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

Area of Science:

• Biotechnology
• Analytical Chemistry
• Environmental Science

Background:

• Chloramphenicol (CAP) is a potent antibiotic with significant side effects, leading to bans in most countries.
• Despite bans, CAP residues persist in food products and the environment, necessitating reliable detection methods.
• Existing aptamer selection methods for CAP often omit crucial chlorine atoms, potentially affecting binding affinity and sensor performance.

Purpose of the Study:

• To develop novel DNA aptamers for chloramphenicol (CAP) detection using a method that preserves the intact molecule.
• To characterize the binding affinity of selected aptamers using biophysical techniques.
• To establish sensitive and selective biosensors for CAP detection in various matrices.

Main Methods:

• DNA aptamers were selected against free, unmodified chloramphenicol using a library-immobilized method.
• Isothermal titration calorimetry (ITC) was used to determine the dissociation constant (Kd) of the aptamers.
• Fluorescent strand-displacement assays and Thioflavin T (ThT) staining were employed for label-free CAP detection.

Main Results:

• Three families of DNA aptamers were identified, with the best aptamer, CAP1, exhibiting a dissociation constant (Kd) of 9.8 μM.
• A fluorescent sensor achieved a limit of detection (LOD) of 14 μM for CAP.
• Label-free detection using Thioflavin T (ThT) staining demonstrated LODs of 1 μM (buffer), 1.8 μM (Lake Ontario water), and 3.6 μM (wastewater sample).

Conclusions:

• The newly developed DNA aptamer CAP1 offers a promising tool for chloramphenicol detection due to its small size and stable structure.
• The selected aptamers provide sensitive detection capabilities, outperforming previously reported aptamers in binding verification.
• This work facilitates the development of aptamer-based biosensors for effective environmental and food safety monitoring of CAP residues.