Related Experiment Video
Updated: May 9, 2025
Primer-Free Aptamer Selection Using A Random DNA Library
Published on: July 26, 2010
Selection and identification of the ssDNA aptamer against polymyxin B sulfate
Zhuxin Chang1, Jiayue Zhou2, Dongmei Li2
1Department of Nutrition and Food Hygiene, School of Public Health, Tianjin Medical University, Tianjin, 300070, China; Military Medical Sciences Academy, Academy of Military Sciences, Tianjin, 300050, China.
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.
Insights
Researchers developed a new aptamer for detecting Polymyxin B Sulfate (PMBS) residues, a critical food safety concern. This aptamer, integrated into a CRISPR/Cas14a biosensor, enables sensitive and specific detection of PMBS in milk.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Food Safety
Background:
- Polymyxin B Sulfate (PMBS) is a widely used veterinary antibiotic.
- Excessive use leads to drug residues, posing significant food safety risks.
- There is an urgent need for sensitive detection methods for PMBS.
Purpose of the Study:
- To develop a specific aptamer for Polymyxin B Sulfate (PMBS).
- To integrate the aptamer into a CRISPR/Cas14a biosensor for PMBS detection.
- To validate the sensor's performance in detecting PMBS in food matrices.
Main Methods:
- Selection of a ssDNA aptamer against PMBS using the Capture-SELEX method.
- Affinity validation using Isothermal Titration Calorimetry (ITC).
- Integration of the aptamer with a CRISPR/Cas14a biosensor for detection.
Main Results:
- A high-affinity aptamer (PMBS24) was identified with a Kd of 3.89 ± 0.46 μM.
- The CRISPR/Cas14a biosensor achieved a Limit of Detection (LOD) of 0.99 ng/mL.
- The sensor demonstrated good specificity and was successfully applied to detect PMBS in milk.
Conclusions:
- A novel aptamer recognition element for PMBS was successfully developed.
- The aptamer-based CRISPR/Cas14a sensor provides amplification-free detection of PMBS.
- This approach offers a promising tool for monitoring PMBS in food safety applications.
