WO2000031101A1 - Verfahren zur elektrochemischen detektion von nukleinsäureoligomerhybriden - Google Patents
Verfahren zur elektrochemischen detektion von nukleinsäureoligomerhybriden Download PDFInfo
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- WO2000031101A1 WO2000031101A1 PCT/EP1999/008888 EP9908888W WO0031101A1 WO 2000031101 A1 WO2000031101 A1 WO 2000031101A1 EP 9908888 W EP9908888 W EP 9908888W WO 0031101 A1 WO0031101 A1 WO 0031101A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6825—Nucleic acid detection involving sensors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
- C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
Definitions
- 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.
- RNA and DNA 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.
- FIG. 1b a DNA fragment with primer is shown in FIG. 1b.
- a DNA-containing solution is divided into four batches and the primer of each batch is covalently modified with a fluorescent dye that emits at different wavelengths.
- deoxyribonucleoside triphosphate of bases A (adenine), T (thymine), C (cytosine), and G (guanine), i.e. dATP, dTTP, dCTP and dGTP is added to each batch to form the single strand , starting from the primer, to be replicated enzymatically by DNA polymerase I.
- each reaction mixture contains enough of the 2 ', 3'-dideoxy analogue (FIG. 1a) of one of these nucleoside triphosphates as a stop base (one of the 4 possible stop bases per approach) to stop replication at all possible binding sites.
- Another optical detection method is based on the attachment of fluorescent dyes such.
- 32 P is incorporated into the phosphate structure of the oligonucleotides, P usually being added at the 5'-hydroxyl end by polynucleotide kinase.
- the labeled DNA is then preferably cleaved at one of the four nucleotide types, under defined conditions, so that an average cleavage occurs per chain. This means that there are chains in the reaction mixture for a certain base type, which extend from the 32 P mark to the position of this base (if the base occurs more than once, chains of different lengths are obtained).
- the four fragment mixtures are then separated by gel electrophoresis on four lanes. An autoradiogram is then made from the gel, from which the sequence can be read immediately.
- connection takes place in an ordered grid of 65 536 test sites, a larger amount of an oligonucleotide combination defining a test site and the position of each individual test site (oligonucleotide combination) being known.
- 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.
- fluorescent dye or 32 P
- the target DNA fragment only binds to the oligomers (in the example to the octamers) whose complementary sequence corresponds exactly to a part (an octamer) of its own sequence.
- oligomer sequences By optical (or autoradiographic) detection of the binding position of the hybridized DNA fragment, all the oligomer sequences (octamer sequences) present in the fragment are thus determined. Due to the overlap of adjacent oligomer sequences, the continuous sequence of the DNA fragment can be determined by suitable mathematical algorithms. The advantages of this method include the miniaturization of the sequencing and thus the enormous amount of data that is recorded simultaneously in one operation. In addition, primers and the gel electrophoretic separation of the DNA fragments can be omitted. This principle is shown by way of example in FIG. 2 for a 13 base long DNA fragment.
- radioactive labels in DNA / RNA sequencing has several disadvantages, such as e.g. B. complex, legally prescribed safety precautions when handling radioactive materials, the radiation exposure, the limited spatial resolution (maximum 1 mm 2 ) and a sensitivity that is only high if the radiation of the radioactive fragments for a correspondingly long (hours to days) an x-ray film.
- the spatial resolution can be increased by additional hardware and software and the detection time can be shortened by the use of ⁇ -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.
- 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.
- 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.
- the object of the present invention is therefore to provide 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 oligonucleotide according to independent claim 1, by the method for producing a modified oligonucleotide according to independent claims 9 and 10, by the modified conductive surface according to independent claim 11, and by the method for producing a modified conductive surface according to independent claim 21 , and solved by the method for electrochemical detection of oligomer hybridization events according to independent claim 27.
- PNA Peptide nucleic acid synthetic DNA or RNA in which the
- N (COCH 2 base) -CH 2 CO unit hybridizes PNA with DNA).
- 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
- nucleic acid refers to any "backbone" of the covalently linked pyrimidine or
- Purine bases such as. B. on the sugar-phosphate backbone
- Essential characteristic 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 dATP deoxyribonucleoside triphosphate of A (DNA unit with base A and two other phosphates to build up a longer DNA fragment or an oligonucleotide).
- dGTP deoxyribonucleoside triphosphate of G DNA unit with base G and two other phosphates to build up a longer DNA fragment or an oligonucleotide).
- dCTP deoxyribonucleoside triphosphate of C DNA unit with base C and two other phosphates to build up a longer DNA fragment or an oligonucleotide).
- dTTP deoxyribonucleoside triphosphate of T DNA unit with base T and two other phosphates to build up a longer DNA fragment or an oligonucleotide).
- Primer start complementary fragment of an oligonucleotide the base length of the primer being only about 4-8 bases. Serves as a starting point for the enzymatic replication of the oligonucleotide.
- 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".
- R any unspecified organic radical as a substituent or side chain.
- alkyl denotes a saturated hydrocarbon radical which is straight-chain or branched (for example ethyl, isopropyl or 2,5-dimethylhexyl etc.).
- alkyl refers to a group with two available valences for covalent linkage (e.g. -CH 2 CH 2 -, -CH 2 CH 2 CH 2 - or -CH 2 C (CH 3 ) 2 CH 2 CH 2 C (CH 3 ) 2 CH 2 - etc.).
- Preferred alkyl groups as substituents or side chains R are those of chain length 1-30 (longest continuous chain of bonded atoms).
- Preferred alkyl groups as linkers or spacers are those of chain length 1-20, in particular chain length 1-14, the chain length being the shortest continuous connection between the structures connected to the linker or spacer.
- Alkenyl alkyl groups in which one or more of the C-C single bonds are replaced by C C double bonds.
- Alkynyl alkyl or alkenyl groups in which one or more of the C-C single or C C double bonds are replaced by C ⁇ C triple bonds.
- Linkers are commercially available as alkyl, alkenyl, alkynyl, hetero-alkyl, hetero-alkenyl or heteroalkynyl chains, the chain being derivatized at two points with (identical or different) reactive groups. These groups form a covalent chemical bond in simple / known chemical reactions with the corresponding reaction partners.
- the reactive groups can also be photoactivatable, ie the reactive groups are only activated by light of certain or any wavelength.
- Preferred linkers are those of chain length 1-20, in particular chain length 1-14, the chain length here being the shortest continuous connection between the structures to be connected, 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.
- 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.
- nx RSS nucleic acid oligomer to which n disulfide functions are connected via spacers in each case -oligo a spacer, with any radical R saturating the disulfide function.
- the spacer for connecting the Disulfide function on the nucleic acid oligomer can each have a different chain length (shortest continuous connection between disulfide function and nucleic acid oligomer), in particular any chain length between 1 and 14 in each case. These spacers can in turn be connected to various molecules naturally present on or through the nucleic acid oligomer Modification attached reactive groups.
- the placeholder "n" is any integer, in particular a number between 1 and 20.
- oligo-Spacer-SS- two identical or different nucleic acid oligomers which are connected to one another via a disulfide bridge, the disulfide bridge being linked via a disulfide bridge any two spacers are attached to the nucleic acid oligomers and the two spacers can have a different chain length (shortest continuous connection between the disulfide bridge and the respective nucleic acid oligomer), in particular any chain length between 1 and 14, and these spacers in turn are connected to different ones naturally Nucleic acid oligomer existing or attached to this by modification attached reactive groups.
- PQQ pyrrolo-quinolino-quinone corresponds to: 4,5-dihydro-4,5-dioxo-1 H-pyrrolo- [2,3-f] -quinoline-2,7,9-tricarboxylic acid)
- Mica muscovite platelets carrier material for applying thin layers.
- the terminal phosphate group of the oligonucleotide is esterified at the 3 'end with (HO- (CH 2 ) 2 -S) 2 to give PO- (CH 2 ) 2 -SS- (CH 2 ) 2 -OH, the SS bond being cleaved homolytically and each causes an Au-SR bond.
- Au-S-ds-oligo-PQQ Au-S-ss-oligo-PQQ which is present in hybridized form with the oligonucleotide complementary to ss-oligo (sequence: TAGTCGGAAGCA).
- Electrode potential that is applied to the working electrode.
- Half-level potential potential in the middle between the current maxima for oxidation and reduction of an electrooxidation or reduction reversible in cyclic voltammetry.
- 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).
- the direction of the potential feed is reversed. The behavior of the products of electrooxidation or reduction is then recorded in the return.
- a nucleic acid oligomer which is modified by chemical attachment of a redox-active substance.
- 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.
- 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.
- nucleic acid oligomer the terms “(probe) oligonucleotide”, “nucleic acid” or “oligomer” are used.
- the redox-active substance can be selectively oxidized and reduced at a potential ⁇ , where ⁇ satisfies the condition 2.0 V> ⁇ > - 2.0 V.
- the potential here relates to the free, unmodified, redox-active substance in a suitable solvent, measured against a normal hydrogen electrode.
- the potential range 1.7 V> ⁇ --1.7 V is preferred, the range 1.4 V> ⁇ > -1.2 V being particularly preferred and the range 0.9 V> ⁇ > -0 , 7 V, in which the redox-active substance of the application example is reduced and reoxidized, is very particularly preferred.
- the present invention relates to a conductive surface to which a nucleic acid oligomer with attached redox-active substance is chemically bound directly or indirectly (via a spacer).
- the present invention relates to a method for producing a modified conductive surface, a modified nucleic acid oligomer being applied to a conductive surface.
- the present invention relates to a method which enables the electrochemical detection of molecular structures, in particular the electrochemical detection of DNA / RNA / PNA fragments in a sample solution by means of sequence-specific nucleic acid-oligomer hybridization. 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 of a sequencing device. Binding of a redox-active substance to a nucleic acid oligomer
- the prerequisite for the method according to the invention is the binding of a redox-active substance to a nucleic acid oligomer.
- any redox-active substance can be used for this as long as it can be selectively oxidized and reduced at a potential ⁇ which satisfies the condition 2.0 V> ⁇ > - 2.0 V.
- the potential here relates to the free, unmodified, redox-active substance in a suitable solvent, measured against a normal hydrogen electrode.
- the potential range 1.7 V> ⁇ > - 1.7 V is preferred, the range 1.4 V> ⁇ > - 1.2 V being particularly preferred and the range 0.9 V> ⁇ > - 0 , 7 V, in which the redox-active substance of the application example is reduced and reoxidized, is very particularly preferred.
- the term “selectively oxidizable and reducible” is understood to mean a redox reaction, that is to say release or acceptance of an electron, which takes place selectively at the location of the redox-active substance.
- no other part of the nucleic acid oligomer is reduced or oxidized by the applied potential, but only the redox-active substance bound to the nucleic acid oligomer.
- redox-active substance means any molecule that can be electro-oxidized / reduced in the electrochemically accessible potential range of the respective carrier surface (electrode) by applying an external voltage to this electrode.
- organic and inorganic redox-active substances such as organic and inorganic redox-active substances such.
- B. hexacyanoferrates, ferrocenes, acridines or phthalocyanines are particularly suitable for binding to the probe oligonucleotide, such as redox-active dyes such.
- R 1 to R 12 are independently H or any alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituent.
- R to R 8 are independently H or any alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituent.
- a redox-active substance is covalently bound to an oligonucleotide by the reaction of the oligonucleotide with the redox-active substance. This binding can be done in three different ways:
- 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.
- the nucleic acid oligomer is via a covalently attached molecular part (spacer) of any composition and chain length (represents the shortest continuous connection between the structures to be connected), in particular chain length 1 to 14, on the oligonucleotide backbone or on a base with a reactive one Group modified.
- the modification is preferably carried out at one of the ends of the oligonucleotide backbone or at a terminal base.
- an alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituent can be used as spacer.
- the nucleic acid oligomer is modified by a redox-active substance which has regions with a predominantly planar, in one plane extended p- ⁇ orbital system, such as, for. B. the PQQ of Example 1 or the quinones of the formula 5 or 7-12 or the porphinoids Structures of the formulas 1 to 4 or the pyridine nucleotides of the general formula 6 or derivatives of these redox-active substances.
- a redox-active substance which has regions with a predominantly planar, in one plane extended p- ⁇ orbital system, such as, for. B. the PQQ of Example 1 or the quinones of the formula 5 or 7-12 or the porphinoids Structures of the formulas 1 to 4 or the pyridine nucleotides of the general formula 6 or derivatives of these redox-active substances.
- the spacer via which the redox-active substance is bound to the nucleic acid oligomer, can be chosen such that the level of the ⁇ orbitals of the redox-active substance is parallel to the p- ⁇ orbitals of the bases adjacent to the redox-active substance of the nucleic acid oligomer can arrange.
- This spatial arrangement of redox-active substance with partially planar p- ⁇ orbitals extended in one plane proves to be particularly favorable.
- the redox-active substance can be bound to the oligonucleotide as described under a) and b) before or after the oligonucleotide is bound to the conductive surface.
- the redox-active substance is then also bound to the oligonucleotide bound on the conductive surface as described under a) and b).
- 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 .
- any doped or undoped semiconductor surfaces of any thickness can also be used. All semiconductors can be used as pure substances or as mixtures. Examples of carbon, silicon, germanium, ⁇ -tin, Cu (l) - and Ag (l) - halides of any crystal structure should be mentioned here as non-restrictive examples. All are also suitable Binary compounds of any composition and structure of the elements of groups 14 and 16, of elements of groups 13 and 15, and of elements of groups 15 and 16. In addition, ternary compounds of any composition and structure of 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.
- an oligonucleotide is linked directly or via a linker / spacer to the support surface atoms or molecules of a conductive support surface of the type described above.
- This binding can be done in three different ways:
- 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 wet-chemically provided with aldehyde or carboxylic acid groups by oxidation. Electrochemically z. B. the possibility by reduction in the presence of aryl diazonium salts the corresponding (functionalized, that is provided with a reactive group) aryl radical or by oxidation in the presence of R'CO 2 H the (functionalized) R 'radical on the graphite Coupling the electrode surface.
- An example of the direct modification of semiconductor surfaces is the derivatization of silicon surfaces to reactive silanols, ie silicon substrates with Si-OR "groups on the surface, where R" as well as R 'represents any functionalized organic residue (e.g. alkyl, Alkenyl, alkynyl, heteroalkyl, heteroalkenyl or heteroalkynyl substituent).
- R represents any functionalized organic residue
- the entire surface can be modified by the covalent attachment of a reactive group of a bifunctional linker, so that a monomolecular layer of any molecule is formed on the surface which preferably contains a reactive group at the end.
- bifunctional linker is understood to mean any molecule of any chain length, in particular chain lengths 2-14, with two identical (homo-bifunctional) or two different (hetero-bifunctional) reactive molecule groups.
- the homo- or hetero-bifunctional linker is a photo-inducible reactive group, ie a group that only becomes reactive 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 covalent attachment of the linker.
- 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.
- 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.
- the redox-active substance can be attached to this second end of the nucleic acid oligomer.
- 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, these reactive groups preferably being in the Located near one end of the nucleic acid oligomer.
- the reactive groups are groups that can react directly with the unmodified surface.
- Examples include: (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.
- 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.
- nucleic acid oligomers which are modified with a plurality of spacer-bridged thiol or disulfide bridges have the advantage that such nucleic acid oligomers have one certain angle of attack against the conductive surface (angle between the surface normal and the helix axis of a double-stranded helical nucleic acid oligomer or between the surface normal and the axis perpendicular to the base pairs of a double-stranded non-helical nucleic acid oligomer) if the thiol or disulfide functions on the spacer linking the nucleic acid oligomer, viewed from one end of the nucleic acid, have an increasing or decreasing chain length.
- 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.
- 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.
- a further reactive group can be bound to the oligonucleotides in the vicinity of their second end, this reactive group, in turn, as described above, being attached directly or via a spacer of any composition and chain length, in particular chain length 1-14.
- the redox-active substance can be attached to this second end of the nucleic acid oligomer.
- the oligonucleotide can alternatively be bound to the conductive surface before or after the redox-active substance has been bound to the oligonucleotide or before or after the spacer provided with a reactive group has been bound to bind the redox-active substance.
- the binding of the already modified oligonucleotide to the conductive surface ie the binding to the surface after the redox-active substance has been bound to the oligonucleotide or after
- the spacer provided with a reactive group is bound to bind the redox-active substance, likewise as described under a) to c) (section "Binding of an Oligonucleotide to the Conductive Surface").
- the hybridized oligonucleotide double strand is then thermally dehybridized, as a result of which a single-strand oligonucleotide-modified carrier surface is produced with a greater distance between the proboligonucleotides.
- the monofunctional linker has a chain length which is identical to the chain length of the spacer between the support surface and the oligonucleotide or which deviates by a maximum of eight chain atoms.
- the hybridized oligonucleotide double-strand is thermally dehybridized after the binding of the double-stranded oligonucleotide and the linker to the carrier surface, as described above under 1.).
- 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.
- a plurality of probe oligonucleotides of different sequences are applied on an oligomer or DNA chip to the sequence of any target oligomer or (fragmented) target DNA to be reliably detected or to detect mutations in the target and to demonstrate them in a sequence-specific manner.
- the carrier surface atoms or molecules of a defined region are linked on a conductive carrier surface with DNA / RNA / PNA oligonucleotides of known but any sequence, as described above.
- the DNA chip can also be derivatized with a single probe oligonucleotide.
- Nucleic acid oligomers (DNA, RNA or PNA fragments) of base length 3 to 50, preferably length 5 to 30, particularly preferably length 7 to 25 are used as probe oligonucleotides.
- a redox-active substance is bound to the probe oligonucleotides either before or after their binding to the conductive surface.
- the already modified probe oligonucleotides are bound to the conductive surface as described above.
- the unmodified probe oligonucleotides bound to the conductive surface at the second, free end of the oligonucleotide chain are modified directly or indirectly via a spacer with a redox-active substance.
- a surface hybrid of the general structure elek-spacer-ss-oligo-spacer redox is formed (FIG. 3).
- the electrical communication between the (conductive) support surface and the redox-active substance (“redox”) bridged by a single-strand oligonucleotide in the general structure elek-spacer-ss-oligo-spacer redox is weak or not available at all.
- the bridges can of course also be carried out without a spacer or with only one spacer (Elek-ss-oligo-Spacer-Redox or Elek-Spacer-ss-oligo-Redox).
- the test sites are brought into contact with the oligonucleotide solution to be examined (target).
- Hybridization only occurs in the case in which the solution contains oligonucleotide strands which are complementary to the probe oligonucleotides bound to the conductive surface, or at least are complementary in a wide range.
- there is an increased conductivity between the support surface and the redox-active substance since these are now bridged via the oligonucleotide consisting of a double strand (shown schematically in FIG. 3 using an example of the electr-spacer-ss-oligo-spacer redox).
- a sequence-specific hybridization event can thus be carried out by electrochemical methods such as, for. B. cyclic voltammetry, amperometry or conductivity measurements can be detected.
- a redox-active substance which has regions with a predominantly planar, p- ⁇ -orbital system which is extended in one plane, such as, for example, B. the PQQ of Example 1 (see FIG. 3), or the quinones of the formula 5 or 7-12 or the porphinoid structures of the formulas 1-4, the pyridine nucleotides of the general formula 6 and derivatives of these redox-active substances.
- the spacer between the nucleic acid oligomer and the redox-active substance is selected such that the level of the ⁇ orbitals of the redox-active substance is parallel to the p- ⁇ orbitals of the base pair of the nucleic acid oligomer hybridized with the complementary strand and adjacent to the redox-active substance can arrange.
- This spatial arrangement of redox-active substance with partially planar p- ⁇ orbitals extended in one plane proves to be particularly favorable for the electrical conductivity of the double-stranded nucleic acid oligomers.
- 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, a maximum current (peak) and then 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 redox-active substance is electro-oxidized (electroreduced) by applying a suitable, constantly kept electrode potential, but the redox-active substance is not reduced (reoxidized) to its original state, but not by changing the electrode potential will, as in the Cyclic voltammetry, but by a suitable reducing agent (oxidizing agent) added to the target solution, whereby the circuit of the entire system is closed.
- reducing agent oxidizing agent
- oxidizing agent oxidizing agent
- Fig. 1 Schematic representation of the Sanger method of oligonucleotide
- Fig. 2 Schematic representation of the oligonucleotide sequencing by
- Fig. 4 Cyclic voltagram of a test site made of Au-S-ss-oligo-PQQ (dotted) compared to an identical test site with a fully hybridized target (Au-S-ds-oligo-PQQ, solid line);
- FIG. 3 An exemplary test site with a hybridized target (Au-S-ds-oligo-PQQ) of the general structure Elek-Spacer-ds-oligo-Spacer-Redox is shown in FIG. 3.
- the carrier surface is a gold electrode.
- 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.
- PQQ tricarboxylic pyrrolo-quinoline-quinone
- one of the three carboxylic acid functions of the PQQ in the example the C-7-CO 2 H function
- amide formation with elimination of water with the terminal amino function of the -CH CH-CO-NH-CH 2 -CH 2 -NH 2 spacer attached to the C-5 position of the 5'-thymine.
- the change in conductivity manifests itself in cyclic voltammetry in a clear current flow between the carrier surface and the redox-active substance (FIG. 4). This makes it possible to sequence-specific hybridization of the target with the probe oligonucleotides by electrochemical methods such as. B. to detect cyclic voltammetry.
- incorrect base pairings can be recognized by a changed cyclic voltammetric characteristic (FIG. 5).
- a mismatch manifests itself in a larger potential distance between the current maxima of the electrical reduction and the electroreoxidation (reversal of the electroreduction with reversed direction of potential feed) or the electrooxidation and electroreduction in a cyclic voltammetrically reversible electron transfer process between the electrically conductive carrier surface and the redox-active substance.
- This fact has a particularly favorable effect in amperometric detection, since the current can be tested there at a potential at which the perfectly hybridizing oligonucleotide target delivers significant current, but not the incorrectly paired oligonucleotide target. In the example in FIG. 5, this is possible at a potential EE 0 of approximately 0.03 V.
- Example 1 Preparation of the oligonucleotide electrode Au-S-ds-oligo-PQQ.
- the production of Au-S-ds-oligo-PQQ is divided into 4 sections, namely representation of the support surface, hybridization of the probe oligonucleotide with the complementary double strand (hybridization step), derivatization of the support surface with the double strand oligonucleotide (incubation step) and binding of the redox active substance (redox step).
- the carrier material for the covalent attachment of the double-strand oligonucleotides is formed by an approximately 100 nm thin gold film on mica (muscovite platelet).
- mica muscovite platelet
- gold (99.99%) was applied in a layer thickness of approx. 100 nm by electrical discharge.
- the gold film with 30% H 2 0 2 , / 70% H 2 S0 4 was freed from surface impurities (oxidation of organic deposits) and immersed in ethanol for about 20 minutes in order to To displace surface adsorbed oxygen.
- a double-modified 12 bp single-strand oligonucleotide of the sequence 5'-TAGTCGGAAGCA-3 ' was used, which is attached to the phosphate group of the 3' end with (HO- (CH 2 ) 2 -S) 2 to the P -0- (CH 2 ) 2 -SS- (CH 2 ) 2 -OH is esterified.
- a 2 x 10 "4 molar solution of this oligonucleotide in the hybridization buffer (10 mM Tris, 1 mM EDTA, pH 7.5 with 0.7 molar addition of TEATFB, see abbreviations) was mixed with a 2 x 10 " 4 molar solution of the (unmodified) complementary strand in the Hybridization buffer hybridized at room temperature for about 2 hours (hybridization step).
- the disulfide spacer P-0- (CH 2 ) 2 -SS- (CH 2 ) 2 -OH of the oligonucleotide was cleaved homolytically during a reaction time of approx. 12-24 h.
- the spacer forms a covalent Au-S bond with the Au atoms on the surface, which leads to a 1: 1 coadsorption of the ds-oligonucleotide and the 2-hydroxy-mercaptoethanol (incubation step).
- the gold electrode modified with a dense (1: 1) monolayer of ds-oligonucleotide and 2-hydroxy-mercaptoethanol was washed with bidistilled water and then with a solution of 3x10 3 molar quinone PQQ, 10 "2 molar EDC and 10 " 2 molar sulfo-NHS wetted in HEPES buffer.
- the -CH CH-CO-NH-CH 2 -CH 2 -NH 2 spacer covalently binds the PQQ (amide formation between the amino group of the spacer and an acid function of the PQQ, redox step).
- Example 2 Preparation of the oligonucleotide electrode Au-S-ss-oligo-PQQ.
- the support surface is derivatized with modified single-strand oligonucleotide, with only the Hybridization of the modified oligonucleotide of the sequence 5'-TAGTCGGAAGCA-3 'with its complementary strand was dispensed with and in the incubation step only the double-modified 12 bp single-strand probe oligonucleotide (see Example 1) as a 1 x 10 ⁇ molar solution in water and in the presence of 10 "2 molar Tris, 10 " 3 molar EDTA and 0.7 molar TEATFB (or 1 molar NaCI) at pH 7.5 was used.
- the redox step was carried out as indicated in Example 1.
- Example 3 Production of the oligonucleotide electrode Au-S-ds-oligo-PQQ with 2-bp mismatches.
- the preparation of a support surface derivatized with modified double-stranded oligonucleotide was carried out analogously to the representation of the system Au-S-ds-oligo-PQQ, with only one complementary strand being used in the hybridization of the modified oligonucleotide of the sequence 5'-TAGTCGGAAGCA-3 '( Sequence: 5'-ATCAG ⁇ 7TTCGT-3 '), in which the actually complementary bases Nos. 6 and 7 (counted from the 5' end) were modified from C to A and from C to 7, respectively, in order to introduce two base pairs of mismatches.
- Example 4 Production of an Au-S-ss-oligo-PQQ oligonucleotide electrode with increased inter-oligonucleotide spacing.
- care must be taken to ensure that there is sufficient space between the attached single strands to enable hybridization with the target oligonucleotide.
- the oligonucleotides can then be returned to the the desired position can be brought about by covalently attaching 2-hydroxy-mercaptoethanol or another thiol or disulfide linker of suitable chain length to the still free surface gold atoms in a second incubation step (before or after attaching the PQQ).
- the electrode which is less densely coated with single-strand oligonucleotide, is modified with an approximately 5 ⁇ 10 2 molar solution of 2-hydroxy before or after modification with PQQ (Au-S-ss-oligo or Au-S-ss-oligo-PQQ) mercaptoethanol or another thiol or disulfide linker of suitable chain length in ethanol or HEPES buffer (or a mixture thereof, depending on the solubility of the thiol) and incubated for 2-24 hours.
- PQQ Au-S-ss-oligo or Au-S-ss-oligo-PQQ
- Example 5 Carrying out the cyclic voltammetric measurements.
- the cyci-voltammetric measurements were carried out using a computer-controlled bipotentiostat (CH Instruments, Model 832) at room temperature in a standard cell with a 3-electrode arrangement.
- the modified gold electrode was used as the working electrode, a platinum wire was used as the auxiliary electrode (counter electrode) and an Ag / AgCI electrode with an internally saturated KCI solution, which was separated from the sample space via a Luggin capillary, was used as the reference electrode.
- 0.7 molar TEATFB or 1 molar NaCI was used as the electrolyte.
- FIG. 4 A cyclovaltagram of the Au-S-ds-oligo-PQQ electrode compared to an Au-S-ss-oligo-PQQ electrode is shown in FIG. 4, the effect of the 2 bp mismatches on the cyclovoltagram of the Au-S-ds-oligo -PQQ electrode is shown in Figure 5.
- the potentials are each given as EE 0 , i.e. relative to the half-wave potential.
- FIG. 4 shows a clearly increased current flow when a double-stranded oligonucleotide is present compared to the non-hybridized form. This enables sequence-specific hybridization events to be detected. It is clear from FIG. 5 that when hybridizing with a target oligonucleotide strand which has 2 base pair mismatches, on the one hand a lower current flows and on the other hand the difference in the current maxima is increased.
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Abstract
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Priority Applications (9)
Application Number | Priority Date | Filing Date | Title |
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PL99348144A PL348144A1 (en) | 1998-11-23 | 1999-11-19 | Method for the electrochemical detection of nucleic acid oligomer hybrids |
KR1020017005877A KR20010080973A (ko) | 1998-11-23 | 1999-11-19 | 핵산 올리고머 하이브리드를 전기화학적으로 검출할 수있는 방법 |
AU13836/00A AU751220B2 (en) | 1998-11-23 | 1999-11-19 | Method for the electrochemical detection of nucleic acid oligomer hybrids |
BR9915526-5A BR9915526A (pt) | 1998-11-23 | 1999-11-19 | OligÈmero de ácido nucléico modificado, processopara a sua produção, superfìcie condutoramodificada, processo para a sua produção, eprocesso para a detecção eletroquìmica deeventos de hibridização de oligÈmero de ácidonucléico |
JP2000583928A JP2002532386A (ja) | 1998-11-23 | 1999-11-19 | 核酸オリゴマーハイブリッドを電気化学的に検出する方法 |
US09/856,543 US7056664B1 (en) | 1998-11-23 | 1999-11-19 | Method of the electrochemical detection of nucleic acid oligomer hybrids |
EP99972637A EP1133514A1 (de) | 1998-11-23 | 1999-11-19 | Verfahren zur elektrochemischen detektion von nukleinsäureoligomerhybriden |
CA002345157A CA2345157A1 (en) | 1998-11-23 | 1999-11-19 | Method for electrochemically detecting nucleic acid oligomer hybrids |
IL14275899A IL142758A0 (en) | 1998-11-23 | 1999-11-19 | Method of electrochemically detecting nucleic acid oligomer hybrids |
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DE19853957.6 | 1998-11-23 | ||
DE19853957 | 1998-11-23 | ||
DE19921940A DE19921940C2 (de) | 1998-11-23 | 1999-04-29 | Verfahren zur elektrochemischen Detektion von Nukleinsäureoligomerhybriden |
DE19921940.0 | 1999-04-29 |
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US (1) | US7056664B1 (de) |
EP (1) | EP1133514A1 (de) |
JP (1) | JP2002532386A (de) |
CN (1) | CN1324365A (de) |
AU (1) | AU751220B2 (de) |
BR (1) | BR9915526A (de) |
CA (1) | CA2345157A1 (de) |
DE (1) | DE19964220C2 (de) |
IL (1) | IL142758A0 (de) |
PL (1) | PL348144A1 (de) |
RU (1) | RU2213095C2 (de) |
TR (1) | TR200101179T2 (de) |
WO (1) | WO2000031101A1 (de) |
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WO2000042217A2 (de) * | 1999-01-18 | 2000-07-20 | Fritz, Biochem Gmbh | Verfahren zur elektrochemischen detektion von nukleinsäure -oligomer- hybridisierungsereignissen |
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DE10049527A1 (de) * | 2000-10-06 | 2001-09-06 | Friz Biochem Gmbh | Thermostabile,photoinduzierbar redoxaktive Einheit zur elektrochemischen Detektion von Nukleinsäure-Oligomer-Hybridisierungsereignissen |
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5312527A (en) * | 1992-10-06 | 1994-05-17 | Concordia University | Voltammetric sequence-selective sensor for target polynucleotide sequences |
WO1995015971A2 (en) * | 1993-12-10 | 1995-06-15 | California Institute Of Technology | Nucleic acid mediated electron transfer |
WO1996040712A1 (en) * | 1995-06-07 | 1996-12-19 | California Institute Of Technology | Nucleic acid mediated electron transfer |
EP0831327A1 (de) * | 1996-09-24 | 1998-03-25 | Roche Diagnostics GmbH | Redoxaktive Verbindungen und deren Anwendung |
WO1999051778A1 (en) * | 1998-04-08 | 1999-10-14 | California Institute Of Technology | Electrochemical sensor using intercalative, redox-active moieties |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3662745A (en) * | 1969-06-30 | 1972-05-16 | Hoffmann La Roche | Metal-metal salt electrode and method for making the same |
US5102798A (en) | 1988-09-08 | 1992-04-07 | Allage Associates | Surface functionalized Langmuir-Blodgett films for immobilization of active moieties |
US5457183A (en) | 1989-03-06 | 1995-10-10 | Board Of Regents, The University Of Texas System | Hydroxylated texaphyrins |
WO1993021530A1 (de) | 1992-04-10 | 1993-10-28 | Messerschmitt-Bölkow-Blohm Gmbh | Verfahren und vorrichtung zur empfindlichkeits- und selektivitätssteigerung bei immuno-assays, molekül-rezeptor-, dna-komplementär-dna- und gast-wirtsmolekül-wechselwirkungs-assays |
US5622821A (en) | 1994-06-29 | 1997-04-22 | The Regents Of The University Of California | Luminescent lanthanide chelates and methods of use |
ATE377751T1 (de) | 1995-05-12 | 2007-11-15 | Novartis Erfind Verwalt Gmbh | Verfahren zur parallelen bestimmung mehrerer analyten mittels evaneszent angeregter lumineszenz |
US5968745A (en) * | 1995-06-27 | 1999-10-19 | The University Of North Carolina At Chapel Hill | Polymer-electrodes for detecting nucleic acid hybridization and method of use thereof |
EP0923595B1 (de) | 1996-06-07 | 2005-11-02 | Amersham Biosciences (SV) Corp. | Nukleinsäurevermittelter elektronentransfer |
CA2270633A1 (en) | 1996-11-05 | 1998-05-14 | Clinical Micro Sensors | Electrodes linked via conductive oligomers to nucleic acids |
US6306584B1 (en) | 1997-01-21 | 2001-10-23 | President And Fellows Of Harvard College | Electronic-property probing of biological molecules at surfaces |
US6060327A (en) * | 1997-05-14 | 2000-05-09 | Keensense, Inc. | Molecular wire injection sensors |
IL121312A (en) | 1997-07-14 | 2001-09-13 | Technion Res & Dev Foundation | Microelectronic components, their manufacture and electronic networks containing them |
DE19964220C2 (de) | 1998-11-23 | 2003-07-03 | Friz Biochem Gmbh | Verfahren zur Herstellung einer modifizierten leitfähigen Oberfläche |
-
1999
- 1999-04-29 DE DE19964220A patent/DE19964220C2/de not_active Expired - Fee Related
- 1999-11-19 PL PL99348144A patent/PL348144A1/xx not_active Application Discontinuation
- 1999-11-19 EP EP99972637A patent/EP1133514A1/de not_active Withdrawn
- 1999-11-19 IL IL14275899A patent/IL142758A0/xx unknown
- 1999-11-19 CN CN99812448A patent/CN1324365A/zh active Pending
- 1999-11-19 JP JP2000583928A patent/JP2002532386A/ja active Pending
- 1999-11-19 AU AU13836/00A patent/AU751220B2/en not_active Ceased
- 1999-11-19 US US09/856,543 patent/US7056664B1/en not_active Expired - Fee Related
- 1999-11-19 WO PCT/EP1999/008888 patent/WO2000031101A1/de not_active Application Discontinuation
- 1999-11-19 TR TR2001/01179T patent/TR200101179T2/xx unknown
- 1999-11-19 CA CA002345157A patent/CA2345157A1/en not_active Abandoned
- 1999-11-19 BR BR9915526-5A patent/BR9915526A/pt not_active IP Right Cessation
- 1999-11-19 RU RU2001114192/04A patent/RU2213095C2/ru not_active IP Right Cessation
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5312527A (en) * | 1992-10-06 | 1994-05-17 | Concordia University | Voltammetric sequence-selective sensor for target polynucleotide sequences |
WO1995015971A2 (en) * | 1993-12-10 | 1995-06-15 | California Institute Of Technology | Nucleic acid mediated electron transfer |
WO1996040712A1 (en) * | 1995-06-07 | 1996-12-19 | California Institute Of Technology | Nucleic acid mediated electron transfer |
EP0831327A1 (de) * | 1996-09-24 | 1998-03-25 | Roche Diagnostics GmbH | Redoxaktive Verbindungen und deren Anwendung |
WO1999051778A1 (en) * | 1998-04-08 | 1999-10-14 | California Institute Of Technology | Electrochemical sensor using intercalative, redox-active moieties |
Non-Patent Citations (5)
Title |
---|
I.WILLNER ET AL.: "Assembly of Functoinalized Monolayers of Redox Proteins on Electrode Surfaces : Novel Bioelectronic and Optobioelectronic systems.", BIOSENSORS AND BIOELECTRONICS, vol. 12, no. 4, 1997, pages 337 - 356, XP000874626 * |
JOHNSTON D H ET AL: "ELECTROCHEMICAL MEASUREMENT OF THE SOLVENT ACCESSIBILITY OF NUCLEOBASES USING ELECTRON TRANSFER BETWEEN DNA AND METAL COMPLEXES", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY,US,AMERICAN CHEMICAL SOCIETY, WASHINGTON, DC, vol. 117, no. 35, 1995, pages 8933 - 8938-8938, XP002900562, ISSN: 0002-7863 * |
KATZ E ET AL: "ELECTROCHEMICAL STUDY OF PYRROLOQUINOLINE QUINONE COVALENTLY IMMOBILIZED AS A MONOLAYER ONTO A CYSTAMINE-MODIFIED GOLD ELECTRODE", JOURNAL OF ELECTROANALYTICAL CHEMISTRY AND INTERFACIAL ELECTROCHEMISTRY,NL,ELSEVIER, AMSTERDAM, vol. 367, 1 January 1994 (1994-01-01), pages 59 - 70, XP000672483, ISSN: 0022-0728 * |
MILLAN K M ET AL: "VOLTAMMETRIC DNA BIOSENSOR FOR CYSTIC FIBROSIS BASED ON A MODIFIED CARBON PASTE ELECTRODE", ANALYTICAL CHEMISTRY,US,AMERICAN CHEMICAL SOCIETY. COLUMBUS, vol. 66, no. 18, 15 September 1994 (1994-09-15), pages 2943 - 2948, XP000478033, ISSN: 0003-2700 * |
N.NAKAMURA ET AL.: "Electrochemical Properties of Coenzyme Pyrroloquinolinequinone Using a Di-(4-pyridyl) Disulfide Modified Gold Electrode under Acidic Conditions.", BULLETIN O FTHE CHEMICAL SOCIETY OF JAPAN, vol. 66, April 1993 (1993-04-01), pages 1289 - 1291, XP000867849 * |
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DE10155054C2 (de) * | 2001-11-09 | 2003-10-23 | Friz Biochem Gmbh | Molekulares elektronisches Bauelement zum Aufbau nanoelektronischer Schaltungen, molekulare elektronische Baugruppe, elektronische Schaltung und Herstellungsverfahren |
WO2003040678A2 (de) * | 2001-11-09 | 2003-05-15 | Friz Biochem Gmbh | Fluoreszenz-quenchen zur detektion von ligat-ligand-assoziationsereignissen in elektrischen feldern |
US8026348B2 (en) | 2003-05-02 | 2011-09-27 | Lesnikowski Zbigniew J | Nucleoside derivative, modified oligonucleotide, method for their synthesis and applications thereof |
US7491496B2 (en) * | 2004-04-01 | 2009-02-17 | Seiko Epson Corporation | Method for immobilizing nucleic acid and method for manufacturing biosensor using the same method |
CN103115954A (zh) * | 2013-01-31 | 2013-05-22 | 中国人民共和国泰安出入境检验检疫局 | 采用蒽醌-2-磺酸钠传感器快速测定食品中三聚氰胺的方法 |
GB2544494A (en) * | 2015-11-17 | 2017-05-24 | Nexeon Ltd | Surface modification |
GB2544494B (en) * | 2015-11-17 | 2018-10-24 | Nexeon Ltd | Functionalised electrochemically active material and method of functionalisation |
US10847790B2 (en) | 2015-11-17 | 2020-11-24 | Nexeon Limited | Functionalised electrochemically active material and method of functionalisation |
US11342558B2 (en) | 2015-11-17 | 2022-05-24 | Nexeon Limited | Surface modification |
US11923544B2 (en) | 2015-11-17 | 2024-03-05 | Nexeon Limited | Surface modification |
Also Published As
Publication number | Publication date |
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AU751220B2 (en) | 2002-08-08 |
PL348144A1 (en) | 2002-05-06 |
CN1324365A (zh) | 2001-11-28 |
DE19964220C2 (de) | 2003-07-03 |
RU2213095C2 (ru) | 2003-09-27 |
BR9915526A (pt) | 2001-11-13 |
AU1383600A (en) | 2000-06-13 |
CA2345157A1 (en) | 2000-06-02 |
TR200101179T2 (tr) | 2001-11-21 |
EP1133514A1 (de) | 2001-09-19 |
JP2002532386A (ja) | 2002-10-02 |
IL142758A0 (en) | 2002-03-10 |
US7056664B1 (en) | 2006-06-06 |
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