Simple assay for screening phytoestrogenic compounds using the oestrogen receptor immobilised magnetite nanoparticles

With increasing interests of phytoestrogens for their potential applications, a rapid and simple tool for screening these phytochemicals is still required. In this study, a simple assay to detect phytoestrogens was developed based on the competition binding between the tested samples and the fluorescently labelled oestrogen (E2) to the human ligand binding domain of oestrogen receptor (LBD-ER) that was immobilised on the magnetite nanoparticles (MNPs). The 40-kDa LBD-ER peptide was produced in an Escherichia coli system. The synthesised 68.7-nm MNPs were silanised and subsequently covalently linked to the C-terminus of LBD-ER peptide. The LBD-ER immobilised MNPs demonstrated the specific binding for the standard E2 with the equilibrium dissociation constant of 9.56 nM and the binding capacity of 0.08 pmol/1 mg of the MNPs. The LBD-ER immobilised MNPs could evaluate oestrogenic activity of the extracts of Asparagus racemosus and Curcuma comosa, the reported phytoestrogenic plants, but not progesterone (P4) and Raphanus sativus extract, the negative controls. The results of this work clearly demonstrated a potential assay for detecting phytoestrogens of crude plant extracts, which is simple and easily adapted to a high throughput format.