Microfluidic paper-based analytical devices (microPADs) are an emerging platform for point-of-care diagnostic tests for use by untrained users with potential applications in healthcare, environmental monitoring, and food safety. These devices can be developed for a multitude of different tests, many of which employ enzymes as catalysts. Without specialized treatment, some enzymes tend to lose their activity when stored on microPADs within 48 hours, which is a major hurdle for taking these types of devices out of the laboratory and into the real world. This work focused on the development of simple methods for stabilizing enzymes by applying polymers to chromatography paper. The longterm stabilization was exlored and SU-8 of various concentrations was found to stabilize horseradish peroxidase for times in excess of two weeks. A variety of microPAD fabrications, enzyme dispensing methods, and substrate delivery techniques were explored.

N,N′-methylenebisacrylamide-crosslinked poly(N-isopropylacrylamide), also known as P(NIPAM), was developed as a fluid delivery system for use with microfluidic paper-based analytical devices (microPADs). MicroPADs are postage-stamp-sized devices made out of paper that can be used as platforms for low-cost, simple-to-use point-of-care diagnostic assays. P(NIPAM) is a thermally responsive polymer that absorbs aqueous solutions at room temperature and will expel the solutions to microPADs when heated. The fluid delivery characteristics of P(NIPAM) were assessed, and P(NIPAM) was able to deliver multiple solutions to microPADs in specific sequences or simultaneously in a laminar-flow configuration. P(NIPAM) was then shown to be suitable for delivering four classes of reagents to microPADs: small molecules, enzymes, antibodies and DNA. P(NIPAM) successfully delivered a series of standard concentrations of glucose (0 – 5 mM) to microPADs equipped to perform a colorimetric glucose assay. The results of these tests were used to produce an external calibration curve, which in turn was used to determine accurately the concentrations of glucose in sample solutions. P(NIPAM) successfully delivered fluorescein-labeled IgG and fluorescein-labeled oligonucleotides (20 base pairs) to microPADs in a variety of concentrations. P(NIPAM) also successfully delivered horseradish peroxidase (HRP) to microPADs, and it was determined that HRP could be stored in P(NIPAM) for 35 days with minimal loss in activity. The combination of P(NIPAM) with microPADs will allow for more complex assays to be performed with minimal user input, will facilitate the preparation of external calibration curves in the field, and may be useful in extending the shelf life of microPADs by stabilizing reagents.