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Metal-organic frameworks (MOFs) as carriers for high-capacity loading of HRP-IgG and gold nanoparticles are introduced, to prepare MOF hybrids with enhanced peroxidase activity. The prepared MOF hybrids were employed to establish an indirect competitive colorimetric immunoassay for chloramphenicol (CAP) detection, in which the limit of detection for CAP is 0.006 µg·L-1, only one-fifth of that of the conventional ELISA using the same antibodies and antigens. The linear range was 0.008-0.108 µg·L-1, and the recovery of spiked milk samples varied in the range 76.0-106.0% through three independent experiments. Our proposed colorimetric immunoassay using the MOF hybrid immunoprobe provides a novel platform for ultra-sensitive determination of CAP residues, and it also could be used as a signal amplification model for the high-performance colorimetric immunoassay in food safety monitoring.
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Nanopartículas del Metal , Estructuras Metalorgánicas , Peroxidasa , Colorimetría , Cloranfenicol , Oro , Peroxidasas , Inmunoensayo , ColorantesRESUMEN
Electrochemical in situ sensing of small signal molecules released from living cells has an increasing significance in early diagnosis, pathological analyses, and drug discovery. Here, a living cell-fixed sensing platform was built using the BC@DNA-Mn3(PO4)2 nanozyme, in which a highly biocompatible bacterial cellulose riveted with very tiny Mn3(PO4)2; it not only delivers high catalytic activity toward superoxide anions but possesses excellent biocompatibility for cell adsorption and growth. Additionally, the experimental results suggested that fixing the living cells on the surface of the sensing platform facilitates tiny Mn3(PO4)2 activity centers to capture and detect O2â¢- very quickly and simultaneously has great potential in miniaturization, cost reduction, and real-time monitoring.
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Materiales Biocompatibles/química , Celulosa/química , ADN/química , Nanoestructuras/química , Compuestos Organometálicos/química , Superóxidos/análisis , Materiales Biocompatibles/síntesis química , Técnicas Biosensibles , Electrodos , Humanos , Tamaño de la Partícula , Superóxidos/metabolismo , Propiedades de Superficie , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
A corn-like CeO2/C coaxial cable textured by a cerium oxide shell and a carbon core was designed to sense NO. The carbon core possesses high electrical conductivity, and the CeO2 surface delivers excellent electrocatalytic activity. The sensor, typically operated at 0.8 V (vs. Ag/AgCl), exhibits a detection limit of 1.7 nM, which is 4-times lower than that of CeO2 nanotubes based one (at S/N = 3). It also displays wide linear response (up to 83 µM), a sensitivity of 0.81 µA µM-1 cm-2, and fast response (2 s). These values are highly competitive to that of a CeO2 tube (0.92 µA µM-1 cm-2 and 2 s). The sensor was used to quantify NO that is released by Aspergillus flavus. Graphical abstractSchematic representation of corn-like CeO2/C which can more sensitively and effectively detect NO released from A. flavus than when using CeO2 nanotubes, benefitting from its unique coaxial cable structure.
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In this work, bamboo fibers are chemically modified with NaOH solution of 1, 4, and 7 wt% concentrations at room temperature, respectively, and subsequently the untreated and treated fibers are prepared with epoxy resin for unidirectional composites by hot pressing molding technique. Tensile and micro-bond tests are conducted on the composite specimens to obtain mechanical properties, such as tensile strength and modulus, elongation at break, and interfacial strength. Besides, scanning electron microscopy (SEM) is employed to perform morphological observations for constituent damages. In addition, the influence of alkali concentration on the thermal performance of epoxy-based composites is examined by using differential scanning calorimetry (DSC) and thermogravimetric (TG) analysis. It is found that composite tensile strength reaches the maximum when the alkali concentration is 4%, increased by 45.24% compared with untreated composites. The composite elongation at break increases on increasing the concentration. Inversely, the composite modulus decreases as the concentration increases. Besides, the results demonstrate that the chemical treatment on the fiber surface could improve interface adhesion, as observed from its topography by SEM. Micro-bond test reveals that there is maximum interfacial shear strength when the alkali concentration is 4%, which increases by 100.30% in comparison with the untreated samples. In case of thermal properties, the DSC analysis indicates that the glass transition temperature is maximized at 4% alkali concentration, which is increased by 12.95%, compared to those from unmodified fibers. In addition, TG results show that the 4% concentration also facilitates thermal stability improvement, indicative of superior interfacial bonding.
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Fenómenos Químicos , Compuestos Epoxi/química , Sasa/química , Resinas Epoxi/química , Fenómenos Mecánicos , Propiedades de Superficie , TermogravimetríaRESUMEN
Human platelet derived growth factor (PDGF) is a major therapeutic protein with great demand in the clinical setting; however, its rate of supply is far from meeting needs. Here, we provide an effective strategy to produce PDGF-BB in large quantities using a transgenic silkworm. The codon-optimized PDGF-B gene regulated by the highly efficient sericin-1 expression system was integrated into the genome of a silkworm. The high transcriptional expression of the PDGF-BB gene in the transgenic silkworm competitively inhibited the transcription expression of the endogenous sericin-1 gene which caused a significant 37.5% decline. The PDGF-BB synthesized in the middle silk gland (MSG) of transgenic silkworms could form a homodimer through intermolecular disulfide bonds, which is then secreted into sericin lumen and finally, distributed in the sericin layer of the cocoon. In this study, a protein quantity of approximately 0.33 mg/g was found in the cocoon. Following a purification process, approximately 150.7 µg of recombinant PDGF-BB with a purity of 82% was purified from 1 g of cocoons. Furthermore, the bioactivity assays showed that the purified recombinant PDGF-BB was able to promote the growth, proliferation and migration of NIH/3T3 cells significantly. These results suggest that the silk gland bioreactor can produce active recombinant PDGF-BB as an efficient mitogen and wound healing agent.
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Becaplermina/metabolismo , Reactores Biológicos , Bombyx/metabolismo , Proteínas Recombinantes/biosíntesis , Animales , Animales Modificados Genéticamente , Becaplermina/genética , Biotecnología/métodos , Bombyx/genética , Humanos , Reproducibilidad de los Resultados , Seda/biosíntesisRESUMEN
Egg albumen is modified by hydrogen peroxide with concentrations of 5%, 10%, 15% and 30% at room temperature. Compared with devices without modification, a memory cell of Ag/10% H2O2-egg albumen/indium tin oxide exhibits obviously enhanced resistive switching memory behavior with a resistance ratio of 104, self-healing switching endurance for 900 cycles and a prolonged retention time for a 104 s @ 200 mV reading voltage after being bent 103 times. The breakage of massive protein chains occurs followed by the recombination of new protein chain networks due to the oxidation of amidogen and the synthesis of disulfide during the hydrogen peroxide modifying egg albumen. Ions such as Fe3+, Na+, K+, which are surrounded by protein chains, are exposed to the outside of protein chains to generate a series of traps during the egg albumen degeneration process. According to the fitting results of the double logarithm I-V curves and the current-sensing atomic force microscopy (CS-AFM) images of the ON and OFF states, the charge transfer from one trap center to its neighboring trap center is responsible for the resistive switching memory phenomena. The results of our work indicate that hydrogen- peroxide-modified egg albumen could open up a new avenue of biomaterial application in nanoelectronic systems.
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Cinnamaldehyde (CIN) is a promising natural preservative and generally recognized as safe for commodities as well as consumers. In this work, the antifungal effects of CIN on Aspergillus flavus were evaluated both in solid and in liquid culture conditions. Our results indicated that CIN effectively inhibited radial growth, spore production, mycelium formation, and aflatoxin B1 biosynthesis by A. flavus in a dose-dependent manner. At the concentration of 104 mg L(-1), CIN exposure was able to completely inhibit fungal growth as well as aflatoxin B1 production. Furthermore, the inhibitory activities of CIN were closely connected with the treatment period and the tested fungal species. Compared with the control strains, CIN dose dependently changed the morphology and ultrastructure of mycelium in different degree. Especially, the reduction of hydrogen peroxide was considered to follow the destruction of mitochondrial. Meanwhile, CIN significantly cut the levels of lipid peroxidation and reduced glutathione. The activity of total superoxide dismutase was significantly inhibited after CIN treatment at the end of incubation, whereas the activities of catalase and glutathione peroxidase were opposite. These results indicated that the inhibitory effect of CIN could attribute to oxidative stress alleviation possibly induced by modifications of cellular structure as well as redox status.
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Acroleína/análogos & derivados , Aflatoxina B1/biosíntesis , Antifúngicos/farmacología , Aspergillus flavus/efectos de los fármacos , Aspergillus flavus/crecimiento & desarrollo , Estrés Oxidativo/efectos de los fármacos , Acroleína/farmacología , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacosRESUMEN
Surface plasmon resonance imaging (SPRi) is one of the powerful tools for immunoassays with advantages of label-free, real-time, and high-throughput; however, it often suffers from limited sensitivity. Herein we report a dual signal amplification strategy utilizing polydopamine (PDA) functionalization of reduced graphene oxide (PDA-rGO) nanosheets for sensitive SPRi immunoassay in serum. The PDA-rGO nanosheet is synthesized by oxidative polymerization of dopamine in a gentle alkaline solution in the presence of graphene oxide (GO) sheets and then is antibody-conjugated via a spontaneous reaction between the protein and the PDA component. In the dual amplification mode, the first signal comes from capture of the antibody-conjugated PDA-rGO to form sandwiched immunocomplexes on the SPRi chip, followed by a PDA-induced spontaneous gold reductive deposition on PDA-rGO to further enhance the SPRi signal. The detection limit as low as 500 pg mL(-1) is achieved on a nonfouling SPRi chip with high specificity and a wide dynamic range for a model biomarker, carcinoembryonic antigen (CEA) in 10% human serum.
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Biomarcadores/química , Grafito/química , Indoles/química , Polímeros/química , Resonancia por Plasmón de Superficie , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Óxidos/químicaRESUMEN
Quantum dot (QD)-incorporated one-dimensional (1D) nanocomposites offer great application potential. However, a facile one-step synthesis of the nanocomposites and fabrication of their free-standing film for sensing has not been accomplished. Herein a rod-shaped nanocomposite is one-pot synthesized via an L-cysteine-assisted hydrothermal approach, in which synthesis parameters including L-cysteine amount, temperature and reaction duration are tailored to control the composite nanostructures. CdTe nanocrystals are incorporated into the L-cystine matrices to form the nanorods, which tangle each other to network an intact film structure via a simple drying process. The free-standing CdTe-cystine nanorod film is directly utilized as a humidity sensor. This work provides a one-pot synthesis approach to grow 1D CdTe incorporated nanocomposites, demonstrating their great potential in film sensing applications.
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Cellular responses to porous tubular structures have recently been investigated in highly ordered ZrO2 nanotube arrays fabricated with anodization. However, the potential applications of the nanotube arrays are hindered by instrument requirements and substrate limitations, as well as by the complicated processes needed for synthesis. In this work, ZrO2 nanotube arrays were synthesized by in situ hydrolysis of zirconium propoxide with a zinc oxide nanorod array-based template. Fibroblast cells were able to grow on the nanotube array surface with produced elongated filopodia. Studies of the capability of cell growth and the expression of adhesion- and proliferation-related genes reveal that ZrO2 nanotube arrays may provide a better environment for cell adhesion and growth than a flat titanium surface. These findings not only provide fundamental insight into cell response to nanostructures but also provide an opportunity to use a unique approach to fabricate ZrO2 nanotube array structures for potential implant applications.
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Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Fibroblastos/fisiología , Nanotubos/química , Óxido de Zinc/química , Circonio/química , Animales , Fibroblastos/efectos de los fármacos , Ratones , Óxido de Zinc/farmacología , Circonio/farmacologíaRESUMEN
Thread-based microfluidic colorimetric sensors have been deemed a potential tool that may be incorporated into textiles for non-invasive sweat analysis. Nevertheless, their poor performance significantly limits their practical uses in sweat glucose detection down to 20 µM. Herein, a microfluidic glucose sensing device containing a salt-responsive thread is developed for the highly sensitive detection of glucose in human sweat. By grafting a zwitterionic polymer brush-which could react to ionic strength by changing the conformation of the polymer chains from the collapsing state to the stretching state-onto the cotton thread, the salt-responsive thread was created. Compared to the pristine cotton thread, the modified thread has better ion-capture capabilities, a more noticeable swelling effect, and a higher ability to absorb water. These enable a significant enrichment of glucose when the saline solution passes through it. The salt-responsive thread was employed to construct a thread/paper-based microfluidic sensing device for the monitoring of glucose in artificial sweat, exhibiting a sensitivity of -0.255 µM-1 and a detection limit of 14.7 µM. In comparison to the pristine cotton thread-based device, the performance is significantly superior. Using a hydrophobic fabric and salt-responsive threads, a glucose-sensing headband was prepared for on-body sweat glucose monitoring. With the use of a smartphone-based image analysis system, the headband can detect the concentration of glucose in a volunteer's perspiration. Using the thread-based salt-responsive zwitterionic polymer brush might offer a novel approach to creating wearable sweat sensors with extremely high sensitivity.
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Técnicas Biosensibles , Sudor , Humanos , Sudor/química , Microfluídica , Automonitorización de la Glucosa Sanguínea , Glucemia/análisis , Glucosa/análisis , PolímerosRESUMEN
Citrus fruits are highly susceptible to pathogenic fungal infections after harvesting, which causes serious economic losses. Therefore, it's necessary to develop new antifungal packaging. In this study, gamma-Decanolactone (DL) was successfully encapsulated in a polycaprolactone (PCL)/ß-cyclodextrin (ß-CD) composite system using electrostatic spinning technology. PCL/ß-CD was compounded in different ratios, the ratio was screened through other indicators such as fiber morphologies and mechanical properties. Then, antifungal mats were prepared by adding different concentrations of DL to the PCL/ß-CD solution. The results showed that when the mixture ratio of PCL/ß-CD was 6:1 and loaded with 6 % DL, the antifungal felt had strong mechanical, significantly inhibiting the growth of three citrus pathogens (P. digitatum, P. italicum and G. candidum), released DL for up to 204 h and effectively reduced the morbidity rate of citrus fruits. Therefore, the antifungal pad prepared in this study has great potential in the field of citrus disease control.
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Vertically oriented and robust polyelectrolyte nanotube arrays with high density, large area and high uniformity were successfully grown on substrates by a ZnO nanowire array-templated layer-by-layer (LbL) self-assembly approach for the first time, and were further used to deliver charged drugs, showing that they not only possess pH-responsive loading property, but also significantly enhance the loading capacity and sustained release time. This work could be extended to fabricate polyelectrolyte nanotube arrays with different polyelectrolyte combinations, including weak polyelectrolyte/weak polyelectrolyte, weak polyelectrolyte/strong polyelectrolyte and strong polyelectrolyte/strong polyelectrolyte. With the great versatility to use various substrates and building blocks, the polyelectrolyte nanotube arrays may have great potential for broad applications such as biosensor arrays, bioreactor arrays and optoelectronics.
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Nanopartículas del Metal/química , Nanopartículas del Metal/ultraestructura , Nanocápsulas/química , Nanotubos/química , Nanotubos/ultraestructura , Óxido de Zinc/química , Cristalización/métodos , Ensayo de Materiales , Tamaño de la Partícula , Electricidad Estática , Propiedades de SuperficieRESUMEN
Superparamagnetic iron oxide nanoparticles (SPIONs) (about 15 nm) were synthesized via a hydrothermal method and characterized by field emission scanning electron microscopy, transmission electron microscopy, dynamic light scattering, x-ray diffraction, and vibrating sample magnetometer. The molecular pathways of SPIONs-induced nanotoxicity was further investigated by protein microarrays on a plastic substrate from evaluation of cell viability, reactive oxygen species (ROS) generation and cell apoptosis. The experimental results reveal that 50 µg ml(-1) or higher levels of SPIONs cause significant loss of cell viability, considerable generation of ROS and cell apoptosis. It is proposed that high level SPIONs could induce cell apoptosis via a mitochondria-mediated intrinsic pathway by activation of caspase 9 and caspase 3, an increase of the Bax/Bcl-2 ratio, and down-regulation of HSP70 and HSP90 survivor factors.
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Nanopartículas de Magnetita/toxicidad , Análisis por Matrices de Proteínas , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Supervivencia Celular/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Nanopartículas de Magnetita/ultraestructura , Células PC12 , Análisis por Matrices de Proteínas/métodos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Great challenges still remain to assemble metal nanoparticles on a substrate with tunability, high density, robust stability, good dispersion and well-retained properties for various applications. Herein a new concept using a polymer thin-film as a sacrificial template is investigated to fabricate highly dense and well-dispersed nanoparticle arrays. In contrast to a conventional "hard" template, the polymer template is a porous multilayered film allowing in situ growth of Au nanoparticles with a restricted ripening mode, and tuning the nanoparticle size and density of the arrays is possible by simply adjusting the loading conditions. The prepared substrate-attached nanoparticle arrays demonstrate good thermal and chemical stability, while offering highly sensitive and tunable localized surface plasmon resonance (LSPR) refractive index sensing with a broad linear dynamic range. This method could be extended to controllably fabricate other robust and "clean" nanoparticle arrays on various substrates for various applications including sensing, catalysis and optoelectronics.
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Silver nanoparticles (AgNPs) is known as one of the most valuable metal nanoparticles in antibacterial and anticancer application. AgNPs-resistant bacteria has been documented, but it is unclear whether cancer cells can also escape the anti-cancer effect of AgNPs. In this study, we aimed to investigate this phenomenon and its underlying mechanism. The antibacterial activity and cytotoxicity of AgNPs were measured in the presence of HeLa cell metabolites. The status of AgNPs in the system associated with metabolites were characterized by UV-Vis, Zetasizer Nano ZS, and transmission electron microscopy. Non-targeted metabolomics was used to reveal the metabolites components that bind with AgNPs. HeLa cells were injected intraperitoneally to establish the tumor-bearing mice model, and the stability of AgNPs in mice serum was analyzed. The results manifested that HeLa cell metabolites inhibited the anticancer and antibacterial effects of AgNPs in a dose-dependent manner by causing AgNPs aggregation. Effective metabolites that inhibited the biological activity of AgNPs were stable in 100 â, insoluble in chloroform, containing sulfur elements, and had a molecular weight less than 1 kDa in molecular weight. There were 115 compounds bound with AgNPs. In vitro experiments showed that AgNPs aggregation occurred only when the concentration of α-ketoglutarate (AKG) and glutathione (GSH) together reached a certain threshold. Interestingly, the concentration of AKG and GSH in HeLa cellular metabolites was 10 and 6 times higher than that in normal cervical epithelial cells, respectively, which explained why the threshold was reached. Furthermore, the stability of AgNPs in the serum of tumor-bearing mice decreased by 20% (P < 0.05) compared with the healthy mice. In conclusion, our study demonstrates that HeLa cells escaped the anti-cancer effect of AgNPs through the synergistic effect of AKG and GSH, suggesting the need to develop strategies to overcome this limitation.
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Nanopartículas del Metal , Plata , Humanos , Animales , Ratones , Células HeLa , Plata/farmacología , Ácidos Cetoglutáricos/farmacología , Antibacterianos/farmacología , Glutatión , Pruebas de Sensibilidad MicrobianaRESUMEN
The yarn-based sweat-activated battery (SAB) is a promising energy source for textile electronics due to its excellent skin compatibility, great weavability, and stable electric output. However, its power density is too low to support real-time monitoring and wireless data transmission. Here, we developed a scalable, high-performance sweat-based yarn biosupercapacitor (SYBSC) with two symmetrically aligned electrodes made by wrapping hydrophilic cotton fibers on polypyrrole/poly (3,4-ethylenedioxythiophene):poly (styrenesulfonate)-modified stainless steel yarns. Once activated with artificial sweat, the SYBSC could offer a high areal capacitance of 343.1 mF cm-2 at 0.5 mA cm-2. After 10,000 times of bending under continuous charge-discharge cycles and 25 cycles of machine washing, the device could retain the capacitance at rates of 68% and 73%, respectively. The SYBSCs were integrated with yarn-shaped SABs to produce hybrid self-charging power units. The hybrid units, pH sensing fibers, and a mini-analyzer were woven into a sweat-activated all-in-one sensing textile, in which the hybrid, self-charging units could power the analyzer for real-time data collection and wireless transmission. The all-in-one electronic textile could be successfully employed to real-time monitor the pH values of the volunteers' sweat during exercise. This work can promote the development of self-charging electronic textiles for monitoring human healthcare and exercise intensity.
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Técnicas Biosensibles , Polímeros , Humanos , Sudor , Pirroles , TextilesRESUMEN
Relationship between mycotoxin-induced hormesis and reactive oxygen species (ROS) has not been systematically investigated due to the lack of an effective analysis method. To monitor cellular release and intracellular level of O2·-, carboxymethyl cellulose-Mn3(PO4)2 nanocomposite was synthesized to fabricate an electrochemical biosensor, which selectively detects O2·- over the range of 57.50 nM â¼ 2.95 µM (R2 = 0.99) with the sensitivity of 78.67 µA µM-1 cm-2 and the detection limit of 8.47 nM. Transient exposure to zearalenone (ZEA) induces the enhancement on cell viability, immediate O2·- release from cells, and reduction of intracellular O2·- level. After post-treatment culture, intracellular O2·- initially increases to a high level and then decreases to the normal level. Concurrently, the ZEA-induced hormesis disappears. Based on the findings, we propose a mechanism, involving the ROS release, increase of succinate dehydrogenase activity and recovery of intracellular ROS, to explain the occurrence and disappearance of hormesis in intestinal porcine enterocytes.
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ZearalenonaRESUMEN
Sweat-activated batteries (SABs) are lightweight, biocompatible energy generators that produce sufficient power for skin-interface electronic devices. However, the fabrication of 1D SABs that are compatible with conventional textile techniques for self-powered wearable electronics remains challenging. In this study, a cotton-yarn-based SAB (CYSAB) with a segmental structure is developed, in which carbon-black-modified, pristine yarn and Zn foil-wrapped segments are prepared to serve as the cathode, salt bridge, and anode, respectively. Upon electrolyte absorption, the CYSAB can be rapidly activated. Its performance is closely related to the ion concentration, infiltrated electrolyte volume, and evaporation rate. The CYSAB can tolerate repeated bending and washing without any significant influence on its power output. Moreover, the CYSABs can be woven into fabrics and connected in series and parallel configurations to produce an energy supplying headband, which can be activated by the sweat secreted from a volunteer during a cycling exercise to power light-emitting diode headlights. The developed CYSAB can also be integrated with yarn-based strain sensors to achieve a smart textile for the self-powered sensing of human motion and breathing. This weavable, washable, and scalable CYSAB is expected to contribute to the manufacturing of self-powered smart textiles for future applications in wearable healthcare monitoring.
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Sudor , Dispositivos Electrónicos Vestibles , Electrodos , Electrónica , Humanos , TextilesRESUMEN
Low-cost, stable and highly efficient oxygen reduction reactions (ORR) electrocatalysts are of great significance for microbial fuel cells to break the limit of the air cathode. The expensive noble metal catalysts are easy to be contaminated due to biofouling, which could damage the catalytic activity significantly. Among the reported non-noble metal catalysts, FeCN materials are promising substitutes that have comparable catalytic activity with Pt/C. In this article, a facile process to obtain N-doped porous carbon fibers (NPCF) with abundant Fe2N moieties from iron based metal organic framework (MOF(Fe)) embedded electrospun fibers has been developed. The fiber structure promotes the in situ conversion of Fe2N sites in embedded MOF(Fe) during pyrolysis under NH3 atmosphere. The abundant Fe2N sites, presence of pyrrolic nitrogen and hierarchical porous structure of obtained Fe2N/NPCF make it possess excellent electrocatalytic activity to ORR with comparable performance (E1/2 = 0.8648 V) and superior long term stability to commercial 20 wt% Pt/C. This work expends the toolbox for design of high performance cathodic catalysts for MFCs and also provides original insights in Fe-N active sites construction for FeNC ORR catalysts.