Multiple Signal Amplification Technology For Electrochemical Detection Of MicroRNA

MicroRNA(miRNA),a class of small and noncoding single-stranded RNA with an approximately length of 22 nucleotides,plays an important role in multiple biological processes such as cell differentiation,cell apoptosis,tumor formation and viras replication.Recently,accumulating evidence has identified the abnormal expression of miRNA in various diseases.Therefore,miRNA is considered as a new class of biomarkers for early diagnosis and prognosis of diseases.Analysis of miRNA has important scientific and practical significance for the exploration of its biological significance and the diagnosis and treatment of diseases.In recent years,a variety of miRNA detection methods have been developed,among which electrochemical biosensors are widely used due to its inherent advantages of miniaturization,convenient operation,low cost and fast response.However,the accurate analysis of miRNA is still challenging because of their intrinsic properties including small size,low expression levels in samples and sequence similarity.It is imperative to develop highly sensitive and selective methods to meet the demands for miRNA detection.To improve the analytical sensitivity,many signal amplification methods including nanomaterial-assisted assays,nuclease-assisted target recycling amrplification and nucleic acid isothermal amplification have been used in bioanalysis.Now,multiple signal ampl:ification technology has become an inevitable trend for a single signal amplification technology still cannot satisfy the requirement for detection of miRNA with an ultralow level.In this study,two highly sensitive electrochemical biosensors based on multiple signal amplification technology were developed for miRNA detection.The details are as follows:(1)Rolling circle amplification-mediated in situ synthesis of palladium nanoparticles for ultrasensitive electrochemical detection of miRNA.The sensor was fabricated by immobilization of dual functionalized hairpin probe(HP)onto the electrode.In the presence of miRNA-21,the specific recognition of miRNA-21 by the HP could trigger the rolling circle amplification(RCA)which produced numerousguanine(G)-rich long single-stranded DNAs(ssDNAs).Based on the interaction of Pd1" ions with the nitrogen atoms from G bases,these G-rich long ssDNAs served as specific templates to in situ synthesize massive palladium nanoparticles(Pd NPs)as label-free electrochemical indicators.With this cascade signal amplification strategy,the developed biosensor achieved a linear range of 50 aM to 100 fM with an ultralow detection limit of 8.6 aM.Furthermore,the developed biosensor exhibited good selectivity and satisfactory feasibility for miRNA-21 detection in human serum samples,ensuring it great potential in disease diagnosis and prognosis applications.(2)DNA self-assembled SiO2@FeNxC nanocatalytic polymeric network for electrochemical detection of miRNA.In the presence of miRNA-21,capture probe(CP)immobilized on the gold electrode was unfolded by the recognition of miRNA-21 resulting in a DNA-RNA heteroduplex.Due to the cleavage preference for DNA in DNA-RNA heteroduplex,duplex-specific nuclease(DSN)hydrolyzed the miRNA-binding part of the CP while liberating miRNA-21 to hybridize with other new CPs.CP residue left on the electrode hybridized with DNA1 which labelled with SiO2@FeNxC nanoparticles,and then DNA1 hybridized with DNA2 in SiO2@FeNxC-DNA1 bioconjugate.Based on DNA1/DNA2 hybridization reaction,SiO2@FeN,C-DNA nanocatalytic polymeric network was constructed through self-assembly of SiO2@FeN,xC-DNA1 bioconjugate and SiO2@FeNxC-DNA2 bioconjugate and was used to catalyze the reduction of H2O2 for electrochemical signals generation.Benefiting from the multiple signal amplification methods,the proposed biosensor exhibited a linear range of 5 fM-1 pM towards target miRNA-21 with a detection limit of 0.43 fM.Furthermore,the proposed assay showed excellent discrimination ability for base-mismatched miRNA sequences and acceptable electrode-to-electrode reproducibility.The proposed assay provided a novel avenue for highly sensitive and selective miRNA detection.