Construction Of Fluorescent Probes Based On Nucleic Acid Functionalized Nanomaterials And Research On Nucleic Acids

Fluorescence probes are constructed to detect trace nucleic acids in living orga n-isms due to their advantages of simple operation,high sensitivity,and good stability.The detection of influenza virus DNA is of great significance in influenza infection,diagnosis and treatment.Messenger RNA is used as a biomarker for tumors(such as breast cancer and cervical cancer),and its specific detection can be used for tumor diagnosis,progress assessment,and treatment response monitoring,has broad appli-cation prospects.Currently,many nanoprobes and DNA nanomachines based on n a-nomaterials,nucleic acid probes and signal amplification technologies have been a p-plied to the detection or imaging of intracellular biomolecules,these methods still have some problems.This thesis mainly studies three different nanoprobes and DNA walkers based on catalytic hairpin self-assembly(CHA)technology,inorganic nano-particles and nucleic acid probes for sensitive and selective detection of influenza v i-rus DNA and mRNA.This thesis contains the following three parts of research:(1)A 3D DNA Walker driven by catalytic hairpin self-assembly for ratiometric fluorescence detection of H5N1 DNAIn this study,2-aminopurine(2-AP)and Thioflavin T(Th T)were used as fluo-rescent signals to construct a ratiometric DNA Walker for H5N1 DNA detection driven by the CHA reaction.The DNA Walker constructed by amide reaction of aminoated silica microspheres(SiO₂-NH₂)and carboxylated H1 hairpin probe is activated by H5N1 DNA to open the H1 hairpin,and cycle through the strand displacement rea c-tion with the H2 hairpin.During these processes,the G-rich sequence on H1 was re-leased and combined with Th T(495 nm)and the fluorescence signal increased,while the fluorescence intensity of 2-AP(363 nm)remained unchanged.The ratiometric DNA Walker is a good solution to the false positive results of single-response DNA Walker,and the designed DNA Walker shows high sensitivity and good specificity,with a detection limit of 60 p M.The DNA Walker has been used to detect H5N1 DNA in actual samples and has great potential in clinical applications.(2)MnO₂ nanosheet-modified mesoporous silica nanoparticles-based nano-probes for simultaneous detection of multiple mRNAsThis study proposes a MnO₂ nanosheet-modified mesoporous silica nanoparti-cles-based nanoprobe for simultaneous detection of multiple mRNAs.The MnO₂nanosheet on the surface of the nanoprobe can be degraded by glutathione(GSH)into Mn(II)ions,resulting in the release of the hairpin probes H1 and H2 loaded in the MSN well.In the presence of target mRNA,survivin mRNA and TK1 mRNA hybri d-ize to the complementary fragments of hairpin probes H1 and H2,respectively,so the distance between the fluorophore in the hairpin probe and the quencher is significantly increased,thereby recovering fluorescence.In this study,the nanoprobe avoids the leakage of the fluorescent probe and obtains an enhanced fluorescent signal to detect multi-component mRNA in living cells,which improves the accuracy of cancer diag-nosis and avoids the appearance of false positive signals.In summary,the proposed method provides a powerful tool for the diagnosis and treatment of mRNA-related cancers.(3)Self-powered 3D DNA Walker based on Y-shaped DNA mediated intramo-lecular catalytic hairpin assembly for mRNA detectionIn this study,a self-powered 3D DNA Walker for mRNA fluorescence detection based on Y-shaped DNA-mediated intramolecular catalytic hairpin assembly was proposed.Y-shaped DNA is immobilized on hollow mesoporous copper sulfide nano-particles(HMCuS NPs)through amide bonds to construct a self-powered 3D DNA Walker.After adding survivin mRNA,the hairpin probe H1 is turned on,and then the adjacent hairpin probe H2 releases survivin mRNA into the n ext cycle through a strand displacement reaction,and the fluorescence intensity is significantly enhanced in the end.The self-powered DNA Walker does not require additional auxiliary chains and metal ions,has high sensitivity and excellent selectivity,so that the self-powered DNA Walker has the potential for clinical application.