Enzyme cascade methods for e-trace assay signal amplification

Abstract

The disclosure also relates to novel methods for detection of biological targets using amplification steps in conjunction with conversion of functional groups attached to a transitional metal complex, resulting in quantifiable electrochemical signal at two unique potentials, E o 1 and E o 2 .

1 - 24 . (canceled) 
     
     
         25 . A method for detecting a target analyte in a test sample, said method comprising:
 a. contacting said test sample with a binding ligand under conditions wherein said target analyte, if present, binds said binding ligand forming a first complex;   b. contacting said first complex with a labeled target analyte, wherein:
 i. said labeled target analyte binds any binding ligands not already bound to said target analyte forming a second complex and the second complex is contacted with an intermediary enzyme of a peroxide generating system comprising a labeled linker to form a third complex; 
 ii. or optionally said labeled target analyte is bound to an intermediary enzyme of a peroxide generating system attached via a labeled linker and binds any binding ligands not already bound to said target analyte forms said third complex; and 
 iii. said labeled target analyte is labeled with a first member of a specific binding pair and said labeled linker is labeled with a second member of said specific binding pair 
   c. isolating said third complex;   d. contacting said third complex with a substrate for said intermediary enzyme under conditions such that product(s) are generated to form a first assay mixture;   e. contacting a peroxide-generating enzyme and optionally a recycling enzyme with first assay mixture under conditions wherein peroxide is generated to form a second assay mixture;   f. contacting the second assay mixture with a first solid support comprising an electrode comprising a covalently attached electroactive moiety (EAM) having a first E 0 , said EAM comprising a transition metal complex comprising a self-immolative moiety (SIM) and a peroxide sensitive moiety (PSM), wherein said peroxide reacts with said PSM to release said SIM from said EAM and result in said EAM having a second E 0 ;   g. measuring the electrochemical properties of said EAM at the first E 0  and at the second E 0 ; and   h. detecting for the presence or the concentration of said target analyte from said electrochemical properties.   
     
     
         26 . A method for detecting a target analyte in a test sample, said method comprising:
 a. contacting said test sample with a first complex comprising a labeled target analyte pre-bound to a binding ligand and an intermediary enzyme of a peroxide generating system attached via a labeled linker to the labeled target analyte under conditions wherein said target analyte, if present, displaces said labeled target analyte, said labeled linker, and said intermediary enzyme from said first complex forming a second complex, wherein said labeled target analyte is labeled with a first member of a specific binding pair and said labeled linker is labeled with a second member of said specific binding pair;   b. isolating said second complex;   c. contacting said second complex with a substrate for said intermediary enzyme under conditions such that product(s) are generated to form a first assay mixture;   d. contacting a peroxide-generating enzyme and optionally a recycling enzyme with first assay mixture under conditions wherein peroxide is generated to form a second assay mixture;   e. contacting the second assay mixture with a first solid support comprising an electrode comprising a covalently attached electroactive moiety (EAM) having a first E 0 , said EAM comprising a transition metal complex comprising a self-immolative moiety (SIM) and a peroxide sensitive moiety (PSM), wherein said peroxide reacts with said PSM to release said SIM from said EAM and result in said EAM having a second E 0 ;   f. measuring the electrochemical properties of said EAM at the first E 0  and at the second E 0 ; and   g. detecting for the presence or the concentration of said target analyte from said electrochemical properties.   
     
     
         27 . A method for detecting a target analyte in a test sample, said method comprising:
 a. contacting said test sample with a first complex comprising a labeled target analyte pre-bound to a binding ligand and an intermediary enzyme of a peroxide generating system attached via a labeled linker to the labeled target analyte under conditions wherein said target analyte, if present, displaces said labeled target analyte, said labeled linker, and said intermediary enzyme from said first complex forming a second complex on the binding ligand and a first solution phase assay mixture containing a third complex comprising the said displaced labeled target analyte with said labeled linker, and said intermediary enzyme, and wherein said labeled target analyte is labeled with a first member of a specific binding pair and said labeled linker is labeled with a second member of said specific binding pair;   b. isolating said third complex;   c. contacting said third complex with a substrate for said intermediary enzyme under conditions such that product(s) are generated to form a second assay mixture;   d. contacting a peroxide-generating enzyme and optionally a recycling enzyme with second assay mixture under conditions wherein peroxide is generated to form a third assay mixture;   e. contacting the third assay mixture with a first solid support comprising an electrode comprising a covalently attached electroactive moiety (EAM) having a first E 0 , said EAM comprising a transition metal complex comprising a self-immolative moiety (SIM) and a peroxide sensitive moiety (PSM), wherein said peroxide reacts with said PSM to release said SIM from said EAM and result in said EAM having a second E 0 ;   f. measuring the electrochemical properties of said EAM at the first E 0  and at the second E 0 ; and   g. detecting for the presence or the concentration of said target analyte from said electrochemical properties.   
     
     
         28 . The method according to claim  1 , wherein said labeled target analyte binds any binding ligands not already bound to said target analyte forming a second complex and the second complex is contacted with an intermediary enzyme of a peroxide generating system comprising a labeled linker to form a third complex. 
     
     
         29 . The method according to claim  1 , wherein the target analyte is a small molecule. 
     
     
         30 . The method according to claim  5 , wherein said target analyte is testosterone. 
     
     
         31 . The method according to claim  1 , wherein said binding ligand is attached to a second solid support. 
     
     
         32 . The method according to claim  7 , wherein said second solid support is chosen from the group consisting of microparticles, magnetic microparticles, beads, and microchannels. 
     
     
         33 . The method according to claim  1 , wherein said specific binding pair is biotin/streptavidin. 
     
     
         34 . The method according to claim  1 , wherein said electrode further comprises a self-assembled monolayer (SAM). 
     
     
         35 . The method according to claim  1 , wherein said substrate for said intermediary enzyme of peroxide-generating system is FADP, NADP. 
     
     
         36 . The method according to claim  1 , wherein said product(s) is a substrate for said peroxide generating enzyme. 
     
     
         37 . The method according to claim  1 , further comprising the presence of a substrate for said peroxide generating enzyme and wherein said product(s) is a cofactor for said peroxide-generating enzyme. 
     
     
         38 . The method according to claim  1 , wherein said recycling enzyme is included to recycle said cofactor. 
     
     
         39 . The method according to claim  1 , wherein said intermediary enzyme of a peroxide generating system is alkaline phosphatase (AP) or another dephosphorylating enzyme. 
     
     
         40 . The method according to claim  1 , wherein said peroxide-generating enzyme is D-amino acid oxidase (DAAO) or another flavin dependent oxidoreductase enzyme. 
     
     
         41 . The method according to claim  17 , wherein said peroxide-generating enzyme is NADH Oxidase and said recycling enzyme is Alcohol Dehydrogenase (ADH). 
     
     
         42 . A method according to claim  1 , wherein said first binding ligand and said second binding ligand are independently chosen from the group consisting of monoclonal antibodies, fragments of monoclonal antibodies, polyclonal antibodies, fragments of polyclonal antibodies, proteins, and peptides. 
     
     
         43 . A method according to claim  1 , wherein said transition metal is chosen from the group consisting of iron, ruthenium, and osmium. 
     
     
         44 . A method according to claim  1 , wherein said EAM is chosen from the group consisting of ferrocene and substituted ferrocene. 
     
     
         45 . A method for detecting a target analyte in a test sample, said method comprising:
 (a) contacting a target specific enzyme, a recycling enzyme and a peroxide-generating enzyme with said target sample under conditions wherein a peroxide is generated in the presence of said target analyte to form an assay mixture;   (b) contacting the assay mixture with a first solid support comprising an electrode comprising a covalently attached electroactive moiety (EAM) having a first E 0 , said EAM comprising a transition metal complex comprising a self-immolative moiety (SIM) and a peroxide sensitive moiety (PSM), wherein said peroxide reacts with said PSM to release said SIM from said EAM and result in said EAM having a second E 0 ;   (c) measuring the electrochemical properties of said EAM at the first E 0  and at the second E 0 ; and   (d) detecting for the presence or concentration of said target analyte from said electrochemical properties.   
     
     
         46 . A method for detecting a target analyte in a test sample, said method comprising:
 (a) contacting said test sample with a first complex or a first binding ligand, wherein:
 i. when said test sample is contacted with the first complex, the first complex comprises a labeled target analyte pre-bound to a binding ligand said test sample under conditions wherein said target analyte from said test sample, if present, displaces said labeled target from said first complex forming a second complex, wherein said labeled target analyte is labeled with a first member of a specific binding pair; and the method further comprises:
 a. contacting said second complex with an intermediary enzyme of a peroxide generating system comprising a labeled linker forming a third complex, wherein said labeled linker is labeled with a second member of said specific binding pair; 
 b. isolating said third complex; and 
 c. contacting said third complex with a substrate for said intermediary enzyme of peroxide-generating system under conditions such that product(s) are generated to form a first assay mixture; 
 
 ii. when said test sample is contacted with the first binding ligand, the first binding ligand is contacted with said test sample under conditions wherein said first binding ligand binds said target analyte, if present, in said test sample to form a first complex, the method further comprises:
 a. contacting said first complex with a second binding ligand under conditions wherein said first complex and said second binding ligand bind to form a second complex, wherein said second binding ligand comprises an intermediary enzyme of a peroxide-generating system and isolating said second complex; and 
 b. contacting said second complex with a substrate for said intermediary enzyme of peroxide-generating system under conditions such that products are generated to form a first assay mixture; 
 
   (b) contacting a peroxide-generating enzyme and optionally a recycling enzyme with first assay mixture under conditions wherein peroxide is generated to form a second assay mixture;   (c) contacting the second, assay mixture with a first solid support comprising an electrode comprising a covalently attached electroactive moiety (EAM) having a first E 0 , said EAM comprising a transition metal complex comprising a self-immolative moiety (SIM) and a peroxide sensitive moiety (PSM), wherein said peroxide reacts with said PSM to release said SIM from said EAM and result in said EAM having a second E 0 ;   (d) measuring the electrochemical properties of said EAM at the first E 0  and at the second E 0 ; and   (e) detecting for the presence or the concentration of said target analyte from said electrochemical properties.