Charge perturbation detection system for DNA and other molecules
First Claim
1. A device for detecting a chemical reaction of a selected reactant with a target molecule, wherein the reaction produces a charge perturbation on the target molecule, comprising:
- (a) a container for containing reaction medium having therein reactants and target molecules;
(b) a polarizable metal electrode in the container adjacent the target molecules, having a potential difference within said electrode between an exposed surface of the polarizable metal electrode which is facing the reaction medium and a side of the polarizable electrode which is not facing the reaction medium;
(c) said amplifying circuit comprising a voltage clamp amplifier having one input attached to a fixed voltage source, and another input attached to the polarizable metal electrode attached to a side of the polarizable metal electrode which is not facing the reaction medium, for maintaining said potential difference in the electrode and generating a signal in response to said charge perturbation, said charge perturbation on the target molecule causing a polarizable metal electrode surface charge perturbation and polarization; and
(d) a detector for detecting the signal, detected by the electrode as a transient current, thereby indicating reaction of the selected reactant with the target molecule.
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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.
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Citations
31 Claims
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1. A device for detecting a chemical reaction of a selected reactant with a target molecule, wherein the reaction produces a charge perturbation on the target molecule, comprising:
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(a) a container for containing reaction medium having therein reactants and target molecules; (b) a polarizable metal electrode in the container adjacent the target molecules, having a potential difference within said electrode between an exposed surface of the polarizable metal electrode which is facing the reaction medium and a side of the polarizable electrode which is not facing the reaction medium; (c) said amplifying circuit comprising a voltage clamp amplifier having one input attached to a fixed voltage source, and another input attached to the polarizable metal electrode attached to a side of the polarizable metal electrode which is not facing the reaction medium, for maintaining said potential difference in the electrode and generating a signal in response to said charge perturbation, said charge perturbation on the target molecule causing a polarizable metal electrode surface charge perturbation and polarization; and (d) a detector for detecting the signal, detected by the electrode as a transient current, thereby indicating reaction of the selected reactant with the target molecule. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)
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13. A device for detecting an enzymatic reaction of a selected reactant with a target molecule, wherein the reaction generates a proton, comprising:
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(a) a container for containing reaction medium having therein reactants and target molecules; (b) a polarizable metal electrode contacting the container adjacent to the target molecules and the reactants, having a potential difference within said metal electrode between an exposed surface of the polarizable electrode that is facing the reaction medium and a side of the polarizable electrode which is not facing the reaction medium and; (c) an amplifying circuit, comprising a voltage clamp amplifier, having an input from the polarizable electrode, attached to a side of the polarizable electrode not facing the reaction medium, a feedback to the input, and an input for a command voltage, thereby generating a signal in response to a difference in the induced charge in the electrode resulting from generation of the proton, said induced charge resulting from a charge perturbation on the target molecule causing a polarizable metal electrode surface charge perturbation and polarization; and (d) a detector for detecting the signal detected by the electrode as a transient current, thereby indicating reaction of the selected reactant with the molecule. - View Dependent Claims (14, 15, 16, 17, 18)
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Specification