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. 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 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 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 ...

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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

Design, Fabrication, and Experimental Characterization of Plasmonic Photoconductive Terahertz Emitters

In this video article we present a detailed demonstration of a highly efficient method for generating terahertz waves. Our technique is based on photoconduction, which has been one of the most commonly used techniques for terahertz generation 1-8. Terahertz generation in a photoconductive emitter is achieved by pumping an ultrafast photoconductor with a pulsed or heterodyned laser illumination. The induced photocurrent, which follows the envelope of the pump laser, is routed to a terahertz radiating antenna connected to the photoconductor contact electrodes to generate terahertz radiation. Although the quantum efficiency of a photoconductive emitter can theoretically reach 100%, the relatively long transport path lengths of photo-generated carriers to the contact electrodes of conventional photoconductors have severely limited their quantum efficiency. Additionally, the carrier screening effect and thermal breakdown strictly limit the maximum output power of conventional photoconductive terahertz sources. To address the quantum efficiency limitations of conventional photoconductive terahertz emitters, we have developed a new photoconductive emitter concept which incorporates a plasmonic contact electrode configuration to offer high quantum-efficiency and ultrafast operation simultaneously. By using nano-scale plasmonic contact electrodes, we significantly reduce the average photo-generated carrier transport path to photoconductor contact electrodes compared to conventional photoconductors 9. Our method also allows increasing photoconductor active area without a considerable increase in the capacitive loading to the antenna, boosting the maximum terahertz radiation power by preventing the carrier screening effect and thermal breakdown at high optical pump powers. By incorporating plasmonic contact electrodes, we demonstrate enhancing the optical-to-terahertz power conversion efficiency of a conventional photoconductive terahertz emitter by a factor of 50 10.

We describe methods for the design, fabrication, and experimental characterization of plasmonic photoconductive emitters, which offer two orders of magnitude higher terahertz power levels compared to conventional photoconductive emitters.

In this video article we present a detailed demonstration of a highly efficient method for generating terahertz waves. Our technique is based on ...

Fabrication and Testing of Microfluidic Optomechanical Oscillators

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including microresonators. However, because of the increased acoustic radiative losses during direct liquid immersion of optomechanical devices, almost all published optomechanical experiments have been performed in solid phase. This paper discusses a recently introduced hollow microfluidic optomechanical resonator. Detailed methodology is provided to fabricate these ultra-high-Q microfluidic resonators, perform optomechanical testing, and measure radiation pressure-driven breathing mode and SBS-driven whispering gallery mode parametric vibrations. By confining liquids inside the capillary resonator, high mechanical- and optical- quality factors are simultaneously maintained.

Parametric optomechanical excitations have recently been experimentally demonstrated in microfluidic optomechanical resonators by means of optical radiation pressure and stimulated Brillouin scattering. This paper describes the fabrication of these microfluidic resonators along with methodologies for generating and verifying optomechanical oscillations.

Cavity optomechanics experiments that parametrically couple the phonon modes and photon modes have been investigated in various optical systems including ...

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. The aggressive scaling of feature sizes, both on devices and interconnects, leads to serious challenges to ensure the required product reliability. Standard reliability tests and post-mortem failure analysis provide only limited information about the physics of failure mechanisms and degradation kinetics. Therefore it is necessary to develop new experimental approaches and procedures to study the TDDB failure mechanisms and degradation kinetics in particular. In this paper, an in situ experimental methodology in the transmission electron microscope (TEM) is demonstrated to investigate the TDDB degradation and failure mechanisms in Cu/ULK interconnect stacks. High quality imaging and chemical analysis are used to study the kinetic process. The in situ electrical test is integrated into the TEM to provide an elevated electrical field to the dielectrics. Electron tomography is utilized to characterize the directed Cu diffusion in the insulating dielectrics. This experimental procedure opens a possibility to study the failure mechanism in interconnect stacks of microelectronic products, and it could also be extended to other structures in active devices.

In Situ Time-dependent Dielectric Breakdown in the Transmission Electron Microscope: A Possibility to Understand the Failure Mechanism in Microelectronic Devices

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. This paper demonstrates the procedure of an in situ TDDB experiment in the transmission electron microscope, which opens a possibility to study the failure mechanism in microelectronic products.

The time-dependent dielectric breakdown (TDDB) in on-chip interconnect stacks is one of the most critical failure mechanisms for microelectronic devices. ...

Thermal Measurement Techniques in Analytical Microfluidic Devices

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

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

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

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

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

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

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

Fabrication of Fully Solution Processed Inorganic Nanocrystal Photovoltaic Devices

We demonstrate a method for the preparation of fully solution processed inorganic solar cells from a spin and spray coating deposition of nanocrystal inks. For the photoactive absorber layer, colloidal CdTe and CdSe nanocrystals (3-5 nm) are synthesized using an inert hot injection technique and cleaned with precipitations to remove excess starting reagents. Similarly, gold nanocrystals (3-5 nm) are synthesized under ambient conditions and dissolved in organic solvents. In addition, precursor solutions for transparent conductive indium tin oxide (ITO) films are prepared from solutions of indium and tin salts paired with a reactive oxidizer. Layer-by-layer, these solutions are deposited onto a glass substrate following annealing (200-400 &#176;C) to build the nanocrystal solar cell (glass/ITO/CdSe/CdTe/Au). Pre-annealing ligand exchange is required for CdSe and CdTe nanocrystals where films are dipped in NH4Cl:methanol to replace long-chain native ligands with small inorganic Cl- anions. NH4Cl(s) was found to act as a catalyst for the sintering reaction (as a non-toxic alternative to the conventional CdCl2(s) treatment) leading to grain growth (136&#177;39 nm) during heating. The thickness and roughness of the prepared films are characterized with SEM and optical profilometry. FTIR is used to determine the degree of ligand exchange prior to sintering, and XRD is used to verify the crystallinity and phase of each material. UV/Vis spectra show high visible light transmission through the ITO layer and a red shift in the absorbance of the cadmium chalcogenide nanocrystals after thermal annealing. Current-voltage curves of completed devices are measured under simulated one sun illumination. Small differences in deposition techniques and reagents employed during ligand exchange have been shown to have a profound influence on the device properties. Here, we examine the effects of chemical (sintering and ligand exchange agents) and physical treatments (solution concentration, spray-pressure, annealing time and annealing temperature) on photovoltaic device performance.

This protocol describes the synthesis and solution deposition of inorganic nanocrystals layer by layer to produce thin film electronics on non-conductive surfaces. Solvent-stabilized inks can produce complete photovoltaic devices on glass substrates via spin and spray coating following post-deposition ligand exchange and sintering.

We demonstrate a method for the preparation of fully solution processed inorganic solar cells from a spin and spray coating deposition of nanocrystal ...

Solvent Bonding for Fabrication of PMMA and COP Microfluidic Devices

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

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

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

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

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

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

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

Design and Fabrication of an Optical Fiber Made of Water

In this report, an optical fiber of which the core is made solely of water, while the cladding is air, is designed and manufactured. In contrast with solid-cladding devices, capillary oscillations are not restricted, allowing the fiber walls to move and vibrate. The fiber is constructed by a high direct current (DC) voltage of several thousand volts (kV) between two water reservoirs that creates a floating water thread, known as a water bridge. Through the choice of micropipettes, it is possible to control the maximal diameter and length of the fiber. Optical fiber couplers, at both sides of the bridge, activate it as an optical waveguide, allowing researchers to monitor the water fiber capillary body waves through transmission modulation and, therefore, deducing changes in surface tension.
Co-confining two important wave types, capillary and electromagnetic, opens a new path of research in the interactions between light and liquid-wall devices. Water-walled microdevices are a million times softer than their solid counterparts, accordingly improving the response to minute forces.

This protocol describes the design and manufacture of a water bridge and its activation as a water fiber. The experiment demonstrates that capillary resonances of the water fiber modulate its optical transmission.

In this report, an optical fiber of which the core is made solely of water, while the cladding is air, is designed and manufactured. In contrast with ...

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 ...