Single, direct detection of hemoglobin A1c percentage using enzyme triggered redox altering chemical elimination (e-trace) immunoassay
First Claim
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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, wherein said first binding ligand is attached to a second solid support;
(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 Eo, 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 Eo;
(f) measuring the electrochemical properties of said EAM at the first Eo and at the second Eo; and
(g) determining the fraction of said glycated protein from said electrochemical properties.
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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, Eo1 and Eo2.
94 Citations
16 Claims
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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:
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(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, wherein said first binding ligand is attached to a second solid support; (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 Eo, 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 Eo; (f) measuring the electrochemical properties of said EAM at the first Eo and at the second Eo; and (g) determining the fraction of said glycated protein from said electrochemical properties. - View Dependent Claims (3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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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:
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(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 Eo, 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 Eo; (f) measuring the electrochemical properties of said EAM at the first Eo and at the second Eo; and (g) determining the fraction of said glycated protein from said electrochemical properties. - View Dependent Claims (5, 6)
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16. A composition comprising:
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(a) a first solid support comprising (i) a covalently attached electroactive 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 Eo when said SIM is covalently attached to said EAM and a second Eo 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, wherein said first binding ligand is attached to a second solid support; (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.
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Specification