Co-immobilization of DNAzyme and horseradish peroxidase via biomineralization for electrochemical sensing of antibiotics

Co-immobilization of horseradish peroxidase (HRP) with G-quadruplex/hemin complex often suffer from low efficiency, intricate procedures, and reduced catalytic performance. To address these challenges, we developed a biomineralization strategy that integrates HRP and G-quadruplex/hemin into the single nanosheet structure. By engineering a split aptamer sequence into a G-rich domain, the nanosheets synergistically combine enhanced catalytic activity with better target-specific molecular recognition. Compared with either free HRP or it biomineralized counterpart alone, the prepared nanosheets exhibited markedly improved peroxidase-like activity. In the presence of enrofloxacin, specific aptamer-target binding facilitated the formation of a ternary complex on the electrode surface, enabling the catalysis of H 2 O 2 -mediated oxidation of TMB. Under the optimized conditions, the electrochemical aptasensor displayed a wide linear detection range of 1 nM–10 μM with a low detection limit of 0.53 nM. Beyond high selectivity, the sensor demonstrated excellent practical applicability in spiked milk and egg white samples. The generality of the developed platform was further confirmed by its successful extension to other antibiotics detection through replacing the split-aptamer pairs.