Single step and mask-free 3D wax printing of microfluidic paper-based analytical devices for glucose and nitrite assays

Microfluidic paper-based analytical devices (μPADs) have recently emerged as a simple, portable, user-friendly, and affordable alternative to more instrument-intensive analytical approaches for point-of-care testing (POCT), food safety analysis, and environmental monitoring. However, most of the existing methods for the fabrication of μPADs still face a great challenge because of different trade-offs among cost, convenience, and the pattern resolution. In this work, we report a facile one-step approach to prepare a μPAD using an affordable, easy-to-build 3D printer to generate patterns of solid wax on laboratory filter paper. The presented wax printing method did not require the use of predesigned masks and an external heat source to form complete hydrophobic wax barrier through the use of a custom-made extruder. The results revealed a strong linear relationship (R2 = 0.985) between the nominal and the printed widths of the wax barriers. The achievable resolution of the wax barrier printed on filter paper was 468 ± 72 µm, which was lower than previously reported minimum barrier feature sizes achieved by wax printing and other wax patterning techniques, such as stamping and screen-printing. The analytical utility of the fabricated μPADs was evaluated for colorimetric nitrite and glucose detection in artificial solutions. It was found that the fabricated μPADs provided adequate accuracy and reproducibility for quantitative determination of nitrite and glucose within concentration ranges relevant to the disease detection in human saliva and urine. The wax printing approach reported here provides a simple, rapid, and cost-effective fabrication method for paper-based microfluidics and may bring benefits to medical diagnostics in the developing world.