Design and testing of pneumatically actuated disposable microfluidic devices for the DxBox: a point-of-care system for multiplexed immunoassay detection in the developing world

Accurate diagnostics are essential to proper treatment of disease. In many low-resource settings lack of infrastructure prevents high quality immunoassays from being utilized. Simple, self-contained multi-analyte detection in low-resource settings could enable diagnosis of co-infection or differentiate among pathogens that share similar presenting symptoms. This dissertation presents a microfluidic point-of-care diagnostic system developed to test for disease-specific antigens and IgM antibodies generated by the host indicative of active infection. The two assay formats are demonstrated for detection of PfHRPII antigen, indicative of malaria infection, and IgM antibodies for the detection of typhoid fever.;Complete sample-to-result immunoassays were performed in 30 minutes inside microfluidic cards. The steps performed on card included: plasma filtration, reagent volume metering, sample dilution, removal of interferent IgG and timed sequential reagent deliveries separated by washes. A novel microfluidic batch mixer operated by inexpensive air pumps was developed to perform the sample preparation steps necessary for IgM detection. The mixer facilitated a 1:100 plasma dilution and continuous batch mixing of the diluted sample in the presence of nanospheres coated with protein G. The IgG interferent was successfully removed without decreasing the concentration of the target analyte, IgM.;Both malaria antigen and typhoid IgM were successfully detected from blood samples in the multi-step multi-analyte diagnostic cards. Characterization of the PfHRPII antigen assay found a sub-nanomolar limit of detection, comparable to the clinical laboratory standard ELISA. A panel of blinded clinical plasma samples was tested for typhi IgM, and the DxBox assay performance was comparable to related bench-top assays and ELISA. This microfluidic diagnostic is a demonstration of an inexpensive pneumatically actuated platform capable of performing antigen and IgM detection applied to the differential diagnosis of fever at the point-of-care. By translating other antigen and IgM assays into this format the platform could be used to perform other differential diagnoses.