We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation  Mammalian cells contain two sources of DNA. While the vast majority of genetic material is contained within the nucleus, mitochondria contain their own small circular dsDNA genome called mitochondrial DNA (mtDNA). The mitochondrial genome is ancestrally bacterial and is typically present at thousands of copies per cell. It encodes only thirteen proteins, all of which are essential subunits of the respiratory chain complexes responsible for the majority of cellular energy transduction . MitochoTransactions between proteins and DNA during DNA replication, gene expression, and DNA packaging disrupt the double helical structure of DNA. During transcription, a translocating RNA polymerase complex creates localised regions of overwinding (positive supercoiling) ahead of the transcription bubble and underwinding (negative supercoiling) behind it ,11. DuriExcessive unresolved negative supercoiling can promote the formation of R-loops and other non-standard DNA structures that can threaten genome stability, while excessive positive supercoiling can inhibit the progression of DNA or RNA polymerase complexes .Topological problems in DNA are solved by topoisomerases, a ubiquitous family of enzymes that untwist and untangle DNA. The human genome contains six topoisomerases. The type IB topoisomerases TOP1 and TOP1MT are swivelases that are able to relieve supercoiling during transcription and replication ,15. TOP1Escherichia coli both type IIA and type IA topoisomerases can contribute to decatenation , then an intact DNA strand is passed through the break, and the break is resealed. This mechanism permits the removal of either intramolecular supercoils (if the broken and passaged strands are from the same DNA molecule) or the decatenation of intermolecular DNA linkages (if the broken and passaged strands are from different DNA molecules). In the eukaryotic nucleus, type IIA topoisomerases are the primary enzymes that act in the removal of chromosome interlinks ,21, whiltenation . The tratenation . The lostenation ,29. TOP3tenation ,31, whertenation .TOP2A and TOP2B are homologous type IIA topoisomerases with essential roles in nuclear DNA maintenance. TOP2A is expressed only in proliferating cells ,34, and In this work, we have assessed the localisation and activity of the six human topoisomerases with a focus on mitochondria and their contributions to mtDNA maintenance. Our results indicate that mitochondria possess one type IA topoisomerase, TOP3A, and one type IB topoisomerase, TOP1MT. Both enzymes contribute to the maintenance of mtDNA topology during DNA replication. TOP3A is also required for mitochondrial transcription, particularly of promoter-distal transcripts. TOP3A and TOP1MT show partial functional redundancy, and based on our results we propose a division of labour between TOP3A and TOP1MT during mtDNA replication and expression.HeLa cells and HEK cells were maintained in Dulbecco's modified Eagle's medium , SH-SY5Y cells were maintained in a 50:50 mixture of minimal essential medium  and Ham's F-12 Nutrient Mixture (Gibco 21765029), K562 cells were maintained in Iscove's modified Dulbecco's medium , and Nalm6 cells were maintained in RPMI 1640 medium (Gibco 61870010). All media were supplemented with 10% foetal bovine serum , 100 U/ml penicillin and 100 μg/ml streptomycin .Transfections using siRNA were carried out using Lipofectamine RNAiMAX (Invitrogen). HeLa cells were passaged and transfected in suspension using 5 nM of each siRNA, and re-transfected after 72 h for a total of six days of transfection. All siRNA oligonucleotides were Silencer Select siRNAs (Ambion), and details are provided in 5 cells per well onto glass coverslips in 6-well plates and left for 16 h prior to transfection. Plasmid transfections were performed for 24–48 h using Lipofectamine 3000 (Invitrogen L3000015), according to the manufacturer's instructions. For each transfection, 2500 ng of DNA was prepared in serum-free OptiMEM media (Thermo Fisher 31985070). Details of plasmid constructs are provided in the section ‘Cloning and DNA templates’.For transient transfections, HeLa cells were seeded at a density of 1 × 10TOP2B exon 1 was targeted with a single guide RNA oligonucleotide (gRNA) (CGCGCCGCAGCCACCCGACT). This guide RNA was cloned into the plasmid vector pSpCas9 (BB)-2A-GFP (PX458) and the resulting construct was transfected into SH-SY5Y, K562 and Nalm6 cells by Nucleofection  using the Amaxa® Cell Line Nucleofector® Kit T  or Amaxa® Cell Line Nucleofector® Kit V  according to the manufacturer's instructions. Cells were selected and sorted based on GFP expression as reported previously  (pSpCas9(BB)-2A-GFP (PX458) was a gift from Feng Zhang  .8 cells by a differential centrifugation method adapted from (g for 5 min. Cell pellets were weighed and resuspended in nine volumes (assuming a cell density of 1.25 g/ml) of hypotonic buffer (20 mM HEPES (pH 8), 5 mM KCl, 1.5 mM MgCl2, 2 mM DTT, 1 mg/ml BSA, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1× protease inhibitors (Thermo Scientific A32965)). After incubation on ice for 10 min, cells were homogenised by 10 strokes of a 15 ml glass Dounce homogeniser with tight-fitting pestle. A 5% sample was retained as the whole cell extract fraction. A two-thirds volume of 2.5× MSH buffer , 5 mM EDTA, 1 mg/ml BSA, 0.2 mM PMSF, 2 mM DTT and 1× protease inhibitors) was added and the lysate was centrifuged at 1600 g for 10 min. The pellet from this step was retained as the nuclear fraction, and the supernatant was centrifuged again at 1600 g for 10 min. The pellet was discarded, and the supernatant centrifuged at 10 000 g for 10 min. The supernatant from this step was retained as the cytosolic fraction, while the pellet was resuspended in 10 ml DNase buffer , 10 mM MgCl2, 2 mM EDTA, 1 mg/ml BSA, 1 mM PMSF and 1× proteinase inhibitors) and treated with 100 U DNase I (Ambion AM2222) for 1 h on a roller at 4°C. EDTA was added to a final concentration of 15 mM and the pellet was washed twice with 1× MSH (DNase buffer omitting MgCl2 and BSA). The pellet was then resuspended in 100 μl of 1× MSH and loaded onto a two-step 1.5/1 M sucrose gradient made up in gradient buffer (10 mM HEPES (pH 7.8), 5 mM EDTA, 2 mM DTT). Sucrose gradients were centrifuged at 117 000 gmax (33 000 rpm in a Beckman Coulter MLS50 rotor) and mitochondria recovered from the interface of the sucrose fractions. Four volumes of gradient buffer were added, mitochondria were washed and pelleted twice with 1× MSH, and then snap frozen in liquid nitrogen. Whole-cell extract and nuclear fractions (200 μl) were treated with 250 U Benzonase for 30 min at 37°C to remove genomic DNA prior to protein extraction.Mitochondria were isolated from ∼1.5 × 10ted from . All steFor western blotting of TOP1MT in crude mitochondrial fractions, mtDNA extraction for 2DNAGE, analyses of mtDNA topology, and whole genome sequencing, the above protocol was followed until the DNase step.g for 15 min. Samples were then resuspended in 1× MSH buffer and divided into two, and one set of samples was treated with 25 μg/ml proteinase K for 30 min at 37°C, then washed and frozen as above.Sucrose gradient purified mitochondria were resuspended in 1× MSH without PMSF or proteinase inhibitors and divided into three equal volumes. Samples were either untreated , or treated with 25 μg/ml proteinase K at 37°C for 30 min in the absence or presence of 1% (v/v) Triton X-100. PMSF was added to proteinase-treated samples to a final concentration of 5 mM, then mitochondria were pelleted and washed twice in 1× MSH and snap frozen. For mitoplast preparations, equal volumes of sucrose gradient purified mitochondria were resuspended in 1× MSH buffer without PMSF or proteinase inhibitors, and digitonin was added to the indicated concentrations in a final volume of 500 μl. Samples were incubated on ice for 10 min and then mitochondria were pelleted at 15 000 C57BL6/J mice were housed in single-sex cages at 20 ± 2°C with a 12 h light/dark cycle. Animal experiments were conducted in compliance with the UK Home Office (PPL P76987201) and the Newcastle University Animal Welfare Ethical Review Board (AWERB 425).2, 1 mM DTT, 0.1% (w/v) BSA, 1× protease inhibitors, pH 7.4) then cut into small pieces using a fresh scalpel. The tissue was transferred into a 15 ml glass Dounce homogeniser in 10 ml of extraction buffer and disrupted with ten strokes of a loose-fitting pestle followed by ten strokes of a tight-fitting pestle. The volume was adjusted to 20 ml with extraction buffer, and a sample retained as the whole cell extract. The remaining sample was centrifuged at 700 g for 10 min at 4°C. The pellet was retained as the nuclear fraction, and the supernatant was centrifuged at 10 000 g for 15 min at 4°C. A sample of the supernatant was retained as the cytosolic sample. The pellet was resuspended in 10 ml of DNase buffer, and DNase treatment and sucrose gradient centrifugation were carried out as described in the section ‘Mitochondrial isolation from cultured cells’.Mitochondria were isolated from fresh mouse liver and spleen tissue according to the method of ,52. BrieThe full-length cDNA sequences for TOP1 (Genscript NM_003286.4), TOP1MT (Genscript NM_052963), TOP3A (Genscript NM_004618.5) and TOP3B (Genscript NM_003935.4) were amplified (omitting the stop codon), incorporating 5′ KpnI and 3′ XhoI restriction sites in the primer sequences. These were then cloned into pcDNA5/FRT/TO in frame with eGFP, which was cloned between the XhoI and ApaI restriction sites. To create the Δ25-TOP3A-eGFP construct, the TOP3A cDNA sequence was amplified from M26 and cloned as above.in vitro assays, the mtDNA region containing LSP  followed by a random DNA sequence upstream of the promoter, were obtained from Eurofins Genomics in the form of linear dsDNA (GeneStrands). LSP-containing DNA fragments were cloned into pUC19 between HindIII and PstI restriction sites (for LSP-A) and PstI and BamHI restriction sites (for LSP-B). In order to confirm whether both cloned promoters have equivalent transcription efficiency, the plasmid was linearised with HindIII and BamHI for use as a template for in vitro transcription reactions. The dual promoter template was used in a supercoiled conformation when the effect of topoisomerases in an in vitro transcription system was assayed. For relaxation experiments, a supercoiled pUC18 plasmid containing the LSP region (positions 1–477 in mtDNA) was used.For For HeLa cells, fixation of cells transfected with topoisomerase-eGFP constructs was performed using 4% (v/v) paraformaldehyde (PFA) with 0.02% (w/v) EM Grade L-Glutaraldehyde (2BScientific). For antibody labelling of TOP2A and TOP2B, optimised fixation was performed using 4% (v/v) PFA and 8% (v/v) PFA respectively. In each case fixation was performed for 10 minutes at room temperature. Permeabilisation was performed in 0.5% (v/v) Triton X‐100 in PBS for 15 min, followed by blocking in 5% (w/v) BSA in PBS for 1 h. Coverslips were then incubated with primary antibodies overnight in a humidified chamber at 4°C. The following day the coverslips were washed extensively with PBS prior to incubation with secondary antibodies (at a dilution of 1:500) for 2 h at room temperature. The details of all primary and secondary antibodies used are provided in de novo transcription products in HeLa cells, cells were pulsed with 2.5 mM BrU  for 1 h prior to fixation using 4% (v/v) PFA with 0.02% (w/v) EM Grade l-glutaraldehyde as described above. Permeabilisation, blocking, labelling and mounting were performed as described earlier for HeLa cells. Incorporated BrU was detected using a primary antibody against BrdU and co-labelled with TOM20. Secondary antibodies were used at a dilution of 1:200 for 2 h at room temperature.For detection of For HeLa cells, Airyscan images were acquired on a Zeiss LSM800 with Airyscan microscope using a 63× 1.4 NA objective. The 405, 488 and 640 nm laser lines were used for image acquisition in conjunction with refractive index‐matched immersion oil (Zeiss). Airyscan processing of the raw images was performed using the Airyscan processing function in the ZEN software.Following image acquisition and subsequent Airyscan processing of HeLa cells following staining of topoisomerases, images were analysed using the object analyser function in Huygens Essential deconvolution software (Scientific Volume Imaging). Specifically, the degree of co-localisation between the topoisomerase signal (or H3 and ATP5I controls) and either the DAPI or TOM20 signal for each cell was measured using the co-localisation analyser tool.xyz) nm was used. The fluorophore Alexa Fluor 594 was excited at 590 nm and Atto647N at 646 nm using a 775 nm laser. Following acquisition, images were deconvolved using Huygens Essential deconvolution software and the object analyser tool was used to determine the number, size and intensity of BrU foci.BrU-labelled HeLa cells were imaged by three-dimensional (3D) STED nanoscopy using a Leica TCS SP8 gSTED 3× microscope (Leica Microsystems) equipped with white light lasers. Images were acquired using a HC PL APO 100×/1.40 Oil STED WHITE objective and a voxel size of (10-20) × (10-20) × 100 nm (®Stranded mRNA Sample Preparation Guide (Illumina). Sequencing (75 bp single end) was performed on the Illumina NextSeq 500 platform.RNA extraction was performed using an RNeasy Mini kit  according to the manufacturer's instructions. The total RNA concentration and RNA integrity number (RIN) of samples were determined using an Agilent 2100 Bioanalyzer System . For SH-SY5Y WT and TOP2B knockout cells, a TruSeq Stranded mRNA kit (Illumina) was used to select polyA+ RNA molecules and libraries were constructed using the High Sample (HS) Protocol of the TruSeqHomo sapiens GRCh38; and quantification was carried out using only coding transcripts. The counts were summarised at gene level using tximport (v.3.13)  was performed on the Illumina NovaSeq6000 platform. Quality checks of raw RNASeq data were made using FastQC (v.0.11.9) and FastQ screen (v0.14.1) ; the qua(v.3.13) . The ann(v.3.13) ,60. NuclCrude mitochondria were isolated from siRNA-treated HeLa cells as described in ‘Mitochondrial isolation from cultured cells’, and DNA was extracted from mitochondrial pellets using a DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's instructions. Isolated DNA (500 ng) was used for library preparation and sequencing, which were carried out by the Newcastle University Genomics Core Facility. Libraries were prepared using an Illumina DNA PCR-Free Prep kit, and sequencing was performed on an Illumina Novaseq6000 at 30× median genome coverage.The quality of sequencing results was assessed using FastQC and MultiQC. Sequences were aligned to the human reference genome with bowtie2  . Alignedg at 4°C and the supernatant retained. Equal amounts of protein (determined by BCA assay) were separated on 4–20% Criterion TGX SDS-PAGE gels (Bio-Rad) and electroblotted onto nitrocellulose membranes. Membranes were blocked in 5% (w/v) milk (Marvel) in PBS at room temperature for 1 h. Primary antibodies were incubated in 5% (w/v) milk in PBS overnight on a roller at 4°C. Membranes were washed three times for 10 min with 0.1% (v/v) Triton X-100 in PBS, then incubated with the corresponding secondary antibodies in 5% (w/v) milk in PBS for 1 h at room temperature. Membranes were washed a further three times as above, then developed using Pierce ECL (Thermo Scientific 32109), SuperSignal West Pico PLUS (Thermo Scientific 34580), or SuperSignal West Femto (Thermo Scientific 34096). Details of all antibodies used are provided in Cell pellets were resuspended in lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 1× protease inhibitors), incubated at 4°C on a roller for 30 min, then centrifuged for 3 min at 11 000 6 cells using a DNeasy Blood and Tissue DNA isolation kit (Qiagen 69504) according to manufacturers' protocol. DNA samples were diluted with water to 10 ng/μl. Quantitative PCR was performed for the nuclear target B2M as well as the mtDNA target MT-ND1 using 2× Taqman universal PCR master mix (Applied Biosystems 4305719). Reactions (20 μl) contained 50 ng of DNA, 300 nM forward and reverse primers and 100 nM probe. Oligo sequences used are provided in DNA was isolated from 0.5–1 × 10MT-ND1 and B2M targets from a single plasmid was run alongside each experiment. Samples were run in technical triplicates and at least three biological replicates. qPCR was performed on a StepOnePlus™ Real-Time PCR System using the following cycling conditions: 2 min at 50°C, 10 min at 95°C, then 40 cycles of 15 s at 95°C and 1 min at 60°C  with α-32P dCTP . Primers for probe synthesis are provided in Crude mitochondria were first isolated from cells as described in ‘Cell Fractionation’. Mitochondria were resuspended in lysis buffer , 0.5% (w/v) SDS, 0.2 mg/ml proteinase K) then DNA was isolated by sequential phenol and chloroform extractions, precipitated using isopropanol and resuspended in TE (pH 8). DNA samples (2 μg) were loaded onto 0.4% (w/v) agarose gels without ethidium bromide and separated at 35 V for 22 h at room temperature. Gels were sequentially incubated in depurination buffer , denaturation buffer  and neutralisation buffer (0.5 M Tris–HCl (pH 7.4), 1.5 M NaCl, 2 × 10 min), then blotted overnight onto nylon membrane. Membranes were UV crosslinked at 1200 mJ/cmTwo-dimensional neutral agarose gel electrophoresis was carried out according to the method of . DNA was2. Probes were made by radiolabelling PCR products using a Prime-It II kit (Agilent 300385) using α-32P dCTP , except for the 5.8S rRNA loading control, which was end-labelled with γ-32P ATP  using T4 polynucleotide kinase (NEB). Primers for probe synthesis are provided in Total RNA was extracted from siRNA-treated cells using an RNeasy Mini kit (Qiagen), and RNA concentrations determined using a Nanodrop spectrophotometer. RNA was mixed with an equal volume of 2× RNA loading buffer  glycerol, 6.5% (v/v) formaldehyde, 50% (v/v) formamide, 10 μg/ml ethidium bromide, 0.05% (w/v) bromophenol blue and 0.05% (w/v) xylene cyanol) and separated on 1.2% (w/v) denaturing agarose gels in 1× NorthernMax (Thermo Scientific) running buffer. Gels were washed in deionised water, blotted onto nylon membranes and then UV crosslinked at 1200 mJ/cmSpodoptera frugiperda Sf9 cells. Cell lysis was carried out in 20 mM Tris–HCl (pH 8), 500 mM NaCl, 10 mM β-mercaptoethanol and 1× proteinase inhibitors with a 1× freeze-thaw cycle using liquid nitrogen. The lysate was centrifuged at 20 000 rpm for 45 min at 4°C using a Sorvall Surespin 630 rotor to remove cell debris. The supernatant was applied to His-Select Nickel Affinity Gel (Sigma-Aldrich) equilibrated with Nickel buffer (25 mM HEPES (pH 7), 400 mM NaCl, 10% glycerol, 10 mM β-mercaptoethanol) containing 5 mM imidazole, then washed with Nickel buffer containing 10 mM imidazole and eluted with Nickel buffer containing 250 mM imidazole. The eluted protein was dialysed in 1 l of Nickel buffer overnight with TEV protease at 4°C. This was then loaded again onto His-Select Nickel Affinity Gel and the flow through was collected. The protein was further purified over HiTrap Heparin HP  equilibrated with Buffer A (25 mM HEPES (pH 7), 200 mM NaCl, 10% glycerol, 1 mM DTT, 0.5 mM EDTA (pH 8), 1 × proteinase inhibitors) and eluted with a linear gradient of Buffer B (25 mM HEPES (pH 7), 1.2 M NaCl, 10% glycerol, 1 mM DTT, 0.5 mM EDTA (pH 8), 1× proteinase inhibitors). The eluate was then loaded onto a Superdex 200 16/600 gel filtration column  and eluted with Buffer C (25 mM HEPES (pH 7), 400 mM NaCl, 10% glycerol, 1 mM DTT, 0.5 mM EDTA (pH 8), 1× proteinase inhibitors). Finally, the protein was loaded onto HiTrap SP HP  equilibrated with Buffer A and eluted with Buffer B as a linear gradient.Recombinant mitochondrial RNA polymerase POLRMT , transcrIn vitro transcription time-course experiments were performed in a reaction buffer consisting of 25 mM Tris–HCl pH 8.0, 10 mM MgCl2, 64 mM NaCl, 100 μg/ml BSA, 1 mM DTT, 400 μM ATP, 150 μM GTP, 150 μM CTP, 10 μM UTP, 0.02 μM α-32P UTP  and 4 U RNase inhibitor Murine (New England Biolabs). 4 nM of DNA template, 20 nM POLRMT, 30 nM TFB2M, 5 nM TEFM and 280 nM TFAM were added to the master-mix. Where topoisomerases are added, reactions were supplemented with either 10 nM of TOP1MT, 40 nM of TOP3A, or 0.125, 0.25 or 0.5 U E. coli TopoI (New England Biolabs). All reactions were assembled on ice, initiated by the addition of ribonucleotides and incubated at 32°C. At the indicated times, 25 μl was removed from the master mix and reactions were ended by the addition of stop buffer  followed by incubation at 42°C for 45 min. Reaction products were purified by ethanol precipitation and pellets were resuspended in loading buffer (98% (v/v) formamide, 10 mM EDTA, 0.025% (w/v) xylene cyanol and 0.025% (w/v) bromophenol blue). Transcripts from supercoiled templates were resolved on denaturing 4% (v/v) polyacrylamide gels, and RNAs synthesised from a linearised dual promoter substrate were resolved on 10% (v/v) polyacrylamide gels. Gels were exposed to phosphorimager screens and imaged using a FLA-7000 scanner.2, 100 μg/ml BSA, 1 mM DTT and 5 nM of supercoiled DNA template. Before the addition of the topoisomerase, DNA substrates were preincubated at 37°C for 10 min in the absence or presence of mtSSB as indicated in the figures , then 25 nM (or 50 nM for the TFAM containing experiment) of TOP1MT was added to the reactions and incubated for 10 min. Reactions were terminated by the addition of stop buffer (10 mM Tris–HCl (pH 8), 1 mM EDTA, and 100 μg/ml proteinase K) and incubated at 42°C for 30 min. DNA SDS loading buffer (Thermo Scientific) was used to resolve the resulting topoisomers in 1× TAE, 0.8% (w/v) agarose gels. Gels were stained with 500 ng/ml ethidium bromide in TAE buffer and visualised in a UV transilluminator.Relaxation experiments (20 μl) contained 25 mM Tris–HCl (pH 8.0), 10 mM MgClThe reliable assignment of mitochondrial localisation to a protein relies upon a combination of multiple complementary techniques, which can include bioinformatic methods  showed a reduction in the level of minor arc mtDNA relative to major arc mtDNA Figure . The prein vitro. TOP1MT was able to relax a negatively supercoiled pUC18 plasmid, with the activity being stimulated by the addition of the abundant and core mtDNA replication protein mtSSB  or negative supercoils (underwinding of DNA) that arise during transcription and DNA replication ,17. TOP3Our results, together with the known substrate specificities of TOP3A and TOP1MT, suggest a division of labour between the two proteins during mitochondrial transcription and DNA replication Figure .in vitro model of converging mitochondrial transcription units  under accession number GSE201426. RNA-seq data from TOP2B knockout cells is available under accession number GSE142383. Whole genome sequencing data has been deposited into the Sequence Read Archive (SRA) under project number PRJNA831603. Raw image data associated with this paper is available at Zenodo and can be accessed using the DOI: 10.5281/zenodo.7115828.gkac857_Supplemental_FilesClick here for additional data file.


This method of fabrication does away with the need for combustible reactants such as hydrogen or oxygen, either as a precursor or as an annealing agent. All previous studies using only ozone could not yield metallic films, and required post treatment using hydrogen or oxygen. In this work, it was discovered that the concentration level of ozone used in the ALD process was critical in determining whether the pure metal film was formed, and whether the metal film was oxidized. By controlling the ozone concentration under a critical limit, the fabrication of these noble metal films was successful. Rhodium thin films were deposited between 200 and 220 °C, whereas palladium thin films were deposited between 180 and 220 °C. A precisely controlled low ozone concentration of 1.22 g m−3 was applied to prevent the oxidation of the noble metallic film, and to ensure fast growth rates of 0.42 Å per cycle for Rh, and 0.22 Å per cycle for Pd. When low-concentration ozone was applied to react with ligand, no excess ozone was available to oxidize the metal products. The surfaces of deposited films obtained the RMS roughness values of 0.30 nm for Rh and 0.13 nm for Pd films. The resistivities of 18 nm Rh and 22 nm Pd thin films were 17 μΩ cm and 63 μΩ cm.Rhodium (Rh) and palladium (Pd) thin films have been fabricated using an atomic layer deposition (ALD) process using Rh(acac) 3 and Pd(hfac)2 precursors, and only low-concentration ozone as co-reactant.Rh and Pd metallic thin films were fabricated by atomic layer deposition using Rh(acac) Rhodium nanoparticles and clusters have also been widely used in gas detection and sensing.5–8 Similarly, Pd is also well-known as a catalyst for oxidation of carbon monoxide and organic compounds.9,10 In addition, Pd is also an important material in fields of hydrogen sensing11–14 and hydrogen storage.15–19 For all the unique properties mentioned above, a larger surface area is required to promote the properties of these noble metals. To achieve this purpose, the coated thin film of nanoparticles with large specific surface area on the substrates or the surface of 3D surface is expected. Since the specific surface area is determined by the nanoparticle size, a controllable nanoparticle size is desirable. Compared with thin film, a bulk material fabricated from noble metals would be less efficient and extremely costly. Besides nanoparticle size, the distribution of it is also a critical factor that influences the performance of the materials. Atomic layer deposition (ALD) has been proven to be a promising method that allows the precise, conformal Rh and Pd deposition with controllable nanoparticle size and size distribution.20–22Noble metals like rhodium (Rh) and palladium (Pd) have drawn academic attention since their properties were realized. Rh is widely used as a catalyst for the hydrogenation of carbon dioxide,iii) acetylacetonate (Rh(acac)3) has been found to be a feasible precursor. In 2005, Aaltonen et al. reported Rh ALD process using Rh(acac)3 as the precursor and oxygen as the co-reactant.23 Another study further proved the feasibility of this process, whose result was consistent with the previous study.24 The use of Rh(acac)3 precursor coordinating with ozone and lower deposition temperature only resulted in Rh2O3 thin films. Partial reduction of Rh2O3 to Rh occurred at 190 °C,25 but no further study has obtained pure Rh thin films by continuing increasing the deposition temperature or trying lower ozone concentration. In a later study, H2 pulse was introduced after each ozone pulse to reduce the oxide products to obtain the metallic Rh thin films.26 However, as an explosive and flammable gas, H2 is so dangerous that it is prohibited by many institutions and prevented from large-scale preparation in industry.Compared with other noble metal ALD processes, Rh ALD is limited, as only rhodium(2 (ref. 3 (ref. 3 is very similar to that of Ir(acac)3, it is expected that there is also a threshold temperature for Rh(acac)3–O3 process, above which pure metallic films will be obtained. Besides temperature, the concentration of oxidizing gas is also an important factor that influences noble metal ALD. It was demonstrated that the concentration of oxygen during the ALD process directly determined the products of reaction between Ru(EtCp)2 and ozone.29 Under high concentration ozone, oxygen could easily diffuse into Ru lattice.30 In previous Rh(acac)3–O3 process, 100 g m−3 high concentration ozone was applied, and only the oxide product was obtained.25 As such, it is postulated that Rh thin film could similarly be achieved at higher temperature with precisely controlled low-concentration ozone.Among noble metal ALD processes using metal acetylacetonate (acac) species as the precursors, Pt(acac)2 ref.  and Ir(a. 3 ref.  could boii) hexafluoroacetylacetonate (Pd(hfac)2) precursor. Pd(hfac)2 is the most widely used precursor in Pd ALD. Depositions of Pd thin film coordinated with H2 (ref. 32) and formalin33,34 on Al2O3 have been proven feasible. However, in those studies, the growth of Pd thin film was hampered by Al(hfac) surface species, leading to a longer nucleation period and growth delay at the beginning of the ALD process.35 Weber et al. attributed the problem of Al(hfac) to the carbon contamination and promoted the process by using another O2 plasma after each H2 plasma to eliminate the contamination.36 As a result, Pd thin films with high uniformity and purity were achieved with Pd(hfac)2. Feng et al. further compared the cycle sequences of Pd(hfac)2–H2, Pd(hfac)2–H2–O2, Pd(hfac)2–O2–H2 and Pd(hfac)2–NH3 in one study.37 However, all these known methods require the use of H2 as a reactant or as an annealing agent. To date, all reported attempts to fabricate Pd thin film with Pd(hfac)2 and another oxidizing agent, such as H2O2, H2O, O2 and ozone38 have failed in achieving pure metallic films.The same process with low concentration ozone was also applied to the Pd ALD process with palladium2 as the precursors and precisely controlled low-concentration ozone as the co-reactant. To figure out magnitude of ozone concentration, the calculation was carried out based the assumption that all the C, H in the precursors reacted with ozone to form CO2 and H2O. In this case, the chemical equations were written as:In this study, Rh and Pd thin films were fabricated by using Rh(acac)3,For Rh(acac)2,For Pd(hfac)−3 for Rh ALD and 1.92 g m−3 for Pd ALD, respectively. During the experiment, different ozone concentration was tested and 1.22 g m−3 was finally selected as the ozone concentration.The calculated ozone concentration was 2.16 g mAll thin films were deposited on sapphire substrates, obtaining low resistivity and uniform surface. The growth of Rh revealed an obvious delay, whereas the growth of Pd was linear from the beginning. The morphology deformation of Pd thin films with deposition temperature was discovered.2 and ozone. The projector-augmented wave (PAW) pseudopotential method39,40 and generalized gradient approximation (GGA) – Perdew–Wang 1991 (PW91) functional41 were executed by Vienna Ab Initio Simulation Package (VASP).42,43 The simulation parameters and models are described in ESI.†The first-principle calculations based on DFT were used in this work to reveal the reaction mechanism between Pd(hfac)iii) acetylacetonate (Rh(acac)3, 97%) was purchased from Sigma Aldrich. Palladium(ii) hexafluoroacetylacetonate (Pd(hfac)2, min, 95%) was purchased from Strem Chemicals. High purity nitrogen (99.999%) was used as the carrier and purging gas. Ozone was obtained by converting high purity oxygen (99.999%) with the ozone generator . 1 × 1 cm2 sapphire (α-Al2O3(0001)) was selected as the substrates.Rhodium(44 whereas others were blown dry with high purity nitrogen flow and stored for Pd fabrication. Rh(acac)3 was loaded in a stainless-steel bubbler , while Pd(hfac)2 was loaded in a glass bubbler. The sublimation temperature for Rh(acac)3 and Pd(hfac)2 was set as 165 °C and 65 °C respectively, to obtain enough vapor pressure. The transfer tube between the bubbler and main chamber was permanently kept at 175 °C and 100 °C for Rh(acac)3 and Pd(hfac)2 respectively to prevent the condensation of the precursors.All experiments have been carried out in a custom-built ALD reactor. 150 sccm nitrogen flow continuously passed through the main chamber to keep a base pressure of 1.0 mbar. The sapphire substrates were washed in deionized water, 95% ethanol (with 5% methanol), acetone and isopropyl alcohol in ultrasonic bath respectively. Each procedure lasted for 5 min. Substrates for Rh fabrication were then kept at 500 °C for 4 h in a tube furnace to create reactive sites for Rh nucleation,t1), pulse of precursor (t2), pulse of ozone (t3) and purging time (t4). Then all the processes were set as t1–t2–t4–t1–t2–t4–t3–t4, and the specific duration of each period is listed in The main process consisted of four main periods: pulse of carrier gas into the precursor holder (λCu-Kα = 1.54 Å) radiation and a grazing angle of 1°. The scan speed of 0.5 seconds and 0.01° per step was applied. The thickness and density of the films were measured by X-ray reflectometry (XRR) on the same diffractometer. The elemental and binding information was characterized using X-ray photoelectron spectroscopy  with an Al Kα source. The surface morphology was characterized with field emission scanning electron microscopy  and atomic force microscopy . The RMS roughness was determined over a 1 × 1 μm2 area by the same AFM.The microstructure of the films was studied using grazing incidence X-ray diffraction  with high intensity (2 and ozone on substrate surface by ALD are investigated using DFT simulations. The ALD process starts by the dissociative chemisorption of Pd(hfac)2 to form the adsorbed Pd(hfac) and hfac (cited as “Pd(hfac)*” and “hfac*” respectively) on substrate surface. As an ozone molecule approaches to Pd(hfac)*, an adsorbed Pd atom (cited as “Pd*”) can be produced by an O atom of the ozone at the bridging C–C site of Pd(hfac)* where the C–C bond is cleaved with an activation barrier of 0.46 eV. The O atom of the Pd–O–C bonds of Pd(hfac)* can be also replaced by an O atom of the ozone at the top Pd site to form the adsorbed Pd(hfac–O) (cited as “Pd(hfac–O)*”) with a lower activation barrier of 0.29 eV. When more ozone molecules are available, an activation barrier of 0.42 eV needs to be overcome for Pd* formation by an O atom of the ozone at the bridging C–C site of Pd(hfac–O)* to cleave the C–C bond. The ozone molecule at the top Pd site can also bond with the Pd atom of Pd(hfac–O)* to form the adsorbed O3–Pd(hfac–O) (cited as “O3–Pd(hfac–O)*”), which is reversible and barrierless. The O3–Pd(hfac–O)* then decomposes into the adsorbed O–Pd(hfac–O) and gaseous oxygen with a high activation barrier of 0.83 eV, meaning that PdO formation is kinetically less favourable. Besides, the formed Pd* can be oxidized by the ozone at the Pd–Si site with an activation barrier of 0.64 eV. Based on activation barriers from DFT calculations, it is possible to prepare palladium without reducing steps when ALD conditions are carefully controlled, especially the control of the ozone concentration. The simulation details are described in ESI.†The reaction mechanisms of direct palladium synthesis using Pd(hfac)3–O3 processes shown in , the thrθ = 40.77°), (200) (47.44°), (202) (59.34°) and (311) (83.68°) revealed the preferred (111) orientation of face-centred cubic Rh. From the full width at half maximum (FWHM) of peak (111), the grain size was determined as 10 ± 1 nm by Debye Scherrer equation. Compared with spectrum at 200 °C, spectrum at 210 °C showed higher intensity, revealing higher crystallinity. However, at 220 °C, the peaks were so blurred that only the peak of (111) can be clearly recognized, implying amorphous Rh or Rh2O3. Since the resistivity of thin films deposited at 220 °C was only 17 μΩ cm, it should be assigned to pure metal. Conversely, the resistivity of Rh2O3 should exceed 6000 μΩ cm.25 Therefore, it was believed that thin films deposited at 220 °C were amorphous Rh. The resistivity of the thin films deposited at 200, 210 and 220 °C was 17 ± 5 μΩ cm, which was slightly higher than those in previous Rh ALD studies.23 The increase in resistivity of noble metal thin films when thickness was less than 20 nm was reported to be caused by the electron-surface and electron-boundary scattering when the electron mean-free-path is close to the grain size and the thin film thickness.45,46GI-XRD measurement with 1° grazing angle was carried out to study the microstructure of the thin films. The XRD spectra in −3, lower than the density of Rh bulk. For Rh thin films deposited on sapphire, the RMS roughness was 0.30 nm  were assigned to metallic Rh5/2 and Rh3/2. Another two minor components at 308.9 and 313.7 eV were assigned to Rh2O3. The existence of oxide component was also verified by the component at 530.4 eV in O 1s spectrum 3.spectrum . Accordiθ = 39.92°), (200) (46.43°), (202) (67.77°) and (311) (81.65°) verified the face-centered cubic Pd. The average grain size was determined as 11.6 nm. As the temperature increased, the intensity of specialized peaks was enhanced, indicating the higher crystallinity at higher deposition temperature. Furthermore, compared with the patterns of the thin film deposited at 200 and 220 °C, the pattern at 180 °C shows a shift to lower 2θ value, implying an isotropic expansion of the lattice. The calculated unit cell dimension is 0.401 nm for 180 °C, larger than 0.391 nm for 200 and 220 °C. The peak shift can be attributed to carbon incorporation in Pd lattice.36,52,53 In this case, the elimination of carbon contamination occurred when the deposition temperature continued increasing. The resistivity of Pd thin films deposited at 180, 200 and 220 °C was 63 ± 5 μΩ cm, which was much higher than 9.8 μΩ cm of pure bulk palladium. Since the electron mean free path of palladium is 25.5 nm,54 which is even longer than the calculated diameter of Pd grain, the electron-grain boundary scattering would be greatly enhanced. Moreover, according to the previous Pd ALD study in which the resistivity was 48 μΩ cm,36 the increase in resistivity was also attributed to the carbon content.All the thin films were determined as metallic palladium by XRD patterns of 22 nm Pd thin films on sapphire deposited at 180, 200 and 220 °C . The peaThe growth of Pd thin film was different from Rh deposition, as the growth shown in 46,55,56 However, in this work, Pd nanoparticles that grew at 200 °C were highly coalesced, while at 220 °C were well-separated. The island coalescence was also reported in other Pt and Pd ALD studies.33,57 It was attributed to the poor nucleation. When the pulses of trimethylaluminum (TMA) were applied prior to the main processes, more nucleation sites were created on the surface, and the nanoparticles became well-separated.58,59 Therefore, in this work, the change in morphology of Pd thin films with increasing temperature indicated an enhancement of nucleation.The change in morphology contradicted the general understanding of the aggregative growth of ALD. It is generally believed that lower temperature results in smaller particle size and narrower particle size distribution, as carbon on the surface prevents nanoparticles from coalescence by suppressing their mobility.3 species partly decomposed when the temperature went beyond 200 °C, and (hfac) species full decomposed at 252 °C.35,60 At 220 °C, the surface could be occupied with CF2, C, F and Pd atoms, rather than (hfac) species. During the ALD process, fluorine was reacted away, and the surface carbon help fix and separate the nanoparticles.Combining with XRD result mentioned above, we proposed that the decomposition of (hfac) species (the poisoning species) at 220 °C enhanced the nucleation of Pd. It has been proved that CF PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O peak at 288.0 eV were identified. As no C–F was detected, it was indicated that fluorine element has been eliminated during the ALD process. Pd 3d spectrum was shown in Δ = 5.26 eV) at 341.1 eV were assigned to metallic Pd5/2 and Pd3/2. Another two minor components at 337.1 and 342.2 eV were assigned to the oxide components. In O 1s spectrum 2 as the precursors, ozone as the co-reactant. The short pulses of low-concentration ozone and the appropriate deposition temperature achieved metallic films with smooth surface, controllable thickness and fast growth rate. For Rh deposition, above the threshold temperature, low concentration ozone was consumed fast by the ligands, leading to the pure metallic surface. For Pd deposition, the metallic films were deposited within the temperature range of 180 to 220 °C. In addition, the enhancement of nucleation was discovered at 220 °C, when (hfac) species partly decomposed. Both deposition temperature and ozone concentration were critical to the formation of high purity metallic Rh and Pd thin films. With this ALD process, only a small amount of ozone joined the process, and no dangerous hydrogen would be used to reduce the metal oxides.In this study, Rh and Pd thin films were obtained by ALD method using Rh(acac)A. I. Y. Tok conceived the project. Y. M. Zou and C. Y. Cheng co-wrote this paper. Y. M. Zou and Y. Y. Guo designed the experiments, performed ALD processes, and characterization. C. Y. Cheng accomplished simulation work. A. J. M. Ong corrected the English. R. Goei performed XPS and SEM characterization. All authors discussed the results, finalized the conclusions, and contributed to the manuscript.There are no conflicts to declare.RA-011-D1RA03942C-s001


Our work aimed to compare different pre-analytical procedures and to evaluate a derivatization step for sphingoid bases quantification, to avoid interferences and improve sensitivity. We tested four protocols for the extraction of sphingolipids from human plasma, at different temperatures and durations, and two derivatization procedures for the conversion of sphingoid bases into phenylthiourea derivatives. Different columns and LC-MS/MS chromatographic conditions were also tested. The protocol that worked better for sphingolipids analysis involved a single-phase extraction in methanol/chloroform mixture  for 1 h at 38 °C, followed by a 2 h alkaline methanolysis at 38 °C, for the suppression of phospholipids signals. The derivatization of sphingoid bases promotes the sensibility of non-phosphorylated species but we proved that it is not superior to a careful choice of the appropriate column and a full-length elution gradient. Our procedure was eventually validated by analyzing plasma and erythrocyte samples of 20 volunteers. While both extraction and methanolysis are pivotal steps, our final consideration is to analyze sphingolipids and sphingoid bases under different chromatographic conditions, minding the interferences.The main concerns in targeted “ Sphingolipids are a ubiquitous class of lipids, whose structure always comprises a long-chain base, usually sphingosine (Sph) or sphinganine. Their name derives from the mythological figure of the sphynx, because of their enigmatic nature . Sphingosphingolipidomics”, and is now standardly carried out through liquid chromatography coupled to mass spectrometry to characterize and differentiate simultaneously the numerous species of sphingolipids belonging to the different subclasses [Due to the various roles that sphingolipids have, any alteration of their metabolism could be part of pathological mechanisms or, sometimes, could be the reason for the diseases themselves ,5,6,7,8.bclasses ,10. Howebclasses . In factbclasses ,13, whicOur work aimed to compare different methods of sample handling and extraction for sphingolipids. Furthermore, we evaluated whether a derivatization step by phenylisothiocyanate (PITC) could improve the detection and analysis of free sphingoid bases.We tested four different extraction protocols  to evaluate whether different conditions could deeply affect the recovery of sphingolipids. As displayed in The effects of times (1/2/4/12 h) and temperatures  on the recovery of sphingolipids from plasma were also considered and the results are graphed in One of the main issues in the analysis of Sph and other sphingoid bases is that their levels in plasma and other biological matrices are not always high enough to allow a precise quantitation. Bearing in mind that the concentrations of sphingoid bases in human plasma range from 0.006 µM to 1.56 µM ,25,26,27In order to fix the issue of Sph-interfering signals, we evaluated whether a derivatization of the extract could be determined. As displayed in 6 cells.When we adopted the final extraction protocol  was made and stored or processed as other samples. Otherwise, the use of individual samples was applied in the study of the sphingolipids’ physiological range in the biological matrix . Blood from twenty volunteers was collected in the fasting state using Kormances , the impormances  and the ormances , a pool l matrix .2PITC) were all at analytical grade and were purchased from Sigma-Aldrich . All aqueous solutions were prepared using purified water at a Milli-Q grade . Lipid standards were purchased from Avanti Polar .The chemicals methanol, chloroform, formic acid, acetic acid, ammonium acetate, ammonium formate, dibutylhydroxytoluene (BHT), phenylisothiocyanate (PITC) and 4-nitrophenylisothiocyanate  coupled to a tandem mass spectrometer AB Sciex 3200 QTRAP  equipped with electrospray ionization TurboIonSpray™ source operating in positive mode (ESI+).The instrument parameters were: CUR 25, GS1 45, GS2 50, capillary voltage 5.5 kV and source temperature 300 °C. Spectra were acquired by multiple reaction monitoring, scanning for each analyte, the transitions reported in The instrument parameters were: CUR 25, GS1 45, GS2 55, capillary voltage 5.5 kV and source temperature 500 °C. Spectra were acquired by multiple reaction monitoring, scanning for each analyte, the transitions reported in Short chromatography (BEH C18). The elution gradient (%B) was set as follows: 0–2 min (20%), 2–4 min (20–99%), 4–7 min (99%), 7–7.5 min (99–20%), held until 10 min [l 10 min . The floLong chromatography (BEH C18). The elution gradient (%B) was set as follows: 0–12 min (70–85%), 12.0–12.2 min (85–99%), 12.2–15.0 min (99%), 15.0–15.2 min (99–70%), held until 20 min. Five microliters of clear aqueous supernatant were directly injected into LC-MS/MS. The flow rate was 0.3 mL/min and the column temperature was set to 30 °C.Long chromatography (Cortecs C18). The elution gradient (%B) was set as follows: 0–12 min (70–85%), 12.0–12.2 min (85–99%), 12.2–15.0 min (99%), 15.0–15.2 min (99–70%), held until 20 min. Three microliters of clear aqueous supernatant were directly injected into LC-MS/MS. The flow rate was 0.2 mL/min and the column temperature was set to 30 °C.2PITC derivative using the transitions reported in The instrument parameters were: CUR 25, GS1 45, GS2 55, capillary voltage 5.5 kV and source temperature 500 °C. Spectra were acquired by multiple reaction monitoring, scanning for each PITC or NOProtocol 1. Plasma (25 µL) was diluted with water (75 µL) before being mixed with a methanol/chloroform solution . The lipids were extracted by ice-sonication and thermo-shaking  of the plasma samples. The organic phase was separated via centrifugation  and evaporated under a stream of nitrogen. The residues were dissolved in 100 µL of methanol + 0.1 mM BHT and withdrawn in a glass vial.Protocol 2. Plasma (25 µL) was diluted with water (75 µL) before being mixed with a methanol/chloroform solution . The lipids were extracted by ice-sonication and thermo-shaking  of the plasma samples. The organic phase was separated via centrifugation  and evaporated under a stream of nitrogen. The residues were dissolved in 100 µL of methanol + 0.1 mM BHT and withdrawn in a glass vial.Protocol 3. Plasma (25 µL) was diluted with water (75 µL) before being mixed with a methanol/chloroform solution . The lipids were extracted by ice-sonication and thermo-shaking  of the plasma samples. They went through alkaline methanolysis  and were then neutralized by the addition of glacial acetic acid (4 µL). The organic phase was separated via centrifugation  and evaporated under a stream of nitrogen. The residues were dissolved in 100 µL of methanol + 0.1 mM BHT and were withdrawn in a glass vial.Protocol 4. Plasma (25 µL) was diluted with water (75 µL) and added with a methanol/chloroform mixture . The lipids were extracted by ice-sonication and thermo-shaking  of the plasma samples. They went through alkaline methanolysis  and were then neutralized by the addition of glacial acetic acid (4 µL). The organic phase was separated via centrifugation  and evaporated under a stream of nitrogen. The residues were dissolved in 100 µL of methanol + 0.1 mM BHT and were withdrawn in a glass vial.v/v). The vial was capped and heated at 80 °C for 1 h. Prior to analysis, pure formic acid (5 µL) was added. The best conditions for derivatization were investigated as reported in 2PITC derivatives were obtained with the same protocol, adding to the final extract (25 µL) a solution of NO2PITC/pyridine . 2PITC derivatives.The amine group reacted with PITC to mainly produce the phenylthiourea  derivatiThe recovery of low abundant sphingolipids is commonly accomplished through alkaline methanolysis which causes the lysis of the ester linkage while retaining the intact amide bond. The percentage of intact phospholipids was used to monitor the reaction over time . The instrument parameters were: CUR 25, GS1 40, GS2 45, capillary voltage 5.5 kV and source temperature 400 °C. Spectra were acquired by multiple reaction monitoring, scanning for DPPC (m/z 734.6 > 184.1) and the internal standard PC d7 (m/z 753.6 > 184.1). To chromatographically isolate the analytes, we used a reverse-phase Acquity BEH C8 column 1.7 μm, 2.1 × 100 mm  equipped with pre-column, using as mobile phases (A) water + 0.2% formic acid + 2 mM ammonium formate and (B) methanol + 0.2% formic acid + 1 mM ammonium formate. The flow rate was 0.3 mL/min and the column temperature was 35 °C. The elution gradient (%B) was set as below: 0–14 min (80–99%), 14–20 min (99%), 20–20.1 min (99–80%), held until 25 min. Five microliters of clear supernatant were directly injected into LC-MS/MS.n = 20).The performances of the operating protocol described in v/v); it was ice-sonicated and extracted by following this scheme: (1) ambient temperature extraction (22 °C) for either 1/2/4 or 12 h; (2) cold extraction (4 °C) for either 1/2/4 or 12 h; (3) warm extraction (38 °C) for either 1/2/4 or 12 h; (4) hot extraction (48 °C) for either 1/2/4 or 12 h. Then, the samples went through alkaline methanolysis  and were then neutralized by the addition of glacial acetic acid (4 µL). The organic phase was separated via centrifugation  and evaporated under a stream of nitrogen. The residues were dissolved in 100 µL of methanol + 0.1 mM BHT and withdrawn in a glass vial.The same plasma pool, already mentioned above (25 µL), was diluted with water (75 µL) before being mixed with a methanol/chloroform solution . The lipids were extracted by ice-sonication and thermo-shaking  of the plasma samples. They went through alkaline methanolysis  and were then neutralized by the addition of glacial acetic acid (4 µL). The organic phase was separated via centrifugation  and evaporated under a stream of nitrogen. The residues were dissolved in 100 µL of methanol + 0.1 mM BHT and withdrawn in a glass vial.Plasma (25 µL) was diluted with water (75 µL) before being mixed with a methanol/chloroform solution  was diluted with water (75 µL) before being mixed with a methanol/chloroform solution . The lipids were extracted by ice-sonication and thermo-shaking . They went through alkaline methanolysis  and were then neutralized by the addition of glacial acetic acid (4 µL). The organic phase was separated via centrifugation  and evaporated under a stream of nitrogen. The residues were dissolved in 100 µL of methanol + 0.1 mM BHT and withdrawn in a glass vial.RBCs (10 µL) were lysed by hypotonic shock in double-distilled water (490 µL). An aliquot of the lysed solution . The precision of the methods was calculated as the coefficient of variation (CV%) by extracting five times the same pool sample in a day (intra-day) and another five times the day after (inter-day).The methods performances were tested using the same plasma pool obtained by combining suitable amounts of each sample . For repeated measures comparison among different groups, repeated measured one-way ANOVA with Dunnett post hoc test was performed. In all tests, In this work, we assessed whether different extraction and analytical protocols could affect the results attained from a targeted sphingolipidomics analysis. The single-phase extraction followed by an alkaline methanolysis seems to be crucial for acquiring as accurate as possible results, while its duration and temperature might not be as significant. Another pivotal aspect in the analyses of sphingolipids is represented by the choice of appropriate columns for distinctively analyzing complex sphingolipids and sphingoid bases. On the other hand, derivatization of the sphingoid bases, while effective on paper, especially on non-phosphorylated species, does not allow a consistent improvement for the analysis of phosphorylated sphingoid bases. For this purpose, the use of a proper column, in this case a Cortecs C18, coupled with a full-length chromatography, seems to be much more convenient, in order to efficiently separate Sph from its interfering peaks and still appreciate all other sphingoid bases.


Moreover, Kir4.1/Kir5.1 heterotetramer plays a key role in determining the expression and activity of thiazide-sensitive Na-Cl cotransport (NCC). In addition to Kir4.1/Kir5.1, Kir1.1 (ROMK) is expressed in the apical membrane of the late DCT (DCT2) and plays a key role in mediating epithelial Na+ channel (ENaC)-dependent K+ excretion. High dietary-K+-intake (HK) stimulates ROMK and inhibits Kir4.1/Kir5.1 in the DCT. Inhibition of Kir4.1/Kir5.1 is essential for HK-induced suppression of NCC whereas the stimulation of ROMK is important for increasing ENaC-dependent K+ excretion during HK. We have now used the patch-clamp-technique to examine whether gender and Cl− content of K+-diet affect HK-induced inhibition of basolateral Kir4.1/Kir5.1 and HK-induced stimulation of ROMK. Single-channel-recording shows that basolateral 40 pS K+ channel (Kir4.1/Kir5.1) activity of the DCT defined by NPo was 1.34 , 0.95 (5% KCl) and 1.03 (5% K+-citrate) in male mice while it was 1.47, 1.02 and 1.05 in female mice. The whole-cell recording shows that Kir4.1/Kir5.1-mediated-K+ current of the early-DCT (DCT1) was 1,170 pA (NK), 725 pA (5% KCl) and 700 pA (5% K+-citrate) in male mice whereas it was 1,125 pA, 674 pA and 700 pA in female mice. Moreover, K+-currents (IK) reversal potential of DCT  was -63 mV (NK), −49 mV (5% KCl) and −49 mV (5% K-citrate) in the male mice whereas it was -63 mV, −50 mV and −50 mV in female mice. Finally, TPNQ-sensitive whole-cell ROMK-currents in the DCT2 /initial-connecting tubule (CNT) were 910 pA (NK), 1,520 pA (5% KCl) and 1,540 pA (5% K+−citrate) in male mice whereas the ROMK-mediated K+ currents were 1,005 pA, 1,590 pA and 1,570 pA in female mice. We conclude that the effect of HK intake on Kir4.1/Kir5.1 of the DCT and ROMK of DCT2/CNT is similar between male and female mice. Also, Cl− content in HK diets has no effect on HK-induced inhibition of Kir4.1/Kir5.1 of the DCT and HK-induced stimulation of ROMK in DCT2/CNT.Basolateral potassium channels in the distal convoluted tubule (DCT) are composed of inwardly-rectifying potassium channel 4.1 (Kir4.1) and Kir5.1. Kir4.1 interacts with Kir5.1 to form a 40 pS K These s the DCT . Thus, K the DCT . In addi the DCT . In addisorption , DCT is xcretion . Long-te and CCD . We have the DCT . Moreove+ intake . Moreove channel . Thus, t− content  Test whether there is a gender difference for the effect of HK intake on Kir4.1/Kir5.1 and ROMK; 2) Assess whether the effect of HK on Kir4.1/Kir5.1 and ROMK is modified by Cl− contents in the HK diet.Although the HK-induced inhibition of the basolateral Kir4.1/Kir5.1 activity of the DCT and HK-induced stimulation of ROMK are well established, it is not investigated whether there is a gender difference regarding the effect of HK on Kir4.1/Kir5.1 and ROMK because animals used in the previous studies are either male only or a mixture population . Also, p content . Thus, i+-citrate for 7 days. For dissecting renal DCT in male and female mice, we euthanized the mice with CO2 inhalation plus cervical dislocation followed by opening the abdomen of the mice to expose the left kidney. The left kidney was then perfused with 2 ml L-15 medium (Life Technology) containing Type 2 collagenase (250 unit/ml). After removing the collagenase-perfused kidney, we cut the renal cortex into small pieces which were further incubated in collagenase-containing L-15 media for 40–50 min at 37°C. We washed the tissue three times with fresh L-15 medium and transferred the renal tissue to an ice-cold chamber for dissection. We placed the isolated DCT on a small cover glass coated with poly-lysine and move the cover glass to a chamber mounted on an inverted microscope.We used 8–10-week-old C57/BL6 mice obtained from Zhuhai campus of Zunyi Medical University. The mice were kept on standard rodent chow containing 1% KCl, 5% KCl or 5% KThe protocol for animal use has been approved by independent Institutional Animal Use and Care Committee at NYMC and by Ethics Committee of Zunyi Medical University Zhuhai Campus.2 and 10 HEPES (titrated with KOH to pH 7.4) and the resistance of the pipette was 5 MΩ (for single channel recording) or 2 MΩ (for whole-cell recording), respectively.We used a Narishige electrode puller  to make the patch-clamp pipettes from Borosilicate glass (1.7-mm OD). The pipette was filled with (in mM) 140 KCl, 1.8 MgCl+ currents (ROMK) and Ba2+-sensitive Kir4.1/Kir5.1-mediated K+ currents were measured with an Axon 200B amplifier. The K+ currents were low-pass filtered at 1 KHz, and digitized by an Axon interface (Digidata 1550B) with sampling rate of 4 KHz. We measured the ROMK-mediated K+ currents with gap-free protocol in split-open DCT2 (last 100 μm DCT before the start of the CNT). We used morphological appearance to determine the end of DCT whose diameter was larger than the CNT. However, we could not exclude the possibility that some experiments were actually performed in the early CNT. Accordingly, we have stated that ROMK-mediated K+ currents were performed in the DCT2/CNT. We used.Whole-cell TPNQ-sensitive K+ currents in early part of the DCT (DCT1) which was identified in the first 100 μm after glomerular attachment. After forming a high resistance seal, we monitored the membrane capacitance until the whole-cell patch configuration was formed. We filled the tip of the pipette with a solution containing (in mM) 140 KCl, 2 MgATP, 1 EGTA, and 10 HEPES (pH 7.4) and then back-filled the pipette with the pipette solution containing amphotericin B (20 μg/0.1 ml). The tubule was perfused in a bath solution contains (in mM) 140 KCl, 2 MgCl2, 1.8 CaCl2 and 10 HEPES (pH = 7.4).Step-protocol or ramp-protocol to measure Kir4.1/Kir5.1 mediated K+ channel currents of the DCT1. The currents were low-pass filtered at 1 KHz, and digitized (sampling rate of 4 KHz) by an Axon interface (Digidata 1550B). For the single channel recording, we used the pipette solution containing (in mM) 140 KCl, 2 MgCl2, 1 EGTA and 10 HEPES (titrated with KOH to pH = 7.4) and the bath solution containing 135 NaCl, 5 KCl, 2 MgCl2, 1.8 CaCl2, 5 glucose and 10 HEPES (titrated with NaOH to pH = 7.4). For determining the channel open probability (Po) and NPo (a product of channel number and open probability), we selected a recording which was at least 5 min long and had less than three current levels. By doing so, we could clearly determine the channel close line thereby calculating Po and NPo . The following equation was used to calculate NPo:i is the fractional open time spent at each of the observed current levels. We determined the channel conductance by measuring the current amplitudes over several voltages.We used an Axon200B amplifier (Axon) to measure single K+ (5%).Amphotericin B was purchased from Sigma-Aldrich and TPNQ was obtained from MedChem Express. Collagenase Type 2 and L-15 medium were purchased from Worthington and Zhejiang Senrui Biotechnology Co., respectively. We obtained normal rodent diet containing 1% KCl , 5% KCl diet catalog #2019102901A) and 5% K-citrate diet  from Beijing Keao Xieli Feed Co. . Both HK diets contain the same amount Kif p-values was smaller than 0.05. Data are presented as the mean ± SEM.The software (Sigma plot 14) was used for the statistical analysis. We used t-test for analyzing the values between two groups and employed paired t-test for comparisons of the values within the same group. One-way ANOVA was used for analyzing results of more than two groups and Holm-Sidak test was used as post-hoc analysis. We considered that the difference was statistically significant + channel activity in the basolateral membrane of the DCT in the male mice on a normal K+ diet (NK), 5% KCl diet or 5% K+-citrate diet for 7 days. We have detected the 40 pS K+ channel in 28 patches from total 42 experiments (patches) in the male mice on NK diet  and open probability (Po) from 6 patches in which a clear channel closed level can be detected (so thus we can determine the channel numbers). + channel activity at 20, 0, and -20 mV. It is apparent that 40 pS K+ channel activity is not voltage dependent since NPo and Po are similar at different holding potentials and they are 1.34 ± 0.08 and 0.37 ± 0.01, respectively. The probability of finding the 40 pS K+ channel was decreased in the DCT of the mice on 5% KCl for 7 days , although the difference was not significant   in comparison to NK. However, from the inspection of +-citrate is statistically not significant.The effect of 5% KCl vs. K+-citrate on Kir4.1/Kir5.1 and ROMK in male mice We first used the single channel recording to examine the 40 pS K NK diet . We havenificant . Moreovees, 53%) . Figures+ currents in the DCT1. Because Kir4.1/Kir5.1 is the only type K+ channels in the DCT1  whereas two HK diets for 7 days decreased the Ba2+-sensitive Kir4.1/Kir5.1-mediated K+ currents equally . Since Kir4.1/Kir5.1 determines the membrane potential of DCT1, a decrease in Kir4.1/Kir5.1 activity is expected to decrease the negativity of the DCT1 membrane potential (depolarization). Thus, we next used the whole-cell recording to measure K+ currents (IK)-reversal potential of the DCT1, an index of the membrane potential. K reversal potential of the DCT1 of the mice on 5% KCl and 5% K+-citrate, respectively. K-reversal potential of the DCT1 in the mice on NK was -63 ± 0.5 mV (n = 7) and it was -49 ± 1.8 mV (n = 6) in the mice on 5% KCl for 7 days and -49 ± 2.3 mV (n = 6) in the mice on 5% K+-citrate. Thus, HK diets with either 5% KCl or 5% K-citrates similarly inhibited the Kir4.1/Kir5.1 activity and depolarized the DCT membrane potential.We have also used the whole-cell recording to examine Kir4.1/Kir5.1-mediated Kthe DCT1 , the Ba2− content in HK diet affects the effect of HK diet on ROMK in the DCT2, we used the whole-cell recording to measure TPNQ-sensitive K+ currents (ROMK) in the split-open DCT2 and initial connecting tubule (CNT). + currents (ROMK) measured at -40 mV using gap-free protocol and n = 5) to 1,520 ± 99 pA  and 1,540 ± 68 pA . Thus, HK-induced stimulation of ROMK in the DCT2/initial CNT is similar in the male mice on 5% KCl or 5% K+-citrate.While Kir4.1/Kir5.1 heterotetramer is expressed in the basolateral membrane of the DCT, the ROMK channel is expressed in the apical membrane of the DCT2 . Previou− content of HK diets plays a role in mediating the effect of increased K+ intake on Kir4.1/Kir5.1 in the DCT of female mice, we first used the single channel recording to examine the 40 pS K+ channel activity in the basolateral membrane of the DCT of the female mice on NK, 5% KCl diet or 5% K+-citrate diet for 7 days. We have detected the 40 pS K+ channel in 8 patches from total 12 experiments (patches) in the female mice on NK diet  but the difference was not significant   in comparison to NK. However, from the inspection of +-citrate is statistically not significant.To examine whether Cl NK diet . Figure nificant . Moreovees, 59%) . Figures+ currents in the DCT1 of female mice on NK and HK diets for 7 days. + currents measured with ramp protocol from -100 to 100 mV in the DCT1 of the female mice on 5% KCl and 5% K+-citrate, respectively. + currents were 1,125 ± 47 pA at -60 mV under control conditions (NK) (n = 6) whereas two HK diets for 7 days decreased the Ba2+-sensitive Kir4.1/Kir5.1-mediated K+ currents equally . We next used the whole-cell recording to measure IK-reversal potential of the DCT1 of the female mice on NK and HK diet for 7 days. K reversal potential of the DCT of the mice on NK and 5% KCl or 5% K+-citrate, respectively. K-reversal potential of the DCT1 in the female mice on NK was -63 ± 1 mV (n = 7) and it was -50 ± 4 mV (n = 4) in the mice on 5% KCl for 7 days and -50 ± 3 mV (n = 5) in the mice on 5% K+-citrate. Thus, similar to male mice, HK diets with either 5% KCl or 5% K+-citrates similarly inhibited the Kir4.1/Kir5.1 activity and depolarized the DCT membrane potential in the female mice.We have next used the whole-cell recording to examine Kir4.1/Kir5.1-mediated K− content in HK diet affects the effect of HK diet on ROMK in the DCT2/initial CNT of the female mice, we used the whole-cell recording to measure TPNQ-sensitive K+ currents (ROMK) in the split-open DCT2 and initial CNT. + currents (ROMK) measured at -40 mV using gap-free protocol and n = 6) to 1,590 ± 112 pA  and 1,570 ± 117 pA . Thus, HK-induced stimulation of ROMK in the DCT2/initial CNT is similar in the female mice on 5% KCl or 5% K+-citrate.To test whether the Cl− content of HK diet does not affect HK-induced inhibition of Kir4.1/Kir5.1 of the DCT and HK-induced stimulation of ROMK in the DCT2/initial CNT, we examined whether gender has an effect on HK-induced inhibition of Kir4.1/Kir5.1 and HK-induced stimulation of ROMK. + channel activity in the male and female mice on NK or HK (including 5% KCl and 5% K+-citrate). The NPo in the male mice on NK is 1.34 ± 0.08 (n = 6), which is not significantly different from female mice on NK . The NPo in the male mice on HK is 0.99 ± 0.04 (n = 11), which is also not significantly different from female mice on HK . 2+-sensitive whole-cell K+ currents at -60 mV were measured in the DCT1 of male and female mice on NK and HK (including 5% KCl and 5% K+-citrate). The Kir4.1/Kir5.1-mediated K+ currents of male mice on NK were 1,170 ± 40 pA (n = 7), which is similar to those in female mice on NK . The Kir4.1/Kir5.1-mediated K+ currents of male mice on HK were 710 ± 40 pA (n = 14), which is similar to those in female mice on HK . K-reversal potential measured in the DCT1 of male and female mice on NK and HK (including 5% KCl and 5% K+-citrate). IK-reversal potential of the DCT1 in male mice on NK is -63 ± 0.5 mV (n = 7) whereas it is also -63 ± 1 mV (n = 7) in female mice on NK. IK-reversal potential of the DCT1 in male mice on HK is -49 ± 1 mV (n = 12) whereas it is -50 ± 2 mV (n = 9) in female mice on HK. Thus, the effect of HK on NPo, Kir4.1/Kir5.1-mediated K+ current and DCT1 membrane potential is similar in male and female mice. Finally, gender also does not affect the HK-induced stimulation of ROMK channels in the DCT2/CNT. + currents (ROMK) at -40 mV were measured in the DCT2/CNT of male and female mice on NK and HK (including 5% KCl and 5% K+-citrate). The ROMK-mediated K+ currents of male mice on NK were 910 ± 32 pA (n = 5), which is similar to those in female mice on NK . The ROMK-mediated K+ currents of male mice on HK were 1,530 ± 59 pA (n = 11), which is similar to those in female mice on HK .After demonstrating that Cl+ channel in the DCT  activity. High WNK activity is expected to increase thiazide-sensitive NCC activity. On the other hand, decreased Kir4.1/Kir5.1 activity in the DCT is expected to raise intracellular Cl− thereby decreasing WNK activity and inhibiting NCC. Indeed, − concentrations of the DCT. The view that basolateral Kir4.1/Kir5.1 activity of the DCT controls NCC activity/expression by regulating [Cli−] is also supported by the observations that an increase in the intracellular Cl− concentration due to depolarization of the membrane voltage are responsible for the suppression of NCC activity in DCT cells which were transfected with loss-function-of-Kir4.1 mutants (Previous studies have convincingly demonstrated that Kir4.1 and Kir5.1 are expressed in the DCT . Moreove the DCT . The rolductance , whereasductance . Furtherductance . Becauseductance , changeschannels . Thus, t mutants .+ intake such that NCC activity is stimulated in the animals on decreased dietary K+ intake whereas NCC activity is inhibited by increased dietary K+ intake (+ intake-induced stimulation of NCC activity is essential for preserving K+ during hypokalemia because high NCC activity should decrease Na+ and volume delivery to the ASDN thereby inhibiting ENaC-dependent K+ excretion. Whereas high-dietary K+ intake-induced inhibition of NCC is important to facilitate ENaC-dependent renal K+ excretion during hyperkalemia (+ delivery to the ASDN thereby enhancing ENaC-dependent renal K+ excretion. + concentration and NCC activity between 3.5 and 4.5 mM plasma K+. This suggests the importance of NCC activity in maintaining K+ homeostasis and renal K+ excretion because plasma K+ concentrations between 3.5 and 4.5 mM falls exactly in physiological relevant ranges.Previous studies have convincingly demonstrated that the NCC activity is regulated by dietary K+ intake . The lowrkalemia , because+ intake depolarizes the membrane of the DCT whereas low K+ intake hyperpolarizes membrane of the DCT (+ intake-induced changes in the basolateral K+ permeability and DCT membrane potential since (+ intake-induced inhibition of Kir4.1/Kir5.1 and DCT membrane potential is well established (− content of HK diet in decreasing the negativity of the DCT membrane potential is not tested. This is due to the fact that the HK diets used in the previous experiments often also contains high Cl− contents (− content in the HK diet plays a role in causing depolarization. Three observations of our present study have suggested that high Cl− content in HK diet did not affect the HK-induced depolarization of DCT membrane potential. First, both 5% KCl and K+ -citrate equally decreased NPo of the 40 pS K+ channel to a similar extend. Secondly, both high K+ diets similarly decreased the whole-cell Kir4.1/Kir5.1-mediated K+ currents of the DCT to the identical levels. Finally, HK intake similarly depolarized the DCT membrane potential regardless the Cl− content. Thus, Cl− content of the HK diet has no effect on HK-induced inhibition of Kir4.1/Kir5.1 in the DCT.By performing electrophysiological studies in the mouse DCT, our previous study has demonstrated that high K the DCT . Furtheral since . Althougablished , the rolcontents . It is n+ channel is the same between male and female mice under the control conditions. Secondly, the baseline whole-cell Kir4.1/Kir5.1-mediated K+ currents are similar between male and female mice. Finally, the DCT membrane potential has the same the negativity in both male and female mice. Thus, the present study excludes the possibility that higher NCC activity in female mice than in male mice is due to higher DCT membrane potential in female mice than male mice.We have also demonstrated that the baseline Kir4.1/Kir5.1 activity and HK-intake-induced inhibition of Kir4.1/Kir5.1 activity in the DCT are similar in the male and female mice. It is well demonstrated that female mice have a higher NCC activity /expression than in male mice . Since K− content of HK diets and that baseline ROMK channel activity and HK-induced stimulation of ROMK are also similar between male and female mice. ROMK channel in the ASDN plays an important role in mediating renal ENaC-dependent K+ excretion (in vivo experiments and mathematic modeling have convincingly demonstrated that DCT2 and CNT are two main nephron segments responsible not only for controlling renal K+ excretion under control conditions but also the main segment for facilitating renal K+ excretion during HK (+ excretion. We conclude that Cl− content of the HK diets has no effect on HK-induced inhibition of Kir4.1/Kir5.1 of the DCT and HK-induced stimulation of ROMK in the DCT2/CNT. Also, baseline Kir4.1/Kir5.1 activity in the DCT and ROMK in the DCT2/CNT and the effect of HK on Kir4.1/Kir5.1 and ROMK are the same between male and female mice.The third finding of the present study is to demonstrate that HK-induced stimulation of ROMK channel activity in DCT2/CNT is not affected by Clxcretion . Moreoveuring HK . This no


Results highlighted that calcination at 900 °C led to a “bi-perovskite heterostructure”, where two different perovskite structures coexist, whereas at higher calcination temperatures a single-phase perovskite was formed. The results showed the effectiveness of the preparation procedures in co-doping the A-site of perovskites with barium and lanthanum as a strategy to optimize the cathode’s properties. The formation of nanometric heterostructure co-doped in the A-site evidenced an improvement in oxygen vacancies’ availability and in the redox properties, which promoted both processes: oxygen adsorption and oxygen ions drift, through the cathode material, to the electrolyte. A reduction in the total resistance was observed in the case of heterostructured material.The electrochemical reduction of molecular oxygen is a fundamental process in Solid Oxide Fuel Cells and requires high efficiency cathode materials. Two La For ensuring satisfying overall resistance at the cathode/electrolyte interface, oxygen anions should be easily incorporated into electrolyte lattices. In fact, oxygen anion formation and mobility require the presence of adequate oxygen lattice vacancy numbers and distribution [In the present energy transition, alternative efficient energy generation is in demand, considering the urgent necessity of lowering carbon dioxide emission. It has been acknowledged that solid oxide fuel cells (SOFCs) provide clean energy with higher efficiency and a lower environmental impact than traditional combustion engines through the exploitation of renewable sources . Today, ribution . Thereforibution ,5.1−xSrxCo1−yFeyO3−δ, LSCF or La1−xSrxFe1−yCoyO3−δ, LSFC) and barium strontium cobaltite  have been widely investigated [0.6Sr0.4Fe0.8Co0.2O3−δ with barium gave rise to a reduction of polarization resistance with respect to samples doped with strontium only.Several strategies have been adopted over the years to improve the cathode material performance and, from a green point of view, low cost and efficient cathode materials are advisable. Among them, perovskite-type oxides such as doped-lanthanum strontium ferro-cobaltite .Shao and Haile , in 2004formance . Severalformance ,13,14,150.5Sr0.5Co0.8Fe0.2O3−δ and La0.6Sr0.4Fe0.8Co0.2O3−δ by ball milling with different volume ratios, it is possible to tune material performance between 500–650 °C. The polarization resistance decreases by increasing the BSCF/LSCF volume ratio and, in general, is always larger than LSCF and BSCF as single phases. Recently, Dey et al. [0.6Sr0.4Co0.8Fe0.2O3−δ and Ba0.6Sr0.4Co0.8Fe0.2O3−δ powders. The authors referred to a decrease in resistance due to the optimization of oxygen vacancies in the heterostructure with respect to BSCF’s and LSCF’s single phases. The affirmation of the importance revested by heterostructure as way for improving the performance of oxide materials for energy devices is relatively recent. Like composite materials, heterostructures differ in the positive synergic effects, for example on catalytic or electrochemical features, due to the intimate connection between the different phases [0.25Ba0.25Sr0.5Co0.8Fe0.2O3−δ has not yet been investigated as evidenced by 0.25Ba0.25Sr0.5Co0.8Fe0.2O3−δ-based materials were prepared by solution combustion synthesis, which is a fast, reproducible, versatile and efficient methodology for the preparation of multicomponent perovskite-type oxides [0.5Sr0.5Co0.8Fe0.2O3−δ), and a BSCF (Ba0.5Sr0.5Co0.8Fe0.2O3−δ), were also prepared using the same experimental conditions. To investigate the effect of Ba introduction in both La0.25Ba0.25Sr0.5Co0.8Fe0.2O3−δ-based systems on the chemical physical properties and to evaluate the oxygen vacancies content and the capability of release oxygen, all the powders were characterized by X-ray Diffraction, Transmission Electron Spectroscopy, X-ray Photoelectron Spectroscopy, Thermo-Gravimetric Analysis and H2-Temperature Programmed Reduction techniques. The powders were deposited onto a Ce0.8Sm0.2O2 (SDC) electrolyte and half-cells were examined by Electrochemical Impedance Spectroscopy at various temperatures.Another strategy adopted in the literature was mixing LSCF and BSCF materials to obtain a superior cathode able to bring peculiar features of each single structure . Clematiy et al.  synthesit phases ,20,21. Te oxides . The use0.25Ba0.25Sr0.5Co0.8Fe0.2O3−δ after thermal treatment at 900 °C (BP-HET) and at 1000 °C (LBSCF) for 5h are shown in 2+ (1.61 Å) ions entered the rhombohedral phase, causing an expansion of the rhombohedral unit cell, while it is likely that La3+ (1.36 Å) ions occupy the A-sites of the cubic phase, leading to a unit cell contraction. Interestingly, it seems that La-doping in the cubic BSCF-type phase of the heterostructure avoids the formation of the typical hexagonal impurity phase that was detected in BSCF900 , and La+3 (1.36 Å) causes an increase in rhombohedral unit cell volume. On the other hand, Szpunar et al. [0.5Ba0.25Sr0.5Co0.8Fe0.2O3−δ, while recently Xie et al. [0.4Ba0.2Sr0.4Co0.2Fe0.8O3−δ by coupled experimental and computational studies, noticed the formation of the rhombohedral lattice structure. In order to reconcile this evidence with our data, it is possible to hypothesize that, beside differences in chemical compositions, different oxidation state of B-site ions in LBSCF contribute to a distortion of the lattice from cubic to rhombohedral. In addition, looking at the unit cell volumes of LBSCF and of BP-HET, it is possible to argue that the rhombohedral phase present in the heterostructure is rich in Ba2+, likely rather close to the nominal composition of 25 wt.%. This result is confirmed by the LSCF1000 volume that is much lower than that of the LSCF-type phase in the heterostructure.The XRD patterns of the powders with the nominal composition La BSCF900 . Such re BSCF900 . Thus, br et al.  observedhkl value corresponded to about 2.22 Å  than its parent LSCF900 structure without Ba (100 nm). This result highlights that nucleation and the growing of the LSCF-type phase was affected by the contemporaneous nucleation and growing of the BSCF-type phase in the BP-HET, leading to highly nanostructured particles. In addition, although the extreme variability observed in dhkl values, The TEM images of BP-HET, LBSCF, LSCF and BSCF treated at 900 °C and 1000 °C at low, medium and high resolutions are reported in  1000 °C  are madeL-SrL); a higher energy component, O 1s 532.2eV and Sr 3d5/2 = 133–134 eV (Sr3d3/2 = 135–135 eV), associated to perovskite surface termination and/or surface secondary phase, here indicated as “surface” component (OS-SrS) [XPS analysis was performed with the intention of evaluating the surface state spread along the first layers of surface, which undeniably affect the oxygen adsorption process. The XPS spectra were recorded on the freshly annealed powders, to minimize the influence of physisorbed species. It is worth underlining that regions corresponding to Ba, Co, and Fe were not investigated for the following reasons: (i) Fe 2p region exhibited a low intensity signal due to low nominal 20 wt.% present in all samples; (ii) Co 2p and Ba 3d regions were overlapped leading to erroneous interpretations. Special attention was paid to O 1s and Sr 3d regions to evaluate differences on these surface states that are correlated with oxygen vacancy content and distribution . As depi(OS-SrS) ,27,28. T(OS-SrS) ,30. LBSC(OS-SrS) , this be(OS-SrS) ,13,26. TConsidering that the amount of vacancies affects both oxygen molecule adsorption and oxygen ion drift along the width of cathode , TGA exp2-TPR measurements, performed from room temperature up to 1050 °C. 3/g, respectively, for the BP-HET and LBSCF specimens. These results are consistent with TGA data and confirm that BP-HET is richer in reactive oxygen species than LBSCF. However, TPR results evidenced that BP-HET also has a higher reducibility compared with the single-phase sample. As referred above, BP-HET is formed by two intimately mixed phases with rhombohedral and cubic structures, respectively, thus the presence of two different lattice arrangements positively influences the oxygen species’ reactivity.A further insight into oxygen ions’ mobility and the amount of oxygen released was carried out through H3+ to Co2+ and Co2+ to Co0, whereas the shoulder at low temperature is ascribable to Co4+/Fe4+ to Co3+/Fe3+ [1−xSrxCo1−yFeyO3−δ perovskite-type oxides, the enlarged lower peak, extended between 100–500 °C with a shoulder at about 160 °C, is due to the simultaneous reduction of Co3+ to Co2+ and Fe3+ to Fe2+, while the higher peak, laying between 550–700 °C with a maximum at 600 °C, is due to Co2+ to Co0. The reduction Fe2+ to Fe0 requires temperatures above 800 °C. In light of these attributions, it is reasonable to associate similar reduction processes for BP-HET, even though the whole composite profile is shifted towards lower temperatures hinting at the presence of more reducible cobalt and iron species and more mobile oxygen ions. It is likely that co-doping at A-site and the co-presence of two phases play a beneficial role in the mobility of the lattice oxygen ions. Considering the single-phase cases in Therefore, to discern the correct binomial reduction processes-reduction peaks it is necessary to compare BSCF900 and LSCF900 a. Prompto3+/Fe3+ . In addio3+/Fe3+  for La1−1−nQ)1/n  [Electrochemical impedance spectroscopy was used to evaluate BP-HET and LBSCF assembled in half-cells over the 500–750 °C temperature range, under static air condition at Open Circuit Voltage (OCV). rameter) . In detarameter) . For therameter)  for an Lrameter) , a speci0.25Ba0.25Sr0.5Co0.8Fe0.2O3−δ, Ba0.5Sr0.5Co0.8Fe0.2O3−δ and La0.5Sr0.5Co0.8Fe0.2O3−δ powders were prepared by solution combustion synthesis using La(NO3)3·6H2O , Ba(NO3)2 (99.99% Sigma-Aldrich), Sr(NO3)2 (≥99.99% Sigma–Aldrich) Co(NO3)2·6H2O (99.99% Sigma-Aldrich and Fe(NO3)3·9H2O (99.99% Sigma–Aldrich) as metal precursors and polyethylene glycol  mixed with sucrose  in 1:1 ratio as combustion fuel mixture. The combustion reactions were carried out under over stochiometric condition, a reducers-to-oxidizers ratio (Φ) = 2.2, with a sucrose-to-metal cations molar ratio equal to 2. To achieve this Φ value, ammonium nitrate  was added to the reaction mixture as oxidant additive. Metal nitrates, fuel and additives were dissolved in aqueous solution in a stainless-steel beaker, and the solution was magnetically stirred at 80 °C to form the gel. By increasing the temperature of the hot plate to about 220 °C, a self-sustaining combustion process was initiated, leaving a grey–black powder. Powders were fired in static air at 900 °C and at 1000 °C for 5 h. La0.25Ba0.25Sr0.5Co0.8Fe0.2O3−δ obtained after firing at 1000 °C/5 h was named after LBSCF, the bi-perovskite heterostructure with nominal composition. La0.25Ba0.25Sr0.5Co0.8Fe0.2O3−δ, obtained after firing at 900 °C/5 h, was named after BP-HET, whereas Ba0.5Sr0.5Co0.8Fe0.2O3−δ and La0.5Sr0.5Co0.8Fe0.2O3−δ were named after BSCF900 and BSCF1000 or LSCF900 and LSCF1000, respectively, depending on the firing temperature used.La0.8Sm0.2O2−x (SDC) powder was synthesized according to a solution combustion synthesis procedure reported elsewhere [® F-127 Sigma-Aldrich) and mixed for 30 min in an ultrasonic bath to obtain a well homogenized suspension. The slurries were deposited by screen printing over both faces of sintered pellets, dried at 160 °C for 1 h for each face, and finally treated at 900 °C for 2 h in static air.Membrane electrode assemblies (MEA) with symmetric cell configuration were assembled to carry out electrochemical impedance spectroscopy measurements. The electrolyte Celsewhere . The SDCX-ray diffraction (XRD) measurements were collected with a Siemens D5005 X-ray powder diffractometer equipped with a curved graphite monochromator on the diffracted beam. The observed range 10–90° 2θ was scanned with a step size of 0.03° 2θ and an integration time of 3 s per step. Rietveld refinement of the diffraction patterns was carried out by using the GSAS-II package version 5365 [TEM investigation was carried out by means of JEOL JEM 3010-UHR, acceleration voltage 300 kV. The powdery samples were dry dispersed on holed carbon film deposited on Cu grid.−8 mbar during data collection. The invariance of the peak shapes and widths at the beginning and at the end of the analyses ensured absence of differential charging. Analyses of the peaks were performed with CasaXPS software, based on non-linear least squares fitting program using a weighted sum of Lorentzian and Gaussian component curves after background subtraction according to Shirley and Sherwood [X-ray photoelectron spectroscopy analyses were performed with a VG-Microtech ESCA 3000Multilab, equipped with a dual Mg/Al anode. The spectra were excited by the unmonochromatized Al Kα source (1486.6 eV) run at 14 kV and 15 mA. The analyzer operated in the constant analyzer energy (CAE) mode. For the individual peak energy regions, a pass energy of 20 eV set across the hemispheres was used. Survey spectra were measured at 50 eV pass energy. The sample powders were analyzed as powder, mounted on a double-sided adhesive tape. The pressure in the analysis chamber was in the range of 10Sherwood ,38. Atom2  at 750 °C for 1 h (step 1), then it was cooled down to room temperature in air  purified from CO2 and H2O (step 2) and purged with N2  for 15 min (step 3). After that, the sample was again treated under N2  by heating (rate 5 °C/min) from room temperature up to 1000 °C (step 4). Step 1 was performed to remove any adsorbed water, oxygen or carbonate species; step 2 aimed to fill by oxygen all the vacancies; step 3 aimed to eliminate all the physisorbed oxygen species; step 4, during which the removal of chemisorbed oxygen species occurred, was taken into account in order to evaluate the oxygen vacancies content of the sample. The evolution of gaseous species occurring during the above four steps was monitored by on line mass quadrupole .Thermogravimetric analyses (TGA) were performed with a TGA/DSC1 STAR system Mettler Toledo. The sample (10 mg) was pretreated in N2/Ar  in the range between room temperature and 1050 °C (heating rate 10 °C/min). Experiments were carried out with a Micromeritics Autochem 2910 instrument equipped with a thermal conductivity detector (TCD) for the evaluation of hydrogen consumption through the use of proper calibration curve. All powders (~0.1 g) were pre-treated in O2/He  at 400 °C for 1 h, in order to remove any impurity, and then cooled down under inert atmosphere (Ar flow).Reduction properties of the oxides were studied by temperature programmed reduction (TPR) measurements in H7 to 10−1 Hz was carried out between 500 and 750 °C employing Methrohm Autolab impedance analyzer (AC voltage of 0.3 V) equipped with a ProboStat from NorECs. After 15 h of equilibration at 750 °C in static air, the impedance spectra were collected from symmetrical cells between 750–500 °C , and analyzed with Nova software version 2.1.5 released by Methrohm Autolab.Complex impedance spectroscopy from 100.25Ba0.25Sr0.5Co0.8Fe0.2O3−δ perovskite has been obtained by solution combustion synthesis and investigated for its chemical–physical properties, with the scope of tuning oxygen vacancies content and availability for its application as an electrocatalyst for oxygen reduction reaction in the intermediate temperature range. The positive effect of Ba was assessed, and a peculiar behavior was evidenced upon thermal treatment. Treatment at 1000 °C in air leads to a complete incorporation of barium ions into the rhombohedral perovskite structure, with a positive effect on the oxygen vacancies distribution, if compared with the analogous perovskite without barium. Treatment at 900 °C in air favors the formation of a bi-perovskite heterostructure, where a lanthanum-enriched BSCF-type cubic phase and a barium-enriched LSCF-type rhombohedral phase coexist. Our results highlighted the potential of a one-pot solution combustion synthesis in producing nanostructured perovskite-type heterostructures with improved properties, as evidenced by temperature programmed reduction, thermal gravimetric analysis and electrochemical impedance spectroscopy. The bi-perovskite heterostructure exhibits available oxygen vacancies on the surface and in the bulk and a lower area specific resistance for the oxygen reduction reaction with respect to the single-phase perovskite with the same nominal composition. Other literature findings on perovskite-type compounds were confirmed and more information on the perovskite-type heterostructures for clean energy applications was added, discussing the effect of the bi-perovskite heterostructure on the structural properties, redox properties and oxygen vacancies. The proposed strategy appears to be an intriguing way for the fine-tuning of electrode materials for IT-SOFCs.A La