Charge perturbation detection method for DNA and other molecules

• US 8,753,812 B2
• Filed: 03/29/2012
• Issued: 06/17/2014
• Est. Priority Date: 11/12/2004
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Pourmand, Nader, Karhanek, Miloslav, Davis, Ronald W.
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US13/434,627
• Publication Number:
  US 20120283107A1
• Time in Patent Office: 810 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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: B82Y 40/00 Manufacture or treatment of nanostructures
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: G01N 27/3275 Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
• CPC Class Codes: G01N 27/3276 being a hybridisation with immobilised receptors using a FET type sensor G01N27/4145; concerning the hybridisation C12Q1/68

Abstract

Methods for direct detection of chemical reactions are provided. Electric charge perturbations of the local environment during enzyme-catalyzed reactions are sensed by an electrode system with an immobilized target molecule. The charge perturbation caused by the polymerase reaction can uniquely identify a DNA sequence. The polymerization process generates local perturbations of charge in the solution near the electrode surface and induces a charge in a polarazible gold electrode. This event is detected as a transient current by a voltage clamp amplifier. Detection of single nucleotides in a sequence can be determined by dispensing individual dNTPs to the electrode solution and detecting the charge perturbations. Alternatively, multiple bases can be determined at the same time using a mix of all dNTPs with subsequent analysis of the resulting signal. This technique may be adapted to other reaction determinations, such as enzymatic reactions, other electrode configurations, and other amplifying circuits.

Claims

1. A method for nucleic acid sequence detection, comprising:

(a) providing a reaction medium including at least one template nucleic acid molecule with components for template polymerization by complimentary addition of one or more nucleotides to the at least one template nucleic acid molecule with a concomitant proton release;

(b) contacting the reaction medium with a polarizable electrode responsive to charge perturbations in the reaction medium;

(c) inducing changes in polarity of the polarizable electrode in response to surface charge perturbations in the polarizable electrode resulting from the concomitant proton release into the reaction medium contacting the polarizable electrode as template polymerization of the at least one template nucleic acid molecule occurs;

(d) generating current signals in the polarizable electrode in response to the changes in polarity induced in step (c) in the polarizable electrode, and amplifying the current signals with an amplifying circuit having an input connected to the polarizable electrode; and

(e) associating amplified current signals generated in step (d) with one or more corresponding nucleotides added to the at least one template nucleic acid to thereby discern at least a portion of the at least one template nucleic acid sequence.

2. The method of claim 1 wherein the reaction medium contacts an insulating layer on the polarizable electrode.

3. The method of claim 2 wherein the insulating layer comprises a self-assembled monolayer.

4. The method of claim 2 further comprising the step of linking the insulating layer to the at least one template nucleic acid.

5. The method of claim 1 wherein the amplifying circuit comprises a differential feedback amplifier comprising a first input from the polarizable electrode and a second input from a fixed voltage, whereby producing the amplified current signals for associating signals with the one or more corresponding nucleotides.

6. The method of claim 1 wherein the polarizable electrode comprises a metallic composition.

7. The method of claim 6 wherein the metallic composition is gold, copper or silver.

8. The method of claim 1 wherein the at least one template nucleic acid molecule is linked to the polarizable electrode.

9. The method of claim 1 wherein the method includes providing the reaction medium to an addressable array of polarizable electrodes by fluid circuits.

10. The method of claim 9 wherein the polarizable electrodes are individually addressable.

11. The method of claim 1 wherein the polarizable electrode is comprised in an integrated circuit on a semiconductor substrate.

12. The method of claim 1 wherein generating the current signals includes applying a voltage to the polarizable electrode.

13. The method of claim 12 further comprising providing a feedback loop to apply the voltage to the polarizable electrode.

14. The method of claim 13 wherein the feedback loop includes a feedback resistor to provide a current path for the generated current signal.

15. The method of claim 1 further comprising:

receiving a reference signal from a reference electrode; and

comparing the current signals to the reference signal to discern at least a portion of the at least one template nucleic acid sequence.

16. The method of claim 15 wherein the at least one template nucleic acid molecule is linked to the polarizable electrode and no template nucleic acid molecule is linked to the reference electrode.

17. The method of claim 1 wherein the one or more nucleotides are label-free.