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Fluorescent Nanoparticle-Based Mitochondrial Labeling and Cell Imaging

Author: YuanZuo
Tutor: HeXiaoXiao；WangKeMin
School: Hunan University
Course: Biomedical Engineering
Keywords: mitochondria fluorescent silica nanoparticles gold nanocluster cell subcellular
CLC: TB383.1
Type: Master's thesis
Year: 2010
Downloads: 223
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Abstract

In recent years, fluorescent nanoparticles have been widely used in fluorescence labeling at cellular and subcellular level in cell biology research as it has excellent fluorescence characteristics, which have provided new technological methods for the biological analysis and the biomedical research. Based on the fluorescent silica nanoparticles’platform in our laboratory, we first carried out the study of using fluorescent silica nanoparticles for subcellular labeling. The results showed that the big diameter of nanoparticles was a block for subcellular targeting. Then, gold clusters with small size were also investigated to be used as a potential fluorescence marker for cell labeling.1. Study on the target interaction between mitochondria and signal peptide functionalized silica nanoparticlesThe mitochondrial signal peptide functionalized fluorescent silica nanoparticles were prepared by conjugated the mitochondrial signal peptide to the nanoparticles through N-(p-Maleimidophenyl)isocyanate (PMPI). The fluorescence spectrophotometer, zeta sizer and TEM have been used to characterize the fluorescent silica nanoparticles. By using the isolated nucleus as a control, the target interaction between the mitochondrial signal peptides modified fluorescent silica nanoparticles and the isolated mitochondria has been investigated by flow cytometry. The results showed that the signal peptides were successfully conjugated to the surface of fluorescent silica nanoparticles, and the size of the fluorescent silica nanoparticles did not change obviously before and after modification. It was revealed that the bioactivity of the signal peptide was well remained after the conjugation with the silica nanoparticles, and the peptides could facilitate the specific recognition and binding between the silica nanoparticles and the isolated mitochondria, which put forwards a new idea for the monitoring and function controlling of the mitochondria.2. Study on the signal peptide functionalized silica nanoparticles targeting of mitochondria in living cellsBased on the above research work, we investigated the mitochondrial targeting ability of signal peptide functionalized silica nanoparticles in living Hela cells. The functionalized fluorescent silica nanoparticles were incubated with Hela cell, and then the subcellular distribution was studied by cofocal microscopy in the situation that the lysosome and mitochondrion were costained by LysoTracker Green and MitoTracker Deep Red. It was found that the functionalized fluorescent silica nanoparticles can enter cells through endocytosis. However, its further movement was prevented by the lysosome. Although the cells were further treated with chloroquine in order to release the nanoparticles into the cytoplasm, experimental results showed that most of nanoparticles were adsorbed in the lysosomal membrane and retained in the lysosome and could not able to reach the mitochondria. The results indicated that the nanoparticles with smaller size, better dispersion and low specific absorption should be prepared to make better use of fluorescent nanoparticles for subcellular labeling.3. Study on the live cells fluorescence imaging by bovine serum albumin-mediated synthesized gold nanoclustersAs discussed aboved, it is necessary to develope nanoparticles with smaller size for cell labeling. In recent years, gold nanoclusters have attracted the attention of scientists in the field of nano-materials because of its improved fluorescent properties and the advantages of small size and nontoxic. In this chapter, the gold nanocluster was synthesized by bovine serum albumin directing, and was characterized by fluorescence spectrophotometer, zeta sizer and non-denaturing polyacrylamide gel electrophoresis. And further study was carried out to investigate the interaction between gold nanoclusters and Hela cells, and the gold nanoclusters were conjugated with biotin to investigate the feasibility of being used as a fluorescent marker.The results showed that the gold nanocluster’s fluorescence signal was strong and stable at different pH. It demonstrated that the gold naoclusters could be successfully uptaken by living cells. The good effects of the fluorescent labeling of living cells can be achieved by using optimal incubation time and gold nanoclusters’concentration. Moreover, the fixation treatment did not change the form of labeling. Furthermore, the gold-nanoclusters can also be used as fluorescent markers to label the biotin and did not affect the interaction between biotin and avidin. These results provide some guidance to the further application of fluorescent gold nanoclusters in the field of live cells labeling.

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