Capture-SELEX for Chloramphenicol Binding Aptamers for Labeled and Label-Free Fluorescence Sensing.

Abstract

Chloramphenicol (CAP) is a potent antibiotic. Due to its side effects, CAP is currently banned in most countries, but it is still found in many food products and in the environment. Developing aptamer-based biosensors for the detection of CAP has interested many researchers. While both RNA and DNA aptamers were previously reported for CAP, they were all obtained by immobilization of the CAP base, which omitted the two chlorine atoms. In this work, DNA aptamers were selected using the library-immobilized method and free unmodified CAP. Three families of aptamers were obtained, and the best one named CAP1 showed a dissociation constant (Kd) of 9.8 μM using isothermal titration calorimetry (ITC). A fluorescent strand-displacement sensor showed a limit of detection (LOD) of 14 μM CAP. Thioflavin T (ThT) staining allowed label-free detection of CAP with a LOD of 1 μM in buffer, 1.8 μM in Lake Ontario water, and 3.6 μM in a wastewater sample. Comparisons were made with previously reported aptamers, and ITC failed to show binding of a previously reported 80-mer aptamer. Due to the small size and well-defined secondary structures of CAP1, this aptamer will find analytical applications for environmental and food monitoring.