3D-printed devices for multiplexed semi-quantitative competitive lateral flow immunoassays

Lateral flow immunoassays (LFIAs) are widely used for the simple and rapid detection of various targets at the point of need. However, LFIAs enabling the simultaneous detection of multiple analytes and the possibility for naked-eye semi-quantitative analysis are facing various challenges, including the requirement of large sample volumes, low efficiency, and accuracy. This is particularly the case for the competitive immunoassay format targeting the detection of low molecular weight compounds, such as, for example, drugs. Due to limited space for multiple reaction zones on a single planar nitrocellulose membrane, conducting multiplexed tests requires the addition of more test strips, which consequently increases the size of the whole device. To overcome these spatial constraints, two 3D-printed devices fitting eight assay lanes of both backed and unbacked nitrocellulose membranes have been designed. For proof of concept, 8-OHdG, caffeine, and acetaminophen were used as model analytes. Inkjet printing was applied to deposit capture reagents in the form of text symbols while controlling the concentration thresholds for text readability to achieve an intuitive result expression. A comparably small sample volume of 350 μL was sufficient to simultaneously visually distinguish 4 concentration levels of caffeine (0, 4, 10, 175 ng mL^-1) and acetaminophen (0, 4, 8, 12 ng mL^-1) in mixed solutions without crosstalk. This study demonstrates the potential of 3D-printed LFIA devices for multiplex and semi-quantitative analyte detection.