Selection and Characterization of DNA Aptamers for Constructing Aptamer-AuNPs Colorimetric Method for Detection of AFM1

Abstract

Aflatoxin M1 (AFM1), one of the most toxic mycotoxins, is a feed and food contaminant of global concern. To isolate the ssDNA aptamer of AFM1, synthesized magnetic graphene oxide nanomaterials, 12 rounds of subtractive systematic evolution of ligands by exponential enrichment (SELEX) selection were carried out. As a result, 24 candidate aptamers were selected, and their sequence similarity exceeded 97%. Their binding affinity and specificity were further examined by fluorescence and biofilm interferometry (BLI) methods. One aptamer (Apt-5) against AFM1 with a high affinity and specificity was isolated and demonstrated to be the optimal aptamer, whose dissociation constant reached the nanomolar level, Kd = 8.12 ± 1.51 nM. Additionally, molecular docking studies were used to predict the possible binding sites and mechanisms of the two. Based on Apt-5, an unlabeled aptamer-AuNPs colorimetric method was established to detect AFM1 in milk with a linear range of 0.078–10 ng/mL, and the actual detection limit was 0.078 ng/mL. These results demonstrated that this detection technique could be useful for the quantitative determination of AFM1 in milk and dairy products.

Aptamer–Protein Interactions: From Regulation to Biomolecular Detection

“…Another optical method that requires the immobilization of one interaction partner is Biolayer interferometry (BLI). , Aptamers are covalently functionalized onto the surface of a fiber-optic biosensor . Upon white light illumination, the two interfaces of optical film layer at the sensor tip will generate two reflected beams that are superimposed to form an interference spectrum. , The association and dissociation of aptamers with target proteins can cause changes in the thickness and density of the film, creating a wavelength shift of the interference spectrum. , By the real-time monitoring of the wavelength shift, BLI is capable of obtaining abundant information about aptamer–protein interactions, including steady-state affinity, kinetic, and thermodynamic parameters …”

Recent advances in colorimetric sensors based on nanozymes with peroxidase-like activity

“…58 Recently, aptamer-based Au NP sensors have been used for competitive colorimetric analysis. 59–61 The aptamers will inhibit the intrinsic peroxidase-mimicking activity of Au NPs by simply adsorbing onto the Au NPs surface. However, when there was a target, the target–aptamer will leave the Au NPs surface due to the high affinity and specificity to the target.…”

Recent Advances in Design and Application of Nanomaterials-Based Colorimetric Biosensors for Agri-food Safety Analysis

“…This method was successfully used for the detection of AFB1 in peanut, corn, and chicken feed samples (Figure C) . Liu et al studied the use of magnetic GO combined with technology to screen a high-affinity aptamer of aflatoxin M1 (AFM1) and established an aptamer-AuNP colorimetric assay using the selected aptamer as a specific recognition probe to effectively monitor the content of AFM1 in milk …”

“…100 Liu et al studied the use of magnetic GO combined with technology to screen a high-affinity aptamer of aflatoxin M1 (AFM1) and established an aptamer-AuNP colorimetric assay using the selected aptamer as a specific recognition probe to effectively monitor the content of AFM1 in milk. 101 Zearalenone (ZEN) is an estrogenic mycotoxin produced by Fusarium oxysporum that threatens the safety of food and agricultural industries. ZEN exerts toxic effects on humans and animals through mutagenicity, teratogenicity, carcinogenicity, nephrotoxicity, immunotoxicity, and genotoxicity.…”