Establishment of a BRET-based fusion protein expression system to detect benzo[a]pyrene among polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants associated with various health risks. They primarily enter the human body through food. The most toxic PAH is benzo[a]pyrene (BaP), which serves as a key marker in food safety regulations. In this study, a NanoBRET-based detection system was developed to evaluate the protein-protein interaction between aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) in response to BaP. BRET vectors encoding NanoLuc-fused AhR and HaloTag-fused ARNT were used to monitor heterodimer formation, and the optimal combination for BaP detection was selected. A cell-free assay was established to assess the biological activity of BaP by quantifying AhR/ARNT heterodimerization. This assay could be used for faster, simpler, and more biologically relevant toxicity evaluation than conventional methods. The assay was optimized to minimize protein consumption and enable high-throughput screening. Thus, this BRET-based platform provides a rapid and accurate method for detecting BaP in food safety applications.