Low-background and visual detection of antibiotic based on target-activated colorimetric split peroxidase DNAzyme coupled with dual nicking enzyme signal amplification

Herein, we have reported the development of a simple, rapid, and low cost colorimetric method for the detection of antibiotic based on target-activated split peroxidase DNAzyme coupled with dual nicking enzyme signal amplification (NESA). To lower background signal in G-quadruplex DNAzyme-based detection, the two split G-rich parts are caged into two different hairpin probes, respectively, preventing the two parts from assembling into the G-quadruplex structure. By the combination of restriction endonuclease-assisted cleavage reaction with the spilt G-quadruplex probes, target-modulated release of the two split G-rich parts is achieved, affording high specificity of antibiotic detection. Our strategy features with several aspects. First, the less background signal produced by the self-assembly of G-quadruplex in the absence of target is effectively eliminated owing to the pre-blocking of the two split G-rich parts. Second, dual NESA coupled G-quadruplex DNAzyme amplification strategy is integrated with colorimetric assay of antibiotic, which significantly improves the detection sensitivity. Third, peroxidase-mimicking DNAzyme is used as biocatalyst in our reaction system, which can catalyze the oxidation of 2,2' - azino - bis (3 - ethylbenzothiozoline - 6 - sulfonic acid) (ABTS2-) mediated by H2O2 to generate the colored radical anion (ABTS•-), allowing to low cost and visual detection of antibiotic by the naked eye. Under optimized conditions, the results revealed the proposed biosensor exhibits excellent specificity and sensitivity toward kanamycin with a detection limit as low as 14.7 pM. Hence, the target-activated split G-quadruplex DNAzyme and dual NESA-based strategy provides a useful and practical platform for antibiotic residues determination and other analytes detection in bio-analysis.