Determination of Trace Metallothioneins at Nanomolar Levels Using Phenanthroline–Copper Coordination by Fluorescence Spectra

Abstract

A direct fluorescence spectra method was applied for the determination of metallothioneins at nanomolar levels. In Britton–Robison (B-R) buffer (pH 7.0), the interaction of bis(1,10-phenanthroline)copper(II) complex cation [Cu(phen)₂]²⁺ and metallothioneins enhanced the fluorescence intensity of system. The fluorescence enhancement at 365 nm was proportional to the concentration of metallothioneins. The mechanism was studied and discussed in terms of the fluorescence and UV-absorption spectra. Under the optimal experimental conditions, at 365 nm, there was a linear relationship between the fluorescence intensity and the concentration of the metallothioneins in the range of 8.30 × 10⁻⁹ – 7.70 × 10⁻⁷ mol L⁻¹. The linear regression equation was ΔF = 8.96 + 38.01c (mol L⁻¹), with a correlation coefficient of r = 0.998 and detection limit 2.50 × 10⁻⁹ mol L⁻¹. The relative standard deviation was 0.47% (n = 11), and the average recovery 97.2%. The proposed method was successfully reliable, selective and sensitive in determining trace metallothioneins in fish visceral organ samples with the results in good agreement with those obtained by HPLC.