Split aptamer-regulated AuNF nanozymes for colorimetric and electrochemical dual-readout kanamycin biosensing

Kanamycin (Kana) residues released into aquatic environments due to its extensive use have raised increasing concerns because of their potential risks to ecosystems and human health. In this work, we report a label-free split aptamer-modified gold nanoflower (AuNF)-based aptasensing platform with colorimetric and electrochemical readouts for the determination of Kana in water. Two thiolated split aptamer fragments (SPA1 and SPA2) were immobilized on AuNFs through Au-S bonds. The flower-like AuNFs provided a high-surface-area scaffold for aptamer loading, while the DNA modification further enhanced the peroxidase-like activity of AuNFs toward TMB oxidation. In the presence of Kana, target-induced assembly of SPA1 and SPA2 promoted the aggregation of AuNFs, which inhibited their catalytic activity toward TMB oxidation, thereby reducing oxTMB formation and the resulting colorimetric and electrochemical signals. Based on this signal-off mechanism, the platform enabled quantitative detection of Kana with linear ranges of 100-3000 nM for colorimetry and 50-2000 nM for chronoamperometry, with limits of detection of 0.51 and 0.074 nM, respectively. The method also showed satisfactory selectivity, reproducibility, and storage stability. In spiked environmental water samples, recoveries ranged from 87.7% to 106.2% with relative standard deviations below 5.0%, and the electrochemical results were in acceptable agreement with those obtained by HPLC-MS. This study provides a simple and effective dual-readout aptasensing strategy for Kana determination in environmental water samples.