Synthesis Of Two-photon Fluorescent Hypochlorous Acid Probe For Bioimaging

Two-photon absorption technology has received wide attentions since it emerged.Two-photon absorption materials have advantages over the one-photon absorption materials,they can be widely applied into many fields,such as microscopy,microfabrication,three-dimentional data storage,optical limiting,up-converting emitting,photo-dynamic therapy and drug release.Two-photon microscopy with the near-infrared laser beam as the light source can be applied for deep-tissue imaging with high spatial resolution and little damage of samples caused by excitation light.However,conventional two-photon absorption materials with large two-photon absorption action cross section usually possess extended conjugated planar structures,which limited their biological application.Therefore,it is highly desired to design some small molecules with large two-photon absorption action cross section for bio-imaging.In this thesis,we designed and synthesized two small two-photon fluorescence molecules and explored their application in bioimaging.The details of the study including:1.A two-photon hypochlorous acid probe OPV-HOCl was designed and synthesized,and then it was applied to the detection of HOCl in live cells and tissues.The probe consists of oligo(phenylene vinylene)group as the two-photon fluorophore,and two oxathiolane moieties as the reaction sites of HOCl.The structure of the probe was characterized by 1H-NMR,13C-NMR and HRMS spectra,and its response to HOCl was studied by UV-Vis and fluorescence spectra.The two-photon absorption cross section of the probe was also measured.Upon addition of hypochlorous acid,the two oxathiolane groups on the probe was oxidized and became aldehyde groups.Due to the strong intramolecular charge transfer,the two-photon absorption cross section of the product displayed a remarkable increase by nearly 15-folds(form 78.9 GM to 1131.5 GM).Furthermore,the probe displayed fast response,high selectivity to HOCl over other ROS in a broad pH range.MTT assay also confirmed the probe with low cytotoxicity.Benefited by these desired sensing properties and biocompatibility,the probe was successfully applied to the determination of hypochlorous acid in living microglia BV-2 cells by two-photon fluorescence imaging.The results showed that the probe can penetrate into the cytoplasm of cells,and response to exogenous and LPS-induced endogenous hypochlorous acid with a “turn-on” signal.2.We prepared polystyrene microspheres loaded with the two-photon squaraine dye SQA-Br,and applied them into the detection of the stroke area.The fluorescence spectra of the two-photon dye-doped polystyrene microspheres showed that these microspheres can provide a relative inert condition for the fluorescent molecule,and make them retain good luminescent properties without aggregation-induced fluorescence quenching.Furthermore,the fluorescence quantum yield and lifetime of the dye increased in the microspheres.These fluorescent microspheres were also injected into the stroke mouse,and the result showed that the fluorescent microspheres spreaded out through the stoke damage areas and accumulated around the peripheral region.