Development of a stable luciferase@nanoflower-based method for the sensitive detection of pesticide residues in milk

Acetylcholinesterase inhibition-luciferase bioluminescence system enables rapid detection of organophosphate and carbamate pesticide residues. However, firefly luciferase (FLuc) suffers from poor stability and short luminescence duration, thereby introducing uncertainties in detection. To address this problem, nanoflower-immobilized FLuc (FLuc@NFs) was prepared using a coprecipitation method, which markedly enhanced stability and activity. FLuc@NFs exhibited intact nanoflower structures and allowed storage at 25 °C and at 4 °C for 2 weeks, overcoming the limitation of free enzyme storage at -20 °C. In the bioluminescence reaction, FLuc@NFs at 1 mg/mL achieved 62.14 % higher luminescence than free FLuc, with stable signals extending from 10 s to 30 min, enabling high-throughput detection. The system achieved detection limits of 5 and 10 ng/mL for chlorpyrifos and carbaryl, respectively, demonstrating improved reliability and sensitivity in pesticide detection.