WO2001021635A2

WO2001021635A2 - Method for electrochemically detecting sequence-specific nucleic acid-oligomer hybridisation events

Info

Publication number: WO2001021635A2
Application number: PCT/DE2000/003016
Authority: WO
Inventors: Gerhard Hartwich
Original assignee: Friz Biochem Gmbh
Priority date: 1999-09-22
Filing date: 2000-09-01
Publication date: 2001-03-29
Also published as: WO2001021635A3; EP1228081A1; DE19945398A1; AU1128501A

Abstract

The present invention relates to a method for electrochemically detecting sequence-specific nucleic acid-oligomer hybridisation events. DNA-/RNA-/PNA oligomer single strands which are bound to a conductive surface at one end and are linked to a catalytic redoxactive unit at the remaining, free end serve as a hybridisation matrix (probe). A proportion of the single strand oligonucleotides are hybridised by means of a treatment with the oligonucleotide solution (target) that is to be examined. The electric communication between the conductive surface and the catalytic redoxactive unit, which is initially non- or barely existent, is increased. A hybridisation event can thus be detected using electrochemical methods such as voltametry, amperometry, potentiometry or conductivity measurement.

Description

Electrochemical detection of sequence-specific nucleic acid oligomer

Hybridization events.

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, in toxicological test methods, in genetic research and development, and in the agricultural and pharmaceutical sector, gel electrophoretic methods with autoradiographic or optical detection are generally used.

In the most important gel-electrophoretic method with optical detection, the Sanger method, a DNA-containing solution is divided into four batches. To distinguish the four approaches, the primer (complementary start sequence for replication) of each approach is covalently modified with a fluorescent dye that emits at different wavelengths. Starting from the primer, each batch is replicated enzymatically by DNA polymerase I. In addition to the necessary deoxyribonucleoside triphosphates of bases A (adenine), T (thymine), C (cytosine) and G (guanine), each reaction mixture contains enough 2 ', 3'-dideoxy analogue of one of these nucleoside triphosphates as a stop base (one of each 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 the length and characterized by fluorescence spectroscopy.

Another optical detection method is based on the attachment of fluorescent dyes such. B. Ethidium bromide on oligonucleotides. In comparison to the free solution of the dye, the fluorescence of such dyes changes drastically when associated with double-stranded DNA or RNA and can therefore be used to detect hybridized DNA or RNA. 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 on 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), S. 114-128 or Bains et al., Theor. Biol. 135, (1988), pp. 303-307). In this method, a complete set of short oligonucleotides or nucleic acid oligomers (probe oligonucleotides), e.g. B. all 65536 possible combinations of bases A, T, C and G of an oligonucleotide octamer are bound to a support material. The connection is made in an ordered grid of 65536 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 ³² P) and hybridized under conditions which only permit specific double-strand formation. As a result, the target DNA fragment only binds to the nucleic acid 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 nucleic acid oligomer sequences (octamer sequences) present in the fragment are thus determined. Due to the overlap of neighboring nucleic acid 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. 1 for a 13 base long DNA fragment. In addition to the so-called de-novo sequencing of previously unknown oligonucleotide sequences, oligonucleotide sequences or DNA fragments which code one or more known genes can also be bound on the oligomer chip described above. So z. B. for each gene of known base sequence searched a sufficient number of oligonucleotide sequences from z. B. 20 bases each, which are complementary to corresponding sequence sections of the gene sought with a known base sequence, are applied to a support material in order to detect this gene with a very high probability. On the other hand, known genes can also be tested for mutations on an oligomer chip, for example by B. the corresponding sequence segments of the genes with and without mutation can be applied to the carrier material.

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 1mm ² ) and a sensitivity that is only high if the radiation of the radioactive fragments for a correspondingly long (hours to days) X-ray film acts. 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, which are usually used to label the DNA, are partly mutagenic (e.g. ethidium bromide) and, like the use of autoradiography, require corresponding safety precautions. In almost all cases, the use of optical detection requires the use of one or more laser systems and thus trained personnel and appropriate safety precautions. The actual detection of fluorescence requires additional hardware, such as. B. optical components for amplification and, at different excitation and interrogation wavelengths as in the Sanger process, a control system. Depending on the required excitation wavelengths and the desired detection power, considerable investment costs can arise. In the case of sequencing by hybridization on the oligomer chip, the detection is even more (expensive) because, in addition to the excitation system, high-resolution CCD cameras (charge coupled device cameras) are required for the 2-dimensional detection of the fluorescence spots.

So, although there are quantitative and extremely sensitive methods for DNA / RNA sequencing, these methods are time-consuming and time-consuming Sample preparation and expensive equipment and are generally not available as portable systems.

Presentation of the invention

The object of the present invention is therefore to create a device and a method for the detection of nucleic acid-oligomer hybrids which do not have the disadvantages of the prior art.

This object is achieved according to the invention by the modified nucleic acid oligomer according to independent patent claim 1, by the method for producing a modified nucleic acid oligomer according to independent claim 22, by the modified conductive surface according to independent patent claim 34, by the method for producing a modified conductive surface according to independent patent claim 34 Claim 49 and a method for the electrochemical detection of nucleic acid-oligomer hybridization events according to independent claim 53.

The following abbreviations and terms are used in the context of the present invention:

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 and uracil

Base A, G, T, C or U Bp base pair

Nucleic acid at least two covalently linked nucleotides or at least two covalently linked pyrimidine (e.g. cytosine, thymine or uracil) or purine bases (e.g. adenine or guanine). The term nucleic acid refers to any "backbone" of the covalently linked pyrimidine or purine bases, such as. B. on the sugar-phosphate backbone of the DNA, cDNA or RNA, on a peptide backbone of the PNA or on analogous structures (e.g. phosphoramide, thio-phosphate or dithio-phosphate backbone). An essential feature of a nucleic acid in the sense of the present invention is that it can bind naturally occurring cDNA or RNA in a sequence-specific manner.

Nucleic acid - nucleic acid of unspecified base length (e.g. oligomeric nucleic acid octamer: a nucleic acid with any backbone in which 8 pyrimidine or purine bases are covalently bonded to one another).

Oligomer equivalent to nucleic acid oligomer.

Oligonucleotide equivalent to oligomer or nucleic acid oligomer, e.g. B. a DNA, PNA or RNA fragment unspecified base length.

Oligo Abbreviation for 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".

SS Single Strand ds double Strand redox-active unit corresponds to a catalytically redox-active unit catalytically a unit referred to in the context of the present invention under the redox-active unit generic term "catalytically redox-active unit" consists of one or more redox-active centers (cofactors, prosthetic groups), hereinafter referred to as electron donors or electron acceptors, and one or more macromolecules binding these redox-active centers. The catalytically redox-active unit thus contains one or more electron-donor molecules and / or one or more electron-acceptor molecules in their appearance-relevant form, whereby this electron-donor molecule (s) and / or this (these) Electron acceptor molecule (s) is / is bound to one or more macromolecules or is embedded in this (s) macromolecule (s). Electron donor (s) and / or electron acceptor (s) can be mutually linked by one or more covalent or ionic bonds, by hydrogen bonds, by van der Waals bridges, by π-π interaction or by coordination be connected to one another by means of electron pair donation and acceptance, it being possible for covalent compounds to be direct or indirect (eg via a spacer but not via a nucleic acid oligomer) compounds. In addition, the electron donor (s) and / or electron acceptor (s) with the macromolecule (s) by covalent attachment to the macromolecule (s), by encapsulation in suitable molecular cavities (binding pockets) of the Macromolecule (of the macromolecules), by ionic bonds, hydrogen bonds, van der Waals bridges, π-π interaction or by coordination by means of electron pair donation and acceptance between the macromolecule (s) and the (n) electron donor molecule (s) and / or the electron acceptor molecule (s). If several macromolecules are part of the catalytically redox-active unit, the binding of the macromolecules to one another can also be covalent, ionic, through hydrogen bonds, van-der-Waals bonds, π-π- Interaction or by coordination by means of electron pair donation and acceptance. In a minimal case, a catalytically redox-active unit can also consist of only one macromolecule, the macromolecule then also acting as an electron donor or acceptor in its manifestation relevant to the invention. It can also consist of only one electron donor or acceptor. In addition, the catalytically redox-active unit can also be formed by spontaneously assembling the component in solution (in situ). In addition to the composition of electron donor (s) and / or electron acceptor (s) and macromolecule (s), essential features of the catalytically redox-active unit are: (i) the unit is in the manifestations relevant to the invention (electron donor (s) and / or electron acceptor (s) in the original or oxidized or reduced state) stable and does not dissociate into its components, (ii) the electrocatalytic activity of the unit (see below), (iii) the unit contains no nucleic acid, (iv) The composition of the unit consisting of electron donor (s) and / or electron acceptor (s) and / or macromolecule (s) can be recognized by a person skilled in the art - regardless of the bond between the components - since the redox-active centers (cofactors, prosthetic Groups) and the associated matrix of macromolecule (s) (e.g. apoprotein in the case of enzymes as an example of a catalytically redox-active unit) can in principle also occur separately from one another. The catalytically redox-active unit can, for. B. any redox-active protein / enzyme from the group of oxidases or reductases, proteins / enzymes from this group of oxidases or reductases modified by protein engineering or gene mutation or an artificially produced unit from one or more redox-active centers (electron donor or . Acceptor) or an artificially produced unit from one or more redox-active centers (electron donor or acceptor) and one or more macromolecules binding these redox-active centers.

Cofactor corresponds to a redox-active center (electron donor or acceptor) of the catalytically redox-active unit prosthetic group corresponds to a redox-active center (electron donor or acceptor) of the catalytically redox-active unit, the redox-active center, the redox-active center of the catalytically redox-active unit, the center, which is distinguished by the fact that it acts as a redox-active unit as a redox-active unit compared to a substrate specific for the catalytically, Donor or acceptor acts. If a catalytically redox-active unit has several redox-active centers (electron donors and / or electron acceptors), a charge transfer can also occur within the catalytically redox-active unit: after the charge transfer there is between the substrate specific for the catalytically redox-active unit and a first redox-active center a further charge transfer between this first redox-active center and a further redox-active center of the same catalytically redox-active unit is possible, this second redox-active center in turn being able to transfer charge to a third redox-active center, etc. So there can be a successive charge transfer within the catalytically redox-active unit, if the catalytically redox-active unit contains several redox-active centers. The process of successive charge transfer is initiated by the presence of the substrate specific for the catalytically redox-active unit (with its property of spontaneously transferring a charge between the substrate and the catalytically redox-active unit).

Electron donor- corresponds to an electron donor. molecule

Elekron acceptor corresponds to an electron acceptor. molecule

Electron donor In the context of the present invention, the term electron donor denotes a component of the catalytically redox-active unit. An electron donor is a molecule that can transfer an electron to an electron acceptor immediately or after exposure to certain external circumstances. Such an external circumstance is e.g. B. the oxidation or reduction of the electron donor or - acceptor of the catalytically redox-active unit by a external oxidizing or reducing agent, so z. B. the transfer of an electron to the electron donor by a reducing agent or the release of an electron by the electron acceptor to an oxidizing agent. These oxidizing or reducing agents can be external redox-active substances, ie they are not covalently linked to the catalytically redox-active unit, the nucleic acid oligomer or the conductive surface, but are associated with these, e.g. B. via the solution added to the modified conductive surface, in contact, in particular the substrates specific for the catalytically redox-active unit can act as external oxidizing or reducing agents. In addition, an external oxidizing or reducing agent can also be covalently linked to the nucleic acid oligomer, the oxidizing or reducing agent being covalently linked to a point on the nucleic acid oligomer that contains at least two covalently linked nucleotides or at least two covalently linked pyrimidine or Purine bases is removed from the covalent attachment site of the redox-active unit, preferably at the end of the oligonucleotide opposite the modification with the catalytically redox-active unit in the vicinity of the conductive surface. In particular, the conductive surface (electrode) can also act as an external oxidizing or reducing agent. The ability to act as an electron donor or acceptor is relative, ie a molecule which acts as an electron donor immediately or after the action of certain external circumstances can act against this molecule under different experimental conditions or against a third molecule same or different experimental conditions also act as an electron acceptor.

Electron acceptor In the context of the present invention, the term electron acceptor denotes a component of a catalytically redox-active unit. An electron acceptor is a molecule that can accept an electron from an electron donor immediately or after exposure to certain external circumstances. Such an outside Circumstance is z. B. the oxidation or reduction of the electron donor or acceptor of the catalytically redox-active unit by an external oxidizing or reducing agent, so z. B. the transfer of an electron to the electron donor by a reducing agent or the release of an electron by the electron acceptor to an oxidizing agent. These oxidizing or reducing agents can be external redox-active substances, ie they are not covalently linked to the catalytically redox-active unit, the nucleic acid oligomer or the conductive surface, but are associated with these, e.g. B. via the solution added to the modified conductive surface, in contact, in particular the substrates specific for the catalytically redox-active unit can act as external oxidizing or reducing agents. In addition, an external oxidizing or reducing agent can also be covalently linked to the nucleic acid oligomer, the oxidizing or reducing agent being covalently linked to a point on the nucleic acid oligomer that contains at least two covalently linked nucleotides or at least two covalently linked pyrimidine or Purine bases is removed from the covalent attachment site of the catalytically redox-active unit, preferably at the end of the oligonucleotide opposite the modification with the redox-active unit in the vicinity of the conductive surface. In particular, the conductive surface (electrode) can also act as an external oxidizing or reducing agent. The ability to act as an electron acceptor or donor is relative, ie a molecule which acts as an electron acceptor immediately or after the action of certain external circumstances can act against this molecule under different experimental conditions or against a third molecule same or different experimental conditions also act as an electron donor.

Oxidizing agent chemical compound (chemical substance), which by taking up electrons from another chemical compound (chemical substance, electron donor, electron acceptor) this other chemical compound (chemical Substance, electron donor, electron acceptor) oxidized. An oxidizing agent behaves analogously to an electron acceptor, but is used in the context of the present invention as a term for an external electron acceptor that does not directly belong to the catalytically redox-active unit. In this context, not directly means that the oxidizing agent is either a substrate specific for the catalytically redox-active unit or a free redox-active substance which is not bound to the nucleic acid oligomer but is in contact with it. In addition, the oxidizing agent can be covalently linked to the nucleic acid oligomer, but at a point on the nucleic acid oligomer that is at least two covalently linked nucleotides or at least two covalently linked pyrimidine or purine bases from the covalent linkage point of the catalytically redox-active unit. In particular, the electrode can also be the oxidizing agent.

Reducing agent chemical compound (chemical substance) which, by donating electrons to another chemical compound (chemical substance, electron donor, electron acceptor), reduces this other chemical compound (chemical substance, electron donor, electron acceptor). A reducing agent behaves analogously to an electron donor, but is used in the context of the present invention as a term for an external electron donor that does not directly belong to the catalytically redox-active unit. In this context, not directly means that the reducing agent is either a substrate specific for the catalytically redox-active unit or a free redox-active substance which is not bound to the nucleic acid oligomer but is in contact therewith or that the reducing agent is covalently to the nucleic acid - The oligomer is attached, but at a point on the nucleic acid oligomer which is at least two covalently linked nucleotides or at least two covalently linked pyrimidine or purine bases from the covalent linkage site of the redox-active unit. In particular, the electrode can also represent the reducing agent. redox active redox active denotes the property of a redox active unit to give electrons to a suitable oxidizing agent under certain external circumstances or to take up electrons from a suitable reducing agent or the property of a redox active substance to give electrons to a suitable electron acceptor under certain external circumstances or from a suitable electron To accept donor electrons.

Analyte corresponds to a substrate

Substrate free, not covalently with the catalytically redox-active unit, the nucleic acid oligomer or the conductive surface composite, but with these, e.g. B. on the modified conductive surface added solution, contacting oxidizing or reducing agent, the substrate z. B. can be a charged or uncharged molecule, any salt, an ion or a redox-active protein or enzyme (oxidoreductase). The substrate is characterized in that it is recognized by the catalytically redox-active unit through the formation of specific interactions between the substrate and the catalytically redox-active unit and can reduce (or oxidize) the donor (or the acceptor) of the catalytically redox-active unit, the catalytic Activity of the catalytically redox-active unit accelerates (catalyzes) this redox reaction of the substrate to the product. Catalytic activity The catalytic activity of the catalytically redox-active unit has an accelerating effect on the specific reaction between the unit and the associated substrate and thus enables a reaction sequence which does not take place or only to an imperceptible extent without the catalytic activity of the unit. This catalytic activity of the redox-active unit is achieved by stabilizing the respective transition state, ie the most energetic species, in the course of the reaction between the catalytically redox-active unit and the associated substrate. electrocatalytic the electrocatalytic activity of the catalytic redox active activity is closely related to the catalytic activity of the Unit. Due to the presence of the catalytically redox-active unit and its integration into the reaction sequence of the electrode reaction of the substrate to the product (sequence of the overall reaction of the electrochemical redox reaction between an electrode and the substrate, ie the release of electrons from the electrode to the substrate or the release of electrons from the substrate to the electrode, via the intermediate stages of redox reaction between substrate and catalytically redox-active unit and redox reaction between redox-active unit and electrode), the electro-chemical conversion of the substrate at the electrode is accelerated. The electrocatalytic activity of a catalytically redox-active unit immobilized on an electrode reduces the activation energy of the electrode reaction of the substrate to the product (energy of the most energetic state for the reaction sequence of the conversion of the substrate into the product at the electrode) and thereby leads to a shift in the for the electrode reaction of the Substrate to the product necessary electrode potential in the direction of the equilibrium potential for these electrodes reaction. The lowering of the activation potential leads to a reduction in the overvoltage necessary for an electrode reaction and thus to an increase in the electron foot between the electrode and the substrate at a certain electrode potential suitable for the electrode reaction (this increase is generally referred to as catalytic current). The main consequence of the electrocatalytic activity is that the electrochemical conversion of the substrate into the product can be carried out in the presence and with the participation of the catalytically redox-active unit at an electrode potential at which no or only very little current flows in the absence of the catalytically redox-active unit.

The specificity of the catalytically redox-active unit acts specifically both with regard to the catalytically active redox-active unit substrate interacting with the catalytically redox-active unit and with regard to the reaction carried out with the respective substrate. In the context of the present invention, redox reactions are the preferred reactions between catalytically redox-active unit and substrate. Initiation process with appropriately chosen external circumstances, the catalytically redox-active unit unfolds its redox activity, ie its property, e.g. B. to give electrons to a suitable oxidizing agent or to take up electrons from a suitable reducing agent, only after an initiation process. Such an initiation process can be the transfer of substrate with its property charge to the catalytically redox-active unit: The reductive property of a catalytically redox-active unit is only through the transfer of electron (s) from the substrate to / an electron donor "D "enables, either in the presence of an oxidizing agent that can oxidize D ^" but not D, or because after successive charge transfer within the catalytically redox-active unit, the electron is transferred from D ^~ to an acceptor "A" (directly or via several electron transfer steps to intermediate Electron acceptors) and an oxidizing agent is present which only accepts electrons from this reduced acceptor "A ^~ " of the catalytically redox-active unit, but not from A. In particular, this oxidizing agent can also be an electrode, for example if the electrode is on an Potential is set at which D ^" (or A ^" ), but not D (or A), ox is identified. On the other hand, the oxidative property of a catalytically redox-active unit is only made possible by the transfer of electron (s) from an electron donor "D" to the substrate, either in the presence of a reducing agent that can reduce D ⁺ but not D or because after successive charge transfer within the catalytically redox-active unit, an electron is transferred from an acceptor "A" to the oxidized donor D ⁺ (directly or via several electron transfer steps of intermediate electron donors) and a reducing agent is present which is only present on this oxidized acceptor "A ⁺ " catalytically redox-active unit emits electrons, but not to A. In particular, this reducing agent can also be an electrode, for. B. if the electrode is set to a potential at which D ⁺ (or A ⁺ ), but not D (or A), is reduced.

Redox active usually consists of so-called apoprotein, which (the)

(Formula 3)

Heme Fe protoporphyrin IX, (Formula 4 with R ₂ = R ₅ = R ₈ = R ₁₀ = H; R ₄ ⁼ Rg ⁼ Rg ⁼ Rι ₂ ⁼ CH ₃ | Ri ⁼ R ₃ ⁼ CH -CH -CO; R ₇ ⁼ Rg ⁼ CH = CH ₂ or a derivative of Fe protoporphyrin (formula 4)

N ⁶ - (2-aminoethyl) - modified flavin adenine dinucleotide, cf. Formula 5 FAD

N ⁶ - (2-aminoethyl) - modified nicotinamide adenine dinucleotide, cf. Formula 6 NAD ⁺

EDTA ethylenediamine tetraacetate (sodium salt) sulfo-NHS N-hydroxysulfosuccinimide

EDC (3-dimethylaminopropyl) carbodiimide

HEPES N- [2-hydroxyethyl] piperazine-N '- [2-ethanesulfonic acid]

Tris tris (hydroxymethyl) aminomethane

Alkyl The term "alkyl" denotes a saturated hydrocarbon group that is straight-chain or branched (eg ethyl, 2,5-dimethylhexyl or isopropyl 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 C (CH ₃ ) ₂ CH ₂ CH ₂ C (CH ₃ ) ₂ CH ₂ - or -CH ₂ CH ₂ CH ₂ - etc.). Preferred alkyl groups as substituents or side chains R are those of chain length 1-30 (longest continuous chain of atoms covalently bonded to one another). Preferred alkyl groups as linkers or spacers 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 connected by the linker or spacer, that is to say between the two molecules or between a surface atom, Surface molecule or a surface molecule group and another molecule.

Alkenyl alkyl groups in which one or more of the C-C 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 by C≡C Triple bonds are replaced.

Hetero-alkyl alkyl groups in which one or more of the C-H bonds or C-C single bonds are replaced by C-N, C = N, C-P, C = P, C-O, C = O, C-S or C = S bonds.

Hetero-alkenyl alkenyl groups in which one or more C-H bonds, C-C single or C = C double bonds are replaced by C-N, C = N, C-P, C = P, C-O, C = O, C-S 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 by CN, C = N, CP, C = P, CO, C = O, CS or C = S bonds are replaced.

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, heteroalkyl, heteroalkenyl or heteroalkynyl chains, the chain being derivatized at two points with (identical or different) reactive groups. These groups form a covalent chemical bond in simple / known chemical reactions with the corresponding reaction partners. The reactive groups can also be photoactivatable, i. H. 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, i.e. between the two molecules or between a surface atom, surface molecule or a surface molecule group and another molecule , represents.

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 1-20, in particular chain length 1-14, the chain length being the shortest continuous connection between the structures to be connected.

(nx HS spacer) - Nucleic acid oligomer to which n thiol functions are linked via a spacer via oligo, the spacers each having a different chain length (shortest continuous connection between thiol function and nucleic acid oligomer), in particular in each case any 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 x R-S-S spacer) - Nucleic acid oligomer to which n disulfide functions are each connected via a spacer via oligo, 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 the disulfide function and nucleic acid oligomer), in particular any chain length between 1 and 14. These spacers can in turn be connected to various naturally on the nucleic acid Oligomeric reactive groups attached or attached to this by modification. The placeholder n is any integer, in particular a number between 1 and 20.

Oligo-Spacer-SS- two identical or different nucleic acid oligomers that are connected to each other via a disulfide bridge, the disulfide bridge being connected to the nucleic acid oligomers via any two spacers and the two spacers having a different chain length (shortest can have a 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.

Mica muscovite platelets, carrier material for thin application Layers.

Au-S- (CH ^ ₂ -ss-oligo- gold film on mica with covalently applied monolayer

Spacer-PQQ-derivatized 12 bp single strand DNA oligonucleotide (sequence:

FAD (GOx) TAGTCGGAAGCA). 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. The terminal base thymine at the 5 'end of the oligonucleotide is modified at the C-5 carbon with -CH = CH-CO-NH-CH ₂ -CH ₂ -NH ₂ , this residue in turn via its free amino group by amide formation with the carboxylic acid group of the PQQ is connected. To another carboxylic acid group of this PQQ, FAD is bound via amide formation, which was previously modified so that it has a reactive amino group, e.g. B. by formation of N ⁶ - (2-aminoethyl) -FAD (Bückmann et al, 1991, European Patent 0.247.537. B1). The FAD is then reconstituted with the apoprotein of the GOx, so that a nucleic acid oligomer covalently attached to the surface is formed, which is additionally - via PQQ as a covalently attached bridge molecule - covalently modified with the complete GOx unit.

Au-S-ζCHd _∑ -ds-oligo-Au-S-fCH ^ ss-oligo-spacer-PQQ-FADfGOx) hybridizes with spacer-PQQ-dem to ss-oligo (sequence: TAGTCGGAAGCA) FAD (GOx) complementary oligonucleotide.

Au-S-fCHJrSS-oligo-gold film on mica with covalently applied monolayer

Spacer-PQQ-NAD ⁺ - derivatized 12 bp single strand DNA oligonucleotide (sequence:

LDH TAGTCGGAAGCA). 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. The terminal base thymine at the 5 'end of the oligonucleotide is modified at the C-5 carbon with -CH = CH-CO-NH-CH ₂ -CH ₂ -NH ₂ , this residue in turn via its free amino group by amide formation with the carboxylic acid group of the PQQ is connected. To another carboxylic acid group of this PQQ, NAD ^{+ is} bound via amide formation, which was previously modified so that it has a reactive amino group, e.g. B. by forming N ⁶ - (2-Aminoethyl) -NAD ⁺ (Bückmann et al, 1991, European Patent 0.247.537.B1). The complete LDH is associated with this terminal NAD ⁺ .

Au-S- (CH ^ ₂ -ds-oligo-Au-S- (CH ^ ₂ -ss-oligo-spacer-PQQ-NAD ⁺ -ADH hybridizes with

Spacer-PQQ-NAD ⁺ - the to ss-oligo (sequence: TAGTCGGAAGCA)

LDH complementary oligonucleotide.

E Electrode potential that is applied to the working electrode.

Equilibrium potential of an electrode reaction

E ° "zero current" potential of an electrode reaction, potential that does not supply a total current (sum of oxidative and reductive current) for a specific electrode reaction. η Overpotential of an electrode reaction

Electrode reaction Redox reaction between a redox-active substance and an electrode (absorption of electrons from the electrode by the redox-active substance or release of electrons from the redox-active substance to the electrode)

Eθx potential at the current maximum of the oxidation of a reversible electro-oxidation or reduction.

^ Red potential at the current maximum of the reduction of a reversible electro-oxidation or reduction.

/ Current density (current per cm ² electrode surface)

Cyclic voltammetry 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 is the potential 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.

Potentiometry Recording an electrode voltage curve depending on the z. B. the substrate consumption. Here, for. B. the potential of a stationary working electrode to the "zero current" potential £ ° of the substrate. When the substrate is used up by the catalytically redox-active unit (in the case of hybridization), the "zero current" potential E ° changes in the direction of the equilibrium potential. = *. Recording the potential as a function of time (~ substrate consumption) thus provides information about the hybridization state.

The present invention relates to a nucleic acid oligomer which is modified by chemical bonding of a catalytically redox-active unit. The catalytically redox-active unit can, after donating an electron to an external oxidizing agent (substrate) from an external reducing agent, e.g. B. an electrode, reduced or after receiving an electron from an external reducing agent (substrate) by an external oxidizing agent, for. B. an electrode, are oxidized.

In the context of the present invention, a nucleic acid oligomer is a compound of at least two covalently linked nucleotides or of 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, is used. In the present invention, 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 backbone structures, such as. B. a thio-phosphate, a dithio-phosphate or a phosphoramide 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. As an alternative to the term “nucleic acid oligomer”, the terms “(probe) oligonucleotide”, “nucleic acid” or “oligomer” are used. The term "electron acceptor" or "electron acceptor molecule" and the term "electron donor" or "electron donor molecule" in the context of the present invention denotes a component (a redox-active center or a cofactor or a prosthetic group) of a catalytically redox-active unit.

A unit referred to in the context of the present invention under the generic term “catalytically redox-active unit” generally consists of one or more redox-active centers (cofactors, prosthetic groups), which are referred to below as electron donors or electron acceptors, and one or more macromolecules binding these redox-active centers. The catalytically redox-active unit therefore contains one or more electron-donor molecules and / or one or more electron-acceptor molecules in their appearance-relevant form, whereby this electron-donor molecule (s) and / or this (these) Electron acceptor molecule (s) are bound to one or more macromolecules or are embedded in this (s) macromolecule (s). Electron donors and / or electron acceptors can be linked to one another by one or more covalent or ionic bonds, by hydrogen bonds, by van der Waals bridges, by π-π interaction or by coordination by means of an electron pair - Donation and - Acceptance can be linked to one another, where covalent compounds can be direct or indirect (eg via a spacer but not via a nucleic acid oligomer) compounds. In addition, the electron donor (s) and / or electron acceptor (s) with the macromolecule (s) by covalent attachment to the macromolecule (s), by encapsulation in suitable molecular cavities (binding pockets) of the Macromolecule (of the macromolecules), by ionic bonds, hydrogen bonds, van der Waals bridges, π-π interaction or by coordination by means of electron pair donation and - acceptance between the macromolecule (s) and the (n) electron donor molecule (s) and / or the electron acceptor molecule (s). If several macromolecules are part of the catalytically redox-active unit, the macromolecules can also be bonded to one another covalently, ionically, by hydrogen bonds, van der Waals bridges, π-π interaction or by coordination by means of electron pair donation and acceptance , In a minimal case, a catalytically redox-active unit can also consist of only one macromolecule, the macromolecule then also acting as an electron donor or acceptor in its manifestation relevant to the invention. It can also consist of only one electron donor or acceptor. In addition, the catalytically redox-active unit can also be formed by spontaneously assembling the component in solution (in situ). The addressed donor and / or acceptor molecules, together with the macromolecules, form a catalytically redox-active unit, ie they are bound to one another directly or via further parts of the molecule. The only restriction of the molecules or parts of molecules connecting the components of the catalytically redox-active unit is the exclusion of nucleic acid oligomers. According to the present invention, the catalytically redox-active unit is bound as a complete unit to the probe oligonucleotide, it being possible, of course, to form several chemical bonds between the oligonucleotide and the redox-active unit. The exclusion of nucleic acid oligomers as the molecules or parts of molecules connecting the constituents of the catalytically redox-active unit is intended to clarify that individual parts of the catalytically redox-active unit are not linked to different locations of the probe oligonucleotide. The probe oligonucleotide therefore explicitly does not represent the connection between the electron donor molecule (s) and the macromolecules and / or the electron acceptor molecule (s) and the macromolecules of the catalytically redox-active unit.

The redox activity of the catalytically redox-active unit, that is to say its property, under certain external circumstances, to give off electrons to a suitable oxidizing agent (or to take up electrons from a suitable reducing agent) is initiated by an initiation process, e.g. B. only unfolded after reduction (or after oxidation) by the substrate. If the external circumstances are selected accordingly, the catalytically redox-active unit only develops its redox activity after the initiation process "adding substrate with the property of transferring charge to the catalytically redox-active unit": the reductive property of a catalytically redox-active unit is only achieved by the transfer of electrons ) from the substrate to the electron donor "D", either in the presence of an external oxidizing agent (e.g. the electrode with a correspondingly selected potential), which can oxidize D ^~ , but not D, or because after successive charge transfer within the catalytically redox-active unit the electron is transferred from D ^~ to an acceptor "A" (directly or via several electron transfer steps to intermediate electron acceptors) and an oxidizing agent is present which only electrons from this reduced acceptor "A ^~ " of the catalytically redox-active unit records ^" , but not from A ( e.g. in the presence of an electrode with an appropriately selected potential). On the other hand, the oxidative property of a catalytically redox-active unit is only made possible by the transfer of electron (s) from an electron donor "D" to the substrate, either in the presence of a reducing agent (e.g. the electrode with the correspondingly chosen potential), the D ⁺ , but cannot reduce D or because after successive charge transfer within the catalytically redox-active unit, an electron from an acceptor "A" to the oxidized donor D ⁺ is transferred (directly or via several electron transfer steps from intermediate electron donors) and a reducing agent is present which only gives off electrons to this oxidized acceptor "A ⁺ " of the catalytically redox-active unit, but not to A (e.g. in the presence of an electrode with correspondingly chosen potential).

In addition to the composition of electron donor (s) and / or electron acceptor (s) and macromolecule (s), essential features of the catalytically redox-active unit are: (i) the unit is in the manifestations relevant to the invention (electron donor (s) and / or electron acceptor (s and macromolecule (s)) in the original or oxidized or reduced state) stable and does not dissociate into their components, (ii) the electrocatalytic activity of the unit (see below), (iii) the unit does not contain any Nucleic acid, (iv) the composition of the unit from electron donors) and / or electron acceptor (s) and macromolecule (s) can be recognized by the person skilled in the art - regardless of the bond between the constituents - since the redox-active centers (cofactors, prosthetic groups) and the associated matrix of macromolecule (s) (e.g. apoprotein in enzymes as an example of a catalytically redox-active unit) can in principle also occur separately.

The substrate specific for a particular catalytically redox-active unit is a free compound that is not covalently bonded to the catalytically redox-active unit, the nucleic acid oligomer or the conductive surface, but with these, e.g. B. on the modified conductive surface added solution, contacting oxidation or

Reducing agent, wherein the substrate z. B. can be a charged or uncharged molecule, any salt, an ion or a redox-active protein or enzyme (oxidoreductase). The substrate is characterized in that it is recognized by the catalytically redox-active unit through the formation of specific interactions between the substrate and the catalytically redox-active unit and the donor

(or the acceptor) of the catalytically redox-active unit can be reduced (or oxidized), the catalytic activity of the catalytically redox-active unit accelerating (catalyzing) this redox reaction of the substrate to the product.

The catalytic activity of the catalytically redox-active unit has an accelerating effect on the specific reaction between the unit and the associated substrate and thus enables a reaction sequence which takes place without the catalytic activity of the unit (e.g. in the form of the substrate and the unbound cofactor in Solution) does not take place or only takes place to an imperceptible extent. This catalytic activity of the redox-active unit is achieved by stabilizing the respective transition state, ie the most energetic species in the course of the reaction between the catalytically redox-active unit and the associated substrate. The electrocatalytic activity of the catalytically redox-active unit is closely related to the catalytic activity of the unit. Due to the presence of the catalytically redox-active unit and its integration into the reaction sequence of the electrode reaction of the substrate to the product (sequence of the overall reaction of the electrochemical redox reaction between an electrode and the substrate, ie the release of electrons from the electrode to the substrate or the release of electrons from the substrate to the electrode, via the intermediate stages of redox reaction between substrate and catalytically redox-active unit and redox reaction between redox-active unit and electrode), the electro-chemical conversion of the substrate at the electrode is accelerated. The electrocatalytic activity of a catalytically redox-active unit immobilized on an electrode reduces the activation energy of the electrode reaction of the substrate to the product (energy of the most energetic state for the reaction sequence of the conversion of the substrate into the product at the electrode) and thereby leads to a shift in the for the electrode reaction of the Substrate to the product necessary electrode potential in the direction of the equilibrium potential for this electrode reaction. The lowering of the activation potential leads to a reduction in the overvoltage necessary for an electrode reaction and thus to an increase in the electron foot between the electrode and the substrate at a certain electrode potential suitable for the electrode reaction (this increase is generally referred to as catalytic current). basics

The consequence of the electrocatalytic activity is that the electrochemical conversion of the substrate into the product can be carried out in the presence and with the participation of the catalytically redox-active unit at an electrode potential at which no or only very little current flows in the absence of the catalytically redox-active unit.

The catalytically redox-active unit acts specifically both with regard to the substrate interacting with the catalytically redox-active unit and with regard to the reaction carried out with the respective substrate. In the context of the present invention, redox reactions are the preferred reactions between catalytically redox-active unit and substrate.

In the context of the present invention, the term “reducing agent” denotes a chemical compound (chemical substance) which, by donating electrons to another chemical compound (chemical substance, electron donor, electron acceptor), this other chemical compound (chemical substance , Electron donor, electron acceptor) reduced. The reducing agent behaves analogously to an electron donor, but is used in the context of the present invention as a term for an external electron donor which does not directly belong to the redox-active unit. In this context, “not immediately” means that the reducing agent is either a free redox-active substance that is not bound to the nucleic acid oligomer, but is in contact with it, or that the reducing agent is covalently bound to the nucleic acid oligomer, but at a point on the nucleic acid oligomer that connects at least two covalently Nucleotides or at least two covalently linked pyrimidine or purine bases are removed from the covalent attachment site of the redox-active unit. In particular, the electrode can represent the reducing agent.

In the context of the present invention, the term “free redox-active substance” is used to mean a free one that is not covalently linked to the redox-active unit, the nucleic acid oligomer or the conductive surface, but with these, e.g. B. on the modified conductive surface added solution, in contact oxidizing or reducing agent called, the free redox active substance z. B. can be an uncharged molecule, any salt, an ion or a redox-active protein or enzyme (oxidoreductase). The free redox-active substance is characterized in that it can reduce (or oxidize) the donor (or the acceptor) of the catalytically redox-active unit. In particular, the specific substrate of the catalytically redox-active unit is a free, redox-active substance.

The modified nucleic acid oligomer is bound directly or indirectly (via a spacer) to a conductive surface. The term “conductive surface” is understood to mean any electrically conductive surface of any thickness, in particular metallic surfaces, surfaces made of metal alloys or doped or undoped semiconductor surfaces, it being possible for all semiconductors to be used as pure substances or as mixtures. In the context of the present invention, the conductive surface can be used alone or on any carrier material, such as, for. B. glass, applied. In the context of the present invention, the term “electrode” is used as an alternative to “conductive surface”.

The term "modified conductive surface" is understood to mean a conductive surface which is modified by binding a nucleic acid oligomer modified with a catalytically redox-active unit. In the context of the present invention, the term “functionalized electrode” is used as an alternative to the term “modified conductive surface”.

According to a further aspect, the present invention relates to a method which enables the electrochemical detection of molecular structures such as e.g. B. the detection of the substrate, but especially the electrochemical detection of DNA / RNA / PNA fragments in a sample solution by sequence-specific nucleic acid oligomer Hybridization enables. The detection of hybridization events by means of electrical signals is a simple and inexpensive method and enables use on site in a battery-operated variant.

In addition, the present invention provides a readout method for the detection of molecular structures, among other things for the parallel detection of hybridization events on an oligomer chip by reading out electrical signals in a microelectrode array. According to the invention, a read-out method that can be controlled via microelectrodes is understood to mean a method in which the detection of molecular structures on a specific electrode within the electrode array functionalized with catalytically redox-active units by electrical control of this electrode, eg. B. is achieved directly or via cMOS technology. Furthermore, parallel detection of hybridization events can also be achieved either by using different catalytically redox-active units for the individual electrodes of the array when building up the different functionalized electrodes of an electrode array, or by using a continuously conductive surface to build up the functionalized electrodes and the distinguishability of molecular structures on a certain area with identical electrode structure (of a certain test site) within the overall system (of the complete oligomer chip) is achieved by using different catalytically redox-active units for the individual test sites Addition of the specific substrate can be addressed. In the latter variant, the electrochemical response of the entire oligomer chip is detected due to the continuous conductive surface, the addressing and the reading out of the electrochemical response of individual test sites is carried out by the selective addition of the substrate specific for this test site.

Furthermore, in the embodiment of an electrode array comprising electrodes that have been functionalized with different catalytically redox-active units, the invention provides a detection method that can be controlled via microelectrodes for the parallel qualitative and quantitative detection of redox-active substances, the respective substrate of the various catalytically redox-active units of the electrodes within of an electrode array. Binding of a catalytically redox-active unit to a nucleic acid oligomer

A prerequisite for the method according to the invention is the binding of a catalytically redox-active unit to a nucleic acid oligomer. The catalytically redox-active unit can, for. B. any redox-active protein / enzyme from the group of oxidases or reductases, proteins / enzymes from this group of oxidases or reductases modified by protein engineering or gene mutation or an artificially produced unit from one or more redox-active centers (electron donor or . Acceptor) or an artificially produced unit from one or more redox-active centers (electron donor or acceptor) and one or more macromolecules binding these redox-active centers.

Examples of a catalytically redox-active unit are:

(i) redox active proteins / enzymes such as e.g. B. the oxidoreeductases, some of which are summarized in Table 1 below. In the context of the present invention, the covalent attachment of the catalytically redox-active unit (the redox-active protein / enzyme) is preferably carried out by covalent attachment of the cofactor with subsequent reconstitution of the apoprotein to the cofactor attached to the nucleic acid oligomer. In the case of catalytically redox-active units (redox-active proteins / enzymes) with several cofactors, one of the cofactors (printed in bold in Table 1 below) is covalently bound to the nucleic acid oligomer and the catalytically redox-active unit is completed by reconstitution with the remaining cofactors and the apoprotein.

Table 1: Selection of some redox-active enzymes (oxidoreductases) as examples of catalytically redox-active units.

(ii) modified redox-active proteins / enzymes as presented under (i), which have been changed by protein engineering or gene mutation and continue to have catalytic or electrocatalytic activity.

(iii) Artificially produced catalytically redox-active units from electron donor (s) and or eletron acceptors and macromolecules which have a catalytic or electrocatalytic activity.

(iv) NAD ⁺ dependent enzymes such as e.g. B. lactate dehydrogenase (LDH, EC 1.1.1.27) or alcohol dehydrogenase (ADH, EC 1.1.1.1). When using NAD ⁺ -dependent enzymes, the catalytically redox-active unit (e.g. LDH or ADH) can be bound to the nucleic acid oligomer by (modified) NAD ⁺ directly or via a spacer (Example 3) covalently to the nucleic acid -Oligomer is bound and the NAD ⁺ -dependent enzyme is then associated with the (modified) NAD ⁺ by noncovalent interaction.

Structure 1: Monomer of glucose oxidase (GOx). The apoprotein consists of α-helical and ß-sheet domains, the coenzyme flavin-adenine dinucleotide (FAD) is shown in the form of the space-filling shallot model. The structure of the FAD is shown in Formula 1. In its native form, the GOx is available as a homodimer.

Formula 1 Formula 2

Formula 3 Formula 4

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

In addition, the catalytically redox-active unit is distinguished according to the invention in that said unit gives off electrons to an oxidizing agent which is likewise covalently bound to the nucleic acid oligomer, or accepts electrons from another reducing agent which is likewise covalently bound to the oligonucleotide, this oxidizing or reducing agent in particular one can be electrically conductive surface (electrode) and the catalytically redox-active unit, in particular the redox-active center of the unit, can be electrooxidized / reduced by applying an external voltage to this electrode in the electrochemically accessible potential range of the electrode.

According to the invention, the catalytically redox-active unit is distinguished by the fact that the redox-active center of the unit (directly or after the specific reaction with the substrate) can be oxidized or reduced on an electrode and the original state of the catalytically redox-active unit - before the oxidation or reduction on the electrode - is restored in a specific catalytic reaction by the specific reaction of the catalytically redox-active unit with the associated substrate. According to the invention, any catalytically redox-active unit can be used for this as long as it or the redox-active center of the catalytically redox-active unit can be oxidized and reduced at a potential φ which satisfies the condition 2.0 V>φ> - 2.0 V. The potential here relates to the free, unmodified, redox-active center of the catalytically redox-active unit in one suitable solvent, measured against normal hydrogen electrode. In the context of the present invention, the potential range 1.7 V>φ> - 1.7 V is 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 centers of the application examples are oxidized (and reduced), is very particularly preferred.

Furthermore, according to the invention, the catalytically redox-active unit is characterized in that, by incorporating the catalytically redox-active unit in the electrochemical oxidation or reduction, the substrate specific for the redox-active center of the unit is oxidized or reduced electrocatalytically on an electrode. H. at a potential at which no or only very little current would flow in the absence of the catalytically redox-active unit, or under the formation of a catalytic (additional) current.

According to the invention, a catalytically redox-active unit is covalently bound to a nucleic acid oligomer by the reaction of the nucleic acid oligomer with the catalytically redox-active unit or parts thereof (see also section “Ways of carrying out the invention”). This binding can be done in five 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 catalytically redox-active unit is either naturally present on the catalytically redox-active unit or is obtained by chemical modification of the catalytically redox-active unit. The connection of the catalytically redox-active unit can take place completely or in parts of the unit with subsequent completion of the catalytically redox-active unit (see below). b) The nucleic acid oligomer is modified via a covalently linked part of the molecule (spacer) of any composition and chain length (longest continuous chain of bonded atoms), in particular chain length 1 to 14, on the oligonucleotide backbone or on a base with a reactive group , The modification is preferably carried out at one of the ends of the oligonucleotide backbone or at a terminal base. For example, an alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituent can be used as spacer. Possible simple reactions for the formation of the covalent bond between the catalytically redox-active unit 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 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. The connection of the catalytically redox-active unit can take place completely or in parts of the catalytically redox-active unit with subsequent completion of the unit (see below).

c) In the case of catalytically redox-active units which have FAD / FADH ₂ as cofactors, a phosphorylated adenine is used as the terminal base in the synthesis of the nucleic acid oligomer and this is fused to a FAD derivative (β-D- by fusion with flavin mononucleotide). 2-deoxiribose-FAD) and the catalytically redox-active unit is completed by reconstitution with the catalytically redox-active unit freed from (a) FAD.

d) When using NAD ⁺ / NADH-dependent enzymes (enzymes from cofactor (s) and apoprotein (s) which, in addition to the specific substrate, also require NAD ⁺ or NADH in order to complete the catalytic reaction cycle, such as lactate dehydrogenase (LDH) or the alcohol dehydrogenase (ADH)), the catalytically redox-active unit (e.g. the LDH or ADH) can be bound to the nucleic acid oligomer by (modified) NAD ⁺ directly (as described here under (a)) or is covalently bound to the nucleic acid oligomer via a spacer (as described here under (b) or in Example 3) and the NAD ⁺ -dependent enzyme is then associated with the (modified) NAD ⁺ - by noncovalent interaction.

e) In the synthesis of the nucleic acid oligomer, a terminal base or a terminal nucleotide is replaced by a cofactor of the catalytically redox-active unit and the catalytically redox-active unit is completed by reconstitution with the catalytically redox-active unit freed from this cofactor (see below). According to the invention, the binding of the catalytically redox-active unit to the nucleic acid oligomer can take place in whole or in part before or after the binding of the nucleic acid oligomer to the conductive surface. In the case of a redox-active protein / enzyme consisting of apoprotein and cofactor (s), instead of the complete catalytically redox-active unit, only the apoprotein, the apoprotein and part of the cofactors or one or more cofactors can be linked and the catalytically redox-active unit is subsequently reconstituted completed with the missing parts.

In the case of several different nucleic acid-oligomer combinations (test sites) on an electrode array, it is advantageous to connect the (covalent) catalytic redox-active unit to the nucleic acid oligomers by suitable choice of the reactive group on the free nucleic acid oligomer ends of the different Standardize electrodes / test sites for the entire surface if the catalytically redox-active unit is to be attached to the surface after immobilization of the nucleic acid oligomer.

If redox-active proteins / enzymes are used as the catalytically redox-active unit, the nucleic acid oligomer can be covalently bound to any naturally occurring or modification-attached reactive group of the protein or - in the case that the redox-active protein / enzyme consists of apoprotein and Cofactor (s) exist - to any reactive group of any arbitrary cofactor that is present naturally or is attached by modification. In the context of the present invention, the covalent attachment to any, naturally present or modification-attached, reactive group of an (arbitrary) cofactor of the protein is preferred. Without wishing to be bound to mechanistic details, one is particularly preferred in the case of a plurality of cofactors that can donate electrons to an external oxidizing agent which is likewise covalently bound to the nucleic acid oligomer or which can take up an external reducing agent which is likewise covalently bound to the nucleic acid oligomer ( see also section "Method for amperometric detection of nucleic acid-oligomer hybrids").

The conductive surface

According to the invention, the term “conductive surface” is understood to mean any carrier with 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.

In addition, any doped or undoped semiconductor surfaces of any thickness can also be used. All semiconductors can be used as pure substances or as mixtures. As non-limiting examples, carbon, silicon, germanium, α-tin, Cu (l) - and Ag (l) - halides of any crystal structure are mentioned here. Also suitable are all binary compounds of any composition and structure from the elements of groups 14 and 16, the elements of groups 13 and 15, and the elements of groups 15 and 16. In addition, ternary compounds of any composition and structure from 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 a nucleic acid oligomer to the conductive surface

According to the invention, a nucleic acid oligomer is linked directly or via a linker / spacer to the surface atoms or molecules of a conductive surface of the type described above. This binding can be done in three different ways:

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'C0 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 on the surface a monomolecular layer of any molecules is formed which preferably contain a reactive group at the end. The term “bifunctional linker” means any molecule of any chain length, in particular chain lengths 2-14, with two identical (homo-bifunctional) or two different (hetero-bifunctional) reactive groups of molecules.

If several different test sites are to be formed on the surface by using the methodology of photolithography, then at least one of the reactive groups of the homo- or hetero-bifunctional linker is a photo-inducible reactive group, i. H. a group that becomes reactive only through light irradiation of a certain or any wavelength. This linker is applied in such a way that the photoactivatable reactive group is available on the surface after the linker has been covalently bound. The nucleic acid oligomers are covalently attached to the surface modified in this way, whereby they themselves are modified with a reactive group via a spacer of any composition and chain length, in particular chain length 1-14, preferably near one end of the nucleic acid oligomer. The reactive group of the oligonucleotide is a group which reacts directly (or indirectly) with the modified surface to form a covalent bond. In addition, a further reactive group can be bound to the nucleic acid oligomers 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 catalytically redox-active unit (complete or components thereof) can be attached to this second end of the nucleic acid oligomer.

b) The nucleic acid oligomer to be applied to the conductive surface is modified with one or more reactive groups via a covalently attached spacer of any composition and chain length, in particular chain length 1-14, the reactive groups preferably being in the Is located near one end of the nucleic acid oligomer. The reactive groups are groups that can react directly with the unmodified surface. Examples of these are: (i) thiol (HS) or disulfide (SS) derivatized nucleic acid oligomers of the general formula (nx HS spacer) oligo, (nx RSS spacer) oligo or oligo spacer SS -Spacer-oligo that react with a gold surface to form gold-sulfur bonds or (ii) amines that attach to platinum or silicon surfaces through chemical or physical sorption. In addition, a further reactive group can be bound to the nucleic acid oligomers in the vicinity of their second end, this reactive group in turn, as described above, directly or is connected 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 catalytically redox-active unit (complete or components thereof) can be attached to this second end of the oligonucleotide. In particular, nucleic acid oligomers which are modified with a plurality of spacer-bridged thiol or disulfide bridges ((nx HS spacer) oligo or (nx RSS spacer) oligo) have the advantage that such nucleic acid oligomers at a certain angle of attack against the conductive surface (angle between the surface normal and the helix axis of a double-stranded helical nucleic acid oligomer or between the surface normal and the axis perpendicular to the base pairs of a double-stranded non-helical nucleic acid oligomer) can be applied if the thiol or disulfide Functions to the nucleic acid oligomer spacer, viewed from one end of the nucleic acid, have an increasing or decreasing chain length.

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 near 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 catalytically redox-active unit (complete or components thereof) can be attached to this second end of the nucleic acid oligomer. The nucleic acid oligomer can be bound to the conductive surface before or after the catalytically redox-active unit has been bound to the nucleic acid oligomer. In the case of a redox-active protein / enzyme consisting of apoprotein and cofactor (s), instead of the complete catalytically redox-active unit, only the apoprotein, the apoprotein with some of the cofactors or one or more of the cofactors can be linked, and the catalytically redox-active unit is obtained by subsequent reconstitution completed with the missing parts. When using a linked (at least bimolecular) electron-donor / electron-acceptor complex as the redox-active unit, the electron-acceptor (or donor), as under b) or c) in the section "Binding a catalytically redox-active unit a nucleic acid oligomer ", be bound to or instead of a terminal base on the nucleic acid oligomer and the electron donor (or acceptor) is bound by subsequent covalent attachment to a reactive group of the electron acceptor (or donor) are or, as described under a) in the section "Binding a catalytically redox-active unit to a nucleic acid oligomer", by subsequent attachment to a terminal reactive group of the nucleic acid oligomer backbone at the same end (see also the section "Ways of carrying out the Invention"). Alternatively, the nucleic acid oligomer can be bound to the conductive surface before or after the spacer provided with a reactive group is bound to bind the catalytically redox-active unit. The binding of the already modified nucleic acid oligomer to the conductive surface, ie the binding to the surface after the connection of the catalytically redox-active unit to the nucleic acid oligomer or after the connection of parts of the catalytically redox-active unit or after the connection with a reactive group provided spacers for binding the catalytically redox-active unit is also carried out as described under a) to c) in this section.

When producing the test sites, when connecting the single-stranded nucleic acid oligomers to the surface, care must be taken to ensure that there is a sufficiently large distance between the individual nucleic acid oligomers, on the one hand, for the hybridization with the target nucleic acid To provide the oligomer with the necessary space and, on the other hand, the space required for the connection of the catalytically redox-active unit. There are three different approaches (and combinations thereof) in particular:

1.) Production of a modified surface by binding a hybridized nucleic acid oligomer, ie a surface derivatization with hybridized probe-nucleic acid oligomer instead of with single-stranded probe oligonucleotide. The nucleic acid oligomer strand used for hybridization is unmodified (the Surface connection is carried out as described under a) - c) in this section). The hybridized nucleic acid-oligomer double strand is then thermally dehybridized, as a result of which a surface modified with single-strand nucleic acid oligomer is produced with a greater distance between the probe-nucleic acid oligomers.

2.) Production of a modified surface by attaching a single strand or double strand nucleic acid oligomer, a suitable monofunctional linker being added during the surface derivatization, which is also bound to the surface in addition to the single strand or double strand nucleic acid oligomer (the surface attachment is carried out as described under a) - c) in this section). According to the invention, the monofunctional linker has a chain length which is identical to the chain length of the spacer between the surface and the nucleic acid oligomer or which deviates by a maximum of four chain atoms. When double-stranded nucleic acid oligomer is used for surface derivatization, the nucleic acid-oligomer double strand is thermally dehybridized after the double stranded nucleic acid oligomer and the linker have been bonded to the surface. The simultaneous attachment of a linker to the surface increases the distance between the single-stranded or double-stranded nucleic acid oligomers likewise bound to the surface. If double-stranded nucleic acid oligomer is used, this effect is further enhanced by the subsequent thermal dehybridization.

3.) Production of a modified surface by connecting a single-strand or double-strand oligonucleotide to which the catalytically redox-active unit is already attached, the catalytically redox-active unit having a diameter of greater than 30 A. When using double-stranded oligonucleotide, the oligonucleotide double-strand is thermally dehybridized after the attachment of the double-stranded oligonucleotide to the surface.

With regard to the individual steps for "binding a catalytically redox-active unit to a nucleic acid oligomer" and for "binding an oligonucleotide to the conductive surface", it should be pointed out that in the section "Ways of Carrying Out the Invention" the various possible combinations of the individual Steps leading to the same end result are demonstrated using an example (Figure 2). Method for the electrochemical detection of nucleic acid-oligomer hybrids

Advantageously, according to the method for the electrochemical detection of nucleic acid-oligomer hybrids, several probe-nucleic acid oligomers of different sequences, in which de novo sequencing ideally all the necessary combinations of the nucleic acid oligomer, are applied to an oligomer (DNA) chip to complete the sequence to detect any target nucleic acid oligomer or a (fragmented) target DNA or to detect mutations in the target and detect them in a sequence-specific manner or to detect the presence of known genes or known nucleic acid oligomers.

For this purpose, an array of microelectrodes is used, which either consists of electrodes that are individually and directly connected to a current / voltage source, or an electrode array is applied by microstructuring on a common surface, in which the individual electrodes are controlled using cMOS technology and can be read out. On the conductive surface of the individual electrodes (a test site), the surface atoms or molecules are linked with DNA / RNA / PNA nucleic acid oligomers of known but any sequence, as described above. In a most general embodiment, however, a single electrode can also be derivatized with a single probe oligonucleotide or a unique type of probe oligonucleotide (with the same base sequence and with the same catalytically redox-active unit). Nucleic acid oligomers (eg DNA, RNA or PNA fragments) with a base length of 3 to 50, preferably a length of 5 to 30, particularly preferably a length of 8 to 25, are used as probe nucleic acid oligomers. According to the invention, a catalytically redox-active unit is or is bound to the probe nucleic acid oligomers, as described below.

Furthermore, parallel detection of hybridization events can also be achieved either by using different catalytically redox-active units for the individual electrodes of the array when building up the different functionalized electrodes of an electrode array, or by using a continuously conductive surface to build up the functionalized electrodes and the distinguishability of molecular structures on a certain area with identical electrode structure (of a certain test site) within the overall system (of the complete oligomer chip) is achieved by using different catalytically redox-active units for the individual test sites Addition of the specific substrate can be addressed. The latter variant is due to the continuous conductive surface, the electrochemical response of the entire oligomer chip is detected, the addressing and reading of the electrochemical response of individual test sites is carried out by the selective addition of the substrate specific for this test site.

The modification of the probe nucleic acid oligomers with a catalytically redox-active unit can take place completely or in components of the catalytically redox-active unit either before or after the binding of the probe oligonucleotide to the conductive surface. The various possible combinations of the individual steps (reaction sequences) are demonstrated with the aid of FIG. 2 using the example of a catalytically redox-active unit bound to an electrode via a probe oligonucleotide in the section “Ways of carrying out the invention”.

Regardless of the respective reaction sequence, a surface hybrid of the general structure elek-spacer-ss-oligo-spacer unit is formed, "unit" being representative of the catalytically redox-active unit. The bridges can of course also be carried out without a spacer or with only one spacer (Elek-ss-oligo-spacer unit or Elek-Spacer-ss-oligo unit). In the example in FIG. 2, the unit is glucose oxidase (GOx), a redox-active enzyme consisting of apoprotein and cofactor. In the example of FIGS. 2, 3 and 4, the GOx is covalently linked to the nucleic acid oligomer via its cofactor flavin-adenine dinucleotide (FAD) in the so-called FAD-protein binding pocket of the GOx. The GOx forms a 1: 1 complex with the cofactor FAD, the GOx occurring in its natural form as a homodimer, but also having a catalytic activity in the form of a monomer relevant to the invention. In the example of FIGS. 5 and 6, the unit is lactate dehydrogenase, an NAD ⁺ -dependent enzyme which, by noncovalent interaction, associates with the (modified) NAD ⁺ covalently bound to the probe oligonucleotide.

The electrochemical communication between the (conductive) surface and the catalytically redox-active unit (“unit”) bridged by a single-strand oligonucleotide in the general structure elek-spacer-ss-oligo-spacer unit is weak or nonexistent.

In a next step, the test sites are brought into contact with the nucleic acid oligomer solution (target) to be examined. Hybridization only occurs in the case in which the solution contains nucleic acid-oligomer strands which are complementary to the probe-nucleic acid oligomers bound to the conductive surface, or at least are complementary in a wide range. In case of Hybridization between probe and target nucleic acid oligomer leads to an increased conductivity between the surface and the catalytically redox-active unit, since this is now bridged by the double-stranded nucleic acid oligomer. Figure 3 shows this schematically using the example of the Elek-Spacer-ss-oligo-Spacer-FAD (GOx). FIG. 4 shows the sequence of the electron transfer steps in Elek-Spacer-ds-oligo-Spacer-FAD (GOx) in detail, while FIG. 5 shows the example of Elek-Spacer-ss-oligo-Spacer-PQQ-NAD ⁺ -LDH schematically shows and Figure 6 shows the sequence of the electron transfer steps in Elek-Spacer-ds-oligo-NAD ⁺ - LDH in detail.

Due to the hybridization of probe-nucleic acid oligomer and the complementary nucleic acid-oligomer strand (target), the electrical communication between the (conductive) surface and the catalytically redox-active unit changes. Thus, a sequence specific hybridization event can be carried out by electrochemical methods such as e.g. B. cyclovoltametry, amperometry, potentiometry or conductivity measurements can be detected.

With cyclic voltammetry, the potential of a stationary working electrode is changed linearly as a function of time. Starting from a potential at which there is no electrooxidation or reduction, 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, which in the current / voltage curve generates an initially rising current, then a maximum current (peak) and finally a gradually decreasing current, the direction of the potential feed is reversed. The behavior of the products of electrooxidation or reduction is then recorded in the return.

An alternative electrical detection method, amperometry, is made possible by the fact that the catalytically redox-active unit can be electro-oxidized (electroreduced) by applying a suitable, constantly kept electrode potential, but the re-reduction (reoxidation) of the catalytically redox-active unit to the original state is not as in the cyclic voltammetry takes place by changing the electrode potential, but by means of a suitable reducing agent (oxidizing agent), the "redox-active substance" added to the target solution, whereby the circuit of the entire system is closed. As long as such reducing agent (oxidizing agent) is present or as long as the used reducing agent (oxidizing agent) is reduced (reoxidized) at the counter electrode, current flows which can be detected amperometrically and which is proportional to the number of hybridization events. This principle of amperometric detection will be explained in more detail using the example of glucose oxidase (see also FIGS. 3 and 4). The probe oligonucleotide covalently bound to the electrode at one end can be functionalized at the other, still free end with the complete enzymatic unit of glucose oxidase, for example by B. the flavin adenine dinucleotide (FAD) cofactor of the enzyme is covalently bound to the probe oligonucleotide and then reconstituted with the glucose oxidase apoprotein (GOx). The resulting surface hybrid of the general structure Elek-Spacer-ss-oligo-Spacer-FAD (GOx) has little or no conductivity between the electrode and the FAD. In the case of hybridization with the target oligonucleotide complementary to "ss-oligo", the conductivity is increased significantly. When the substrate glucose is added to the target oligonucleotide solution, the FAD of gucose oxidase (FAD (GOx)) is reduced to FADH ₂ of glucose oxidase (FADH ₂ (GOx)), whereby glucose is oxidized to gluconic acid. If a suitable external potential is now present at the electrode, so that electrons from FADH ₂ (GOx) are released to the electrode via the hybridized oligonucleotide and thus FADH ₂ (GOx) is reoxidized to FAD (GOx) (but neither glucose nor gluconic acid is added this potential can be electrooxidized or reduced) flows in the system Elek-Spacer-ds-oligo-Spacer-FAD (GOx) as long as current such as FAD (GOx) is reduced by free glucose, ie until all of the glucose has been consumed or for the case that there is a potential at the counter electrode at which gluconic acid can be reduced to glucose, as long as gluconic acid is reduced at the counter electrode. This current can be detected amperometrically and is proportional to the number of hybridization events.

In the potentiometric detection of hybridization events, the course of the electrode potential depending on the z. B. recorded the substrate consumption. Here, for. B. set the potential of a stationary working electrode to the "zero current" potential E ° of the substrate. When substrate is used up by the catalytically redox-active unit (in the case of hybridization), the "zero current" potential E ° changes in the direction of the equilibrium potential E ^. Recording the potential as a function of time (~ substrate consumption) thus provides information about the hybridization state. 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 oligonucleotide sequencing by hybridization on a chip;

Fig. 2 Different reaction sequences for the production of the surface hybrid Elek-Spacer-ss-oligo-Spacer-PQQ-FAD (GOx). The catalytically redox-active unit in this surface hybrid is glucose oxidase (GOx) consisting of apoprotein and flavin adenine dinucleotide (FAD) cofactor. The GOx is covalently linked to the oligonucleotide via its cofactor FAD via PQQ and a spacer;

3 shows a schematic representation of the amperometric measurement method using the example of the surface hybrid elek-spacer-ss-oligo-spacer-PQQ-FAD (GOx) from FIG. 2 (injection: addition (injection) of the substrate glucose);

Fig. 4 Detailed schematic representation of the surface hybrid Au-S (CH ₂ ) ₂ -ds-oligo-spacer-FAD (GOx) of Figure 3 with gold as a surface material, mercaptoethanol as a spacer (-S-CH ₂ CH ₂ - spacer) between Electrode and oligonucleotide and -CH ₂ -CH = CH-CO-NH-CH ₂ - CH ₂ -NH-PQQ-NH-CH ₂ -CH ₂ - as a spacer between the cofactor FAD and oligonucleotide and the representation of the sequence by the Substrate induced electron transfer steps. The apx protein of the GOx is only indicated as a shell (solid line) (see structure 1). The 12 bp probe oligonucleotide of the exemplary sequence 5'-TAGTCGGAAGCA-3 'is shown as a section in the hybridized state;

5 shows a schematic representation of the amperometric measurement method using the example of the surface hybrid Elek-Spacer-ss-oligo-Spacer-PQQ-NAD ⁺ -LDH (Inj .: addition (injection) of the substrate lactate);

Fig. 6 Detailed schematic representation of the surface hybrid Au-S (CH ₂ ) ₂ -ds-oligo-spacer-PQQ-NAD ⁺ -LDH of Figure 3 with gold as the surface material, mercaptoethanol as a spacer (-S-CH ₂ CH ₂ - spacer ) between electrode and oligonucleotide and and -CH ₂ -CH = CH-CO-NH-CH ₂ -CH ₂ -NH-PQQ-NH-CH ₂ -CH ₂ - is associated as a spacer between the NAD ⁺ and oligonucleotide to the ADH as well the representation of the sequence of the electron transfer steps induced by the substrate. The 12 bp probe oligonucleotide of the exemplary sequence 5'-TAGTCGGAAGCA-3 'is shown as a section in the hybridized state;

Ways of Carrying Out the Invention

A formation unit of an exemplary test site with hybridized target, Au-S (CH ₂ ) ₂ -ds-oligo-spacer-PQQ-FAD (GOx) of the general structure elek-spacer-ds-oligo-spacer unit is shown in FIG. 4 shown. In the context of the present invention, formation unit is understood to mean the smallest repeating unit of a test site or of a functionalized electrode within the electrode array. In the example in FIG. 4, the surface is a gold electrode. The connection between gold electrode and probe oligonucleotide was established with the linker (HO- (CH ₂ ) ₂ -S) ₂ , which with the terminal phosphate group at the 3 'end to P-0- (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 co-adsorbed on the surface. The catalytically redox-active unit in the example in FIG. 4 is glucose oxidase (GOx), a redox-active enzyme consisting of apoprotein and FAD cofactor (s). In the application example, the GOx is covalently linked to the oligonucleotide via its FAD cofactor, free FAD first being provided to a reactive amino group (see Example 1), then free FAD being covalently linked to the probe oligonucleotide via this amino group ( Amide formation with elimination of water with a carboxylic acid group of the PQQ which is linked to the terminal amino function of the -CH = CH-CO-NH-CH ₂ -CH ₂ - NH ₂ linker at the C-5 position of the 5th with another carboxylic acid group '-Thymins of the probe oligonucleotide is bound) and finally the apoprotein of the GOx was reconstituted at FAD.

As already mentioned above, the modification of the probe oligonucleotides with the complete or with a component of the catalytically redox-active unit can take place either before or after the binding of the probe oligonucleotide to the conductive surface. The various possible combinations of the individual steps, which in principle lead to the same educational unit of a test site or one Functionalized electrode within the electrode array, should be shown below with the help of Figure 2 using the example of the surface hybrid Au-S (CH ₂ ) ₂ -ss-oligo-spacer-PQQ-FAD (GOx) or in its more general form as Elek -Spacer-ss- oligo-Spacer-PQQ-FAD (GOx) are shown.

The GOx can be freed from the FAD cofactor by simple manipulation (see Example 4), so that GOx can be broken down into two components, FAD and apoprotein. The probe oligonucleotide is provided with (identical or different) reactive groups in the vicinity of the two ends via an (arbitrary) spacer. In a reaction sequence "1", the probe oligonucleotide modified in this way can covalently bind together with the monofunctional linker in the presence of a monofunctional linker (corresponding to points a) - c) and 2.) in the section "Binding an oligonucleotide to the conductive surface" the electrode is connected, taking care that enough monofunctional linker of suitable chain length is added in order to provide sufficient space between the individual probe oligonucleotides for hybridization with the target oligonucleotide and for the connection of the catalytically redox-active unit. The free, spacer-bridged, reactive group of the probe oligonucleotide PQQ and then N ⁶ - (2-aminoethyl) -FAD (formula 5 or example 1) are bound to it. The binding is carried out as described under a) or b) in the section “Binding a catalytically redox-active unit to a nucleic acid oligomer” or in Example 4. In the last step of this reaction sequence "1", the apoprotein of GOx is then reconstituted on the (modified) FAD cofactor as described in Example 9. In a variant to this (reaction sequence "2"), the probe oligonucleotide (modified with spacers and reactive groups) can first be covalently bound to the electrode without a free, monofunctional linker (spacer), resulting in a flat attachment of the oligonucleotide. The free, monofunctional linker (spacer) is then covalently bound to the electrode. A further possibility (reaction sequence "3") consists in first modifying the probe oligonucleotide (with spacers and reactive groups) with PQQ and FAD, then covalently binding it to the electrode in the presence of free, monofunctional linker (spacer) and then to reconstitute with the GOx apoprotein. Finally, in a reaction sequence "4", the probe oligonucleotide (modified with spacers and reactive groups) can first be modified with PQQ and FAD, in order to then reconstitute it with the GOx apoprotein and then covalently bind it to the electrode. If, as in the case of the GOx, the catalytically redox-active unit has a substantially larger diameter than the hybridized ds-oligonucleotide (larger than 30 A), the covalent attachment of a suitable free, monofunctional linker (spacer) to the electrode can be dispensed with. otherwise it happens Functionalized electrode within the electrode array, should be shown below with the help of Figure 2 using the example of the surface hybrid Au-S (CH ₂ ) ₂ -ss-oligo-spacer-PQQ-FAD (GOx) or in its more general form as Elek -Spacer-ss- oligo-Spacer-PQQ-FAD (GOx) are shown.

The GOx can be freed from the FAD cofactor by simple manipulation (see Example 4), so that GOx can be broken down into two components, FAD and apoprotein. The probe oligonucleotide is provided with (identical or different) reactive groups in the vicinity of the two ends via an (arbitrary) spacer. In a reaction sequence "1", the probe oligonucleotide modified in this way can covalently attach to the monofunctional linker in the presence of a monofunctional linker (corresponding to points a) - c) and 2.) in the section "Binding an oligonucleotide to the conductive surface") the electrode is connected, taking care that enough monofunctional linker of suitable chain length is added in order to provide sufficient space between the individual probe oligonucleotides for hybridization with the target oligonucleotide and for the connection of the catalytically redox-active unit. The free, spacer-bridged, reactive group of the probe oligonucleotide PQQ and then N ⁶ - (2-aminoethyl) -FAD (formula 5 or example 1) are bound to it. The binding is carried out as described under a) or b) in the section “Binding a catalytically redox-active unit to a nucleic acid oligomer” or in Example 4. In the last step of this reaction sequence "1", the apoprotein of GOx is then reconstituted on the (modified) FAD cofactor as described in Example 9. In a variant to this (reaction sequence "2"), the probe oligonucleotide (modified with spacers and reactive groups) can first be covalently bound to the electrode without a free, monofunctional linker (spacer), resulting in a flat attachment of the oligonucleotide. The free, monofunctional linker (spacer) is then covalently bound to the electrode. Another possibility (reaction sequence "3") is to first modify the probe oligonucleotide (with spacers and reactive groups) using PQQ and FAD, then covalently attach it to the electrode in the presence of free, monofunctional linker (spacer) and then to reconstitute with the GOx apoprotein. Finally, in a reaction sequence "4", the probe oligonucleotide (modified with spacers and reactive groups) can first be modified with PQQ and FAD, in order to then reconstitute it with the GOx apoprotein and then covalently bind it to the electrode. If, as in the case of the GOx, the catalytically redox-active unit has a substantially larger diameter than the hybridized ds-oligonucleotide (larger than 30 A), the covalent attachment of a suitable free, monofunctional linker (spacer) to the electrode can be dispensed with. otherwise it happens Detect amperometry. The individual electron transfer steps which are triggered by the substrate in the surface hybrid Au-S (CH ₂ ) ₂ -ds-oligo-spacer-PQQ-FAD (GOx) are shown in FIG. 4. In principle, the surface hybrid Au-S (CH ₂ ) ₂ -ds-oligo-spacer-PQQ-FAD (GOx) can also be reversed under suitable external circumstances, so that FAD is reduced by the electrode and reduced FAD (FAD ^~ or FAD ^2- or FADH ₂ ) is oxidized by a suitable substrate in a catalytic reaction.

Another test site or another functionalized electrode within the electrode array, Au-S (CH ₂ ) ₂ -ds-oligo-spacer-PQQ-NAD ⁺ -LDH, of the general structure Elek-Spacer-ss-oligo- Spacer unit is shown in Figure 5. By adding the ADH substrate lactate, electrons are transferred to the FMN cofactor of the LDH and from this reduced FMN (FMN- or FMN ² or FMNH ₂ ) directly or with the participation of other cofactors of the LDH to NAD ⁺ and finally from reduced NAD ⁺ (NAD, NAD- or NADH) can be transferred to the electrode. In the case of the probe oligonucleotide not hybridized with the target oligonucleotide, there is still no current flow between the reduced NAD ⁺ (NAD, NAD ^" or NADH) and the electrode, since the conductivity of the ss oligonucleotide in Au-S (CH ₂ ) ₂ -ss-oligo-spacer-PQQ-NAD ⁺ -LDH is very low or nonexistent, but in the hybridized state (Au-S (CH ₂ ) ₂ - ds-oligo-spacer-PQQ-NAD ⁺ -LDH) it is High conductivity, electrons can be transferred from the reduced NAD ⁺ (NAD, NAD ^" or NADH) to the electrode (with the formation of NAD ⁺ ). This is expressed amperometrically in a clear current flow between the electrode and the catalytically redox-active unit (FIG. 5). This makes it possible to detect the sequence-specific hybridization of the target with the probe oligonucleotides by amperometry. The individual electron transfer steps which are triggered by the substrate in the surface hybrid Au-S (CH ₂ ) ₂ -ds-oligo-spacer-PQQ-NAD ⁺ -ADH are shown in FIG. 6. In principle, the surface hybrid Au-S (CH ₂ ) ₂ -ds-oligo-spacer-PQQ-NAD ^{+ "} -LDH can also be reversed under suitable external circumstances, so that NAD ⁺ is reduced by the electrode and reduced NAD ⁺ (NAD ^" NAD ^" or NADH) is oxidized by a suitable substrate (e.g. acetaldehyde) in a catalytic reaction.

Since the redox activity of the catalytically redox-active unit - even with a suitable electrode potential - is only triggered by the addition of the specific substrate and is maintained for a maximum of as long as the substrate is present, this can be exploited according to the invention in that a specific test site or a specific test site Group of an oligomer chip is spatially resolved by different catalytically redox-active units for the different test sites (or Test site groups) can be used. This has the advantage according to the invention that the different test sites (nucleic acid-oligomer combinations) of an oligomer chip can be applied to a common, continuous, electrically conductive surface and a specific test site or a specific test site group can be easily applied can be addressed and added by amperometric detection by adding the respective specific substrates. The various test sites therefore do not have to be applied to individual (micro) electrodes which are electrically insulated from one another and can be individually controlled to apply a potential and read out the current.

In addition, faulty base pairings (base pair mismatches) can be recognized by a changed cyclic voltammetric characteristic. A mismatch manifests itself in a larger potential distance between the current maxima of the electroreduction and the electroreoxidation (reversal of the electroreduction with reversed direction of potential feed) or the electrooxidation and electroreduction in a cyclovoltammetrically reversible electron transfer between the electrically conductive surface and the catalytically redox-active unit. 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.

Example 1: Modification of the FAD to the frf-β-aminoethyl -FAD, formula 5, or of the NAD * to the f-β-aminoethyO-NAD ^* : N ⁶ - (2-aminoethyl) -FAD is prepared by alkylating the N-1 position of the adenine unit of FAD with aziridine (acazyclopropane) under mild, aqueous conditions (pH = 3.2 - pH = 5.5) and subsequent intramolecular Dimroth rearrangement under mild aqueous conditions (pH = 6 - 6, 5, 50 ° C) according to the regulation in Bückmann et al, 1991, European Patent 0.247.537.B1. Alternatively, the method of Morris et al Anal. Chem. 53 (1991) 658-665 or Zapelli et al. Eur. J. Biochem. 89 (1978) 491-499. The N ⁶ - (2-aminoethyl) -NAD ⁺ can also be prepared under the same reaction conditions if NAD ^{+ is used} as the starting substance instead of FAD.

Example 2: Production of the oligonucleotide electrode Au-S-fCH ^^ ss-oligo-spacer-PQQ-FAD (GOx): The production of Au-S- (CH ₂ ) ₂ -ss-oligo-spacer-PQQ-FAD (GOx) is divided into 5 sections, namely the representation of the conductive surface, the derivatization of the surface with the probe oligonucleotide in the presence of a suitable monofunctional linker (incubation step), the covalent attachment Test site groups) can be used. This has the advantage according to the invention that the different test sites (nucleic acid-oligomer combinations) of an oligomer chip can be applied to a common, continuous, electrically conductive surface and a specific test site or a specific test site group can be easily applied can be addressed and added by amperometric detection by adding the respective specific substrates. The various test sites therefore do not have to be applied to individual (micro) electrodes which are electrically insulated from one another and can be individually controlled to apply a potential and read out the current.

Example 1: Modification of the FAD to If-ß-Aminoethylj-FAD, Formula 5, or of NAD ⁺ to f-fi-AminoethyO-NAD *: N ⁶ - (2-aminoethyl) -FAD is prepared by alkylating the N-1 position of the adenine unit of FAD with aziridine (acazyclopropane) under mild, aqueous conditions (pH = 3.2 - pH = 5.5) and subsequent intramolecular Dimroth rearrangement under mild aqueous conditions (pH = 6 - 6, 5, 50 ° C) according to the regulation in Bückmann et al, 1991, European Patent 0.247.537.B1. Alternatively, the method of Morris et al Anal. Chem. 53 (1991) 658-665 or Zapelli et al. Eur. J. Biochem. 89 (1978) 491-499. The N ⁶ - (2-aminoethyl) -NAD ⁺ can also be prepared under the same reaction conditions if NAD ^{+ is used} as the starting substance instead of FAD.

Example 2: Preparation of the oligonucleotide electrode

FAD (GOx): The production of Au-S-fCH ^ ss-oligo-spacer-PQQ-FADfGOx) is divided into 5 sections, namely the representation of the conductive surface, the derivatization of the surface with the probe oligonucleotide in the presence of a suitable one monofunctional linker (incubation step), the covalent linkage the PQQ (redox step I), the binding of the N ⁶ - (2-aminoethyl) -FAD (redox step II) and the reconstitution of the apoprotein the GOx (reconstitution 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 approx. 100 nm by electrical discharge. The gold film was then freed from surface impurities (oxidation of organic deposits) with 30% H ₂ 0 ₂ , / 70% H ₂ S0 ₄ and immersed in ethanol for about 20 minutes in order to displace oxygen adsorbed on the surface. After rinsing the surface with bidistilled water, a previously prepared IxlO ^"4 molar solution of the (modified) double-stranded oligonucleotide is applied to the horizontally stored surface so that the entire gold surface is wetted (incubation step, see also below).

For the incubation, a double-modified 12 bp single-stranded oligonucleotide of the sequence 5'-TAGTCGGAAGCA-3 'was used, which at the phosphate group of the 3' end with (HO- (CH ₂ ) ₂ -S) ₂ to P-0- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH is esterified. At the 5 'end, the terminal base thymine of the oligonucleotide on the C-5 carbon is modified with -CH = CH-CO-NH-CH ₂ -CH ₂ -NH ₂ . About 10 ^"4 to 10 ^{" 1} molar 2-hydroxy-mercaptoethanol was added to a 2x10 ^" molar solution of this oligonucleotide in HEPES buffer (0.1 molar in water, pH 7.5 with 0.7 molar addition of TEATFB, see abbreviations) ( or another thiol or disulfide linker of suitable chain length) and the gold surface of a test site completely wetted and incubated for 2-24 hours, during which the disulfide spacer P-0- (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 a 1: 1 coadsorption of the ss oligonucleotide and the cleaved 2-hydroxy-mercaptoethanol Free 2-hydroxy-mercaptoethanol that is simultaneously present in the incubation solution is also adsorbed by forming an Au-S bond (incubation step).

The gold electrode modified with a monolayer of ss-oligonucleotide and 2-hydroxy-mercaptoethanol was washed with bidistilled water and then with a solution of 3x10 ³ molar quinone PQQ, 10 ^"2 molar EDC and 10 ^{" 2} molar sulfo-NHS in HEPES buffer wetted. After a reaction time of approx. 1-4 h, the -CH = CH-CO-NH-CH ₂ -CH ₂ -NH ₂ spacer and the PQQ form a covalent bond (amide formation between the amino group of the spacer and the C-7 carboxylic acid function of the PQQ, redox step I). The gold electrode modified in this way was then washed with bidistilled water and with a solution of 1-10 x 10 ^"3 molar quinone N ⁶ - (2-aminoethyl) - FAD, 1-5 x 10 ^{" 2} molar EDC and 10 ^"2 molar sulfo -NHS wetted in HEPES buffer After a reaction time of about 1-4 h, PQQ and the N ⁶ - (2-aminoethyl) -FAD form a covalent bond (amide formation between the amino group of the spacer and the C-2-carboxylic acid function of the the oligonucleotide-bound PQQ, redox step II)

Finally, the gold electrode modified in this way was washed with double-distilled water and with a solution of approx. 5x10 ^'5 molar FAD-free GOx in 100 mM phosphate buffer, pH = 7, with 0.5 molar addition of TEATFB at approx. 25 ° C for approx 4 h and then approx. 12 h at 4 ° C to reconstitute the apoprotein of GOx to the oligonucleotide-bound N ⁶ - (2-aminoethyl) -FAD and finally with approx. 4 ° C cold buffer solution (100 mM Phosphate, pH = 7 with 0.5 molar addition of TEATFB) washed (reconstitution step).

Example 3: Preparation of the oligonucleotide electrode Au-S- (CH ^ ₂ -ss-oligo-spacer-PQQ NAD * LDH: The production of Au-S- (CH ₂₎ ₂ -ss-oligo-spacer-PQQ -NAD ⁺ -LDH is divided into 5 sections, namely the representation of the conductive surface, the derivatization of the surface with the probe oligonucleotide in the presence of a suitable monofunctional linker (incubation step), the covalent attachment of the PQQ (redox step I), the attachment of the N ⁶ - (2-Aminoethyl) -NAD ⁺ (redox step II) and the association with LDH (association step).

A double-modified 12 bp single-strand oligonucleotide of the sequence 5'-TAGTCGGAAGCA-3 'was used for the incubation, which was attached to the phosphate group of the 3' end is esterified with (HO- (CH ₂ ) ₂ -S) ₂ to give P-0- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH. At the 5 'end, the terminal base thymine of the oligonucleotide on the C-5 carbon is modified with -CH = CH-CO-NH-CH ₂ -CH ₂ -NH ₂ . About 10 ^"4 to 10 ^'1 molar 2-hydroxy-mercaptoethanol was added to a 2x10 ^{" 4} molar solution of this oligonucleotide in HEPES buffer (0.1 molar in water, pH 7.5 with 0.7 molar addition of TEATFB, see abbreviations) (or another thiol or disulfide linker of suitable chain length) and the gold surface of a test site completely wetted and incubated for 2-24 hours. During this reaction time, the disulfide spacer P-0- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH of the oligonucleotide is cleaved homolytically. The spacer forms a covalent Au-S bond with the Au atoms on the surface, which leads to a 1: 1 coadsorption of the SS oligonucleotide and the split off 2-hydroxy-mercaptoethanol. The free 2-hydroxy-mercaptoethanol which is simultaneously present in the incubation solution is also adsorbed by forming an Au-S bond (incubation step).

The gold electrode modified with a monolayer of ss-oligonucleotide and 2-hydroxy-mercaptoethanol was washed with double-distilled water and then with a solution of 3x10 ^"3 molar quinone PQQ, 10 ^{" 2} molar EDC and 10 ^"2 molar sulfo-NHS in HEPES After a reaction time of about 1 - 4 h, the -CH = CH-CO-NH-CH ₂ -CH ₂ -NH ₂ spacer and the PQQ form a covalent bond (amide formation between the amino group of the spacer and the C- 7- carboxylic acid function of PQQ, redox step I).

The gold electrode modified in this way was then washed with bidistilled water and with a solution of 1-10 x 10 ^"3 molar quinone N ⁶ - (2-aminoethyl) - NAD ⁺ , 1-5 x 10 ^{" 2} molar EDC and 10 ^"2 molar sulfo-NHS wetted in HEPES buffer After a reaction time of about 1 to 4 h, PQQ and the N ⁶ - (2-aminoethyl) -NAD ^{+ form} a covalent bond (amide formation between the amino group of the spacer and the C-2-carboxylic acid function of the PQQ bound to the oligonucleotide, redox step II)

Finally, the gold electrode modified in this way was washed with bidistilled water and incubated with a solution of LDH (5 mg / mL) in 10 mM Tris, pH = 7, with 0.7 molar addition of TEATFB at approx. 4 ° C. for approx. 1 h Associate LDH with the oligonucleotide-bound N ⁶ - (2-aminoethyl) -NAD ⁺ and finally wash with approx. 4 ° C cold buffer solution (10 mM Tris, pH = 7 with 0.7 molar addition of TEATFB) (association step).

EXAMPLE 4 Illustration of the Apoprotein from Glucose Oxidase / Extraction of the FAD from Glucose Oxidase: Glucose Oxidase (GOx) in Phosphate Buffer (80mg GOx in 14mL Buffer) with the Addition of 6mL Glycerol is Cooled to -4 ° C. in a Salt / Ice Bath, vigorously stirred and gradually mixed with 2.5% H ₂ S0 ₄ (v / v) until the pH drops to pH = 1.4 and is thus incubated on the ice bath for 2.5 h. The apoprotein is then obtained by column chromatography. For this purpose, a Sephadex G-50 column with 30% glycerol in water (v / v) is equilibrated and with conc. H ₂ S0 _{4 brought} to pH = 1, 4, cooled to 4 ° C and the incubation solution applied. The protein peak is eluted at a flow rate of 1.3 mUmin and collected in a storage container with 0.4 molar phosphate buffer (4 ml with the addition of 200 mg bovine serum albumin and 400 mg activated carbon) (the protein peak can be recognized by UV absorption of the eluate) , The protein-containing eluate is adjusted to pH = 7 and the activated carbon is removed by filtration (0.8 μm and then 0.22 μm Millipore filter). Finally 10% sodium azide in water (w / v) is added until the total concentration of sodium azide is 0.1% (w / v).

Claims

claims

1.) By linking a catalytically redox-active unit modified nucleic acid oligomer, characterized in that the catalytically redox-active unit has one or more electron donor molecules and / or one or more electron

Contains acceptor molecules and additionally one or more macromolecules.

2.) Modified nucleic acid oligomer according to claim 1, characterized in that the catalytically redox-active unit is covalently linked.

3.) Modified nucleic acid oligomer according to claim 1 or 2, characterized in that the catalytically redox-active unit via one or more

Electron donor molecules are covalently attached.

4.) Modified nucleic acid oligomer according to one of claims 1 to 3, characterized in that the catalytically redox-active unit is covalently linked via one or more electron acceptor molecules.

5.) Modified nucleic acid oligomer according to one of the preceding claims, characterized in that the catalytically redox-active unit is covalently linked via one or more of the macromolecules.

6.) Modified nucleic acid oligomer according to one of the preceding claims, characterized in that the catalytically redox-active unit is a redox-active enzyme.

7.) Modified nucleic acid oligomer according to claim 6, characterized in that the catalytically redox-active unit is the native or modified glucose oxidase.

8.) Modified nucleic acid oligomer according to claim 6, characterized in that the catalytically redox-active unit is the native or modified

Alcohol dehydrogenase or fructose dehydrogenase.

9.) Modified nucleic acid oligomer claim 6, characterized in that the catalytically redox-active unit is the native or modified lactate dehydrogenase.

10.) Modified nucleic acid oligomer according to claim 6, characterized in that the catalytically redox-active unit is a native or modified peroxidase.

11.) Modified nucleic acid oligomer according to one of claims 1 to 5, characterized in that one or more of the electron donor and / or electron

Acceptor molecule (s) are dyes, especially flavins, or (metallo-) porphyrins or derivatives thereof.

12.) Modified nucleic acid oligomer according to one of claims 1 to 5, characterized in that one or more of the electron donor and / or electron acceptor molecule (s) are nicotinamides or quinones, in particular pyrrolo-

Quinoline quinones (PQQ) or derivatives thereof.

13.) Modified nucleic acid oligomer according to one of the preceding claims, characterized in that the modified nucleic acid oligomer can bind sequence-specific single-stranded DNA, RNA and / or PNA.

14.) Modified nucleic acid oligomer according to claim 13, characterized in that the modified nucleic acid oligomer is a deoxyribonucleic acid, ribonucleic acid, a peptide nucleic acid oligomer or a nucleic acid oligomer with a structurally analogous backbone.

15.) Modified nucleic acid oligomer according to any one of the preceding claims, characterized in that the catalytically redox-active unit alternatively covalently to one of the phosphoric acid, carboxylic acid or amine groups or to a sugar, in particular to a sugar-hydroxy group, the Nucleic acid-

Oligomer backbone is bound.

16.) Modified nucleic acid oligomer according to one of claims 1-14, characterized in that the catalytically redox-active unit alternatively covalently to one

Thiol, hydroxy, carboxylic acid or amine group of a modified base of the nucleic acid oligomer is attached.

17.) Modified nucleic acid oligomer according to claim 16, characterized in that the reactive thiol, hydroxy, carboxylic acid or amine group of the base covalently via a branched or unbranched part of the molecule

Composition and chain length is bound to the base, the shortest continuous connection between the thiol, hydroxy, or carboxylic acid Amine group and the base is a branched or unbranched part of the molecule with a chain length of 1-20 atoms, in particular 1-14 atoms.

18.) Modified nucleic acid oligomer according to one of claims 15 to 17, characterized in that the catalytically redox-active unit is attached to one end of the nucleic acid oligomer backbone or to a terminal, modified base.

19.) Modified nucleic acid oligomer according to one of the preceding claims, characterized in that the catalytically redox-active unit has catalytic activity after binding to the nucleic acid oligomer.

20.) Modified nucleic acid oligomer according to one of the preceding claims, characterized in that the catalytically redox-active unit has electrocatalytic activity after binding to the nucleic acid oligomer.

21.) Modified nucleic acid oligomer according to one of the preceding claims, characterized in that several catalytically redox-active units are linked to the nucleic acid oligomer.

22.) Method for producing a modified nucleic acid oligomer as defined in one of the preceding claims, characterized in that a catalytically redox-active unit is covalently linked to a nucleic acid oligomer.

23.) Method for producing a modified nucleic acid oligomer according to claim 22, characterized in that the catalytically redox-active unit is bound to a nucleic acid oligomer by covalent attachment of one or more electron donor molecule (s).

24.) Method for producing a modified nucleic acid oligomer according to claim 23, characterized in that the catalytically redox-active unit by adding one or more electron acceptor molecule (s) and / or one or more macromolecules and / or one or more Proteins is completed.

25.) Method for producing a modified nucleic acid oligomer according to claim 22, characterized in that the catalytically redox-active unit is bound to a nucleic acid oligomer by covalent attachment of one or more electron acceptor molecules.

26.) Method for producing a modified nucleic acid oligomer according to

Claim 25, characterized in that the catalytically redox-active unit is completed by adding one or more electron donor molecule (s) and / or one or more macromolecules and / or one or more proteins.

27.) Method for producing a modified nucleic acid oligomer according to claim 22, characterized in that the catalytically redox-active unit by covalently attaching one or more macromolecules to one

Nucleic acid oligomer is attached.

28.) Method for producing a modified nucleic acid oligomer according to

Claim 27, characterized in that the catalytically redox-active unit by adding one or more electron acceptor molecule (s) and / or one or more electron donor molecule (s), one or more

Macromolecules is completed.

29.) Method for producing a modified nucleic acid oligomer according to claim 22, characterized in that the catalytically redox-active unit is linked to a nucleic acid oligomer by covalent attachment of one or more proteins.

30.) Method for producing a modified nucleic acid oligomer according to

Claim 29, characterized in that the catalytically redox-active unit is completed by adding one or more electron acceptor molecule (s) and / or one or more electron donor molecule (s) and / or one or more macromolecules.

31.) Method for producing a modified nucleic acid oligomer according to one of claims 22 to 30, characterized in that the nucleic acid oligomer alternatively by one or more amide formations with amine or with acid groups of the catalytically redox-active unit, by one or more Ester formation with alcohol or with acid groups of the catalytically redox-active

Unit, by thioester formation with thio-alcohol or with acid groups of the catalytically redox-active unit or by condensation of one or more amine groups of the nucleic acid oligomer with aldehyde groups of the catalytically redox-active unit and subsequent reduction of the resulting carbon-nitrogen double bond is bound to the catalytically redox-active unit.

32.) Method for producing a modified nucleic acid oligomer according to one of claims 22 to 30, characterized in that one or more branched or unbranched covalently to the catalytically redox-active unit

Molecular parts of any composition and chain length is attached and the branched or unbranched molecular parts alternatively have a reactive amine, hydroxy, thiol, acid or aldehyde group for covalent attachment to a nucleic acid oligomer.

33.) Method for producing a modified nucleic acid oligomer according to claim 32, characterized in that the shortest continuous connection between the nucleic acid oligomer and the catalytically redox-active unit is a branched or unbranched part of the molecule with a chain length of 1-20 atoms, in particular of 1 - 14 atoms, is.

34.) Modified conductive surface, characterized in that one or more types of modified nucleic acid oligomers according to one of claims 1 to 21 are bound to a conductive surface.

35.) Modified conductive surface according to claim 34, characterized in that the surface consists 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.

36.) Modified conductive surface according to claim 34, characterized in that the surface consists of a semiconductor, in particular a semiconductor selected from the group of carbon, silicon, germanium and tin.

37.) Modified conductive surface according to claim 34, characterized in that the surface consists 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 of the elements of groups 15 and 16, or a binary compound of the elements of groups 11 and 17, in particular of a Cu (l) halide or an Ag (l) halide.

38.) Modified conductive surface according to claim 34, characterized in that the surface consists of a temporary connection of the elements of groups 11, 13 and 16 or a ternary connection of elements of groups 12, 13 and 16.

39.) Modified conductive surface according to claims 34 to 38, characterized in that the connection of the modified nucleic acid oligomers to the conductive surface is carried out covalently or by chemical or physisorption.

40.) Modified conductive surface according to one of claims 34 to 39, characterized in that alternatively one of the phosphoric acid, carboxylic acid, amine or a sugar group, in particular a sugar-hydroxy group, the

Nucleic acid oligomer backbone is bound covalently or by chemical or physical sorption to the conductive surface.

41.) Modified conductive surface according to one of claims 34 to 39, characterized in that alternatively a thiol, hydroxy, carboxylic acid or amine group of a modified base of the nucleic acid oligomer covalently or by

Chemical or physisorption is bound to the conductive surface.

42.) Modified conductive surface according to claims 40 or 41, characterized in that the modified nucleic acid oligomer is bonded to the conductive surface via a group at the end of the nucleic acid oligomer backbone or via a group of a terminal, modified base.

43.) Modified conductive surface according to claims 34 to 42, characterized in that branched or unbranched on the conductive surface

Molecular parts of any composition and chain length covalently or through

Chemi- or physisorption are attached and the modified nucleic acid oligomers are covalently attached to these parts of the molecule.

44.) Modified conductive surface according to claim 43, characterized in that the shortest continuous connection between the conductive surface and the nucleic acid oligomer is a branched or unbranched part of the molecule with a chain length of 1 to 20 atoms, in particular 1 to 12 atoms.

45.) Modified conductive surface according to claims 43 or 44, characterized in that the branched or unbranched part of the molecule alternatively to a phosphoric acid, carboxylic acid, an amine or a sugar group, in particular a sugar-hydroxyl group of the nucleic acid oligomer backbone or a thiol, hydroxyl, carboxylic acid or amine group of a modified base of the nucleic acid oligomer is attached.

46.) Modified conductive surface according to claim 45, characterized in that the branched or unbranched part of the molecule to a phosphoric acid,

Sugar hydroxy, carboxylic acid or amine group at the end of the nucleic acid

Oligomer backbone or a thiol, hydroxy, carboxylic acid or amine group of a terminal, modified base is bound.

47.) Modified conductive surface according to one of claims 34 to 46, characterized in that in each case predominantly one type of modified

Nucleic acid oligomers is bound in a spatially limited area of the conductive surface.

48.) Modified conductive surface according to one of claims 34 to 46, characterized in that in each case only one type of modified nucleic acid oligomers in a spatially limited area of the conductive

Surface is attached.

49.) Method for producing a modified conductive surface as defined in claims 34 to 48, characterized in that one or more types of modified nucleic acid oligomers are applied to a conductive surface.

50.) Process for producing a modified conductive surface as in the

Claims 34 to 48 defined, characterized in that one or more

Types of nucleic acid oligomers are applied to a conductive surface and then a modification of the nucleic acid oligomers is carried out by a method according to claims 22 to 33.

51.) Process for producing a modified conductive surface according to

Claim 49 or 50, characterized in that the nucleic acid oligomers or the modified nucleic acid oligomers are hybridized with the complementary nucleic acid oligomer strand in each case and are applied to the conductive surface in the form of the double strand hybrid.

52.) Method for producing a modified conductive surface according to claims 49 or 50, characterized in that the nucleic acid Oligomer or the modified nucleic acid oligomer is applied to the conductive surface in the presence of further chemical compounds, which are also bound to the conductive surface.

53.) Method for the electrochemical detection of oligomer hybridization events, characterized in that one or more modified conductive surfaces, as defined in claims 34 to 48, are brought into contact with nucleic acid oligomers and then a detection of the electrical communication between the catalytic redox-active unit and the respective conductive surface.

54.) Method according to claim 53, characterized in that the detection is carried out cyclovoltametric, amperometric, potentiometric or by conductivity measurement.

55.) Method for electrochemical detection according to claims 53 or 54, characterized in that the electrochemical detection is started by adding a substrate to the catalytically redox-active unit bound to the conductive surface via a nucleic acid oligomer.

56.) Method according to claim 55, characterized in that the addition of

Substrate to which the catalytically redox-active unit bound to the conductive surface via a nucleic acid oligomer is limited to a region of the conductive surface with one or more modified nucleic acid oligomer types.

57.) Method according to claim 55 or 56, characterized in that the substrate is a free redox-active substance which is not bound to a nucleic acid oligomer but is in contact with the nucleic acid oligomer and is selectively oxidizable and reducible at a potential φ , the potential of

Condition 2.0 V> φ> - 2.0 V, measured against a normal hydrogen electrode, is sufficient.