Cascaded antibody directionality relying on a zinc-based nanocomposite for performance enhancement of multiplex lateral flow immunoassay

The quantity and orientation of antibodies are key elements affecting the bioactivity of immunoprobes, which have important influences on the sensitivity and specificity of immuno-biosensors. In this work, a multiplex lateral flow immunoassay (mLFIA) mediated by a powerful cascaded antibody directionality (CAbD) strategy was constructed for the simultaneous monitoring of nitrofurazone and furazolidone metabolites in food samples. Here the CAbD strategy innovatively integrated three elements: antibody staining by Coomassie brilliant blue R-250, directional anchoring of second antibodies (Ab2) by the amorphous ZnO-based nanocomposites (ZnO-CBB-Ab2, ZnO-NCMs), and orientation of detection antibodies (mAb1) by the Ab2. The ZnO-NCMs play a double role in antibody orientation and signaling to exhibit. Thanks to the difference in surface electrical properties of Fc and Fab segments in immunoglobulin G, the excessive R-250 stained Ab2 was anchored in ZnO-NCMs to form a multidetection signal tag with colorimetric signal brightness. Compared with immobilization strategies of directly directional anchor-hold or post-adsorption of mAb1, this strategy can reduce the mAb1 consumption, better ensure the activity and utilization efficiency of mAb1, and shorten the construction time of the mLFIA system. As expected, the mLFIA is given satisfactory performances with a calculated sensitivity of 0.106 and 0.060 ng mL-1 for nitrofurazone and furazolidone metabolites, respectively. The developed multiplex immunosensor ultimately shows its application potential for point-of-care testing by performing well in fish and honey samples with accredited recoveries. It's worth emphasizing that the CAbD strategy can be a universal and scatheless antibodydirected approach, especially in multiplex immunoassays for ultrasensitive food safety monitoring.