Clinical detection of Hepatitis C viral infection by yeast-secreted HCV-core:Gold-binding-peptide

Abstract Access to affordable and field deployable diagnostics are key barriers to the control and eradication of many endemic and emerging infectious diseases. While cost, accuracy, and usability have all improved in recent years, there remains a pressing need for even less expensive and more scalable technologies. To that end, we explored new methods to inexpensively produce and couple protein-based biosensing molecules (affinity reagents) with scalable electrochemical sensors. Previous whole-cell constructs resulted in confounding measurements in clinical testing due to significant cross-reactivity when probing for host-immune (antibody) response to infection. To address this, we developed two complimentary strategies based on either the release of surface displayed or secretion of fusion proteins. These dual affinity biosensing elements couple antibody recognition (using antigen) and sensor surface adhesion (using gold-binding peptide-GBP) to allow single-step reagent production, purification, and biosensor assembly. As a proof-of-concept, we developed Hepatitis C virus (HCV)-core antigen-GBP fusion proteins. These constructs were first tested and optimized for consistent surface adhesion then the assembled immunosensors were tested for cross-reactivity and evaluated for performance in vitro . We observed loss of function of the released reagents while secreted constructs performed well in in vitro testing with 2 orders of dynamic range, and a limit of detection of 32 nM. Finally, we validated the secreted platform with clinical isolates (n = 3) with statistically significant differentiation of positive vs. non-infected serum (p