Electrochemical assay for the detection of enzymes

• US 8,802,390 B2
• Filed: 01/18/2012
• Issued: 08/12/2014
• Est. Priority Date: 10/17/2007
• Status: Active Grant

• Current Assignee: Ohmx Corporation
• Original Assignee: Ohmx Corporation
• Inventors: Bertin, Paul A., Ahrens, Michael, Georganopoulou, Dimitra, Gray, Harry B., Meade, Thomas J., Wunder, Markus Franz
• Primary Examiner(s): Shen, Bin
• Application Number:
  US13/353,001
• Publication Number:
  US 20120156709A1
• Time in Patent Office: 937 Days
• Field of Search: 435/4, 435/23
• US Class Current: 435/23
• 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 for the presence of a protease in a test sample, said method comprising:

(a) adding a test sample to 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 with an E⁰, wherein said EAM comprises a ferrocene; and

(iii) a plurality of proteins attached to said electrode, wherein said proteins are arranged so that the EAM is at least partially shielded by the proteins from exposure to a solution and comprise a cleavage site of said protease, wherein the cleavage site is near the height of said EAM;

(b) cleaving a plurality of said proteins with said protease, if said protease is present, such that when the proteins are cleaved at the cleavage site, the EAM is exposed to the solution; and

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

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

3. The method according to claim 2, wherein said endopeptidase neurotoxin is selected from the group consisting of Clostridium botulinum A, B or E.

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

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

6. The method according to claim 1, wherein the proteins are polypeptides, peptides or oligopeptides.

7. The method according to claim 1, wherein the proteins contain non-naturally occurring amino acids, amino acid analogs and/or peptidomimetic structures.

8. A method for detecting for the presence of a protease in a test sample, said method comprising:

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

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

(ii) a covalently attached electroactive 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 are arranged so that the EAM is at least partially shielded by the proteins from exposure to a solution and comprises a cleavage site of said protease, wherein the cleavage site is near the height of said EAM;

(b) cleaving a plurality of said proteins with said protease, if the protease is present, such that when the proteins are cleaved at the cleavage site, the EAM is exposed to the solution; and

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

9. The method according to claim 8, wherein said protease is an endopeptidase neurotoxin.

10. The method according to claim 9, wherein said endopeptidase neurotoxin is selected from the group consisting of Clostridium botulinum A, B or E.

11. The method according to claim 8, wherein said EAM and said proteins are attached separately to said electrode.

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

13. The method according to claim 8, wherein the proteins are polypeptides, peptides, or oligopeptides.

14. The method according to claim 8, wherein the proteins contain non-naturally occurring amino acids, amino acid analogs and/or peptidomimetic structures.