A site-specifically labeled split-NanoLuc biosensor for rapid homogeneous detection of 3-phenoxybenzoic acid

Split-NanoLuc biosensors for homogeneous small-molecule detection are often limited by structural heterogeneity arising from random conjugation, which compromises enzyme complementation efficiency and analytical reproducibility. Here, a site-specifically labeled split-NanoLuc biosensor was developed for rapid detection of 3-phenoxybenzoic acid (3-PBA), a representative biomarker of pyrethroid insecticide exposure. A site-specific chemoenzymatic labeling strategy integrating sortase A-mediated ligation with oxime-based bioorthogonal chemistry was employed to generate a structurally homogeneous LgBiT-3-PBA tracer. Combined with a SmBiT-nanobody recognition probe, the system enabled efficient proximity-induced NanoLuc complementation in a competitive assay format. Structure-informed linker optimization yielded a signal-to-noise ratio > 28, a half-maximal inhibitory concentration (IC₅₀) of 2.3 ng/mL, and a detection limit of 0.41 ng/mL, with a total assay time of 10 min and no washing steps required. This represents an approximately seven-fold improvement in sensitivity compared with the previously reported 3-PBA homogeneous detection method. The sensor demonstrated satisfactory recoveries in urine and water samples and good agreement with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) (R² = 0.98). This work establishes a site-specific conjugation strategy for constructing homogeneous, high-performance split-NanoLuc biosensors, which holds potential for broader applications in small-molecule detection.