Design, Synthesis And Application Fluorescent Probes For Thrombin And Tyrosinase Detection

In this thesis, we employed fluorescent probes for thrombin and tyrosinase detection. In the thrombin detection, the probe was designed based on the structure of thrombin, whereas we focused on the biological function of tyrosinase in the detection.Thrombin is a specific serine endoprotease that plays a critical role in hemostasis. It naturally serves as a blood clotting factor that solidifies the fibrin clot in terms of strength and elasticity. We designed and synthesized compound1and2for its detection. In the aggregation-induced quenching experiment, compound1exhibited more sensitivity than compound2, so compound1was more suitable for thrombin detection. Then, we employed a turn-on switching thrombin detection on the basis of compound1and a thrombin-binding aptamer (TBA). The detection mechanism includes three steps.1) Strong fluorescent emission is observed due to the monomeric form of compound1probe in aqueous solution.2) When TBA is added into the solution, the fluorescence is almost quenched by aggregation-induced quenching effect.3) Upon the addition of thrombin, it binds to TBA and induces the formation of an anti-parallel G-quadruplex conformation. The specific binding pattern weakens the interaction between compound1and the aptamer and releases the probe, leading to fluorescence recovery. Our detection system is endowed with high thrombin selectivity against other proteins and can detect at least50nM thrombin without signal amplification or fluorophore labeling. Furthermore, the emission changes in the whole detection process could be visualized by the naked eye.Tyrosinase is a copper-containing enzyme that can accelerate the hydroxylation of phenol derivatives to relevant catechol derivative, followed by further conversion to the respective orthoquinone oxidation products. Owing to its elevated expression level, tyrosinase is acknowledged as a biomarker of melanoma cells. Herein, we designed and synthesized two novel tyrosinase-detecting probes that combine phenol group as enzyme substrate and compound44or32as activatable signal reporters. In this approach, compound3,4,5,6and7were synthesized with satisfactory yield. Compound5is not stable, so it could not be used as the probe. Meanwhile, compound4and7exhibited minor fluorescent change, taking sensitivity into consideration, compound3and6were more suitable for tyrosinase detection in comparison with compound4and7. The fluorescence emission of them mainly depends on the enzyme activity, accordingly the emission peaks of them gradually rose along with increasing concentration of tyrosinase and displayed nearly12-fold enhancement. Next, we assessed the feasibility of compound6as a two-photon probe for tyrosinase detection in living cells. B16F1cells that incubated with compound6displayed bright-green fluorescence in cytoplasm, whereas HeLa cells with similar treatment elicited relatively weak responses. Therefore, the probe compound6can be utilized for the visualization of endogenous tyrosinase activity.