Immunoassay

This thesis aims to address the challenges of the development of cost-effective and rapid assays for the accurate counting of CD4+ T cells and quantification of HIV-1 viral load for resource-constrained settings. The lack of such assays has severely affected people living in disease prevalent areas. CD4+ T cells count information plays a vital role in the effective management of HIV-1 disease. Here, we present a flow-free magnetic actuation platform that uses antibody-coated magnetic beads to efficiently capture CD4+ T cells from a 30 μL drop of whole blood. On-chip cell lysate electrical impedance spectroscopy has been utilized to quantify the isolated CD4 cells. The developed assay has a limit of detection of 25 cells per μL and provides accurate CD4 counts in the range of 25–800 cells per μL. The whole immunoassay along with the enumeration process is very rapid and provides CD4 quantification results within 5 min time frame. The assay does not require off-chip sample preparation steps and minimizes human involvement to a greater extent. The developed impedance-based immunoassay has the potential to significantly improve the CD4 enumeration process especially for POC settings.

Microcystin-LR (MCLR) is hepatotoxic to animals and humans with disruption of liver structure causing cytoskeletal damage, necrosis and pooling of blood in the liver, leading to large increase in liver weight. It is also a strong liver tumor promoter and protein phosphatase inhibitor. Microcysin-LR binds protein phosphatases 1 and 2A, and influences regulation of cellular protein phosphorylation. In the present study, a colloidal gold based immunoassay test strip was developed for Microcystin-LR detection. The detection limit was found to be 1 ng/mL. 5 nm colloidal gold test strips exhibits more efficient for detection, compared with 20 nm colloidal gold test strips. The interaction between Microcystin-LR antibody (immunoglobulin G) and colloidal gold nanoparticles was investigated by various analytical methods, including Ultraviolet/Visible (UV/VIS), Fourier Transform Infrared (FTIR) and Fluorescence spectroscopy as well as transmission electron microscopy (TEM).