2-Amino-3-hydroxylpyridine-H2O2-Horseradish Peroxidase Voltammetric Enzyme-Linked Immunoassay for Detection of Carcionembryonic Antigen in Human Serum

2-Amino-3-hydroxylpyridine-H2O2-horseradish peroxidase (HRP) voltammetric enzyme-linked immunoassay based on N-heterocyclic substrate has been successfully applied for the detection of carcionembryonic antigen (CEA) in human serum. 2-Amino-3-hydroxylpyridine is oxidized with H2O2 catalyzed by HRP, and the resulting electroactive product produces a sensitive voltammetric peak at the potential of 0.36 V (vs. SCE) in Britton-Robinson (B-R) buffer solution. By this voltammetric peak, free HRP can be measured and immunoassay of HRP label can be developed. The enzyme-catalyzed reaction conditions and voltammetric detection conditions have been optimized, and the electrode procedure of the enzymatic product was investigated. The selected optimum reaction conditions were that the reaction medium was pH 6.0 B-R buffer solution for 10 mL reaction solution containing 1.0 mL of 0.2 mol/L B-R buffer solution, 3.0 mL of 8.0 mmol/L 2-amino-3-hydroxylpyridine solution and 1.5 mL of 0.5 mmol/L H2O2 solution, and the reaction time was 30 min at 37 degrees C. The optimum detection conditions were that the supporting electrolyte was pH 7.0 B-R buffer solution for 10 mL of the overall detection solution containing 5 mL of reaction solution and 1.0 mL of 0.2 mol/L B-R buffer solution. The optimum instrumental conditions for the detection were chosen as follows: the initial potential, 0.00 V; the final potential, -0.80 V; the potential scanning rate, 400 mV/s; the mercury drop standing time, 7 s. Under the optimum conditions, the linear range for detection of free HRP was 4.0 x 10(-4) - 1.0 mu g/L with a detection limit of 0.12 mu g/L. Based on the new immunoassay system, the linear range of the detection to CEA was 0.50 - 80 mu g/L and the detection limit was 0.50 mu g/L, which is 10 times lower than that of traditional spectrophotometric enzyme-linked immunosorbent assay method. The 2-Amino-3-hydroxylpyridine-H2O2-HRP voltammetric enzyme-linked immunoassay new system exhibits excellent performance having wider linear range and lower detection limit. The new method is inexpensive and simple. It has a great potential in clinical diagnosis.