AVS 60th International Symposium and Exhibition
    Biomaterial Interfaces Thursday Sessions
       Session BI+AS+BA+NS+SS-ThA

Paper BI+AS+BA+NS+SS-ThA12
Microfluidic Extraction and Labeling of Methylated DNA from Small Cell Populations for Single-Molecule Analysis

Thursday, October 31, 2013, 5:40 pm, Room 102 B

Session: Biomolecules at Interfaces
Presenter: J. Benitez, Cornell University
Authors: J. Benitez, Cornell University
J. Topolancik, Cornell University
H. Tian, Cornell University
C. Wallin, Cornell University
V. Adiga, Cornell University
P. Murphy, Cornell University
J. Hagarman, Cornell University
P. Soloway, Cornell University
H.G. Craighead, Cornell University
Correspondent: Click to Email
We describe a microfluidic device for the extraction, labeling, and purification of human chromosomal DNA from single cells and small cell populations. The extracted and labeled material was quantified using single-molecule fluorescence analysis in nanofluidic channels. A two-dimensional array of micropillars in a microfluidic polydimethylsiloxane (PDMS) channel was designed to capture cells. Megabase-long DNA strands released from the cell upon lysis are trapped in the micropillar array and stretched under optimal hydrodynamic flow conditions. Chromosomal DNA is immobilized in the array, while other cellular components are washed away from the channel. To assess DNA methylation, genomic DNA from different cell types was extracted using the device and labeled on-chip with methyl-CpG binding domain 1 (MBD1) protein. MBD1-bound DNA was released from the device and directly transferred to a nanofluidic channel for single-molecule detection of MBD1 molecules. Individual DNA fragments and MBD1 proteins were driven electrophoretically through the nanofluidic channels. The photon counts obtained from each MBD1 detection event are directly proportional to the total number of MBD1 molecules. By quantifying the amount of bound MBD1 molecules, the DNA methylation abundance of each cell type can be assessed and compared. This methodology provides a means for epigenetic fluorescence analysis of small cell populations with single-molecule resolution, extendable to single cells.