Abstract
In recent years, small molecule fluorescent turn-on probes are getting attention as they allow for sensitive, simple, and specific detection with high signal-to-background ratios. Currently most of the small-molecule fluorescent turn-on probes are designed for monitoring enzyme activities, for example, glycosidases, proteases. Typically, their fluorescence turn-on mechanism is based on an enzymatic reaction with the chemical probes to convert a non-fluorescent substrate into a fluorescent product. On the other hand, the design of fluorescence probes for non-enzymatic proteins remains a challenging task. Herein, we introduce a new type of fluorescent turn-on probes, where a small molecule ligand is conjugated to an environment-sensitive fluorophore, for the selective detection of both enzymes and non-enzymatic proteins. The fluorescent turn-on mechanism is based on the binding of the ligand to a hydrophobic ligand binding domain of the target protein whereby the close proximity to the hydrophobic environment can influence the environment-sensitive fluorophore to exhibit stronger fluorescence. We successfully established a SBD type model for specific detection of hCAII, trypsin and avidin with fluorescent turn-on ratios of up to 17-fold. We also demonstrated that these fluorescent probes can be employed to visualize carbonic anhydrase expressed on the cell surface, and applied on several sulfa drugs detection by tunable ligand affinity. Furthermore, we also extended our protein detection strategy, in Cy5 rotor model, the proteins selectivity remain to be improved, but the novel design and characteristics of our detection method is worth to be developed by adapting fluorescent molecular rotor based on Cy5.