Cai Shixin, Shi Ke, Ji Aohua, Long Yilin, Wang Xiaoli, Zhang Yuting, Zhou Nandi
School of Biotechnology and Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, Jiangnan University, Wuxi 214122, China.
Anal Chem. 2025 Aug 26;97(33):18343-18354. doi: 10.1021/acs.analchem.5c03822. Epub 2025 Aug 15.
Methylation level of circulating tumor DNA (ctDNA) is widely recognized as a pivotal biomarker in precision oncology for tumor characterization and risk assessment. However, current point-of-care testing platforms for methylation analysis are constrained in achieving sufficient detection specificity due to their limitations in single-locus detection and their dependence on complex instrumentation. In this study, to overcome the above limitations, a smartphone-integrated, microfluidic chip technology-based biosensing platform was engineered for simultaneous analysis of multiple methylation loci in ctDNA. In this platform, whole blood samples from suspected individuals underwent GlaI methylation-sensitive enzyme digestion and target-specific primer hybridization, followed by microfluidic chip loading for on-chip colorimetric reactions. Methylation levels at specific loci were quantified through chamber-specific absorbance measurements by using the developed portable biosensing platform. The biosensor achieved simultaneous detection of three specific methylation loci (CpG sites) on the Septin 9 gene, which are associated with colorectal cancer. The detection limits (LODs) for the individual methylation loci are 0.25, 0.4, and 0.525 fM, respectively. This platform demonstrates high accuracy in detecting methylation signatures within blood samples from colorectal cancer patients, showing a significant correlation with conventional diagnostic methods. With its low cost and portable features, the developed biosensing platform may provide an economical solution for early-stage screening of cancer for the healthcare system in remote and under-resourced regions.
循环肿瘤DNA（ctDNA）的甲基化水平被广泛认为是精准肿瘤学中用于肿瘤特征描述和风险评估的关键生物标志物。然而，由于当前用于甲基化分析的即时检测平台在单基因座检测方面存在局限性且依赖复杂仪器，其在实现足够的检测特异性方面受到限制。在本研究中，为克服上述局限性，设计了一种基于智能手机集成的微流控芯片技术的生物传感平台，用于同时分析ctDNA中的多个甲基化基因座。在该平台中，疑似个体的全血样本先进行GlaI甲基化敏感酶消化和靶标特异性引物杂交，然后加载到微流控芯片上进行芯片比色反应。通过使用开发的便携式生物传感平台，通过特定腔室的吸光度测量对特定位点的甲基化水平进行定量。该生物传感器实现了对与结直肠癌相关的Septin 9基因上三个特定甲基化基因座（CpG位点）的同时检测。各个甲基化基因座的检测限分别为0.25、0.4和0.525 fM。该平台在检测结直肠癌患者血液样本中的甲基化特征方面显示出高精度，与传统诊断方法具有显著相关性。凭借其低成本和便携特性，开发的生物传感平台可为资源匮乏的偏远地区医疗系统提供一种经济的癌症早期筛查解决方案。
