Electrochemical detection of HbA1c, a maker for diabetes, using a flow immunoassay system

An on-chip electrochemical flow immunoassay system for the detection of hemoglobin A1c (HbA1c) 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 HbA1c 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 HbA1c. The assay procedure included immunoreactions with Fc–IgG and HbA1c, separation of immunocomplexes by boronate affinity, and electrochemical detection of Fc–IgG–HbA1c immunocomplexes. The immunoreaction mixtures were injected onto a boronate-affinity column. HbA1c–antibody complexes were then trapped onto the column by the affinity of HbA1c to boronic acid. Subsequently, elution buffer containing sorbitol was applied to elute HbA1c–antibody complexes and a current was detected by applying 600 mV versus Ag/AgCl. The elution signal was an estimation of the HbA1c amount. A linear correlation between the increase of current and HbA1c concentration was obtained up to an HbA1c concentration of 500 μg/ml. The HbA1c 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 HbA1c.