Escherichia coli showed that these mAbs recognized a total of seven (1–7) linear B cell epitopes. These mAbs did not show significant neutralizing activity. Epitopes 1 (249HKPHQSKPIL258), 2 (69PVGFEYENKV77), 5 (195VNGNSLDEYSS205), and 7 (223GYKHLVGQEV233) are novel. Sequence alignment analysis revealed that the identified epitopes were highly conserved among 27 ASFV strains from nine genotypes. Preliminary screening using known positive and negative sera indicated the diagnostic potential of mAb-2B8D7. The results provide new insights into the antigenic regions of ASFV p72 and will inform the diagnosis of ASFV.African swine fever virus (ASFV) causes a highly lethal hemorrhagic viral disease (ASF) of pigs that results in serious losses in China and elsewhere. The development of a vaccine and diagnosis technology for ASFV is essential to prevent and control the spread of ASF. The p72 protein of ASFV is highly immunogenic and reactive, and is a dominant antigen in ASF vaccine and diagnostic research. In this study, 17 p72 monoclonal antibodies (mAbs) were generated. Epitope mapping by a series of overlapping peptides expressed in  The virus is the only member of the rbovirus . The ASFrbovirus . It encorbovirus . Recent rbovirus . From ourbovirus .ASF cases were first reported in Kenya in 1921, with subsequent spread to western Europe and Latin America . In 2007The p72 protein is encoded by the B646L gene and is the main structural protein of ASFV, accounting for 31 to 33% of the virus particle . p72 forDetailed analyses of epitopes provide a deep understanding of the roles of amino acids in the epitopes and viral replication . HoweverIn the present study, we obtained 17 monoclonal antibodies (mAbs) against p72, which did not show significant neutralizing activity. In addition, four novel and highly conserved linear epitopes of p72 were identified. We developed a competitive ELISA (cELISA) based on mAb-2B8D7. This cELISA method could specifically distinguish ASF-negative and positive sera. These results significantly increase the understanding of the epitopes of p72 and provide valuable information for the development of diagnostic methods and subunit vaccines.2.The animal experiments on mice were approved by the Animal Care and Use Committee of Lanzhou Veterinary Research Institute. The experimental procedures were performed in strict accordance with the recommendations in the nursing Guide, Laboratory Animals and Use, Ministry of Science and Technology, PRC.3.3.1.BALB/c, 6- to 8-week-old, female, specific pathogen-free mice were purchased from the Laboratory Animal Center of Lanzhou Veterinary Research Institute. The ASFV/II/CN/SC/2019 strain was preserved in the P3 biosafety laboratory of Lanzhou Veterinary Research Institute. SP2/0 and SF9 were stored in our laboratory. Commercially available primary porcine alveolar macrophages (PAMs) isolated from lung tissues of healthy pigs according to 3.2.Escherichia coli BL21(DE3). A single colony that developed was incubated in LB medium at 37°C with shaking at 200 rpm until the optical density at 600 nm (OD600) was approximately 0.6. Then, isopropyl β-d-1-thiogalactopyranosid (IPTG) was added  to induce the expression of p72 protein during a 16 h incubation at 20°C. Bacteria were recovered by centrifugation at 5,000 g for 10 min. To purify p72, the bacteria were resuspended in inclusion body buffer  and sonicated on ice. After centrifugation, the supernatant was removed, and the pelleted inclusion bodies were resuspended in IB solubilization buffer  and spun at 4°C overnight. Supernatants were harvested following centrifugation at 10,000 × g for 20 min. Each supernatant was purified using Ni-NTA and the eluted protein was analyzed for purity by SDS-PAGE. The purified protein was dialyzed for renaturation. In brief, the purified protein was placed in a dialysis bag  and renaturated in renaturation buffer 1  for 12 h at 4°C. The dialysis bag was then transferred to renaturation buffer 2  for 12 h followed by renaturation buffer 3  for 12 h.The p72 gene was retrieved from the GenBank database (GenBank MH766894.1), optimized for synthesis, and cloned into a pET-SUMO vector. The recombinant plasmid was transferred into 3.3.The mice were immunized with 20 μg renaturated p72 protein emulsified in equal proportions with Freund’s complete adjuvant or Freund’s incomplete adjuvant . The immunizations were performed four times with 2-week intervals. Three days after the fourth immunization, spleen cells were harvested and fused with SP2/0 cells. Confluent cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing HAT Supplement (Sigma-Aldrich) and 20% fetal bovine serum (FBS). After 8 days, supernatants from confluent cells were collected and detected by indirect ELISA using wells coated with p72 recombinant protein. Positive wells were selected and subcloned three times by limited dilution. The positive clones were cultured in DMEM containing 20% FBS and 1% penicillin–streptomycin.6 hybridoma cells were injected into the abdominal cavity of the mice. Ascites was collected after 9–10 days and purified using affinity chromatography . The purified antibodies were determined using indirect ELISA. Subtypes of mAbs were identified by SBA Clonotyping System-HRP Kit . Specificity of mAbs against recombinant proteins and viruses was evaluated by western blotting and immunofluorescence assay.To prepare ascites containing antibodies against the p72 protein, 0.5 ml of ascites adjuvant  was injected into the abdominal cavity of 12-week-old female Balb/c mice. After 15 days, approximately 1.0 × 103.4.6 cells/well; Corning, New York, NY, United States) cultured with RPMI-1640  containing 15% FBS. Twelve hours after seeding, PAMs were infected with ASFV/II/CN/SC/2019 at a multiplicity of infection of 0.1 for 72 h. The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 1 h. After washing three times with PBS, the cells were permeabilized with 0.25% Triton X-100 for 10 min. After blocking with 5% BSA for 2 h, the cells were incubated with the hybridoma supernatant (1:10 dilution) at 37°C for 1 h. After washing with PBS, a 1:1000 dilution of tetramethyl rhodamine isothiocyanate (TRITC) conjugated goat anti-mouse IgG  was added and incubated for 1 h. After washing three times with PBS, the cells were observed by inverted fluorescence microscopy using a model DM16000B microscope .PAMs were seeded in 24-well plates (1.0 × 103.5.E. coli BL21 (DE3), expressed as described above, and finally analyzed by SDS-PAGE and western blot analysis.To locate the epitopes recognized by each mAb, a series of overlapping peptides were designed based on the full-length p72 gene . A total3.6.To verify the conservation of the seven epitopes in different ASFV genotypes, the representative sequences of 27 isolates from nine different genotypes were aligned using Clustal W to determine whether the mutation occurred in these epitopes.3.7.Samples were mixed with 4× loading buffer and heated at 100°C for 10 min. Proteins in each sample were separated by 12% SDS-PAGE. After the gel was stained with Coomassie blue staining solution for 10 min, the bands were decolorized with decolorization solution and analyzed. For western blot analysis, protein samples were electro-transferred to polyvinylidene difluoride (PVDF) membranes . After blocking with 5% skim milk powder at 37°C for 2 h, hybridoma cell supernatant dilution (1:10 dilution) was added and incubated overnight at 4°C. After washing three times with PBS-Tween (PBST), a 1:5000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG  was added and incubated at 37°C for 1 h. After washing three times with PBST, enhanced chemiluminescence (ECL) reagent  was added. Blots were scanned and analyzed using the Odyssey CLx imaging system .3.8.2S04 was added, and OD values were measured at 450 nm using a microplate reader (Thermo Fisher Scientific).Wells of 96-well plates were coated with p72 recombinant protein diluted in CBS to a final concentration of 0.25 μg/ml at 4°C overnight. Plates were washed three times and blocked with 5% skim milk at 37°C for 2 h. Hybridoma cell supernatant and ascites were diluted and added to the plate in a volume of 100 μl/well and incubated at 37°C for 30 min. After washing three times with PBST, goat anti-mouse IgG-HRP (1:10000 dilution) was added as the secondary antibody and incubated at 37°C for 30 min. After washing three times with PBST, 50 μl of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution  was added and incubated at 37°C for 10–15 min. Fifty microliter of 2 M H3.9.6/mL were added to wells of a 24-well culture plate and cultured in RPMI-1640 medium containing 15% FBS in an atmosphere of 5% CO2 at 37°C for 12 h. The purified mAbs, diluted to a final concentration of 5 μg/ml, were mixed with ASFV CN/SC/2019 (median tissue culture infectious dose [TCID50] of 200) and incubated at 4°C overnight. The mixture of virus and mAbs was inoculated into wells containing PAMs. After 1 h, the mixture solution was discarded. After washing three times with PBS, RPMI-1640 medium containing 5% FBS was added and cultured for 72 h. After three cycles of freezing and thawing in a culture plate, the ASFV genomes of each well were extracted using a virus genome extraction kit  and amplified by qPCR .PAMs adjusted to 1 × 10The inactivated ASFV CN/SC/2019 was used as a template for PCR amplification using primer p72-F (5′-GGAATTCCATATGGCATCAGGAGGAGC-3′) and p72-R (5′-CCGCTCGAGTTCTTAAACCCCGCAAAT-3′). PCR amplification and pET-28a vector were digested with BamH I and Xho I enzymes, then ligated with T4 ligase to form the recombinant plasmid pET28a-p72. The recombinant plasmid diluted 10 times continuously was used as template for qPCR amplification. The logarithmic value of plasmid concentration and Ct value were analyzed by linear regression. The copy number of the B646L gene in each sample was calculated according to the Ct value and the established standard curve. The virus neutralization rate (%) was calculated as:3.10.2SO4 was added, and OD values were measured at 450 nm using a microplate reader (Thermo Fisher Scientific). Using the determined optimized coating concentrations of antigens, dilutions of mAbs, and serum dilution, the ratios of negative serum to positive serum (N/P) were calculated. A greater N/P ratio indicated that ASFV-infected sera was better able to block the binding of mAbs to p72 protein.To verify the diagnostic feasibility of the 17 mAbs, preliminary screening was performed by cELISA. In brief, wells of 96-well plates were coated with p72 recombinant protein diluted with CBS to a final concentration of 0.125–8 μg/ml at 4°C overnight. Wells were washed three times using PBST and then blocked with 5% skim milk at 37°C for 2 h. ASF standard positive and negative sera were diluted (1:5–1:640) and added to the wells (100 μl/well) and incubated at 37°C for 1 h. After washing three times with PBST, mAbs were diluted  and added to the wells and incubated at 37°C for 30 min. After washing the wells three times with PBST, goat anti-mouse IgG-HRP (1:10000 dilution) was added and incubated at 37°C for 30 min. After washing three times with PBST, 50 μl of TMB substrate solution (Surmodics) was added and incubated at 37°C for 10–15 min. Fifty microliter of 50 μl 2 M H4.4.1.E. coli. SDS-PAGE confirmed that the expressed form of the protein was an inclusion body. As expected, the apparent molecular weight was approximately 100 kDa  had poor reactivity with ASFV . We furt4.3.E. coli expression system and analyzed by western blotting. The recognized epitopes were located at p72-2 (6H2C5), p72-4 (2C9D6), p72-5 (4F6D4), p72-4 and − 72-5 (3D4A9), p72-6 , and p72-5 and p72-6  spanning p72 (1–646 amino acids) were expressed using an by 4F6D4 ,C. The eby 4F6D4 .4.4.Sequence alignment of 27 ASFV isolates from nine genotypes revealed that the seven epitopes identified by the mAbs obtained in this study were highly conserved .4.5.2 = 0.9923). According to the curve, seven mAbs recognizing seven epitopes (divided into four groups) were incubated with ASFV (200 TCID50) and inoculated into PAMs. The copy number of the B646L gene in each sample was calculated according to the Ct value and the established standard curve. Each group was repeated three times. The data were expressed as mean ± standard error of the mean, and the significance of the differences between groups was calculated by GraphPad Prism Software . p > 0.05 indicated no significant difference . None of the anti-p72 mAbs displayed significant neutralizing activity against ASFV ] × 100%. The results showed that the PI values of six positive sera were 93.56, 95.13, 92.61, 91.25, 90.15, and 88.63%, and PI values of negative serum were 9.34%. Therefore, the preliminary results show that mAb-2B8D7 could distinguish negative and positive sera.To determine whether the 17 mAbs could be used in diagnosis, these mAbs were preliminarily selected according to their N/P ratio in the same conditions of an antigen coating concentration of 0.25 μg/ml, hybridoma cell supernatant dilution of 1:5, and serum dilution of 1:10 . Only fo5.ASFV has a history of more than 100 years worldwide . Yet, cu the ASF . p72 dis the ASF  and is u the ASF . Identif158VDPFGRPIV166) recognized by mAb 2C9D6, epitope 6 (271TNPKFLSQHF280) recognized by mAbs 2B8F7 and 2B8B9, and epitope 3 (281PENSHNIQTA290) recognized by mAbs 6D6D9, 2B7B6, 5C9E3, 5C9D6, and 2B8D7 were similar to the study by Heimerman et al.  recognized by mAb 6H2C5, epitope 7 (223GYKHLVGQEV233) recognized by mAb 4F6D4, epitope 5 (195VNGNSLDEYSS205) recognized by mAb 3D4A9, and epitope 1 (249HKPHQSKPIL258) recognized by mAbs 4E10B3, 4E10C9, 4F6B8, 4D4C8, 4E10B2, and 6B10E8 have never been reported in previous studies. Furthermore, four linear epitopes  have been reported by To obtain more comprehensive mAbs and expand the epitope map of p72, the full-length p72 protein was presently expressed using prokaryotic system to immunize the mice. Seven linear B cell epitopes (epitopes 1–7) were identified using the 17 mAbs obtained. Epitope 4 . Four of the linear epitopes  are novel. These epitopes are highly conserved among the nine genotypes of ASFV. In addition, mAb-2B8D7 that recognizes epitope 3  was screThe datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by Animal Ethics Committee of LVRI, CAAS.HC and JS performed the conceptualization and funding acquisition. WL performed the formal analysis, review and editing, and funding acquisition. CM and SY performed the methodology, formal analysis, clinical sample detection methodology, and writing-original draft. GZ, YM, SW, ZH, and DP performed the material preparation investigation. All authors contributed to the article and approved the submitted version.This study was financially supported by the National Natural Science Foundation of China (32273038) and the National Key Research and Development Program of China (2021YFD11801402).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fmicb.2023.1126794/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.


By measuring five Bragg reflections from a single isolated microcrystal at ambient conditions, we compare the full three-dimensional (3D) strain state before corrosion and the strain along the [111] direction throughout the corrosion process. We find that the strained surface layer of the crystal dissolves to leave a progressively less strained surface. Interestingly, the average strain closer to the centre of the crystal increases during the corrosion process. We determine the localised corrosion rate from BCDI data, revealing the preferential dissolution of facets more exposed to the acid stream, highlighting an experimental geometry effect. These results bring new perspectives to understanding the interplay between crystal strain, morphology, and corrosion; a prerequisite for the design of more corrosion-resistant materials.Corrosion is a major concern for many industries, as corrosive environments can induce structural and morphological changes that lead to material dissolution and accelerate material failure. The progression of corrosion depends on nanoscale morphology, stress, and defects present. Experimentally monitoring this complex interplay is challenging. Here we implement  in situ Bragg coherent X-ray diffraction imaging.Morphology, 3D lattice strain, and dissolution of a Co–Fe microcrystal was monitored using  However, often the corrosion rates reported in the laboratory setting and in the field are inconsistent, despite well-controlled extrinsic conditions of the experiment.4–6 Frequently, the cause for the corrosion rate discrepancy is based on intrinsic factors related to the properties of the sample,4,5 for example, morphological features and lattice defects are not accounted for in the bulk dissolution rate determined from mineral powders. Crystals possess heterogeneous spatial reactivity on the crystal surface,1,4,5 as reaction rates tend to differ along facets, edges, and vertices.7,8 In addition to these morphological features, dissolution rates can be influenced by internal features such as crystal defects. For example, dislocation-induced etch pits9,10 are a commonly reported result of the interaction between defect-induced strain fields and corrosion. This highlights the importance of studying defect-mediated corrosion and the effects of lattice strain on local nanoscale corrosion. Consolidating our understanding of these processes will aid in the design of more corrosion resistant materials.Corrosion processes are important in a wide variety fields such as geology,11 atomic-force-microscopy (AFM),12 digital holographic microscopy,13 and phase-shift interferometry14 are used to probe surface dissolution rates. However, these methods primarily focus on measuring upwards-facing sample features at the surface. Liquid cell transmission electron microscopy (LCTEM) techniques have been used to study pitting corrosion,15 but these studies focus on thin samples where surface effects are dominant, and are prone to bubble formation due to radiolysis.16Ab initio molecular dynamics simulations (AIMD) have been used to predict the energy barriers of dissolution for crystal edges, corners, and kinks,17 but we note that the direct determination of dissolution rate for spatially resolved surface sites remains uncommon in the laboratory. Three-dimensional localised dissolution information has only been recently reported using transmission X-ray microscopy (TXM)18 and three-dimensional (3D) X-ray microtomography (XMT).6,8 Although TXM and XMT provide a spatial resolution on the order of 50 nm, they are limited to probing morphological features.Corrosion mechanisms start with the interaction of the crystal surface with a corrosive medium. Techniques such as interferometry (VSI),19 Areas having dislocations, grain boundaries, and high surface roughness dissolve faster than defect-free regions.1 Indeed, it was through the appearance of etch pits that dislocations were first visualised.20 Experiments have shown that crystal lattice strain can noticeably increase the dissolution rate for calcite19,21 and sodium chlorate crystals22 compared to theoretical values. A full picture of crystal corrosion mechanisms can therefore only be obtained by also studying how the evolution of defect networks, lattice strains within the crystal and local morphology modify the local corrosion characteristics.Crystal lattice defects beneath the crystal surface must also be considered when investigating corrosion. Defects can induce strain in the crystal lattice, which changes the activation barrier for dissolution near the defect.23,24 BCDI allows 3D-resolved, nanoscale strain measurements with a 3D spatial resolution of 10–30 nm and a strain resolution on the order of ∼2 × 10–4,25 making it well suited to probing crystal defects and morphology. Currently, at third generation synchrotron light sources, several minutes are required to collect a BCDI dataset. At these timescales, BCDI allows the in situ probing of slow dynamic changes during the corrosion process. Several in situ BCDI experiments have been reported, for example, Pt nanocrystals during methane oxidation,26 silver–gold alloys undergoing nitric acid-induced dealloying,23,24 and magnetite reacting with hydrochloric acid (HCl).27To elucidate how both crystal strain and morphology influence localised corrosion, we use Bragg coherent X-ray diffraction imaging (BCDI) to study the evolution of a Co–Fe alloy microcrystal. A binary alloy was chosen for this study to identify how strain distributions can be affected by the presence of another element during corrosion.hkl reflection, the diffraction pattern is collected on a pixelated area detector positioned perpendicular to the outgoing wave vector in the Fraunhofer diffraction regime. By rotating the sample through the Bragg condition, a 3D coherent X-ray diffraction pattern (CXDP) is collected as different parts of the 3D Bragg peak sequentially intersect the Ewald sphere in reciprocal space, which is projected onto the detector.BCDI involves illuminating a single crystal sample with a coherent X-ray beam. The sample must be sufficiently small to fit within the coherence volume of the beam, which typically restricts the sample dimensions to less than 1 μm at third generation synchrotron sources. Once the Bragg condition is met for a specific  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="Fscr" format="PBM" resolution="300" x-size="16" y-size="11" xml:space="preserve">00000011111111100000010000100000000010010100000000010010010000000001010011011000000010011111000000000011110000000000001100000000011000100000000001001110000000000011100000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="22.363636pt" height="16.000000pt" viewBox="0 0 22.363636 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M480 840 l0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -80 0 -80 40 0 40 0 0 -40 0 -40 40 0 40 0 0 40 0 40 40 0 40 0 0 40 0 40 40 0 40 0 0 40 0 40 40 0 40 0 0 40 0 40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 80 0 80 40 0 40 0 0 40 0 40 40 0 40 0 0 40 0 40 160 0 160 0 0 -40 0 -40 -40 0 -40 0 0 -80 0 -80 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -40 0 -40 -40 0 -40 0 0 -120 0 -120 -80 0 -80 0 0 -40 0 -40 -80 0 -80 0 0 40 0 40 40 0 40 0 0 40 0 40 -80 0 -80 0 0 -80 0 -80 40 0 40 0 0 -40 0 -40 120 0 120 0 0 40 0 40 80 0 80 0 0 80 0 80 40 0 40 0 0 40 0 40 80 0 80 0 0 40 0 40 80 0 80 0 0 40 0 40 40 0 40 0 0 40 0 40 -80 0 -80 0 0 -40 0 -40 -40 0 -40 0 0 120 0 120 40 0 40 0 0 40 0 40 160 0 160 0 0 40 0 40 -360 0 -360 0 0 -40z"/></g></svg>, of the complex electron density, ρ(r), where r is the position vector, of the crystalline volume for a particular crystal reflection. The phase, ψ(r), corresponds to the projection of the lattice displacement field, u(r), onto the Bragg vector, Qhkl, of the crystal reflection under consideration:The intensity of the diffraction data is proportional to the magnitude of the Fourier transform, Ihkl of the CXDP, as Ihkl ∝ |(ρ(r)eiψhkl(r))|.2 In order to facilitate recovery of the phase, the CXDP must be oversampled by at least twice the Nyquist frequency,28 at least 4 pixels per fringe period. Iterative phase retrieval algorithms that apply constraints in real and reciprocal space are then used to recover the phase.29 The result is transformed from detector conjugated space to orthogonal lab or sample space.30However, the phase is lost because we only record the square of the amplitude in the intensity, in situ BCDI. Prior to corrosion, we measured five crystal reflections to determine the full lattice strain and rotation tensor, providing a clearer understanding of the defects initially present inside the crystal. During the corrosion, we measure the (111) reflection at 2.6, 4 and 6 hours after the start of acid exposure to examine how the 3D strain, crystal morphology, and local variations in dissolution rate evolve as a function of corrosion. The experiment provides a new perspective for the analysis of corrosion, by probing how both strain and morphology affect the localised dissolution of the crystal.Here, we image the spatial and temporal evolution of a Co–Fe alloy microcrystal submerged in an aqueous HCl solution (pH = 0.65) using 22.1C-plane orientation). The thickness of the film was 100 nm. The thin film was dewetted in a vacuum furnace purged with a 5% hydrogen, balance argon, gas mixture at 1250 °C for 16 hours. The resulting crystals exhibit a face-centred cubic structure, range from 100 nm to 1 μm in size,  analysis was used to determine the elemental composition of the crystal (The samples were coated with 10 nm of amorphous carbon to assist with scanning electron microscope (SEM) imaging. The coating was applied through thermal evaporation using a Leica ACE600 Coater. SEM images were taken on a ZEISS Auriga focused ion beam (FIB)-SEM equipped with an in-lens detector with an accelerating voltage of 7 kV. To facilitate reliable measurement of multiple reflections from a specific microcrystal, Ga ion FIB was used to remove the surrounding crystals within a 20 μm radius using a current of 150 pA – 6 nA. The isolated crystal is shown in  crystal . All ele2.231Synchrotron X-ray diffraction measurements were performed at the Advanced Photon Source (APS), Argonne National Lab, USA. Prior to BCDI measurements, micro-beam Laue diffraction at beamline 34-ID-E was used to determine the lattice orientation of the crystal. This served to pre-align the crystal for BCDI measurements at beamline 34-ID-C, as previously described.λ = 0.138 nm), with a bandwidth of δλ/λ ≈ 1.3 × 10−4 from a Si(111) monochromator. The X-ray beam was focused to a size of 740 nm × 680 nm  using Kirkpatrick–Baez (KB) mirrors. Beam defining slits were used to select the coherent portion of the beam at the entrance to the KB mirrors. For beamline 34-ID-C, the transverse coherence is ξh > 10 μm and the longitudinal coherence is ξw ≈ 0.7 μm at an energy of 9 keV.32The substrate was attached to the fluid flow cell  providedp, of 55 μm × 55 μm positioned at 0.75 m from the sample to ensure oversampling. CXDPs were recorded by rotating the crystal through an angular range of 0.7° and recording an image every 0.005° with 0.1 s exposure time and 35 accumulations at each angle. The lower bound for the detector distance was determined by 2dp/λ = 0.50 m, where d is the sample size.CXDPs were collected on a 256 × 256 pixel Timepix area detector (Amsterdam Scientific Instruments) with a GaAs sensor and pixel size, 33,34 CXDPs were corrected for dead-time, darkfield, and whitefield prior to cross-correlation alignment. CXDPs from the following reflections were collected (the number of repeat scans that were averaged is noted in  brackets): (111) [10], (1̄1̄1) [9], (11̄1̄) [9], (200) [10], (002) [10]. Details regarding the recovery of the real space images using phase retrieval algorithms can be found in appendix A and the computation of the strain can be found in appendix B.To optimise the signal to noise ratio and increase the dynamic range of the CXDPs, multiple repeated scans of each reflection were performed at ambient conditions and aligned to maximise their cross-correlation. Once aligned, the minimum acceptable Pearson cross-correlation for summation of CXDPs from a specific Bragg reflection was chosen to be 0.981, similar to other BCDI studies.2.3−1, pH = 0.65) was injected at a flow rate of 40 mL min−1 during the full 6 hours. Single scans for the (111) reflection for the crystal were measured at 2.6, 4 and 6 hours after the start of acid injection. CXDPs were recorded and processed using the same procedure as before corrosion.The fluid flow cell was covered with a 0.0508 mm thick mylar film. HCl solution . Furthermore, since the crystal remained attached to the substrate while measurements were performed, all the reconstructions were aligned such that the bottom surface of the crystal remained in the same position.−1. From 2.6–4 h, the average corrosion rate approximately doubles to 0.0074 nm s−1. Finally, from 4–6 h the corrosion rate decreases to 0.0033 nm s−1.et al. shows that the corrosion of 316 stainless steel coated with 200 nm of carbon has a corrosion current density two orders of magnitude smaller and a higher corrosion potential in a prototypical fuel cell environment with a pH of 2.38 Thus the lower initial localised dissolution rate from 0–2.6 h .ex situ measurements, repeated scans are typically aligned and summed to improve reconstruction quality. However, due to the constant evolution of the crystal, only slow changes in corrosion can be probed. Pre-corrosion measurements showed no evidence of beam induced sample damage based on the high cross-correlation of repeated scans. We therefore attribute subsequent changes of the sample during in situ corrosion entirely to the corrosion of the sample itself. X-ray radiation can cause excess heat and the ionisation of water,43 both of which can accelerate the corrosion rate. This must be considered when using in situ BCDI to study wet chemistry processes.Here, the BCDI rocking scans required just under 10 minutes to collect. For 5This prototypical study demonstrates the feasibility of using BCDI to understand how strain and morphology contribute to nanoscale corrosion rate. We note that analysing localised dissolution is critical to understanding how defects effect the nature of corrosion. Here, we observe that localised corrosion rates can be up to two times higher than the average corrosion rate of the crystal. Regions such as vertices and edges are shown to corrode faster than facets, leading to a progressively rounder crystal morphology. Furthermore, the average lattice strain on the surface is lower than the strain just below the surface of the crystal, suggesting that corrosion leads to a relaxation of surface strain. These findings would not be apparent when analysing changes in average facet size and average facet-normal distance. Finally, we demonstrate that corrosion at the sub-micron level appears to be heavily dependent on environmental conditions, as the top facets that are more exposed to the flowing fluid corrode more quickly than bottom facets.in situ study of how corrosion progresses and interacts with the crystal morphology, defects and strain distribution. This insight to transient structures is not accessible via any other technique and can be crucial in understanding the driving mechanisms of corrosion. In turn, this may enable the design of better corrosion-resistant materials in the future, e.g. by enabling the selection of corrosion-resistant textures in polycrystalline samples.BCDI uniquely allows the 610.5281/zenodo.5548084.The processed diffraction patterns, final reconstructions, and data analysis scripts are publicly available at AThe reconstruction process of CXDPs was executed in stages, using the output from the previous stage to seed the next phasing stage (listed below). Initially, CXDPs were padded to a size of 256 × 256 × 144 voxels.44 with 40 individuals and four generations was used with a geometric average breeding mode. For each generation and population, a block of 20 error reduction (ER) and 180 hybrid input–output (HIO) iterations, with β = 0.9, was repeated three times. This was followed by 20 ER iterations to return the final object. The shrinkwrap algorithm45 with a threshold of 0.1 was used to update the real-space support every iteration. The σ for the Gaussian kernel shrinkwrap smoothing for each generation was σ = 2.0, 1.5, 1.0, 1.0 respectively. The best reconstruction was determined using a sharpness criterion, as it is the most appropriate metric for crystals containing defects.46 The two worst reconstructions were removed after each generation.1. Each reconstruction was seeded with a random guess. A guided phasing approach47 Starting from the 50th iteration, the MCF was updated every 50 iterations using 20 iterations of the Richardson–Lucy deconvolution algorithm.482. The reconstruction was seeded with the output from stage 1. This stage was identical to stage 1, except now we account for the longitudinal and transverse partial coherence using a mutual coherence function (MCF).β = 0.9, was repeated 15 times, followed by 1000 ER iterations. The final 50 iterates were averaged to produce the final image.3. The reconstruction was seeded with the output from stage 2. This stage was identical to stage 2, except now we use a guided phasing approach with 10 individuals and four generations. For each generation and population, a block of 20 error reduction (ER) and 180 hybrid input–output (HIO) iterations, with The average 3D spatial resolution was 30 nm. This was determined by differentiating the electron density amplitude across the crystal/acid interface for the five reflection directions and fitting a Gaussian to each of the profiles. The reported spatial resolution is the averaged full width at half maximum of the Gaussian profiles. The average 3D spatial resolution for each reconstruction during corrosion was 23, 31, 30 and 38 nm at 0, 2.6, 4 and 6 h respectively.B3.We accounted for arbitrary phase offsets by establishing a cubic volume  centred about the centre of mass as the zero-phase reference for every reconstruction. The resulting reconstructions were transformed from detector conjugated space to orthogonal sample space with a voxel size of 5 × 5 × 5 nmε(r), projected along the hkl Bragg vector was found by differentiation,Phase ramps were eliminated by centring the Fourier transform of the complex electron density in detector conjugated space and sample space. The strain, ε(r), and rotation tensor, ω(r), are obtained by49u(r). With a single BCDI measurement, we can determine one component of the displacement field. For multi-reflection BDCI (MBCDI), if at least three linearly independent reflections are measured, u(r) can be determined by minimising the least-squares error,31,50et al.,34 in which case, the squared error between phase gradients is minimised:j corresponds to the spatial x, y, or z coordinate, to find ∇u(r) directly for the computation of ε(r), and ω(r) in The full 3D lattice strain tensor, CDEA brief description of each ESIx–y plane slices through the lattice strain and rotation tensors in • SV1y–z plane slices through the lattice strain and rotation tensors in • SV2z–x plane slices through the lattice strain and rotation tensors in • SV3• SV4x–y plane slices through the crystal at various time points during corrosion, based on • SV5• SV6• SV7There are no conflicts to declare.CE-024-D1CE01586A-s001CE-024-D1CE01586A-s002CE-024-D1CE01586A-s003CE-024-D1CE01586A-s004CE-024-D1CE01586A-s005CE-024-D1CE01586A-s006CE-024-D1CE01586A-s007


The full characterization using X-ray diffraction, FT-IR spectroscopy, 13C cross-polarization magic-angle spinning NMR spectroscopy, zeta-potentials, BET surface and pore analysis indicated that these COMs and ImI@TpBd-(SO3)2 exhibited different morphologies, porosities, and potentials. The effects of the type of dye, adsorption time, initial dye concentration, and pH on the adsorption of dyes on ImI@TpBd-(SO3)2 were systematically investigated, respectively. The results revealed that ImI@TpBd-(SO3)2 possessed good adsorption performance for nine different cationic dyes with adsorption capacities in the range from 2865.3 mg g−1 for methylene blue (MB) to 597.9 mg g−1 for basic orange 2 (BO), but little adsorption for anionic and neutral dyes, revealing charge selectivity. The adsorption ratio of ImI@TpBd-(SO3)2 for MB was as high as 74.0% at 10 min by using 1.0 mg material, owing to the post modification of TpBd-(SO3)2 with imidazole. The adsorption of MB on ImI@TpBd-(SO3)2 was pH dependent. The adsorption isotherm and kinetics fitted well with the Freundlich and pseudo second-order kinetic model, respectively. Finally, the very outstanding advantages of superior selective adsorption, desorption, convenient preparation, and low density of ImI@TpBd-(SO3)2 predicted its research and application potential in dye wastewater recovery.The discharge of industrial printing and dyeing wastewater is one of the main reasons for the increasing water shortage and deterioration. The treatment of dyestuff wastewater is an issue and needs to be urgently solved. In this work, anionic ionic liquid functional covalent organic materials (COMs) were firstly synthesized and used for the selective adsorption of cationic dyes. First, a series of sulfonic acid group (SO Anionic ionic liquids@covalent organic materials for the selective adsorption of cationic dyes. Recently, Wang9 synthesized anionic microporous organic polymers (MOPs) to selectively adsorb cationic dyes. Yang10 reported poly(ionic liquid)-polyoxometalate hybrids for the selective removal of anionic dyes, proving the function of electrostatic interactions in dye binding. However, these materials possessed shortcomings in practical applications, such as limited capacity, metal ion leakage, limited stability, complicated preparation processes, and poor selective adsorption ability. Hence, developing selective adsorbents with large adsorption capacity synthesized by a simple method is attractive.Wastewater caused by the large amount of dye consumption in textile and paper manufacturing industry poses serious threat to mankind and aquatic living organisms because dyes are highly toxic, resistant to degradation, and visible even in trace amounts.11 they are constructed by covalent bonds between the organic building units with two and three dimensions. COMs include amorphous porous organic polymers (POPs),12 polymers with intrinsic microporosity (PIM),13 covalent organic polymers (COPs)14 and crystalline ordered covalent organic frameworks (COFs).15,16 Due to their unique advantages of various construct structures, functional sites, and diverse pore sizes, COM-based adsorbents are applied to adsorb metal ions,17 oil,18 bisphenol A,19 marine toxins,20 and dyes.21,22In recent years, covalent organic materials (COMs), an exciting new type of porous material, have aroused great interest;23 ILs@COMs adsorbent solves the problem of the lack of selectivity to targets of conventional COMs.24 In spite of the progress in research on cationic COMs, their complicated preparation procedures or high toxicities of the currently used cationic building units have greatly restricted their practical applications. Besides, the incorporation of anion in the COMs to produce anionic ILs@COMs is a rarely explored area.Ionic liquids (ILs) possess the potential to selectively absorb objects efficiently due to their unique properties of limitless structural tunability and multiple interactions on adsorbate, such as electrostatic interaction, hydrogen bond, van der Waals force, and π–π stacking.3)2 with green synthesis method, and used them as adsorbents to selectively adsorb cationic dyes from aqueous solutions (3)2 include pH, initial concentration, contact time, competitive anionic dyes, and cationic dyes. The adsorption isotherm and kinetic parameter are provided, respectively.Herein, we synthesized novel anionic ILs@TpBd- in KBr were recorded on a Vector22 spectrometer . The elemental  contents of the materials were determined using a Vario MACRO cube Elementar . Scanning electron microscopy (SEM) images were obtained with a Hitachi SU-1510 . Field emission transmission electron microscopy (TEM) images were obtained with Jem-2100F . Thermogravimetric analysis (TGA) was carried out using a thermo-gravimetric analyzer  at a heating rate of 10 °C min−1 up to 800 °C. Powder X-ray diffraction (PXRD) measurement was acquired on a Bruker AXS D8 Advance diffractometer  equipped with Cu Kα radiation (λ = 1.5418 Å). 13C cross-polarization magic-angle spinning (CP-MAS) was recorded in a Bruker 600 MHz NMR spectrometer. BET surface and pore analysis of the samples were carried out using a Quantochrome Autosorb instrument . Zeta potential was measured using a zeta potential analyzer by electrophoresis . The electronic analytical balance (model EX125DZH) was obtained from Ohaus International Trading . Chromatographic analysis was carried out in an HPLC instrument equipped with a photo-diode array (PDA) . HPLC-MS containing a 6410 triple quad mass spectrometry and Agilent 1200 series HPLC instrument was used for HPLC-MS analysis. Multiple positive mode (MRM) was adopted for MS acquisition. The source conditions were as follows: capillary voltage, 4000 V; nebulizer, 40 psi; gas temperature, 350 °C; and gas flow, 10 L min−1.Fourier transform-infrared spectra (FT-IR) (4000–400 cm3 was synthesized by a reflux procedure under nitrogen (N2) pressure.25 Typically, Tp (0.15 mmol) and Pa (0.225 mmol) were dissolved in 25.0 mL ethanol, separately, and then mixed in a 100 mL three-neck round bottom flask equipped with a vacuum link head and N2. The flask was vacuumed, and then flushed with N2. After stirring for 4 h at 90 °C, the sanguineous suspension TpPa-SO3 was obtained. Afterwards, it was washed with DMF and ethanol, respectively, followed by vacuum drying. As for TpBd-(SO3)2, the reagents Tp (0.075 mmol) and 2,2′-benzidinedisulfonic acid  were kept for 8 h at 90 °C.TpPa-SO3)2, Tp (0.0.375 mmol) and CR (0.375 mmol) were, separately, dissolved in a mixture of 25.0 mL DMF and mesitylene (v : v 1 : 1). After stirring for 4 h at 105 °C, the black suspension TpCR-(SO3)2 was obtained, and it was washed with DMF, DMF : mesitylene (v : v) and ethanol, respectively.As for TpCR-2 was dispersed in ethanol (10.0 mL), which was followed by the addition of 34.0 mg imidazole, 62.1 mg 1-butylimidazole, and 72.1 mg 1-phenylimidazole, separately. The mixture was stirred at room temperature with a rotation speed of 600 rpm. RImI@TpBd-(SO3)2 was collected by centrifugation and washed with ethanol.Acid–base neutralization is well established for the synthesis of ILs or to introduce functional IL groups in materials.The materials (1.0 mg) were immersed in the dye solutions (35.0 mL) and incubated at room temperature for 18 h. For pH adjustment, 1 M HCl or 1 M NaOH was used. After centrifugation, the supernatants were collected respectively, and the responding materials were washed firstly with water and then the eluents, separately. Finally, in single and binary systems, the initial concentration, final concentration in the supernatants, and elution concentration of dyes were analyzed by an UV-vis spectrophotometer, respectively. In the adsorption experiment of a nine-component system, the initial concentration and final concentration of the dyes in the supernatants were analyzed by HPLC-MS, except that of BBR, which was analysed by HPLC. The cationic dye molecules and their properties used in the HPLC-MS analysis including ion pair, fragment ion, and collision energy are displayed in Table S1.†Q (mg g−1), elution efficiency of dyes E , and separation efficiency S  were calculated on the basis of the following formula:27,28Ci and Cf (mg L−1) are the dye concentrations before adsorption and after adsorption, respectively. Ce (mg L−1) is the elution concentration of dyes. Vi and Ve (L) are the volumes of the adsorption solution and elution solution, respectively. M (g) is the quality of dry material.The adsorbed amounts The maximum absorption wavelengths of the dyes were 533 nm for reactive brilliant red K-2BP (RBR), 464 nm for methyl orange (MO), 663 nm for methylene blue (MB), 451 nm for basic orange 2 (BO), 430 nm for auramine O (AO), 460 nm for Bismarck brown R (BBR), 657 nm for Azure A (AZA), 607 nm for Azure B (AZB), 611 nm for Azure C (AZC), 530 nm for basic red 5 (BR), and 581 nm for crystal violet (CV), respectively.18 column  at a flow rate of 0.8 mL min−1 at 30 °C, with the PDA detector at the wavelength of 451 nm and the injection volume of 50 μL. The mobile phases were 0.1% formic acid in water (A), and ACN (B), respectively. The initial gradient conditions were set as 0–12 min, 20–85% B, 13–25 min, 85% B, 25–26 min, 85–20% B, 27–35 min, 20% B.The analytes were separated using a C18 column . The mobile phases were 5 mmol L−1 ammonium acetate and 0.2% formic acid in water (A), and ACN (B), respectively. The initial gradient conditions were set as 0–8.2 min, 20% B with a flow rate of 0.2 mL min−1; 8.2–15.0 min, 20–60% B with a flow rate of 0.1 mL min−1; 15.0–18.0 min, 60–85% B with a flow rate of 0.2 mL min−1; 18.0–19.0 min, 85–20% B with a flow rate of 0.2 mL min−1; 20–21 min, 20–20% B, with a flow rate of 0.2 mL min−1; the injection volume was 3 μL.The dyes were separated on a ZORBAX XDB-C−1, respectively, and agitated at 180 rpm for 18 h at room temperature. Then, 2.0 mL dye solution was pipetted to measure the dye concentration and uptake. The Langmuir and Freundlich isotherm model were applied to fit the experimental data, respectively.27,28 The Langmuir isotherm model could be expressed by formula (4)The material (1.0 mg) was immersed in the dye solution (35.0 mL) with the initial concentrations was varied from 20 to 130 mg Lce (mg L−1) and qe (mg g−1) are the equilibrium concentration of MB and equilibrium adsorption capacity, respectively. KL (L mg−1) is the Langmuir constant. KF (mg g−1) and bF are the Freundlich isotherm constants.The Freundlich isotherm model adsorption equation is represented as.−1, 35.0 mL) and agitated at 180 rpm at room temperature. Then 2.0 mL dye solution was pipetted at different time intervals ranging from 10 min to 18 h to measure the dye concentration and uptake. Pseudo-first-order and pseudo-second-order model were utilized to fit the adsorption data, respectively, and they could be described by formula (8)qt and qe (mg g−1) are the adsorption capacities of the dye at time t and at equilibrium, respectively. K1 (min−1) and K2 (g mg−1 min−1) are the rate constants of pseudo-first-order and pseudo-second-order model, respectively.The material (1.0 mg) was immersed in the dye solution (100 mg L3)2 (1.0 mg) was incubated with the dye solution (2.0 mL) for 10 min. After centrifugation, the supernatant was collected and the responding material was washed firstly with water and then the eluent of methanol + 2 mmol L−1 ammonium acetate + 0.1% formic acid . The same operation was repeated five times. The adsorbed amount Q (mg g−1) was calculated based on formula ImI@TpBd- in van der Waals (VDW) styles.30Stable molecular structures of Bd, CR, imidazole, 1-phenylimidazole, 1-butylimidazole, and nine kinds of cationic dyes were calculated, respectively, using Gaussian 09 software under density functional theory (DFT).3 exhibited a weak diffraction signal at the low angle of ∼4.8° corresponding to the (100) reflection plane, while TpBd-(SO3)2 and TpCR-(SO3)2 exhibited no diffraction peak in the region (<5°). The results revealed the long-range molecular ordering of TpPa-SO3 (3)2 and TpCR-(SO3)2.14 The reason might be that the benzene rings in Bd and CR were not planar, which caused the distortion and interpenetration of network during the formation of the polymers.33 All anionic COMs displayed a broad diffraction peak at 22.5–27.5°, ascribed to the π–π stacking of benzene rings between adjacent layers. As shown in 3 was further simulated using Material Studio 8.0, and an AA stacking structure of the P1 space group with a = 22.8 Å, b = 22.77 Å, c = 4.52 Å, α = 90°, β = 90°, and γ = 120° resulted in a PXRD pattern that was in accordance with the experimentally observed pattern 2 are presented in 3, TpBd-(SO3)2, and TpBd-(SO3)2 exhibited bands at 1240, 1280, and 1296 cm−1, respectively, which were assigned to C–N stretching vibration. The bands at about 1580 cm−1 were attributed to C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>C stretching vibration.31,33 The bands at 1231, 1194, and 1062 cm−1 were owing to SO2 stretching vibration.34,35 Compared with that in TpBd-(SO3)2, the new bands at 2978–2965 and 750 cm−1 in ImI@TpBd-(SO3)2 and BuImI@TpBd-(SO3)2 corresponded to imidazole C–H stretching vibration and imidazole C–H out-plane flexural vibration, respectively.36The FT-IR and NMR spectra can be applied to analyze the functional groups of materials and determine whether the modification is successful. The FT-IR spectra of anionic COMs and ILs@TpBd-2 can be attributed to CN in imidazole, indicating the successful modification of TpBd-(SO3)2 using imidazole.25 BuImI@TpBd-(SO3)2 possessed an extra peak at 13.0 ppm ascribed to the CH3 carbon atoms.The 3)2, and the results are presented in 3, TpBd-(SO3)2, and TpCR-(SO3)2 displayed different morphologies, indicating that linear diamines with different lengths had an obvious effect on the morphology. TpPa-SO3 was composed of uniform nanofibers with widths of about 20 nm and lengths in the range of 100 nm to tens of micrometres. In the HR-TEM image, some fringes were observed. TpCR-(SO3)2 was a mixture of micrometre particles and nano-particles, which might be caused by phase separation. TpBd-(SO3)2 and ILs@TpBd-(SO3)2 were composed of micrometre rods.SEM and HR-TEM were used to observe the surface morphology of anionic COMs and ILs@TpBd-(SO3)2 and TpBd-(SO3)2 indicated different C, N, H, and S contents, which confirmed the successful modification of TpBd-(SO3)2 with ILs. The zeta-potential values of anionic COMs and ImI@TpBd-(SO3)2 are shown in 3H made the materials negatively charged, which was beneficial to interact with positively charged guests through electrostatic interaction.37 The TGA curves of the materials are compared in 2 up to 260 °C.As presented in Table S3,2 adsorption–desorption experiments of COMs and ILs@TpBd-(SO3)2 were conducted at 77 K and one bar pressure. The samples were activated by degasifying them at 150 °C under vacuum for 8 h. The surface area, pore volume and pore size are shown in Table S4.3 possessed both micropores and mesopores with pore sizes of 1.7 and 2.9 nm, respectively, while TpBd-(SO3)2 and TpCR-(SO3)2 predominantly possessed mesopores of 9.3 and 2.9 nm, respectively. The pore size of TpPa-SO3 (1.7 nm) was close to the theoretical value provided by the refinement of the PXRD simulation , pore size changed irregularly, which was probably due to the amorphous structure of TpBd-(SO3)2 and TpCR-(SO3)2. The surface area and pore volume of TpPa-SO3 and TpBd-(SO3)2 were 70.8 m2 g−1, 0.1709 cm3 g−1 and 60.5 m2 g−1, 0.1711 cm3 g−1, respectively, which were much higher than that of TpCR-(SO3)2 (9.6 m2 g−1 and 0.0190 cm3 g−1). SO3H-functionalized COMs displayed lower BET surface area and pore volume in comparison with the previously reported TpPa and TpBd series, partly because of the introduction of bulky SO3H groups in the pore.32 The sizes of imidazole, 1-butylimidazole, and 1-phenylimidazole were smaller than the pore size of TpBd-(SO3)2 2 have been measured to be 61.8 cm3 g−1 and 0.1722 nm, respectively, which were almost the same as that of TpBd-(SO3)2 . However, the pore size of ImI@TpBd-(SO3)2 was 14.5 and 23.8 nm, which were about 1.6 times that of TpBd-(SO3)2 (9.3 nm). The reason might be that imidazole changed the arrangement of amorphous TpBd-(SO3)2. The modification of TpBd-(SO3)2 with 1-butylimidazole and 1-phenylimidazole obviously decreased the BET surface area and pore volume, probably due to the occupation of these molecules in the pore.Nmulation . In crys2 Fig. S1, suggest3)2 for MB adsorption at pH 9.0 is presented in 3)2 and TpPa-SO3 showed the maximum and minimum adsorption capacity (2653.3 and 1806.9 mg g−1) for MB, respectively. This obvious difference might be due to the following reasons. On the condition of approximate negative zeta-potential values, although TpPa-SO3 showed a greater BET specific surface area, TpPa-SO3 displayed the minimum adsorption capacity for MB. In contrast, the pore size of TpBd-(SO3)2 was five times larger than that of TpPa-SO3, and TpBd-(SO3)2 showed the maximum adsorption capacity for MB, indicating the important effect of mesopores in TpBd-(SO3)2 in improving the adsorption capacity. The adsorption could be speculated: more free space in the mesopores of TpBd-(SO3)2 provided the incoming dye molecules a greater area for interaction, accommodated the dyes and facilitated the transport of dyes. In contrast, diffusion was hindered by the narrower pore size of TpPa-SO3.14 After the modification of TpBd-(SO3)2 with ILs, all ILs@TpBd-(SO3)2 displayed outstanding adsorption performance for MB; especially, ImI@TpBd-(SO3)2 showed the maximum adsorption capacity of 2841.5 mg g−1 at pH 9.0, which could compete with any of the other materials reported 2 and ImI@TpBd-(SO3)2 was carried out by adjusting the pH in the range of 4.0–9.0 with 1 M HCl and 1 M NaOH, respectively. As shown in 3)2 and ImI@TpBd-(SO3)2 for MB was pH dependent. With pH increasing from 4.0 to 9.0, the adsorption capacity increased, and reached the maximum at pH 9.0. Positively charged MB with N+(CH3)3 group was adsorbed in negatively charged TpBd-(SO3)2 and ImI@TpBd-(SO3)2 with SO3 group functional through electrostatic interaction. With pH increasing, the adsorption capacity of TpBd-(SO3)2 and ImI@TpBd-(SO3)2 for MB increased. The reason might be that the deprotonation of SO3 group from the materials increased as pH increased.39The pH-dependence experiment for MB adsorption on TpBd-(SO3)2 and ImI@TpBd-(SO3)2, respectively. As shown in −1, the adsorption capacity increased, and the saturation capacity of TpBd-(SO3)2 and ImI@TpBd-(SO3)2 was achieved at the MB concentration of 100 mg L−1. Fitting the adsorption data using the Langmuir model caused very low correlation coefficients 2 and ImI@TpBd-(SO3)2, with correlation coefficients 0.9496 and 0.9297, respectively. The maximum adsorption capacities of TpBd-(SO3)2 and ImI@TpBd-(SO3)2 for MB were calculated to be 2641.2 and 2854.9 mg g−1, respectively. These results indicated the multilayer adsorption of MB on heterogeneous surfaces.Adsorption equilibrium experiment was used to investigate the adsorption mechanism of MB in TpBd-2 (74.0%) revealed a larger adsorption ratio than that of native TpBd-(SO3)2 (56.9%) at 10 min due to the engineering with imidazole. Besides, ImI@TpBd-(SO3)2 exhibited a slightly elevated MB adsorption equilibrium capacity (2865.3 mg g−1) compared with TpBd-(SO3)2 (2645.0 mg g−1). The fitting plots of the kinetic models and the relative parameters are exhibited in Fig. S4, S5 and Table S7.3)2 and ImI@TpBd-(SO3)2 fitted the pseudo-second-order kinetic model very well with R2 , indicating that the chemical adsorption of MB on the materials was the rate limiting step. In comparison with TpBd-(SO3)2, ImI@TpBd-(SO3)2 further increased the adsorption capacity and adsorption ratio for MB, which could be speculated to be due to the following factors: (i) multiple interactions such as the hydrogen bond and π–π interaction of imidazole counter cation of ImI@TpBd-(SO3)2 strengthened the interaction with MB;40,41 (ii) the post modification of TpBd-(SO3)2 by imidazole led to the exposure of more favourable adsorption sites and increase in mesopore sizes 2, three kinds of dye molecules with different sizes and charges were chosen as model compounds, including anionic dyes (MO (monoanionic), RBR (dianionic)), neutral dyes , and cationic dyes , BO (dicationic), BR (dicationic), AO (tricationic), CV (tricationic), BBR (forcationic)) (To explore the selective adsorption of charged dyes on ImI@TpBd-(SOtionic)) .3)2 for anionic, neutral, and cationic dye single and mixed systems was evaluated in water at pH 5.5. As shown in 3)2 indicated high adsorption capacity for all the cationic dyes with different charges and sizes. In contrast, ImI@TpBd-(SO3)2 showed no adsorption capacity for anionic dyes of small size (MO) and large size (RBR). Also, neither neutral dyes of small size (NR) nor those of large size (CA) were adsorbed. In comparison, ImI@TpBd-(SO3)2 indicated excellent selective separation performance for cationic dye molecules. The reason was mainly speculated as follows: plenty of SO3 groups endowed the material with negative charge on the mesopore walls. In the case that the molecular sizes of all the dyes were lesser than the pore size of ImI@TpBd-(SO3)2, the difference in the structure of the dye molecules caused different results 2 for cationic dyes was in the range from 1662.6 mg g−1 of MB to 597.9 mg g−1 of BO at pH 5.5, and decreased in the sequence of MB (1662.6 mg g−1) > AZB (1015 mg g−1) > BBR (974.1 mg g−1) > AZC (936.4 mg g−1) > AZA (763.1 mg g−1) > AO (672.3 mg g−1) > CV (671.1 mg g−1) > BR (632.6 mg g−1) > BO (597.9 mg g−1). Cationic dyes were adsorbed mainly through electrostatic attraction; so, dyes with more positive charges possessed higher adsorption, such as the adsorption capacity of for cationic dyes BBR > tricationic dyes CV > dicationic dyes BR. On the other hand, restricted by the limited specific surface area and pore volume of the material, dyes with a smaller size had a higher adsorption capacity, such as adsorption capacity of dicationic dyes MB > forcationic dyes BBR. The larger BBR molecules might occupy more surface area and pore volume of the material in achieving adsorption saturation, so less BBR was adsorbed. From these results, it could be inferred that the absorption of cationic dyes in ImI@TpBd-(SO3)2 was mainly driven by electrostatic interaction, and limited by pore volume and specific surface area. The adsorption capacities of ImI@TpBd-(SO3)2 for MB, CV, and AO were higher than that of most of the reported adsorbents 2 towards dye mixture was also evaluated. To preclude the effect of dye concentration difference, the blue-colored mixture of MB + RBR with a mass ratio of 1 : 1 at an initial pH of 5.5 was treated with ImI@TpBd-(SO3)2. As shown in −1 and 641.7 mg g−1, respectively. The percent of MB in the MB + RBR mixture adsorbed in ImI@TpBd-(SO3)2 was 92.7%, indicating the charge-selective separation of the cationic dye from the binary system. The separation mechanism was speculated as follows: in the beginning of the adsorption experiment of the material for the mixture of MB and RBR, the positively charged MB could replace the imidazole counter cations to fit in the channels. Then the MB molecules were immobilized into the pores of ImI@TpBd-(SO3)2 due to the strong electrostatic interaction between the negatively charged pore walls and cationic MB molecules. A small part of RBR was attracted into ImI@TpBd-(SO3)2 through electrostatic interaction between MB and RBR. However, although the mesopore could accommodate MB + RBR at the same time, most RBR was rejected from the negatively charged material due to strong electrostatic repulsion.24 So the adsorption capacity of ImI@TpBd-(SO3)2 for RBR slightly increased. The excellent separation of ImI@TpBd-(SO3)2 for cationic MB from mixtures of MB + RBR was mainly controlled by the electrostatic interaction.The selective adsorption of specific dyes can not only improve the recovery of dyes but also reduce environmental pollution. So, the separation ability of ImI@TpBd-(SO3)2 was carried out using the nine-component mixture with a mass ratio of 1 : 1. As shown in 3)2 displayed good adsorption capacities for cationic dyes MB, BBR, AZC, AO, BR, and BO in the range from 88.9 mg g−1 of BBR to 610.5 mg g−1 of AO. However, ImI@TpBd-(SO3)2 displayed no adsorption capacities for AZA, AZB and CV. The results clearly showed that the affinity of different dyes to the adsorbed site was different, suggesting a wider application of ImI@TpBd-(SO3)2 in separation areas such as cationic dyes pre-treatment and chromatographic separation.As a kind of typical fine industrial products, various cationic dyes are used for industrial dyeing, which has led to the complex composition of cationic dye production wastewater. So, a nine-component system of cationic dyes with different charges and sizes was tested in the research. To exclude the effect of dye concentration difference, the adsorption of cationic dyes on ImI@TpBd-(SO3)2 facilitated the recovery of dyes and their application in industry. So, the dye desorption from ImI@TpBd-(SO3)2 was further established by immersing the samples into six kinds of eluents at 25 °C. As can be seen from 3)2, while an excellent elution of MB with 92.5% efficiency was achieved by a mixed solution of methanol + 2 mmol L−1 ammonium acetate + 0.1% formic acid .Efficient elution of the adsorbed dyes from ImI@TpBd-(SO3)2.The successive experiments indicated that after five cycles the adsorption capacity remained almost constant Fig. S6, which i3)2 was firstly prepared through the convenient modification of TpBd-(SO3)2 with imidazole, and used for the selective adsorption of cationic dyes of different sizes and charges with superb adsorption capacities. Such factors as the type of material, solution pH, adsorption time, initial dye concentration, and type of dye were systematically investigated to determine the optimal adsorption. As a consequence, the maximum adsorption capacity of ImI@TpBd-(SO3)2 for MB was 2865.3 mg g−1 at pH 9.0. ImI@TpBd-(SO3)2 displayed super adsorption performance for nine kinds of cationic dyes with maximum adsorption capacity in the range from 1662.6 mg g−1 of MB to 597.9 mg g−1 for BO at pH 5.5, which was among the highest capacities in cationic dye adsorption. Besides, the study on the adsorption mechanism indicated that the Freundlich model displayed a better fit to adsorption isotherms, and the pseudo-second order model fitted well with adsorption kinetics. Furthermore, ImI@TpBd-(SO3)2 could selectively adsorb cationic dyes in cationic and anionic binary dye systems, and it showed favorable adsorption performance for nine-component dye mixture. Finally, the convenient preparation, selective adsorption performance, excellent adsorption capacity, desorption, advantages of low density, and very fast adsorption in the initial phase revealed the potential application of ImI@TpBd-(SO3)2 in dye wastewater separation and recycling.ImI@TpBd-(SOThere are no conflicts to declare.RA-010-C9RA10035K-s001


Lymphocytic leukemia (LL) is a primary malignant tumor of hematopoietic tissue, which seriously affects the health of children and the elderly. The study aims to establish a new detection method for screening acute/chronic LL using time-resolved fluorescence immunoassay (TRFIA) via quantitative detection of S100 calcium binding protein A8 (S100A8) and leucine-rich alpha-2-glycoprotein 1 (LRG1) in serum.3+)/samarium(III) (Sm3+) chelates. Finally time resolved fluorometry measured the fluorescence intensity.Here a sandwich TRFIA was optimized and established: Anti-S100A8/LRG1 caputre antibodies immobilized on 96-well plates captured S100A8/LRG1, and then banded together with the anti-S100A8/LRG1 detection antibodies labeled with Europium(III) , and 3.2 ng/mL for LRG1 . The intra-assay and inter-assay CVs were low, ranging from 5.75% to 8.23% for S100A8 and 5.30% to 9.45% for LRG1 with high specificity and affinity in serum samples. Bland–Altman plots indicated TRFIA and ELISA kits have good agreement in clinical serum samples. Additionally, the cutoff values for S100A8 and LRG1 were 1849.18 ng/mL and 588.08 ng/mL, respectively.The sensitivity of S100A8 was 1.15 ng/mL(LogY = 3.4027 + 0.4091 × LogX, The present TRFIA method could be used for the quantitative detection of S100A8 and LRG1 in serum, and it has high sensitivity, accuracy and specificity. Clinically, this TRFIA method could be suitable for screening of LL via the quantitative detection of S100A8 and LRG1. Lymphocytic leukemia (LL) is roughly divided into acute and chronic lymphocytic leukemia, and its lesions involve the surrounding blood, lymph nodes and organs throughout the body . Acute LS100 calcium binding protein A8 (S100A8) is a member of the S100 calcium-binding protein family, it associated with myeloid cell differentiation/apoptosis, adhesion of neutrophils, and chemotactic activities etc. , 5. The Leucine-rich alpha-2-glycoprotein-1 (LRG1) is a serum glycoprotein and have been increasingly recognized as biomarkers for certain diseases including microbial infections and cancer . LRG1 waBased on the above information, we hypothesized that S100A8 and LRG1 are specific biomarkers for acute/chronic LL. Therefore, we aimed to establish a new method to screen acute/chronic LL via the quantitative detection of S100A8 and LRG1. Time-resolved fluorescence immunoassay (TRFIA) is a promising detection technique, and has been widely used in various fields, including antigen, antibody and viruses detection , 20. In 3+ labels and S100A8 detection antibody: 100 μg:1 mg, and the optimal ratio of Sm3+ labels and LRG1 detection antibody: 150 μg:1 mg; the sample volume: 30 μL/well; Eu3+/Sm3+ labeled antibodies volume: 100 μL/well Eu3+-labeled antibody + 100 μL/well Sm3+-labeled antibody; enhancement solution volume: 200 μL/well, and the immunoreaction time was 1 h , LRG1: LogY = 3.3009 + 0.4082 × LogX . Graphical estimation indicates the LOD of S100A8 was 1.15 ng/mL, and LOD of LRG1 was 3.2 ng/mL .Hook effect was seen when the range exceeded 10,000 ng/mL  with high concentrations, and the results are shown in Table As presented in Table The comparison results are shown in Fig. P < 0.001, Fig. For 120 clinically healthy serum samples, the mean S100A8 concentration was 1499.48 ng/mL, the SD was 178.42, while the mean LRG1 concentration was 437.83 ng/mL, and the SD was 76.66, so the cutoff value of S100A8 was 1849.18 ng/mL and 588.08 ng/mL for LRG1, which meant that when the concentration of S100A8 was higher than 1849.18 ng/mL and the LRG1 concentration was higher than 588.08 ng/mL, the provider of this serum sample may be suffering from lymphocytc leukemia. Additionally, statistical analysis of the test results of 120 healthy serum samples and 59 serum samples with LL found that the concentrations of S100A8 and LRG1 in the lymphocytc leukemia patients was significantly higher than that of healthy human , while this TRFIA only needs approximately 70 min , which will save approximately 3 h in detection time. Additionally, this TRFIA kit can detect two indicators (S100A8 and LRG1) at once, simultaneous detection of multiple indicators can more accurately assist the diagnosis results, reduce diagnosis time and save money. Moreover, the given cutoff values and reference intervals of S100A8/LRG1 can facilitate the assessment of the patient's physical condition. Although the sensitivity of TRFIA is not as good as that of ELISA, it is because TRFIA is designed according to the actual level of S100A8/LRG1 in clinical serum samples, so that the obtained serum samples can be directly used for detection without dilution, which simplifies the detection steps.In this study, we optimized and established a sandwich TRFIA method with high sensitivity, specificity, and accuracy for the quantitative detection of S100A8 and LRG1 in serum. The sensitivity of S100A8 was 1.15 ng/mL, and 3.2 ng/mL for LRG1. The intra-assay and inter-assay CVs were low with high specificity and affinity in serum samples. There was a high In conclusion, a dual-label TRFIA with high sensitivity, specificity, and accuracy was developed for the quantitative detection of S100A8 and LRG1 in serum. Clinically, this TRFIA method could be suitable for screening of LL via the quantitative detection of S100A8 and LRG1. However, although we have established a dual-label TRFIA detection method for S100A8 and LRG1 in serum, its clinical application still needs more clinical samples verification, and other clinical diagnostic methods are needed to confirm the diagnosis of LL.The S100A8 antigen, LRG1 antigen, and their capture and detection antibodies were purchased from Yidenuo Bio-technology . 59 clinical serum samples with acute/chronic LL and 120 healthy serum samples came from the Baoshan Branch of Renji Hospital Affiliated to Shanghai Jiaotong University, and all serum samples were stored at − 80 °C. All participants or their guardians gave written informed consent, and this study was approved by the Institutional Review Board of Baoshan Branch of Renji Hospital Affiliated to Shanghai Jiaotong University.3+) and samarium (III) (Sm3+) labels were purchased from PerkinElmer . Tween-20, bovine serum albumin (BSA), triton X-100, β-naphthoyltrifluoroacetate and tri-n-octylphosphine oxide were procured from Sigma-Aldrich . Other chemicals and reagents used were analytical grade. Sephadex G50 column was purchased from GE Healthcare Life Sciences . 96-well plates and time-resolved analyzer (Auto 1420) were purchased from PerkinElmer . Washing buffer: 20 mmol/L Tris-HCl (pH 8.0) containing 0.05% Tween-20 and 0.9% NaCl. Blocking buffer: 50 mmol/L PBS (pH 7.4) containing 4% BSA. Labeling buffer: 50 mmol/L Na2HCO3-Na2CO3 (pH 9.0) containing 0.9% NaCl. Assay bufer: 50 mM Tris-HCl (pH 7.8) supplemented with 0.05% Tween-20 and 0.02% Proclin300. Enhancement solution: 0.1 mol/L acetate-phthalate buffer (pH 3.2) supplemented with 0.1% Triton X-100, 30 μmol/L β-naphthoyltrifluoroacetate, 60 μmol/L tri-n-octylphosphine oxide and 0.5% glacial acetic acid.Europium(III) . Briefly, serum sample, standard or controls, and Eu3+/Sm3+ labeled detection antibodies were added into the 96-well plates, and then incubated the plates at room temperature. After washed the wells three times, added the enhancement solution into the wells, and shaken gently for another 5 min. Finally, time-resolved analyzer measured the fluorescence. The scheme of the dual-label TRFIA for S100A8 and LRG1 is shown in Fig. Optimized assay procedure using the one-step procedure, including the sample volume, EuThe standard curve was established using serial dilutions of S100A8  and LRG1  protein. The limit of the blank (LOB) was defined as the mean of the blank assayed in 20 independent measurements. The limit of detection (LOD) was determined by adding two standard deviations (SD) to the LOB, that means LOD = LOB + 2 *SD .High concentrations of tumor necrosis factor-α (TNF-α), high sensitivity C-reactive protein (hs-CRP), carcino-embryonic antgen (CEA), Human transferrin (TRF), and human serum albumin (HSA) were selected for specificity assay. Data were obtained from eight independent experiments. High concentrations of interferences mentioned above were added into the healthy control serum and detected by this TRFIA. TNF-α and hs-CRP are the common and highly expressed inflammatory factors in the LL patients’ serum. CEA and TRF are the most commonly used tumor marker with highly expression in the LL patients’ serum. HSA was used to evaluate whether the serum matrix has an effect on the specificity of this assay.By adding different concentrations of S100A8 and LRG1 standards into the serum samples, we evaluated the accuracy and recovery of this method. Ten independent experiments were performed, and the intra-assay and interassay variations were obtained. The formula of recovery is: Recovery (%) =  × 100.59 clinical serum samples with acute/chronic LL were simultaneously measured by this TRFIA method, human S100A8 ELISA kit  and LRG1 ELISA kit . Bland–Altman plots were used to compare the consistency of the TRFIA and the ELISA kits.Serum samples from 120 healthy human was used to identify the reference intervals. The S100A8 and LRG1 concentrations of the 120 healthy human were detected by this dual-label TRFIA method. The S100A8 and LRG1 concentrations were performed a normality test with SPSS 17.0 respectively, and applied the one-sided upper limit of the 95% reference interval range to identify the reference intervals. Therefore, the cutoff values were calculated using the following formula: cutoff value = mean + 1.96SD .Data were statistically analyzed using SPSS 17.0, and graphed using GraphPad Prism 5  and Origin 8.0 . The data are expressed as the mean ± SD. Correlations among different methods were calculated with Pearson correlation.


Dehydroepiandrosterone (DHEA) is an FDA-approved food supplement used as an assisted reproductive sex hormone. The bioavailability is severely limited by its poor solubility (23 µg/mL). Herein, we aimed to modulate its solubility through cocrystallization. Eight cocrystals of DHEA with pyrocatechol (CAT), hydroquinone (HQ), resorcinol (RES), phloroglucinol (PG), 1,5-dihydroxy naphthalene (DHN), p-hydroxybenzoic acid (PHBA), gallic acid (GA), and 5-hydroxyisophthalic acid (5HIPA) were designed and synthesized. Some basic characterization tools, including powder X-ray diffraction, thermogravimetric analysis, differential scanning calorimetry, and Fourier transform infrared spectroscopy, were also applied in our work for basic analyses of cocrystals. It is indicated that DHEA-GA exhibits its superiority in dissolution and pharmacokinetic behaviors. While the area under the curve values of DHEA-GA is improved at the ratio of 2.2, the corresponding bioavailability of DHEA is expected to be accordingly increased. Diminished ovarian reserve (DOR) is the third leading cause of infertility after multiple factors and endometriosis at a ratio of 13.0% ,3. ThougDHEA is a functional food supplement in the USA and has been applied as an assisted reproductive sex hormone in recent decades. It’s an endogenous precursor for androgen and estrogen to assist in regulating the endocrine system and improving the physiological state of gonadal tissue . HoweverSince 1987, when Donald J. Cram, Jean-Marie Lehn, and Charles J. Pedersen were awarded the Nobel Prize in Chemistry for their development and use of molecules with structure-specific interactions of high selectivity, the concept of supramolecular chemistry was developed and widely applied as a cross-discipline in many fields like organic chemistry, physical chemistry, coordination chemistry, material sciences, biological science, and pharmaceutical chemistry ,21. SuprAccording to its steroid-based four-linked cyclopentanophenanthrene ring structure with a hydroxyl group at ring A and a ketone carbonyl group at ring D, we can see that the C=O···HO-Ar heterosynthon can be the most competitive synthon in cocrystallizing steroids. Therefore, a series of phenolic compounds as coformers were selected for cocrystal construction. Ultimately, crystallization attempts resulted in eight novel DHEA cocrystals: DHEA-CAT, DHEA-HQ, DHEA-RES, DHEA-PG-DH, DHEA-DHN, DHEA-PHBA, DHEA-GA, and DHEA-5HIPA . ComprehDHEA (Form I)   and n-hexane (30%), and the generated clear solution was kept at −20 °C for a few days. The single crystal was finally available under this condition. As regards the DHEA-PG-DH cocrystal, about 200 mg sample of DHEA and PG at a stoichiometric ratio of 1:1 was dissolved in 4 mL acetonitrile. Then the single crystal of DHEA-PG-DH was available under a slow crystallization process for half a month.PXRD patterns were collected on a Bruker D8 Advance X-ray diffractometer with Cu Kα radiation. The voltage and current of the generator were set to 40 kV and 40 mA, respectively. The scanning range was 3–40° 2θ with the scanning speed at 0.1 s/step under the ambient temperature. Then the data was imaged and integrated with the help of RINT Rapid, while the final peak analysis was conducted using Jade 6.0 from Rigaku.All X-ray diffraction collections of the single crystal sample were performed on a Bruker D8 Venture diffractometer with Mo Kα radiation (λ = 0.71073 Å) under 150 K, or 170 K. Intensity data were integrated and scaled on Program SAINT. At the same time, this was influenced by the adsorption effect and was further refined and adjusted using SADABS. The Olex 2 software was utilized to solve and refine the crystal structures of all multi-component forms. The crystal structures were solved directly on SHELXTL and further refined with the full-matrix least-squares technique on SHELX-2017 software. All non-hydrogen atoms were refined with isotopic atomic displacement parameters, while all hydrogen atoms were located in calculated positions and subsequently refined with the riding model. Crystallographic data in cif format has been deposited in the Cambridge Crystallographic Data Center, CCDC N0 2212357-2212364. −1. About 5 mg sample in a sealed non-hermetic aluminum pan was heated at the heating rate of 10 °C·min−1. The instrument was calibrated by the two-point calibration using indium and tin.All the experiments were carried out on a DSC Q2000 instrument  with the nitrogen gas flow rate at 50 mL·min−1. The sample was placed in an open aluminum oxide pan and heated at the rate of 10 °C·min−1 from 25 °C to 410 °C.Thermogravimetric analysis was performed on a TGA 55 equipment . The nitrogen was applied as purge gas at 20 mL·min−1 in the range from 4000 to 400 cm−1 using a ThermoFisher Nicolet iS50 FT-IR spectrometer. For each spectrum, thirty-two scans were acquired.The cocrystal sample was dispersed in KBr and scanned at a resolution of 4 cmConsidering the effect of particle size on the results of solubility and powder dissolution tests, all samples were sieved through 100-mesh sieves. Solubility test was carried out in pH 2.0, pH 4.5, and pH 6.8 buffer with 0.5% PVP K13-18 and water, respectively. About 20 mg sample was added into 1 mL different buffer solution and then stirred at the rate of 250 rpm for 12 h under the ambient temperature. Then the suspension was centrifuged at 12,000 rpm for 5 min. The supernatant was filtered through the 0.45 µm nylon filter and finally submitted to HPLC analysis, while the PXRD pattern of residue powder after vacuum drying was further collected.A mini-Bath dissolution device equipped with a Julabo-5 heater/circulator was applied for powder dissolution experiments. The experiment temperature was set at 37 °C, and the rotation speed was 25 rpm. About 50 mg sample was placed in a container with 15 mL buffer. Subsequently, about 0.5 mL sample was collected and filtered at different time points through a 0.45 µm nylon filter and then analyzed by HPLC. The collection time points were set at 5, 15, 30, 60, 90, 120, and 180 min. The residue powder in the container was filtered and dried for PXRD analysis.All pharmacokinetic experiments were supported by the Institutional Animal Care and Use Committee of the Shanghai Institute of Material Medica and conformed to the Guide for Care and Use of Laboratory Animals. Six male Sprague-Dawley rats (200–250 g) were set as a group and fasted overnight with only water available before drug administration. The dosage was set as 21 mg/kg in male rats . The suspension samples for oral administration were prepared by uniformly dispersing the solid samples into the solution, which contained 0.5% (wt%) CMC-Na and 0.5% (wt%) PVP K13-18. Since DHEA was an endogenous steroid hormone, the blood samples before dosing were collected as initial blank serum samples to investigate the effect of exogenous DHEA on plasma concentration. After dosing, 700 µL blood samples were collected from the orbital sinus at specific time points and placed into heparinized tubes. The time points were set at 0, 5, 10, 15, 30, 60, 120, 240, and 360 min. After centrifugation at 14,000 rpm for 10 min, supernatants of collected blood samples were transferred and stored at −80 °C until HPLC-MS/MS analysis. Additionally, the fodder was available for rats again when the blood samples of 240 min were collected.v/v) as the mobile phase was performed with the flow rate at 1 mL/min. The retention time point for DHEA was 4 min.The accurate concentration of DHEA was analyzed on an Agilent 1260 series HPLC  equipped with a quaternary pump (G1311C), a diode-array detector (G1315D) and a 4.6 × 150 mm, 5 µm Agilent ZORBAX Eclipse Plus C18 column. The injected volume was set to 50 µL, while the UV-vis wavelength was set to 210 nm. The column temperature was 35 °C. An isometric elution method using methanol and deionized water at the ratio of 75:25 (Acetonitrile (ACN) was used to extract DHEA from serum and denature the protein. Firstly, ACN (400 µL) and an internal standard solution of abiraterone  were added to a 200 µL thawing serum sample. After thoroughly blending in an ultrasonic apparatus for 15 min, the mixture was further centrifuged for 10 min at 12,000 rpm. Then 200 µL supernatant was transferred into a 96-well plate and reacted with 400 µL hydroxylamine hydrochloride solution (0.1 mol/L) for further derivatization. The reaction was conducted at 60 °C for an hour, and the resultant solution was submitted for HPLC-MS/MS analysis directly. The quantification of DHEA in rat plasma after oral administration was carried out by HPLC-MS/MS. A SCIEX Triple QuadTM 4500 LC-MS instrument with a Polaris 3 C18-A column (2.0 × 50 mm) was applied to detect DHEA in plasma. The column temperature was set to 40 °C. The mobile phase consisted of A: 1% acetic acid in water and B: methanol. The gradient elution method started with 55% A and linearly decreased to 20% in 2 min, then held for 1 min, and finally returned to 55% in half a minute and held for one and a half minutes. The flow rate was set to 0.6 mL/min with the injection volume at 10 µL. The detection wavelength was 210 nm. Detection and quantification were employed in multiple reaction monitoring modes (MRM), with m/z 304.4 → 253.2 for DHEA pharmacokinetic analysis. p-value < 0.05 or less. GraphPad Prism 8.0 was applied for the statistical analyses. The results are significant if the Crystal Explorer 21.5 software was utilized to calculate the Hirshfeld Surface of DHEA cocrystals . For MolDHEA is a steroid hormone molecule with a hydroxyl group and a ketone carbonyl group. From the crystal structure of DHEA, it can be seen that the alcohol hydroxyl group and the ketone carbonyl group on it form a tight and regular 1D chain in a head-to-tail manner . To breaTwelve typical coformers are chosen and calculated for MEPs analyses . Via visDuring the screening process, PXRD analysis is considered a predominant method to detect the formation of a new multi-component phase and identify the purity of the new solid substance. All experimental and simulated PXRD patterns of these eight cocrystals are exhibited in The key crystallographic parameters of DHEA cocrystals are summarized in P212121 space group with Z = 4 . The ASU of DHEA-CAT contains one DHEA molecule and one CAT molecule . The DHEA-CAT crystalizes in the orthorhombic molecule . Two molP212121 space group with Z = 4. One DHEA molecule and one DHN molecule are presented in the ASU of DHEA-DHN. Similar to that of DHEA-CAT, DHEA and DHN molecules are connected through C=O···HO-Ar synthon O2···H3A-O3 (2.736 Å) as expected. Additionally, each DHEA-DHN unit is linked via O4-H4···O1 (2.739 Å) interaction, forming the 1D infinite hydrogen-bonding chain in DHEA-DHN cocrystal (1:1). The DHEA-DHN takes the orthorhombic P212121). As the way we designed (C=O···HO-Ar synthon), DHEA interacts with HQ through O2···H4-O4 (2.833 Å), forming a basic structural unit. Subsequently, DHEA-HQ unit links each other via O3-H3A···O1 (2.777 Å) interactions to form a 1D supramolecular chain, then O1-H1···O4 (2.881 Å) interaction contributes the final formation of 2D infinite chain in DHEA-HQ cocrystal (1:1). The ASU of DHEA-HQ contains one DHEA molecule and one HQ molecule  interaction is also observed in DHEA-PHBA. To construct the 2D framework, O4-H4·· O1 (2.584 Å) interaction (blue line) and O1-H1···O3 (2.893 Å) interaction (purple line) are regarded as the bridge of infinite 1D chain and following 2D skeleton, respectively . The structure of DHEA-PHBA is very similar to that of DHEA-HQ. The space group is ectively b.P212121 space group with Z = 4. The ASU of DHEA-RES contains two DHEA molecules and one RES molecule. A supramolecular three-fold helix is formed via continuous O5-H5···O1 (2.671 Å), O1-H1···O3 (2.859 Å) and O3-H3A···O5 (2.858 Å) interactions as shown in DHEA-RES cocrystal (2:1). The DHEA-RES takes the orthorhombic P21 space group with Z = 2. The ASU of DHEA-PG-DH contains two DHEA molecules, one PG molecule, and two water molecules. There is still a head-to-tail O1-H1···O2 (3.001 Å) interaction as that in DHEA . The crystal structure of DHEA-PG-DH is solved in the monoclinic  in DHEA a. The ot in DHEA b. Two 1D in DHEA c. These  in DHEA d.P21 space group with DHEA and GA in 2:1 stoichiometry. Six hydrogen-bonds were formed for every GA molecule (O6-H6A···O3 (2.630 Å), O1-H1···O5 (2.763 Å), O9-H9A···O2 (2.706 Å), O7-H7···O4 (2.721 Å), O3-H3···O8 (2.984 Å) and O8-H8A···O1 (2.726 Å)) as shown in DHEA-GA cocrystal (2:1). The crystal structure belongs to the monoclinic ractions b. IntereP1 space group with DHEA and 5HIPA in 1:1 stoichiometry. Two DHEA and two 5HIPA molecules first connected by O8−H8A···O1 (2.597 Å), O9−H9A···O4 (2.766 Å), O10−H10···O3 (2.548 Å) and O14−H14A···O2 (2.753 Å) interactions in DHEA-5HIPA cocrystal (1:1). The crystal structure belongs to the triclinic c chains b. Polyme2.701 Å) c.Additionally, the thermal performances of prepared samples are accessed via DSC and TGA methods to confirm the formation of new solid phases and control their quality. As for the DSC analytical result of multi-component substance, the single and sharp endothermic peak in the thermogram often indicates the formation of a single phase with high purity. Moreover, the TGA technique can also determine the content of residue solvent adsorbed to the surface or existing in the lattice. The corresponding analytical results are displayed in −1 is assigned to carboxyl C=O stretching vibration  and bioavailability (3.1% in the rhesus monkey) of DHEA . In viewWith the collapse of the crystal structure, the solubility of DHEA-GA and DHEA-PHBA are both improved. Compared with DHEA-PHBA, DHEA-GA exhibits greater enhancement in solubility under pH 2.0 conditions at 2-fold. As well as the results of the solubility test, DHEA-GA also exhibits its excellent advantage in enhancing the saturated solubility and maintaining the high supersaturated state of DHEA by 2-fold for three hours under powder dissolution experiments. These results are also in accord with that of the Hirshfeld Surface analysis. As for DHEA-GA, the higher proportion of H···O/O···H interactions (28.2%) indicates the strong H-bond interactions between DHEA-GA and aqueous solvent to some extent during the process of solvation, which promotes the improvement of solubility. With regard to DHEA-PHBA, the further precipitation after 60 min might also be another reason for its limited enhancement in solubility. When the coformer escapes from the lattice too quickly for its excellent solubility in the medium, the supersaturated DHEA will precipitate to give loosely aggregated clusters in some cases, along with the disappearance of superiority in solubility .Besides, all PXRD results in max, AUC0–t, and Tmax) are displayed in 0–t of DHEA-GA is 2.2 times that of DHEA (p < 0.05), and the Cmax of cocrystal is 2.8 times that of DHEA (p < 0.05). The enhancement of relative oral bioavailability may be attributed to enhanced solubility and adsorption of DHEA via creating the supersaturated state of DHEA molecules in the gastrointestinal tract. However, as the substrate of hepatic metabolic enzymes, DHEA is metabolized quickly and eliminated immediately with Tmax at 10 min. The rapid adsorption process of DHEA-GA also results in the fading of the advantage in adsorption after 60 min.Oral absorption of crystalline DHEA and its cocrystal was evaluated in male rats at 21 mg/kg. The profiles of plasma concentration versus time and correlative characteristic parameters (CIn this study, eight cocrystals of DHEA were successfully prepared through multiple methods, and the single crystal structures were determined by SCXRD. To improve the solubility of DHEA, a functional food supplement used as an assisted reproductive sex hormone, eight new cocrystals of DHEA were designed and synthesized. These cocrystals share a common supramolecular motif C=O···H-O-Ar. Corresponding properties, including physicochemical properties, solubility, and dissolution performance in-vitro and in-vivo, were investigated in this paper. For these two edible cocrystals, DHEA-GA exhibits its superiority in solubility with approximately 2-fold enhancement in both pH 2.0 and pH 4.5 buffer solution. This superiority in in-vitro dissolution behavior is further reflected in its great enhancement in in-vivo bioavailability. The AUC of DHEA-GA was about 2.2 times that of pure DHEA. The advantages of DHEA-GA make it a promising alternative to pure DHEA to be developed in the future.