Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor
Xixia Liu,
Qi Lu,
Sirui Chen,
Fang Wang,
Jianjun Hou,
Zhenlin Xu,
Chen Meng,
Tianyuan Hu,
Yaoyao Hou
Affiliations
Xixia Liu
Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Cihu Road, Huangshigang District, Huangshi 435002, China
Qi Lu
Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Cihu Road, Huangshigang District, Huangshi 435002, China
Sirui Chen
Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Cihu Road, Huangshigang District, Huangshi 435002, China
Fang Wang
Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Cihu Road, Huangshigang District, Huangshi 435002, China
Jianjun Hou
Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Cihu Road, Huangshigang District, Huangshi 435002, China
Zhenlin Xu
Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, China
Chen Meng
Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Cihu Road, Huangshigang District, Huangshi 435002, China
Tianyuan Hu
Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Cihu Road, Huangshigang District, Huangshi 435002, China
Yaoyao Hou
Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Cihu Road, Huangshigang District, Huangshi 435002, China
We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5′-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33–97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.
