Tetrahedral DNA nanostructure-corbelled click chemistry-based large-scale assembly of nanozymes for ratiometric fluorescence assay of DNA methyltransferase activity†

Ligation efficiency in a surface-based DNA click chemistry (CuAAC) reaction is extremely restricted by the orientation and density of probes arranged on a heterogeneous surface. Herein, we engineer DNA tetrahedral nanostructure (DTN)-corbelled click chemistry to trigger a hybridization chain reaction (HCR) assembling a large-scale of nanozymes for ratiometric fluorescence detection of DNA adenine methyltransferase (Dam). In this study, a DNA tetrahedron structure with an alkynyl modifying pendant DNA probe (Alk-DTN) is designed and assembled on a magnetic bead (MB) as a scaffold for click chemistry. When a CuO NP-encoded magnetic nanoparticle (CuO-MNP) substrate was methylated by Dam, CuO NPs were released and turned into a mass of Cu⁺. The Cu⁺ droves azido modifying lDNA (azide-lDNA) to connect with the Alk-DTN probe on the MB through the click reaction, forming an intact primer to initiate the HCR. The HCR product, a rigid structure double-stranded DNA, periodically assembles glucose oxidase mimicking gold nanoparticles (GNPs) into a large-scale of nanozymes for catalyzing the oxidation of glucose to H₂O₂. NH₂-MIL-101 MOFs, a fluorescent indicator and a biomimetic catalyst, activated the product H₂O₂ to oxidize o-phenylenediamine (oPD) into visually detectable 2,3-diaminophenazine (DAP). The change of the signal ratio between DAP and NH₂-MIL-101 is proportional to the methylation event corresponding to the MTase activity. In this study, the DTN enhances the efficiency of the surface-based DNA click reaction and maintains the catalytic activities of gold nanoparticle nanozymes due to the intrinsic nature of mechanical rigidity and well-controlled orientation and well-adjusted size. Large-scale assembly of nanozymes circumvents the loss of natural enzyme activity caused by chemical modification and greatly improves the amplification efficiency. The proposed biosensor displayed a low detection limit of 0.001 U mL⁻¹ for Dam MTase due to multiple amplification and was effective in real samples and methylation inhibitor screening, providing a promising modular platform for bioanalysis.