Selection and Identification of Novel Aptamers Specific for Clenbuterol Based on ssDNA Library Immobilized SELEX and Gold Nanoparticles Biosensor

Abstract

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.

A label-free aptasensor for rapid detection of clenbuterol based on SYBR Green I

“…Pork was purchased from a local supermarket and pretreated according to Liu's procedure 26 with slight modifications. Firstly, 20 mL of the corresponding concentration of clenbuterol was added to one gram homogenized pork sample and treated with ultrasound for 10 min.…”

“…13 Liu et al developed a simple colorimetric sensor to determine clenbuterol by using DNA aptamers and gold nanoparticles. 26 Duan et al designed a surface enhanced Raman scattering (SERS) aptasensor for the detection of clenbuterol by using Fe 3 O 4 @Au@AgNPs as the SERS probe. 27 These methods have excellent sensitivity and selectivity, but the labeled probe or the complex nanomaterial synthesis process is costly and time-consuming.…”

Selection of Aptamers Specific for DEHP Based on ssDNA Library Immobilized SELEX and Development of Electrochemical Impedance Spectroscopy Aptasensor

“…The DEHP standards and analogs were purchased from TMRM Ltd. (Beijing, China Table S1, according to our previous report [36], were synthesized by Nanjing Genscript Biotechnology Ltd. (Nanjing, China). The aptamers were synthesized from Suzhou Hongxun Biotechnology Ltd. (Suzhou, China).…”

Preparation and Characterization of Aptamers Against O,p’-DDT

“…The selection process was performed as previously described with modifications [37]. In brief, in the first round, the synthetic ssDNA oligonucleotide library (library Lib3_bank, Table 1, 1 OD) was dissolved in binding buffer (1mM CaCl 2 , 2.5 mM KCl, 1.5 mM KH 2 PO 4 , 0.5 mM MgCl 2 •6H 2 O, 137 mM NaCl, 8mM Na 2 HPO 4 , pH 7.2).…”

“…The concentration of AuNPs in ultrapure water was determined to be 8.76 nM using UV-vis spectroscopy according to the previously reported method [40]. The assay was performed as previously described with optimizations [37]. In brief, 50 µL of aptamer at 0.48 µM and 50 µL of AuNPs from the stock were incubated for 45 min at room temperature.…”