The control of particle size is challenging, andmost methods produce polydisperse Fe3C nanoparticles of20–100 nm in diameter. Given the wide range of applicationsof Fe3C nanoparticles, it is essential that we understandthe evolution of the system during the synthesis. Here, we reportan in situ synchrotron total scattering study ofthe formation of Fe3C from gelatin and iron nitrate sol–gelprecursors. A pair distribution function analysis reveals a dramaticincrease in local ordering between 300 and 350 °C, indicatingrapid nucleation and growth of iron oxide nanoparticles. The oxideintermediate remains stable until the emergence of Fe3Nat 600 °C. Structural refinement of the high-temperature datarevealed local distortion of the NFe6 octahedra, resultingin a change in the twist angle suggestive of a carbonitride intermediate.This work demonstrates the importance of intermediate phases in controllingthe particle size of a sol–gel product. It is also, to thebest of our knowledge, the first example of in situ total scattering analysis of a sol–gel system.The sol–gelsynthesis of iron carbide (Fe In situ total scatteringanalysis has uncoveredthe importance of iron oxide as an intermediate phase in the synthesisof Fe3C nanoparticles from sol−gel precursors. Asudden increase in order is observed between 300 and 350 °C,associated with the crystallization of iron oxide nanoparticles. Thelocal structure of the Fe3N intermediate is also investigated. These 3N andFe3C, it is important to have controlled routes to nanoparticlesof these materials.7 Various routes havebeen proposed to achieve this goal, including laser ablation, ammonolysisof iron oxide nanoparticles, nanocasting,9 solvothermal synthesis10 and sol–gelchemistry. Sol–gel chemistry has the advantage of being relativelysimple both in terms of the precursors and processing. In general,sol–gel synthesis of Fe3N or Fe3C nanoparticlesis achieved by mixing aqueous iron salts with organic molecules such as urea11 orgelatin12 as well as with CTAB and melamine.7 The resulting “gel” is dried andpyrolyzed in an inert atmosphere to produce nanoparticles of the requiredproduct. While sol–gel chemistry is simple and scalable, itis difficult to achieve significant control over the particle size.It is also difficult to isolate pure nitride or carbide phases andsmall changes in experimental conditions can have a large effect onthe product composition (Fe3N/Fe3C/Fe).13 In order to maximize the beneficial catalyticproperties of iron nitrides and carbides and fully explore their potential,it is essential to gain a better understanding of how they are formed.In order to fully exploit the potential of Fein situ synchrotron X-ray diffraction studies,we showed that the sol–gel route to Fe3C proceedsvia several intermediates . This is consistent with transmissionelectron microscopy (TEM) images that show iron oxide nanoparticlesembedded in a carbon matrix. From 560 °C, sharp Fe3N peaks emerged and from 610 °C, sharp Fe3C peakswere observed, produced by carbothermal reduction and nitridationof the iron oxide intermediate by the surrounding nitrogen-doped carbonmatrix. The Scherrer analysis indicated larger crystallite diametersof 30 nm (Fe3N) and 60 nm (Fe3C), again consistentwith the TEM images. The Fe3N to Fe3C transitionwas believed to proceed via carbon diffusion into the nitride (forminga carbonitride intermediate), based on observations of a peak shiftin the Fe3N phase.In mediates 1.12 In ain situ synchrotron total scatteringstudy of the sol–gel synthesis of Fe3C. Total scatteringand the pair distribution function (PDF) have been widely used ex situ to study the local order in crystalline and amorphousmaterials. They have also been used in situ to studynanoparticles formed in solvo/hydrothermal synthesis, showing theevolution of local and long-range structures.15 These systems, however, are comparatively simple as they involveonly two or three precursor phases that evolve to a single phase suspendedin a solvent. Total scattering has enormous potential to aid the understandingof sol–gel synthesis of materials. It offers information aboutlocal structural details that may be missed in Bragg scattering. Italso allows us to examine the evolution of particle size and crystallinityat lower temperatures where no long-range order is present. The datain this study specifically demonstrate the very fast crystallizationof the iron oxide intermediate during Fe3C synthesis. Italso offers insight into the formation and structure of the Fe3N intermediate. To the best of our knowledge, this study isthe first example of an in situ total scatteringstudy of a sol–gel process. This is particularly significantas it shows that PDF analysis can be used to extract useful informationfrom complex systems where there are multiple crystalline and amorphouscomponents.We now report an 22.112 A hot aqueous solution of gelatin  with aqueous iron nitrate 3·9H2O) formed aviscous orange gel. The orange gel was dried in air at 70 °Cto produce a brittle orange-brown foam.The synthesis of the gelatinprecursor was performed as described in the previous literature.2.22 was blownover the open end of the capillary to prevent oxidation of the sample.A temperature calibration was performed using a Si-Al2O3 standard.16 Data were collectedfrom 150 to 400 °C in 50 °C increments and at 500 and 600°C, with a heat rate of 10 °C min–1. Eachdata collection was 10 min in length. During heating, the samplesunderwent expansion due to the release of gases from gelatin decomposition,so the samples were occasionally repacked with a thin wire.X-ray totalscattering data were collected using a wavelength of λ = 0.16167Å. Samples of the orange-brown foam were ground and loaded into1 mm diameter fused silica capillaries  at theDiamond Light Source beamline I15-1. A hot air blower was used forthe variable temperature experiments, and pure N2.318 The starting modelswere derived from the following sources: FeOx (refined with the fixed stoichiometry of FeO) from Fjellvåg et al.,19 Fe3C fromWood et al.,20 and Fe3N from Jacobs et al.21 Backgrounds were described using sixth-order Chebyshev polynomialsand with the scans of the empty fused silica capillaries collectedat similar temperatures, where a refined scale factor was included.Peak shapes were described using the Thompson–Cox–Hastingspseudo-Voight function. Additionally, a zero-point parameter was refined.A range of 1.5 ≤ 2θ ≤ 20° was used. Refinementswere performed against Bragg scattering obtained from the total scatteringexperiments described above for temperatures of 350, 400, 450, 500,and 600 °C. Attempts to perform Rietveld refinements againstthe data for 200–300 °C were made, but it was found thatthe entirety of the Bragg scattering can be described by the emptycapillary backgrounds . As the sample at 200 °C showed no Bragg scattering,no Rietveld analysis was attempted at 150 °C.Rietveld refinementswere performed using TOPAS v6.2.422 The PDF data were obtained using GudrunX23 version 5 to produce D(r) data (as defined by Keen).24 The D(r) data were produced using Qmax = 20 Å–1. A Lorch25 correction function was used to remove Fourierripples generated from the limited Qmax. The broadening power of the function was set to 0.03 Å. Forthe refinement performed against the 600 °C data, the same phasesused in the Rietveld refinement were included in the PDF refinements.Additionally, two amorphous carbon phases were modeled using graphite(starting model obtained from Trucano and Chen)26 for sp2 carbon and diamond(starting model obtained from Yamanaka and Morimoto)27 to model sp3 carbon. A functionavailable in TOPAS22 that removes correlationsat ranges of r = 5 Å was applied to these phasesto model them as amorphous. Additionally, a further Fe3N phase with symmetry lowered from P6322 to P63 was included. The P63 phase was limited to contributing to the PDF at r <4.1 Å, while P6322was limited to contributing at r >4.1 Å tosimulatelocal ordering.Small-boxPDF refinements were performed using TOPAS v6.33.1Figure S1). For data collected from 350 to 600 °C, Rietveld analysiswas used in order to estimate the compositions of crystalline componentsto produce the PDF data. T ≤ 500 °C, thereis a very little change in the composition of the sample. FeOx (wüstite) is the only crystalline phasepresent and the broad peaks are consistent with the previous synchrotron diffraction data.12 At T = 600 °C, there isa dramatic change in the pattern where Fe3N becomes themajor phase, with some FeOx still presentand Fe3C beginning to form. Several peaks that arise fromFe3C are in similar positions to those that are from Fe3N, which can potentially result in Rietveld refinement softwarefitting the background using the structural parameters of Fe3C. Despite the weak intensity arising from Fe3C, thereis evidence to suggest the presence of crystalline Fe3C.The peak at Q = 3.3 Å–1 arisesfrom Fe3C and is unaccounted for in refinements where Fe3C was excluded. Additionally, our previous in situ synchrotron diffraction experiment,12 which was performed using a high Bragg resolution instrument, alsoshowed Fe3C beginning to form at 600 °C. The calculatedweight percentages for FeOx, Fe3N, and Fe3C are approximately 29, 48, and 22%, respectively.It is likely that the calculated weight percentage for Fe3C is higher than the real weight percentage. The “excess”Fe3C that is calculated here is calculated at the expenseof Fe3N due to the large overlap of potential Bragg peaks.However, for the purpose of this study, which was to obtain an approximatecomposition for processing the PDF data, the effect is negligible.This is due to the very similar X-ray scattering lengths and densitiesof Fe3N and Fe3C. The peaks arising from Fe3N are also far more noticeable than those from Fe3C due to the higher symmetry of Fe3N (P6322 compared to Pnma). The peak sharpnessalso indicates that the Fe3N phase is more crystallineand has bigger particles than the FeOx phase.29 The synchrotron XRD experiment showed Fe3O4 at low temperatures, which is not observed inthis new data, presumably due to the longer scan times  required for collecting data of sufficientquality for PDF analysis. Indeed, our previous laboratory studieshave also shown that the onset point of the Fe3O4 to FeOx transition is dependent on experimentalconditions.13 It is also possible thatthe experimental setup (N2 gas flowing over the end ofa closed capillary as opposed to through the capillary via a retort)caused this slight variation in the system.The data from 200to 300 °C produced negligible Bragg scattering and so were fittedusing only the empty capillary background S1. For dAs Bragg scattering canonly be produced by materials with a long-rangeorder , it cannot tell us about the natureof any amorphous phases present. However, as carbon is likely to makeup most of the amorphous fraction of the sample, the contributionof the amorphous phases to the total scattering factor will be negligiblecompared to the much more electron-dense crystalline iron compounds.Therefore, the phase compositions extracted from the Rietveld refinementsrepresent a good approximation for use in the total scattering processingto produce the PDFs.3.23C and Fe3N, we also processed the data using Rietveldrefinements where Fe3C was excluded. The resultant PDFsat 600 °C were nearly identical , indicating that even if the Fe3C composition has beenoverestimated this will not impact the conclusions from the PDF analysis. 3O4 at this temperature, based on previous studies12 has a very short-range order with no correlationsabove r = 6 Å. This is an even shorter range of order than isobserved in amorphous carbon, which typically shows correlations inthe range of 10 ≤ r ≤ 20 Å.31 Fe3O4 has been shown to produce highly crystallinenanoparticles when synthesized via a solution route, with correlationsextending nearly through the entire nanoparticle.32 The very short-range order in our system therefore suggeststhat the sample is completely amorphous up to 300 °C rather thancontaining very small crystalline iron oxide nucleation clusters.Sol–gel methods have for many years been promoted as routesthat maximize homogeneity in solid-state precursors33 and these PDF data are direct evidence that this is infact the case.Inorder to probe thelocal structure, the PDFs from 150 to 600 °C were produced. Dueto the similar peak positions and X-ray scattering of Fedentical S2, indicr ≤ 2.6 Å that shifts and broadens.This region corresponds to the average Fe–O distances in Fe3O4 (1.89 and 2.06 Å) and FeOx (2.16 Å).34 and the shift in thelarge peak from ∼2.0 to ∼2.1 Å suggests the carbothermalreduction of amorphous Fe3O4 to FeOx. It is possible that the shift is due to the finaldecomposition of the iron nitrate precursor. However, there is alsoa further peak broadening from 250 to 300 °C at 3.0 ≤ r ≤ 3.8 Å, corresponding to the first Fe–Fedistance in Fe3O4 and FeOx, which provides further evidence that an amorphous Fe3O4 phase is being converted to FeOx.Between 150 and 200 °C,there is a peak in the region of 2.0≤ x, showing the onsetof crystallization of FeOx nanoparticlesfrom the amorphous precursor. The local structures at 300 and 350°C are similar at <5 Å  and a graphite phase with correlations at r >5Å removed. This was found to provide a better fit  than with short-range diamondor a diamond/graphite combination. This fit was not satisfactory for r <4 Å, so another phase of FeOx with a lower P4 symmetry was added toaccount for local breaking of symmetry . Having a lower symmetry in the local structure whilehaving a higher symmetry long-range structure due to disorder is commonlyobserved in oxide materials such as Ba2In2O5 and La2Mo2O9.37 While this did improve the fit (Rwp =19.150% and χ2 = 0.151), there were still some discrepancieswith the peak at r = 1.4 Å, corresponding tothe carbon phases, and the peak at r = 2.1 Å,corresponding to the nearest neighbor Fe–O distance. FeOx is known to have a highly defective (and oftenoxygen-deficient) structure containing Frenkel defects.19 This fact, combined with the complexity of theoverall system, means that there could be many factors contributingto peak broadening and shifting.An attemptwas made to refine the local structure at 350 °Cusing FeOx nanoparticles. Between 500 and 600 °C, thereis another dramatic change, and in this case, there is a substantialshift in peak intensities and positions, correlating to the observationof Fe3N peaks in the Bragg scattering. At 600 °C,there are correlations up to r = 50 Å, whichis the maximum distance that the PDFs were processed to. This indicatesa growth in the crystallite size during the FeOx to Fe3N transition.From 350 to 500 °C, thereis a very little change in the PDF,suggesting that there is no significant growth in the FeO3C, Fe3N, and FeOx using TOPAS. The raw data and the resulting fitare shown in 22 however, it also permits theuse of functions that scale calculated contributions as an arbitraryfunction of distance. This allows amorphous phases with a very short-rangeorder to be approximated. Therefore, both diamond and graphite wereincluded in our refinement (with correlations for r >5 Å removed) to model the mixture of sp2 andsp3 carbons that result from the decomposition of gelatin.38 The C–C distances found in sp2 and sp3 carbons range from 1.4 to 1.5 Å, correspondingto the first peak in the PDF at ∼1.42 Å. As the majorityof the scattering is produced by the Fe-containing phases and theprimary purpose of this study is the structure of the Fe3N nanoparticles, we propose that this approximation for the carbonphase is sufficient. The cell parameters of all included phases wererefined according to their long-range symmetry as well as sphericalatomic displacement parameters. Atomic coordinates were only refinedfor Fe3N as it was the primary phase, whereas Fe3C constituted only an estimated 22% by weight of the sample at thistemperature, so there was not enough sensitivity in the PDF to accuratelyrefine the coordinates.In order to fullycharacterize the PDF data for the complex mixtureof components present at 600 °C, structural refinements wereperformed for Fe3N withits long-range space group of P6322. Whilethis resulted in a near-satisfactory fit (Rwp = 12.318%and χ2 = 0.099), the first two peaks, correspondingto the amorphous carbon phases and the nearest-neighbor Fe–Ndistance in Fe3N, do not fit. They were instead shiftedas a result of incorrect cell parameters. It is not uncommon for materialsto locally break symmetry, such as in Ba2In2O5 and La2Mo2O9.37 In order to address this, a lower-symmetry P63 phase of Fe3N was used in addition to the P6322 phase. The P63 space group is a maximal subgroup of P6322, where the twofold rotational axes parallel and perpendicularto the x and y axes have been removed.This provides more degrees of freedom to the Fe atoms as they movefrom the 6g Wyckoff position  in the P6322 phase to the general6c Wyckoff position . It also provides additional degreesof freedom to the N atoms as the z coordinate isallowed to be refined in the P63 phase. There is some intensity in the calculatedcurve at r < rmin;this is due to TOPAS broadening the peak shape function.Initial refinements used a singlephase of Fe63 phase1 and 2. 6 octahedra is adjusted in the P63 phase. The twist angle, φ, is a parameterused in coordination chemistry to describe how trigonal-prismaticor octahedral in nature a sixfold coordinate polyhedron is, whereφ = 0° is a perfect trigonal prism and φ = 60°is a perfect octahedron.40 In the long-range order,the average structure of Fe3N with a P6322 symmetry, φ1 = 57.96°,21 is an almost perfect octahedron. The CFe6 polyhedra in Fe3C are trigonal prisms, so a gradualshift in the local structure could be expected if the nitride to carbidetransformation occurs via gradual replacement of N atoms with C.42 The twist angle in the P63 phase inour system was found to be φ2 = 50.85(2)°. Thedifference between the two structures is illustrated in 6 polyhedra are distortedand more trigonal prismatic in nature compared to the average structure.Given that iron carbonitride phases are known to exist,43 it is plausible that the distortion in the octahedra could be dueto the incorporation of C into the structure as Fe3N reactswith the surrounding carbon during the formation of Fe3C. A detailed ex situ total scattering study ofthese systems would be necessary to establish whether the distortionis indeed due to carbon diffusion or whether it is an intrinsic featureof Fe3N. Distinguishing between carbon and nitrogen throughatomic form factors alone is challenging in total scattering but ispossible by comparing the bond lengths.Due to the increaseddegrees of freedom of the Fe and N atoms,the twist angle of the NFe4In situ total scattering has been used to probethe evolution of FeOx and Fe3N nanoparticles from a Fe(NO3)3/gelatin sol–gelprecursor. Despite the complex, multicomponent nature of the system,we were able to extract valuable information about the intermediatephases and phase transitions. The onset of crystallization is veryfast. Correlations in the PDFs are only observed at <6 Å at300 °C, indicating a highly amorphous structure. At 350 °C,however, there are correlations up to ∼40 Å and this isonly 5 min further on in the synthesis. This lack of change in theshort-range order during this transition indicates that crystallizationof FeOx nanoparticles occurs from thelocal structural rearrangement of the atoms. Given that the size andnature of intermediate oxide phases in the sol–gel synthesiscan dramatically affect the nature and morphology of a ceramic product,44 this ability to observe early nucleation stages in situ could enable us to tune the synthesis conditionsin our system to achieve more control over the particle size. Ourresults also offer insight into the Fe3N phase. At 600°C, when Fe3N becomes the dominant phase, the nanoparticleshave a longer-range order, suggesting a larger particle size. Structuralrefinements reveal that the NFe6 octahedra present in theFe3N phase at 600 °C are in fact distorted, resultingin symmetry lowering in the local structure from P6322 to P63. The distortionsto the NFe6 octahedra may be caused by carbon beginningto replace N within the Fe lattice as the structure of Fe3C consists of CFe6 trigonal metaprisms. In summary, thestudy has offered us a unique insight into the mechanism of Fe3C nanoparticle formation by sol–gel chemistry. Giventhat controlling the particle size is very important in metal carbidechemistry, these results suggest that focusing on the distributionof amorphous metal oxides in the precursor material will be crucialin reducing the particle size of the final carbide.


Tissue engineering of cartilage requires the selection of an appropriate artificial scaffold. Polylactic acid (PLA) honeycomb films are expected to be highly biodegradable and cell adhesive due to their high porosity. The purpose of this study was to determine the optimal pore size of honeycomb PLA films for in vitro cartilage formation using synovial mesenchymal stem cells (MSCs). μm or 20 μm pores) and then observed by scanning electron microscopy. The numbers of cells remaining in the film and passing through the film were quantified. One day after plating, the medium was switched to chondrogenic induction medium, and the films were time-lapse imaged and observed histologically.  Suspensions of human synovial MSCs were plated on PLA films with different pore sizes (no pores, or with 5 μm pore film showed MSCs with pseudopodia that extended between several pores, while the 20 μm pore film showed MSC bodies submerged into the pores. The number of adhered MSCs was significantly lower for the film without pores, while the number of MSCs that passed through the film was significantly higher for the 20 μm pore film. MSCs that were induced to form cartilage peeled off as a sheet from the poreless film after one day. MSCs formed thicker cartilage at two weeks when growing on the 5 μm pore films than on the 20 μm pore films.  The 5 μm pores were suitable for in vitro cartilage formation by synovial MSCs. Honeycomb PLA films with 5  Tissue engineering of cartilage requires the appropriate selection of cells . SeveralThe appropriate selection of artificial material is also important for cartilage engineering  in additPLA can be prepared in the form of honeycomb-like sheets, and these are expected to show highly biodegradability and improved cell adhesion due to their high porosity . Porous All methods were carried out in accordance with relevant guidelines and regulations. All procedures performed in the study involving human participants were in accordance with the Declaration of Helsinki . This stμm cell strainer . The obtained nucleated cells were cultured in 150 cm2 culture dishes  in 18 mL alpha minimum essential medium  containing 10% fetal bovine serum . The cells were treated with 0.25% trypsin-EDTA (Thermo Fisher Scientific) at 37°C for 5 min, harvested, and cryopreserved as passage 0. For cell culture, the frozen cells were slowly thawed, plated, and incubated for 4 days as passage 1. These passage 1 cells were then replated at 50 cells/cm2, cultured for 14 days, and the resulting passage 2 cells were used for analyses.Human synovium was harvested from the knees of patients with osteoarthritis who underwent total knee arthroplasty operations, and cell culture was performed according to the method established in our previous reports , 14, 15.5 synovial MSCs in 0.5 mL chondrogenic induction medium consisting of Dulbecco's modified Eagle's medium  containing 10 ng/mL transforming growth factor-β3 , 500 ng/mL bone morphogenetic protein 2 , 40 μg/mL proline (Sigma-Aldrich), 100 nM dexamethasone , 100 μg/mL pyruvate (Sigma-Aldrich), 50 μg/mL ascorbate-2-phosphate , and 50 mg/mL 1% ITS Premix . The cells were pelleted by centrifugation at 500 × g for 10 min and then cultured for 21 days. After centrifugation, the pellets were sectioned and stained with toluidine blue  for morphological analysis. Adipogenesis was determined by suspending 100 synovial MSCs in a 60 cm2 dish and culturing in culture medium for 14 days to produce cell colonies. The adherent cells were cultured for a further 21 days in adipogenic induction medium consisting of α-MEM supplemented with 100 nM dexamethasone, 0.5 mM isobutylmethylxanthine (Sigma-Aldrich), and 50 mM indomethacin . Adipocytes were stained with oil red O .The differentiation potential of the MSCs was evaluated as in previous studies –17. Chon2 dish and culturing for 14 days in culture medium to allow formation of cell colonies. The adherent cells were further cultured in a calcification induction medium consisting of α-MEM supplemented with 50 μg/mL ascorbic acid 2-phosphate, 10 nM dexamethasone, and 10 mM β-glycerophosphate (Sigma-Aldrich). After 21 days, calcification was assessed by alizarin red staining .Calcification was studied by plating 100 synovial MSCs in a 60 cmμm were prepared as three types: a PLA film without any pores (0 μm), one with 5 μm pores, and one with 20 μm pores. Before cell plating, the films were immersed in 70% ethanol, washed with phosphate-buffered saline  for hydrophilization, and incubated with FBS overnight at 4°C.Circular PLA films with a diameter of 6 mm and thickness of 5 4 cells) were suspended in 500 μL αMEM with 10% FBS and plated on PLA films in a 24-well plate. After 2 h, the films were fixed in 2.5% glutaraldehyde  for 2 h and washed overnight in 0.1 M PBS at 4°C. Each film specimen was then postfixed with 1% osmium tetroxide (TAAB Laboratories and Equipment Ltd.) for 2 h at 4°C and dehydrated in graded ethanol solutions . After exchanging with 3-methyl butyl acetate  and critical point drying, the specimen was coated with platinum and the surface was observed by SEM  [Synovial MSCs  .5 cells) at passage 3 in 500 μL αMEM containing 10% FBS were dropped onto films in the wells of a 24-well plate. A 1 mL volume of αMEM containing 10% FBS was added, and the cells were incubated for 24 h at 37°C and 5% CO2. Adherent cells were then detached by treating with 0.25% trypsin-EDTA for 5 min. The cells were suspended in Hank's balanced salt solution  at a density of 5 × 105 cells/mL and treated for 30 minutes on ice with antibodies for CD14, CD29 (integrin β1), CD31, CD44, CD45, CD49c (integrin α3), CD49f (integrin α6), CD51/CD61 (integrin αvβ3), CD61, CD73, CD106, and CD146 . The data were analyzed by FACS Verse (BD) and FlowJo software . Cells positively stained with Ghost Dye Violet 510  were removed as dead cells. Isotype controls were prepared as negative controls (BD). The expressions of CD29, CD49c, CD49f, and CD51/CD61 by cells seeded on plastic dishes (just before seeding on the film) were examined in the same manner.Synovial MSCs . The MSCs remaining on the PLA films and the MSCs passing through the film to the bottom of the plates were observed, and the numbers of the cells were counted from the images obtained with a microscope analysis system . Partial images of 4 × 4 area of film and a 10 × 10 area of bottom were taken at 4x magnification of the objective lens and automatically combined using software . The regions with a fluorescence intensity above a fixed value within the film or on the bottom were defined as cells, and the number of regions was counted using imaging software . The numbers of nonadherent cells were calculated from the numbers of adherent cells on film and bottoms.Synovial MSCs were stained with DiI (Thermo Fisher Scientific) and 3 × 10μL volume of αMEM with 10% FBS containing 5 × 105 synovial MSCs was dropped onto films in the wells of a 24-well plate. A further 1 mL αMEM with 10% FBS was added, and the cells were incubated for 24 h. The medium was then switched to chondrogenic induction medium (this was considered time 0). The medium was exchanged every 1–3days.A 500 Synovial MSCs seeded on PLA films were observed with a microscope  at time 0, 1, 3, and 5 days. The film and cell sheet areas at each time point were defined manually using ImageJ software , and the area ratio was calculated.Synovial MSCs seeded on PLA films were washed with PBS, fixed in 4% paraformaldehyde  at 4°C for 1 h, and embedded in 2% agarose gel . The framework of the PLA film was then removed, and the cylindrical agarose gel holding the PLA film was embedded in paraffin, sliced, stained with safranin-o/fast green , and observed with a microscope (BZ-X810).μm on both sides of that midpoint. The midpoints of both ends of the cartilage were determined on each vertical line, and the minimum width through these points was determined. Finally, an average thickness of the cartilage and the coefficient of variance for cartilage thickness at 7 points were calculated using ImageJ.The cartilage thickness was measured by drawing a single line along the long axis of the cartilage, determining the midpoint of both ends of the cartilage on that line, and then drawing seven perpendicular lines 500 P > 0.05). The analysis between the two groups were calculated by a paired Student's t test. Cell counts and time courses were statistically analyzed by two-way analysis of variance (ANOVA) with Tukey's multiple comparisons test using GraphPad Prism 8 software . All statistical analysis methods are described in the figure legends. Two-tailed P values of <0.05 were considered statistically significant.The Shapiro-Wilk test was used to confirm the normality of the data (μm), MSCs showed extended pseudopodia and attachment to the film (μm pore film also showed pseudopodia that extended and adhered to several pores. MSCs on the 20 μm pore film showed submergence of the cell body into the pore and pseudopodia extending around the pore.Synovial MSCs showed multipotency for chondrogenesis, adipogenesis, and calcification . At 2 h the film . MSCs onμm, the MSCs in each film expressed 100% of the positive MSC markers CD44, CD73, and CD90, and less than 5% of the negative MSC markers CD14, CD45, CD106, and CD146 (α6 was significantly lower in MSCs on films than in MSCs cultured in plastic dishes (One day after seeding onto films with pore sizes of 0, 5, and 20 nd CD146 . The expc dishes .μm pores than dishes containing the other film types (μm pore film or 1100 ± 120 cells for the 20 μm pore film (μm pore film, which was significantly lower than 170 ± 110 cells noted for the 20 μm pore film. The number of cells not adhered to the film or the bottom of the dish was 2200 ± 100 cells for the film with no pores and was significantly higher than the 1900 ± 60 cells obtained with the 5 μm pore film. The value was also significantly higher than 1700 ± 130 cells obtained with the 20 μm pore film.One day after seeding the MSCs , fewer clm types . The numore film . The numμm pore films remained sheet-like and did not peel off the film. Quantitative evaluation showed that the area of the sheet of MSCs plated on the film without pores significantly decreased to 20 ± 10% at 1 day, while the area of the sheet of MSCs plated on the film with 5 and 20 μm pores was maintained at 100% for 5 days (During in vitro cartilage formation by MSCs, the cell sheet formed by the plated MSCs peeled off the film without pores at one day, assumed a round shape at three days, and was maintained as a cartilage mass at five days . By contr 5 days .μm pore film (μm and was significantly thicker than the 460 ± 100 μm thick sheet-like cartilage formed by plating MSCs on the 20 μm pore film (μm pore film and was significantly lower than the 0.15 ± 0.11 thickness measured for MSCs seeded on the 20 μm pore film. This suggests the formation of a more even cartilage by MSCs seeded on 5 μm pore film than on 20 μm pore film.Synovial MSCs were plated on the three types of PLA films, cultured in the chondrogenic induction medium for two weeks, and then observed histologically . MSCs plore film  was 630 ore film . The coeμm pore PLA films and cultured in the chondrogenic induction medium for three weeks showed multilayered cells above the film and a small number of cells below the film (μm at 3 weeks. The thickness ratio of the cartilage above and below the film was approximately 3 : 1.Synovial MSCs plated on 5 the film . A few lSynovial MSCs seeded on PLA films without pores exhibited extended pseudopodia. Some reports have indicated that the pseudopodia perform the function of cell adhesion , 19; howα6 was reduced compared to cells cultured on plastic dishes. The PLA film we used is a thin sheet of less than 10 μm thickness and has softer physical properties than a plastic dish. The expression level of integrin α6 might be altered when MSCs adhere to soft PLA materials, since the expression levels of other integrins depend on mechanosensing of the rigidity of the materials by the adhering cells [The expression of surface markers by the synovial MSCs that adhered to the film did not differ with the presence or size of the pores, but the expression of integrin ng cells –24.Chondrogenic induction was observed in MSCs plated on the PLA films without pores, but the cell sheet peeled off from the films after one day and formed a spherical cartilage mass after two weeks. The ascorbate-2-phosphate contained in the chondrogenic induction medium promotes the production of extracellular matrix and the aggregation of MSCs . The obsμm pore films formed the thickest cartilage among the three types of films used in this study. This is because the MSCs did not fall through the pores after plating; instead, they firmly adhered to the pores via their pseudopodia. This firm adhesion prevented the cells from peeling off after chondrogenic differentiation. The chondrogenic differentiation of MSCs using scaffolds is affected by the size of the scaffold pores [μm pore size was the best of the three tested films for cartilage formation by synovial MSCs under the conditions used here. MSCs seeded on 20 μm pore films formed thinner and more uneven cartilage compared to MSCs seeded on 5 μm pore films. The number of cells that adhered to the film one day after seeding was the same, but the number of cells that passed through the film and fell to the bottom was higher for the 20 μm pore films.MSCs plated on 5 ld pores , 27. In μm pores, probably because the MSC body is less than 20 μm in diameter [μm and 20 μm pores, even though the number of cells remaining in the film at 1 day was the same, might be that cells fell through the 20 μm pores from 1 day to 2 weeks after seeding, especially in the early period before the cartilage matrix was fully formed.Although the MSCs extended their pseudopodia and were caught in the pores, some of the cells passed through the 20 diameter  and therμm, 3.2 μm, and 4.7 μm pores and reported that the number of adherent bone marrow MSCs increased as the pore size increased [μm.The pore size in honeycomb films can be adjusted in a range from several tens of nanometers to tens of micrometers by controlling the template water droplets by the breath figure method. The honeycomb films have the added advantage of forming uniformly sized pores when compared to conventional porous scaffolds with size-distributed pores . The porncreased . This fiμm pore honeycomb PLA films or whether the MSCs will require further ex vivo differentiation into cartilage sheets. We also do not know whether the PLA film will be absorbed after implantation, or when this will occur. Another point that requires clarification is whether a PLA film will be recognized as a foreign substance in the joint and cause inflammation.One limitation of our study is that we did not conduct in vivo investigations of the effectiveness of implanting honeycomb PLA films plated with MSCs into cartilage defects. Therefore, we cannot say at this time whether cartilage defects will be repaired when transplanted with undifferentiated MSCs adhering to 5 μm pores. MSCs on the 5 μm pore film showed pseudopodia that extended out to several pores. MSCs on the 20 μm pore film showed cell bodies submerged in the pores. MSCs plated on 5 μm pore films formed the thickest and most even cartilage layer among the three types of films. Honeycomb PLA films with 5 μm pores were therefore considered suitable for in vitro cartilage formation by synovial MSCs.We compared in vitro cartilage sheet formation by synovial MSCs using honeycomb PLA films with 0, 5, and 20


Knowing this, the authors choose a bulky alkyl side chain containing molecule (named L8‐BO) as the acceptor, which shows slower photo bleaching and performance decay rates. A combination of ZnO surface passivation with phenylethanethiol (PET) yields a high efficiency of 17% and an estimated long T80 and Ts80 of 5140 and 6170 h, respectively. The results indicate functionalization of the β‐position of the thiophene unit is an effective way to improve device stability of the NFA.Despite the tremendous efforts in developing non‐fullerene acceptor (NFA) for polymer solar cells (PSCs), only few researches are done on studying the NFA molecular structure dependent stability of PSCs, and long‐term stable PSCs are only reported for the cells with low efficiency. Herein, the authors compare the stability of inverted PM6:NFA solar cells using ITIC, IT‐4F, Y6, and N3 as the NFA, and a decay rate order of IT‐4F > Y6 ≈ N3 > ITIC is measured. Quantum chemical calculations reveal that fluorine substitution weakens the C═C bond and enhances the interaction between NFA and ZnO, whereas the  β‐alkyl chains on the thiophene unit next to the C═C linker improves the stability of acceptor molecules by forming protecting atomic cage. When using PET‐treated ZnO and L8‐BO as the electron acceptor, T80 was estimated over 5000 h.Fluorine substitution increases the interaction with ZnO and accelerates the photon decomposition of A‐D‐A type (nonfullerene acceptor) NFA, while he  In fact, the breakthrough of A‐D‐A type non‐fullerene acceptors are the key to the leap in device PCE. Through donor−acceptor structure modifications, functional group substitution, and side‐chain engineering, various high‐performance NFAs were developed and studied. DC‐IDT2T was first reported by Zhan et al. as the A‐D‐A type NFA in PSC, where the indacenodithiophene (IDT) unit was introduced as the π‐conjugated central donor moiety and 1,1‐dicyanomethylene‐3‐indanone as the terminal acceptor unit . Fusing IDT with two thiophene at both sides yielded a new acceptor molecule ITIC . Owing to the electron deficient nature of the BT core unit, Y6 has low lying HOMO/LUMO energy levels, and a high PCE of over 15% was achieved. By replacing the 2‐ethylhexyl with 3‐ethylheptyl on the pyrrole motif of Y6, Yan et al. synthesized a new acceptor N3, which has improved solubility and the consequently PCE in PSC. Very recently, functionalization of Y6 on the β‐position of the thiophene unit next to the C═C bond was performed and high performance NFA, including Y6‐1O by attaching one alkoxy chain on the Y6, L8 series, with which a high PCE of over 18% were reported.With the advantages of being light‐weight, flexibility, and solution processability, polymer solar cells (PSC) have received widespread attention and become a promising new generation of solar cells.1 DuriF Figure .[5c] photon dimerization of fullerene molecules, interfacial photochemical reaction of polymer with MoO3, as well as nanomorphology changes of the photoactive layer under operation were clarified, and methods to improve the stability of the polymer:fullerene solar cells were developed. As for the NFA molecules, especially for the A‐D‐A type NFAs that have a C═C linker between the donor core and the accepter end group, more efforts were made on the understanding of the intrinsic stability of NFA molecules at different conditions. For example, Zhou et al. reported that the A‐D‐A type NFA molecules can react with polyethylenimine (PEI), yielding the disruption of the exocyclic C═C linker. Kim et al. compared the photon stability of IDTBR and IDFBR in air, and revealed that the IDFBR is more prone to degradation owing to the twisted π‐conjugated core unit that initiates fast photooxidation of the IDFBR. Du et al. compared the stability of ITIC derivatives with different substitutions  on the terminal acceptor unit, and results demonstrated that fluorination of the end‐group stabilizes molecules against light soaking, while methyl groups show an opposite trend. Koster et al. found that stability of the ITIC derivatives based cells is both depended on the NFA molecular structure as well as the device structure, that is fluorine substitution accelerates degradation in conventional device, while methyl substitution slows down degradation in the same device structure. All these reports provide a qualitative understanding of the stability of NFA molecules and the corresponding solar cells. However, there is a lack of deep insight of detailed degradation mechanism of the NFA solar cells and the specific influence of molecular structure on these degradation processes.With the rapid development of PCE of PSC, stability of PSCs also received much attention in the last few years. In this aspect, polymer:fullerene solar cells were well investigated and different degradation pathways including crystallization of fullerene molecules,14 pho Park and Son demonstrated also that ITIC underwent interfacial photon decomposition on ZnO surface, making the ITIC based cell less stable than fullerene‐based cells. By comparing the stability difference of the IT‐4F cells with different ZnO interlayer, we recently proved that photon generated hydroxyl radicals on the ZnO surface is the chemical reactive species that causes the breaking of the C═C bonds. With the better understanding on the detailed degradation mechanism of the NFA cells, methods to improve device stability, including blending with fullerene molecules,surface treatment with Lewis acids, a thin PEI layer, or a self‐assembled monolayer were reported, supporting the proposed degradation mechanism. Despite these excellent research works in improving the stability of the cells, these cells showed lower initial efficiencies less than the optimized cell performance, and most of the cells showed a PCE lower than 15%.Zhou et al. first reported that IT‐4F tend to decompose on the ZnO surface under UV light illumination, which is originally due to the photocatalytic activity of ZnO and consequently leads to the poor performance stability of IT‐4F based cells under light illumination.23 Parβ‐position of the thiophene ring as the electron acceptor in the fabrication of PSCs. In combination with the phenylethanethiol‐treated ZnO layer, a L8‐BO cell with PCE over 17% and long T80 and Ts80 of 5140 and 6170 h were achieved, demonstrating a successful way to simultaneously achieving high PCE and high device stability.To create a guideline for choosing a better NFA molecule for PSCs, in this paper, we systematically investigate the molecular structure–stability relationship of NFA molecules. We compared the performance decay rates of the ITIC, IT‐4F, Y6, and N3 based cells and correlated the results to the photobleaching processes on the ZnO surface. And the effect of fluorine substitution (ITIC versus IT‐4F), the core electron donor structure (IT‐4F versus Y6/N3), as well as the side‐chain substitution (Y6 versus N3) on the device stability were clearly clarified. DFT calculations and NMR measurements were performed to fully understand how the molecular structure influences the interaction between ZnO and NFA molecules. With this useful guideline, we choose the L8‐BO, which has a 2‐butyloctyl chain on the 22.13/Al  characteristics and external quantum efficiency spectra (EQE) of the PSCs under simulated AM 1.5G solar illumination are shown in Figures Table  whereas the Y6 and N3 based cells showed a highest PCE of 15%, which are comparable to the literature reported PCE for the corresponding cell with the same inverted device structure. All these cells were then aged at the maximum power point (mpp) inside the glovebox with continuous white light illumination. Figure τ, α, and PCE(∞) represent the mean lifetime, pre‐exponential factor (degradation amplitudes), and the intercept (the saturated PCE over a long time aging), respectively. The stretching exponent β is in the range 0 < β ≤ 1, which indicates the complexity of the decay process. The fitting decay curves are shown in Figure To understand the influence of NFA on the degradation processes of the PSC, we fabricated and tested the PSCs with an inverted structure of ITO/ZnO/PM6:NFA/MoOl Figure . Four A‐es Table 30 whe2.2VOC), short circuit current (JSC), fill factor (FF), and PCE  aged under LED white light. Figure  and Y6/N3 component (600–950 nm) decreases gradually with the increase in light‐soaking time, suggesting that these acceptor molecules decomposed gradually on the ZnO surface. Figure The comprehensive comparison of the decays of open‐circuit voltage VOC, short2.3Figure + of the NFA molecule. After light illumination for 10 h, decomposition of IT‐4F to mono‐ and bis‐aldehyde products with m/z of 1287.2 and 1075.2 were measured, confirming the breaking of the exocyclic C═C bond of NFA molecules. Similarly, m/z peak sets of 1250.1/1075.2, 1239.2/1027.2, and 1267.8/1055.2 were measured for ITIC, Y6, and N3, which correspond to the mono‐ and bis‐aldehyde products as well, confirming the decoupling of the core–donor and terminal acceptor moieties for all these three compounds during aging. For the aged IT‐4F sample, the M+H+ signal is very weak, while the m/z signal of 1075.2 is the most intensive one, indicating a large percent of decomposition of IT‐4F. In contrast, weaker m/z signals were measured for the ITIC, Y6, and N3 samples. These results are in good accordance with the ultraviolet–visible (UV–vis) absorption decay dynamic results . s Figure , confirm2.4 A faster decomposition rate of fluorine atom‐containing acceptor molecules  than fluorine‐free ITIC indicates that the terminal F substitution is harmful for NFA molecules in terms of stability. To better understand the detailed mechanism, the interactions of F atoms with ZnO were investigated by comparing the adsorption energy (Ead) of 1,1‐dicyanomethylene‐3‐indanone (IC) and 5,6‐difluoro‐1,1‐dicyanomethylene‐3‐indanone (IC‐2F) on ZnO surface using a DFT calculation. ZnO (101) face was chosen as it is the most stable face for ZnO, and two different surface states, Zn—O—H and oxygen vacancy (VO+‐Zn) were selected for this simulation. Figure Ead of the organic moiety to different ZnO surfaces were calculated and are listed in Figure VO+‐Zn) through the C—F···H—O—Zn and C—F···VO+—Zn interaction with an Ead of −0.92 and −0.70 eV, respectively. In comparison, the F‐free IC unit interacts only with H—O—Zn but with a lower Ead of −0.62 eV. The more intensive interaction of F atoms with ZnO brings the NFA molecule closer to the ZnO surface, which could be one of the reasons for the faster degradation rate of the fluorine conation NFA molecules.Our previous results demonstrated that photon‐induced formation of hydroxyl radicals on the ZnO surface is the main reactive species causing the decomposition of NFA molecules.25 A f and the net electro population of the C═C bond, calculated by PBE0/def2TZVP method with Gaussian09 program, are found to be different among NFA molecules  as well as the C═C bond. It can be seen from the 1D 1H NMR spectra and Y6 structure  are numbered 3 and 5 in sequence. Besides, the hydrogen atoms of the alkyl chain (—C8H17) on the central N atoms are numbered 2 and 4. As shown in Figure β‐alkyl side chain plays an important role in determining the stability of the NFA molecules, which could be the direction for further development of high‐performance NFA molecules.Note that both Y6 and N3 have an alkyl side chain on the y Figure 4a, which /PM6:L8‐BO/MoO3/Al was fabricated, which showed the highest PCE of 17.02%  are shown in Figure JSC, 98% for VOC, and 94% for FF), indicating an excellent stability for this cell. Especially, there is almost no JSC decay measured of the cells, indicating that the interfacial decomposition of NFA on ZnO surface was dramatically reduced with the combination of using PET‐treated ZnO and L8‐BO as the electron acceptor. Since fast VOC and FF decays were measured for this cells, the performance decay was then ascribed to the interfacial degradation at the polymer/MoO3 interface. Fitting the PCE decay curve of the cell to a stretched exponential model according to Equation  of 971 h, and a PCE (∞) of 0.89. The mean lifetime is 6 times higher than Y6/N3 cells ,27 can3β‐position of the thiophene unit next to the C═C bond can significantly improve the stability of the cells, which was attributed to the formation of atoms cages, which prevent the attacking of hydroxyl radical to the C═C bond. The new NFA molecule L8‐BO with a branched alkyl side chain on the β‐position of the thiophene unit showed improved device stability, which showed a high device performance with 17.02% and a high performance stability (with T80 over 5000 h). The current work pointed out that functionalization on the β‐position of the thiophene unit next to the C═C bond is the most feasible way to improve the stability of the NFA cells.In summary, we systematically investigated the influence of NFA molecular structure on the stability of solar cells. Results showed that UV–vis absorption analyses confirmed that the PCE decay rates of these cells are directly correlated with the photobleaching process of the NFAs films on the ZnO surface, indicating that photon‐induced decomposition of NFA molecules on the ZnO surface is the main reason for the PV performance decay. The introduction of fluorine atoms on the terminal electron‐accepting moiety decreases the stability, which was mainly due to the strong intermolecular action between ZnO and fluorinated NFA molecules. More importantly, the introduction of an alkyl side chain on the 42, TMAH, and chlorobenzene  were purchased from J&K Scientific Ltd. 1,8‐diiodooctane (DIO) and 1‐chloronaphthalene (CN) was purchased from sigma‐Aldrich. Molybdenum (VI) oxide (MoO3) was purchased from Sterm Chemicals. All materials were used as received without further purification. ZnO was prepared through the reaction of TMAH and Zn(OAc)2 in DMSO as reported by Qian et al.PM6 (PBDB‐T‐2F) and IT‐4F, ITIC, Y6, and N3 were purchased from Solarmer Materials Inc., Beijing. L8‐BO was purchased from Guangzhou Chasing Light Technology Co., Ltd. 2‐phenylethylmercaptan (PET), Zn(OAc)an et al.33trans‐2‐[3‐(4‐tert‐Butylphenyl)‐2‐methyl‐2‐propenylidene]malononitrile (DCTB) as the matrix. NFAs were spin‐coated on the glass substrates and degraded in white LED light for 5 and 10 h. Then the degraded films were dissolved with methylene chloride (CH2Cl2) and dried under vacuum. 1H NMR spectra were recorded on a Bruker Ascend 400 MHZ NMR spectrometer at room temperature with deuterated chloroform as the solvent.The UV–vis absorption spectra of ZnO and NFAs films were measured with a PerkinElmer Lambada 750 at room temperature. All the films were spin‐coated on the glass substrates and aged in a glove box under white LED light. The mass spectra of NFAs were measured with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) using 2 flow and then treated in a UV–ozone oven for 30 min. First, ZnO NPs (10 mg mL−1 in ethanol) was spin‐coated on the ITO substrates at 2000 rpm for 60 s and then were annealed at 130 °C for 10 min on a hot plate in a glove box filled with N2. The solution of PM6:IT‐4F and PM6:ITIC (10 mg mL−1 for each compound) blended in chlorobenzene (CB) with 0.5 vol% DIO was spin‐coated on the top of the ZnO electron transportation layer at 2000 rpm for 60 s and then were annealed at 130 °C for 10 min on a hot plate in a glove box filled with N2. The mixed solution of PM6:Y6 together with 0.5 vol% CN was dissolved in CF with concentrations of 7 and 8.4 mg mL−1, respectively. The blend solution of PM6:L8‐BO was prepared by dissolving PM6 and L8‐BO in chloroform solvent with 0.5 vol% CN. The total concentration of PM6:L8‐BO solution was maintained at 15.4 mg mL−1. Finally, MoO3 (20 nm) as the hole‐extraction layer and Al (100 nm) as the anode were sequentially vacuum deposited on the top of the active layer respectively. The effective photovoltaic area, defined by the geometrical overlap between the bottom cathode electrode and the top anode, was 0.09 cm2. Note that the performance decay of the PM6:IT‐4F cells was reported in our previous paper already. For better comparison, however, we fabricated a new set of the PM6:IT‐4F cells and tested the decay dynamics simultaneously with other cells.ITO substrates were sequentially cleaned by detergent, deionized water, acetone, and isopropanol in an ultrasound cleaner. Then they were put in the isopropanol for storage. Before using them, they were first dried by N already.25 ForVOC, JSC, and FF were measured using a Keithley 2400 source meter under illumination with simulated AM 1.5G sunlight  in a glove box filled with N2. The EQE spectra were recorded by EQE system which was built in home and the light from a 150 W tungsten halogen lamp (Osram 64 610) was used as a probe light and was modulated with a mechanical chopper before passing through the monochromator  to select the wavelength. The response was recorded as the voltage by an I–V converter , using a lock‐in amplifier (Stanford Research Systems SR 830) with a stand silicon cell as the reference before testing the devices.The PV parameters of the cells including https://www.ccdc.cam.ac.uk/).The single crystal structure data used in the calculation comes from CCDC  and continuously illuminated with white LED light  is as same as that measured under standard conditions by AM 1.5G. For monitoring changes in illumination light intensity, it was monitored by a photodiode (Hamamtsu S1336‐8BQ). J–V characters of the devices were checked periodically, and the photovoltaic performances data  were calculated automatically according to the J–V curves. In between the J–V tests, an external resistor load matching the maximum power output point (Rmpp = Vmax/Imax), was attached to the cell. The external load can dynamically change according to the J–V results, the measured performance decay curves is therefore close to the decay behaviors of the cells under a real operation. The stability test method of using passive resistor fits the highest recommended testing protocol ISOS‐L3.The temperature of the cells is thermostatically controlled at room temperature 25 °C by temperature control equipment.The long‐term stability of un‐encapsulated devices was conducted by multi‐channel solar cell performance decay test system  under a testing condition in accordance with ISOS‐L‐1 in the glove box. The cells were put inside a glove box filled with NThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Hypoxia‐inducible factors (HIFs) have been evaluated in various cancers and diseases. However, the specific role of hypoxia‐inducible factor 3 alpha (HIF3A) in non‐small cell lung cancer (NSCLC) remains controversial.We investigated HIF3A mRNA expression in the plasma and tumor tissues of patients with NSCLC and explored its clinical significance. Plasma samples from 103 cases of lung adenocarcinoma (LUAD) and 96 cases of lung squamous cell carcinoma (LUSC), and tumor‐adjacent normal tissues from 58 LUAD and 62 LUSC cases were retrospectively evaluated at the No.8 People's Hospital of Qing Dao. HIF3A expression was explored using RT‐qPCR. The clinical significance of HIF3A was evaluated in the plasma and tumor tissues using the receiver operating curve (ROC) and the area under the curve (AUC).Hypoxia‐inducible factor 3 alpha expression was notably downregulated in the plasma or tumor tissues of patients with LUAD and LUSC, compared with the healthy control group or adjacent normal tissues. Furthermore, HIF3A expression had a significant positive correlation in the plasma and tumor tissues of LUAD and LUSC patients. Meanwhile, the ROC‐AUCs achieved a significantly higher range, from 0.84 to 0.93, with the plasma or tumor tissues of NSCLC patients. Thus, HIF3A expression was not only correlated with plasma and tumor tissues, but also showed potential significance in NSCLC.Hypoxia‐inducible factor 3 alpha is aberrantly detectable in NSCLC patients in the plasma and tumor tissues. HIF3A may be involved in hypoxic responses during the development and occurrence of NSCLC. 2 (TPM +1) of each gene in HIF3A signature gene set. The red box indicates the tumor samples while the gray one represents the normal adjacent tissues. The HIF3A signature score level in NSCLC is significantly lower than normal tissues.Bioinformatics‐based differential signature scores analysis in box plots by using GEPIA2 tool. The signature score is calculated by the mean value of log Previous studies have discovered the tumor‐suppressive activities of HIF1A and HIF2A in renal cell carcinoma and lung cancer.22.1Plasma samples from 103 cases of LUAD and 96 cases of LUSC, and tumor‐adjacent normal tissues from 58 cases of LUAD and 62 cases of LUSC were retrospectively collected from patients undergoing primary tumor resection surgery at the No.8 People's Hospital of Qing Dao. Paired plasma samples were also collected from the 58 LUAD and 62 LUSC patients from whom tissue samples were obtained. The patients were recruited from May 2019 to September 2020. All patients signed informed consent forms during their hospital stay. The study was approved by the ethics committee of the No.8 People's Hospital of Qing Dao and was conducted following the guidelines from the Declaration of Helsinki.2.2Total RNA was extracted using phenol‐chloroform solutions and then homogenized using guanidine isothiocyanate . RNA concentration was evaluated using a NanoDrop spectrophotometer ND1000 (NanoDrop Technologies Inc.).2.3−△△Ct was used for the relative quantitative analysis of the data.The total RNA in the plasma and tissue samples after transfection was extracted with TRIzol reagent, according to the manufacturer's instructions. Thereafter, the total RNA was reverse‐transcribed into cDNA using a reverse transcription kit . The primers used were as follows: HIFA3,5′‐F: AGGCGCCAGAGGCACCATGGAC‐3′, R: 5′‐CATCCTGTGCGTTGGCTGCC‐3′,U6 F: 5′‐GAAGGTGAAGGTCGGAGTC‐3′, R: 5′‐GAAGATGGTGATGGGATTT‐3′. The PCR reaction conditions were as follows: A: pre‐denaturation at 95°C for 10 min, B: denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and elongation at 72°C for 20 s, for a total of 40 cycles; and C: 72°C for 15 min. The reaction was terminated at 4°C. Three replicates were set for each sample, and 22.4http://gepia2.cancer‐pku.cn/). The data used were from TCGA normal and GTEx data.Bioinformatics were performed using GEPIA 2 and the area under the curve (AUC). Statistical significance was set at p < 0.05.SPSS 20.0 (SPSS Inc.) was used for all statistical analyses in the study, and data were expressed as mean ± SD. Clinical information of the patients in Tables 33.1A total of 199 NSCLC patients were enrolled to evaluate HIF3A expression in plasma samples, consisting of 103 LUAD and 96 LUSC patients with higher or lower expression of HIF3A. Furthermore, the patients were characterized according to clinical parameters such as age, sex, smoking history, differentiation, tumor size, and lymph node (LN) metastasis. Among the LUAD and LUSC groups, the tumor size and LN metastasis were statistically significant compared with the other parameters Table .3.2p < 0.05). Furthermore, in 58 patients with LUAD, the tumor tissues had significantly low expression of HIF3A mRNA . Overall, the results demonstrated that HIF3A expression levels in both groups were significantly lower in tumor tissues than in adjacent normal tissues . Subsequently, a significant positive correlation was observed between the plasma and tumor tissues in LUSC patients . Therefore, the expression level of HIF3A was directly correlated between the plasma and tumor tissues of NSCLC patients.The correlation between plasma and tumor tissues with respect to HIF3A expression in 58 LUAD and 62 LUSC patients is shown by scatter plots. Here, the LUAD patients showed a significantly moderate positive relationship between plasma and tumor tissues for HIF3A expression  Figure , whereas4The HIF3A gene is located on chromosome 19q13.2, which is 43 kb long and comprises 19 exons.Human HIF3A may play an opposing role to HIF‐1 and HIF‐2, which have a significant role in the development and progression of cancers. Jang et al.p < 0.05).The exact molecular mechanisms of HIF3A in human cancer, specifically in NSCLC, remain unknown. HIF1 α and HIF2 α affect tumor progression by directly regulating distinctive and allocated target genes such as SCGB3A1 in NSCLC,In recent years, the prognosis and survival rates of NSCLC patients are closely associated with clinical tumor stages, representing a keen drop from approximately 70%–90% in early‐stage (I/II) patients to 5%–10% in patients with advanced stages (III/IV).Currently, there are no related studies that can prove the diagnostic value of HIF3A in LUAD or LUSC, and as this study had a smaller sample size, to confirm our findings, a larger number of samples are required. The present study did not record whether patients received any chemotherapy or radiotherapy treatments; therefore, subsequent validation is essential. HIF3A could act as a novel target to be comprehensively studied in hypoxic cancer biology. Further studies are needed to increase our understanding of the role of HIF3A in the occurrence and development of NSCLC.5Hypoxia‐inducible factor 3 alpha is aberrantly detectable in plasma and tumor tissues of NSCLC patients and is a potentially discoverable target gene that might play an effective role in LUAD and LUSC. HIF3A expression was notably downregulated in the plasma or tumor tissues of LUAD and LUSC patients compared with the plasma of the healthy control group or adjacent tumor tissues. The present study validated the expression of HIF3A and its clinical significance in NSCLC. HIF3A may be involved in hypoxic responses during tumor occurrence and development.The authors declare that they have no potential conflicts of interest.


E. coli ribonuclease HI in complex with Mg2+ ions or with a Zn2+ ion reveal the pivotal role of a highly conserved histidine residue.X-ray crystal structures of  Escherichia coli) in the flexible loop around the active site remains poorly understood, although an important role was suggested by NMR analyses. Here, novel high-resolution X-ray crystal structures of E. coli RNase HI are described, with a particular focus on the interactions of divalent cations with His124 oriented towards the active site. The enzyme–Mg2+ complex contains two metal ions in the active site, one of which has previously been observed. The second ion lies alongside the first and binds to His124 in an octahedral coordination scheme. In the enzyme–Zn2+ complex a single metal ion was found to bind to the active site, showing a tetrahedral coordination geometry with the surrounding atoms, including His124. These results provide structural evidence that His124 plays a crucial role in the catalytic activity of RNase HI by interacting weakly and transiently with metal ions in the catalytic center.The ribonuclease (RNase) H family of enzymes catalyze the specific cleavage of RNA strands of RNA/DNA hybrid duplexes and play an important role in DNA replication and repair. Since the first report of the crystal structure of RNase HI, its catalytic mechanisms, which require metal ions, have been discussed based on numerous structural and functional analyses, including X-ray crystallography. In contrast, the function of the conserved histidine residue (His124 in  The cell debris was removed by centrifugation and the supernatant was loaded onto a SP Sepharose Fast Flow cation-exchange column (Cytiva) pre-equilibrated with the same buffer. The column was washed with 20 mM Tris–HCl pH 7.0, 100 mM NaCl, 1 mM EDTA, 1 mM DTT and the sample was eluted with a linear gradient of 100–300 mM NaCl. The eluted fractions containing RNase HI were pooled and loaded onto a HiLoad 26/600 Superdex 75 pg size-exclusion chromatography column pre-equilibrated with phosphate-buffered saline (PBS). The peak fractions containing RNase HI were dialyzed against 20 mM PIPES–NaOH pH 7.2 and were concentrated with Amicon Ultra-4 centrifugal filter units (Millipore). All purification procedures were performed at 4°C. The purity of all samples was checked with SDS–PAGE.The cells were thawed and resuspended in 20 m2.2.–1 in 20 mM PIPES–NaOH pH 7.2, 1 mM DTT with 50 mM MgSO4 or 1.5–2 mM ZnCl2. The original target of the research was to capture the transient bound state of RNase HI with adenosine monophosphate (AMP) or oligonucleotides; these substrates were also added to the sample in an RNase HI:substrate molar ratio of 1:5 or 1:10. Crystallization conditions were first screened with commercial screening kits  by the sitting-drop vapor-diffusion method at 20°C. 1 µl of the RNase HI sample was mixed with the same volume of reservoir solution and equilibrated against reservoir solution. The crystallization conditions were mainly optimized by adjusting the precipitant concentration. Optimization was carried out using either the hanging-drop or the sitting-drop method at 20°C by equilibrating a mixture of 2 µl RNase HI sample and 2 µl reservoir solution. Crystals appeared in 2–4 days and reached equilibrium in about one week. Most of the crystals had a thin needle-like shape with a typical thickness of around 25–50 µm. Although a significant amount of precipitation was observed when mixing RNase HI with the ZnCl2 solution, diffraction-quality crystals were obtained from several crystallization conditions. The optimized crystallization conditions are summarized in Table 1Concentrated RNase HI was diluted to a final concentration of 8 mg ml2.3.v/v) glycerol and flash-cooled in liquid nitrogen. X-ray diffraction data were collected on BL45XU at the SPring-8 synchrotron-radiation facility  were performed on an iTC200 . ZnCl2 solution (1 mM) was titrated into RNase HI solution (0.1 mM) in 20 mM Tris–HCl pH 7.2 using a 40 µl syringe. Each titration consisted of a preliminary 0.4 µl injection followed by subsequent additions of 2.0 µl. Each corrected heat was divided by the number of moles of Zn2+ injected and was analyzed on the basis of the ‘one set of sites’ model using the MicroCal Origin 5.0 software supplied by the manufacturer. The binding stoichiometry (n), equilibrium dissociation constant (Kd) and binding enthalpy change (ΔH) were directly calculated from the fitting procedure.Isothermal titration calorimetry (ITC) experiments . Based on the X-ray diffraction patterns, these crystals were found to belong to similar but different crystal forms with distinct unit-cell dimensions. In comparison with previously reported crystals, the molecular arrangements within the crystals showed a common feature in which the enzyme molecules form a similar dimer related by a twofold axis, as reported previously . Metal B was coordinated by four water molecules and the carbonyl groups of Asp10 and Glu48, while metal A was coordinated by three water molecules and the carbonyl groups of Asp10, Asp134 and Gly11. Two of the water molecules coordinating to the Mg2+ ions were shared. The distances between metal B and its coordination ligands were in the range 2.0–2.2 Å, whereas those of metal A ranged from 2.4 to 3.5 Å. A tightly bound metal B could be vital for precise binding to the phosphoryl group of a substrate. Conversely, loose interactions of metal A, which are reported to be pivotal to the nucleophilic attack of the coordinated water molecule, seem to correspond to mobility, which possibly benefits product release and the recruitment of a new Mg2+ ion. When comparing the behaviors of the Asp and Glu48 carboxyl side chains involved in Mg2+ coordination, that of the latter appears to be more functionally profound; the longer and more flexible side chain of Glu may allow metal B to form a more ideal octahedral coordination.We obtained a total of five subunits from the three crystal forms. The root-mean-square distances among them for 155 corresponding Cc) shows a structural comparison of the observed doubly Mg2+-bound RNase HI (2Mg2+-RNase HI) with the previously reported singly Mg2+-bound enzyme. The Mg2+ ion present in the singly bound state is in a similar position to metal B in the doubly bound state in RNH_Mg_3, at a distance of 1.43 Å. The side chain of His124 observed in the present research is in a different orientation and Nɛ of the imidazole group is located only 3.45 Å from the Mg2+-coordinated water molecule. Moreover, the unambiguous electron density of the His124 side chain observed in this research also proves the existence of the Nɛ–H2O interaction .Fig. 2on Fig. 2b.3.2.2+-bound enzyme were determined in tetragonal and orthorhombic crystal forms. In both forms, the RNase HI–Zn2+ complex structures indicated that a Zn2+ atom is bound to the active site . Consistent with this structural observation, ITC measurements indicated that the enzyme binds one Zn2+ ion per molecule, with an estimated Kd of 1.09 µM .Structures of the Zn2+ ion in both structures was in a tetrahedral coordination with the imidazole group of His124, the carboxyl groups of Asp10 and Asp70 and a water molecule. However, the orientations of the coordinated His124 and Asp10 were different from each other. In the RNH_Zn_1 structure  the Nɛ atom of the His124 imidazole group forms a coordination bond with the Zn2+ ion, whereas in the case of RNH_Zn_2  the Nδ atom participates in the tetrahedral coordination.The Znre Fig. 3a the Nɛ_2 Fig. 3b the NδKa of E. coli RNase HI His124 in the metal-free state was reported to be around 7, according to the results of 1H-NMR analysis . The side chain of His124 is also located close to Asp70 and H2Oδ−. It has been reported that the resonance corresponding to C2H of His124 shifts significantly on the addition of Mg2+ in 1H-NMR analysis , even though His124 is located in a flexible loop structure. Therefore, it is reasonable to infer that this side chain is firmly fixed by adjacent amino-acid residues or water molecules. H2Oδ− made relatively strong interactions with Mg2+ at the B position and Asp70, with O–X distances of 2.20 and 2.61 Å, respectively, while the O–N distance of 3.45 Å between the imidazole group of His124 and H2Oδ− suggests a weaker hydrogen bond. These detailed views are in good agreement with a scenario involving a His124→H2O→Mg2+→H2O→PO4 carboxylate–hydroxyl relay mechanism , as proposed by the previous NMR analyses /DNA8, indicating inhibition of product release. The present crystal structures in the absence of substrate show that the single Zn2+ ion is located at a considerably different position from the two Mg2+ sites . Thus, it appears to be too difficult to propose even a tentative catalytic scheme, while the Zn2+ ion certainly plays a critical roles in the cleavage reaction. However, one water molecule coordinated to the Zn2+ ion is intriguing in terms of its possible involvement in substrate hydrolysis, for instance, as a candidate for a nucleophilic molecule . Further analyses would be required to propose a clearer catalytic mechanism for Zn2+-bound RNase HI, including knowledge of coordination chemistry based on ternary-complex structure analyses.The Znes Fig. 3b. Thus,ts Fig. 4b. Furth4.3.2+, Mn2+ or Zn2+  showed obvious differences in the Me2+  at the A position adjacent to Asp134 and His124, while the Me2+ at the B position near Glu48 did not substantially change in the case of Mn2+ or Mg2+. It is particularly notable that the imidazole group of His124 has the possibility of interacting either directly with Zn2+ or indirectly with Mn2+ and Mg2+. This differing binding property can be ascribed to coordination-bond formation and electrostatic interaction abilities. Although RNase HI is not a sequence-specific endonuclease, cleavage sites of the same substrate vary depending on the types of metal cations . This result contrasts with the finding that wild-type RNase H never produces such intermediates at some 10 mM level concentrations of Mg2+. Presumably, the His124–H2Oδ− interaction could maintain the Mg2+–His124 interaction with moderate affinity without preventing product release. This notion highlights that the mobility of metal ions during the cleavage reaction may be a critical factor which could be determined by the surrounding protein atoms in the active site.Structural comparison of RNase HI in complex with Mg2+ Fig. 3c showed4.4.E. coli RNase HI is not conserved in B. halodurans RNase H , 7vsbcomplex with one ZnPDB reference: 2+ ion, 7vsccomplex with one MgPDB reference: 7vsdPDB reference: 2+ ion (His124 Nɛ binding), 7vsecomplex with one ZnPDB reference: 10.1107/S2059798322000870/ji5026sup1.pdfSupplementary Figures. DOI:


Herein, the growth of high‐quality phase pure CsPbBr3 perovskite single crystals at room temperature by a humidity controlled solvent evaporation method is reported first. Meanwhile, the room temperature phase transition process from three dimensional (3D) cubic CsPbBr3 to two dimensional (2D) layered tetragonal CsPb2Br5 and the detailed mechanism induced by humidity are revealed. Moreover, compared with the organic–inorganic perovskite, the prepared CsPbBr3 single crystals are much more stable under high humidity, which satisfies the long‐term working conditions of X‐ray detectors. The X‐ray detectors based on CsPbBr3 single crystals show a high sensitivity and a low detection limit of 1.89 μGyair s–1, all of which meet the needs of medical diagnosis.Generally, growing phase pure CsPbBr 2 are rich and diverse, which is a phenomenon of people scratching their heads. In the humidity controlled low‐temperature preparation process, the mechanism of its diversity is uncovered through the transformations of products under the influence of different conditions and a valuable guidance is provided for the subsequent crystal acquisition and growth.The mixed products of CsBr and PbBr Perovskite single crystals can be prepared by the low‐temperature solution method. Compared with the traditional semiconductor preparation technology, it has the advantages of the simple fabrication method, the controllable cost and the reduced complexity of device integration. Moreover, single crystals of perovskites have better air stability than the thin films prepared by the spin‐coating method, which is more suitable for the long‐term application of devices.The application of lead halide perovskites in the field of optoelectronics has attracted extensive research interest in recent years. As a new semiconductor photoelectric conversion material, it has plenty of advantages, such as high light absorption coefficient,1, 2 The direct‐detection semiconductor X‐ray detector has a better sensitivity and energy resolution than the one using a scintillator. Generally, high‐performance direct‐detection semiconductor X‐ray detectors need to have high carrier mobility and charge carrier lifetime product for ensuring the holes and electrons have sufficient drift length, and high resistivity at room temperature. Furthermore, it also has a large average atomic number (Z) to ensure good absorption of radiation. In the case of certain X‐ray photon energy (E), the absorption coefficient (α) of X‐ray is determined by Z4/E3. The adjustability of elements in perovskite makes it have a large material system and also helps to adjust the average atomic number for X‐ray detection.As a conversion device to convert an X‐ray photon signal into an electrical signal, the X‐ray detector is a key part of the X‐ray application system and widely used in medical diagnostic radiotherapy, industrial flaw detection, safety detection, aerospace navigation and material analysis of scientific research, et al.13 The The nature of all‐inorganic perovskite CsPbBr3 that without the volatile organic molecules, which migrate easily under the electric field, makes its long‐term stability much enhanced. Meanwhile, the average atomic number of the all‐inorganic perovskite is larger compared to that of the organic–inorganic hybrid perovskite, which means the all‐inorganic perovskite has a higher absorption coefficient to X‐ray. Therefore, all‐inorganic perovskites like CsPbBr3 have been extensively studied for X‐ray detection. Amid them, the sensitivity of the X‐ray detector using CsPbBr3 thin film with the vertical structure of Au/film/ITO can reach 1700 µC Gyair–1 cm–2, while the highest sensitivity of the vertical Au/CsPbBr3 single crystal/Au X‐ray detector can reach 918 µC Gyair–1 cm–2. Though CsPbBr3 based X‐ray detectors have achieved much progress, most CsPbBr3 single crystals are grown by the high‐temperature melting method and many by‐products (CsPb2Br5 or Cs4PbBr6) appear during the growth process of the low‐temperature solution method, which lead to some difficulties in single crystal growth. Thus, changing the factors that affect its growth to simplify the growth method and controlling the by‐product formation is worth investigating by researchers.As for the direct detection X‐ray detector, the lead‐based perovskite semiconductor has made a series of progress due to its advantages of the high X‐ray absorption coefficient and the high carrier collection efficiency, which proves the great potential of the perovskite in the field of X‐ray detection.15 The3 perovskite single crystals under the room temperature condition. The experimental results show that CsPbBr3 single crystals of different phases can be obtained from the precursor solution under different environmental humidity conditions. Meanwhile, phase pure transparent 2D tetragonal CsPb2Br5 single crystals could be obtained in the solution by this method under extremely high humidity conditions. While for yellow hexagonal Cs4PbBr6 single crystal (emitting green light under 365 nm light‐emitting diode (LED)), it can be prepared at the preparation temperature of 90–100°C. Finally, the planar X‐ray detector based on orthorhombic CsPbBr3 perovskite single crystals shows a high sensitivity of 6021.99 µC Gyair–1 cm–2, a low detection limit of 1.89 μGyair s–1, all of which meet the needs of medical diagnosis. Comparing with the organic–inorganic perovskite, CsPbBr3 single crystals are much stable under high humidity, which is convenient for the material to satisfy the long‐term working conditions of X‐ray detectors.In this work, we utilize a humidity controlled solvent evaporation method to synthesize various CsPbBr23 3D perovskite single crystals were synthesized by a simple solvent evaporation method.Figure  When the process continues, the crystal nucleus overcomes the buoyancy, sinks to the bottom of the solution and keeps growing. After 15 d, the CsPbBr3 single crystal of mm‐sized was obtained. It is worth noting that the ambient humidity could affect the crystal growth behavior of CsPbBr3 single crystal. To investigate this strange phenomenon, XRD tests were performed on two shapes of CsPbBr3 single crystals. Under the condition of low humidity (RH ≈ 20%), the single crystal that grows toward the strip shape is cubic CsPbBr3 . Further to increases the humidity (RH > 80%), the orange CsPbBr3 single crystals gradually turn into opaque white crystals . Moreover, none of the other peaks are detected in the XRD patterns, which confirms its high phase purity for the fabricated cubic CsPbBr3. According to the thermodynamic stability properties of the phase structure reported by Stoumpos et al., the crystal structure of CsPbBr3 is orthorhombic at room temperature, and it could convert to tetragonal phase at 88 ℃ and cubic phase at 130 °C. However, the single‐crystal XRD result of orange CsPbBr3 shows that it belongs to a typical cubic system, with the centrosymmetric space group Pm‐3m , and the crystal direction is along (001) and (110) directions. The single‐crystal XRD result of square CsPbBr3 shows that it belongs to the orthorhombic system, with the centrosymmetric space group Pnma . The single‐crystal XRD result of the CsPb2Br5 crystals shows that it belongs to the tetragonal system, with the centrosymmetric space group I4/mcm  is contributed by Pb 5s orbit and the valence band maximum (VBM) is contributed by Br 5p orbit. Nevertheless, different from the direct band structures of the CsPbBr3 (cubic and orthorhombic phases), CsPb2Br5 shows an indirect band structure, which indicates a lower absorption at the wavelength of around 350 nm. There are also many reports on the crystal formation and degradation of perovskites under the influence of water. Density functional theory (DFT) calculation is used to evaluate the formation energy to further verify the phase transition of crystal growth. The CsBr and PbBr2 unit cells are set as the initial system, and the total energy is 0. First, the formation energies (ΔH) of the cubic CsPbBr3 (ΔHc) and the orthorhombic CsPbBr3 (ΔHo) are calculated to be −0.22 and −0.20 eV, respectively. It shows that the initial system can proceed spontaneously in the direction of forming CsPbBr3, which corresponds to the process (i) and (ii) in Figure 2 are dissolved in dimethyl sulfoxide (DMSO) to grow single crystals. The ΔHc and ΔHo that dissolved in DMSO are calculated to be −0.76 and −0.68 eV, respectively. Clearly, ΔHc is smaller than ΔHo, indicating that the cubic CsPbBr3 is easier to form in DMSO. This result corresponds to processes (iv) and (v) in Figure 2O) molecule is added in this process, the ΔHo is the lowest ). It shows that the crystal growth process involving H2O could greatly affect the crystal phase, which reduces the ΔHo of the orthorhombic CsPbBr3 in DMSO and promotes the reaction to proceed in this direction.In order to further explore the differences of the three structures, their band structures were calculated by Perdew–Burke–Ernzerhof functional within the generalized gradient approximation (GGA‐PBE), as shown in Figure of water.33 Den3, orthorhombic CsPbBr3 and tetragonal CsPb2Br5 are further calculated by DFT. The potential barrier (ΔEp) for the direct phase transition from cubic CsPbBr3 to orthorhombic CsPbBr3 is 0.78 eV . The reason is that Cs+ and Br– combine with OH– and H+ respectively after absorbing H2O, thereby changing the bond length. Without the participation of H2O, two processes are involved when the orthorhombic CsPbBr3 transforms to tetragonal CsPb2Br5: one is the decomposition of CsPbBr3 , and the other is the combination of CsPbBr3 and PbBr2 in the DMSO . However, with the participation of H2O, this process uses CsPbBr3 as a catalyst for the photolysis of water to produce H3O+ and OH– to replace part of the Cs+ and Br– in the crystal structure, thereby extracting hydrates and producing CsBr. The ΔEp required for this process is only 0.03 eV (process V). This result also corresponds to the experimental result that no phase transition occurred in the control group in the oven. Namely, in the process of transforming CsPbBr3 to CsPb2Br5, it is first transformed into orthorhombic CsPbBr3 with the participation of H2O, and then into CsPb2Br5. In addition, the transparent CsPb2Br5 single crystal will not decompose like a white opaque crystal when being heated at 40–120 ℃, which can be explained by the inverse process of (III), (IV), and (V) in Figure Ep of 0.03 eV (corresponding to the electron temperature of 0.026 eV at room temperature (300k)). It is easy to overcome and can occur with mild heating. The CsPb2Br5 single crystal undergoes the reverse process of (IV), and the energy required to overcome the ΔEp is far from being achieved under heating conditions of 40–120 ℃. Therefore, the humidity and heat stability of CsPb2Br5 is good. The reaction process and formula involved in Figure In order to explore the transformation process of orange crystals in DMSO to white opaque crystals, the conversion barriers between cubic CsPbBrarrier ΔEp for thearrier ΔEp for thearrier ΔEp for thearrier ΔEp for thearrier ΔEp for thearrier ΔEp for the4PbBr6 by single‐crystal XRD test . LED with a wavelength of 365 nm is used to illuminate hexagonal Cs4PbBr6 single crystal, which emits strong green light, while cubic CsPbBr3, orthorhombic CsPbBr3 and tetragonal CsPb2Br5 single crystals have no luminescence phenomenon under the same condition  is approximately at the wavelength of 390 nm, corresponding to an optical bandgap of ≈3.18 eV  peak position of orthorhombic CsPbBr3 single crystal is located at 512 nm under a 375 nm laser excitation  is 16.96 ns, in which the fast decay lifetime (t1) and the slow decay life (t2) are 24.3 ns and 2.3 ns, respectively, accounting for 66.6% and 33.4%. The SEM images of orthorhombic CsPbBr3, cubic CsPbBr3, CsPb2Br5 and Cs4PbBr6 single crystals are shown in Figure 3 at the scales of 5 µm and 1 mm, respectively, confirming that the CsPbBr3 single crystals grown by the solvent evaporation method at the low temperature have high quality. The size of the whole CsPb2Br5 crystal is small, but its quality is better, as shown in SEM  product is also one of the factors affecting the X‐ray detection capability of devices, and the value reflects the electrons extraction ability of materials. By using the modified Hecht equation (supporting information equation 1), the photoconductivity curve of CsPbBr3 single‐crystal vertical device can be fitted that its µτ product value is about 5.14 × 10–3 cm2·V–1, as shown in Figure 3 single crystals prepared by melt growth method, which reflects the high quality of orthorhombic CsPbBr3 single crystal grown by low‐temperature solvent evaporation method is realized. The planar device structure is utilized for the measurement to obtain the X‐ray response characteristics. Figure Idark–V diagram of reverse sweep from 50 to −50 V in the dark state of the device. The Idark–t diagram of the device under 5 V bias shows that the Idark of the device does not drift with the time change, and its value is stable around 21 pA, as shown in Figure I–V curves of the CsPbBr3 single‐crystal X‐ray detector measured with different dose rates from 103.14 to 5293 μGyair s−1. The I–V curves of the device are smooth under different X‐ray dose rate irradiations and increase gradually with the augments of X‐ray dose rate (with a positive correlation).In general, the higher the average atomic number of a material, the more X‐ray absorbability it has. The X‐ray absorption coefficients of CsPbBrrealized.24 The3 single‐crystal X‐ray detector under different voltage biases (from 50 to 5 V) and dose rates (from 852.75 to 103.14 Gyair s–1) to explore the device sensitivity. The ON/OFF current response of CsPbBr3 single crystal devices at 50 V bias voltage is shown in Figure air s–1. The device continues to switch at 50 V high voltage, maintaining good switching characteristics. The relationship between the gain current density and the radiation dose rate of the CsPbBr3 single‐crystal X‐ray detector, tested at 5, 10, 20, 30, 40, 50 V bias voltage, is shown in Figure 3 single‐crystal X‐ray detector device at a low voltage bias of 5 V can reach 2549.36 µC Gyair–1 cm–2. When the applied voltage is increased to 50 V, the sensitivity can reach 6021.99 µC Gyair–1 cm–2. The high sensitivity of CsPbBr3 is not only related to the structural symmetry of lead‐based perovskite itself, but also due to the unique electronic configuration ns2np0 of Pb2+. Furthermore, compared with vertical or horizontal structure devices based on different materials, this parameter should be among the highest values .  and a steady current under a fast switching test at low bias voltage (5 V) and high dose rate (5292.9 μGyair s–1). Figure 3 single‐crystal X‐ray detector has good operating stability and current remains steady at 5293.9 μGyair s–1 with ± 5 V bias voltage. These two figures show that the device performance is not degraded and still maintains good performance under high‐dose X‐ray irradiation. To sum up, CsPbBr3 is a promising perovskite material for X‐ray detectors.The X‐ray radiation can cause radiation damage to creatures. Therefore, another requirement for commercial X‐ray detectors is to pursue extremely low detection limits to reduce radiation loss. Many pieces of literature employ signal‐to‐noise ratio (SNR) = 3 recommended by IUPAC as the signal detection limit.42 Bas33 perovskite single crystals were synthesized by a simple solvent evaporation method at room temperature. Under high humidity (80%) condition, 3D cubic CsPbBr3 perovskite can be transformed into CsPb2Br5 hydrate during growth, which is thermally unstable. 2D tetragonal CsPb2Br5 single crystal grown directly under RH 80% condition has good humidity and heat stability. Yellow hexagonal Cs4PbBr6 single crystal (emitting green light under 365nm LED) can be prepared at the preparation temperature of 90–100 °C. Most importantly, the planar‐structured CsPbBr3 perovskite single crystal X‐ray detector shows high sensitivity  and a low detection limit of 1.89 μGyair s–1 . Compare with the organic–inorganic perovskite, CsPbBr3 single crystals are still not decomposed under high humidity and have better air stability, which is convenient for the material to satisfy the long‐term use conditions of X‐ray detectors.High‐quality 3D CsPbBr43 3D perovskite single crystals were synthesized by a simple solvent evaporation method. 1.5975g CsBr  and 2.7525g PbBr2  were dissolved in 30 ml DMSO  at a molar ratio of 1:1 to generate 0.25 m CsPbBr3 precursor. The precursors were stirred constantly in the atmospheric environment for 24 h at room temperature (25 °C). After filtration with a 0.45 µm organic filter head, the precursor solution was clarified and transferred to a precleaned crystallizing dish. The crystallizing dish was covered to prevent dust or impurities from falling contamination and to reduce the evaporation rate of the solution to obtain higher quality crystals. After that, the whole unit was placed in the oven and kept at 27 °C. Several days later, CsPbBr3 3D perovskite single crystals were obtained with a size of a few millimeters. The cubic CsPbBr3 single crystals (tended to be a long strip) were grown in the low humidity growing environment. However, with increasing humidity, the growth tended to be an orthorhombic phase crystal structure .CsPbBr3, orthorhombic CsPbBr3, CsPb2Br5, and Cs4PbBr6 single‐crystal XRD measurements were performed with Bruker D8 Venture with Mo Kα X‐rays. SEM images of cubic CsPbBr3, orthorhombic CsPbBr3, CsPb2Br5, and Cs4PbBr6 were collected with a tungsten filament scanning electron microscope (HITACHI SU‐3500). Absorption spectra were measured using a Perkin–Elmer Lambda 950 UV–vis–NIR spectrophotometer. Steady‐state and time‐resolved PL measurements of cubic CsPbBr3 and orthorhombic CsPbBr3 were taken using a PicoQuant FT‐300 and FT‐100, with 375 nm excitation wavelength. All material characterizations were measured in the air without encapsulation.XRD was performed with a Bruker D8 Discover X‐ray diffractometer with a conventional Cu target X‐ray tube set to 40 kV and 40 mA. Cubic CsPbBr3 grown in a planar shape was simpler than the cubic CsPbBr3 grown in a stripe shape to make a planar X‐ray detector electrode. The X‐ray detector prepared later used the orthorhombic CsPbBr3. Two kinds of X‐ray devices were prepared by gold electrodes deposition on orthorhombic CsPbBr3 perovskite single crystal for testing. Au/CsPbBr3/Au vertical structure devices  dosimeter. All X‐ray response characterizations were performed directly in the dark air with optical and electrical shielding to reduce electromagnetic and ambient light interference. All measurements were performed at room temperature.The orthorhombic CsPbBr The exchange‐correction functional was described by the Generalized Gradient Approximation with the Perdew–Burke–Ernzerhof functional. The transformation barriers were calculated using the climbing image nudged elastic band (CI‐NEB) method through the VTST tools. The plane‐wave cutoff energy was set to be 400 eV. The Monkhorst−Pack k‐point mesh is sampled with a separation of 0.05 and 0.015 Å−1 in the Brillouin zone. All structures were relaxed until the residual force on each atom was less than 0.01 eV Å−1. The self‐consistent convergence accuracy was set at 10–5 eV per atom in the structural calculation, and 10–7 eV per atom in the CI‐NEB calculation. The formation energies were obtained from the total energy variations of the chemical processes listed in the Supporting Information.All calculations were carried out by using density functional theory based on the projector‐augmented wave method implemented in the VASP code.56 TheThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.