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

Area of Science:

• Nanotechnology
• Biochemistry
• Biosensing

Background:

• Gold nanoparticles (AuNPs) are versatile for biosensing due to biocompatibility and surface properties.
• Protease activity assays are crucial in diagnostics and research.
• Developing sensitive and efficient protease detection methods is an ongoing challenge.

Purpose of the Study:

• To develop a novel protease activity assay utilizing gold nanoparticles (AuNPs) and a luminescent reporter.
• To investigate the immobilization of a genetically engineered fusion protein onto AuNPs.
• To evaluate different measurement strategies for enhanced sensitivity and workflow simplification.

Main Methods:

• Engineered a fusion protein comprising NanoLuc luciferase (NLuc), a gold-binding peptide (AuBP1), and a TEV protease (TEVp) recognition sequence.
• Immobilized the fusion protein onto AuNPs via the AuBP1.
• Assessed protease activity by measuring luminescence changes upon TEVp-mediated cleavage and release of NLuc.
• Compared centrifugation-based separation with a non-separation method for signal detection.

Main Results:

• Successful immobilization of fusion proteins onto AuNPs.
• TEVp cleavage led to a measurable increase in luminescence intensity.
• The centrifugation-based separation method demonstrated a broader dynamic range and higher sensitivity.
• The non-separation method offered a simplified workflow with adequate luminescence response.

Conclusions:

• Demonstrated the feasibility of combining recombinant protein technology with AuNPs for protease biosensing.
• The developed AuNP-based assay is a practical and adaptable tool for protease activity detection.
• This approach holds potential for various bioanalytical applications requiring sensitive protease quantification.