Programmable microfluidic flow for automatic multistep digital assay in a single-sheet 3-dimensional paper-based microfluidic device

Abstract A single-sheet 3-dimensional paper-based microfluidic analytical device (3D-μPAD) has a user-friendly compact format that eliminates any potential for user alignment error compared to the stacking and folding format. However, achieving sophisticated analysis is still a challenge because forming a complex 3D microfluidic network sufficient for performing analysis in a single sheet of paper is difficult. Here, we present the first report of automatic multistep digital analysis in a single-sheet 3D-μPAD created by controlling the penetration of different wax inks into paper. Simple printing of different wax inks with different spreading properties produces different dimension microchannels in paper. This controllable change in the dimensions of the microchannels results in different flow rates of fluid in each microchannel. Based on the spatial resolution of the microchannels, our strategy enables programming of microfluidic flow through manipulation of the fluidic time delay. This approach creates 3D-μPADs capable of sequential fluid delivery and multistep biochemical reactions with single loading of a sample solution. The methodology can be further expanded to combination with digital assays to measure the concentration of a target analyte by simply counting the number of colored bars at a fixed time. It does not require any external instruments to analyze the measurement of the target analyte concentration. The significance of this work lies in the promising potential for automatic multistep digital assays in a low-cost and easy-to-use paper-based format that can be integrated with a roll-to-roll process for commercial-scale manufacturing.