Analyst, January 1996, Vol. 121 15N Conference Report Analytical Applications of Biosensors: February 23,1995 The analytical applications of biosensors were examined at a symposium organized by the Joint Pharmaceutical Analysis Group in collaboration with the Royal Society of Chemistry Analytical Division (East Anglia and South East Regions) and the M25 Measurement and Sensors Club on February 23, 1995. Analytical measurements are being performed more and more in situ than in the laboratory. As a result, biosensors are being developed as a subset of chemical sensors using enzymes and antibodies. The new approaches towards electrochemically- based biosensors were introduced by Professor H. A. 0. Hill (University of Oxford). Professor Hill’s team began research with the electrochemistry of the cytochrome-c.Near the surface of the molecule is the heme group which is involved in electron transfer. The development of so-called ‘promoters’ of the electron transfer marked a singular discovery. The detection and measurement of such electron transfer reactions forms the basis of amperometric biosensors. This fundamental research was followed in 1982 by key work involving glucose oxidase, using a ferrocene as the basis of an oxygen-insensitive enzyme electrode. This was developed by Professor Hill’s team and Medisense (Oxford) to form a home-test biosensor. The development of the glucose test kit for clinical use was discussed by Dr. Gordon Sanghera (Medisense). He described a biosensor as being the marriage of a biological component, i.e.a redox protein, enzyme or antibody, with a transduction system using cyclic voltammetry, which allows measurement and (if required) amplification of the signal. The market for blood glucose monitoring is worth more than $800 million worldwide with an estimated 12 million diabetics in the US alone. It was recognized that in the self-monitoring of blood glucose the major source of error came from the user and therefore there was a clear need for simpler and less user-dependent systems. Such systems should achieve less than 10% variability at glucose concentrations of 3040 milligrams per decilitre. Dr. Sanghera suggested, that a biosensor which uses a direct reaction is a simpler device and should have significant advantages over products generally available which use a colorimetric secondary reaction using a dye system.Having developed a commercial test kit for blood glucose, research and development work is continuing to produce similar kits for cholesterol, alcohol, and paracetamol measurement. The subject of the presentation given by Dr. Elizabeth Hall (University of Cambridge) was the use of optics and optrodes for biosensing. Dr. Hall explained how fibre optic technology may be used for biosensors with measurement being made at the interface. The recognition layer, i.e. that layer which is specific for the substance of interest, may be applied directly to the surface of the sensor or alternatively indirectly onto a modulation layer, i.e. silane. Dr. Hall said a small change in the modulation layer may cause a large change in measurement which in turn leads to calibration difficulties.In recent years a polymer deposition process has been developed for the biorecognition molecules, and photolithography allows depo- sition of different layers. Where the biorecognition molecules are antibodies, Dr. Hall explained that the amount of antibody was important for sensitivity of the biosensor and if applied directly onto the surface of the sensor it was difficult to manipulate the concentration and density on the surface. Dr. Hall discussed other uses for optical sensors aside from pharmaceutical analysis and explained how they were suitable for measuring air quality and in particular for the determination of nitrogen oxides. The practical applications of biosensors in the pharmaceut- ical industry was discussed by Dr.Jennifer Hall (Cranfield University). Within the pharmaceutical industry, biosensors have a wide range of uses, which include research and development; process monitoring and control; quality control; and monitoring of the work environment. Within research and development two key areas were of particular importance; the analysis of biomolecular interactions and the monitoring of the physiological effects of a drug, hormone or other ligand. Of the bimolecular interactions which may be investigated, Dr. Hall highlighted antigen-antibody and hormone-receptor inter- actions and a very elegant technology for the monitoring of these, surface plasmon resonance (SPR). This technique detects the changes in the refractive index of the surface layer of a solution in contact with a sensor chip.This sensor chip being a very thin metallic strip over which there is a hydrogel material to which the biomolecules are attached. Changes in refractive index can be caused by a variation of the mass on the surface of the chip due to interactions of biomoIecules. A number of SPR instruments are available from which various types of informa- tion may be obtained including specificity of binding, affinity, kinetics, and relative binding pattern. Applications of the SPR system which have recently been published include the detection and measurement of cyclosporin and peptide ana- logues. The recent work undertaken at Cranfield in the field of DNA liquid crystals was discussed by Dr. Hall. She emphasized the work undertaken by herself and her co-workers, particularly Professor A.P. F. Turner (in collaboration with Professor Y. Yevdokiniov, Institute of Molecular Biology in Moscow). Deoxyribonucleic acid (DNA) liquid crystals are based on the formation of cholesteric liquid-crystalline dispersions of DNA which have anomalous optical activity. Different optical properties are associated with ordered and non-ordered packing and only linear double-stranded DNA undergo ordered packing. This optical activity may then be measured by circular dichroism or polarization microscopy. However, they were working towards a simpler instrument for monitoring the optical properties which could be used as an analytical tool for measuring compounds which may in quantifiable manner, disrupt, these DNA liquid-crystalline dispersions. This was exciting new work which Dr.Hall felt could lead to the development of new biosensors and instrumentation. Moving on to methodology for the monitoring of physiological interactions, Dr. Hall introduced the technology of the light addressable potentiometric sensor (LAPS) device. She ex- plained the LAPS system was based on an insulated semi- conductor device that responds to surface potentials at an electrolyte/solid interface. Based on this technology a com- mercially available microphysiometer had recently been used to measure the extracellular proton flux from isolated gastric gland; the in vitro analyses of ocular irritancy; and the activity of muscarinic receptors. The particular uses of biosensors in process monitoring and control has also been the subject of research at Cranfield.In situ monitoring and automated off-line monitoring, for example in fermentation processes, have been examined. In the area of pharmaceutical quality control the analysis of DNA contamination can cause significant problems.16N Analyst, January 1996, Vol. 121 The use of LAPS technology had been applied in this area and using this technique it had been reported that 2 picograms of DNA could be detected within a couple of hours. Concluding her presentation, Dr. Hall turned to gas- and vapour analysis in the control of the workplace environment. Over the past few years Dr. Hall and her colleagues have been developing biosensors for the direct measurement of gases and vapours such as phenol and sulfur dioxide, which they believe will lead to the development of a range of gas sensors including personal monitors.Professor Brian Birch (University of Luton) introduced the subject of disposable and permanent biosensors based on thick- film technology. About ten years ago thick-film technology was identified as a valuable means of producing biosensors using processes taken from the electronics industry based on precision screen printing and the furnace firing of inks containing metals. Using this technique, a variety of precise, repeatable sensors could be manufactured which could be used particularly for electrochemically based devices. The capillary fill device (CFD) which consists of two parallel plates separated by a small air gap has been used to produce a generic range of disposable sensors.Liquid sample is drawn into the device by capillary action where it reacts with solid materials deposited in the device during manufacture. This technique makes the CFD an ideal device for the rapid and accurate sampling of liquids stated Professor Birch, although he warned that high viscosity liquids and air bubbles can affect the electrode causing false results, unless a thin layer of surfactant is sprayed to both internal surfaces. Sensors are available for the measurement of various parameters using electrochemical techniques such as potentio- metry, controlled potential coulometry and controlled current coulometry. The potentiometric CFD for example, is designed to measure the redox potentials of a solution and may be used to determine the concentration of a species in the sample solution.This type of sensor consists of a working redox electrode usually made of carbon or gold and of a solid reference silver/ silver halide electrode. It is the manufacture of the reference electrode which is an important part of the CFD concept emphasised Professor Birch. They may be manufactured either by electrochemical means or by the chemical oxidation of silver in the presence of chloride ions. In either case electrodes of equally reliable results are produced. The coulometric CFD utilizes the measurement of the charge produced by an electrochemical reaction at the surface of an electrode, where the potential of the electrode is kept constant, or varied in controlled manner. In principle three electrodes are required for coulometric experiments, these being a reference electrode, a working electrode and a counter electrode which used to complete the electrical circuit.In this case it is important for the working electrode to be relatively inert and that it allows only electron transfer between the solution and the electrode. In all types of CFD three of the important kinetic rates in electro- chemical reactions are the rate of the heterogeneous electron- transfer process; the rate of diffusion of the electrode reactants and products; and the rate of any chemical reaction coupled to the electron transfer. However, in the simplest electrode processes and experiments only the diffusional event needs to be considered. Through time, sideways diffusion effects can occur as molecules from the outer solution penetrate further in from the edges of the electrode.To counter this effect (which can distort the results) a guard electrode has been developed which surrounds the working electrode. It is maintained at the same potential as the working electrode although on separate electrical circuit. Since the CFD had been developed it had been used for a variety of measurements including glucose in blood, amino acids as well as nitrate and trace heavy metals. Professor Birch concluded his presentation by considering biosensors in its widest interpretation and discussed the role of permanent sensors in the water industry. This has been undertaken as a joint project between Siemens and the University of South- ampton and has led to the development of an integrated solid- state device for the measurement of temperature, conductivity, pH and dissolved oxygen in the monitoring of drinking and wastewater.Thus far, he said, these sensors had proved to be robust, have remained free from major fouling and have a working life in excess of one month. The success of this project has now led to a second project being undertaken with the approval of the Department of Trade and Industry for the development of sensors to measure chlorine, trace metals and ammonia. Biosensors have proved an attractive area for basic research in recent years and it is only with the recognition of an imperative to generate a viable measurement capability has research shifted to addressing practical and pragmatic issues. This was the belief of Professor Pankaj Vadgama (University of Manchester) in his presentation on the optimization of bio- sensors in the hospital environment. It was important to remember that there existed not only a host of competing technologies, but also high expectations about the de-skilling of analysis. Biosensors clearly provided technological benefits in that they promise simplified construction, ready miniaturization and direct electrical read-out.However, the end-user in hospital requires ease of operation independent of any measurement principle, together with low cost and reliability. Professor Vadgama highlighted the near-unique ability of biosensors to measure optically-opaque samples and a capability for con- tinuous measurement, which can confer a competitive ad- vantage.In the hospital environment the measurement of samples at the bedside is an attractive proposition, which would overcome the problems of sample storage and stability as well as effecting a fast turn-around time. The closer monitoring with biosensors would considerably aid the understanding of bio- chemical variability during chronic disease states which could lead to improved medical treatment. The ability of biosensors to provide real-time continuous monitoring could be of immense value in the acutely ill, diabetic or those receiving intravenous nutrition. Professor Vadgama identified the considerable bene- fits which could arise from the implantation of miniaturized biosensors at selected locations in the body to provide organ specific biochemical information.He believed differences existed between tissues and blood for many metabolites and drugs, and knowledge of local concentrations in specific organs may provide especially valuable insights for tailoring therapy. Although the most successful approach in the development of biosensors had involved electrochemical devices, the major challenge remains one of translating some interesting or novel chemical/transduction concept into a practical system capable of operating in blood, tissue or urine. He suggested that it appeared membranes such as those of microporous poly- carbonate, isopropyl myristate incorporated porous supports, or diamond-like carbon, provided the requisite generic technology for interfacing biosensors. These confer improved biocompati- bility; extended linear range; selectivity against interferent species (e.g., platelets and fibrin); a non-toxic/non-immuno- genic interface; and mechanical stability. In vivo monitoring requires not only the special adaptation of biosensors in terms of miniaturization and biocompatibility, he states, but must also be fail-safe. Concluding his presentation Professor Vadgama stressed that biosensors were a potentially powerful tool for biochemical monitoring in the hospital environment, but the realization of their potential required a close appreciation of the materials required for interfacing needs. The institution of an integrated approach that combines engineering with chemistry in both academic and industrial effort is vital if devices are to be of practical value in hospital medicine. Dr. Peter Harrowing Bristol Royal Infirmary Bristol, UK