Name: three-dimensional printing of thermoplastic materials to create automated syringe pumps with feedback control for microfluidic applications
Uploaded: 2018-08-30T12:30:00
Description: Watch this Scientific Journal Video about Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications at JoVE.com

The authors acknowledge support from the Office of Naval Research awards N00014-17-12306 and N00014-15-1-2502, as well as from the Air Force Office of Scientific Research award FA9550-13-1-0108 and the National Science Foundation Grant No. 1709238.

1. 3D-printing and Assembly of Syringe Pump
<ol>
	<li>Prepare and 3D-print the syringe pump components
	<ol>
		<li>Download the .STL design files from the Supplemental Files of this paper. 
		NOTE: There are six .STL files, titled &#39;JoVE_Syringe_Clamp_10mL_Size.stl&#39;, &#39;JoVE_Syringe_Platform.stl&#39;, &#39;JoVE_Syringe_Plunger_Connectors.stl&#39;, &#39;JoVE_Syringe_Pump_End_Stop.stl&#39;, &#39;JoVE_Syringe_Pump_Motor_Connector.stl&#39;, and &#39;JoVE_Syringe_Pump_Traveler...

<ol>
	<li>Sackmann, E. K., Fulton, A. L., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+present+and+future+role+of+microfluidics+in+biomedical+research.">The present and future role of microfluidics in biomedical research.</a> Nature. 507 (7491), 181-189 (2014).</li><li>Duncombe, T. A., Tentori, A. M., Herr, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics:+reframing+biological+enquiry.">Microfluidics: reframing biological enquiry.</a> Nature Reviews Molecular Cell Biology. 16 (9), 554-567 (2015).</li><li>Prakadan, S. M., Shalek, A. K., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+by+shrinking:+empowering+single-cell+'omics'+with+microfluidic+devices.">Scaling by shrinking: empowering single-cell 'omics' with microfluidic devices.</a> Nature Reviews Genetics. 18 (6), (2017).</li><li>Kim, Y., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+in+nanomedicine.">Microfluidics in nanomedicine.</a> Reviews in Cell Biology and Molecular Medicine. 1, 127-152 (2015).</li><li>Rusconi, R., Garren, M., Stocker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+expanding+the+frontiers+of+microbial+ecology.">Microfluidics expanding the frontiers of microbial ecology.</a> Annual Review of Biophysics. 43, 65-91 (2014).</li><li>Skafte-Pedersen, P., Sabourin, D., Dufva, M., Snakenborg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-channel+peristaltic+pump+for+microfluidic+applications+featuring+monolithic+PDMS+inlay.">Multi-channel peristaltic pump for microfluidic applications featuring monolithic PDMS inlay.</a> Lab on a Chip. 9 (20), 3003-3006 (2009).</li><li>Heo, Y. J., Kang, J., Kim, M. J., Chung, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning-free+controller+to+accurately+regulate+flow+rates+in+a+microfluidic+network.">Tuning-free controller to accurately regulate flow rates in a microfluidic network.</a> Scientific Reports. 6, 23273 (2016).</li><li>Kuczenski, B., LeDuc, P. R., Messner, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-driven+spatiotemporal+control+of+the+laminar+flow+interface+in+a+microfluidic+network.">Pressure-driven spatiotemporal control of the laminar flow interface in a microfluidic network.</a> Lab on a Chip. 7 (5), 647-649 (2007).</li><li>Lake, J. R., Heyde, K. C., Ruder, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+feedback-controlled+syringe+pressure+pumps+for+microfluidics+applications.">Low-cost feedback-controlled syringe pressure pumps for microfluidics applications.</a> PLoS One. 12 (4), (2017).</li><li>Kong, D. S., 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=Open-source,+community-driven+microfluidics+with+metafluidics.">Open-source, community-driven microfluidics with metafluidics.</a> Nature Biotechnology. 35 (6), 523-529 (2017).</li><li>Wijnen, B., Hunt, E. J., Anzalone, G. C., Pearce, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-source+syringe+pump+library.">Open-source syringe pump library.</a> PLoS One. 9 (9), e107216 (2014).</li><li>Ferry, M. S., Razinkov, I. A., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+synthetic+biology:+from+design+to+execution.">Microfluidics for synthetic biology: from design to execution.</a> Methods in Enzymology. , 295-372 (2011).</li><li>. Arduino Libraries for Timer.h Available from: <a target="_blank" href="https://github.com/JChristensen/Timer">https://github.com/JChristensen/Timer</a> (2018)</li><li>. Arduino Libraries for AccelStepper.h Available from: <a target="_blank" href="https://github.com/adafruit/AccelStepper">https://github.com/adafruit/AccelStepper</a> (2018)</li><li>Lin, F., 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=Generation+of+dynamic+temporal+and+spatial+concentration+gradients+using+microfluidic+devices.">Generation of dynamic temporal and spatial concentration gradients using microfluidic devices.</a> Lab on a Chip. 4 (3), 164-167 (2004).</li><li>Korczyk, P. M., Cybulski, O., Makulska, S., Garstecki, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+unsteadiness+of+the+rates+of+flow+on+the+dynamics+of+formation+of+droplets+in+microfluidic+systems.">Effects of unsteadiness of the rates of flow on the dynamics of formation of droplets in microfluidic systems.</a> Lab on a Chip. 11 (1), 173-175 (2011).</li></ol>

Here, we presented a new design for a syringe pump system with closed-loop pressure control. This was accomplished by integrating a 3D-printed syringe pump with a piezoresistive pressure sensor and an open-source microcontroller. By employing a PID controller, we were able to precisely control the inlet pressure and provide fast response times while simultaneously maintaining the stability about a set point.
Many experiments using microfluidic devices require a precise fluidic control and expl...

Microfluidic tools have become useful for scientists in biological and chemical research. Due to the low volume utilization, rapid measurement capabilities, and well-defined flow profiles, microfluidics has gained traction in genomic and proteomic research, high-throughput screening, medical diagnostics, nanotechnology, and single-cell analysis1,2,3,4. Furthermore, the flexibility of microfluidic device design readily enables basic science research, such as investigating the spatiotemporal dynamics of cultured bacterial colonies5.
Many types of fluid injection systems have been developed to accurately deliver flow to microfluidic devices. Examples of such injection systems include peristaltic and recirculation pumps6, pressure-controller systems7, and syringe pumps8. These injection systems, including syringe pumps, are often composed of expensive precision engineered components. Augmenting these systems with closed-loop feedback control of pressure in the output flow adds to the cost of these systems. In response, we previously developed a robust, low-cost syringe pump system that uses closed-loop feedback control to regulate outputted flow pressure. By using closed-loop pressure control, the need for expensive precision-engineered components is abrogated9.
The combination of affordable 3D-printing hardware and a significant growth in associated open-source software has made the design and fabrication of microfluidic devices increasingly accessible to researchers from a variety of disciplines10. However, the systems used to drive fluid through these devices remain expensive. To address this need for a low-cost fluid control system, we developed a design that can be fabricated by researchers in the lab, requiring only a small number of assembly steps. Despite its low-cost and straightforward assembly, this system can provide precise flow control and provides an alternative to commercially available, closed-loop syringe pump systems, which can be prohibitively expensive.
Here, we provide protocols for the construction and use of the closed-loop controlled syringe pump system we developed (Figure 1). The fluid handling system is composed of a physical syringe pump inspired by a previous study11, a microcontroller, and a piezoresistive pressure sensor. When assembled and programmed with a proportional-integral-derivative (PID) controller, the system is capable of delivering a well-regulated, pressure-driven flow to microfluidic devices. This provides a low-cost and flexible alternative to high-cost commercial products, enabling a broader group of researchers to use microfluidics in their work.

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			<td height="21" style="height:21px;width:289px;">Arduino IDE</td>
			<td style="width:245px;">Arduino.org</td>
			<td style="width:169px;"></td>
			<td style="width:565px;">Arduino Uno R3 control software</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Header Connector, 2 Positions</td>
			<td>Digi-Key</td>
			<td>WM4000-ND</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Header Connector, 3 Positions</td>
			<td>Digi-Key</td>
			<td>WM4001-ND</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Header Connector, 4 Positions</td>
			<td>Digi-Key</td>
			<td>WM4002-ND</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hook-up Wire, 22 Gauge, Black</td>
			<td>Digi-Key</td>
			<td>1528-1752-ND</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hook-up Wire, 22 Gauge, Blue</td>
			<td>Digi-Key</td>
			<td>1528-1757-ND</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hook-up Wire, 22 Gauge, Red</td>
			<td>Digi-Key</td>
			<td>1528-1750-ND</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hook-up Wire, 22 Gauge, White</td>
			<td>Digi-Key</td>
			<td>1528-1768-ND</td>
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			<td height="21" style="height:21px;">Hook-up Wire, 22 Gauge, Yellow</td>
			<td>Digi-Key</td>
			<td>1528-1751-ND</td>
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			<td height="21" style="height:21px;">Instrumentation Amplifier</td>
			<td>Texas Instruments</td>
			<td>INA122P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcontroller, Arduino Uno R3</td>
			<td>Arduino.org</td>
			<td>A000066</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mini Breadboard</td>
			<td>Amazon</td>
			<td>B01IMS0II0</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Power Supply</td>
			<td>BK Precision</td>
			<td>1550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pressure Sensor</td>
			<td>PendoTech</td>
			<td>PRESS-S-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rectangular Connectors, Housings</td>
			<td>Digi-Key</td>
			<td>WM2802-ND</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rectangular Connectors, Male</td>
			<td>Digi-Key</td>
			<td>WM2565CT-ND</td>
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			<td height="21" style="height:21px;">Resistors, 10k Ohm&#160;</td>
			<td>Digi-Key</td>
			<td>1135-1174-1-ND</td>
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			<td height="21" style="height:21px;">Resistors, 330 Ohm&#160;</td>
			<td>Digi-Key</td>
			<td>330ADCT-ND</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Stepper Motor Driver, EasyDriver</td>
			<td>Digi-Key</td>
			<td>1568-1108-ND</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">USB 2.0 Cable, A-Male to B-Male</td>
			<td>Amazon</td>
			<td>PC045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3D Printed Material, Z-ABS&#160;</td>
			<td>Zortrax</td>
			<td></td>
			<td>A variety of colors are available</td>
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		<tr height="21">
			<td height="21" style="height:21px;">3D Printer</td>
			<td>Zortrax</td>
			<td>M200</td>
			<td>Printing out the syringe pump components</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ball Bearing, 17x6x6mm</td>
			<td>Amazon</td>
			<td>B008X18NWK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hex Machine Screws, M3x16mm&#160;</td>
			<td>Amazon</td>
			<td>B00W97MTII</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hex Machine Screws, M3x35mm&#160;</td>
			<td>Amazon</td>
			<td>B00W97N2UW</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hex Nut, M3 0.5&#160;</td>
			<td>Amazon</td>
			<td>B012U6PKMO</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Hex Nut, M5&#160;</td>
			<td>Amazon</td>
			<td>B012T3C8YQ</td>
			<td></td>
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			<td height="21" style="height:21px;">Lathe Round Rod</td>
			<td>Amazon</td>
			<td>B00AUB73HW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Linear Ball Bearing</td>
			<td>Amazon</td>
			<td>B01IDKG1WO</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Linear Flexible Coupler</td>
			<td>Amazon</td>
			<td>B010MZ8SQU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Steel Lock Nut, M3 0.5</td>
			<td>Amazon</td>
			<td>B000NBKLOQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stepper Motor, NEMA-17, 1.8o/step</td>
			<td>Digi-Key</td>
			<td>1568-1105-ND</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe, 10mL, Luer-Lok Tip</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Threaded Rod</td>
			<td>Amazon</td>
			<td>B01MA5XREY</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1H,1H,2H,2H-Perfluorooctyltrichlorosilane</td>
			<td>FisherScientific</td>
			<td>AAL1660609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera Module</td>
			<td>Raspberry Pi Foundation</td>
			<td>V2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Compact Oven</td>
			<td>FisherScientific</td>
			<td>PR305220G</td>
			<td>Baking PDMS pre-polymer mixture and the device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dispensing Needle, 22 Gauge</td>
			<td>McMaster-Carr</td>
			<td>75165A682</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dispensing Needle, 23 Gauge</td>
			<td>McMaster-Carr</td>
			<td>75165A684</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand Premium Cover Glasses</td>
			<td>FisherScientific</td>
			<td>12-548-5C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Culture Petri Dish, 130x25mm</td>
			<td>American Educational Products</td>
			<td>7-1500-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-32G</td>
			<td>Binding the cover glass with the PDMS device</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor Blades</td>
			<td>FisherScientific</td>
			<td>7071A141&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scotch Magic Tape</td>
			<td>Amazon</td>
			<td>B00RB1YAL6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Single-board Computer</td>
			<td>Raspberry Pi Foundation</td>
			<td>Raspberry Pi 2 model B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Smart Spatula</td>
			<td>FisherScientific</td>
			<td>EW-06265-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sylgard 184 Silicone Elastomer Kit</td>
			<td>FisherScientific</td>
			<td>NC9644388</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe Filters</td>
			<td>Thermo Scientific</td>
			<td>7252520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tygon Tubing</td>
			<td>ColeParmer&#160;</td>
			<td>EW-06419-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vacuum Desiccator</td>
			<td>FisherScientific</td>
			<td>08-594-15C</td>
			<td>Degasing PDMS pre-polymer mixture and coating fluorosilane on the master mold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weighing Dishes</td>
			<td>FisherScientific</td>
			<td>S67090A</td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional printing of thermoplastic materials to create automated syringe pumps with feedback control for microfluidic applications

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Here, we present a protocol for the construction of a feedback-controlled syringe pump system and demonstrate its potential uses for microfluidic applications. Figure 1 shows the connected system of the syringe pump, pressure sensor, microfluidic device, microcontroller, pressure sensor circuit, and stepper motor driver. Detailed callouts for the syringe pump assembly are shown in Figure 2 and the electronic circuit schematic for...

Watch this Scientific Journal Video about Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications at JoVE.com

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an&#160;additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic ...

This method can help enable research in any field requiring precision liquid handling including molecular, cell, and systems biology, chemical engineering and microfabrication. The main advantage of this approach is that we are able to fabricate low-cost pressure-regulated syringe pumps. This technique should save money across a wide range of disciplines.This technique showcases how we can couple additive manufacturing with open-source electronics to make a useful piece of laboratory instrumentation. To begin, download the STL design files from the supplemental files of this publication. Prepare these files for printing by opening them in a software package dedicated to the conversion of STL model files to executable instruction sets for the 3D printer being used.Ensure that the proper software is being used as some printers will require proprietary software whereas others may be able to print directly from the STL file. Now print the plastic components using acrylonitrile butadiene styrene with a high-quality 3D printer setting. If other common 3D printing materials are being used such as polylactic acid or other thermoplastic elastomers, make sure that the finished mechanical properties are comparable.Once complete, detach the printed parts from the printing platform of the 3D printer. Remove the printed supporting structure from the finished parts. Smooth the printed components by sanding any rough edges using sandpaper.For best results, use sandpaper with a grit size below 220. Make sure all components are smooth before assembling and ensure that all seven parts have been printed. Fasten the stepper motor to a threaded rod using a motor shaft z-axis flexible coupler with set screws.Before continuing, make sure that rotating the stepper motor shaft drives the threaded rod without slippage. Connect the syringe platform to the motor connector by firmly pressing the syringe platform's connection pegs into the mating holes on top of the motor connector. Attach the two parts by fastening four 16 millimeter screws through the motor connector.Now insert two linear ball bearings and a 0.8 millimeter hex nut into the openings located on the bottom of the traveler push. Align the threaded rod on the motor connector through the 0.8 millimeter hex nut in the traveler push. Then insert the two linear shafts through the traveler push in the motor connector.Now place two hex nuts in the hexagonal spaces of the motor connector piece and then use two 16 millimeter screws to tighten the connections securing the linear shafts from moving. Insert the ball bearing into the middle opening of the end stop. Connect the end stop with the assembled components.Place two hex nuts in the hexagonal spaces of the end stop piece and then use two 16 millimeter screws to tighten the connections to affix the end stop to the assembly. Next, attach the syringe plunger female connector piece to the traveler push piece using two steel locknuts and two 16 millimeter screws. Place a 10 milliliter syringe on the top of the pump.Ensure the head of the plunger is aligned into the notch of the syringe plunger female connector piece and that the top of the syringe barrel is fixed into the slot of the motor connector. Now insert the syringe plunger male connector piece into the syringe plunger female connector. Connect the syringe clamp to the syringe platform using two hex nuts and two 35 millimeter screws ensuring the syringe barrel is fixed into the slot of the syringe clamp.Ensure that there is a tight fit between the male and female components securing the plunger in place. Proceed to microfluidic device preparation as detailed in the text protocol. To begin the syringe pump system assembly, remove 80%of the length of the wire insulation and shielding from the pressure sensor's electrical cable using a razor.Be gentle when cutting to ensure the wires are not compromised above the desired length. Once the insulation and shielding are removed, connect the wires to male rectangular connectors. Using a similar approach, remove one to two centimeters of the wire insulation from a stepper motor's leads and connect the wires to male rectangular connectors.Now affix the syringe onto the inlet side of the pressure sensor. Connect the 22 gauge dispensing needle onto the outlet side of the pressure sensor. Slide one end of 0.51 centimeter diameter tubing over the 22 gauge dispensing needle attached to the pressure sensor.Slide the other end of the tubing over a 22 gauge dispensing needle that can be connected to the microfluidic device. Then connect the needle to the inlet port of the microfluidic device. Connect the outlet port of the microfluidic device to a waste disposal reservoir using a 22 gauge needle and 0.51 centimeter diameter tubing similar to the inlet port's connection.Next, assemble the electronic circuit on a prototyping breadboard according to the diagram in the text protocol. Connect the wires from the stepper motor with the stepper motor driver. Then connect the wires from the pressure sensor and the stepper motor driver to the breadboard.Now connect the output signal from the breadboard with the analog input pin on the microcontroller. Connect the logic input pins from the stepper motor driver with the digital pins on the microcontroller. The step input on the stepper motor driver is connected with a pulse width modulated port of digital pins on the microcontroller denoted by a tilde sign.Connect the power supply to the breadboard as shown in the text. Then set the power supply to 10 volts for the breadboard and the stepper motor driver. To control the syringe pressure pumps, first open the Integrated Development Environment or IDE for the open-source microcontroller.Download the Timer. h and ExcelStepper. h libraries to the microcontroller's IDE library directory.Download the controller code titled Dual Pump PID Control.INO. This code is used to control the feedback-controlled syringe pump system with two pumps. Program the controller code so that it fits the experiment being conducted.Modify the control parameters for the timing parameters to fit the desired response and duration of the experiment. Compile and upload the code to the microcontroller before running the experiment. Turn on the power supply for the syringe pump system.Finally, set the voltage to 10 volts for the stepper motor power supply. Shown here are representative results showing how control parameters may be tuned. Modifying the proportional coefficients as shown here lead to different response profiles for the pressure signals.Modifying the integral and differential coefficients also lead to changes in the response profiles. Parameters may be optimized for different experimental designs. Shown here are representative results showing the control and modulation of a laminar flow profile in a microfluidic device.By controlling the inlet pressure at two inlet ports of a Y-shaped microfluidic device, the interface position may be precisely controlled downstream. Following this procedure, other techniques such as cell culturing and microscopy can be performed in order to answer questions related to synthetic gene network regulatory dynamics, cell physiology and biological regulatory networks. These syringe pump systems have enabled more rapid development of microfluidic procedures within our lab group.These devices are an inexpensive and time-efficient way for groups to control flow through many different microfluidic devices. We hope that researchers across a wide range of disciplines will be able to use and extend these pressure-regulated syringe pumps for their microfluidic needs.

three-dimensional-printing-thermoplastic-materials-to-create

Research

JoVE Journal

Bioengineering

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

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 ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. Thus, many novel biomaterials are being designed to mimic specific environments required for biomedical applications such as tissue engineering and controlled drug delivery. The development of materials with improved properties for the immobilization of cells or enzymes is also a current research topic in bioprocess engineering. However, one of the most desirable properties of a material in these applications is the antimicrobial capacity to avoid any undesirable infections. For this, we present easy-to-follow protocols for the antimicrobial characterization of materials based on (i) the agar disk diffusion test (diffusion method) and (ii) the ISO 22196:2007 norm to measure the antimicrobial activity on material surfaces (contact method). This protocol must be performed using Gram-positive and Gram-negative bacteria and yeast to cover a broad range of microorganisms. As an example, 4 materials with different chemical natures are tested following this protocol against Staphylococcus aureus, Escherichia coli, and Candida albicans.The results of these tests exhibit non-antimicrobial activity for the first material and increasing antibacterial activity against Gram-positive and Gram-negative bacteria for the other 3 materials. However, none of the 4 materials are able to inhibit the growth of Candida albicans.

We present a protocol for the antimicrobial characterization of advanced materials. Here, the antimicrobial activity on material surfaces is measured by two methods that complement each other: one is based on the agar disk diffusion test, and the other is a standard procedure based on the ISO 22196:2007 norm.

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. ...