Purpose: In this study, we developed an aptamer-based dipstick assay for the rapid and simple detection of AFB1 and validated the dipstick assay with corn samples artificially spiked with known concentration of AFB1.
Methods: A cy5-modified DNA probes and biotin-modified aptamer specific to AFB1 were designed and used to develop an aptamer-based dipstick assay for AFB1. The format of the dipstick assay was based on a competitive assay. AFB1 competes with a cy5-modified DNA probes to bind a biotin-modified aptamer specific to AFB1. The optimization of the assay was performed by testing key parameters such as the length of DNA probe, amount of the biotin-modified aptamer and cy5-modified DNA probe, working buffer, and incubation step, time and temperature. The specificity and sensitivity of the dipstick assay was tested. Sample preparation to minimize matrix effect was investigated, and corn samples spiked with AFB1 at 0, 0.1, 0.5, 1, 5, and 10 ng/g were extracted and analyzed by the assay.
Results: The aptamer-based dipstick assay for AFB1 determination was successfully developed. cy5-modified DNA with 14 mer length was selected a competitor against AFB1 to bind biotin-modified aptamer and produced sufficient fluorescence on the dipstick assay. The limits of detection for the dipstick assay were 0.1 ng/ml AFB1 in buffer and 0.5 ng/g AFB1 in a corn sample. The method was confirmed to be highly specific to AFB1, and the entire process of the assay can be completed within 30 min. Aqueous methanol (20%) provided a good extraction efficiency, and the matrix influence from corn extracts was successfully reduced through 2-fold dilution.
Significance: Recently, since aptamers have been considered as a good candidate to replace antibodies which are used in other immunoassays, the aptamer-based dipstick assay developed in this study is superior to other immunoassays with respect to its setting speed and stability. In this study, we firstly reported the development of an aptamer-based dipstick assay, and the results provide great opportunities to apply the aptamer to the development of dipstick assays.