Entropy-Engineered Aptamer Truncation Enables Dual-Mode LRET/SERS Aptasensing for Multiplex Detection of Phalloidin and α-Amanitin in Mushroom Samples.

Phalloidin (PHD) and α-amanitin (α-AMT) are structurally constrained cyclic mushroom toxins whose rigid structures and limited accessible recognition surfaces challenge high-affinity molecular recognition and sensitive multiplex detection. Here, we report an entropy-guided aptamer engineering strategy that removes redundant regions while preserving essential stem-loop recognition motifs, optimizing the thermodynamic balance of target binding, and yielding truncated aptamers with 3.3- and 3.6-fold improved SGI-derived affinities toward PHD and α-AMT, respectively. The engineered aptamers were integrated into a dual-mode luminescence resonance energy transfer (LRET)/surface-enhanced Raman scattering (SERS) platform comprising upconversion nanoparticles (UCNPs), and Fe3O4@AuNS. A single target-binding-driven aptamer-cDNA structural-switching event synchronously regulated both optical outputs, enabling internally cross-validated multiplex quantification with low-pg/mL detection limits over 0.01-100 ng/mL (R2 > 0.99), recoveries of 92.0-99.2%, and relative standard deviations below 5% in mushroom samples. This strategy provides a generalizable framework for sensitive detection of structurally constrained food toxins and multiplex food safety analysis.