A coumarin-based fluorescent chemosensor as a Sn indicator and a fluorescent cellular imaging agent

Abstract

In the present study, fluorogenic coumarin-based probes (1-3) through condensation of 4-hydroxy coumarin with malondialdehyde bis(diethyl acetal)/triethyl orthoformate were prepared. The absorption and fluorescence emission properties of 2b and 3 in different solvents were studied, and a considerable solvatochromic effect was observed. The sensitivity of chemosensors 2b and 3 toward various cations and anions was investigated. It was revealed that compound 3 had a distinct selectivity toward Sn²⁺, possibly via a chelation enhanced quenching mechanism. The fluorescence signal was quenched over the concentration range of 6.6-120 μM, with an LOD value of 3.89 μM. The cytotoxicity evaluation of 3 against breast cancer cell lines demonstrated that the chemosensor was nontoxic and could be used successfully in cellular imaging. The probe responded to tin ions not only via fluorescence quenching, but also through colorimetric signal change. The change in optical properties was observed in ambient conditions and inside living cells.

Scheme 1. Synthesis route of compounds 1–3.

Fig. 1. (A) Fluorescence emission of 2b (5.0 × 10⁻⁵ M) in the presence of different ions (10 eq.), 2b was solubilized in DMF–H₂O (1/5, v/v) with λ_ex of 470 nm; (B) fluorescence emission of 3 (5.0 × 10⁻⁵ M) in response to various ions (10 eq.), 3 was solubilized in EtOH–H₂O (2/3, v/v) with λ_ex of 480 nm. The concentration of sodium and potassium ions is 34 mM and 120 mM, respectively, equal to the values found in normal lymphocytes.

Fig. 2. (A) Fluorescence spectra of compound 3 (2.0 × 10⁻⁴ M) with the addition of various concentrations of Sn²⁺ (0–0.2 mM, λ_ex = 480 nm). (B) The SnCl₂ concentration effect on fluorescence quenching.

Fig. 3. (A) Fluorescence quenching behavior of 3, (B) the absorbance spectra of 3 and 3 + Sn²⁺, (C) the difference spectrum of 3 and 3 + Sn²⁺.

Fig. 4. Job's plot for the complex formation between 3 with Sn²⁺.

Fig. 5. Cytotoxicity of compound 1–3 against MCF-7 cells.

Fig. 6. Bright-field microscopy images of the probe in MCF7 cells (A). The cells were incubated with a probe (2.5 × 10⁻⁵ M) for 60 min at 37 °C and washed with PBS three times (B). DAPI staining (C). Red emission was collected at 645 ± 75 nm; Blue emission was collected at 470 ± 40 nm. Digitally merged image (D).

Fig. 7. The MCF7 cells with (2.5 × 10⁻⁵ M) probe were further incubated with SnCl₂ 1.0 × 10⁻⁴ M (A). 10 to 30 min later, the fluorescence images were obtained on a fluorescent microscope with an objective lens (×200). A significant decrease in the probe's fluorescence signal was observed after incubating cells with SnCl₂ (B and C).

Fig. 8. Bright field microscopic images of the probe in MCF7 cells. The cells were incubated with probe (2.5 × 10⁻⁴ M) (A) for 60 minutes at 37 °C and washed with PBS 3 times. Then the cells were treated and incubated with SnCl₂ ions 1.0 × 10⁻⁴ M for 15 minutes at 37 °C (B).

Fig. 9. Fluorescence emission of 3 and its complexes with Sn²⁺, Li⁺, Na⁺, and Cl⁻ ions in EtOH–H₂O (2/3) solvent.

Fig. 10. Real-space representation of hole (blue) and electron (green) distributions for the main transitions of 3 and its complexes with Sn²⁺, Li⁺, Na⁺, and Cl⁻ (isovalue = 0.001).