Mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration, including Alzheimer's disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signaling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterized. Here, we present phage display generated antibody single-chain variable fragments (scFvs) to human TREM2 immunoglobulin-like domain. Co-crystal structures revealed the binding of two scFvs to an epitope on the TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species, which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterization of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.  •scFvs were generated against the immunoglobulin-like domain of the receptor TREM2•Crystal structures revealed scFv binding to epitopes outside the TREM2 CDRs•Oligomeric scFv species reduced levels of shed TREM2 ectodomain in a HEK293 model•The scFvs form renewable structural and functional biology tools for TREM2 research TREM2 variants are associated with an increased risk of dementia. Szykowska et al. generated scFv antibody fragments to TREM2 immunoglobulin-like domain and solved crystal structures of scFv-TREM2 complexes to reveal their binding modes. Oligomeric scFv species showed enhanced functional activity reducing levels of shed TREM2 ectodomain in a HEK293 model. Late-onset Alzheimer's disease (LOAD) is the most common type of dementia, characterized by accumulation of extracellular amyloid-β (Aβ) aggregates and intracellular neurofibrillary tangles of hyper-phosphorylated tau, with a long prodromal phase followed by cognitive decline. Despite the urgency and necessity to develop therapeutics, there are currently no approved drugs which cure or slow the underlying progression of Alzheimer's disease (AD) and patients are still reliant on symptomatic treatments discovered in the mid-last century .TREM2 (Triggering Receptor Expressed on Myeloid cells), which encodes a single transmembrane receptor expressed in myeloid-derived cells, including microglia in the central nervous system (CNS)  linked genes coding for components of the immune response to AD, neuro-inflammation has become an area of intense research for therapeutics . Among tem (CNS) . Homozygem (CNS) . Since tem (CNS) . The mosem (CNS) . A stronem (CNS) .TREM2 has numerous putative functions, including regulation of lipid and cholesterol metabolism, phagocytosis of myelin and Aβ, and generation of a microglial barrier around Aβ plaques . TREM2 hTREM2 contains an immunoglobulin-like (Ig-like) domain followed by a flexible stalk region, a transmembrane domain, and a short cytoplasmic tail. The stalk region can be cleaved by ADAM10/17 proteases to generate a soluble TREM2 fragment (sTREM2), while the C-terminal intramembranous domain is further cleaved by gamma-secretase . sTREM2 There is minimal published structural data for TREM2 ectodomain motifs responsible for ligand engagement or regulation of TREM2 functions . While tin vitro, and also lead to a significant reduction in amyloid plaques in 6-month-old amyloid-beta precursor protein knockin mice  against the human TREM2 ectodomain with which to study TREM2 structure and function. Agonist antibodies have already been reported in recently published work . The grokin mice . Antibodkin mice .There are currently no published structures of antibodies in complex with TREM2. Here, we present our work on the generation and characterization of four scFvs selected against the Ig-like domain (herein Ig domain) of human TREM2. The co-crystal structures of two of these antibody fragments, scFv-2 and scFv-4, have been solved in complex with TREM2, revealing interactions with epitopes distal to the putative ligand-binding site. Two antibody fragments, scFv-3 and scFv-4, reduced the shedding of sTREM2 from HEK293 cells.Together, these data identify scFv antibody fragments as valuable reagents for cellular and structural studies of the TREM2 receptor and identify TREM2/ligand interfaces that may be used to modulate TREM2 function.E. coli and, therefore, scFvs were prepared by secretion from Sf9 insect cells, which allowed for higher yields, increased solubility and a reduction in endotoxin contamination  > 100 nM  to establish their relative binding affinities , which has been structurally characterized previously , as wellk region . Both TRDiffracting crystals were first obtained for the complexes of the smaller TREM2 Ig domain with both scFv-2 and scFv-4, enabling their structure determination at 3.36 and 3.07 Å resolution, respectively see  and, des5ELI and 5UD7), except for some variability in the flexible loops  and variable light (VL) domains of scFv-4 displayed canonical immunoglobulin folds tethered by an engineered glycine-serine-rich linker that was disordered in the refined structure. The binding of scFv-4 to TREM2 was centered on the CDR3 loop of the scFv-4 VH domain (herein CDR-H3), flanked by interactions from CDRs-H1 and H2 and by the VL domain CDR-L2 A and 4B.The resolution of the scFv-4 complex structure enabled a detailed analysis of the side chain interactions in the binding interface A and 4B.While the primary scFv-4 interaction was mediated by the TREM2 Ig domain as described above, we identified crystal contacts between the TREM2 stalk and CDR-H1 from a neighboring scFv-4 subunit. This additional packing allowed the stalk to be traced in the electron density maps up to Asp137 in the C terminus and several hydrogen bond interactions were observed to support the stalk interaction .The binding of scFv-2 to TREM2 was mediated by the three CDR loops of the VH domain , as well as by CDR-L3 from the light domain A and 5B.Together the scFv-2 and scFv-4 complex structures show the potential value of these antibody fragments as crystallization chaperones. More importantly, however, they define the epitopes of these scFvs, allowing for the interpretation of functional properties in the context of the binding site on the surface of TREM2, which has been poorly described for other anti-TREM2 antibodies.The H157Y polymorphism, which leads to accelerated cleavage of TREM2 and increases AD risk, suggests that regulation of shedding of sTREM2 might be important in human biology . Based oOligomerization of scFvs has been observed and can be exploited to enhance the properties of selected molecules . We deciFinally, to demonstrate selective binding to TREM2, we conjugated the dimeric fractions of scFv-3 and scFv-4 to Alexa Fluor 647 and repeated the HEK293 treatment protocol with subsequent imaging of the fluorescent scFvs . LabelinIn this paper, we have presented our work on the structural characterization of scFvs raised against human TREM2 by phage display. We screened a library of scFv antibody fragments against fully folded and glycosylated TREM2 Ig domain, which resulted in the generation of three successful candidates. Despite challenging purification procedures, we were successful in producing recombinant antibody fragments and demonstrated them to be suitable for X-ray crystallography. We have solved the structures of two scFvs bound to TREM2 and provide the first crystallographic structures of antibody interactions with TREM2. These are also the first antibody fragments reported to unambiguously bind to the TREM2 globular Ig domain. In addition, we have shown that higher-MW oligomeric species of scFvs are able to display enhanced functional activity, suggesting parallels with the activity of bivalent mAbs and diabodies.Since TREM2 is a potential therapeutic target for AD, the pharmaceutical industry has initiated several projects aimed at developing TREM2 antibody therapeutics, with at least two candidates having commenced clinical trials. Data have been reported for two, AL002 and 4D9, which allow for an interesting comparison with scFv-4 . Other fAs both AL002 and 4D9 are stalk binding antibodies, it is possible that they might affect cleavage of TREM2 by virtue of sterically hindering access by ADAMs. Consistent with that hypothesis, both full-length antibodies (4D9 and AL002) have been shown to reduce shedding of sTREM2. This interesting property has been confirmed for the first time in human with AL002 . This fuAt present, the mechanism of action for the inhibition of shedding by all of these antibody reagents is unclear. It might occur either by inhibition of cleavage of TREM2 at the cell surface or by promoting internalization of full-length TREM2, thereby removing substrate availability. Schlepckow et al. propose that 4D9 exerts some of its effects by stabilizing TREM2 at the plasma membrane, explaining the potentiation of signaling in response to liposomes by 4D9. However, it is also possible that reduction of sTREM2 production by bivalent antibodies or oligomeric scFvs is due to crosslinking of receptor and internalization, thereby reducing cell surface substrate for shedding. It is noteworthy that a bivalent antibody is necessary for 4D9 functional effects and, to date, we have not observed any functional responses to monomeric scFv-4, only observing activity in preparations containing appreciable dimeric species. Furthermore, Alexa Fluor-labeled scFv-4 and scFv-3 label HEK293 cells in a TREM2 expression-dependent manner, leading to intracellular immunofluorescence. Interestingly, the scFv-2 antibody fragment has the lowest propensity to form oligomeric species and did not show any inhibitory activity in the sTREM2 shedding assay. Our data are therefore consistent with the effect of scFv-3 and scFv-4 upon shedding of sTREM2 being due to promoting the internalization of TREM2.in vitro. AL002 elicits TREM2-mediated secondary intracellular signaling, demonstrated using an NFAT reporter cell line and phosphorylation of a protein of equal MW to DAP12 in bone-derived macrophages . Similarly to the mAbs discussed above, multivalent scFvs will have the potential to crosslink TREM2 ectodomains into higher-order structures, thereby creating significant steric hindrance to proteolytic complexes, altering their characteristics as substrates, or triggering endocytosis.By using phage display to raise recombinant scFvs we have assured that the source of the molecules is renewable and can be easily shared. During our studies, we have encountered difficulties caused by the use of commercially available antibodies with unknown TREM2 epitopes, especially when used in ELISA, pull-down studies or immunocytochemistry. The presented scFvs bind away from the proposed ligand recognition site, qualifying them for development into additional tools for the study of TREM2-ligand interactions and receptor processing.Our data represent the first crystallographic characterization of binding interactions between functional antibody fragments and TREM2 and we hope that these additional tools will help elucidate the complex pathophysiology of TREM2 in AD.alex.bullock@cmd.ox.ac.uk)Further information and requests for reagents and resources may be directed to and will be fulfilled by the Lead Contact Alex N. Bullock, (Plasmids generated in this study will be deposited to Addgene and made available on request.6YYE (TREM2-scFv-2), 6Y6C (TREM2-scFv-4) and 6YMQ (TREM2-scFv-4).The coordinates and structure factors for the crystal structures reported in this article have been deposited in the PDB with accession codes E. coli XL1-blue .All plasmids were generated in MACH1 or DH5α (Thermofisher Scientific C862003) cells grown in LB media at 37°C and bacmid DNA for insect cell expression and baculovirus generation were generated in DH10Bac (Thermofisher Scientific 10361012). For phage display, the enriched phages were amplified overnight by infection of 2.Baculoviral protein expression was performed in Sf9 cells (Thermofisher Scientific 11496015) grown at 27°C in SF900 II-SFM media. Mammalian protein expression was performed in Expi293F™ cells  in Expi293™ expression medium (Thermofisher Scientific) at 37°C, 8% CO2. The cell culture medium for the HEK293 TREM2-DAP12 cells was supplemented with 1 mg/mL puromycin hydrochloride .WT HEK293  and HEK293 cells stably expressing WT hTREM2 and hDAP12 were cultured in DMEM (GIBCO) with 10% heat-inactivated foetal bovine serum (FBS) (Gibco) at 37°C, 5% CODNA encoding human TREM2  used for antigen generation and TREM2 (a.a. 19-138) for ELISA validation were cloned into the baculovirus secretion plasmid pFB-Sec-Bio5, which provides a C-terminal Avi-tag sequence for biotinylation. The stalk-containing TREM2 (a.a. 19-174) used for ELISA validation was cloned into baculovirus secretion plasmid pFB-Sec-NH which provides a N-terminal hexahistidine tag. See A TREM2 construct containing the native signal peptide (a.a. 1-174) was cloned into the pTT5 vector, followed by an Avi tag and TEV-cleavable hexahistidine tag (GLNDIFEAQKIEWHEGSENLYFQSHHHHHH) at its C-terminus. The SP sequence was cloned from pHL-sec vector into pTT5 DNA inserts .For structural studies, human TREM2 (a.a. 19–131) was cloned into the mammalian expression vector pTT5, following an N-terminal signal peptide (MGILPSPGMPALLSLVSLLSVLLMGCVAETG) with hexahistidine tag preceeded by additional linker (a.a. GTK) at its C-terminus.The longer construct TREM2 (a.a. 19-174) containing the stalk was inserted by ligation independent cloning (LIC)  into theScFv antibody fragments identified by phage display were cloned for baculovirus expression with scaffold specific primers into the vector pFB-Sec-NH.6 in adherent culture were transfected for 4 hours (h) at 27°C followed by medium exchange to SF900 II-SFM containing 0.1% penicillin and streptomycin (Thermofisher Scientific) with 2% FBS (Thermofisher Scientific). P0 viruses were harvested after 7 days at 27°C and 120 μL was used to infect 3 mL of Sf9 cells for P1 virus amplification (72 h and 450 rpm shaking and throw 6 mm). Following another round of viral amplification, 0.75 L of Sf9 cells in SF900 II-SFM media were infected with virus P2 at 7 mL/L at density 2x106 cells/mL in glass flasks .TREM2 viruses for baculovirus expression were generated by transfection with bacmid DNA:JetPrime (Polyplus) complexes. Sf9 cells at a density of 0.2x10in vitro biotinylated using recombinant BirA enzyme . The reaction was performed in a buffer containing 10 mM Tris-HCl, 50 mM NaCl and 10 mM MgOAc, pH 8.0 for 14 h, at 4°C in the presence of 0.1 mM biotin and 10 mM ATP. Subsequently, the buffer was exchanged by dialysis to 20 mM HEPES, 300 mM NaCl, 5% glycerol and 0.5 mM TCEP, pH 7.5The clarified supernatants were loaded onto 5 mL Ni-NTA (Qiagen)/per 1 L at 10 mL/min. Resin was washed with 20 column volumes (CV) of 50 mM HEPES pH 7.4 and 300 mM NaCl, 5% glycerol, 5 mM imidazole followed by 10 CV of 10 mM imidazole and 600 mM NaCl containing buffer. Proteins were eluted with buffer containing 500 mM imidazole. Proteins were purified further by size exclusion chromatography  and TREM2 (His19-Ala138) and (His19-Ser174) for ELISA validation were purified in the same manner but were stored at -80°C after size exclusion.g and immediately frozen in liquid nitrogen and used for the assay within a week.Viruses for scFvs were generated as above. Immobilised metal affinity chromatography (IMAC) was performed as for TREM2, except for a change to pH 6.8 which decreased precipitation. Eluted proteins were cleaved with TEV protease overnight (O/N) at 4°C followed by loading onto a 1 mL Protein A FF  column at 3 mL/min. Resin was washed with 20 CV of base buffer  followed by a wash with 10 CV of BB containing 0.05% Triton-X. Proteins were eluted with 0.1 M acetic acid pH 3 at 0.5 mL/min and immediately buffer exchanged on PD10 columns  and frozen until further use. Later, scFv-3 and scFv-4 oligomers were separated by double SEC on Superdex 200 16/60 in 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5% glycerol. Separated oligomers were pooled and buffer exchanged to PBS using vivaspin 2 10 MWCO concentrators . Proteins were clarified by centrifugation at 30,000 x 6 were transfected with a mixture of 1 mg of pTT5:TREM2 (19-131) DNA to 6 mg L-PEI 25 K (Polysciences) per 1 L of culture. 50 mL of DNA and 50 mL of L-PEI in Opti-MEM (Thermofisher Scientific) was mixed and incubated for 15 min before addition to the culture. 1 mg/L kifunensine (Carbosynth) was added followed by 1 mM valproic acid (VPA), 20 h post-transfection. Cells were grown in vented flasks at 100 rpm (throw 25 mm), 37°C, 8% CO2 for 72 h.For crystallisation studies of TREM2 (a.a. 1-131), Expi293F™ cells (Thermofisher Scientific A14527) in Expi293™ expression medium (Thermofisher Scientific) at 2.5x10Culture supernatants were collected and TREM2 (a.a 19-131) was purified by affinity chromatography  in buffers containing 20 mM Tris-HCl pH 8, 500 mM NaCl, 5% glycerol followed by size exclusion chromatography . The purified protein was deglycosylated with EndoH  in 50 mM sodium citrate pH 5.5 overnight at 4°C, at the ratio of 1 mg protein: 2000 U EndoH. EndoH was removed using a second Ni-NTA affinity column, and the purified protein was stored in buffer containing 50 mM Tris pH 8.5, 200 mM NaCl, and 5% glycerol at 10 mg/mL concentration.The longer TREM2 (a.a. 19-174) for crystallisation was expressed and purified similarly but transfection was performed with DNA to L-PEI 1:3 ratio and cells were grown in 2 L roller bottles (Greiner Bio-one). Affinity chromatography was performed in base buffer: 20 mM HEPES 7.4, 300 mM NaCl, 5% glycerol and proteins were immediately deglycosylated in solution adjusted with sodium citrate . Size exclusion was performed in 20 mM HEPES 7.4, 150 mM NaCl, 5% glycerol on Superdex 200 16/60 .2. TREM2 was further purified by a second Ni-NTA affinity column and stored in buffer containing 50 mM Tris pH 8.5, 200 mM NaCl, and 5% glycerol.Fully glycosylated biotinylated pTT5-TREM2 (19-174) protein for SPR studies was purified as described for TREM2 (a.a. 19-131) without the deglycosylation step. The Ni-NTA purified protein was biotinylated overnight at 4°C in the presence of 0.5 mM biotin, BirA (1:50), 1 mM ATP, and 7.5 mM MgClin vitro biotinylated TREM2A (19-131) from Sf9 cells as antigen and a human synthetic single-chain fragment variable (scFv) library  were mixed at molar ratio 1:1.1 and buffer exchanged into 20 mM Tricine pH 8, 200 mM NaCl, 200 mM L-arginine, 200 mM L-glutamic acid. The complex was concentrated to 14 mg/mL and crystals were grown by sitting drop vapour diffusion in solution containing 39% MPD, 0.2 M ammonium acetate, 0.1 M citrate pH 5.5 at 20°C. Crystals were mounted directly from the drop and vitrified in liquid nitrogen.g spin for 20 min. Protein crystals appeared after 1 day using a reservoir solution containing 0.2 M potassium chloride, 35% pentaerythritol propoxylate 5/4, 0.1 M HEPES pH 7.5 at 20°C. Crystals were cryo-protected after addition of reservoir solution containing 20% ethylene glycol and vitrified in liquid nitrogen.Antibody scFv-4 was buffer exchanged into 25 mM HEPES, 300 mM NaCl and 5% glycerol and mixed with TREM2 (a.a. 1-174) at molar ratio 1:1.1. The complex was concentrated to 12.5 mg/mL and crystal plates were set up after 32,000 x Protein A purified antibody scFv-2 in 100 mM citrate buffer and 250 mM L-arginine and 50 mM L-glutamic acid was mixed with TREM2 (a.a 19-131) at a molar ratio 1.3:1 and buffer exchanged on PD10 column to 60 mM Tricine 8, 200 mM NaCl containing 200 mM L-arginine, 200 mM L-glutamic acid. The complex was concentrated to 16 mg/mL and precipitation was removed by centrifugation. Crystals were grown in 15 mM nickel chloride, 0.1 M Tris pH 8.5, 2.2 M ammonium acetate at 20°C. Crystals were cryo-protected with reservoir containing 20% ethylene glycol before vitrification.For scFv-4 structures , data were collected on beamline I24 at the Diamond Light Source using the X-ray wavelength of 0.9688Å and processed using Xia2 package and DIALS G. .For the lower resolution scFv-2 dataset (6YYE), data were collected on beamline I03 at the Diamond Light Source using the X-ray wavelength 0.9763 Å. The initial model was built using PhenixRefine and coot modelling using Xia and DIALS dataset. Later the model was used to refine against AutoProc processed dataset with ellipsoidal truncation by Staraniso , 2018. T6YYE) was phased using the structure of another scFv (PDB: 6G8R) generated from the same library and solved previously (5UD7 (6YYE) was used for phasing of PDB 6YMQ. Phenix molecular replacement was used to phase the data and coot to build a model .WT HEK293 and HEK293 cells stably expressing WT hTREM2 and hDAP12 were cultured in T-175 flasks (Greiner Bio-one) in DMEM (GIBCO) with 10% heat-inactivated foetal bovine serum (FBS) (Gibco) at 37°C, 5% CO3 cells per well, grown for 24 h, washed with culture medium and treated with antibodies for 5 h. After antibody incubation, the medium was collected and transferred into a new plate, centrifuged at 1,000 x g followed by supernatant transfer into a fresh plate that was stored at -80°C.HEK293 transfected with TREM2-DAP12 were plated in 96-well plates  at 30x10TREM2 ELISA was performed by coating high bind 96-well plates  with 8 μg/mL TREM2 antibody  O/N at 4°C followed by 1 h blocking using PBS with 1% BSA (Sigma). 100 μL of the defrosted cell supernatants were added at the appropriate dilution in DMEM + 1% BSA alongside a standard curve with TREM2 peptide  and incubated for 2 h, at RT. 25 μL of the biotinylated detection antibody  at 1.5 μg/mL was added for an hour followed by the addition of HRP conjugated streptavidin diluted at 1:30,000 . TMB ELISA substrate (ThermoFisher Scientific 34028) was added for 20 min followed by 2 M sulphuric acid (Immuno Chemistry technologies). Results were obtained by measuring optical density (OD) at 450 nm using a plate reader (SpectraMax M2). OD measurements in the linear range (0.8-1.9) were chosen for analysis and TREM2 concentration was interpolated from the TREM2 standard curve. To ensure that scFv binding to TREM2 did not interfere with the assay antibodies, the standard peptide  was mixed with each one of the scFvs in DMEM +1% BSA and results were compared to the standard curve only. Also, as another control, to assess TREM2 epitope recognition by the ELISA antibodies, TREM2 proteins (His19-Ala138 and His19-Ser174) were diluted to 2441 pg/mL in DMEM +1% BSA using five-point serial dilutions and analysed using the protocol described above. Additionally, the medium conditioned in cells for 5 h was spiked with scFvs and ELISA was performed in comparison to non-spiked sample (data not shown).Purified scFv-3 and scFv-4 oligomers and control non-TREM2 scFvs were incubated with Protein A and Alexa Fluor 647 NHS ester (Thermofisher Scientific A37573) at 1:10 molar ratio for 20 h, at 4°C and excess dye was removed by NAP5 columns . HEK293 and HEK293 transfected with TREM2-DAP12 were prepared as for the ELISA and treated with scFv-4 for 5 h. Cells were fixed in 4% paraformaldehyde in PBS (Santa Cruz Biotechnology) for 15 min and kept at 4°C until imaging. Nuclei were stained with Hoechst 33342 for 1 h in the dark and cells were imaged using an OperaPhenix confocal microscope with 40 x magnification, 405/456 nm and 640/706 nm, excitation/emission respectively.50 was interpolated using non-linear regression (inhibitor vs. response- four parameters) in GraphPad Prism Software 8.4.2. Each statistical test was performed on raw data normalised to cell number or signal before normalisation to the percentage of control was performed. One-way ANOVA with Dunnett’s test comparison to control untreated cells was utilised to calculate significance. p values are indicated in the respective figure legends with symbols .All statistical analyses were carried out using three independent biological replicates and two or three technical repeats unless specifically stated otherwise. Error bars are standard deviation (SD) and EC


Flutriafol (FTF) is a triazole fungicide that can cause liver toxicity through the ingestion of its residues in food and water. However, little is known about the liver toxicity of FTF, particularly nonalcoholic fatty liver disease (NAFLD) in humans. Therefore, the purpose of this study was to investigate whether FTF induces NAFLD in human liver cells and animal liver. HepG2 cells and Sprague Dawley (SD) rats were treated with FTF at doses of 0–640 µM for 24 h and 0–150 mg/kg bw/day for 28 days, respectively. FTF  treatment to cells induced lipid accumulation. FTF (80 and 160 µM)-treated cells had higher levels of cytochrome P450 enzymes and reactive oxygen species and increased mitochondrial membrane potential loss than the control. FTF also increased the mRNA levels of antioxidant enzymes through oxidative stress and nuclear factor erythroid 2-related factor 2 pathways in HepG2 cells. However, a higher level of FTF (320 µM) induced apoptosis. The treatment of SD rats with FTF (2.5–150 mg/kg bw/day) induced fatty infiltration in the liver by impairing liver metabolism and inducing apoptosis. Therefore, our data suggest that human exposure to FTF residues may be a risk factor for liver diseases, such as NAFLD. Fungicides have been widely used in agriculture since the early 1900s to control fungal diseases and improve crop yield and quality . The useFlutriafol (FTF), (RS)-2,4-difluoro-α- benzyl alcohol, is a systemic triazole fungicide used to treat various grains and seeds . FTF actFTF absorbed through the gastrointestinal tract is transported to the liver through the hepatic portal vein. In the liver, the detoxification process occurs in three metabolic phases  . In partNonalcoholic fatty liver disease (NAFLD) is a chronic liver illness associated with metabolic syndrome, with a global prevalence rate of 25% ,15. AlthAlthough there are risks of hepatotoxicity associated with human ingestion of residual FTF present in crops and water, the liver toxicity of FTF, such as NAFLD, and the underlying mechanisms have not been investigated. Therefore, the aim of this study was to investigate the risk of FTF on NAFLD development and progression, particularly lipid accumulation and the underlying cellular mechanisms using the human liver cell line HepG2 and a rat model.FTF, Pierce bicinchoninic acid protein assay kit, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), and 2, 2, 2-Tribromoethanol were obtained from Sigma-Aldrich . Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, and 0.05% trypsin/0.53 mM ethylenediaminetetraacetic acid (EDTA) solution were provided by WELGENE Inc. . Phosphate-buffered saline (PBS) was supplied by Lonza . T-25 flasks, 6-well plates, and 96-well plates were obtained from SPL Life Sciences . Dimethyl sulfoxide (DMSO) and N-acetylcysteine (NAC) were obtained from Amresco . The nitrocellulose membrane was supplied by GE Healthcare Bio-Sciences . Enhanced chemiluminescence was purchased from Thermo Fisher Scientific  and Advansta . Antibodies against nuclear factor erythroid 2-related factor 2 (Nrf2) were obtained from Abcam . Antibodies against Caspase-3 and B-cell lymphoma 2 (Bcl-2) were supplied by Cell Signaling Technology . Antibodies against Bcl-2-associated X protein (Bax) and proliferating cell nuclear antigen (PCNA) were purchased from Santa Cruz Biotechnology Inc. . Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from EMD Millipore , and goat anti-rabbit IgG-HRP was obtained from Enzo Life Science .v/v) in a humidified carbon dioxide (CO2) incubator supplied with 5% CO2 at 37 °C. The medium was replaced every 2 days and the cells were subcultured using 0.05% trypsin/0.53 mM EDTA solution. The cells were seeded in 6-well or 96-well plates at 4 days before FTF exposure (0–640 µM) or 0.004-0.016% DMSO (vehicle control) for 24 h.The human liver cell line HepG2 was obtained from the American Type Culture Collection. The cells were cultured in DMEM added with 10% FBS and 1% penicillin/streptomycin , according to the manufacturer’s protocol. HepG2 cells were seeded at a density of 1 × 104 cells/well in 96-well plates and exposed to FTF  or DMSO  for 24 h (n = 3 wells per group). Next, 10× lysis solution (10 µL per 100 µL) was added to the wells for determining maximum LDH release 45 min prior to the end of FTF treatment. Supernatants (50 µL) from the original 96-well plate were moved into a new 96-well plate, and a 50 µL CytoTox 96® reagent was added to each well. After incubation for 30 min at room temperature in the dark, the stop solution (50 µL) was added to each well and the optical density (OD) was measured at 490 nm using an Epoch spectrophotometer . The released LDH percentage was calculated using the following formula:Lactate dehydrogenase (LDH) release was evaluated using the Cytox 964 cells/well in 96-well plates and exposed to FTF  or DMSO  for 24 h (n = 3 wells per group). The cells were replaced with fresh medium and then incubated with 10 µL MTT solution (5 mg/mL in PBS) at 37 °C for 3 h. After discarding 90 µL of the medium, 180 µL of acidic isopropanol was added to each well to solubilize the insoluble formazan crystals. Next, the 96-well plate was incubated at 37 °C for 1 h, and the OD was measured at 570 nm and 630 nm using an Epoch spectrophotometer. The OD at 630 nm was subtracted from the OD at 570 nm. The percentage of cell viability was calculated using the following formula:Cell viability was determined using 3--2,5-diphenyl-2H-tetrazolium bromide . HepG2 cells were seeded at a density of 1 × 105 cells/well into 6-well plates and treated with 80, 160, and 320 µM FTF or 0.008% DMSO for 24 h (n = 3 wells per group). The cells were fixed with 10% formalin of 2.4 mL for 1 h at room temperature, and the fixed cells were rinsed with 60% isopropanol. Then, Oil red O staining solution of 1 mL was added to each well. After incubation for 10 min, the cells were washed with deionized distilled water thrice to remove the unbounded dye. Images were visualized and captured using a Nikon Eclipse Ti2-U and Nikon Elipse Ts2R camera . Then, the images were quantified using ImageJ software .Intracellular neutral lipid such as triglycerides and cholesterol esters were evaluated using Oil red O dye . HepG2 cells were seeded at a density of 2 × 10n = 3 wells per group). The cells were then treated with 10 µM DCFDA and incubated at 37 °C in a 5% CO2 incubator for 30 min. The cells were then washed thrice with PBS. The green fluorescence area stained with DCFDA was imaged using Nikon Eclipse Ti2-U and the fluorescence intensity was quantified using ImageJ software.Intracellular ROS levels were estimated using 2,7-dichlorofluorescein diacetate . HepG2 cells were grown in 6-well plates and exposed to 80 and 160 µM FTF or 0.004% DMSO (vehicle control) for 24 h with or without pretreatment with NAC  for 1 h  and treated with 80 and 160 µM FTF or 0.004% DMSO (vehicle control) for 24 h (n = 3 wells per group). The cells were stained with 50 µL of JC-10 dye-loading solution. After incubation at room temperature for 45 min in the dark, 50 µL assay buffer B was added to each well. The integrity of MMP was determined as the ratio of the fluorescence of JC-10 aggregates (Ex/Em = 490/525 nm) to monomeric JC-10 (Ex/Em = 540/590 nm). The fluorescence intensity was determined using a SpectraMax Gemini EM Microplate Reader . The fluorescence ratio was calculated using the following formula:Mitochondrial membrane potential  was evaluated using the JC-10 Mitochondrial Membrane Potential Assay Kit , according to the manufacturer’s protocol. HepG2 cells were grown in Corningn = 3 wells per group). RNA was obtained from the cells using TRIzol reagent . Reverse transcription was conducted using the TOPscript RT DryMIX kit  to synthesize cDNA. The mRNA expression level was estimated by using an RT-PCR system  with a total volume of 20 µL of PCR reaction mixture containing 10 µL of 2× Real-Time PCR Smart mix , 8 µL of RNase-free water, 1 µL of cDNA sample, and 0.5 µL each of 10 µM forward and reverse primers . The thermal condition of PCR reaction consisted of 60 cycles of 95 °C for 15 min, followed by 60 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and extension at 72 °C for 10 s. The mRNA expression level was quantified by the 2−ΔΔCt method, and GAPDH was used as an internal control. Primer sequences were designed using the AmplifX software . HepG2 cells were grown in 6-well plates and then treated with 80 and 160 µM FTF or 0.004% DMSO (vehicle control) for 24 h with or without pretreatment with NAC  for 1 h (software .n = 3 wells per group). Cells were lysed in hypotonic buffer containing 20 mM Tris (pH 7.4), 10 mM sodium chloride (NaCl), 3 mM magnesium chloride (MgCl2), and protease inhibitor mixture . Next, 10% Triton X-100 was added to extract the cytosolic proteins, and the supernatants were eliminated through centrifugation at 3000× g for 10 min at 4 °C. The cell pellets were resuspended in cell extraction buffer containing 100 mM Tris (pH 7.4), 2 mM sodium orthovanadate (Na3VO4), 100 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 0.1% SDS, 20 mM tetrasodium pyrophosphate (Na4P2O7), and a protease inhibitor mixture. The homogenates were centrifuged at 14,000× g for 20 min at 4 °C, and the supernatants were used for Western blotting. For immunofluorescence staining, HepG2 cells were grown in 6-well plates and exposed to 160 µM FTF or 0.004% DMSO (vehicle control) for 24 h with or without pretreatment with NAC  for 1 h (n = 3 wells per group). The cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized using 0.1% Triton X-100 in PBS for 10 min. Next, a blocking buffer consisting of 3% bovine serum albumin and 2% normal donkey serum was used to block the cell monolayers, and the cells were incubated with anti-Nrf2 diluted in blocking buffer at 4 °C for 24 h. After washing with PBS, the cells were incubated with DyLightTM -488-conjugated anti-IgG at room temperature for 1 h and washed thrice with PBS, followed by fixation with 4% paraformaldehyde. Subsequently, the cell nuclei were stained with DAPI (1 µg/mL) for 30 min and washed thrice with PBS. The fluorescence areas were photographed using a Nikon Eclipse Ti2-U microscope.Nuclear translocation of Nrf2 was determined by nuclear fractionation and immunofluorescence staining. HepG2 cells were grown in 6-well plates and exposed to 160 µM FTF or 0.004% DMSO (vehicle control) for 24 h with or without pretreatment with NAC  for 1 h (n = 3 wells per group). The medium was collected in a 15 mL tube after the end of the FTF treatment, and the cells were washed twice with PBS. Next, the cells were harvested and pelleted by centrifugation at 3000× g for 5 min at 4 °C. Subsequently, the cells were washed with 4 °CPBS and resuspended in 1× binding buffer. Next, the cells were stained with Annexin V-fluorescein isothiocyanate (1.25 µL) and propidium iodide (10 µL) and incubated in the dark for 10 min at room temperature. The stained samples were analyzed using a CytoFlex Flow Cytometry Analyzer .Cell death was determined using the EzWay Annexin V-FITC Apoptosis Detection Kit  according to the manufacturer’s instructions. HepG2 cells were grown in 6-well plates and exposed to 320 µM FTF or 0.008% DMSO (vehicle control) for 24 h with or without pretreatment with NAC  for 1 h (n = 5); (2) FTF 0.25 mg/kg bw/day (n = 5); (3) FTF 2.5 mg/kg bw/day (n = 5); (4) FTF 25 mg/kg bw/day (n = 5); (5) FTF 75 mg/kg bw/day (n = 5); (6) FTF 150 mg/kg bw/day (n = 5). The dose of FTF (2.5 mg/kg bw/day) for rats was determined with the consumer chronic risk value in agriculture commodities (0.5 mg/kg) using body surface area (BSA) [The animal experiments were carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) of the Ethics Committee of Konkuk University , and the animals were treated humanely to alleviate distress and discomfort. Eight-week-old female Sprague Dawley (SD) rats (220–250 g) were purchased from Orient Bio Inc.  and acclimatized for 1 week. The rats were nurtured under controlled constant temperature (22–25 °C) and humidity (40–70%) with 12 h light–dark cycles. All the animals were provided free access to feed and water ad libitum. Six groups of 30 animals each were used in this study: (1) FTF 0 mg/kg bw/day (ea (BSA) . Body su® K2EDTA tubes  through cardiac puncture. Blood samples were centrifuged at 3000× g for 10 min at 4 °C, and the supernatants were stored at –80 °C for further experiments. Aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), albumin (ALB), urea nitrogen (BUN), and total protein (TP) were evaluated as biochemical parameters.The biochemical profiles of the plasma samples were analyzed using an automated dry chemistry analyzer . Blood was collected into BD VacutainerLiver samples were collected and fixed in 10% formalin for histopathological analysis. Thereafter, the tissue samples were dehydrated in ascending grades of ethanol solution and de-waxed with xylene. The tissue pieces were cut into 3.5 µm sections using a microtome. Next, the sections were stained with hematoxylin and eosin, and histological alterations were mainly examined for fatty infiltration. The slides were photographed using a Nikon Eclipse Ti2-U and Nikon Eclipse Ts2R camera.g for 20 min at 4 °C to eliminate tissue debris. The supernatants were analyzed using a bicinchoninic acid protein assay kit. Western blot protein samples (20 µg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 3% nonfat milk buffer for 1.5 h at room temperature and incubated with primary antibodies overnight at 4 °C. The membrane was washed thrice with Tris-buffered saline with Tween 20 and then incubated with horseradish peroxidase-conjugated secondary antibody for 1.5 h at room temperature. The protein bands were visualized by enhanced chemiluminescence Western blot detection reagents and quantified using ImageJ software. In addition, the nitrocellulose membrane was used again after the stripping process as follows: the nitrocellulose membrane was incubated in stripping buffer for 30 min at 57 °C. The membrane was washed thrice with distilled water for 5 min, followed by three washes with Tris-buffered saline with Tween 20 for 10 min. Subsequently, the membrane was blocked again for Western blot analysis.Protein expressions of Nrf2 and apoptosis-related markers were determined by Western blotting. Rat liver samples stored at −80 °C were immersed in liquid nitrogen and crushed for further experiments. Next, the liver tissue protein (0.01 g) was lysed using a 1 mL radioimmunoprecipitation assay buffer  containing a protease inhibitor cocktail. The tissue lysates were sonicated at 30 kHz using a probe sonicator  and centrifuged at 18,000× P < 0.05.The experimental data are presented as mean ± standard error of the mean (SEM). Statistical significance was analyzed using one-way ANOVA with post hoc Dunnett’s test and Student’s t-test. The tests were performed using SPSS-PASW statistics software version 18.0 for Windows . Statistical difference was determined at P < 0.001; P < 0.001; P < 0.001; The cells treated with FTF at a concentration of 640 µM for 24 h had significantly higher LDH release levels than the control cells . These results showed a correlation between the activation of CYP and the generation of cellular oxidative stress in human liver cells.The cells treated with FTF  had significantly higher mRNA expression of CYP1A2, CYP2C9, CYP2C19, CYP3A4, CYP51A1, and CYP2E1 than control cells . Furthermore, the fluorescence microscopy images were confirmed that Nrf2 proteins were translocated from the cytosol to the nucleus in FTF-treated cells, which was reversed by NAC treatment .Cells treated with FTF  showed significantly increased transfer of Nrf2 from the cytosol to the nucleus, compared with the control cells . Furthermore, the flow cytometry data displayed that the apoptotic ratio (early apoptotic and late apoptotic cells) increased after treatment with 320 µM FTF for 24 h  showed significantly lower cell viability than the control cells , there was no statistically significant difference in body weight among the treatment groups from 3 weeks (P < 0.05), the feed intake levels recovered after 1 week . Regarding biochemical parameters, there were no significant differences in the rats treated with FTF (0–150 mg/kg bw/day) and the control rats, with regard to the plasma levels of AST, ALT, ALP, and ALB (P < 0.01).An animal study was performed with rats to provide further evidence for FTF-induced liver metabolism and apoptosis. Although the rats treated with FTF (2.5 mg/kg bw/day) had significantly higher body weight than control rats at 1 and 2 weeks .Rat liver samples were histologically evaluated by hematoxylin and eosin staining A. The liPesticides, such as insecticides, herbicides, and fungicides, are widely used to increase agricultural productivity. However, because the excessive use of pesticides results in the accumulation of residues in the soil, water, and food products, humans are routinely exposed to residual pesticides . EuropeaIn our study, cell membrane integrity and cell viability assays were performed for human liver cells. FTF at concentrations of up to 320 µM did not affect cell membrane integrity, whereas 320 and 640 µM FTF significantly decreased cell viability up to 83% and 63% A,B. TherUncontrolled ROS production is known to activate Nrf2 as the main cellular defense mechanism against oxidative stress . Nrf2 isThe expression of antioxidant enzymes through Nrf2 activation maintains redox homeostasis. Nevertheless, excessive ROS production, which is not controlled by antioxidant enzymes, leads to apoptosis . The apoThe experimental dose of FTF (2.5 mg/kg bw/day) in the animal study was chosen based on the maximum consumer risk assessment of FTF (0.5 mg/kg) reported by the European Food Safety Authority, which was translated from humans to rats using body surface area normalization . FurtherNAFLD is a liver disease related to metabolic disorders . This diIn conclusion, our data revealed that FTF can cause lipid accumulation in human liver cells and rat liver through oxidative stress and apoptosis. Although further studies may be needed to identify the role of FTF on NAFLD in humans, our data suggest that chronic exposure of residual FTF to humans may be a potential risk factor for the pathogenesis of NAFLD. According to the current regulation by European Food Safety Authority, the maximum residual limits of FTF are legally tolerated up to 0.4 mg/kg in pome fruit, 0.5 mg/kg in cereals, and 1 mg/kg in grapes [


The compounds of interest were obtained by arc meltingand characterized by differential thermal analysis, scanning electronmicroscopy, and X-ray diffraction both on powder and on a single crystal, to ensure the high quality of the samples andaccurate crystallographic data. Chemical bonding was studied by analyzingthe electronic structure and effective QTAIM charges of La2Pd3Ge5. A significant charge transfer mainlyoccurs from La to Pd so that Ge species assume tiny negative charges.This result, together with the -(I)COHP analysis, suggests that, inaddition to the expected homopolar Ge bonds within zigzag chains,heteropolar interactions between Ge and the surrounding La and Pdoccur with multicenter character. Covalent La–Pd interactionsincrease the complexity of chemical bonding, which could not be adequatelydescribed by the simplified, formally obeyed, Zintl–Klemm scheme.Electric resistivity, specific heat, magnetization, and magnetic susceptibilityas a function of temperature indicate for both compounds a metallic-likebehavior. For Nd2Pd3Ge5, two low-temperaturephase transitions are detected, leading to an antiferromagnetic groundstate.The two La R2Pd3Ge5 (R = La and Nd) intermetallics are presented. La2Pd3Ge5 shows polar Ge−Pd/La multicenter interactionsin addition to covalent Ge−Ge bonds. The bonding scenario isfurther complicated by the fact that Pd and La are also covalentlyinteracting. For Nd2Pd3Ge5, an antiferromagneticground state is established after a long-range magnetic ordering (at∼7.5 K) followed by a spin-reorientation transition (at ∼6.2K).The chemical bonding and physical propertiesof the twoisotypic  When the electronegativitydifference is decreased by substituting an alkali with an alkaline-earthmetal, the reduced charge transfer leads to covalent interactionsamong cations and the anionic partial structures. In these cases,although the Zintl–Klemm rules are frequently fulfilled, theyare, indeed, not sufficient to account for all of the different kindsof interactions taking place.4 Modern quantumchemical approaches based on QTAIM5 andelectron localizability ELI-D8 have been applied to achieve moreinsights into chemical bonding features.9When the electronegativitydifference is remarkable , an almost complete charge transfer can be assumedand covalently bonded anionic Ge fragments form. For the Ge-richestcompositions, the so-called “intermetallic clathrates”feature infinite frameworks composed by four-bonded (4R) and furthermore by adding a third element, for example, of the d-block.10 In these compounds,due to a less pronounced charge transfer, many different kinds ofGe fragments are observed, such as (3b)Ge infinitenetworks (as in RGe2 and their derivatives)10 or corrugated layers ,13 (2b)Ge zigzag chains with differenttopologies , and simpler isolated fragmentslike cis-Ge4 (as in Lu5Pd4Ge820) and Ge–Gedumbbells . The occurrence of these Ge motifs with peculiargeometries mainly depends on two chemical factors: the compositionand the nature of Ge–metal and metal–metal interactions.In fact, a higher content of electropositive metals causes a largernumber of valence electrons formally transferred to Ge, so simplerGe fragments, characterized by a reduced number of Ge–Ge bonds,occur. The geometry of the resulting fragments is strongly dependenton the presence of stabilizing bonding interactions, more complexthan the ionic–covalent ones described by the Zintl model.Therefore, the application of the Zintl–Klemm concept to thesecompounds usually fails.Anotherwide group of germanides forms when the electropositivecounterpart is represented by a rare-earth metal 13 and Y2PdGe6 applying theQTAIM and ELI-D position-space techniques. In all cases, in additionto the expected Ge–Ge covalent bonds, the Ge–La/M  interactionswere described as polar–covalent. The increased complexityof the bonding situation when moving from the main group to the transitionmetal analogues was also revealed by the presence of La–M heteropolar interactions, traceable when deeply analyzedby means of the ELI-D fine structure based on its relative Laplacian.Recently, chemical bonding in the Ge-rich R2M3Ge5. Numerous studies previouslyexamined the crystal structure and physical properties of these intermetallics,24 also because many of them formeasily, and high-yield samples can be obtained by simple arc or inductionmelting with no further thermal treatments. Nevertheless, chemicalbonding studies of these germanides are almost absent. To fill thisgap, chemical bonding in La2Pd3Ge5 was studied, and the results are reported in this work togetherwith some measurements of physical properties.Another large family of ternary germanides with a lower Ge contentis that with a general formula of 22.1R20.0Pd30.0Ge50.0 (R = La or Nd)were prepared starting from stoichiometric amounts of the pure constituents,all with nominal purities of >99.9 mass %. Elemental lanthanumorneodymium  andpalladium and germanium were arc melted on a water-cooled copper hearth with a tungsten electrodeunder an Ar atmosphere, yielding very brittle ingots of ∼0.8g that proved to be stable in air. Mass losses were <0.5%.Samples with a nominal composition of For metallographic analysis, some fragments of each alloy were embeddedin a phenolic resin with graphite filler, employing an automatic hotcompression mounting press . The sample surfaceswere smoothed with an automatic grinding and polishing machine . A multistep grinding and polishing procedure was performedusing SiC papers, with grain sizes from 600 to 1200 mesh with runningwater as the lubricant, and finally diamond pastes from 6 to 1 μmwith an alcohol-based lubricant. Petroleum ether was employed aftereach polishing step to clean samples in an ultrasonic bath for a fewminutes.To examine microstructures and measure phase compositions,a scanningelectron microscope  was used, equipped with an energy dispersive X-ray (EDX)spectroscope from Oxford Instruments (INCA X-ACT). The compositionalanalysis was performed on the basis of the characteristic X-ray intensitiesof each element. A Co standard was used for calibration. The errorin the measured EDXS composition is ∼0.5 atom % for each element.2.22Pd3Ge5 was selected from the crushed alloy.Its shape and composition were checked by SEM-EDXS prior to X-rayanalysis (see the Supporting Information).A single crystal of La25The data set was obtained in a routine fashion under ambientconditions on a Bruker Kappa APEXII diffractometer operating in theω-scan mode, equipped with a CCD area detector and graphitemonochromatized Mo Kα (λ = 0.071073 Å) radiation.The crystal, glued on a glass fiber, was mounted on the goniometrichead, and intensity data were collected over the reciprocal spaceup to ∼30° in θ with exposures of 30 s per frame.Semiempirical absorption corrections based on a multipolar sphericalharmonic expansion of equivalent intensities were applied to all databy the SADABS software.26 showing excellent residuals.The corresponding CIF file has been deposited at the Cambridge StructuralDatabase as entry 1917376. Selected crystallographic data and structure refinementparameters are listed in Tables S1 and S2.The structuralmodel was easily found by direct methods and successivelyrefined using full-matrix least-squares methods with the SHELX-14package2Pd3Ge5 is isostructuralwithother orthorhombic R2Pd3Ge5 compounds: space group Ibam, oI40-U2Co3Ge5, a =10.1914(6) Å, b = 12.2082(7) Å, c = 6.1901(4) Å.La15X-ray powder diffraction (XRPD)analyses were conducted on samplesby means of a Philips X’Pert MPD diffractometer with a θ:2θBragg–Brentano geometry . Powder patterns were collected in the 2θrange of 10–100°, with a scanning step of 0.02° anda time per step of 15 s. Lattice parameters of the studied phases,refined by a least-squares routine, match well with those obtainedby single-crystal analyses.2.3Tm) of the titlecompounds was measured by differential thermal analysis (DTA) usinga LABSYS EVO instrument ,equipped with type S (Pt-PtRh 10%) thermocouples. Measurements wereconducted in the temperature range of 25–1200 °C witha heating/cooling rate of 5 °C/min under an Ar flow (20 mL/min).Custom-made Ta crucibles were used as both sample containers (∼200mg) and references. The loaded crucibles were arc-sealed under anAr atmosphere after being cooled with liquid nitrogen to avoid undesiredreactions. The recorded thermograms were evaluated and analyzed withthe aid of Calisto, supplied by SETARAM. To calibrate the equipment,the measured melting temperatures of standard materials  and their tabulated values27 were employed.Temperatures were defined from the onset point of the heating curvepeaks, determined through an extrapolation procedure.The melting temperature by means of a homemade quasi-adiabaticstep heating system in the temperature range of 2.2–15 K and(ii) with a Quantum Design PPMS relaxation-type calorimeter in theextended interval of 2–280 K. For the measurements of the electricalresistivity, a common four-probe ac technique with spot-welded goldcontacts on a bar-shaped sample in the temperature range of 2–300K was applied. In the temperature range of 4.2–10 K, the measurementwas performed in steps of 0.05 K.2.52Pd3Ge5 was studiedwith TB-LMTO-ASA version 4.7c,28 employingan exchange and correlation potential, according to Barth and Hedin,29 within the local density approximation (LDA).For this purpose, the experimental structural data presented in thiswork were used. The addition of empty spheres, to meet the minimumoverlapping criterion, was not necessary, yielding the following atomicsphere radii: r(La) = 2.156 Å, r(Ge1) = 1.558 Å, r(Ge2) = 1.518 Å, r(Ge3) = 1.464 Å, r(Pd1) = 1.398 Å,and r(Pd2) = 1.507 Å. The self-consistent calculationwas performed with a basis set that included La-6s/(6p)/5d/4f, Ge-4s/4p/(4d), and Pd-5s/5p/4d/(4f); parentheses indicate orbitals treated according to a downfoldingprocedure. The Brillouin zone was integrated with the tetrahedronmethod using 280 irreducible k-points from a totalof 1728.The electronicstructure of La30 andthe corresponding integrated values up to the Fermi level (ICOHP).Plots of densities of states (DOS) and COHP curves were generatedusing wxDragon,31 setting the Fermi energyat 0 eV as a reference point. The electron density (ED) was calculatedon an equidistant grid of ∼0.05 Bohr using an implemented module32 within the FPLO software.33 For the sake of consistency, the same exchange and correlationpotential applied in the previous calculations was employed samplingthe Brillouin zone with an 8 × 8 × 8 k-pointmesh. The ED was analyzed according to Bader’s quantum theoryof atoms in molecules (QTAIM)5 using DGrid.34 As a result, the crystal space is separatedinto non-overlapping and space-filling regions called atomic basins.The integration of the electron density within QTAIM basins yieldstheir average electronic populations; their subtraction from atomicnumbers gives the effective charges. The ED and QTAIM atomic basinswere visualized within the unit cell using ParaView.36Chemical bonding was studied on the basis of crystalorbital Hamilton populations (COHPs)2Mg3Ge5”. Its structure was simplygenerated by introducing Mg into Pd sites and geometrically optimizedwith the all-electron DFT-based FHI-aims package.38 To validate this computational procedure, also the La2Pd3Ge5 structure was relaxed under the samesetup: Perdew and Zunger (LDA)39 exchangeand correlation potential, (4 4 8) k-point mesh forBrillouin zone sampling, and predefined default “light”basis set for all atomic species. In addition, scalar relativisticeffects were included within the “atomic ZORA” approximation;a Gaussian smearing of 0.01 eV was set up. The relaxed La2Pd3Ge5 unit cell parameters are in very goodagreement with the experimental ones (see the Supporting Information).To thoroughly investigate the role of Pd, a comparative chemicalbonding analysis was conducted considering the hypothetical “Lap)DOS and COHP curves for selected interactionsand ICOHP were calculated for relaxed La2Mg3Ge5 with the TB-LMTO-ASA (same setup as for La2Pd3Ge5). More details about the latter calculationand structural data of the relaxed La2Mg3Ge5 compound are reported in the Supporting Information.Partial (3Supporting Information). Both are almost single-phase R2Pd3Ge5; a small amount of the eutectic (Ge+PdGe) mixturecan be detected only by electron microscopy. The average microprobecompositions of the title phases, listed in R3+ ionic radiuspreviously reported for the R2Pd3Ge5 series.15The quality of the prepared samples was carefully checked by SEM-EDXSand XRPD (see the 2Pd3Ge5) and 974 °C (Nd2Pd3Ge5). For both samples, the small peak at∼725 °C indicates the melting of the (Ge+PdGe) eutecticmixture.40Two endothermic effects were recorded during DTA measurements,as shown in 3.1R2Pd3Ge5 intermetallics,15 also in La2Pd3Ge5 the Ge2 (8g) and Ge3 (8j) speciesare likely covalently bonded, forming infinite chains perpendicularto (001) planes, whereas Ge1 (4a) has no homocontacts.For the sake of simplicity, these species will be further labeledas two-bonded (2b) and zero-bonded (0b).A quick glance atinteratomic distances see 2 confirmb)Ge and (0b)Gecould be accounted forby assuming a charge transfer from La and Pd to Ge, giving an ionicformula of (La3+)2(Pd2+)3[(2b)Ge2–]4[(0b)Ge4–]. Nevertheless, only a partialcharge transfer is more probable considering the relatively smallelectronegativity difference between Ge and Pd referring to differentscales .According to the Zintl–Klemm approach,the presence of Ge2– and (0b)Ge4–. The tiny negativecharges indicate that interactions of Ge with the surrounding La andPd should be interpreted as covalent rather than ionic. Additionalinformation can be extracted from the shape of QTAIM atoms. In fact,La is rather spherical, which is typical for cations in similar compounds,47 and both Ge and Pd basins possess a polyhedral shape.What is more, not only the shared faces between Ge2 and Ge3 are flat,which is characteristic for homopolar bonds, but also those betweenGe and Pd.The lowest negative charges are associated with Pd species: Pd2(−0.62) is less negative than Pd1 (−0.71), which canbe explained by the presence of Pd2–Pd2 contacts, with distancesof ∼3.09 Å see 2, wherea2Pd3Ge5 couldnot be adequately described by the simplified Zintl–Klemm scheme.This hypothesis is also borne out by the measured properties, presentedin 48Hence, from the QTAIM effective charges, it turnsout that thebonding scenario of LapDOS, respectively) are shownin These preliminary findings motivated usto give more insight intothe chemical interactions among the constituents. Thus, the TB-LMTO-ASAmethod was applied and the obtained total and projected densitiesof states Ge chains is coherentwith the corresponding −COHP curve and −ICOHP valueof 1.73 eV Ge2– chains (with 2e lone pairs per atom according to the Lewis formula), one may expectmore occupied antibonding states close to the Fermi level. The observedfeatures suggest rather the existence of polar–covalent bondsbetween Ge species and the surrounding metals.The presence ofthe Ge chains in the Mg-simulated analogue Ge2– chains. This is also reflected in the reduced−ICOHP value of 1.29 eV  along with the increased Ge–Gedistance: 2.78 Å (La2Mg3Ge5)versus 2.63 Å (La2Pd3Ge5).To corroborate this interpretation,a comparative chemical bondinganalysis was conducted on the hypothetical “Laanalogue 4b shows EF in the case of Ge1/Ge2 andessentially nonbonding down to approximately −2 eV for Ge3.The Ge–Pd bonds are the strongest in the compound, rangingfrom 2.38 eV/bond for the shortest (2b)Ge3–Pd1contact (2.46 Å) to 1.18 eV/bond for the (2b)Ge2–Pd2 contact (2.78 Å). The situation is differentfor the Ge–La interactions, which are weaker (1.03–0.70eV) and not saturated, bonding also above EF.The Ge–metal bonding would be better depicted by the analysisof the corresponding −COHP curves 4 c-e. ThoP8-FeAs,49 the average Ge–Pddistance is 2.56 Å, giving a −ICOHP of 1.76 eV. Upon comparisonof these values with those for each Ge species in La2Pd3Ge5 , the presence of strongpolar covalent Ge–Pd bonds could be deduced. The same is alsotrue for Ge–La interactions, which can be compared with analogousones in La2ZnGe6, featuring very similar distances,COHP curves, and −ICOHP values.11 These findings have also been recently confirmed by position-spacebonding analysis.13 In conclusion, becauseeach Ge is bonded to the surrounding metals, which are always morenumerous than the additionally realizable 2c–2e interactions ,the overall Ge–Pd/La bonding features a multicenter character.With the aim of correctly discussing the magnitude of Ge–Pdand Ge–La ICOHP, the same values obtained for chemically relatedcompounds may be used as references. For example, in GePd, 2Pd3Ge5, La–Pd and Pd–Pd bonds must also betaken into account. The Pd1 and Pd2 species are surrounded by fiveand four La atoms, respectively. As shown in panels a and b of EF, revealingtheir stabilizing role. The covalent nature of La–Pd interactionswas also recently highlighted by means of the electron localizabilityindicator (ELI-D) and its “fine structure”, based onits relative Laplacian, in the LaPdGe3 and La2PdGe6 compounds,13 where Pdis coordinated by La in a topologically similar way to the title intermetallic.To complete the bonding analysis for La2Pd2Ge,50 where the Pd–Pd distance is 2.87 Åwith a −ICOHP of 0.99 eV/bond. However, in La2Pd3Ge5, Pd atoms are remarkably less interactive,because their COHP curve , of La2Pd3Ge5 and Nd2Pd3Ge5 are displayed in The temperature-dependentelectrical resistivities, ρ is likely torefer to some degree of disorder at the grain boundaries. The low-temperatureresistivity of Nd2Pd3Ge5 increasesto ∼6.3 K and then exhibits two anomalies: (i) a distinct dropfollowed by (ii) a weak increase, which typically results from anantiferromagnetic phase transition via superzone boundary scattering.The simple metallic behavior of La2Pd3Ge5 is well in line with the specific heat data (T < 6 K) follow the C(T) = γΤ + βΤ3 function with an electronic Sommerfeld coefficient γof 9.1(2) mJ mol–1 K–2 and a Debyetemperature θD of 305(5) K of the lattice vibrations[β = 6.84(8) × 10–4 J mol–1 K–4]. The Sommerfeld coefficient extracted fromthe calculated DOS at EF (compare –1 K–2) is in very goodagreement with the experimental value.Their moderately largeresidual resistivity  = SNd(T) – SLa(T), associated with the magnetic specificheat anomaly reaches Smag = R ln 2 = 5.6 J mol–1 K–1 at 8.4K, i.e., which conforms to the ordering of Nd moments in their crystallineelectric field ground state doublet. The antiferromagnetic natureof the magnetic phase transition is confirmed by temperature-dependentac and dc magnetic susceptibility data depicted in TN of 7.5 K .The occurrence of two-phase transitions and the formation of anantiferromagnetic ground state of the Nd magnetic moments in Nd3Ge5 see 7 displayT < TN and an applied magneticfield near 3.5–5.5 T  of 3.27 μB/Nd and refers to the specificcrystalline electric field ground state of Nd2Pd3Ge5.The spin-reorientation transitionat 6.2 K relates to a distinctdrop in the magnetic susceptibility when entering the magnetic groundstate. At higher magnetic fields, as revealed by the 3 T dc magneticsusceptibility in .5 T see 9. The ma2Pd3Ge5 in TN. To analyzethe temperature dependence of the inverse susceptibility (3 T data),displayed in 0 + C/(T – θp), was applied, where χ0 accounts for a temperature-independent Pauli susceptibility, θp is the paramagnetic Curie–Weiss temperature, and C is the Curie constant. The least-squares fitting yieldsa χ0 of 1.07 × 10–3 emu/mol,a θp of ≅0 K, and a Curie constant C of 2.75 emu K mol–1 and accordinglyan effective magnetic moment μeff of 3.32 μB/Nd, which is reasonably close to the theoretical free ionvalue of Nd3+, i.e., 3.62 μB. The moderatereduction of the observed effective moment is attributed to the crystallineelectric field splitting of the J = 9/2 multiplet of the Nd ions. The magnetic behavior of Nd2Pd3Ge5 is closely related to the evenmore complex magnetism as seen in a single-crystal study of Pr2Pd3Ge5,51 whereas isotypic Ce2Pd3Ge5 revealedsimple antiferromagnetic order below a TN of 3.8 K.52Temperature- and field-dependentmagnetization data of Nd42Pd3Ge5 and Nd2Pd3Ge5 intermetallic compounds (oI40-U2Co3Ge5) were investigated,enriching the available data for the numerous R2Pd3Ge5 series and enhancing our comprehensionof their structure–bonding–property relationships.Thechemical bonding and physical properties of the two isotypicLa2Pd3Ge5) and 974 °C (Nd2Pd3Ge5). Refined structural data for the La-containing analogue,essential for the chemical bonding investigations, were obtained bysingle-crystal X-ray diffraction analysis.Single-phase samples of the title compounds were obtained afterarc melting. Differential thermal analysis measurements indicate thatthey melt congruently at 1058 °C  followed by a spin-reorientation transition(at ∼6.2 K).The findings of chemicalbonding studies are consistent with themeasurements of physical properties: in fact, electrical resistivityas a function of temperature shows that both compounds are metallic.While La


In this work, Li-stabilized Na-β″-alumina electrolytes were doped with 3d transition metal oxides, namely TiO2, Mn3O4, and NiO, in order to improve their ionic conductivity and fracture strength. Due to XRD and EDX measurements, it was concluded that Mn- and Ni-ions are incorporated into the crystal lattice of Na-β″-alumina. In contrast, TiO2 doping results in the formation of secondary phases that enable liquid-assisted sintering at temperatures as low as 1500 °C. All dopants increased the characteristic fracture strength of the electrolytes; 1.5 wt% of NiO doping proved to be most efficient and led to a maximal characteristic fracture strength of 296 MPa. Regarding the ionic conductivity, TiO2 doping showed the uppermost value of up to 0.30 S cm−1 at 300 °C. In contrast to the other dopants, TiO2 doping lowered the sintering temperature needed to obtain a dense, stable, and highly conductive Na-β″-alumina electrolyte suitable for applications in Na based batteries.Na-β″-alumina is the commercially most successful solid electrolyte due to its application in ZEBRA and NAS The excThe research on Na-β″-alumina electrolytes has so far mainly focused on different production techniques ,5,6,7,8 The high ionic conductivity of the Na-β″-alumina electrolyte lowers the overall resistance of an electrochemical cell and increases, therefore, its efficiency. The fracture strength is important because Na-β″-alumina electrolytes have to withstand temperature and pressure variations within a cell . Further2+ or Li+ to prevent decomposition of Na-β″-alumina at the commonly applied sintering temperatures of about 1600 °C [1.67Mg0.67Al10.33O17 or Na1.67Li0.33Al10.67O17 [Na-β″-alumina is typically doped with Mg 1600 °C ,13,14. T10.67O17 ,16.2 is a common dopant that inhibits extensive grain growth of Na-β″-alumina grains by forming intergranular ZrO2 particles. However, the addition of isolating ZrO2 is known to lower the ionic conductivity of Na-β″-alumina electrolytes [Additionally, up to 15 vol.% ZrOtrolytes ,17,18.2, Mn3O4, and NiO, are of interest in this work.3d transition metal doping has shown a high potential to promote ionic conductivity and characteristic fracture strength ,20,21,222Ti3O7, Na8Ti5O14, or Na2Ti6O13. The liquid phase could form at temperatures of about 1300 °C and promote the densification process. Furthermore, an increased number of Al3+ vacancies could promote diffusion and thereby improve the sintering behavior. This mechanism is widely accepted for the increased densification of TiO2-doped Al2O3 [Compared to other 3d dopants, Ti doping was under investigation in literature repeatedly ,21,23,24ed Al2O3 ,29.This process also might be possible for Mn- or Ni-doped Na-β″-alumina.2+ stabilized Na-β″-alumina with 1, 2, and 4 wt% Mn(NO3)2 and found an increase of the ionic conductivity from 0.018 to 0.025 S cm−1 at 300 °C for a doping amount of 4 wt%. The characteristic fracture strength and the influence of different sintering regimes were not analyzed. Lu et al. [3)2, Ti(OCH(CH3)2)4, and ZrO2). The ionic conductivity or the characteristic fracture strength of Mn(NO3)2-single-doped Na-β″-alumina was not reported, but Lu et al. noted a slightly increased shrinkage while sintering Mn(NO3)2-doped Na-β″-alumina.A comprehensive evaluation of Mn-doped Na-β″-alumina has not been published so far. Kennedy et al.  doped Mgu et al.  tested c−1 at 350 °C), while Kennedy et al. found a steady increase of the conductivity up to the maximum tested doping amount of 4 wt% Ni(NO3)2 (0.047 S cm−1 at 300 °C). Both reported ionic conductivities are in comparison to undoped Na-β″-alumina in other publications well below the average [Zhu et al.  and Kenn average .2 doping. Furthermore, it investigates the key characteristics, such as conductivity and characteristic fracture strength of Mn3O4 and NiO-doped Na-β″-alumina electrolytes at different doping amounts and sintering temperatures. In contrast to previous studies, various dopants, doping amounts, and sintering temperatures were tested. Density, SEM–EDX, and XRD studies were carried out to further investigate the so far poorly addressed mechanisms of 3d transition metal doping of Na-β″-alumina.Here, we close the lack of comparable and comprehensive data regarding 3d transition metal doping of Na-β″-alumina. Therefore, this work cites and extends some results of a former publication  of the a2CO3 , and Li2CO3  were mixed in the stoichiometry Na1.67Al10.67Li0.33O17. Afterwards, the powder was calcined at 1280 °C / 2 h in MgO crucibles. The obtained white powder was mixed with TiO2  or NiO  without any further treatment. MnO2  was oxidized to Mn3O4 (650 °C/5 h) before using it as a dopant. The powder mixture was then suspended in EtOH and ball milled in ZrO2 beakers for 0.5 h. After drying (80 °C/12 h), granulating (with an organic binder), and pressing (up to 110 kN), the ceramic green bodies were sintered under air atmosphere at 1500 °C, 1600 °C, or 1700 °C for 0.5 h. This resulted in transition-metal-doped solid electrolyte bodies. To calculate the molar ratio presented in 1.67Al10.67Li0.33O17. The mass fraction of the dopants is always given in percentage.AlO(OH) , NaXRD patterns were measured from grounded disks . The quantitative XRD evaluation was performed by the Rietveld refinement method (Autoquan 2.8.0.2).Density analysis was carried out via Archimedes’ principle in toluene.The absolute density was measured by a He-Pycnometer .To take SEM images , fractured disks were used. SEM–EDX-scans  were taken from polished disks.The fracture strength was tested on ten sintered tablets per doping level by ball-on-three-balls method . The data were evaluated by Weibull statistics (maximum likelihood estimation).b) and the grain boundary resistance (Rgb) were selected from the regression analysis curve to calculate the resistance (R) of the sample Equation (1).Ionic conductivities were measured from bar-shaped samples. The measurements were carried out at two separately sintered bars of each sample by impedance spectroscopy  at a temperature of 300 °C. The range of the applied frequencies was from 1 MHz to 9 Hz. The sinus amplitude was set to 10 mV. To calculate the ionic conductivity (σ), Equation (2) was used. L represents the length of the sample and A, the cross-sectional area.sgb) was done by Equation (3).The calculation of the specific grain boundary resistance  and lower sintering temperatures (≤1600 °C) do not indicate the formation of any secondary phases besides Na-β″-alumina, NaAlO2, and some negligible amounts of Na-β-alumina. XRD patterns of highly doped samples and/or samples sintered at 1700 °C clearly show the formation of two additional phases next to NaAlO2 and Na-β″-alumina, namely NaLiTi3O7 and Na1.97Al1.82Ti6.15O16. Those secondary phases might also be present in samples from sintering at lower temperatures, but they are not detectible by XRD due to a lower crystalline fraction of small crystallites. The latter phenomenon would lead to spread out reflexes, which cannot be detected under the noise (compare XRD amorphousness). 2 and a sintering temperature of 1700 °C, with clearly visible secondary phases beside NaAlO2. Additionally, the same section of an XRD pattern taken from a sample with the same doping amount but a sintering temperature of only 1600 °C is depicted. Here, no clear secondary phases besides NaAlO2 are detected.Diffraction patterns of samples with lower TiO2, while Yi et al. [2 or sintering temperatures of 1700 °C were not described in those publications.Wei et al.  found ani et al.  reportedi et al. ,23,27,32The lattice parameters of the Na-β″-alumina phase were calculated for all doped samples sintered at 1600 °C from XRD data. In 2-doped samples do not support the assumption that Ti-ions substitute Al-ions under the synthesis conditions tested in this work.Boilot et al.  proposed3O4- and NiO-doped Na-β″-alumina electrolytes reveal no secondary phases besides NaAlO2 and minor amounts of Na-β-alumina. They also reveal a high agreement with the literature diffractogram of Na-β″-alumina, as 3O4- and NiO doping do not heavily affect the Na-β″-alumina phase content. Zhu et al. [2O3 or NaAlO2 were not taken into account.The XRD measurements of Mnu et al.  found an3O4- and NiO-doped samples . A temperature of 1600 °C leads to slightly lower densities with a maximum at 1 wt% doping (98.1%/3.14 g cm−3). Even higher doping amounts lead to a density drop. Similar behavior was found at sintering temperatures of 1700 °C. The maximum is reached at 1 wt% TiO2, but density reaches only 96.4%/3.09 g cm−3. Hence, 1 wt% of TiO2 doping leads to a density maximum regardless of the sintering temperature. Higher doping amounts and sintering temperatures lead to relatively lower densities. The higher density hints towards a liquid-assisted sintering process at a sintering temperature of 1500 °C. The porosity increased most likely in consequence of oversintering, caused by pore agglomeration, and increased sublimation of Na2O (sublimating point 1275 °C) [1275 °C) .3O4 doping on the relative densities of Na-β″-alumina. Samples sintered at 1600 °C or 1700 °C show relative densities of about 97.5% and 93.0%, respectively, not depending on the doping amount. The density of samples sintered at 1500 °C drops from 98.6%/3.16 g cm−3 to 96.0%/3.11 g cm−3 due to 0.5 wt% of Mn3O4 doping.3O4-doped samples can be observed; 0.5 wt% of NiO doping reduces the relative density from 98.6%/3.16 g cm−3 to 96.0%/3.11 g cm−3.The relative density of NiO-doped Na-β″-alumina c has a m3O4 and NiO doping shows a slight influence on the relative density at sintering temperatures of 1600 °C and 1700 °C, while the relative density is lowered at sintering temperatures of 1500 °C.In summary, MnTo analyze the influence of 3d transition metal doping and the sintering temperature on the microstructure, SEM studies were carried out. Subsequently, an EDX mapping was performed to investigate the dopant distribution within the microstructure.2 doping on Na-β″-alumina samples for different sintering regimes. For undoped samples, a drastic microstructure changes from sintering temperatures of 1500 °C to 1700 °C is obvious  gave a characteristic Al signal, while point X3 (Na2.0Al0.1Ti3.0O7) displays only signals of Ti, Na, and O. The small Al peak is most likely caused by the surrounding area of darker grains. It should be mentioned that areas with the composition of X3 are more often than those similar to X2. Point X1 (Na1.63Al10.8O17) is directly placed on a dark grain, which shows the typical composition of pure Na-β″-alumina but no Ti. The results strongly indicate that Ti-ions are not replacing Al-ions within the Na-β″-alumina crystal structure but are located in secondary phases. This postulate is supported by an unchanged lattice constant for Na-β″-alumina despite TiO2 doping. Furthermore, the strong influence of TiO2 doping on the microstructure and densification of Na-β″-alumina, which is shown in this and previous studies [3O7 or Na1.97Al1.82Ti6.15O16 . The ch3O4 doping on Na-β″-alumina strongly differs from TiO2 doping. The microstructure of samples doped with 1.0 wt% Mn3O4 and sintered at 1500 °C or 1600 °C predominantly consist of small grains, while sintering at 1700 °C results in excessive grain growth. Higher doping amounts do not promote the formation of large grains  and Na2CO3 were used in this work. Especially the Al source influences the Na-β″-alumina ceramic´s fracture strength [NiO is the most efficient dopant in terms of increasing fracture strength. Doping with 1.5 wt% NiO increased the characteristic fracture strength from 162 MPa to 296 MPa after sintering at 1600 °C, because it prevents extensive grain growth. The impact of Ni-ion incorporation into the crystal lattice on the fracture strength is not clear yet. However, it seems likely that very high doping amounts can destabilize the crystal lattice. Zhu et al.  also dopstrength ,37.b and Rgb.High ionic conductivity of the electrolyte is crucial for a highly efficient cell system. Several factors are influential for the ionic conductivity of polycrystalline Na-β″-alumina electrolytes. First, grain size and extensive grain growth play an important role . Large g−1 over 0.21 S cm−1 to 0.25 S cm−1 with an increasing sintering temperature from 1500 °C over 1600 °C to 1700 °C. This increase can be addressed to the increasing grain size since the phase content of the sample stays nearly constant  show resistance of 0.38 Ω cm and 0.34 Ω cm because Na-ions must cross more grain boundaries. Potentially, impedance spectroscopy can be used to get an impression of the microstructure of Na-β″-alumina electrolytes. It is already done for other materials [The specific grain boundary resistances Rsgb see a are in aterials ,40.3O4 and NiO doping on the overall ionic conductivity , granulated, pressed, and sintered at different temperatures. For the first time, the mechanism from TiO2 doping of Li-stabilized Na-β″-alumina was clarified and understood. It was found that TiO2 doping promotes the formation of secondary phases such as NaLiTi3O7 and Na1.97Al1.82Ti6.15O16. This assumption was supported by results from SEM–EDX measurements, which clearly proved the absence of Ti inside the Na-β″-alumina grains. Since the melting point of Na2Al2Ti6O16 amounts to 1300 °C [−1 at 300 °C) and the formation of a coarse-grained microstructure even at a very low sintering temperature of only 1500 °C. It is not clear yet to what extent the secondary phases can influence the long-term stability of Na-β″-alumina regarding electrochemical stability and dendrites formation.Li-stabilized Na-β″-alumina powder was doped with three different 3d transition metal oxides .2 is the dopant that affects the Na-β″-alumina properties most intensively. It has the potential to reduce the sintering temperature, increase fracture strength, and enhance ionic conductivity. A good balance of those parameters was found at a doping amount of 1.0 wt% TiO2 and a sintering temperature of 1500 °C. The low sintering temperature decreases the energy consumption of the manufacturing process. Simultaneously, the characteristic fracture strength was increased from 193 to 249 MPa. The ionic conductivity at 300 °C amounts to 0.22 S cm−1 for this material.From the comparative studies, it can be summarized that TiO2 and NiO to evaluate if an even higher characteristic fracture strength can be achieved at low sintering temperatures. Furthermore, tests on Na/NiCl2-cells in order to test the long-term stability of doped Na-β″-alumina electrolytes are pending.The reported results suggest that 3d transition metal doping represent an effective method to adjust the electrolyte properties on the one hand and optimize the energy consumption for electrolyte production on the other hand. By targeted doping, the production of thin-walled, stable, and highly conductive Na-β″-alumina electrolytes for Na-based batteries becomes feasible. Future research will address the mixture of different dopants such as TiO


CdBPTC owned three types of channels with dimensions of approximately 8.4 × 8.3 Å, 6.0 × 5.2 Å, 9.7 × 8.4 Å along a, b, and c axis, respectively. This MOF has high selectivity to ferric ions and shows excellent anti-inference ability toward many other cations. The results indicate that the fluorescence quenching efficiency of CdBPTC is close to 100% when the concentration of Fe3+ reaches 1.0 × 10−3 mol·L−1. Moreover, the luminescent intensity at 427 nm presents a linear relationship at a concentration range of 2.0 × 10−4~7.0 × 10−4 mol·L−1, which can be quantitatively expressed by the linear Stern–Volmer equation I0/I = 8489 [Fe3+] − 0.1400, which is comparable to the previously reported better-performing materials. Competitive energy absorption and ion exchange may be responsible for the variation in fluorescence intensity of CdBPTC in different Fe3+ concentrations.Fluorescent metal–organic frameworks (MOFs) are ideal materials for sensors because of their adjustable pore size and functional groups, which provide them with favorable metal ion selective recognition. In this paper, a new cadmium-based MOF was synthesized using Cd(NO Iron ions play an irreplaceable role in living systems, and the lack or excess of ferrum in the human body causes serious dysfunction. Iron ions are an important component of hemoglobin and myoglobin but are also related to transferrin. In addition, the level of iron ions content has a profound impact on industrial and agricultural production, as well as daily life ,2,3,4,5.3+, thus forming a “turn-off” sensor. For instance, Lv. R. et al. 2 [Cd(BPTC)]∙6H2O)}n, calculated (%): C 37.60, H 5.36, N 4.38; found (%): C 37.72, H 5.29, N 4.67.A mixture of Cd(NOλ = 1.54178 Å). The structure was solved by direct methods and refined by full-matrix least-squares refinements based on F2. Anisotropic thermal parameters were applied to all non-hydrogen atoms. The hydrogen atoms were generated geometrically. The crystallographic calculations were performed using the SHELXL programs [2NH2]+ and H2O were further verified by the FTIR spectra, elemental analysis, and TGA data, obtaining the final chemical formulas of {[Me2NH2]2[CdBPTC]·6H2O}n.A suitable size and high-quality crystal of CdBPTC was mounted at the end of glass fiber for single-crystal X-ray diffraction analysis. Data collection was performed by a Rigaku XtaLAB PRO MM007-DW diffractometer system, which was equipped with an RA-Micro7HF-MR-DW(Cu/Mo) X-ray generator and a Pilatus3R-200K-A detector x aqueous solutions were added to the above suspensions. Then, the suspensions were loaded in the cuvettes for collecting the photoluminescence spectra. The fluorescence intensity of the suspension in DMF was measured as the blank sample.In metal ions sensing, 3 mg of CdBPTC was ground to a fine powder and ultrasonically dispersed into 3 mL DMF to prepare the 1 mg·mL−4~7.0 × 10−4 mol·L−1. The Ksv was about 8489 L·mol−1, which was obtained from the linear Stern–Volmer equation. This MOF exhibited a fluorescence “turn-off” effect for Fe2+ and Fe3+, which may be attributed to the competitive absorption of excitation source energy between ferric ions and CdBPTC. This type of MOFs can be developed as a potential candidate for the detection of Fe3+ in complex environments and can also be used for the determination of iron ion concentration in environmental hygiene, food safety, medical health, soil, and other media.In summary, we successfully synthesized an anionic microporous MOF, CdBPTC, with good thermal and chemical stability. CdBPTC was proved to be highly selective for the discrimination of ferric ions and showed excellent anti-inference ability toward many other cations. The luminescent intensity presented a linear relationship at a concentration range of 2.0 × 10


Structural and electrochemical characterizations indicate that the significant enhancement is correlated to the high charge transfer efficiency from nitriding nanosheet products Fe3N. Our report here may provide new insight on the development of high-performance, low-cost and binder-free supercapacitor electrodes for flexible and portable electronic device applications with multiple anions.In this work, we report the fabrication and enhanced supercapacitive performance of nitrogen-doped nanoporous stainless steel foils, which have been prepared by electrochemical anodization and subsequent thermal annealing in ammonia atmosphere. The nanoporous oxide layers are grown on type-304 stainless steel foil with optimal thickness ~11.9 μm. The N-doped sample exhibits high average areal capacitance of 321.3 mF·cm With rapid growth of global population and high spread of electronic devices, the demanding for energy storage devices and technologies have stimulated high interests towards research and development from academia and industry sectors ,2. As th2O3 [3O4 [−2 in NaOH electrolyte. Deshmukh et al. [−2 average capacitance. Long et al. [Although much research has been devoted to the usage of SS served as substrate or collector ,7,8, lit2O3  or Fe3O42O3 [3O4 . Among t2O3 [3O4  made wirh et al.  thermallg et al.  exploredRecently, mixed-anion compounds, conductive polymer compounds or compounding metals with multiple anions beyond the single-oxide ion, offered a new platform for the superior functional materials ,15,16. TConsidering the issues raised above, we fabricated an ultra-thick nanoporous oxide layer on the surface of type-304 stainless steel foil by pulsed anodic oxidation method with different applied voltages . A nitro2H6O2 99.5%, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), 0.37 g ammonium fluoride  and 0.18 g H2O. To optimize the supercapacitive performance of NASS, three different potentials, 30, 50 and 70 V were applied with the oxidation time of 2 h at room temperature. The synthesized NASS samples were rinsed in acetone and DI water several times, and then annealed at 500 °C in air for 2 h to stabilize their nanoporous structure. Some NASS samples were further treated under ammonia atmosphere at 500 °C for 1 h to fabricate the nitrogen doped (NASS-N) samples.Nanoporous anodized stainless steel (NASS) sample was synthesized through anodic oxidation method. Prior to oxidation process, the SS foils  with dimensions of 10.0 mm × 20.0 mm × 0.1 mm were ultrasonicated in acetone, ethanol, and DI water for 10 min, respectively. The anodic oxidation was performed in a two-electrode electrochemical cell with the SS foil as the working electrode and a graphite plate as the counter electrode. The electrolyte was made by a mixture of 111 g ethylene glycol . A small amount of powder was scratched from stainless-based samples. The powder was further mixed with ethanol followed by 15 mins’ ultrasonic dispersion, forming a homogeneous solution for further transmission electron microscope  observation. The crystal structures were tested by an X-ray diffraction  in the 2θ range of 10–70 degree with CuKα (λ = 1.54 Å) radiation. The chemical analysis was evaluated under an X-ray photoelectron spectroscopy . The elemental compositions and species were studied through an energy dispersive spectrometry  equipped in the same SEM.2SO4 neutral aqueous solution was served as electrolyte in the electrochemical measurements.The supercapacitive performance of NASS and NASS-N samples was examined by cyclic voltammetry (CV), electrochemical impedance spectra (EIS) and galvanostatic charge-discharge (GCD) tests employing an electrochemical workstation . All the tests were under a standard three-electrode system, where the fabricated samples, platinum mesh and Ag/AgCl were used as working, counter and reference electrode, respectively. 1.0 M Na3O4 phase (0.489 nm) [3N phase (0.269 nm) [3N crystal structure..489 nm)  from the.269 nm) . The res2O3 at 24.1° (012), 33.2°(104), 41.2°(113), 49.3° (024) and 54.3° (116) (JCPDS No. 33-0664). After the nitrogen doping process (NASS-N), several new peaks appeared at 28.7°, 30.3° and 57.5°, corresponding to (101), (110) and (112) of Fe3N (JCPDS No. 49-1663). These data provide the evidence that some stable Fe3N crystals were generated during nitridation process, which shows a good agreement with the observation result from TEM. Moreover, the peak intensity of Fe3O4 at 18.5° (111), 37.4° (222) and 57.5° (511) (JSPDS No. 75-0449) increased significantly after nitridation. This can be explained as the production of Fe3O4 from Fe2O3 by the reduction of ammonia at a relatively high temperature of 500 °C. Further information on the chemical composition and surface electronic state of NASS and NASS-N was acquired from XPS. As shown in 3+, while the satellite peak suggesting the presence of Fe3N after nitrogen doping [The phase and composition of NASS-N are characterized by SEM-EDS, XRD and XPS. The SEM-EDS elemental mapping of NASS-N is shown in ), 41.2°1, 49.3°  characteristic of the electrodes [−2 are presented in −2, 90.0 mF·cm−2 and 62.5 mF·cm−2  calculated from GCD curves [Electrochemical Measurements were carried out to evaluate the capacitive performance of NASS samples with different anodic fabrication potentials. ectrodes ,30. MoreD curves . This re−1. No obvious redox peak was found even at low scan rate, indicating its EDLC characteristic. Furthermore, the good symmetrical characteristic of CV curves proved reliable electrochemical reversibility of NASS-N samples. −2. The nearly isosceles triangle shape and no obvious potential plateau further confirmed that the EDLC characteristic of NASS-N sample. The areal specific capacitance decreased with increasing current density due to the limited ion diffusion. At a commonly used current density (1.0 mA·cm−2) [−2 (discharge time was 257 s). −2 to 8 mA·cm−2, the capacitance is dropped from 330.7 mF·cm−2 to 250.3 mF·cm−2, with 75.7% of the capacitance is retained. The result shows its good rate capability. A series of comparison of electrochemical measurements between NASS and NASS-N sample have been recorded and shown in −2, while NASS-N samples had the value of 90 mF·cm−2. The IR drop was also obtained from GCD curves [ct = 5.23 Ω), which can be contributed due to its improved conductivity by nitrogen doping process. As shown the intercept of the real axis in the high frequency range, both of the samples had relatively small equivalent series resistance of around 3.5 Ω), benefited from the good adhesion of SS substrate with active materials, and their nanopouors structure.The nitrogen doping processes were performed on the optimal 50 V anodic NASS samples reported above. mA·cm−2) ,33, the D curves . For NASD curves . The NASD curves . In the −2. A degradation in cyclic stability was observed by nitrogen doping process. After 500 cycles, 70% of the areal capacitance of NASS-N samples was preserved, while the residual capacitance of NASS samples was more than 84%. The inset in 3N nanosheets in NASS-N sample. In the ammonia atmosphere environment with high temperature, part of Fe2O3 in the nanoporous structure was reduced to Fe3O4, and was further transformed to Fe3N. Compared with Fe2O3 or Fe3O4, Fe3N had relatively low molecular weight and can transfer three electrons per formula unit as anode materials [3N also has high conductivity and good ionic diffusion due to the vacancies within their crystal structures [3N nanosheets were planted in the nanoporous structure tightly, the charge carrier mobility of NASS sample can be improved accordingly, leading to smaller impedance in the Nyquist plots, smaller IR drops, as well as larger capacitance value. However, the excess oxygen vacancies caused by nitrogen doping process may lead to the poor cyclic stability [The NASS sample shows a relatively high capacitance value compared with other report on Fe oxides ,12,13,38aterials , and resructures . Since Ftability ,41. The 3N nanosheets were existed in the oxide layer without any damage on its nanoporous structure. The nitrogen doped sample exhibited excellent supercapacitor performance with an areal capacitance as high as 321.3 mF·cm−2 at 1.0 mA·cm−2, which was 3.6 times than the figure of untreated sample, owing to improved charge transfer efficiency from Fe3N nanosheets. The observations and discussions reported in this article may contribute to a better understanding of how to fabricate the high-performance, binder-free SS-based electrodes for supercapacitor usage in the view of constructing multiple anions compounds.In summary, nanoporous oxide layers have been successfully fabricated on the surface of SS foil through anodization method for supercapacitor applications. The electrochemical anodization potential was optimized and 50 V oxidized sample showed the highest average areal capacitance. High temperature ammonia treatment was adopted for supercapacitive performance enhancement. The optimal thickness of nitrogen doped sample is ~11.9 μm. The morphological and structural characterization such as SEM, TEM, XRD and XPS concluded that Fe