Method for the determination of glycated hemoglobin
A method of determining the percentage of glycated hemoglobin in a blood sample is disclosed wherein at least one of the assay steps is performed electrochemically. The method includes determining the total amount of hemoglobin in a sample by electrochemically measuring, in an oxygen electroreduction reaction at a cathode, the amount of oxygen in the sample. Because the amount of oxygen dissolved in the sample is known, the total hemoglobin is determined by subtracting the amount of free oxygen from the total oxygen measured, recognizing the fast equilibrium Hb+O 2 ⇄HbO 2 . This can be followed by determining the amount of glycated hemoglobin in the sample. The cathode reaction is accomplished by contacting the sample with an enzyme, the enzyme being a copper-containing enzyme having four copper ions per active unit. The family of these enzymes includes, for example, laccases and bilirubin oxidases.
1. A method of assaying glycated hemoglobin in a sample, comprising:
electrochemically determining a total amount of hemoglobin in a blood, blood-containing, or blood-derived sample, comprising:
contacting the sample in an electrochemical cell with an enzyme capable of determining an oxygen content of the sample; and
determining the total amount of hemoglobin from the oxygen content;
determining an amount of glycated hemoglobin in the sample; and
determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample.
2. The method of claim 1 , wherein the enzyme is a copper-containing enzyme.
3. The method of claim 1 , wherein the enzyme is selected from of laccase, bilirubin oxidase, and ascorbate oxidase.
4. The method of claim 1 , wherein determining the amount of glycated hemoglobin comprises spectrophotometrically determining the amount of glycated hemoglobin.
5. The method of claim 1 , wherein determining the amount of glycated hemoglobin comprises electrochemically determining the amount of glycated hemoglobin.
6. The method of claim 1 , wherein determining the amount of glycated hemoglobin comprises determining the amount of glycated hemoglobin via direct enzymatic action.
7. The method of claim 2 , wherein determining the amount of glycated hemoglobin comprises determining the amount of glycated hemoglobin via direct enzymatic reaction.
8. The method of claim 1 , wherein determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample comprises calculating a ratio or a percentage of the amount of glycated hemoglobin to the total amount of hemoglobin in the sample.
9. A method of assaying glycated hemoglobin in a sample, comprising:
electrochemically determining a total amount of hemoglobin in a blood, blood-containing, or blood-derived sample;
spectrophotometrically determining an amount of glycated hemoglobin in the sample; and
determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample.
10. The method of claim 9 , wherein electrochemically determining the total amount of hemoglobin comprises:
contacting the sample in an electrochemical cell with an enzyme capable of determining an oxygen content of the sample; and
determining the total amount of hemoglobin from the oxygen content.
11. The method of claim 10 , wherein the enzyme is a copper-containing enzyme.
12. The method of claim 10 , wherein the enzyme is selected from laccase, bilirubin oxidase, and ascorbate oxidase.
13. The method of claim 9 , wherein determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample comprises calculating a ratio or a percentage of the amount of glycated hemoglobin to the total amount of hemoglobin in the sample.
14. A method of assaying glycated hemoglobin in a sample, comprising:
electrochemically determining a total amount of hemoglobin in a blood, blood-containing, or blood-derived sample;
separating glycated hemoglobin from the sample and from non-glycated hemoglobin to form a second sample;
determining an amount of glycated hemoglobin in the second sample; and
determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample.
15. The method of claim 14 , wherein determining the amount of glycated hemoglobin in the second sample comprises electrochemically determining the amount of glycated hemoglobin in the second sample.
16. The method of claim 14 , wherein determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample comprises calculating a ratio or a percentage of the amount of glycated hemoglobin to the total amount of hemoglobin in the sample.
17. A method of assaying glycated hemoglobin in a sample, comprising:
electrochemically determining a total amount of hemoglobin in a blood, blood-containing, or blood-derived sample, by exposing the sample to an enzyme associated with a cathode, determining an amount of oxygen in the sample via an enzymatic redox reaction, and determining the total amount of hemoglobin in the sample from the amount of oxygen;
determining an amount of glycated hemoglobin in the sample; and
determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample.
18. The method of claim 17 , wherein determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample comprises calculating a ratio or a percentage of the amount of glycated hemoglobin to the total amount of hemoglobin in the sample.
19. The method of claim 14 , wherein determining an amount of glycated hemoglobin in the second sample comprises measuring the amount of glycated hemoglobin in the second sample.
20. The method of claim 14 , wherein determining an amount of glycated hemoglobin in the second sample comprises measuring hemoglobin in the sample after said separating.
21. A method of assaying glycated hemoglobin in a sample, comprising:
electrochemically determining a total amount of hemoglobin in a blood, blood-containing, or blood-derived sample;
separating glycated hemoglobin from the sample to form a second sample;
spectrophotometrically determining an amount of glycated hemoglobin in the second sample; and
determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample.
22. The method of claim 21 , wherein determining an amount of glycated hemoglobin in the second sample comprises measuring the amount of glycated hemoglobin in the second sample.
23. The method of claim 21 , wherein determining an amount of glycated hemoglobin in the second sample comprises measuring hemoglobin in the sample after said separating.
24. The method of claim 21 , wherein determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample comprises calculating a ratio or a percentage of the amount of glycated hemoglobin to the total amount of hemoglobin in the sample.
25. A method of assaying glycated hemoglobin in a sample, comprising:
electrochemically determining a total amount of hemoglobin in a blood, blood-containing, or blood-derived sample;
separating glycated hemoglobin from the sample and from non-glycated hemoglobin to form a second sample;
spectrophotometrically determining an amount of glycated hemoglobin in the second sample; and
determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample.
26. The method of claim 25 , wherein determining an amount of glycated hemoglobin in the second sample comprises measuring the amount of glycated hemoglobin in the second sample.
27. The method of claim 25 , wherein determining an amount of glycated hemoglobin in the second sample comprises measuring hemoglobin in the sample after said separating.
28. The method of claim 25 , wherein determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample comprises calculating a ratio or a percentage of the amount of glycated hemoglobin to the total amount of hemoglobin in the sample.
29. A method of assaying glycated hemoglobin in a sample, comprising:
electrochemically determining a total amount of hemoglobin in a blood, blood-containing, or blood-derived sample;
separating glycated hemoglobin from non-glycated hemoglobin to form a second sample;
determining an amount of glycated hemoglobin in the second sample via direct oxidation or enzymatic electrooxidation; and
determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample.
30. The method of claim 29 , wherein determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample comprises direct oxidation via a cross-linked poly(N-vinyl imidazole)-based redox polymer.
31. The method of claim 29 , wherein determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample comprises enzymatic electrooxidation via an enzyme selected from a fructosyl amine oxidase or a copper-containing enzyme.
32. The method of claim 29 , wherein determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample comprises enzymatic electrooxidation via an enzyme selected from laccase, bilirubin oxidase, and ascorbate oxidase.
33. The method of claim 29 , wherein determining the amount of glycated hemoglobin relative to the total amount of hemoglobin in the sample comprises calculating a ratio or a percentage of the amount of glycated hemoglobin to the total amount of hemoglobin in the sample.