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合成了辣根过氧化物酶的５种荧光底物，它们分别是：Ｎ，Ｎ′—二氰甲基邻苯二胺（ＤＣＭ－ＯＰＡ），３，４—二氧喹喔啉－２（１Ｈ）－酮（ＤＨＱ），３－甲基－３，４－二氢喹喔啉－２（１Ｈ）－酮（ＭＤＨＱ），３，４－二氢喹喔啉－２（１Ｈ）－酮－６－羧酸（ＤＨＱ－６－Ａ）及３－甲基－３，４－二氢喹喔啉－２（１Ｈ）－酮－６－羧酸（ＭＤＨＱ－６－Ａ）。其中荧光底物ＤＣＭ－ＯＰＡ，ＤＨＱ和ＭＤＨＱ都可以很容易地进行大量纯品的制备。我们用流动注射分析法对合成底物和已有的最佳荧光底物在ＨＲＰ及其模拟酶氯化血红素催化体系中的性能进行了比较研究。结果表明，ＤＣＭ－ＯＰＡ和ＭＤＨＱ是合成的５种底物中最好的两个，而对羟基苯丙酸（ｐ－ＨＰＰＡ）和对羟基苯乙酸（ｐ－ＨＰＡ）优于高香草酸（ＨＶＡ）和酪氨。底物ｐ－ＨＰＰＡ和ｐ－ＨＰＡ与合成底物ＤＣＭ－ＯＰＡ和ＭＤＨＱ体系在对过氧化氢的分析方法性能相当，最低检测限都在１～１０ｎｍｏｌ／Ｌ之间。对酶的检测，ＤＣＭ－ＯＰＡ是所有研究的底物中最灵敏的，上述两种合成底物保存在４℃的冰箱中都可以稳定一个月以上。
Five kinds of fluorescent substrates of horseradish peroxidase were synthesized. They are N, N’-dicyanomethyl o-phenylenediamine (DCM-OPA), 3,4-dioxoquinoxaline- (DHQ), 3-methyl-3,4-dihydroquinoxalin-2 (1H) -one (MDHQ), 3,4- dihydroquinoxalin-2 (1H) Carboxylic acid (DHQ-6-A) and 3-methyl-3,4-dihydroquinoxalin-2 (1H) -one-6-carboxylic acid (MDHQ-6-A). Among them, fluorescent substrates DCM-OPA, DHQ and MDHQ can be easily prepared for a large number of pure products. Flow injection analysis was used to compare the performance of the synthesized substrate and the best available fluorogenic substrate in HRP and its mimic enzyme hemin. The results showed that DCM-OPA and MDHQ were the best two of the five synthetic substrates, while p-HPPA and p-HPA were superior to homovanillic acid (HVA ) And tyramine. The substrates p-HPPA and p-HPA are comparable to the synthetic substrates DCM-OPA and MDHQ in the analysis of hydrogen peroxide. The minimum detection limits are between 1 and 10 nmol / L. For enzyme detection, DCM-OPA was the most sensitive of all studied substrates, and the two synthetic substrates were stable in a refrigerator at 4 ° C for more than a month.