Site-Specific Immobilization Boosts the Performance of a Galectin-1 Biosensor

Area of Science:

• Biochemistry
• Biotechnology
• Analytical Chemistry

Background:

• Protein-bound glycan analysis is crucial for understanding physiological and pathological processes.
• Current glycan analysis methods face challenges due to similar physicochemical properties of carbohydrates.
• Lectin-based biosensors offer an alternative for glycan detection but require optimal protein immobilization.

Purpose of the Study:

• To develop a directed immobilization strategy for lectin biosensors using noncanonical amino acids and bioorthogonal chemistry.
• To enhance the sensitivity and selectivity of lectin biosensors through controlled protein orientation.
• To improve the performance of diagnostic tools for glycan analysis.

Main Methods:

• Site-specific incorporation of a reactive noncanonical amino acid (Nε-((2-azidoethoxy)carbonyl)-l-lysine) into a cysteine-less single-chain variant of human galectin-1 (scCSGal-1).
• Utilized strain-promoted azide-alkyne cycloaddition for directed lectin immobilization on a biosensor surface.
• Employed biolayer interferometry to assess binding sensitivity.

Main Results:

• Directed immobilization of scCSGal-1 via bioorthogonal chemistry resulted in a 12-fold enhancement in binding sensitivity to glycosylated von Willebrand factor compared to random immobilization.
• Demonstrated successful site-specific incorporation of the reactive noncanonical amino acid.
• Validated the effectiveness of controlled protein orientation in improving biosensor performance.

Conclusions:

• Controlled, site-specific protein orientation is critical for optimizing lectin biosensor sensitivity and selectivity.
• The combination of noncanonical amino acid incorporation and bioorthogonal chemistry provides a powerful platform for developing advanced diagnostic tools.
• This strategy significantly improves the performance of lectin-based glycan detection systems.