Construction And Application Of Novel Detection Methods For Tumor Exosomes Based On Responsive DNA Nanostructures

Exosomes are bilayer vesicles containing specific proteins,nucleic acids and lipids.Studies have shown that exosomes produced by cancer cells can promote cell growth and increase tumor invasiveness and metastasis as a means of intercellular communication.In recent years,liquid biopsy as a non-invasive detection method has attracted wide attention.As potential biomarkers in liquid biopsy,exosomes have a strong clinical application prospect in the diagnosis and treatment of cancer.Since exosome surface proteins are not completely heterogeneous,leading to the same protein markers among exosomes from different sources,the accurate detection and analysis of exosomes remain a great challenge.In addition,analyzing the types and expression levels of exosome surface proteins and exploring the protein combinations of potential tumor markers will contribute to the early diagnosis and prognostic treatment of cancer.In this paper,two new biosensors for exosome detection were constructed based on DNA nanostructures,combined with isothermal nucleic acid amplification technology and CRISPR/Cas system.The contents of this paper are as follows:Part Ⅰ: A specific detection and analysis method without complicated separation and purification of exosomes was proposed.This method constructed DNA hydrogel by double roller ring amplification to capture exosomes in the supernatant of cells,thus minimizing the damage of exosomes and realizing the non-separation and purification of samples.The exosomes released later trigger CHA reaction through bi-specific recognition and binding,and form a large amount of ds DNA,which can be used as the target of CRISPR/Cas12 a,activate its unique trans-cutting activity,and cut fluorescent probes to achieve specific sensitive detection of the target.In addition,this strategy can realize the detection of exosomes from different sources only by changing the aptamer sequence,to obtain the protein map.This method has the advantages of free separation and purification,simple operation,and a short experimental period,and is expected to be a powerful tool for early disease diagnosis and accurate analysis of exosome subgroups.Part Ⅱ: Flow cytometry is a kind of instrument for multi-parameter statistical and quantitative analysis of single particles such as exosomes and cells,which is widely used in the basic research of rapid analysis of single cell surface proteins.However,the minimum detection size of the traditional flow cytometry was 200 nm,and the exosome particles were small,much lower than the detection size.Based on this,an RCA-mediated flow cytometry method was constructed to detect exosomes quickly and analyze surface proteins.In the presence of tumor-derived exosomes,two single chains with aptamer sequences bind to exosome proteins,and double recognition proximity mediates the generation of a ligation probe,which then triggers the rolling ring amplification reaction.The product is a G-rich sequence,which can form G four-strand bodies,which can be embedded with fluorescent groups to generate amplified fluorescence signals for fluorescence detection.Because RCA occurs on the surface of exosomes,the size of detected particles is increased to a certain extent,so that it can meet the requirements of flow detection,which provides a new idea for cancer diagnosis.Theoretically,different exosome surface proteins can be analyzed by changing the protein recognition sequence,so this method has the potential to achieve high throughput detection.