A triple-signal-amplified colorimetric biosensor based on 3D-DNA-walker and hybridization chain reaction for sensitive detection of tetracycline residues.

Tetracycline residues in food matrices remain a concern for food safety, making sensitive detection of tetracycline important for residue monitoring. Herein, we developed a triple-amplification colorimetric biosensor for parent tetracycline (TET) detection by integrating aptamer-based recognition, a three-dimensional DNA walker, hybridization chain reaction (HCR), and G-quadruplex DNAzyme catalysis. Upon target binding, the aptamer releases multiple complementary DNA strands, which initiate strand displacement and activate the DNA walker for continuous signal production. The generated strands subsequently trigger HCR to form G-quadruplex-rich nanostructures. After hemin incorporation, the resulting DNAzymes catalyze the oxidation of 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) by H2O2, producing a distinct color change for visual observation and absorbance-based quantification. Under optimized conditions, the biosensor exhibited a linear response to TET concentrations ranging from 0.005 to 10 ng/mL, with a detection limit of 0.00326 ng/mL. In spiked raw milk samples, recoveries of 98.8-104.0% with relative standard deviations (RSDs) of 0.1-1.5% were obtained. The designed triple-amplification colorimetric biosensor provides a sensitive method for parent TET determination in spiked raw milk samples.