CRISPR/Cas12a-drived fluorescent and electrochemical signal-off/on dual-mode biosensors for ultrasensitive detection of EGFR 19del mutation

The mutation status of epidermal growth factor receptor exon 19 (EGFR 19) is critical in predicting the susceptibility to tyrosine kinase inhibitors. Herein, we develop CRISPR/Cas12a-drived fluorescent and electrochemical dual-mode biosensors for detection of EGFR 19 deletion (EGFR 19del). By introducing a padlock probe with two endonuclease sites for target recognition, the rolling-circle strand displacement amplification (RC-SDA) is triggered to achieve preamplification and CRISPR/Cas12a reaction. When EGFR 19del is present, no amplified products activate the CRISPR/Cas12a system, resulting in the fluorescent signal remaining in the "off" state. To improve the detection accuracy and sensitivity, we have ingeniously constructed an electrochemical biosensor that converts the "signal-off" state to the "signal-on" state. In this design, the inactive Cas12a does not cleave the electrochemical probe, which contains a T-rich sequence with a 5'methylene blue modification. With the assistance of Hg2+, the electrochemical probe can form a "hairpin-like" of Thymine-Hg-Thymine complex, generating an enhanced current. Experimental results demonstrate that the limit of detection (LOD) for the fluorescent biosensor is 18.62 fM, while the electrochemical biosensor achieves an LOD of 0.13 fM. Furthermore, the biosensors can discriminate 0.5% and 0.1% mutation, respectively. They also exhibit the capability to differentiate between wild-type and mutant genes from various cells. The obtained results are identical to those obtained by qPCR method, indicating their broad application prospects in drug development and disease surveillance.