The authors thank Stratasys (Rehovot, Israel) for three-dimensional printing of the device. We are grateful to Microdyne-Nadir (Germany) for the membrane samples. This research was supported by The Israel Science Foundation (Grant 1474-13) to C.J.A.

1. Design and Fabrication of the Microfluidic Test System
<ol>
	<li>Design microfluidic device as two separate parts: a top part and bottom part (Figure 1) in a CAD program.</li>
	<li>Start making the bottom part by using the rectangle tool to draw a 40 mm by 60 mm rectangle.</li>
	<li>At one corner with the circle tool create a 6.2 mm diameter circle centered 10 mm from edges. With the linear pattern tool replicate the holes across the rectangle with 20 mm spacing for a total of 6 holes.</li>
	<li>Using the fillet tool fillet the rectangles with a radius of 1 mm.</li>
	<li>Extrude the part 10 mm with the extrude tool.</li>
	<li>In the center of the top face, with the rectangle tool create a rectangle 30 mm by 1 mm and with the extrude cut tool cut 0.2 mm for the flow channel.</li>
	<li>Using the circle tool make a 1 mm diameter circle at the end of the flow channel. Then with the line tool construct a path connecting the circle to the nearest 40 mm by 10 mm face, including a 4 mm radius made with the fillet tool. Make a cut along this path with the swept cut tool.</li>
	<li>With the circle tool create a 3.9 mm diameter circle at the center of the flow path and cut 8 mm with the extrude cut tool to allow for fittings.</li>
	<li>Repeat steps 1.7 and 1.8 for the opposite side of the flow channel.</li>
	<li>With the top part repeat steps 1.2-1.5. Then in center of the top face create a permeate channel using the rectangle tool to create a rectangle 30 mm by 1 mm and cut 0.5 mm using the extrude cut tool.</li>
	<li>Use the circle tool to make a 1 mm circle centered in the permeate channel 5 mm from an end. With the line tool construct a path connecting the circle to one of the 1 cm by 6 cm faces, including a 4 mm radius made with the fillet tool. Make a cut along the path with the swept cut tool.</li>
	<li>With the circle tool create additional 3.9 mm diameter circle with its center on the permeate path and cut 8 mm with the extrude cut tool.</li>
	<li>At the parts top 40 mm edges, with the rectangle tool, create rectangles 40 mm by 5 mm adding 4 mm radii with the fillet tool. Use the extrude tool to extrude 3 mm downward for handles.</li>
	<li>Print parts with a multi-material photopolymer 3-D printer using a hard transparent polymer, including 0.05 mm overcoating with a soft rubbery polymer on the face of each part that contains the channel. Use manufacturer&#8217;s standard protocol, calibration and settings.</li>
	<li>Tap threads (M5) into feed, retentate and permeate orifices. Use plumber&#8217;s tape to connect 1/8&#8221; fittings to the feed and retentate and 1/16&#8221; fittings to the permeate.</li>
	<li>Connect microfluidic devices to pump, valves, pressure transducer and backpressure regulator with 1/8&#8221; tubing (Figure 2).</li>
	<li>Connect 0.45 &#956;m filters to inlet tubes.</li>
	<li>Discharge permeate to flow-meter and beakers on balances with 1/16&#8221; tubing.</li>
	<li>Attach continuous rotation servo to backpressure regulator with screws and standard servo to 3-way valve with tie-wire.</li>
	<li>Connect servos and power supply to servo shield.</li>
	<li>Connect pressure transducer, switches and servo shield to microcontroller.</li>
	<li>Connect microcontroller, balances, flow-meter and pump to a PC for data logging and system control.</li>
	<li>Configure balances to print data to their serial port.</li>
</ol>
2. Prepare Membranes to Be Tested
<ol>
	<li>Cut membranes to 40 mm x 8 mm.</li>
	<li>Soak membranes in ultrapure water (3 x 10 min) with sonication.</li>
	<li>Then soak the membranes in 50/50 ultrapure water/ethanol for 1 hr.</li>
	<li>Rinse the membranes with ultrapure water and store in ultrapure water at 4 &#176;C.8</li>
</ol>
3. Prepare Solutions to Be Tested with Nanofiltration Membranes
<ol>
	<li>Add 500 ml of ultrapure water to an Erlenmeyer flask. Then add 0.04 g of BSA and 0.29 g of NaCl.</li>
	<li>Add 500 ml of ultrapure water to a separate Erlenmeyer flask. Then add 0.6 g of MgSO4.</li>
	<li>Add 500 ml of ultrapure water to a third Erlenmeyer flask. Then add 0.29 g of NaCl.</li>
	<li>Insert stir bars into each flask and place flasks on stir plates. Mix for 5 min at 500 rpm.</li>
</ol>
4. Perform a Nanofiltration Fouling Experiment
Note: Perform the experiment at RT (ca. 24 &#176;C). First configure the system for measuring a single membrane by closing valves to flow cells not connected to the flow-meter.
<ol>
	<li>Insert one pump inlet tube into the ultrapure water reservoir and the other inlet tube into the MgSO4 solution (Figure 2).</li>
	<li>Use a syringe to draw water and MgSO4 solution through tubing so as to remove all air bubbles in the system.</li>
	<li>Insert a nanofiltration membrane on the bottom part of the flow cell, with the active side towards the feed channel, and place on the top part of the flow cell.</li>
	<li>Fasten nuts by hand and then tighten evenly with a wrench so as to minimize leakage.</li>
	<li>Select the ultrapure water with the reservoir selector switch.</li>
	<li>Set pump flow rate to 2 ml/min and start the pump.</li>
	<li>Adjust pressure regulator to 4 bar.</li>
	<li>Set experimental parameters to switch reservoirs every 45 min starting with the water reservoir.</li>
	<li>Set reservoir switch to auto, and start experiment.</li>
	<li>At 60 min collect MgSO4 permeate in a tube for next 30 min.</li>
	<li>At 91 min replace MgSO4 flask with flask containing the solution of BSA and NaCl.</li>
	<li>Quickly stop pump and use a syringe to draw BSA solution through the inlet tube to remove MgSO4 leftover in tubing. Then start pump again.</li>
	<li>At 150 min collect BSA permeate in a tube for next 30 min.</li>
	<li>After 225 min, shut down the system and remove nanofiltration membrane from the flow cell.</li>
	<li>Using a syringe, flush out test solution inlet tube with ultrapure water.</li>
	<li>Repeat steps 4.1-4.15 for each additional membrane tested.</li>
	<li>For NaCl only tests, repeat steps 4.1-4.10, and 4.14-4.16 replacing MgSO4 solution with NaCl solution and ending the experiment after 90 min instead of 225 min.</li>
</ol>
5. Calculate Salt Rejection of Nanofiltration Membranes
<ol>
	<li>Rinse electrodes of the potentiostat test cell with ultrapure water.</li>
	<li>With a pipette, deposit 5 &#956;l of MgSO4 solution onto the test cell electrodes.</li>
	<li>Record resistance of the solution.</li>
	<li>Repeat steps 5.1-5.3 four more times and calculate the average value.</li>
	<li>Repeat steps 5.1-5.4 for the NaCl and BSA/NaCl solutions as well as for each permeate solution collected.</li>
	<li>Calculate salt rejection with Equation 1: 
	<img alt="Eq 1" src="/files/ftp_upload/53556/53556eq1.jpg" /> 
	where &#937;s is the resistance of the test solution and &#937;p is the resistance of the permeate. The resistance is inversely proportional to conductivity of a solution, which directly correlates to salt concentration.</li>
</ol>
6. Prepare Solution to Be Tested with Ultrafiltration Membranes
<ol>
	<li>Add 1 L of ultrapure water to a 4 L beaker. Then add 0.32 g of BSA.</li>
	<li>Insert stir bar into beaker and place on a stir plate. Mix for 5 min at 500 rpm.</li>
	<li>Add additional 3 L of ultrapure water to beaker and mix again for 5 min at 500 rpm.</li>
</ol>
7. Perform an Ultrafiltration Fouling Experiment
Note: Perform an experiment at RT (ca. 24 &#176;C). First configure the system to measure 4 membranes in parallel by opening all valves to flow cells.
<ol>
	<li>Place one pump inlet tube into the ultrapure water reservoir and other inlet tube into the BSA solution (Figure 2).</li>
	<li>Use a syringe to draw the water and the BSA solution through the tubing so as to remove all air bubbles in the system.</li>
	<li>Insert ultrafiltration membranes on the bottom part of the flow cells, with the active sides towards the feed channels, and close the cells with the top halves of the microfluidic device.</li>
	<li>Fasten nuts by hand, then tighten evenly with a wrench. Improper tightening may lead to water leakage.</li>
	<li>Select ultrapure water with reservoir switch.</li>
	<li>Set pump flow rate to 8 ml/min and start the pump.</li>
	<li>Adjust pressure regulator to 0.4 bar.</li>
	<li>Monitor flux values of membranes with data acquisition software according to manufacturer&#8217;s protocol.</li>
	<li>Adjust pressure regulator until average flux is 200 LMH &#177; 10%.</li>
	<li>Replace individual membrane if flux is not 200 LMH &#177; 20%.</li>
	<li>Enter experimental run parameters. First select the ultrapure water reservoir for 60 min with a constant flux of 200 &#177; 20 LMH. Then, select the BSA reservoir for 420 min with manual control of the pressure regulator. Finally, select the ultrapure water reservoir for 15 min with manual control of the pressure regulator to the flush system at the end of experiment.</li>
	<li>Set reservoir switch to auto, and start experiment.</li>
	<li>After run completion, shut system down and remove membranes from flow cells.</li>
	<li>With a syringe, flush pump inlet tube with ultrapure water.</li>
</ol>

<ol>
	<li>Guo, W., Ngo, H. -. H., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mini-review+on+membrane+fouling.">A mini-review on membrane fouling.</a> Bioresource technol. 122, 27-34 (2012).</li><li>Fane, A. G., Fell, C. J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+fouling+and+fouling+control+in+ultrafiltration.">A review of fouling and fouling control in ultrafiltration.</a> Desalination. 62, 117-136 (1987).</li><li>Tang, C. Y., Chong, T. H., Fane, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloidal+interactions+and+fouling+of+NF+and+RO+membranes:+a+review.">Colloidal interactions and fouling of NF and RO membranes: a review.</a> Adv. colloid interfac. 164 (1-2), 126-143 (2011).</li><li>De Jong, J., Lammertink, R. G. H., Wessling, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membranes+and+microfluidics:+a+review.">Membranes and microfluidics: a review.</a> Lab on a chip. 6 (9), 1125-1139 (2006).</li><li>Haas, R., Gutman, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosphingolipids+Enhance+Bacterial+Attachment+and+Fouling+of+Nanofiltration+Membranes.">Glycosphingolipids Enhance Bacterial Attachment and Fouling of Nanofiltration Membranes.</a> Environ. Sci. Technol. Lett. 2, (2015).</li><li>Nabe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+of+polysulfone+ultrafiltration+membranes+and+fouling+by+BSA+solutions.">Surface modification of polysulfone ultrafiltration membranes and fouling by BSA solutions.</a> J. Membr. Sci. 133 (1), 57-72 (1997).</li><li>Ang, W., Elimelech, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+(BSA)+fouling+of+reverse+osmosis+membranes:+Implications+for+wastewater+reclamation.">Protein (BSA) fouling of reverse osmosis membranes: Implications for wastewater reclamation.</a> J. Membr. Sci. 296 (1-2), 83-92 (2007).</li><li>Bernstein, R., Belfer, S., Freger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+of+dense+membranes+using+radical+graft+polymerization+enhanced+by+monomer+filtration.">Surface modification of dense membranes using radical graft polymerization enhanced by monomer filtration.</a> Langmuir. 26 (14), 12358-12365 (2010).</li><li>Kaufman, Y., Kasher, R., Lammertink, R. G. H., Freger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+NF/RO+separation:+Cell+design,+performance+and+application.">Microfluidic NF/RO separation: Cell design, performance and application.</a> J. Membr. Sci. 396, 67-73 (2012).</li><li>Kaufman, Y., 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=Towards+supported+bolaamphiphile+membranes+for+water+filtration:+Roles+of+lipid+and+substrate.">Towards supported bolaamphiphile membranes for water filtration: Roles of lipid and substrate.</a> J. Membr. Sci. 457, 50-61 (2014).</li></ol>

The authors have nothing to disclose.

This protocol describes the design of a three-dimensionally printed microfluidic cross-flow device for testing of nanofiltration and ultrafiltration membranes. Recently, we have shown the success of a variation of this protocol with nanofiltration membrane conditioning and fouling with glycosphingolipids and lipopolysaccharides and membrane performance differences with subsequent bacterial culture injection.5 Future applications employing this technique could be used to evaluate membrane performance changes with different foulants. Compared to larger flow cells this microfluidic device requires much less test solution and can significantly reduce the costs for foulants and compounds, especially those that are only available in limited quantities. The small format also makes it convenient for laboratory scale testing and may be amenable to high throughput testing.
Design of the microfluidic cross-flow device was achieved through iterative prototyping, which is the major advantage of three-dimensional printing. General device design features were based on a previously published microfluidic cross-flow device used for nanofiltration membrane applications.9,10 The most significant design differences were that the feed and permeate channels were not offset but directly overlaying each other, and the thickness of the parts, and the water sealing method. The prevention of water leakages was the major problem that was overcome in the design process through printing the device with a multi-material photopolymer 3-D printer. This allowed a thin soft polymer to be on the surfaces of the device that were in contact with the membrane. After placement of the membrane in the device, and evenly tightening with 6 nuts (M6), water leakages were prevented. Other potential areas for water leakages are the feed inlet and retentate outlet tubing connection points, and may be prevented using plumber&#8217;s tape and being careful not to over tighten the connection of the tubing, which would damage the threading. The device has been pressure tested up to 5 bar without leakages.
It is important that ultra-pure water is used for preparation of all the solutions. Water from other sources may contain unknown fouling substances that would cause decrease in membrane performance. Also, a filter (0.45 mm) is attached to the feed tubing to ensure the absence of particulate matter in the system. A permeate flow meter was used in order to more accurately measure low flux values in the representative experiment using a nanofiltration membrane. A fixed pressure of 4 bars was chosen based on a previous glycosphingolipids study.5 Repeat measurements using different membrane coupons were averaged. In a representative experiment using ultrafiltration membranes, the initial pure water flux of the membrane was measured using a pressure of 0.4 bar. Permeate flux from membrane to membrane can vary greatly therefore each membrane&#8217;s flux was checked to ensure that the flux differences were not larger than &#177; 20%. Membranes falling outside the desired initial flux values were replaced with new membrane coupons. In fouling studies a constant flux may be preferred over a constant pressure because membranes tested with different initial fluxes may foul at different rates. Pressure was then adjusted for the desired average initial permeate flux of 200 LMH &#177; 10%, however, these initial starting conditions may be chosen according to required experimental conditions. Subsequent changes in feed solution composition, and monitoring the flux changes will give valuable insight into the performance characteristics of the membrane.

Membrane technology is integral to industrial and other processes requiring the separation of solutes from a bulk solution, however, membrane fouling is a major ongoing challenge.1 Common examples where membrane fouling occurs include the use of ultrafiltration membranes for the size based separation of wastewater,2 and thin film composite membranes for the separation of ions and larger solutes from brackish or seawater.3 Characteristic indications of fouling include an increase in transmembrane pressure and a decline in flux. This decreases the productivity of the membrane and shortens its lifetime due to chemical or other cleaning protocols. Therefore membrane performance is a good indicator to assess fouling and to understand the mechanisms and effects of fouling, biofouling and biofilm formation on membranes. Also, performance assessment is important in the design or modification of new membranes.

Interest in the use of membranes in microfluidic devices has been growing over the last decade.4 Recently, we studied the effect of microbial components lipopolysaccharide, and glycosphingolipid on fouling the surface of a nanofiltration membrane, and the subsequent susceptibility of the conditioned surface to microbial attachment.5 A microfluidic cross-flow device was used to assess the performance of nanofiltration membranes. This allowed the use of special non-commercial lipid components only available in small quantities for membrane surface fouling because the membrane surface area was small. The system size allowed efficient use of membrane materials and low volumes of solutions. In this protocol, we describe the design and fabrication of the microfluidic device for membrane performance testing, and outline the incorporation of the device into a pressure flow system. Demonstration of the device is shown by testing the performance of ultrafiltration membranes and nanofiltration membranes using a model foulant, BSA.6,7

<table><tbody><tr><td>BSA</td><td>SIGMA-ALDRICH</td><td>A6003</td><td></td></tr><tr><td>NaCl</td><td>DAEJUNG</td><td>7548-4100</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMSURE</td><td>1058861000</td><td></td></tr><tr><td>NF Membrane</td><td>Filmtec</td><td>NF200</td><td></td></tr><tr><td>30 kDa UF Membrane</td><td>MICRODYN NADIR</td><td>UH030</td><td></td></tr><tr><td>50 kDa UF Membrane</td><td>MICRODYN NADIR</td><td>UH050</td><td></td></tr><tr><td>Pressure Transducer</td><td>Midas</td><td>43006711</td><td></td></tr><tr><td>Ball Valves</td><td>AV-RF</td><td>Q91SA-PN6.4</td><td></td></tr><tr><td>3-way Valve</td><td>iLife Medical Devices</td><td>902.071</td><td></td></tr><tr><td>Pressure Regulator</td><td>Swagelok</td><td>KCB1G0A2A5P20000</td><td></td></tr><tr><td>Flow-meter</td><td>Bronkhorst</td><td>L01-AGD-99-0-70S</td><td></td></tr><tr><td>Balances</td><td>MRC</td><td>BBA-1200</td><td></td></tr><tr><td>Pump</td><td>Cole-Parmer</td><td>EW-00354-JI</td><td></td></tr><tr><td>1/8&quot; Tubing</td><td>Cole-Parmer</td><td>EW-06605-27</td><td></td></tr><tr><td>1/16&quot; Tubing</td><td>Cole-Parmer</td><td>EW-06407-41</td><td></td></tr><tr><td>1/16&quot; Fittings</td><td>Cole-Parmer</td><td>EW-30486-70</td><td></td></tr><tr><td>1/8&quot; Fittings</td><td>Kiowa</td><td>QSM-B-M5-3-20</td><td></td></tr><tr><td>Microcontroller</td><td>Adafruit</td><td>50</td><td>Arduino UNO R3</td></tr><tr><td>Continuous Rotation Servo</td><td>Adafruit</td><td>154</td><td></td></tr><tr><td>Standard Servo</td><td>Adafruit</td><td>1142</td><td></td></tr><tr><td>Power Supply</td><td>Adafruit</td><td>658</td><td></td></tr><tr><td>Servo Shield</td><td>SainSmart</td><td>20-011-905</td><td></td></tr><tr><td>Switches</td><td>Parts Express</td><td>060-376</td><td></td></tr><tr><td>0.45 Micron Filters</td><td>EMD Millipore</td><td>SLHV033RS</td><td></td></tr><tr><td>Potentiostat</td><td>Gamry</td><td>PCI4</td><td></td></tr><tr><td>Sonicator</td><td>MRC</td><td>DC-150H</td><td></td></tr><tr><td>Connex 3D Printer</td><td>Stratasys</td><td>Objet Connex</td><td></td></tr><tr><td>Veroclear&amp;nbsp;</td><td>Stratasys</td><td>RGD810&amp;nbsp;</td><td>transparent polymer for printing flow cell</td></tr><tr><td>Tangoblack-plus</td><td>Stratasys</td><td>FLX980</td><td>soft rubbery polymer for gasket layers on flow cell</td></tr></tbody></table>

three-dimensionally printed microfluidic cross-flow system for ultrafiltration/nanofiltration membrane performance testing

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane technology. Understanding the fouling process could lead to optimization and higher efficiency of membrane based filtration. Here we show the design and fabrication of an automated three-dimensionally (3-D) printed microfluidic cross-flow filtration system that can test up to 4 membranes in parallel. The microfluidic cells were printed using multi-material photopolymer 3-D printing technology, which used a transparent hard polymer for the microfluidic cell body and incorporated a thin rubber-like polymer layer, which prevents leakages during operation. The performance of ultrafiltration (UF), and nanofiltration (NF) membranes were tested and membrane fouling could be observed with a model foulant bovine serum albumin (BSA). Feed solutions containing BSA showed flux decline of the membrane. This protocol may be extended to measure fouling or biofouling with many other organic, inorganic or microbial containing solutions. The microfluidic design is especially advantageous for testing materials that are costly or only available in small quantities, for example polysaccharides, proteins, or lipids due to the small surface area of the membrane being tested. This modular system may also be easily expanded for high throughput testing of membranes.&#160;

The microfluidic flow cells were designed using a CAD program and printed using a multi-material photopolymer three-dimensional (3-D) printer. This cell was designed in two parts, so that membranes could be easily inserted and removed from the device (Figure 1). Each part was 1 cm thick, printed from a hard, clear polymer for structural integrity, and the sides facing the membrane were overcoated with a very thin 50 &#181;m layer of rubber-like polymer. The overcoating was performed to provide the cell with a sealing capability, which prevents water leakage. A flow channel was designed at 0.2 mm deep, 1 mm wide and 30 mm long to test a 30 mm2 area of membrane. After cutting the membranes to 40 mm by 8 mm and washing protocol, a test membrane was inserted into the device. Six stainless steel bolts and nuts (M6) were used to tighten the device and it was connected to the system (Figure 2). In this manner, the cell is permanently connected to the system, while membranes may be easily replaced. One cell was operated for nanofiltration membrane experiments, and four cells were operated in parallel for ultrafiltration membrane experiments.
For nanofiltration membranes, a flow meter was connected to measure the permeate flux. To perform an experiment, pure water at a rate of 2 ml/min. was initiated and the pressure was adjusted to 4 bar. This resulted in a permeate flux of ~40 LMH (Figure 3), and corresponded to ~10 LMH/bar. After equilibration and observation of a constant flux (ca. 45 min), the solution was changed to MgSO&#173;4&#173; (10 mM) to test for rejection and verify the integrity of the membrane, and permeate was collected. The resistivity of this solution was measured which is inversely proportional to conductivity. At the salt concentrations tested, the conductivity is linearly proportional to the concentration and the % salt rejection may be calculated. The membranes tested in the present experiment gave 83% &#177; 4%, and 64% &#177; 3% rejections of MgSO4 and NaCl, respectively. The system feed was then returned to pure water until a stable flux was achieved, and then changed to an aqueous solution of BSA (0.08 g/L) in NaCl (10 mM). The decrease in flux compared to the flux of a control membrane under the conditions of 10 mM NaCl indicated membrane fouling due to BSA.
For ultrafiltration membranes, four microfluidic devices were connected in parallel, with permeate flux measured using balances. These balances were connected to the computer and facilitated continuous data collection. Using a pure water feed rate of 8 ml/min for the system, which is 2 ml/min per flow cell, the pressure was adjusted in order to obtain an average flux of 200 LMH (Figure 4). The flux of each membrane was assessed, and the membrane was replaced if the flux difference was &#62; 20% from the initial chosen flux average of 200 LMH. The solution was changed to BSA (0.08 g/L) and the flux decrease was monitored. The feed solution was then changed back to pure water. For representative results, we compared 30 and 50 kDa hydrophilic polyethersulfone ultrafiltration membranes, and observed that although 50 kDa membrane had a higher normalized flux at the termination of the experiment (26.5% of initial flux) compared with 23% for the 30 kDa membrane, the difference was not significant.
<img alt="Figure 1" src="/files/ftp_upload/53556/53556fig1.jpg" />
Figure 1. Design and image of the microfluidic device used. The design was made using a CAD program and printed using a three-dimensional photopolymer printer. (A) Bottom part containing the feed channel (top view). (B) Top part containing the permeate channel (top view). (C) Assembly of the device (side view). (D) Image of the functional device including a membrane coupon, the parts fastened together with nuts and bolts. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53556/53556fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53556/53556fig2.jpg" />
Figure 2. Schematic representation of the system. The nanofiltration membrane testing was performed using Flow cell 1. The ultrafiltration membrane testing was performed using all 4 flow cells in parallel. Computer data logging not shown. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53556/53556fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53556/53556fig3.jpg" />
Figure 3. Performance and fouling of a nanofiltration membrane under cross-flow conditions. Experimental conditions for full run (black square): i) ultrapure water, 2 ml/min, 4 bar. ii) 10 mM MgSO&#173;4&#173;, 2 ml/min, 4 bar. iii) ultrapure water, 2 ml/min, 4 bar. iv) BSA (0.08 g/L) in 10 mM NaCl, 2 ml/min, 4 bar. v) ultrapure water, 2 ml/min, 4 bar. Control membrane 10 mM NaCl, 2 ml/min, 4 bar (blue circle). Error bars denote the standard deviation. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53556/53556fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53556/53556fig4.jpg" />
Figure 4. Fouling of ultrafiltration membranes 30 kDa (red square) and 50 kDa (blue diamond) under cross-flow conditions. i) pressure was adjusted so that average initial pure water permeate flux was 200 LMH. ii) BSA (0.08 g/L) 2 ml/min. Error bars denote the standard deviation. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53556/53556fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing at JoVE.com

Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

Design and fabrication of a three-dimensionally (3-D) printed microfluidic cross-flow filtration system is demonstrated. The system is used to test performance and observe fouling of ultrafiltration and nanofiltration (thin film composite) membranes.

Minimization and management of membrane fouling is a formidable challenge in diverse industrial processes and other practices that utilize membrane ...

three-dimensionally-printed-microfluidic-cross-flow-system-for

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

11.2K Views.  Ben-Gurion University of the Negev, Sede-Boqer Campus. Design and fabrication of a three-dimensionally (3-D) printed microfluidic cross-flow filtration system is demonstrated. The system is used to test performance and observe fouling of ultrafiltration and nanofiltration (thin film composite) membranes.

Video: Three-Dimensionally Printed Microfluidic Cross-flow System for Ultrafiltration/Nanofiltration Membrane Performance Testing

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, ...

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes. The device consists of three layers: first layer contains an array of microscale wells (500 &mu;m diameter and 150 &mu;m depth) that help to immobilize the islets while exposed to flow and maximize the exposed surface area of the islets; the second layer contains a circular perifusion chamber (3 mm deep, 7 mm diameter); and the third layer contains an inlet-mixing channel that fans out before injection into the perifusion chamber (2 mm in width, 19 mm in length, and 500 &mu;m in height) for optimizing the mixing efficiency prior to entering the perifusion chamber. The creation of various glucose gradients including a linear, bell shape, and square shapes also can be created in the microfluidic perifusion network and is demonstrated.

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence imaging of calcium influx and mitochondrial potential changes.

A microfluidic islet perifusion device was developed for the assessment of dynamic insulin secretion of multiple islets and simultaneous fluorescence ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

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 ...

Engineering

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

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

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

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

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

A Reconfigurable Barrier Tissue Platform with Open-Well and Microfluidic Modes

Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. However, these platforms have not gained widespread adoption in bioscience laboratories where open-well, membrane-based approaches serve as the gold standard for mimicking tissue barriers, despite lacking fluid flow capabilities. This issue can be primarily attributed to the incompatibility of existing microphysiological systems with standard protocols and tools developed for open-well systems.
Here, we present a protocol for creating a reconfigurable membrane-based platform with an open-well structure, flow enhancement capability, and compatibility with conventional protocols. This system utilizes a magnetic assembly approach that enables reversible switching between open-well and microfluidic modes. With this approach, users have the flexibility to begin an experiment in the open-well format using standard protocols and add or remove flow capabilities as needed. To demonstrate the practical usage of this system and its compatibility with standard techniques, an endothelial cell monolayer was established in an open-well format. The system was reconfigured to introduce fluid flow and then switched to the open-well format to conduct immunostaining and RNA extraction. Due to its compatibility with conventional open-well protocols and flow enhancement capability, this reconfigurable design is expected to be adopted by both engineering and bioscience laboratories.

Author Spotlight: Developing a Unique Modular Microphysiological System to Mimic Human Barrier Tissue

This protocol describes a reconfigurable membrane-based cell culture platform that integrates the open-well format with fluid flow capabilities. This platform is compatible with standard protocols and allows for reversible transitions between open-well and microfluidic culture modes, accommodating the needs of both engineering and bioscience laboratories.

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. ...