Targeted screening of natural thrombin inhibitors from herbal extracts using an enzyme-immobilized microfluidic...

An enzyme-immobilized microfluidic reactor (EIMR) is a highly efficient platform for in vitro screening of catalysts and inhibitors. Herein, an EIMR integrated in a glass-PDMS hybrid microfluidic chip (HMC) was developed for in situ screening of potential thrombin inhibitors from complex herbal extracts. Thrombin was selectively immobilized in situ on a gold nanoparticle-functionalized porous silica film (AuNPs@FPSF)-coated ITO glass slide using the DNA aptamer TBA₁₅, while the microfluidic part of the EIMR, comprising a microchannel and microfluidic connections, was formed in a polydimethylsiloxane (PDMS) plate. The EIMR was compatible with dual-model detection: fluorescence analysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). A customized fluorescent substrate was synthesized for in situ monitoring of the thrombin reaction using fluorescence microscopy, and its enzymatic hydrolysate was detected and quantified using MALDI MS. The reproducibility of the EIMR was significantly improved by optimizing conditions for thrombin immobilization and the concentration of a fluorescent substrate. As a result, eight potential thrombin inhibitors were discovered from five herbal extracts, and their inhibitory activities were determined using MALDI MS. Overall, the developed EIMR exhibited the advantages of high sensitivity, ease of use, and fast-response, facilitating the screening of candidate enzyme inhibitors from complex herbal plants.