A novel water-soluble naked-eye probe with a large Stokes shift for selective optical sensing of Hg2+ and its application in water samples and living cells

Abstract

A water-soluble and colorimetric fluorescent probe with a large Stokes shift (139 nm) for rapidly detecting Hg²⁺, namely Hcy-mP, was synthesized by using an indole derivative and 2,4-dihydroxybenzaldehyde as starting materials. This probe demonstrates good selectivity for Hg²⁺ over other metal ions including Ag⁺, Pb²⁺, Cd²⁺, Cr³⁺, Zn²⁺, Fe³⁺, Co²⁺, Ni²⁺, Cu²⁺, K⁺, Na⁺, Mg²⁺, and Ca²⁺ in aqueous solution. With the increase in concentration of Hg²⁺, the color of the solution changed from pale yellow to pink and the fluorescence intensity decreased slightly. When 5-equivalents of EDTA were added to the solution with Hg²⁺, the fluorescence intensity of this probe was restored. The probe has been applied to the detection of Hg²⁺ in real water samples. Moreover, this probe was confirmed to have low cytotoxicity and excellent cell membrane permeability. The effect of Hcy-mP-Hg²⁺ towards living cells by confocal fluorescence was also investigated.

Scheme 1. The synthetic routes of probe Hcy-mP.

Fig. 1. Absorption and emission spectra of Hcy-mP (λ_ex = 378 nm, λ_em = 517 nm, Stokes shift = 139 nm).

Fig. 2. (a) UV-vis spectra of Hcy-mP (10 μM) upon the addition of different metal ions (10 μM) in 100% HEPES buffer solution (pH = 7.0); (b) fluorescence spectra of Hcy-mP (10 μM) upon the addition of different metal ions (10 μM) in HEPES buffer solution (λ_ex = 378 nm, silt = 10 nm).

Fig. 3. Fluorescence intensity of Hcy-mP (10 μM) upon addition of metal ions (20 μM) and addition of Hg²⁺ (10 μM) in HEPES buffer solution (λ_ex = 378 nm, silt = 10 nm).

Fig. 4. (a) Effects of different concentrations of Hg²⁺ on the fluorescence spectra of Hcy-mP (10 μM) at different Hg²⁺ concentrations (from top to bottom Hg²⁺ concentrations are 0–25 μmol L⁻¹). (b) and (c) Naked-eye color of Hcy-mP solution in HEPES buffer in the absence (left, bright) or presence (right, fluorescence) of Hg²⁺ (20 μM). (d) The scatter diagram of the fluorescence titration experiment.

Fig. 5. Effect of reaction time on fluorescence emission spectra of Hcy-mP and Hcy-mP with Hg²⁺ (20 μmol L⁻¹).

Fig. 6. (a) Absorption and (b) emission spectral changes of Hcy-mP (10 μM), after addition of Hg²⁺ (10 μM) and followed by EDTA (10 μM); (c) fluorescence intensity changes of Hcy-mP (10 μM) at 517 nm under addition of compounds (Hg²⁺ and EDTA) for 4 cycles (λ_ex = 378 nm).

Scheme 2. The structure of Hcy-pP.

Fig. 7. Fluorescence spectra of Hcy-pP (10 μM) upon the addition of different metal ions (20 μM) in HEPES buffer solution.

Fig. 8. Job plot of the Hcy-mP–Hg²⁺ mixture in HEPES (pH = 7.0) solution, keeping the total concentration of Hcy-mP and Hg²⁺ at 10 μM. The observed wavelength was 517 nm.

Fig. 9. The ¹H NMR for Hcy-mP and Hcy-mP + Hg²⁺ (2, 5 equiv.) in DMSO.

Scheme 3. Proposed sensing mechanism of Hcy-mP towards Hg²⁺.

Fig. 10. Cell viability HeLa cells.

Fig. 11. Confocal microscopy images of 10 μM Hcy-mP in HeLa cells at pH 7.4,3.0 and 11.0 (b, e and h) for 30 min Fluorescence images of cells were then further incubated with Hg²⁺ (10 μM) for 30 min (k). Cells were further treated with EDTA (10 μM) for 30 min. Bright field (a, d, g, j and m), fluorescence (b, e, h, k and n) and merged field (c, f, i, l and o). λ_ex = 378 nm.