Selection and Application of ssDNA Aptamers against Clenbuterol Hydrochloride Based on ssDNA Library Immobilized SELEX

Clenbuterol hydrochloride (CLB) is often abused as additive feed for livestock to decrease adipose tissue deposition and to increase growth rate. It raises a potential risk to human health through the consumption of animal product. In this study, aptamers with higher affinity and specificity were screened through 16 selection rounds based on the ssDNA library immobilized systematic evolution of ligands by exponential enrichment (SELEX) technique. After cloning and sequencing, five aptamer candidates were picked out for affinity and specificity assays based on a graphene oxide (GO) adsorption method. The results showed that the aptamer CLB-2 binds specifically against CLB with a dissociation constant, <i>K</i>_d, value of 76.61 ± 12.70 nM. In addition, an aptamer-based fluorescence bioassay was established for CLB analysis. The correlation between the CLB concentration and fluorescent signal was found to be linear within the range of 0.10 to 50 ng/mL with a limit of detection of 0.07 ng/mL. It has been further applied for the determination of CLB in pork samples, showing its great potential for sensitive analysis in food safety control

Simultaneous Aptasensor for Multiplex Pathogenic Bacteria Detection Based on Multicolor Upconversion Nanoparticles Labels

A highly sensitive and specific multiplex method for the simultaneous detection of three pathogenic bacteria was fabricated using multicolor upconversion nanoparticles (UCNPs) as luminescence labels coupled with aptamers as the molecular recognition elements. Multicolor UCNPs were synthesized via doping with various rare-earth ions to obtain well-separated emission peaks. The aptamer sequences were selected using the systematic evolution of ligands by exponential enrichment (SELEX) strategy for <i>Staphylococcus aureus</i>, <i>Vibrio parahemolyticus</i>, and <i>Salmonella typhimurium</i>. When applied in this method, aptamers can be used for the specific recognition of the bacteria from complex mixtures, including those found in real food matrixes. Aptamers and multicolor UCNPs were employed to selectively capture and simultaneously quantify the three target bacteria on the basis of the independent peaks. Under optimal conditions, the correlation between the concentration of three bacteria and the luminescence signal was found to be linear from 50–10⁶ cfu mL^–1. Improved by the magnetic separation and concentration effect of Fe₃O₄ magnetic nanoparticles, the limits of detection of the developed method were found to be 25, 10, and 15 cfu mL^–1 for <i>S. aureus</i>, <i>V. parahemolyticus</i>, and <i>S. typhimurium</i>, respectively. The capability of the bioassay in real food samples was also investigated, and the results were consistent with experimental results obtained from plate-counting methods. This proposed method for the detection of various pathogenic bacteria based on multicolor UCNPs has great potential in the application of food safety and multiplex nanosensors

Fe–Co-Based Metal–Organic Frameworks as Peroxidase Mimics for Sensitive Colorimetric Detection and Efficient Degradation of Aflatoxin B₁

Building multifunctional platforms for integrating the detection and control of hazards has great significance in food safety and environment protection. Herein, bimetallic Fe–Co-based metal–organic frameworks (Fe–Co-MOFs) peroxidase mimics are prepared and applied to develop a bifunctional platform for the synergetic sensitive detection and controllable degradation of aflatoxin B1 (AFB1). On the one hand, Fe–Co-MOFs with excellent peroxidase-like activity are combined with target-induced catalyzed hairpin assembly (CHA) to construct a colorimetric aptasensor for the detection of AFB1. Specifically, the binding of aptamer with AFB1 releases the prelocked Trigger to initiate the CHA cycle between hairpin H2-modified Fe–Co-MOFs and hairpin H1-tethered magnetic nanoparticles to form complexes. After magnetic separation, the colorimetric signal of the supernatant in the presence of TMB and H2O2 is inversely proportional to the target contents. Under optimal conditions, this biosensor enables the analysis of AFB1 with a limit of detection of 6.44 pg/mL, and high selectivity and satisfactory recovery in real samples are obtained. On the other hand, Fe–Co-MOFs with remarkable Fenton-like catalytic degradation performance for organic contaminants are further used for the detoxification of AFB1 after colorimetric detection. The AFB1 is almost completely removed within 120 min. Overall, the introduction of CHA improves the sensing sensitivity; efficient postcolorimetric-detection degradation of AFB1 reduces the secondary contamination and risk to the experimental environment and operators. This strategy is expected to provide ideas for designing other multifunctional platforms to integrate the detection and degradation of various hazards

Multiplexed Fluorescence Resonance Energy Transfer Aptasensor between Upconversion Nanoparticles and Graphene Oxide for the Simultaneous Determination of Mycotoxins

We presented a new aptasensor for mycotoxins, which was based on multiplexed fluorescence resonance energy transfer (FRET) between multicolor upconversion fluorescent nanoparticles (UCNPs) as donors and graphene oxide (GO) as the entire and effective acceptor. BaY_0.78F₅:Yb_0.2, Er_0.02 and BaY_0.78F₅:Yb_0.7, Tm_0.02 upconversion nanoparticles were synthesized and functionalized, respectively, with immobilized ochratoxin A (OTA)-aptamers and fumonisin B₁ (FB₁)-aptamers. On the basis of the strong π–π stacking effect between the nucleobases of the aptamers and the sp² atoms of GO, the aptamer modified-UCNPs can be brought in close proximity to the GO surface. The strong upconversion fluorescence both of BaY_0.78F₅:Yb_0.2, Er_0.02 and BaY_0.78F₅:Yb_0.2, Tm_0.02 can be completely quenched by the GO, because of a good overlap between the fluorescence emission of multicolor UCNPs and the absorption spectrum of GO. In contrast, in the presence of OTA and FB₁, the aptamers preferred to bind to their corresponding mycotoxins, which led to changes in the formation of aptamers, and therefore, aptamer modified-UCNPs were far away from the GO surface. Our study results showed that the fluorescence intensity of BaYF₅:Yb Er and BaYF₅:Yb Tm were related to the concentration of OTA and FB₁. We therefore developed a sensitive and simple platform for the simultaneous detection of OTA and FB₁ with multicolor UCNPs and GO as the FRET pair. The aptasensor provided a linear range from 0.05 to 100 ng·mL^–1 for OTA and 0.1 to 500 ng·mL^–1 for FB₁; the detection limit of OTA was 0.02 ng·mL^–1 and FB₁ was 0.1 ng·mL^–1. As a practical application, the aptasensor was used to monitor OTA and FB₁ level in naturally contaminated maize samples with the results consistent with that of a classic ELISA method. More importantly, the novel multiplexed FRET was established for the first time based on multiplexed energy donors to the entire energy acceptor; this work was expected to open up a new field of FRET system applications for various targets

Selection and Characterization of Aptamers against Salmonella typhimurium Using Whole-Bacterium Systemic Evolution of Ligands by Exponential Enrichment (SELEX)

In this paper, a high-affinity ssDNA aptamer binding to Salmonella typhimurium was obtained by a whole-bacterium-based Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure. After nine rounds of selection with <i>S. typhimurium</i> as the target, a highly enriched oligonucleotide pool was sequenced and then grouped into different families based on primary sequence homology and secondary structure similarity. Eleven sequences from different families were selected for further characterization via flow cytometry analysis. The results showed that the sequence ST2P demonstrates affinity for <i>S. typhimurium</i> much more strongly and specifically than other sequences tested. The estimated <i>K</i>_d value of this particularly promising aptamer was 6.33 ± 0.58 nM. To demonstrate the potential use of the aptamers in the quantitative determination of <i>S. typhimurium</i>, a fluorescent bioassay with the aptamer ST2P was prepared. Under optimal conditions, the correlation between the concentration of <i>S. typhimurium</i> and fluorescent signal was found to be linear within the range of 50–10⁶ cfu/mL (<i>R</i>² = 0.9957). The limit of detection (LOD) of the developed method was found to be 25 cfu/mL. This work demonstrates that this aptamer could potentially be used to improve the detection of <i>S. typhimurium</i>