Highly Sensitive Analysis Of MicroRNAs And Circulating Exosomes

MicroRNAs(miRNAs)are a class of small non-coding RNAs,which can regulate the gene expression by binding with target mRNA to inhibit protein translation or induce mRNA degradation,and play vital roles in various biological processes.Nowadays,many studies have shown that the expression levels of miRNA in cells are closely related to many human diseases,especially cancers.So the miRNA can be used as the biomarkers for cancer diagnosis and prognosis analysis.Generally,a specific cancer is typically associated with a simultaneous change in the expression level of several miRNA species.Simultaneous detection of multiple miRNAs can greatly improve the accuracy of cancer diagnosis.Therefore,the development of highly sensitive method for multiplexed detection of miRNAs is of great significance for informative disease diagnostics and treatment of cancers as well as for the research of the mechanism of tumorigenesis.Exosomes are a class of membrane-enclosed biological nanoparticles with a diameter of 30?200 nm.Various types of cells can release exosomes into the extracellular environment under normal or pathological conditions,and exosomes are present in various body fluids.Exosomes carry lots of key molecular information from parental cells to be good carriers in intercellular communication and play important roles in the occurrence,development,invasion and metastasis of tumors.Accumulating evidences have shown that the circulating exosomes can be used as important biomarkers for early diagnosis and prognosis of diseases,so the development of convenient,sensitive and reliable methods for the analysis of circulating exosomes is urgently needed.In this paper,a sensitive method for multiplexed detection of target miRNAs was established by encoding probe extension in improved cDNA library.And highly sensitive methods for the analysis of circulating exosomes were established by combining single microbead(MB)based separation technology with in situ fluorescence imaging.(1)By adding an enzymatic digestion step to digest the excessive adaptor,avoiding the generation of adaptor dimer,we firstly improved the method to construct cDNA library of miRNAs,which greatly improved the cDNA yield.The cDNAs in the library were used as the templates for single base extension(SBE)reaction.By specifically designing the SBE-DNA probes with different lengths to hybridize with the cDNAs at key positions and extend different fluorophore-labelled ddNTPs during SBE,different miRNA could produce different SBE products with unique code,which can be separated and detected by capillary electrophoresis.Thus,a sensitive method was developed for multiple miRNA detection.With double encoding technology,the method has greatly enhanced the high-throughput capacity of simultaneous detecting multiple miRNA targets,and 17 types of miRNAs could be simultaneously detected.Meanwhile,by specifically designing of SBE DNA probes,the method achieved the high specificity for miRNA detection at single-base resolution.More importantly,the construction of cDNA library for all miRNAs in sample provided powerful capability for multiplexed detection of wanted miRNAs.(2)A single MB was used as the reaction carrier,and firstly modified with the DNA aptamers for CD63 protein of exosomes to specifically recognize and enrich the exosomes from sample.Owing to the exosomal membrane structure,the fluorescent DNA probes labeled with cholesterol spontaneously insert into the exosomal membrane,so that the exosomes on the MB were labeled with fluorescent molecules.By using the laser confocal microscope for in situ fluorescent imaging of the single MB,The fluorescence intensity on the single MB was directly detected.Therefore,a simple and sensitive method for detection of exosomes was developed.This detection system realized sensitive exosome detection without the requirement of other signal amplification routes,and as low as 4.9×104 particles/?L of exosomes could be simply detected.Additionally,the method has been successfully applied for direct detection of the exosomes from small amounts of the clinical plasma samples.(3)Tumor-derived exosomes carry lots of tumor-specific proteins,which can be used as an ideal biomarker for early diagnosis and prognosis analysis of cancer.Herein,we developed an ultrasensitive method for detection of tumor-specific exosomal proteins with a single MB based aptasensor.Firstly,a single MB was used as the reaction carrier and modified with antibodies against generic membrane proteins of exosomes(anti-CD63 and anti-CD81)to effectively enrich total bulk exosomes in sample.Then,the DNA aptamers immobilized on gold nanoparticles(AuNPs)were employed to specifically bind to the tumor-specific exosomal proteins,so that,each target protein could capture many aptamers.Subsequently,each aptamer can be catalyzed by terminal deoxynucleotidyl transferase(TdT)and generated a long poly(dT)tail for hybridization with a lot of fluorescein-labeled poly(dA)probes.As a result,lots of fluorescent molecules were enriched on the surface of the MB for high efficiency signal amplification.With this strategy,tumor-specific exosomal protein can be sensitively detected from the sample containing 55 particles/?L of tumor-derived exosomes.Furthermore,this proposed method has been successfully applied for direct detection of tumor-specific exosomal protein markers in plasma microsamples.