DE4433980C2 - Process and biosensor hit for investigating the interaction of biomolecules by means of surface plasma resonance - Google Patents

Description

The invention relates to the study of biospecific interaction of molecules.

The analysis of intermolecular interactions of Macromolecules, e.g. B. (poly) peptide (poly) peptide or (Poly) peptide-DNA interactions have been demonstrated in the Past in general under Equilibrium conditions take place. The investigation until recently, kinetic parameter could be with few exceptions, not or only with difficulty be carried out because they are larger quantities and one a higher degree of purity of the macromolecules required or because the existing methods like that Affinity chromatography or immunological methods, are not fast enough to be biospecific To track interactions.

A recently developed method is based on the optical phenomenon of surface plasmon resonance ("Surface Plasmon Resonance", SPR). The evaluation of optical signals with changes in the Refractive index at the biospecific interface correlate and that with changes in concentration of the Macromolecules, e.g. B. by binding the Reactants to each other, are obtained now enables this analysis in real time perform. This can be done with smaller amounts Macromolecules without radioactive or fluorescent Markings will be worked, and there will be too the purity of the macromolecule is less high Requirements. This method has been used. a. in the PCT applications WO 90/05295 and WO 90/05303 revealed.

The most widespread system, which is based on the "surface plasmon resonance" method and is commercially available (BIACore ^TM , Pharmacia Biosensor), has as one of the main elements, in addition to the optical system and the sample transport devices, a biosensor unit in the form of a so-called biosensor. Chips on. This consists of a glass carrier which has a gold layer on one side, to which a hydrogel matrix made of carboxymethyl-dextran is covalently bound via a barrier layer provided with linker groups. The hydrogel matrix serves on the one hand to immobilize one of the reaction partners and on the other hand to provide the environment required for the analysis of the biospecific interaction of the immobilized macromolecule with its reaction partner (Stenberg et al., 1991).

To immobilize one of the reaction partners, which is in the is generally a (poly) peptide attached to the hydrogel matrix So far, the following methods have been used:

• 1. Direct, irreversible immobilization to the Hydrogel surface of the biosensor chip by means of chemical methods.
• 2. Direct, irreversible immobilization of the biotinylated reactant hydrogel-bound streptavidin or avidin.
• 3. Indirect, reversible immobilization by binding via a hydrogel-bound antibody of Reaction partner.

However, these methods have several disadvantages on: Procedure 1) is fraught with the risk that due to the non-directed chemical reaction between hydrogel-forming substance and the (Poly) peptide, which, depending on the used Method, preferably on primary amino groups, Carbohydrate groups or free thiol groups of  (Poly) peptide takes place, its biological and / or biophysical properties in a non or only difficult to define. That can result in the immobilized (poly) peptide not or not completely in its native, biologically active form because z. B. the Section of the molecule associated with the reactant to interact through a chemical grouping blocked or due to a change in the conformation of the Molecule has become inaccessible. As for the Fundamentally biotinylation of macromolecules Similar methods as for the coupling of (Poly) peptides are used to meet these Disadvantages and thus the limitations of the procedure also to the procedure mentioned under 2). Both Methods have the additional disadvantage that a regeneration of the hydrogel surface, the one essential simplification of the procedure at Carrying out series analyzes with the same Would represent (poly) peptide while maintaining the biological and biophysical activity very much is difficult.

These drawbacks can be overcome by using antibodies (Procedure 3) are avoided, in principle the same procedures are used as for Sandwich immunoassays, e.g. B. in the ELISA (enzyme linked Immunosorbent Assay) technique. The application of this However, the procedure is required by the Availability of monoclonal, high affinity antibodies restricted with specificity for the (poly) peptide. A another limitation is that the antibodies are not allowed to interact with epitopes for which Interaction with those to be examined Reaction partners are important.

The present invention was based on the object an improved method and means for studying the interaction of (Poly) peptides with reactants using SPR to provide.

This object is solved by the features of claims 1 and 17, respectively.

There are various methods available for producing proteins whose sequence has been elucidated using genetic engineering methods as fusion proteins with sequence segments which have high affinity for a ligand (so-called affinity peptides). Immobilized metal chelate affinity chromatography (IMAC) is a widely used method for the purification of proteins and peptides, in which such fusion proteins are bound to immobilized metal chelates by means of affinity peptide. Among the various chelating agents from the group of iminodiacetic acid derivatives, nitrilotriacetic acid derivatives show particularly advantageous properties, to which the extremely high affinity for certain metal ions, e.g. B. Cu ²⁺ , Ni ²⁺ or Zn ²⁺ heard. So far, this method has been widely used using nickel as metal ion and nitrilotriacetic acid as complexing agent in the purification of recombinant fusion proteins which have an affinity peptide bound directly or indirectly to at least one histidine residue. Such purification methods of recombinant proteins have been disclosed, inter alia, in European patent applications No. 184 355 (using iminodiacetic acid as a complexing agent) and No. 282 042 (describing nitrilotriacetic acid and proteins with an affinity peptide with at least two adjacent histidine residues).

To solve the problem within the scope of the present invention task was posed by consideration assumed that the metal chelate Affinity chromatography known principle for the SPR Method to exploit.

The present invention thus relates to a method to study the interaction of a (poly) peptide with a reactant using a Biosensor unit, in which the through the Interaction triggered surface plasmon resonance in a metallic layer at the interface of two media permeable to electromagnetic radiation is determined with a different refractive index, the medium with a lower refractive index is aqueous medium in which the (poly) peptide in immobilized form and with the Reaction partner is brought into contact. The The method is characterized in that the (Poly) peptide is immobilized via a metal chelate.

The invention relates to use in the method according to a of claims 1 to 8 in a further aspect Biosensor unit for the investigation of Interaction of a (poly) peptide with one Reaction partner by determining the surface Plasmon resonance in a metallic layer on the Interface two for electromagnetic radiation permeable media with different Refractive index, the medium being lower Refractive index is an aqueous medium. The Biosensor unit is characterized in that its surface facing the aqueous medium Chelating agent is bound.  

In a preferred aspect, the aqueous phase a biocompatible porous matrix, especially a Hydrogel. With regard to the hydrogel formers basically no restriction, provided their suitability for the SPR process, especially with regard to the required diffusion of the biomolecules in the Hydrogel matrix, is basically given. Examples suitable hydrogel formers are polysaccharides, such as Agarose, dextran, carragen, alginates, starch, cellulose or derivatives of these polysaccharides, such as. B. Carboxymethyl derivatives, or water-swellable organic Polymers, such as polyvinyl alcohol, polyacrylic acid, Polyacrylamide or polyethylene glycol.

A particularly suitable hydrogel consists of dextran, which with regard to the covalent bond of the (Poly) peptide with reactive groups, e.g. B. hydroxyl, Carboxyl, amino, aldehyde, carbonyl, epoxy or Vinyl groups, is provided. The execution of the Hydrogel layer and its binding to the metal layer, which may have an organic barrier layer was done u. a. in PCT application WO 90/05303 as well as by Löfas and Johnsson, 1990. In the preferred embodiment of the invention is the Chelating agents to the reactive groups of the hydrogel bound.

In a particularly preferred embodiment, the Hydrogel former is a dextran that acts as reactive groups Has carboxymethyl groups.

In a further preferred embodiment, this is (Poly) peptide is a fusion (poly) peptide that is next to its biologically active section an affinity peptide has at least two adjacent histidine residues contains. In this case the chelating agent is on  Nitrilotriacetic acid (NTA) derivative of the general Formula Y-R-CH (COOH) -N (CH₂COO-) ₂, where R is a aliphatic or aromatic group or a May be oxyethylene group and selected accordingly is that on the one hand the ability to complex in As little as possible compared to the unbound NTA influenced and on the other hand the distance to the surface the biosensor unit is sufficiently small to prevent the SPR Not to irritate phenomena. Y is a reactive one Group with which the chelating agent reaches the surface of the Biosensor unit, in particular to those in the Hydrogel matrix containing reactive groups, bound becomes. The reactive group of the chelating agent thus becomes on the reactive groups surface of the Biosensor unit, in particular the hydrogel matrix switched off; is particularly preferred as a reactive group Y is an NH₂ group that the modified Carboxymethyl groups of dextran binds. Other reactive groups Y, which are suitable for covalent bonding are, SH groups are in a stable Thioether bond can be transferred. These Thioether bond is below the disulfide bond Taking into account stability in the present reducing agents, such as. B. mercaptoethanol, the often in the purification or synthesis of (Poly) peptides used is superior.

The chelating agent can be via its reactive groups known for the coupling of (poly) peptides Methods, e.g. B. using N-hydroxysuccinimide (NHS) and N-ethyl-N ′ - (dimethylaminopropyl) carbodiimide (EDC) (see e.g. B. Cuatrecasas and Parikh, 1972) to the hydrogel be bound.

Suitable in the context of the present invention Chelating agents are described in US-A-4,877,830  EP-A 253 303, WO 90/12803 and in the work of Hochuli and Piesecki, 1992, and Yokoyama et al., 1993, described. Nickel as the metal ion is preferred However, cobalt and manganese or copper ions in question.

Particularly within the scope of the present invention the preferred NTA derivative is that of Hochuli et al., 1987, N- (5-amino-1-carboxypentyl) described iminodiacetic acid and as a complexed metal ion Nickel.

Regarding the production of the fusion (poly) peptides, with regard to the ability to bind to the preferred metal chelate at least two adjacent Histidine residues and to which the present Invention in its preferred embodiment is applied (so-called "His-Tag proteins"), is applied to the European patent application EP-A-282 042; Examples of this are (His) ₆ proteins in which the Affinity peptide six histidine residues side by side having.

In principle, a direct coupling of the Chelating agent on the metal surface of the Biosensor chips, possibly via a reactive one Group-containing organic barrier layer possible. With regard to such barrier layers, reference is made to the Disclosure of WO 90/05303 referenced.

Immobilization of the (poly) peptide in the However, hydrogel matrix is generally preferred, especially because because of their structure they physiological conditions inside the cell represents a similar structure and thus for the Investigation of the interaction of biomolecules natural environment is approximated.  

The present invention has the crucial point Advantage in that the biosensor surface is complete is regenerable. This makes it possible to carry out series tests to be carried out under comparable conditions.

The biosensor unit according to the invention fulfills the following conditions for regeneration:

• 1. The (poly) peptide bound to the metal chelate can be completely removed.
• 2. When reloaded, the surface of the Biosensor unit no binding capacity for one too immobilizing (poly) peptide.
• 3. The binding properties of the immobilized (Poly) peptides are not affected.

For the regeneration of a metal ion-saturated chelate Surface containing a ligand, e.g. B. a His (₆) - Protein has bound, there are several options Available. On the one hand, the ligand can pass through Acid treatment (e.g. 10 mM acetic acid) removed be, the metal ion loading, depending on used metal ion, remains largely stable. If necessary, the bound protein can be put together with the metal ion by adding another strong one Chelating agents such as B. 100 mM EDTA, from the immobilized chelation are removed. The Stability of the metal ion bond to the chelating agent must be determined in individual cases and is u. a. of the Stability of the ligand-metal ion complex dependent. The procedure for regeneration of the chelate surface with other chelating agents, with due regard to the Reproducibility of the process, the first method preferable.  

In individual cases it should also be checked whether the regeneration of a sandwich-like surface, e.g. B. Metal Chelate Ligand I Ligand 2 (where Ligand 1 is a (poly) peptide with high affinity for the metal chelate surface and Ligand 2 is a macromolecule with affinity for Ligand 1 but not for the metal chelate surface), under conditions that remove of ligand 2 without affecting the binding of ligand 1 to the chelate surface. In particular, solutions with high salt concentrations can be used for this, as are used in classic affinity chromatography.

The present invention can be used in addition to the classic Areas of application of the SPR method advantageous for biochemical cleanings are used to Fractions containing the protein searched for to verify. This procedure is classic Cleaning procedures not only in terms of Precision and speed but also regarding superior to simplicity many times over.

In a further aspect, the invention relates to a Biosensor kit for the study of the interaction a (poly) peptide with a reactant SPR. The kit contains an SPR in a first container Biosensor unit, one in another container Chelating agent, salt in another container one for complexation with the chelating agent suitable metal, in one or more other Contain the reagents for activation of the Surface of the biosensor unit.  

The kit expediently contains a reagent in further containers to deactivate the surface, a reagent to regenerate the Surface as well as one or more comparison proteins.

A surface of the biosensor unit is preferred a hydrogel layer.

The chelating agent is expediently in the form of a frozen solution, taking concentration and Buffer solution with regard to the coupling to the Surface of the biosensor unit are turned off. Preferred chelating agents are nitrilotriacetic acid Derivatives, especially N- (5-amino-1-carboxypentyl) iminodiacetic acid.

The metal salt is preferably nickel sulfate, preferably in the form of a stock solution designed for the desired degree of loading of the surface diluted can be.

In the preferred embodiment of the invention, at one surface of the biosensor unit from one Hydrogel with reactive groups, especially from one carboxymethylated dextran, there are Reagents to activate the biosensor surface N-hydroxysuccinimide and N-ethyl-N ′ - (dimethylaminopropyl) carbodiimide, the preferably in the form of frozen solutions, with regard to concentration and buffer solution Activation are suitable. In the preferred The embodiment is the reagent for deactivating the after coupling of the (poly) peptide remaining N- Hydroxysuccinimide groups on the biosensor surface Ethanolamine in suitable concentration and Buffer solution, preferably also in frozen Shape.  

The reagent to regenerate the biosensor surface is preferably a chelating agent, especially EDTA.

Any existing in other containers Test proteins preferably have an affinity peptide with several histidine residues and lie as Standard solutions in concentrations and buffer solutions to check the loading of the biosensor Surface are suitable.

In the context of the present invention experiments carried out according to the preferred Embodiment and for the sake of simplicity Coupling made in the hydrogel matrix because of the commercially available biosensor units by themselves have a dextran matrix and for the direct coupling necessary removal of this Hydrogel layer from the biosensor unit in one reproducible way difficult to carry out appears.

For binding the chelating agent N- (5-Amino-1-carboxypentyl) iminodiacetic acid to the Biosensor hydrogel surface became the coupling method used with N-hydroxysuccinimide. The Chelating agent, which is a free, primary amino group contains, to the modified dextran hydrogel surface a commercially available biosensor unit (BIACore) covalently coupled. As a chelator synthesized a derivative of nitrilotriacetic acid, the can be used directly for immobilization. The Synthesis of the derivative largely corresponds to that published method (Hochuli et al., 1987; and European Patent 339 389) with which Difference that to split off the protecting group instead of the described hydrogenation with Pd / C as  Catalyst with an acid cleavage Trifluoroacetic acid / trifluoromethanesulfonic acid was made. The material obtained in this way can it is largely free of inorganic contaminants is directly for coupling to the surface of the Biosensor unit can be used.

The chelating agent was immobilized according to the method recommended by the manufacturer of the biosensor chips. Since the very low relative molecular weight of the nitrilotriacetic acid derivative (M _r 295) means that it is not possible to determine the immobilized amount directly due to the instrument, the relative amount was detected indirectly by determining the binding capacity for a protein. Commercially available bovine serum albumin (BSA) has proven suitable for this.

It was found that the affinity of the test (Poly) peptide for the nickel-free, with chelating agent modified biosensor chip surface is very small, there was no bond to the modified surface too observe. The binding capacity of the surface for the test protein is, as shown in Example 4, correlates directly with the nickel ion concentration, the is used for loading. The setting of the Nickel ion concentration enables easy Matching the binding capacity to the experimental requirements.

Figure overview

Fig. 1 Immobilization of N- (5-amino-1-carboxypentyl) iminodiacetic acid to the dextran surface of a biosensor,

Fig. 2 Indirect determination of the loading of the dextran with N- (5-amino-1-carboxypentyl) iminodiacetic acid by means of bovine serum albumin,

Fig. 3 regeneration of the laden with bovine serum albumin biosensor surface by means of EDTA,

Fig. 4 determine the nonspecific binding of a protein to the biosensor surface,

Fig. 5 Determination of the binding ability of a protein to the biosensor surface as a function of the nickel concentration,

Fig. 6: Binding ability of various proteins to the loaded N- (5-amino-1-carboxypentyl) iminodiacetic acid / nickel biosensor surface.

The invention is illustrated by the following examples explains:

example 1 Synthesis of N- (5-amino-1-carboxypentyl) iminodiacetic acid

4.17 g of bromoacetic acid (Aldrich) were dissolved in 15 ml of 2M NaOH dissolved and cooled to 0 ° C. To this solution with stirring, a solution of 4.2 g of N-benzyloxycarbonyl L-Lysine (Fluka) in 22.5 ml of 2 M NaOH was slowly added dropwise. After 2 hours the solution was at room temperature warmed and stirred overnight. Subsequently the solution was heated to 50 ° C for 2 hours and then slowly added 45 ml of 1 M HCl. The resulting one Precipitate was centrifuged off with 0.1 M HCl  washed and dried (5 g product, theoretical Yield 5.9 g).

0.87 g of the derivative obtained above were in 10 ml Trifluoroacetic acid (Merck) dissolved. For a clear solution 1 ml of trifluoromethanesulfonic acid (Merck) slowly added dropwise and for 1 hour at room temperature touched. The precipitate formed was separated off, the solution was mixed with 30 ml of water and almost to evaporated to dryness. An aliquot of this solution was made on a PD10 column (Pharmacia) with water as Solvent separated. The ninhydrin positive, neutral fractions were pooled and lyophilized.

Example 2 Coupling of N- (5-amino-1-carboxypentyl) iminodiacetic acid to the dextran surface of an SPR Biosensor unit

All steps of immobilization were carried out in the BIAcorgerät (Pharmacia) with HBS buffer (10 mM HEPES, 150 mM NaCl and 5 mM MgCl₂, pH 7.4) at 25 ° C carried out.

A CM5 biosensor chip (Pharmacia Biosensor, Certified Grade) was used for immobilization. (This biosensor unit has a hydrogel surface made of carboxymethylated dextran.) The activation of the hydrogel surface was carried out by injecting 35 μl of a 0.05 M N-hydroxysuccininimide (NHS) /0.2 M N-ethyl-N ′ - (dimethylaminopropyl) carbodiimide (EDC) solution (Flow rate 5 ml / min). To couple the N- (5-amino-1-carboxypentyl) iminodiacetic acid described in Example 1 to the activated surface, 35 μl of a solution of 15 mg / ml N- (5-amino-1-carboxypentyl) iminodiacetic acid in 1 M NaOH were added at a flow rate of 3 µl / min. Unsaturated binding sites on the surface were saturated by injecting 35 µl of a 1 M solution of ethanolamine (pH 8.5) (flow rate 5 µl / min). To condition the surface, 10 μl of a 100 mM EDTA solution (pH 8 adjusted with NaOH) were injected and the surface was loaded with 4 μl of a 0.1 M NiSO₄ / 0.2 M CH₃-COONa solution (flow rate 5 μl / min). The course of this immobilization is shown in FIG. 1 on the basis of the change in the surface plasmon resonance. The individual steps of immobilization are identified in the figure. Since, due to its low relative molar mass (M _r 295), the loading of the surface with the chelating agent cannot be determined directly, the loading was determined indirectly by 35 µl of a solution of 1 g bovine serum albumin (BSA.) Under constant flow (5 µl / min) , Sigma) was injected into 100 ml running buffer. The sensorgram ( Fig. 2) shows significant binding of the protein to the surface. The reproducibility of the surface mobilization was confirmed on the basis of series tests with a standardized solution of this protein.

Example 3 Regenerability of the N- (5-amino-1- carboxypentyl) iminodiacetic acid surface

In order to investigate the regenerability of the N- (5-amino-1-carboxypentyl) iminodiacetic acid surface, the surface was loaded with Ni ²⁺ twenty times in succession under constant flow (5 μl / min), as described in Example 2, 35 µl of a solution of 1 g bovine serum albumin (BSA, Sigma) injected in 100 ml running buffer and regenerated with EDTA. To calculate the data shown in Fig. 3 (baseline, injection maximum and bound protein t = 1), the resonance was 30 sec before injection of bovine serum albumin (baseline), 30 sec before the end of injection of bovine serum albumin (injection maximum) and 30 sec after injection of Bovine serum albumin (bound protein t = 1) determined.

EDTA (100 mM, pH 8) was found to be the most suitable for removing the bound protein. As shown in FIG. 3, neither a loss of activity of the surface when reloaded (injection maximum, bound protein t = 1) nor a persistence of the bound protein after regeneration (baseline) can be determined. Since EDTA removes the chelated nickel, Ni ²⁺ must be loaded again after the surface has been regenerated.

Example 4 Effect of Ni ²⁺ concentration on protein binding ability

To determine the non-specific binding of the protein to the surface, the surface described in Example 1 was regenerated with EDTA, as described in Example 3, and 20 μl of a test protein solution were injected at a flow rate of 5 μl / min. After renewed regeneration of the surface with EDTA and loading with 4 ul of a 0.1 M NiSO₄ / 0.2 M CH₃-COONa solution (flow rate 5 ul / min) again 20 ul of the test protein solution were injected. As shown in FIG. 4, no binding to the surface can be observed in the absence of Ni ²⁺ ; after loading the surface with Ni 2+, a saturation of the surface with test protein can be found.

In order to determine the binding capacity of the surface as a function of the Ni ²⁺ concentration, the Ni ²⁺ -N- (5-amino-1-carboxypentyl) iminodiacetic acid surface described in Example 1 was regenerated with EDTA as described in Example 3 and with each Load 4 µl of a Ni ²⁺ solution (0.1 M NiSO₄ / 0.2 M CH₃-COONa, flow rate 5 µl / min) of the given concentrations. After loading with nickel, protein was injected and the resonance at t = 1 was determined. The plot of the measured resonance against the concentration of the nickel solution ( FIG. 5) shows that a complete loading of the surface with 4 μl of a 100 μM Ni ²⁺ solution is achieved. Depending on the concentration, lower concentrations only show incomplete loading of the surface.

Example 5 Binding ability of various proteins to the N- (5- Amino-1-carboxypentyl) iminodiacetic acid surface

To investigate the different binding behavior of different proteins, 40 µl of solutions (10 µg / ml in running buffer) of a chicken protein of the sequence shown in the sequence listing, modified with His (₆) ((His) ₆-SCF), bovine serum albumin (Sigma) and of lysozyme from egg white (Sigma) injected onto a biosensor surface synthesized according to Example 2. (The chicken protein was produced and purified after expression in a commercially available expression vector (pQE5O, Diagen GmbH) according to the cleaning scheme specified by the manufacturer of the system (The QIAexpressionist, Diagen GmbH, Protocol 3, p. 35 and Protocol 7, p. 45). For further purification, the protein was purified on an HQ / M anion exchange column (Perseptive Biosystems, Freiburg) and the protein concentration was determined using the Bradford protein assay [BioRad). The binding behavior of the individual proteins is shown in FIG. 6: lysozyme (M _r 14300) and bovine serum albumin (M _r 68000) show a low resonance, despite the relatively high concentration; the His ₍₆₎ -modified chicken protein (M _r 22000), however, binds to the surface many times more. Furthermore, it can be seen that lysozyme and bovine serum albumin, in contrast to the His (₆) - modified chicken protein, achieve a saturation of the surface. The surface loaded with the His ₍₆₎ -modified chicken protein for saturation can be used to search for proteins interacting with this protein, e.g. B. cell culture supernatants from chicken cell lines can be used by injecting cell culture supernatants from different cell lines. The binding of an interacting partner can be demonstrated by increasing the resonance after the injection.

literature

Cuatrecasas, P. and Parikh, 1972, I. Biochemistry 11, 2291.
Hochuli, E., H. Döbel and Schacher, A., 1987, J. Chromatography 411, 177-184.
Hochuli, E. and Piesecki, S., 1992, Methods 4 (San Diego), 66-72.
Löfas, S. and Johnsson, B., 1990, J. Chem Soc., Chem. Communications 1526.
Stenberg, E. et al., 1991, L. of Colloid and Interface Science, 143, 513.
Yokoyama, T., Sigeko, A. and Masatoshi, K., 1993, Chem. Lett. 2, 383-386.

SEQUENCE LOG

Claims (20)

1. Method for investigating the interaction of a (poly) peptide with a reaction partner by means of a biosensor unit, in which the surface-plasmon resonance triggered by the interaction is determined in a metallic layer at the interface of two media with a different refractive index that are permeable to electromagnetic radiation, wherein the medium with a lower refractive index is an aqueous medium in which the (poly) peptide is in immobilized form and is brought into contact with the reactant, characterized in that the (poly) peptide is immobilized by a metal chelate. 2. The method according to claim 1, characterized in that that the aqueous medium is a biocompatible porous Matrix, especially a hydrogel. 3. The method according to claim 2, characterized in that that the chelating agent has reactive groups of the Hydrogels to the surface of the biosensor unit is bound. 4. The method according to claim 2 or 3, characterized characterized in that the hydrogel is a dextran. 5. The method according to claim 4, characterized in that the dextran as reactive groups Has carboxymethyl groups. 6. The method according to any one of the preceding claims characterized in that the (poly) peptide Fusion (poly) peptide is next to its  biologically active section an affinity peptide having at least one histidine residue and the Chelating agent is an iminodiacetic acid derivative. 7. The method according to claim 6, characterized in that the (poly) peptide has an affinity peptide with at least two adjacent histidine residues and the chelating agent Nitrilotriacetic acid derivative of the general formula Y-R-CH (COOH) -N (CH₂COO-) ₂, wherein R is a aliphatic or aromatic group or a Oxyethylene group and Y is a reactive group, in particular an NH₂ or an SH group. 8. The method according to claim 7, characterized in that that the nitrilotriacetic acid derivative N- (5-amino-1- carboxypentyl) iminodiacetic acid and the metal Is nickel. 9. Biosensor unit for use in the method according to one of claims 1 to 8 for the investigation of Interaction of a (poly) peptide with one Reaction partner by determining the Surface plasmon resonance in a metallic Layer at the interface of two for electromagnetic radiation from permeable media with different refractive index, the Medium with a lower refractive index is an aqueous one Medium is characterized in that to the surface facing the aqueous medium Biosensor unit bound to a chelating agent is. 10. Biosensor unit according to claim 9, characterized characterized in that the chelating agent to the  reactive groups of a biocompatible porous Matrix, in particular a hydrogel, is bound. 11. Biosensor unit according to claim 10, characterized characterized in that the hydrogel is a dextran. 12. Biosensor unit according to claim 11, characterized characterized in that the dextran as reactive Has groups carboxymethyl groups. 13. Biosensor unit according to one of claims 9 to 12, characterized in that the chelating agent is a Iminodiacetic acid derivative. 14. Biosensor unit according to claim 13, characterized characterized that a chelating agent Nitrilotriacetic acid derivative of the general formula Y-R-CH (COOH) -N (CH₂COO-) ₂, wherein R is a aliphatic or aromatic group or a Oxyethylene group and Y is a reactive group, in particular an NH₂ or an SH group. 15. Biosensor unit according to claim 14, characterized characterized in that the nitrilotriacetic acid Derivative N- (5-amino-1- carboxypentyl) iminodiacetic acid. 16. Biosensor unit according to one of claims 9 to 15, characterized in that the chelating agent in with a metal ion of the most complex form. 17. Biosensor kit for the investigation of Interaction of a (poly) peptide with one Reaction partner by means of SPR, contained in one first container an SPR biosensor unit, in one another container a chelating agent, in one another container a salt one for complexation with the chelating agent suitable metal, as well as in one or more other containers of reagents  for the activation of the surface of the Biosensor unit. 18. Biosensor kit according to claim 17, comprising in one another container a reagent to deactivate the Surface. 19. Biosensor kit according to claim 16 or 17 containing in another container a reagent for Regeneration of the surface. 20. biosensor kit according to one of claims 16 to 19, containing one or more others Containers of one or more reference proteins.