pH-Directed Capture-SELEX for Nanomolar Affinity Aptamers for Kanamycin Detection

Abstract

Kanamycin A is a widely used antibiotic, although it has a narrow therapeutic window demanding highly accurate monitoring. The sensing of kanamycin A using aptamers is of great interest since aptamers can be used for continuous monitoring with a rapid response. While kanamycin has been the target for at least four previous aptamer selections, the binding affinities of the reported DNA aptamers are still sub-optimal. All the previous aptamer selections were performed at pH 7.5 or higher. Given that kanamycin A has four amino groups with pK_a values close to 7, we herein selected DNA aptamers for kanamycin A at both pH 6 and pH 8. The selection at pH 6 enriched aptamers although the pH 8 selection library remained highly diverse in the end. The best aptamer named KAN6-1 showed a dissociation constant of around 320 nM measured using isothermal titration calorimetry in the selection buffer. In buffers without salt, binding can happen from pH 6 to 8. Specific binding was confirmed using mutation studies. A strand displacement assay was developed with a limit of detection (LOD) of 100 nM in buffer. Similar LOD values were also obtained in lake water and in 10 % human serum. Comparisons were also made with some previously reported DNA aptamers. This study shows the importance of pH value on the selection of aptamers and provides a new aptamer for kanamycin A detection.