Biosensors4 (1989) 393-402

Short Communication

A New Method for Enzyme Membrane Preparation Based

on Polyurethane Technology: Electrode Modification for
Sensor ~ve~opment

The new method developed for enzyme membrane preparation is based on

cross-linking poly(viny1 alcohol) (PVAL) with triisocyanate (TIC) in the
presence of enzyme. Dimethylsulfoxide (DMSO) was the only solvent
found to dissolve PVAL, TIC and enzyme at room temperature, without
completely denaturing the latter. The rate of ge~tion to form the desired
network membrane can be controlled by the amount of solvent used. All
the enzymes tested (alkaline phosphatase and alcohol, cholesterol and
glucose oxidases) dissolved in DMSO and retained sufjicient activity for
use in electrochemical sensors. Membranes were formed on both graphite
and platinized graphite electrodes. The resulting prototype sensors were
examined with regard to fe~ibili~ of preparation, adhesion of the gels to
the electrode surfaces, swelling properties of the gels in DMSO and
aqueous buglers, and their electrochemical properties.

Key words: biosensor, dimethylsulfoxide, enzyme electrode, isocyan-

ates, ~lyurethane technology, poly(viny1 alcohol).

INTRODUCTION

Numerous researchers have explored the concept of immobilization of

biomotecules of analytical interest into several different kinds of mem-
branes either by chemical cross-linking of the enzyme or by topological
entrapment (Lubrano & Guilbault, 1978; Thevenot et al., 1979; Carr &
Bowers, 1980; Coulet & Gautheron, 1981; Mascini et al., 1983). The
polymer poly(viny1 aIcoho1) (PVAL) has been used for the formation of
such enzyme-containing membranes (~ascini et al., 1986; Ichimura &
393
Biosensors0265928x/s9/$03.50@ 1989Elsevier Science Publishers Ltd, England. Printed
in Great Britain
394 C. Galiatsatos et al.

Komatsu, 1987; Galiatsatos et al., 1988), primarily due to its biocompati-

bility. The methods employed in these papers include either irradiation of
PVAL in the presence of an enzyme or co-cross-linking of PVAL with
substrates such as collagen.
Polyurethane technology makes use of several isocyanates and their
addition products (Frisch & Vogt, 1984). The basic reactions i,nvolve chain
extension (from diisocyanates) and network formation (triisocyanates)
from the addition of hydroxyl groups to isocyanate groups. A whole series
of diisocyanates is reported in the literature (Frisch & Vogt, 1984).
Aromatic triisocyanates have been employed to prepare model net-
works of the polyurethane-type by end-linking hydroxyl-terminated
chains, for example poly(e-caprolactone) (Sung & Mark, 1980). The same
aromatic triisocyanate has been used in this study, primarily because it is
highly pure compared to commercially available di- and triisocyanates. By
this method, both the cross-link functionality as well as the average
molecular weight between cross-links can be controlled. We have found
that mixing PVAL with different amounts of the triisocyanate in the
presence of enzymes leads to stable, active gels, which also adhere strongly
to both graphite and platinized graphite electrode substrates leading to the
construction of electrodes with specific analytical properties.
This method has not been used before in the preparation of enzyme-
based biosensors, possibly because of the difficulty in finding a common
solvent for polymer, TIC, and enzyme which does not denature the latter,
We have found that dimethylsulfoxide (DMSO) is a good solvent for this
system. Both PVAL and TIC are soluble in DMSO, and their reaction rate
can be controlled by controlling their concentrations. Also, DMSO dis-
solves all the enzymes tested in this study without destroying their activity,
a fact which has also been reported in the literature (Douglas & Singer,
1956; Rammler, 1967; Lineback & Sayeed, 1971; Rammler, 1971; Hein-
rich, 1972; Griffin & Fogarty, 1973; Kashkin & Silina, 1973; Adams et al.,
1979; Jones & Pliura, 1981; Srivastava, 1985; Kralisz & Kotelba-Witkows-
ka, 1986). Major problems which arise from the use of DMSO are
primarily some enzyme activity inhibition, its toxicity (Uranuma, 1960;
Caujolle et al., 1964; Haeusler & Jahn, 1966; Morris, 1966), its low
volatility, and the exothermicity of its mixing with water (Lindberg &
Pietila, 1962) which may in turn affect the enzymatic activity.

EXPERIMENTAL

A 3-O% solution of PVAL (mol. wt 86 000, Aldrich Chemical Company,

Milwaukee, Wisconsin) in DMSO (b.p. 189C, Fisher Scientific, Cincinna-
 A new method for enzyme membrane preparation 395

ti, Ohio) was prepared by heating 80.0 ml of DMSO, slowly adding 3-O g of
PVAL by continuous stirring and adjusting the volume to 100-O ml.
Overheating should be avoided; PVAL decomposes at 150C and the
solution turns yellow. A l-3 ml sample of the resulting solution was mixed
with O-09 g of TIC in O-5 ml of DMSO (TIC was specially prepared and
generously provided by Dr D. H. Chadwick of the Mobay Chemical
Company, New Martinsville, West Virginia). Its structure is shown in Fig.
1. Both PVAL and TIC were dried by using a desiccant. Under the
conditions mentioned above, gels form in approximately 15 s at room
temperature (about 23C). When slower reaction times are desirable,
lower concentrations of both reactants are used.

N-C=0

N=C=O

Fig. 1. The structure of TIC.

Several enzymes were tested in terms of their solubility and extent of

retention of their activity in DMSO. For alkaline phosphatase (ALP)
(from calf intestine, Boeringer Mannheim, Indianapolis, Indiana) a col-
orimetric method similar to the one reported in the literature (Mossner et
al., 1980) was followed. For all the oxidases used cholesterol oxidase
(CHOLOX) (f rom nocardia erythropolis, Yellow Springs Instrument Co.
(YSI), Yellow Spring, Ohio), alcohol oxidase (ALCOX) (from pichia
postoris, YSI) and glucose oxidase (GO) (from Aspergillus niger, Sigma,
St Louis, Missouri) a linked-calorimetric assay was used for the activity
measurements as described previously (Galiatsatos et al., 1988). PVAL-
TIC and PVAL-TIC-enzyme membranes were swollen in DMSO and
data on per cent gel and extent of swelling were obtained (Collins et al.,
1974). They were subsequently placed into water where they swelled
again, but to a lesser extent.
The same approach was used for electrode modification. Graphite
electrodes (Poco Graphite, Dallas, Texas) of average area of O-17 cm2
396 C. Guliatsatos et al.

were polished with 600 grit silicon-carbide paper and sonicated for
10.0 min in distilled water. A 13 ~1 sample of the 3.0% PVAL in DMSO
solution and 5 ~1 of the TIC solution were successively applied to the
surface of the electrode while it was spinning. The gel forms after 10-20 s.
The electrodes were left to dry in a dust-free air atmosphere for 16 h. This
step was omitted as more knowledge of the film properties was procured.
Instead the electrodes were immersed in cold, stirred 0.1 M phosphate
buffer of pH 7.0 for 5-10min to ensure extraction of DMSO and un-
reacted materials, as well as to examine both the adhesion and the swelling
of the gel. This procedure enables modified electrodes to be prepared in a
considerably shorter period of time.
Cyclic voltammetry and chronoamperometry, which were the tech-
niques chosen for the electrochemical determination of the efficiency of
the resulting sensors, were performed with a CV-27 Voltammograph
(Bioanalytical Systems, Inc., West Lafayette, Indiana).

RESULTS AND DISCUSSION

Both PVAL-TIC and PVAL-TIC-enzyme membranes swell to a very

significant extent upon placement in DMSO. The extent of the cross-
linking reaction ranges from 27 to 43%) depending on the nature and the
quantity of the enzyme. The results are summarized in Table 1. It should
also be pointed out that all gels swell when placed in water, but to a lesser
extent than in DMSO.
Since DMSO was the only solvent found to dissolve both PVAL and
TIC, the retention of enzyme activity in DMSO becomes a vital issue. A
series of measurements of enzymatic activity was taken, based on the
initial rates of the color-developing reaction. As shown in Table 2, alkaline
phosphatase (ALP) retains as much as 85% of its activity under the

TABLE 1
Gel and Sol Fractions as Expressed by Extent of Reaction (%)

Gelation system Extent of reaction

(%)

TICYPVAL 32.4 67.6

TKYPVALKHOLOX 27.3 72.7
TIC/PVAL/GO 42-7 57.3
TIC~VA~ALP 34.4 656

Data obtained according to Collins et al., 1974.

 A new method for enzyme membrane preparation 397

TABLE 2
Initial Rates of Enzymes in Different Solvent Systems

Enzyme Solvent Change in absorbancelmin

GO l-01
GO DMSO o-03
ALCOX Hz0 0406 5
ALCOX H*O/DMSO (l/S) 0405 3
CHOLOX Hz0 0-25
CHOLOX DMSO o-021
CHOLOX H,O/DMSO (l/5) o-15
ALP 0.1 M (NH&CO&Ig*+ l-18
pH g/H20
ALP 0.1 M (NH4)2C03h4g2+/DMS0 1.10

Mg*+ concentration 1-OmM

experimental conditions used. The amount for ALCOX is 80%, while in

the case of CHOLOX it drops to 60%. The effect of DMSO on the
enzymatic activity becomes more pronounced when no water is used: only
3% of the glucose oxidase remains active under these conditions, again as
evidenced by the initial rates. CHOLOX activity is now only 8%. The
same trend is observed for all the other enzymes. The key point is that dry
conditions are required for the formation of the gels by preventing
reaction of water with TIC, while at the same time small amounts of water
are desirable for greater retention of enzymatic activity. We are currently
working on optimizing these conditions. It has also been noted that in
some cases the enzymatic activity is in part regained when the enzymes are
placed in aqueous solution, since the inhibition may be, to some extent,
reversible (Rees & Singer, 1956).
When the gels are formed on electrode surfaces, the adhesion of the
films to both graphite and platinized graphite electrode substrates has
been found to be excellent. The high porosity of the surfaces may be a
significant contributing factor, as noted previously (Coury & Heineman,
1988).
The background cyclic voltammogram of the PVAL-TIC coated elec-
trode in a pH 7-O phosphate buffer shows no distinctive peaks; however
the residual current is somewhat increased as compared to the bare
electrode. Notwithstanding, electrochemical experiments can still be per-
formed using these electrodes, as seen in Fig. 2. Of great importance is also
the fact that upon addition of 5.0 mM ferricyanide solution in the phos-
phate buffer, the reversible electrochemistry of the ferri/ferro couple can
398 C. Galiatsatos et al.

EW,vs.AglAgCI)

Fig. 2. Cyclic voltammograms of a PVAGTIC electrode in 0.1 M phosphate buffer of pH

7.0 (----) and in 50 mM Fe(CN)d- in the same buffer (-). Scan rate = 100 mV/s.

be seen. This indicates that the swollen films, although relatively thick (c.
0.5 mm), do not block access to the electrode surface of species dissolved
in solution. The network structure is rather open, allowing small mole-
cules such as Fe(CN)64-3- to diffuse through the membrane.
As it was pointed out earlier in this report, one major advantage of this
method is the fact that the reaction rate can be controlled by varying
reactant concentrations in a manner that could result in gels acquiring
different and known average molecular weights between cross-links. This
feature of the method was put into practice by applying different TIC
concentrations to the same amounts of PVAL. As the concentration of
TIC solution increased not only was a shorter time required for gel
formation, but also thicker films were obtained.
Two major electrochemical interferences, uric acid and acetaminophen
were examined on the modified electrodes made by the introduction of
TIC concentrations required for lO-25% theoretical degree of cross-
linking, Table 3. The electrode responses are represented in the form of
current densities at O-5 min and 5.0 min after the introduction of 1 mM of
the above mentioned interference solutions. As the degree of cross-linking
decreases (lOO-25% theoretical), the corresponding current density in-
creases. It should also be pointed out that in all of the above cases a
significant attenuation (9840%) of the interfering signal was observed on
these chemically modified electrodes as compared with a bare unmodified
graphite electrode.
The amperometric enzyme sensors were tested by holding the potential
at a constant value, injecting various concentrations of the substrate and
monitoring the current generated from the enzymatic reaction. A typical
response of the ALP-based sensor is shown in Fig. 3, where the enzymati-
 A new method for enzyme membrane preparation 399

tally generated p-aminophenol (from the substrate p-aminophenyl phos-

phate) is detected amperometrically at 200 mV vs Ag/AgCl (Tang et al.,
1988). The electrode response to different concentrations of substrate
leads to the construction of the calibration curve shown in Fig. 4. The
five-point calibration curve features a slope of 2.02 pA/rnM and an inter-
cept of 0.0394 PA with a correlation coefficient being O-996. The calcu-
lated errors were O-0226 ,uA in the intercept and 0.108 pA/mM in the
slope. The ALP-based sensors provide stable and reproducible responses
to the addition of 1 mM substrate concentration for 15 days with the

TABLE 3
Effect of Degree of Cross-Linking on Uric Acid and Acetaminophen Electrochemical
Response

Cross-linking % Current density (0.5 min) Current density (5 min)

(theoretical) @cm2 pAlOT?

Uric acid
100 12.87 19.95
75 20.69 34.62
50 65.2 70.5
25 86.17 -
Bare electrodea 693.26 -

Acetaminophen
100 - -
75 29,98 28.55
50 75.14 94.41
25 94.1 -
Bare electrode 515.05 -

Average response from three unmodified electrodes.

21
0 5 10
TIME (mid

Fig. 3. Typical response of a PVAL-TIC-ALP electrode to the addition (indicated by *) of

1.0 mM substrate p-amino-phenylphosphate, in O-1 M (NH&C03/1.0 mM M2+ buffer,
pH = 9.0.
400 C. Galiatsatos et al.

5
SUBSTRATE CONCENTRATION (mM)

Fig. 4. Chronoamperometric calibration curve for the ALP-based prototype sensor upon
injection of different concentrations of p-aminophenylphosphate.

coefficient of variation being in the order of 6% for two electrodes. The

response time for several substrate conversions on these modified elec-
trodes is about 2 min. The reproducibility of the response for the addition
of 1 mM ~~~~-aminophenylphosphate (PAPP) solution on to one elec-
trode is 7.6% (n = 3). Also a coefficient of variation of @0-@058 PA was
obtained for three different electrodes upon the introduction of various
substrate concentrations. These small variations are believed to arise from
differences in the surface areas of the spectrographic graphite electrodes
(Coury & Heineman, 1988), which can be corrected for by measuring the
surface areas of each individual electrode.

CONCLUSIONS

The proposed method is potentially useful for several reasons: (1) It is a

general method with a wide range of possible applications both in the
preparative macro- as well as sensor micro-levels. (2) It is fast and rather
straightforward. When all of the starting materials are available a sensor
can be made in 5-10 min and used immediately for the intended applica-
tions, while for most of the immobilization methods that we are aware of,
such as covalent attachment of proteins to the electrode surface and
radiation immobilization of enzymes, at least several hours are needed to
obtain a sensor ready for use. (3) It is relatively inexpensive. (4) It brings
together for the first time all the knowledge and skill of polyurethane
technology into the area of bioelectrochemistry. The latter is particularly
advantageous in terms of creating model networks where both the cross-
link functionality as well as the average molecular weight between cross-
links are known, so desirable size-exclusion properties can be achieved.
 A new method for enzyme membranepreparation 401

ACKNOWLEDGEMENTS

The authors acknowledge support of this research by the Edison Sensor

Technology Center. Christos Galiatsatos gratefully acknowledges the
support of the University of Cincinnati Research Council.

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ChristosGaliatsatos, Kiamars Hajizadeh,

James E. Mark & William R. Heineman*
Edison Sensor Technology Center,
Department of Chemistry,
~~~versi~ of Cincinna~,
Cincinnati, Ohio 45221-0172
USA

(Received 16 June 1988; revised version received 16 December 1988; accepted 4

January 1989)

*To whom correspondence should be addressed.