Mimotope-Driven lucCage-lucKey Homogeneous Bioluminescent Immunosensor for the Small Molecule Fumonisin B₁

Homogeneous immunoassays offer advantages of operational simplicity, high sensitivity, and automation potential, yet their application to small-molecule detection remains challenging due to the inherent structural limitations of small molecules and the complexity of chemical conjugation for constructing labeled components. Herein, we present a novel mimotope-driven lucCage-lucKey biosensor strategy that enables a fully biosynthetic homogeneous bioluminescent immunoassay for the mycotoxin Fumonisin B₁ (FB₁), without requiring modification of existing antibodies or development of new ones. Using Rosetta computational design and AlphaFold3 structural prediction, FB₁ mimotopes were grafted onto lucCage in single-end and double-terminal configurations, yielding inverse and positive bioluminescent signal responses, respectively, through competitive binding with anti-FB₁ monoclonal antibodies. The optimized single-end sensor (Cb-lucCageF1_342) achieved a limit of detection (LOD) of 2.99 ng/mL with a linear range of 6.73-107.60 ng/mL, while the optimized double-terminal sensor (NCb-lucCageF1_S_L2) attained an LOD of 9.73 ng/mL. Both sensors demonstrated specificity with no cross-reactivity to AFB₁, DON, OTA, or ZEN. This modular platform can be readily extended to other small molecules by grafting different mimotopes, offering a versatile and simplified approach for small-molecule homogeneous immunoassays.