Peptide molecular lock-engineered nanobodies enable an oriented dual-modal immunoassay for reliable detection of Cronobacter sakazakii.

Pengyu Chen; Jiahao Zhang; Yuxia Luo; Sijie Liu; Yuechun Li; Bingzhi Li; Jing Sun; Guo Hao; Jianlong Wang; Yanru Wang

doi:10.1016/j.foodchem.2026.150387

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Peptide molecular lock-engineered nanobodies enable an oriented dual-modal immunoassay for reliable detection of Cronobacter sakazakii.

Pengyu Chen·Jiahao Zhang·Yuxia Luo·Sijie Liu·Yuechun Li·Bingzhi Li·Jing Sun·Guo Hao·Jianlong Wang·Yanru Wang

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Abstract

Conventional nanobody ELISAs for trace Cronobacter sakazakii in powdered infant formula suffer from random orientation and low signal output. We developed an oriented dual-modal immunoassay that combines site-specific biotinylation via a C-terminal AviTag and a peptide molecular lock, enabling controlled surface orientation while preserving nanobody structural integrity. This strategy was further integrated with phage-displayed nanobodies for multivalent amplification and both fluorescent and colorimetric readouts. The assay exhibited a broad linear range of 103-106 CFU/mL, with limits of detection (LODs) of 6.70 × 102 CFU/mL for fluorescence and 1.55 × 103 CFU/mL for colorimetry, showing improved sensitivity compared with the conventional passive adsorption-based Nb-ELISA evaluated in this study. XGBoost-based multimodal fusion improved quantitative accuracy, and SHAP analysis elucidated modality contributions. In spiked powdered infant formula samples, recoveries ranged from 92.1% to 118% with coefficients of variation below 5.98%, confirming acceptable matrix tolerance and analytical reliability.

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Cronobacter sakazakii Food Contamination Infant Formula Single-Domain Antibodies Immunoassay Humans Peptides Limit of Detection Enzyme-Linked Immunosorbent Assay Dual-modal immunoassay Foodborne pathogen Machine learning Nanobody Journal Article Evaluation Study

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Pengyu Chen, Jiahao Zhang, Yuxia Luo, Sijie Liu, Yuechun Li, Bingzhi Li, Jing Sun, Guo Hao, Jianlong Wang, Yanru Wang. 2026-07-11. Peptide molecular lock-engineered nanobodies enable an oriented dual-modal immunoassay for reliable detection of Cronobacter sakazakii.. https://doi.org/10.1016/j.foodchem.2026.150387

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