Nuclear DNA is the target responsible for anticancer activity of platinum anticancer drugs. Their activity is mediated by altered signals related to programmed cell death and the activation of various signaling pathways. An example is activation of nuclear factor kappaB (NF-κB). Binding of NF-κB proteins to their consensus sequences in DNA (κB sites) is the key biochemical activity responsible for the biological functions of NF-κB. Using gel-mobility-shift assays and surface plasmon resonance spectroscopy we examined the interactions of NF-κB proteins with oligodeoxyribonucleotide duplexes containing κB site damaged by DNA adducts of three platinum complexes. These complexes markedly differed in their toxic effects in tumor cells and comprised highly cytotoxic trinuclear platinum(II) complex BBR3464, less cytotoxic conventional cisplatin and ineffective transplatin. The results indicate that structurally different DNA adducts of these platinum complexes exhibit a different efficiency to affect the affinity of the platinated DNA (κB sites) to NF-κB proteins. Our results support the hypothesis that structural perturbations induced in DNA by platinum(II) complexes correlate with their higher efficiency to inhibit binding of NF-κB proteins to their κB sites and cytotoxicity as well. However, the full generalization of this hypothesis will require to evaluate a larger series of platinum(II) complexes. This cob = 0.023, 0.045, or 0.091 [rb is defined as the number of molecules (not platinum atoms) of the platinum complex bound per nucleotide residue]. The samples containing these duplexes were incubated with NF-κB proteins  at protein to duplex molar ratio of 10. To avoid nonspecific interactions, an excess of unplatinated synthetic double-helical poly(dI-dC).poly(dI-dC) was also added to the reactions as the nonspecific competitor. Samples were then analyzed by native PAA gel electrophoresis (PAGE)  and the DUPLEX-κB globally modified by BBR3464 and by cisplatin or clinically ineffective transplatin determined previously under identical experimental conditionsThe experiments described in the preceding section were performed with purified recombinant proteins p50 and p65 in a cell-free medium. To correlate with cellular behavior, binding of native NF-κB present in whole cell extract to DNA containing the Ig-κB site modified by BBR3464 was also examined. Native NF-κB proteins present in nuclear extracts consist of both Rel and non-Rel subunits that comprise multiple protein complexes with different gene activation specificities24This result can be interpreted to mean that the complex between oligonucleotide duplex containing consensus site and native NF-κB proteins was formed. The fact that identical, but cold (unlabeled) oligonucleotide duplex was able to compete away the induced bands confirmed the specificity of these bands. Moreover, an additional shift was observed when antibodies against the p50 and p65 subunits of NF-κB were added, which also confirmed the specificity of the bands corresponding to the DNA-protein complexes. The changes in mobility also indicated that the upper two more slowly migrating bands contained p65/p65 homodimer and p50/p65 heterodimer consistent with previous reports24b = 0.023, 0.045 or 0.091 reduced the yield of the DNA-protein complexes formed as a results of incubation with the whole cell extract stimulated by IL-1α  globally modified by BBR3464 and by cisplatin or clinically ineffective transplatin determined previously under identical experimental conditionsFurther experiments were aimed at examining binding of NF-κB proteins to the 22-bp oligonucleotide duplex lacking the consensus κB site  technology. SPR spectroscopy makes possible real-time detection and the determination of binding kinetics and affinities of biomacromolecular interactions27To delineate more precisely the binding properties of NF-κB proteins with DNA containing κB-sites modified by platinum complexes including information on the dynamics of binding of NF-κB proteins, we have carried out binding studies using We studied binding of p50/p50 homodimer, as a representative of NF-κB proteins, which binds specifically to sites recognized by NF-κBThe biotin-labeled DNA duplexes [DUPLEX-κB(SPR-BIO) or Scrambled DUPLEX(SPR-BIO)] were first immobilized on the surface of SA chip which was coated with streptavidin. The p50/p50 homodimer samples were then injected and flowed through the channels on the chip and interacted with the bound duplexes. The details of these procedures are described in the section Materials and methods. Upon injection through the SPR flow cell, the protein solution is replenished within the system so the protein concentration effectively remains constant at the initial value. When the association phase is long enough the binding reaction reaches equilibrium. There is a linear relationship between the amount of bound material and response units (RU).The typical sensorgrams are shown in The sensorgrams were analyzed by BIAevaluation 3.1 software. After blank subtraction, values of response units (RU) at 400 s were read and plotted against protein concentration . At protPlatination of DUPLEX-κB(SPR-BIO) resulted in a decrease of the plateau level indicating inhibition of the formation of the complex between p50/p50 homodimer and DUPLEX-κB(SPR-BIO). This decrease was most pronounced when the duplex was modified by BBR3464 (decrease to 21%) whereas modification of the duplex by ineffective transplatin was only small (decrease to only 88%) . Thus, tBinding of p50/p50 homodimer to the Scrambled DUPLEX(SPR-BIO) (lacking the consensus κB site) which was modified by BBR3464, cisplatin or transplatin under identical experimental conditions as described for DUPLEX-κB(SPR-BIO) was examined as well. After immobilizing the biotinylated duplex Scrambled DUPLEX(SPR-BIO) on the surface of SA chip coated with streptavidin, p50/p50 homodimer was injected over the surfaces. No increase of RU typical for the association sensorgrams was noticed, which indicates that complex between the NF-κB protein and DNA duplex lacking κB site was not formed. Thus, also these results of SPR spectroscopic analysis demonstrate that adducts of BBR3464 formed on DNA do not represent a structural motif which is recognized by NF-κB proteins.KD and Gibbs free energy change (ΔG0310)  by platinum complexes tested in this work reduced the duplex thermodynamic stability. The efficiency of DNA adducts formed by transplatin, cisplatin and BBR3464 to reduce the thermodynamic stability differed; the trend was transplatin < cisplatin < BBR3464.The SPR sensorgrams were further analyzed to estimate the association and dissociation rate constants as well as the dissociation equilibrium constant (ΔG0310) . InspectExamination of the association and dissociation phases of SPR sensorgrams shows that the kinetic parameters responsible for the affinity decrease are different for duplexes containing κB sites [DUPLEXes-κB(SPR-BIO)] and modified by transplatin, cisplatin or BBR3464 b = 0.023, 0.045, or 0.091, i.e. 2.3, 4.5, or 9.1 molecules of the platinum complex was bound per 100 base residues in average. Considering a probability of distribution of the platinum molecules bound to the duplex, the samples used for these experiments could contain also a fraction of unplatinated molecules (as well as a certain fraction of oligonucleotide molecules bearing more than one platinum adduct), which could affect the resulting response. Therefore, the sample of the oligonucleotide duplex (DUPLEX-κB) containing a single adduct of BBR3464 and no unplatinated duplexes was prepared and tested for its inhibitory effect. The bottom strand of κB-site containing oligonucleotide duplex  in the dark for 24 h. The platinated oligonucleotide was purified by HPLC.The oligonucleotide duplexes used in the EMSA experiments described in 4 (0.1 M). The resulting duplex was further purified on 12% native PAA gel so that the sample only encompassed oligonucleotide duplexes which contained just 1 adduct of BBR3464 . On the other hand, it cannot be excluded that these samples also contained a small fraction of duplexes in which the single adduct was formed outside the κB site. An EMSA experiment was performed with this sample under the same experimental condition as described for globally modified oligonucleotides; concentration of the oligonucleotide probe was 1 nM and the concentration of p50/p50 was 10 nM. For other details, see the section Materials and methods.The HPLC profile of bottom strand of κB-site containing oligonucleotide is shown in Similarly to the globally modified κB-site containing oligonucleotide , presencTo determine the effect of BBR3464 modification of the кB site on the DNA binding activity of NF-кB proteins in cells, the decoy strategy has been employed as already described in our recent studyb = 0.021, the decoy activity was inhibited by 73% 293 cell line containing a stably integrated luciferase-reporter gene driven by a NF-кB-responsive promoter with multiple κB elements was transfected with platinated or nonplatinated dumbbells and luciferase expression was measured in these transfected cells. The specific inhibition of transcriptional activity of NF-кB in HEK293 cells by the DUMBBELL-κB decoy was confirmed in the same way as described in our previous reportd by 73% . These din cellulo experiments were in full agreement with the conclusions drawn on the basis of the experiments performed in cell-free media with recombinant p50 or p65 proteins or with whole cell extracts prepared from the stimulated cells in which NF-κB signal transduction pathway was activated.In the present investigation, electrophoretic mobility shift assay and SPR spectroscopy were used to examine the binding affinity of NF-κB proteins to DNA duplexes containing κB site which was damaged by DNA adducts of antitumor trinuclear platinum(II) complex BBR3464. The effects of these DNA adducts were compared with those of less cytotoxic cisplatin and ineffective transplatin. We observed a strong inhibitory effect of DNA adducts of BBR3464 on the binding affinity of native NF-κB proteins or NF-κB proteins reconstituted from purified p50 and p65 proteins. Inhibition of the formation of the complex between NF-κB proteins and DNA duplexes containing κB site by DNA adducts of cisplatin or antitumor-inactive transplatin was significantly less, being least effective for transplatin. Examination of the association and dissociation phases of SPR sensorgrams yielded the kinetic parameters responsible for the decrease of affinity of NF-κB proteins to platinated κB sites in DNA. These parameters indicated that this decrease was related mainly to variation of the association step. Importantly, the effect of modification of the кB site by BBR3464, cisplatin or transplatin on the DNA binding activity of NF-кB proteins in cells was also studied employing the decoy strategy. The results of these The κB sites in DNA contain several sites at which adducts of platinum complexes tested in the present work are formed. In the κB sites, the platinum complexes form adducts, which disturb DNA secondary structure. We suggest that the result of these perturbances is that the tuned steric fit required for the formation and stability of the complex of NF-κB protein with the κB site cannot be attained so that NF-κB protein binds to its platinated consensus sequence in DNA (κB site) with a reduced affinity. Transplatin, cisplatin and BBR3464 form discrete adducts in DNA which disturb DNA secondary structure to a different extent2132in vitro and in cellulo studies thus demonstrate that affinity of NF-κB proteins to DNA modified by the bifunctional PtII complexes tested decreases with their growing cytotoxic effectiveness. Our findings suggest that mechanisms of toxicity of antitumor platinum complexes in tumor cells may involve interference with NF-κB signaling pathways leading to its antiapoptotic effects. This interference might be connected with efficiency of platinum drugs to form on DNA consensus sequences of these transcription factors the adducts which effectively distort conformation DNA. The results of this work are consistent with the hypothesis that a control of protein recognition and pathways leading to cell death by properly designed DNA adducts may be an interesting approach to predict more efficient anticancer metallodrugs. The confirmation of this hypothesis and its full generalization will require to evaluate a larger series of platinum(II) complexes.These It can be expected that in a near future new transition metal-based compounds will be designed, synthesized and tested for their biological effects, which will be able to form on DNA the adducts distorting conformation of this nucleic acid still more than DNA adducts of conventional antitumor platinum compounds. It is also reasonable to predict that DNA adducts of these new complexes will inhibit binding of NF-κB protein to its κB site more in comparison with the adducts of platinum compounds so far tested and concomitantly antitumor effects of these new complexes will be potentiated.32P]ATP was from Amersham . Streptavidin coated SA sensor chips were from BIAcore AB . T4 DNA ligase and Exonuclease III were from New England Biolabs .BBR3464   was prep33luc2P HEK293 cell line was obtained from Promega . Cells were grown at 37 °C with 5% CO2 in DMEM supplemented with fetal bovine serum (10%), hygromycin B (50 μg mL−1), penicillin (100 U mL−1) and streptomycin (100 μg mL−1).Stable human NF-κB-RE-32P ATP; T4-polynucleotide kinase). The unincorporated γ-32P ATP was disposed using Sephadex G-50 fine columns. To anneal the radiolabeled top strands with equimolar amounts of corresponding bottom strands the samples were heated to 65 °C for 10 min in a solution containing Tris.HCl , MgCl2 (10 mM), and DTT (1 mM), and then allowed to cool down to 4 °C for 4 h. It was verified by loading the samples onto 15% native PAA gels that they contained no non-annealed single-stranded oligonucleotide. It is to be understood that in the following text, the molar concentrations of the single-stranded oligonucleotide or oligonucleotide duplexes are related to the oligomers or double-stranded molecules, respectively (not to the monomer content).If not stated otherwise, the top strands of the duplexes used in the electrophoretic mobility shift assay (EMSA) experiments were radioactively labeled at their 5′-end (γ-4 (10 mM) at 37 °C for 24 h in the dark, followed by precipitation by ethanol to remove free, unbound platinum. The levels of modification (average number of platinum molecules coordinated per nucleotide residue) were determined using FAAS and UV spectrophotometry.Oligonucleotide duplexes were platinated in NaClO−1), NP-40 (1%), glycerol (5%), poly(dI-dC).poly(dI-dC) (4.1 × 10−5 M related to the phosphorus content), radiolabeled oligonucleotide duplex , purified proteins p50/p50 (0.2 pmol), p65/p65 (0.3 pmol), heterodimer p50/p65 (0.3 pmol) or IL-1α stimulated HeLa whole-cell extract . To form the p50/p65 heterodimer equal amounts of p50 and p65 were incubated at 37 °C for 60 min . Following the incubation at 20 °C for 45 min, the samples were loaded onto 6% PAA gels in 0.5xTris-borate-EDTA . The electrophoresis was run under the same conditions for 1.5 h and the gels were dried and exposed to a molecular imaging plate. The autoradiograms were recorded using a FUJIFILM bioimaging analyzer and processed with AIDA image analyzer software.EMSAs were performed as described in several previously published papers1036The following equation was employed to calculate the percentage of DNA bound to proteinOligonucleotide duplex bound (%) = [protein-DNA complex/total DNA] × 100% = [protein-DNA complex/(free DNA + protein-DNA complex)] × 100%4 (0.02 M) at 65 °C for 10 min and then allowed to cool down to 4 °C for 4 h. These duplexes, which contained no biotinylated part of the bottom strand, were then incubated with BBR3464, cisplatin or transplatin in the ratio of one molecule of the platinum complex per one molecule of the duplex for 24 h. The platinated duplexes were subsequently exhaustively dialyzed to remove any residual platinum complexes which were not coordinatively bound to the duplex. The platinated duplexes were annealed with 3′-end biotinylated bottom strand (12-mer) in Tris.HCl (10 mM) and NaCl (100 mM) for 4 h at 4 °C. An excess of duplex was used at this step so that no biotinylated single-stranded oligonucleotides remained in the mixture. This precaution was used to prevent streptavidin coated chips to bind to single-stranded biotinylated oligonucleotides.The oligonucleotide duplexes were prepared as schematically shown in −1. The injection was stopped when the amount of bound DNA corresponded to the signal increase of 50 RU. The overall increase in signal for all samples ranged from 50 to 52 RU. The protein p50 was injected at a flow rate of 20 μLmin−1 in running buffer . Two washes of 10 μL of running buffer containing SDS (0.04%) were used to regenerate the surface after each injection of p50. Binding of each duplex was assayed at concentrations of p50 ranging from 0 to 200 nM. To obtain kinetic parameters, late association phases and early dissociation phases of the resulting sensorgrams were fit to the Langmuir binding model using BIAevaluation software (version 3.1). After blank subtraction RU values at 400 s  were read and plotted against protein concentration.SPR measurements were performed on a BIAcore 3000 at 20 °C using a streptavidin coated chips and oligonucleotide DNA duplexes containing a 3′-biotin on the bottom strand. Biotinylated oligonucleotide constructions were bound to the streptavidin-coated SA sensors by injecting Tris.HCl , EDTA (3 mM), NaCl (0.63 M), glycerol (2%), surfactant P-20 (0.005%) and 1 nM biotinylated duplexes at a flow rate of 10 μLminHow to cite this article: Brabec, V. et al. Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes. Sci. Rep.6, 28474; doi: 10.1038/srep28474 (2016).


This requires the design of alloys which allow for a very high H/M ratio. Transition metal hydrides typically have a maximum H/M ratio of 2 and higher ratios can only be obtained in alloys based on rare-earth elements. In this study we demonstrate, for the first time to the best of our knowledge, that a high entropy alloy of TiVZrNbHf can absorb much higher amounts of hydrogen than its constituents and reach an H/M ratio of 2.5. We propose that the large hydrogen-storage capacity is due to the lattice strain in the alloy that makes it favourable to absorb hydrogen in both tetrahedral and octahedral interstitial sites. This observation suggests that high entropy alloys have future potential for use as hydrogen storage materials.Metal hydrides (MH Consequently, there is a need for new concepts to identify more efficient hydrogen storage alloysHydrogen has a strong potential for use as an alternative fuel provided that it can be stored in a safe and efficient way. One possibility is to store hydrogen as a solid hydride using suitable metals or alloys. Metal hydrides have been widely studied as storage materials but most alloys are unable to fulfil the requirements of a competitive hydrogen storage unit that can be exploited in practical applications. A problem with hydrides based only on transition metals is their limited capacity with an H/M ratio ≤ 2. Metal hydrides used for applications today  suggest a significant lattice distortion that could favour a more efficient hydrogen storage capacity. The aim of the present work has been to investigate hydride formation in TiZrHfNbV and to evaluate its potential for use as a hydrogen storage material.Equimolar TiVZrNbHf samples were synthesized by arc-melting stoichiometric amounts of Ti , V , Zr . Nb  and Hf . To ensure homogeneity, the sample was re-melted five times and turned over between each melting step. Oxygen contamination was minimized by flushing the furnace three times with high purity Ar and by melting a Ti getter for 5 minutes prior to each cycle of alloy melting. The total weight loss from the sample was less than 1%. The chemical composition and microstructure was studied using a Zeiss 1550 scanning electron microscope (SEM) equipped with a secondary electron (SE) detector, a backscatter electron (BSE) detector and an energy dispersive X-ray spectrometer (EDS).x was synthesized by subjecting powdered TiVZrNbHf  to 20 bar H2 at 400 °C for 48 h in an alumina crucible placed in a purpose built high pressure furnace. The chamber was flushed three times with H2 prior to the hydrogenation experiments. The mass of the samples were measured carefully before and after hydrogen absorption to calculate the storage capacity. The pressure-composition-isotherm (PCT) measurements were performed on the TiVZrNbHf in powder form using the SETARAM PCTPRO volumetric instrument. The absorption isotherm at 299 °C (±1) was recorded by stepwise increase of hydrogen pressure up to around 50 bar. The absorption kinetics is quite slow and the equilibrium time is set to the maximum 10 h per isotherm point. The hydrogen sorption performances were repeatable under similar pressure and temperature conditions. The thermal desorption spectroscopy (TDS) measurements have been performed by the help of a homemade instrument coupled to a quadruple mass spectrometer (MKS MicroVision Plus RGA) working under high vacuum. A constant heating ramp of 5 °C/min has been used.(TiVZrNbHf)Hx was performed with X-ray powder diffraction (XRD) with either a Bruker D8 Advance equipped with a Lynx-eye XE position sensitive detector using CuKα radiation or a Bruker D8 using a Johansson monochromator (CuKα1) equipped with a Lynx-eye detector. The samples for diffraction were prepared by sieving powder onto zero background single crystal silicon sample holders. The structure of the sample as-synthesized was refined from the X-ray diffraction data using the Rietveld methodth order Chebychev polynomial and a Thompson-Cox-Hastings pseudo-VoigtStructural characterization of TiVZrNbHf and (TiVZrNbHf)HAs can be seen from In the gravimetric measurements made using the high pressure furnace, the TiVZrNbHf sample absorbed hydrogen easily above 200 °C. This was observed as a decrease in the hydrogen pressure in the reactor. Treatment at 400 °C for 48 h yielded a completely hydrogenated sample with a BCT lattice (a = 3.2183(4) Å, c = 4.6556(16) Å) , right. 2) at 299 °C (see 2), which is equivalent to 2.7 wt% hydrogen.Isothermal hydrogen absorption showed that TiVZrNbHf absorbs hydrogen readily with a plateau pressure of 0.1 bar (H9 °C see . The plaHydrogen desorption was investigated using TDS and it shows that hydrogen readily desorbs (with a maximum in pressure) at temperatures above 400 °C  when heaThe results presented confirm our hypothesis that an HEA can have excellent hydrogen absorption properties. It is striking that the hydrogen content in this alloy is significantly higher than in any of the binary hydrides of the constituent elements141516172 and ZrH2, the hydrogen is placed in tetrahedral interstitial sites. The observed H/M ratio of 2.5 in the hydrogenated TiVZrNbHf alloy requires that both tetrahedral sites and about 50% of the octahedral sites are filled with hydrogen. This behaviour is unique and has never been observed before in pure transition metal hydrides. Alloys with both transition metals and rare earth (RE) metals (lanthanides and yttrium) are known to form hydrides with H/M > 2 but our results indicate that using an HEA could be a feasible and useful strategy to avoid inclusion of RE elements in hydrogen storage materials.In binary hydrides with a cubic close packed (FCC) structure such as TiH2). However for intermediate hydrogen content, a distorted BCC phase has been observed in many transition metal-hydrogen systems (BCC → distorted BCC (BCT) → FCC)3192 phase with hydrogen in the tetrahedral sites. In the case of Ti, Zr and Hf, a tetragonal distortion of the FCC lattice (with c/a < 1) is observed for H concentrations above the critical values but with H/M < 2et al.2 lattice using electronic structure calculations. They found a stronger Zr-Zr bond in the FCT lattice compared to the FCC lattice that is explained by a planar type crystal field splitting in the FCT lattice and occupancy of the degenerate dyz and dxz orbitals.The results above clearly show that the TiVZrNbHf alloy has a different behaviour to that of ‘normal’ transition metals. The archetypical structural transition upon creating a hydride is the formation of a FCC lattice in the fully hydrogenated form .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


DYSFUNCTIONAL TAPETUM1 (DYT1) was expressed in tapetum preferentially. Previous study had confirmed that a “CTCC” sequence within DYT1 promoter was indispensable for correct DYT1 expression. However, precise analysis on the function of each nucleotide of this sequence still lacks. Here we employed site mutation assay to identify the function roles of the nucleotides. As a result, the “T” and final “C” of “CTCC” were found essential for the temporal and spatial specificity of DYT1 expression, whereas the other two “C” nucleotides exhibited substitutable somewhat. The substitutes of two flanking nucleotides of “CTCC,” however, hardly affected the normal promoter function, suggesting that the “CTCC” sequence as a whole did meet the standard of a canonical cis-element by definition. In addition, it was found that as short as 497 bp DYT1 promoter was sufficient for tissue-specific expression, while longer 505 bp DYT1 promoter sequence was sufficient for species-specific expression.As a core regulatory gene of the anther development,  Arabidopsis anther during meiosis is a four-lobed structure comprised of concentric outer epidermis, endothecium, middle fibrous layer, tapetum and pollen mother cell , DEFECTIVE IN TAPETAL DEVELOPMENT AND FUNCTION1 (TDF1), MYB103/MYB80, ABORTED MICROSPORE (AMS), MALE STERILITY1 (MS1), etc.  abolished DYT1 expression completely at the anther stage 6, suggesting that the “CTCC” sequence was indispensable for normal DYT1 expression  cell suspensions to identify which regions of DYT1 promoter were essential for tissue, and further species specificity of DYT1 expression.In our previous study, it had been confirmed that as short as 513 bp sequence in front of the transcription start site (TSS) of Arabidopsis thaliana ecotype Col-0 was used in all of the transformation and promoter analysis in this study. The plants were cultivated under 16 h light/8 h dark photoperiod with 300 Es−1m−2 illumination intensity, at 22 ± 1°C. The seeds were stratified at 4°C for 4 days prior to growth.Nicotiana tabacum L. cv Bright Yellow 2, BY2) was cultivated in a modified liquid Murashige and Skoog (MS) medium  primers with the sequence listed in Table Total RNA was extracted from the transgenic BY2 cell suspension and performed reverse transcription according to Zhou et al. . Then GFAnthers were stripped and collected from transgenic plants flower bud just around male meiosis (anther stage 4–9) on a microscopy slides. Added one drop of sterile water on the anthers and covered a slide carefully without squeezing. Then the sample was observed and photographed under Zeiss LSM-710 confocal microscope  and Leica DM2500 fluorescence microscope. As for semi-quantification of the fluorescence intensity, randomly 10 sites on fluorescence images were selected and the intensity was measured and normalized by the SMART software. Statistics of at least 15 anthers per line, 10 independent T1 generation transgenic lines were counted for each construct transformation. As for BY2 cell suspension, at least 100 cells per callus ancestor were observed, and 10 callus were counted for each construct transformation.DYT1 promoter could faithfully regenerate the temporal and spatial profile of native DYT1 expression , and the first and third nucleotide substitutes from “C” to “T” in the “CTCC” imposed no effect on the expression pattern of GFP , but also in the connective and epidermis tissues  expression profile, whereas the nucleotide replaces just out of “CTCC” imposed little effect on either tissue or species specificity, confirmed that the “CTCC” sequence did satisfy the definition of a canonical cis-element, and play as the core role in determining DYT1 expression profile. Furthermore, with more “CTCC” flanking sequences truncated from DYT1 promoter, the extent of gene expression specificity became weaker, reflected in the facts that the species specificity was lost firstly, then the expression region extended from the central locule to the connective tissue and epidermis in the Arabidopsis anther, resulting in a constitutive pattern at last. Thus it was suggested that DYT1 promoter was a functional unit comprised of multiple parts whose absence would lead to expression specificity attenuation, from both species and tissue-specific to only tissue-specific, and finally to constitutive. In other words, the core motif “CTCC” and its flanking sequences need work together to restrict the driven gene expressed precisely in specific tissues, and furthermore in specific species.The findings of this study that the substitutes of the “T” and final “C” nucleotides in the “CTCC” sequence dramatically changed the driven gene  which recognizes and binds to the “CTCC” cis-element, and finally activates DYT1 expression. As mentioned before, among the known DYT1 upstream regulatory factors which are involved in transcription regulation, SPL8 participates in the small RNA signaling in cell differentiation regulation in anther. As a SBP domain factor, the DNA-binding motif of SPL8 is zinc-binding motif rather than “CTCC” (Xing et al., cis-element of DYT1 promoter still needs to be characterized in future work. This unknown factor might be unable to activate DYT1 expression alone. Conversely it would associate with SPL/NZZ and/or LFR to form an active transcription complex to trigger DYT1 expression.With the key WZ and SZ designed all experiments, analyzed data, and wrote the manuscript. HZ performed experiments on transgenic expression assays. RL worked on the transgenic lines. QH performed experiments on construction of transformation vector. YL performed analysis of promoter function element. QX performed statistical analysis of fluorescence intensity.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The BL is prepared through a simple spray deposition onto a fluorine-doped tin oxide (FTO) conducting glass substrate before the growth of a α-Fe2O3 nanorods via a hydrothermal process. Insertion of the hematite BL between the FTO and the nanorods markedly enhances the generated photocurrent, by limiting undesired losses of photogenerated charges at the FTO||electrolyte interface. The proposed approach warrants a marked improvement of material performances, with no additional thermal treatment and no use/dispersion of rare or toxic species, in agreement with the principles of green chemistry.The effect of a thin α-Fe High charge carrier recombination in hematite is one of the critical problems to face. To reduce the recombination rate of photogenerated charges, the use of quasi 1D structures, such as nanorods or nanowires, has been proposed. This approach would favor the holes to reach the oxide||electrolyte interface, oxidizing the water before the recombination takes place81215Bard and Hardee13451-xMoxO4Doping of hematite NRs with different materials, such as Mn18x, TiO2, Nb2O5, or Ga2O3 between the FTO and the metal oxide semiconductor increases the delivered photocurrent1323et al.2O3:Ti underlayer to simultaneously reduce bulk, interface and surface recombination phenomena, without morphology variation of the NRs and limited self-diffusion doping2 blocking layer to reduce the recombination of charges and a thermal treatment for enhancing the photoconversion rate (resulting again in self doping of hematite).It has been also demonstrated that the deposition of dense underlayers of SiO2O3 layer to physically insulate the FTO from the electrolyte, hence exploiting a strategy already proved highly beneficial in electrodes for excitonic solar cells28In the present investigation, a simple electrode modification is proposed, consisting of the formation of a thin compact hematite buffer layer (BL) over the FTO substrate, inhibiting the undesired losses of charges at the FTO||electrolyte interface. The hematite BL is inserted in between the FTO and the active α-Fein situ hydrothermal synthesis  and maghemite (671 cm−1) phases may be foundRaman analysis  confirme−1 . Sivula et al.−1 in thin films of hematite that was initially attributed to the presence of magnetite phase, but later excluded by Mossbauer analysis. Bersani et al.−1 is possibly due to the disorder-induced activation of the longitudinal optical Eu IR-active mode present at the same wavenumber3435In previous literature many hematite samples have been found to show a peak in the region between 600 and 700 cm−1 previously reported in literature at 400 nm37Hydrothermal growth of the hematite nanorods layer on BL results in a remarkable enhancement of absorption (red line). Optical band gap was calculated by Tauc plot  curves of FTO/hematite-NRs and FTO/hematite-BL/hematite-NRs photoanodes, respectively, recorded in 1 M NaOH. The response of jph-E curve reported in on, the current jumps to a large value but soon decays towards a (lower) steady state value defined by the relative rates of charge recombination and transfer to redox species. At the successive light interruption (off state), an overshoot of opposite sign occurs, that is associated to the filling of surface states emptied during illumination. This transient decays with a law similar to that observed for the on period. The shift towards negative potentials of the cathodic current  we obtain for both types of electrodes a marked increase of the photocurrent  and iodide-rich solutions (0.1 M NaOH + 0.2 M LiI), 1131Further photospectra were recorded at electrodes with BL in 0.1 M NaOH + 0.2 M LiI , illumin− concentration exceeding the concentration of OH−, whereas a lower relative concentration has negligible effects. We can conclude, along the same lines of the mentioned paper, that: a significant concentration of iodide promotes the photocurrent, presumably acting as a hole scavenger with the role of reducing recombination at surface states; the effect of iodide is suppressed by a much larger concentration of OH−, competing for adsorption on the same surface sites. It should be however remarked that for the hematite nanocrystalline films of the mentioned literature investigations, the IPCEs recorded under EE irradiation geometry were 2 orders of magnitude smaller than those observed under SE irradiation geometry, on which basis the Authors concluded that photogenerated charge collection through the nanocrystallites was particularly inefficientThe main observations are in qualitative agreement with early findings of Grätzel and collaborators on nanocrystalline hematite filmset al.−/I−) around 1/2, i.e. with an important presence of OH− ions. This implies that after exciton generation, electrons are effectively drawn to the back contact along the NRs, with a remarkable positive effect exerted by the hematite BL in limiting the charge losses at the FTO||electrolyte interface.A similar result is described in the work by Beerman The higher photocurrent yield obtained for back side illumination (SE) with photons of wavelength λ < 550 nm (hν > 2.25 eV) indicates that charge carriers produced close to the back contact are collected comparatively more efficiently. Conversely, photons of wavelength close to the photocurrent onset (λ > 550 nm), absorbed deep in the film “bulk”, show similar photoresponse for EE and SE illumination, as shown in Transient absorption spectroscopy has been recently adopted as a powerful tool to study and understand charge transfer processes taking place in metal oxide photoelectrodes upon photoexcitation495049505152Hematite shows a broad time-resolved spectrum right after the excitation, with a maximum centered at 580 nm ,b, attri52Minor difference was observed between samples featuring the same architecture, as well as by measuring the same sample at different spots. The difference was within 20–30% range and can be attributed to sample inhomogeneity over the large area. Importantly, within this accuracy interval, there was virtually no difference between samples with and without BL as presented in We presented a simple and fast treatment able to enhance the photocurrent of hematite photoelectrodes. We demonstrated that a relevant increase of photocurrent delivered from a forest of hematite nanorods can be achieved through physical insulation of FTO from the electrolyte by a thin layer of the same material.Results appear of interest as they were obtained with a little additional cost in terms of both materials and procedures: no complicated or expensive further processing, no doping or decoration with costly species, no production/dispersion of harmful materials. The compliance with similar criteria of green chemistryThis study represents an example of how a rational, although simple, electrode engineering strategy holds the potential to significantly push forward the functional features of a material otherwise poorly performing: the path to fabricate efficient hematite-based photoelectrodes is still long but may consist of several significant steps like the one presented here.5H7O2)3) (≥97%) were purchased from Sigma Aldrich and used without further purification. Solutions for photoelectrochemical measurements were prepared from deionized water (ρ > 15 MΩ cm) and high purity chemicals . FTO glasses were purchased from XOP Glass (sheet resistance 15 Ω/cm2).Iron(III) Chloride (≥97%), sodium Nitrate (≥99%), ethanol (≥99.8%), acetylacetone (≥99%), Iron(III) tris(acetylacetonate) (Fe(C5H7O2)3  on the substrate (T 400 °C). Hematite NRs are then grown by applying a standard hydrothermal procedure3 containing 1M NaNO3 (pH is set at 1.5 using HCl) and heated in an oven at 95 °C for 24 h. After cooling down at room temperature, the sample is rinsed several times with distilled water and then dried at 60 °C in an oven. Electrodes are eventually annealed at 450 °C (1 h) to carry out the phase transition from β-FeOOH to α-Fe2O3.Hematite BL is deposited on FTO glasses by spraying a 0.15 mM ethanolic solution of Fe(CUV-Vis adsorption spectroscopy UV-Vis spectra were recorded in a T80 PG Instruments in the range 1100–350 nm (resolution of 2 nm).−1.All Raman spectra were collected using a modular micro-Raman confocal system from Horiba equipped with a single monochromator (ihr320mst3) and a Peltier-cooled CCD camera. A solid state laser at 532 nm and 30 mW was used as the excitation source, along with interference filters on the laser lines and edge filters on the signal. The spectra were collected by a 100x objective operating with 1800 l/mm grating, with 60 s integration time, over a spectral range 150–1100 cmAFM measurements were performed by means of a Bruker-Thermomicroscope CP-Research microscope working in non-contact mode at atmospheric pressure with a Si cantilever with 10 nm of radius.SEM analysis was carried out by a field-emission LEO 1525 microscope, equipped with an In-Lens detector for secondary electron imaging.vs RHE.The TAS instrument and data handling procedures are described elsewhereExperiments were performed in a pyrex electrochemical cell, with a three-electrode configuration, equipped with a quartz optical flat on the side. Experiments were recorded with a standard photoelectrochemical set up: 150 W Xe lamp and monochromator (Applied Photophysics); filters (Schott); light chopper ; potentiostat and function generator ; lock-in amplifier (Perkin-Elmer model 5210). All potentials were measured versus a Hg/HgO, 1 M NaOH electrode and are referred in text and figures to the reversible hydrogen electrode (RHE):−1) under white light illumination interrupted by the chopper (0.5 Hz). Photocurrent spectra were recorded feeding the reference signal from the chopper (2 Hz) and the output signal from the potentiostat to the lock-in amplifier. Signals were acquired in digital form after conversion by a multipurpose laboratory interface (Vernier). In some experiments the working electrode was illuminated through the electrolyte/electrode interface (EE experiments) or through the substrate/electrode interface (SE experiments), according to a literature terminologyThe pH value of 13.6 was attributed to 1.0 M NaOH solutionHow to cite this article: Milan, R. et al. Compact hematite buffer layer as a promoter of nanorod photoanode performances. Sci. Rep. 6, 35049; doi: 10.1038/srep35049 (2016).


Fundamental molecular-level understanding of functional properties of liquid solutions provides an important basis for designing optimized electrolytes for numerous applications. In particular, exhaustive knowledge of solvation structure, stability, and transport properties is critical for developing stable electrolytes for fast-charging and high-energy-density next-generation energy storage systems. Accordingly, there is growing interest in the rational design of electrolytes for beyond lithium-ion systems by tuning the molecular-level interactions of solvate species present in the electrolytes. Here we present a review of the solvation structure of multivalent electrolytes and its impact on the electrochemical performance of these batteries. A direct correlation between solvate species present in the solution and macroscopic properties of electrolytes is sparse for multivalent electrolytes and contradictory results have been reported in the literature. This review aims to illustrate the current understanding, compare results, and highlight future needs and directions to enable the deep understanding needed for the rational design of improved multivalent electrolytes. A key challenge of modern society is the sustainability of energy supply with increasing demand and depleting fossil resources. We are on the verge of a power revolution, where we desire enhanced energy storage devices with higher energy density, faster charge/discharge capability, and longer life compared to existing technologies. Access to low-cost and environmentally benign energy storage devices will not only transform the world’s energy economy but also build a foundation for a carbon-free society. Li-ion batteries are considered a linchpin technology of energy storage because of their ubiquitous use in electric vehicles and electronic devices. However, current demands are pushing Li-ion technology to the limit of its capacity, which is raising serious safety concerns . Even wi2+, Ca2+, Zn2+, Al3+, Y3+,…) proffer more than one electron per redox center, they are capable of delivering significantly higher energy density as compared to monovalent Li-ion and Na-ion batteries. Even though the concept of both divalent and trivalent batteries dates back to 1970s, rechargeable trivalent batteries remain in the realm of scientific curiosities −) .2+, Mg2+, and Zn2+ systems. We also discuss key advancements, major hurdles, and possible future directions in the research of multivalent electrolytes.It is well known that the structures of the active species in solution can significantly affect the kinetics of metal deposition and dynamics of charge-carrying species. In conventional electrolytes, the formation of ion pairs and AGGs are generally assumed to negatively affect the mobility of ions and the transference number, which in turn decreases the conductivity . On the 2, H2O, CO2, and traditional non-aqueous electrolytes result in the formation of a passivation layer that is both electronically and ionically insulating. Furthermore, in contrast to Li-ion intercalation chemistry, very few solids are known to reversibly intercalate Mg2+ ions and no electrolytes have demonstrated the stability and conductivity required to enable a > 4.0-V electrochemical window while exhibiting reversible Mg metal stripping and deposition. Today’s Mg electrolytes are limited by their (1) insufficient anodic stability, which makes them incompatible with high-voltage cathode materials, (2) inability for reversible electrodeposition, and (3) low conductivity. Hence, intense efforts have been devoted to developing novel electrolytes to enable high-energy–density Mg-ion batteries. The high negative potential and activity of Mg metal render aqueous Mg electrolytes impractical, resulting in a preference for non-aqueous Mg electrolyte solutions. The difference in bonding and coordination environment of Mg ions in organomagnesium salts and simple Mg salts such as Mg(TFSI)2 and Mg(BH4)2 impacts the performance. For example, it was observed that the electrolyte exhibiting the highest degree of covalent bonding with Mg2+ such as C2H5MgCl results in the formation of complex species that are capable of reversible electrodeposition at the metal anode. On the other hand, ionically bonded compounds such Mg(ClO4)2 and Mg(BF4)2 result in the formation of CIPs and AGGs, which were found to be capable of supporting intercalation in most solvents but fail to show reversible electrodeposition of Mg2+ (m−2n)−) but also affects the dynamics of ionic species in the solution as well as kinetics/energetics for desolvation at an electrode–electrolyte interface 2+ cationic species for a concentration range of 0–1.6 M using Raman spectroscopy and DFT at the B3LYP/6-311 + G** level. The strong chelating effect of neutral glyme molecules on metal cations reduces the cation–anion interaction, which promotes SSIPs, as observed previously for Li salts . For a weakly Lewis basis anion such as TFSI−, M(Glyme)m(TFSI)n solutions behave as a typical ionic liquid with [M(Glyme)m]+ as the cationic species and exhibit properties similar to those of ionic liquids such as high oxidative stability, high thermal conductivity, high ionic conductivity, etc. n)n]z−n)2 in short-chain glymes and acetonitrile and SSIPs in DMSO and tetraglyme, but the diffusion constant of ionic species was found to be faster in AN and short-chain glymes  charges . Hence tmes Fig. c 30]. T. Tn]m]m−2n)−2 from MD simulations by Rajput et al. in acetonitrile and monoglyme solutions where AGGs were observed + and the [TFSI]_ anion 2/THF solution + complex.Several recent studies have reported enhancement in electrochemical performance through the current density and coulombic efficiency for reversible Mg deposition with the addition of MgClg(TFSI)2 . Althougion Fig.  in the ssolution , it is l4)2 is an example of another simple and non-corrosive inorganic Mg salt that has gained much popularity over the last few years due to its compatibility with a Mg metal anode [HCB9H91−]2 which form SSIPs as observed from 11B NMR spectrum and X-ray diffraction study with three DME molecules coordinated with Mg2+ in the solid state. Monocarborane is known to be chemically inert and weakly coordinating − and [MgCl]+ as the possible electroactive species present in the DCC electrolytes [2ClAl-Cl-AlClEt2]− and [MgCl]+, while neutral complexes [MgCl2] formed at 1:1 ratio decrease the conductivity of the electrolyte solution. Many experimental studies including Raman spectroscopy, nuclear magnetic resonance (NMR) in solution phase, and X-ray diffraction (XRD) on the crystallized samples, Mg K-edge near X-ray absorption fine structure (NEXAFS) identified various species in multiple equilibria. These species include charged complexes such as [Mg2Cl3]+ and [MgCl]+ as the most general cation species where the inorganic ligand bonds directly to Mg and Mg is always found to be a hexa-coordinated. The majority anion species are of the form AlCln4−Rn− (1 ≤ n ≤ 3) and exhibit organic ligands which reside primarily bound to Al with tetrahedral coordination. Finally, neutral complexes, MgCl2 and AlCln3−Rn (1 ≤ n ≤ 3), are also evidenced [2Cl3]+ exist in the solution through an equilibrium between MgCl+, and MgCl2. DFT calculations suggest that since the charged complex [AlCl4]− possess a low-lying HOMO frontier orbitals, the ion pair complex between [Mg2(μ-Cl3)THF6]+ and [AlCl4]− should have high anodic stability [2(μ-Cl3)THF6]+ adopts a pseudo-D3h symmetry where two magnesium atoms share three chlorine atoms and each magnesium has three THF ligands attached 22Cl3+ and MgCl+ in solution by using MgCl2 as a non-nucleophilic source of Mg2+ with an aluminum Lewis acid such as  2/THF with BuMgCl/THF and Mg(ClO4)2/H2O, it was concluded that the coordination number of Mg and Al are 2/3 in Mg(AlCl2EtBu)2/THF and BuMgCl/THF compared to those in aqueous Mg(ClO4)2/H2O and Al(NO3)2 solutions. The presence of second and third solvation shell for Mg indicated the formation of oligomers while monomers were observed for Al complexes. Contrary to previous reports, they observed tetrahedral coordination for both Mg and Al, where the coordination number of Al varies between 4 and 6 depending on the pH of the solution within the range of 3 < pH < 7 2+, (R2AlCl2)−, and (R2AlCl3)− as the active ionic species present in the solution responsible for the electrochemical performance of the DCC electrolytes. The first-principle molecular dynamics (FPMD) simulations based studies using DFT (PBE-GGA) by Wan et al. also found the sixfold coordination Mg structures energetically unstable 2DME4]2+ when dimethoxyethane (DME) was used as a solvent instead of THF for which they observed six-coordinated cation complex, [Mg2(μ-Cl)3THF6]+ 2Cl3+ ands Mg-THF . However+ . The tetulations .Fig. 12CxCly+  as the majority Mg species, where Mg is always six-coordinated and AlCln4−Phn− (n = 0–4) and Ph4Al− as the major anionic species features consistently tetra-coordinated Al + and [Al(OR)4]−, but these electrolytes still exhibit a highly corrosive nature due to the high chlorine content + and [Mg2(OPh3)Cl(THF)2]+, where magnesium is five-coordinated. The anion complex was identified as [Al(Ph)4]- with Al in tetrahedral coordination.The electrochemical window of the DCC electrolyte was still narrow and limited by the relatively weak Al-C bond, which breaks through a β-H elimination reaction , 78. To nated Al , 79. Neullectors , 67. Howdditives , 81. Panfraction . Hence, orrosive , 83. To  content , 85. Nel3THF6]+ and [HMDSAlCl3]− as the anion complex ammonium bis(trifluoromethanesulfonyl)imide (DEME-TFSI) can enhance the ionic conductivity of Mg(HMDS)2-MgCl2/THF electrolyte − and forming a weak ion pair between and [TFSI]−. Such rapid exchange of ions can possibly result in structural diffusion, leading to enhanced ionic conductivity and current density of the solution. It should be noted that the structure of the cation complex observed here ([{(THF)3MgCl}2-µ-Cl]+) is different than the typical dimer cation complex observed in the previous study for organometallic electrolyte where three chlorine ions are shared by two magnesium ions, whereas here only one chlorine forms the bridge between two magnesium ions.In 2011, Kim et al. proposed a strategy using non-nucleophilic Hauser bases salt comprising hexamethyldisilazide magnesium chloride (HMDSMgCl) electrolyte which demonstrates good compatibility with the electrophilic sulfur cathode . Similarure Fig.  86]. Re. Re3THF6ctrolyte . The hig3B) and PhMgCl in THF. Even though exact intermediates species present in the solution are unclear, based on the single-crystal XRD, NMR, Raman and fluorescence spectra analyses, they reported the same cation complexes  as observed in DCC, APC and HMDSMgCl electrolytes as the main cationic species and Ph2Mg and [Mes3BPh]− as anionic species present in the solution. The XRD results suggested the presence of ion paired [Mg2Cl3-THF6]+ [Mes3BPh]−, where the anion is tetrahedrally coordinated, while the cation shows a typical bridged structure of magnesium atoms hexa-coordinated by three chlorine and six THF molecules 2 electrolyte solution.Organic boron based Mg complex (OMBCs) salts were initially studied by Gregory in 1990 and later by Aurbach in 2002, but they were found to be limited by low cycling efficiency and anodic stability , 66. Recles Fig. . Further2 and AlCl3 in ethereal solutions, namely the magnesium aluminum chloride complex electrolytes (MACC). The MACC electrolyte shows a high anodic stability of ~ 3.3 V with good reversible deposition/dissolution, however, the reported enhanced performance after conditioning remains a mystery and the performance deteriorates after the few cycles   . It was 4− being more easily immobilized than TFSI−. At both EO:Mg ratios, 64 and 32, the relative proportion of AGGs and CIPs decrease while ion dissociation increased (shown by Raman) as compared to a system with a lower concentration of borate ester group  (Emim = 1-ethyl-3-methylimidazolium) complexes at the B3LYP/6-31G(d) level of theory suggested that zinc forms a homoleptic octahedral complex with all the TFSI− anions bound to Zn in the cis conformation /tetraglyme, the anodic limit is set by the solvent [4]2/DME electrolyte with excellent coulombic efficiency (> 98%) and conductivity (6.8 mS cm−1) the anodic stability (4.3 V vs. Mg) is limited by the solvent + as the active Mg2+ species and the electrochemical performance and stability varies with the choice of anion which often exists as part of an anionic aggregate in the solution. However, a few studies also reported four- and five-coordination for Mg2+ in the aggregates, but in all cases two coordinated Mg2+ ions are bridged by three chlorine atoms and form a complex ion pair with the counter anion aggregate/complex. Contradictory results in the literature regarding the coordination number of Mg may be due to inherent approximations in simulation methods, ex situ experimental results and interpretation. Classical molecular dynamics simulations have been widely used to study the solvation structure of ions in solution; however, the accuracy of the predicted coordination environment strongly depends on the force field parameters. For example, a +2 charge on cation in the case of Mg2+ and − 1 charge on anion results in hexacoordination of Mg ions, while reduced charges for cations and anions obtained from ab initio calculations results in tetra-coordinated Mg ions [4)2 and MgCl2 have been reported. Ion-pairing reduces the hydration numbers, which in turn affects the electrolyte electrochemical properties as aqueous systems often operate by co-intercalation of water. While smaller hydration numbers exhibit less detrimental impact on the electrode materials, it was demonstrated that some hydration may benefit intercalation in oxides. The solvation environment of Mg salts in PEO-type polymer electrolytes is not only concentration dependent, but greatly influenced by the anion present, due to varying strengths of attraction between the cation and anion. When a MgTFSI2 PEO system was compared to the analogous Li system, Mg was found to be more highly coordinated by oxygen, which is consistent with the higher charge density. Although PEO-type solvents are capable of solvating and at least partially dissociating multivalent salts, the conductivity remains low due to the strong ether oxygen cation attraction which limits polymer segmental motion. Similar to Li systems, anions are hypothesized to carry the majority of the current. As mentioned, classical molecular dynamics simulations and other ab initio calculations help develop a more comprehensive picture of solvation structure accessing spatial and temporal scales difficult to attain purely through experiment. To the best of our knowledge, there are no such studies on Mg PEO systems. Calcium secondary batteries are relatively underdeveloped, and as a result, the solvation structure of Ca nonaqueous electrolytes is relatively unexplored and should be further investigated. Nuclear magnetic resonance (NMR) has been used widely to study the solvation structure of electrolytes; however, it is limited by the temporal scale, which makes it difficult to de-convolute the peaks and assign them to specific speciation present in the solution using experiments alone, especially in novel electrolyte systems. Also, any change in the charge density localization in the complexes in the solution alters the screening effect experienced by each NMR nucleus resulting in a different NMR response [The high charge density of multivalent ions typically results in greater tendency to form ion pairs and aggregates in most simple inorganic salts and complexes in organometallic solutions. For example, for simple inorganic Mg and Zn salts, a strong tendency of ion pairing has been observed in most solvents at moderate concentrations, however some high dielectric solvents such as DMSO and some low dielectric solvents with high oxygen donor denticity such as glymes result in solvent separated ion pairs at similar concentrations. The interaction energy between cation and anion tends to be slightly stronger for Zn as compared to Mg ion pairs, with possible ramifications on the relative extent of ion pairing and corresponding effects on conductivity and viscosity of the electrolyte. While MD simulations show significant ion pairing at 0.4 M and 0.5 M in many nonaqueous electrolytes for both Mg and Zn, it may be beneficial to compare the solvation structure more closely in terms of the differences in interaction energies for coordination complexes formed in these electrolytes. Anions with bulky structure and more dispersed charge such as TFSI Mg ions . Accordiresponse . NMR pea


Here we describe an enzymatically active, ultra-thin, biomimetic membrane enabling CO2 capture and separation under ambient pressure and temperature conditions. The membrane comprises a ~18-nm-thick close-packed array of 8 nm diameter hydrophilic pores that stabilize water by capillary condensation and precisely accommodate the metalloenzyme carbonic anhydrase (CA). CA catalyzes the rapid interconversion of CO2 and water into carbonic acid. By minimizing diffusional constraints, stabilizing and concentrating CA within the nanopore array to a concentration 10× greater than achievable in solution, our enzymatic liquid membrane separates CO2 at room temperature and atmospheric pressure at a rate of 2600 GPU with CO2/N2 and CO2/H2 selectivities as high as 788 and 1500, respectively, the highest combined flux and selectivity yet reported for ambient condition operation.The limited flux and selectivities of current carbon dioxide membranes and the high costs associated with conventional absorption-based CO 2 separation and capture, but are currently limited by a trade-off between permeance and selectivity. Here, the authors fabricate a bio-inspired, ultra-thin enzymatic liquid membrane that displays exceptional CO2 permeability and selectivity under ambient conditions.Porous membranes show great promise for CO According to the 2014 report of the World Meteorological Organization4, atmospheric CO2 reached 142% of its pre-industrial level in 2013, primarily because of emissions from the combustion of fossil fuels and production of cement. In November 2016, the Paris Accord was ratified with the goal of maintaining a global temperature rise of only 2 °C above pre-industrial levels during this century. However, the realization of this goal is imperiled by the cost of CO2 sequestration. Seventy percent of the cost of capturing of CO2 involves separation from other gases.Carbon dioxide  or flue gas  could be of great economic value8. An efficient membrane should have both high permeance and selectivity. Permeance is the flux of a specific gas through the membrane, typically reported in Gas Permeation Units (GPUs) (1 GPU = 10−6 cm3 (STP) cm−2 s−1 cm−1 Hg−1). Selectivity is the capacity to separate two or more gases, typically reported as a dimensionless ratio of flux. Porous membranes usually exhibit a high CO2 flux, but due to pore size variability, they often display a poor selectivity. Notable exceptions are zeolite membranes whose sub-nanometer pore size is defined by the zeolite crystallographic lattice and is monodisperse. Recently Korelskiy et al. reported an H-ZSM-5 zeolite membrane exhibiting a CO2/H2 selectivity of ca. 200 and a CO2 permeance of ca. 17,000 when operated at 9 bars and −43 °C10. Dense membranes, typically polymers, exhibit moderate selectivity, but the CO2 flux is usually low because of the low solubility and diffusivity of CO2. In general, most existing membranes exhibit a sharp trade-off between flux and selectivity and are so far impractical for CO2 capture applications12.The conventional process for CO2 in particular, the respiratory system of vertebrates is an excellent case in point. Red blood cells employ carbonic anhydrase (CA) enzymes to rapidly and selectively dissolve the CO2 produced by tissues and regenerate the CO2 exhaled from the lung. CAs represent a family of metalloenzymes that catalyze the rapid interconversion of CO2 and water into carbonic acid H2CO3 (Eq. 1), which dissociates to bicarbonate (HCO3–) and protons according to the prevailing species concentrations  how fast the species to be separated can enter into or exit from the membrane, (2) how selectively it can enter into or exit from the membrane, and (3) how fast it can be transported through the thickness of the membrane. Not surprisingly, biological systems maximize the combination of these factors as separation processes typically take place in an ultra-thin liquid layer aided by enzymes that catalyze the selective and rapid dissolution and regeneration of the target species (increasing solubility and selectivity), and short diffusion distances combined with higher diffusivity within liquid vs. solid media maximize transport rates. For CO how fast2 separation was first reported by Ward and Robb who impregnated a cellulose acetate film with a potassium bicarbonate solution containing CA and observed a factor of six increase in CO2 permeability over potassium bicarbonate alone15. Based on a similar concept, Carbozyme Inc. encapsulated an aqueous CA solution within a microporous polypropylene hollow fiber membrane15 and achieved a five times higher CO2 permeability compared to Ward and Robb’s membrane. However, the CO2 flux (18.9 GPU16) still fell far short of that needed for practical CO2 sequestration, since a CO2 capture cost below $20–40 per ton is required by the U.S. Department of Energy17, which translates into a CO2/N2 selectivity higher than 30–50 as well as a CO2 permeance higher than 300–3000 GPU18. Inherent problems/limitations of CA membranes developed to date are thickness , which establishes the diffusion length and limits flux, and CA concentration, which governs the CO2 dissolution and regeneration rates, but is limited in by the enzyme solubility .The concept of employing CA for CO2 membranes and exceed the DOE requirements for CO2 sequestration, we have developed an ultra-thin, CA-catalyzed, liquid membrane nano-stabilized via capillary forces for CO2 separation , whose effective thickness  is defined by oxygen plasma treatment to be ~18 nm. Through capillary condensation, the pores are filled with water plus CA enzymes confined and stabilized to high pressures by nanoconfinement  exerted by water within a hydrophilic 18 nm diameter nanopore). Due to the exceptional thinness of the membrane and the high effective concentration of CA within the close-packed arrangement of nanopores, we demonstrate (under approximately ambient conditions of pressure and temperature) unprecedented values of combined CO2 flux (as high as 2600 GPU) and CO2/N2 selectivity (as high as 788). Because the CO2 selectivity derives from that of the confined CA enzyme, the enzymatic liquid membrane also exhibits high CO2/H2 selectivity (as high as 1500).Here, in order to overcome the limitations of current CO21, see Methods). In this process, the Anodisc pore channels are filled to a depth of about 1 µm with a cylindrical hexagonal P123/silica mesophase (space group p6mm), which when confined to a cylindrical channel orients parallel to the channel axis  within a confined water layer and small enough to spontaneously fill with water above ~75% relative humidity (RH) (vide infra). However, the thickness of the resulting nano-stabilized liquid membrane would be ~1 µm far exceeding that of natural membranes. In order to reduce the effective thickness of the nano-stabilized liquid membrane, we conducted two steps of surface modification 3. In step 3, we then exposed the membrane to a remote oxygen plasma for 5 s to re-convert hydrophobic trimethylsilyl groups to hydrophilic silanol groups at the immediate membrane surface. The mechanism of this plasma-nanopore-modification has been described by us previously23. Briefly, reactive radicals generated in a low-pressure oxygen plasma are mainly charged ions that cannot penetrate deeply into the nanoporous support, because the plasma Debye length (~20 cm under our conditions) is much larger than the pore size (~8 nm). In order to confirm the hydrophilicity of the plasma-modified nanoporous membrane surface and the hydrophobicity of the HMDS-modified surface, the water contact angle was measured with a Biolin Scientific Theta Optical Tensiometer. Fig. 24. In comparison, the water contact angle of the HMDS-modified surface was ~150° consistent with a superhydrophobic surface stemming from the hydrophobic surface chemistry plus nanoscale roughness25.The enzymatic liquid membrane was fabricated using a four step-process Figs.  and 4. Ssee Fig. . Calcinasee Fig.  whose po2 ALD on the original hydrophilic mesoporous silica membrane with TiO2 ALD on the HMDS plus oxygen plasma-modified ‘amphiphilic’ membrane, using conventional TiCl4 and H2O vapor as the TiO2 ALD precursors. It is well established that TiO2 ALD requires a hydrophilic  surface to initiate deposition; therefore, the formation of TiO2 can be used to ‘map’ the hydrophilic surface chemistry. Fig. In order to estimate the depth of the hydrophilic plasma-modified surface layer, we compared TiOHaving successfully fabricated an ultra-thin hydrophilic nanoporous layer on the hydrophobic support, we next introduced CA enzymes into the hydrophilic nanopores by simple immersion of the sample in an aqueous enzyme solution with a CA concentration of 0.05 mM Step 4, Fig. , Fig. 4 2O adsorption isotherms of nanoporous silica films processed before and after plasma processing, where 0% RH corresponds to samples purged using pure dry N2 for more than 1 h. For the original HMDS/TMCS-treated hydrophobic nanoporous silica membrane  and using the geometric surface area of 4.91 cm2 for the 25 mm diameter QCM sensor, we calculate the corresponding water layer thickness to be 19.6 nm, which is in reasonable agreement with the 18 nm thickness observed according to the TiO2-ALD control experiments  to measure the mass of water adsorbed within the nanoporous membrane deposited onto the active area of the QCM and processed identically to the membrane deposited on the Anodisc support, i.e., by HMDS/TMCS ALD followed by plasma processing. To confirm the structural similarity of the films deposited on the QCM and AO support surfaces, we performed grazing-incidence small-angle scattering (GISAXS). Fig. nts Fig. .Fig. 6GI2 (maintained at 95% RH) through the membrane (prepared as described above) was measured using a bubble flow rate meter for a 1 atm pressure difference. The permeance of N2 through the membrane was almost undetectable, whereas, in contrast, the N2 permeance through the completely hydrophobic sample  was measured to be 340 sc cm cm−2 atm−1. As a further control, we also measured the permeance of CO2 (maintained at 95% RH) through the membrane prepared as described above, but without the CA enzymes, i.e., through the ultra-thin stabilized water layer. In this case the CO2 permeance was undetectable. These results indicate that the ultra-thin CA containing liquid membrane is continuous and essentially defect-free. One conceivable concern might be how to ensure that the liquid membrane is stable and will not ‘dry out’ in real-world applications. As previously discussed, this concern is alleviated by maintaining the membrane at a sufficient relative humidity where, due to capillary condensation, the uniformly sized hydrophilic nanopores remain water-filled. According to the Kelvin equation, capillary condensation for a hydrophilic pore occurs at a relative humidity RH defined by: ln(RH) = −(2γVm/rRT), where γ and Vm are the surface tension and the molar volume of water, r is the pore radius, T is the temperature in Kelvin, and the R constant (8.32 J mol−1 K−1). For the 8 nm diameter pores of our membrane, the Kelvin equation predicts condensation to occur at an RH equal to or exceeding 75%, which is consistent with the water adsorption ‘step’ observed in Fig. 2O if it is from a coal-fired plant and 14.6 wt% H2O if from a gas-fired plant. Both are much higher than the saturated water vapor concentration at 40 °C (~50 g H2O kg–1 air or 0.5 wt% H2O). Therefore, the humidity requirement to maintain membrane stability can be easily satisfied if the membrane is used to capture CO2 from power plant flue gas or used in any moderate humidity environment . For water confined within 8 nm diameter hydrophilic pores, where the contact angle θ equals zero, the capillary pressure is about 35 atm  followed by diffusion across the 18-nm-thick liquid membrane and ex-solution (reverse of Eq. 1) at the hydrophobic interface is temperature dependent and, as expected, is maximized at mammalian body temperature, 30–40 °C. For membranes containing the Desulfovibrio vulgaris CA enzyme, the CO2 permeance is practically temperature independent exceeding that of the bovine CA membrane at low and high temperatures, but found to be less than that of bovine CA at 30–40 °C. Our observed temperature dependent CA activity is in good agreement with that reported by Hooks and Rehm26. Fig. 2 permeance as a function of pH for bovine CA membranes and Desulfovibrio vulgaris CA membranes. Similar to the temperature dependence, the bovine CA membranes performed best at neutral pH, whereas, the Desulfovibrio vulgaris CA membranes exhibited a very moderate pH dependence over the pH range 2–10 and exhibited higher CO2 permeance at both lower and higher pHs.Figure 2 separation and permeance performance of our ultra-thin enzymatic liquid membrane to that of other classes of CO2 membranes. The liquid membrane was operated at 37 °C and pH 7.5 with only a chemical potential driving force. The feed gas composition was 20 vol% CO2 in N2 maintained near ambient pressure (36 cm Hg (0.48 bar)) and the collection side comprised a Ca(OH)2 aqueous solution to capture CO2 and maintain a constant chemical potential driving force  and high CO2/N2 selectivity  an2 capture (step I), HCO3− transport (step II), and CO2 release (step III). In our case, CA enzymes catalyze the selective and rapid dissolution and regeneration of the target species (steps I and III); short diffusion distances combined with the inherent three orders of magnitude higher diffusivity within liquid vs. polymers commonly used for CO2 membranes14 maximize transport rates (step II). Liquid membranes containing CA have been reported previously for CO2 separation by Ward and Robb in the 1960s15 and more recently by Carbozyme Inc27. However, the inherent mechanical weakness of the water layer in their membrane configurations limited their membrane thicknesses, to be only as thin as 10–100 microns, about a hundred times thicker than most polymer membranes; therefore, negating the potential advantage of the liquid membrane compared to a polymer membrane. Here, through nanoconfinement, we have created a mechanically stable liquid membrane only ~18-nm-thick. Furthermore, compared to the Ward and Robb and Carbozyme membranes another advantage of our membrane is the high enzyme concentration achieved by confinement within the close-packed array of hydrophilic nanopores  and Ward and Robb were able to use a CA concentration of only 0.06 mM (2 g l–1). In contrast, the high density of hydrophilic nanopores , or, an effective areal density of 8.0 × 1011 CA cm–2. This CA concentration is ten times greater than that achievable in solution (~10 mg ml–1) and correspondingly accelerates the rates of selective CO2 dissolution and release from the membrane.In order to explain the enhanced performance of our CA catalytic membrane, we must reconsider the three steps governing the flux and the selectivity of any membrane considered previously: COsee Fig. . CA enzy2 permeance was then estimated via the calculated CA enzyme areal density. Here we first considered which step, among steps I–III, is rate-limiting for CO2 permeance. Based on the equilibrium described by Eq. 1, CAs catalyze the dissolution of CO2 from the feed side to form bicarbonates that dissociate into carbonic acids and diffuse through the water layer and eventually be enzymatically converted back to CO2 on the ‘downstream’ side. Hence, the CO2 capture (step I) as well as the CO2 release (step III) are both dependent on the activity of CA enzymes, whereas the HCO3− transport (step II) does not and is a function of the diffusion coefficient of carbonate species in water. In regards to the diffusion transport (step II), given the known CO2 permeability in pure water15 (210 × 10–9 cm3 (STP) cm sec–1 cm–2 cm–1 Hg–1), the permeance of the designed 20-nm-thick water-membrane should be of 210 × 10–9 cm3 (STP) cm sec–1 cm–2 cm–1 Hg–1 divided by the thickness, which is 0.1 cm3 s–1 cm–2 cm–1 Hg–1, or 105 GPU. Now, since a CO2 permeance of 105 GPU is much larger than the permeance observed  assuming the native enzymatic activity of CA (106 reactions per second15). This results in a calculated CO2 permeance of 8 × 1017 molecules sec–1 cm–2 corresponding to a volumetric flux of 0.03 cm3 sec–1 cm–2. At the 36 cm Hg driving pressure (see Methods), this corresponds to a CO2 permeance of 833 GPU, which is within the measured data range .We reasoned that the variability in the measured permeance and the discrepancy between the calculated ‘theoretical’ value and the highest measured CO29. However, adsorbed CA enzymes retain some mobility and are able to move along the silica surface. Different portions of the enzyme contact the pore walls at different times with the active site remaining accessible to the solution and permitting substrate and product molecules to readily diffuse in and out. The structure of the CA enzyme in the nanopore is highly robust, as shown by the root-mean-squared deviation (RMSD) of the backbone and active site atoms compared to the CA crystal structure . These results indicate that the enzymatic activity of CA confined within silica nanopores should not be diminished by adsorption and/or crowding. Further, we cannot rule out the possibility that the (effective) CA enzymatic-specific activity could be increased due to molecular crowding in the nanopores as experimentally observed for other confined enzymes29, or that the effective binding affinity could be enhanced (decreased Michaelis constant) due to excluded volume effects30. This may explain the generally higher levels of CO2 flux measured experimentally compared to values calculated assuming native enzymatic activities.The simulations revealed that, initially placed in the center of the nanopore, the CA enzymes rapidly (<100 ns) diffuse toward the walls of the pore Fig.  and formure Fig. , and doeure Fig. , even fo2 capture and separation, we have fabricated an ultra-thin, enzymatic, nano-stabilized liquid membrane. By using nature’s design principles of ultra-thin membranes and enzymatic aqueous media, we achieved a combination CO2 flux and CO2/N2 selectivity under ambient pressure and temperature conditions greatly exceeding that of conventional polymer or inorganic membranes. The membrane design employs a regular array of close-packed 8 nm diameter hydrophilic nanopores whose depth is only 18 nm to confine and stabilize water plus CA enzymes. The high density of CA-filled nanopores establishes an effective CA concentration ten times greater than possible in aqueous solution. At low pressure, the CA enzyme array catalyzes the rapid and selective capture of CO2 via dissolution to form carbonic acid H2CO3 on the upstream side and the conversion of bicarbonate (HCO3–) to CO2, which is released on the downstream side. The short water-filled channels minimize diffusional constraints. Altogether, this design maximizes the three steps governing the flux and the selectivity of a CO2 membrane: CO2 capture (step I), HCO3− transport (step II), and CO2 release (step III) and enables our enzymatic liquid membrane to exceed Department of Energy standards for CO2 sequestration technologies. Because selectivity is dependent on the exquisite catalytic activity of CA, it should effectively separate CO2 from any gas or gas mixture as we demonstrated for H2/CO2. By simple replacement of CA enzymes with alternate enzymes, we propose that our ultra-thin, enzymatic, nano-stabilized liquid membrane concept could be readily adapted to other separation processes.Separation processes in natural biological systems typically take place in an aqueous environment at ambient pressure driven by the prevailing chemical potential gradient. Oftentimes separations are aided by enzyme catalysis, and the thickness of biological membranes is normally on the nanometer scale. To implement these natural design strategies for CO2 capture from flue gas, where the gas pressure is typically less than several atmospheres. The small uniform pore size also confers environmental stability. Based on the Kelvin equation, the membrane should remain water-filled if the RH is maintained above 75%, which is less than that of flue gas streams, which are typically oversaturated in water. For mammalian-derived CA enzymes, the optimal operation temperature would be ca. 37 °C (compatible with flue gas CO2 sequestration), but extremophile enzymes could enable higher temperature operation albeit with less efficiency. Finally, we should consider whether the membrane would be de-activated by impurities known to be present in flue gas. Here, Lu et al.31 employed CA to promote the adsorption of CO2 from a gas stream containing major flue gas impurities into a polycarbonate solution and concluded that the concentrations of up to 0.9 mol l−1 SO2−, 0.2 mol l−1 NO3−, and 0.7 mol l−1 Cl−,  did not influence the kinetics of absorption from a CA-loaded potassium carbonate solution. Taken together these considerations suggest that the enzymatic liquid membrane is stable enough for use in CO2 capture from flue gas. Additionally, based on its low pressure/temperature performance, it could be considered for other applications like CO2 sequestration in manned space flights.Concerning stability, the membrane is predicted to be mechanically stable because the capillary pressure of water condensed within uniform hydrophilic 8 nm nanopores is ~35 atmospheres. This should prevent water displacement under operations like CO35, ALD37, and plasma processing36 are all scaleable and used today in the microelectronics industry and in roll-to-roll printing operations (see for example ref. 38). For demonstration purposes and to compare with other reported membranes, we used a costly commercial 25 mm diameter anodic alumina substrate (Anodisc) for our support. Further, to rigorously control chemistry, we employed calcination to remove surfactant templates and multiple steps of ALD to modify (hydrophobize) the mesoporous silica pore surfaces. To achieve scaleability and reduce costs, the Anodisc could be replaced with tubular alumina supports as employed previously by us for microporous silica membranes40, and by Korelskiy et al. for zeolite membranes10. Here it is noteworthy that based on their high flux and selectivity, modules of zeolite membranes prepared on tubular alumina supports were found to be 33% cheaper than a commercial spiral-wound polymer membrane unit for separation of 300 tons of CO2 per day during operation at 10 bars and room temperature. Our membranes have ten times lower flux, but ten times greater selectivity and operate at atmospheric pressure, so similar cost reductions might be expected. However, by replacing calcination with oxygen plasma treatment22, and ALD with CVD or other large-scale vapor phase methods, it is conceivable that enzymatic liquid membranes could be processed on low cost hollow fiber polymer supports, which would dramatically reduce the cost of CO2 capture technologies.Concerning cost and scaleability, the unit operations of our membrane synthesis, viz, EISA of ordered mesoporous silica films via dip-coating or spin-coating© Anodisc porous anodic alumina disc supports purchased from Whatman International Ltd. Bovine CA enzymes were purchased from Sigma-Aldrich, St Louis MO and the Desulfovibrio vulgaris CA enzyme was provided from Codexis, Inc.The membranes were fabricated on Whatman© Anodisc porous support is 50-µm-thick and is composed of oriented asymmetric vertical channels that are perpendicular to the disc surface. The channel diameters taper from 200 nm in diameter on the bottom surface to 50–100 nm in diameter on the top surface  by spin-coating; this film was then processed in an identical manner as the Anodisc supported P123-templated film described above and loaded with CA.Focused ion beam and scanning electron microscopy (FIB/SEM) experiments were carried out on a FEI Q3D dual beam FIB/SEM system, with 30 kV/3 nA initial voltage/current followed by 8 kV/25 pA final polishing voltage/current for ion beam mode, and 5 kV/24 pA for scanning electron microscopy mode. Transmission electron microscopy images were acquired using a JEOL2010F HRTEM, and Ti-mapping was acquired using the same TEM with a Gatan EELS system. GISAXS was performed using a Bruker Nanostar on samples prepared on Anodisc substrates fabricated as indicated above or on Si substrates prepared as described for Fourier-transform infrared analysis (vide infra). Quartz crystal microbalance analyses were performed using a QCM200-5MHz QCM manufactured by Stanford Research Systems. A home-built, air-tight environmental chamber equipped with gas flow controllers to perform the H2 permeance and CO2/N2 or CO2/H2 selectivity measurements were performed using a home-made test cell designed to accommodate a 25 mm diameter sample and to be immerged into a water bath for needed temperature control. The feed gas was first introduced through a water bubbler heated at 90 °C to achieve a saturated humidity. In the permeance vs. temperature and pH measurements  into a calibrated Inficon 3000 Micro GC gas analyzer for quantitative measurement discrimination.COnts Fig. , the fee27. The CHARMM36 force field42 was used to model the bovine CA enzyme (Protein Data Bank accession number 1V9E43) under different conditions relevant to CO2 separation, including interaction with silica nanopores. The silica nanopore atoms were modeled with the CHARMM36-compatible INTERFACE force field45. Protonation states of amino acids of the CA enzyme were selected according to the results of PROPKA analysis at pH 746. A rectangular silica nanopore was built based on the structure of the alpha-cristobalite unit cell. The pore’s outer dimensions are 11 × 12 × 10 nm and its internal dimensions are 8 × 8 × 10 nm. The average surface silanol density of the pore is 5.9 Si–OH nm–2, which provides a reasonable model of the amorphous silica surface used in the experimental membranes48. A percentage (16.5%) of the surface silanols were ionized to match the pH 7 conditions. Sodium (Na+) ions were added to counter the negative charge of the ionized silanol groups. No additional salt molecules, either Na+ or Cl−, were added to the simulation, except to produce an overall neutral charge simulation system. A vertical water-filled space exists between periodic images of the simulation cell of height 6 nm, giving the CA enzyme the ability to exit the nanopore. Three CA-nanopore systems were simulated with one, two, and four enzymes within the pore to observe possible crowding effects. A free CA enzyme in solution was also simulated for reference. All systems were simulated at room temperature (298 K) for 300 ns using a Nose–Hoover thermostat. The simulation volume for pore systems was adjusted during the early stages of the simulation to obtain an average pressure of 1 atm, and subsequently simulated at constant volume. The free CA enzyme was simulated at constant 1 atm pressure using a Parrinello–Rahman barostat.The simulations were performed with the GROMACS software packageAll relevant data are available from the authors on request.Supplementary InformationPeer Review File


Given the need for access to patient-facing materials in multiple languages, this study aimed to develop and pilot test an accurate and understandable translation of CollaboRATE, a three-item patient-reported measure of shared decision-making, for Spanish-speaking patients in the United States (US).We followed the Translate, Review, Adjudicate, Pre-test, Document (TRAPD) survey translation protocol. Cognitive interviews were conducted with Spanish-speaking adults within an urban Massachusetts internal medicine clinic. For the pilot test, all patients with weekday appointments between May 1 and May 29, 2015 were invited to complete CollaboRATE in either English or Spanish upon exit. We calculated the proportion of respondents giving the best score possible on CollaboRATE and compared scores across key patient subgroups.Four rounds of cognitive interviews with 26 people were completed between January and April 2015. Extensive, iterative refinements to survey items between interview rounds led to final items that were generally understood by participants with diverse educational backgrounds. Pilot data collection achieved an overall response rate of 73 percent, with 606 (49%) patients completing Spanish CollaboRATE questionnaires and 624 (51%) patients completing English CollaboRATE questionnaires. The proportion of respondents giving the best score possible on CollaboRATE was the same (86%) for both the English and Spanish versions of the instrument.Our translation method, guided by emerging best practices in survey and health measurement translation, encompassed multiple levels of review. By conducting four rounds of cognitive interviews with iterative item refinement between each round, we arrived at a Spanish language version of CollaboRATE that was understandable to a majority of cognitive interview participants and was completed by more than 600 pilot questionnaire respondents. Making efforts to engage and activate patients without addressing diverse patient needs with regard to racial, ethnic, and linguistic differences can exacerbate existing health disparities–4. It isGiven the importance of rigorous translation and evaluation procedures paired with a need for access to patient resources in multiple languages, this study aimed to develop and pilot test a Spanish translation of CollaboRATE for use by Spanish-speaking patients in the US.This project consisted of three phases: 1) translation and review of the English version of CollaboRATE by professional bilingual translators, 2) refinement of a Spanish language version of CollaboRATE through interviews with end users, and 3) pilot testing of the Spanish language questionnaire with adult patients in an internal medicine clinic.Questionnaires fielded in multiple languages are susceptible to measurement bias resulting from inconsistencies between questionnaire versions–16. TranWhile back-translation as recommended by Brislin has beenIn keeping with the TRAPD protocol, we engaged two professional translators to independently draft Latin American Spanish translations of the CollaboRATE questions and 10-point response scale intended for Spanish-speaking individuals in the United States. A third independent translator from the same vendor reviewed those translations and incorporated favored components of the initial drafts into a third version. A bilingual member of the research team with subject matter expertise in shared decision-making (DP) reviewed the third draft and, after making refinements, established a final version for cognitive interview pre-testing.Cognitive interviews play an important role in evaluating survey instruments, particularly when translation is involved,24. CognThe Spanish interview guide  was adapInterviews were conducted at the adult internal medicine clinic of an urban Massachusetts community health center.A native speaker of both English and Spanish approached individuals in the waiting room and staff areas of the health center’s adult internal medicine clinic inviting them to participate in the study. All Spanish-speaking adults (18 years or older) within the clinic were eligible to participate. Participants reviewed a Spanish-language information sheet and provided verbal consent. Participants were compensated with $10 grocery store gift cards.Analysis focused on participants’ understanding and interpretation of each item. As detailed by Levin, we usedPilot data collection took place within the same adult internal medicine clinic where cognitive interviews were conducted.Adult patients with weekday visits at the internal medicine clinic between May 1 and May 29, 2015 were eligible to participate. Patients with visits on Saturdays were excluded as the clinic is minimally staffed for only urgent appointments on weekends.Following visits, bilingual medical assistants (MAs) provided each patient with a one-page survey  along wiWe assessed the proportion of respondents across key subgroups including gender, age, questionnaire language, and interpreter use. We also examined these demographics by questionnaire language. The CollaboRATE score represents the proportion of respondents giving the best score possible on CollaboRATE. This scoring technique is a strategy for aiding interpretation notwithstanding potential ceiling effects common in patient-reported measurement of clinician performance,25. CollDartmouth College’s Committee for the Protection of Human Subjects reviewed and approved this study and its consent procedures. Partners Healthcare Institutional Review Board reviewed this study and considered it exempt from further review. To minimize the identifiable participant information collected in this minimal risk research study, written consent requirements were waived. Prospective interview participants were given an information sheet  and askeThe reviewer created a third version of the questionnaire by incorporating elements of each previous version and making modifications as appropriate. While item 1 was wholly adopted from independent translation 2, the reviewer adapted items 2 and 3 from independent translation 2 to refer to ‘mayor importancia’ rather than ‘más importancia’ when asking about the ‘things that matter most’.In the adjudication stage, modifications to the response scale labels were recommended to maintain consistency in structure and vocabulary between the two extremes of the scale. Additional changes at this stage included a transition in the third item from ‘lo de mayor importancia para usted’ to ‘lo que a usted le importa más’ to describe that which is most important to the respondent.Participants’ demographic characteristics are presented in Four rounds of cognitive interview were completed between January and April 2015. After three rounds of interviews, we reached consensus on items 1 and 2. One additional round of interviews was required for item 3. In round 1 of interviews, item 1 was understood as intended by four of the eight participants. Of those who did not understand the question, two cited the term ‘esfuerzo’ (effort) as problematic. As such, ‘esfuerzo’ was replaced with ‘trató’ (tried) for interview round 2. Three out of eight participants favored the phrase ‘problemas de salud’  in this round, while five preferred ‘estado de salud’ . Accordingly, both phrases were tested again in round 2.In round 2, the refined version of item 1 was understood as intended by only one participant (1/6 participants). Quasi-homophones, or words which are pronounced similarly but confer different meanings, impeded comprehension for two participants (2/6 participants), where ‘cuándo’ was mistaken for ‘cuánto’ and ‘tardo’ for ‘trato’. Thus, alternative wording to ‘cuánto se trató’ was presented in round 3. Two additional participants cited confusion about the grammatical subject of the question (2/6 participants) as the translation retains ambiguity surrounding whose effort each item is intended to assess. Three participants preferred the phrase ‘problemas de salud’ over ‘estado de salud’ (3/6 participants), while three cited no preference (3/6 participants). Despite an overall lack of clarity among most participants, the phrase ‘ayudarle a entender’ was well understood by participants to mean ‘help you understand’.Five of the six round 3 participants found item 1b to be clear and understandable. Item 1a posed problems in that some participants understood ‘trato’ to mean ‘treatment’ rather than ‘trying’; therefore item 1b was adopted as the final version for pilot testing.Item 2 was well understood by five of eight participants in round 1, though two participants again cited ‘esfuerzo’ as a problematic term. There was also confusion on whether it was the patient or the provider who was expected to ‘escuchar a las cosas que más le importan a usted’ (listen to the things that matter most to you). As the original English version of the questionnaire included intended ambiguity around the subject of each CollaboRATE question, this fiIn round 2, while item 2 was well understood by three participants (3/6 participants), two expressed concern with the length of the question. The phrase ‘las cosas que más le importan sobre su estado/sus problemas de salud’ in particular was considered too long. Accordingly, round 3 adopted the shorter phrase ‘lo que más le importa’ to replace ‘las cosas que más le importan’.All round 3 participants found item 2 clear and understandable (6/6 participants). As both 2a and 2b had equally acceptable comprehension, 2b was adopted as the final version for pilot testing to maintain consistency with item 1.In round 1, item 3 was well understood by only one participant (1/8 participants). Three cited the phrase ‘elección que hacer a continuación’ (choosing what to do next) as unclear, attributing the lack of clarity to both sentence structure and improper use of the word ‘elección’ (choice). Round 2 therefore replaced ‘elección’ with ‘al escoger’.Item 3 was again poorly understood in round 2, with five participants finding the item confusing (5/6 participants) despite three participants understanding that it refers to a situation in the future (3/6 participants). While the term ‘incluir’ contributed to the confusion, ‘al escoger cómo seguir adelante’ was well understood. Additionally, the verb structure ‘se hizo’ was reintroduced in round 3 based on a participant’s suggestion.Round 3 saw improved comprehension, though three participants (3/6 participants) misinterpreted the phrase ‘seguir adelante’ to refer to an ‘effort’ or ‘fight’ rather than the intended ‘next steps’. As such, item 3 was rephrased to refer instead to ‘el siguiente paso’ and tested in a fourth round of interviews.In round 4, a majority of respondents reported item 3 to be clear and understandable (4/6 participants). Despite some confusion with the phrase ‘cuando se escogió el siguiente paso’, three of the six participants expressed an understanding of the phrase that was consistent with our intent. Item 3 was therefore adopted at this stage for pilot testing.Of the four response scale options presented, participants in round 1 favored ‘ningún’ (none) as the lower anchor by a slight margin (3/8 participants), and half (4/8 participants) preferred ‘un gran esfuerzo’ (a large effort) as the upper anchor. As use of the term ‘esfuerzo’ was considered problematic elsewhere in the questionnaire, round 2 adopted the term ‘se trató’ in place of ‘esfuerzo’.In round 2, participants favored ‘no se trató’ (2/6 participants) as the lower anchor and ‘se trató lo mejor que se pudo’ (3/6 participants) as the corresponding upper anchor. As round 3 introduced ‘se hizo’ as an alternate phrasing in items 1 through 3, this new language was also tested as part of the response scale in round 3.Participants in round 3 favored ‘no se hizo’ (2/6 participants) as the lower anchor and equally preferred ‘bastante’ (2/6 participants) and ‘se hizo lo mejor que se pudo’ (2/6 participants) as upper anchors. To maintain structural consistency with the lower anchor, ‘se hizo’ was maintained in the final version of the response scale. However, to simplify syntax, the phrase ‘se hizo lo mejor posible’ was adopted as the final version for pilot testing.Of the 1687 eligible patients during the study period, 1230 (73%) completed the survey. 606 patients (49%) completed the Spanish survey, while the remaining 624 (51%) completed the English survey. A large proportion of eligible patients with Spanish documented in clinic records as their primary language completed a Spanish questionnaire . χ2 = 0.015, p = 0.901).CollaboRATE scores did not vary between questionnaire languages, with 86% of respondents to both the English and Spanish questionnaires providing highest-possible scores  provides preliminary evidence of acceptability for routine CollaboRATE administration among Spanish-speaking patients. Additionally, as only one respondent to the Spanish-language questionnaire submitted an incomplete CollaboRATE response, the high rate of completion suggests that the Spanish translation is easy for this population to complete.www.collaboratescore.org.High response rates for the Spanish-language CollaboRATE survey support its acceptability as a patient-reported measure of shared decision-making. However, more research is needed to assess the generalizability of this translation to other US Spanish-speaking populations and confirm the psychometric properties of the Spanish translation. Further testing of this version is currently underway in a large multi-center trial. Further iterations of this translation are available at S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.