An all-in-one enzyme-free fluorescent aptasensor integrating localized catalyzed hairpin assembly for sensing antibiotics in food with improved detection efficiency.

Enzyme-free signal amplification fluorescent aptasensors depending on multi-component freely diffusing probes have become indispensable tools for antibiotic detection in food, but they suffer from low detection efficiency and tedious operation steps. Herein, an all-in-one enzyme-free fluorescent aptasensor integrating localized catalyzed hairpin assembly (L-CHA) was designed for antibiotic detection with improved detection efficiency. In the designed aptasensor, a double-stranded DNA reactant containing an antibiotic aptamer and a primer as well as two paired hairpin DNA reactants were immobilized on one spatial-confinement DNA scaffold (that is a DNA tetrahedron). Upon addition of the target antibiotic kanamycin, the activated primer initiated L-CHA, generating an amplified fluorescence signal. Compared with previously reported enzyme-free signal amplification fluorescent aptasensors, the designed aptasensor integrated the functions of target recognition, signal transduction, and L-CHA signal amplification into a single probe. In this all-in-one design, the reactants in this aptasensor were confined to a compact space for a higher local concentration, which improved detection efficiency. In particular, this aptasensor achieved sensitive detection of kanamycin within 60 min with a low detection limit of 0.019 ng mL. Additionally, the designed aptasensor depended on a single probe rather than multi-component probes, leading to simplified operation steps. Furthermore, this aptasensor was employed for detecting kanamycin in spiked milk samples with recoveries of 96.00% to 108.60%, indicating an acceptable accuracy. Therefore, this L-CHA-based all-in-one enzyme-free fluorescent aptasensor offers a prospective tool for antibiotic detection in the field of food safety.