Fluorescence Nanoprobes For High Sensitivity Detection And Imaging Of Reactive Small Molecules And Mirna In Living Cells And In Vivo

The active molecules are important components of the cells,and it can be divided into two categories: active small molecules and active macromolecules.Active small molecules mainly include free radicals(reactive oxygen species and reactive nitrogen species),endogenous gas molecules(CO/NO/H2S/O2),antioxidants(glutathione and VC),H+,metal cations and anions.Active macromolecules include proteins and nucleotides.These active molecules play a crucial role in the life of organism and is essential for physiological function of live cells.At present,the detection of intracellular active molecules is widely concerned and studied extensively,but there are still shortcomings such as high detection limit,low sensitivity and poor selectivity,which will greatly limit our ability to accurately explore the role of active molecules in cell physiological and pathological activities and can not make an accurate judgment of its function.Therefore,the development of high sensitivity and high selectivity analysis method to achieve their detection is of great significance.DNA nanotechnology is a technology developed in recent decades,which is based on deoxyribonucleic acid as a structural unit with a nanostructure of nanoscale.In addition to being a gene carrier,DNA itself is a one-dimensional nanowire,which can be assembled to construct two-dimensional planar structure,three-dimensional tube and polyhedron and other structures.In these three-dimensional structures,DNA tetrahedron structure has been widely used in the field of biosensing and therapy due to its simple synthesis and easy modification.The DNA tetrahedron is composed of six DNA double strands and four vertices.Each edge and each vertex can be functionalized dye or other nucleotides.Moreover,the DNA tetrahedronal structure can enter the cell easily and have good biocompatibility,high nuclease stability,which is widely applied in biological research.Graphene oxide nanosheets are two-dimensional single-layer sp2 carbon layer structure material.They have good water solubility and biocompatibility.Some hydrophobic molecules containing aromatic ring can be absorbed on the surface by ?-? stacking and hydrogen bonding.The surface can modify with functional groups such as hydroxyl,carboxyl and amino groups,which can be possible to further functional modification of other substances.Graphene oxide is a good fluorescence quenching agent due to a wide absorption band.The fluorescence of dye molecules can be quenched on the surface of graphene oxide.Moreover,the size is suitable for a carrier to carry dye molecules or drug into cells.In this thesis,two kinds of fluorescent nanoprobes were designed and synthesized based on DNA tetrahedron and graphene oxide as carriers,which were used to detect reactive oxygen species and miRNA in living cells and in vivo.This paper mainly carried out from two aspects:1.A DNA tetrahedron nanoprobe control the dye distance for simultaneous detection of multiple analytes in living cell and in vivo.The current multiple detection strategy may cause fluorescence self-quenching and reduce the sensitivity of detection.To solve this problem,we develop a DNA tetrahedral nanoprobe to control the dye distance for detection of multiple analytes under a single wavelength excitation.DNA tetrahedron can be assembled by four designed isometric single strands.Fluorescein and hydroethidine as the pH and O2·-responsive units were conjugated with the vertexes of the tetrahedron to achieve simultaneous determination of pH and O2·-.Compare to mesoporous silica nanoparticle based nanoprobes,the DNA tetrahedron nanoprobes display enhanced fluorescence intensities due to partially avoiding the fluorescence resonance wnergy transfer.Confocal fluorescence images shows that the nanoprobes are capable of detecting and visualizing pH and O2·-in living cells under a single wavelength excitation.In a mice inflammation model,the nanoprobes simultaneously image the down-regulation of pH and up-regulation of O2·-.2.Based on graphene oxide(GO)and molecular beacon(MB),we design and synthesize a highly sensitive method for the detection of miRNA in living cells and in vivo.Two Cy5 molecules were connected with both ends of the MB.When the MB forms the stem-loop structure,the fluorescence of two Cy5 molecules is quenched due to the close distance.Moreover,when the MB was absorbed on the GO,the fluorescence of Cy5 was further quenched.Compared to previous reported probe based on one dye,connecting two dyes can reduce the fluorescence background.Meawhile,when meeting target molecules,the design of two dye molecules can achieve two molecules fluorescent recovery,but the design of one dye molecule only get one molecule fluorescent recovery.So the method that we designed can improve the sensitivity of detection.The assay of fluorescence recovery can be seen in the chemical system that the background fluorescence of designed nanoprobe was lower than one MB,but the fluorescence signal recovery was stronger.Moreover,the detection limit is decreased by more than 10 times.So the designed nanoprobe is feasible to detect miRNA with high sensitivity.Different concentrations of miRNAs in different cancer cells were distinguished in cell experiments,which further demonstrated the high sensitivity of the probe.