Autonomous positive-feedback DNA walker cascade for ultrasensitive detection of DNA repair glycosylases

Currently, DNA walker-based biosensors used for bioanalysis often suffer from limited amplification efficiency due to irreversible walker consumption and a restricted walking distance. Herein, we report an autonomous positive-feedback DNA walker biosensor for the ultrasensitive electrochemical detection of uracil-DNA glycosylase (UDG) and human alkyladenine DNA glycosylase (hAAG). The platform employs a single bifunctional hairpin probe that integrates target recognition and signal transduction. Through the synergistic action of terminal deoxynucleotidyl transferase (TdT) and exonuclease III (Exo III), a positive-feedback "cleavage-extension-recutting" loop is established, enabling continuous walker regeneration and signal amplification. Benefiting from this autonomous positive-feedback mechanism, the proposed biosensor achieves detection limits of 6.8 × 10^-7 U/mL for UDG and 1.5 × 10^-6 U/mL for hAAG, representing a reduction of over two orders of magnitude compared with conventional feed-forward DNA walker systems. The sensor exhibits excellent selectivity toward the target glycosylases over structurally similar enzymes and potential interferents, and shows recoveries ranging from 97.8% to 105.4% in serum spike-recovery tests. Furthermore, the platform successfully discriminates cancer cells from normal cells and differentiates serum samples from breast cancer patients and healthy individuals. These results demonstrate that the proposed autonomous positive-feedback DNA walker strategy holds great promise as a versatile and highly sensitive platform for DNA repair enzyme analysis and disease-related biomarker detection.