Cellular responses to patterned poly(acrylic acid) brushes

We use patterned poly(acrylic acid) (PAA) polymer brushes to explore the effects of surface chemistry and topography on cell-surface interactions. Most past studies of surface topography effects on cell adhesion have focused on patterned feature sizes that are larger than the dimensions of a cell, and PAA brushes have been characterized as cell repellent. Here we report cell adhesion studies for RBL mast cells incubated on PAA brush surfaces patterned with a variety of different feature sizes. We find that when patterned at subcellular dimensions on silicon surfaces, PAA brushes that are 30 or 15 nm thick facilitate cell adhesion. This appears to be mediated by fibronectin, which is secreted by the cells, adsorbing to the brushes and then engaging cell-surface integrins. The result is detectable accumulation of plasma membrane within the brushes, and this involves cytoskeletal remodeling at the cell-surface interface. By decreasing brush thickness, we find that PAA can be 'tuned' to promote cell adhesion with down-modulated membrane accumulation. We exemplify the utility of patterned PAA brush arrays for spatially controlling the activation of cells by modifying brushes with ligands that specifically engage IgE bound to high-affinity receptors on mast cells.     

Selective placement of carbon nanotubes on metal-oxide surfaces

We describe a method to selectively position carbon nanotubes on Al2O3 and HfO2 surfaces. The method exploits the selective binding of alkylphosphonic acids to oxide surfaces with large isoelectric points (i.e. basic rather than acidic surfaces). We have patterned oxide surfaces with acids using both microcontact printing and conventional lithography. With proper choice of the functional end group (e.g., -CH3 or -NH2), nanotube adhesion to the surface can be either prevented or enhanced.     

Micropatterning neuronal cells on polyelectrolyte multilayers

This paper describes an approach to adhere retinal cells on micropatterned polyelectrolyte multilayer (PEM) lines adsorbed on poly(dimethylsiloxane) (PDMS) surfaces using microfluidic networks. PEMs were patterned on flat, oxidized PDMS surfaces by sequentially flowing polyions through a microchannel network that was placed in contact with the PDMS surface. Polyethyleneimine (PEI) and poly(allylamine hydrochloride) (PAH) were the polyions used as the top layer cellular adhesion material. The microfluidic network was lifted off after the patterning was completed and retinal cells were seeded on the PEM/PDMS surfaces. The traditional practice of using blocking agents to prevent the adhesion of cells on unpatterned areas was avoided by allowing the PDMS surface to return to its uncharged state after the patterning was completed. The adhesion of rat retinal cells on the patterned PEMs was observed 5 h after seeding. Cell viability and morphology on the patterned PEMs were assayed. These materials proved to be nontoxic to the cells used in this study regardless of the number of stacked PEM layers. Phalloidin staining of the cytoskeleton revealed no apparent morphological differences in retinal cells compared with those plated on polystyrene or the larger regions of PEI and PAH; however, cells were relatively more elongated when cultured on the PEM lines. Cell-to-cell communication between cells on adjacent PEM lines was observed as interconnecting tubes containing actin that were a few hundred nanometers in diameter and up to 55 microm in length. This approach provides a simple, fast, and inexpensive method of patterning cells onto micrometer-scale features.     

Adhesion of bioinspired micropatterned surfaces: effects of pillar radius, aspect ratio, and preload

Inspired by biological attachment systems, micropatterned elastomeric surfaces with pillars of different heights (between 2.5 and 80 microm) and radii (between 2.5 and 25 microm) were fabricated. Their adhesion properties were systematically tested and compared with flat controls. Micropatterned surfaces with aspect ratios above 0.5 were found to be more compliant than flat surfaces. The adhesion significantly increases with decreasing pillar radius and increasing aspect ratio of the patterned features. A preload dependence of the adhesion force has been identified and demonstrated to be crucial for obtaining adhesives with tunable adherence.     

Simultaneous targeted immobilization of anti-human IgG-coated nanotubes and anti-mouse IgG-coated nanotubes on the complementary antigen-patterned surfaces via biological molecular recognition

Introduction of self-assembly in nanometer-sized building blocks is expected to accomplish bottom-up fabrications in a more reproducible, efficient, and economic manner; however, it is necessary to selectively place multiple types of nano-building blocks (e.g., metal nanotubes and semiconductor nanotubes) at specific locations on surfaces with high precision and reproducibility for more complex nanometer-scale device assemblies. Biological molecular recognition such as antibody-antigen bindings may be suitable to use in the building-block assembly since nature always assembles materials with complex functions and structures at room temperature reproducibly. Our approach is to immobilize antibody-coated nanotubes at specific complementary binding positions patterned on surfaces. To demonstrate this hypothesis, two types of nanotubes coated with different antibodies were anchored selectively onto their complementary antigen areas, patterned by tips of atomic force microscope (AFM). Because those nanotubes can be coated by various metals and semiconductors with controlled morphologies, this outcome opens the possibility to accomplish the proposed unconventional device fabrication methodology that antibody nanotubes coated with different types of metals/semiconductors can be self-assembled on antigen-patterned surfaces via biological molecular recognition.     

The deformation and adhesion of randomly rough and patterned surfaces

Using a surface forces apparatus (SFA) and an atomic force microscope (AFM) we have studied the effects of surface roughness (root-mean-square (RMS) roughness between 0.3 and 220 nm) on the "contact mechanics", which describes the deformations and loading and unloading adhesion forces, of various polymeric surfaces. For randomly rough, moderately stiff, elastomeric surfaces, the force-distance curves on approach and separation are nearly reversible and almost perfectly exponentially repulsive, with an adhesion on separation that decreases only slightly with increasing RMS. Additionally, the magnitude of the preload force is seen to play a large role in determining the measured adhesion. The exponential repulsion likely arises from the local compressions (fine-grained nano- or submicron-scale deformations) of the surface asperities. The resulting characteristic decay lengths of the repulsion scale with the RMS roughness and correlate very well with a simple finite element method (FEM) analysis based on actual AFM topographical images of the surfaces. For "patterned" surfaces, with a nonrandom terraced structure, no similar exponential repulsion is observed, suggesting that asperity height variability or random roughness is required for the exponential behavior. However, the adhesion force or energy between two "patterned" surfaces fell off dramatically and roughly exponentially as the RMS increased, likely owing to a significant decrease in the contact area which in turn determines their adhesion. For both types of rough surfaces, random and patterned, the coarse-grained (global, meso- or macroscopic) deformations of the initially curved surfaces appear to be Hertzian.     

表面微图案化聚乳酸膜的制备及其细胞亲和性评价

利用溶剂-非溶剂法(SNS)制备表面具有微孔图案的聚乳酸(PLA)膜和聚苯乙烯(PS)膜,并以微孔PS膜为模板,构建表面具有微岛图案的PLA膜.以此为基础,对所制备的微图案表面对PLA膜亲/疏水性及成骨细胞粘附与增殖性能的影响进行研究.结果显示微图案的存在显著增强了PLA膜的表面疏水性(水接触角90°);成骨细胞在微图案表面具有良好的铺展性,其黏附数量明显高于光滑PLA膜,但细胞的生长曲线相对较平缓,显示微图案表面虽有利于细胞在PLA膜表面的粘附与铺展,但对促进细胞的增殖无贡献.     

Electro-osmotic flows in a microchannel with patterned hydrodynamic slip walls

We present an analysis of the electro-osmotic flow of electrolytic solutions in a microchannel with patterned hydrodynamic slippage on channel walls. A set of governing equations is formulated to account for the effects of small variations in hydrodynamic slippage over the microchannel walls on the electro-osmotic flow. These equations are then solved analytically by using the perturbation method. Two frequently encountered surface patterns, (i) cosine wave variation and (ii) square wave variation in slip length, are considered in our analyses. The results show that patterned slippage over microchannel walls can induce complex flow patterns (such as vortical flows) in otherwise plug-like electro-osmotic flows, which suggests potential applications of such flows in microfluidic mixers.     

Covalent microcontact printing of proteins for cell patterning

We describe a straightforward approach to the covalent immobilization of cytophilic proteins by microcontact printing, which can be used to pattern cells on substrates. Cytophilic proteins are printed in micropatterns on reactive self-assembled monolayers by using imine chemistry. An aldehyde-terminated monolayer on glass or on gold was obtained by the reaction between an amino-terminated monolayer and terephthaldialdehyde. The aldehyde monolayer was employed as a substrate for the direct microcontact printing of bioengineered, collagen-like proteins by using an oxidized poly(dimethylsiloxane) (PDMS) stamp. After immobilization of the proteins into adhesive "islands", the remaining areas were blocked with amino-poly(ethylene glycol), which forms a layer that is resistant to cell adhesion. Human malignant carcinoma (HeLa) cells were seeded and incubated onto the patterned substrate. It was found that these cells adhere to and spread selectively on the protein islands, and avoid the poly(ethylene glycol) (PEG) zones. These findings illustrate the importance of microcontact printing as a method for positioning proteins at surfaces and demonstrate the scope of controlled surface chemistry to direct cell adhesion.     

TOF-SIMS analysis of structured surfaces biofunctionalized by a one-step coupling of a spacer-linked GRGDS peptide

Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) was applied to validate GRGDS peptide patterned surfaces. The structuring of the surfaces included several steps: micro contact printing (μCP), chemical etching and aminofunctionalization followed by chemical coupling of spacer-linked GRGDS peptides via an isothiocyanate anchor. TOF-SIMS analysis of characteristic ions and molecular fragments with a lateral resolution of 100 nm allowed proving the change in chemical properties of the surface with each step during the structuring process. We found that the application of polydimethylsiloxane as stamp material resulted in the contamination of the surface with this polymer. TOF-SIMS investigations, however, also showed that during the preparation process the contaminations were removed and do not influence the bio functionality of the surface patterns. The results of the surface analysis carried out with TOF-SIMS were confirmed by complementary cell adhesion experiments with murine fibroblasts. As a result, specific cell adhesion restricted to GRGDS peptide functionalized areas was obvious by the formation of focal adhesion contacts in the fibroblasts. Thus, TOF-SIMS is the method of choice in chemical characterization of surfaces in structuring and functionalization processes, because it offers the opportunity to follow surface contamination during the preparation process and to assess the influence of the contamination on the applicability of the final substrate.     

Cellular microfabrication: observing intercellular interactions using lithographically-defined DNA capture sequences

Previous reports have shown that synthetic DNA strands can be attached to the plasma membrane of living cells to equip them with artificial adhesion "receptors" that bind to complementary strands extending from material surfaces. This approach is compatible with a wide range of cell types, offers excellent capture efficiency, and can potentially be used to create complex multicellular arrangements through the use of multiple capture sequences. In this work, we apply an aluminum "lift off" lithography method to allow the efficient generation of complex patterns comprising different DNA sequences. The resulting surfaces are then demonstrated to be able to capture up to three distinct types of living cells in specific locations. The utility of this approach is demonstrated through the observation of patterned cells as they communicate by diffusion-based paracrine signaling. It is anticipated that the ability of this technique to create virtually any type of 2D heterogeneous cell pattern should prove highly useful for the examination of key questions in cell signaling, including stem cell differentiation and cancer metastasis.     

Thin-film models of liquid displacement on chemically patterned surfaces for lithographic printing processes

We examine in this work a model problem relevant to the liquid displacement that occurs in lithographic printing processes. The model problem consists of two stratified thin liquid films confined between parallel plates, one of which is chemically heterogeneous. The films are assumed to be thin enough so that intermolecular forces are important and the lubrication approximation can be invoked. Both linear stability analysis and nonlinear simulations are applied to a partial differential equation governing the behavior of the liquid-liquid interface. The results provide physical insights into and numerical estimates of the smallest and largest feature sizes that can be printed, as well as the minimum spacing between feature sizes that can be tolerated. The results also provide insight into experimental observations on a closely related process, wire-wound rod coating on chemically patterned surfaces. The work presented here has important implications for the production of electronic devices and displays by lithographic printing, as well as for other processes that rely on coating and printing on chemically patterned surfaces.     

Contact shape controls adhesion of bioinspired fibrillar surfaces

Following a recent bioinspired paradigm, patterned surfaces can exhibit better adhesion than flat contacts. Previous studies have verified that finer contact structures give rise to higher adhesion forces. In this study, we report on the effect of the tip shape, which was varied systematically in fibrillar PDMS surfaces, produced by lithographic and soft-molding methods. For fiber radii between 2.5 and 25 microm, it is found that shape exerts a stronger effect on adhesion than size. The highest adhesion is measured for mushroom-like and spatular terminals, which attain adhesion values 30 times in excess of the flat controls and similar to a gecko toe. These results explain the shapes commonly found in biological systems, and help in the exploration of the parameter space for artificial attachment systems.     

Macroscopic-wetting anisotropy on the line-patterned surface of fluoroalkylsilane monolayers

Micropatterned fluoroalkylsilane monolayer surfaces with liquidphilic/liquidphobic area (line width 1-20 microm) were prepared with few defects by vacuum ultraviolet (VUV) photolithography. The anisotropic wetting of a macroscopic droplet with a 0.5-5 mm diameter on the micropatterned surfaces was investigated. The strong anisotropy of the contact angle and the sliding angle and droplet distortion for fluoroalkylsilane/silanol patterned surfaces was attributed to the difference in the energy barrier of wetting between parallel and orthogonal lines. The wetting anisotropy decreased with decreases in the liquidphilic area. Fluoroalkylsilane/alkylsilane patterned surfaces with small differences in the surface free energies of the components showed anisotropic wetting only for the low-surface-tension liquids.     

Tuning microchannel wettability and fabrication of multiple-step Laplace valves

By using characteristics of titania nanoparticles, a patterning and tuning method of microchannel surface wettability was developed for microfluid control. Titania modification of a microchannel provided a nanometer-sized surface roughness and the subsequent hydrophobic treatment made the surface superhydrophobic. Photocatalytic decomposition of the coated hydrophobic molecules was used to pattern the surface wettability which was tuned in the range from superhydrophobic to superhydrophilic under controlled photoirradiation. Four-step wettability-based Laplace valves working as passive stop valves (6.8-12.5 kPa pressure barrier) were prepared by using the patterned and tuned surface. As a demonstration, a batch operation system consisting of two sub-nL dispensers and a reaction chamber was constructed. Fundamental liquid manipulations required for the batch operation were successfully conducted, including liquid measurement (390 and 770 pL), transportation, injection into the chamber, and retention in the chamber. To verify the quantitative operation, the system was applied to a fluorescence quenching experiment as an example of volumetric analyses. The present method provides flexible patterning in a wide range of tuned wettability surfaces in microchannels even after channel fabrication and it can be applied to various two- or multi-phase microfluidic systems.     

Soft lithography microfabrication of functionalized thermoplastics by solvent casting

A soft lithographic method is described for casting functional thermoplastic devices with microscale features without the need for specialized tools or equipment. In the thermoplastic soft lithography process, termed solvent casting, low temperature supersaturated solutions of thermoplastic are poured over solvent permeable PDMS molds which allow omnidirectional solvent removal as they template functional microstructures into the thermoplastic layers. Rapid gelation of supersaturated solutions enables the deposition of multiple patterned layers of varying composition, with self‐adhesion of the solvent‐laden thermoplastic ensuring intimate bonding between adjacent layers. This latter feature is further used in this work to realize sealed thermoplastic microfluidic devices with high fidelity replication of microchannel features with negligible channel deformation. The incorporation of functional dopants into patterned thermoplastic layers allows the fabrication of thermoplastic devices with embedded fluorescent sensors and integrated conductive elements. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015 , 53, 1315–1323     

Groove-spanning behavior of lipid membranes on microfabricated silicon substrates

We report on a spreading behavior of phospholipid membranes that arise from a lump of phospholipid (a lipid source) on topographically patterned substrates immersed in an aqueous solution. Microgrooves with well-defined shapes were prepared on Si111 surfaces by anisotropic etching in an alkaline solution. A spreading front that consists of membrane lobes and a single lipid bilayer was observed on the patterned silicon substrates by utilizing fluorescence interference contrast (FLIC) microscopy. FLIC images indicate that the membrane lobes span the microgrooves, while the underlying single lipid bilayer spread along the surface of the microgrooves. In fact, fluorescent polystyrene nanoparticles could be encapsulated in the microgrooves that were completely covered with the membrane lobes. The groove-spanning behavior of membrane lobes is discussed in terms of a balance between adhesion and bending energies of lipid bilayers.     

Effect of fluorosurfactant on capillary instabilities in nanoimprinted polymer patterns

Surface forces play a paramount role in most aspects of Nanoimprint Lithography. In particular, subjecting nanoimprinted patterns to moderate heating allows surface tension to smooth out undesirable roughness and defects in the patterns, but this “thermal reflow” treatment can induce structural decay or even collapse of the patterns by capillary instability if this process is not carefully controlled. Adhesion between the mold and polymer film can also cause the imprinted structure to tear or fracture. Fluorinated surfactants (FS) are attractive for reducing mold adhesion, yet the effects of these additives on nanostructure stability during thermal reflow are not well understood. Here we present thermal stability studies of line-space grating patterns created by Thermal Embossing Nanoimprint Lithography (TENIL) on model polystyrene (PS) films with FS additives. As expected by energy considerations, FS segregates to the air interface, where it seems to facilitate mold release. This also reduces the surface energy and thus reduces the driving force for pattern “slumping” (height decay). However, the beneficial effects of the surfactant are counterbalanced by the fact that the FS decreases the effective film viscosity, which accelerates nanopattern leveling. The net effect is that the pattern height decay is strongly a function of FS concentration. This enhanced film fluidity in the presence of FS also makes the pattern more susceptible to an undulatory capillary instability under thermal reflow conditions. Surface phase segregation of FS and PS is also observed in conjunction with both slumping and lateral capillary instabilities, which may be useful for producing chemically patterned surfaces. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 47: 2591–2600, 2009     

Detection of mismatched DNA on partially negatively charged diamond surfaces by optical and potentiometric methods

The effects of surface charge density on DNA hybridization have been investigated on a mixture of hydrogen-, oxygen-, and amine-terminated diamond surfaces. A difference in the hybridization efficiencies of complementary and mismatched DNA was clearly observed by fluorescence and potentiometric observations at a particular coverage of oxygen. In the fluorescence observation, singly mismatched DNA was detected with high contrast after appropriate hybridization on the surface with 10-20% oxygen coverage. The amount of oxygen in the form of C-O(-) (deprotonated C-OH) producing the surface negative-charge density was estimated by X-ray photoelectron spectroscopy. Electrolyte solution gate field-effect transistors (SGFETs) were used for potentiometric observations. The signal difference (change in gate potential) on the SGFET, which was as large as approximately 20 mV, was caused by the difference in the hybridization efficiencies of complementary target DNA (cDNA) and singly mismatched (1MM) target DNA with a common probe DNA immobilized on the same SGFET. The reversible change in gate potential caused by the hybridization and denaturation cycles and discriminating between the complementary and 1MM DNA targets was very stable throughout the cyclical detections. Moreover, the ratio of signals caused by hybridization of the cDNA and 1MM DNA targets with the probe DNA immobilized on the SGFET was determined to be 3:1 when hybridization had occurred (after 15 min on SGFET), as determined by real-time measurements. From the viewpoint of hybridization kinetics, the rate constant for hybridization of singly mismatched DNA was a factor of approximately 3 smaller than that of cDNA on this functionalized (oxidized and aminated) diamond surface.     

Local control of protein binding and cell adhesion by patterned organic thin films

Control of the cell adhesion and growth on chemically patterned surfaces is important in an increasing number of applications in biotechnology and medicine, for example implants, in-vitro cellular assays, and biochips. This review covers patterning techniques for organic thin films suitable for site-directed guidance of cell adhesion to surfaces. Available surface patterning techniques are critically evaluated, with special emphasis on surface chemistry that can be switched in time and space during cultivation of cells. Examples from the authors’ laboratory include the use of cell-repellent self-assembled monolayers (SAM) terminated by oligoethylene glycol (OEG) units and the lifting of the cell repellent properties by use of electrogenerated Br₂/HOBr which can be performed with positionable microelectrodes. Structural changes of the SAM were analyzed by polarization-modulated infrared reflection absorption spectroscopy (PM IRRAS). Use of a soft array system of individually addressable microelectrodes enables formation of flexible and complex patterns in a short time and has the potential for further acceleration of probe-induced local manipulation of cell adhesion.