Rational Truncation of Aptamer for Ultrasensitive Aptasensing of Chloramphenicol: Studies Using Bio-Layer Interferometry

Abstract

Aptamers are excellent choices for selective detection of small molecules. However, the previously reported aptamer for chloramphenicol suffers from low affinity, probably as a result of steric hin-drance due to its bulky nature (80 nucleotides) leading to lower sensitivity in analytical assays. The present work was aimed at improving this binding affinity by truncating the aptamer without compromising its stability and three-dimensional folding. Shorter aptamer sequences were designed by systematically removing bases from each or both ends of the original aptamer. Thermodynamic factors were evaluated computationally to give insights into the stability and folding patterns of the modified aptamers. Binding affinities were evaluated by bio-layer interferometry. Among the 11 sequences generated, one aptamer was selected based on its low dissociation constant, length and regression of model fitting with association and dissociation curves. The dissociation constant could be lowered by 86.93% by truncating 30 bases from the 3’ end of the previously reported aptamer. The selected aptamer was used for gold nanospheres based colorimetric detection of chloram-phenicol in real samples. The detection limit could be reduced 32.87 times (1.673 pg mL-1) using the modified length aptamer, indicating its improved affinity as well as suitability in real sample analysis for ultrasensitive detection of chloramphenicol.