In traditional LFA experiments, sample mixtures containing a total of 12.5% v/v human serum was shown to produce the greatest signal-to-noise, as well as the least variation between measured test line signals (Figure ). AmpliFold sample mixtures containing a total 12.5% v/v were similarly shown to result in the greatest signal-to-noise, when compared to AmpliFold experiments conducted solely in running buffer conditions (Supplementary Information Figure ), and negligible false positive test line signals (Supplementary Information Figure ). In both LFA formats, increasing the percentage of human serum in sample mixtures was noted to have a detrimental impact of test line signal intensities for HER2-positive (500 ng/mL) tests as well as increased variation in the signals generated. We theorised that the detrimental impact on test line signal for HER2positive tests could reflect the non-specific interference of FluoroPerAuNP and FabHERbiotin conjugate anti-HER2 binding sites by human serum components, such as albumins, hormones, enzymes, and IgG antibodies. Although endogenous free thiols could also play a role in the observed matrix effects -particularly regarding the undesirable cleavable of FabHER-biotin conjugates -these would be expected to be present at drastically lower concentrations than DTT in these experiments. Ultimately, for a fair comparison of the traditional and AmpliFold LFA formats, the percentage of human serum in assay sample mixtures for both formats was kept the same, following this study, in order to match and matrix effects in the two formats as best as possible. In subsequent dose-response experiments, a dilution series of HER2 antigen spiked into human serum was prepared. A total human serum concentration of 12.5% v/v in assay sample mixtures was achieved by prediluting the typical antigen volume by half, prior to mixing in with FabHER-biotin (4) and 150 nm FluoroPerAuNP conjugate volumes. When testing serial dilutions of HER2 antigen in human serum, our AmpliFold assay was shown to detect the target analyte with a LOD of 3.35 ng/mL (95% CI of 2.37 to 4.74 ng/mL) (Figure ). This was shown to be a 12-fold enhancement in sensitivity when compared to traditional LFA formats where the LOD for HER2 antigen detection was determined to be 38. Overall, in both running buffer and real matrix conditions, our AmpliFold approach was shown to greatly enhance LFA performance when using large 150 nm FluoroPerAuNP labels. Despite this, the magnitude of sensitivity enhancement observed in these studies (ca. 12-fold) was lesser than that of our 40 nm FluoroPerAuNP model example where a 16-fold improvement was exhibited at minimal capture efficiency (Figure ). To rationalise this, we noted that the signal distribution of capture strips featuring 150 nm FluoroPerAuNPs (Figure ) remained comparatively uneven, when compared to the ideal uniform distribution obtained under low receptor density capture regimes (Figure ). We anticipate that larger particle systems, beyond what we shown, would tend towards more evenly distributed capture regimes based on diffusivity arguments. This further highlights the potential of our 'capture-and-release' AmpliFold approach in combination with existing LFA enhancements, such as using flurorescent nanodiamonds or latex microspheres, where larger particles play key roles in obtaining higher sensitivity. We envisage that there is also scope for future work to further optimise the workflows presented in this work, particularly with a focus on reducing the number of wash steps and the overall need for manual operation steps. Addressing these limitations are key towards affording faster assay runtimes and greater ease-of-use for future generations of AmpliFold design, ultimately bringing the technology closer towards viability at the point-of-care.