DE102004038163A1 - Fluorescence-based assays for the rapid, quantitative analysis of biomolecules (proteins and nucleic acids) by enrichment on cells or beads - Google Patents

Abstract

The present invention relates to a method for detecting at least one molecule (212) in a solution in a reaction reservoir of a microfluidic chip with a bottom, wherein DOLLAR A a) the molecule to be detected with coated with suitable antibodies beads (210) and with dye-labeled secondary antibodies (214) DOLLAR A b) where the molecule to be detected binds with said substances, DOLLAR A c) forming a complex (216) of molecule to be detected, beads and dye-labeled secondary antibodies and DOLLAR A d) whereby this complex sediments and is detected on the ground.

Description

Territory of invention

The The present invention relates to a method for rapid, quantitative Detection of at least one molecule, in particular a biomolecule, in solution, being the molecule brought into contact with at least two other modified substances and the resulting complex is formed by enrichment Cells or beads is detected.

For the detection of certain antigens or antibodies, mainly the ELISA (enzyme-linked immunosorbent assay) is used. In general, a capture molecule (antigen or antibody) is immobilized unspecifically on the bottom of a microtiter plate (usually adsorption on glass surfaces). After addition of the sample to be examined (incubation time up to hours), the surface is washed at least once to remove unspecifically adsorbed molecules. In the next step, the incubation is followed by a so-called "detector molecule" (usually secondary antibodies labeled with an enzyme, eg horseradish peroxidase) which also binds specifically to the molecule of interest in a sandwich configuration This substance is converted by the enzyme on the detector molecule, eg by oxidation, into a colored compound Finally, the colouration in the supernatant of the sample is measured, which is directly correlated with the presence of the substance to be detected Amplification step (each enzyme forms many colored molecules) ELISA's achieve high sensitivity in the range of 10 ^-9 to 10 ^-11 M target molecule Disadvantages are the many washing steps, the long duration (several hours) and the unspecific adsorption on the glass surfaces.

To the Detection of specific DNA sequences there are various standard methods all on the hybridization of a complementary, dye-labeled oligonucleotide based. For DNA chips, the sample is usually also several times washed to nonspecifically adsorbed nucleic acids and the excess to remove dye-labeled oligonucleotide.

The hitherto used methods are therefore not for use, for example, in one Microfluidic chip suitable in the washing steps difficult to are realizing.

The The invention therefore has as its object, a method for detection of at least one molecule to provide what is specific and sensitive enough, a fast, Quantitative detection of the molecule on a variety of support surfaces too guarantee.

solution

These The object is achieved by the inventions having the features of the independent claims. advantageous Further developments of the inventions are characterized in the subclaims. The wording of all claims is hereby incorporated by reference into the content of this specification.

The specific recognition and activity of biomolecules (nucleic acids, antibodies, enzymes etc.) can be coupled with the concentration of the detected molecules on z. B. beads in an observation volume, and that this coupling is a fast, quantitative analysis of a z. B. allows positive fluorescence signal.

in the The following individual process steps are described in more detail. The steps do not have to necessarily be performed in the order given, and the method to be described can also other, not mentioned Steps have.

It is a method for detecting at least one molecule in one solution in a jar with one Soil proposed. For this purpose, the molecule to be detected with at least a modified first substance and at least one dye-labeled second substance brought into contact. The molecule to be detected binds on the modified first substance and the dye-labeled second substance. This creates a complex of detectable Molecule, modified first substance and dye-labeled second substance. This complex sediments at least in part and can settle on the ground be detected.

This method is suitable, for example, for use in a microfluidic chip which absorbs the solution to be analyzed (eg blood serum, secretion, etc.) by means of capillary forces and into a variable number of reaction vessels or reaction reservoirs (eg 96 ), in which the specific detection reactions take place. The modified first substance and the dye-labeled second substance, which indicate the presence of the molecule to be detected by, for example, a marked increase in fluorescence, are already present in the reaction reservoirs prior to the entry of the solution to be analyzed. These substances are in usually dried at the bottom of the reaction reservoir. By entering the solution to be examined they are released from the ground.

In contrast to most microtiter plates used so far, the specific detection reactions take place in solution, without the use of any washing steps. The detection sensitivities of the method are in the micro (10 ^-6 M) to picomolar (10 ^-12 M) range, depending on the particular assay.

According to one preferred embodiment of Inventions, the molecule to be detected is a biomolecule especially a nucleic acid, an antigen, an antibody and / or an enzyme. Other biomolecules, which detected using the method according to the invention can be are familiar to the expert, and you can For example, the group of peptides, proteins, carbohydrates, etc., but also include more complex structures such as viruses, viroids, prions, etc.

In a further preferred embodiment the modified first substance is a bead with a diameter of about 50 nm to 50 microns, preferably about 1 to 10 microns, in particular approx. 2 to 3 μm. These beads can out all, for the Use of the method according to the invention advantageous materials, provided they comply with your have their planned application modified. As a particularly advantageous have beads made of polystyrene, in addition may also have an iron core. By using such beads with a z. B. iron oxide core can additional Concentration effects are made at the bottom of the reservoir, for example, by using a magnet.

In a further preferred embodiment becomes the surface the modified first substance modified with specific anchor molecules by coating. The specific anchor molecules are it according to one another preferred embodiment to antibodies, Antigens, peptides, carbohydrates, nucleic acids and / or cells. This does not provide a final List of possible anchor molecules and further anchor molecules accessible to the method according to the invention (eg. Viruses, viroids, prions, etc.) are familiar to the expert and are also encompassed by the present invention.

In a particularly preferred embodiment is the dye-labeled second substance to dye-labeled secondary antibodies, substances, which become fluorescent by binding to the molecule to be detected, z. B. conformable flexible smart probes, and / or complementary oligonucleotides. The conformationally flexible, so-called "smart probes" (see, for example, WO 01/36668 A1) the signal to background ratio and can For example, be used as fluorescence-labeled probes, the by attachment to the target molecule their fluorescence intensity increase dramatically. This can be higher Probe concentrations, without increasing the Background fluorescence, achieve. The so far successfully developed "Smart Probes "allow the detection of certain DNA target sequences and certain tumor-associated antibodies directly in, for example Blood samples from patients.

there can according to a another preferred embodiment different molecules to be detected with different dye-labeled Substances interact, preferably bind specifically with them. This specific interaction may then be at least two, preferably cause several color reactions, where these are then recorded and evaluated separately by type of color can be.

It can for the Detection of different molecules to be detected also used different, each specific binding substances be, the substances each with spectroscopically distinguishable Dyes are marked. Advantageously, it is in the dyes to fluorescent dyes, which are spectroscopically are distinguishable. Spectroscopically, dyes can be distinguished for example, due to the absorption spectrum, the emission spectrum, fluorescence lifetime, triplet lifetime, etc.

The Efficiency of this technique can be used by optimizing the Fluorescent dyes are further increased. That's the way it is Example possible, with the help of different fluorescent dyes, for example Detect prions and viruses on or in cells. One can further at this possibility also by using fluorescent dyes with different Absorption wavelengths Carry out multiple markings. To the solution of the problem posed can be a mixed homogeneous / heterogeneous based Procedures are used.

For the detection of certain proteolytic enzymes (proteases), tryptophan residue-modified and dye-labeled protease substrates (short peptides) can be immobilized on the beads. The dye fluorescence is efficiently quenched by the proximity to the Tryptophanrestes. In the presence of the protease, the amino acid sequence between the dye and the tryptophan moiety is hydrolyzed (cut), whereby the spatial proximity is abolished and a strong Fluorescence increase results. If the part of the protease substrate which carries the dye remains immobilized on the bead after the hydrolysis, the protease concentration can be determined by quantitatively reading the fluorescence on the spherical surface.

Similar Dye-labeled peptides may as well for the specific detection of certain antibodies used directly in the blood serum become. This is the fluorescence-quenching influence of Tryptophanrestes, which is triggered by contact formation to the dye, by the binding of the Peptids to the antibody and the associated conformational change in the peptide prevents causing a fluorescence increase (see for example WO 1003/014742 A2). By binding the appropriate, dye-labeled peptide on the surface of the globule can the antibody detected by an increase in the fluorescence signal on the bead become.

Certain nucleic acid segments (specific for are an antibiotic resistance or a viral disease) in the same assay format through the use of DNA hairpins ("Smart Probes ", see z. B. WO 01/36668 A1) who demonstrated the. Here, the to be examined Sample amplified using PCR and a biotinylated primer. In the reaction reservoir are streptavidin coated beads and for the respective DNA sequence specific DNA hairpins. DNA hairpins are single-stranded oligonucleotides, because of the complementarity the DNA building blocks occupy a stem / loop structure at both ends. In the coming to use DNA hairpins is a suitable Fluorescent dye attached to one end of the oligonucleotide, the by guanosine in the complementary other end in the closed state of the DNA hairpin is deleted. The DNA hairpin finds the DNA sequence complementary to its loop on the bead, it spontaneously hybridizes to form the thermodynamically more favorable Double strand. This destroys the trunk / loop structure and simultaneously the fluorescence quenching induced by contact with the guanosine residue Dye lifted.

According to one another preferred embodiment the process of the invention is characterized characterized by that for the detection of the molecule to be detected, the fluorescent dye by means of Laser and / or light-emitting diodes excited at least one suitable wavelength becomes. The quantitative readout of the fluorescence signals can thereby carried out after homogeneous excitation of the individual reaction vessels. It can be a spatially limited Excitations of the fluorescent dye take place in the z direction, ie in a direction perpendicular to the bottom of the vessel, on which the binding complexes sediment. By the additional spatially limited Excitation of the reaction vessels in the z-direction specifically, only the fluorescence at the bottom of the reaction reservoir can be specific read out and thus achieve even better discrimination become.

In a preferred embodiment is the detection of the molecule to be detected using a CCD camera and / or optical tweezers. This can happen the optical tweezers act around a pulsed infrared laser. It is possible, the beads with optical tweezers due to the radiation pressure in the laser focus to capture and selectively read only the fluorescence on the spherical surface. This can be achieved by using a pulsed infrared laser (with a wavelength between about 800 and about 1100 nm), on the one hand the beads begins and simultaneously the fluorescence of the dyes by two-photon excitation generated. The optics used is due to the complementary application unaffected by single- and two-photon microscopy.

to Parallelization of technology can be new Multi-jet systems with, for example, 8 × 8 Laser beams are used advantageously. Here, the excitation laser beam through a combination of beam splitters and mirrors in 64 beams disassembled. The rays can then either using a piezo scanner mirror quickly (i.e., in the range of milliseconds) over the Bottom of the reservoir was scanned and a fluorescence microscope Image generated on the CCD chip or stationary for trapping globules with simultaneous reading of fluorescence can be used.

The Detection principle also allows the use of different excitation wavelengths for simultaneous Determination of different target molecules per reservoir ("multiplexing") the probes with two spectrally different dyes and the globule modified with different catcher molecules become. With simultaneous excitation with two laser wavelengths for the case the one-photon excitation can the signals of the two dyes spectrally separated by a dichroic beam splitters simultaneously on different areas of the CCD chip are read out. In the case of two-photon excitation can dyes be selected with the same laser wavelength efficient can be stimulated but clearly different in their emission spectrum. Of the Use of one- or two-photon excitation is ultimately through the number of parallel to be detected target molecules and the for that disposal controlled costs.

In a particularly preferred embodiment the detection of the molecule to be detected takes place in the red spectral range. In the red spectral range, there are hardly any natural substances or molecules that To absorb light efficiently and to fluoresce it, so that to reduce autofluorescence in biological samples the fluorescence excitation of the sample preferably in this spectral range he follows.

• (1) In der ersten Variante befinden sich bestimmte nicht fluoreszenzmarkierte Fängermoleküle auf den Kügelchen. Fängermoleküle sind Antikörper, Antigene, Peptide, Kohlenhydrate, Nukleinsäuren etc. Alternativ können aber auch Zellen, die bestimmte antigene Oberflächenstrukturen besitzen, z. B. Erythrozyten zur Bestimmung der Blutgruppen, eingesetzt werden. Wichtig ist nur, dass die Kügelchen, bzw. Zellen, so groß sind, dass sie schnell (Sekunden bis Minuten) sedimentieren. Bei Verwendung von Polystyrolkügelchen kann zusätzlich ein Eisenkern verwendet werden, damit die Kügelchen mit magnetischen Kräften manipuliert werden können. Andererseits können die Kügelchen oder Zellen mittels optischen Kräften gefangen und bewegt werden. Zusätzlich werden Detektormoleküle (farbstoffmarkierte Sekundärantikörper oder komplementäre Oligonukleotide) benötigt, die ebenfalls bei Vorhandensein an die Zielmoleküle binden. Durch die Aufkonzentrierung des Fluoreszenzsignals auf der Kügelchenoberfläche und Sedimentation der Kügelchen kann eine einfache und quantitative Detektion bestimmter Zielmoleküle erreicht werden. Das Verfahren kann ohne große Umstände direkt für die meisten gängigen ELISA's und jede nachzuweisende DNA-Sequenz sofort eingesetzt werden.
• (2) In der zweiten Variante können farbstoffgelöschte Proteasesubstrate oder Peptid-Epitope oder DNA-Hairpins auf der Kügelchenoberfläche immobilisiert werden. In Gegenwart des Zielmoleküls kommt es zur Anbindung an die Oberfläche und zu einem Signalanstieg, der quantitativ ausgelesen werden kann.
• (3) In der dritten Variante kann für den Nachweis bestimmter DNA-Sequenzen die vorher immer durchgeführte PCR-Amplifizierung bspw. mit einem biotinylierten Primer durchgeführt werden. Nach Denaturierung der PCR-Produkte werden sie zu mit Streptavidin beschichteten Kügelchen und DNA-Hairpins (Smart Probes, Gen-Pins, siehe oben) oder normalen farbstoffmarkierten Oligonukleotiden gegeben. Die PCR-Produkte binden auf der Oberfläche der Kügelchen und die Sonden hybridisieren in Gegenwart der komplementären Zielsequenz. Somit kommt es ebenso zu einer Anreicherung der Fluoreszenzsignals auf der Oberfläche, die nach Sedimentation der Kügelchen leicht quantitativ ausgelesen werden kann.

The described method can be characterized as follows:

• (1) In the first variant, certain non-fluorescently labeled capture molecules are located on the beads. Catcher molecules are antibodies, antigens, peptides, carbohydrates, nucleic acids, etc. Alternatively, however, cells which have certain antigenic surface structures, for. B. erythrocytes for the determination of blood groups, are used. The important thing is that the beads, or cells, are so large that they sediment quickly (seconds to minutes). When using polystyrene beads, an iron core may additionally be used to allow the beads to be manipulated with magnetic forces. On the other hand, the beads or cells can be caught and moved by optical forces. In addition, detector molecules (dye-labeled secondary antibodies or complementary oligonucleotides) are needed which also bind to the target molecules when present. By concentrating the fluorescence signal on the bead surface and sedimentation of the beads, a simple and quantitative detection of certain target molecules can be achieved. The procedure can readily be used immediately for most common ELISA's and any DNA sequence to be detected.
• (2) In the second variant, dye-deleted protease substrates or peptide epitopes or DNA hairpins can be immobilized on the bead surface. In the presence of the target molecule it comes to the connection to the surface and an increase in signal, which can be read quantitatively.
• (3) In the third variant, the previously always performed PCR amplification can be carried out, for example, with a biotinylated primer for the detection of specific DNA sequences. After denaturation of the PCR products, they are added to streptavidin-coated beads and DNA hairpins (smart probes, gene pins, supra) or normal dye-labeled oligonucleotides. The PCR products bind to the surface of the beads and the probes hybridize in the presence of the complementary target sequence. Thus, there is also an accumulation of the fluorescence signal on the surface, which can be easily read quantitatively after sedimentation of the beads.

• a) farbstoffmarkierten Sekundärantikörper, und/oder
• b) Substanzen, die durch die Bindung an das zu detektierende Molekül fluoreszenzfähig werden.

For the detection of at least one molecule in a solution is a kit with beads having a diameter of about 50 nm to 50 .mu.m, preferably about 1 to 10 .mu.m, in particular about 2 to 3 .mu.m, whose surface with specific anchor molecules for the was modified to be detected molecule. Further, the kit contains

• a) dye-labeled secondary antibodies, and / or
• b) substances that become fluorescent by binding to the molecule to be detected.

• a) farbstoffmarkierte Sekundärantikörper, die das zu detektierende Molekül binden können; und/oder
• b) Substanzen, die durch die Bindung an das zu detektierende Molekül fluoreszenzfähig werden.

For the detection, a microfluidic chip can be used with reaction reservoirs containing the following: beads with a diameter of about 50 nm to 50 .mu.m, preferably about 1 to 10 .mu.m, in particular about 2 to 3 .mu.m, the surface with specific Ankermolekülen was modified for the molecule to be detected. Furthermore, the reaction reservoirs contain

• a) dye-labeled secondary antibodies that can bind the molecule to be detected; and or
• b) substances that become fluorescent by binding to the molecule to be detected.

• (1) Immunhämatologie (z. B. Blutgruppenbestimmung)
• (2) Nachweis von Tumormarkern (Proteasen, p53, Antikörper)
• (3) Bestimmung genetischer Dispositionen (Antibiotika-Resistenz, HIV, HCV)
• (4) Bestimmung von Blutgerinnung und Kardiakmarkern.

Potential applications are z. B .:

• (1) Immunohematology (eg, blood group determination)
• (2) detection of tumor markers (proteases, p53, antibodies)
• (3) Determination of genetic dispositions (antibiotic resistance, HIV, HCV)
• (4) Determination of blood coagulation and cardiac markers.

Further Details and features of the invention will become apparent from the following Description of the representation of the method according to the invention and of preferred Embodiments in Connection with the subclaims. Here you can the respective characteristics for be realized alone or in combination with each other.

In the pictures shows:

1 a schematic representation of the microfluidic chip;

2 a schematic representation of the detection method;

3 an exemplary measurement result for the detection of anti-heparin / PF4 antibodies in blood sera; and

4 a schematic representation of a dye-labeled peptide with tryptophan residue; and

5 a schematic representation of the detection of a protease.

example 1

The basic principle of the detection method is in the 1 for the detection of certain antigens in a microfluidic chip 110 schematically summarized.

In the reaction reservoirs 112 are (dried) globules 210 (please refer 2 ) with a diameter of 2-3 μm whose surface has been modified with specific anchor molecules (eg antibodies) so that the target molecules 212 (Antigens) specifically bind in the presence. In addition, there are dye-labeled probes 214 (eg, secondary antibodies) in each reaction reservoir 112 which are also specific to the target molecules 212 tie.

The microfluidic chips 110 have filling reservoirs 114 on, in the sample or solution to be examined, for. B. blood serum, is dropped. From the filling reservoirs 114 is the sample by capillary forces through Füllungskapillaren 116 to the reaction reservoirs 112 directed. The air in the reaction reservoir 112 escapes through vent capillary 118 , The microfluidic chip is through a foil 120 covered.

When filling the reaction reservoir 112 with the sample, there is a strong mixing of the sample with the beads presented 210 and probes 214 and in the presence of the target molecules 212 for connection to the probes 214 and the beads 210 , It forms a "sandwich" complex 216 out.

The beads 210 as well as the complex 216 Within a few minutes, they sediment quantitatively to the bottom of the reaction reservoir, where they can easily be imaged by fluorescence microscopy (see 3 left half). For a negative sample (see 3 right half) show the sedimented beads 210 no fluorescence. The due to the dye-labeled probes 214 resulting background fluorescence is on fluorescence microscopy imaging of the bottom surface of the reservoir 112 very low.

• (a) die Sedimentation der Kügelchen, d.h. die Möglichkeit der lokalen mikroskopischen Abbildung der Oberfläche und
• (b) die Aufkonzentrierung des Fluoreszenzsignals auf der Kugeloberfläche zur eindeutigen Diskriminierung vom Hintergrundsignal.

The procedure uses

• (a) the sedimentation of the beads, ie the possibility of local microscopic imaging of the surface and
• (b) the concentration of the fluorescence signal on the spherical surface for clear discrimination from the background signal.

3 shows as an example the fluorescence image for the detection of anti-heparin / PF4 antibodies in blood sera. For this purpose, heparin / PF4 was immobilized on 2.7 μm polystyrene beads and a secondary antibody (goat-anti-human) with a dye absorbing in the red spectral region (Alexa 633) was marked.

The secondary antibody and the modified beads were then mixed with various blood sera, and finally a drop of the mixture was placed on a microscope cover glass and with a standard fluorescence microscope - with a mercury lamp as excitation source - with a suitable filter set and a CCD camera Fluorescence at the bottom of the coverslip without washing steps. In 3 on the left the positive result is shown. The right half of 3 shows a negative test solution. The fluorescence of the dye-labeled secondary antibodies in the supernatant only marginally interferes with the detection since the signal on the surface of the beads concentrates and the beads fall quantitatively onto the bottom of the cover glass within a few minutes. In addition, the signal / background ratio can be easily increased by the use of imaging (ie, scanning) confocal fluorescence microscopy, which has a better z-sharpness. The same tests were also used successfully for the determination of blood groups and other antigens.

Example 2

Under the 20 of course occurring amino acids there are similar as with the DNA bases only one amino acid, namely tryptophan, which fluoresces selected Dyes are selectively deleted. This extinguishing principle may also be used to develop peptide-based probes for detection of antibodies be exploited.

The following will be on 4 Referenced. 4 shows in the upper part of an equilibrium reaction between two conformations of a peptide. The peptide has a first fluorescently labeled substrand 412 and a second sub-string 414 containing a tryptophan residue.

Due to the conformational flexibility of the peptide 410 the tryptophan residue may come in contact with the dye. This is the in 4 right above conformation. In this conformation is the fluorescence intensity of the peptide probe 410 greatly reduced.

When the amino acid sequence between dye and tryptophan residue is an epitope for an antibody, binding of the antibody results in the conformational flexibility of the peptide 410 so severely limited that there is no more contact between dye and tryptophan, ie the presence of the antibody results in a fluorescence increase. This technique was successfully used to detect p53 autoantibodies, a universal tumor marker, directly in patient sera (see, for example, WO 2003/014742 A2). When the dye-labeled epitopes are immobilized on beads, binding of the antibodies to the peptides on the bead surface results in a strong increase in fluorescence that can be used to detect the antibodies.

Example 3

The selective fluorescence quenching of some dyes in tryptophan-containing peptides 410 can be equally successful for the detection of various proteases, eg. As tumor-associated proteases, are used when the amino acid sequence between dye 412 and tryptophan residue 414 contains a recognition sequence for the protease, which allows the protease to "cut through" the peptide on this recognition sequence, ie to hydrolyze. This is in 5 clarified.

Experiments in homogeneous solution (see, for example, WO 02/081509 A2) allow reliable detection of very low protease concentrations (10 ^-12 -10 ^-15 M) by simply reading out the fluorescence intensity.

In the variant relevant for the method presented here, the peptides become 410 so immobilized on the spherical surface that after cutting through the protease of the dye carrying peptide residue 412 remains on the surface and thus the beads have a comparatively strong fluorescence intensity after sedimentation.

110

Microfluidic chip

112

reaction reservoir

114

Befüllreservoir

116

Befüllungskapillare

118

deaerating

120

foil

210

globule

212

targets

214

dye-labeled probe

216

"Sandwich" complex

410

fluorescently labeled peptide

412

first fluorescence-labeled partial strand of the fluorescence

labeled peptide 410

414

second substrand of the fluorescently labeled peptide 410 .

of the contains a tryptophan residue

Claims (22)

Method for detecting at least one molecule ( 212 ) in a solution in a vessel ( 112 ) with a bottom, a) wherein the molecule to be detected ( 212 ) with at least one modified first substance ( 210 ) and at least one dye-labeled second substance ( 214 ) is brought into contact; b) wherein the molecule to be detected ( 212 ) on the modified first substance ( 210 ) and the dye-labeled second substance ( 214 ) binds; c) where a complex ( 216 ) from the molecule to be detected ( 212 ), modified first substance ( 210 ) and dye-labeled second substance ( 214 ) forms; and d) where this complex ( 216 ) sedimented at least in part and detected on the ground. Method according to the preceding claim, characterized in that the molecule to be detected ( 212 ) is a biomolecule, in particular a nucleic acid, an antigen, an antibody and / or an enzyme. Method according to one of the preceding claims, characterized in that the modified first substance is beads ( 210 ) having a diameter of about 50 nm to 50 microns, preferably about 1 to 10 microns, in particular about 2 to 3 microns, is. Process according to the preceding claim, characterized in that the beads ( 210 ) is polystyrene beads. Process according to the preceding claim, characterized in that the polystyrene beads ( 210 ) have an iron core. Method according to one of the preceding claims, characterized in that the surface of the modified first substance ( 210 ) was modified with specific anchor molecules. Method according to the preceding claim, characterized in that the specific anchor molecules are antibodies, antigens, peptides, carbohydrates, nucleic acids and / or cells. Method according to one of the preceding claims, characterized in that it is in the dye-labeled second substance ( 214 ) to a) dye-labeled secondary antibodies, and / or b) to substances which become fluorescent by binding to the molecule to be detected, and / or c) complementary oligonucleotides. Method according to one of the preceding claims, thereby in that for the detection of different molecules to be detected different, in each case specific binding substances are used, in which the substances each with spectroscopically distinguishable dyes be marked. Method according to the preceding claim, characterized characterized in that the specific interactions are at least two, preferably more color reactions leads and these by type Color can be recorded separately and evaluated. Method according to one of the 3 preceding claims, thereby in that the dye is a fluorescent dye acting; that for the detection of the molecule to be detected, the fluorescent dye by means of laser and / or LEDs at least one suitable wavelength is stimulated; and that a spatially limited suggestion of the Fluorescent dye is carried out in the direction perpendicular to the ground. Method according to one of the preceding claims, characterized characterized in that the detection of the molecule to be detected under Use of a CCD camera and / or optical tweezers takes place. Method according to the preceding claim, characterized in that the optical tweezers are a pulsed infrared laser is. Method according to one of the preceding claims, characterized characterized in that the detection of the molecule to be detected in the red Spectral range takes place. Kit for the detection of at least one molecule ( 212 ) in a solution with beads ( 210 ) having a diameter of about 50 nm to 50 microns, preferably about 1 to 10 microns, in particular about 2 to 3 microns, whose surface has been modified with specific anchor molecules for the molecule to be detected; and with a) dye-labeled secondary antibody ( 214 ), and / or with b) substances which become fluorescent by binding to the molecule to be detected. Microfluidic chip ( 110 ) with reaction reservoirs ( 112 ) containing: globules ( 210 ) having a diameter of about 50 nm to 50 microns, preferably about 1 to 10 microns, in particular about 2 to 3 microns, whose surface has been modified with specific anchor molecules for the molecule to be detected; and a) dye-labeled secondary antibodies ( 214 ), which can bind the molecule to be detected, and / or b) substances which become fluorescent by binding to the molecule to be detected. Method for detecting an antibody in a solution, in a vessel ( 112 ) with a bottom, a) wherein at least one epitope of an antigen is selected such that the antibody to be detected and the epitope can bind to each other; b) wherein the epitope and a fluorophore are selected such that the epitope has at least one tryptophan residue (quencher) capable of quenching the luminescence of the fluorophore with sufficient spatial proximity between the fluorophore and the tryptophan residue; c) wherein the epitope is labeled with the fluorophore; d) wherein the epitope is immobilized on a first substance; e) wherein the antibody and the first substance bound labeled epitope are contacted in the solution; f) wherein the antibody to be detected binds to the epitope; g) forming a complex of antibody to be detected, the first substance and the dye-labeled epitope; h) wherein this complex is at least partially sedimented and detected on the ground. A kit for the detection of at least one antibody having a) at least one epitope of an antigen selected such that the antibody to be detected and the epitope can bind to each other; b) a fluorophore, wherein the epitope and the fluorophore are selected such that the epitope has at least one tryptophan residue (quencher) which, when sufficiently close to the space between the fluorophore and the fluorophore, luminesces the fluorophore Quenching tryptophan residue, wherein the epitope is labeled with the fluorophore; and with c) beads having a diameter of about 50 nm to 50 μm, preferably about 1 to 10 μm, in particular about 2 to 3 μm, whose surface has been modified with the dye-labeled epitope. Microfluidic chip ( 110 ) for the detection of at least one antibody with reaction reservoirs ( 112 ) containing the kit according to the preceding claim. Kit for the detection of an endopeptidase (enzyme) with I) a dye-labeled Peptide with the following properties: a) the peptide contains a Recognition sequence of endopeptidase (enzyme) and therefore can be used for the enzyme act as a substrate, with an interface between two amino acids at the recognition sequence in the peptide; b) the peptide contains at least a quencher and at least one fluorophore; c) the quencher is the amino acid tryptophan; d) the fluorophore is a fluorescent dye; e) the fluorescent dye is covalently coupled to peptides or proteins; f) the fluorescence of the fluorescent dye can be determined by tryptophan deleted become; g) the sequence of the peptide is formed as follows: H) the quencher is located on one side of the interface; i) the fluorophore is located on the other side of the interface; j) a sufficient spatial Close between Quencher and fluorophore is given as long as the substrate is not yet by the enzyme at the interface between quencher and fluorophore was cut to the possibility at least partial deletion to ensure the emission of the fluorophore; k) after the Cutting the substrate by the enzyme at the interface between Quencher and fluorophore is a significant increase in emission the fluorophore possible; and with II) beads with a diameter of about 50 nm to 50 microns, preferably about 1 to 10 microns, in particular about 2 to 3 μm, on their surface the peptide is immobilized in such a way that the fluorophore after a Cut immobilized by the enzyme to be detected on the surface stays while the quencher is no longer immobilized on the surface. Microfluidic chip ( 110 ) for the detection of an endopeptidase with reaction reservoirs ( 112 ) containing the kit according to the preceding claim. Method for detecting an endopeptidase (enzyme) with the following steps: mix the kit according to claim 20 and the enzyme to be detected in a solution, the beads sediment at least in part and the strength of the fluorescence signal on the surface the sedimented globules is detected.