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 wi...

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 border="0" cellpadding="0" cellspacing="0" width="915">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">BSA</td>
			<td style="width:148px;">SIGMA-ALDRICH</td>
			<td style="width:143px;">A6003</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>DAEJUNG</td>
			<td>7548-4100</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4</td>
			<td>EMSURE</td>
			<td>1058861000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NF Membrane</td>
			<td>Filmtec</td>
			<td>NF200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30 kDa UF Membrane</td>
			<td>MICRODYN NADIR</td>
			<td>UH030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 kDa UF Membrane</td>
			<td>MICRODYN NADIR</td>
			<td>UH050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pressure Transducer</td>
			<td>Midas</td>
			<td>43006711</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ball Valves</td>
			<td>AV-RF</td>
			<td>Q91SA-PN6.4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-way Valve</td>
			<td>iLife Medical Devices</td>
			<td>902.071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pressure Regulator</td>
			<td>Swagelok</td>
			<td>KCB1G0A2A5P20000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow-meter</td>
			<td>Bronkhorst</td>
			<td>L01-AGD-99-0-70S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Balances</td>
			<td>MRC</td>
			<td>BBA-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pump</td>
			<td>Cole-Parmer</td>
			<td style="width:143px;">EW-00354-JI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1/8&#34; Tubing</td>
			<td>Cole-Parmer</td>
			<td>EW-06605-27</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1/16&#34; Tubing</td>
			<td>Cole-Parmer</td>
			<td>EW-06407-41</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1/16&#34; Fittings</td>
			<td>Cole-Parmer</td>
			<td>EW-30486-70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1/8&#34; Fittings</td>
			<td>Kiowa</td>
			<td>QSM-B-M5-3-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcontroller</td>
			<td>Adafruit</td>
			<td>50</td>
			<td>Arduino UNO R3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Continuous Rotation Servo</td>
			<td>Adafruit</td>
			<td>154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Servo</td>
			<td>Adafruit</td>
			<td>1142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Power Supply</td>
			<td>Adafruit</td>
			<td>658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Servo Shield</td>
			<td>SainSmart</td>
			<td>20-011-905</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Switches</td>
			<td>Parts Express</td>
			<td>060-376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 Micron Filters</td>
			<td>EMD Millipore</td>
			<td>SLHV033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potentiostat</td>
			<td>Gamry</td>
			<td>PCI4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sonicator</td>
			<td>MRC</td>
			<td>DC-150H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Connex 3D Printer</td>
			<td>Stratasys</td>
			<td>Objet Connex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Veroclear&#160;</td>
			<td>Stratasys</td>
			<td>RGD810&#160;</td>
			<td>transparent polymer for printing flow cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 w...

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 ...

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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 Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

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.

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 ...

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality that shows great potential for both preclinical and clinical applications. Conventional optical parametric oscillator-based PAT (OPO-PAT) systems are bulky and expensive and cannot provide high-speed imaging. Recently, pulsed-laser diodes (PLDs) have been successfully demonstrated as an alternative excitation source for PAT. Pulsed-laser diode PAT (PLD-PAT) has been successfully demonstrated for high-speed imaging on photoacoustic phantoms and biological tissues. This work provides a visualized experimental protocol for in vivo brain imaging using PLD-PAT. The protocol includes the compact PLD-PAT system configuration and its description, animal preparation for brain imaging, and a typical experimental procedure for 2D cross-sectional rat brain imaging. The PLD-PAT system is compact and cost-effective and can provide high-speed, high-quality imaging. Brain images collected in vivo at various scan speeds are presented.

A High-performance Compact Photoacoustic Tomography System for In Vivo Small-animal Brain Imaging

A compact pulsed-laser diode-based photoacoustic tomography (PLD-PAT) system for high-speed in vivo brain imaging in small animals is demonstrated.

In vivo small-animal imaging has an important role to play in preclinical studies. Photoacoustic tomography (PAT) is an emerging hybrid imaging modality ...

A Performance-testing Platform for a Conduction Micropump with an FR-4 Copper-clad Electrode Plate

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is fabricated. It is used to investigate the influence of chamber dimensions on the performance of a conduction micropump and to determine the reliability of the conduction pump when acetone is used as the working fluid. A testing platform is set up to evaluate conduction micropump performance under different conditions. When the chamber height is 0.2 mm, the pump pressure reaches its peak value.

This paper presents a protocol for the fabrication of a conduction micropump using symmetric planar electrodes on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) to test the influence of chamber dimensions on the performance of a conduction micropump.

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is ...

A Millimeter Scale Flexural Testing System for Measuring the Mechanical Properties of Marine Sponge Spicules

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the flexural behavior of these LBBSs is important for understanding the origins of their remarkable mechanical functions.
We describe a protocol for performing three-point bending tests using a custom-built mechanical testing device that can measure forces ranging from 10-5 to 101 N and displacements ranging from 10-7 to 10-2 m. The primary advantage of this mechanical testing device is that the force and displacement capacities can be easily adjusted for different LBBSs. The device&#39;s operating principle is similar to that of an atomic force microscope. Namely, force is applied to the LBBS by a load point that is attached to the end of a cantilever. The load point displacement is measured by a fiber optic displacement sensor and converted into a force using the measured cantilever stiffness. The device&#39;s force range can be adjusted by using cantilevers of different stiffnesses.
The device&#39;s capabilities are demonstrated by performing three-point bending tests on the skeletal elements of the marine sponge Euplectella aspergillum. The skeletal elements&#8212;known as spicules&#8212;are silica fibers that are approximately 50 &#181;m in diameter. We describe the procedures for calibrating the mechanical testing device, mounting the spicules on a three-point bending fixture with a &#8776;1.3 mm span, and performing a bending test. The force applied to the spicule and its deflection at the location of the applied force are measured.

We present a protocol for performing three-point bending tests on sub-millimeter scale fibers using a custom-built mechanical testing device. The device can measure forces ranging from 20 &#181;N up to 10 N and can therefore accommodate a variety of fiber sizes.

Many load bearing biological structures (LBBSs)&#8212;such as feather rachises and spicules&#8212;are small (&#60;1 mm) but not microscopic. Measuring the ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...