Research On Exosomes Liquid Biopsy Technology Based On Antibody Microbeads Filled Microfluidic Chip

Liquid biopsy is a technology for disease diagnosis and monitoring through the sampling and analysis of bodily fluids,providing advantages such as convenient detection,multiple detectability,safety,non-invasiveness,and early detection.Exosomes,a subtype of extracellular vesicles(EVs)secreted by most cells,are characterized by a lipid bilayer membrane and can serve as stable carriers of genetic material and proteins from their source cells,thereby reflecting the occurrence and development of cancer.And exosomes exist in large amounts in blood and other body fluids,it is an ideal detection analyte for liquid biopsy.Exosome-based liquid biopsy technology has important application value and potential in cancer diagnosis,prognosis,and treatment evaluation.However,the current exosome-based liquid biopsy method has limitations in terms of exosomes capture and enrichment,and detection,such as complicated operation,long time-consuming,large sample volume,and the influence of chemical reagents on the analysis results.Fully meet the needs of clinical rapid detection and point of care test(POCT)applications.Therefore,there is an urgent need to develop a rapid,sample-saving,simple-to-operate,and integrated exosomes liquid biopsy method.In this study,we developed a continuous,rapid,and efficient microfluidic exosomes liquid biopsy method based on transparent antibody-conjugated microbeads.The optical transparent antibody-conjugated microbeads in the microfluidic chip captured and enriched exosomes,disrupting the laminar flow to increase the mass transfer efficiency,the specific surface area of the reaction,and the fluorescent signal for improving the detection sensitivity.Under the optimized detection conditions,the microfluidic detection method successfully realized the rapid quantitative detection of pure exosomes,exosomes in the cell culture supernatant,and tumor-associated exosomes in plasma.Specifically,it mainly includes the following parts:Firstly,a microfluidic chip based on transparent antibody-conjugated microbeads was designed and fabricated using pressure-sensitive adhesives(PSA).The detection chamber was filled with glass microbeads functionalized with surface-modified monoclonal antibodies for the enrichment and detection of exosomes.Second,the biological functions of the microfluidic chip were characterized and verified.Pure exosomes and cell culture supernatants extracted by commercial kits were used as experimental samples.Compared with the control group without exosomes,more obvious fluorescence was detected in the experimental group,indicating that the method of this study can be effectively used for the detection of exosomes.Then,we optimized the optical parameters of the detection method and the conditions of the immune binding reaction through systematic experiments to achieve the best detection effect.The evaluation results of various biological parameters of the microfluidic detection method demonstrate its excellent reproducibility,stability,and specificity.Finally,the microfluidic chip was employed to detect the total amount of exosomes,EpCAM-positive exosomes,and MUC1-positive exosomes in the plasma samples of breast cancer patients and healthy volunteers.The results revealed that the blood samples of cancer patients and healthy volunteers can be accurately distinguished by detecting these exosomes,illustrating the potential of this method in the application of breast cancer liquid biopsy.The receiver operating characteristic(ROC)curve was used to evaluate the diagnostic ability of biomarkers,and the results showed that the combined evaluation of multiple biomarkers had extremely high sensitivity and specificity.The study of this method and the development of the system have provided a novel avenue for liquid biopsy technology based on circulating exosomes and their biomarkers and provided a fresh approach to the early detection and diagnosis of diseases based on exosomes biomarkers.