Browsing by Author "Singh, Raunak"

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    The Electrochemistry of Ferrocenyl-thiolate Self-Assembled Monolayers for use in a Toll-like Receptor 4-based sensor
    (2021-08-20) Singh, Raunak; Birss, Viola Ingrid; MacCallum, Justin Laine; Sutherland, Todd Christopher
    Detection of biological agents by traditional bench-top instrumentation is insufficient for purposes of real time on-site analysis, which is imperative for biodefense applications. Unlike radiological and chemical warfare agents, sophisticated detection systems have not yet been designed for immediate sensing of biological agents. Development of field-deployable biosensors is a response to fulfill this gap, specifically, the demand for immediate recognition of samples of unknown origin requiring broad-based detection capabilities. Our research group has been pursuing this venture by creating an electrochemical biosensor that is able to detect the bacterial endotoxin, lipopolysaccharide (LPS), produced by gram negative bacteria. Our biosensor utilizes Toll-like Receptor 4 (TLR4) immunoproteins as the biorecognition element. These immunoproteins are surface immobilized via ternary alkanethiolate self-assembled monolayers (SAMs) deposited on polycrystalline Au surfaces. These SAMs possess a uniquely low interfacial resistance (? 1 k?) due to the presence of a Ferrocenyl-thiolate component, which mediates electron transfer from dissolved Fe(CN)64- to the underlying Au substrate. The work presented in this thesis focuses on the problem, and its subsequent solution, encountered with the SAM electrochemistry portion of the TLR4 sensor assembly. The issue was initially detected by false positive responses of the sensor in the absence of LPS-containing bacterial lysate, exhibiting an increase in resistance of similar intensity in both the absence and presence of heat-killed Salmonella typhimurium (HKST) bacterial lysate. The primary cause of the TLR4 sensor drift was determined here to be the instability of the Fc+ group, which is generated repeatedly during the mediation of the Fe(CN)64- oxidation process. This is consistent with past literature that alluded to the irreversible redox deactivation of the Fc+ group under non-optimal conditions. This problem was remedied in this work by replacing phosphate ions from the buffer solution by perchlorate, but as proteins such as TLR4 should be kept in neutral conditions, another solution was sought. This involved replacing the original Fc-thiol molecule (i.e., 11-mercaptodecyl-ferrocenylcarboxamide (Fc-amide)) with 11-(ferrocenyl)undecanethiol (FcC¬11).These novel ternary SAMs containing the stable FcC11 component were then used to prepare a new generation of TLR4-based sensors, which showed negligible drift and were able to reliably detect LPS from 0.0128 to 5000 µg/mL. This demonstrates that utilizing Fc-thiolate SAMs is now a viable method to prepare low-resistance biosensors for use as field-deployable systems.