Electronic measurements of monolayers following homogeneous reactions of their components

• US 9,404,883 B2
• Filed: 07/26/2013
• Issued: 08/02/2016
• Est. Priority Date: 07/27/2012
• Status: Active Grant

• Current Assignee: Ohmx Corporation
• Original Assignee: Ohmx Corporation
• Inventors: Bao, Yijia Paul, Gaustad, Adam G.
• Primary Examiner(s): Noguerola, Alexander
• Application Number:
  US13/952,215
• Publication Number:
  US 20140027309A1
• Time in Patent Office: 1,103 Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/28 involving peroxidase
• CPC Class Codes: G01N 2333/904 acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
• CPC Class Codes: G01N 2333/916 acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
• CPC Class Codes: G01N 2610/00 Assays involving self-assembled monolayers [SAMs]
• CPC Class Codes: G01N 27/3276 being a hybridisation with immobilised receptors using a FET type sensor G01N27/4145; concerning the hybridisation C12Q1/68
• 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 33/5438 Electrodes

Abstract

The disclosure relates to novel methods for performing a solution based assay reaction with an electroactive active moiety (EAM) that subsequently forms a self-assembled monolayer (SAM) utilizing the advantages of faster solution reaction kinetics, SAM protected electrode and surface based electrochemistry for electronic measurement.

Claims

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

(a) contacting a test sample and a capture binding ligand that binds to a target analyte, under conditions wherein said capture binding ligand binds said target analyte, if present, in said test sample to form a first complex, said capture binding ligand bound to a first solid support;

(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 product(s) are generated to form a first assay mixture;

(e) contacting a peroxide-generating enzyme with said first assay mixture under conditions wherein peroxide is generated to form a peroxide-containing second assay mixture;

(f) contacting said peroxide containing second assay mixture with an electroactive moiety (EAM) comprising a transition metal complex, a self-immolative moiety (SIM), and a peroxide sensitive moiety (PSM), wherein said SIM joins the PSM to the transition metal complex and wherein said EAM has a first E⁰, to form a third assay mixture wherein said peroxide reacts in the solution phase with said PSM of said EAM to release said SIM from said EAM and result in said EAM having a second E⁰;

(g) contacting said third assay mixture with a second solid support comprising an electrode under conditions such that a covalently attached self-assembled monolayer (SAM) forms comprising said EAM with said first E⁰ and with said second E⁰; and

(h) detecting for a change between the first E⁰ and the second E⁰ of said EAM, wherein said change is an indication of the presence of said target analyte.

2. A method according to claim 1 wherein the target analyte is a protein.

3. A method according to claim 1, wherein said first solid support is chosen from the group consisting of microparticles, magnetic microparticles, beads, and microchannels.

4. A method according to claim 1, wherein said product(s) is a substrate for said peroxide-generating enzyme.

5. A 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.

6. A method according to claim 1, wherein said intermediary enzyme of a peroxide-generating system is a dephosphorylating enzyme.

7. A method according to claim 6, wherein said intermediary enzyme of a peroxide-generating system is alkaline phosphatase.

8. A method according to claim 1, wherein said peroxide-generating enzyme is a flavin dependent oxidoreductase enzyme.

9. A method according to claim 8, wherein said peroxide-generating enzyme is D-amino acid oxidase.

10. A method according to claim 1, wherein said intermediary enzyme of a peroxide generating system is an oxidase enzyme.

11. A method according to claim 10, wherein said intermediary enzyme of a peroxide generating system is glucose oxidase.

12. 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.

13. A method according to claim 1, wherein said peroxide is hydrogen peroxide.

14. A method according to claim 1, wherein said EAM comprises a transition metal.

15. A method according to claim 14, wherein said transition metal is chosen from the group consisting of iron, ruthenium and osmium.

16. A method according to claim 1, wherein said transition metal complex is chosen from the group consisting of ferrocene and substituted ferrocene.

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

(a) contacting said target analyte with a peroxide-generating enzyme, under conditions wherein said target, if present, acts as a substrate for said peroxide-generating enzyme and peroxide is generated forming a first assay mixture;

(b) contacting said peroxide-containing first assay mixture with an electroactive moiety (EAM), said EAM comprising a transition metal complex, a self-immolative moiety (SIM), and a peroxide sensitive moiety (PSM), wherein said SIM joins the PSM to the transition metal complex and wherein said EAM has a first E⁰ to form a second assay mixture wherein said peroxide reacts in the solution phase with said PSM of said EAM to release said SIM from said EAM and result in said EAM having a second E⁰;

(c) contacting said second assay mixture with a first solid support comprising an electrode under conditions that a covalently attached self-assembled monolayer (SAM) forms comprising said EAM with said first E⁰ and with said second E⁰;

(d) detecting for a change between the first E⁰ and the second E⁰ of said EAM, wherein said change is an indication of the presence of said target analyte.

18. A method according to claim 17 wherein the target analyte is a small molecule.