Enzymatically Cyclic Activated Biosensor Based on a Tetrahedral DNA Framework for Precise Tumor <i>in Situ</i> Molecular Imaging

The development of stimulus-responsive and amplification-based strategies is crucial for achieving improved spatial specificity and enhanced sensitivity in tumor molecular imaging, addressing challenges such as off-tumor signal leakage and limited biomarker content. Therefore, a cyclically activated enzymatic biosensor based on the modification of an AP site within a tetrahedral framework DNA (AP-tFNA) was rationally developed for tumor cell-specific molecular imaging using the endogenous enzyme apurinic/apyrimidinic endonuclease 1 (APE1) as a target, exhibiting superior spatial specificity and high sensitivity. APE1, which predominantly localizes within the nucleus in normal cells but exhibits cytosolic and nucleus expression in cancer cells, can specifically recognize and cleave the AP site in AP-tFNA, resulting in the separation of the fluorophore and quenching group, thereby inducing a fluorescence signal. Additionally, upon completion of the excision of one AP site in AP-tFNA, APE1 is released, thereby initiating a subsequent cycle of hydrolytic cleavage reactions. The experimental results demonstrated that AP-tFNA enables precise differentiation of tumor cells both <i>in vitro</i> and <i>in vivo</i>. In particular, the AP-tFNA can monitor drug resistance in neuroblastoma cells and classify the risk for neuroblastoma patients at the clinical plasma level.