Target-initiated autonomous synthesis of metal-ion dependent DNAzymes for label-free and amplified fluorescence detection of kanamycin in milk samples

Accurate and sensitive monitoring of the abused antibiotics is vital because excessive antibiotics in human body can cause toxicity to kidney or lead to potential loss of hearing. In this work, we described a label-free and highly sensitive fluorescent aptasensing platform for detecting kanamycin in milk samples based on the synchronization signal amplification of primer exchange reaction (PER) and metal-ion dependent DNAzyme. The target kanamycin binds the aptamer sequence hybridized on a hairpin template and initiates PER for autonomous synthesis of Mg2+-dependent DNAzyme sequences with aid of Bst-DNA polymerase at isothermal conditions. Such a synthesis process can be repeated many times to produce lots of DNAzymes to cyclically cleave the rA site in the signal hairpin substrates under the assistance of Mg2+ cofactor to liberate numerous free G-quadruplex fragments. The organic dye thioflavin T (ThT) further associates with these G-quadruplex fragments to yield substantially intensified fluorescence for sensitive detection of kanamycin with a low detection limit of 0.36 nM. In addition, the developed aptamer sensing method also shows a good selectivity for kanamycin against other interfering antibiotics, and can realize the monitoring of kanamycin added in milk samples, highlighting its potential for sensitive monitoring of trace amount of kanamycin for food safety applications.