The Synthesis Of Fluorescent Probe And Application For Proteins Disulfenylation Detection

Fluorescent probes contain many advantages such as the high sensitivity, excellent selectivity, fast response, in-situ and real-time detection of analytes, non-cytotoxicity, good biocompatibility, etc. Therefore, they play an important role in biochemistry and living cells detection. In recent years, fluorescent probes have accomplished albeit substantial progress for detection of DNA and protein. However, the enhanced-fluorescent probes have attracted increasing interest due to low background signal and high sensitivity. Currently, only less small molecular fluorescent probes that can combine with target protein specifically. Simultaneously, the lack of methods for design and synthesis also limit the further development in biological application. Therefore, it is important to design a novel enhanced-fluorescence probe for detection of protein with high sensitivity and specificity. In this paper, we have studied the synthesis of enhanced-fluorescence probe and biological application. The details are as follows:1. Firstly, we made a brief introduction to the definition, structure and function of protein. Thioredoxin(Trx) is one kind of protein, we expounded the structure and redox mechanism of Trx. Secondly, we also summarized the design principle, response mechanism and unique advantages of fluorescent probes. We discussed the application of biological analysis based on pyrene fluorescence probe due to its excited singlet of pyrene possesses long lifetime and easy to form excimer.2. The design, synthesis and characterization of the fluorescent probe 2, 4-cyclohexanedione-1--propyl-pyrene. We have synthesized fluorescent probe 2, 4-cyclohexanedione-1--propyl-pyrene(TAR), which could combine Trx specifically. A series of characterization of TAR such as H NMR spectroscopy, C NMR spectroscopy and high resolution mass spectrum were represented. Moreover, we employed fluorescent spectrometry to analyze the performance. Results confirmed that TAR exhibited stable fluorescence characteristics in various redox environments. Cell viability was tested by using a standard methyl thiazolyl tetrazolium(MTT) assay, the cell survival rate remained at 85% even the TAR concentration increased to 100 μM, which indicated TAR have very low cytotoxicity.3. TAR-based fluorescent probe detect protein disulfenylation and the application in living cells. As a novel signal enhanced-fluorescence probe, pyrene could be applied in fluorescence sensor for vicinal dithiol proteins assay. With hydrogen peroxide in the solution, vicinal dithiol of Trx could be oxidized to sulfenic acids and combine with TAR specifically. Which resulted in fluorescence enhancement, this was due to the pyrene molecules of TAR closed to each other and form dimers. We obtained different degrees of fluorescence intensity in 480 nm by varying concentrations of Trx in the solution with high sensitivity and selectivity. TAR was introduced into living cells, the cell imaging and colocalization analysis demonstrated that Trx located mainly in the mitochondria. In conclusion, we have designed a novel enhanced-fluorescent probes TAR for Trx detection and further application in cell imaging.