Generating signals at converging liquid fronts to create line-format readouts of soluble assay products in three-dimensional paper-based devices

The correct interpretation of the result from a point-of-care device is crucial for an accurate and rapid diagnosis and to guide subsequent treatment. Lateral flow tests (LFTs) use a well- established format that was designed to simplify the user experience. However, the LFT device architecture is inherently limited to detecting analytes that can be captured by molecular recognition. Microfluidic paper-based analytical devices (μPADs), like LFTs, have the potential to be used in diagnostic applications at the point of care. However, μPADs have not gained significant traction outside of academic laboratories, in part, because they have often demonstrated a lack of homogeneous shape or color in signal outputs, which consequently can lead to inaccurate interpretation of results by users. Here, we demonstrate a new class of μPADs that generate colorimetric signals at the interfaces of converging liquid fronts (i.e., lines) to control where colorimetric signals are formed without relying on capture techniques. We demonstrate our approach by developing assays for three classes of analytes—an ion, an enzyme, and a small molecule—using iron (III), acetylcholinesterase, and lactate, respectively. Additionally, we show these devices have the potential to support multiplexed assays by generating multiple lines in a common readout zone. These results highlight the ability of this new paper-based device architecture to aid the interpretation of assays that create soluble products by using flow to constrain those colorimetric products in a familiar, line-format output.