Split-GFP-based homogeneous open sandwich immunoassay for rapid and simple detection of daidzin and genistin in soybeans.

This study reports the first split-GFP-based homogeneous open sandwich immunoassay for the convenient detection of major soy isoflavone glycosides, daidzin (DZ) and genistin (GEN). GFP1-10 and GFP11 were fused to the heavy-chain variable (VH) and light-chain variable (VL) domains of a DZ-recognizing monoclonal antibody with cross-reactivity toward GEN. The presence of DZ or GEN promoted VH-VL interaction, enabling GFP reconstitution and fluorescence activation without immobilization, blocking, or washing. The N-terminal GFP11 fusion variant exhibited superior performance. The immunoassay was completed within 2 h, with a working range of 0.039-10.0 μg mL-1, a limit of detection of 27.9 ng mL-1, intra- and inter-day coefficients of variation of ≤ 14.6% and ≤ 11.2%, respectively, spike recoveries of 95.8-111.7%, and a strong linear association with HPLC results (R2 = 0.970) in soy and soy-derived products. This homogeneous split-GFP immunoassay provides a simple approach for compositional assessment of combined DZ and GEN-equivalent content in food products.