Synergistic powering of DNA walker movement by endogenous dual enzymes for constructing dual-mode biosensors

To achieve highly sensitive and reliable detection of apurinic/apyrimidinic endonuclease 1 (APE1), a critical cancer diagnostic biomarker, we designed a DNA walker-based dual-mode biosensor, utilizing cellular endogenous dual enzymes (APE 1 and Flap endonuclease 1 (FEN 1)) to collaborate in activating and propelling DNA walker motion on DNA-functionalized Au nanoparticles. Incorporating both fluorescence and electrochemical detection modes, this system leverages signal amplification from DNA walker movement and cascade amplification through tandem hybridization chain reactions (HCR), achieving highly sensitive detection of APE 1. In the fluorescence mode, continuous DNA walker movement, initiated by APE1 and driven by FEN1, generates a robust signal response within a concentration range of 0.01-500 U mL^-1, presenting a good linearity in the concentration range of 0.01-10 U mL^-1, with a detection limit of 0.01 U mL^-1. In the electrochemical detection module, the cascade upstream DNA walker and downstream HCR dual signal amplification strategy further enhances the sensitivity of APE1 detection, extending the linear range to 0.01-50 U mL^-1 and reducing the detection limit to 0.002 U mL^-1. Rigorous validation demonstrates the biosensor's specificity and anti-interference capability against multiple enzymes. Moreover, it effectively distinguishes cancer cells from normal cell lysates, exhibiting excellent stability and consistency in the dual-modes. Overall, our findings underscore the efficacy of the developed dual-mode biosensor for detecting APE1 in serum and cell lysates samples, indicating its potential for clinical applications in disease diagnosis.