Electrochemical detection of HbA_1c, a maker for diabetes, using a flow immunoassay system

An on-chip electrochemical flow immunoassay system for the detection of hemoglobin A_1c (HbA_1c) was developed using anti-human hemoglobin (Hb) IgG labeled with ferrocene monocarboxylic acid (Fc-COOH) and boronate-affinity chromatography. An on-chip column packed with boronate-activated agarose beads was used for the separation of HbA_1c from both non-glycated Hb and free antibody. Anti-human Hb IgG conjugated to Fc-COOH (Fc-IgG) was used for the electrochemical detection of HbA_1c. The assay procedure included immunoreactions with Fc-IgG and HbA_1c, separation of immunocomplexes by boronate affinity, and electrochemical detection of Fc-IgG-HbA_1c immunocomplexes. The immunoreaction mixtures were injected onto a boronate-affinity column. HbA_1c-antibody complexes were then trapped onto the column by the affinity of HbA_1c to boronic acid. Subsequently, elution buffer containing sorbitol was applied to elute HbA_1c-antibody complexes and a current was detected by applying 600 mV versus Ag/AgCl. The elution signal was an estimation of the HbA_1c amount. A linear correlation between the increase of current and HbA_1c concentration was obtained up to an HbA_1c concentration of 500 μg/ml. The HbA_1c flow immunoassay was successfully achieved using hemolysates. This electrochemical flow immunoassay system enabled us to construct a novel point-of-care testing device for the monitoring of glycated proteins including HbA_1c.