Rational design of hairpin aptamer using intrinsic disorder mechanism to enhance sensitivity of aptamer folding-based electrochemical sensor for tobramycin

Folding-based electrochemical sensors employing electrode-immobilized, structure-switching aptamers represent a promising platform for rapid and selective detection of a variety of target analytes. Sensitivity of this class of sensors usually needs, however, to be improved through re-engineering the parent aptamer probes to undergo large conformation change upon target binding. Here, we report the development of a sensitive folding-based electrochemical sensor for tobramycin by re-engineering a DNA hairpin aptamer based on the concept of intrinsic disorder. Through incorporating disordered poly-thymine base spacers of various lengths into the loop of the parent hairpin aptamer, structure-switching aptamers exhibiting large target binding-induced conformation change for high signal sensitivity were re-engineered and an optimal hairpin aptamer containing a disordered spacer of 24 thymine bases (probe T24) was obtained. Folding-based electrochemical sensor modified with the probe T24 is sensitive for tobramycin with a maximum percent signal change of 767.1% for signal-saturating target concentration compared to 54.7% obtained using the parent aptamer. This sensor is also selective enough and applicable to detection of tobramycin in real samples with satisfactory results. We believe that the intrinsic disorder-based concept for re-engineering of structure-switching aptamers will help guide further design and application of highly sensitive aptamer folding-based electrochemical sensors.