Preparation And Application Of Nucleic Acid Fluorescent Nanoprobe

Nucleic acid is a kind of biological macromolecular compound composed of many nucleotides.It is responsible for storing and transmitting genetic information and is one of the most basic substances in life activities.Nucleic acids are widely present in all animal and plant cells,as well as microorganisms.They often bind to proteins in organisms to form nuclear proteins.Depending on the chemical composition of nucleic acid,it can be divided into ribonucleic acid?RNA?and deoxyribonucleic acid?DNA?.DNA is the most important material basis for storing,reproducing and transmitting genetic information.RNA plays a decisive role in protein synthesis.The messenger ribonucleic acid,named as mRNA,is transcribed from DNA and a template for protein synthesis.microRNA?abbreviated miRNA?is a class of small non-coding RNA molecules?containing about 22 nucleotides?that function in RNA silencing and post-transcriptional regulation of gene expression.With the development of disease molecular biology,especially the breakthrough of human genome project,people realize that human major diseases such as cardiovascular disease,cancers and diabetes are directly or indirectly associated with abnormal gene expression.In practical clinical application,dysregulated genes such as microRNA-21 and thymidine kinase 1?TK1?mRNA have been considered as the biomarkers of related diseases.The disease diagnosis and therapeutic effect evaluation can be achieved by monitoring the changes in the expression levels of these biomarkers in vitro or in situ.Unlike the initial determination of phosphorus and pentose quantitation methods based on the most basic composition of nucleic acid molecules,nucleic acid detection can be achieved by using ultraviolet spectrophotometry,fluorescent dye method,real-time quantitative polymerase chain reaction?RT-qPCR?and flow cytometry technique.However,most of them require expensive equipments,professional technical operators or cumbersome steps,which limits their clinical application.Recently,many research groups have designed a series of fluorescent nanoprobes for nucleic acid detection by utilizing the complementary property of DNA sequences.These fluorescent nanoprobes have simple preparation process and can conveniently realize in situ nucleic acid detection with high-throughput,high-sensitivity and high-selectivity,which endows the nanoprobes with great application potential in nucleic acid detection.In this thesis,two nanoprobes for detection of nucleic acid molecules were designed.The main research contents are as follows:?1?In the second chapter of the thesis,we synthesized molybdenum disulfide quantum dots?MoS₂ QDs?with photoluminescence properties,and covalently modified the dye-labeled DNA strand on the surface of MoS₂ QDs to prepare fluorescent nanoprobes for detection of tumor biomarker microRNA-21.We detailed the principle of the nanoprobe for detection of nucleic acid.Various analytical methods such as transmission electron microscopy?TEM?,X-ray photoelectron spectroscopy?XPS?,zeta potential,and ultraviolet-visible?UV-Vis?absorption spectroscopy were applied to characterize the nanoprobe.We also investigated the ability of this nanoprobe to detect microRNA-21 in buffered and complex matrices,as well as the ability to distinguish the tumor cells from normal cells.?2?In the third chapter of the thesis,we used Y-shaped DNA?Y-DNA?as the biological template to prepare a novel Y-DNA@Cu₃?PO₄?₂ hybrid nanoflower through mild biomineralization for nucleic acid detection.We discussed the mechanism of nanoflower formation and introduced the principle of nucleic acid detection in detail.The morphologies and composition of nanoflowers were characterized by scanning electron microscopy?SEM?,TEM,X-ray diffraction?XRD?and XPS.We also investigated the ability of hybrid nanoflowers as nucleic acid probes to detect intracellular tumor biomarker TK1 mRNA,the ability to distinguish of tumor cells from normal cells,and the ability to evaluate drug treatment effects.