Imaging Surface Immobilization Chemistry: Correlation with Cell Patterning on Non‐Adhesive Hydrogel Thin Films

High‐fidelity surface functional group (e.g., N‐hydroxysuccinimide (NHS) reactive ester) patterning is readily and reliably achieved on commercial poly(ethylene glycol) (PEG)‐based polymer films already known to exhibit high performance non‐fouling properties in full serum and in cell culture conditions. NHS coupling chemistry co‐patterned with methoxy‐capped PEG using photolithographic methods is directly spatially imaged using imaging time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) and principal components statistical analysis. Patterned NHS surface reactive zones are clearly resolved at high sensitivity despite the complexity of the polymer matrix chemistry. ToF‐SIMS imaging also reveals the presence of photo‐resist residue remaining from typical photolithography processing methods. High cross‐correlation between various ion‐derived ToF‐SIMS images is observed, providing sensitive chemical corroboration of pattern chemistry and biological reactivity in complex milieu. Surface‐specific protein coupling is observed first by site‐selective reaction of streptavidin with NHS patterns, followed by identical patterns of biotinylated Alexa‐labeled albumin coupling. This suggests that streptavidin immobilized on the patterns remains bioactive. Fluorescently labeled full serum is shown to react selectively with NHS‐reactive regions, with minimal signal from methoxy‐capped regions. Insufficient serum is adsorbed under any conditions to these surfaces to support cell attachment in serum‐containing media. This reflects the high intrinsic non‐adsorptive nature of this chemistry. Fibroblasts attach and proliferate in serum culture only when a cell adhesion peptide (RGD) is first grafted to NHS regions on the PEG‐based surfaces. Longer‐term serum‐based cell culture retains high cell‐pattern fidelity that correlates with chemical imaging of both the NHS and RGD patterns and also lack of cell adhesion to methoxy‐capped regions. Cell staining shows orientation of adherent cells within the narrow patterned areas. Cell patterns are consistently retained beyond 15 days in serum media.     

Nanostructured Polymer Brushes by UV‐Assisted Imprint Lithography and Surface‐Initiated Polymerization for Biological Functions

Functional polymer brush nanostructures are obtained by combining step‐and‐flash imprint lithography (SFIL) with controlled, surface‐initiated polymerization (CSIP). Patterning is achieved at length scales such that the smallest elements have dimensions in the sub‐100 nm range. The patterns exhibit different shapes, including lines and pillars, over large surface areas. The platforms obtained are used to selectively immobilize functional biomacromolecules. Acrylate‐based polymer resist films patterned by SFIL are first used for the selective immobilization of ATRP silane‐based initiators, which are coupled to unprotected domains of silicon substrates. These selectively deposited initiators are then utilized in the controlled radical SIP of poly(ethylene glycol)methacrylates (PEGMA). Nanostructured brush surfaces are then obtained by removal of the resist material. The areas previously protected by the SFIL resist are passivated by inert, PEG‐based silane monolayers following resist removal. PEGMA brush nanostructures are finally functionalized with biotin units in order to provide selective attachment points for streptavidin proteins. Atomic force microscopy and fluorescence spectroscopy confirm the successful immobilization of streptavidin molecules on the polymer grafts. Finally, it is demontrated that this fabrication method allows the immobilization of a few tens of protein chains attached selectively to brush nanostructures, which are surrounded by nonfouling PEG‐functionalized areas.     

Photoclickable Surfaces for Profluorescent Covalent Polymer Coatings

The nitrile imine‐mediated tetrazole‐ene cycloaddition reaction (NITEC) is introduced as a powerful and versatile conjugation tool to covalently ligate macromolecules onto variable (bio)surfaces. The NITEC approach is initiated by UV irradiation and proceeds rapidly at ambient temperature yielding a highly fluorescent linkage. Initially, the formation of block copolymers by the NITEC methodology is studied to evidence its efficacy as a macromolecular conjugation tool. The grafting of polymers onto inorganic (silicon) and bioorganic (cellulose) surfaces is subsequently carried out employing the optimized reaction conditions obtained from the macromolecular ligation experiments and evidenced by surface characterization techniques, including X‐ray photoelectron spectroscopy and FT‐IR microscopy. In addition, the patterned immobilization of variable polymer chains onto profluorescent cellulose is achieved through a simple masking process during the irradiation.     

Photochemical Generation of Light Responsive Surfaces

The preparation of patterned photoswitchable surfaces by employing the nitrile imine‐mediated tetrazole ene cycloaddition (NITEC) photoinduced reaction in the presence of dipolarophiles based on photoresponsive azobenzene moieties is reported. The dipolarophile used is a maleimide carrying either an azobenzene unit or a first generation dendron containing two azobenzene units. X‐ray photoelectron spectroscopy (XPS) is employed to analyze the functionalized silicon wafers, while time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) evidences the spatial control of the functionalization of the surface achieved by using a micropatterned shadow mask. Water contact angle measurements and optical inspection observing the behavior of a water droplet demonstrate the photoinduced change on wettability of the structured functionalized surfaces due to the reversible trans‐to‐cis isomerization of the azobenzene moities.     

Versatile Functionalization of the Micropatterned Hydrogel of Hyperbranched Poly(ether amine) Based on “Thiol‐yne” Chemistry

The functionalization of a hydrogel with target molecules is one of the key steps in its various applications. Here, a versatile approach is demonstrated to functionalize a micropatterned hydrogel, which is formed by “thiol‐yne” photo‐click reaction between the yne‐ended hyperbranched poly(ether amine) (hPEA‐yne) and thiol‐containing polyhedral oligomeric silsesquioxane (PEG‐POSS‐SH). By controlling the molar ratio between hPEA‐yne and PEG‐POSS‐SH, patterned hydrogels containing thiol or yne groups are obtained. A series of thiol‐based click chemistry such as “thiol‐epoxy”, “thiol‐halogen”, “thiol‐ene”, and “thiol‐isocyanate” are used to functionalize the thiol‐containing hydrogel (Gel‐1), while the yne‐containing hydrogel (Gel‐2) is functionalized through a typical copper‐catalysed alkyne‐azide reaction (CuAAC). FTIR, UV‐vis spectra and confocal laser scanning microscopy (CLSM) are used to trace these click reactions. Due to the selective adsorption to the hydrophilic dyes, the obtained patterned hydrogel of hPEA modified with fluorescence dye is further demonstrated in application for the recognition of guest molecules.     

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.     

Cover Picture: An Epoxy Photoresist Modified by Luminescent Nanocrystals for the Fabrication of 3D High‐Aspect‐Ratio Microstructures (Adv. Funct. Mater. 13/2007)

M. Lucia Curri and co‐workers report on p. 2009 an epoxy‐based negative tone photoresist that can be functionalized with red emitting CdSe@ZnS core/shell type nanocrystals and patterned by UV lithography. The 3D high aspect ratio of the microfabricated structures proves that lithographic properties of the functional nanocomposite are retained and the nanocrystals properties conveyed into the resist. The emitting nanocomposite represents a convenient model for material functionalization expandable to nanocrystals with different properties. An epoxy‐based negative‐tone photoresist, which is known as a suitable material for high‐aspect‐ratio surface micromachining, is functionalized with red‐light‐emitting CdSe@ZnS nanocrystals (NCs). The proper selection of a common solvent for the NCs and the resist is found to be critical for the efficient incorporation of the NCs in the epoxy matrix. The NC‐modified resist can be patterned by standard UV lithography down to micrometer‐scale resolution, and high‐aspect‐ratio structures have been successfully fabricated on a 100 mm scaled wafer. The “as‐fabricated”, 3D, epoxy‐based surface microstructures show the characteristic luminescent properties of the embedded NCs, as verified by fluorescence microscopy. This issue demonstrates that the NC emission properties can be conveniently conveyed into the polymer matrix without deteriorating the lithographic performance of the latter. The dimensions, the resolution, and the surface morphology of the NC‐modified‐epoxy microstructures exhibit only minor deviations with respect to that of the unmodified reference material, as examined by means of microscopic and metrologic investigations. The proposed approach of the incorporation of emitting and non‐bleachable NCs into a photoresist opens novel routes for surface patterning of integrated microsystems with inherent photonic functionality at the micro‐ and nanometer‐scale for light sensing and emitting applications.     

Affinity‐Based Protein Surface Pattern Formation by Ligand Self‐Selection from Mixed Protein Solutions

Photolithographically prepared surface patterns of two affinity ligands (biotin and chloroalkane) specific for two proteins (streptavidin and HaloTag, respectively) are used to spontaneously form high‐fidelity surface patterns of the two proteins from their mixed solution. High affinity protein‐surface self‐selection onto patterned ligands on surfaces exhibiting low non‐specific adsorption rapidly yields the patterned protein surfaces. Fluorescence images after protein immobilization show high specificity of the target proteins to their respective surface patterned ligands. Time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) imaging further supports the chemical specificity of streptavidin and HaloTag for their surface patterned ligands from mixed protein solutions. However, ToF‐SIMS did detect some non‐specific adsorption of bovine serum albumin, a masking protein present in excess in the adsorbing solutions, on the patterned surfaces. Protein amino acid composition, surface coverage, density, and orientation are important parameters that determine the relative ToF‐SIMS fragmentation pattern yields. ToF‐SIMS amino acid‐derived ion fragment yields summed to produce surface images can reliably determine which patterned surface regions contain bound proteins, but do not readily discriminate between different co‐planar protein regions. Principal component analysis (PCA) of these ToF‐SIMS data, however, improves discrimination of ions specific to each protein, facilitating surface protein pattern identification and image contrast.     

Photoinduced Tetrazole‐Based Functionalization of Off‐Stoichiometric Clickable Microparticles

We report the preparation of tetrazole‐containing step‐growth microparticles and the subsequent use of photoinduced nitrile imine‐mediated tetrazole‐ene cycloaddition (NITEC) reactions on the particles with spatiotemporal control. Microparticles with an average diameter of 4.1 µm and with inherent tetrazole‐ene dual functionality are prepared by a one‐pot off‐stoichiometric thiol‐Michael addition dispersion polymerization. The NITEC reaction is performed efficiently in the solid phase by UV irradiation, leading to the formation of fluorescent pyrozoline adducts, with an estimated quantum yield of 0.7. Particle concentration‐independent reaction kinetics are observed and full conversion is reached within 10 min of UV exposure at an intensity of 8 mW cm^?2. Temporal control is demonstrated with either UV or rooftop sunlight irradiation of variable duration. By using two‐photon writing with a laser centered around 700 nm wavelength, spatial control is demonstrated with micrometer‐scale resolution via surface patterning of the microparticles. Further, microparticles with exclusive tetrazole functionality are prepared by a one‐pot, two‐step thiol‐Michael addition dispersion polymerization. The NITEC reaction between tetrazole‐functional particles and acrylates in solution is examined at various tetrazole/alkene molar ratios, and a 10:1 excess of alkenes in solution is found necessary for efficient functionalization.     

Screening Mesenchymal Stem Cell Attachment and Differentiation on Porous Silicon Gradients

The profound effects that nanoscale surface topography exerts on cell behavior are highly relevant to the development of advanced biomaterials and to advances in tissue engineering and regenerative medicine. Here, an asymmetric anodization procedure is used to produce n‐type porous silicon (pSi) gradients with pore sizes ranging from tens to hundreds of nanometers in diameter and changes in the ridge nanoroughness from a few to tens nanometers. Rat mesenchymal stem cells (rMSCs) adhere poorly at the regions with small pore size but high ridge roughness. Cell adhesion is increased gradually towards the large pore size but low ridge roughness end of the pSi gradients. Surface topography influences cell differentiation, but not cell proliferation. Osteogenesis of rMSCs is enhanced by porous topography with a ridge roughness lower than 10 nm, while adipogenesis of rMSCs is enhanced on the entire pSi gradient compared with flat Si substrates. The results demonstrate that the gradient format allows in‐depth screening of surface parameters that are important for the control of mammalian cell behavior, thereby advancing the development of new and improved biomaterials for orthopaedic and tissue engineering applications.     

Enhanced Osteoblast Adhesion to Epoxide‐Functionalized Surfaces

As an alternative to expensive extracellular matrix (ECM) proteins generally applied as coatings in Petri dishes used for cell binding, an innovative system based on epoxide‐functionalized monolayers capable of protein binding is proposed. Since cells bind to material surfaces through proteins, protein‐binding surfaces should also promote cell binding. Here we investigate how the cell‐binding properties of an epoxide‐functionalized surface compares with ECM protein gel coated surfaces and tissue culture polystyrene control surfaces. Glass surfaces are functionalized with glycidoxypropyltriethoxysilane (GOPS), which results in an epoxide‐functionalized surface capable of binding proteins through an epoxide–amine reaction. Advancing contact angle measurements and atomic force microscopy measurements confirm the formation of a homogeneous GOPS monolayer. This monolayer is micropatterned with fluorescein‐labeled ECM protein gel by microcontact printing (µCP). Confocal laser scanning microscopy (CLSM) shows accurately transferred ECM protein gel micropatterns. Osteoblasts that are seeded on these micropatterned substrates show a clear preference for adhering to the epoxide‐functionalized areas. The morphology of these cultured osteoblasts is needle‐like with high aspect ratios. As controls, osteoblasts are cultured on GOPS‐functionalized surfaces, unstructured ECM protein gel surfaces, and tissue culture polystyrene (TCPS). The GOPS surfaces demonstrate a drastic increase in cell adhesion after 2 h, whilst the other tests show no adverse effects of this surface on the osteoblasts as compared to ECM and TCPS. CLSM shows healthy cell morphologies on each surface. It is demonstrated for the first time that epoxide groups outperform ECM protein gel in cell adhesion, thereby providing new routes for cost‐effective coatings that improve biocompatibility as well as exciting, new methodologies to control and direct cell adhesion.     

Porous Silicon Nanodiscs for Targeted Drug Delivery

There is a strong demand for techniques that allow the fabrication of biocompatible porous nanoparticles for drug delivery applications. In this work, a new method to fabricate size‐ and shape‐controlled porous silicon (pSi) nanodiscs is described. The process relies on a combination of colloidal lithography and metal‐assisted chemical etching. Height and diameter of the pSi nanodiscs can be easily adjusted. The nanodiscs are degradable in physiological milieu and are nontoxic to mammalian cells. In order to highlight the potential of the pSi nanodiscs in drug delivery, an in vitro investigation that involved loading of nanodiscs with the anticancer agent camptothecin and functionalization of the nanodisc periphery with an antibody that targets receptors on the surface of neuroblastoma cells is carried out. The thus‐prepared nanocarriers are found to selectively attach to and kill cancer cells.     

Facile Preparation of a Patterned,Aminated Polymer Surface by UV‐Light‐Induced Surface Aminolysis

Introducing amine functional groups on polymer surfaces is extremely important for studying various processes that involve polymer surfaces. We report a novel and extremely simple method for preparing a tertiary‐amine‐terminated poly(ethylene terephthalate) (PET) surface by using a UV‐light‐induced surface aminolysis reaction. X‐ray photoelectron spectroscopy and attenuated total‐reflection infrared spectroscopy give direct evidence of the incorporation of tertiary amine functionalities and the possible reaction mechanism behind this technique. Tertiary amines are easily protonated, so we have developed an extremely simple method for immobilizing and patterning biomolecules on a soft surface by the electrostatic self‐assembly of proteins, such as immunoglobulin (IgG) and horseradish peroxide (HRP), onto a patterned, aminated surface. An enzyme–substrate reaction, which is followed optically by observing the resulting precipitation on the surface, is used to reveal the patterned immobilization of HRP, where 3‐amino‐9‐ethylcarbazole, as a substrate for HRP, is deposited on the aminated surface after HRP adsorption. Fluorescein isothiocyanate‐labeled IgG (FITC‐IgG) has been immobilized electrostatically onto the ordered aminated spots, and the fluorescence intensity ratio of the IgG‐immobilized region (inside the spot) to the background (outside the spot) is about 5:1, as calculated from a fluorescence image and fluorescence spectra obtained by microlaser confocal Raman spectroscopy. We have found that the background intensity is mainly caused by the autofluorescence of virgin PET, and after subtracting this value from the measured intensity inside and outside the spot, respectively, a much higher intensity ratio between the spot and the background is obtained (about 22:1). The patterned immobilization of FITC‐IgG has been further proven by examining the change in intensity inside the spot after photobleaching the fluorophore.     

Biofunctional Polyelectrolyte Multilayers and Microcapsules: Control of Non‐Specific and Bio‐Specific Protein Adsorption

Layers of the polyelectrolytes poly(allylamine hydrochloride) (PAH, polycationic) and poly(styrene sulfonate) (PSS, polyanionic) are consecutively adsorbed on flat silicon oxide surfaces, forming stable, ultrathin multilayer films. Subsequently, a final monolayer of the polycationic copolymer poly(L ‐lysine)‐graft‐poly(ethylene glycol) (PLL‐g‐PEG) is adsorbed onto the PSS‐terminated multilayer in order to impart protein resistance to the surface. The growth of each of the polyelectrolyte layers and the protein resistance of the resulting [PAH/PPS]_n(PLL‐g‐PEG) multilayer (n = 1–4) are followed quantitatively ex situ using X‐ray photoelectron spectroscopy and in situ using real‐time optical‐waveguide lightmode spectroscopy. In a second approach, the same type of [PAH/PSS]_n(PLL‐g‐PEG) multilayer coatings are successfully formed on the surface of colloidal particles in order to produce surface‐functionalized, hollow microcapsules after dissolution of the core materials (melamine formaldehyde (MF) and poly(lactic acid) (PLA; colloid diameters: 1.2–20 μm). Microelectrophoresis and confocal laser scanning microscopy are used to study multilayer formation on the colloids and protein resistance of the final capsule. The quality of the PLL‐g‐PEG layer on the microcapsules depends on both the type of core material and the dissolution protocols used. The greatest protein resistance is achieved using PLA cores and coating the polyelectrolyte microcapsules with PLL‐g‐PEG after dissolution of the cores. Protein adsorption from full serum on [PAH/PPS]_n(PLL‐g‐PEG) multilayers (on both flat substrates and microcapsules) decreases by three orders of magnitude in comparison to the standard [PAH/PPS]_n layer. Finally, biofunctional capsules of the type [PAH/PPS]_n(PLL‐g‐PEG/PEG‐biotin) (top copolymer layer with a fraction of the PEG chains end‐functionalized with biotin) are produced which allow for specific recognition and immobilization of controlled amounts of streptavidin at the surface of the capsules. Biofunctional multilayer films and capsules are believed to have a potential for future applications as novel platforms for biotechnological applications such as biosensors and carriers for targeted drug delivery.     

Lewis Acid Induced Functionalization of Photoluminescent 2D Silicon Nanosheets for the Fabrication of Functional Hybrid Films

Various Lewis acids are found to efficiently catalyze hydrosilylation reactions of hydride‐terminated 2D silicon nanosheets at room temperature. The hydride‐terminated nanosheets can be functionalized with a variety of unsaturated functional substrates and still possess their unique characteristic (opto)electronic properties (e.g., photoluminescence). This is demonstrated by successfully implementing the readily functionalized materials into new silicon/semiconducting polymer‐based field‐effect transistors (FETs). Surface modification of the freestanding silicon nanosheets opens new possibilities to form highly homogeneous blends with the already broadly used conventional polymers poly(3‐hexylthiophene‐2,5‐diyl). The consequential combination of the different properties of the materials enables the enhancement of the sensitivity of the solution‐gated FETs and increases the transconductance of the operating device.     

On‐the‐Spot Immobilization of Quantum Dots,Graphene Oxide,and Proteins via Hydrophobins

Class I hydrophobin Vmh2, a peculiar surface active and versatile fungal protein, is known to self‐assemble into chemically stable amphiphilic films, to be able to change wettability of surfaces, and to strongly adsorb other proteins. Herein, a fast, highly homogeneous and efficient glass functionalization by spontaneous self‐assembling of Vmh2 at liquid–solid interfaces is achieved (in 2 min). The Vmh2‐coated glass slides are proven to immobilize not only proteins but also nanomaterials such as graphene oxide (GO) and quantum dots (QDs). As models, bovine serum albumin labeled with Alexa 555 fluorophore, anti‐immunoglobulin G antibodies, and cadmium telluride QDs are patterned in a microarray fashion in order to demonstrate functionality, reproducibility, and versatility of the proposed substrate. Additionally, a GO layer is effectively and homogeneously self‐assembled onto the studied functionalized surface. This approach offers a quick and simple alternative to immobilize nanomaterials and proteins, which is appealing for new bioanalytical and nanobioenabled applications.     

Hydrogel−Solid Hybrid Materials for Biomedical Applications Enabled by Surface‐Embedded Radicals

Combinations of hydrogels and solids provide high level functionality for devices such as tissue engineering scaffolds and soft machines. However, the weak bonding between hydrogels and solids hampers functionality. Here, a versatile strategy to develop mechanically robust solid?hydrogel hybrid materials using surface embedded radicals generated through plasma immersion ion implantation (PIII) of polymeric surfaces is reported. Evidence is provided that the reactive radicals play a dual role: inducing surface‐initiated, spontaneous polymerization of hydrogels; and binding the hydrogels to the surfaces. Acrylamide and silk hydrogels are formed and covalently attached through spontaneous reactions with the radicals on PIII activated polymer surfaces without cross‐linking agents or initiators. The hydrogel amount increases with incubation time, monomer concentration, and temperature. Stability tests indicate that 95% of the hydrogel is retained even after 4 months in PBS solution. T‐peel tests show that failure occurs at the tape?hydrogel interface and the hydrogel‐PIII‐treated PTFE interfacial adhesion strength is over 300 N m^?1. Cell assays show no adhesion to the as‐synthesized hydrogels; however, hydrogels synthesized with fibronectin enable cell adhesion and spreading. These results show that polymers functionalized with surface‐embedded radicals provide excellent solid platforms for the generation of robust solid?hydrogel hybrid structures for biomedical applications.     

A Solution‐Processed UV‐Sensitive Photodiode Produced Using a New Silicon Nanocrystal Ink

This article presents a simple and effective method of functionalizing hydrogen‐terminated silicon (Si) nanocrystals (NCs) to form a high‐quality colloidal Si NC ink with short ligands that allow charge transport in nanocrystal solid films. Si NCs fabricated by laser‐pyrolysis and acid etching are passivated with allyl disulfide via ultraviolet (UV)‐initiated hydrosilylation to form a stable colloidal Si NC ink. Then a Si NC‐based photodiode is directly fabricated in air from this ink. Only a solution‐processed poly(3,4‐ethylenedioxy‐thiophene):poly(styrene sulfonate) (PEDOT: PSS) electron blocking layer and top‐ and bottom‐contacts are needed along with the Si NC layer to construct the device. A Schottky‐junction at the interface between the Si NC absorber layer and aluminum (Al) back electrode drives charge separation in the device under illumination. The unpackaged Si NC‐based photodiode exhibites a peak photoresponse of 0.02 A W^?1 to UV light in air, within an order of magnitude of the response of commercially available gallium phosphide (GaP), gallium nitride (GaN), and silicon carbide (SiC) based photodetectors. This provides a new pathway to large‐area, low‐cost solution‐processed UV photodetectors on flexible substrates and demonstrates the potential of this new silicon nanocrystal ink for broader applications in solution‐processed optoelectronics.     

Highly Ordered Nanometer‐Scale Chemical and Protein Patterns by Binary Colloidal Crystal Lithography Combined with Plasma Polymerization

Surfaces with micro‐ and nanometer‐scale patterns have many potential applications, particularly in lifescience. This article reports on a versatile, straightforward, and inexpensive approach for the creation of chemical patterns using fabricated binary colloid crystals, consisting of small and large particles, as masks for the deposition of an amino‐functionalised ultrathin film by plasma polymerization. After removal of the binary colloidal mask, the characterization techniques [scanning electron microscopy (SEM) and atomic force microscopy (AFM)] reveal a surface contrast that depicts an ability of the small particles to allow diffusion of the plasma to the substrate. A plasma‐polymer film is created under the small particles and the region of substrate in direct contact with the large particle remains uncoated. Numerous types of patterns and feature heights can be produced with good fidelity over areas of several cm² by appropriate tuning of the binary colloid crystal mask morphology and the plasma‐polymer deposition time. Finally, the amine groups of the patterned surface are used for covalent grafting poly(ethylene glycol) propionaldehyde (PEG‐PALD) by reductive amination under conditions of reduced solubility to produce a patterned surface for directed adsorption of protein. AFM investigations show that the proteins are preferentially attached to the nanometer‐scale regions of the pattern without PEG‐PALD.     

Fabrication of Robust Biomolecular Patterns by Reactive Microcontact Printing on N‐Hydroxysuccinimide Ester‐Containing Polymer Films

The fabrication of robust biomolecule microarrays by reactive microcontact printing (μCP) on spin‐coated thin films of poly(N‐hydroxysuccinimidyl methacrylate) (PNHSMA) on oxidized silicon and glass is described. The approach combines the advantages of activated polymer thin films as coupling layers, characterized by high reactivity and high molecular loading, with the versatility and flexibility of soft lithography. The transfer of amino end‐functionalized poly(ethylene glycol) (PEG) from oxidized poly(dimethylsiloxane) elastomer stamps to PNHSMA films is shown by Fourier transform infrared spectroscopy, X‐ray photoelectron spectroscopy, fluorescence microscopy, and ellipsometry measurements to result in covalent coupling and identical grafting densities, as found previously for coupling from solution. The PEG‐protected areas effectively inhibit the adsorption of fluoresceinamine, bovine serum albumin, as well as 25‐mer DNA, while the unreacted N‐hydroxysuccinimidyl methacrylate ester groups retain their reactivity towards primary amino groups. Biomolecule microarrays have been thus conveniently fabricated in a two‐step procedure. The hybridization of target DNA to immobilized probe DNA in micropatterns proves the concept of reactive μCP on activated polymer films for obtaining robust platforms for biomolecule immobilization and screening.