Enzyme cascade methods for E-TRACE assay signal amplification

• US 9,250,203 B2
• Filed: 01/09/2013
• Issued: 02/02/2016
• Est. Priority Date: 01/09/2012
• Status: Active Grant

• Current Assignee: Ohmx Corporation
• Original Assignee: Ohmx Corporation
• Inventors: Bertin, Paul A., Ahrens, Michael J.
• Primary Examiner(s): Ball, J. Christopher
• Application Number:
  US13/737,634
• Publication Number:
  US 20130264220A1
• Time in Patent Office: 1,119 Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: G01N 27/26 by investigating electrochemical variables; by using electrolysis or electrophoresis
• CPC Class Codes: G01N 27/27 Association of two or more measuring systems or cells, each measuring a different parameter, where the measurement results may be either used independently, the systems or cells being physically associated, or combined to produce a value for a further parameter
• CPC Class Codes: G01N 27/3271 Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood amperometry per se G01N27/49; aspects concerning the enzyme reagent C12Q1/001
• CPC Class Codes: G01N 27/3273 Devices therefor, e.g. test element readers, circuitry details not specific to biochemical electrodes G01N33/4875
• CPC Class Codes: G01N 27/3277 being a redox reaction, e.g. detection by cyclic voltammetry voltammetry per se G01N27/42, G01N27/48
• CPC Class Codes: G01N 27/4166 measuring a particular property of an electrolyte
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)

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°₁ and E°₂.

Claims

1. 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 E⁰, 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 E⁰;

(c) measuring the electrochemical properties of said EAM at the first E⁰ and at the second E⁰; and

(d) detecting for the presence or concentration of said target analyte from said electrochemical properties.

13. A method according to claim 1, wherein said target analyte is reduced Nicotinamide Adenine Dinucleotide (NADH).

14. A method according to claim 13, wherein said recycling enzyme is Alcohol Dehydrogenase (ADH), said target specific enzyme is NADH Oxidase, and said peroxide generating enzyme is NADH oxidase.

15. A method according to claim 1, wherein said target analyte is Adenosine Triphosphate (ATP).

16. A method according to claim 15, wherein said target specific enzyme is Glycerol Kinase (GK), said recycling enzyme is Creatine Kinase (CK) or Glycerol 3-Phosphate Dehydrogenase (GPDH), and said peroxide generating enzyme is Glycerol-3-Phosphate Oxidase (G3PO).

29. The method according to claim 1, wherein the target analyte is a small molecule.

2. 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, with 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;

(b) 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;

(c) isolating said third complex;

(d) 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;

(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 E⁰, 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 E⁰;

(g) measuring the electrochemical properties of said EAM at the first E⁰ and at the second E⁰; and

(h) detecting for the presence or the concentration of said target analyte from said electrochemical properties.

3. The method according to claim 2, wherein said specific binding pair is biotin/streptavidin.

6. The method according to any of claim 2 or 4, wherein said binding ligand is attached to a second solid support.

7. A method according to claim 6, wherein said second solid support is chosen from the group consisting of microparticles, magnetic microparticles, beads, and microchannels.

8. A method according to any of claim 2 or 4, wherein said electrode further comprises a self-assembled monolayer (SAM).

9. The method according to any of claim 2 or 4, wherein the target analyte is a small molecule.

10. The method according to any of claim 2 or 4, wherein said product(s) is a substrate for said peroxide generating enzyme.

11. The method according to any of claim 2 or 4, 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.

12. The method according to claim 11, wherein said recycling enzyme is included to recycle said cofactor.

17. The method according to any of claim 2 or 4, wherein said intermediary enzyme of a peroxide generating system is alkaline phosphatase (AP) or any other dephosphorylating enzyme.

18. The method according to any of claim 2 or 4, wherein said peroxide-generating enzyme is selected from the group consisting of D-amino acid oxidase (DAAO), or any flavin dependent oxidoreductase enzyme.

19. The method according to any of claim 2 or 4, wherein said peroxide-generating enzyme is NADH Oxidase and said optional recycling enzyme is Alcohol Dehydrogenase (ADH).

20. A method according to any of claim 2 or 4, where the target analyte is testosterone.

21. A method according to any of claims 2 or 4, 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.

22. A method according to any of claim 2 or 4, wherein said EAM comprises a transition metal.

23. A method according to claim 22, wherein said transition metal is chosen from the group consisting of iron, ruthenium and osmium.

24. A method according to any of claim 2 or 4, wherein said EAM is chosen from the group consisting of ferrocene and substituted ferrocene.

25. A method according to claim 2, wherein the target analyte is a nucleic acid.

27. A method according to claim 25, wherein the binding ligand is a nucleic acid.

4. A method for detecting a target analyte in a test sample, said method comprising:

(a) contacting a first binding ligand 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;

(b) 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;

(c) isolating said second complex;

(d) 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;

(e) contacting a peroxide-generating enzyme and optionally a recycling enzyme with a 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 E⁰, 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 E⁰;

(g) measuring the electrochemical properties of said EAM at the first E⁰ and at the second E⁰; and

(h) detecting for the presence or the concentration of said target analyte from said electrochemical properties.

5. The method according to claim 4, wherein the target analyte is a protein.

26. A method according to claim 4, wherein the target analyte is a nucleic acid.

28. A method according to claim 26, wherein the first binding ligand is a nucleic acid.