Biosensom & Bioel~mnicr 6 (1991) 183-191
Optical immunosensing systems meeting the market needs G. A. Robinson Serono Diagnostics Ltd, 21 Woking Business Park, Albert Drive, Woking, Surrey GU21 5JY, UK (Received 4 May 1990, accepted 14 February 1991)
Ahstr&r Optical immunosensors and sensing systems are biosensors which produce a quantitative measure of the amount of antibody, antigen or hapten present in a complex sample such as serum or whole blood. The market needs for such devices and their associated instrumentation are reviewed. A brief history of the development of optical immunosensors is presented and the performance of the most well-developed optical immunosensors for meeting these market needs is reviewed. One device, the fluorescent capillary fill device (FCFD) is reviewed in detail with respect to it fultilling the market needs for an optical immunosensor. Areas for the future development of such sensing systems are also discussed. Keywords: optical immunosensor, antibody, antigen, surface plasmon resonance, fluorescence, direct immunosensor. indirect immunosensor, evanescent field, market needs.
INTRODUCTION Ever since the pioneering work of Yalow and Berson during the 1950s (Yalow & Berson, 1959) the technique of immunoassay has been providing scientists and clinicians with precise, sensitive and specific measurements of the amount of antibody or antigen present in a sample over a wide range of analyte sizes and concentration ranges in complex media such as whole blood and other biological fluids. The immunodiagnostics industry is based upon these techniques and is now a multi-million dollar international business supplying clinicians with many thousands of test results per day. The advent of the biosensor at the beginning of the 1960s (Clark & Lyons, 1962) offered the possibility of rapid testing without the need for complex assay protocols. The major thrust of the initial research into biosensors was aimed at
enzymatic analysis. Now there are commercially available biosensors which are capable of performing such enzymatic analyses on clinical samples (e.g. the ExacTech” (Medisense Inc., Boston, MA) blood glucose sensor). Conventional immunoassays require the accurate dispensing of reagents and sample in a set sequence in order to perform the analysis and, until recently, radioisotopes have been the major signal generation system for such assays. Despite the move to non-radioactive assay labels such as enzymes, many immunoassays still require much skill in order to obtain a reliable analytical result. As one response to removing the skill from immunoassays, automated analysers for the carrying out of such assays have been developed. Another solution to the deskilling of immunoassays is the development of biosensors which exploit the immunological binding event (immunosensors). In recent years much research effort has
183 Biosen.w~& Bioelmtnmics 095~5663/91/SO3..5OQ 1991 Elsevier Science Publishers Ltd. England. Printed in Great Britain
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been directed at immunosensing techniques. Many technologies have been evaluated in attempts to produce commercially viable immunosensing systems including electrochemistry (e.g. Robinson ef al., 1986), solid state devices (e.g. Janata & Blackbum, 1984)and optical techniques (e.g. North, 1985).Whilst immunosensors based upon both electrochemistry and solid state devices have been shown to produce doseresponse curves, they frequently lack the sensitivity and/or dynamic range to be commercially useful. In order to overcome these problems many researchers have used optical techniques as the basis for immunosensors because such methods have a number of advantages including well characterised starting materials for device construction (e.g. glass, polymeric materials), sensitive techniques for interrogating the signal and rapid signal generation and reading times. Also, the rapidly expanding telecommunications industry has resulted in the performance of optoelectronic components increasing whilst their cost deceases. The development of integrated optical devices offers promise for the construction of even more sophisticated immunosensors in the future (Flanagan et al., 1988). The function of optical immunosensors and immunosensing systems is to produce a measure of the amount of an antibody, antigen or hapten present in a sample. Such systems use optical techniques to convert an immunological binding event into a measurable electrical signal and are capable of detecting a wide variety of analytes which can range in size from small haptens (e.g. the hormone metabolite estrone-3glucuronide (E3G) (molecular weight = 468)) to intact virus particles (e.g. the influenza virus (molecular weight = 178 X 106)).Non-clinical analytes such as pesticides can also be detected using immunosensors (e.g. Stewart, 1986).Theoretically at least, as long as an antibody can be raised to an analyte, an immunosensor should be capable of detecting the analyte. An optical immunosensor consists of either an antibody or an antigen immobilised onto a suitable surface. Upon incubation with the appropriate target molecule in the sample, the target molecule binds onto the immobilised immunological component and the resulting complex is interrogated optically. Changes in the optical properties of the antibody: antigen: sensor surface are a measure of the amount of target molecule present in the sample. Suitable surfaces
G. A. Robinson
include planar optical waveguides (e.g. the fluorescent capillary fill device (Badley et al., 1987)), optical fibres (e.g. Bluestein, 1989)or, in the case of surface plasmon resonance immunosensors, metallised prisms or diffraction gratings (e.g. Nylander et al., 1982 and Parry et al., 1988 respectively). Usually these systems need some form of associated instrumentation to interrogate the biologically active surface; however, immunosensors which can be read visually have been described in the literature (Giaever, 1973).
OPTICAL IMMUNOSENSORS - MARRET ADVANTAGES Immunosensors are interesting commercially because they offer a number of potential advantages over conventional assay techniques including user convenience as no wash, separation or pippetting steps are needed in order to perform the assay; they allow the possibility of performing immunoassays in remote locations (e.g. the doctor’s surgery or beside a production line); such sensors have the ability to measure simultaneously more than one analyte in a sample, and, perhaps most importantly, the use of immunosensors can decrease the time between the taking of a sample and obtaining the result of the analysis. Such speed can be of the utmost importance in critical care and process control environments where a rapid, accurate result needs to be made to allow a decision upon an appropriate course of action. However, consumers will not purchase immunosensors only for the sake of either their technological elegance or their use of leading edge technology. In order for there to be a market for such sensors they must be commercially viable in terms of unit cost per test and their ease of use as well as their sensitivity and specifwity compared to conventional immunoassay techniques. Furthermore, the instrumentation associated with the sensor must also be robust, easy to use, have low maintenance and calibration requirements and be low-cost. OPTICAL IMMUNOSENSORS - MARKET NEEDS For the clinical market, immunosensors must be capable of handling a wide variety of samples including whole blood, serum, plasma, urine and
Optical immunosensing systems - meering the market needs
saliva and the device itself should be designed in such a way that no measurement of the volume of sample applied to the device is needed. This is important both in terms of the level of skill required to operate the sensor and its ease of use as the operator should not require great expertise to use the device in order to obtain a reliable assay result. Ideally, the sensitivity of an immunosensor should be better than picomolar and the assay result must be available within 10 min of application of the sample to the sensor. In order to perform the immunoassay there must be no addition of reagents, either to the device or to the sample, prior to analysis. If reagents are required by the sensor (e.g. in the case of an indirect immunosensor) they must be packaged within the device thus making it appear reagentless in use. The biologically active components of the immunosensor must have a shelf-life of at least six months at room temperature. Any instrumentation required to interrogate the sensor has to be small and low-cost, ideally with no moving parts, thereby making it reliable and easy to maintain in the field. A more stringent requirement for the system is to incorporate some form of calibration technique within it. Calibration could take the form of a dual channel measurement (i.e. one channel for the assay and the other to run a calibrator) with the performance of the calibrator being compared with a batch calibration produced on the production line (Nakamura, 1988). This will allow for any degradation of the sensor’s performance on storage to be compensated for when calculating the assay result. Such calibration systems will be essential for the commercial success of immunosensors as the end user will need to guarantee the analytical performance of each sensor. Additionally, immunosensing systems targeted towards non-clinical markets will require the instrumentation associated with the system to be robust, portable and waterproof whilst the sensor itself must be capable of coping with extreme types of sample (e.g. samples with high or low pH or a high viscosity) without the need for any sample pretreatment. Assuming that technologies can be found which allow all the above requirements to be fulfilled, the manufacturing process for the immunosensor will also place some constraints upon the device’s design and commercial viability. The fabrication processes involved must allow for the throughput to be readily increased from the
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laboratory to industrial scale whilst remaining a low cost and high yield operation. The use of stable reagents will facilitate the increase in scale of sensor manufacture. Furthermore, the manufacturing process must allow for easy inprocess QC testing, this being important if a high yield of devices is to be obtained from the process. Also, the techniques involved in the large scale manufacture of immunosensors should not require the extensive development of new technology for their implementation as the cost of such technology will be reflected in the unit manufacturing cost and, therefore, in the profit margin available for the sensors.
OPTICAL IMMUNOSENSORS AND SENSING SYSTEMS Optical immunosensing systems fall into two categories: direct and indirect. The direct forms of sensor rely solely upon the binding between antibody and antigen to modulate the signal being measured whilst the indirect types depend on the use of a label (e.g. a fluorophore) to visualise the immunological binding event. Some of the advantages and disadvantages of direct and indirect optical sensors are summarised in Table 1. Surface plasmon resonance (SPR) is an example of a direct optical immunosensing method whilst most of the evanescent wave techniques are examples of indirect immunosensing systems as they frequently utilise fluorescent labels to allow the assay to be measured; however, such systems can still be packaged in such a way that they appear reagentless to the user. Whilst a wide variety of techniques have been examined for their applicability to immunosensor development, optical methods currently appear very attractive in terms of both their technical and commercial viability. The use of optical methods to study proteins has been known for a long time. The application of ellipsometry to such studes was described by Vroman in 1969 who used the technique to examine the exchange of plasma proteins at a solid/liquid interface. Recently there have been attempts to commercialise ellipsometerbased immunosensing systems as a result of the development of the greatly simplified IsoscopeW (Sagax Instruments) ellipsometer (Nygren et al., 1985). The first true optical immunosensor was described by Giaever in 1973. Giaever’s very
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TABLE1 Comparisons between direct and indirect optical immunosensors Direct
“Reagentless”
Instrumentation may be simpler
Sensitivity may be limited by NSB
Indirect Label improves sensitivity and selectivity Packaged to appear reagentless Label reduces NSB Normal assay formats Needs a labelled reagent
Analyte size limits the use of standard assay formats
simple device consisted of islets of indium deposited upon a suitable substrate (e.g. silicon). Protein (bovine serum albumin, BSA) was coated onto the indium by adsorption and the slide was incubated with rabbit antiserum against BSA. The appearance of the original slide was dark brown when viewed in scattered light and, after coating with BSA, the slide appeared darker; upon further incubation with anti-BSA antiserum the slide took on a still darker appearance. When used in this manner the sensor was able to detect 10lug/ml of anti-BSA protein and was also used to detect both Hepatitis B antigen and anti-Hepatitis antibodies present in human serum. The next major breakthrough in the field of optical immunosensing was the application of total internal reflection spectroscopy (IRS) to fluorescent immunoassays (Kronick & Little, 1974)which was followed by the reporting of the application of surface plasmon resonance to biosensing (Nylander et al., 1982). SURFACE PLASMON RESONANCE The phenomenon of surface plasmon resonance (SPR) was first reported at the beginning of this century (Wood, 1902) but a detailed theoretical understanding of SPR had to await the development of quantum physics. The theory of SPR is now well understood and documented (see, for example, Raether (1982)). The first applications of SPR as a sensing technique were directed towards the study of thin films deposited upon metal surfaces (e.g. Pockrand et al., 1977)with its
application of SPR to biosensing not being reported until several years later (Nylander et al., 1982). In an SPR immuno~n~r, the binding between antibody and antigen at the surface of a thin metal film alters the effective refractive index at the interface between the sample and the metal film upon which the immunological binding partner is immobilised, resulting in a change in the properties of light reflected off the metal surface. SPR, when applied to biosensing, can use one of two basic types of transducer, these transducers being either prisms coated with a thin (-55 nm thick) film of metal (this format is frequently known as the Kretschmann configuration) (e.g. Flanagan & Pantell, 1984; Daniels et al., 1988) or metallised diffraction gratings (Cullen et al., 1988; Parry et al., 1988) where the metal thickness can be very much greater (up to 150 nm thick (Robinson, 1990)). The mode of operation of SPR biosensors has been adequately described many times (e.g. Daniels et al., 1988) and will not be discussed here. The detection of both large analytes (e.g. antibodies in serum (Parry et al., 1988)) and small analytes has been demonstrated with SPR and such immunosensor systems are currently under commercial development (Frew, 1988). TOTAL INTERNAL REFLECTION SPECTROSCOPY (IRS) IRS and its role in the excitation of surface-bound fluorophores was reported in the late 1960s and early 1970s (e.g. Harrick & Loeb, 1973). Kronick
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Optkal immunosensing systems - meeting the market neea3
Photodetector I’
Bound tluorophore \
0
Optical waveguide -
. direct detection of fhofeseence Bound fluorophore
Photodetector 0-b
/
Optical waveguide -
exciytion and detection of fluorescence $$tphore
Photodetector /
\
Optical waveguide -Tt* Direct etitation fl
I/
aLLd‘ewwwent Excitation
light
detection fl
of fluorescence
Emitted light
Fig. 1. Diagrammatic representation of the methods of evanescent excitation and detection of sutjbcejluorescence.
and Little (1974) realised that the use of this method could result in a fluoroimmunoassay in which there was no need for a wash or separation step prior to reading the assay as the IRS technique would allow bound fluorophore label to be measured in the presence of the free (unbound) label. Using a fluorescently-labelled antibody, Kronick and Little were able to detect 02 pr&itre of morphine in a competition assay. There are three variations of evanescent optics which are important and involve various combinations of direct and evanescent excitation of bound fluorophore with direct or evanescent detection of the emitted fluorescence (Fig. 1). These variations are the evanescent excitation of the fluorophore with the emitted light being detected perpendicularly to the waveguide surface (e.g. Kronick & Little, 1974); excitation of the fluorophore by illuminating the fluorophore directly and collecting the emitted light via the evanescent field of the optical waveguide (e.g. Badley et al., 1987); and, thirdly, using the evanescent field to both excite the fluorophore
and collect the light emitted by the fluorophore (Bluestein, 1989). Evanescent techniques involve the use of two optically transparent media of different refractive indices. Light is introduced into the medium of greater refractive index (n 1) and is reflected off the interface between the two media, allowing the change in intensity of the light with its wavelength to be examined. At the interface between the two media all the energy associated with the light beam is not confined within the high refractive index material and some energy is lost into the medium with the lower refractive index (nz). The intensity of this energy rapidly decays away from the interface between the two media and is dissipated over a distance comparable to the wavelength of light used (this energy field is known as the evanescent field). Such techniques are well suited to the investigation of layers close to the surface of a waveguide as material which is present in the bulk of the solution (i.e. at distances greater than several hundreds of nanometres away from the waveguide surface) is not inter-
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G. A. Robinson TABLE 2 Sensitivities achieved by a range of optical immunosensing
systems
Analyte
Concentration
Assay conditions
Source
HCG E3G Digoxin Ferritin
3 ng/ml O-5ng/ml 05 q/ml 5 &ml 08 @ml
No wash No wash No wash Premixed reagents Wash step
Deacon et al. (1990) Robinson (1990) Bluestein (1989) Bluestein (1989) Sutherland et al. (1984)
w
rogated by the evanescent field. Thus, when using the evanescent field to excite fluorophores, only those molecules which are very close to the surface of the optical waveguide will be illuminated. Similarly, if collecting emitted light via the evanescent field, only light originating very close to the waveguide surface will be detected. This allows the fluorophore label which is bound by the formation of an antibody:antigen complex to be measured without the need for the removal of the unbound label. The detailed physics of the effect have been adequately described elsewhere (e.g. Harrick, 1967; Place et al., 1985). Both optical fibres and planar waveguides have been used to exploit evanescent optics for immunosensing with assays being demonstrated for human chorionic gonadotrophin (HCG) (Deacon et al., V&O), e&one-3glucuronide (Robinson, 1990), Rubella antibody (Parry et al., 1990) and IgG and methotrexate (Sutherland et al., 1984) when using planar devices and digoxin and ferritin with optical waveguides (Bluestein, 1989) (Table 2).
THE FLUORESCENT FILL DEVICE
CAPILLARY
A planar evanescent device that is undergoing commercial development is the fluorescent capillary till device (FCFD) (Badley et al., 1987) (Fig. 2). This device consists of two parallel glass plates held apart by a gap, O-1 mm wide. This narrow gap allows a sample to enter the device by capillarity with the dimensions of the device metering the volume of sample applied. The narrow gap within the FCFD also allows rapid assay times as the reagents needed for the assay only have to diffuse over a very short distance (i.e. the distance of the gap between the plates) to reach the assay’s solid phase. This means that the rates of reaction of assays performed within the
Fluorescently labelled
Opaque lid
aeaay reagents \
I Optical edge
/
\
l
eee
100 urn I
Optical wavebuide baseplate
C&ture
sap
antibody
Fig. 2. Diagrammatic representation of the fluorescent capillary filI device (FCFD).
device are limited by the kinetics of antibody/ antigen binding and not by the kinetics of diffusion. This means that the device can achieve very rapid immunoassays (e.g. an assay for E3G in urine will reach equilibrium within 5 min of sample addition (Robinson, MO)). All the reagents needed for the immunoassay are contained within the device, either, as in the case of the assay’s capture system, covalently coupled to the lower plate of the device (the baseplate), which also functions as an optical waveguide, or dosed onto the upper plate in such a way that they dissolve when sample enters the device. Upon completion of the antibody:antigen binding reaction, all the fluorophore within the FCFD is excited directly. The light arising from the bound fluorophore is discriminated from the free by the evanescent optics of the baseplate waveguide. This allows the assay to be read without the need for any wash or separation steps. The instrumentation needed to interrogate the FCFD is essentially simple and consists of a suitably filtered low-cost light source (e.g. a photographic flash tube) which is focused onto the baseplate of the device. Light leaving the optical edge of the baseplate of the device is collected via an aperture to reject the solution signal arising from the unbound fluorophore (i.e.
189
Optical immunosensing systems - meeting the market needs
strated in the FCFD are Rubella antibody in serum (Fig. 4; Parry d al., l!#O), E3G in urine (Robinson, 1990) and human chorionic gonadotrophin (Deacon et al., 1990)(Table 2). Sensitivities of 75 X lo-” and 1.1 X 10B9 moles/We have been obtained for HCG in serum and E3G in urine respectively. The FCFD is a good example of an immunosensor that meets most of the clinical market requirements as it can operate on a range of samples (e.g. whole blood, serum, plasma and urine), there is no need to measure the volume of sample applied to it as capillarity controls the filling of the device and meters the sample volume, assays can be read within 10 min of sample application and the associated instrumentation is simple and will be low-cost. Furthermore, all the reagents needed to perform the assay are contained within the device, making it appear reagentless, and no skill is required for its use. To date, a shelf-life of 6 months at 45°C for the immunosensing devices has been achieved. One important area still to be addressed for the device, however, is that ofcalibration as it still relies upon the batch calibration of the sensor during the manufacturing process.
Focussing ootice FCFD cell
Filter
/
-Low cost, white light source (e.g. photographic flash tube)
Fig. 3. Schema& representation of the instrumentation required to interrogate theJluowscent capillaryf 11device.
light emerging from the waveguide at angles greater than +44” and less than -44” to the plane of the device for serum samples) onto a photodetector. The placing of an aperture in the path of the light leaving the device allows the construction of a simple, low-cost instrument with no moving parts (Fig 3). Amongst the assays which have been demon-
Signal (units) 12
0
2
Zne n = podtive
10
8
4 serum
12
14
(mind pool:
+
l
nogatlvo serum pool
Fig. 4. Dose-tvsponse curvefor an assayfor Rubella antibody in serum pe#onned using thefluorescent capillaryfir device.
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TECHNICAL CHALLENGES FOR OPTICAL IMMUNOSENSORS Whilst optical immunosensing systems have been demonstrated which are very close to commercial reality, there are a number of areas within their development which still require technical progress to be made. The first such area is that of calibration of both the sensor and its instrument. Manufacturers will need to incorporate controls into their immunosensors so that the user has absolute confidence in the assay result. This area of work is especially important for both the quantitative and qualitative clinical applications of such devices. Enzyme-based biosensors can be calibrated by exposing them to known concentrations of the analyte of interest and comparing this data with results obtained when the sensor is incubated with sample. Unfortunately this technique is not directly applicable to immunosensom because of the irreversible nature of the antibody:antigen bond. Although such binding can be reversed (indeed, the ability to break such bonds is essential to the widespread technique of afftnity chromatography), there is always a risk of the immunological activity of the sensor degrading as a result of eluting the bound calibrator, resulting in compromise of either the sensor’s performance or the reliability of the calibration procedure. Another area that requires further development is the chemistry used to immobilise biological molecules to surfaces and the results of such chemistries on the non-specific binding (NSB) of proteins to surfaces. This is important if the ultimate sensitivity of both direct and indirect immunosensors is not to be limited by the NSB of proteins onto the active surface of the sensor. Furthermore, a rigorous understanding of the chemistries involved in both protein immobilisation and non-specific binding may result in the ability to increase the biological capacity of the reactive surface of a sensor thereby improving its performance. For certain applications (e.g. process control) the re-use of a sensor may be important, therefore a means of achieving reuse without compromising the performance of the sensor must be found. One way of addressing this problem might be to use antibodies that have a low affinity for the target antigen which will, therefore, allow the bound antigen to be readily eluted off the antibody. Such an approach may, however, compromise the
G. A. Robinson
sensitivity, specificity and speed of the sensor. Also important for some applications (e.g. certain types of process control, in-vivo sensing) is the sterility of the sensor. The problem of fabricating a sterile device without compromising the biological activity of the protein immobilised on the device is most challenging. Finally, the problems of the fluid handling associated with sensors needs careful optimisation, both to make such sample handling systems easy to use and to prevent limiting the sensor’s sensitivity by analyte depletion. When immersed in a static solution the effective volume of sample interrogated by an immunosensor is partially governed by the kinetics of mass transport of the analyte to the immunologically active surface of the sensor. Fluid handling systems will greatly increase the effective volume of sample interrogated by the sensor, resulting in increased sensitivity for the device; the penalty being the cost of the fluid handling system. The development of optical immunosensors has resulted in a greater understanding of the underlying physics of such devices and of the specific requirements for a device designed to monitor an immune reaction. With industrial organisations now undertaking the development of such devices to address specific market niches, the commercial future for optical immunosensors looks assured. To conclude, much technical progress has been made in the development of optical immunosensing systems with several technologies under intense commercial development and the next few years should see the commercialisation of at least one of these exciting analytical systems.
REFERENCES Badley, R A., Drake, R. A. L., Shanks, I. A., Smith, k M. & Stephenson, P. R. (1987). Optical biosensom for immunoassays: the fluorescence capillary till device. Phil. Trans. Roy. Sac Land., B 316,143~60. Bluestein, B. I. (1989). Antibody coated fibre optic transducers. Rvc. 2nd Int. Biosensors ‘89Symposium, New Orleans, October 1989. Clarke, L. C. & Lyons, C. (1962). Electrode systems for continuous monitoring in cardiovascular surgery. Ann. N. Y. Acad Sci., 192, 29-45. Cullen, D. C., Brown, R G. W. & Lowe, C. R (1988). Detection of immune-complex formation via surface plasmon resonance on gold coated diffraction gratings. Biosensors, 3, 21 l-25.
Optical immunosensing systems - meeting the market needs Daniels, P. B., Deacon, J. K., Eddowes, M. J. & Pedley, D. G. (1988). Surface plasmon resonance applied to immunosensing. Sens. Act, 15, 11-18. Deacon, J. K, Thomson, A M., Page, A L., Stops, J. E., Roberts, P. R, Whiteley, S. W., Attridge, J. W., Love, C. A, Robinson, G. A & Davidson, G. P. (1991). An assay for human chorionic gonadotrophin using the fluorescence capillary till device. Biosens. Bioelectmn.. 6, 193-199. Flanagan, M. T. & Pantell, R H. (1984). Surface plasmon resonance and immunosensors. EZmnicr L&&Q 20,968-70. Flanagan, M. T., Sloper, A N. & Ashworth, R H. (1988). From electronic to opto-electronic biosensors: an engineering view. Anal. Chim. Acta, 213,23-33. Frew, J. E. (1988). Biosensors in Medical Diagnostics, PJB Publications Ltd., Richmond, UK Giaever, I. (1973). The antibody:antigen reaction: a visual observation. J. Zmmunol., 110, 1424-6. Harrick, N. J. (1967). Internal Rejection Spectmscopy, Interscience, New York Ha&k, N. J. & Loeb, G. I. (1973). Multiple internal reflection spectroscopy. Anal. Chem., 45,687-91. Janata, J. & Blackbum, G. F. (1984). Immunochemical potentiometric sensors. Ann. N. Y. Acad. Sci., 428, 286-92. Kmnick, M. N. & Little, W. A (1974). A new immunoassay based on fluorescent excitation by internal reflection spectroscopy. Ptwc. Nat1 Acad. Sci. US.4, 71,4553-T Nakamura, R M. (1988). Clinical/medical applications of biosensors. kceedings of the International Biosenso~ ‘88Symposium Institute for International Research Inc., Washington DC. North, J. R (1985). Immunosensors: antibody based biosensors. TIX&S in Biotechnofogv, 3, 180-6. Nygren, B. H., Sandstrom, T., Stenberg, J. E. & Stilbert, L. B. (1985). Method and member for detecting and/or measuring the concentration of a chemical substance, US Patent 4 558 012. Nylander, C., Liedberg, B. & Lind, T. (1982/83). Gas detection by means of surface plasmon resonance. Sens. Act., 3, 79-88.
191 Parry, R, Deacon, J. K., Robinson, G. A, Skehel, J. J. & Forrest, G. C. (1988). Surface plasmon resonance immunosensors. In Rapid Methods andAutomation in Microbiology and Immunology. ed. A Balows, R C. Tilton, A TIuano. Brixia Academic Press, Brescia, pp. 641-8. Parry, R P., Love, C. A & Robinson, G. A (1990). Detection of Rubella antibody using an optical immunosensor. A fi&. Methoak 27,39-48. Place, J. F., Sutherland, R M. & Dahne, C. (1985). Optoelectronic immunosensors: a review of optical immunoassays at continuous surfaces. Biosensors. 1,321-54. Pockrand, I., Swalen, J. D., Gordon, J. G. & Philpott, M. R (1977). Surface plasmon spectroscopy of organic monolayer assemblies. Surface Science, 74, 237-44. Raether, H. (1982). Dispersion relation of surface plasmons on gold and silver gratings. Optics Comm.. 42,217-22. Robinson, G. A (1990). Optical immunosensors - an overview. Adv. BiosenL, in press. Robinson, G. A, Cole, V. M., Rattle, S. J. & Forrest, G. C. (1986). Bioelectrochemical immunoassay for human chorionic gonadotrophin in serum using an electrode-immobilised capture antibody. Biosensots, 2,45-57. Stewart, W. J. (1986). Optical assay, UK Patent Application No. 2 173 895. Sutherland, R M., Dahne, C., Place, J. F. & Ringrose, A R (1984). Optical detection of antibody-antigen reactions at a glass-liquid interface. Clin. Chem., 30, 1533-8. Vroman, L. & Adams, A L. (1969). Findings with the recording ellipsometer suggesting rapid exchange of specific plasma proteins at solid/liquid interfaces. Sut$ Sci., 16,438-46. Wood, R W. (1902). On the remarkable case of uneven distribution oflight in diffraction grating spectrum. Phil. Msg., 4, 3%-402. Yalow, R S. & Berson, S. A (1959). Assay of plasma insulin in human subjects by immunological methods. Nature 184, 1648-9.
Optical immunosensing systems meeting the market needs G. A. Robinson Serono Diagnostics Ltd, 21 Woking Business Park, Albert Drive, Woking, Surrey GU21 5JY, UK (Received 4 May 1990, accepted 14 February 1991)
Ahstr&r Optical immunosensors and sensing systems are biosensors which produce a quantitative measure of the amount of antibody, antigen or hapten present in a complex sample such as serum or whole blood. The market needs for such devices and their associated instrumentation are reviewed. A brief history of the development of optical immunosensors is presented and the performance of the most well-developed optical immunosensors for meeting these market needs is reviewed. One device, the fluorescent capillary fill device (FCFD) is reviewed in detail with respect to it fultilling the market needs for an optical immunosensor. Areas for the future development of such sensing systems are also discussed. Keywords: optical immunosensor, antibody, antigen, surface plasmon resonance, fluorescence, direct immunosensor. indirect immunosensor, evanescent field, market needs.
INTRODUCTION Ever since the pioneering work of Yalow and Berson during the 1950s (Yalow & Berson, 1959) the technique of immunoassay has been providing scientists and clinicians with precise, sensitive and specific measurements of the amount of antibody or antigen present in a sample over a wide range of analyte sizes and concentration ranges in complex media such as whole blood and other biological fluids. The immunodiagnostics industry is based upon these techniques and is now a multi-million dollar international business supplying clinicians with many thousands of test results per day. The advent of the biosensor at the beginning of the 1960s (Clark & Lyons, 1962) offered the possibility of rapid testing without the need for complex assay protocols. The major thrust of the initial research into biosensors was aimed at
enzymatic analysis. Now there are commercially available biosensors which are capable of performing such enzymatic analyses on clinical samples (e.g. the ExacTech” (Medisense Inc., Boston, MA) blood glucose sensor). Conventional immunoassays require the accurate dispensing of reagents and sample in a set sequence in order to perform the analysis and, until recently, radioisotopes have been the major signal generation system for such assays. Despite the move to non-radioactive assay labels such as enzymes, many immunoassays still require much skill in order to obtain a reliable analytical result. As one response to removing the skill from immunoassays, automated analysers for the carrying out of such assays have been developed. Another solution to the deskilling of immunoassays is the development of biosensors which exploit the immunological binding event (immunosensors). In recent years much research effort has
183 Biosen.w~& Bioelmtnmics 095~5663/91/SO3..5OQ 1991 Elsevier Science Publishers Ltd. England. Printed in Great Britain
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been directed at immunosensing techniques. Many technologies have been evaluated in attempts to produce commercially viable immunosensing systems including electrochemistry (e.g. Robinson ef al., 1986), solid state devices (e.g. Janata & Blackbum, 1984)and optical techniques (e.g. North, 1985).Whilst immunosensors based upon both electrochemistry and solid state devices have been shown to produce doseresponse curves, they frequently lack the sensitivity and/or dynamic range to be commercially useful. In order to overcome these problems many researchers have used optical techniques as the basis for immunosensors because such methods have a number of advantages including well characterised starting materials for device construction (e.g. glass, polymeric materials), sensitive techniques for interrogating the signal and rapid signal generation and reading times. Also, the rapidly expanding telecommunications industry has resulted in the performance of optoelectronic components increasing whilst their cost deceases. The development of integrated optical devices offers promise for the construction of even more sophisticated immunosensors in the future (Flanagan et al., 1988). The function of optical immunosensors and immunosensing systems is to produce a measure of the amount of an antibody, antigen or hapten present in a sample. Such systems use optical techniques to convert an immunological binding event into a measurable electrical signal and are capable of detecting a wide variety of analytes which can range in size from small haptens (e.g. the hormone metabolite estrone-3glucuronide (E3G) (molecular weight = 468)) to intact virus particles (e.g. the influenza virus (molecular weight = 178 X 106)).Non-clinical analytes such as pesticides can also be detected using immunosensors (e.g. Stewart, 1986).Theoretically at least, as long as an antibody can be raised to an analyte, an immunosensor should be capable of detecting the analyte. An optical immunosensor consists of either an antibody or an antigen immobilised onto a suitable surface. Upon incubation with the appropriate target molecule in the sample, the target molecule binds onto the immobilised immunological component and the resulting complex is interrogated optically. Changes in the optical properties of the antibody: antigen: sensor surface are a measure of the amount of target molecule present in the sample. Suitable surfaces
G. A. Robinson
include planar optical waveguides (e.g. the fluorescent capillary fill device (Badley et al., 1987)), optical fibres (e.g. Bluestein, 1989)or, in the case of surface plasmon resonance immunosensors, metallised prisms or diffraction gratings (e.g. Nylander et al., 1982 and Parry et al., 1988 respectively). Usually these systems need some form of associated instrumentation to interrogate the biologically active surface; however, immunosensors which can be read visually have been described in the literature (Giaever, 1973).
OPTICAL IMMUNOSENSORS - MARRET ADVANTAGES Immunosensors are interesting commercially because they offer a number of potential advantages over conventional assay techniques including user convenience as no wash, separation or pippetting steps are needed in order to perform the assay; they allow the possibility of performing immunoassays in remote locations (e.g. the doctor’s surgery or beside a production line); such sensors have the ability to measure simultaneously more than one analyte in a sample, and, perhaps most importantly, the use of immunosensors can decrease the time between the taking of a sample and obtaining the result of the analysis. Such speed can be of the utmost importance in critical care and process control environments where a rapid, accurate result needs to be made to allow a decision upon an appropriate course of action. However, consumers will not purchase immunosensors only for the sake of either their technological elegance or their use of leading edge technology. In order for there to be a market for such sensors they must be commercially viable in terms of unit cost per test and their ease of use as well as their sensitivity and specifwity compared to conventional immunoassay techniques. Furthermore, the instrumentation associated with the sensor must also be robust, easy to use, have low maintenance and calibration requirements and be low-cost. OPTICAL IMMUNOSENSORS - MARKET NEEDS For the clinical market, immunosensors must be capable of handling a wide variety of samples including whole blood, serum, plasma, urine and
Optical immunosensing systems - meering the market needs
saliva and the device itself should be designed in such a way that no measurement of the volume of sample applied to the device is needed. This is important both in terms of the level of skill required to operate the sensor and its ease of use as the operator should not require great expertise to use the device in order to obtain a reliable assay result. Ideally, the sensitivity of an immunosensor should be better than picomolar and the assay result must be available within 10 min of application of the sample to the sensor. In order to perform the immunoassay there must be no addition of reagents, either to the device or to the sample, prior to analysis. If reagents are required by the sensor (e.g. in the case of an indirect immunosensor) they must be packaged within the device thus making it appear reagentless in use. The biologically active components of the immunosensor must have a shelf-life of at least six months at room temperature. Any instrumentation required to interrogate the sensor has to be small and low-cost, ideally with no moving parts, thereby making it reliable and easy to maintain in the field. A more stringent requirement for the system is to incorporate some form of calibration technique within it. Calibration could take the form of a dual channel measurement (i.e. one channel for the assay and the other to run a calibrator) with the performance of the calibrator being compared with a batch calibration produced on the production line (Nakamura, 1988). This will allow for any degradation of the sensor’s performance on storage to be compensated for when calculating the assay result. Such calibration systems will be essential for the commercial success of immunosensors as the end user will need to guarantee the analytical performance of each sensor. Additionally, immunosensing systems targeted towards non-clinical markets will require the instrumentation associated with the system to be robust, portable and waterproof whilst the sensor itself must be capable of coping with extreme types of sample (e.g. samples with high or low pH or a high viscosity) without the need for any sample pretreatment. Assuming that technologies can be found which allow all the above requirements to be fulfilled, the manufacturing process for the immunosensor will also place some constraints upon the device’s design and commercial viability. The fabrication processes involved must allow for the throughput to be readily increased from the
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laboratory to industrial scale whilst remaining a low cost and high yield operation. The use of stable reagents will facilitate the increase in scale of sensor manufacture. Furthermore, the manufacturing process must allow for easy inprocess QC testing, this being important if a high yield of devices is to be obtained from the process. Also, the techniques involved in the large scale manufacture of immunosensors should not require the extensive development of new technology for their implementation as the cost of such technology will be reflected in the unit manufacturing cost and, therefore, in the profit margin available for the sensors.
OPTICAL IMMUNOSENSORS AND SENSING SYSTEMS Optical immunosensing systems fall into two categories: direct and indirect. The direct forms of sensor rely solely upon the binding between antibody and antigen to modulate the signal being measured whilst the indirect types depend on the use of a label (e.g. a fluorophore) to visualise the immunological binding event. Some of the advantages and disadvantages of direct and indirect optical sensors are summarised in Table 1. Surface plasmon resonance (SPR) is an example of a direct optical immunosensing method whilst most of the evanescent wave techniques are examples of indirect immunosensing systems as they frequently utilise fluorescent labels to allow the assay to be measured; however, such systems can still be packaged in such a way that they appear reagentless to the user. Whilst a wide variety of techniques have been examined for their applicability to immunosensor development, optical methods currently appear very attractive in terms of both their technical and commercial viability. The use of optical methods to study proteins has been known for a long time. The application of ellipsometry to such studes was described by Vroman in 1969 who used the technique to examine the exchange of plasma proteins at a solid/liquid interface. Recently there have been attempts to commercialise ellipsometerbased immunosensing systems as a result of the development of the greatly simplified IsoscopeW (Sagax Instruments) ellipsometer (Nygren et al., 1985). The first true optical immunosensor was described by Giaever in 1973. Giaever’s very
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TABLE1 Comparisons between direct and indirect optical immunosensors Direct
“Reagentless”
Instrumentation may be simpler
Sensitivity may be limited by NSB
Indirect Label improves sensitivity and selectivity Packaged to appear reagentless Label reduces NSB Normal assay formats Needs a labelled reagent
Analyte size limits the use of standard assay formats
simple device consisted of islets of indium deposited upon a suitable substrate (e.g. silicon). Protein (bovine serum albumin, BSA) was coated onto the indium by adsorption and the slide was incubated with rabbit antiserum against BSA. The appearance of the original slide was dark brown when viewed in scattered light and, after coating with BSA, the slide appeared darker; upon further incubation with anti-BSA antiserum the slide took on a still darker appearance. When used in this manner the sensor was able to detect 10lug/ml of anti-BSA protein and was also used to detect both Hepatitis B antigen and anti-Hepatitis antibodies present in human serum. The next major breakthrough in the field of optical immunosensing was the application of total internal reflection spectroscopy (IRS) to fluorescent immunoassays (Kronick & Little, 1974)which was followed by the reporting of the application of surface plasmon resonance to biosensing (Nylander et al., 1982). SURFACE PLASMON RESONANCE The phenomenon of surface plasmon resonance (SPR) was first reported at the beginning of this century (Wood, 1902) but a detailed theoretical understanding of SPR had to await the development of quantum physics. The theory of SPR is now well understood and documented (see, for example, Raether (1982)). The first applications of SPR as a sensing technique were directed towards the study of thin films deposited upon metal surfaces (e.g. Pockrand et al., 1977)with its
application of SPR to biosensing not being reported until several years later (Nylander et al., 1982). In an SPR immuno~n~r, the binding between antibody and antigen at the surface of a thin metal film alters the effective refractive index at the interface between the sample and the metal film upon which the immunological binding partner is immobilised, resulting in a change in the properties of light reflected off the metal surface. SPR, when applied to biosensing, can use one of two basic types of transducer, these transducers being either prisms coated with a thin (-55 nm thick) film of metal (this format is frequently known as the Kretschmann configuration) (e.g. Flanagan & Pantell, 1984; Daniels et al., 1988) or metallised diffraction gratings (Cullen et al., 1988; Parry et al., 1988) where the metal thickness can be very much greater (up to 150 nm thick (Robinson, 1990)). The mode of operation of SPR biosensors has been adequately described many times (e.g. Daniels et al., 1988) and will not be discussed here. The detection of both large analytes (e.g. antibodies in serum (Parry et al., 1988)) and small analytes has been demonstrated with SPR and such immunosensor systems are currently under commercial development (Frew, 1988). TOTAL INTERNAL REFLECTION SPECTROSCOPY (IRS) IRS and its role in the excitation of surface-bound fluorophores was reported in the late 1960s and early 1970s (e.g. Harrick & Loeb, 1973). Kronick
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Optkal immunosensing systems - meeting the market neea3
Photodetector I’
Bound tluorophore \
0
Optical waveguide -
. direct detection of fhofeseence Bound fluorophore
Photodetector 0-b
/
Optical waveguide -
exciytion and detection of fluorescence $$tphore
Photodetector /
\
Optical waveguide -Tt* Direct etitation fl
I/
aLLd‘ewwwent Excitation
light
detection fl
of fluorescence
Emitted light
Fig. 1. Diagrammatic representation of the methods of evanescent excitation and detection of sutjbcejluorescence.
and Little (1974) realised that the use of this method could result in a fluoroimmunoassay in which there was no need for a wash or separation step prior to reading the assay as the IRS technique would allow bound fluorophore label to be measured in the presence of the free (unbound) label. Using a fluorescently-labelled antibody, Kronick and Little were able to detect 02 pr&itre of morphine in a competition assay. There are three variations of evanescent optics which are important and involve various combinations of direct and evanescent excitation of bound fluorophore with direct or evanescent detection of the emitted fluorescence (Fig. 1). These variations are the evanescent excitation of the fluorophore with the emitted light being detected perpendicularly to the waveguide surface (e.g. Kronick & Little, 1974); excitation of the fluorophore by illuminating the fluorophore directly and collecting the emitted light via the evanescent field of the optical waveguide (e.g. Badley et al., 1987); and, thirdly, using the evanescent field to both excite the fluorophore
and collect the light emitted by the fluorophore (Bluestein, 1989). Evanescent techniques involve the use of two optically transparent media of different refractive indices. Light is introduced into the medium of greater refractive index (n 1) and is reflected off the interface between the two media, allowing the change in intensity of the light with its wavelength to be examined. At the interface between the two media all the energy associated with the light beam is not confined within the high refractive index material and some energy is lost into the medium with the lower refractive index (nz). The intensity of this energy rapidly decays away from the interface between the two media and is dissipated over a distance comparable to the wavelength of light used (this energy field is known as the evanescent field). Such techniques are well suited to the investigation of layers close to the surface of a waveguide as material which is present in the bulk of the solution (i.e. at distances greater than several hundreds of nanometres away from the waveguide surface) is not inter-
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G. A. Robinson TABLE 2 Sensitivities achieved by a range of optical immunosensing
systems
Analyte
Concentration
Assay conditions
Source
HCG E3G Digoxin Ferritin
3 ng/ml O-5ng/ml 05 q/ml 5 &ml 08 @ml
No wash No wash No wash Premixed reagents Wash step
Deacon et al. (1990) Robinson (1990) Bluestein (1989) Bluestein (1989) Sutherland et al. (1984)
w
rogated by the evanescent field. Thus, when using the evanescent field to excite fluorophores, only those molecules which are very close to the surface of the optical waveguide will be illuminated. Similarly, if collecting emitted light via the evanescent field, only light originating very close to the waveguide surface will be detected. This allows the fluorophore label which is bound by the formation of an antibody:antigen complex to be measured without the need for the removal of the unbound label. The detailed physics of the effect have been adequately described elsewhere (e.g. Harrick, 1967; Place et al., 1985). Both optical fibres and planar waveguides have been used to exploit evanescent optics for immunosensing with assays being demonstrated for human chorionic gonadotrophin (HCG) (Deacon et al., V&O), e&one-3glucuronide (Robinson, 1990), Rubella antibody (Parry et al., 1990) and IgG and methotrexate (Sutherland et al., 1984) when using planar devices and digoxin and ferritin with optical waveguides (Bluestein, 1989) (Table 2).
THE FLUORESCENT FILL DEVICE
CAPILLARY
A planar evanescent device that is undergoing commercial development is the fluorescent capillary till device (FCFD) (Badley et al., 1987) (Fig. 2). This device consists of two parallel glass plates held apart by a gap, O-1 mm wide. This narrow gap allows a sample to enter the device by capillarity with the dimensions of the device metering the volume of sample applied. The narrow gap within the FCFD also allows rapid assay times as the reagents needed for the assay only have to diffuse over a very short distance (i.e. the distance of the gap between the plates) to reach the assay’s solid phase. This means that the rates of reaction of assays performed within the
Fluorescently labelled
Opaque lid
aeaay reagents \
I Optical edge
/
\
l
eee
100 urn I
Optical wavebuide baseplate
C&ture
sap
antibody
Fig. 2. Diagrammatic representation of the fluorescent capillary filI device (FCFD).
device are limited by the kinetics of antibody/ antigen binding and not by the kinetics of diffusion. This means that the device can achieve very rapid immunoassays (e.g. an assay for E3G in urine will reach equilibrium within 5 min of sample addition (Robinson, MO)). All the reagents needed for the immunoassay are contained within the device, either, as in the case of the assay’s capture system, covalently coupled to the lower plate of the device (the baseplate), which also functions as an optical waveguide, or dosed onto the upper plate in such a way that they dissolve when sample enters the device. Upon completion of the antibody:antigen binding reaction, all the fluorophore within the FCFD is excited directly. The light arising from the bound fluorophore is discriminated from the free by the evanescent optics of the baseplate waveguide. This allows the assay to be read without the need for any wash or separation steps. The instrumentation needed to interrogate the FCFD is essentially simple and consists of a suitably filtered low-cost light source (e.g. a photographic flash tube) which is focused onto the baseplate of the device. Light leaving the optical edge of the baseplate of the device is collected via an aperture to reject the solution signal arising from the unbound fluorophore (i.e.
189
Optical immunosensing systems - meeting the market needs
strated in the FCFD are Rubella antibody in serum (Fig. 4; Parry d al., l!#O), E3G in urine (Robinson, 1990) and human chorionic gonadotrophin (Deacon et al., 1990)(Table 2). Sensitivities of 75 X lo-” and 1.1 X 10B9 moles/We have been obtained for HCG in serum and E3G in urine respectively. The FCFD is a good example of an immunosensor that meets most of the clinical market requirements as it can operate on a range of samples (e.g. whole blood, serum, plasma and urine), there is no need to measure the volume of sample applied to it as capillarity controls the filling of the device and meters the sample volume, assays can be read within 10 min of sample application and the associated instrumentation is simple and will be low-cost. Furthermore, all the reagents needed to perform the assay are contained within the device, making it appear reagentless, and no skill is required for its use. To date, a shelf-life of 6 months at 45°C for the immunosensing devices has been achieved. One important area still to be addressed for the device, however, is that ofcalibration as it still relies upon the batch calibration of the sensor during the manufacturing process.
Focussing ootice FCFD cell
Filter
/
-Low cost, white light source (e.g. photographic flash tube)
Fig. 3. Schema& representation of the instrumentation required to interrogate theJluowscent capillaryf 11device.
light emerging from the waveguide at angles greater than +44” and less than -44” to the plane of the device for serum samples) onto a photodetector. The placing of an aperture in the path of the light leaving the device allows the construction of a simple, low-cost instrument with no moving parts (Fig 3). Amongst the assays which have been demon-
Signal (units) 12
0
2
Zne n = podtive
10
8
4 serum
12
14
(mind pool:
+
l
nogatlvo serum pool
Fig. 4. Dose-tvsponse curvefor an assayfor Rubella antibody in serum pe#onned using thefluorescent capillaryfir device.
190
TECHNICAL CHALLENGES FOR OPTICAL IMMUNOSENSORS Whilst optical immunosensing systems have been demonstrated which are very close to commercial reality, there are a number of areas within their development which still require technical progress to be made. The first such area is that of calibration of both the sensor and its instrument. Manufacturers will need to incorporate controls into their immunosensors so that the user has absolute confidence in the assay result. This area of work is especially important for both the quantitative and qualitative clinical applications of such devices. Enzyme-based biosensors can be calibrated by exposing them to known concentrations of the analyte of interest and comparing this data with results obtained when the sensor is incubated with sample. Unfortunately this technique is not directly applicable to immunosensom because of the irreversible nature of the antibody:antigen bond. Although such binding can be reversed (indeed, the ability to break such bonds is essential to the widespread technique of afftnity chromatography), there is always a risk of the immunological activity of the sensor degrading as a result of eluting the bound calibrator, resulting in compromise of either the sensor’s performance or the reliability of the calibration procedure. Another area that requires further development is the chemistry used to immobilise biological molecules to surfaces and the results of such chemistries on the non-specific binding (NSB) of proteins to surfaces. This is important if the ultimate sensitivity of both direct and indirect immunosensors is not to be limited by the NSB of proteins onto the active surface of the sensor. Furthermore, a rigorous understanding of the chemistries involved in both protein immobilisation and non-specific binding may result in the ability to increase the biological capacity of the reactive surface of a sensor thereby improving its performance. For certain applications (e.g. process control) the re-use of a sensor may be important, therefore a means of achieving reuse without compromising the performance of the sensor must be found. One way of addressing this problem might be to use antibodies that have a low affinity for the target antigen which will, therefore, allow the bound antigen to be readily eluted off the antibody. Such an approach may, however, compromise the
G. A. Robinson
sensitivity, specificity and speed of the sensor. Also important for some applications (e.g. certain types of process control, in-vivo sensing) is the sterility of the sensor. The problem of fabricating a sterile device without compromising the biological activity of the protein immobilised on the device is most challenging. Finally, the problems of the fluid handling associated with sensors needs careful optimisation, both to make such sample handling systems easy to use and to prevent limiting the sensor’s sensitivity by analyte depletion. When immersed in a static solution the effective volume of sample interrogated by an immunosensor is partially governed by the kinetics of mass transport of the analyte to the immunologically active surface of the sensor. Fluid handling systems will greatly increase the effective volume of sample interrogated by the sensor, resulting in increased sensitivity for the device; the penalty being the cost of the fluid handling system. The development of optical immunosensors has resulted in a greater understanding of the underlying physics of such devices and of the specific requirements for a device designed to monitor an immune reaction. With industrial organisations now undertaking the development of such devices to address specific market niches, the commercial future for optical immunosensors looks assured. To conclude, much technical progress has been made in the development of optical immunosensing systems with several technologies under intense commercial development and the next few years should see the commercialisation of at least one of these exciting analytical systems.
REFERENCES Badley, R A., Drake, R. A. L., Shanks, I. A., Smith, k M. & Stephenson, P. R. (1987). Optical biosensom for immunoassays: the fluorescence capillary till device. Phil. Trans. Roy. Sac Land., B 316,143~60. Bluestein, B. I. (1989). Antibody coated fibre optic transducers. Rvc. 2nd Int. Biosensors ‘89Symposium, New Orleans, October 1989. Clarke, L. C. & Lyons, C. (1962). Electrode systems for continuous monitoring in cardiovascular surgery. Ann. N. Y. Acad Sci., 192, 29-45. Cullen, D. C., Brown, R G. W. & Lowe, C. R (1988). Detection of immune-complex formation via surface plasmon resonance on gold coated diffraction gratings. Biosensors, 3, 21 l-25.
Optical immunosensing systems - meeting the market needs Daniels, P. B., Deacon, J. K., Eddowes, M. J. & Pedley, D. G. (1988). Surface plasmon resonance applied to immunosensing. Sens. Act, 15, 11-18. Deacon, J. K, Thomson, A M., Page, A L., Stops, J. E., Roberts, P. R, Whiteley, S. W., Attridge, J. W., Love, C. A, Robinson, G. A & Davidson, G. P. (1991). An assay for human chorionic gonadotrophin using the fluorescence capillary till device. Biosens. Bioelectmn.. 6, 193-199. Flanagan, M. T. & Pantell, R H. (1984). Surface plasmon resonance and immunosensors. EZmnicr L&&Q 20,968-70. Flanagan, M. T., Sloper, A N. & Ashworth, R H. (1988). From electronic to opto-electronic biosensors: an engineering view. Anal. Chim. Acta, 213,23-33. Frew, J. E. (1988). Biosensors in Medical Diagnostics, PJB Publications Ltd., Richmond, UK Giaever, I. (1973). The antibody:antigen reaction: a visual observation. J. Zmmunol., 110, 1424-6. Harrick, N. J. (1967). Internal Rejection Spectmscopy, Interscience, New York Ha&k, N. J. & Loeb, G. I. (1973). Multiple internal reflection spectroscopy. Anal. Chem., 45,687-91. Janata, J. & Blackbum, G. F. (1984). Immunochemical potentiometric sensors. Ann. N. Y. Acad. Sci., 428, 286-92. Kmnick, M. N. & Little, W. A (1974). A new immunoassay based on fluorescent excitation by internal reflection spectroscopy. Ptwc. Nat1 Acad. Sci. US.4, 71,4553-T Nakamura, R M. (1988). Clinical/medical applications of biosensors. kceedings of the International Biosenso~ ‘88Symposium Institute for International Research Inc., Washington DC. North, J. R (1985). Immunosensors: antibody based biosensors. TIX&S in Biotechnofogv, 3, 180-6. Nygren, B. H., Sandstrom, T., Stenberg, J. E. & Stilbert, L. B. (1985). Method and member for detecting and/or measuring the concentration of a chemical substance, US Patent 4 558 012. Nylander, C., Liedberg, B. & Lind, T. (1982/83). Gas detection by means of surface plasmon resonance. Sens. Act., 3, 79-88.
191 Parry, R, Deacon, J. K., Robinson, G. A, Skehel, J. J. & Forrest, G. C. (1988). Surface plasmon resonance immunosensors. In Rapid Methods andAutomation in Microbiology and Immunology. ed. A Balows, R C. Tilton, A TIuano. Brixia Academic Press, Brescia, pp. 641-8. Parry, R P., Love, C. A & Robinson, G. A (1990). Detection of Rubella antibody using an optical immunosensor. A fi&. Methoak 27,39-48. Place, J. F., Sutherland, R M. & Dahne, C. (1985). Optoelectronic immunosensors: a review of optical immunoassays at continuous surfaces. Biosensors. 1,321-54. Pockrand, I., Swalen, J. D., Gordon, J. G. & Philpott, M. R (1977). Surface plasmon spectroscopy of organic monolayer assemblies. Surface Science, 74, 237-44. Raether, H. (1982). Dispersion relation of surface plasmons on gold and silver gratings. Optics Comm.. 42,217-22. Robinson, G. A (1990). Optical immunosensors - an overview. Adv. BiosenL, in press. Robinson, G. A, Cole, V. M., Rattle, S. J. & Forrest, G. C. (1986). Bioelectrochemical immunoassay for human chorionic gonadotrophin in serum using an electrode-immobilised capture antibody. Biosensots, 2,45-57. Stewart, W. J. (1986). Optical assay, UK Patent Application No. 2 173 895. Sutherland, R M., Dahne, C., Place, J. F. & Ringrose, A R (1984). Optical detection of antibody-antigen reactions at a glass-liquid interface. Clin. Chem., 30, 1533-8. Vroman, L. & Adams, A L. (1969). Findings with the recording ellipsometer suggesting rapid exchange of specific plasma proteins at solid/liquid interfaces. Sut$ Sci., 16,438-46. Wood, R W. (1902). On the remarkable case of uneven distribution oflight in diffraction grating spectrum. Phil. Msg., 4, 3%-402. Yalow, R S. & Berson, S. A (1959). Assay of plasma insulin in human subjects by immunological methods. Nature 184, 1648-9.
