ANALYTICA

CHIMICA
ACIA
EISEVIER Analytica Chimica Acta 323 (1996) 87-96

Parameters which influence the optimal immobilisation of oxidase

type enzymes on methacrylate copolymers as demonstrated for
amperometric biosensors
C.E. Hall, D. Datta I, E.A.H. Hall *
University of Cambridge. Institute of Biotechnology, Tennis Court Road, Cambridge, UK

Received 2.5September 1995; revised 20 November 1995; accepted 1 December 1995

Abstract

Optimisation of the covalent attachment of enzymes to copolymers of methyl and glycidyl methacrylates is explored. The
importance of ionic strength and pH were shown to influence the immobilisation efficiency, as well as enzyme concentration
and immobilisation time. It was seen that for the case of glucose oxidase, the protein immobilisation efficiency correlated
with the isoelectric point, whereas the immobilised enzyme activity, measured in terms of an amperometric response to
glucose correlated with an immobilisation pH of maximum enzyme activity. Conditions for the maximum reactivity of the
glycidyl group were also considered but found not to be the major determinant of an optimum immobilised enzyme. The
conditions were evaluated for five oxidase type enzymes: glucose oxidase, lactate tyrosinase (polyphenol oxidase),
cholesterol oxidase and alcohol oxidase in terms of their immobilisation efficiency. The immobilisation process has also
been shown to stabilise the enzyme with respect to activity loss with time of storage, especially in the case of lactate
oxidase.

Keywords: Enzymatic methods; Biosensors; Amperometry; Methacrylate copolymers

1. Introduction Mass deposition of this active component, as well as

The basic element of an amperometric biosensor
is the biocatalyst which produces a chemical signal
that can be determined electrochemically. The “ac-
tive ’ ’ component of such a sensor, which in this
instance is an enzyme, can be immobilised in differ-
ent ways, dependent upon its intended use [ 1,2].
l Corresponding author.
’ Permanent address: Department of Inorganic Chemistry, In-
dian Association for the Cultivation of Science, Jadavdur, Cal-
cutta-700 032, India.
any cofactors, mediators [3] or other reagents, in
biosensor designs in which all components are im-
mobilised or entrapped in one step [4,5] is of funda-
mental importance to the success of the device.
Much research has been directed at deposition partic-
ularly by ink-jet printing [6], and photolithographic
techniques [7,8] (which has resulted in a reduction in
the overall size of the sensing device [9-l 111, but
these advances require compatible immobilisation
strategies which provide a stable environment for the
recognition protein.
In previous papers we described an immobilisa-

0003-2670/%/$15.00 0 1996 Elsevier Science B.V. All rights reserved

SSDI 0003-2670(95)00617-6
88 C.E. Hall et al./Analytica
tion system based on the covalent binding of glucose
oxidase to methacrylate copolymers, which we ex-
plored in the development of amperometric sensors
[12-141. These methacrylate polymers were selected
because they were proven materials in photolithogra-
phy [15-171 and could be produced with glycidyl
groups which provide a convenient covalent immo-
bilisation site f18,19]. Although the effects of certain
polymer characteristics, such as molecular weight,
copolymer ratio and hydrophobicity, were explored,
and the feasibility of tuning these parameters for
operation in different measurement environments was
proposed, the optimal conditions were not identified,
for covalent attachment of the enzyme and retention
of maximum enzyme activity. In this respect, subse-
quent reports have disagreed about the ideal condi-
tions for efficient enzyme attachment via the gly-
cidyl group [ 16,17,20].
The criteria which will be of importance here will
either concern the normal conditions for enhanced
epoxide ring opening (acid or base catalysed) or an
optimum pH environment for the enzyme. In this
paper we have thus considered the factors which
influence the efficiency of the immobilisation reac-
tion with respect to the amount of enzyme bound to
the polymer and subsequently the activity of that
bound enzyme, measured as the current response to
substrate additions, of enzyme modified polymer
coated electrodes in an amperometric setup.
2. Experimental
2.1. Materials
Glucose oxidase EC 1.1.3.4 (type VII from As-
pergillus Niger), lactate oxidase EC 1.1.3.2 (from
Pediococcus sp.), cholesterol oxidase EC 1.1.3.6
(from Brevibacterium sp.), alcohol oxidase EC
1.1.3.13 (from Hansenula sp.) and tyrosinase EC
1.14.18.1 (from mushroom) were supplied by Sigma.
Buffer solutions were prepared from AnalaR grade
reagents and deionised water. The polymers were
prepared and purified in the manner described below
using HPLC grade solvents. The monomers, methyl
methacrylate and glycidyl methacrylate were ob-
tained from Aldrich and used without further purifi-
cation. The initiator cy, cr’azoisobutyronitrile (AIBN)
Chimica Acta 323 (1996) 87-96
from Fluka, was recrystallised from methanol prior
to use.
2.2. Polymer preparation
Polymers were prepared as described previously
[12,13], using AIBN as the initiator. The molecular
weights of the polymers were determined by GPC
(quoted in polystyrene units) by RAPRA Technol-
ogy, the polymer characterisation centre provided by
the Materials Committee of the SERC. The chro-
matographic conditions were as follows: columns,
PLgel 2 X mixed bed-B, 30 cm, 10 ,um; solvent,
tetrahydrofuran; flow rate, 1.O ml/min; temperature,
ambient; detector, RI. Sample solutions were pre-
pared by dissolving 20 mg of polymer in 10 ml of
solvent overnight, 1,Zdichlorobenzene was added as
an internal marker and the solution was filtered
through a 0.2 pm filter. Typically these polymers
had the following characteristics; M, = 10000, M,
= 16000 and a polydispersity of 1.6.
2.3. Enzyme immobilisation on polymers
The covalent immobilisation of enzymes to a
substrate has been extensively reviewed [1,2,21]. The
basic protocol of the enzyme immobilisation has
been described previously [12], and involves the
mixing of the polymer (125 mg) with enzyme solu-
tion (5 ml) and shaking for up to 96 h. Subsequently
the polymer is filtered, washed with buffer solution
and shaken in a 1 mM glycine solution (5 ml) for 24
h to destroy residual epoxide groups. Finally the
modified polymer is filtered and washed sequentially
with buffer (2 X 10 ml), NaCl solution (2 X 5 ml,
0.5 M in buffer), and buffer solution (2 X 10 ml)
before drying under reduced pressure for 1 h.
In this paper the effects of the ionic strength of
the immobilisation medium are reported between
0.05 and 1.0 M, the pH between 4.0 and 9.0, the
enzyme concentration, which was varied between 0.5
and 2.0 mg/ml, and the temperature of the immobil-
isation reaction, either 4°C or 25°C. The amount of
enzyme immobilised on the polymer was estimated
by difference between the concentration of the en-
zyme solution used and the total enzyme concentra-
tion of the collected washings. The protein determi-
nation was carried out using Coomassie Protein
 C.E. Hall et al./Analytica Chimica Acta 323 (1996) 87-96 89

Reagent (Sigma) on a Perkin-Elmer Lambda 5 UV- 2.5. Electrochemical measurements

visible spectrophotometer. It should be pointed out
that although the lower detection limit of this method
was found to be 10 pg/ml with an error of 15%, the
assay is very approximate and the results can only be
considered relative to each other to identify trends.
Determination of residual enzyme activity after the
immobilisation procedure is not possible using exist-
ing spectroscopic assays because of interference from
the polymer particles. However, electrochemically
determined activities have been calculated and are
expressed in terms of current density per pg of
immobilised enzyme and are therefore subject to the
same error range as the original protein determina-
tion.
2.4. Electrode preparation
In this study the substrates were measured indi-
rectly via the electrochemical determination of hy-
drogen peroxide, the common co-product of these
enzyme reactions. This was carried out at a potential
of 0.65 V (vs. SCE), in 0.05 M potassium perchlo-
rate-O.05 M potassium phosphate buffer solution at
pH 5.5 for glucose and pH 7.0 for lactate, using
electrodes coated with enzyme modified polymer.
Alternative measurements employing artificial medi-
ators which require lower measuring potentials were
considered, but this was precluded by the high de-
gree of partitioning previously observed [13]. The
electrochemical measurements were made using an
EG& G 273A potentiostat/galvanostat in conjunc-
tion with a Gould Y-t recorder.

Electrodes were prepared by coating platinum 3. Results and discussion

wire electrodes (length 1.5 cm, diameter 0.05 cm,
area 0.25 cm21 with the enzyme modified polymer.
This was achieved by dripping a dichloromethane
solution of the polymer (5 mg/150 ~1) down the
length of the electrode. In this way reproducible
coatings of modified polymer of known weight (1 +
0.2 mg) were obtained. This reproducibility has been
discussed previously [ 131. Before use the electrodes
were soaked in buffer solution (0.05 M potassium
perchlorate-0.05 M potassium phosphate pH 5.5) for
24 h.
3.1. Glucose oxidase
The successful commercial development of a
biosensor will depend on a number of factors which
can be directly influenced by the choice of immobili-
sation system including the efficiency of the immo-
bilisation procedure, manufacturing reproducibility
and the stability and shelf life of the sensor device
1221. In preliminary papers glucose oxidase was co-
valently bound to a range of copolymers which could

Table 1
lmmobilisation data at different buffer concentrations
Buffer lmmobilisation Bound Polymer Enzyme
concentration efficiency ‘, enzyme b, normal&d current normal&d
CM) %(Yt3) % ( f 0.07) response at current response
40 mM glucose at 40 mM glucose
hA/cm’) (nA/pg/cm*)

0.05 28.0 0.57 731 128

0.1 29.1 0.58 804 139
0.3 27.4 0.55 862 157
0.5 30.3 0.61 853 140
1.0 26.2 0.52 619 119

a Based on the uptake of enzyme from a solution containing 0.5 mg/ml of glucose oxidase.
b Calculated from the mass of enzyme consumed divided by the mass of polymer used.
90 C.E. Hall et al./Analytica Chimica Acta 323 (1996) 87-96

be deposited using thin-layer technologies in a “nor- 3.1 .I. Ionic strength

mal” fabrication environment and the effects of
polymer loading and conditioning time on the magni-
tude of the current response, studied for glucose
additions on a GOD modified polymer coated elec-
trode [12]. The reproducibility of the enzyme immo-
bilisation and electrode fabrication steps were shown
to deviate by a maximum of 7% at 40 mM glucose
for non-automated hand deposition, thus illustrating
the potential reproducibility of the process [13].
A number of factors have been shown to affect
the efficiency of protein immobilisation on solid
supports [23]. These include enzyme purity, the ionic
strength of the immobilisation medium, the pH of the
immobilisation medium, enzyme concentration, tem-
perature and length of the procedure. This study has
focused on the effects of ionic strength, pH, enzyme
concentration, temperature and reaction time on the
amount of enzyme immobilised and the subsequent
activity shown by the enzyme modified polymer.
Variation in the ionic strength of the immobilisa-
tion medium had little effect on the amount of
enzyme immobilised on the polymer (Table 1). How-
ever, in terms of the performance of electrodes pre-
pared from these polymers, it was seen that the
optimum current response to glucose additions was
obtained between 0.3 and 0.5 M ionic strength (Ta-
ble 1 and Fig. 1). At lower ionic strength the re-
sponse gradually decreased, showing a 15% reduc-
tion in signal from polymers prepared in 0.05 M
buffer solutions, while at higher ionic strength there
was a significant and rapid loss of apparent activity
above 0.5 M resulting in a 25% reduction in signal
output for 1.O M ionic strength modified polymers. It
is known that the conformation of an enzyme and
hence its activity is affected by ionic strength [24],
this suggests that the conformation in which the
enzyme is covalently attached and hence the en-
zyme’s activity thereafter can be manipulated by

Table 2
Immobilisation data at different temperatures and enzyme concentrations
Temperature Sample Enzyme Enzyme Immobilisation Bound Polymer Enzyme
(“0 time cont. consumed efficiency a, enzyme b, normalised normalised
(days) (mg/ml) (mg) %(+3) % ( f 0.08) current current
response at response at
40 mM glucose. 40 mM glucose.
hA/cm*) (nA/pg/cm’)
4 1 0.5 0.32 13.1 0.24 253 105
4 2 0.5 0.56 22.8 0.47 936 199
4 3 0.5 0.55 22.2 0.46 769 167
4 4 0.5 0.52 21.1 0.41 661 161
4 7 0.5 0.47 19.2 0.39 208 53
4 1 1.0 0.58 10.9 0.45 432 96
4 2 1.0 0.66 12.4 0.49 599 122
4 3 1.0 0.69 13.0 0.57 659 116
4 4 1.o 0.77 14.5 0.63 648 71
4 7 1.o 0.77 14.5 0.60 524 87
4 1 2.0 0.24 3.5 0.19 678 357
4 2 2.0 0.55 7.9 0.43 820 191
4 3 2.0 0.72 10.4 0.55 777 141
4 6 2.0 0.72 10.4 0.51 594 116
4 7 2.0 0.61 8.7 0.48 309 64
25 0.66 1.o 1.02 20.8 0.84 329 39.2
25 1 1.0 1.11 22.1 0.88 154 17.5
25 2 1.0 1.14 22.7 0.94 238 25.3
25 3 1.0 1.17 23.3 0.94 319 33.9
25 4 1.o 1.17 23.3 0.95 277 29.2

a Based on the uptake of enzyme from a solution of glucose oxidase.

b Calculated from the mass of enzyme consumed divided by the mass of polymer used.
 C.E. Hall et al./Analytica Chimica Acta 323 (1996) 87-96 91

tration, within the range OS-2 mg/ml, and gener-

ally was reached in two to three days (Table 2). This
infers that the reaction is not limited by enzyme as
far as consumption of starting material is concerned
600
and the efficiency of the process with respect to
oow enzyme immobilised was highest when the enzyme
400
- 01H
concentration was lowest. At 25°C the same trend
- 034
was observed, so that it can be deduced that tempera-
200
- O.SM
ture influences all the elementary steps of the reac-
- I.oM
op..,‘...‘.. I...I,..I...’ tion mechanism in a similar manner.
0 10 20 30 40 50 60 Electrodes coated with these same enzyme modi-
[GLUCOSE] (mM) fied polymers gave a current response to glucose
Fig. 1. Plot of current response versus glucose concentration for
(Fig. 2) consistent with the trends above for protein
polymers modified at various buffer concentrations between 0.05 loading, if the enzyme immobilisation was termi-
and 1.0 M whilst maintaining the following conditions; 0.5 mg/ml nated prior to maximum loading being achieved (i.e.,
glucose oxidase, 4°C and 72 h immohilisation time. l-3 days, see Table 2). However, the response
rapidly diminished for immobilisation times exceed-
varying the ionic strength of the immobilisation solu- ing four days as shown in Fig. 3. This has been seen
tion. previously and is generally expected since the activ-
ity of an enzyme is reduced after prolonged shaking,
3.1.2. Enzyme concentration, time and temperature and no longer correlates with the apparent protein
At 4°C the maximum immobilisation level ob- loading on the polymer.
tained was independent of the initial enzyme concen- In fact, even within the initial immobilisation

DA” 1 b
DAY 2
DAY 3
DAY 4

[;;*

0 10 20 30 40 50 0 10 20 30 40 )

[GLUCOSE!) (mM) [GLUCOSE] (mM)

1003, 400,
+
600.: ‘.
I
600 _

0 10 20 30 40 50 0 10 20 30 40 50

[GLUCOSE) (mM) [GLUCOSE] (mM)

Fig. 2. Plot of current response versus glucose concentration for polymers modified at various temperatures/glucose oxidase concentrations:
(a) 4°C. 0.5 rug/ml; (b) 4°C. 1.O mg/ml; (c) 4°C. 2.0 mg/ml; (d) 25°C. 1.O mg/ml
92 C.E. Hall et al./Analytica Chimica Acta 323 (1996) 87-96

to be independent of enzyme concentration within

the first two days, the current response to glucose of
the resultant polymer favoured the low enzyme con-
centration (Fig. 3). In any case, as can be seen from
these results enzyme concentration during immobili-
sation is important in determining the electrochemi-
cal response to glucose of these copolymer materials.

0 2 4 6
IMMOSILISATION TIME (DAYS)
Fig. 3. Plot of cunent response at 20 mM glucose versus immobil-
isation time for polymers modified at various glucose oxidase
concentrations.
period the resultant current response to glucose from
the polymer cannot be directly calculated from just
the total enzyme loading, but is a complex relation-
ship between the amount of protein in solution and
the immobilisation time. The latter presumably ac-
counting for the loss of activity during the shaking.
Assuming that the immobilisation kinetics are not
limited by enzyme concentration (see above), then it
would be predicted that the optimum immobilisation
protocol would use the minimum initial enzyme
concentration, or would add the enzyme gradually
during the immobilisation period, such that aggrega-
tion of protein in solution is minimised and the
opportunity for protein denaturing prior to attach-
ment to the polymer is reduced. Indeed, it is seen
that although the enzyme loading at 4°C is estimated
3.1.3. pH effects
6 The choice of pH for the immobilisation reaction
must take into consideration the optimum pH re-
quired for reaction of the epoxide group and the pH
range over which a particular enzyme is stable, or its
activity loss is negligible. The epoxide group under-
goes reaction across a wide range of pH values from
very acidic to very basic, but the most efficient ring
opening is obtained at the extremes of PH. However,
most enzymes are unstable at such pH values, and
therefore it is more usual to carry out this reaction at
pH 7.0, where it has been shown to occur readily
118,231. In the case of glucose oxidase typical load-
ings of 0.57% are obtained with an efficiency of
28% at pH 7.0 [12,13]. Variation of the pH of the
immobilisation reaction should enhance the ring
opening reaction and thereby increase the enzyme
loading and efficiency of the process. Since the pH
of the immobilisation solution will also affect the
conformation and therefore activity of the enzyme,
significant changes should also be observed in the
response of glucose oxidase modified, polymer
coated electrodes to glucose additions.
The variation in the pH of the immobilisation

Table 3
lmmobilisation data at different pH values

PH lmmobilisation Bound enzyme b, Polymer Enzyme

efficiency 8, % ( * 0.07) normalised current normalised
%(+_3) response at current response
40 mM glucose at 40 mM glucose
(nA/cm*) (nA/pg/cm*)
4 51.9 1.15 1230 107
5 51.4 1.16 3040 262
6 41.1 0.81 2680 331
7 26.9 0.58 1870 322
8 18.3 0.44 1490 339
9 14.0 0.30 750 250

’ Based on the uptake of enzyme from a solution containing 0.5 mg/ml of glucose oxidase.
b Calculated from the mass of enzyme consumed divided by the mass of polymer used.
 C.E. Hall et al./Analytica
medium did indeed influence the amount of enzyme
immobilised on the polymer (Table 3). At pH 4.0 the
immobilisation efficiency was 51.9% compared to
14.0% at pH 9.0. However this may also be due to
differences in the protein solubility and interactions
with the polymer as the pH was varied. At low pH
values, approaching the isoelectric point of glucose
oxidase (pH 4.21, the solubility of the enzyme falls
and the protein has a tendency to aggregate and
adsorb onto surfaces due to increasing surface-
surface interaction as the net charge tends to zero
[24]. At high pH values the high net electric charge
on the protein structure inhibits aggregation and the
large water envelope around the protein inhibits reac-
tion between the protein and the polymer. Since the
pH for efficient epoxide ring opening and the iso-
electric point coincide these two influences cannot
be separated for glucose oxidase.
Electrodes prepared from these polymers demon-
strated a wide variation in the current response to
sequential glucose additions (Fig. 4), which again
was consistent with the protein loading data in Table
3 except at very low pH values. The maximum
current at pH 4.0 was only 40% of that obtained at
pH 5.0, indicating a significant degree of enzyme
inactivation during the immobilisation process. This
was also seen in the current response normalised to
enzyme loading (final column, Table 3). Above pH
8.0 the current response fell in line with the protein
loading data and reached a minimum for the polymer
prepared at pH 9.0. This data shows that the optimal
pH for the immobilisation of glucose oxidase is
between 5 and 6 with respect to maximising the
amperometric response of the polymer-enzyme con-
jugate, although the electrochemically determined
Table 4
Enzyme modification data for a 3: 1 copolymer of methyl and glycidyl methacrylates with various enzymes
Polymer Enzyme
25G75M-2 Glucose oxidase
25G75M-2 Lactate oxidase
25G75M-2 Cholesterol oxidase
25G75M-2 Alcohol oxidase
25G75M-2 Tyrosinase
’ Based on the uptake of enzyme.
b Calculated from the mass of enzyme consumed divided by the mass of polymer used.
Chimica Acta 323 (1996) 87-96 93
enzyme activities are similar between pH 6 and 8.
This corresponds to the pH of maximum enzyme
activity for the free enzyme, suggesting that the
optimal immobilisation pH should be close to that of
the enzyme activity maximum rather than the epox-
ide reactivity maximum. This is a reasonable obser-
vation as the conformation of the immobilised en-
zyme will be determined during the immobilisation,
so that immobilisation at a pH at which the enzyme
is in a conformation resulting in maximum activity
should result in the polymer enzyme conjugate hav-
ing its maximum activity.
If this assumption is correct then the conditions
should be predicted, whereby optimal immobilisation
efficiency and/or optimal enzyme activity for other
enzymes is achieved, depending on the pH profile
for the enzyme. The integrity of this result was thus
tested on other oxidase systems.
3.1.4. Other oxidase enzymes
Table 4 illustrates the immobilisation efficiency at
pH 7.0 for five different enzymes of the oxidase
class; glucose, lactate, cholesterol, alcohol and
polyphenol. A clear distinction can be seen in this
data between glucose oxidase, 28% immobilisation
efficiency, and the other enzymes for which the
efficiency was 100%. This correlates with differ-
ences in their isoelectric points, and it may appear
therefore that the surface charge of the protein is
more critical than the optimum pH for epoxide at-
tack. Lactate oxidase and polyphenol oxidase possess
isoelectric points of 7.2 and 6.9, respectively, and the
reaction with these two enzymes goes to 100% com-
pletion in a pH 7.0 buffer solution. In contrast
glucose oxidase, with an isoelectric point of 4.2,
Enzyme Immobilisation Bound
concentration efficiency ’ enzyme b
(mg/ml) %(f3) % ( f 0.07)
0.5 28.0 0.57
0.5 100 2.08
0.5 100 2.04
0.5 100 1.93
0.3 100 1.19
94 C.E. Hall er al./Analytica Chimica Acta 323 (1996) 87-96
0
0 10 20 30 40 50
[GLUCOSE] (mM)
Fig. 4. Plot of current response versus glucose concentration for
polymers modified by varying the pH between 4.0 and 9.0 whilst
maintaining the following conditions: 0.1 M buffer solution, 0.5
mg/ml glucose oxidase, 4°C and 72 h immobilisation time.
only attains 28% efficiency at pH 7.0 but approaches
52% efficiency at pH 4.0. However, as was seen in
the previous section where the maximum loading of
glucose oxidase was obtained at pH 4.0, this did not
correspond to the maximum electrochemical signal
which was found at the pH for maximum enzyme
activity, between pH 5.0 and 6.0.
3.2. Application to lactate oxidase
Using the results deduced above, electrodes were
fabricated from lactate oxidase modified 25G75M-2,
prepared near the isoelectric point for the enzyme
(and at the optimal pH for enzyme activity) as in
Table 4, in a comparable manner to those prepared
from glucose oxidase modified polymer. The electro-
chemical response of these electrodes to sequential
lactate additions are shown in Fig. 5, for two sepa-
rate preparations of lactate oxidase modified poly-
mer. The figure shows a 25% difference in the
response obtained, which is a reflection of the differ-
ence in activity in solution assays of the enzyme
samples employed (19% variation measured in a
spectroscopic assay) and hence the reproducibility is
comparable with that obtained for glucose oxidase-
polymer conjugates.
Of special interest to the amperometric enzyme
sensor format used here to demonstrate the immobil-
isation layer are the partitioning characteristics of the
cofactors and substrates within the polymer which
modulate the amplitude of the signal and thus change
the apparent linear response range. The data shows a
significant increase in the range for the determina-
tion of lactate over that of the free enzyme in
solution, which has a K, of 2-3 mM lactate, possi-
bly indicating the effect of the polymers’ transport
kinetics, although this could also suggest that a
change in the enzyme caused by the immobilisation.
The kinetic behaviour of a bound enzyme can differ
significantly from that of the same enzyme in free
solution. These changes may be due to conforma-
tional alterations within the enzyme resulting from
the immobilisation protocol, or to the presence and
nature of the immobilisation support. The kinetic
constants (e.g., K,, V,,) of an enzyme can be
modified by suitable choice of the immobilisation
protocol. However, the same method may have ap-
preciably different effects on different enzymes [2].
It has already been seen that the apparent K, of
glucose oxidase immobilised on these polymers is
approximately ten times greater than that of the free
enzyme. This was also found in the case of lactate
oxidase. Such duplication may suggest that the in-
creases in the apparent K, of these enzymes is due
entirely to the transport kinetics of the immobilisa-
tion support and not to conformational changes in
the enzymes during immobilisation. However, since
the substrates for these enzymes will themselves
behave differently under the given experimental con-
ditions this similarity should be discounted as coinci-
dence. In the case of lactate oxidase the immobilisa-
tion and enzyme activity optima coincide, so that the
system appears ideally suited for this enzyme. This is
substantiated in the following stability data.
3.3. Stability
An earlier study demonstrated the stability of the
glucose oxidase modified polymer over a short pe-.
riod of a few weeks [12]. Fig. 6a shows a compara-
ble study employing glucose oxidase modified
25G75M-2, as in Table 4. The data shows the cur-
rent response to glucose additions of electrodes fab-
ricated from this polymer over a period of one year,
in which 37% of the maximum activity was lost. The
glucose oxidase modified polymer was stored at 4°C
during this period indicating that the enzyme-poly-
 C.E. Hall et al./Analytica Chimica Acta 323 (1996) 87-96 9.5

mer conjugate can be seen to be relatively stable

consistent with the renowned stability of glucose
oxidase.
Lactate oxidase is known to be an inherently
unstable enzyme, undergoing rapid hydrolysis in
aqueous environments [25]. Fig. 6b shows two iden-
tical measurements made with electrodes fabricated
from the same lactate oxidase-polymer conjugate,
but prepared six months apart. In this instance 23%
of activity was lost over the storage period, during
(IACTATE] (mM)
which time the polymer conjugate was stored at 4°C.
This compares to a 100% loss of activity (measured
Fig. 5. Plot of current response versus lactate concentration for
lactate oxidase modified polymer electrodes prepared from two
spectroscopically) for a sample of the free enzyme
samples of lactate oxidase modified polymer. from the batch used in the polymer preparation and
thus represents a significant improvement in the
enzyme stability. This stability is consistent with the
15W
hydrophobic storage environment associated with the
- DQl a
- DQ7
polymer which inhibits the hydrolysing degradative
action of water.

4. Conclusions

During the immobilisation of enzyme low temper-

atures give lower enzyme loadings but the best cur-
rent response to glucose additions. Very low concen-
Or trations of enzyme are required to achieve maximum
material efficiency in the immobilisation process.
[GLUCOSE] (mM)
Higher concentrations showed only slight improve-
ment in protein loading, and displayed lower current
responses to glucose than those modified at lower
enzyme concentrations. This result is of particular
importance if more expensive enzymes are to be
immobilised in this manner.
The ionic strength of the immobilisation medium
was also shown to influence the current response to
5 loo- glucose additions of electrodes fabricated from glu-
cose oxidase modified polymers, although the en-
zyme loadings were unaffected. More importantly,
the pH of the buffer solution can significantly affect
both the immobilisation efficiency and the level of
enzyme activity retained by the enzyme-polymer
0 10 20 30 40 conjugate. Carrying out the immobilisation at, or
[LACTATE] (mM)
near the isoelectric point of the enzyme, increases
Fig. 6. The effect of storage on the elcctrochemically determined
the efficiency of the immobilisation procedure,
enzyme activity. (a) Plot of current response versus glucose
concentration for glucose oxidase modified polymers. (b) Plot of
whereas utilising a pH close to that for maximum
current response versus lactate concentration for lactate oxidase enzyme activity of the enzyme in question, leads to
modified polymers. higher amperometric responses and residual enzyme
% C.E. Hall et al./Analytica
activities in the subsequent enzyme-polymer elec-
trodes. It was thus seen that optimisation of condi-
tions with respect to the enzyme was more important
than with respect to the epoxide group of the poly-
mer.
These conclusions were tested on four other en-
zymes: lactate, cholesterol, alcohol and polyphenol
oxidase which were successfully immobilised onto a
25% glycidyl-75% methyl methacrylate polymer
with 100% efficiency (in terms of the enzyme con-
sumed during the reaction) at a pH close to that for
maximum enzyme activity for all four enzymes as
well as being close to their isoelectric points where
these are known.
The stability of the enzyme-polymer conjugate
has been demonstrated for both the glucose oxidase
and lactate oxidase systems. In the former case 37%
of the maximum activity was lost in the course of a
year with no special storage precautions. In the case
of lactate oxidase, 23% of activity was lost over six
months of storage at 4°C compared to 100% for the
free enzyme. This latter result represents a signifi-
cant improvement in the enzyme stability.
In summary, therefore, the data presented herein
have identified some of the critical parameters for
the optimisation of enzyme immobilisation onto a
methacrylate copolymer, and shown that under these
conditions enhanced enzyme stability may be at-
tained. These results may be projected beyond this
application, so long as the biological component is
able to withstand the mechanics of the immobilisa-
tion process.
Acknowledgements
We are grateful to the U.K. Engineering and
Physical Sciences Research Council for the financial
support for this project.
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