Abstract
Lateral flow assay (LFA) is one of the most prevalent commercially available techniques for point-of-care tests due to its simplicity, celerity, low cost and robust operation. However, conventional colorimetric LFAs have inferior limits of detection (LODs) compared to sophisticated laboratory-based assays. Here, we report a simple strategy of test-zone pre-enrichment to improve the LOD of LFA by loading samples before the conjugate pad assembly. The developed method enables visual LODs of miR-210 mimic and human chorionic gonadotropin protein, to be improved by 10–100 fold compared with a conventional LFA setup without introducing any additional instrument and reagent except for phosphate running buffer, while no obvious difference occurred for Aflatoxin B1 (AFB1). It takes about 6–8 min to enrich every 50 μL of sample diluted with phosphate running buffer, therefore we can get visual results within 20 min. We identified a parameter by modeling the entire process, the concentration of probe-analyte conjugate at test zone when signaling unit being loaded, to be important for the improvement of visual limit of detection. In addition, the test-zone pre-enrichment did not impair the selectivity when miR-210 mimic was adopted as target. Integrated with other optimization, amplification and modification of LFAs, the developed test-zone pre-enrichment method can be applied to further improve LOD of LFAs.
Highlights
There are two strategies to load signaling unit in the conventional Lateral flow assay (LFA)
Higher sensitivity could be obtained due to the higher capturing rate of DP-AuNPs. We applied this strategy with miR-210 mimic, human chorionic gonadotropin (HCG) protein and Aflatoxin B1 (AFB1) as model targets
The limit of detection (LOD) of miR-210 mimic were improved by 10–100 fold compared with the conventional LFAs, and those of HCG were improved by 10 fold
Summary
There are two strategies to load signaling unit in the conventional LFA. The first one is the direct sampling method with drying signaling unit on the conjugate pad fixed between the nitrocellulose (NC) membrane and the sample pad before sampling. In sandwich format and competitive format I of LFA, the samples would be pre-enriched and captured at test zone with the proposed method, causing much higher concentration of probe-analyte conjugate at test zone than with direct sampling method. We explored a sandwich-like format and two competitive formats for miR210 detection to develop the test-zone pre-enrichment sampling method, compared the performance of different sampling methods and studied the volume effect.
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