WO2003040679A2 - Reversibly binding a fluorophore to a surface for the purpose of detecting ligate/ligand association events by fluorescence quenching - Google Patents

Abstract

The invention relates to a method for detecting ligate/ligand association events by fluorescence quenching, which comprises as a first step the provision of a modified surface. The modification of the surface consists in the binding of at least one type of modified ligates 201, wherein said ligates 201 are modified by at least one type of fluorophore 102 bound to it, said fluorophore 102 establishing a reversible bond with the modified surface. The further steps of the inventive method are: providing a sample that includes ligands, contacting the sample with the modified surface, detecting the fluorescence of the fluorophore and comparing the detected fluorescence intensity with reference values.

Description

Reversible binding of a fluorophore to a surface for the detection of ligate-ligand association events by fluorescence quenching

Technical field

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

State of the art

Immunoassays and increasingly sequence analysis of DNA and RNA are used in disease diagnosis, in toxicological test procedures, in genetic research and development, as well as in the agricultural and pharmaceutical sectors. In addition to the known serial methods with autoradiographic or optical detection, parallel detection methods using array technology, so-called DNA or protein chips, are increasingly being used. With these parallel methods, too, 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 one 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

Evidence of the corresponding hybridization events can be identified on the chip. In general, the analysis is carried out using optical (or autoradiographic)

Detection method using target oligonucleotides with a

Radio label (e.g. ³² P) or a fluorescent dye (e.g. fluorescein, Cy3 ™ or

Cy5 ™) are marked. The use of fluorescent labels and corresponding

Fluorescence scanners are increasingly using radio labels. The fluorescence scanners currently available on the market enable the detection of

Fluorophore in the subattomole range. However, using labeled targets to detect hybridization events has some drawbacks. On the one hand, the marking must take place before the actual measurement, which means an additional synthesis step and thus additional work. It is also difficult to ensure homogeneous marking of the sample material. In addition, stringent washing conditions are necessary in order to remove non-specific or non-specifically bound material following hybridization.

For both protein and DNA analysis, it is therefore desirable and advantageous for the user if the targets (antibodies or antigen or DNA fragment) do not have to be modified with a detection label and after the hybridization no complicated washing steps are necessary are.

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

In addition to organic molecules (such as DABCYL), gold nanoparticles are also used as efficient quenchers (cf. Nature Biotech. Vol. 19, 2001, page 365). The quenching of fluorescence by metals is based primarily on a radiationless energy transfer from the dye molecule to the metal. A greater sensitivity is observed when using gold nanoparticles than with organic quenchers. In addition, dyes are efficiently quenched into the near infrared range. A disadvantage of this method, however, is that gold nanoparticles at temperatures are no longer stable above 50 ° C. Another disadvantage is that this method is limited to the investigation of solutions and therefore only a few sequences can be examined at the same time, so the degree of parallelization of this approach is low.

Although there are many detection options for ligate-ligand associates, the need for simple, inexpensive, easy-to-perform and reliable detection principles is particularly 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, for example for so-called POC (Point of Care) systems or for high throughput screening (HTS systems) high.

Presentation of the invention

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

This object is achieved according to the invention by the method according to independent patent claim 1 and the kit according to independent patent claim 24.

Further advantageous details, aspects and configurations 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, PNA hybridizes with DNA.)

A adenine

G guanine

C cytosine

T thymine and uracil

Base A, G, T, C or U

Bp base pair

Nucleic acid contains two or more 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, e.g. 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, for example a DNA, PNA or RNA fragment of a base length not specified in more detail. 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 the base A (or C) of one strand forms hydrogen bonds with the base T (or G) of the other strand (for RNA, T is by uracil ) replaced. 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

Substrate, cofactor, complex binding partner of a protein (enzyme). coenzyme

Antibody complex binding partner of an antigen.

Antigen complex binding partner of an antibody.

Receptor complex binding partner of a hormone. Association constant for the association of ligate and ligand.

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 usually shifted by 15 to 40 nm (Stokes shift).

FP Fluorophore Cy3 ™ δ.δ'-disulfo-l .l ^' di ^ carbopenteny -SSS ^' ^ ^' -tetramethyl-indodicarbocyanine

(Fluorescent dye from Amersham Life Science, Inc.)

Cy5 TM 5.5 \ 7.7'-tetrasulfo-1, 1 'd -carbopenteny -SS ^ .S etramethyl- benzindodicarbocyanin

(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 of radiationless energy transfer

Fluorescence Quenching Fluorescence Quenching

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).

Left 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, hetero-alkyl, hetero-alkenyl or hetero-alkynyl chains, the chain being derivatized at two points with (identical or different) reactive groups. These groups form a covalent one in simple / known chemical reactions with the corresponding reaction partner chemical bond. 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 1-60, in particular chain length 5-40, the chain length here being the shortest continuous connection between the structures to be connected, i.e. between the two molecules or between a surface atom, a surface molecule or a surface molecule group and another Molecule.

Spacer linker which is covalently bonded via the reactive groups to one or both of the structures to be connected (see linker). Preferred spacers are those of chain length 1-60, in particular chain length 5-40, the chain length being the shortest continuous connection between the structures to be connected.

Modified surfaces / electrodes

Au-S- (CH ₂ ) ₂ -ss-oligo- gold surface with covalently applied monolayer made from FP 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 Cy3 ™, Cy5 ™, Texas Red®, Rhodamine 6G, fluorescein etc.

Au-S- (CH ₂ ) ₂ -ds-oligo-Au-S- (CH ₂ ) ₂ -ss-oligo-FP hybridizes with the oligonucleotide complementary to ss-oligo FP.

The present invention relates to a method 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 the attachment of at least one type of modified ligate, the ligates being modified by attachment of at least one type of fluorophore, the Fluorophore forms a reversible bond with the modified surface. The further steps of the method according to the invention are providing a sample with ligands, bringing the sample into contact with the modified surface, detection of the fluorescence of the fluorophore and comparison of the detected fluorescence intensity with reference values.

A comparison of the detected fluorescence intensities with reference values is required in the method according to the invention. These reference values can already exist from previous measurements and therefore do not need to be detected in the most general case in the course of the method according to the invention. However, since the reference values should ideally be determined under exactly the same external conditions as the actual measured values of the fluorescence intensities, according to a preferred embodiment of the present invention, a first detection of the fluorescence of the fluorophore is carried out before the targets (sample) come into contact with the modified surface , The values obtained in this way are then used as reference values.

According to the particularly preferred embodiments of the present invention described below, a normalization measurement is also carried out. In these cases, sites are applied to the modified surface to which a very specific degree of association can be assigned after adding the sample. The signal obtained during the detection is then characteristic of this particular degree of association and can be used to normalize the signals from the test sites.

The present invention namely also encompasses methods in which a modified surface is used which has been modified by binding at least two types of modified ligates. The different types of ligates are bound to the surface in spatially essentially separated areas. "Substantially separated areas" are understood to mean areas of the surface that are largely modified by binding a certain type of ligate. Only in areas in which two such essentially separated areas adjoin one another can spatial mixing of different types of ligate occur. In the method preferred in the context of the present invention, the ligand is added to the sample before the sample is brought into contact with the modified surface, the ligand being a binding partner with a high association constant with a particular type of ligate, which is used in a particular Area (Site T ₁₀₀ ) is bound to the surface. The ligand is added to the sample in an amount that is greater than the amount of ligand that is necessary to fully associate the ligates of the T ₁₀₀ sites. The last step of this method is the comparison of the values obtained in the detection of the fluorescence of the fluorophore with the value obtained for the region T ₁₀ o. The value obtained for the area T ₁₀₀ thus corresponds to the value when the association is complete (100%).

According to a particularly preferred embodiment, a modified surface is used which has been modified by attaching at least three types of ligates. The different types of ligates are bound to the surface in spatially essentially separated areas. In this case, at least one type of ligate is bound to the surface in a certain area (site T ₀ ), from which it is known that there is no binding partner with a high association constant in the sample, that is to say the corresponding association partner or ligand does not appear in the sample , In this particularly preferred method, too, a ligand is added to the sample before the sample is brought into contact with the modified surface, the ligand being a binding partner with a high association constant with a certain type of ligate, which occurs in a certain region (site T ₁₀ o) is bound to the surface. The ligand is added to the sample in an amount that is greater than the amount of ligand that is necessary to fully associate the ligates of the T ₁₀ o sites. The last step of this method is the comparison of the values obtained in the detection of the fluorescence of the fluorophore with the value obtained for the area T ₁₀₀ and with the value obtained for the area T ₀ . The value obtained for the area To corresponds to the value in the absence of association (0%).

According to a very particularly preferred embodiment of the methods described above, at least one further type of ligand is added to the sample before the sample is brought into contact with the modified surface, it being known that this ligand is not contained in the original sample. This further type of ligand has an association constant> 0 to a type of ligate that is bound to the surface in a certain region (site T _n ). The ligand is added to the sample in such an amount that after contacting the sample with the modified surface, n% of the ligates of the site T _{n are} in an associated form. The last step of this method is the comparison of the values obtained in the detection of the fluorescence of the fluorophore with the value obtained for the area T ₁₀₀ , with the value obtained for the area T ₀ and with those for the areas T _n values obtained. The value obtained for a particular test site T _n thus corresponds to the value in the presence of n% ligate-ligand associates based on the total number of ligates of the type.

The amount of ligand which has to be brought into contact with the modified surface in order to bring about an n% association at the site T _n can be determined by the person skilled in the art by simple routine tests. For this purpose, for example after detection of the values for T ₀ and T ₀ o, a calibrated measurement is carried out, in which the signal intensity is determined by (different) detection labels with which the ligate and the ligand are equipped. The ratio of the intensities of the ligand label signal to the ligate label signal corresponds to n%.

If a sufficient number of sites T _{n are} applied to the modified surface, a normalization curve can be recorded with high accuracy. Standardizing the measurements of the actual test sites using this standardization curve significantly improves the reproducibility of the analysis using chip technology.

It should be pointed out that finding a ligand that is not contained in the sample does not pose any problems, in particular if nucleic acid oligomers are used as ligands and ligates, since even the most extensive genomes still have a sufficient selection of nonexistent ones Offer sequences. In the event that the non-existing sequence differs from a sequence present only by a base, the hybridization step must be carried out under stringent conditions. However, preference is given to using sequences which differ significantly, that is to say in several bases, from the sequences present in the sample. Particularly good results are achieved if oligonucleotides with the same or at least a similar number of bases are used for the test sites and for the standardization sites.

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 comprises a modified surface, the modification being the attachment of at least one type of modified ligate, the ligates being modified by attachment of at least one type of fluorophore and the fluorophore having a reversible bond with the modified surface. According to a preferred embodiment, the kit comprises a modified surface, the modification consisting in the attachment of at least one type of modified ligate, the ligates being modified by attachment of at least one type of fluorophore and the fluorophore being modified by attachment of at least one chemical group, which forms a reversible bond with the modified surface.

According to a preferred embodiment, the reference values are already included in the kit, so that the fluorescence of the fluorophore only has to be detected once by the end user. The values obtained in this detection then only need to be compared with the existing reference values.

According to a preferred embodiment of the present invention, the kit comprises a modified surface which has at least one area T ₀ and at least one area T-100. Embodiments are particularly preferred, according to which the modified surface additionally comprises at least one region T _n .

If there are no groups on the fluorophore in its unmodified form which have sufficient binding capacity with the modified surface, then modified fluorophores can also be used in the process according to the invention. According to a preferred embodiment, fluorophores are therefore used which are modified by attachment of at least one chemical group, the chemical group forming a reversible bond with the modified surface.

A chemical group that forms a reversible bond with the modified surface can not only be attached directly to the fluorophore, but also to the ligates. According to a preferred embodiment of the present invention, ligates are used which are additionally modified by attachment of at least one chemical group, the chemical group forming a reversible bond with the modified surface. Particularly favorable conditions for detecting the difference in the fluorescence intensity of ligand / ligate associates and ligates exist when the fluorophore is as close as possible to the modified surface. This condition is met in particular if ligates are used which are additionally modified by attachment of at least one chemical group, the chemical group entering into a reversible bond with the modified surface and the chemical group, for example, through 10 chemical bonds from the fluorophore is separated. In this case, the chemical group is in spatial proximity to the fluorophore, which in turn means that after the reversible bond between the chemical group and the modified surface has been formed, the fluorophore is in spatial proximity to the modified surface.

Particularly favorable conditions are to be expected if the modified surface is additionally modified by attachment of chemical groups and at the same time the ligates are modified with chemical groups, because then the chemical groups bound to the surface with the chemical groups attached to the ligates have a reversible bond can enter into.

The quench surface

The term "surface" denotes any carrier material which is suitable for carrying fluorophore-derivatized ligates directly or after appropriate chemical modification, which are covalently immobilized on the surface and whose fluorescence is close to the surface (in approx. 1 to 50 Å Distance to the surface) is significantly reduced by fluorescence quenching (radiation-free energy transfer between the fluorophore as emitter and the surface as absorber) (> 10% of the expected fluorescence intensity of the fluorophore in the absence of the surface under otherwise identical conditions). Gold and silver are particularly suitable as quench surface material. The term surface is independent of the spatial dimensions of the surface and also includes nanoparticles (particles or clusters of a few individual to several hundred thousand surface atoms or molecules). The surface can also be attached to a solid support such as e.g. Glass, metal or plastic are bound.

Binding of the ligate to the surface

Methods for immobilizing ligate molecules, in particular biopolymers such as nucleic acid oligomers or antigens or antibodies, on a surface are known to the person skilled in the art. The immobilization can be carried out, for example, covalently via hydroxyl, epoxy, amino or carboxy groups of the support material with thiol, hydroxyl, amino or carboxyl groups which are naturally present on the ligate or are attached to the ligate by derivatization. The ligate can be used directly or via a linker / spacer are connected to the surface atoms or molecules of a surface. In addition, the ligate can be anchored by the methods customary in immunoassays, for example by using biotinylated ligates for non-covalent immobilization on avidin or streptavidin-modified surfaces. If nucleic acid oligomers are used as ligates, the chemical modification of the probe nucleic acid oligomers with a surface anchor group can already be introduced in the course of automated solid phase synthesis or in separate reaction steps. The nucleic acid oligomer is also linked directly or via a linker / spacer to the surface atoms or molecules of a surface of the type described above. This binding can be carried out in various ways known from the prior art (cf. for example Hartwich, G .: ELECTROCHEMICAL DETECTION OF

SEQUENCE-SPECIFIC NUCLEIC ACID OLIGOMER HYBRIDIZATION EVENTS (1999), WO 00/42217).

When attaching the nucleic acid oligomers to the surfaces, a particularly important point is important. Fundamentally, there are particularly favorable conditions for detecting the difference in the fluorescence intensity of hybridized nucleic acid oligomers and single-stranded nucleic acid oligomers if the fluorophore is "hybridized" or "not hybridized" as close as possible to the modified surface in only one of the states. As is known, the extent of the quench process changes with a higher (third to sixth) power of the distance between the quench surface and the fluorophore. Such particularly favorable conditions can only be achieved with special connection technologies. When connecting the nucleic acid oligomers, care must be taken that they are either bound to the surface completely without additional co-adsorbate or, if a co-adsorbate appears necessary, that it forms a layer as thin as possible above the surface. Either a direct attachment of the nucleic acid oligomer to the surface must be carried out or it must be coated together with short-chain co-adsorbates such as e.g. short chain thiols. Co-adsorbates of chain length 1 to 30, particularly preferably chain length 1 to 20, in particular chain length 1 to 10 are preferred.

A particular disadvantage in this connection is the binding of the nucleic acid oligomers in the form of a surface-biotin-avidin-biotin-oligomer compound. With such a connection, the fluorophore is always covered by a very thick layer of biotin Avidin-biotin is shielded from the surface, which is associated with corresponding disadvantages in the detection of fluorescence.

Ligands (targets)

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

fluorophores

Commercially available fluorescent dyes such as e.g. Texas Red®, rhodamine dyes, cyanine dyes such as Cy3 ™, Cy5 ™, fluorescein etc (see Fluka, Amersham and Molecular Probes catalog) are used.

fluorescence quenching

Fluorescence quenching is the deactivation of an electronically excited species via a radiationless process. Deactivation can take place by means of impacts or by radiation-free energy transfer to metals. The energy released is dissipated as thermal energy. Gold is an example of a metal that has the ability to quench fluorescence. The quenching has a strong dependence on the distance of the fluorophore from the surface functioning as a fluorescence quencher (inversely proportional to a higher (third to sixth) power of the distance). The effect of fluorescence quenching can therefore only be measured at distances of less than 100 to 200 A. In the range greater than approx. 200 A, further changes in distance no longer lead to a measurable increase in the fluorescence intensity. Brief description of the drawings

The invention will be explained in more detail below using exemplary embodiments in conjunction with the drawings. Show it

1 shows a schematic representation of the detection of nucleic acid oligomer hybridization events by means of “molecular beacons”;

Fig. 2 is a schematic representation of the detection of nucleic acid-oligomer hybridization events by specific interactions between groups on the fluorophore and the modified surface.

LIST OF REFERENCE NUMBERS

101: probe in the form of a hairpin

102: fluorophore

103: fluorescence quencher

104: Target

105: fluorescence quenching 106: fluorescence

201: single-stranded oligomer probe

202: probe hybridizes with target

203: surface (e.g. gold)

204: Distance between fluorophore and gold surface before hybridization 205: Distance between fluorophore and gold surface after hybridization

206: Molecular group on the fluorophore that enters into specific interactions with corresponding groups on the surface

207: Molecular group on the surface that enters into specific interactions with corresponding groups on the fluorophore

FIG. 1 shows a schematic representation of the detection of nucleic acid-oligomer hybridization events known from the prior art by means of “molecular beacons”. The single-stranded oligonucleotides have a hairpin structure 101 (hairpin, stem-and-loop), carry a fluorophore at one end of the sequence

102 (e.g. Fluorescein, TexasRed®) and at the other end of the sequence a suitable fluorescence quencher 103 (e.g. DABCYL). Due to the special geometric arrangement, the fluorescent group and the unit that leads to the quenching of the fluorescence are located in close proximity to one another. Therefore the probes show only an extremely low fluorescence. In the presence of the corresponding sequence 104 (target) complementary to the sequence of the loop, the hybridization takes place in this area. This leads to a change in the conformation and the separation of fluorophore and quencher, which can be observed as a strong increase in fluorescence 106.

FIG. 2 shows a schematic representation of the detection of nucleic acid-oligomer hybridization events by specific interactions between groups 206 on the fluorophore 102 and the modified surface. Due to specific interactions between groups 206 on the fluorophore 102 and corresponding groups 207 on the surface 203, the single-stranded probe nucleic acid oligomer 201 is in a compressed form. The hybridization with the complementary nucleic acid oligomer strand 202 (target) increases the distance 205 between the fluorescent dye molecule 102 and the metal surface 203 functioning as a quencher. This leads to a strong increase in the fluorescence intensity (see bar chart in FIG. 2). ,

Ways of Carrying Out the Invention

The method according to the invention for the detection of ligate / ligand associates is explained below 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, the described method can readily be applied to the detection of other ligate / ligand associates, as mentioned in the section "Ligands (Targets)".

To apply the advantages of DNA chip technology to the detection of nucleic acid-oligomer hybrids by modulating fluorescence quenching, various modified nucleic acid-oligomer probes with different sequences are used tied to a support using the immobilization techniques described above. With the arrangement of the nucleic acid oligomer probes of known sequence at each position on the surface, a DNA array, the hybridization event of any target nucleic acid oligomer or (fragmented) target DNA is to be detected, for example in order to detect mutations in the target and to be demonstrated in a sequence-specific manner. For this purpose, the surface atoms or molecules of a defined area (a test site) are linked on a surface with DNA / RNA / PNA nucleic acid oligomers of known but any sequence, as described above. In a most general embodiment, the DNA chip can also be derivatized with a single probe oligonucleotide. Nucleic acid oligomers (eg DNA, RNA or PNA fragments) with a base length of 3 to 70, preferably a length of 5 to 60, particularly preferably a length of 10 to 50, particularly preferably a length of 12 to 40, are used as probe nucleic acid oligomers used.

At this point it should be mentioned that the target oligonucleotides can also comprise a larger number of bases, ie they can be longer than the probe oligonucleotides. In this case, the expression “nucleic acid oligomer complementary to the probe oligonucleotide” is understood to mean a nucleic acid oligomer which has a base sequence which is complementary to the probe oligonucleotide in a partial region. The remaining portion (s) of the nucleic acid oligomer then protrude at the end (s) of the probe oligonucleotide beyond its base chain.

On the surfaces thus provided with immobilized and fluorophore-labeled probe oligonucleotides, a reference measurement, e.g. with a fluorescence scanner, the fluorescence intensity of the fluorophore-labeled probe oligonucleotides is determined in the single-stranded state.

In the next step, the (as concentrated as possible) test solution with target oligonucleotide (s) is added to the surface with immobilized probe oligonucleotides. Hybridization occurs only in the case in which the solution contains target nucleic acid oligomer strands which are complementary to the probe-nucleic acid oligomers bound to the surface or at least in many areas complementary. After the hybridization between probe and target, the fluorescence intensity in the hybridized, double-stranded state is determined in a second fluorescence measurement.

Due to the hybridization of probe-nucleic acid oligomer and the complementary nucleic acid-oligomer strand (target), the distance between the fluorescent dye molecule and the metal surface acting as quencher changes. Due to the changed distance, the extent of the quench process and thus the intensity of the fluorescence also undergoes a major change. Thus, a sequence-specific hybridization event can be performed by fluorescence-based methods such as e.g. Fluorescence microscopy or measurements with fluorescence scanners can be detected.

The difference between the reference measurement and the second measurement for each test site is proportional to the number of complementary (or in many areas complementary) target oligonucleotides originally present in the test solution for the respective test site.

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

A specific geometric arrangement of the probe nucleic acid oligomer relative to the modified surface can be achieved through specific interactions between groups on the dye that are naturally already present on the dye or that are introduced by chemical modification, and the (optionally modified) surface. A geometrical arrangement can be realized by attaching suitable molecular groups to the dye (e.g. complexing groups such as bipyridyl groups or diamino groups), which can enter into a specific interaction that is stable under suitable conditions (e.g. complex formation) with corresponding groups. where the fluorophore is close to the surface in the non-hybridized state. Before hybridization, the single-stranded probe nucleic acid oligomer is therefore in a form which is characterized by a small distance between the fluorophore and the quenching metal surface (low fluorescence intensity). The distance changes as a result of the hybridization with the complementary nucleic acid oligomer strand (target) between the fluorescent dye molecule and the metal surface acting as quencher in such a way that the distance increases, the quenching decreases and a higher intensity of the fluorescence can be observed after the hybridization (see FIG. 2).

embodiments

The n nucleotide (nt) long nucleic acid probe (DNA, RNA or PNA, for example a 20 nucleotide long oligo) is near one of its ends (3 'or 5' end) directly or via any spacer a reactive group for covalent anchoring to the surface, e.g. 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. from amine-modified ligate oligonucleotide, which is used for anchoring to surface-bonded carboxylic acid functions (e.g. via acid-functionalized thiols such as mercaptopropionic acid and activation e.g. as an active ester). In the vicinity of the other terminus of the probe oligonucleotide, a fluorophore is covalently attached (see Example 1), which is modified with a functional group (e.g. a fatty acid, a bipyridyl group or a diamino group). The nucleic acid probe modified in this way is

(i) dissolved in buffer (e.g. 10 - 500 mM phosphate buffer, pH = 7, 1mM EDTA) brought into contact with the surface and there via the reactive group of

Probe nucleic acid oligomers attached to the - or, if appropriate, derivatized - surface

(ii) in the presence of a bifunctional linker (for example a suitable diamino thiol compound or a mercaptopropionic acid activated as an active ester and then reacting with a corresponding triamino compound) in buffer (for example 100 mM phosphate buffer, pH = 7.1 mM EDTA, 0.1 - 1 M NaCI) dissolved in contact with the surface and there via the reactive group of the probe nucleic acid oligomer together with the bifunctional linker to the - if appropriate derivatized -

Tied surface, taking care that enough bifunctional linker of 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 target oligonucleotide or

(iii) dissolved in buffer (e.g. 10-350 mM phosphate buffer, pH = 7.1 mM EDTA) brought into contact with the surface and bound there via the reactive group of the probe nucleic acid oligomer to the - if appropriate derivatized - surface. Subsequently, the surface modified in this way is brought into contact with the corresponding bifunctional linker in solution (for example a suitable diaminothiol compound or a mercaptopropionic acid activated as an active ester and subsequent reaction with a corresponding triamino compound in phosphate buffer / EtOH mixtures in the case of thiol-modified probe oligonucleotides) binds the bifunctional linker via 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. by fluorescence measurement with a fluorescence scanner using specific interactions between functional groups on the dye and, if appropriate, correspondingly functionalized groups on the gold surface. One embodiment is e.g. bipyridyl groups attached to the dye molecule or in its vicinity, which enter into relatively stable interactions with the gold surface under suitable conditions. A further embodiment are diamino groups attached to or in the vicinity of the dye molecule, which enter into a relatively stable interaction via a complex bond generated by copper (II) or nickel (II) to corresponding diamino groups attached to the gold surface.

A further embodiment are hydrophobic alkyl chains attached to the dye molecule (for example by binding a fatty acid), which enter into relatively stable interactions via hydrophobic interactions with the likewise hydrophobic gold surface. The dissolved target is then added, potential hybridization events are made possible under suitable conditions known to the person skilled in the art (arbitrary, freely selectable stringency conditions of the parameters potential temperature / salt / chaotropic salts etc. for the hybridization) and the measurement is repeated to detect the fluorophore.

The difference in the measurement signal (increase in fluorescence intensity after hybridization, 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.

The described method can be used for a target type (eg a specific target oligonucleotide type with a known sequence) on a surface or - if different probe types are used for each test site - for several target types (same ligand groups as eg several different target Oligonucleotide types or different antibody types, antigen types etc., but also mixtures thereof) can be used.

example 1

Representation of the amino-modified oligonucleotides for anchoring on modified gold surfaces as ligate or probe oligonucleotides

The synthesis of the oligonucleotides takes place in an automatic oligonucleotide synthesizer (Expedite 8909; ABI 384 DNA / RNA synthesizer) according to the synthesis protocols recommended by the manufacturer for a 1.0 μmol synthesis. In the syntheses with the 1-O-dimethoxytrityl-propyl-disulfide-CPG carrier (Glen Research 20-2933), the oxidation steps are carried out with a 0.02 M iodine solution in order to avoid oxidative 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 built into the sequences with the respective standard protocol. The coupling efficiencies are determined online during the synthesis via the DMT cation concentration photometrically or conductometrically. The oligonucleotides are deprotected with concentrated ammonia (30%) at 37 ° C for 16 h. The oligonucleotides are purified using RP-HPL chromatography according to standard protocols (eluent: 0.1 M triethylammonium acetate buffer, acetonitrile), and the characterization is carried out using MALDI-TOF MS. The amine-modified oligonucleotides are coupled to the corresponding activated fluorophores (eg fluorescein isothiocyanate) in accordance with conditions known to the person skilled in the art. The coupling can take place both before and after the oligonucleotides have been bound to the surface.

Example 2

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

The synthesis of the oligonucleotides takes place in an automatic oligonucleotide synthesizer (Expedite 8909; ABI 384 DNA / RNA synthesizer) according to the synthesis protocols recommended by the manufacturer for a 1.0 μmol synthesis. In the syntheses with the 1-O-dimethoxytrityl-propyl-disulfide-CPG carrier (Glen Research 20-2933), the oxidation steps are carried out with a 0.02 M iodine solution in order to avoid oxidative cleavage of the disulfide bridge. Modifications to the 5 'position of the oligonucleotides are carried out with a coupling step which is extended to 5 min. The fluorophores are incorporated in the synthesizer in the penultimate coupling step as phosphoramidites (fluorescein-phosphoramidites Glen Research 10-1963) in the sequences with the respective standard protocol. The amino modifier C2 dT (Glen Research 10-1037) is incorporated in the sequences with the respective standard protocol in the last coupling step. The coupling efficiencies are determined online during the synthesis via the DMT cation concentration photometrically or conductometrically.

Subsequently, a fatty acid or an ethylenediamine function (for example as ethylenediamine-N, N'-diacetic acid) or the bipyridyl group (for example as 2,2'-bipyridine-4,4'-dicarboxylic acid) is attached to the amino group via appropriate active ester processes , Example 3

Preparation 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 with (HO- (CH ₂ ) ₂ -S) ₂ esterified to PO- (CH ₂ ) ₂ - SS- (CH ₂ ) ₂ -OH; modification 2: at the 5 'end is a fluorophore and an ethylenediamine group (see Ex. 2) installed according to the respective standard protocol) in 5x10 ^"5 molar buffer solution (phosphate buffer, 0.5 molar in water, pH 7) with the addition of approx. 10 ^{" 5} to 10 ^"1 molar diaminothiol compound (or other suitable thiols or disulfides suitable chain length, such as mercaptopropionic acid activated as an active ester, to which a triamino group is then coupled) for 0.5-24 h during which the disulfide spacer becomes PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH of the oligonucleotide is cleaved homolytically, whereby the spacer forms a covalent Au-S bond with Au atoms on the surface h the ss oligonucleotide and the cleaved 2-hydroxy-mercaptoethanol are coadjusted. The free bifunctional thiol present at the same time 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.

A state is then achieved by adding soluble nickel salts (e.g. Ni (II) chloride) or other suitable metal ions (such as copper) through the formation of a complex using the complex formation properties of the diamino group on the fluorophore and the diamino group on the surface , in which the fluorophore is in a geometric arrangement close to the surface.

Example 4

Alternative production of the oligonucleotide electrode Au-SfCH ^ _∑ -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 linker (subsequent coating 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 with (HO- (CH ₂ ) ₂ -S) ₂ esterified to PO- (CH ₂ ) ₂ - SS- (CH ₂ ) ₂ -OH; modification 2: at the 5 'end is a modified fluorophore and an ethylenediamine group (see example 2) Incorporated according to the respective standard protocol) in 5x10 ^"5 molar buffer solution (phosphate buffer, 0.5 molar in water, pH 7) for 0.5 - 24 h. During this reaction time the disulfide spacer PO- (CH ₂ ) ₂ -SS - (CH ₂ ) ₂ -OH of the oligonucleotide cleaved homolytically, the spacer forming a covalent Au-S bond with Au atoms on the surface, which leads to coadsorption of the ss-oligonucleotide and the cleaved 2-hydroxy-mercaptoethanol. Instead of the single-strand oligonucleotide, this single strand can also be hybridized with its complementary strand.

The gold surface thus modified is then completed with an approximately 10 ^"5 to 10 ^{" 1} molar diaminothiol compound (or other suitable thiols or disulfides of suitable chain length, such as mercaptopropionic acid activated as an active ester, to which a triamino group is then coupled) wetted and incubated for 0.5 - 24 h. The free thiol compound covers the free gold surface remaining after the incubation step by forming an Au-S bond.

Then, by adding soluble nickel salts (e.g. Ni (II) chloride) or other suitable metal ions (such as copper), a state is reached in which the fluorophore is present in a geometrical arrangement close to the surface (see Example 4 ).

Example 5

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

An alternative production of Au-S (CH ₂ ) ₂ -ss-oligo-FP consists in the derivatization of the gold surface with the probe oligonucleotide (incubation step) without subsequent loading. 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 with (HO- (CH ₂ ) ₂ -S) ₂ esterified to PO- (CH ₂ ) ₂ - SS- (CH ₂ ) ₂ -OH; modification 2: at the 5 'end is a modified fluorophore and a bipyridyl group (see example 2) Incorporated according to the respective standard protocol) in 5x10 ^"5 molar buffer solution (phosphate buffer, 0.5 molar in water, pH 7) for 0.5 - 24 h. During this reaction time the disulfide spacer PO- (CH ₂ ) ₂ -SS - (CH ₂ ) ₂ -OH of the oligonucleotide cleaved homolytically, the spacer forming a covalent Au-S bond with Au atoms on the surface, which leads to coadsorption of the ss-oligonucleotide and the cleaved 2-hydroxy-mercaptoethanol. Instead of the single-strand oligonucleotide, this single strand can also be hybridized with its complementary strand.

The bipyridyl group attached to the fluorophore or in its vicinity interacts with the unmodified gold surface, whereby a geometrical arrangement is achieved in which the fluorophore is present close to the surface.

Example 6

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

Another alternative preparation of Au-S (CH ₂ ) ₂ -ss-oligo-FP consists in the derivatization of the gold surface with the probe oligonucleotide (incubation step) without subsequent loading.

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 with (HO- (CH ₂ ) ₂ -S) ₂ esterified to PO- (CH ₂ ) ₂ - SS- (CH ₂ ) ₂ -OH; modification 2: at the 5 'end is a modified fluorophore and an alkyl chain (e.g. as a fatty acid unit, cf. Example 2) built in according to the respective standard protocol) in 5x10 ^"5 molar buffer solution (phosphate buffer, 0.5 molar in water, pH 7) for 0.5-24 h. During this reaction time the disulfide spacer PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH of the oligonucleotide cleaved homolytically, whereby the spacer forms a covalent Au-S bond with Au atoms on the surface, which leads to coadsorption of the ss-oligonucleotide and the cleaved 2-hydroxy- mercaptoethanols is coming. Instead of the single-strand oligonucleotide, this single strand can also be hybridized with its complementary strand.

As an alternative to this, the gold surface can also be correspondingly hydrophobically modified by coadsorption (see Example 3) or subsequent coating (see Example 4) with hydrophobic alkylthiols (e.g. propanethiol or other thiols or disulfides of a suitable chain length).

The fatty acid unit attached to or in the vicinity of the fluorophore interacts with the hydrophobic gold surface, whereby a geometric arrangement is achieved in which the fluorophore is present close to the surface.

Example 7

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

The probe surface is produced analogously to Examples 3-6. 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 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 for the measurement of fluorescence in the presence of liquid media 150 μl of the medium are placed on the gold surface and then covered with a cover glass, or alternatively Hybriwells or imaging chambers can be used.

Claims

claims 1. A method for detecting 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 (201) and the ligate (201) being modified by attachment of at least one type of fluorophore (102), the fluorophore with the modified surface reversible binding occurs, b) provision of a sample with ligands, c) contacting the sample with the modified surface, d) detection of the fluorescence of the fluorophore (102), e) comparison of the fluorescence intensity detected in step d) with reference values. 2. The method of claim 1, wherein after step a) and before step c) the step b ₃ ) first detection of the fluorescence of the fluorophore (102) is carried out and in step π the values obtained in step d) are compared with the reference values obtained in step b ₃ ). 3. The method according to claims 1 or 2, wherein as step a) the step a) providing a modified surface, the modification consisting in the attachment of at least two types of modified ligates (201) and the different types of modified ligates (201) being bound to the surface in spatially essentially separated areas, and wherein the ligates ( 201) are modified by binding at least one type of fluorophore, the fluorophore (102) entering into a reversible bond with the modified surface, is carried out after step b) and before step c) the steps bi) addition of a ligand to the sample, the ligand being a binding partner with a high association constant of a modified ligate (201) bound to the surface in a certain region T ₁₀ o, the ligand being added in an amount which is greater than the amount of ligand that is necessary to fully associate the modified ligates (201) of the T ₁₀ o sites and b ₃ ) first detection of the fluorescence of the fluorophore (102) are carried out and in step e) the values obtained in step d) are compared with the value obtained in step b ₃ ) for the region T ₁₀₀ and the reference values obtained in step b ₃ ). 4. The method according to claim 3, wherein as step a) the step a) providing a modified surface, the modification consisting in the connection of at least three types of modified ligates (201) and the different types of modified ligates being bound to the surface in spatially essentially separated areas, at least one type of modified ligate (201) is bound to the surface in a certain region T ₀ , and in the sample there is no binding partner with a high association constant to this modified ligate, and the ligates are modified by binding at least one type of fluorophore (102), where the fluorophore (102) forms a reversible bond with the modified surface, is carried out and in step e) the values obtained in step d) with the value obtained in step b ₃ ) for the area T ₁₀₀ , with the value obtained in step b ₃ ) for the area T ₀ and with the value in step b ₃ ) reference values obtained are compared. 5. The method according to claim 3 or 4, wherein before step c) the step b ₂ ) addition of at least one further type of ligand to the sample, the ligand not being contained in the sample provided in step b) and the ligand having an association constant> 0 to a modified ligate bound to the surface in a certain region T _n wherein the ligand is added in an amount such that after step c) n% of the modified ligates in the region T _{n are} in an associated form is performed, and in step e) in step d) values obtained with the B in step ₃₎ for the area T I ₀₀ value obtained, with which, in step b ₃₎ for the area T ₀ value obtained with the b in Step ₃ ) values obtained for the areas T _n and with the reference values obtained in step b ₃ ). 6. The method according to any one of the preceding claims, wherein the fluorophore (102) is modified by linking at least one chemical group (206) which forms a reversible bond with the modified surface. 7. The method according to any one of the preceding claims, wherein the ligates (201) are additionally modified by binding at least one chemical group which forms a reversible bond with the modified surface. 8. The method according to claim 7, wherein the modified surface is additionally modified by binding chemical groups (207), the chemical groups (207) bound to the surface forming a reversible bond with the chemical groups bound to the ligates (201). 9. The method according to any one of the preceding claims, wherein the modified surface is additionally modified by binding chemical groups (207), wherein the chemical groups (207) bound to the surface form a reversible bond with the fluorophore (102). 10. The method according to claim 9, wherein the chemical groups (207) bound to the surface form a reversible bond with the chemical groups (206) bound to the fluorophore. 11. The method according to any one of the preceding claims, wherein the fluorophore is modified by linking at least one bipyridyl group or by linking at least one diamino group. 12. The method according to any one of the preceding claims, wherein the ligate is additionally modified by attaching at least one bipyridyl group or by attaching at least one diamino group. 13. The method according to claims 8 to 12, wherein the modified surface is additionally modified by linking diamino groups. 14. The method according to claim 13, wherein after step a) and before step c) the step a-i) Contacting a solution of a copper (II) or nickel (II) salt with the modified surface is carried out. 15. 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 (resorcin phthalein), or a cyanine dye, especially 5.5-disulfo- 1, di (-carbopentenyl) -3.3.3 \ 3 etramethyl-indodicarbocyanine (Cy3 ™) or 5.5 \ 7.7 ^" - tetrasulfo- 1, 1 "d-carbopenteny -SS ^ .S'-tetramethyl-benzindodicarbocyanin (Cy5 ™) is used. 16. The method according to any one of the preceding claims, wherein the modified surface is an at least one modification-bearing surface made of a metal or a metal alloy, in particular a metal selected from the group consisting of gold, silver, copper and mixtures thereof. 17. The method according to any one of the preceding claims, wherein substrates, cofactors or coenzymes are used as ligands and proteins or enzymes are used as ligates. 18. The method according to any one of claims 1 to 16, wherein antibodies are used as ligands and antigens are used as ligates. 19. The method according to any one of claims 1 to 16, wherein antigens are used as ligands and antibodies are used as ligates. 20. The method according to any one of claims 1 to 16, wherein receptors are used as ligands and hormones are used as ligates. 21. The method according to any one of claims 1 to 16, wherein hormones are used as ligands and receptors are used as ligates. 22. The method according to any one of claims 1 to 16, wherein nucleic acid oligomers as ligands and nucleic acid oligomers complementary thereto are used as ligates. 23. The method according to claim 22, wherein the nucleic acid oligomers used as ligates comprise 3 to 70 bases, in particular 5 to 60 bases, particularly preferably 10 to 50 bases, particularly preferably 12 to 40 bases. 24. Kit for carrying out a method according to claims 1 to 23 comprising a modified surface, the modification consisting in connecting at least one type of modified ligate (201), the ligate (201) by connecting at least one type of fluorophore (102 ) are modified and the fluorophore (102) forms a reversible bond with the modified surface. 25. The kit of claim 24, wherein the fluorophore (102) is modified by attachment of at least one chemical group (206) that forms a reversible bond with the modified surface. 26. The kit of claim 24 or 25, wherein the kit additionally comprises a copper (II) or nickel (II) salt or a solution of a copper (II) or nickel (II) salt. 27. Kit according to one of claims 24 to 26, wherein the kit additionally reference values for comparison with those in the detection of the fluorescence of the fluorophore (102) in one Method according to claims 1 to 23 values obtained. 28. Kit according to one of claims 24 to 27, wherein the modified surface comprises at least one area T ₀ and at least one area T ₁₀₀ . 29. The kit of claim 28, wherein the modified surface additionally comprises at least one area T _n .