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 syste...

<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. -. 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W., Jackman, R. J., Firebaugh, S. L., Schmidt, M. A., Jensen, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+microfluidic+devices+for+multiphase+mixing+and+reaction.">Design and fabrication of microfluidic devices for multiphase mixing and reaction.</a> Journal of Microelectromechanical Systems. 11 (6), 709-717 (2002).</li><li>Kobayashi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+device+for+conducting+gas-liquid-solid+hydrogenation+reactions.">A microfluidic device for conducting gas-liquid-solid hydrogenation reactions.</a> Science. 304 (5675), 1305-1308 (2004).</li><li>Shuler, M. 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. 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+compact+low-cost+low-maintenance+open+architecture+mask+aligner+for+fabrication+of+multilayer+microfluidics+devices.">A compact low-cost low-maintenance open architecture mask aligner for fabrication of multilayer microfluidics devices.</a> Biomicrofluidics. 12 (4), 044119 (2018).</li><li>Ravi, T., Ranganathan, R., Shunmugam, M. S., Kanthababu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topology+and+build+path+optimization+for+reducing+cost+in+FDM+uPrint+SE.">Topology and build path optimization for reducing cost in FDM uPrint SE.</a> Advances in Additive Manufacturing and Joining. , 189-198 (2019).</li><li>SU-8 Permanent Negative Epoxy Photoresist. Kayaku Advanced Materials Available from: <a target="_blank" href="https://kayakuam.com/wp-content/uploads/2020/09/KAM-SU-8-50-100-Datasheet-9.3.20-Final.pdf">https://kayakuam.com/wp-content/uploads/2020/09/KAM-SU-8-50-100-Datasheet-9.3.20-Final.pdf</a> (2020)</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#34; x 8&#34;, 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(1H,1H,2H,2H-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...

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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 ...

This protocol is significant as it allows for the generation of a cost-effective and easy to use platform that aids in the production of precise multilayer microfluidic master molds. The main advantage of the technique is that it only requires the use of the 3D printed platform and standard laboratory equipment commonly found in laboratories that produce microfluidic devices. Visual demonstration of this protocol is critical to demonstrate how to customize and use the 3D printed microscope mask alignment adapter.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. Measure the diameter of the inner circular rim, the inner height of the UV light emission systems tray, the total width and length of the tray. Using a computer design application, apply these dimensions to customize the wafer holder to fit within the UV light emission systems tray.Measure the length between the screws and the width of the screws on the available upright microscope stage that hold the slide holder in place. Using a computer design application, apply these dimensions to customize the magnetic holder to fit the available microscope to allow for easy and precise fixation of the MMAA to the microscope. Use a four inch silicon wafer with you appropriate photo resist to create the first layer of the master mold, ensuring that the thickness is greater than the subsequent layers for easy identification of the alignment markers.Use a light colored marker pen to color the first layer's alignment markers on all four sides. Using the photo resist manufacturer's instructions, initiate the second layer of the master mold by spin coating the photo resist onto the wafer and performing the soft bake. Insert the coated wafer into the wafer holder of the MMAA and fix the coated wafer to the MMAA using tape.Attach the wafer holder to the available upright microscope using the magnetic microscope fastener. 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. Remove the wafer holder from the microscope stage and insert the second layer photo mask into the wafer holder on top of the coated wafer.Ensure that the first layer's colored alignment markers can be partially seen through the alignment markers on the photo mask and that the straight edge of the photo mask is superimposed with the straight edge of the silicon wafer. Attach the wafer holder back onto the microscope stage and attach the photo mask to a scissor lift through one of the side cutouts with tape. Use the scissor lift to adjust the Z direction position of the photo mask until it lies right above the coated wafer.While keeping the photo mask still, look through the microscope lens and identify the first layer's colored alignment markers beneath the alignment markers of the photo mask using the X and Y direction knobs of the microscope stage to move the position of the MMAA. Adjust the position of the MMAA until the alignment marker on the photo mask is superimposed with the colored alignment marker on the first layer. Carefully apply a slight force to the photo mask and use tape to secure the photo mask in place on top of the coated wafer.Detach the photo mask from the scissor lift and ensure all four alignment markers on the photo mask are in alignment with the four alignment markers on the first layer. Post alignment, carefully detach the wafer holder from the microscope stage. Insert the glass top plate on top of the wafer and photo mask to decrease the gap between the two pieces.Place the entire wafer holder into the available UV light exposure system and perform exposure of the second layer. Remove the wafer holder from the UV light exposure system, then remove the coated wafer from the wafer holder and detach the photo mask from the wafer. Complete the post-bake and developing of the second layer following the photo resist manufacturer's instructions.Retrieve the master mold and place it on the stage of the upright microscope to determine the gap distance between the first layer and second layer. Measure the distance by which the second layer is shifted and misaligned from the first layer on the microchannel structures. 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. Through the optimization and use of the MMAA, multilayer master molds with minimal alignment error were fabricated. This system and the described protocol were used for the alignment of the markers on the photo mask with the markers on the initial layer of the master mold.The double layer SU-8 master mold for a microfluidic device with a herringbone pattern was fabricated and shown to have a gap distance of less than five micrometres between the two layers. The two layer master mold was then used to fabricate PDMS microchips. Scanning electron microscope images show that the microfluidic device with the herringbone pattern contains clear edges, straight channel walls, and well-aligned layers, which are essential for proper device functionality.In addition, a four layer master mold with simple circular features was created using the MMAA to show successful alignment of a multilayer master mold. Profilometer data confirms the four distinct layers of the master mold. It is important to have patience and work slowly when aligning the first and second layers'alignment markers and while fixing the photo mask to the coated wafer.This procedure can be used for the production of many different multilayer master molds, allowing researchers from smaller labs to explore more complex microfluidic device designs.

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2.6K visualizações.  Texas Tech University. Este protocolo é significativo, pois permite a geração de uma plataforma econômica e fácil de usar que auxilia na produção de moldes mestres microfluidos multicamadas precisos. A principal vantagem da técnica é que ela requer apenas o uso da plataforma impressa 3D e equipamentos de laboratório padrão comumente encontrados em laboratórios que produzem dispositivos microfluidos. A demonstração visual deste protocolo é fundamental para demonstrar como personalizar e usar o adaptad...

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