A Highly Sensitive Electrochemical Aptasensor for Kanamycin: Leveraging RecJf Exonuclease-Assisted Target Recycling and Hybridization Chain Reaction Signal Amplification

A highly sensitive electrochemical aptasensor was developed for the detection of kanamycin, employing a DNA signal amplification strategy that combines RecJf exonuclease-assisted target recycling with a hybridization chain reaction (HCR). The sensing platform is constructed by covalently immobilizing double-stranded DNA (dsDNA) comprised of a kanamycin-specific aptamer and its thiol-modified complementary strand (SH-CDNA) onto a gold electrode. In the presence of kanamycin and RecJf exonuclease, the aptamer selectively binds to kanamycin, dissociating from the dsDNA complex. The RecJf exonuclease then cleaves the aptamer, releasing kanamycin and initiating a cycle of repetitive binding and release. The residual SH-CDNA on the electrode triggers an HCR between two types of ferrocene-labeled hairpin DNA, forming an elongated stable dsDNA nanostructure. This results in an amplified electrochemical signal proportional to the logarithm of kanamycin concentration over a range of 0.01-10 nmol/L, with a remarkable detection limit of 1.8 pmol/L. The aptasensor's performance was validated by analyzing spiked kanamycin in pharmaceutical eye drops and milk samples, yielding recovery rates between 95.6% and 104.8% and relative standard deviations from 1.4% to 4.2%. With its exceptional selectivity and sensitivity, this aptasensor offers a compelling alternative to traditional HPLC for rapid on-site detection of kanamycin. Capitalizing on the specificity of aptamers, the sensor design presented herein serves as a valuable blueprint for engineering detectors of other molecules, with significant implications for analytical chemistry and food safety monitoring.