In conventional LFA design, antigen binding can typically be described as a two-antibody system which relies on orthogonal epitopes to simultaneously capture and label the target analyte. In our AmpliFold approach, illustrated in Figure , 'capture-and-release' is performed entirely across a lateral flow architecture -comprising a capture and detection strip -and utilises additional affinity interactions to accomplish orthogonal binding mechanisms beyond that of a traditional immunoassay. In the first instance, the selection of polystreptavidin (PSA) as a capture receptor presented opportunities to develop a 'capture-and-release' mechanism via the medium of a cleavable linker. Here, the utilisation of a cleavable linker provides a discrete alternative from initiating analyte release directly from the antibody-antigen complex itself, which often requires harsh immunodisruption conditions. Aside from its exceptional affinity for capturing biotinlabelled targets, the choice of PSA also represents a commonly employed LFA receptor commercially regarded for its stability and robustness against thermal and chemical influences. In a pre-assay step, the model target analyte, HER2 antigen, was pre-mixed with, and bound by, a modified anti-HER2 Fab fragment (FabHER) and a fluoresceintagged anti-HER2 antibody-AuNP conjugate, forming a sandwich immunocomplex structure. When run up AmpliFold capture strips, antigen-bound sandwich immunocomplexes are initially sequestered by PSA test line receptors due to chemical modification of anti-HER2 Fab fragments (FabHER) with a cleavable linker bearing a biotin tag. The cleavable linker features a disulfide bond, which can undergo triggered cleavage using reducing reagents, thus enabling the release and elution of captured sandwich immunocomplexes from initial PSA capture. Disulfide-based cleavage was selected as a promising candidate as it is well-validated within cleavable antibody-drug applications and benefits from a prevalence of commercial disulfide-containing linkers capable of enabling facile protein biotinylation. Beyond this, disulfide-based cleavage chemistry was anticipated to provide suitably fast cleavage kinetics for developing a capture-and-release strategy, which could be performed under a paper microfluidic flow regime and in a comparable timeframe to typical LFAs (ca. 10-20 minutes). 26