Journal of Bioteehnology, 15 (1990) 229-238
229
Elsevier BIOTEC 00552
Amperometric biosensors S.P. H e n d r y , I.J. Higgins a n d J.V. Bannister Biotechnology Centre, Cranfield Institute of Technology, Cranfield, U.K.
(Received 21 February 1990; accepted5 April 1990)
Sensors; Amperometric; Oxygen electrode; Mediator
Introduction
Electrochemical, optical piezoelectric and calorimetric routes linking biology to electronics have demonstrated the basis for a range of biosensors (Turner et al., 1987a). A biosensor is therefore defined as an analytic device incorporating a biological or biologically devised sensing element either intimately associated with or integrated within a physiochemical transducer. Biosensors are distinguished from bioprobes because the latter are sensors used for in vivo monitoring (Thompson and Krull, 1986). The diversity of biological elements incorporated into biosensors has expanded steadily since the pioneering work of Clark (1987) leading to the construction of enzyme electrodes. Intact microorganisms (Karube, 1987), tissues (Sidewell and Rechnitz, 1986) and antibodies (K_ress-Rogers and Turner, 1988) are increasingly being reported in the literature on a biosensor configuration. Recently attention has also focussed on the use of DNA probes for the detection of human genetic disorders and pathogens. Enzyme labels such as horseradish peroxidase, alkaline phosphatase and glucose oxidase which are commonly used for immunoassays have also been used for the electrochemical detection of DNA-DNA hybridisation (Downs et al., 1987). The literature on electrochemical biosensors has been recently extensively reviewed (Turner et al., 1987; Smyth and Vos, 1986; Higgins and Lowe, 1987; Guilbault and Mascini, 1987) and the reader is referred to these publications for a historical appreciation of the field. Indirect amperometric and potentiometric biosensors in which the natural product or substrate of a biologically catalysed reaction Correspondence to: J.V. Bannister, BiotechnologyCentre, Cranfield Institute of Technology, Cranfield
MK43 0AL, U.K. 0168-1656/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (Biomedical Division)
230
is detected by a discrete electrochemical transducer continue to dominate the literature. Microfabrication techniques are also being used for the construction of both potentiometric (Gotoh et al., 1986a, b; Anzai et al., 1986; Karube et al., 1986; Nakako et al., 1986) and amperometric biosensors (Murakami et al., 1986).
Biosensors based on the o x y g e n electrode
The various publications describing variation in the theme of biocatalyst immobilised at an oxygen electrode were reviewed in detail by Hopkins (1985). It appears that an enzyme electrode can be constructed for any analyte provided a suitable oxidase is available. The principle of the technique broadens the range of the simple and effective Clark oxygen electrode by incorporating a biocatalyst which utilises oxygen in the presence of a specific substrate (Clark, 1987). The presence of the analyte causes a decrease in the current obtained from the enzyme electrode. The classical example is the reaction catalysed by glucose oxidase: Glucose + 0 2 ~ Gluconic acid + H 2 0 2
(1)
The electrode response is dependent on the oxygen concentration in the sample and it ceases to function in anoxic and anaerobic situations (Karube, 1987). In addition, the hydrogen peroxide produced may inactivate the enzyme and interfere with the base transducer giving a positive signal which tends to counteract the decrease in signal due to the consumption of oxygen (Tse et al., 1987) despite the presence of a hydrophobic membrane designed to minimise the problem. Campanella et al. (1985) recently described the use of an oxygen electrode for the determination of cholic acids in human bile. NAD-dependent hydroxysteroid dehydrogenases were coupled to an oxygen electrode using peroxidase to catalyse the oxidation of N A D H : Hydroxysteroid + N A D + ~ Ketosteroid + H ++ N A D H 2 N A D H + 2 H + + 02 -+ N A D + + 2H20
(2)
Ethanol and lactate levels in blood could also be measured via the peroxidase catalysed oxidation of N A D H (Cheng and Christian, 1978, 1979). Rahni et al. (1986) described the use of salicylate hydroxylase immobilised at an oxygen electrode to determine salicylate (the hydrolysis product of aspirin) in blood serum. The reaction consumes oxygen in the presence of substrate and required reducing equivalents in the form of N A D H and N A D P H . Thus cofactor must be added to the assay solution. The electrode was found to respond linearly to salicylate over the range of 1 × 10 .5 to 6.9 × 10 - 4 M and is saturated at 1.87 × 10 .3 M. The electrode output took 1 to 6 rain to reach steady state and the values obtained correlated well with the established analytical methods (correlation coefficient = 0.99, n = 24). Some novel adaptations of the technology of immobilising oxygen utilising 02 liberating enzymes at an electrode have recently appeared in the literature. Watanabe et al. (1986) expanded their investigations on multi-enzyme electrodes for the determination of fish freshness to a microprocessor based system. These oxygen
231 electrode-base enzyme electrodes were used in conjunction with a fourth base oxygen electrode for compensation of the changes in the oxygen concentration in the samples. The three enzyme electrodes responded to inosine monophosphate, inosine and hypoxanthine respectively. Simultaneous measurement of the three chemial indicators was considered to give a rapid (15 rain) and reliable indication of the freshness of a range of fish species. An enzyme electrode constructed by immobilising choline oxidase at an oxygen electrode was used in conjunction with phospholipase D in free solution for the determination of lecithin in food and drugs (Campanella et al., 1985, 1986). Lecithin in these samples was measured with high precision and accuracy compared to the enzymatic methods which use spectrophotometric detection. Detection of aspartame, the low calorie sweetener was studied by Renneberg et al. (1985) who used intact aspartame-grown Bacillus subtilis cells immobilised at an oxygen electrode. The sensor responded linearly to aspartamate over the range of 0.07 to 0.6 x 10 -3 M with a response time to steady state of 1 min. The use of B. subtilis in a biosensor configuration was also applied for the determination of glutamic acid (Riedel and Scheller, 1987). A thin layer of B. subtilis cells on a paper support were retained at an oxygen electrode using dialysis membrane. The selectivity of the biosensor to glutamic acid in the presence of glucose was increased by treating the cells with low concentrations of p-chloromercuribenzoate for 20 rain and followed by determinations of glutamic acid in the presence of sodium fluoride. The sensor was f o u n d to be highly selective for glutamic acid and responded linearly over the range 0 to 3.7 x 10 -3 M. The respiratory chain isolated from Escherichia coli grown in medium supplemented with L-lactate was immobilised at an oxygen electrode to measure L-lactate in yoghurt, wine and blood (Adamowicz and Burnstein, 1987). K u b o and Karube (1986) reported performance from a hybrid creatinine sensor by using a substituted polymethylglutamate membrane. The sensor was constructed from creatinine deiminase immobilised on the membrane with immobilised nitrifying bacteria at an oxygen electrode. The electrode was found to have greater sensitivity and an improved response time. Macholan and Chemlikova (1986) measured ascorbic acid by using the mesocarp obtained from zucchini or cucumber retained at an oxygen electrode. The tissue slice from these vegetables is a rich source of ascorbate oxidase and the sensor was found to be relatively insensitive to other organic acids, phenols, amino acids and glucose. It responded linearly to ascorbic acid over the range 0.02 to 2 x 10 -3 M and reached saturation at a concentration of 0.57 x 10 -3 M. The response time to steady state was 70 to 90 s and the sensor was used to measure ascorbic acid in fruit juices and vitamin tablets.
Biosensors based on hydrogen peroxide detection The detection of hydrogen peroxide has also become an established approach to the construction of biosensors based on electrodes containing immobilised oxidases. Reactions catalysed by oxidases produce hydrogen peroxide which can be readily detected with a platinum electrode poised at + 60 to + 700 mV versus a saturated
232 calomel electrode (SCE). The major advantage of hydrogen peroxide over the oxygen electrode based sensors is high sensitivity and the ability to use the platinum electrode in whole blood obviating the need to remove the red cells. However, sensors based on hydrogen peroxide detection are still affected by change in the oxygen concentration since the gas is a cosubstrate of oxidase catalysed reactions. A sensitive method for the detection of glucose using glucose oxidase to oxidise glucose and peroxidase adsorbed to a carbon electrode' to measure the hydrogen peroxide produced has been reported (Iwai and Akihama, 1986). The same protocol was used for the detection of uric acid and cholesterol using uricase and cholesterol oxidase respectively in place of glucose oxidase. Delaney et al. (1986) investigated a number of carbon substrates for the covalent immobilisation of glucose oxidase with subsequent detection of hydrogen peroxide. The most successful carbon electrode material, platinised carbon, responded to glucose in the range of 0-33 mM. While most of these investigations give details regarding the response to glucose there still remains the problem of interference by substances present in physiological samples. Due to the relatively high overpotential required to dismute hydrogen peroxide (+ 600 - + 700 mV vs SCE), various substances such as acetaminophen, cysteine, ascorbic acid and many other potential blood constituents may significantly interfere with hydrogen peroxide based sensors used in conjunction with blood. These interferences can be circumvented by the use of selective membranes such as cellulose acetate (Scheller et al., 1987). Alternatively it is possible to lower the potential at which hydrogen peroxide oxidation occurs. Bennetto et al. (1987) claim that the overpotential required to reduce hydrogen proxide can be lowered to around + 400 mV vs SCE by using a platinised carbon electrode. A significant reduction in the electrode potential would greatly enhance the practical application of such devices. Other applications of hydrogen peroxide based sensors have included the measurement of lactate. Monitoring of the metabolite was reported by Vadgama et al. (1986) and by Mullen et al. (1986) using lactate dehydrogenase and lactate oxidase respectively with hydrogen peroxide detection. The electrode reported by Mullen et al. (1986) had an organsilane treated microporous membrane over an enzymatically active polycarbonate membrane. In this configuration a calibration curve linear to 18 mM L-lactate with one response time of 1-3 rain was obtained. Bardeletti et al. (1986) have reported a very sensitive lactate oxidase membrane electrode with a detection limit of 1.25 x 10-7 M with a response time of < 2 min. Several other devices exploiting the detection of hydrogen peroxide produced by the action of various oxidases have been reported. These include pyruvate oxidase (Mascini and Mazzei, 1987), oxalate oxidase (Fonong, 1986a) and sulphite oxidase (Fonong, 1986b).
Mediated amperometric sensors
Modified and mediated amperometric sensors have received much attention in recent years and are currently beginning to supersede hydrogen peroxide-based sensors as the main thrust of enzyme electrode research. The main advantages of
233
mediated amperometric biosensors over hydrogen peroxide sensors is the lack of dependence on oxygen as the mediator replaces oxygen as the electron acceptor. Also it is possible to employ mediators which have a low redox potential. The resultant enzyme electrode can therefore be operated at a potential much lower than that required to dismute hydrogen peroxide reducing the possibility of interference from various components present in physiological fluids. Amperometry is preferred to potentiometry by many investigators as the need for an accurate reference electrode is obviated. Organo-conducting compounds have been the centre of attention from electrochemists for several years. The natural progression of the various investigations is their incorporation into biosensors. Organo-conducting compounds are polymers which exhibit electrical conductivity (Bryce and Murphy, 1984). One such typical compound which is by far the best characterised is tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) (Jaeger and Bard, 1979). Kulys et al. (1980) and Kulys (1986) reported the use of this polymer in a biosensor configuration. Cytochrome b 2, glucose oxidase, xanthine oxidase and peroxidase were utilised to form sensors for L-lactate, glucose, hypoxanthine and hydrogen peroxide, respectively. The electrocatalytic oxidation of NADH by these polymers is also discussed. The use of tetracyanoquinodimethane oxylyemedipyridyl (TCNQ-PXDP) for the analysis of glucose has been reported (Yaropolov and Ghindilis, 1987). Immobilisation of the enzyme resulted in a 50% increase of the specific activity compared to the natural enzyme and a 10-fold increase in enzyme stability. Galactose oxidase was also reported to be compatible with this system. Dehydrogenases represent the largest group of redox enzymes known. Approximately 250 dehydrogenases are dependent on the N A D / N A D H couple. In the absence of direct electron transfer from the enzyme to the electrode, the N A D / N A D H couple can be utilised in the same manner as a mediator. The various applications of this system have been reviewed by Gorton (1986). Laval et al. (1987) demonstrated that they achieved a yield of 99.97% for electrochemical regeneration of NAD representing 3000 potential regenerating cycles. These results demonstrate that electrochemical oxidation is an alternative to enzymatic regeneration for use in conjunction with dehydrogenases. The area of electron transfer from proteins in the context of biosensors has been discussed by Cardosi and Turner (1987). A comprehensive review by Fultz and Durst (1982) gives a list of some 69 mediators compatible with various biological redox systems. Jonsson and Gorton (1985) have described a membrane-free glucose sensor using covalently bound glucose oxidase. The electrode was poised at + 50 mV vs SCE and N-methyl-phenazinium methyl sulphate was used as the electron mediator between glucose oxidase and the graphite electrode. The use of a novel electron-transfer mediator, tetrathiafulvalene (TTF) between glucose oxidase and a carbon electron has been reported (Turner et al., 1987b). The glucose oxidase was covalently attached to a graphite laminate which had been modified with TTF. The enzyme electrode demonstrated linearity in the range 0-25 mM glucose saturating at 70 mM and could function both anaerobically and aerobically. Hendry and Turner (1987) also described a glucose sensor that utilised tetracyanoquinodimethane (TCNQ).
234 However, following the pioneering work of Cass et al. (1984) ferrocene appears to be the mediator of choice. In this system electron transfer was observed between glucose and glucose oxidase was observed in the presence of ferrocenes. Petersson (1986) reported the use of a ferrocene modified platinum electrode for the determination of ascorbic acid (Vitamin C). The required overpotential for the oxidation reaction of p H 2.2 was reduced by 150 mV compared with the oxidation potential of ca 550 mV vs SCE at a bare platinum electrode. The ferrocene-modified electrode was used to determine the vitamin C content of fresh grapefruit and orange juice. The system gave good results which correlated well with titrimetrically obtained values. Dicks et al. (1986) described three mediated amperometric biosensors for D-galactose, glycolate and L-amino acids. The preferred mediator was 1,1-dimethylferrocene. The appropriate oxidase was retained behind a dialysis membrane at a carbon paste electrode containing the mediator. This mediator was also utilised for the construction of miniature amperometric glucose sensors (Claremont et al., 1986). Ferrocene derivatives were also used as mediators for the measurement of cholesterol (Ball et al., 1986). Another area which has received a great deal of attention in the past few years is the development of amperometric methods for the detection of microbial biomass in liquid samples such as milk, water, urine etc. The method developed by Turner et al. (1986) utilised a cocktail of soluble mediators to direct electrons arising from the respiratory chains of microorganisms to an electrode which is held at a constant preset potential. This causes the reoxidation of the mediators and the concomitant production of a current response. The rate of change of current thus produced over a 2 min test period is proportional to the number of bacteria present in the stored solution and to their metabolic activity. Finally mediated systems are also being applied to antigen-antibody reactions. McNeil et al. (1988) have developed an amperometric assay for alkaline phosphatase using a specially synthesised substrate (N-ferrocenoyl)-4-aminophenyl phosphate. In the presence of alkaline phosphatase the substrate is converted to (N-ferrocenoyl)-4-aminophenyl which could be detected at + 180 mV vs SCE. The change in peak current at this potential was found to be related to the alkaline phosphatase concentration. The method was successfully applied to an ELISA method for measuring estriol using alkaline phosphatase as the marker enzyme. Another approach is an amperometric immunoassay based on enzyme amplification (Stanley et al., 1988). A sandwich immunoassay is performed in which the second antibody is conjugated to alkaline phosphatase. Digestion of N A D P by alkaline phosphatase leads to the formation of N A D which enters a redox cycle involving alcohol dehydrogenase and diaphorase. The redox cycle causes the reduction of a mediator which is then detected amperometrically.
Conclusions
Amperometric biosensors present a novel approach to analysis. Amperometric detection methods have a much wider dynamic range than current optical methods, similar or improved sensitivity and greatly reduced instrumentation costs.
235
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236 Gotoh, M., Tamiya, E. and Karube, I. (1986a) Polyvinylbutyral resin membrane for enzyme immobilisation to an ISFET microbiosensor, J. Mol. Catal. 37, 133-139. Gotoh, M., Tamiya, E., Karube, I. and Kagawa, Y. (1986b) A microsensor for adenosine-5-triphosphate pH-sensitive field effect transistors. Anal. Chim. Acta 187, 287-291. Guilbault, G.G. and Mascini, M. (1987) (Eds) Analytical uses of immobilised biological compounds for detection, medical and industrial uses. Proc. NATO ARW Workshop, Reidel, Dordrecht, Holland. Hendry, S.P. and Turner, A.P.F. (1987) A glucose sensor utilising tetracyanoquinodimethane as a mediator. Hormone and Metabolic Research Implantable glucose sensors - The State of the Art 20, 37-40. Higgins, I.J. and Lowe, C.R. (1987) Introduction to the principles and applications of biosensors. In: Akhtar, M., Higgins, I.J. and Lowe, C.R. (Eds), Biosensors, Royal Society, London, pp. 3-11. Hopkins, T.A. (1985) A multi-purpose enzyme sensor based on alcohol oxidase. Am. Biotech. Lab. 3, 32-35. Iwai, H. and Akihama, S. (1986) Electric microassays of glucose, uric acid and cholesterol using peroxidase adsorbed on a carbon electrode. Chim. Pharm. Bull. 34, 3471-3474. Jaeger, C.D. and Bard, A.J. (1979) Electrochemical behaviour of tetrathifulvalene-tetracyano-quinodimethane electrodes in aqueous media. Am. Chem. Soc. 101, 1690-1699. Jonsson, G. and Gorton, L. (1985) An amperometric glucose sensor made by modification of a graphite electrode surface with immobilised glucose oxidase and adsorbed mediator. Biosensors 1, 355-368. Karube, I. (1987) Micro-organisms based sensors. In: Turner, A.P.F., Karube, I. and Wilson, G.S. (Eds), Biosensors: Fundamentals and Applications, Oxford University Press, Oxford, pp. 13-29. Karube I., Tamiya, E., Dicks, J.M. and Gotoh, M. (1986) A microsensor for urea based on an ion-selective field effect transistor. Anal. Claim. Acta 185, 195-200. Kress-Rogers, E. and Turner, A.P.F. (1988) Immunosensors based on acoustic, optical and bioelectrochemical devices and techniques. In: Clifford, M.N., Jackman, R., Morris, B.A. and Morris, J.A. (Eds) Advances in immunoassays for veterinary and food analysis, Elsevier, Barking, pp. 227-253. Kubo, I. and Karube, I. (1986) Immobilisation of creatinine deaminase on a substituted poly(methylglutaminate) membrane and its use in a creatinine sensor. Anal. Chim. Acta 187, 31-37. Kulys, J.J., Samalius, A.S. and Svimickas, G-J.S. (1980) Electron exchange between the enzyme active centre and organic metals. FEBS Lett. 14, 7-10. Kulys, J.J. (1986) Enzyme electrodes based on organic metals. Biosensors 2, 3-13. Laval, J-M., Bourdillon, C. and Moiroux, J. (1987) The electrochemical regeneration of NAD revisited. In: Biotechnology and Bioengineering 30, John Wiley & Sons, New York, pp. 157-159. Macholan, L. and Chemlikova, B. (1986) Plant tissue based membrane biosensor for L-ascorbic acid. Anal. Claim. Acta 185, 187-193. Mascini, M. and Mazzei, F. (1987) Amperometric sensor for pyruvate with immobilised pyrnvate oxidase. Anal. Chim. Acta 192, 9. McNeil, C.J., Higgins, I.J. and Bannister, J.V. (1988) Amperometric determination of alkaline phosphatase activity: application to enzyme immunoassay. Biosensors 3, 199-209. Mullen, W.H., Churchouse, S.J., Keedy, F.H. and Vadgama, P.M. (1986) Enzyme electrode for the measurement of lactate in undiluted blood. Clin. Chem. Acta 157, 191-196. Murakami, T., Nakamoto, S., Kimura, J., Kuriyama, T. and Karube, I. (1986) A micro planar amperometric glucose sensor using an ISFET as a reference electrode. Anal. Lett. 19, 1973-1976. Nakako, M., Hanazato, Y., Maeda, M. and Shiono, S. (1986) Neutral lipid enzyme electrode based on ion-sensitive field effect transistors. Anal. Chim. Acta 185, 179-186. Petersson, M. (1986) Electrocatalytic oxidation of ascorbic acid and voltammetric determination with a ferrocene-modified platinum electrode. Anal. Chim. Acta 187, 333-338. Rahni, M.A.N., Guilbault, G. and De Oliveira, G.N. (1986) Immobilised enzyme electrode for the determination a salicylate in blood serum. Anal. Chim. Acta 181, 219-225. Renneberg, R. Riedel, K. and Scheller, F. (1985) Microbial sensor for aspartame. Appl. Microbiol. Biotechnol. 21, 180-181. Riedel, K. and Schelier, F.W. (1987) Inhibitor-treated microbial sensor for the selective determination of glutamic acid. Analyst 112, 341-342. Scheller, F.W., Pfeiffer, D., Schubert, F., Renneberg, R. and Kirstein, D. (1987) The application of
237 enzyme-based amperometric biosensors to the analysis of "real" samples. In: Turner, A.P.F, Karube, I. and Wilson, G.S. (Eds), Biosensors: Fundamentals and Applications, Oxford University Press, Oxford, pp. 315-346. Sidewell, J.S. and Rechnitz, G.A. (1986) Progress and challenges for biosensors using plant tissue materials. Biosensors 2, 221-233. Smyth, M.R. and Vos, J.G. (Eds) (1986) Electrochemistry, Sensors and Analysis, Elsevier, Amsterdam. Stanley, C.J., Cox, R., Cardosi, M.F. and Turner, A.P.F. (1988) Electrochemical enzyme amplified immunoassay for human prostatic acid phosphatase. J. Immunol. Methods 112, 153-161. Thompson, M. and Krull, U.J. (1986) The Chemoreceptor-transducer interface in the development of biosensors. In: Smyth, M.R. and Vos J.G. (Eds), Electrochemistry Sensors and Analysis, Elsevier, Amsterdam, pp. 247-262. Turner, A.P.F., Karube, I. and Wilson, G.S. (Eds) (1987a) Biosensors: Fundamentals and Applications, Oxford University Press, Oxford. Turner, A.P.F., Hendry, S.P. and Cardosi, M.F. (1987b) Tetrathiafulvalene: a new mediator for amperometric biosensors. In: The World Biotech. Report on Biosensors, Instrumentation and Processing, I, Online Publications, Pinner, UK, pp. 125-137. Turner, A.P.F., Cardosi, M.F., Ramsay, G., Schneider, B.H. and Swain, A. (1986) Biosensors for use in the food industry: A new bioreactivity monitor. In: Biotechnology in the Food Industry, Online Publications, Pinner, pp. 97-116. Tse, P.H.S., Leypoldt, J.K. and Gough, D.A. (1987) Determination of the intrinsic kinetic constants of immobilised glucose oxidase and catalase. Biotech. Bioeng. 29, 696-704. Vadgama, P., Covington, A.K. and Alberti, K.G.M.M. (1986) Enzyme electrode systems for extra corporeal lactate monitoring based on lactate dehydrogenase. Analyst 111, 803-807. Watanabe, E., Endo, H., Hayashi, T. and Toyama, K. (1986) Simultaneous determination of hypocanthine and inosine with an enzyme sensor. Biosensors 2, 235-244. Yaropolov, A.I. and Ghindilis, A.L. (1987) Immobilised biocatalysts in electroconductive TCNQ-polymer matrices for enzyme electrodes. In: Proc. 4th European Congress in Biotechnology 3, 104.
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Elsevier BIOTEC 00552
Amperometric biosensors S.P. H e n d r y , I.J. Higgins a n d J.V. Bannister Biotechnology Centre, Cranfield Institute of Technology, Cranfield, U.K.
(Received 21 February 1990; accepted5 April 1990)
Sensors; Amperometric; Oxygen electrode; Mediator
Introduction
Electrochemical, optical piezoelectric and calorimetric routes linking biology to electronics have demonstrated the basis for a range of biosensors (Turner et al., 1987a). A biosensor is therefore defined as an analytic device incorporating a biological or biologically devised sensing element either intimately associated with or integrated within a physiochemical transducer. Biosensors are distinguished from bioprobes because the latter are sensors used for in vivo monitoring (Thompson and Krull, 1986). The diversity of biological elements incorporated into biosensors has expanded steadily since the pioneering work of Clark (1987) leading to the construction of enzyme electrodes. Intact microorganisms (Karube, 1987), tissues (Sidewell and Rechnitz, 1986) and antibodies (K_ress-Rogers and Turner, 1988) are increasingly being reported in the literature on a biosensor configuration. Recently attention has also focussed on the use of DNA probes for the detection of human genetic disorders and pathogens. Enzyme labels such as horseradish peroxidase, alkaline phosphatase and glucose oxidase which are commonly used for immunoassays have also been used for the electrochemical detection of DNA-DNA hybridisation (Downs et al., 1987). The literature on electrochemical biosensors has been recently extensively reviewed (Turner et al., 1987; Smyth and Vos, 1986; Higgins and Lowe, 1987; Guilbault and Mascini, 1987) and the reader is referred to these publications for a historical appreciation of the field. Indirect amperometric and potentiometric biosensors in which the natural product or substrate of a biologically catalysed reaction Correspondence to: J.V. Bannister, BiotechnologyCentre, Cranfield Institute of Technology, Cranfield
MK43 0AL, U.K. 0168-1656/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (Biomedical Division)
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is detected by a discrete electrochemical transducer continue to dominate the literature. Microfabrication techniques are also being used for the construction of both potentiometric (Gotoh et al., 1986a, b; Anzai et al., 1986; Karube et al., 1986; Nakako et al., 1986) and amperometric biosensors (Murakami et al., 1986).
Biosensors based on the o x y g e n electrode
The various publications describing variation in the theme of biocatalyst immobilised at an oxygen electrode were reviewed in detail by Hopkins (1985). It appears that an enzyme electrode can be constructed for any analyte provided a suitable oxidase is available. The principle of the technique broadens the range of the simple and effective Clark oxygen electrode by incorporating a biocatalyst which utilises oxygen in the presence of a specific substrate (Clark, 1987). The presence of the analyte causes a decrease in the current obtained from the enzyme electrode. The classical example is the reaction catalysed by glucose oxidase: Glucose + 0 2 ~ Gluconic acid + H 2 0 2
(1)
The electrode response is dependent on the oxygen concentration in the sample and it ceases to function in anoxic and anaerobic situations (Karube, 1987). In addition, the hydrogen peroxide produced may inactivate the enzyme and interfere with the base transducer giving a positive signal which tends to counteract the decrease in signal due to the consumption of oxygen (Tse et al., 1987) despite the presence of a hydrophobic membrane designed to minimise the problem. Campanella et al. (1985) recently described the use of an oxygen electrode for the determination of cholic acids in human bile. NAD-dependent hydroxysteroid dehydrogenases were coupled to an oxygen electrode using peroxidase to catalyse the oxidation of N A D H : Hydroxysteroid + N A D + ~ Ketosteroid + H ++ N A D H 2 N A D H + 2 H + + 02 -+ N A D + + 2H20
(2)
Ethanol and lactate levels in blood could also be measured via the peroxidase catalysed oxidation of N A D H (Cheng and Christian, 1978, 1979). Rahni et al. (1986) described the use of salicylate hydroxylase immobilised at an oxygen electrode to determine salicylate (the hydrolysis product of aspirin) in blood serum. The reaction consumes oxygen in the presence of substrate and required reducing equivalents in the form of N A D H and N A D P H . Thus cofactor must be added to the assay solution. The electrode was found to respond linearly to salicylate over the range of 1 × 10 .5 to 6.9 × 10 - 4 M and is saturated at 1.87 × 10 .3 M. The electrode output took 1 to 6 rain to reach steady state and the values obtained correlated well with the established analytical methods (correlation coefficient = 0.99, n = 24). Some novel adaptations of the technology of immobilising oxygen utilising 02 liberating enzymes at an electrode have recently appeared in the literature. Watanabe et al. (1986) expanded their investigations on multi-enzyme electrodes for the determination of fish freshness to a microprocessor based system. These oxygen
231 electrode-base enzyme electrodes were used in conjunction with a fourth base oxygen electrode for compensation of the changes in the oxygen concentration in the samples. The three enzyme electrodes responded to inosine monophosphate, inosine and hypoxanthine respectively. Simultaneous measurement of the three chemial indicators was considered to give a rapid (15 rain) and reliable indication of the freshness of a range of fish species. An enzyme electrode constructed by immobilising choline oxidase at an oxygen electrode was used in conjunction with phospholipase D in free solution for the determination of lecithin in food and drugs (Campanella et al., 1985, 1986). Lecithin in these samples was measured with high precision and accuracy compared to the enzymatic methods which use spectrophotometric detection. Detection of aspartame, the low calorie sweetener was studied by Renneberg et al. (1985) who used intact aspartame-grown Bacillus subtilis cells immobilised at an oxygen electrode. The sensor responded linearly to aspartamate over the range of 0.07 to 0.6 x 10 -3 M with a response time to steady state of 1 min. The use of B. subtilis in a biosensor configuration was also applied for the determination of glutamic acid (Riedel and Scheller, 1987). A thin layer of B. subtilis cells on a paper support were retained at an oxygen electrode using dialysis membrane. The selectivity of the biosensor to glutamic acid in the presence of glucose was increased by treating the cells with low concentrations of p-chloromercuribenzoate for 20 rain and followed by determinations of glutamic acid in the presence of sodium fluoride. The sensor was f o u n d to be highly selective for glutamic acid and responded linearly over the range 0 to 3.7 x 10 -3 M. The respiratory chain isolated from Escherichia coli grown in medium supplemented with L-lactate was immobilised at an oxygen electrode to measure L-lactate in yoghurt, wine and blood (Adamowicz and Burnstein, 1987). K u b o and Karube (1986) reported performance from a hybrid creatinine sensor by using a substituted polymethylglutamate membrane. The sensor was constructed from creatinine deiminase immobilised on the membrane with immobilised nitrifying bacteria at an oxygen electrode. The electrode was found to have greater sensitivity and an improved response time. Macholan and Chemlikova (1986) measured ascorbic acid by using the mesocarp obtained from zucchini or cucumber retained at an oxygen electrode. The tissue slice from these vegetables is a rich source of ascorbate oxidase and the sensor was found to be relatively insensitive to other organic acids, phenols, amino acids and glucose. It responded linearly to ascorbic acid over the range 0.02 to 2 x 10 -3 M and reached saturation at a concentration of 0.57 x 10 -3 M. The response time to steady state was 70 to 90 s and the sensor was used to measure ascorbic acid in fruit juices and vitamin tablets.
Biosensors based on hydrogen peroxide detection The detection of hydrogen peroxide has also become an established approach to the construction of biosensors based on electrodes containing immobilised oxidases. Reactions catalysed by oxidases produce hydrogen peroxide which can be readily detected with a platinum electrode poised at + 60 to + 700 mV versus a saturated
232 calomel electrode (SCE). The major advantage of hydrogen peroxide over the oxygen electrode based sensors is high sensitivity and the ability to use the platinum electrode in whole blood obviating the need to remove the red cells. However, sensors based on hydrogen peroxide detection are still affected by change in the oxygen concentration since the gas is a cosubstrate of oxidase catalysed reactions. A sensitive method for the detection of glucose using glucose oxidase to oxidise glucose and peroxidase adsorbed to a carbon electrode' to measure the hydrogen peroxide produced has been reported (Iwai and Akihama, 1986). The same protocol was used for the detection of uric acid and cholesterol using uricase and cholesterol oxidase respectively in place of glucose oxidase. Delaney et al. (1986) investigated a number of carbon substrates for the covalent immobilisation of glucose oxidase with subsequent detection of hydrogen peroxide. The most successful carbon electrode material, platinised carbon, responded to glucose in the range of 0-33 mM. While most of these investigations give details regarding the response to glucose there still remains the problem of interference by substances present in physiological samples. Due to the relatively high overpotential required to dismute hydrogen peroxide (+ 600 - + 700 mV vs SCE), various substances such as acetaminophen, cysteine, ascorbic acid and many other potential blood constituents may significantly interfere with hydrogen peroxide based sensors used in conjunction with blood. These interferences can be circumvented by the use of selective membranes such as cellulose acetate (Scheller et al., 1987). Alternatively it is possible to lower the potential at which hydrogen peroxide oxidation occurs. Bennetto et al. (1987) claim that the overpotential required to reduce hydrogen proxide can be lowered to around + 400 mV vs SCE by using a platinised carbon electrode. A significant reduction in the electrode potential would greatly enhance the practical application of such devices. Other applications of hydrogen peroxide based sensors have included the measurement of lactate. Monitoring of the metabolite was reported by Vadgama et al. (1986) and by Mullen et al. (1986) using lactate dehydrogenase and lactate oxidase respectively with hydrogen peroxide detection. The electrode reported by Mullen et al. (1986) had an organsilane treated microporous membrane over an enzymatically active polycarbonate membrane. In this configuration a calibration curve linear to 18 mM L-lactate with one response time of 1-3 rain was obtained. Bardeletti et al. (1986) have reported a very sensitive lactate oxidase membrane electrode with a detection limit of 1.25 x 10-7 M with a response time of < 2 min. Several other devices exploiting the detection of hydrogen peroxide produced by the action of various oxidases have been reported. These include pyruvate oxidase (Mascini and Mazzei, 1987), oxalate oxidase (Fonong, 1986a) and sulphite oxidase (Fonong, 1986b).
Mediated amperometric sensors
Modified and mediated amperometric sensors have received much attention in recent years and are currently beginning to supersede hydrogen peroxide-based sensors as the main thrust of enzyme electrode research. The main advantages of
233
mediated amperometric biosensors over hydrogen peroxide sensors is the lack of dependence on oxygen as the mediator replaces oxygen as the electron acceptor. Also it is possible to employ mediators which have a low redox potential. The resultant enzyme electrode can therefore be operated at a potential much lower than that required to dismute hydrogen peroxide reducing the possibility of interference from various components present in physiological fluids. Amperometry is preferred to potentiometry by many investigators as the need for an accurate reference electrode is obviated. Organo-conducting compounds have been the centre of attention from electrochemists for several years. The natural progression of the various investigations is their incorporation into biosensors. Organo-conducting compounds are polymers which exhibit electrical conductivity (Bryce and Murphy, 1984). One such typical compound which is by far the best characterised is tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) (Jaeger and Bard, 1979). Kulys et al. (1980) and Kulys (1986) reported the use of this polymer in a biosensor configuration. Cytochrome b 2, glucose oxidase, xanthine oxidase and peroxidase were utilised to form sensors for L-lactate, glucose, hypoxanthine and hydrogen peroxide, respectively. The electrocatalytic oxidation of NADH by these polymers is also discussed. The use of tetracyanoquinodimethane oxylyemedipyridyl (TCNQ-PXDP) for the analysis of glucose has been reported (Yaropolov and Ghindilis, 1987). Immobilisation of the enzyme resulted in a 50% increase of the specific activity compared to the natural enzyme and a 10-fold increase in enzyme stability. Galactose oxidase was also reported to be compatible with this system. Dehydrogenases represent the largest group of redox enzymes known. Approximately 250 dehydrogenases are dependent on the N A D / N A D H couple. In the absence of direct electron transfer from the enzyme to the electrode, the N A D / N A D H couple can be utilised in the same manner as a mediator. The various applications of this system have been reviewed by Gorton (1986). Laval et al. (1987) demonstrated that they achieved a yield of 99.97% for electrochemical regeneration of NAD representing 3000 potential regenerating cycles. These results demonstrate that electrochemical oxidation is an alternative to enzymatic regeneration for use in conjunction with dehydrogenases. The area of electron transfer from proteins in the context of biosensors has been discussed by Cardosi and Turner (1987). A comprehensive review by Fultz and Durst (1982) gives a list of some 69 mediators compatible with various biological redox systems. Jonsson and Gorton (1985) have described a membrane-free glucose sensor using covalently bound glucose oxidase. The electrode was poised at + 50 mV vs SCE and N-methyl-phenazinium methyl sulphate was used as the electron mediator between glucose oxidase and the graphite electrode. The use of a novel electron-transfer mediator, tetrathiafulvalene (TTF) between glucose oxidase and a carbon electron has been reported (Turner et al., 1987b). The glucose oxidase was covalently attached to a graphite laminate which had been modified with TTF. The enzyme electrode demonstrated linearity in the range 0-25 mM glucose saturating at 70 mM and could function both anaerobically and aerobically. Hendry and Turner (1987) also described a glucose sensor that utilised tetracyanoquinodimethane (TCNQ).
234 However, following the pioneering work of Cass et al. (1984) ferrocene appears to be the mediator of choice. In this system electron transfer was observed between glucose and glucose oxidase was observed in the presence of ferrocenes. Petersson (1986) reported the use of a ferrocene modified platinum electrode for the determination of ascorbic acid (Vitamin C). The required overpotential for the oxidation reaction of p H 2.2 was reduced by 150 mV compared with the oxidation potential of ca 550 mV vs SCE at a bare platinum electrode. The ferrocene-modified electrode was used to determine the vitamin C content of fresh grapefruit and orange juice. The system gave good results which correlated well with titrimetrically obtained values. Dicks et al. (1986) described three mediated amperometric biosensors for D-galactose, glycolate and L-amino acids. The preferred mediator was 1,1-dimethylferrocene. The appropriate oxidase was retained behind a dialysis membrane at a carbon paste electrode containing the mediator. This mediator was also utilised for the construction of miniature amperometric glucose sensors (Claremont et al., 1986). Ferrocene derivatives were also used as mediators for the measurement of cholesterol (Ball et al., 1986). Another area which has received a great deal of attention in the past few years is the development of amperometric methods for the detection of microbial biomass in liquid samples such as milk, water, urine etc. The method developed by Turner et al. (1986) utilised a cocktail of soluble mediators to direct electrons arising from the respiratory chains of microorganisms to an electrode which is held at a constant preset potential. This causes the reoxidation of the mediators and the concomitant production of a current response. The rate of change of current thus produced over a 2 min test period is proportional to the number of bacteria present in the stored solution and to their metabolic activity. Finally mediated systems are also being applied to antigen-antibody reactions. McNeil et al. (1988) have developed an amperometric assay for alkaline phosphatase using a specially synthesised substrate (N-ferrocenoyl)-4-aminophenyl phosphate. In the presence of alkaline phosphatase the substrate is converted to (N-ferrocenoyl)-4-aminophenyl which could be detected at + 180 mV vs SCE. The change in peak current at this potential was found to be related to the alkaline phosphatase concentration. The method was successfully applied to an ELISA method for measuring estriol using alkaline phosphatase as the marker enzyme. Another approach is an amperometric immunoassay based on enzyme amplification (Stanley et al., 1988). A sandwich immunoassay is performed in which the second antibody is conjugated to alkaline phosphatase. Digestion of N A D P by alkaline phosphatase leads to the formation of N A D which enters a redox cycle involving alcohol dehydrogenase and diaphorase. The redox cycle causes the reduction of a mediator which is then detected amperometrically.
Conclusions
Amperometric biosensors present a novel approach to analysis. Amperometric detection methods have a much wider dynamic range than current optical methods, similar or improved sensitivity and greatly reduced instrumentation costs.
235
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