Time-Resolved "Barbecue" Förster Resonance Energy Transfer for Sensitive and Homogenous Detection of Alginate

Area of Science:

• Glycoscience
• Analytical Chemistry
• Biochemistry

Background:

• Carbohydrate analysis is challenging due to structural complexity and limited methodologies.
• Carbohydrate-binding modules (CBMs) and carbohydrate-active enzymes (CAZymes) offer specific carbohydrate recognition.
• Existing assays for polysaccharides like alginate have limitations in sensitivity and simplicity.

Purpose of the Study:

• To develop the first homogeneous assays for alginate quantification.
• To leverage CBMs and CAZymes for sensitive and specific polysaccharide detection.
• To introduce a novel "Barbecue" assay based on time-resolved Förster resonance energy transfer (TR-FRET).

Main Methods:

• Utilized orthogonally labeled CBMs/lyases with CoraFluor-1 (CRF) and Cy5 for TR-FRET detection.
• Developed a homogeneous, single-step "Barbecue" assay design without wash steps.
• Applied the assay to quantify alginate in various sample types.

Main Results:

• Achieved a lower limit of quantification as low as 0.4 ng·mL⁻¹ for alginate.
• Demonstrated a ≥100-fold improvement in sensitivity compared to conventional microarray assays.
• Validated the assay's broad applicability in biomedical, food, and cosmetics samples.

Conclusions:

• The "Barbecue" assay provides a sensitive, rapid, and simple method for alginate quantification.
• The TR-FRET approach is generalizable for developing assays for other polysaccharides.
• This work advances glycoscience by overcoming limitations in carbohydrate analysis.