Electrochemical Detection Of Tumor Exosomes And Exosomal MicroRNA Based On HCR Signal Amplification

Exosome is a lipid bilayer vesicle body with a diameter of about 30-150 nm,which can communicate between cells and can transport some functional information molecules to adjacent or distant cells.In addition,there are many different proteins on the membrane of exosomes,and these proteins have sites that can specifically bind to antibodies or nucleic acid aptamers,which plays an important role in the detection of exosome.Micro RNA(mi RNA)plays a key role in post-transcriptional regulation of gene expression in many biological processes(such as cell differentiation and apoptosis).Compared with free mi RNAs in biological fluids,mi RNAs in exosomes are very stable.Abnormal expression of mi RNA can be found in exosomes secreted by different types of tumor cells.Therefore,in the process of early tumor diagnosis and prognosis,there is an urgent need to develop an efficient and sensitive method for detecting exosome mi RNA.In recent years,the application of hybrid chain reaction(HCR)in biosensing has undergone tremendous development.With the advantages of enzyme-free,efficient isothermal amplification and high sensitivity,the hybrid chain reaction has become a powerful molecular tool.Electrochemical nucleic acid sensors based on hybrid strand reactions as signal amplifiers have attracted widespread attention in the scientific community.In this paper,based on the HCR,two electrochemical sensors were prepared for sensitive detection of exosomes and exosome mi RNA,respectively.1.Electrochemical detection of tumor exosomes based on HCR signal amplification.Taking the exosomes derived from CCRF-CEM tumor cells as a model,the DNA chain structure changes caused by the specific binding of DNA containing the hairpin structure of PTK7 aptamers to membrane proteins,and endoncleaes was added to assists the circulation.Finally,the use of HCR signal amplification to achieve high-sensitivity electrochemical detection of CCRF-CEM exosomes.The detection linear range of CCRF-CEM exosomes is 4×10~3-1.6×10~7 particles/?L,and the detection limit reaches 500 exosomes per microliter.2.Electrochemical detection of tumor exosomes mi RNA based on HCR signal amplification.In this study,by simultaneously fixing the probe DNA and the short12-nucleotide single-stranded DNA(S-12),the precise control of the probe DNA density on the electrode surface was achieved and a higher HCR efficiency was achieved.More importantly,before introducing the target mi RNA-122,the application of exonuclease I(Exo I)to reduce or completely eliminate false positive signals.Under optimal conditions,the detection limit of this method for mi RNA-122reached the level of a M,with a linear range of nine orders of magnitude and the specificity of single mismatch recognition.This highly sensitive electrochemical test can successfully assess the concentration of mi RNA-122 in exosomes derived from different tumor cells,indicating its potential use in cancer diagnosis.