Suspension culture is superior to planar culture in efficiently growing large numbers of cells. It also greatly improves cell functions by mimicking the in vivo environment. Although several methods are available to differentiate human induced pluripotent stem (iPS) cells into hepatocyte-like cells, their differentiation by suspension culture by using functional polymers remains unreported. We developed a suspension culture system using the functional polymers FP001 and FP003 to generate hepatocyte-like cells with superior phenotypes than human iPS cell-derived hepatocyte-like cells generated in planar culture. Specifically, optimizing the initial spheroid size and utilizing FP003 effectively maintained appropriate spheroid structures, resulting in higher albumin production compared to FP001. In 3D sphere culture, the mRNA expression of albumin (ALB), drug-metabolizing enzymes and transporters, and the activity of CYP1A2 were increased in the presence of 0.01% FP003. Additionally, immunofluorescence analysis confirmed uniform hepatic differentiation throughout the spheroids. These findings suggest that differentiation in 3D sphere culture with the functional polymer FP003 improved the function of human iPS cell-derived hepatocyte-like cells. Therefore, we conclude that this system may be an effective strategy to generate human iPS cell-derived hepatocyte-like cells with enhanced functions.
Rapid, accurate, and on-site detection of foodborne pathogens remains a critical challenge in ensuring food safety. Traditional microbiological methods, including culture-based assays, ELISA, and PCR, often require long processing times, specialized equipment, and trained personnel, limiting their practical application in real-time monitoring. Electrochemical biosensors have emerged as promising alternatives due to their portability, fast response, low cost, and compatibility with complex food matrices. Molecularly imprinted polymers (MIPs) offer a robust synthetic recognition layer for these sensors, providing high chemical stability and tenable selectivity while overcoming limitations of biological recognition elements such as antibodies and enzymes. However, conventional MIP fabrication often relies on empirical trial-and-error approaches, which can reduce reproducibility and slow sensor development. This review summarizes recent advances in computationally guided MIP design for electrochemical biosensing of key foodborne pathogens, including Salmonella spp., Listeria monocytogenes, Escherichia coli O157:H7, Staphylococcus aureus, and Pseudomonas aeruginosa. We discuss how molecular docking, density functional theory, and molecular dynamics simulations can predict template-monomer interactions, optimize cavity formation, and guide polymer properties, thereby improving sensor sensitivity, selectivity, stability, and response time. By integrating computational modeling with experimental electrochemical methods, these approaches enable a mechanism-driven development of portable, high-performance biosensors for microbial detection. This convergence of computational and microbiological methods provides a pathway toward next-generation, ready-to-use devices for rapid foodborne pathogen monitoring.
The chiral ligand (R)-3-(1-carboxyethoxy)benzoic acid [(R)-H2cea], was synthesized by modifying an aromatic carboxylic acid with lactic acid. This ligand was used to react with Zn2+ and Cd2+ ions under hydrothermal conditions in the presence of N-containing heterocyclic auxiliary ligands [1,4-bis(2-methyl-1H-imidazol-1-yl)benzene (1,4-bmib) and 1,1'-(2,5-dimethyl-1,4-phenylene)bis(1H-imidazole) (2,5-dpb)], successfully constructing two chiral coordination polymers (CPs), namely, poly[[μ-1,4-bis(2-methyl-1H-imidazol-1-yl)benzene-κ2N:N'][μ-(R)-3-(1-carboxylatoethoxy)benzoato-κ2O:O']zinc(II)], [Zn(C10H8O5)(C14H14N4)]n or HCU40-R, and poly[[μ-1,4-bis(1H-imidazol-1-yl)-2,5-dimethylbenzene-κ2N:N'][μ-(R)-3-(1-carboxylatoethoxy)benzoato-κ4O,O':O'',O''']cadmium(II)], [Cd(C10H8O5)(C14H14N4)]n or HCU41-R. Both CPs crystallize in the chiral space group P21, and the central chirality from the (R)-cea2- anion is transferred and amplified into supramolecular helical chirality. In HCU40-R, the Zn2+ ions adopt a tetrahedral geometry, forming a one-dimensional right-handed helical chain that further extends into a two-dimensional helical layer. In HCU41-R, the Cd2+ ions exhibit an octahedral geometry, displaying a double-helical chain and a twofold interpenetrated two-dimensional layered structure. Powder X-ray diffraction and elemental analysis confirmed the phase purity of the samples. Thermogravimetric analysis revealed thermal stability up to 350 °C for HCU40-R and 300 °C for HCU41-R. Fluorescence measurements showed emission peaks at 451 nm for HCU40-R and 491 nm for HCU41-R. Furthermore, both compounds exhibited second-order nonlinear optical (NLO) effects, with second harmonic generation (SHG) intensities of 0.38 and 0.35 times that of KDP, respectively.
Mechanically guided 3D assembly has emerged as a powerful approach for constructing complex mesostructures, but conventional strategies rely on selective chemical bonding between 2D precursors and elastomeric substrates, restricting material compatibility and scalability. Here, we introduce a physical anchoring-based, buckling-guided assembly platform that decouples 3D structure formation from chemical adhesion. The platform employs an elastomeric substrate patterned with an array of rods to achieve robust mechanical fixation, enabling the integration of diverse functional materials, including polymers, metals, water-soluble polymers, and stimuli-responsive systems, independent of their interfacial or mechanical properties. Oxygen plasma surface treatment further reduces adhesion, allowing reliable assembly of freestanding and large-area architectures. As a proof-of-concept, we integrate liquid crystalline networks (LCNs) to demonstrate light-responsive soft robotic systems capable of remote actuation. This physically anchored assembly strategy establishes a general and scalable framework for mesoscale fabrication, expanding material and geometric design freedom for applications in soft robotics, bioelectronics, and multi-material integration.
Cyclopentane is used to make high-performance plastics and composites. Cyclohexane is a polymer that has several specialized applications due to its unique properties, such as good chemical resistance, low moisture absorption, and high thermal stability. Despite their industrial relevance, a systematic comparative analysis of degree-based and neighborhood degree-based topological indices and their predictive capability for chemical properties remains limited in the existing literature. In this study, we compute and analyze several fundamental molecular descriptors, including the Zagreb indices, Randić index, atom bond connectivity index, geometric arithmetic index, sum connectivity index, and augmented Zagreb index for the molecular graphs networks of Cyclopentane and Cyclohexane. These indices can be used to correlate various properties of the polymers with the topological indices. The obtained topological indices are employed to establish quantitative structure-property relationships (QSPR) between molecular structure and physicochemical properties of these polymers. To address the lack of predictive modeling in earlier graph-theoretical studies, a machine-learning-based regression models are developed and applied to evaluate the strength of correlation between molecular descriptors and experimental property data. This approach provides a computationally efficient framework for molecular property estimation and offers a foundation for extending advanced learning models to more complex polymer networks.
Microtubules are dynamic cytoskeletal polymers whose lattice architecture regulates force generation, nucleotide hydrolysis, and recognition by motor proteins and microtubule-associated proteins (MAPs). Microtubule-stabilizing agents (MSAs), including taxanes and laulimalide/peloruside-site ligands, suppress depolymerization by binding to defined lattice sites, yet stabilization is not structurally neutral. How ligand chemistry reshapes lattice organization and function remains unresolved. Here, we address three mechanistic questions. First, do distinct ligand classes induce defined lattice states? Using X-ray fiber diffraction, we show that MSAs selectively stabilize two preferred longitudinal conformations, a compact state (~4.06 nm monomer rise) and an expanded state (~4.17 nm), while modulating lateral organization reflected in shifts in mean MT radius. These axial spacings cluster around discrete values across chemotypes, indicating stabilization of preexisting conformational minima rather than continuous distortion. Second, are these states interconvertible upon changes in ligand occupancy? Time-resolved diffraction reveals that longitudinal transitions occur within seconds of ligand addition even at substoichiometric occupancy, whereas, lateral equilibration proceeds slower, consistent with redistribution within heterogeneous protofilament organizations. Third, do such structural states alter nucleotide hydrolysis and motor/MAP behavior? Expanded lattices are associated with reduced apparent GTP hydrolysis rates under steady-state assembly conditions and altered kinesin motility, whereas compact lattices preferentially promote tau binding and distinct motor interaction profiles. Together, these findings establish longitudinal lattice conformation as a regulatory parameter and position MSAs as chemical tools that bias a dynamic structural landscape with predictable catalytic and transport consequences.
This study focuses on the CO2 capture performance of poly(N,N-dimethylaminopropyl acrylamide) (PDMAPAm) and poly(N-[3-(dimethylamino)propyl]-acrylamide)-b-poly(methyl methacrylate) (PDMAPAm-b-PMMA) diblock copolymers for fabrication of CO2-responsive membrane adsorbers. By systematically varying the block composition of the diblock copolymer PDMAPAm-b-PMMA, optimal compositions for maximizing CO2 adsorption capacity are identified. The adsorption mechanisms were characterized under both dry and humid conditions, revealing distinct physisorption and chemisorption pathways. The first major novelty of this work is the creation of a unified kinetic model that, for the first time, integrates polymerization kinetics with adsorption kinetics, allowing the CO2 uptake capacity of membrane adsorbers to be directly predicted from the underlying polymer properties. A second key innovation is the use of this unified model to rationally design and fabricate a polymer membrane adsorber that achieves a CO2 uptake capacity of 6 mmol g-1, substantially exceeding the performance of commercially available polymer-based sorbents.
Co-precipitation of poly(hydroxy butyrate), a biobased and biodegradable polymer produced through bacterial fermentation, and hen egg white lysozyme, an enzyme with antimicrobial activity, is a viable route towards non-covalent enzyme immobilization. Ultrasonication during the co-precipitation process results in spherical particles with a well-defined size distribution. The amount of immobilized enzyme can be controlled through variation of its concentration in the antisolvent (aqueous) phase. The presence and density of the enzyme bound to the particles have a decisive influence on the internal order (chain arrangement) and thermal properties of the biopolymer. The fraction of amorphous (glassy) material increases with higher enzyme content. When the actual amount of enzyme bound to the particles is accounted for, limited detrimental effects of the immobilization procedure on the antimicrobial activity are observed, compared to the free enzyme. Immobilization also improves the stability of the antimicrobial activity when exposed to elevated temperatures. The activity of the immobilized enzyme doubles at 45 °C, compared to 25 °C, while that of the free enzyme decreases by 10%. Co-precipitation of enzymes such as hen egg white lysozyme in the presence of biopolymers is a straightforward approach to prepare fully biobased, functional (e.g., antimicrobial) materials with beneficial properties. This enzyme immobilization approach results in improved stability against harsh environmental conditions (e.g., elevated temperatures) which is advantageous for applications such as shelf-life extending packaging.
Core-shell nanoformulations from linear (PEG-b-PCL) and branched (PEG2-b-PCL) polymers containing a multi-potent drug interact with human serum proteins. These interactions are affected by PEG shell density, leading to enhanced lipophilic cargo release without drug aggregation.
By combining amphiphilic polymers with natural biomolecules, layer-by-layer (LbL) surface modification technology provides a versatile strategy to tailor the interfacial properties of self-assembled fullerene nanostructures for biomedical applications. Herein, we developed LbL surface-modified supramolecular fullerene microrods (FMR) to investigate tunable cell-material interactions associated with a cell-feeding phenomenon. Using LbL surface modification, FMR (average length 55 ± 8 μm and diameter 1.7 ± 0.6 μm) prepared by the liquid-liquid interfacial precipitation (LLIP) method was endowed with a layered polymer structure, thereby forming multilayer-coated fullerene microrods (FMR-P/G) with enhanced surface hydrophilicity. After 12 h of gelatin cross-linking, the resulting FMR-P/G_12 h sample exhibited layered structures, with Pluronic and gelatin layers stacked with thicknesses of ∼19 ± 6 and ∼31 ± 4 nm, respectively. The contact angle of FMR decreased from 104° to 44°, reflecting a pronounced enhancement in surface wettability. The impact of the surface-modified FMR-P/G_12 h sample on the biological behavior of NIH/3T3 fibroblasts was explored to assess its potential for biomedical applications. When FMR-P/G_12 h was used to treat NIH/3T3 fibroblasts, the cells exhibited markedly enhanced early stage viability (∼152% at 1-day culture and ∼349% at 3-day culture), while showing comparable survival levels during extended culture (cell viability ∼323% at 14-day culture). This effect may be attributed to the improved hydrophilicity and interfacial properties of the polymer-modified fullerene microrods, which facilitate favorable cell-material interactions during the early culture stage. These results suggest that FMR-P/G_12 h can modulate cellular responses through surface-engineered interfaces, highlighting the feasibility of LbL surface-modified self-assembled fullerene nanostructures for biointerface-related applications. Overall, this study demonstrates the potential of LbL-engineered fullerene constructs for interface-driven biomedical material design.
The behavior and mechanisms of marine biofouling─the process by which marine organisms attach onto surfaces in the ocean─have been extensively studied using various biofoulers. Barnacles are often used as model hard fouling organisms, which deposit a proteinaceous adhesive from the shell edge that is later calcified; the side plates and base plates remain on some surfaces even after death. In nature and laboratory settings, barnacles can settle as larvae (cyprids), juveniles, and adults. Much work on barnacle biofouling has focused on characterizing its influence on surface behavior, including inducing crevice corrosion, lowered surface modulus, and dehydration. However, details of the adhesive biomolecules' interaction with fouling surfaces are still under exploration. In this paper, we employed sum frequency generation (SFG) vibrational spectroscopy, a surface-sensitive optical technique that can elucidate molecular behavior at the buried interface between a barnacle and a fouling substrate. Several barnacle/substrate interfaces were studied, using juvenile and adult barnacles, and hydrophobic and hydrophilic substrates with varying complexity. Our results indicated that adhesive proteins were present in both juvenile and adult systems, and the strength, ordering, and structure of these proteins differed between surfaces. Interfacial dehydration occurred when barnacles attached to the nonantifouling surfaces, while surface hydration induced antifouling/fouling-release behavior on zwitterionic coatings. In this study, SFG successfully probed buried barnacle/polymer interfaces, providing vital insight into the hydration mechanisms that are necessary for promoting and deterring bioadhesion and leading the way for future studies between fouling organisms and highly functionalized nonfouling polymers.
Microbial aerosols pose a serious threat to public health and safety due to their pathogenicity and need to be monitored quantitatively. Although evaluating airborne particulate pollution by size/number is the gold standard for atmospheric assessment, it cannot be applied to microbial aerosol detection because impurities of similar size interfere. Therefore, we propose an innovative detection method that amplifies the size signal of bacterial cells via colloidal encapsulation to count microbial aerosols in the "clean-size-range". This method can detect total microbial aerosols after cell surface engineering and detect special microbial aerosols by grafting bacteria-templated polymers. The detection sensitivity can reach 50 CFU/mL in water and 6 CFU/m3 in air. This work provides an innovative approach to the total and specific detection of microbial aerosols by "transforming complex microbial aerosols detection into simple polymer particle counting". This innovative approach can achieve precise microbial detection in complex particulate environments.
Ganoderma lucidum polysaccharides exhibit various biological activities, but conventional extraction is biased toward high-molecular-weight (Mw) polymers. This study aimed to explore the structure and bioactivity of low-Mw polysaccharides from G. lucidum (AGLP1). A low-Mw heteroglucan from G. lucidum was purified and identified as a heteroglucan mainly composed of a (1 → 3)-linked glucopyranosyl backbone with (1 → 6)-linked glucopyranosyl side chains. In vivo investigations revealed that AGLP1 significantly enhanced the spleen index, macrophage phagocytic activity and proliferation of T and B lymphocytes. These immunomodulatory effects were gut microbiota-dependent, as evidenced by their abolition in microbiota depleted mice via antibiotic (ABX) treatment. Mechanistically, AGLP1 reshaped the gut microbiota and serum metabolite profile by enriching potentially beneficial bacteria such as Ruminococcaceae and Eubacterium brachy group, as well as associated metabolites including decanoic acid, stearic acid and pimelic acid. In vitro fermentation using a human fecal model showed that AGLP1 also consistently increased the abundances of Ruminococcaceae and Eubacterium brachy group. These findings demonstrate that the immunomodulatory and microbiota-reshaping effects of AGLP1, observed in animal models, were also replicated in a human gut simulation. This underscores its potential as a functional food ingredient.
Coal gangue is the main solid waste generated during the mining and processing of coal. Its resource utilization is a pressing issue that needs to be addressed urgently at present. Meanwhile, lead pollution has become one of the major environmental challenges faced by global soil and water bodies. This study utilized the bacterium (Bacillus megaterium) to conduct microbial modification of coal gangue, with the aim of enhancing its application performance in the remediation of heavy metal pollution. By evaluating the tolerance of the strain to different Pb2+ concentrations, the adsorption behavior of the modified coal gangue (CG-W) was systematically investigated, and the adsorption mechanism was revealed by combining techniques such as XRD, XPS and FT-IR. The results show that Bacillus megaterium can still grow normally under the condition of 800 mg/L Pb2+, demonstrating a high tolerance; the modified coal gangue achieved a removal rate of 94% for Pb2+ under the optimal adsorption conditions (initial pH 6, temperature 40°C, and time 60 min) with an initial Pb2+ concentration of 200 mg/L. Under the optimal modification conditions (bacterial concentration of 5.88×1012CFU/mL, load temperature of 30 °C, and coal gangue pH of 6.0-8.0), the repair mechanism mainly results from the complexation effect of extracellular polymers secreted by microorganisms on Pb2+, as well as the ion exchange process involving mineral components. This study provides an effective approach for the resource utilization of coal gangue and the treatment of heavy metal pollution.
Microplastic (MP) pollution has been increasingly documented in remote high-altitude environments worldwide, including the European Alps, Himalayas, Andes, and polar snowfields, where atmospheric transport delivers plastic particles even to regions far from emission sources. Despite this growing body of evidence, the occurrence of MPs in the mountainous regions of Türkiye remains unknown. This study presents the first nationwide investigation of MP contamination in alpine snow, conducted across eleven sites, including Mount Ağrı, Süphan, Kaçkar, Erciyes, Uludağ, and Sandras. MPs were detected at all locations, with an average concentration of 286 ± 91 MPs/L with the highest concentration detected at Sandras Mountain (908 MPs/L) and the lowest at Uzundere-Uzunkavak, Erzurum (16 MPs/L). Across the eleven sites, the mean relative shape composition was 71.2 ± 6.1% fibres, 23.8 ± 5.8% fragments, and 5.0 ± 4.5% films (summing to 100% as percentages of all detected MPs). Polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), nylon (PA), and polymethyl methacrylate (PMMA) were identified as the primary polymers. Spatial variability suggested both atmospheric deposition and local human activities contributed to contamination. Backward trajectory analysis indicated that microplastic deposition in snow was influenced by both long-range atmospheric transport from Africa and the Mediterranean and short-range local emissions, highlighting the combined impact of transboundary and regional processes on microplastic distribution. These first-season findings provide a preliminary baseline that even isolated alpine snowpacks of Türkiye can receive measurable MP inputs of both regional and long-range origin, highlighting the need for sustained, multi-season monitoring of high-mountain ecosystems within national and transboundary mitigation frameworks.
Biosensing technologies play a critical role across the healthcare, environmental monitoring, and food safety sectors. The in vivo sensing of biomolecules is challenging due to the non-biocompatibility of nano-microelectrodes. In this regard, lignocellulosic materials will have a significant impact on sensors owing to their outstanding properties. Although lignocellulose lacks conductivity, it can be modified with other metal nanoparticles or conductive polymers to improve its conductivity. By leveraging functionally applied nanomaterials with lignocellulose, promising flexible biosensors can be developed with enhanced sensitivity, selectivity, and versatility. This integration of lignocellulosic materials with nanomaterials enables advanced biosensors with improved performance, facilitated by their high surface area-to-volume ratios and suitability for biomolecule immobilization. Lignocellulosic nanofibrils exhibit thermal stability, absorption in the ultraviolet-visible (UV-vis) region, water stability, and reduced moisture sensitivity and enhance sensor performance. Lignocellulosic materials have emerged as promising substrates for the development of next-generation biosensors. This review explores the suitability of lignocellulose for biosensing applications. Here, we discuss how plant-based materials have been used for biomolecule sensing. Lignocellulose has outstanding mechanical properties, which is why it can be used as a base material and sensing electrode to fabricate brain-on-chip and organ-on-chip devices. Because it is a plant-derived material, it also exhibits microfluidic properties. A cellulose skin-substituted natural polymer shows promise as a substrate for wearable sensors.
In this study, four zein-ECG polymer conjugates were prepared by grafting two epicatechin gallate (ECG)-derived polymers (BP and EP) onto zein using alkaline and radical approaches. The effects of ECG polymerization and grafting strategies on conjugate properties were compared. SDS-PAGE, FT-IR, CD, and fluorescence spectroscopy confirmed covalent-modification-reduced migration, an α-helix- to-β-sheet triggered transition, and quenched zein fluorescence. SEM revealed that the alkaline conjugates formed rod-like aggregates, whereas the radical conjugates retained their spherical morphology. The BP-zein conjugates exhibited the maximum grafting efficiency and total phenolic content. Alkaline modification significantly improved solubility, while covalent bonding enhanced interfacial diffusion and rearrangement. Correlation analysis confirmed a strong positive correlation between the total phenolic content and grafting efficiency, both of which governed the performance of the conjugate. Overall, the modification method and ECG polymer type collectively determined the structure, physicochemical properties, and biological activity of the zein conjugates.
Bone loss occurs in astronauts during prolonged spaceflight, thus indicating the sensitivity of skeletal homeostasis to altered gravitational environments. Previous studies have shown that microgravity affects osteoclast differentiation and bone resorption, which suggests that osteoclasts possess mechanisms to sense and respond to gravity-generated mechanical forces. For testing of the related mechanisms, hypergravity can be experimentally reproduced with use of a centrifuge. In the present study, osteoclasts derived from mouse bone marrow were subjected to hypergravity under three conditions: 30G exposure using a non-CO2 centrifuge system, and short- or long-term exposure to 3G or 5G using an incubator-compatible centrifuge system. Cytoskeletal organization and resorptive function were assessed using TRAP (tartrate-resistant acid phosphatase) staining, F-actin visualization, and dentin pit assays. In addition, phosphoproteomic analysis was performed after short-term exposure to 5G hypergravity. Hypergravity exposure for as brief as 30 minutes compromised F-actin ring integrity, reduced fluorescence intensity, and promoted nuclear repositioning toward actin rings, whereas tubulin and vinculin localization remained unchanged, and the structural alterations corresponded to attenuated resorption pit formation. Quantitative phosphoproteomic profiling revealed coordinated hypergravity-dependent changes in phosphorylation across multiple cellular modules, including cytoskeletal organization, membrane trafficking, intracellular signaling, and nuclear regulatory pathways. Together, these results indicate that osteoclasts are sensitive to gravity-generated mechanical loading, with hypergravity rapidly modifying F-actin-associated cytoskeleton properties and reprogramming phosphorylation-dependent signaling networks, ultimately attenuating bone-resorptive activity. These findings provide mechanistic insight into how osteoclasts respond to altered gravitational loading conditions and have implications for skeletal adaptation during spaceflight and under altered mechanical loading conditions on Earth.
We report a complete neutron-scattering and molecular dynamics investigation of the structure and dynamics of monomer and polymer phases of C60 carbon peapods. Above ∼250 K, the physics of the confined chains can fully be described without accounting for the nanotube, the latter merely playing the role of a container for a 1D system─the system can be described as an unpinned state in the extended Frenkel-Kontorova framework. As the temperature is lowered below about 250 K, we observe a progressive damping of the longitudinal acoustic phonons, measured in both monomer and polymer data. As a set of experimental observations suggest that this damping can be attributed neither to a 3D ordering of the chains nor to a transition driven by rotation-rotation-translation coupling, we attribute it to an increase in the chain-nanotube interaction. This translates into a progressive pinning of the C60 chains on the nanotube lattice as the temperature is lowered and explains the observed low-temperature damping.
N-doped graphene (NG) is a promising electrocatalyst for the oxygen reduction reaction (ORR) at the cathodes of Polymer Electrolyte Fuel Cells (PEFCs). The NG catalyst is generally supported on an electrode substrate composed of carbon materials. However, the influence of the carbon substrate on the ORR performance of the NG catalyst has not been systematically explored. In this study, we have systematically investigated the impact of the carbon substrate on the ORR activity of the NG catalyst using first-principles calculations based on density functional theory. Specifically, we have examined the ORR activity of the NG catalyst on graphene-based substrates with a van der Waals (vdW) interface. It has been revealed that the difference in work function between the substrate and the NG catalyst dominates the ORR activity; the maximum electrode potential (UMax) depends linearly on the work function difference. Such a linear relationship is derived from the fact that the net charge of the NG catalyst scales linearly with the work function of the substrate. Furthermore, UMax was predicted to show a volcano trend with respect to the work function difference. This is because the reaction step that determines UMax switches due to the change in the free energies of reaction intermediates by charge transfer across a vdW gap, indicating an optimal work function difference that maximizes the ORR activity of the NG catalyst supported on graphene-based substrates. The establishment of experimental techniques to control the work function difference at the substrate/NG catalyst interface will be key to achieving superior catalytic performance.