Department of Chemistry, Bangladesh University of Engineering and Technology (BUET), Dhaka 1000, Bangladesh.
Queensland Micro- and Nanotechnology Centre (QMNC), Griffith University, Nathan Campus, QLD 4111, Australia.
Analyst. 2021 Jun 7;146(11):3654-3665. doi: 10.1039/d1an00345c. Epub 2021 May 5.
The quantification of global 5-methylcytosine (5mC) content has emerged as a promising approach for the diagnosis and prognosis of cancers. However, conventional methods for the global 5mC analysis require large quantities of DNA and may not be useful for liquid biopsy applications, where the amount of DNA available is limited. Herein, we report magnetic nanoparticles-assisted methylated DNA immunoprecipitation (e-MagnetoMethyl IP) coupled with electrochemical quantification of global DNA methylation. Carboxyl (-COOH) group-functionalized iron oxide nanoparticles (C-IONPs) synthesized by a novel starch-assisted gel formation method were conjugated with anti-5mC antibodies through EDC/NHS coupling (anti-5mC/C-IONPs). Anti-5mC/C-IONPs were subsequently mixed with DNA samples, in which they acted as dispersible capture agents to selectively bind 5mC residues and capture the methylated fraction of genomic DNA. The target-bound Anti-5mC/C-IONPs were magnetically separated and directly adsorbed onto the gold electrode surface using gold-DNA affinity interaction. The amount of DNA adsorbed on the electrode surface, which corresponds to the DNA methylation level in the sample, was electrochemically estimated by differential pulse voltammetric (DPV) study of an electroactive indicator [Ru(NH)] bound to the surface-adsorbed DNA. Using a 200 ng DNA sample, the assay could successfully detect differences as low as 5% in global DNA methylation levels with high reproducibility (relative standard deviation (% RSD) = <5% for n = 3). The method could also reproducibly analyze various levels of global DNA methylation in synthetic samples as well as in cell lines. The method avoids bisulfite treatment, does not rely on enzymes for signal generation, and can detect global DNA methylation using clinically relevant quantities of sample DNA without PCR amplification. We believe that this proof-of-concept method could potentially find applications for liquid biopsy-based global DNA methylation analysis in point-of-care settings.
全球 5-甲基胞嘧啶（5mC）含量的定量分析已成为癌症诊断和预后的一种有前途的方法。然而，传统的全基因组 5mC 分析方法需要大量的 DNA，并且可能不适用于液体活检应用，因为可用的 DNA 量有限。在此，我们报告了一种基于磁性纳米粒子辅助的甲基化 DNA 免疫沉淀（e-MagnetoMethyl IP）与电化学定量检测全基因组 DNA 甲基化相结合的方法。采用一种新型的淀粉辅助凝胶形成方法合成的羧基（-COOH）功能化氧化铁纳米粒子（C-IONPs）通过 EDC/NHS 偶联（anti-5mC/C-IONPs）与抗 5mC 抗体偶联。随后，将抗 5mC/C-IONPs 与 DNA 样品混合，在其中它们作为可分散的捕获剂选择性地结合 5mC 残基并捕获基因组 DNA 的甲基化部分。目标结合的抗 5mC/C-IONPs 通过金-DNA 亲和相互作用被磁性分离，并直接吸附到金电极表面。通过与表面吸附的 DNA 结合的电化学活性指示剂[Ru(NH3)6]2+的差分脉冲伏安法（DPV）研究，定量估计吸附在电极表面上的 DNA 量，该量对应于样品中的 DNA 甲基化水平。使用 200ng DNA 样品，该测定法可以成功地检测到低至 5%的全基因组 DNA 甲基化水平的差异，并且具有高重现性（n = 3，相对标准偏差（%RSD）<5%）。该方法还可以在合成样品以及细胞系中重复分析各种水平的全基因组 DNA 甲基化。该方法避免了亚硫酸氢盐处理，不依赖于酶产生信号，并且可以在不进行 PCR 扩增的情况下使用临床相关量的样本 DNA 检测全基因组 DNA 甲基化。我们相信，这种概念验证方法有可能在即时护理环境中用于基于液体活检的全基因组 DNA 甲基化分析。
