This work is supported by the NSF-EPSCoR (grant #0814194) and the Department of Bioengineering.

1. Equipment Set-up
<ol>
	<li>Assemble dispensing system and connect valve controller and syringe barrel to the pneumatic source, via a pressure regulator, to set the pressure at 15 psi to dispense the polymer solution from the needle tip at a flow rate of 2.45 &#181;l/min.</li>
	<li>Insert the 3-axis robot and dispensing system into a thermal enclosure to ensure a stable working environment (Figure 2).</li>
	<li>Install Joint-Robot Control Points (JR-C Points) software provided by the system manufacturer and connect 3-axis robot to a computer via serial communication port.</li>
	<li>Mount dispensing valve to 3-axis robot and install needle tip onto the valve.</li>
	<li>Level the robotic stage platen according to the manufacturer&#8217;s guidelines to ensure flatness with respect to the height of the valve tip15.</li>
	<li>Assemble a feedback-controlled heater in the enclosure to add environmental control.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/52834/52834fig2.jpg" /> 
Figure 2.&#160;(A) 3-Axis dispensing robot with required accessories housed inside an enclosure; and, (B) close up image of the dispensing valve with attached USB microscope for visualization. <a href="https://www.jove.com/files/ftp_upload/52834/52834fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Experimental Materials and Controlling Factors
<ol>
	<li>Controlling factors and their combination.
	<ol>
		<li>Vary feed rate from 1% speed (5 mm/sec) up to 100% (500 mm/sec), by the JR C-Points software. For this application, use a 2% speed (10 mm/sec) to fabricate the fiber structures presented in this work.</li>
		<li>Vary polymer solution concentrations to attain specific viscosity, surface tension and volatility parameters required for desired application. For this application, use a 24% polymethylmethacrylate (PMMA) in Chlorobenzene solution to fabricate the fiber structures presented in this work.</li>
		<li>Vary control valve dispense time from 0.02 sec to 1 sec in order to eject solutions via pneumatic air. For this application, use 0.02 sec to fabricate the fiber structures presented in this work.</li>
		<li>Select a gauge size of high precision needle tips to dispense polymer solutions. For this application, use a 30 G (inner diameter (ID) = 152.4 &#181;m) tip to fabricate the fiber structures presented in this work.</li>
		<li>Set the operating temperature range between 70 &#176;F to 100 &#176;F in the thermally insulated box to maintain a constant evaporation rate of the polymer sample. For this application, use a temperature of 70 &#176;F to fabricate the fiber structures presented in this work.</li>
	</ol>
	</li>
	<li>Preparation of polymer solutions.
	<ol>
		<li>Mix polymer resins of polymethyl methacrylate (PMMA; 0.72 g) with the solvent Chlorobenzene (2.28 g) under a chemical flow hood.</li>
		<li>Calculate the weight of the polymer (PMMA) and its solvent (chlorobenzene) to achieve the desired concentration of polymer in solution. For this application, use a 24% polymethylmethacrylate (PMMA) in Chlorobenzene solution to fabricate the fiber structures presented in this work.</li>
		<li>Place desired amount of polymer powder/resin into glass vial. For this application, use 0.72 g of PMMA resin to attain a 24% concentration of PMMA.</li>
		<li>Transfer solvent onto polymer within vial by pipette until the total weight of 3 g is reached.</li>
		<li>Mix the vial for 1 min using a vortex shaker and ultrasonically process them for 5 hr to completely dissolve the polymer powder/resin.</li>
		<li>Check the transparency of solutions, if there is any cloudiness or opaqueness to sample continue to sonicate until clear.</li>
	</ol>
	</li>
	<li>Viscosity measurement of polymer solutions.
	<ol>
		<li>Measure solution viscosities via cone-and-plate viscometer (e.g., LVDV-II+ and RVDV-II+)16.</li>
		<li>Load 0.5 ml of known viscosity standard sample (a glycerin-based and water mixture) provided by the viscometer manufacturer to calibrate the viscometer in order to minimize errors in the viscosity measurements. The manufacturer provided a variety of standard samples for calibrating the viscometer. Use the glycerin-based standard sample with a viscosity of 100,000 cP.</li>
		<li>Connect water jacket to viscometer to maintain the test fluid at a constant operating temperature.</li>
		<li>Start test fluid measurement by moving the On/Off switch to the On position to initiate rotation of the spindle. Once the value of the torque on the display panel has stabilized, record the torque and calculate the final viscosities using the relationship between torque, spindle multiplier constant and speed of spindle16 
		<img alt="Equation 1" src="/files/ftp_upload/52834/52834eq1.jpg" /> 
		where is viscosity, RPM is the speed of the spindle affixed to cone, TK is the torque (0.09373 for LVDV-II+, 1 for RVDV-II+), SMC is the spindle multiplier constant that depends on the specific spindle being used during the viscosity measurement. For this study, the CP-52 spindle was used which has a spindle multiplier constant of 9.83.</li>
	</ol>
	</li>
	<li>Surface tension measurement of polymer solutions.
	<ol>
		<li>Measurement method refers to the Wilhelmy technique.14</li>
		<li>Place 1 ml test solutions in a glass vial onto a high-resolution balance (scale resolution = 0.001 g). Dip a glass rod of known diameter into test solutions.</li>
		<li>Control the position of rod via a servo or stepper controlled linear actuator to touch surface and partially immerse into test solutions with zero contact.</li>
		<li>Monitor and record the change in mass measurement on the balance when removing rod from solution surface.</li>
		<li>Calculate surface tension by force change, perimeter of rod and contact angle of fluid on the rod. 
		<img alt="Equation 2" src="/files/ftp_upload/52834/52834eq2.jpg" /> 
		where is the wetted perimeter of rod (l = 10.05 mm with diameter = 3.2 mm), &#952; is contact angle of fluid on the rod, F is force change due to surface tension of test fluid on the tip of rod.</li>
	</ol>
	</li>
	<li>Mass transfer coefficient measurement of solutions.
	<ol>
		<li>Measure mass transfer coefficients of polymer solutions by thermogravimetric analysis.14</li>
		<li>Load 30 &#181;l of the 24% (by weight) PMMA polymer solution onto a platinum plate before taring the balance.</li>
		<li>Vent the chamber, program the device to run at desired operating temperature (70 &#176;F) for 2 hr to monitor mass of target solutions.</li>
		<li>Calculate the mass transfer coefficients by changes in the solution&#8217;s mass, area of solution/air interface and density. 
		<img alt="Equation 3" src="/files/ftp_upload/52834/52834eq3.jpg" /> 
		 
		where m(t) is the mass of PMMA solutions, A is the area of the solution/air interface (equals 78.5 mm2 for standard plate), mPOLYMER is the polymer mass in the solution and is the density of the solution.</li>
	</ol>
	</li>
</ol>
3. Direct Write Experiment Procedure
<ol>
	<li>Dispensing system sample loading protocol.
	<ol>
		<li>Load 3 ml of polymer solution into the syringe barrel, place piston into syringe barrel to eliminate inconsistent pneumatic pressure distribution.</li>
		<li>Twist the inlet line adapter on the syringe barrel, connect it to the source line air tube.</li>
		<li>Choose desired gauge size of the precision needle tip to perform experiment.</li>
		<li>On the dispense controller panel, switch to purge state and click &#8220;cycle&#8221; button to fill the dispense valve with polymer solution until discharged from the needle tip.</li>
		<li>Wipe residual polymer solution from the tip in preparation of a programmed fiber writing procedure.</li>
	</ol>
	</li>
	<li>Direct write polymer fibers with 3-axis robot and dispensing system.
	<ol>
		<li>Determine the robot stage offset to reposition the tip of the dispensing needle from the default home position to the initiation point of the prescribed micro/sub-micron fiber structure to be drawn. Mount a USB microscope (magnification = 200X) to the dispensing system valve bracket, which translates along the Z-axis. Manually focus on the dispenser tip by adjusting the focusing knob on the USB microscope that assists in precisely positioning the valve tip to the desired location, prefabricated substrate or device.</li>
		<li>Create/design the desired fiber structure pattern (Figures 3A, 4A, and 5A) using a CAD software package. Input the spatial coordinates (x, y, z) in the robot JR-C controlling software for all initiation and termination points in sequential order with respect to the desired pattern created within the CAD program (Figure 3B, 4B, and 5B). 15</li>
		<li>Transfer the completed fiber structure design program from the computer to the robot by clicking &#8220;Send C&#38;T Data&#8221; under the Robot menu within the JR-C software15.</li>
		<li>Load 3 ml of PMMA solution sample of known concentration (24%) into syringe barrel, purge valve and needle, set all the dispensing parameters for the valve controller and robot controlling software.</li>
		<li>Place prefabricated substrate onto the robot stage platen, close the thermal enclosure doors to prevent ambient air flow, which can induce unstable evaporation of the polymer solution.</li>
		<li>Start writing the fibers onto the substrate by clicking the robot menu from JR-C software and selecting &#8220;test running&#8221;15.</li>
		<li>DC sputter coat a conductive gold metal layer for 2 min until a 2 nm thick layer of gold is deposited onto the drawn fibers to allow visualization of the fibers in the scanning electron microscope.</li>
		<li>Measure the diameter and structure of fibers via scanning electron microscope17. Microscope parameters: High voltage level: 2.00 kV; Objective: InLens, Working Distance (9.0 mm).</li>
	</ol>
	</li>
	<li>Perform the dispensing system cleaning procedure under a chemical flow hood.
	<ol>
		<li>Place the dispensing system in a chemical flow hood to perform the cleaning procedure.</li>
		<li>Disassemble needle valve according to manufacturer&#8217;s protocol.</li>
		<li>Place all the metal parts into a beaker, pour acetone into the beaker until all parts are immersed.</li>
		<li>Place beaker in an ultrasonic bath for 30 min to remove all polymer remnants.</li>
		<li>Rinse all parts under flowing DI water, then use air gun to blow them dry.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/52834/52834fig3.jpg" /> 
Figure 3.&#160;(A) Example of designed &#8220;quad&#8221; web structure on a 10 mm x 10 mm frame, (B) sequential point-to-point spatial instructions to be inputted into JR-C point software. <a href="https://www.jove.com/files/ftp_upload/52834/52834fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52834/52834fig4.jpg" /> 
Figure 4. (A) Example of designed &#8220;symmetric&#8221; web structure on a 10 mm x 10 mm frame, (B) sequential point-to-point spatial instructions to be inputted into JR-C point software. <a href="https://www.jove.com/files/ftp_upload/52834/52834fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52834/52834fig5.jpg" /> 
Figure 5.&#160;(A) Example of designed &#8220;dual chevron&#8221; web structure on a 10 mm x 10 mm frame, (B) sequential point-to-point spatial instructions to be inputted into JR-C point software. <a href="https://www.jove.com/files/ftp_upload/52834/52834fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose.

Before attempting each trial, it is critical that the viscosity, mass transfer coefficients and surface tension test of the polymer solutions be accurately measured in order to determine whether the robot and dispensing system is capable of processing the desired polymer. As described previously by our group, the polymer solutions must retain adequate: 1) surface tension to enable the formation of liquid filaments into micron/sub-micron structures; 2) viscosity to withstand capillary break up; and, 3) evaporation rate to enhance fiber solidification18. The synergy between these parameters is key to successfully produce fibers over a specified range of diameters. At the same time, instability in any of these parameters prevents the formation of micron/sub-micron scale fibers. To maintain the synergy between these parameters during fiber fabrication, it is important to ensure that the needle and needle valve are thoroughly cleaned after a direct-write session to prevent: 1) contamination of the solution; 2) a reduction in the polymer solution flow rate through the needle; and, 3) excessive growth in the polymer bead at the tip of the needle. In addition, the temperature-controller on the heater must be set to the desired temperature to maintain a constant evaporation rate of the polymer solution.
The bifurcated branch fibers were 59%, 41% and 24% larger in diameter than that of the support structures in Figures 6-8, respectively, using the 24% PMMA solution. This is primarily due to the distance at which the fibers are drawn. Specifically, the support structures are drawn across the entire width of the substrate (10.0 mm in X and Y directions; 14.4 mm diagonally). As a result, these are the longest fibers of the overall suspended structures. The bifurcated branch structures are significantly shorter, ranging from 7 mm maximum length down to 2.5 mm. This shorter fiber drawing length does not effectively induce the fiber strain required during the fiber thinning process in order to produce the small diameter fibers. On the other hand, larger diameter wires are required to serve as the support fibers in order to effectively sustain the tugging and deformation induced during the bifurcated branch drawing process. As the bifurcation branches are drawn across the support fibers, a remodeling of the support fiber geometry can occur due to the drawing forces as well as a localized dissolution of the PMMA polymer at the interface between the support and branched fibers from the solvent present in the polymer solution. Thus, in some instances, the support fibers may need to be fabricated from a polymer solution consisting of a higher concentration of polymer to produce larger diameter and mechanically stronger fibers.
There are primarily three effective methods for modifying the existing protocol to generate a wider range of support and branched fiber diameters: 1) initially dispense the polymer from a larger needle tip (e.g., 25 G; ID = 254 &#956;m) to generate the support fibers and then exchange for a smaller needle tip (e.g., 32 G; ID = 101.6 &#956;m) to fabricate the smaller branched fibers; 2) as noted above, use multiple polymer concentrations; and/or, 3) adjust the feed rate, i.e., the speed at which the stage traverses, where increasing the feed rate produces smaller diameter fibers and decreasing the feed rate creates larger diameter fibers. To date, we have been able to successfully fabricate fibers as small as 90 nm; however, the fiber yield at this dimension is low due to capillary breakup.
One limitation of the automated direct write process is that only one concentration of polymer solution can be dispensed at a time. This restricts the level of complexity of the suspended structures to be developed without having to: 1) add a second dispensing valve to the robot; or, 2) remove the existing valve and perform the cleaning protocol (Section 3.4) before dispensing the second polymer solution, which takes additional time. A second limitation is the feed rate (or print speed), where the maximum feed rate that the system is capable of achieving is 500 mm/sec. However, there is a tradeoff between feed rate and fiber formation. Specifically, if the inertial forces (forces due to the feed rate) are greater than the surface tension forces and evaporation rate of the polymer solution, fiber formation does not occur. On the other hand, if the feed rate is too low, fibers will fracture due to excessive evaporation before and during the elongation process. Thirdly, fiber and structure dimensions are limited to the operating range of the stage of the robot, i.e., 200 mm, 200 mm and 25 mm (with a 10 &#956;m positional accuracy) in the x, y and z directions, respectively. Nonetheless, this process does enable the formation of high aspect ratio (fiber length:diameter) fibers. Strategically varying the dispensing tip size and the polymer solution concentration would allow for a broader fiber diameter range, creating the ability to generate freely suspended structures of higher complexity.
By following the protocols stated above, micron and submicron diameter polymer fibers can be generated with a high level of spatial control by exploiting the surface tension-driven fluid mechanics of the polymer solutions, which cannot be controlled with any other wet, dry or electrospinning processes. From prior work8,19, we know that this technique can be utilized to fabricate intricate micro/submicron fluidic devices19 and bioengineered scaffolds8. This cheap and easy technique has its own advantages over traditional planer fabrication methods in many ways.

Over the past several decades, a variety of fabrication techniques, such as wet spinning, dry spinning and electrospinning, have been employed to create novel polymer fiber structures with diverse and robust biological, chemical, electrical and mechanical properties1-12. Although these spinning techniques are capable of generating suspended three-dimensional fibers, they are limited in their ability to precisely control fiber orientation in three dimensions since fiber deposition via these processes are random in nature. In addition, these techniques are restricted in their dimensional range for fiber fabrication; specifically, fibers produced via wet and dry spinning vary in diameter from tens to hundreds of microns, while electrospinning yields fibers with diameters ranging from tens of nanometers to a single micron13.
To provide more precise control of fiber orientation in 3-D space, our group developed a self-assemble or &#8220;direct-write&#8221; fiber fabrication process that directly ejects a polymeric material out of a hollow capillary and then draws individual filaments which thin and solidify into predictable fiber diameters by exploiting surface tension-driven fluid mechanics14. Our initial direct-write system for increasing the level of control of fiber position and diameter consisted of a custom fabricated spring loaded syringe dispensing system attached to the head of a custom made Ultra-High Precision Micromilling Machine (Figure 1). The UHPMM had a stage with a positional resolution of 1.25 nm in the X and Y directions and 20 nm in the Z direction that was programmatically controlled to create micron and sub-micron scale wires and structures. One limitation of this particular direct write system was the lack of flow control of the polymer solution through the needle tip. Although the spring loaded dispensing system successfully generated constant flow through the tip, a continuously expanding spherical bead of polymer solution was created at the outlet of the syringe tip, which varied in size and volume depending on environmental conditions.
<img alt="Figure 1" src="/files/ftp_upload/52834/52834fig1.jpg" /> 
Figure 1. Image of Ultra High Precision Micromilling Machine: the first direct write system employed in fabricating micron/sub-micron scale structures. <a href="https://www.jove.com/files/ftp_upload/52834/52834fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The inconsistency of this source bead impacted the ability of the system to repeatedly fabricate wires of a prescribed diameter. Albeit, structures were successfully generated using this direct write process, enhancement of the process by increasing control of the flow of polymer solution would allow for more precise, prescribed fiber diameters through regulation of the bead size at the syringe tip. Thus, this work describes the implementation of a 3-axis automated dispensing system with pneumatically actuated dispenser valve to precisely control the polymer solution flow rate and tip bead size to create prescribed, micron/sub-micron suspended structures.

<table><tbody><tr><td>ROBOT DR2203N 3 AXES 200MM X 200MM</td><td>Nordson EFD</td><td>7023145</td><td>3-Axis Robot</td></tr><tr><td>CONTROLLER 7100, DISPENSE VALVE</td><td>Nordson EFD</td><td>7015340</td><td>Valve Controller</td></tr><tr><td>MICRODOT VALVE</td><td>Nordson EFD</td><td>7021233</td><td>Microdot Valve</td></tr><tr><td>ROBOT ACC FIXTURE PLATE 200MM</td><td>Nordson EFD</td><td>7028276</td><td>Fixture Platen</td></tr><tr><td>ROBOT ACC DRN / DSRN POINTS SOFTWAR</td><td>Nordson EFD</td><td>7023144</td><td>JR-C Software</td></tr><tr><td>ROBOT MOUNT VALVE UNIVERSAL</td><td>Nordson EFD</td><td>7028273</td><td>Microdot Valve Mount</td></tr><tr><td>15 PSI BARREL PRESS. REGULATOR</td><td>Nordson EFD</td><td>7020585</td><td>Barrel Regulator</td></tr><tr><td>KIT O BRL/PIST 5CC CL/WH 40</td><td>Nordson EFD</td><td>7012096</td><td>5CC Barrels with Pistons</td></tr><tr><td>ADAPTER ASM O 5CC BL</td><td>Nordson EFD</td><td>7012054</td><td>Pneumatic Barrel Adapter</td></tr><tr><td>TIP 30GA .006X.25 LAVNDR 50PC</td><td>Nordson EFD</td><td>7018424</td><td>30 gauge Needle Tip (0.250&quot; length)</td></tr><tr><td>Electric Baseboard Heater (500&amp;nbsp;W, 30&quot; length)</td><td>Cadet</td><td>2F500</td><td>Heater</td></tr><tr><td>Temperature Controller with Timer</td><td>Control Company</td><td>130726596</td><td>Temperature Controller</td></tr><tr><td>eScope USB Microscope</td><td>OiTez</td><td>DP-M02</td><td>200X USB Microscope</td></tr><tr><td>Poly(methyl methacrylate)</td><td>Aldrich</td><td>182265-500G</td><td>PMMA Powder</td></tr><tr><td>Chlorobenzene</td><td>Sigma Aldrich</td><td>284513</td><td>Solvent to dissolve PMMA</td></tr></tbody></table>

prescribed 3-d direct writing of suspended micron/sub-micron scale fiber structures via a robotic dispensing system

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer fiber length and orientation is defined by the spatial positioning of the dispensing system 3-axis stages. The fiber diameter is defined by the prescribed dispense time of the dispensing system valve, the feed rate (the speed at which the stage traverses from an initiating to a terminating position), the gauge diameter of the dispensing tip, the viscosity and surface tension of the polymer solution, and the programmed drawing length. The stage feed rate affects the polymer solution&#8217;s evaporation rate and capillary breakup of the filaments. The dispensing system consists of a pneumatic valve controller, a droplet-dispensing valve and a dispensing tip. Characterization of the direct write process to determine the optimum combination of factors leads to repeatedly acquiring the desired range of fiber diameters. The advantage of this robotic dispensing system is the ease of obtaining a precise range of micron/sub-micron fibers onto a desired, programmed location via automated process control. Here, the discussed self-assembled micron/sub-micron scale 3D structures have been employed to fabricate suspended structures to create micron/sub-micron fluidic devices and bioengineered scaffolds.

Images of the actual structures produced via the 3-axis robot and direct write method for each of the respective designs and JR C-Point algorithms defined in Figures 3-5A &#38; B above are shown in Figures 6A, 7A &#38; 8A. As can be seen in the resulting images, three-dimensional, freely suspended fibers have been successfully &#8220;written&#8221; onto the substrates by precisely manipulating the dispenser tip to prescribed spatial locations including the initiating/terminating points as well as points of intersection. The insets of these figures magnify the intersection points of suspended fibers, which demonstrate the system&#8217;s ability to precisely control fiber orientation (Figures 6B, 7B &#38; 8B) in 3-D space.
<img alt="Figure 6" src="/files/ftp_upload/52834/52834fig6.jpg" /> 
Figure 6.&#160;(A) Optical image of fabricated &#8220;quad&#8221; suspended web structure having 2 support fibers (diagonals) and 12 bifurcated branched PMMA fibers (15X optical magnification; scale bar = 1 mm), (B) SEM image of a single bifurcated fiber (289X magnification; scale bar = 100 &#956;m). <a href="https://www.jove.com/files/ftp_upload/52834/52834fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/52834/52834fig7.jpg" /> 
Figure 7.&#160;(A) Optical image of fabricated &#8220;symmetric&#8221; suspended web structure having 1 support fiber (horizontal) and 11 bifurcated branched PMMA fibers (15X optical magnification; scale bar = 1 mm), (B) SEM image of a single bifurcated fiber (107X magnification; scale bar = 100 &#956;m). <a href="https://www.jove.com/files/ftp_upload/52834/52834fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/52834/52834fig8.jpg" /> 
Figure 8.&#160;(A) Optical image of fabricated &#8220;dual chevron&#8221; suspended web structure having 1 support fiber (horizontal) and 22 bifurcated branched PMMA fibers (15X optical magnification; scale bar = 1 mm), (B) SEM image of a single bifurcated fiber (80X magnification; scale bar = 100 &#956;m). <a href="https://www.jove.com/files/ftp_upload/52834/52834fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 9 displays close up view of the sequential point-to-point&#160;order of fiber fabrication at which the 3-axis robot and dispensing system can generate the freely suspended web-like structures. The polymer contact points displayed with numbers correspond to the programmed initiating and terminating points in the JR-C software referenced in Figure 3B above. The arrows represent robot trajectory. Figure 10 displays micron and submicron polymer fibers, which demonstrates the direct write system&#8217;s ability to fabricate wires of varying sizes.
<img alt="Figure 9" src="/files/ftp_upload/52834/52834fig9.jpg" /> 
Figure 9. SEM image illustrating the sequential point-to-point fabrication order for drawing the micron and submicron polymer fibers (29X magnification; scale bar = 200 &#956;m). <a href="https://www.jove.com/files/ftp_upload/52834/52834fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/52834/52834fig10.jpg" /> 
Figure 10. SEM image of micron (6.5 &#181;m) and sub-micron (555 nm) fibers drawn using a 20% concentration PMMA polymer solution (2,270X magnification; scale bar = 2 &#956;m). <a href="https://www.jove.com/files/ftp_upload/52834/52834fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Polymer fiber length and diameter were measured using scanning electron microscopy. Table 1 shows the average wire diameter for each unique structure presented in Figures 6-8 above corresponding to the bifurcated branch fibers and the support fibers. Table 2 displays the polymer parameters that were measured from the 24% PMMA solutions that were utilized to fabricate the structures noted above.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>Structure diameter in Figure 6</td>
			<td>Structure diameter in Figure 7</td>
			<td>Structure diameter in Figure 8</td>
		</tr>
		<tr>
			<td>Support fibers&#160;</td>
			<td>8.65 &#177; 1.43 &#181;m</td>
			<td>9.39 &#177; 1.23&#160; &#181;m</td>
			<td>9.31 &#177; 1.65&#160; &#181;m</td>
		</tr>
		<tr>
			<td>Branch fibers</td>
			<td>20.96 &#177; 3.35 &#181;m</td>
			<td>15.92 &#177; 1.44&#160; &#181;m</td>
			<td>12.24 &#177; 5.42&#160; &#181;m</td>
		</tr>
	</tbody>
</table>
Table 1. Average diameters of the suspended support and branch fibers shown in Figures 6-8 for a 24% PMMA solution.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>24% PMMA</td>
		</tr>
		<tr>
			<td>Viscosity (Pa*sec)</td>
			<td>35.19</td>
		</tr>
		<tr>
			<td>Surface Tension (mN/m)</td>
			<td>262.01</td>
		</tr>
		<tr>
			<td>Mass Transfer Coefficient (m/sec)</td>
			<td>8.59 x 10-8</td>
		</tr>
	</tbody>
</table>
Table 2. Polymer parameters of the 24% PMMA solutions that were utilized to fabricate the fiber structures presented in this work.

Watch this Scientific Journal Video about Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System at JoVE.com

Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System

Here, we present a protocol to fabricate freely-suspended, micron/sub-micron scale polymer fibers and &#8220;web-like&#8221; structures generated via automated direct writing procedure by means of a 3-axis dispensing system.

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer ...

prescribed-3-d-direct-writing-suspended-micronsub-micron-scale-fiber

Research

JoVE Journal

Engineering

8.0K Views.  University of Louisville.  Hier presenteren we een protocol om vrij-zwevende, micron / sub-micron schaal polymeer vezels en "web-achtige" structuren gegenereerd via geautomatiseerde direct schriftelijk procedure door middel van een 3-assige doseersysteem fabriceren. 

Video: Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System

Fabrication, Densification, and Replica Molding of 3D Carbon Nanotube Microstructures

The introduction of new materials and processes to microfabrication has, in large part, enabled many important advances in microsystems, lab-on-a-chip devices, and their applications. In particular, capabilities for cost-effective fabrication of polymer microstructures were transformed by the advent of soft lithography and other micromolding techniques 1, 2, and this led a revolution in applications of microfabrication to biomedical engineering and biology. Nevertheless, it remains challenging to fabricate microstructures with well-defined nanoscale surface textures, and to fabricate arbitrary 3D shapes at the micro-scale. Robustness of master molds and maintenance of shape integrity is especially important to achieve high fidelity replication of complex structures and preserving their nanoscale surface texture. The combination of hierarchical textures, and heterogeneous shapes, is a profound challenge to existing microfabrication methods that largely rely upon top-down etching using fixed mask templates. On the other hand, the bottom-up synthesis of nanostructures such as nanotubes and nanowires can offer new capabilities to microfabrication, in particular by taking advantage of the collective self-organization of nanostructures, and local control of their growth behavior with respect to microfabricated patterns. 
Our goal is to introduce vertically aligned carbon nanotubes (CNTs), which we refer to as CNT "forests", as a new microfabrication material. We present details of a suite of related methods recently developed by our group: fabrication of CNT forest microstructures by thermal CVD from lithographically patterned catalyst thin films; self-directed elastocapillary densification of CNT microstructures; and replica molding of polymer microstructures using CNT composite master molds. In particular, our work shows that self-directed capillary densification ("capillary forming"), which is performed by condensation of a solvent onto the substrate with CNT microstructures, significantly increases the packing density of CNTs. This process enables directed transformation of vertical CNT microstructures into straight, inclined, and twisted shapes, which have robust mechanical properties exceeding those of typical microfabrication polymers. This in turn enables formation of nanocomposite CNT master molds by capillary-driven infiltration of polymers. The replica structures exhibit the anisotropic nanoscale texture of the aligned CNTs, and can have walls with sub-micron thickness and aspect ratios exceeding 50:1. Integration of CNT microstructures in fabrication offers further opportunity to exploit the electrical and thermal properties of CNTs, and diverse capabilities for chemical and biochemical functionalization 3.

We present methods for fabrication of patterned microstructures of vertically aligned carbon nanotubes (CNTs), and their use as master molds for production of polymer microstructures with organized nanoscale surface texture. The CNT forests are densified by condensation of solvent onto the substrate, which significantly increases their packing density and enables self-directed formation of 3D shapes.

The introduction of new materials and processes to microfabrication has, in large part, enabled many important advances in microsystems, lab-on-a-chip ...

A Method to Fabricate Disconnected Silver Nanostructures in 3D

The standard nanofabrication toolkit includes techniques primarily aimed at creating 2D patterns in dielectric media. Creating metal patterns on a submicron scale requires a combination of nanofabrication tools and several material processing steps. For example, steps to create planar metal structures using ultraviolet photolithography and electron-beam lithography can include sample exposure, sample development, metal deposition, and metal liftoff. To create 3D metal structures, the sequence is repeated multiple times. The complexity and difficulty of stacking and aligning multiple layers limits practical implementations of 3D metal structuring using standard nanofabrication tools. Femtosecond-laser direct-writing has emerged as a pre-eminent technique for 3D nanofabrication.1,2 Femtosecond lasers are frequently used to create 3D patterns in polymers and glasses.3-7 However, 3D metal direct-writing remains a challenge. Here, we describe a method to fabricate silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm. The method enables the fabrication of patterns not feasible using other techniques, such as 3D arrays of disconnected silver voxels.8 Disconnected 3D metal patterns are useful for metamaterials where unit cells are not in contact with each other,9 such as coupled metal dot10,11or coupled metal rod12,13 resonators. Potential applications include negative index metamaterials, invisibility cloaks, and perfect lenses.
In femtosecond-laser direct-writing, the laser wavelength is chosen such that photons are not linearly absorbed in the target medium. When the laser pulse duration is compressed to the femtosecond time scale and the radiation is tightly focused inside the target, the extremely high intensity induces nonlinear absorption. Multiple photons are absorbed simultaneously to cause electronic transitions that lead to material modification within the focused region. Using this approach, one can form structures in the bulk of a material rather than on its surface.
Most work on 3D direct metal writing has focused on creating self-supported metal structures.14-16 The method described here yields sub-micrometer silver structures that do not need to be self-supported because they are embedded inside a matrix. A doped polymer matrix is prepared using a mixture of silver nitrate (AgNO3), polyvinylpyrrolidone (PVP) and water (H2O). Samples are then patterned by irradiation with an 11-MHz femtosecond laser producing 50-fs pulses. During irradiation, photoreduction of silver ions is induced through nonlinear absorption, creating an aggregate of silver nanoparticles in the focal region. Using this approach we create silver patterns embedded in a doped PVP matrix. Adding 3D translation of the sample extends the patterning to three dimensions.

Femtosecond-laser direct-writing is frequently used to create three-dimensional (3D) patterns in polymers and glasses. However, patterning metals in 3D remains a challenge. We describe a method for fabricating silver nanostructures embedded inside a polymer matrix using a femtosecond laser centered at 800 nm.

The standard nanofabrication toolkit includes  techniques primarily aimed at creating 2D patterns in dielectric media. Creating  metal patterns on a ...

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material fabrication techniques are able to mimic the complexity of these structures while maintaining scalability. The Vaporization of a Sacrificial Component (VaSC) process is able to do so. This process uses sacrificial fibers as a template to form hollow, cylindrical microchannels embedded within a matrix. Tin (II) oxalate (SnOx) is embedded within poly(lactic) acid (PLA) fibers which facilitates the use of this process. The SnOx catalyzes the depolymerization of the PLA fibers at lower temperatures. The lactic acid monomers are gaseous at these temperatures and can be removed from the embedded matrix at temperatures that do not damage the matrix. Here we show a method for aligning these fibers using micromachined plates and a tensioning device to create complex patterns of three-dimensionally arrayed microchannels. The process allows the exploration of virtually any arrangement of fiber topologies and structures.

The Vaporization of a Sacrificial Component (VaSC) process is used to fabricate microvascular structures. This procedure uses sacrificial poly(lactic) acid fibers to form hollow microchannels with precise 3D geometric positioning provided by laser micromachined guide plates.

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material ...

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 ...

Bioengineering

Manufacturing of Three-dimensionally Microstructured Nanocomposites through Microfluidic Infiltration

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite in&#64257;ltration of 3D interconnected microfluidic networks. The manufacturing of the reinforced beams begins with the fabrication of microfluidic networks, which involves layer-by-layer deposition of fugitive ink filaments using a dispensing robot, filling the empty space between filaments using a low viscosity resin, curing the resin and finally removing the ink.&#160;Self-supported 3D structures with other geometries and many layers (e.g.&#160;a few hundreds layers) could be built using this method. The resulting tubular micro&#64258;uidic networks are then infiltrated with thermosetting nanocomposite suspensions containing nanofillers (e.g.&#160;single-walled carbon nanotubes), and subsequently cured. The infiltration is done by applying a pressure gradient between two ends of the empty network (either by applying a vacuum or vacuum-assisted microinjection). Prior to the infiltration, the nanocomposite suspensions are prepared by dispersing nanofillers into polymer matrices using ultrasonication and three-roll mixing methods. The nanocomposites (i.e.&#160;materials infiltrated) are then solidified under UV exposure/heat cure, resulting in a 3D-reinforced composite structure. The technique presented here enables the design of functional nanocomposite macroscopic products for microengineering applications such as actuators and sensors.

Three-dimensional (3D) microstructured composite beams are fabricated through the directed and localized infiltration of nanocomposites into 3D porous microfluidic networks. The flexibility of this manufacturing method enables the utilization of different thermosetting materials and nanofillers in order to achieve a variety of functional 3D reinforced nanocomposite macroscopic products.

Microstructured composite beams reinforced with complex three-dimensionally (3D) patterned nanocomposite microfilaments are fabricated via nanocomposite ...

Chemistry

Laser-induced Forward Transfer of Ag Nanopaste

Over the past decade, there has been much development of non-lithographic methods1-3 for printing metallic inks or other functional materials. Many of these processes such as inkjet3 and laser-induced forward transfer (LIFT)4 have become increasingly popular as interest in printable electronics and maskless patterning has grown. These additive manufacturing processes are inexpensive, environmentally friendly, and well suited for rapid prototyping, when compared to more traditional semiconductor processing techniques. While most direct-write processes are confined to two-dimensional structures and cannot handle materials with high viscosity (particularly inkjet), LIFT can transcend both constraints if performed properly. Congruent transfer of three dimensional pixels (called voxels), also referred to as laser decal transfer (LDT)5-9, has recently been demonstrated with the LIFT technique using highly viscous Ag nanopastes to fabricate freestanding interconnects, complex voxel shapes, and high-aspect-ratio structures. In this paper, we demonstrate a simple yet versatile process for fabricating a variety of micro- and macroscale Ag structures. Structures include simple shapes for patterning electrical contacts, bridging and cantilever structures, high-aspect-ratio structures, and single-shot, large area transfers using a commercial digital micromirror device (DMD) chip.

We demonstrate the use of the Laser-induced forward transfer technique (LIFT) for the printing of high-viscosity Ag paste. This technique offers a simple, low temperature, robust process for non-lithographically printing microscale 2D and 3D structures.

Over the past decade, there has been much development of non-lithographic methods1-3 for printing metallic inks or other functional materials. Many of ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Using Carbon Nanofiber Arrays for Efficient Biomolecule and Dye Delivery to Plants

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to plants. As exciting as these new tools are, they have yet to be widely applied. Nanomaterials fabricated on rigid substrate (backing) are particularly difficult to successfully apply to curved plant structures. This study describes the process for microfabricating vertically aligned carbon nanofiber arrays and transferring them from a rigid to a flexible substrate. We detail and demonstrate how these fibers (on either rigid or flexible substrates) can be used for transient transformation or dye (e.g., fluorescein) delivery to plants. We show how VACNFs can be transferred from rigid silicon substrate to a flexible SU-8 epoxy substrate to form flexible VACNF arrays. To overcome the hydrophobic nature of SU-8, fibers in the flexible film were coated with a thin silicon oxide layer (2-3 nm). To use these fibers for delivery to curved plant organs, we deposit a 1 &#181;L droplet of dye or DNA solution on the fiber side of VACNF films, wait 10 min, place the films on the plant organ and employ a swab with a rolling motion to drive fibers into plant cells. With this method, we have achieved dye and DNA delivery in plant organs with curved surfaces.

Author Spotlight: Unlocking Plant Transformation by Innovating with Carbon Nanofiber Arrays 

Here we describe methods for microfabricating vertically aligned carbon nanofibers (VACNFs), transferring VACNFs to flexible substrates, and applying VACNFs on both rigid and flexible substrates to plants for biomolecule and dye delivery.

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to ...

3D Human Myocardial Tissue Generation Using Melt Electrospinning Writing of Polycaprolactone Scaffolds and hiPSC-Derived Cardiac Cells

The development of functional human cardiac tissues holds significant promise for advancing applications in drug screening, disease modeling, and regenerative medicine. This protocol describes the stepwise fabrication of 3D myocardial tissues with advanced mimicry of native cardiac structure by combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds with fibrin hydrogels and human induced pluripotent stem cell (hiPSC)-derived cardiac cells. The process involves embedding a mixture of cardiomyocytes (hiPSC-CMs) and cardiac fibroblasts (hiPSC-CFs) within a fibrin matrix to create mini-tissues, with structural support provided by MEW-generated scaffolds. These fibrillar scaffolds are fabricated at the micro- to nanoscale, allowing for precise control over fiber architecture, which plays a key role in organizing cell distribution and alignment. Meanwhile, the fibrin matrix promotes cell viability and mimics the extracellular environment. Characterization of the generated tissues reveals well-organized sarcomeres within hiPSC-CMs, along with stable contractile activity. The tissues demonstrate consistent spontaneous beating as early as two days post-seeding, with sustained functionality over time. The combination of hiPSC-CFs with hiPSC-CMs enhances the structural integrity of the tissues while supporting long-term cell viability. This approach offers a reproducible, adaptable, and scalable method for creating biomimetic cardiac tissue models, providing a versatile platform for preclinical drug testing, mechanistic studies of cardiac disease, and potential regenerative therapies.

A reproducible method is presented to generate 3D myocardial tissues combining melt electrospinning writing (MEW) polycaprolactone (PCL) scaffolds and fibrin hydrogels with hiPSC-derived cardiomyocytes and fibroblasts. This technique offers precise control over scaffold architecture and can be applied in preclinical drug testing and cardiac disease modeling.

Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System

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