| Exosomes are nano-scale vesicles(30-150 nm)with a double-layer membrane structure and actively secrete by cells,which can directly reflect the physiological and functional status of secreted cells and carry out material transport and information communication between cells.They not only participate in a variety of physiological and pathological processes,including the occurrence,development and metastasis of tumors,but also carry rich and potential biomarkers.They can provide valuable molecular pathological information and they are clinically valuable liquid biopsy specimen.Therefore,it is significant to visualize real-time monitoring exosomes secretion for basic and applied research on its mechanism,biological function,disease correlation,etc.At present,most analytical methods quantified exosomes which have been secreted by cells.They were impossible to obtain the specific cell source of different exosomes.This article focused on the dynamic process of exosomes secretion by cells.Based on the DNA tetrahedron,combined with cell membrane modification technology and fluorescence imaging technology,we designed and constructed a simple,easy-to-operate and high-stability DNA tetrahedron biosensor on cell membrane.It applied for real-time monitoring of exosomes secretion by different cell types.The main research content includes the following two aspects:1.Design and construction of cell membrane-anchored DNA tetrahedron sensorDNA tetrahedron nanostructure has the advantages of adjustable size,easy connection of biomolecules,high stability and low cytotoxicity.It has attracted great attention in the field of analytical sensing and biomedicine.In order to stably anchor the DNA tetrahedron onto the cell membrane,we employed a high-affinity cholesterol hydrophobic anchor to mediate the anchoring of the DNA tetrahedral sensor.We designed three anchoring strategies.The first strategy was to directly modify cholesterol on the three vertices of the DNA tetrahedron.The other two strategies were the use of cholesterol-modified single strands DNA to mediate DNA tetrahedral anchoring,of which the second was that the single strand was firstly bound to the DNA tetrahedron before anchoring.The third was the single-strand DNA anchored on cell membrane before binding to the DNA tetrahedron.The results showed that the sensors of the first two strategies would aggregate during the anchoring process which impacted the anchoring the DNA tetrahedron.The third anchoring strategy could make the DNA tetrahedral anchor on cell membrane successfully.The scaffold structure had good cell surface retention and high stability under cell culture conditions.2.Cell membrane-anchored DNA tetrahedron sensor for real-time monitoring of exosomes secretionAim at the exosomal membrane surface protein CD63,we integrated the CD63 aptamer sequence into the DNA tetrahedron scaffold to construct a DNA tetrahedron sensor.We could monitor the changes of fluorescence in the membrane surface by specifically capturing the exosomes secretion,and then analysed the secretion of exosomes on the cell membrane surface.Cholesterol-modified single-stranded DNA was anchored to the cell membrane by hydrophobic action and hybridized the three extended toes on the three vertices of the DNA tetrahedron,thereby anchoring the sensor to the cell membrane.A signal detection system was designed on the other vertex of the DNA tetrahedron,which was consisted of a fluorophore-modified CD63 aptamer sequence,a quenchor-modified quenchor probe,and a block probe.The sensor was activated by adding a trigger probe to replace the block probe.When the cells secreted exosomes,the CD63 aptamer specifically binds to the exosomes,thereby releasing the quenchor probe,and the fluorescent signal on the sensor was restored.The intensity of the fluorescent signal was positively correlated with the amount of exosomes.Therefore,we could analyze the secretion of exosomes by monitoring the changes of fluorescent signal on cell membrane. |
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