Detection of KRAS G12D point mutation level by anchor-like DNA electrochemical biosensor
Detection of KRAS G12D point mutation level by anchor-like DNA electrochemical biosensor
复制标题
锚样DNA电化学生物传感器检测KRAS G12D点突变水平
DOI：
10.1016/j.talanta.2019.01.105
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发表时间：
2019
期刊：
影响因子：
6.1
通讯作者：
Xiang Juan
中科院分区：
文献类型：
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作者：
Zeng Ni;Xiang Juan
KRAS G12D点突变在非小细胞肺癌（NSCLC）以及结直肠癌、胰腺癌和乳腺癌的发病中起重要作用。在这里，我们开发了一种新型锚样DNA（alDNA）电化学传感器，用于检测KRAS点突变水平（特定KRAS点突变DNA（M-DNA）与携带相同等位基因的突变型和野生型DNA的总DNA（t-DNA）的浓度比）。与传统的基于连接的DNA传感器相比，所提出的alDNA传感器实现了野生型和突变型DNA的一步捕获，并在一块芯片上进行后续检测，有效减少了由于界面结构和微环境变化的不确定差异而产生的系统误差，提高了检测精度。此外，使用甲酰胺-NaCl溶液，包括双链DNA变性的整个检测过程在温和的温度条件（~40°C）下进行。大大降低了成本，提高了方法的重现性和可行性。在此基础上，分别实现了t-DNA在0.1pM至10nM范围内和M-DNA在100pM至10nM范围内与浓度对数的线性相关性。对 t-DNA 和 M-DNA 掺入的人血清样品的分析表明该方法具有很高的准确性。这种alDNA传感器方便、低成本、省时，具有宽动态范围、高灵敏度和选择性。能够满足临床对血样中KRAS点突变水平的检测需求，对于NSCLC的早期诊断、个体化治疗和疗效评价具有重要意义。
KRAS G12D point mutation plays an important role in the incidence of non-small-cell lung cancer (NSCLC) as well as colorectal cancer, pancreatic cancer and breast cancer. Here, we developed a novel anchor-like DNA (alDNA) electrochemical sensor for the detection of KRAS point mutation level (the concentration ratio of the specific KRAS point mutant DNA (M-DNA) to the total DNA (t-DNA) of both mutant and wild-type DNAs carrying the same allele). Compared to those conventional ligation-based DNA sensors, the proposed alDNA sensor achieved the one-step capture of both wild-type and mutant DNA, and the subsequent detection on one chip, which effectively decreased systematic errors from the uncertain differences on the interfacial structural and microenvironmental changes, improved the detection accuracy. Also, with the usage of formamide-NaCl solution, the whole detection process including the denaturation of double-stranded DNA was performed under mild temperature conditions (~40 °C). It greatly decreased the cost and improved the reproducibility and feasibility of the method. Based on it, the linear correlations with the logarithm of concentration in wide range from 0.1 pM to 10 nM for t-DNA and from 100 pM to 10 nM for M-DNA were achieved, respectively. The analysis of t-DNA- and M-DNA-spiked human serum samples revealed the high accuracy of the method. This alDNA sensor is convenient, low-cost and time-saving, with broad dynamic range, high sensitivity and selectivity. It can meet the demands of the clinical detection on the KRAS point mutation level in blood samples, which means significant for the earlier NSCLC diagnosis, individualized treatment, and therapeutic efficacy evaluation.