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

• US 9,194,836 B2
• Filed: 03/11/2013
• Issued: 11/24/2015
• Est. Priority Date: 08/07/2009
• Status: Expired due to Fees

• Current Assignee: Ohmx Corporation
• Original Assignee: Ohmx Corporation
• Inventors: Bertin, Paul A.
• Primary Examiner(s): BROWN, MELANIE YU
• Application Number:
  US13/793,752
• Publication Number:
  US 20130236909A1
• Time in Patent Office: 988 Days
• US Class Current: 1/1
• 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 moiety (EAM) comprising a transition metal complex comprising a self-immolative moiety and a peroxide sensitive moiety (PSM), wherein said EAM has a first E°

,

(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 an enzyme that catalyzes the generation of a cofactor from a phosphorylated precursor cofactor for a soluble apo-oxidase enzyme to form a second complex;

(d) adding said phosphorylated precursor cofactor, said soluble apo-oxidase enzyme, and a substrate of the soluble apo-oxidase enzyme to said second complex under conditions that peroxide is generated and said self-immolative moiety is removed such that said EAM has a second E⁰; and

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

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

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

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

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

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

7. A method according to claim 1, wherein said EAM is a ferrocene.

8. A method according to claim 1, further comprising detecting a change between the first E⁰ and the second E⁰ of said EAM, and wherein said change is an indication of the presence of said target.