An ssDNA library immobilized SELEX technique for selection of an aptamer against ractopamine

Nuo Duan; Wenhui Gong; Shijia Wu; Zhouping Wang

doi:10.1016/j.aca.2017.01.008

An ssDNA library immobilized SELEX technique for selection of an aptamer against ractopamine

Anal Chim Acta. 2017 Apr 8:961:100-105. doi: 10.1016/j.aca.2017.01.008. Epub 2017 Jan 23.

Authors

Nuo Duan¹, Wenhui Gong², Shijia Wu³, Zhouping Wang⁴

Affiliations

¹ State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China.
² Market Supervision and Administration Bureau, Taicang, 215400, China.
³ State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China; School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 212013, China.
⁴ State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China; National Engineering Research Center of Seafood, School of Food Science and Technology, Dalian Polytechnic University, Dalian, 116034, China. Electronic address: wangzp@jiangnan.edu.cn.

PMID: 28224901
DOI: 10.1016/j.aca.2017.01.008

Abstract

An improved SELEX technique was developed for selecting aptamers against ractopamine (RAC) by immobilizing ssDNA library on the magnetic beads. After sixteen selection rounds, a highly enriched ssDNA pool was sequenced and nine families were grouped according to their homology and secondary structures analysis. One representative aptamer candidate from each family was picked out for binding affinity identification by graphene oxide (GO) adsorption platform. The aptamer RAC-6 was demonstrated as the optimal aptamer with high specificity and dissociation constant (K_d) value of 54.22 ± 8.02 nM. To prove the potential application of aptamer RAC-6 in the quantitative determination of RAC, a fluorescent bioassay with aptamer RAC-6 was developed. The linear range for RAC was from 0.10 ng/mL to 100 ng/mL and the limit of detection was as low as 0.04 ng/mL. Furthermore, the method was validated for the analysis of RAC spiked real samples, and the recoveries were between 82.57% and 104.65%.

Keywords: Aptamer; Graphene oxide; Library immobilization; Ractopamine; SELEX.

MeSH terms

Aptamers, Nucleotide / chemistry*
DNA, Single-Stranded / chemistry*
Phenethylamines / chemistry*
SELEX Aptamer Technique*

Substances

Aptamers, Nucleotide
DNA, Single-Stranded
Phenethylamines
ractopamine