基于连接酶链式反应的超灵敏电化学生物传感器检测非小细胞肺癌EGFR药物靶点的研究

Non small cell lung cancer (NSCLC) is a serious hazard to human health. Tyrosine kinase inhibitors (TKIs), a kind of targeted drug for the treatment of NSCLC, are targeted for epidermal growth factor receptor (EGFR). For scientifically selecting a reasonable individualized-medication plan, guidelines for the treatment of NSCLC recommend the detection of EGFR gene mutations prior to TKIs’ therapy. However, the major testing technologies including DNA sequencing, amplification refractory mutation system, polymerase chain reaction, due to low sensitivity and accuracy, high cost, and time consuming, respectively, limit their clinical application. This project presents a new electrochemical biosensor based on ligase chain reaction (LCR) amplification and double-strand DNA-assembling mode. A large quantity of dsDNAs generated by homogeneous LCR were preciously controlled on the electrode surface to produce electrochemical signals, with a detection limit of 15 copies of DNA, a wide linear range from 1.0×10-16 to 1.5×10-11 M, as well as the ability to analyze single nucleotide polymorphism (SNP). Therefore, the proposed method possessing both ultra-high sensitivity and ultra-high selectivity showed excellent detection performance and clinical perspectives. With the advantages of high sensitivity, accuracy, simplicity and low cost for detection of EGFR gene mutations for patients with NSCLC, it is expected to establish a new avenue to meet the clinically urgent demands of NSCLC and provide importantly scientific guidance for clinically precision medicine and individualized medication.

非小细胞肺癌（NSCLC）严重危害人类健康。小分子酪氨酸激酶抑制剂（TKIs）为治疗NSCLC的分子靶向药物，其药物靶点为表皮生长因子受体（EGFR）。肺癌治疗指南规定进行分子靶向药物治疗前须进行EGFR药物靶点的基因突变检测，以科学选择合理的个体化用药方案。EGFR基因突变检测方法如DNA测序法和PCR等技术，分别存在灵敏度或准确度低、操作繁琐、耗时等问题，限制了在临床中的应用。本项目首次提出一种基于连接酶链式反应（LCR）的超灵敏双链组装型电化学生物传感器，通过均相LCR扩增产生的大量短dsDNA在电极表面精确组装产生电化学信号，其检测限可低至15个目标DNA分子，且能实现单核苷酸多态性的分析。本新方法兼具超高灵敏度和特异性，显示极好的临床应用前景。有望针对NSCLC患者，建立超灵敏、准确、简便经济的EGFR基因突变检测新方法，为临床精准医疗及个体化用药提供重要的科学指导。

非小细胞肺癌（NSCLC）严重危害人类健康。小分子酪氨酸激酶抑制剂（TKIs）为治疗NSCLC的分子靶向药物，其药物靶点为表皮生长因子受体（EGFR）。肺癌治疗指南规定进行分子靶向药物治疗前须进行EGFR药物靶点的基因突变检测，以科学选择合理的个体化用药方案。EGFR基因突变检测方法如DNA测序法和PCR等技术，分别存在灵敏度或准确度低、操作繁琐、耗时等问题，限制了在临床中的应用。本项目通过对EGFR基因探针的设计，结合连接酶链式反应与核酸外切酶辅助目标链循环反应两种信号放大技术，构建了多种新型电化学生物传感器，在优化的实验条件下，建立了用于EGRF基因检测的新方法。该方法灵敏准确、简便经济、重现性和稳定性好，将为应用于临床EGFR药物靶点的基因突变检测提供科学研究基础，并为临床精准医疗及个体化用药提供重要的科学指导。