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

Aptamers are an excellent choice for the selective detection of small molecules. However,
the previously reported aptamer for chloramphenicol suffers from low affinity, probably as
a result of steric hindrance 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 provide insight into the stability
and folding patterns of the modified aptamers. Binding affinities were evaluated using bio-layer
interferometry. Among the eleven 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 30 end of the
previously reported aptamer. The selected aptamer was used for the detection of chloramphenicol in
honey samples, based on a visible color change upon the aggregation of gold nanospheres caused
by aptamer desorption. The detection limit could be reduced 32.87 times (1.673 pg mL