4-羟基苯乙烯基吡啶为辣根过氧化物酶底物的酶荧光免疫传感体系测定布氏杆菌抗体

A Fluorometric Enzyme-linked Immunosensing System Using 4-(p-Hydroxystyryl)pyridine As a New Substrate of Horseradish Peroxidase for Brucella melitensis Antibody Assay

合成了一种新型辣根过氧化物酶(HRP)荧光底物-4-羟基苯乙基吡啶(pHSP),并首次将它运用于酶联荧光免疫传感体系.对pHSP化学性质的研究证实,pHSP在空气中较稳定,对HRP,H2O2的荧光响应性能优于传统HRP荧光底物,如对羟苯乙酸、Amplex Red和佳味醇等.在pH5.8的Britton-Robinson缓冲溶液中,pHSP本身只有极弱的荧光,在HRP-IgG催化下可被H2O2氧化成二聚体产物,该二聚体在300nm的激发光下能发射波长为437nm的强荧光,并且反应体系的荧光增加与HRP量在一定浓度范围内成线性相关.根据此原理,建立了兔布氏杆菌抗体的酶联荧光传感分析新方法.运用制备的传感装置测定兔布氏杆菌抗体的线性范围为6.3×10-11～1×10-8mol·L-1,抗体检出限为6.3×10-11mol·L-1,相对标准偏差为4.1%(n=11).pHSP的二聚体产物水溶性很低,利用设计的装置较好地解决了传统测定溶液体系方法灵敏度不高的问题.

A novel substrate, 4-（p-hydroxystyryl）pyridine （pHSP） was synthesized and firstly used in a horseradish peroxidase （HRP） based fluorometric enzyme-linked immunosensing system. The results of the property investigation of pHSP demonstrate the advantages in stability and reactivity with HRP over conventional substrates such as p-hydroxyphenylacetic acid （p-HPA）, Amplex red and chavicol. In a pH 5.8 Britton-Robinson buffer solution, HRP-antibody conjugate （HRP-IgG） could catalyze the oxidation reaction of pHSP by H202, and the pHSP was converted to significantly fluorescent dimers. The increase of the fluorescence intensity （excitation： 300 nm, emission： 437 nm） of the HRP enzymatic product is proportional to the concentration of HRP-IgG binding to the Brucella melitensis antigen modified matrix. An enzyme-linked immunosensing method based on this principle was developed. The linear range of determination is 6.3× 10^-11～1× 10^-8 mol·L^-1 with the relative standard deviation of 4.1%. The detection limit is 6.3 × 10^-11 mol·L^-1. The another obvious advantage of the proposed procedure is the sensitivity deriving from the solution of the insoluble substrate determination.