Enzyme-free dual-amplification assay for colorimetric detection of tetracycline based on Mg2+-dependent DNAzyme assisted catalytic hairpin assembly

Detection of ultralow concentration of tetracycline (TC) plays a key role in food safety and human health. Herein, we fabricated an enzyme-free dual-amplification assay for sensitive detection of TC in milk. The sensing system ingeniously combined Mg 2+ -dependent DNAzyme (MNAzyme) cleavage and catalytic hairpin assembly (CHA). Through the binding of TC and specific aptamer (Apt), DNA1 was released from the Apt-DNA1 complex. Then the separated DNA1 would hybridize with DNA2 to activate the catalytic activity of MNAzyme, which subsequently cleaved the substrate H0 and generated a new unit to trigger the following CHA reaction between H1 and H2, thereby releasing the G-rich sequence of H1 to induce the formation of G-quadruplex–hemin DNAzyme as a colorimetric signal readout. Under optimal conditions, the absorption intensity exhibited a significant linear correlation with the logarithm of the target TC concentration over a range of 4 orders of magnitude dynamic range from 1 pM to 10 nM. The limit of detection was calculated to be 0.89 pM and the method showed high selectivity for TC. More impressively, the proposed method was employed for TC determination in the milk sample.