A label-free ratiometric fluorescent aptasensor for highly sensitive detection of metronidazole

Antibiotic contamination in the environment poses significant threats to ecosystems and public health. However, conventional methods for detecting metronidazole (MNZ) are often unsuitable for field-deployable or real-time applications due to high equipment costs, labor-intensive sample preparation, and the need for specialized expertise. To address these limitations, we developed a novel label-free ratiometric fluorescent aptasensor for the sensitive and accurate detection of MNZ in complex samples. The sensor integrates three key components: a fluorescent dye (ethyl violet, EV), an MNZ-specific aptamer (Apt_MNZ), and an internal reference fluorophore (fluorescein sodium, FS). EV and Apt_MNZ form a complex with enhanced red fluorescence emission. Upon MNZ binding, the fluorescence intensity of EV decreases in a dose-dependent manner, while the signal from FS remains stable, enabling precise ratiometric quantification. The platform exhibits high sensitivity, with a detection limit of 1.11 μM, and is cost-effective (∼$0.14 per analysis), making it suitable for trace-level MNZ detection in various samples. Successful validation in real-world samples confirms its potential for environmental monitoring, contamination assessment, and regulatory surveillance.