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

•Aptamers are single-strand oligonucleotides that offer advantages over antibodies.•DNAzymes can be synthesized and stored for long periods in solution compared to HRP.•Aptamer linked DNAzymes is first used to develop an assay for aflatoxin B1 analysis.

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.