Development of a Novel Aptamer-Antibody Sandwich Chemiluminescent Biosensor and Its Application in the Detection of Aflatoxin B₁

In addressing the challenges posed by high costs, low accuracy, and cumbersome operations in mycotoxin detection, a novel aptamer-antibody sandwich chemiluminescent biosensor for detecting aflatoxin B₁ (AFB₁) was developed. The indirect competition between AFB₁, aflatoxin B₁-ovomucoid complete antigen (AFB₁-OVA), and rabbit anti-ovomucoid (OVA) antibody results in the formation of a sandwich complex. This sandwich assay is linked to a horseradish peroxidase-labeled antibody, which catalyzes luminol chemiluminescence for the indirect detection of AFB₁. The biosensor was designed to operate with high precision, low cost, and a low detection limit for AFB₁, which is contingent upon experimental conditions such as pH, reagent concentration, temperature, and incubation time. The optimization of pH, aptamer concentration, competitive incubation time, competitive incubation temperature, and HRP-labeled antibody concentration was instrumental in achieving these objectives. Experimental findings demonstrated that the sensor’s detection limit was 0.067 ng/mL, exhibiting excellent linearity (R² = 0.99679) within the concentration range of 0.25–10 ng/mL. The recovery rate of spiked samples ranged from 94.4% to 108.05%. This sensor boasts a low detection limit, straightforward operation, and minimal cost, thus offering a novel solution for developing cost-effective, high-precision mycotoxin detection methods.