Self-oxidation and proximity crosslinking of G-quadruplex in G-quadruplex /hemin peroxidase system

G-quadruplex (G4)/hemin peroxidase has emerged as a robust platform for signal amplification in biosensors. While offering distinct advantages over protein enzymes, its susceptibility to inactivation often results in low apparent catalytic activity, thereby limiting broader applications. To address this, we investigated the side reactions occurring during the catalytic process of G4/hemin DNAzyme in both N-acetyl-cysteine (NAC) and H₂O₂ systems. Notably, high-molecular-weight products were observed when the hemin/G4 ratio exceeded 10. Further analysis revealed that these products primarily resulted from the self-oxidation of G4 sequences or proximity cross-linking with hemin fractions, substrates, and coexisting DNA sequences. Our findings provide critical insights into the inactivation mechanism and offer guidance for enhancing the catalytic efficiency of G4-based biosensors.