The Performance of Antibodies as Catalytic Readout for Sensor Applications
Infectious diseases are a leading threat to global health, accounting for an estimated 15.5 million physicians’ appointments annually in the US. They also present the danger of escalating to pandemics, which, as COVID-19 has demonstrated, can affect a broad range of societal functions and industries. Accordingly, infectious disease monitoring is a critical tool for public healthcare infrastructure. This monitoring is frequently done by Enzyme-Linked Immunosorbent Assays (ELISAs), in which antibodies for a target disease are specifically captured on an antigen-coated surface. These antibodies are detected using a tagged secondary, which binds to the subject’s antibodies in a species-dependent manner. This secondary step can introduce time and resource costs and present limitations for adaptive infectious disease monitoring, particularly in the event of zoonotic transfer. This research explores a novel antibody detection protocol that bypasses these limitations. The protocol utilizes the inherent catalytic ability of antibodies to convert water to hydrogen peroxide through a process known as the Antibody-Catalyzed Water Oxidation Pathway (ACWOP). An ACWOP-based sensor would allow for the direct detection of immobilized antibodies, eliminating the need for secondaries and associated time and resource costs. Because all antibodies perform ACWOP, such a sensor could be applied for many diseases and could provide a consistent technical detection system across different species, allowing for more adaptive monitoring of zoonotic transfer. This thesis details the development and optimization of such a readout protocol, which relies on commercially available reagents to scale more readily for monitoring and point-of-care applications. It then investigates some of the major parameters for a generalized ACWOP-based immunosensor, such as 1) substrate necessities like high surface area and pore size, 2) the impact of sodium azide, and 3) design constraints associated with blocking agents and antigen choice. Regarding the latter, proteins containing tryptophan, including BSA and antigens like the C6 peptide, can also produce hydrogen peroxide under these conditions, substantially increasing background signal. This research explores possible methods for mitigating these issues, such as increased illumination time and intentional oxidation of the blocking agent prior to antibody analysis. Finally, the research concludes with a discussion of regulatory and pedagogical approaches to address the harms of racial biases associated with artificial intelligence technologies.