The Study On The New Analytical Methods For Isolation And Detection Of Exosomes Based On Aptamers

Exosomes are nanometer vesicles secreted by most cells.They are between 30 and 150 nm in diameter and carry information such as functional nucleic acids and proteins from the cells they originate.They were originally considered to be a kind of excretion waste.In recent years,more and more studies have shown that exosome is a carrier of intercellular communication,which is related to many physiological and pathological functions of cells.In addition,exosomes are involved in the formation of tumor microenvironment.Therefore,exosomes play a pivotal role in the diagnosis,treatment and prognosis of diseases.At present,there is no standardized and unified technology for exosomes separation and detection.These methods all have different disadvantages in some respects.Therefore,in order to solve the problems existing in the process of exosomes separation and detection,this paper has developed two kinds of methods for exosomes detection.One of the method was to design a dual signal amplification method for exosome detection with high-sensitivity in serum samples,and another innovative method applying the rolling circle amplification technique to the separation of exosomes realized the integration of exosome separation,detection and protein analysis,which provided a new view for exosomes research.The main research contents are as follows:1.Detection of HepG2-derived exosomes with high sensitivity using dual signal amplification techniques.In this work,we took advantage of the facile adaptability of aptamers to design an exosome quantitative method,which converted exosome capture event to nucleic acids detection.With the help of hairpin DNA cascade reaction(HDCR)and easy accessibility of DNA dendrimer self-assembly,dual signal amplification was achieved.CD63 aptamer linked via a DNA probe to magnetic beads acted as the capture component.In the presence of target exosomes,aptamers identified and combined with exosomes,releasing the DNA probe as a trigger to initiate HDCR(the first signal amplification process)by opening hairpin DNA(HP 1)bound to gold nanoparticles(AuNPs).Fluorescently-labeled DNA dendrimers concatenated with HP1 as the second signal amplification process to increase signal-to-noise ratio.Under optimal conditions,our method achieved good linear response for HepG2 cell-derived exosomes in a concentration range from 1.75×103 to 7.0×106 particles/?L with a detection limit of 1.16×103 particles/?L.It also showed a good performance for detection of exosomes in biological sample.2.Construction a DNA-AuNP-Exo network to achieve the integration of exosome separation,detection and protein analysis with low centrifugal speed.Using a rolling circle amplification reaction(RCA)generated a long-chain DNA hairpin structure comprising a plurality of functional domains,such as multiple CD63 aptamer regions,junction regions,and spacer regions and the base pairs was complement each other to form a hairpin structure.When the CD63 aptamer bound to the exosome,the hairpin structure changed its conformation,exposing the junction regions(AuNPs binding sequence).Then the probe on the surface of AuNPs bound to the long-chain DNA by toehold-mediated strand displacement reaction,releasing the fluorescent labeled complementary probe which generated a detection signal and simultaneously formed a DNA-AuNP-Exo network.Thus,exosomes can be enriched by low-speed centrifugation.The formation of the DNA-AuNP-Exo network structure was confirmed by transmission electron microscopy and confocal fluorescence microscopy.In addition,we established a standard curve of the fluorescence ratio and the logarithm of exosome concentration,and performed protein profile analysis of the isolated exosomes via LC/MS,which showed that the amount of protein obtained by these two methods was very close.