期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2014
卷号：111
期号：42
页码：14985-14989
DOI：10.1073/pnas.1417351111
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceEpigenetic modifications, including DNA methylation, govern gene expression. Aberrant methylation by DNA methyltransferases can lead to tumorigenesis, so that efficient detection of methyltransferase activity provides an early cancer diagnostic. Current methods, requiring fluorescence or radioactivity, are cumbersome; electrochemical platforms, in contrast, offer high portability, sensitivity, and ease of use. We have developed a label-free electrochemical platform to detect the activity of the most abundant human methyltransferase, DNA(cytosine-5)-methyltransferase1 (DNMT1), and have applied this method in detecting DNMT1 in crude lysates from both cultured human colorectal cancer cells (HCT116) and colorectal tissue samples. The role of abnormal DNA methyltransferase activity in the development and progression of cancer is an essential and rapidly growing area of research, both for improved diagnosis and treatment. However, current technologies for the assessment of methyltransferase activity, particularly from crude tumor samples, limit this work because they rely on radioactivity or fluorescence and require bulky instrumentation. Here, we report an electrochemical platform that overcomes these limitations for the label-free detection of human DNA(cytosine-5)-methyltransferase1 (DNMT1) methyltransferase activity, enabling measurements from crude cultured colorectal cancer cell lysates (HCT116) and biopsied tumor tissues. Our multiplexed detection system involving patterning and detection from a secondary electrode array combines low-density DNA monolayer patterning and electrocatalytically amplified DNA charge transport chemistry to measure selectively and sensitively DNMT1 activity within these complex and congested cellular samples. Based on differences in DNMT1 activity measured with this assay, we distinguish colorectal tumor tissue from healthy adjacent tissue, illustrating the effectiveness of this two-electrode platform for clinical applications.
