Biopolym. Cell. 2002; 18(1):13-25.
Amperometric biosensors. Key work principles and features of transducers of different generations
1Dzyadevych S. V.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680


The key electrochemical principles connected with application of amperometric method in bioanalytical practice were considered, and the possible measuring electrode circuits were shown. Amperometric biosensors were classified on three groups such as unmediated, mediated and based on direct transfer of electrones, which were described in detail, and their advantages and disadvantages were shown.


[1] Clark MC Jr. Monitor and control of blood and tissue oxygen tensions. Trans Am Soc Artif Int Organs. 1956; 2: 41-48.
[2] Clark LC Jr, Lyons C. Electrode systems for continuous monitoring in cardiovascular surgery. Ann N Y Acad Sci. 1962;102:29-45.
[3] Updike SJ, Hicks GP. The enzyme electrode. Nature. 1967;214(5092):986-8.
[4] Koryta J, Dvorak J, Bohackova V. Electrochemistry. Methuen & Co. Ltd., London, England, 1970, 1970. 350 p.
[5] Hall E. Biosensors. Cambridge: Open Univ. press, 1991.
[6] White SF, Turner APF. Mediated amperometric biosensors. Handbook of biosensors and electronic noses: medecine, food, and environment. Ed. E. Kress-Rogers. New York: CRC press, 1997: 227-44.
[7] Davis G. Electrochemical techniques for the development of amperometric biosensors. Biosensors. 1985; 1(2): 161-8.
[8] Warsinke A. Biosensors for food analysis. In: Frontiers in Biosensorics II. Practical Applications. Eds F. W. Scheller, F. Schubert, J. Fedrowitz. Basel: Birkhauser, 1997: 121-39.
[9] Tran Minh C. Biosensors. London: Chapman & Hall, 1993.
[10] Pfeiffer D. Commercial biosensors for medical application. In: Frontiers in Biosensorics II. Practical Applications. Eds F. W. Scheller, F. Schubert, J. Fedrowitz. Basel: Birkhauser, 1997: 149-60.
[11] Bardeletti G, Sechaud F, R. Coulet P. A reliable l-lactate electrode with a new membrane for enzyme immobilization for amperometric assay of lactate. Anal Chim Acta. 1986;187:47–54.
[12] Mizutani F, Yamanaka T, Tanabe Y, Tsuda K. An enzyme electrode forl-lactate with a chemically-amplified response. Anal Chim Acta. 1985;177:153–66.
[13] Matsumoto K, Seijo H, Karube I, Suzuki S. Amperometric determination of choline with use of immobilized choline oxidase. Biotechnol Bioeng. 1980;22(5):1071–86.
[14] Xin Q, Wightman RM. Enzyme modified amperometric sensors for choline and acetylcholine with tetrathiafulvalene tetracyanoquinodimethane as the electron-transfer mediator. Anal Chim Acta. 1997;341(1):43–51.
[15] Verduyn C, Van Dijken JP, Scheffers WA. A simple, sensitive, and accurate alcohol electrode. Biotechnol Bioeng. 1983;25(4):1049-55.
[16] Boujtita M, Hart JP, Pittson R. Development of a disposable ethanol biosensor based on a chemically modified screen-printed electrode coated with alcohol oxidase for the analysis of beer. Biosens Bioelectron. 2000;15(5-6):257-63.
[17] Clark LC Jr. A family of polarographic enzyme electrodes and the measurement of alcohol. Biotechol Bioeng. 1972; 3:377-94.
[18] Belghith H, Romette JL, Thomas D. An enzyme electrode for on-line determination of ethanol and methanol. Biotechnol Bioeng. 1987;30(9):1001-5.
[19] Korpan YaI, Dzyadevich SV, Arkhipova VN, Gonchar MV, Gibson TD, Jaffrezic-Renault N, Martelet C, Soldatkin AP. Enzyme-based electrochemical sensors for formaldehyde detection. Sensors and Materials. 2000; 12(2):79-86.
[20] Wollenberger U, Scheller FW, B?hmer A, Passarge M, M?ller H-G. A specific enzyme electrode for l-glutamate-development and application. Biosensors.1989;4(6):381–91.
[21] Simonian AL, Rainina EI, Wild J, Fitzpatrick PF. A Biosensor for L-tryptophan determination based on recombinant pseudomonas savastanoi tryptophan-2-monooxygenase. Anal Lett. 1995;28(10):1751–61.
[22] Saurina J, Hern?ndez-Cassou S, Alegret S, F?bregas E. Amperometric determination of lysine using a lysine oxidase biosensor based on rigid-conducting composites. Biosens Bioelectron. 1999;14(2):211-20.
[23] Vrbova E, Marek M, Ralys E. Biosensor for determination of L-lysin. Anal Chim Acta. 1992; 270(1): 131-6.
[24] Niu J, Lee JY. Renewable-surface graphite–ceramic enzyme sensors for the determination of hypoxanthine in fish meat. Anal Commun. 1999;36(3):81–3.
[25] Eggins BR. Biosensors: an introduction. Chichester-Stuttgart: John Wiley and Sons and Teubner, 1996: 31-51.
[26] Dziadevich SV, Doldatkin AP, Rossokhaty? VK, Shram NF, Shul'ga AA, Strikha VI. [Amperometric enzyme biosensor with a glucose oxidase-polyaniline membrane]. Ukr Biokhim Zh. 1994;66(3):54-60.
[27] Lorenzo E, Pariente F, Hern?ndez L, Tobalina F, Darder M, Wu Q, et al. Analytical strategies for amperometric biosensors based on chemically modified electrodes. Biosens Bioelectron. 1998;13(3-4):319–32.
[28] Albery WJ., Bartlett PN, Cass AEG, Sim KW. Amperometric enzyme electrodes. Part IV. An enzyme electrode for ethanol. J Electroanal Chem. 1987;218(1-2):127-34.
[29] Miyamoto S, Murakami T, Saito A, Kimura J. Development of an amperometric alcohol sensor based on immobilized alcohol dehydrogenase and entrapped NAD+. Biosens Bioelectron. 1991;6(7):563–7.
[30] Blaedel WJ, Engstrom RC. Reagentless enzyme electrodes for ethanol, lactate, and malate. Anal chem.1980;52(11):1691–7.
[31] Jaraba P, Ag??? L, Y??ez-Sede?o P, Pingarr?n J. NADH amperometric sensor based on poly(3-methylthiophene)-coated cylindrical carbon fiber microelectrodes: application to the enzymatic determination of L-lactate. Electrochim Acta. 1998;43(23):3555–65.
[32] Bartlett PN, Whitaker RG. Strategies for the development of amperometric enzyme electrodes. Biosensors. 1987;3(6):359–79.
[33] Albery WJ, Bartlett PN, Cass AE. Amperometric enzyme electrodes. Philos Trans R Soc Lond B Biol Sci. 1987;316(1176):107-19.
[34] Wilson GS, Thevenot DR. Unmediated amperometric enzyme electrodes. Biosensors. A practical approach. Ed. A. E. G. Cass. Oxford: Oxford Univ. press, 1990: 1-17.
[35] Gajovic N, Warsinke A, Scheller FW. A novel multienzyme electrode for the determination of citrate. J Chem Technol Biotechnol. 1995;63(4):337–44.
[36] Matsumoto K, Tsukatani T, Okajima Y. Amperometric flow-injection determination of citric acid in food using free citrate lyase and co-immobilized oxalacetate decarboxylase and pyruvate oxidase. Electroanalysis . Wiley-Blackwell; 1995;7(6):527–30.
[37] Scheller F, Karsten C. A combination of invertase reactor and glucose oxidase electrode for the successive determination of glucose and sucrose. Anal Chim Acta.1983;155:29–36.
[38] Filipiak M, Fludra K, Go?cimi?ska E. Enzymatic membranes for determination of some disaccharides by means of an oxygen electrode. Biosens Bioelectron. 1996;11(4):355-64.
[39] V?radi M, Ad?nyi N, Nagy G, Rezessy-Szab? J. Studying the bienzyme reaction with amperometric detection for measuring maltose. Biosens Bioelectron. 1993;8(6):339-45.
[40] Pfeiffer D, Ralis EV, Makower A, Scheller FW. Amperometric bi-enzyme based biosensor for the detection of lactose--characterization and application. J Chem Technol Biotechnol. 1990;49(3):255-65.
[41] Xu Y, Guilbault GG, Kuan SS. Fast responding lactose enzyme electrode. Enzyme Microb Technol. 1990;12(2):104–8.
[42] Larsson N, Ruzgas T, Gorton L, Kokaia M, Kissinger P, Cs?regi E. Design and development of an amperometric biosensor for acetylcholine determination in brain microdialysates. Electrochim Acta. 1998;43(23):3541–54.
[43] Guilbault GG, Nanjo M. A phosphate-selective electrode based on immobilized alkaline phosphatase and glucose oxidase. Anal Chim Acta. 1975;78(1):69-80.
[44] Campanella L, Pacifici F, Sammartino MP, Tomassetti M. A new organic phase bienzymatic electrode for lecithin analysis in food products. Bioelectrochem Bioenerg. 1998;47(1):25–38.
[45] Albery WJ, Hillman AR. Transport and kinetics in modified electrodes. J Electroanal Chem Interfacial Electrochem. 1984;170(1-2):27–49.
[46] Li Q-S, Ye B-C, Liu B-X, Zhong J-J. Improvement of the performance of H2O2 oxidation at low working potential by incorporating TTF-TCNQ into a platinum wire electrode for glucose determination. Biosens Bioelectron. 1999;14(3):327–34.
[47] Albery WJ, Craston DH. Amperometric enzyme electrodes: Theory and experiment. Biosensors. Fundamentals and Applications. Eds A. P. F. Turner, I. Karube, G. S. Wilson. Oxford: Oxford Univ. press, 1987: 180-210.
[48] Cosnier S. Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films. A review. Biosens Bioelectron. 1999;14(5):443–56.
[49] Albery WJ, Bartlett PN. An organic conductor electrode for the oxidation of NADH. J Chem Soc Chem Commun. 1984; 4:234-6.
[50] Albery WJ, Bartlett PN, Bycroft M, Craston DH, Driscoll BJ. Amperometric enzyme electrodes. J Electroanal Chem Interfacial Electrochem. 1987;218(1-2):119–26.
[51] Albareda-Sirvent M, Merko?i A, Alegret S. Configurations used in the design of screen-printed enzymatic biosensors. A review. Sensors and Actuators B: Chemical. 2000;69(1-2):153–63.
[52] Eddowes M. Theoretical methods for analysing biosensor performance. Biosensors. A practical approach. Ed. A. E. G. Cass. Oxford: Oxford Univ. press, 1990: 211-63.
[53] Thevenot DR, Sternberg R, Coulet PR, Laurent J, Gautheron DC. Enzyme collagen membrane for electrochemical determination of glucose. Anal Chem. 1979;51(1):96–100.
[54] Reddy SM, Vadgama PM. Membranes to improve amperometric sensor characteristics. Handbook of biosensors and electronic noses: medicine, food, and environment. Ed. E. Kress-Rogers. New York: CRC press, 1997: 111-35.
[55] Cass AE, Davis G, Francis GD, Hill HA, Aston WJ, Higgins IJ, Plotkin EV, Scott LD, Turner AP. Ferrocene-mediated enzyme electrode for amperometric determination of glucose. Anal Chem. 1984;56(4):667-71.
[56] Spinas GA, Andres UR, Heinzinger T, Berger W. [Evaluation of Pen meters for blood glucose analysis in ambulatory diabetics]. Schweiz Med Wochenschr. 1990;120(5):125-8.
[57] Bradley J, Schmid RD. Optimisation of the biosensor for in situ fermentation monitoring of glucose concentration. Biosens Bioelectron. 1991;6(8):669–74.
[58] Stredansky M, Pizzariello A, Stredanska S, Miertus? S, Miertus? S. Determination of D-fructose in foodstuffs by an improved amperometric biosensor based on a solid binding matrix. Anal Commun. 1999;36(2):57–61.
[59] Xie X, Kuan SS, Guilbault GG. A simplified fructose biosensor. Biosens Bioelectron. 1991;6(1):49–54.
[60] White SF, Higgins IJ, DCosta E, Bradley J, Schmid RD. Amperometric detection of lactate: a comparison between mediated and platinised carbon electrodes. Biosensors: Fundamental technologies and application. Eds F. Scheller, R. D. Schmid. Weinheim: VCH Publishers, 1992: 403-21.
[61] Kulys J, Schuhmann W, Schmidt H-L. Carbon-paste electrodes with incorporated lactate oxidase and mediators. Anal Lett. 1992;25(6):1011–24.
[62] Hale PD, Lee H-S, Okamoto Y, Skotheim TA. Glutamate biosensors based on electrical communication between L-glutamate oxidase and a flexible redox polymer. Anal Lett. 1991;24(3):345–56.
[63] Vahjen W, Bradley J, Bilitewski U, Schmid RD. Mediated enzyme electrode for the determination of L-glutamate. Anal Lett. 1991;24(8):1445–52.
[64] Dempsey E, Wang J, Wollenberger U, Ozsoz M, Smyth MR. A lysine dehydrogenase-based electrode for biosensing of L-lysine. Biosens Bioelectron. 1992;7(5):323-7.
[65] Yon Hin BFY, Lowe CR. Catalytic oxidation of reduced nicotinamide adenine dinucleotide at hexacyanoferrate-modified nickel electrodes. Anal Chem. 1987;59(17):2111–5.
[66] Holt PJ, Stephens LD, Bruce NC, Lowe CR. An amperometric opiate assay. Biosens Bioelectron. 1995 Summer;10(6-7):517-26.
[67] Pat USA 3,838,033. Enzyme electrode. W. Mindt, P. Racine, P. Schlapfer. Publ. 1974.
[68] Cass AE, Davis G, Francis GD, Hill HA, Aston WJ, Higgins IJ, Plotkin EV, Scott LD, Turner AP. Ferrocene-mediated enzyme electrode for amperometric determination of glucose. Anal Chem. 1984;56(4):667-71.
[69] Dicks JM, Hattori S, Karube I, Turner AP, Yokozawa T. Ferrocene modified polypyrrole with immobilised glucose oxidase and its application in amperometric glucose microbiosensors. Ann Biol Clin (Paris). 1989;47(10):607-19.
[70] Rosen-Margalit I, Rishpon J. Novel approaches for the use of mediators in enzyme electrodes. Biosens Bioelectron. 1993;8(6):315-23.
[71] Marcus RA, Sutin N. Electron transfer in inorganic, organic and biological systems. Adv Chem. 1991. 228;1-13.
[72] Marcus RA, Sutin N. Electron transfers in chemistry and biology. Biochim Biophys Acta.1985;811(3):265–322.
[73] Yeh P, Kuwana T. Reversible electrode reaction of cytochrome C. Chem Lett. 1977;(10):1145–8.
[74] Eddows MJ, Hill HAO. Novel method for the investigation of the electrochemistry of metalloproteins: cytochrome C. J Chem Soc Chem Commun. 1977; 21: 770-2.
[75] Niki K, Yagi T, Inokuchi H, Kimura K. Electrochemical behavior of cytochrome c3 of Desulfovibrio vulgaris, strain Miyazaki, on the mercury electrode. J Am Chem Soc. 1979;101(12):3335–40.
[76] Armstrong FA, Hill HAO, Walton NJ. Direct electrochemistry of redox proteins. Acc Chem Res. 1988;21(11):407–13.
[77] Armstrong FA. Dynamic electrochemistry of iron—sulfur proteins. Adv Inor Chem.1992;117–63.
[78] Tarasevich MR, Yaropolov AI, Bogdanovskaya VA, Varfolomeev SD. 293 - Electrocatalysis of a cathodic oxygen reduction by laccase. Bioelectrochem Bioenerg. 1979;6(3):393–403.
[79] Berezin IV, Varfolomeev SD, Lomonosov MV. Principles of Bioelectrocatalysis. Enzyme Engineering. 1980;95–100.
[80] Varfolomeev SD, BErezin IV. Bioelectrocatalysis: accelerating electrode reactions with enzymes. Advances in Physical Chemistry. Ed. YaM. Kolotyrkin. M: Mir, 1982: 60-95.
[81] Lee C-W, Gray HB, Anson FC, Malmstr?m BG. Catalysis of the reduction of dioxygen at graphite electrodes coated with fungal laccase A. J Electroanal Chem Interfacial Electrochem. 1984;172(1-2):289–300.
[82] Yaropolov AI, Malovik B, Varfolomeev SD, Berezin IV. Bioelectrocatalysis. Direct electron transfer from the active site from peroxidase to the electrode. Dokl Akad Nauk SSSR. 1979; 249: 1399-1402.
[83] J?nsson G, Gorton L. An electrochemical sensor for hydrogen peroxide based on peroxidase adsorbed on a spectrographic graphite electrode. Electroanalysis. 1989;1(5):465–8.
[84] Wollenberger U, Bogdanovskaya V, Bobrin S, Scheller F, Tarasevich M. Enzyme electrodes using bioelectrocatalytic reduction of hydrogen peroxide. Anal Lett. 1990;23(10):1795–808.
[85] Zhao J, Henkens RW, Stonehuerner J, O’Daly JP, Crumbliss AL. Direct electron transfer at horseradish peroxidase—colloidal gold modified electrodes. J Electroanal Chem. 1992;327(1-2):109–19.
[86] Ho WO, Athey D, McNeil CJ, Hager HJ, Evans GP, Mullen WH. Mediatorless horseradish peroxidase enzyme electrodes based on activated carbon: potential application to specific binding assay. J Electroanal Chem. 1993;351(1-2):185–97.
[87] Ruzgas T, Gorton L, Emn?us J, Marko-Varga G. Kinetic models of horseradish peroxidase action on a graphite electrode. J Electroanal Chem. 1995;391(1-2):41–9.
[88] McNeil CJ, Athey D, Ho WO. Direct electron transfer bioelectronic interfaces: application to clinical analysis. Biosens Bioelectron. 1995;10(1-2):75-83.
[89] Armstrong FA, Lannon AM. Fast interfacial electron transfer between cytochrome c peroxidase and graphite electrodes promoted by aminoglycosides: novel electroenzymic catalysis of hydrogen peroxide reduction. J Am Chem Soc . 1987;109(23):7211–2.
[90] Paddock RM, Bowden EF. Electrocatalytic reduction of hydrogen peroxide via direct electron transfer from pyrolytic graphite electrodes to irreversibly adsorbed cytochrome c peroxidase. J Electroanal Chem Interfacial Electrochem. 1989;260(2):487–94.
[91] Scott DL, Paddock RM, Bowden EF. The electrocatalytic enzyme function of adsorbed cytochrome c peroxidase on pyrolytic graphite. J Electroanal Chem. 1992;341(1-2):307–21.
[92] Degani Y, Heller A. Direct electrical communication between chemically modified enzymes and metal electrodes. I. Electron transfer from glucose oxidase to metal electrodes via electron relays, bound covalently to the enzyme. J Phys Chem. 1987;91(6):1285–9.
[93] Heller A. Electrical wiring of redox enzymes. Acc Chem Res.1990;23(5):128–34.
[94] Gregg BA, Heller A. Cross-linked redox gels containing glucose oxidase for amperometric biosensor applications. Anal Chem. 1990;62(3):258-63.
[95] Heller A. Electrical connection of enzyme redox centers to electrodes. J Phys Chem. 1992;96(9):3579–87.
[96] Katz E, Heleg-Shabtai V, Willner B, Willner I, B?ckmann AF. Electrical contact of redox enzymes with electrodes: novel approaches for amperometric biosensors. Bioelectrochem Bioenerg. 1997;42(1):95–104.
[97] Schmid RD, Karube I. Biosensors and Bioelectronics. Biotechnology. Eds H. J. Rehm, G. Reed. Weinheim: VCH Verlagsgesellschaft, 1988. Vol. 6b: 317-65.
[98] MediSense Inc. Blood Glucose Sensor Electrodes. Product Information. New York, 1990.
[99] Riklin A, Katz E, Willner I, Stocker A, B?ckmann AF. Improving enzyme-electrode contacts by redox modification of cofactors. Nature. 1995;376(6542):672-5.