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-(d illydroxy phosphoryloxyl) 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 significantly in droplets within 5 min, the enzyme-catalyzed fluorescent product (7-hydroxycotunarin-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 enzymelinked 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.