A Label‐Free Bioluminescent scFv‐NLuc Fusion Probe Enabling a Smartphone‐Based Sensitive Immunoassay Platform Using Lomefloxacin as a Model Analyte
Mingyue Ding, Hao Wen, Shixiang Wu, Shiyun Han, Linwei Zhang, Hongfei Yang, Xu Wang, Long Li, Dapeng PengABSTRACT
Enzymes as labeling agents enhance lateral flow immunoassays (LFIA). However, their biological instability and reliance on labor‐intensive chemical conjugation hinder practical high‐throughput screening of food contaminants. To address this, we report a rapid, label‐free luciferase‐based platform for quantifying lomefloxacin (LOM) in food. The assay uses an engineered immunoprobe: a high‐affinity single‐chain variable fragment antibody (3G9‐scFv) specific to LOM, combined with Nanoluciferase (NLuc) as an intrinsic signal generator without covalent labeling. NLuc catalyzes furimazine oxidation, producing stable bioluminescence inversely proportional to LOM concentration. This label‐free design eliminates conjugation variability and simplifies protocol execution. Smartphone‐based luminescence measurement enables sensitive field detection, with a limit of quantification of 0.12 ng/mL. Furthermore, homology modeling and molecular docking elucidated the structural basis for the dual functionality of 3G9‐scFv, validating the rational use of unlabeled enzymatic tracers. The visual detection limit was determined to be 1 ng/mL. Recovery rates ranged from 79.0% to 113.0%, with coefficients of variation (CVs) between 2.3% and 10.9%, indicating high accuracy and reproducibility. This system offers a novel rapid detection platform for monitoring hazardous residues in food safety.