Chemiluminescence competitive aptamer assay for the detection of aflatoxin B1 in corn samples

In this study, we developed a chemiluminescence competitive aptamer assay for aflatoxin B1 (AFB1) using a hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-DNAzyme) linked with an aptamer specific to AFB1. Single, double, and triple HRP-DNAzymes coupled to the AFB1 aptamer were tested, and the AFB1 aptamer linked with double HRP-DNAzymes that produced sufficient chemiluminescence (CL) values when binding to AFB1-ovalbumin (OVA) used as a coating antigen, was selected. Under conditions optimized by testing key parameters, the aptamer assay exhibited a wide dynamic range from 0.1 to 10 ng/mL and showed a limit of detection of 0.11 ng/mL. Cross-reaction to aflatoxin G1, aflatoxin M1, and zearalenone was observed but no cross-reaction to other mycotoxins or the herbicide (atrazine) was observed. Aqueous methanol (20%) gave a good extraction efficiency and the matrix influence from corn extracts was successfully reduced through 4-fold dilution with water. Recovery from spiked corn samples averaged from 60.4 to 105.5%. Thus, the aptamer linked with HRP-DNAzymes can be useful as a reagent in the development of a biosensor for the rapid and simple detection of AFB1. Results from this study provide the basis for research into the development of various aptasensors for AFB1 analysis in foods.