The project aims to develop ratiometric near-infrared (NIR) fluorescent probes for the precise and
quantitative analysis of lysosomal and mitochondrial pH changes in live cells, and for effective monitoring of the
mitochondrial delivery to the lysosomes during mitophagy caused by nutrient starvation, neurotoxic
aminochrome and drug treatment. We will develop ratiometric near-infrared fluorescent probes for sensitive
ratiometric detection of intracellular pH changes by electrically conjugating TPE donors to near-infrared
hemicyanine acceptors with spirolactam on-off switches based on a TBET (through-bond energy transfer)
strategy, or by incorporating hemicyanine or cyanine dyes into rhodol dyes with spirolactam or spiropyran rings
on-off switches based on a π-conjugation modulation strategy. The water solubility and biocompatibility of the
probes will be achieved by introducing long oligo(ethylene glycol) residues or mannose residues through
oligo(ethylene glycol) tethered spacers to the probes. Morpholine and triphenylphosphonium residues through
an oligo(ethylene glycol) spacer will be attached to fluorescent probes for specific targeting of lysosomes and
mitochondria in live cells, respectively. Ratiometric near-infrared fluorescent probes based on the TBET strategy
will only display TPE donor fluorescence under neutral and basic pH conditions because the hemicyanine
acceptors are non-fluorescent with closed spirolactam ring structures in mitochondrial slightly basic condition
(pH 8.0). A lysosomal acidic environment will trigger the opening of the acceptor spirolactam ring structures,
significantly increase hemicyanine acceptor fluorescence at 740 nm, and considerably reduce TPE donor
fluorescence at 640 nm through high efficiency of through-bond energy transfer from the TPE donors to the
hemicyanine acceptors. The ratiometric near-infrared fluorescent probes based on π-conjugation strategy will
only show fluorescence of hemicyanine moieties under neutral and basic pH conditions since rhodol moieties
will keep closed spirolactam or spiropyran ring structures in mitochondrial slightly basic condition. A lysosomal
acidic environment will trigger the opening of the spirolactam or spiropyran rings ring structures of rhodol
moieties, significantly enhance π-conjugation between hemicyanine and rhodol moieties, and result in
ratiometric fluorescence responses to pH changes. The probes will possess advantages such as deep tissue
penetration, minimum cell damage and interference from biological autofluorescence, large pseudo-Stokes shifts
to overcome measurement errors by excitation and scattered lights, self-calibration ratiometric responses with
dual-emission capability, excellent water solubility, high stability, high cell permeability, good biocompatibility,
excellent intracellular retention, high selectivity, sensitivity, fast and reversible responses to pH changes. We
will apply these fluorescent probes for the precise and quantitative analysis of lysosomal and mitochondrial pH
changes in HEK293 cells, neuronal cells, HeLa, and breast cancer cells to investigate pH changes and monitor
delivery process of the mitochondria to the lysosome during the mitophagy for insightful understanding of
physiological and pathological processes.
