Enantioselective Profiling of d -Amino Acids in Food Matrices via a DAAO/Nanozyme Colorimetric Cascade Sensing Platform

Abstract d-Amino acids (d-AAs) are crucial biomarkers for food quality and fermentation, yet their enantioselective multiplex profiling in complex food matrices remains challenging. Herein, we report a stereoselective colorimetric cascade platform integrating d-amino acid oxidase (DAAO) and vanadium-doped Fe3O4 (V–Fe3O4) nanozymes. DAAO stereospecifically oxidizes d-AAs to produce H2O2, which triggers peroxidase-mimetic V–Fe3O4 to oxidize 3,3′,5,5′-tetramethylbenzidine (TMB), generating target-specific triple-wavelength optical fingerprints (370, 652, and 880 nm) governed by DAAO’s substrate specificity. Coupled with linear discriminant analysis (LDA), the platform achieved 100% discrimination accuracy for five representative d-AAs against 100-fold l-enantiomer excess and resolved complex binary, ternary, and five-component mixtures at both high and trace levels. For quantitative evaluation, detection limits for target d-AAs ranged from 0.01 to 0.045 mM. Validated in apple juice, skim milk, fresh tea leaves, and authentic vinegar containing endogenous d-AAs, this cascade provides a robust, cost-effective, and versatile paradigm for chiral analysis and food safety monitoring.