Microscale control of cell contact and spacing via three-component surface patterning

The complexity of micropatterned cell constructs has been limited by difficulties in patterning more than two surface components on a culture substrate. Photolithography using multiple aligned masks is well established for generalized multicomponent patterning, but is often too harsh for biomolecules. We report a two-mask photolithographic process that is tuned to preserve bioactivity in patterns composed of covalently coupled poly(ethylene glycol) (PEG), adsorbed extracellular matrix protein (e.g., collagen I), and adsorbed serum proteins (e.g., vitronectin). Thereby, we pattern two cell types-primary hepatocytes and 3T3 fibroblasts-demonstrating control over contact and spacing (20-200 microm) between the two cell types for over one week. This method is applicable to the study of intercellular communication in cell biology and tissue engineering.     

Protein adsorption and surface patterning

Surface patterning has become an important discipline of biologically oriented surface science over the past decades. Many methods have been developed that allow the formation of patterns on the micro- and nanoscale. This Opinion discusses the role of protein adsorption in patterning technologies, highlighting how it can be used as an integrated part of the patterning process, how it can be controlled by patterns with appropriate properties, and how it may lead to disruption of formed patterns if not properly accounted for. Recent examples from literature are used to emphasize some of the most interesting developments in the field, such as novel surface chemistries only allowing specific protein adsorption, directed self-sorting adsorption of proteins on patterned surfaces, and control of protein adsorption through nanopatterning.     

Sequential patterning of two fluorescent streptavidins assisted by photoactivatable biotin on an aminodextran-coated surface

Sequential patterning of two fluorescent streptavidins (SAvs) was carried out using photopatterning of photoactivatable biotin (photobiotin) on an aminodextran surface, which was crucial for the minimization of non-specific binding. Photobiotin was bound by photoreaction to the amine groups of aminodextran. Water contact angle at each step during the preparation of the aminodextran surface was measured to investigate the hydrophilicity of the surfaces. The specific and nonspecific binding of a fluorescent SAv was investigated for the aminodextran surface and the amine-silane surface. The aminodextran surface almost entirely prevented nonspecific binding of a fluorescent SAv and was successfully used for sequential patterning of two fluorescent SAvs. The addition of ethanolamine (40 mM) in the photobiotin solution diminished blurring of pattern shape. To decrease pattern size, the UV light was focused on the aminodextran surface in an inverted microscope system. Under optimized conditions, two fluorescent SAvs array of approximately 25 μm size was obtained using a shadow mask of 100 μm hole size in the inverted microscope system.     

Facile patterning of upconversion NaYF4:Yb,Er nanoparticles

Different kinds of highly ordered patterns of NaYF(4):Yb,Er nanoparticles on gold substrates were fabricated using a simple method combining micro-contact printing and "breath figures" techniques. Ordered arrays of water droplets were first formed in the hydrophilic regions of patterned self-assembled monolayers (SAMs). This was subsequently submerged in a chloroform solution of NaYF(4):Yb,Er nanoparticles. The particles were spontaneously assembled at the interface of chloroform/water droplet surface, leading to different kinds of uniform patterns after solvent evaporation. The structures of NaYF(4):Yb,Er particles patterns depended on the dimension of the substrate, the concentration of the NaYF(4):Yb,Er nanoparticles and the water condensation process.     

Functional nanostructures from surface chemistry patterning

Nanoscale devices are expected to provide important advances for a number of applications. While many methods to generate nanoscale patterns exist, their use is confined to a relatively narrow range of materials. To fabricate nanoscale structures of a material with useful properties, the most convenient route is to transfer the geometry of an existing pattern into another material. Methods to achieve this pattern transfer are summarized and organized in this review. Methods to generate the original patterns, as well as applications of the final structure are also described.     

Light driving force for surface patterning on azobenzene-containing polymers

In this paper, we investigated the effect of light driving force induced surface deformation on azobenzene-containing polymers. The surface deformation is attributed to light-induced mass migration inside the polymers. Circular cap arrays are firstly fabricated by high power laser ablation via polarization controlled three-beam interference. The circular caps are subsequently exposed to polarization controlled two-beam interfering field. The results illuminate that when the interfering laser beams are both set to P polarization, the circular caps are deformed. While the laser beams are of other interfering modes like (S, S) and (+45° , -45°), the caps are seldom deformed. The circular caps are also exposed to single intensity-homogeneous linearly polarized laser beam. The deformation of the caps keeps the same direction as the irradiating polarization. A model based on the focusing effect of the circular caps is addressed to explain the origin of the light driving force for mass migration in azopolymers. The all-optical approach for the production of deformed caps can be used to generate aspherical lens, which may be applied to many domains.     

Facile method for fabrication of nanocomposite films with an ordered porous surface

This paper presents a novel and facile method for the fabrication of nanocomposite films with ordered porous surface structures. In this approach, a water-borne poly(styrene-co-butyl acrylate-co-acrylic acid)/silica nanocomposite dispersion was synthesized in situ by surfactant-free emulsion polymerization by using 3-allyloxy-2-hydroxy-1-propanesulfonate as a polymerizable surfactant. When this dispersion was dried to form a film at a certain temperature, an ordered porous structure could be directly obtained on the surface of the nanocomposite film. SEM, TEM, and AFM were employed to observe the morphology, and XPS and particle analyzer were used to analyze the surface composition of the ordered porous nanocomposite film and the particle size, respectively.     

Protein surface patterning using nanoscale PEG hydrogels

We have used focused electron-beam cross-linking to create nanosized hydrogels and thus present a new method with which to bring the attractive biocompatibility associated with macroscopic hydrogels into the submicron length-scale regime. Using amine-terminated poly(ethylene glycol) thin films on silicon substrates, we generate nanohydrogels with lateral dimensions of order 200 nm which can swell by a factor of at least five, depending on the radiative dose. With the focused electron beam, high-density arrays of such nanohydrogels can be flexibly patterned onto silicon surfaces. Significantly, the amine groups remain functional after e-beam exposure, and we show that they can be used to covalently bind proteins and other molecules. We use bovine serum albumin to amplify the number of amine groups, and we further demonstrate that different proteins can be covalently bound to different hydrogel pads on the same substrate to create multifunctional surfaces useful in emerging bio/proteomic and sensor technologies.     

Fluorescent aromatic platforms for cell patterning

This paper describes a simple experimental method of patterning fluorescent organic dyes, fluorescein, and rhodamine on gold substrates by microcontact printing techniques. The development of this step-by-step protocol has allowed us to prepare striped and squared micropatterns with poly(ethylene glycol) (PEG) linkers terminated by these fluorophores using a fast, easy, and inexpensive technique. Although the rest of the surface was covered with aliphatic molecules (methyl terminated), human fibroblasts demonstrated an unexpected response, aligning themselves according to the aromatic patterns, despite the presence of PEG, which is a cell resistant molecule, in the fluorescent regions.     

Plasma stencilling methods for cell patterning

In this paper we describe plasma stencilling techniques for patterning 10 mammalian cell lines on hydrophobic and cell repellent poly(dimethylsiloxane) (PDMS), methylated glass and bacterial grade polystyrene surfaces. An air plasma produced with a Tesla generator operating at atmospheric pressure was used with microengineered stencils for patterned surface oxidation, selectively transforming the surface to a hydrophilic state to enable cell adhesion and growth. Plasma stencilling obviates the need for directly patterning cell adhesion molecules. Instead, during cell culture, adhesion proteins from the media assemble in a bioactive form on the hydrophilic regions. Critically, the removal of protein patterning prior to cell culture provides the option to also use PDMS–PDMS plasma bonding to incorporate cell patterns within microfluidic systems. Linear patterns were generated using PDMS microchannel stencils, and polyimide stencils with through holes were used for the production of cellular arrays. For the production of smaller cellular arrays, a novel microcapillary-based dielectric barrier discharge system was developed. A numerical method to characterise the cell patterns is also introduced and was used to demonstrate that plasma stencilling is highly effective, with complete patterns confined during long term cell culture (>10 days). In summary, plasma stencilling is simple, rapid, inexpensive, reproducible and a potentially universal cell line patterning capability.

The construction of an individually addressable cell array for selective patterning and electroporation

In basic cell biology research and drug discovery, it is important to rapidly introduce genes, proteins or drug compounds into cells without permanent damage. Here, we report a three dimensional SU-8 micro-well structure sandwiched with an indium tin oxide (ITO) electrode-covered slide from the top and an individually addressable array of microelectrodes on the bottom to allow parallel delivery of exogenous molecules into various cells in a spatially specific manner. A positive dielectrophoretic force was selectively applied by energizing appropriate electrodes to capture the dispersed cells at the bottom electrode, while the micro-wells were designed to confine cells in situ when the positive dielectrophoretic force is removed. The combination of spatial positive dielectrophoresis (pDEP) and micro-wells made it possible to construct cell microarrays with specific patterns. Once the cells become attached to the electrodes, different plasmids can be introduced sequentially for selective electroporation. The present cell arraying-assisted electroporation chip integrates a pDEP-assisted cell positioning function with selective electroporation to provide a simple and efficient method for gene transfer. This platform is ideal for high throughput screening of compounds in parallel and thus holds promise for applications in cellular and molecular research.     

Dip-pen patterning and surface assembly of peptide amphiphiles

This paper presents results on controlling the surface morphology of evaporation-driven self-assembly of peptide amphiphile (PA) nanofibers by dip-pen nanolithography. These PA nanofibers, which measure only a few nanometers in diameter, can be oriented perpendicularly to the receding edge of a solution. Dragging a meniscus of PA ink with an atomic force microscope (AFM) tip creates reproducibly aligned arrays of isolated and close-packed PA nanofiber patterns on silicon substrates, utilizing surface coating of poly(ethylene glycol) to suppress the self-assembly of nanofibers on AFM tips. We also demonstrate the ability to construct double-layer patterns of differing nanofiber orientations at the same position. This result could be important in producing a complex, multilayer pattern of these peptide-based supramolecular nanostructures.     

Confinement of surface patterning in azo-polymer thin films

Azobenzene polymer thin films are known to spontaneously generate surface patterns in response to incident light gradients. This peculiar process is investigated in terms of the dynamics of the various azobenzene photomotions, which occur on different length scales. In particular, the formation and thermal erasure of surface relief gratings are measured as a function of film thickness and by using combinatorial samples with thickness gradients. The thermal erasure of gratings in this system provides a direct measure of the glass-transition temperature, which is found to deviate substantially from the bulk value. Thin azo films exhibit a glass transition up to 50 K higher than the bulk. These dynamical measurements allow the authors to probe the length scale of mass transport, which is found to be approximately 150 nm. Furthermore, surface mass transport is completely arrested in thin films<40 nm. According to these results, mass transport involves the coordinated motion of many polymer chains in the depth of the sample, rather than surface diffusion of individual chains.     

Facile fabrication of a superamphiphobic surface on the copper substrate

A simple solution-immersion technique was developed for the fabrication of a superamphiphobic surface on the copper sheet. Hierarchical structure composed of nanorod arrays and microflowers was formed on the copper surface by an alkali assistant oxidation process; after fluorination, the surface became super-repellent toward water and several organic liquids possessing much lower surface tension than that of water, such as hexadecane. Such superamphiphobicity is attributed to the synergistic effect of their special surface chemicals and microscopic structures, which allows for the formation of a composite interface with all probing liquids tested. We also discuss the effects of surface chemical constituent and geometrical structure on hydrophobicity and oleophobicity; such information allows us to engineer surfaces with specific oleophobic behavior. Additionally, the stability of the composite interface on the created superamphiphobic surface is studied by the compression and immersion test.     

Photolabile carboxylic acid protected terpolymers for surface patterning. Part 2: Photocleavage and film patterning

The surface properties of films made of p-methoxyphenacyl derivative terpolymers, associated with photocleavage by UV irradiation, and their optical patterning are investigated. The deprotection reaction has been monitored by UV and FTIR spectroscopy, contact angle measurements, and X-ray photoelectron spectroscopy, revealing the photoremoval of the protecting p-methoxyphenacyl group in high yields under mild conditions. Parallel and serial patterning of the films has been performed by selective irradiation through optical masks and by laser irradiation via fiber tips of a scanning near-field optical microscope, respectively. By irradiation of photolabile protected functional groups, free carboxylic groups become exposed to the surface with which fluorescent dyes and proteins can be associated specifically.     

Direct patterning of copper on polyimide using ion exchangeable surface templates generated by site-selective surface modification

We demonstrate site-selective chemical surface modification by dispensing potassium hydroxide solution onto polyimide, which confines source metallic ions that can subsequently be used in resist- and mask-free fabrication of copper circuit patterns. Metallization can be achieved by a wet chemical method, providing control over metal/polymer interfacial structures. Because the approach is compatible with other existing printing technologies and much simpler than conventional lithography-based methods, we propose that the present surface template method may be of general application in fabrication of metallized polymers as well as in development of integrated circuits with a variety of electronic circuit elements.     

Effects of electrode surface modification with chlorotoxin on patterning single glioma cells

A microchip patterned with arrays of single cancer cells can be an effective platform for the study of tumor biology, medical diagnostics, and drug screening. However, patterning and retaining viable single cancer cells on defined sites of the microarray can be challenging. In this study we used a tumor cell-specific peptide, chlorotoxin (CTX), to mediate glioma cell adhesion on arrays of gold microelectrodes and investigated the effects of three surface modification schemes for conjugation of CTX to the microelectrodes on single cell patterning, which include physical adsorption, covalent bonding mediated by N-hydroxysuccinimide (NHS), and covalent bonding via crosslinking succinimidyl iodoacetate and Traut's (SIA-Traut) reagents. The CTX immobilization to microelectrodes was confirmed by high-resolution X-ray photoelectron spectroscopy. Physically adsorbed CTX showed better support for cell adhesion and is more effective in confining adhered cells on the electrodes than covalently-bound CTX. Furthermore, cell adhesion and spreading on microelectrodes were quantified in real-time by impedance measurements, which revealed an impedance signal from physically adsorbed CTX electrodes four times greater than the signal from covalently-bound CTX electrodes.