Patent ID: 8673646
Filing Date: 2014-03-18
Classification: C12Q,G01N

Abstract:
1. A method of measuring percentage of glycated hemoglobin (HbA1c) in a sample, comprising: a) applying a blood sample to a sample receiving well of an electrode device, wherein the blood sample has been treated with 1) a lysing buffer which releases hemoglobin from red blood cells in the blood sample; 2) a first oxidizing agent which selectively oxidizes a low molecular weight reducing substance 3) a second oxidizing agent which selectively oxidizes a high molecular weight reducing substance, and 4) a protease which digests glycated hemoglobin into glycated peptides or glycated amino acids; wherein the electrode device comprises: i) an electrode support; ii) a working electrode placed on the electrode support, wherein a fructosyl amino acid oxidase is placed on or near the working electrode; iii) a counter electrode placed on the electrode support; iv) a reference electrode placed on the electrode support, wherein the working, counter and reference electrodes are spaced from each other; wherein the working electrode and/or the counter electrode further comprise a catalyst which catalyzes the redox reaction of hydrogen peroxide; and v) an insulation/covering layer defining an enclosed space over the working electrode, the counter electrode, and the reference electrode, wherein said insulation/covering layer forms a well for receiving a sample, wherein the sample well is located on the electrodes or near the electrodes; wherein (i) the second oxidizing agent is a tetrazolium salt which is 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt or 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt; (ii) the fructosyl amino acid oxidase comprises the amino acid sequence of SEQ ID NO:1; or (iii) the protease generates a glycated peptide from about 2 to about 30 amino acid residues; b) applying an electrical potential between the working electrode and the reference electrode suitable for monitoring the hydrogen peroxide generated from the fructosyl amino acid oxidase-catalyzed reaction; and c) measuring an electrical current between the working electrode and the counter electrode or a charge passed at the working electrode; whereby the percentage of glycated hemoglobin in the sample is determined based on the measured current or charge as compared to a reference without measuring the total hemoglobin in the blood sample separately.