水溶性香豆素荧光底物的制备及在液滴数字式检测中的应用

4-甲基伞形酮磷酸酯(4-MUP)是一类重要的荧光底物, 由于具有较高的疏水性, 荧光信号易在液滴间扩散而限制了其在液滴微流控芯片领域中的应用. 本文首先通过修饰7-羟基香豆素-4-乙酸, 制备了具有较高水溶性的新型底物分子7-二羟基磷酸酯香豆素-4-乙酸甲酯; 进而以7-二羟基磷酸酯香豆素-4-乙酸甲酯为底物, 以球刷酶(荷载大量碱性磷酸酶的聚电解质纳米颗粒, SP-AKP)为模式酶, 建立了基于液滴微流控的单SP-AKP数字式检测体系. 结果表明, 该水溶性香豆素荧光底物具有与传统4-MUP底物相似的荧光光谱和酶催化性能. 传统4-MUP酶促荧光产物5 min即在液滴中发生明显扩散, 而该水溶性香豆素荧光底物酶催化后产生的荧光产物7-羟基香豆素-4-乙酸甲酯在24 h后仍未观察到明显扩散现象, 具有优异的抑制荧光扩散性能. 在基于液滴微流控芯片的单SP-AKP数字式检测中, 对SP-AKP的检测限可达29.9 amol/L, 同时有效提升了检测时间的可操作性与数字式信号读取的准确性. 新型水溶性香豆素荧光底物有望替代4-MUP应用于以基于液滴数字式超敏生物检测为代表, 在液滴分区实现酶促反应进行超灵敏检测的众多检测领域中.

Preparation of Water-soluble Coumarin Fluorescent Substrate and Its Application in Droplet Based Digital Detection

4-Methylumbelliferyl phosphate is one of the most important fluorescent substrate， but its application in the droplet based microfluidic field is limited by its high hydrophobicity and diffusion property between droplets. In this paper， a novel high water solubility fluorescent substrate 7-（dihydroxyphosphoryloxyl） coumarin-4-acetic acid methyl ester was synthesized by modifying 7-hydroxycoumarin-4-acetic acid. The droplet microfluidics based single SP-AKP digital detection system was established with prepared 7-（dihydroxyphosphoryloxyl） coumarin-4-acetic acid methyl ester as the fluorescent substrate， and the spherical brush enzyme（polyelectrolyte nanoparticals loaded with a large number of alkaline phosphatases， SP-AKP） prepared by our research group as the model enzyme. The results showed that the new water-soluble coumarin fluorescent substrate had similar fluorescence spectroscopy and enzyme catalytic performance to traditional 4-MUP substrate. Compared with the enzyme-catalyzed fluorescent product of traditional substrate 4-MUP， which diffused signi- ficantly in droplets within 5 min， the enzyme-catalyzed fluorescent product（7-hydroxycoumarin-4-acetic acid） of the new fluorescent substrate had excellent diffusion suppression performance， which was stable in the droplet within 24 h with no obvious diffusion phenomenon. Further application in droplet based digital enzyme-linked immunosorbent assay of single SP-AKP indicated this new substrate could reach a low limit of detection of 29.9 amol/L， with flexible detection time and high signal accuracy. This new water-soluble coumarin fluorescent substrate was expected to replace 4-MUP in the applications such as ultrasensitive droplet based biological detection and many other fields that require enzymatic reaction in the droplet partitions.