A Competitive‐Equilibrium Fluorescent Aptasensor for Label‐Free Kanamycin Detection With Integrated Recognition and Signal Output

Kanamycin residues pose potential risks to food safety and public health, highlighting the need for rapid, low‐cost, and sensitive detection methods. However, developing label‐free aptasensors that integrate target recognition and signal output remains challenging. Herein, a label‐free fluorescent aptasensor was developed using a SMART‐derived fluorescent aptamer and a competitive‐equilibrium biosensing mechanism. The fluorescent aptamer MC‐DA‐LG was employed as an integrated signal‐output and kanamycin‐responsive aptamer module. MC‐DA‐LG generated a fluorescence signal upon binding to the fluorescent molecule, whereas the addition of kanamycin resulted in a concentration‐dependent fluorescence decrease, suggesting a shift in the aptamer–dye binding equilibrium. Circular dichroism spectroscopy indicated conformational changes in MC‐DA‐LG upon the addition of kanamycin or malachite green, while molecular docking suggested partially overlapping binding regions for the two small molecules, collectively supporting the proposed mechanism. Under optimized conditions, the biosensor showed a linear response from 0.1 to 10 μM, with an ultralow detection limit of 400 pM. Moreover, the biosensor exhibited favorable selectivity over other antibiotics and satisfactory recoveries of 94.1%–97.3% in milk and fetal bovine serum samples. This simple, low‐cost, rapid, and ultrasensitive assay enables direct fluorescence detection without labeling, immobilization, or complex procedures, demonstrating the potential of SMART‐tailored fluorescent aptamers for integrated label‐free biosensing of kanamycin.