Construction And Spectral Properties Of A FRET-based Ratiometric Fluorescent Thiol Probe

Chemical fluorescent probes have the advantages of fast response, high sensitivity, uncomplicated operation, reproducible performance and simple equipment. And high-spatial resolution of rapid analysis for the detection of a specific analyte by fluorescence imaging with almost no damage. The chemical fluorescent probes have attracted considerable interests in recent years, because of their wide range of applications of the identification and the detection of metal ions, small organic molecules, biological macromolecules, polarity, viscosity, pH, cell activity, etc. In this paper, we have rationally designed and constructed the first novel FRET-based ratiometric thiol probe, applied to ratiometric imaging in living cells successfully based on the native chemical ligation reaction.Currently some fluorescent probes for thiols have been developed. However, a vast majority of them respond to thiols with optical signal changes only in the fluorescent intensity to express the information. These were almost no fluorescence ratio probes, let alone the reports of fluorescence ratiometric imaging for thiol in biological systems. Therefore, the development of probes, which have selectivity for thiols in biological systems and can be applied within the ratio of fluorescence ratiometric imaging have a very great significance.For this reason, we designed the FRET-based ratiometric thiol probe, based on the native chemical ligation reaction. To achieve this goal, we have designed two systems: dansyl chloride-Rhodamine energy transfer system and BODIPY-rhodamine energy transfer system. In the dansyl chloride-Rhodamine energy transfer system, we totally designed two synthetic routes and a 4-step consecutive synthesis to get two target molecules. However, the study of the spectral properties of the two target molecules, which their principle based on FRET, found they had low sensitivity, emission spectra overlap seriously and poor resolution. Therefore, we used the BODIPY-rhodamine energy transfer system, re-designed ratiometric fluorescent thiol probe. This time, we totally designed three synthetic routes and modified and discussed after many failures. Ultimately, we completed the synthesis route and through a 12-step consecutive synthesis to obtain the objective Probe. Firstly, ratiometric fluorescent probes allow the measurement of emission intensities at two wavelengths, which should provide a built-in correction for environmental effects.Second, the probe is in favour of quantitative analysis of the concentration of thiol of analytes. Finally, the probe has a wide range of practical applications, we have successfully applied to the probe in the quantitative detection of the concentration of thiols in neonatal calf serum, the concentration of thiols in human urine, as well as ratiometric imaging in living cells. The favorable features of the probe include high stability and functioning well at physiological pH, high selectivity, high sensitivity, a large ratio signal variation, and a large linear dynamic range. Importantly, the probe has been applied for ratiometric detection of thiols in biological fluids successfully. Thus, we expect that the probe will be a useful molecular tool for diverse biological applications including the determination of thiol levels in biological fluids, fluorescence labelling of proteins, and the assaying of enzymes with a thiol as a product for enzyme-inhibitor screening. Furthermore, the NCL-based thiol probe design concept should be widely applicable for construction of ratiometric thiol probes.