Thiolactone-Functional Pullulan for In Situ Forming Biogels.

Gelation in the presence of cells with minimum cytotoxicity is highly desirable for materials with applications in tissue engineering. Herein, the naturally occurring polysaccharide pullulan is functionalized with thiolactones that undergo ring-opening addition of amines. As a result, the modified pullulan can be cross-linked with diamines and/or amine-containing biological substrates enhancing the system's versatility (e.g., gelatin and cell-binding ligands GHK/GRGDS). Thiolactone degrees of substitution of 2.5 or 5.0 mol % are achieved, and respective hydrogels exhibit mesh sizes of 27.8 to 49.1 nm. Cell proliferation studies on chosen gels (G' ≅ 500 Pa, over 14 days) demonstrate that for normal human dermal fibroblasts (NHDFs), both gelatin and GRGDS equally support cell proliferation, while in the case of hepatocytes (HepG2), the presence of GRGDS and GHK improve cell proliferation 10-fold compared to gelatin. Cells remain viable and in one instance were successfully encapsulated by in situ gelation, altogether confirming the mild and biocompatible nature of this strategy to produce biogels using biologically active substrates as cross-linkers.

A Layer-by-Layer Single-Cell Coating Technique To Produce Injectable Beating Mini Heart Tissues via Microfluidics.

Human induced pluripotent stem cells (hiPSCs) are used as an alternative for human embryonic stem cells. Cardiomyocytes derived from hiPSCs are employed in cardiac tissue regeneration constructs due to the heart's low regeneration capacity after infarction. A coculture of hiPSC-CM and primary dermal fibroblasts is encapsulated in injectable poly(ethylene glycol)-based microgels via microfluidics to enhance the efficiency of regenerative cell transplantations. The microgels are prepared via Michael-type addition of multi-arm PEG-based molecules with an enzymatically degradable peptide as a cross-linker and modified with a cell-adhesive peptide. Cell-cell interactions and, consequently, cell viability are improved by a thin extracellular matrix (ECM) coating formed on the cell surfaces via layer-by-layer (LbL) deposition. The beating strength of encapsulated cardiomyocytes (∼60 BPM) increases by 2-fold compared to noncoated cells. The combination of microfluidics with the LbL technique offers a new technology to fabricate functional cardiac mini tissues for cell transplantation therapies.

Development of Endothelial Cell Networks in 3D Tissues by Combination of Melt Electrospinning Writing with Cell-Accumulation Technology.

A remaining challenge in tissue engineering approaches is the in vitro vascularization of engineered constructs or tissues. Current approaches in engineered vascularized constructs are often limited in the control of initial vascular network geometry, which is crucial to ensure full functionality of these constructs with regard to cell survival, metabolic activity, and potential differentiation ability. Herein, the combination of 3D-printed poly-ε-caprolactone scaffolds via melt electrospinning writing with the cell-accumulation technique to enable the formation and control of capillary-like network structures is reported. The cell-accumulation technique is already proven itself to be a powerful tool in obtaining thick (50 µm) tissues and its main advantage is the rapid production of tissues and its ease of performance. However, the applied combination yields tissue thicknesses that are doubled, which is of outstanding importance for an improved handling of the scaffolds and the generation of clinically relevant sample volumes. Moreover, a correlation of increasing vascular endothelial growth factor secretion to hypoxic conditions with increasing pore sizes and an assessment of the formation of neovascular like structures are included.

Control of vascular network location in millimeter-sized 3D-tissues by micrometer-sized collagen coated cells.

Engineering three-dimensional (3D) vascularized constructs remains a central challenge because capillary network structures are important for sufficient oxygen and nutrient exchange to sustain the viability of engineered constructs. However, construction of 3D-tissues at single cell level has yet to be reported. Previously, we established a collagen coating method for fabricating a micrometer-sized collagen matrix on cell surfaces to control cell distance or cell densities inside tissues. In this study, a simple fabrication method is presented for constructing vascular networks in 3D-tissues over micrometer-sized or even millimeter-sized with controlled cell densities. From the results, well vascularized 3D network structures can be observed with a fluorescence label method mixing collagen coated cells and endothelia cells, indicating that constructed ECM rich tissues have the potential for vascularization, which opens up the possibility for various applications in pharmaceutical or tissue engineering fields.

Construction and myogenic differentiation of 3D myoblast tissues fabricated by fibronectin-gelatin nanofilm coating.

In this study, we used a recently developed approach of coating the cells with fibronectin-gelatin nanofilms to build 3D skeletal muscle tissue models. We constructed the microtissues from C2C12 myoblasts and subsequently differentiated them to form muscle-like tissue. The thickness of the constructs could be successfully controlled by altering the number of seeded cells. We were able to build up to ∼76 µm thick 3D constructs that formed multinucleated myotubes. We also found that Rho-kinase inhibitor Y27632 improved myotube formation in thick constructs. Our approach makes it possible to rapidly form 3D muscle tissues and is promising for the in vitro construction of physiologically relevant human skeletal muscle tissue models.

Three-Dimensional Tissue Models Constructed by Cells with Nanometer- or Micrometer-Sized Films on the Surfaces.

Living tissues or organ modules consist of different types of highly organized cells and extracellular matrices (ECMs) in a hierarchical manner, such as the multilayered structure of blood vessels and the radial structures of hepatic lobules. Due to animal examinations being banned in the EU since 2013 and a shortage in the demand for tissue repair or organ transplantation, the creation of artificial 3D tissues possessing specific structures and functions similar to natural tissues are key challenges in tissue engineering. To date, we have developed a simple but unique bottom-up approach, a hierarchical cell manipulation technique, with a nanometer-sized ECM matrix consisting of fibronectin (FN) and gelatin (G) on cell surfaces. About 10 nm thick FN/G ECM films on cell surfaces were coated successfully by using layer-by-layer coating methodology. Various 3D constructs with higher cell density with different types of cells were successfully constructed. In addition to the construction of tissues with higher cell densities, other tissues, such as cartilage or skin tissues, with different cell densities are also important tissue models for tissue engineering and pharmaceutical industries. Thus, we recently developed other methodologies, the collagen coating method and multiple coating method, to fabricate micrometer-sized level ECM layers on cell surfaces. Various micro- or millimeter-sized 3D constructs with lower cell densities were constructed successfully. By using these two methods, cell distances in 2D or 3D views can be controlled by different thicknesses of ECM layers on cell surfaces at the single-cell level. Both FN/G and the collagen coating method resulted in homogenous 3D tissues with a controlled layer numbers, cell type, cell location, and properties; these will be promising to achieve different goals in tissue engineering.

Three-dimensional human arterial wall models for in vitro permeability assessment of drug and nanocarriers.

Monolayers of endothelial cells (1L-ECs) have been generally used as in vitro vascular wall models to study the vascular mechanisms and transport of substances. However, these two-dimensional (2D-) system cannot represent the properties of native vascular walls which have a 3D-structure and are composed of not only ECs, but also smooth muscle cells (SMCs) and other surrounding tissues. Here in, 5-layered (5L) 3D-arterial wall models (5L-AWMs) composed of EC monolayer and 4-layered SMCs were constructed by hierarchical cell manipulation. We applied the 5L-AWMs to evaluate their barrier function and permeability to nano-materials in order to analyze drug, or drug nanocarrier permeability to the blood vessel in vitro. Barrier property of the 3D-AWMs was confirmed by Zonula occludens (ZO-1) staining and their transendothelial electrical resistance (TEER), which was comparable to 1L-ECs, while the SMCs showed close to zero. The effect of substance size to permeability across the 5L-AWMs was clearly observed from dextrans with various molecular weights, which agreed well with the known phenomena of the in vivo blood vessels. Importantly, transport of nano-materials could be observed across the depth of 5L-AWMs, suggesting the advantage of 3D-AWMs over general 2D-systems. By using this system, we evaluate the transport of 35 nm phenylalanine-modified poly(γ-Glutamic Acid) nanoparticles (γ-PGA-Phe NPs) as a candidate of biodegradable drug carrier. Interestingly, despite of having comparable size to dextran-2000 k (28 nm), the γ-PGA-Phe NPs distinctly showed approximately 20 times faster transport across the 5L-AWMs, suggesting the effect of intrinsic properties of the substance on the transport. This in vitro evaluation system using the 3D-AWMs is therefore useful for the design and development of nano-drug carriers for treatment of vascular diseases, such as atherosclerosis.

Stereocomplex Film Using Triblock Copolymers of Polylactide and Poly(ethylene glycol) Retain Paxlitaxel on Substrates by an Aqueous Inkjet System.

The stereocomplex formation of poly(L,L-lactide) (PLLA) and poly(D,D-lactide) (PDLA) using an inkjet system was expanded to the amphiphilic copolymers, using poly(ethylene glycol) (PEG) as a hydrophilic polymer. The diblock copolymers, which are composed of PEG and PLLA (MPEG-co-PLLA) and PEG and PDLA (MPEG-co-PDLA), were employed for thin-film preparation using an aqueous inkjet system. The solvent and temperature conditions were optimized for the stereocomplex formation between MPEG-co-PLLA and MPEG-co- PDLA. As a result, the stereocomplex was adequately formed in acetonitrile/water (1:1, v/v) at 40 °C. The aqueous conditions improved the stereocomplex film preparation, which have suffered from clogging when using the organic solvents in previous work. The triblock copolymers, PLLA-co-PEG-co-PLLA and PDLA-co-PEG-co-PDLA, were employed for square patterning with the inkjet system, which produced thin films. The amphiphilic polymer film was able to retain hydrophobic compounds inside. The present result contributed to the rapid film preparation by inkjet, retaining drugs with difficult solubility in water, such as paclitaxel within the films.

Biomineral/Agarose Composite Gels Enhance Proliferation of Mesenchymal Stem Cells with Osteogenic Capability.

Hydroxyapatite (HA) or calcium carbonate (CaCO3) formed on an organic polymer of agarose gel is a biomaterial that can be used for bone tissue regeneration. However, in critical bone defects, the regeneration capability of these materials is limited. Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into bone forming osteoblasts. In this study, we loaded MSCs on HA- or CaCO3-formed agarose gel and cultured them with dexamethasone, which triggers the osteogenic differentiation of MSCs. High alkaline phosphatase activity was detected on both the HA- and CaCO3-formed agarose gels; however, basal activity was only detected on bare agarose gel. Bone-specific osteocalcin content was detected on CaCO3-formed agarose gel on Day 14 of culture, and levels subsequently increased over time. Similar osteocalcin content was detected on HA-formed agarose on Day 21 and levels increased on Day 28. In contrast, only small amounts of osteocalcin were found on bare agarose gel. Consequently, osteogenic capability of MSCs was enhanced on CaCO3-formed agarose at an early stage, and both HA- and CaCO3-formed agarose gels well supported the capability at a later stage. Therefore, MSCs loaded on either HA- or CaCO3-formed agarose could potentially be employed for the repair of critical bone defects.

Inkjet printing of layer-by-layer assembled poly(lactide) stereocomplex with encapsulated proteins.

Inkjet printing, a technique that precisely deposits liquid droplets in picoliter-volume ranges on a substrate, has received increased attention for its novelty and ability to produce functional materials. This technology is considered one of the most promising methods for the controlled deposition of different polymers. In our previous study, a poly(lactide) (PLA) stereocomplex was fabricated using inkjet printing on a substrate. The stereocomplex was formed by the layer-by-layer (LbL) stepwise deposition of poly(l-lactide) (PLLA) and poly(d-lactide) (PDLA). Multiple inkjet passes could conclusively improve the PLAs crystal structure with solvent evaporation (solidification) and dissolution of PLA. We suggested that this technique may also be applicable for fabricating polymer composites with drugs, such as peptides, proteins, and nanoparticles, which is incompatible with the PLA. Here, we report the utilization of this technique to create a PLA stereocomplex with drugs as a drug carrier/reservoir. The three components of PLLA, PDLA, and model drugs (an 8-mer peptide, ovalbumin, and protein-encapsulating nanoparticles) were alternately overprinted onto the substrate without an intermediate rinsing step. Inkjet printing was used successfully to form PLA stereocomplex composites with drugs by the LbL deposition of polymers and functioned as drug carriers/reservoirs. The sustained release of the drugs was observed from the PLLA/PDLA/drug composites. By varying the crystalline structure of PLAs-drug composites, the release kinetics of drugs could be altered and controlled efficiently. Moreover, a high drug loading content (wt %) of PLA stereocomplex composites was achieved up to 100 wt % loading, and the composites with 50 wt % of drug loading content were available for sustained-release formulation. This fabrication technique would provide a platform for creating protein/vaccine/gene delivery carriers with the desired release profiles by controlling the microphase-separated structure and drug distribution within the composites.

Cyclodextrin polymers as highly effective adsorbents for removal and recovery of polychlorobiphenyl (PCB) contaminants in insulating oil.

A total of 179 countries (parties) ratified the Stockholm Convention on persistent organic pollutants (POPs) and agreed to destroy polychlorobiphenyls (PCBs) and develop a sound management plan by 2028. Currently, still 3 million tons of PCB-contaminated oil and equipment need to be managed under the Stockholm Convention. Thus, the development of a facile and environmentally benign method to treat large amounts of oil stockpiles contaminated with PCBs is a crucial subject. Herein, we report that cyclodextrin (CD) polymers, which are easily prepared by cross-linking the renewable cyclic oligosaccharide γ-cyclodextrin (γ-CD) with dibasic acid dichlorides, are a new selective and powerful adsorbent to remove PCB contaminants in oil. When PCB (100 ppm)-contaminated oil was passed through a column packed with the terephthaloyl-cross-linked γ-CD polymer (TP-γ-CD polymer) at 80-110 °C, the PCB contaminants were completely removed from the oil. Additionally, methyl esterification of the free carboxylic groups of the TP-γ-CD polymer enabled the complete recovery of the PCBs adsorbed on the polymer (with >99.9% recovery efficiency) by simply washing with acetone. The methyl-esterified TP-γ-CD polymer could be recycled at least 10 times for PCB adsorption without any loss in the adsorption capability. These results revealed that the γ-CD polymers can function as highly effective and powerful adsorbents for the removal and recovery of PCBs from PCB-contaminated oil and, thus, significantly contribute to the protection of the global environment.

Recent Developments in Layer-by-Layer Assembly for Drug Delivery and Tissue Engineering Applications.

Surfaces with biological functionalities are of great interest for biomaterials, tissue engineering, biophysics, and for controlling biological processes. The layer-by-layer (LbL) assembly is a highly versatile methodology introduced 30 years ago, which consists of assembling complementary polyelectrolytes or biomolecules in a stepwise manner to form thin self-assembled films. In view of its simplicity, compatibility with biological molecules, and adaptability to any kind of supporting material carrier, this technology has undergone major developments over the past decades. Specific applications have emerged in different biomedical fields owing to the possibility to load or immobilize biomolecules with preserved bioactivity, to use an extremely broad range of biomolecules and supporting carriers, and to modify the film's mechanical properties via crosslinking. In this review, the focus is on the recent developments regarding LbL films formed as 2D or 3D objects for applications in drug delivery and tissue engineering. Possible applications in the fields of vaccinology, 3D biomimetic tissue models, as well as bone and cardiovascular tissue engineering are highlighted. In addition, the most recent technological developments in the field of film construction, such as high-content liquid handling or machine learning, which are expected to open new perspectives in the future developments of LbL, are presented.

Cultivation and Transplantation of Three-Dimensional Skins with Laser-Processed Biodegradable Membranes.

For the treatment of irreversible, extensive skin damage, artificial skins or cultured skins are useful when allogeneic skins are unavailable. However, most of them lack vasculature, causing delayed perfusion and hence delay or failure in engraftment of the tissues. We previously developed a prevascularized three-dimensional (3D) cultured skin based on the layer-by-layer cell coating technique (LbL-3D skin), in which cells are seeded and laminated on a porous polymer membrane for medium supply to the thick cultured tissue. Recent animal studies have demonstrated that LbL-3D skin can achieve rapid perfusion and high graft survival after transplantation. However, there were practical issues with separating LbL-3D skins from the membranes before transplantation and the handling separated LbL-3D skins for transplantation. To address these problems, in this study, we examined the use of biodegradable porous polymer membranes that enabled the transplantation of LbL-3D skins together with the membranes, which could be decomposed after transplantation. Thin films made from poly (lactic-co-glycolic acid) (PLGA) were irradiated with femtosecond laser pulses to create micro through-holes, producing porous membranes. We designed and fabricated culture inserts with the PLGA membranes and cultivated LbL-3D skins with 2 × 106 neonatal normal human dermal fibroblasts and 1 × 104 human umbilical vein endothelial cells in the dermis of 20 cell layers and 1 × 105 neonatal human epidermal keratinocytes in the epidermis. Histological analyses revealed that the skins cultured on the PLGA membranes had thickness of about 400 µm and that there were no defects in the quality of the skins cultured on the PLGA membranes when compared with those cultured on the conventional (nonbiodegradable) commercial membranes. The cultured LbL-3D skins were then transplanted together with the PLGA membranes onto full-thickness excisional wounds in mice. At 7 days posttransplantation onto a mouse, the tissues above and below the membrane were connected through the holes with collagen-positive fibers that appeared to migrate from both the host and donor sides, and favorable reepithelization was observed throughout the transplanted skin region. However, insufficient engraftment was observed in some cases. Thus, further optimization of the membrane conditions would be needed to improve the transplantation outcome.

Formation of unimer nanoparticles by controlling the self-association of hydrophobically modified poly(amino acid)s.

Amphiphilic block or graft copolymers have been demonstrated to form a variety of self-assembled nano/microstructures in selective solvents. In this study, the self-association behavior of biodegradable graft copolymers composed of poly(γ-glutamic acid) (γ-PGA) as the hydrophilic segment and L-phenylalanine (Phe) as the hydrophobic segment in aqueous solution was investigated. The association behavior and unimer nanoparticle formation of these γ-PGA-graft-Phe (γ-PGA-Phe) copolymers in aqueous solution were characterized with a focus on the effect of the Phe grafting degree on the intra- and interpolymer association of γ-PGA-Phe. The particle size and number of polymer aggregates (N(agg)) in one particle of the γ-PGA-Phe depended on the Phe grafting degree. The size of γ-PGA-Phe with 12, 27, 35, or 42% Phe grafting (γ-PGA-Phe-12, -27, -35, or -42) was about 8-14 nm and the N(agg) was about 1, supporting the presence of a unimolecular graft copolymer in PBS. The pyrene fluorescence data indicated that γ-PGA-Phe-35 and -42 have hydrophobic domains formed by the intrapolymer association of Phe attached to γ-PGA. These results suggest that the Phe grafting degree is critical to the association behavior of γ-PGA-Phe and that γ-PGA-Phe-35 and -42 could form unimer nanoparticles. Moreover, when γ-PGA-Phe-42 dissolved in DMSO was added to various concentrations of NaCl solution, the particle size and N(agg) could be easily controlled by changing the NaCl concentration during the formation of the particles. These results suggest that biodegradable γ-PGA-Phe is useful for the fabrication of very small nanoparticles. It is expected that γ-PGA-Phe nanoparticles, including unimer particles, will have great potential as multifunctional carriers for pharmaceutical and biomedical applications, such as drug and vaccine delivery systems.

Controlled release using a polymer stereocomplex capsule through the selective extraction and incorporation of one capsule shell component.

Isotactic poly(methyl methacrylate) (it-PMMA)/syndiotactic poly(methacrylic acid) (st-PMAA) stereocomplex hollow capsules were fabricated by the deposition of stereocomplex films of it-PMMA and st-PMAA on silica particles by alternate layer-by-layer assembly and the subsequent removal of the silica particles with aqueous HF. The selective extraction of st-PMAA from the it-PMMA/st-PMAA stereocomplex capsule shells was successfully carried out by immersion in a pH 6-9 aqueous solution. The incorporation of st-PMAA into the resulting porous capsule shells was performed by immersion in an acetonitrile/water (1/1) solution of st-PMAA. The controlled release of an encapsulated dye from the it-PMMA/st-PMAA hollow capsules was achieved by combining the selective extraction of st-PMAA from the capsule shells and the incorporation of st-PMAA into the resulting porous shells.

Fusion of porous isotactic poly(methyl methacrylate) thin films fabricated on silica nanoparticles with stepwise stereocomplex assembly.

Structures of silica nanoparticles coated with stereocomplex thin films composed of isotactic (it) poly(methyl methacrylate) (PMMA) and syndiotactic (st) poly(methacrylic acid) (PMAA) and with porous it-PMMA thin films under gentle stirring or static conditions were analyzed by dynamic light scattering (DLS) and scanning electron microscopy (SEM), respectively. The porous it-PMMA films were fabricated by stepwise stereocomplex assembling of it-PMMA and st-PMAA, and subsequent extraction of the st-PMAA from the films. From DLS results, an evident difference was not observed between the it-PMMA films and the stereocomplex films, whereas the it-PMMA films after 10 h of stirring in acetonitrile/water (4/6, v/v) and drying on a SEM stage fused to form nanostructured networks. The fusion of the it-PMMA films on the silica nanoparticles occurred not by the dissolution of it-PMMA in the mixed solvent, but rather by an interaction of the it-PMMA chains driven by the slight solvation of acetonitrile without dissolution. Thus, leaving the solution at rest would be important for film fusion on the particles, and multiple spherical substrates could promote the crosslinking of the it-PMMA chains on the particles.

Interpenetrating polymer networks of poly(N-vinylacetamide) and stimuli responsive polymers applied to novel amphiphilic gel.

The swelling behaviors of IPN with poly(N-vinylacetamide) (PNVA), which possibly converts from nonionic gel to cationic gel, and the stimuli responsive polymers, such as poly(acrylic acid) (PAAc) and poly(N-isopropylacrylamide) (PNIPAm) were investigated in order to prepare the stimuli responsive amphiphilic gel. When the monomer concentrations were uniformed at the IPN preparation, the obtained PNVA/PAAc IPN showed the pH responsivity with around 100 of swelling ratio at pH 4 to around 1 of swelling ratio at pH 2, although it lost the amphiphilicity due to the lack of swelling in ethanol. On the other hand, the gelation of N-vinylacetamide at 2 M in PNIPAM gel resulted in thermosensitive and amphiphilic hydrogel, that the swelling ratio in EtOH/water (3/7, v/v) also decreased, compared to the value in water at 25 degrees C.

Control of cell surface and functions by layer-by-layer nanofilms.

Various nanometer-sized multilayers were directly prepared onto the surface of mouse L929 fibroblast cells by a layer-by-layer (LbL) assembly technique to control the cell surface microenvironment and cell functions, such as viability, morphology, and proliferation. The species of LbL nanofilms strongly affected the cell morphology and growth. Polyelectrolyte (PE) multilayers induced a round-shaped morphology of the adhered cells, although each component of the multilayers had high cytocompatibility, whereas fibronectin (FN)-gelatin (G) and -dextran sulfate (DS) multilayers with FN-binding domain interactions (FN films) showed extended morphologies of the cells similar to that of control cells (without films). A clear difference in cell proliferation was observed for PE and FN films. The cells with FN films on their surfaces showed good proliferation profiles independent of the film thickness, but cell proliferation was not observed using the PE films although the cells survived during the culture period. Fluorescence microscopic and scanning electron microscopic observations clearly suggested a nanometer-sized meshwork morphology of the FN films on the cell surface after 24 h of incubation, whereas the PE films showed homogeneous film morphologies on the cell surface. These nanomeshwork morphologies seemed to be similar to the fibrous structure of the natural extracellular matrix. The results of this study demonstrated that the components, charge, and morphology of LbL nanofilms prepared directly on the cell surface strongly affected cell functions, and the effects of these LbL nanofilms on cell functions differed vastly as compared to PE films prepared on a substrate. The preparation of LbL nanofilms onto a cell surface might be a novel and interesting technique to control cell functions.

Cell proliferation on stereoregular isotactic-poly(propylene oxide) as a bulk substrate.

Stereoregular isotactic-poly(propylene oxide) (it-PPO) was investigated for use as a biomaterial surface. The conventional characteristic of nonstereoregular atactic PPO was altered to a hydrophobic solid nature (contact angle: 95.6 ± 3.8°), which resulted in the potential solid surface applications. The high crystallinity of it-PPO created both smooth and microsized, random crater-shaped surfaces using the spin coating and dip coating approaches, respectively. The results of protein adsorption with bovine serum albumin (BSA), bovine gamma globulin (BγG), and bovine plasma fibrinogen (BPF) showed multilayered adsorption onto it-PPO. Mouse fibroblast L929 cells adhered onto the it-PPO surfaces, and cultured well as compared with commercially available Cell Desk LF1 as a control surface. These unique physical characteristics of it-PPO were due to the configuration of the polymer chain backbone structure, which maintained the polyether chemical structure.

Polyelectrolyte multilayers-modified polystyrene plate improves conventional immunoassay: full covering of the blocking reagent.

In this study, we fabricated polyelectrolyte multilayers (PEMs) on a polystyrene (PS) plate by a simple and novel alternate drop coating process (Acta Biomaterialia 2008, 4, 1255-1262), leading to the construction of a functional platform for improving conventional enzyme-linked immunosorbent assay (ELISA) systems. Poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS) were used as cationic and anionic polyelectrolytes, and then positively or negatively charged surfaces were obtained on the PEMs. The PDDA/PSS PEMs on the PS plate had the following favorable characteristics. On the positive PEMs, the coverage of the blocking reagent (ovalbumin from egg white: OVA) was over 100% by electrostatic interaction between the protein and PEMs, hence, nonspecific adsorption from the secondary antibody was efficiently suppressed. Moreover, the positive PEMs showed higher sensitivity on the ELISA than the negative PEMs, including the PS plate. Regularly oscillating behavior for sensitivity (specific signal-to-noise ratio) was observed on 1-10-step assemblies. The calibration curves for antigen detection on the positive PEMs had wide range of concentration from 0.002 to 5 microg/mL, and largest change in signal as compared with the negative PEMs and the PS plate. In summary, we discovered that positive PEMs possessed excellent performance for ELISA systems, and PEMs could easily improve the immunoassay with a convenient process and diverse substrates.