Dong Dong, Zhang Jizhao, Zhang Runshi, Li Fang, Li Yueguo, Jia Yunfang
Department of Laboratory, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, National Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China.
College of Electronic Information and Optical Engineering, Nankai University, Tianjin 300071, China.
ACS Omega. 2020 Jun 25;5(26):16228-16237. doi: 10.1021/acsomega.0c02022. eCollection 2020 Jul 7.
The hypermethylation in the promoter region of the gene is associated with the development of colorectal cancer (CRC). Although its clinical significance for early diagnosis and screening of CRC has been demonstrated, the tedious operations in the conventional DNA methylation (DNAm) detection hinder its wide application. Herein, an electronic method for determining methylation in CRC patients is proposed by using the carbon dot-modified liquid exfoliated graphene field effect transistor (CDs-LEG-FET) as the DNAm sensor, the specifically designed probes to capture the gene and the immunologic recognition to recognize 5-methylcytosine (5mC) positions on the anchored sequences. The identification and nanomorphology of the as-prepared materials and devices are executed first by the characterizations of UV-vis, Raman, atomic force microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and electronic measurements. Then, the role of CDs in enhancing DNAm sensitivity of CD-LEG-FET is manifested by comparing it with that of CD-free LEG-FET. Third, the captured genes on CD-LEG-FETs by different probes are evaluated, and the optimized temperature for hybridizing the target ssDNA sequences is determined to be 48 °C. Furthermore, the detection sensitivity for the low-quantity of DNA samples is demonstrated to be as low as 2 ng. Finally, the methylation degree of the tumor and corresponding noncancerous tissue DNA samples were examined by the proposed electric method and methylight assay in parallel. The diagnostic value of the electrical assay is confirmed by using the receiver operating characteristic curves; meanwhile, the superiority of the CD-LEG-FET platform is found to present a methylation panorama of the target gene.
该基因启动子区域的高甲基化与结直肠癌（CRC）的发生发展相关。尽管其在CRC早期诊断和筛查中的临床意义已得到证实，但传统DNA甲基化（DNAm）检测操作繁琐，阻碍了其广泛应用。在此，提出了一种用于测定CRC患者DNA甲基化的电子方法，该方法使用碳点修饰的液相剥离石墨烯场效应晶体管（CDs-LEG-FET）作为DNAm传感器，通过专门设计的探针捕获该基因，并通过免疫识别来识别锚定序列上的5-甲基胞嘧啶（5mC）位点。首先通过紫外可见光谱、拉曼光谱、原子力显微镜、傅里叶变换红外光谱、X射线光电子能谱和电学测量等表征手段对所制备的材料和器件进行识别和纳米形态分析。然后，通过将其与无CD的LEG-FET进行比较，揭示了CD在增强CD-LEG-FET的DNAm检测灵敏度方面的作用。第三，评估了不同探针在CD-LEG-FET上捕获的该基因，确定目标单链DNA序列杂交的最佳温度为48℃。此外，还证明了对低量DNA样品的检测灵敏度低至2 ng。最后，通过所提出的电学方法和甲基化特异性荧光定量PCR分析并行检测肿瘤组织和相应癌旁组织DNA样品的甲基化程度。通过绘制受试者工作特征曲线证实了电学检测的诊断价值；同时，发现CD-LEG-FET平台的优势在于能够呈现目标基因的甲基化全景图。
