Single, Direct Detection Of Hemoglobin A1C Percentage Using Enzyme Triggered Redox Altering Chemical Elimination (E-Trace) Immunoassay

• US 20130098777A1
• Filed: 10/17/2012
• Published: 04/25/2013
• Est. Priority Date: 08/07/2009
• Status: Active Grant

• Current Assignee: Ohmx Corporation
• Original Assignee: Ohmx Corporation
• Inventors: GAUSTAD, Adam
• Granted Patent: US 9,624,522 B2
• Time in Patent Office: Days
• US Class Current: 205/777.5
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• 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 1/26 involving oxidoreductase
• CPC Class Codes: G01N 2400/00 Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin

Abstract

The disclosure also relates to novel compositions and methods for the single, direct detection of Hemoglobin A1c percentage in a sample, using conversion of functional groups attached to a transitional metal complex, resulting in quantifiable electrochemical signal at two unique potentials, E^o₁ and E^o₂.

Claims

1. A single, direct measurement method for measuring the fraction of a protein that is a glycated protein in a test sample comprising said protein and said glycated protein, said method comprising:

(a) contacting a first binding ligand with said test sample under conditions wherein said first binding ligand binds selectively and equivalently to said protein and said glycated protein in direct proportion to the fraction of said glycated protein in said test sample to form a first complex;

(b) contacting said sample containing first complex, with a second binding ligand under conditions wherein said first complex and said second binding ligand bind to form a second complex, wherein said second binding ligand comprising a peroxide-generating system and wherein said second binding ligand binds specifically to said glycated protein of said first complex;

(c) isolating said second complex;

(d) contacting said second complex with a substrate for said peroxide-generating system under conditions wherein a peroxide is generated to form an assay mixture;

(e) contacting the assay mixture with a first solid support comprising an electrode comprising a covalently attached electroactive moiety (EAM) having a first E⁰, said EAM comprising a transition metal complex comprising a self-immolative moiety (SIM) and a peroxide sensitive moiety (PSM), wherein said peroxide reacts with said PSM to release said SIM from said EAM and result in said EAM having a second E⁰;

(f) measuring the electrochemical properties of said EAM at the first E⁰ and at the second E⁰; and

(g) determining the fraction of said glycated protein from said electrochemical properties.

3. A method according to claim 1 or 2, wherein said electrode further comprises a self-assembled monolayer (SAM).

4. A method according to claim 1 or 2, wherein said first binding ligand is attached to a second solid support.

5. A method according to claim 4, wherein said second solid support is chosen from the group consisting of microparticles, magnetic microparticles, beads, and microchannels.

8. A method according to claim 1 or 2, wherein said peroxide-generating system is an oxidase enzyme.

9. The method according to claim 8, wherein said oxidase enzyme is selected from the group consisting of glucose oxidase, glucose oxidase, acyl CoA oxidases, alcohol oxidases, aldehyde oxidases, D-amino acid oxidase (DAAO), choline oxidase, and acyl CoA oxidases.

10. A method according to claim 1 or 2, wherein said peroxide-generating system is an intermediary enzyme component of a peroxide generating system, such as alkaline phosphatase or any other phosphatase.

11. A method according to claim 1 or 2, wherein said first binding ligand and said second binding ligand are independently chosen from the group consisting of monoclonal antibodies, fragments of monoclonal antibodies, polyclonal antibodies, fragments of polyclonal antibodies, proteins, and peptides.

12. A method according to claim 1 or 2, wherein said EAM comprises a transition metal.

13. A method according to claim 12, wherein said transition metal is chosen from the group consisting of iron, ruthenium and osmium.

14. A method according to claim 12, wherein said EAM is chosen from the group consisting of ferrocene and substituted ferrocene.

15. A method according to claim 1 or 2, wherein said protein is hemoglobin and said glycated protein is glycated hemoglobin.

16. A method according to claim 1 or 2, wherein said protein is hemoglobin and said glycated protein is hemoglobin A1c.

17. A method according to claim 1 or 2, wherein said glycated protein is glycated serum protein, fructosamine and glycated albumin.

2. A single, direct measurement method for measuring the fraction of a protein that is a glycated protein in a test sample comprising said protein and said glycated protein, said method comprising:

(a) contacting a first binding ligand comprising a peroxide-generating system with a test sample under conditions wherein first binding ligand binds specifically to said glycated protein to form a first complex;

(b) contacting said sample containing first complex, with a second binding ligand under conditions wherein said second binding ligand binds selectively and equivalently to said protein and said glycated protein of said first complex in direct proportion to the fraction of said glycated protein in said test sample to form a second complex;

(c) isolating said second complex;

(d) contacting said second complex with a substrate for said peroxide-generating system under conditions wherein a peroxide is generated to form an assay mixture;

(e) contacting the assay mixture with a first solid support comprising an electrode comprising a covalently attached electroactive moiety (EAM) having a first E⁰, said EAM comprising a transition metal complex comprising a self-immolative moiety (SIM) and a peroxide sensitive moiety (PSM), wherein said peroxide reacts with said PSM to release said SIM from said EAM and result in said EAM having a second E⁰;

(f) measuring the electrochemical properties of said EAM at the first E⁰ and at the second E⁰; and

(g) determining the fraction of said glycated protein from said electrochemical properties

6. A method according to claim 2, wherein said second binding ligand is attached to a second solid support.

7. A method according to claim 6, wherein said second solid support is chosen from the group consisting of microparticles, magnetic microparticles, beads, and microchannels.

18. A composition comprising:

(a) a first solid support comprising

(i) a covalently attached electroactive active moiety (EAM) comprising a transition metal complex comprising a self-immolative moiety (SIM) and a peroxide sensitive moiety (PSM), wherein said EAM has a first E^o when said SIM is covalently attached to said EAM and a second E^o when said SIM is absent;

(ii) optional self-assembled monolayer (SAM);

(b) a first binding ligand that binds selectively and equivalently to a protein and a glycated protein in direct proportion to the fraction of glycated protein in a test sample;

(c) a second binding ligand that specifically binds to said glycated protein, said second binding ligand comprising a peroxide-generating system and optionally bound to a second solid support; and

(d) optional substrate for said peroxide-generating system.