ELECTRIC MEASUREMENT OF MONOLAYERS FOLLOWING PRO-CLEAVE DETECTION OF PRESENCE AND ACTIVITY OF ENZYMES AND OTHER TARGET ANALYTES

• US 20140027310A1
• Filed: 07/26/2013
• Published: 01/30/2014
• Est. Priority Date: 07/27/2012
• Status: Active Grant

• Current Assignee: Ohmx Corporation
• Original Assignee: Ohmx Corporation
• Inventors: Gaustad, Adam G., Ahrens, Michael J., Bertin, Paul A.
• Granted Patent: US 9,416,390 B2
• Time in Patent Office: Days
• US Class Current: 205/777.5
• 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: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2610/00 Assays involving self-assembled monolayers [SAMs]
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

This invention describes methods for detecting target analytes by utilizing an electroactive moiety (EAM) that functions as a substrate for a specific enzyme. If target analyte is present, a functional group is enzymatically removed from a transition metal complex resulting in quantifiable electrochemical signal at two unique potentials, E^o₁ and E^o₂ that is detected through a self-assembled monolayer (SAM) on an electrode.

Claims

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

(a) contacting a test sample with an electroactive moiety (EAM), said EAM comprising a transition metal complex and an enzyme reactive moiety (ERM) and having a first E^o, under conditions wherein said ERM is removed from at least a portion of said EAM provided in the presence of at least one target analyte in the test sample and results in said EAM having a second E^o;

(b) detecting for a change between the first E^o and the second E^o of said EAM, wherein said change is an indication of the presence of said at least one target analyte.

2. The method according to claim 1, wherein said at least one target analyte is an enzyme that removes said ERM from said EAM.

3. The method according to claim 1, wherein said at least one target analyte is bound between (i) a capture binding ligand bound to a first solid support and (ii) a soluble binding ligand comprising a surrogate enzyme, said surrogate enzyme is an enzyme that removes said ERM from said EAM.

4. The method according to claim 3, wherein said capture binding ligand and said soluble binding ligand are independently selected from the group consisting of monoclonal antibodies, fragments of monoclonal antibodies, polyclonal antibodies, fragments of polyclonal antibodies, proteins and peptides.

5. The method according to claim 1, wherein an assay mixture in a solution phase is formed in step (a) and prior to step (b), further comprising step (al) contacting said assay mixture with a second solid support comprising an electrode or an array of electrodes, under conditions such that a self-assembled monolayer (SAM) forms on said electrode, said EAM having said first E^o and said EAM having said second E^o.

6. The method according to claim 1, wherein said EAM is covalently attached to an electrode or array of electrodes on a second solid support as a self-assembled monolayer (SAM).

7. The method according to claim 1, wherein said EAM further comprising a self-immolative moiety (SIM) which joins said ERM to said transition metal complex.

8. The method according to claim 1, wherein said enzyme reactive moiety (ERM) is selected from the group consisting of an amino acid, peptide, acetate, lipid, carbohydrate, phosphate, or other functional group.

9. The method according to claim 1, wherein said at least one target analyte is selected from the group consisting of proteases, peptidases, phosphatases, oxidases, hydrolases, lyases transferases, isomerase, and ligases.

10. The method according to claim 3, wherein said surrogate enzyme is selected from the group consisting of proteases, peptidases, phosphatases, oxidases, hydrolases, lyases, transferases, isomerase, and ligases.

11. The method according to claim 1, wherein said target analyte is Prostate Specific Antigen (PSA).

12. The method according to claim 1, wherein said transition metal complex includes a transition metal selected from the group consisting of iron, ruthenium and osmium.

13. The method according to claim 1, wherein said transition metal complex comprises a ferrocene and substituted ferrocene.

14. The method according to claim 1, wherein said EAM comprises a flexible oligomer anchor tethering said transition metal complex to said electrode, said flexible anchor being an oligomers with polar or charged functional groups in their main chain or side chains examples including poly acrylic acids, polyethylene glycol (PEG), poly vinyl alcohol, polymethacrylate, poly vinyl-pyrrolidinone, acrylamide, maleic anhydride, and poly vinylpyridine, allylamine, ethyleneimine, oxazoline, and other hydrophobic oligomers with side chains that limit intermolecular hydrophobic interactions and prevent organization and rigidity.

15. The method according to claim 5, wherein each electrode in said array of electrodes is modified with a SAM comprising a unique EAM, each EAM comprising a unique ERM for a specific target analyte or said surrogate enzyme such that two or more different target analytes may be detected in said test sample.

16. A composition comprising a solid support comprising:

an electrode comprising;

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

(ii) a covalently attached electroactive active moiety (EAM) comprising a transition metal complex and an enzyme reactive moiety (ERM), wherein said EAM has a first E⁰ when said ERM is present and a second E⁰ when said ERM is absent.

17. The composition of claim 16, wherein the EAM further comprises a self-immolative moiety (SIM) that joins the ERM to the transition metal complex.

18. The composition according to claim 16, wherein said transition metal is selected from the group consisting of iron, ruthenium and osmium.

19. The composition according to claim claim 16, wherein said transition metal complex is a ferrocene or a substituted ferrocene.

20. The composition according to claim 16, wherein said EAM comprises a flexible oligomer anchor tethering said transition metal complex to said electrode, said flexible anchor being an oligomers with polar or charged functional groups in their main chain or side chains examples including poly acrylic acids, polyethylene glycol (PEG), poly vinyl alcohol, polymethacrylate, poly vinylpyrrolidinone, acrylamide, maleic anhydride, and poly vinylpyridine, allylamine, ethyleneimine, oxazoline, and other hydrophobic oligomers with side chains that limit intermolecular hydrophobic interactions and prevent organization and rigidity.

21. The composition according to claim 16, wherein said solid support comprises an array of electrodes.

22. The composition according to claim 16, wherein each electrode in said array of electrodes is modified with a SAM comprising a unique EAM, each EAM comprising a unique ERM for a specific target analyte or said surrogate enzyme such that at two or more different target analytes may be detected in said test sample.

23. A kit for detecting at least one target analyte in a test sample, the kit comprising the composition of claim 16.

24. A method for detecting at least one target analyte in a test sample, said method comprising:

(a) contacting a test sample with an electroactive moiety (EAM) to form an assay mixture in solution phase, said EAM having a first E^o and comprising a transition metal complex and an enzyme reactive moiety (ERM), under conditions wherein said ERM is removed from at least a portion of said EAM provided in the presence of at least one target analyte in the test sample and results in said EAM having a second E^o;

(b) contacting said assay mixture with a solid support comprising an electrode or an array of electrodes under conditions such that a self-assembled monolayer (SAM) forms on said electrode, said SAM comprising said EAM having said first E^o and said EAM having said second E^o; and

(c) detecting for a change between the first E^o and the second E^o of said EAM, wherein said change is an indication of the presence of said at least one target analyte.

25. A method for detecting at least one target analyte in a test sample, said method comprising:

(a) contacting a test sample with a solid support, said solid support comprising an electrode or array of electrodes, said electrode comprising;

(i) a self-assembled monolayer; and

(ii) a covalently attached electroactive moiety (EAM), said EAM having a first E^o and comprising a transition metal complex and an enzyme reactive moiety (ERM),

under conditions wherein said ERM is removed from at least a portion of said EAM provided in the presence of at least one target analyte in the test sample and results in said EAM having a second E^o;

(b) detecting for a change between the first E^o and the second E^o of said EAM, wherein said change is an indication of the presence of said at least one target analyte.

26. A method for detecting at least one target analyte in a test sample, said method comprising:

(a) contacting a test sample with a capture binding ligand bound to a first solid support under conditions so as to form a first complex in the presence of at least one target analyte;

(b) contacting the first complex with a soluble binding ligand comprising a surrogate enzyme under conditions so as to form a second complex on said first solid support;

(c) contacting the second complex with an electroactive moiety (EAM), said EAM comprising a transition metal complex and an enzyme reactive moiety (ERM) and having a first E⁰, under conditions wherein said ERM is removed from at least a portion of said EAM provided in the presence of at least one target analyte in the test sample and results in said EAM having a second E⁰; and

(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 at least one target analyte, wherein said surrogate enzyme is an enzyme that removes said ERM from said EAM.

27. The method according to claim 26, wherein a wash step is included between steps (a) and (b).

28. The method according to claim 26, wherein a wash step is included between steps (b) and (c).

29. The method according to claim 26, wherein an assay mixture in a solution phase is formed in step (c) and further comprising step (c1) contacting said assay mixture with a second solid support comprising an electrode or an array of electrodes, under conditions such that a self-assembled monolayer (SAM) forms on said electrode, said EAM having said first E⁰ and said EAM having said second E⁰.

30. The method according to claim 26, wherein said EAM is covalently attached to an electrode or array of electrodes on a second solid support as a self-assembled monolayer (SAM).