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 area by drawing a 4&#34; circle. Wafer designs (Figure 4, Additional Resources) are provided as an example.</li>
	<li>Inside the 4&#34; wafer outline, place device borders using 300 &#181;m polyline rectangles. Use these device borders for alignment during photolithography.</li>
	<li>Create different layers for each different height or photoresist needed for the final design (i.e., flow round, flow low, flow high, and control in the design) using the Layers panel.
	<ol>
		<li>Design features of a particular desired height on the corresponding layer. The example design shows 4 different active layers, each with its own color (Figure 4). 
		NOTE: Device borders, global text, and the wafer outline should be made on their own layer (i.e., 1-Negative in the designs), which, later, will appear on all layers for global alignment. Features of different photoresist (such as fully sealable valves that must be fabricated with positive resist) must appear on different layers, regardless of height.</li>
	</ol>
	</li>
	<li>Using closed zero-width polylines, design device features within device borders.
	<ol>
		<li>Consider design parameters in Table 1 to increase chances of successful fabrication.</li>
		<li>For each height, select that layer in the Layers panel and add all features of that height.</li>
	</ol>
	</li>
	<li>Ready designs for transparency film printing using the Basic Mask File (Additional Resources) where each 4&#34; wafer circle is inserted within a 5&#34; rectangular border. Each layer will be printed on a separate transparency film for sequential addition of each photoresist layer. 
	NOTE: This Basic Mask File represents the final designs used for printing.
	<ol>
		<li>To complete design, turn all Layers off except 1-Negative and the AZ50 XT valve layer. Copy the entire wafer with the active layer (i.e., valves) and global features (i.e., device borders).</li>
		<li>Open the Basic Mask File and paste this design into the rectangle entitled AZ50 XT valves. Use the outer wafer border for alignment and subsequently delete it after pasting.</li>
		<li>Repeat for the rest of the layers (e.g., in the example design: flow square low, flow square high, and control). Example transparency files are provided (Additional Resources).</li>
		<li>Send files to a commercial printing company (e.g., FineLine Imaging) for printing on transparency film. Use 32,000 DPI for printing &#62;10 &#181;m features and up to 50,000 DPI for smaller features. If features less than 7 &#181;m are needed, order a Chrome Mask instead of a transparency film.</li>
	</ol>
	</li>
</ol>
Table 1: Design Parameters and Suggestions. Design considerations to avoid common pitfalls during the CAD design process of microfluidic devices. <a href="http://cloudfront.jove.com/files/ftp_upload/55276/Jove_Table_1.xlsx" target="_blank">Please click here to view this table. (Right-click to download.)</a>
2. Preparing a Wafer for Photolithography
NOTE: These steps additionally appear in table-format in Table 2.
<ol>
	<li>In cleanroom or designated clean area, clean and dehydrate a 4&#34; test-grade silicon wafer (single-side polished).
	<ol>
		<li>Rinse the wafer well with methanol. 
		NOTE: No further cleaning steps are needed if using the SU-8 adhesion layer described below. Other adhesion layers that deviate from this protocol (e.g., HMDS) often require more thorough cleaning, such as piranha etching.</li>
		<li>Blow dry with N2 or compressed air.</li>
		<li>Bake on an aluminum hotplate at 95 &#176;C for 10 min to fully evaporate solvent.</li>
	</ol>
	</li>
	<li>Fabricate a uniform 5 &#181;m thick layer of SU-8 2005 to improve adhesion for subsequent photoresist layers.
	<ol>
		<li>Place the cleaned wafer on a spin coater, turn on the vacuum to affix it to the spin chuck, and blow away dust with N2 or compressed air.</li>
		<li>Apply 1-2 ml of SU-8 2005 negative photoresist in center of wafer and spin as follows: spread: 500 rpm, 10 sec, 133 rpm/s acceleration; cast: 3,000 rpm, 40 sec, 266 rpm/s acceleration.</li>
		<li>Remove wafer and soft bake by switching wafer between two hotplates set at 65 &#176;C and 95 &#176;C according to the following program: 65 &#176;C: 2 min, 95 &#176;C: 3 min, 65 &#176;C: 2 min.</li>
		<li>Allow wafer to cool to RT.</li>
		<li>Place wafer in the chuck of a UV mask aligner and expose without a mask (&#39;flood exposure&#39;) for a total energy deposition of 124 mJ (here, 20 sec at ~6.2 mW/cm2 lamp intensity). If available, select hard contact mode to achieve a 300 &#181;m wafer: mask separation.</li>
		<li>Remove wafer and post-exposure bake by switching the wafer between two hotplates set at 65 &#176;C and 95 &#176;C as follows: 65 &#176;C: 2 min, 95 &#176;C: 4 min, 65 &#176;C: 2 min.</li>
	</ol>
	</li>
</ol>
Fabricating Rounded Valves
<ol start="3">
	<li>Use online AZ50 XT valve predictor resource26 to plan spin speeds for desired valve dimensions and heights. 
	NOTE: The following steps will deposit a 55 &#181;m layer of positive photoresist for valve definition and reflow rounding.</li>
	<li>Place the wafer on a spin coater, turn on vacuum to affix it to the spin chuck, and blow away dust with N2 or compressed air.</li>
	<li>Apply 2-3 mL of AZ50 XT positive photoresist to center of the wafer. Spin as follows: spread: 200 rpm, 10 s, 133 rpm/s acceleration; cast: 1,200 rpm, 40 sec, 266 rpm/s acceleration; Snap spin to remove edge bead: 3,400 rpm, 1 sec, 3,400 rpm/s acceleration.</li>
	<li>In a 5&#34; Petri dish, lay down the wafer carefully and let relax for 20 min.</li>
	<li>Soft bake the wafer on a hotplate: 65 &#176;C - 112 &#176;C, 22 min, 450 &#176;C/h ramping speed.</li>
	<li>Remove the wafer and let rest overnight at RT in a Petri dish for ambient rehydration.</li>
	<li>Tape Flow Round transparency mask to 5&#34; glass plate print-side down (closest to wafer) and load into mask positioner of the UV mask aligner. Expose the wafer to 930 mJ of UV in 6 cycles (e.g., 6 cycles of 25 sec at ~6.2 mW/cm2 lamp intensity, 30 s wait time between exposures).</li>
	<li>Develop wafer immediately by immersing in a stirred bath of 25 mL of AZ500k 1:3 Developer in 6&#34; glass dish for 3-5 min or until bath turns purple and features emerge.
	<ol>
		<li>Remove the wafer and rinse well with DI water.</li>
		<li>Assess pre-reflow height using a profilometer (stylus force of 10.5 mg). 
		NOTE: Operate the profilometer according to manufacturer instructions, carefully positioning the force stylus next to a feature channel on the desired layer before profiling. Settings used throughout this protocol were the following: stylus force 10.5 mg, length 1,000 &#181;m, speed 200 &#181;m/s, regime down-up.</li>
	</ol>
	</li>
	<li>Reflow hard bake the wafer to melt and round valve features as follows: 65 &#176;C - 190 &#176;C, 15 hr, 10 &#176;C/hr ramping speed.</li>
	<li>Let the wafer cool to RT. Assess post-reflow height using a profilometer (stylus force of 10.5 mg). Heights of 55 &#181;m &#177; 2 &#181;m should be expected for this device geometry.</li>
</ol>
3. Fabricating Variable Height Features in Tandem
<ol>
	<li>Proceed to variable height fabrication with the developed wafer with Flow Low, Flow High and Herringbone Mixer transparencies of the Bead Synthesizer design.</li>
	<li>To adjust protocol for the designs, use manufacture data sheets25 to determine exposure energy, spin speeds and bake time parameters, allowing for &#177; 5% tolerance. 
	NOTE: This protocol fabricates a 55 &#181;m tall Flow Low layer using SU-8 2050 negative photoresist spun over the valve features.</li>
	<li>Place the cleaned wafer on spin coater, turn on the vacuum to affix it to the spin chuck, and blow away dust with N2 or compressed air.
	<ol>
		<li>Apply 1-2 ml of SU-8 2050 negative photoresist to center of the wafer and spin as follows: spread: 500 rpm, 10 sec, 133 rpm/sec acceleration; cast: 3,000 rpm, 40 sec, 266 rpm/sec acceleration. Spin photoresist over developed valve features.</li>
	</ol>
	</li>
	<li>Carefully place the spun wafer in 5&#34; Petri dish and let relax for 20 min on flat surface or until any streaking patterns fade.</li>
	<li>Remove the wafer and soft bake by placing on two hotplates set at 65 &#176;C and 95 &#176;C as follows: 65 &#176;C: 2 min, 95 &#176;C: 8 min, 65 &#176;C: 2 min.</li>
	<li>Allow the wafer to cool to RT.</li>
	<li>Tape the Flow Low transparency mask to a quartz 5&#34; glass plate print-side down (closest to the wafer) and load into the mask positioner of the UV mask aligner.</li>
	<li>Place the wafer in UV mask aligner chuck and, using microscope eyepiece or camera, carefully align new Flow Low layer features to Flow Round valve layer features. Begin by aligning horizontal, vertical and tilt axes of device borders to the device border features on mask. Next, align cross-hair features between layers. Finally, confirm that valve features intersect Flow Low features where applicable.</li>
	<li>Expose to 170 mJ UV deposition (28 sec at ~6.2 mW/cm2).</li>
	<li>Remove the wafer and post-exposure bake by switching between two hotplates set at 65 &#176;C and 95 &#176;C as follows: 65 &#176;C: 2 min, 95 &#176;C: 9 min, 65 &#176;C: 2 min.</li>
	<li>Without developing, allow the wafer to cool to RT and proceed to fabrication of Flow High layer. This Flow High layer will add 30 &#181;m of photoresist to the undeveloped 55 &#181;m photoresist layer to yield 85 &#181;m features in previously unexposed locations.</li>
	<li>Repeat steps 3.3 to 3.10 using SU-8 2025 and the Flow High layer mask with these modifications for the spin coat settings: spread: 500 rpm, 10 sec, 133 rpm/s acceleration; cast: 3,500 rpm, 40 sec, 266 rpm/sec acceleration.
	<ol>
		<li>Expose to 198 mJ UV deposition (32 s at ~6.2 mW/cm2).</li>
	</ol>
	</li>
	<li>Without developing, allow the wafer to cool to RT and proceed to fabrication of Chaotic Mixer Herringbone layer. Final features in this layer will have a total height of 125 &#181;m: 55 &#181;m from the Flow Low layer, 30 &#181;m from the Flow Square layer, and 40 &#181;m from this Chaotic Mixer Herringbone layer (see Figure 3) and include 35 &#181;m herringbone grooves.</li>
	<li>Repeat steps 3.3 to 3.10 using SU-8 2025 and the Herringbone layer mask with the following modifications, ensuring that herringbone grooves are completely within Flow High channel outlines.
	<ol>
		<li>Use the following soft bake program: 65 &#176;C: 2 min, 95 &#176;C: 7 min, 65 &#176;C: 2 min.</li>
		<li>Expose to 148 mJ UV deposition (24 s at ~6.2 mW/cm2).</li>
	</ol>
	</li>
	<li>After all layers have been completed, develop by immersing the wafer in a stirred bath of 25 ml of SU-8 developer in a 6&#34; glass dish for 3.5 min or until features clearly emerge. Check that features have clear, defined feature boundaries using a stereoscope.</li>
	<li>Hard bake the wafer to stabilize all photoresist features on a hot plate as follows: 65 &#176;C - 165 &#176;C, 2 hr 30 min, 120 &#176;C/h ramping speed.</li>
	<li>Assess feature height in all layers using a profilometer (stylus force of 10.5 mg).</li>
</ol>
4. Control Wafer Fabrication
<ol>
	<li>Clean, dehydrate, and fabricate a 5 &#181;m adhesion layer on a new 4&#34; silicon wafer as in Section 4.</li>
	<li>Fabricate a 25 &#181;m Control Layer using SU-8 2025 negative photoresist.</li>
	<li>Place the wafer on a spin coater, turn on vacuum to affix it to the spin chuck, and blow away dust with N2 or compressed air.</li>
	<li>Apply 1-2 ml of SU-8 2025 negative photoresist in the center of the wafer and spin as follows: spread: 500 rpm, 10 sec, 133 rpm/s acceleration; cast: 3,500 rpm, 40 sec, 266 rpm/sec acceleration.</li>
	<li>Remove the wafer and soft bake by switching between two hotplates set at 65 &#176;C and 95 &#176;C as follows: 65 &#176;C: 2 min, 95 &#176;C: 5 min, 65 &#176;C: 2 min.</li>
	<li>Allow the wafer to cool to RT.</li>
	<li>Align the control transparency mask to a 5&#34; glass plate and load into UV mask aligner.</li>
	<li>Place the wafer in chuck of UV mask aligner and expose to 155 mJ UV deposition (25 sec at ~6.2 mW/cm2 lamp intensity).</li>
	<li>Remove the wafer and post-exposure bake by switching between two hotplates set at 65 &#176;C and 95 &#176;C as follows: 65 &#176;C: 2 min, 95 &#176;C: 6 min, 65 &#176;C: 2 min.</li>
	<li>Develop by immersing the wafer in a stirred bath of 25 ml of SU-8 Developer in 6&#34; glass dish for 1 min or until features emerge. Check features using a stereoscope.</li>
	<li>Hard bake the wafer to stabilize photoresist features as follows: 65 &#176;C - 165 &#176;C, 2 hr 30 min, 120 &#176;C/hr ramping speed.</li>
</ol>
5. Silane Wafer Treatment for Easy PDMS Lift-off
<ol>
	<li>Place the completed wafers in wafer rack within a bell-jar vacuum desiccator inside a fume hood free of water or water-soluble reagents.</li>
	<li>Under the hood, use a dropper to apply 1 drop of trichloro(1H,1H,2H,2H-perfluorooctyl) silane (PFOTS) to a glass slide and place inside the desiccator.</li>
	<li>Close the desiccator lid and apply vacuum for 1 min.</li>
	<li>After 1 min, turn off vacuum without re-pressurizing or evacuating bell jar.</li>
	<li>Let the mixture sit for 10 min while aerosolized PFOTS coats wafer surface.</li>
	<li>Open the bell jar lid and remove wafer using tweezers. Place into a Petri dish for PDMS replica molding. Dispose of silane-coated slides in proper hazardous waste. 
	NOTE: Wafers coated with fluorinated silanes can be used hundreds to thousands of times without re-treatment. A sacrificial layer of 1:10 PDMS can be cast on wafers, cured, and discarded after the first silane treatment to remove excess silane groups from wafer surface.</li>
</ol>
6. PDMS Replica Molding
<ol>
	<li>Fabricate multilayer microfluidic devices in a &#34;push up&#34; geometry on glass according to existing open-access protocols16. 
	NOTE: A detailed protocol can additionally be found on the website27.</li>
	<li>By visual inspection, ensure all valves are aligned properly to control lines and all inlets (on both the flow and control layers) are punched fully before proceeding.</li>
</ol>
7. Production of Hydrogel Beads from Droplets
<ol>
	<li>Connect tubing (e.g., Tygon) loaded with water to a flow control system (e.g., syringe pumps, fluidic controllers, or an open-source solenoid valve array with reservoirs28).</li>
	<li>Connect metal pins to tubing and connect to device ports at control line inlets. Pressurize device control lines by setting the flow control system of choice to 25 psi for each line. Ensure that valves close and re-open by inspection under the microscope. 
	NOTE: Follow manufacturer instructions for the flow control system of choice. In this work, a custom software-controlled pneumatic system applies pressure to each line using solenoid valves that toggle between 25 psi compressed air (pressurized) and atmospheric pressure (depressurized). Details on this system can be found in Discussion.</li>
	<li>Prepare custom microfluidic pressure vessels for reagent and oil loading.
	<ol>
		<li>Using a push-pin, punch two holes in the top of a cryogenic vial tube, insert capillary PEEK tubing into one hole, and insert a metal pin connected to the tubing into the second hole.</li>
		<li>Seal tubing in place with epoxy. Let dry for 1 hr.</li>
	</ol>
	</li>
	<li>While waiting, in a microcentrifuge tube, suspend 3.9 mg of LAP photoinitiator into 100 &#181;l of DI water ([LAP] = 39 mg/ml) to prepare photoinitiator solution used for polymerizing droplets to hydrogel beads. Protect from light.</li>
	<li>In a second microcentrifuge tube, add 132 &#181;l DI water, 172 &#181;L PEG diacrylate, 12 &#181;l LAP solution, and 85 &#181;L HEPES buffer to make hydrogel droplet solution.</li>
	<li>Transfer the hydrogel droplet solution to the completed cryogenic tube vessel. 
	NOTE: Additives for other applications such as nanocrystals, magnetic particles or biological molecules can be included within the HEPES component.</li>
	<li>Connect the tubing of the cryogenic tube vessel to a controllable pressure source and connect the PEEK tubing to the device reagent inlet.</li>
	<li>Prepare 10 mL of light mineral oil with 2% v/v nonionic surfactant (e.g., Span 80) and 0.05% EM90 for oil droplet emulsion. Filter using a 0.22 &#181;m syringe filter and load 1 ml into a second cryogenic tube vessel.</li>
	<li>Insert PEEK tubing at the device outlet for collection of droplets.</li>
	<li>Remove air bubbles from the device by pressurizing oil, water, or PEG mixture inlets (4 psi operational pressure). Turn on all valves. Sequentially turn off each valve in a fluid pathway after 1 min or until air bubbles have permeated through the PDMS device. For instance, to de-bubble herringbone mixers, turn on valves Inlet 1, Mix 1 out, and Mix Waste. Then depressurize Inlet 1, Mix 1 out, and Mix Waste until all bubbles are gone.</li>
	<li>When the device is repressurized after debubbling, depressurize Ro1 oil valve and set oil pressure to 10 psi.</li>
	<li>Set PEG mixture pressure to 9 psi, depressure upstream valves (Inlet 1, Drops 1) and adjust as necessary to produce droplets of the desired size. Droplet size can be determined via microscopy using a camera with 50 fps or higher.</li>
	<li>When the droplets have stabilized, position a 5 mm spot from a UV light source (e.g., a UV spot curing system with liquid light guide (LLG) or a focused UV LED) over the polymerization region of the device and apply 100 mW/cm2 UV (365 nm) from the UV source.</li>
	<li>Pressurize bead sieve valve to watch polymerized beads collect and ensure that droplets have hardened into beads. Adjust LLG as necessary to achieve full polymerization.</li>
	<li>Depressurize bead sieve valve and collect beads into tube through outlet PEEK tubing.</li>
</ol>

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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. J., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+multi-layer+SU-8+microstructures.">Fabrication of multi-layer SU-8 microstructures.</a> JMM. 16 (2), 276 (2006).</li><li>. Rafael's Microfluidics Site Available from: <a target="_blank" href="https://sites.google.com/site/rafaelsmicrofluidicspage/valve-controllers">https://sites.google.com/site/rafaelsmicrofluidicspage/valve-controllers</a> (2016)</li><li>Wanat, S., Plass, R., Sison, E., Zhuang, H., Lu, P. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Thick+Film+Processing+for+Bumping+Layers.">Optimized Thick Film Processing for Bumping Layers.</a> Proc. SPIE. , 1281-1288 (2003).</li></ol>

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><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mylar Transparency Masks, 5&quot;</td><td>FineLine Plotting</td><td></td><td></td></tr><tr><td>5&quot; Quartz Plates</td><td>United Silica&amp;nbsp;</td><td>Custom</td><td></td></tr><tr><td>4&quot; Silicon Wafers, Test Grade</td><td>University Wafer</td><td>452</td><td></td></tr><tr><td>SU8 2005, 2025, 2050 photoresist</td><td>Microchem</td><td>Y111045, Y111069, Y111072</td><td></td></tr><tr><td>Az50XT&amp;nbsp;</td><td>Integrated Micromaterials</td><td>AZ50XT-Q</td><td></td></tr><tr><td>SU8 Developer</td><td>Microchem</td><td>Y020100</td><td></td></tr><tr><td>AZ400K 1:3 Developer</td><td>Integrated Micromaterials</td><td>AZ400K1:3-CS</td><td></td></tr><tr><td>Pyrex 150 mm glass dish</td><td>Sigma-Aldrich</td><td>CLS3140150-1EA</td><td></td></tr><tr><td>Wafer Petri Dishes, 150 mm</td><td>VWR</td><td>25384-326</td><td></td></tr><tr><td>Wafer Tweezers&amp;nbsp;</td><td>Electron Microscopy Sciences (EMS)</td><td>78410-2W</td><td></td></tr><tr><td>Trichloro(1H,1H,2H,2H-perfluorooctyl)silane (PFOTS)</td><td>Sigma-Aldrich</td><td>448931-10G</td><td></td></tr><tr><td>2&quot; x 3&quot; glass slides</td><td>Thomas Scientific&amp;nbsp;</td><td>6686K20</td><td></td></tr><tr><td>RTV 615 elastomeric base and curing agent PDMS set</td><td>Momentive&amp;nbsp;</td><td>RTV615-1P</td><td></td></tr><tr><td>Tygon Tubing, 0.02&quot; O.D.&amp;nbsp;</td><td>Fischer Scientific&amp;nbsp;</td><td>14-171-284</td><td></td></tr><tr><td>Capillary PEEK tubing, 510 &amp;#956;m OD, 125 &amp;#956;m ID</td><td>Zeus</td><td>Custom</td><td>360 &amp;#956;m PEEK is readily available by Idex (catalog number: 1571)</td></tr><tr><td>Cyro 4 ml tube</td><td>Greiner Bio-One</td><td>127279</td><td></td></tr><tr><td>Epoxy, 30 min</td><td>Permatex</td><td>84107</td><td></td></tr><tr><td>Metal Pins, 0.025&quot; OD, .013&quot; ID</td><td>New England Small Tube</td><td>NE-1310-02</td><td></td></tr><tr><td>Poly(ethylene glycol) diacrylate, Mn 700</td><td>Sigma-Aldrich</td><td>455008-100ML</td><td></td></tr><tr><td>Lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate photoinitator&amp;nbsp;</td><td>Tokyo Chemical Industry Co.</td><td>L0290</td><td>We typically synthesize LAP in-house.&amp;nbsp;</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034-25G</td><td></td></tr><tr><td>Light mineral oil</td><td>Sigma-Aldrich</td><td>330779-1L</td><td></td></tr><tr><td>Span-80</td><td>Sigma-Aldrich</td><td>85548</td><td></td></tr><tr><td>ABIL EM 90</td><td>UPI Chem</td><td>420095</td><td>&amp;nbsp;&lt;br /&gt;&amp;nbsp;</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;&amp;nbsp;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td>Equivalent equiptment or homebuilt setups will work equally as well</td><td></td><td></td></tr><tr><td>Mask Aligner</td><td>Karl Suss</td><td>MA6</td><td></td></tr><tr><td>Profilometer</td><td>KLA-Tencor</td><td>Alpha-Step D500</td><td></td></tr><tr><td>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><td>Vacuum Dessicator, Bell-Jar Style</td><td>Bel-Art</td><td>420100000</td><td></td></tr><tr><td>Oven</td><td>Cole-Palmer</td><td>WU-52120-02</td><td></td></tr><tr><td>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><td>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><td>Automated control scripting</td><td>MATLAB</td><td></td><td></td></tr><tr><td>Hotplate</td><td>Tory Pines Scientific</td><td>HP30</td><td>Any hotplate with uniform heating (&lt;em&gt;i.e.&lt;/em&gt;, aluminum or ceramic plates) will suffice.</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 ...

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

Research

JoVE Journal

Bioengineering

14.3K Views.  Stanford University. 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. 

Video: Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

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

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

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

Biology

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

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

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

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

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

Engineering

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

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

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

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

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

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

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

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

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

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

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

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

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...

Setup of a Microfluidic Device for Combinatorial Plug  Production

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Author Spotlight: Integrating Computational and Experimental Approaches in Precision Oncology

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

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