Modulation of lymphatic distribution of subcutaneously injected poloxamer 407-coated nanospheres: the effect of the ethylene oxide chain configuration

Lymphatic distribution of interstitially injected poloxamer 407-coated nanospheres (45 nm in diameter) is controlled by surface configuration of the ethylene oxide (EO) segments of the adsorbed copolymer. At low poloxamer surface coverage, EO tails spread laterally on a nanosphere surface and assume a ‘flat or mushroom-like’ configuration. Such entities drain rapidly from the subcutaneous site of injection into the initial lymphatic, when compared to uncoated nanospheres, and subsequently are captured by scavengers of the regional lymph nodes. In vitro experiments have also confirmed that such entities are prone to phagocytosis. When the equilibrium poloxamer concentration is at 75 μg/ml or greater the EO chains become more closely packed and project outward from the nanosphere surface. These surface-engineered nanospheres drain faster than those with EO chains in mushroom configurations into the initial lymphatic, escape clearance by lymph node macrophages, reach the systemic circulation, and remain in the blood for prolonged periods. These experiments provide a rational approach for the design and engineering of nano-vehicles for optimal lymphatic targeting and are discussed.     

Poly(organo phosphazene) nanoparticles surface modified with poly(ethylene oxide)

The use of biodegradable derivatives of poly(organo phosphazenes) for the preparation of nanoparticles and their surface modification with the novel poly(ethylene oxide) derivative of poly(organo phosphazene) has been assessed using a range of in vitro characterization methods. The nanoparticles were produced by the precipitation solvent evaporation method from the derivative co-substituted with phenylalanine and glycine ethyl ester side groups. A reduction in particle size to less than 200 nm was achieved by an increase in pH of the preparation medium. The formation (and colloidal stability) of these nanoparticles seems to be controlled by two opposite effects: attractive hydrophobic interactions between phenylalanine ester groups and electrostatic repulsions arising from the carboxyl groups formed due to (partial) hydrolysis of the ester bond(s) at the high pH of the preparation medium. The poly[(glycine ethyl ester)phosphazene] derivative containing 5000-Da poly(ethylene oxide) as 5% of the side groups was used for the surface modification of nanoparticles. Adsorbed onto the particles, the polymer produced a thick coating layer of approximately 35 nm. The coated nanoparticles exhibited reduced surface negative potential and improved colloidal stability toward electrolyte-induced flocculation, relative to the uncoated system. However, the steric stabilization provided was less effective than that of a Poloxamine 908 coating. This difference in effectiveness of the steric stabilization might indicate that, although both the stabilizing polymers possess a 5000-Da poly(ethylene oxide) moiety, there is a difference in the arrangements of these poly(ethylene oxide) chains at the particle surface. (c) 1996 John Wiley & Sons, Inc.     

Adsorption of proteins onto glass surfaces and its effect on the intensity of circular dichroism spectra

Errors in analyzing CD spectra of proteins arising from adsorption loss onto glass surfaces were examined for six proteins: apolipoproteins A-I and E, fibronectin, bovine serum albumin, insulin, and glucagon. Among these, the glycoproteins, apolipoprotein E and fibronectin, adsorbed most onto glass surfaces. Their CD intensities decreased by about 50% when proteins were diluted serially from 1 to 0.01 mg/ml in regular glass-ware and CD was measured in uncoated cells. The other proteins, except glucagon, also showed a certain degree of adsorption. Thus, adsorption loss of proteins onto glass surfaces is common and may lead to serious errors in experimental results. Adsorption can be minimized by using plastic containers and pipet tips, coating the cell with silicone, and wetting the cell before adding the protein solution.     

Multilayer binding of proteins to polymer chains grafted onto porous hollow-fiber membranes containing different anion-exchange groups

Various anion-exchange groups were introduced into the polymer chains grafted onto a porous hollow-fiber membrane for protein recovery by radiation-induced graft polymerization and subsequent functionalization of a monomer containing an epoxy group. The graft chains extended from the pore surface toward the pore interior, resulting in the multilayer binding of proteins to the graft chains. Combinations of three anion-exchange groups, namely, amino (AM), ethylamino (EA), and diethylamino (DEA) groups, and three proteins, namely, beta-lactoglobulin, bovine serum albumin, and urease, were examined to evaluate the degree of multilayer binding of protein to the graft chains in the permeation mode. Multilayer binding was observed for hollow-fiber membranes containing EA and DEA groups, with conversions of epoxy groups to EA or DEA groups of higher than 80%. The amount of adsorbed protein remained constant irrespective of the conversion for the hollow-fiber membrane containing an AM group. The dependence of the flux on the conversion was consistent with that of the degree of multilayer binding to the graft chains.     

Quercetin modifies reactive oxygen levels but exerts only partial protection against oxidative stress within HL-60 cells

Neutron specular reflection has been used to study the structure of a monolayer of dimyristoylphosphatidylcholine (DMPC) deposited using the Langmuir-Blodgett technique onto a silicon oxide substrate. A self-assembled monolayer of octadecyltrichlorosilane with a deuterated alkyl chain (d-OTS) had been previously bonded onto this silicon oxide substrate which rendered it hydrophobic. In the system under study, the alkyl chains of the phospholipid were found to penetrate extensively into the d-OTS layer with the mixed chain region (d-OTS and DMPC) having a total thickness of 30.5 A. This mixed region was divided into two halves for analysis; the 'lower half' (nearest to the substrate surface) was found to comprise anchored d-OTS chains mixed with the lipid chains in the volume ratio approx. 0.60:0.35. The corresponding volume ratio in the 'upper half' of this region was determined to be approx. 0.50:0.40. The thicknesses of these regions were found to be 17.9 A (incorporating approx. 6% solvent) and 12.6 A (incorporating approx. 9% solvent) for the lower and upper halves respectively. The DMPC head groups were found to be confined to the most external layer (furthest away from the silicon substrate). This layer was found to have a thickness of 9.4 A and included a small fraction of the lipid alkyl chains with approx. 47% solvent.     

Glyceryl Monooleate/Poloxamer 407 Cubic Nanoparticles as Oral Drug Delivery Systems: I. In Vitro Evaluation and Enhanced Oral Bioavailability of the Poorly Water-Soluble Drug Simvastatin

Glyceryl monooleate (GMO)/poloxamer 407 cubic nanoparticles were investigated as potential oral drug delivery systems to enhance the bioavailability of the water-insoluble model drug simvastatin. The simvastatin-loaded cubic nanoparticles were prepared through fragmentation of the GMO/poloxamer 407 bulk cubic-phase gel using high-pressure homogenization. The internal structure of the cubic nanoparticles was identified by cryo-transmission electron microscopy. The mean diameter of the cubic nanoparticles varied within the range of 100–150 nm, and both GMO/poloxamer 407 ratio and theoretical drug loading had no significant effect on particle size and distribution. Almost complete entrapment with efficiency over 98% was achieved due to the high affinity of simvastatin to the hydrophobic regions of the cubic phase. Release of simvastatin from the cubic nanoparticles was limited both in 0.1 M hydrochloride solution containing 0.2% sodium lauryl sulfate and fasted-state simulated intestinal fluid with a total release of <3.0% at 10 h. Pharmacokinetic profiles in beagle dogs showed sustained plasma levels of simvastatin for cubic nanoparticles over 12 h. The relative oral bioavailability of simvastatin cubic nanoparticles calculated on the basis of area under the curve was 241% compared to simvastatin crystal powder. The enhancement of simvastatin bioavailability was possibly attributable to facilitated absorption by lipids in the formulation rather than improved release.     

Poly(ethylene oxide) layers grafted to dopamine-melanin anchoring layer: stability and resistance to protein adsorption

In this study, we propose substrate-independent modification for creating a protein-repellent surface based on dopamine-melanin anchoring layer used for subsequent binding of poly(ethylene oxide) (PEO) from melt. We verified that the dopamine-melanin layer can be formed on literally any substrate and could serve as the anchoring layer for subsequent grafting of PEO chains. Grafting of PEO from melt in a temperature range 70-110 °C produces densely packed PEO layers showing exceptionally low protein adsorption when exposed to the whole blood serum or plasma. The PEO layers prepared from melt at 110 °C retained the protein repellent properties for as long as 10 days after their exposure to physiological-like conditions. The PEO-dopamine-melanin modification represents a simple and universal surface modification method for the preparation of protein repellent surfaces that could serve as a nonfouling background in various applications, such as optical biosensors and tissue engineering.     

Protein adsorption at polymer-grafted surfaces: comparison between a mixture of saliva proteins and some well-defined model proteins

Grafting a dense layer of soluble polymers onto a surface is a well-established method for controlling protein adsorption. In the present study, polyethylene oxide (PEO) layers of three different grafting densities were prepared, i.e. 10-15 nm2, 5.5 nm2 and 4 nm2 per polymer chain, respectively. The adsorption of different proteins on the PEO grafted surfaces was measured in real time by reflectometry. Furthermore, the change of the zeta-potential of such surfaces resulting from adsorption of the proteins was determined using the streaming potential method. Both the protein adsorption and the zeta-potential were monitored for 1 h after exposure of the protein solution to the surface. The adsorption pattern for a mixture of saliva proteins was compared to those observed for a number of well-defined model-proteins (lysozyme, human serum albumin, beta-lactoglobulin and ovalbumin). The results of the adsorption kinetics and streaming potential measurements indicate that the effect of the PEO layer on protein adsorption primarily depends on the size and the charge of the protein molecules. The saliva proteins are strongly blocked for adsorption, whereas the change in the zeta-potential is larger than for the other proteins (except lysozyme). It is concluded that positively charged protein molecules, having dimensions larger than those of lysozyme, are involved in the initial stage of adsorption from saliva onto a negatively charged surface.     

Translocation of poly(ethylene glycol-co-hexadecyl)cyanoacrylate nanoparticles into rat brain endothelial cells: role of apolipoproteins in receptor-mediated endocytosis

Previous in vivo observations in rats have shown that poly(ethylene glycol) polyhexadecylcyanoacrylate (PEG-PHDCA) nanoparticles could translocate into the brain after intravenous injection, which polyhexadecylcyanoacrylate (PHDCA) nanoparticles did not. Through the detailed analysis of the plasma protein adsorption onto the surface of PEG-PHDCA nanoparticles, the present study aimed at clarifying the mechanism by which nanoparticles could penetrate into rat brain endothelial cells (RBEC). Two-dimensional polyacrylamide gel electrophoresis and Western blotting revealed that, after incubation with rat serum, apolipoprotein E (ApoE) adsorbed more onto PEG-PHDCA than on PHDCA nanoparticles. Adsorption of apolipoprotein B-100 (ApoB-100) onto PEG-PHDCA nanoparticles was demonstrated by capillary electrophoresis experiments. Moreover, only when ApoE or ApoB-100 were preadsorbed onto PEG-PHDCA nanoparticles, nanoparticles were found to be more efficient than control nanoparticles for penetrating into RBEC, suggesting the involvement of a low density lipoprotein receptor in this process. Thus, these data clearly demonstrate the involvement of apolipoproteins in the brain transport of PEG-PHDCA nanoparticles, which may open interesting prospects for brain drug delivery.     

Protein Adsorption at Polymer-grafted Surfaces: Comparison between a Mixture of Saliva Proteins and some Well-defined Model Proteins

Grafting a dense layer of soluble polymers onto a surface is a well-established method for controlling protein adsorption. In the present study, polyethylene oxide (PEO) layers of three different grafting densities were prepared, i.e. 10?–?15 nm², 5.5 nm² and 4 nm² per polymer chain, respectively. The adsorption of different proteins on the PEO grafted surfaces was measured in real time by reflectometry. Furthermore, the change of the zeta-potential of such surfaces resulting from adsorption of the proteins was determined using the streaming potential method. Both the protein adsorption and the zeta-potential were monitored for 1?h after exposure of the protein solution to the surface. The adsorption pattern for a mixture of saliva proteins was compared to those observed for a number of well-defined model-proteins (lysozyme, human serum albumin, β-lactoglobulin and ovalbumin). The results of the adsorption kinetics and streaming potential measurements indicate that the effect of the PEO layer on protein adsorption primarily depends on the size and the charge of the protein molecules. The saliva proteins are strongly blocked for adsorption, whereas the change in the zeta-potential is larger than for the other proteins (except lysozyme). It is concluded that positively charged protein molecules, having dimensions larger than those of lysozyme, are involved in the initial stage of adsorption from saliva onto a negatively charged surface.     

Patterned protein films on poly(lipid) bilayers by microcontact printing

The use of polymerized lipid bilayers as substrates for microcontact printing (muCP) of protein films was investigated. We have previously shown that vesicle fusion of bis-SorbPC, a dienoate lipid, on glass and silica substrates, followed by redox-initiated radical polymerization, produces a planar supported lipid bilayer (PSLB) that is ultrastable(1a) [Ross, E. E.; Rozanski, L. J.; Spratt, T.; Liu, S.; O'Brien, D. F.; Saavedra, S. S. Langmuir 2003, 19, 1752] and highly resistant to nonspecific adsorption of dissolved proteins [Ross, E. E.; Spratt, T.; Liu, S.; Rozanski, L. J.; O'Brien, D. F.; Saavedra, S. S. Langmuir 2003, 19, 1766].(1b) Here we demonstrate that muCP of bovine serum albumin (BSA) onto a dried poly(bis-SorbPC) PSLB from a poly(dimethylsiloxane) (PDMS) stamp produces a layer of strongly adsorbed protein, comparable in surface coverage to films printed on glass surfaces. Immobilization of proteins on poly(PSLB)s has potential applications in biosensing, and this work shows that direct muCP of proteins is a technically simple approach to create immobilized monolayers, as well as multilayers of different proteins.     

Real-time evidence of surface modification at polystyrene lattices by poloxamine 908 in the presence of serum: in vivo conversion of macrophage-prone nanoparticles to stealth entities by poloxamine 908

Intravenously injected polystyrene nanoparticles, which are prone to rapid sequestration by professional phagocytes, are converted to stealth entities by prior bolus intravenous injection of poloxamine 908. This behaviour is not due to alteration in macrophage phagocytic activity. Laser Doppler anemometry and surface plasmon resonance were used to unravel the mechanisms fundamental to generation of such stealth entities in vivo by poloxamine 908. Electrophoretic mobility of poloxamine pre-coated monodisperse polystyrene nanoparticles in serum, which behave as stealth entities in vivo, was similar to that of uncoated nanoparticles incubated in poloxamine pre-treated serum. This observation supported the notion that poloxamine in serum can modify the surface of nanoparticles with similar topography to that of stealth poloxamine pre-coated particles, i.e. with polyoxyethylene chains projected from the surface. Surface plasmon resonance optical phenomenon was used for real-time monitoring of protein-poloxamine interactions and adsorption at the polystyrene interface. It was found that poloxamine can not only adsorb to a serum-modified surface but in addition poloxamine in serum can form macromolecular complexes with high affinity for adsorption to a polystyrene lattice. A role for serum albumin in surface modification of nanoparticles by poloxamine 908 is also identified. Hence, our biophysical observations correlate precisely with the in vivo longevity of uncoated polystyrene nanoparticles in poloxamine pre-treated rats. This rational and sensitive biophysical approach has unravelled the probable mechanism fundamental to generation of stealth entities in vivo and therefore has application in the design and nano-engineering of stealth colloidal carriers for optimal biological performance.     

Development and estimation of nanosomal rifampicin]

A lab-scale method for preparation of rifampicin-loaded polybutylcyanoacrylate nanoparticles (nanosames) was developed. The biodistribution of the nanosome-entrapped rifampicin after its intravenous administration was studied on healthy mice. The nanoparticles provided significant liver and spleen accumulation of rifampicin. Modification of the nanoparticles surface with poloxamer 407 or poloxamine 908 led to optimization of the biodistribution: the concentrations of rifampicin in the lungs and plasma increased, whereas the liver accumulation decreased vs. the unmodified nanoparticles. The increased lung accumulation of rifampicin enhanced bacterial clearance in the lungs of the mice infected with M. tuberculosis. The results showed that the use of the nanoparticles for optimization of the drug biodistribution was effective for increasing the efficacy of antiinfective chemotherapy.     

Investigation of Bovine Serum Albumin (BSA) Attachment onto Self-Assembled Monolayers (SAMs) Using Combinatorial Quartz Crystal Microbalance with Dissipation (QCM-D) and Spectroscopic Ellipsometry (SE)

Understanding protein adsorption kinetics to surfaces is of importance for various environmental and biomedical applications. Adsorption of bovine serum albumin to various self-assembled monolayer surfaces including neutral and charged hydrophilic and hydrophobic surfaces was investigated using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. Adsorption of bovine serum albumin varied as a function of surface properties, bovine serum albumin concentration and pH value. Charged surfaces exhibited a greater quantity of bovine serum albumin adsorption, a larger bovine serum albumin layer thickness, and increased density of bovine serum albumin protein compared to neutral surfaces at neutral pH value. The quantity of adsorbed bovine serum albumin protein increased with increasing bovine serum albumin concentration. After equilibrium sorption was reached at pH 7.0, desorption of bovine serum albumin occurred when pH was lowered to 2.0, which is below the isoelectric point of bovine serum albumin. Our data provide further evidence that combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry is a sensitive analytical tool to evaluate attachment and detachment of adsorbed proteins in systems with environmental implications.     

Protein interactions with nanosized hydrotalcites of different composition

Nanosized hydrotalcite-like compounds (HTlc) with different chemical composition were prepared and used to study protein adsorption. Two soft proteins, myoglobin (Mb) and bovine serum albumin (BSA), were chosen to investigate the nature of the forces controlling the adsorption and how these depend on the chemical composition of the support. Both proteins strongly interact with HTlc exhibiting in most cases a Langmuir-type adsorption. Mb showed a higher affinity for Nickel Chromium (NiCr-HTlc) than for Nickel Aluminum (NiAl-HTlc), while for BSA no significant differences between supports were found. Adsorption experiments in the presence of additives showed that proteins exhibited different types of interactions onto the same HTlc surface and that the adsorption was strongly suppressed by the addition of disodium hydrogen phosphate (Na₂HPO₄). Atomic force microscopy images showed that the adsorption of both proteins onto nanoparticles was followed by the aggregation of biocomposites, with a more disordered structure for BSA. Fluorescence measurements for adsorbed Mb showed that the inorganic nanoparticles induced conformational changes in the biomolecules; in particular, the interactions with HTlc surface quenched the tryptophan fluorescence and this process was particularly efficient for NiCr-HTlc. The adsorption of BSA onto the HTlc nanoparticles induced a selective quenching of the exposed fluorescent residues, as indicated by the blue-shift of the emission spectra of tryptophan residues and by the shortening of the fluorescence decay times.     

The antibody–antigen interaction at an aqueous–solid interface: A study by means of polarized infrared ATR spectroscopy

The determination of structural changes in antibodies due to their specific interaction with antigenic proteins is an important problem in understanding immunological responses. The method of polarized ATR infrared spectroscopy applied to protein films adsorbed on an appropriate solid surface can give information about the conformation of the polypeptide chains, as well as their orientation with respect to the surface. The adsorption of anti-rabbit serum albumin onto monomolecular films of rabbit serum albumin, bovine serum albumin, and ovalbumin, and of anti-ovalbumin onto films of rabbit serum albumin and ovalbumin at a Ge-aqueous interface have been studied by this technique. The intensity of the amide I absorption indicates that the strengths of binding of these three albumin proteins with anti-rabbit serum albumin is, under appropriate conditions, in the order rabbit > bovine ? ovalbumin; with anti-ovalbumin, it is ovalbumin ? rabbit. Since the frequencies of the amide I band appear near 1655 cm^?1 for all the proteins and protein complexes studied, the major contributions to their conformation comes from α-helix and random-coil structures. The average orientation of the transition moments of the amide I and A bands has been shown to be about 75° with respect to the surface normal. This indicates that the polypeptides chains are on the average approximately parallel to the surface for all the systems studied. Consequently, the effect of the specific antibody-antigen interaction on the conformation and orientation of the former seems negligible in these films.     

Microchip reactor packed with metal-ion chelated magnetic silica microspheres for highly efficient proteolysis

An easily replaceable and regenerable protease microreactor with metal-ion chelated adsorption of enzyme has been fabricated on chip. Magnetic microspheres with small size (approximately 200 nm in diameter) and strong magnetism were synthesized and were modified with tetraethyl orthosilicate. The metal chelating agent of iminodiacetic acid was then reacted with glycidoxypropyltrimethoxysilane before its immobilization onto the surface of magnetic silica microspheres (MS microspheres). The metal ion of copper and enzyme were subsequently adsorbed onto the surface. The prepared MS microspheres were then locally packed into the microchannel by the application of a strong magnetic field using a magnet to form an on-chip enzymatic microreactor. Capability of the proteolytic microreactor was demonstrated by cytochrome c and bovine serum albumin as model proteins. The digestion products were characterized using MALDI-TOF/TOF MS with sequence coverage of 77% and 21% observed, respectively. This microreactor was also applied to the analysis of one RPLC fraction of rat liver extract. After a database search, 23 unique peptides corresponding to 7 proteins were identified when one RPLC fraction of rat liver extract was digested by the microreactor. This opens a route for its future application in top-down proteomic analysis.     

Structure and reactivity of adsorbed fibronectin films on mica

Understanding the interactions of adsorbed fibronectin (Fn) with other biomolecules is important for many biomedical applications. Fn is found in almost all body fluids, in the extracellular matrix, and plays a fundamental role in many biological processes. This study found that the structure (conformation, orientation) and reactivity of Fn adsorbed onto mica is dependent on the Fn surface concentration. Atomic force microscopy and x-ray photoelectron spectroscopy were used to determine the surface coverage of adsorbed Fn from isolated molecules at low surface coverage to full monolayers at high surface coverage. Both methods showed that the thickness of Fn film continued to increase after the mica surface was completely covered, consistent with Fn adsorbed in a more upright conformation at the highest surface-Fn concentrations. Time-of-flight secondary ion mass spectrometry showed that relative intensities of both sulfur-containing (cystine, methionine) and hydrophobic (glycine, leucine/isoleucine) amino acids varied with changing Fn surface coverage, indicating that the conformation of adsorbed Fn depended on surface coverage. Single-molecule force spectroscopy with collagen-related peptides immobilized onto the atomic force microscope tip showed that the specific interaction force between the peptide and Fn increases with increasing Fn surface coverage.     

A quantitative and selective chromatography method for determining coverages of multiple proteins on surfaces

Competitive protein adsorption plays a key role in the surface hemocompatibility of biological implants. We describe a quantitative chromatography method to measure the coverage of multiple proteins physisorbed to surfaces. In this method adsorbed proteins are displaced by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and then analyzed by high performance liquid chromatography to separate and quantify the individual proteins, in this case bovine serum albumin (BSA) and bovine fibrinogen (Fg). CHAPS displaced over 95% of the adsorbed proteins and was easily removed from solution by dialysis. This method was tested by measuring the coverage of BSA, 66 kDa, and Fg, 340 kDa, simultaneously adsorbed from solutions with concentration of 20 microg/ml, on bare and dextranized silicon. Relative to silicon, the dextranized surfaces were found to strongly inhibit protein adsorption, decreasing BSA and Fg coverages by 76 and 60%, respectively.     

Functionalization of poly(oligo(ethylene glycol) methacrylate) films on gold and Si/SiO2 for immobilization of proteins and cells: SPR and QCM studies

Thin films of a biocompatible and nonbiofouling poly(oligo(ethylene glycol) methacrylate) ( pOEGMA) with various thicknesses were formed on gold and Si/SiO 2 substrates by a combination of the formation of self-assembled monolayers (SAMs) terminating in bromoester-an initiator of atom transfer radical polymerization (ATRP)-and surface-initiated ATRP. After the formation of the pOEGMA films, terminal hydroxyl groups of side chains divergent from the methacrylate backbones were activated with N, N'-disuccinimidyl carbonate (DSC), and the DSC-activated pOEGMA films were reacted with (+)-biotinyl-3,6,9-trioxaundecanediamine (Biotin-NH 2) to form biotinylated pOEGMA films. By surface plasmon resonance experiments with the target protein (streptavidin) and model proteins (fibrinogen and lysozyme), we verified that the resulting films showed the enhanced signal-to-noise ratio ( approximately 10-fold enhancement) for the biospecific binding of streptavidin compared with the biotinylated substrate prepared from carboxylic acid-terminated SAMs. Quartz crystal microbalance measurements were also carried out to obtain the surface coverage of streptavidin and fibrinogen adsorbed onto the biotinylated pOEGMA films with various thicknesses and to investigate the effect of film thicknesses on the biospecific binding of streptavidin. Both the binding capacity of streptavidin and the signal-to-noise ratio of streptavidin/fibrinogen were found to be saturated at the 20 nm thick pOEGMA film. In addition, to demonstrate a wide applicability of the pOEGMA films, we constructed micropatterns of streptavidin and cells by microcontact-printing biotin-NH 2 and poly- l-lysine onto the DSC-activated pOEGMA films, respectively.