Electrochemical sensor using intercalative, redox-active moieties

• US 7,202,037 B2
• Filed: 08/14/2003
• Issued: 04/10/2007
• 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., Boon, Elizabeth M., Kelley, Shana O., Hill, Michael G.
• Primary Examiner(s): Brusca; John S.
• Assistant Examiner(s): DeJong; Eric S.
• Application Number:
  US10/641,884
• Publication Number:
  US 20040063126A1
• Time in Patent Office: 1,335 Days
• Field of Search: None
• US Class Current: 435/6.11
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• 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, common DNA lesions 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 base stacking perturbations in a duplex nucleic acid sequence associated with protein binding to the duplex nucleic acid sequence, comprising:

a) contacting a first single stranded derivatized nucleic acid sequence, wherein the first single stranded nucleic acid sequence contains a protein binding sequence and an electrochemical probe binding sequence, with a second single stranded nucleic acid sequence to form a duplex, wherein the second single stranded nucleic acid sequence is hybridized to a monolayer of first single stranded nucleic acid sequences on an electrode, wherein the monolayer is prepared by first attaching a double stranded nucleic acid sequence comprising the first single stranded nucleic acid sequence to the electrode, and dehybridizing the double stranded nucleic acid sequence, such that the first single stranded nucleic acid sequence remains attached to the electrode, and wherein a hybrid of the first single stranded nucleic acid sequence and the second single stranded sequence form a duplex-modified film;

b) adding an electrochemical probe to the duplex modified film, wherein the electrochemical probe binds to the electrochemical probe binding sequence of the first single stranded nucleic acid sequence;

c) adding a protein to the duplex-modified film, wherein the protein binds to the protein binding sequence of the first single stranded nucleic acid sequence; and

d) measuring a difference in reduction of the electrical current or charges of the electrochemical probe between the duplex-modified film of step (c) and a control duplex-modified film without protein, wherein the difference in the electrical current or charge from the duplex not having the bound protein compared to the duplex having the bound protein is indicative of the presence or absence of one or more base stacking perturbations in the duplex.

2. The method of claim 1, wherein the first single stranded derivatized nucleic acid sequence is an oligonucleotide with a thiol-terminated alkyl chain on the 5′

end.

3. The method of claim 2, wherein the thiol-terminated oligonucleotide is a DNA sequence.

4. The method of claim 1, wherein the electrode is gold.

5. The method of claim 1, wherein Mg²⁺ is added to the double stranded nucleic acid before the double stranded nucleic acid is deposited onto the electrode.

6. The method of claim 1, wherein Mg²⁺ is not added to the double stranded nucleic acid before the double stranded nucleic acid is deposited onto the electrode.

7. The method of claim 6, wherein the electrode surface is backfilled with mercaptohexanol after the double stranded nucleic acid is deposited onto the electrode.

8. The method of claim 1, wherein the electrochemical probe is daunomycin (DM).

9. The method of claim 8, wherein the daunomycin is covalently crosslinked to a guanine residue near the 5′

terminus of the first single stranded nucleic acid sequence, which terminus is attached to the electrode.

10. The method of claim 1, wherein the protein is TATA-box binding protein (TBP) or restriction endonuclease PvuII (R.PvuII).