WO2005036171A1

WO2005036171A1 - Method and system for detection of a target analyte

Info

Publication number: WO2005036171A1
Application number: PCT/IB2004/051961
Authority: WO
Inventors: Henk Stapert; Joke Orsel; Nico Willard
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2003-10-09
Filing date: 2004-10-04
Publication date: 2005-04-21

Abstract

The present invention relates to a method for detection of a target molecule or a particle having several different recognition sites in a complex mixture of different biological compounds, comprising the steps of: a) immobilizing, as primary affinity probes, at least two different biorecognition probes selected from the group consisting of (photo)aptamers, carbohydrates, peptides, lipids, metabolites, proteins, cofactors, hormones, cytokines, cells, micro-organisms, virus, drugs, pesticides, herbicides, fungicides or vitamins in a homogeneously mixed manner on a solid substrate, each probe being specific for the same or a different recognition site of the same target molecule or particle, b) exposing the substrate to the complex mixture to allow association between target molecules or particles and the probes to occur, c) optionally removing the complex mixture, d) optionally rinsing the substrate to remove non-specifically bound material, and e) specifically assaying the probe-bound target molecules or particle; and to a biorecognition probe system for detection of a target molecule or particle having several different recognition sites, comprising at least two different biorecognition probes which are immobilized in a homogeneously mixed manner on a solid substrate, each probe being specific for the same or a different recognition site of the same target molecule or particle.

Description

METHOD AND SYSTEM FOR DETECTION OF A TARGET ANALYTE

The present invention relates to a method for detection of a target molecule or a target particle and biorecognition probes suitable for that method.

Nature has developed ways to increase the interactions between different biological species. For example the simultaneous formation of multiple protein- carbohydrate interactions is one binding mode that can be exploited to achieve the necessary avidity, i.e. the binding strength with which a multivalent antibody binds a multivalent antigen. In physiological settings, saccharide epitopes and their protein receptors are arranged such that multiple binding events can occur simultaneously. Naturally occurring carbohydrate displays are widespread: examples include highly glycosylated proteins (e.g. mucins), the carbohydrate surfaces of bacteria and other pathogens, and the outer membranes of mammalian cells. The interaction of multivalent presentations can result in the formation of numerous simultaneous complexation events that proceed to afford a high observed affinity and a high functional affinity (refs: Bertozzi, C.R., Kiessling, L.L., Science, 291, 2357 (2001) and Mammen, M., Choi, S.-K., Whitesides, G.M., Angew. Chem. Int. Ed., 37, 2754 (1998). Also, prior art is known describing multivalent glycoconjugates as well as carbohydrate assays, wherein more than one glycan is coupled to a polymer, which in turn is bound to a surface. The affinities towards the cell adhesion molecules P- and L-selectins reported for the surfaces displaying the multivalent ligands, are five to six fold better than the affinities for a surface modified with the corresponding monovalexit ligand. Further, calixarenes are reported with four peptide loops as an antibody mimic and other approaches to artificial receptor design, see D. Wang et al., Nature Biotechnology, 20, 275-281(2002), L. L. Kiessling and C. W. Cairo, Nature Biotechnology, 20, 234-235(2002), J. E. Gestwicki et al., Anal. Biochem., 305, 149- 155(2002); R. Roy, Curr. Opin. Struct. Biol., 1996, 6:692-702; Y. Hamuro et al., Angew. Chem. Int. Ed. Engl., 1997, 36, 23, 2680-2683; A. D. Hamilton and P. Kazanjian, Tetrahedron Lett., 26,47, 5735-5738 (1985); M. W. Peczuh and A. D. Hamilton, Chem. Rev. 2000, 100, 2479-2494; M. S. Goodman et al., J. Am. Chem. Soc. 1995, 117, 11610-11611. Aptamers are macromolecules composed of nucleic acid, such as RNA or DNA, that associate with a specific target molecule. Like all nucleic acids, a particular aptamer may be described by a linear sequence of nucleotides or nucleotide derivatives. The chain of nucleotides may form intramolecular interactions that fold the molecule into a complex three-dimensional shape. The shape of the aptamer allows it to associate with the surface of its target molecule. Because an extraordinary diversity of molecular shapes exists for all possible nucleotide sequences, aptamers may be obtained for a wide array of molecular targets, including proteins, drugs, metabolites and most other small molecules. For many applications in molecular diagnostics it is highly desirable to be able to measure low concentrations of a target molecule in complex mixtures. Such target molecules may be of any origin and may include proteins, cofactors, metabolites, drugs and most other small molecules. Current analytical methods are able to detect ^■ down to pmole/liter concentrations of a target molecule, but these methods require expensive analytical instruments and highly specific capture probes to be able to associate with the target molecule in a sufficiently strong manner. The specificity of a biorecognition probe is given by its affinity constant K_a (also called equilibrium binding constant). Detection of concentrations lower than 100 pM is often limited by the absence of suitable recognition probes with a high enough affinity. At concentrations lower than pM, the detection limit of currently used detection principles, such as fluorescence intensity measurements, is another limiting factor. Therefore, measuring low concentrations (below ng/ml) of different target molecules in complex mixtures is severely limited by the low affinity constants of biorecognition probes . Another drawback of the prior art is the relatively poor stability of the biorecognition probes, such as proteins (antibodies). The affinity of antibodies usually decreases significantly when they become immobilized to a surface. Furthermore, the biological activity of proteins decreases over time and is very sensitive to changes in temperature, relative humidity, ion strength, pH, etc. A further drawback of the prior art is that antibodies cannot be produced synthetically, i.e. without the use of bacteria or cells. Another drawback of the prior art is that for single aptamers made against larger molecules, such as larger proteins, a high affinity is not often achieved.

It is an object of the present invention, to provide a method for detection of a target molecule or particle in a complex mixture of different biological compounds, which overcomes the drawbacks of the prior art, which especially enables a detection of a target molecule in very low concentrations. Further, it is an object of the present invention to provide biorecognition probes, which may be used in the method of the invention. The first object is achieved by a method for detection of a target molecule or a target particle having several different recognition sites in a complex mixture of different biological compounds, comprising the steps of: a) immobilizing, as primary affinity probes, at least two different biorecognition probes selected from the group consisting of (photo)aptamers, carbohydrates, peptides, lipids, metabolites, proteins, cofactors, hormones, cytokines, cells, micro-organisms, virus, drugs, pesticides, herbicides, fungicides and vitamins, in a homogeneously mixed manner on a solid substrate, each probe being specific for the same or a different recognition site of the same target molecule or particle, b) exposing the substrate to the complex mixture to allow association between target molecules or particles and the probes to occur, c) optionally removing the complex mixture, d) optionally rinsing the substrate to remove non-specifically bound material, and e) specifically assaying the probe-bound target molecules or particles, or f) detecting the probe-bound target or probe-target complex. Preferably, the method comprises: el) specifically binding at least one secondary affinity probe to the probe-bound target molecule or the target-primary probe complex, e2) optionally removing unbound secondary affinity probes, and e3) detecting the secondary affinity probes or the complexes of secondary affinity probe and primary probe-bound target molecule. Preferably, the primary affinity probes are two different (photo)aptamers. More preferably, the secondary affinity probe is chosen from the group of the primary affinity probes and/or the secondary affinity probe carries a label, such as a (chemo)luminescent, electroluminescent, magnetic, redox-active or radio-active label or an enzyme. Preferred secondary affinity probes are antibodies and (photo)aptamers. Further, a method is preferred, wherein at least one of the different biorecognition probes is a (photo)aptamer. There are several variants of the sandwich assay known, which may influence the sequence of steps a) to e) as well as slightly changing the steps. Notably, one may first expose the complex mixture to secondary affinity probes to "fish" for the targets in the complex mixture and subsequently lead the complexes over the substrate with the different primary affinity probes, after which steps el), e2), e3) are preformed. Another variant is to use a tertiary (labelled) affinity probe after the sandwich of primary probe, target and secondary probe has been formed. This tertiary affinity probe has a high affinity for all secondary affinity probes. Other variants of the sandwich assay are a displacement assay and competitive assay. It will be appreciated that the person skilled in the art is able to design and perform such an assay based on the primary objective of the invention. In another alternative embodiment of the invention a method is proposed, wherein, as primary affinity probes, at least two different biorecognition probes are selected from the group consisting of (photo)aptamers, carbohydrates, peptides and lipids, antibodies, proteins, vitamins and other small capture probes and step e) comprises: el') specifically staining the probe-bound target molecule or particles, e2') optionally removing un-reacted stain, and e3') detecting stained probe-bound target molecules or particles. In this embodiment it is necessary that the primary affinity probes are not reactive towards the stain. To this end groups reactive towards the stain may optionally be blocked in the primary and/or secondary affinity probes before being brought into contact with the analyte. The non-specific stain should be chosen such that it does not react with the primary affinity probe (aptamers, lipids, carbohydrates, etc.). The stain itself may be a luminophore, a redox-active compound, en enzyme, a luminescent particle, a (super)paramagnetic particle or a ferro-electric particle. One could target primary amine groups or carboxylic groups or sulfurhydryl and/or alcohol groups, which are present in bound proteins. It is for the person skilled in the art possible to choose the right nonspecific stain. Preferably, the different probes are two different (photo)aptamers, two different carbohydrates, such as oligosaccharides, one (photo)aptamer and one carbohydrate, or the like. Still preferably, the two different probes are two different (photo)aptamers. It will be appreciated that the substrate may consist of several specially separated areas such that each area contains different biorecognition probes for a specific target molecule, being the same or a different target molecule. In this way one can build-in redundancy to improve the analytical accuracy for a measurement of a certain target and one can detect for different target molecules on the same substrate as well. The location of the immobilized probes will allow for identification of a certain target. The exposure of the substrate to the complex mixture can be achieved by immersing it into the complex mixture. Often the substrate is mounted in a flow cell chamber in which the complex mixture is injected and subsequently spreads over the substrate. The flowing of liquid can be directed by (microfluidic) channels, if necessary. Usually, the complex mixture will be brought to the substrate. In practice it is unlikely that more than five different probes, especially aptamers may bind to one target molecule at the same time. Preferably the target molecule or particle is a protein, cofactor, hormone, peptide, (poly)saccharide, lipid, pesticide, herbicide, fungicide, cell, micro-organism, virus, drug, toxin, metabolite or any other small molecule, or any combination thereof (e.g. protein with bound ligand, or bound nucleic acids (ribosome), or carbohydrate- protein-complex) . Furthermore the complex mixture of different biological compounds may be a bodily fluid, such as blood, urine, saliva, lung fluid, cerebrospinal fluid, cell extract, plasma or serum. Alternatively the complex mixture of different biological compounds is waste water, any fluid in industrial processing, milk, drinking water, surface water or any other food product or solution thereof. In one embodiment the complex mixture is undiluted or diluted with a solvent, wherein an appropriate solvent may be easily chosen by someone skilled in the art. In a further embodiment the presence of the target molecule(s) or particle(s) is qualitatively determined and/or the concentration of the target molecule or particle in the mixture is determined. Depending on the application a quantitative and/or qualitative determination of target molecule(s) is (are) preferred. For example, when detecting hCG hormone a qualitative test suffices to diagnose pregnancy. In many clinical chemical tests however it is necessary to measure (or even monitor) quantitative amounts of target molecules in order to determine the (changing) health status of a patient. The concentration of the target molecule or particle may be about one millimole/liter, preferably below one nanomole/liter, most preferably below 10 picomole/liter. Further the substrate may be flat. The substrate, including an optional surface coating, may contain flat recessed or elevated parts. An example for a flat layer is a self assembled monolayer of (mixtures of) thiolates on gold or (mixtures of) siloxanes on glass. The monolayer preferably has a functional group accessible for the immobilization of the probe molecule.

Alternatively the surface can exist of a polymer absorbed or bound to a flat or pre- structured substrate, e.g. plastic, metal layer (e.g. gold), glass, silicon, silicon nitride, metal-oxide layer (e.g. aluminium oxides, titanium dioxide, tantalum oxides, silicon oxides, indium tin oxides, indium zinc oxides) etc. and combinations of such materials. The polymer can be, but is not limited to, a (block)copolymer of e.g. polyacrylamide and polyacrylic acid or a graft copolymer of polylysine and polyethylene glycol. Preferably the surface is modified in such a manner as to minimize unspecific adsorption of compounds present in the complex mixture. Alternatively, the substrate is a three-dimensional network, such as a cross-linked polymer network or a hydrogel, wherein a swollen organic network is preferred. For example the hydrogel is a dextran, functionalised with e.g. primary amine groups or carboxylic acid groups. Still alternatively, the substrate is an organic, metallic or inorganic microporous or nanoporous substrate or membrane with small average pore sizes, preferably below 100 μm, more preferably below 10 μm, most preferably below 1 μm. For example, the microporous membrane can be an Al₂O₃ membrane, polycarbonate, aluminium oxide or silicon oxide with small three-dimensional pores with a diameter of for example 0.2 micrometers. Alternatively, the substrate may be a micro-sized or nano-sized particle with diameters ranging from 1 nm to 5 μm. For example the particle is a colloidal gold particle with sizes ranging from 20 to 40 nm; or a super-paramagnetic particle with sizes ranging from 35 nm to 2.8 μm; or a luminescent inorganic semiconducting particle ("quantum dot") with a size ranging from 2 to 15 nm. In one embodiment the spatial arrangement of the probes on the substrate corresponds to the spatial arrangement of the respective recognition sites on the target molecule. Preferably a fitting spatial arrangement of the probes is achieved by attachment of the probes to the substrate via a flexible linker, such as oligomethylene or oligo(ethylene glycol), preferably with a length of more than 6 atoms. In one embodiment the immobilized probes contain two or more target binding sites, either in separate molecules or combined in one molecule, which are derived from probes with an affinity for different parts of the target molecule or particle. In an alternative embodiment the immobilized probes contain two or more target binding sites, either in separate molecules or combined in one molecule, which are derived from probes with different on and off rates for identical recognition sites of a (multivalent) target molecule or particle. Preferably the captured target molecule or particle is detected using luminescence intensity, luminescence lifetime or luminescence polarisation measurements, amperometric measurements, voltammetric measurements, magnetic measurements, surface acoustic waves, impedimetric measurements, dielectric measurements, radio-active measurements, electroluminescent measurements, chemoluminescent measurements, absorption measurements, interferometric measurements, reflective measurement, colorimetric measurements, quartz crystal microbalance measurements, surface plasmon resonance and/or other evanescent field techniques. It will be immediately clear for the expert in the field that certain labels are used in combination with a specific detection technology. For example when detecting luminescent intensity, the target molecules can be labelled with a luminophore, quantum dot, luminescent beads, etc. For example when detecting magnetically, the target molecules can be labelled with super paramagnetic nanoparticles or microparticles. Surface plasmon resonance, surface acoustic waves, impedimetric measurements, dielectric measurements, absorption measurement, interferometric measurements, reflective measurements and quartz crystal micro balance measurements may be performed with or without using a label. The second object is achieved by a biorecognition probe system for detection of a target molecule or particle having several different recognition sites, comprising at least two different biorecognition probes which are immobilized in a homogeneously mixed manner on a solid substrate, each probe being specific for the same or a different recognition site of the same target molecule or particle. In one embodiment, the immobilized probes are specific for the same recognition site on a multivalent target molecule, but with different on and off rates. The combination of probes, preferably aptamers, used contains preferably at least one aptamer with a high on rate (very fast association with target molecule) and at least one aptamer with a very low off rate (very slow dissociation from target molecule). In yet another embodiment, a molecule is immobilized that contains the target recognition sites from different probes used in the two previous embodiments, a so-called "multi-probe". The recognition sites in the "multi-probe" are preferably spaced in a manner to best complement the spatial layout of the recognition sites on the target molecule or particle. Possible methods to construct a "multi-probe" could be by synthesizing two or more different probes, preferably aptamers, single chain antibodies; peptides; sugars or mixtures of the mentioned bioprobe types (e.g. an aptamer and a single chain antibody) each terminally modified with a (flexible) linker and covalently linking the ends of the linkers. An example would be to allow formation of a disulfϊde bridge between two thiol-modified linker-aptamer molecules and then chemisorption of the disulfide bridge onto gold, effectively causing immobilization of two different aptamers in close proximity. An alternative example is the attachment of two different aptamers each on one side of a (flexible) linker and containing multiple attachment points, and which harbours a moiety that can react with the substrate used. Preferably, the substrate is flat. The substrate, including an optional surface coating may contain flat recessed or elevated parts. Alternatively, the substrate is a three-dimensional network, such as a cross-linked polymer network or a hydrogel. The three-dimensional network may be immobilized on one or more flat substrates described above. Still alternatively, the substrate is an organic, metallic or inorganic microporous or nanoporous substrate or membrane with small average pore size, preferably below 100 μm, more preferably below 10 μm, most preferably below 1 μm. Alternatively, the substrate may be a microsized or nanosized particle with sizes ranging from 1 nm to 5 μm. For example the particle is a colloidal gold particle with a size of 20-40 nm; or a super paramagnetic particle with sizes ranging from 35 nm to 2.8 μm; or a luminescent inorganic semiconducting particle ("quantum dot") with a size ranging from 2 to 10 nm. Preferably, the biorecognition probes have a high on and low off rate. Aptamers with a high on and high off rate are preferred if it results in a high equilibrium affinity constant in combination with a second aptamer that has a low on and off rate. The inclusion of probes with relatively high on and off rates in combination with other probes with relatively low on and off rates is not known in the prior art, since currently probes are selected on their low off rates only, in order to increase the overall affinity. According to the invention, probes, especially aptamers, with different off rates for the same recognition sites, or different specificities for the same target molecule or particle are generated. The selection process for generation of probes can be modified to select specifically for such probes. For example for the generation of an. aptamer with a low off rate, in each round of the selection process aptamer-target complexes can be subjected to prolonged washing under continuous flow. To generate different aptamers against the same target, aptamers surviving after for example two or three rounds of selection could for example be cloned and each separately subjected to following selection rounds. Additionally, to generate probes specific for different sites on the same target molecule or particle, separate selection processes can be performed with different parts of the target molecule or particle. For example, to generate aptamers for different parts of a protein, separate selection processes can be performed with peptides identical to different parts of the protein or with different fragments of the protein, for example isolated after a tryptic digest of the protein. Finally, the biorecognition probe systems according to the present invention may be used in a method according to the present invention. Surprisingly it was found, that it is possible to increase the affinity for binding of target molecules, such as proteins, to capture molecules by using more than one specific biorecognition probe supported on a substrate for a single target molecule. Therefore, the target molecule may be detected with very low concentrations. The present invention makes preferably use of (more than one) aptamers , peptides, lipids, saccharides or small molecules, such as arylsulfonamide, as biorecognition probes. Aptamers, peptides, lipids, saccharides or small molecules are much more stable than proteins and hardly loose their biological affinity upon binding to a surface. Furthermore, when more than one probe is used for one target molecule or particle the overall stability increases because the chance that two different probes loose (part of) their affinity is smaller than for only one probe. Aptamers can be more easily produced in larger quantities than antibodies, can more easily be remade and will have less batch-to-batch variation. The present invention is more effective for detection larger molecules and provides a solution for the low aptamer affinities found for larger molecules. The affinity constant of each of the different capture probe aptamers is lower than the overall affinity constant of a co-operative association. According to the present invention especially proteins are suitable target molecules, since proteins are molecules that are large enough to have several different recognition sites. The present invention can be seen as an analogue to antibody avidity. Immunoglobulin G (IgG) antibodies is the class of antibodies used most in immunoassays. These each have two identical target recognition sites (paratopes) and are thus bivalent. Because the two paratopes will influence each other, it is difficult to calculate the individual affinities. Therefore, the strength with which a multivalent antibody binds a multivalent antigen is called avidity. The avidity of a bivalent antibody (IgG) for a multivalent antigen can be a thousand fold stronger than the affinity of only one of the paratopes. When probes are immobilized on a surface, they may have a relatively strong apparent affinity constant for multivalent analytes, as the immobilization may cause more than one probe to bind the same target. The present invention allows the same principle to be applied to targets that are not multivalent. Besides using a mixture of at least two different probes with different specificities, for multivalent targets the use of at least two different probes with the same specificity, but with different on and off rates is preferred, preferably at least one probe with a high on rate (very fast association with target) combined with at least one probe with a low off rate (very slow dissociation from target). In this way the best characteristics of two different probes, each with a possibly relatively low affinity of their own, can be combined to achieve a very high overall affinity. For the strength of this synergistic or cooperative association of aptamers and/or other (synthetic) capture molecules with a target, the term "capvidity" is used herein. The resulting capvidity constant (Kcapv) may be estimated according to the following: Consider a protein P that can be bound by aptamer 1 (Ai) and aptamer 2 (A₂). The complex (Ai A₂P) formation at equilibrium between the three molecules can be described by four different affinities Ki, K₂. Kι₂ and K₂ι according to the following scheme:

with Ki = k_on,ι/koff,ι etc. We can now define the overall capvidity constant as: K_capv = [A₁A₂P]/[A₁][A₂][P] (1) And the affinity constants as: Kι = [A!P]/[Aι][P] (2) K₂ = [A₂P]/[A₂][P] (3) K₁₂ = [AιA₂P]/[A₁P][A₂] (4) K₂₁ = [A2A,P]/[A2P][Aι] (5) Rearranging eq. (1) to (5) gives: Kcapv = k_on,capv koff, capv ⁼ (Kι.K₂.Kι₂.K₂ι) ' (6) K will be greater than 1. When we assume that the binding constant of a second aptamer is not influenced by the first aptamer we can then write Ki = K₂ι and K₂ = Kι₂. Eq. 6 then reduces to: Kcapv = Kι.K₂ (7) From this equation it is immediately clear that Kca v will be greater than

Ki and K₂. From eq. 6 it can also be seen that K_capv will be larger than or the same as

Ki and K₂ when Kι₂ > Ki and K₂ι > K₂ (i.e. when the binding of the second aptamer is positively influenced by the binding of the first) and that Kc_apv will be larger than or the same as Ki and K₂ when only Kι₂ ≥ Ki or K₂ι > K₂ (i.e. under conditions when one particular aptamer should bind first to only positively influence the binding of the second). Eq. (6) then reduces to: Kcapv ⁼ K] .K₁₂ = K₂.K₂ι (8) The increase of Kcapv as compared to the single affinity coefficients Ki and K₂ can thus be expected for any relevant Kj and K . The above description can be easily adopted for a complex formation of more than three molecules by those skilled in the art. k₀n,capv is primarily a function of the orientation of the complementary binding sites and will therefore not be much influenced by the number of different aptamers on the substrate that can bind to one target molecule or particle. Indeed it is found that among most biorecognition probes k_on does not differ much. However, k_on may be influenced by the surface density of the probes, k_m may decrease with increasing surface densities due to lateral sterical hindrance. The off rate (k_off._cap_v) however, will be greatly influenced by the strength of the association, caused by the number and strength of interactions between the aptamer and its target. Therefore, the apparent k₀ff,capv for a combination of aptamers will be mainly depending on the product of the individual off rates. It is clear for a person skilled in the art that this description of co-operative binding holds also for other bioprobes (such as peptides, saccharides, lipids, and other small molecules), or combinations thereof. It has to be noted that in the co-operative assay using the method of the present invention it is not absolutely necessary for the detection of, for example proteins, to use at least one additional incubation with a target-specific (labelled) secondary capture probe to detect binding of the analyte to the first capture probe, in case, for example, (photo)aptamers, carbohydrates, peptides and lipids are utilized as biorecognition probes. Further advantages and features of the present invention will now be described in detail with reference to the accompanied drawings, in which

Fig. 1 shows a first embodiment of a biorecognition probe system according to the invention; and Fig. 2 shows a second embodiment of a biorecognition probe system according to the invention.

Figure 1 shows a biorecognition probe system comprising a flat support 1, onto which, exemplarily, two probes 2, 3 are supported. The probe 2 has an affinity

K_ai and the probe 3 has an affinity K^. for a specific target molecule, such as a protein.

As can be seen in figure 1, a target molecule 4 is bound via its recognition sites to both probes 2 and 3. Preferably, both probes are different aptamers, lipids, saccharides, peptides or other small capture probes, or combinations thereof. The inventive use of at least two aptamers with respective affinity constants for detecting a target molecule having several different recognition sites greatly improves the concentration range, in which the target molecule may be detected.

With the method according to the invention, target molecules in low concentrations below 1 nanomole/liter, preferably below 10 picomole/liter may be determined. The minimum detectable number of surface bound analyte molecules is set by the detection limit of a given detection technology. Figure 2 shows a further biorecognition probe system according to the present invention, wherein a three-dimensional support 10, for example a hydrogel, is used. Two different probes 20 and 30, preferably aptamers, are immobilized in that three-dimensional network, such that the spatial distance of the probes 20, 30 is small enough for co-operative binding. A protein 40 having respective specific recognition sites is bound to those probes 20, 30. The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Claims

CLAIMS:

1. Method for detection of a target molecule or a target particle having several different recognition sites in a complex mixture of different biological compounds, comprising the steps of: a) immobilizing, as primary affinity probes, at least two different biorecognition probes selected from the group consisting of (photo)aptamers, carbohydrates, peptides, lipids, metabolites, proteins, cofactors, hormones, cytokines, cells, micro-organisms, virus, drugs, pesticides, herbicides, fungicides and vitamins, in a homogeneously mixed manner on a solid substrate, each probe being specific for the same or a different recognition site of the same target molecule or particle, b) exposing the substrate to the complex mixture to allow association between target molecules or particles and the probes to occur, c) optionally removing the complex mixture, d) optionally rinsing the substrate to remove non- specifically bound material, and e) specifically assaying the probe-bound target molecules or particles, or f) detecting the probe-bound target or probe-target complex.

2. Method according to claim 1, wherein step e) comprises: el) specifically binding at least one secondary affinity probe to the probe-bound target molecule or the target-primary probe complex, e2) optionally removing unbound secondary affinity probes, and e3) detecting the secondary affinity probes or the complexes of secondary affinity probe and primary probe-bound target molecule.

3. Method according to claim 1 or 2, wherein the primary affinity probes are two different (photo)aptamers.

4. Method according to claim 2 or 3, wherein the secondary affinity probe is chosen from the group of the primary affinity probes and/or the secondary affinity probe carries a label, such as a (chemo)luminescent, electroluminescent, magnetic, redox-active or radio-active label or an enzyme.

5. Method according to claim 2, 3 or 4, wherein at least one of the different biorecognition probes is a (photo)aptamer.

6. Method according to claim 1, wherein, as primary affinity probes, at least two different biorecognition probes are selected from the group consisting of (photo)aptamers, carbohydrates, peptides and lipids, antibodies, proteins, vitamins and other small capture probes, and step e) comprises: el') specifically staining the probe- bound target molecules or particles, e2') optionally removing un-reacted stain, and e3') detecting stained probe-bound target molecules or particles.

7. Method according to claim 6, wherein the different probes are two different (photo)aptamers, two different carbohydrates, such as oligosaccharides, one (photo)aptamer and one carbohydrate, or the like.

8. Method according to claim 7, wherein the two different probes are two different (photo)aptamers.

9. Method according to any of the preceding claims, wherein the target molecule or particle is a protein, cofactor, hormone, peptide, (poly)saccharide, lipid, pesticide, herbicide, fungicide, cell, micro-organism, virus, drug, toxin, metabolite or any other small molecule or any combination thereof.

10. Method according to any of the preceding claims, wherein the complex mixture of different biological compounds is a bodily fluid, such as blood, urine, saliva, lung fluid, cerebrospinal fluid, cell extract, plasma or serum.

11. Method according to any of the preceding claims 1 to 9, wherein the complex mixture of different biological compounds is waste water, any fluid in industrial processing, milk, drinking water, surface water or any other food product or solution thereof.

12. Method according to claim 10 or 11, wherein the complex mixture is undiluted or diluted with a solvent.

13. Method according to any of the preceding claims, wherein the target molecule or particle is qualitatively determined and/or the concentration of the target molecule or particle in the mixture is determined.

14. Method according to claim 13, wherein the concentration of the target molecule or particle is about one millimole/liter, preferably below one nanomole/liter, most preferably below 10 picomole/liter.

15. Method according to any of the preceding claims, wherein the substrate is flat.

16. Method according to any of the preceding claims 1 to 14, wherein the substrate is a three-dimensional network, such as a cross-linked polymer network or a hydrogel.

17. Method according to any of the preceding claims 1 to 14, wherein the substrate is an organic, metallic or inorganic microporous or nanoporous substrate or membrane with small average pore sizes, preferably below 100 μm. more preferably below 10 μim, most preferably below 1 μm.

18. Method according to any of the preceding claims 1 to 14, wherein the substrate is a micro-sized or nano-sized particle with diameters ranging from 1 nm to 5 μm.

19. Method according to any of the preceding claims, wherein the spatial arrangement of the probes on the substrate corresponds to the spatial arrangement of the respective recognition sites on the target molecule.

20. Method according to claim 19, wherein a fitting spatial arrangement of the probes is achieved by attachment of the probes to the substrate via a flexible linker, such as oligomethylene or oligo(ethyleneglycol), preferably having a length of more than 6 atoms.

21. Method according to any of the preceding claims, wherein the immobilized probes contain two or more target binding sites, either in separate molecules or combined in one molecule, which are derived from probes with an affinity for different parts of the target molecule or particle.

22. Method according to any of the preceding claims 1 to 20, wherein the immobilized probes contain two or more target binding sites, either in separate molecules or combined in one molecule, which are derived from probes with different on and off rates for identical recognition sites of a (multivalent) target molecule or particle.

23. Method according to any of the preceding claims, wherein the captured target molecule or particle is detected using luminescence intensity, luminescence lifetime or luminescence polarization measurements, amperometric measurements, voltammetric measurements, magnetic measurements, surface acoustic waves, impedimetric measurements, dielectric measurements, radio-active measurements, electroluminescent measurements, chemoluminescent measurements, absorption measurements, interferometric measurements, reflective measurement, colorimetric measurements, quartz crystal microbalance measurements, surface plasmon resonance and/or other evanescent field techniques.

24. Biorecognition probe system for detection of a target molecule or particle having several different recognition sites, comprising at least two different biorecognition probes which are immobilized in a homogeneously mixed manner on a solid substrate, each probe being specific for the same or a different recognition site of the same target molecule or particle.

25. Biorecognition probe system according to claim 24, wherein the substrate is flat.

26. Biorecognition probe system according to claim 24, wherein the substrate is a three-dimensional network, such as a cross-linked polymer network or a hydrogel.

27. Biorecognition probe system according to claim 24, wherein the substrate is an organic, metallic or inorganic microporous or nanoporous substrate or membrane with small average pore size, preferably below 100 μm, more preferably below 10 μm, most preferably below 1 μm.

28. Biorecognition probe system according to claim 24, wherein the substrate is a micro-sized or nano-sized particle with diameters ranging from 1 nm to 5 μm.

29. Biorecognition probe system according to any of the claims 24 to 28, wherein the biorecognition probes have a high on and low off rate.

30. Use of a biorecognition probe system according to any of the claims 24 to 29 in a method according to any of the claims 1 to 23.