An aggregation-induced emission (AIE) cyanine-based fluorescent cassette with a large pseudo-Stokes shift was designed and prepared to sensitively image pH changes in live cells via through-bond energy transfer (TBET) from a tetraphenylethene (TPE) donor to a cyanine acceptor.

The specificity of carbohydrate transporters towards their substrates poses a significant challenge for the development of molecular probes to monitor sugar uptake in cells for biochemical and biomedical applications. Herein we report a new set of coumarin-based fluorescent sugar conjugates applicable for the analysis of fructose uptake due to their free passage through the fructose-specific transporter GLUT5. The reported probes cover a broad range of the fluorescence spectrum providing essential tools for the evaluation of fructose transport capacity in live cells.

In this paper, we present three ratiometric near-infrared fluorescent probes (A-C) for accurate, ratiometric detection of intracellular pH changes in live cells. Probe A consists of a tetraphenylethene (TPE) donor and near-infrared hemicyanine acceptor in a through-bond energy transfer (TBET) strategy, while probes B and C are composed of TPE and hemicyanine moieties through single and double sp carbon-carbon bond connections in a π-conjugation modulation strategy. The specific targeting of the probes to lysosomes in live cells was achieved by introducing morpholine residues to the hemicyanine moieties to form closed spirolactam ring structures. Probe A shows aggregation-induced emission (AIE) property at neutral or basic pH, while probes B and C lack AIE properties. At basic or neutral pH, the probes only show fluorescence of TPE moieties with closed spirolactam forms of hemicyanine moieties, and effectively avoid blind fluorescence imaging spots, an issue which typical intensity-based pH fluorescent probes encounter. Three probes show ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with TPE fluorescence decreases and hemicyanine fluorescence increases, because acidic pH makes the spirolactam rings open to enhance π-conjugation of hemicyanine moieties. However, probe A shows much more sensitive ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with remarkable ratio increase of TPE fluorescence to hemicyanine fluorescence up to 238-fold than probes B and C because of its high efficiency of energy transfer from TPE donor to the hemicyanine acceptor in the TBET strategy. The probe offers dual Stokes shifts with a large pseudo-Stokes shift of 361 nm and well-defined dual emissions, and allows for colocalization of the imaging readouts of visible and near-infrared fluorescence channels to achieve more precisely double-checked ratiometric fluorescence imaging. These platforms could be employed to develop a variety of novel ratiometric fluorescent probes for accurate detection of different analytes in applications of chemical and biological sensing, imaging, and diagnostics by introducing appropriate sensing ligands to hemicyanine moieties to form on-off spirolactam switches.

Near-infrared hybrid rhodol dyes (probes A and B) for sensitive ratiometric visualization of pH changes were prepared by incorporating hemicyanine dyes into traditional rhodol dyes. This approach was based on π-conjugation changes involving a rhodol hydroxyl group as a spiropyran switch upon pH changes. Probes A-2 and B-2 consist of a rhodol fluorophore and an N,O-disubstituted hemiaminal ether residue linked through a cyclic carbon–carbon double-bonded connection. Electronic spectra of probes A-2 and B-2 contain sharp absorption peaks at 535 nm and fluorescence peaks at 558 nm with similar π-conjugation and a closed spiropyran form at a basic pH of 10.2. However, acidic pH conditions break down the hemiaminal ether groups, leading to indolenium moieties and significantly extending the π-conjugation within the rhodol fluorophores, resulting in additional near-infrared emissions for probes A-1 and B-1. As a result, probes A and B exhibit gradual decreases of the absorption peaks at 535 nm and gradual increases in absorption peaks at 609 and 622 nm upon transition from basic to acidic pH, respectively. Both probes display ratiometric fluorescence sensing responses to pH downgrades from 10.2 to 3.6 with visible fluorescence decreases at 558 nm, as well as corresponding increases of the near-infrared fluorescence peaks at 688 and 698 nm, respectively. They exhibit fast, sensitive, and selective fluorescence responses with clearly defined ratiometric features to pH changes and show low cytotoxicity and excellent cell permeability. Our probes were successfully applied to ratiometrically detect pH changes in mitochondria, D. melanogaster first-instar larvae, and to visualize the mitophagy process caused by either cell nutrient starvation or drug treatment.

A fluorescence resonance energy transfer (FRET)-based near-infrared fluorescent probe (B+) for double-checked sensitive detection of intracellular pH changes has been synthesized by binding a near-infrared rhodamine donor to a near-infrared cyanine acceptor through robust C-N bonds via a nucleophilic substitution reaction. To demonstrate the double-checked advantages of probe B+, a near-infrared probe (A) was also prepared by modification of a near-infrared rhodamine dye with ethylenediamine to produce a closed spirolactam residue. Under basic conditions, probe B+ shows only weak fluorescence from the cyanine acceptor while probe A displays nonfluorescence due to retention of the closed spirolactam form of the rhodamine moiety. Upon decrease in solution pH level, probe B+ exhibits a gradual fluorescence increase from rhodamine and cyanine constituents at 623 nm and 743 nm respectively, whereas probe A displays fluorescence increase at 623 nm on the rhodamine moiety as acidic conditions leads to the rupture of the probe spirolactam rings. Probes A and B+ have successfully been used to monitor intracellular pH alternations and possess pKa values of 5.15 and 7.80, respectively.

New near-infrared rhodamine dyes with large Stokes shifts were developed and applied for sensitive detection of cellular pH changes and fluctuations by incorporating an additional amine group with fused rings into the rhodamine dyes to enhance the electron donating ability of amine groups and improve the spectroscopic properties of the dyes.

Three near-infrared ratiometric fluorescent probes with different pKa values were synthesized to achieve sensitive ratiometric visualization of pH variations in lysosomes.

We report two ratiometric fluorescent probes based on π-conjugation modulation between coumarin and hemicyanine moieties for sensitive ratiometric detection of pH alterations in live cells by monitoring visible and near-infrared fluorescence changes. In a π-conjugation modulation strategy, a coumarin dye was conjugated to a near-infrared hemicyanine dye via a vinyl connection while lysosome-targeting morpholine ligand and o-phenylenediamine residue were introduced to the hemicyanine dye to form closed spirolactam ring structures in probes A and B, respectively. The probes show only visible fluorescence of the coumarin moiety under physiological and basic conditions because the hemicyanine moieties retain their closed spirolactam ring structures. However, decrease of pH to acidic condition causes spirolactam ring opening, and significantly enhances π-conjugation within the probes, thus generating new near-infrared fluorescence peaks of the hemicyanine at 755 nm and 740 nm for probes A and B, respectively. Moreover, the probes display ratiometric fluorescence response to pH with decreases of the coumarin fluorescence and increases of the hemicyanine fluorescence when pH changes from 7.4 to 2.5. The probes are fully capable of imaging pH changes in live cells with good ratiometric responses in visible and near-infrared channels, and effectively avoid fluorescence blind spots under neutral and basic pH conditions - an issue that typical intensity-based pH fluorescent probes run into. The probe design platform reported herein can be easily applied to prepare a variety of ratiometric fluorescent probes for detection of biological thiols, metal ions, reactive oxygen and nitrogen species by introducing appropriate functional groups to hemicyanine moiety.