Zebrafish (Danio rerio) TRβ- and TTR-based electrochemical biosensors: Construction and application for the evaluation of thyroid-disrupting activity of bisphenols.

Thyroid-disrupting chemicals (TDCs) have received increasing concerns because of their negative health impacts on both wildlife and humans. This study aimed to develop in vitro screening assays for TDCs based on thyroid hormone receptor β (TRβ) and transthyretin (TTR) proteins. Firstly, the recombinant ligand-binding domain of TRβ (TRβ-LBD) and TTR proteins of zebrafish were produced by eukaryotic expression system and then used as bio-recognition components to construct electrochemical biosensors. In the biosensors, the supported bilayer lipid membrane (s-BLM) was used as a matrix to immobilize proteins, and gold nanoflowers (AuNFs) were used to improve the sensitivity by increasing electroactive surface area. Under the optimizing conditions, the zfTRβ-LBD/AuNFs/s-BLM/GCE biosensor had a detection range of 0.23 nM-1.92 μM and a detection limit of 0.07 nM for triiodothyronine (T3), while the zfTTR/AuNFs/s-BLM/GCE biosensor had a detection range of 0.46 nM-3.84 μM, with a detection limit of 0.13 nM. Based on the constructed biosensors, the order of T3 equivalent concentrations of bisphenols was BPA ≈ BPS > BPF > BPAF ≈ BPAP > BPZ, which was similar to the results of recombinant TRβ two-hybrid yeast assay. Furthermore, the reliability of the biosensors was validated by molecular docking, in which BPA and BPS showed higher binding affinity to zfTRβ-LBD. Therefore, this study provided a valuable tool for efficiently screening TDCs.