Low Nonspecific Adsorption Research Articles

Performance of Protein-A-Based Affinity Membranes for Antibody Purification

The preparation of affinity membranes for application in antibody purification studies is described here. Protein-A-attached poly(hydroxyethyl methacrylate-N-methacryloyl-L-alanine) (PHEMAAL) membranes were produced by a photopolymerization technique and then characterized by swelling tests, surface area measurements, contact angle and scanning electron microscopy (SEM) studies. The water swelling ratio of the PHEMAAL membrane was 133.2%. PHEMAAL membranes have large pores with a size in the range of 5–10 μm. Protein A was covalently attached onto the PHEMAAL membranes via cyanogen bromide (CNBr) activation. Maximum protein A loading was 4.7 mg/g. There was a very low non-specific IgG adsorption onto the PHEMAAL membranes, about 0.38 mg/g. The maximum IgG adsorption on the PHEMAAL–protein A membrane was found to be 9.8 mg/g at pH 7.4 from aqueous solutions. Higher adsorption amount was observed from human plasma (up to 37.3 mg/g). Adsorbed IgG was eluted using 0.1 M glycine-HCl buffer (pH 3.5) with a purity of 93%. PHEMAAL–protein A membrane was used for repetitive adsorption/elution of IgG without noticeable loss in IgG adsorption amount after 10 cycles. The PHEMAAL–protein A membrane showed several advantages, such as simpler preparation procedure, good selectivity for IgG purification from human plasma and good stability throughout repeated adsorption–elution cycles.

Improvement of Accuracy in Flow Immunosensor System by Introduction ofPoly-2-[3-(methacryloylamino)propylammonio]ethyl 3-aminopropyl Phosphate

In order to improve the accuracy of immunosensor systems,poly-2-[3-(methacryloylamino)propylammonio]ethyl 3-aminopropyl phosphate (poly-3MAm3AP), which includes both phosphorylcholine and amino groups, was synthesized and applied to the preparation of antibody-immobilized beads. Acting as an antibody-immobilizing material,poly-3MAm3AP is expected to significantly lower nonspecific adsorption due to the presence of the phosphorylcholine group and recognize large numbers of analytes due to the increase in antibody-immobilizing sites. The elimination of nonspecific adsorption was compared between the formation of a blocking layer on antibody-immobilized beads and the introduction of a material to combine antibody with beads. Determination with specific and nonspecific antibodies was then investigated for the estimation of signal-to-noise ratio. Signal intensities with superior signal-to-noise ratios were obtained whenpoly-3MAm3AP was introduced. This may be due to the increase in antibody-immobilizing sites and the extended space for antigen-antibody interaction resulting from the electrostatic repulsion ofpoly-3MAm3AP. Thus, the application ofpoly-3MAm3AP coatings to immunoassay beads was able to improve the accuracy of flow immunosensor systems.

Silanization of silica and glass slides for DNA microarrays by impregnation and gas phase protocols: A comparative study

Surface immobilization of oligonucleotide probes (oligoprobes) is a key issue in the development of DNA-chips. The immobilization protocol should guarantee good availability of the probes, low non-specific adsorption and reproducibility. We have previously reported a silanization protocol with tert-butyl-11-(dimethylamino)silylundecanoate performed by impregnation (Impregnation Protocol, IP) of silica substrates from dilute silane solutions, leading to surfaces bearing carboxylic groups. In this paper, the Impregnation protocol is compared with a Gas phase Protocol (GP) which is more suited to industrial requirements such as reliable and robust processing, cost efficiency, etc.… The morphology of the oligoprobe films at the nanoscale (characterized by Atomic Force Microscopy) and the reproducibility of subsequent oligoprobes immobilization steps have been investigated for the two protocols on thermal silica (Si/SiO2) and glass slide substrates. IP leads to smooth surfaces whereas GP induces the formation of islands features suggesting a non-continuous silane layer. The reproducibility of the overall surface layer (18.75mm²) has been evaluated through the covalent immobilization of a fluorescent oligoprobes. Average fluorescent signals of 6 (a.u.) and 4 (a.u.) were observed for IP and GP, respectively, with a standard deviation of 1 for both protocols. Thus, despite a morphological difference of the silane layer at the nanometer scale, the density of the immobilized probes remained similar.

Lysine–poly(2-hydroxyethyl methacrylate) modified polyurethane surface with high lysine density and fibrinolytic activity

We have developed a potentially fibrinolytic surface in which a bioinert polymer is used as a spacer to immobilize lysine such that the ε-amino group is free to capture plasminogen when in contact with blood. Adsorbed plasminogen can be activated to plasmin and potentially dissolve nascent clots formed on the surface. In previous work lysine was immobilized through a poly(ethylene glycol) (PEG) spacer; however, the graft density of PEG was limited and the resulting adsorbed quantity of plasminogen was insufficient to dissolve clots efficiently. The aim of the present work was to optimize the surface using graft-polymerized poly(2-hydroxyethyl methacrylate) (poly(HEMA)) as a spacer to increase the grafting density of lysine. Such a poly(HEMA)–lysine modified polyurethane (PU) surface is expected to have increased plasminogen binding capacity and clot lysing efficiency compared with PEG–lysine modified PU. A lysine density of 2.81nmolcm−2 was measured on the PU–poly(HEMA)–Lys surface vs. 0.76nmolcm−2 on a comparable PU–PEG–Lys surface reported previously. The poly(HEMA)–lysine-modified surface was shown to reduce non-specific (fibrinogen) adsorption while binding plasminogen from plasma with high affinity. With increased plasminogen binding capacity these surfaces showed more rapid clot lysis (20min) in a standard in vitro assay than the corresponding PEG–lysine system (40min). The data suggest that poly(HEMA) is superior to PEG when used as a spacer in the immobilization of bioactive molecules at high density. This method of modification may also provide a generic approach for preparing bioactive PU surfaces of high activity and low non-specific adsorption of proteins.

An activated medium with high durability and low nonspecific adsorption: Application to protein A chromatography

Activated media allow the user to easily synthesize a variety of affinity media. We have developed a novel activated medium based on porous silica modified with phosphorylcholine (PC) and N-hydroxysuccinimide (NHS) groups for the purpose of high-throughput purification and reducing nonspecific protein adsorption. The PC groups function as suppressors of nonspecific protein adsorption, whereas the NHS groups are able to covalently bind to the primary amino groups of ligands. Because protein A affinity medium is the most frequently used affinity medium, we prepared protein A media in which a recombinant protein A was bound to the NHS groups of the activated media and evaluated its utility. After optimizing various factors in the synthetic process, the resultant protein A medium showed improved durability at a high flow rate over 300 purification cycles and reduced nonspecific protein adsorption compared with commercially available protein A media.

Automated Printing of Electrochemical Immunoassay Microarrays: Studies of Conditions Affecting Alkaline Phosphatase Enzyme Label Activity

Microarray printing of components of an enzyme-linked immunosorbant assay (ELISA) is attractive for constructing miniaturized platforms to simultaneously and quantitatively analyze multiple chemical species in clinical, environmental, and forensic applications. A truncated version of an electrochemical ELISA was used to address a major challenge of this approach, namely the effect of evaporation on assay activity. An antibody-alkaline phosphatase conjugate (Ab-AP) was printed with a microarrayer onto an N-hydroxysuccinimide-ester-terminated self-assembled monolayer on gold. The activity of the enzyme label AP to convert the substrate p-aminophenyl phosphate to electroactive p-aminophenol was followed as a function of different conditions via detection by cyclic voltammetry at microband electrodes. It was found that enzyme activity decreased by 60% from 30 min to 4 h after evaporation. However, printing in ambient humidity ("dry") as opposed to printing in a humidified chamber, resulted in the highest assay signals observed and low nonspecific adsorption.

Engineering the Polymer Backbone To Strengthen Nonfouling Sulfobetaine Hydrogels

We have demonstrated that molecularly engineering the chemical structure of a monomer can lead to hydrogels with improved mechanical strength. In this case, hydrogels from zwitterionic sulfobetaine methacrylate monomers were compared to sulfobetaine vinylimidazole (pSBVI) hydrogels. We show that the introduction of the vinylimidazole backbone improves the tensile and compressive mechanical properties of the sulfobetaine hydrogel by an order of magnitude over the same properties of a methacrylate hydrogel. Zwitterionic groups have been shown to create surface coating materials with ultralow fouling properties, and we demonstrate here that the presence of the imidazole group does not compromise the nonfouling properties attributed to the zwitterionic sulfobetaine: surfaces coated with pSBVI exhibited exceptionally low nonspecific protein adsorption, and cell adhesion was reduced by 97% relative to low-fouling poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels.

Studies on the interactions between bovine β-lactoglobulin and chitosan at the solid–liquid interface

Chitosan ultrathin films have been formed on polycrystalline Au substrates using the LbL technique with the purpose of studying its interaction with bovine β-lactoglobulin (β-LG) at the solid–liquid interface. The immobilization of chitosan was followed by Quartz Crystal Microbalance with energy dissipation (QCM-D), Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS). The behavior of the chitosan films in the presence of β-LG solutions with different bulk concentrations ([β-LG]), ionic strength ( I), and pH has been investigated using the same techniques plus Atomic Force Microscopy (AFM). The results showed that for pHs lower than protein's p I, weak intermolecular forces (H bonding, Van der Waals, hydrophobic, etc.) are established between β-LG and chitosan (especially close to the p I) leading to low coverage nonspecific adsorption. On the contrary when pH > p I, strong ionic bonding through attractive electrostatic interactions lead to high coverage adsorbed phases composed of large β-LG aggregates. The adsorption process was shown to consist of a relatively fast step (in which these interactions are predominant) which is followed, once the β-LG monolayer is exceeded, by the slow formation of thicker and increasingly viscoelastic films through β-LG self-aggregation. QCM-D and AFM experiments unveiled the role of [β-LG] and I on the formation of these aggregates. The adsorption isotherm built from impedance data in the medium-low [β-LG] range (0.001–0.3 mg mL −1), showed good fitting to the Langmuir model confirming that the formation of one β-LG monolayer is achieved in this concentration range.

Mechanical Properties of Ultrahigh Molecular Weight PHEMA Hydrogels Synthesized Using Initiated Chemical Vapor Deposition

In this work, poly(2-hydroxyethyl methacrylate) (PHEMA), a widely used hydrogel, is synthesized using initiated chemical vapor deposition (iCVD), a one-step surface polymerization that does not use any solvents. iCVD synthesis is capable of producing linear stoichiometric polymers that are free from entrained unreacted monomer or solvent and, thus, do not require additional purification steps. The resulting films, therefore, are found to be noncytotoxic and also have low nonspecific protein adsorption. The kinetics of iCVD polymerization are tuned so as to achieve rapid deposition rates ( approximately 1.5 microm/min), which in turn yield ultrahigh molecular weight polymer films that are mechanically robust with good water transport and swellability. The films have an extremely high degree of physical chain entanglement giving rise to high tensile modulus and storage modulus without the need for chemical cross-linking that compromises hydrophilicity.

Protein patterning on polycrystalline silicon–germanium via standard UV lithography for bioMEMS applications

Polycrystalline silicon–germanium (poly-SiGe) is a promising structural material for the post-processing of micro electro-mechanical systems (MEMS) on top of complementary metal-oxide-semiconductor (CMOS) substrates. Combining MEMS and CMOS allows for the development of high-performance devices. We present for the first time selective protein immobilization on top of poly-SiGe surfaces, an enabling technique for the development of novel poly-SiGe based MEMS biosensors. Active regions made of 3-aminopropyl-triethoxysilane (APTES) were defined using silane deposition onto photoresist patterns followed by lift-off in organic solvents. Subsequently, proteins were covalently bound on the created APTES patterns. Fluorescein-labeled human serum albumin (HSA) was used to verify the immobilization procedure while the binding capability of the protein layer was tested by an antigen-labeled antibody pair. Inspection by fluorescence microscopy showed protein immobilization inside the desired bioactive areas and low non-specific adsorption outside the APTES pattern. Furthermore, the quality of the silane patches was investigated by treatment with 30 nm-diameter gold nanoparticles and scanning electron microscope observation. The developed technique is therefore a promising first step towards the realization of poly-SiGe based biosensors.

Solid phase single-molecule counting of antibody binding to supported protein layers surface with low nonspecific adsorption

On the basis of the supported protein layers (SPLs) substrate, the study presented an ultrasensitive and highly specific platform for single-molecule fluorescence detection of antibody using quantum dots (QDs) as probes. To construct the SPLs surface platform for antibody immobilization, bovine serum albumin (BSA), anti-BSA, and protein G were firstly attached to carboxyl-terminated substrate surfaces by turns. Then nonspecific adsorption of single antibody molecules on SPLs surfaces was investigated. Through the irreversible interaction between streptavidin and biotin, streptavidin-QD conjugates were employed to conjugate with biotinylated antibody, producing QD-antibody conjugates for generating fluorescent signals in fluorescent imaging. Epi-fluorescence microscopy equipped with an electron multiplying charge-coupled device was chosen as the tool for single-molecule fluorescence detection here. The concentration of antibody is quantified based on the direct counting of individual fluorescent spots, one by one. The generated fluorescent signals increased with the increasing concentration of immobilized antibody and were found to be proportional to antibody concentrations. The better brightness and photostability of QDs, and slower increase in the number of counted molecules make a large linear dynamic range of 1.0 × 10 −14 to 3.0 × 10 −12 mol L −1 between the number of single molecules and antibody concentrations, which is comparable to the previously reported surface-based SMD analysis.

Magnetic Silica‐Coated Sub‐Microspheres with Immobilized Metal Ions for the Selective Removal of Bovine Hemoglobin from Bovine Blood

Magnetic silica-coated magnetite (Fe(3)O(4)) sub-microspheres with immobilized metal-affinity ligands are prepared for protein adsorption. First, magnetite sub-microspheres were synthesized by a hydrothermal method. Then silica was coated on the surface of Fe(3)O(4) particles using a sol-gel method to obtain magnetic silica sub-microspheres with core-shell morphology. Next, the trichloro(4-chloromethylphenyl) silane was immobilized on them, reacted with iminodiacetic acid (IDA), and charged with Cu(2+). The obtained magnetic silica sub-microspheres with immobilized Cu(2+) were applied for the absorption of bovine hemoglobin (BHb) and the removal of BHb from bovine blood. The size, morphology, and magnetic properties of the resulting magnetic micro(nano) spheres were investigated by using scanning microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and a vibrating sample magnetometer (VSM). The measurements showed that the magnetic sub-microspheres are spherical in shape, very uniform in size with a core-shell, and are almost superparamagnetic. The saturation magnetization of silica-coated magnetite (Fe(3)O(4)) sub-microspheres reached about 33 emu g(-1). Protein adsorption results showed that the sub-microspheres had a high adsorption capacity for BHb (418.6 mg g(-1)), low nonspecific adsorption, and good removal of BHb from bovine blood. This opens a novel route for future applications in removing abundant proteins in proteomic analysis.

Small and Stable Sulfobetaine Zwitterionic Quantum Dots for Functional Live-Cell Imaging

We have developed a novel surface coating for semiconductor quantum dots (QDs) based on a heterobifunctional ligand that overcomes most of the previous limits of these fluorescent probes in bioimaging applications. Here we show that QDs capped with bidentate zwitterionic dihydrolipoic acid-sulfobetaine (DHLA-SB) ligands are a favorable alternative to polyethylene glycol-coated nanoparticles since they combine small sizes, low nonspecific adsorption, preserved optical properties, and excellent stability over time and a wide range of pH and salinity. Additionally, these QDs can easily be functionalized with biomolecules such as streptavidin (SA) and biotin. We applied streptavidin-functionalized DHLA-SB QDs to track the intracellular recycling of cannabinoid receptor 1 (CB1R) in live cells. These QDs selectively recognized the pool of receptors at the cell surface via SA-biotin interactions with negligible nonspecific adsorption. The QDs retained their optical properties, allowing the internalization of CB1R into endosomes to be followed. Moreover, the cellular activity was apparently unaffected by the probe.

Multifunctional Dendrimer-templated Antibody Presentation on Biosensor Surfaces for Improved Biomarker Detection.

Dendrimers, with their well-defined globular shape and a high density of functional groups, are ideal nanoscale materials for templating sensor surfaces. This work exploits dendrimers as a versatile platform for capturing biomarkers with improved sensitivity and specificity. Synthesis, characterization, fabrication, and functional validation of the dendrimer-based assay platform are described. Bifunctional hydroxyl/thiol functionalized G4-polyamidoamine (PAMAM) dendrimer is synthesized and immobilized on to the polyethylene-glycol (PEG)-functionalized assay plate by coupling PEG-maleimide and dendrimer thiol groups. Simultaneously, part of the dendrimer thiol groups are converted to hydrazide functionalities. The resulting dendrimer-modified surface is coupled to the capture antibody in the Fc region of the oxidized antibody. This preserves the orientation flexibility of the antigen binding region (Fv) of the antibody. To validate the approach, the fabricated plates are further used as a solid phase for developing a sandwich type ELISA to detect IL-6 and IL-1β, important biomarkers for early stages of chorioamnionitis. The dendrimer-modified plate provides assays with significantly enhanced sensitivity, lower nonspecific adsorption, and a detection limit of 0.13 pg ml-1 for IL-6 luminol detection and 1.15 pg ml-1 for IL-1β TMB detection, which are significantly better than those for the traditional ELISA. The assays were validated in human serum samples from normal (non-pregnant) woman and pregnant women with pyelonephritis. The specificity and the improved sensitivity of the dendrimer-based capture strategy could have significant implications for the detection of a wide range of cytokines and biomarkers since the capture strategy could be applied to multiplex microbead assays, conductometric immunosensors and field effect biosensors.

Lysine-attached anodic aluminum oxide (AAO)–silica affinity membrane for bilirubin removal

Anodic aluminum oxide (AAO)–silica composite membrane was prepared by depositing silica onto the AAO membrane through sol–gel method. The obtained composite membrane exhibited excellent uniformities, low nonspecific adsorption and good hydraulic permeability. Lysine (Lys) as ligand was immobilized onto the AAO–silica composite membranes by activating with glutaraldehyde. The content of Lys of Lys-attached affinity membranes was ∼5.6 mg/g membrane. Such Lys-attached affinity membranes were used to adsorb bilirubin from the bilirubin–phosphate solution and bilirubin–albumin solution. The adsorption mechanism of bilirubin and the effects of temperature, initial concentration of bilirubin, albumin concentration and ionic strength on adsorption were investigated by batch experiments. The results showed that the adsorption capacity increased with increasing the temperature while decreased with increasing the NaCl concentration and albumin concentration, and the adsorption isotherm fitted the Langmuir model well. The result of dynamic experiment showed Lys-attached affinity membranes can well remove the bilirubin from the bilirubin–albumin solution.

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 pattern discrimination and contrast.

Electrospun polyethersulfone affinity membrane: Membrane preparation and performance evaluation

Non-woven polyethersulfone (PES) membranes were prepared by electrospinning. After heat treatment and surface activation, the membranes were covalently functionalized with ligands to be used as affinity membranes. The membranes were characterized in terms of fiber diameter, porosity, specific area, pore size, ligand density and binding capacities. To evaluate the binding efficiency of the membrane, dynamic adsorption of bovine serum albumin (BSA) on the Cibacron blue F3GA (CB) functionalized PES membrane was studied. Experimental breakthrough curves were fitted with the theoretical curves based on the plate model to estimate plate height ( H p ) of the affinity membrane. The high value of H p (1.6–8 cm) of the affinity membrane implied a poor dynamic binding efficiency, which can be explained by the intrinsic microstructures of the material. Although the electrospun membrane might not be an ideal candidate for the preparative affinity membrane chromatography for large-scale production, it still can be used for fast small-scale protein purification in which a highly efficient binding is not required. Spin columns packed with protein A/G immobilized PES membranes were demonstrated to be capable of binding IgG specifically. SDS-PAGE results demonstrated that the PES affinity membrane had high specific binding selectivity for IgG molecules and low non-specific protein adsorption. Compared with other reported affinity membranes, the PES affinity membrane had a comparable IgG binding capacity of 4.5 mg/ml, and had a lower flow through pressure drop due to its larger pore size. In conclusion, the novel PES affinity membrane is an ideal spin column packing material for fast protein purification.

Preparation of novel bovine hemoglobin surface-imprinted polystyrene nanoparticles with magnetic susceptibility

In this research, a surface imprinting strategy has been adopted in protein imprinting. Bovine hemoglobin surface-imprinted polystyrene (PS) nanoparticles with magnetic susceptibility have been synthesized through multistage core-shell polymerization system using 3-aminophenylboronic acid (APBA) as functional and cross-linking monomers. Superparamagnetic molecularly imprinted polystyrene nanospheres with poly(APBA) thin films have been synthesized and used for the first time for protein molecular imprinting in an aqueous solution. The magnetic susceptibility is imparted through the successful encapsulation of Fe3O4 nanoparticles. The morphology, adsorption, and recognition properties of superparamagnetic molecularly imprinted polymers (MIPs) have been investigated using transmission electron microscopy, X-ray diffraction, thermogravimetric analysis, and vibrating sample magnetometer. Rebinding experimental results show that poly(APBA) MIPs-coated superparamagnetic PS nanoparticles have high adsorption capacity for template protein bovine hemoglobin and comparatively low nonspecific adsorption. The imprinted superparamagnetic nanoparticles could easily reach the adsorption equilibrium and achieve magnetic separation in an external magnetic field, thus avoiding some problems of the bulk polymer.

Novel Polymer Monolithic Column for Hydrophilic Compounds

A hydrophilic polymer-based monolithic column was prepared from water-soluble crosslinking agents for liquid chromatography. The column media were prepared with a diacrylate monomer, aqueous polyethylene glycol, methanol, and a water-soluble radical initiator. A fused silica capillary column, which was significantly modified by vinyl groups, was utilized as the outer column in order to control polymer shrinkage. We optimized the modification of the capillary column, composition of the monomer and porogen, and polymerization condition. The optimized column exhibited high hydrophilicity and achieved the baseline separation of nucleobases by using only a buffered solution as the mobile phase. Additionally, another column prepared from binary water-soluble crosslinking agents exhibited lower nonspecific adsorption of several proteins. Basically, we would show the possibility of a new type of polymer monolith prepared from water-soluble crosslinker and water-soluble porogenic solvent, and they can be used for chromatographic separation without non-specific hydrophobic interaction.

Multifunctional Polymer Coatings for Cell Microarray Applications

Biocompatible coatings with suitable chemistries for the immobilization of biomolecules are increasingly in demand, as they can be applied in a wide range of biomedical applications. In particular, multifunctional polymer coatings displaying reactive functional groups for the immobilization of specific biological factors that can influence the cellular response while at the same time exhibiting low nonspecific protein adsorption and cell attachment properties have the potential to significantly advance the fields of biomaterials and regenerative medicine. In this study, multifunctional polymer surface chemistries were developed for a cell microarray application with the aim of screening cellular interactions with surface immobilized factors. Coatings were prepared by the deposition of an allylamine plasma polymer pinning layer followed by the deposition of random copolymers of glycidyl methacrylate (GMA) and poly(ethylene glycol) methacrylate (PEGMA). Coatings were characterized by X-ray photoelectron spectroscopy (XPS), infrared spectroscopy, ellipsometry, and contact angle measurements. A variety of proteins as well as synthetic polymers were printed onto copolymer-coated slides using a high-precision contact microarrayer. Printing conditions were optimized for a fluorescently labeled model protein in regard to the temperature, humidity, pin geometry, concentration, and pH of the printing solution. Finally, the suitability of the surface chemistry for the evaluation of cellular responses to surface immobilized factors in a microarray format was demonstrated using HeLa cells.