Abstract
AbstractMelamine is an illegal additive in milk products. Mel‐34 is an aptamer specific for melamine. In this paper, we studied the base‐pair function in the stem regions of Mel‐34 using label‐free thiazole orange (TO) staining assay. Then, we used isothermal titration calorimetry (ITC) to verify the binding activity, and the fluorescence resonance energy transfer (FRET) biosensor based molecule‐beacon was established to detect melamine in yogurt sample. We found one of the mutants (mut6) has four folds higher affinity than Mel‐34, which indicated its critical binding domain was G18‐C25 base pair. We detected melamine in 1 % yogurt sample using mut6 as recognition molecule, the linear range was below 20 μM and the limit of detection was 4.09 μM which is below the melamine threshold (1.0 mg/kg) in milk products. This work calls for carefully study the critical binding domain to discover higher affinity aptamer specific for small molecule using label‐free method.
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