CRISPR/Cas12a cleavage triggered nanoflower for fluorescence-free and target amplification-free biosensing of ctDNA in the terahertz frequencies

The detection of tumor biomarkers in liquid biopsies requires high sensitivity and low-cost biosensing strategies. However, few traditional techniques can satisfy the requirements of target amplification-free and fluorescence-free at the same time. In this study, we have proposed a novel strategy for ctDNA detection with the combination of terahertz spectroscopy and the CRISPR/Cas12 system. The CRISPR/Cas12a system is activated by the target ctDNA, resulting in a series of reactions leading to the formation of an Au-Fe complex. This complex is easily extracted with magnets and when dropped onto the terahertz metamaterial sensor, it can enhance the frequency shift, providing sensitive and selective sensing of the target ctDNA. Results show that the proposed terahertz biosensor exhibits a relatively low detection limit of 0.8 fM and a good selectivity over interference species. This detection limit is improved by three orders of magnitude compared with traditional biosensing methods using terahertz waves. Furthermore, a ctDNA concentration of 100 fM has been successfully detected in bovine serum (corresponding to 50 fM in the final reaction system) without amplification.