An aptazyme driven dual-FRET nanoplatform for simultaneous detection of chlorpyrifos and lead ions

The co-occurrence of pesticides and heavy metal ions in water resources poses significant health risks, necessitating the development of advanced detection methods capable of simultaneously monitoring these contaminants. Sometimes, traditional biosensors for multiple target detection may face challenges in detecting chemically distinct analytes due to their signal interference and interdependent detection mechanisms. To overcome these challenges, a simple dual-target optical biosensor was fabricated for sensitive and efficient measurement of chlorpyrifos and lead ions (Pb²⁺) by utilizing a Förster resonance energy transfer (FRET) based dual recognition enzyme beacon (DRAB) nano-machine. In our design, the self-blocked DRAB contains a 6-carboxy-X-rhodamine (ROX)-labeled chlorpyrifos aptamer at one terminal, with its fluorescence quenched by BHQ-2 strategically positioned in the central region of DRAB where the aptamer terminates. Simultaneously, the Pb²⁺ dependent substrate strand labeled with FAM is quenched by BHQ-1, which is located in the terminal region of the DRAB complementary to the substrate strand. This assay facilitates simultaneous and independent detection of chlorpyrifos and Pb²⁺. Upon the addition of chlorpyrifos, the hairpin-locked aptamer undergoes conformational opening, activating the system and restoring fluorescence at the FAM emission peak (520 nm). Similarly, the introduction of Pb²⁺ ions triggers the cleavage of the substrate strand, leading to fluorescence activation at the ROX emission peak (620 nm). This dual target sensing mechanism ensures high specificity and sensitivity, and the LOD for chlorpyrifos is 2.9 nM and the LOD for Pb²⁺ is 0.2 nM. The biosensor enables the generation of independent fluorescence signals for each analyte, ensuring precise detection of chlorpyrifos and Pb²⁺ in complex matrices.