| The cyclic redox of methionine is an antioxidant defense mechanism in living organisms,and methionine sulfoxide reductases（Msrs）play an important role in this process.Msrs are very significant as reactive oxygen species scavengers in vivo for cellular defense against oxidative damage,and their abnormal activities may generate oxidative damage to certain key proteins in the brain,further leading to the development of neurodegenerative diseases.Therefore,it is of great practical importance to develop quantitative assays of Msrs to further improve our understanding of the mechanisms of resistance to oxidative damage in organisms.To date,a number of assays for Msrs have been reported.Among them,fluorescence assays have been widely adopted because of high sensitivity,high selectivity,etc.However,the implementation of intracellular quantitative detection of Msrs still has certain challenges.On the one hand,the lack of high-performance ratiometric probes to accurately achieve quantitative detection in living cells;on the other hand,the organism is a very complex environmental system,especially the presence of bioabsorption and autofluorescence would cause interference in the detection.Therefore,there is an urgent need for probes that are less interfered by the physiological environment.In this thesis,the following studies are carried out on the above scientific issues:In Chapter 1,the role of Msrs in organisms is introduced and its association with neurological diseases is aslo pointed out.Then,various detection methods（including colorimetric,fluorescent methods,etc.）are reviewed,and the limitations of the current detection methods of Msrs were discussed.Finally,some strategies to overcome these limitations are proposed.In Chapter 2,an integrated intramolecular charge transfer and fluorescence resonance energy transfer（ICT-FRET）unimolecular platform（CPSO）was designed and synthesized for two-photon ratiometric fluorescence sensing of Msrs.After recognition by Msrs,the ICT process in the probe is turned on,which further activates a FRET process,leading to ratiometric spectral changes in the fluorescence spectrum.Two-photon excitation was used,which greatly reduce photo damage to cells in subsequent biological applications.The probe CPSO is successfully used to detect and image the fluctuation of Msrs level in live neurons and mouse brain tissue,and the results indicate an upregulation of Msrs activity in neurons under oxidative stress.In Chapter 3,a supramolecular room temperature phosphorescent（RTP）probe1（?）CB[8] is designed and synthesized,which is used to detect Msrbased on the variation of the phosphorescent lifetime.On the one hand,similar to the ratiometric fluorescence signal,the lifetime of the probe is independent of the concentration of the probe or the power of the laser source.On the other hand,as a output signal,phosphorescent lifetime is not only sensitive,but also anti-interference,which can avoid the influence of short-life biological background fluorescence.Therefore,both advantages can minish the influence of the physiological environment during detection.Moreover,it could be applied to the phosphorescence imaging and detection Msrs in AD rat brain sections,providing a powerful tool for understanding the physiological and pathological conditions associated with Msrs. |
