Electrochemical biosensor for direct determination of percentage of glycated hemoglobin
First Claim
Patent Images
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.
3 Assignments
0 Petitions
Accused Products
Abstract
The invention provides electrochemical biosensors for direct determination of percentage of glycated hemoglobin in blood samples without the need of a separated measurement of total hemoglobin content in blood samples. The invention provides methods for using the electrochemical biosensors.
-
Citations
56 Claims
-
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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
-
-
21. 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 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, and contains a first oxidizing agent which selectively oxidizes a low molecular weight reducing substance, a second oxidizing agent which selectively oxidizes a high molecular weight reducing substance, one or more proteases which digest glycated hemoglobin into glycated peptides or glycated amino acids, and a detergent that lyses red blood cells in a blood sample and releases hemoglobin; and vi) a sample transfer path between said sample well and said working electrode; 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 said working electrode and said 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. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38)
-
-
39. A method of measuring percentage of glycated hemoglobin (HbA1c) in a sample, comprising:
-
a) applying a blood sample to a working electrode 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 if the first oxidizing agent is not placed on or near the working electrode, or a second oxidizing agent which selectively oxidizes a high molecular weight reducing substance if the second oxidizing agent is not placed on or near the working electrode, and
3) 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, and wherein a first oxidizing agent which selectively oxidizes a low molecular weight reducing substance or a second oxidizing agent which selectively oxidizes a high molecular weight reducing substance is placed on or near the working electrode; iii) a counter electrode placed on the electrode support; and iv) a reference electrode placed on the electrode support, wherein the working, counter and reference electrodes are spaced from each other; 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 said working electrode and said 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. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56)
-
Specification