Selection and identification of the ssDNA aptamer against polymyxin B sulfate

Abstract

Background

Polymyxin B (PMB) is a potent antibiotic, and its sulfate form, Polymyxin B Sulfate (PMBS), is widely used. When PMBS is used as a veterinary drug, excessive use can lead to drug residues in animal bodies, which is a significant food safety issue and raises serious concerns. Therefore, there is an urgent need to strengthen research in this area. In this study, we obtained a ssDNA aptamer against PMBS using the Capture-SELEX method, which has notable advantages in small molecule selection. Then, we integrated the obtained aptamer with a biosensor based on the CRISPR/Cas14a system for a series of validations.

Results

In this study, specific aptamers against PMBS were discovered via the Capture-SELEX process. The selection process consisted of ten rounds. The affinity of the candidate sequences was determined by Isothermal Titration Calorimetry (ITC), and the PMBS24 aptamer with the highest affinity was ultimately identified, with a dissociation constant (Kd) of 3.89 ± 0.46 μM. To further obtain high-affinity aptamers, we attempted to truncate the PMBS24 aptamer. However, the results demonstrated that the aptamer with the full-length sequence exhibited superior affinity. Subsequently, it was applied in the CRISPR/Cas14a-based sensor for the detection of PMBS. This method had a LOD of 0.99 ng mL⁻¹, with good specificity, and was successfully used for the detection in milk.

Significance

An important recognition element of PMBS has been successfully obtained, filling a part of the gap in the detection of PMBS. Moreover, the aptamer has been applied to the CRISPR/Cas14a-based sensor for amplification-free PMBS detection, yielding favorable detection results. This also opens up the possibility of applying the aptamer to other similar sensors.