Modular Avi-Tag Linkers Provide Predictable Protein Orientation for Generic Biosensor Construction
Yiyu Chen1,2, Huiting Lian1,3, Guangming Liu2,4
1College of Materials Science and Engineering, Huaqiao University, Xiamen 361021, China.
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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.
