Dear Ammar Ibrahim

I hope this will help you. Designing your experiment with the Octet® R8 system to study the binding kinetics between nanoparticles (with free NH2 and NH groups) and a protein involves several considerations based on the characteristics of your analytes (nanoparticles and protein) and the technical specifications of AR2G biosensors as detailed in the technical note you've provided. Here's a step-by-step guide tailored to your scenario:

1. Determine the Ligand and Analyte:

Given that the nanoparticles are larger than the protein, the usual approach would be to immobilize the smaller molecule (the protein) to the biosensor. However, if the nanoparticles have specific functional groups (like NH2 and NH) that allow for stable covalent immobilization without affecting their binding properties or causing steric hindrance, you could consider immobilizing them instead.

2. Immobilization:

• Protein as Ligand: If immobilizing the protein, follow the standard protocol where the protein is immobilized on the biosensor using EDC/NHS chemistry, which forms a covalent bond with amine groups on the protein.
• Nanoparticles as Ligand: If immobilizing the nanoparticles, ensure their chemical groups are compatible with EDC/NHS chemistry for stable immobilization. You may need to optimize the immobilization conditions, considering the nanoparticles' unique surface properties and ensuring that they are stable and retain their functional binding sites post-immobilization.

3. Running Buffer Considerations:

• BSA in Running Buffer: Adding BSA (Bovine Serum Albumin) to the running buffer can help reduce non-specific binding by blocking potential nonspecific adsorption sites on the biosensor surface. However, ensure that BSA does not interfere with the specific interaction between your nanoparticle and the protein.

4. Assay Flow:

• If you decide to immobilize the nanoparticles, the assay flow will indeed be flipped compared to the standard procedure:
  • Loading Step: Nanoparticles will be immobilized on the biosensor.
  • Association Step: The protein will be introduced to study the binding kinetics.
• Ensure to include a reference biosensor with immobilized nanoparticles but without exposure to the protein to account for non-specific binding or drift during the assay.

5. Experiment Design Steps:

Special Considerations:

• Size of Nanoparticles: Ensure that the size difference does not introduce mass transport limitations or steric hindrance that could affect the binding kinetics interpretation.
• Regeneration: Developing effective regeneration conditions that remove bound analyte without damaging the immobilized ligand will be crucial, especially if you plan to reuse the biosensors.

the standard protocol recommends immobilizing proteins due to their well-understood chemistry with EDC/NHS, immobilizing nanoparticles is feasible if they have compatible functional groups and don't introduce significant complications to the assay. Optimization and careful experimental design are key to successfully measuring the binding kinetics between your nanoparticles and the protein.

Thanks