Modular Avi-Tag Linkers Provide Predictable Protein Orientation for Generic Biosensor Construction.

Current multiplexed IgE assays suffer from random protein orientation, which reduces epitope accessibility. Here, we address this limitation by genetically fusing oyster allergens (Cra a 1 and Cra a 2) to an Avi-tag for site-specific biotinylation. The oriented capture probes, integrated with RCA-enhanced cell-free transcription, enable the simultaneous detection of two IgE targets with limits of detection of 1.5 and 27 pg/mL. Circular dichroism and epitope mapping reveal that Avi-tag conjugation maintains the native protein conformation, resulting in 2.3-fold higher IgE-binding activity compared with random NHS-biotinylation. Pilot testing with 20 serum samples shows 90% concordance with ELISA, demonstrating the potential for component-resolved allergy diagnosis.