Aptamer-Based Fluorescence Quenching Approach for Detection of Aflatoxin M1 in Milk

Abstract

Aflatoxin M1 (AFM1), one of the most toxic mycotoxins, is a feed and food contaminant of global concern. In this study, we developed a fast and simple method for detection of AFM1 based on a structure-switching signaling aptamer. This aptasensor is based on the change in fluorescence signal due to formation of an AFM1/aptamer complex. To generate the aptasensor, the specific aptamer was modified with FAM (carboxyfluorescein), and their complementary DNAs (cDNA) were modified with a carboxytetramethylrhodamine (TAMRA) quenching group. In the absence of AFM1, the aptamers were hybridized with cDNA, resulting in quenching of the aptamer fluorescence due to the proximity of the aptamer’s fluorophore to the quenching group on the cDNA. On the other hand, in the presence of AFM1, a structural switch in the aptamer was induced by formation of an AFM1/aptamer complex. Changes in the structure of the aptamer led to the release of the cDNA, causing the generation of a fluorescence signal. Thus, AFM1 concentrations could be quantitatively monitored based on the changes in fluorescences. Under optimized conditions, this assay exhibited a linear response to AFM1 in the range of 1–100 ng/mL and a limit of detection of 0.5 ng/mL was calculated. This proposed aptasensor was applied to milk samples spiked with a dilution series of AFM1, yielding satisfactory recoveries from 93.4 to 101.3%. These results demonstrated that this detection technique could be useful for high-throughput and quantitative determination of mycotoxin levels in milk and dairy products.

Fluorescent Aptasensors: Design Strategies and Applications in Analyzing Chemical Contamination of Food

“…A typical example is shown in Figure B­(a), where a fluorophore-labeled aptamer (FDNA) and a quencher-labeled cDNA (QDNA) form a duplex structure through DNA hybridization, resulting in F-Q proximity and concomitant fluorescence quenching of the fluorophore. When the target specifically binds to FDNA, QDNA is released and the fluorescence intensity increases . The same target-dependent unquenching principle can also be applied to the dual-strand switch with F and Q labeled on both termini of FDNA and QDNA (Figure B­(b)) .…”

“…The two F-Q pairs in the dual-terminal proximity structure are applied to transduce the molecular recognition, which is usually more sensitive than a single pair transduction. Different F-Q pairs are available, such as carboxyfluorescein (FAM) and black hole quencher 1 (FAM-BHQ1) or FAM and carboxytetramethylrhodamine (FAM/TAMRA). , Triple-DNA strand structure switches comprise one aptamer DNA, one FDNA, and one QDNA to accomplish F-Q proximity. Upon aptamer-target binding, the triple-DNA is disassembled, resulting in free diffusion of FDNA and QDNA in the solution (long distance between F and Q), and the fluorescence is restored (Figure B­(c)) .…”

A Competitive Assay Based on Dual-Mode Au@Pt-DNA Biosensors for On-Site Sensitive Determination of Carbendazim Fungicide in Agricultural Products

“…Currently, the rapid immunoassay for detecting CBZ with high sensitivity and specificity (6) overcomes the pitfalls of the traditional analytical methods (7). However, the antibodies are prone to degradation and denaturation during field applications (8), resulting in difficultly in specifically recognizing the target (9). Moreover, antibodies are more complicated, and the manufacturing process is costly and time consuming (10).…”

Antibiotics Resistance and Virulence of Staphylococcus aureus Isolates Isolated from Raw Milk from Handmade Dairy Retail Stores in Hefei City, China

“…Dairy products account for a certain proportion of all reported SFP cases [ 6 ]. Milk and dairy products are favored by consumers as an essential source of protein, calcium, and vitamins [ 7 , 8 ]. Of these, milk powder, rich in micronutrients, is used worldwide as the main ingredient in infant formula [ 9 ].…”