DE19921940A1 - Method for the electrochemical detection of nucleic acid oligomer hybrids - Google Patents

Abstract

The invention relates to a method for the electrochemical detection of sequence-specific nucleic acid oligomer hybridization events. To this end single DNA/RNA/PNA oligomer strands which at one end are covalently joined to a support surface and at the other, free end, covalently linked to a redox pair, are used as hybridization matrix (probe). As a result of treatment with the olignucleotide solution (target) to be examined, the electric communication between the conductive support surface and the redox pair bridged by the single-strand oligonucleotide, which communication initially is either absent or very weak, is modified. In case of hybridization, electric communication between the surface support and the redox pair, which is now bridged by a hybridized double-strand oligonucleotide, is increased. This permits the detection of a hybridization event by electrochemical methods such as cyclic voltametry, amperometry or conductivity measurement.

Description

Technical field

The present invention relates to a modified nucleic acid oligomer and a Method for the electrochemical detection of sequence-specific nucleic acid Oligomer hybridization events.

State of the art

For sequence analysis of DNA and RNA, e.g. B. in disease diagnosis toxicological test procedures, in genetic research and development, as well as in the agricultural and pharmaceutical sector, are generally gel electrophoretic processes with autoradiographic or optical detection used.

To illustrate the most important gel electrophoretic method with optical detection (Sanger method), a DNA fragment with primer is shown in FIG. 1b. In the Sanger process, a DNA-containing solution is divided into four batches and the primer of each batch is covalently modified with a fluorescent dye that emits at different wavelengths. As shown in FIG. 1b, deoxyribonucleoside triphosphate of bases A (adenine), T (thymine), C (cytosine), and G (guanine), i.e. dATP, dTTP, dCTP and dGTP, is added to each batch to form the single strand , starting from the primer, to be replicated enzymatically by DNA polymerase I. In addition to the four deoxyribonucleoside triphosphates, each reaction mixture contains enough of the 2 ', 3'-dideoxy analogue ( Fig. 1a) of one of these nucleoside triphosphates as a stop base (one of the 4 possible stop bases per approach) to stop replication at all possible binding sites . After combining the four approaches, replicated DNA fragments of all lengths with stop-base-specific fluorescence are formed, which can be sorted by gel electrophoresis according to their length and characterized by fluorescence spectroscopy ( FIG. 1c).

Another optical detection method is based on the addition of Fluorescent dyes such. B. Ethidium bromide on oligonucleotides. The Fluorescence of such dyes increases compared to the free solution of the dye by about 20 times if they are attached to double-stranded DNA or RNA  attach and can therefore be used to detect hybridized DNA or RNA become.

In the case of radioactive labeling, ³² P is incorporated into the phosphate skeleton of the oligonucleotides, ³² P usually being added at the 5'-hydroxyl end by polynucleotide kinase. The labeled DNA is then preferably cleaved at one of the four nucleotide types, under defined conditions, so that an average cleavage occurs per chain. This means that there are chains in the reaction mixture for a certain base type, which extend from the ³² P mark to the position of this base (if the base occurs more than once, chains of different lengths are obtained). The four fragment mixtures are then separated by gel electrophoresis on four lanes. An autoradiogram is then made from the gel, from which the sequence can be read immediately.

A few years ago, a further method for DNA sequencing based on optical (or autoradiographic) detection was developed, namely sequencing by oligomer hybridization (cf. e.g. Drmanac et al., Genomics 4, (1989), p. 114- 128 or Bains et al., Theor. Biol. 135, (1988), pp. 303-307). In this method, a complete set of short oligonucleotides or oligomers (probe oligonucleotides), e.g. B. all 65 536 possible combinations of bases A, T, C and G of an oligonucleotide octamer are bound to a support material. The connection takes place in an ordered grid of 65 536 test sites, a larger amount of an oligonucleotide combination defining a test site and the position of each individual test site (oligonucleotide combination) being known. On such a hybridization matrix, the oligomer chip, a DNA fragment, the sequence of which is to be determined, the target is labeled with fluorescent dye (or 32P) and hybridized under conditions which only permit specific double-strand formation. As a result, the target DNA fragment only binds to the oligomers (in the example to the octamers) whose complementary sequence corresponds exactly to a part (an octamer) of its own sequence. By optical (or autoradiographic) detection of the binding position of the hybridized DNA fragment, all the oligomer sequences (octamer sequences) present in the fragment are thus determined. Due to the overlap of adjacent oligomer sequences, the continuous sequence of the DNA fragment can be determined by suitable mathematical algorithms. The advantages of this method include the miniaturization of the sequencing and thus the enormous amount of data that is recorded simultaneously in one operation. In addition, primers and the gel electrophoretic separation of the DNA fragments can be dispensed with. This principle is shown by way of example in FIG. 2 for a 13-fragment-long DNA fragment.

The use of radioactive labels in DNA / RNA sequencing has several disadvantages, such as e.g. B. complex, legally prescribed safety precautions when handling radioactive materials, the radiation exposure, the limited spatial resolution (maximum 1 mm ² ) and a sensitivity that is only high if the radiation of the radioactive fragments for a correspondingly long (hours to days) an x-ray film. Although the spatial resolution can be increased by additional hardware and software and the detection time can be shortened by using β scanners, both are associated with considerable additional costs.

The fluorescent dyes that are commonly used to label DNA are partly mutagenic (e.g. ethidium bromide) and require, just like the Use of autoradiography, corresponding safety precautions. Almost In all cases, the use of optical detection requires the use of one or several laser systems and thus trained personnel and corresponding Safety precautions. The actual detection of fluorescence requires additional hardware, such as B. optical components for amplification and different excitation and interrogation wavelengths as in the Sanger method Control system. Depending on the required excitation wavelengths and the Desired detection performance can thus result in significant investment costs arise. When sequencing by hybridization on the oligomer chip is the Detection even more (costly) because, in addition to the excitation system, dimensional detection of fluorescent spots high resolution CCD cameras (Charge Coupled Device Cameras) are required.

So although there are quantitative and extremely sensitive methods for DNA / RNA Sequencing out there, these methods are time consuming, involve time consuming Sample preparation and expensive equipment and are generally not considered portable systems available.

Presentation of the invention

The object of the present invention is therefore a device and a To provide methods for the detection of nucleic acid oligomer hybrids which the Not have disadvantages of the prior art.  

According to the invention, this object is achieved by the modified oligonucleotide independent claim 1, by the method for producing a modified oligonucleotide according to independent claims 9 and 10, by which modified conductive surface according to independent claim 11, the Process for producing a modified conductive surface according to independent claim 21, and by the method for electrochemical detection of oligomer hybridization events according to independent claim 27 solved.

Within the scope of the present invention the following abbreviations and Terms used:

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
Base: A, G, T or C
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 that it can bind naturally occurring cDNA or RNA in a sequence-specific manner.
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).
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.
dATP: deoxyribonucleoside triphosphate of A (DNA unit with base A and two other phosphates to build up a longer DNA fragment or an oligonucleotide).
dGTP: deoxyribonucleoside triphosphate of G (DNA unit with base G and two other phosphates to build up a longer DNA fragment or an oligonucleotide).
dCTP: deoxyribonucleoside triphosphate of C (DNA unit with base C and two other phosphates to build up a longer DNA fragment or an oligonucleotide).
dTTP: deoxyribonucleoside triphosphate of T (DNA unit with base T and two other phosphates to build up a longer DNA fragment or an oligonucleotide).
Primer: start complementary fragment of an oligonucleotide, the base length of the primer being only about 4-8 bases. Serves as a starting point for the enzymatic replication of the 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 (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".
ds: double strand (double strand)
ss: single strand

Chemical substances / groups

R: any unspecified organic radical as a substituent or side chain.
Redox: redox-active substance
Alkyl: The term "alkyl" denotes a saturated hydrocarbon radical that is straight-chain or branched (e.g. ethyl, isopropyl or 2,5-dimethylhexyl etc.). When "alkyl" is used to refer to a linker or spacer, the term refers to a group with two available valences for covalent linkage (e.g. -CH ₂

CH ₂

-, -CH ₂

CH ₂

CH ₂

- or -CH ₂

C (CH ₃

) ₂

CH ₂

CH ₂

C (CH ₃

) ₂

CH ₂

- Etc.). Preferred alkyl groups as substituents or side chains R are those of chain length 1-30 (longest continuous chain of bonded atoms). Preferred alkyl groups as linkers or 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 connected to the linker or spacer.
Alkenyl: alkyl groups in which one or more of the CC single bonds are replaced by C = C double bonds.
Alkynyl: alkyl or alkenyl groups in which one or more of the CC single or C = C double bonds are replaced by C∼C triple bonds.
Hetero-alkyl: alkyl groups in which one or more of the CH bonds or CC single bonds are replaced by CN, C = N, CP, C = P, CO, C = O, CS or C = S bonds.
Hetero-alkenyl: alkenyl groups in which one or more CH bonds, CC single or C = C double bonds are replaced by CN, C = N, CP, C = P, CO, C = O, CS or C = S bonds.
Hetero-alkynyl: alkynyl groups in which one or more of the CH bonds, CC single, C = C double or C∼C triple bond through CN, C = N, CP, C = P, CO, C = O, CS or C = S bonds are replaced.
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, hetero-alkyl, hetero-alkenyl 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 partners. 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, surface molecule or a surface molecule group and another molecule , represents.
Spacer: Linker that is covalently linked via the reactive groups to one or both of the structures to be connected (see linker). 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.
(n × HS spacer) oligo: nucleic acid oligomer to which n thiol functions are linked via a spacer, the spacers each having a different chain length (shortest continuous connection between thiol function and nucleic acid oligomer), in particular any one Chain length between 1 and 14. These spacers can in turn be bound to various reactive groups that are naturally present on the nucleic acid oligomer or are attached to it by modification and “n” is any integer, in particular a number between 1 and 20.
(n × RSS spacer) oligo: nucleic acid oligomer to which n disulfide functions are each connected via a spacer, any residue R saturating the disulfide function. The spacer for connecting the disulfide function to the nucleic acid oligomer can each have a different chain length (shortest continuous connection between disulfide function and nucleic acid oligomer), in particular any chain length between 1 and 14. These spacers can in turn be attached to different ones of course on the nucleic acid oligomer existing or attached to this by modification attached reactive groups. The placeholder "n" is any integer, especially a number between 1 and 20.
oligo-spacer-SS-spacer-oligo: two identical or different nucleic acid oligomers which are connected to one another via a disulfide bridge, the disulfide bridge being linked to the nucleic acid oligomers via any two spacers and the two spacers having a different chain length ( have the shortest continuous connection between the disulfide bridge and the respective nucleic acid oligomer), in particular any chain length between 1 and 14, and these spacers can in turn be bound to various reactive groups that are naturally present on the nucleic acid oligomer or are attached to them by modification.
PQQ: pyrrolo-quinolino-quinone, corresponds to: 4,5-dihydro-4,5-dioxo-1H pyrrolo- [2,3-f] -quinoline-2,7,9-tricarboxylic acid)
TEATFB: tetraethylammonium tetrafluoroborate
sulfo-NHS: N-hydroxysulfosuccinimide
EDC: (3-dimethylaminopropyl) carbodiimide
HEPES: N- [2-hydroxyethyl] piperazine-N '- [2-ethanesulfonic acid]
Tris: tris (hydroxymethyl) aminomethane
EDTA: ethylenediamine tetraacetate (sodium salt)
Cystamine: (H ₂

N-CH ₂

-CH ₂

-S-) ₂

Modified surfaces / electrodes

Mica: muscovite platelets, carrier material for applying thin layers.
Au-S-ss-oligo-PQQ: Gold film on mica with covalently applied monolayer of derivatized 12 bp single-strand oligonucleotide (sequence: TAGTCGGAAGCA). The terminal phosphate group of the oligonucleotide is at the 3 'end with (HO- (CH ₂

) ₂

-S) ₂

to PO- (CH ₂

) ₂

-SS- (CH ₂

) ₂

-OH esterified, the SS bond being cleaved homolytically and each causing an Au-SR bond. The terminal base thymine at the 5 'end of the oligonucleotide is at the C-5 carbon with -CH = CH-CO-NH-CH ₂

-CH ₂

-NH _2nd

modified and this residue in turn is linked via its free amino group by amide formation to a carboxylic acid group of the PQQ.
Au-S-ds-oligo-PQQ: Au-S-ss-oligo-PQQ, which is present hybridized with the oligonucleotide complementary to ss-oligo (sequence: TAGTCGGAAGCA).

Electrochemistry

E: Electrode potential that is applied to the working electrode.
E ₀

: Half-step potential, potential in the middle between the current maxima for oxidation and reduction of an electrooxidation or reduction reversible in cyclic voltammetry.
i: current density (current per cm ²

Electrode surface)
Cyclic voltametry: recording a current / voltage curve. The potential of a stationary working electrode is changed linearly as a function of time, starting from a potential at which no electrooxidation or reduction takes place up to a potential at which a dissolved or adsorbed species is oxidized or reduced (i.e. current flows). After passing through the oxidation or reduction process, which generates an initially increasing current in the current / voltage curve and a gradually decreasing current after reaching a maximum, the direction of the potential feed is reversed. The behavior of the products of electrooxidation or reduction is then recorded in the return.
Amperometry: recording a current / time curve. Here, the potential of a stationary working electrode z. B. is set by a potential jump to a potential at which the electro-oxidation or reduction of a dissolved or adsorbed species takes place and the flowing current is recorded as a function of time.

The present invention relates to a nucleic acid oligomer by chemical Connection of a redox-active substance is modified. As a nucleic acid oligomer is within the scope of the present invention a connection of at least two covalently linked nucleotides or from at least two covalently linked Pyrimidine (e.g. cytosine, thymine or uracil) or purine bases (e.g. adenine or Guanine), preferably a DNA, RNA or PNA fragment. In the In the present invention, the term nucleic acid refers to any "Backbone" of the covalently linked pyrimidine or purine bases, such as. B. on that Sugar-phosphate backbone of DNA, cDNA or RNA, on a peptide backbone of PNA or on analog backbone structures, such as. B. a thio phosphate, a dithio Phosphate or a phosphoramide backbone. Essential characteristic of a Nucleic acid in the sense of the present invention is that it is natural occurring cDNA or RNA can bind sequence-specifically. Alternatively to that The term “nucleic acid oligomer” means the terms “(probe) oligonucleotide”, "Nucleic acid" or "oligomer" used.

The redox-active substance can be selectively oxidized at a potential cp reducible, where ϕ satisfies the condition 2.0 V <ϕ <-2.0 V. The potential relates focus on the free, unmodified, redox-active substance in a suitable Solvent, measured against normal hydrogen electrode. As part of the In the present invention, the potential range 1.7 V ≧ ϕ ≧ -1.7 V is preferred, wherein the range 1.4 V ≧ ϕ ≧ -1.2 V is particularly preferred and the range 0.9 V ≧ ϕ ≧ -0.7 V, in which the redox-active substance of the application example is reduced and is reoxidized, is particularly preferred. In addition, the present concerns Invention a conductive surface to which directly or indirectly (via a spacer) a nucleic acid oligomer with attached redox-active substance chemically is bound. The present invention also relates to a method for Manufacture of a modified conductive surface, a modified Nucleic acid oligomer is applied to a conductive surface. According to In another aspect, the present invention relates to a method that electrochemical detection of molecular structures, especially the electrochemical detection of DNA / RNA-IPNA fragments in one Sample solution by sequence-specific nucleic acid-oligomer hybridization enables. The detection of hybridization events by electrical signals is a simple and inexpensive method and enables in one battery-operated variant of a sequencer used on site.  

Binding of a redox-active substance to a nucleic acid oligomer The prerequisite for the method according to the invention is the binding of a redox-active substance to a nucleic acid oligomer. According to the invention any redox active substance can be used as long as it has a potential ϕ that the condition 2.0 V ≧ ϕ ≧ -2.0 V is sufficient, selectively oxidizable and reducible. The potential here relates to the free, unmodified, redox-active substance in a suitable solvent, measured against normal hydrogen electrode. In the context of the present invention, the potential range is 1.7 V ≧ ϕ ≧ -1.7 V preferred, the range 1.4 V ≧ ϕ ≧ -1.2 V being particularly preferred and the Range 0.9 V ≧ ϕ ≧ -0.7 V, in which the redox-active substance of Application example is reduced and reoxidized, is very particularly preferred. The term "selectively oxidizable and reducible" is used in the context of Present invention a redox reaction, so delivery or uptake Electrons understood, which takes place selectively at the location of the redox-active substance. Ultimately, no other part of the Nucleic acid oligomers reduced or oxidized, but only those attached to the Nucleic acid oligomer bound redox active substance.

According to the invention, redox-active substance means any molecule that can be electro-oxidized / reduced in the electrochemically accessible potential range of the respective carrier surface (electrode) by applying an external voltage to this electrode. In addition to the usual organic and inorganic redox-active substances such. B. hexacyanoferrates, ferrocenes, acridines or phthalocyanines are particularly suitable for binding to the probe oligonucleotide, such as redox-active dyes such. B. (metallo) porphyrins of the general formula 1, (metallo) chlorophylls of the general formula 2 or (metallo) bacteriochlorophylls of the general formula 3, (colored) naturally occurring oxidation agents such as. B. flavins of general formula 4, pyridine nucleotides of general formula 5 or pyrrolo-quinoline quinones (PQQ) of general formula 6 or other quinones such as. B 1,4-benzoquinones of the general formula 7, 1,2-benzoquinones of the general formula 8, 1,4-naphthoquinones of the general formula 9, 1,2-naphthoquinones of the general formula 10 or 9,10-anthraquinones of the general formula 11 .

M = 2H, Mg, Zn, Cu, Ni, Pd, Co, Cd, Mn, Fe, Sn, Pt etc .; R ₁ to R ₁₂ are independently H or any alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituent.

R ₁ to R ₈ are independently H or any alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituent.

According to the invention, a redox-active substance becomes covalent to an oligonucleotide bound by the reaction of the oligonucleotide with the redox-active substance. This binding can be done in three different ways:

a) A reactive group for binding formation on the nucleic acid oligomer is a free phosphoric acid, sugar-C-3-hydroxy, carboxylic acid or amine group of the oligonucleotide backbone, in particular a group at one of the two ends of the oligonucleotide backbone, used. The free, terminal phosphoric acid, sugar-C-3-hydroxy, carboxylic acid or amine groups have an increased reactivity and are therefore easily typical reactions such. B. amide formation with (primary or secondary) amino groups or with acid groups, ester formation with (primary, secondary or tertiary) alcohols or with acid groups, thioester formation with (primary, secondary or tertiary) thio alcohols or with acid groups or the condensation of Amine and aldehyde with subsequent reduction of the resulting CH = N bond to the CH ₂ NH bond. The coupling group (acid, amine, alcohol, thioalcohol or aldehyde function) required for the covalent attachment of the redox-active substance is either naturally present on the redox-active substance or is obtained by chemical modification of the redox-active substance.

b) The nucleic acid oligomer is via a covalently attached molecular part (spacer) of any composition and chain length (represents the shortest continuous connection between the structures to be connected), in particular chain length 1 to 14, on the oligonucleotide backbone or on a base with a reactive one Group modified. The modification is preferably carried out at one of the ends of the oligonucleotide backbone or at a terminal base. As a spacer z. B. an alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituent can be used. Possible simple reactions for the formation of the covalent bond between the redox-active substance and the nucleic acid oligomer modified in this way are as described under a), the amide formation from the acid and amino group, the ester formation from the acid and alcohol group, the thioester formation from the acid and thio alcohol group or the condensation of aldehyde and amine with subsequent reduction of the resulting CH = N bond to the CH ₂ NH bond.

According to a preferred embodiment, the nucleic acid oligomer is through modified a redox-active substance, the areas with a predominantly planar, in one plane extended p-π orbital system, such as. B. that PQQ of Example 1 or the quinones of the formula 5 or 7-12 or the porphinoids  Structures of the formulas 1-4 or the pyridine nucleotides of the general formula 6 or derivatives of these redox-active substances. In this case, the spacer can over which the redox-active substance is bound to the nucleic acid oligomer, so be chosen so that the level of the π orbitals of the redox-active substance parallel to the p-π orbitals of the bases adjacent to the redox-active substance of the nucleic acid oligomer can arrange. This spatial arrangement of redox-active substance with partially planar p-π- Orbitals prove to be particularly cheap.

c) In the synthesis of the nucleic acid oligomer, a terminal base is replaced by the redox-active substance replaced.

According to the invention, the binding of the redox-active substance to the Oligonucleotide as described under a) and b) before or after the binding of the Oligonucleotide to the conductive surface. The connection of the redox-active substance to that bound on the conductive surface The oligonucleotide is then also carried out as described under a) and b).

With several different oligonucleotide combinations (test sites) on one common surface, it is advantageous to connect the (covalent) redox-active substance to the probe oligonucleotides by suitable choice of reactive group on the free probe oligonucleotide ends of the different Unify test sites for the entire interface.

The conductive surface

According to the invention, the term “conductive surface” includes each carrier understood an electrically conductive surface of any thickness, in particular Surfaces made of platinum, palladium, gold, cadmium, mercury, nickel, zinc, Carbon, silver, copper, iron, lead, aluminum and manganese. As part of the In the present invention, the terms "electrode" and "conductive (carrier) Surface "used as an alternative to" conductive surface ".

In addition, any doped or undoped semiconductor surfaces can also be used any thickness can be used. All semiconductors can be used as pure substances or used as mixtures. Non-limiting examples at this point let be carbon, silicon, germanium, α-tin, Cu (I) - and Ag (I) - Halides of any crystal structure called. All are also suitable  binary compounds of any composition and structure of the Elements of groups 14 and 16, elements of groups 13 and 15, and the Elements of groups 15 and 16. In addition, ternary compounds can be any Composition and any structure of the elements of groups 11, 13 and 16 or the elements of groups 12, 13 and 16 can be used. The Names of the groups in the Periodic Table of the Elements refer to the 1985 IUPAC recommendation.

Binding of an oligonucleotide to the conductive surface

According to the invention, an oligonucleotide is added directly or via a linker / spacer the support surface atoms or molecules of a conductive support surface linked of the type described above. This bond can be made in three different ways Types are carried out:

a) The surface is modified so that a reactive group of molecules is accessible. This can be done by direct derivatization of the surface molecules, e.g. B. done by wet chemical or electrochemical oxidation / reduction. So z. B. the surface of graphite electrodes can be provided by wet chemical oxidation with aldehyde or carboxylic acid groups. Electrochemically z. B. the possibility by reduction in the presence of aryl diazonium salts the corresponding (functionalized, that is provided with a reactive group) aryl radical or by oxidation in the presence of R'CO ₂ H the (functionalized) R 'radical on the graphite Coupling the electrode surface. An example of the direct modification of semiconductor surfaces is the derivatization of silicon surfaces to reactive silanols, ie silicon substrates with Si-OR "groups on the surface, where R" as well as R 'represents any functionalized organic residue (e.g. Alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituent). Alternatively, the entire surface can be modified by the covalent attachment of a reactive group of a bifunctional linker, so that a monomolecular layer of any molecule is formed on the surface which preferably contains a reactive group at the end. The term “bifunctional linker” is understood to mean any molecule of any chain length, in particular chain lengths 2-14, with two identical (homo-bifunctional) or two different (hetero-bifunctional) reactive molecule groups.

Are supposed to have several different test sites on the surface by taking advantage of the Methodology of photolithography are formed, so is at least one of the  reactive groups of the homo- or hetero-bifunctional linker one photo-inducible reactive group, d. H. one determined only by exposure to light or any wavelength reactive group. This linker will be like this applied that the / a photoactivatable reactive group after the covalent Linker link is available on the interface. To that modified surface, the nucleic acid oligomers are covalently attached, these themselves using a spacer of any composition and Chain length, especially chain length 1-14, with a reactive group are modified, preferably near one end of the nucleic acid oligomer. The reactive group of the oligonucleotide is a group that directly (or indirectly) with the modified surface to form a covalent Bond react. In addition, the nucleic acid oligomers close to their second, another reactive group may be bound, this being reactive Group in turn, as described above, either directly or via a spacer Composition and chain length, in particular chain length 1-14, is connected. Furthermore, the redox-active substance can be used as an alternative to this another reactive group, at this second end of the nucleic acid oligomer be connected.

b) The nucleic acid oligomer that is applied to the conductive surface is, is via a covalently attached spacer of any composition and chain length, in particular chain length 1-14, with one or more modified reactive group, these reactive groups preferably in the Located near one end of the nucleic acid oligomer. With the reactive groups are groups that react directly with the unmodified surface can. Examples include: (i) thiol (HS) or disulfide (S-S) derivatized Nucleic acid oligomers of the general formula (n × HS spacer) oligo, (n × R-S-S- Spacer) -oligo or oligo-spacer-S-S-spacer-oligo, with a gold surface underneath Formation of gold-sulfur bonds react or (ii) amines, which are characterized by Add chemical or physical sorption to platinum or silicon surfaces. Besides can add another to the nucleic acid oligomers near their second end be reactive group, this reactive group in turn, as above described, directly or via a spacer of any composition and Chain length, in particular chain length 1-14, is connected. Furthermore can the redox-active substance as an alternative to this further reactive group be attached to this second end of the oligonucleotide. In particular Nucleic acid oligomers which are bridged with several spacers or Disulfide bridges are modified ((n × HS spacer) oligo or (n × R-S-S spacer) - oligo) have the advantage that such nucleic acid oligomers under one  certain angle of attack against the conductive surface (angle between the Surface normal and the helix axis of a double-stranded helical Nucleic acid oligomers or between the surface normal and the axis perpendicular to the base pairs of a double-stranded non-helical nucleic acid Oligomers) can be applied if the thiol or disulfide Functions to the nucleic acid oligomer spacer, from one end considered the nucleic acid forth, an increasing or decreasing chain length have.

c) The reactive group on the probe nucleic acid oligomer is the phosphoric acid, sugar-C-3-hydroxy, carboxylic acid or amine groups of the oligonucleotide backbone, in particular terminal groups. The phosphoric acid, sugar-C-3-hydroxy, carboxylic acid or amine groups have an increased reactivity and are therefore easy to carry out typical reactions such. B. amide formation with (primary or secondary) amino or acid groups, ester formation with (primary, secondary or tertiary) alcohols or acid groups, thioester formation with (primary, secondary or tertiary) thio alcohols or acid groups or the condensation of amine and Aldehyde with subsequent reduction of the resulting CH = N bond to the CH ₂ -NH bond. In this case, the coupling group required for covalent attachment to the phosphoric acid, sugar-C-3-hydroxy, carboxylic acid or amine group is part of the surface derivatization with a (monomolecular) layer of any molecular length, as under a) in described in this section, or the phosphoric acid, sugar-C-3-hydroxy, carboxylic acid or amine group can react directly with the unmodified surface, as described under b) in this section. In addition, a further reactive group can be bound to the oligonucleotides in the vicinity of their second end, this reactive group, in turn, as described above, being attached directly or via a spacer of any composition and chain length, in particular chain length 1-14. Furthermore, as an alternative to this further reactive group, the redox-active substance can be attached to this second end of the nucleic acid oligomer.

The binding of the oligonucleotide to the conductive surface can alternatively be before or after binding the redox-active substance to the oligonucleotide or before or after attaching the spacer provided with a reactive group to bind the redox-active substance. The binding of the already modified Oligonucleotide to the conductive surface, i.e. H. the bond to the surface after binding the redox-active substance to the oligonucleotide or after the  Attachment of the spacer provided with a reactive group to bind the redox-active substance, is also carried out as under a) to c) (section "binding of an oligonucleotide to the conductive surface ").

When producing the test sites, the single-strand Oligonucleotides on the surface be careful that between the individual oligonucleotides a sufficiently large distance remains to the one Hybridization with the target oligonucleotide provides the necessary free space put. There are two different ways of doing this:

1) Production of a modified carrier surface by connecting a hybridized oligonucleotide, i.e. a carrier surface derivatization with hybridized probe oligonucleotide instead of single stranded probe oligonucleotide. The oligonucleotide strand used for hybridization is unmodified (the Surface connection is carried out as under a) - c) in the section "Binding of an oligonucleotide to the conductive surface ") the hybridized oligonucleotide duplex is thermally dehybridized, whereby a single strand oligonucleotide modified support surface with a larger Distance between the proboligonucleotides is established.

2) Production of a modified carrier surface by connecting a Single-stranded or double-stranded oligonucleotide, during which A suitable monofunctional linker was added to the carrier surface derivatization which, in addition to the single-stranded or double-stranded oligonucleotide, is also attached to the Surface is bound (the surface connection is carried out as under a) - c) in the section "binding of an oligonucleotide to the conductive surface" described). According to the invention, the monofunctional linker has a chain length that of the chain length of the spacer between the support surface and the oligonucleotide is identical or deviates by a maximum of eight chain atoms. When using Double-stranded oligonucleotide for carrier surface derivatization is after the Attachment of the double-stranded oligonucleotide and the linker to the Carrier surface of the hybridized oligonucleotide double strand thermal dehybridized as described above under 1.). Through the simultaneous connection of a linker to the surface, the distance between the also to the Surface bound single or double stranded nucleic acid oligomers enlarged. In case of using double stranded nucleic acid oligomer this effect is further reinforced by the subsequent thermal dehybridization.

Method for the electrochemical detection of nucleic acid oligomer hybrids

Advantageously, according to the method for electrochemical detection several probe oligonucleotides of different sequences, ideally all necessary combinations of the nucleic acid oligomer, on an oligomer or DNA chip applied to the sequence of any target oligomer or a (fragmented) target DNA safely to detect or mutations in Tracking down the target and demonstrating it in a sequence-specific manner. For this, be on a conductive support surface the support surface atoms or molecules of a defined area (a test site) with DNA-IRNA-IPNA oligonucleotides known, but any sequence, as described above, linked. In a but the most general embodiment, the DNA chip can also with a single Probe oligonucleotide can be derivatized. As probe oligonucleotides Nucleic acid oligomers (DNA, RNA or PNA fragments) with a base length of 3 to 50, preferably length 5 to 30, particularly preferably length 7 to 25 used. According to the probe oligonucleotides either before or a redox-active substance after binding to the conductive surface bound.

The probe oligonucleotides are modified before binding to the conductive one Surface, so the already modified probe oligonucleotides as above described bound to the conductive surface. Alternatively, they won't modified probe oligonucleotides bound to the conductive surface second, free end of the oligonucleotide chain directly or indirectly via a spacer modified with a redox-active substance.

In both cases, a surface hybrid of the general structure elek-spacer-ss-oligo-spacer redox is formed ( FIG. 3). The electrical communication between the (conductive) support surface and the redox-active substance (“redox”) bridged by a single-strand oligonucleotide in the general structure elek-spacer-ss-oligo-spacer redox is weak or not available at all. The bridges can of course also be carried out without a spacer or with only one spacer (Elek-ss-oligo-Spacer-Redox or Elek-Spacer-ss-oligo-Redox).

In a next step, the test sites are brought into contact with the oligonucleotide solution to be examined (target). Hybridization only occurs in the case in which the solution contains oligonucleotide strands which are complementary to the probe oligonucleotides bound to the conductive surface, or at least are complementary in a wide range. In the case of hybridization between probe and target oligonucleotide, there is an increased conductivity between the support surface and the redox-active substance, since these are now bridged via the oligonucleotide consisting of a double strand (in FIG. 3 using an example of the electrod spacer ss-oligo spacer redox shown schematically).

By changing the electrical communication between the (conductive) Carrier surface and the redox-active substance due to the hybridization of Probe oligonucleotide and the complementary oligonucleotide strand (Target) can thus execute a sequence-specific hybridization event electrochemical processes such. B. cyclic voltammetry, amperometry or Conductivity measurements can be detected.

In a particularly preferred embodiment of the present invention, a redox-active substance is used which has regions with a predominantly planar, p-π-orbital system which is extended in one plane, such as, for example, B. the PQQ of Example 1 (see FIG. 3), or the quinones of the formula 5 or 7-12 or the porphinoid structures of the formulas 1-4, the pyridine nucleotides of the general formula 6 and derivatives of these redox-active substances. In this case, the spacer between the nucleic acid oligomer and the redox-active substance is selected such that the level of the π orbitals of the redox-active substance is parallel to the p-π orbitals of the base pair of the nucleic acid oligomer hybridized with the complementary strand and adjacent to the redox-active substance can arrange. This spatial arrangement of redox-active substance with partially planar p-π orbitals extended in one plane proves to be particularly favorable for the electrical conductivity of the double-stranded nucleic acid oligomers.

With cyclic voltammetry, the potential of a stationary working electrode becomes time-dependent changed linearly. Starting from a potential where no electrooxidation or -reduction takes place, the potential is changed until the redox-active substance is oxidized or reduced (i.e. current flows). After passing through the oxidation or Reduction process that initially increases in the current / voltage curve Current, a maximum current (peak) and then a gradually decreasing current, the direction of the potential feed is reversed. Behavior then becomes in the return of the products of electro-oxidation or reduction.

This creates an alternative electrical detection method, amperometry enables the redox-active substance by applying an appropriate, constant held electrode potential is electrooxidized (electroreduced), the Reduction (reoxidation) of the redox-active substance to its original state but is not achieved by changing the electrode potential, as in the  Cyclic voltammetry, but by a suitable one added to the target solution Reducing agent (oxidizing agent), which creates the circuit of the overall system is closed. As long as reducing agent (oxidizing agent) is present or as long as used reducing agent (oxidizing agent) on the counter electrode is reduced (reoxidized), current flows which can be detected amperometrically and which is proportional to the number of hybridization events.

Brief description of the drawings

The invention is based on an embodiment in Connection with the drawings will be explained in more detail. Show it

Fig. 1 Schematic representation of the Sanger method of sequencing oligonucleotide;

Fig. 2 Schematic representation of the oligonucleotide sequencing by hybridization on a chip;

Fig. 3 Schematic diagram of the surface hybrid having the general structure elec-spacer-ss-oligo-spacer-redox probe with a 12 bp oligonucleotide of the exemplary sequence 5'-TAGTCGGAAGCA-3 '(left) and Au-S-ss- oligo-PQQ in the hybridized state as an exemplary embodiment of an electro-spacer-ss-oligo-spacer redox, only a part of the probe oligonucleotide with hybridized complementary strand being shown (right) and the attachment of the oligonucleotide to the surface via an -S- CH ₂ CH ₂ spacer and the connection of the redox-active substance PQQ via the spacer -CH ₂ - CH = CH-CO-NH-CH ₂ -CH ₂ -NH-;

Fig. 4 Cyclic voltagram of a test site made of Au-S-ss-oligo-PQQ (dotted) compared to an identical test site with a fully hybridized target (Au-S-ds-oligo-PQQ, solid line);

Fig. 5 cyclogram a test site with fully hybridized target (Au-S-ds-oligo-PQQ) (solid line) compared to a test site with hybridized target, the 2 base pair mismatches has (Au-S-ds- oligo-PQQ with 2 bp mismatches, dashed).

Ways of Carrying Out the Invention

An exemplary test site with a hybridized target (Au-S-ds-oligo-PQQ) of the general structure Elek-Spacer-ds-oligo-Spacer-Redox is shown in FIG. 3. In the example of FIG. 3, the carrier surface is a gold electrode. The connection between the gold electrode and the probe oligonucleotide was established with the linker (HO- (CH ₂ ) 2 S) 2, which with the terminal phosphate group at the 3 'end leads to PO- (CH ₂ ) 2 SS- (CH ₂ ) 2 OH was esterified and after homolytic cleavage of the SS bond on the gold surface each caused an Au-S bond, with which 2-hydroxy mercaptoethanol and mercaptoethanol-bridged oligonucleotide was adsorbed on the surface. The redox-active substance in the example in FIG. 3 is tricarboxylic pyrrolo-quinoline-quinone (PQQ), one of the three carboxylic acid functions of the PQQ (in the example the C-7-CO ₂ H function) for covalently linking the PQQ to the probe. Oligonucleotide was used (amide formation with elimination of water with the terminal amino function of the -CH = CH-CO-NH-CH ₂ -CH ₂ -NH ₂ spacer linked to the C-5 position of the 5'-thymine). Both free, unmodified PQQ and a short spacer of chain length 1-6, such as. B. -S- (CH ₂ ) 2 NH-, or PQQ bridged with the support surface via (modified) double-stranded oligonucleotide, e.g. B. in HEPES buffer with 0.7 molar addition of TEATFB (see abbreviations), in the potential range 0.7 V ≧ ϕ ≧ 0.0 V, measured against normal hydrogen electrode, selectively reduced and reoxidized.

The electrical communication between the (conductive) support surface and the redox pair bridged by single-strand oligonucleotide in the general structure elek-spacer-ds-oligo-spacer redox is weak or not present at all. For the exemplary test site Au-S-ss-oligo-PQQ (with 12 bp probe oligonucleotides), this is shown using cyclic voltammetry ( FIG. 4). Without wishing to be bound by a theoretical description, it is assumed that the negative charges of the phosphate skeleton cause mutual repulsion of the oligonucleotide single strands and thus a structure of the spacer-ds-oligo-spacer redox chain (in the direction of the helix axis) under one Force angle ϕ = 70 ° to the normal of the support surface ("standing tubes"). The (hybridized) test site Au-S ds-oligo-PQQ of FIG. 3 has a structure with ϕ = 30 °. Due to the length of the spacer-ds-oligo-spacer redox chain (e.g. approx. 40 Å length of a 12-base pair oligonucleotide; the spacers and the attached PQQ are approx. 10 Å long) arise at ϕ = 70 ° a distance of <17 Å between the carrier surface and the redox-active substance. This means that direct electron or hole transfer between the carrier surface and the redox-active substance can be excluded. By treating the test site (s) with an oligonucleotide solution to be investigated, in the case of hybridization between probe and target, there is an increased conductivity between the support surface and the redox couple bridged by the double-stranded oligonucleotide. The change in conductivity manifests itself cyclically in a clear current flow between the carrier surface and the redox-active substance ( FIG. 4). This makes it possible to sequence-specific hybridization of the target with the probe oligonucleotides by electrochemical methods such as. B. to detect cyclic voltammetry.

In addition, incorrect base pairings (base pair mismatches) can be recognized by a changed cyclovoltametric characteristic ( FIG. 5). A mismatch manifests itself in a larger potential distance between the current maxima of the electrical reduction and the electroreoxidation (reversal of the electroreduction with reversed direction of potential feed) or the electrooxidation and electroreduction in a cyclovoltametrically reversible electron transfer process between the electrically conductive carrier surface and the redox-active substance. This fact has a particularly favorable effect in amperometric detection, since the current can be tested there at a potential at which the perfectly hybridizing oligonucleotide target delivers significant current, but not the incorrectly paired oligonucleotide target. In the example in FIG. 5, this is possible at a potential EE ₀ of approximately 0.03 V.

example 1 Preparation of the Au-S-ds-oligo-PQQ oligonucleotide electrode

The production of Au-S-ds-oligo-PQQ is divided into 4 sections, namely Representation of the carrier surface, hybridization of the probe oligonucleotide with the complementary double strand (hybridization step), derivatization of the Carrier surface with the double-stranded oligonucleotide (incubation step) and Connection of the redox-active substance (redox step).

The carrier material for the covalent attachment of the double-stranded oligonucleotides is formed by an approximately 100 nm thin gold film on mica (muscovite platelet). For this purpose, freshly split mica was cleaned with an argon ion plasma in an electrical discharge chamber and gold (99.99%) was applied in a layer thickness of approximately 100 nm by electrical discharge. The gold film was then freed from surface impurities (oxidation of organic deposits) using 30% H ₂ O ₂ /70% H ₂ SO ₄ and immersed in ethanol for about 20 minutes in order to displace oxygen adsorbed on the surface. After rinsing the support surface with bidistilled water, a previously prepared 1 × 10 ^-4 molar solution of the (modified) double-stranded oligonucleotide is applied to the horizontally stored support surface, so that the entire support surface is wetted (incubation step, see also below).

To prepare the ds-oligonucleotide solution, a double-modified 12 bp single-strand oligonucleotide of the sequence 5'-TAGTCGGAAGCA-3 'was used, which is attached to the phosphate group of the 3' end with (HO- (CH ₂ ) ₂ -S) ₂ PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH is esterified. At the 5 'end, the terminal base of the oligonucleotide, thymine, at the C-5 carbon is modified with -CH = CH-CO-NH-CH ₂ -CH ₂ -NH ₂ . A 2 × 10 ^-4 molar solution of this oligonucleotide in the hybridization buffer (10 mM Tris, 1 mM EDTA, pH 7.5 with 0.7 molar addition of TEATFB, see abbreviations) was mixed with a 2 × 10 ^-4 molar solution of the (unmodified) complementary strand hybridized in the hybridization buffer at room temperature for about 2 h (hybridization step). The disulfide spacer PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH of the oligonucleotide was cleaved homolytically during a reaction time of approx. 12-24 h. The spacer forms a covalent Au-S bond with the Au atoms on the surface, which leads to a 1: 1 coadsorption of the ds-oligonucleotide and the 2-hydroxymercaptoethanol (incubation step).

The gold electrode modified with a dense (1: 1) monolayer of ds-oligonucleotide and 2-hydroxy mercaptoethanol was washed with bidistilled water and then with a solution of 3 × 10 ^-3 molar quinone PQQ, 10 ^-2 molar EDC and 10 ^-2 molar sulfo-NHS wetted in HEPES buffer. After a reaction time of approx. 1 h, the -CH = CH-CO-NH-CH ₂ -CH ₂ -NH ₂ spacer covalently binds the PQQ (amide formation between the amino group of the spacer and an acid function of the PQQ, redox step).

The clarification of the surface properties with XPS (X-Ray photoelectron spectroscopy) revealed a maximally densely packed monolayer of 1: 1 adsorbed ds-oligonucleotide and 2-hydroxy-mercaptoethanol (4.7 × 10 ¹² ds-oligonucleotides / cm ² ), whereby the long axis (direction of the helix axis) of the ds-oligonucleotides forms an angle of ϕ ≈ 30 ° with the surface normal of the gold surface.

Example 2 Preparation of the Au-S-ss-oligo-PQQ oligonucleotide electrode

Analogous to the representation of the Au-S-ds-oligo-PQQ system, the carrier surface is derivatized with modified single-strand oligonucleotide, only the hybridization of the modified oligonucleotide of sequence 5'-TAGTCGGAAGCA-3 'with its complementary strand being dispensed with and in the incubation step only the double modified 12 bp single-stranded probe oligonucleotide (see example 1) as a 1 × 10 ^-4 molar solution in water and in the presence of 10 ^-2 molar Tris, 10 ^-3 molar EDTA and 0.7 molar TEATFB (or 1 molar NaCl) was used at pH 7.5. The redox step was carried out as indicated in Example 1.

Example 3 Production of the Oligonucleotide Electrode Au-S-ds-oligo-PQQ with 2-Bp- Mismatches

The production of a carrier surface derivatized with modified Double-stranded oligonucleotide was analogous to the representation of the Au-S-ds system oligo-PQQ performed, only in the hybridization of the modified Oligonucleotides of sequence 5'-TAGTCGGAAGCA-3 'a complementary strand was used (sequence: 5'-ATCAGATTTCGT-3 '), in which the actually complementary bases Nos. 6 and 7 (counted from the 5 'end) from C to A and from C, respectively were modified after T so as to introduce two base pairs of mismatches.

Example 4 Production of an oligonucleotide electrode using Au-S-ss-oligo-PQQ increased inter-oligonucleotide distance

When producing the test sites, when derivatizing the support surface with single-strand probe oligonucleotide, care must be taken to ensure that there is sufficient space between the attached single strands to enable hybridization with the target oligonucleotide. There are three different procedures for this: (a) Production of an Au-S-ds-oligo-PQQ electrode as described in Example 1 with subsequent thermal dehybridization of the double strands at temperatures of T <40 ° C. (b) Preparation of an Au-ss-oligo-PQQ electrode as described in Example 2, but in the incubation step to derivatize the gold surface with (double-derivatized) single-strand oligonucleotide, 2-hydroxy mercaptoethanol or another thiol or disulfide linker of suitable chain length 10 ^-5 - to 10 ^-1 molar added (depending on the desired inter-oligonucleotide distance), which co-adsorbs to the gold surface together with the single strand of oligonucleotide. (c) Preparation of an Au-ss-oligo-PQQ electrode as described in Example 2, but with the omission of the 0.7 molar addition of electrolytes (in the example TEATFB) in the incubation step to derivatize the gold surface with (double-derivatized) single-strand oligonucleotide. The phosphate groups and base nitrogen atoms of the oligonucleotide are not electrostatically shielded by the absence of the salt and interact strongly with the gold surface. This leads to a flat accumulation of the oligonucleotides on the electrode surface (ϕ <60 °) and significantly fewer oligonucleotides are bound per unit area. The oligonucleotides can then be brought back into the desired position by covalently attaching 2-hydroxy-mercaptoethanol or another thiol or disulfide linker of suitable chain length to the still free surface gold atoms in a second incubation step (before or after attaching the PQQ) is connected. For this purpose, the electrode, which is less densely covered with single-stranded oligonucleotide, is modified with an approx. 5 × 10 ^-2 molar solution of the 2nd -Hydroxy mercaptoethanol or another thiol or disulfide linker of suitable chain length in ethanol or HEPES buffer (or a mixture thereof, depending on the solubility of the thiol) wetted and incubated for 2-24 h.

Example 5 Carrying out the cyclovoltametric measurements

The cyclic voltametric measurements were measured with a computer-controlled bipotentiostat (CH Instruments, Model 832) at room temperature in a standard cell with a 3-electrode arrangement. The modified gold electrode was used as the working electrode, a platinum wire was used as the auxiliary electrode (counter electrode) and an Ag / AgCl electrode with an internally saturated KCl solution, which was separated from the sample space via a Luggin capillary, was used as the reference electrode. 0.7 molar TEATFB or 1 molar NaCl was used as the electrolyte. A cyclovoltagram of the Au-S-ds-oligo-PQQ electrode compared to an Au-S-ss-oligo-PQQ electrode is shown in FIG. 4, the effect of the 2 bp mismatches on the cyclovoltagram of the Au-S-ds- oligo-PQQ electrode is shown in Fig. 5. The potentials are each given as EE ₀ , i.e. relative to the half-wave potential.

From Fig. 4, a significantly increased current flow in case of a double-stranded oligonucleotide to the non-hybridized form can be seen. This enables sequence-specific hybridization events to be detected. It is clear from FIG. 5 that when hybridizing with a target oligonucleotide strand which has 2 base pair mismatches, on the one hand a lower current flows, on the other hand the difference in the current maxima is increased.

Claims (28)

1. A nucleic acid oligomer modified by attaching a redox-active substance, characterized in that the redox-active substance can be selectively oxidized and reduced at a potential cp, the condition being 2.0 V ≧ ϕ ≧ -2.0 V, measured against normal hydrogen electrode, enough. 2. Modified nucleic acid oligomer according to claim 1, wherein as a redox active Substance a dye is used, in particular a flavin derivative Porphyrin derivative, a chlorophyl derivative or a bacteriochlorophyl derivative. 3. Modified nucleic acid oligomer according to claim 1, wherein as a redox active Substance a quinone, especially a pyrrolo-quinoline quinone (PQQ) 1,4-benzoquinone, a 1,2-naphthoquinone, a 1,4-naphthoquinone or a 9,10- Anthraquinone is used. 4. Modified nucleic acid oligomer according to one of the preceding Claims, wherein the redox-active substance alternatively covalently to one of the Phosphoric acid, carboxylic acid or amine units to one of the sugar Units or is attached to one of the bases of the nucleic acid oligomer, in particular to a terminal unit of the nucleic acid oligomer. 5. Modified nucleic acid oligomer according to one of claims 1 to 3, wherein the redox-active substance covalently to a branched or unbranched Molecular part of any composition and chain length is attached and the branched or unbranched part of the molecule alternatively to one of the Phosphoric acid, carboxylic acid or amine units to one of the sugar Units or is attached to one of the bases of the nucleic acid oligomer, especially to a separate unit of the nucleic acid oligomer. 6. Modified nucleic acid oligomer according to claim 5, wherein the redox active Substance covalently to a branched or unbranched part of the molecule is connected, the shortest continuous connection between the connected structures comprises 1 to 14 atoms. 7. Modified nucleic acid oligomer according to one of the preceding Claims, wherein the modified nucleic acid oligomer is sequence specific Single-stranded DNA, RNA and / or PNA can bind.   8. A modified nucleic acid oligomer according to claim 7, wherein the modified Nucleic acid oligomer is a deoxyribonucleic acid, ribonucleic acid Peptide nucleic acid oligomer or a nucleic acid oligomer with structural analog backbone. 9. A method for producing a modified nucleic acid oligomer according to claims 1 to 4, characterized in that the redox-active substance is bound to a nucleic acid oligomer, the binding to a Phosphoric acid or carboxylic acid group of the nucleic acid oligomer Amide formation with (primary or secondary) an amino group of the redox-active Substance, by ester formation with a (primary, secondary or tertiary) Alcohol group of the redox-active substance, or by thioester formation with a (primary, secondary or tertiary) thio alcohol group of redox-active substance or by condensation of an amine group of Nucleic acid oligomers with an aldehyde group of the redox-active substance he follows. 10. The method for producing a modified nucleic acid oligomer according to claims 5 to 8, characterized in that the redox active Any substance to a branched or unbranched part of the molecule Composition and chain length is covalently attached, the Connection to a phosphoric acid or carboxylic acid group of the branched or unbranched part of the molecule through amide formation with (primary or secondary) an amino group of the redox-active substance, by ester formation with a (primary, secondary or tertiary) alcohol group redox-active substance, or by thioester formation with a (primary, secondary or tertiary) thio-alcohol group of the redox-active substance or by condensation of an amine group of the branched or unbranched part of the molecule with an aldehyde group of the redox-active Substance occurs. 11. Modified conductive surface, characterized in that one or several types of modified nucleic acid oligomers according to the Claims 1 to 8 are connected to a conductive surface. 12. Modified conductive surface according to claim 11, wherein the surface of a metal or a metal alloy, in particular a metal selected from the group platinum, palladium, gold, cadmium, mercury,  Nickel, zinc, carbon, silver, copper, iron, lead, aluminum, manganese and their mixtures. 13. Modified conductive surface according to claim 11, wherein the surface of a semiconductor, in particular a semiconductor selected from the Group carbon, silicon, germanium and α-tin. 14. Modified conductive surface according to claim 11, wherein the surface of a binary connection of the elements of groups 14 and 16, a binary Connection of the elements of groups 13 and 15, a binary connection the elements of groups 15 and 16, or a binary combination of the Elements of groups 11 and 17 consists, in particular, of a Cu (I) - Halide or an Ag (I) halide. 15. Modified conductive surface according to claim 11, wherein the surface of a ternary combination of the elements of groups 11, 13 and 16 or one ternary compound elements of groups 12, 13 and 16. 16. Modified conductive surface according to claims 11 to 15, wherein the modified nucleic acid oligomers to the conductive surface covalently or are bound by physisorption. 17. Modified conductive surface according to claim 16, wherein alternatively one of the Phosphoric acid, carboxylic acid or amine units, one of the sugar units or one of the bases of the nucleic acid oligomer covalently or through Physisorption is bound to the conductive surface, especially one terminal unit of the nucleic acid oligomer. 18. Modified conductive surface according to one of claims 11 to 15, wherein branched or unbranched parts of the molecule on the conductive surface any composition and chain length covalently or by Physisorption are linked and the modified nucleic acid oligomers are covalently attached to these molecular parts. 19. Modified conductive surface according to claim 18, wherein the branched or unbranched part of the molecule a shortest continuous connection between the connected structures comprises from 1 to 14 atoms.   20. Modified conductive surface according to claims 18 and 19, wherein the branched or unbranched part of the molecule alternatively to one of the phosphoric acid Carboxylic acid or amine units, to one of the sugar units or to one the bases of the nucleic acid oligomer is covalently bound, in particular to a terminal unit of the nucleic acid oligomer. 21. Process for producing a modified conductive surface according to Claims 11 to 20, wherein one or more types of modified Nucleic acid oligomers according to claims 1 to 8 on a conductive Surface to be applied. 22. A method for producing a modified conductive surface according to Claims 11 to 20, wherein one or more types of nucleic acid Oligomers are bound to a conductive surface and exclusively the nucleic acid oligomers bound to the conductive surface Linking a redox-active substance to the nucleic acid oligomers be modified. 23. Method for producing a modified conductive surface according to Claim 22, wherein the binding of the redox-active substance to the Nucleic acid oligomer by reaction of the redox-active substance with a Phosphoric acid unit, a sugar unit or one of the bases of the Nucleic acid oligomers takes place, in particular by reaction with a terminal unit of the nucleic acid oligomer. 24. A method for manufacturing a modified conductive surface according to Claim 22, wherein the redox-active substance is covalently attached to a branch or unbranched molecular part of any composition and chain length is attached and the branched or unbranched part of the molecule alternatively to one of the phosphoric acid, carboxylic acid or amine units, to one of the Sugar units or to one of the bases of the nucleic acid oligomer is attached, in particular to a terminal unit of the nucleic acid Oligomers. 25. Process for producing a modified conductive surface according to Claims 21 to 24, wherein the nucleic acid oligomer or the modified Nucleic acid oligomer with the complementary nucleic acid Oligomer strand is hybridized and in the form of the double strand hybrid on the conductive surface is applied.   26. Process for producing a modified conductive surface according to Claims 21 to 25, wherein the nucleic acid oligomer or the modified Nucleic acid oligomer in the presence of other chemical compounds, which are also attached to the conductive surface on which conductive surface is applied. 27. Method for the electrochemical detection of nucleic acid oligomer Hybridization events, characterized in that a conductive Surface as defined in claims 11 to 20 with nucleic acid Oligomers are contacted with nucleic acid oligomers be brought and then a detection of the electrical Communication between the redox-active unit and the respective one conductive surface. 28. The method according to claim 27, wherein the detection is cyclovoltametric, done amperometrically or by conductivity measurement.