Dual DNAzyme-catalytic assembly of G-quadruplexes for inducing the aggregation of gold nanoparticles and developing a novel antibiotic assay method.

By utilizing a target biorecognition reaction to induce the self-assembly of G-quadruplexes and the aggregation of gold nanoparticles (Au NPs), this work develops a novel colorimetric biosensing method for kanamycin (Kana) antibiotic detection. The compact G-quadruplex structure was assembled from its two half-split sequences which were designed in two hairpin substrates of the Mg2+-dependent DNAzyme (MNAzyme). Besides hybridizing with the aptamer strand, the MNAzyme sequence was also split into two half fragments to be designed in the two substrates. Upon the aptamer-recognition reaction toward Kana, the MNAzyme strand could be quantitatively released to cause the exposure of the split G-quadruplex-sequences on two hairpin substrate-modified Au NPs and simultaneous release of two half fragments of the MNAzyme-sequence. Thus, the K+-assisted self-folding of G-quadruplexes causes the cross-linking of the two Au NPs to realize the Au NP aggregation-based colorimetric signal output (measured at the largest absorption peak near 520 nm). Meanwhile, the self-assembled formation of the second MNAzyme drastically amplified the signal response. Under the optimal conditions, a wide linear range from 0.1 pg mL-1 to 10 ng mL-1 and an ultrahigh sensitivity with the detection limit of 76 fg mL-1 were obtained. The dose-recovery experiments in real samples showed satisfactory results with recoveries from 98.4 to 105.4% and relative errors compared with the ELISA method less than 4.1%. Due to the high selectivity, excellent repeatability and stability, and simple manipulation, this method indicates a promising potential for practical applications. A novel homogeneous biosensing method was developed for the convenient detection of the kanamycin antibiotic. The target biorecognition-induced and dual DNAzyme-catalytic assembly of G-quadruplexes enabled the amplified aggregation of gold nanoparticles for the simple, cheap, stable, and ultrasensitive colorimetric signal transduction of the method.