Barrier-Free Microfluidic Paper Analytical Devices for Multiplex Colorimetric Detection of Analytes

In recent years, microfluidic paper analytical devices (μPADs) have been extensively utilized to conduct multiplex colorimetric assays. Despite their simple and user-friendly operation, the need for patterning paper with wax or other physical barriers to create flow channels makes large-scale manufacturing cumbersome. Moreover, convection of rehydrated reagents in the test zones leads to nonuniform colorimetric signals, which makes quantification difficult. To overcome these challenges, we present a device called a barrier-free μPAD (BF-μPAD) that consists of a stack of two paper membranes having different wicking rates-the top layer acting as a fluid distributing layer and the bottom layer containing reagents for colorimetric detection. Multiple analytes can be detected using this assembly without the need to pattern either layer with wax or other barriers. In one embodiment, a device is capable of delivering the sample fluid to 20 distinct dried reagent spots stored on an 8 cm × 2 cm membrane in as few as 30 s. The multiplexing feature of the BF-μPAD is demonstrated for colorimetric detection of salivary thiocyanate, protein, glucose, and nitrite. Most importantly, the device improves the limit of detection of colorimetric assays performed on conventional μPADs by more than 3.5×. To understand fluid imbibition in the paper assembly, the device geometry is modeled in COMSOL Multiphysics using the Richards equation; the results obtained provide insights into the nonintuitive flow pattern producing perfectly uniform signals in the barrier-free assembly.