AVS 58th Annual International Symposium and Exhibition
    Biomaterial Interfaces Division Monday Sessions
       Session BI-MoA

Paper BI-MoA11
Microfluidic Extraction of Human Chromosomal DNA from Single Cells

Monday, October 31, 2011, 5:20 pm, Room 108

Session: Sensors and Fluidics for Biomedical Applications
Presenter: Juraj Topolancik, Cornell University
Authors: J. Topolancik, Cornell University
H.C. Tian, Cornell University
C.B. Wallin, Cornell University
D.R. Latulippe, Cornell University
J.J. Benítez, Cornell University
B.R. Cipriany, Cornell University
P.J. Murphy, Cornell University
P.D. Soloway, Cornell University
H.G. Craighead, Cornell University
Correspondent: Click to Email
Genome-wide analysis of single cells is important in life science research and modern medicine in applications ranging from cancer diagnosis to understanding tissue development. Microfluidic devices have been explored as a promising platform for single cell studies, providing superior handling of minute sample and reagent volumes in engineered microstructures. Isolation of nucleic acids from biological samples is an essential step of every type of genetic analysis. While numerous extraction methods have been explored, it remains rather challenging to isolate and analyze genomic DNA from small cell populations and individual cells. Traditional microfluidic devices utilize solid phase extraction (SPE), a method based on binding of DNA to chemically functionalized solid phase matrices for separation of nucleic acids from cell lysates. The binding affinity is sensitive to factors such as pH, temperature, and buffer composition which must be controlled, often dynamically, to minimize DNA losses. Even when the extraction process is optimized, it is difficult to ensure that all of the DNA fragments are adsorbed on the solid phase matrix and that the whole genome is represented in the purified extracts. An appreciable fraction of genomic DNA is often lost during the purification process when the cell debris is washed away. Additional DNA losses can be caused by incomplete elution. State-of-the-art microfluidic devices for DNA separation from cell lysates exhibit rather modest extraction efficiencies of 60-85%. This is sufficient for genetic analysis of cell populations because multiple copies of every gene are present in the extract, which statistically guarantees complete genome coverage, but such losses are hardly acceptable when single-copy genes in a single cell need to be investigated. This work describes a valveless two-port microfluidic device for highly-efficient isolation and fluorescent analysis of DNA contents of single cells. Long strands of human chromosomal DNA released from the cell by chemical lysis loop around PDMS micropillars and are physically retained while the remaining cellular contents are washed away under hydrodynamic flow. DNA fragmentation is minimized by operating at low flow rates. Hydrodynamic entrapment of DNA in non-functionalized obstacle arrays allows separation of very large genomic DNA from cell debris and components such as proteins and membrane fragments as well as from much smaller mitochondrial DNA and RNA. The purified DNA was subsequently released from the device by enzymatic fragmentation with restriction endonucleases under continuous flow and collected for fragment-size analysis and evaluation of the extraction efficiency. Fluorospectrometric measurements indicate that the microdevice extracts >95% of genomic DNA, which outperforms all alternative microchip-based extraction methods.