Detection Of MicroRNA-21 Based On Electrochemical DNA Biosensor

MicroRNAs?miRNAs?are a class of endogenous single-stranded small RNAs of non-encoding and about 18-25 nucleotides in length.They regulate the expression of protein-coding genes and participate in various physiological processes,such as cell proliferation,differentiation,and apoptosis,as well as inflammatory and tumor diseases.Compared with other detection methods,electrochemical detection has been widely used in biological detection due to its advantages including high sensitivity,low background signal,low cost and simple operation.Nanomaterials have the advantages of large specific surface area and excellent conductive properties,which have been attracted wide attention in the construction of biosensors.This thesis aims to construct new electrochemical DNA biosensors by using different kinds of nanomaterials and amplification strategies to realize the purpose of wide range and high sensitive detection of miRNA.The detailed content were showed as following:1.A molybdenum disulfide-based electrochemical DNA sensor was constructed.The gold nanoparticle-thionine functionalized molybdenum disulfide nanocomposite?MoS₂-Thi-AuNP?was prepared by microwave-assisted method.A large number of gold nanoparticles loaded on the nanocomposite surface,which provides large numbers of sites to load many probe DNA and increase the conductivity.Thionine?Thi?in the nanocomposite acts as an electroactive substance,providing the possibility of label-free detection.Under the optimal conditions,the signal descreasement of peak currents of Thi has a linear relationship with the logarithm of miRNA-21 concentration ranging from 1.0 pM to 10.0 nM and the limit of detection was calcaulated as 0.26 pM.Moreover,such MoS₂-based sensor also had good selectivity and stability,which could be used to detect miRNA-21 in real samples.2.A label-free and enzymatic-amplification electrochemical DNA sensor was designed by using the different affinities of MoS₂ toward single-stranded DNA?ssDNA?and double-stranded DNA?dsDNA?.Using[Fe?CN?₆]^3-/4-as an electrochemical signal molecule,the electrochemical sensor could detect as low as 0.692 pM miRNA-21 with the help of endonuclease?DSN?,which could specifically cleave DNA strands in DNA-mi RNA double system.Meanwhile,this sensor also had good selectivity for miRNA detection.3.A multiple amplification electrochemical miRNA sensor based on flower-like gold nanostructures was builded.Flower-like gold nanostructures deposited on ITO possessed a large specific surface area,which can effectively increase the loading of DNA probes and make detection signal amplifying.Gold nanoprobe assembled with many initiation DNA strands,which provided enough reaction site for the HCR reaction.Long double DNA strands formed from HCR reaction could absorbed a large number of electrochemical signal molecules[Ru?NH₃?₆]³⁺via electrostatic interaction,making the electrochemical signal further amplifying.Therefore,the experimental results showed this sensors had a wide linear range from 1 fM to 1 nM and a low detection limit of 0.12 fM?S/N=3?.Moreover,this sensor had good selectivity,which could be used to determine miRNA-21 in the real samples.All data proved that the sensor had potential application in the early detection of diseases.