, n = 21,
p < 0.02). Thus, approximately 80% of the GOx was
inactivated by the electropolymerization process (Fig. 1
and Table 1), either by being denatured, blocked from
access to solution glucose, or lost from the surface com-
pletely. The increase in average KM(G) caused by thePPD suggests that the enzyme remaining active in the
198 C.P. McMahon et al. / Journal of Electpolymer layer has a diminished anity (k1) for glucose(Eq. (5)) that can be represented schematically (see
Fig. 2, top left). Thus, with a thickness that has been
determined to be in the range 10 and 30 nm for the insu-
lating form of the polymer generated at neutral pH
[34,49,58,59], it is likely that PPD hinders a population
of surface GOx molecules (9 nm diameter ) that be-come distributed within the polymer matrix. More de-
tailed trends in KM(G) values are examined below.
3.3. Glucose response of biosensors with dierent
geometries and enzyme congurations
The Jmax values for six combinations of geometry and
enzyme distribution are compared in Table 1. A numberof trends are evident. The largest mean value of Jmax was
achieved for disk biosensors containing no permselective
polymer, PtD/GOxGA. When compared with the cor-
responding cylinder design, PtC/GOxGA, a seven times
lower Jmax-value was obtained, demonstrating again the
eectiveness of the disk shape in dip-evaporation en-
zyme deposition. Electropolymerization after GOx
immobilization on the bare disk, as observed for the cyl-inders, led to a substantial reduction in glucose sensitiv-
ity, that was counteracted when enzyme was deposited
over the PPD-coated metal, i.e., for PtD/PPDBSA/
The overall pattern, therefore, in the data presented
in Fig. 1 and Table 1 is that more active enzyme can
be deposited on disks, compared with cylinders, under
the same conditions of immobilization. The GOx wasmost active when present on a surface (either bare metal
or polymer) compared with biosensors in which the PPD
was generated after the enzyme. These ndings are in
line with expectation and consistent with the schematic
models given in Fig. 2, but only reect the amount of ac-
tive GOx in each design. Equally important, are the cor-
responding KM(G) values, because this is a key
parameter in determining both the biosensors sensitivityto glucose in the linear response region (Jmax/KM(G))and the extent of the linear range (1/2KM(G)). Wetherefore carried out a detailed correlation analysis of
the dependence of KM(G) on enzyme loading, both with-
in and between designs.
3.4. Correlation analyses for KM(G) vs. Jmax
Fig. 3 (top) shows the correlation between KM(G)
and Jmax (enzyme loading) for the two PtC-based biosen-
sor designs incorporating PPD. Unexpectedly, KM(G)
values for PtC/GOx/PPDBSA showed a statistically sig-
nicant negative correlation (R2 = 0.37, p < 0.01) with
Jmax, ranging from 15 mM for electrodes with the low-est GOx loading to 5 mM for those with the highestloading. Thus, the change in KM(G) here cannot be as-cribed to changes in oxygen demand as this factor would
lytical Chemistry 580 (2005) 193202have the opposite eect . Instead, an explanation is
0 10 20 30 40 500
slope = 0.026 0.023R2 = 0.14 (n=10)
p > 0.28
slope = -1.15 0.36R2 = 0.37 (n=19)
p < 0.01
Jmax / A cm-2
C.P. McMahon et al. / Journal of Elecprovided by the model of the GOx/PPD matrix shown in
Fig. 2. For biosensors of this design with very low active
enzyme levels, much of the GOx is buried in the PPD
that hinders binding by glucose, decreasing k1 and there-fore increasing KM(G); see Eq. (5). As the Jmax increases,
a greater fraction of the GOx lies towards the surface of
the polymer so that the average enzymesubstrate bind-
ing barrier diminishes, decreasing KM(G). Although this
interpretation is speculative, it is strongly supported by
the behavior of KM(G) vs. Jmax for the other cylinder
PPD-based design: PtC/PPDBSA/GOx; see Fig. 3
(top). Here, the GOx was placed over the polymer andthe observed values of KM(G) are all similar, i.e.,
independent of Jmax over a four times larger range of
Jmax than for the PtC/GOx/PPDBSA design. Moreover,
the values of KM(G) for the GOx-on-surface congura-
tions clustered around the minimum value observed
for the PtC/GOx/PPDBSA design: 5.0 1.4 mM
0 100 200 300 400 500 6000
slope = 0.035 0.004R2 = 0.766 (n=30)
p < 0.001
Jmax / A cm-2
) / m
Fig. 3. KM(G) as a function of enzyme loading and distribution.
Correlation plots of the apparent MichaelisMenten parameters (Eq.
(4)), KM(G) vs. Jmax for Pt/PPD electrodes incorporating the enzyme
GOx in a variety of congurations: as 125-lm diameter cylinders (PtC)and disks (PtD); and with the GOx immobilized either before (GOx/
PPD) or after (PPD/GOx) deposition of the PPD polymer. (Top)
Cylinder biosensors showing the dierent correlation behaviors of the
two enzyme congurations. (Bottom) Systematic increase in KM(G)
with enzyme loading (Jmax) for PtD-based biosensors with surface GOx
compared with the cylinder geometry behavior, as detailed above
(top).(PtC/GOxGA, n = 6, R2 vs. Jmax = 0.16, p > 0.42); and
6.4 0.3 mM (PtC/PPDBSA/GOxGA, n = 10, R2 vs.
Jmax = 0.14, p > 0.28), values also consistent with litera-
ture KM(G) for unhindered GOx molecules .
Table 1 shows that there were some similarities be-
tween the average KM(G) values for cylinder and disktype biosensors. Thus, there were no dierences be-
tween: the high values of KM(G) observed for enzyme-
hindered PtC/GOx/PPDBSA and PtD/GOx/PPDBSA
designs (15 1 mM, p > 0.94; see also Fig. 1) that have
been reported previously for GOx trapped in polymers
[63,64]; the low values for the exposed enzyme in
PtC/PPDBSA/GOxGA (6.4 0.3 mM) and PtD/
PPDBSA/GOxGA (7.1 1.1 mM, p > 0.40); or thosefor PtC/GOxGA (5.0 1.4 mM) and PtD/GOxGA
(9.0 1.4 mM, p > 0.20).
Because the values for PtD/PPDBSA/GOxGA and
PtD/GOxGA electrodes were on the high side of the
averages, it is possible that the very high GOx loading
for these biosensor designs, discussed above, might be
aecting KM(G) as observed previously . Therefore,
correlation analyses (KM(G) vs. Jmax) were performedfor disk-based designs showing high average Jmax values
(GOx exposed on the surface) to compare with cylinder
results (Fig. 3). There was no dierence between the
linear regression correlation slopes calculated for
PtD/GOxGA (31 4 lM lA1 cm2, R2 = 0.963,
p < 0.02) and PtD/PPDBSA/GOxGA (36 4 lMlA1 cm2, R2 = 0.772, p < 0.001) designs (p > 0.63).The correlation points for these two sensor types weretherefore pooled to compare with cylinder behavior.
The positive correlation between KM(G) and Jmax for
the pooled disk-based biosensor designs (35 4
lM lA1 cm2, n = 30, R2 = 0.766, p < 0.001) was verydierent to that for the cylinder designs (negative or zero
correlation); see Fig. 3 (bottom). It appears, therefore,
that the extensive build-up of enzyme on the disk sur-
face, possibly through a stacking arrangement previ-ously observed , led to the signicant positive
correlation between KM(G) and Jmax for these PtD-based
biosensors, associated with a reduction in GOx e-
ciency. This loss of eciency could be due to either
the development of a barrier to glucose binding by
GOx itself as the enzyme loading increased, or to a lim-
itation in oxygen supply with excess loading. This issue
is addressed in Section 3.6.
3.5. Linear region glucose sensitivity
Having established patterns in enzyme loading (Jmax)and KM(G) for the six biosensor designs listed in Table 1,
it is now possible to compare rationally the analytically
more relevant parameter of LR glucose slope which, in
the limit as glucose concentration approaches zero, is
Jmax/KM(G). Based on the analyses described above, it
lytical Chemistry 580 (2005) 193202 199was not surprising that the lowest LR slope, determined
PtD/PPD-BSA/GOx in 1 mM glucose
0 20 40 60 800
R2 = 0.996
[O2] / M
Fig. 4. Example of raw data for the response (current density) of a
PtD/PPDBSA/GOx biosensor to 1 mM glucose as a function of
continuously changing concentration of dissolved oxygen. The curve
was generated using non-linear regression and Eq. (6) (R2 = 0.996).
The KM(O2) values obtained for dierent concentrations of glucose are
plotted in Fig. 5.
Oxygen dependence of PtD/PPD-BSA/GOx
0.0 0.5 1.0 1.5 2.00
[glucose] / mM
Fig. 5. Values of KM(O2) for the PtD/PPDBSA/GOx design
(means SD, n = 4; see Fig. 4) plotted as a function of glucose
concentration. The improved experimental design allowed more
reproducible results at low concentrations of glucose and a clear
demonstration that KM(O2) is a linear function in glucose
(R2 = 0.9995) with an intercept at the origin. The slopes of similar
plots for dierent biosensor designs were used to compare the O2
roanalytical Chemistry 580 (2005) 193202experimentally by calibration in the LR, was for the cyl-
inder design with hindered enzyme (PtC/GOx/PPDBSA,
0.4 0.1 lA cm2 mM1; Table 1), whereas the highestvalue was that for a disk-based design with surface en-
zyme (PtD/GOxGA, 21 2 lA cm2 mM1), a value
50 times more sensitive than the cylinder design usedfor neurochemical monitoring in vivo [23,38,48]. How-
ever, the absence of the permselective PPD barrier in
the PtD/GOxGA sensors makes them unsuitable for
applications in real samples containing interference
species, such as ascorbic and uric acids. The most sensi-
tive biosensor involving the PPD layer was PtD/PPD
BSA/GOx (11 2 lA cm2 mM1) that, with a 25times greater sensitivity (LR current density slope), anda 30 times smaller area, than the standard PtC/GOx/
PPDBSA design, could be very useful for in vivo anal-
ysis provided its oxygen dependence was suitable for
The limit of detection (LOD) for the dierent biosen-
sor congurations was determined as 3r of the corre-sponding background currents. The values ranged
from 19 6 lM for the lowest LR sensitivity design(PtC/GOx/PPDBSA) to 3 1 lM for the highest sensi-tivity implantable biosensor (PtD/PPDBSA/GOx).
Thus, all these congurations were suitable for applica-
tions involving low glucose levels, for example, in brain
ECF (as low as 350 lM ). Other GOx designs, suchas those reported recently involving platinization of Pt
cylinders, can show excellent sensitivity, but much
higher LOD values .With a correspondingly higher glucose turnover rate
for the disk-based designs compared to PtC/GOx/
PPDBSA sensors that have previously been shown
not to be limited by oxygen over relevant physiological
ranges for both glucose and pO2 , it was important
to evaluate and compare the oxygen dependence of the
four variations of PPD-containing biosensors. Such a
comparison should also reveal whether oxygen supplylimitations are responsible for the positive correlation
of KM(G) with GOx loading observed in Fig. 3 for
3.6. Oxygen dependence
The experimental protocol for determination of oxy-
gen dependence was similar to that reported recently. Fig. 4 shows the variation of Jgluc, recorded with
a PtD/PPDBSA/GOx biosensor in 1 mM glucose, as a
function of solution oxygen concentration that was
monitored simultaneously using the self-calibrating
CellOx probe. The data tted the adapted Michaelis
Menten equation well (Eq. (6), R2 = 0.996) and provided
KM(O2) values that can be used as an index of oxygen
dependence, with lower values of KM(O2) beingpreferred. In the previous study that characterized
200 C.P. McMahon et al. / Journal of ElectPtC/GOx/PPDBSA electrodes , the lowest glucoseconcentration for KM(O2) studies was 0.5 mM, and a
linear relationship for KM(O2) vs. glucose concentration
suggested based on an R2 value of 0.96. Because baseline
glucose levels in certain brain regions are below 0.5 mM
[62,66,67], this linearity was re-investigated down to a
concentration of 0.2 mM. Fig. 5 conrms this importantlinearity through the origin (R2 = 0.9995), and allows
one to interpolate a value of KM(O2) for any
concentration of glucose for the corresponding biosen-
Using this protocol and analysis (Fig. 5) for a range
of discrete concentrations of glucose, the slope of
KM(O2) vs. glucose concentration was determined forsensitivities of the diverse devices.
each of the four PPD-containing biosensor types. This
slope (lM O2 per mM glucose) was remarkably similarfor all four designs: PtC/GOx/PPDBSA (12.2 1.3,
n = 12); PtC/PPDBSA/GOx (15.7 1.0, n = 12);
PtD/GOx/PPDBSA (13.3 0.6, n = 4); and PtD/PPD
BSA/GOx (12.1 0.6, n = 4). These results are surpris-ing given the very dierent LR glucose slopes for the
four designs (Table 1) and the corresponding greater
oxygen demand of the disk-based devices, even in the
LR of glucose response.
The trends in these average KM(O2) values can, how-
ever, be understood better in terms of the correlation be-
tween KM(O2) and KM(G) for individual sensors. Fig. 6
(top) shows that, not only did the KM(O2) not increaseas the LR glucose slope increased, there was a statisti-
cally signicant, albeit modest, decrease in KM(O2) for
sensors with higher glucose sensitivity. A possible cause
for this behavior is revealed in Fig. 6 (bottom). There
was a parallel weak negative correlation between
KM(O2) and KM(G). Thus, the sensors that had high
GOx loading had high LR slope (Table 1), but also a
higher KM(G). There was more enzyme in the biosensinglayer, but each GOx molecule had a lower anity for
glucose and a lower turnover rate that lowered oxygen
can therefore be understood, not as a limitation in oxy-gen supply, but as a decrease of the anity of glucose
oxygen interference , it remains to be seen whether
the more active designs with surface enzyme can with-
20 slope = -0.240.09R2 = 0.213 (n=30)p < 0.02
C.P. McMahon et al. / Journal of Electroana0 3 6 9 12 1510
glucose LR slope / A cm-2 mM -1
4 6 8 10 12 14 16 1810
20 slope = -0.270.14R2 = 0.20 (n=18)p = 0.06
KM(G)/ mM glucose
Fig. 6. Correlation analyses for oxygen dependence (KM(O2) slope; see
Fig. 5) vs. glucose sensitivity quantied as both the linear region
glucose slope (top) and the KM(G), the apparent Michaelis constant
for glucose (bottom). There was no evidence for increased oxygen
dependence as the glucose sensitivity increased; moreover, there was a
slight statistically signicant trend for the oxygen dependence todecrease with increased glucose sensitivity.stand the conditions of direct tissue contact in in vivo
monitoring, or whether a protective barrier such as a
microdialysis membrane will be needed for protection.
In either case, the PtD/PPDBSA/GOx biosensor will
now enable us to explore glucose concentration
dynamics of smaller and layered brain regions withgood sensitivity and minimal interference from uctu-
ations in tissue pO2.
This work was funded in part by Science Foundation
Ireland (04/BR/C0198). We thank Enterprise Ireland fora postgraduate scholarship (CMcM), and UCD forfor GOx (Eq. (5)) associated with steric hindrance by
neighboring enzyme molecules. A similar, but more
pronounced, trend has been reported recently for gluta-
mate oxidase in PPD-based biosensors, where glutamate
binding was even more sensitive to enzyme loading, an
eect due, at least in part, to electrostatic interactions
between the anionic substrate and the polyanionicenzyme .
The glucose sensitivity and oxygen dependence of a
variety of implantable GOx-based biosensors incorpo-
rating a PPDBSA polymer permselective barrier weremeasured and compared. Dip-evaporation of the en-
zyme was more ecient on disks than on cylinders. Be-
cause polymerization after GOx deposition deactivated
a large fraction of the immobilized enzyme, the most
sensitive biosensor design studied was PtD/PPDBSA/
GOx in the linear region of glucose response, that was
20 times more sensitive than the more widely usedPtC/GOx/PPDBSA conguration. The oxygen depen-dence, KM(O2), of both active and less active designs
was surprisingly similar, a nding that could be rational-
ized in terms of an increase in KM(G) with increased en-
Although a basic PPD-GOx polymerenzyme com-
posite biosensor design has been used for continuous
recording over weeks in the harsh electrochemical
environment of brain tissue  without signicantdemand on a molecular level. For sensors with high glu-
cose sensitivity, the enzyme layer as a whole had a high
oxygen demand, but this was spread out, possibly over a
3D stacking arrangement of the GOx . The positive
correlation for KM(G) vs. Jmax shown in Fig. 3 (bottom)
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Design variations of a polymer -- enzyme composite biosensor for glucose: Enhanced analyte sensitivity without increased oxygen dependenceIntroductionMaterials and methodsChemicals and solutionsInstrumentation and softwareWorking electrode preparationAmperometric experimentsData analysis
Results and discussionGlucose response of a cylinder- and disk-based polymer ndash enzyme configurationGlucose response of cylinder-based biosensors with different enzyme configurationsGlucose response of biosensors with different geometries and enzyme configurationsCorrelation analyses for KM(G) vs. JmaxLinear region glucose sensitivityOxygen dependence