RESUMEN
Improving the stability of lead halide perovskite solar cells (PSCs) for industrialization is currently a major challenge. It is shown that moisture induces changes in global PSC performance, altering the nature of the absorber through phase transition or segregation. Understanding how the material evolves in a wet environment is crucial for optimizing device performance and stability. Here, the chemical and structural evolution of state-of-the-art hybrid perovskite thin-film Cs0.05 (MA0.15 FA0.85 )0.95 Pb(I0.84 Br0.16 )3 (CsMAFA) is investigated after aging under controlled humidity with analytical characterization techniques. The analysis is performed at different scales through Photoluminescence, X-ray Diffraction Spectroscopy, Cathodoluminescence, Selected Area Electron Diffraction, and Energy Dispersive X-ray Spectroscopy. From the analysis of the degradation products from the perovskite layer and by the correlation of their optical and chemical properties at a microscopic level, different phases such as lead-iodide (PbI2 ), inorganic mixed halide CsPb(I0.9 Br0.1 )3 and lead-rich CsPb2 (I0.74 Br0.26 )5 perovskite are evidenced. These phases demonstrate a high degree of crystallinity that induces unique geometrical shapes and drastically affects the optoelectronic properties of the thin film. By identifying the precise nature of these specific species, the multi-scale approach provides insights into the degradation mechanisms of hybrid perovskite materials, which can be used to improve PSC stability.
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Lipopolysaccharides are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex. Detailed insights into the architecture of LptDE transporter complexes have been derived from X-ray crystallography. However, no structure of a laterally open LptD transporter, a transient state that occurs during LPS release, is available to date. Here, we report a cryo-EM structure of a partially opened LptDE transporter in complex with rigid chaperones derived from nanobodies, at 3.4 Å resolution. In addition, a subset of particles allows to model a structure of a laterally fully opened LptDE complex. Our work offers insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a highly rigid chaperone scaffold to enable structural biology of challenging protein targets.
Asunto(s)
Proteínas de Escherichia coli , Lipopolisacáridos , Proteínas de la Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Microscopía por Crioelectrón , Cristalografía por Rayos X , Proteínas de Escherichia coli/metabolismo , Bacterias Gramnegativas/metabolismo , Lipopolisacáridos/metabolismoRESUMEN
The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100- and 1,000-fold increase in neutralization potency, respectively. Cryo-EM of the sybody-spike complex additionally reveals a novel up-out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.
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Tratamiento Farmacológico de COVID-19 , Anticuerpos de Dominio Único , Anticuerpos Neutralizantes , Anticuerpos Antivirales/metabolismo , Resistencia a Medicamentos , Humanos , Pandemias , Unión Proteica , SARS-CoV-2/genética , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/farmacología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
To upscale the emerging perovskite photovoltaic technology to larger-size modules, industrially relevant deposition techniques need to be developed. In this work, the deposition of tin oxide used as an electron extraction layer is established using chemical bath deposition (CBD), a low-cost and solution-based fabrication process. Applying this simple low-temperature deposition method, highly homogeneous SnO2 films are obtained in a reproducible manner. Moreover, the perovskite layer is prepared by sequentially slot-die coating on top of the n-type contact. The symbiosis of these two industrially relevant deposition techniques allows for the growth of high-quality dense perovskite layers with large grains. The uniformity of the perovskite film is further confirmed by scanning electron microscopy (SEM)/scanning transmission electron microscopy (STEM) analysis coupled with energy dispersive X-ray spectroscopy (EDX) and cathodoluminescence measurements allowing us to probe the elemental composition at the nanoscale. Perovskite solar cells fabricated from CBD SnO2 and slot-die-coated perovskite show power conversion efficiencies up to 19.2%. Furthermore, mini-modules with an aperture area of 40 cm2 demonstrate efficiencies of 17% (18.1% on active area).
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A decade after the report of the first efficient perovskite-based solar cell, development of novel hole-transporting materials (HTMs) is still one of the main topics in this research field. Two of the main advance vectors of this topic lie in obtaining materials with enhanced hole-extracting capability and in easing their synthetic cost. The use of anthra[1,9-bc:5,10-b'c']dithiophene (ADT) as a flat π-conjugated frame for bearing arylamine electroactive moieties allows obtaining two novel highly efficient HTMs from very cheap precursors. The solar cells fabricated making use of the mixed composition (FAPbI3)0.85(MAPbBr3)0.15 perovskite and the novel ADT-based HTMs show power conversion efficiencies up to 17.6% under 1 sun illumination compared to the 18.1% observed when using the benchmark compound 2,2',7,7'-tetrakis(N,N-di-p-methoxyphenylamine)-9,9'-spirobifluorene (spiro-OMeTAD). Detailed density functional theory calculations allow rationalization of the observed opto-electrochemical properties and predict a flat molecular structure with a low reorganization energy that supports the high conductivity measured for the best-performing HTM.
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The human glycine transporter 1 (GlyT1) regulates glycine-mediated neuronal excitation and inhibition through the sodium- and chloride-dependent reuptake of glycine1-3. Inhibition of GlyT1 prolongs neurotransmitter signalling, and has long been a key strategy in the development of therapies for a broad range of disorders of the central nervous system, including schizophrenia and cognitive impairments4. Here, using a synthetic single-domain antibody (sybody) and serial synchrotron crystallography, we have determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 Å resolution. We find that the inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine-release site. The inhibitor is likely to reach GlyT1 from the cytoplasmic leaflet of the plasma membrane. Our results define the mechanism of inhibition and enable the rational design of new, clinically efficacious GlyT1 inhibitors.
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Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Glicina en la Membrana Plasmática/química , Glicina/metabolismo , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Cristalografía , Humanos , Modelos Moleculares , Piperazinas/química , Piperazinas/farmacología , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Anticuerpos de Dominio Único , Sulfonas/química , Sulfonas/farmacología , SincrotronesRESUMEN
The surface, interface, and bulk properties are a few of the most critical factors that influence the performance of perovskite solar cells. The photoelectron spectroscopy (PES) is used as a technique to analyze these properties. However, the information depth of PES is limited to 10-20 nm, which makes it not suitable to study the complete devices, which have a thickness of â¼1 µm. Here, we introduce a novel and simple technique of PES on a tapered cross section (TCS-PES). It provides both lateral and vertical resolutions compared to the conventional PES so that it is suitable to study a complete perovskite solar cell. It offers many benefits over conventional PES methods such as the chemical composition in the micrometer scale from the surface to the bulk and the electronic properties at the multiple interfaces. The chemical natures of different layers of the perovskite-based solar cells [(FAPbI3)0.85(MAPbBr3)0.15] can be identified precisely for the first time using the TCS-PES method. We found that the perovskite layer has higher iodine concentration at the Spiro/perovskite interface and higher bromine concentration at the TiO2/perovskite interface. UPS measurements on the tapered cross section revealed that the perovskite is n-type, and the solar cell studied here is a p-n-n structure type device. The unique possibilities to analyze the complete solar cell by XPS and UPS allow us to estimate the band bending in a working solar cell. Moreover, this technique can further be used to study the device under operating conditions, and it can be applied in other solid-state devices like solid electrolyte Li-ion batteries, LEDs, or photoelectrodes.
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Intracellular replication of the deadly pathogen Mycobacterium tuberculosis relies on the production of small organic molecules called siderophores that scavenge iron from host proteins1. M. tuberculosis produces two classes of siderophore, lipid-bound mycobactin and water-soluble carboxymycobactin2,3. Functional studies have revealed that iron-loaded carboxymycobactin is imported into the cytoplasm by the ATP binding cassette (ABC) transporter IrtAB4, which features an additional cytoplasmic siderophore interaction domain5. However, the predicted ABC exporter fold of IrtAB is seemingly contradictory to its import function. Here we show that membrane-reconstituted IrtAB is sufficient to import mycobactins, which are then reduced by the siderophore interaction domain to facilitate iron release. Structure determination by X-ray crystallography and cryo-electron microscopy not only confirms that IrtAB has an ABC exporter fold, but also reveals structural peculiarities at the transmembrane region of IrtAB that result in a partially collapsed inward-facing substrate-binding cavity. The siderophore interaction domain is positioned in close proximity to the inner membrane leaflet, enabling the reduction of membrane-inserted mycobactin. Enzymatic ATPase activity and in vivo growth assays show that IrtAB has a preference for mycobactin over carboxymycobactin as its substrate. Our study provides insights into an unusual ABC exporter that evolved as highly specialized siderophore-import machinery in mycobacteria.
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Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium smegmatis/metabolismo , Sideróforos/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Microscopía por Crioelectrón , Cristalografía por Rayos X , Modelos Moleculares , Mycobacterium smegmatis/química , Mycobacterium smegmatis/genética , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Here, we provide a protocol to generate synthetic nanobodies, known as sybodies, against any purified protein or protein complex within a 3-week period. Unlike methods that require animals for antibody generation, sybody selections are carried out entirely in vitro under controlled experimental conditions. This is particularly relevant for the generation of conformation-specific binders against labile membrane proteins or protein complexes and allows selections in the presence of non-covalent ligands. Sybodies are especially suited for cases where binder generation via immune libraries fails due to high sequence conservation, toxicity or insufficient stability of the target protein. The procedure entails a single round of ribosome display using the sybody libraries encoded by mRNA, followed by two rounds of phage display and binder identification by ELISA. The protocol is optimized to avoid undesired reduction in binder diversity and enrichment of non-specific binders to ensure the best possible selection outcome. Using the efficient fragment exchange (FX) cloning method, the sybody sequences are transferred from the phagemid to different expression vectors without the need to amplify them by PCR, which avoids unintentional shuffling of complementary determining regions. Using quantitative PCR (qPCR), the efficiency of each selection round is monitored to provide immediate feedback and guide troubleshooting. Our protocol can be carried out by any trained biochemist or molecular biologist using commercially available reagents and typically gives rise to 10-30 unique sybodies exhibiting binding affinities in the range of 500 pM-500 nM.
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Técnicas de Química Sintética/métodos , Anticuerpos de Dominio Único/química , Bacteriófagos/química , Ribosomas/químicaRESUMEN
The selective immobilization of proteins represents an essential step in the selection of binding proteins such as antibodies. The immobilization strategy determines how the target protein is presented to the binders and thereby directly affects the experimental outcome. This poses specific challenges for membrane proteins due to their inherent lack of stability and limited exposed hydrophilic surfaces. Here we detail methodologies for the selective immobilization of membrane proteins based on the strong biotin-avidin interaction and with a specific focus on its application for the selection of nanobodies and sybodies. We discuss the challenges in generating and benefits of obtaining an equimolar biotin to target-protein ratio.
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Avidina/metabolismo , Biotina/metabolismo , Biotinilación/métodos , Proteínas de la Membrana/metabolismo , Anticuerpos de Dominio Único/aislamiento & purificación , Secuencia de Aminoácidos , Avidina/química , Biotina/química , Ligasas de Carbono-Nitrógeno/química , Ligasas de Carbono-Nitrógeno/metabolismo , Técnicas de Visualización de Superficie Celular/métodos , Clonación Molecular/métodos , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Klebsiella pneumoniae , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Unión Proteica , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/metabolismo , Estreptavidina/química , Estreptavidina/metabolismoRESUMEN
Three hole-transporting materials (HTMs) were prepared following a straightforward synthetic route by cross-linking arylamine-based ligands with a simple thieno[3,2-b]thiophene (TbT) core. The novel HTMs were fully characterized with standard techniques to gain insight into their optical and electrochemical properties and were incorporated in solution-processed mesoporous (FAPbI3)0.85(MAPbBr3)0.15 perovskite-based solar cells. The similar molecular structure of the synthesized HTMs was leveraged to investigate the role that the bridging units between the conjugated TbT core and the peripheral arylamine units plays on their properties and thereby on the photovoltaic response. A remarkable power conversion efficiency exceeding 18% was achieved for one of the TbT derivatives, which was slightly higher than the value measured for the benchmark spiro-OMeTAD.
RESUMEN
ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 Å crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter's conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening result in a comparable equilibrium shift and strongly reduce ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state.
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Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Simulación de Dinámica Molecular , Dominio AAA/genética , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Espectroscopía de Resonancia por Spin del Electrón , Mutación , Multimerización de Proteína , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Thermotoga maritimaRESUMEN
The insertase BamA is an essential protein of the bacterial outer membrane. Its 16-stranded transmembrane ß-barrel contains a lateral gate as a key functional element. This gate is formed by the C-terminal half of the last ß-strand. The BamA barrel was previously found to sample different conformations in aqueous solution, as well as different gate-open, gate-closed, and collapsed conformations in X-ray crystallography and cryo-electron microscopy structures. Here, we report the successful identification of conformation-selective nanobodies that stabilize BamA in specific conformations. While the initial candidate generation and selection protocol was based on established alpaca immunization and phage display selection procedures, the final selection of nanobodies was enhanced by a solution NMR-based screening step to shortlist the targets for crystallization. In this way, three crystal structures of BamA-nanobody complexes were efficiently obtained, showing two types of nanobodies that indeed stabilized BamA in two different conformations, i.e., with open and closed lateral gate, respectively. Then, by correlating the structural data with high resolution NMR spectra, we could for the first time assign the BamA conformational solution ensemble to defined structural states. The new nanobodies will be valuable tools towards understanding the client insertion mechanism of BamA and towards developing improved antibiotics.
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Proteínas de la Membrana Bacteriana Externa/química , Modelos Moleculares , Conformación Proteica , Anticuerpos de Dominio Único/química , Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Anticuerpos de Dominio Único/farmacología , SolucionesRESUMEN
Binding protein generation typically relies on laborious screening cascades that process candidate molecules individually. We have developed NestLink, a binder selection and identification technology able to biophysically characterize thousands of library members at once without the need to handle individual clones at any stage of the process. NestLink uses genetically encoded barcoding peptides termed flycodes, which were designed for maximal detectability by mass spectrometry and support accurate deep sequencing. We demonstrate NestLink's capacity to overcome the current limitations of binder-generation methods in three applications. First, we show that hundreds of binder candidates can be simultaneously ranked according to kinetic parameters. Next, we demonstrate deep mining of a nanobody immune repertoire for membrane protein binders, carried out entirely in solution without target immobilization. Finally, we identify rare binders against an integral membrane protein directly in the cellular environment of a human pathogen. NestLink opens avenues for the selection of tailored binder characteristics directly in tissues or in living organisms.
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Proteínas Portadoras/genética , Código de Barras del ADN Taxonómico/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biblioteca de Péptidos , Proteínas de la Membrana Bacteriana Externa/genética , Cromatografía Liquida , Legionella pneumophila/genética , Proteínas de la Membrana/genética , Espectrometría de Masas en TándemRESUMEN
Selective export and retrieval of proteins between the endoplasmic reticulum (ER) and Golgi apparatus is indispensable for eukaryotic cell function. An essential step in the retrieval of ER luminal proteins from the Golgi is the pH-dependent recognition of a carboxyl-terminal Lys-Asp-Glu-Leu (KDEL) signal by the KDEL receptor. Here, we present crystal structures of the chicken KDEL receptor in the apo ER state, KDEL-bound Golgi state, and in complex with an antagonistic synthetic nanobody (sybody). These structures show a transporter-like architecture that undergoes conformational changes upon KDEL binding and reveal a pH-dependent interaction network crucial for recognition of the carboxyl terminus of the KDEL signal. Complementary in vitro binding and in vivo cell localization data explain how these features create a pH-dependent retrieval system in the secretory pathway.
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Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Receptores de Péptidos/química , Animales , Pollos , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Ratones , Conformación Proteica , Receptores de Péptidos/metabolismoRESUMEN
Organic-inorganic hybrid perovskites have attracted great attention over the last few years as potential light-harvesting materials for efficient and cost-effective solar cells. However, the use of lead iodide in state-of-the-art perovskite devices may demonstrate an obstacle for future commercialization due to toxicity of lead. Herein we report on the synthesis and characterization of low dimensional tin-based perovskites. We found that the use of symmetrical imidazolium-based cations such as benzimidazolium (Bn) and benzodiimidazolium (Bdi) allow the formation of 2D perovskites with relatively narrow band gaps compared to traditional -NH3 + amino groups, with optical band gap values of 1.81â eV and 1.79â eV for Bn2 SnI4 and BdiSnI4 respectively. Furthermore, we demonstrate that the optical properties in this class of perovskites can be tuned by formation of a quasi 2D perovskite with the formula Bn2 FASn2 I7 . Additionally, we investigate the change in band gap in the mixed Sn/Pb solid solution Bn2 Snx Pbx-1 I4 . Devices fabricated with Bn2 SnI4 show promising efficiencies of around 2.3 %.
RESUMEN
Hybrid perovskite solar cells have been capturing an enormous research interest in the energy sector due to their extraordinary performances and ease of fabrication. However, low device lifetime, mainly due to material and device degradation upon water exposure, challenges their near-future commercialization. Here, we synthesized a new fluorous organic cation used as organic spacer to form a low-dimensional perovskite (LDP) with an enhanced water-resistant character. The LDP is integrated with three-dimensional (3D) perovskite absorbers in the form of MA0.9FA0.1PbI3 (FA = NH2CH = NH2+, MA = CH3NH3+) and Cs0.1FA0.74MA0.13PbI2.48Br0.39. In both cases, a LDP layer self-assembles as a thin capping layer on the top of the 3D bulk, making the perovskite surface hydrophobic. Our easy and robust approach, validated for different perovskite compositions, limits the interface deterioration in perovskite solar cells yielding to >20% power conversion efficient solar cells with improved stability, especially pronounced in the first hours of functioning under environmental conditions. As a consequence, single and multijunction perovskite devices, such as tandem solar cells, can benefit from the use of the waterproof stabilization here demonstrated, a concept which can be further expanded in the perovskite optoelectronic industry beyond photovoltaics.
RESUMEN
Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.
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Transportadoras de Casetes de Unión a ATP/aislamiento & purificación , Tranportador Equilibrativo 1 de Nucleósido/aislamiento & purificación , Proteínas de Transporte de Glicina en la Membrana Plasmática/aislamiento & purificación , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/inmunología , Transportadoras de Casetes de Unión a ATP/metabolismo , Técnicas de Visualización de Superficie Celular , Tranportador Equilibrativo 1 de Nucleósido/química , Tranportador Equilibrativo 1 de Nucleósido/inmunología , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Proteínas de Transporte de Glicina en la Membrana Plasmática/química , Proteínas de Transporte de Glicina en la Membrana Plasmática/inmunología , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Humanos , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Anticuerpos de Dominio Único/genéticaRESUMEN
The straightforward synthesis and photophysical properties of a new series of heteroleptic iridium(III) bis(2-arylimidazole) picolinate complexes are reported. Each complex has been characterized by nuclear magnetic resonance, UV-vis, cyclic voltammetry, and photoluminescent angle dependency, and the emissive properties of each are described. The preferred orientation of transition dipoles in emitter/host thin films indicated more preferred orientation than homoleptic complex Ir(ppy)3.
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Compositional engineering of a mixed cation/mixed halide perovskite in the form of (FAPbI3)0.85(MAPbBr3)0.15 is one of the most effective strategies to obtain record-efficiency perovskite solar cells. However, the perovskite self-organization upon crystallization and the final elemental distribution, which are paramount for device optimization, are still poorly understood. Here we map the nanoscale charge carrier and elemental distribution of mixed perovskite films yielding 20% efficient devices. Combining a novel in-house-developed high-resolution helium ion microscope coupled with a secondary ion mass spectrometer (HIM-SIMS) with Kelvin probe force microscopy (KPFM), we demonstrate that part of the mixed perovskite film intrinsically segregates into iodide-rich perovskite nanodomains on a length scale of up to a few hundred nanometers. Thus, the homogeneity of the film is disrupted, leading to a variation in the optical properties at the micrometer scale. Our results provide unprecedented understanding of the nanoscale perovskite composition.