Green Enzyme-Linked Immunosorbent Assay Based on the Single-Stranded Binding Protein-Assisted Aptamer for the Detection of Mycotoxin

In this work, a green enzyme–linked immunosorbent assay (ELISA) based on the single-stranded binding protein (SSB)-assisted aptamer was designed for biosensing applications. Combined with the biotin–streptavidin (SA) system and the high catalytic activity of horseradish peroxidase (HRP), this SSB-assisted aptamer sensor was applied for the detection of aflatoxin B1, ochratoxin A, and zearalenone. In this novel ELISA, mycotoxin–protein conjugations were replaced by SSB to avoid the hazard of mycotoxin, whereas antibodies were replaced by aptamer to avoid the complex and tedious preparation of antibodies. In the absence of target mycotoxins, SSB can bind the aptamer–biotin specifically. Detection was performed using the strong combination of biotin and SA after adding SA–HRP and substrate/chromogen solution, thereby resulting in a strong yellow color signal. In the presence of target mycotoxins, the aptamer–biotin cannot bind to the SSB, thereby leading to a weak yellow color signal. Under optimal conditions, the designed method was successfully applied for the determination of real sample and exhibited high specificity and low limits of detection in corn (112 ng L–1 for aflatoxin B1, 319 ng L–1 for ochratoxin A, and 377 ng L–1 for zearalenone). The green ELISA may also be extended to the detection of other biohazardous targets by changing the aptamer.