Synthesis Of Several Organic Fluorescent Sensors And Their Application In Endogenous Enzymes And Thiols Detection

In recent years, organic fluorescent sensors have been widely applied in chemical, environmental and biological fields. That’s because organic fluorescent sensors are generally easy to synthesize, low-cost and versatile to use. Furthermore, for organic fluorescent sensors, the detection process is very simple, there is no need of complicated sample pretreatment process, and real-time detection can be achieved with good cell membrane permeability for live-cell or tissue imaging. Therefore they display quite potential for future applications.In this study, three kinds of organic fluorescent sensors have been designed and synthesized for endogenous enzyme and small molecule biothiols quantitative detection and bioimaging based on different fluorescence response mechanisms.Firstly, we synthesized a fluorescent probe for detecting ALP via excited state intramolecular proton transfer(ESIPT). The probe was synthesized by coupling a phosphate group onto flavone-based fluorophore. For this probe, the phosphate group serves both as the blocking agent for ESIPT and the responsive moiety toward ALP. Due to the shielding of the hydroxyl group by the phosphate moiety, no ESIPT process occurs, the tautomer emission of the probe is therefore quenched; while in the presence of ALP, ALP cleaves phosphate group and generates hydroxyl group, thereby enabling the ESIPT and generating strong ESIPT-based emission. This probe exhibits high selectivity for ALP detection with a very low detection limit of 0.032 U/L. It is capable of detecting ALP in biological fluid like serum. Furthermore, it is of little cytotoxicity and can be easily internalized into cells for endogenous ALP imaging.Secondly, a facile ratiometric fluorescent sensing system for HAase has been developed, which is based on hyaluronan-induced formation of red-light emitting excimers and can realize sensitive detection of HAase with a detection limit of 0.007 U/mL. A positively-charged pyrene analogue(N-Py) has been synthesized and then mixed with the negatively-charged HA, due to electrostatic interaction between the two components, aggregation along with the N-Py excimers readily form which emits red light. While in the presence of HAase, the enzyme catalyzes the hydrolysis of HA into small fragments, which in turn triggers disassembly of excimers; consequently the N-Py excimer emission turns into monomer emission. The emission ratio resulted from the excimer-monomer transition can be used as the sensing signal for detecting HAase. The probe features visible-light excitation and red light emission(excimer), which is conducive to reducing possible interference from autofluorescence of biological samples. Furthermore, the assay system can be successfully used to determine HAase in human urine samples with satisfactory accuracy. This strategy may provide a suitable sensitive and accurate assay for HAase as well as an effective approach for developing fluorescent ratiometric assays for other enzymes.Finally, we developed a fluorescent probe through coupling two fluorophores with biothiol-sensitive linker, which has two biothiol-responsive and distinctly-separated emissions via independent visible-light excitations, for simultaneously discriminating GSH and Cys/Hcy with near-infrared and green emissions. For this probe, a cyanine-based fluorophore(NR-OH) is attached to 7-nitrobenzofurazan(NBD) by an ether linker, which serves as the reactive site for GSH and Cys/Hcy. The probe is non-fluorescent due to intramolecular charge transfer(ICT) from NBD moiety to NR-OH moiety. In the presence of GSH, the probe system will exhibit the emission of NR-OH(716 nm), while the resultant NBD-GSH is nonfluorescent due to strong quenching ability of the sulfur ether moiety in the molecular structure. While Cys or Hcy would induce an intramolecular rearrangement cascade reaction, thus the emission of NBD moiety can also be observed at 550 nm. GSH cannot induce this intramolecular rearrangement reaction for the lack of proximal amine group. The two distinctly-separated emissions(716 and 550 nm) due to two independent excitations(470 and 670 nm) can be used for simultaneous discrimination of GSH from Cys/Hcy. The probe is operable in human serum samples and shows low cytotoxicity and function well in complex biological milieu as inside living cells imaging. Therefore, it would provide a useful method for further elucidating the roles of biothiols as well as for conducting pathological analysis for diseases involving biothiols.