Three-Dimensional DNA Nanostructure-Assisted Multiplexed Aptamer Assay for Simultaneous Detection of...

This study developed a multiplexed aptasensor for the simultaneous detection of an endocrine-disrupting chemical (EDC) cluster-specifically, a mixture of bisphenol A (BPA), phthalic acid esters (PAEs), and nonylphenol (NP)-utilizing a three-dimensional DNA nanostructure framework. A tetrahedral 3D-DNA nanostructure was constructed, and its formation and morphology were verified via fluorescence-based assessments, gel electrophoresis, atomic force microscopy, and dynamic light scattering analysis. The reaction complex was fabricated by anchoring multiple aptamers to 3D-DNA nanostructures immobilized onto magnetic beads. This design enabled the quantitative detection of NP, PAEs, and BPA by measuring the signal decrease from DNA probes conjugated to specific quantum dots (QD₅₆₅, QD₆₀₅, and QD₆₅₅). To validate the multiplexing capability, signal crosstalk and the dissociation of the QD-probe DNA upon target binding were examined. The quantitative performance of the multiplexed EDC detection was assessed in terms of selectivity against potential interferences, dynamic range, and limit of quantification (LOQ). The assay demonstrated dynamic ranges of 1-100 ng/mL for NP, 0.001-100 ng/mL for di-n-butyl phthalate (DBP), and 0.0005-100 ng/mL for BPA, with corresponding LOQs of 1 ng/mL (ppb), 1 pg/mL (ppt), and 0.5 pg/mL (ppt), respectively. Finally, the assay was applied to EDC mixtures spiked into real-world sample matrices (tap water and beverages), and recovery rates were evaluated to demonstrate its practical applicability.