Enzyme triggered redox altering chemical elimination (E-trace) immunoassay
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.
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 E 0 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 E 0 when said self-immolative moiety is absent; and
(e) detecting said second E 0 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 E 0 when said self-immolative moiety is present and a second E 0 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.