A hydrophobic wax barrier (so-called a “wax gate”) combined with the use of surfactants was developed as a valving mechanism in paper-based microfluidic systems to enable the control of delays in reagent addition in the device. This mechanism allowed the delay of reagent delivery and assisted multistep analysis on microfluidic paper-based analytical device (μPADs). Specifically, the hydrophobicity of porous wax gates prevented liquid imbibition until the “locked gate” had been trigged by rehydrated surfactant (so-called “chemical key”). In this work, three geometries of the wax gates including line, arc and triangle were investigated. Evidently, the triangle and arc gates performed significantly better than the line gate as hydrophobic barriers. Using the triangle wax gate, the wax gate's actuation time could be controlled in the range of approximately 2–21 min depending on the concentration of the surfactants. In contrast to previous approaches, temporal control could be extended by the end-users on demand due to the gate's actuation independency from delay channels and sample solution. Finally, the integration of the triangle wax gate into prototype multistep sandwich immunosensors allowed sensitive detection of mouse-IgG and SARS-CoV2 antibody.
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