Detection Of Micro Rna By Isothermal Amplification Of Nucleic Acid Based On Dynamic Light Scattering Technique

As a cancer marker,microRNA(miRNA)plays an important role in many biological processes.It is a class of endogenous non coding single strandedRNA in eukaryotic cells,which is involved in the regulation of more than half of protein-coding genes in human body.Studies have shown that abnormal expression of miRNAs in human tissues and blood may contribute to the occurrence,development and drug resistance of cancer.Therefore,the sensitive detection of small miRNA molecules will help us better predict and timely treat human diseases.This paper is mainly divided into the following parts:(1)Rolling circle amplification(RCA)-based self-assembly DNA network was developed for ultrasensitive and specific detection of microRNA(miRNA)with dynamic light scattering(DLS)technology.A dumbbell-shaped padlock probe was designed,which could be connected with itself as a template under the action of T4 DNA ligase to form a dumbbell-shaped sealed probe.In the presence of the target miRNA,the miRNA bound to the toehold domain of the sealed probe.The sealed probe was activated into a circular probe to start RCA reaction by a spontaneous chain displacement reaction.The DNA functionalized Au NPs probe was hybridized with the RCA products to form RCA-DNA-Au NPs polymer with network,whose diameter was measured by DLS.Not only did this method greatly improve the sensitivity of detection,but the specificity of detection increased by using sealed probe as well.The limit of detection(LOD)was 0.11 f M.Furthermore,this strategy has been successfully used to analyze target miRNA in cancer cell samples,which confirmed that RCA-DLS analysis has broad application prospects in early clinical diagnosis.(2)Using the characteristics of the continuous mechanical movement of the enzymatic DNA(DNAzyme)walker,a miRNA detection strategy based on the "enzyme digestion and walking" of the DNAzyme walker guided by the hairpin probe is established.DNAzyme is initially locked by the locked strand and cannot perform its function.When miRNA is present,the miRNA binds to the toehold domain of the locked strand.The DNAzyme is replaced by a strand substitution reaction and walks freely on the hairpin probe on the surface of the gold nanoparticle.When the ions are present,the r A base is specifically digested,causing the partial sequence of the hairpin probe to fall off the surface of the gold nanoparticle,and the exposed 5ends of the DNAzyme Walker continue to walk on to the next hairpin probe for digestion.DNAzyme Walker continuously cuts and walks to induce the sustained release of hairpin fragments.The remaining hairpin sequences on the surface of the gold nanoparticles are complementary to each other,leading to the aggregation of gold nano ions.This strategy uses the enzymatic DNA cleavage feature to carry out enzymatic signal amplification,avoiding the problem of expensive and easily degraded protein enzymes used in the ordinary signal amplification process.Secondly,the autonomous movement mechanism of the enzymatic DNA walker accelerates the reaction process and improves the efficiency of the reaction.Using DLS technology as the signal output system of the DNAzyme Walker experiment,ultra-sensitive detection of signals is realized.The LOD is 3.58 f M.In addition,this strategy has been successfully used to analyze target miRNAs in cancer cell samples.