EP1350107A2 - Procede et dispositif de caracterisation et/ou d'identification d'un complexe de liaison - Google Patents

Procede et dispositif de caracterisation et/ou d'identification d'un complexe de liaison

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Publication number
EP1350107A2
EP1350107A2 EP01974149A EP01974149A EP1350107A2 EP 1350107 A2 EP1350107 A2 EP 1350107A2 EP 01974149 A EP01974149 A EP 01974149A EP 01974149 A EP01974149 A EP 01974149A EP 1350107 A2 EP1350107 A2 EP 1350107A2
Authority
EP
European Patent Office
Prior art keywords
binding partner
binding
complex
force
holding device
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01974149A
Other languages
German (de)
English (en)
Inventor
Hermann Gaub
Christian Albrecht
Filipp Oesterhelt
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Arrowhead Pharmaceuticals Inc
Original Assignee
Nanotype GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE10051143A external-priority patent/DE10051143C2/de
Priority claimed from DE10117866A external-priority patent/DE10117866A1/de
Application filed by Nanotype GmbH filed Critical Nanotype GmbH
Publication of EP1350107A2 publication Critical patent/EP1350107A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots

Definitions

  • the invention relates to a method and an apparatus for characterizing and / or for detecting a binding complex, in particular by means of differentiating molecular separation forces by means of a differential force test.
  • 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 smaller than covalent bonds that are formed or released by chemical reactions.
  • Non-covalent interactions between binding partners with a high degree of selective binding properties are the prerequisite for molecular recognition, which is used in chemical analysis and diagnostics. In the following, such interactions are referred to as specific interactions.
  • the methods for the detection or characterization of biochemical molecules are mostly binding tests.
  • a binding test is based on the formation of a binding complex through the specific interactions of a ligand with a receptor and the detection of this complex.
  • the diagnostic binding test depends on the detection of a known substance in a sample:
  • the receptors are usually antibodies or antibody derivatives with which antigens in the form of proteins, low-molecular substances, but also viruses and whole cells can be detected.
  • the receptor is referred to as a probe that consists of a nucleic acid such as DNA or RNA and that is used to detect nucleic acids in a sample.
  • the most common immunodiagnostic test is the enzyme linked immunosorbent assay (ELISA).
  • ELISA enzyme linked immunosorbent assay
  • a first antibody is immobilized on a surface. It selectively binds an antigen of an added sample mixture via a first binding site (epitope).
  • a second antibody which is labeled and is in free solution, binds to a second epitope of the antigen.
  • the fraction of the labeled antibody that has not bound to the antigen is separated off.
  • the sandwich complexes of the first antibody, antigen and second antibody remaining on the surface are detected using the label.
  • the marking can bind an enzyme that develops the signal by forming a dye. The dye is ultimately the measure of the amount of antigen in the sample examined.
  • a typical molecular diagnostic test is Southern hybridization. It is based on the interaction of a known nucleic acid molecule, the probe, with a complementary nucleic acid of a sample mixture. The sample is immobilized on a surface and the labeled probe is 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 from probe and sample molecules are quantified using the label.
  • ELISA and Southern hybridization have in common that the binding event is detected by a label and that one of the binding partners is bound on a surface. Another property that they have in common with all common binding tests is that the binding properties of two binding partners to one another are characterized on the basis of the size of the binding energy in the resulting binding complex.
  • WO99 / 45142 An analytical method in which a force component is also used is described by WO99 / 45142.
  • a nucleic acid complex is separated as soon as it is connected to a tensile force by the addition of a test substance.
  • the separation of this complex leads to a fluorescence signal due to the spatial separation of two fluorophores.
  • thermodynamics The chemical interaction between two binding partners can be described using different models.
  • the classic theoretical framework is thermodynamics.
  • 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 the basis of macroscopically measurable quantities.
  • binding energy which is defined as the energy required to release a bond.
  • the binding energy of two binding partners to each other can be derived by measuring the concentrations of free and bound binding partners using the equilibrium constant.
  • a fundamentally different concept for the characterization of molecular interaction was developed by force measurement, e.g. with the Atomic Force Microscope, on individual molecules.
  • the forces that are formed between the atoms of two binding partners can be determined experimentally by the separating force to be used.
  • the binding potential of a binding complex can be reconstructed from the rate dependence of the separation forces.
  • the disintegration of the complex in a force measurement is not due to thermal excitation, but to a manipulative intervention.
  • a binding complex is not only characterized by its binding energy. It also has a characteristic activation barrier, which 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.
  • the ligand sits at the minimum of the potential.
  • the ligand must be pulled out of the binding pocket or out of the binding potential via the potential barrier. The force required for this is determined by deriving 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 ⁇ G.
  • a force applied to the complex lowers the one still to be overcome Activation barrier. With every 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:
  • the binding width ⁇ x characteristic of the binding complex and the decay rate can be derived from this of the complex.
  • the first example of such a device is the "surface force apparatus” (SFA) which was developed by Israelachvili in 1976 (patent US5861954, 1999, Israelachvili).
  • SFA surface force apparatus
  • each of the test substances is applied to a cylindrically curved mica sheet, which ideally should be brought into contact at only one point.
  • a precisely controlled movement of the mica surfaces towards each other creates forces between the molecular layers. If one measures the movement of the surfaces in relation to one another as a function of the applied force, one obtains a statement about the adhesive forces between the two molecular layers.
  • the second method for measuring molecular forces is the "atomic force microscope” (AFM).
  • AFM the first determination of the separating force was achieved weak interaction of a biological receptor-ligand pair, the biotin-streptavidin system (E.-L. Florin, VT Moy and HE Gaub, Science 264, 415 (1994)).
  • the biotin-streptavidin system E.-L. Florin, VT Moy and HE Gaub, Science 264, 415 (1994)
  • no macroscopic surfaces are brought into contact with the AFM.
  • the tip of an AFM is only a few nanometers in size and, ideally, can tip at the end of a single atom.
  • a single molecule for example a DNA double strand, can be suspended between an AFM tip and a second surface. If the tip is now pulled away from the surface, the molecule becomes tensioned and the AFM cantilever bends, which allows the molecular binding forces to be measured if the cantilever is known
  • a third method for characterizing molecular forces is based on the use of microscopic magnetic spheres.
  • a receptor is bound to a magnetic ball and this is allowed to form a binding complex with a ligand, which in turn is bound to a surface. If you expose the magnetic ball to a defined magnetic field, you can apply a defined tensile force to the binding complex between the ball and the surface. If you observe the position of the magnetic ball in the direction of the tensile force while varying it until the binding complex is separated, you can determine the separating force that is required to tear apart the receptor and ligand.
  • the fourth method is a force measurement with "optical tweezers" (Optical Tweezers).
  • the basics of this technique go back to Arthur Ashkin.
  • the pulling force is exerted here by the movement of a highly focused laser beam that can capture and move a microscopic particle.
  • An application of the optical tweezers for force measurement for diagnostic purposes has been described by Kishore. (Patent US5620857, 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 a surface which is coated with a probe.
  • stamp conformal pillar
  • separating forces can be measured which serve to identify the sample (patent EP0962759; 1999, Delamarche et al.).
  • Characterizing binding complexes based on separation forces instead of binding energies brings some significant advantages. Via the potential width of the bond, a new independent parameter for characterizing the bond is obtained, which allows different binding modes that have the same binding energies to be distinguished from one another. This applies in particular to the distinction between unspecific and specific bindings in proteins, as well as to the discrimination between nucleic acid duplexes that are completely complementary or have mismatches.
  • An object of the present invention is to provide a method and a device which enables a simple test.
  • Another object of the present invention is to make the described advantages of binding tests, which are based on the differentiation of separating forces, accessible to a broad field of application and to commercial use, which was not possible due to the prior art.
  • the invention has advantages over conventional force discrimination methods.
  • the traditional methods of molecular force measurement are further developments of methods that originally served a completely different purpose.
  • the SFA was developed for surface forces, AFM for the imaging of surfaces, magnetic balls for the separation of molecules and the conformal pillar method as a preparative method for structuring surfaces.
  • Another object of the present invention is to provide a strength test that Can test binding properties of a binding complex by simultaneously testing many non-cooperative individual events.
  • Methods with magnetic beads usually result in Separation forces, which are based on several cooperative events, since several binding complexes are attached to a sphere.
  • Another object of the present invention is a force 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 degree of flexibility in the selection of the detection method.
  • Another object of the present invention is a force test in which tensile forces can be realized which are far above that of the optical tweezers and that of the magnetic balls.
  • Another object of the present invention is to provide faster binding kinetics of the binding partners than is possible in a method such as the ELISA, in which the kinetics are limited by the rate of diffusion of the reactants in free solution.
  • the present invention is based on a
  • the main advantage of the Atomic force Microscope is its high force resolution.
  • apparatus construction leads to high initial costs and the handling, which requires an expert, makes this device unsuitable for use outside of basic research.
  • Another limitation is that a statistically reliable measurement result requires a large number of sequential experiments and is therefore time-consuming.
  • the AFM also has an inherent disadvantage with regard to one of the most important requirements for a diagnostic measurement method, the parallel measurement of many different test substances.
  • the present invention can have the following advantageous properties: A simple and inexpensive apparatus structure Simple handling
  • One of the main advantages of the invention is the possibility of testing many identical sample complexes simultaneously during a measurement process, the result for each complex being independent of the other complexes, that is to say being non-cooperative.
  • the described methods with the conformal pillar or with magnetic balls are always cooperative events, since the separation of some of the complexes affects the probability of separation of the remaining complexes. The information about the individual events is lost.
  • Suspension connection of a binding partner to 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 separating force as a reference value or 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 similar 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.
  • binding complex 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 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.
  • Coupling Connection of the two holding devices via a chain.
  • Coupling partner two elements that bind together and thus create a coupling.
  • Holding device means by which a force can be applied to the chain.
  • Coupling number Number of couplings actually made in one test run.
  • Coupling efficiency The quotient of the number of couplings actually made (number of couplings) and the number of the maximum possible couplings:
  • Fig. 2 shows the principle of the differential force test. After exerting a tensile force on the conjugate of Bl and B2, Bl is broken if Ft ⁇ F 2 or B2 is broken if Fi> F 2 ,
  • FIG. 6 shows the simultaneous execution of a comparative force test on five independent, similar binding complexes in the capture format.
  • the sample has the binding partner BP1 and is bound to surface 1.
  • the conjugate of BP2 and BP3 is labeled. Since F ! > F 2 , predominantly tear the binding complexes B2.
  • FIG. 7 shows a possible embodiment of a stamp apparatus which is suitable for carrying out the method according to the invention. A more detailed description of a possible embodiment can be found in Experimental Example 1.
  • FIG. 8 shows representations of a base (8A) and a stamp (8B) after a strength test for comparing 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 pad in experiment 2a
  • FIG. 9B shows the pad in experiment 2a
  • FIG. 9C shows the pad in experiment 2b
  • FIG. 9D shows the pad in experiment 2b.
  • 10 shows the results of the evaluation of the base and the stamp after a force comparison, a DNA duplex being compared with an identical duplex (IOC and D) and a further duplex (10A and B) (see Experimental Example 2).
  • 10A Document for experiment 2a
  • 10B stamp in experiment 2a
  • IOC document for experiment 2b
  • 10D stamp in experiment 2b.
  • FIG 11 shows schematically the distribution of the conjugate with complete (A) or partial (B and C) coupling.
  • Fig. 12 shows the principle of reverse stamping.
  • a and C each show the merged surfaces during stamping, the marked sample being bound once on the left side (A) and once on the right side (B).
  • the binding partners marked with "X” do not form a coupling efficiency of 2/3 Bond to the other surface. You also do not go into the distribution between the surfaces when separating.
  • Second binding partner BP2 Second binding partner BP2
  • the present invention is a method and an apparatus for carrying out this method, which is completely different from the conventional strength tests has different principle of determining separation forces.
  • a differential strength test consists of two binding complexes that are linked together. When a force is applied that is at least above the separating force of one of the two binding complexes, one of the two binding complexes tears. The binding complex with the higher separation force remains intact. If one knows the separating force of one of the two binding complexes, one can conclude in this way whether the separating force of the second binding complex is greater or less than that of the first.
  • the differential force test can be used for a variety of diagnostic applications.
  • the invention is particularly suitable as a method for diagnostic detection or for characterizing the binding properties of biochemical molecules or molecules with a high degree of specific molecular recognition.
  • the binding properties of binding partners are characterized on the basis of the separating force which is necessary for separating their binding complex.
  • the differential force test according to the invention can be carried out on a single chain. However, it is preferred that several similar chains are used in a force test. If certain components of the chaining and / or process steps are mentioned in the singular in the present application (e.g. binding partner, binding complex, conjugate, chaining, coupling partner, sample, etc.), this does not mean that the invention is based on force tests on individual chains is limited. Rather, this also includes force tests with multiple chains. The use of the singular only serves to improve the clarity of the representation. It is known to the person skilled in the art that a test is generally carried out on many molecules, binding partners, complexes etc. in practice.
  • the characterization of the separating force F 1; which must be used for the separation of a binding complex B1 (5) is carried out by comparison with the reference separating force F 2 , which must be used for the separation of a second binding complex B2 (6).
  • Both binding complexes are linked to form a chain, on the two sides of which a tensile force is applied.
  • Figure 2 shows the principle of the force test.
  • the binding complex B 1 (5) is usually a sample complex, i.e. 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 around a reference complex, i.e. a binding complex, the binding properties of which dictate a size, in particular a separating force, with which the sample complex is compared.
  • the sample complex usually comprises the first binding partner BP1 and the second binding partner BP2, the reference complex the third binding partner BP3 and the fourth binding partner BP4. According to the present invention, however, the sample complex can also include the third binding partner BP3 and the fourth binding partner BP4, the reference complex the first binding partner BP1 and the second binding partner BP2.
  • the principle described above is not a “measurement” in the narrower sense, but rather a “teaching".
  • the term “teaching” describes a test method in which the object to be tested is compared with a known size of another object. In this sense, the separating force of the binding complex B1 is also compared with a second known separating force in the present invention.
  • “Measuring”, on the other hand, is a test method in which the quantity to be determined gives a concrete numerical value on the measuring scale. This corresponds to the situation of a force measurement with the AFM or with one of the other methods of the prior art.
  • each sample complex to be tested is assigned a force gauge on a nanoscopic scale, the reference complex.
  • Each sample complex is tested independently; the result of many individual tests ultimately results in the measurement result.
  • a special feature that follows from this principle is the possibility of separating the binding complexes and the detection in time to be able to execute separately.
  • 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 separation 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 Fi> F 2 or F ! ⁇ F 2 is. This is done by simultaneously exposing many of the same binding complexes to the same separation force and determining how many of the binding complexes B 1 and how many of the binding complexes 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 Fi 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 can be crucial 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 together with the suspension of the first binding partner BP1 and the fourth binding partner BP4.
  • the invention takes into account the number of couplings or the efficiency with which a chain consisting of the binding partners BP1, BP2, BP3 and BP4 is coupled between the two holding devices. Particularly when comparing two similarly large separating forces, it is advantageous to include the number of couplings and / or the coupling efficiency in the evaluation of the experiment in order to be able to determine the actual ratio of the separating forces.
  • a central problem of binding tests in which a first binding partner is immobilized on a surface is the non-specific background signal.
  • the non-specific background signal is caused by molecules from a second binding partner that were added in free solution and bound non-specifically to the surface.
  • the specific signal that is to say the signal of those molecules of the second binding partner which have specifically bound to the first binding partner, is thus superimposed. Since non-specific and specific interactions can bind with similarly large binding energies, it is difficult to distinguish them by a binding test based on the discrimination of binding energies.
  • Figure 3 shows a differential force test to differentiate between non-specific and specific binding.
  • the conjugate of BP2 and BP3 binds non-specifically 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.
  • the separating force F 2 of the binding pair B2 is greater than the separating force of the non-specific binding of the binding partner BP2 to the surface.
  • the specific separation force Fj of BP2 and BPl is greater than F 2 . After the tensile force has been applied, the non-specifically bound conjugate is therefore separated, but not the specifically bound conjugate.
  • a major problem in differentiating nucleic acid sequence variants by reverse southern hybridization is to distinguish nucleic acid sample molecules that have bound to the immobilized probe according to whether they have bound completely complementarily or have a single-base mismatch.
  • single base mismatches can also be distinguished from complete pairings based on the binding energies.
  • the hybridization is carried out close to the melting temperature of the fully paired complex. Under these conditions is the mismatch unstable.
  • this possibility does not exist in the case of an arrangement of several probes of different sequences, as is usually the case with reverse hybridization.
  • FIG. 4 shows a distinction between a complete base pairing and a single base mismatch.
  • the invention is implemented using the following means:
  • the pulling force is a mechanical, macroscopic pull.
  • the chain between two bodies is attached and moved away from each other until one of the two complexes is separated.
  • the bodies can be nanoscopically small, but they can also be macroscopically large surfaces.
  • the tensile forces are caused by magnetic particles that are attached to the chain and act on a magnetic field. They can be two different particles, each attached to one end of the chain and which in one case have diamagnetic properties in the other case.
  • the possibility is used to connect the chain with large molecules or polymers and to use their resistance to build up tensile forces in a liquid flow. Dynamic tensile forces can be built up if the chain is bound between particles into which sound waves, in particular ultrasound, can be coupled.
  • the influence of an electric field on charged molecules is used, as is the case with an electrophoretic process.
  • the chain is connected at least at one end to a charged molecule, preferably a multiply charged polymer. If both ends are linked with a charged molecule, the molecules are oppositely charged.
  • the force is applied by shortening a polymer that forms the suspensions of the binding partners BP1 and BP2 or the connection between BP2 and BP3. The shortening is based on a change in the conformation of the polymer, which is caused by a change in the chemical environment, for example the pH or a salt concentration.
  • the rate of force with which a molecule is pulled is determined by two parameters. On the one hand it is the spring constant of the suspensions of the binding partners BP1 and BP4 or the spring constant of the connection between BP2 and BP3, on the other hand it is the pulling speed. To vary the force rate, either choose a different spring constant or a different pulling speed.
  • the preferred embodiment for varying the spring constant of a suspension or a conjugate is by varying the length of a polymer that forms the suspension or connection.
  • the purpose of the detection is to determine which of the two binding complexes of a chain has been separated after the application of a tensile force. This can be done indirectly or directly.
  • Indirect evidence is directed to a free binding partner BP that was part of a binding complex B before the tensile force was applied. This is achieved by adding a probe that is directed against the free binding partner. Is it e.g. to separate the binding complex B1, BP1 and / or BP2 can be detected.
  • Direct detection shows which of the two pulling directions the conjugate of the binding partners BP3 and BP2 was moved to after tearing.
  • the conjugate of BP3 and BP2 is provided with a label.
  • the determination of which of the two complexes has been separated can be made by determining the amount of conjugate from BP2 and BP3 that is on one of the surfaces or holding devices after the application of the force and after the separation.
  • the determination can also done by determining the amount of conjugate from BP2 and BP3 that is after the application of the force and after the separation on the first holding device and the amount of conjugate from BP2 and BP3 that is determined after the application of the force and after separation is located on the second holding means.
  • the label is a fluorescent molecule.
  • FRET Fluorescence Resonance Energy Transfer
  • FRET Fluorescence Resonance Energy Transfer
  • Another modification is to provide one of the two binding partners of a binding complex with a fluorophore, the other with a molecule that quenches the fluorescence of the fluorophore. When the binding partners are separated, the extinction of the fluorophore is canceled, so the signal is amplified.
  • Nanoscale, colloidal semiconductor particles are used as preferred fluorophores.
  • Other options are radioactive labeling, labeling with an affinity marker to which an enzyme binds which amplifies the signal, chemiluminescence, electrochemical labeling or mass spectroscopy.
  • the differential force test described here can be used for a wide range of samples. These are preferably proteins, generally antibodies, antigens, haptens or natural and artificial nucleic acids. However, they can also be viruses, phages, cell components or whole cells. Complex-forming substances such as chelators are also suitable.
  • a binding partner of a reference complex can itself be part of a sample, as is the case with sandwich format.
  • a reference complex can be made up of the same components as 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.
  • the conjugate of BP2 and BP3 can only be formed after an interaction with BP1 and BP4 has occurred.
  • Coupling means the connection of a chain to the two holding devices.
  • the two elements involved in the coupling are referred to as coupling partners (KP1 and KP2).
  • the coupling partners can be binding partners, as is the case with case 1 (see below). In other cases, however, the coupling partners are different from the binding partners (see cases 2 and 3).
  • the coupling is to be discussed here using the example of a preferred embodiment: When using two surfaces as holding devices, the macroscopic pull to exert forces and a marking on binding partner 2 or 3 or on the conjugate, the coupling can be carried out in various ways; 14 schematically illustrates the following cases:
  • BPl is bound to the first surface.
  • the conjugate of BP2 and BP3 is incubated on the first surface, the complex B 1 being formed from BP 1 and BP2.
  • the second surface is approximated to the first, whereby B2 is formed from BP3 and BP4.
  • the formation of B2 achieves both a chaining of the binding partners 1 to 4 and a coupling of the chaining with the two surfaces.
  • BP3 and BP4 are therefore also the coupling partners.
  • BPl is bound to the first surface.
  • the conjugate of BP2 and BP3 and BP4 are incubated on the first surface in such a way that a chain is formed.
  • the second surface is approximated to the first, whereby BP4 binds to the second surface.
  • the chaining already pre-formed on the first surface is coupled.
  • BP4 is connected to a coupling partner, which binds to a second coupling partner, which is bound on the second surface.
  • BPl is bound to the first surface.
  • BP2 is incubated on the first surface, causing B 1 to form.
  • BP4 is bound on the second surface.
  • BP3 is incubated on the second surface, causing B2 to form.
  • the second surface is approximated to the first, and the conjugate of BP2 and BP3 is formed.
  • both the formation of the linkage and the coupling of the linkage to the two surfaces take place.
  • BP2 and BP3 are each connected to one of the coupling partners.
  • the ratio of the separation forces of B 1 and B2 could be determined directly from the amount of conjugate that remained on the first surface after the test was performed and the amount of conjugate that was transferred to the second surface. In practice, however, these values are more or less falsified if it is not possible to couple almost all of the marked binding partners. 11 shows this clearly.
  • the transfer of the labeled conjugate to the other surface depends on the force ratio of Bl and B2, as well as on the amount of linked chains (coupling number).
  • a small carryover of e.g. B. the first to the second surface can indicate that the separation force of B 1 is greater than that of B2, as well as that only a small number of linkages have been coupled to the second surface.
  • the amount of labeled conjugates remaining on the first surface is increased and thus falsified by those conjugates which are not coupled, i.e. were also not subjected to a force comparison.
  • the coupling efficiency is obtained.
  • the number of the maximum possible couplings is limited by the number of those of the two coupling partners who are in the minority. According to the invention it is therefore possible to determine at least approximately the coupling number or the coupling efficiency, namely the quotient of the number of couplings actually formed and the number of the maximum possible couplings, and, if appropriate, the coupling number or the coupling efficiency when determining whether the sample complex or the reference complex is separated was taken into account.
  • a reference experiment in addition to the actual force comparison, a reference experiment is carried out.
  • the labeled conjugate is incubated with a first binding partner (e.g. BP1), which is bound on a first surface, and the transfer to the second surface, which is decorated with a further binding partner (e.g. BP4), is determined.
  • BP1 and BP4 are different binding partners whose separation force ratio is to be determined.
  • the reference test is carried out in the same way, the binding partners on both surfaces being identical to the binding partner BP4 (ie the binding partner of the force comparison on the second surface).
  • BP4 binding partner of the force comparison on the second surface.
  • the labeled conjugate is incubated in a first implementation with BP1, which is bound on the first surface, and the transfer to surface 2 is determined with BP4.
  • the conjugate is incubated with BP4 on the second surface and the transfer to the first surface is determined.
  • the invention accordingly also relates to a method in which, in a first implementation (i), the first binding partner BP1 and the conjugate comprising the second binding partner BP2 and the third binding partner BP3 are bound on a first holding device, the fourth on a second holding device Binding partner BP4 is immobilized, the two holding devices are approximated so that the third binding partner BP3 and the fourth binding partner BP4 can bind to one another, the amount of conjugate comprising the second binding partner BP2 and the third binding partner BP3, which is determined after the separation from the first Holding device was transferred to the second holding device, and / or the amount of conjugate comprising the second binding partner BP2 and the third binding partner BP3 is determined, which was not transferred from the first holding device to the second holding device after the separation; and in a further implementation (ii) the fourth binding partner BP4 and the conjugate comprising the second binding partner BP2 and the third binding partner BP3 are bound on the second holding device, the first binding partner BP1 is immobil
  • a second marking is used, the transfer of which to the second surface corresponds to the coupling number.
  • An implementation of this procedure is described in Experiment Example 3.
  • B1 and B2 are pre-formed together on the first surface, the conjugate and BP4 being provided with distinguishable markings.
  • the coupling occurs as soon as the one connected to BP4 Coupling partner binds by approaching the two surfaces to the second surface, which carries the second coupling partner.
  • the amount of BP4 that has bound to the second surface thus corresponds to the coupling number.
  • the amount of the labeled conjugate that has been transferred to the second surface or that has been detached from the first is now determined.
  • the conjugate of second and third binding partners, the second binding partner or the third binding partner is preferably provided with a first label, and the first or fourth binding partner is provided with a second label, the second label being different from the first label.
  • the separation point is then detected by determining the amount of the first marking that is bound to one of the holding devices, the amount of second marking that is bound to the same holding device, and comparing the determined values with one another and / or with one another relates. From the ratio of the amounts of the first and second markings, which are bound, for example, to the stamp or the base, a statement about the extent of the separation of samples or Reference complex can be taken.
  • the chaining which comprises the first, the second, the third and the fourth binding partner, is first formed on a first holding device and then in a second step the coupling is carried out with a second holding device.
  • At least one of the binding partners can comprise a nucleic acid, in particular DNA. At least two of the binding partners preferably comprise a natural or artificial nucleic acid.
  • the tensile force is applied by a mechanical, macroscopic pull, the detection takes place directly via a marking.
  • 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 one of the binding partners BP4 (4), which is bound on the second surface (14).
  • the sample interacts with BPl 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.
  • 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).
  • Both surfaces are brought into contact, which leads to the interaction of BPl with BP2. If the surfaces are now pulled apart, the weaker of the two complexes breaks, ie either the complex of BP1 with BP2 or the complex of BP3 with BP4. The distribution of the conjugate of BP2 and BP3 between the two surfaces is determined and provides information about which of the two complexes was the more stable.
  • 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: i) a consumable consisting of two surfaces for binding the reactants ii) the binding partners bound on the surfaces iii) a device for bringing the two surfaces into contact and to separate them again after the molecular interaction of the binding partners has occurred. iv) A label of the conjugate of the binding partners BP2 and BP3, on the basis of which the distribution between the two surfaces can be determined. v) A device for detecting the label
  • the experiment shows that the differential force of the complexes biotin / streptavidin and iminobiotin / streptavidin can be determined by a differential force test.
  • Biotin and iminobiotin are bound to a support. Fluorescence-labeled streptavidin is bound to the immobilized haptens. A stamp which is coated with biotin is approximated to the base such that the biotin bound to the stamp can bind the streptavidin coupled to the base via the haptens. Subsequently the stamp is removed. Finally, it is determined what proportion of the streptavidin has been transferred from the iminobiotin of the support to the biotin of the stamp and what proportion of the streptavidin from the biotin of the support to the biotin of the stamp.
  • a micro-structured stamp was made from PDMS (polydimethylsiloxane).
  • the structures consisted of stamp feet of approx. 100 x 100 ⁇ m, which were separated by depressions approx. 25 ⁇ m wide and 1 ⁇ m deep.
  • the contact between the stamp and the base assumes that the buffer located between them is displaced. 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 surface.
  • microstructure Another advantage of the microstructure is that no molecules are "stamped away” on the base in mirror image to the stamp grooves.
  • the intensity value of the remaining "grids” represents the density of the molecules before stamping and can thus be used as a reference value in the evaluation.
  • a batch of a 1:10 mixture of silicone elastomer and crosslinking reagent (Sylgard 184, Dow Corning) was poured after repeated degassing between a correspondingly structured silicon wafer and a smooth plexiglass plate and incubated vertically for 24 hours at room temperature.
  • the structured surface of the stamp was exposed to H 2 O plasma at 15 mbar in a plasma furnace.
  • the oxidized surface was incubated with 3% aminosilane (3-aminopropyldimethylethoxysilane; ABCR, Düsseldorf) 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 end had a biotin group.
  • 20 ⁇ l of a solution with 2 mg / 100 ⁇ l 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.
  • a glass slide was cleaned by treating with saturated KOH-ethanol solution for 100 minutes.
  • the cleaned surface was incubated with 3% aminosilane (3-aminopropyldimethyl-ethoxysilane; ABCR, Düsseldorf) 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, Shearwater, Huntsville). The tBoc protective group was then cleaved off with trifluoroacetic acid.
  • NHS biotin and NHS iminobiotin were each diluted with PBS (phosphate buffered saline, Sigma) starting from a 50 niM stock solution in DMSO to a final concentration of 5 mM. This solution was used to bind biotin or iminobiotin to the amino-functionalized glass base. This was incubated for one hour in a saturated water atmosphere, washed with ultrapure water and blown dry with nitrogen.
  • a solution with a concentration of 0.1 mg / ml in a glycine / NaOH buffer (pH 10) was prepared to bind the streptavidin-AlexaFluor®-546 conjugate (Molecular Probes, Eugene).
  • the support was incubated with the solution for 20 min, then washed in this buffer for 5 min and blown dry with nitrogen.
  • 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 padding (4), the stamp head (5) and the stamp padding (6) (see FIG. 7).
  • the base plate, the guide rods and the stamp slide can be made of metal.
  • the stamp head cushion can be made from one Foam rubber and the stamp head are 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 example, of a particularly soft PDMS.
  • the base is placed on the base plate and the stamp on the stamp pad. Both are covered with buffer (pH 10).
  • the slide is inserted into the guide rods and lowered by hand until the stamp comes into contact with the surface. The separation is also done by hand.
  • the stamp head cushion has the function of bringing the stamp head into an exactly parallel position to the base when the stamp is placed on it.
  • the stamp pad is used to compensate for slight unevenness between the stamp and the base.
  • the surfaces were brought together so that the biotin bound on the stamp could interact with the streptavidin of the support. After an incubation period of 30 minutes, the surfaces were separated from one another.
  • the stamp and base were scanned with a laser scanner (Perkin Elmer GeneTac LS IV) using the Alexa-Fluor®-546 marker.
  • biotin or iminobiotin to biotin reflects the different mechanical stability of the streptavidin-hapten complexes.
  • the transfer from biotin to biotin corresponds to half of the actual coupling events, i.e. the coupling number corresponds to the double carry.
  • FIGS. 8A and 8B show a representation of the base or of the stamp after stamping. Measurements with the AFM force spectrometer showed 160pN ⁇ 20pN for the receptor-ligand pair biotin / avidin 160pN 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 to the same result qualitatively.
  • the experiment shows that the differential force of the complexes biotin / streptavidin and desthiobiotin / streptavidin can be determined by a differential force test with reverse stamping.
  • a micro-structured stamp was made from PDMS (polydimethylsiloxane).
  • the structures consisted of stamp feet of approx. 100 x 100 ⁇ m, which were separated by depressions approx. 25 ⁇ m wide and 1 ⁇ m deep.
  • the contact between the stamp and the base assumes that the buffer located between them is displaced. 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 surface.
  • microstructure Another advantage of the microstructure is that no molecules are "stamped away” on the base in mirror image to the stamp grooves.
  • the intensity value of the remaining "grids” represents the density of the molecules before stamping and can thus be used as a reference value in the evaluation.
  • the pad also consisted of 1 mm thick PDMS. However, the document was not structured. For production, the mixture was poured between two vertical plexiglass plates and also incubated at room temperature for 24 hours.
  • the polymerized structured and unstructured PDMS plates were cut to a size of 1 cm 2 .
  • the pieces were then exposed to H 2 O plasma in a plasma oven at 2 mbar for 30 s.
  • the oxidized surface was incubated with a solution of 2% aldehyde silane (4-triethoxysilylbutanal, Amchro, Hattersheim, Germany) in 10% HO and 88% ethanol for 30 min.
  • the silanized surface was washed with ethanol and ultrapure water and blown dry with nitrogen.
  • the functionalized documents and stamps were incubated overnight in PBS (phosphate buffered saline; Sigma, St. Louis, USA) with 2% BSA (bovine serum albumin; Roth, Düsseldorf, Germany). In doing so, BSA binds with its amino groups to the surface aldehyde groups. For stabilization, the resulting bases were reduced with 1% sodium borohydride for 15 min. Biotin and desthiobiotin (Sigma, St. Louis, USA) activated by NHS were then bound to unused amino groups of the BSA. For this, 50 mM stock solutions were prepared in DMSO.
  • the stamping procedure can be carried out with a simple apparatus, as is shown, for example, in FIG. 7.
  • the apparatus consists of a base plate (1), two guide rods (2), a stamp carriage (3), a stamp head padding (4), the stamp head (5) and the stamp padding (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 can be square and in this embodiment has the area of one cm 2 and a thickness of 1 mm.
  • the stamp pad has the same dimensions, but is smooth on both sides and consists, for example, of particularly soft PDMS.
  • the stamp head cushion has the function of bringing the stamp head into an exactly parallel position to the base when the stamp is placed on it.
  • the stamp pad is used to compensate for slight unevenness between the stamp and the base.
  • the base is placed on the base plate and the stamp on the stamp pad. Both are covered with buffer.
  • the slide is inserted into the guide rods and lowered by hand until the stamp comes into contact with the surface. The separation is also done by hand.
  • the surfaces were brought together so that the free binding partner on one surface can interact with the complex of streptavidin and the other binding partner on the other surface. After an incubation period of 30 minutes, the surfaces were separated from one another.
  • streptavidin to a base or stamp must lead to the same result with the same surfaces and therefore serves as a control.
  • the stamp and the base were scanned with a laser scanner (GenePix 4000B, Axon Instruments Inc., USA) according to the AlexaFluor®-647 marker.
  • duplex 1 is a 20 base pair long double strand
  • duplex 2 is a 30 base pair long double strand.
  • Oligo2 and Oligo3 are labeled with different fluorophores.
  • Oligo3 is also 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 oligol with Oligo2 and Oligo3, either the sample complex or the reference complex is torn.
  • the sample complex is a DNA duplex of 20bp.
  • the reference complex consists of a DNA duplex of 30 bp, 20 of which are identical to those of the sample complex.
  • a second experiment is carried out in which the sample and reference complex are 20bp long and have the same GC content.
  • the force test cannot be limited to marking only Oligo2 in order to determine the ratio of the separation forces of To calculate sample and reference complex.
  • FRET fluorescence resonance transfer
  • Oligol 5 'NH2-AAA ⁇ AAAAAA TCTCCGGCTTTACGGCGTAT (SEQ ID NO: 1)
  • Oligol 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 ⁇ l each of a mixture of 25 ⁇ M oligol 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 base.
  • 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.
  • SDS sodium dodecyl sulfate
  • the oligos 2a and 2b have a CyS ⁇ marking (Cyanine3, Amersham-Pharmacia Biotech) at the 5 'end.
  • Oligo 3 has a Cy5 ® label (Cyanine5, Amersham-Pharmacia) at the 5 'end (all oligos from metabion, Martinsried).
  • a 1mm thick, microstructured stamp was made from PDMS (polydimethylsiloxane).
  • the structures consisted of stamp feet of approx. 100 x 100 ⁇ m, which were separated by depressions approx. 25 ⁇ m wide and 1 ⁇ m deep.
  • a mixture consisting of a 1:10 mixture of silicone elastomer and cross-linking reagent (Sylgard 184, Dow Corning) was poured between several degassings between an appropriately structured silicon wafer and a smooth plexiglass plate and incubated for 24 hours at RT. After the polymerization, the structured surface of the stamp was exposed to H 2 O plasma at 15 mbar in a plasma furnace.
  • the oxidized surface was incubated with 3% aminosilane (3-aminopropyldimethylethoxysilane; ABCR, Düsseldorf) in 10% H 2 O and 87% ethanol for 30 min.
  • the silanized surface was washed first with ethanol, then 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 ⁇ l of a solution with 20 mg / ml 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 .
  • a freshly prepared stamp and a base were pressed onto the spots with the attached oligos (Oligol + Oligo2 + Oligo3) using a solution of 15 mM NaCl under a pressure of 400 g / cm 2 . After 30 minutes the stamp was lifted very slowly. The pad and stamp were washed with ultrapure water and with nitrogen blown dry.
  • the stamp and base were scanned for the markers Cy3 and Cy5 using a two-color laser scanner (Perkin Elmer GeneTac LS IV). In order to ensure comparability of the measurement results, the stamps from the experiment and the reference experiment as well as the documents from the experiment and the reference experiment were scanned with the same laser intensities.
  • the stamp and the base were evaluated for each experiment.
  • the force comparison of the 20bp duplex with another 20bp duplex in Experiment 2b should be used as a reference for comparing the force of the 30bp duplex with the 20bp duplex in Experiment 2a.
  • the Q B determined here 0.36 is to be expected in comparison with a calibrated measurement
  • FIGS. 9A and 9B show a representation of the base or the stamp after stamping in experiment 2a.
  • FIGS. 9C and 9D show a representation of the base or the stamp after stamping in experiment 2b. Only the dye Cy3 is shown in each case.
  • FIGS. 10A and IOC show the result of the underlay after a force comparison with oligonucleotide 2a and 2b, respectively.
  • Figures 10B and 10D show the result for the stamps. These are sections of fluorescence profiles. The course of the profiles is indicated by the arrow in FIG. 9C.
  • the maxima of graphs 10A and IOC correspond to the light grid lines, which represent unstamped areas of the base. 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.
  • the minima of the graphs in Figures 10B and 10D correspond to the dark grid lines on the stamps to which no fluorophore was transferred.
  • the maxima correspond to the bright quadrants, to which oligos were transferred to the stamp as a result of contact with the base.

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Abstract

La présente invention concerne un procédé de caractérisation et/ou d'identification d'un complexe de liaison comprenant les étapes suivantes: mise à disposition d'un premier partenaire de liaison et d'un conjugué constitué d'un deuxième et d'un troisième partenaire de liaison, et mise à disposition d'un quatrième partenaire de liaison; formation d'un enchaînement des partenaires de liaison, le premier partenaire de liaison formant un complexe échantillon avec le deuxième partenaire de liaison, et le troisième partenaire de liaison formant un complexe de référence avec le quatrième partenaire de liaison; application d'une force sur la chaîne, ladite force conduisant à la séparation du complexe échantillon ou du complexe de référence; et détermination duquel des deux complexes a subit la séparation.
EP01974149A 2000-08-11 2001-08-09 Procede et dispositif de caracterisation et/ou d'identification d'un complexe de liaison Withdrawn EP1350107A2 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
DE10039393 2000-08-11
DE10039393 2000-08-11
DE10051143A DE10051143C2 (de) 2000-08-11 2000-10-16 Verfahren und Vorrichtung zur Charakterisierung und/oder zum Nachweis eines Bindungskomplexes
DE10051143 2000-10-16
DE10117866 2001-04-10
DE10117866A DE10117866A1 (de) 2001-04-10 2001-04-10 Verfahren und Vorrichtung zur Charakterisierung und/oder zum Nachweis eines Bindungskomplexes
PCT/EP2001/009206 WO2002014862A2 (fr) 2000-08-11 2001-08-09 Procede et dispositif de caracterisation et/ou d'identification d'un complexe de liaison

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EP1350107A2 true EP1350107A2 (fr) 2003-10-08

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CA2418861A1 (fr) 2002-02-21
WO2002014862A2 (fr) 2002-02-21
IL154069A0 (en) 2003-07-31
JP2004525341A (ja) 2004-08-19
WO2002014862A8 (fr) 2004-03-25
US20040086883A1 (en) 2004-05-06
AU2001293747A1 (en) 2002-02-25
WO2002014862A3 (fr) 2003-05-30

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