Screening of Single-Stranded DNA Aptamer Specific for Florfenicol and Application in Detection of Food Safety

In this work, the single-stranded DNA (ssDNA) aptamers specific to florfenicol (FF) and having a high binding affinity were prepared using the magnetic bead-based systematic evolution of ligands by the exponential enrichment technique (MB-SELEX). After 10 rounds of the MB-SELEX screening, aptamers t...

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Bibliographic Details
Published in:Biosensors (Basel) 2022-09, Vol.12 (9), p.701
Main Authors: Shi, Minghui, Liu, Ruobing, Zhang, Fuyuan, Chitrakar, Bimal, Wang, Xianghong
Format: Article
Language:English
Subjects:
Acids
Antibiotic residues
Antibiotics
Antibodies
aptamer
Aptamers
AuNPs
Binding sites
Colorimetry
Contamination
Deoxyribonucleic acid
DNA
Farm produce
Florfenicol
Food
Food safety
Graphene
Livestock
Magnetic fields
Measurement
Metabolites
Methods
Molecular docking
Nanoparticles
Nucleic acid aptamers
Poultry
Protein structure
Safety and security measures
Screening
Secondary structure
SELEX
Single-stranded DNA
Structural analysis
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Summary:In this work, the single-stranded DNA (ssDNA) aptamers specific to florfenicol (FF) and having a high binding affinity were prepared using the magnetic bead-based systematic evolution of ligands by the exponential enrichment technique (MB-SELEX). After 10 rounds of the MB-SELEX screening, aptamers that can simultaneously recognize FF and its metabolite florfenicol amine (FFA) were obtained. The aptamer with the lowest dissociation constant (Kd) was truncated and optimized based on a secondary structure analysis. The optimal aptamer selected was Apt-14t, with a length of 43 nt, and its dissociation constant was 4.66 ± 0.75 nM, which was about 7 times higher than that of the full-length sequence. The potential binding sites and interactions with FF were demonstrated by molecular docking simulations. In addition, a colorimetric strategy for nanogold aptamers was constructed. The linear detection range of this method was 0.00128–500 ng/mL and the actual detection limit was 0.00128 ng/mL. Using this strategy to detect florfenicol in actual milk and eggs samples, the spiked recoveries were 88.9–123.1% and 84.0–112.2%, respectively, and the relative standard deviation was less than 5.6%, showing high accuracy.
ISSN:2079-6374
2079-6374
DOI:10.3390/bios12090701