Research On MicroRNA Electrochemiluminescence Analysis Method Based On Composite Silica Nanoparticles And Nano-gold Sensing

MicroRNA(miRNA)is a class of endogenous,non-protein coding small RNA that is approximately 22 nucleotides in length,and regulates the expression of target genes at the post-transcriptional level by either translational repression or degradation of miRNA.Recently,many studies have also shown that the abnormal expression level of miRNA in the cell or blood samples is associated with lots of human diseases,such as cancer and diabetes,especially the latter.This also indicates that miRNA could be as an ideal biomarker for diagnosis and treatment of cancer.Therefore,it is very necessary to develop an accurate method for assay of expression level of miRNA in the cell or blood samples.However,it is still a tough challenge to develop an approach for highly sensitive and selective detection of miRNA due to their short length,low abundance and sequence homology among the miRNA family.In addition to the tranditional assay methods,some optical,electrochemical and electrochemiluminescence(ECL)techniques have been applied for highly sensitive detect of target miRNA in the conection with signal amplification stragegies.Though enzyme catalysis and DNA assembling technologies can make the sensitivities of miRNA ECL assay greatly be improved,they are high cost,and often need some harsh reaction conditions or DNA probes need be specially designed.Generally,the nanomaterials-based signal amplification stragegies have a promising prospect in biosensing and bioimaging.Among them,silica nanoparticles and gold nanoparticles(AuNPs)have been widely applied because of low cost and mature preparation methods.Some fluorescent or electroactive molecules could be doped into the silica nanoparticles matrix,resulting in the better optical characteristics compared with that of free molecules.Moreover,their mass transfer channels also could be improved by changing pore sizes.Due to their optical properties and good conductivity,AuNPs have been greatly paid attention.Especially,AuNPs could well be applied in the optical and electrochemical sensing fields for different target analytes by changing their sizes,surface modification,charge characters and existential states.This thesis consists of two parts,and the first part is a review that mainly introduces the research progress of electrochemical sensing miRNAs and focuses on the signal amplification-based voltammetry and ECL assay for miRNAs.The second part is research works.Based on the different interaction of ss-DNA and ss-DNA/miRNA heteroduplex with crystal violet,composite silica nanoparticles and AuNPs,three ECL sensing methods for miRNAs are developed combing with the selective concentration effect of interacted products at the modified electrode surface.This part focuses on the following three aspects:1.Efficient Quenching of the Electrochemiluminescence of porous Ru(bpy)32+-doped silica nanoparticles by crystal violet and its application to Sensitive Assay of MicroRNAIt was found that the interaction of crystal violet with ss-DNA probes or ss-DNA/miRNA heteroduplex exhibited a great difference,forming different numbers of free crystal violet.Thus,different numbers of free crystal violet molecules could be concentrated at the etched Ru(bpy)32+-doped silica nanoparticles(ERuSiNPs)surface to efficiently quench the ECL of ERuSiNPs.Based on these observations,an enzyme-free and label-free ECL approach was developed for assay of miRNA.In this sensing stragegies,r-GO/Nafion-ERuSiNPs modified electrode was used as the sensing interface,and crystal violet,tripropylamine and let-7a as the hybridization indicator,the ECL co-reactant and the target model,respectively.Compared with the unetched composite nanoparticles by alkali,the proposed sensing stragegy presents the higher sensitivy using ERuSiNPs as the sesing indicator of signals.The developed protocol might provide an attractive platform for label-free and sensitive detection of other specific miRNAs.2.The preparation of Hemin/PEI/SiO2 composite nanoparticles for Label-free Electrochemiluminescene Detection of MicroRNAPEI-doped silica nanoparticles(PEI/SiO2NPs)were firstly synthesised by reverse microemulsion method.Hemin could be efficiently assembled into the resulting PEI/SiO2NPs due to the supramolecular interaction between them.Thus,we successfully prepared a novel type of composite silica nanoparticle,Hemin/PEI/SiO2NPs(HPSiNPs)that own both the good biocompatibility and electrocatalytic activity.It was found that their existential states presented an obvious difference after HPSiNPs interacted with ss-DNA and ss-DNA/miRNA heteroduplex,respectively.Additionally,Nafion-carbon nanotubes(Nafion-CNTs)modified electrode could selectively adsorb HPSiNPs in the different existing-states and hemin embedded inside HPSiNPs could significantly catalyze the ECL reaction of luminol-hydrogen peroxide system.Based on the above-mentioned finding,we established a label-free ECL method for sening miRNA.Compared with rolling circle and DNA assembling technologies amplification stageries,the proposed probes not need be specially designed and labeled.3.Electrochemiluminescence based detection of MicroRNA using target-guided assembly of AuNPs at the Nafion-carbon nanotubes-polyvinylpyrrolidone interfaceIn this work,a biosensing interface is constructed by sequential assembly of Nafion-CNTs and polyvinylpyrrolidone(PVP)at the glass carbon electrode.The sensing interface is capable of selectively adsorbing AuNPs that interact with ss-DNA/miRNA heteroduplex.Combing with the catalytic effect of AuNPs to luminol ECL reaction,we present a novel method for label-free ECL detection of miRNA.Carbon nanotubes at the modified electrode surface can not only improve the electron transfer efficiency,but also obtain more PVP active sites for adsorbing AuNPs.On the one hand,the introducing of PVP into the sensing interface reduces the ECL background signal,resulting in a higer sensitivy.On the other hand,it can provide a microenvironment for bonding AuNPs.These results suggest that this sensing interface will be promising in cost-effective and sensitive assay of clinically cancer biomarkers.