A coumarin derivative-Cu2+ complex-based fluorescent chemosensor for detection of biothiols

Abstract

Herein, a novel fluorescent sensor has been developed for the detection of biothiols based on theoretical calculations of the stability constant of the complex between a Cu²⁺ ion and (E)-3-((2-(benzo[d]thiazol-2-yl)hydrazono)methyl)-7-(diethylamino) coumarin (BDC) as a fluorescent ligand. In this study, on the basis of density functional theory method, the Gibbs free energy of ligand-exchange reaction and the solvation model were carried out using thermodynamic cycles. The obtained results are in good agreement with the experimental data. The BDC-Cu²⁺ complex can be used as a fluorescent sensor for the detection of biothiols in the presence of non-thiol containing amino acids, with a detection limit for cysteine at 0.3 μM. Moreover, theoretical calculations of excited states were used to elucidate variations in the fluorescence properties. The computed results show that the excited doublet states D₂ and D₁ are dark doublet states, which quench the fluorescence of the complex.

Scheme 1. Thermodynamic cycle for the calculation of the Gibbs free energy of a ligand-exchange reaction in an aqueous solution, ΔG_aq.

Fig. 1. Schematic of the synthesis route of BDC.

Fig. 2. (a) Fluorescence spectra of BDC after the addition of 1 equiv of various metal ions, i.e. Na⁺, K⁺, Ca²⁺, Mg²⁺, Fe³⁺, Co²⁺, Ni²⁺, Zn²⁺, Pb²⁺, Cd²⁺, Hg²⁺, and Cu²⁺ ions, and (b) fluorescence titration spectra of BDC with the gradual addition of Cu²⁺ ions (BDC: 5 μM in ethanol/HEPES, pH: 7.4; 1/1, v/v; and excitation wavelength: 460 nm).

Fig. 3. Optimized geometries of BDC at the PBE0/6-31+G(d) level of theory.

Fig. 4. Optimized geometries of the BDC–Cu²⁺ complex at the PBE0/6-31+G(d) level of theory.

Fig. 5. Nonlinear curve-fitting method for the determination of the complexation equilibrium constants in the aqueous solution of the [CuL]²⁺ complex. C_M is the total concentration of the Cu²⁺ ion added to the solution: 0–10 μM; F₀ is the fluorescence intensity of the free BDC solution (C_L = 5 μM, in ethanol/HEPES, pH: 7.4, 1/1, v/v); F is the fluorescence intensity of the BDC solution at the time when the concentration of Cu²⁺ ions was C_M; excitation wavelength: 460 nm; and emission wavelength: 536 nm.

Fig. 6. (a) Fluorescence titration spectra of BDC–Cu²⁺ with the gradual addition of Cys and (b) variation in the fluorescence intensity of BDC–Cu²⁺ with the gradual addition of Cys at the emission wavelength of 536 nm. BDC–Cu²⁺: 5 μM, in ethanol/HEPES, pH: 7.4, 1/1, v/v; Cys: 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 13.0, 17.0, and 20.0 μM; and excitation: 460 nm.

Fig. 7. Schematic of the reaction between the BDC–Cu²⁺ complex and Cys.

Fig. 8. Fluorescence spectra of BDC (5 μM); BDC (5 μM)+Cu²⁺ (5 μM); BDC (5 μM)+ Cu²⁺ (5 μM) + Cys/Hcy/GSH/Ala, Asp, Arg, Gly, Glu, Ile, Leu, Lys, Met, Thr, Ser, Tyr, Trp, Val, His, and H₂S (10 μM).

Fig. 9. The characteristics of the main transition between the electron excited states and ground states of BDC and BDC–Cu²⁺ at the PBE0/6-31+G(d) level.