This work was supported by the PRESTO "Design and Control of Cellular Functions" research area of the Japan Science and Technology Agency (JST), a Grant-in-Aid for Scientific Research of Innovative Areas "Molecular Robotics" (Project No. 24104002) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, Grant-in-Aid for Young Scientists (A) (Project No. 24680033) and Scientific Research (B) (Project No. 26280097) from the Japan Society for the Promotion of Science (JSPS), and the Creative Design for Bioscience and Biotechnology course of the School of Bioscience and Biotechnology at Tokyo Tech.

1. Fabrication of a Capillary-based Microfluidic Device
<ol>
	<li>Set up of the holders 
	Note: The holder design is presented in Figure 2A.
	<ol>
		<li>Cut out each of the four discs of the holders (Figure 2A(i)-(iv)) from 2-mm-thick polyacetal plastic plate using a milling machine. Use the following dimensions for each of the four discs of the holder: (i) disc 1 diameter 8.5 mm, capillary hole (CH) diameter 1.3 mm, screw hole (SH) diameter 1.8 mm; (ii) disc 2 di...

<ol>
	<li>Song, H., Chen, D. L., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactions+in+droplets+in+microfluidic+channels.">Reactions in droplets in microfluidic channels.</a> Angew. Chem., Int. Ed. 45 (44), 7336-7356 (2006).</li><li>Huebner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdroplets:+a+sea+of+applications?.">Microdroplets: a sea of applications?.</a> Lab Chip. 8, 1244-1254 (2008).</li><li>Taly, V., Kelly, B. T., Griffiths, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplets+as+microreactors+for+highthroughput+biology.">Droplets as microreactors for highthroughput biology.</a> ChemBioChem. 8 (3), 263-272 (2007).</li><li>Teh, S. Y., Lin, R., Hung, L. H., Lee, A. P. . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+Droplet+microfluidics."> Droplet microfluidics.</a> Lab Chip . 8, 198-220 (2008).</li><li>Takinoue, M., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+microfluidics+for+the+study+of+artificial+cells.">Droplet microfluidics for the study of artificial cells.</a> Anal. Bioanal. Chem. 400 (6), 1705-1716 (2011).</li><li>Hase, M., Yamada, A., Hamada, T., Baigl, D., Yoshikawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulation+of+cell-sized+phospholipid-coated+microdroplets+and+their+use+as+biochemical+microreactors.">Manipulation of cell-sized phospholipid-coated microdroplets and their use as biochemical microreactors.</a> Langmuir. 23 (2), 348-352 (2007).</li><li>Zheng, B., Tice, J. D., Roach, L. S., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Droplet-Based,+Composite+PDMS/Glass+Capillary+Microfluidic+System+for+Evaluating+Protein+Crystallization+Conditions+by+Microbatch+and+Vapor-Diffusion+Methods+with+On-Chip+X-Ray+Diffraction.">A Droplet-Based, Composite PDMS/Glass Capillary Microfluidic System for Evaluating Protein Crystallization Conditions by Microbatch and Vapor-Diffusion Methods with On-Chip X-Ray Diffraction.</a> Angew. Chem., Int. Ed. 43 (19), 2508-2511 (2004).</li><li>Nakano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+PCR+using+water-in-oil+emulsion.">Single-molecule PCR using water-in-oil emulsion.</a> J. Biotechnol. 102 (2), 117-124 (2003).</li><li>Diehl, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEAMing:+single-molecule+PCR+on+microparticles+in+water-in-oil+emulsions.">BEAMing: single-molecule PCR on microparticles in water-in-oil emulsions.</a> Nat. Methods. 3, 551-559 (2006).</li><li>He, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+encapsulation+of+single+cells+and+subcellular+organelles+into+picoliter-+and+femtoliter-volume+droplets.">Selective encapsulation of single cells and subcellular organelles into picoliter- and femtoliter-volume droplets.</a> Anal. Chem. 77 (6), 1539-1544 (2005).</li><li>Baroud, C., Gallaire, F., Dangla, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+microfluidic+droplets.">Dynamics of microfluidic droplets.</a> Lab Chip. 10, 2032-2045 (2010).</li><li>Utada, A. S., Nieves, A. F., Stone, H. A., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dripping+to+jetting+transitions+in+coflowing+liquid+streams.">Dripping to jetting transitions in coflowing liquid streams.</a> Phys. Rev. Lett. 99 (9), 094502 (2007).</li><li>Cramer, C., Fischer, P., Windhab, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drop+formation+in+a+co-flowing+ambient+fluid.">Drop formation in a co-flowing ambient fluid.</a> Chem. Eng. Sci. 59 (15), 3045-3058 (2004).</li><li>Anna, S. L., Bontoux, N., Stone, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+dispersions+using+&amp;#34;flow-focusing&amp;#34;+in+microchannels.">Formation of dispersions using &amp;#34;flow-focusing&amp;#34; in microchannels.</a> Appl. Phys. Lett. 82, 364-366 (2003).</li><li>Takeuchi, S., Garstecki, P., Weibel, D. B., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+axisymmetric+flow-focusing+microfluidic+device.">An axisymmetric flow-focusing microfluidic device.</a> Adv. Mater. 17 (8), 1067-1072 (2005).</li><li>Thorsen, T., Roberts, R. W., Arnold, F. H., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+pattern+formation+in+a+vesicle-generating+microfluidic+device.">Dynamic pattern formation in a vesicle-generating microfluidic device.</a> Phys. Rev. Lett. 86 (18), 4163-4166 (2001).</li><li>Yamashita, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+monodisperse+cell-sized+microdroplets+using+a+centrifuge-based+axisymmetric+co-flowing+microfluidic+device.">Generation of monodisperse cell-sized microdroplets using a centrifuge-based axisymmetric co-flowing microfluidic device.</a> J. Biosci. Biotech. 119 (4), 492-495 (2015).</li><li>Maeda, K., Onoe, H., Takinoue, M., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+synthesis+of+3D+multi-compartmental+particles+with+centrifuge-based+microdroplet+formation+from+a+multi-barrelled+capillary.">Controlled synthesis of 3D multi-compartmental particles with centrifuge-based microdroplet formation from a multi-barrelled capillary.</a> Adv. Mater. 24 (10), 1340-1346 (2012).</li></ol>

Using this device, the monodisperse W/O microdroplets were generated by Plateau-Rayleigh instability of a jet-flow17. Microscopic examination did not reveal the presence of satellite droplets. In the fabrication of the device, three critical steps are essential to successfully generate monodisperse W/O microdroplets. First, to supply a straight flow of oil containing surfactant and aqueous solution, the capillary holes of four discs must be arranged in a concentric pattern. Second, the inner capillary was care...

W/O microdroplets1-5 have many important applications for the study of biochemistry and bioengineering, including protein synthesis6, protein crystallization7, emulsion PCR8,9, cell encapsulation10, and construction of artificial cell-like systems5,6. To produce W/O microdroplets for these applications, important criteria are control of size and monodispersibility of the W/O microdroplets. Microfluidic devices for making monodisperse, size-controllable W/O microdroplets11 are based on the co-flowing method12,13, flow-focusing method14,15, and the T-junction method16 in microchannels. Although these methods produce highly monodisperse W/O microdroplets, the microfabrication process requires complicated handling and specialized techniques for the preparation of microchannels, and also requires a large amount of sample solution (at least several hundred &#181;l) because of the inevitable dead volume in the syringe pumps and tubes that conduct the sample solution to the microchannels. Thus, an easy-to-use and low-dead-volume method to generate monodisperse W/O microdroplets is needed.
This paper, along with videos of experimental procedures, describes a centrifugal capillary-based axisymmetric co-flowing microfluidic device17 for generating cell-sized, monodisperse W/O microdroplets (Figure 1). This simple method achieves size monodispersity and size controllability. It requires just a tabletop mini-centrifuge and a capillary-based axisymmetric co-flowing microfluidic device fixed in a sampling microtube. Our method needs only a very small volume (0.1 &#181;l), and does not waste any significant volume of the sample.

<table border="0" cellpadding="0" cellspacing="0" width="747">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2-mm-thick polyacetal plastic plate</td>
			<td style="width:71px;">Tool</td>
			<td style="width:119px;">Nikkyo Technos, Co., Ltd. (Japan)</td>
			<td style="width:167px;">244-6432-08</td>
			<td style="width:175px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Milling machine</td>
			<td style="width:71px;">Tool</td>
			<td style="width:119px;">Roland DG Co., Ltd. (Japan)</td>
			<td>MDX-40A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">End Mill RSE230-0.5*2.5</td>
			<td style="width:71px;">Tool</td>
			<td style="width:119px;">NS Tool Co., Ltd. (Japan)</td>
			<td>01-00644-00501</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">M2*40 screws</td>
			<td style="width:71px;">Tool</td>
			<td style="width:119px;">Jujo Synthetic Chemistry Labo. (Japan)</td>
			<td>0001-024</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass Capillry Puller</td>
			<td style="width:71px;">Tool</td>
			<td style="width:119px;">Narishige (Japan)</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microforge</td>
			<td style="width:71px;">Tool</td>
			<td style="width:119px;">Narishige (Japan)</td>
			<td>MF-900</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inner Glass Capillary</td>
			<td style="width:71px;">Tool</td>
			<td style="width:119px;">Narishige (Japan)</td>
			<td>G-1</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Outer Glass Capillary</td>
			<td style="width:71px;">Tool</td>
			<td style="width:119px;">World Precision Instruments Inc. (USA)</td>
			<td>1B200-6</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1.5 ml Sample tube</td>
			<td style="width:71px;">Tool</td>
			<td style="width:119px;">INA OPTIKA CO.,LTD (Japan)</td>
			<td>ST-0150F</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Hexadecane</td>
			<td style="width:71px;">Reagent</td>
			<td style="width:119px;">Wako Pure Chemical Industries Ltd. (Japan)</td>
			<td>080-03685&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sorbitan monooleate (Span 80)</td>
			<td style="width:71px;">Reagent</td>
			<td style="width:119px;">Tokyo Chemical Industry Co., Ltd. (Japan)</td>
			<td>S0060</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Milli Q system</td>
			<td style="width:71px;">Reagent</td>
			<td style="width:119px;">Merck Millipore Corporation (Germany)</td>
			<td>ZRQSVP030</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Swinging-out-type Mini-centrifuge</td>
			<td style="width:71px;">Tool</td>
			<td style="width:119px;">Hitech Co., Ltd. (Japan)</td>
			<td>ATT101</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Digital Microscope</td>
			<td style="width:71px;">Tool</td>
			<td style="width:119px;">KEYENCE Corporation (Japan)</td>
			<td>VHX-2001</td>
			<td></td>
		</tr>
	</tbody>
</table>

capillary-based centrifugal microfluidic device for size-controllable formation of monodisperse microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

In this study, we present a simple method for the generation of cell-sized W/O microdroplets by using a capillary-based centrifugal microfluidic device (Figure 1). The microfluidic device was composed of a capillary holder (Figure 2B), two glass capillaries (inner and outer glass capillaries in Figure 3C), and a microtube containing an oil including surfactant. We injected 0.1 &#181;l of sample solution into the inner glass capillary and ...

Watch this Scientific Journal Video about Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets at JoVE.com

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

The overall goal of this procedure is to generate monodispersed water and oil microdroplets for biological studies using a capillary based centrifugal axi-symmetric co-flowing microfluidic device. This method can help produce monodispersed microdroplet for the biochemical field to advance the study of work in synthesis, single-molecule PCR, single encapsultation, and the simple modeling of living cells. The main advantages of this technique are that it is easy and adjustable.It requires less than a microliter of sample solution and it can produce monodispersed microdroplets of the required size. Demonstrating the procedure will be Morita, a postdoc from my laboratory. The capillary based microfluidic assembly begins with preparing the capillary holder.Begin with milling four discs from two millimeter thick polyacetyl plastic plates using a milling machine. The discs are used to form a column supported by rods and screws. Using M.2 screws, assemble the plates.First, from discs one and two, assemble the bottom of the holder. Then, shorten the screws by cutting them in the threaded sections. Connect three of the four holes to secure the two discs making a 0.9 centimeter stack.Similarly, assemble the top part of the holder from discs three and four. Use two of the holes in each plate to connect the plates. Make the stack 0.7 centimeters tall.Now, use a long screw to join the two empty holes in the top part of the holder with the two empty holes in the bottom part of the holder. The long screw should put a gap between the holders that is about two to three millimeters and the head of the screw should overhang the completed holder by about 2.2 centimeters. Each experiment uses a new set of capillaries fabricated from two capillaries.From a 90 millimeter capillary, make two inner capillaries using a glass cutter. Make two equally-sized sections. From a 150 millimeter capillary, make three inner capillary sections.Next, using a glass puller, sharpen each capillary section. Set the puller weight to its maximum and set the heat level to 60-70 degrees for the outer capillary or 70-80 for the inner capillary. Then, carefully sharpen the glass.Keep the length of the constricted parts of the capillaries within the advised limits. If the length of the tips is too long or short, adjust the heating element until the correct length is achieved. Now, prepare to cut the tips.First, use some clear tape to secure a capillary to a microscope stage. Overhang the tip under the objective. Second, using a platinum wire covered by glass beads, cut the tip off in three steps.First, put the capillary where the break should occur in contact with a cold bead. Second, heat the wire for one to two seconds. Then third, cut the tip off by cooling the platinum wire.This operation should be performed using a foot pedal control to heat the wire. Now, using a microforge, adjust the diameters of the capillary orifices. For the two outer glass capillaries, make their orifices 60 microns.For the three inner capillaries make orifices having diameters of five, 10 and 20 microns. To generate the microdroplets, first fill an outer glass capillary with an oil-containing surfactant such as hexadecane containing 2%sorbitin monooleate by weight. Load 10 microliters of the solution into the capillary.Then, secure the loaded copillary to the bottom part of the holder. Next, using capillary action, load about 0.1 microliters of aqueous solution into an inner glass capillary. Position the loaded inner capillary in the upper part of the holder.Now, insert the inner capillary across the holder and into the outer capillary. While observing the tip through a microscope, turn the long screw of the holder to slowly and carefully advance in the inner capillary into the outer capillary. Advance the inner capillary to where the outer capillary's diameter is between 100 and 150 microns.Now, introduce 100 microliters of the same oil with surfactant into the bottom of a 1.5 milliliter microtube. This will serve as a microdoplet collection tube. Carefully position the holder and capillaries into the loaded microtube.Make sure the outer capillary is not in contact with the solution in the microtube. Then, load the tube in to a tabletop swing-out type centrifuge. To generate the microdroplets, run the centrifuge at 1, 600 times gravity for one or two seconds.Then, remove the capillaries and holder from the tube and take an aliquot of the water-oil microdroplets collected in the tube. Eject the microdroplets onto a glass slide and image them for analysis. Using this protocol, various water-oil microdroplets where made from hexadecane containing 2%sorbitin monooleate.Droplets made using an inner pipette with an orifice diameter of five microns were measured to be 8.3 microns in diameter on average. Droplets made using an inner pipette with an orifice diameter of 10 microns were measured to be 12.7 microns in diameter on average. Droplets made using an inner pipette with a 20 microns orifice were about 17.9 microns in diameter.Thus, this method produces monodispersed microdroplets that have a diameter close to the size of the inner capillary orifice. After watching this video, you should have a good understanding of how to produce and control the size of monodispersed water and oil microdroplets for quantitative chemical experiments. Once mastered, this technique can be done in five to 10 minutes, if it is performed properly.

capillary-based-centrifugal-microfluidic-device-for-size-controllable

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10.1K Views.  Tokyo Institute of Technology. The overall goal of this procedure is to generate monodispersed water and oil microdroplets for biological studies using a capillary based centrifugal axi-symmetric co-flowing microfluidic device. This method can help produce monodispersed microdroplet for the biochemical field to advance the study of work in synthesis, single-molecule PCR, single encapsultation, and the simple modeling of living cells. The main advantages of this technique are that it is easy and adjustable.It require...