Protein Micropatterns Research Articles

Spatially well-defined binary brushes of poly(ethylene glycol)s for micropatterning of active proteins on anti-fouling surfaces

We report a novel method for micropatterning of active proteins on anti-fouling surfaces via spatially well-defined and dense binary poly(ethylene glycol)s (PEGs) brushes with controllable protein-docking sites. Binary brushes of poly(poly(ethylene glycol) methacrylate- co-poly(ethylene glycol)methyl ether methacrylate), or P(PEGMA- co-PEGMEMA), and poly(poly(ethylene glycol)methyl ether methacrylate), or P(PEGMEMA), were prepared via consecutive surface-initiated atom transfer radical polymerizations (SI-ATRPs) from a resist-micropatterned Si(1 0 0) wafer surface. The terminal hydroxyl groups on the side chains of PEGMA units in the P(PEGMA- co-PEGMEMA) microdomains were activated directly by 1,1′-carbonyldiimidazole (CDI) for the covalent coupling of human immunoglobulin (IgG) (as a model active protein). The resulting IgG-coupled PEG microdomains interact only and specifically with target anti-IgG, while the other PEG microregions effectively prevent specific and non-specific protein fouling. When extended to other active biomolecules, microarrays for specific and non-specific analyte interactions with a high signal-to-noise ratio could be readily tailored.

Use of Photolithography to Encode Cell Adhesive Domains into Protein Microarrays

Protein microarrays are rapidly emerging as valuable tools in creating combinatorial cell culture systems where inducers of cellular differentiation can be identified in a rapid and multiplexed fashion. In the present study, protein microarraying was combined with photoresist lithography to enable printing of extracellular matrix (ECM) protein arrays while precisely controlling "on-the-spot" cell-cell interactions. In this surface engineering approach, the micropatterned photoresist layer formed on a glass substrate served as a temporary stencil during the microarray printing, defining the micrometer-scale dimensions and the geometry of the cell-adhesion domains within the printed protein spots. After removal of the photoresist, the glass substrates contained micrometer-scale cell-adhesive regions that were encoded within 300 or 500 microm diameter protein domains. Fluorescence microscopy and atomic force microscopy (AFM) were employed to characterize protein micropatterns. When incubated with micropatterned surfaces, hepatic (HepG2) cells attached on 300 or 500 mum diameter protein spots; however, the extent of cell-cell contacts within each spot varied in accordance with dimensions of the photoresist stencil, from single cells attaching on 30 microm diameter features to multicell clusters residing on 100 or 200 microm diameter regions. Importantly, the photoresist removal process was shown to have no detrimental effects on the ability of several ECM proteins (collagens I, II, and IV and laminin) to support functional hepatic cultures. The micropatterning approach described here allows for a small cell population seeded onto a single cell culture substrate to be exposed to multiple scenarios of cell-cell and cell-surface interactions in parallel. This technology will be particularly useful for high-throughput screening of biological stimuli required for tissue specification of stem cells or for maintenance of differentiated phenotype in scarce primary cells.

Micropatterning of proteins on the surface of three-dimensional poly(ethylene glycol) hydrogel microstructures

This paper describes micropatterning of proteins on the surface of three-dimensional hydrogel microstructures. Poly(ethylene glycol) (PEG)-based hydrogel microstructures were fabricated on a glass substrate using a poly(dimethylsiloxane) (PDMS) replica as a molding insert and photolithography. The lateral dimension and height of the hydrogel microstructures were easily controlled by the feature size of the photomask and depth of the PDMS replica, respectively. Bovine serum albumin (BSA), a model protein, was covalently immobilized to the surface of the hydrogel microstructure via a 5-azidonitrobenzoyloxy N-hydroxysuccinimide bifunctional linker at a surface density of 1.48 mg cm −2. The immobilization of BSA on the PEG hydrogel surface was demonstrated with XPS by confirming the formation of a new nitrogen peak, and the selective immobilization of fluorescent-labeled BSA on the outer region of the three-dimensional hydrogel micropattern was demonstrated by fluorescence. A hydrogel microstructure could immobilize two different enzymes separately, and sequential bienzymatic reaction was demonstrated by reacting glucose and Amplex Red with a hydrogel microstructure where glucose oxidase was immobilized on the surface and peroxidase was encapsulated. Activity of immobilized glucose oxidase was 16.5 U mg −1, and different glucose concentration ranged from 0.1 to 20 mM could be successfully detected.

Use of hydrogel microstructures as templates for protein immobilization

AbstractIn this study, protein micropatterns were created on the surface of three-dimensional hydrogel microstructures. Poly(ethylene glycol)(PEG)-based hydrogel microstructures were fabricated on a glass substrate using a poly(dimethylsiloxane) (PDMS) replica as a molding insert and photolithography. The lateral dimension and height of the hydrogel microstructures were easily controlled by the feature size of the photomask and depth of the PDMS replica, respectively. Bovine serum albumin (BSA), a model protein, was covalently immobilized to the surface of the hydrogel microstructure via a 5-azidonitrobenzoyloxy N-hydroxysuccinimide bifunctional linker, which has a phenyl azide group and a protein-binding N-hydroxysuccinimide group on either end. The immobilization of BSA on the PEG hydrogel surface was demonstrated with XPS by confirming the formation of a new nitrogen peak, and the selective immobilization of fluorescent-labeled BSA on the outer region of the three-dimensional hydrogel micropattern was demonstrated by fluorescence. A hydrogel microstructure could immobilize two different enzymes separately, and sequential bienzymatic reaction was demonstrated by reacting glucose and Amplex Red with a hydrogel microstructure where glucose oxidase was immobilized on the surface and peroxidase was encapsulated.

Photopatterned Surfaces for Site-Specific and Functional Immobilization of Proteins

Photosensitive silanes containing nitroveratryl (Nvoc)-caged amine groups and protein repellent tetraethylene glycol units were synthesized and used for modification of silica surfaces. Functional surface layers containing different densities of caged amine groups were prepared and activated by UV-irradiation of the surface. The performance of these layers for functional and site-selective immobilization of proteins was tested. For this purpose, biotin and tris-nitrilotriacetic acid (tris-NTA) were fist coupled to the activated surface, and the interaction of streptavidin and His-tagged proteins with the functionalized surfaces was monitored by real-time label-free detection. After optimizing the coupling protocols, highly selective functionalization of the deprotected amine groups was possible. Furthermore, the degree of functionalization (and therefore the amount of immobilized protein) was controlled by diluting the surface concentration of the amine-functionalized silane with a nonreactive (OMe-terminated) tetraethylene glycol silane. Immobilized proteins were highly functional on these surfaces, as demonstrated by protein-protein interaction assays with the type I interferon receptor. Protein micropatterns were successfully generated after masked irradiation and functionalization of the caged surface following the optimized coupling protocols.

The effects of proteoglycan surface patterning on neuronal pathfinding.

Protein micropatterning techniques are increasingly applied in cell choice assays to investigate fundamental biological phenomena that contribute to the host response to implanted biomaterials, and to explore the effects of protein stability and biological activity on cell behavior for in vitro cell studies. In the area of neuronal regeneration the protein micropatterning and cell choice assays are used to improve our understanding of the mechanisms directing nervous system during development and regenerative failure in the central nervous system (CNS) wound healing environment. In these cell assays, protein micropatterns need to be characterized for protein stability, bioactivity, and spatial distribution and then correlated with observed mammalian cell behavior using appropriate model system for CNS development and repair. This review provides the background on protein micropatterning for cell choice assays and describes some novel patterns that were developed to interrogate neuronal adaptation to inhibitory signals encountered in CNS injuries.

Protein patterning on self-assembled polyelectrolyte thin films

This study demonstrated a simple micropatterning method using engineered surface and microcontact printing (μCP). For the selection of appropriate surface, several representative surfaces modified with various functional materials including aldehyde, epoxide, poly- l-lysine, amine, and self-assembled polyelectrolyte multilayers (PEL) were investigated. The PEL coated surface endowing electrostatic interaction force showed most high functionality in point of homogeneous patterning of proteins with high density and preservation of inherent three-dimensional structure of proteins. The printed sizes of protein micropatterns were almost identical to the used microstamp, which proves the μCP method is independent of diffusion of the transferring proteins. At last, the immunoassay as a model system of protein–protein interaction showed good linearity over a range from 100 to 25 μg/ml, indicating the feasibility of a quantitative measurement of the concentration of target proteins in sample. Our proposed approach will be useful for the development of biomedical microdevices because of green chemistry, simple process, and high potential of various applications.

Preparation of micropatterned hydrogel substrate via surface graft polymerization combined with photolithography for biosensor application

This paper presents a simple method to create protein micropattern on the poly(ethylene glycol) (PEG) hydrogels through the surface graft polymerization and photolithography. The modification of the protein-repellent PEG hydrogel surface was achieved by a two-step process using immobilization of benzophenone on the PEG hydrogel as surface initiator and subsequent surface-initiated polymerization of acrylic acid by UV irradiation. Surface modification of PEG hydrogels was demonstrated with FTIR/ATR spectroscopy and XPS by confirming the presence of carboxyl groups in the poly(acrylic acid) (PAA). The photograft polymerization through the designed photomask produced well-defined, pH-responsive PAA micropatterns with diameters ranging from 50 to 300 μm on the PEG hydrogels. The size of PAA micropatterns was controlled by changing the environmental pH, such that a 300 μm diameter and 17 μm thick PAA micropattern at pH 4 swelled to 480 μm diameter and 80 μm thick at pH 7. Activation of the carboxyl groups in PAA allowed covalent immobilization of proteins only on the PAA micropatterns due to the nonadhesivity of PEG. Based on these results, biotin was micropatterned on the PEG hydrogels and binding of streptavidin was qualitatively and quantitatively investigated, demonstrating the possibility of micropatterned PEG hydrogels for various biosensor systems.

Optimal Micropattern Dimensions Enhance Neurite Outgrowth Rates, Lengths, and Orientations

Micropattern dimensions can significantly influence neurite outgrowth orientation, rate, and length. Laminin micropatterns of various widths from 10 to 50 microm at 10 microm intervals separated by 40 microm spaces were generated on poly(methyl methacrylate) surfaces using microscale plasma-initiated patterning (microPIP). Dissociated dorsal root ganglion (DRG) neurons were seeded on the micropatterned surfaces and cultured for 24 h in serum-free media. Neurite outgrowth numbers, lengths, rates, and orientations were measured on all micropatterned substrates. The results indicated that the dimension of the laminin pattern influenced the neurite outgrowth length, rate, and orientation, but not the numbers of neurite outgrowth. Neurons on more than 30 microm wide laminin pattern showed faster neurite outgrowth compared to other dimensions, and relatively low orientation at 50 microm pattern dimensions. Neurites at 40 microm laminin pattern widths demonstrated the fastest outgrowth rates and were highly oriented. The 40 microm laminin dimension is wide enough to provide sufficient laminin amounts for neuron growth and narrow enough to efficiently guide neurites. Based on these results, adhesive protein micropatterns of 40 microm dimensions are recommended when investigating DRG neurons.

Protein Micropatterning on Bifunctional Organic−Inorganic Sol−Gel Hybrid Materials

Active protein micropatterns and microarrays made by selective localization are popular candidates for medical diagnostics, such as biosensors, bioMEMS, and basic protein studies. In this paper, we present a simple fabrication process of thick (approximately 20 microm) protein micropatterning using capillary force lithography with bifunctional sol-gel hybrid materials. Because bifunctional sol-gel hybrid material can have both an amine function for linking with protein and a methacryl function for photocuring, proteins such as streptavidin can be immobilized directly on thick bifunctional sol-gel hybrid micropatterns. Another advantage of the bifunctional sol-gel hybrid materials is the high selective stability of the amine group on bifunctional sol-gel hybrid patterns. Because amine function is regularly contained in each siloxane oligomers, immobilizing sites for streptavidin are widely distributed on the surface of thick hybrid micropatterns. The micropatterning processes of active proteins using efficient bifunctional sol-gel hybrid materials will be useful for the development of future bioengineered systems because they can save several processing steps and reduce costs.

Micropatterning of Proteins on 3D Porous Polymer Film Fabricated by Using the Breath‐Figure Method

Micropatterning of Proteins on 3D Porous Polymer Film Fabricated by Using the Breath‐Figure Method

A novel approach to the micropatterning of proteins using dewetting of polymer bilayers

The ability to control protein and cell positioning on a microscopic scale is crucial in many biomedical and bioengineering applications, such as tissue engineering and the development of biosensors. We propose here a novel, simple, and versatile method for the micropatterning of proteins. Micropatterned substrates are produced by the dewetting of a metastable polymer film on top of another polymer film. Selective adsorption, or micropatterning, of proteins can be achieved on such substrates by choosing pairs of polymers which differ in protein affinity. In this study, patterns were produced in bilayers of poly(methylmethacrylate) (PMMA) and polystyrene (PS), and of PMMA and octadecyltrichlorosilane (OTS). Fluorescence microscopy and atomic force microscopy (AFM) provide evidence that model proteins adsorb preferentially on isolated bio-adhesive (PS and OTS) micropatches in a protein-resistant (PMMA) matrix. "Inverse" protein patterns, containing non-adhesive (PMMA) islands in a protein-adhesive (PS) matrix can also be produced. Such micropatterned substrates could potentially be used in the development of biosensors and bioassays, and in the study of cell growth and motility.

On-Chip Micropatterning of Plastic (Cylic Olefin Copolymer, COC) Microfluidic Channels for the Fabrication of Biomolecule Microarrays Using Photografting Methods

This paper reports on the surface modification of plastic microfluidic channels to prepare different biomolecule micropatterns using ultraviolet (UV) photografting methods. The linkage chemistry is based upon UV photopolymerization of acryl monomers to generate thin films (0.01-6 microm) chemically linked to the organic backbone of the plastic surface. The commodity thermoplastic, cyclic olefin copolymer (COC) was selected to build microfluidic chips because of its significant UV transparency and easiness for microfabrication by molding techniques. Once the polyacrylic films were grafted on the COC surface using photomasks, micropatterns of proteins, DNA, and biotinlated conjugates were readily obtained by surface chemical reactions in one or two subsequent steps. The thickness of the photografted films can be tuned from several nanometers up to several micrometers, depending on the reaction conditions. The micropatterned films can be prepared inside the microfluidic channel (on-chip) or on open COC surfaces (off-chip) with densities of functional groups about 10(-7) mol/cm2. Characterization of these films was performed by attenuated-total-reflectance IR spectroscopy, fluorescence microscopy, profilometry, atomic force microscopy, and electrokinetic methods.

Neurite guidance on protein micropatterns generated by a piezoelectric microdispenser

In this study, we developed a microdispenser technique in order to create protein patterns for guidance of neurites from cultured adult mouse dorsal root ganglia (DRG). The microdispenser is a micromachined silicon device that ejects 100 picolitre droplets and has the ability to position the droplets with a precision of 6–8 μm. Laminin and bovine serum albumin (BSA) was used to create adhesive and non-adhesive protein lines on polystyrene surfaces (cell culture dishes). Whole-mounted DRGs were then positioned close to the patterns and neurite outgrowth was monitored. The neurites preferred to grow on laminin lines as compared to the unpatterned plastic. When patterns were made from BSA the neurites preferred to grow in between the lines on the unpatterned plastic surface. We conclude that microdispensing can be used for guidance of sensory neurites. The advantages of microdispensing is that it is fast, flexible, allows deposition of different protein concentrations and enables patterning on delicate surfaces due to its non-contact mode of operation. It is conceivable that microdispensing can be utilized for the creation of protein patterns for guiding neurites to obtain in vitro neural networks, in tissue engineering or rapid screening for guiding proteins.

Protein and cell micropatterning and its integration with micro/nanoparticles assembly

Micropatterning of proteins and cells has become very popular over the past decade due to its importance in the development of biosensors, microarrays, tissue engineering and cellular studies. This article reviews the techniques developed for protein and cell micropatterning and its biomedical applications. The prospect of integrating micro and nanoparticles with protein and cell micropatterning is discussed. The micro/nanoparticles are assembled into patterns and form the substrate for proteins and cell attachment. The assembled particles create a micro or nanotopography, depending on the size of the particles employed. The nonplanar structure can increase the surface area for biomolecules attachment and therefore enhance the sensitivity for detection in biosensors. Furthermore, a nanostructured substrate can influence the conformation and functionality of protein attached to it, while cellular response in terms of morphology, adhesion, proliferation, differentiation, etc. can be affected by a surface expressing micro or nanoscale structures. Proteins and cells tend to lose their normal functions upon attachment to substrate. By recognizing the types of topography that are favourable for preserving proteins and cell behaviour, and integrating it with micropattering will lead to the development of functional protein and cell patterns.

Micropatterning of proteins on nanospheres

Currently micropatterning of proteins is mainly carried out on a planar substrate, which involves multi-step surface modifications directly on the substrate. Efficiency of chemical reactions is usually low, resulting in low signal-to-noise (S/N) ratio and poor repeatability of results. Here we presented a micropatterning method using polystyrene nanospheres with non-planar surface as a solid support for attaching proteins, which introduces many advantages. The patterning of proteins was carried out in two approaches: one was to dispense polystyrene nanospheres into an array of microwells and then attach proteins onto the nanospheres, and another was to coat polystyrene nanospheres with proteins first and then deposit the spheres into the microwells. For both approaches, a uniform pattern of proteins was generated. The amount of proteins attached via nanospheres was much higher than that on planar surface.

Nanoparticle-assisted micropatterning of active proteins on solid substrate

Micropatterning of proteins on silica substrate was achieved using a new method. Proteins were first immobilized onto silica nanoparticles which were then dispensed into arrayed microwells on silicon. Atomic force microscopy (AFM), fluorescence microscopy and Fourier transform infrared (FTIR) spectroscopy were used to characterize the samples. The results showed that, compared to a planar surface, curved surfaces of nanoparticles provide more space for attaching proteins and thus increases the intensity of fluorescence signal. Furthermore, after attaching to silica nanoparticles, bovine serum albumin (BSA) maintains its major structure and the cytokine IFN-gamma maintains its ability to bind to its antibody. Use of this method can be extended to micropatterning of other biomolecules, such as DNA and enzymes.

Protein Micropatterns Using a pH-Responsive Polymer and Light

Protein and peptide microarrays are popular candidates for medical diagnostics because of the possibility for high sensitivity and simultaneous marker screening. To realize the potential of these arrays, new strategies for ligand patterning are needed. We report a method for patterning proteins that utilizes a pH-responsive polymer, deep ultraviolet (DUV) light, and a photoacid generator (PAG). Poly(3,3'-diethoxypropyl methacrylate) (PDEPMA) contains reactive acetal side chains which are converted to aldehydes following treatment with acid. PDEPMA was spin-coated onto Si-SiO(2) substrates and was either chemically deprotected with 1 M HCl or photochemically deprotected by exposure to DUV in the presence of triphenylsulfonium triflate. Conversion to aldehyde groups was confirmed with Purpald and by reaction with a green fluorescent hydroxylamine. Protein microarrays were demonstrated by incubating photochemically patterned surfaces with an aldehyde-reactive biotin followed by red fluorescent streptavidin. This methodology provides a new substrate for the precise patterning of both peptides and proteins for various biological applications including medical sensors.

Protein Micropatterning Using Surfaces Modified by Self-Assembled Polystyrene Microspheres

A technique for micropatterning of proteins on a nonplanar surface to improve the coverage and functionality of biomolecules is demonstrated. A nonplanar microstructure is created by the self-assembly of polystyrene microspheres into an array of microwells on a silicon wafer to allow the integration of a nonplanar spot on a planar chip. After the microspheres were deposited into the microwells, they were conjugated with proteins. The curve surfaces of the microspheres present more surface area for attaching biomolecules which will increase the density of biomolecules and, hence, the sensitivity for detection. Moreover, proteins immobilized on a curved surface can retain their native structures and function better than on a planar surface because of a smaller area of interaction between the protein and the substrate. Patterning of biomolecules was tested with two model fluorescent proteins. The results show that precise patterning of biomolecules on a nonplanar spot can be achieved with this technique.

Microcontact printing of multiproteins on the modified mica substrate and study of immunoassays

Microcontact printing (µCP) is an easy and efficient way of producing patterns of self-assembled monolayers (SAM) containing different functional groups. We have developed a simple and convenient µCP-based technique for the modification of a mica substrate with 3-aminopropyltriethoxysilane (APTES) and the micropatterning of proteins (chicken IgG and rabbit IgG) on the modified mica surface. Our approach provides a quick and easy way to produce protein patterns on solid surfaces. The printed immunoglobulin patterns were detected by exposing the substrate to solutions containing fluorescently labeled complementary anti-IgGs, and the formed immunoassays were studied using fluorescence microscopy. Copyright © 2005 John Wiley & Sons, Ltd.