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. Analysing OTA in food and monitoring its presence in biological samples is recommended to assess individual exposure to the mycotoxin. The primary technique used for OTA detection in biological samples is LC-MS/MS. However, biosensors provide a viable alternative for mycotoxin detection due to their high portability potential and ease of use. Various types of biosensors have been developed for OTA detection, relying on the specific recognition of OTA by DNA aptamers. DNA's engineering versatility makes it a powerful and programmable element for constructing microscale systems that find numerous applications in biosensing. In our study, we present a DNA-based biosensor for detecting OTA in urine. The sensor consists of a DNA-based capture system and a detection system. We created paramagnetic microbeads carrying a capture aptamer for OTA, enabling its specific capture in liquid samples. A detection complex, which triggers an isothermal rolling circle amplification (RCA), was assembled using the same aptamer annealed to a circularized probe. This complex was used to detect the occurrence of toxin capture. We designed the RCA to generate autocatalytic units with peroxidase activity (DNAzyme). In the presence of OTA, the circular DNA initiates its isothermal amplification at 30°C, producing a single-stranded and tandemly repeated long homologous copy of its sequence. Within the amplified DNA strand, a peroxidase self-catalytic structure induces a colour reaction that is visible to the naked eye. The resulting biosensor exhibited high sensitivity and selectivity for detecting OTA, with a limit of detection as low as 1.09×10-12 ng/ml. Furthermore, we tested the biosensor for OTA detection in naturally contaminated urine. Accuracy and repeatability data obtained from recovery experiments showed recoveries exceeding 95%, with relative standard deviations ranging from 3.6 to 15%. For the first time, an aptasensor has been successfully applied to detect OTA in biological fluids. It can be used for mycotoxin biomonitoring and assessment of individual exposure.