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 using any software that allows for drawing lines and shapes.
	<ol>
		<li>Draw each layer separately using black, solid lines and shapes devoid of shades. Line thickness of 6 or more points are recommended. At this stage, the dimensions of channel and chamber features are less important than the overall proportions.</li>
		<li>Use the copy-and-paste function when creating features and superimposing layers. See Figure 1B for examples of layer drawings.</li>
	</ol>
	</li>
	<li>Import each layer into the craft cutter software (Figure 1C). Do this by making a screen capture of the drawn design and using a drag and drop approach.
	<ol>
		<li>Create a new document in the craft cutter software (free download). Drop the image file on the mat displayed. The software will recognize most image files.</li>
		<li>Enlarge the image to facilitate processing by pulling from a corner. The design can now be recognized by the software using the trace function. 
		NOTE: Users may produce de novo designs directly on this software (use drawing tools in design palette).</li>
	</ol>
	</li>
	<li>To trace the design, select the Trace icon (shape of a butterfly) on the right side of the window and completely select the imported designs.
	<ol>
		<li>Select the Trace Preview option labeled outline. Adjust (if needed) the Threshold and Scale settings to adjust the yellow trace to match the design.</li>
		<li>Select Trace from the Trace menu once the yellow trace matches the design. The channels are now shown as a red contour. If the red contour matches the design, the imported image can be selected and deleted. The design is now imported and ready for sizing.</li>
	</ol>
	</li>
	<li>Size the device by selecting the traced design and by using the grid provided by the software. Pull to change the width and length of channels and chambers. 
	NOTE: The software provides measurements, and small lines can be drawn temporarily (use design palette on the left side of the window) to measure dimensions within the device. Functional channel-width dimensions range from 100 &#181;m to 900 &#181;m. Dimensions may have to be adjusted after testing initial prototypes. It is important that all layers are sized proportionally, to ensure proper alignment during assembly.
	<ol>
		<li>After the design is properly sized, select the square tool on the shape drawing menu to draw a square/rectangle around each layer of the device. This shape should be of the same size for all layers. See Figure 1C for examples.</li>
	</ol>
	</li>
	<li>Create a separate top layer containing access ports to the channels. Simple designs will consist of a main (middle) channel layer, a bottom sealing layer (often glass) and a top layer that should contain circular perforations to access the channels (inlets/outlets). 
	NOTE: Designs containing more than three layers will require inlet/outlet perforations in multiple layers (See Figure 1C, Figure 5A). These perforations may be already included in the design, or can be added at this time.
	<ol>
		<li>Select the drawing tool on the left side of the screen. Draw circles over the inlet and outlet ports of the design.</li>
		<li>Copy and paste both the original design and the circles. Erase the channels from the underlying device. 
		NOTE: This leaves the inlet/outlet ports in the right position corresponding to the original design. Shapes may also be added to the periphery of each layer to aid with aligning.</li>
	</ol>
	</li>
	<li>Arrange all layers to be cut on the displayed mat. The device is now ready for cutting.</li>
</ol>
2. Cutting
<ol>
	<li>Apply a single PET/EVA film (or other material) of preferred thickness (3 mil is standard) onto the adhesive cutting mat. Make sure the adhesive (matte) side faces up and the plastic (shiny) side faces down. 
	NOTE: Use clean gloves to avoid introducing oils and microparticles to the layers.</li>
	<li>Flatten the film against the mat (Figure 1D), removing all air that may have been trapped. This can be done using gloved hands or a roller.</li>
	<li>Align the edge of the cutting mat to the line indicated on the cutter. Load the mat by pressing Load Mat on the cutter. Keep the setting on the cutting blade between 3 and 5, depending on the film thickness.</li>
	<li>Connect the cutter USB cable to the computer.
	<ol>
		<li>Select the SEND tab and select a cutting setting. 
		NOTE: A multitude of settings are available on the cascade menu. The -Sticker Paper, Clear- is a setting that works well with PET/EVA film that has a thickness of 3&#8211;5 mil (75&#8211;125 &#956;m). Modify settings for different materials and save custom settings for future use.</li>
	</ol>
	</li>
	<li>Click Send. Cutting will begin (Figure 1E). Make sure there is enough room in the back of the cutter for the mat to move unimpeded. When the cutter is finished, unload the mat by selecting Unload on the cutter. Do not pull the mat out before unloading.</li>
</ol>
3. Alignment
<ol>
	<li>Place the cutting mat next to a clean surface. With gloved hands, use a pair of tweezers to lift each layer of the microfluidics device off the cut mat (Figure 1F). Be especially careful around turns and bends in the channel; these are especially delicate and susceptible to tearing and warping.</li>
	<li>Place the layers of the microfluidics device onto a clean surface. Order them according to their top-to-bottom position in the device (Figure 1G, Figure 2A, Figure 5A and Figure 7A).</li>
	<li>Cut small (~3 mm x 10 mm) pieces of double-sided tape that will be used to temporarily attach the layers together.</li>
	<li>Superimpose the layers one by one, starting with the bottom layer. Add a small piece of double-sided tape to a corner between the layers, away from any channels or inlets/outlets (Figure 1G, arrow). The tape, although not required, immobilizes the layers and assures they will not shift during lamination. Use a wire jig to facilitate the alignment of layers in devices with more than 4 layers (Supplementary Figure 3).</li>
	<li>Ensure that the adhesive (matte-EVA) side of the film always faces the inside (within-layers portion) of the device. 
	CAUTION: Exposed adhesive will melt against the internal parts of the laminator and adhere to them, resulting not only in the loss of the device but also affecting the future performance of the laminator.</li>
	<li>Once all layers have been superimposed, inspect the device. There should be at least one EVA side in between all layers, and no EVA should be exposed. When introducing non-EVA coated materials (e.g., polyvinyl chloride (PVC) film, glass), a film coated with EVA on both sides might be needed, particularly in the case of more complex devices (Figure 5).</li>
</ol>
4. Lamination
<ol>
	<li>Turn on and set the laminator to the desired thickness setting. Some laminators offer 3 and 5 mil settings, while some do not. For any device with 4 or more layers, use the 5-mil setting.</li>
	<li>Once the laminator is ready, run the device through the laminating rollers (Figure 1H&#8211;I). Place the end to which double sided tape has been added for best results. 
	NOTE: When fabricating devices of five or more layers, they may be run through the laminator more than once.</li>
	<li>Recover the laminated device. 
	NOTE: It is advisable for devices to be large enough as to make easy their recovery from the laminator. This consideration does not affect the size of the channels or chip architecture, it simply calls for a &#8220;frame&#8221; that can easily go through the laminator without remaining inside.</li>
</ol>
5. Inlet/outlet ports
<ol>
	<li>Use a rotary tool and a 1/32 in. drill bit to cut a small hole through the center of a furniture bumper. Alternatively, use a 1 mm biopsy punch to perforate the bumpers. 
	NOTE: A drill press is recommended. Although sizes vary, 2 mm x 6 mm-diameter bumpers are recommended. Avoid simply &#8220;stabbing&#8221; the bumper. Unless material is removed, the bumper will seal again (Supplementary Figure 1). The perforations as indicated above are meant to interface with polyetheretherketone (PEEK) tubing, a pipette and tip, or a blunt needle (16&#8211;18 G). Larger perforations can be achieved using revolving punch pliers (Supplementary Figure 1). These are useful when the bumper is used as a &#8220;reservoir&#8221; for liquids or other biologicals.</li>
	<li>Make sure that the orifice is completely clear by removing any debris (caused by drilling or punching) with a pair of small tweezers.</li>
	<li>After the inlet/outlet ports are successfully cleared, carefully align the bumpers with the inlet/outlet ports on the laminated device (Figure 1J&#8211;K). This step is essential to having proper flow of liquids in and out of the device. Hold the bumper behind the device, position the adhesive face facing the open inlet/outlet on the device, then align and adhere. Device assembly is now completed.</li>
</ol>
6. Testing
<ol>
	<li>Access the channel/chamber architectures through the perforated bumpers (ports). There are several options regarding how to introduce fluids and biologicals into the devices.</li>
	<li>Use laboratory or medical/surgical tubing by attaching it to a plastic connector (e.g., Luer adaptors) or to a blunt needle. A standard pipette and tip or PEEK tubing without adaptors can also be used (Supplementary Figure 2).</li>
	<li>Perform infusion or drawing of fluids with syringes and tubing using syringes or peristaltic pumps. 
	NOTE: There are many options in the market, starting at ~300 USD at the time of writing.</li>
	<li>Set different flow rate settings according to the device and experiment. 
	NOTE: We routinely use flow rate settings in the range of 0.01&#8211;100 &#181;L/min, but other rates can be used.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60307/60307fig01.jpg" /> 
Figure 1: Fabrication.&#160;(A) An office laminator and a craft cutter are the only two pieces of equipment required for fabrication. Both are available online or at crafts/office supplies stores. Other required tools include scissors and tweezers. (B) Channel and chamber architectures can be composed digitally using any software program that includes drawing tools (vector graphics may be preferred by some users, but are not required). Lines and shapes are drawn in black with a white background. The file or a screen capture of the design can be imported into the craft cutter software by dragging and dropping. (C) Craft cutter software is available free to download and is required to control the cutter. The software acquires the design and allows for modifications, like sizing. It also provides drawing tools. (D) The cutting mat carries the film for cutting. It is slightly adhesive, allowing for immobilization of the materials to be cut. The figure shows four different materials ready for loading: 3 mil-thick PET/EVA film (top), 5 mil-thick PET/EVA film (middle), 6 mil-thick EVA/PET/EVA (bottom left) and PVC film (bottom right). (E) Cutter is open to display blade (in black) unit and loaded mat. (F) After cutting, individual layers are lifted using tweezers. Cut outs of channels and chambers remain attached to the mat and are later removed and discarded. (G) Individual layers are aligned and superimposed for lamination. Small pieces of double-sided tape (arrow) are often used to aid in aligning and prevent layer shifting during lamination. (H, I) The device is fed at the top of the laminator and recovered through the slot. Lamination provides a robust seal, leaving channel paths open. (J, K) In order to access the channels, it is necessary to add perforated, self-adhesive vinyl bumpers. Image in (J) displays the &#8220;reverse&#8221; approach for alignment, in which the bumper is placed from the back, allowing visual alignment of the inlet/outlet with the bumper perforation. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60307/60307fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOFT+LITHOGRAPHY.">SOFT LITHOGRAPHY.</a> Annual Review of Materials Science. 28 (1), 153-184 (1998).</li><li>Beebe, D. J., Mensing, G. A., Walker, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physics+and+Applications+of+Microfluidics+in+Biology.">Physics and Applications of Microfluidics in Biology.</a> Annual Review of Biomedical Engineering. 4 (1), 261-286 (2002).</li><li>Whitesides, G. M., Ostuni, E., Takayama, S., Jiang, X., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+Lithography+in+Biology+and+Biochemistry.">Soft Lithography in Biology and Biochemistry.</a> Annual Review of Biomedical Engineering. 3 (1), 335-373 (2001).</li><li>Sackmann, E. K., Fulton, A. L., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+present+and+future+role+of+microfluidics+in+biomedical+research.">The present and future role of microfluidics in biomedical research.</a> Nature. 507 (7491), 181-189 (2014).</li><li>Berthier, E., Young, E. W. 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>

Fernando Ontiveros is in the process of launching PETL FLUIDICS (LLC), a company that will commercialize and provide consulting services for this technology. The co-authors have nothing to disclose.

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 &amp;amp; Transcendia</td><td>TP3854-200,TP5854-100 &amp;amp; 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&quot; 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 &amp;amp; tweezers</td><td>Any</td><td>Fiskars-Inch-Titanium-Softgrip-Scissors |Cole-Parmer&amp;nbsp;&amp;ndash;# 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 ...

rapid-fabrication-custom-microfluidic-devices-for-research

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.6K Views.  University of Notre Dame. 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.

Video: Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.

The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.

Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.

The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.

We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays.  ...

Biology

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

One-Step Approach to Fabricating Polydimethylsiloxane Microfluidic Channels of Different Geometric Sections by Sequential Wet Etching Processes

Polydimethylsiloxane (PDMS) materials are substantially exploited to fabricate microfluidic devices by using soft lithography replica molding techniques. Customized channel layout designs are necessary for specific functions and integrated performance of microfluidic devices in numerous biomedical and chemical applications (e.g., cell culture, biosensing, chemical synthesis, and liquid handling). Owing to the nature of molding approaches using silicon wafers with photoresist layers patterned by photolithography as master molds, the microfluidic channels commonly have regular cross sections of rectangular shapes with identical heights. Typically, channels with multiple heights or different geometric sections are designed to possess particular functions and to perform in various microfluidic applications (e.g., hydrophoresis is used for sorting particles and in continuous flows for separating blood cells6,7,8,9). Therefore, a great deal of effort has been made in constructing channels with various sections through multiple-step approaches like photolithography using several photoresist layers and assembly of different PDMS thin sheets. Nevertheless, such multiple-step approaches usually involve tedious procedures and extensive instrumentation. Furthermore, the fabricated devices may not perform consistently and the resulted experimental data may be unpredictable. Here, a one-step approach is developed for the straightforward fabrication of microfluidic channels with different geometric cross sections through PDMS sequential wet etching processes, that introduces etchant into channels of planned single-layer layouts embedded in PDMS materials. Compared to the existing methods for manufacturing PDMS microfluidic channels with different geometries, the developed one-step approach can significantly simplify the process to fabricate channels with non-rectangular sections or various heights. Consequently, the technique is a way of constructing complex microfluidic channels, which provides a fabrication solution for the advancement of innovative microfluidic systems.

Several methods are available for the fabrication of channels of non-rectangular sections embedded in polydimethylsiloxane microfluidic devices. Most of them involve multistep manufacturing and extensive alignment. In this paper, a one-step approach is reported for fabricating microfluidic channels of different geometric cross sections by polydimethylsiloxane sequential wet etching.

Polydimethylsiloxane (PDMS) materials are substantially exploited to fabricate microfluidic devices by using soft lithography replica molding techniques. ...

Engineering

Fabrication of Refractive-index-matched Devices for Biomedical Microfluidics

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices can be implemented as part of a robust platform capable of making simultaneous complementary measurements. The primary challenge created by the combination of these two techniques is the mismatch in refractive index between the materials traditionally used to make microfluidic devices and the aqueous solutions typically used in biomedicine. This mismatch can create optical artifacts near the channel or device edges. One solution is to reduce the refractive index of the material used to fabricate the device by using a fluorinated polymer such as MY133-V2000 whose refractive index is similar to that of water (n = 1.33). Here, the construction of a microfluidic device made out of MY133-V2000 using soft lithography techniques is demonstrated, using O2 plasma in conjunction with an acrylic holder to increase the adhesion between the MY133-V2000 fabricated device and the polydimethylsiloxane (PDMS) substrate. The device is then tested by incubating it filled with cell culture media for 24 h to demonstrate the ability of the device to maintain cell culture conditions during the course of a typical imaging experiment. Finally, quantitative phase microscopy (QPM) is used to measure the distribution of mass within the live adherent cells in the microchannel. This way, the increased precision, enabled by fabricating the device from a low index of refraction polymer such as MY133-V2000 in lieu of traditional soft lithography materials such as PDMS, is demonstrated. Overall, this approach for fabricating microfluidic devices can be readily integrated into existing soft lithography workflows in order to reduce optical artifacts and increase measurement precision.

This protocol describes the fabrication of microfluidic devices from MY133-V2000 to eliminate artifacts that often arise in microchannels due to the mismatching refractive indices between microchannel structures and an aqueous solution. This protocol uses an acrylic holder to compress the encapsulated device, improving adhesion both chemically and mechanically.

The use of microfluidic devices has emerged as a defining tool for biomedical applications. When combined with modern microscopy techniques, these devices ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Design and Development of a Three-Dimensionally Printed Microscope Mask Alignment Adapter for the Fabrication of Multilayer Microfluidic Devices

This project aims to develop an easy-to-use and cost-effective platform for the fabrication of precise, multilayer microfluidic devices, which typically can only be achieved using costly equipment in a clean room setting. The key part of the platform is a three dimensionally (3D) printed microscope mask alignment adapter (MMAA) compatible with regular optical microscopes and ultraviolet (UV) light exposure systems. The overall process of creating the device has been vastly simplified because of the work done to optimize the device design. The process entails finding the proper dimensions for the equipment available in the laboratory and 3D-printing the MMAA with the optimized specifications. Experimental results show that the optimized MMAA designed and manufactured by 3D printing performs well with a common microscope and light exposure system. Using a master mold prepared by the 3D-printed MMAA, the resulting microfluidic devices with multilayered structures contain alignment errors of &lt;10 &#181;m, which is sufficient for common microchips. Although human error through transportation of the device to the UV light exposure system can cause larger fabrication errors, the minimal errors achieved in this study are attainable with practice and care. Furthermore, the MMAA can be customized to fit any microscope and UV exposure system by making changes to the modeling file in the 3D printing system. This project provides smaller laboratories with a useful research tool as it only requires the use of equipment that is typically already available to laboratories that produce and use microfluidic devices. The following detailed protocol outlines the design and 3D printing process for the MMAA. In addition, the steps for procuring a multilayer master mold using the MMAA and producing poly(dimethylsiloxane) (PDMS) microfluidic chips is also described herein.

This project allows small laboratories to develop an easy-to-use platform for the fabrication of precise multilayer microfluidic devices. The platform consists of a three-dimensionally printed microscope mask alignment adapter using which multilayer microfluidic devices with alignment errors of &lt;10 &#181;m were achieved.

This project aims to develop an easy-to-use and cost-effective platform for the fabrication of precise, multilayer microfluidic devices, which typically ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...