Highly sensitive detection of doxycycline residues in food using lysozyme and trypsin as protein-based fluorescence...

Lysozyme and trypsin were separately used as fluorescent probes for the sensitive detection of doxycycline, owing to the stable intrinsic fluorescence, excellent biocompatibility, low toxicity, and low cost of the two proteins. Fluorescence quenching of both lysozyme and trypsin induced by doxycycline was verified to be static by the stern-Volmer equation and time-resolved fluorescence measurements. Synchronous fluorescence and three-dimensional (3D) fluorescence spectra of the proteins exhibited no obvious peak shift upon the addition of doxycycline, indicating that the microenvironment surrounding tyrosine (Tyr) and tryptophan (Trp) residues was barely disturbed. Circular dichroism (CD) and Fourier transform infrared (FT-IR) spectra demonstrated that doxycycline modified the secondary structure of the proteins. Binding constants (K_a) and binding sites (n) were obtained at various temperatures. Molecular docking and thermodynamic analysis revealed that electrostatic forces played a dominant role in the intermolecular interactions. The static quenching process was accompanied by non-radiative energy transfer. Interference experiments demonstrated that the proposed method exhibited good selectivity. For the lysozyme and trypsin systems, the linear calibration equations were F₀/F = 0.1005c + 0.9911 and F₀/F = 0.0884c + 0.9708 over the concentration ranges of 6.2-1.6 × 10⁴ ng mL^-1 and 8.2-1.8 × 10⁴ ng mL^-1, with limits of detection (LOD) of 0.58 and 0.89 ng mL^-1, respectively. The proposed method was applied to the determination of doxycycline in fish, pork, and chicken samples, and satisfactory recoveries verified its accuracy and feasibility.