Development of a solid-phase receptor-based assay for the detection of cyclic imines using a microsphere-flow...

Area of Science:

• Marine biology
• Analytical chemistry
• Toxicology

Background:

• Cyclic imines, specifically spirolides, are potent marine toxins first identified in Canadian aquaculture in the 1990s.
• These lipophilic toxins exhibit rapid toxicity in mouse bioassays, posing a risk to seafood safety.
• Existing detection methods for marine toxins can be limited in sensitivity or dynamic range.

Purpose of the Study:

• To develop a novel receptor-based detection method for spirolides and related cyclic imines.
• To utilize a microsphere/flow cytometry Luminex system for sensitive and rapid toxin quantification.
• To establish a screening assay suitable for routine analysis of shellfish contamination.

Main Methods:

• Two binding proteins, nicotinic acetylcholine receptor (nAChR) and Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP), were evaluated.
• A receptor-based inhibition assay was designed using microsphere immobilization and competition with biotin-α-bungarotoxin (α-BTX).
• Quantification was performed using phycoerythrin-labeled streptavidin and analyzed via Luminex 200 flow cytometry.

Main Results:

• Both nAChR and Ls-AChBP effectively bound 13-desmethyl spirolide C.
• The nAChR demonstrated a cross-reactivity profile aligning with known toxic potencies of spirolides and gymnodimine.
• A simple acetone extraction protocol showed no matrix interference in shellfish samples (mussels, scallops, clams).

Conclusions:

• The nAChR-based microsphere assay is a sensitive and rapid method for detecting cyclic imines in shellfish.
• The assay exhibits a wider dynamic range and higher sensitivity compared to previous receptor-based methods.
• This method is suitable for multiplexing, enabling simultaneous detection of multiple marine toxins for enhanced food safety monitoring.