A two-color fluorescence sensing strategy based on functionalized tetrahedral DNAzyme nanotweezers for ochratoxin A detection.

A two-color fluorescent sensing strategy based on a functionalized tetrahedral DNAzyme nanotweezer (FTDN) was developed to detect ochratoxin A (OTA) utilizing the multifunctional properties of DNA nanotechnology. The FTDN enables rapid OTA detection directly through a Cy5 fluorescent group, modified to respond to the target signal. Additionally, FTDN exhibits DNAzyme cutting activity in the presence of Mg2⁺ ions, enabling it to traverse DNA nanoflower-functionalized magnetic beads. This process results in the continuous cleavage of DNA nanoflowers labeled with numerous FAM fluorescent groups, thereby amplifying the detection signal and enhancing OTA sensitivity. The linear ranges for the Cy5 and FAM signals in response to OTA were 5-1000 ng/mL and 0.05-100 ng/mL, respectively, with corresponding limits of detection (LOD) of 1.59 ng/mL and 0.03 ng/mL. This study demonstrates that dual-color fluorescence via Cy5 and FAM can effectively verify OTA detection in food, significantly reducing false-positive and false-negative rates. The proposed platform offers sensitive and accurate detection of mycotoxins in food and can be adapted for monitoring other trace contaminants by simply altering the aptamer.