Soda Narshone, Gonzaga Zennia Jean, Pannu Amandeep Singh, Kashaninejad Navid, Kline Richard, Salomon Carlos, Nguyen Nam-Trung, Sonar Prashant, Rehm Bernd H A, Shiddiky Muhammad J A
School of Environment and Science (ESC), Griffith University, Nathan Campus, Nathan, QLD 4111, Australia.
Queensland Micro- and Nanotechnology Centre (QMNC), Griffith University, Nathan Campus, Nathan, QLD 4111, Australia.
Cancers (Basel). 2021 Jul 27;13(15):3787. doi: 10.3390/cancers13153787.
DNA methylation is a cell-type-specific epigenetic marker that is essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. It is also responsible for the pathogenesis of many diseases, including cancers. Herein, we present a simple approach for quantifying global DNA methylation in ovarian cancer patient plasma samples based on a new class of biopolymer nanobeads. Our approach utilises the immune capture of target DNA and electrochemical quantification of global DNA methylation level within the targets in a three-step strategy that involves (i) initial preparation of target single-stranded DNA (ss-DNA) from the plasma of the patients' samples, (ii) direct adsorption of polymer nanobeads on the surface of a bare screen-printed gold electrode (SPE-Au) followed by the immobilisation of 5-methylcytosine (5mC)-horseradish peroxidase (HRP) antibody, and (iii) immune capture of target ss-DNA onto the electrode-bound PHB/5mC-HRP antibody conjugates and their subsequent qualification using the hydrogen peroxide/horseradish peroxidase/hydroquinone (HO/HRP/HQ) redox cycling system. In the presence of methylated DNA, the enzymatically produced (in situ) metabolites, i.e., benzoquinone (BQ), binds irreversibly to cellular DNA resulting in the unstable formation of DNA adducts and induced oxidative DNA strand breakage. These events reduce the available BQ in the system to support the redox cycling process and sequel DNA saturation on the platform, subsequently causing high Coulombic repulsion between BQ and negatively charged nucleotide strands. Thus, the increase in methylation levels on the electrode surface is inversely proportional to the current response. The method could successfully detect as low as 5% methylation level. In addition, the assay showed good reproducibility (% RSD ≤ 5%) and specificity by analysing various levels of methylation in cell lines and plasma DNA samples from patients with ovarian cancer. We envision that our bioengineered polymer nanobeads with high surface modification versatility could be a useful alternative platform for the electrochemical detection of varying molecular biomarkers.
DNA甲基化是一种细胞类型特异性的表观遗传标记，对转录调控、重复DNA沉默和基因组印记至关重要。它也与包括癌症在内的许多疾病的发病机制有关。在此，我们基于一类新型生物聚合物纳米珠，提出了一种简单的方法来定量卵巢癌患者血浆样本中的整体DNA甲基化。我们的方法采用三步策略，通过免疫捕获目标DNA并对目标中的整体DNA甲基化水平进行电化学定量，这三步包括：(i) 从患者样本的血浆中初步制备目标单链DNA (ss-DNA)；(ii) 将聚合物纳米珠直接吸附在裸丝网印刷金电极 (SPE-Au) 表面，随后固定5-甲基胞嘧啶 (5mC)-辣根过氧化物酶 (HRP) 抗体；(iii) 将目标ss-DNA免疫捕获到电极结合的PHB/5mC-HRP抗体偶联物上，并随后使用过氧化氢/辣根过氧化物酶/对苯二酚 (HO/HRP/HQ) 氧化还原循环系统对其进行定量。在存在甲基化DNA的情况下，酶促产生的（原位）代谢产物，即苯醌 (BQ)，与细胞DNA不可逆地结合，导致DNA加合物的不稳定形成并诱导氧化性DNA链断裂。这些事件减少了系统中可用于支持氧化还原循环过程的可用BQ，并导致平台上的DNA饱和，随后导致BQ与带负电荷的核苷酸链之间产生高库仑排斥。因此，电极表面甲基化水平的增加与电流响应成反比。该方法能够成功检测低至5% 的甲基化水平。此外，通过分析卵巢癌患者细胞系和血浆DNA样本中的各种甲基化水平，该检测方法显示出良好的重现性（% RSD ≤ 5%）和特异性。我们设想，我们具有高表面修饰通用性的生物工程聚合物纳米珠可能是用于电化学检测各种分子生物标志物的有用替代平台。
