Charge perturbation detection system for DNA and other molecules

• US 8,012,756 B2
• Filed: 06/23/2010
• Issued: 09/06/2011
• Est. Priority Date: 11/12/2004
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Davis, Ronald W., Karhanek, Miloslav, Pourmand, Nader
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US12/821,809
• Publication Number:
  US 20100261619A1
• Time in Patent Office: 440 Days
• Field of Search: None
• US Class Current: 436/6
• 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 and apparatus for direct detection of chemical reactions are provided. In a preferred embodiment, electric charge perturbations of the local environment during enzyme-catalyzed reactions are sensed by an electrode system with an immobilized target molecule. The target molecule is preferably DNA. 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. The initial enzyme attachment to the DNA molecule can be detected prior to polymerization, with electrode capacitance measurement using the same voltage-clamp amplifier. This technique and device may be adapted to other reaction determinations, such as enzymatic reactions, other electrode configurations, and other amplifying circuits.

Claims

1. A device for sequencing a nucleic acid, comprising:

(a) a single stranded DNA (ssDNA) modified measurement electrode; and

(b) a voltage clamp amplifying circuit for maintaining a set potential in the measurement electrode and generating a signal in response to a charge perturbation in the vicinity of the measurement electrode, the signal indicative of the charge perturbation of the ssDNA modified electrode;

(c) a detector for detecting the signal, thereby indicating reaction of a selected reactant with a target molecule; and

(d) microfluidic channels for delivering reactants to the ssDNA modified measurement electrodes.

3. The device of claim 1 or 2, further comprising a target molecule immobilized on the measurement electrode, the target molecule comprising a nucleic acid.

4. The device of claim 3, wherein the nucleic acid is chemically linked to the measurement electrode.

5. The device of claim 1 or 2, wherein the reactants comprise a nucleotide triphosphate (NTP).

7. The device of claim 1 or 6, wherein the microfluidic channels comprise a polymer.

2. A device for determining the sequence of a target molecule, comprising:

(a) a container for containing reaction medium having therein reactants and the target molecule;

(b) a measurement electrode and reference electrode, the measurement electrode adjacent the target molecule;

(c) a voltage clamp amplifying circuit for maintaining a set potential in the measurement electrode and generating a transient current in response to a charge perturbation in the vicinity of the measurement electrode; and

(d) a detector for detecting the signal, thereby indicating reaction of the selected reactant with the target molecule.

6. The device of claim 2, further comprising microfluidic channels for delivering reactants to the measurement electrode.