Department of Chemistry, University of Patras, 26504, Rio, Patras, Greece.
Department of Anatomy-Histology-Embryology, Medical School, University of Patras, 26504, Patras, Greece.
Anal Chim Acta. 2021 Jun 8;1163:338470. doi: 10.1016/j.aca.2021.338470. Epub 2021 Apr 12.
Liquid biopsy, as a minimally invasive method that allows real-time monitoring of the tumor genome, represents a competing approach for cancer diagnosis, prognosis and therapy decision making. Liquid biopsy in cancer patients mainly includes analysis of circulating tumor cells (CTC) and cell-free circulating tumor DNA (ctDNA). ctDNA is the tumor-derived fraction of the cell-free DNA present in the blood. ctDNA is detected based on cancer-specific genomic aberrations (mainly mutations) and represents a challenging analyte due to high fragmentation and low concentration. Several methodologies have been developed for ctDNA analysis in cancer patients but many of these technologies are too time-intensive, complicated and expensive for implementation in diagnostic testing. Herein, we developed a novel lateral flow strip assay for mutational analysis of ctDNA in blood samples and visual detection that is based on gold nanoparticles as reporters. As a model, common single-point mutations of the KRAS gene, related to colorectal cancer (CRC), have been selected for method development. The proposed DNA biosensor has been successfully applied for the detection of three KRAS mutations (KRAS G12D/A/V), along with the wild-type KRAS gene in synthetic DNA targets, cancer cell lines and cfDNA from blood samples of healthy individuals and CRC patients. The main advantages of the proposed lateral flow assay are simplicity, rapid analysis time (∼10 min) and visual detection without the requirement of special instrumentation. The assay is also cost-effective with high detectability, specificity and reproducibility and has the potential to be used as a portable and universal device. In conclusion, the proposed assay offers a rapid diagnostic strip test for visual genotyping, as an alternative approach for liquid biopsy applications.
液体活检作为一种微创方法，可以实时监测肿瘤基因组，是癌症诊断、预后和治疗决策的一种竞争方法。癌症患者的液体活检主要包括循环肿瘤细胞（CTC）和游离循环肿瘤 DNA（ctDNA）的分析。ctDNA 是血液中存在的无细胞游离 DNA 中肿瘤来源的部分。ctDNA 是基于癌症特异性基因组异常（主要是突变）检测到的，由于高度碎片化和低浓度，它是一种具有挑战性的分析物。已经开发了几种用于癌症患者 ctDNA 分析的方法，但这些技术中的许多方法对于诊断测试来说太耗时、复杂和昂贵。在此，我们开发了一种新的侧向流动条带分析用于血液样本中 ctDNA 的突变分析和基于金纳米粒子作为报告物的可视化检测。作为模型，选择了与结直肠癌（CRC）相关的 KRAS 基因的常见单点突变用于方法开发。所提出的 DNA 生物传感器已成功应用于检测三种 KRAS 突变（KRAS G12D/A/V）以及合成 DNA 靶标、癌细胞系和来自健康个体和 CRC 患者血液样本的 cfDNA 中的野生型 KRAS 基因。该侧向流动分析的主要优点是简单、快速的分析时间（约 10 分钟）和可视化检测，无需特殊仪器。该测定还具有成本效益，具有高检测能力、特异性和重现性，并且有可能用作便携式通用设备。总之，该测定提供了一种用于视觉基因分型的快速诊断条带测试，是液体活检应用的一种替代方法。
