Citation: RAN Xu-Dong, WU Yuan-Gen. Screening Aptamers and Development of Colorimetric Detection Method of Paraquat Pesticide[J]. Chinese Journal of Analytical Chemistry, ;2019, 47(4): 567-575. doi: 10.19756/j.issn.0253-3820.191013 shu

Screening Aptamers and Development of Colorimetric Detection Method of Paraquat Pesticide

RAN Xu-Dong ^1, ,
WU Yuan-Gen ^1,2

1.
Guizhou Province Key Laboratory of Fermentation Engineering and Biopharmacy, Guizhou University, Guiyang 550025, China;
2.
School of Liquor and Food Engineering, Guizhou University, Guiyang 550025, China

Received Date: 6 January 2019
Revised Date: 21 February 2019

Fund Project: This work was supported by the National Natural Science Foundation of China (Nos.21565009, 31760486), the Natural Science Foundation of Guizhou Province (No. [2016]1403) and the Natural Science Project of Guizhou Provincial Education Department (No. KY[2014]261).

Aptamers have similar functions as antibodies, and have attracted increasing attentions in the field of specific recognition and detection of target of interest. Small molecule substances are usually closely related to environmental pollution and human health. It is particularly difficult to screen aptamers for specifically binding to those small molecule targets, because they have less active sites for immobilization as well as have little difference in properties to nucleic acid libraries. Herein, the random nucleic acid libraries were firstly immobilized on the surface of streptavidin-coated magnetic beads through hydrogen bonds complementary to a biotin-labeled sequence with 12 bases. Subsequently, the magnetic beads were incubated with different concentrations of paraquat and the ssDNA sequences with high affinity to paraquat molecules were obtained by elution. The enriched ssDNA libraries for the next screening were purified from the products of above sequences after asymmetric PCR amplification. After 9 rounds of screening, 15 ssDNA sequences with specific affinity to paraquat were obtained through cloning and sequencing. 6 ssDNA sequences were selected to determine their affinity to paraquat based on the cluster analysis and secondary structure prediction of the above 15 sequences. These results showed that PQ-15 aptamer sequence had the highest affinity to paraquat with a dissociation constant (K_d) of (54±4) nmol/L. Finally, a novel aptasensor for colorimetric detection of paraquat pesticides was established for the first time, in which PQ-15 aptamer sequence was used as the target specific recognition molecule and the aggregation of gold nanoparticles mediated by cationic compound was employed as the signal transmit element. The proposed aptasensor had a detection limit as low as 25.2 nmol/L for paraquat, and displayed high selectivity against other competitive pesticides. Moreover, the aptasensor also exhibited excellent accuracy in detection of paraquat in real samples with recoveries from 91.13% to 99.60%.
- Aptamer,
- Paraquat,
- Screening,
- Colorimetric detection,
- Gold nanoparticles
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