| Fluorescent probes, achieving the qualitative and quantitative analysis of the specific objectives mainly through its changes of color or fluorescence, have a series of advantages, like high sensitivity, high selectivity, short reaction time, fast operation and so on. Fluorescent probes play a more and more important role in environmental pollution detection and life sciences area application. Compared with One-photon microsopy, Two-photon microscopy (TPM), which employs two photons for the excitation in the near-infrared wavelength (ca.700-1100 nm), provides an opportunity to overcome the problems originated from the single-photon fluorescence technology, such as excitation with short-wavelength light (ca.350-550 nm), photo-bleaching, photo-damage, and cellular auto fluorescence. It has becoming hot topics and research areas, attracting more and more researchers today.As a major source of cellular reactive sulphur species (RSS) and reactive oxygen species (ROS), mitochondria play a central role in RSS and ROS biology. Sulfur dioxide (SO2) in mitochondrial has been considered to significantly reduce myocardial injury and isopropylarterenol-induced mitochondrial swelling and deformation. And mitochondrial biogenesis increases after exposure to SO2 in the brain. Thus, monitoring SO2 derivatives in mitochondria is particularly meaningful and valuable. On the other hand, Hypochlorite (ClO-), a type of reactive oxygen species (ROS), is a powerful antimicrobial agent that plays an important role in the immune system. Studies have suggested that abnormal levels of ClO- are involved in several diseases, such as Parkinson’s disease, Alzheimer’s disease, multiple sclerosis and cancer. Therefore, it’s of great importance to develop the efficient method for monitoring the level of ClO- in mitochondria.After reading lots of literatures and research, we designed two types of carbazole-based mitochondrial-targeted two-photon fluorescence probe in this paper. The structure of the compounds were confirmed by 1H NMR,13C NMR spectra and ESI-TOF mass spectrometry characterization. We further study their optical properties, recognition performance and mitochondria-targeted two-photon imaging properties in living cells, a series of positive results have been achieved.1. Introduced Two-photon microscopy briefly, and an intro of mitochondria-targeted fluorescence probes.2. In this work, two carbazole-based two-photon fluorescent probes PCB and MPCB for SO2 derivatives detection utilizing the Michael-addition mechanism were developed by incorporating pyridine/methyl pyridinium and methylbenzothiazolium moiety to the carbazole framework. The electron-donating carbazole and electron-withdrawing pyridine/cationic pyridinum moieties formed a novel donor-π-acceptor (D-π-A) structure and endowed the probes with outstanding two-photon properties. The proposed probes showed excellent selectivity toward SO2 derivatives over varieties of other species, with rapid response, low detection limits, and large Stokes shifts. As expected, probe MPCB possessed the red-shifted spectra properties, better water-solubility, larger two-photon excitation action cross-section than PCB due to the introduce of a methyl pyridinium group. More importantly, MPCB has been successfully applied to the mitochondria-targeted two-photon imaging of SO2 derivatives in living HeLa cells, due to the utility of the methyl pyridinium moiety as a mitochondria-targeted functional group to realize the subcellular localization. Co-staining experiments of MPCB and MitoTracker Red FM (co-localization coefficient: 0.89) revealed that MPCB was predominantly present in mitochondria.3. we developed a diaminmaleonitrile-based Schiff base derivative HCCN, as a mitochondria-targeted two-photon fluorescent probe for CIO- detection with rapid response, low detection limits, large Stokes shift and two-photon excitation action cross-section, which utilized the de-diaminmaleonitrile reaction by ClO- to the corresponding aldehyde derivative. We found HCCN is highly selective for ClO-detection without the interference of other metal ions (especially for Cu2+) in CH3CN/H2O and other mixed solvent systems. We speculated that HCCN may give a remarkable fluorescence "turn-on" response to ClO- due to the enhanced ICT efficiency, which could be clearly elucidated by DFT/TDDFT calculation. The high selectivity of HCCN for ClO- detection without the interference of other metal ions (especially for Cu2+) may also be explained by calculation results. Moreover, HCCN contains the methyl pyridium moiety which has been extensively utilized as a mitochondria-targeted functional group. Bioimaging experiments revealed that HCCN can be used for the real-time sensing and bioimaging of ClO- in mitochondria under two-photon excitation, owing to the large two-photon action cross sections and good biocompatibility. |
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