Name: Generation of Size-controlled Poly (ethylene Glycol) Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices (Video) | JoVE Journal
Uploaded: 2018-07-03T12:30:00.000+00:00
Duration: 11 min 8 s
Description: Learn to fabricate semi-3D flow-focusing microfluidic chips for producing size-controlled PEG diacrylate droplets. This method enables efficient droplet formation with minimal sample consumption for biochemical applications.

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5 minutes

This work was supported by the Shenzhen fundamental research funding (Grant No. JCYJ 20150630170146829, JCYJ20160531195439665 and JCYJ20160317152359560). The authors would like to thank Prof. Y. Chen at the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences for supports.

1. Mold Fabrication
<ol>
	<li>Design two photomasks using a drawing software. Describe the outline of microchannel structure and use two separate layers for mask 1 and 2 in the same drawing file, so ensure all connections between different channels. Print different layers independently to chrome plate on the glass by a vendor with 1 &#181;m resolution. Ensure that the photomasks are dark with transparent designed structures, as a negative polarity. 
	Note: The mask 1 contains the dispersed phase inlet channel and an orifice. The mask 2 contains the continuous phase inlet channel, the filter, and the outlet.</li>
	<li>In designated photolithography lab, clean a 3-inch diameter silicon wafer. Place the wafer on a spin coater, turn on the vacuum to affix the wafer to the spin chuck. Spin-coat 2-3 mL of SU-8 2025 negative photoresist onto the wafer for 10 s at 1,000 rpm, then 30 s at 3,000 rpm, providing the first layer thickness of 20 &#181;m.</li>
	<li>Soft bake on a 95 &#176;C hotplate for 6 min. After the coated wafer cools to room temperature (RT), expose it via the mask 1 under a collimated 15 mW/cm2, 365 nm of UV for 18 s. Post-exposure, bake on a 95 &#176;C&#160;hotplate for 6 min, then allow the wafer to cool to RT.</li>
	<li>Repeat the spin-coating process. Apply 2-3 mL of SU-8 2100 negative photoresist onto the wafer for 10 s at 1,000 rpm, then 30 s at 2,000 rpm, providing the second layer thickness of 130 &#181;m. After soft bake on a 95 &#176;C&#160;hotplate for 35 min, place the mask 2 on the second layer photoresist to expose for 30 s, which was aligned with the dispersed phase channel layer by a UV aligner. Post-exposure, bake on a 95 &#176;C&#160;hotplate for 7 min.</li>
	<li>Develop the wafer by immersing in a stirred bath of 50 mL of propylene glycol methyl ether acetate until features become clear on the wafer, then wash it with ethyl alcohol. Finally, place the wafer on the thermostatic platform for hard-baking for 2 h.</li>
</ol>
2. Semi-3D Flow-focusing Microfluidic Chip Fabrication
<ol>
	<li>Mix PDMS monomer and its curing agent in a slightly different weight ratio for the top and the bottom layers, typically 10:1 for the top layer, and 8:1 for the bottom layer, using automatic ointment agitator for 4 min. 
	Note: The top and bottom PDMS slabs are prepared in a slightly different ratio (10:1 and 8:1 respectively) of PDMS base to curing agent, enhancing the bonding strength. When 5:1 ratio is chosen for the bottom layer, the stiffer bottom PDMS slab makes alignment difficult and reduces bonding strength due to lack of flexibility. In addition, the thickness of the bottom PDMS slab is about 1 mm for adapting the working distance of microscope. The chip easily deforms under high pressure when 15:1 ratio is chosen.</li>
	<li>Pour the mixture into the completed silicon mold in a 90-mm petri dish and provide a thickness of 2~3 mm. Place it in a vacuum chamber and degas until all air bubbles disappear. Cure at 80 &#176;C&#160;for 1 h in an oven.</li>
	<li>Allow the PDMS to cool to RT. Use a scalpel to cut the device at least 3 mm away from the features and slowly peel off the PDMS layer from the silicon wafer. Punch the dispersed phase inlet, the continuous phase inlet and the outlet in the top PDMS layer using a 0.75 mm diameter punch.</li>
	<li>Clean the PDMS with adhesive tape to remove dust particles. Plasma treat both the top and bottom PDMS layers simultaneously for 2 min in a 300 W plasma cleaner. Place the top layer on the bottom layer surface and slide the surfaces relatively until features are aligned viewing through a stereo microscope.</li>
	<li>Cure the device in an oven at 120 &#176;C&#160;for one day to enhance the strength and complete bonding.</li>
</ol>
3. Reagents Preparation
<ol>
	<li>Prepare the solution ofthe continuous phase: Hexadecane by dissolving 18 vol% silicone-based nonionic surfactant.</li>
	<li>Prepare the solution of the dispersed phase: hydrophilic poly (ethylene glycol) diacrylate (PEGDA, MW 255,) containing 2-hydroxy-4&#39;-(2-hydroxyethoxy)-2-methylpropiophenone (98%, MW 224) at a concentration of 5 mg/mL as the photo-initiator, and rhodamine B (95%, MW 479.01) at a concentration of 1 mmol/L as the fluorescent dyes.</li>
	<li>Fill the 1 mL reservoirs of pneumatic pressure controller with the continuous phase. Fill the 200 &#181;L gel loading tip with the dispersed phase.</li>
</ol>
4. System Preparation
<ol>
	<li>Place the semi-3D microfluidic device on the stage of an inverted optical microscope, viewing with a high-speed camera.</li>
	<li>Connect the fluorinated ethylene propylene (FEP) tube to the punched hole of the continuous phase by attaching a short stainless-steel tube, then insert the end of a gel loading tip into the punched hole of the dispersed phase. Insert a 20-cm length of FEP tube into the outlet in the device and place the end in a 15-mL centrifuge tube. 
	Note: The configuration of the system is illustrated in Figure 1.</li>
</ol>
5. Droplets Formation
<ol>
	<li>Place the device on the workbench of an inverted microscope, and ensure that the junction of different channels is roughly located in the position of the light source of the microscope. Focus the inverted microscope on a region that contains two-phases intersection, the orifice region, and the downstream channel.</li>
	<li>Set pressures of two phases using pneumatic pressure controller to deliver fluid slowly to the intersecting region, with 15 mbar for the dispersed phase and 30 mbar for the continuous phase. Wait 3 min for stabilization and equilibration until stable fluid flow carrying no bubbles and PDMS residue.</li>
	<li>Input the parameters on the user interface of the control software. Set the pressure of the dispersed phase as the base-level pressure of the system to a constant, for example, maintain it at 45 mbar. Increase the pressure of continuous phase until the emulsion breakup mode is changed from the jetting to the tip-streaming mode, then wait 5 min for stabilization.</li>
	<li>Place the FEP tube end connecting the outlet hole to the centrifuge tube to collect the droplets.</li>
</ol>
6. PEGDA Particles Collection and Characterization
<ol>
	<li>Fix the centrifuge tube aslant in the fixture. Obtain the PEGDA particles through the rapid solidification by UV exposure when liquid phase flow to the tube.</li>
	<li>When finishing the collecting process, sample and observe the PEGDA particles through the fluorescent microscope with 20X or 60X objects respectively. 
	Note.The digital fluorescent images captured by the camera are analyzed by a custom-made software routine. The image is first deconvolution based on the R-L algorithm17&#160;to eliminate the blurring effect of out-of-focusing light, and the sphere objects in the image are extracted based on the Canny edge detection method; finally, the diameter of each sphere object can be calculated using Hough transformation18. As a result, mean and standard deviation of the diameters of sphere objects in each image can be estimated.</li>
</ol>

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The authors have nothing to disclose.

The generation of droplets in the flow-focusing mode using 2D and semi-3D microfluidic device has previously been developed in a variety of reports8,9,15,19,20,21. In these systems, the aqueous liquid that could not be solidified was chosen as the dispersed phase, such as deionized water8,<s...

Droplet-based microfluidic systems have the ability to produce highly monodisperse droplets from nanometer to micrometer diameter range1 and hold great potential in the high-throughput drug discovery2, synthesis of biomolecules3,4, and the diagnostic testing5. Due to the unique advantages of smaller droplets, such as the greater surface area to volume ratio and the large-scale applications with consuming a few microliters of sample, the technology has attracted extensive interest in a broad range of fields. The emulsification of two immiscible liquids is one of the most typical methods to generate droplet. In previous reports in the field, researchers have developed a variety of different droplet formation geometries, including T-junction, flow-focusing and co-flowing geometries. In the T-junction geometry, the dispersed phase is delivered through a perpendicular channel into the main channel, in which the continuous phase flows6,7. In the typical two-dimensional (2D) flow-focusing8,9&#160;geometry, the dispersed phase flow is sheared from the lateral; and for the co-flowing geometry10,11, on the other hand, a capillary introducing the dispersed phase flow is placed co-axially inside a bigger capillary for co-flowing geometry, so that the dispersed phase flow is sheared from all directions.
The droplet size is controlled by adjusting channel size and flow rate ratio, and the minimum size produced by co-flowing or T-junction is limited to dozens of micrometers. For flow-focusing droplet formation system, three modes of droplet breakup form by adjusting the pressure ratio of two-phase and surfactant concentration, including the dripping regime, the jetting regime, and tip-streaming15. Tip-streaming mode is also called thread formation, and the appearance of a thin the thread drawing out from the tip of dispersed phase flow cone will be observed.Previous studies have demonstrated droplets less than a few micrometers could be generated though tip-streaming process in 2D or semi-3D flow-focusing device8,12. However, as an aqueous solution containing a very low concentration of PEGDA was used as the dispersed phase, the shrinkage ratio of PEGDA particles was about 60% of the original droplets in diameter after photo-polymerization, while PEGDA without dilution as the dispersed phase led to unstable tip-streaming mode12. Interfacial tension is an important parameter of emulsion process and it will decrease due to the addition of the surfactant into the continuous phase liquid,leading to decrease in droplet size, higher generation frequency13, highly curved tip, and preventing instability14. Furthermore, when the bulk surfactant concentration is much higher than the critical micelle concentration, the interfacial tension is approximately invariable in the saturated state13 and the tip-streaming mode can occur15.
Based on the above observations, in this paper, we developed a facile approach for PEGDA droplets generation using a semi-3D flow-focusing microfluidic device, fabricated by multi-layer soft lithography method. Different from the typical 2D flow-focusing device, the semi-3D flow-focusing device has a shallow dispersed phase channel and a deep continuous phase channel, so that the dispersed phase can be sheared from up and down beside lateral. This provides larger adjusting range for flow-focusing mode by reducing the energy and pressure required for droplet breakup. Different from the previous report12, the dispersed phase is pure PEGDAcontaining photo-initiator, making sure that the shrinkage ratio of PEGDA particles is lower than 10%16; and the continuous phase is the mixture of hexadecane dissolving with a high bulk concentration of the silicone-based nonionic surfactant. Size-controllable and uniform droplets were produced by adjusting the pressure ratio of two phases. The diameter of the droplets changes from 80 &#181;m to 1 &#181;m as the droplet breakup processes changes from the jetting mode to tip-streaming mode. In addition, the PEGDA particle was synthesized through photo-polymerization process under UV exposure. The droplet generation microfluidic system with ease of fabrication will provide more possibilities for biological applications.

<table><tbody><tr><td>Silicon wafer</td><td>Huashi Co., Ltd</td><td></td><td></td></tr><tr><td>SU-8 2025, 2100</td><td>Microchem Co.</td><td>Y111069</td><td></td></tr><tr><td>SU-8 developer</td><td>Microchem Co.</td><td>Y020100</td><td></td></tr><tr><td>Chromium mask</td><td>Qingyi Precision Mask Making Co., Ltd</td><td></td><td></td></tr><tr><td>polydimethylsiloxane(PDMS)</td><td>Dow Corning</td><td>Sylgard 184</td><td></td></tr><tr><td>poly(ethylene glycol) diacrylate (PEGDA)</td><td>Sigma</td><td>26570-48-9</td><td></td></tr><tr><td>2-hydroxy-40-(2-hydroxyethoxy)-2-methylpropiophenone</td><td>TCI</td><td>H1361-5G</td><td>photoinitiator</td></tr><tr><td>Hexadecane</td><td>Sigma</td><td>544-76- 3</td><td></td></tr><tr><td>ABIL EM 90</td><td>CHT</td><td>144243-53-8</td><td>surfactants</td></tr><tr><td>Rhodamine B</td><td>Aladdin</td><td>81-88-9</td><td>fluorescent dye</td></tr><tr><td>Spin Coater</td><td></td><td></td><td></td></tr><tr><td>Lithography machine</td><td></td><td></td><td></td></tr><tr><td>Automatic ointment agitator</td><td>Thinky</td><td>ARV-310</td><td></td></tr><tr><td>Oven</td><td>BluePard</td><td></td><td></td></tr><tr><td>Optical microscope</td><td>OLYMPUS</td><td>IX71</td><td></td></tr><tr><td>High-speed camera</td><td>Hamamatsu, Japan</td><td>ORCA-flash</td><td></td></tr><tr><td>MAESFLO Microfluidic Fluid Control System</td><td>FLUIGENT</td><td>MFCS-EZ</td><td></td></tr><tr><td>UV lamp</td><td>FUTANSI</td><td></td><td>365 nm UV light, 8000 MW/CM&lt;sup&gt;2&lt;/sup&gt;</td></tr></tbody></table>

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

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.

The semi-3D flow-focusing microfluidic chip was fabricated using multi-layer soft lithography techniques as described above. The fabrication process and results for master mold in the protocolare shown in Figure 2. The first layer, which provides a 65 µm wide channel for introducing the dispersed phase and a 50 µm wide orifice (Figure 2a), is 20 µm in thickness. An addition 130 µm thickness layer is used to provi...

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. ...

generation-size-controlled-poly-ethylene-glycol-diacrylate-droplets

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Learn to fabricate semi-3D flow-focusing microfluidic chips for producing size-controlled PEG diacrylate droplets. This method enables efficient droplet formation with minimal sample consumption for biochemical applications.

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

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

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