Name: expanding nanopatterned substrates using stitch technique for nanotopographical modulation of cell behavior
Uploaded: 2016-12-08T14:00:00
Description: Watch this Scientific Journal Video about Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior at JoVE.com

This work was partly supported by NSF CBET 1227766, NSF CBET 1511759, and Byars-Tarnay Endowment. We gratefully acknowledge use of the West Virginia University Shared Research Facilities which are supported, in part, by NSF EPS-1003907.

1. Replication of PDMS Molds from an EBL Mold
<ol>
	<li>Fabricate silicon mold29
	<ol>
		<li>Spin coat 200 &#956;l polymethyl methacrylate (PMMA) solution on a 1&#160;&#215; 1 cm silicon (Si) substrate at 2,500 rpm for 1 min to form a thin film.</li>
		<li>Bake the PMMA film on the Si substrate at 180 &#176;C for 2 min.</li>
		<li>Write the designed nanopattern on the PMMA film by using a focused electron beam at an area dose of 300 &#181;C/cm2.</li>
		<li>Develop the PMMA nanopattern in developer...

<ol>
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We present a simple, affordable, yet versatile method to generate a large area of nanopatterned substrate. To faithfully expand highly defined nanopatterns, great attention should be paid to several critical steps. The first one is to trim the multiple PDMS molds. Unpatterned areas of the PDMS molds need to be removed. Additionally, the sidewalls of the molds should be cut vertically as perfect as possible to minimize the gaps between the molds. Collectively, the portion of unpatterned areas in the final stitch mold can ...

A number of recent findings reveal that substrate nanotopography has pronounced influence on cell behavior, from cell adhesion, spreading and migration, to proliferation and differentiation1-6. For instance, a smaller cell size and lower proliferation rate have been observed in cells cultured on deep nanogratings, even leading to apoptosis although the cell alignment, elongation and migration were enhanced, compared with the flat controls2,7-10. Moreover, nanotopography has been shown to facilitate the differentiation of stem cells into certain lineages such as neuron2,11,12, muscle13, and bone3,4. In addition, because of increasing concerns on the toxicity of engineered nanomaterials14,15, there is a need to incorporate nanotopography into physiologically relevant in vitro models for accurate risk assessment of nanomaterials. To fulfil the biochemical and molecular biology analyses, enough cells are needed to be grown on a large area of nanopatterned substrate. However, conventional nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area.
Self-assembly including colloid lithography16 and polymer demixing17 can readily generate large-area nanostructures at low costs. Because self-assembly relies on interactions between the assembling elements such as colloidal particles and macromolecules, and possible interactions between these elements and substrate, it cannot be a stand-alone method of producing nanostructures with precise spatial positioning and arbitrary shapes18. The accompanied high density of defects is also a drawback. Precise spatial control of nanopatterns can be achieved by employing templated self-assembly, which uses top-down lithographic approaches to provide the topographical and/or chemical template to guide the bottom-up assembly of the assembling elements19-21. Alternative nanofabrication techniques such as step-and-flash lithography22 and a roll-to-roll nanoimprinting lithography23 have been developed but have limited use because of their sophisticated process or the requirement of specialized equipment. Nevertheless, a template or a master mold with defined nanoscale patterns is needed for templated or alternative nanofabrication techniques. 
Such templates and master molds are conventionally generated by using focused electron, ion, or photon beam lithography. For instance, electron beam lithography (EBL)24 and focused ion beam lithography25 can generate defined patterns with a sub-5 nm resolution. Two-photon lithography has demonstrated a feature size as small as 30 nm26. Although the focused beam lithography techniques enable generation of well-defined nanoscale structures, the capital investment and the time-consuming, costly process restrict their widespread use in academic research27. Therefore, it is highly desirable to develop enabling yet affordable techniques to produce a large area of nanopatterned surfaces with high fidelity.
We have reported a simple, cost-effective stitch technique for generating a large area of nanopatterned surface from a small well-defined mold28. This protocol provides step-by-step procedure from replication of poly(dimethylsiloxane) (PDMS) molds using an EBL-written pattern, to assembly of multiple PDMS molds into a single large mold, to generation of a master mold on polymeric such as polystyrene (PS) substrates, to production of working substrates. With the expanded nanopatterned substrates, we demonstrated nanotopographical modulation of cell spreading.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>JEOL field emission SEM</td>
			<td>JEOL</td>
			<td>JSM-7600F</td>
			<td>EBL</td>
		</tr>
		<tr>
			<td>E-beam evaporator</td>
			<td>Kurt J. Lesker</td>
			<td>Model: LAB 18 e-beam evaporator</td>
			<td>nickel deposition</td>
		</tr>
		<tr>
			<td>Trion Minilock III ICP/RIE</td>
			<td>Trion technology</td>
			<td>Model: Minilock-phantom III</td>
			<td></td>
		</tr>
		<tr>
			<td>Press machine</td>
			<td>PHI Hydraulic Press</td>
			<td>Molde: SQ-230H</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Laurell Technologies</td>
			<td>Modle: WS-400A-6NPP-LITE</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 critical dryer</td>
			<td>Tousimis</td>
			<td>Modle: Autosamdri-815</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>University Wafer</td>
			<td>1080</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum plates</td>
			<td>McMaster-carr</td>
			<td>9057K123</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon sheets</td>
			<td>McMaster-carr</td>
			<td>8711K92</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>FALCON</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm petri dish</td>
			<td>FALCON</td>
			<td>353004</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Fisher Scientific</td>
			<td>12-550-34</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable weighing boats</td>
			<td>Fisher Scientific</td>
			<td>13-735-743</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass desiccator</td>
			<td>Fisher Scientific</td>
			<td>02-913-360</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic desiccator</td>
			<td>Bel-Art Products</td>
			<td>F42025-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>Fisher Scientific</td>
			<td>1110049SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Ted Pella, inc.</td>
			<td>5726</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade</td>
			<td>Fisher Scientific</td>
			<td>S17302</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal blocks</td>
			<td>McMaster-carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Punch</td>
			<td>Brettuns Village Leather Craft Supplies</td>
			<td>Arch punch</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(methyl methacrylate)</td>
			<td>MicroChem</td>
			<td>495 PMMA A4</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning</td>
			<td>Sylgard 184 kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene</td>
			<td>Dow Chemical</td>
			<td>Styron 685D</td>
			<td></td>
		</tr>
		<tr>
			<td>1H,1H,2H,2H-perfluorooctylmethyldichlorosilane</td>
			<td>Oakwood Chemical</td>
			<td>7142</td>
			<td></td>
		</tr>
		<tr>
			<td>Developer</td>
			<td>MicroChem</td>
			<td>MIBK/IPA at 1: 3 ratio</td>
			<td></td>
		</tr>
		<tr>
			<td>Remover</td>
			<td>MicroChem</td>
			<td>Remover PG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Fisher Scientific</td>
			<td>T324-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified eagle medium</td>
			<td>Sigma Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microsopy Science</td>
			<td>15712-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde&#160;</td>
			<td>Fisher Chemical</td>
			<td>G151-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>ATCC CCL-185</td>
			<td></td>
		</tr>
	</tbody>
</table>

expanding nanopatterned substrates using stitch technique for nanotopographical modulation of cell behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

The stitch technique can generate a large area of nanopatterned substrates with high fidelity. Figure 1a and 1b display the large area of nanopatterns transferred from the stitched PDMS mold to PS plate and PS thin film on a Si substrate, respectively. The comparison between the original EBL-written mold (Figure 1c) and the final PDMS working substrate (Figure 1d) confirms that the EBL-written nanopatterns can be faithful...

Watch this Scientific Journal Video about Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior at JoVE.com

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

The overall goal of this procedure is to produce a large area of nanopatterned substrate from small nanopatterned molds by using a simple and affordable yet versatile stitch technique. This method can help study substrate nanotopography modulation of cell phenotype and function at cellular and molecular levels, such as expression of focal adhesion proteins. The main advantage of this technique is that it is simple and cost effective.To begin, prepare the silanized silicon mold in the PDMS prepolymer, as described in the text protocol. Put the silanized silicon mold in a 60 millimeter Petri dish. Pour the PDMS prepolymer on the silicon mold in the Petri dish.Place the Petri dish in a plastic desiccator and degas for about 10 minutes, until all bubbles disappear. Transfer the Petri dish to a hot plate and cure the PDMS prepolymer at 70 degrees Celsius for four hours. Carefully peel off the PDMS mold from the silicon mold by using tweezers.Determine the orientation of anisotropic PDMS nanopatterns, such as nanogratings, under an optical microscope, and mark it on the backside of the PDMS molds with a marker pen. Clean the silicon substrate with ethanol in a fume hood, and then dry it with compressed air. Trim off the unpatterned areas of each PDMS mold with a blade.The PDMS molds should be aligned as close as possible, but not touching the surrounding mold, to minimize the unpatterned area and to avoid touching to the nanopattern deformation when the compressor force is applied. Place the trimmed PDMS mold with the nanopattern face-down on the mirror side of the silicon substrate, and then align other molds close to but not touching the surrounding molds. It is critical to try to cure the PDMS at the basal layer because uncured PDMS may flow through the gap between the multiple molds and damage the nanopatterns.However, the completely cured PDMS cannot bond the multiple molds together. To prepare a PDMS adhesive layer, cast one gram of degassed PDMS prepolymer on a clean glass slide to form a 0.5 millimeter thick layer. Bake the PDMS layer at 100 degrees Celsius on a hot plate for three to five minutes.Use a needle to touch the layer and ensure that the layer is partially but not completely cured. Place the PDMS layer on the backside of the aligned PDMS molds, quickly invert this assembly, and transfer it to the hot plate. Apply a compressive force using a metal block on top of the assembly to ensure good contact between the PDMS adhesive layer and the backside of the PDMS molds.Cure the PDMS adhesive layer at 100 degrees Celsius for one hour. Turn off the hot plate, remove the metal block, and peel off the single-stiched PDMS mold from the silicon substrate. To nanoimprint the stitched PDMS mold into the PS plate, place the PS plate in an aluminum spacer set on a three inch silicon wafer.Heat the PS plate on a hot plate at 250 degrees Celsius for 30 minutes. Then, place the stitched PDMS mold, with nanopatterns face-down, on the molten PS plate. Next, place an aluminum plate on the glass slide of the stitched PDMS mold.Apply a compressive pressure by using metal blocks on the aluminum plate, and wait for three minutes. Lift and replace the metal block from the aluminum plate, and increase the compressive pressure to 25 kilopascals. Then, repeat this step with the pressure increased to 50 kilopascals.Maintain the temperature of the hot plate at between 240 and 250 degrees Celsius under consistent pressure of 50 kilopascals for 15 minutes. Turn off the hot plate, and cool down the whole setup. Remove the metal blocks after the temperature is below 50 degrees Celsius, and carefully peel off the stitched PDMS mold from the PS plate.To nanoimprint the PDMS mold on a PS thin film, place the stitched PDMS mold with nanotopography face-down on the PS thin film, which is set on a hot plate. Apply compressive pressure of 12 kilopascals on the PDMS mold by using metal blocks on the glass slide of the PDMS mold. Increase the temperature of the hot plate to 180 degrees Celsius, and maintain it for 15 minutes.Turn off the hot plate, and cool down the whole setup. Remove the metal blocks after the temperature drops below 50 degrees Celsius, and carefully peel off the stitched PDMS mold from the PS film. Using a hollow steel arch punch, cut the nanopatterned PDMS substrates into discs to fit the configuration of a specific multi-well plate.Then, use tweezers to place the PDMS discs into the wells of the multi-well plate. Sterilize the PDMS substrates by using 70%ethanol for 30 minutes. Afterwards, aspirate the ethanol, and follow this by UV exposure for 30 minutes.Wash the PDMS substrates with 1X sterile phosphate-buffered saline three times. Next, coat the PDMS substrates with extracellular matrix protein for 30 minutes at room temperature. Rinse the PDMS substrates three times with sterile PBS, each for five minutes.Suspend human A549 lung cancer cells in Dulbecco's Modified Eagle Medium with 10%fetal bovine serum, and count the cells using a hemocytometer. Plate the cells at a seeding density of 2, 000 cells per square centimeter on the PDMS substrates. Culture the cells at 37 degrees Celsius in a humidified atmosphere containing 5%carbon dioxide for one day, and observe them afterwards.The generated nanopattern on the PS plate and the thin film are shown here. The arrows indicate the polymer raise in the interstices of the stitched PDMS molds. These SEM images demonstrate that PDMS-working substrates are faithfully transferred from the electron beam lithography-written nanopattern by applying the stitch technique.Representative SEM images of A549 cells grown on nanogratings and nanopillars are shown here. The cells show aligned cell morphology on the nanogratings, while the cells spread randomly on the nanopillars. After watching this video, you should have a good understanding of how to generate a large area of nanopatterned substrate by stitching multiple, small defined nanopatterned molds.Don't forget that working with toluene and paraformaldehyde can be extremely hazardous, and always wear appropriate personal protection equipment while performing the procedures.

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