Enzyme and other

biosensors:
Evolution of a technoloav
by Seamus F? J. Higson, Subrayal M. Reddy
and Pankaj M. Vadgama
Since 1962, when thefirst enzyme electrode was reported, biosensors have been thefocus ofintensive research and
have captured the interest and imagination ofboth the wider scient& and lay communities. They have a wide
potential applicability encompacsing both biomedical and industrial areas and oJer a unique route to simplified,
reagentless analysis. Financial savitgs in analysis are an important motivating forcefor biosensor development; this
is no more so than in medical diagnostics, where, especially in the West andjapan, an upward demandfor
biomedical testing has contributed to escalating health care costs. More recently, it has been recognised that with
biosensor based monitoring ofbiotechnological processes used in thefood and drink industries, a substantial
enhancement in e@ciency is possible.

Introduction creation of devices which remain experimental in

nature, and which are difficult to use practically. It is
now apparent that for rapid progress the end user as well

A
biosensor is a chemical sensing device in
which a biologically derived recognition
entity is coupled to a transducer, to allow the
quantitative determination of some com-
plex biochemical parameter. This self-contained sensor
or probe relies on the specificity of the biological
component to achieve reliable recognition ofan analyte
in a mixed sample. The subsequent transduction
produces a signal that is preferably electrical and which
may be simply related back to the analyte concentra-
tion. Practical sensors have been realised by successful
coupling and exploitation of principles derived from
physics and biology as well as chemistry, the result being
a multidisciplinary field in its own right.
Initial progress has been difficult, but following three
decades of intensive research in biosensors, occasionally
punctuated by interest from the popular media,
biosensors have at last encroached upon the applied
disciplines, and thus they have begun to be used, for
example, in hospital departments.
It cannot be overstated that for the development of
practical sensors, adequate consideration needs to be
given to the needs and requirements of the ultimate
end user. All too often the academic curiosity of a
scientist, albeit fully justified intellectually,hac led to the
as to some extent application engineers should be
participants in biosensor research at the earliest stage
possible. At the very least, in this way a clear remit for
sensor requirements can be established prior to any
major research effort being expended.
Fig. 1 shows some of the possible applications for
biosensors, though this emphasises their clinical use, as
this is the area for which the maximum research effort
has been made to date and where most experience
resides. At one extreme is a biosensor, conventionally
in the form of a needle, designed for in vivo use, which
nust be able to operate in an intimate way with a
reactive body (blood/tissue) environment with
minimal loss of sensor performance over a prespecified
time period. In adhtion, ethical and safety considera-
tions enter into this equation, but certainly the sensor
must not induce a significant inflammatory response or
clotting, and must be noninmunogrnic and nontoxic'.
At the other extreme, a 'one shot' biosensor designed
for disposable single use in the doctor's surgery or even
the home may be primarily required to have an
extended shelf life, while its resistance to biofouling is
not crucial and its operational lifetime is permitted to
be of the order of seconds. Alternatively, operating

ENGINEERING SCIENCE AND EDUCATION JOURNAL FEBKAUKY l W 4

41
Fig. 1 Potential scope of
biosensors: applications
and environments

n
long-term
mplantoble
-
- cllnlcal

short-term
mvasive
%
blosensors

single shot
nonclintcd

multi-omlyses
h
single analysis reactive
monitormg

conditions for biosensors for use within the food
industry and processing industries may demand
stability over widely varying solution conmtions (pH,
ionic strength) and temperatures with analyte
measurement ranges that may extend well beyond
those required for medical applications.
Even with careful prior design, unexpected and
often peripheral requirements may affect practical
viability In one successful system, the Medisense
‘ExacTech’ pen’ (Fig. 2 4 for home glucose monitor-
ing, although acceptable biomedical function was
achieved, it became apparent only after marketing that
many diabetic patients with poor eyesight could not
differentiate the readings on the small LCD display
provided. Accordingly, the sensor has now been
redesigned with an enlarged &splay in the shape of a
credit card, the ‘Companion’ system (Fig. 2b), which
still maintains portability.
The first 30 years: establishment of some
basic ground rules
Historically, the advent of the biosensor era was
heralded in 1962 by Clarke and Lyons” report of the
first enzyme electrode for the measurement of blood
glucose. This device employed an enzyme, glucose
oxidase (GOD), to catalyse the reaction for the
oxidation of glucose to gluconic acid and hydrogen
peroxide:
(GOD)
glucose + 0 2 + H2O + hydrogen peroxide
(H202) + gluconic acid (1)
electrode made of an inert material, at which oxygen
may be detected by the imposition of a negative
voltage, eqn. 2. The electrode is covered by a suitable
gas permeable membrane to protect the electro-
chemical surface. A p H electrode was suggested as an
alternative to monitor p H change due to the
production of gluconic acid by the enzyme, whereas
the oxygen electrode enabled detection of changes in
the local oxygen partial pressure ( p 0 z ) due to oxygen
consumption by the enzymatic reaction (eqn. 1).
Changes in p H are vulnerable to buffering effects, and
0 2 monitoring is subject to fluctuations in ambient
p02. Nevertheless measurement of the latter is quite
convenient electrochemically:
- hO0 inV vs Ag/AgCI
0 2 + 2H20 + 4e- 4 4 0 H - (2)
Later, Updike and Hicks’ utilised a gel (acrylamidej to
entrap glucose oxidase over dual cathodic
ampemmetric oxygen sensors. Here, one cathode was
coated with active enzyme and the other with
inactivated (heat denatured) enzyme; the latter acted as
a reference/control electrode to compensate for
background PO?flucturations.
Hydrogen peroxide (H202j produced from the
enzynuc reaction (eqn. 1) can also be monitored in a
simple transduction step in which the generation of a
current is made directly proportional to glucose
concentration’,h:
(3)

A thin layer of the enzyme in solution was entrapped This method needs neither background 0 2 correction
between two polymeric membranes and then placed nor the use of twin differential electrodes.
over either a conventional pH or an oxygen electrode. All members of the group of enzymes known as the
An oxygen electrode here can simply comprise an oxidases act as catalysts for the production of H202 in
Table 1: Peroxide-based oxidase sensors the presence of specific substrates (analytes). This
Enzvme Substrate Bio-samde fandy of enzymes has therefore provided a generic
glucose oxidase glucose5 blood
method of constructing a variety of enzyme electrodec,
lactase oxidase lactate’ blood each of which is highly specific for a particular analyte
oxalate oxidase oxalates bloodhrine or analytes. A few examples of some peroxide-based
ascorbate oxidase a~orbate~ blood oxidase sensors that have ~uccessfullydeveloped are
alcohol oxidase alcoholso blood yhown in Table 1.

ENGINEERING SCIENCE AND EI>UCATION JOURNAL FEUIWAKY 1YY4

42

To date the vast majority of biosensor research has
centred around the oxidase enzymes and in particular
glucose oxidase. This can be attributed largely to the
stability of glucose oxidase, its stability in water and
even organic solvents, and the practical need for sensing
technologies due to the prevalence of mabetes. In the
United States diabetes has been increasing for many
years, with an estimated 2% of the population now
suf+ing from this disease".
Fig. 3 shows a schematic representation of a typical
glucose oxidase membrane based sensor". This sensor
relies upon the chenucal immobihsation ofthe enzyme
glucose oxidase (GOD) within an enzyme/membrane
laminate. Entrapment of the enzyme could also be
achieved by encapsulation or physical adsorption
techniques, but chemical immobilisation gives more
~tability'~.The outer covering membrane functionally
acts as the outer surface interface with a bulk analyte
solution, but also frequently serves to provide a
substrate diffusion limiting barrier. In this way the
enzyme encounters a locally diminished analyte
concentration so avoiding enzyme saturation at the
higher concentrations and linearising the sensor signal
output. However, an outer membrane that also allows
free passage of oxygen would be advantageous as it
is an absolute requirement for the enzymic reaction
(eqn. 1).
In the hostile environment frequently encountered
lmmobillsed enzymelglutaraldehyde
GOD
p D glucose + 02- glucanolactone
1
underlying permselective membrane,
02 +, 2e-+ 2H*
PI worklng electrode(+650 r n V vs AglAgCI)
ENCINEEKING SCIENCE ANI) E1)UCATION JOURNAL
Fig. 2 (a) Medisense ExacTech glucose 'pen'; (b)
home monitoring of blood glucose levels using
the Medisense ExacTech 'companion'
within biological fluids such as blood or urine, a major
cause for loss of sensor performance is that due to
biofouling by the adhesion of proteins, platelets and
other cellular components to the outer membrane
surface. This deposition alters the total membrane
permeability and constitutes an unpredictable and
additional difision barrier that detracts and impedes
reliable sensor performance. This is somewhat
equivalent to an optical window gradually cloudmg
over. The search for ever more biocompatible materials
to minimise surface fouling at the outer membrane
surface has of necessity therefore been itself an
important area of research".
An inner membrane needs to be located between
the enzyme layer and the workmg electrode surface for
the classical peroxide type device. The peroxide
produced is then able to traverse this inner membrane
where it is amperometrically interrogated at the
electrode surface. With the right membrane in place,
other species present in biological solution (for
example ascorbate), which may also be electro-
chemically active at typical operating polarisation
potentials, can be screened out. To this effect various
'permselective' membrane materials have been
reported, and their properties tailored to prevent the
passage of particular interferents while allowing the
passage of the small uncharged Hz02 molecule. A
membrane with perfect selectivity for H202 is of
Fig. 3 Schematic
diagram of glucose
coverlng outer membrane oxidase membrane-
based enzyme laminate
electrode
+ H202 inner
/membrane
- I
7
H2 02
1
FEBl<UAl<Y 1994
43

sulpho~ies~’ have been employed with some
suc~essCharge exclusion of anions rehes on
the repulsion of negatively charged
interferent species from an (aniomc) polymer
matruc providing fixed negative charges
Fortunately, inany problematic interferents in
solution are anionic or negatively charged so
allowing screening of the worhng electrode
However, an electrocheimcally active
intederent can be less easlly differentiated by
charge if it is only partially ionised under typical
measurement conditions. One such example is the
drug paracetamol, which may be present in blood in
quite high concentrations and which, probably because
of its neutral form, is di5cult to select against. Despite
this, sonie electropolymerised phenolic nienibranes
deposited directly over the workmg electrode provide
examples of effktive screening layers against this
drug“.
The working electrode is usually a noble nietal
(typically gold or silver), but may be carbon, and is
anodically polarised to allow the oxidation of H202
with the generation of an anodic current proportional
to the analyte concentration. This process also allows
the generation of 0 2 (eqn. 3) which is then free to
diffuse back into the enzyme layer, a process that may
significantly augment the local pOz level preventing
low ambient pOz levels that could compronllse sensor
performance. Careful choice of an underlying
iriembrane that allows relatively free passage of 0 2 is
again a key des@ consideration“.
This structure for the glucose oxidase membrane
laminate electrode forms the basis for the Yellow
Springs (Yellow Springs Instrument Corporation,
Ohio 43587, USA) glucose analyser, Fig. 4, which has
been used extensivelyfor blood glucose deternlinations
over the past twenty years within clinical biochemistry
laboratories. As a ‘pre’ stage, for research purposes,
enzyme laminates may be evaluated within a simple
ENGINEEKING SCIENCE AND EIlUCAT10N JOUKNAL
44
Fig. 4 Yellow Springs
glucose analyser
bench-top electrode assembly (Fig. 5).
Another generic approach to the exclusion of
interferent signals, in oxidase enzyme electrodes, has
been the use of chemical memators. A medlator is a
chemical species which fachtates electron transfer &om
the enzyme active site to the worlung electrode, so
allowing the interrogation of the enzyme reaction at
lowered polarisation potentials and preventing the
oxidation ofother electrochemically active interferents.
O f these, ferrocene, was the first to be reported*“ in
1984, and to date together with its derivatives remains
the most extensively used mediator compound.
Typically ferrocene will allow electron transfer h m
glucose oxidase at approximately +240 niV vs
Ag/AgCI (cf +650 mV vs Ag/AgC1 for H202). It
should be noted that as ferrocene now acts as the
oxidant for the reduced form of the enzyme, 0 2 is no
longer a necessity for the enzyme action, so
circumventing the limitations imposed by near zero
p 0 2 levels.
Direct electron transfer from the enzyme to the
electrode surface has been attempted, but proved
di6cult since the active site of the enzyme is deeply
embedded on the molecular scale within the protein
macromolecule, the latter acting as an insulator
attenuating electron exchange between the active site
and the electrode surface”.
A mediator, however, may also affect the oxidation
of other interferent species. It has been shown that an
FEBRUARY 1994
formance for either type
cannot be simply determined
theoretically and requires
practical evaluation for a
given sample matrix. Despite
signal
possible disadvantages im-
posed by the use of a
mediator, the Medisense elect rode
biosensors, based on ferro-
Y
placed on the electrode strip, Fig. 2b; this instrument
thereby allows a rapid (130 s) determination of blood
glucose without careful timing or sample preparation.
Monitoring of blood glucose is thus sufficiently
simplified for patient use, obviating the need for a fully
equipped laboratory or coniplicated wet chemistry
techniques.
Continuing research efforts
Although the present exploitation of biosenson is
centred around oxidase enzyme electrodes, consider-
able research activity has involved the development of
other concepts which could also lead to viable sensors
for the future.
Multi-ana@ monitors
Recently it has been demonstrated that more than
one enzyme may be used within the same sensor to
allow the multiple determination of analytes. In
practice this may be achieved by the use ofone or more
enzymes whose reaction depends on the presence of a
co-factoot; this is a compound required to give the
enzyme its catalytic activity. In one arrangement a co-
factor-consuming enzyme E1 that degrades one analyte
A could be held in front of a second layer containing
two different enzymes E1 and E2 for analytes A and B,
but without co-factors. O n addition of A and B a
combined signal is generated, but that due to A can be
subtracted out when the co-factor is included (Fig. 6).
In this way the co-factor operates as a switch to switch
on and off the outer enzyme, which then determines
whether the analyte A can reach the second layer. A
specific example is the hexakinase, glucoamylase/
glucose oxidase sensof', for the determination of
either glucose or maltose, where hexalunase uses the
co-factor adenosine triphosphate (ATP) to degrade
ENGINEERING SCIENCE AND EDUCATION JOURNAL
45
through ink-jet printer
technology".
P
Unquestionably, the de-
velopment of micro-elec-
tronic5 and silicon tech-
possibilities for the future
development of biosensor
arrays with associated in situ
Y "
stability, high redundancy,
and self interrogation for signal drift. The inherent
redundancy could also be exploited to provide
elements individually optimised for particular
environmental conditions (e.g. pH, temperature and
analyte levels), so further improving sensor
performance".
In vivo monitoriq
A further application for miniaturised sensors has
been in the development of invasive sensors for in vivo
monitoring. The most favoured approach to date has
been to miniaturise conventional electrodes in the
form of implantable needles'", e.g. for subcutaneous
insertion. A compromise with respect to electrode
materials may be needed ifsuch devices are not to pose
a hazard to the patient. Thus, appropriate coating with
polymer layers may be needed to avoid adverse
effects".*', and certainly a toxic material such as silvei'
for the reference electrode may need to be replaced by
a less toxic alternative such as stainless steel".
Sterilisation frequently involves heat, radiation or
chemical treatment. As enzymes are biologically
derived components their activity is often destroyed
under such condltions, but nevertheless implantation
demands sterility. To overcome this problem, research
has necessarily been directed towards developing
specifically tailored procedures and protocols to allow
adequate effective sterilisation (e.g. using ethylene
oxide'"), while maintaining an acceptable degree of
enzyme activity'"-".
Though not a biosensorin the formal sense, localised
monitoring of biochemical parameters in vivo may be
possible through optical interrogation of a naturally
present biomolecule. Ultiniately this could be an
enzyme, and offers the benefits of non-invasive
biosensing. An example ofthis approach is provided by
contemporary research on cerebral oxygenation. Here,
FEBRUARY 1994
near-infra-red (750-1000 nm) light is passed through
cerebral tissue, and with absorbance measurement set
at appropriate wavelengths, the population of
oxygenated and deoxygenated blood in the cerebral
circulation can be determined3j.
Other biosensor types
Enzyme electrodes rely on the catalytic conversion
of an analyte to a product, but affinity properties of
other biomolecules which do not induce analyte
degradation are also usable. Thus for example
antibodies for antigens, or cell membrane receptors
for neurotransmitters are all potentially exploitable bio-
recognition systems. Because of the lack of a depletion
product, however, transduction may be difficult to
achieve. One convenient mechanism for immuno-
sensing is to use a piezoelectric microgravimetric mass
detector coated with antibody; specific antigen binding
to the immobilised antibody at the surface of an AT cut
quartz crystal"' then altes the mass and this can be the
means of transduction. The basis of the 'transduction'
of weight is that the crystal oscillates at a characteristic
frequencyf; dependent on its mass when stimulated by
an externally applied oscdlating voltage, causing
deformation by relative motion of two parallel crystal
suriaces. The minute gravimetric change of the crystal
upon surface antigedantibody binding can then be
related to the measured change, Af; in the vibrational
frequency O f biomedcal importance, piezoelectric
crystals are now being developed for the determination
of proteins in complex media using crystal bound
immunochemical binding reactions. The greatest
problenls encountered by these techniques have been
errors imposed by nonspecific surface binding by
proteins and colloids. Again this shortfall could well be
addressed for the future, using multiple arrays with
systematically varied surface properties.
Though not stressed in this brief review, optical
fibres with biosensors at the tip'5,36 or optical
waveguides mounted with bioreagent at the surface"'
and interacting with the evanescent wave provide a
powerful means of transducing biomolecule changes
on analyte binding.
A further field of research, now made possible by the
exploitation of modern nlicroelectronics. has been the
development of ion-selective field effect transistors
(ISFETs)lX.The device essentially comprises an r~pn
transistor in which the metal gate is covered by an ion-
sensitive coating. If a proton-selective surface such as
silicon dioxide (SiOz) or silicon nitride (Si3N4) is
present, then changes in adjacent solution p H
modulate charge within this layer. This in turn
regulates the flow of current through thep region from
the source and drain n type elements. In this way the
modulated current may be readily related to solution
pH. Any surface coated enzyme which changes net H'
concentration can then be exploited for substrate
analysis. Such enzyme-based ISFETs have been termed
ENFETs"; their responses are directly related to
enzymic behaviour and avoid inherent problems of
ENGINEERING SCIENCE AND EDUCATION JOURNAL
46
electrochemically active interferents as is the case with
amperometry (e.g. peroxide measuring devices). As
with any technique, however, there are disadvantages:
an ENFET requires a highly stable reference electrode
which is also difficult to miniaturise using micro-
electronic technology. Furthermore, problems associ-
ated with gate poisoning when using biological samples
remain to be addressed properly
Enzyme catalysed reactions are usually exothermic
(heat generating) and the heat produced may be
measured and related to analyte concentration. Such
enzyme thermistor combinahons, e.g. in a thermal
enzyme probe where the thermistor is directly coated
with an appropriate enzyme, have wide applicabihty.
As with ENFETs. however, background correction is
typically required for surrounding temperature
fluctuations and they have a low sensitivity. Therefore
calorimetric"' sensors have been reported where the
thermistor is at the end of an enzyme reaction column;
here, tnuch more heat accumulates, and many
metabolites have been monitored, including plucose" ,
alcohol? and oxalate'3.
There are at present many different approaches being
actively explored for the development of even more
ingenious and elegant sensors with specific applications
in mind. Although this brief description of present
research efforts is by no means intended to be
comprehensive, some of the major approaches that are
currently being explored have been given
consideration, to give the reader insight into the
current state of the art.
A view to the future?
It is impossible to predict what the future holds for
biosensors, and how their current status will dictate
their development through this decade and into the
next century. However, it is certain that with an ever-
increasing public awareness of environmental issues,
such as river pollution, and the care and quality control
of food production, simple, effective monitoring of
pollutants, industrial products and foodstuEs will be
added to the present application areas. This need for
practical biosensors may provide impetus for the
dmction of future research; whereas in the over-
optimistic early days exaggerated claims for the hture
lead to disappointment and subsequent pressure from
funding bodies for 'instant success', now there is an era
of realism which provides the best guarantee for
success.
Academic research has often been aimed at showing
that a novel idea may be used as opposed to necessarily
producing a viable sensor, with applications being the
dominant consideration. Something of a gap has
opened up therefore, with industry frequently only
desiring to develop a product that requires nunimal
input; the gap is currently closing and product
development for Feveral biosensor systems is likely in
the next few years.
As emphasised earlier, ideally an end-user should be
FEBRUARY 1994

identified and consulted at the conceputal phase, with
early integration of academic research effort being
vitally important for this burgeoning multidisciplinary
area. Another important point to be considered is that
if a sensor is to enjoy widespread commercial success,
an end-user must first be satisfied as to the reliability of
the new technique and then be sufficiently motivated
to invest in the new technology. Hospital pathology
laboratories, for example, have frequently invested
considerable capital expenditure for the purchase of
automated analysers (Fig. 7). Here, even with financial
savings that biosensors niay offer, the purchaser may
need to be convinced that the new equipment warrants
the capital outlay involved. Accordingly it will be also
necessary to ‘niche’ market devices, especially for
extra-laboratory and field use.
It is hoped that as ideas become ever more tried and
tested, those that prove viable will become more
evident, leading to the realisation of more practicable
and exploitable sensors. However it should be
appreciated that biosensors, above all in the context of
the effort put into other biochenlical techniques, are at
an early stage of development, and a review in a few
years time may well portray a very different story, one
of much wider dversity with emphasis on practical
exploitation.
Acknowledgment
We would like to thank the UK SERC for financial
support to SI’JH and SMR.
References
1 WIESE, D. A., BOWEN, T. P., and KOST, G .J.: ‘Enzyme
electrode for glucose measurement in whole-blood with a
ENGINEERING SCIENCE AND EDUCATION JOURNAL
47
Fig. 7 Modern clinical
biochemistry laboratoryI with an
auto-analyser
critical care profiling instrument’, Clinical Chemistry, 1989,
35, ( 6 ) ,p.1098
2 MEDISENSE ‘ExacTech’ blood glucose testing system
(users’ guide), 1989, p.9
3 CLARK, L. C., and LYONS, L.: ‘Electrode system for
continuous monitoring in cardovascular surgery’, A n n .
N Y Acad. SII., IY62, 102, pp.29-45
4 UPDIKE, S. J., and HICKS, G. P.: ‘The enzymr electrodr’,
Natirre, 1967, 214. pp.986-988
5 TANG, L. X., and VADGAMA, P: ‘Optinusation of
enzyme electrodes’,Med. G Bid. Big. G Cornprrr., 1990, 28,
(3). pp.B18-B24
6 MULLEN, W. H.. KEEDY, E H., CHUKCHOUSE, S.J.,
and VAIIGAMA, F! M.: ‘Glucose enzyme rlectrodc with
extended linearity-Application to undiluted blood
measurements’, Anal. Chim. A d a , 1Y86, 183, pp.5946
7 KULYS, J., WANG, L. Z . , and MASKIMOVIENNE, A.:
‘L-lactase oxidase electrode based on methylene grren and
carbon pdste’, Anal. Clz~m.Acta, 1993, 274, (1). pp.5358
R BRADLEY, C. K., andKECHNITZ, G. A.: ‘Comparison
of oxalate enzyme electrodes for urinary oxalate
deternunation’, Analyti~aal h t f e n , 1986, 19, (1&2),
pp. 1.5 1-1 62
9 DAILY, S.. ARMFIELD, S.J., HAGGETT, B. G. D., and
DOWNS. M. E. A.: ‘Automated enzyme packed-bed
system for thr deterimnation of vitamin C in foodxuffs’,
Aiialyst, 1991. 116, pp.569-572
10 GIULUAULT, G. C., DANIELSSON, B., MANDENIUS,
C. E, and MOSBACH, K.: ‘Enzyme electrode and
thernustor probcs for determination of alcohols with
alcohol oxidace’,Anal. Chern., 1983, 55, (9). pp.1582-1585
11 HALL, E. A. H., in ‘Biosensors, IYYO’, Open University
Press Biotechnology Serie5, p.223
12 HIGSON, S. P.J., and VADGAMA, 1.’ M.: ’Diamond-like
carbon coated microporous polycarbonate as a composite
barrier for a glucosr enzyme electrode’, Anal. Cliim. Acta.,
1993,271, pp.125-133
13 VADGAMA, F?: ‘Biosensors’ in WILLIAMS, D. L., and
MARK, V. (Eds.): ‘Principles of clinical biochemistry’
(Hrinenmann Medical Books, Oxford and London, 198X)
FEBRUARY I994
 14 TANG, L. X., KOOCHAKI, Z . B., and VADGAMA, E:
‘Composite liquid membrane for enzyme electrode
construction’, Anal. Chim. Acta, 1990,232, (2),pp.357-365
15 GORTON, L., KARAN, H. I., HALE, P. D., INAGAKI,
T., OKAMOTO, Y., and SKOTHEIM, T. A.: ‘A glucose
electrode based on carbon pastes chemically modified with
a ferrocene-containing siloxane polymer and glucose
oxidase, coated with a poly(ester-sulfonic acid) cation
exchanger’, Anal. Chim. Aita, 1990,228, (l), pp.23-30
16 GONON, E G., FOMBARLET, C. M., BUDA, M. J., and
PUJOL, T. E: ‘Electrochemical treatment of pyrolytic
carbon fibre electrodes’, Anal. Chem., 1981, 53, (9,
pp. 1386-1389
17 VADGAMA, P., SPOORS, J., TANG, L. X., and
BATTERSBY, C.: ‘The needle glucose electrodein vitro
performance and optimisation for implantation,’, Biomed.
Biochim. Acta, 1989, 48, pp.935-942
18 CHRISTIE, I., VADGAMA, P., and LLOYD, S.:
‘Modification ofelectrode surfaces with oxidised phenols to
confer selectivity to amperometric biosensors for glucose
deterininations’. Anal. Chim. Acta, 1993, 274, pp.191-199
19 HIGSON, S. P J., DESAI, M., KOOCHAKI, Z., and
VADGAMA, P.: ‘Glucose oxidase enzyme electrode:
relation between inner membrane permeability and
substrate response’, Anal. Chim. Acta, 1993, 276,
pp.335-34n
20 CASS, A. E. G., DAVIS, G., FRANCIS, G. D., HILL,
H. A. O., ASTON, W. J., HIGGINS, I. J., PLOTKIN,
E. V., SCOTT, L. D. L., and T U R N E R , A. P. E:
‘Ferrocene-medated enzyme electrode for amperometric
determination of glucose’, Anal. Chem., 1984, 56, (4),
pp.667471
21 SCHELLER, E, PFEIFER, D., HINTSCHE, R . ,
DRANSFIELD, I., WOLLENBERGER, U,, and
SCHUBERT, E: ‘Analytical aspects of internal signal-
processing in biosensors’, Ann. N.Y Acad. SCI.,1990, 613,
pp.68-78
22 DAUTARTUS, M. E, and EVANS, J. E: ‘EC catalysis of
ascorbic acid oxidation using plasma polymerised
vinylferrocene film electrodes’. J. Electroanalytiial Chem.,
1980, 109, pp.301-312
23 SCHELLER, E, WARSINKE. A., LUTTER, I.,
RENNEBERG, R., and SCHUBERT, E: ‘Multi-enzyme
sensors’, in ACS symposium series, 487, ‘Biosensors and
chemical sensors’, 1992, Amer. Chem. Soc., Washington
DC
24 NEWMAN, J. D., T U R N E R , A. P E, and MARRAZZA,
G.: ‘Inkjet printing for the fabrication of amperometric
glucose biosensors’, Anal. Clrim. Acta, 1992, 262, (1).
pp.13-17
2s CONNOLY, P., CLARK, I?, CURTIS, A. S. G., DOW,
J. A. T., and WILKINSON, C. D. W.: ‘An extracellular
microelectrode array for monitoring electrogenic cells in
culture’. Biosensors G. Bioelecrroniu, 1990, 5, pp.223-234
26 WEISS, T, and CANMANN, K.: ‘An amperometric
glucosc sensor with combined enzyme layers’, Hormone
Metab. Res. Suppl., 1988, 20, pp.23-25
27 WOODWARD, S. C.: ‘How fibroblasts and giant cells
encapsulate implants-considerations in design of glucose
sensors’, Diabetes Care, 1982, 5, (3).pp.278-281
28 CHURCHOUSE, S.J., BATTERSBY, C. M., MULLEN,
W. H., and VADGAMA, P.: ‘Needs enzyme electrodes for
biological studies’, Biosensors, 1986, 2, (6), pp.325-342
29 IACKSON, W. E, and DULING, U. R.: ‘Toxic effects of
silver/silver chloride electrodes on vascualr smooth muscle’,
Circular Research, 1983, 53, (1). pp.105-108
ENGINEERING SCIENCE AND EDUCATION JOURNAL
48
30 WILSON, G. S., REACH, G., and THEVENOT, D. R.:
‘Biosensors for intracorporeal measuremen~-problem
and strategies’, Biochemical Soc. Earn., 19, (l),pp.9-11
31 BINDRA, D. S., ZHANG, Y. N., WILSON, G. S.,
STERNBERG, R., THEVENOT, D. R., MOATTI, D.,
and REACH, G.: ‘Design and in virro studies of a needle
type glucose sensor for subcutaneous monitoring’, Anal.
Chem., 1991, 63, (17), pp.1692-1696
32 MOATTISIRAT, D., CAPRON, E., POITOUT, V,
REACH, G., BINDRA, D. S., ZHANG, Y., WILSON,
G. S., and THEVENOT, D. R.: ‘Towards continuous
glucose monitoring-in vivo evaluation of a miniaturised
glucose sensor implanted for several days in rat subcutaneous
tissue’, Diabetolo& 1992, 35, (3), pp.224-230
33 ROLFE, E , WICKRAMASINGHE, Y. A. B. D.,
THORNILEY, M. S., FARIS, F., HOUSTON, R . , KAI,
Z., YAKAMOSHI, K., OBRIEN, S., DOYLE, M.,
PALMER, K., and SPENCER, S.: ‘Fetal and neonatal
cerebral oxygen monitoring with NIRS-Theory and
practice’, Early Human Development, 1992, 29, (1-3),
pp.269-273
34 LUONG, J. H. T., and GUILBAULT, G. G.: ‘Analytical
applications of piezoelectric crystal biosensors’, in BLUM,
L. J., and COULET, P. R . (E&.): ‘Biosensorprinciples and
applications’ (Marcel Deker, New York, 1991)
35 MORENOBONDI, M. C., WOLFBEIS, 0.S., LEINER,
M. J. P., and SCHAFFAR, B. P H.: ‘Oxygen optrode for
use in a fiberoptic glucose biosensor’, Anal. Chem., 1990,
62, (21), pp.2377-2380
36 SCHEPER, T., BRANDES, W., GRAU, C.,
HUNDECK, H. G., REINHARD. B., RUTHER, E,
PLOTZ, E, SCHELP, C., SCHUGERL, K.,
SCHNEIDER, K. H., GIFFHORN, E, REHR, B., and
SAHM, H.: ‘Applications of biosensor systems for
bioproccss monitoring’, Anal. Chim. Aita, 1991, 249, (1).
pp.25-34
37 ROGERS, K. R., CAO, C. J., VALDES, J. J.,
ELDERFRAWI, A. T., and ELDERFRAWI, M. E.:
‘Acetylcholinesterasefiberoptic biosensor for detection of
anticholinesterases’, Fundamental and Applied Toxicology,
1991, 16, (4), pp.81&820
38 BERGVELD, F!: ‘Development, operation and application
of the ion-sensitivr field effect transistor as a tool for
electrophysiology’,IEEE Fans., 1972, BME-19, p.342
39 BERGVELD, P.: ‘The development and application of
FET-based biosensors’, Biosensors, 1986, 2, (l), pp.15-33
40 DANIELSON, B.: ‘Enzyme thermistor devices’, in BLUM,
L. J., and COULET, P. R. (Eds.): ‘Biosensor principles and
applications’ (Marcel Dekker, New York, 1991)
41 SUGIER, H., and KURCZWSKI, J.: ‘Glucose assay with
extended concentration range by flow-through enzyme
enthalpymeter’, Starch-Slarke, 1992, 44, (4), pp.153-156
42 GUILBAULT, G. G., DANIELSSON, B., MANDENIUS,
C. E, and MOSUACH, K.: ‘Enzyme electrode and
thermistor probes for determination of alcohols with
alcohol oxidase’, Anal. Chem., 1983,55, (9,pp.1582-1585
43 WINQUIST, E , DANIELSON, B., MALPOTE, J. Y.,
P E R S O N , L., and LARSSON, M. B.: ‘Determination of
oxalate with immobilised oxalate oxidase in an enzyme
thernllstor’, Anal. Lrtt. E., 1985, 18, ( S ) , pp.573-588
0 IEE: 1994
The authors are with the Department of Medicine (Section
of Clinical Biochemistry), University of Manchester, Hope
Hospital, Eccles Old Road, Manchester M 6 8HD, UK.
FEBRUARY 1994