Fluorescent Probes Based on π-Conjugation Modulation between Hemicyanine and Coumarin Moieties for Ratiometric Detection of pH Changes in Live Cells with Visible and Near-infrared Channels

Abstract

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.

Keywords: Fluorescent probe; Ratiometric imaging; live cells; near-infrared emission; pH.

Figure 1

Absorbance spectra of 5 μM intermediate 7 (left), probe A (middle) and probe B (right) in 10 mM citrate buffer containing 40% EtOH.

Figure 2

Fluorescence spectra of 5 μM probe A in 10 mM citrate buffer containing 40% EtOH at Fluorescence excitation of 420 nm (left), 420 nm (right) and 680 nm (right), respectively; pH dependent fluorescence ratios of hemicyanine acceptor (I_{755 nm}) to coumarin donor (I_{528 nm}) at excitation of 420 nm (middle).

Figure 3

Fluorescence spectra of 5 μM probe B in 10 mM citrate buffer containing 40% EtOH at excitation of 420 nm (left), 420 nm (right) and 680 nm (right), respectively; pH dependent fluorescence ratios of hemicyanine acceptor (I_{740 nm}) to coumarin donor (I_{515 nm}) at excitation of 420 nm (middle).

Figure 4

Plots of fluorescent intensity of 5 μM probe A (left) at 755 nm and probe B (right) at 740 nm versus pH at 680 nm excitation with three repeated measurements.

Figure 5

Cytotoxicity and cell proliferation of probes A and B conducted by MTS assay. HeLa cells were incubated with 2, 5, 10, 15, and 20 μM of probes for 48 h, and cell viability was measured by adding MTS reagent and measuring at 490 nm. The absorbance measured at 490 nm was directly proportional to the cell viability and was normalized to control cells in the absence of probes.

Figure 6

Fluorescence images of HeLa cells incubated with different concentrations of probe A in the presence of LysoTracker Red. Images were acquired using the confocal fluorescence microscope at 60 X magnification. Scale Bar: 50 μM

Figure 7

Enlarged fluorescence images of HeLa cells incubated with different concentrations of probe A in the presence of LysoTracker Red.

Figure 8

Fluorescence images of HeLa cells incubated with 15 μM fluorescent probe A. HeLa cells were incubated with 15 μM probe A at the pH ranging from pH 3.0 to 7.0 in presence of 5 μg/mL nigericin. Images were acquired using the confocal fluorescence microscope at 60 X magnification. Scale Bar: 50 μM

Figure 9

Fluorescence images of HeLa cells incubated with 15 μM fluorescent probe B. HeLa cells were incubated with 15 μM probe B at the pH ranging from pH 3.0 to 7.0 in presence of 5 μg/mL nigericin. Images were acquired using the confocal fluorescence microscope at 60 X magnification. Scale Bar: 50 μM

Figure 10

Fluorescence intensity of probe A (left) and probe B (right) in the HeLa cells obtained from statistical analysis of the confocal imaging in figures 8 and 9.

Scheme 1

Chemical structure response of fluorescent probes to pH changes with π-conjugation changes.

Scheme 2

Synthetic strategy to prepare a ratiometric fluorescent probes based on π-conjugation modulation between coumarin and hemicyanine dyes.