O10: A DNA-based biosensor for the fast and sensitive detection of ochratoxin A in urine with isothermal rolling circle DNA amplification

Ochratoxin A (OTA) is a toxic and teratogenic metabolite produced by fungal species of the genera Penicillium and Aspergillus, necessitating careful analysis in food and monitoring its presence in biological samples to assess individual exposure to the mycotoxin. While LC-MS/MS remains the primary technique for OTA detection in biological samples, biosensors emerge as a promising alternative due to their portability potential and ease of use. We introduce a novel DNA-based biosensor designed for detecting OTA in urine. The sensor comprises a DNA-based capture system and a detection system. Paramagnetic microbeads loaded with capture aptamers for OTA and a detection system capable of isothermal rolling circle amplification (RCA). The RCA was strategically designed to generate autocatalytic units with peroxidase activity (DNAzyme). In the presence of OTA, the circular DNA initiated its isothermal amplification at 30°C, yielding a single-stranded, tandemly repeated long homologous copy of its sequence. Within the amplified DNA strand, a peroxidase self-catalytic structure induced a visible color reaction discernible to the naked eye. The developed biosensor demonstrated remarkable sensitivity and selectivity for detecting OTA, boasting a low limit of detection at 10 ng/mL. Furthermore, we conducted tests for OTA detection in naturally contaminated rat urine. The accuracy and repeatability data derived from recovery experiments revealed recoveries exceeding 95%, with relative standard deviations ranging from 3.6% to 15%¹. This innovative DNA-based biosensor showcases its potential for practical application in OTA detection, emphasizing the significance of DNA origami technology in enhancing biosensing capabilities.