MicroRNA Detection Based On DNA-templated Fluorescent Metal Nanoprobes Combined With Target Cycle Amplification

MicroRNAs(miRNAs)are a class of endogenously expressed and short non-coding RNAs that act as significant post-transcriptional regulators of gene expression and disease biomarkers.Hence,it was of great importance for miRNAs detection with high sensitivity and specifity,which could supply valuable information for biomedical research,clinical early diagnosis and therapy.However,the traditional probes using organic fluorescent dye as signal output was unsatisfactory for poor photostability,short fluorescence lifetimes,easy photobleaching and toxicity.In recent years,with the development of nanotechnology,a series of novel fluorescent probes with excellent physical and chemical properties have been developed.Among them,DNA-stabilized fluorescent metal nanoclusters as a promising type of label-free fluorescent probes are of great interest in biochemical analysis,due to their good water solubility,low biological toxicity,simple operation and flexible design.In particular,benifiting from the nucleic acid based template,a strategy that combinated of DNA-stabilized fluorescent metal nanoclusters and nucleic acid amplification technology could be well developed,which could provide good opportunities for highly sensitive miRNA detection.In this thesis,aiming at developing new methods for miRNA detection with high selectivity and high sensitivity,two kinds of DNA-stabilized fluorescent metal nanoclusters combined with target cycle amplification strategies have been developed.The detailed description is listed as follows:1.Label-free detection and cyclic amplification assay of miRNA based on"super long" poly(thymine)-hosted fluorescent copper nanoparticles combined with graphene oxide-DNase IA novel label-free and cyclic amplification detection method of miRNA was developed based on "superlong" poly(thymine)-hosted fluorescent copper nanoparticles by combining the different adsorption performance and nucleic acid enzyme protective effect of graphene oxide(GO)toward ssDNA and dsDNA,and deoxyribonuclease I(DNase I)that can selectively shearing DNA.In this method,a capture DNA probe was first designed and adsorbed on the surface of GO.When the target let-7a presents,it allows hybridization with the capture DNA probe through base pairing,thus releasing from the surface of GO for the weak adsorption capacity of GO towards the double chain.Upon adding of DNase I,DNase I selectively cleaves the DNA of DNA/RNA into multiple fragments,releasing miRNA into the next cycle.After the reaction was completed,GO was removed by centrifugation and the supernatant was extracted,and then TdT and dTTP solutions were added for poly T polymerization.The obtained superlong poly T products can be used as templates to synthesize fluorescent copper nanoparticles under the assistant of Cu2+ and ascorbic acid,thus achieving the detection of miRNA.Using let-7a as a target,both fluorescence analysis and electrophoresis have demonstrated good feasibility of cyclic amplification and good linear correlation in the range of 50 pM-10 nM with a detection limit of 50 pM.Moreover,the method has good selectivity and can be successfully used for the detection of target miRNA in serum samples,providing a promising platform for miRNA detection methods in early clinical diagnosis.2.Highly sensitive detection of miRNA based on activatable DNA-Ag NCs fluorescent probe combined with catalytic hairpin self-assembly Using the DNA-AgNCs with high fluorescence intensity as signal output,an activatable fluorescence probe was successfully constructed by introducing blocking DNA probe labeled with BHQ1.The fluorescence quenching efficiency and activation efficiency of the fluorescent probe are as high as 90%and 92%,respectively.Further,combining with catalytic hairpin self-assembly(CHA)amplification technology,a new method for miRNA cycle amplification detection was developed and the target was designed as a trigger.In this strategy,two hairpin probes:H1 and H2 were rationally designed.For H1,the DNA sequence that can activate the activatable fluorescent probe was enclosed in the stem,which can't hybridize to H2 in the absence of targets.While,in the presence of targets,the CHA was initiated by forming H1-miRNA-21 intermediate,and catalyzed the formation of H1-H2 duplex accompanying the release of miRNA-21 into the next cycle.Here,miRNA-21 was chose as a model target.Fluorescence analysis and electrophoresis experiments have confirmed the highly sensitive and specific detection of target miRNA after the systematic optimization of the CHA system,including sequence,buffer,and the ratio of activated fluorescent probes.The strategy shows advantages in simple operation,enzyme-free,and washing-free,which is expected to provide a new platform for high selectivity and high sensitivity analysis of miRNAs.