The authors would like to acknowledge the Center for Transformative Undergraduate Experiences from Texas Tech University for providing funding for this project. The authors would also like to acknowledge support from the Chemical Engineering Department at Texas Tech University.

1. Designing the MMAA
<ol>
	<li>Obtain the dimensions of the tray of the available UV light emission system to be the upper bound for the dimensions of the wafer holder (or UV exposure unit) shown in Figure 1. As shown in Figure 2A, measure the diameter (d) of the inner circular rim, the inner height (h) of the UV light emission system&#39;s tray, the total width (w), and length (l) of the tray. 
	NOTE: As an example, the available UV light exposure system had inner tray dimensions of 5 inch (&#34;) x 5&#34; x 0.25&#34; with a 4&#34; circular cut-out. The dimensions of the MMAA were then designed to be no greater than the inner tray dimensions to fit properly and sit flat within the tray of the system as shown in Figure 2B. See Figure 3 for the 3D-printed pieces of the MMAA: photoresist-coated silicon wafer and a fastener to fix the setup to the microscope.</li>
	<li>Measure the length between the screws on the available upright microscope stage that hold the slide holder in place. Additionally, measure the width of the screws. Apply these dimensions to customize the magnetic holder (Figure 1) to fit the available microscope to allow for easy and precise fixation of the MMAA to the microscope (Figure 4A).</li>
	<li>Using an available computer design application, customize the wafer holder and magnetic microscope fastener to fit within the measured dimensions. Design the height, width, and length of the wafer holder to be no greater than the height (h), width (w), and length (l) of the UV light emission system&#39;s tray. In addition, include the circular cut-out at the bottom of the wafer holder with the same diameter (d) as the UV light emission system&#39;s tray. Generate STL or CAD files for the two pieces of the MMAA to be used for 3D printing of the device (see Supplemental Material).</li>
</ol>
2. 3D Printing the MMAA
<ol>
	<li>Upload the generated STL or CAD files to the available 3D-printing software. 3D-Print the two pieces of the MMAA by following the appropriate procedure for the 3D process and printer used. Complete the pieces by following any required post-printing steps (e.g., removal of support material, removal of uncured resin, additional washing or curing steps). Alternatively, use an available 3D printing facility to have the designed pieces printed and completed elsewhere.</li>
	<li>Ensure the wafer holder fits well and sits flat inside the tray of the available UV light exposure system (Figure 2B). Additionally, ensure that the microscope fastener is attached to the microscope stage and can be moved easily using the knobs that control the x- and y- positions of the microscope stage (Figure 4A).</li>
	<li>Once the pieces have been finalized, insert and fix the magnets into the wafer holder and microscope fastener (Figure 3A), using super glue or any other fixing substance. Allow for the glue to dry before testing the system. 
	&#8203;NOTE: If desired, a protype piece can first be printed using a Fused Deposition Modeling (FDM) 3D printer to save resources and money15. This protype can then be assessed for accurate fit to the available equipment, and the design can then be modified, if needed. The final device can then be printed using a more accurate process (e.g., Stereolithography) for better precision. The final device can also be printed with a translucent finish for optimal use under the microscope.</li>
</ol>
3. Experimental testing of the MMAA
<ol>
	<li>Design and printing of the microfluidic device photomasks with alignment markers
	<ol>
		<li>Use a computer design application to design photomasks for the desired bilayer microfluidic device.</li>
		<li>Include additional structures on the side of the microfluidic device channel structures that will act as alignment markers (closer towards the edge of the photomask/master mold) as shown in Figure 5A,B. Ensure there is one alignment marker on each side of the microfluidic device (for a total of at least four). In addition, ensure the photomask contains a straight edge that can align perfectly with the straight edge of the silicon wafer. 
		NOTE: The higher intricacy of the alignment marker structure will allow for greater alignment accuracy of the additional layers. At the least, a simple cross structure with measurements of 1 mm x 1 mm should be used (Figure 6A). An example of the alignment markers can be seen in the corners and bottom middle edge of Figure 5A,B, which depict the first- and second-layer photomasks used to generate a double-layer master mold.</li>
		<li>Print the photomasks either through a commercial vendor or through other accessible facilities</li>
	</ol>
	</li>
	<li>Creation of the bilayer master mold using the MMAA (photolithography)
	<ol>
		<li>Using standard photolithography techniques and the photoresist manufacturer&#39;s instructions, create the first layer of the master mold using the first layer photomask16. Use a 4&#34; silicon wafer with the appropriate photoresist (i.e., SU-8) to create the desired layer thickness. Ensure the first layer thickness is greater than the subsequent layers for easy identification of the alignment markers.</li>
		<li>Use a light-colored marker pen (e.g., gold) to color the first layer&#39;s alignment markers on all four sides.</li>
		<li>Using the photoresist manufacturer&#39;s instructions, initiate the second layer of the master mold by spin-coating the photoresist onto the wafer and performing the soft bake16. Insert the coated wafer into the wafer holder of the MMAA (Figure 3B) and fix the coated wafer to the MMAA using tape.</li>
		<li>Attach the wafer holder to the available upright microscope using the magnetic microscope fastener (Figure 4A). Move the position of the MMAA using the x- and y-direction knobs of the microscope stage until one of the colored alignment markers on the wafer is in view through the microscope lens.</li>
		<li>Insert the second-layer photomask into the wafer holder, on top of the coated wafer (Figure 3C). Ensure that the first-layer&#39;s&#160;colored alignment markers can be partially seen through the alignment markers on the photomask.&#160;</li>
		<li>Attach the photomask to a scissor lift (also known as a support jack) through one of the side cut-outs (Figure 4B) with tape. Use the scissor lift to adjust the z-direction position of the photomask until it lies right above the coated wafer (Figure 3C). 
		&#8203;NOTE: The scissor lift allows for fine adjustment of the z-position of the photomask, as the scissor lift can be used to move the position of the attached photomask in the z-direction.</li>
		<li>While keeping the photomask still, look through the microscope lens and identify the first-layer&#8217;s colored alignment markers beneath the alignment markers of the photomask. Use the x- and y-direction knobs of the microscope stage to move the position of the MMAA (Figure 4D). Adjust the position of the MMAA until the alignment marker on the photomask is superimposed with the colored alignment marker on the first layer (Figure 6A,B) by observing the position of the alignment markers through the microscope lens.</li>
		<li>Carefully apply a slight force to the photomask and use tape to secure the photomask in place on top of the coated wafer. Detach the photomask from the scissor lift. Ensure all four alignment markers on the photomask are in alignment with the four alignment markers on the first layer.</li>
		<li>Once the alignment is achieved, carefully detach the wafer holder from the microscope stage. Insert the glass top plate on top of the wafer and photomask to decrease the gap between the two pieces (Figure 1). Place the entire wafer holder into the available UV light exposure system as shown in Figure 4E. Expose the second layer for the appropriate time and light intensity as described in the photoresist manufacturer&#39;s instructions16.</li>
		<li>Remove the wafer holder from the UV light exposure system. Remove the coated wafer from the wafer holder and detach the photomask from the wafer. Complete the processing of the second layer (e.g., post-bake, developing, and rinse and dry) as per the photoresist manufacturer&#39;s instructions16. 
		&#8203;NOTE: The exact spin-coating, soft baking, exposing, post-baking, and developing conditions (time, temperature) will vary based on the photoresist being used and the desired layer thickness. The actual conditions and exact photolithography procedure should be based on the photoresist manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>Preparation of a microfluidic device using the master mold (soft lithography)
	<ol>
		<li>Retrieve the master mold and secure it in the middle of a 150 mm x 15 mm plastic Petri dish with tape.</li>
		<li>Prepare ~15-20 g of PDMS based on the manufacturer&#39;s instructions. Place the PDMS in a vacuum chamber or let it rest until free of any bubbles. Pour the PDMS into the Petri dish containing the master mold.</li>
		<li>Let the Petri dish with the master mold rest on the countertop until the PDMS is free of any bubbles. Place the Petri dish in an oven at 65 &#176;C until the PDMS is fully cured (at least 3 h).</li>
		<li>Cut out the PDMS to reveal the microchannel structures. Cut the PDMS around the microchannel structures into separate microchips and create the inlet and outlet holes for the microfluidic device. Use tape to gently remove any small particulates that may lie on the PDMS surface.</li>
		<li>Complete the microchip fabrication by bonding the PDMS chip to the PDMS or a microscope slide by plasma-treating the PDMS chip and the additional substrate.</li>
	</ol>
	</li>
	<li>Determination of the alignment error
	<ol>
		<li>Retrieve the master mold and use the upright microscope to determine the gap distance (alignment error) between the first layer and second layer. Do this by simply measuring the distance by which the second layer is shifted and misaligned from the first layer on the microchannel structures (see Figure 5D for an example of a measured gap distance).</li>
		<li>Use the upright microscope to determine whether the PDMS chip contains channel walls that are straight with clear device edges. Additionally, check the PDMS chip for any possible defects that may hinder device functionality. 
		NOTE: The master mold fabrication (sections 3.2 and 3.3) may need to be repeated to achieve a lower alignment error. Repeated practice using the MMAA is shown to enhance the user&#39;s ability to create a well-aligned master mold. In addition, images can be obtained by scanning electron microscopy (SEM) (Figure 7) to confirm the alignment error.</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+gene+synthesis+in+a+microfluidic+device.">Parallel gene synthesis in a microfluidic device.</a> Nucleic Acids Research. 35 (8), 61 (2007).</li><li>Yang, M., Li, C. -. W., Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+docking+and+on-chip+monitoring+of+cellular+reactions+with+a+controlled+concentration+gradient+on+a+microfluidic+device.">Cell docking and on-chip monitoring of cellular reactions with a controlled concentration gradient on a microfluidic device.</a> Analytical Chemistry. 74 (16), 3991-4001 (2002).</li><li>Keles, 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=Development+of+a+robust+and+reusable+microreactor+employing+laser+based+mid-IR+chemical+imaging+for+the+automated+quantification+of+reaction+kinetics.">Development of a robust and reusable microreactor employing laser based mid-IR chemical imaging for the automated quantification of reaction kinetics.</a> Organic Process Research &amp; Development. 21 (11), 1761-1768 (2017).</li><li>Losey, M. 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L. <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-,+body-,+and+disease-on-a-chip+systems.">Advances in organ-, body-, and disease-on-a-chip systems.</a> Lab on a Chip. 19 (1), 9-10 (2019).</li><li>Kimura, H., Sakai, Y., Fujii, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ/body-on-a-chip+based+on+microfluidic+technology+for+drug+discovery.">Organ/body-on-a-chip based on microfluidic technology for drug discovery.</a> Drug Metabolism and Pharmacokinetics. 33 (1), 43-48 (2018).</li><li>Lee, 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=A+pumpless+Multi-Organ-on-a-Chip+(MOC)+combined+with+a+Pharmacokinetic-Pharmacodynamic+(PK-PD)+model.">A pumpless Multi-Organ-on-a-Chip (MOC) combined with a Pharmacokinetic-Pharmacodynamic (PK-PD) model.</a> Biotechnology and Bioengineering. 114 (2), 432-443 (2017).</li><li>Kang, S. -. W., Wang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+soft+lithography+for+nano+functional+devices.">Application of soft lithography for nano functional devices.</a> Lithography. , 403-426 (2010).</li><li>Challa, P. K., Kartanas, T., Charmet, J., Knowles, T. P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+fabricated+using+fast+wafer-scale+LED-lithography+patterning.">Microfluidic devices fabricated using fast wafer-scale LED-lithography patterning.</a> Biomicrofluidics. 11, 014113 (2017).</li><li>Li, X., 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=Desktop+aligner+for+fabrication+of+multilayer+microfluidic+devices.">Desktop aligner for fabrication of multilayer microfluidic devices.</a> Review of Scientific Instruments. 86 (7), 075008 (2015).</li><li>Pham, Q. L., Tong, N. -. A. N., Mathew, A., Voronov, R. 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The authors have nothing to disclose.

The aforementioned protocol outlines the procedure for 3D-printing an MMAA and using the system to create a precise, multilayer, microfluidic device master mold. Although the device is easy to use, there are critical steps within the protocol that require practice and care to ensure proper alignment of the master mold layers. The first critical step is the design of the MMAA. It is essential when designing the MMAA to determine the exact measurements for the device that will allow for a proper fit inside the UV light exp...

A well-developed and promising field in engineering research is microfabrication because of the vast expanse of applications employing microfluidic platforms. Microfabrication is a process wherein structures are produced with &#181;m- or smaller-sized features using different chemical compounds. As microfluidic research has developed over the last 30 years, soft lithography has become the most popular microfabrication technique with which to produce microchips made from poly(dimethylsiloxane) (PDMS) or similar substances. These microchips have been widely used for the miniaturization of common laboratory practices1,2,3,4 and have become powerful research tools for engineers to mimic reaction processes5,6,7, study reaction mechanisms, and mimic organs found in the human body in vitro (e.g., organ-on-a-chip)8,9,10. However, as the complexity of the application increases, it is typical that a more complex microfluidic device design allows for better replication of the real-life system it is intended to imitate.
The basic soft lithography procedure involves coating a substrate with a photoresist substance and placing a photomask over the coated substrate before subjecting the substrate to UV light11. The photomask has transparent regions that mimic the desired pattern of the microfluidic device channels. When subjecting the coated substrate to UV light, the transparent regions allow the UV light to penetrate through the photomask, causing the photoresist to be crosslinked. After the exposure step, the un-crosslinked photoresist is washed away using a developer, leaving solid structures with the intended pattern. As the complexity of the microfluidic devices becomes greater, they require multiple-layer construction with extremely precise dimensions. The process of multilayer microfabrication is much more difficult compared to single-layer microfabrication.
Multilayer microfabrication requires precise alignment of the first layer features with the designs on the second mask. Normally, this process is performed using a commercial mask aligner, which is expensive and requires training to operate the machinery. Thus, the process of multilayer microfabrication is typically unattainable for smaller laboratories that lack the funds or time for such endeavors. While several other custom-built mask aligners have been developed, these systems often require the purchase and assembly of many different parts and can still be quite complex12,13,14. This is not only expensive for smaller laboratories, but also requires time and training to build, understand, and use the system. The mask aligner detailed in this paper sought to alleviate these issues as there is no need for the purchase of additional equipment, only requiring equipment that is typically already present in laboratories that produce and use microfluidic devices. In addition, the mask aligner is fabricated by 3D printing, which with the recent advancement of 3D printing technology, has become readily available to most laboratories and universities at an affordable cost.
The protocol detailed in this paper aims to create a cost-effective and easy-operation alternative mask aligner. The mask aligner detailed herein can make multilayer microfabrication feasible for research laboratories without conventional fabrication facilities. Using the microscope mask alignment adapter (MMAA), functional microchips with complex features can be achieved using a regular UV light source, optical microscope, and common laboratory equipment. The results show that the MMAA performs well with an example system using an upright microscope and a UV light-exposure box. The MMAA produced using the 3D printing process was used to acquire a bilayer master mold of a herringbone microfluidic device with minimal alignment errors. Using the master mold fabricated with a 3D-printed MMAA, microfluidic devices were prepared with multilayered structures containing alignment errors of &#60;10 &#181;m. The alignment error of &#60;10 &#181;m is minimal enough to not hinder the application of the microfluidic device.
In addition, the successful alignment of a four-layer master mold produced using the MMAA was confirmed, and alignment errors were determined to be &#60;10 &#181;m. The functionality of the microfluidic device and minimal alignment errors validate the successful application of the MMAA in creating multilayer microfluidic devices. The MMAA can be customized to fit any microscope and UV exposure system by making minor changes to the file in the 3D printer. The following protocol outlines the steps necessary to fine-tune the MMAA to fit the equipment available in each laboratory and 3D-print the MMAA with the required specifications. In addition, the protocol details how to develop a multilayer master mold using the system and subsequently produce PDMS microfluidic devices using the master mold. Generation of the master mold and microfluidic chips then allows the user to test the effectiveness of the system.

<table><tbody><tr><td>Acrylonitrile Butadiene Styrene (ABS), 3D Printing Filament</td><td></td><td></td><td>Provided by the Texas Tech University 3D printing facility</td></tr><tr><td>BX53, Upright Microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Form 2, Stereolithography 3D printer</td><td>Formlabs</td><td></td><td></td></tr><tr><td>Advanced Hot Plate Stirrer</td><td>VWR</td><td>97042-642</td><td></td></tr><tr><td>Isoproyl Alcohol, 70% (v/v)</td><td>VWR</td><td>BDH7999-4</td><td></td></tr><tr><td>Light Colored Marker</td><td>Sharpie</td><td></td><td></td></tr><tr><td>Magnets, 3 mm x 3 mm</td><td>WOTOY</td><td></td><td>ASIN #: B075PLVW8W</td></tr><tr><td>SYLGARD 184 Silicone Elastomer Kit</td><td>DOW</td><td>4019862</td><td></td></tr><tr><td>Petri Dish, 150 mm x 15 mm</td><td>VWR</td><td>25384-326</td><td></td></tr><tr><td>Printed Photomasks</td><td>CAD/Art Services, Inc.</td><td></td><td></td></tr><tr><td>Aluminum Support Jack - 8&quot; x 8&quot;, Scissor Lift</td><td>VWR</td><td>12620-904</td><td></td></tr><tr><td>Silicon Wafer</td><td>University Wafer</td><td>452</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>VWR</td><td></td><td></td></tr><tr><td>Sonication Bath</td><td>Branson</td><td>CPX3800H</td><td></td></tr><tr><td>Spin Coater</td><td>Laurell Technologies Corporation</td><td>Model WS-650MZ-23NPPB</td><td></td></tr><tr><td>STRATASYS SR-30</td><td>MakerBot Industries, LLC</td><td>SR-30</td><td>Dissolvable support material for 3D printing</td></tr><tr><td>Stratasys uPrint SE 3D Printer</td><td>Computer Aided Technology, LLC</td><td></td><td></td></tr><tr><td>SU-8 50</td><td>Kayaku</td><td>Y131269 0500L1GL</td><td></td></tr><tr><td>SU-8 100</td><td>Kayaku</td><td>Y131273 0500L1GL</td><td></td></tr><tr><td>SU-8 Developer</td><td>Kayaku</td><td>Y020100 4000L1PE</td><td></td></tr><tr><td>Super glue</td><td>Gorilla Glue</td><td></td><td></td></tr><tr><td>Trichloro(1&lt;em&gt;H&lt;/em&gt;,1&lt;em&gt;H&lt;/em&gt;,2&lt;em&gt;H&lt;/em&gt;,2&lt;em&gt;H&lt;/em&gt;-perfluorooctyl)silane</td><td>Sigma-Aldrich</td><td>448931-10G</td><td></td></tr><tr><td>Tape</td><td>Scotch</td><td></td><td></td></tr><tr><td>Form Cure, UV Curing Chamber</td><td>Formlabs</td><td>FH-CU-01</td><td></td></tr><tr><td>UV-KUB2, UV Light-Exposure Box</td><td>Kloe</td><td>UV-KUB2</td><td></td></tr></tbody></table>

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.

Through the optimization and use of the MMAA (Figure 1), multilayer master molds with minimal alignment error were fabricated. The final MMAA was fabricated using the fused filament fabrication (FFF) 3D-printing process (Figure 2).&#160;The FFF process confers increased accuracy for the desired device dimensions. The MMAA consists of two main pieces (Figure 3): the base piece and the custom fastener. The base piece consists of the U...

Watch this Scientific Journal Video about Design and Development of a Three-Dimensionally Printed Microscope Mask Alignment Adapter for the Fabrication of Multilayer Microfluidic Devices at JoVE.com

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

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 ...

design-development-three-dimensionally-printed-microscope-mask

Research

JoVE Journal

Engineering

2.7K Views.  Texas Tech University. 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 

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

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Bioengineering

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Medicine

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 ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

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 ...

Biology

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Soft Lithographic Procedure for Producing Plastic Microfluidic Devices with View-ports Transparent to Visible and Infrared Light

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable microfluidic devices. Here, a protocol for the fabrication of plastic microfluidic devices is demonstrated, where soft lithographic techniques are used to embed transparent Calcium Fluoride (CaF2) view-ports in connection with observation chamber(s). The method is based on a replica casting approach, where a polydimethylsiloxane (PDMS) mold is produced through standard lithographic procedures and then used as the template to produce a plastic device. The plastic device features ultraviolet/visible/infrared (UV/Vis/IR) -transparent windows made of CaF2 to allow for direct observation with visible and IR light. The advantages of the proposed method include: a reduced need for accessing a clean room micro-fabrication facility, multiple view-ports, an easy and versatile connection to an external pumping system through the plastic body, flexibility of the design, e.g., open/closed channels configuration, and the possibility to add sophisticated features such as nanoporous membranes.

A protocol for the fabrication of plastic microfluidic devices with transparent view-ports for visible and infrared light imaging is described.

Infrared (IR) spectro-microscopy of living biological samples is hampered by the absorption of water in the mid-IR range and by the lack of suitable ...

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an&#160;additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic ...