Ratiometric fluorescent aptasensor based on DNA-gated FeO@Uio-66-NH and Exo I-assisted signal amplification.

Background: Ochratoxin A (OTA) is toxic secondary metabolites produced by fungi and can pose a serious threat to food safety and human health. Due to the high stability and toxicity, OTA contamination in agricultural products is of great concern. Therefore, the development of a highly sensitive and reliable OTA detection method is crucial to ensure food safety. Although fluorescent sensors have been widely used for OTA detection, they still face challenges such as false positives and insufficient sensitivity, thus further improvement of the detection performance is necessary.

Results: Herein, a ratiometric biosensor based on DNA-gated Fe₃O₄@Uio-66-NH₂ nanocomposites and Nucleic Acid Exonuclease I (Exo I)-assisted signal amplification was constructed for the sensitive detection of OTA. This functional nanocomposite combines magnetic, porous and fluorescent properties. Fe₃O₄ of the core enables efficient magnetic separation, while the porous structure of Uio-66-NH₂ encapsulates the fluorescent dye rhodamine 6G (Rho 6G). In addition, a label-free DNA gating strategy was introduced to control the release of Rho 6G. The sensor performs OTA detection by the fluorescence signal ratio of FeO@Uio-66-NH and Rho 6G, which effectively avoids false positives of the sensor. Accelerated release of Rho 6G using Exo I greatly improves the sensitivity of the sensor. The sensor has a low LOD (0.308 ng mL) and the recoveries for OTA detection in real samples were 92.4%-116.0 %.

Significance: The development of this ratiometric fluorescent aptasensor highlights its potential for highly sensitive OTA detection, offering significant advantages in selectivity and accuracy due to its unique DNA-gated mechanism and the dual fluorescence signal ratio strategy. Additionally, the use of Fe₃O₄@Uio-66-NH₂ enables effective magnetic separation, while the Exo I-assisted signal amplification enhances sensitivity, making this sensor a powerful tool for detecting OTA in complex sample matrices with high reliability.