Preliminary Investigation Of Exosomes As Liquid Biopsy Biomarker For Tumor Diagnosis

Exosomes are extracellular vesicles with a diameter of 30～150 nm secreted by cells,which are widely distributed in various body fluids and carry biological information such as proteins,lipids and nucleic acids of their parent cells.They can stably exist in blood circulation and become a new biomarker of liquid biopsy.Tumor exosomes are closely related to the occurrence and development of tumors and the detection of tumor exosomal characteristic protein is more reliable than soluble protein in serum.However,conventional methods for the analysis of exosome protein generally have problems of complex detection process and low sensitivity.Therefore,development of novel methods for the analysis of tumor exosomal protein is of great significance for its use in tumor diagnosis.Based on the understanding of tumor characteristic proteins on the surface of exosomes,this dissertation used surface plasmon resonance technology and the property of photonic crystal signal amplification to develop two analytical methods for the specific detection of tumor exosomes,providing a new strategy for the non-invasive application of exosomes in clinical early cancer diagnosis.Main contents and results are as follows:1.Biochip based on surface plasmon resonance for detecting PD-L1⁺exosomesThe expression of PD-L1 protein on the surface of tumor exosomes is significantly correlated with tumor immunotherapy response.To solve the problem that it is difficult to detecting the glycosylated PD-L1 protein on the surface of exosomes,a biochip based on SPR for analyzing exosomal PD-L1⁺(termed SPR-Exo_PD-L1)is designed,which utilizes the property of nucleic acid aptamer,such as small molecular weight,chemosynthesis and easy modification.Specifically,PD-L1 aptamer was firstly conjugated to the surface of biochip.Compared with the antibody,the aptamer is smaller in size,and it is easier to recognize and detect glycosylated PD-L1 protein.The aptamer specifically recognizes the PD-L1 membrane protein of exosomes and PD-L1⁺exosomes is captured,increasing the surface quality of the SPR chip,then causing the change of refractive index.The generated SPR signal is positively correlated with the concentration of PD-L1⁺exosomes,thus realizing sensitive detection of PD-L1⁺exosomes.There is a strong affinity between PD-L1 aptamer and exosomes,with a constant of 7.98 n M.Biochip based on SPR had a wide detection range,which can detect exosomes in the range of 0.63～7.50 n M,and limit of detection was 44.50 p M.This method could distinguish PD-L1⁺exosomes from PD-L1^-exosomes.Compared with commercial enzyme-linked immunosorbent assay kits,this method could better distinguish exosomes derived from melanoma cells with different PD-L1 expression levels.This method may provide a new strategy for clinical evaluation of cancer immune efficacy.2.Signal amplifying diagnostic chip based on biomimetic periodic nanostructures for detecting cancer exosomesIn view of the relatively small number of tumor exosomes in the early stage of cancer,a diagnostic biochip based on photonic crystals（DBPCs）is developed,realizing the ultra-sensitive and specific detection of GPC1⁺tumor exosomes.Specifically,GPC1 antibody labeled with quantum dots is used to recognize GPC1protein on the surface of exosomes,and then the ultrasensitive detection of tumor exosomes is realized using the signal amplification of photonic crystals.The prepared photonic crystal chip is able to specifically reflect the fluorescence with emission wavelength around 525 nm,and have the abality of signal amplification.Under the optimized parameters（dilution ratio of polystyrene spheres,binding ratio of quantum dots to antibodies,reaction temperature and time,etc.）,DBPCs have a good dynamic detection range（1.00×10⁷～1.00×10⁹ particles/m L）,and the limit of detection is as low as 4.00×10⁶ particles/m L.DBPCs could not only distinguish exosomes of pancreatic cancer cell line Panc-1 from normal cell line MCF10A,but also distinguish exosomes of pancreatic cancer cell line Panc-1 from melanoma cell line B16F10.In addition,the method could also detect exosomes in complex samples and successfully distinguish mimetic serum of healthy controls and pancreatic cancer patients.DBPCS allows the quantitative analysis of other disease-specific surface proteins on exosomes by changing biomarkers,providing a new idea of the realization of exosomes as liquid biopsy biomarkers for clinical tumor diagnosis.