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Many existing synthetic hydrogels are inappropriate for repetitive motions because of large hysteresis, and their mechanical properties in warm and saline physiological conditions remain understudied. In this study, a stretch-rate-independent, hysteresis-free, elastic, and tough nanocomposite hydrogel that can maintain its mechanical properties in phosphate-buffered saline of 37 °C similar to warm and saline conditions of the human body is developed. The strength, stiffness, and toughness of the hydrogel are simultaneously reinforced by biomimetic silica nanoparticles with a surface of embedded circular polyamine chains. Such distinctive surfaces form robust interfacial interactions by local topological folding/entanglement with the polymer chains of the matrix. Load transfer from the soft polymer matrix to stiff nanoparticles, along with the elastic sliding/unfolding/disentanglement of polymer chains, overcomes the traditional trade-off between strength/stiffness and toughness and allows for hysteresis-free, strain-rate-independent, and elastic behavior. This robust reinforcement is sustained in warm phosphate-buffered saline. These properties demonstrate the application potential of the developed hydrogel as a soft, elastic, and tough bio-strain sensor that can detect dynamic motions across various deformation speeds and ranges. The findings provide a simple yet effective approach to developing practical hydrogels with a desirable combination of strength/stiffness and toughness, in a fully swollen and equilibrated state.
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The efficient evolution of gaseous hydrogen and oxygen from water is required to realize sustainable energy conversion systems. To address the sluggish kinetics of the multielectron transfer reaction, bifunctional catalyst materials for both the hydrogen evolution reaction (HER) and the oxygen evolution reaction (OER) should be developed. Herein, a tailored combination of atomically minimized iridium catalysts and highly conductive black WO3- x nanofiber supports are developed for the bifunctional electrolyzer system. Atomic Ir catalysts, particularly those that activate the OER, minimize the utilization of precious metals. The oxygen-deficient black WO3- x NF support, which boosts the HER, offers increased electronic conductivity and favorable nucleation sites for Ir loading. The Ir-black WO3- x NFs exhibit increased double-layer capacitance, a significantly reduced onset potential, lower Tafel slope, and stable cyclability for both the OER and HER, compared to large-sized Ir catalysts loaded on white WO3 nanofibers. This study offers a strategy for developing an optimal catalyst material with suitable supports for high-performance and economical water electrolysis systems for achieving carbon-negative targets.
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Anion exchange membrane water electrolysis (AEMWE) offers a sustainable path for hydrogen production with advantages such as high current density, dynamic responsiveness, and low-cost electrocatalysts. However, the development of efficient and durable oxygen evolution reaction (OER) electrocatalysts under operating conditions is crucial for achieving the AEMWE. This study systematically investigated Fe-Co-Ni ternary amorphous electrocatalysts for the OER in AEMWE through a comprehensive material library system comprising 21 composition series. The study aims to explore the relationship between composition, degree of crystallinity, and electrocatalytic activity using ternary contours and binary plots to derive optimal catalysts. The findings reveal that higher Co and lower Fe contents lead to increased structural disorder within the Fe-Co-Ni system, whereas an appropriate amount of Fe addition is necessary for OER activity. It is concluded that the amorphous structure of Fe-Co3-Ni possesses an optimal ternary composition and degree of crystallinity to facilitate the OER. Post-OER analyses reveal that the optimized ternary amorphous structure induces structural reconstruction into an OER-favorable OOH-rich surface. The Fe-Co3-Ni electrocatalysts exhibit outstanding performances in both half-cells and single-cells, with an overpotential of 256 mV at 10 mA cm- 2 and a current density of 2.0 A cm- 2 at 1.89 V, respectively.
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We present an innovative solvent-free micromolding technique for rapidly fabricating complex polymer microparticles with three-dimensional (3D) shapes utilizing a surface tension-induced dipping process. Our fabrication process involves loading a photocurable solution into micromolds through mold dipping. The loaded solution, induced by surface tension, undergoes spatial deformation upon mold removal caused by surface forces, ultimately acquiring an anisotropic shape before photopolymerization. Results show that the amount of photocurable solution loaded depends on the degree of capillary penetration, which can be adjusted by varying the dipping time and mold height. It enables the production of polymer particles with precisely controlled 3D shapes without diluting them with volatile organic solvents. Sequential micromolding enables the spatial stacking of the polymer domain through a bottom-up approach, facilitating the creation of complex multicompartmental microparticles with independently controlled compartments. Finally, we demonstrated the successful simultaneous conjugation of multiple model-fluorescent proteins through the biofunctionalization of microparticles, indicating functional stability and effective conjugation of hydrophilic molecules such as proteins. We also extend our capacity to create bicompartmental microparticles with distinct functionalities in each compartment, revealing spatially controlled functional structures. In summary, these findings demonstrate a straightforward, rapid, and reliable method for producing highly uniform complex particles with precise control over the 3D shape and compartmentalization, all accomplished without the use of organic solvents.
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BACKGROUND: Wastewater treatment plants contribute approximately 6% of anthropogenic methane emissions. Methanotrophs, capable of converting methane into polyhydroxybutyrate (PHB), offer a promising solution for utilizing methane as a carbon source, using activated sludge as a seed culture for PHB production. However, maintaining and enriching PHB-accumulating methanotrophic communities poses challenges. RESULTS: This study investigated the potential of Methylosinus trichosporium OB3b to bioaugment PHB-accumulating methanotrophic consortium within activated sludge to enhance PHB production. Waste-activated sludges with varying ratios of M. trichosporium OB3b (1:0, 1:1, 1:4, and 0:1) were cultivated. The results revealed substantial growth and methane consumption in waste-activated sludge with M. trichosporium OB3b-amended cultures, particularly in a 1:1 ratio. Enhanced PHB accumulation, reaching 37.1% in the same ratio culture, indicates the dominance of Type II methanotrophs. Quantification of methanotrophs by digital polymerase chain reaction showed gradual increases in Type II methanotrophs, correlating with increased PHB production. However, while initial bioaugmentation of M. trichosporium OB3b was observed, its presence decreased in subsequent cycles, indicating the dominance of other Type II methanotrophs. Microbial community analysis highlighted the successful enrichment of Type II methanotrophs-dominated cultures due to the addition of M. trichosporium OB3b, outcompeting Type I methanotrophs. Methylocystis and Methylophilus spp. were the most abundant in M. trichosporium OB3b-amended cultures. CONCLUSIONS: Bioaugmentation strategies, leveraging M. trichosporium OB3b could significantly enhance PHB production and foster the enrichment of PHB-accumulating methanotrophs in activated sludge. These findings contribute to integrating PHB production in wastewater treatment plants, providing a sustainable solution for resource recovery.
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Hidroxibutiratos , Metano , Methylosinus trichosporium , Esgotos , Esgotos/microbiologia , Methylosinus trichosporium/metabolismo , Hidroxibutiratos/metabolismo , Metano/metabolismo , Poliésteres/metabolismo , Biodegradação Ambiental , Águas Residuárias/microbiologia , Poli-HidroxibutiratosRESUMO
Staphylococcus aureus is a major pathogen that causes infections and life-threatening diseases. Although antibiotics, such as methicillin, have been used, methicillin-resistant S. aureus (MRSA) causes high morbidity and mortality rates, and conventional detection methods are difficult to be used because of time-consuming process. To control the spread of S. aureus, a development of a rapid and simple detection method is required. In this study, we generated a fluorescent anti-S. aureus antibody, and established a novel fluorescence-linked immunosorbent assay (FLISA)-based S. aureus detection method. The method showed high sensitivity and low limit of detection toward MRSA detection. The assay time for FLISA was 5 h, which was faster than that of conventional enzyme-linked immunosorbent assay (ELISA) or rapid ELISA. Moreover, the FLISA-based detection method was applied to diagnose clinically isolated MRSA samples that required only 5.3 h of preincubation. The FLISA method developed in this study can be widely applied as a useful tool for convenient S. aureus detection. KEY POINTS: ⢠A fluorescence-linked immunosorbent assay-based S. aureus detection method ⢠Simultaneous quantification of a maximum of 96 samples within 5 h ⢠Application of the novel system to diagnosis clinical isolates.
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Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Humanos , Imunoadsorventes , Staphylococcus aureus , Ensaio de Imunoadsorção Enzimática , Infecções Estafilocócicas/diagnóstico , AnticorposRESUMO
Prediction of protein-ligand binding poses is an essential component for understanding protein-ligand interactions and computer-aided drug design. Various proteins involve prosthetic groups such as heme for their functions, and adequate consideration of the prosthetic groups is vital for protein-ligand docking. Here, we extend the GalaxyDock2 protein-ligand docking algorithm to handle ligand docking to heme proteins. Docking to heme proteins involves increased complexity because the interaction of heme iron and ligand has covalent nature. GalaxyDock2-HEME, a new protein-ligand docking program for heme proteins, has been developed based on GalaxyDock2 by adding an orientation-dependent scoring term to describe heme iron-ligand coordination interaction. This new docking program performs better than other noncommercial docking programs such as EADock with MMBP, AutoDock Vina, PLANTS, LeDock, and GalaxyDock2 on a heme protein-ligand docking benchmark set in which ligands are known to bind iron. In addition, docking results on two other sets of heme protein-ligand complexes in which ligands do not bind iron show that GalaxyDock2-HEME does not have a high bias toward iron binding compared to other docking programs. This implies that the new docking program can distinguish iron binders from noniron binders for heme proteins.
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Hemeproteínas , Ligantes , Heme , Simulação de Acoplamento Molecular , Ligação Proteica , AlgoritmosRESUMO
Alkaline water electrolysis (AWE) is considered a promising technology for green hydrogen (H2 ) production. Conventional diaphragm-type porous membranes have a high risk of explosion owing to their high gas crossover, while nonporous anion exchange membranes lack mechanical and thermochemical stability, limiting their practical application. Herein, a thin film composite (TFC) membrane is proposed as a new category of AWE membranes. The TFC membrane consists of an ultrathin quaternary ammonium (QA) selective layer formed via Menshutkin reaction-based interfacial polymerization on a porous polyethylene (PE) support. The dense, alkaline-stable, and highly anion-conductive QA layer prevents gas crossover while promoting anion transport. The PE support reinforces the mechanical and thermochemical properties, while its highly porous and thin structure reduces mass transport resistance across the TFC membrane. Consequently, the TFC membrane exhibits unprecedentedly high AWE performance (1.16 A cm-2 at 1.8 V) using nonprecious group metal electrodes with a potassium hydroxide (25 wt%) aqueous solution at 80 °C, significantly outperforming commercial and other lab-made AWE membranes. Moreover, the TFC membrane demonstrates remarkably low gas crossover, long-term stability, and stack cell operability, thereby ensuring its commercial viability for green H2 production. This strategy provides an advanced material platform for energy and environmental applications.
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We report a simple platform for the fabrication of nonspherical alginate hydrogel particles using a dripping method. Hydrogel particles with novel morphologies, such as vortex ring, teardrop, disk, sphere, and mushroom, are fabricated by controlling various parameters. We monitored the deformation process of the hydrogel particles after they penetrated the crosslinking solution using a high-speed camera. Then, we proposed a mechanism showing a unique morphological transformation from a spherical to a disk shape. We demonstrated how controlling the collecting height that causes the drop impact force against the crosslinking solution surface was critical to producing hydrogel particles with these intriguing shapes. In particular, disk-shaped alginate particles show their ability as potential platforms for culturing mouse adrenocortical tumor cells (Y1) and a hippocampal neuronal cell (HT-22). To modify alginate particles, cell-adhesive gelatin is incorporated into the alginate matrix and then alginate particles are coated with poly(allylamine hydrochloride). Two modified alginate particles show good adhesion and proliferation rates on their surfaces. In particular, the hybrid hydrogel particles provide great potential to be developed into promising materials for cell culture, drug delivery, and tissue engineering.
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Alginatos , Hidrogéis , Animais , Camundongos , Engenharia Tecidual , Técnicas de Cultura de Células , GelatinaRESUMO
OBJECTIVES: S100A8 is highly expressed in several inflammatory and oncological conditions. To address the current lack of a reliable and sensitive detection method for S100A8, we generated a monoclonal antibody with a high binding affinity to human S100A8 to enable early disease diagnosis. RESULTS: A soluble recombinant S100A8 protein with a high yield and purity was produced using Escherichia coli. Next, mice were immunized with recombinant S100A8 to obtain anti-human S100A8 monoclonal antibodies using hybridoma technology. Lastly, the high binding activity of the antibody was confirmed and its sequence was identified. CONCLUSIONS: This method, including the production of antigens and antibodies, will be useful for the generation of hybridoma cell lines that produce anti-S100A8 monoclonal antibodies. Moreover, the sequence information of the antibody can be used to develop a recombinant antibody for use in various research and clinical applications.
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Anticorpos Monoclonais , Calgranulina A , Animais , Camundongos , Anticorpos Monoclonais/química , Hibridomas , Linhagem Celular , Proteínas Recombinantes/genética , BiomarcadoresRESUMO
Nitroreductase (NTR) has the ability to activate nitro group-containing prodrugs and decompose explosives; thus, the evaluation of NTR activity is specifically important in pharmaceutical and environmental areas. Numerous studies have verified effective fluorescent methods to detect and image NTR activity; however, near-infrared (NIR) fluorescence probes for biological applications are lacking. Thus, in this study, we synthesized novel NIR probes (NIR-HCy-NO2 1-3) by introducing a nitro group to the hemicyanine skeleton to obtain fluorescence images of NTR activity. Additionally, this study was also designed to propose a different water solubility and investigate the catalytic efficiency of NTR. NIR-HCy-NO2 inherently exhibited a low fluorescence background due to the interference of intramolecular charge transfer (ICT) by the nitro group. The conversion from the nitro to amine group by NTR induced a change in the absorbance spectra and lead to the intense enhancement of the fluorescence spectra. When assessing the catalytic efficiency and the limit of detection (LOD), including NTR activity imaging, it was demonstrated that NIR-HCy-NO2 1 was superior to the other two probes. Moreover, we found that NIR-HCy-NO2 1 reacted with type I mitochondrial NTR in live cell imaging. Conclusively, NIR-HCy-NO2 demonstrated a great potential for application in various NTR-related fields, including NTR activity for cell imaging in vivo.
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Corantes Fluorescentes , Dióxido de Nitrogênio , Corantes Fluorescentes/farmacologia , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Nitrorredutases/metabolismoRESUMO
Pseudomonas aeruginosa (P. aeruginosa) is a major pathogen that causes nosocomial infections and often exhibits antibiotic resistance. Therefore, the development of an accurate method for detecting P. aeruginosa is required to control P. aeruginosa-related outbreaks. In this study, we established an enzyme-linked immunosorbent assay method for the sensitive detection of three P. aeruginosa strains, UCBPP PA14, ATCC 27853, and multidrug-resistant ATCC BAA-2108. We produced a recombinant antibody (rAb) against P. aeruginosa V-antigen (PcrV), which is a needle tip protein of the type III secretion system of P. aeruginosa using mammalian cells with high yield and purity, and confirmed its P. aeruginosa binding efficiency. The rAb was paired with commercial anti-P. aeruginosa Ab for a sandwich ELISA, resulting in an antigen-concentration-dependent response with a limit of detection value of 230 CFU/mL. These results suggest that the rAb produced herein can be used for the sensitive detection of P. aeruginosa with a wide range of applications in clinical diagnosis and point-of-care testing.
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Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Anticorpos Antibacterianos/metabolismo , Antígenos de Bactérias , Ensaio de Imunoadsorção Enzimática , Humanos , Mamíferos , Infecções por Pseudomonas/diagnósticoRESUMO
We have successfully produced a recombinant human matrix metalloproteinase 9 (hMMP9) antigen with high yield and purity and used it to generate a hybridoma cell-culture-based monoclonal anti-hMMP9 antibody. We selected the most effective antibody for binding antigens and successfully identified its nucleotide sequence. The entire antigen and antibody developmental procedures described herein can be a practical approach for producing large amounts of monoclonal antibodies against hMMP9 and other antigens of interest. Additionally, the nucleotide sequence information of the anti-hMMP9 monoclonal antibody revealed herein will be useful for the generation of recombinant antibodies or antibody fragments against hMMP9.
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Anticorpos Monoclonais/genética , Metaloproteinase 9 da Matriz/genética , Proteínas Recombinantes/genética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Sequência de Bases , Técnicas de Cultura de Células , Regulação da Expressão Gênica , Humanos , Hibridomas/citologia , Fragmentos de Imunoglobulinas/química , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/imunologia , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , SolubilidadeRESUMO
Bone homeostasis plays a major role in supporting and protecting various organs as well as a body structure by maintaining the balance of activities of the osteoblasts and osteoclasts. Unbalanced differentiation and functions of these cells result in various skeletal diseases, such as osteoporosis, osteopetrosis, and Paget's disease. Although various synthetic nanomaterials have been developed for bone imaging and therapy through the chemical conjugation, they are associated with serious drawbacks, including heterogeneity and random orientation, in turn resulting in low efficiency. Here, we report the synthesis of bone-targeting ferritin nanoparticles for bone imaging. Ferritin, which is a globular protein composed of 24 subunits, was employed as a carrier molecule. Bone-targeting peptides that have been reported to specifically bind to osteoblast and hydroxyapatite were genetically fused to the N-terminus of the heavy subunit of human ferritin in such a way that the peptides faced outwards. Ferritin nanoparticles with fused bone-targeting peptides were also conjugated with fluorescent dyes to assess their binding ability using osteoblast imaging and a hydroxyapatite binding assay; the results showed their specific binding with osteoblasts and hydroxyapatite. Using in vivo analysis, a specific fluorescent signal from the lower limb was observed, demonstrating a highly selective affinity of the modified nanoparticles for the bone tissue. These promising results indicate a specific binding ability of the nanoscale targeting system to the bone tissue, which might potentially be used for bone disease therapy in future clinical applications.
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Ferritinas/genética , Nanopartículas Metálicas/química , Osteoblastos/efeitos dos fármacos , Peptídeos/genética , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/ultraestrutura , Diferenciação Celular/efeitos dos fármacos , Durapatita/química , Ferritinas/química , Ferritinas/farmacologia , Humanos , Imagem Molecular , Osteoblastos/ultraestrutura , Osteoclastos/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologiaRESUMO
AIM: This cross-sectional study aimed to examine the diagnostic ability of salivary matrix metalloproteinase (MMP)-9 lateral flow test (LFT) point-of-care (POC) kit and develop an algorithm for diagnosis of periodontitis. MATERIALS AND METHODS: Through Seoul National Dental Hospital, 137 participants (46 LFT negatives, 91 LFT positives) were recruited. For salivary diagnostics, 150 µl of the unstimulated saliva was applied to LFT-POC kit. To make a diagnosis of periodontitis, stage II-IV in modified new international classification system was used. Covariates encompassing age, sex, smoking and obesity were evaluated through face-to-face interview. Enzyme-linked immunosorbent assay was used for quantification of salivary MMP-9. To develop a diagnostic algorithm, multivariable logistic regression analysis was used. Receiver operating characteristic curve was applied for evaluating diagnostic ability. RESULTS: Diagnostic ability of salivary MMP-9 LFT-POC test was 0.82 (sensitivity of 0.92, specificity of 0.72) in total participants. Diagnostic algorithm using POC test resulted in a response equation, that is algorithm score = -3.675 + 2.877*LFT + 0.034*age + 0.121*sex + 0.372*smoking + 0.192*obesity. Diagnostic ability of the algorithm was 0.88 (sensitivity of 0.92, specificity of 0.85) with cut-off score of 0.589. CONCLUSIONS: Salivary MMP-9 LFT-POC kit showed appropriate diagnostic ability for periodontitis and would be an efficient tool for screening of periodontitis.
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Metaloproteinase 9 da Matriz , Periodontite , Biomarcadores , Estudos Transversais , Humanos , Lactente , Metaloproteinase 8 da Matriz , Periodontite/diagnóstico , Testes Imediatos , SalivaRESUMO
We have achieved heteroepitaxial stacking of a van der Waals ( vdW) monolayer metal, 1T'-WTe2, and a semiconductor, 2H-WSe2, in which a distinctively low contact barrier was established across a clean epitaxial vdW gap. Our epitaxial 1T'-WTe2 films were identified as a semimetal by low temperature transport and showed the robust breakdown current density of 5.0 × 107 A/cm2. In comparison with a series of planar metal contacts, our epitaxial vdW contact was identified to possess intrinsic Schottky barrier heights below 100 meV for both electron and hole injections, contributing to superior ambipolar field-effect transistor (FET) characteristics, i.e., higher FET mobilities and higher on-off current ratios at smaller threshold gate voltages. We discuss our observations around the critical roles of the epitaxial vdW heterointerfaces, such as incommensurate stacking sequences and absence of extrinsic interfacial defects that are inaccessible by other contact methods.
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A biosimilar is a biological medicinal product that is highly similar to an authorized biological product in terms of quality, biological activity, safety and efficacy. SB5 was developed by Samsung Bioepis as a biosimilar referencing adalimumab, and was authorized by the European Commission (EC) in August 2017 (Imraldi®). Extensive characterization studies were performed to demonstrate functional similarity of SB5 to reference adalimumab (Humira®, AbbVie Inc. and AbbVie Deutschland GmbH & Co. KG). SB5 and Humira® showed highly similar soluble TNF-α binding and neutralizing activity, as well as transmembrane TNF-α binding activity and reverse signaling induced in the membrane TNF-α expressing cell line. Both products exhibited similar binding of the Fc gamma receptors and Fc-related effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In addition, additional mechanisms of action induced by TNF-α, such as cytokine release and expression of adhesion molecules, were analyzed and shown to be similar between SB5 and Humira®. Taken together, our results demonstrate that SB5 and Humira® are highly similar in terms of their functional characteristics.
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Adalimumab , Medicamentos Biossimilares , Adalimumab/farmacocinética , Adalimumab/farmacologia , Medicamentos Biossimilares/farmacocinética , Medicamentos Biossimilares/farmacologia , Medicamentos Biossimilares/normas , Humanos , Células Jurkat , Equivalência TerapêuticaRESUMO
The Lewis acid-base adduct approach has been widely used to form uniform perovskite films, which has provided a methodological base for the development of high-performance perovskite solar cells. However, its incompatibility with formamidinium (FA)-based perovskites has impeded further enhancement of photovoltaic performance and stability. Here, we report an efficient and reproducible method to fabricate highly uniform FAPbI3 films via the adduct approach. Replacement of the typical Lewis base dimethyl sulfoxide (DMSO) with N-methyl-2-pyrrolidone (NMP) enabled the formation of a stable intermediate adduct phase, which can be converted into a uniform and pinhole-free FAPbI3 film. Infrared and computational analyses revealed a stronger interaction between NMP with the FA cation than DMSO, which facilitates the formation of a stable FAI·PbI2·NMP adduct. On the basis of the molecular interactions with different Lewis bases, we proposed criteria for selecting the Lewis bases. Owed to the high film quality, perovskite solar cells with the highest PCE over 20% (stabilized PCE of 19.34%) and average PCE of 18.83 ± 0.73% were demonstrated.
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Aluminum nanoparticles are considered promising as alternatives to conventional ink materials, replacing silver and copper nanoparticles, due to their extremely low cost and low melting temperature. However, a serious obstacle to realizing their use as conductive ink materials is the oxidation of aluminum. In this research, we synthesized the oxide-free aluminum nanoparticles using catalytic decomposition and an oleic acid coating method, and these materials were applied to conductive ink for the first time. The injection time of oleic acid determines the size of the aluminum nanoparticles by forming a self-assembled monolayer on the nanoparticles instead of allowing the formation of an oxide phase. Fabricated nanoparticles were analyzed by transmission electron microscopy and x-ray photoelectron spectroscopy to verify their structural and chemical composition. In addition, conductive inks made of these nanoparticles exhibit electrical properties when they are sintered at over 300 °C in a reducing atmosphere. This result shows that aluminum nanoparticles can be used as an alternative conductive material in printed electronics and can solve the cost issues associated with noble metals.
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Paper-based analytical devices (PADs) have been widely used in many fields because they are affordable and portable. For reproducible quantitative analysis, it is crucial to strongly immobilize proteins on PADs. Conventional techniques for immobilizing proteins on PADs are based on physical adsorption, but proteins can be easily removed by weak physical forces. Therefore, it is difficult to ensure the reproducibility of the analytical results of PADs using physical adsorption. To overcome this limitation, in this study, we showed a method of covalent binding of proteins to cellulose paper. This method consists of three steps, which include periodate oxidation of paper, the formation of a Schiff base, and reductive amination. We identified aldehyde and imine groups formed on paper using FT-IR analysis. This covalent bonding approach enhanced the binding force and binding capacity of proteins. We confirmed the activity of an immobilized antibody through a sandwich immunoassay. We expect that this immobilization method will contribute to the commercialization of PADs with high reproducibility and sensitivity.