Study Of Assay Methods For Exosomes Based On Porous Anodic Aluminum Film

Tumor is one of the major diseases that threaten human survival and health.Early detection and treatment are essential for cancer patients to improve patient survival rate and prognosis.Therefore,early diagnosis and treatment of cancer are becoming increasingly crucial in clinic.Exosomes are natural nano-scale extracellular vesicles.They are widely distributed in body fluids and tissues,and contain a variety of genetic information of the parental cells.Exosomes can participate in inter-cell information exchange and substance exchange,which play a critical role in the physiological and pathological processes of cells.For tumors,especially for malignant tumors,exosomes participate in the composition and regulation of the local microenvironment,affecting the growth and development of the tumors.Therefore,exosomes are expected to be a new type of biomarker for early diagnosis of cancer.Analysis of exosomes in clinic,however,remains a difficult task,primarily due to the lack of sensitive,specific,rapid and convenient detection methods.The anodic aluminum film has the advantages of adjustable nanopore diameter,favorable nanochannel array and feasible functionalization on the surface of nanopores,and can be used to construct biosensors with high sensitivity and specificity for the analysis of multiple targets.In view of this,this thesis utilizes anodic aluminum film as a carrier to fabricate detection devices of exosomes.The surface of the barrier layer of porous anodic aluminum film is modified to achieve the capture of exosomes,and use the subsequent amplification reaction to achieve signal amplification so as to improve the sensitivity of detection.The 2 specific contents are as follows:1.Dual Aptamer-Modified Porous Anodic Aluminum Film for Highly Specific Capture and Sensitive Detection of ExosomesBecause exosomes carry various genetic information of their parental cells,they are expected to become a new non-invasive biomarker for early diagnosis of cancer.However,how to accurately detect exosomes is a problem that needs to be solved urgently.In this part,we propose a dual aptamer-modified porous anodic aluminum film for rapid and accurate analysis of exosomes.Two aptamers(Apt-CD63 and AptEp CAM)were simultaneously immobilized on the surface of the barrier layer of porous anodic alumina film to combine with proteins overexpressed on the exosomes membrane.As such,exosomes could be specifically captured by the dual aptamers.The captured exosomes effectively covered the surface of the barrier layer,leading to remarkable reduction in ion current.Based on this principle,the captured exosomes could be sensitively and accurately detected with electrochemical workstations.The study found that the two aptamers greatly increased the capture efficiency of exosomes compared to that using a single aptamer.The results show that when the exosomes concentration is in the range of 1.33×104 ~ 1.33×108 particles/m L,the currents and the logarithm of the exosomes concentration has a good linear correlation with the potential at-1.2 V,and the lowest detection limit can reach 4.43×103 particles/m L,achieving simple,fast and sensitive detection of exosomes.2.Ultrasensitive Detection of Exosomes based on in situ Amplification of Aptamers on Porous Anodic Aluminum FilmMore and more evidences indicate that tumor-derived exosomes can enhance tumor migration and invasion through the modulation of tumor microenvironment.However,due to limitations of detection technology,how to detect exosomes very sensitively is still a pressing issue.In this work of the thesis,we propose a new method that can sensitively detect exosomes.This method is based on in situ amplification of terminal deoxynucleotidyl transferase(Td T)on the surface of PAA films to achieve the purpose of sensitive detection of exosomes.Specifically,Apt-CD63 containing aldehyde groups was modified on the surface of the barrier layer of PAA film.When exosomes are present,Apt-CD63 can selectively bind to the transmembrane receptor protein CD63,which is highly expressed on the exosomes membrane,and this specific recognition can selectively capture exosomes.At this time,the second specific aptamer Apt-Ep CAM was added,then the Apt-Ep CAM combined with exosomes,followed by the subsequent Td T polymerization reaction for signal amplification.In this way,highly sensitive and specific detection of exosomes can be achieved.The experimental results show that the exosomes can be quantitatively analyzed in the concentration range of 2.06×103 ~ 2.06×108 particles/m L,and the detection limit is down to 2.57×102 particles/m L,it is expected to be used for early clinical diagnosis of tumors.