Based on our previous work, we have constructed a new electrochemical biosensor to detect epidermal growth factor receptor (EGFR) gene mutation, which was a significant therapeutic effect predictor of target drugs for non-small cell lung cancer. In order to lower the detection limit to detect the small amount of EGFR gene status, we have employed lambda exonuclease (λ-Exo) to form a hybridization-digestion cycle. The reaction stages are depicted as follows: the target DNA hybridized with auxiliary DNA which had been modified with the λ-Exo recognition site; then, the double strands were cleaved by λ-Exo. The target DNA was released completely, and continued to hybridize with remaining auxiliary DNA, which formed a recycle for target reutilization. Finally, we detected the remaining auxiliary DNA to evaluate the amount or status of the EGFR gene. The reutilization of target DNA will help to achieve an enlarged signal with a small amount of target DNA, and the detection limit of the biosensor decreased down to 10 pM. Meanwhile, our assay can differentiate wild genes from the mutational gene of EGFR with excellent specificity. Our signal amplification method provides a research foundation for the detection system of the electrochemical biosensor by employing exonuclease, and impels the biosensor to be developed as a suitable method for EGFR detection in clinical applications.
Keywords: DNA electrochemical biosensor; epidermal growth factor receptor; hybridization–digestion cycles; lambda exonuclease; signal amplification.