Direct detection for concentration ratio of HbA1c to total hemoglobin by using potentiometric immunosensor with simple process of denaturing HbA1c

•A direct detection method for HbA1c (%), which is the concentration ratio of HbA1c to total hemoglobin (Hb), is proposed with a simple process of denaturing HbA1c.•This method is based on a sandwich immunoassay combing anti-Hemoglobin and anti-HbA1c antibodies.•By adding the denaturing process of HbA1c, the direct measurement of HbA1c (%) was successfully performed in the clinically relevant range. A direct measurement method for HbA1c (%), which is the concentration ratio of HbA1c to total hemoglobin (Hb), was investigate with a potentiometric immunosensor. The direct measurement method is based on sandwich immunoassay that combines anti-Hb and enzyme-labeled anti-HbA1c antibodies. While the anti-Hb antibodies capture both Hb and HbA1c maintaining HbA1c (%) in blood sample, the enzyme-labeled anti-HbA1c antibodies bind only to HbA1c in the total captured Hb. Though we tried the direct detection of HbA1c (%) with a combination of four anti-Hb antibodies and two anti-HbA1c antibodies, the obtained result showed lower sensitivity and reproducibility. We presumed that the binding site of HbA1c for anti-HbA1c antibody hides inside the folding structure of HbA1c and anti-HbA1c antibody is not able to bind to HbA1c efficiently resulting in lower sensitivity. As a result of examination of both the type and concentration of the surfactant, it was found that the process of denaturing HbA1c with 0.2% dodecyltrimethylammonium bromide was added to enable it to be accessible to anti-HbA1c antibodies and could enhance the sensitivity. By adding the simple process of denaturing HbA1c, the direct measurement of HbA1c (%) was successfully performed with this immunosensor. The results showed a good correlation (correlation efficiency, r2 = 0.975) between the certified and experimental values of HbA1c (%) in the clinically relevant range (from 5.6% to 10.6%).