A Label‐Free Bioluminescent scFv‐NLuc Fusion Probe Enabling a Smartphone‐Based Sensitive Immunoassay Platform Using Lomefloxacin as a Model Analyte

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.