Methods for the electrochemical detection of multiple target compounds

Abstract

A method of detecting two different target molecules through a single electrode is carried out by (a) providing a conductive oxidation-reduction reaction detection electrode; (b) contacting a sample suspected of containing a first and second target molecule to the electrode under conditions in which the first and second target molecules are deposited on the electrode, wherein the first target molecule comprises a first label and the second target molecule comprises a second label; (c) contacting to the electrode a first transition metal complex that oxidizes the first preselected label in a first oxidation-reduction reaction and a second transition metal complex that oxidizes the first and second labels in a second oxidation-reduction reaction, with the first and second oxidation-reduction reactions producing different detectable signals; (d) detecting the presence of the first target molecule by detecting the first oxidation-reduction reaction; and(e) detecting the presence of the second target molecule by detecting the second oxidation-reduction reaction. Devices for carrying out the method are also described.

1 - 22 . (canceled)  
   
   
       23 . A microelectronic device useful for the electrochemical detection of at least two different members of at least two different binding pairs, said device comprising: 
 a microelectronic substrate;    a conductive oxidation-reduction detection electrode on said substrate;    a first member of a first specific binding pair immobilized on a non-conductive layer, which first member binds with a second member of the first specific binding pair present in a sample, said first member of said first binding pair being adjacent said detection electrode so that an oxidation-reduction reaction occurring upon application of a potential to the detection electrode is detectable; and    a first member of a second specific binding pair immobilized on a non-conductive layer that binds with a second member of the second specific binding pair present in a sample, said first member of said second binding pair being adjacent said detection electrode so that an oxidation-reduction reaction occurring upon application of a potential to the detection electrode is detectable;    wherein said first member of said first binding pair and said first member of said second binding pair are different.    
   
   
       24 . The device according to  claim 23 , further comprising: 
 a first member of a third specific binding pair immobilized on a non-conductive layer that binds with a second member of the third specific binding pair present in a sample, said first member of said third binding pair being adjacent said detection electrode so that an oxidation-reduction reaction occurring upon application of a potential to the detection electrode is detectable;    wherein said first member of said first binding pair, said first member of said second binding pair, and said first member of said third binding pair are different.    
   
   
       25 . The device according to  claim 23 , wherein said first member of said first binding pair and said first member of said second binding pair are oligonucleotides.  
   
   
       26 . The device according to  claim 23 , wherein said first member of said first binding pair and said first member of said second binding pair are peptides or proteins.  
   
   
       27 . The device according to  claim 23 , wherein: 
 said first member of said first binding pair is an oligonucleotide; and    said first member of said second binding pair is a protein or peptide.    
   
   
       28 . The device according to  claim 23 , wherein the microelectronic substrate comprises a sample container, which comprises said conductive oxidation-reduction detection electrode and said immobilized first member of said first and second binding pair.  
   
   
       29 . The device according to  claim 28 , wherein the microelectronic substrate comprises said sample container which comprises a plurality of conductive oxidation-reduction detection electrodes and a plurality of immobilized first members of said first and second binding pairs.  
   
   
       30 . The device according to  claim 29 , further comprising a conductive reference electrode comprising a conducting metal.  
   
   
       31 . The device according to  claim 29 , further comprising a conductive auxiliary electrode comprising a conducting metal.  
   
   
       32 . The device according to  claim 29 , wherein the oxidation-reduction reaction is detectable via an electrical connection from each conductive oxidation-reduction detection electrode.  
   
   
       33 . The device according to  claim 23 , wherein said substrate is silicon.  
   
   
       34 . The device according to  claim 23 , wherein said substrate is glass.  
   
   
       35 . The device according to  claim 23 , further comprising an oxidation-reduction reaction detector.  
   
   
       36 . A method of detecting at least two different hybridization events through a common electrode, comprising the steps of: 
 (a) providing a device comprising: 
 a microelectronic substrate;  
 a conductive oxidation-reduction detection electrode on said substrate;  
 a first member of a first specific binding pair immobilized on a non-conductive layer that binds with a second member of the first specific binding pair present in a sample, said first member of said first binding pair being adjacent said detection electrode so that an oxidation-reduction reaction occurring upon application of a potential to the detection electrode is detectable; and  
 a first member of a second specific binding pair immobilized on a non-conductive layer that binds with a second member of the second specific binding pair present in a sample, said first member of said second binding pair being adjacent said detection electrode so that an oxidation-reduction reaction occurring upon application of a potential to the detection electrode is detectable;  
 wherein said first member of said first binding pair and said first member of said second binding pair are different;  
 wherein said second member of said first binding pair comprises a first preselected label and said second member of said second binding pair comprises a second preselected label;  
 and wherein said first and second preselected labels are different;  
   (b) contacting a sample suspected of containing said second member of said first binding pair and said second member of said second binding pair;    (c) simultaneously contacting to said substrate (i) a first transition metal complex that oxidizes said first preselected label in an oxidation-reduction reaction under conditions that cause a first oxidation-reduction reaction between the first transition metal complex and the first preselected label and (ii) a second transition metal complex that oxidizes said first and second preselected labels in an oxidation-reduction reaction under conditions that cause a second oxidation-reduction reaction between said second transition metal catalyst and the second preselected label, from which preselected labels there is electron transfer to the transition metal complex, resulting in regeneration of the reduced form of the corresponding transition metal complex as part of a catalytic cycle, and with said first and second oxidation-reduction reactions producing different detectable signals;    (d) detecting the presence of said second member of said first binding pair from the detection of said first oxidation-reduction reaction; and    (e) detecting the presence of said second member of said second binding pair from the detection of said second oxidation-reduction reaction.    
   
   
       37 . The method according to  claim 36 , wherein said transition metal complex is selected from the group consisting of Ru(bpy) 3   2+ , Ru(Me 2 -bpy) 3   2+ , Ru(Me 2 -phen) 3   2+ , Fe(bpy) 3   2+ , Fe(5-Cl-phen) 3   2+ , Os(5-Cl-phen) 3   2+ , and ReO 2 (py) 4   1+ .  
   
   
       38 . The method according to  claim 36 , wherein said first and second preselected labels are selected from the group consisting of adenine, guanine, and 6-mercaptoguanine.  
   
   
       39 . The method according to  claim 36 , wherein said second member of said first binding pair and said second member of said second binding pair are proteins or peptides.  
   
   
       40 . The method according to  claim 36 , wherein said second member of said first binding pair and said second member of said second binding pair are oligonucleotides.  
   
   
       41 . The method according to  claim 36 , wherein: 
 said second member of said first binding pair is a protein or peptide; and    said second member of said second binding pair is an oligonucleotide.    
   
   
       42 . The method according to  claim 36 , wherein at least one of said second member of said first binding pair and said second member of said second binding pair is a DNA.  
   
   
       43 . The method according to  claim 42 , further comprising the step of amplifying said DNA prior to said contacting step.  
   
   
       44 . The method according to  claim 43 , wherein said step of amplifying said DNA sample is carried out by polymerase chain reaction, strand displacement amplification, ligase chain reaction, or nucleic acid sequence-based amplification.  
   
   
       45 . The method according to  claim 36 , wherein said contacting step is carried out by sandwich assay.  
   
   
       46 . The method according to  claim 36 , wherein said contacting step is carried out by competitive assay.  
   
   
       47 . The method according to  claim 36 , wherein said contacting step is carried out by direct assay.  
   
   
       48 . The method according to  claim 36 , wherein said contacting step is carried out by competitive assay for an immobilized target substance.  
   
   
       49 . The method according to  claim 36 , wherein said contacting step is carried out by binding interaction assay.