Copper-based nanozyme-linked immunosorbent assay for quantitative detection of fumonisin B1

Fumonisin B₁ (FB₁) is a Group 2B carcinogen. It is vital to develop an ultra-sensitive, low-cost, and rapid screening method for FB₁ for food safety risk control. In this study, a novel copper-based nanozyme was synthesized. The anti-FB₁ monoclonal antibody (mAb) was conjugated with copper-based nanozyme through physical adsorption to probe construction. The probe could compete with the limited antigen and trigger a color change when employing the 3,3’,5,5’-tetramethylbenzidine (TMB) as chromogenic substrate in the presence of H₂O₂. Several experimental conditions were optimized, including the concentration of coating antigen, the concentration of mAb coupled to the nanozyme, incubation time, and methanol concentration. Under optimal conditions, a direct competitive nanozyme-linked immunosorbent assay (NLISA) has been established with a limit of detection (LOD) of 6.52 pg/mL. The developed method presented good accuracy and precision. The spiked recoveries ranged from 97.368 % to 120.916 %, with coefficient of variation (CV) below 10 %. In addition, there was a high consistency (R²=0.9820 for corn; R²=0.9936 for wheat) between the developed detection method and the reference method, liquid chromatography/mass spectrometry (LC-MS/MS). This research can expand the application areas of copper-based nanozymes and improve the sensitivity and efficiency of FB₁ monitoring.