Study On The Construction Of Quantum Dot-ligand Fluorescent Probes And Application In Cell Imaging

Quantum dots?QDs?are an important class of biophotonic nanomaterial,possessing unique optical properties such as high quantum yield,broad-band absorption,narrow-band and tunable emission and high resistance to photobleaching,electronic properties of charge/energy transfer,and chemical property of facile surface functionalization.The QDs based Fluorescent probes,with significant advantages in terms of high sensitivity,good selectivity,low cost and good visualization capability,have been widely developed and applied in biosensing and bioimaging in recent years.In this dissertation,we developed the QD-Ligand-type nanoprobes by suramolecularly assembling newly synthesized ligands onto single-emission QD or dual-emission QDs nanohybrids.Owing to high affinity and selectivity of the ligand toward the targets,the probes were successfully used to image biotin receptor?BR?on cell membrane and G-quadruplex in nucleus.The main contents are as follows:1.A dual emission ratiometric fluorescent nanoprobe is developed for high-contrast imaging of avidin-type biotin receptor?AV-type BR?on cell surface.The nanoprobe comprises a dual emission quantum dots?QDs?nanohybrid wherein a silica-encapsulated red-emission QD?rQD@SiO₂?is used as“core”and green-emission QDs?gQDs?are used as“satellites”,with newly synthesized BR ligands PB,a conjugate of phenanthroline and biotin through an amino linker.The nanoprobe shows intense rQD emission,with the gQD emission quenched by PB.The PB possesses the“love-hate”characteristics?the biotin moiety“love”BRs with high affinity,and the phenanthroline moiety“hate”BRs by clashing with the protein surface?,conferring the nanoprobe with high recognition selectivity toward AV-type BR versus its isoform streptavidin-type BR.During imaging,the rQD emission stays constant,while the emission intensity of gQD increases as the abundance of AV-type BR increases on the cell surface.This results in an overall fluorescence color change of the cell surface from red to yellow,and then to green.We refer such color change as a traffic light pattern.With the AV-type BR as the target,the distinct traffic light pattern is demonstrated to be fully qualified for differentiation of normal cell/cancer cell,and differentiation of cancer cell types including BR-positive cervical carcinoma,breast cancer and hepatoma carcinoma cells,BR-negative colon cancer cells,and even the subtypes?MDA-MB-231 and SKBr3?of breast cancer cells.The fluorescent traffic light nanoprobe is further used to image the subtle change in expression level of AV-type BR during a cell cycle,capable of differentiating between the G1/S phase and the?G2/M?phase of HeLa cells.Together with the ligand-governed specific recognition,the fluorescent traffic light nanoprobe facilitates the visualization of cell surface biomarker with high imaging contrast and accuracy,holding high potential in applications for clinic cancer diagnosis and basic biology research.2.By complexing a non-ionic G-quadruplex lilgand with dual-emission quantum dots?QDs?nanohybrid,a ratiometric fluorescent nanoprobe?QDs-DI?is developed for G-quadruplex?G4?detection in a sensitive and specific manner.The QDs nanohybrid comprised of a green-emission QD?gQD?and multiple red-emission QDs?rQDs?inside and outside of a silica shell,respectively,is utilized as the signal displaying unit.Only the presence of G4 can displace the ligand from QDs,breaking up the QDs-DI complexation,and inducing the restoration of the rQDs fluorescence.Since the fluorescence of embedded gQD stays constant,variations of the dual-emission intensity ratios display continuous color changes from green to bright orange,which can be clearly observed by naked eyes.Furthermore,by utilizing competitive binding of a cationic ligand versus the non-ionic ligand toward G-quadruplex,the nanoprobe is demonstrated to be applicable for assessing the affinity of a G4-targeted anti-cancer drug candidate,exhibiting ratiometric fluorescence signals?reverse to that for G4 detection?.By functionalizing with a nuclear localization peptide,the nanoprobe can be used for visualization of G4 in the nucleus of human cells.Furthermore,via the G4 imaging,the nanoprobe is demonstrated to be applicable for differentiation of normal cell/cancer cell.3.A fluorescent nanoprobe?QD@ZnPc?is developed for in situ imaging of G4in nucleus.The green-emission QD is first functionized with PEG,folic acid?FA?and TAT peptide for enhanced membrane-permeability and nucleus-targeting.A G4 ligand,Zn???N-methylpyridyloxyphthalocyanine?ZnPc?,is assembled onto the surface of bio-functionized gQD via ultrasonic-assisted electrostatic interaction.The developed fluorescent probe features small size,neutrality,biocompatibility,high stability and nucleus-targeting ability.The presence of G4 in nucleus can break the gQD@ZnPc complexation,and form the G4/ZnPc complexes,which emit red emission.The fluorescent probe shows high specificity toward G4 against other DNA/RNA forms.During imaging,the gQD emission can be used for tracking the internalization of the nanoprobe into the cells,while the red ZnPc emission is used to image G4 in the nucleus with high resolution.Furthermore,via the G4 imaging,the nanoprobe is demonstrated to be applicable for monitoring of a cell cycle of HepG2 cell.