ELECTROCHEMICAL BIOSENSOR FOR DIRECT DETERMINATION OF PERCENTAGE OF GLYCATED HEMOGLOBIN

• US 20100025264A1
• Filed: 05/13/2009
• Published: 02/04/2010
• Est. Priority Date: 05/13/2008
• Status: Active Grant

• Current Assignee: Diazyme Laboratories, Inc. (General Atomics, Inc.)
• Original Assignee: General Atomics, Inc.
• Inventors: Fang, Lei, Blue, Neal K., Yuan, Chong-Sheng, Datta, Abhijit, Liu, Limin
• Granted Patent: US 8,673,646 B2
• Time in Patent Office: Days
• US Class Current: 205/777.500
• 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: G01N 2333/906 acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin

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.

Claims

1. An electrode device for use in an electrochemical sensor for measuring percentage of glycated hemoglobin (HbA1c) in a sample, comprising:

an electrode support;

a working electrode placed on the electrode support, wherein a fructosyl amino acid oxidase is placed on or near the working electrode;

a counter electrode placed on the electrode support; and

a reference electrode placed on the electrode support, wherein the working, counter and reference electrodes are spaced from each other.

2. The device of claim 1, further comprising 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.

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

4. The device 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.

5. The device of claim 4, wherein the mediator comprises one or more agents selected from the group consisting of ferrocene, Prussian Blue, metal phthalocyonine, and tetrathiafulvalene (TTF).

6. The device of claim 1, wherein the working electrode and/or the counter electrode further comprise a catalyst which catalyzes the redox reaction of hydrogen peroxide.

7. The device of claim 6, wherein the catalyst comprises one or more agents selected from the group consisting of Ir-carbon, Rh-carbon, and Ru-carbon.

8. The device of claim 7, wherein said Ir-carbon, Rh-carbon, and Ru-carbon are nanoparticles.

9. The device of claim 1, wherein a first oxidizing agent which selectively oxidizes a low molecular weight reducing substance and a second oxidizing agent which selectively oxidizes a high molecular weight reducing substance are placed on or near the working electrode.

10. The device of claim 9, wherein one or more proteases which digest glycated hemoglobin into glycated peptides or glycated amino acids are placed on or near the working electrode.

11. The device of claim 10, wherein a detergent that lyses red blood cells in a blood sample and releases hemoglobin is placed on or near the working electrode.

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

13. The device of claim 12, wherein one or more proteases which digest glycated hemoglobin into glycated peptides or glycated amino acids are formulated in a matrix and are placed on the top of the matrix containing the fructosyl amino acid oxidase on the working electrode.

14. The device of claim 12, wherein the matrix comprises an enzyme immobilizing agent and a thickening polymer.

15. The device of claim 2, further comprising a sample transfer path between said sample well and said working electrode.

16. The device of claim 15, wherein the sample well contains a first oxidizing agent which selectively oxidizes a low molecular weight reducing substance and a second oxidizing agent which selectively oxidizes a high molecular weight reducing substance.

17. The device of claim 16, wherein the sample well further contains one or more proteases which digest glycated hemoglobin into glycated peptides or glycated amino acids.

18. The device of claim 17, wherein the sample well further contains a detergent that lyses red blood cells in a blood sample and releases hemoglobin.

19. The device 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.

20. The device of claim 19, wherein Ir-carbon, Rh-carbon, and Ru-carbon are nanoparticles.

21. The device of claim 1, wherein the reference electrode is Ag/AgCl.

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

23. The device of claim 1, further comprising a second working electrode and a glucose oxidase or a glucose dehydrogenase to measure both HbA1c and glucose using a single device.

24. An electrochemical biosensor comprising an electrode device according to claim 1, a detection meter for the electrode device, wherein the detection meter comprises a wireless data connection that transfers the test results to a data base or to one or more email addresses.

25. A method of measuring percentage of glycated hemoglobin (HbA1c) in a sample, comprising:

a) applying a blood sample to the sample receiving well of the electrode device of claim 2, 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;

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;

c) measuring an electrical current between the working electrode and the counter or a charge passed at the working electrode; and

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.

26. The method of claim 25, 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.

27. The method of claim 25, wherein the electrical potential is applied between the working electrode and the reference electrode in a time delayed manner.

28. The method of claim 25, wherein the reference is a calibration curve.

29. A method of measuring percentage of glycated hemoglobin (HbA1c) in a sample, comprising:

a) applying a blood sample to the sample receiving well of the electrode device of claim 18;

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;

c) measuring an electrical current between the working electrode and the counter electrode or a charge passed at the working electrode; and

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.

30. The method of claim 29, 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.

31. The method of claim 29, wherein the electrical potential is applied between the working electrode and the reference electrode in a time delayed manner.

32. The method of claim 29, wherein the reference is a calibration curve.

33. A method of measuring percentage of glycated hemoglobin (HbA1c) in a sample, comprising:

a) applying a blood sample to the working electrode of the electrode device of claim 1, 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;

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;

c) measuring an electrical current between the working electrode and the counter electrode or a charge passed at the working electrode; and

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.

34. The method of claim 33, wherein the treated blood sample is pipetted onto the working electrode.

35. The method of claim 33, 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.