Imprecise control of fluid flows in paper-based devices is a major challenge in pushing the innovations in this area towards societal implementation. Assays on paper tend to have low reaction yield and reproducibility issues that lead to poor sensitivity and detection limits. Understanding and addressing these issues is key to improving the performance of paper-based devices. In this work, we use colorimetric analysis to observe the mixing behaviour of molecules from two parallel flow streams in unobstructed (on unpatterned paper) and constricted flow (through the gap of a patterned hourglass structure). The model system used for characterization of mixing involved the reaction of Fe3+ with SCN- to form the coloured, soluble complex Fe(SCN)2+. At all tested concentrations (equal concentrations of 50.0 mM, 25.0 mM or 12.5 mM for KSCN and FeCl3 in each experiment), the reaction yield increases (higher colorimetric signal) and better mixing is obtained (lower relative standard deviation) as the gap of the flow constriction becomes smaller (4.69-0.32 mm). This indicates enhanced passive mixing of reagents. A transition window of gap widths exhibiting no mixing enhancement (about 2 mm) to gap widths exhibiting complete mixing (0.5 mm) is defined. The implementation of gap sizes that are smaller than 0.5 mm (below the transition window) for passive mixing is suggested as a good strategy to obtain complete mixing and reproducible reaction yields on paper. In addition, the hourglass structure was used to define the ratio of reagents to be mixed (2 : 1, 1 : 1 and 1 : 2 HCl-NaOH) by simply varying the width ratio of the input channels of the paper. This allows easy adaptation of the device to reaction stoichiometry.
We propose the use of water-based alkyl ketene dimer (AKD) ink for fast and user-friendly patterning of paper microfluidic devices either manually or using an inexpensive XY-plotter. The ink was produced by dissolving hydrophobic AKD in chloroform and emulsifying the solution in water. The emulsification was performed in a warm water bath, which led to an increased rate of the evaporation of chloroform. Subsequent cooling led to the final product, an aqueous suspension of fine AKD particles. The effects of surfactant and AKD concentrations, emulsification procedure, and cooling approach on final ink properties are presented, along with an optimized protocol for its formulation. This hydrophobic agent was applied onto paper using a plotter pen, after which the paper was heated to allow spreading of AKD molecules and chemical bonding with cellulose. A paper surface patterned with the ink (10 g L-1 AKD) yielded a contact angle of 135.6° for water. Unlike organic solvent-based solutions of AKD, this AKD ink does not require a fume hood for its use. Moreover, it is compatible with plastic patterning tools, due to the effective removal of chloroform in the production process to less than 2% of the total volume. Furthermore, this water-based ink is easy to prepare and use. Finally, the AKD ink can also be used for the fabrication of so-called selectively permeable barriers for use in paper microfluidic networks. These are barriers that stop the flow of water through paper, but are permeable to solvents with lower surface energies. We applied the AKD ink to confine and preconcentrate sample on paper, and demonstrated the use of this approach to achieve higher detection sensitivities in paper spray ionization-mass spectrometry (PSI-MS). Our patterning approach can be employed outside of the analytical lab or machine workshop for fast prototyping and small-scale production of paper-based analytical tools, for use in limited-resource labs or in the field.