Mitigating hook effect in one-step quantitative sandwich lateral flow assay by timed conjugate release

Sandwich lateral flow assay (LFA) is one of the most successfully commercialized paper-based biosensors, which offers a rapid, low-cost, one-step assay. Despite its advantages, conventional sandwich LFA is fundamentally limited by the high-dose “hook” effect—a phenomenon that occurs at very high analyte concentrations and results in false-negative results. In this paper, we present a novel strategy of automatic timed detection antibody release to mitigate the hook effect in sandwich LFA without additional manual steps. We introduced an intermediate pad treated with saturated sucrose solution to regulate the flow between the nitrocellulose membrane and the conjugate pad in order to delay the reaction between detection antibodies and analytes. Using C-reactive protein (CRP) as a representative analyte, we demonstrated that our strategy exhibited a range of detection 10 times wider than that of our conventional LFA, without sacrificing the limit of detection. Comparing to other published strategies, our work could offer a one-step, cost-effective approach that is closely unified with the benefits of the LFA.