Covalent immobilization of protein monolayers for biosensor applications

Area of Science:

• Biomaterials Science
• Surface Chemistry
• Biotechnology

Background:

• Protein immobilization is essential for various sensor technologies.
• Achieving a stable, active monolayer of proteins on surfaces remains a challenge.
• Covalent immobilization methods often suffer from significant non-specific protein adsorption.

Purpose of the Study:

• To evaluate and optimize methods for covalent protein immobilization on silicon nitride surfaces.
• To minimize non-specific protein adsorption during immobilization.
• To achieve a high surface density of active immobilized proteins.

Main Methods:

• Evaluated five protein immobilization strategies involving surface and protein linker attachment (CNBr, silanization, amine, carboxyl, aromatic ring binding).
• Assessed immobilization efficiency and protein adsorption using alkaline phosphatase and radiolabeled monoclonal antibodies (125I-MAb).
• Optimized immobilization conditions by adjusting buffer pH, ionic strength, and incorporating detergents like Tween 60.

Main Results:

• Initially, approximately 75% of immobilized protein was adsorbed, not covalently bound.
• Varying buffer conditions and adding chaotropes/competitors partially reduced adsorption.
• Incorporating 0.5% Tween 60 detergent during immobilization significantly reduced adsorption to ~10%.
• The optimized method achieved a high surface loading of 1.20 x 10^12 MAb/cm2, consistent with a monolayer.
• Immobilized antibodies retained activity, demonstrated by specific antigen binding and release of radioactive material.

Conclusions:

• Optimized immobilization using detergents like Tween 60 drastically reduces non-specific protein adsorption.
• Achieved a high-density, active protein monolayer suitable for sensor applications.
• This improved method enhances the reliability and performance of protein-based sensors.