CRISPR/Cas13a And Liposome Assisted Amplification Magnetic Relaxation Sensing For Direct Detection Of MiRNA And Pathogenic Bacteria

Cancer and infectious diseases are serious threats to human health.Many studies have demonstrated that the expression of micro RNA（miRNA）is related to tumor growth,invasion,angiogenesis,and immune evasion.Therefore,mi RNA is a potential important indicator for tumor diagnosis and prognosis evaluation of cancer,and the rapid and accurate detection of mi RNA is of great significance.Pathogenic bacteria such as Staphylococcus aureus（S.aureus）are important causes of sepsis,pneumonia,endocarditis and other infectious diseases,and the sensitively and quickly detection of pathogenic bacteria is of important clinical research and practical significance.Due to the strong background interference signals from the various components in complex biological samples,colorimetric,fluorescence,electrochemical and other methods exhibit deficiencies of direct and rapid detection of the targets in the complex samples.Therefore,it is urgent to develop rapid and accurate methods for the detection of targets（such as mi RNA and pathogenic bacteria）in complex biological samples（such as blood）,which is of great significance to the diagnosis and treatment of the cancer and infectious diseases.Magnetic relaxation sensing（MRS）could quantify substances by changing the relaxation time of the water proton as analytical signals.MRS assay does not depend on the light signal and can directly analyze target without pre-treatment of samples,which is simple and convenient,non-destructive,and anti-interference.In this paper,the CRISPR/Cas13a system,liposome enhanced magnetic relaxation sensing signals for the sensitive and accurate directly detection of the targets（mi RNA and pathogenic bacteria）in complex biological samples.The main contents of this thesis are as follows:Part I:CRISPR/Cas13a system enhanced magnetic relaxation signals of Fe₃O₄magnetic nanoparticles for detecting mi RNA.The T₂ signal probe(MB₃₀-RNA₁-MM₁₀₀₀)was prepared by co-modified the RNA₁ with the carboxylation Fe₃O₄magnetic nanoparticles(with size of 30 nm,MB₃₀)and streptavidin functionalized magnetic microspheres(with the particle size of 1000 nm,MM₁₀₀₀).The cr RNA（CRISPR RNA,cr RNA）in the CRISPR/Cas13a system could specifically recognize mi RNA-21（mi R-21）,triggering enzyme activation reactions.The CRISPR/Cas13a system cut off the signal probe(MB₃₀-RNA₁-MM₁₀₀₀)and released MB_30.After magnetic separation,the magnetic relaxation signal（T₂）of free MB₃₀ in the sample was measured to finish the quantitative detection of mi R-21.The research shows that the detection range of this method is 1 p M～50 n M,the linear equation is y=142.25x+33.69,R²=0.9930 and the limit of detection is 0.22 p M.Non-target mi RNA-15（mi R-15）,single base mismatch mi R-21（Smi R-21）,double base mismatch mi R-21（Dmi R-21）do not interfere with the detection of mi R-21.Further research shows that this strategy combined with CRISPR/Cas13a signal amplification and MRS assay has been successfully used the directly analysis of mi RNA in serum samples.And compared with q RT-PCR,the results of the MRS assay for detecting mi R-21 is closer to the real value.PartⅡ:Liposome enhanced magnetic relaxation sensing signals of Fe²⁺/Fe³⁺for detecting S.aureus.In this research,the recognition probes of vancomycin（Van）modified 200nm magnetic beads(MB₂₀₀-Van)and biotinylated immunoglobulin（Biotin-Immunoglobulin,Biotin-Ig G）captured S.aureus by sandwich method.Biotinylated liposomes loaded with glucose molecules（Biotin-liposome@Glu）could label S.aureus by affinity with streptavidin（SA）.Then,the Biotin-liposome@Glu could release glucose molecules under the 1%Triton-X-100.The hydrogen peroxide（H₂O₂）was produced with the action of glucose and glucose oxidase（Glucose oxidase,Gox）that could oxidize Fe²⁺into Fe³⁺.the change of magnetic relaxation signal caused by the valence state change of Fe²⁺.The T₂change value（ΔT₂）was used to analyze the content of S.aureus in the blood sample.The research shows that the detection range of this method is 10^{^2}-10^{^7}cfu·m L^-1 and the limit of detection is 10^{^2} cfu·m L^-1.Further research shows that this strategy combined with liposome signal amplification and MRS has been successfully used the directly analysis of S.aureus in whole blood samples...