Highly-selective detection of EGFR mutation gene in lung cancer based on surface enhanced Raman spectroscopy and asymmetric PCR
Highly-selective detection of EGFR mutation gene in lung cancer based on surface enhanced Raman spectroscopy and asymmetric PCR
复制标题
基于表面增强拉曼光谱和不对称PCR的肺癌EGFR突变基因高选择性检测
DOI：
10.1016/j.jpba.2020.113522
复制
发表时间：
2020-10-25
影响因子：
3.4
通讯作者：
Wang, Lei
中科院分区：
文献类型：
--
作者：
Guo, Ting;Li, Weimiao;Wang, Lei
检测肺癌患者外周血中肿瘤DNA（ctDNA）中EGFR突变基因是肺癌诊断的关键。表面增强拉曼散射（Sers）技术在DNA或RNA的痕量检测中具有潜在的应用价值。现有方法的检出率不能满足临床需要。将不对称聚合酶链反应（PCR）与表面增强拉曼光谱（Sers）相结合，建立了一种高选择性检测肺癌EGFR突变基因的方法。采用银晶种法合成了海胆状Au纳米团簇。直径为120 nm的AuNC覆盖有79个纳米棒（20 nm）。然后，EGFR突变特异性分子信标（MBs）与Cy 3标记的AuNCs的表面涂覆。在一个AuNC上MB的负载量计算为5720 +/-740。这些AuNCs探针具有良好的效率（平衡时间：20分钟）、高灵敏度（检测限：5.8 nM）、高特异性（能够识别单碱基错配）和对核酸酶的良好稳定性。随后，进行不对称PCR以获得大量单链DNA（ssDNA，E746-A750 del）。将ssDNA与AuNCs探针一起孵育，并基于AuNCs探针的Sers信号进行定量测试。这种组合的不对称PCR-SERS方法具有非常高的检测阈值（4.24 fM）。在另外15个临床血液样本中，不对称PCR-SERS方法的总体灵敏度为75%，特异性为100%。这种方法被证明是有前途的非侵入性和敏感的检测EGFR突变的ctDNA。(C)2020爱思唯尔B. V.保留所有权利。
The evaluation of EGFR mutation genes in circulating tumor DNA (ctDNA) in blood sample is key for patients with lung cancer. Surface-enhanced Raman scattering (SERS) has potential for trace detection of DNA or RNA. The detection rate offered by current methods can not meet clinical demand. By combining asymmetric polymerase chain reaction (PCR) and SERS, a highly-selective detection for EGFR mutation genes in lung cancer was developed. Sea-urchin like Au nanoclusters (AuNCs) were synthesized via Ag seed-mediated growth. AuNCs with a diameter of 120 nm were covered with 79 nanopricks (20 nm). Then, EGFR mutation specific molecular beacons (MBs) labeled with Cy3 were coated on the surface of AuNCs. The loading amount of MBs was calculated as 5720 +/- 740 on one AuNCs. These AuNCs probes had good efficiency (equilibrium time: 20 minutes) with high sensitivity (detection limit: 5.8 nM), high specificity (capable of single-base mismatch recognition) and good stability against nucleases. Following this, asymmetric PCR was performed to obtain large numbers of single-stranded DNA (ssDNA, E746-A750del). The ssDNA was incubated with the AuNCs probes and tested quantitatively based on the SERS signals of the AuNCs probes. This combined asymmetric PCR-SERS method had a very high detection threshold (4.24 fM). The asymmetric PCR-SERS method was shown to have an overall sensitivity of 75% and specificity of 100% in a further 15 clinical blood samples. This method is proved to be promising for non-invasive and sensitive detection of EGFR mutations in ctDNA. (C) 2020 Elsevier B.V. All rights reserved.