Second Flowing Type Lateral Flow Assay for High-Fidelity Detection

Lateral flow assay is a strip-based method, in which the loaded sample can flow across the strip autonomously and induce test line and/or control line coloration for visual determination of target of interest. It has the advantages of portable assay, easy operation, and no analytical instruments. A universally applicable way is the target-sandwiched linking of reporter elements to capture units on the test line. However, a common concern therein is the possibility to generate false-positive signals, which can result from either the high similarity of analogues to target or the residue interaction between reporter elements and capture units in the absence of target. Herein, we propose a second flowing-type lateral flow assay, by which the second flow is utilized to test and correct the result of first flow. The test line that originally develops color during the first flow will retain color if target is indeed present while get discolored if target is absent, after the second flowing of designed displacing strands, to achieve a high-fidelity detection. We have demonstrated the discrimination of single base mutation in a 31-base length DNA oligonucleotide, as well as the differentiation of low concentration of adenosine triphosphate (ATP) from background interference by the second flowing-type lateral flow assay, both of which are prone to misjudging by false-positive signal in the one-time flowing. The strategy can also identify muted sites or base types that are unknown a priori and restricted to the region complementary to the displacing strand. The second flowing-type lateral flow assay provides an effective and feasible way to ensure detection accuracy and reliability in the universally adopted target-sandwiched lateral flow detection.