Duplexed aptamer-isothermal amplification-based nucleic acid-templated copper nanoparticles for fluorescent detection of okadaic acid

Okadaic acid (OA) is a major marine toxin that can cause diarrheic shellfish poisoning (DSP). Because of food safety concerns, simple and reliable field assays are in high demand. Herein, a highly sensitive fluorescent assay for OA in seafood was reported. For this purpose, a duplexed aptamer (DA) based on a conformational change upon binding OA was designed and immobilized on the surface of streptavidin-conjugated magnetic beads (SMBs). With the addition of OA, the DAs melted, and ss-aptamers were released into the solution. After magnetic separation, a terminal deoxynucleotidyl transferase (TdT)-mediated isothermal amplification system was formed via a free ss-aptamer acting as a template. This product with a poly(thymine) tail was directly added to a fluorescent copper nanoparticle (CuNP) formation system, and the ultralong consecutive T-rich sequence provided deposition sites for copper. Finally, the OA concentration was determined by recording fluorescence spectra and calculating the intensity change. This duplexed aptamer-isothermal amplification-based nucleic acid CuNP (DAIA-CuNP) sensor can achieve ultrahigh-sensitivity detection of OA compared to LC-MS and electrochemical methods. This DAIA-CuNP sensor can detect OA with a limit of detection (LOD) of 1.1 ng/L and a good linear range from 1.0 ng/L to 50.0 μg/L in shellfish extract. Additionally, this method is expected to be compatible with a handheld fluorescence reader and can meet field detection needs.