Selection of a High-Affinity Aptamer for Phalloidin and Application in a Dual-Mode LRET/Colorimetric Aptasensor

Phalloidin (PHD), a potent bicyclic peptide from Amanita species, poses severe food safety risks when misidentified as edible mushrooms. However, no aptamer-based detection system has been reported to date. Here, we report the isolation of a high-affinity and highly specific DNA aptamer (Apt-PHD) selected through 15 rounds of Capture-SELEX using a structured 97-nt ssDNA library. High-throughput sequencing identified PHD-3 as the optimal candidate, exhibiting dissociation constants of 0.292 ± 0.0034 μM (isothermal titration calorimetry) and 42.7 ± 6.7 nM (SYBR Green I fluorescence assay). Mechanistic insights from circular dichroism spectroscopy and molecular docking revealed target binding at the stem-loop junction, stabilized by hydrogen bonding, van der Waals forces, and hydrophobic interactions (docking score = −15.9 kcal/mol). Leveraging Apt-PHD, a dual-mode lanthanide resonance energy transfer (LRET)/colorimetric aptasensor was developed using upconversion nanoparticles (UCNPs@SiO₂–NH₂) as the luminescence donor and gold nanoparticles (AuNPs) as both a quencher and a colorimetric reporter. Under optimized conditions, the sensor achieved limits of detection of 12.6 pg/mL (LRET) and 18.0 pg/mL (colorimetric), with wide dynamic ranges of 0.01–1000 ng/mL and 0.01–100 ng/mL, respectively. The platform demonstrated excellent reproducibility (RSD = 3.56% and 3.20%), high selectivity over related mycotoxins, and accurate recovery in spiked mushroom samples (90.5–97.5% and 91.8–96.6%). This study establishes the first aptamer-based recognition system for phalloidin and introduces a versatile dual-mode optical sensing platform, enabling rapid, reliable, and field-deployable detection of small-molecule toxins for enhanced food safety surveillance.