Fluorescence Lifetime Imaging And Molecular Mechanism Analysis Of Oxidative Stress-related Substances

As a common physiological activity in cells,oxidative stress has always been playing an important role in controlling life and health.Oxidative stress is defined as an interference in the balance between intracellular reactive oxygen species?ROS?and antioxidant defense.With the increasing understanding of oxidative stress,it has been found that it plays a key role in regulating cell activities.Appropriate ROS production is conducive to cell proliferation and differentiation,also immune function enhancement and signal transduction between cells.However,once oxidative stress balance is broken,such as the outbreak of ROS,it will gradually lead to severe oxidative damage,which aggravates cell apoptosis and even necrosis and further leads to a series of diseases,especially neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.Therefore,the analysis and detection of oxidative stress-related substances is an important way to study the corresponding process,which is also one of the bases to further explore many physiological and pathological processes.Many people are committed to the above research,and significant findings are appeared.However,there still has some inherent challenges:1.oxidative stress is a pretty complicated event and many substances are related to it also these substances on the other hand are closely related to each other.Thus,the detection of a single substance obviously cannot meet the research requirements.However,due to the uneven distribution,it is difficult for the mixed probes to accurately analyze the objects with the same location;2.The intracellular oxidative stress process mainly happens in mitochondria,so it is necessary to further develop a probe that can target mitochondria;3.The concentration of substances related to oxidative stress is in a close dynamic change,and real-time monitoring of the corresponding changes is conducive to accurate understanding of the whole process which means that the probe must be highly selective and accurate.Therefore,to sum up,it is of great significance to develop a single probe with multiple detection,high selectivity and accuracy,which actually allows to analyze the dynamic changes and mechanism of molecules related to oxidative stress process!At present,many methods have been reported for the detection and analysis of these substances,among which fluorescence imaging microscopy is widely used in the imaging of cells and even in vivo due to its high spatial-temporal resolution.Compared with ordinary fluorescence imaging technology,fluorescence lifetime imaging takes more significant advantages.As a matter of fact,fluorescence imaging depends on the signal of fluorescence intensity,which is easily interfered by excitation light intensity,probe concentration and photobleaching.However,fluorescence lifetime imaging determines the change of fluorescence lifetime,which mainly depends on the property changes of the probe.It avoids the susceptibilities of the excitation light intensity,probe concentration as well as photobleaching.Combined with the key problems to be solved and the technical advantages to be achieved,my master's career mainly includes the following two works:In the first work,a single fluorescence probe?TFP?was designed and synthesized for the simultaneous detection of hydrogen peroxide?H₂O₂?and adenosine triphosphate?ATP?in mitochondria.This probe has high selectivity and accuracy,which exhibits no fluorescence resonance energy transfer?FRET?and crosstalk in the detection of the two molecules.The TFP probe demonstrates real-time fluorescence lifetime imaging and quantitative analysis of H₂O₂ and ATP in mitochondria of neurons.It is found that the level changes of H₂O₂ and ATP in mitochondria are closely related to each other under different states.Meanwhile,under superoxide anion(O₂^·–)stimulation for different times,mitochondrial H₂O₂ and ATP exhibits different and changeable status.However,with the increasing stimulation time,it eventually leads to unrecoverable oxidative damage and even neuron death.Then in the second work,it was proposed to construct a DNA tetrahedral fluorescent nanoprobe for the simultaneous determination of pH,hypochlorite?HClO?and glutathione?GSH?.By modifying three molecules which specifically response to above substances and a mitochondria-targeting molecule TPP to the four vertex positions of DNA tetrahedron,a whole nanoprobe is formed.On the one hand,since the DNA strand length is 12 nm,there is no FRET between the three fluorescent molecules.On the other hand,the fluorescence lifetime ranges of the three probes can be well distinguished,which means the they can meet the detections independently.At present,only some in vitro data,such as the fluorescence spectral properties,the high accuracy and selectivity of three probes and the synthesis of DNA nanoprobe have been completed.The following work will mainly focus on the simultaneous determination of three objects to be measured by the DNA nanaoprobe,the cytotoxicity,mitochondria-targeting ability and the neuron imaging experiments upon oxidative stress.