New Generation of Fluorogenic Substrate for Ultrahigh Throughput Screening of Phosphotriesterases

• 1、School of life sciences and biotechnology, Shanghai Jiao Tong University，Shanghai，200240

Abstract： In vitro compartmentalization based fluorescence-activated droplet sorting (IVC-FACS) is a power tool for enzyme high throughput screening. It is crucial for an IVC-FACS system to keep the stability of the fluorescent signal inside a particular microreactor, so that the information for the reaction progress can be readily maintained during several hours of screening. Coumarin derived fluorogenic substrates are commonly used for the detection of many enzymes. However, the rapid diffusion of coumarin between droplets severely hampered its application in IVC-FACS. Here, a hydrophilic carboxylic acid group modified coumarin, 7-Hydroxycoumarin-3-carboxylic acid (HCCA), was reported with good retention in droplet. An HCCA-phosphotriester substrate was synthesized for the IVC-FACS screening of phosphotriesterase. The new substrate could diffuse into the w/o/w double emulsion droplets and react with the enzyme, producing fluorescent product which could be confined well in droplets. Also, the enzyme activity could be faithfully reflected in droplet assays. The model screening assay revealed that enzyme-expressing E.coli cells could be effectively enriched as high as 900-folds from large accesses of background cells, further demonstrating the suitability of the HCCA derived substrate in the application of IVC-FACS system.

Keywords： fluorogenic substrate ultrahigh throughput screening phosphotriesterase droplet