A ratiometric fluorescent pH probe based on keto-enol tautomerization for imaging of living cells in extreme acidity.

6-(Diethylamino)-2,3-dihydro-1H-xanthene-4-carbaldehyde (DDXC), a reported synthetic intermediate for near-infrared fluorescent dyes, was developed into a fluorescent pH probe for extreme acidity. The unique sensing mechanism of DDXC for pH is based on the reversible protonation of the carbonyl oxygen followed by keto-enol tautomerization. The probe displays a linear ratiometric fluorescence response (I512/I580) to H+ over the extremely acidic range of pH 2.0-4.0 with a pKa of 3.11, and features high fluorescence quantum yield (Φ = 0.60) and excellent selectivity. More importantly, the probe can be applied to ratiometric fluorescence imaging of pH changes in living cells, making it a potential molecular tool for pH-related cell biology study.

A novel two-photon fluorescent probe with a long Stokes shift and a high signal-to-background ratio for human NAD(P)H:quinone oxidoreductase 1 (hNQO1) detection and imaging in living cells and tissues.

In recent years, many activatable fluorescent probes have been developed for hNQO1 detection. However, most of the reported fluorescent probes are susceptible to the interferences of endogenous fluorescence and have the drawback of inadequate penetration depth. Very recently, researchers have reported a two-photon excitation (TPE) fluorescent probe for hNQO1 detection. Nevertheless, this probe only exhibits a compromised signal-to-background ratio, and has not been applied to image hNQO1 in living tissues. Herein, a novel TPE fluorescent probe, trimethyl locked quinone caged Acedan (Q3CA-P), has been developed for hNQO1 detection and imaging in living cells and tissues. Q3CA-P displays over 25-fold enhancement in fluorescence intensity toward hNQO1 with a Stokes shift over 100 nm in one-photon excitation and exhibits a very low detection limit of 5.6 ng mL-1. The imaging experiments performed in tumour cells and tissue slices using Q3CA-P demonstrate that Q3CA-P could image the endogenous hNQO1 with high selectivity and sensitivity with a TPE probing depth of 120 µm. Thus, our probe may have great potential for use in cancer diagnosis and image-guided surgery.

A novel off-on fluorescent probe for sensitive imaging of mitochondria-specific nitroreductase activity in living tumor cells.

Sensitive and selective detection and imaging of nitroreductase (NTR) in cancer cells is of great importance for better understanding their biological functions. Since there are a few fluorescent probes concerning NTR activity specifically located in mitochondria, we developed a novel fluorescent benzoindocyanine probe (BICP) for mitochondrial NTR activity monitoring and imaging via extending a benzoindole moiety into a benzoindocyanine based fluorophore (BICF) with a strong intramolecular charge transfer (ICT) effect and incorporating 4-nitrobenzyl as a fluorescence-quenching and enzyme-responsive moiety. Live cell imaging of HeLa and A549 demonstrates that the developed BICP is able to realize sensitive and selective mitochondrial NTR activity probing with high-contrast "off-on" fluorescence. These findings implied the great potential of the developed probe for monitoring mitochondrial-specific NTR activities in living cells and related applications in cell biology.

A novel mitochondria-targeted near-infrared fluorescence probe for ultrafast and ratiometric detection of SO2 derivatives in live cells.

A novel mitochondria-targeted ratiometric near-infrared fluorescence probe NDMBT for Sulfur dioxide (SO2) derivatives was constructed based on the SO2 derivatives-triggered Michael addition reaction. It displayed ultrafast response time (within 10s), large hypsochromic shift (260nm), high photostability, excellent selectivity and high sensitivity in aqueous media with a detection limit of 43nM. More importantly, it was successfully applied to imaging of the enzymatically generated SO2 derivatives in mitochondria of live cells.

A novel fluorescent probe for sensitive detection and imaging of hydrazine in living cells.

A turn-on fluorescent probe (Naphsulf-O) for hydrazine was developed by protecting the hydroxy group of the fluorophore 6-acetyl-2-hydroxynaphthalene via O-4-nitrobenzenesulfonylation, where 4-nitrobenzene was used as a fluorescence quenching moiety as well as an electrophile. Upon nucleophilic aromatic substitution (NAS) reaction of hydrazine toward the probe, the protecting group was removed and fluorophore was released. The probe exhibits a large Stokes shift, excellent selectivity and high sensitivity for hydrazine detection in aqueous solution with a detection limit of 0.716 ppb (22nM), which is of great importance in both environmental and biological system. Furthermore, it was successfully applied to imaging of hydrazine in living cells.

Developing Activity Localization Fluorescence Peptide Probe Using Thiol-Ene Click Reaction for Spatially Resolved Imaging of Caspase-8 in Live Cells.

Small molecule probes suitable for high-resolution fluorescence imaging of enzyme activity pose a challenge in chemical biology. We developed a novel design of activity localization fluorescence (ALF) peptide probe, which enables spatially resolved, highly sensitive imaging of peptidase in live cells. The ALF probe was synthesized by a facile thiol-ene click reaction of a cysteine-appended peptide with an acryloylated fluorophore. Upon cleavage by peptidase, the probe undergoes a seven-membered intramolecular cyclization and releases the fluorophore with the excited-state intramolecular photon transfer (ESIPT) effect. A highly fluorescent, insoluble aggregate was formed around the enzyme, which facilitates high-sensitivity and high-resolution imaging. This design is demonstrated for detection of caspase-8 activation. The results show that our design allows easy, high-yield synthesis of the probe, and the probe affords high sensitivity for caspase-8 detection. Live cell imaging reveals that the probe is able to render highly localized and high-contrast fluorescence signal for caspase-8. Our design holds the potential as a generally applicable strategy for developing high-sensitivity and high-resolution imaging peptide probes in cell biology and diagnostics.