Silica Encapsulated Quantum Dot-encoded Microbeads For Multiplexed G4-DNA Detection

G-Quadruplex(G4-DNA)is a special secondary structure of DNA.It is a G-rich short chain,Four g base self assemble into G-tetrads by Hoogsteen Hydrogen bond.Multiple G-tetrads self assemble into G-quadruplex by ?-? stacking effect.There are 376000 G4-DNA in human genome,which distributed in chromosome telomeres region,the promoter region of the gene,immune protein switch area and exon region.G4-DNA involved cell proliferation,gene expression and regulation,protein translation and other biological functions.So it has important research significance to find an analysis and diagnostic methods which is high sensitivity high accuracy and fast.In recent years,fluorescent microsphere technology has made great progress,can be widely used in nucleic acid,protein,carbohydrate and other biological components of high-throughput,high sensitivity,rapid analysis.The quantum dot fluorescence coding microspheres not only make use of the excellent optical properties of quantum dots,but also make coding more diverse decoding more convenient.However,in most cases,the detection sensitivity can not meet the requirements of clinical analysis,the need for further integration of signal amplification technology.Rolling circle amplification is an efficient method for amplification of nucleic acid detection signals,with the advantages of low cost,simple operation,good repeatability and no need to change the temperature.In this paper,G4-DNA of G4-DNA in different regions of human genome was highly sensitive and multivariate by rolling circle amplification reaction on the surface of silica-coated quantum dot fluorescent microspheres.The main contents of the thesis are as follows:CdSe @ ZnS quantum dots with emission peaks at 513 nm(green)and 582 nm(orange)were synthesized by high temperature hot injection.The polystyrene microspheres were synthesized by disperse polymerization method.PSDM mesoporous microspheres were synthesized by seed polymerization.Using the"swell-volatilization" method to encapsulate two kinds of quantum dots of orange and green to form quantum dot fluorescently encoded microspheres.Silica-coated quantum dot-coded microspheres(Qbead@SiO2)were prepared by Stober method.The constructed Qbead@SiO2 has excellent fluorescence stability.Qbead@SiO2-RCA analysis was developed for high sensitivity detection of G4-DNA with a detection limit of 10 fM.The main steps of this analysis include:coupling the capture probe sequence on the surface of Qbead@SiO2-COOH by carbonyl diimine reaction,immobilizing the Padlock DNA sequence by hybridization;the target G4-DNA hybridizes with Padlock DNA,and the Padlock DNA is linked to the T4 ligase And then amplified by enzyme catalysis.ZnPc was used to stain the RCA product.The fluorescence signal of ZnPc was quantitatively detected by flow cytometry.Based on three Qbead@SiO2 fluorescent codes,the simultaneous detection of three G4-DNA sequences was achieved by Qbead @ SiO2-RCA method,and the specificity of detection was better.