Name: rapid fabrication of custom microfluidic devices for research and educational applications
Uploaded: 2019-11-20T13:00:00
Description: Watch this Scientific Journal Video about Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications at JoVE.com

The work in this manuscript was supported in part by the National Science Foundation (NSF) (Grant No. CBET-1553826) (and associated ROA supplement) and the National Institutes of Health (NIH) (Grant No. R35GM124935) to J.Z., and the Notre Dame Melchor Visiting Faculty fund to F.O. We would like to thank Jenna Sjoerdsma and Basar Bilgi&#231;er for providing mammalian cells and culture protocols and Fabio Sacco for assistance with supplementary figures.

1. Design
<ol>
	<li>Identify an application for the devices and list the channel/chamber components that will be required. 
	NOTE: All devices will require input and output channels. Devices used for microscopy will require an imaging chamber. More complex devices will require channels and chambers situated in multiple layers.</li>
	<li>Start by hand-drawing each layer, considering how the device&#8217;s functionality is affected by the superposition of the layers.</li>
	<li>Draw the final designs on a computer us...

<ol>
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K., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineers+are+from+PDMS-land,+Biologists+are+from+Polystyrenia.">Engineers are from PDMS-land, Biologists are from Polystyrenia.</a> Lab on a Chip. 12 (7), 1224 (2012).</li><li>Zhang, B., Korolj, A., Lai, B. F. L., Radisic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+organ-on-a-chip+engineering.">Advances in organ-on-a-chip engineering.</a> Nature Reviews Materials. 3 (8), 257-278 (2018).</li><li>Bartholomeusz, D. A., Boutte, R. W., Andrade, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xurography:+rapid+prototyping+of+microstructures+using+a+cutting+plotter.">Xurography: rapid prototyping of microstructures using a cutting plotter.</a> Journal of Microelectromechanical Systems. 14 (6), 1364-1374 (2005).</li><li>Mart&#237;nez-Hern&#225;ndez, K. J., Rovira-Figueroa, N. D., Ontiveros, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Implementation and Assessment of Student-Made Microfluidic Devices in the General Chemistry Laboratory. , (2016).</li><li>Levis, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+on+the+fly:+Inexpensive+rapid+fabrication+of+thermally+laminated+microfluidic+devices+for+live+imaging+and+multimodal+perturbations+of+multicellular+systems.">Microfluidics on the fly: Inexpensive rapid fabrication of thermally laminated microfluidic devices for live imaging and multimodal perturbations of multicellular systems.</a> Biomicrofluidics. 13 (2), 024111 (2019).</li><li>Subramaniam, A., Sethuraman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+18+-+Biomedical+Applications+of+Nondegradable+Polymers.">Chapter 18 - Biomedical Applications of Nondegradable Polymers.</a> Natural and Synthetic Biomedical Polymers. , 301-308 (2014).</li><li>Yuen, P. K., Goral, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+rapid+prototyping+of+flexible+microfluidic+devices+using+a+desktop+digital+craft+cutter.">Low-cost rapid prototyping of flexible microfluidic devices using a desktop digital craft cutter.</a> Lab Chip. 10 (3), 384-387 (2010).</li><li>Oya, K., 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=Surface+Characteristics+of+Polyethylene+Terephthalate+(PET)+Film+Exposed+to+Active+Oxygen+Species+Generated+via+Ultraviolet+(UV)+Lights+Irradiation+in+High+and+Low+Humidity+Conditions.">Surface Characteristics of Polyethylene Terephthalate (PET) Film Exposed to Active Oxygen Species Generated via Ultraviolet (UV) Lights Irradiation in High and Low Humidity Conditions.</a> Journal of Photopolymer Science and Technology. 27 (3), 409-414 (2014).</li><li>Narciso, C. E., Contento, N. M., Storey, T. J., Hoelzle, D. J., Zartman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+Applied+Mechanical+Loading+Stimulates+Intercellular+Calcium+Waves+in+Drosophila+Wing+Discs.">Release of Applied Mechanical Loading Stimulates Intercellular Calcium Waves in Drosophila Wing Discs.</a> Biophysical Journal. 113 (2), 491-501 (2017).</li><li>Suh, Y. K., Kang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Review+on+Mixing+in+Microfluidics.">A Review on Mixing in Microfluidics.</a> Micromachines. 1 (3), 82-111 (2010).</li><li>Jahn, A., Vreeland, W. N., Gaitan, M., Locascio, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+Vesicle+Self-Assembly+in+Microfluidic+Channels+with+Hydrodynamic+Focusing.">Controlled Vesicle Self-Assembly in Microfluidic Channels with Hydrodynamic Focusing.</a> Journal of the American Chemical Society. 126 (9), 2674-2675 (2004).</li><li>Weibel, D., Whitesides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+microfluidics+in+chemical+biology.">Applications of microfluidics in chemical biology.</a> Current Opinion in Chemical Biology. 10 (6), 584-591 (2006).</li><li>Chen, X., Li, T., Shen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CO2+Laser+Ablation+of+Microchannel+on+PMMA+Substrate+for+Effective+Fabrication+of+Microfluidic+Chips.">CO2 Laser Ablation of Microchannel on PMMA Substrate for Effective Fabrication of Microfluidic Chips.</a> International Polymer Processing. 31 (2), 233-238 (2016).</li><li>Chen, X., Shen, J., Zhou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+fabrication+of+a+four-layer+PMMA-based+microfluidic+chip+using+CO2-laser+micromachining+and+thermal+bonding.">Rapid fabrication of a four-layer PMMA-based microfluidic chip using CO2-laser micromachining and thermal bonding.</a> Journal of Micromechanics and Microengineering. 26 (10), 107001 (2016).</li></ol>

While microfluidics are increasingly present in the toolbox of laboratories around the world, the pace of adoption has been disappointing, given the potential for its positive impact16. Low cost and high efficiency of microfluidic device fabrication are essential to accelerate adoption of this technology in the average research laboratory. The method described here uses multiple film layers to create two and three-dimensional devices at a fraction of the time and cost required by lithographic meth...

Microfluidics enables fluid control at small scales, with volumes ranging from microliters (1 x 10-6 L) to picoliters (1 x 10-12 L). This control has been made possible in part due to the application of microfabrication techniques borrowed from the microprocessor industry1. The use of micro-sized networks of channels and chambers allows the user to take advantage of the distinct physical phenomena characteristic of small dimensions. For example, at the micrometer scale, fluids can be manipulated using laminar flow, where viscous forces dominate inertial forces. As a result, diffusive transport becomes the prominent feature of microfluidics, and can be studied quantitatively and experimentally. These systems can be properly understood using Fick&#8217;s laws, Brownian motion theory, the heat equation, and/or the Navier-Stokes equations, which are important derivations in the fields of fluid mechanics and transport phenomena2.
Because many groups in the biological sciences study complex systems at the microscopic level, it was originally thought that microfluidic devices would have an immediate and significant impact on research applications in biology2,3. This is due to diffusion being dominant in the transport of small molecules across membranes or within a cell, and the dimensions of cells and microorganisms are an ideal match for sub-millimeter systems and devices. Therefore, there was significant potential for enhancing the way in which cellular and molecular experimentation is conducted. However, wide adoption of microfluidic technologies by biologists has lagged behind expectations4. A simple reason for the lack of technology transfer may be the disciplinary boundaries separating engineers and biologists. Custom device design and fabrication have remained just outside of the capabilities of most biological research groups, making them dependent on external expertise and facilities. Lack of familiarity with potential applications, cost, and the time required for design-iteration are also significant barriers for new adopters. It is likely that these barriers have had the effect of disrupting innovation and preventing the widespread application of microfluidics to address challenges in the biological sciences.
A case in point: Since the late 1990&#8217;s soft-photolithography has been the method of choice for the fabrication of microfluidic devices. PDMS (polydimethylsiloxane, a silicone-based organic polymer) is a widely used material because of its physical properties, such as transparency, deformability, and biocompatibility5. The technique has enjoyed great success, with lab-on-a-chip and organ-on-a-chip devices continually being developed on this platform6. Most of the groups working on these technologies, however, are found in engineering departments or have strong ties to them4. Lithography usually requires clean-rooms for the fabrication of molds and specialized bonding equipment. For many groups, this makes standard PDMS devices less than ideal due their capital costs and lead-time, particularly when there is a need to make repeated design modifications. Furthermore, the technology is mostly inaccessible to the average biologist and to students without access to specialized engineering laboratories. It has been proposed that for microfluidic devices to be widely adopted, they must mimic some of the qualities of materials commonly used by biologists. For example, polystyrene used for cell culture and bioassays is inexpensive, disposable, and amenable to mass production. In contrast, industrial manufacturing of PDMS-based microfluidics has never been realized because of its mechanical softness, surface treatment instability, and gas permeability5. Because of these limitations, and with the goal of solving technical challenges using customized devices built &#8220;in-house&#8221;, we describe an alternative method that utilizes xurography7,8,9 protocols and thermal lamination. This method can be adopted with little capital and time investment.
PETLs are fabricated using polyethylene terephthalate (PET) film, coated with the thermoadhesive ethylene-vinyl acetate (EVA). Both materials are widely used in consumer products, are biocompatible and are readily available at minimal cost10. PET/EVA film can be obtained in the form of laminating pouches or rolls. Using a computer-controlled craft cutter commonly found in hobbyist or craft stores, channels are cut out of a single film sheet to define the device&#8217;s architecture11. The channels are then sealed by applying additional film (or glass) layers that are bonded using an (office) thermal laminator (Figure 1A). Perforated, self-adhesive vinyl bumpers are added to facilitate access to the channels. Fabrication times range from 5 to 15 min, which allows rapid design iteration. All the equipment and materials used to make PETLs are commercially accessible and affordable (&#60;350 USD starting cost, compared to thousands of USDs for lithography). Therefore, PETLs provide a novel solution to two main problems posed by conventional microfluidics: affordability and time effectiveness (See PDMS/PETL comparison in Supplementary Tables 1, 2).
In addition to providing researchers with the opportunity to design and fabricate their own devices, PETLs can be easily adopted in the classroom because they are simple and intuitive to use. PETLs can be included in high school and college curricula8, where they are used to help students better understand physical, chemical and biological concepts, like diffusion, laminar flow, micromixing, nanoparticle synthesis, gradient formation and chemotaxis.
In this work we illustrate the overall workflow for the fabrication of model PETLs chips with different levels of complexity. The first device is used to facilitate imaging of cells and micro-organs in a small chamber. The second, more complex device consists of several layers and materials, and is used for research in mechanobiology9. Lastly, we built a device that displays several fluid dynamics concepts (hydrodynamic focusing, laminar flow, diffusive transport and micromixing) for educational purposes. The workflow and device designs presented here can be easily tailored for a large range of purposes in both the research and classroom settings.

<table>
	<tbody>
		<tr>
			<td>Biopsy punch (1mm)</td>
			<td>Miltex</td>
			<td>33-31AA</td>
			<td>Optional, replaces rotary tool set up</td>
		</tr>
		<tr>
			<td>Blunt needles</td>
			<td>Janel, Inc.</td>
			<td>JEN JG18-0.5X-90</td>
			<td>Remove plastic and attach to Tygon tubing</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Any</td>
			<td></td>
			<td>24 x 60 mm are preferred</td>
		</tr>
		<tr>
			<td>Cutting Mat and blades</td>
			<td>Silhouette America or Nicapa</td>
			<td>www.silhouetteamerica.com/shop/blades-and-mats</td>
			<td>Re-use/Disposables</td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch/3M</td>
			<td>667</td>
			<td>Small amounts, any width or brand</td>
		</tr>
		<tr>
			<td>PEEK tubing</td>
			<td>IDEX/any</td>
			<td>1581L</td>
			<td>Different configurations available. Consider using Tygon tubing intead, if not already using PEEK</td>
		</tr>
		<tr>
			<td>PET/EVA thermal laminate film</td>
			<td>Scotch/3M &#38; Transcendia</td>
			<td>TP3854-200,TP5854-100 &#38; transcendia.com/products/trans-kote-pet</td>
			<td>3 - 6 mil (mil = 1/1000 inch) laminating pouches or rolls.</td>
		</tr>
		<tr>
			<td>PVC film - Cling Wrap</td>
			<td>Glad / Any</td>
			<td></td>
			<td>Food wrapping</td>
		</tr>
		<tr>
			<td>Rotary tool-drill</td>
			<td>Dremel/Any</td>
			<td>200-121 or other</td>
			<td>1/32 and 3/64&#34; drill bits from Dremel recommended</td>
		</tr>
		<tr>
			<td>Rubber Roller</td>
			<td>Speedball</td>
			<td>4126</td>
			<td>To facilitate adhesion, any brand will work</td>
		</tr>
		<tr>
			<td>Scissors &#38; tweezers</td>
			<td>Any</td>
			<td>Fiskars-Inch-Titanium-Softgrip-Scissors |Cole-Parmer&#160;&#8211;# UX-07387-12</td>
			<td>Quality brands are recommended</td>
		</tr>
		<tr>
			<td>Silhouette CAMEO Craft cutter</td>
			<td>Silhouette America</td>
			<td>www.silhouetteamerica.com/shop/cameo/SILHOUETTE-CAMEO-3-4T</td>
			<td>Preferred craft cutter</td>
		</tr>
		<tr>
			<td>Silhouette Studio software</td>
			<td>Silhouette America</td>
			<td>www.silhouetteamerica.com/software</td>
			<td>Controls the craft cutter and provides drawing tools (free download MAC and PC)</td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Harvard Apparatus or New Era</td>
			<td>70-4504 or NE-300</td>
			<td>Pumps are ideal, pipettes or burettes can be used.</td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Any</td>
			<td></td>
			<td>1-3mL</td>
		</tr>
		<tr>
			<td>Thermal laminator</td>
			<td>Scotch/3M</td>
			<td>TL906</td>
			<td>Standard home/office model</td>
		</tr>
		<tr>
			<td>Tygon tubing (E-3603)</td>
			<td>Cole-Parmer</td>
			<td>EW-06407-70</td>
			<td>Use with blunt needle tips</td>
		</tr>
		<tr>
			<td>Vinyl furniture bumpers</td>
			<td>DerBlue/3M/ Everbilt</td>
			<td>Clear, self-adhesive (6 x 2 mm and 8 x 3 mm)</td>
			<td>Round bumpers are recommended</td>
		</tr>
	</tbody>
</table>

rapid fabrication of custom microfluidic devices for research and educational applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

In addition to low cost and rapid iteration, PETL technology can be easily customized to solve specific challenges. First, we describe a simple device consisting of a glass coverslip, a chamber layer, a channel layer, and an inlet/outlet layer (Figure 2). This device was designed to facilitate the imaging of cells and micro-organs under constant flow. Culture medium is replenished at low flow rates to encourage nutrient and gas exchange. The round chamber features a glass bottom, which allow...

Watch this Scientific Journal Video about Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications at JoVE.com

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to ...

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