DE10155031A1 - Fluorescence quenching for the detection of ligate-ligand association events in electric fields - Google Patents

Abstract

A method is described for the detection of ligate-ligand association events by fluorescence quenching, which comprises providing a modified surface as a first step. The modification of the surface consists in attaching at least one type of modified ligate, the ligates being modified by attaching at least one type of fluorophore, the further steps of the method according to the invention are providing a sample with ligands, applying an electric field and setting a defined one Strength of the electric field at the location of the modified ligates, contacting the sample with the modified surface, detection of the fluorescence of the fluorophore and comparison of the detected fluorescence intensities with reference values.

Description

Technical field

The present invention relates to a method for the detection of ligate ligand Association events by fluorescence quenching.

State of the art

In disease diagnosis, in toxicological test procedures, in genetic Research and development, as well as in the agricultural and pharmaceutical sector become immunoassays and increasingly also the sequence analysis of DNA and RNA used. In addition to the known serial procedures with autoradiographic or optical detection increasingly find parallel detection methods using array Technology, so-called DNA or protein chips, use. Even with these parallel methods the detection is based on optical, radiographic, mass spectrometric or electrochemical methods.

For gene analysis on a chip, a library of known DNA sequences ("probe oligonucleotides") is fixed in an ordered grid on a surface, so that the position of each individual DNA sequence is known. Fragments of active genes ("target oligonucleotides") present in the test solution, the sequences of which are complementary to certain probe oligonucleotides on the chip, can be identified by detecting the corresponding hybridization events on the chip. In general, the analysis is carried out via optical (or autoradiographic) detection methods using target oligonucleotides which are labeled with a radio label (e.g. ³² P) or a fluorescent dye (e.g. fluorescein, Cy3 or Cy5). The use of fluorescent labels and corresponding fluorescence scanners is increasingly prevalent over the use of radio labels. The fluorescence scanners currently available on the market enable the detection of fluorophores in the subattomole range.

The use of labeled targets for the detection of hybridization events however has some drawbacks. On the one hand, the marking must be in front of the actual one Measurement take place, which is an additional synthesis step and thus additional Workload means. It is also difficult to make a homogeneous marking of the Ensure sample material. There are also stringent washing conditions necessary to be non-specific or non-specific following a hybridization remove bound material.

It is therefore desirable for both protein and DNA analysis advantageous for the user if the targets (antibodies or antigen or DNA Fragment) do not have to be modified with a detection label and according to the Hybridization does not require complicated washing steps.

The disadvantages of labeling the sample material with radioactive elements or Fluorescent dyes can be avoided if instead of the targets Probe molecules are labeled with appropriate fluorescent dyes. To The so-called molecular lights (molecular beacons). These single-stranded oligonucleotides have a hairpin structure (hairpin, stem-and-loop) and carry a fluorophore at one end of the sequence (e.g. Fluorescein, TexasRed®) and a suitable one at the other end of the sequence Fluorescence quencher (e.g. DABCYL). Due to the special geometric arrangement are the fluorescent group and the unit that is used to extinguish the Fluorescence results in close proximity to one another. Therefore, the probes only show one extremely low fluorescence. In the presence of the corresponding to the sequence of Loop complementary sequence (target) hybridization takes place in this Area. This leads to a change in the conformation and the separation of Fluorophore and Quencher, which is observed as a sharp increase in fluorescence can be.

In addition to organic molecules (such as DABCYL), gold nanoparticles are also considered efficient quenchers are used (Nature Biotech. Vol. 19, 2001, page 365). The Quenching the fluorescence through metals is based primarily on a radiationless one Energy transfer from the dye molecule to the metal. When using gold A greater sensitivity is observed for nanoparticles than for organic quenchers. In addition, dyes are efficiently quenched into the near infrared range. On However, the disadvantage of this method is that gold nanoparticles are included Temperatures above 50 ° C are no longer stable. Another disadvantage is that this method is limited to examining solutions and therefore only few sequences can be examined at the same time that The degree of parallelization of this approach is therefore low.

So although there are many detection options for ligate-ligand associates, the need for simple, inexpensive, easy-to-use and reliable detection principles is especially in the area of less dense array technologies (DNA and protein chips with a few individual or up to several hundred thousand test sites per cm ^2, e.g. for so-called POC (Point of Care) systems or for high throughput screening (HTS systems).

Presentation of the invention

The object of the present invention is therefore to provide a method for the detection of Create ligate-ligand association events by fluorescence quenching, which does not have the disadvantages of the prior art.

This object is achieved by the method according to independent Claim 1, by use according to independent claim 25 and solved the kit according to independent claim 26.

Further advantageous details, aspects and refinements of the present invention result from the dependent claims, the description, the figures and the Examples.

The following abbreviations and terms are used in the context of the present invention: Genetics DNA: deoxyribonucleic acid
RNA: ribonucleic acid
PNA: peptide nucleic acid (synthetic DNA or RNA in which the sugar-phosphate unit is replaced by an amino acid. When the sugar-phosphate unit is replaced by the -NH- (CH ₂ ) ₂ - N (COCH ₂ base) -CH ₂ CO unit hybridizes PNA with DNA.)
A: Adenine
G: Guanine
C: Cytosine
T: thymine
U: uracil
Base: A, G, T, C or U
Bp: base pair
Nucleic acid: at least two covalently linked nucleotides or at least two covalently linked pyrimidine (e.g. cytosine, thymine or uracil) or purine bases (e.g. adenine or guanine). The term nucleic acid refers to any "backbone" of the covalently linked pyrimidine or purine bases, such as. B. on the sugar-phosphate backbone of the DNA, cDNA or RNA, on a peptide backbone of the PNA or on analogous structures (e.g. phosphoramide, thio-phosphate or dithio-phosphate backbone). An essential feature of a nucleic acid in the sense of the present invention is the ability for sequence-specific binding of naturally occurring cDNA or RNA.
nt: nucleotide
Nucleic acid oligomer: Nucleic acid of unspecified base length (e.g. nucleic acid octamer: A nucleic acid with any backbone in which 8 pyrimidine or purine bases are covalently bound to one another).
ns oligomer: nucleic acid oligomer
Oligomer: Equivalent to nucleic acid oligomer.
Oligonucleotide: Equivalent to oligomer or nucleic acid oligomer. B. a DNA, PNA or RNA fragment unspecified base length.
Oligo: Abbreviation for oligonucleotide.
Mismatch: To form the Watson Crick structure of double-stranded oligonucleotides, the two single strands hybridize in such a way that base A (or C) of one strand forms hydrogen bonds with base T (or G) of the other strand (in RNA, T is replaced by uracil ). Any other base pairing does not form hydrogen bonds, distorts the structure and is referred to as a "mismatch".
ss: single strand
ds: double strand (double strand) substances fluorophore: chemical compound (chemical substance) that is able to emit a longer-wave (red-shifted) fluorescent light when excited with light. Fluorophores (fluorescent dyes) can absorb light in a wavelength range from ultraviolet (UV) to the visible (VIS) to the infrared (IR) range. The absorption and emission maxima are shifted by 15 to 40 nm (Stokes shift).
FP: fluorophore
Cy3 ™: 5,5'-disulfo-1,1'di (-carbopentenyl) -3,3,3 ', 3'-tetramethylindodicarbocyanine (fluorescent dye from Amersham Life Science, Inc.)
Cy5 ™: 5,5 ', 7,7'-tetrasulfo-1,1'di (-carbopentenyl) -3,3,3', 3'-tetramethyl-benzindodicarbocyanine (fluorescent dye from Amersham Life Science, Inc.)
Fluorescein: resorcin phthalein (fluorescent dye)
Rhodamine 6G: Basic Red 1 (fluorescent dye)
Texas Red®: fluorescent dye from Molecular Probes, Inc.
DABCYL: 4 - ((4 '- (Dimethylamino) phenyl) azo) benzoic acid
Fluorescence quenching: radiation-free energy transfer
Fluorescence quenching: quenching of fluorescence
Quench surface: conductive (metal) surface that can quench fluorescence through an energy transfer (especially gold, silver, copper surfaces etc.)
EDTA: ethylenediamine tetraacetate (sodium salt)
Ligand: term for molecules that are specifically bound by ligates; Examples of ligands in the context of the present invention are substrates, cofactors or coenzymes of a protein (enzyme), antibodies (as ligand of an antigen), antigens (as ligand of an antibody), receptors (as ligand of a hormone), hormones (as ligand of a receptor) ) or nucleic acid oligomers (as ligand of the complementary nucleic acid oligomer.
Ligate: Term for (macro) molecule with specific recognition and binding sites for the formation of a complex with a ligand (template).
Linker: molecular connection between two molecules or between a surface atom, surface molecule or a surface molecule group and another molecule. As a rule, linkers are commercially available as alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl chains, the chain being derivatized at two points with (identical or different) reactive groups. These groups form a covalent chemical bond in simple / known chemical reactions with the corresponding reaction partner. The reactive groups can also be photoactivatable, ie the reactive groups are only activated by light of certain or any wavelength. Preferred linkers are those of chain length 1-20, in particular chain length 1-14, the chain length here being the shortest continuous connection between the structures to be connected, that is to say between the two molecules or between a surface atom, a surface molecule or a surface molecule group and another Molecule.
Spacer: Linker that is covalently attached to one or both of the structures to be connected (see linker) via the reactive groups. Preferred spacers are those of chain length 1-20, in particular chain length 1-14, the chain length being the shortest continuous connection between the structures to be connected. Modified surfaces / electrodes Au-S- (CH ₂ ) ₂ -ss-oligo-FP: Gold surface with covalently applied monolayer made of derivatized single-strand oligonucleotide. The terminal phosphate group of the oligonucleotide is esterified at the 3 'end with (HO- (CH ₂ ) ₂ -S) ₂ to PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH, the SS bond being cleaved homolytically and each causes an Au-SR bond. At the free end, the probe oligonucleotide carries a covalently linked fluorophore (FP) such as e.g. B. Cy3 ™, Cy5 ™, Texas Red®, Rhodamine 6G, Fluorescein etc.
Au-S- (CH ₂ ) ₂ -ds-oligo-FP: Au-S- (CH ₂ ) ₂ -ss-ollgo-FP hybridizes with the oligonucleotide complementary to ss-oligo. Electrochemistry E: electrode potential applied to the working electrode.
^g E _ss : Geometric change potential of the surface-bound single-stranded probe: Potential, when it passes through in the positive direction, the single-stranded probe undergoes a change from a more elongated conformation protruding into the solution to a more compressed form pressed against the surface. (Change of conformation from "standing" to "lying")
^g E _ds : Geometric change potential of the surface-bound double-stranded probe / target: Potential, when it passes through in the positive direction, the double-stranded probe / target undergoes a change from a more elongated conformation protruding into the solution to a more compressed form pressed against the surface. (Change of conformation from "standing" to "lying")

The present invention relates to a method for the detection of ligate ligand Association events by fluorescence quenching, the first step being the Providing a modified surface includes. Modification of the surface consists in the connection of at least one type of modified ligate, where the ligates are modified by binding at least one type of fluorophore. The further steps of the method according to the invention include providing a sample with Ligands, application of an electric field and setting a defined strength of the electric field at the location of the modified ligates, contacting the sample with the modified surface, detection of the fluorescence of the fluorophore and comparison of the detected fluorescence intensities with reference values.

The present invention also relates to the use of substrates, Cofactors, coenzymes, proteins, enzymes, antibodies, antigens, receptors, Hormones and nucleic acid oligomers as ligates and / or ligands in one Method for the detection of ligate-ligand association events by fluorescence Quenching.

In addition, the present invention also includes a kit for carrying out a Method for the detection of ligate-ligand association events by fluorescence Quenching. The kit includes a modified surface, the modification in the There is binding of at least one type of modified ligate and the Ligates are modified by binding at least one type of fluorophore.

In the method according to the invention there is a comparison of the detected Fluorescence intensities with reference values required. These reference values can from previous measurements are already available and therefore need in most general case not detected in the course of the method according to the invention become. Ideally, however, since the reference values are under exactly the same external ones Conditions should be determined like the actual measured values of the Fluorescence intensities, according to a preferred embodiment of the present invention after applying an electric field and adjusting a defined strength of the electric field at the location of the modified ligates and before contacting the target (sample) with the modified surface first detection of the fluorescence of the fluorophore was carried out. The values thus obtained are then used as reference values.

Alternatively, the reference values can also be measured at the same time as the Fluorescence intensity can be determined, ie after the targets have been brought into contact (Sample) with the modified surface. This can be done in two different ways respectively. Either an area of the modified surface is known, which in the carries essentially no ligands or it is a region of the modified surface known, which carries essentially only ligand / ligate associates. So it has to in the first case, ensure that no ligands are present in the sample, those with the ligates present in said area of the modified surface Can form ligate / ligand associates. In the second case, the other way round ensure that there is a sufficient amount of ligand in the sample that are with essentially all of the amount in the defined range the ligates / ligate associations present. That in these two ways certain reference values reflect the fluorescence intensity with 0% association formation or with 100% association formation again and can be used for the quantitative determination of the Association formation in the other areas of the modified surface serve. According to a particularly preferred embodiment of the present invention the two reference measurements described are carried out. So it gets after contacting the sample with the modified surface, both the Fluorescence intensity determined in a region of the modified surface, which in the carries essentially no ligands, as well as in a range of modified Surface which essentially only carries ligand / ligate associates. Through the The determination of the two extreme values is a quantitative detection with a higher one Accuracy required. It is understood that in this case the ligates of the must distinguish the first region from the ligates of the second region.

The quench surface

The term “surface” denotes any carrier material that is suitable fluorophore-derivatized directly or after appropriate chemical modification To wear ligates that are covalently immobilized on the surface and their Fluorescence close to the surface (approx. 1 to 50 angstroms from the surface) by quenching fluorescence (radiation-free energy transfer between the fluorophore as emitter and the surface as absorber) significantly (> 10% of the expected Fluorescence intensity of the fluorophore in the absence of the surface otherwise same conditions) is reduced. In particular, gold and silver are quenched Suitable surface material. The designation is independent of the surface spatial dimensions of the surface and also includes nanoparticles (particles or clusters of a few single to several hundred thousand surface atoms or molecules). The surface can also be attached to a solid support such. B. glass, Metal or plastic bound.

Binding of the ligate to the surface

Process for immobilizing ligate molecules, especially biopolymers such as Nucleic acid oligomers or antigens or antibodies are on a surface known to the expert. The immobilization can e.g. B. covalently via hydroxyl, Epoxy, amino, or carboxy groups of the carrier material with naturally on Ligate present or attached to the ligate by derivatization of thiol, hydroxy, Amino or carboxyl groups take place. The ligate can be directly or via a Linker / spacer connected to the surface atoms or molecules of a surface become. In addition, the ligate can be made using the methods customary in immunoassays be anchored such as B. by using biotinylated ligates noncovalent immobilization on avidin or streptavidin-modified surfaces. at Use of nucleic acid oligomers as ligates can modify the chemical of the probe nucleic acid oligomers with a surface anchor group already in the Course of automated solid phase synthesis or in separate Reaction steps are introduced. It also becomes the nucleic acid oligomer directly or via a linker / spacer with the surface atoms or molecules of one Surface of the type described above linked. This bond can be on various types known from the prior art are carried out (cf. z. B. Hartwich, G .: ELECTROCHEMICAL DETECTION OF SEQUENCE-SPECIFIC NUCLEIC ACID OLIGOMER HYBRIDIZATION EVENTS (1999), WO 00/42217).

Ligands (targets)

Molecules are referred to as ligands that are specific to those on a surface immobilized ligates (probe) interact to form a complex. Examples of ligands in the sense of the present invention are substrates, cofactors or coenzymes as complex binding partners of a protein (enzyme), antibodies (as Complex binding partner of an antigen), antigens (as complex binding partner of an Antibody), receptors (as a complex binding partner of a hormone), hormones (as Complex binding partner of a receptor) or nucleic acid oligomers (as Complex binding partner of the complementary nucleic acid oligomer).

fluorophores

As fluorophores, commercially available fluorescent dyes such. B. Texas Red®, rhodamine dyes, cyanine dyes such as B. Cy3 ™, Cy5 ™, Fluorescein etc (see Fluka, Amersham and Molecular Probes catalog).

fluorescence quenching

Fluorescence quenching is the deactivation of an electronically excited species referred to as a radiationless process. Deactivation can be caused by bumps or also by radiation-free energy transfer to metals. The released energy is dissipated as thermal energy. Gold is an example of one Metal that has the ability to quench fluorescence. The deletion has one strong dependence on the distance of the fluorophore from that as a fluorescence quencher acting surface (inversely proportional to the sixth power of the Distance). The effect of fluorescence quenching is therefore only at distances less than 100 measurable up to 200 Å. In the area larger than approx. 200 Å there are others Distance changes no longer lead to a measurable increase in Fluorescence intensity.

Brief description of the drawings

In the following, the invention will be described in connection with exemplary embodiments are explained in more detail with the drawings. Show it

Fig. 1 is a schematic representation of the detection of nucleic acid oligomer hybridization events by modulation of the fluorescence quenching of the quench surfaces;

Fig. 2 is a schematic representation of the detection of nucleic acid oligomer hybridization events by E-field assisted modulation of the fluorescence quenching of the quench surfaces;

Fig. 3 is a schematic representation of the detection of nucleic acid oligomer hybridization events by "Immobilized ion" -assisted modulation of fluorescence quenching of the quench surfaces. LIST OF REFERENCE NUMBERS 102 fluorophore
201 single-stranded oligomer probe
202 probe hybridizes to target
203 surface (e.g. gold)
204 Distance of the fluorophore to the gold surface before hybridization
205 Distance of the fluorophore to the gold surface after hybridization
301 Device for applying an electric field
401 ionic groups (anions) immobilized on the surface
402 ionic groups (cations) immobilized on the surface

Fig. 1 is a schematic representation showing the detection of nucleic acid-oligomer hybridization events by modulation of the fluorescence quenching of the quench surfaces. According to Fig. 1A is prior to hybridization, the single-stranded probe nucleic acid oligomer 201 in a form which is characterized by a large distance 204 of fluorophore 102 and quenching metal surface. The hybridization with the complementary nucleic acid oligomer strand 202 (target) reduces the distance 205 between the fluorescent dye molecule and the metal surface 203 functioning as a quencher. This leads to a decrease in the fluorescence intensity (bar diagram in FIG. 1A). According to FIG. 1B, the single-stranded probe nucleic acid oligomer 201 exists prior to hybridization in a form which is characterized by a small distance 204 between fluorophore 102 and quenching metal surface. The hybridization with the complementary nucleic acid oligomer strand 202 (target) increases the distance 205 between the fluorescent dye molecule and the metal surface functioning as a quencher. This leads to an increase in the fluorescence intensity (bar chart of FIG. 1B).

Fig. 2 is a schematic representation showing the detection of nucleic acid-oligomer hybridization events by E-field assisted modulation of the fluorescence quenching of the quench surfaces. An electrical field is generated at the location of the probe nucleic acid oligomer 201 by an electrical potential applied to the modified surface. FIG. 2A shows the case in which the single-stranded probe nucleic acid oligomer 201 is in an elongated form due to the electric field. The hybridization with the complementary nucleic acid oligomer strand 202 (target) reduces the distance 205 between the fluorescent dye molecule and the metal surface functioning as a quencher. This leads to a decrease in the fluorescence intensity (bar diagram in FIG. 2A). FIG. 2B shows the case in which the single-stranded probe nucleic acid oligomer 201 is in a compressed form due to the electric field. The hybridization with the complementary nucleic acid oligomer strand 202 (target) increases the distance 205 between the fluorescent dye molecule and the metal surface functioning as a quencher. This leads to an increase in the fluorescence intensity (bar chart of FIG. 2B).

Fig. 3 is a schematic representation showing the detection of nucleic acid-oligomer hybridization events by "Immobilized ion" -assisted modulation of fluorescence quenching of the quench surfaces. Referring to FIG. 3A, the single-stranded probe nucleic acid oligomer 201 is in a stretched shape due to the repulsive interaction between the negatively charged probe, and the immobilized anions four hundred and first The hybridization with the complementary nucleic acid oligomer strand 202 (target) reduces the distance 205 between the fluorescent dye molecule and the metal surface functioning as a quencher. This leads to a decrease in the fluorescence intensity (bar diagram in FIG. 3A). FIG. 3B shows the case where the single-stranded probe nucleic acid oligomer 201 is in a compressed form due to the attractive effect between the negatively charged probe and the immobilized cations 402 . The hybridization with the complementary nucleic acid oligomer strand 202 (target) increases the distance 205 between the fluorescent dye molecule and the metal surface functioning as a quencher. This leads to an increase in the fluorescence intensity (bar chart of FIG. 3B).

Ways of Carrying Out the Invention

The following are two particularly preferred alternatives to the invention Method for the detection of ligate-ligand association events by fluorescence Quenching using the example of nucleic acid hybridization (ligate: probe oligonucleotide; Ligand: DNA section complementary to the probe oligonucleotide). For the person skilled in the art, these alternative methods are straightforward for detection other ligate / ligand associates as described in the section "Ligands / Signal Ligands" were mentioned to transfer.

To reap the benefits of DNA chip technology on the detection of nucleic acid Use oligomer hybrids by modulating fluorescence quenching, different modified nucleic acid oligomer probes become different Sequence with the immobilization techniques described above to a carrier bound. With the arrangement of the nucleic acid oligomer probes of known sequence at every position on the surface, a DNA array, the hybridization event should any target nucleic acid oligomer or a (fragmented) target DNA can be detected, e.g. B. to detect mutations in the target and to be demonstrated in a sequence-specific manner. To do this, the Surface atoms or molecules of a defined area (a test site) with DNA / RNA / PNA nucleic acid oligomers of known but any sequence, as above described, linked. In a most general embodiment, the DNA chip can also be derivatized with a single probe oligonucleotide. As a probe Nucleic acid oligomers become nucleic acid oligomers (e.g. DNA, RNA or PNA Fragments) of base length 3 to 50, preferably length 5 to 40, particularly preferably the length 12 to 30 used.

On the surfaces thus provided with immobilized and fluorophore-labeled Probe oligonucleotides are measured in a reference measurement, e.g. B. with a fluorescent Scanner, the fluorescence intensity of the fluorophore-labeled probe oligonucleotides determined in the single-stranded state.

In the next step, the (as concentrated as possible) test solution with target Oligonucleotide (s) to the surface with immobilized probe oligonucleotides given. Hybridization only occurs in the case where the solution Contains target nucleic acid oligomer strands leading to the surface bound probe nucleic acid oligomers complementary, or at least in wide areas are complementary.

After the hybridization between probe and target is carried out in a second Fluorescence measurement the fluorescence intensity in the hybridized, double-stranded Condition determined.

The difference between the reference measurement and the second measurement per test site is proportional the number of originally in the test solution for the respective test site existing complementary (or in many areas complementary) target Oligonucleotides.

In an alternative, the reference measurement can be omitted if the size of the reference signal beforehand (e.g. due to previous measurements etc.) is known exactly.

Because of the hybridization of probe nucleic acid oligomer and the related complementary nucleic acid oligomer strand (target) changes the distance between the fluorescent dye molecule and the one that acts as a quencher Metal surface. Due to the changed distance, the extent of the Quench process and thus the intensity of the fluorescence a major change. Thus, a sequence-specific hybridization event by fluorescence-based Methods such as B. fluorescence microscopy or measurements with fluorescence scanner can be detected.

• a) Vor der Hybridisierung liegt das einsträngige Sonden-Nukleinsäure-Oligomer 201 in einer Form vor, die durch einen großen Abstand 204 von Fluorophor 102 und quenchender Metalloberfläche 203 charakterisiert ist (hohe Fluoreszenzintensität). Durch die Hybridisierung mit dem dazu komplementären Nukleinsäure-Oligomer-Strang 202 (Target) verändert sich der Abstand 205 zwischen dem fluoreszierenden Farbstoffmolekül 102 und der als Quencher fungierenden Metalloberfläche 203 dahingehend, dass es durch die Verringerung des Abstands zu einer Vermehrung des Quenchen kommt und eine geringere Intensität der Fluoreszenz nach der Hybridisierung beobachtet werden kann (siehe Fig. 1A).
• b) Vor der Hybridisierung liegt das einsträngige Sonden-Nukleinsäure-Oligomer 201 in einer Form vor, die durch einen geringen Abstand 204 von Fluorophor 102 und quenchender Metalloberfläche 203 charakterisiert ist (geringe Fluoreszenzintensität). Durch die Hybridisierung mit dem dazu komplementären Nukleinsäure-Oligomer-Strang 202 (Target) verändert sich der Abstand zwischen dem fluoreszierenden Farbstoffmolekül 102 und der als Quencher fungierenden Metalloberfläche 203 dahingehend, dass es durch die Vergrößerung des Abstands 205 zu einer Verringerung des Quenchen kommt und eine höhere Intensität der Fluoreszenz nach der Hybridisierung beobachtet werden kann (siehe Fig. 1B).

By specifically influencing the strength of the electric field, in the area of which the fluorophore-modified probe oligonucleotides are located, two different spatial arrangements can be realized for the single-stranded probe-nucleic acid oligomer:

• a) Before the hybridization, the single-stranded probe nucleic acid oligomer 201 is in a form which is characterized by a large distance 204 from the fluorophore 102 and the quenching metal surface 203 (high fluorescence intensity). As a result of the hybridization with the complementary nucleic acid oligomer strand 202 (target), the distance 205 between the fluorescent dye molecule 102 and the metal surface 203 functioning as a quencher changes in such a way that the distance increases and the quenching increases lower fluorescence intensity can be observed after hybridization (see FIG. 1A).
• b) Before the hybridization, the single-stranded probe nucleic acid oligomer 201 is in a form which is characterized by a small distance 204 from the fluorophore 102 and the quenching metal surface 203 (low fluorescence intensity). The hybridization with the complementary nucleic acid oligomer strand 202 (target) changes the distance between the fluorescent dye molecule 102 and the metal surface 203 functioning as a quencher in such a way that increasing the distance 205 leads to a reduction in the quenching and one higher fluorescence intensity can be observed after hybridization (see FIG. 1B).

The two geometrical arrangements shown above can be chosen suitable conditions can be achieved, namely by creating an external electric field or by ions immobilized on the modified surface:

Apply an external electric field

Different geometrical arrangements can be realized by applying an external electrical field. In principle, three potential ranges can be distinguished, the exact location of which depends on the selected measurement conditions (e.g. ionic strength, chain length). With negative to slightly positive potentials (E <0.2 V against silver wire), both single and double-stranded oligomers are in a rather elongated conformation due to the repulsion of the negatively charged DNA backbone (see FIG. 2A). A decrease in the fluorescence intensity is observed due to the hybridization.

In an intermediate range with slightly positive potentials (0.2 V <E <0.4 V against silver wire), single-stranded oligomers are present in a more compressed form due to their greater mobility. In this area, double-stranded oligomers are oriented rather perpendicular to the surface due to the rigid helical structure (see FIG. 2B). An increase in the fluorescence intensity is observed due to the hybridization.

With more positive potentials (E> 0.4 V against silver wire) the double-stranded oligomers are also pulled closer to the surface. Therefore, the hybridization results in only a slight change in the fluorescence intensity. The potential at which the single strand experiences a change from "standing" to "lying" (approx. 0.2 V) is defined as the geometric change potential of the single strand ^g E _ss . The potential at which the double strand experiences a change from "standing" to "lying" (approx. 0.4 V) is defined as the geometric change potential of the double strand ^g E _ds .

The application of an external electrical field is usually done by the application potential on the modified surface. From the present Invention, however, are also embodiments according to which, for. B. by Placing the modified surface in a capacitor at the location of the modified Ligates an electric field is generated.

Ions immobilized on the modified surface

Various geometrical arrangements can be realized by immobilizing ions on the modified surface. In principle, two states can be distinguished. Anions 401 immobilized on the surface (e.g. via corresponding bifunctional molecules with carboxylic acid or sulfonic acid functions which are present as surface-bound anions in a certain pH range) lead to an elongated conformation of the single strand 201 because of the repulsion of the negatively charged phosphate backbone. Because of the greater mobility, the single-stranded probe is stretched more than the double-stranded probe / target (see FIG. 3A). A decrease in the fluorescence intensity is observed due to the hybridization.

Cations 402 immobilized on the surface (e.g. via corresponding bifunctional molecules with amine or ammonium functions, which are present as surface-bound cations in a certain pH range) lead to a rather compressed conformation of the single strand because of the attraction of the negatively charged phosphate backbone 201 . Because of the lower mobility, the double-stranded probe / target is compressed less than the single-stranded probe (see FIG. 3B). An increase in the fluorescence intensity is observed due to the hybridization.

embodiments E-field assisted modulation of fluorescence quenching by hybridization

• a) in Puffer (z. B. 10-500 mM Phosphat-Puffer, pH = 7,1 mM EDTA) gelöst mit der Oberfläche in Kontakt gebracht und dort über die reaktive Gruppe des Sonden-Nukleinsäureoligomers an die - gegebenenfalls entsprechend derivatisierte - Oberfläche angebunden oder
• b) in Gegenwart eines monofunktionalen Linkers in Puffer (z. B. 100 mM Phosphat-Puffer, pH = 7,1 mM EDTA, 0,1-1 M NaCl) gelöst mit der Oberfläche in Kontakt gebracht und dort über die reaktive Gruppe des Sonden-Nukleinsäureoligomers gemeinsam mit dem monofunktionalen Linker an die - gegebenenfalls entsprechend derivatisierte - Oberfläche angebunden, wobei darauf geachtet wird, dass genügend monofunktionaler Linker geeigneter Kettenlänge zugesetzt wird (etwa 0.1 bis 10 facher Überschuss), um zwischen den einzelnen Sonden-Oligonukleotiden genügend Freiraum für eine Hybridisierung mit den Signal-Liganden bzw. dem Target-Oligonukleotid zur Verfügung zu stellen oder
• c) in Puffer (z. B. 10-350 mM Phosphat-Puffer, pH = 7,1 mM EDTA) gelöst mit der Oberfläche in Kontakt gebracht und dort über die reaktive Gruppe des Sonden-Nukleinsäureoligomers an die - gegebenenfalls entsprechend derivatisierte - Oberfläche angebunden und anschließend wird die so modifizierte Oberfläche mit dem entsprechenden monofunktionalen Linker in Lösung (z. B. Alkanthiole oder ω-Hydroxy-Alkanthiole in Phosphat- Puffer/EtOH-Mischungen bei Thiol-modifizierten Sondenoligonukleotiden) in Kontakt gebracht, wobei der monofunktionale Linker über seine reaktive Gruppe an die - gegebenenfalls entsprechend derivatisierte - Oberfläche anbindet (vgl. Abschnitt "Bindung des Ligaten an die Oberfläche").

The n nucleotide (nt) long nucleic acid probe (DNA, RNA or PNA, e.g. a 20 nucleotide long oligo) is near one of its ends (3 'or 5' end) directly or via one (any ) Provide spacers with a reactive group for covalent anchoring to the surface, e.g. B. as a 3'-thiol-modified probe oligonucleotide, in which the terminal thiol modification serves as a reactive group for binding to gold. Other covalent anchoring options arise e.g. B. from amine-modified ligate oligonucleotide, which is used for anchoring to surface-bonded carboxylic acid functions (z. B. via acid-functionalized thiols such as mercaptopropionic acid and an activation z. B. as an active ester). A fluorophore is covalently attached in the vicinity of the other terminus of the probe oligonucleotide (cf. Example 1). The nucleic acid probe modified in this way is

• a) dissolved in buffer (for example 10-500 mM phosphate buffer, pH = 7.1 mM EDTA) brought into contact with the surface and there via the reactive group of the probe nucleic acid oligomer to the - if appropriate derivatized - surface tied up or
• b) in the presence of a monofunctional linker in buffer (z. B. 100 mM phosphate buffer, pH = 7.1 mM EDTA, 0.1-1 M NaCl) brought into contact with the surface and there via the reactive group of Probe nucleic acid oligomers are bound together with the monofunctional linker to the surface, which may be correspondingly derivatized, taking care that enough monofunctional linker of suitable chain length is added (about 0.1 to 10-fold excess) in order to allow sufficient space between the individual probe oligonucleotides to provide a hybridization with the signal ligand or the target oligonucleotide or
• c) dissolved in buffer (for example 10-350 mM phosphate buffer, pH = 7.1 mM EDTA) brought into contact with the surface and there via the reactive group of the probe nucleic acid oligomer to the - if appropriate derivatized - surface attached and then the surface modified in this way is brought into contact with the corresponding monofunctional linker in solution (for example alkanethiols or ω-hydroxyalkanethiols in phosphate buffer / EtOH mixtures in the case of thiol-modified probe oligonucleotides), the monofunctional linker being brought into contact attaches its reactive group to the surface, which may be correspondingly derivatized (see section "Binding of the Ligate to the Surface").

The (residual) fluorescence of the fluorophore on the probe oligonucleotide is detected by a suitable method, e.g. B. by fluorescence measurement with a fluorescence scanner in the presence of an applied electric field. At potentials E smaller than the geometric change potential of the single strand ^g E _ss (E < ^g E _ss ), a decrease in the fluorescence intensity is observed due to the hybridization. At potentials E in the range between the geometric change potential of the single strand ^g E _ss and the geometric change potential of the double strand ^g E _ds ( ^g E _ss <E < ^g E _ds ), an increase in the fluorescence intensity is observed due to the hybridization. The location of the potentials ^g E _ss and ^g E _ds depends on the selected conditions. With 100 mM chloride, the potential ^g E _ss is approx. 0.2 V (against silver wire as a reference electrode) and the potential ^g E _ds is approx. 0.4 V (against silver wire as a reference electrode).

The dissolved target is then added, potential hybridization events are made possible under suitable conditions known to those skilled in the art (any freely selectable stringency conditions for the parameters Potential / temperature / salt / chaotropic salts etc. for hybridization) and the measurement for detection of the fluorophore in the presence of the applied electric field repeated.

The difference in the measurement signal (decrease or increase, depending on the measurement method, cf. FIG. 2) is proportional to the number of hybridization events between probe nucleic acid oligomer on the surface and suitable target nucleic acid oligomer in the test solution.

"Immobilized-ion" -supported modulation of the fluorescence quenching by hybridization

• a) in Gegenwart eines bifunktionalen Linkers zur Erzeugung von immobilisierten Ionen (z. B. ω-Carboxy-, Amin- oder Sulfonsäure-Alkanthiole) in Puffer (z. B. 100 mM Phosphat-Puffer, pH = 7,1 mM EDTA, 0,1-1 M NaCl) gelöst mit der Oberfläche in Kontakt gebracht und dort über die reaktive Gruppe des Sonden-Nukleinsäureoligomers gemeinsam mit dem bifunktionalen Linker an die - gegebenenfalls entsprechend derivatisierte - Oberfläche angebunden, wobei darauf geachtet wird, dass genügend bifunktionaler Linker geeigneter Kettenlänge zugesetzt wird (etwa 0.1 bis 10 facher Überschuss), um zwischen den einzelnen Sonden-Oligonukleotiden genügend Freiraum für eine Hybridisierung mit den Signal-Liganden bzw. dem Target-Oligonukleotid zur Verfügung zu stellen oder
• b) in Puffer (z. B. 10-350 mM Phosphat-Puffer, pH = 7,1 mM EDTA) gelöst mit der Oberfläche in Kontakt gebracht und dort über die reaktive Gruppe des Sonden-Nukleinsäureoligomers an die - gegebenenfalls entsprechend derivatisierte - Oberfläche angebunden und anschließend wird die so modifizierte Oberfläche zur Erzeugung von immobilisierten Ionen mit dem entsprechenden bifunktionalem Linker in Lösung (z. B. ω-Carboxy-, Amin- oder Sulfonsäure-Alkanthiole in Phosphat-Puffer/EtOH-Mischungen bei Thiolmodifizierten Sondenoligonukleotiden) in Kontakt gebracht, wobei der monofunktionale Linker über seine reaktive Gruppe an die - gegebenenfalls entsprechend derivatisierte - Oberfläche anbindet (vgl. Abschnitt "Bindung des Ligaten an die Oberfläche").

The n nucleotide (nt) long nucleic acid probe (DNA, RNA or PNA, e.g. a 20 nucleotide long oligo) is near one of its ends (3 'or 5' end) directly or via one (any ) Provide spacers with a reactive group for covalent anchoring to the surface, e.g. B. as a 3'-thiol-modified probe oligonucleotide, in which the terminal thiol modification serves as a reactive group for binding to gold. Other covalent anchoring options arise e.g. B. from amine-modified ligate oligonucleotide, which is used for anchoring to surface-bonded carboxylic acid functions (z. B. via acid-functionalized thiols such as mercaptopropionic acid and an activation z. B. as an active ester). A fluorophore is covalently attached in the vicinity of the other terminus of the probe oligonucleotide (cf. Example 1). The nucleic acid probe modified in this way is

• a) in the presence of a bifunctional linker to generate immobilized ions (e.g. ω-carboxy-, amine- or sulfonic acid alkanethiols) in buffer (e.g. 100 mM phosphate buffer, pH = 7.1 mM EDTA, 0.1-1 M NaCl) dissolved in contact with the surface and bonded there via the reactive group of the probe nucleic acid oligomer together with the bifunctional linker to the - if appropriate derivatized - surface, taking care that sufficient bifunctional linkers are more suitable Chain length is added (about 0.1 to 10-fold excess) in order to provide sufficient space between the individual probe oligonucleotides for hybridization with the signal ligands or the target oligonucleotide or
• b) dissolved in buffer (for example 10-350 mM phosphate buffer, pH = 7.1 mM EDTA) brought into contact with the surface and there via the reactive group of the probe nucleic acid oligomer to the - if appropriate derivatized - surface attached and then the surface modified in this way to generate immobilized ions is contacted with the corresponding bifunctional linker in solution (e.g. ω-carboxy, amine or sulfonic acid alkanethiols in phosphate buffer / EtOH mixtures in the case of thiol-modified probe oligonucleotides) brought, wherein the monofunctional linker binds via its reactive group to the - if appropriate derivatized - surface (see section "binding of the ligate to the surface").

The (residual) fluorescence of the fluorophore on the probe oligonucleotide is indicated by a suitable method is detected, e.g. B. by fluorescence measurement with a Fluorescence scanner. Then the dissolved target is added, potential Hybridization events are carried out under suitable, known to those skilled in the art Conditions enabled (any freely selectable stringency conditions for the parameters Potential / temperature / salt / chaotropic salts etc. for hybridization) and the measurement repeated for detection of the fluorophore.

The difference in the measurement signal (decrease or increase, depending on the measurement method, cf. FIG. 3) is proportional to the number of hybridization events between probe nucleic acid oligomer on the surface and suitable target nucleic acid oligomer in the test solution.

The embodiments described above can be used for a target type (e.g. a certain target oligonucleotide type with known sequence) on a surface or - when using different probe types per test site - for several target types (same ligand groups as e.g. several different target oligonucleotide types or different antibody types, antigen types etc., but also mixtures thereof) be applied.

embodiments example 1 Representation of the amino-modified oligonucleotides for anchoring on modified Gold surfaces as ligate or probe oligonucleotides

The oligonucleotides are synthesized in an automatic oligonucleotide Synthesizer (Expedite 8909; ABI 384 DNA / RNA synthesizer) according to the manufacturer recommended synthesis protocols for a 1.0 µmol synthesis. With the syntheses with the 1-O-dimethoxytrityl-propyl-disulfide-CPG carrier (Glen Research 20-2933) The oxidation steps are performed with a 0.02 M iodine solution to make an oxidative Avoid cleavage of the disulfide bridge. Modifications to the 5 'position of the Oligonucleotides are carried out with a coupling step that is extended to 5 min. The amino Modifier C2 dT (Glen Research 10-1037) is included in the sequences with the respective Standard protocol built in. The coupling efficiencies are during the synthesis online via the DMT cation concentration photometrically or conductometrically certainly.

The oligonucleotides are concentrated ammonia (30%) at 37 ° C for 16 h deprotected. The oligonucleotides are purified using RP-HPL chromatography according to standard protocols (eluent: 0.1 M triethylammonium acetate buffer, Acetonitrile), characterization using MALDI-TOF MS. The amine modified Oligonucleotides are attached to the correspondingly activated fluorophores (e.g. Fluoresceinisothiocyanat) coupled according to conditions known to those skilled in the art. The coupling can take place both before and after the oligonucleotides have been attached to the Surface.

Example 2 Representation of the fluorophore-modified oligonucleotides for anchoring modified gold surfaces as ligate or probe oligonucleotides

The oligonucleotides are synthesized in an automatic oligonucleotide Synthesizer (Expedite 8909; ABI 384 DNA / RNA synthesizer) according to the manufacturer recommended synthesis protocols for a 1.0 µmol synthesis. With the syntheses with the 1-O-dimethoxytrityl-propyl-disulfide-CPG carrier (Glen Research 20-2933) The oxidation steps are performed with a 0.02 M iodine solution to make an oxidative Avoid cleavage of the disulfide bridge. Modifications to the 5 'position of the Oligonucleotides are carried out with a coupling step that is extended to 5 min. The In the last coupling step, fluorophores are used as phosphoramidites on the synthesizer (Glen Research 10-1037) into the sequences with the respective standard protocol built-in. The coupling efficiencies are checked online during the synthesis via the DMT cation concentration determined photometrically or conductometrically.

Example 3 Production of the oligonucleotide electrode Au-S (CH ₂ ) - ₂ -ss-oligo-FP

The quench surface (here: gold plate) is treated with a double-modified 20 bp single-strand oligonucleotide of the sequence 5'-AGC GGA TAA CAC AGT CAC CT-3 '(modification 1: the phosphate group of the 3' end is marked with (HO- (CH ₂ ) ₂ -S) ₂ esterified to PO- (CH ₂ ) ₂ -S- S- (CH ₂ ) ₂ -OH; modification 2: at the 5 'end is the fluorescein modifier fluorescein phosphoramidite (proligo biochemistry GmbH) installed according to the respective standard protocol) in 5 × 10 ^-5 molar buffer solution (phosphate buffer, 0.5 molar in water, pH 7) with the addition of approx. 10 ^-5 to 10 ^-1 molar propanethiol (or other thiols or disulfides) Chain length) incubated for 2-24 h. During this reaction time, the disulfide spacer PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH of the oligonucleotide is cleaved homolytically. The spacer forms a covalent Au-S bond with Au atoms on the surface, which leads to a 1: 1 coadsorption of the SS oligonucleotide and the split off 2-hydroxy-mercaptoethanol. The free propanethiol present in the incubation solution is also co-adsorbed by forming an Au-S bond (incubation step). Instead of the single strand oligonucleotide, this single strand can also be hybridized with its complementary strand.

Example 4 Alternative production of the oligonucleotide electrode Au-S (CH ₂ ) ₂ -ss-oligo-FP

The alternative production of Au-S (CH ₂ ) ₂ -ss-oligo-FP is divided into two sections, namely the derivatization of the gold surface with the probe oligonucleotide (incubation step) and the subsequent coating of the electrode modified in this way with a suitable monofunctional Left (re-assignment step).

The quench surface (here: gold plate) is treated with a double-modified 20 bp single-strand oligonucleotide of the sequence 5'-AGC GGA TAA CAC AGT CAC CT-3 '(modification 1: the phosphate group of the 3' end is marked with (HO- (CH ₂ ) ₂ -S) ₂ esterified to PO- (CH ₂ ) ₂ -S- S- (CH ₂ ) ₂ -OH; modification 2: at the 5 'end is the fluorescein modifier fluorescein phosphoramidite (proligo biochemistry GmbH) according to the respective standard protocol) in 5 × 10 ^-5 molar buffer solution (phosphate buffer, 0.5 molar in water, pH 7) incubated for 2-24 h. During this reaction time, the disulfide spacer PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH of the oligonucleotide is cleaved homolytically. The spacer forms a covalent Au-S bond with the Au atoms on the surface, which leads to a 1: 1 coadsorption of the ss-oligonucleotide and the split off 2-hydroxy-mercaptoethanol. Instead of the single-strand oligonucleotide, this single strand can also be hybridized with its complementary strand.

The gold surface modified in this way is then completely wetted with an approximately 10 ^-5 to 10 ^-1 molar propanethiol solution (in water or buffer, pH 7-7.5) and incubated for 2-24 h. The free propanethiol covers the free gold surface remaining after the incubation step by forming an Au-S bond. As an alternative to propanethiol, another thiol or disulfide of suitable chain length can also be used.

Example 5 Alternative preparation of the oligonucleotide electrode Au-S (CH ₂ ) ₂ -ss-oligo-FP

The alternative production of Au-S (CH ₂ ) ₂ -ss-oligo-FP is divided into two sections, namely the derivatization of the gold surface with the probe oligonucleotide (incubation step) and the subsequent coating of the electrode modified in this way with a suitable bifunctional Left (re-assignment step).

The quench surface (here: gold plate) is treated with a double-modified 20 bp single-strand oligonucleotide of the sequence 5'-AGC GGA TAA CAC AGT CAC CT-3 '(modification 1: the phosphate group of the 3' end is marked with (HO- (CH ₂ ) ₂ -S) ₂ esterified to PO- (CH ₂ ) ₂ -S- S- (CH ₂ ) ₂ -OH; modification 2: at the 5 'end is fluorescein modifier fluorescein phosphoramidite (Proligo Biochemie GmbH ) built in according to the respective standard protocol) in 5 × 10 ^-5 molar buffer solution (phosphate buffer, 0.5 molar in water, pH 7) for 2-24 h. During this reaction time, the disulfide spacer PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH of the oligonucleotide is cleaved homolytically. The spacer forms a covalent Au-S bond with Au atoms on the surface, which leads to a 1: 1 coadsorption of the ss-oligonucleotide and the cleaved 2-hydroxymercaptoethanol. Instead of the single strand oligonucleotide, this single strand can also be hybridized with its complementary strand.

Then the modified gold surface with an approximately 10 ^-5 to 10 ^-1 molar solution of z. B. mercaptopropionic acid, mercaptoethanesulfonic acid or cysteamine (in water or buffer, pH 7-7.5 or in ethanol) completely wetted and incubated for 2-24 h. The free thiol (mercaptopropionic acid, mercaptoethanesulfonic acid, cysteamine) covers the free gold surface remaining after the incubation step by forming an Au-S bond. Alternatively, other functional thiols or disulfides of suitable chain length with the same or different functional groups can also be used.

Example 6 Fluorescence intensity measurements on the system Au-ss-oligo-Fluorescein or on Au-ds-oligo-fluorescein system in the presence of liquid media

The probe surface is produced analogously to Example 4. For this purpose, a modified oligonucleotide of the sequence 5'-fluorescein-AGC GGA TAA CAC AGT CAC CT-3 '[C ₃ - SSC ₃ -OH] is immobilized on gold (50 μmol oligonucleotide in phosphate buffer (K ₂ HPO ₄ / KH ₂ PO ₄ 500 mM, pH 7, subsequent coating with propanethiol 1 mM in water) and in the form Au-S (CH ₂ ) ₂ -ss-oligo-fluorescein the fluorescence intensity of the surface was determined with a fluorescence scanner from Lavision Biotech To measure the fluorescence, a potential of 0.3 V is applied against silver wire. In the presence of liquid media (100 mmol chloride), 150 µl of the medium is placed on the gold surface and then covered with a cover glass. Alternatively, Hybriwells or imaging chambers can also be used ,

Example 7 Fluorescence intensity measurements on the system Au-ss-oligo-Fluorescein or on Au-ds-oligo-fluorescein system in the presence of surface-immobilized anions and liquid media

The probe surface is produced analogously to Example 5. For this purpose, a modified oligonucleotide of the sequence 5'-fluorescein-AGC GGA TAA CAC AGT CAC CT-3 '[C ₃ - SSC ₃ -OH] is immobilized on gold (50 μmol oligonucleotide in phosphate buffer (K ₂ HPO ₄ / KH ₂ PO ₄ 500 mM, pH 7 and subsequent subsequent coating with an approx. 10 ^-5 to 10 ^-1 molar solution of e.g. mercaptopropionic acid, which covers the remaining free gold surface) and in the form Au-S (CH ₂ ) ₂ -ss-oligo-fluorescein determines the fluorescence intensity of the surface with a fluorescence scanner from Lavision Biotech To measure the fluorescence in the presence of liquid media, 150 µl of the medium is placed on the gold surface and then covered with a cover glass Hybriwells or imaging chambers can be used.

Claims (26)

1. A method for the detection of ligate-ligand association events by fluorescence quenching comprising the steps a) providing a modified surface, the modification consisting in the attachment of at least one type of modified ligate and the ligates ( 201 ) being modified by attachment of at least one type of fluorophore ( 102 ), b) providing a sample with ligands, c) applying an electric field and setting a defined strength of the electric field at the location of the modified ligates ( 201 ), d) bringing the sample into contact with the modified surface, e) detection of the fluorescence of the fluorophore ( 102 ), f) Comparison of the fluorescence intensities detected in step e) with reference values. 2. The method according to claim 1, wherein after step c) and before step d) step c1) first detection of the fluorescence of the fluorophore ( 102 ) is carried out and in step f) the values obtained in step e) with the values in step c1) obtained reference values are compared. 3. The method according to claim 1, wherein as step e) the step a) detection of the fluorescence of the fluorophore ( 102 ) and detection of reference values is carried out and in step f) the values obtained in step e) are compared with the reference values likewise obtained in step e). 4. The method according to claim 3, wherein the detection of the reference values in step e) by detecting the fluorescence of an area of the modified surface takes place in which essentially no ligands are attached. 5. The method according to claim 3, wherein the detection of the reference values in step e) by detecting the fluorescence of an area of the modified surface takes place in which essentially only ligand / ligate complexes are bound are present. 6. The method according to claim 3, wherein the detection of the reference values in step e) by detecting the fluorescence of an area of the modified surface takes place in which essentially no ligands are attached, and in addition by detection of a second area of the modified surface, in which is essentially exclusively bound to ligand / ligate complexes are. 7. The method according to any one of the preceding claims, wherein the setting a defined strength of the electric field in step c) by applying a external electrical field on the modified surface. 8. The method of claim 7, wherein a potential of E <0.2 V (measured against Silver wire) is applied to the modified surface. 9. The method of claim 7, wherein a potential of 0.2 V <E <0.4 V (measured against silver wire) is applied to the modified surface. 10. The method according to any one of claims 1 to 6, wherein the setting of a defined strength of the electric field in step c) is carried out by immobilizing ions ( 401 , 402 ) on the modified surface. 11. The method according to claim 10, wherein setting a defined strength of the electric field in step c) by immobilization of bifunctional molecules with carboxylic acid or sulfonic acid functions on the modified surface he follows. 12. The method of claim 11, wherein setting a defined strength of the electric field in step c) by immobilization of an ω-carboxy-alkanethiol or by immobilizing a sulfonic acid alkanethiol. 13. The method of claim 10, wherein setting a defined strength of the electric field in step c) by immobilization of bifunctional molecules with amine or ammonium functions on the modified surface. 14. The method of claim 13, wherein setting a defined strength of the electric field in step c) by immobilization of an amine alkanethiol he follows. 15. The method according to any one of the preceding claims, wherein a fluorescent dye is used as the fluorophore ( 102 ). 16. The method according to any one of the preceding claims, wherein as the fluorophore ( 102 ) Texas Red, a rhodamine dye, in particular Rhodamine G6 (Basic Red 1), fluorescein (Resorcinphtalein), or a cyanine dye, in particular 5,5'-disulfo- , 1'di (-carbopentenyl) -3,3,3 ', 3'-tetramethyl-indodicarbocyanine (Cy3 ™) or 5,5', 7,7'-tetrasulfo-1,1'di (-carbopentenyl) -3 , 3,3 ', 3'-tetramethylbenzindodicarbocyanin (Cy5 ™) is used. 17. The method according to any one of the preceding claims, wherein it is in the modified surface around a surface bearing a modification from a Metal or a metal alloy, in particular from a metal selected from the group consisting of gold, silver, copper and their Mixtures. 18. The method according to any one of the preceding claims, wherein as a ligand Substrates, cofactors or coenzymes and as ligates proteins or enzymes be used. 19. The method according to any one of claims 1 to 17, wherein as ligand antibodies and antigens can be used as ligates. 20. The method according to any one of claims 1 to 17, wherein as ligands antigens and as Ligate antibodies can be used. 21. The method according to any one of claims 1 to 17, wherein receptors and as ligands hormones are used as ligates. 22. The method according to any one of claims 1 to 17, wherein hormones and as ligands can be used as ligate receptors. 23. The method according to any one of claims 1 to 17, wherein as the ligand nucleic acid Oligomers and nucleic acid oligomers complementary thereto as ligates be used. 24. The method according to claim 23, wherein the nucleic acid used as ligates Oligomers 3 to 50 bases, in particular 5 to 40 bases, particularly preferably 12 comprise up to 30 bases. 25. Use of substrates, cofactors, coenzymes, proteins, enzymes, Antibodies, antigens, receptors, hormones and nucleic acid oligomers as Ligates and / or ligands in a process according to claims 1 to 17. 26. Kit for carrying out a method according to claims 1 to 17 comprising a modified surface, the modification consisting in the attachment of at least one type of modified ligate ( 201 ) and the ligate ( 201 ) by attachment of at least one type of fluorophore ( 102 ) are modified.