Electrochemical biosensor based on bipedal DNA walker for highly sensitive amplification detection of apurinic/apyrimidinic endonuclease 1

DNA walker has been widely used in various types of biosensors for signal-amplification detection of low abundance analytes. However, the usual design of unipedal walker provides limited signal amplification effect with poor sensitivity. Here, we designed an electrochemical biosensor based on bipedal DNA walker allowing for highly sensitive detection of apurinic/apyrimidinic endonuclease 1 (APE 1), a significant base excision repair enzyme, which is over-expressed in multiple cancer cells emerging as a promising biomarker for cancer diagnostics. The bipedal DNA walker can be released after APE1 cleaving apurinic/apyrimidinic (AP) sites, and then perform the catalytic hairpin assembly (CHA) process, triggering the downstream hybridization chain reaction (HCR) to achieve a dual-amplified current signal. This bipedal walker presents dramatically increased catalytic efficiency in the upstream CHA process, and finally contributes almost 5 times synergistic enhanced current signal compared to unipedal walker. We successfully realized the highly sensitive detection of APE 1 with a wide linear range from 0.001 U mL−1 to 1 U mL−1 reaching a detection limit of 0.001 U mL−1. This biosensor showed good specificity in discrimination of APE1 from other interfering enzymes. It was successfully applied to investigate APE 1 expression level in cell lysate, demonstrating its potential diagnostics applications.