BRET Nano Q-Body: A Nanobody-Based Ratiometric Bioluminescent Immunosensor for Point-of-Care Testing

Area of Science:

• Biotechnology
• Immunosensors
• Point-of-Care Diagnostics

Background:

• Nanobody-based biosensors offer high specificity and stability.
• Bioluminescence Resonance Energy Transfer (BRET) enables sensitive detection.
• Point-of-care testing (POCT) requires rapid, portable, and stable diagnostic tools.

Purpose of the Study:

• To develop a nanobody-based homogeneous bioluminescent immunosensor for one-pot POCT.
• To create a BRET nano Q-body for sensitive and stable detection.
• To demonstrate the applicability of the BRET nano Q-body in biological fluids and on paper devices.

Main Methods:

• Fused NanoLuc luciferase and a cysteine-containing tag to an MTX-recognizing nanobody.
• Labeled the nanobody with a fluorescent dye via thiol-maleimide Michael addition.
• Optimized dye/linker and tested stability, sensitivity, and performance in biological matrices and lyophilized paper devices.

Main Results:

• The BRET nano Q-body showed a >7-fold increase in emission ratio (TAMRA/NanoLuc) dose-dependently.
• Demonstrated superior thermostability and tolerance to organic solvents, reducing agents, and detergents.
• Achieved limits of detection of 0.50 nM in milk, 1.6 nM in serum, and 3.7 nM in whole blood without dilution.
• Lyophilized paper devices maintained function for over a month at 25 °C and performed well in undiluted biological fluids.

Conclusions:

• The BRET nano Q-body is a powerful tool for POCT, applicable in various biological fluids.
• Paper device fabrication enhances portability and storage, enabling on-site therapeutic drug monitoring.
• This technology holds transformative potential for in situ detection in therapeutic, diagnostic, and environmental applications.