Citation: CAI Xian-Hong, SUN Qiang, WU Qian, YE Hao-Nan, WU Qin-Fang, OU Wen-Hui, CHEN Jin-Can, CHEN Lan-Mei. Screening of Virginiamycin M₁ Aptamer Based on Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Application[J]. Chinese Journal of Analytical Chemistry, ;2022, 50(5): 728-738. doi: 10.19756/j.issn.0253-3820.210827 shu

Screening of Virginiamycin M₁ Aptamer Based on Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Application

CAI Xian-Hong ¹ ,
SUN Qiang ¹ ,
WU Qian ¹ ,
YE Hao-Nan ¹ ,
WU Qin-Fang ¹ ,
OU Wen-Hui ¹ ,
CHEN Jin-Can ^2,3,4,, ,
CHEN Lan-Mei ^1,,

1.
School of Pharmacy, Guangdong Medical University, Zhanjiang 524023, China;
2.
The Marine Biomedical Research Institute, Guangdong Medical University, Zhanjiang 524023, China;
3.
The Marine Biomedical Research Institute of Guangdong Zhanjiang, Zhanjiang 524023, China;
4.
Southern Marine Science and Engineering Guangdong Laboratory, Zhanjiang 524023, China

Corresponding author: CHEN Jin-Can, CHEN Lan-Mei,
Received Date: 4 November 2021
Revised Date: 12 February 2022

Fund Project: Supported by the Fund of Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang) (No.ZJW-2019-007), the Science and Technology Program of Zhanjiang (No.2021A05037) and the University Student Innovation Experiment Program (No.202110571028).

Capillary electrophoresis (CE) has been considered as an efficient method for aptamer screening due to its high separation efficiency, less sample consumption and low screening cost. In this study, an aptamer screening method was established for virginiamycin M₁ (VGM-M₁) based on capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX). The initial ssDNA library was mixed and incubated with VGM-M₁, and the conjugate was separated and collected by capillary electrophoresis-ultraviolet detection (CE-UV). The next sub-library ssDNA was obtained by polymerase chain reaction (PCR) amplification and single stranded DNA (ssDNA) preparation. After four rounds of screening, the first 10 candidate aptamer sequences with high enrichment were obtained, and the first 4 candidate aptamer sequences with the highest enrichment were selected for affinity and specificity evaluation. The VGM-M₁ aptamer Seq 1 with the highest affinity and the strongest specificity was obtained, and its equilibrium constant value (K_d) was 49 nmol/L. The aptamer Seq 1 was modified to obtain optimized candidate aptamer sequences and their affinity and specificity were evaluated. The results showed that the modified aptamer Seq 1-1 had the highest affinity and specificity, and its K_d was as low as 45 nmol/L. Competitive enzyme-linked aptamer visual detection method was used to detect VGM-M₁, and the minimum detection limit was as low as 51 ng/mL, which was far below the maximum residue limit of 100 ng/mL for VGM-M₁. Further standard addition and recovery experiments were carried out on VGM-M₁ in milk, and the recoveries reached 97.8%.8-103.2%. The above research results showed that the aptamer Seq 1-1 screened by CE-SELEX could specifically recognize VGM-M₁ and could be applied to the detection of VGM-M₁ in real samples.
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Screening of Virginiamycin M1 Aptamer Based on Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Application

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通讯作者: 陈斌, bchen63@163.com

Screening of Virginiamycin M₁ Aptamer Based on Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Application