Nanotechnology for detection of DNA methylation in cancer.

摘要

Methylation of cytosines often occurs at tumor suppressor genes (TSG) and is correlated with an increased risk for tumorigenesis. Such epigenetic alterations precede the formation of tumors and, therefore, can serve as potential biomarkers for use in early cancer detection, prognostic assessment of tumor, and responsiveness to therapy. This creates a tremendous need for a robust epigenetic assay that can be used in research laboratories and allow the transition of such tests into clinical management of cancer. Current available methods to detect methylation lack the sensitivity for direct screening of challenging clinical samples (such as serum, sputum, polyps, PanINs) where target DNA quantity is low. To overcome the above obstacles, this thesis discusses the development of new methylation detection technologies that detects methylation status through quantum dots (QDs)-mediated fluorescence resonance energy transfer (FRET) technology (QD-FRET) and analyzes methylation density through fluorescence measurements. In addition, with the use of silica superparamagnetic beads (SSBs), the thesis presents a way to integrate the disjoint processes involved in methylation detection (DNA extraction, bisulfite conversion, methylation analysis) into a single-tube process that could be compatible with robotic or microfluidic-based analysis. The thesis then illustrates another technique wherein methylation density information can be obtained through fluorescence detection. The new technological innovations aim to enable ultrasensitive DNA methylation detection in challenging clinical samples and simple single-tube analysis. Many of these methods are applied to detecting DNA methylation in clinical samples (sputum, serum, plasma). The preliminary studies from these patients highlight the applicability of the developed detection techniques in clinic.