Screening of specific aptamers against chlorpromazine and construction of novel ratiometric fluorescent aptasensor based on metal-organic framework.

Chlorpromazine is a phenothiazine representative drug that can be used to anesthetize and calm animals. However, chlorpromazine excess may lead to residual persistence in edible tissues, which is potentially harmful to human health and animal production. In this work, high affinity and specificity aptamers against chlorpromazine were screened out based on Capture-SELEX. After ten rounds of screening, the candidate aptamers were obtained. The optimal aptamer of CHL-3 was obtained by isothermal titration calorimetry (ITC) and SYBR Green I (SGI) fluorescence-competition methods. The Kd value of CHL-3 was 69.8 ± 9.81 nM. Subsequently, the Uio-66-NH2 material was prepared, filled with rhodamine 6G (Rho 6G) dye into the pore, and sealed with CHL-3, and fluorescence probes were obtained. The ratiometric fluorescence detection method was established to detect the concentration of chlorpromazine. A linear relationship was obtained in a range of 1-100 nM, with a lower detection limit of 0.67 nM. Meanwhile, a good recovery was shown in spiked food samples, such as eggs and milk. These results indicate that the constructed ratiometric fluorescence strategy can be successfully applied in food detection.

Selection of aptamer targeting levamisole and development of a colorimetric and SERS dual-mode aptasensor based on AuNPs/Cu-TCPP(Fe) nanosheets.

Levamisole (LEV) is a veterinary drug that often remains in animal food. Consuming products containing high levels of LEV will cause a series of harmful reactions to human health. This work describes the Capture-SELEX (Capture-systematic evolution of ligands by exponential enrichment) screening strategy of LEV aptamers, using streptavidin modified agarose beads as a solid phase medium to separate target-bound and unbound ssDNA. The affinity and specificity of candidate aptamers were determined by SYBR Green I (SGI) dye and isothermal titration calorimetry (ITC), in which LEV-5 showed good binding affinity and specificity, and the dissociation constant was 66.15 ± 11.86 nM. Circular dichroism (CD) was used to characterize aptamer conformational changes before and after target binding, including increased helicity and enhanced base stacking. To evaluate whether this aptamer can be used for LEV detection, a colorimetric-surface-enhanced Raman spectroscopy (colorimetric-SERS) dual-mode aptasensor was constructed based on the peroxidase-like activity and SERS effect of AuNPs/Cu-TCPP(Fe) nanosheets. The detection limits of this dual-mode aptasensor for LEV were 5 nM and 1.12 nM, respectively. This aptamer-based method was further successfully used to detect LEV in milk, with recoveries ranging from 94.95% to 111.2%, providing a potential application for the detection of harmful substances in food.

Deoxynivalenol fluorescence aptasensor based on AuCu bimetallic nanoclusters and MoS2.

Aptamers against deoxynivalenol (DON) were selected through capture-systematic evolution of ligands by exponential enrichment. Through isothermal titration calorimetry and fluorimetric assay, aptamer candidate DN-2 demonstrated good affinity to DON with Kd value of 40.36 ± 6.32 nM. Accordingly, a Forster resonance energy transfer aptasensor was fabricated by using the aptamer DN-2 combined with AuCu bimetallic nanoclusters as energy donor and MoS2 nanosheets as energy acceptor. Under the optimal conditions, the fluorescence response was utilized for DON quantitative determination ranging from 5 to 100 ng/mL with a detection limit of 1.87 ng/mL. The practical application of this method was verified in maize flour samples and demonstrated a satisfied recovery of 94.6 ~ 103.1%. The obtained aptamers and their application in DON determination provide a new tool for DON monitoring in various foodstuff.

Signal amplification of SiO2 nanoparticle loaded horseradish peroxidase for colorimetric detection of lead ions in water.

In this work, we developed an aptamer-based optical assay for the analysis of Pb2+, a hazardous heavy metal that may be present in the food chain and harmful to human health. An aptamer targeted against Pb2+ was immobilized onto the microplate as the capture probe. SiO2 nanoparticles (NPs) were synthesized and used as carriers of the signaling horseradish peroxidase (HRP) to achieve amplification of the optical signal. Complementary DNA (cDNA) of the aptamer was also linked to the above mentioned SiO2 nanoparticle (NPs) as the signal probe. The aptamers were found to be able to capture Pb2+, and the unbound aptamers were subsequently hybridized with cDNA-HRP-SiO2 conjugates. As a result, the addition of TMB-H2O2 promoted the formation of blue products in the catalytic system. The assay adopting SiO2 NPs as an enhancer resulted in higher sensitivity with an LOD of 2.5 nM compared to normal procedures. The feasibility of the aptamer-based colorimetric assay was verified by successful detection of Pb2+ in water samples with recoveries in the range of 97.4-103.52%.

Effectively Selecting Aptamers for Targeting Aromatic Biogenic Amines and Their Application in Aptasensing Establishment.

It is necessary to detect the biogenic amine (BA) content in food due to their toxicological effects and their role as an index of freshness for protein-rich foods. Aptamer-based techniques have the potential to provide alternative methods for sensitive and efficient monitoring of BAs. Herein, we described the selection and characterization of DNA aptamers for tyramine (TYR) and ß-phenethylamine (PHE) using a one-pot coupled with separate selection strategy. During the selection process, melting curve analysis was developed to monitor the enrichment of the aptamer species, and a saturation of the selection was found at the 14th round. Based on the fluorescence assay, aptamers TYR-2 and PHE-2 showed high affinity to TYR and PHE with the dissociation constant values of 64.28 ± 10.4 and 71.64 ± 11.47 nM, respectively. The circular dichromatic and molecular docking technologies were employed for the preliminary binding mechanism analysis. The obtained aptamers TYR-2 and PHE-2 were used in a fluorescence method for the TYR and PHE determination with limits of detection of 0.34 and 0.39 ng/mL, respectively. In addition, the developed aptasensor was further applied to the TYR and PHE detection in pork and beer samples, and the recovery rate was between 95.6 and 104.2%. It was demonstrated that the selected aptamers had enormous potential as a molecular probe for the identification and determination of BAs.