Quantification of metallothioneins in the earthworm by lomefloxacin–europium(iii) fluorescent probe

Abstract

A new fluorimetric method was established for the determination of trace amounts of metallothioneins (MT) in earthworm, using a lomefloxacin–europium(III) (LMLX–Eu³⁺) complex as a fluorescent probe. In a pH 6.5 Tris–HCl buffer solution, MT can markedly decrease the fluorescence intensity of LMLX–Eu³⁺ at λ = 613 nm, and the magnitude of the decrease in this intensity was in direct proportion to the concentration of MT. The linear range was 0.08–20 mg L⁻¹ with a detection limit of 0.022 mg L⁻¹, and the recovery was in the range of 91.9–104.4%. The results show that the fluorimetric method is relatively accurate and sensitive to measurements of concentration for MT over a wide range. This method has been successfully applied to the determination of the concentration of MT induced by heavy metal ions (Cd²⁺, Pb²⁺, Cu²⁺, Zn²⁺) in Eisenia andrei. The amount of MT increased significantly in a dose-dependent manner to the heavy-metal exposure, and these proteins can be used as biomarkers to assess the impact of heavy-metal contamination in soils. The method offered high sensitivity as well as accuracy with simple instrumentation and is suitable for direct quantification of total MT in Eisenia andrei.