Fu, ElainThiessen, Greg2014-10-202015-12-142014-10-202014Thiessen_washington_0250O_13704.pdfhttp://hdl.handle.net/1773/26970Thesis (Master's)--University of Washington, 2014Current newborn screening programs for phenylketonuria (PKU) do not reach many individuals, worldwide, who would benefit from testing. Existing PKU tests require a high-resource laboratory, as well as a base level of local infrastructure, which prohibits their use in low-resource settings. In this report, we demonstrate a paper-based PKU test, based on a well-known enzyme colorimetric assay (ECA), which is applicable to low-resource settings. Phenylalanine dehydrogenase (PheDH) catalyzes the reaction between phenylalane (Phe) and NAD+ to produce phenylpyruvate, NH4, and NADH. Subsequently, NADH reacts with nitroblue tetrazolium salt (NBT) and phenazine methosulfate (PMS), an intermediate electron carrier, to produce NAD+ and a purple formazan dye. We characterized the enzymatic and colorimetric reactions in solution before characterizing their performance in porous substrates. The enzymatic reaction was translated into a glass fiber substrate and the colorimetric reaction into a cellulose substrate. Short-term dry storage of reagents within the porous media was examined. Finally, we demonstrated an easy-to-use, fully disposable test card with a limit of detection near 1 mg/dL phenylalanine with a time to result of 10 minutes. A test like this has potential for the application of newborn PKU screening in areas of the world that are not currently reached by existing screening programs.application/pdfen-USCopyright is held by the individual authors.Enzyme colorimetric assay; Paper-based test; PheDH; Phenylalanine; Phenylketonuria; PKUBiomedical engineeringbioengineeringThesis