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 (K d) 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.

Electrostatic-Mediated Binding of DNA to Lysozymes: Evaluation of Aptamer-Based Assays for Highly Positively Charged Targets

“…Nucleic acid-binding dyes can be used as efficient indicators of aptamer-target interactions . Different fluorescent dyes, such as SGI and thioflavin T (ThT), have been used to develop label-free biosensors and verify aptamer-target binding. – It was hypothesized that the interaction between an aptamer and its target can either foster dye binding or displace the dye, leading to either an increase or decrease in fluorescence, respectively.…”

“…In this assay, a short DNA with a quencher label (Q-DNA) was hybridized to a FAM-labeled DNA aptamer, resulting in quenched fluorescence. For our purpose, we used an aptamer of chloramphenicol as a rDNA control . We did not test any lysozyme aptamers for this reaction because there is no reliable DNA aptamer with a straightforward secondary structure available for this test.…”

Capture‐SELEX for a short aptamer for label‐free detection of salicylic acid

“…For example, the classical adenosine aptamer has a K d of 6 μM, [36] and a recently reported chloramphenicol aptamer has a K d of 9.8 μM. [37] Both are more complex molecules compared to SA.…”

Screening Environmental Pollutants Binding Aptamers through Capture-SELEX for Biosensing Assays: Challenges and Opportunities

“…5−8 In this study, Liu and colleagues utilized the capture-SELEX method to screen a few short DNA aptamers for the recognition of chloramphenicol (CAP). 9 Capture-SELEX is a selection process variant that immobilizes the oligonucleotides instead of the target molecules, making it ideal for isolating aptamers against small molecules. When the target is recognized, the library folds into a hairpin structure, causing it to detach from the previously immobilized beads.…”

“…Scheme of capture-SELEX for CAP binding aptamers for labeled and label-free fluorescence sensing. Reproduced from ref . Copyright 2023 American Chemical Society.…”