Biosensors: Past, Present and Future
Professor Anthony P F Turner
© Cranfield University 1996
This paper reviews the principal milestones in the history of biosensor development. The current success of glucose biosensors is attributed to the extraordinary demands of diabetes and the ability of biosensors to offer a convenient, hygienic and compact method of personal monitoring. Biosensors offer enormous potential to detect a wide range of analytes in health care, the food industry and environmental monitoring. Five key areas of development needed to facilitate the widespread adoption of this technology are identified and Cranfield's work under each of these topics is briefly discussed.
The term biosensor has been variously applied to a number of devices either used to monitor living systems or incorporating biotic elements. A recent IUPAC committee has been trying to unravel a literature that, at one time or another, has used the term to describe a thermometer, a mass spectrometer, daphnia in pond water, electrophysiology equipment, chemical labels for imaging and ion-selective electrodes. The consensus, however, is that the term should be reserved for use in its modern context of a sensor incorporating a biological element such as an enzyme, antibody, nucleic acid, microorganism or cell; this decision is readily endorsed by a cursory examination of a data base such as Chemabs. For the purposes of this article then, a biosensor will be defined as a compact analytical device incorporating a biological or biologically-derived sensing element either integrated within or intimately associated with a physicochemical transducer. The usual aim of a biosensor is to produce either discrete or continuous digital electronic signals which are proportional to a single analyte or a related group of analytes(1).
The above clarification of scope allows us to identify clearly Professor Leland C Clark Jnr. as the father of the biosensor concept. In 1956, Clark published his definitive paper on the oxygen electrode (2). Based on this experience and addressing his desire to expand the range of analytes that could be measured in the body, he made a landmark address in 1962 at a New York Academy of Sciences symposium in which he described how "to make electrochemical sensors (pH, polarographic, potentiometric or conductometric) more intelligent" by adding "enzyme transducers as membrane enclosed sandwiches". The concept was illustrated by an experiment in which glucose oxidase was entrapped at a Clark oxygen electrode using dialysis membrane. The decrease in measured oxygen concentration was proportional to glucose concentration. In the published paper (3), Clark and Lyons coined the term enzyme electrode, which many reviewers have mistakenly attributed to Updike and Hicks4, who expanded on the experimental detail necessary to build functional enzyme electrodes for glucose. Guilbault and Montalvo (5) were the first to detail a potentiometric enzyme electrode. They described a urea sensor based on urease immobilised at an ammonium-selective liquid membrane electrode. Clark's ideas became commercial reality in 1975 with the successful re-launch (first launch 1973) of the Yellow Springs Instrument Company (Ohio) glucose analyser based on the amperometric detection of hydrogen peroxide. This was the first of many biosensor-based laboratory analysers to be built by companies around the world.
The use of thermal transducers for biosensors was proposed in 1974 and the new devices were christened thermal enzyme probes(6) and enzyme thermistors (Mosbach)(7), respectively. The biosensor took a further fresh evolutionary route in 1975, when Divis (8) suggested that bacteria could be harnessed as the biological element in microbial electrodes for the measurement of alcohol. This paper marked the beginning of a major research effort in Japan and elsewhere into biotechnological and environmental applications of biosensors. Lubbers and Opitz (9) coined the term optode in 1975 to describe a fibre-optic sensor with immobilised indicator to measure carbon dioxide or oxygen. They extended the concept to make an optical biosensor for alcohol by immobilising alcohol oxidase on the end of a fibre-optic oxygen sensor (10). Commercial optodes are now showing excellent performance for in vivo measurement of pH, pCO2 and pO2, but enzyme optodes are not yet widely available. In 1976, Clemens et al.(11) incorporated an electrochemical glucose biosensor in a bedside artificial pancreas and this was later marketed by Miles (Elkhart) as the Biostator. Although the Biostator is no longer commercially available, a new semi-continuous catheter-based blood glucose analyser has recently been introduced by VIA Medical (San Diego). In the same year, La Roche (Switzerland) introduced the Lactate Analyser LA 640 in which the soluble mediator, hexacyanoferrate, was used to shuttle electrons from lactate dehydrogenase to an electrode. Although this was not a commercial success at the time, it turned out in retrospect to be an important forerunner of a new generation of mediated-biosensors and of lactate analysers for sports and clinical applications. A major advance in the in vivo application of glucose biosensors was reported by Shichiri et al.(12) who described the first needle-type enzyme electrode for subcutaneous implantation in 1982. Companies are still pursuing this possibility, but no device for general use is available yet. The idea of building direct immunosensors by fixing antibodies to a piezoelectric or potentiometric transducer had been explored since the early 70's, but it was a paper by Liedberg et al. (13) that was to pave the way for commercial success. They described the use of surface plasmon resonance to monitor affinity reactions in real time. The BIAcore (Pharmacia, Sweden) launched in 1990 is based on this technology. In 1984, we published a much cited paper on the use of ferrocene and its derivatives as an immobilised mediator for use with oxidoreductases (14) in the construction of inexpensive enzyme electrodes. This formed the basis for the screen-printed enzyme electrodes launched by MediSense (Cambridge, USA) in 1987 with a pen-sized meter for home blood-glucose monitoring. The electronics were redesigned into popular credit-card and computer-mouse style formats, and MediSense's sales showed exponential growth reaching US$175 million by 1996 when they were purchased by Abbott. Boehringer Mannheim and Bayer now have competing mediated biosensors and the combined sales of the three companies dominate 85% of the world market for biosensors and are rapidly displacing conventional reflectance photometry technology for home diagnostics.
Academic journals now contain descriptions of a wide variety of devices exploiting enzymes, nucleic acids, cell receptors, antibodies and intact cells, in combination with electrochemical, optical, piezoelectric and thermometric transducers1, (15). Within each permutation lies a myriad of alternative transduction strategies and each approach can be applied to numerous analytical problems in health care (16), food and drink (17), the process industries (18), environmental monitoring (19), defence and security. Generic goals may be identified which underpin more applied biosensor programmes and tackle some of the principal hurdles to the more widespread adoption of biosensor technology for analysis. The design of integrated systems, approaches to patterning sensitive elements and methods to improve the sensitivity, stability and selectivity of biosensors are key areas.
Biosensor technologists strive for the simplest possible solution to measurement in complex matrices. While notable success has been achieved with individual sensors, pragmatic solutions to many problems involve the construction of a sensor system in which the carefully optimised performance of the sensor is supported by associated electronics, fluidics and separation technology. In process monitoring, for example, the process must remain inviolate while the sensor frequently requires protection from the process and its products. We have designed an integrated system (20) comprising a rotary aseptic sampling system with flow-injection analysis incorporating a reusable, screen-printed electrode. The enzyme electrode utilised glucose oxidase immobilised in a hydrophilic gel and detected hydrogen peroxide at a catalytic electrode made of rhodinised carbon. While the enzyme electrode alone exhibited enhanced stability and interference characteristics, a complete solution of the monitoring problem demanded the optimisation of the whole system. There are increasing demands for a systems orientated approach in other sectors; environmental monitoring places demands on sensor technology that in many cases are unlikely to be met by isolated sensors, and in clinical monitoring microdialysis offers a useful way forward for measurement in vivo. The sensor/sampling system biointerface is a key target for further investigation and we are using evanescent wave techniques and atomic force microscopy to further our understanding of protein interactions (21). Work on in vivo sensing systems for both glucose and lactate (22) has confirmed the effectiveness of phospholipid copolymers in improving haemocompatability. Immunosensors offer a further general example where micro separations, using for example immuno-chromatographic methods, can be coupled with electrochemical or optical detectors to yield simple dip-stick style devices combining the speed and convenience of sensors with the specificity and sensitivity of immunoassays. The advent of micromachining makes these and other hyphenated techniques amenable to such a high degree of miniaturisation that the distinction between sensor and analytical instrument becomes hazy.
The success of single analyte sensors has been followed by the formulation of arrays of sensors to offer menus relevant to particular locations or situations. The most obvious example is in critical care where commercially-available hand-held instruments provide clinicians with information on the concentration of six key analytes in blood samples and bench-top instruments on the ward can measure sixteen analytes. These instruments feature biosensors for glucose, lactate, urea and creatinine. The dual demands for increased range of analytes and decreased size are driving biosensors towards micro- or even nano-arrays. Thinking in this area is being stimulated by the demands of the pharmaceutical industry where high volume, high throughput drug screening is essential for survival. We are already working with 12 channel Mach Zhender interferommeters (23) built on 1cm2 pieces of silicon and 244 individually addressable electrodes can be fabricated on a similar area. Advanced ink-jet printing technology (24) is being used to deposit fractions of a nanolitre on three dimensional surfaces with a production line moving at 6 m/sec. In the longer term, however, arrays of a million sensors/cm2 are a realistic target. Photolithography, microcontact printing and/or self assembly techniques offer routes to high density arrays, but laser desorption is particularly promising and offers the ability to "write" proteins to surfaces with very high resolution.
Clinicians, food technologists and environmentalists all have an interest in generally increased sensitivity and limits of detection for a range of analytes. While the precise demands to meet today's requirements may be modest in these respects, few would contest the longer term benefits of reliable detection of trace amounts of various indicators, additives or contaminants. With the advent of atomic force microscopy we can consider single molecule detection in the research laboratory, but great strides have also been made with conventional sensors. Enzyme electrodes have been designed which preconcentrate the analyte of interest (25). We have reported a gas-phase microbiosensor for phenol, for example, in which polyphenol oxidase was immobilised in a glycerol gel on an interdigitated microelectrode array (26). Phenol vapour partitioned directly into the gel where it was oxidised to quinone. Signal amplification was enhanced by redox recycling of the quinone/catechol couple resulting in a sensor able to measure 30 ppb phenol. Detection limits of parts per trillion volatile organic carbons are feasible with this approach. Ultra-low detection limits are achievable with affinity sensors and electrochemical detection may be readily integrated with chromatographic techniques to yield user-friendly devices (27). In an alternative approach, double-stranded DNA may be used as a receptor element. "Sandwich"-type biosensors based on liquid-crystalline dispersions formed from DNA-polycation complexes may find application in the determination of a range of compounds and physical factors that affect the ability of a given polycation molecule to maintain intermolecular crosslinks between neighbouring DNA molecules (28). In the case of liquid-crystalline dispersions formed from DNA-protamine complexes, the lowest concentration detectable of the hydrolytic enzyme trypsin was 10-14M. Elimination of the cross links caused an increase in the distance between the DNA molecules which resulted in the appearance of an intense band in the circular dichroism spectrum and a "fingerprint" (cholesteric) texture. Work is in progress to develop mass-producible films and inexpensive instrumentation.
Arguably the most obvious disadvantage in exploiting the exquisite specificity and sensitivity of complex biological molecules is their inherent instability. Many strategies may be employed to restrain or modify the structure of biological receptors to enhance their longevity. We have recently confirmed the effectiveness of sol gels as an immobilisation matrix in an optode for glucose using simultaneous fluorescence quenching of two indicators, (2,2'-bipyridyl)ruthenium(II) chloride hexahydrate and 1-hydroxypyrene-3,6,8-trisulphonic acid. In addition to the excellent optical properties of the gel, enhanced stability of the glucose oxidase catalyst was clearly evident (29). Some desirable activities, however, remain beyond the reach of current technology. Methane monooxygenase is one such case where, despite reports of enhanced stability in the literature, the demands of hydrocarbon detection require stability far beyond that exhibited by the enzyme. In these cases it is valuable to resort to biomimicry to retain the essence of the biocatalytic activity, but to house this within a smaller and more robust structure. For example, we have developed a simple and rapid method for quantifying a range of toxic organohalides based on their electrocatalytic reaction with a metalloporphyrin catalyst. This approach can be used to measure Lindane and carbon tetrachloride (representative of haloalkane compounds) perchloroethylene (a typical haloalkene) 2,4D and pentachlorophenol (aromatics) and the insecticide DDT (30).
Improvement in the selectivity of biosensors may be sought at two levels; the interface between the transducer and the biological receptor may be made more exclusive thus reducing interference, and new receptors can be developed with improved or new affinities. The use of mediators as a strategy to improve performance in amperometric biosensors has proved extremely popular and we have continued to explore these possibilities. A recent publication (31) describes the use of pyrroloquinoline quinone as a "natural" mediator, but used with glucose oxidase in an enzyme electrode for the measurement of sugar in drinks. Alternatively, electrocatalytic detection of the products of enzymatic reactions may be enhanced by the use of chemically modified electrodes such as rhodinised (32) or hexacyanoferrate-modified (33) carbon. The latter method results in a Prussian Blue coating on the electrode which may then be used for amperometric detection of hydrogen peroxide at both oxidative and reductive potentials in enzyme electrodes for lactate (34) and glucose (33). Arguably a more elegant solution is to seek connection of the redox centre of an enzyme to an electrode via a molecular wire. Much has been published about so called "wired" enzymes, but these papers have generally been concerned with immobilised mediators on various polymer backbones. We have sought to use molecular wires in their pure sense for long distance electron transfer effected via a single molecule with delocalised electrons. Novel heteroarene oligomers, consisting of two pyridinium groups, linked by thiophene units of variable length (thienoviologens) are promising candidates for such conducting molecular wires and may be used in conjunction with self-assembly techniques to produce an insulated electrode which transfers electrons specifically along predetermined molecular paths (35). This design should produce enzyme electrodes free from electrochemical interference. Advances in computational techniques now allow us to model both electron transfer reactions and receptor binding interactions with increasing accuracy. This not only enhances our understanding of the receptor/transducer interface, but allows us to consider designing new receptors based on biological molecules. To obtain improved binding ligands for use in an optical sensor for glycohemoglogin (HbA1c), a novel synthetic peptide library composed of one million L-amino acid hexapeptides was constructed from ten amino acids using combinatorial chemistry (36). The hexapeptide library was screened against HbA1c, HbA1b, HbAF and HbA0, and selected ligands sequenced. Individual ligands or arrays of ligands in conjunction with pattern recognition techniques will be used to design a sensor with improved selectivity.
Conclusions The indications are that analytical chemistry, and sensor technology in particular, could follow the same trends as microelectronics. As chemical analysis becomes simpler and more widely available, we can expect to see a proliferation of uses in conjunction with microprocessors and telecommunications equipment. Equipment capable of acquiring data as well as processing it could find wide application in monitoring personal health, the food we eat and our environment. In order to facilitate this analytical revolution, biosensor technologists must resolve the remaining problems hindering the exploitation of biological molecules and their analogues in conjunction with microelectronics. With continued progress, we can expect the limiting factors to be shifted from technological problems to the identification of sensible exploitation of an immense analytical capability.
1) Turner, A.P.F., Karube, I. and Wilson, G.S. (1987) Biosensors: Fundamentals and Applications. Oxford University Press, Oxford. 770p.
2) Clark, L.C. Jnr. Trans. Am. Soc. Artif. Intern. Organs 2, 41-48 (1956).
3) Clark, L.C. Jnr. Ann. NY Acad. Sci. 102, 29-45 (1962).
4) Updike, S.J. and Hicks, J.P. Nature 214, 986-988 (1967).
5) Guilbault, G.G. and Montalvo, J. JACS 91, 2164-2569 (1969).
6) Cooney, C.L., Weaver, J.C., Tannebaum, S.R., Faller, S.R., Shields, D.V. and Jahnke, M. In: "Enzyme Engineering" (Eds. E.K. Pye and L.B. Wingard Jnr.) 2, 411-417. Plenum, New York. (1974).
7) Mosbach, K. and Danielsson, B. Biochim. Biophys. Acta. 364, 140-145 (1974).
8) Divis, C. Annals of Microbiology 126A, 175-186 (1975).
9) Lubbers, D.W. and Opitz, N. Z. Naturforsch. C: Biosci. 30c, 532-533 (1975).
10) Voelkl, K.P., Opitz, N. and Lubbers, D.W. Fres. Z. Anal. Chem. 301, 162-163 (1980).
11) Clemens, A.H., Chang, P.H. and Myers, R.W. Proc. Journes Ann. de Diabtologie de l'Htel-Dieu, Paris (1976).
12) Shichiri, M., Kawamori, R., Yamaski, R., Hakai, Y. and Abe, H. Lancet ii, 1129-1131 (1982).
13) Liedberg, B., Nylander, C. and Lundstrm, I. Sensors and Actuators 4, 299-304 (1983).
14) Cass, A.E.G., Francis, D.G., Hill, H.A.O., Aston, W.J., Higgins, I.J., Plotkin, E.V., Scott, L.D.L. and Turner, A.P.F. Anal. Chem. 56, 667-671 (1984).
15) Turner, A.P.F. "Advances in Biosensors", I; II; Suppl. I; III. JAI Press, London, UK, 1991; 1992; 1993; 1995.
16) Alcock, S.J. and Turner, A.P.F. IEEE Engineering in Medicine and Biology, June/July, 319 (1994).
17) Kress-Rogers, E. "Handbook of Biosensors and Electronic Noses: Medicine, Food and the Environment", CRC Press, Boca Raton, USA, 1996.
18) White, S.F. and Turner, A.P.F. In: "Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis and Bioseparation"(Eds. M C Flickinger and S W Drew). Wiley, New York, USA, 1997.
19) Dennison, M.J. and Turner, A.P.F. Biotechnol. Adv., 13, 1 (1995).
20) Tothill, I.E., Newman, J.D., White, S.F. and Turner, A.P.F. Enzyme and Microbial Technol., 1996
21) Cullen, D.C. "4th World Congress on Biosensors", 29-31 May, Bangkok, Thailand 1996. Elsevier Applied Science, Oxford, UK. p. 50.
22) Fisher, U., Alcock, S.J. and Turner, A.P.F. Biosen. Bioelectron., 10, xxiii (1995)
23) Turner, A.P.F., Newman, J.D. and RICKMAN, A. "Optics for Environmental and Public Safety", Munich, Germany, 19-23 June 1995, Europto Series, Berlin, Germany.
24) Newman, J.D., Marazza, G. and Turner, A.P.F. Anal. Chim. Acta 262, 13 (1992).
25) Saini, S. and TURNER, A.P.F. Trends Anal. Chem., 14, 304 (1995)
26) Dennison, M.J., Hall, J.M. and Turner, A.P.F. Anal. Chem., 67, 3922 (1995).
27) Alcock, S.J., White, S.F., Turner, A.P.F., Setford, S., Tothill, I.E., Dicks, J.M., Stephens, S., Hall, J.M. and Warner, P.J. British Patent Application 9416002.5, 1994.
28) Skuridin, S.G., Yevdokimov, Y.M., Efimov, V.S., Hall, J.M. and Turner, A.P.F. Biosensors Bioelectron., 11, 903 (1996).
29) Psoma, S. and Turner, A.P.F. "3rd World Congress on Biosensors", 1-3 June 1994, New Orleans, USA. Elsevier Applied Science, Oxford, UK.
30) Dobson, D.J., Turner, A.P.F. and Saini, S. Anal. Chem.
31) Loughran, M.G., Hall, J.M. and Turner, A.P.F.> Electroanal.7, 1 (1996).
32) Newman, J.D., White, S.F., Tothill, I.E. and Turner, A.P.F. Anal. Chem., 67, 4594 (1995).
33) Jaffari, S.A. and Turner, A.P.F. UK Patent Application GB9402591.3 (1994) & Biosen. Bioelectron., (1996).
34) Selkirk, J.Y., Turner, A.P.F. and Saini, S. "4th World Congress on Biosensors", 29-31 May, Bangkok, Thailand 1996. Elsevier Applied Science, Oxford, UK. p. 198.
35) Albers, W.M., Lekkala, J.O., Jeuken, L., Canters, G.W. and Turner, A.P.F. Bioelectrochem. Bioenerg. (1996).
36) Chen, B. and Turner, A.P.F. "24th Symposium European Peptide Society", Edinburgh, Scotland, September 1996.