In Situ Detection Method Based On Extracellular Vesicles Of Tumor Cells

Extracellular vesicles are found in large numbers in various biological fluids and are important vehicles for maintaining cell-to-cell communication and transferring substances because they naturally carry many biomolecules(e.g.,proteins,nucleic acids,lipids,etc.)from the parent cell.Extracellular vesicles carrying multiple signaling molecules can bind to target cells through direct fusion of phospholipid membranes or specific binding of ligands-receptors,and then release the contents to the target cells to exert specific biological effects.Notably,extracellular vesicles secreted by tumor cells can assist in modifying the tumor and its microenvironment,mediating the process of cancer metastasis and recurrence.In this paper,two new methods of in situ fluorescence detection were constructed for tumor-derived extracellular vesicles and applied to the pre-invasive in vitro analysis and detection of tumor markers.The details of the study are as follows:1.Detection of extracellular vesicles of tumor origin based on aptamer recognition and hybridization chain reaction amplification signal: A new method for extracellular vesicle detection integrating capture and detection is proposed.The aptamer is modified on polystyrene microspheres for the purpose of identifying and capturing extracellular vesicles.The MUC1 membrane protein receptor and CD63 membrane protein receptor were selected as recognition sites due to their high expression on MCF-7 cells.The capture of extracellular vesicles by polystyrene microspheres increases their nanoparticle weight and thus promotes enrichment,an aspect that also increases the sensitivity of the assay.In addition,a hybridization chain reaction was introduced in this assay system to achieve fluorescence detection.2.In situ detection of miRNA-21 in MCF-7 cell-derived extracellular vesicles using erythrocyte membrane vesicle strategy: The traditional method for detecting miRNA-21 in extracellular vesicles mainly involves disrupting the membrane structure of extracellular vesicles and releasing their internal biomolecules before detecting miRNA-21,which cannot satisfy the detection in the restricted space.Therefore,an innovative strategy of membrane fusion using erythrocyte membrane vesicles with extracellular vesicles is proposed to deliver fluorescent detection probes into the extracellular vesicles without destroying the membrane vesicle structure,and to achieve in situ fluorescence detection of miRNA-21 in the restricted space.This nanoconfined space detection strategy greatly improves the collision probability between the probe and the detection target.Therefore,the improved efficiency of DNA self-assembly reaction also improves the sensitivity of miRNA-21 in situ fluorescence detection.In short,this new detection method designed in this work can be used for in situ fluorescence detection of miRNA-21 within extracellular vesicles using erythrocyte membrane vesicles in a confined space.In conclusion,the goal of this work was to perform a highly sensitive quantitative detection of extracellular vesicles of tumor origin or biomolecules within them.On the one hand,the specific capture of MCF-7 cell-derived extracellular vesicles by an aptamer-modified polystyrene microsphere and the amplification of the fluorescence signal in combination with a hybridization chain reaction were achieved.On the other hand,in situ detection of miRNA-21,a nucleic acid molecule carried inside extracellular vesicles,within the restricted space was further achieved.