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 i...

<ol>
	<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 on a chip. 8, 198-220 (2008).</li><li>Xiao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+multifunctional+NaYF4:Er3+,Yb3+/PEGDA+hybrid+microspheres:+NIR-light-activated+photopolymerization+and+drug+delivery.">Novel multifunctional NaYF4:Er3+,Yb3+/PEGDA hybrid microspheres: NIR-light-activated photopolymerization and drug delivery.</a> Chemical communications. 49, 1527-1529 (2013).</li><li>Pan, M., Kim, M., Blauch, L., Tang, S. K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface-functionalizable+amphiphilic+nanoparticles+for+pickering+emulsions+with+designer+fluid-fluid+interfaces.">Surface-functionalizable amphiphilic nanoparticles for pickering emulsions with designer fluid-fluid interfaces.</a> RSC Adv. 6, 39926-39932 (2016).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+synthesis+of+rigid+nanovesicles+for+hydrophilic+reagents+delivery.">Microfluidic synthesis of rigid nanovesicles for hydrophilic reagents delivery.</a> Angewandte Chemie. 54, 3952-3956 (2015).</li><li>Shembekar, N., Chaipan, C., Utharala, R., Merten, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet-based+microfluidics+in+drug+discovery,+transcriptomics+and+high-throughput+molecular+genetics.">Droplet-based microfluidics in drug discovery, transcriptomics and high-throughput molecular genetics.</a> Lab on a chip. 16, 1314-1331 (2016).</li><li>Soh, G. Y., Yeoh, G. H., Timchenko, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numerical+investigation+on+the+velocity+fields+during+droplet+formation+in+a+microfluidic+T-junction.">Numerical investigation on the velocity fields during droplet formation in a microfluidic T-junction.</a> Chemical Engineering Science. 139, 99-108 (2016).</li><li>Chiarello, E., Derzsi, L., Pierno, M., Mistura, G., Piccin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Oil+Droplets+in+a+Non-Newtonian+Liquid+Using+a+Microfluidic+T-Junction.">Generation of Oil Droplets in a Non-Newtonian Liquid Using a Microfluidic T-Junction.</a> Micromachines. 6, 1825-1835 (2015).</li><li>Moyle, T. M., Walker, L. M., Anna, S. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water-in-Water+Droplets+by+Passive+Microfluidic+Flow+Focusing.">Water-in-Water Droplets by Passive Microfluidic Flow Focusing.</a> Analytical chemistry. 88, 3982-3989 (2016).</li><li>Zhu, P., Tang, X., Wang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+generation+in+co-flow+microfluidic+channels+with+vibration.">Droplet generation in co-flow microfluidic channels with vibration.</a> Microfluidics and Nanofluidics. , 20 (2016).</li><li>Kim, S. H., Kim, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+formation+of+double-emulsion+drops+in+sudden+expansion+channels.">Controlled formation of double-emulsion drops in sudden expansion channels.</a> Journal of colloid and interface science. 415, 26-31 (2014).</li><li>Jeong, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+generation+of+submicron+emulsion+droplets+via+highly+stable+tip-streaming+mode+in+microfluidic+devices.">Controlled generation of submicron emulsion droplets via highly stable tip-streaming mode in microfluidic devices.</a> Lab on a chip. 12, 1446-1453 (2012).</li><li>Peng, L., Yang, M., Guo, S. S., Liu, W., Zhao, X. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+interfacial+tension+on+droplet+formation+in+flow-focusing+microfluidic+device.">The effect of interfacial tension on droplet formation in flow-focusing microfluidic device.</a> Biomedical microdevices. 13, 559-564 (2011).</li><li>Nunes, J. K., Tsai, S. S., 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 (2013).</li><li>Anna, S. L., Mayer, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+tipstreaming+in+a+microfluidic+flow+focusing+device.">Microscale tipstreaming in a microfluidic flow focusing device.</a> Physics of Fluids. 18, 121512 (2006).</li><li>Liu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+synthesis+of+QD-encoded+PEGDA+microspheres+for+suspension+assay.">Microfluidic synthesis of QD-encoded PEGDA microspheres for suspension assay.</a> J. Mater. Chem. B. 4, 482-488 (2016).</li><li>Richardson, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bayesian-based+iterative+method+of+image+restoration.">Bayesian-based iterative method of image restoration.</a> Journal of the Optical Society of America. 62, 55-59 (1972).</li><li>Mukhopadhyay, P., Chaudhuri, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+Hough+Transform.">A survey of Hough Transform.</a> Pattern Recognition. 48, 993-1010 (2015).</li><li>Ward, T., Faivre, M., 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=Drop+production+and+tip-streaming+phenomenon+in+a+microfluidic+flow-focusing+device+via+an+interfacial+chemical+reaction.">Drop production and tip-streaming phenomenon in a microfluidic flow-focusing device via an interfacial chemical reaction.</a> Langmuir: the ACS journal of surfaces and colloids. 26, 9233-9239 (2010).</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+"flow+focusing"+in+microchannels.">Formation of dispersions using "flow focusing" in microchannels.</a> Applied Physics Letters. 82, 364 (2003).</li><li>Kim, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+production+of+emulsion+drops+using+an+electric+field+in+a+flow-focusing+microfluidic+device.">Controlled production of emulsion drops using an electric field in a flow-focusing microfluidic device.</a> Applied Physics Letters. 91, 133106 (2007).</li><li>Dangla, R., Gallaire, F., Baroud, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microchannel+deformations+due+to+solvent-induced+PDMS+swelling.">Microchannel deformations due to solvent-induced PDMS swelling.</a> Lab on a chip. 10, 2972-2978 (2010).</li><li>Chiu, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universally+applicable+three-dimensional+hydrodynamic+microfluidic+flow+focusing.">Universally applicable three-dimensional hydrodynamic microfluidic flow focusing.</a> Lab on a chip. 13, 1803-1809 (2013).</li><li>Oh, K. W., Lee, K., Ahn, B., Furlani, E. 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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 border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
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	</colgroup>
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">Silicon wafer</td>
			<td style="width:71px;">Huashi Co., Ltd</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">SU-8 2025, 2100</td>
			<td style="width:71px;">Microchem Co.</td>
			<td style="width:119px;">Y111069</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">SU-8 developer</td>
			<td style="width:71px;">Microchem Co.</td>
			<td style="width:119px;">Y020100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="91">
			<td height="91" style="height:91px;width:216px;">Chromium mask</td>
			<td style="width:71px;">Qingyi Precision Mask Making Co., Ltd</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">polydimethylsiloxane(PDMS)</td>
			<td style="width:71px;">Dow Corning</td>
			<td style="width:119px;">Sylgard 184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">poly(ethylene glycol) diacrylate (PEGDA)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">26570-48-9</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">2-hydroxy-40-(2-hydroxyethoxy)-2-methylpropiophenone</td>
			<td style="width:71px;">TCI</td>
			<td style="width:119px;">H1361-5G</td>
			<td style="width:167px;">photoinitiator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hexadecane</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">544-76- 3</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ABIL EM 90</td>
			<td style="width:71px;">CHT</td>
			<td style="width:119px;">144243-53-8</td>
			<td style="width:167px;">surfactants</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rhodamine B</td>
			<td style="width:71px;">Aladdin</td>
			<td style="width:119px;">81-88-9</td>
			<td style="width:167px;">fluorescent dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Spin Coater</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lithography machine</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Automatic ointment agitator</td>
			<td style="width:71px;">Thinky</td>
			<td style="width:119px;">ARV-310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oven</td>
			<td style="width:71px;">BluePard</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Optical microscope</td>
			<td style="width:71px;">OLYMPUS</td>
			<td style="width:119px;">IX71</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">High-speed camera</td>
			<td style="width:71px;">Hamamatsu, Japan</td>
			<td style="width:119px;">ORCA-flash</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:216px;">MAESFLO Microfluidic Fluid Control System</td>
			<td style="width:71px;">FLUIGENT</td>
			<td style="width:119px;">MFCS-EZ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">UV lamp</td>
			<td style="width:71px;">FUTANSI</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">365 nm UV light, 8000 MW/CM2</td>
		</tr>
	</tbody>
</table>

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 &#181;m wide channel for introducing the dispersed phase and a 50 &#181;m wide orifice (Figure 2a), is 20 &#181;m in thickness. An addition 130 &#181;m thickness la...

Watch this Scientific Journal Video about Generation of Size-controlled Poly ethylene Glycol Diacrylate Droplets via Semi-3-Dimensional Flow Focusing Microfluidic Devices at JoVE.com

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.

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 method can help to answer the key questions in the microfluidic droplet formation field, including fabricating the semi-three dimensional flow focusing PDMS chip, and producing the pure PGD droplets. The main advantage of this tactic is that the semi-3D flow focusing device provides larger testing grounds for flow focusing mold. Pure PGD makes sure that the ratio of PGD particles is lower than 10%The implications of this technique can be extended to many biochemical implications due to the greater surface area to volume ratio.Nozzle, large-scale applications will be benefit for this technique with consuming a few microliters of sample. Now this method can provide insight into PGD droplet formation. It can also be applied to other systems such as gas-equipped flow focusing analyzing.Generally, individuals new to this method will struggle because of clogging in the orifice and the instability of tip streaming mold. Of this method is critical, as semi-3d flow focusing chip fabrication is difficult to learn because the alignment of top and bottom PDMS layers to fabricate semifluidic chip by hands is not reviewed. Design the two photo masks using standard techniques.Use two separate layers for mask one and two in the same drawing file to ensure that all connections line up between the different channels. Mask one contains the dispersed phased inlet channel and an orifice, while mask two contains the continuous phase inlet channel, the filter, and the outlet. Use the two alignment marks and print the different layers independently to a chrome plate on glass.Next, in a designated photolithography lab, place a clean, 3-inch diameter silicon wafer on a spin coater and turn on the vacuum to affix the wafer to the spin chuck. Spin coat two to three milliliters of SU-8 2025 negative photoresist onto the wafer for 10 seconds at 1, 000 rpm. Then change the speed to 3, 000 rpms for 30 seconds.This will provide a layer thickness of 20 microns for the first layer. Soft bake the first layer by placing it on a 95 degrees celsius hot plate for six minutes. And then, remove the wafer and let it cool back to room temperature.Expose the soft-baked wafer to mask number one under a collimated UV light for 18 seconds. Following UV exposure, post bake the wafer on a 95 degrees celsius hot plate for six minutes. Afterwards, allow the wafer to cool back to room temperature.Repeat the spincoating process a second time using two to three milliliters of SU-8 2100 negative photoresist. Spin the wafer for 10 seconds at 1, 000 rpm, and then at 2, 000 rpms for 30 seconds. This provides a second layer thickness of 130 microns.After soft baking the wafer on a 95 degrees celsius hot plate for 35 minutes, line up mask number two on the second layer photoresist, and expose it to UV light for 30 seconds. Then, post bake it on a 95 degree celsius hot plate for seven minutes. Once cooled back to room temperature, develop the wafer by immersing in a stirred bath of 50 milliliters of propylene glycol methyl ether acetate until features become clear on the wafer.Then, wash the wafer with ethyl alcohol. Finally, place the wafer on the thermostatic platform and hard bake it at 95 degrees celsius for two hours. Mix a PDMS monomer and its curing agent for four minutes at a 10:1 ratio for the top layer and an 8:1 ratio for the bottom layer.Using an automatic ointment agitator, place the silicon wafer into a 90 millimeter Petri dish, and then pour the 8:1 ratio PDMS mixture over the mold to a thickness of two to three millimeters. Then, degas the PDMS in a vacuum chamber until all the bubbles disappear. Place the Petri dish in an oven and cure the PDMS at 80 degrees celsius for one hour.Once cooled back to room temperature, use a scalpel to cut the device at least three millimeters away from the features, and slowly peel the PDMS layer from the silicon wafer. Then, punch the dispersed phase inlet, the continuous phase inlet, and the outlet in the top PDMS layer using a 0.75 millimeter diameter punch. Clean the PDMS surface with adhesive tape to remove any dust particles.Then, place both the top and bottom PDMS layers simultaneously into a plasma cleaner, and expose to plasma for two minutes at 300 watts. Immediately following plasma exposure, place the top layer on the bottom layer and slide the surfaces relatively until features are aligned viewing through a stereo microscope. Cure the device in an oven at 120 degrees celsius for one day to enhance its strength and complete the bonding.To begin, prepare the continuous phase solution by dissolving 18%of a silicone-based non-ionic surfactant in hexadecane. Next, prepare the solution of the dispersed phase by starting with a hydrophilic peg diacrylate, one millimole per liter of a fluorescent dye, and adding five milligrams per milliliter of a photo initiator. Fill the one milliliter reservoirs of a pneumatic pressure controller with the continuous phase.Then, fill a 200 microliter gel-loading tip with the dispersed phase. Place the semi-3D microfluidic device on the stage of an inverted optical microscope equipped with a high speed camera. Next, connect a fluorinated ethylene propylene tube to the punched hole of the continuous phase by first attaching it to a short, stainless steel tube, inserting the tube into the end of a gel loading tip, and placing the tip into the punched hole of the dispersed phase.Then, insert a 20 cm long FEP tube into the outlet of the device, and place the end into a Petri dish. Focus the inverted microscope on a region that contains the two phase intersection, the orifice region, and the downstream channel. Then, set pressures of the two phases using a connected pneumatic pressure controller.Set the dispersed phase at 15 millibar, and the continuous phase at 30 millibar. Wait three minutes for the pressure to stabilize, and stable fluid flow is reached, carrying no bubbles or PDMS residue. Next, set the pressure of the dispersed phase as the base level pressure of the system to 45 millibar.Increase the pressure of the continuous phase until the emulsion breakup mode is changed from jetting to the tip streaming mode. Then, wait five minutes for it to stabilize. Once set up, place the tube end connecting the outlet hole to Petri dish, and collect the droplets.Expose them to UV light where they will rapidly solidify into spheres. Because of the difference of depth of the dispersed phase channel, and the continuous phase channel, the dispersed phase flow is squeezed on all directions by the continuous phase flow. As a result, symmetric conical liquid tip forms to produce droplets continuously.The size of droplets is changed by the pressure ratio of dispersed and continuous phases flow. Here, the pressure of the continuous phase is modified to affect the sheering force so that the droplet breakup processes change from the jetting mode to the tip streaming mode. The droplets are solidified by photopolymerization forming particles upon exiting the chip.The droplets shown here were formed with different pressure ratios. The resulting droplet radius has an inverse relationship with the continuous phase to dispersed phase pressure ratio. This technique paves the way for researchers in the field of biological applications and provides a powerful tool for drug discovery, synthesis of biomolecules and the diagnostic testing.After watching this video, you should have a good understanding of how to fabricate semi-three dimensional microfluidic chips, and how to produce a few micro PGD droplets. Don't forget that working with photoresist PGMEA has a can be extremely hazardous and precautions such as wearing disposable gloves and a mask should always be taken while performing this procedure.

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

Research

JoVE Journal

Engineering

7.6K ビュー.  Tsinghua University. This method can help to answer the key questions in the microfluidic droplet formation field, including fabricating the semi-three dimensional flow focusing PDMS chip, and producing the pure PGD droplets. The main advantage of this tactic is that the semi-3D flow focusing device provides larger testing grounds for flow focusing mold. Pure PGD makes sure that the ratio of PGD particles is lower than 10%The implications of this technique can be extended to many biochemical implications due to t...

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

Determining 3D Flow Fields via Multi-camera Light Field Imaging

In the field of fluid mechanics, the resolution of computational schemes has outpaced experimental methods and widened the gap between predicted and observed phenomena in fluid flows. Thus, a need exists for an accessible method capable of resolving three-dimensional (3D) data sets for a range of problems. We present a novel technique for performing quantitative 3D imaging of many types of flow fields. The 3D technique enables investigation of complicated velocity fields and bubbly flows. Measurements of these types present a variety of challenges to the instrument. For instance, optically dense bubbly multiphase flows cannot be readily imaged by traditional, non-invasive flow measurement techniques due to the bubbles occluding optical access to the interior regions of the volume of interest. By using Light Field Imaging we are able to reparameterize images captured by an array of cameras to reconstruct a 3D volumetric map for every time instance, despite partial occlusions in the volume. The technique makes use of an algorithm known as synthetic aperture (SA) refocusing, whereby a 3D focal stack is generated by combining images from several cameras post-capture 1. Light Field Imaging allows for the capture of angular as well as spatial information about the light rays, and hence enables 3D scene reconstruction. Quantitative information can then be extracted from the 3D reconstructions using a variety of processing algorithms. In particular, we have developed measurement methods based on Light Field Imaging for performing 3D particle image velocimetry (PIV), extracting bubbles in a 3D field and tracking the boundary of a flickering flame. We present the fundamentals of the Light Field Imaging methodology in the context of our setup for performing 3DPIV of the airflow passing over a set of synthetic vocal folds, and show representative results from application of the technique to a bubble-entraining plunging jet.

A technique for performing quantitative three-dimensional (3D) imaging for a range of fluid flows is presented. Using concepts from the area of Light Field Imaging, we reconstruct 3D volumes from arrays of images. Our 3D results span a broad range including velocity fields and multi-phase bubble size distributions.

In the field of fluid mechanics, the resolution of computational schemes has outpaced experimental methods and widened the gap between predicted and ...

Thermal Measurement Techniques in Analytical Microfluidic Devices

Thermal measurement techniques have been used for many applications such as thermal characterization of materials and chemical reaction detection. Micromachining techniques allow reduction of the thermal mass of fabricated structures and introduce the possibility to perform high sensitivity thermal measurements in the micro-scale and nano-scale devices. Combining thermal measurement techniques with microfluidic devices allows performing different analytical measurements with low sample consumption and reduced measurement time by integrating the miniaturized system on a single chip. The procedures of thermal measurement techniques for particle detection, material characterization, and chemical detection are introduced in this paper.

Here, we present three protocols for thermal measurements in microfluidic devices.

Thermal measurement techniques have been used for many applications such as thermal characterization of materials and chemical reaction detection. ...

Fabrication and Operation of Acoustofluidic Devices Supporting Bulk Acoustic Standing Waves for Sheathless Focusing of Particles

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must be smaller than the incident wavelength of sound and the width of the fluidic channels are typically tens to hundreds of micrometers across, acoustofluidic devices typically use ultrasonic waves generated from a piezoelectric transducer pulsating at high frequencies (in the megahertz range). At characteristic frequencies that depend on the geometry of the device, it is possible to induce the formation of standing waves that can focus particles along desired fluidic streamlines within a bulk flow. Here, we describe a method for the fabrication of acoustophoretic devices from common materials and clean room equipment. We show representative results for the focusing of particles with positive or negative acoustic contrast factors, which move towards the pressure nodes or antinodes of the standing waves, respectively. These devices offer enormous practical utility for precisely positioning large numbers of microscopic entities (e.g., cells) in stationary or flowing fluids for applications ranging from cytometry to assembly.

Acoustofluidic devices use ultrasonic waves within microfluidic channels to manipulate, concentrate and isolate suspended micro and nanoscopic entities. This protocol describes the fabrication and operation of such a device supporting bulk acoustic standing waves to focus particles in a central streamline without the aid of sheath fluids.

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must ...

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

Preparation and 3D Tracking of Catalytic Swimming Devices

We report a method to prepare catalytically active Janus colloids that "swim" in fluids and describe how to determine their 3D motion using fluorescence microscopy. One commonly deployed method for catalytically active colloids to produce enhanced motion is via an asymmetrical distribution of catalyst. Here this is achieved by spin coating a dispersed layer of fluorescent polymeric colloids onto a flat planar substrate, and then using directional platinum vapor deposition to half coat the exposed colloid surface, making a two faced "Janus" structure. The Janus colloids are then re-suspended from the planar substrate into an aqueous solution containing hydrogen peroxide. Hydrogen peroxide serves as a fuel for the platinum catalyst, which is decomposed into water and oxygen, but only on one side of the colloid. The asymmetry results in gradients that produce enhanced motion, or "swimming". A fluorescence microscope, together with a video camera is used to record the motion of individual colloids. The center of the fluorescent emission is found using image analysis to provide an x and y coordinate for each frame of the video. While keeping the microscope focal position fixed, the fluorescence emission from the colloid produces a characteristic concentric ring pattern which is subject to image analysis to determine the particles relative z position. In this way 3D trajectories for the swimming colloid are obtained, allowing swimming velocity to be accurately measured, and physical phenomena such as gravitaxis, which may bias the colloids motion to be detected.

A method to prepare catalytically active Janus colloids that can "swim" in fluids and determine their 3D trajectories is presented.

We report a method to prepare catalytically active Janus colloids that "swim" in fluids and describe how to determine their 3D motion using fluorescence ...

Solvent Bonding for Fabrication of PMMA and COP Microfluidic Devices

Thermoplastic microfluidic devices offer many advantages over those made from silicone elastomers, but bonding procedures must be developed for each thermoplastic of interest. Solvent bonding is a simple and versatile method that can be used to fabricate devices from a variety of plastics. An appropriate solvent is added between two device layers to be bonded, and heat and pressure are applied to the device to facilitate the bonding. By using an appropriate combination of solvent, plastic, heat, and pressure, the device can be sealed with a high quality bond, characterized as having high bond coverage, bond strength, optical clarity, durability over time, and low deformation or damage to microfeature geometry. We describe the procedure for bonding devices made from two popular thermoplastics, poly(methyl-methacrylate) (PMMA), and cyclo-olefin polymer (COP), as well as a variety of methods to characterize the quality of the resulting bonds, and strategies to troubleshoot low quality bonds. These methods can be used to develop new solvent bonding protocols for other plastic-solvent systems.

Solvent bonding is a simple and versatile method for fabricating thermoplastic microfluidic devices with high quality bonds. We describe a protocol to achieve strong, optically clear bonds in PMMA and COP microfluidic devices that preserve microfeature details, by a judicious combination of pressure, temperature, an appropriate solvent, and device geometry.

Thermoplastic microfluidic devices offer many advantages over those made from silicone elastomers, but bonding procedures must be developed for each ...

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the main trend in the advanced development of flow cytometry. Notably, adding high-resolution imaging capabilities allows for the complex morphological analysis of cellular/sub-cellular structures. This is not possible with standard flow cytometers. However, it is valuable for advancing our knowledge of cellular functions and can benefit life science research, clinical diagnostics, and environmental monitoring. Incorporating imaging capabilities into flow cytometry compromises the assay throughput, primarily due to the limitations on speed and sensitivity in the camera technologies. To overcome this speed or throughput challenge facing imaging flow cytometry while preserving the image quality, asymmetric-detection time-stretch optical microscopy (ATOM) has been demonstrated to enable high-contrast, single-cell imaging with sub-cellular resolution, at an imaging throughput as high as 100,000 cells/s. Based on the imaging concept of conventional time-stretch imaging, which relies on all-optical image encoding and retrieval through the use of ultrafast broadband laser pulses, ATOM further advances imaging performance by enhancing the image contrast of unlabeled/unstained cells. This is achieved by accessing the phase-gradient information of the cells, which is spectrally encoded into single-shot broadband pulses. Hence, ATOM is particularly advantageous in high-throughput measurements of single-cell morphology and texture &#8211; information indicative of cell types, states, and even functions. Ultimately, this could become a powerful imaging flow cytometry platform for the biophysical phenotyping of cells, complementing the current state-of-the-art biochemical-marker-based cellular assay. This work describes a protocol to establish the key modules of an ATOM system (from optical frontend to data processing and visualization backend), as well as the workflow of imaging flow cytometry based on ATOM, using human cells and micro-algae as the examples.

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

This protocol describes the implementation of an asymmetric-detection time-stretch optical microscopy system for single-cell imaging in ultrafast microfluidic flow and its applications in imaging flow cytometry.

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the ...

Flow-assisted Dielectrophoresis: A Low Cost Method for the Fabrication of High Performance Solution-processable Nanowire Devices

Flow-assisted dielectrophoresis (DEP) is an efficient self-assembly method for the controllable and reproducible positioning, alignment, and selection of nanowires. DEP is used for nanowire analysis, characterization, and for solution-based fabrication of semiconducting devices. The method works by applying an alternating electric field between metallic electrodes. The nanowire formulation is then dropped onto the electrodes which are on an inclined surface to create a flow of the formulation using gravity. The nanowires then align along the gradient of the electric field and in the direction of the liquid flow. The frequency of the field can be adjusted to select nanowires with superior conductivity and lower trap density. 
In this work, flow-assisted DEP is used to create nanowire field effect transistors. Flow-assisted DEP has several advantages: it allows selection of nanowire electrical properties; control of nanowire length; placement of nanowires in specific areas; control of orientation of nanowires; and control of nanowire density in the device. 
The technique can be expanded to many other applications such as gas sensors and microwave switches. The technique is efficient, quick, reproducible, and it uses a minimal amount of dilute solution making it ideal for the testing of novel nanomaterials. Wafer scale assembly of nanowire devices can also be achieved using this technique, allowing large numbers of samples for testing and large-area electronic applications.

In this paper, flow assisted dielectrophoresis is demonstrated for the self-assembly of nanowire devices. The fabrication of a silicon nanowire field effect transistor is shown as an example.

Flow-assisted dielectrophoresis (DEP) is an efficient self-assembly method for the controllable and reproducible positioning, alignment, and selection of ...

Experimental Investigation of the Flow Structure over a Delta Wing Via Flow Visualization Methods

It is well known that the flow field over a delta wing is dominated by a pair of counter rotating leading edge vortices (LEV). However, their mechanism is not well understood. The flow visualization technique is a promising non-intrusive method to illustrate the complex flow field spatially and temporally. A basic flow visualization setup consists of a high-powered laser and optic lenses to generate the laser sheet, a camera, a tracer particle generator, and a data processor. The wind tunnel setup, the specifications of devices involved, and the corresponding parameter settings are dependent on the flow features to be obtained.
Normal smoke wire flow visualization uses a smoke wire to demonstrate the flow streaklines. However, the performance of this method is limited by poor spatial resolution when it is conducted in a complex flow field. Therefore, an improved smoke flow visualization technique has been developed. This technique illustrates the large-scale global LEV flow field and the small-scale shear layer flow structure at the same time, providing a valuable reference for later detailed particle image velocimetry (PIV) measurement.
In this paper, the application of the improved smoke flow visualization and PIV measurement to study the unsteady flow phenomena over a delta wing is demonstrated. The procedure and cautions for conducting the experiment are listed, including wind tunnel setup, data acquisition, and data processing. The representative results show that these two flow visualization methods are effective techniques for investigating the three-dimensional flow field qualitatively and quantitatively.

Here, we present a protocol to observe unsteady vortical flows over a delta wing using a modified smoke flow visualization technique and investigate the mechanism responsible for the oscillations of the leading-edge vortex breakdown locations.

It is well known that the flow field over a delta wing is dominated by a pair of counter rotating leading edge vortices (LEV). However, their mechanism is ...

Safe Experimentation in Optical Levitation of Charged Droplets Using Remote Labs

The work presents an experiment that allows the study of many fundamental physical processes, such as photon pressure, diffraction of light or the motion of charged particles in electrical fields. In this experiment, a focused laser beam pointing upwards levitate liquid droplets. The droplets are levitated by the photon pressure of the focused laser beam which balances the gravitational force. The diffraction pattern created when illuminated with laser light can help measure the size of a trapped droplet. The charge of the trapped droplet can be determined by studying its motion when a vertically directed electrical field is applied. There are several reasons motivating this experiment to be remotely controlled. The investments required for the setup exceeds the amount normally available in undergraduate teaching laboratories. The experiment requires a laser of Class 4, which is harmful to both skin and eyes and the experiment uses voltages that are harmful.

Optical levitation is a method for levitating micrometer-sized dielectric objects using laser light. Utilizing computers and automation systems, an experiment on optical levitation can be controlled remotely. Here, we present a remotely controlled optical levitation system that is used both for educational and research purposes.

The work presents an experiment that allows the study of many fundamental physical processes, such as photon pressure, diffraction of light or the motion ...