Enzyme-Amplified Redox Micoarray Detection Process

Abstract

There is disclosed a process and an array for assaying for binding of target molecules to capture molecules on micro array devices, wherein the microarray 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 micro array wherein the reaction is detected by current changes detected on the addressable electrode.

What is claimed is: 
     
         1 . A process for reading micro array devices having addressable electrodes to determine binding between a capture probe and a target molecule comprising:
 (a) providing an array having a plurality of addressable electrodes and a plurality of capture molecules at sites corresponding to the addressable electrodes;   (b) non-specifically attaching an oxidation/reduction enzymatic moiety to one or more target molecules;   (c) administering the one or more target molecules to the array and allowing for binding of the one or more target molecules to the plurality of capture molecules;   (d) adding a substrate to the array that will create a local voltage signal or a current signal when catalyzed by the oxidation/reduction enzymatic moiety through local generation of electrochemical reagents;   (e) waiting a period of time for the current signal to level off; and   (f) measuring for the presence or absence of a voltage signal or a current signal generated locally by electrochemical reagents at one or more of the plurality of addressable electrodes.   
     
     
         2 . The process of  claim 1 , where the array further comprises a porous reaction layer covering the plurality of addressable electrodes. 
     
     
         3 . The process of  claim 2 , where the porous reaction layer has a thickness of between:
 a lower limit of approximately 0.1 microns; and   an upper limit of approximately 10 microns.   
     
     
         4 . The process of  claim 2 , where the array is generated by a technique selected from the group consisting of in situ synthesis with electrochemical techniques, spotting the plurality of capture molecules, ink-jet printing the plurality of capture molecules, and in situ synthesis through photolithography techniques. 
     
     
         5 . The process of  claim 2 , where the oxidation/reduction enzymatic moiety is selected from the group consisting of laccase, horseradish peroxidase, β-galactosidase, glucose oxidase, alkaline phosphatase, dehydrogenases, and combinations thereof. 
     
     
         6 . The process of  claim 2 , where the oxidation/reduction enzymatic moiety is attached to the target molecule through an antibody and an anti-idiotype antibody combination. 
     
     
         7 . The process of  claim 2 , where the oxidation/reduction enzymatic moiety is attached to the target molecule through a biotin and a streptavidin combination. 
     
     
         8 . The process of  claim 2 , where the oxidation/reduction enzymatic moiety is attached to the target molecule through a biotin and an avidin combination. 
     
     
         9 . The process of  claim 2 , where the plurality of capture molecules is selected from the group consisting of oligonucleotides, polypeptides, antibodies, glycosylated polypeptides, polysaccharides, and combinations thereof. 
     
     
         10 . The process of  claim 2 , where the one or more target molecules are 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, salt cations, and combinations thereof. 
     
     
         11 . A microarray device for detecting binding of a target molecule to a capture probe, comprising:
 (a) an array having a plurality of addressable electrodes and a plurality of capture molecules at sites corresponding to the addressable electrodes;   (b) an oxidation/reduction enzymatic moiety bound to one or more target molecules in a sample for analysis, where 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 or current signal when catalyzed by the oxidation/reduction enzymatic moiety through local generation of electrochemical reagents;   (d) a voltage signal or a current signal measuring device electrically connected to each electrode on the array; and   (e) a time course monitoring device adapted to wait a period of time for the current signal measuring device to level off before measuring the voltage signal or the current signal at one or more of the plurality of addressable electrodes.   
     
     
         12 . The microarray device of  claim 11 , where the array further comprises a porous reaction layer covering the plurality of addressable electrodes. 
     
     
         13 . The microarray device of  claim 12 , where the porous reaction layer has a thickness of between:
 a lower limit of approximately 0.1 microns; and   an upper limit of approximately 10 microns.   
     
     
         14 . The microarray device of  claim 12 , where the array 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 photolithography techniques. 
     
     
         15 . The microarray device of  claim 12 , where the oxidation/reduction enzymatic moiety is selected from the group consisting of laccase, horseradish peroxidase, β-galactosidase, glucose oxidase, alkaline phosphatase, dehydrogenases, and combinations thereof. 
     
     
         16 . The microarray device of  claim 12 , where the oxidation/reduction enzymatic moiety is attached to the target molecule through an antibody and an anti-idiotype antibody combination. 
     
     
         17 . The microarray device of  claim 12 , where the oxidation/reduction enzymatic moiety is attached to the target molecule through a biotin and a streptavidin combination. 
     
     
         18 . The microarray device of  claim 12 , where the oxidation/reduction enzymatic moiety is attached to the target molecule through a biotin and an avidin combination. 
     
     
         19 . The microarray device of  claim 12 , where the plurality of capture molecules is selected from the group consisting of oligonucleotides, polypeptides, antibodies, glycosylated polypeptides, polysaccharides, and combinations thereof. 
     
     
         20 . The microarray device of  claim 13 , where the one or more target molecules are 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, salt cations, and combinations thereof.