Modular Avi-Tag Linkers Provide Predictable Protein Orientation for Generic Biosensor Construction.
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| Main Authors: | , , , , |
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| Format: | Artículo científico |
| Language: | en |
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Analytical chemistry
2026
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| _version_ | 1868266041730138113 |
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| author | Chen, Yiyu Lian, Huiting Liu, Guangming Liu, Bin Wei, Xiaofeng |
| author_facet | Chen, Yiyu Lian, Huiting Liu, Guangming Liu, Bin Wei, Xiaofeng Chen, Yiyu Lian, Huiting Liu, Guangming Liu, Bin Wei, Xiaofeng |
| collection | PubMed - marine biology |
| contents | Modular Avi-Tag Linkers Provide Predictable Protein Orientation for Generic Biosensor Construction. Chen, Yiyu Lian, Huiting Liu, Guangming Liu, Bin Wei, Xiaofeng Biosensing Techniques Animals Allergens Immunoglobulin E Humans Biotinylation Epitope Mapping Circular Dichroism 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. |
| format | Artículo científico |
| id | pubmed_42233754 |
| institution | PubMed |
| language | en |
| publishDate | 2026 |
| publisher | Analytical chemistry |
| record_format | pubmed |
| spellingShingle | Modular Avi-Tag Linkers Provide Predictable Protein Orientation for Generic Biosensor Construction. Chen, Yiyu Lian, Huiting Liu, Guangming Liu, Bin Wei, Xiaofeng Biosensing Techniques Animals Allergens Immunoglobulin E Humans Biotinylation Epitope Mapping Circular Dichroism Modular Avi-Tag Linkers Provide Predictable Protein Orientation for Generic Biosensor Construction. Chen, Yiyu Lian, Huiting Liu, Guangming Liu, Bin Wei, Xiaofeng Biosensing Techniques Animals Allergens Immunoglobulin E Humans Biotinylation Epitope Mapping Circular Dichroism 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. |
| title | Modular Avi-Tag Linkers Provide Predictable Protein Orientation for Generic Biosensor Construction. |
| topic | Biosensing Techniques Animals Allergens Immunoglobulin E Humans Biotinylation Epitope Mapping Circular Dichroism |
| url | https://pubmed.ncbi.nlm.nih.gov/42233754/ |