Fluorescence Imaging And Real-Time Sensing In Neurons Under Oxidative Stress

Oxidative stress is an important physiological process in life,and its level is closely related to many diseases.For example,cancer,neurological diseases,and cardiovascular and cerebrovascular diseases.Therefore,it is of great significance to realize the research on the monitoring of oxidative stress-related processes.The important way to realize the monitoring of oxidative stress-related processes is the analysis and detection of substances closely related to oxidative stress.A variety of methods have been reported for the detection and analysis of oxidative stress-related substances,and the fluorescence method has been widely used for in situ and dynamic monitoring of changes in living or intracellular substances.However,real-time,dynamic,high-selection,and highly sensitive fluorescence analysis of these related substances remains challenging.The current challenges are mainly in three aspects:?1?There are many coexisting substances and complex components in living organisms or cells,the fluorescence detection and analysis of oxidative stress-related substances in living or cellular levels require high selectivity for probes.?2?The concentration of substances closely related to oxidative stress is in dynamic changes,and real-time and dynamic monitoring of changes in these substances can more accurately understand the oxidative stress-related processes.This puts higher requirements on the time resolution and accuracy of the probe,so it is necessary to prepare probes with fast response dynamics.?3?There is a correlation between different substances,and simultaneous detection and analysis of multiple substances can better understand oxidative stress.However,the dynamic of mixed probes entering living cells or cells are different and unevenly distributed.On the other hand,the co-modification of different fluorescent probes onto the same nanomaterials easily leads to fluorescence resonance energy transfer and cross-talk.Therefore,it is necessary to develop high-efficiency probes for simultaneous detection of multiple substances.In order to solve the above key scientific problems,my doctoral thesis mainly carried out the following three aspects:?1?To improve the selectivity of the probe,I have synthesized organic ligand molecules specifically recognized by Fe²⁺or Ca²⁺,and further conjugated these molecules onto metal nanoclusters to construct inorganic-organic composite nanoprobes to achieve rapid response kinetics of probes,thereby increasing the time resolution of the probes.In addition,the reference molecule was further modified to construct a ratiometric fluorescent probe,realizing real-time imaging and analysis of Fe²⁺and Ca²⁺in neurons with high-selectivity and high-accuracy.We found that Fe²⁺distribution in neurons was inhomogeneous,and ischemia caused a significant increase in Fe²⁺concentration in neurons.On the other hand,it was found that histamine-induced Ca²⁺increase in neurons was different at different regions of neuron.?2?I have summarized and studied the factors affecting the fluorescence of carbon quantum dots.It is found that the factors affecting the fluorescence of carbon quantum dots mainly include two aspects,namely internal factors and external factors.Internal factors mainly include size effect and doping effect.With the increase size of carbon quantum dot,the fluorescence emission peak of carbon quantum dots is red-shifted;doping can significantly improve the fluorescence quantum yield of carbon quantum dots.The external factors mainly refer to the factors affecting the surface state of carbon quantum dots.It is found that the electron donating group has the greatest influence on the fluorescence of carbon quantum dots.The stronger the electron donating ability of the surface modification molecules of carbon quantum dots,the stronger the fluorescence of carbon quantum dots.This work supports the analysis of carbon quantum dot fluorescence mechanisms and provides the suppoting for the design and fabrication of functionalized carbon quantum dots.?3?Based on the analyzing the fluorescence enhancement mechanism of carbon quantum dots,a new Ca²⁺highly selective fluorescent probe based on carbon quantum dots was constructed,and DNA tetrahedral nanostructures with side length of¹2 nm were introduced,which were combined with pH response molecules,reference fluorescent molecules and the mitochondrial targeting molecule simultaneously.The DNA composite nanoprobe is constructed to avoid the possible fluorescence resonance energy transfer and cross talk between different response probes,and the simultaneous imaging and quantitation of pH and Ca²⁺in the mitochondria of the neuron is realized.I have found that mitochondrial pH is associated with Ca²⁺,and superoxide anion and A?protein-induced neuronal death are closely related to acidosis-induced mitochondrial Ca²⁺overload.These results indicate that Fe²⁺,Ca²⁺and pH are closely related to oxidative stress,and provide a new method for analyzing the role and molecular mechanism of oxidative stress in related brain diseases from the cellular level.