Tuning Library Length Revealing High Affinity Melamine Aptamers with Opposite Fluorescence Responses for Highly Selective Detection

The adulteration of dairy products with melamine has created an urgent need for reliable, sensitive, and convenient detection methods. Since melamine can form extensive hydrogen bonding and stacking interactions with DNA bases, aptamers represent an attractive platform for melamine sensing. In this work, capture-SELEX was employed using two DNA libraries containing either 30 or 36 randomized nucleotides. The longer library generated a greater diversity of sequence families, including a dominant family featuring more than 30 conserved nucleotides, a structural motif unattainable with the shorter library. Comparison was made with previous length-dependent SELEX experiments. The highest affinity aptamers exhibited a dissociation constant ( K d ) of ∼60 nM, as determined by isothermal titration calorimetry. Screening aptamer responses using a thioflavin T fluorescence assay revealed a pair of aptamers with opposite signal readouts, enabling melamine detection with limits of detection of 61 nM and 208 nM, respectively. In 5% ( v / v ) milk samples, limits of detection of 89 and 315 nM were achieved, respectively. Overall, this study advances understanding of the impact of library length on aptamer selection and provides a set of high affinity aptamers along with a robust, label-free strategy for melamine detection.