Orientational probes-powered sensitive and accurate immunochromatographic assays with reduced antibody consumption and diminished matrix interference.

Rapid monitoring of pesticides using gold nanoparticle-based immunochromatographic assays (AuNPs-ICA) is desirable but is limited by two major obstacles: insufficient utilization of monoclonal antibodies (mAbs) and matrix interference, which critically affect the sensitivity and accuracy of immunoassay. Herein, oriented labeling at the Fc fragment of mAbs (OLAb)-based ICAs were developed for sensitive detection of carbofuran (CBF) and 3-hydroxycarbofuran (3-OH-CBF) in nine high-interference fruits and vegetables. Streptavidin, protein A, and goat anti-mouse immunoglobulin were employed to functionalize AuNPs, carbon nanoparticles, and time-resolved fluorescent microspheres, yielding nine OLAb probes with enhanced signals and superb bioactivity. These probes reduced mAbs consumption by 50-92.9% and maximized antigen-binding fragment exposure, enhancing sensitivity by 1.07-5-fold. Importantly, by minimizing nonspecific matrix protein binding, the platform required only 0.3- to 1.5-fold the original extraction volumes and achieved dramatically lower detection limits (0.1-1.81 μg/kg), which represented a 2.9-50.8-fold improvement over conventional AuNPs-ICA.