To provide a reliable integration of components within a solid oxide electrolysis cell stack, it is fundamental to evaluate the mechanical properties of the glass–ceramic sealing materials, as well as the stability of the metal–glass–ceramic interface. In this work, the mechanical behavior of two previously developed glass–ceramic sealants joined to Crofer22APU steel is investigated at room temperature, 650 °C, and 850 °C under shear load. The mechanical properties of both the glass–ceramics showed temperature dependence. The shear strength of Crofer22APU/glass–ceramic/Crofer22APU joints ranged from 14.1 MPa (20 °C) to 1.8 MPa (850 °C). The elastic modulus of both glass–ceramics also reduced with temperature. The volume fraction of the crystalline phases in the glass–ceramics was the key factor for controlling the mechanical properties and fracture, especially above the glass-transition temperature. Solid oxide electrolysis cells (SOECs) are a promising technology to produce hydrogen through the electrolysis of water. Among the various components of an SOEC stack, the stability and performance of the glass–ceramic sealants are key factors to determine and control the overall efficiency of the system ,2. BecaugT) [2O3–Al2O3–SiO2 glass–ceramic joined to the Crofer22H specimen, at room temperature and at 800 °C under shear and tensile loading. They evaluated the effect of ageing and pre-oxidation of the Crofer22H and found that the tensile joint strength is lower if the fracture involves delamination at the interface between the steel substrate and BaCrO4 layer, formed by the reaction between BaO from the glass and Cr from the steel. Anyhow, by increasing the testing temperature, the shear strength reduced from 7 MPa (25 °C) to 4 MPa (800 °C) [Due to cyclic temperature working conditions, there is a possibility of stress generation at the Crofer22APU/glass–ceramic interface and/or within the glass–ceramic joint. If the stresses increase to a critical level, either debonding at the Crofer22APU/glass–ceramic sealant interface or within the glass–ceramic can occur, thus causing gas leakage. Therefore, besides the thermal, chemical, thermomechanical and electrical stability of glass sealants, it is also important to analyze their response to mechanical loading under conditions corresponding to operational ones ,7,8,9,10Tg) ,12,13. RTg) ,19,20,21Tg)  employedIn a paper by López et al. , the fleIn our work, the shear strength of the Crofer22APU/glass–ceramic/Crofer22APU joined samples was studied by using two SrO-containing glass-based systems, designed for a working temperature of 850 °C. The Crofer22APU/glass–ceramic/Crofer22APU samples were investigated under shear load at room temperature, 650 °C and 850 °C. The elastic modulus of the glass–ceramics was also measured from room temperature to 650 °C by vibration method. gT of the HJ3 and HJ4 glasses was 722 °C and 736 °C, respectively, as measured by differential thermal analysis (DTA). Previously performed XRD analysis showed the presence of Sr2Al2SiO7, Ca0.75Sr0.2Mg1.05(Si2O6) and Ca2Mg(Si2O7) phases in the HJ3 glass–ceramic, while the HJ4 glass–ceramic had SrSiO3 and SiO2 phases after joining [Two previously developed glass–ceramic systems, further labeled as HJ3 and HJ4, are employed to investigate their mechanical behavior in contact with the Crofer22APU interconnect. The  joining . The coefficients of thermal expansion (CTE) and the softening behavior of both the glass–ceramics were measured by dilatometer , at a heating rate of 5 °C/min. The dilatometer analyses were performed on the glass–ceramic pellets (diameter 1 cm), prepared by pressing the glass powder in a steel mold, followed by a heat treatment in static air. Quantitative XRD analyses based on the Rietveld method were not feasible for the as-joined HJ3 and HJ4 glass–ceramics due to the complex crystalline phases and the corresponding XRD patterns. Consequently, in order to determine the relative quantities of the crystalline phases in the HJ3 and HJ4 as-joined glass–ceramics, an estimation could be made on the relative weight balance between the crystals in the glass–ceramics and the internal standard (ZnO), introduced in a defined quantity (20 wt.%). Therefore, the semi-quantitative analysis was performed by means of a Match software package , operating based on the reference intensity ratio method (RIR method) . For mechanical characterization under quasistatic shear loading conditions, the Crofer22APU/glass–ceramic/Crofer22APU joined samples were prepared. Quasistatic shear testing was carried out at a constant machine cross-head rate of 50 µm/min. The loading fixture developed for the experiments is also shown in The elastic modulus of the pure glass–ceramics was measured by vibration method, at temperatures ranging from room temperature to 650 °C, during the heating and cooling cycles. For this purpose, thin rectangular samples of glass–ceramics with dimensions of 20 mm × 2 mm × 2 mm were prepared. The high-temperature impulse excitation technique, HT1600 system  was applied for these analyses. The elastic modulus was determined by measuring the resonant frequency of the sample at the given temperature and then calculated from the specimen dimensions and density. The previously performed DTA on these glass systems showed a significant difference in crystallization behaviors. No crystallization peak was detected during the DTA analysis of the HJ4 glass, thus indicating that the crystallization was probably not sufficient to be detected. However, the DTA analysis of the HJ3 glass showed a crystallization peak due to the sufficient crystallization in that system . −6 K−1 and 9.3 × 10−6 K−1 respectively, in the temperature range of 200 °C–500 °C. The dilatometer curve of the HJ3 as-joined glass–ceramic is quite linear, thus indicating that the HJ3 glass–ceramic did not become soft up to 1000 °C. Nevertheless, the dilatometer curve of the HJ3 glass–ceramic shows a slight change in the slope around 680 °C, which is most likely due to the gT of the residual glassy phase. On the other hand, the dilatometer analysis of the HJ4 glass–ceramic shows that the HJ4 glass–ceramic became soft at around 800 °C with a gT around 700 °C. These results further support the hypothesis that the as-joined HJ4 glass–ceramic has more residual glass than the as-joined HJ3 glass–ceramic. The dilatometer curves of the HJ3 and HJ4 glass–ceramics are shown in gT), the fracture occurred in the linear mode once the applied stress reached maximum shear strength. This behavior reflects the almost elastic deformation and brittle response of the glass–ceramic joint. However, as expected, at 850 °C (T > gT), the joints showed enhanced displacement under the applied load and extensive non-linear behavior. This effect is due to the stress relaxation and the softening and viscous flow of the residual glassy phase above gT, as observed by Zhao et al. [o et al. . Althougo et al.  observedThe shear strength values as determined and calculated from the stress displacement curves shown above  are giveIt is worth highlighting that the HJ3-based joints fractured cohesively (fracture occurred within the glass–ceramic joint), while the HJ4-based joints fractured in an adhesive manner (fracture occurred at the Crofer22APU/glass–ceramic interface) for all the three testing temperatures. Furthermore, overall thermal expansion coefficient (CTE) of a glass–ceramic, the CTE of individual crystal phases in that glass–ceramic system can play a key role in controlling the fracture behavior as they may create the stress concentration regimes, either within the glass–ceramics and/or at interface.−6 K−1 that is closely matching CTE of the Crofer22APU (12 ×10−6 K−1) [2Al2SiO7 as main crystalline phase with a CTE of 1.1 ×10−6 K−1 [2Al2SiO7 is not present).The HJ3 glass–ceramic has CTE of 10.2 ×100−6 K−1) ; however10−6 K−1  that cou3 as main crystalline phase having a CTE of 10.9 ×10−6 K−1 [−6 K−1 which is lower than that of Crofer22APU, therefore, it can lead to generate stresses at Crofer22APU/HJ4 glass–ceramic interface and make adhesive fracture more favorable under the externally applied load. The HJ4 system has SrSiO10−6 K−1 , thus reFurthermore, the presence of defects (pores or bubbles etc.) within the glass–ceramics can also lead to initiation of crack. The high degree of devitrification in the HJ3 glass–ceramic increases the possibility to generate residual micro porosity due to enhanced viscosity cause by crystallization. These micro pores could be another possible reason to initiate the crack within the HJ3 glass–ceramic. Hasanabadi et al.  also tes2) phase in the HJ4 glass–ceramic [The lower mechanical strength for HJ4 with respect to HJ3 at 650 °C could be attributed to the presence of cristobalite  phase in the HJ4 glass–ceramic [The elastic moduli of the as-joined glass–ceramics for both the glass systems are shown in –ceramic ,32. Neve–ceramic ,31,33,34gT).Mechanical testing in shear conditions performed at room temperature, 650 °C and 850 °C enabled to quantify the temperature dependence of the investigated shear parameters. The mechanical strength of both glass–ceramic-joined samples tested at 650 °C was retained up to 60–70% of the RT values, while a substantial decrease was observed at 850 °C (T > Similarly, as expected the elastic modulus of both the glass–ceramics also showed nearly linear reduction with temperature. The volume fraction of the crystalline phases has been found to be an important factor controlling the mechanical properties at high temperatures. The higher amount of residual glassy phase in the glass–ceramic reduces the high-temperature mechanical properties of the sealant but promotes the stress relaxation possibilities due to softening, which is beneficial to release the thermal stresses at high temperature.This study provides insights for design and development of glass–ceramic sealants for SOEC working at 850 °C.


This work represents the first fabrication of freestanding 2D gold with a sub‐nanometer thickness, opens up an innovative pathway toward atomically‐thin metal nanomaterials that can serve as model systems for inspiring fundamental advances in materials science, and holds potential across a wide region of applications.2D metal nanomaterials offer exciting prospects in terms of their properties and functions. However, the ambient aqueous synthesis of atomically‐thin, 2D metallic nanomaterials represents a significant challenge. Herein, freestanding and atomically‐thin gold nanosheets with a thickness of only 0.47 nm (two atomic layers thick) are synthesized via a one‐step aqueous approach at 20 °C, using methyl orange as a confining agent. Owing to the high surface‐area‐to‐volume ratio, abundance of unsaturated atoms exposed on the surface and large interfacial areas arising from their ultrathin 2D nature, the as‐prepared Au nanosheets demonstrate excellent catalysis performance in the model reaction of 4‐nitrophenol reduction, and remarkable peroxidase‐mimicking activity, which enables a highly sensitive colorimetric sensing of H Sub‐nanometer gold nanosheets (thickness: 0.47 ± 0.01 nm) are successfully prepared via a facile aqueous synthesis at an ambient temperature. These nanosheets represent the first free‐standing 2D gold nanostructure with a sub‐nanometer thickness, and demonstrate excellent catalysis performance in the model reaction of 4‐nitrophenol reduction, coupled with remarkable peroxidase‐mimicking activity, which enables a highly sensitive colorimetric detection of H2O2. The obtained gold nanoseaweeds (AuNSWs) exhibit high efficiency as heterogeneous nanocatalysts and peroxidase‐mimicking nanoenzymes.4 and Na3C6H5O7  were sequentially added into an aqueous solution of MO (0.21 × 10−3m) at 20 °C. The resultant solution was kept undisturbed at this temperature for 12 h  and X‐ray diffraction (XRD). The HRTEM image of AuNSW Figure2a exhiFigureIn order to explore the formation mechanism of the AuNSWs, we carried out simulation and a series of control experiments. First, we performed a control experiment of synthesizing gold nanostructures in the absence of the confining agent MO whilst keeping all other reaction parameters unaltered. The products showed a quasi‐spherical shape Figure3a with−1, respectively. These results suggest that the interfacial interaction between Au (111) surface and MO is the strongest among three surface models. The stronger affinity of MO molecules on the basal (111) planes of Au nanosheets restricts the growth along the [111] direction and contributes to directing the formation of 2D structure. This confinement effect based on the preferential adsorption is similar to that in the carbon monoxide‐confined growth strategy for preparing freestanding ultrathin Pd or Rh nanosheets, whereby the strong adsorption of carbon monoxide on the (111) surface of PdGiven the reported simulation work regarding preferred adsorption structures for benzene on metal surfaces,tween Au 1 surface−3m) to elucidate the function of the above‐mentioned 2D molecular stacking of MO in regulating the growth of AuNSWs. The inability of MO to form 2D stacking at low concentration (below 0.20 × 10−3m)−3m MO, whist keeping all the reaction parameters the same with those in the preparation of AuNSWs, generated mostly quasi‐spherical nanoparticles and limited number of small nanoflakes . To study whether the failure of growing a 2D gold nanostructure with low concentration (0. 07 × 10−3m) of MO arises from the absence of 2D stacking or insufficient coverage of MO on the (111) surface, we further carried out synthesis with 0. 07 × 10−3m MO, 0. 167 × 10−3m HAuCl4 and 33.3 × 10−3m SC for keeping the ratio of MO: HAuCl4: SC identical to that in the synthesis of AuNSWs to confer effective surface coverage. The products  are predominantly the aggregates of nanoflakes and nanoparticles, i.e., the growth of 2D nanocrystal occurred to some extent due to the preferential adsorption of MO, but not well‐confined in a 2D space to produce the final product of Au nanosheets, which implies that 2D planar stacking of MO is also a contributing factor for creating 2D gold sheets. Taken together, MO has dual roles in regulating the formation of <111> oriented AuNSW: the effect of soft template provided by the 2D planar stacking and preferential adsorption on Au (111) for restricting the growth along the [111] direction.In addition, we have conducted control experiments of syntheses with lower concentration of MO  Growth of primary nucleus into Au nanoflakes of small lateral size, as confined by MO molecules; 3) Selective oriented attachment of small Au nanoflakes onto the side facets to minimize their high surface energy.We also characterized the products at different stages during formation using TEM Figure c–f and U4, a well‐established benchmark reaction to assess the catalytic activity of various noble metal nanocatalysts.4, the 400 nm peak showed negligible change during 2 h  using pseudo‐first‐order kineticsCt/C0) and reaction time showed a linear fit  associated with AuNSWs and AuNPs were calculated to be 1.1 × 105 and 9.2 × 103 min−1 g−1 respectively, which reveals that the catalytic activity of AuNSWs is 10 times larger than that of AuNPs in this model reaction. Notably, AuNSWs exhibit a superior catalytic activity to most reported noble metal nanocatalysts , and maintain ≈90% of such a high activity after 6 repeated cycles , to determine Michaelis–Menten constant (Km) and maximum initial velocity (Vmax), with results shown in Table S2 (Supporting Information). Km is a parameter indicating the enzyme affinity with substrates, and a low Km value represents a strong affinity. The apparent Km value of AuNSWs with TMB as the substrate was about 1/3 of that of HRP, revealing that AuNSWs have a higher affinity with TMB than HRP. The apparent Km of AuNSWs with H2O2 as the substrate was remarkably larger than that for HRP, suggesting that a higher H2O2 concentration was required to reach maximal activity, in accordance with previously reported observations regarding artificial enzyme mimics.Kcat values of AuNSWs and HRP with H2O2 substrate are respectively 2.24 × 105and 3.48 × 103 s−1,2O2. As can be seen in Figure 2O2 concentration from 0.1 to 1000 × 10−6m. Figure 2O2 dose‐response curve, with the inset showing a linear range between 0.1 and 10 × 10−6m . The limit of detection (LOD) is 0.11 × 10−6m, lower than that of other reported nanoenzymes .Motivated by the excellent catalytic performance of the AuNSWs shown in the model reaction above, we further explored the applicability of AuNSWs as nanoenzymes. Nanomaterials with enzyme‐mimicking catalytic properties, that is, nanoenzymes, have recently emerged as promising artificial enzymes, which exhibit unique catalytic activities, while circumventing the high cost and low stability of natural enzymes. In addition, nanoenzymes have several advantages over molecular artificial enzymes, including diverse functions beyond catalysis and large surface areas facilitating bioconjugation.2O2. It is also demonstrated that these sub‐nanometer thick AuNSWs can be readily exploited in the sensing systems based on peroxidase‐mimicking activity, such as for the highly sensitive colorimetric detection of H2O2. We envisage that the ease of production and unique structural properties of AuNSWs endow them with a variety of potential applications in catalysis, sensors, biomedicine and so on. Our synthesis strategy opens new avenues to the bottom‐up preparation of ultrathin 2D metal nanostructures with enhanced performance and multiple functionalities, which would potentially extend our understanding of the underlying fundamental science and may lead to the unveiling of unprecedented phenomena and properties.In summary, atomically‐thin 2D AuNSWs have been successfully prepared by a simple aqueous synthesis at an ambient temperature. This synthesis strategy relies on the use of MO molecules to provide confinement during nanocrystal formation. The resultant AuNSWs represent the first free‐standing 2D gold with a sub‐nanometer thickness, and display pronounced catalytic performance toward the reduction of 4‐niotrophenoal as well as the degradation of Hhttps://doi.org/10.5518/464.Experimental details and any associated references are available in the Supporting Information of this communication. The data presented in this article will be openly available from the University of Leeds data repository S.Y. and S.D.E. have filed a patent (Application number: 1818923.3) relating to 2D metal nanomaterials.SupplementaryClick here for additional data file.


N,N‐diethylamino benzaldehyde (DEAB) linked to a fluorinated pyridine ring either via an amide or amine linkage. Of the focused library of compounds evaluated, N‐ethyl‐6‐(fluoro)‐N‐(4‐formylbenzyl)nicotinamide 4 b was found to have excellent affinity and isozyme selectivity for ALDH1A1 in vitro. Following 18F‐fluorination, [18F]4 b was taken up by colorectal tumor cells and trapped through the conversion to its 18F‐labeled carboxylate product under the action of ALDH. In vivo positron emission tomography revealed high uptake of [18F]4 b in the lungs and liver, with radioactivity cleared through the urinary tract. Oxidation of [18F]4 b, however, was observed in vivo, which may limit the tissue penetration of this first‐in‐class radiotracer.Aldehyde dehydrogenases (ALDHs) catalyze the oxidation of aldehydes to carboxylic acids. Elevated ALDH expression in human cancers is linked to metastases and poor overall survival. Despite ALDH being a poor prognostic factor, the non‐invasive assessment of ALDH activity in vivo has not been possible due to a lack of sensitive and translational imaging agents. Presented in this report are the synthesis and biological evaluation of ALDH1A1‐selective chemical probes composed of an aromatic aldehyde derived from  The measurement of chemoresistance through ALDH imaging could potentially enable the clinician to select the most suitable therapeutic intervention for the individual patient (e.g. chemotherapy versus immunotherapy) with the possibility to improve outcomes and reduce unnecessary treatment. Currently, the in vitro assessment of ALDH activity has been restricted to fluorescence‐based assays.ALDH1A1 chemical probes were designed to have a) an aldehyde that can serve as a substrate for ALDH1A1; b) contain a (radio)fluorine atom that would allow for detection via gamma counting/PET imaging; c) a suitable hydrophobic‐hydrophilic balance which would allow for passive diffusion in and out of cells, and importantly; d) subsequent trapping of the in situ generated carboxylic acid product within the cytosol as a result of the acquired negative charge  1, which is a well‐known inhibitor of ALDH1A1 .1 in to a substrate whilst maintaining suitable ALDH1A1 selectivity over other commonly expressed ALDH isozymes. 1 is thought to form a stalled acyl‐enzyme intermediate as a result of the delocalization of the electron lone pair of the para‐substituted amine into the aromatic ring.1 from an inhibitor to a substrate, we uncoupled the amine nitrogen from the aromatic π‐system through the introduction of a methylene linker to give 2 3 and diethylamine in DCE afforded the crude reductive amination product which was directly treated with aqueous hydrochloric acid in THF to furnish 3 a  and catalytic efficiency (Vmax/KM) for ALDH1A1 over both ALDH2 and ALDH3A1 . In comparison to ALDH2 and ALDH3A1, ALDH1A1 has the largest access tunnel to the active site residue , which suggests that bulkier and more rigid substrates would be preferentially turned over by ALDH1A1. Tunnel topography may also explain why reducing the amide in compound 4 a to a flexible tertiary amine in compound 5, resulted in increased affinity for ALDH2 and ALDH3A1. Compounds 4 a and 4 b displayed similar binding modes as 2 to ALDH1A1 in our in silico model, as shown in Figure 4 a, 4 b, 5, and 6 occupied a similar site to that of compound 2; however, the pyridyl‐ring was predicted to make additional contacts, presumably via π‐stacking between the pyridine and the tyrosine‐Y296 of ALDH1A1 ‐N‐(4‐formylbenzyl)nicotinamide was chosen as our lead candidate radiotracer. To access [18F]4 b, a nucleophilic aromatic substitution (SNAr) was performed on the 6‐chloronicotinamide precursor 7, which was prepared from the acid chloride of 6‐chloronicotinic acid 20 and amine A  decay‐corrected radiochemical yield (RCY) after reverse phase semi‐preparative HPLC purification following manual radiolabeling (Scheme n=3). Starting the synthesis with ≈1.0 GBq of [18F]fluoride, the radiotracer was obtained with a radiochemical purity of 99 % (see Supporting Information) and a molar activity of up to 4.4 GBq μmol−1  and up to 7.2 GBq μmol−1 (automated).Compound 8 and alcohol 9, were prepared by the KMnO4 mediated oxidation of 4 b, and the NaBH4 mediated reduction of aldehdye 4 b, respectively (Scheme 18F]4 b could provide a readout of ALDH activity in tumor cells grown in culture. We used the HCT116 KrasG13D/− mutant (HCT116 mut) human colorectal cancer cell line as a model of aggressive, therapy‐resistant cancer. HCT116 mut lines displayed high ALDH activity, which was specifically inhibited through the incubation of cells with the ALDH inhibitor DEAB . Treatment of cells with DEAB resulted in an 83 % reduction in cell‐associated radioactivity to 1.0±0.1 % radiotracer dose mg−1 protein , indicating ALDH‐specific intracellular trapping of either [18F]4 b or its products , confirmed through co‐injection with non‐radioactive [19F]8 (See Supporting Information for details). Incubation of cells with DEAB resulted in a substantial reduction in the production of [18F]8, with >90 % of radioactivity present as the parent compound . Analysis of media samples by radio‐HPLC following cell incubation showed a progressive increase in the levels of [18F]8 and the subsequent appearance of the alcohol [18F]9 , confirming that the conversion to the carboxylate was cell‐mediated. Together, these data show [18F]4 b to be specific and sensitive marker of ALDH activity in tumor cells.Whilst the levels of 8F4 b in 18F]4 b can measure ALDH activity in HCT116 mut cells with high sensitivity and specificity, we next explored [18F]4 b's in vivo biodistribution. Dynamic microPET imaging following intravenous injection of [18F]4 b into healthy balb/c mice . [18F]4 b clearance was initially via the kidneys and bladder to afford high contrast images with low uptake in background tissue . However, hepatobiliary excretion was evident by 30 min p.i., as shown by radiotracer uptake in the gallbladder and gastrointestinal (GI) tract, indicating possible metabolism of the parent compound at this time point. [18F]4 b was further cleared from all other organs other than gallbladder and GI over the remainder of the imaging time course. For time activity curves for organs with high [18F]4 b uptake, see Figure S4.Given that [e Figure  revealed18F]4 b. Radio‐HPLC analysis of plasma taken by terminal exsanguination from anesthetized mice following tail vein intravenous (i.v.) injection of [18F]4 b revealed complete conversion to [18F]8 by 2 min . The appearance of a second, unknown peak of similar retention time to [18F]8 (Supporting Information chromatogram S7) was evident by 5 min, increasing to ≈40 % total radioactivity in the blood by 60 min, which may account for the mixed routes of excretion observed at later imaging time points. Importantly, the rapid metabolism of the free aldehyde may limit the in vivo tissue penetration of [18F]4 b.The combined renal and hepatobiliary excretion observed at 30 min p.i., prompted us to assess the in vivo stability of [N‐Ethyl‐6‐(fluoro)‐N‐(4‐formylbenzyl)nicotinamide 4 b was taken forward for radiolabeling and evaluation in both tumor cells and in mice. [18F]4 b was rapidly taken up by ALDH‐expressing colorectal cancer cells in culture and intracellularly trapped through ALDH‐specific conversion to the corresponding carboxylic acid. In vivo, high radiotracer uptake in the lung and liver were observed, combined with rapid clearance from background tissues and excretion via the urinary tract. Rapid oxidation of this lead compound in vivo however highlights a potential limitation of aldehyde‐based radiotracers for ALDH imaging. Future strategies will focus on the development of second generation ALDH1A1 radiotracers with improved in vivo stability to image drug resistance in animal models of cancer.In conclusion, we have developed a focused library of ALDH substrates based on the well‐known inhibitor DEAB. The addition of a fluoronicotinamide dramatically increased ALDH1A1 isoform specificity, thought to result from π‐stacking interactions with a tyrosine residue proximal to the active site. See Supporting Information for detailed synthetic, radiochemical methods, enzyme assays, cellular and studies in mice.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.


The capability of electrical stimulation (ES) in promoting bone regeneration has already been addressed in clinical studies. However, its mechanism is still being investigated and discussed. This study aims to investigate the responses of macrophages (J774A.1) and preosteoblasts (MC3T3‐E1) to ES and the faradic by‐products from ES. It is found that pH of the culture media was not significantly changed, whereas the average hydrogen peroxide concentration was increased by 3.6 and 5.4 µM after 1 and 2 hr of ES, respectively. The upregulation of Bmp2 and Spp1 messenger RNAs was observed after 3 days of stimulation, which is consistent among two cell types. It is also found that Spp1 expression of macrophages was partially enhanced by faradic by‐products. Osteogenic differentiation of preosteoblasts was not observed during the early stage of ES as the level of Runx2 expression remains unchanged. However, cell proliferation was impaired by the excessive current density from the electrodes, and also faradic by‐products in the case of macrophages. This study shows that macrophages could respond to ES and potentially contribute to the bone formation alongside preosteoblasts. The upregulation of Bmp2 and Spp1 expressions induced by ES could be one of the mechanisms behind the electrically stimulated osteogenesis. In this study, the responses of macrophages and preosteoblasts to electrical stimulation (ES) were investigated. The mRNA expression from each cell type was measured after being exposed to ES. The effects of faradic by‐products were also investigated by treating the cells with electrically stimulated media. It is found in this study that Bmp2 and Spp1 mRNA expressions from both cells were upregulated by ES, whilst Spp1 mRNA expression from macrophages was partially influenced by the faradic by‐products. Cells were incubated at 37°C, 5% CO2, and atmospheric O2 concentration. All reagents were purchased from Sigma‐Aldrich, UK, unless stated otherwise. In the experiments, cells were seeded into six‐well plates at the density of 50,000 cells per well for macrophages and 100,000 cells per well for preosteoblasts, and the media volume was kept at 3 ml. Media change was carried out on the following day after seeding before applying ES. Optical images of the cells were taken using EVOS™ XL Imaging System (Life Technologies).J774A.1 murine macrophage and phenotypically heterogeneous MC3T3‐E1 murine preosteoblastic cell lines were supplied from The European Collection of Authenticated Cell Cultures and used as received. The phenotype of J774A.1 cells used in this study were 91.4% M1 (CD11c positive), 0.6% M2 (CD206 positive), and the rest were M0 (neither CD11c nor CD206 positive)  was used for measuring the pH of culture media. pH probe was calibrated with pH buffer between pH 4.0 and pH 10.0  and washed with deionized water before use. Acellular media were incubated overnight before starting the experiment. The data were obtained by submerging the probe into the acellular culture media right after finishing the stimulation.2.42O2 concentration was calculated using the calibration curve from standard H2O2 solution supplied with the kit.Fluorimetric Hydrogen Peroxide Assay Kit  was used in this study. The reagent preparation was carried out following the supplier's instructions. Acellular media samples were incubated overnight before applying ES, and the assay was performed immediately after stimulation. The fluorescence intensity of the samples was measured after 30 min of room‐temperature incubation with the working solution at excitation wavelength of 544 nm and emission wavelength of 590 nm using microplate reader . The measured intensity was subtracted by background readings from nonstimulated media. H2.5Samples were incubated with 1 ml of 10% resazurin solution  diluted in the complete media for 1 hr. The fluorescence intensity of the supernatant was measured by microplate reader at excitation wavelength of 544 nm and emission wavelength of 590 nm. The background readings were subsequently subtracted from the measured intensity before analysis. The measurement was carried out the day after the final stimulation or ES media treatment.2.6t methods relative to Gapdh and control samples. The details of each primer are shown in Table Samples were washed three times with Dulbecco's phosphate‐buffered saline (DPBS) and collected immediately after the final stimulation or after 2 hr of the final ES media treatment at similar exposure time as its 2‐hr direct ES counterpart. Total RNA were extracted from the samples using RNeasy Mini Kit  following the supplier's instructions. The concentration and purity of the extracted RNA samples were measured by NanoDrop™ Lite Spectrophotometer . Complementary DNA were synthesized from 9 ng of total RNA and undergone thermal cycling following the supplier's instructions using QuantiFast SYBR® Green RT‐PCR Kit and QuantiTect Primer Assay  alongside StepOnePlus™ Real‐Time PCR System . Melt curves of the amplified samples were analyzed using the supplier's software. The messenger RNA (mRNA) fold expression level was calculated by comparative ΔΔC2.7Escherichia coli O111:B4 (100 ng/ml) on the following day after seeding. Tumor necrosis factor‐α (Tnfα) mRNA expression was measured from the samples collected immediately after the first 2 hr of ES, LPS treatment, and LPS treatment with simultaneous ES as this time point was shown to exhibit the maximal level of Tnfα mRNA expression induced by LPS . The measured H2O2 concentration of the media after stimulation is shown in Figure 2O2 has increased by 3.6 and 5.4 µM after 1 and 2 hr of ES, respectively. Although the average H2O2 concentration after 2 hr of ES are around 1.5 times higher than 1 hr, the difference was not statistically significant (p = .06).Figure 3.2The metabolic activity of macrophages and preosteoblasts measured by resazurin assay after 3 days of stimulation are shown in Figure 3.3The optical images of two cell types after 3 days of 2‐hr daily ES are shown in Figure 3.4The expression of mRNAs from macrophages and preosteoblasts, which are responsible for translating Bmp2, Hmgb1, Vegfa, and Spp1 proteins, were evaluated in this study as well as the expression of Runx2, from preosteoblasts. It is found that Spp1 expression was upregulated after 3 days of 1‐hr daily ES in both cell types, as shown in Figure 3.5p = .06). Furthermore, the metabolic activity of both cell types after ES media treatment were significantly different from direct ES group.The influence of faradic by‐products as a whole was investigated by treating the cells with ES media, in which the presence of electric field, potential, and current were eliminated. The cellular responses after being exposed to the ES media in terms of the overall metabolic activity are shown in Figure Changes in mRNA expressions induced by faradic by‐products are shown in Figure 4−) and H2O2. OH− is associated with pH of the culture media, whereas H2O2 is one of the ROS that could either be beneficial for cell signaling or inducing cell oxidative stress  and those generated from ES (<10 μM) (M. Cho et al., It has been reported that Bmp2 is essential for the early stage of bone healing, which subsequently enhances differentiation of osteoprogenitor cells and their migration towards the injury site as well as bone formation (Knippenberg, Helder, Doulabi, Wuisman, & Klein‐Nulend, Furthermore, the level of Runx2 expression was not significantly changed after 3 days of stimulation, which indicates that there was no change in cellular activities related to the maturation of preosteoblasts during this period Komori, . This suThe upregulation of Bmp2 expression after direct ES is also consistent with previous in vivo study. It was reported that the fractured bone tissue expresses Bmp2 after applying direct ES, and the expression of Vegf was not increased (Fredericks et al., 2O2, faradic by‐products also include H2 generation, O2 reduction, and any other surrounding molecules generated by redox reactions during ES (Bodamyali et al., 2O2 alone, or other faradic by‐products, or both, which triggered this response based on our current results. Despite the discussion that faradic by‐products could possibly be behind the upregulation of Bmp2 mRNA expression after ES, we clearly demonstrate that the increased Bmp2 mRNA expression is more likely a result of ES than the by‐products (Fredericks et al., Interestingly, this study shows that Bmp2 and Spp1 mRNA expressions are primarily a result of ES, whereas faradic by‐products have partially contributed to an increase in Spp1 expression from macrophages, potentially involving 4EPB‐1 translation or nuclear factor‐κB and AP‐1 transcription (Lyle et al., 4.3Macrophages are well known for triggering inflammatory responses; however, they could also be beneficial for wound healing and tissue regeneration depending on their phenotypes. The proinflammatory phenotype is M1 macrophages and wound‐healing phenotype is M2, and the differentiation towards these two phenotypes is dependent on their microenvironment and stimuli Murray, . It is uIt is found from this study that direct ES reduced Tnfα mRNA expression from macrophages, which is a proinflammatory and anti‐M2 macrophage marker that could be triggered by LPS (Huang et al., In addition to the findings from this study, the recent in vivo study also reports that ES has significantly reduced the inflammation during bone injury, which is consistent with the conclusion from an analysis of clinical studies suggesting that ES is effective in reducing pain and radiographic nonunion‐healing rate (Aleem et al., 4.42O2 generation (Kalbacova et al., 2, and exceeding this range would result in tissue or cell necrosis (Spadaro & Becker, It is seen from this study that although the majority of cellular responses are induced by ES, the faradic by‐products also have an effect on cells. Therefore, further characterization of the ES media may be requisite to explore the unidentified faradic by‐products and their effects on cellular activities, such as the radicals present during the process of H5ES could promote bone regeneration by inducing Bmp2 and Spp1 mRNA expressions from macrophages and preosteoblasts. Preosteoblasts did not respond to faradic by‐products in terms of Bmp2 and Spp1 mRNA expressions, whereas macrophages responded by increasing their Spp1 expression. We demonstrated that the roles of faradic by‐products are not dominant, and the by‐products alone would be less effective in promoting bone formation without the presence of ES. The findings from this study also imply that cellular responses from preosteoblasts and macrophages to ES are predominantly triggered by the mechanism involving electric field, potential, and/or current. On the contrary, Vegfa and Hmgb1 mRNA expressions from both types of cells and Runx2 expression from preosteoblasts are not affected by the ES regime used in this study. In addition, we showed that this ES system may need further optimization to reduce the current density near the electrodes, which locally impairs cell viability and proliferation.The authors declare that there are no conflict of interests.Supplementary informationClick here for additional data file.


To this end, SnO2 nanoparticles that are prepared using the industrially adopted flame spray pyrolysis (FSP) technique as active catalysts are reported for the conversion of CO2 to formate (HCOO−), exhibiting a FEHCOO− of 85% with a current density of −23.7 mA cm−2 at an applied potential of −1.1 V versus reversible hydrogen electrode. Through tuning of the flame synthesis conditions, the amount of oxygen hole center  is synthetically manipulated, which plays a vital role in CO2 activation and thereby governing the high activity displayed by the FSP‐SnO2 catalysts for formate production. The controlled generation of defects through a simple, scalable fabrication technique presents an ideal approach for rationally designing active CO2 reduction reactions catalysts.The large‐scale application of electrochemical reduction of CO In addition to developing catalysts with suitable active sites (for higher selectivity), it is also of utmost importance to improve the binding of CO2 reactants on electrocatalysts  as this step is widely reported as one of the limiting factors for CO2RR.2 activation.2RR to formate in prior reports,2RR. In this regard, the use of a facile technique to systematically vary defect sites to improve reactant binding for selective CO2RR to formate is desired.Solar powered electrochemical CO2 nanoparticles prepared using the commercialized flame spray pyrolysis (FSP) process as a viable catalyst for the electrocatalytic reduction of CO2 to formate. The high temperature facilitated by the FSP process allows precise control over the formation of nonstoichiometric atomic arrangements and vacancies that are commonly ascribed as beneficial for heterogeneous catalysis.2RR activity. Our extensive ex situ characterization (electron paramagnetic resonance (EPR), X‐ray absorption spectroscopy (XAS), and high‐energy X‐ray diffraction (HE‐XRD) coupled to pair distribution function (PDF) analysis) as well as in situ characterization (Raman) reveal that apart from the traditionally ascribed active sites, surface defects play a crucial role in activating CO2 on Sn‐based catalysts, providing further insights into designing cost‐effective electrocatalysts for CO2RR.Here, we report SnO22, FSP was utilized. By varying the precursor feed rate to the flame, the flame conditions can be controlled and thus allow for the tuning of material properties such as particle size and morphology, crystallinity, and defects.2 catalysts were synthesized at a feed‐rate of 3, 5, and 7 mL min−1 and are denoted as FSP‐SnO2‐3, FSP‐SnO2‐5, and FSP‐SnO2‐7, respectively .In order to systematically control the morphology and surface properties of SnOFigure2 (at 5 mL min−1 precursor feed rate). It is evident that the FSP results in the formation of highly crystalline  facets within the material.2 preparation, is clearly evident in TEM images . It can be observed that a higher feed‐rate during FSP results in increasing particle sizes (from ≈9 to ≈14 nm as the feed rate increases from 3 to 7 mL min−1) of the FSP‐SnO2 which is consistent with the X‐ray diffraction (XRD) and N2 adsorption‐desorption results, vide infra.Figure2 g−1 for FSP‐SnO2‐3 to 81.4 m2 g−1 for FSP‐SnO2‐7) with a corresponding increase in the BET equivalent diameter, dBET . This increase in surface area is consistent with the literature and arises from the changing flame conditions.2 adsorption‐desorption isotherms  indicate an IUPAC Type III isotherm consistent with a nonporous material.2 samples obtained was then analyzed with the aid of XRD , (101), and (310) facets of SnO2 respectively (JCPDS 41‐1445).As shown in 2 was investigated using hydrogen temperature programmed reduction (H2‐TPR). As indicated in Figure 2‐TPR profiles exhibit two distinct major peaks, one at low temperature region (200–300 °C) that corresponds to reduction of the SnO2 surface and possibly subsurface, and at high temperature region (640–700 °C) that can be ascribed to the reduction of the bulk SnO2.2‐3 is evident, as well as a shoulder for FSP‐SnO2‐5. The decreasing intensity of this additional peak with increasing feed rate during FSP clearly suggests that increasing feed‐rate leads to an increase in oxygen vacancy (facilitated by a decrease in complete oxidation) and this phenomenon can be explained using the fuel equivalence ratio. The fuel equivalence ratio, Φ, displayed in Figure 2 catalysts are Sn4+, as only two major peaks can be observed at binding energy of 487.6 and 496.8 eV corresponding to Sn 3d5/2 and Sn 3d3/2, respectively.a), ≈532 eV (Ob), and ≈533 eV (Oc) corresponding to the presence of Sn4+‐O, adsorbed oxygen and O atoms adjacent to oxygen vacancies, respectively.2‐5. Collectively, these characterization results indicate the controlled systematic tuning of the physiochemical properties of SnO2, empowering us to undertake a thorough investigation into the role of SnO2 as well as defects in defining CO2RR performance. A clear, and anticipated trend, with decreasing surface area with increasing precursor feed rate was evident along with an increase in oxygen vacancies.The reduction behavior of SnO2 toward CO2RR was evaluated using 2 h long potentiostatic studies carried out in CO2 saturated 0.1 m KHCO3. The polarization curves, presented in Figurej) attained with FSP‐SnO2‐5 was higher compared to FSP‐SnO2‐3 and FSP‐SnO2‐7 electrodes. At the applied potential of −1.1 V, the attained j with FSP‐SnO2‐5 was −23.7 mA cm−2 compared to −13.3 and −12.9 mA cm−2 observed with FSP‐SnO2‐3 and FSP‐SnO2‐7. Moreover, two gas phase products (H2 and CO) were detected using gas chromatography and the dependence of the Faradaic efficiency for H2(FEH2) and CO (FECO) on the applied potential is illustrated in Figure FEH2 , which is beneficial as more charge can be consumed toward CO2RR rather than the competing HER.CO attained with FSP‐SnO2‐3, FSP‐SnO2‐5, and FSP‐SnO2‐7 was 8%, 13%, and 17% at −0.7, −1.0, and −1.0 V, respectively. Nuclear magnetic resonance (NMR) revealed that the only liquid product generated during CO2RR was the target formate (HCOO−) with all the FSP catalysts exhibiting a strong potential dependence for FEHCOO−. Of these catalysts, FSP‐SnO2‐5 was the most active toward CO2RR to formate, requiring an onset overpotential of 375 mV whilst both FSP‐SnO2‐3 and FSP‐SnO2‐7 required 575 and 475 mV of additional overpotential before formate was detected. At all potentials, FSP‐SnO2‐5 exhibited better selectivity toward HCOO− production, attaining a maximum FEHCOO− of 85% at −1.1 V whereas the maximum FEHCOO− attained with FSP‐SnO2‐3 and FSP‐SnO2‐7 were 60% and 79% at −0.9 and −1.1 V, respectively. We also carried out mechanistic Tafel analysis  and found that the Tafel slope for FSP‐SnO2‐5 is much smaller, indicating faster reaction kinetics on the catalyst for CO2RR. The overall enhanced catalytic activity as well as notable stability  of FSP‐SnO2‐5 is amongst the highest for CO2RR to formate , thereby endorsing its suitability for potential large‐scale applications.The activity of FSP‐SnO2‐3) does not demonstrate a higher partial current density for formate production . To further validate this, volumetric CO2 adsorption measurements were carried out , which clearly demonstrated that physical CO2 adsorption on the catalysts do not dictate the CO2RR selectivity trends. Interestingly, both XRD and electrochemical impedance spectroscopy  measurements suggested similar conductivity of the FSP‐SnO2 catalysts and thereby cannot be used to explain the CO2RR trends outlined above. The above results alongside the electrochemical surface area measurements  reveal the physical changes of the FSP catalysts do not have a direct correlation with their varied selectivity.Combining the structure‐performance results for the FSP catalysts, it is clear that the particle size/surface area variation of the catalysts (as a result of the varying feed rates) is not governing the overall production rate for the FSP catalysts for formate production as the catalyst with the higher surface area  was maximized for FSP‐SnO2‐5 (Idefect/IA1g = 1.36) followed by FSP‐SnO2‐7 (Idefect/IA1g = 1.25) and the lowest was for FSP‐SnO2‐3 (Idefect/IA1g = 1.02). These results are consistent with the trends observed with CO2RR where FSP‐SnO2‐5 demonstrated the highest activity. UV–vis carried out with FSP‐SnO2‐5 further indicated that these defects are not changing the electronic states of the catalyst .2 catalysts. It can be seen from Figure g values of 2.158 and 4.278 are observed which can be attributed to the formation of oxygen hole centers  and Sn3+ associated with oxygen vacancy species, respectively.3+ associated with oxygen vacancy can be generated through incomplete oxidation during FSP synthesis.2 reactants,2 adsorption as well as lowering the activation energy for CO2RR.2 catalysts with the amount of OHC defects. The electrocatalytic results show a direct dependence on the amounts of defects . For instance, FSP‐SnO2‐5 with the highest OHC defects demonstrated the highest FEHCOO− followed by FSP‐SnO2‐7 with the second highest concentration of OHC defects and lastly FSP‐SnO2‐3 which contained the lowest amount of OHC defects. To further confirm the role of these defects, we carried out a control experiment where the active FSP‐SnO2‐5 was thermally annealed in air (referred as HT‐FSP‐SnO2‐5) to reduce the amount of OHC. This control sample was then evaluated for CO2RR  and we can clearly demonstrate that the removal of defects  leads to decreased formate production, in line with our hypothesis.After ruling out the physical properties, the role of chemical state and electronic properties of the catalysts in dictating COts Figure4a demo2 , which strongly suggests that the majority of the Sn share similar electronic configuration. The lack of sensitivity to defect structures in the XANES (as well as EXAFS) is likely due to a combination of core‐hole broadening that occurs in heavier elements and relative concentration of surface defects in material.2 catalysts, suggesting that a similar atomic environment is shared among the Sn species . The fitted EXAFS results  for the FSP‐SnO2 catalysts clearly indicate that the coordination number is ≈5.1 for the oxygen first‐shell, with a fitted bond length of 2.06 Å. These results collectively suggest that the defects within FSP‐SnO2 are on the surface of the catalysts and thereby the low concentration is not easily observable from Sn K‐edge EXAFS measurements.Additionally, we investigated the electronic configuration of the FSP catalysts with the aid of X‐ray absorption spectroscopy. The X‐ray absorption near‐edge spectra (XANES) showcase very similar edge features for all FSP samples that are characteristic of SnO2, with minor structural variations found throughout the PDF. The relative magnitude of the Sn–O first coordination sphere suggests disordered surface structure caused by defect formation that may not have been directly observed via EXAFS.2‐7 indicates a higher degree of longer‐range order under these synthetic conditions , likely due to a decrease in defects and an overall increase in particle size. Crystallographic modeling techniques were implemented to quantify structural changes that occur between the samples synthesized under different FSP conditions . The resulting a and c lattice parameters in Table S4 (Supporting Information) showcase a general trend to unit cell expansion with increasing feed rate, from 4.7213 Å for FSP‐SnO2‐3 to 4.7234 Å for FSP‐SnO2‐7 in the a lattice parameter, corresponding well with the formation of oxygen vacancies which are reported to cause lattice expansion.c lattice parameter shows less of a change with FSP feed rate, with values of 3.1774, 3.1780, and 3.1772 Å for FSP‐SnO2‐3, FSP‐SnO2‐5, and FSP‐SnO2‐7 respectively. In addition to lattice constant changes, anisotropic temperature factors can be used to better understand how defects influence overall structure, as a defective moiety represents an increase in atomic position uncertainty from a crystallographic viewpoint. Shown in Table S5 (Supporting Information), the anisotropic temperature factors for O atoms are ≈5× bigger than for Sn atoms, showcasing the impact of formation of O defects and terminal O atoms at the catalyst surface.2‐5 exhibited the highest O anisotropic temperature factor of 0.04221, followed by FSP‐SnO2‐3 and FSP‐SnO2‐7 (0.04166 and 0.03852 respectively). While being sensitive to defects, anisotropic temperature factors are inversely related to nanoparticle size as well, as an increase in surface atoms become crystallographically dissimilar from the bulk lattice. Given that the surface area decreases between FSP‐SnO2‐3 and FSP‐SnO2‐5, the increase in anisotropic temperature factor between the catalysts is evidence of greater formation of defects within FSP‐SnO2‐5. In contrast, FSP‐SnO2‐7 exhibited a large decrease in anisotropic temperature factor as a result of the lowest surface area presented by the catalyst .Additional synthesis‐dependent structural information was obtained using atomic pair distribution function analysis Figure c. Atomic2RR with SnO2 catalysts leads to partial reduction of oxide layers to generate SnOx sites.2 lattice during in situ reduction of the catalyst results in increasing the oxygen vacancies. To understand this changing defect structure under applied potentials, in situ Raman spectroscopy  was carried out with FSP‐SnO2‐5. In the absence of an applied potential, the Raman spectra  for the catalyst demonstrated peaks typical for SnO2 vibration modes as well as that of surface defects  and defects during CO2RR.It is well established that the application of bias during CO2 catalysts demonstrate a clear dependence of selectivity toward HCOO− on the defects , highlighting for the first time the modulation of CO2RR activity through defect engineering. The collective characterization results confirm the formation of highest amounts of defects in FSP‐SnO2‐5 . Thus, the maxima of surface defects of the FSP‐SnO2‐5 sample can be attributed to the tradeoff between the surface area and oxygen vacancies. Ultimately, these factors play a synergistic role to maximize the surface defects on FSP‐SnO2‐5. For instance, FSP‐SnO2‐3 contained a smaller amount of defects (indicated by Raman and EPR) but demonstrated a high anisotropic temperature factor because of its higher surface area. However, in the case of FSP‐SnO2‐5, the surface area decreases but we observe a maximum for the anisotropic temperature factor which is suggesting a significantly higher amount of defects . A further increase in feed‐rate  is found to lead to a slight decrease in defects but a much larger decrease in anisotropic temperature factor as a result of lower surface area exhibited by FSP‐SnO2‐7. On the basis of the above findings, it is suggested that the CO2 reactant molecules are initially adsorbed on the defects present on the surface of the optimized FSP‐SnO2, allowing the subsequent conversion of CO2 to formate on the SnO2 active sites. The presence of defects is known to increase the charge density along the valence band maximum, improving CO2 activation ,2 to formate as well as lowering onset overpotentials. Most importantly, these findings illustrate the ability to potentially tune CO2RR properties through synthetic manipulation of FSP conditions to optimizing for surface area and defect density.The collective structure–activity relationship for the FSP‐SnO32 catalysts for the large‐scale CO2 reduction to formate. Through extensive characterizations, we have revealed the decisive role of OHC in activating CO2 reactants, allowing higher activity toward formate production. Through control of the fabrication technique, we were able to modulate the amount of the defects, allowing us to achieve a high a FEHCOO− of 85% with a current density of −23.7 mA cm−2 at an applied potential of −1.1 V with the FSP‐SnO2 catalysts.In summary, we have developed and demonstrated an effective approach for scalable fabrication and implementation of defective SnO4Catalyst Preparation: Three SnO2 samples were prepared via flame spray pyrolysis in a setup described previously.m. This solution was fed to the FSP flame at three different flow rates  with a syringe pump, to give three separate SnO2 samples. The precursor solution was dispersed by a constant 5 L min−1 flow of oxygen . The flame was ignited and maintained with a supporting flame mixture which consisted of 3.2 L min−1 oxygen and 1.5 L min−1 methane . The flame was directed with the aid of a 5 L min−1 flow of oxygen and a vacuum pump toward a glass fiber filter, where the SnO2 nanoparticles were deposited and collected.2 MeasurementsElectrochemical Reduction of CO: Catalyst inks were prepared by dispersing 5 mg of FSP‐SnO2 catalysts in 0.5 mL deionized water and ethanol solution  followed by the addition of 25 µL of Nafion solution (Sigma‐Aldrich). The sonicated homogenous inks were then drop‐casted on carbon fiber paper (loading: 1 mg cm−2) to yield the working electrodes. The working electrodes were then placed with a saturated calomel reference electrode (SCE) in the cathodic compartment of a customized gas‐tight H‐cell whereas the anodic compartment consisted of a Pt wire as the counter electrode. The two compartments of the H‐cell were separated by a glass frit to ensure that the reduction products from the cathode did not oxidize in the anode. The electrolyte utilized in this study was 0.1 m KHCO3 and prior to every experiment, the cathodic part of the H‐cell was purged with CO2 for duration of 30 min and the pH of the saturated 0.1 m KHCO3 solution was 6.8. All potentials measured in this study were converted to the reversible hydrogen electrode (RHE) reference for the purpose of comparison using the following equation: ERHE (V) = ESCE (V) + 0.245 + 0.059 × pH. The electrochemical measurements were carried out using a CHI 760E  electrochemical workstation. Potentiostatic studies were carried out at various potentials for a duration of 2 h and the experiments repeated and the results presented are the averaged values. For each potential, the results for the first hour of the first testing is discarded as the working electrode required conditioning.Product Detection: The gas phase products were quantified after 3600 and 7200 s during the 2 h electrolysis session using a gas chromatograph  equipped with both thermal conductivity detector (TCD) and flame ionization detector (FID) detectors. To detect the liquid products, 0.5 mL of aliquot was collected at the end of each bulk electrolysis at fixed potentials and was mixed with 0.1 mL of D2O and 7.143 ppm of internal standard dimethyl sulfoxide  and was analyzed using a 600 MHz 1H 1 D liquid NMR spectrometer (Bruker Advance) at 25 °C. The 1D 1H spectrum was measured with water suppression with a pre‐saturation method . The amounts of products were calculated by comparing the integral areas of the observed formate product with that of the DMSO. The peak positions were calibrated using formic acid  dissolved in 0.1 m KHCO3 solution containing the internal standard solution.Physical Characterization: Surface morphology of the catalysts were studied using TEM with a Phillips CM 200 microscopy that was operated at 200 kV. Surface chemical composition was evaluated using X‐ray photoelectron spectroscopy with a Thermo ESCALAB250i X‐ray photoelectron spectrometer. UV–vis absorption spectra of the samples were acquired using a Shimadzu UV‐3600 UV–vis–NIR spectrophotometer with BaSO4 as the reference. The structural characterization was studied using powder X‐Ray Diffraction pattern using PANalytical X'Pert instrument using Cu K∝ radiation (λ = 1.54 Å) and scan range from 10° to 90°. BET isotherms were measured on a Micrometrics Tristar 3030 using nitrogen adsorption at 77 K. Raman spectroscopy was carried out using Renishaw inVia Raman Microscope (514 nm laser). In situ Raman spectra were carried out with FSP‐SnO2‐5 catalyst drop‐casted on glassy carbon electrode and was placed in CO2 saturated 0.1 m KHCO3 solution in the custom cell where SCE reference and Pt counter electrode were utilized. Raman spectra was first obtained when no potential was applied to establish the background. Subsequently, Raman measurements were carried out when the applied potential was −1.5 V (for 300 s) versus RHE to study the change in surface chemical states for the catalyst during CO2RR. The presence of defects was evaluated using electron paramagnetic resonance spectroscopy on a Bruker EMX‐plus X‐Band EPR spectrometer. The EPR measurements were conducted at 9.41 GHz (X‐band) at 120 K with the microwave power set at 2 mW and the modulation amplitude at 5G. Hydrogen temperature programmed reduction measurements were conducted using a Micromeritics Autochem II 2920 instrument to study reducibility of the SnO2 surface and bulk. ≈50 mg of sample was placed into a U‐shaped quartz sample tube, on a plug of quartz wool, and heated to 150 °C under 20 mL min−1 Ar  at a heating rate of 10 °C min−1 and held for 30 min. Following pre‐treatment, the sample was cooled to 50 °C. The TPR analysis was conducted under a 10% H2‐Ar (20 mL min−1) flow with the temperature ramped from 50 to 700 °C at 10 °C min−1. High‐energy X‐ray diffraction experiments were performed at the 11‐IB‐B beamline of the advanced photon source (APS). Powders were loaded into Kapton capillaries and examined using 86.7 keV X‐rays. A sample to detector distance of 220 mm was used to obtain a qmax of ≈32 Å−1, which as calibrated with a reference CeO2 powder. HE‐XRD patterns were background corrected , transformed into reduced structure factors , and Fourier transformed into atomic PDFs using the program RAD.2.2 powders were uniformly spread across Kapton tape and examined using a fluorescence geometry. Spectra were obtained from 200 eV below the Sn K‐edge (29.2 keV) up to 1000 eV past the edge. Subsequent data processing and EXAFS modeling was performed using the Demeter software package.02 value of 0.879 as obtained from modeling a metallic Sn reference foil.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


Haplodiploidy has evolved repeatedly among invertebrates, and appears to be associated with inbreeding. Evolutionary biologists have long debated the possible benefits for females in diplodiploid species to produce haploid sons–beginning their population's transition to haplodiploidy–and whether inbreeding promotes or inhibits this transition. However, little attention has been given to what makes a haploid individual male rather than female, and whether the mechanism of sex determination may modulate the costs and benefits of male haploidy. We remedy this by performing a theoretical analysis of the origin and invasion of male haploidy across the full range of sex‐determination mechanisms and sib‐mating rates. We find that male haploidy is facilitated by three different mechanisms of sex determination–all involving male heterogamety–and impeded by the others. We also find that inbreeding does not pose an obvious evolutionary barrier, on account of a previously neglected sex‐ratio effect whereby the production of haploid sons leads to an abundance of granddaughters that is advantageous in the context of inbreeding. We find empirical support for these predictions in a survey of sex determination and inbreeding across haplodiploids and their sister taxa. This article deals with an important outstanding question: why is reproduction, an essential feature of all living systems, so variable across the tree of life? Haplodiploidy––an unusual reproductive system in which females develop from fertilised (diploid) eggs in the usual way but males develop from unfertilised (haploid) eggs–is one of the most common alternative reproductive modes among animals, and has attracted huge and sustained attention from evolutionary biologists for decades, particularly in relation to its association with eusocial insect societies.Evolutionary biologists have long debated the possible benefits for females in diplodiploid species to start producing haploid sons, and thereby begin their population's transition to haplodiploidy. However, little attention has been given to what makes haploid individuals male rather than female in the first place, and how the mechanism of sex determination affects the costs and benefits of male haploidy. Moreover, there has been a longstanding debate as to whether inbreeding tends to promote or inhibit the evolution of haplodiploidy, with the majority view tending to favour an inhibitory role for inbreeding, on theoretical grounds, despite an apparently strong positive association between inbreeding and haplodiploidy in nature.We address these problems by means of a theoretical analysis of the evolution of male haploidy across the full range of sex‐determination mechanisms and inbreeding rates. This reveals that male haploidy is facilitated by three different mechanisms of sex determination and is impeded by others, and that inbreeding does not pose the evolutionary barrier that had previously been imagined. We also conduct an empirical survey of all known independent origins of haploidiploidy, finding support for our theoretical predictions.Sexual reproduction, whereby each offspring receives genetic material from two parents, is by far the most common mode of reproduction across the animal kingdom. Yet, in a sizeable minority of extant animal species ∼12%), whilst daughters are produced sexually and receive a genome from both their mother and their father, sons are produced asexually from unfertilised eggs and have just one haploid genome that they derive from their mother Normark . Such ha2%, whilsThe transition from standard diplodiploid sexual reproduction to haplodiploidy requires that some mothers start producing haploid sons, and a long‐running strand of evolutionary theory has explored the possible benefits of doing so Table . As the However, whilst existing theory has considered the costs and benefits of producing a haploid male, relatively little attention has been given to the circumstances under which haploid individuals are expected to develop as males as opposed to females in the first place. That is, the sex determination (SD) mechanism employed by a given diplodiploid species may dictate whether it is even possible for that species to transition to haplodiploidy, irrespective of the costs and benefits involved. Moreover, whilst existing theory frames the mother's decision in terms of producing haploid sons versus diploid sons, of more relevance might be the tradeoff between producing haploid offspring versus diploid offspring, with the mother having little direct control of their sex. For example, if diploid offspring are equally likely to develop as sons or daughters, the production of haploid males might come at the expense of biasing the overall sex ratio toward males, with potential consequences for sex‐ratio selection. Furthermore, the SD mechanism will likely influence the sex of a haploid male's own offspring. For example, under classic haplodiploid inheritance, haploid males father only daughters and not sons. This too may have consequences for sex‐ratio selection, particularly in the context of an inbreeding lifestyle that favours female‐biased sex allocation.Here, we investigate the origin and evolutionary invasibility of haploid males in diploid populations for a range of different SD mechanisms and sib‐mating rates. First, we identify which SD mechanisms–including different types of male (X0 and XY) and female (ZW and Z0) heterogamety–lead haploid individuals to develop as males versus females, with reference to published data on the molecular mechanisms underpinning SD in different taxonomic groups. Second, for those SD mechanisms that do robustly allow for the production of haploid males, we develop mathematical population models to quantify the likelihood of invasion of male haploidy from rarity, as a function of sib‐mating rate. Third, we compare the resulting theoretical predictions with the scant empirical data that exist in relation to mechanisms of SD across haplodiploids and their sister taxa, and consider the implications of our theoretical analysis for the longstanding puzzle as to whether inbreeding preceded or followed the evolution of haplodiploidy.First, we investigate how the SD mechanism employed in a diploid population will determine the sex of rare haploid individuals developing from unfertilised eggs. We exclude from our analysis those mechanisms of SD that are “environmental”, that is, where the haploid individual's genomic constitution does not determine its sex, but consider the full range of known–and unknown but feasible–“genetic” mechanisms of SD. Second, narrowing our attention to those SD mechanisms under which haploid individuals robustly develop as males, we investigate the invasibility of male haploidy in an evolutionary, population model.a the probability that any two eggs chosen at random from the same patch were laid by the same foundress. In a departure from Gardner and Ross's .The larger the viability cost c* when sex is determined by X‐chromosome count or a paternal‐origin X‐linked feminizer as follows (see Methods for model assumptions). Consider a population in which foundresses who produce haploid sons are vanishingly rare, and fasten attention upon a focal foundress who leaves a proportion δ of her eggs unfertilized. If the total number of eggs produced by all foundresses on her patch is N and her eggs constitute a proportion x of this total, then she gives rise to m1 = Nxδ(1‐c) viable haploid sons, m2 = Nx(1‐δ)/2 diploid daughters and m3 = Nx(1‐δ)/2 diploid sons, whilst her cofoundresses collectively produce m4 = N(1‐x)/2 diploid daughters and m5 = N(1‐x)/2 diploid sons. All of the female offspring produced on this patch experience exactly the same mating environment, with a proportion α = m1/(m1+m3+m5) of their mates being haploid sons of the focal foundress, a proportion β = m3/(m1+m3+m5) of their mates being diploid sons of the focal foundress, and a proportion γ = m5/(m1+m3+m5) of their mates being diploid sons of the cofoundresses.We derive the potential for male haploidy Ny and the number of eggs that her cofoundresses produce N(1‐y). This daughter of the focal foundress produces n1 = Nyα diploid daughters fathered by her haploid brothers, n2 = Nyβ/2 diploid daughters fathered by her diploid brothers, n3 = Nyβ/2 diploid sons fathered by her diploid brothers, n4 = Nyγ/2 diploid daughters fathered by unrelated diploid males and n5 = Nyγ/2 diploid sons fathered by unrelated diploid males, whilst the other foundresses on her patch collectively produce n6 = N(1‐y)/2 diploid daughters and n7 = N(1‐y)/2 diploid sons. The ratio of females to males on the patch is therefore F = (n1+n2+n4+n6)/(n3+n5+n7).Now consider a patch founded by a daughter of the focal foundress, and denote the number of eggs that she produces Nz and the number of eggs that her cofoundresses produce N(1‐z). This daughter of a cofoundress of the focal foundress produces n8 = Nzα diploid daughters fathered by haploid sons of the focal foundress, n9 = Nzβ/2 diploid daughters fathered by diploid sons of the focal foundress, n10 = Nzβ/2 diploid sons fathered by diploid sons of the focal foundress, n11 = Nzγ/2 diploid daughters fathered by diploid sons of a cofoundress of the focal foundress and n12 = Nzγ/2 diploid sons fathered by diploid sons of a cofoundress of the focal foundress, whilst the other foundresses on her patch collectively produce n13 = N(1‐z)/2 diploid daughters and n14 = N(1‐z)/2 diploid sons. The ratio of females to males on the patch is therefore G = (n8+n9+n11+n13)/(n10+n12+n14).Finally, consider a patch founded by a daughter of a cofoundress of the focal foundress, and denote the number of eggs that she produces H = E(m2(n1p1 + n2p2 + n3F p3 + n4p4 + n5F p5 + n6p6 + n7F p7) + m4(n8p8 + n9p9 + n10G p10 + n11p11 + n12G p12 + n13p13)), where p denotes the consanguinity of the focal foundress to each type of grandoffspring and E denotes an expectation taken over the values x, y, and z. The consanguinities are given by p1 = (3+5f)/8, p2 = (1+3f)/4, p3 = (1+3f)/4, p4 = (1+3f)/8, p5 = (3+5f)/8, p6 = 0, p7 = 0, p8 = (1+f)/4, p9 = (1+3f)/8, p10 = (1+3f)/8, p11 = 0, p12 = 0, and p13 = 0, where f is the consanguinity between mating partners. Note that f = a((1/4) × (1+f)/2 + (1/2) × f + (1/4) × f), which rearranges as f = a/(8‐7a). Also note that E(x) = E(y) = E(z) = ∑i= 1T(1/T)ix = σ and E(x2) = E(y2) = E(z2) = ∑i=1T(1/T)ix2 = τ, where ix denotes the proportion of a patch's total egg production that derives from the ith foundress among the T foundresses that contribute eggs to the patch, and that τ/σ = a.The inclusive fitness Hamilton  of the fH/dδ > 0, decreases her inclusive fitness by increasing her investment into haploid sons if dH/dδ < 0, and breaks even if dH/dδ = 0. Accordingly, the threshold viability cost c* that defines the potential for male haploidy satisfies dH/dδ|c=c* = 0, and is given by c* = (2‐4a+5a2‐2a3)/(4‐5a+5a2‐2a3), as reported in the Results.The focal foundress increases her inclusive fitness by increasing her investment into haploid sons if dTo empirically assess our key theoretical predictions, we survey sex chromosome karyotype data available on the “Tree of Sex” database  diploid individuals carrying a maternal‐origin Zm chromosome have karyotype ZpZm and would be male whereas diploid individuals lacking a maternal‐origin Zm chromsome have karyotype Zp0 or ZpWm and would be female. Half of the haploid individuals will have karyotype Zm and hence would be male, but the other half will have karyotype 0 or Wm and hence would be female .Fourth, parent‐of‐origin effects could in principle determine sex Fig. . Whilst Apis mellifera), “femaleness” is determined by heterozygosity at the csd locus directing female‐specific splicing of the fem gene , the potential for male haploidy is c* = ½, recovering Bull's (a < 1) leads to a slightly reduced potential for male haploidy ; and under full sib‐mating (a = 1), the potential for male haploidy is c* = ½. The complexity of the X‐linked CSD scenario means that it resists analytical solution, but numerical analysis–performed under the assumption that males homozygous at the SD locus are inviable –reveals a very similar U‐shaped relationship: in the extremes of absent or full sib‐mating (a = 0 or 1), the potential for male haploidy is c* = ½, while an intermediate level of sib‐mating (0 < a < 1) leads to a reduced potential for male haploidy  assumes a patch‐structured diplodiploid population, in which the rate of sib‐mating is described by a parameter g Bull's  predictia = 1), a mother gains no transmission advantage from haploid male production , nor does she lose grandoffspring owing to haploid‐male inviability among her sons (since her surviving sons inseminate her daughters just as surely). Instead, leaving some eggs unfertilized reduces her production of daughters  while increasing the rate at which her remaining daughters produce daughters of their own (since her haploid sons only sire daughters), and these two effects exactly cancel when c = 1/2 . That is, inbreeding does not necessarily inhibit the evolution of male haploidy.Why is the potential for male haploidy under full inbreeding exactly the same as in a fully outbred population? Under full sib‐mating , but that they may do so under some forms of male heterogamety (i.e. X0 and XY systems)–which might itself be viewed as a partial form of male haploidy, as males are haploid with respect to their X‐linked genes. Despite the scarcity of relevant data, we have found support for this theoretical prediction in our empirical survey.Drosophila males that lack a Y chromosome are viable but sterile . Sadly, there appears to be no information about the actual mechanism of SD in any X0 species (Beukeboom and Perrin  Bridges ). RecentHaving narrowed the space of possibilities to three SD mechanisms that lead haploid individuals to develop as males, we considered the subsequent evolutionary dynamics with a particular focus on how the invasion success of haploid males is modulated by sib‐mating. The haploid condition is initially expected to be associated with substantial viability costs–as recessive deleterious mutations are exposed in haploid males and male fertility is likely reduced during the transition from meiotic to mitotic spermatogenesis–and so there has been a longstanding search for compensating benefits. Previously, the maternal‐transmission‐advantage hypothesis has emphasised the benefit accruing to a female who produces a haploid as opposed to a diploid offspring owing to her monopolising her offspring's reproductive value, rather than sharing half of this with her mate, and this benefit diminishes when mating partners are genetically related Brown . Howeverc < c* for male haploidy to be evolutionary favoured either by altering the threshold viability cost c*  or else by altering the viability cost c itself (as explored in the simulation study of Smith Our analysis reveals how these two opposing effects of inbreeding quantitatively balance out in the context of X‐chromosome counting, a paternal‐origin X‐linked feminizer or X‐linked CSD. We have found that, in contrast to the expectation of many researchers who have investigated this topic, inbreeding does not necessarily inhibit the evolution of male haploidy, and indeed male haploidy appears to evolve just as readily under full sib‐mating as it does in a fully outbred population. One aspect of inbreeding not explicitly captured in our model is the concomitant purging of recessive deleterious alleles, which could reduce the viability cost incurred by haploid males, further promoting the transition to haplodiploidy. That is, inbreeding could alter the stringency of the condition Associate Editor: K. LythgoeSupplementary MaterialClick here for additional data file.Table S1. Overview of all taxonomic groups with haplodiploidy and their diploid sister clades.Click here for additional data file.