Electrochemical sensor using intercalative, redox-active moieties

• US 6,221,586 B1
• Filed: 04/08/1998
• Issued: 04/24/2001
• Est. Priority Date: 04/09/1997
• Status: Expired due to Term

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Barton, Jacqueline K., Kelley, Shana O., Hill, Michael G.
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): Lundgren, Jeffrey S.
• Application Number:
  US09/056,995
• Time in Patent Office: 1,112 Days
• Field of Search: 435/6, 536/23.1
• US Class Current: 435/6.11
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

Compositions and methods for electrochemical detection and localization of genetic point mutations and other base-stacking perturbations within oligonucleotide duplexes adsorbed onto electrodes and their use in biosensing technologies are described. An intercalative, redox-active moiety (such as an intercalator or nucleic acid-binding protein) is adhered and/or crosslinked to immobilized DNA duplexes at different separations from an electrode and probed electrochemically in the presence or absence of a non-intercalative, redox-active moiety. Interruptions in DNA-mediated electron-transfer caused by base-stacking perturbations, such as mutations or binding of a protein to its recognition site are reflected in a difference in electrical current, charge and/or potential.

Claims

1. A composition comprising:

a first single stranded nucleic acid hybridized to a second single stranded nucleic acid forming a nucleic acid duplex, said nucleic acid duplex containing one or more electron donor moieties and one or more electron acceptor moieties combined therewith, wherein one of the electron donor or acceptor moieties is an electrode and the other electron donor or acceptor moiety is an intercalating intercalative, redox-active moiety, wherein said nucleic acid duplex comprises one or more base stacking perturbations, and wherein the presence of the one or more base stacking perturbations reduces charge transport between the intercalator and the electrode, as compared to the charge transport that occurs in the absence of the base stacking perturbations.

2. The composition of claim 1, wherein said first single stranded nucleic acid is derivatized on one end with a functionalized linker.

3. The composition of claim 1, wherein said intercalating intercalative, redox-active moiety is either noncovalently adsorbed or covalently attached to the nucleic acid duplex.

4. The composition of claim 1, wherein said intercalating intercalative, redox-active moiety is selected from the group consisting of phenanthridines, phenothiazines, phenazines, acridines, and anthraquinones.

5. The composition of claim 1, wherein said intercalating intercalative, redox-active moiety is daunomycin.

16. The composition of claim 1, wherein said electrode comprises part of a multielectrode array.

17. The composition of claim 16, wherein said multielectrode array is addressable.

6. A sensor comprising:

(a) an electrode;

(b) a nucleic acid duplex deposited onto at least one surface of said electrode, wherein said nucleic acid duplex is comprised of a first single-stranded nucleic acid hybridized to a second single-stranded nucleic acid forming a nucleic acid duplex which comprises one or more base-stacking perturbations; and

(c) an intercalating intercalative, redox-active moiety contacting said nucleic acid duplex, wherein the presence of the one or more base stacking perturbations reduces charge transport between the intercalator and the electrode, as compared to the charge transport that occurs in the absence of the base stacking perturbations.

7. The sensor of claim 6, wherein said intercalating intercalative, redox-active moiety is either noncovalently adsorbed or crosslinked to said nucleic acid duplex.

8. The sensor of claim 6, wherein said intercalating intercalative, redox-active moiety is selected from the group consisting of phenanthridines, phenothiazines, phenazines, acridines, and anthraquinones.

9. The sensor of claim 6, wherein said intercalating intercalative, redox-active moiety is daunomycin.

10. The sensor of claim 6, wherein said electrode is gold.

11. The sensor of claim 6, wherein said electrode is carbon.

12. The sensor of claim 6, wherein one of said nucleic acids is derivatized with a functionalized linker.

13. The sensor of claim 12, wherein said functionalized linker is comprised of 5 to 20σ

bonds.

14. The sensor of claim 12, wherein said functionalized linker is thiol-terminated.

15. The sensor of claim 12, wherein said functionalized linker is amine-terminated.

18. The composition of claim 6, wherein said electrode comprises part of a multielectrode array.

19. The composition of claim 18, wherein said multielectrode array is addressable.