On-chip type cation-exchange chromatography with ferrocene-labeled anti-hemoglobin antibody and electrochemical detector for determination of hemoglobin A1c level

An on-chip type cation-exchange chromatography system with electrochemical detection of HbA1c, which is one of the most important diabetes marker protein, was developed using ferrocene-conjugated anti-human hemoglobin (Hb) monoclonal antibody (FcAb). The FcAb was used as an electrochemical probe for the detection of each Hb. The system contains syringe pump, on-chip type cation-exchange column consisted of PDMS and cation-exchange resin beads, and a three-electrode flow-cell system. The separation conditions of HbA1c in blood calibrator samples from other Hbs, e.g. HbA0, HbA1a or HbA1b, were optimized using the on-chip type system. The electrochemical oxidation current from FcAb reacting with each Hb was measured at 350 mV (vs. Ag/AgCl). Hbs including HbA1a and HbA1b, HbA1c and HbA0 fractions were eluted in this order. A linear relationship between HbA1c levels and electrochemical oxidation currents was obtained in the range from 4.0% to 12.6% HbA1c. All procedure including antigen–antibody reaction was completed in 15 min. Furthermore, a good correlation was obtained between KO500 method (HPLC) and our proposed method. These results indicate that the on-chip type system with electrochemical detection can be applied to a novel POCT device for rapid and precise detection of HbA1c.