Charge Perturbation Detection System for DNA and Other Molecules

Abstract

Methods and apparatus 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.

What is claimed is: 
     
         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 electrode in the container adjacent the target molecules;   (c) an amplifying circuit for maintaining a set potential in the electrode and generating a signal in response to said charge perturbation; and   (d) a detector for detecting the signal, thereby indicating reaction of the selected reactant with the target molecule.   
     
     
         2 . The device of  claim 1  further comprising a self assembled monolayer on the surface of the electrode for preventing nonspecific adhesion of the test molecule to the electrode. 
     
     
         3 . The device of  claim 2  wherein the self assembled monolayer is linked to the target molecule. 
     
     
         4 . The device of  claim 1  wherein the amplifying circuit comprises a differential feedback amplifier having one input at a fixed voltage and another input attached to the polarizable electrode. 
     
     
         5 . The device of  claim 4  wherein the amplifying circuit has its negative input connected to the polarizable electrode. 
     
     
         6 . The device of  claim 1  wherein the polarizable electrode is gold, copper or silver. 
     
     
         7 . The device of  claim 6  wherein the polarizable electrode comprises an insulating layer between the electrode and the reaction mixture. 
     
     
         8 . The device of  claim 1  wherein the target molecule is a polynucleic acid linked to the polarizable electrode. 
     
     
         9 . The device of  claim 1  comprising an addressable array of electrodes and fluid circuits addressing the array collectively. 
     
     
         10 . The device of  claim 9  formed on a semiconductor substrate provided with integrated circuits for connections to electrodes and for the amplifying circuits and channels for the fluid circuits. 
     
     
         11 . The device of  claim 10  wherein the channels are defined by a solid, shaped polymer. 
     
     
         12 . The device of  claim 11  wherein the polymer is selected from the group consisting of poly(dimethylsiloxane) (PDMS) and Poly (silarylene siloxane) (PSS). 
     
     
         13 . The device of  claim 1  further comprising a polynucleic acid template immobilized adjacent to the electrode. 
     
     
         14 . A device for detecting an enzymatic reaction of a selected reactant with a target molecule, wherein the reaction generates a proton, comprising:
 (a) a container for containing reaction medium having therein reactants and target molecules;   (b) a polarizable electrode contacting the container adjacent to the target molecules and the reactants;   (c) an amplifying circuit having an input from the polarizable electrode, 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; and   (d) a detector for detecting the signal, thereby indicating reaction of the selected reactant with the molecule.   
     
     
         15 . The device of  claim 14  further comprising a capacitance meter for detecting enzyme attachment to a target molecule. 
     
     
         16 . The device of  claim 14  further comprising a filter blocking signals at 5-10 kHz. 
     
     
         17 . The device of  claim 14  further comprising an immobilized target molecule which is a polynucleic acid, the reactants comprising NTPs, whereby a sequence of the target molecule is reflected in the signal. 
     
     
         18 . The device of  claim 17  wherein the nucleic acid a chemically linked to the electrode. 
     
     
         19 . The device of  claim 14  wherein each electrode is linked to between 1 and 10 fmol of a given polynucleic acid target. 
     
     
         20 . A method for detecting a chemical reaction of a selected reactant with a molecule, wherein the reaction generates a charged particle, comprising:
 (a) providing a reaction medium having therein reactants and target molecules;   (b) contacting the reaction medium with a polarizable electrode within the container in the vicinity of the target molecules and the reactants;   (c) inducing a polarity in the electrode;   (d) generating a signal in response to a difference in the induced charge in the electrode resulting from generation of the charged particle and its interaction with the electrode when a reactant reacts with the molecule; and   (e) detecting a signal indicating reaction of the selected reactant with the molecule.