Self-Assembled Dual-Functional Anti-Idiotypic Nanobody on IMX313 Nanoscaffolds for a Mycotoxin Zearalenone Environmentally Friendly Immunoassay

Competitive immunoassays for small molecules are limited by complex antigen synthesis and multistep signal transduction. Here, the anti-idiotypic nanobody was employed to enable eco-friendly detection. Furthermore, dual-label protein nanoscaffolds were used to achieve the design of programmable dual-functional antigen substitutes and one-step signal transduction in immunoassays. To determine the application potential, the pattern target zearalenone (ZEN) was selected as a case in this study. In detail, anti-idiotypic nanobody Z6 has been reported as a ZEN biosynthesis mimic. The Z6SC3-ST3IDT-DCN (Z6-SpyCatcher003-SpyTag003-IMX313-DogTag-DogCatcher-NanoLuc) was constructed to achieve the one-step bioluminescence detection of ZEN. To further streamline the workflow, we developed a magnetic-bead-based bifunctional programmable scaffold bioluminescent enzyme immunoassay (Mb-BPs-BLEI) using Z6SC3-ST3IDT-DCN, achieving a detection limit of 0.1 ng/mL for ZEN. This dual-tag design allows the modular replacement of recognition or signaling elements, providing a programmable platform for customizable immunoassays.