Highly sensitive biomolecular interaction detection method using optical bound/free separation with grating-coupled...

Area of Science:

• Biophotonics
• Analytical Chemistry
• Biomolecular Engineering

Background:

• Immunoassays require high sensitivity for detecting low-affinity biomaterials.
• Conventional methods struggle with accurate quantification of weak biomolecular interactions.
• Surface plasmon resonance (SPR) is a common technique but has limitations.

Purpose of the Study:

• To develop a highly sensitive immunoassay system using grating-coupled surface plasmon field-enhanced fluorescence spectroscopy (GC-SPFS).
• To implement an optical bound/free (B/F) separation technique for enhanced detection.
• To evaluate the system's performance for low-affinity biomaterials, specifically anti-lysozyme single-domain antibodies (sdAb).

Main Methods:

• Fabrication of a plastic sensor chip using nanoimprinting.
• Construction of a GC-SPFS system utilizing polarized surface plasmon-coupled emission (SPCE).
• Comparison of optical B/F separation with conventional washing B/F separation for immunoassay.

Main Results:

• The GC-SPFS immunoassay with optical B/F separation achieved a detection limit (LOD) of 1.2 ng/ml for a low-affinity mutant sdAb (KD ~10-7 to 10-6 M).
• This represents a significant improvement over the conventional washing B/F separation method, which yielded an LOD of 9.4 ng/ml.
• The system demonstrated higher sensitivity than the conventional Biacore SPR system, even with single amino acid mutations affecting ligand affinity.

Conclusions:

• GC-SPFS with optical B/F separation enables highly sensitive immunoassays, irrespective of ligand affinity.
• This technique facilitates the re-evaluation and utilization of low-affinity biomaterials.
• The developed method offers novel opportunities for drug discovery and diagnostic development.