Fabrication of Biofunctionalized Quasi‐Three‐Dimensional Microstructures of a Nonfouling Comb Polymer Using Soft Lithography

This paper describes a simple set of patterning methods that are applicable to diverse substrates and allow the routine and rapid fabrication of protein patterns embedded within a background that consists of quasi‐three‐dimensional microstructures of a cell‐resistant polymer. The ensemble of methods reported here utilizes three components to create topographically nonfouling polymeric structures that present cell‐adhesive protein patterns in the regions between the microstructures: the first component is an amphiphilic comb polymer that is comprised of a methyl methacrylate backbone and pendant oligo(ethylene glycol) moieties along the side chain, physically deposited films of which are protein‐ and cell‐resistant. The second component of the fabrication methodology involves the use of different variants of soft lithography, such as microcontact printing to create nonfouling topographical features of the comb polymer that demarcate cell‐adhesive regions of the third component: a cell‐adhesive extracellular protein or peptide. The ensemble of methods reported in this paper was used to fabricate quasi‐three‐dimensional patterns that present topographical and biochemical cues on a variety of substrates, and was shown to successfully maintain cellular patterns for up to two months in serum‐containing medium. We believe that this, and other such methods under development that allow independent and systematic control of chemistry, topography and substrate compliance will provide versatile “test‐beds” for fundamental studies in cell biology as well as allow the discovery of rational design principles for the development of biomaterials and tissue‐engineering scaffolds.     

Soft Lithography of Ceramic Patterns

Polymer‐based precursor solutions are patterned using a soft‐lithographic patterning technique to yield sub‐micrometer‐sized ceramic patterns. By using a polymer–metal‐nitrate solution as a lithographic resist, we demonstrate a micromolding procedure using a simple rubber stamp that yields a patterned precursor layer. A subsequent high‐temperature annealing step degrades the polymer giving rise to a patterned metal oxide film. This procedure is demonstrated for three different ceramic materials: Al₂O₃, ZnO, and PbTiO₃. Al₂O₃ initially forms an amorphous phase that is subsequently converted into a polycrystalline material upon electron irradiation. The formed ZnO and PbTiO₃ are polycrystalline. PbTiO₃ exhibits epitaxial alignment when cast onto a SrTiO₃(001) surface that matches its lattice periodicity. This epitaxial alignment is maintained when the PbTiO₃ phase is patterned by micromolding, giving rise to epitaxially grown PbTiO₃ patterns with feature sizes down to 300 nm.     

Multiphoton Lithography of Unconstrained Three‐Dimensional Protein Microstructures

Multiphoton lithography (MPL) is a highly versatile strategy for creating 3D microscale objects with complex geometrical arrangements, including nested boxes, interlocking blocks, and braided threads. Of the various chemistries used to produce solid forms in MPL, protein photocrosslinking has been of particular value in biological applications, yielding materials with high porosity, tunable elasticity, and a diverse set of chemical and biochemical properties. Unfortunately, the potential for object drift, and consequent distortion, during this direct‐write process has required that microforms be constructed in integral contact with an immobile surface, precluding fabrication of protein‐based objects that retain rotational and translational degrees of freedom. Here, the development of a high‐viscosity protein‐based reagent that can be used to fabricate complex 3D microstructures that are not adhered to a surface, including chains of Möbius strips, paddlewheels, and unconstrained (free‐floating) probes for bacterial motility, is reported.     

Primary Neuron/Astrocyte Co‐Culture on Polyelectrolyte Multilayer Films: A Template for Studying Astrocyte‐Mediated Oxidative Stress in Neurons

We engineered patterned co‐cultures of primary neurons and astrocytes on polyelectrolyte multilayer (PEM) films without the aid of adhesive proteins/ligands to study the oxidative stress mediated by astrocytes on neuronal cells. A number of studies have explored engineering co‐culture of neurons and astrocytes predominantly using cell lines rather than primary cells owing to the difficulties involved in attaching primary cells onto synthetic surfaces. To our knowledge this is the first demonstration of patterned co‐culture of primary neurons and astrocytes for studying neuronal metabolism. In our study, we used synthetic polymers, namely poly(diallyldimethylammoniumchloride) (PDAC) and sulfonated poly(styrene) (SPS) as the polycation and polyanion, respectively, to build the multilayers. Primary neurons attached and spread preferentially on SPS surfaces, while primary astrocytes attached to both SPS and PDAC surfaces. SPS patterns were formed on PEM surfaces, either by microcontact printing SPS onto PDAC surfaces or vice‐versa, to obtain patterns of primary neurons and patterned co‐cultures of primary neurons and astrocytes. We further used the patterned co‐culture system to study the neuronal response to elevated levels of free fatty acids as compared to the response in separated monoculture by measuring the level of reactive oxygen species (ROS; a widely accepted marker of oxidative stress). The elevation in the ROS levels was observed to occur earlier in the patterned co‐culture system than in the separated monoculture system. The results suggest that this technique may provide a useful tool for engineering neuronal co‐culture systems, that may more accurately capture neuronal function and metabolism, and thus could be used to obtain valuable insights into neuronal cell function and perhaps even the pathogenesis of neurodegenerative diseases.     

High‐Strain Shape‐Memory Polymers

Shape‐memory polymers (SMPs) are self‐adjusting, smart materials in which shape changes can be accurately controlled at specific, tailored temperatures. In this study, the glass transition temperature (T_g) is adjusted between 28 and 55 °C through synthesis of copolymers of methyl acrylate (MA), methyl methacrylate (MMA), and isobornyl acrylate (IBoA). Acrylate compositions with both crosslinker densities and photoinitiator concentrations optimized at fractions of a mole percent demonstrate fully recoverable strains at 807% for a T_g of 28 °C, at 663% for a T_g of 37 °C, and at 553% for a T_g of 55 °C. A new compound, 4,4′‐di(acryloyloxy)benzil (referred to hereafter as Xini) in which both polymerizable and initiating functionalities are incorporated in the same molecule, was synthesized and polymerized into acrylate shape‐memory polymers, which were thermomechanically characterized yielding fully recoverable strains above 500%. The materials synthesized in this work were compared to an industry standard thermoplastic SMP, Mitsubishi's MM5510, which showed failure strains of similar magnitude, but without full shape recovery: residual strain after a single shape‐memory cycle caused large‐scale disfiguration. The materials in this study are intended to enable future applications where both recoverable high‐strain capacity and the ability to accurately and independently position T_g are required.     

Fabrication of stamps for microcontact printing by injection molding

Microcontact printing has been shown to be a viable lithographic technique for the fabrication of a variety of microelectronic components, including source/drain and gate electrodes for organic field effect transistors. Future manufacturing efforts may require a means of mass producing stamps for this process. In the present work, stamps for microcontact printing were rapidly produced by injection molding a commercial polyurethane resin, using a silicon master as the mold insert. The performance of these stamps was evaluated by microcontact printing gold coated silicon surfaces with a fluorinated alkanethiol. Etching of the stamped surface protected by the patterned alkanethiol revealed excellent replication of the submicron linear features of the micromold. The use of injection molding as a standard method for the production of stamps for microcontact printing is proposed and may have advantages for future nanotechnology applications that require mass production of stamps. Because a wide range of polymers may be injection molded, this method may make possible the fabrication of stamps with improved mechanical and chemical properties compared to polydimethylsiloxane based stamps.     

3D Microstructured Surfaces Obtained by Soft‐Lithography Using Fast‐Crosslinking Elastomeric Precursors and 2D Masters

A new method for the fabrication of microstructured polymer surfaces possessing features with different 3D geometries is reported. Controlled micromolding using masters with 2D topographies and fluid elastomeric precursors with various viscosities and crosslinking kinetics yielded homogeneously structured surfaces possessing microtubes and concave and convex hemispheres with defined dimensions. This fabrication strategy does not require sophisticated 3D structuring equipment and can be extended to other materials, dimensions and geometries.     

Photoresponsive Ferroelectric Liquid‐Crystalline Polymers

The photoresponse of ferroelectric smectic side‐chain liquid‐crystalline (LC) polymers containing a photoisomerizable azobenzene derivative as a covalently linked photochromic side group is investigated. By static measurements in different photostationary states, the effect of trans–cis isomerization on the material's phase‐transition temperatures and its ferroelectric properties (spontaneous electric polarization P_S and director tilt angle θ) are analyzed. It turns out that the Curie temperature (transition S_C* to S_A) can be reversibly shifted by up to 17 °C. The molecular mechanism of this “photoferroelectric effect” is studied in detail using time‐resolved measurements of the dye's optical absorbance, the director tilt angle, and the spontaneous polarization, which show a direct response of the ferroelectric parameters to the molecular isomerization. The kinetics of the thermal reisomerization of the azo dye in the LC matrix are evaluated. A comparison to the reisomerization reaction in isotropic solution (toluene) reveals a faster thermal relaxation of the dye in the LC phase.     

Enhanced Electro‐Optic Behavior for Shaped Polymer Cholesteric Liquid‐Crystal Flakes Made Using Soft Lithography

Polymer cholesteric liquid‐crystal (PCLC) flakes were investigated for their electro‐optical behavior under an applied alternating‐current field. Shaped flakes, fabricated using soft lithography and suspended in dielectric‐fluid‐filled cells, reoriented more uniformly than randomly shaped flakes made by fracturing of PCLC films. Extensive characterization found shaped flakes to be smooth and uniform in size, shape, and thickness. Reorientation in applied fields as low as tens of mV_rms μm^–1 was fastest for flakes with lateral aspect ratios greater than 1:1, confirming theoretical predictions based on Maxwell–Wagner polarization. Brilliant reflective colors and inherent polarization make shaped PCLC flakes of interest for particle displays.     

Transforming One‐Dimensional Nanowalls to Long‐Range Ordered Two‐Dimensional Nanowaves: Exploiting Buckling Instability and Nanofibers Effect in Holographic Lithography

Two‐dimensional nanowaves with long‐range order are fabricated by exploiting swelling‐induced buckling of one‐dimensional (1D) nanowalls with nanofibers formed in‐between during holographic lithography of the negative‐tone photoresist SU‐8. The 1D film goes through a constrained swelling in the development stage, and becomes buckled above the critical threshold. The degree of lateral undulation can be controlled by tuning the pattern aspect ratio (height/width) and exposure dosage. At a high aspect ratio (e.g., 6) and a high exposure dosage, nanofibers (30–50 nm in diameter) are formed between the nanowalls as a result of overlapping of low crosslinking density regions. By comparing experimental results with finite‐element analysis, the buckling mechanism is investigated, which confirms that the nanofibers prevent the deformed nanowalls from recovery to their original state, thus, leading to long‐range ordered two‐dimensional (2D) wavy structures. The film with nanowaves show weaker reflecting color under an ambient light and lower transmittance compared to the straight nanowalls. Using double exposure through a photomask, patterns consisting of both nanowaves and nanowalls for optical display are created.     

Fabrication of Organic Thin‐Film Transistors on Three‐Dimensional Substrates Using Free‐Standing Polymeric Masks Based on Soft Lithography

Here, a novel fabrication technique for integrated organic devices on substrates with complex structure is presented. For this work, free‐standing polymeric masks with stencil‐patterns are fabricated using an ultra‐violet (UV) curable polyurethaneacrylate (PUA) mixture, and used as shadow masks for thermal evaporation. High flexibility and adhesive properties of the free‐standing PUA masks ensure conformal contact with various materials such as glass, silicon (Si), and polymer, and thus can also be utilized as patterning masks for solution‐based deposition methods, such as spin‐coating and drop‐casting. Based on this technique, a number of integrated organic transistors are fabricated simultaneously on a cylindrical glass bottle with high curvature, as well as on a flat silicon wafer. It is anticipated that these results will be applied to the development of various integrated organic devices on complex‐structured substrates, which can lead to further applications.     

Cover Picture: Enhanced Electro‐Optic Behavior for Shaped Polymer Cholesteric Liquid‐Crystal Flakes Made Using Soft Lithography (Adv. Funct. Mater. 2/2005)

The cover shows a variety of shaped flakes fabricated from polymer cholesteric liquid‐crystal material using soft lithography. In work reported by Jacobs and co‐workers on p. 217, the micrometer‐sized flakes exhibit brilliant circularly polarized selective reflection colors without polarizers or color filters when placed in a fluid‐filled electro‐optic cell. With the application of a low‐magnitude alternating current field, the flakes reorient in hundreds of milliseconds and the colors disappear. Polymer cholesteric liquid‐crystal (PCLC) flakes were investigated for their electro‐optical behavior under an applied alternating‐current field. Shaped flakes, fabricated using soft lithography and suspended in dielectric‐fluid‐filled cells, reoriented more uniformly than randomly shaped flakes made by fracturing of PCLC films. Extensive characterization found shaped flakes to be smooth and uniform in size, shape, and thickness. Reorientation in applied fields as low as tens of mV_rms μm^–1 was fastest for flakes with lateral aspect ratios greater than 1:1, confirming theoretical predictions based on Maxwell–Wagner polarization. Brilliant reflective colors and inherent polarization make shaped PCLC flakes of interest for particle displays.     

Conducting‐Polymer Microcontainers: Controlled Syntheses and Potential Applications

We have demonstrated the controlled synthesis of conducting‐polymer microcontainers through the electrochemical generation of surfactant (i.e., β‐naphthalenesulfonic acid, β‐NSA)‐stabilized H₂ gas bubbles on the working electrode, followed by electrochemical polymerization of pyrrole around the wall of the “soap‐bubble” template. It was noticed that the density, shape, and wall thickness of the polypyrrole microcontainers thus prepared could be regulated by controlling the electrochemical potential applied for the generation of H₂ gas and the experimental conditions (e.g., the surfactant concentration, number of the cyclic voltammetric scanning) for the electropolymerization of pyrrole. By pre‐patterning the working electrode surface with non‐conducting polymers using microcontact printing (μCP) or plasma patterning, we have also produced conducting‐polymer microcontainers in a patterned fashion. Furthermore, potential applications of the patterned and non‐patterned conducting‐polymer microcontainers have been demonstrated; for example, through the encapsulation of appropriate fluorescence‐labeled molecules (e.g., fluorescein cadaverin) into the conducting‐polymer microcontainers by sealing their opened mouths with sequential electropolymerization of pyrrole. The resulting closed microcontainers could then be used for controlled releases.     

Patterned Self‐Assembled Monolayers on Silicon Oxide Prepared by Nanoimprint Lithography and Their Applications in Nanofabrication

Nanoimprint lithography (NIL) is used as a tool to pattern self‐assembled monolayers (SAMs) on silicon substrates because of its ability to pattern in the micrometer and nanometer ranges. The polymer template behaves as a physical barrier preventing the formation of a SAM in the covered areas of the substrate. After polymer removal, SAM patterns are obtained. The versatility of the method is shown in various nanofabrication schemes. Substrates are functionalized with a second type of silane adsorbate. Pattern enhancement via selective electrostatic attachment of carboxylate‐functionalized particles is achieved. Further applications of the NIL‐patterned substrates include template‐directed adsorption of particles, as well as the fabrication of electrodes on top of a SAM.     

Reversible Diffraction Efficiency of Photochromic Polymer Gratings Related to Photoinduced Dimensional Changes

In this Full Paper, the possibility of reversibly changing the diffraction efficiency of gratings, fabricated by soft molding lithography on polymer films, containing photochromic molecules, is demonstrated. In particular, alternating UV and visible laser irradiation of the gratings causes the doped photochromic molecules to undergo transformations, which induce reversible dimensional changes to the samples. As a result, reversible changes are monitored in the intensity of the beams of a diode laser, transmitted and diffracted from the gratings. These changes affect the diffraction efficiency, which is increased upon irradiation with UV and decreased after irradiation with visible laser light. Such gratings are promising candidates for the fabrication of modern optical components such as optical switching devices.     

3D Electrophoresis‐Assisted Lithography (3DEAL): 3D Molecular Printing to Create Functional Patterns and Anisotropic Hydrogels

The ability to easily generate anisotropic hydrogel environments made from functional molecules with microscale resolution is an exciting possibility for the biomaterials community. This study reports a novel 3D electrophoresis‐assisted lithography (3DEAL) platform that combines elements from proteomics, biotechnology, and microfabrication to print well‐defined 3D molecular patterns within hydrogels. The potential of the 3DEAL platform is assessed by patterning immunoglobulin G, fibronectin, and elastin within nine widely used hydrogels and characterizing pattern depth, resolution, and aspect ratio. Furthermore, the technique's versatility is demonstrated by fabricating complex patterns including parallel and perpendicular columns, curved lines, gradients of molecular composition, and patterns of multiple proteins ranging from tens of micrometers to centimeters in size and depth. The functionality of the printed molecules is assessed by culturing NIH‐3T3 cells on a fibronectin‐patterned polyacrylamide‐collagen hydrogel and selectively supporting cell growth. 3DEAL is a simple, accessible, and versatile hydrogel‐patterning platform based on controlled molecular printing that may enable the development of tunable, chemically anisotropic, and hierarchical 3D environments.     

Three‐Dimensional Printing of Elastomeric,Cellular Architectures with Negative Stiffness

Three‐dimensional printing of viscoelastic inks to create porous, elastomeric architectures with mechanical properties governed by the ordered arrangement of their sub‐millimeter struts is reported. Two layouts are patterned, one resembling a “simple cubic” (SC)‐like structure and another akin to a “face‐centered tetragonal” (FCT) configuration. These structures exhibit markedly distinct load response with directionally dependent behavior, including negative stiffness. More broadly, these findings suggest the ability to independently tailor mechanical response in cellular solids via micro‐architected design. Such ordered materials may one day replace random foams in mechanical energy absorption applications.     

Pattern‐Transfer Fidelity in Soft Lithography: The Role of Pattern Density and Aspect Ratio

Using high‐aspect‐ratio nanostructures fabricated via two‐photon laser‐scanning lithography, we examine the deformation of elastomeric stamps used in soft nanolithography and the fidelity of patterns and replicas made using these stamps. Two‐photon laser‐scanning lithography enables us to systematically regulate the aspect ratio and pattern density of the nanostructures by varying laser‐scanning parameters such as the intensity of the laser beam, the scanning speed, the focal depth inside the resist, and the scanning‐line spacing. Two commercially available stamp/mold materials with different moduli have been investigated. We find that the pattern‐transfer fidelity is strongly affected by the pattern density. In addition, we demonstrate that true three‐dimensional structures can be successfully replicated because of the flexible nature of elastomeric poly(dimethylsiloxane).     

Inverted‐Colloidal‐Crystal Hydrogel Matrices as Three‐Dimensional Cell Scaffolds

Successful engineering of functional tissues requires the development of three‐dimensional (3D) scaffolds that can provide an optimum microenvironment for tissue growth and regeneration. A new class of 3D scaffolds with a high degree of organization and unique topography is fabricated from polyacrylamide hydrogel. The hydrogel matrix is molded by inverted colloidal crystals made from 104 μm poly(methyl methacrylate) spheres. The topography of the scaffold can be described as hexagonally packed 97 μm spherical cavities interconnected by a network of channels. The scale of the long‐range ordering of the cavities exceeds several millimeters. In contrast to analogous material in the bulk, hydrogel shaped as an inverted opal exhibits much higher swelling ratios; its swelling kinetics is an order of magnitude faster as well. The engineered scaffold possesses desirable mechanical and optical properties that can facilitate tissue regeneration while allowing for continuous high‐resolution optical monitoring of cell proliferation and cell–cell interaction within the scaffold. The scaffold biocompatibility as well as cellular growth and infiltration within the scaffold were observed for two distinct human cell lines which were seeded on the scaffold and were tracked microscopically up to a depth of 250 μm within the scaffold for a duration of up to five weeks. Ease of production, a unique 3D structure, biocompatibility, and optical transparency make this new type of hydrogel scaffold suitable for most challenging tasks in tissue engineering.