Electrochemical Assay for the Detection of Enzymes

• US 20100003710A1
• Filed: 10/17/2008
• Published: 01/07/2010
• Est. Priority Date: 10/17/2007
• Status: Abandoned Application

• Current Assignee: Ohmx Corporation
• Original Assignee: Ohmx Corporation
• Inventors: Meade, Thomas J., Ahrens, Michael, Georganopoulou, Dimitra, Wunder, Markus Franz, Bertin, Paul A., Gray, Harry B.
• Application Number:
  US12/253,828
• Publication Number:
  US 20100003710A1
• Time in Patent Office: Days
• US Class Current: 435/24
• CPC Class Codes: C12Q 1/003 Functionalisation
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 27/30 Electrodes, e.g. test electrodes; Half-cells G01N27/414 takes precedence
• CPC Class Codes: Y10T 29/49117 Conductor or circuit manufacturing

Abstract

The invention relates to novel compositions and methods for the detection of enzymes using the nuclear reorganization energy, λ, of an electron transfer process.

Claims

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

(a) adding a test sample comprising a protease to a solid support comprising an electrode comprising;

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

(ii) a covalently attached eletroactive active moiety (EAM) comprising a transition metal complex with an E⁰, wherein said EAM comprises a ferrocene; and

(iii) a plurality of proteins attached to said electrode, wherein said proteins comprise a cleavage site of said protease;

(b) cleaving a plurality of said proteins with said protease; and

(c) determining the presence of said protease by measuring a change of said E⁰.

5. The method according to claim 1, wherein said protease is an endopeptidase neurotoxin.

6. The method according to claim 5, wherein said endopeptidase neurotoxin is selected from the group consisting of Clostridium botulinum A, B or E.

7. The method according to claim 1, wherein said EAM and said substrates are attached separately to said electrode.

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

2-4. -4. (canceled)

9. (canceled)

10. A method for detecting a protease in a test sample, said method comprising:

(a) adding a test sample comprising a protease to a solid support comprising an electrode, said electrode comprising;

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

(ii) a covalently attached eletroactive active moiety (EAM) comprising a transition metal complex with an E⁰, wherein said EAM comprises osmium and at least one cyano ligand; and

(iii) a plurality of proteins attached to said electrode, wherein said proteins comprises a cleavage site of said protease;

(b) cleaving a plurality of said proteins with said protease; and

(c) determining the presence of said protease by measuring a change of said E⁰.

11. The method according to claim 10, wherein said protease is an endopeptidase neurotoxin.

18. The method according to claim 11, wherein said endopeptidase neurotoxin is selected from the group consisting of Clostridium botulinum A, B or E.

19. The method according to claim 10, wherein said EAM and said substrates are attached separately to said electrode.

20. The method according to claim 10, wherein said solid support comprises an array of electrodes.

12. A method for detecting a kinase in a test sample, said method comprising the steps of:

(a) adding a test sample comprises a kinase protease to a solid support comprising an electrode, said electrode comprising;

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

(ii) a covalently attached eletroactive active moiety (EAM) comprising a transition metal complex with an E⁰; and

(iii) a plurality of proteins attached to said electrode, wherein said proteins are first substrates of said kinase;

(b) phosphorylating said proteins with said kinase and a second kinase substrate so that said second kinase substrate is covalently attached to said protein; and

(c) determining the presence of said kinase by measuring a change of said E⁰.

13. The method according to claim 12, wherein said EAM comprises at least one cyano ligand.

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

15. The method according to claim 12, wherein said EAM and said substrates are attached separately to said electrode.

16. The method according to claim 12, wherein said solid support comprises an array of electrodes.

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