Construction And Application Of Biosensor Platform On Cell Surface

Synaptic transmission is an important physiological process in organism.Monitoring synaptic transmission in real time and clarifying its biological mechanism are of great benefit to the diagnosis and treatment of many neurological diseases.In the process of synaptic transmission,synaptic vesicles first are fused with presynaptic membrane,then release neurotransmitters rapidly,and then immediately return to a state that is not fused with presynaptic membrane to maintain sustained neuronal activity.In this process,the p H value of internal lumen of synaptic vesicles usually changes from p H 7.4 to p H 5.6 and then to p H 7.4.Therefore,the purpose of real-time monitoring synaptic transmission can be achieved by monitoring the changes of p H in the internal lumen of synaptic vesicles in the process of neural signal transmission.Based on this,a series of fluorescent probes have been developed to label synaptic vesicles,which are sensitive to environmental p H changes,including the ratio fluorescent protein phlurins and the amphiphilic styryl dyes on the market.However,these probes are still insufficient and are not ideal for the study of the dynamics of synaptic vesicles in the process of nerve signal transmission.Therefore,the development of selective labeling of synaptic vesicle fluorescent probes with the advantages of high reliability,good biocompatibility,good p H sensitivity and easy operation has become one of the most important research topics.And DNA nanotetrahedron has a promising prospect in biological research,especially in neuroscience because of its superior mechanical properties,relatively stable structure,excellent biocompatibility,easy synthesis and easy modification.Therefore,in this paper,we intend to use DNA nanotetrahedron as a tool to label synaptic vesicles.And in order to solve the problem of low surface efficiency of anchoring cells when DNA nanotetrahedron structures are applied in organism,we combine DNA nanotetrahedron with lipid conjugated nucleic acid(DNA-lipid)to develop a fluorescence ratio type(f _FAM/f _TAMRA)DNA nanotetrahedron probe(pHadtnps),which is sensitive to the p H change of environmental system.Then we apply this probe to label the synaptic vesicles inside the synaptic structure of neurons to achieve monitoring synaptic transmission in real time by observing the fluorescence of the biosensor platform.(1)In order to synthesize DNA nanotetrahedron,in the second chapter,we synthesized a series of DNA nanotetrahedra modified with different numbers of FAM,TAMRA and cholesterol molecules.Then we used nondenaturing polyacrylamide gel electrophoresis to verify that the DNA tetrahedron nanostructures were successfully synthesized.In addition,we have successfully synthesized DNA tetrahedrons modified with 0,1,2,3 cholesterol molecules.Then we measured the effect of p H change on the fluorescence properties of DNA nanotetrahedron in Tyrode buffer system with different p H,and found that four FAM fluorescence groups and one TAMRA fluorescence group were modified in pHadtnps.(2)In order to determine the modification of cholesterol molecules in pHadtnps and whether pHadtnps can be used to label synaptic vesicles in synaptosomes,we applied DNA tetrahedron modified with different amount of cholesterol to CCRF-CEM cell system and synaptosome system,and determined that the modification of pHadtnps was:one DNA tetrahedron modified with four FAM fluorophores,one TAMRA fluorophore and three cholesterol molecules.It was also found that phadtnps could be labeled on on synaptic membrane and synaptic vesicles in synaptic structure.It is also verified that we can simulate the conduction of neural signal by changing the concentration of K⁺in the environmental system and DNase I can reduce the background fluorescence signal.(3)In order to detect the effect of pHadtnps on labeling synaptic vesicles in the synaptic structure of actual neurons,in the fourth chapter,we applied pHadtnps to mouse neuron system and found that pHadtnps could label synaptic vesicles in the synaptic structure of neurons.At the same time,immunocytochemistry and standard dye FM4-64 colocation experiment were used to verify that pHadtnps can label synaptic vesicles in neuron system with high standard.Finally,we simulated the neural signal transduction by changing K⁺concentration in the microenvironment and found that pHadtnps can be applied to real-time monitoring of synaptic transmission,which is an important physiological process.At the same time,we found that the experimental results are basically consistent with the experimental results based on the standard fluorescent dye FM4-64 and the previous experimental results,which indicates that we have successfully developed a highly efficient,reliable and biocompatible method and tool for labeling synaptic vesicles.