A Small Extracellular Vesicle-associated MicroRNAs Electrochemical Biosensor Based On Localized DNA Tetrahedron-assisted Catalytic Hairpin Assembly

ObjectiveThe profling of small extracellular vesicle-associated microRNAs(sEV-miRNAs)plays a vital role in cancer diagnosis and monitoring.However,detecting sEV-miRNAs with low expression in clinical samples remains challenging.Herein,we propose a novel electrochemical biosensor using localized DNA tetrahedron-assisted catalytic hairpin assembly(LDT-CHA)for sEV-miRNA determination.The LDT-CHA contained Localized DNA tetrahedrons with CHA substrates,leveraging an efficient localized reaction to enable sensitive and rapid sEV-miRNA measurement.Methods1.Successful assembly of the LDT-CHA signal amplification strategy was verified by polyacrylamide gel electrophoresis(PAGE),atomic force microscopy,dynamic light scattering(DLS)and zeta potential;2.The feasibility of LDT-CHA to generate amplified signal was verified by the detection of electrochemical signal and the detection of fluorescence signal;3.The interfacial assembly of the electrochemical sensor was characterized by electrochemical impedance spectroscopy(EIS)and square wave voltammetry(SWV);4.The biosensor was optimized according to the influencing factors such as the reaction temperature and the reaction time.To verify its performance in signal amplification,such as sensitivity,specificity,etc.5.The constructed LDT-CHA electrochemical biosensor was used to detect plasma-derived sEV-miRNA,and compared it with qRT-PCR results;6.Furthermore,this method detect tumor-derived sEV-miRNAs in plasma samples to establish disease diagnosis models.Results1.We designed a novel electrochemical platform based on Localized DNA tetrahedron-assisted catalytic hairpin assembly(LDT-CHA)for sensitive sEV-miRNA detection.Compared with other metastable hairpin-driven amplifcation strategies,our methods have multiple advantages.Firstly,DNA tetrahedron-assisted catalytic hairpin assembly(DT-CHA)could increase the local concentrations of CHA substrates by confung hairpins in a compact space,which dramatically improve the amplifcation efficiency.Secondly,the LDT-CHA contained a number of DT-CHAs as successive reactants in a confned space,which could minimize leakage of CHA by the precise-control space of each DT-CHA.Thirdly,The LDT could act as programmed track,once the driving motor is triggered by a target sEV-miRNA,the sEV-miRNA would move along the LDT-CHA to produce cascaded signal output.2.Based on the LDT-CHA,the proposed platform can quantitatively detect sEV-miRNA down to 25 aM in 30 min with outstanding specificity.3.For accurate diagnosis of gastric cancer patients,a combination of LDT-CHA and a panel of four sEV-miRNAs(sEV-miR-1246,sEV-miR-21,sEV-miR-183-5P,and sEV-miR-142-5P)was employed in a gastric cancer cohort.Compared with diagnosis with single sEV-miRNA,the proposed platform demonstrated a higher accuracy of 88.3%for early gastric tumor diagnoses with higher efciency(AUC:0.883)and great potential for treatment monitoring.Conclusions1.The electrochemical platform based on LDT-CHA enables rapid,sensitive and specific detection of sEV-miRNA with a linear range of 100 aM-100 pM,detection limit as low as 25 aM and detection time of 30 min.2.The sEV-miRNA detection results of this platform show good agreement with the qRT-PCR results and are expected to be used clinically for the detection of sEV-miRNA.3.For accurate diagnosis of gastric cancer patients,a combination of LDT-CHA and a panel of four sEV-miRNAs(sEV-miR-1246,sEV-miR-21,sEV-miR-183-5P,and sEV-miR-142-5P)was employed in a gastric cancer cohort.Compared with diagnosis with single sEV-miRNA,the proposed platform demonstrated a higher accuracy for early gastric tumor diagnoses and great potential for treatment monitoring.Thus,this study provides a promising method for the bioanalysis and determination of the clinical applications of LDT-CHA.