Development of a Modular APEX2-based Lateral Flow Signaling Platform for Virus Diagnostics

Rapid point-of-care (POC) diagnostics require sensitive, stable, and cost-effective reporter systems. While lateral flow assays (LFAs) are widely used, their sensitivity is often limited by the use of colloidal gold, whereas enzymatic amplification using horseradish peroxidase (HRP) is constrained by the difficulties of recombinant production in bacterial hosts. In this study, we present a protein engineering framework utilizing APEX2, a robust engineered peroxidase, as a next-generation signaling element. We optimized the expression of APEX2-His6 in E. coli , achieving high yields (64 mg/L) of a functional apoenzyme that was efficiently reconstituted in vitro (RZ value: 2.1). Furthermore, we investigated the development of a bifunctional reporter by genetically fusing APEX2 to a single-domain antibody (sdAb). While the production of the fusion protein encountered significant solubility challenges in E. coli (yielding ∼0.5 µg/liter culture), our results confirm that APEX2 retains high catalytic activity and stability when integrated into an LFA format. Using West Nile Virus (WNV) as a model system, we demonstrate that APEX2-mediated enzymatic amplification provides a clear colorimetric signal on nitrocellulose membranes. This work establishes APEX2 as a commercially viable, recombinantly accessible alternative to traditional peroxidases, providing a versatile platform for the development of high-sensitivity diagnostic tools.