A label-free electrochemical platform for the detection of antibiotics based on cascade enzymatic amplification coupled with a split G-quadruplex DNAzyme

Herein, a split G-quadruplex DNAzyme as a signal reporter was integrated into an electrochemical sensing platform for the detection of antibiotics with specificity and sensitivity. To improve the signal-to-noise ratio, two G-rich oligonucleotide sequences (G1 and G2) were blocked into two different hairpin probes, preventing the two segments from assembling into a spilt G-quadruplex structure. Moreover, we designed a double-arch probe, consisting of an aptamer as the recognition element and two-step enzymatic signal amplification. Concretely, the first is the Nt.BbvCI-assisted nicking cyclic reaction activated by target-aptamer binding, and the second is exonuclease III-aided cyclic amplification for generating abundant G1 and G2. The modified capture probe on the electrode was used to combine G1 and G2 to form the spilt G-quadruplex/hemin when K+ and hemin were present. This complex plays the role of DNAzyme with superior horseradish peroxidase activity in catalyzing the decomposition of H2O2. Under optimal conditions, this biosensor showed an excellent performance for sensing kanamycin with a detection limit of 83 fM for kanamycin concentrations ranging from 100 fM to 1 nM. Hence, the proposed strategy has potential as an efficient and actual platform for small molecule analysis.