College of Chemistry and Material Science, Shandong Agricultural University, 271018, Taian, Shandong, People's Republic of China.
College of Resource and Environment, Shandong Agriculture University, 271018, Taian, Shandong, People's Republic of China.
Mikrochim Acta. 2020 Feb 5;187(3):156. doi: 10.1007/s00604-020-4131-0.
A photoelectrochemical method was proposed for DNA hydroxymethylation determination using black TiO (B-TiO) nanosphere as photoactive material and ZnO as photoactivity inhibitor. After hydroxymethylated DNA (5hmC-DNA) was captured on the probe modified B-TiO/ITO electrode surface through hybridization, a glycosyl can be then transferred from uridine diphosphoglucose to 5hmC-DNA and formed a covalent structure with -CHOH in the presence of T4 β-glucosyltransferase (β-GT). Afterwards, based on a series of covalent reaction, amino functionalized ZnO nanoparticles are further immobilized to the surface of the electrode. Due to the capacity to expend the irradiation light and the photogenerated electron of electron donor, the modified ZnO nanoparticles can result in a decreased photocurrent. The developed method shows wide linear ranges from 0.05-200 nM for hydroxymethylated DNA and 1-220 unit·mL for T4-β-glucosyltransferase. The corresponding determination limits were 0.013 nM and 0.24 unit·mL, respectively. The enzyme activity inhibited by 4-phenylimidazole was evaluated. This photoelectrochemical method shows high specificity for 5hmC-DNA (compared to 5fC, 5mC, m6A, control) and β-GT (compared to β-AGT, UGT2B7), and shows excellent stability for testing 5hmC (RSD = 2.75%). Graphical abstractSchematic representation of photoelectrochemical method for DNA hydroxymethylation and β-glucosyltransferase detection based on the glycosylation reaction of -CHOH in 5-hydroxymethylcytosine and the inhibition activity of ZnO to the photoactivity of black TiO nanospheres.
提出了一种光电化学方法，用于使用黑 TiO（B-TiO）纳米球作为光活性材料和 ZnO 作为光活性抑制剂来测定 DNA 羟甲基化。在通过杂交将羟甲基化 DNA（5hmC-DNA）捕获到探针修饰的 B-TiO/ITO 电极表面之后，在 T4β-葡萄糖基转移酶（β-GT）的存在下，糖苷可以从尿苷二磷酸葡萄糖转移到 5hmC-DNA 并与-CHOH 形成共价结构。之后，基于一系列共价反应，进一步将氨基功能化的 ZnO 纳米颗粒固定到电极表面。由于可以扩展辐照光的范围并消耗电子供体的光生电子，因此修饰的 ZnO 纳米颗粒会导致光电流减小。开发的方法对于羟甲基化 DNA 的线性范围从 0.05-200 nM 宽，对于 T4-β-葡萄糖基转移酶的线性范围从 1-220 单位·mL-1。相应的测定下限分别为 0.013 nM 和 0.24 单位·mL-1。评估了被 4-苯并咪唑抑制的酶活性。与 5fC、5mC、m6A、对照相比，该光电化学方法对 5hmC-DNA 具有高特异性（与 5fC、5mC、m6A、对照相比），并且对β-GT（与β-AGT、UGT2B7 相比）具有优异的稳定性，用于检测 5hmC（RSD=2.75%）。示意图表示基于-CHOH 在 5-羟甲基胞嘧啶中的糖基化反应和 ZnO 对黑 TiO 纳米球光活性的抑制活性的用于 DNA 羟甲基化和β-葡萄糖基转移酶检测的光电化学方法。
