CAJ | 학술논문

以氮杂环化合物为电化学分析底物的2-氨基-3-羟基吡啶-H2O2-辣根过氧化物酶(HRP)伏安酶联免疫体系测定人血清癌胚抗原(CEA).HRP催化H2O2氧化2-氨基-3-羟基吡啶的酶促反应产物,在缓冲液中-0.36 V处产生一个灵敏的伏安还原峰,借助此峰可以测定游离的HRP,进而可用于以HRP为标记物的酶联免疫分析.对酶促反应条件和测定条件的优化反应条件为:以B-R缓冲液(pH 6.0)为反应介质,在10 mL总反应液中含有1.0 mL 0.2 mol/L B-R缓冲液、3.0 mL 8.0 mmol/L 2-氨基-3-羟基吡啶溶液以及1.5 mL 0.5 mmol/L H2O2溶液,反应温度37 ℃,反应时间30 min.最佳测定条件为:B-R缓冲液(pH 7.0)为支持电解质,在10 mL总测定溶液中含有5 mL上述总反应液、1.0 mL 0.2 mol/L B-R缓冲液.测定仪器条件:起始电位0.00 V,终止电位-0.80 V,电位扫描速度400 mV/s,滴汞静止时间7 s.在最佳的反应条件和测定条件下,新体系测定游离HRP的线性范围为4.0×10-4～1.0 μg/L; 对HRP的检出限为0.12 ng/L.新体系对CEA测定的线性范围为0.50～80.0 μg/L; 检出限为0.50 μg/L.为经典ELISA法的检出限的1/10.
이담잡배화합물위전화학분석저물적2-안기-3-간기필정-H2O2-랄근과양화물매(HRP)복안매련면역체계측정인혈청암배항원(CEA).HRP최화H2O2양화2-안기-3-간기필정적매촉반응산물,재완충액중-0.36 V처산생일개령민적복안환원봉,차조차봉가이측정유리적HRP,진이가용우이HRP위표기물적매련면역분석.대매촉반응조건화측정조건적우화반응조건위:이B-R완충액(pH 6.0)위반응개질,재10 mL총반응액중함유1.0 mL 0.2 mol/L B-R완충액、3.0 mL 8.0 mmol/L 2-안기-3-간기필정용액이급1.5 mL 0.5 mmol/L H2O2용액,반응온도37 ℃,반응시간30 min.최가측정조건위:B-R완충액(pH 7.0)위지지전해질,재10 mL총측정용액중함유5 mL상술총반응액、1.0 mL 0.2 mol/L B-R완충액.측정의기조건:기시전위0.00 V,종지전위-0.80 V,전위소묘속도400 mV/s,적홍정지시간7 s.재최가적반응조건화측정조건하,신체계측정유리HRP적선성범위위4.0×10-4～1.0 μg/L; 대HRP적검출한위0.12 ng/L.신체계대CEA측정적선성범위위0.50～80.0 μg/L; 검출한위0.50 μg/L.위경전ELISA법적검출한적1/10.
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 ℃. 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 4.0×10-4-1.0 μg/L with a detection limit of 0.12 μg/L. Based on the new immunoassay system, the linear range of the detection to CEA was 0.50-80 μg/L and the detection limit was 0.50 μg/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.