Purpose: In this study, we developed a chemiluminescence competitive aptamer assay of AFB1 using an aptamer linked with hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) and validated the aptamer assay with corn samples artificially spiked with known concentration of AFB1.
Methods: Three lengths of oligonucleotides as aptamers linked with HRP-DNAzyme were designed and used for the development of chemiluminescence competitive aptamer assay of AFB1. The optimization of the aptamer assay was carried out by testing key parameters such as selection of coating antigen (AFB1-BSA and AFB1-OVA), kind of blocking reagents (1% BSA and 1% skim milk), and selection of optimum length and concentration of aptamer linked with different numbers of HRP-DNAzyme. The specificity and sensitivity of the aptamer assay was tested. Sample preparation to minimize matrix effect was investigated, and corn samples spiked with AFB1 at 0, 0.5, 1, 5, and 10 ng/g were extracted and analyzed by the aptamer assay.
Results: The AFB1 aptamer linked with the double HRP-DNAzyme that produced sufficient chemiluminescence (CL) values when binding to AFB1-OVA used as a coating antigen was selected. Under conditions optimized by testing key parameters, the aptamer assay exhibited wide dynamic range from 0.1 to 1000 ng/ml and showed the limit of detection of 0.11 ng/ml. Cross reaction to aflatoxin G1 and zearalenone was observed but no cross-reaction to other mycotoxins and herbicide (atrazine) was shown. Aqueous methanol (20%) gave good extraction efficiencies and matrix influence from corn extracts was successfully reduced by 4-fold dilution with water. The recovery, as a practical application, from spiked corn samples averaged from 60.4 to 105.5%.
Significance: The aptamer assay developed in this study is the first application of aptamer for the detection of AFB1. This study provides great opportunities to apply the aptamer toward AFB1 to the development of biosensors and aptamer assays.