Microfluidic paper based analytical devices (microPADs) are a novel platform for point of care (POC) diagnostics. Limitations of reagent shelf life have been overcome with the introduction of reagent pencils as a method for solid-based reagent deposition. While useful, little work has been reported on the characterization and optimization of reagent pencils. Herein, an investigation on reagent pencil composition and efficiency is conducted via colorimetric release profile tests utilizing an erioglaucine disodium salt that yields a quantifiable blue colored product in the presence of water. Within this work, an investigation on the molecular weight dependence, polymer chain end functionality, and polymer-graphite ratio was conducted to determine the most desirable parameters in reagent pencil composition. Further, the effects of enzyme stability in the presence of poly(ethylene glycol) (PEG) is investigated. To show the versatility of reagent pencils, a novel reagent pencil incorporating a stimuli responsive polymer, poly(N-isporopylacrylamide) (PNIPAM) was developed. In this work, PNIPAM’s lower critical solution temperature (LCST) was manipulated with various salt solutions to control fluid flow both laterally and vertically through various microPAD designs. It was found that, while PNIPAM successfully blocked or retarded fluid flow in microPADs, the effect was limited when DI H2O wash solutions were run prior to salt solutions. To counteract this, PNIPAM was successfully covalently bound to alkene modified chromatography paper via thiolene click chemistry to reinforce solution wash tolerance.

3-D paper-based microfluidic devices (micoPADs) are small and portable devices made out of paper that offer a promising platform for diagnostic applications outside of a laboratory. These devices are easy to use, low cost, require no power source, and capable of detecting multiple targets simultaneously. The work in this thesis demonstrated the ability of a 3-D paper-microfluidic platform to simultaneously detect 5 targets. Rubber cord stock was used in conjunction with an acrylic housing unit to apply pressure along the edge of the channel. The indirect pressure application was successful in promoting vertical fluid flow between layers. Average channel development times were recorded between 110 seconds and 150 seconds. The implementation of the 3-D paper-microfluidic platform as a diagnostic device was validated with a colorimetric glucose assay. In a novel application, reagents were deposited onto the 3-D platform via a glucose reagent pencil created by Martinez et al. A visual signal was observed for the successful detection of glucose at a concentration of 1.2 mM. These results offer promise for future work in combing new reagent deposition techniques with a multi-layer paper-microfluidic platform. Overall, this research made advancements in the design of a paper-microfluidic platform capable of the simultaneous detection of 5 targets.