| Fluorescence microscopy imaging because of its high sensitivity and spatial resolution, has become a widely used technology in biological sensing and imaging. However, this intensity-based technology is affected by the change of excitation power, concentration of probe and short-lived autofluorescence. Thus it can only qualitatively reflect the fluorescence intensity of organisms somewhere, but can not accurately obtain information in organisms. In contrast, Luminescence lifetime is independent of these factors that can been utilized for application of bioimaging via time-resolved luminescence imaging. Especially, long- lived sensor can be distinguished from short- lived background fluorescence via time-resolved photoluminescence imaging techniques, which can greatly improve the sensitivity and accuracy in biosensing. In several important long lifetime fluorescent probes, phosphorescent transition metal complexes have been attracting much interest in intracellular imaging and sensing because of their high photostability, high phosphorescence quantum yields, the ability for wide spectral tuning and longer lifetime. Silica nanoparticles exhibit optically transparent, good hydrophilic, biocompatible and easy functionalized that make them as an ideal platform for fluorescent probes. In this work, we have combined the silica nanopaticles with iridium(III) complexes to construct two-channel phosphorescent silica nanoprobe for detection of biological analytes(fluoride, hypochlorite and Oxygen Levels) in live cells via ratiometric and time-resolved luminescence imaging. The main work is as follows:1. Using strong affinity between silicon and fluorine, A yellow-emitting Iridium complexes 1 with an excellent selectivity and sensitivity to fluoride has been designed and synthesized. A blue-emitting and long- lived Ir complex 2 which is unsensitive to fluoride was used as the reference signal doped in solid core. Next, Iridium complexes 1 was covalently linked to the surface of silica nanopaticles. This reaction-based two-channel phosphorescent nanosensor has fast response speed(< 10 s) and high selectivity to F- in the pure water system and living cells. In addition, compared with the probe based on the single strength changes, the ratio method can effectively reduce the interference of the environmental factors to improve the accuracy of detection. Notably, this nanoprobe has been used for ratiometric and time-resolved luminescence imaging of intracellular F- based on the F-- induced variation in phosphorescent intensity and lifetime.2. Core-shell structure nanoparticles with uniform particle size and good water-dispersion have been developed, which provides a simple and effective method for the construction of luminescent ratiometric nanoprobes. The Ir complex 1 doped in solid core was protected by outer shell from oxygen quenching, the large surface area of mesoporous shells to ensure high effeciency of the entrapped Ir complex 2 to detection of ClO-. Combined with ratio method and time-resolved technique, the dual phosphorescent core-shell phosphorescence probes has used to detection of hypochlorite in pure water system and living cells.3. We developed dual phosphorescent nanoprobes for sensing of Oxygen Levels based on core-shell structure nanostructure. Rhodamine B with high fluorescence quantum yield and short lifetime was acted as the interference fluorescence. Taking advantage of long lifetimes of two phosphorescent transition metal complexes, ratiometric and lifetime-based measurements of nanoprobe under different oxygen levels were performed. Compared to intensity-based luminescent probe, ratiometric and time-resolved luminescence imaging are less affected by external influence and short- lived background interference fluorescence, which can greatly improve the detection accuracy and sensitivity. In addition, we realize the quantitative detection of the oxygen levels in living cells. |
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