以葛根素为辣根过氧化物酶底物的新体系及其分析应用

以一种天然活性成分葛根素(Puerarin)为辣根过氧化物酶(HRP)底物建立了葛根素-辣根过氧化物酶-过氧化氢反应新体系.在反应体系中HRP催化H2O2氧化葛根素(弱荧光)形成二聚体产物(强荧光),该产物在315 nm的激发光下能发射波长为478 nm的强荧光,并且反应体系荧光强度增加与HRP量在一定浓度范围内呈线性相关.根据此关系和竞争型免疫定量原理,以兔布氏杆菌抗体为分析对象建立了基于葛根素的酶联荧光免疫传感分析新方法.对葛根素性质的研究结果证实,葛根素在空气中稳定、对温度稳定,对H2O2+HRP敏感性优于传统底物如对羟基苯乙酸、Amplex Red和高香草酸.优化了酶联荧光免疫传感分析方法的实验条件如HRP-BrAb用量、温度等.运用新体系测定了兔血清样品的布氏杆菌抗体,该方法线性范围为1.3～120 ng/mL,检测限为1.3 ng/mL(3σ),相对标准偏差为3.8%.

An Enzyme-Catalyzed Reaction System Using Puerarin as Substrates for Horseradish Peroxidase and Its Application in Immunoassays

An enzyme-catalyzed reaction system puerarin-horseradish peroxidase(HRP)-hydrogen perox-ide,which used puerarin as substrates for HRP,was developed.In enzymatic reaction procedure,HRP cata-lyze the polymerization of puerarin by H2O2,and the puerarin is partly converted to dimers,a significantly fluorescent species.The increase of the fluorescence of the HRP-enzymatic reaction solution at emission of 478 nm(excitation: 315 nm) is proportional to the concentration of HRP.The enzyme-linked fluoroimmu-noassay based on the principle mentioned above and competitive immunoassay model for the determination of Brucella melitensis antibodies(BrAb) was established.The results of the property investigation of puer-arin demonstrate the advantages in stability and reactivity with HRP over conventional substrates such as p-hydroxyphenylacetic acid(p-HPA),Amplex red and homovanillic acid.The experimental parameters such as BrAb-HRP,temperature were optimized.The proposed method has been successfully used for analysis of BrAb in rabbit plasma sample.The linear range of determination for BrAb is 1.3～120 ng/mL with the rela-tive standard deviation of 3.8%.The detection limit is 1.3 ng/mL(3σ).