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. OTA analysis in food and its biomonitoring in biological samples are recommended to assess individual exposure to the mycotoxin. The main technique used for OTA detection in biological samples is LC-MS/MS. Unlike chromatographic techniques, biosensors represent a valid alternative for mycotoxin detection due to their high portability potential and ease of use. For OTA detection, different types of biosensors have been developed, based on the specific recognition of OTA by DNA aptamers. The engineering versatility of DNA makes it a powerful and programmable element for the construction of micro-scale systems that find many applications in biosensing. In our study, we describe a DNA-based biosensor for the detection of OTA in urine. The sensor is composed with a DNA-based capture-system and detection-system. We assembled paramagnetic microbeads carrying a capture-aptamer for OTA that allows its specific capture in liquid samples. A detection complex triggering an isothermal rolling circle amplification (RCA) was assembled using the same aptamer, annealed to a circularized probe, and used to detect the toxin capture event. We designed the RCA to generate autocatalytic units with peroxidase activity (DNAzyme). In the presence of OTA, the circular DNA triggers its isothermal amplification at 30°C, producing a single-stranded and tandem repeated long homologous copy of its sequence. In the amplified DNA strand, a peroxidase self-catalytic structure induces the development of colour reaction that is visible at naked eye. The resulting biosensor showed high sensitivity and selectivity for the detection of OTA as low as 1.09×10 ng/mL. Moreover, the biosensor was used for the detection of OTA in naturally contaminated urine. Accuracy and repeatability data obtained in recovery experiments showed recoveries >95%, and relative standard deviations in the range 3.6-15%.