Ultrasensitive and Efficient CYFRA21-1 Nanosensor Empowered by CRISPR/Cas12a Nanopipette Technology

The abnormal expression of CYFRA21-1 is closely related to the development of non-small cell lung cancer (NSCLC), making its detection crucial for early diagnosis. In this study, we successfully constructed an electrochemical sensor based on nanopipettes and CRISPR/Cas12a for the detection of CYFRA21-1. We utilized an electrochemical three-electrode system to quantify the target by monitoring changes in electrochemical signals at the single-cell level. The small size of the nanopipettes, combined with the trans-cleavage activity of Cas12a, ensures reaction specificity while detecting CYFRA21-1 at the single-cell level. Experimental results demonstrated that the sensor exhibited a linear relationship with T-DNA concentration over the range of 1 fM to 1 μM, with a detection limit of 0.362 nM, indicating high sensitivity. The nanopipettes successfully detected CYFRA21-1 at the single-cell level, showing distinct signaling changes before and after the assay. Additionally, the sensor exhibited high specificity for CYFRA21-1 and was not affected by single-base or three-base mismatched DNA. In stability tests, the sensor demonstrated good reproducibility and stability. These properties render this electrochemical sensor a promising tool for important applications in disease diagnosis and monitoring.