Name: fabricating high-viscosity droplets using microfluidic capillary device with phase-inversion co-flow structure
Uploaded: 2018-04-17T14:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure at JoVE.com

This work was supported by the National Natural Science Foundation of China (Nos. 51420105006 and 51322501). We thank Daniel for his helpful discussion on the high viscosity ideas.

1. Producing a Phase-Inversion Co-Flow Capillary Device for Observing the Generation Process of Aqueous, High-Viscosity Droplets with a Diameter of ~500 &#956;m.
NOTE: The square outer tube used here is for taking images of the generation process of the high-viscosity droplets. If there is no need to take images, a simplified version of the device can be made according to protocol step 2.
<ol>
	<li>Prepare three glass tubes with different sizes for the assembly of the capillary device.
	<ol>
		<li>Take a square glass tube with an inner size of 1.05 mm, and cut a piece of the tube ~4 cm in length. This will be the outer tube of the device.</li>
		<li>Take a round glass tube with an inner diameter (I.D.) of 580 &#956;m and an outer diameter (O.D.) of 1 mm, and cut a piece of the tube ~3 cm in length. This will be the middle tube of the device.</li>
		<li>Take a round glass tube with I.D. = 200 &#956;m and O.D. = 330 &#956;m, and cut a piece of the tube ~2 cm in length. This will be the inner tube of the device.</li>
	</ol>
	</li>
	<li>Modify the surface wettability of one end of the middle tube to be hydrophobic.
	<ol>
		<li>Take a 1 mL glass vial, and add 0.3 mL of trichloro(octadecyl)silane (OTS) into the glass vial.</li>
		<li>Take the middle tube with I.D. = 580 &#956;m prepared in protocol step 1.1.2, and dip one end of it into the OTS in the glass vial for ~10 s.</li>
		<li>Take out the middle tube, and flush the tube with nitrogen gas from the untreated end.</li>
	</ol>
	</li>
	<li>Prepare the needles for the inlets of the capillary device.
	<ol>
		<li>Take a 20G blunt tip dispensing needle, and cut a slot with ~0.5 mm x 0.5 mm on the edge of the plastic Luer hub with a blade. 
		NOTE: This needle will serve as the inlet for the low-viscosity oil phase.</li>
		<li>Take another 20G blunt tip dispensing needle, and cut two slots on the edge of the plastic Luer hub. Align the two slots in a line passing the diameter of the Luer hub. 
		NOTE: One slot has a size of ~0.5 mm x 0.5 mm, while the other slot has a size of ~1.0 mm x 1.0 mm. This needle will serve as the inlet for the high-viscosity aqueous phase.</li>
		<li>Take another 20G blunt tip dispensing needle, and cut two slots on the edge of the plastic Luer hub. Align the two slots in a line passing the diameter of the Luer hub. 
		NOTE: One slot has a size of ~1.5 mm x 1.5 mm; while the other slot has a size of ~1.0 mm x 1.0 mm. This needle will serve as the inlet for cleaning purposes.</li>
	</ol>
	</li>
	<li>Assemble the glass tubes according to Figure 1A.
	<ol>
		<li>Take a regular 7.62 cm x 2.54 cm glass slide as the substrate of the capillary device.</li>
		<li>Put the outer tube with I.D. = 1.05 mm, prepared in protocol step 1.1.1, on the glass slide with ~1 cm extruding the short edge of the glass slide.</li>
		<li>Take the middle tube with I.D. = 580 &#956;m, prepared in protocol step 1.2, and insert the hydrophobic end of the middle tube into the outer tube from the end on the glass slide, and keep ~1 cm of the middle tube outside the outer tube.</li>
		<li>Take the inner tube with I.D. = 200 &#956;m, prepared in protocol step 1.1.3, and insert one end of the inner tube into the middle tube, and keep ~1 cm of the inner tube outside the middle tube.</li>
		<li>Use epoxy glue to fix the three tubes in position along the centerline of the glass slide. Then wait ~5 min or longer for the glue to solidify completely.</li>
	</ol>
	</li>
	<li>Assemble the inlets on the capillary device.
	<ol>
		<li>Take the inlet needle for the low-viscosity oil phase, prepared in protocol step 1.3.1, and let the Luer hub cover the end of the inner tube on the substrate, and then use epoxy glue to fix the Luer hub on the substrate.</li>
		<li>Take the inlet needle for the high-viscosity aqueous phase, prepared in protocol step 1.3.2, and let the Luer hub cover the junction between the inner tube and the middle tube, and then use epoxy glue to fix the Luer hub onto the substrate.</li>
		<li>Take the inlet needle, prepared in protocol step 1.3.3, and, for cleaning, let the Luer hub cover the junction between the middle tube and the outer tube, and then use epoxy glue to fix the Luer hub on the substrate.</li>
		<li>Wait ~5 min or longer for the glue to solidify completely.</li>
		<li>Use epoxy glue to seal the Luer hubs of the needles on the substrate.</li>
	</ol>
	</li>
	<li>Wait ~30 min or longer for the glue to solidify completely, and then the device is ready to use.</li>
</ol>
2. Make a Phase-Inversion, Co-Flow Capillary Device for Fabricating Aqueous High-Viscosity Droplets with a Diameter of ~500 &#956;m.
NOTE: The device made here is a simplified version of the device in protocol step 1.
<ol>
	<li>Prepare two glass tubes with different sizes for the assembly of the capillary device.
	<ol>
		<li>Take a round glass tube with I.D. = 580 &#956;m and O.D. = 1 mm, and cut a piece of the tube with ~ 3 cm in length. This will be the middle tube of the device.</li>
		<li>Take a round glass tube with I.D. = 200 &#956;m and O.D. = 330 &#956;m, and cut a piece of the tube with ~ 2 cm in length. This will be the inner tube of the device.</li>
	</ol>
	</li>
	<li>Modify the surface wettability of one end of the middle tube to be hydrophobic.
	<ol>
		<li>Add 0.3 mL of OTS into a 1 mL glass vial.</li>
		<li>Take the middle tube with I.D. = 580 &#956;m, prepared in protocol step 2.1.1, and dip one end of it into the OTS in the glass vial for ~10 s.</li>
		<li>Take out the middle tube, and then flush the tube with nitrogen gas from the untreated end.</li>
	</ol>
	</li>
	<li>Prepare the needles for the inlets of the capillary device.
	<ol>
		<li>Prepare a 20G blunt tip dispensing needle, which will serve as the inlet for the low-viscosity oil phase. Then, cut a slot of ~0.5 mm x 0.5 mm with a blade on the edge of the plastic Luer hub.</li>
		<li>Take another 20G blunt tip dispensing needle, and cut two slots on the edge of the plastic Luer hub. Align the two slots in a line passing the diameter of the Luer hub. 
		NOTE: One slot has a size of ~0.5 mm x 0.5 mm, while the other slot has a size of ~1.0 mm x 1.0 mm. This second needle will serve as the inlet for the high-viscosity aqueous phase.</li>
	</ol>
	</li>
	<li>Assemble the glass tubes according to Figure 1A.
	<ol>
		<li>Take a regular 7.62 cm x 2.54 cm glass slide as the substrate of the capillary device.</li>
		<li>Put the middle tube with I.D. = 580 &#956;m, prepared in protocol step 2.2, on the glass slide with the hydrophobic end extruding ~1 cm over the short edge of the glass slide.</li>
		<li>Take the inner tube with I.D. = 200 &#956;m, prepared in protocol step 2.1.2, and insert one end of the inner tube into the middle tube from the untreated end on the glass slide, and keep ~ 1 cm of the inner tube outside the middle tube.</li>
		<li>Use epoxy glue to fix the two tubes in position along the centerline of the glass slide.</li>
		<li>Wait for ~5 min or longer for the glue to solidify completely.</li>
	</ol>
	</li>
	<li>Assemble the inlets on the capillary device.
	<ol>
		<li>Take the inlet needle for the low-viscosity oil phase, prepared in protocol step 2.3.1, and let the Luer hub cover the end of the inner tube on the substrate, and then use epoxy glue to fix the Luer hub on the substrate.</li>
		<li>Take the inlet needle for the high-viscosity aqueous phase, prepared in protocol step 2.3.2, and let the Luer hub cover the junction between the inner tube and the middle tube, and then use epoxy glue to fix the Luer hub on the substrate. 
		NOTE: The other end of the middle tube is the outlet of the device.</li>
		<li>Wait ~5 min or longer for the glue to solidify completely.</li>
		<li>Use epoxy glue to seal the Luer hubs of the needles on the substrate.</li>
	</ol>
	</li>
	<li>Wait ~30 min or longer for the glue to solidify completely.</li>
	<li>Connect the free end of the middle tube with the outlet tubing, i.e., polyethylene tubing with I.D. = 0.86 mm and ~20 mm in length. 
	NOTE: The slight deformation of the outer tubing will ensure the seal of the connection, so that no glue is needed here. The outlet tubing acts as a wider outer tube for the phase inversion. At this point, the device is ready to use.</li>
</ol>
3. Make Phase-Inversion Co-Flow Capillary Device for Observing the Generation Process of Aqueous High-Viscosity Droplets with a Diameter of ~200 &#956;m.
NOTE: The device made here is a smaller version of the device of protocol step 1 to make smaller droplets.
<ol>
	<li>Prepare three glass tubes with different sizes for the assembly of the capillary device.
	<ol>
		<li>Take a square glass tube with I.D. = 400 &#956;m, and cut a piece of the tube ~4 cm in length, which will be the outer tube of the device.</li>
		<li>Take a round glass tube with I.D. = 200 &#956;m and O.D. = 330 &#956;m, and cut a piece of the tube ~3 cm in length, which will be the middle tube of the device.</li>
		<li>Take a round glass tube with I.D. = 100 &#956;m and O.D. = 170 &#956;m, and cut a piece of the tube ~2 cm in length, which will be the inner tube of the device.</li>
	</ol>
	</li>
	<li>Modify the surface wettability of one end of the middle tube to be hydrophobic.
	<ol>
		<li>Take a 1 mL glass vial, and add 0.3 mL of OTS into the glass vial.</li>
		<li>Take the middle tube with I.D. = 200 &#956;m, prepared in protocol step 3.1.2, and dip one end of it into the OTS in the glass vial for ~10 s.</li>
		<li>Take out the middle tube, and then flush the tube with nitrogen gas from the untreated end.</li>
	</ol>
	</li>
	<li>Prepare the needles for the inlets of the capillary device.
	<ol>
		<li>Prepare a 20G blunt tip dispensing needle, which will serve as the inlet for the low-viscosity oil phase. Then, with a blade, cut a slot ~0.2 mm x 0.2 mm on the edge of the plastic Luer hub.</li>
		<li>Prepare another 20G blunt tip dispensing needle, and cut two slots on the edge of the plastic Luer hub. Align the two slots in a line passing the diameter of the Luer hub. 
		NOTE: One slot has a size of ~0.2 mm x 0.2 mm, while the other slot has a size of ~0.4 mm x 0.4 mm. This second needle will serve as the inlet for the high-viscosity aqueous phase.</li>
		<li>Take another 20G blunt tip dispensing needle, and cut two slots on the edge of the plastic Luer hub. The two slots are aligned in a line passing the diameter of the Luer hub. 
		NOTE: One slot has a size of ~0.8 mm x 0.8 mm, while the other slot has a size of ~0.4 mm x 0.4 mm. This third needle will serve as an inlet for cleaning purposes.</li>
	</ol>
	</li>
	<li>Follow protocol steps 1.4 - 1.6 to finish the device, using the glass tubes prepared in protocol step 3.1 instead of those prepared in protocol step 1.1, and using the needles prepared in protocol step 3.3 instead of those prepared in protocol step 1.3.</li>
</ol>
4. Observing the Generation of Glycerol Droplets in Liquid Paraffin
NOTE: For taking the images shown in Figures 1B - D, use the device prepared in protocol step 1; for taking images shown in Figure 3, use the device prepared in protocol step 3.
<ol>
	<li>Prepare solutions to be used in the experiment.
	<ol>
		<li>Use glycerol as the high-viscosity aqueous phase, and add 0.5 w.t.% Toluidine Blue O to dye it blue.</li>
		<li>Use liquid paraffin as the low-viscosity oil phase, and add 1% w.t. Span 80 in it as surfactant.</li>
	</ol>
	</li>
	<li>Prepare three 1 mL syringes and three syringe pumps. 
	NOTE: Three syringes for the fluids prepared in protocol step 4.1: one for injecting the high-viscosity glycerol, prepared in protocol step 4.1.1, and the other two for injecting the low-viscosity liquid paraffin, prepared in protocol step 4.1.2, respectively.
	<ol>
		<li>Connect the syringe containing glycerol to the inlet to the middle tube.</li>
		<li>Connect one syringe containing liquid paraffin to the inlet of the inner tube, while connecting the other to the inlet for cleaning purposes.</li>
	</ol>
	</li>
	<li>Place the device prepared in protocol step 1 on an inverted microscope, and place a piece of a Kimwipe under the outlet of the outer tube to absorb the leaked fluid. 
	CAUTION: Do not let the fluid leak outside the Kimwipe area.</li>
	<li>Set the flow rates of the syringe pumps. 
	NOTE: Use the syringe pump connected to the outer tube for cleaning purposes when there are trapped bubbles or droplets around the exit of the middle tube. Otherwise, just leave the pump stopped.
	<ol>
		<li>Set the flow rate of glycerol injecting to the middle tube of Qw = 10 &#956;L/min.</li>
		<li>Set the flow rate of liquid paraffin injecting to the inner tube of Qo = 30 &#956;L/min.</li>
		<li>Run the two pumps to generate glycerol droplets.</li>
	</ol>
	</li>
	<li>Wait for ~0.5 min until the flows are stabilized and the glycerol droplets are uniformly generated at the exits of the middle tube. Then, take videos or images of the droplet generation process. 
	NOTE: Images in Figures 1B- C can be taken using the device prepared in protocol step 1, while images in Figure 3A can be taken using the device prepared in protocol step 3. Stop&#160;all the pumps as soon as videos or images are taken, and take the device off the microscope right away.</li>
	<li>Prepare for collecting the high-viscosity droplets.
	<ol>
		<li>Place the device in a vertical plane with the outlet pointed down, and put a petri dish under the outlet. Use tape to fix the device with the outlet ~2 mm above the bottom of the petri dish.</li>
		<li>Pour some liquid paraffin prepared in protocol step 4.1.2 into the petri dish, and just immerse the outlet of the device.</li>
	</ol>
	</li>
	<li>Run the two syringe pumps again at Qw = 10 &#956;L/min and Qo = 30 &#956;L/min, and collect the glycerol droplets in the petri dish. 
	NOTE: Wait for ~1 min until the flows are stabilized and the glycerol droplets are uniformly generated at the exits of the outer tube, the image of the droplets in the petri dish can be taken, as shown in Figure 1D for the device prepared in protocol 1, or Figure 3B for the device prepared in protocol step 3.</li>
</ol>
5. Generating and Collecting the Glycerol Droplets in Liquid Paraffin with the Simplified Device Prepared in Step 2.
NOTE: This is for taking images of the glycerol droplets generated under different flow rate ratio of Qo/Qw, and measuring the corresponding size variation of the droplets for the data points in Figure 2.
<ol>
	<li>Prepare solutions to be used in the experiment by following protocol step 4.1.</li>
	<li>Prepare two 1 mL syringes and two syringe pumps. 
	NOTE: Two syringes for the fluids prepared in protocol step 4.1: One for injecting the high-viscosity glycerol, prepared in protocol step 4.1.1, and the other for injecting the low-viscosity liquid paraffin, prepared in protocol step 4.1.2, respectively.
	<ol>
		<li>Connect the syringe containing 0.8 mL glycerol to the inlet to the middle tube.</li>
		<li>Connect the syringe containing 0.8 mL liquid paraffin to the inlet of the inner tube.</li>
	</ol>
	</li>
	<li>Prepare for collecting the high-viscosity droplets.
	<ol>
		<li>Place the device in a vertical plane with the outlet pointed down, and put a 35 mm Petri dish under the outlet. Use tape to fix the device with the outlet ~2 mm above the bottom of the petri dish.</li>
		<li>Pour some liquid paraffin prepared in protocol step 4.1.2 into the petri dish, and just immerse the outlet of the device.</li>
	</ol>
	</li>
	<li>Set the flow rates of the syringe pumps. 
	NOTE: For each flow rate ratio in Figure 2, fix the flow rate of glycerol Qw = 2 &#956;L/min, while increasing the flow rate of liquid paraffin Qo to different values according to the required flow rate ratios of Qo/Qw. For each flow rate ratio, wait ~1 min until the flows are stabilized and uniform glycerol droplets are collected in the petri dish, then take images of the droplets.
	<ol>
		<li>Set the flow rate of glycerol injected into the middle tube of Qw = 2 &#956;L/min.</li>
		<li>Set the flow rate of liquid paraffin injected into the inner tube of Qo = 6 &#956;L/min.</li>
		<li>Run the two pumps to generate glycerol droplets. 
		NOTE: The generation process of the droplets can be directly observed with a cellphone camera, or a digital camera mounted on a tripod.</li>
	</ol>
	</li>
	<li>Wait ~1 min until the flows are stabilized, and change a new petri dish for collecting uniform glycerol droplets.</li>
</ol>
6. Generate Other High-Viscosity Droplets in Liquid Paraffin Using the Phase-Inversion Co-Flow Device.
NOTE: This is for the images in Figure 4. All the low-viscosity oil phase used in the experiments is the same as used in protocol step 4.1.2.
<ol>
	<li>Use pure honey as the high-viscosity aqueous phase for Figure 4A.</li>
	<li>Prepare 6 w.t.% starch solution for Figure 4B. 
	CAUTION: Use a proper high-temperature glass media bottle and a high-temperature cap. Wear heat resistant gloves.
	<ol>
		<li>Add 47 g of water in a 100 mL glass media bottle and put a stir bar in the bottle.</li>
		<li>Put the bottle in a water bath and set the temperature to 100 &#176;C.</li>
		<li>Add 3 g of starch powder into the hot water after the water bath reaches 100 &#176;C.</li>
		<li>Cover the cap of the bottle and keep stirring for ~4 h until the solution is clear.</li>
		<li>Wait until the solution cools down to room temperature before use.</li>
	</ol>
	</li>
	<li>Prepare 10 w.t.% PVA-124 solution for Figure 4C. 
	CAUTION: Use a proper high-temperature glass media bottle and a high-temperature cap. Wear heat resistant gloves.
	<ol>
		<li>Add 45 g of water in a 100 mL glass media bottle and put a stir bar in the bottle.</li>
		<li>Put the bottle in a water bath and set the temperature to 70 &#176;C.</li>
		<li>Add 5 g of PVA-124 powder into the bottle after the water bath reaches 70 &#176;C.</li>
		<li>Cover the cap of the bottle and keep stirring for ~1 h until the solution is clear.</li>
		<li>Wait until the solution cools down to room temperature before use.</li>
	</ol>
	</li>
	<li>Generate high-viscosity droplets in liquid paraffin.
	<ol>
		<li>Follow protocol step 5 using the high-viscosity fluids prepared in step 6.1 - 6.3, instead of the glycerol in protocol step 5.</li>
		<li>Use the flow rate settings of Qw = 1 &#956;L/min and Qo = 5 &#956;L/min for Figure 4.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Shah, R. K., Shum, H. C., Rowat, A. C., Lee, D., Agresti, J. J., Utada, A. S., Chu, L. Y., Kim, J. W., Fernandez-Nieves, A., Martinez, C. J., 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=Designer+emulsions+using+microfluidics.">Designer emulsions using microfluidics.</a> Mater. Today. 11, 18-27 (2008).</li><li>Park, J. I., Saffari, A., Kumar, S., G&#252;nther, A., Kumacheva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+synthesis+of+polymer+and+inorganic+particulate+materials.">Microfluidic synthesis of polymer and inorganic particulate materials.</a> Annu. Rev. Mater. Res. 40, 415-443 (2010).</li><li>Heath, J. R., Ribas, A., Mischel, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis+tools+for+drug+discovery+and+development.">Single-cell analysis tools for drug discovery and development.</a> Nat. Rev. Drug Discovery. 15, 204-216 (2016).</li><li>Murphy, S. V., Atala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Bioprinting+of+tissues+and+organs.">3D Bioprinting of tissues and organs.</a> Nat. Biotechnol. 32, 773-785 (2014).</li><li>Du, G., Fang, Q., den Toonder, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+cell-based+high+throughput+screening+platforms-a+review.">Microfluidics for cell-based high throughput screening platforms-a review.</a> Anal. Chim. Acta. 903, 36-50 (2016).</li><li>Ushikubo, F. Y., Oliveira, D. R. B., Michelon, M., Cunha, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+food+structure+using+microfluidics.">Designing food structure using microfluidics.</a> Food Eng. Rev. 7, 393-416 (2015).</li><li>Xu, J. H., Li, S. W., Tan, J., Wang, Y. J., Luo, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+highly+monodisperse+droplet+in+a+T-junction+microfluidic+device.">Preparation of highly monodisperse droplet in a T-junction microfluidic device.</a> AIChE Journal. 52, 3005-3010 (2006).</li><li>van Steijn, V., Kleijn, C. R., Kreutzer, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flows+around+confined+bubbles+and+their+importance+in+triggering+pinch-off.">Flows around confined bubbles and their importance in triggering pinch-off.</a> Phys. Rev. Lett. 103, 214501 (2009).</li><li>Utada, A. S., Fernandez-Nieves, A., 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, 094502 (2007).</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>Utada, A. S., Lorenceau, E., Link, D. R., Kaplan, P. D., 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=Monodisperse+double+emulsions+generated+from+a+microcapillary+device.">Monodisperse double emulsions generated from a microcapillary device.</a> Science. 308, 537-541 (2005).</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>Nunes, J. K., Tsai, S. S. H., Wan, J., 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=Dripping+and+jetting+in+microfluidic+multiphase+flows+applied+to+particle+and+fiber+synthesis.">Dripping and jetting in microfluidic multiphase flows applied to particle and fiber synthesis.</a> J. Phys. D: Appl. Phys. 46, 114002 (2013).</li><li>Cubaud, T., Mason, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+threads+and+viscous+droplets+in+square+microchannels.">Capillary threads and viscous droplets in square microchannels.</a> Phys. Fluids. 20, 053302 (2008).</li><li>Shestopalov, I., Tice, J. D., 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=Multi-step+synthesis+of+nanoparticles+performed+on+millisecond+time+scale+in+a+microfluidic+droplet-based+system.">Multi-step synthesis of nanoparticles performed on millisecond time scale in a microfluidic droplet-based system.</a> Lab Chip. 4, 316-321 (2004).</li><li>Zheng, B., 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=Screening+of+protein+crystallization+conditions+on+a+microfluidic+chip+using+nanoliter-size+droplets.">Screening of protein crystallization conditions on a microfluidic chip using nanoliter-size droplets.</a> J. Am. Chem. Soc. 125, 11170-11171 (2003).</li><li>Nie, Z. H., Xu, S. Q., Seo, M., Lewis, P. C., Kumacheva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+production+of+biopolymer+microcapsules+with+controlled+morphology.">Microfluidic production of biopolymer microcapsules with controlled morphology.</a> J. Am. Chem. Soc. 127, 8058-8063 (2005).</li><li>Abate, A. R., Kutsovsky, M., Seiffert, S., Windbergs, M., Pinto, L. F., Rotem, A., Utada, A. S., 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=Synthesis+of+monodisperse+microparticles+from+non-Newtonian+polymer+solutions+with+microfluidic+devices.">Synthesis of monodisperse microparticles from non-Newtonian polymer solutions with microfluidic devices.</a> Adv. Mater. 23, 1757-1760 (2011).</li><li>Seo, M., Nie, Z., Xu, S., Mok, M., Lewis, P. C., Graham, R., Kumacheva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+microfluidic+reactors+for+polymer+particles.">Continuous microfluidic reactors for polymer particles.</a> Langmuir. 21, 11614-11622 (2005).</li><li>Duncanson, W. J., Lin, T., Abate, A. R., Seiffert, S., Shah, R. K., 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=Microfluidic+synthesis+of+advanced+microparticles+for+encapsulation+and+controlled+release.">Microfluidic synthesis of advanced microparticles for encapsulation and controlled release.</a> Lab Chip. 12, 2135-2145 (2012).</li><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, 7336-7356 (2006).</li><li>Chen, H., Zhao, Y., Li, J., Guo, M., Wan, J., Weitz, D. A., 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=Reactions+in+double+emulsions+by+flow-controlled+coalescence+of+encapsulated+drops.">Reactions in double emulsions by flow-controlled coalescence of encapsulated drops.</a> Lab Chip. 11, 2312-2315 (2011).</li><li>Wang, P., Li, J., Nunes, J., Hao, S., Liu, B., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+micro-reactor+for+internal+gelation+to+fabricate+ZrO2+ceramic+microspheres.">Droplet micro-reactor for internal gelation to fabricate ZrO2 ceramic microspheres.</a> J. Am. Ceram. Soc. 100, 41-48 (2017).</li><li>Chen, H., Man, J., Li, Z., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+generation+of+high-viscosity+droplets+by+surface-controlled+breakup+of+segment+flow.">Microfluidic generation of high-viscosity droplets by surface-controlled breakup of segment flow.</a> ACS Appl. Mater. Interfaces. 9, 21059-21064 (2017).</li><li>Man, J., Li, Z., Li, J., Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+inversion+of+slug+flow+on+step+surface+to+form+high+viscosity+droplets+in+microchannel.">Phase inversion of slug flow on step surface to form high viscosity droplets in microchannel.</a> Appl. Phys. Lett. 110, 181601 (2017).</li></ol>

The authors have nothing to disclose.

The phase-inversion co-flow device provides a simple and straight forward method to generate monodisperse high-viscosity droplets. This device has a similar structure to common co-flow devices, as the basic co-flow structure consists of an inner tube inserted in the middle tube, the exit of which is connected to outlet tubing. However, there are two main differences between the phase-inversion co-flow device and common co-flow device for the generation of high-viscosity droplets with a viscosity of &#951;&#160;&...

The generation of droplets is becoming a key technology in a variety of applications, such as drug delivery, materials synthesis, 3D bioprinting, cell assays, and food engineering1,2,3,4,5,6. Microfluidic devices with T-junction7,8, co-flow1,9, or flow-focusing10,11 structures are widely used to generate monodisperse single emulsion droplets. Selection of a more viscous continuous phase will facilitate the formation of droplets12, and the viscosities of both the continuous and dispersed fluids are commonly below 0.1 Pas in droplet microfluidics13. However, in many applications, the dispersed phase may have a viscosity several hundred times higher than that of water, such as glycerol14, solutions containing nanoparticles15, proteins16, or polymers17,18,19, while it is difficult to achieve monodisperse droplets directly from high-viscosity fluids in a stable dripping regime11 in microfluidic devices, especially for fluids with a viscosity of &#951;&#160;&#62;&#160;1&#160;Pa&#183;s14,17,18,19. Furthermore, it has been reported13,18 that typical microfluidic methods for droplet formation require fluids with a relatively low viscosity and moderate interfacial tension to form uniform droplets in a stable dripping regime.
For a dispersed phase with a viscosity slightly larger than 0.1 Pas, there are several possible approaches to facilitate the droplet formation with typical T-junction, co-flow, or flow-focusing microfluidic devices: (1) decrease the viscosity of the dispersed phase by diluting it in an volatile solvent11,20; (2) decrease the dispersed-to-continuous viscosity ratio by increasing the viscosity of the continuous phase1,11; (3) decrease the flow rate of the dispersed phase to an extremely low value, while keeping a high continuous-to-dispersed flow rate ratio 14,19. However, these approaches are not practical for fluids with much higher viscosity, as they will significantly lower the production rate while dramatically raising the consumption of the volatile solvent or the continuous phase. In addtion, it has been reported that some high viscosity polymer solutions with &#951;&#160;&#62;&#160;1&#160;Pa&#183;s still did not break up into droplets with the approaches mentioned above17,19.
There are also several improved designs of microfluidic devices which introduce a third phase of fluid into the system, which facilitates the generation of high-viscosity droplets. Innovations include: bubbles introduced to cut a jetting thread into droplets21, an immiscible chaperoning fluid with moderate viscosity, introduced as the middle phase between the dipsersed phase and the continuous phase18, and microreactors introduced to generate high-viscosity droplets from two low-viscosity precursors21,22,23. However, as one more fluid is involved in the process, the system becomes more complicated, and the devices usually work in a much narrower flow regime than the typical devices for the generation of single emulsion droplets.
To generate monodisperse droplets directly from a high-viscosity fluid with &#951;&#160;&#62;&#160;1&#160;Pa&#183;s, surface-controlled phase-inversion methods have been investigated24. As the generation of low-viscosity droplets is much easier than that of high-viscosity droplets12, elongated low-viscosity droplets in a high-viscosity continuous phase are first generated using a typical co-flow structure, and then are broken up due to the change of surface wettability downstream of the co-flow structure. The released low-viscosity fluid inversely encapsulates the downstream high-viscosity fluid into droplets so that phase inversion is completed. According to the phase inversion mechanism, monodisperse high-viscosity droplets can be generated based on a typical co-flow device, while the exit of the co-flow device is treated to be wetted by the low-viscosity fluid, and then connected to a wider tube24,25.

<table><tbody><tr><td>VitroTubes Glass Tubing</td><td>VitroCom</td><td>8240</td><td>Square - Miniature Hollow Glass Tubing, I.D.=0.4mm, OD=0.8mm</td></tr><tr><td>VitroTubes Glass Tubing</td><td>VitroCom</td><td>CV2033</td><td>Round - Miniature Hollow Glass Tubing, I.D.=0.2mm, O.D.=0.33mm</td></tr><tr><td>VitroTubes Glass Tubing</td><td>VitroCom</td><td>CV1017</td><td>Round - Miniature Hollow Glass Tubing, I.D.=0.1mm, O.D.=0.17mm</td></tr><tr><td>VitroTubes Glass Tubing</td><td>VitroCom</td><td>Q14606</td><td>Square - Miniature Hollow Glass Tubing, I.D.=1.05mm+0.1/-0, OD=1.5mm</td></tr><tr><td>Standard Glass Capillaries</td><td>WPI</td><td>1B100-6</td><td>Round - Glass Tubing, I.D.=0.58mm, O.D.=1.00mm</td></tr><tr><td>Glycerol</td><td>Sinopharm Chemical Reagent Beijing</td><td>10010618</td><td></td></tr><tr><td>Paraffin Liquid</td><td>Sinopharm Chemical Reagent Beijing</td><td>30139828</td><td></td></tr><tr><td>Poly(vinyl alcohol), PVA-124</td><td>Sinopharm Chemical Reagent Beijing</td><td>30153084</td><td></td></tr><tr><td>Span 80</td><td>Sigma-Aldrich</td><td>85548</td><td></td></tr><tr><td>Starch</td><td>Sigma-Aldrich</td><td>S9765</td><td></td></tr><tr><td>Trichloro(octadecyl)silane</td><td>Sigma-Aldrich</td><td>104817</td><td></td></tr><tr><td>Toluidine Blue O</td><td>Sigma-Aldrich</td><td>T3260</td><td></td></tr><tr><td>Honey</td><td></td><td></td><td>Chaste tree honey, common food product purchased from supermarket</td></tr><tr><td>DEVCON 5 Minute Epoxy</td><td>ITW&amp;nbsp;</td><td></td><td>Epoxy glue</td></tr><tr><td>Blunt Tip Stainless Steel Dispensing Needles (Luer Lock)</td><td>Suzhou Lanbo Needle, China</td><td>LTA820050</td><td>20G x 1/2&quot;&amp;nbsp;</td></tr><tr><td>Tungsten/Carbide Scriber</td><td>Ullman</td><td>1830</td><td>For cutting glass tubing</td></tr><tr><td>Microscope Slides</td><td>Sail Brand</td><td>7101</td><td>76.2 mm x 25.4 mm, Thickness 1 - 1.2 mm</td></tr><tr><td>Polyethylene Tubing</td><td>Scientific Commodities</td><td>BB31695-PE/5</td><td>I.D. = 0.86 mm, O.D. = 1.32 mm</td></tr><tr><td>Syringe Pumps</td><td>Longer Pump, China</td><td>LSP01-1A</td><td>3 pumps needed for the experiments</td></tr></tbody></table>

fabricating high-viscosity droplets using microfluidic capillary device with phase-inversion co-flow structure

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion co-flow device to generate uniform high-viscosity droplets in a low-viscosity fluid. The microfluidic capillary device has a common co-flow structure with its exit connecting to a wider tube. Elongated droplets of the low-viscosity fluid are first encapsulated by the high-viscosity fluid in the co-flow structure. As the elongated low-viscosity droplets flow through the exit, which is treated to be wetted by the low-viscosity fluid, phase inversion is then induced by the adhesion of the low viscosity droplets to the tip of the exit, which results in the subsequent inverse encapsulation of the high-viscosity fluid. The size of the resultant high-viscosity droplets can be adjusted by changing the flow rate ratio of the low-viscosity fluid to the high-viscosity fluid. We demonstrate several typical examples of the generation of high-viscosity droplets with a viscosity up to 11.9 Pas, such as glycerol, honey, starch, and polymer solution. The method provides a simple and straightforward approach to generate monodisperse high-viscosity droplets, which may be used in a variety of droplet-based applications, such as materials synthesis, drug delivery, cell assay, bioengineering, and food engineering.

A microfluidic capillary device with a phase-inversion, co-flow structure was designed to generate monodisperse aqueous high-viscosity droplets, as shown in Figure 1A. In Figure 1, the high-viscosity aqueous phase was glycerol, which has a viscosity of &#951;w = 1.4 Pas; the low-viscosity oil phase was liquid paraffin, which has a viscosity of &#951;o = 0.029 Pas; the surface tension betwee...

Watch this Scientific Journal Video about Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure at JoVE.com

Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure

A phase-inversion co-flow device is demonstrated to generate monodisperse high-viscosity droplets above 1 Pas, which is difficult to realize in droplet microfluidics.

The generation of monodisperse droplets with high viscosity has always been a challenge in droplet microfluidics. Here, we demonstrate a phase-inversion ...

fabricating-high-viscosity-droplets-using-microfluidic-capillary

Research

Education

JoVE Journal

JoVE Core

Chemistry

Liquids, Solids, and Intermolecular Forces

SCIENTISTS IN ACTION

10.3K Views.  University of Science and Technology Beijing. A phase-inversion co-flow device is demonstrated to generate monodisperse high-viscosity droplets above 1 Pas, which is difficult to realize in droplet microfluidics.

Video: Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Bioengineering

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

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.

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 ...

Engineering

Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. This paper proposes a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation. The polydimethylsiloxane (PDMS) chip was fabricated using the multi-layer soft lithography method. Hexadecane containing surfactant was used as the continuous phase, and PEGDA with the ultraviolet (UV) photo-initiator was the dispersed phase. Surfactants allowed the local surface tension to drop and formed a more cusped tip which promoted breaking into tiny micro-droplets. As the pressure of dispersed phase was constant, the size of droplets became smaller with increasing continuous phase pressure before dispersed phase flow was broken off. As a result, droplets with size variation from 1 &#181;m to 80 &#181;m in diameter could be selectively achieved by changing the pressure ratio in two inlet channels, and the average coefficient of variation was estimated to be below 7%. Furthermore, droplets could turn into micro-beads by UV exposure for photo-polymerization. Conjugating biomolecules on such micro-beads surface have many potential applications in the fields of biology and chemistry.

Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices

Here, we present a protocol to illustrate the fabrication processes and verifying experiments of a semi-three-dimensional (semi-3D) flow-focusing microfluidic chip for droplet formation.

Uniform and size-controllable poly (ethylene glycol) diacrylate (PEGDA) droplets could be produced via the flow focusing process in a microfluidic device. ...

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

Biology

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Glass-Based Devices to Generate Drops and Emulsions

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. The first device is built for the generation of simple drops driven by gravity. The second device is designed to generate emulsion drops in a coflowing scheme. The third device is an extension of the coflowing device with the addition of a third liquid that acts as an electric ground, allowing the formation of electrified drops that subsequently discharge. In this setup, two of the three liquids have an appreciable electrical conductivity. The third liquid mediates between these two and is a dielectric. A&#160;voltage difference applied between the two conducting liquids creates an electric field that couples with hydrodynamic stresses of the coflowing liquids, affecting the jet and drop formation process. The addition of the electric field provides a path to generate smaller drops than in simple coflow devices and for generating particles and fibers with a wide range of sizes.

Here, a protocol to manufacture glass-based microfluidic devices used for generating highly monodisperse emulsions with controlled drop size is presented.

In this manuscript, three different step-by-step protocols to generate highly monodisperse emulsion drops using glass-based microfluidics are described. ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Setup of a Microfluidic Device for Combinatorial Plug  Production

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Author Spotlight: Integrating Computational and Experimental Approaches in Precision Oncology

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...