CA2183204A1 - Optical solid-phase biosensor based on streptavidin and biotin - Google Patents

Abstract

A new optical solid-phase biosensor based on the Förster energy transfer betweena fluorescent dye F1 and F2, for example in the form of a test strip, consists of a) a transparent support, b) an adjacent multilayer of polyanions and polycations which, as uppermost layer, contains biotinylated polylysine hydrobromide, c) a covering of the uppermost ionic layer by streptavidin, which is bound to the biotinylated layer, and d) further biotinylated biomolecules as receptors, it being possible for the fluorescent dye F1 to be bound to the streptavidin or to other biomolecules.

The analyte is furnished with the dye F2 or competes with an analyte furnished with F2 and bound to the biosensor.

Description

Le A 29 864-US / Ha /ngb/S-P - 1 - 2 1 8 3 2 0 4 Optical Solid-Phase Biosensor Based on Strentavidin and Biotin Back~round of the Invention 1. Field of the Invention The present invention relates to an optical biosensor for the detection of dissolved molecules (in the following called analytes) which can be labeled with a fluores- -cent dye and for which a biomolecule (in the following called receptor) specifi-cally recognizing these exists. What is concerned in this case is a solid-phase 10 sensor with fluorescent dye which, via an energy-transfer process to a molecule to be detected labeled with a second fluorescent dye, allows the determination of its presence and amount. Even the determination of unlabeled analytes is possible via a displacement or a sandwich reaction.

2. Description of the Related Art 15 There are various methods of detecting analytes, such as hormones, enzymes, other proteins, carbohydrates, nucleic acids, pharmacological active compounds, toxins and others, in liquid samples of biological origin. Among the known methods, immunoassays and methods related thereto stand out as a sensitive detection method for the determination of very small amounts of organic sub-20 stances. Immunoassay methods are generally based on the capability of a receptormolecule, for example an antibody, specifically to recognize the structure and molecular org~ni7~tion of a ligand molecule, be it defined by nonpolar and/or polar interactions, and to bind this molecule very specifically in such a manner.

Immunoassays are carried out using various methods. These include the use of 25 various labeling techniques, which often aim at a quantification of the analyte by means of radioactive, enzyme-coupled and fluorescent labels (E.F. Ulman, P.L. Khanna, Methods in Enzymology, 74 (1981) 28-60). Radiation-free fluorescence energy transfer (Forster energy transfer or resonant energy transfer, RET) can be considered as a special case of the last-mentioned method, using 30 which the relative geometric position of two fluorescent dyes can be measured if the mutual distance is at most a few nm. Thus the immediate interaction of a receptor/ligand pair can be directly detected (L Stryer, Annual Reviews in Biochemistry 47 (1978) 819-846). This principle has been repeatedly mentioned in Le A 29 864-US 2 1 8 3 ~ 0 4 the technology of immunoassays and biosensor systems (S.M. Barnard, D.R. Walt, Science 251, 927 (1991), EP 150 905, DE 3938598).

Moreover, the present invention relates to the immobilization of biomolecules inmolecularly thin, well-ordered layer structures which are particularly suitable for 5 an optical biosensor with Forster energy transfer as a detection principle. The immobilization of receptors on a solid phase is of crucial importance for biosensors. According to present technique, proteins are usually bound to surfaces - -adsorptively via ionic or hydrophobic interactions or they are coupled via covalent bonds using auxiliary reagents. As a meanwhile classic example of the latter 10 procedure to be mentioned, the activation of glass by 3-aminopropyl-triethoxysilane and the subsequent binding of protein with glutaraldehyde with reduction of the resulting Schiff base by sodium borohydride may be mentioned.
A review of methods used in immunoassays is found, for example, in P. Tijssen, "Practice and Theory of Enzyme Immunoassays", pp. 297-328 (Elsevier, 15 Amsterdam 1987). For biotechnological methods, in addition, encapsulation processes for enzymes in permeable polymers or membranes are customary.

While the processes which work by adsorption have the disadvantage of lack of stability of protein immobilization, covalent binding with coupling or activation reagents often requires a relatively high number of process steps, the use of highly 20 pure, in some cases unstable reagents or the use of reaction conditions underwhich not all proteins are stable. Most customary immobilization techniques havethe problem in common that the receptors are not bound regiospecifically, so that the entities important for subsequent reactions are sterically blocked in a highpercentage of the receptors. The efficiency of protein immobilization is often also 25 deficient, either due to protein denaturation or due to too low a coating of the surface with proteins. Some activating reagents are furthermore capable of crosslinking, as a result of which poorly defined surfaces result. The reproducibility of immobilization is thus also very poor. For the quantification- of the analyte concentrations by means of Forster energy transfer, in this case it turns 30 out to be unfavorable that the distance between energy donor and acceptor varies irregularly according to local surface composition, which is generally accompanied by an increase in the system-related measurement accuracy.

One possibility of solving the abovementioned system-related problems is to coatthe support with molecularly well-defined films. This can be carried out by Le A 29 864-US
- 2 1 ~3204 coating the support with a thin film-forming copolymer which, besides structure-forming units, contains comonomers having reactive groups in the side chain, which are capable of covalent bonding of the protein to be immobilized, as is described, for example, in DE 43 19 037. A disadvantage of this process is that S the number of reactive groups is restricted, as a rule, by the limited proportion of the reactive monomer in the polymer. As a result, the coating thickness of the receptors on the surface is often too low.

A further disadvantage in the concept of biosensors is the often nonspecific inter-action of proteins with the solid-phase surface. This leads to adsorption by means of hydrophobic or ionic interactions, is undesirable, and leads to nonreproducible results and to the reduction of the measuring accuracy.
Summarv of the Invention The invention relates to an optical solid-phase biosensor with biomolecules as receptors, for the specific recognition of analytes using the Forster energy transfer between two fluorescent dyes Fl and F2, consisting of a) a transparent support, b) an adj acent multilayer which consists alternately of polyanions and polycations and, as uppermost layer, contains a biotinylated polycation, the degree of biotinylation being 20-80 mol%, preferably 30-70 mol%, particu-larly preferably 40-60 mol%, based on the number of equivalent cationic groups, c) a covering of the uppermost biotinylated cationic layer by streptavidin, which is bonded to this biotinylated layer, d) further biotinylated biomolecules as receptors, preferably antibodies, which can bind to analytes labeled with a fluorescent dye F2, it being possible for the fluorescent dye Fl to be bound to the polyionic base layers, to the streptavidin or to the biomolecules binding further antibodies or to the antibodies.

Le A 29 864-US 2 1 8 3 2 Q 4 Brief Description of the Drawin~s The accompanying drawings show schematically the multilayer construction of the biosensor (Fig. 1), X-ray reflectograms of the layer structure (Fig. 2), results of ELISA measurements on sensor surfaces (Fig. 3), and the results of a biosensor 5 measurement on a sample (Fig. 4).

Detailed Description of the Invention In a preferred embodiment, to the layer described in c) are bound biotinylated receptors which, for their part, can immobilize antibodies by means of a specific recognition reaction.

10 The present invention accordingly relates to the immobilization of biomolecules, in particular of receptors or antibodies, on a solid phase, the immobilization being of permanent and directed nature and a high coating thickness of the surface with the receptor being achieved. The binding of the analyte is detected by Forster energy transfer and is reproducible and regular in its concentration-dependence due to a 15 molecularly well-defined mutual arrangement of energy donor and acceptor. Thesurface is simultaneously passivated against nonspecific adsorption of proteins. In the present invention, these requirements are followed and the abovementioned problems are solved by the immobilization of organic and biological components in molecularly well-defined layers using the natural system biotin/streptavidin.20 Streptavidin is a protein having four binding sites for biotin (vitamin H). It can therefore be used as a matrix for the coupling of biotinylated biomolecules. As the binding constant of biotin to streptavidin is ~ 10l5 M-l, the binding of biotin to streptavidin is almost irreversible.

The invention is especially characterized by the use of polycations and polyanions 25 for the construction of the multilayer.

This invention is realized by the coating of a transparent solid support, as a rule float glass or quartz glass or o-ganic polymers such as, for example, polyester,polycarbonate or polyethylene terephthalate or other transparent, nonfluorescentsolids, with a multilayer by means of the self-assembly (SA) technique by conse-30 cutive physisorption of anionic and cationic polymers. This process is described indetail in EP 472 990. In the present invention, the last layer of the multilayer LeA29 864-US 21 83204 which is physisorbed is a polycation which is biotinylated on amino groups. The polycations are biotinylated with biotin-N-hydroxysuccinimide ester or other reactive esters to 20-80 mol%, preferably to 30-70 mol%, particularly preferably to 40-60 mol%, relative to the number of equivalents of cationic groups, according to the process described in EP-A 0 472 990. Polycations which are suitable for the invention are, for example, polylysine, polyallylamine, polyvinylamine, poly-(4-vinylpyridine), polyacrylamide, polymethacrylamide, polyarginine, polyasparagine, polyglutamine, polyethylenimine and copolymers of the underlying monomers, -preferably polylysine and polyallylamine. These polycations, in which, for example, N atoms are present as ammonium groups which carry 2 or 3 H atoms, can carry, for example, as counterions: halide, such as chloride or bromide, sulfate, hydrogen sulfate, nitrate, nitrite, carbonate, hydrogen carbonate, phosphate, hydrogen phosphate, and aliphatic carboxylic acid anions, such as formate, acetate, trifluoroacetate or trichloroacetate. Possible biotinylatable polycations according to the invention are: polylysine, polyarginine, polyglutamine, polyasparagine, poly-acrylamide, polymethacrylamide, polyallylamine and copolymers of the underlying monomers, preferably polylysine and polyallylamine. Polyanions are, for example,polystyrenesulfonate (PSS), polyacrylic acid, polymethacrylic acid, poly-(2-acryl-amido-2-methyl-1-propanesulfonic acid), polyvinylsulfonic acid, polyvinyl sulfate, dextran sulfate, cellulose sulfate and copolymers of the underlying monomers, preferably polystyrene sulfonate. Countercations in the polyanions are, for example, H+, Na+, K+ and NH4+, preferably Na+ or K+. The amount of the biotinylated cation equivalent can be adjusted via the stoichiometry of the desired requirements. As a result of the incubation with streptavidin, streptavidin is bound to the biotinylated polymer layer. The surface is then almost completely and, asfluorescence experiments show, uninterruptedly coated with streptavidin. Such a system exhibits the reqirements and advantages demanded above of a biosensor.
On the one hand, the biosensor surface is screened off against nonspecific inter-actions by a coating with the protein streptavidin, which is as thick as possible, and, on the other hand, makes available a universal binding matrix for function-ali-zation of the solid interface for use as a biosensor. Owing to the labeling of the protein streptavidin with a fluorescent dye F 1 (e.g. with fluorescein isothio-cyanate), which is adequately known to the person skilled in the art, the donor dye is made available for Forster energy transfer.

After the immobilization of the streptavidin on the biotinylated polymer surface, the streptavidin with its still uncoated binding sites serves as a matrix for the ~` LeA29 864-US 21 83204 binding of further biotinylated biomolecules as receptors, in particular for thebinding of antibodies. A plurality of preferred variants for the binding of anti-bodies are possible. Thus one embodiment of the present invention is that of thecoupling of biotinylated protein A or biotinylated protein G to the streptavidin5 matrix. The biotinylation of protein A by means of N-hydroxysuccinimide or viaother reactive esters is easily possible and familiar to the person skilled in the art.
Protein A is a protein from the cell wall of the bacterium Staphylococcus aureus.
It is capable of binding immunoglobulins of the IgG type specifically on their Fc - -portion. This method moreover has the advantage of regiospecific immobilization 10 of antibodies. The Fab portion of the antibodies remains free. A reduction inimmunological activity due to blockage of the antigen binding site does not takeplace. Alternatively to binding of F1 to streptavidin, F1 can also be bound to the other biomolecules described above; binding to streptavidin, however, is preferred.

Optical solid-phase biosensors of this type can be employed, for example, in the15 form of test strips.

Another embodiment of the immunosensor comprises the binding of biotinylated antibodies to the streptavidin matrix. In this context, in turn, two embodimentsaccording to the invention are conceivable. On the one hand, the antibodies can be biotinylated by means of biotin-N-hydroxysuccinimide or via other reactive esters.
20 Such a form of biotinylation of antibodies, which is well known to the personskilled in the art, has the disadvantage that the biotinylation does not take place regiospecifically. One part of the IgG molecule is also biotinylated on or near the antigen-binding site, so that steric blocking of the latter takes place and the immunological activity of the antibodies and thus the sensitivity of the sensor 25 decreases. In another embodiment according to the invention, biotin derivatives having hydrazide reactive groups are used which react with oxidized antibodies.
During the oxidation of the antibodies, the carbohydrates located on the Fc portion of the IgG are cleaved in a reaction known to the person skilled in the art (glycol cleavage) and produce aldehydes. These react with the hydrazide groups of the 30 biotin derivatives with formation of hydrazones. The biotin groups are thereby regiospecifically bound to the Fc portion of the antibodies. The biotinylated anti-bodies are coupled to the streptavidin matrix and complete the test strip surface of the immunosensor without coupling via biotinylated protein A being necessary.

LeA29 864-US 21 83204 The test strip is then capable of recognizing and quantifying the analyte provided with a suitable acceptor dye F2 (e.g. rhodamine isothiocyanate) as a result of interaction of the receptor, in particular of the antibody, with the analyte. The analyte is detected by simple bringing into contact of the coated support with the 5 solution in which a molecule is suspected as an analyte (sample solution) and sub-sequent fluorescence measurement. The fluorescence of the donor dye (F1) and of the acceptor dye (F2) is measured. If there is an analyte labeled with the acceptor dye F2 in the test liquid (sample solution), after specific binding thereof to- the - -immobilized antibodies, as a result of Forster energy transfer the intensity of the 10 acceptor fluorescence is increased and that of the donor is decreased compared to the unbound state. Alternatively, if unlabeled analyte is to be determined by means of a displacement reaction, the test strip is first equilibrated with an acceptor-labeled analog of the analyte concerned. In this state, the acceptor fluorescence of F2 then outweighs the donor fluorescence of Fl. If unlabeled analyte from the test 15 liquid then comes into contact with the equilibrated test strip, after the displacement reaction the Forster energy transfer is interrupted so that an increase in the donor fluorescence of Fl and a decrease in the acceptor fluorescence of F2 signals the binding of the unlabeled analyte. In both cases, the change in acceptor and donor fluorescence is clearly connected with the concentration of the analyte.
20 The Forster energy transfer can be measured in customary fluorescence spectro-meters, but also in specially designed apparatuses for an energy-transfer immuno-sensor. By means of suitable calibration curves, the concentration of the analyte in the analysis liquid can then be determined. In a further embodiment, a specifically binding molecule labeled with the fluorescent dye F2 which competes in the 25 presence of the analyte to be detected with this for binding to the uppermost layer of the biosensor is added in a known amount to the analysis liquid in which the presence of the analyte to be detected is suspected. The concentration of the analyte to be detected is then measured by means of the dependence of the fluorescence intensities of F2 or of Fl or the ratio of the two intensities.

30 Another embodiment according to the invention of the immunosensor comprises the binding of the donor dye Fl to the receptor, preferably the antibody, preferably via hydrazide reactive groups of oxidized antibodies in the form described above.
In this embodiment, the efficiency of Forster energy transfer is increased by a decrease in the average distance between donor (F1) dye and acceptor (F2) dye, 35 which can be used to increase the sensitivity of the test method.

Le A 29 864-US 2 1 8 3 2 0 4 The invention is illustrated in greater detail by the following figures:

Figure 1 shows schematically, and approximately to scale, the multilayer construc-tion of the test strips for the Forster energy transfer immunosensor (at the top), and the detection principle (at the bottom).

Fi~ure 1: Schematic construction and function of the immuno-sensor. The molecular components are drawn ~~
approximately to scale. The support I is much thicker than shown.

At the top: Test strip, consisting of transparent support I (e.g. glass), a polyelectrolyte multilayer II (for simplification of the figure only the last biotinylated layer is shown), a layer of strepta-vidin fluorescence-labeled with F1 III, a layer of biotinylated protein A IV and the antibody V immobilized thereon.

At the bottom: After wetting with test liquid, the wavelength of the fluores-cence observed depends, after excitation of the fluorescent dye F1 coupled to streptavidin, on whether direct emission is present (dotted arrow) or whether the excitation energy after energy transfer (RET = dashed arrow) to the labeled analyte is emitted with a red shift (full arrow). The ratio of red-shifted to direct emission intensity is clearly dependent on the number of bound analyte molecules per unit area.

Figure 2 shows X-ray reflectograms of the layer structure in various stages of preparation.

Fi~ure 2: X-ray reflectograms of the layer structure (support material: silicon) in various stages of preparation.
The various sets of data were in each case shifted by a factor of 100 compared with one another. From the bottom to the top, the curves show first the poly-electrolyte multilayer, then the latter, coated with streptavidin, subsequently still coated with bio-tinylated protein A and f1nally provided with IgG

Le A 29 864-US 2 1 8 3 2 0 4 g antibody. The pulse transfer Qz is plotted in ~~1 on the abscissa and the X-ray intensity I on the ordinate.

The measurement shows for the present example that the surface is coated with aninterface layer of organic material grown regularly in the various preparation steps 5 and in each step the preparation remains smooth in molecular terms. This meansin particular that the surface functionalization does not lead to the coating of the solid phase interface with laterally inhomogeneous structures, i.e. droplet forma- - ~
tion does not take place even on the nanometer linear scale.

Figure 3 shows the results of ELISA measurements on sensor surfaces which were 10 prepared analogously to the methods indicated in Figure 1 and recorded in Figure 2 by means of X-ray reflectivity measurements.

Fi~ure 3: Titration of various sensor surfaces with antigen.
Specific binding was detected by means of ELISA. A
preparation in which IgG was adsorbed electrostati-cally on a PSS layer (white diamonds) was compared with a sensor which was prepared by the technique described here (black diamonds). The antigen concen-tration CAG in mol/l is given on the abscissa and the optical density A at ~ = 414 nm on the ordinate.

20 Figure 4 shows the results of a biosensor measurement on a sample which was prepared as the sample described in Figure 3.

Fi~ure 4: Titration of a sensor surface with antigen (black dia-monds). Specific binding was detected by means of energy transfer. The antigen was present in the culture supernatant at about 2-fold dilution. Control (white diamonds): antigen adsorption on a test Skip which was only built up to the streptavidin layer (without protein A and receptor layer). The abscissa shows the concentration CAG in the culture super-natant in mol/l. The ordinate shows the intensity ratio of the fluorescences of the fluorescent dyes Fl and F2 at 577 and 530 nm.

~- LeA29 864-US 21 83204 Examples:

1. Chemicals used Polymers: Polystyrenesulfonate, sodium salt (PSS), MW = 70,000, Aldrich Polyallylamine hydrochloride (PAH), MW = 50,000 -60,000, Aldrich Polyethylenimine (PEI), MW = 50,000, 50 % strength --solution in H2O, Aldrich Poly-L-lysine hydrobromide (PL), MW < 50,000, Bachem-I 0 Biochemica The PSS was dialyzed against very pure water for two days in aqueous solution ina VISKING27/32 dialysis tube from Roth and then freeze-dried.

Proteins: Streptavidin, Boehringer-Mannheim Protein A, Pharmacia Rabbit IgG, polyclonal, specificity: anti-mouse IgG, Immunol. Institute Univ. Mainz Bovine Serum Albumin (BSA), Sigma Antigens: Mouse IgG, monoclonal, culture supernatant, Immunol.
Institute Univ. Mainz Horseradish peroxidase (HPO)-coupled mouse IgG, affinity purified, Jackson Immuno-Research Laboratories, Dianova, U.S.A.

Fluorophores: Rhodamine B isothiocyanate (RITC), Sigma Fluorescein isothiocyanate isomer I (FITC), Sigma 25 The labeling of the streptavidin was on average 1.4 FITC per protein molecule and that of the mouse IgG on average 3 RITC per protein molecule.

Biotin active ester: Biotinamidocaproyl-N-hydroxysuccinimide ester, Sigma To biotinylate protein A, the active ester and the protein were weighed in a molecular ratio of 12:1.

LeA29 864-US 21 83204 Detergent: Polyoxyethylene sorbitan monolaurate (TWEEN 20), Sigma PBS buffer: Sodium dihydrogen phosphate, monohydrate, p.a., Merck Disodium hydrogen phosphate, p.a., Merck Sodium chloride, p.a., Merck 5Citrate buffer: Disodium hydrogen phosphate, p.a., Merck Citric acid monohydrate, Sigma - - -Potassium chloride, p.a., Merck Magnesium chloride, hexahydrate, p.a., Merck ABTS: 2,2'-Azino-bis(3-ethyl-benzothiazoline)-sulfonic acid, Sigma Glass substrate: (38x12) mm2 and (1-1.2) mm thick microscope slide, Gebr.
Rettberg GmbH.

2. Cleaning of support The support was cleaned according to a standard procedure (W. Kern, D.A. Puotinen, RCA Review, 31 (1970), 187) 3. Preparation of support All solutions were prepared using distilled water. The water-wetted supports were placed in PEI solution (diluted to 2.2 mg/ml) for 30 minutes and then washed in 10 ml of water three times for about 30 seconds in each case and then blown dry in a gentle stream of nitrogen. The sample was then placed in a PSS solution (20 mg of PSS in 10 ml of a 2 M NaCl solution) for about 20 minutes and washed and dried as described above. The support was placed in a PAH solution (20 mg of PAH in 10 ml of 2 M NaCl solution) for a further 20 minutes and again washed and dried. As described above, a further PSS layer, a PAH layer and, in turn, a PSS layer were adsorbed on the support. For functionalization with biotin, the support was placed in a solution of biotinylated polylysine hydro-bromide (PLB) (5 mg/10 ml in 0.4 M NaCl) for 20 minutes and then washed and dried again. The coated supports were stored at a temperature of 4C until use.

4. Building-up of the protein heteromultilayers:
PLB-coated supports were placed for about 30 minutes in an FITC-streptavidin solution (10-7 mol/l of FITC-streptavidin in 10 mM PBS buffer, pH = 7.2, Le A 29 864-US 21 83204 150 mM NaCl), washed three times with 10 ml of water and then placed for a further 40 minutes in a solution of biotinylated protein A (5x10-7 mol/l, PBS
buffer as above) and washed three times with the pure PBS buffer. Drying steps between the individual protein coatings were dispensed with. The support coated 5 with protein A was placed for 40 minutes in a rabbit IgG solution (specificity:
anti-mouse IgG, 5x10-7 mol/l, PBS buffer as above) and washed three times with the pure buffer. Up to binding of the antigen, the support was stored in PBS
buffer. - -Figure 2 shows X-ray refiectograms which were measured (dry sample) between 10 the various adsorption steps during the preparation of a test strip (support material silicon). These results confirm that during the adsorption process a coherent layer structure is formed which is uniform on a linear molecular scale, that the layerthickness increments in each case correspond to the molecular dimensions of the absorbents and that the surface remains smooth in molecular terms after each 15 adsorption step. An exception is the terminal antibody layer, which because of the elongated molecular form of the antibody and the regioselective binding by protein A contains a high amount of aqueous buffer. This layer collapses in the drying process during measurement and after this appears significantly thinner than is to be expected from the molecular dimensions. The experimental data from Figure 2 20 are evaluated quantitatively in Table 1.

Table 1: Molecular dimensions of the active interface layer of the immuno-sensor Coating steps Layer Surface roughness thickness org. interface/air (A) increment (A) 6 molecular polyelectrolyte layers 203 6 (incl. PLB) Streptavidin layer on polyelectrolyte 56 11 film/PLB
Biotinylated protein A on streptavidin 7 11 layer Rabbit IgG (anti-mouse) on protein A 13 17 Le A 29 864-US 2 1 8 3 2 0 4 5. Incubation with antigen, including controls a. ELISA measurements Samples were prepared on silicon as described above. 10 mg/ml of BSA were dissolved in PBS buffer containing 0.1 % TWEEN 20. This protein solution was 5 used for ELISA measurements for the preparation of a dilution series of the HPO-labeled antigen (mouse IgG-HPO), whose concentration was between 10-l3 and 10-7 mol/l. After incubation with the antigen, the samples were developed in --citrate buffer with 3 g/l of ABTS and 0.0075 % H2O2 and measured. Figure 3 shows a representative result for a preparation which was prepared by the techno-10 logy presented here. Compared with this is the titration of a preparation in whichIgG was only adsorbed electrostatically on a silicon interface which was coated with a thin molecular layer of PSS. The higher sensitivity, better linearity andlower nonspecific adsorption which distinguish the preparation prepared by the new technique are clearly visible in the figure.

15 b. Fluorescence measurements Samples were prepared on float glass as described above. 10 mg/ml of BSA were dissolved in PBS buffer. This protein solution was used to prepare a dilution series of the RITC-labeled antigen. In this dilution series, the concentration of the antigen was between 2.5xlO-9 and 5x10-6 mol/l. The supports were placed for 20 about 40 minutes into the solution of a certain antigen concentration and then washed 5 times for about 1 minute in a PBS buffer (buffer composition as above) treated with 0.1 % TWEEN 20. The supports were blown dry in a stream of nitrogen and stored in darkness at 4C until measurement.

To quantify the nonspecific interaction of the antigen with the substrate, supports 25 were coated in the same sequence up to and including the streptavidin layer (but without protein A and IgG layers) and placed for about 40 minutes in an antigen-containing solution of the dilution series in each case and, as described above,washed and dried.

The sample and reference supports were measured both in a conventional 30 fluorescence spectrometer and a specially designed apparatus for the measurement of fluorescence energy transfer in the dry state. Figure 4 shows representative results.

Claims (15)

1) An optical solid-phase biosensor with biomolecules as receptors, for the specific recognition of analytes using Förster energy transfer between two fluorescent dyes F1 and F2, consisting of a) a transparent support, b) an adjacent multilayer which consists alternately of polyanions and polycations and, as uppermost layer, contains a biotinylated polycation, the degree of biotinylation being 20-80 mol%, based on the number of equivalents of cationic groups, c) a covering of the uppermost biotinylated cationic layer by strepta-vidin, which is bonded to this biotinylated layer, d) further biotinylated biomolecules as receptors, which can bind to analytes labeled with a fluorescent dye F2, it being possible for the fluorescent dye F1 to be bound to the polyionic base layers, to the streptavidin or to the biomolecules binding further antibodies or to the antibodies.

2) The optical biosensor of claim 1, wherein according to b) the degree of biotinylation is 30-70 mol%, based on the number of equivalents of cationic groups.

3) The optical biosensor of claim 2, wherein according to b) the degree of biotinylation is 40-60 mol%, based on the number of equivalents of cationic groups.

4) The optical biosensor of claim 1, wherein according to d) the further bio-tinylated biomolecules are antibodies.

5) The optical biosensor of claim 1, in which the donor dye F1 is bound to streptavidin.

6) The optical biosensor of claim 1, in which the receptors are bound to the streptavidin layer by means of biotinylated biomolecules which themselves are not antibodies.

7) The optical biosensor of claim 6, in which the receptors, which are bound to the streptavidin layer by means of biotinylated biomolecules which themselves are not antibodies, are antibodies.

8) The optical biosensor of claim 6, in which the receptors are bound to the streptavidin layer via protein A or protein G.

9) The optical biosensor of claim 7, in which the antibodies are bound to the streptavidin layer via protein A or protein G.

10) The optical biosensor of claim 1, in which the receptors which are biotiny-lated are bound to the streptavidin layer.

11) The optical biosensor of claim 10, in which the receptors are antibodies.

12) The optical biosensor of claim 1, in which the analyte molecule displaces another molecule bound to the uppermost layer and labeled with the fluorescent dye F2, which is not the analyte, from the uppermost layer and the concentration of the finally bound analyte molecule is measured as a function of the decrease in the fluorescence intensity of F2 or the increase in the fluorescence intensity of F1 or the change in the ratio of the two intensities.

13) The optical biosensor of claim 1, in which the analyte molecule competes with a specifically binding molecule labeled with the fluorescent dye F2, which is not the analyte and which is added to the analysis solution in a known amount, for binding to the uppermost layer of the biosensor, and the concentration of the analyte is measured as a function of the fluorescence intensity of F2 or of F1 or the ratio of the two intensities.

14. An optical biosensor of claim 1, in which the support material employed is a transparent, nonfluorescent solid.

15. An optical biosensor of claim 1, in which the support material employed is float glass, quartz glass, polyester, polyethylene terephthalate or polycarbonate.