摘要
提出了间氨基酚(MAP)-H_2O_2-辣根过氧化物酶(HRP)伏安酶联免疫分析新体系.本方法以线性扫描二阶导数伏安法检测HRP催化H_2O_2氧化MAP的产物,用于游离HRP和各种HRP标记物的测定,灵敏度均高于经典的ELISA显色光度法.测定游离HRP的线性范围为1.0×10^(-8)～1.0×10^(-6)g/L,检测限达3.8×10^(-9) g/L.制备出了HRP催化H_2O_2氧化MAP的产物纯品,并应用电化学分析,高效液相色谱,元素分析,紫外-可见光谱,红外光谱,~1H核磁共振谱,^(13)C核磁共振谱及质谱等技术对体系酶促反应进行了深入的研究.在选择的酶促反应条件下,生成的产物为2-氨基-5-[(3-羟苯基)氨基]-2,5-环己二烯基-l,4-二酮.提出了酶催化反应机理及其产物的电极还原过程.
A voltammetric enzyme - linked immunoassay based on a new system of m - aminophenol (MAP) - H2O2 - horseradish peroxidase (HRP) has firstly been developed and used for the detection of HRP and labelled HRP. HRP or labelled HRP catalyzes the oxidation reaction of MAP with H2O2, the product of which yields a sensitive voltammetric peak at potential of - 0.46V ( vs. SCE) in Britton -Robinson (B - R) buffer solution. By using this voltammetric peak, HRP can be measured with adetection limit of 3.8 × 10-9g/L and a linear range of 1.0×10-8~1.0×10-6g/L. The pure product of H2O2 oxidizingMAP catalyzed by HRP was prepared with chemical method. The enzyme - catalyzed reaction has been investigated with electroanalytical chemistry , elemental analysis , UV - vis , IR , 1H NMR, 13C NMR and MS spectroscopy. Under the selected enzyme- catalyzed reaction conditions, the oxidation product of MAP with H2O2 catalyzed by HRP is 2 - amino - 5 - [ (3 - hydroxyphenyl) amino] - 2,5 - cyclohexadiene - 1,4 - dione. The processes of the enzyme - catalyzed reaction and the electroreduction of the product of the enzyme - catalyzed reaction are described.
出处
《化学学报》
SCIE
CAS
CSCD
北大核心
1999年第8期922-930,共9页
Acta Chimica Sinica
基金
国家自然科学基金(29775012)资助项目
关键词
免疫分析
酶联免疫分析
间氨基酚
HRP
伏安法
m - aminophenol, horseradish peroxidase ( HRP) , enzyme - catalysis, voltammetry
