An on-chip type cation-exchange chromatography system with electrochemical detection of HbA(1c), 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 HbA(1c) in blood calibrator samples from other Hbs, e.g. HbA(0), HbA(1a) or HbA(1b), 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 HbA(1a) and HbA(1b), HbA(1c) and HbA(0) fractions were eluted in this order. A linear relationship between HbA(1c) levels and electrochemical oxidation currents was obtained in the range from 4.0% to 12.6% HbA(1c). 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 HbA(1c).
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