Development of a non-instrumented point-of-care (POC) HIV-1 diagnostic device

There is a growing need for sensitive, simple, affordable and rapid detection of infectious disease biomarkers for faster access to test results and improved patient outcomes. The development of rapid, portable and accurate POC clinical diagnostic devices could lower transmission rates, especially in cases of recent HIV infection in Resource Limited Settings (RLS) by providing timely access to intervention. Self-powered assays will be affordable, dependable and efficient for RLS with minimal user training. The clinical impact of HIV rapid testing technologies in patient care will be to achieve earlier, faster infection identification, allowing immediate counseling and treatment using Antiretrovirals (ARVs).;In this thesis, we developed an inexpensive and rapid diagnostics module for the capture and enrichment of an analyte from pooled human plasma samples. We have developed a flow-through membrane based system combining covalently conjugated polymer-antibodies and detection conjugates in a simultaneous binding, capture, and detection assay with potential adaptation for low-resource settings. This flow through assay module provides immunocomplex capture and detection of HIV-1 p24 antigen on a porous membrane surface for visualization and semi-quantification. We investigated two formats of rapid immunocomplex capture where the visual signal is developed after surface capture of an enzyme detection sandwich and addition of precipitating substrate, and the second, where a signal was directly visualized with gold colloid nanoparticles (AuNPs). This former system was optimized to provide two-step immunocomplex capture and detection for application to HIV-1 p24 antigen using a prototype disc device. Using this flow-through system, we detected 10pg/ml of p24 antigen in 50% human plasma as positive signal above background, reproducible with a coefficient of variation of <10%. Finally, the later membrane p24 assay system was optimized to enrich picomolar concentrations of HIV-1 p24 antigen in milliliter volume samples using a non-instrumented self-powered prototype syringe device with gold nanoparticles as the detection system. The final assay system has been adapted for RLSs using a non-instrumented device with heat generation capabilities. In this self-powered heating device, we use the exothermic reaction of sodium acetate trihydrate and water to generate heat. The development of the prototype non-instrumented flow-through immunoassay-heating device represents the first example of using an exothermic chemical reaction to generate the heat needed for thermally mediated immunoassays in building a fully functional and electricity-free diagnostic device for HIV. Using this flow-through system with incorporated device, we detected 12.5pg/ml of p24 antigen in 20% human plasma as positive signal above background, reproducible with a coefficient of variation of <15%. This thesis presents an initial prototype non-instrumented flow-through immunoassay device for HIV p24 antigen capable of detecting picogram per milliliter from milliliter plasma samples yielding results in less than 30minutes.