Nutrient replete conditions prevailed at the beginning of the experiment and light was provided at in situ levels upon reaching pCO2 target levels. A diatom-dominated bloom developed in all treatments with Skeletonema costatum as the dominant species but with an increased abundance and biomass contribution of larger diatom species in the Greenhouse treatment. Conditions in the Greenhouse treatment accelerated bloom development with faster utilization of inorganic nutrients and an earlier peak in phytoplankton biomass compared to the Control and High CO2 but no difference in maximum concentration of particulate organic matter (POM) between treatments. Loss of POM in the Greenhouse treatment, however, was twice as high as in the Control and High CO2 treatment at the end of the experiment, most likely due to an increased proportion of larger diatom species in that treatment. We hypothesize that the combination of warming and acidification can induce shifts in diatom species composition with potential feedbacks on biogeochemical element cycling.An indoor mesocosm experiment was carried out to investigate the combined effects of ocean acidification and warming on the species composition and biogeochemical element cycling during a winter/spring bloom with a natural phytoplankton assemblage from the Kiel fjord, Germany. The experimental setup consisted of a “Control” (ambient temperature of ~4.8 °C and ~535 ± 25 μatm  Climate change, driven primarily by anthropogenic utilization of fossil fuels over the past 250 years, is changing sea surface temperatures (i.e. ocean warming) and seawater carbonate chemistry (i.e. ocean acidification). The IPCC 2014 projects an additional increase in sea surface temperature of 1–6 °C and a decrease in surface pH of ~0.3 units by the end of this century  with potIndirect effects of warming are expected through enhanced stratification of the water column and consequent alteration of nutrient supply and light availability, potentially affecting growth at the base of the marine food web and modulating energy transfer to higher trophic levels  which is2 on PP (+) concentration particularly on calcifying phytoplankton  and combined acidification and warming (i.e. “Greenhouse”) conditions. The increases in temperature and pCO2 applied in our experiment are within the range expected by the end of this century as projected by the IPCC 2014  around noon , “High CO2” treatment  and “Greenhouse” treatment . Elevated temperature and CO2 scenarios were chosen according to IPCC 2014 projections for the end of this century (The indoor mesocosm experiment was carried out between 12 January and 24 February 2012 at the GEOMAR Helmholtz Centre for Ocean Research Kiel. Six mesocosms (see end of section for details) with a volume of 1400 L each  were set up in temperature controlled rooms and filled simultaneously with seawater from Kiel Bight containing a representative late winter/early spring plankton community of bacteria, phytoplankton and microzooplankton. Although mesozooplankton was present in the water column at this time of year, most of it was probably trapped on the fine-sand filter collecting large detritus particles before filling the mesocosms. Dissolved inorganic nutrients at the beginning of the experiment were naturally available and therefore not manipulated. Each mesocosm had a computer-controlled light system equipped with full spectrum light bulbs  covering the full natural spectrum of photosynthetically active radiation (PAR: 400–700 nm). The light programme allowed for simulation of a daily triangular light curve with maximum light intensity . Samples for biogeochemical parameters and phytoplankton analysis were taken daily at 9 a.m. during the bloom period (Days 12–20) and every other day otherwise. Samples were taken from an intermediate depth (0.5 m below surface) either with a membrane pump directly into measuring vials or with a silicone tube by gravity into sample canisters. The canisters were gently mixed before any subsampling took place to ensure representative homogenous samples.Low replication in the experimental setup (i.e. duplicates instead of triplicates per treatment) prevented us from conducting statistical analysis on our data. The low replication stemmed from unforeseen technical issues (i.e. propellers and light system failing) that, although immediately addressed during the experiment, could not be solved. The unbalanced design of the experiment  was due to logistical constraints of the available infrastructure. In view of this, we have applied a rather conservative approach in our data interpretation, remaining mostly descriptive and pointing out treatment effects only in cases where differences among treatments are rather obvious (i.e. no overlapping standard deviation). Nonetheless, we still believe our results provide valuable information about changes in community composition due to changing environmental conditions.in situ conditions) was attained within 12 hrs after filling the mesocosms and remained constant throughout the experiment. For the CO2 manipulation, 25 L of seawater (same water used for filling the mesocosms) were aerated with pure CO2 (99.99%) for ~24 hrs before the manipulation. The CO2-saturated seawater was used to manipulate total dissolved inorganic carbon (DIC) in the High CO2 and Greenhouse treatments while leaving total alkalinity (TA) unaffected. The CO2-saturated seawater was added to the mesocosms at an intermediate depth with a silicone tube and the manipulation performed gradually over 2 days to avoid rapid changes and acute stress for the plankton community. During the CO2 manipulation, in all mesocosms, light conditions during the day were kept at 10% (17–20 μmol photons m−2 s−1) of the maximum surface irradiance to acclimate to the new carbonate chemistry conditions. Once target CO2 levels were attained (Experiment day 3), the regular daily light regime was started. Transparent floating lids  were placed on the water surface for the duration of the experiment to minimize gas exchange with the atmosphere. A gas tracer (N2O) was used to estimate gas exchange fluxes (2O was only added to one mesocosm per treatment and any corrections (see below) were applied also to the replicate mesocosm.Target temperature for the Greenhouse treatment  with ~2 μmol kg−1 precision. N2O was used as a gas tracer to estimate air/water gas exchange of CO2 and the estimated loss/gain in CO2 was used to calculate the biologically driven changes (i.e. photosynthesis and respiration) on DIC concentrations (i.e. DICbio). DIC concentrations presented in this study are therefore representative for biological consumption, unless stated otherwise.DIC samples were sterile filtered (0.2 μm) with a membrane pump at a flow rate of ~50 mL min4 and Si(OH)4), using the dissociation constants by Samples for TA were filtered through GF/F filters and measured within 2 days in a Metrohm Basic Titrino 794 titration device according to 3−), phosphate (PO43−) and silicic acid (Si(OH)4) as well as dissolved organic carbon (DOC), nitrogen (DON) and phosphorus (DOP) were filtered through pre-combusted  GF/F filters with a membrane pump at a rate of ~100 mL min−1 directly into vials. Except for DOC samples, which were fixed with 85% phosphoric acid  and refrigerated, all samples were frozen at −20 °C until analysis. Nutrients were measured on a segmented flow autoanalyzer (SEAL QuAAtro) according to Dissolved inorganic nutrients nitrate (NOSamples for particulate organic carbon (POC), nitrogen (PON) and phosphorus (POP) were filtered at low vacuum (200 mbar) onto pre-combusted  GF/F filters and stored at −20 °C until analysis. Filters for POC/PON were fumed with hydrochloric acid (37% HCl) for 2 hrs to remove all inorganic carbon and subsequently dried overnight at 60 °C. Samples were analysed with a EuroEA analyzer according to pCO2 levels were established) from measurements at each experimental day. In most cases, maximum POM build-up was observed after nutrient depletion and maximum levels lasted for more than 2 experimental days.Initial POM values were calculated by averaging concentrations of all treatments on the first 2 days of the experiment (±standard deviation). Build-up of POM was calculated by subtracting the average of the first 2 days (once target a (Chl a) according to the presence and amounts of marker pigments.Phytoplankton species composition was analysed by pigment analysis [high-pressure liquid chromatography (HPLC)], size categorization by flow cytometry and Utermöhl microscopy. HPLC samples were filtered onto pre-combusted  GF/F filters and stored at −80 °C until extraction. Photosynthetic pigments were separated via HPLC with 100% acetone (HPLC grade) according to the method of v/v final concentration) and immediately frozen at −80 °C until analysis. Samples were thawed for 10 min and mixed gently before measuring with an Accuri C6 flow cytometer (BD Biosciences). Autotrophic organisms were detected by their red fluorescence (fluorescence channel FL3). Using forward scatter as an indicator, three size classes were determined. Group I represented the smallest phytoplankton, approximately in the pico size range (<2 μm). Groups II and III represented phytoplankton approximately in the nano (2–20 μm) and micro size class (>20 μm), respectively. The contribution of each group to total Chl a fluorescence was calculated by multiplying the cell abundance within a gate and its corresponding average red fluorescence (fluorescence channel FL3).Samples for flow cytometry were fixed with 25% glutaraldehyde , while Group III included diatoms >20 μm such as Thalassionema nitzschioides, Thalassiosira sp, Nitzschia longissima, Navicula transitans, Fragilaria sp, Guinardia sp and Chaetoceros sp (>20 μm).Phytoplankton larger than 2 μm were counted using the inverted microscopy method according to pCO2 levels  in the High CO2 and Greenhouse treatments  were established within the first 3 days of the experiment and remained relatively constant for ~10 days. Thereafter, pCO2 levels decreased to an averaged minimum of 40 ± 10 μatm in all treatments  were 7.93 ± 0.02 in the Control, 7.67 ± 0.02 in the High CO2 and 7.69 ± 0.02 in the Greenhouse treatment. pHtotal increased up to maximum values of 8.98 ± 0.04 in the Control, 8.94 ± 0.02 in the High CO2 and 8.87 ± 0.02 in the Greenhouse treatment  was observed along with decreasing inorganic nutrients and increasing Chl nts Fig. D–F. The −1 to maximum average concentrations of 14.1 ± 0.65 μmol N L−1 in all treatments. PON concentrations at the end of the experiment were on average 5.9 ± 1.7 μmol L−1 in the Control, 5.8 ± 0.83 μmol L−1 in the High CO2 and 3.5 ± 0.77 μmol L−1 in the Greenhouse treatment.PON increased from an initial average of 1.6 ± 0.22 μmol N L−1 to maximum concentrations of 0.59 ± 0.07 μmol P L−1 in the Control, 0.58 ± 0.04 μmol P L−1 in the High CO2 and 0.54 ± 0.04 μmol P L−1 in the Greenhouse treatment. Concentrations of POP at the end of the experiment were almost twice as high in the Control (0.37 ± 0.07 μmol P L−1) and High CO2 (0.32 ± 0.07 μmol P L−1) compared to the Greenhouse treatment (0.15 ± 0.05 μmol P L−1). After PO4 exhaustion, DOP slightly increased to maximum concentrations of 0.16 ± 0.07 μmol P L−1 in all treatments and remained stable throughout the experiment with no distinct difference between treatments (data not shown).POP increased on average from 0.05 ± 0.01 μmol P La from the beginning of the experiment up to the peak of the bloom. BSi increased on average from 1.0 ± 0.1 μmol L−1 to maximum concentrations of 28.5 ± 1.2 μmol L−1 in the Control, 27.9 ± 1.0 μmol L−1 in the High CO2 and 30.0 ± 0.6 μmol L−1 in the Greenhouse treatment. BSi concentrations remained relatively high after the peak of the bloom while the concentrations of pigments associated to diatoms decreased similarly to Chl a  compared to the Greenhouse treatment (4.9 ± 1.9 μmol L−1).BSi, indicative for diatom biomass, followed the development of Chl 2 out-gassing did not exceed 12 μmol L−1 d−1 and during the second half of the experiment (Days 20–44) calculated CO2 in-gassing did not exceed 20 μmol L−1 d−1. Higher out-gassing occurred in the High CO2 and Greenhouse treatments, where CO2 gradients were larger. However, the lid in one of the Control mesocosms was not properly covering the water surface and a small opening was created near the propeller, increasing gas exchange in this mesocosm towards the end of the experiment. DIC concentrations in this Control steadily increased after Day 20 of the experiment, most likely from in-gassing, since no difference in POC or DOC concentrations was observed between this and the replicate mesocosm  calculated CO Days 20–4 calcula−1 to 1657.6 ± 72 μmol L−1 in the Control and from 2113.1 ± 2 μmol L−1 to 1627 ± 4 μmol L−1 in the High CO2 and 2095.5 ± 1 μmol L−1 to 1633.4 ± 10 μmol L−1 in the Greenhouse treatments  compared to the Control and High CO2 treatments  increased at the beginning of the experiment up to the peak of the bloom and decreased rapidly thereafter, similarly to the results reported by 2 , developed in our experiments under all treatment conditions  was observed for the largest species. This was hypothesized to be related to an alleviation of carbon limitation in larger cells which have a thicker diffusive boundary layer compared to smaller cells.While physiological studies with single species give valuable information on tolerance ranges and help us understand underlying mechanisms for the response of species to different stressors  was more pronounced in the combined treatment (“Greenhouse”), although the absolute diatom abundances were three times lower than in the High CO2. Additionally, incubation experiments with a natural plankton community from the subtropical North Atlantic showed that if the community was initially dominated by diatoms, elevated CO2 tended to favour the larger diatom species  and biomass build-up of POC, PON, POP, and BSi. Once inorganic nutrients were exhausted between Days 15 and 18 (depending on treatment), peak concentrations in the respective particulate pools were reached  was consistently observed in mesocosm experiments investigating warming  followed a typical natural diatom-dominated spring bloom exhibiting rapid drawdown of inorganic nutrients and accompanied by exponential autotrophic growth  pools, faster degradation at elevated temperature was unlikely the cause. Thus, two possible mechanisms remain that could explain these results, either the initiation of aggregation processes and therefore the earlier sinking/loss of POM from the water column (Towards the end of the experiment and after the phase of stable POM concentrations, the loss of POM in the Greenhouse treatment was about twice as fast as in the Control and High CODiatoms are known to be important organic matter exporters as they often contribute a high fraction of the biomass produced during spring blooms (2 treatment and while we did not have a warming treatment alone, based on evidence from similar experiments investigating warming and CO2 separately, we hypothesize that the combination of both factors was important for the shift in size. The potential for warming and CO2 induced changes in species composition and size distribution during bloom events in the future ocean is likely to influence biogeochemical element cycling with consequences for CO2 partitioning between ocean and atmosphere.It was recently suggested that the interactive effects of environmental drivers, rather than the sum of the individual effects, best explained plankton responses in aquatic ecosystems (


We find that the Im structure of XeC2 is the semiconducting phase. Accurate electronic structures of tetragonal XeC2 have been calculated using a hybrid density functionals HSE06, which gives larger more accurate band gap than a GGA–PBE exchange-correlation functional. Specifically, we find that the Im structure of XeC2 is a dynamically stable structure at high pressure. We also predict that the P6/mmm structure of XeC2 is the superconducting phase with a critical temperature of 38 K at 200 GPa. The ground-state structure of xenon carbides is of critical importance for understanding in the missing Xe. We discuss the inference of the stable structures of XeC2. The accumulation of electrons between Xe and C led to the stability by investigating electron localization function (ELF).The ‘missing Xe paradox’ is one of the phenomena at the Earth’s atmosphere. Studying the ‘missing Xe paradox’ will provide insights into a chemical reaction of Xe with C. We search the ground–state structure candidates of xenon carbides using the Universal Structure Predictor: Evolutionary Xtallography (USPEX) code, which has been successfully applied to a variety of systems. We predict that XeC The several models for a Xe basin have been proposed since the missing Xe could be contained in the Earth’s inner core has not yet been responded. Attempts to capture Xe in reactivity with elemental metals is observed. Zhu el al.4 have elucidated that Xe can capture in the Earth’s inner core and have proposed that it would have to form chemically stable compounds with Fe/Ni. They have predicted Xe with Fe/Ni using the Crystal Structure Analysis by Particle Swarm Optimization (CALYPSO) and the ab initio random structure searching (AIRSS). They also show that the Xe can capture with with Fe/Ni. In addition, they suggested that the revealed reactivity of Xe with Fe/Ni under the conditions in the Earth’s core not only assists in deciding the missing Xe paradox. Moreover, the revealed reactivity of Xe with O has been found5. Two oxides, Xe3 O2 and Xe2 O5 phases have synthesized by laser-heated diamond anvil cells as well as ab initio calculations coupled with the AIRSS calculation successfully predicted their structures and stability. This may be a general trend in compounds formed under high compression. The understanding of the Earth’s inner core is accepted to be composed originally 8–12% of other light elements 6. This is suggested that there might be a significant amount of C in the Earth’s inner core, and the likely stoichiometries of the most stable Fe–C compounds at high pressure7.The ‘missing Xe paradox’ is one of the most charming in the Earth’s atmosphere, scientists have been ongoing to great effort to finding the cause of the missing Xe. It is known that the possibility of Xe escaping from the Earth’s atmosphereIn this study, we select Xe–C compound as an example to address these problems. We report a predicted high-pressure stabilization of Xe–C compound by the Universal Structure Predictor: Evolutionary Xtallography (USPEX) code. A variety of advance computational methods have been used to identify the thermodynamics stability, dynamics stability, and electronic structure. In addition, a chemical reaction of Xe with C is given way to the relatively open the existence of Xe through the electron localization function. The methods can observe the missing Xe. This is a unique case that Xe is found to be stable in Xe–C compound.2, Xe2 C and, XeC4 have found to be stable Xe-C compounds. While structures on the revealed by dash line are meta-stable structure. Xe3 C, Xe5 C2, Xe3 C2, XeC, Xe2 C3, and XeC3 have found to be the meta–stable Xe–C compounds. We find that XeC2 with space group Im is the most stable structure at 50 GPa.Based on our ground-state searches, we predicted stable structure in Xe–C compound as shown in Fig. b symmetry site , Xe atoms located at 4e symmetry site  and C atoms located at 8i symmetry site , and C atoms located at 4 d symmetry site  and at pressure 200 GPa, the optimized structural parameters for the hexagonal structure are a = 2.539 Å and c = 4.421 Å with Xe atoms located at 1 a symmetry site  and C atoms located at 2d symmetry site  as can be obtained from a convex hull of comparative stability.At pressure 50 GPa, the optimized structural parameters for the tetragonal structure are a = 3.471 Å and c = 18.196 Å with Xe atoms located at 22 is studied, we found that it is a semiconductor with a band gap of 1.32 eV using the GGA–PBE semi–local density functional at 50 GPa  distributes over the entire this frequency range as can be seen Fig. λ of the P6/mmm structure is 0.38 and the calculated logarithmic average of phonon frequency ωlog is 622 K. By using the Coulomb pseudopotential μ* = 0.10 for estimation superconducting transition temperature Tc, the P6/mmm structure predicts to give the maximum Tc of around 38 K at 200 GPa.To investigate the dynamically stable of XeCGPa Fig. . We stro2. Xe may be hidden in the interior of the Earth as the Earth’s inner core is accepted to be composed originally 8–12% of other light elements. Interestingly, C is one of other light elements and C can exist in a diverse number of forms due to its capability to form the spn  hybridized bonds. C changes its hybridization state with increasing pressure. The formation of spn hybridized bonds can be explained by transformation of carbon’s sp2 hybridization in the graphite structure. The p electron transfered from the sp2 (graphite structure) into the sp3 (the diamond structure) hybridizations under high pressure. Moreover, superhard graphite is shown that a half of C atoms converted the σ bond to π bonds8. Hence the tendency of C to change hybridization evident also in carbon compounds. In Fig. sp3 hybridized bonds by analyzing the electron localization function (ELF) in the (100) plane. On pressure increase, 75 GPa, the ELF in the (100) plane shown the formation of sp3 hybridized bonds of C the formation of a bonding between C and Xe atoms which there is a weak bonding between Xe atoms forming the C layer. Moreover, we have obtained the P6/mmm structure from the ground–state search at 200 GPa. We now present the P6/mmm structure by considering the ELF in the (100) plane  can be formed into tetragonal XeCane Fig. . It indiab initio calculations combined with the USPEX. The tetragonal XeC2 structure is predicted to be ground state structure and dynamically stable at 50 GPa. The formation of spn hybridized bonds indicates the formation of sp3 hybridized bonds of C the formation of a bonding between C and Xe atoms. This is because a tendency of carbon to change hybridization is also evident in carbon compounds, which is required for xenon. The ‘missing Xe paradox’ can be formed in carbon compounds under high pressure. Using the HSE06 for band gap calculation gives larger more accurate band gap than the GGA–PBE. We show that the formation of XeC2 under pressure is common and examine the possibility of superconductivity in relation to fundamental phases of materials science. These observations are of fundamental importance of the semiconducting–superconducting phase transition.In conclusion, we predict a new high-pressure phase of Xe–C compound by the 10. To get the most stable structure of Xe–C compound, we searched for its lower enthalpy’s phase based on USPEX code in combined with Vienna ab initio simulation package (VASP) package. The GGA–PBE11 for the exchange-correlation functional to density functional theory. We employed the projector augmented wave (PAW) method12. The PAW potential with an 8-electron (5 s2 5p6) for Xe and 4-electron (2 s2 2p2) for C have been employed. The pseudocore radius of Xe is 2.5 bohr and of C is 1.5 bohr, which are small enough that the overlap of spheres will not occur. We performed the prediction simulations using USPEX code for Xe-C compound  containing one, two, three, and four formula units in the simulation cell at 50, 100, 150, and 200 GPa, respectively. To get the most stable structure of Xe–C compound, a plane waves basis set up to a cutoff energy of 800 eV and the initial BZ sampling grid of spacing 2 π × 0.01 Å−1. The parameters of all structures are fully relaxed using a conjugate gradient scheme. All structures are relaxed at each pressure until the Hellman-Feynman forces became less than 10−3 eV/Å. We predicted the stable structure of Xe–C compound in the form of a convex hull of comparative stability by examining the enthalpy H of each formula unit under the relevant pressure. All enthalpies H are given at the same pressure and zero temperature. At a given pressure, the Xe–C compound located on the convex hull are thermodynamically stable against decomposition to any other binaries or the elements, while the compounds above the convex hull are the meta-stable structure.We presented a novel phase using a structural prediction USPEX code13 combined with VASP code. To look at the semiconducting phase of Xe–C compound, one has to go beyond a standard DFT in order to get well defined excited state properties. We employed the hybrid functional of Heyd, Scuseria, and Ernzerhof (HSE06)15 to verify the results of band structures with the GGA-PBE for the exchange-correlation functional. A plane wave basis set 800 eV and the initial BZ sampling grid of spacing 2 π × 0.04 Å−1 are used.The phonon calculations are carried out by using a supercell approach within the PHONOPY code16. The plan waves basis set is expanded with a kinetic energy cutoff of 60 Ry. The calculation studies presented here are based on the GGA–PBE. We employed GGA–PBE method as implemented in Quantum Espresso (QE)17. The BZ integrations in the electronic and phonon calculations are performed using MP meshes. Both the meshes of k-points for electronic states and the meshes of phonons are used in these calculation. For the P6/mmm structure, individual phonon calculations are performed on the first BZ on 4 × 4 × 2 q-meshes with a 12 × 12 × 8 k-points mesh. The EPC matrix elements are computed in the first BZ on 4 × 4 × 2 q-meshes using individual EPC matrices obtained with a 24 × 24 × 16 k-points mesh.ωlog is the averaged phonon frequency. We used effective Coulomb interaction parameter μ* = 0.10. It is assumed by the original Allen-Dynes formula18.We calculated the EPC with density functional perturbation theory19.LaTeX formats citations and references automatically using the bibliography records in your.bib file, which you can edit via the project menu. Use the cite command for an inline citation, e.g.


Herein described is a photoredox flow reaction for 18F‐difluoromethylation of N‐heteroaromatics that are widely used in medicinal chemistry. Following the two‐step synthesis for a new 18F‐difluoromethylation reagent, the photoredox reaction is completed within two minutes and proceeds by C−H activation, circumventing the need for pre‐functionalization of the substrate. The method is operationally simple and affords straightforward access to radiolabeled N‐heteroaromatics with high molar activity suitable for biological in vivo studies and clinical application.Despite a growing interest in CHF Make a trace: A new flow photoredox methodology allows the 18F‐difluoromethylation of heteroaromatics by C−H activation. The method is applicable to a wide range of heteroaromatics and can be used for the preparation of radiotracers with high molar activities that are suitable for positron emission tomography studies. SNAr substitution reactions with [18F]fluoride is still the most prevalent method for the labeling of aromatics. This method is however limited to reactive electron‐deficient aromatics. Recently, a number of new synthetic methodologies have been developed for the incorporation of fluorine‐18 into electron‐neutral and electron‐rich aromatics to overcome this limitation, expanding significantly the chemical space accessible to radiolabeling.3.2 in medicinal chemistry. The difluoromethyl group (CHF2) is less lipophilic than the trifluoromethyl one and has been successfully used to block oxidative metabolism because of aldehyde oxidase.2 can serve as a hydrogen‐bond donor and thus can act as a lipophilic bioisoster of the hydroxy group.18F into druglike compounds have been reported 2.2CHF2)2 for C−H difluoromethylation of N‐heteroaromatics.2 on heteroaromatics.2.2.Several reagents have been described for the C−H functionalization of heteroaromatics with CHFHowever, all these described reagents are not available yet for fluorine‐18 application, either because they are not easy to label, or because they are difficult to purify and analyze (such as anionic compounds).2 to alkenes, coupled to cyclization would serve our purpose well. The CHF2 radical can be produced under neutral conditions through a catalytic photoredox reaction. In addition, the opportunity to introduce fluorine‐18 through a halogen exchange  reaction and the excellent solubility of this reagent in organic solvent, as well as its easy purification, are attractive for PET chemistry. Below, we describe the labeling of Hu's reagent, as well as a novel methodology for the CHF18F labeling of N‐heteroaromatics under neutral conditions by a photoredox reaction in flow chemistry.We envisioned that the benzothiazole sulfone reagent, described by Hu and co‐workers18F]3 . Unless otherwise specified, all the RCYs are based on HPLC and TLC analysis of the crude reaction mixture (see the Supporting Information). Lower temperature slightly decreased the RCY. Conducting the reaction in either DMSO or DCE either did not give the desired product or led to a lower RCY (entries 1 and 2). Increasing the quantity of 1, or replacement of the base, did not afford any improvement (entries 5 and 6). [18F]2 was directly engaged in the second step. Oxidation of [18F]2 to the corresponding sulfone [18F]3 using RuCl3, x H2O, and NaIO4 in water on a tC18 cartridge proceeded readily and quantitatively with a RCY of 13.4±0.4 % over the two steps (entry 7). No catalyst resulted in no reaction, and a lower amount of oxidant translated into a lower RCY (entries 8 and 9). After semi‐preparative HPLC purification, the molar activity of [18F]3 was 81.4±11.1 GBq μmol−1  at the end‐of‐bombardment (EOB), affording one of the best molar activities for CHF18F addition. The duration of the synthesis of [18F]3, including HPLC purification was about 45 minutes. With these encouraging results in hand, we started the exploration of the reaction of [18F]3 with N‐heteroaromatics in the presence of a photoredox catalyst.We first turned our attention to the preparation of the labeled benzothiazole sulfone 8F3 , an anti‐herpetic drug . Performing the reaction in DMSO with Ir(ppy)3 as a catalyst (0.01 μmol), under irradiation with blue LEDs  at 35 °C turned out to be optimal (entry 3). Under these reaction conditions, the desired difluoromethylated acyclovir [18F]5 was formed in only 2 minutes with an excellent RCY of 70±7 % (n=7). A slightly higher temperature , or a lower reaction time  decreased the RCY. Changing the solvent and switching from DMSO to DMF also decreased the RCY from 70 to 44 %. The low solubility of acyclovir in MeCN and DCE prevented the use of these solvents. Interestingly, running the reaction with 0.001 μmol of Ir catalyst still afforded [18F]5 but in a lower RCY. In contrast, Ru(bpy)3 did not lead to any product at all, whereas benzophenone was less effective than the Ir(ppy)3 catalyst. It is worth mentioning that the presence of water  had a negative impact on the RCY. The use of dried DMSO and minimizing the presence of water within the reagent [18F]3 is recommended (see the Supporting Information). After HPLC purification, [18F]5 was obtained with a very satisfactory RCY of 42±4 %. Gratifyingly, the molar activity of [18F]5 was 44.4±11.1 GBq μmol−1 (1.2±0.3 Ci μmol−1 dc) at the EOB (around 90 min for the three steps). The estimated RCY for the three‐step synthesis is 3–4 % (dc). The desired labeled product [18F]5 was not formed without either a catalyst or light supporting the photoredox mechanism (entries 10 and 11). In addition, no reaction was observed with TEMPO (entry 12), suggesting that the reaction takes place through a radical mechanism. The mechanism of the reaction is likely to be similar to the one put forward by the group of MacMillan18F radical (see the Supporting Information).The ug Table . All rea18F‐difluoromethylation can be carried out on a broad range of heteroaromatics such as indole, benzimidazole, azaindole, pyridine, pyrimidine. 18F‐difluoromethylated compounds were obtained in low to excellent RCYs (from 18 to 75 % RCY). A higher amount of catalyst and a longer residence time were sometimes needed to complete the reaction ([18F]16–[18F]18). It should be noted that more than one isomer was usually observed when several C−H bonds were available for functionalization. However, isomers were readily separated and isolated by PREP‐HPLC (see [18F]31). We also observed that some substrates in Figure 18F]20–[18F]22). The methodology could also be used to label nucleic acid bases such as uracil, cytosine, and adenine ([18F]23–[18F]25). Guanine could not be used in flow because of its limited solubility in DMSO. However, guanosine and other nucleosides such as uridine, cytidine, and adenosine ([18F]26–[18F]29) were readily labeled under standard reaction conditions.With the optimal reaction conditions in hand, the scope of the photochemical reaction was investigated Figure . Pleasin18F analogue of moxonidine . Moxonidine,36(see Section 1d in the Supporting Information), CHF18F was readily introduced in place of the methyl group under the optimal reaction conditions described above. [18F]30was obtained with 65±4 % RCY.To further demonstrate the benefit of the new methodology, the new procedure was applied to the preparation of the CHF2A is the molecular target of the antiepileptic drugs Keppra  gave a mixture of three isomers, [18F]31 a, [18F]31 b, and [18F]31 c, which were readily separated by HPLC. After separation, [18F]31 b was obtained with a RCY of 1.5 %.SVA Figure . Our obj2A radiotracer with affinity similar to that of UCB‐J  suitable for biological in vivo studies and clinical application, affording a significant advantage over existing methods. The protocol is operationally simple, and the use of a flow process for the photoredox reaction enables very short reaction time for the labeling step (less than 2 min). The reaction proceeds under mild reaction conditions and displays high functional‐group tolerance. Most functional groups used in medicinal chemistry, such as free amine, alcohol, nitrile, chlorine, amide and ether, as well as aminals in sugars appear to be compatible with reaction conditions. This method does not require pre‐functionalization of substrates and can be realized at the very last step of the synthesis as demonstrated by the late‐stage labeling of four drugs  and a SV2A ligand. Furthermore, our flow method holds potential for automation. The development of a fully automated process is currently underway and will be reported in due course. We anticipate that this novel methodology will be very useful for late‐stage labeling of druglike compounds and the discovery of new PET tracers.We have disclosed the development of a new methodology for the direct C−H 18F‐difluoromethylation. A solution of the substrate (20 μmol), Ir(ppy)3 (0.01 μmol) in DMSO (200 μL) was prepared. Then [18F]3 in solution in DMSO  was added. The solution was injected in a 100 μL microchip, pumped with DMSO at a flow rate of 50 μL min−1 (residence time of 2 min) and irradiated under blue LED , at a temperature of 35 °C. The exited solution was analyzed by Radio‐TLC and Radio‐UPLC for radiochemical yield (RCY) determination.General radiochemical procedure for The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


In the original article, there were a small number of transcription errors in There are:The label “Norway 6” (at position 6 o'clock) was incorrectly “positioned” with isolate WM_18.31 (ST45) and should have been positioned with “Norway 5.” This is now corrected.Isolates Taiwan 1 and Taiwan 2 should be positioned together. In addition, the isolate WM_18.57 was inadvertently omitted from the ST16 “bubble.”Isolate WM_18.51 should have been placed in the group under ST46 instead of ST59. The corrected The authors apologize for these errors and state that they do not change the scientific findings of the article in any way. The original article has been updated.


Using selective substitution of lanthanum with strontium and nickel with iron to tune the extent to which transition metal and oxygen valence bands hybridize, we demonstrate remarkable catalytic activity of 10 mA cm−2 at a 360 mV overpotential and mass activity of 1930 mA mg−1ox at 1.63 V via a mechanism that utilizes lattice oxygen. This work demonstrates that Ruddlesden–Popper materials can be utilized as active catalysts for oxygen evolution through rational design of structural and electronic configurations that are unattainable in many other crystalline metal oxide phases.The electrolysis of water is of global importance to store renewable energy and the methodical design of next-generation oxygen evolution catalysts requires a greater understanding of the structural and electronic contributions that give rise to increased activities. Herein, we report a series of Ruddlesden–Popper La Water electrolysis provides a potential means to large-scale renewable fuel generation, although sluggish oxygen evolution kinetics challenges progress. Here, authors report on Ruddlesden–Popper oxides as active oxygen evolution electrocatalysts that provide impetus for overcoming kinetic barriers. While precious metals such as Ir and Ru have become standard OER catalysts when working at low pH, alkaline electrolytes allow for the use of abundant, less expensive metals. For example, Ni–M oxyhydroxides such as Nix1−FexOOH are known to be very active for the OER; however, lack of long-range order and uncontrolled electronic structure stemming from different synthetic methods2 complicates elucidation of the mechanism(s) by which the OER activity is improved5. In fact, recent reports question whether Fe is part of the catalytic cycle or if it promotes partial charge transfer between Ni and Fe metal centers6. Additionally, recent work on Ni–Fe oxyhydroxides demonstrated that a significant portion of the measured OER current may be due to other processes and highlighted the need for careful electrochemical analysis of what reactions are contributing to the high activities reported7. Collectively this means that while Nix1−FexOOH materials have been reported as highly active catalysts, the large variations in electronic configuration and the resulting catalytic activity in these studies complicate establishment of precise structure–property correlations for Ni–Fe oxyhydroxides8.Increasing global energy demand requires greater efficiency in water electrolyzers to generate hydrogen at a low cost as well as in other devices for energy storage such as rechargeable metal-air batteries. Given that the efficiencies of devices for these types of energy storage are limited largely by the slow kinetics of the oxygen evolution reaction  reaction pathway, consistent with prior reports and theory16. This LOM mechanism does not require the redox switching of transition metal sites, but rather utilizes lattice oxygen in the OER when the Fermi level d–O 2p hybridized bands. This results in ligand holes that activate lattice oxygen which may combine with chemisorbed OH to produce O2−. This mechanism has since been confirmed by others using isotopic labeling experiments to detect exchanged oxygen from the lattice of SrCoO317. In a separate report, SrNiO3 perovskite was predicted to be more covalent, even more prone to oxygen deficiency, and thus be more catalytically active than SrCoO318. Unfortunately, this prediction is hard to validate experimentally as SrNiO3 adopts a non-perovskite structure based on a hexagonal close packing of Sr and O atoms19. In addition, it is known that the substitution of Sr into LaNiO3 is limited to ~5–20% after which phase impurities appear that result in poor catalytic activity21.In contrast, perovskite oxides with the nominal formula ABO3 that promotes high OER activity but does not suffer from limited Sr solubility and can stabilize highly oxidized, covalently bound Ni. The Ruddlesden–Popper (RP) crystal structure is represented as An+1BnOn+13 or equivalently (AO)(ABO3±δ)n, wherein perovskite layers with a thickness of n (BO6) octahedra are separated by rocksalt (AO)(OA) double layers. This RP phase can accommodate all the elemental substitutions available to perovskites as well as additional compositions that are not stable in the perovskite structure22. While Ruddlesden–Popper materials have been explored as solid oxide fuel-cell cathodes owing to their chemical flexibility and labile lattice oxygen, they have not been fully examined for room temperature water oxidation29.To overcome this limitation, we investigate an alternative crystal structure to the perovskite-phase SrNiO0.5Sr1.5Nix1−FexOδ4±  OER catalysts that have enabled us to achieve exceptionally high catalytic activities at low overpotentials with small OER Tafel slopes. We show that Sr substitution promotes high catalytic activity by further oxidizing Ni via charge compensation, enhancing Ni–O covalency and electronic conductivity. Chemical substitution of Fe for Ni introduces and tunes the overlap between the Ni and Fe 3d bands and the O 2p band. Density functional theory (DFT) modeling confirms that cross-gap hybridization between eg(Ni), p(O) and eg(Fe) bands across the Fermi level enhances charge transfer interactions across Fe–O–Ni bridges and the bandwidth available for electrode-adsorbate electron transfer. The increased covalency and cross-gap hybridization30 of transition metal 3d states and O 2p orbitals are an effective new catalyst design criteria for improving OER activity that supports oxygen evolution taking place via the LOM mechanism, a mechanism which has already been demonstrated to apply to other catalyst materials as well. Furthermore, our work illustrates the remarkable catalytic activity of the RP LSNF series that encompasses a range of chemical substitutions and electronic configurations not accessible in other crystalline metal oxide phases and that enables the elucidation of crucial structural–chemical–electronic relationships and the OER mechanism that has not been possible with Ni–M oxyhydroxides and other reported metal oxide catalysts for OER.Herein, we report a series of La0.5Sr1.5Nix1−FexOδ4±  samples were synthesized using a modified Pechini method31 followed by crystallization and annealing, details of which are found in the Methods section. Electron diffraction and powder X-ray diffraction (PXRD) patterns correspond to a body-centered tetragonal unit cell with the I4/mmm space group, characteristic of the n = 1 Ruddlesden–Popper (RP) crystal structure  = 0.645 Å, r = 0.585 Å, r = 0.56 Å, r = 0.48 Å)32. The ratio of the apical to equatorial Ni/Fe–O distances increases from 1.038 in LSN to 1.073 in LSF, reflecting slight apical elongation of the (Ni/Fe)O6 octahedra. The I4/mmm crystal structure is also directly viewed with annular bright field scanning transmission electron microscopy   layers and the rocksalt (AO)(OA)  layers without stacking faults and the crystal structure propagates to the surface without amorphization.Laers Fig.  and uniters Fig.  increase2 g−1  and Brunauer–Emmet–Teller (BET) surface area analysis were performed and the results, presented in Supplementary Figure 0.5Sr1.5Nix1−FexOδ4± (LNSF) series was supported on Vulcan carbon (VC) at 30 wt% and tested for the OER with the results presented in Fig. 33.The La−2oxide) by over an order of magnitude at 1.63 V vs the reversible hydrogen electrode corrected for electrolyte resistance (RHE-iR) as seen in Fig. −2oxide and 1930 mA mg−1oxide at 1.63 V, over 20 and 40 times higher than the respective values for LSN, with a Tafel slope of 44 mV dec−1 and achieves the common benchmark of 10 mA cm−2geo at only a 360 mV overpotential which is remarkable considering the relatively small mass loading used  and over an order of magnitude better than IrO2 (173 mA mg−1oxide), a leading precious metal oxide benchmark catalyst. The LSNF30 composition generates three times as much current per surface area as SrCoO2.7 (32.7 vs 9.2 mA cm−2oxide) and over an order of magnitude more than IrO2 (1.2 mA cm−2oxide) at 1.63 V  of Ni2+/3+ oxidation as documented with Ni–Fe oxyhydroxides35. Integration of the oxidation waves  transferred during oxidation/intercalation consistently decreases upon continued replacement of Ni with Fe, with the exception of the initial introduction of Fe in LSNF15  by strongly hybridizing with the O 2p and Ni 3d states which facilitates cation oxidation and redox processes28. The mixed Fe 3d and Ni 3d bands pinned at EF in close proximity to the O 2p band should give rise to a mixture of Ni3+/4+ and Fe3+/4+ where the relative proportion of oxidation states changes with the amount of Fe substitution41. This was confirmed by ex situ room temperature Mössbauer spectroscopy  and the average B-site oxidation state. The increase in the oxidation state of Ni is further demonstrated by the observation that the Ni 3p spectrum obtained by X-ray photoelectron spectroscopy (XPS) shifts to higher binding energies with increasing Fe content  series by DFT. While the SNF system was not investigated experimentally, it was modeled to confirm that the same trends in electronic structure evolution that occur in LSNF also take place in a sample with a different A-site composition. Additionally, modeling the SNF series also eliminates a possible source of variation between the atomic arrangements of A-site elements in the modeled LSNF system vs the arrangements in the synthesized samples and verifies our results on a simplified catalyst system.To further understand the electronic structure evolution across the LSNF series and its implications for the OER activity, as well as confirm the results of the Mössbauer spectroscopy and iodometric titrations, we model the bulk phase of LSNF and a simplified Sr0.5Sr1.5NiO4 (LSN), La0.5Sr1.5Ni0.75Fe0.25O4 (LSNF25), LSNF50, LSNF75, and LSF. The SNF series was modeled using the same Ni and Fe ratios, and details regarding cell formation and magnetic investigation are given in the Methods and Supplementary Note 2. Figure 42. Using this La/Sr distribution we determine the minimum energy Ni/Fe ordering which results in two key features governing the Fe arrangement in LSNF O2 layers has an equal number of Fe cations in order to prevent an unbalanced charge distribution between them. Second, the Fe cations in each B(B′)O2 plane are distributed such that the number of Fe–O–Ni bridges is maximized2. This arrangement mitigates the effects of the induced O 2p electron hole at Ni–O–Ni bridges due to more effective electron donation by Fe at Fe–O–Ni bridges, which increases stability18. Compared with Fe–O–Fe bridges, the Fe–O–Ni bridges induce shorter Fe–O bond lengths and indicate higher Fe oxidation states, a trend that also occurs in the simplified SNF system. This observation agrees with the experimentally observed predominance of Fe4+ over Fe3+ at low Fe substitution as well as the increase in unit cell parameters as Fe content increases , driving the latter towards eg states are relevant for surface chemisorption, the increasing bandwidth around 28 at the adsorbate-catalyst interface, and therefore should increase the rate of reaction according the Gerischer–Marcus model of charge transfer45. The increased Fe concentrations in LSNF75 and SNF75, however, drive Band I (Ni) into the similar distribution of Band II (Fe) and this reduces the bandwidth  of the Fe–O–Ni bridges in both LSNF and SNF confirm that the Ni cations are in the low-spin state with fully occupied ies Fig. , in agre EF Fig.  and decrmentclass2pt{minimdth Fig. . This su2. Conesa calculated the DOS of one such polymorph, FeNi7O8(OH)8 in the 2HC structure with Fe in O4(OH)2 coordination, and their result is strikingly similar to that of LSNF in that Ni and Fe 3d bands are strongly hybridized just above EF. They concluded that Fe4+ is stabilized by induced charge transfer between Ni sites, which is also seen in Ni–Fe oxyhydroxides6, and this explanation agrees with our observation of Fe in the 4+ oxidation state in LSNF , Srx2−LaxMOδ4±(ref.50), and Ba0.5Sr0.5Co0.75Fe0.25Oδ3± (BSCF)51. Merkle calculated the band diagram for BSCF and found a similar electronic configuration as in LSNF, and Schmidt and co-workers saw that the catalytic activity of Ba0.5Sr0.5Co0.8Fe0.2Oδ3± was much higher than that of Ba0.5Sr0.5CoOδ3±(ref.34). Thus it is possible that the high activity of BSCF, SrNb0.1Co0.7Fe0.2Oδ3−52 as well as others with similar mixed transition metal sites may be rationalized not just by the covalent bonding, but also by triply-overlapping Co/Ni and Fe 3d states with O 2p near EF similar to the LSNF series, which has largely been overlooked up to now. Furthermore, the similarities between the band structures of LSNF materials and these other active catalysts of varying structures, some of them amorphous, reinforces how the principle of cross-gap hybridization may apply to many different metal oxide catalysts and applies to the LSNF series even in the event of surface restructuring.The band structure of LSNF also resembles the computed band structures of LaCo18. In the AEM, chemisorbed intermediates undergo a series of electrochemical oxidations as the transition metal active site undergoes oxidation and reduction, and these redox reactions are the most energetically intensive and thus rate limiting steps14. The LOM OER mechanism, on the other hand, does not require significant redox switching of transition metal sites but instead requires the participation of lattice oxygen in the OER. Ligand holes that arise when transition metal 3d bands are highly covalent with O 2p bands and exist around 2−. In previous studies we found that the DFT-computed Ovac formation energy in bulk ABO3 perovskites . The decrease in oxygen vacancy formation energy in these materials occurred in concert with an increase in oxygen diffusion rates and OER activity14. This proposed mechanistic switch was experimentally confirmed by a recent report from the Shao Horn group who used isotopic labeling of lattice oxygen to observe a transition from the AEM on LaCoO3 to the LOM on La0.5Sr0.5CoOδ3− and SrCoOδ3−17.As we have discussed in our previous work, the OER can proceed by the adsorbate exchange mechanism (AEM) or the LOM mechanismeg(Ni) and eg(Fe) bands with the p(O) band across 3 and La0.5Sr0.5CoOδ3−18 while eg bandwidth through Ni–O–Fe cross-gap hybridization. Altogether this demonstrates how the LOM applies to a new series of materials and solidifies the importance of both increased covalency that results in oxygen electron holes, as well as the cross-gap hybridization that causes broadening of the overall bandwidth near EF in the design and optimization of oxide catalysts.The same type of band overlap and resulting covalency that is a requirement of the LOM mechanism and that occurs in the previously reported materials discussed above also occurs in LSNF. The hybridization of d–O 2p–Fe 3d bonds that give rise to exceptional OER activity. Sr substitution into the perovskite LaNiO3 has previously been proposed as an avenue to increase the oxidation state if Ni and Ni–O covalency but has not been fully realized. In this work we were able to avoid inactive secondary phases resulting from poor solubility of Sr in the perovskite phase through utilization of the RP crystal structure. Additionally, by using La0.5Sr1.5NiOδ4± as the host lattice we achieved complete substitution of Fe for Ni across the entire compositional range. The crystalline RP structure and the high degree of phase purity enabled precise study of the impact of Fe substitution on the chemical and electronic properties of the LSNF series, something that has not been possible up to this point with other, amorphous materials. Iodometric titrations coupled with Mössbauer spectroscopy indicate that the average Ni oxidation state in the LSNF series increases from +3.46 to +3.95 with increasing Fe substitution while the Ni2+/3+ redox peaks also shift to more positive potentials, consistent with Ni developing a more oxidized character. The influence of Fe substitution extends beyond increasing the oxidative strength of Ni, however, as the electrocatalytic activity increases by over an order of magnitude from LSN to LSNF30 despite possessing statistically equivalent Ni oxidation states. DFT calculations reveal that Fe substitution results in cross-gap hybridization where the Fe 3d eg band is hybridized with both the Ni 3d eg and the top of the O 2p density of states across the Fermi level. The increased covalency of the Ni–O bonds as well as facile charge transfer through Fe–O–Ni bridges due to incorporation of Fe explains the enhanced catalytic activity going from LSN to LSNF30. Furthermore, the increased covalency demonstrated via calculated partial density of states for LSNF and a simplified SNF that is a requirement for the OER to take place via a LOM mechanism resembles the band structure of other highly active OER catalysts with varying crystal or amorphous structures for which this mechanism has already been verified. Decreased oxygen vacancy formation energies and increased rates of oxygen diffusion, descriptors already accepted in the literature as indications of the LOM, are seen on LSNF and further support the hypothesis that the OER proceeds via a LOM mechanism on LSNF. This methodology of selective A- and B-site substitution to promote cross-gap hybridization in RP oxides reveals important fundamental aspects related to their structure and the exceptional electrocatalytic activities of these materials as well as other metal oxide catalysts.We have precisely synthesized a series of RP catalysts having highly oxidized and covalent Ni 32 powder were obtained from Sigma-Aldrich. Millipore high-purity water  was used in all experiments and synthesis and research grade, 99.999% purity oxygen and argon gases were purchased from Praxair. Vulcan XC72 carbon (VC) was purchased from Cabot Corporation and ball-milled prior to use.All chemicals were used as they were received from manufacturers. Lanthanum(III) nitrate hexahydrate (99.995%), strontium(II) nitrate hexahydrate (99%), nickel(II) nitrate hexahydrate (99%), iron(III) nitrate non-ahydrate (99.99%), tetramethylammonium hydroxide pentahydrate , diethylene glycol , citric acid monohydrate, 2-propanol, ethylenediaminetetraacetic acid, and potassium hydroxide (KOH) were purchased from Fisher Scientific. Absolute 200 proof ethanol was obtained from Aaper Alcohol. 5 wt % Nafion solution in lower alcohols and IrO0.5Sr1.5Nix1−FexOδ4±  samples were synthesized using a modified Pechini method29 followed by crystallization and annealing. A- and B-site nitrate salts, in stoichiometric ratios, were dissolved in water to create a solution in which the total concentration of metal salts was 0.1 M. Citric acid and EDTA were added, each at a concentration of 0.1 M, and then 1 M TMAOH was added dropwise until the pH had reached 7.5 in order to deprotonate and dissolve the EDTA. DEG was then injected to reach a concentration of 0.067 M and the solution was heated to 85 °C while stirring. The EDTA and citric acid were both added to ensure complete chelation of the metal cations, preventing agglomerations or particle formations that may lead to catalyst inhomogeneity. DEG and heat were added to the solution to drive a dehydration reaction between the polyhydroxyl alcohol and the carboxylic acid groups of the chelates to form a polyester gel. Once all the water had been evaporated the gel was combusted on a hot plate at 350 °C to form mixed metal oxide precursor particles. This step was performed on a hot place and not in a sealed tube furnace to avoid possible explosions from rapid evolution of gasses upon combustion. Finally, precursor particles were crystallized at 950 °C (heated at 20 °C min−1) for 5 h, then cooled to 400 °C and left to anneal for 6 h in a tube furnace. The entire crystallization and annealing routine was done under pure O2 flowing at 200 mL min−1. Catalysts were recovered and immediately stored under Ar gas to prevent catalyst surface amorphization. Total catalyst yields per synthesis ranged from 500 to 750 mg. More details regarding the synthesis can be found in Kakihana et al.31La2.7 and LaNiO3 were synthesized via a coprecipitation method previously reported elsewhere46 in which A and B-site nitrate salts in a 1:1 ratio were dissolved in a 1 wt% solution of TMAOH containing an equimolar amount of TPAB to the total moles of metal cations in order to form mixed metal hydroxide precursor particles. These solutions were flash-frozen on a rotating metal drum that had been cooled to cryogenic temperatures and was collected before lyophilization to remove water. The precursor particles were then calcined at 700 °C (LaNiO3) or 950 °C (SrCoO2.7) to form the perovskite phase.SrCoO47 and bath sonicated for at least 1 h. Ten microliters of catalyst ink was drop cast onto 5 mm glassy carbon electrodes (GCE) from Pine Instruments and dried in air overnight. All GCEs were polished before drop casting via sonication in a solution of equal parts water and ethanol followed by polishing using 0.05 μm alumina powder after which they were again sonicated in a fresh solution of water and ethanol and dried in ambient air. This method was used to clean all GCEs prior to drop casting, after which a composite catalyst loading of 51 μgtotal cm−2geo, yielding 15.3 μgoxide cm−2geo was achieved for catalysis and intercalation tests.To make catalyst inks 2 mg of catalyst powder were added to 2 mL of NaOH neutralized 0.05 wt% Nafion solutionCH Instruments CHI832a or Metrohm Autolab PGSTAT302N potentiostats were used to carry out all electrochemical testing. Potentiostats were equipped with high-speed rotators from Pine Instruments. Experiments were performed at room temperature in 0.1 M KOH (measured pH ≈ 12.8). Electrolyte resistance was measured using current interrupt and positive feedback methods (46 Ω) and iR compensation was applied to all electrochemical measurements after testing unless otherwise noted. A 3 electrode cell was used for all experiments. The reference electrode was a CH Instruments Hg/HgO (1 M KOH) reference electrode, a Au wire served as the counter electrode, and GCEs drop cast with catalyst ink and dried as described above were used as the working electrode. All potentials were measured vs Hg/HgO but were reported vs the reversible hydrogen electrode (RHE), which was determined experimentally to be +0.8976 V vs. Hg/HgO (1 M KOH). Supplementary Figure oxide cm−2geo) or unsupported catalyst. CV scans were performed from 1 to 2 V vs RHE at a scan rate of 10 mV s−1 while rotating at 1600 rpm in O2-saturated 0.1 M KOH. The anodic and cathodic scans were averaged and iR corrected, and the current at 1.63 V vs RHE-iR was used for comparison of OER activities. Scatter in the data at high current densities is due to oxygen bubble formation and desorption on the electrode surface. Data presented in this report are the average of at least three tests on fresh electrodes. OER testing using Ni foam supports were performed under the same electrolyte conditions but dropcast at 1 mg cm−2 and held stationary.All testing for OER activities was performed on newly dropcast electrodes which had not undergone previous testing, dropcast with 30 wt% catalyst on VC  to limit the amount of oxygen generated a minimize bubble formation which reduces collection efficiency. Additional information regarding the RRDE calibration data can be found in Supplementary Figure All RRDE tests were performed in argon-saturated, 0.1 M KOH on fresh 30 wt% oxide films. LSNF30 was cycled at 10 mV s−1 until the oxidative peak potential did not change upon further cycling. Chronoamperometry was then performed at potentials 10 mV more anodic than Ep to ensure diffusion-limited intercalation. Linear regression was performed as described elsewhere49 to determine the diffusion rate. Particle sizes were estimated using the surface area calculated from BET measurements and densities determined by Rietveld analysis. All values reported are the average of at least three tests.All oxygen diffusion tests were performed in argon-saturated, 1 M KOH on fresh 85 wt% oxide films. Each LSNF composition was cycled at 20 mV s13. In this procedure 3 mL of 2 M KI solution that had previously been deoxygenated were combined in a flask with 15–20 mg of perovskite and stirred for 3 min under argon, after which 25 mL of 1 M HCl was added to dissolve the perovskite. The solution was then titrated with a solution of ~26 mM solution of Na2S2O3 that had been pre-standardized with 0.1 N KIO3 until a faint golden color was reached, after which a starch indicator was injected and the titration was continued until the solution was clear, marking the end point.Iodometric titrations were performed following the procedure referenced aboveFresh, Ar-sealed catalysts were ball milled for 3 min using a Wig-L-Bug spectroscopic grinding mill. To support the catalysts onto VC, the correct amounts of pre-ground catalyst and pre-ground VC were measured into the grinding mill’s vial and ball milled for 3 min.θ in 0.01° increments with a dwell time of 0.35 s per step. PXRD patterns for the Rietveld refinement were taken with a Huber G670 Guinier diffractometer  monochromator; image plate). The refinement was performed with the JANA2006 package50. As the electron diffraction patterns of all compositions revealed no deviation from the RP n = 1 I4/mmm structure, the La2NiO4 structure51 was used as a starting model. The La/Sr occupancy factors of the A positions were refined; the Fe/Ni occupancy factors for the B positions were assigned according to the results of the EDX analysis. The crystallographic data, positional and atomic displacement parameters, interatomic distances, and reliability factors are listed in Supplementary Table Catalyst structure was examined by performing X-ray diffraction with a Rigaku MiniFlex600 Diffractometer. Measurements were performed at 298 K in ambient conditions while the instrument operated at 40 kV and 15 mA using Cu Kα radiation (1.54 Å wavelength). For all tests, argon-sealed catalyst powder was exposed to ambient air and scanned over 10–100° 2P/P0) of 0.05 to 0.30, with a minimum R2 of 0.995 and C value of 20 was used to calculate the specific surface area using the BET method.Catalyst surface areas were measured via nitrogen sorption analysis which was performed at a temperature of 77 K on a Quantachrome Instruments NOVA 2000 high-speed surface area BET analyzer. Before BET experiments were performed, all catalyst materials were ball milled for 3 min followed by degassing under vacuum at room temperature for at least 12 h. Only data in the relative pressure range  spectra were collected using an aberration-corrected Titan G57Fe Mössbauer spectra were collected using a constant acceleration spectrometer MS1104  in transmission mode with a 57Co/Rh γ-ray source. Calibration of the spectrometer was performed using standard sodium nitroprusside absorbers. All isomer shift values (IS) are referred to α-Fe at room temperature and spectra evaluation was performed using “UnivemMS”  and custom least squares fitting software with Lorentzian–Gaussian line shapes. Details regarding the deconvolution of the spectra are found in the text accompanying Supplementary Figure 52, using the PAW pseudopotentials and the exchange-correlation functional of Perdew–Burke–Ernzerhof (PBE), with the effective Hubbard Ueff of 5.3 (Fe) and 6.2 eV (Ni), respectively. The Ruddlesden–Popper bulk phase is modeled by fully relaxing a 2 × 2 × 1 primitive unit cell, La(Sr)16Ni(Fe)8O32, with a plane wave cutoff energy of 520 eV, Monkhorst-Pack k-point of 4 × 4 × 2, forces convergence criterion of 10–4 eV Å−1 and Gaussian smearing, where the smearing width is 0.1 eV. All models follow ferromagnetic ordering, as our calculations indicate its lower energy than those of nonmagnetic and antiferromagnetic orderings . Details regarding the determinations of effective Hubbard Ueff, atomic, magnetic and electronic structures, and oxygen hyperstoichiometry effects are provided Supplementary Note 2.The Vienna Ab initio Simulation Package (VASP) was used to do spin polarized calculationsp spectrum are found in the text accompanying Supplementary Figure Characterization of the chemical states was done using a Kratos AXIS Ultra DLD XPS and a monochromatic Al X-ray source , scanning in 0.1 eV steps with a dwell time of 4 s per step. Charge compensation was used for all samples. Binding energies were for all spectra were calibrated against the adventitious carbon peak at 285 eV. CasaXPS was used for all data analysis and deconvolution. Details regarding the deconvolution of the Ni 33+/4+. Supplementary Figure Together, the iodometric titrations and the Mössbauer spectroscopy enable the calculation of Ni’s average oxidation state and the relative percentage of FeThe authors declare that all data collected and discussed in support of the findings of this study are available within the paper and its Supplementary Information.Supplementary Information


CHO cells are the preferred host for the production of complex pharmaceutical proteins in the biopharmaceutical industry, and genome engineering of CHO cells would benefit product yield and stability. Here, we demonstrated the efficacy of a Dnmt3a‐deficient CHO cell line created by CRISPR/Cas9 genome editing technology through gene disruptions in Dnmt3a, which encode the proteins involved in DNA methyltransferases. The transgenes, which were driven by the 2 commonly used CMV and EF1α promoters, were evaluated for their expression level and stability. The methylation levels of CpG sites in the promoter regions and the global DNA were compared in the transfected cells. The Dnmt3a‐deficent CHO cell line based on Dnmt3a KO displayed an enhanced long‐term stability of transgene expression under the control of the CMV promoter in transfected cells in over 60 passages. Under the CMV promoter, the Dnmt3a‐deficent cell line with a high transgene expression displayed a low methylation rate in the promoter region and global DNA. Under the EF1α promoter, the Dnmt3a‐deficient and normal cell lines with low transgene expression exhibited high DNA methylation rates. These findings provide insight into cell line modification and design for improved recombinant protein production in CHO and other mammalian cells. A possible reason for the decrease in transgene expression is the gradual loss of gene copies, which result in decreased transcripts Previous studies on the effect of DNA methylation on the transgene expression in CHO cells have mainly focused on promoter modification, such as mutating the cytosine within promoters,22.1A DNA fragment 3a3 + 4 of Dnmt3a NW_003613640.1) containing exon1 was amplified by polymerase chain reaction (PCR) from the genomic DNA of CHO‐K1 cells using primers  using the Lipofectamine® 3000 Transfection Reagent  in accordance with the manufacturer's protocol. After being transfected for 8 hours, the cells in the plate were recovered for 48 hours in fresh medium and the single clones were screened. The transfected cells were subjected to gradient and limiting dilution to select the single clones of Dnmt3a KO cells. These single putative clones were identified through PCR amplification using the sequencing primers  were grown in Dulbecco's modified Eagle's medium + F12 , supplemented with 10% foetal bovine serum , 1% penicillin and streptomycin . The cells were incubated in a humidified incubator at 37°C under 5% COQuantitative detection in vitro was performed using DNMT3A ELISA kit  to determine the concentrations of Dnmt3A in the Dnmt3a KO CHO‐K1 cells. After the stop solution was added, the blue colour changed to yellow, and the intensity was measured using a spectrophotometer at 450 nm. The calibration standards were also assayed. The concentrations of Dnmt3A in the cellular samples were determined by comparing the OD of the samples to the standard curve.2.3™ II 1st Strand cDNA Synthesis Kit (TaKaRa) according to the manufacturer's instructions. qRT‐PCR was performed with SYBR Green using a SYBR Premix EX Taq™ (TaKaRa) in a final reaction volume of 10 μL: 4 μL of template cDNA (0.05 μg/μL), 5 μL of SYBR Premix Ex Taq (2×), 0.2 μL (10 μmol/L) of forward primer, 0.2 μL (10 μmol/L) of reverse primer and 0.6 μL of deionized water. The qPCR was carried out on a PikoReal™ 96 Real‐Time PCR System  equipped with PikoReal Software 2.2 (ThermoFisher Scientific). The amplification conditions were as follows: 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 20 seconds and 72°C for 10 seconds, and a final extension at 72°C for 5 minutes. Finally, relative expression levels of Dnmt3a mRNA were calculated using the 2ΔΔCt method, with normalization to GAPDH mRNA levels as an internal control. The primers used in this study are shown in Table qPCR was conducted to determine the expression levels of Dnmt3a mRNA in the Dnmt3a‐deficient and normal control CHO‐K1 cells. Briefly, total RNAs were extracted from the cells using RNAiso Plus (TaKaRa) and reverse transcribed with the PrimeScript2.43VO4 20 mmol/L). A protease inhibitors cocktail (Sigma) was added before extraction. Cell lysates of the samples were separated by 10% SDS‐PAGE gel (Mini‐PROTEAN TGX gels) and transferred to nitrocellulose membrane . Western blot was performed using primary antibodies anti‐DNMT3a  and anti‐GAPDH  at dilutions 1:1000. Secondary antibodies conjugated with HRP were used at a dilution of 1:2000, and the reaction was detected using Amersham ECL Prime Western Blotting Detection Reagent by GE Healthcare Life Sciences, according to the manufacturer's instructions.Western blot was used to identify the protein levels of Dnmt3a in the Dnmt3a‐deficient and normal control CHO‐K1 cells. Briefly, the pellets of the cells were lysed on ice using the extraction buffer  in accordance with the manufacturer's protocol. At 48‐hour post‐transfection, the transfection efficiency and the transient expression were analysed by assessing the fluorescence intensity of the transfected cells through fluorescence microscopy .The pWTY‐CMV and pWTY‐EF1α vectors, which contained either the CMV (589 bp) or the EF1α (1335 bp) promoters, were constructed by cloning eGFP as a report gene from pEGFP‐C1  into the pIRES‐neo vector (Clontech).2.63 cells/well. One plate was removed at the same time every day after the cells adhered to the wells. Absorbance was measured with a microplate reader at 450 nm. All experiments were performed in triplicate. Cell cycle and the apoptosis rate were analysed through flow cytometric analysis. Cells were seeded into a 35‐mm dish at a density of 1 × 105 cells/dish. After 72 hours, the harvested cells were washed with cold PBS thrice and fixed in 70% ethyl alcohol at 4°C overnight. For apoptosis analysis, the cells were stained using Annexin V‐FITC Apoptosis Detection Kit  in accordance with the manufacturer's instructions. Flow cytometric analysis was performed using the Guava EasyCyte 6‐2L system , and data were analysed by Guava InCyte (GuavaSoft™ 3.2).Cell viability was measured using Cell Counting Kit‐8  in accordance with the manufacturer's instructions. Briefly, the CHO‐K1 cells, the deficient cells and the cells stably transfected with CMV or EF1α were seeded in four 96‐well plates at a density of 2 × 10tD) was determined to evaluate the growth characteristics of the normal CHO‐K1 cells, the Dnmt3a‐deficient cells and the cells stably transfected with CMV or EF1α. Briefly, the cells were seeded into 6‐well plates at 1 × 105 cells/mL and allowed to grow for 2, 4, 6, 10, 24 and 48 hours at 37°C under 5% CO2 prior to cell counting using a haemocytometer measurement tool and trypan blue staining assay. The tD value was determined by the formula tD = (t2−t1) × (log(2)/log(C2/C1)), where C2 and C1 are the cell concentrations at times t2 and t1, respectively, under a constant growth rate.The cell doubling time (2.7+) or the absence (G418−) of G418 for 60 generations. The mean fluorescent intensity (MFI) values of each cell line at 0, 10, 20, 30, 40, 50 and 60 passages were measured with a FACS Calibur cytometer . Detailed protocols for stability testing were based on a previous report.Stably transfected cell polycolonies were selected by adding Geneticin  to the medium at a final concentration of 800 μg/mL for 14 days. Cells stably transfected with CMV or EF1α were split into 2 and further passaged in either the presence  ELISA Easy Kit  in accordance with the manufacturer's instruction. Briefly, the methylated DNA was detected by using capture and detection antibodies to (5‐mC) and then quantified calorimetrically using SpectraMax® i3 Platform  to read the absorbance at 450 nm. The amount of methylated DNA was proportional to the measured OD intensity. A standard curve was used to quantify the absolute amount of methylated DNA (the percentage of 5‐mC) in the total genomic DNA. Each sample was assessed in duplicate.Genomic DNA was extracted from stably transfected cells using the TIANamp Genomic DNA Kit  and the Tissue and Cell Genomic DNA Purification Kit (GeneMark). Global DNA methylation levels were determined using MethylFlash2.9Quantitative methylation changes in CMV and EF1α in the transfected cells were compared using high‐throughput mass spectrometry on the matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass array. Briefly, genomic DNA was extracted from CHO cell polycolonies stably transfected with the vectors containing CMV or EF1α at the start and at 50 passages of stability testing. Isolated genomic DNA was treated with bisulphite using the EZ DNA Methylation Kit . We designed primers for CMV and EF1α to cover the regions with the main CpG sites . Unincorporated dinucleotide triphosphates were removed by inducing shrimp alkaline phosphatase (SEQUENOM) treatment. The T Cleavage Transcription/RNase Cocktail (5 μL) contained 3.15 μL of RNase Free‐ddH2O, 0.89 μL of 5× T7 polymerase buffer, 0.24 μL of T Cleavage mix, 0.22 μL of 100 mmol/L DTT, 0.44 μL of T7 RNA and DNA polymerase (22 units/reaction) and 0.06 μL of RNase A. Afterward, 2 μL of the PCR product (as the template) and the aforementioned 5 μL of MassCLEAVE mix were added to one well of the 384‐well plate for the transcription reaction at 37°C for 3 hours. The samples were diluted with H2O to a final volume of 27 μL. The phosphate backbone was conditioned by adding 6 mg of Clean Resin (SEQUENOM) prior to MALDI‐TOF MS analysis. Exactly 22 μL of the RNase‐A‐treated product was robotically dispensed onto silicon matrix preloaded chips . Mass spectra were obtained using MassARRAY Compact MALDI‐TOF (SEQUENOM), and methylation ratios of the spectra were generated using EpiTYPER software v1.0 (SEQUENOM).T Cleavage assay was performed using the “T” Cleavage MassCLEAVE2.10t test was used for statistical analysis when only 2 groups were tested. P < .05 was considered statistically significant. All experiments were performed at least thrice, and all samples were tested in triplicate.The data were expressed as means ± standard deviation. All statistical analyses were performed on the SPSS 18.0 . One‐way ANOVA was used to compare multiple groups. Paired‐sample Student's 33.1The CRISPR/Cas9 system was facilitated to generate Dnmt3a KO in CHO‐K1 cells. Basing on the coding conservation among different transcripts, we designed 2 pairs of single‐guide RNAs (sgRNAs), which targeted the conserved exon1 of the Dnmt3a transcript. Following the limiting dilution of genetically manipulated cells, PCR amplification was used to screen for monoclonal mutant cells. As shown in Figure P < .05). These results indicated that Dnmt 3a gene was knocked out in CHO‐K1 cells.PCR productions from 2 monoclones (3a‐30 and 40) were sequenced to validate the gene KO. The sequencing results revealed that the frame shift mutation occurred in the target region of the Dnmt3a gene Figure B. The ex3.2The detection of cell proliferation and apoptosis indicated that Dnmt3a KO did not alter the cell morphology and the growth status  or absence (G418−) of G418 for 60 passages. The MFIs were detected to evaluate the intensity values of the expressed eGFP at 10, 20, 30, 40, 50 and 60 passages. The Dnmt3a‐deficient 3a‐30 cell line transfected with CMV exhibited the most stable and the highest expression levels regardless of the presence or absence of G418 . For EF1α, the percentages of the DNA methylation of the global genomic DNA were (2.24 ± 0.21)% and (1.74 ± 0.19)% in the stably transfected 3a‐30 cells and normal control CHO‐K1 cells, respectively.To determine whether DNA methylation affected the long‐term transgene expression in CHO cell lines, we detected the DNA methylation of the global genomic DNA and the CMV or EF1α contained in the stably transfected CHO cells. The global DNA methylation percentage was (0.84 ± 0.08)% in untransfected 3a‐30 and (3.05 ± 0.13)% in CHO‐K1 cells. For CMV, the percentages of the DNA methylation of the global genomic DNA were (0.79 ± 0.11)% and (3.14 ± 0.17)% in the stably transfected 3a‐30 cells and normal CHO‐K1 cells, respectively, suggesting that the rate of global DNA methylation was significantly decreased in the Dnmt3a‐deficient cells  was high in the natural CHO cell lines but low at the start and at 50 passages in the 3a‐30 cell lines, thus verifying the high stability of transgene expression during long‐term culture Figure A. By con4Previous reports have shown that the DNA hypermethylation in the promoter region can cause instability in stable recombinant CHO cell lines.The CRISPR/Cas9 genome editing technology has been extensively applied for modifying a single gene into a genome‐wide regulation of genes through the rapid, easy and efficient engineering of mammalian genomes.Cellular growth characteristics of Dnmt3a‐deficient and normal CHO‐K1 cell lines before and after transfection with CMV or EF1α were evaluated. For this purpose, the cell proliferation, apoptosis and doubling times were determined for the original Dnmt3a‐deficient, normal CHO‐K1 cells and stably transfected cells. In this work, the deletion of the Dnmt3a gene did not significantly affect the doubling times of the mutant CHOs and transfected cells. Characteristics of cell proliferation and apoptosis showed no differences in the cell morphology and growth status of the mutant and normal CHO cells. Challen et alThe hCMV‐MIE provides high levels of transgene expression; however, its loss of productivity has been reported with prolonged cultivation time.To investigate whether Dnmt3a KO could affect the methylation levels at CpG sites within the promoter region, we measured the methylation levels of CpG sites in the CMV and EF1α regions through bisulphite conversion coupled with PCR amplification and MALDI‐TOF Mass Array. We observed a distinct methylation pattern at 50 passages in the CMV promoter region of the normal transfected control CHO cells, which showed the highest rate of DNA methylation. In particular, we detected low DNA methylation in the promoter region at start and at 50 passages in the transfected 3a‐30 cells, which showed high stability of transgene expression during long‐term culture. Moritz et alIn summary, Dnmt3a‐dificent CHO cells are a potential alternative expression system to traditional methods. This approach offers a new platform for improving the transgene expression stability in recombinant CHO cells. Obviously, further studies on Dnmt3a‐deficient CHO cells and DNA methylation may help elucidate the underlying molecular mechanisms. Our results may be promoter‐specific and follow‐up studies that consider more promoters could provide additional insight into the effects of Dnmt3a KO on the long‐term expression stability for the large‐scale production of recombinant expression in CHO and other mammalian cells. Therefore, we believe that the development of the Dnmt3a‐deficient CHO cell line will be greatly beneficial to a broad community of users.The authors declare that they have no competing interests.W.T.Y. and J.Y.L. conceived and designed the study and analysed the results. G.X., L.J.T. and Q.L.L. performed the experiments. G.X., J.Y.L. and W.T.Y. and analysed the data. J.Y.L. and W.T.Y. wrote the paper. All authors read, edited and approved the final manuscript.


Even-denominator fractional quantum Hall (FQH) states, such as 5/2 and 7/2, have been well known in a two-dimensional electron gas (2DEG) for decades and are still investigated as candidates of non-Abelian statistics. In this paper, we present the observation of a 3/2 FQH plateau in a single-layer 2DEG with lateral confinement at a bulk filling factor of 5/3. The 3/2 FQH plateau is quantized at  ν = 5/3 quantum Hall state has a 3/2 plateau in the diagonal resistance that has not been captured by existing models.Fractional quantum Hall states in 2D electron gases arise due to strong electron-electron interactions, which makes a general theoretical understanding difficult. Fu et al. present data showing the  Most of the observed fractional states are odd-denominator states3 and can be explained under the framework of the composite fermion theory4. For example, the 1/3 FQH state can be mapped into the filling factor ν = 1 integer quantum Hall (IQH) state of composite fermions carrying fractional charge of e/3, which leads to a fractional N = 0 Landau level , the absence of FQH states is attributed to the formation of Fermi sea of composite fermions5. The discovery of the first even-denominator state, the 5/2 FQH state7, viewed as a paired state of composite fermions with e/4 fractional charge8, further enriched the knowledge of interacting electrons. Recent studies such as phase transition to the stripe phase9, and even-denominator states in other two-dimensional systems11, keep providing interesting physics at even-denominator filling factors.The fractional quantum Hall (FQH) effect has been investigated to extend our understanding of strongly interacting particles in two dimensions since its discovery14, which is related to the boundary of a realistic experimental system. Theoretical treatments for the FQH effect usually consider an infinite 2DEG, but experiments can only measure finite samples with boundaries. The quantum Hall edge originating from the boundary was discussed15 and later described by chiral Luttinger liquids16. Experiments based on the FQH edge physics have provided many exciting results and led to more efforts, such as the measurement of fractional charge17, weak quasi-particle tunneling between edge currents19 and the search of the neutral modes20. Theoretically, a 2DEG confined in an interferometer is proposed to test the non-Abelian statistics directly21, and the interference of the edge current has been measured22. Although the realistic boundary of a many-body system may complicate the theoretical treatment23, the confinement can be a powerful experimental approach to manipulate the system. Even though the FQH effect has been studied for decades, systems of interacting electrons with confinement are still an open area for exploration.The excitations in the FQH system carry fractional charge owing to the strong correlation between electrons. Besides, there is another equally famous way of charge fractionalization through topological soliton and topological phase transitionHere we show an even-denominator FQH plateau quantized at RD across the confined region at zero magnetic field and T = 6 K. RD increases quickly from near zero to about 140 Ω when the gate voltage is swept from −1.0 V to −1.3 V, and below −1.3 V RD increases slowly, indicating the electrons underneath the gates are depleted when the gate voltage is not positive than −1.3 V. By estimating the transverse modes through the confined region, 140 Ω corresponds to a lateral confinement of ~2.1 μm, close to the lithography length. In order to achieve the confined region, Vgate = −4.5 V was applied to the gates to deplete the electrons underneath and around the gates. This gate voltage was kept for more than 6 h above 4 K and during the cooling to 18 mK. This procedure named gate annealing is similar to that used in quasi-particle tunneling experiments for uniform density25. At 18 mK, we kept gate voltage at −4.5 V, and measured the Hall traces between filling factor 1 and 2. Both the Hall resistance RXY in the bulk of the Hall bar and the diagonal resistance RD develop two IQH plateaus and several FQH plateaus, which indicates that well-defined compressible edge states and incompressible bulk states appear in the confined region, and the density inside the confined region and outside the confined region is nearly uniform (Fig. The Hall bar samples were made from a wafer of GaAs/AlGaAs heterostructures. The 2DEG is about 200 nm below the sample surface. Two separate gates were deposited on the surface for the confined region. A sketch of the Hall bar and measurement setup are shown in Fig. orm Fig. .Fig. 1SaRXY does not change. Unexpectedly, the diagonal resistance RD develops an even-denominator FQH plateau quantized at RD and the 5/3 FQH plateau of RXY center at almost the same magnetic field, and the width of these two plateaus are similar at 18 mK. Analogous to conventional FQH states, the width of the 3/2 plateau decreases with increasing temperature. Interestingly, the 3/2 FQH plateau is even wider than that of the 5/3 FQH plateau at 100 mK (Fig. In our samples, the confined region is formed and controlled by the gate voltages. Changing the gate voltage will tune the lateral confinement but not influence the bulk properties. As shown in Fig.  mK Fig. , and thementclass2pt{minimmentclass2pt{minimVgate = −2.8 V, and becomes larger with less negative gate voltage. When the gate voltage is less negative than or equal to −2.0 V, the 3/2 FQH plateau appears. The gate voltage dependence of the 3/2 FQH plateau can also be demonstrated by the relationship between the gate voltage and the diagonal resistance. As shown in Fig. The critical parameter to trigger the appearance of the 3/2 FQH plateau is the gate voltage. The diagonal resistance traces with different gate voltages at 18 mK are shown in Fig. Vgate = −1.3 V is sufficient for the depletion of the electrons underneath the gates, as shown in Fig. Vgate = −4.5 V. If the gates are annealed at -1.5 V, even though enough to achieve the confined region, the 3/2 FQH plateau will not appear (Supplementary Fig. Another essential condition for the observation of the 3/2 FQH plateau is the gate annealing procedure with appropriate negative gate voltage. In our samples, Vgate = −4.5 V, and the measurement was carried out at bulk filling factor 5/3 and T = 18 mK. As shown in Fig. In order to demonstrate the full depletion underneath the confinement gates, we fabricated a sample with a single top gate crossing the entire Hall bar. This gate was also annealed at Vgate = −4.5 V is applied to the gates, the electrons both underneath and around the gates will be depleted. The annealing procedure may adjust the charge distribution of the donor layer at T > 4 K, which causes a very sharp potential in the confined region at the measurement temperature range, and supports the 5/3 FQH state in the confined region (Fig. In the confined region, a possible result caused by the annealing procedure and gate voltage change is quasi-particle tunneling. When ion Fig. . When th26, when the diagonal resistance develops the 3/2 FQH plateau, the conductance Vgate = −2.8 V and at Vgate = −1.3 V. The conductance in the bulk of the Hall bar should be From the Landauer-Büttiker theory27. In their measurements, the 3/2 FQH plateaus appeared in samples with both uniform density and non-uniform density. From the corresponding R-B features between RD and RXY in Fig. In general, the density of the gate-defined confinement region is smaller than that of the bulk, and affected by the gate voltage. However, the gate annealing procedure can keep uniform density for the whole sample in our measurements. This can be verified by the magnetic field dependence of the IQH and FQH plateaus in Hall traces in Fig. 29. At ν = 5/3 FQH state, the edge state could be complicated due to potential edge reconstruction and neutral mode30. The formation of the 3/2 FQH plateau could be caused by a more complicated edge reconstruction with lateral confinement at ν = 5/3. In our system, the disorders induced by the residual potential in the confined region could also help with the collapse of the 5/3 FQH state and the formation of intriguing edge structure and excitation with fractional charge. The 3/2 FQH plateau has been found in the single-layer two-dimensional system of ZnO where Landau level crossing induced by tilted magnetic field is needed10, while there is no in-plane magnetic field in our measurements. The exact origin of the 3/2 FQH plateau remains an open theoretical question to be investigated.Disorder can drive the collapse of energy gap and then induce phase transition from the FQH state to another state7 cm2 V−1 s−1 at 20 mK. A 150 μm wide Hall bar was shaped by wet etching. Ohmic contacts were made of Pt/Au/Ge alloy deposited by e-beam evaporation and annealed at 550 °C for 100 s. Two separate Cr/Au gates were deposited on the surface of the sample to form the confined region. In this experiment, samples with different confined region sizes were fabricated and measured. The dimensions of these geometries are 1 × 2 μm2, 0.3 × 2 μm2 and 1.5 × 3 μm2, respectively. The confined region on the samples used in the main text is the size of 1 × 2 μm2. The data from other samples are shown in the Supplementary Figs. The samples were made from a wafer of GaAs/AlGaAs heterostructures. The center of the 30 nm quantum well is 210 nm below the surface, with modulated Si doping at ~100 nm below and above the quantum well center. The mobility is higher than 1.0 × 10RXY and the diagonal resistance RD were measured simultaneously using lock-in techniques at 6.47 Hz with 1 nA. The two-terminal conductance measurements were also carried out using lock-in techniques at 6.47 Hz with voltage excitations smaller than 35 μV.The measurements were carried out in a dilution refrigerator with a base refrigerator temperature below 6 mK. The temperatures given in this work are electron temperatures. In the four-terminal resistance measurements, the Hall resistance Supplementary InformationSource Data


This article  was repuThe image presented for the DMSO/SZ1 experiment in Fig 3A was duplicated from a previous publication .In Fig 4, duplicate images were shown for SZ1 treated with DMSO and 5μM GSI IX, and the wrong image was shown for the 20μM panel.In Fig 5, duplicate images were presented in the “Snail” and “E cadherin” panels of Fig 5A and Fig 5B, and the molecular weight labels for these panels in Fig 5B were incorrect.The University of Tuebingen investigated these issues and did not find evidence of misconduct.The authors apologize for the errors in the original publication  and havePlease download this article again to view the correct version.S1 File(PPT)Click here for additional data file.