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long-term mplantoble
n
short-term mvasive
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 monitoring, 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.
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 electrochemical 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:
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:
(1)
(3)
A thin layer of the enzyme in solution was entrapped between two polymeric membranes and then placed over either a conventional pH or an oxygen electrode. An oxygen electrode here can simply comprise an
Table 1: Peroxide-based oxidase sensors Enzvme Substrate glucose oxidase glucose5 lactase oxidase lactate oxalate oxidase oxalates ascorbate oxidase a~orbate~ alcohol oxidase alcoholso Bio-samde blood blood bloodhrine blood blood
This method needs neither background 0 2 correction nor the use of twin differential electrodes. All members of the group of enzymes known as the oxidases act as catalysts for the production of H202 in the presence of specific substrates (analytes). This fandy of enzymes has therefore provided a generic method of constructing a variety of enzyme electrodec, each of which is highly specific for a particular analyte or analytes. A few examples of some peroxide-based oxidase sensors that have ~uccessfullydeveloped are yhown in Table 1.
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Fig. 2 (a) Medisense ExacTech glucose 'pen'; (b) home monitoring of blood glucose levels using the Medisense ExacTech 'companion'
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
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 electrochemically 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 oxidase membranebased enzyme laminate electrode
D glucose
0 2 -
GOD
glucanolactone
+ H202
+, 2e-+ 2H*
inner /membrane
7
H2 02
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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
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
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formance for either type cannot be simply determined theoretically and requires practical evaluation for a given sample matrix. Despite possible disadvantages imposed by the use of a mediator, the Medisense biosensors, based on ferro-
through ink-jet printer technology". Unquestionably, the development of micro-electronic5 and silicon techpossibilities for the future development of biosensor arrays with associated in situ
signal
elect rode
"
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.
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,
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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 cofactor-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
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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 biorecognition systems. Because of the lack of a depletion product, however, transduction may be difficult to achieve. One convenient mechanism for immunosensing 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 ionsensitive 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
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 microelectronic technology. Furthermore, problems associated 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.
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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.
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Acknowledgment
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We would like to thank the UK SERC for financial support to SIJH and SMR.
References
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1 WIESE, D. A., BOWEN, T. P., and KOST, G .J.: Enzyme
critical care profiling instrument, Clinical Chemistry, 1989, 35, ( 6 ) ,p.1098 MEDISENSE ExacTech blood glucose testing system (users guide), 1989, p.9 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 UPDIKE, S. J., and HICKS, G. P.: The enzymr electrodr, Natirre, 1967, 214. pp.986-988 TANG, L. X., and VADGAMA, P: Optinusation of enzyme electrodes,Med. G Bid. Big. G Cornprrr., 1990, 28, (3). pp.B18-B24 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 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 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 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 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 HALL, E. A. H., in Biosensors, IYYO, Open University Press Biotechnology Serie5, p.223 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 VADGAMA, F?: Biosensors in WILLIAMS, D. L., and MARK, V. (Eds.): Principles of clinical biochemistry (Hrinenmann Medical Books, Oxford and London, 198X) FEBRUARY I994
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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 . D., INAGAKI, 15 GORTON, L., KARAN, H. I., HALE, P 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 signalprocessing 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
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 pp.1582-1585 alcohol oxidase, Anal. Chem., 1983,55, (9, 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
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