Electrochemical biosensor for direct determination of percentage of glycated hemoglobin

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.

2. The method of claim 1 , wherein the hydrogen peroxide generated by the fructosyl amino acid oxidase-catalyzed reaction is electrochemically detected directly or indirectly via a mediator or a catalyst.

3. The method of claim 1 , wherein the electrical potential is applied between the working electrode and the reference electrode in a time delayed manner.

4. The method of claim 1 , wherein the reference is a calibration curve.

5. The method of claim 1 , wherein the electrode device further comprises a capillary-fill channel for receiving a sample through capillary action, wherein one end of the channel is in contact with the working electrode.

6. The method of claim 1 , wherein the working electrode and the counter electrode further comprise a mediator which shuttles electrons between the hydrogen peroxide generated from said fructosyl amino acid oxidase-catalyzed reaction and the working electrode to create a current representative of the amount of the glycated hemoglobin in the sample.

7. The method of claim 6 , wherein the mediator comprises one or more agents selected from the group consisting of ferrocene, Prussian Blue, metal phthalocyonine, and tetrathiafulvalene (TTF).

8. The method of claim 1 , wherein the catalyst comprises one or more agents selected from the group consisting of Ir-carbon, Rh-carbon, and Ru-carbon.

9. The method of claim 8 , wherein said Ir-carbon, Rh-carbon, and Ru-carbon are nanoparticles.

10. The method of claim 1 , wherein the fructosyl amino acid oxidase is formulated in a matrix and is placed on or near the working electrode.

11. The method of claim 10 , wherein the matrix comprises an enzyme immobilizing agent and a thickening polymer.

12. The method of claim 1 , wherein the electrode device further comprises a sample transfer path between said sample well and said working electrode.

13. The method of claim 1 , wherein the working electrode and/or the counter electrode comprise a metallized carbon selected from the group consisting of Ir-carbon, Rh-carbon, and Ru-carbon.

14. The method of claim 13 , wherein Ir-carbon, Rh-carbon, and Ru-carbon are nanoparticles.

15. The method of claim 1 , wherein the reference electrode is Ag/AgCl.

16. The method of claim 1 , wherein the electrodes are screen-printed using thick film technology or using thin film lithography.

17. The method of claim 1 , wherein the first oxidizing agent is selected from the group consisting of Dess-Martin periodinane, N-ethyl maleimide, sodium iodoacetate, sodium periodate, and Chloramine-T.

18. The method of claim 1 , wherein the tetrazolium salt 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.

19. The method of claim 1 , wherein the fructosyl amino acid oxidase comprises the amino acid sequence of SEQ ID NO:1.

20. The method of claim 1 , wherein the protease generates a glycated peptide from about 2 to about 30 amino acid residues.

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.

22. The method of claim 21 , wherein the hydrogen peroxide generated by the fructosyl amino acid oxidase-catalyzed reaction is electrochemically detected directly or indirectly via a mediator or a catalyst.

23. The method of claim 21 , wherein the electrical potential is applied between the working electrode and the reference electrode in a time delayed manner.

24. The method of claim 21 , wherein the reference is a calibration curve.

25. The method of claim 21 , wherein the working electrode and the counter electrode further comprise a mediator which shuttles electrons between the hydrogen peroxide generated from said fructosyl amino acid oxidase-catalyzed reaction and the working electrode to create a current representative of the amount of the glycated hemoglobin in the sample.

26. The method of claim 25 , wherein the mediator comprises one or more agents selected from the group consisting of ferrocene, Prussian Blue, metal phthalocyonine, and tetrathiafulvalene (TTF).

27. The method of claim 21 , wherein the catalyst comprises one or more agents selected from the group consisting of Ir-carbon, Rh-carbon, and Ru-carbon.

28. The method of claim 27 , wherein said Ir-carbon, Rh-carbon, and Ru-carbon are nanoparticles.

29. The method of claim 21 , wherein the fructosyl amino acid oxidase is formulated in a matrix and is placed on or near the working electrode.

30. The method of claim 29 , wherein the matrix comprises an enzyme immobilizing agent and a thickening polymer.

31. The method of claim 21 , wherein the working electrode and/or the counter electrode comprise a metallized carbon selected from the group consisting of Ir-carbon, Rh-carbon, and Ru-carbon.

32. The method of claim 31 , wherein Ir-carbon, Rh-carbon, and Ru-carbon are nanoparticles.

33. The method of claim 21 , wherein the reference electrode is Ag/AgCl.

34. The method of claim 21 , wherein the electrodes are screen-printed using thick film technology or using thin film lithography.

35. The method of claim 21 , wherein the first oxidizing agent is selected from the group consisting of Dess-Martin periodinane, N-ethyl maleimide, sodium iodoacetate, sodium periodate, and Chloramine-T.

36. The method of claim 21 , wherein the tetrazolium salt 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.

37. The method of claim 21 , wherein the fructosyl amino acid oxidase comprises the amino acid sequence of SEQ ID NO:1.

38. The method of claim 21 , wherein the protease generates a glycated peptide from about 2to about 30 amino acid residues.

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.

40. The method of claim 39 , wherein the treated blood sample is pipetted onto the working electrode.

41. The method of claim 39 , wherein the electrode device comprises the capillary-fill channel for receiving a sample through capillary action and one end of the channel is in contact with the working electrode, and wherein the treated blood sample is applied to the working electrode through a capillary-fill channel.

42. The method of claim 39 , wherein the working electrode and the counter electrode further comprise a mediator which shuttles electrons between the hydrogen peroxide generated from said fructosyl amino acid oxidase-catalyzed reaction and the working electrode to create a current representative of the amount of the glycated hemoglobin in the sample.

43. The method of claim 42 , wherein the mediator comprises one or more agents selected from the group consisting of ferrocene, Prussian Blue, metal phthalocyonine, and tetrathiafulvalene (TTF).

44. The method of claim 39 , wherein the working electrode and/or the counter electrode further comprise a catalyst which catalyzes the redox reaction of hydrogen peroxide.

45. The method of claim 44 , wherein the catalyst comprises one or more agents selected from the group consisting of Ir-carbon, Rh-carbon, and Ru-carbon.

46. The method of claim 45 , wherein said Ir-carbon, Rh-carbon, and Ru-carbon are nanoparticles.

47. The method of claim 39 , wherein the fructosyl amino acid oxidase is formulated in a matrix and is placed on or near the working electrode.

48. The method of claim 47 , wherein the matrix comprises an enzyme immobilizing agent and a thickening polymer.

49. The method of claim 39 , wherein the working electrode and/or the counter electrode comprise a metallized carbon selected from the group consisting of Ir-carbon, Rh-carbon, and Ru-carbon.

50. The method of claim 49 , wherein Ir-carbon, Rh-carbon, and Ru-carbon are nanoparticles.

51. The method of claim 39 , wherein the reference electrode is Ag/AgCl.

52. The method of claim 39 , wherein the electrodes are screen-printed using thick film technology or using thin film lithography.

53. The method of claim 39 , wherein the first oxidizing agent is selected from the group consisting of Dess-Martin periodinane, N-ethyl maleimide, sodium iodoacetate, sodium periodate, and Chloramine-T.

54. The method of claim 39 , wherein the tetrazolium salt 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.

55. The method of claim 39 , wherein the fructosyl amino acid oxidase comprises the amino acid sequence of SEQ ID NO:1.

56. The method of claim 39 , wherein the protease generates a glycated peptide from about 2 to about 30 amino acid residues.