Construction Of An Exosomal MicroRNA Electrochemical Biosensor Based On Multifunctional DNA Tetrahedron-assisted Catalytic Hairpin Assembly Technology

OBJECTIVE:Exosomal microRNA(exo-miRNA)holds great potential as biomarker of tumor diagnosis.However,developing a rapid and accurate method for exo-miRNA detection in clinical application is challengeable.Herein,this work proposes a novel signal amplification strategy that uses multifunctional DNA tetrahedrons assisted catalytic hairpin assembly(MDTs-CHA)to construct a rapid,sensitive and efficient electrochemical biosensor for exo-miRNA detection.Methods:The feasibility of the MDTs-CHA signal amplification strategy was verified by polyacrylamide gel electrophoresis(PAGE),fluorescence spectroscopy and differential pulse voltammetry(DPV).Electrochemical impedance spectroscopy(EIS),square wave voltammetry(SWV)and cyclic voltammetry(CV)were used to characterize the interface construction of the electrochemical biosensor.The signal amplification performance,sensitivity and selectivity of this strategy were demonstrated via DPV signal.The constructed MDTs-CHA electrochemical biosensor was used to detect plasma-derived exo-miRNA,and the results were evaluated according to consistency analysis with qRT-PCR results.Furthermore,this method detect tumor-derived exo-miRNA in plasma samples to establish disease diagnosis models.Results:1.MDTs-CHA is composed of two DNA tetrahedrons(T1,T2).Each tetrahedron can not only be used as carriers of DNA hairpin structures,but also used as signal labels by loading RuHex molecules.2.The DNA tetrahedron are synthesized via "single-step" of high temperature denaturation,and DNA hairpins attaches to DNA tetrahedron through complementary sequence at room temperature.3.In the presence of the target exo-miRNA,the two-layer MDTs-CHA reaction was initiated rapidly to obtain large numbers of RuHex-labeled DNA tetrahedrons,resulting in significant electrochemical signal.Therefore,the MDTs-CHA strategy ensure high sensitivity of electrochemical biosensor with a ultralow detection limit of 7.2 aM and linear range from 10 aM to 100 pM.4.The miRNA detection results from MDTs-CHA strategy and qRT-PCR are consistent.5.This platform showed high effeciency(AUC:0.931)and high sensitivity of 89.9%for breast tumors diagnosis.Conclusions:1.The electrochemical platform based on MDTs-CHA can quantify exo-miRNA down to 7.2 aM with linear range from 10 aM to 100 pM.Meanwhile,the established biosensor displays excellent sensitivity and selectivity.2.The miRNA detection results of MDTs-CHA electrochemical biosensor have much consistency with qRT-PCR,and it is desirable to be used in clinical detection of exo-miRNA.Therefore,electrochemical biosensor based on the MDTs-CHA strategy will be a powerful tool for exo-miRNA clinical detection.