From Cross-Reactivity to Specificity: A SELEX-Derived Aptamer Sensor Array for Aristolochic Acid I and Aristolactam I

Aptamer selection often yields multiple sequence families that recognize different epitopes or bind through distinct molecular interactions. Rather than using a single aptamer for detection, we demonstrate that these complementary aptamers can be integrated into a cross-reactive sensor array to enhance molecular discrimination. Aristolochic acids, a family of structurally similar nephrotoxic compounds in herbal products, were chosen as a model system. Three DNA aptamers against the most toxic analog, aristolochic acid I (AAI), were isolated by capture-SELEX and exhibited sub-micromolar affinities, as characterized by thioflavin T fluorescence assays and isothermal titration calorimetry. Using a fluorescence strand-displacement format, the aptamers displayed distinct but overlapping response profiles toward AAI, its major metabolite aristolactam I (ALI), and related analogs, generating characteristic multiaptamer response fingerprints. By matching the dynamic ranges of the individual sensors and applying chemometric partial least-squares regression, these fingerprints were deconvoluted to achieve selective discrimination and simultaneous quantification of AAI and ALI over 0.025-4 μg mL-1. The sensor array showed good interday reproducibility and achieved recoveries of 85-108% in synthetic herbal matrices and 80-118% in artificial urine. This work establishes a general strategy for transforming aptamer cross-reactivity into analytical specificity through chemometric analysis, providing a versatile platform for the rapid detection of structurally similar small-molecule toxins.