A need for rapid and sensitive point-of-care diagnostics has emerged in the context of global health concerns. It is desirable that assays be simple, rapid, portable, and robust and be able to perform as well as standard laboratory assays. Microfluidic technology has the capacity to improve the speed, sample processing, waste generation, and performance of rapid diagnostic assays. Immobilization strategies based upon the phase-transition properties of poly(N-isopropylacrylamide) (pNIPAAm) allow solution phase sample interrogation prior to immobilization in the microfluidic device. Combining the sample handling capabilities of microfluidic card assays with separation and enrichment capacities of phase-transition polymers is the focus of this dissertation.
Covalent antibody conjugates were prepared with amine-reactive semi-telechelic pNIPAAm. Such antibodies were found to be precipitable and bind antigen following conjugation. Using a chain transfer agent-bound surface, brush-like pNIPAAm grafts were grafted from nylon membranes using the reversible addition-fragmentation chain transfer (RAFT) technique of polymerization. By making small changes in the system temperature, efficient and reversible capture of antibody conjugates of pNIPAAm at porous pNIPAAm-grafted surfaces was possible. This capture and release system was integrated into a microfluidic card design and applied to antigen and full-stack immunocomplexes with detection antibodies. A full stack assay for Plasmodium falciparum PfHRP2 antigen spiked into human plasma was demonstrated. For a rapid single surface capture and assay system, gold nanoparticle detection antibody conjugates resulted in a visible signal accumulation at the membrane corresponding to increasing amounts of PfHRP2 antigen. This assay system for Plasmodium falciparum PfHRP2 antigen was applied to human plasma samples using recombinant antigen and to clinical plasma samples obtained from a malaria-endemic area.
|School:||University of Washington|
|School Location:||United States -- Washington|
|Source:||DAI-B 71/05, Dissertation Abstracts International|
|Keywords:||Analyte enrichment, Immunoassays, Polymer conjugates|
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