A Higher Affinity Melamine Binding Aptamer Mutant for More Sensitive Detection

Graphical Mel-34 is a aptamer for melamine. In this paper, we carefully studied the base-pair (A−T, G−C) function in the stem regions 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. Melamine 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.