Enzyme triggered redox altering chemical elimination (E-trace) immunoassay

• US 8,530,170 B2
• Filed: 08/09/2010
• Issued: 09/10/2013
• Est. Priority Date: 08/07/2009
• Status: Active Grant

• Current Assignee: Ohmx Corporation
• Original Assignee: Ohmx Corporation
• Inventors: Bertin, Paul A.
• Primary Examiner(s): BROWN, MELANIE YU
• Application Number:
  US12/853,204
• Publication Number:
  US 20110033869A1
• Time in Patent Office: 1,128 Days
• Field of Search: 435/7.1, 435/7.9, 435/7.91, 436/518, 436/524, 436/525
• US Class Current: 435/7.1
• CPC Class Codes: G01N 2610/00 Assays involving self-assembled monolayers [SAMs]
• CPC Class Codes: G01N 27/26 by investigating electrochemical variables; by using electrolysis or electrophoresis
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

The present invention is directed to electronic methods of detecting target analytes such that upon binding of the target analyte a shift in electrochemical potential is seen. This assay relies on the use of an electroactive moiety (“EAM”) that is attached to an electrode and comprises a self-immolative moiety, whose presence gives the EAM a first E0, and whose absence, upon irreversible cleavage gives the EAM a second E0. This difference is detected, and if such change occurs, it is an indication of the presence of a target analyte.

Claims

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

(a) providing a solid support comprising an electrode comprising;

(i) a self-assembled monolayer (SAM);

(ii) a covalently attached electroactive active moiety (EAM) comprising a transition metal complex, a self-immolative moiety and a peroxide sensitive moiety (PSM), wherein said EAM has a first E⁰ when said self-immolative moiety is present;

(iii) a capture binding ligand that binds said analyte;

(b) contacting said target analyte and said solid support, under conditions wherein said target analyte binds said capture binding ligand to form a first complex;

(c) contacting said first complex with a soluble capture ligand that binds said target analyte, wherein said soluble capture ligand comprises a peroxide generating moiety to form a second complex;

(d) adding a peroxide substrate to said second complex under conditions such that peroxide is generated and said self-immolative moiety is removed such that said EAM has a second E⁰ when said self-immolative moiety is absent; and

(e) detecting said second E⁰ as an indication of the presence of said target.

2. The method according to claim 1, wherein prior to said step (b), a washing step is performed.

3. The method according to claim 1, wherein prior to step (c), a washing step is performed.

4. The method according to claim 1, wherein said steps (b) and (c) are done simultaneously.

5. The method according to claim 1, wherein said peroxide generating moiety is a glucose oxidase enzyme.

6. The method according to claim 1, wherein said solid support comprises an array of electrodes.

7. The method according to claim 1, wherein said transition metal is selected from the group consisting of iron, ruthenium and osmium.

8. The method according to claim 1, wherein said EAM is a ferrocene.

9. A composition comprising a solid support comprising:

(a) an electrode comprising;

(i) a self-assembled monolayer (SAM);

(ii) a covalently attached electroactive active moiety (EAM) comprising a transition metal complex, a self-immolative moiety and a peroxide sensitive moiety (PSM), wherein said EAM has a first E⁰ when said self-immolative moiety is present and a second E⁰ when said self-immolative moiety is absent;

(iii) a capture binding ligand that binds to a target analyte; and

(b) a soluble capture ligand that binds said target analyte, wherein said soluble capture ligand comprises a peroxide generating moiety.

10. The composition according to claim 9, wherein said peroxide generating moiety is a glucose oxidase enzyme.

11. The composition according to claim 9, wherein said solid support comprises an array of electrodes.

12. The composition according to claim 9, wherein said transition metal is selected from the group consisting of iron, ruthenium and osmium.

13. The composition according to claim 9, wherein said EAM is a ferrocene.