This material is based on work supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2017R1A2B4008053) and the Ministry of Trade, Industry and Energy (MOTIE, Korea) under Industrial Technology Innovation Program No. 10052802 and the Korea Institute for the Advancement of Technology (KIAT) through the Encouragement Program for The Industries of Economic Cooperation Region (N0002310).

1. Fabrication of Nano-Micro Multiscale Structure Surface Using an AAO Filter (Figure 1)
<ol>
	<li>Purchase an AAO filter with a pore size, height, and diameter of 200 nm, 60 &#181;m, and 25 mm, respectively.</li>
	<li>1.2. Clean the surface of the Polyethylene terephthalate (PET) film having a thickness of 100 &#956;m to use acetone with 99.8% and isopropyl alcohol (IPA) with 99.9% for 5 min, and completely dry for 3 min using an air gun.</li>
	<li>Place the PET film on a flat surface without contaminants, and add a 0.1 mL drop of UV-curable polyurethane-acrylate (PUA)-type resin with a viscosity of 257.4 cPs onto the surface.</li>
	<li>Place the AAO filter on the resin and press uniformly, using a rubber roller with a diameter of 32 mm. The spread of the resin is visually confirmed, so the roller must be repeatedly and carefully pushed when pressing. 
	CAUTION: The AAO filter is brittle and may break if excessive force is applied.</li>
	<li>After rolling, expose the specimen made with the PET and AAO filter (attached using the resin) to UV light with a wavelength of 365 nm for 30 s to cure the resin.</li>
	<li>Immerse the cured specimen in 100 mL of 2 M NaOH solution for 10 min to dissolve the filter. 
	NOTE: The SEM images show the cross-section and surface of the structure (Figure 2).</li>
	<li>Clean the specimen with DI water, then completely dry it for 3 min using an air gun. 
	NOTE: EDX analysis confirmed that Na and Al were not detected and were completely etched (Figure 3).</li>
</ol>
2. Surface Treatments
<ol>
	<li>UV ozone treatment</li>
	<li>Clean the specimen with nano-micro multiscale structures using IPA and DI water for 5 min, then dry it using an air gun for 3 min.</li>
	<li>Irradiate the side of the multiscale structures of the specimen (the side with multiscale structures) using UV rays (wavelength of 185-254 nm) for 60 min. 
	NOTE: The UV ozone equipment has an intensity of 25 mW/cm2.</li>
	<li>Octadecyltrichlorosilane (OTS) self-assembly</li>
	<li>Place a hot plate inside the glove box and maintain a N2 environment for a vapor deposition process.</li>
	<li>Fix the edge of the specimen on a glass or flat plate using adhesive tape. Ensure that the size of the glass or plate is large enough to cover the top of a beaker (with 8 mm of diameter and 13 mm of height).</li>
	<li>Place the&#160;beaker on the hot plate with 5&#34;x7&#34;&#160;and add 2&#160;mL of OTS solution tothe beaker using a pipette.</li>
	<li>Cover the beaker with the glass or plate face-on, with the specimen facing downward into the beaker.</li>
	<li>Process for 60 min at 100 &#176;C, then remove the specimen from the glove box. 
	NOTE: After the OTS-coating process, the beaker and glove box must be cleaned.</li>
</ol>
3. Fabrication of Functional Surface by Injecting Lubricants
<ol>
	<li>Deposit approximately 0.2&#160;mL&#160;of perfluorocarbon (PFC) liquid onto the OTS-coated self-aggregated nanofiber assembly.</li>
	<li>Observe the wetting process of PFC&#160;using an optical microscope with an objective lens at 5X-20X.</li>
	<li>Remove the excess PFC liquid by placing the samples in a vertical position for a few hours.</li>
</ol>

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The authors have no competing financial interests to disclose.

The key step in fabrication of the self-aggregated nanofiber assembly is to ensure that the brittle AAO filter does not break when applying the resin with the rubber rollers. In fact, it should be ensured that the AAO filter does not break at any point before the etching step. Because the AAO filter is 25 mm in diameter, the size of the substrate is approximately 30 x 30 mm.
The self-aggregated nanofiber assembly allows us to provide various functional surfaces through the proper surface treatment. After imprinting, the primary surface is hydrophilic, but it can be modified and become hydrophobic by being subjected to UV ozone treatment and surface energy change after OTS coating. In addition, the proposed multiscale porous structures can be converted to a slippery surface via the liquid lubricant-infusion process.
The surface with nano-micro multiscale structures is opaque, possibly owing to the irregularity of the aggregated nanofibers, and this characteristic can be employed in optical applications. Thus, in subsequent studies, we will investigate the optical characteristics of the substrate using a UV-Vis-IR spectrometer. We expect that the optical properties of such surfaces can be applied to industries that require diffused reflection of light.

Nanoscale textured structures such as nanoparticles, nanotubes, and nanofibers have attracted attention in the scientific community, as they demonstrate unique characteristics in various applications including electrical, biomedical, optical, and surface engineering1,2,3,4,5,6,7,8. In particular, nanofibers are widely used in stretchable and transparent electrodes9, wearable sensors10,11, interconnections12,13, and nano-optics applications14. Among the various methods of fabricating nanoscale structures, such as sol-gel methods, self-assembly, lithography, and replication15,16,17,18,19,20, direct replication using a template is currently considered a promising method because it is simple, cost-effective, and applicable to various curable materials21,22,23,24,25,26.
Owing to its multiscale structure having a large number of nano-scale pores and micro-scale height, AAO is widely used as the template for fabrication of nanofibers and nanotubes with a high aspect ratio27,28,29,30. However, because of the surface tension at such a high aspect ratio, nanofibers tend to easily aggregate31,32,33. Existing research has proved that nanofibers having an aspect ratio greater than 15:1 do not stand upright but instead aggregate, whereas those having a ratio less than 5:1 are individually isolated without aggregation33,34. Capillary force and surface tension play an important role upon the removal of alumina using an etchant, which is one of the processes during nanofiber fabrication. When aspect ratio increases, surface tension among nanofibers tends to pull them closer to one another, causing aggregation. Several studies have focused on methods to prevent such aggregation35, which is particularly observed in polymer and metallic nanofibers. Among these, hydration of the nanofiber surface may reduce the agglomeration because when a liquid occupies the spaces between nanofibers, surface tension decreases. Further, the freeze-drying method may also reduce aggregation by decreasing surface tension between nanofibers. However, despite various efforts, the straightening of nanofibers with a high aspect ratio remains a challenge.
To this end, we report a unique method for fabricating multiscale structures of tangled nanofiber by exploiting the aggregation phenomenon in a positive manner. Here, the nanofiber structure is imprinted using an AAO filter and polyurethane-acrylate (PUA)-type resins with a viscosity of 257.4 cP. After UV nano imprint lithography (UV-NIL) is performed, the mold is etched with a&#160;NaOH solution. To characterize the proposed multiscale structures, we investigate the pattern behaviors of the sample with aggregated nanofibers and the surface wettability after proper surface treatments such as coating with a self-assembled monolayer and UV ozone treatment. Furthermore, we propose that the multiscale porous surface can be converted simply to a slippery surface using a lubricant-infused process.

<table><tbody><tr><td>MINS 511RM</td><td>Minuta Tech</td><td></td><td>UV curable resin</td></tr><tr><td>Octadecyltrichlorosilane (OTS)</td><td>Aldrich</td><td></td><td>Surface treatment</td></tr><tr><td>Sodium oxidanide</td><td>SAMCHUN</td><td></td><td>Etching solution</td></tr><tr><td>Anopore Inoganic Membranes</td><td>Whatman</td><td></td><td>25mm/0.2&amp;micro;m</td></tr><tr><td>MT-UV-A 47</td><td>Meiji Techno</td><td></td><td>UV curing equipment</td></tr><tr><td>UVC-30</td><td>Jaesung Engineering</td><td></td><td>UVO treatment equipment</td></tr><tr><td>Smart Drop Plus</td><td>FEMTOFAB</td><td></td><td>Contact angle measurement</td></tr><tr><td>Fluorinert FC-70</td><td>3M</td><td></td><td>liquid mixture of completely fluorinated aliphatic compounds</td></tr><tr><td>Polyethylene terephthalate film</td><td>Sunchem</td><td></td><td>Substrate</td></tr><tr><td>Acetone (99.8%)</td><td>Daejung</td><td></td><td>Cleaning solution</td></tr><tr><td>Isopropyl alcohol (99.9%)</td><td>Daejung</td><td></td><td>Cleaning solution</td></tr><tr><td>Rubber roller</td><td>Hwahong</td><td></td><td>For application of resin</td></tr><tr><td>Corning Stirring Hot Plates</td><td>Corning</td><td></td><td>Hot plate equipment (5&quot; x 7&quot;)</td></tr></tbody></table>

multiscale structures aggregated by imprinted nanofibers for functional surfaces

Multiscale surface structures have attracted increasing interest owing to several potential applications in surface devices. However, an existing challenge in the field is the fabrication of hybrid micro-nano structures using a facile, cost-effective, and high-throughput method. To overcome these challenges, this paper proposes a protocol to fabricate multiscale structures using only an imprint process with an anodic aluminum oxide (AAO) filter and an evaporative self-aggregation process of nanofibers. Unlike previous attempts that have aimed to straighten nanofibers, we demonstrate a unique fabrication method for multiscale aggregated nanofibers with high aspect ratios. Furthermore, the surface morphology and wettability of these structures on various liquids were investigated to facilitate their use in multifunctional surfaces.

We demonstrated a fast and simple method for the fabrication of multiscale nano-micro hybrid structures using an AAO filter as an imprinting mold. The entire process took 30 min (Figure 4). It was noted that after undergoing the etching process using NaOH, the resultant surface exhibited an opaque color similar to the original AAO filter, owing to the aggregated nanofiber assembly caused by surface tension. Further, the results of the EDX analysis confirmed that the AAO filter was completely removed by wet chemical etching (Figure 3).
The surface characteristics were determined by measuring the contact angle by dropping onto the surface of specimen 5 &#956;L of water droplets. Because the materials used in the AAO-filter-mediated imprint process exhibit superhydrophilicity, and the fabricated multiscale structures have highly porous networks due to self-aggregated nanofibers, the water droplets tend to be instantaneously absorbed into the substrates. However, the hydrophilicity can be modified to hydrophobicity using proper surface treatments. We demonstrated, as shown in Figure 5, that the surface of the imprinted multiscale structures was modified to a hydrophobic surface with a contact angle of approximately 117&#176; after OTS coating. In addition, UV ozone treatment can further increase the contact angle of the surface by approximately 10&#176; (Figure 6). After sequentially performing OTS coating and UV ozone treatment onto the imprinted surface, it was confirmed that the resulting contact angle increased to 134&#176; (Figure 7).
The surface and cross-section of the specimen with OTS coating show aggregation of the nano-fibers (Figure 5), which results in a dimple structure. The size and orientation of this dimple structure is irregular; however, this phenomenon occurred throughout the entire surface of the specimen. The surface of the specimen became smooth after it was subjected to the UV ozone treatment process36 (Figure 6 and Figure 7). This is also why the contact angle of the surface increased after the UV ozone treatment process. This phenomenon also occurred uniformly on the surface of the specimen, and the error of the contact angle was less than 3&#176;.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58356/58356fig1.jpg" /> 
Figure 1: Procedure for fabricating a structure with soluble aluminum oxide. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58356/58356fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58356/58356fig2.jpg" /> 
Figure 2: SEM images of a nano-micro multiscale structure after the etching process, showing the surface and cross-section. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58356/58356fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58356/58356fig3.jpg" /> 
Figure 3: Result of EDX analysis after the etching of nano-micro multiscale structures fabricated using an AAO filter. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58356/58356fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58356/58356fig4.jpg" /> 
Figure 4: Schematic for the fabrication of a nano-micro multiscale structure by aggregation of nanofibers after complete etching. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58356/58356fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58356/58356fig5.jpg" /> 
Figure 5: Contact angle after OTS coating on the surface and nano-micro structure. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58356/58356fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58356/58356fig6.jpg" /> 
Figure 6: Contact angle after UV ozone treatment on the surface and nano-micro structure. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58356/58356fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58356/58356fig7.jpg" /> 
Figure 7: Contact angle after sequentially performing OTS coating and UV ozone treatment on the surface and nano-micro structure. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58356/58356fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Multiscale Structures Aggregated by Imprinted Nanofibers for Functional Surfaces at JoVE.com

Multiscale Structures Aggregated by Imprinted Nanofibers for Functional Surfaces

Presented is an easy method to fabricate nano-micro multiscale structures, for functional surfaces, by aggregating nanofibers fabricated using an anodic aluminum oxide filter.

Multiscale surface structures have attracted increasing interest owing to several potential applications in surface devices. However, an existing ...

multiscale-structures-aggregated-imprinted-nanofibers-for-functional

Nanyang Technological University

Research

JoVE Journal

Engineering

6.5K Views.  Changwon National University. Presented is an easy method to fabricate nano-micro multiscale structures, for functional surfaces, by aggregating nanofibers fabricated using an anodic aluminum oxide filter.

Video: Multiscale Structures Aggregated by Imprinted Nanofibers for Functional Surfaces

Nanomoulding of Functional Materials, a Versatile Complementary Pattern Replication Method to Nanoimprinting

We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding combined with layer transfer enables the replication of arbitrary surface patterns from a master structure onto the functional material. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes. In particular we demonstrate the fabrication of patterned transparent zinc oxide electrodes for light trapping applications in solar cells.

We describe a nanomoulding technique which allows low-cost nanoscale patterning of functional materials, materials stacks and full devices. Nanomoulding can be performed on any nanoimprinting setup and can be applied to a wide range of materials and deposition processes.

We describe a nanomoulding technique which  allows low-cost nanoscale patterning of functional materials, materials stacks  and full devices. Nanomoulding ...

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

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Bioengineering

Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl dimethyl azlactone) (PGMA-b-PVDMA), are presented. Due to the high reactivity of azlactone groups towards amine, thiol, and hydroxyl groups, PGMA-b-PVDMA surfaces can be modified with secondary molecules to create chemically or biologically functionalized interfaces for a variety of applications. Previous reports of patterned PGMA-b-PVDMA interfaces have used traditional top-down patterning techniques that generate non-uniform films and poorly controlled background chemistries. Here, we describe customized patterning techniques that enable precise deposition of highly uniform PGMA-b-PVDMA films in backgrounds that are chemically inert or that have biomolecule-repellent properties. Importantly, these methods are designed to deposit PGMA-b-PVDMA films in a manner that completely preserves azlactone functionality through each processing step. Patterned films show well-controlled thicknesses that correspond to polymer brushes (~90 nm) or to highly crosslinked structures (~1-10 &#956;m). Brush patterns are generated using either the parylene lift-off or interface directed assembly methods described and are useful for precise modulation of overall chemical surface reactivity by adjusting either the PGMA-b-PVDMA pattern density or the length of the VDMA block. In contrast, the thick, crosslinked PGMA-b-PVDMA patterns are obtained using a customized micro-contact printing technique and offer the benefit of higher loading or capture of secondary material due to higher surface area to volume ratios. Detailed experimental steps, critical film characterizations, and trouble-shooting guides for each fabrication method are discussed.

Surface fabrication methods for patterned deposition of nanometer thick brushes or micron thick, crosslinked films of an azlactone block co-polymer are reported. Critical experimental steps, representative results, and limitations of each method are discussed. These methods are useful for creating functional interfaces with tailored physical features and tunable surface reactivity.

In this paper, fabrication methods that generate novel surfaces using the azlactone-based block co-polymer, poly (glycidyl methacrylate)-block-poly (vinyl ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

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.

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 ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

Procedure for Fabricating Biofunctional Nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, &alpha;,&omega;-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of &alpha;-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The &alpha;,&omega;-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-&isin;-amino caproic acid, by DCC coupling, resulting in the formation of &alpha;,&omega;-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, 1H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups ...

Biology

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 ...