Methotrexate (MTX) is a widely used antimetabolite drug, mainly used in the treatment of a variety of cancer. Given the low therapeutic index and significant individual variability of MTX, it was critical to perform therapeutic drug monitoring (TDM) to minimize the side effects. Here, we designed a rapid and sensitive fluorescence/colorimetric assay for the detection of MTX in diluted human serum. After the aptamer binds specifically to MTX, the Walking strand cleaves the Hairpin strand and releases a large amount of fluorescent signal, and the color of the gold nanoparticles changes after the addition of sodium chloride. The change of color could be visualized by eyes to achieve point-of-care TDM of MTX. The quantitative detection of MTX concentration is carried out through the collection of fluorescent signals or absorptions. The fluorescence method can detect MTX in the range of 0.05–1000 μM with the detection limit of 0.0243 μM, meanwhile, the colorimetric method can detect MTX in the range of 0.01–100 μM with the detection limit of 0.0097 μM. The magnetic separation-assisted DNAzyme walker-based nanosensor exhibited good sensitivity, selectivity and stability for detecting MTX in serum and achieve point-of-care methods of TDM of MTX and the nanocarriers has demonstrated significant potential for clinical application.
Kanamycin residue has attracted much attention due to its potential risk to the environment and human health. Herein, an electrochemical platform with superior performance was proposed based on enzyme-assisted target recycling with a strand displacement amplification (SDA) reaction and a DNAzyme-derived DNA walker. Our sensing system consists of an SDA amplification system and a DNA walker system. In the DNA walker system, a DNAzyme walker (DW)–locking probe (LP) and signal hairpin (HP) were assembled on the surface of a gold electrode (AuE) through a gold sulfur bond (Au–S). The specific binding of kanamycin with its aptamer induced an enzyme-assisted SDA process, leading to the generation of output probes (OP) which can open the locked DW-LP. And then, in the presence of Mn2+, the unlocked DW walked along the HP on the electrode surface cutting the loop of HP, so that the HP was consumed to form a G-quadruplex (G4) in the presence of K+. G4/hemin has peroxidase-like activity and can catalyze the reduction of H2O2 to produce obvious electrical signals. Upon the optimization of the analytical conditions, the proposed biosensor showed a wide linear range from 100 fM–5 nM with a limit of detection (LOD) of 87.1 fM. In addition, the aptasensor also had good stability and specificity, showing good prospects in practical applications.
In this study, a novel dual-switch fluorescent biosensor based on a bipedal DNAzyme walker was developed for the highly sensitive detection of the tumour biomarker miRNA-141. By exploiting the typically elevated expression levels of miRNA-141 and apurinic/apyrimidinic endonuclease 1 (APE1) in tumour cells, an endogenous dual-gating system was constructed. The presence of both targets was required to initiate an enzyme-free strand displacement reaction, enabling target recycling and the subsequent release of bipedal DNAzyme walker components (D1/D2), which were efficiently isolated using streptavidin-modified magnetic beads. In the presence of Mg2+, the activated DNAzyme walker specifically cleaved fluorophore-labelled substrate strands on gold nanoparticles (AuNPs). Through repeated“walking–cleavage”cycles, fluorescence signals were progressively restored and amplified. By integrating AuNPs as carriers and magnetic separation technology, a cascade dual-amplification system based on non-enzymatic strand displacement and bipedal DNAzyme walking was established, markedly enhancing the detection sensitivity. The biosensor exhibited dual-switch-specific activation, effectively suppressing interference from non-target RNAs and other nucleases, and maintained a low background signal. It demonstrated excellent discrimination between tumour cells and normal cells, and showed good anti-interference performance in spiked serum samples. This study provides a new strategy for the development of high-precision tumour biomarker detection, offering potential for trace analysis in complex biological samples.
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