The authors thank Scott Longwell for helpful comments and edits to the manuscript and Robert Puccinelli for device photography. The authors acknowledge generous support from a Beckman Institute Technology Development Grant. K.B. is supported by a NSF GFRP fellowship and the TLI component of the Stanford Clinical and Translational Science Award to Spectrum (NIH TL1 TR 001084); P.F. acknowledges a McCormick and Gabilan Faculty Fellowship.

1. Multi-layer Device Design
NOTE: Features of different heights and/or photoresists must be added sequentially to the wafer during different fabrication steps to create final composite features. Therefore, designs for each separate height and photoresist to be included on a wafer must be printed on their own mask (Figure 4).
<ol>
	<li>Download a computer-assisted design (CAD) drafting program (e.g., AutoCAD Educational Version).</li>
	<li>Define the 4&#34; wafer ar...

<ol>
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Biotech. 28 (9), (2010).</li><li>Fan, R., 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=Integrated+barcode+chips+for+rapid,+multiplexed+analysis+of+proteins+in+microliter+quantities+of+blood.">Integrated barcode chips for rapid, multiplexed analysis of proteins in microliter quantities of blood.</a> Nat. Biotech. 26 (12), (2008).</li><li>Kovarik, M. L., Gach, P. C., Ornoff, D. M., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro+total+analysis+systems+for+cell+biology+and+biochemical+assays.">Micro total analysis systems for cell biology and biochemical assays.</a> Anal. Chem. , (2011).</li><li>Stroock, A. D., Dertinger, S. K. W., Ajdari, A., Mezi&#263;, I., Stone, H. A., Whitesides, G. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Large-Scale+Integration.">Microfluidic Large-Scale Integration.</a> Science. 298 (5593), (2002).</li><li>Li, N., Sip, C., Folch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Chips+Controlled+with+Elastomeric+Microvalve+Arrays.">Microfluidic Chips Controlled with Elastomeric Microvalve Arrays.</a> JoVE. (8), e296 (2007).</li><li>Kim, P., 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=Soft+lithography+for+microfluidics:+a+review.">Soft lithography for microfluidics: a review.</a> Biochip. J. 2 (1), 1-11 (2008).</li><li>Studer, V., Hang, G., Pandolfi, A., Ortiz, M., Anderson, W. F., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+properties+of+a+low-actuation+pressure+microfluidic+valve.">Scaling properties of a low-actuation pressure microfluidic valve.</a> J. Appl. Phys. 95 (1), 393-398 (2004).</li><li>Kartalov, E. P., Scherer, A., Quake, S. R., Taylor, C. R., Anderson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimentally+validated+quantitative+linear+model+for+the+device+physics+of+elastomeric+microfluidic+valves.">Experimentally validated quantitative linear model for the device physics of elastomeric microfluidic valves.</a> J. Appl. Phys. 101 (6), 064505 (2007).</li><li>Gerver, R. E., G&#243;mez-Sj&#246;berg, R., 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=Programmable+microfluidic+synthesis+of+spectrally+encoded+microspheres.">Programmable microfluidic synthesis of spectrally encoded microspheres.</a> Lab. Chip. 12 (22), 4716-4723 (2012).</li><li>Fordyce, P. M., Diaz-Botia, C. A., DeRisi, J. L., G&#243;mez-Sj&#246;berg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+characterization+of+feature+dimensions+and+closing+pressures+for+microfluidic+valves+produced+via+photoresist+reflow.">Systematic characterization of feature dimensions and closing pressures for microfluidic valves produced via photoresist reflow.</a> Lab. Chip. 12 (21), 4287-4295 (2012).</li><li>Li, C. -. W., Cheung, C. N., Yang, J., Tzang, C. H., Yang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS-based+microfluidic+device+with+multi-height+structures+fabricated+by+single-step+photolithography+using+printed+circuit+board+as+masters.">PDMS-based microfluidic device with multi-height structures fabricated by single-step photolithography using printed circuit board as masters.</a> The Analyst. 128 (9), 1137-1142 (2003).</li><li>Romanowsky, M. B., Abate, A. R., Rotem, A., Holtze, C., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+production+of+single+core+double+emulsions+in+a+parallelized+microfluidic+device.">High throughput production of single core double emulsions in a parallelized microfluidic device.</a> Lab. Chip. 12 (4), 802-807 (2012).</li><li>Mata, A., Fleischman, A. 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The authors declare that they have no competing financial interests.

This work demonstrates a complete multi-step photolithography protocol for a multilayer microfluidic device with valves and variable height geometry that can be tuned for any application with simple modifications to fabrication parameters based on our online tool26 and manufacturer instructions25. This protocol is intended to demystify multilayer photolithography for researchers wishing to construct microfluidic devices beyond simple, passive one-layer molds.
<p class="...

For the past 15 years, microfluidics as a field has undergone rapid growth, with an explosion of new technologies enabling the manipulation of fluids at the micrometer scale1. Microfluidic systems are attractive platforms for wet laboratory functionality because the small volumes have the potential to realize increased speed and sensitivity while at the same time dramatically increasing throughput and reducing cost by leveraging economies of scale2,3. Multilayer microfluidic systems have made particularly significant impacts in high-throughput biochemical analysis applications such as single cell analysis4,5,6, single molecule analysis (e.g., digital PCR7), protein crystallography8, transcription factor binding assays9,10, and cellular screening11.
A central goal of microfluidics has been the development of &#34;lab on a chip&#34; devices capable of performing complex fluidic manipulations within a single device for total biochemical analysis12. The development of multi-layer soft lithography techniques has helped realize this goal by enabling creation of on-chip valves, mixers, and pumps for actively controlling fluids within small volumes13,14,15. Despite their advantages and demonstrated applications, many of these microfluidic technologies remain largely unharnessed by non-specialist users. Widespread adoption has been challenging in part due to limited access to microfabrication facilities, but also due to inadequate communication of fabrication techniques. This is especially true for multilayer microfluidic devices featuring structures for valves or complex geometries: the paucity of detailed, practical information about important design parameters and fabrication techniques often deters new researchers from embarking on projects involving the design and creation of these devices.
This article aims to address this knowledge gap by presenting a complete protocol for making multilayer microfluidic devices with valves and variable height features, starting from design parameters and moving through all fabrication steps. By focusing on the initial photolithography steps of fabrication, this protocol complements other microfluidics protocols16 that describe downstream steps of casting devices from molds and running specific experiments.
Microfluidic devices with monolithic on-chip valves are composed of two layers: a &#34;flow&#34; layer, where the fluid of interest is manipulated in microchannels, and a &#34;control&#34; layer, where microchannels containing air or water can selectively modulate fluid flow in the flow layer14. These two layers are each fabricated on a separate silicon molding master, which is subsequently used for polydimethylsiloxane (PDMS) replica molding in a process called &#34;soft lithography17.&#34; To form a multilayer device, each of the PDMS layers are cast on their respective molding masters and then aligned to one another, thereby forming a composite PDMS device with channels in each layer. Valves are formed at locations where flow and control channels cross one another and are separated by only a thin membrane; pressurization of the control channel deflects this membrane to occlude the flow channel and locally displace the fluid (Figure 1).
Active on-chip valves can be fabricated in multiple ways, depending on the desired final application. Valves can be configured in either a &#34;push down&#34; or &#34;push up&#34; geometry, depending on whether the control layer is above or below the flow layer (Figure 1)15. &#34;Push up&#34; geometries allow for lower closing pressures and higher device stability against delamination, while &#34;push down&#34; geometries allow for the flow channels to be in direct contact with the bonded substrate, conferring the advantage of selective functionalization or patterning of the substrate surface for later functionality18,19.
Valves can also be either intentionally leaky &#34;sieve&#34; valves or fully sealable, depending on the cross-sectional profile of the flow channel. Sieve valves are useful for trapping beads, cells or other macroanalytes1, and are fabricated via the use of typical negative photoresists (i.e., SU-8 series), which have rectangular profiles. When a control channel is pressurized over these valve regions, the PDMS membrane between the control and flow layer deflects isotropically into the rectangular profile of the valve without sealing the corners, permitting fluid flow but trapping macro scale particles (Figure 1). Conversely, fully-sealable microfluidic valves are fabricated by including a small patch of rounded photoresist at valve locations. With this geometry, pressurization of the control channel deflects the membrane against the rounded flow layer to completely seal the channel, halting fluid flow. Rounded profiles in the flow layer are generated via the melting and reflow of positive photoresist (e.g., AZ50 XT or SPR 220) after typical photolithography steps. We have previously demonstrated that post-reflow heights of valve regions depend on chosen feature dimensions21. This protocol demonstrates the fabrication of both valve geometries within a bead synthesis device.
<img alt="Figure 1" src="/files/ftp_upload/55276/55276fig1.jpg" /> 
Figure 1: Multilayer Microfluidic Valve Geometries. Typical &#34;push up&#34; device architectures for sieve and fully sealable valves before (top) and after (bottom) pressurization. <a href="http://cloudflare.jove.com/files/ftp_upload/55276/55276fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Devices can also include complex passive features such as chaotic mixers13 and on-chip resistors20 that require features of multiple different heights within a single flow layer. To achieve a variable height flow layer, different groups have employed many methods including printed circuit board etching22, multilayer PDMS relief alignment23, or multi-step photolithography24. Our group has found multi-step photolithography on a single molding master to be an effective and reproducible method. To do this, a simple photolithography technique of building thick channels of negative photoresist (e.g., SU-8 series photoresists) in layers without development in between application of each layer is employed. Each layer is spun in negative photoresist according to its thickness using manufacturer instructions25 on the silicon master. Features of this height are then patterned onto the layer using a specific transparency mask (Figure 2) affixed to a glass mask plate and aligned to the previously spun layer before exposure. In multi-step photolithography, precise alignment between layers is critical in forming a complete variable height flow channel. After alignment, each layer is subjected to a thickness-dependent post-exposure bake. Without development, the next layer is similarly patterned. In this way, tall features can be built up on a single flow wafer layer-by-layer via the use of multiple masks. By skipping development between each step, previous photoresist layers can be used to generate composite height features (i.e., two 25 &#181;m layers can make a 50 &#181;m feature)24. Additionally, channel floor features such as chaotic mixer herringbone grooves13 can be made using layers with previously exposed features. A final development step completes the process, creating a single flow wafer with features of variable height (Figure 3).
Here, a complete protocol for multi-step photolithography that includes examples of all procedures necessary to fabricate on-chip valves and flow channels with multiple heights is provided. This fabrication protocol is presented in the context of a multi-layer microfluidic bead synthesizer that requires valves and variable-height features for its functionality. This device includes T-junctions for generating water droplets in an oil sheath, on-chip resistors to modulate flow rates through controlling Poiseuille resistance, a chaotic mixer for homogenizing droplet components, and both fully sealing and sieve valves to enable automated workflows involving multiple reagent inputs. Using multi-step photolithography, these features are each fabricated on a different layer according to height or photoresist; the following layers are constructed in this protocol: (1) Flow Round valve layer (55 &#181;m, AZ50 XT) (2) Flow Low layer (55 &#181;m, SU-8 2050) (3) Flow High layer (85 &#181;m, SU-8 2025, 30 &#181;m additive height), and (4) Herringbone Grooves (125 &#181;m, SU-8 2025, 40 &#181;m additive height) (Figure 3).
Hydrogel beads can be used for a variety of applications including selective surface functionalization for downstream assays, drug encapsulation, radiotracing and imaging assays, and cell incorporation; we previously used a more complex version of these devices to produce spectrally encoded PEG hydrogel beads containing lanthanide nanophosphors20. The designs discussed here are included in Additional Resources for any lab to use in their research efforts if desired. We anticipate that this protocol will provide an open resource for specialists and non-specialists alike interested in making multi-layer microfluidic devices with valves or complex geometries to lower the barrier to entry in microfluidics and increase the chances of fabrication success.

<table border="0" cellpadding="0" cellspacing="0" width="1709">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Materials</td>
			<td style="width:332px;"></td>
			<td style="width:259px;"></td>
			<td style="width:659px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mylar Transparency Masks, 5&#34;</td>
			<td>FineLine Plotting</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5&#34; Quartz Plates</td>
			<td>United Silica&#160;</td>
			<td>Custom</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#34; Silicon Wafers, Test Grade</td>
			<td>University Wafer</td>
			<td>452</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SU8 2005, 2025, 2050 photoresist</td>
			<td>Microchem</td>
			<td>Y111045, Y111069, Y111072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Az50XT&#160;</td>
			<td>Integrated Micromaterials</td>
			<td>AZ50XT-Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SU8 Developer</td>
			<td>Microchem</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AZ400K 1:3 Developer</td>
			<td>Integrated Micromaterials</td>
			<td>AZ400K1:3-CS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyrex 150 mm glass dish</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3140150-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wafer Petri Dishes, 150 mm</td>
			<td>VWR</td>
			<td>25384-326</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wafer Tweezers&#160;</td>
			<td>Electron Microscopy Sciences (EMS)</td>
			<td>78410-2W</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFOTS)</td>
			<td>Sigma-Aldrich</td>
			<td>448931-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2&#34; x 3&#34; glass slides</td>
			<td>Thomas Scientific&#160;</td>
			<td>6686K20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RTV 615 elastomeric base and curing agent PDMS set</td>
			<td>Momentive&#160;</td>
			<td>RTV615-1P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tygon Tubing, 0.02&#34; O.D.&#160;</td>
			<td>Fischer Scientific&#160;</td>
			<td>14-171-284</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary PEEK tubing,&#160; 510 um OD, 125 um ID</td>
			<td>Zeus</td>
			<td>Custom</td>
			<td>360 um PEEK is readily available by Idex (catalog number: 1571)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyro 4 mL tube</td>
			<td>Greiner Bio-One</td>
			<td>127279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epoxy, 30-minute</td>
			<td>Permatex</td>
			<td>84107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Metal Pins, 0.025&#34; OD, .013&#34; ID</td>
			<td>New England Small Tube</td>
			<td>NE-1310-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(ethylene glycol) diacrylate, Mn 700</td>
			<td>Sigma-Aldrich</td>
			<td>455008-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate photoinitator&#160;</td>
			<td>Tokyo Chemical Industry Co.</td>
			<td>L0290</td>
			<td>We typically synthesize LAP in-house.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034-25G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>330779-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Span-80</td>
			<td>Sigma-Aldrich</td>
			<td>85548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ABIL EM 90</td>
			<td>UPI Chem</td>
			<td>420095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name&#160;</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:459px;">Equipment</td>
			<td>Equivalent equiptment or homebuilt setups will work equally as well</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mask Aligner</td>
			<td>Karl Suss</td>
			<td>MA6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Profilometer</td>
			<td>KLA-Tencor</td>
			<td>Alpha-Step D500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spin Coater</td>
			<td>Laurell Technologies</td>
			<td>WS-650-23</td>
			<td>Any spincoater can be used that accepts 100 mm wafers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vacuum Dessicator, Bell-Jar Style</td>
			<td>Bel-Art</td>
			<td>420100000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oven</td>
			<td>Cole-Palmer</td>
			<td>WU-52120-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV Spot Curing System with 3 mm LLG option</td>
			<td>Dymax</td>
			<td>41015</td>
			<td>UV LEDs, Xenon Arc Lamps, or other UV sources of the same intensity work equally as well</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MFCS Microfluidic Fluid Control System</td>
			<td>Fluidgent</td>
			<td>MFCS-EZ</td>
			<td>Syringe pumps, custom pneumatics or other control systems can also be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Automated control scripting</td>
			<td>MATLAB</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hotplate</td>
			<td>Tory Pines Scientific</td>
			<td>HP30</td>
			<td>Any hotplate with uniform heating (i.e. aluminum or ceramic plates) will suffice.&#160;</td>
		</tr>
	</tbody>
</table>

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.

Here, we demonstrate the fabrication of valved, variable height multilayer microfluidic molds by making devices capable of generating poly ethylene glycol (PEG) hydrogel beads from droplets (Figure 2). An overview of the complete fabrication process is included in Figure 3. Using design elements from previous work, the bead synthesizer employs 4 heights in its flow layer including (1) rounded AZ50 XT valves for laminar flow modulation (55...

Watch this Scientific Journal Video about Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices at JoVE.com

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

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 ...

The overall goal of this video protocol is to demonstrate complete multi-step photolithography of microfluidic master molds with on-chip valves and multiple height features tunable for any application. This method is a complete overview for how to fabricate master molds with complex geometries, including on-chip membrane valves, for microfluidic devices. The main advantage of this technique is that it makes it possible to easily control flow in microfluidic devices, overcoming a major barrier to entry to microfluidics in biological applications.Visual demonstration of this technique is critical, as the steps for photolithography are often difficult for beginners to master. Proper alignment, development, and exposure rely on visual cues and clean room experience. To begin, design your device and prepare the individual photo masks for the multilayer geometries.Additionally, prepare about four wafers with a five-micron layer of SU-8 2050 negative photoresist, and flood expose as described in the accompanying text protocol. Place the coated wafer on a spin coater, and turn on the vacuum to affix it to the spin chuck. Use nitrogen or compressed air to blow away any dust from the surface.Then, apply two to three milliliters of AZ 50XT positive photoresist to the center of the wafer. Spin coat the photoresist to create a 55-micron layer. Once coated, lay down the wafer carefully in a five-inch Petri dish, and let relax for 20 minutes.Next, soft bake the wafer on a hotplate for 22 minutes while ramping the temperature from 65 degrees Celsius to 112 degrees Celsius at a rate of 450 degrees Celsius per hour. Then, remove the wafer and let it rest overnight at room temperature in a Petri dish for ambient rehydration. Tape the flow round transparency mask to the five-inch glass plate so that the print side is closest to the wafer, and load into the mask positioner of the UV mask aligner.Expose the wager to 930 milliJoules of UV in six cycles. Develop the wafer immediately by immersing it in a stirred bath of developer for three to five minutes, or until the bath turns purple and the features emerge. Once developed, remove the wafer and rinse it well with deionized water.Then, hard bake the wafer to melt and round valve features. Ramp the temperature from 65 to 190 degrees Celsius over the course of 15 hours at a rate of 10 degrees Celsius per hour. Once finished, turn off the hotplate and let the wafer cool to room temperature.The features on the wafer are now rounded. This hard bake is critical to properly reflow rectangular valve features into rounded valve profiles. Shorter times may result in cracking or instability.In order to fabricate a device with variable height features, place the cleaned wafer on a spin coater as previously shown. Apply one to two milliliters of SU-8 2050 negative photoresist to the center of the wafer, and spin the photoresist over the developed valve features. Then, carefully place the spun wafer in a five-inch Petri dish, and let it relax for 20 minutes on a flat surface, or until any streaking patterns fade.Next, preheat two hotplates to 65 degrees Celsius and 95 degrees Celsius, and then set the wafer on the 65-degree-Celsius plate for two minutes, the 95-degree-Celsius plate for eight minutes, and the 65-degree-Celsius plate for two additional minutes to soft bake the wafer. Once the wafer cools back to room temperature, tape the flow low transparency mask to a quartz five-inch glass plate so that the print side is closest to the wafer, and load it into the mask positioner of the UV mask aligner. Then, place the wafer in a UV mask aligner chuck, and using the microscope eyepiece or camera, carefully align the new flow low layer features to the flow round valve layer features.Begin by aligning the horizontal, vertical, and tilt axes of device borders to the device border features on the mask. Next, align crosshair features between the layers. Finally, confirm that the valve features intersect the flow low features where desired.Next, expose the wafer to 170 milliJoule UV deposition. When finished, remove the wafer, and bake it post-exposure by switching between the two hotplates set at 65 degrees Celsius and 95 degrees Celsius. Without developing the wafer, allow it to cool to room temperature, and then sequentially add the flow high layer and then the chaotic mixer herringbone layer using SU-8 2025 as described in the accompanying text protocol.After all layers have been completed, develop the features by immersing the wafer in a stirred bath containing 25 milliliters of SU-8 developer for 3.5 minutes, or until the features clearly emerge. Use a stereoscope to verify that the features have clear, defined feature boundaries. During development, be sure to check every 20 seconds to see that features have become fully defined and resist has washed away.Overdevelopment may result in damaging the features, especially on complex mold designs. Then, hard bake the wafer to stabilize all of the photoresist features. Subsequently, fabricate the control layer as described in the accompanying text protocol.Fabricate multilayer microfluidic devices in a pushup geometry on glass according to existing open-access protocols, and use visual inspection to ensure that all of the valves are properly aligned to control lines, and that all of the inlets are punched fully before proceeding. Connect Tygon tubing loaded with water to a flow control system, such as a syringe pump, fluidic controllers, or an open-source solenoid valve array with reservoirs. Next, connect metal pins to the tubing and the metal pins to the device ports at control line inlets.Then, set the flow control system to 25 PSI for each line to pressurize the device control lines. Ensure that valves close and reopen by inspection under the microscope. In a microcenrifuge tube, suspend 3.9 milligrams of photoinitiator in 100 microliters of DI water to prepare the photoinitiator solution used to polymerize droplets into hydrogel beads.Cover the solution to protect it from light. In a second microcentrifuge tube, add 132 microliters of deionized water, 172 microliters of PEG diacrylate, 12 microliters of the photoinitiator solution, and 85 microliters of HEPES buffer to make the hydrogel droplet solution. Transfer the hydrogel droplet solution to a customized cryogenic tube vessel.Then, connect the tubing of the cryogenic tube vessel to a controllable pressure source, and connect the PEEK tubing to the device reagent inlet. Next, insert the PEEK tubing at the device outlet in order to collect the droplets. Remove air bubbles from the device, repressurize the system, and then depressurize the RO1 oil valve, and set the oil pressure to 10 PSI.Next, set the PEG mixture pressure to nine PSI, depressurize the upstream valves, and adjust the pressure as necessary to produce droplets of the desired size. Determine the droplet size via microscopy using a camera with 50 FPS or higher. When the droplets have stabilized, position a UV light source over the polymerization region of the device, and apply 100 milliWatts per square centimeter of 365 nanometer light from the source onto a five millimeter spot.Pressurize the bead sieve valve to watch polymerized beads collect and ensure that droplets have hardened into beads. Finally, depressurize the bead sieve valve and collect beads into a tube through the PEEK outlet tubing. This protocol starts by demonstrating a method for rounding flow valves.Here, a profilometer was used to determine the typical post-reflow valve rounding profile resulting from this method, showing a height of approximately 55 microns. In the image on the left, the valve is off, and liquid can pass through the channels. Once activated by pressurizing the valves, the flow through these valves is cut off.Here, one can see the bead synthesizer device in operation, producing hydrogel droplets in an oil emulsion at the T junction droplet generator. By partially closing a downstream flow using a sieve valve, the fluid can continue to flow, but the beads are trapped behind the valve. The resulting beads produced using this process averaged 52.6 microns in diameter with a standard deviation of only 1.6 microns.Out of nearly 3, 000 beads, less than 1%were off by more than three standard deviations. Once mastered, this technique can be completed in three days from design to testing. This allows for rapid design iteration.Following this procedure, even researchers with little fabrication experience can build their own complex microfluidic devices and apply them to their own biological problems. After watching this video, you should have a good understanding of how to perform the photolithography steps necessary to make microfluidic devices of any level of complexity, including devices with complex variable height features or valves.

multi-step-variable-height-photolithography-for-valved-multilayer

Research

JoVE Journal

Bioengineering

14.2K 조회수.  Stanford University. The overall goal of this video protocol is to demonstrate complete multi-step photolithography of microfluidic master molds with on-chip valves and multiple height features tunable for any application. This method is a complete overview for how to fabricate master molds with complex geometries, including on-chip membrane valves, for microfluidic devices. The main advantage of this technique is that it makes it possible to easily control flow in microfluidic devices, overcoming a major barrier...