Tailoring Fluorescent Probes for Organelle-Specific Imaging and Sensing

Abstract: Exploring and visualizing biological and pathological processes in living cells are useful for both fundamental research and applications. An understanding on the molecular level for these processes with focus on tracking biomarkers, collecting information about their surroundings, and uncovering essential molecular pathways and functions in live cells are of vital interest for cell biological study and clinical diagnosis application. Fluorescent imaging technologies have become essential tools in cell biology studies, providing dynamic information about the localization and quantity of the analytes in living systems that we can not see by our naked eyes. Since the discovery of organic fluorescent dyes, unremitting efforts have been made to visualize the behaviors of specific targets by using these fluorophores as labels. Today, a variety of probes and nanoprobes have been developed for specific targeting and sensing. However, the current probes and nanoprobes still show some inherent deficiencies, such as poor specificity, strong photobleaching, high toxicity, low signal-to-noise ratio, poor cell membrane penetration, etc. To conquer these limitations, this thesis will focus on developing fluorescent platforms for bioimaging and biosensing with improved sensitivity, selectivity, specificity, and stability.A red-emitting fluorescent probe is first proposed that not only tracks the dynamic changes in real-time, during migration and fusion of lipid droplets but also monitors starvation-induced lipophagy (Paper I). In addition to organic fluorophores, hybrid fluorescent silica nanoparticles (SiNPs) are very promising for bioimaging/sensing owing to the advantages of low toxicity, high biocompatibility, multifunctionality, hydrophilicity and accessible surface functionality. Nonetheless, to apply SiNPs for such purposes, it is mandatory to address common problems of poor cell penetration and lack of specificity. Therefore, in this thesis, an efficient membrane-penetration SiNP is tailored with the intention to enable subcellular imaging/sensing. The proposed SiNPs are characterized by rapid cellular uptake (˂ 30 min) and specific subcellular targeting capabilities with surface modification. Finally, real-time tracking of dynamic changes in mitochondria and lysosomes during autophagy process is successfully performed (Paper II). Through the rational design of new functionalities on the established SiNPs, mitochondria- and lysosome-specific pH nanoprobes are further tailored for real-time monitoring of pH variations under various stimuli (Paper III and IV). Another ratiometric nanoprobe is developed for quantitative indication of lysosomal adenosine 5'-triphosphate (ATP) levels in living cells. The nanoprobe enables a deeper understanding of the interplay between energy metabolism and autophagy (Paper V).In conclusion, throughout the studies in this thesis, the fabrication and utilization of fluorescent molecular probes and SiNP-based nanoprobes for cellular probing have been investigated and analyzed. The strategies and the fabricated SiNPs can facilitate our deeper understanding of cellular pathological processes and provide basic knowledge for the development of other functional materials for life science applications.