Increasing Aptamer Affinity from Millimolar to Nanomolar by Forming a Covalent Adduct for Detecting Acrylamide

Being a neurotoxin and carcinogen, acrylamide has been an important target for developing biosensors. DNA aptamers are attractive for making biosensors due to their programmable structure, low cost, and ease of modification. However, DNA aptamers have poor affinities to low-binding epitope target molecules such as acrylamide. In this work, an aptamer for acrylamide was isolated with an apparent Kd of 10.5 mM using a thioflavin T fluorescence assay and 4.7 mM using the fluorescence strand-displacement assay. To improve binding affinity, acrylamide was reacted with xanthydrol to form a covalent adduct, and a new aptamer selected for this adduct achieved a Kd of 20 nM using the strand-displacement assay, representing an improvement of 235,000-fold. Using the strand-displacement biosensor, a limit of detection of 4.2 nM was achieved for the adduct. This work demonstrates a practical route to convert low epitope targets to high-affinity targets for aptamer binding and bioanalytical applications.