Cyto2015_emulsion_sorting
1
30 th Congress of the International Society for Advancement of Cytometry, June 26-30, 2015 Glasgow, Scotland 366/B235 Water-in-Oil Emulsion Sorting for Digital PCR and Single Cell Analysis Using Air Pressure Pulse Flow in Disposable Microfluidic Chip Jin Akagi * , Yuu Fujimura, Yoshie Kawase, Yohsuke Bansho, Kazuo Takeda On-chip Biotechnologies Co., Ltd., Tokyo, Japan Positive Negative Before sorting After sorting Purity: 94% Yield: 84% Fig.3 Disposable microfluidic chip based cell sorter (On-chip Sort) On-chip Sort TM Disposable chip Fig.4 Emulsion droplets generated by On-chip Droplet Generator On-chip Droplet Generator TM 80μm Fig.6 Cell lysis in W/O droplets Digital polymerase chain reaction (dPCR) is a compartmentalization concept for high sensitivity assay of nucleic acids of specific cells, free DNA or free RNA of low concentration suspended in sample containing high concentration of non-target particles 1 . The method using water-in-oil (W/O) emulsion droplets allows for detection of, for instance, extremely rare cells blended in 1 million other cells. A sorting technique of post-PCR W/O droplets according to fluorescence signal intensity using a commercial cell sorter was proposed in 2004, where W/O emulsion droplets are converted into a double emulsion droplets (i.e. W/O/W emulsion) prior to running them through a cell sorter 2 . Notwithstanding that, this method is not ideal for collecting DNA from sorted emulsion droplets or analyzing amplified DNA because conventional cell sorter is a source of DNA contamination. Collection of emulsions in oil within a microfluidic device using dielectrophoresis was also demonstrated in 2006, but an instrument that integrated such technology is not yet commercialized 3 . Herein, we report the success on W/O emulsion droplet sorting within a disposable microfluidic chip. This is the first emulsion sorting technology that resolved the problem of DNA contamination by the use of disposable microfluidic chip. The chip is made of polymeric material using injection molding process, and is already integrated in our cell sorting system, On- chip Sort, for gentle sorting of cells using pneumatic actuated liquid pulses 4,5 (Fig.1). The current sorting speed of On-chip Sort reaches 300 targets per second (Please refer to poster B242 in CYTO2015). On-chip Sort enables collection of emulsion droplets using oil as sheath liquid (Fig.2). 1. Vogelstein. B., Kinzler K. W.; Digital PCR, Proc. Natl. Acad. Sci. USA. 96, 9236–9241, 1999 2. Bernath K., Hai M., Mastrobattista E., Griffiths A. D., Magdassi S., Tawfika D. S.; In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting, Anal. Biochem. 325, 151–157, 2004 3. Ahn K., Kerbage C., Hunt T. P., Westervelt R. M., Link D.R., Weitz D. A.；Dielectrophoretic manipulation of drops for high-speed microfluidic sorting devices. Appl. Phys. Lett. 88, 024104−024104, 2006 4. Takeda K.; Disposable chip flow cell and cell sorter using same. PCT/JP2011/050270 5. Watanabe M., Serizawa M., Sawada T., Takeda K., Takahashi T., Yamamoto N., KoizumiF., Koh Y.; A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of rare tumor cells in blood. J Transl Med. 12, 143, 2014 Acknowledgement: This work was supported by a grant program of Japan Science and Technology Agency. Fig.1 Disposable microfluidic chip based cell sorter (On-chip Sort). Realization of contamination free W/O droplet sorting. Fig.2 Sorting of post-PCR W/O droplet Sample DNA: KRAS Reference Standard template 1ng/20μL, Primer Forward 1 μM, Primer Reverse 1μM, SsoFast EvaGreen Supermix (Biorad) Oli: Mineral Oil (Light Oil) 95% The optimal size for droplets in emulsion dPCR varies depending on the goal of the analysis. For example, small droplet size is preferred for detection of extremely rare cell-free DNA, while large droplet size is ideal for accommodating cells. For this reason, we have developed an emulsion droplet generator that allows for droplet size adjustment (On-chip Droplet Generator, Fig.3). Monodisperse droplets of diameters between 20 to 100 μm can be generated, and thus it would be very useful for single cell analysis (Fig.4). Fig.5 shows sorting of W/O droplets containing GFP expressing E.coli. For single cell DNA analysis, cell lysis was performed within a W/O droplet using Proteinase K. Fig.6 is a photograph of Hoechst stained PC9 in a droplet. Cells were completely lysed after droplet was heated at 55℃ for 4 hours. We are currently conducting PCR experiments on droplets containing lysed cell. Droplets (size 25μm) Droplets (size 100μm) 100μm 100μm E. coli expressing GFP Before Sort After Sort 40 μm Fig.5 Sorting of W/O droplets containing GFP expressing E.coli. Intensity of FS FL2 (GFP) Negative GFP positive 40 μm 0 hour 4 hour DNA DNA Cell: PC9 stained with Hoechst, Proteinase K Oil: mineral oil, Temperature: 55℃ Target: 2.4% *Correspondence: [email protected]
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Transcript of Cyto2015_emulsion_sorting
30th Congress of the International Society for Advancement of Cytometry, June 26-30, 2015 Glasgow, Scotland
366/B235
Water-in-Oil Emulsion Sorting for Digital PCR and Single Cell AnalysisUsing Air Pressure Pulse Flow in Disposable Microfluidic Chip
Jin Akagi*, Yuu Fujimura, Yoshie Kawase, Yohsuke Bansho, Kazuo Takeda
On-chip Biotechnologies Co., Ltd., Tokyo, Japan
Positive
Negative
Before sorting
After sorting
Purity: 94% Yield: 84%
Fig.3 Disposable microfluidic chip based cell sorter (On-chip Sort)
On-chip Sort TM
Disposable chip
Fig.4 Emulsion droplets generated by On-chip Droplet Generator
On-chip DropletGenerator TM
80μm
Fig.6 Cell lysis in W/O droplets
Digital polymerase chain reaction (dPCR) is a compartmentalization concept for high sensitivity assay of nucleic acids of
specific cells, free DNA or free RNA of low concentration suspended in sample containing high concentration of non-target
particles1. The method using water-in-oil (W/O) emulsion droplets allows for detection of, for instance, extremely rare cells
blended in 1 million other cells.
A sorting technique of post-PCR W/O droplets according to fluorescence signal intensity using a commercial cell sorter was
proposed in 2004, where W/O emulsion droplets are converted into a double emulsion droplets (i.e. W/O/W emulsion) prior to
running them through a cell sorter2. Notwithstanding that, this method is not ideal for collecting DNA from sorted emulsion
droplets or analyzing amplified DNA because conventional cell sorter is a source of DNA contamination. Collection of
emulsions in oil within a microfluidic device using dielectrophoresis was also demonstrated in 2006, but an instrument that
integrated such technology is not yet commercialized3.
Herein, we report the success on W/O emulsion droplet sorting within a disposable microfluidic chip. This is the first
emulsion sorting technology that resolved the problem of DNA contamination by the use of disposable microfluidic chip. The
chip is made of polymeric material using injection molding process, and is already integrated in our cell sorting system, On-
chip Sort, for gentle sorting of cells using pneumatic actuated liquid pulses4,5 (Fig.1). The current sorting speed of On-chip
Sort reaches 300 targets per second (Please refer to poster B242 in CYTO2015). On-chip Sort enables collection of emulsion
droplets using oil as sheath liquid (Fig.2).
1. Vogelstein. B., Kinzler K. W.; Digital PCR, Proc. Natl. Acad. Sci. USA. 96, 9236–9241, 19992. Bernath K., Hai M., Mastrobattista E., Griffiths A. D., Magdassi S., Tawfika D. S.; In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting, Anal. Biochem. 325, 151–157, 20043. Ahn K., Kerbage C., Hunt T. P., Westervelt R. M., Link D.R., Weitz D. A.；Dielectrophoretic manipulation of drops for high-speed microfluidic sorting devices. Appl. Phys. Lett. 88, 024104−024104, 20064. Takeda K.; Disposable chip flow cell and cell sorter using same. PCT/JP2011/0502705. Watanabe M., Serizawa M., Sawada T., Takeda K., Takahashi T., Yamamoto N., KoizumiF., Koh Y.; A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of rare tumor cells in blood. J Transl Med.
12, 143, 2014
Acknowledgement: This work was supported by a grant program of Japan Science and Technology Agency.
Fig.1 Disposable microfluidic chip based cell sorter (On-chip Sort).
Realization of contamination free W/O droplet sorting.
Fig.2 Sorting of post-PCR W/O dropletSample DNA: KRAS Reference Standard template 1ng/20μL,
Primer Forward 1 μM, Primer Reverse 1μM, SsoFast EvaGreen Supermix (Biorad)
Oli: Mineral Oil (Light Oil) 95%
The optimal size for droplets in emulsion dPCR varies depending on the goal of the analysis. For example, small droplet
size is preferred for detection of extremely rare cell-free DNA, while large droplet size is ideal for accommodating cells. For
this reason, we have developed an emulsion droplet generator that allows for droplet size adjustment (On-chip Droplet
Generator, Fig.3). Monodisperse droplets of diameters between 20 to 100 μm can be generated, and thus it would be very
useful for single cell analysis (Fig.4). Fig.5 shows sorting of W/O droplets containing GFP expressing E.coli. For single cell
DNA analysis, cell lysis was performed within a W/O droplet using Proteinase K. Fig.6 is a photograph of Hoechst stained
PC9 in a droplet. Cells were completely lysed after droplet was heated at 55℃ for 4 hours. We are currently conducting PCR
experiments on droplets containing lysed cell.
Droplets (size 25μm) Droplets (size 100μm)
100μm100μm
E. coli expressing GFP
Before Sort After Sort
40 µm
Fig.5 Sorting of W/O droplets containing GFP expressing E.coli.
Intensity of FS
FL2
(G
FP)
Negative
GFP
positive
40 µm
0 hour 4 hour
DNA
DNA
Cell: PC9 stained with Hoechst, Proteinase K
Oil: mineral oil, Temperature: 55℃
Target: 2.4%
*Correspondence: [email protected]
