We propose the design of engineered antibodies which harbor a redox-sensitive domain by employing an anti-epidermal growth factor receptor (EGFR) single chain variable fragment (scFv) and heme c containing electron transfer protein. Antibodies are commonly employed in biosensing applications due to their high sensitivity and selectivity towards specific targets. Antibodies typically exhibit binding constants within the low micromolar to nanomolar range, with some demonstrate affinity in the picomolar range. While high specificity is an attractive feature of biosensors which aim to detect proteins of interest in low physiological concentrations in complex biological samples, the tight binding of antibodies to their targets presents a challenge in designing regenerable sensor surfaces. Regeneration of affinity biosensors typically involves adding detergents or buffers of variable pH to completely strip the sensor surface of the antibody-target complex. However, this approach is not feasible for continuous monitoring. To work towards in situ regeneration of antibodies immobilized on an electrode surface, modulation of binding through an external physical or chemical signal is necessary. To this end, we constructed a fusion molecule consisting of an anti-EGFR scFv fused with cytochrome c-like electron transfer protein derived from bacterial glucose-3-dehyrogenase, using Escherichia coli as a host microorganism. Anti-EGFR scFv was chosen as a model target for this system as this receptor plays important roles in regulating cell development (proliferation, survival, differentiation, etc). The fusion protein showed recognition of EGFR, demonstrating the cytochrome c domain does not impact EGFR affinity. The characterization of thus created antibody fusion for EGFR sensing will be presented.

Creation and characterization of engineered antibodies harboring a redox-sensitive domain

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