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

Abstract

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.