DE10117866A1 - Detecting and characterizing binding complexes, useful e.g. for diagnosis, comprises forming sample and reference complexes and determining force required for separation - Google Patents

Abstract

Characterizing and/or detecting a binding complex comprising: (a) forming a linkage of binding partners so that binding partner (B1) and binding partner (B2) form a sample complex (SC) and binding partners (B3 and 4) form a reference complex (RC); (b) applying a force to the linkages so that SC or RC is separated; and (c) determining which complex has been separated, is new. Characterizing and/or detecting a binding complex comprising: (a) providing a first binding partner (B1), a conjugate (C) of second and third binding partners (B2, B3) and a fourth binding partner (B4); (b) forming a linkage of binding partners so that B1 and 2 form a sample complex (SC) and B3 and 4 form a reference complex (RC); (c) applying a force to the linkages so that SC or RC is separated and (d) determining which complex has been separated, is new. Independent claims are also included for the following: (1) an apparatus for the process; and (2) kit for the performing the method or for use with the apparatus of (1).

Description

The invention relates to a method and an apparatus for characterization and / or for the detection of a binding complex, in particular by means of molecular differentiation Separation forces through a differential force test

Binding tests based on equilibrium constants

Non-covalent interactions between molecules are based on the atomic Interaction of binding partners through hydrogen bonds, ionic, hydrophobic and van der-Waals forces. Weak interactions are orders of magnitude less than covalent bonds that are formed or broken by chemical reactions.

Non-covalent interactions between binding partners with a high degree selective binding properties are the prerequisite for the molecular recognition that one makes use of chemical analysis and diagnostics. Below are such interactions are referred to as specific interactions. In the process  it is mostly for the detection or characterization of biochemical molecules about binding tests.

A binding test is based on the formation of a binding complex by the specific one Interactions of a ligand with a receptor and the detection of this complex.

Diagnostic binding testing is about detecting a known substance in a sample:

• - Determine the identity of a test substance
• - Detect a test substance in a complex test mixture
• - to distinguish variants of a test substance
• - Determine the concentration of a test substance

In the case of binding tests for the development of new diagnostic or therapeutic agents, it is important to find a suitable, yet unknown second binding partner for a specific binding partner, ie:

• - To identify those from a large number of test substances that are associated with one specific receptor binds
• - Determine the binding constant of the test substance to the receptor
• - Other binding properties such as the rate of association (on rate) or To determine dissociation (off rate)

In immunodiagnostics, the receptors are i. d. R. for antibodies or Antibody derivatives with which antigens of proteins, but low molecular weight substances viruses and whole cells can also be detected.

In molecular diagnostics, the receptor is called a probe that consists of a There is nucleic acid such as DNA or RNA and with the nucleic acids in a sample be detected.

The most common immunodiagnostic test is the enzyme linked immunosorbent assay (ELISA) In the ELISA, a first antibody is immobilized on a surface. He selectively binds an antigen of an added sample mixture via a first binding site (Epitope). A second antibody that is labeled and that is in free Solution located binds to a second epitope of the antigen. In a separation step, the fraction of the labeled antibody is separated which is not bound to the antigen Has. The sandwich complexes of the first antibody remaining on the surface,  The antigen and the second antibody are detected using the label. To the Marking can i. d. R. to bind an enzyme that by forming a dye Signal developed. The dye is ultimately the measure of the amount of antigen in the examined sample.

A typical molecular diagnostic test is Southern hybridization. It is based on the interaction of a known nucleic acid molecule, the probe, with one complementary nucleic acid of a sample mixture. The sample is on a surface immobilized and the labeled probe added. With suitable buffer and Temperature conditions bind the probe molecules to complementary sequences of the sample. After separation of the unbound probe, the nucleic acid duplexes formed are removed Probe and sample molecules quantified using the label.

In reverse Southern hybridization, the basis for the highly parallel Nucleic acid analysis on miniaturized probe arrays (DNA microarrays) the probe molecules bound to the surface and labeled sample molecules in free Solution added.

ELISA and Southern hybridization have in common that the binding event is triggered by a Marking is detected and that one of the binding partners on a surface is bound. Another property they share with all common binding tests have that one has binding properties of two binding partners to each other on the Characterized the size of the binding energy in the resulting binding complex.

Molecular interaction model

The chemical interaction between two binding partners can be seen from describe different models. The classic theoretical framework is that Thermodynamics. For the development of thermodynamics it was crucial that it It has long been impossible to measure molecular interactions on individual molecules. That is why it describes the interaction of particles on a macroscopic basis measurable sizes. This results in the concept of binding energy, which is considered to be that of releasing energy required for a bond is defined. The binding energy of two Binding partners to one another can be measured by measuring the concentrations of free and bound binding partners can be derived from the equilibrium constant.

A fundamentally different concept for characterizing molecular interaction was developed by force measurement, e.g. B. with the Atomic Force Microscope, on individual molecules  allows. The forces formed between the atoms of two binding partners can be determined experimentally by the separation force to be used. Out the rate dependence of the separating forces can do this under certain conditions The binding potential of a binding complex can be reconstructed. In contrast to one Characterization of a binding complex based on its binding energies is the decay of the Complexities in a force measurement not by thermal excitation but by one manipulative intervention.

To explain the binding potential, here is a greatly simplified binding model described.

A binding complex is not only characterized by its binding energy. It also has a characteristic activation barrier that determines its binding and disintegration probability. Each binding complex also has a defined spatial structure. A characteristic variable describing this structure is the bond length or potential width. These quantities can be summarized in the following simple model of the binding potential, which is shown in FIG. 1.

In the bound state, the ligand sits at the minimum of the potential. To the complex the ligand must be broken open via the potential barrier from the binding pocket or from the Binding potential can be extracted. The force required for this is determined by the Derivation of the potential (slope).

A spontaneous disintegration of the complex by thermal activation is superimposed on the mechanical breakup. This spontaneous decay depends on the activation barrier ΔG. A force applied to the complex lowers the activation barrier still to be overcome. With each applied force, the complex has a certain probability of disintegrating within a certain time. The force at which a complex actually disintegrates also depends on how quickly the force is applied. If you increase the applied force continuously with a fixed force rate r until the complex disintegrates, you get the following average separation force for the complex depending on the applied force rate:

If the mean separation force is determined under different force rates, the bond width Δx characteristic of the binding complex and the decay rate k _{decay of} the complex can be determined _therefrom .

Measuring separating forces therefore represents a new approach to the characterization of Binding complexes.

Methods of molecular force measurement

Although the vast majority of binding tests are based on selective interactions Evidence of binding energies, there are examples of methods that can take advantage of to make characteristic separation forces of the binding complexes their own.

The first example of such a device is the "surface force apparatus" (SFA) Developed by Israelachvili in 1976 (Patent US 5861954, 1999, Israelachvili). With this The adhesive forces of two molecular layers can be measured with respect to one another. each the test substance is placed on a cylindrically curved mica sheet applied, which are ideally brought into contact in only one point. By a precisely controlled movement of the mica surfaces towards one another forces between the molecular layers. If one measures the movement of the surfaces towards one another Depending on the applied force, you get a statement about the adhesive forces between the two molecular layers.

The second method for measuring molecular forces is the "atomic force microscope "(AFM). With the AFM the first determination of the separation force of a weak interaction of a biological receptor-ligand pair, the biotin Streptavidin system (E.-L. Florin, V.T. Moy and H.E. Gaub, Science 264, 415 (1994)). In contrast to the SFA, no macroscopic surfaces are in contact with the AFM brought. The tip of an AFM is only a few nanometers in size and can end up ideally pointed to a single atom. At best, you can choose between an AFM Tip and a second surface single molecule, e.g. B. a DNA double strand, hanging. If the tip is now pulled away from the surface, tension arises of the molecule and for the bending of the AFM cantilever, which is known when the spring constant is known of the cantilever allows a measurement of the molecular binding forces.  

A modification of the AFM method, which is aimed at diagnostic applications in patent US 5992226; Lee et al. described. Here the sample is full on a flat Surface bound between a tapered projection and the AFM tip, or a surface on which the AFM cantilever is bound.

A third method of characterizing molecular forces is based on use microscopic magnetic balls. You bind a receptor on one magnetic ball and leaves it with a ligand, which in turn is on a surface is bound to form a binding complex. If you put the magnetic ball in a defined position magnetic field, you can apply a defined tensile force to the binding complex place between ball and surface. Observing the position of the magnetic ball in Direction of traction, while varying it until it separates Binding complex comes, so you can determine the separation force that is needed to Tearing apart receptor and ligand.

The detection of separating forces with magnetic balls is based on an immunological Sandwich test from, in which the forces only for the separation of unbound and bound Ligand can be used (patents US 5445970 and US 5445971, 1995, Rohr). On further approach provides for fine-tuning the tensile forces, so that in principle weak specific binding of an antigen to an antibody of strong can be distinguished (Patent WO 9936577, 1999, Lee).

The fourth method is a force measurement with "optical tweezers" (Optical Tweezers). The basics of this technique go back to Arthur Ashkin. The The pulling force is exerted here by the movement of a highly focused laser beam exercised, which can capture and move a microscopic particle.

An application of the Optical Tweezers for force measurement for diagnostic purposes has been made described by Kishore. (Patent US 5620857, 1997, Kishore et al.)

The fifth method is based on the adhesion of an elastic stamp (conformal pillar), which is coated with a test substance to form a surface which is covered with a probe is coated. When the stamp is detached from the surface, separation forces can occur are measured, which serve to identify the sample (patent EP 0962759; 1999, Delamarche et al.). Binding complexes based on separation forces instead of on binding energies characterizing brings some significant advantages. Receives about the potential range of the bond a new independent parameter to characterize the bond that allows it  different binding modes that have the same binding energies from each other differ. This applies in particular to the distinction between non-specific and specific bindings for proteins, as well as for discrimination between Nucleic acid duplexes that are completely complementary or have mismatches. Another advantage is that it determines association rates and dissociation rates can be, which is difficult with conventional binding tests.

An object of the present invention is a method and an apparatus to provide, which enables a simple test.

This object is achieved with the features of the claims.

Another object of the present invention is the described advantages of binding tests, which are based on the differentiation of separating forces for a wide range of applications and to make commercial use accessible through what has been done to date was not possible.

The invention has advantages over conventional force discrimination methods. The conventional methods of molecular force measurement are further developments of Methods that originally served a completely different purpose. The SFA was created for Surface forces, AFM for the imaging of surfaces, magnetic balls for the Separation of molecules and the conformal pillar method for structuring Surfaces developed.

Another object of the present invention is to provide a strength test that Binding properties of a binding complex by simultaneous testing of many do not can test cooperative individual events.

In methods with magnetic beads (magnetic beads) i. d. R. separation forces, the are based on several cooperative events, since several binding complexes on one Ball to be attached.

Another object of the present invention is a strength test in which the separation of the Binding complex and the detection of the result are separated in time (once). This results in a simple apparatus structure and a high flexibility in the selection the detection method.  

Another object of the present invention is a strength test that is not complex apparatus construction required and its implementation requires no experts. This is comparable to conformal pillar.

Another object of the present invention is a force test that has a high parallel Allows measurement of many different samples.

Another object of the present invention is a force test in which tensile forces can be realized are far above that of optical tweezers and magnetic balls.

Another object of the present invention is faster binding kinetics of the To provide binding partners than is possible with a method such as the ELISA the kinetics by the rate of diffusion of those in free solution Reaction partner is limited.

In contrast to the methods described, the present invention is concerned a technique that has been used from the beginning for the determination of binding potentials was conceived, and which offers significant advantages over the prior art. The Surface Force Apparatus from Israelachvili is not for the analysis of biomolecules suitable. The construction of the equipment is extremely complex and expensive and therefore for diagnostic applications hardly suitable. A parallel measurement of different Substances are not described and may be difficult to implement.

The main advantage of the Atomic force Microscope is its high force resolution. The complex apparatus construction, however, leads to high acquisition costs and also the handling that requires an expert, making this device unsuitable for use outside of the Basic research. Another limitation is that it is statistical assured measurement result requires a large number of sequential experiments and therefore takes a lot of time. Also regarding one of the most important Requirements for a diagnostic measurement method, the parallel measurement of many of different test substances, the AFM has an inherent disadvantage.

The use of magnetic balls is problematic in the exercise of Forces. Practical particle sizes and field strengths do not allow forces that are capable  would be a DNA duplex to pull apart what this method of use in the excludes molecular diagnostics. The same problem applies to the use of Optical tweezers. The realizable tensile forces are well below 100 pN and are thus far too low to be able to test a DNA duplex.

The conformal pillar method uses small stamps with one of the Binding partners are coated and which also serve as light guides for the detection of Detach the stamp from the second surface. The miniaturization of the Stamp is therefore limited because it is at least the size of the wavelength of the must have used light. Another disadvantage of the conformal pillar is that that the tearing of the binding complexes is not independent, i. H. cooperatively what happens leads to a deterioration in the measurement statistics.

The present invention combines several properties which no conventional method can have in this way
A simple and inexpensive apparatus construction
Easy handling
A high force resolution with simultaneous measurement of a large number of binding events
Unlimited traction
Non-cooperative simultaneous testing of many binding complexes

The possibility of many similar sample complexes during one measurement process test simultaneously, with the result for each complex independent of the others One of the main advantages of the invention is complex failure, i.e. non-cooperative to watch. In the described methods with the conformal pillar or with magnetic Spheres are always cooperative events because some of the Complexes affects the probability of separation of the remaining complexes. The Information about the individual events is lost.

The terms used to describe the invention are defined as follows:
Suspension: Connection of a binding partner with a holding device.
Binding properties: ratio of two binding partners to each other such as: no binding; Binding affinity; Binding mode.
Binding complex: complex of several binding partners; Molecules or bodies or bodies and molecules that interact with each other and that can be separated by a tensile force.
Binding partner: Part of a binding complex that can be separated from another binding partner by tensile forces. Binding partners can interact with each other specifically or non-specifically. The interaction is non-covalent.
Biomolecules: Molecules that are obtained from biological systems or artificial molecules that are similar to those from biological systems.
Conjugate: connection of two binding partners.
Connection: connecting element of a conjugate.
Reference complex: binding complex with a known separating force.
Ligand: One of the binding partners of a specific binding complex.
Average separation force: Arithmetic mean of the separation forces of several identical binding complexes, whose individual separation force varies due to the thermal excitation.
Sample (target): Molecule, polymer, etc. that can form a sample complex.
Sample complex: binding complex to be characterized / demonstrated. Either there are two known binding partners whose binding properties are to be determined or it is a known binding partner. It can be an unknown or a known separation force.
Receptor: One of the binding partners of a specific binding complex.
Separation: Separate binding partners who have not formed a binding complex from those who have formed a binding complex.
Specific interaction: Molecular interaction between two binding partners of a binding complex, which is characterized by a high degree of molecular recognition.
Unbinding force: maximum force required to mechanically separate a binding complex.
Linking: linear arrangement of a first binding partner that binds to a second binding partner of a conjugate and a third binding partner that is part of the conjugate and that binds a fourth binding partner.

The invention is explained in more detail below with the aid of examples and the drawings.

Show it

Fig. 1 is a simplified potential binding of a binding complex,

Fig. 2 shows the principle of the differential force test. After exerting a tensile force on the conjugate from B1 and B2, B1 breaks if F ₁ <F ₂ or B2 breaks if F ₁ <F ₂ ,

Fig. 3 shows the distinction between non-specific interactions with a body and specific interactions with a binding partner,

Fig. 4 shows the differentiation of a fully paired nucleic acid duplex of an incompletely mated,

Fig. 5, the simultaneous execution of a compare strength tests on five independent, similar binding complexes in the sandwich format. In this case, the sample has the binding partners BP2 and BP3. The sample is marked. Since F ₁ <F ₂ , the binding complexes B2, and tend to tear

Fig. 6, the simultaneous execution of a comparative strength tests on five independent, similar binding complexes in the capture format. In this case the sample is bound to the surface 1 . The conjugate from R ₁ and R ₂ is provided with a label. Since F ₁ <F ₂ , the binding complexes B2 mostly tear.

Fig. 7 shows a punch apparatus suitable for carrying out the inventive method. A more detailed description can be found in Experimental Example 1.

Fig. 8 shows representations of a base (8A) and a punch (8B) to a power test for comparison of the complexes biotin / streptavidin and iminobiotin / streptavidin (see Experimental Example 1).

Fig. 9 shows representations of a base (9A and 9C) and a stamp (9B and 9D) after force tests to compare two DNA duplexes (see Experimental Example 2). FIG. 9A shows the substrate in Experiment 2a, Fig. 9B shows the punch in Experiment 2a, Fig. 9C shows the base in experiment 2b, Fig. 9D shows the punch in Experiment 2b.

Fig. 10 shows the results of evaluation of substrate and stamp by a motor comparison, wherein a DNA duplex was compared with an identical duplex (10C and D) and a further Duplex (10A and B) (see Experimental Example 2).

10A: Document for experiment 2a; 10B: stamp in experiment 2a; 10C: Document at Experiment 2b; 10D: stamp in experiment 2b. They are excerpts from Fluorescence profiles.

• 1. First binding partner BP1
• 2. Second binding partner BP2
• 3. Third binding partner BP3
• 4. Fourth binding partner BP4
• 5. Binding complex 1 (B1)
• 6. Binding complex 2 (B2)
• 7. Conjugate from B1 and B2
• 8. Conjugate of the second binding partner BP2 and the third binding partner BP3
• 9. Suspension of the first binding partner BP1
• 10. Suspension of the fourth binding partner BP4
• 11. Connection of the second binding partner BP2 and the third binding partner BP3
• 12. Vector of the speed
• 13. Surface 1
• 14. Surface 2
• 15 samples
• 16. Sample with the second binding partner BP2 and the third binding partner BP3
• 17. Suspension of the sample
• 18 mark

The present invention is a method which is compared to the conventional strength tests a completely different principle of determining Has separating forces.

A differential strength test consists of two binding complexes that are linked together are. When applying a force that exceeds the separation forces of the two binding complexes one of the two binding complexes breaks. The binding complex with the higher separation force remains intact. Do you know the separation force of one of the two Binding complexes, it can be concluded in this way whether the separating force of the  second binding complex is larger or smaller than that of the first. The differential Strength testing can be used for a variety of diagnostic applications. The The invention is particularly suitable as a method for diagnostic detection or Characterization of the binding properties of biochemical molecules or molecules with a high degree of specific molecular recognition.

In the present invention, binding properties are characterized by Binding partners based on the separating force required for separating their binding complex necessary is.

To characterize the separating force F ₁ which must be used for the separation of a binding complex B1 ( 5 ), by comparison with the known separating force F ₂ which must be used for the separation of a second binding complex B2 ( 6 ). Both binding complexes are connected to a conjugate, on both sides of which a tensile force is applied. It should be emphasized that the tensile force on the two serially arranged binding complexes is the same. If the tensile force exceeds a value which is greater than one of the separating forces (F ₁ or F ₂ ), the binding partners of the binding complex whose separating force is lower are separated. Is it z. B. to separate the binding complex B1 it follows that F ₁ <F ₂ . If B2 was separated, it follows that F ₁ <F ₂ . Fig. 2 shows the principle of the force test.

The binding complex B1 ( 5 ) is generally a sample complex, ie a binding complex whose binding properties are to be characterized or in which a binding partner is to be detected by means of a known binding property with another binding partner. However, it can also be an undefined, non-specific interaction between two binding partners or between a binding partner and a body.

B2 ( 6 ) is usually a reference complex, ie a binding complex whose binding properties, in particular a separating force, are known.

The principle described above is not a "measurement" in the narrower sense, but rather a "teaching". According to the German industry standard the term "teaching" describes a test procedure in which the object to be tested also includes compared to a known size of another item. With this in mind also in the present invention, the separation force of the binding complex B1 with a second known separation force compared. In contrast, "measuring" is a Test method in which the size to be determined has a concrete numerical value on the  Measurement scale results. This corresponds to the situation of a force measurement with the AFM or with one of the other methods of the prior art.

The peculiarity of the present invention is that everyone to be tested Sample complex a force theory on a nanoscopic scale, the reference complex assigned. Each sample complex is tested independently, the result of many individual tests finally gives the measurement result. A peculiarity that follows from this principle is that Possibility of separating the binding complexes and the detection separately to be able to execute.

The invention particularly takes thermal excitation into account. From the model of molecular interaction ( FIG. 1) discussed at the beginning, it can be seen that the separating force which is required to separate the binding complex B1 or a further binding complex B2 varies, since the interaction between the binding partners is subject to thermal excitation. Therefore, according to the present invention, the comparison of the two pairs of bonds is preferably carried out several times in order to be able to form a statistically reliable mean, the average separating force, which states whether F ₁ <F ₂ or F ₁ <F ₂ . This is done by simultaneously exposing many similar binding complexes to the same separation force and determining how much of the binding partner of B1 and how much of B2 have been separated.

The invention additionally or alternatively takes into account the force rate dependency. An important parameter for the execution of a differential force test is the rate of the tensile force applied, since the separating forces F ₁ and F ₂ can vary greatly at different force rates. In order to be able to repeat a differential force test according to the principle described above, it is important to work with only a certain force rate.

The rate of force is determined by the speed of the tensile force and the elasticity of the Conjugate of the two binding complexes including the suspension of the first binding partner BP1 and the fourth binding partner BP4.

Depending on the application of the differential force test, you can also consciously consider the force rate vary in order to obtain certain information about a binding complex.

Application examples for the invention 1. Differentiation of binding modes 1.1. Differentiation between specific and non-specific interactions

A central problem of binding tests in which a first binding partner on one Surface immobilization is the non-specific background signal. The unspecific Background signal is caused by molecules of the second binding partner that are in free Solution were added and bound non-specifically to the surface. It is coming thus a superposition of the specific signal, those molecules of the second Binding partners who have bound specifically to the first binding partner. Because unspecific and can bind specific interactions with similarly large binding energies, it is difficult with a binding test based on discrimination of binding energies differ.

Fig. 3 shows a differential force test to differentiate between non-specific and specific binding. The conjugate of BP2 and BP3 binds unspecifically on the surface and specifically with the binding partner BP1 immobilized on the surface with which it forms the binding complex B1. BP4 forms a binding complex B2 with BP3. For a certain force rate, the separation force F _{2 of} the binding pair B2 is greater than the non-specific separation force of the complex to the surface. However, the specific separation force F ₁ of BP2 and BP1 is greater than F ₂ . After the tensile force is applied, the unspecifically bound complex is separated, but not the specifically bound one.

1.2. Distinguishing weaker specific bonds from stronger specific ones Binding using the example: Differentiation of a single base mismatch in one Nucleic acid duplex from a complete pairing

A major problem in differentiating between nucleic acid sequence variants reverse southern hybridization consists of nucleic acid sample molecules attached to the immobilized probe bound to distinguish whether it is complete have complementary bound or have a single base mismatch. During a test with only one specific probe of approx. 15-25 base pairs Single base mismatches of complete pairings also due to the binding energies be distinguished. To do this, the hybridization is carried out close to the melting temperature of the fully paired complex through. Under these conditions is the mismatch unstable. If several probes of different sequence are arranged, as is the case with one reverse hybridization is usually the case, this possibility does not exist.  

FIG. 4 shows a distinction between a complete base pairing and a single base mismatch

2. Determination of dissociation rates

Dissociation rates (off rates) of two binding partners of a sample complex can can be determined when using a sample complex against several reference complexes tested different separation forces at different force rates.

The invention is implemented using the following means:

1. Exercise of strength

The application of tensile forces on the conjugate of the two binding complexes B1 and B2 can be done in very different ways, but it doesn't matter of what nature the applied force is.

In the preferred embodiment, the tensile force is one mechanical, macroscopic train. For this, the conjugate from B1 and B2 is between fixed two bodies and moved them away from each other until it separated the Complex comes. The bodies can be nanoscopic, as is the case with the AFM's cantilever is the case. However, it can also be macroscopically large Act surfaces.

In the second case, the tensile forces are caused by magnetic particles which are attached to the conjugate ( 7 ) from B1 and B2 and on which a magnetic field acts. They can be two different particles, each attached to one end of the complex and which in one case have diamagnetic properties in the other case.

In a third case, one uses the possibility of using the conjugate from B1 and B2 with large Connect molecules or polymers and their resistance in one Liquid flow to build tensile forces. Dynamic tractive forces can be built up if the conjugate from B1 and B2 is bound between particles into which sound waves, in particular let ultrasound couple in.

In a fourth case, we use the influence of an electric field on charged molecules, as is the case with an electrophoretic process. The conjugate from B1 and B2 is for this purpose at least at one end with a charged molecule, preferably one  multiply charged polymer linked. Both ends are loaded with a molecule linked, they are oppositely charged molecules.

In a fifth case, the force is applied by shortening a polymer, that the suspensions of the binding partners BP1 and BP2 or the connection between BP2 and forms BP3. The shortening is based on a change in the conformation of the polymer, caused by a change in the chemical environment, e.g. B. the pH or one Salt concentration is caused.

2. Variation of the force rate

The rate of force with which a molecule is pulled is determined by two parameters. Firstly, it is the spring constant of the suspensions between the binding partner BP1 and BP4 or the spring constant of the connection between BP2 and BP3, the second is the pulling speed. To vary the rate of force, either choose another Spring constant or other pulling speed.

The preferred embodiment for varying the spring constant of a suspension or one Conjugate is done by varying the length of a polymer, the suspension or the Connection forms.

3. Detection

The purpose of the detection is to determine which of the two binding complexes one has Conjugates from B1 and B2 were separated after applying a tensile force. This can done indirectly or directly.

Indirect proof is directed to a free binding partner BP, who is in front of the Applying the tensile force was part of a binding complex B. This is achieved by adding a probe directed against the free binding partner. Is it z. B. for separation of the binding complex B1, BP1 and / or BP2 can be detected.

Direct detection shows which of the two pulling directions the conjugate is in the binding partner BP1 and BP2 was displaced after tearing. To this For this purpose, the conjugate from BP1 and BP2 is provided with a label.

To prove which of the binding complexes has been separated, two can also be used different markings can be used. For this purpose, the conjugate is preferably made from second and third binding partner, the second binding partner or the third Attach a first marker to the binding partner, and the first or fourth  Provide binding partner with a second label, the second label from the first mark is different.

In one embodiment, the separation point is then detected by looking at the amount of the first marker, which is bound to one of the holding devices, determines the amount of second marker determined, which is bound to the same holding device, and compares the determined values with one another and / or relates them to one another. From the Ratio of the quantities of first and second markings, for example stamps or Document, a statement about the extent of the separation of sample or reference complex.

To detect the probe the indirect or the label of the direct detection can Various methods of the prior art can be used. With the preferred In one embodiment, the label is a fluorescent molecule. Here FRET (Fluorescence Resonance Energy Transfer) can also be used by the two binding partners of a binding complex with two different ones Fluorophores, between which a resonance transfer takes place. When separating the the fluorescence signal is lost in both fluorophores. Another variation exists in one of the two binding partners of a binding complex with a fluorophore provided the other with a molecule that quenches the fluorescence of the fluorophore (Quenching). When the binding partners are separated, the extinction of the fluorophore canceled, so there is a reduction in the signal. As preferred fluorophores nanoscale, colloidal semiconductor particles (quantum dots) are used. Further Options are radioactive labeling, labeling with an affinity marker to which an enzyme binds that amplifies the signal, the chemiluminescence, electrochemical markings or mass spectroscopy.

4. Samples

The differential force test described here can be used for a wide range of samples be used. These are preferably proteins, generally antibodies, Antigens, haptens or around natural and artificial nucleic acids. However, it can also be around Viruses, phages, cell components or whole cells act. Furthermore come too complex-forming substances such as chelators into consideration.  

5. Reference complexes

A binding partner of a reference complex can itself be part of a sample, such as it is the case with the sandwich format. In principle, a reference complex can consist of the same Components be built up, like a sample complex.

6. Sequence of chaining

The binding partners BP2 ( 2 ) and BP3 ( 3 ) can be combined to form a conjugate ( 8 ) before the interaction of BP2 and BP3 with BP1 or BP4 occurs.

Alternatively, the conjugate of BP2 and BP3 can only be formed after one Interaction with BP1 and BP4 has occurred.

Preferred embodiment of the invention

In the first preferred embodiment of the invention in which the conjugate from B1 and B2 is attached between two surfaces, the tensile force is determined by a mechanical, macroscopic train applied, the detection takes place directly via a marker.

This embodiment is discussed here using the two formats, which can also be implemented by all other embodiments, the sandwich and the capture format:
In the sandwich format of the differential force test ( FIG. 5), the conjugate of BP2 and BP3 is the sample ( 15 ). A binding partner BP2 ( 2 ) of the sample is specific for a binding partner BP1 ( 1 ) which is bound on the first surface ( 13 ). Another binding partner BP3 ( 3 ) is specific for the binding partner BP4 ( 4 ), which is bound on the second surface ( 14 ). If the two surfaces are brought into contact, the sample interacts with BP1 and BP4, whereby the surfaces ( 13 , 14 ) are linked by means of the resulting binding complexes B1 and B2. If the surfaces are now pulled apart, then that of the two binding complexes that has the lower separation force tends to break. The sample adheres to the surface to which an intact binding complex still exists.

In the capture format of the differential force test ( FIG. 6), the sample ( 15 ) is immobilized on the first surface ( 13 ). The sample has the first binding partner BP1 ( 1 ), which is specific for the second binding partner BP2 ( 2 ). BP2 is linked to BP3 ( 3 ) to form a conjugate, with BP3 binding a fourth binding partner BP4, which is bound on the second surface ( 14 ).

One brings both surfaces into contact, causing BP1 to interact with BP2 comes. If you pull the surfaces apart, the weaker one breaks  two complexes, d. H. either the complex of BP1 with BP2 or the complex of BP3 with BP4. The distribution of the conjugate from BP2 and BP3 between the two surfaces is determined and provides information about which of the two complexes was the more stable.

The connection of the sample ( 15 ) in the capture format can take place covalently or via weak interactions.

The preferred device for carrying out the present invention consists of:

• a) A consumable that consists of two surfaces for binding the reactants consists
• b) The binding partners that are bound on the surfaces
• c) A device to bring the two surfaces in contact and close them again separate after the molecular interaction of the binding partners has occurred.
• d) A label of the conjugate of the binding partners BP2 and BP3, based on which n Distribution between the two surfaces can be determined
• e) A device for detecting the marking

Experimental example 1 Strength test to compare the complexes biotin / streptavidin and iminobiotin / streptavidin

The experiment shows that a differential force test shows the differences in the Separation power of the complexes biotin / streptavidin and iminobiotin / streptavidin can be determined can.

principle

Biotin and iminobiotin are bound to a support. fluorescence-labeled Streptavidin is bound to the immobilized haptens. A stamp made with biotin is coated, is approximated to the base so that the biotin bound to the stamp which can bind streptavidin coupled to the substrate via the haptens. Subsequently the stamp is removed. In the end it is determined which part of the  Streptavidins from iminobiotin of the pad to the biotin of the stamp and what proportion of streptavidin was transferred from the biotin of the pad to the biotin of the stamp.

When separating the surfaces, one of the two bonds must be released. The ligand remains either bound to the stamp or the pad, depending on which of the bindings is mechanically more stable.

execution 1. Coating the stamp

A micro-structured stamp was made from PDMS (polydimethylsiloxane). The Structures consisted of stamp feet of approx. 100 × 100 µm, which through Wells of approximately 25 µm wide and 1 µm deep were separated. Bring that in contact of the stamp and the base assumes that the buffer in between is ousted. This is extremely difficult or slow to do on smooth surfaces. The grooves in the stamp ensure the rapid drainage of the buffer and complete contact of the stamp feet with the pad.

Another advantage of the microstructure is that it is mirrored on the base no molecules are "stamped away" to the stamp grooves. The intensity value of the remaining "grid" represents the density of the molecules before stamping and can can thus be used as a reference value in the evaluation.

To produce the microstructured stamp, a mixture of a 1:10 mixture of silicone elastomer and crosslinking reagent (Sylgard 184, Dow Corning) was poured onto an appropriately structured silicone wafer after multiple degassing and incubated for 24 h at RT. After the polymerization, the structured surface of the stamp was exposed to H ₂ O plasma at 1 mbar in a plasma oven for 15 s. The oxidized surface was incubated with 3% aminosilane (3-aminopropyldimethylethoxysilane; ABCR, Karlsruhe) in 10% H ₂ O and 87% ethanol for 30 min. The silanized surface was washed with ultrapure water and blown dry with nitrogen.

A bifunctional PEG was attached to the amino groups of the silane, one end of which had a carboxy group activated by NHS and the other had a biotin group. 20 μl of a solution with 2 mg / 100 μl of NHS-PEG-Biotin (Shearwater, Huntsville) were incubated under a cover glass for 1 h on a stamp with an area of 1 cm ² . It was washed with ultrapure water and blown dry with nitrogen.

2. Coating the underlay

A glass slide was cleaned by treatment with saturated KOH-ethanol solution for 100 minutes. The cleaned surface was incubated with 3% aminosilane (3-aminopropyldimethyl-ethoxysilane; ABCR, Karlsruhe) in 10% H ₂ O and 87% ethanol for 30 min. The silanized surface was washed with ultrapure water and blown dry with nitrogen. A bifunctional PEG was attached to the amino groups of the silane, one end of which had an NHS-activated carboxy group and the other had a t-Boc-protected amino group (NHS-PEG-NH-tBoc, Shearewater, Huntsville). The tBoc protective group was then cleaved off with trifluoroacetic acid. Biotin and iminobiotin (Sigma, St. Louis) were extracted from a PBS solution (Phosphate Saline Buffer; Sigma) with a concentration of 5 mg / ml with 5 mg / ml EDC (1-ethyl-3- (3-dimethylamino-propyl) carbiimide; Sigma) and 5 mg / ml NHS (N-hydroxysuccinimide; Sigma, St. Louis) bound to the amino groups of the deprotected PEG. The pad was incubated in a saturated H ₂ O atmosphere for 1 h, rinsed with ultrapure water and blown dry with nitrogen. The pad with the immobilized haptens was incubated with 0.1 mg / ml Alexa-Fluor®-546-streptavidin conjugate, rinsed with ultrapure water and blown dry with nitrogen.

3. Stamp

The stamping procedure can be carried out using a simple apparatus such as that shown in FIG. 7. The apparatus consists of a base plate ( 1 ), two guide rods ( 2 ), a stamp carriage ( 3 ), a stamp head pad ( 4 ), the stamp head ( 5 ) and the stamp pad ( 6 ) (see Fig. 7). The base plate, the guide rods and the stamp slide can be made of metal. The stamp head pad can consist of a foam rubber and the stamp head made of plexiglass. The stamp is square and has the area of one cm ² and a thickness of 1 mm. The stamp poster has the same dimensions, but is smooth on both sides and consists, for. B. from a particularly soft PDMS.

For stamping, the base is placed on the base plate and the stamp on the  Stamp pad placed. Both are covered with buffer. The sledge is in the Guide rods inserted and lowered by hand until the stamp with the Pad comes into contact. The separation is also done by hand.

The stamp head cushion has the function of the stamp head when the stamp is placed in bring an exactly parallel position to the base. The stamp pad is used for this compensate for slight unevenness between the stamp and the base.

The surfaces were brought together so that the one bound on the stamp Biotin can interact with the streptavidin of the pad. After an incubation period the surfaces were separated from one another for 30 min.

4. Measurement

The stamp and base were scanned with a laser scanner (Perkin Elmer GeneTac LS IV) scanned for the marker Alexa-Fluor®-546.

Evaluation and result

• 1. The different transfer of biotin or iminobiotin to biotin reflects that different mechanical stability of the streptavidin-hapten complexes again. The Transfer from biotin to biotin provides a 50% reference from which the density of the Coupling events can be determined in the approximate state.
• 2. 15% of the streptavidin bound to biotin is coated by the biotin Stamp taken. The efficiency of the coupling during stamping is therefore included 30%. 30% is transferred from iminobiotin to biotin. The streptavidin biotin Binding is therefore stronger than the streptavidin-iminobiotin binding, so that when separated The streptavidin is always bound to the biotin from the stamp and the underlay while it is releases the bond to iminobiotin.

Fig. 8A and 8B show a representation of the base or of the punch after stamping. Measurements with the AFM force spectrometer showed 160 pN ± 20 pN for the receptor-ligand pair biotin / avidin and 85 ± 15 for iminobiotin / avidin (Florin, EL, Moy VT and Gaub HE Science April 15, 1994, Vol. 264, pp 415-417: "Adhesion Forces Between Individual Ligand-Receptor Pairs"). The force comparison between biotin / streptavidin and iminobiotin / streptavidin comes qualitatively to the result. It can be seen on the support in FIG. 8A that considerably less streptavidin was bound to the iminobiotin spots in the left row than in the biotin spots in the right row. This image can be found in mirror image on the stamp in FIG. 8B, the spots in the left row, in which a concatenation of the species biotin / streptavidin / biotin took place, significantly less streptavidin transferred to the stamp than in the right row, which corresponds to a chain of iminobiotin / streptavidin / biotin.

Experimental example 2 Strength test to compare two DNA duplexes

The experiment shows that the separation forces of two DNA duplexes can be compared using a differential force test. In this example, duplex 1 is a 20 base pair long double strand, duplex 3 is a 30 base pair long double strand.

principle

Oligonucleotide 1 (= Oligo1) is terminally bound to a support. Oligonucleotide 2 (= Oligo2) is hybridized with Oligo1, whereby a sample complex is formed. Oligonucleotide 3 (= Oligo3) is hybridized with Oligo2, whereby a reference complex is formed. Oligo1 and Oligo2 are labeled with different fluorophores. Oligo3 is labeled with a biotin. A stamp, which is coated with streptavidin, is pressed onto the base with the three hybridized oligos. The biotin of Oligo3 is bound to the streptavidin of the stamp. The stamp is removed, and in a chain of Oligo1 with Oligo2 and Oligo3, either the sample complex or the reference complex is torn.

The sample complex is a 20 bp DNA duplex. The Reference complex consists of a DNA duplex of 30 bp, 20 of which match those of the Sample complex are identical. As a reference, a second experiment is carried out at the sample and reference complex are 20 bp long and have the same GC content.  

Because the efficiency with which chains are formed via the biotin-streptavidin bond can only be controlled to a limited extent when stamping, you cannot rely on it during the strength test restrict only Oligo2 to mark by means of the extent of its enrichment on the The ratio of the separation forces of the sample and reference complex to to calculate. You need a second label on Oligo3. One determines from the two marks, how much Oligo2 is enriched in relation to Oligo3 on the stamp, or was depleted on the pad, you get a measure of whether Oligo2 a has greater separation force to Oligo1 or Oligo3.

Care must be taken to ensure that the oligos are not falsified by fluorescent labeling the measurement result by a fluorescence resonance transfer (FRET) between the Markings takes place. Because of a small amount of FRET between the marks a chaining is often unavoidable, make sure that the markings inside a chain in experiment and reference experiment the same distance from each other to have.

Experiment: 20 bp against 30 bp (= experiment 2a)

Reference experiment: 20 bp against 20 bp (= experiment 2b)

execution

• 1. Coating of the underlay:
  Glass slides functionalized with aldehyde groups (Telechem, Atlanta: Superaldehyde Slides) were used as the base. As a passivation against the absorption of the streptavidin of the stamp, polyethylene glycol (PEG) was covalently attached to the glass surface. For this purpose, 2 mg of bifunctional PEG (Shearwater, Huntsville; molecular weight = 3400 g / mol), each with a terminal amino and a terminal carboxy group, were dissolved in 100 μl PBS (Phosphate Saline Buffer; Sigma, St. Louis) and placed under a cover slip for Incubated for 2 hours on the slide. The Schiff bases resulting from the reaction of the amino with the aldehyde groups were reduced for 5 min with a 1% NaBH ₄ solution. The pad was then washed with ultrapure water and blown dry with an oil-free nitrogen jet.
• 2. Connection of Oligo1:
  Oligo1 has an amino label at the 5 'end and a spacer made of 10 adenines. The other 20 bases form the sample complex with Oligo2. Several drops of 1 µl each of a mixture of 25 µM Oligo1 with 5 mg / ml EDC (1-ethyl-3- (3-dimethylamino-propyl) carbiimide; Sigma, St. Louis) and 5 mg / ml NHS (N-hydroxy succinimide; Sigma, St. Louis) in PBS (Phosphate Saline Buffer; Sigma, St. Louis) were spotted on the coated pad. The pad was incubated in a saturated H ₂ O atmosphere for 1 h, washed with 0.2% SDS (sodium dodecyl sulfate; Sigma St. Louis), rinsed with ultrapure water and blown dry with nitrogen.
• 3. Hybridization:
  The oligos 2a and 2b have a Cy3® label (Cyanine3, Amersham-Pharmacia Biotech) at the 5 'end. Oligo3 has a Cy5® label (Cyanine5, Amersham-Pharmacia) at the 5 'end (all oligos from metabion, Martinsried).
  5 µl of a mixture of Oligo2a (1 µM) and Oligo3 (2 µM) in a citrate buffer (pH = 7.2) with 750 mM NaCl were placed under a cover glass over the spots of the attached Oligo1. The hybridization mixture was incubated in a saturated H ₂ O atmosphere at 80 ° C. for 15 min, then allowed to cool at room temperature (RT) for 30 min. The pad was washed once with 15 mM NaCl / O, 2% SDS and a second time with 15 mM NaCl at room temperature and stored in 15 mM NaCl. The same procedure was used for the reference experiment, with Oligo2a being replaced by Oligo2b.
• 4. Coating of the stamp:
  A micro-structured stamp was made from PDMS (polydimethylsiloxane). The structures consisted of stamp feet of approx. 100 × 100 µm, which were separated by depressions approx. 25 µm wide and 1 µm deep. For this purpose, a mixture of a 1:10 mixture of silicone elastomer and crosslinking reagent (Sylgard 184, Dow Corning) was poured onto a correspondingly structured silicone wafer after multiple degassing and incubated for 24 h at RT. After the polymerization, the structured surface of the stamp was exposed to H ₂ O plasma at 1 mbar in a plasma oven for 15 s.
  The oxidized surface was incubated with 3% aminosilane (3-aminopropyldimethylethoxysilane; ABCR, Karlsruhe) in 10% H ₂ O and 87% ethanol for 30 min. The silanized surface was washed with ultrapure water and blown dry with nitrogen. A bifunctional PEG was attached to the amino groups of the silane, one end of which had a carboxy group activated by NHS and the other had a biotin group. 20 ul of a solution with 2 mg / 100 ul NHS-PEG-Biotin (Shearwater, Huntsville) were incubated under a cover glass for 1 h on a stamp with an area of 1 cm ² . It was washed with ultrapure water and blown dry with nitrogen. 0.1 mg / ml streptavidin (Sigma) in PBS was added to the now biotinylated surface and incubated for 30 min. It was washed with ultrapure water and blown dry with nitrogen.
• 5. Stamp:
  A freshly prepared stamp and a base were pressed onto the spots with the attached oligos (Oligo1 + Oligo2 + Oligo3) with a solution of 15 mM NaCl under a pressure of 400 g / cm ² . After 30 minutes, the stamp was raised very slowly. The pad and stamp were washed with ultrapure water and blown dry with nitrogen.
• 6. The stamp and the base were checked with a two-color laser scanner (Perkin Elmer GeneTac LS IV) scanned for the markers Cy3 and Cy5. To make the The stamp of experiment and to ensure measurement results Reference experiment as well as the documents from experiment and reference experiment with same laser intensities scanned.

evaluation

The stamp and the base were evaluated for each experiment.

Evaluation of the documents

The differences ΔCy3 _Unt and ΔCy5 _{Unt were} formed by subtracting the intensities of the stamped areas from the intensities of the non-stamped grooves. The quotient Q _AUnt = ΔCy3 _Unt / ΔCy5 _{Unt was} formed from the two differences for experiment A (Oligo2a), and the quotient Q _BUnt = ΔCy3 _Unt / ΔCy5 _Unt from the differences for experiment B (Oligo2b). The quotient Q _Unt = Q _AUnt / Q _{BUnt was} formed as a measure of the difference in the depletion of Oligo2a and Oligo2b.

Evaluation of the stamp

By subtracting the intensities of the grooves to which no oligos were transferred from the intensities of the stamp surfaces, the differences ΔCy3 _St and ΔCy5 _{St were} formed. The quotient Q _ASt = ΔCy3 _St / ΔCy5 _{St was formed} from the two differences for Experiment A (Oligo2a), and the quotient Q _BSt = ΔC _y 3 _St / ΔCy5 _St from the differences for Experiment B (Oligo2b). The quotient Q _St = Q _ASt / Q _{BSt was} formed as a measure of the difference between the enrichment of Oligo2a and Oligo2b on the stamp.

Result

For the base of experiment 2b, the value Q _BUnt = 0.36 ± 0.08 was found for the base of 2a Q _AUnt = 0.69 ± 0.09. This resulted in Q _Unt = Q _AUnt / Q _BUnt = 1.92.

For the stamp of 2b there was Q _BSt = 0.61 ± 0.08, for the stamp of 2a Q _ASt = 1.20 ± 0.08. This resulted in Q _St = Q _ASt / Q _BSt = 1.97.

Experiment 2a was a force comparison of a 30 bp duplex on the side of the stamp and a 20 bp duplex on the side of the base. If one looks at the result for 2a (Q _AUnt = 0.69, Q _ASt = 1.2) in isolation, no statement can be made as to whether one of the two duplexes was more stable or which of the two when separating the stamp from the Pad torn more than the other. Such a statement from experiment 2a alone would only be possible if the actual amounts of Cy3 and Cy5 could be calculated from the measured fluorescence intensities. Since corresponding calibration values are not available in the present experiment, the force comparison of the 20 bp duplex with another 20 bp duplex in experiment 2b should be used as a reference for the force comparison of the 30 bp duplex with the 20 bp duplex in experiment 2a.

The quotients from experiment 2a and reference experiment 2b give Q _Unt = Q _AUnt / Q _BUnt = 1.92 for the base and Q _St = Q _ASt / Q _BSt = 1.97 for the stamp. This means that about 2 times as much Cy3-labeled oligo was stamped away on the support 2a as for 2b and that about 2 times as much Cy3-labeled oligo was transferred onto the pad as in 2b.

For a calibrated measurement, in which the fluorescence intensities would be directly proportional to the amounts of Cy3 and Cy5, in reference experiment 2b one would use a Q _BUnt = 0.5 and a Q _BSt = 0 due to the same stability of the two 20 bp duplexes. 5 expect. The Q _BUnt = 0.36 determined here is too small by a factor of 1.39 compared to the result to be expected in a calibrated measurement. If one _corrects Q _BUnt by this factor to 0.5, one gets for the correction of experiment 2a

Q _AUcorr. = Q _AUnt × 1.39 = 0.69 × 1.39 = 0.96.

When correcting the values for the stamp, the following is obtained analogously:

Q _BStcorr. = 0.5

Q _BSt = 0.61 = 1.22 × 0.5

Q _Corr. = Q _ASt / 1.22 = Q _ASt = 1.20 / 1.22 = 0.98

From this it can be concluded that there is a depletion of the Oligo2a from the base or an enrichment of Oligo2a on the stamp of approx. 97% is coming.

FIG. 9A and 9B show a representation of the base or of the punch after stamping in Experiment 2a. Fig. 9C and 9D show a representation of the base or of the punch after stamping in Experiment 2b.

FIG. 10A to 10D show the result of evaluation of substrate and stamp by force compared with oligonucleotide or oligonucleotide 2a 2b. These are sections of fluorescence profiles. The course of the profiles is indicated by the arrow in Fig. 9C. The maxima of the graphs correspond to the light grid lines, which represent unstamped areas. The minima between the peaks correspond to the dark squares, from which oligos were stamped away as a result of contact with the streptavidin bound on the stamping feet.

Q _St and Q _Unt showed that the duplex between Oligo2a and Oligo3 has at least 50% greater separation force than the duplex between Oligo2b and Oligo3.

SEQUENCE LISTING

Claims (90)

1. A method for characterizing and / or detecting a binding complex, comprising the steps:
Providing a first binding partner and a conjugate of a second and a third binding partner and providing a fourth binding partner forming a linkage of the binding partners, the first binding partner forming a sample complex with the second binding partner and the third binding partner forming a reference complex with the fourth binding partner,
Applying a force to the chain which leads to the separation of the sample complex or the reference complex and
Determine which of the two binding complexes has been separated. 2. The method according to claim 1, wherein first the conjugate from the second and third Binding partner is provided and then the sample complex and / or the Reference complex is formed. 3. The method according to claim 1, wherein first the sample complex and / or the Reference complex is formed and then the conjugate by connecting the second and third binding partner is provided. 4. The method according to claim 3, comprising the steps of immobilizing the first Binding partner on a first holding device, immobilizing the fourth Binding partner on a second holding device, production of the sample complex by bringing the immobilized first binding partner into contact with the second Binding partner, production of the reference complex by bringing the immobilized fourth binding partner with the third binding partner, approximating the first and second holding means, the second and third binding partner can interact with each other. 5. The method according to any one of claims 1 to 4, wherein the first binding partner and the fourth binding partners are the same.   6. The method according to any one of claims 1 to 5, wherein it is the first and the second binding partner is a ligand and a receptor that is specific bind together. 7. The method according to any one of claims 1 to 5, wherein the sample complex on a non-specific interaction. 8. The method according to claims 1 to 7, wherein the reference complex on a specific or an unspecific interaction. 9. The method according to claims 1 to 8, wherein at least one of the binding partners preferably the sample complex is a body. 10. The method according to any one of the preceding claims, wherein at least one of the Binding partner of the sample complex is a biomolecule. 11. The method according to any one of the preceding claims, wherein the first and / or second Binding partners are attached to a first or second holding device, the preferably each have a body. 12. The method according to any one of claims 9 to 11, wherein at least one body Has macroscopically large surface with which preferably several Sample complexes and / or reference complexes are connectable. 13. The method according to any one of claims 9 to 12, wherein at least one body is nanoscopically small, preferably selected from a group consisting of particles, magnetic particles, paramagnetic particles, diamagnetic particles, colloids, Molecules, charged molecules, polymers, and multiply charged polymers. 14. The method according to any one of the preceding claims, wherein the force to separate the Chaining is applied by a macroscopic train. 15. The method according to any one of the preceding claims, wherein the force by a magnetic field is applied.   16. The method according to any one of the preceding claims, wherein the force by a hydrodynamic flow is applied. 17. The method according to any one of the preceding claims, wherein the force by coupling of sound waves, preferably ultrasonic waves. 18. The method according to any one of the preceding claims, wherein the force by application electrostatic forces is built up. 19. The method according to any one of the preceding claims, wherein the force by molecular Conformational changes of suspensions and / or the connections applied becomes. 20. The method according to any one of the preceding claims, wherein a force in compliance a certain force rate is applied. 21. The method of claim 20, wherein adjusting the force rate over the Pulling speed takes place with which the holding devices are separated. 22. The method of claim 20 or 21, wherein the setting of the force rate on the Spring constant of the chain with its connections. 23. The method of claim 22, wherein adjusting the spring constant via the variation the length of a polymer from which the ligand bonds or the compound, that connects the receptors. 24. The method according to any one of the preceding claims, wherein a sample complex and / or a reference complex has at least one component that is selected from a group that contains low molecular weight substances, polymers, proteins, antibodies, Antigens, haptens, natural or artificial nucleic acids, particles, viruses, phages, Has cells, cell components and / or complex-forming substances such as chelators. 25. The method according to any one of claims 1 to 24, with the steps of immobilizing the first binding partner on a first holding device, immobilizing the fourth Binding partner at a second holding device, providing a conjugate the second and third binding partners representing the sample, the second  Binding partner can bind to the first binding partner and the third Binding partner can bind to the fourth binding partner, approaching the first and the second holding device, the binding partner of the sample with the two other associated binding partners can interact. 26. The method according to any one of claims 1 to 24, with the steps of immobilizing the first binding partner, which is the sample, on a first holding device, Immobilizing the fourth binding partner on a second holding device, Providing a conjugate of the second and third binding partners, wherein the second binding partner can bind to the sample, and the third binding partner can bind the fourth binding partner, approximating the first and second Holding device, the associated binding partners interacting can. 27. The method according to any one of the preceding claims with the step of marking the Sample complex and / or the reference complex, preferably at least one Binding partner and indirect or direct detection of the separation point, which is after the Applying the strength results. 28. The method of claim 27, wherein the conjugate of second and third Binding partner, the second binding partner or the third binding partner with one the first marker, and the first or the fourth binding partner with be provided with a second marking which is different from the first marking is. 29. The method according to claim 28, wherein the detection of the separation point takes place in that one determines the amount of the first marking that is sent to one of the holding devices is bound, the amount of second label determined to the same Holding device is bound, and the determined values are compared and / or relates to each other. 30. The method according to any one of claims 27 to 29, wherein the marking by fluorescent molecules.   31. The method according to claim 30, wherein a binding partner of a binding complex with a first fluorophore and the second binding partner with a second fluorophore between which a fluorescence resonance transfer (FRET) takes place. 32. The method of claim 30, wherein a binding partner of a binding complex with a fluorophore and the second binding partner is provided with a molecule which quenching the fluorescence of the fluorophore. 33. The method according to any one of claims 27 to 29, wherein the marking is fluorescent nanoscopic semiconductor particles (quantum dots). 34. The method according to any one of claims 27 to 29, which is a radioactive Mark is. 35. The method according to any one of claims 27 to 29, wherein the marking is a Enzyme or an affinity marker to which an enzyme can bind, which can by a reaction develops a signal substance. 36. The method of claim 35, wherein the activation of the separation of a complex is coupled to an enzyme that develops a detectable signal. 37. The method according to any one of claims 27 to 29, wherein the marking is a Molecule of electroluminescence, an electrochemically detectable molecule or around is a mass tag that can be detected by mass spectroscopy can. 38. The method according to any one of claims 1 to 37, wherein the method during a Measurement process is carried out on many similar chains. 39. The method of claim 38, wherein only one type of concatenation at only one force rate is tested, the chaining always the same sample complex and always the contains the same reference complex. 40. The method of claim 39, wherein only one type of concatenation at different Force rates is tested, the chaining always the same sample complex and always contains the same reference complex.   41. The method of claim 38, wherein different linkages at only one force rate be tested, whereby the chains always the same sample complex contains different reference complexes with different separating forces. 42. The method of claim 38, wherein different concatenations at different Force rates are tested, the linkages always the same sample complex but contain different reference complexes with different separating forces. 43. The method according to any one of claims 40 to 42, wherein the linkages are serial to only one approach. 44. The method according to any one of claims 40 to 42, wherein the different linkages or force rates in separate approaches are preferably tested in parallel. 45. Device for characterizing and / or detecting a binding complex, with:
a first binding partner and a conjugate of a second and a third binding partner and a fourth binding partner,
a device for linking the binding partners, the first binding partner forming a sample complex with the second binding partner and the third binding partner forming a reference complex with the fourth binding partner,
a device for applying a force to the chain which leads to the separation of the sample complex or the reference complex and
a device for determining which of the two binding complexes has been separated. 46. The apparatus of claim 45, wherein first the conjugate from the second and third binding partner is provided and then the sample complex and / or the reference complex is formed. 47. Apparatus according to claim 45, wherein first the sample complex and / or the Reference complex is formed and then the conjugate by connecting the second and third binding partner is provided. 48. The apparatus of claim 47, wherein the first binding partner to a first Holding device is immobilized, the fourth binding partner to a second  Holding device is immobilized, the second binding partner with the first Binding partner forms the sample complex, and the third binding partner with the fourth binding partner forms the reference complex, and with a facility for Approaching the first and second holding means, the second and third Binding partners can interact. 49. Device according to one of claims 45 to 48, wherein the first binding partner and the fourth binding partner is the same. 50. Device according to one of claims 45 to 49, wherein it is the first and the second binding partner is a ligand and a receptor that is specific bind together. 51. Device according to one of claims 45 to 49, wherein the sample complex on a non-specific interaction. 52. Apparatus according to claims 45 to 51, wherein the reference complex on a specific or non-specific interaction. 53. Device according to claims 45 to 52, wherein at least one of the binding partners preferably the sample complex is a body. 54. Device according to one of claims 45 to 53, wherein at least one of the Binding partner of the sample complex is a biomolecule. 55. Device according to one of claims 45 to 54, wherein the first and / or second Binding partners are attached to a first or second holding device, the preferably each have a body. 56. Device according to one of claims 53 to 55, wherein at least one body Has macroscopically large surface with which preferably several Sample complexes and / or reference complexes are connectable. 57. Device according to one of claims 53 to 56, wherein at least one body is nanoscopically small, preferably selected from a group consisting of particles,  magnetic particles, paramagnetic particles, diamagnetic particles, colloids, Molecules, charged molecules, polymers, and multiply charged polymers. 58. Device according to one of claims 45 to 57, wherein the force device for Separation of the chaining means for applying a macroscopic pull having. 59. Device according to one of claims 45 to 58, with a device for application of magnetic forces. 60. Device according to one of claims 45 to 59, with a device for application of hydrodynamic forces. 61. Device according to one of claims 45 to 60, with a device for coupling of sound waves, preferably ultrasonic waves. 62. Device according to one of claims 45 to 61, with a device for application of electrostatic forces. 63. Device according to one of claims 45 to 62, with a device for effecting of molecular conformational changes of suspensions and / or the Links. 64. Device according to one of claims 45 to 63, wherein the force while maintaining a certain force rate is applied. 65. Device according to claim 64, with a device for adjusting the force rate, preferably the pulling speed at which the holding devices are separated. 66. The apparatus of claim 64 or 65, wherein the force rate on the spring constant of Chaining with their connections is adjustable. 67. The apparatus of claim 66, wherein the spring constant over the variation in length of a polymer from which the bonds of the ligands or the compound that the Connects receptors, is adjustable.   68. Device according to one of claims 45 to 67, wherein a sample complex and / or Reference complex has at least one component that is selected from a Group containing low molecular weight substances, polymers, proteins, antibodies, antigens, Haptens, natural or artificial nucleic acids, particles, viruses, phages, cells, Has cell components and / or complex-forming substances such as chelators. 69. Device according to one of claims 45 to 68, wherein the first binding partner a first holding device is immobilized, the fourth binding partner on one is immobilized, a conjugate of the second and third Binding partner exists, which is the sample, with the second binding partner on can bind the first binding partner and the third binding partner to the fourth Binding partner can bind, and with a facility to approximate the first and the second holding device, the binding partner of the sample with the two other associated binding partners can interact. 70. Device according to one of claims 45 to 68, wherein the first binding partner, the represents the sample, is immobilized on a first holding device, the fourth Binding partner is immobilized on a second holding device, a conjugate the second and the third binding partner, the second binding partner can bind to the sample, and the third binding partner to the fourth binding partner can bind, and with a device for approximating the first and the second Holding device, the associated binding partners interacting can. 71. Device according to one of claims 45 to 70 with a marking on the Sample complex and / or the reference complex, preferably at least one Binding partner and a device for indirect or direct detection of the Separation point that results after the application of the force. 72. The apparatus of claim 71 with a first label on the conjugate from the second and third binding partners and a second label on the first or fourth binding partner, the second marker from the first marker is different.   73. The apparatus of claim 71 or 72, wherein the marking by fluorescent Molecules. 74. The apparatus of claim 73, wherein a binding partner of a binding complex with a first fluorophore and the second binding partner with a second fluorophore between which a fluorescence resonance transfer (FRET) takes place. 75. The apparatus of claim 73, wherein a binding partner of a binding complex with a fluorophore and the second binding partner is provided with a molecule which quenching the fluorescence of the fluorophore. 76. Apparatus according to claim 71 or 72, wherein the marking is fluorescent nanoscopic semiconductor particles (quantum dots). 77. The apparatus of claim 71 or 72, wherein the label is radioactive. 78. The apparatus of claim 71 or 72, wherein the label is an enzyme or Affinity marker to which an enzyme can bind, which by a reaction a Signal substance developed. 79. The apparatus of claim 78, wherein activation of the separation of a complex is coupled to an enzyme that develops a detectable signal. 80. The apparatus of claim 71 or 72, wherein the marking is a Molecule of electroluminescence, an electrochemically detectable molecule or around is a mass tag that can be detected by mass spectroscopy can. 81. Device according to one of claims 45 to 80, with many identical chains, on which the measuring process is carried out. 82. Apparatus according to claim 81, wherein only one type of concatenation at only one force rate is tested, the chaining always the same sample complex and always the contains the same reference complex.   83. The apparatus of claim 81, wherein only one type of concatenation at different Force rates is tested, the chaining always the same sample complex and always contains the same reference complex. 84. The apparatus of claim 81, wherein different linkages at only one force rate be tested, whereby the chains always the same sample complex contains different reference complexes with different separating forces. 85. The apparatus of claim 81, wherein different concatenations at different Force rates are tested, the linkages always the same sample complex but contain different reference complexes with different separating forces. 86. Device according to one of claims 83 to 85, wherein the concatenations in series to only one approach. 87. Device according to one of claims 83 to 85, wherein the different linkages or force rates in separate approaches are preferably tested in parallel. 88. Device according to one of claims 45 to 87, wherein the separating forces of Reference complexes are chosen so that the separation force of the sample complex can be determined approximately. 89. Kit for the detection of a binding complex for carrying out a method according to at least one of claims 1 to 44. 90. Kit for the detection of a binding complex with the facilities according to at least any of claims 45 to 88.