Electrochemical sensor using intercalative, redox-active moieties

• US 6,461,820 B1
• Filed: 12/29/2000
• Issued: 10/08/2002
• Est. Priority Date: 04/09/1997
• Status: Expired due to Fees

• 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.
• Application Number:
  US09/753,362
• Publication Number:
  US 20020055103A1
• Time in Patent Office: 648 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 method of detecting one or more base-stacking perturbations in a target sequence comprising:

(a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex;

(b) depositing said first complex onto an electrode or an addressable multielectorde array;

(c) adding an intercalative, redox-active moiety to said first complex to form a second complex; and

(d) measuring an electron transfer event between said electrode or addressable multielectrode array and said intercalative, redox-active moiety as an indication for the presence or absence of said base-stacking perturbations.

2. A method according to claim 1, wherein said base-stacking perturbations are point mutations, protein-DNA adducts, adducts between any chemical entity and said target sequence, or combinations thereof.

3. A method according to claim 1, wherein said intercalative, redox-active moiety is either noncovalently adsorbed or crosslinked to said first complex.

4. A method according to claim 1, wherein said intercalative, redox-active moiety is an intercalator.

5. A method according to claim 1, wherein said intercalative, redox-active moiety is an intercalator selected from the group consisting of phenanthridines, phenothiazines, phenazines, acridines, and anthraquinones.

6. A method according to claim 1, wherein said intercalative, redox-active moiety is daunomycin.

7. A method according to claim 1, wherein said intercalative, redox-active moiety is a part of a protein.

8. A method according to claim 1, wherein said intercalative, redox-active moiety is mut Y.

9. A method according to claim 1, wherein said electrode or addressable multielectrode array is gold.

10. A method according to claim 1, wherein said electrode or addressable multielectrode array is carbon.

11. A method according to claim 1, wherein one of said single-stranded nucleic acids is derivatized with a functionalized linker.

12. A method according to claim 11, wherein said functionalized linker is comprised of 5 to 20 σ

bonds.

13. A method according to claim 11, wherein said functionalized linker is thiol-terminated.

14. A method according to claim 11, wherein said functionalized linker is amine-terminated.

15. A method according to claim 1, wherein said addressable multielectrode array is comprised of a monolayer of oligonucleotide duplexes of 5 to 10 base-pairs in length deposited onto said array, wherein each of said oligonucleotide duplexes is derivatized on one end with a functionalized linker and on the opposite end with a first single-stranded overhang of known sequence composition, and wherein one of said single-stranded nucleic acids contains a second single-stranded overhang complementary to said first single-stranded overhang on said electrode or addressable multielectrode array.

16. A method of detecting one or more base-stacking perturbations in a target sequence comprising:

(a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex, wherein said nucleic acids are comprised of 12 to 25 nucleotides, and wherein one of said single-stranded nucleic acids is derivatized with a thiol-terminated linker comprised of 5 to 20 σ

bonds;

(b) depositing said first complex onto an addressable gold multielectrode array;

(c) adding daumomycin to said electrode-bound first complex to form a second complex; and

(d) measuring an electron transfer event between said addressable gold multielectrode array and daunomycin as an indication for the presence or absence of said base-stacking perturbations.

17. A method according to claim 16, wherein said base-stacking perturbations are point mutations, protein-DNA adducts, adducts between any chemical entity and said target sequence, or combinations thereof.

18. A method of detecting one or more base-stacking perturbations in a target sequence comprising:

(a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex, wherein said nucleic acids are comprised of 12 to 25 nucleotides, and wherein one of said single-stranded nucleic acids is derivatized with a amine-terminated linker comprised of 5 to 20 σ

bonds;

(b) depositing said first complex onto an addressable carbon multielectrode array;

(c) adding daunomycin to said electrode-bound first complex to form a second complex; and

(d) measuring an electron transfer event between said addressable carbon multielectrode array and daumonycin as an indication for the presence or absence of said base-stacking perturbations.

19. A method according to claim 18, wherein said base-stacking perturbations are point mutations, protein-DNA adducts, adducts between any chemical entity and said target sequence, or combinations thereof.

20. A method of detecting one or more base-stacking perturbations in a target sequence comprising:

(a) hybridizing a first single-stranded nucleic acid to a second single-stranded nucleic acid to form a first complex of 12 to 25 nucleotides in length, wherein said first complex contains a first single-stranded overhang of known sequence composition, and wherein said first single-stranded overhang can be the same or different;

(b) depositing said first complex onto an addressable multielectrode array, wherein said addressable multielectrode array is comprised of a monolayer of oligonucleotide duplexes of 5 to 10 base-pairs in length deposited onto said array, wherein each of said oligonucleotide duplexes is derivatized on one end with a functionalized linker and on the opposite end with a second single-stranded overhang complementary to said first single-stranded overhang;

(c) adding daumomycin to said electrode-bound first complex to form a second complex; and

(d) measuring an electron transfer event between said addressable multielectrode array and daunomycin as an indication for the presence or absence of said base-stacking perturbations.

21. A method according to claim 20, wherein said base-stacking perturbations are point mutations, protein-DNA adducts, adducts between any chemical entity and said target sequence, or combinations thereof.

22. A method of detecting one or more base-stacking perturbations electrocatalytically in a target sequence comprising:

(a) hybridizing a first single stranded nucleic acid to a second single stranded nucleic acid to form a first complex;

(b) depositing said first complex onto an electrode or an addressable multielectrode array to form a second complex;

(c) immersing said second complex in a solution comprising an intercalative, redox-active species and a non-intercalative, redox-active species; and

(d) measuring an electron transfer event as an indication for the presence or absence of said base-stacking perturbations.

23. A method according to claim 22, wherein said base-stacking perturbations are point mutations, protein-DNA adducts, adducts between any chemical entity and said target sequence, or combinations thereof.

24. A method according to claim 22, wherein said intercalative, redox-active moiety is an intercalator.

25. A method according to claim 22, wherein said intercalative, redox-active moiety is an intercalator selected from the group consisting of phenanthridines, phenothiazines, phenazines, acridines, and anthraquinones.

26. A method according to claim 22, wherein said non-intercalative, redox-active moiety is selected from the group consisting of ferricyanide, ferrocenes, hexacyanoruthenate, and hexacyanoosmate.

27. A method according to claim 22, wherein said intercalative, redox-active moiety is a protein.

28. A method according to claim 22, wherein said intercalative, redox-active moiety is methylene blue, and wherein said non-intercalative, redox-active moiety is ferricyanide.

29. A method according to claim 22, wherein said electrode or addressable multielectrode array is gold.

30. A method according to claim 22, wherein said electrode or addressable multielectrode array is carbon.

31. A method according to claim 22, wherein one of said single-stranded nucleic acids is derivatized with a functionalized linker.

32. A method according to claim 31, wherein said functionalized linker is comprised of 5 to 20 σ

bonds.

33. A method according to claim 31, wherein said functionalized linker is thiol-terminated.

34. A method according to claim 31, wherein said functionalized linker is amine-terminated.

35. A method according to claim 22, wherein said addressable multielectrode array is comprised of a monolayer of oligonucleotide duplexes of 5 to 10 base-pairs in length deposited onto said array, wherein each of said oligonucleotide duplexes are derivatized on one end with a functionalized linker and on the opposite end with a first single-stranded overhang of distinct sequence composition, and wherein one of said single-stranded nucleic acids contains a second single-stranded overhang complementary to said first single-stranded overhang on said electrode or addressable multielectrode array.

36. A method of detecting one or more point mutations electrocatalytically within the p53 gene, comprising:

(a) forming a set of oligonucleotide duplexes of approximately 20 base-pairs in length corresponding to the approximately 600 base pair long region within exons 5 through 8 of the p53 gene, wherein said oligonucleotide duplexes are derivatized with a thiol-terminated linker comprised of 5 to 20 σ

bonds;

(b) depositing said oligonucleotide duplexes onto an addressable gold multielectrode array;

(c) denaturing said oligonucleotide duplexes by immersing them in aqueous solution at elevated temperatures for 1 minute and removing the complementary strands to form a single-stranded monolayer;

(d) exposing said single-stranded monolayer to a first sample comprising PCR-amplified and fragmented p53 gene DNA under hybridizing conditions to form a first complex;

(e) rinsing said electrode-bound first complex to remove any unhybridized material;

(f) immersing said electrode-bound first complex into a dilute solution comprised of 1.0 μ

M methylene blue and 1.0 mM ferricyanide;

(g) measuring an electron transfer event as an indication for the presence or absence of said point mutations;

(h) denaturing said electrode-bound first complex by immersing it in an aqueous solution at elevated temperatures for 1 minute, and regenerating said single-stranded monolayer;

(i) exposing said single-stranded monolayer to a second sample containing PCR-amplified and fragmented p53 gene DNA under hybridizing conditions to form a second complex; and

(k) repeating steps (e) through (h) using several sample solutions.