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. Lateral flow assay (LFA), a paper-based in-situ detection platform, has been widely used in diverse fields such as biomedicine, environmental health, quality control and food safety due to its rapid, inexpensive, portable measurements 1. Basing on affinity interactions such as hybridization of oligonucleotide or aptamer-target, antibody-antigen or biotin-streptavidin, the signal of the test zone on the LFA test strip changes with the concentration of a particular analyte of interest when a liquid sample is applied 2,3. There are two basic formats of LFA: sandwich format and competitive format. In addition, the competitive format could be designed in the following two ways: fixing the capture probe of target at test zone (competitive format I) or fixing the competitive analogue at test zone (competitive format II). Generally, 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. This strategy is the most widely used and easily handled one and the results usually show significant concentration dependence. However, the limit of detection (LOD) is usually poor, which is the main drawback of this conventional LFA. The other strategy is the pre-mixing/pre-incubation sampling method with mixing signaling unit with sample solution to load together onto the sample pad 4. Signaling unit is greatly diluted by sample solution in this strategy, resulting in a prolonged interaction between signaling unit and capture molecules at test zone and ...
