Development of colloidal gold-labeled dual-probe lateral flow immunoassay for sensitive aflatoxin B1 detection by a signal amplification strategy

Abstract

In this study, a dual-probe LFIA was constructed to realize sensitive and naked-eye determination of aflatoxin B1 (AFB1) by introducing a signal amplification strategy. Colloidal gold (CG) with merit of easily synthesis was adopted as the signal material and used to absorb antibodies to form two probes of the CG-labeled monoclonal antibody (CG-mAb) and the CG-labeled goat anti-mouse IgG antibody (CG-GAMA). The achievement of the signal amplification strategy benefits from the immune recognition between CG-mAb and CG-GAMA, which combined with each other to form the amplified signal complexes. In the single-probe LFIA, detection relies solely on CG-mAb for both signal generation and target capture. In contrast, the dual-probe LFIA strategically redistributes these functions, CG-mAb maintains its primary capture role while its signal contribution is partially attenuated, with CG-GAMA serving as a supplementary signal reporter. By introducing CG-GAMA, the required amount of CG-mAb compared to the single-probe LFIA was relatively reduced. This simple design, which eliminates the need for additional pads, blocking steps, or enhancing reagents, enables the dual-probe LFIA to achieve superior sensitivity for detection with low sample volume while maintaining clear naked-eye readability. This dual-probe LFIA with the visual limit of detection (vLOD) of 0.5 ng mL⁻¹ was improved by 4 times than the single-probe LFIA (vLOD of 2 ng mL⁻¹). Besides, the capability of the dual-probe LFIA was successfully verified in various grains (oatmeal, rice, and corn) and validated by HPLC-FLD. The proposed strip could be extended for other small molecule targets in the field of food safety.