Precision Medicine Center, Beilun People's Hospital, Zhejiang University School of Medicine First Affiliated Hospital Beilun Branch, Ningbo, Zhejiang 315806, P. R. China.
Analyst. 2020 Jul 7;145(13):4671-4679. doi: 10.1039/d0an00591f. Epub 2020 May 27.
Genotyping of the epidermal growth factor receptor (EGFR) mutation status is of great importance in the screening of appropriate patients with advanced non-small cell lung carcinoma (NSCLC) to receive superior tyrosine kinase inhibitor (TKIs) therapy. Yet conventional assays are generally costly with a relatively long turnaround time for obtaining results, which can lead to a bottleneck for immediately starting TKI therapy in late-staged patients. In this study, we propose an on-site electrochemical platform for sensitive simultaneous genotyping of the two major EGFR mutations (19del and L858R) through plasma ctDNA based on tetrahedral DNA nanostructure decorated screen-printed electrodes (SPE). Linear-after-the-exponential (LATE)-PCR combined with the amplification refractory mutation system (ARMS) was adopted to produce abundant biotin-labeled single-stranded DNA with high amplification efficiency and specificity. Disposable SPE decorated with self-assembled tetrahedral nanostructured DNA probes that showed ordered orientation and good target accessibility enabled the highly efficient hybridization of the specific amplicons through a sandwich-type and quantitatively translated the interfacial hybridization event into electrochemical signals via enzymatic amplification. Taking advantage of the ARMS-based LATE-PCR and the tetrahedral nanostructure-decorated SPE platform, we achieved the accurate detection of around 30 pg DNA of 19del or L858R, or as low as 0.1% of them in the presence of wild-type DNA. Moreover, the EGFR mutation profiles of 13 NSCLC patients we enlisted were accurately genotyped by our electrochemical platform, the results of which were in good agreement with those of commercial genetic detection methods.
表皮生长因子受体 (EGFR) 基因突变的基因分型对于筛选晚期非小细胞肺癌 (NSCLC) 患者接受更优的酪氨酸激酶抑制剂 (TKI) 治疗非常重要。然而，传统的检测方法通常成本较高，获得结果的周转时间相对较长，这可能导致晚期患者无法立即开始 TKI 治疗。在这项研究中，我们提出了一种基于等离子体 ctDNA 的现场电化学平台，通过四面体 DNA 纳米结构修饰的丝网印刷电极 (SPE) 对两种主要 EGFR 突变 (19del 和 L858R) 进行敏感的同时基因分型。线性后指数 (LATE)-PCR 结合扩增不可避免突变系统 (ARMS) 被用来产生丰富的生物素标记的单链 DNA，具有高扩增效率和特异性。通过自组装四面体纳米结构 DNA 探针修饰的一次性 SPE 显示出有序的取向和良好的靶标可及性，通过三明治型高效杂交特异性扩增子，并通过酶促扩增定量转化界面杂交事件为电化学信号。利用基于 ARMS 的 LATE-PCR 和四面体纳米结构修饰的 SPE 平台，我们实现了约 30pg 19del 或 L858R 或其在野生型 DNA 中低至 0.1%的 DNA 的准确检测。此外，我们通过电化学平台准确地对 13 名 NSCLC 患者的 EGFR 突变谱进行了基因分型，结果与商业遗传检测方法一致。
