The Research On Optical Biosensor Based On DNA Self-assembly And Isothermal Signal Amplification Technology

As an endogenous molecule in organisms,DNA is the carrier of genetic material to determine the traits of species.In addition,under the clever design,through the construction of DNA,it is also widely used in biochemical analysis,DNA nano Technology,medical diagnosis and other fields.DNA self-assembly technology is to precisely hybridize between DNA molecules with the help of Watson-Crick and other base complementary pairing characteristics,and form double-stranded or even triple-stranded structures from single-stranded DNA,and then continuously amplify these double-stranded structures Combine to form new two-dimensional structure and three-dimensional structure.Isothermal signal amplification technology can achieve rapid amplification of DNA fragments under isothermal reaction conditions in a short time.Compared with polymerase chain reaction(Polymerease Chain reaction,PCR),the biggest advantage is that it does not require repeated temperature changes and higher Equipment requirements and simple operation.At present,DNA self-assembly technology is widely used in various biosensors,which greatly enhances the specificity of biosensors,and the addition of isothermal signal technology has further improved the sensitivity of biosensors.Therefore,the combination of these two technologies has very good application prospects in clinical testing and diagnosis,environmental sanitation and food safety testing.This paper mainly uses DNA self-assembly to form different structures,and uses nucleic acid isothermal amplification technologies such as catalytic hairpin self-assembly(CHA),rolling circle amplification(RCA),hybrid chain reaction(HCR),and endonuclease amplification.Next,three convenient and efficient fluorescent biosensor strategies were designed and applied to the activity of DNA methyltransferase(Dam MTase)and ultra-sensitive detection of Microrna(miRNAs)two disease markers.The main content of the research paper is summarized as follows:In the first part,a novel fluorescent biosensor technology based on catalytic hairpin self-assembly(CHA)coupled with endonuclease-assisted circulation amplification was constructed and applied in the detection of DNA methyltransferase(Dam MTase)activity.In this strategy,in the presence of the target,it can induce the methylation of the specific sequence of the hairpin.The methylated hairpin is specifically recognized and cleaved by theendonuclease DpnI,and the short chain released by cleavage triggers the CHA reaction.Generate a three-pass structure while achieving loop amplification of short chains.In another cycle amplification process,the endonuclease Endo IV was used to design an AP site to embed a double-labeled signal probe for assembly to the three ends of the three-path structure.The endonuclease IV(Endo IV)is used to catalyze the cleavage of the AP site,and the signal probe is released due to the inability to stably hybridize.Fluorescence signal.Among the two amplification techniques used in this method,CHA is an enzyme-free loop amplification technique performed autonomously at a constant temperature,which improves detection sensitivity and achieves simple and rapid loop amplification.The introduced endonuclease amplification technology can make more signal probes in the system combine with the three pathways,which greatly improves the amplification efficiency and reduces the background signal.Based on the above aspects,ultra-sensitive detection of Dam MTase activity was achieved with a detection limit(LOD)as low as 0.00024 U / ?L.In the second part,this paper develops a novel fluorescent biosensor technology mediated by DNA triple helix molecular switch-mediated rolling circle amplification(RCA)and applies it to miRNA detection.The DNA triple helix molecule designed in this strategy The switch(THMS)is marked with a fluorophore / quencher and is also cleverly designed with a specific endonuclease recognition sequence,the target's complementary sequence and the RCA product's complementary sequence.Starting from the attachment of the target to the THMS,the conversion of the THMS conformation is induced,and the "extension-cleavage" enzymatic amplification process occurs at the same time,and multiple primers are induced to continue to trigger the exponential RCA response.In addition,the obtained RCA product of the first round contains many tandem repeats that can hybridize with THMS,can hybridize with a large number of THMS,release a large number of primers and start the second round of running RCA.This scheme forms THMS under DNA self-assembly and combines with RCA isothermal amplification technology,which enables the fluorescent biosensor to detect miRNAs with high sensitivity,improved LOD as low as 1.1 aM,and detection in actual samples.In the third part,a novel fluorescent biosensing technology based on G-quadruplexDNAzymes regulating AuNPs/ DNA-AgNCs fluorescence resonance energy transfer is constructed and applied to the detection of miRNA.The biosensor's clever design uses a combination of DNA-AgNCs chains and AuNPs with fluorescence quenching capability,and G-quadruplex DNAzymes.With the help of hybrid chain reaction(HCR),the labeling of miRNA-122,Ultra-sensitive detection without enzymes.When there is no target in the reaction system,cysteine ??can promote the reunion of AuNPs through the carboxylation reaction;when the target is present,it can initiate the HCR reaction,and a large number of G-quadruplexes are formed by DNA self-assembly after heme is added DNAzymes,G-quadruplex DNAzymes can oxidize cysteine ??to cystine,and cystine cannot agglomerate AuNPs.Therefore,AuNPs adsorb DNA-AgNCs single-stranded chains,resulting in fluorescence quenching.Ultra-sensitive detection of targets.Under optimal reaction conditions,this fluorescent biosensor can detect miRNAs with high sensitivity.The minimum detection line is 9 aM.By changing the target recognition sequence,this scheme can also be used for the detection of different targets.