New Methods For Analysis Of Reactive Oxygen Species And Its Related Molecular Mechanism

Under normal physiological conditions,the production and consumption of reactive oxygen species?ROS?in cells is in a state of dynamic equilibrium.At this time,the generated ROS with an appropriate amount has important physiological significances for signal transduction and immunity in the body cells.However,under some exogenous or endogenous stimuli,this dynamic balance will be broken,causing a rapid increase in ROS,and resulting in the body's inability to clear in time,leading to oxidative stress.The occurrence of oxidative stress is accompanied by a series of negative effects:excessive production of ROS can cause irreversible damage to proteins,lipids,DNA and RNA,and accelerating the aging process of organisms;dynamic imbalance of ROS can lead to the changes of intracellular pH and cause the body's internal environment to lose its acid-base balance,resulting in acid-base balance disorders;ROS imbalance can also lead to a range of diseases,such as respiratory diseases,cancer and neurodegenerative diseases.Therefore,it is of great importance to develop analytical methods with high sensitivity,good selectivity,good reproducibility and fast response to detect the concentrations and changes of various related molecules in the process of oxidative stress,and to understand the oxidative stress processes and the molecular mechanisms of physiological diseases.This thesis provides new research methods for studying ROS and its related molecules,as well as the molecular mechanism of oxidative stress.The first topic in this thesis is“A ratiometric fluorescent probe for bioimaging and biosensing of HBrO in mitochondria upon oxidative stress”.The second topic of this thesis is“Mitochondria-targeted ratiometric fluorescent nanosensor for simultaneous biosensing and imaging of O₂^·–and pH in live cells”.This nanosenor enabled us understanding the relationship between O₂^·–and pH in the mitochondria.The third topic of this thesis is“Direct observation of O₂^·-generated in Cu-A?-catalyzed redox processes”.The details are as follows:In the first work,a fluorescent probe for imaging mitochondrial HBrO with excellent selectivity,high accuracy,good reproducibility and high sensitivity was developed on the basis of the ability of HBrO to catalyze the cyclization reaction between the amino group and S-methyl group and the mitochondria targeting ability of triphenylphosphonium cation.Further studies found that O₂^·--induced oxidative stress triggered the burst of HBrO in mitochondria.In the second work,a ratiometric fluorescent nanosensor for simultaneous detection of mitochondrial O₂^·-and pH was developed on the basis of the pH sensing ability of FITC,the sensing ability of HE to O₂^·-,the excellent fluorescence stability of SiO₂@QD and the mitochondria targeting ability of triphenylphosphonium cation.In vitro experiments showed that the ratiometric fluorescent nanosensor had good selectivity and sensitivity to O₂^·-and pH,and can be used not only for separately sensing O₂^·-and pH,but also for simultaneous detection of O₂^·-and pH.The results of cellular experiments showed that the ratiometric fluorescent nanosensor had mitochondrial targeting function and was successfully applied to fluorescence imaging and biosensing of intracellular O₂^·-and pH.Further studies found that O₂^·-entered cells through anion channels on the cell membrane,which caused an increase in the concentration of O₂^·-in the mitochondria and an increase in pH;a decrease in the pH value in the mitochondria caused the increase of O₂^·-.In the third work,three different methods including biochemical methods?indirect methods?,electron paramagnetic resonance spectroscopy?direct method?and transient absorption spectroscopy?direct method?were used to thoroughly study the generation of ROS during the Cu-A?-catalyzed redox processes.The results obtained by all these methods indicated that Cu-A?in the biological reducing environment was not only able to catalyze the production of H₂O₂ and·OH,but also to generate free O₂^·-.We also found that the amount of ROS produced was closely related to the state of A?bounded to Cu²⁺.This work had comprehensively evaluated the radical species in the oxidative stress,not only in mechanism insights,but more importantly in providing an alternative approach to cure AD through inhibition of the generated free O2^·-.