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 devic...

<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. 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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. 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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 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&#160;</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&#34;&#160;</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 ...

The overall goal of this procedure is to generate monodispersed high-viscosity droplets with viscosities above one pascal seconds and a low viscosity continuous phase via phase-inversion in a simple co-flow microfluidic device. This method can solve the key problem in the droplet microfluidics field about manipulation of inherently high-viscosity fluids such as high glycerol polymer solutions and nanoparticle suspensions. The main advantage of this technique is that it initially generates low-viscosity droplets which are easier to form and control than high-viscosity droplets.The subsequent phasing version to generate high-viscosity droplets is induced when the low-viscosity droplets flow from the exit of a co-flow structure. Demonstrating the procedure will be Mr.Zhongnan Li, a graduate student from Tsinghua University. To begin preparing the device, use a tungsten carbide scriber to cut a three centimeter length of a round glass tube with an inner and outer diameter of 580 micrometers and one millimeter respectively.This will form the middle tube of the device. To make the inner tube of the device, cut a two centimeter length of a round glass tube with an inner and outer diameter of 200 and 330 micrometers respectively. Place one milliliter of ODTS in a 1.5 milliliter centrifuge tube.Immerse one end of the middle tube in the ODTS for 10 seconds. Then flush the tube with nitrogen gas from the untreated end until the tube is dry. Next, use a blade to cut a 0.5 millimeter by 0.5 millimeter notch.On the edge of the plastic Luer hub of a 20 gauge half-inch blunt needle, cut a 0.5 millimeter by 0.5 millimeter notch and a one millimeter by one millimeter notch directly across from each other on the hub of a second needle. Then place the middle tube lengthwise on a standard glass microscope slide with the hydrophobic ODTS-coated end extending about one centimeter passed the narrow end of the slide. Insert the inner tube into the untreated end of the middle tube leaving about one centimeter of the inner tube outside the middle tube.Use epoxy to fix the tubes in place along the center line of the slide. For better performance, adjust the position of the inner tube to be approximately concentrate to the middle tube. Once the epoxy has set, place the singly-notched needle over the end of the inner tube so that the tube fits in the notch.Fix the needle in place with epoxy to form the low-viscosity oil inlet. Then fix the double-notched needle over the junction between the inner and middle tubes to form the high-viscosity aqueous solution inlet. Use epoxy to seal the needle hubs both around the tubes and to the glass substrate.To ensure that the high-viscosity inlet needle sits firmly on the substrate, fit the middle tube into the big notch and the inner tube into the small notch. After the epoxy has dried, fit a 20 millimeter length of polyethylene tubing with an inner diameter of 0.86 millimeter over the hydrophobic end of the middle tube to complete the device. To begin the process, draw 0.8 milliliters of glycerol tinted with blue dye into a one milliliter syringe.Draw 0.8 milliliters of light paraffin oil into a second one milliliter syringe. Connect the glycerol syringe to the high-viscosity aqueous solution inlet of the device via polyethylene tubing with an inner diameter of 0.86 millimeters. Connect the liquid paraffin syringe to the low-viscosity oil inlet.Mount both syringes on syringe pumps. Then use a two-finger clamp and a lab stand to fix the device vertically over a 35 millimeter Petri dish. Adjust the device position so that the end of the outlet tubing is about two millimeters above the bottom of the dish.Pour enough liquid paraffin into the Petri dish to just immerse the device outlet. Add the same quantity of liquid paraffin to a second 35 millimeter Petri dish. Set the glycerol syringe pump flow rate to two microliters per minute.Set the liquid paraffin syringe pump flow rate to six microliters per minute. Run both pumps to begin generating the glycerol droplets. Monitor the droplet generation with a camera if desired.Wait about one minute for the glycerol and liquid paraffin flows to stabilize sufficiently to form uniform glycerol droplets. Then exchange the Petri dish under the device outlet for the second liquid paraffin-filled dish to collect the uniform droplets. Monodispersed glycerol droplets were generated by phase-inversion co-flow devices with middle tubes having diameters of either 200 or 500 micrometers.Monodispersed droplets were also generated from other high-viscosity fluids including honey, a starch solution, and a polyvinyl alcohol solution. Glycerol droplets generated by the 500 micrometer device with oil and glycerol flow rates of 30 and 10 microliters per minute respectively had an average diameter of 521 micrometers. Glycerol droplets generated by the 200 micrometer device at the same flow rates had an average diameter of 212 micrometers.The size of the glycerol droplets was found to change with variations in the ratio of the oil flow rate to the glycerol flow rate. Increasing the oil flow rate while holding the glycerol flow rate constant resulted in a decrease in particle size. After watching this video, you should have a good understanding of how to make monodispersed high-viscosity droplets with a phase-inversion co-flow device.In contrast to common co-flow devices, you do not need to taper the inner glass capillary to a sharp tip to make the phase-inversion co-flow device. The phase-inversion co-flow device can be used to generate high-viscosity droplets with dynamic viscosities of up to 12 pascal seconds. Once mastered, this technique can be done in 30 minutes if it is performed properly.

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Liquids, Solids, and Intermolecular Forces

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10.1K ビュー.  University of Science and Technology Beijing. The overall goal of this procedure is to generate monodispersed high-viscosity droplets with viscosities above one pascal seconds and a low viscosity continuous phase via phase-inversion in a simple co-flow microfluidic device. This method can solve the key problem in the droplet microfluidics field about manipulation of inherently high-viscosity fluids such as high glycerol polymer solutions and nanoparticle suspensions. The main advantage of this technique is that it initially generates low...

ビデオ: 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...

double emulsion generation using a polydimethylsiloxane (pdms) co-axial flow focus device

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...

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

generation of dynamical environmental conditions using a high-throughput microfluidic device

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in...

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

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...

generation of size-controlled poly (ethylene glycol) diacrylate droplets via semi-3-dimensional flow focusing microfluidic devices

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...

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

fabricating highly open porous microspheres (hopms) via microfluidic technology

Fabricating Highly Open Porous Microspheres HOPMs via Microfluidic Technology

The present protocol describes the fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (HOPMs) via the single-emulsion formulation based facile microfluidic technology. These microspheres have potential applications in tissue engineering and drug...

Fabricating Highly Open Porous Microspheres (HOPMs) via Microfluidic Technology

cell co-culture patterning using aqueous two-phase systems

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer...

generating neuromuscular junctions using a microfluidic device

The video demonstrates the formation of neuromuscular junctions using a microfluidic device. Neural precursor cells and muscle progenitor cells are seeded into different wells within the device and left to mature into motor neurons and myotubes, respectively. The creation of a volume and chemical gradient leads to the development of active neuromuscular...

Generating Neuromuscular Junctions Using a Microfluidic Device

co-culture of living microbiome with microengineered human intestinal villi in a gut-on-a-chip microfluidic device

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological...

bilayer microfluidic device for combinatorial plug production

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.

Bilayer Microfluidic Device for Combinatorial Plug Production

single step isolation of extracellular vesicles from large-volume samples with a bifurcated a4f microfluidic device

Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

Microfluidic Device for Continuous Extracellular Vesicle Isolation from Human Urine

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synthesis and characterization of multi-modal phase-change porphyrin droplets

Synthesis and Characterization of Multi-Modal Phase-Change Porphyrin Droplets

In this protocol, methods for synthesizing and characterizing multi-modal phase-change porphyrin droplets are...

amplification of escherichia coli in a continuous-flow-pcr microfluidic chip and its detection with a capillary electrophoresis system

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

This protocol describes how to build a continuous-flow-polymerase chain system based on a microfluidic chip and how to build a capillary electrophoresis system in the lab. It presents a simple method for the analysis of nucleic acids in the lab.

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

cell capture using a microfluidic device

Cell Capture Using a Microfluidic Device

production of sirna-loaded lipid nanoparticles using a microfluidic device

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named...

perfusable vascular network with a tissue model in a microfluidic device

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional...

protocol for biofilm streamer formation in a microfluidic device with micro-pillars

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Pause for a few seconds to allow the cell suspension to flow through the channel before adding the remaining half of the cell suspension in the lower well. Use a pen to mark the seeded side as NPC or equivalent for easy orientation of the device without a microscope and incubate for five minutes for cell attachment. Next, slowly top up the two NPC-seeded wells with an additional day 10 motor neuron medium to a total volume of 200 microliters per well. Using a 200-microliter pipette, remove DPBS...

a microfluidic device with groove patterns for studying cellular behavior

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture.  In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can...

establishing single-cell based co-cultures in a deterministic manner with a microfluidic chip

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be...

Fabricating Nanogaps by Nanoskiving

There are several methods of fabricating nanogaps with controlled spacings, but the precise control over the sub-nanometer spacing between two electrodes-and generating them in practical quantities-is still challenging. The preparation of nanogap electrodes using nanoskiving, which is a form of edge lithography, is a fast, simple and powerful technique. This method is an entirely mechanical process which does not include any photo- or electron-beam lithographic steps and does not require any special equipment or infrastructure such as clean rooms. Nanoskiving is used to fabricate electrically addressable nanogaps with control over all three dimensions; the smallest dimension of these structures is defined by the thickness of the sacrificial layer (Al or Ag) or self-assembled monolayers. These wires can be manually positioned by transporting them on drops of water and are directly electrically-addressable; no further lithography is required to connect them to an electrometer.

The fabrication of electrically addressable, high-aspect-ratio (> 1000:1) metal nanowires separated by gaps of single nanometers using either sacrificial layers of aluminum and silver or self-assembled monolayers as templates is described. These nanogap structures are fabricated without a clean room or any photo- or electron-beam lithographic processes by a form of edge lithography known as nanoskiving.

There are several methods of fabricating nanogaps with controlled spacings, but the precise control over the sub-nanometer spacing between two ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

Monitoring the Effects of Illumination on the Structure of Conjugated Polymer Gels Using Neutron Scattering

We demonstrate a protocol to effectively monitor the gelation process of a high concentration solution of conjugated polymer both in the presence and absence of white light exposure. By instituting a controlled temperature ramp, the gelation of these materials can be precisely monitored as they proceed through this structural evolution, which effectively mirrors the conditions experienced during the solution deposition phase of organic electronic device fabrication. Using small angle neutron scattering (SANS) and ultra-small angle neutron scattering (USANS) along with appropriate fitting protocols we quantify the evolution of select structural parameters throughout this process. Thorough analysis indicates that continued light exposure throughout the gelation process significantly alters the structure of the ultimately formed gel. Specifically, the aggregation process of poly(3-hexylthiophene-2,5-diyl) (P3HT) nano-scale aggregates is negatively affected by the presence of illumination, ultimately resulting in the retardation of growth in conjugated polymer microstructures and the formation of smaller scale macro-aggregate clusters.

A protocol for the analysis of gels formed from the optoelectronic conjugated polymer poly(3-hexylthiophene-2,5-diyl) (P3HT) using small and ultra-small angle neutron scattering in both the presence and absence of illumination is presented.

We demonstrate a protocol to effectively monitor the gelation process of a high concentration solution of conjugated polymer both in the presence and ...

Preparation of Hollow Polystyrene Particles and Microcapsules by Radical Polymerization of Janus Droplets Consisting of Hydrocarbon and Fluorocarbon Oils

In this article, we have demonstrated a method for producing hollow particles and microcapsules using oil droplets consisting of hydrocarbon oil (styrene) and fluorocarbon oil (perfluoro-n-octane, PFO) in aqueous surfactant (sodium dodecylsulfate, SDS) solutions. Since fluorocarbon oils are immiscible with hydrocarbon oils, the two oils are separated. Emulsions are prepared by stirring styrene/PFO/aqueous SDS solution mixtures at 80 &#176;C. The type of emulsions and the morphology of droplets in the emulsions are observed by light microscope and scanning confocal fluorescence microscope. It is found that oil droplets with Janus-type morphologies consisting of mutually immiscible styrene and PFO are formed in aqueous SDS solutions. Polystyrene particles are fabricated by radical polymerization of the ternary mixtures of styrene/PFO/aqueous SDS solution at 80 &#176;C. The morphologies of the polystyrene are confirmed by scanning electron microscopy and scanning transmission electron microscopy observations. These observations show the preparation of hollow polystyrene particles with a single hole on the surface. To our knowledge, this method is a novel strategy using the immiscibility of hydrocarbon and fluorocarbon oils. The hollow particles can also be applied to the preparation of microcapsules.

A protocol for the fabrication of hollow polymer particles and microcapsules by radical polymerization using emulsions consisting of styrene, perfluoro-n-octane, and aqueous SDS (sodium dodecylsulfate) solution is presented.

In this article, we have demonstrated a method for producing hollow particles and microcapsules using oil droplets consisting of hydrocarbon oil (styrene) ...

Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal structure and probing the hydrogen-bond (H-bond) dynamics of the water molecules adsorbed on solid surfaces are fundamental issues of water science, which remains a great challenge owing to the light mass and small size of hydrogen. Scanning tunneling microscopy (STM) is a promising tool for attacking these problems, thanks to its capabilities of sub-&#197;ngstr&#246;m spatial resolution, single-bond vibrational sensitivity, and atomic/molecular manipulation. The designed experimental system consists of a Cl-terminated tip and a sample fabricated by dosing water molecules in situ onto the Au(111)-supported NaCl(001) surfaces. The insulating NaCl films electronically decouple the water from the metal substrates, so the intrinsic frontier orbitals of water molecules are preserved. The Cl-tip facilitates the manipulation of the single water molecules, as well as gating the orbitals of water to the proximity of Fermi level (EF) via tip-water coupling. This paper outlines the detailed methods of submolecular resolution imaging, molecular/atomic manipulation, and single-bond vibrational spectroscopy of interfacial water. These studies open up a new route for investigating the H-bonded systems at the atomic scale.

Here, we present a protocol to investigate the structure and dynamics of interfacial water at the atomic scale, in terms of submolecular resolution imaging, molecular manipulation, and single-bond vibrational spectroscopy.

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Synthesis and Structure Determination of &amp;#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...