Synthesis And Characterization Of Fluorescent Probes Of Cuprous Ion And Study On The Effect Of Copper Ion Deficiency On Mitochondrial Function And Neurodevelopment

Copper ion plays an important role in physiology.They participate in the physiological processes of the body.Especially copper ion plays an important role in energy metabolism and nervous system development.Imbalance of copper ions can cause serious diseases.For example,the lack of copper ions will directly affect the synthesis of neurotransmitters and neuropeptides,as well as neuromodulation and the development of the nervous system.Mitochondria are the"copper pool"of the cell.Insufficient copper ions can also lead to impaired mitochondrial function,which can lead to energy failure in severe cases,and ultimately lead to neurodegenerative diseases.The main reason is the deficiency of copper ion,which leads to the decrease of the activity of copper containing protease COX in mitochondrial respiratory chain,and then affects the function of mitochondria.Therefore,it is of great significance to be able to detect the changes of copper ions in organisms sensitively,visually and non-invasively.Small molecule fluorescent probe imaging has the above advantages.In addition,copper ions mostly exist in the form of Cu~+due to the reducibility of the intracellular environment.Based on this,we designed two Cu~+fluorescent probes,which can detect Cu~+sensitively and detect cells and zebras with the help of a laser confocal microscope.The Cu~+content in fishes,the effects of Cu~+deficiency on mitochondrial function and the development of the zebrafish nervous system were studied using respirometers,etc.The main studies are as follows:1.Using rhodamine as the luminophore and polysulfide as the Cu~+recognition group,the fluorescent probe Mito-OFCu was synthesized.The study on it in the solution showed that the probe was saturated with Cu~+in a p H buffer solution of 7.5.The maximum absorption peak and emission peak are 555nm and 590nm,the fluorescence quantum yield is 45%,and the Cu~+response multiple is 11 times.Studies in cells have shown that the probe has good biocompatibility,localizes to mitochondria,and the Pearson colocalization coefficient is as high as 0.96;it can sensitively detect the change of Cu~+content in mitochondria,and has good light stability in cells.Used for cell imaging for a long time.2.The study of mitochondrial Cu~+deficiency in cells showed that we introduced exogenous si RNA to inhibit the copper transporter SLC25A3 on the inner mitochondrial membrane of Hela cells,and combined with WB technology proved the effectiveness of the inhibition.Afterwards,we used the silicon-based rhodamine probe Mito-Si RCu to detect the decrease of Cu~+content in the mitochondria after transfection,indicating that we successfully constructed a mitochondrial copper-deficient cell model.Combined with WB again,it was proved that the lack of Cu~+also led to a decrease in the expression of COX subunits COX1 and COX2.In addition,cell respiration experiments proved that the COX activity of the mitochondrial copper-deficient cell model constructed by transfection was reduced.3.Using a laser confocal microscope and a cell respirator,the effects of Cu~+deficiency on zebrafish mitochondrial function and nervous system development were studied.The results showed that zebrafish with mutations in the copper transporter ATP7A allele showed phenotypic changes such as melanin loss,notochord curve,and pericardial effusion.The body Cu~+deficiency of mutant zebrafish was detected by the probe Mito-Si RCu.Breathing experiment found that zebrafish with gene mutations have reduced COX activity,impaired mitochondrial function,and weakened respiration.Imaging shows that zebrafish lacking Cu~+ions in the whole body will significantly affect the neurodevelopment of the brain and eyes.The number of neurons is reduced,the expression range becomes smaller,and degenerative changes occur.