A mercury complex-based fluorescent sensor for biological thiols

Abstract

A novel fluorescent sensor, Hg(DST)₂, was developed for the selective detection of biological thiols, including glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), in fully aqueous solutions at pH 7.2. The sensor exhibited significant fluorescence quenching upon coordination with Hg²⁺, which was reversibly restored in the presence of thiols due to the formation of thermodynamically favored Hg-thiol complexes. The OFF-ON fluorescence mechanism of the sensor was elucidated using DFT calculations. Fluorescence titration experiments revealed a strong linear correlation (R ² ≈ 0.998) between fluorescence intensity and thiol concentrations within the ranges of 0.34-8.00 μM for GSH, 0.47-10.00 μM for Cys, and 0.26-8.00 μM for Hcy, with corresponding limits of detection (LOD) of 0.34, 0.47, and 0.26 μM, respectively. The sensor demonstrated high selectivity toward thiols in the presence of common amino acids, metal ions, and anions, with interference from Ag⁺, Cu²⁺, Co²⁺, and Ni²⁺ mitigated using 1,10-phenanthroline (PHEN). Owing to its high sensitivity, selectivity, and water solubility, Hg(DST)₂ represents a promising tool for thiol quantification in biological and environmental matrices.

Fig. 1. The reaction scheme for the synthesis of the DST fluorescent compound.

Fig. 2. (a) The fluorescence titration spectra of DST solution (10 μM) with Hg²⁺ at concentrations ranging from 0 to 6.5 μM; (b) Job's plot of the interaction between Hg²⁺ and DST (in pH 7.2 phosphate buffer, with an excitation wavelength of 368 nm, an emission wavelength of 445 nm).

Fig. 3. The stable structure of Hg(DST)₂ sensor at the DFT/PBE0/lanl2dz.

Fig. 4. Diagram illustrating the singlet electron transition processes in the excited state for DST and Hg(DST)₂.

Fig. 5. Nonlinear curve fitting for determining the complexation equilibrium constants of the Hg(DST)₂ complex in an aqueous solution. Where y = CM represents the total concentration of Hg²⁺ ions added to the solution, with values of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 μM. The initial concentration of DST is C_L = 20 μM. F₀ and F are the fluorescence intensities of the DST solution when the Hg²⁺ concentration is 0 and C_M, respectively. The reaction occurs in a pH 7.2 phosphate buffer, with an excitation wavelength of 368 nm and an emission wavelength of 445 nm.

Fig. 6. Fluorescence titration results of the Hg(DST)₂ complex sensor solution (5 μM) by the thiols GSH, Cys, and Hcy (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20 μM) in a pH 7.2 phosphate buffer, with an excitation wavelength of 368 nm and an emission wavelength of 445 nm. GSH (a and b), Cys (c and d), and Hcy (e and f).

Fig. 7. Fluorescence spectra of the Hg(DST)₂ solution (5 μM) upon addition of thiols GSH, Cys, and Hcy (20 μM each), as well as amino acids Ala, Arg, Asp, Glu, Gly, His, Ile, Leu, Lys, Met, Ser, Thr, Trp, Tyr and Val (20 μM each), in a pH 7.2 phosphate buffer, with an excitation wavelength of 368 nm and an emission wavelength of 445 nm.

Fig. 8. Fluorescence intensity of solutions: DST (20 μM); Hg(DST)₂ (10 μM); Hg(DST)₂ (10 μM) + GSH (10 μM); Hg(DST)₂ (10 μM) + GSH (10 μM) + metal ions or anions (20 μM); Hg(DST)₂ (10 μM) + GSH (10 μM) + Cu²⁺, Co²⁺or Ni²⁺ (20 μM) + PHEN (1500 μM); in a pH 7.2 phosphate buffer, with an excitation wavelength of 368 nm and an emission wavelength of 445 nm.

Scheme 1. Diagram Illustrating the operating principle of the Hg(DST)₂ sensor with biothiols.