Enzyme-amplified redox microarray detection process

Abstract

There is disclosed a process and an array for assaying for binding of target molecules to capture molecules on microarray devices, wherein the mircoarray devices contain electrodes. Specifically, there is disclosed a binding (including nucleotide hybridization) process to detect binding on a microarray wherein the microarray contains electronically addressable electrode devices. There is further disclosed an enzymatically catalyzed oxidation/reduction reaction to take place within a “virtual flask” region of a microarray wherein the reaction is detected by current changes detected on the addressable electrode.

I claim:  
     
         1 . A process for reading microarray devices having addressable electrodes to determine binding between a capture probe and a target molecule, comprising: 
 (a) providing an array having a plurality of electrodes and a plurality of capture molecules at sites corresponding to the electrodes;    (b) non-specifically attaching an oxidation/reduction enzymatic moiety to one or a plurality of target molecules in a sample for analysis to create a prepped target sample;    (c) administering the prepped target sample to the array and allowing for binding of target molecules to capture molecules;    (d) adding a substrate to the array that will create a local voltage signal when catalyzed by the oxidation/reduction enzyme through local generation of electrochemical reagents; and    (e) measuring for the presence or absence of a voltage signal generated locally by electrochemical reagents at each electrode having a capture molecule attached thereto.    
     
     
         2 . The process for reading microarray devices having addressable electrodes to determine binding between a capture probe and a target molecule of  claim 1 , wherein the array having a plurality of electrodes and capture molecules corresponding to the electrodes is generated by a technique selected from the group consisting of in situ synthesis with electrochemical techniques, spotting the capture molecules, ink-jet printing the capture molecules, and in situ synthesis through photolighography techniques.  
     
     
         3 . The process for reading microarray devices having addressable electrodes to determine binding between a capture probe and a target molecule of  claim 2 , wherein the array having a plurality of electrodes and capture molecules corresponding to the electrodes is formed by in situ synthesis with electrochemical techniques.  
     
     
         4 . The process for reading microarray devices having addressable electrodes to determine binding between a capture probe and a target molecule of  claim 1 , wherein the oxidation/reduction enzyme is selected from the group consisting of laccase, horseradish peroxidase, β-galactosidase, glucose oxidase, alkaline phosphatase, dehydrogenases, and combinations thereof.  
     
     
         5 . The process for reading microarray devices having addressable electrodes to determine binding between a capture probe and a target molecule of  claim 1 , wherein the oxidation/reduction enzyme is attached to the target molecule(s) through an antibody and anti-idiotype antibody combination or through a biotin and streptavidin (or avidin) binding combination.  
     
     
         6 . The process for reading microarray devices having addressable electrodes to determine binding between a capture probe and a target molecule of  claim 1 , wherein the array having a plurality of electrodes further comprises a porous reaction layer covering the electrodes, wherein the porous reaction layer has a thickness of from about 0.1 microns to about 10 microns and whereby the porous reaction layer functions to block diffusion of oxidation/reduction activity products such that there is little lateral signal being picked up at an adjacent electrode.  
     
     
         7 . The process for reading microarray devices having addressable electrodes to determine binding between a capture probe and a target molecule of  claim 6 , wherein the porous reaction layer is made from a polymeric material selected from the group consisting of polyvinyl alcohol, polyvinyl acetate, polyvinyl alcohol, tricellulose acetate, polyurethane, agarose, controlled porosity glass with a PTFE resin, dextran, epoxy-based polymers, and combinations thereof.  
     
     
         8 . The process for reading microarray devices having addressable electrodes to determine binding between a capture probe and a target molecule of  claim 1 , wherein the capture molecule is a molecule from the class of molecules selected from the group consisting of oligonucleotides, polypeptides, antibodies, glycosylated polypeptides, polysaccharides, and mixed molecules having monomers from a plurality of the foregoing molecules.  
     
     
         9 . The process for reading microarray devices having addressable electrodes to determine binding between a capture probe and a target molecule of  claim 1 , wherein a target molecule is from a class of molecules selected from the group consisting of DNA, RNA, single-stranded DNA, ribosomal RNA, mitochondrial DNA, cellular receptors, glycosylated membrane-bound proteins, non-glycosylated membrane-bound proteins, polypeptides, glycosylated polypeptides, antibodies, cellular antigenic determinants, organic molecules, metal ions, salt anions and cations, and combinations thereof.  
     
     
         10 . A mircoarray device for detecting binding of a target molecule to a capture probe, comprising: 
 (a) an array having a plurality of electrodes and a plurality of capture molecules at sites corresponding to the electrodes;    (b) an oxidation/reduction enzymatic moiety bound to one or a plurality of target molecules in a sample for analysis, wherein the oxidation/reduction enzymatic moiety bound to the target molecules is incubated with the capture molecules on the array such that binding between capture molecules and target molecules that bind, will occur;    (c) a substrate molecule that will create a local voltage signal when catalyzed by the oxidation/reduction enzyme through local generation of electrochemical reagents; and    (e) a voltage signal measuring device electrically connected to each electrode on the array.    
     
     
         11 . The mircoarray device for detecting binding of a target molecule to a capture probe of  claim 10  wherein the array having a plurality of electrodes and capture molecules corresponding to the electrodes is generated by a technique selected from the group consisting of in situ synthesis with electrochemical techniques, spotting the capture molecules, ink-jet printing the capture molecules, and in situ synthesis through photolighography techniques.  
     
     
         12 . The mircoarray device for detecting binding of a target molecule to a capture probe of  claim 11  wherein the array having a plurality of electrodes and capture molecules corresponding to the electrodes is formed by in situ synthesis with electrochemical techniques.  
     
     
         13 . The mircoarray device for detecting binding of a target molecule to a capture probe of  claim 10  wherein the oxidation/reduction enzyme is selected from the group consisting of laccase, horseradish peroxidase, β-galactosidase, glucose oxidase, alkaline phosphatase, dehydrogenases, and combinations thereof.  
     
     
         14 . The mircoarray device for detecting binding of a target molecule to a capture probe of  claim 10  wherein the oxidation/reduction enzyme is attached to the target molecule(s) through an antibody and anti-idiotype antibody combination or through a biotin and streptavidin (or avidin) binding combination.  
     
     
         15 . The mircoarray device for detecting binding of a target molecule to a capture probe of  claim 10  wherein the array having a plurality of electrodes further comprises a porous reaction layer covering the electrodes, wherein the porous reaction layer has a thickness of from about 0.1 microns to about 10 microns and whereby the porous reaction layer functions to block diffusion of oxidation/reduction activity products such that there is little lateral signal being picked up at an adjacent electrode.  
     
     
         16 . The mircoarray device for detecting binding of a target molecule to a capture probe of  claim 15  wherein the porous reaction layer is made from a polymeric material selected from the group consisting of polyvinyl alcohol, polyvinyl acetate, polyvinyl alcohol, tricellulose acetate, polyurethane, agarose, controlled porosity glass with a PTFE resin, dextran, epoxy-based polymers, and combinations thereof.  
     
     
         17 . The mircoarray device for detecting binding of a target molecule to a capture probe of  claim 10  wherein the capture molecule is a molecule from the class of molecules selected from the group consisting of oligonucleotides, polypeptides, antibodies, glycosylated polypeptides, polysaccharides, and mixed molecules having monomers from a plurality of the foregoing molecules.  
     
     
         18 . The mircoarray device for detecting binding of a target molecule to a capture probe of  claim 10  wherein a target molecule is from a class of molecules selected from the group consisting of DNA, RNA, single-stranded DNA, ribosomal RNA, mitochondrial DNA, cellular receptors (i.e., glycosylated or non-glycosylated membrane-bound proteins), polypeptides, glycosylated polypeptides, antibodies, cellular antigenic determinants, organic molecules, metal ions, salt anions and cations, and combinations thereof.