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

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.