Cas12a/crRNA recognition initiated self-priming mediated chain extension for colorimetric cell-free DNA (cfDNA) analysis

Cell-free DNA (cfDNA) has attracted increasing attention as a promising biomarker in liquid biopsy due to its crucial role in disease diagnosis. However, previous cfDNA detection methods are commonly based on the development of target-specific primers and integrated signal amplification strategies, which may induce false-positive results. This paper presents a sensitive yet accurate method for cfDNA detection that combines phosphorothioated-terminal hairpin creation with a self-priming extension process. This approach initiates a self-priming mediated chain extension-based signal cycle following the trans-cleavage of H0@MBs when the CRISPR-Cas12a complex is activated by target cfDNA, resulting in the production of a substantial quantity of pyrophosphate. A pyrophosphate sensing probe (pp probe) was utilized, facilitating both high-efficiency and stable colorimetric signaling. This innovative technique for colorimetric detection of target cfDNA demonstrated exceptional sensitivity with a low limit of detection of 1.04 fM and greatly enhanced selectivity, with the complete detection process taking around 60 min. In addition, this technique is capable of detecting cfDNA from the culture medium of HEK293 cells, indicating its clinical application potential. Compared with the previous CRISPR-Cas system-based cfDNA method that necessitates an amplification step before detection, Cas12a was directly used to identify a target sequence that can avoid false target amplification. This technique is simple, accurate, and rapid, engineered to identify cancer-associated cfDNA via a highly sensitive colorimetric change, which is expected to be beneficial for applications requiring point-of-care cancer detection.