Polymer-electrodes for detecting nucleic acid hybridization and method of use thereof

• US 5,968,745 A
• Filed: 10/14/1997
• Issued: 10/19/1999
• Est. Priority Date: 06/27/1995
• Status: Expired due to Term

• Current Assignee: Xanthon, Inc. (Nokia Corporation), University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: Xanthon, Inc. (Nokia Corporation), University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Thorp, H. Holden, Napier, Mary E., Loomis, Carson R.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/950,503
• Time in Patent Office: 735 Days
• Field of Search: 435/6, 435/91.2, 435/5
• US Class Current: 435/6.11
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety

Abstract

A polymer-electrode including (a) a substrate having a conductive working surface; and (b) a polymer layer on the conductive working surface. The polymer layer has a plurality of microfluidic reaction openings distributed throughout the layer. An oligonucleotide probe can be attached to the polymer layer and is available to capture target nucleic acid. A soluble mediator can diffuse freely and transfer electrons from the preselected base in the hybridized nucleic acid to the conductive working surface of the substrate. An electronic signal generated from the electron transfer reaction is detected and quantitated.

Claims

1. A polymer-electrode comprising:

(a) a substrate having a conductive working surface; and

(b) a nonconductive outer polymer layer on said conductive working surface, said polymer layer having a plurality of microfluidic reaction openings distributed throughout the layer, and through which layer a transition metal complex can transfer electrons to the conductive working surface.

2. The polymer-electrode according to claim 1, further comprising an oligonucleotide probe attached to the polymer layer.

3. The polymer-electrode according to claim 1, further comprising a protein-binding substance attached to the polymer layer.

4. The polymer-electrode according to claim 1, wherein the polymer layer is activated with a coupling agent.

5. The polymer-electrode according to claim 4, wherein the coupling agent is a carbodiimide.

6. The polymer-electrode according to claim 1, wherein the substrate is selected from the group consisting of metallic substrates and non-metallic substrates.

7. The polymer-electrode according to claim 1, wherein the conductive working surface is indium-tin oxide and the polymer layer is a polyethylene terephthalate.

23. The polymer-electrode according to claim 3, wherein the protein-binding substance comprises a protein.

8. An apparatus comprising:

(a) a sample container for holding a fluid sample;

(b) a polymer-electrode in electronic communication with the sample container, said polymer-electrode comprising a substrate having a conductive working surface, and a nonconductive outer polymer layer on the conductive working surface, said polymer layer having a plurality of microfluidic reaction openings, and through which layer a transition metal complex can transfer electrons to the conductive working surface; and

(c) a potentiostat in electronic communication with the polymer-electrode.

24. The apparatus according to claim 8, further comprising an oligonucleotide probe attached to the polymer layer.

25. The apparatus according to claim 8, further comprising a protein-binding substance attached to the polymer layer.

9. A method of determining the presence of a target in a sample, comprising:

(a) contacting a nonconductive polymer layer with a label-bearing target which is capable of being oxidized in an oxidation-reduction reaction, said polymer layer having a plurality of microfluidic reaction openings and through which layer a transition metal complex can transfer electrons to the conductive working surface as recited in step (e) below, said polymer layer having immobilized thereon a binding molecule capable of binding to the target so that the immobilized binding molecule and the target form a target complex on the polymer layer;

(b) reacting the target complex with a transition metal complex capable of oxidizing the label-bearing target in an oxidation-reduction reaction;

(c) detecting the oxidation-reduction reaction;

(d) determining the presence or absence of the target from the detected oxidation-reduction reaction; and

(e) contacting the polymer layer with a conductive working surface of a substrate at any time prior to detecting the oxidation-reduction reaction.

10. The method according to claim 9, wherein the sample is selected from the group consisting of:

synthetic or natural oligonucleotides, surgical specimens, specimens used for medical diagnostics, specimens used for genetic testing, environmental specimens, food specimens, dental specimens and veterinary specimens.

11. The method of claim 9, wherein the label-bearing target is a nucleic acid containing guanine and the immobilized binding molecule is a nucleic acid hybridizable with said target to form a hybridized target complex.

12. The method according to claim 11, wherein the polymer layer is brought into contact with the conductive working surface of the substrate after reacting the hybridized target complex with the transition metal complex.

13. The method according to claim 11, wherein the sample is selected from the group consisting of:

synthetic or natural oligonucleotides, surgical specimens, specimens used for medical diagnostics, specimens used for genetic testing, environmental specimens, food specimens, dental specimens and veterinary specimens.

26. The method according to claim 11, further comprising amplifying the target nucleic acid to produce an amplified nucleic acid solution prior to contacting the polymer layer.

32. The method according to claim 26, wherein the amplification is carried out by a method selected from the group consisting of polymerase chain reaction, strand displacement amplification, ligase chain reaction, and nucleic acid sequence-based amplification.

33. The method according to claim 26, wherein the polymer layer is brought into contact with the conductive working surface of the substrate after reacting the hybridized target complex with the transition metal complex.

14. A method of preparing a polymer-electrode, comprising:

(a) providing a substrate having a conductive working surface;

(b) preparing a nonconductive polymer layer for attachment thereto of a binding molecule capable of binding to a substance of interest, said polymer layer having a plurality of microfluidic reaction openings, and wherein a transition metal complex can transfer electrons to the conductive working surface through the polymer layer;

(c) attaching a binding molecule to the polymer layer; and

(d) contacting the polymer layer to the conductive working surface of the substrate.

15. The method according to claim 14, wherein preparing the polymer layer includes providing of carboxylate moieties.

16. The method according to claim 15, wherein the carboxylate moieties are activated with a coupling agent.

17. The method according to claim 16, wherein the coupling agent is water soluble carbodiimide.

18. The method according to claim 14, wherein the binding molecule for which the polymer layer in step (b) is prepared is an oligonucleotide.

19. The method according to claim 18, further comprising coupling to the polymer-layer an oligonucleotide which hybridizes with a nucleic acid as the substance of interest.

27. The method according to claim 14, wherein the binding molecule for which the polymer layer in step (b) is prepared is a protein.

28. The method according to claim 27, further comprising coupling to the polymer-electrode a protein which binds to the substance of interest.

20. A method of determining the presence of a target protein in a sample, comprising:

(a) providing a protein-binding substance attached to a nonconductive polymer layer having a plurality of microfluidic reaction openings wherein a transition metal complex can transfer electrons through the polymer layer to a conductive working surface when the polymer layer is contacted with the conductive working surface as recited in step (g) below;

(b) exposing the protein-binding substance to the sample;

(c) exposing the polymer layer to a second protein-binding substance capable of binding the target protein and which has been modified to contain a label;

(d) reacting the bound target protein with a transition metal complex capable of oxidizing the label in an oxidation-reduction reaction;

(e) detecting the oxidation-reduction reaction;

(f) determining the presence or absence of the protein from the detected oxidation-reduction reaction; and

(g) contacting the polymer layer with a conductive working surface of a substrate prior to detecting the oxidation-reduction reaction.

21. The method according to claim 20, wherein the polymer layer is brought into contact with the conductive working surface of the substrate after reacting the polymer layer with the transition metal complex.

22. The method according to claim 20, wherein the label on the second protein-binding substance is an oligonucleotide.

29. A method of determining the presence of a target protein in a sample, comprising:

(a) providing a binding molecule attached to a nonconductive polymer layer having a plurality of microfluidic reaction openings, wherein a transition metal complex can transfer electrons through the polymer layer to a conductive working surface when the polymer layer is contacted with the conductive working surface as recited in step (f) below;

(b) forming a bound complex by exposing the binding molecule to a sample that has been modified to contain a label;

(c) reacting the bound complex with a transition metal complex capable of oxidizing the label in an oxidation-reduction reaction;

(d) detecting the oxidation-reduction reaction;

(e) determining the presence or absence of the protein from the detected oxidation-reduction reaction; and

(f) contacting the polymer layer with a conductive working surface of a substrate prior to detecting the oxidation-reduction reaction.

30. The method of claim 29, wherein the binding molecule is a protein-binding substance.

31. The method of claim 29, wherein the binding molecule is an oligonucleotide.