Study On Signal Amplification Strategy Of Nucleic Acid Molecular Probe And A New Method For Biological Analysis

Micro RNAs(mi RNAs)are a group of short endogenous noncoding RNAs that can bind to the 3′ untranslated region of m RNAs to regulate the expression of target genes in cell proliferation,differentiation,and tumorigenesis.Recently,mi RNAs have been emerged as new biomarkers for early diagnosis of diseases including cancer.What’s worse,for the detection of mi RNAs,their small size,high sequence similarity,low abundance and difficult extraction from cells impose great challenges in the analysis.In recent years,considerable effort has been challenged into fabricating fluorescent sensors for nucleic acid detection owing to the inherent advantages which they exhibit.These advantages include ease of operation,high sensitivity,and in situ imaging properties.In this paper,several novel fluorescent nanomaterials were prepared,and a fluorescent biosensor was designed based on the target multiple amplification strategy to achieve the high sensitivity detection of various mi RNAs nucleic acid molecules.This paper mainly studies the following aspects.1.In this experiment,the structure of two stem rings with endonuclease sites was designed skillfully,and the process of multiple circular amplification based on enzyme-assisted target-recycle was realized.In this process,ds DNA nanostructures based on double-cycle drive can induce two successive amplification stages and stimulate regeneration,resulting in a large amount of single-stranded DNA accumulation,thus amplifying the signal.The carboxylic carbon quantum dots(c CQD)with small particle size,good biocompatibility,low cytotoxicity and good stabilitywere synthesized.Fluorescence probe(FAM-probe)was used as signal probe,carboxylic carbon quantum dots(c CQD)was used as fluorescence quenching device,and fluorescence labeled ss DNA of probe FAM-probe was adsorbed on carboxylic carbon quantum dots(c CQD),which resulted in fluorescence quenching.When the fluorescence probe FAM-probe hybridized with its complementary ss DNA to form ds DNA,the interaction between ds DNA and carboxyl carbon quantum dot c CQD was very weak,which made the ds DNA far away from the surface of c CQD,leading to fluorescence recovery and high sensitive detection of Mi RNA-34.2.The aim of this study was based on the circular amplification strategy of the target under the catalysis of enzyme.The DNA reticular nanostructures were successfully constructed by using circular products and polymerase via two kinds of stem ring structure HAPs.The cyclic shear template DNA-1/DNA-2 was firstly prepared,and then the target hybridized with single strand of the DNA template.Then the target extends along the template under the action of the polymerase,replacing the DNA-2,and at the same time,the enzyme splits the resulting cutting site,and the 3’end of the incision continues to extend under the action of the polymerase.Then the DNA-2 was replaced,and the process was repeated,which resulted in a large accumulation of DNA-2.The circular product DNA-2 first opened the stem ring structure HAP1,which further opened the stem ring HAP2 and extended under the action of polymerase.The open HAP2 continues to open the HAP1 by complementing the HAP1 fragments,so the DNA reticular nanostructures are assembled through the HAPs hybridization process.By inlaying fluorescence dye into the double-strand DNA of the nanostructures,highly sensitive fluorescence detection of Micro RNA-21 was achieved.The detection limit is 5.4 f M.The nucleic acid nano-assembly will be widely used in the construction of biosensors and functional nanomaterials.3.In this study,we designed a fluorescence biosensor platform to detect Micro RNA based on target cross-chain displacement cyclic amplification strategy by using photoinduced electron transfer distance dependence.In this experiment,the cross structure of DNA was designed cleverly,and the restriction site was first protected.The amplification reaction of target cycle and chain replacement reaction based on DNA cross-configuration was carried out under the catalysis of primer,polymerase and cutting enzyme.A large number of amplification products,ss DNA(S1 and S2)can be exported after the input and transfer of single target mi RNA by cyclic amplification.Based on the high affinity of Ag to the base cytosine,fluorescentDNA/Ag NCs was synthesized by ss DNA sequence rich in cytosine C and single-stranded DNA sequence rich in guanine was used to synthesize G-quadruplex complex,and used as the electron receptor and donor in the subsequent PET process,respectively.The amplified product S1 and S2 hybridized with the flexible COM1 and COM2.By forming rigid double-stranded DNA to inhibit the quenching of fluorescence in the PET process,the corresponding fluorescence was recovered.The amplified products based on mi RNA-182-5p can lead to the change of the fluorescence specifically,and the changes increases with increasing concentration.The proposed fluorescent biosensor can be applied to the quantitative determination of mi RNA-182-5p,which has great potential in the early clinical diagnosis of mi RNAs related diseases.