Cascade DNA Circuits Mediated CRISPR-Cas12a Fluorescent Aptasensor based on Multifunctional Fe₃O₄@hollow-TiO₂@MoS₂ Nanochains for Tetracycline Determination

Herein, for the first time, the CRISPR-Cas12a system is combined with aptamer, cascaded dynamic DNA network circuits, and Fe₃O₄@hollow-TiO₂@MoS₂ nanochains (Fe₃O₄@h-TiO₂@MoS₂ NCs) to construct an efficient sensing platform for tetracycline (TC) analysis. In this strategy, specific recognition of the target is transduced and amplified into H1-H2 duplexes containing the specific sequence of Cas12a-crRNA through an aptamer recognition module and the dual amplification dynamic DNA network. Subsequently, the obtained activated Cas12a protein non-specifically cleaves the adjacent reporter gene ssDNA-FAM to dissociate the FAM molecule from the quencher Fe₃O₄@h-TiO₂@MoS₂ NCs, resulting in the recovery of the fluorescence signal and further signal amplification. Particularly, the synthesized multifunctional Fe₃O₄@h-TiO₂@MoS₂ NCs composites also exhibit superb magnetic separability and photocatalytic degradation ability. Under optimal conditions, the aptasensor displays a distinct linear relationship with the logarithm of TC concentration, and the limit of detection is as low as 0.384 pg mL⁻¹. Furthermore, the results of spiked recovery confirm the viability of the proposed aptasensor for TC quantification in real samples. This study extends the application of the CRISPR-Cas12a system in the field of analytical sensing and contributes new insights into the exploration of reliable tools for monitoring and treating hazards in food and environment.