Label-free electrochemical split-aptamer aptasensor with signal amplification via flower-like gold nanostructures for ultrasensitive detection of kanamycin in water

Residual kanamycin (Kana) in water bodies threatens ecological safety and human health, driving demand for straightforward and sensitive detection tools. In this work, we present a label-free electrochemical sandwich aptasensor that achieves ultrasensitive Kana detection by synergizing split-aptamer recognition with signal amplification through flower-like gold nanostructures (AuNFs). The split aptamer fragments (SPA1/SPA2) remain separate in the absence of target, minimizing background from nonspecific folding. Upon Kana introduction, a SPA1–Kana–SPA2 ternary complex assembles on the AuNF-modified ITO electrode, providing a scaffold for intercalation of the redox indicator ferrocene–naphthalimide (FND). The consequent accumulation of FND results in a markedly enhanced differential pulse voltammetry response. The three-dimensional AuNFs offer a large electroactive surface for high-density probe immobilization and efficient electron transfer. The sensor exhibits a linear response for Kana from 5 nM to 2 μM with a detection limit of 0.2 nM (3 σ / k ). It also shows excellent selectivity over analogous aminoglycosides, good reproducibility, and acceptable storage stability. The aptasensor was successfully applied to the analysis of real water samples, yielding recoveries of 86.4–116.8%, consistent with HPLC-MS measurements. This work provides a sensitive, selective, and reliable platform for on-site monitoring of Kana in water.