以对硝基磷酸苯酯为底物电化学分析法检测碱性磷酸酯酶

A System of p-Nitrophenylphosphate-Alkaline Phosphatase for Voltammetric Enzyme-linked Immunoassay

研究了对硝基磷酸苯酯（ＰＮＰＰ）为底物伏安法测定碱性磷酸酯酶（ＡＬＰ）的方法。ＰＮＰＰ在ＡＬＰ的催化作用下水解生成对硝基苯酚（ＰＮＰ）。ＰＮＰ在玻碳电极上＋１．０２Ｖ（ｖｓＡｇ／ＡｇＣｌ）左右产生氧化峰，借助此氧化电流可以测定ＡＬＰ，并进而可用于以ＡＬＰ为标记物的酶免疫分析。用微分脉冲伏安法对酶催化反应条件和酶催化反应产物的测定条件进行了详细的研究，测定ＡＬＰ的线性范围是４．０×１０２～１．０×１０６ｍＵ／Ｌ；检测限为２．８ ×１０２ｍＵ／Ｌ。

A new electrochemical voltammetric enzyme immunoassay system was investigated based on p-nitrophenyl phosphate (PNPP)-alkaline phosphatase (ALP) coupling reaction. PNPP is electro-inactive in the potential range of 0.4 similar to 1.8 V, it can be enzymatic-catalyzed hydrolysis and the product is p-nitrophenol (PNP), which can be oxidized at + 1.02 V (vs. Ag/AgCl) on glassy carbon electrode (GCE). So the differential pulse voltammetry (DPV) was chosen to detect the enzymatic-generated PNP. The conditions for ALP enzymatic-catalyzed reaction and electrochemical detection were carefully studied with a CHI 832 electrochemical analyzer. According to this method, the oxidative peak current of PNP is in linear with the ALP concentration from 4.0 x 10(2) to 1.0 x 10(6) mU/L and the detection limit for ALP is 2.8 x 10(2) mU/L. So this system can be further used in ALP-labeled electrochemical enzyme immunoassay.