DROPLET MICROFLUIDICS FOR COMPARTMENTALIZED CELL …Philip Zimny1, David Juncker1, Walter Reisner2...
Transcript of DROPLET MICROFLUIDICS FOR COMPARTMENTALIZED CELL …Philip Zimny1, David Juncker1, Walter Reisner2...
Philip Zimny1, David Juncker1, Walter Reisner2
DROPLET MICROFLUIDICS FOR COMPARTMENTALIZED CELL LYSIS
AND SINGLE CELL DNA MAPPING
1 Department of Biomedical Engineering, 2 Department of Physics, McGill University, Montreal, Canada, [email protected]
Acknowledgements
Philip would like to acknowledge the assistance of Milad Dagher for his guidance and consultation with the droplet microfluidics, Susan Amin for assisting with gathering on-chip cell lysis data, and Donald MacNearney for advising on the poster template. Philip received funding from NSERC and a Travel Award from the ISS program at McGill.
References
[1] Reisner, W. et al. “Single-molecule denaturation mapping of DNA in nanofluidic channels”, PNAS. vol. 107, pp. 13294– 13299, 2010.
[2] Welch, R. L., Sladek, R., Dewar, K. & Reisner, W. W. “Denaturation mapping of Saccharomyces cerevisiae”, Lab Chip vol 12, pp. 3314, 2012.
Background
10 μm
10 μm
Above Right: Time series of cell lysis
and chromatin extension using a
dedicated microfluidic device.
Chromatin is imaged using an
intercalating fluorescent dye.
Right: Chromatin strands imaged using
an 100x objective. Dashed red line
indicates the same field of view.
Cells are lysed in a microfluidic
chamber. Chromatin strands are
extended using hydraulic actuation via
application of negative pressure.
ConclusionWe validated hydrogel microsphere encapsulation of single, cultured, human lymphoblast cells as a sample preparation protocol for whole single cell genomes.
We also demonstrate in-alginate lysis using time course analysis of fluorescence imaging
of chromatin.
The present results show that single cell genome content can be handled off-chip using
alginate microspheres before introduction into a device for single molecule sensing.
Alginate microsphere droplet generation
Above: (a) Water in oil droplet generation of alginate microspheres.
(b) Sample preparation (cell lysis) is carried out in a centrifuge tube following extraction from oil.
(c) Microspheres loaded with intact single cell genomes are delivered to the microfluidics chip.
a) b) c)
Right: Alginate microspheres containing intact cells with a membrane-permeable
fluorescent nuclear stain. Select microspheres containing cells are indicated by the arrows.
o
w
Mbp-length DNA extraction on a microscope slide
Above: (a) Microspheres containing cellular chromatin are deposited onto a microscope slide.
(b) Solution containing an intercalating agent is added to the solution to disrupt the polymer network of the microspheres.
Right: Stretched chromatin reaching a length of 1mm.
Below: Chromatin strands imaged using an 100x objective. Dashed
red line indicates the same field of view.
(c-d) A coverslip is placed over the sample and drawn
across to induce fluid shear to separate and stretch
chromatin strands.
10 μm
a) b) c) d)
Mammalian cell lysis inside alginate microspheres
Below: Time series of mammalian cell lysis inside of a trapped alginate microsphere.
Right: Traps for alginate microspheres in a PDMS (polydimethylsiloxane) microfluidic
device. Trapping regions are indicated by the arrows.
Cells are initially labelled with membrane-permeable
fluorescent dye for identification.
Lysis buffer is added at t = 0 seconds. It contains a
membrane-impermeable fluorescent intercalator.
Membrane lysis was identified by an increase in
fluorescence intensity due to intercalator binding.
Yellow dashed line indicates alginate microsphere200 μm
IntroductionOur previous development of a single molecule, single cell mapping technology relies on nanofluidic confinement1, and demonstrated usage on whole yeast genomes derived from gel plugs2.
Single cell sample handling remains problematic in a microfluidic context.
We present a simplified hydrogel
microsphere encapsulation protocol for
single cell handling in bulk intended to:
(i) bypass the physical limitations of mass
transport in a microfluidic environment and
(ii) shield long molecules from shearing
forces during sample manipulation.