Dual CHA-mediated high-efficient formation of a tripedal DNA walker for constructing a novel proteinase-free...

DNA walkers have been recognized as a type of powerful signal amplification tool for biosensors, but how to adopt a proper strategy to increase their amplification efficiency is still highly desirable. Herein we design a dual-catalytic hairpin assembly (CHA)-mediated strategy for the high-efficient formation of a tripedal Mg²⁺-dependent DNAzyme (MNAzyme)-DNA walker, and thus develop a novel proteinase-free dual-mode biosensing method for the kanamycin (Kana) antibiotic assay. The first CHA is initiated by a target-biorecognition reaction, which can produce the DNA walker and also induce the target recycling. The second CHA is initiated by a special base sequence designed as a one-half substrate of the MNAzyme. Upon the first CHA-triggered DNA walking at a magnetic bead (MB) track, this "pseudo-target" sequence can be released to induce another CHA-cycle for the formation of the same DNA walker. Meanwhile, the other one-half substrate strand exposed on the MB surface will trigger the quantitative hybridization chain reaction (HCR)-assembly of a G-quadruplex DNAzyme (G-DNAzyme)-enriched double-stranded DNA polymer. So the enzymatic reaction of G-DNAzymes enabled the convenient colorimetric and photoelectrochemical dual-mode signal transduction of the method. Due to the dual-CHA facilitation to the tripedal and three-dimensional DNA walking and synergetic signal amplification of HCR, this method exhibits very low detection limits of 9.4 and 0.55 fg mL^-1, respectively. In combination with its wide linear range, automated manipulation, and excellent selectivity, repeatability and reliability, the proposed method is expected to be used for the convenient semiquantitative screening and accurate determination of possible antibiotic residues in complicated matrices.