Chemical sensors comprising reversible competitive recognition units consisting of receptors and their ligands

FIELD OF THE INVENTION

The present invention is in the field of chemical and biochemical sensors, i.e. solid-phase analytical devices that respond continuously to the amount of a specific analyte in a tested liquid or gaseous-phase sample. The specific response to a certain analyte is obtained by an appropriate combination of a molecular recognition system for the analyte and a suitable transducer.

Biosensor is a sensor in which the recognition system is based on biological species such as the interaction between an antigen Ag, as analyte, with its antibody Ab immobilized on a sensing surface.

Chemical and biochemical sensors have ,a great potential for application in various fields such as medicine, industry and defence. In medicine, biosensors are useful, for example, for monitoring in vivo and in real time clinically and biochemically important analytes. Sensors for .continuously monitoring analytes in real time are also of great importance in industry, e.g. for controlling and regulating chemical process, as well as in defence for monitoring biological and biochemical hazards.

LIST OF REFERENCES

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8. Lim C.S., Miller J.N. and Bridges J.W.: EnergyTransfer Immunoassay: A Study of the Experimental Parameters in an Assay for Human Serum Albumin. Anal. Biochan. 108, 176-184 (1980).

9. Bright F.V. and McGown L.B.: Homogeneous Immunoassay of Phenobarbital by Phase-Resolved Fluorescence Spectroscopy Taianta 32(1) 15-18 (1985).

10. Tahboub Y.R. and McGown L.B.: Phase-Resolved Fluoroimmunoassay of Human Serum Albumin Anal. Chia. Acta 182, 185-191 (1986).

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13. Li T.M., Benovic J.L. and Burd J.F.: Serum Theophylline Determination by Fluorescence Polarization Immunoassay Utilizing an Umbel liferone Derivative as a Fluorescent Label Anal. Biochem. 118, 102-107 (1981).

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15. Sutherland R.M., Dahne C., Place J.F. and Ringrose A.R.: Immunoassays at a Quartz-Liquid Interface: Theory, Instrumentation and Preliminary Application to the Fluorescent Immunoassay of Human Immunoglobulin G. J. Immunological Methods, 74, 253265 (1984).

16. Andrade J.D., Vanwagenen R.A., Gregonis D.E., Newby K. and Lin J.N.: Remote Fiber-Optic Biosensors Based on Evanescent-Excited Fluoro-Immunoassay: Concept and Progress IEEE Transactions an Electron Devices ED32(7) 1175-1179 (1985).

17. Axelrod, D., Burghardt, T.P. and Thompson, N.L. Total Internal Reflection Fluorescence. Ann. Rev. Biophys. Bioeng. 13: 247-268 (1984).

18. Watkins, R.W. and Robertson, C.R.: A total Internal Reflection Technique for the Examination of Protein Adsorption. J. Blamed. Mater. Res. 11: 915-938 (1977).

19. Ullamn, F., Schwarzberg, M. and Rubinstein, K.: Fluorescent Excitation Transfer Immunoassay - A General Method for Determination of Antigens. J. Biol. Chen. 251: 4172-4178 (1976).

20. Tromberg, B.J. and Sepaniak, M.J.: Fiber-Optic Chemical Sensors for Competitive Binding Fluoroimmuno assay. Anal. Chem. 59:1226-1230 (1987).

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23. Felix, A.M., Heimer, E.P., Lambros, T.J., Tzougraki, C. and Maienhofer, J.: Rapid Removal of Protecting Groups from Peptides by Catalytic Transfer Hydrogeneration with 1,4-Cyclohexadiene. J. Qrg. Chen. 43: 4194-4196 (1978).

24. McGregor, A.R., Crookall-Greening, J.0., Landon, J. and Smith, D.S.: Polar iz at in Fluoroimmunoassay of Phenytoin. Clin. Chin, acta 83: 161-166 (1978).

25. Parini, C., Bacigalupo, M.A. Colombi, S., Ferrara, L., Franceschetti, F. and Saita, R.: Two New Fluorescent Derivatives of Progestrone to Use in Fluoroimmunoassay. Steoids 46: 903-913 (1985).

26. Bodanszky M. and Vigneaud, V.D: A Method of Synthesis of Long Peptide Chains Using a Synthesis of Oxytocin as an Example J. Amer. Chem. Soc. 81:56881959) 5691־).

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28. Claassen, E., Doorsma D.M., Kors N. and Van Rooijen N.: Double-Enzyme Conjugates, Producing an Intermediate Color, for Simultaneous and Direct Detection of Three Different Intracellular Immunoglobin Determinants With Only Two Enzymes. J. Histochem. Cytochem. 34: 423-428 (1986).

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J.H., Calvert, J.M., Bredehorst, R. and Ligler, F.S.: Use of Thiol- Terminal Silanes and Heterobifunctional Cross-linkers for Immobilization of Antibodies on Silica surfaces. Anal. Biochem. 178: 408-413 (1989).

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BACKGROUND OF THE INVENTION AND PRIOR ART

Interest in chemical and biochemical sensors has expanded rapidly in recent years. For the purpose of the present invention the term chemical sensor, as distinct from a probe, is meant to describe a device which responds continuously and reversibly to changing concentrations of an analyte in a test medium. One of the more recent developments has been the attempt to develop immunosensors based on optical fibres, which are usually characterized by their high specificity and high sensitivity. These and other developments focused on an attempt to adapt the principles of solid-phase immunoassay to continuous monitoring of various substances e.g. drugs, hormones etc. in body fluids. Immunosensors developed for these purposes are usually based on the conventional competitive immunoassay reaction between an immobilized receptor present in a solid phase on the one hand and a mobile ligand (analyte) and a mobile labelled analogue of the latter present in a liquid phase on the other hand. Such a reaction may be described by the following equation:

Ab + Ag + Ag*-------> Ab-Ag + Ab-Ag*

In this equation Ab is the receptor and stands for an antibody and Ag and Ag* are the ligands and stand for an antigen analyte and a labelled derivative of the same antigen or of an analogue thereof, respectively.

According to the elementary immunoassay method for the determination of an analyte Ag in a test liquid, a sample of the tested liquid containing a known amount of labelled antigen Ag* and an unknown amount of the analyte Ag is introduced into a vessel holding a solid phase containing a given amount of an appropriate antibody Ab. Upon contact, the analyte antigen Ag present in the sample and the added labelled antigen Ag* compete for the limited amount of antibody Ab in the solid phase. After a certain incubation period the liquid and solid phases are separated from each other, the solid phase is washed and the analyte concentration is determined by following the labelled component, either as Ag* in the liquid phase or as the conjugate Ab-Ag* in the solid phase.

In known solid-phase immunoassays only one component of the competitive recognition system (usually the antibody Ab) is immobilized on the sensing area, while the labelled antigen Ag* is introduced ad hoc into the liquid sample which is tested for the antigen Ag.

The above-described immunoassay method is static and unsuitable for the continuous determination of analyte in real or nearly real time. A further development in the field of immunoassay was achieved by the employment of probes which enable discrete measurements of analytes, although not in real time. In one version of these probes the sensing tip, with predetermined amounts of immobilized receptors, is dipped in a test liquid containing a known amount of labelled ligand and unknown amount of analyte ligand. From the relative amount of the labelled ligand which conjugated to the receptors, present on the sensing tip, the concentration of the analyte is determined. By a sequence of such discrete measurements the change in concentration of the analyte with time may be determined in close approximation.

In sensors in which a labelled antigen or a 1 abelled receptor (one of them is an analogue to the analyte) is used, the concentration of the labelled species has to be kept constant in order to serve as a reliable reference for the continuously changing concentration of the analyte. Subject to this it is further required that the receptor-ligand affinity interaction be specific and reversible so that the system will responds continuously and reversably to changes of analyte concentration.

Various approaches have been proposed to solve the latter problem. Some of these proposals are based on a compromise in that a degree of reversibility is obtained by so selecting the receptor that the affinity between receptors and ligands is moderated. However, by its very nature such a compromise solution is not quite satisfactory as the sensing is less specific and it is liable to yield false results. Consequently, efforts are continuously made to find better solutions for the desired reversibility while keeping high specificity of the competitive recognition system.

Recently, some approaches, based mainly on encapsulated reagents, have been proposed (Refs. 1-5). Thus, for example, Mansouri and Schultz (Ref. 2) have developed an optical affinity glucose sensor, in which a labelled glucose analogue in the form of high molecular weight fluoresceindextran is entrapped within a dialysis fibre having an outer membrane permeable to glucose. A given amount of concanavalin-A is covalently attached to the inner lumen of the dialysis fibre, and the entrapped fluoresceinated dextran and permeating glucose compete for conjugation therewith. At equilibrium the level of free fluorescein in the hollow fibre lumen is measured via the optical fibre and is correlated to the concentration of glucose. The higher the glucose concentration, the higher the recorded fluorescence.

Similar biosensors have been proposed by Meadows and Schultz (Ref. 3) and by Anderson and Miller (Ref. 4). In these systems, which are based on fluorescence energy transfer, the specific receptor and the high molecular weight labelled ligand are enclosed within a dialysis membrane, permeable to the low molecular weight unlabelled ligand such as glucose or phenytoin.

A further system, based on potentiometric response, has been proposed by Bush and Rechnitz (Ref. 5). A monoclonal antibody Ab is trapped between two membranes while its antigen (DNP) is immobilized on one of the membranes.

In all the known biosensor systems according to Reference 1 to 5, one or more of the competitive reagents is enclosed within a membrane permeable to the low molecular weight analyte molecules, but not permeable to their labelled analogues. This is essential in order to meet the requirement for a constant level of labelled analyte analogues within the sensing zone. The result is that all these biosensing systems are suitable only for low molecular weight analytes.

A further disadvantage of the above prior art sensors is that since the analyte has to diffuse through the membrane, the response time is increased. In addition sensors of this type are less suitable for parenteral application due to the fragile structure of the dialysis membrane which might be damaged upon penetration through the skin tissue. Consequently, the prior art sensors may not be easily applied for in vivo testings.

Recently, Davis and Leary (1989), Ref. 6, have developed piezoelectric biosensor for kinetic immunoassay which is based on the change of crystal frequency as a function of analyte concentration. Within the definitions herein, this piezoelectric device is a probe and not a sensor. Although Davis's system does not require the use of a labelled antigen and consequently no semipermable membrane, and although it measures continuously for a certain time, it is not reversible. In this piezoelectrical system, whenever a new sample has to be measured the chemical sensing tip has to be restroed to activity by washing at pH 3 at which the conjugates are dissociated.

It is an object of the present invention to provide a new concept for chemical and biochemical sensors which combines effectively the features of stability, specificity and reversibility without restriction on the size of the analyte.

Another object of the invention is to provide a sensor which does not need a semipermeable membrane while still keeping a constant predetermined amount of analogue receptors or analogue ligands (labelled or unlabelled) in the sensing area. This type of sensor is more suitable for in-vivo applications.

GLOSSARY

The following are the meanings of some of the terms used in the description and claims herein:

Species - molecules, radicals, cells and ions;

Receptor - any species capable of conjugating with specific ligands via affinity interaction;

Ligand - any species capable of conjugating specifically with a receptor via affinity interaction;

Competitive Recognition System - A system comprising as constituent components receptors and ligands whose concentrations are constant;

Reversible Competitive Recognition Unit (RCRU) - A unit which is composed of at least two constituent components, namely at least one !receptor and at least one ligand (labelled or unlabelled) wherein one of them is an analogue of the analyte and the other is a specific recognizer of the analyte.

Analyte - a ligand or a receptor whose concentration in the sample has to be determined.

Analyte analog!!*» ־ A constituent component of the RCRU which is a labelled or unlabelled ligand or receptor whose specific affinity interaction with the recognizer is the same as or similar to that between the recognizer and the analyte.

Recognizer - A constituent component of RCRU which is a receptor or ligand whose specific affinity interaction with the analyte is the same as or similar to that with the analyte analogue.

Affinity band - a non-covalent bond between two species;

RIi-Conjugate - an association between a receptor and a ligand held together by an affinity bond;

Chemical sensor - any type of solid phase recognition system bearing immobilized receptors and ligands, operating by the reversible formation of RL-conjugates, regardless of the chemical nature of any of the recognizer, analyte analogue and analyte;

Sensing surface - a portion of a chemical sensor that bears Reversible Competitive Recognition Units;

GENERAL DESCRIPTION OF THE INVENTION

In the following the invention will occasionally be specifically described with reference to immunosensors, it being understood that the invention is in no way confined thereto and covers quite generally chemical sensors within the meaning of this term as defined hereinabove.

From the foregoing description of the background of the invention it follows that in prior art chemical sensors the components of the competitive chemical recognition systems (the receptor and the analogue ligand) are completely separated from each other when the receptor is conjugated with the analyte ligands. Also in the prior art sensor systems, only one component of the competitive recognition system (the receptor or the ligand) is immobilized on the sensor while the other, although entrapped within a space enclosed by a semipermeable membrane, is relatively mobile in the liquid phase within that space that contains the analyte.

The present invention is based on an entirely new concept. According to this invention, the sensing surface is coated with reversible comptetitive recognition units (RCRUs) each of which contains as constituent components at least one receptor and at least one ligand, one of which components is in an analyte analogue. In these RCRUs the receptor and the ligand are a priori connected to each other, directly or indirectly, in such configuration that even when, for example, an analyte analogue ligand is displaced from the receptor by an analyte ligand, the analogue will still be retained in relatively close proximity to the receptor. In addition, the relative positions of the receptor and the ligand in the RCRU are such that when no analytes are present or when the analyte concentration is at a low level, they are affinity conjugated by the specific affinity interaction.

According to the present invention, the RCRU which is immobilized on the sensing surface, is the basic chemical sensing unit which enables the real time continuous and reversible monitoring of the analyte in a tested sample; As will be explained below, the unique features of the RCRU. are gained as a result of its unique structure and configuration.

The invention provides an analyte specific chemical sensor for determining an analyte in a test medium, comprising a carrier body with a sensing surface having attached thereto a plurality of RCRUs each comprising as constituent components at least one receptor and one ligand arranged in such relationship that in the absence of analyte the receptor( s) and ligand( s) of each RCRU associate to form an inner conjugate which conjugate is capable of dissociating in the presence of the analyte and reassociating when the analyte concentration in the test medium is low.

If desired the constituent receptor(s) and ligand(s), of each RCRU, are bound directly to the sensing surface (hereinafter direct linkage). Preferably, however, the binding is indirect, via spacer molecules which are interposed between each of the constituent components and the sensing surface (hereinafter indirect linkage).

In accordance with one embodiment of the indirect linkage each of the receptor(s) and ligand(s) of each RCRU are bound via a separate spacer molecule.

In accordance with other embodiments the constituent components of the RCRU are interconnected by a common spacer molecule (or directly, in case the species are large enough) to form a molecular construct with the receptor and the ligand moieties, which construct is linked to the sensing surface, either directly or by means of a further spacer molecule.

In the indirect linkage configuration of the RCRUs of chemical sensors according to the invention it is possible by Judicious selection of the spacer molecules based on general common knowledge of the chemical and stereo-chemical properties of the RCRU-receptors, the RCRUligands and spacer molecules, to optimize the distance between the receptor and the ligand thereby to ensure a stable yet reversible affinity bond between the constituent components in an RCRU.

The new concept underlying the present invention is of a universal character and applies to various specific receptors, for example receptors of hormones, vitamins, neurotransmitters, toxins etc. Also it is applicable to other receptor-ligand systems such as antibody-antigen; glycoconjugation-lectins; metal interacting macromoleculesmetal ions; etc.

The new chemical sensors according to the invention can be applied for the determination of a great variety of different types of analytes, e.g. low molecular weight compounds such as drugs and hormones; high molecular weight compounds such as proteins, enzymes, DNA and RNA molecules; cells; and many more in either a liquid or gaseous test sample. The preferred configuration of a sensor according to the invention to be used for a particular case is determined with due regard to the various parameters of the analyte under consideration.

The fluctuations of analyte concentrations in the tested sample affect the chemical occurrences at the RCRUs, and. consequently the physico-chemical characteristics of the components thereof. The changes in the analyte concentration are monitored, according to the present invention, by continuously measuring the changes of the above physicochemical characteristics induced by occurrences at the RCRUs, namely the inner association and dissociation thereof. Such physico-chemical characteristics may be, for example, photochemical, e.g. light absorption, light emission, light scattering and light polarization; electrochemical; and piezoelectrical.

Hence, a chemical sensor according to the invention functions as a transducer that converts chemical occurrences at the RCRUs into detectable physical phenomena, e.g. by producing an optical, electrochemical or piezoelectrical response, if desired, the carrier body may form part of the transducing system.

According to one embodiment of the present invention there is provided a chemical sensor as defined herein, wherein each RCRU comprises an electrochemical group, which reacts upon association and/or dissociation of an R-L conjugate, to produce an electric signal. In such embodiments the carrier body may be an electrode or a field effect transistor (FET).

In sensors with luminophore labelled analyte analogues, e.g. ligands, the luminescence characteristics are under certain excitation conditions correlated to . the concentration of the analyte in the tested sample. This is the preferred mode of detection in accordance with the invention.

Generally, the physical characteristics of light which is emitted by a labelled analyte analogue is different in the conjugated and the non-conjugated states. Accordingly the changes in the concentration of analyte may be monitored by observing the alterations of the luminescent emission.

One way of causing and detecting luminescence in a sensor according to the invention, is to design the carrier body as a waveguide so that the evanescent light wave propagating at the solid phase/liquid or gaseus phase interface excites the luminophore analyte analogue and the emitted luminiscence is conducted via the waveguide to an appropriate detector.

If desired the distinction between the inner conjugated and npn-conjugated states of the RCRU components may be sharpened by fitting each recognizer, e.g. receptor, of the RCRUs with a luminescence quencher group while the analogue analyte, e.g. ligand, contains a luminophore group. In this way luminescence in the state of inner conjugation between the receptors and labelled ligands of an RCRUs which occurs in the absence of analyte, is eliminated or minimized due to the energy transfer between the luminophore group of the labelled ligand and the quencher group of the receptor. When the fluorophore labelled ligand is displaced from the conjugate by a competing analyte, the energy transfer between the group of the labelled ligand and the quencher group of the receptor is minimized, and as a result the luminescence increases. Similarly, each recognizer may be fitted with a luminescence enhancer, and in such a case, the luminescence increases upon formation of the R-L conjugate and decreases upon dissaciation of the conjugate.

Where the physicochemical characteristic of the RCRU is piezoelectrical, the carrier body is a crystal whose frequency is modulated by fluctuations of analyte in the test medium.

Due to the reversible nature of the RCRUs of a sensor according to the invention, there is a real time response to fluctuations of analyte concentrations in the test medium with concomitant continuous modulations of the intensity of the detected phenomena which are recorded by suitable detection instrumentation, for example of the kind described by Sutherland et al. (Ref. 15), Tromberg et al. (Ref. 20), and Bush and Rechnitz (Ref. 5).

The invention also provides an apparatus for measuring the concentration of an analyte either in liquid or gaseous phase, comprising a probing vessel fitted with a sensor of the kind specified herein and transducing means which transduce the receptor-ligand interactions into a measurable physico-chemical phenomenon. If desired, such apparatus may be designed as monitor for continuous operation with said probing vessel being adapted for the continuous throughflow of liquid or gaseous test fluid.

In accordance with a further aspect of the present invention there is provided a method for preparing a chemical sensor of the kind specified, which method comprises interacting the constituent components of an RCRUs of the kind specified herein, to form at least one affinity bond between them, and then attaching the RCRUs directly or indirectly to the carrier body by covalent bonds and/or affinity bonds.

In accordance with a modification of this method one of the constituent components of each RCRU is first attached to the carrier body and is then interacted with the remaining constituent components of the RCRUs by covalent or affinity interactions to yield a conjugate and thereafter the said remaining constituent components are attached to the carrier body.

DESCRIPTION OF THE DRAWINGS

For better understanding the invention will now be described, by way of example only, with reference to the annexed drawings to which it is not limited. In the drawings:

Fig. 1 is a conceptual illustration of the functioning k of a prior art solid phase competitive immunoprobe;

Fig. 2 shows conceptually six different embodiments of

RCRU of a chemical sensor according to the invention;

Fig. 3 shows conceptually several forms of linkage of a labelled ligand In the RCRU;

Figs. 4-7 are conceptual representations of the functioning of different chemical sensors according to the invention;

Figs. 8-10 show different reaction schemes for the formation of immobilized RCRUs on a sensing surface of a sensor according to the invention;

Fig. 11 is a diagrammatic illustration of a sensor according to the invention in which the carrier body is an optical fibre;

Fig. 12 is a diagrammatic illustration of a monitor embodying a sensor according to the invention;

Fig. 13 is a diagrammatic illustration of the sensor used in the monitor of Fig. 12, drawn to a larger scale; and

Fig. 14 is a diagrammatic illustration of another type of monitor embodying a sensor according to the invention.

Figs. 15 to 31 are various formulae and reaction schemes relating to matters described in the Examples herein.

DESCRIPTION OF SPECIFIC EMBODIMQiTS

Fig. 1 is a conceptual illustration of the functioning of a prior art solid phase immunoprobe in which the receptors sesrve as recognizers and are immobilized on the sensing surface of the probe body, while the labelled ligands, which serve as analyte analogues, are dispersed, together with the analyte, in the liquid sample to be tested. As shown, a probe 1 has a sensing surface 2 to which are covalently bonded receptors 3. The labelled ligands 4 are dispersed in the tested liquid sample together with the analytes 5 and in the course of testing the labelled ligands 4 and analytes 5 compete for free receptors 3 as shown on the right-hand side of Fig. 1. Each labelled ligand 4 has a labelling group L. At the end of the analysis the solid phase is separated from the liquid phase, washed and the concentration of the analyte 5 in the solution is computed from the determination of the amount of labelling groups L on the probe.

Attention is now directed to Fig. 2 which illustrates conceptually six different embodiments of RCRUs in sensors according to the invention. The conceptual illustrations are with reference to immuno-sensors, it being understood that the invention is not confined thereto. In Fig. 2 Ab designates an antibody which in this case is the receptor, Ag* designates an antigen analogue which is the RCRU-ligand. Full lines represent covalent bonds and dashed lines represent affinity bonds.

Fig. 2(a) shows ths simplest form of. a RCRU according to the invention. As shown the RCRU 8 is anchored on the sensing surface 9 of a carrier body (not shown). It comprises one Ab and one Ag* molecule linked each separately to the sensing surface 9 by means of spacer molecules Ml and M2, respectively, which may be the same or different. The bonds between each of Ab and Ag* and the associated spacer molecule Ml and M2 and between these latter and the sensing surface 9 are all covalent. It is further seen that normally, when no analyte is present, Ab and Ag* of each RCRUs are associated by an affinity bond to form an Ab-Ag* conjugate.

The RCRU 10 of Fig. 2(b) is similar but in this case each of Ab and Ag* is associated with two spacer molecules M3 and M4 and M5 and M6, respectively, with the bonds between M3 and M4 and between M5 and M6 being affinity bonds while those between Ab and M3, Ag* and M5 and each of M4 and M6 and the sensing surface 11 being covalent. Conceptually the RCRUs of Figs. 2(a) and 2(b) are similar.

In the embodiment of Fig. 2(c) Ab and Ag* (of the RCRU 12) are linked to the two opposite ends of a spacer molecule M7 which in turn is linked via another spacer molecule M8 to the sensing surface 13. All bonds which lead from Ab and Ag* to sensing surface 13 are covalent.

In accordance with the embodiment of Fig. 2(d), Ab and Ag* (of the RCRU 14) are linked covalently to spacer molecules M9 and MIO respectively, the latter are linked by affinity bonds to another spacer molecule Mil which in turn is linked covalently to yet another spacer molecule M12 which in turn is linked covalently to the sensing surface

15. Conceptually this RCRUs is similar to that of Fig. 2(c).

In accordance with Fig. 2(e), the RCRU 16 contains two Ag* species which are associated with one Ab which is positioned between them. The two Ag* species are linked to the two ends of a spacer molecule M13 to which Ab is also linked via another spacer molecule M14. M13 is linked to a further spacer molecule M15 which in turn is linked to the sensing surface 17. All bonds which lead from the two Ag* species and the Ab to the sensing surface 17 are covalent.

The RCRU 18, of the embodiment described in Fig. 2(f) also contains two Ag* species associated with one Ab. The two Ag* species and Ab are linked covalently to sparer molecules M16, M17 and M18 respectively, all of which are linked via affinity bonds to another spacer molecule M19 which in turn is bonded covalently to a spacer molecule M20, the latter being covalently bonded to the sensing surface

19.

In all the embodiments of Fig. 2(a) to 2(f) the verj pus spacer molecules M are so selected that the distance between each Ab and an associated Ag* is optimized so as to enable formation of a specific affinity bond between the Ab and the Ag* of the RCRU, in the absence or in relatively low concentration of analyte species. In addition, when this specific inter RCRU conjugate dissociates, for example under competitive interactions of the analyte antigens, the displaced RCRU-antigen, although being still connected to the receptor (by the spacer molecules) behaves as if it were free in the solution.

As it has been already mentioned, the chemical components of the RCRU may contain labelled or unlabelled groups. An example of applying a sensor, according to the present invention, without using a label group is the case when the the sensing is based on a piezoelectrical effect. »

In this case the sensing will be more suitable for analytes of the higher molecular weights.

When Ag* contains a label group, L, it can be fitted into a RCRU in various ways as shown in Fig. 3. In that Figure three different modifications are shown with reference to the RCRU embodiment of Fig. 2(c) and the same reference signs are used. In all of Figs. 3(a), 3(b) and 3(c), Ag* of Fig. 2(c) is replaced by Ag-L which in Fig. 3(a) is -Ag-L, in Fig. 3(b) is -L-Ag and in Fig. 3(c) is

Accordingly, in Fig. 3(a) L is linked to M7 via Ag, in Fig. 3(b) Ag is linked to M7 via L and in Fig. 3(c) Ag and L are linked In parallel to a juncture 22 which is linked to M7. Which of these modifications will be used in practice will depend on the nature of the analytes and labelling. Assuming L to be a fluorophore group, the embodiment of Fig. 3(a) will, as a rule, be used for high molecular weight antigens and/or small labelling groups while those of Fig. 3(b) will as a rule, be preferred when Ag is of low molecular weight and L is a high molecular weight fluorophore such as phycobiliprotein.

The embodiment of Fig. 3(c) is suitable in cases where L and Ag are of low molecular weight.

Figs. 4 to 7 illustrate conceptually the functioning of sensors according to the invention having RCRUs of the kind illustrated conceptually in Figs. 3(a), 3(b) and 3(c), respectively. In these drawings all spacer molecules are for the sake of simplicity of illustration shown by way of drawn-out lines. Thus in Fig. 4, the configuration of the RCRUs 23 which are immobilized on the sensing surface 24, is in accordance with Fig. 3a. With no analyte Ag or a very low concentration thereof in the liquid phase the equilibrium is shifted to internal conjugation with each Ab in a RCRU conjugating with the associated Ag-L. When the concentration of the analyte Ag in the tested sample increases the equilibrium is shifted to the right with the result shown on the right-hand side of Fig. 4. As seen in that state some of the Ab/Ag-L affinity bonds are severed and the labelled antigens Ag-L are displaced by analytes Ag. With a decrease of the concentration of the analyte Ag the equilibrium again shifts to the left; and so forth. When groups L are, for example, fluorophore they are of such a nature that there is a significant difference between their light emission in the conjugated and nonconjugated state and the ensuing fluorescent light modulations are recorded by a suitable recorder.

Fig. 5 is a similar conceptual representation with the configuration of the RCRUs 25, which are immobilized on the sensing surface 26 being in accordance with Fig. 3(b). Likewise Fig. 6 is a similar conceptual representation with the configuration of the RCRUs 27, immobilized on a sensing surface 28, being in accordance with Fig. 3(c).

In the conceptual representation of Fig. 7 in which the manner of presentation is similar to that in Figs.

to 6 each Ab comprises a quenching group Q which in the inner conjugation state shown on the left-hand side of the Figure is in close affinity to the luminofluorophore group L of the associated labelled antigen analogue Ag-L, and in consequence the luminescence of the group L is quenched. When the equilibrium is shifted to the right due to the presence of the analyte Ag some of the luminescent groups L are set free with the intensity of the ensuing luminescence being proportional to the concentration of the analyte Ag in the tested sample.

Figs. 8 to 10 show conceptually some manners of preparation of an immunosensor according to the invention with immobilized RCRUs according to Fig. 2(a). Similar as in Fig. 2 continuous lines signify covalent bonds and dashed lines affinity bonds.

Referring first to Fig. 8, it is seen that in a first step each of Ab and Ag* is reacted with a bifunctional spacer molecule Ml and M2 to yield Ab-Ml and Ag*-M2, respectively. In a second step Ab-Ml and Ag*-M2 are conjugated and in a third step the resulting conjugate is immobilized on the sensing surface of the sensor by reaction of a terminal functional group of each of Ml and M2 with suitable complementary functional group on the sensing surface.

Fig. 9 illustrates conceptually the preparation of an immunosensor according to the invention with immobilized RCRUs having the configuration of Fig. 2(c). As shown, in a first step two polyalanine molecules with terminal functional groups X and Y are bound jointly to a spacer molecule M anchored in the sensing surface. In a second step Ag* is reacted with the polyalanine-X molecule to yield a polyalanine-Ag* construct which in a third step is conjugated with a receptor antibody Ab. In a fourth step the resulting product is subjected to conditions at which Ab reacts with the neighbouring polyalanine-Y grouping to yield the final RCRUs. Other variations are also possible. Thus, for example, steps 2-4 may be performed first and groups X and Y are then bound to the spacer molecule M.

Fig. 10 illustrates conceptually the preparation of an immuno-sensor according to the invention in which the immobilized sensing groups have the configuration of Fig. 2(d). As shown in a first step, streptavidin is linked to the sensing surface by reaction with a pre-activated hydroxyl group thereof. Independently Ag* is reacted with a compound MB where M is a spacer molecule and B is biotin, to produce Ag*-M-B. Similarly and again independently, Ab is reacted with MB to produce Ab-M-B. In a fourth step Ag*-M-B and Ab-M-B are admixed under conditions inducive of the formation of an affinity bond between Ag* and Ab and the resulting product is reacted with the immobilized streptavidin group to yield a RCRUs having the configuration of Fig. 2(d).

As will be readily understood by those versed in the art, reactants other than those specifically mentioned by way of example with reference to Figs. 9 and 10 may be used. Furthermore, by similar approaches RCRUs of different configurations may be produced.

Fig. 11 shows diagrammatically a fraction of a cuvette 30 forming part of an analyzer apparatus and having

%.

an opening 31 for the ingress of a test fluid, and another opening (not shown) for the egress thereof. Mounted within the cuvette 30 is an optical fibre 32 having its free end goyppori by a mirror 33 while the other end is associated with a light source (not shown). On the sensing surface of the optical fibre 32 there axe anchored a plurality of RCRUs 33 comprising each a receptor antibody molecule Ab bearing a quenching group Q and a labelled ligand consisting of the analyte-type antigen molecule Ag bearing a luminous fluorophore group L. In operation, light travels forth and back within the optical fibre 32 producing an evanescent light wave in zone 34. This evanescence excites the L groups present on the labelled antigen. Due to the presence of quencher groups Q on the labelled antibody, the measured intensity of the emitted fluorescence is correlated to the relative proportions of the conjugated and unconjugated labelled antigens. The higher the relative proportion of the Q-Ab Ag-L conjugate, the lower the intensity of the emitted fluorescence.

Figs, 12 and 13 illustrate diagrarranatically an apparatus for continuously monitoring the presence of an analyte Ag in a fluid and embodying an immunosensor according to the invention in which the carrier body is an optical fibre. As shown, the instrument comprises a laser light source 36 with associated laser line filter 37, a first mirror 38 and a second, dichroic mirror 39 which is transparent to the laser light and reflects fluorescent light. A condensing lens 40 focuses the incoming laser light into a first optical fibre 41 which is coupled to the sensor 42 by means of a fibre coupler 43. The waveguide 42 rrmpr-i spa an unclad sensing portion 44 which is located within a cuvette 45 having an inlet 46 and outlet 47 for the ingress and egress, respectively, of the test fluid. The evanescence produced within cuvette 45 around the sensing portion 44 of sensor 42 at the solid phase/fluid interface excites the luminous groups of RCRUs 48 which may, for example, have the configuration shown in Fig. 7.

The role of mirror 49 is to double the path of -light propagating inside the fibre and thus to amplify the optical signal. The resulting omnidirectional fluorescence is conducted via lens 40, mirror 39 and a further lens 61 into a monochromator 62 and from there via a photomultiplier 63 to a detector 64. The latter comprises a microprocessor and a printer to produce a tracing showing the modulation of the fluorescence with time. If desired, the detected light modulations may be computed into concentration fluctuations of Ag so that the printout will show directly the latter.

Another embodiment of a device embodying an immunosensor according to the invention is shown in Fig. 14. Laser light is produced by a generator 50 associated with a laser light filter 51 and is reflected by means of mirror 52 and conducted via a prism 53 into an immunosensor according to the invention 54, comprising a waveguide slide whose upper face constitutes the sensing surface and comprises a solid phase coat with a plurality of RCRUs anchored therein. The slide waveguide 54 is enclosed within a cuvette 55 with inlet 56 and outlet 57 for ingress and egress, respectively, of the tested sample. At the edge opposite to that at which the light enters, the slide waveguide 54 is associated with another prism 58 which directs the fluorescent beam resulting from the excitation of luminous groups of the RCUs by the laser beam, to a filter 59 and from there via a photomultiplier unit 60 to further processing and recording as known per se. The laser light coming out of the waveguide 54 is directed away by prism 58 so as not to impinge on the pulse moderator beam.

Working examples

The following examples are provided to illustrate, but in no way to limit the present invention, which is defined by the claims following the description. The following are the meanings of the abbreviations and symbols in the examples below, and in the figures which relate to matter described in the examples, i.e. Figs. 15 to 31:.

<img file="IL92955A_D0002.tif" />

ABBREVIATIONS

<td> Ab</td><td> antibody</td>

<td> Ab*</td><td> labelled or analogue antibody</td>

<td> Ag</td><td> antigen</td>

<td> Ag*</td><td> labelled or analogue antigen</td>

<td> BOC</td><td> t-butoxy carbonyl (C^HgOCO-)</td>

<td> B</td><td> biotin</td>

<td> CBZ</td><td> benzyloxycarbonyl (CgHgCI^OCO-)</td>

<td> DCC</td><td> dicyclohexylcarbodiimide (^βΗ^Ν^-ΝΟ^Η!!)</td>

<td> DMF</td><td> N, N-dimethylformamide</td>

<td> F</td><td> fluorophore</td>

<td> FITC</td><td> fluoresceinisothiocyanate</td>

<td>^mF(ab’)₂^HSAF(ab₂(׳</td><td> F(ab')₂ fragment for morphine F(ab')₂ fragment for HSA</td>

<td> GMBS</td><td> N—maleimidebutyryloxy succinimide ester</td>

<td> HSA</td><td> human serum albumin</td>

<td> CIU</td><td> competitive immuno unit</td>

<td> L</td><td> label species</td>

<td> LG1</td><td> Ν-ε -BOC-lysylglycine-t-butylester*HCl</td>

<td> MDS</td><td> mercaptomethyl-dimethylethoxysilane</td>

<td> M2</td><td> O^-carboxymethyl morphine</td>

<td> NHS</td><td> N-hydroxysuccinimide</td>

<td> PBS</td><td> phosphate buffered saline</td>

<td> P.A1</td><td> d.1.-polyalanine</td>

t».-A Λ

<td> Q</td><td> quencher</td>

<td> R1</td><td> N⁷-CBZ-heptanoic-succinimidylester</td>

<td> RCIU</td><td> reversible competitive immuno unit</td>

<td> RCRU</td><td> reversible competitive recognition unit</td>

<td> Rh2</td><td> tetramethylrhodamine-5-isothiocyanate</td>

<td> StAv</td><td> streptavidin</td>

<td> TR</td><td> texas red</td>

<td> *</td><td> single covalent bond</td>

<td> ΛΛΛ/ΙλΠ</td><td> spacer group (connected covalently)</td>

<td> min</td><td> specific affinity interaction</td>

<td> \\\\\</td><td> sensing surface</td>

Example 1 t

Preparation of immobilized bis-polyalanine on the sensing surface (fused silica) for immunosensor of the type illustrated in Fig. 2(c). Specifically, the RCRAs have the configuration of Fig. 15.

Strategy

1.1 - Preparation of the bis-polyalanine.

1.2 - Activation of the silica surface.

1.3 - Immobilization of the bis-polyalanine on the silica surface.

1.1 Preparation of ־Hie bis-polyalanine (^LG-Bis-P.Al.)

This preparation includes several steps, using conventional procedures, described in the literature. The preparation process is depicted in the scheme of Fig. 16.

a) Product LG2: N-e-BOC-lysylglycine methylester-hydrochloride (LG1) was reacted with N⁷-CBZ-heptanoicsuccineimidyl ester (Rl) under similar conditions used by Anderson et al (1964), (21), to obtain the product LG2.

b) Product LG3: Saponification of LG2, under basic conditions (0.1N NaOH) as is known in the art, yielded LG3.

c) Product LG4: Product LG3 was reacted with N-hydroxy- succinimide in anhydrous dimethoxyethane solution in the presence of dicyclo-hexylcarbodiimide to obtain LG4 (Anderson G.W. et al. (1964), (21)).

d) product ¹LG-P.A1.: Product ¹LG-P.A1. was reacted with the amino methylester of DL-polyalanine (H₂N-P.Al.-COOMe). in dimethoxyethane solution and in the presence of sodium bicarbonate solution (Anderson G.W. et al. (1964), (21)).

_e) Product ²LG-P.A1.: The BOC group of ¹LG-P.A1. was cleaved by trifluoroacetic acid (Trabell D.S. et al. (1972), (22)) to obtain ²LG-P.A1.

f) Product ¹LC-Bis P.A1.: ²LG-P.A1. was reacted with the N-hydroxy succinimide of the polyalanine derivative in anhydrous dimethoxyethane solution in the presence of DCC (Anderson G.W. et al. (1964), (21)), to obtain ¹LG-Bis-P.Al.

g) Product ²LG-Bis-P.Al. (free amine): The CBZ protecting group in ¹LG-Bis-P.Al. was then cleaved by the catalytic transfer by hydrogenation with 1,4 cyclohexadiene in DMF, following the procedure described by Felix A.M. et al (1978), (23), to yield ²LG-Bis-P.Al.

1.2 activation of the silica surface (as epoxide)

A fiber (fused silica) having 600 pm core diameter was stripped of approximately 25 mm of cladding. Using (3glycidoxypropyl) trimethylsilane (GOPS) the quartz core was activated to epoxide and then to aldehyde following the procedure described by Tromberg B.J. and Sepaniak M.J. (1987), (20). The same procedure was used for quartz microscope slide (75x25x2 mm).

1.3 Immobilization of the Mino bis-polyalanine derivative (²LG-Bis-P.Al.) to the active surface

a) Following the procedure described by Tromberg and Sepaniak (1987), (20), the free amine on the bis-polyalanine diravteive (²LG-Bis-P.Al.) was covalently bound to the surface aldehyde, to obtain the immobilized bis-polyalanine spacers, ¹S-Bis-P.Al. having the formula shown in Fig. 17.

b) Removal of the BOC amino protecting group, to obtain the free amine.

This was done by using trifluoroacetic acid following the procedure described by Trabell D.S. et al. (1972), (22) to obtain the product ²S-Bis-P.Al. having the formula shown in Fig. 18.

Example 2 ρτ-eparafi nn of the reversible immuno competitive unit for morphine using fluoresceine as the fluorophor (donor) and Rhodamine as the quencher (acceptor).

Strategy:

2.1 - Preparation of Morphine fluoresceine conjugate of the type shown in Fig. 19.

2.2 - Coupling of the Morphine-Fluoresceine conjugate to the immobilized bis-polyalanine species (product S-bis- 40 P.A1.).

2.3 - Tagging the specific antibody ^mF(ab')2 fragments to the quencher.

2.4 - Coupling the tagged ^raF(ab’>2 fragments to the sensing surface; production of the complete immuno competitive unit.

2.1 Preparation of Lys.Gly. morphine-fluoresceine conjugate (F-LG3-M)

The preparation includes several steps by using conventional procedures found in the literature. This preparation process is depictged in the scheme of Fig. 20.

a. Product F-LG1: FITC and LG1 (see Fig. 20), were reacted in pyridine/water/triethylamine (11:8:1, v/v/v) solution, under similar conditions described by McGregor

A.R. et al. (1978), (24).

b. Product F-LG2: The amino protecting group (BOC) was cleaved by trifluroacetic acid-H20 under conditions the same as described by Trabell D.S. et al. (1972), (22).

c. Product M3: - carboxymethy !morphine was reacted with N-hydroxy-succinimide in anhydrous DMF, and in the presence of dicyclohexyl-carbodiimide (DCC) using the conventional experimental conditions as described by Parini C. et al.

(1985), (25).

d. Product F-LG2-M: The activated carboxy group of M3 was reacted with the amine F-LG2 under the similar conditions described by Parini C. et al. (1985), (25).

e. Product F-LG3-M: Saponification of F-LG2-M, under basic conditions (0.1N NaOH), as is well known in the art, turned to F-LG3-M.

2.2 Coupling of the orphine-Fluoreceine conjugate (FG3) to the immdbxlized bis-polyalanine species (product ²S-Bis-P.Al.

This coupling process is depicted in the scheme of Fig. 21.

a. Product F-LG4-M: F-LG3-M was reacted with N-hydroxysuccunimide in anhydrous dimethoxyethane solution in the presence of DCC, under conditions described by Anderson G.W. et al. (1964), (21).

b. Product ²S-Bis-P.Al.: The activated carboxy group of FLG4-M was reacted with the amine ²S-Bis-P.Al under similar conditions described by Parini C. et al. (1985), (25), to obtain ²S-Bis-P.Al.-FM.

c. Product ³S-Bis-P.Al.-FM: Saponification of ²S-Bis-P.Al.-

FM, under basic conditions (0.1 N NaOH), as is well known in the art, yielded ³S-Bis-P.Al.-FM.

d. Product ^S-Bis-P.Al.-FM: The carboxy group of ³S-Bis-

P.A1.-FM was activated as the p-nitrophenylester, using DCC as the coupler, following similar procedure as described by

Bodanszky M. and Vigneaud V. (1959), (26).

2.3 Coupling of the quencher (Rhodamine) to the morphine specific antibody fragments (®F(ab' )2)» obtain (^mF(ab')₂h)

The quencher (Rh2) was conjugated to the ^raF(ab' )2 fragment at room temperature, in acetone solution at pH 9.5 tp 10.0 following similar procedure to those described by Ullman E.F. et al. 1976), (18), to obtain the product ^mF(ab')₂-Rh.

2.4 Coupling of ^fl^(ab^,)2־^Rh to sensing surface; producing the complete Immuno competitive unit (Product (Rh)^mF(ab^,)2⁴־S־FM). This coupling was done in two stages. The reactrions described in Example 2.4 are depicted in the reaction schemes of Fig. 22.

Product (Rh)^mF(ab*)2-⁴S-FM: was obtained by incubation of the activated ester product, which included the labelled morphine (⁴S-Bis-P.Al.-FW), with a specific immunological reagent, (Rh)^mF(ab')2.

The product is obtained in two stages. Practically, during the first stage (Fig. 22 (a)) ⁴S-FM||||||^mF(ab’)2(Rh), which is immuno-affinity conjugate, is obtained. At the second stage (scheme 2.4(b)) the amino group of ^mF(ab')2 interact with the activated carboxy to produce the amide bond by which the (Rh)^mF(ab')2 is covaletly bound to the polyalanine spacer. In the product of this stage, (Rh)^raF(ab' )2-⁴S-FM, the immuno components ((Rh)^mF(ab* )₂ and the immobilized morphine) are kept conjugated by affinity interaction.

The incubation, for a period of about 1 h was carried out in

DMF solution and trimethylamine (Bodanszky M. and Vigneaud

V. (1959), (26)). The product was then washed with phosphate buffer solution, pH 8.0, and kept in cold (4°C) until used.

Preparation of the reversible immuno competitive unit for HSA, using Texas-Red as the fluorescent label.

Strategy

3.1 - Preparation of the labelled HSA (labelled with-Texas

Red).

3.2 - Immobilization of ¹®^AF(ab’)2 fragments to the sensing surfaces.

3.3 - Covalent binding of the labelled HSA to the polyalanine spacer; production of the complete immuno competitive unit.

(a) -Conjugation of the labeled HSA to the immobilized ^HSAF(ab’)2_׳ by affinity interaction.

(b) -Covalent binding of the affinity conjugated HSA to the polyalanine spacer; production of the complete immuno competitive unit.

3.1 Preparation of HSA-Texas-Red conjugate. This reaction is depicted in Fig. 23.

Procedure: similar to those described by Chan M.A.

et al. (1987) Ref. 27.

Five mg HSA were dissolved in 30 ml of 0.1 mol/1 carbonate buffer (pH 9.1). Seven mg Texas Red sulfonylchloride solution (dissolved in 200 μΐ of DMF) was added (while stirring) at room temperature, for one hour. The reaction mixture was then dialyzed three times against 5 liters of 0.1 mol/1 NaCO₃ solution, containing 0.25 g sodium azide per liter.

3.2 Immobilization of ®^F(ab’)2 fragments to the immobilized bis-polyalanine This procedure is depicted in the scheme of Fig. 24.

a) Product ³S-Bis-P.Al.: Saponification of ²S-Bis-P.Al. under basic conditions (0.1N NaOH) as is well known in the art, yielded product ³S-Bis-P.Al.

b) Product ⁴S-Bis-P.Al.: This activated product was obtained by interaction of product ³S-Bis-P.Al. with the hetrobifunctional reagent N-hydroxysuccinimide ester of N- (4-carboxycyclohexyl methyl )-maleimide. The reaction was carried out in sodium phosphate buffer (0.1 M, pH 7.5) in DMF solution, under similar conditions to those described by

Λ

Claassen et al. (1986), (28).

The amino group on the derivitized silica (²S-Bis-P.Al.) was reacted with succinimidyl 4-maleimidylbutyrate in 0.1 M sodium phosphate buffer, pH 7.0 at 30’C. Reaction condition were similar to those described for proteins (Claassen E. et al. (1986), (28)).

c) Product ⁴S-Bis-P.Al.: The carboxy group in ³S-Bis-P.Al. was activated by N-hydroxysuccinimide in DMF solution, under conditions similar to those described by Anderson G.W. et al. (1964), (21).

d) Product ¹S-Bis-P.Al-¹³¹⁸^ (ah' )2: The ^HSAF(ab')2 was covalently bound to the derivatized active surface (⁴S-BisP.A1.) in PBS solution, pH 7.4, using similar conditions to those described by Claassen E. et al (1986), (28).

3.3 Immobilization of the labelled HSA to the polyalanine spacer;

Preparation of the immobilized immuno competitive unit (¹SBis-P.Al.(^HSAF(ab')₂)(HSA-TR)). This procedure is depicted in the scheme of Fig. 25.

Product ¹S-Bis-P.Al. (^HSAF(ab’)2)(HSA-TR): -was obtained by incubation of the activated maleimide product ¹S-BisP.Al.^HSAF(ab')2 with the labelled HSA,(HSA-TR).

The product is obtained in two stages during incubation time) Practically, during the first stage (Fig. 25 (a)) ¹S-Bis-P.Al.-^HSAF(ab2( ׳|| || (|HS^A־TR, which is immuno-affinity conjugate, is obtained) At the second stage (Fig. 25 (b)) the labelled HSA is bound covalently to the polyalanine spacer by interaction between the HSA thiol group to the maleimide) Incubation was carried out in sodium phosphate buffer (0.1 M pH 7.5) for 1 h at room temperature and then for 24 h at 4’C. The immobilized unit, (¹S-Bis-P.Al. ^HSAF(ab')2(HSA-TR)) was then well washed in PBS solution and kept at 4’C until use,

Example 4

Preparation of immobilized immuno competitive unit for HSA, based on StAv-Biotin affinity interaction.

Strategy

4.1 - Immobilization of Streptavidin (StAv) on the silica surface)

4.2 - Preparation of Biotinylated HSA-־fluor.

4.3 - Preparation of Biotinylated ^HSAF(ab')2־Rh.

4.4 - Preparation of the Biotinylated type Fig. 2(d); immuno competitive unit for HSA (conjugate Rh-F(ab' )₂ HSAfluor.).

4.5 - Immobilization of the Bis-biotinylated immuno competi- tive unit to the surface (via StAv).

4.1 Immobilizatio Streptavidin on the silica surface This procedure is depicted in the scheme of Fig. 27.

Procedures

a) Activation of the silica surface

A fiber (fused silica) having 600 pm core diameter was stripped of approximately 25 mm of cladding. The activation of the silica surface was done by MDS and GMBS following the procedure described by Bhatia et al (1989), (29).

b) Immobilization of StAv

A solution of the StAv in PBS was placed on silica coated with silane and the crosslinker (S-Su.Im.) and allowed to incubate for 1 h, at room temperature under similar conditions described by Bhatia et al (1989), (29), to obtain S-StAv.

4.2 Preparation of biotinylated HSA-F1 This procedure is depicted in the scheme of Fig. 28

a) Product HSA-F1? 4 ’-5'-Dimethoxy-5(and 6)carboxyfluorescelne -N-hydroxy succinimide ester, was reacted with HSA in phosphate buffer, pH 8.0, under experimental conditions, similar to those described by Khanna and Ullman (1980), (7).

b. Product B-P.A1.: This product was obtained by the conventi ו»מס procedure of condensation between the activated biotin and the amine group of the polyalanine (Anderson et al. (1964), 21.

c. Product B-P.ai.-diazl: This biotin derivative was prepared by a procedure similar to those described by Bayer E.A. and Wilchek M. (1980), (30).

d. Product B-P.A1.-HSA-F(ab')₂: B-P.Al.-diaz.l freshly prepared (just before use) was added to the labelled HSA (HSA-F1) in PBS solution (pH 7.5) and incubated for 1 h at room temperature, to get B-P.A1.-HSA-F1. This reaction is based on the reaction of tyrosine groups, present on the HSA protein, with the diazo reagent; Bayer E.A. and Wilchek M. (1980), (30).

4.3 Preparation of biotinylated ^H®^aF(ab')2”Hb

This procedure is depicted in the scheme of Fig. 29.

a) ⁰⁵®Fiab ’) 2Rh: N, N, N', N' -tetramethylrhodamine-6-carboxylic acid NHS ester was reacted with ^HSAF(ab')₂ in phosphate buffer, pH 8.0 under conditions similar to those described by Khanna and Ullman (1980), (7).

b) B-P.Al.-^HSaF(ab’)2־Rh: This product was obtained by reacting the labelled specific antibody ⁰®^(ab* )2 fragment with the freshly prepared diazo reagent (Bayer E.A. and Wilchek (1980), (30)).

Λ

4.4 Preparation of bis-biotinylated iamino competitive unit for HSA (conjugate B-(^HS&amp;F(ab^l )₂~Rh) 111 (HSA-Fl)-B).

. The product was obtained by incubation of the two biotinylated immunological reagents, B-P.Al-H^Fiab* )2^-Rhand B-P.A1.-HSA-F1. in PBS solution (pH 8.0) for 1 h at room temperature. Schematic description of this affinity interaction is depicted in Fig. 30.

4.5 immobilization of the biotinylated HS&amp; immuno rrpwpefritive unit on the surface This reaction is depicted in Fig. 31.

Procedure

The StAv substrate (the silica fiber or the microscope slide; (product S-StAv) was incubated for 1 h, in phosphate buffer solution (pH 8.0), with the biotinylated immuno competitive conj ugate (B- ( ^hsaf (ab') 2^-Rh) || || || (HSA-F1) -B) to get the product: (F1)HSA-8|) || || StAv |||[|| 8-^^(35¹ )₂(Kh).

w»ample 5 ״.Reversible biosensor of morphine

Fiber optic for morphine was treated as described in Example 1.2 and immobilized with morphine immuno competitive unit as described in Example 2. It has been found that the surface capacity on the fiber core (600 pm diameter, 25 mm long) was 3*10“^^ _m0!_{e O}f morphine immuno competitive units (morph.-ICU). The monitoring conditions of morphine, with the biosensor, relies on the fluorescence energy transfer technique) The immuno competitive unit in this system (Example 2 product (Rh)^mF(ab')2-⁴S-FM) containing labelled fluorescent morphine, where fluoresceine acts as the donor and (Rh)^mF(ab')2 where the rhodamine acts as the acceptor (quencher).

Schematic presentation of the sensing system is shown in Fig. 12. In this case argon laser was directed into the fiber, through the 490 nm laser line filter, to excite the immobilized fluoresceine, and fluorescence was detected at 520 nm.

All the morphine measurements were carried out in phosphate buffer (0.01 M, pH 7.5) which was prepared from NaH₂P0₄ and Na₂HPO₄, containing 1% sodium chloride and 0.1% sodium azide as a preservative) The fluorescence signal of the buffer (background) was substracted from all measurements.

Example 6 -Reversible biosensor for human serum albumin (HSA)

Immuno competitive units for HSA, as those presented in Example 4, product (Fl)HSA-B111StAv111B-^HSAF(ab ₂( י(Rh), were immobilized on the fiber optic in a similar way to that described in Example 4.

The surface capacity on the fiber core (600 pm dia1 2 meter, 25 mm long) was determined 2.10 x 10^-J־ mole of the HSA immuno competitive units (HSA-ICU).

The continuous monitoring of HSA, with this biosensor, also relies on the fluorescence energy transfer technique) The immuno competitive unit in this immuno competitive unit, contains 4,5-dimethoxy-5- (and - 6-)carboxyfluorescein labelled HSA, which acts as the energy donor, and Ν,Ν,Ν’,Ν’,tetramethylrhodamine-6-carboxyrhodamine, labelled ^^^AF(ab')₂wherein the rhodamine derivative acts as the acceptor (quencher).

Schematic presentation of the sensing system is shown in Fig. 12. The optical detection system, including excitation and fluorescence wavelength were identical to those described in Example 5 (*excitation ⁴⁹⁰־ *emission ⁵²⁰ ־nm). All measurements of HSA standard solutions were carried out in phosphate buffer (0.01 M, pH 7.5), which was prepared from NaH₂P04 and Na2HP0₄, containing 1% sodium chloride and 0.1% sodium azide as a preservative)