Electrochemical assay for the detection of enzymes

Abstract

The invention relates to novel compositions and methods for the detection of enzymes using the nuclear reorganization energy, λ, of an electron transfer process.

We claim: 
     
         1 . A method for detecting a target enzyme in a test sample, said method comprising:
 (a) adding a test sample comprising a target enzyme to a solid support comprising an electrode comprising:
 (i) a self-assembled monolayer (SAM); 
 (ii) a covalently attached eletroactive active moiety (EAM) comprising a transition metal complex with an E 0 ; and 
 (iii) a plurality of substrates attached to said electrode, wherein said substrates are substrates of said enzyme; 
   (b) contacting said target enzyme and said substrates to form a plurality of reactants; and   (c) determining the presence of said enzyme by measuring a change of said E 0 .   
     
     
         2 . A method according to  claim 1 , wherein said enzyme is a protease. 
     
     
         3 . A method according to  claim 1 , wherein said enzyme is a kinase. 
     
     
         4 . A method according to  claim 1 , wherein said EAM comprises at least one cyano ligand. 
     
     
         5 . A method according to  claim 1 , wherein said protease is an endopeptidase neurotoxin. 
     
     
         6 . A method according to  claim 5  wherein said endopeptidase neurotoxin is selected from the group consisting of  Clostridium botulinum  A, B or E. 
     
     
         7 . A method according to  claim 1 , wherein said EAM and said substrates are attached separately to said electrode. 
     
     
         8 . A method according to  claim 1 , wherein said solid support comprises an array of electrodes. 
     
     
         9 . A method according to  claim 1 , wherein said transition metal is selected from the group consisting of iron, ruthenium and osmium. 
     
     
         10 . A method for detecting a protease in a test sample, said method comprising:
 (a) adding a test sample comprising a protease to a solid support comprising an electrode, said electrode comprising:
 (i) a self-assembled monolayer (SAM); 
 (ii) a covalently attached eletroactive active moiety (EAM) comprising a transition metal complex with an E 0 ; and 
 (iii) a plurality of proteins attached to said electrode, wherein said proteins comprises a cleavage site of said protease; 
   (b) cleaving a plurality of said proteins with said protease; and   (c) determining the presence of said protease by measuring a change of said E 0 .   
     
     
         11 . A method according to  claim 10 , wherein said EAM comprises at least one cyano ligand. 
     
     
         12 . A method for detecting a kinase in a test sample, said method comprising the steps of:
 (a) adding a test sample comprises a kinase protease to a solid support comprising an electrode, said electrode comprising:
 (i) a self-assembled monolayer (SAM); 
 (ii) a covalently attached eletroactive active moiety (EAM) comprising a transition metal complex with an E 0 ; and 
 (iii) a plurality of proteins attached to said electrode, wherein said proteins are first substrates of said kinase; 
   (b) phosphorylating said proteins with said kinase and a second kinase substrate so that said second kinase substrate is covalently attached to said protein; and   (c) determining the presence of said kinase by measuring a change of said E 0 .   
     
     
         13 . A method according to  claim 12 , wherein said EAM comprises at least one cyano ligand.