A protein-nanoparticle conjugate platform for simplified and sensitive protease activity assays.

Gold nanoparticles (AuNPs) have attracted increasing attention as functional platforms for biosensing due to their high biocompatibility and tunable surface properties. In this study, we developed a protease activity assay using NanoLuc luciferase (NLuc) as a luminescent reporter, genetically fused to a gold-binding peptide (AuBP1) and a TEV protease (TEVp) recognition sequence. The fusion proteins were immobilized onto AuNPs via the gold-binding peptide. Upon TEVp treatment, NLuc was cleaved and released from the AuNP surface, resulting in a measurable increase in luminescence intensity. Two measurement strategies were evaluated: one involving centrifugation-based separation after cleavage, and another without separation. The separation method showed a broader dynamic range and higher sensitivity, while the non-separation method enabled a simplified workflow with sufficient luminescence response. The results demonstrate the feasibility of combining recombinant protein technology with AuNPs to construct functional biosensors for protease assays. This approach may offer a practical and adaptable tool for further development in bioanalytical applications.