A simple method for controlled immobilization of proteins on modified SAMs

Area of Science:

• Biomaterials Science
• Surface Chemistry
• Protein Immobilization

Background:

• Self-assembled monolayers (SAMs) are crucial for surface functionalization.
• Immobilizing histidine-tagged proteins often requires complex synthesis and can lead to surface issues.
• Nitrilotriacetic acid (NTA) is a common chelator for histidine-tagged proteins.

Purpose of the Study:

• To develop a simplified and efficient method for modifying SAMs with NTA for protein immobilization.
• To improve upon existing methods by performing reactions in situ.
• To enable controlled protein loading and maintain protein functionality.

Main Methods:

• Formation of a carboxyl-terminated SAM using mercaptohexadecanoic acid on a gold surface.
• In situ condensation of the carboxyl group with an NTA derivative to form a peptide bond.
• Chelation of Ni(2+) ions and subsequent immobilization of hexahistidine-tagged proteins.

Main Results:

• A homogeneous and well-ordered NTA-modified SAM was successfully created.
• In situ modification avoided complex synthesis and phase segregation issues.
• Controlled NTA loading allowed for optimal and rapid protein immobilization.
• Immobilized proteins, including (His)(6) enhanced green fluorescent protein, were shown to be functional and reversibly bound.
• Surface plasmon resonance and cyclic voltammetry confirmed specific and reversible protein immobilization.

Conclusions:

• The in situ NTA modification of SAMs offers a streamlined approach for protein immobilization.
• This method enhances control over surface density and prevents molecular crowding, leading to improved protein loading.
• The functional and reversible immobilization of histidine-tagged proteins is demonstrated, with potential applications in biosensing and biomaterials.