The traditional nucleic acid-modified electrode in room-temperature manner suffers from the drawback of time-consuming and uneven distribution. In this work, we adopted the freeze-thawing method to construct a nicking enzyme-powered DNA walking machine on the gold electrode. In the absence of kanamycin, the intact substrate strand can trigger the self-assembly and form G4 nanowires on the electrode surface. After adding hemin, a significant catalytic current of the TMB oxidization can be observed. By contrast, once the specific binding between aptamer and kanamycin occurs, the activated walking strand processes a consecutive binding-cleavage cycle with the substrate strand on the electrode surface. The residue substrate fragment is unable to trigger the subsequent G-quadruplex assembly, resulting in a decrease in catalytic current. Under optimal experimental conditions, a linear relationship between the kanamycin concentration and current decrease was obtained in the range from 5 to 100 pM, and the limit of detection was 1.44 pM. This method has also demonstrated practical performance in the spiked milk and honey with satisfactory recoveries of 107.1 % to 114.3 % and 94.3 % to 101.5 %, respectively.
A DNAzyme-powered DNA walking machine was constructed to develop the fluorescence aptasensing for sensitive detection of kanamycin. The aptamer for kanamycin is partially hybridized with complementary DNA (cDNA) modified on magnetic beads (MBs). The specific interaction of target and aptamer triggered the cDNA to be free tentatively, which captured walker DNA. Then the autonomous motion of DNA walker on MBs surface was propelled via DNAzyme digestion of recognition sites. The signal probe was separated, and the amplified fluorescence signal was achieved by the accumulation of the signal probe. Kanamycin was used as a model analyte, and the developed assay achieves a detection limit of 0.00039 ng·mL −1 (S/ N = 3) within a linear detection range from 0.001 to 2000 ng·mL −1 . This aptasensing strategy can be extended for detection of other antibiotics by adapting corresponding target recognition aptamer sequence.
Graphical abstract The fluorescence aptasensing for sensitive detection of kanamycin based on DNAzyme-powered DNA walking machine was constructed.
Deoxynivalenol (DON) contamination in cereals is a widespread issue with global implications, necessitating the development of efficient detection methods. Here, a fluorescent aptasensor integrating target-responsive DNA three-way junction (TWJ) and DNA walking machine was developed to detect DON. The DON-specific aptamer (Apt) and the walker (Walker DNA) are integrated into TWJs. TWJs initiate the DNA walking machine with the assistance of Nb. Bbv CI to provide signal amplification for sensing events. The constructed fluorescent aptasensor selectively recognizes and detects DON. Additionally, the sensing platform explored the quantitative relationship between fluorescence signal changes and DON concentration, achieving a limit of detection (LOD) of 0.009 ng/mL. Ultimately, the method performed well in the detection of cornmeal samples with recoveries ranging from 95.49 % to 100.82 %. The method demonstrates good stability and is expected to be a potential candidate for the accurate detection of DON in grains.
危险品化学品经营许可证（带存储）