Selection Of Aptamers Against Extracellular Vesicles And Its Application In Detection Of Lung Cancer

Lung cancer is one of the most common cancers in the world.Early diagnosis of lung cancer usually shows promising rate of survival.The failures of detection at early stages will result in high death rate.Therefore,new approaches for effective early detection of lung cancer are priority to develop.Imageological diagnosis,tracheobronchoscopy,mediastinoscopy and thoracoscopy,puncture and exhalation detection were used as clinical diagnosis.However,these methods have some disadvantages.Imaging examination with low sensitivity is difficult to detect small pulmonary nodules.The patients will suffer from some damage by these invasive diagnoses,such as tracheobronchoscopy,mediastinoscopy and thoracoscopy,puncture.So the methods seem not suitable for repeated examination and diagnosis.Some volatile organic compounds in exhaled air lack the normal reference value for comparison and the stability of this method is poor.Liquid biopsy can be used as an auxiliary method for early diagnosis and prognosis of cancers owing to its minimally invasive performance.Tumor biomarkers available for liquid biopsy are circulating tumor cells(CTCs),circulating tumor DNA(ctDNA),microRNA(miRNA),extracellular vesicles(EVs)and so on.CTCs are living cells shed from tumors,with low detecting rate,less number and has certain tissue heterogeneity.It is only suitable for patients with tumor metastasis.ctDNA is a genetic material from dead cells,with low content,low abundance of genetic mutations,easy to be masked by healthy DNA,low detecting sensitivity,and needs to be used together with other methods.EVs released from cells are more abundant,easy to be enriched,highly stable,and can be stored for a long time.EVs have lipid bilayer membranes and usually contain the proteins,lipids,RNA,and DNA,reflecting the information of the parent cell.These performances make EVs biomarkers ideal for clinical diagnosis.The proteins on the surface of EVs can also be used as tumor markers.Looking for the tumor markers for early diagnosis,treatment guidance,efficacy evaluation and prognosis is the key to solve the problems of the clinical diagnosis and treatment.Using this tumor marker to establish high sensitive and specific detecting method is also very important.Recently,aptamers have been widely used for sensitive detection of proteins due to their unique characteristics.Therefore,in this paper two methods were established for screening aptamers of surface proteins on EVs(EVs-SELEX)based on the traditional“Systematic Evolution of Ligands by Exponential Enrichment,SELEX”technology and the aptamers were successfully acquired.Two highly sensitive and specific methods for detecting EVs in lung cancer were established based on the aptamers.This study laid a foundation for the clinical application of EVs as a target of liquid biopsy.The research contents of this paper are as follows.(1)Selection and identification of EVs-specific DNA aptamers based on immunomagnetic separation.We established the method of EVs-SELEX based on traditional SELEX method to select specific aptamers against A549-EVs.EVs were extracted from the supernatant of cells by Total exosome isolation kit(TEIkit)successfully,and the EVs were characterized by TEM,NTA,immunogold labeling and Western-blot.The extracted EVs were round or oval membranous vesicles with a diameter range of 70 nm-250 nm.They were bound to immunogold particles and expressed CD63 protein and CD9 proteins but not Calnexin protein.Aptamers were selected using EVs-SELEX based on immunomagnetic beads and specific aptamers(Ap3)were obtained.The aptamer was analysed and characterized by flow cytometry.The results showed that the Ap3 could recognize A549-EVs specifically.The value of Kd is 2.59±2.77 nM.Ap3 could be combined with A549-EVs at different temperatures:4℃,25℃and 37℃.The Ap3 would be used as an ideal detecting probe for recognizing of A549-EVs.(2)Selection and identification of EVs-specific DNA aptamers based on Size-Selective Method(SSM).EVs were extracted from the supernatant of cells by SSM successfully,and the EVs were characterized by transmission TEM,NTA,immunogold labeling and Western-blot.The extracted EVs were round or oval membranous vesicles with a diameter range of 30 nm-150 nm.They were bound to immunogold particles and expressed CD63 protein and CD9 proteins but not Calnexin protein.SSM was used as the separating method to select aptamer of A549-EVs by EVs-SELEX.Specific aptamers(Ap6)were obtained successfully.Ap6 was analysed and characterized by flow cytometry.The results showed that the Ap6 could recognize A549-EVs specifically.The value of Kd is 2.93±1.29nM.Ap6 could be combined with A549-EVs at different temperatures:4℃,25℃and 37℃.The Ap6 would be used as an ideal detecting probe for following experiments.(3)Detecting method and clinical application of EVs secreted from lung cancer by padlock probe-based exponential rolling circle amplification(P-ERCA).In order to detect EVs sensitively,a new highly specific and sensitive fluorescence scheme based on P-ERCA assay for detecting EVs was reported.Ap6 was linked to a primer sequence(Ap6-primer complex)that was complementary to padlock probe.The padlock probe was composed of a nicking site for nicking endonuclease.At the presence of target EVs,Ap6-primer complex could trigger linear rolling circle amplification(LRCA)under isothermal conditions to enhance the fluorescence signal.The method exhibits high specific and sensitivity to lung cancer EVs with low detection limit of 4.222×10~4 particles/mL,the linear range is 5×10~4 particles/mL-1.2×10~6 particles/mL.Furthermore,this method could successfully distinguish the signal of EVs in real human serum sample between 17 lung cancer patients and 17 healthy persons(P<0.001).(4)Detecting method and clinical application of EVs secreted from lung cancer by hybridization chain reaction(HCR).In order to solve the problems of the former detecting method,such as narrow detection range and several enzymes were used,we established a fluorescence aptasensor based on HCR for detecting EVs of lung cancer.We first designed an Ap6-initiator probe which can specifically bind to the target EVs.Secondly,two short sequences,H1 and H2 were synthesized.H1 and H2 could coexist stably at the absence of target EVs.When the target EVs were present,a hybrid chain reaction would be initiated to self-assemble and produce the double-stranded DNA,generating amplified fluorescence signals,and realizing the highly sensitive and selective detection of the target EVs.The results showed that the detection range of the fluorescence sensor was 8×10~6 particles/mL-8×10~9 particles/mL and the detection limit was 7.79×10~6 particles/mL.The fluorescence signal of the human serums of patients with lung cancer had statistically distinguishment between lung cancer patients and healthy persons(P<0.0001).