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 generaPenicillium and Aspergillus. Analysing OTA in food and monitoring its presence in biological samples isrecommended to assess individual exposure to the mycotoxin. The primary technique used for OTAdetection in biological samples is LC-MS/MS. However, biosensors provide a viable alternative formycotoxin detection due to their high portability potential and ease of use. Various types of biosensorshave 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 microscalesystems that find numerous applications in biosensing. In our study, we present a DNA-basedbiosensor for detecting OTA in urine. The sensor consists of a DNA-based capture system and adetection system. We created paramagnetic microbeads carrying a capture aptamer for OTA, enablingits specific capture in liquid samples. A detection complex, which triggers an isothermal rolling circleamplification (RCA), was assembled using the same aptamer annealed to a circularized probe. Thiscomplex was used to detect the occurrence of toxin capture. We designed the RCA to generateautocatalytic units with peroxidase activity (DNAzyme). In the presence of OTA, the circular DNAinitiates 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 structureinduces a colour reaction that is visible to the naked eye. The resulting biosensor exhibited highsensitivity 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 andrepeatability data obtained from recovery experiments showed recoveries exceeding 95%, with relativestandard deviations ranging from 3.6 to 15%. For the first time, an aptasensor has been successfullyapplied to detect OTA in biological fluids. It can be used for mycotoxin biomonitoring and assessmentof individual exposure.