MXPA01003985A - Method for the electrochemical detection of nucleic acid oligomer hybrids - Google Patents

Abstract

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

Description

METHOD TO DETECT ELECTROLYTICALLY HYBRID NUCLEIC ACIDS FIELD OF THE INVENTION The present invention is directed to a modified nucleic acid oligomer, as well as to a method for electrolytically detecting hybridization events of specific sequence nucleic acid oligomers.

BACKGROUND OF THE INVENTION In general, gel electrophoresis methods with autoradiographic or optical detection are used for the analysis of the DNA and RNA sequence, for example, in the diagnosis of diseases, in toxicological test procedures, in research and development in genetics, as well as in the agricultural and pharmaceutical sectors. To illustrate the gel electrophoresis method with more significant optical detection (Sanger method), Figure Ib shows a DNA fragment with primer. In the Sanger method, a solution containing DNA is divided into four samples and the primer of each sample is covalently modified with a fluorescent dye that emits at a different wavelength. As illustrated in Figure Ib, the deoxyribonucleoside triphosphate of bases A (adenine), T (thymine), C (cytosine) and G (guanine), ie, dATP, dTTP, dCTP and dGTP is added to each sample to enzymatically replicate the chain alone, starting at the primer, by means of DNA polymerase I. In addition to the four deoxyribonucleoside triphosphates, each reaction mixture also contains sufficient 2 ', 3' -dideoxy analogue (FIG. one of these nucleoside triphosphates as a blocking base (one of each of the four possible blocking bases per sample) to terminate replication at all possible binding sites. After combining the four samples, all the lengths of replicated DNA fragments having the specific fluorescence of the blocking base result and can be classified by gel electrophoresis, in accordance with the length and characterized using fluorescent spectroscopy (Figure 1). Another method of optical detection is based on the accumulation of fluorescent dyes, such as for example ethidium bromide in oligonucleotides. The fluorescence of these dyes increases approximately 20-fold with the free solution of the dye when they accumulate in double-stranded DNA or in RNA and, therefore, can be used to detect hybridized DNA or RNA. In radiolabelling, 32P is incorporated into the phosphate backbone of the oligonucleotides, where 32P will normally be added to the 5'-hydroxyl end by means of polynucleotide kinase. After this, the tagged DNA is preferably cleaved, under defined conditions, into one of each of the four nucleotide types, such that it results in an average of one strand per strand. Thus, for a given base type, chains extending from the 32P label to the position of that base are present in the reaction mixture (if there are multiple appearances of the base, in accordance with these, length chains will result). variables). The four mixtures of fragments are then separated by gel electrophoresis in four bands. After this, an autoradiogram of the gel is prepared, from which the sequence can be read directly. A few years ago, an additional method for DNA sequencing was developed on the basis of optical (or autoradiographic) detection, namely, sequencing by means of oligomer hybridization (see, for example, Drmanac et al., Genomics 4, (1989), pp. 114-128 or Bains et al., Theor. Biol. 135, (1988), pp. 303-307). In this method, a complete set of short oligonucleotides or oligomers (probe oligonucleotides), for example, all 65,536 possible combinations of bases A, T, C and G of an oligonucleotide octamer are attached to a support. This connection occurs in an ordered grid consisting of 65,536 test sites, wherein each major amount of a combination of oligonucleotides defines a test site and the position of each individual test site (combination of oligonucleotides) is known. In this hybridization matrix, the oligomer part, a DNA fragment whose sequence is to be determined, the target, is labeled with a fluorescent dye (or 32P) and hybridized under conditions that allow only the formation of a specific double chain. In this way, the target DNA fragment binds only to the oligomers (in this example, to the octamers) whose complementary sequence corresponds exactly to a portion (an octamer) of its own sequence. In this way, all the oligomer sequences (octamer sequences) present in the fragment are determined by means of optical (or autoradiographic) detection of the binding position of the hybridized DNA fragment. Due to the overlap of neighboring oligomeric sequences, the continuous sequence of the DNA fragment can be determined, using suitable mathematical algorithms. The advantages of this method lie, among other things, in the miniaturization of the sequencing and, thus, in the enormous amount of data that can be captured simultaneously in an operation. The primer and separation by gel electrophoresis of the DNA fragments can be provided. This principle is demonstrated by the example of Figure 2 for a DNA fragment of 13 bases long. The use of radioactive labels in the DNA / RNA sequencing has several disadvantages associated, such as, for example, elaborating the safety precautions legally required when dealing with radioactive materials, radiation, a spatially limited resolution capacity (maximum 1 mm2). ) and sensitivity that is only high when radiation from the radioactive fragments act on an X-ray film for an appropriately long time (from hours to days). Although spatial resolution can be increased by additional hardware and software and the detection time can be reduced by means of ß scanners, these two options include considerable additional costs. Some of the fluorescent dyes that are commonly used to label DNA (eg, ethidium bromide) are mutagenic and require appropriate safety precautions, as required by the use of autoradiography. In almost every case, the use of optical detection requires the use of one or more laser systems and, in this way, of experienced and experienced personnel. 52/109 ^ g ^^ i appropriate safety precautions. The actual detection of fluorescence requires additional hardware, such as, for example, optical components for the amplification and, in the case of variable stimulation and query wavelengths as in the Sanger method, a control system. Thus, depending on the stimulation wavelengths required and the desired detection performance, they can result in considerable investment costs. During sequencing through hybridization in the oligomeric part, detection is even more expensive, because, in addition to the stimulation system, high-resolution CCD cameras (cameras with charge-coupled devices) are needed to detect two-dimensional zones fluorescent In this way, although for the sequencing of DNA / RNA There are quantitative and extremely sensitive methods, these methods are slow, require elaborate sample preparation and expensive equipment and generally do not exist as portable systems.

DESCRIPTION OF THE INVENTION Therefore, the object of the present invention is to create, to detect oligomeric nucleic acid hybrids, an apparatus and a method that do not have the disadvantages of the state of the art.

In accordance with the present invention, this object is solved by the modified oligonucleotide, according to independent claim 1, by the method of producing a modified oligonucleotide, according to the independent claims 9 and 10, by means of the modified conductive surface, of according to independent claim 11, the method for producing a modified conductive surface, in accordance with independent claim 21, and a method for electrochemically detecting hybridization events of oligomers, according to independent claim 27. following abbreviations and terms: 52/109 guanine adenine cytosine thymine base A, G, T, or C bp base pair or base pairs nucleic acid At least two nucleotides covalently linked or at least two pyrimidine bases (eg, cytosine, thymine or uracil) or purines (eg, adenine or guanine) covalently linked. The term "nucleic acid" refers to any major chain of the pyrimidinic or choricly linked bases, such as, for example, the sugar-phosphate backbone of the DNA, cDNA, or RNA, to a NPC peptide backbone, or to analogous structures. (for example, a main chain of phosphoramide, thiophosphate or dithiophosphate). The essential feature of a nucleic acid according to the present invention is that it can bind, specifically in sequence, with natural cDNAs or RNAs. 52/109 aah? É &fc ^ ** ^ to * tf '^ t', A ^^, .... &, ^ i¿ ^ &. ^^^. ^^ a ^ oligomer of base-length nucleic acid that does not Nucleic acids are additionally specified (e.g., nucleic acid octamer: a nucleic acid having any backbone in which 8 pyrimidine or purine bases are covalently linked together). oligomer Equivalent to nucleic acid oligomer. oligonucleotide Equivalent to oligomer or oligomer of nucleic acids, thus, for example, a fragment of DNA, APN, or RNA with base length that is not further specified. oligo Abbreviation for oligonucleotide dATP Deoxyribonucleoside triphosphate from A (portion of DNA with base A and two additional phosphates to create a longer DNA or oligonucleotide fragment). dGTP G deoxyribonucleoside triphosphate (portion of DNA with base G and two additional phosphates to create a longer DNA or oligonucleotide fragment). 52/109 -^^^^ ^ and ^ ^^ - í 52/109 mgjH! ^^ Are C-H bonds, single C-C bonds, C = C double bonds or C = C triple bonds replaced by C-N bonds?, C = N, C-P, C = P, C-O, C = 0, C-S, or C = S. Binding A molecular bond between two molecules or between a surface atom, surface molecule or molecular group of surface and another molecule. The linkers can usually be purchased in the form of alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl chains, the chain will be derived in two places with reactive groups (identical or different). These groups form a covalent chemical bond in simple / known chemical reactions with the appropriate reaction partner. The reactive groups can also be photoactivatable, ie the reactive groups are activated only by light of a specific or random wavelength. Preferred binders are those having a chain length of 1 to 20, especially a chain length of 1 to 14, the chain length 52/109"s ** J ^ l ^ l ^ - ^ «r * ^ 52/109 that are naturally present in the oligomer of nucleic acids or that have been fixed thereto by means of modification and "n" is any integer, especially a number between 1 and 20. (nx RSS- An oligomer of acids nuclei to which separator) -oligo are joined each of the n disulfide functions by means of a separator and any R residue saturates the disulfide function. Each separator for the connection of the disulfide function with the oligomer of nucleic acids can have a different chain length (the shortest continuous link between the disulfide function and the nucleic acid oligomer), especially any chain length between 1 and 14 each one. These separators can, in turn, be linked to several reactive groups that are naturally present in the oligomer of nucleic acids or that have been fixed thereto by modification. The literal "n" is any integer, especially a number between 1 and 20. oligo-separator- Two nucleic acid oligomers 52/109 ^ ¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿¿SaM? Íái SS-separador- different or identical that are united oligo to each other by means of a disulfide bridge, the disulfide bridge is linked to the oligomers of nucleic acids by means of any two separators and the two spacers have potentially different chain lengths (the shortest continuous link between the disulfide bridge and the respective nucleic acid oligomer), especially any chain length between 1 and 14 and these spacers, in turn, potentially will bind to several reactive groups which are naturally present in the nucleic acid oligomer or which have been fixed thereto by means of modification. PQQ pyrroloquinoline quinone; corresponds to 4,5-dihydro-4,5-dioxo-lH-pyrrolo- [2, 3-f] -quinoline-2,7,9-tricarboxylic acid) TEATFB tetraethylammonium tetrafluoroborate sulfo-NHS N-hydroxysulfosuccinimide EDC (3-dimethylaminopropyl) -carbodiimide HEPES N- [2-hydroxyethyl] piperazine-N '- [2-ethanesulonic acid] Tris trishydroxymethylamino methane 52/109 EDTA ethylenediamine tetraacetate (sodium salt) cystamine (H2N-CH2-CH2-S-): Modified Surfaces / Mica Electrodes Platelets of Muskovite platelets, support for the application of thin layers. Au-S-ss-oligo- Gold film on mica having a covalently applied monolayer PQQ of the oligonucleotide derived from a single 12-bp chain (sequence: TAGTCGGAAGCA). Here, the terminal phosphate group of the oligonucleotide at the 3 'end is esterified with (HO- (CH2) 2-S) 2 to P-0- (CH2) 2- SS- (CH2) 2-OH, homolytically cleaving the bond SS and producing an Au-SR link each. The terminal thymine base at the 5 'end of the oligonucleotide is modified at carbon C-5 with -CH = CH-CO-NH-CH2-CH2-NH2 a and the residue is in turn bound via its amino group free with a carboxylic acid group of the PQQ by means of amination. Au-S-ds -oligo- Au-S-ss-oligo-PQQ that hybridizes with the PQQ oligonucleotide complementary to the ss-oligo (sequence: TAGTCGGAAGCA). 52/109 t £ 52/109 r¡ffitlrfft? rftitriPfaa- "« > > * - »* & * ^ g á ^ .¿;., The present invention is directed to an oligomer of nucleic acids that is modified by means of the chemical connection or union of a substance with redox activity. According to the present invention, the nucleic acid oligomer is a compound consisting of at least two covalently linked nucleotides or at least two pyrimidine bases (eg, cytosine, thymine or uracil) or pyrrhus (eg, adenine or guanine) covalently linked, preferably a DNA, RNA or NPC fragment. As used in the 52/109 present, the term "nucleic acid" refers to any major chain of the pyrimidine or chorionic acid bases covalently linked, such as, for example, to the sugar-phosphate backbone of the DNA, CDNA or RNA, to an APN peptide backbone or to analogous backbone structures such as, for example, a thiophosphate, a dithiophosphate or a phosphoramide backbone. The essential feature of a nucleic acid, according to the present invention, is that it can bind, in a specific manner with the sequence, with natural cDNAs or RNAs. The terms "(probe of) oligonucleotide", "nucleic acid" and "oligomer" are used as alternatives to the term "oligomer of nucleic acids". The substance with redox activity can be oxidized and selectively reduced to a potential f, where f satisfies the condition 2.0 V = f = - 2.0 V. The potential here refers to the substance with redox activity, free and unmodified in a adequate solvent, measured against the normal hydrogen electrode. In accordance with the present invention, the potential range 1.7 V = f = - 1.7 V, the interval 1.4 V >is preferred.; f > - 1.2 V is particularly preferred and the 0.9 V > f > - 0.7 V, in which the substances with redox activity of the application example are reduced and reoxidized, particularly that which has the highest preference. In addition, the present invention is directed to a conductive surface to which a nucleic acid oligomer having a substance with redox activity is bound, either directly or indirectly (via a separator). In addition, the present invention is directed to a method for producing a modified conductive surface, wherein a modified nucleic acid oligomer is applied to a conductive surface. According to a further aspect, the present invention is directed to a method that allows the electrochemical detection of molecular structures, especially the electrochemical detection of DNA / RNA / NPC fragments in a probe solution by means of the hybridization of acid oligomers. nucleic of specific sequence. The detection of hybridization events by means of electrical signals is a simple and inexpensive method and, in a variation of a battery operated sequencing device, allows on-site application.

Binding of a portion with redox activity to a nucleic acid oligomer For the method of the present invention, it is necessary to bind a substance with redox activity to a nucleic acid oligomer. 52/109 t ^ g ^ gjg ^^^ Ij ^ ggBg ^^ oligomer of nucleic acids. In accordance with the present invention, any substance with redox activity can be used for this purpose so long as it is selectively oxidizable and reducible to a potential f satisfying condition 2.0 V > f = - 2.0 V. The potential here refers to the substance with redox activity, free and unmodified, in a suitable solvent, measured against the normal hydrogen electrode. In accordance with the present invention, the potential range 1.7 V > f > - 1.7 V, particularly the potential interval 1.4 V = f = - 1.2 V is preferred and the interval that has the highest preference is the 0.9 V > f > - 0.7 V, in which the substances with redox activity of the application example are reduced and reoxidized. In accordance with the present invention, it is understood that the term "selectively oxidizable and reducible" refers to a redox reaction, ie, that it yields or takes an electron, which occurs selectively at the location of the substance with redox activity . In this way, finally, no other part of the nucleic acid oligomer is reduced or oxidized by means of the applied potential, but rather, exclusively the substance with redox activity bound to the nucleic acid oligomer. In accordance with the present invention, it is understood that a substance with redox activity refers to any molecule which, in the range of electrochemically accessible potential of the respective support surface (electrode), can be electro-oxidized / electroreduced by the application of a voltage external to that electrode. In addition to the common substances with organic and inorganic redox activity, such as, for example, hexacyanoferrates, ferrocenes, acridines, or phthalocyanines, dyes with redox activity, such as for example, (metallo-) porphyrins of the general formula 1, metallo-) chlorophylls of general formula 2, or the (metallo-) bacteriochlorophylls of general formula 3, natural (colored) oxidation agents, such as, for example, flavins of general formula 4, pyridine-nucleotides of general formula 5 or pyrrolo- quinoline quinones (PQQ) of the general formula 6 or other quinones, such as, for example, 1,4-benzoquinones of the general formula 7, 1, 2-benzoquinones of the general formula 8, 1,4-naphthoquinones of the general formula 9,1 2-naphthoquinones of general formula 10 or 9, 10-anthraquinones of general formula 11, are particularly suitable for binding to the probe oligonucleotide. 52/109? Gu? ££ ^^^ M ^ i! J ^ Sj2g¡? ^^ Formula 1 Formula 2 Formula 3 Where M = 2H, Mg, Zn, Cu, Ni, Pd, Co, Cd, Mn, Fe, Sn, Pt, etc .; R a R 12 are, independently of each other, H or any alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituents. 52/109. , ^ ^^? ^^ .., ^. ^ -, r jilfili ^^ Formula 4 Formula 5 Formula 6 Formula 7 Formula 9 Formula 10 Formula 11 -._ S & -t - ^^ - ¿«. ' - • - * ** - < ** e * ~ 8 *** ~ * Ri to R8 are, independently of each other, H or any alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituents. In accordance with the present invention, a substance with redox activity is covalently linked to an oligonucleotide by means of the oligonucleotide which reacts with the substance with redox activity. This linkage can be achieved in three different ways: a) the reactive group to form a linkage in the nucleic acid oligomer is a phosphoric acid, sugar-C-3-hydroxy, free carboxylic acid or amino group of the oligonucleotide backbone, especially a group at one of the two ends of the oligonucleotide backbone. The free groups phosphoric acid, C-3-hydroxy, carboxylic acid or amino terminal sugars show an increase in reactivity and, thus, easily undergo typical reactions, such as, for example, amidation with amino groups (primary or secondary) or with acid groups, esterification with alcohols (primary, secondary or tertiary) or with acid groups, the formation of thioester with thioalcohols (primary, secondary or tertiary) or with acid groups, or the condensation of amine and aldehyde with the subsequent reduction of the CH = N bond resulting in a bond or CH-NH bond. The coupling group required for the union 52/109 covalent of the substance with redox activity (acid function, amine, alcohol, thioalcohol or aldehyde) or is present naturally in the substance with redox activity or is obtained by means of chemical modification of the substance with redox activity. b) The nucleic acid oligomer is modified with a reactive group on the oligonucleotide backbone or on a base by means of a covalently linked molecular moiety (spacer) of any composition and chain length (representing the shortest continuous link between the structures to be joined), especially with chain length from 1 to 14. The modification preferably occurs at one end of the oligonucleotide backbone or at a terminal base. The spacers can be, for example, an alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituent. The possible simple reactions for the formation of the covalent bond between the substance with redox activity and the oligomer of nucleic acids thus modified are, as described in a), the amidation from an acid and an amino group, the esterification from of an acid and an alcohol group, the formation of thioester from an acid and a thioalcohol group or the condensation of aldehyde and amine with the subsequent reduction of the CH = N bond resulting in a 52/109 '^^ fa * ^, J¿, IM, < ** CH2-NH binding. According to a preferred embodiment, the nucleic acid oligomer is modified using a substance with redox activity that exhibits regions that have a predominantly planar, flattened, orbital pp-system, such as, for example, the PQQ of Example 1 or the quinones of Formulas 5 or 7 to 12 or the porfinoid structures of Formulas 1 to 4 or the pyridine nucleotides of general formula 6, or the derivatives of these substances with redox activity. In this case, the separator by means of which the substance with redox activity is bound to the oligomer of nucleic acids can be selected in such a way that the plane of the orbitals p of the substance with redox activity can autoacomodarse in parallel to the p-p orbital bases of the nucleic acid oligomer that limit the substance with redox activity. This arrangement or spatial arrangement of the substance with redox activity with partially flat p-p orbitals extended in a flat proves to be particularly favorable. c) In synthesizing the nucleic acid oligomer, a terminal base will be replaced by the substance with redox activity. In accordance with the present invention, the binding of the substance with redox activity to 52/109 oligonucleotide, as described in a) and b) may occur before or after the oligonucleotide is attached to the conductive surface. The connection of the substance with redox activity to the oligonucleotide attached to the conductive surface then occurs in the same way as described in a) and b). If there are several combinations of different oligonucleotides (test sites) on a common surface, it is advantageous to normalize or standardize the (covalent) binding of the substance with redox activity to the probe oligonucleotides over the entire surface by the appropriate choice of the reactive group in the free ends of the probe oligonucleotide from the various test sites.

The conductive surface In accordance with the present invention, the term "conductive surface" refers to any support having a surface of any thickness, electrically conductive, especially surfaces made of platinum, palladium, gold, cadmium, mercury, nickel, zinc , coal, silver, copper, iron, lead, aluminum and manganese. In accordance with the present invention, the terms "electrode" and "conductive surface (support)" are used as alternatives to "conductive surface". In addition, any 52/109 ..? A .. ^ ,, a. * ¿? ^^^ - i - ti - i semiconducting surfaces doped or undoped of any thickness. All semiconductors are useful in the form of pure substances or as mixtures. Examples include, but are not limited to, carbon, silicon, germanium, tin a, and halogenides of Cu (I) and Ag (I) of any crystal structure. In the same way, all binary compounds of any composition and any structure of the elements of groups 14 and 16, of the elements of groups 13 and 15, and of the elements of groups 15 and 16 are suitable. the ternary compounds of any composition and any structure of the elements of groups 11, 13 and 16 or of the elements of groups 12, 13 and 16 are used. The designations of the groups of the periodic system refer to the recommendation of the IUPAC from 1985.

Binding of an oligonucleotide to the conductive surface In accordance with the present invention, a nucleotide is directly linked or by a linker / separator to the atoms or molecules of the support surface of a conductive support surface of the type described above. This connection can be made in three different ways: a) the surface is modified in such a way that a reactive molecular group is accessible. This 52/109 fflrr ir ^, &r? * t -U? * km ° ^ - may occur by direct derivation of surface molecules, for example, by chemical oxidation / reduction in wet or electrochemical . Thus, for example, the surface of graphite electrodes can be wet chemically supplied with aldehyde or carboxylic acid groups by means of oxidation. Electrochemically it is possible, for example, by means of the reduction in the presence of aryl diazonium salts to couple the corresponding aryl radical (with some function, that is, provided with a reactive group) or by means of oxidation in the presence of R '. C02H for coupling the radical R '(with function) to the surface of the graphite electrode. An example of the direct modification of semiconductor surfaces is the derivation of silicon surfaces in reactive silanols, that is, silicon supports that on the surface have Si-OR '' groups where both R "and R 'are any organic waste. with function (for example, substituent to alkyl, alkenyl, alkynyl, heteroalkenyl or heteroalkynyl). Alternatively, the entire surface can be modified by covalently joining the reactive group of a bifunctional linker, such that a monomolecular layer consisting of any molecules and containing a reactive, preferably terminal, group on the surface results. It is understood that the term "bifunctional linker" is 52/109 refers to any molecule of any chain length, especially with chain lengths of 2 to 14, which have two identical reactive (homobifunctional) or two different (heterobifunctional) reactive molecular groups. If several different test sites are going to form on the surface using the methodology of photolithography, then at least one of the reactive groups of the homobifunctional or heterobifunctional linkers is a reactive photoinducible group, that is, a group that it becomes reactive only with irradiation with light of a specific or random wavelength. This binder is applied in such a way that the photoactivatable reactive group is available after the binder was covalently bound to the surface. The oligomers of nucleic acids are covalently bound to the surface thus modified and are themselves modified with a reactive group, preferably near one end of the nucleic acid oligomer, by a separator of any composition and chain length, especially with length of chain from 1 to 14. The reactive group of the oligonucleotide is a group that reacts directly (or indirectly) with the modified surface to form a covalent bond. In addition, an additional reactive group can be linked to nucleic acid oligomers near its second end, this group 52/109 reagent, in turn, will be bonded, as described above, directly or through a separator of any composition and chain length, especially with chain length from 1 to 14. In addition, as an alternative to this additional reactive group , the substance with redox activity, can bind to this second end of the nucleic acid oligomer. b) The oligomer of nucleic acids that will be applied to the conductive surface is modified with one or more reactive groups by means of a covalently bound separator, of any composition and chain length, especially with chain length from 1 to 14, these groups reagents will preferably be located near one end of the nucleic acid oligomer. The reactive groups are groups that can react directly with the unmodified surface. Some examples are: (i) oligomer of nucleic acids derived with thiol- (HS-) or disulfide- (SS-) of general formula (nx HS-separator) -oligo, (n X RSS-separator) -oligo or oligo- separator-SS-separator-oligo that reacts with a gold surface to form gold-sulfur bonds or (ii) amines that accumulate on platinum or silicon surfaces by means of chemisorption or fisisorption. In addition, an additional reactive group can be linked to nucleic acid oligomers near its second end. 52/109 This reactive group, in turn, will be bonded, as described above, directly or by means of a separator of any composition and chain length, especially with chain length of 1-14. In addition, as an alternative to this additional reactive group, the substance with redox activity can be attached at this second end of the oligonucleotide. Particularly, oligomers of nucleic acids that are modified with several bridges of thiol or disulphide bridged with separator ((nx HS-10 separator) -oligo or (nx RSS-separator) -oligo) have the advantage that these oligomers of nucleic acids can be applied to the conductive surface at a particular fixation angle (the angle between the normal surface and the helix axis of a nucleic acid oligomer helical double-stranded or between the normal surface and the axis perpendicular to the base pairs of a non-helical and double-stranded nucleic acid oligomer) if the separators that bind the thiol or disulfide functions with the oligomer of nucleic acids possess a increase or decrease in chain length, as observed from one end of the nucleic acid. c) The phosphoric acid, C-3-hydroxy, carboxylic acid or amine groups of the oligonucleotide backbone, especially the terminal groups, are used as the reactive group in the oligomer of 52/109 probe nucleic acids. The groups phosphoric acid, sugar-C-3-hydroxy, carboxylic acid or amine show a higher reactivity and, thus, easily suffer typical reactions, such as for example, amination with amino groups (primary or secondary) or acid, esterification with alcohols (primary, secondary, or tertiary) or acid groups, the formation of thioester with thioalcohols (primary, secondary or tertiary) or acid groups, or the condensation of amine and aldehyde with the subsequent reduction of the resulting CH = N bond in a CH2-NH bond. In this case, the coupling group required for the covalent attachment of the phosphoric acid group, C-3-hydroxy sugar, carboxylic acid or amine is part of the surface derivatization with a (monomolecular) layer of any molecule length, as described in a) of this section or the phosphoric acid, C-3-hydroxy, carboxylic acid or amine group can react directly with the unmodified surface, as described in b) in this section. In addition, an additional reactive group can be attached to the oligonucleotides near its second end, this reactive group, in turn, will be bound, as described above, directly or through a separator of any composition and chain length, especially with length of chain 1 to 14. Also, as an alternative to this reactive group 52/109 3 & In addition, the substance with redox activity can be bound to this second end of the nucleic acid oligomer. Alternatively, binding of the oligonucleotide to the conductive surface can occur before or after binding the substance with redox activity to the oligonucleotide or before or after binding the separator provided with a reactive group for the binding of the substance with redox activity. Linking the already modified oligonucleotide to the conductive surface, ie, the binding to the surface, after binding the substance with redox activity to the oligonucleotide or after joining the separator provided with a reactive group for the binding of the substance with redox activity, occurs in the same way as described in a) to c) (in the section "binding of an oligonucleotide to the conductive surface"). During the production of the test sites, care must be taken when joining the single-stranded oligonucleotides to the surface because between the individual oligonucleotides a distance large enough remains to provide the necessary space for hybridization with the target oligonucleotide. To this end, among others, two different methods are presented to proceed: 1.) produce a modified support surface by joining a hybridized oligonucleotide, ie 52/109 deriving the support surface with a hybridized probe oligonucleotide, rather than with a single-stranded probe oligonucleotide. The oligonucleotide chain used for hybridization is not modified (the connection to the surface is effected as described in a) - c) in the section "binding an oligonucleotide to the conductive surface"). After this, the double strand of the hybridized oligonucleotide is thermally inhibited, thereby producing a modified single-stranded oligonucleotide bearing surface having a greater distance between probe oligonucleotides. 2.) Produce a modified support surface by joining a single-stranded or double-stranded oligonucleotide and adding during the derivation of the support surface a suitable monofunctional linker which, in addition to the single-stranded or double-stranded oligonucleotide, is also one to the surface (the surface bond is made as described in a) - c) in the section "binding an oligonucleotide to the conductive surface"). In accordance with the present invention, the monofunctional linker has a chain length which is identical to the chain length of the separator between the support surface and the oligonucleotide or which differs by a maximum of eight chain atoms. If it is used 52/109 double-stranded oligonucleotide for the derivatization of the support surface, the double strand of the hybridized oligonucleotide is thermally inhibited after joining the double-stranded oligonucleotide and the binder to the support surface, as described in the previous 1 .). By simultaneously joining a binder to the surface, the distance between the single stranded or double stranded nucleic acid oligomers that are likely to bind to the surface is increased. If a double stranded nucleic acid oligomer is used, this effect is further increased by the subsequent thermal dehybridization.

Method for electrolytically detecting hybrids of nucleic acid oligomers Advantageously, in accordance with the method of electrochemical detection, various probe oligonucleotides that vary in sequence, ideally all necessary combinations of the nucleic acid oligomer, apply to an oligomeric part or part of DNA to reliably detect the sequence of any white oligomer or of a white DNA (fragmented) or to search and detect in a specific manner with the sequence, mutations in the target. To this end, the atoms or molecules of the support surface of a defined area 52/109 .t - ^ - ^ 'I- ^ s- ^' aaia-A-s .., - ^ ... ^ and JÉÉÉ¡llftÍ | fe (a test site) are linked to AD / RNA / APN oligonucleotides with known sequence, although random, on a conductive support surface, as described above. However, in the more general mode, the DNA part can also be derived with a single probe oligonucleotide. Preferred probe oligonucleotides are oligomers of nucleic acids (fragments of DNA, RNA or PNA) with a base length of 3 to 50, preferably with a length of 5 to 30, particularly preferred with a length of 7 to 25. In accordance with present invention, a substance with redox activity is attached to the probe oligonucleotides, either before or after they are attached to the conductive surface. If the modification of the probe oligonucleotides occurs before binding to the conductive surface, then the modified probe oligonucleotides are bound to the conductive surface as described above. Alternatively, the unmodified probe oligonucleotides attached to the conductive surface are modified with a substance with redox activity, at the second free end of the oligonucleotide chain, directly or indirectly by means of a separator. In both cases, it results a surface hybrid with a general structure elec-separator-ss-oligo-separator- 52/109 ^ s ^ n? É ^., .. ^^,. ^, ^. ^, ^ iÉ ^ a ^ redox (Figure 3). The electrical communication between the support surface (conductive) and the substance with redox activity ("redox") bridged by means of a single-stranded oligonucleotide in the general structure 5 elec-separator-ss-oligo-separator-redox is weak or it is not present at all. Of course, bridges can also be made without separators or only with a separator (elec-ss-oligo-separator-redox or elec-separator-ss-oligo-redox). 10 In a subsequent step, the test sites are contacted with the oligonucleotide solution (blank) to be examined. Hybridization will occur only if the solution contains oligonucleotide chains that are complementary to the probe oligonucleotides attached to the conductive surface or at least broadly complementary. In the case of hybridization between the probe oligonucleotide and the target, there will be an increase in conductivity between the support surface and the substance with redox activity, because they are now bridged by means of the oligonucleotide composed of a double chain (shown schematically in Figure 3, using the example of the elec-separator-ss-oligo-separator-redox). Due to the change in electrical communication between the supporting surface (conductive) and the substance 52/109 If there is redox activity due to the hybridization of the probe oligonucleotide and the oligonucleotide chain (target) complementary to it, a sequence specific hybridization event can be detected using methods electrochemical, such as, for example, cyclic voltammetry, amperometry or conductivity measurements. In a particularly preferred embodiment of the present invention, a substance with redox activity is used which shows regions having a pp orbital system, predominantly flat, extending in a plane, such as, for example, the PQQ of Example 1 (cf. Figure 3), or the quinones of Formula 5 or 7 to 12 or the porfinoid structures of Formulas 1 to 4, the pyridine nucleotides of general formula 6 and the derivatives of these substances with redox activity. In this case, the separator between the oligomer of nucleic acids and the substance with redox activity is selected in such a way that the plane of the p orbitals of the substance with redox activity can be arranged in parallel to the pp orbitals of the pair of bases of the oligomer of nucleic acids hybridized with the complementary chain and that limits the substance with redox activity. This arrangement or spatial arrangement of the substance with redox activity with partially planar p-p orbitals, 52/109 2 * t? Spread in a plane proves to be particularly favorable for the electrical conductivity of double stranded nucleic acid oligomers. In cyclic voltammetry, the potential of a stationary work electrode changes linearly as a function of time. Starting at a potential in which there is no electrooxidation or reduction, the potential changes until the substance with redox activity is oxidized or reduced (ie, current flows). After passing through the oxidation or reduction operation, which in the current-voltage curve produces an initially increasing current, a maximum current (peak) and then the gradual reduction of the current, the direction of the fed potential is reversed. Then, in an inverted run, the behavior of the electrooxidation or electroreduction products is recorded. An alternative method of electrical detection, the amperometry, is possible thanks to the fact that the substance with redox activity is electrooxidated (electroreduce) applying an adequate constant electrode potential, although the re-reduction (reoxidation) of the substance is achieved with redox activity to its original state, without changing the electrode potential as in the cyclic voltammetry, but rather, by means of an appropriate reducing agent (oxidizing agent), added to the 52/109 white solution, closing the current circuit of the entire system. As long as the reducing agent (oxidizing agent) is present, or as long as the reducing agent (oxidizing agent) consumed is reduced again (re-oxidized) in the counter electrode, a current flows that can be amperometrically detected and that is proportional to the number of hybridization events.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be explained in more detail using the following application example and the accompanying drawings.

Fig. 1 shows a schematic illustration of the Sanger method of oligonucleotide sequencing; Fig. 2 shows a schematic illustration of the oligonucleotide sequencing by means of hybridization in one part; 52/109 Fig. 3 shows a schematic illustration of the surface hybrid of general structure elec-separator-ss-oligo-separator-redox with a 12-bp probe oligonucleotide of the exemplary sequence 5'- TAGTCGGAAGCA-3 '(left ) and Au-S-ss-oligo-PQQ in the hybridized state as an example of the mode of an elec-separator-ss-oligo-separator-redox; only a portion of the probe oligonucleotide having a complementary hybridized strand (right) is shown, the oligonucleotide binding to the surface occurs by means of a -S-CH2CH2- separator, and the connection or binding of the substance with PQQ redox activity occurs by means of the separator -CH2-CH = CH-CO-NH-CH2-CH2-NH-; Fig. 4 Shows a cyclic voltammogram of a test site consisting of Au-S-ss-oligo-PQQ (dotted line) as compared to an identical test site with fully hybridized target (Au-S-ds-oligo-PQQ , continuous line; 52/109 t *. -MOriKIt *** »* * ...... ^. ^ .. .... > ^ ..., .., ^ j Ü & ^^^^^ ^ ^ ^ ^ ^ ^^^^^ A ^ ^ Fig. 5 Shows a cyclic voltammogram of a completely hybridized test site (Au-S-ds-oligo-PQQ) (solid line) in comparison with a hybridized test site showing 2 mismatches of the base pair (Au-S-ds-oligo-PQQ with 2 bp mismatches, interrupted line).

Best Form for Carrying Out the Invention Figure 3 shows an exemplary test site with hybridized target (Au-S-ds-oligo-PQQ) with general structure elec-separator-ds-oligo-separator-redox. In the example of Figure 3, the support surface is a gold electrode. The bond between the gold electrode and the probe oligonucleotide is formed with the linker (HO- (CH2) 2-S) 2 / which was esterified to P-0- (CH2) 2-SS- (CH2) 2-OH with the terminal phosphate group at the 3 'end and, then, the homolytic cleavage of the SS bond on the gold surface, each produced an Au-S bond, with which the 2-hydroxy-mercaptoethanol and the oligonucleotide bridged with mercaptoethanol they co-adsorbed on the surface. The substance with redox activity in the example of Figure 3 is pyrrolo-quinoline quinone tricarboxylic (PQQ) and one of the three carboxylic acid functions of the PQQ (in the example, the C-7-C02H function) was used to covalently connect the PQQ with the oligonucleotide 52/109 * > s *? *to? > ** ~ **** probe (amidation and dehydration with the amino terminal function of the separator -CH = CH-CO-NH-CH2-CH2-NH2 attached at the C-5 position of the 5 'thymine). The two free PQQ, which is unchanged and bridged with the support surface by means of a short separator with chain length from 1 to 6, such as, for example, -S- (CH2) 2-NH- or by means of a double-stranded oligonucleotide (modified), for example, in HEPES buffer with the addition of 0.7 molar TEATFB (see abbreviations), it is selectively reduced and oxidized in the potential range 0.7 V > f = 0.0 V, measured against the normal hydrogen electrode. The electrical communication between the support surface (conductive) and the redox pair bridged by means of the single-stranded oligonucleotide in the general structure elec-separator-ss-oligo-separator-redox is weak or does not exist at all. For the exemplary test site Au-S-ss-oligo-PQQ (with 12-bp probe oligonucleotides), this is shown with the cyclic voltammetry (Figure 4). Without wishing to be bound to a theoretical description, it is supposed that the negative charges of the phosphate skeleton cause the mutual repulsion of the simple chains of oligonucleotide and in this way, force the formation of the chain-separator-ds-oligo-separator- redox (in the direction of the propeller shaft) at an angle f <; 70 ° 52/109 with the normal support ("vertical tubes"). The test site (hybridized) Au-S-ds-oligo-PQQ of Figure 3 shows a formation having a f = 30 °. Due to the length of the -separator-ds-oligo-separator-redox chain (for example, approximately 40 A in length of an oligonucleotide with 21 base pairs, the separators and the bound PQQ are approximately 10 A long), yes f < 70 °, between the support of the surface and the substance with redox activity results a distance of > 17 Á. As a result, the possibility of the transfer of an electron or direct orifice between the support and the substance with redox activity can be excluded. When treating the test sites with a solution of the oligonucleotide to be examined, in the case of hybridization between the probe and the target, there will be an increase in conductivity between the supporting surface and the bridged redox pair by means of a double oligonucleotide. chain. The change in conductivity manifests itself in a voltammetrically cyclic form in a significant current flow between the support surface and the substance with redox activity (Figure 4). Thus, it is possible to detect the specific hybridization of the target sequence with the probe oligonucleotides, using electrochemical methods, such as for example, cyclic voltammetry. 52/109 In addition, the mating of defective bases (mismatches of the base pair) can be recognized by means of a modified cyclic voltammetric characteristic (Figure 5). A mismatch manifests itself in a greater potential difference between the current maximums of the electroreduction and the electroremoxidation (the inversion of the electroreduction when the power supply direction is reversed) or the electrooxidation and electrorereduction in a reversible electronic transfer process voltammetrically cyclic between the electrically conductive support surface and the substance with redox activity. This fact has an advantageous effect mainly on the amperometric detection, because there, the current can be tested at a potential in which the perfect hybridization of the target oligonucleotide supplies a significant current, although the target oligonucleotide with defective matches does not. In the example of Figure 5, this is possible at a potential E-E0 of approximately 0.03 V.

Example 1: Production of the Au-S-ds-oligo-PQQ electrode oligonucleotide. The production of the Au-S-ds-oligo-PQQ is divided into 4 subsections, namely the production of the support surface, the hybridization of the 52/109 probe oligonucleotide with the complementary double strand (step of hybridization), the derivation of the support surface with the double-stranded oligonucleotide (incubation step) and the connection or binding of the substance with redox activity (step of redox). A gold film approximately 100 nm thick on mica (muscovite platelets) forms the support for the covalent attachment of the double-stranded oligonucleotides. To this end, the freshly excised mica was purified with argon ion plasma in an electric discharge chamber and gold (99.99%) was applied, by means of electric discharges, in a layer thickness of approximately 100 nm. After this, the gold film was freed of the surface impurities (the oxidation of the organic accumulations) with 30% H2O2, / 70% H2SO4 and immersed in ethanol for approximately 20 minutes to remove any amount of oxygen adsorbed on it. the surface. After rinsing the support surface with bidistilled water, a previously prepared lxlO-4 molar solution of the double-stranded (modified) oligonucleotide was applied to the horizontally placed surface, so that the entire support surface was wetted ( incubation step, see also below). To prepare the oligonucleotide solution of ds, the single-stranded oligonucleotide with 12 52/109 bp and modified twice from the sequence 5 '-TAGTCGGAAGCA-3', which was esterified in P-0- (CH2) 2-SS- (CH2) 2-OH with (HO- (CH2) 2-S ) 2 in the phosphate group at the 3 'end. At the 5 'end, the terminal base of the oligonucleotide, thymine, is modified in carbon C-5 with -CH = CH-CO-NH-CH2-CH2-NH2. A 2xl0 ~ 4 molar solution of this oligonucleotide in the hybridization buffer (10 mM Tris, 1 mM EDTA, pH 7.5 with the addition of 0.7 molar TEATFB, see abbreviations) was hybridized with 2 × 10 -4 molar solution of the complementary strand ( modified) in the hybridization buffer at room temperature for about 2 hours (hybridization step). During a reaction time of about 12 to 24 hours, the bisulfide separator P-O- (CH2) 2-S-S- (CH2) 2-0H of the oligonucleotide was homolytically excised. In this process, the separator forms an Au-S covalent bond with the Au atoms on the surface, thus causing a 1: 1 co-adsorption of the ds oligonucleotide and the 2-hydroxy-mercaptoethanol. The gold electrode modified in this way with a dense (1: 1) monolayer consisting of ds and 2-hydroxy-mercaptoethanol oligonucleotide was washed with bidistilled water and subsequently moistened with a 3 x 10"3 molar solution of the quinone PQQ, EDC 10"2 molar, and sulfo-NHS in HEPES 10" 2 molar buffer After a reaction time of approximately 1 hour, the separator -CH = CH-CO- 52/109 NH-CH2-CH2-NH2 is covalently bound to the PQQ (amidation between the amino group of the separator and an acid function of the PQQ, redox step). The resolution of the surface composition with XPS (X-ray photoelectron spectroscopy) showed a monolayer with the maximum packing density of 1: 1 co-absorbed ds oligonucleotide and 2-hydroxy-mercaptoethanol (4.7 x 10 ds / cm oligonucleotide), the longitudinal axis (direction of the helix axis) of the oligonucleotides of ds form an angle of f * 30 ° with the normal surface of the gold surface.

Example 2: Production of the Au-S-ss-oligo-PQQ electrode oligonucleotide. In a manner analogous to the production of the Au-S-ds-oligo-PQQ system, the support surface was derived with modified single-stranded oligonucleotide, providing only the hybridization of the modified oligonucleotide of the 5'-TAGTCGGAAGCA-3 'sequence with its complementary chain and, in the incubation step, using only the 12 bp single chain probe oligonucleotide modified 2 times (see Example 1) in the form of a 1 x 10"molar solution in water and in the presence of Tris 10" molar, EDTA 10"3 molar and TEATFB 0.7 molar (or 1 molar NaCl) at pH 7.5 The redox step was carried out as indicated in Example 1. 52/109 «***** .. ^ ** -, ** ti * '**** ^ *' --- 8 '- Example 3: Production of the electrode Au-S-ds-oligo-PQQ oligonucleotide that it has unpaired 2 bp. The production of a support surface derived with modified double-stranded oligonucleotide was carried out analogously to the production of the Au-S-ds-oligo-PQQ system, although only in the hybridization of the modified oligonucleotide of the 5 'sequence -TAGTCGGAAGCA- 3 'a complementary strand was used (sequence: 5' -ATCAGATTTCGT-3 '), in which the bases do not. 6 and 7 (counted from the 5 'end), which are really complementary, were modified from C for A or from C for T to introduce two mismatches of the base pair.

Example 4: Production of an electrode Au-S-ss-oligo-PQQ oligonucleotide having a greater distance between oligonucleotides. During the production of the test sites, care must be taken that, when deriving the support surface with single-stranded probe oligonucleotide, there remains sufficient space between the joined single strands to allow hybridization with the target oligonucleotide. To this end, three different methods are presented to proceed: (a) Production of an Au-S-ds-oligo-PQQ electrode as described in the Example 1, with the subsequent thermal dehybridization of the double chains at temperatures of T > 40 ° C. (b) Production of a 52/109 Au-ss-oligo-PQQ electrode, as described in Example 2, although in the incubation step to derive the gold surface with single-stranded oligonucleotide (double derivative), 10 to 10 were added. "molar of 2-5 hydroxy-mercaptoethanol or other thiol or bisulfide linker with suitable chain length (depending on the distance between desired oligonucleotides) and coadsorbed on the gold surface together with the single-stranded oligonucleotide. (c) Production of a Au-ss-oligo-PQQ electrode, as described in Example 2, although omitting the 0.7 molar electrolyte (TEATFB in the Example) was omitted in the incubation step to derive the gold surface with single-stranded oligonucleotide (double-derivative). Due to the absence of salt, The phosphate groups and the nitrogen base atoms of the oligonucleotide are not electrostatically protected and interact in a very important way with the gold surface. Due to this, a surface accumulation of oligonucleotides (f > 60 °) results in the surface of the electrode A signifi- cantly smaller amount of oligonucleotides is bound per unit area. After this, the oligonucleotides can be returned to the desired position by joining covalently, in a second incubation step (before or after the binding of the PQQ), a 2-hydroxy-mercaptoethanol or other thiol binder or 52/109 tmíi ^? ^ t ^ S? ma ^^^^ i ^ ámßs ^ ^ ak í? ^^ - i -... »- > »-.: -.,. x - ¿H ... bisulfide with chain length adequate to the surface of gold atoms that are still free. To do this, the electrode that is less densely covered with the single-stranded oligonucleotide is wetted, before or after the modification with PQQ (Au-S-ss-oligo or Au-S-ss-oligo-PQQ) / with a approximately 5x10"molar solution of 2-hydroxy-mercaptoethanol or other thiol or bisulfide linker with suitable chain length in ethanol or HEPES buffer (or a mixture thereof, depending on the solubility of the thiol) and incubated for 2 to 24 hours .

Example 5: Carrying out the cyclic voltammetry measurements. The cyclic voltammetry measurements were made using a computer controlled bipotensostat (CH Instruments, Model 832) at room temperature in a standard cell having a 3 electrode configuration. The modified gold electrode was used as the working electrode, a platinum wire served as the auxiliary electrode (against electrode), and an Ag / AgCl electrode with internal solution of saturated KCl, separated from the space of the probe by means of a Luggin capillary, was used as the reference electrode to determine the potential. Electrolyte served 0.7 molar TEATFB or 1 molar NaCl. Figure 4 shows a voltammogram of the Au-S-ds-oligo-PQQ electrode in 52/109 comparison with the Au-S-ss-oligo-PQQ electrode, and the effect of the mismatches of the 2 bp on the cyclic voltammogram of the Au-S-ds-oligo-PQQ electrode is shown in Figure 5. The Potentials are each indicated as E-E0, that is, with respect to the half-wave potential. In Figure 4, a significantly greater current flow compared to the unhybridized form is clearly shown, which is very evident when a double-stranded oligonucleotide is present. This allows it to be detect sequence-specific hybridization events. From Figure 5 it is clear that, in the case of hybridization with a white oligonucleotide chain that exhibits a mismatch of 2 base pairs, on the one hand, a weaker current flows and, on the other, increases the difference of the current maximums. 52/109

Claims (55)

1. t X CLAIMS: 1. A nucleic acid oligomer modified by the union of a substance with redox activity, characterized in that the substance with redox activity is a compound having a predominantly flat pp orbital system, namely a 1,2-naphthoquinone with structure general or a 1,4-naphthoquinone with general structure or a 9, 10-anthraquinone with general structure 52/109 or a pyrrolo-quinoline quinone with general structure wherein Rx to R8 are, independently of each other, H or any alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, or heteroalkynyl substituents.
2. 2. The modified nucleic acid oligomer according to claim 1, wherein the substance with redox activity is a pyrrolo-quinoline quinone with general structure 52/109 where R2, R = H and Ri, R3, R5 = COOH.
3. 3. The modified nucleic acid oligomer according to claim 1 or 2, wherein the substance with redox activity is covalently bound to one of the phosphoric acid, carboxylic acid, or amine portions, to one of the sugar portions or to one of the bases of the nucleic acid oligomer, especially to a terminal portion of the nucleic acid oligomer.
4. 4. The modified nucleic acid oligomer according to claim 1 or 2, wherein the substance with redox activity is covalently bound to a branched or linear molecular portion of any composition and chain length and the branched or linear molecular portion is attached to one of the phosphoric acid, carboxylic acid or amine portions, to one of the sugar portions or to one of the bases of the nucleic acid oligomer, especially to a terminal portion of the nucleic acid oligomer.
5. 5. The modified nucleic acid oligomer 52/109? t-and according to claim 4, wherein the substance with redox activity is covalently bound to a branched or linear molecular proportion whose shorter continuous bond between the attached structures comprises from 1 to 14 atoms.
6. 6. The modified nucleic acid oligomer according to any of the preceding claims, wherein the modified nucleic acid oligomer can bind, specifically with the single-stranded DNA sequence, RNA, and / or APN.
7. 7. The modified nucleic acid oligomer according to claim 6, wherein the modified nucleic acid oligomer is an oligomer of deoxyribonucleic acid, an oligomer of ribonucleic acid, an oligomer of nucleic acid peptide, or a nucleic acid oligomer having a nucleic acid oligomer. structurally analogous main chain.
8. The method for producing a modified nucleic acid oligomer according to claims 1 to 3, characterized in that the substance with redox activity is bound to a nucleic acid oligomer, the binding occurs in a phosphoric acid or carboxylic acid group of the acid oligomer nucleic acids by means of amidation with an amino group (primary or secondary) of the substance with redox activity, by means of esterification with a 52/109 alcohol group (primary, secondary or tertiary) of the substance with redox activity, by means of the formation of a thioester with a thioalcohol group (primary, secondary or tertiary) of the substance with redox activity, or by means of the condensation of an amino group of the oligomer of nucleic acids with an aldehyde group of the substance with redox activity.
9. 9. The method for producing a modified nucleic acid oligomer according to claims 4 to 7, characterized in that the substance with redox activity is covalently bound to a branched or linear molecular portion of any composition and chain length, the binding occurs in a group phosphoric acid or carboxylic acid of the branched or linear molecular portion by means of amidation with a group (primary or secondary) of the substance with redox activity, by means of esterification with an alcohol group (primary, secondary or tertiary) of the substance with redox activity, by means of the formation of a thioester with a thioalcohol group (primary, secondary or tertiary) of the substance with redox activity, or by means of the condensation of an amino group of the branched or linear molecular portion with a aldehyde group of the substance with redox activity.
10. 10. A modified conductive surface, characterized in that the conductive surface is 52/109 linked to one or more types of modified nucleic acid oligomers, according to claims 1 to 7.
11. The modified conductive surface according to claim 10, wherein the surface consists of a 5 metal or a metal alloy, especially a metal selected from the group of platinum, palladium, gold, cadmium, mercury, nickel, zinc, carbon, silver, copper, iron, lead, aluminum, manganese and their compounds.
12. 12. The modified conductive surface according to claim 10, wherein the surface consists of a semiconductor, especially a semiconductor selected from the group of carbon, silicon, germanium and tin a.
13. 13. The modified conductive surface according to claim 10, wherein the surface consists of a binary composite of the elements of groups 14 and 16, a binary composite of the elements of groups 13 and 15, a binary composite of the elements of groups 15 and 16, or a binary compound of the elements of groups 20 and 17, especially a Cu (I) halide or an Ag (I) halide.
14. The modified conductive surface according to claim 10, wherein the surface consists of a ternary composite of the elements of groups 11, 13, and 25 16, or a ternary compound of the elements of the groups 52/109 , ^. ^^^,. ^^^ - rf ^^ ^ .. ^^^^? ^ ai ^ iii ^ m?. ^ | ^ fl ^ | T ^^ * •! & 12, 13 and 16.
15. 15. The modified conductive surface according to one of claims 10 to 14, wherein the modified nucleic acid oligomers are attached to the conductive surface covalently or by means of the physisorption.
16. The modified conductive surface according to claim 15, wherein one of the portions of phosphoric acid, carboxylic acid, or amine, one of the sugar portions or one of the bases of the nucleic acid oligomer is bonded to the conductive surface in covalent form or by means of physisorption, especially with a terminal portion of the nucleic acid oligomer.
17. 17. The modified conductive surface according to one of claims 10 to 14, wherein the branched or linear molecular portions of any composition and chain length are attached to the conductive surface, covalently or by physisorption, and the acid oligomers Modified nuclei are covalently bound to these molecular moieties.
18. 18. The modified conductive surface according to claim 17, wherein the branched or linear molecular portion comprises a shorter continuous bond of 1 to 14 atoms between the bonded structures.
19. 19. The conductive surface modified according to 52/109 one of claims 17 or 18, wherein the branched or linear molecular portion is covalently linked to one of the phosphoric acid, carboxylic acid or amine portions, to one of the sugar portions or to one of the bases 5 of the oligomer of nucleic acids, especially to a terminal portion of the nucleic acid oligomer.
20. A method for producing a modified conductive surface according to one of claims 10 to 19, wherein one or more types of modified nucleic acid oligomers are applied to a conductive surface according to claims 1 to 7.
21. 21. The method for producing a modified conductive surface according to one of claims 10 to 19, wherein one is attached to the conductive surface 15 or more types of nucleic acid oligomers and only the oligomers of nucleic acids attached to the conductive surface are modified by the binding of a substance with redox activity to the nucleic acid oligomers.
22. 22. The method for producing a modified conductive surface according to claim 21, wherein the binding of the substance with redox activity to the oligomer of nucleic acids occurs by means of the reaction of the substance with redox activity with a phosphoric acid moiety, a sugar portion, or one of the bases of the nucleic acid oligomer, especially by means of 52/109 ^ g ^ j ^^^ S ^^^ = ^^ gg¡ «£ fcÉ ^ ¿ißi¿i ^ i? & = i, the reaction with a terminal portion of the nucleic acid oligomer.
23. 23. The method for producing a modified conductive surface according to claim 21, wherein 5 the substance with redox activity is covalently bound to a branched or linear molecular portion of any composition and chain length and the branched or linear molecular moiety is attached to one of the phosphoric acid, carboxylic acid or amine portions, at one of 10 sugar portions or one of the bases of the nucleic acid oligomer, especially to a terminal portion of the nucleic acid oligomer.
24. 24. The method for producing a modified conductive surface according to one of the claims 20 15 to 23, wherein the nucleic acid oligomer or the modified nucleic acid oligomer hybridizes with the oligomeric nucleic acid strand complementary thereto and is applied to the conducting surface in the form of a double stranded hybrid.
25. 25. The method for producing a modified conductive surface according to one of claims 20 to 24, wherein the nucleic acid oligomer or the modified nucleic acid oligomer is applied to the conductive surface in the presence of chemical compounds 25 additional ones that are linked in the same way to the surface 52/109 , * a > ^ tf ^ fyrffft ^. ^^^ conductive.
26. 26. A method for electrochemically detecting nucleic acid oligomer hybridization events, characterized in that a conductive surface, as defined in claims 10 to 19, is contacted with the nucleic acid oligomers and, after this, the detection of the change in electrical communication between the portion with redox activity and the respective conductive surface, resulting from the hybridization of the nucleic acid oligomers with the modified nucleic acid oligomers.
27. The method according to claim 26, wherein detection occurs by means of cyclic voltammetry, amperometry or conductivity measurement.
28. 28. A method for producing a modified conductive surface, wherein an oligomer of nucleic acids or a nucleic acid oligomer modified by the binding of a substance with redox activity that is oxidizable and selectively reducible to a potential f with 2.0 V > f > - 2.0 V, measured against the normal hydrogen electrode, is hybridized with the oligomeric nucleic acid chain complementary to it and applied to a conductive surface in the form of the double-stranded hybrid.
29. 29. The method according to claim 28, wherein the double-stranded hybrid is thermally dehybridized 52/109 after application to the conductive surface.
30. 30. The method according to claim 28 or 29, wherein the double-stranded hybrid is applied to the conductive surface in the presence of additional chemical compounds that are similarly bonded to the conductive surface.
31. 31. A method for producing a modified conductive surface, wherein an oligomer of nucleic acids or a nucleic acid oligomer modified by the binding of a substance with redox activity that is oxidizable and selectively reducible to a potential f with 2.0 V = f > - 2.0 V, measured against a normal hydrogen electrode, is applied to the conductive surface in the presence of additional chemical compounds that are linked in the same way to the conductive surface.
32. 32. A method for producing a modified conducting surface, wherein a nucleic acid oligomer or a nucleic acid oligomer modified by the binding of a substance with redox activity that is oxidizable and selectively reducible to a potential f, with 2.0 V > f > - 2.0 V, measured against a normal hydrogen electrode, is applied to the conductive surface in a buffer with the addition of non-conductive salt, to reduce the electrostatic protection of the nucleic acid oligomer and, thereafter, to the surface 52/109 additional chemical compounds are applied which are bonded in the same way to the conductive surface.
33. 33. The method according to one of claims 30 to 32, wherein the chemical compounds are alkyl, alkenyl, alkynyl heteroalkyl, heteroalkenyl and heteroalkynyl chains.
34. 34. The method according to claim 33, wherein the alkyl, alkenyl, alkynyl heteroalkyl, heteroalkenyl or heteroalkynyl chains have a chain length of 1 to 20 atoms.
35. 35. The method according to claim 34, wherein the alkyl, alkenyl, alkynyl heteroalkyl, heteroalkenyl or heteroalkynyl chains have a chain length of 1 to 14 atoms.
36. 36. The method according to one of the claims 28 to 35, wherein the nucleic acid oligomers or the modified nucleic acid oligomers are attached to the conductive surface covalently or by means of the physisorption.
37. 37. The method according to claim 36, wherein one of the portions phosphoric acid, carboxylic acid or amine, one of the sugar portions or one of the bases of the nucleic acid oligomer or the modified nucleic acid oligomer is bound to the surface conductive covalently or by means of the 52/109 fisisorption, especially with a terminal portion of the nucleic acid oligomer or the modified nucleic acid oligomer.
38. 38. The method according to one of claims 28 to 35, wherein the nucleic acid oligomers or the modified nucleic acid oligomers are covalently linked to branched or linear molecular portions of any composition and chain length and these molecular portions are attached to the conducting surface covalently or by means of the physisorption.
39. 39. The method according to claim 38, wherein the chain length of the branched or linear molecular portion is from 1 to 14 atoms.
40. 40. The method according to claim 38 or 39, wherein the branched or linear molecular portion is covalently linked to one of the phosphoric acid, carboxylic acid or amine portions, to one of the sugar portions or to one of the bases of the acid oligomer nucleic acids, especially to a terminal portion of the nucleic acid oligomer.
41. 41. The method according to one of claims 30 to 40, wherein the chain length of the additional chemical compound and the chain length of the branched or linear molecular portion differ by a maximum of 8. 52/109 atoms.
42. 42. The method according to claim 41, wherein the chemical compound and the branched or linear molecular portion have the same chain length.
43. 43. The method according to one of the claims 28 to 42, wherein one or more types of nucleic acid oligomers in the form of the double-stranded hybrid are attached to a conductive surface and only the oligomers of nucleic acids attached to the conductive surface are modified by the binding of a substance with activity. redox to nucleic acid oligomers.
44. 44. The method according to claim 43, wherein the binding of the substance with redox activity to the oligomer of nucleic acids occurs by means of the reaction of the substance with redox activity with a phosphoric acid portion, a sugar portion or one of the bases of the oligomer of nucleic acids, especially by means of the reaction with a terminal portion of the nucleic acid oligomer.
45. 45. The method according to claim 44, wherein the substance with redox activity is covalently bound to a branched or linear molecular portion of any composition and chain length and the branched or linear molecular portion is attached to one of the phosphoric acid moieties, carboxylic acid or amine, to a 52/109 l ^^^^^^^ g ^^^^^^^^^ of the sugar portions or one of the bases of the nucleic acid oligomer, especially to a terminal portion of the nucleic acid oligomer.
46. 46. The method according to one of claims 5 to 45, wherein the substance with redox activity is a dye, especially a flavin derivative, a porphyrin derivative, a chlorophyll derivative or a bacteriochlorophyll derivative.
47. 47. The method according to one of claims 10 to 45, wherein the substance with redox activity is a quinone, especially a pyrrolo-quinoline quinone (PQQ), a 1,4-benzoquinone, a 1,2-naphthoquinone, a 1,4-naphthoquinone or a 9, 10-anthraquinone.
48. 48. The method according to claim 46 or 47, wherein the substance with redox activity is covalently bound to one of the phosphoric acid, carboxylic acid or amine portions, to one of the sugar portions or to one of the bases of the acid oligomer nucleic acids, especially to a terminal portion of the nucleic acid oligomer.
49. 49. The method according to claim 46 or 47, wherein the substance with redox activity is covalently bound to a branched or linear molecular portion of any composition and chain length and the portion Molecular or linear molecule is attached to one of the 52/109 ~ ** th, »'n ^" - ^^. ^^ m ^^^^ ^^^^^ portions phosphoric acid, carboxylic acid or amine, to one of the sugar portions or to one of the bases of the acid oligomer nucleic acids, especially to a terminal portion of the nucleic acid oligomer
50. 50. The method according to claim 49, wherein the substance with redox activity is covalently bound to a branched or linear molecular portion, whose shorter continuous bond between the attached structures comprises from 1 to 14 atoms
51. 51. The method according to one of the claims 28 to 50, wherein the modified nucleic acid oligomer is an oligomer of deoxyribonucleic acid, an oligomer of ribonucleic acid, an oligomer of nucleic acid peptide or a oligomer of nucleic acid having a structurally analogous backbone.
52. 52. The method according to one of claims 28 to 51, wherein the conductive surface consists of a metal or a metal alloy, especially a metal selected from the group of platinum, palladium, gold, cadmium, mercury, nickel, zinc, carbon, silver, copper, iron, lead, aluminum, manganese and their compounds.
53. 53. The method according to one of claims 28 to 51, wherein the conductive surface consists of a semiconductor, especially a semiconductor selected from the group of carbon, silicon, germanium and 52/109 tin a.
54. 54. The method according to one of claims 28 to 51, wherein the conductive surface consists of a binary composite of the elements of groups 14 and 16, a binary composite of the elements of groups 13 and 15, a binary compound of the elements of groups 15 and 16 or a binary compound of the elements of groups 11 and 17, especially a Cu (I) halide or an Ag (I) halide.
55. 55. The method according to one of claims 28 to 51, wherein the conductive surface consists of a ternary compound of the elements of groups 11, 13, and 16, or a ternary compound of the elements of groups 12, 13 and 16. 52/109 | | & i 8Agitt & asi. < \ MBte-jdMiM-l-