(Invited) Engineering Antibodies Fragments for Continuous Protein Biosensing

Developing new approaches to monitor critical analytes in complex biological environments is key to the development of new diagnostic tools for application in a range of fields including the biomedical, environmental and food sciences. While reversible ion and metabolite sensing can be achieved through a variety of approaches including ionophore-based techniques, there is a lack of approaches to achieve real-time protein monitoring. Immunoassays (e.g. ELISA) are robust approaches to achieve endpoint measurements, however the multi-step nature of the technique is not amenable to reversible biosensing. One approach to simplify immunoassay chemistry is to reduce the number of assay steps required, which could lead to simpler manual assays, less reliance on laboratory infrastructure, or simpler POC designs. We have developed a biosensor approach based on direct detection of antigen-antibody binding in a single step. Using a site-specific approach to incorporate fluorescent dyes into antibody fragments, we screened a range of positional mutants and identified several that showed antigen-dependent spectral changes. These changes were observed to be dose-dependent in both buffer and diluted plasma samples. We then applied a site-directed mutagenesis approach to generate antibody fragments capable of reversible protein interactions and reversible sensing. These fragments can be used in homogenous assays or immobilised to surfaces, and in this presentation we will present our most recent results on epidermal growth factor receptor (EGFR) and cardiac troponin I (cTnI) monitoring.