四面体 DNA 纳米结构-基于点击化学的纳米酶大规模组装，用于 DNA 甲基转移酶活性的比例荧光测定,Journal of Materials Chemistry B

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四面体 DNA 纳米结构-基于点击化学的纳米酶大规模组装，用于 DNA 甲基转移酶活性的比例荧光测定
Journal of Materials Chemistry B ( IF 6.1 ) Pub Date : 2023-10-03 , DOI: 10.1039/d3tb01795h
Guohui Cao ₁ , Huiying Jia ₁ , Shuling Xu ₁ , Ensheng Xu ₁ , Pin Wang ₂ , Qingwang Xue ₁ , Huaisheng Wang ₁

Affiliation

基于表面的 DNA 点击化学 (CuAAC) 反应中的连接效率受到异质表面上排列的探针的方向和密度的极大限制。在此，我们设计了DNA四面体纳米结构（DTN）-corbelled点击化学来触发杂交链式反应（HCR），组装大规模纳米酶，用于DNA腺嘌呤甲基转移酶（Dam）的比率荧光检测。在这项研究中，设计了带有炔基修饰悬垂 DNA 探针 (Alk-DTN) 的 DNA 四面体结构，并将其组装在磁珠 (MB) 上，作为点击化学的支架。当 CuO NP 编码的磁性纳米颗粒 (CuO-MNP) 基质被 Dam 甲基化时，CuO NP 被释放并转化为大量 Cu ⁺。Cu ⁺驱使叠氮修饰l DNA（叠氮-l DNA）通过点击反应与MB上的Alk-DTN探针连接，形成完整的引物以启动HCR。HCR产物是一种刚性结构的双链DNA，周期性地将模拟金纳米颗粒(GNP)的葡萄糖氧化酶组装成大规模的纳米酶，用于催化葡萄糖氧化成H ₂ O ₂。NH ₂ -MIL-101 MOFs 是一种荧光指示剂和仿生催化剂，可激活产物 H ₂ O ₂将邻苯二胺 ( o PD)氧化成肉眼可检测的 2,3-二氨基吩嗪 (DAP)。_{DAP和NH 2} -MIL-101之间信号比的变化与MTase活性对应的甲基化事件成正比。在本研究中，由于机械刚性、良好控制的方向和良好调整的尺寸的固有性质，DTN提高了基于表面的DNA点击反应的效率，并保持了金纳米颗粒纳米酶的催化活性。纳米酶的大规模组装避免了化学修饰造成的天然酶活性损失，大大提高了扩增效率。由于多重扩增，所提出的生物传感器对 Dam MTase显示出 0.001 U mL ^-1的低检测限，并且在实际样品和甲基化抑制剂筛选中有效，为生物分析提供了一个有前景的模块化平台。

"点击查看英文标题和摘要"

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.

更新日期：2023-10-03

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