Liu_2020_Mikrochim.Acta_187_313

A low-molecular-weight molecule (4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl-benzoate, DDPB) has been developed. The organic framework possesses very weak fluorescence . The feasibility of the signal transduction has been performed via fluorometric titrations in solution. DDPB gives rise to responses to carboxylesterase 2 (CES2) based on "off-on" responses. The red emission at 670 nm has been derived from the enzyme-induced hydrolysis of ester linkages, thus suppressing the intramolecular charge transfer (ICT) effect and thereby generating the fluorescent segment. The optical excitation window for this probe is extended to the visible light range (lambdaex = 516 nm), and it will induce less harmful influence on biological substances. The detection limit for the measurement of CES2 concentration is as low as 2.33 mU/mL. The conventional studies concerning the activation process are generally performed within only a single liveing cell system. In this study, it is the first time that expression of carboxylesterase 2 in five kinds of cell lines (HeLa > C1498 > active T cell > Jurkat > unactive T cell) has been clarified by flow cytometry, Western blotting, and confocal microscopy analysis. The elucidation of CES2 and its variability in a variety of cells will open new ways for drug metabolism and disease prevention. Graphical abstract We reported a new "substrate-mediated light-on" strategy based on an ester bond cleavage reaction. Most of prepared nanomaterials and organic fluorophores possessed short wavelength emissions in the blue or green region which will not be difficult for cellular imaging. In this study, a novel functional molecule (DDPB) was considered as the substrate for CES2 and the optical "off-on" response was realized. DDPB was cell permeable and possessed very low cytotoxicity. Moreover, the identification of CES2 and their subtle changes in five different cells afforded the sequence for carboxylesterase-2 as Hela > C1498 > Active T cell > Jurkat > Unactive T cell. Inhibition studies showed that the hydrolysis of DDPB was effectively suppressed by bis-p-nitrophenyl phosphate and the cellular tracking results firmly supported this point. To our knowledge, the inter-individual variability for the CES2 expressions in five different cell lines has never been reported via the substrate induced optical changes.