Method of the electrochemical detection of nucleic acid oligomer hybrids

• US 7,056,664 B1
• Filed: 11/19/1999
• Issued: 06/06/2006
• Est. Priority Date: 11/23/1998
• Status: Expired due to Fees

• Current Assignee: FRIZ Biochem Gesellschaft für Bioanalytik mbH
• Original Assignee: Fritz Biochem Gesellschaft Fur Bioanalytik mbH
• Inventors: Heller, Adam, Hartwich, Gerhard
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/856,543
• Time in Patent Office: 2,391 Days
• Field of Search: 435/6, 536/23.1
• US Class Current: 435/6.12
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

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 oligonucleotide 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, the electric communication between the support surface 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.

Claims

1. A method of producing a modified conductive surface, wherein a nucleic acid oligomer or a nucleic acid oligomer modified by attaching a redox-active substance that is selectively oxidizable and reducible at a potential φ

with 2.0 V≧

φ

≦

−

2.0 V, measured against a normal hydrogen electrode, is hybridized with the nucleic acid oligomer strand which is complementary to the nucleic acid oligomer or the modified nucleic acid oligomer and applied to a conductive surface and is in the form of the double-strand hybrid; and

one or more kinds of nucleic acid oligomers in the form of the double-strand hybrid are bound to a conductive surface and only the nucleic acid oligomers bound to the conductive surface are modified by attaching a redox-active substance to the nucleic acid oligomers.

2. A method of producing a modified conductive surface, wherein a nucleic acid oligomer or a nucleic acid oligomer modified by attaching a redox-active substance that is selectively oxidizable and reducible at a potential φ

with 2.0 V≧

φ

≧

−

2.0 V, measured against a normal hydrogen electrode, is hybridized with the nucleic acid oligomer strand which Is complementary to the nucleic acid oligomer or the modified nucleic acid oligomer and applied to a conductive surface and is in the form of the double-strand hybrid, which is thermally dehybridized following application to the conductive surface.

3. The method according to claim 2, wherein the double-strand hybrid is applied to the conductive surface in the presence of further chemical compounds also attached to the conductive surface.

4. The method according to claim 2, wherein the nucleic acid oligomers or the modified nucleic acid oligomers are attached to the conductive surface covalently or by means of physisorption.

5. The method according to claim 2, wherein the nucleic acid oligomers or the modified nucleic add oligomers are covalently attached to branched or linear molecular moieties of any composition and chain length and these molecular moieties are attached to the conductive surface covalently or by mean so physisorption.

6. The method according to claim 2, wherein one or more kinds of nucleic acid oligomers in the form of the double-strand hybrid are bound to a conductive surface and only the nucleic acid oligomers bound to the conductive surface are modified by attaching a redox-active substance to the nucleic acid oligomers.