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

Area of Science:

• Biochemistry
• Biosensor Technology
• Immunotechnology

Background:

• Homogeneous immunoassays offer operational simplicity and high sensitivity but struggle with small-molecule detection due to molecular limitations and complex conjugation.
• Existing methods for small-molecule immunoassays often require antibody modification or new antibody development, limiting their versatility.

Purpose of the Study:

• To develop a novel, fully biosynthetic homogeneous bioluminescent immunoassay for Fumonisin B1 (FB1) detection.
• To present a mimotope-driven lucCage-lucKey biosensor strategy that bypasses the need for antibody modification.

Main Methods:

• Utilized Rosetta computational design and AlphaFold3 structural prediction to engineer FB1 mimotopes.
• Grafted FB1 mimotopes onto lucCage in single-end and double-terminal configurations for competitive binding assays.
• Developed and optimized two sensor configurations: Cb-lucCageF1_342 (single-end) and NCb-lucCageF1_S_L2 (double-terminal).

Main Results:

• The 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.
• The double-terminal sensor (NCb-lucCageF1_S_L2) achieved an LOD of 9.73 ng/mL.
• Both sensors demonstrated high specificity, with no cross-reactivity to other mycotoxins (AFB1, DON, OTA, ZEN).

Conclusions:

• The novel mimotope-driven lucCage-lucKey biosensor strategy enables a versatile and simplified homogeneous immunoassay for small molecules.
• This modular platform can be extended to detect other small molecules by incorporating different mimotopes, offering a significant advancement in biosensing.
• The developed biosensors provide sensitive and specific detection of Fumonisin B1 without requiring antibody engineering.