Nontoxic Homogeneous Immunoassay Ultrasensitive Detection of Saxitoxin in Shellfish and Water by Peptide Antigen-CDs/COF System with Dual-Trigger Mode

Accurate, nontoxic, and time-saving detection of saxitoxin (STX) is crucial for maintaining seafood and water safety and preventing outbreaks of foodborne poisoning. Fluorescent homogeneous analysis is recognized as an easy-to-operate detection method. However, the poor antidisturbance ability of the signal is a major pain point in the development of fluorescent homogeneous analysis. Herein, a Förster resonance energy transfer (FRET)-based homogeneous immunoassay was developed for the detection of STX using carbon dots/covalent organic framework (CDs/COF)-conjugated anti-STX antibody as energy donors and FITC-labeled mimotope (FITC-M) as energy acceptors. FRET transfer characteristics and the adsorption force of STX can be well enhanced by the intensity of the CDs/COF fluorescent signal, utilizing the high bandgap framework of COF to load CDs. The spectra of FITC-M and CDs/COF-rmAb overlapped, making the possibility of FRET in a homogeneous immunoassay. Especially, the hydrogen bonding and electrostatic interactions between the carboxyl group on the surface of CDs/COF and the guanidine group of STX triggered a decrease in fluorescence intensity at the same time, forming a dual-trigger mode. The assay can detect STX with a linear range of 0.1-400 ng/mL and a limit of detection (LOD) of 0.04 ng/mL. Moreover, the entire detection process was about 90 min shorter than a traditional immunoassay. Furthermore, high specificity toward six common PST analogues and successful utilization in shellfish and water were observed, demonstrating good antidisturbance properties. The expandability of the homogeneous strategy renders it a promising candidate for nontoxic and rapid detection of biotoxins.