Exposure to perfluoroalkyl substances may affect offspring immune development and thereby increase risk of childhood asthma, but the underlying mechanisms and asthma phenotype affected by such exposure is unknown.In the Danish COPSAC2010 cohort of 738 unselected pregnant women and their children plasma PFOS and PFOA concentrations were semi-quantified by untargeted metabolomics analyses and calibrated using a targeted pipeline in mothers (gestation week 24 and 1 week postpartum) and children . We examined associations between pregnancy and childhood PFOS and PFOA exposure and childhood infections, asthma, allergic sensitization, atopic dermatitis, and lung function measures, and studied potential mechanisms by integrating data on systemic low-grade inflammation (hs-CRP), functional immune responses, and epigenetics.Higher maternal PFOS and PFOA exposure during pregnancy showed association with a non-atopic asthma phenotype by age 6, a protection against sensitization, and no association with atopic asthma or lung function, or atopic dermatitis. The effect was primarily driven by prenatal exposure. There was no association with infection proneness, low-grade inflammation, altered immune responses or epigenetic changes.Prenatal exposure to PFOS and PFOA, but not childhood exposure, specifically increased the risk of low prevalent non-atopic asthma, whereas there was no effect on atopic asthma, lung function, or atopic dermatitis.www.copsac.com. The 10.13039/501100003554Lundbeck Foundation (Grant no R16-A1694); The 10.13039/501100009708Novo Nordic Foundation ; The Ministry of Health (Grant no 903516); 10.13039/100007398Danish Council for Strategic Research (Grant no 0603-00280B); and The Capital Region Research Foundation have provided core support to the COPSAC research center. COPSAC acknowledges the National Facility for Exposomics  for supporting calibration of the untargeted metabolomics PFAS data. BC and AS has received funding for this project from the 10.13039/501100007601European Union’s Horizon 2020 research and innovation programme .All funding received by COPSAC are listed on  Perfluoroalkyl substances are persistent and bioaccumulative exogenous chemicals in the human body with a range of suspected negative health effects. It is hypothesised that exposure during prenatal and early postnatal life might have particularly detrimental effects on the developing immune system, which could affect the risk of asthma in childhood. We did a systematic search using various combinations of the keywords: Perfluorooctane sulfonate (PFOS); perfluorooctanoic acid (PFOA); child; mother; longitudinal; asthma; sensitization; allergy.Both PFOS and PFOA exposure have previously been investigated in relation to childhood asthma and related outcomes, and a recent meta-analysis found PFOS to increase the risk of atopic dermatitis, and PFOA to increase the risk of allergic rhinitis, but no consistent associations to asthma and no stratification on asthma phenotypes.In a longitudinal mother-child cohort we found prenatal exposure to PFOS and PFOA, but not childhood exposure, specifically increased the risk of non-atopic asthma, whereas there was no effect on atopic asthma, lung function, or atopic dermatitis.Conflicting results on effect of perfluoroalkyl substances and risk of asthma could be explained by lack of consistency in timing of exposure and phenotypic asthma definition.Childhood asthma and atopic diseases are early, prevalent immune diseases with complex etiology. Diagnostic criteria for childhood asthma are complex and multiple phenotypes of asthma exists.,,Environmental exposure to a broader range and higher levels of xenobiotics, i.e., chemicals found in an organism that are extrinsic to the normal metabolism, has increased in recent generationsBoth PFOS and PFOA exposure have previously been investigated in relation to childhood asthma and related outcomes, and a recent meta-analysis found PFOS to increase the risk of atopic dermatitis, and PFOA to increase the risk of allergic rhinitis, but no consistent associations to asthma.The maturation of the immune system in early life creates a window of susceptibility, explaining why early life environmental exposures such as PFASs are of particular interest for immune programming of later health and disease.The Copenhagen Prospective Studies of Asthma in Childhood 2010 (COPSAC2010) is a single-centre longitudinal clinical mother-child cohort with extensive phenotyping of childhood asthma and related diseases during childhood. PFOA and PFOS has been assessed longitudinally with measures in mothers in pregnancy and 1 week postpartum, and in children at ages ½, 1½ and 6 years. Thereby the COPSAC2010 cohort is a unique data source for exploratory investigations on the associations between PFASs and asthma. With this study we aim to utilize the COPSAC2010 cohort to make exploratory investigations on the association between PFOS and PFOA exposure in pregnancy and early childhood on atopic and non-atopic asthma phenotypes at age 6 defined by various comorbidities and biomarkers and prospectively collected data among COPSAC2010 participants, where underlying mechanisms are investigated by integrating data on systemic low-grade inflammation (hs-CRP), functional immune responses, and epigenetics.This study is part of the ongoing longitudinal population-based COPSAC2010 mother-child cohort, in which 738 women were enrolled at pregnancy week 24. Pregnant women were recruited by a monthly surveillance of reimbursement to general practitioners for the mandatory pregnancy visit. They received an invitation by posted mail to contact the clinic during 2008–2010. Exclusion criteria were gestational age above week 26; daily intake of more than 600 IU vitamin D during pregnancy; or having any endocrine, heart, or kidney disorders. Women who contacted the COPSAC clinic by phone received detailed verbal information. Those who were still interested and qualifying for the study received comprehensive study information by posted mail. Finally, the women attended the clinical research unit within pregnancy weeks 22–26 for a visit in the research clinic with detailed information and enrolment into the pregnancy cohort. The pregnant women and their children, including five twin pairs, attended 14 scheduled clinical visits plus acute care visits in the first 6 years of life. All the children are followed closely at the COPSAC research clinic and all diagnoses and treatment for asthma, allergy and atopic dermatitis follow predefined and validated algorithms. The COPSAC paediatricians act as the family practitioner for all participants avoiding the heterogeneity of diagnoses and treatments in the medical community.All information was obtained by personal interview at clinical visits by medical doctors (MDs) and research assistants both with paediatric training. Medical, familial, environmental, and socio-economic histories were assessed by predefined questions and closed response categories and entered online into a dedicated database running an audit trail (Oracle database on a Novel SQL server). Data on sex was self-reported by the parents. Analyses included both males and females. Objective information and measurements were entered into the database, subsequently double-checked against source data, and finally locked for further editing. Quality assurance followed ‘Good Clinical Practice’ guidelines. Standard operating procedures (SOPs) were predefined for all study and database registration procedures.The persons involved in the study participate voluntarily and after written informed consent by both parents. The participating families are informed that the study is unlikely to benefit their child and that discoveries will only benefit future generations. The analyses of the biobank are anonymized, and the results are not provided to the families.http://copsac.com/available-data/.A detailed list of clinical information and exposures is presented at the COPSAC website: Relative abundance of PFOS and PFOA were measured in the blood of pregnant woman at gestational week 24 and 1 week postpartum (i.e. reflecting late pregnancy) and in the child at ages ½, 1½ and 6 years using untargeted plasma metabolomic profiling from the HD4 platform Metabolon, Inc. .Batch normalization was performed to compensate for inter-day variation. Specifically, each metabolite was corrected in run-day blocks by registering the medians to equal one (1.00) and correcting each variable accordingly. Thereafter, the datasets from all the four platforms were imported into RStudio  for statistical analysis. The maternal and child age 18 months plasma samples were analyzed at a later stage compared to the child age 6 months and 6 years samples. Therefore, we used 20 replicated samples, which were analyzed with the 6 months and 6 years batch (t1) and with the maternal and child age 18 months batch (t2). For each compound we calculated a ratio between t2 and t1 for each 20 replicated sample and the median of the ratios was used as a correction factor for the maternal and child age 18 months dataset.A selection of 48 samples were subsequently quantified for PFOS and PFOA concentrations using a targeted re-analysis and calibration pipeline. Samples were selected from children having all study measurements and included samples from children with both low and high concentrations of PFOS and PFOA. The quantitative data were used to calibrate the relative measures for PFOS (sum of branched and linear) and PFOA in all the samples to generate semi-quantitative measures using a targeted method for perfluoroalkyl acids modified from Glynn et al. (2012).g for 5 min. The supernatant was transferred to a new 15 mL polypropylene tube and concentrated with nitrogen gas at 30 °C to a volume of 0.2 mL. The extract was then diluted to 1 mL to a 50:50 methanol:water solvent composition before undergoing dispersive clean-up. For the clean-up, the extract was transferred to a 1.5 mL tube containing 0.025 g of bulk graphitized carbon , that had been acidified with 50 μL of glacial acetic acid and vortexed for 10 s. The sample was then centrifuged for 10 min at 20,800×g and the top 0.5 mL was filtered with 0.2 μm nylon centrifuge filters . An aliquot of 0.2 mL of the filtered extract was transferred to a glass auto-sampler vial for analysis.Briefly, an aliquot of 0.2 mL of plasma was placed in a 15 mL conical polypropylene centrifuge tube and spiked at a concentration of 1 ng/mL with 13 labelled internal standards . The plasma was extracted by protein precipitation with 4 mL of acetonitrile (ACN), followed by sonication at room temperature for 10 min and centrifugation at 700×m/z, 120,000 resolution FWHM at 200 m/z) and four MS2 data-independent acquisition (DIA) scans  with variable m/z precursor windows. A 10 μL sample volume was injected onto an ACQUITY UPLC BEH C18 analytical column  equipped with a ACQUITY BEH C18 1.7 μM VANGUARD Pre-Column at 40 °C. Upstream of the injector, one ACQUITY UPLC BEH C18 analytical column  was in place to separate instrumental background PFOS and PFOA from the analytes in the sample. A binary gradient elution was used, including (A) 2 mM ammonium acetate, and (B) 100% methanol at 0.35 mL/min.Analysis was performed by ultra-high pressure liquid chromatography  coupled to a HRMS Q Exactive Orbitrap HF-X  with electrospray ionization (ESI). The mass spectrometer was operated in negative ESI mode and alternated between a full MS scan . Quantification was performed using an external solvent-based calibration curve with the internal standard method.When extrapolating the semi quantitative measures to target quantification values, several child PFOA measures were extrapolated to negative values. For this reason, child PFOA values are offset in analyses where log transform is used.•Common infections registered in daily diary cards at age 0–3 years.•Childhood asthma diagnosed from a validated algorithm that included recordings of five episodes of troublesome lung symptoms for at least 3 consecutive days in the diary within the preceding 6 months (i.e. recurrent wheeze); symptoms typical of asthma; the rescue use of inhaled beta2-agonist; and response to a 3-month course of inhaled glucocorticoids followed by relapse after the end of treatment. Remission was defined as a period of 12 months without relapse. Current asthma at age 6 was used as outcome.•Atopic dermatitis was diagnosed prospectively until age 6 years of life according to the criteria of Hanifin and Rajka and with a clinical assessment.•Sensitization to inhalant allergens by specific-IgE or skin prick test measured at age 6. For no sensitization children needed to be tested against minimum 80% of aeroallergens in both tests.•Blood eosinophils at age 6 was categorized by count below vs. above 0.3.•versus asthma at age 6 and never atopic dermatitis, no aeroallergen sensitization and blood eosinophil count below 0.3. We also investigated asthma with vs. without each of the components one by one.Atopic and non-atopic asthma phenotypes are defined as asthma at age 6 years and aeroallergen sensitization, lifetime occurrence of atopic dermatitis and blood eosinophil count above 0.3 •Severe asthma exacerbations 0–6 years were based on acute care visits or medical record checks and diagnosed if the child 1) needed oral prednisolone or high-dose inhaled corticosteroid (ICS) for acute asthma-like symptoms, 2) were hospitalized due to such.•Lung function and airway inflammation measures at age 6 including FeNO; height and sex calibrated FVC, FEV1, FEV1/FVC ratio, MMEF, sRaw; and methacholine provocation test.The primary outcomes of this study are asthma and allergy outcomes, whereas secondary outcomes are early life infections, and lung function measures.•C-reactive protein (CRP) levels were determined in maternal blood at pregnancy week 24 and 1 week postpartum, and in the children at age 6 months by a high sensitivity CRP electrochemiluminescence-based assay from MesoScale Discovery .•Functional immune profiling of response to viral- and bacterial-derived ligands, as well as selective T cell stimulators, totalling 186 parameters were determined from whole blood from 541 18-month-old infants.•DNA-methylation (DNAm) was assessed in respiratory  epithelium at 6 years.Mechanisms of potential associations were sought investigating potential mediation using the following data.Covariates were chosen a priori as factors with association to PFOS/PFOA,https://dawadocs.dataforsyningen.dk/), the longitude and latitude were obtained. These were linked to other publicly available data for land use classification (https://land.copernicus.eu/pan-european/corine-land-cover).Birth address of participants was obtained at interviews. Via a public database with all Danish addresses  was performed to capture co-variance of PFOS and PFOA. Additionally, a PCA of all time point measures was performed.The effect of PFOS and PFOA were investigated individually for each time point, with maternal 1 week postpartum reflecting late pregnancy and breastmilk exposure. In all models, PFOS and PFOA were included as continuous non-transformed concentrations, giving effect estimates per ng/mL, except for models of principal components. For outcomes at age 6, principal components 1 and 2 (PC1 and PC2) of all measurements of PFOS and PFOA were additionally investigated. As sensitivity analyses log-linear effects are also investigated.All analyses are adjusted for confounders listed above in regression models. Time-to-events outcomes were analyzed in Cox regression models. Binary outcomes in logistic regression models; count outcomes in negative binomial models; continuous outcomes in linear regression models. In all analyses interaction with child sex was investigated. In tables all outcomes are on their original scale, except for lung functions measures which are height and sex calibrated. As both multiple exposures and outcome measurements are correlated no multiple test corrections were performed. Mediation was investigated with the R package “mediation”.DNA-methylation (DNAm) was assessed in respiratory  epithelium at 6 years. We included 517 individuals in the DNAm analysis following the exclusion of subjects that did not pass quality control for the DNAm protocol (n = 45).Of the total 866,836 probes on the Illumina Methylation EPIC array, we removed 23,172 with detection p-value of >0.01 in 90% of the samples, 18,695 located on sex chromosomes and 120,903 that overlapped a known common SNP  or mapped to multiple positions because of cross-hybridizing. In total, 703,565 CpGs passed these QC thresholds and were used in downstream analysis. Normalization was performed using the SWAN algorithm from the R package minfi (version 1.26) (10.1093/bioinformatics/btu049) and quantile normalization from the R package lumi (version 2.36) (10.1093/bioinformatics/btn224). We used M-values in analysis, as they provide better sensitivity to slight changes in methylation.Principal component analysis was used to identify the associations with possible confounding variables on DNAm. DNA concentration, array and sampling location significantly correlated with at least one of the first ten principal components and array and sampling location were regressed out using ComBat (part of sva R package version 3.36) For more details on the data procedure see Morin et al., 2020.Latent factors were estimated after protecting for the PFOx using the CorrConf methodAs a sensitivity analysis data are imputed using the method described by van Buuren (2011) using the mice package in R.Ignoring the longitudinal aspect of data , a wide data frame is imputed 20 times, excluding variables which are derived from other data . Default imputation settings are used when possible, however many of the numeric data could not converge using predictive mean matching and were therefore imputed with the cartesian method. Pooled estimates of 20 complete data analyses are presented in The clinical investigations of the children and collection of biobank materials have been approved by the local Committee on Health Research Ethics and the Danish Data Protection Agency , ensuring that all personal data are handled according to GDPR standards and Danish law. All participating parents/caregivers have provided verbal and written informed consent for the participation of their children and use of the biobank samples for metabolomics research, genetics, and other measurements in the project. Participants can withdraw that consent at any time and without any further explanation.The funding agencies did not have any role in design and conduct of the study; collection, management, and interpretation of the data; or preparation, review, or approval of the manuscript.At pregnancy week 24 and 1 week postpartum, a total of 727 and 684 women, respectively, had a valid measurement of both PFOS and PFOA. In the children at age ½, 1½ and 6 years, 602, 606 and 513 had a measurement, respectively. Median values of serum concentrations of PFOS and PFOA are presented in Maternal concentrations were highly correlated (Spearman R > 0.9), and child early life concentrations were similarly correlated both within child and between child and mother. At age 6 years, the child's serum concentrations were no longer strongly correlated to neither previous child measurements nor maternal concentrations , p < 0.01 . We founMaternal and child 6 months measurement of PFOS and PFOA were not associated to increased risk of childhood infections online .The tendency of increased risk of non-atopic phenotypes by maternal PFAS concentrations was confirmed in imputed data, although these results were not statistically significant .There was an isolated statistically significant cross–sectional association between maternal PFOA and maternal hs-CRP (log transformed) at pregnancy week 24, beta-coefficient 0.15 , p = 0.028, and borderline association to PFOS 0.03 (0.01), p = 0.071, but no associations to child's hs-CRP at age 6 months (data not shown). Therefore, hs-CRP at week 24 was tested for any potential mediating effect to offspring non-atopic asthma, which was not significant: mediation effect −1.73e-05 , p = 0.78.The relation between PFOS and PFOA concentrations and innate immune responsiveness towards viral and bacterial ligands and T cell stimulations were weak (|r| < ∼0.15) with none of the signals passing multiple test corrections online .Further, PFOS and PFOA concentrations during pregnancy and childhood showed no association with the changes to DNAm of nasal epithelial cells at age 6 years online .Repeated prenatal and early childhood serum concentrations of bio accumulative PFOS and PFOA in a Danish cohort of 738 pregnant women and their children showed that increasing prenatal exposure was associated with increased risk of a specific low prevalent non-atopic asthma phenotype by age 6 years, but not atopic asthma, asthma exacerbations, atopic dermatitis, common infections, or lung function. Paralleled to increased non-atopic asthma, prenatal PFOS was associated with a decreased risk of sensitization to inhalant allergens. These findings point towards potential prenatal programming but not a childhood effect of PFAS exposure, which was not explained by systemic low-grade inflammation, aberrant functional immune responsiveness towards viral and bacterial ligands and T cell stimulations nor epigenetic alterations.It is a main strength of this study that it is part of the ongoing single-center COPSAC2010 mother-child cohort study and uses meticulously collected and validated longitudinal clinical data. Thus, all data was collected at several time points and manually compared for consistency. All data was double-checked and locked, which minimized the risk of incorrect registrations in the database. The families were followed by trained staff using predefined standard operating procedures. All clinical staff and physicians had paediatric training, ensuring high quality of data collection, validation, and homogenous diagnostic procedures. Further, asthma was diagnosed following a standardized algorithmIt is a limitation that when childhood asthma is divided in phenotypes, the number of subjects investigated are very low. We cannot rule out that some associations reported are chance findings. Further it is a limitation that only PFOS and PFOA exposure was assessed. A recent report showed higher transplacental transfer of other PFAS compounds, i.e., chlorinated polyfluorinated ether sulfonates, Cl-PFESAs for PFOS and perfluorobutanoic acid, PFBA for PFOA, highlighting the need to investigate additional PFAS compounds.,The serum concentrations of PFOS and PFOA measured in this Danish cohort from the Zealand region are approximately three times lower than reported Faroese concentrations measured in a cohort of similar age.,The accumulation of PFOS and PFOA in the human body is tissue specific, i.e., they do not accumulate well in adipose tissues but are present in blood and highly perfused tissues, including liver, kidney and lung tissue.The COPSAC2010 is an asthma-dedicated clinical cohort, with deep phenotyping of childhood asthma and related outcomes, which provides a very homogenous asthma diagnosis and means for subtyping asthma. Further, we included repeated measurements of PFOS and PFOA through pregnancy and early childhood. Thus, in our cohort we were able to observe an association between prenatal PFAS exposure and a specific non-atopic asthma phenotype at age 6 years, but not atopic asthma or lung function, atopic dermatitis or infection proneness. This increased risk specific for non-atopic asthma is a novel contribution to the literature of detrimental effects of PFAS exposure and may explain previous ambiguous findings with respect to asthma. Interestingly, our longitudinal exposure data further revealed that this association was specific to prenatal PFAS exposure, i.e., a suggested in utero programming, while no such association existed for childhood PFAS concentrations. Still, the results should be interpreted with caution as the analyses of the atopic and non-atopic phenotypes were limited by a low number of cases. However, we observed consistent results in auxiliary analyses exploring a range of additional atopic and non-atopic phenotypes, where there were only associations with non-atopic asthma phenotypes.A recent NHANES study in adults found positive associations between PFAS and FeNO,,Low-grade systemic inflammation, determined by blood hs-CRP, is elevated in pregnant women as compared to non-pregnant women.Finally, we did not detect any adverse effects of PFAS exposure on either immune responsiveness to bacterial and viral ligands or T cell stimulations at 18 months or the nasal epithelial DNA methylation pattern at age 6 years.The longitudinal mother-child cohort data from COPSAC2010 on prenatal and early childhood PFOS and PFOA exposure showed that maternal exposure, but not childhood exposure, specifically was associated with an increased risk of low prevalent non-atopic asthma phenotypes at age 6 years. There were no associations between PFAS exposure and risk of atopic asthma, infections, lung function, or atopic dermatitis, and no alterations in innate immune responsiveness towards viral and bacterial ligands and T cell stimulations nor epithelial DNA methylation. These findings suggest an adverse asthma subtype-specific prenatal programming effect, which adds to the list of detrimental health effects of PFAS exposure.The guarantors of the study are HB and BC, from conception and design to conduct of the study and acquisition of data. AS drafted the manuscript. AS, CETP, GG, MAR performed analyses. KS and JM calibrated the semi quantitative data. JS acquired and analyzed data on drinking water. AMo and CO QCed the methylation data. AS and BC have verified the underlying data. AS, CETP, GG, MAR, JALS, AMo, CO, JS, AMMS, KB, BC, and HB have provided important intellectual input and contributed considerably to the analyses and interpretation of the data. All authors guarantee that the accuracy and integrity of any part of the work have been appropriately investigated and resolved and all have approved the final version of the manuscript. The corresponding author had full access to the data and had final responsibility for the decision to submit for publication. No honorarium, grant, or other form of payment was given to any of the authors to produce this manuscript.All data that supports the findings in this study, including clinical data, are available from the corresponding author upon reasonable request: participant-level personally identifiable data are protected under the Danish Data Protection Act and European Regulation 2016/679 of the European Parliament and of the Council (GDPR) that prohibit distribution even in pseudo-anonymized form, but can be made available under a data transfer agreement as a collaboration effort.JWM declares expert witness testimony for plaintiffs’ executive committee representing communities impacted by PFAS and AFFF contamination in the United States . AMMS was a paid speaker for ThermoFischer Scientific. All other authors declare no potential, perceived, or real conflict of interest regarding the content of this manuscript. No pharmaceutical company was involved in the study.


The interactions between liquid droplets and solid surfacesduringwetting and phase change are important to many applications and arerelated to the physicochemical properties of the substrate and thefluid. In this work, we investigate experimentally the evaporationof pure water, pure ethanol, and their binary mixture droplets, accessinga wide range of surface tensions, on hydrophobic micro-pillared surfacesvarying the spacing between the pillars. Results show that on structuredsurfaces, droplets evaporate following three classical evaporativebehaviors: constant contact radius/pinning, stick–slip, ormixed mode. In addition, we report two further droplet evaporationmodes, which are a mixed stick–slip mode where the contactangle increases while the contact radius decreases in a stick–slipfashion and a mixed stick–slip mode where both the contactangle and the contact radius decrease in a stick–slip fashion.We name these evaporation modes not yet reported in the literatureas the increasing and decreasing contact angle mixed stick–slipmodes, respectively. The former ensues because the fluid surface tensionincreases as the most volatile fluid evaporates coupled to the presenceof structures, whereas the latter is due to the presence of structuresfor either fluid. The duration of each evaporation mode is dissimilarand depends on the surface tension and on the spacing between structures.Pure water yields longer initial pinning times on all surfaces beforestick–slip ensues, whereas for binary mixtures and pure ethanol,initial pinning ensues mainly on short spacing structures due to thedifferent wetting regimes displayed. Meanwhile, mixed stick–slipmodes ensue mainly for high ethanol concentrations and/or pure ethanolindependent of the solid fraction and for low ethanol concentrationson large spacing. Contact line jumps, changes in contact angle andpinning forces are also presented and discussed. This investigationprovides guidelines for tailoring the evaporation of a wide rangeof surface tension fluids on structured surfaces for inkjet printing,DNA patterning, or microfluidics applications. They concluded that for pure liquids andhigh water concentration mixtures, the evaporation process ensuesin the CCA mode with a monotonic decrease in the contact radius duringmost of the droplet lifetime, while in the case of the binary mixtureswith a lower water concentration, droplets evaporate following threedifferent stages. In these binary mixtures, a mode of evaporationdifferent from the CCA or CCR modes is observed in which the contactangle increases while the radius decreases as the most volatile andlowest surface tension fluid evaporates.26 The first stage of the evaporation of binarymixtures is governed by the evaporation of the most volatile fluidand the last stage by the less volatile fluid, while the intermediatestage shows different quantiative behavior depending on the ethanol–waterconcentration and initial wetting states.20 More recently, the increase in contact angle coupled with the decreasein the contact radius was also reported during the evaporation ofpure ethanol droplets in humid air as a consequence of the adsorption-absorptionand/or condensation of water vapor onto the surface, which modifiesthe droplet surface tension locally.15The use of pure fluids has been widely studied and addressedinthe literature; nonetheless, these pure fluids provide rather limitedspecific values of thermophysical properties, including surface tension.For binary mixtures, several works address wetting and evaporationon smooth surfaces.29 which in turn play an importantrole in the static and the dynamics of the triple contact line duringwetting and evaporation, i.e., the evaporative modes.30 For pure fluids, the presence of structures prevents the occurrenceof the CCA mode of evaporation in favor of either the CCR or the stick–slipmodes as the additional structural sites present locations for thedroplet contact line to pin.30 McHale et al. showed experimentally that pure water droplets initiallyevaporate in the CCR mode followed by the stick–slip mode onSU-8 textured surfaces with apparent contact angles above 140°.31 More recently, Chen et al.found out that water droplets exhibit three distinctive evaporationmodes on hydrophobic structured surfaces, namely, CCR, CCA, and mixedmodes.32 They further showed, experimentally,that the contact line dynamics of evaporating droplets can be controlledby changing the geometric arrangement of the substrate, i.e., the pillar-to-pillar spacing.32 Xu et al. also noticed these three classical evaporation modes;CCR, CCA, and mixed mode, when using hydrophobic micro-pillared surfaces.17 Their study clearly shows that the evaporationmodes depend highly on the morphology of the structures. When increasingthe spacing between pillars, a longer duration of the CCA mode isobserved when compared to the CCR mode. Consequently, the durationof each evaporation mode depends highly on the nature of the surfaceand the liquid used,33 and its controlis crucial to many applications such as spray cooling and biosensors.10Additionally,in the presence of structures, the wettability andsurface structures strongly influence wetting, spreading, and thefinal shape of the droplet,10 investigatedthe effect of pure water dropletson a superhydrophobic structured surface and found that by increasingthe droplet volume, the evaporation mode can vary accordingly. Xu et al.17 also investigated theevaporation of pure water droplets on smooth and on four differentmicro-pillared surfaces having different spacing between pillars, i.e., different solid fractions. Results further supportedthe similar distinctive evaporation modes to those already reportedin the literature on rough surfaces, where droplets begin evaporatingin the CCR mode, then transition to the CCA mode, and vanish withthe mixed mode. They proposed that as the spacing between pillarsincreases, the duration of the CCR decreases, and the CCA mode durationincreases with similar behavior to that on the smooth hydrophobiccounterpart, while the duration of the mixed mode increases as spacingdecreases.17 Presumably, distinctive stick–slipbehavior must ensue during their reported CCA owing to the presenceof structures underneath the evaporating droplets, which was not reportedas per the low spatial and temporal resolution of their data.17 We highlight that most of the works on structuredsurfaces reported evaporation in the CCA mode and overlooked providingthorough details and analysis on the occurrence of the stick–slipphenomenon during their evaporation, as per the lack of temporal andspatial resolution of the measurements.In addition to the surface structure andfluid type, Dash and Garimella35 nonetheless, there is still a lack of complete andbeneficial understanding of the topic. Using binary mixtures on structuredsurfaces has a significant effect on wetting27 and evaporation26 characteristics. Feng et al. reported on the evaporation of squared and octagonlow ethanol–water concentration droplets (below 30%) on micropyramidcavity substrates looking at the contact angle from two differentazimuthal directions with differences within 12°.36 Droplets were found to evaporate in the CCRmode for most of the droplet lifetime, i.e., from40 to 60% of the time, with no qualitative apparent differences inthe evaporation regimes displayed and their duration when lookingat the droplet from different azimuthal directions. On micropyramidsubstrates and in the presence of surfactants, droplets displayinggeometrical features such as a square or octagon remained pinned duringmost of the droplet lifetime.37 Chiangand Lu made use of water–methanol binary mixtures, at two differentlow methanol mole fractions, on superhydrophobic copper surfaces decoratedwith high-aspect-ratio nanostructured and on smooth ones for comparison.25 Low methanol mole fraction binary mixture dropletson nanostructured surfaces were found to evaporate in the CCR modefollowed by the CCA mode and ending with the mixed mode, while nostick–slip mode was observed. Evaporation behaviors resembledthose of pure water as per the rather low methanol concentrationsstudied. These findings are in agreement with the work of Yu et al.,35 who used ethanol–waterbinary mixtures at three different concentrations on a single PDMSmicrostructured surface configuration. By increasing the ethanol concentrationof the liquid or by using pure ethanol, the evaporation behavior ensuesfollowing two distinctive evaporation modes instead of three. Evaporationoccurs in the CCA mode for almost 80% of the initial evaporation lifetime,and droplets then vanish in the mixed mode without stick–slip.He et al. also investigated the full evaporationprocess of binary mixtures with a high ethanol concentration or pureethanol.33 The duration of the evaporationprocess could be shortened by elongating the droplet contact lineby making use of pillars and binary mixtures.33 In the same line of research, Wang et al. foundout that pure liquid droplets evaporate following the two main evaporationmodes (CCR and CCA modes) while the binary mixture additionally displaysa mixed mode at the end of the evaporation lifetime on a PMMA surface.21Only recently, the evaporationof binary mixtures on structuredsurfaces has received dedicated attention;i.e., the solid fraction. The findings envisagedhere provide needful guidelines to predict the evaporative behavior,duration of the different modes as well as the characteristics ofthe stick-slip mode in terms of contact line jumps, changes in contactangles and pinning forces for fluids varying in surface tension andalso the function of the structure of the surface.Despite the wealth of research on thistopic, binary mixture wettingand evaporation on smooth and/or on structured surfaces have beeninvestigated independently for either a smooth/fixed structural parameterwith varying surface tension, for a single fluid (mostly pure water),or for a limited range of binary mixture concentrations also on alimited range of solid fraction structures. In this work, we providea thorough and systematic study addressing a wide range of surfacetensions, including pure fluids and their binary mixtures on a widerange of microstructured surfaces with an equal microstructure aspectratio (fixed pillar diameter and height) varying the spacing betweenpillars, Tamb = 22 ± 3° withan ambient relative humidity of 35 ± 8% and at ambient pressure.Care was taken to ensure that preferential evaporation of the mostvolatile components was minimized. 27 To note here is the gradual increase in the surface tensionfrom 100% ethanol to 20% ethanol, while a sharp increase where thesurface tension almost doubles ensues between 20% and pure water.By referring to this trend, we can better understand and explain theeffect of surface tension during the evaporation of binary mixturedroplets, which will be discussed in the following sections.Pure liquids, namely, deionizedwater (W) and pure ethanol (E) (Sigma-Aldrich), and also their binarymixtures were used in this study. The binary mixtures were preparedon a volume basis as follows: 80% W-20% E, 60% W-40% E, 40% W-60%E, and 20% W-80% E. This allows the effect of the concentration and/orfluid surface tension to be studied. For brevity, the term concentrationis used as a proxy for volume percentage of ethanol in some graphsand descriptions below. The surface tension of the different fluidsused was measured using the pendant drop method in air in a drop shapeanalyzer device DSA30  at ambienttemperature h, and 10 μm indiameter, d, for an aspect ratio h/d = 1. Meanwhile, the spacing between pillars s varies as s = 5, 10, 20, 40, 80, and160 μm and the corresponding solid fractions equal 0.34, 0.20,0.09, 0.031, 0.009, 0.002, respectively. Microstructured surfaceswere fabricated on a silicon wafer, purchased from Si-Mat , via deep reactive ion etching (DRIE),and further coated with a hydrophobic self-assembled monolayer atthe Scottish Microelectronic Centre (SMC) at the University of Edinburgh.A total ofsix different microstructured surfaces were fabricated and used inthis study. Microstructured surfaces have similar cylindrical pillarswith 10 μm in height, s = 5 μm (top left), s = 10 μm (top middle), s = 20 μm(top right), s = 40 μm (down left), s = 80 μm (down middle), and s =160 μm (down right) captured by scanning electron microscopy(SEM), in a JSM-IT100 InTouchScope scanning electron microscope fromJEOL Ltd., (Japan) with an accelerating voltage of 20 kV, probe current(PC) of 50 a.u., and tilting angle of 30°. The inset in s = 20 μm.Tamb = 22 ±3° with an ambient relative humidity of 35 ± 8% and at ambientpressure. The apparent contact angles, θ (°),on the differently structured surfaces and for the different pureand binary mixture fluids studied in this work are presented in et al.27 The different contactangles reported in et al.27 We note here that inthe presence of a high ethanol concentration equal or above 60%, i.e., fluid surface tension equal or below 30 mN/m and shortpillar spacing equal or below s ≤ 20 μm,droplets display a shape other than symmetric as reported in the workof Al Balushi et al.27 Higher standard deviations were reported for high ethanol concentrations(60% or above) droplets on surfaces with short spacing (below 40 μm)where asymmetrical droplets were observed. The high standard deviationsreported support the different contact angles of asymmetric droplets.Dropletswith defined volumes of 3 ± 0.2 μL for the different liquidsdescribed above were gently deposited at the center of each microstructuredsurface using a drop shape analyzer device DSA 100 . Needles with inner and outer diameters of 0.23and 0.41 mm, respectively, were purchased from Octoinkjet Ltd. (UK)and attached to a 2 mL syringe placed in the DSA100 automatic dosingsystem utilized to produce the droplets. All experiments were performedat ambient temperature θ (°),the volume V (μL), the height h (mm), and the base radius R (mm) of the dropletswere then extracted in time t (mins) using the dropshape analyzer DSA100 and the DSA1 v1.9 software from KRÜSS. Evaporation behavior reportedin terms of contact angle θ (°) and baseradius R (mm) in time t (mins) includesthe average and standard deviation of at least three independent experiments.While the apparent contact angle measurements were carried out fromdifferen azimuthal directions, in the present work, since we focuson the different evaporation regimes ensuing rather than on the asymmetryof the droplets displayed, observations of droplet evaporation werecarried from one single azimuthal direction. To note is that in thework of Feng et al., looking at asymetric dropletevaproation, no major qualitative differences in the evaporation modesand/or quantiative differences on the duration of the different evaporationmodes were reported, which supports adopting one single visualizationangle.36After being deposited on the substrate, droplets wereleft to evaporatefully, and the complete evaporation process was recorded from theside and top views. The contact angle t and t0 as the droplet evaporationtime and the total droplet evaporation lifetime, respectively , on various pillar spacings s = 5, 40, and160 μm for the following pure and binary mixtures 100% W, 80%W-20% E, 40% W-60% E, and 100% E. In addition, top view snapshotsof the droplets at t/t0 = 0 are included to represent the initial droplet shape for eachcase. To note is that only binary mixtures with ethanol concentrationsequal or above 60% on structured surfaces with a spacing below 40μm were found to display asymmetrical shapes. From First, we report experimentalobservations of the droplet evaporation profile. θ, and contact radius, R, versusnormalized time, t/t0, extracted from the same cases represented in s = 5 μm (left) (shortest spacing in this study), s = 40 μm (middle), and s = 160 μm(right) (longest spacing in this study) microstructured surfaces.Depending on the structure spacing and the nature of the liquid (interms of surface tension), different evaporative behaviors ensue,namely, the constant contact radius (CCR), the stick–slip (SS),and the different mixed stick–slip (MSS) modes, as representedin D between 0.005 and 0.02 mm with 0.002 mm as the error on the measurements.The droplet shape and apparent contact angle also vary depending onthe structure spacing and the nature of the liquid, with contact anglesranging between 41 and 148°. The dynamics of the contact angleand contact radius are reported as the average value in solid linesand standard deviation, of at least three independent experiments,in shaded areas. The evaporation behavior for each specific mixtureevaporating on a smooth hydrophobic substrate is also included forcomparison along with the experimental results on s = 160 μm. Both the qualitative evaporation mode/s and thequantitative duration of the modes looking from one single azimuthalangle shall be representative of the evaporation even for dropletsdisplaying asymmetric shapes, although the magnitude of the contactangle and contact radius may differ.36 Ofnote is the rather low standard deviation in the case of evaporationon the smooth hydrophobic surface, i.e., the smallestof the shaded areas. The complete droplet evaporative behaviors forthe full range of binary mixtures and solid structures utilized inthis study can be retrieved within the accompanying Supporting Information. In what follows, the detailed experimentalresults are presented and discussed.To provide further qualitative and quantitativeanalysis on thedifferent evaporation dynamics, s = 5 μm, whereas on larger spacingsurfaces, i.e., 40, 80, and 160 μm, contactangles range between 110 and 112°, which are similar contactangles to those reported on the smooth hydrophobic surface. Dropletsof pure water on all surfaces used have a perfect spherical cap andcircular footprint with ±1° difference in contact angleswhen measured from different azimuthal directions.27 On the shortest spacing s = 5 μmsurfaces represented in s = 10 μm), purewater droplets evaporate following the CCR mode for more than halfof the droplet lifetime followed by the stick–slip mode. Duringthis stick–slip mode, the contact angle remains within a ratherconstant range while the contact radius decreases until the end ofthe evaporation lifetime, similar to the stick–slip evaporationreported upon the addition of nanoparticles to a fluid.9 On larger spacing s = 40 and160 μm represented in s = 5 μm;however, there are obvious differences in the duration of these evaporationmodes, which will be further discussed in subsequent subsections.In addition, when looking more closely into the mixed mode near theend of the droplet lifetime, the contact angle and the contact radiusboth decrease following a stick–slip fashion, which is differentfrom the mixed mode where both the contact angle and contact radiusmonotonically decrease in time. We coin this mode as “the decreasingcontact angle mixed stick–slip” mode. It is worth notinghere that such a mode has not been reported in the literature, presumablydue to the lack of resolution and accuracy on the droplet shape analysisat the late stages of droplet evaporation. In the case of pure wateron structured surfaces, the larger duration of the CCR mode and theoccurrence of stick–slip mode instead of the CCA mode are highlightedas the main differences when comparing to the evaporation of dropletson smooth hydrophobic surfaces represented in When looking into the wettability and contactangles of pure water on the different structured surfaces studied,a maximum apparent contact angle of 148° is reported on the shortestspacing 40 The reason for the quicker ethanol evaporation is its highervapor pressure when compared to water under ambient conditions. Threedistinctive phases including the transition phase have been proposedas phase I: ethanol evaporates at the beginning of the droplet evaporation;phase II: or transition phase, where the strength of ethanol evaporationdiminishes in favor of that of water evaporation; and phase III: wherethere is mainly pure water left to evaporate. When looking into thedroplet profile for ethanol–water binary mixtures, as the mostvolatile, i.e., lowest surface tension fluid, ethanolevaporates, there is a local increase in surface tension as the concentrationof water increases and so does the contact angle of the evaporatingdroplets. Presumably, most of the ethanol will have evaporated atthe point at which the contact angle reaches the highest of the contactangles observed during evaporation.15 Hence,to estimate and delimit the different phases, we studied the evaporationresults of the different binary mixtures on the smooth hydrophobicsurface, and we established a transition threshold between phase Iand phase III at the point at which the contact angle reaches thehighest of the contact angle reported, which is represented with avertical dashed black line in et al. made use of smart sensors to record the concentrationof ethanol during the evaporation of ethanol–water binary mixtureson hydrophilic surfaces.41 They addressedthe evaporation of ethanol–water binary mixtures with concentrationsranging between 0 and 100%. They observed that for 70% ethanol–waterbinary mixtures, the ethanol concentration drops to nearly 5% or lesswithin 15–40% of the droplet lifetime.In the case of binary mixtures, theapparent contact angles reported sit between those of pure water andpure ethanol for each of the independent surface structure configurationsaddressed, as presented in 27 On 5 μm spacing  and finishes with the decreasing contact anglemixed stick–slip mode.However, on larger spacing micropillaredsurfaces When looking into a larger spacingof 160 μm in θ is considerably different,which will be further considered and discussed in the next subsections.For the binary mixture,40% W-60% E on short spacing surfaces of 5 μm, droplets displayan octagonal/squared polygonal shape with an apparent contact angleof around 122° and a standard deviation of 5° when consideringthe different azimuthal directions. The evaporation behavior for thiscase is represented in i.e., increasingcontact angle mixed stick–slip mode, followed by the traditionalstick–slip mode; and the droplet eventually vanishes followingthe decreasing contact angle mixed stick–slip mode. The durationof each of the different evaporative modes is approximately 30% ofthe droplet lifetime for each of these modes. Considering now thelargest of the spacing of 160 μm represented in When considering the intermediate 40 μm spacing representedin s = 5 and 160 μm, respectively. For pure ethanol onshort spacing structures s = 5 μm, despitethe polygonal shape displayed, the evaporation behavior resembles,to some extent, that of water and the other binary mixtures representedin s = 40 μm representedin 15For pure ethanol droplets, the apparentcontact angles vary between 86 and 41° as the spacings vary between s = 160 μm represented in i.e., 12% of the droplet lifetime, and then transitions into the traditionalstick–slip mode. At the end of the evaporation, first, a decreasingcontact angle mixed stick–slip mode ensues followed by an increasingcontact angle mixed stick–slip mode also due to the presenceof adsorbed-absorbed and/or condensed water onto thde droplet duringpure ethanol evaporation. Pure ethanol droplets on a smooth hydrophobicsurface show similar behavior to those on a structured surface when s = 160 μm, especially when looking at the decreasein the radius throughout most of the evaporation lifetime. Of noteis the absence of the CCR mode on the smooth surface, the increasein contact angle, and the decrease in radius taking place on the smoothsurface as earlier reported here and in the literature owed to theadsorption-absorption and/or condensation of water onto the pure ethanoldroplet15 when compared to the traditional stick–slipensuing on the structured surface, this latter being attributed tothe presence of pillars as mentioned before.On large spacing D, and contact angle changes, δθ, as well as evaluation of the pinning force, δF, for each of the configuration studiedover the entire evaporation process.Next, we quantify and discuss the differentevaporation modes and mechanisms taking place, which were presentedand introduced in the previous subsection. In addition, we providesome general guidelines on the initial and main evaporation modesunderpinning the phase change of the wide range of fluid surface tensionson a wide range of structured surfaces. To understand how the structuredsurfaces and fluid binary mixture affect the evaporation dynamics,we first evaluate the duration of each evaporation mode as normalizedtime introduced in the previous section. Second, we additionally assessand compare the magnitudes of the droplet contact line jumps or slips,δ42 mode during evaporation on structuredsurfaces; nonethless, structures induce a certain degree of pinning, i.e., CCR, stick–slip, or mixed stick–slipmodes, and as such, the CCA mode does not ensue in the present caseand as such is not reported within i.e., durations above 60% of the droplet lifetime as wellas the presence of other less prevalent regimes of shorter durationfrom 0 to 20, 20 to 40, and 40 to 60%, ensuing among the CCR, stick–slipand mixed modes, respectively.Looking at the experimentalcontact angle and base diameter or radius, the duration of each ofthe different evaporation modes as normalized times (NT) is calculatedfor at least three independent evaporating droplets under the sameconditions. The percentage duration of the three main distinctiveevaporative behaviors reported in this work, namely, CCR mode (pinning),stick–slip mode, and mixed stick–slip modes (this lattermode including both increasing contact angle mixed stick–slipmode and decreasing contact angle mixed stick–slip mode), foreach fluid composition and for the different spacings studied, ispresented in i.e., pure water, pure ethanol, and their mixtures, on short pillar spacing, i.e., s = 5 and 10 μm, while thegreatest pinning durations are found for pure water. The CCR modeduration ranges between 20 and 70% of the total droplet evaporationlifetime for these cases. However, pure water shows higher pinningtimes on all spacings, particularly on s = 5 μm,where the longest pinning time occurs for around 70% of the dropletlifetime. As the spacing between structures increases or the densityof pillars decreases, i.e., s ≥20 μm, pinning times below 20% of the droplet lifetime are reportedfor pure ethanol and the binary mixtures studied while longer pinningtimes between 20 and 50% are still ensuing in the case of pure water.Thereafter, the evaporation behavior transitions into the stick–slipmode or one of the mixed stick–slip modes.Independently of the droplet compositionand spacing between pillars, most/all of the evaporating behaviorsbegin in the CCR (pinning) mode, as reported in i.e., for binary mixtureson intermediate spacing. Looking closely at each case, for low ethanolconcentration binary mixtures ≤40% E, the stick–slipmode ranges between 40 and 60% of the droplet lifetime on all surfaceswith different spacings. Meanwhile, for high ethanol concentrations≥60% E, the duration of the stick–slip mode is the longeston surfaces with s ≥ 20 μm, while forshort spacing s < 20 μm, the stick–slipmode duration was the shortest. For the cases of pure water droplets,unlike pure ethanol, stick–slip behavior takes place between33 and 56% of the evaporation lifetime independently of the spacing.For pure ethanol, the stick–slip behavior occurred on the surfaceswith large spacing s ≥ 40 μm for theduration between 51 and 71% of the droplet lifetime.When examining the stick–slipmode in s ≥ 40 μm. In thecase of pure water, the duration of the mixed stick–slip modewith respect to the droplet lifetime was the shortest. The short durationof the mixed stick–slip mode also ensues for low ethanol concentrationdroplets ≤40% E on surfaces with short spacing s ≤ 20 μm, which lasts for less than 20% of the evaporationlifetime and/or does not ensue on particular cases.The rest of the evaporation,in fact, ensues in a stick–slip mode behavior coupled withthe decrease or increase in the contact angle, or as defined earlierin the mixed stick–slip mode, which has been represented in s = 40 and160 μm spacing, as represented in s ≥ 20 μm as it can be retrieved from the Supporting Information. For pure ethanol, thismode was observed both at the middle and at the end of the evaporationlifetime without clear indication or relation to the droplet size.On one hand,looking closely at the mixed stick–slip evaporation mode, 26 and hence the contact angle increase coupled withstick–slip behavior due to the presence of the structures.For pure ethanol, this mode occurs due to the adsorption-absorptionand/or condensation of water vapor onto the surface and hence thetransition from pure ethanol to a binary mixture and/or to pure water,which eventually changes locally the droplet surface tension.15On the other hand, the increasing contact angle mixed stick–slipmode mainly occurs for binary mixture droplets on large spacing, althoughwith a duration shorter than 30% of the droplet lifetime. The increasingcontact angle mixed stick–slip mode ensues just after the CCRmode at the beginning of the droplet evaporation, and this is dueto the evaporation of ethanol resulting in the droplet surface tensionincreaseD, and the average changes in contactangle, δθ, respectively, for the differentconcentrations and different structured surfaces investigated. Sincefor binary mixtures and for pure ethanol, the droplet concentrationchanges in time, to provide deeper insights on the different stick–slipevaporating behavior, the magnitude of the jumps and that of changesin contact angle are differentiated whether ethanol preferentiallyevaporates (E) or for the complete binary mixture evaporation includingboth water, ethanol and transition evaporation periods or stages(M). Hence, black columns (E) represent phase I assuming sole ethanolevaporation, which typically occurs at the first stage of the binarymixture evaporation due to the greater volatility of ethanol whencompared to water,40 and red columns (M) representthe average values over the complete binary mixture evaporation processwithout distinguishing the evaporating component, i.e., ethanol and/or water evaporating. We note here that results onthe average distance of the contact line jumps, δD, and the average change in contact angle, δθ, assuming that only water evaporates, i.e., phaseII, are in good quantitative agreement with those results representedfor the mixture (M). Hence, for simplicity, we only represent resultsfor ethanol and for the mixture within subsequent Figures S7 and S8 .This section focuses on the discussion of the contactline behavior during the different pinning/depinning events takingplace in the stick–slip evaporating mode excluding the CCRmode. s + d, accounts for the expected jump distanceto overcome one row of micropillars so that the contact line sitsat the subsequent micropillar row, which is mainly function of thepillar spacing s and is represented as a horizontaldashed lines in s = 5, 10, and 20 μm)substrates shown in D, is highly dependent of the spacingbetween pillars with jumps of similar values as s + d for both the pure fluids and the binary mixturesstudied. Of note is the remarkable agreement between the magnitudeof the jumps for pure water (W) and for the binary mixture (M) withconcentrations of 40 and 60% E evaporating and the expected jump distance s + d.The spacing plus the pillar diameter, calculated as i.e., s ≥ 40 μm, the number/densityofmicrostructures or sites available for contact line pinning decreases,and the contact line is able to move more freely with the consequentmismatch between the expected jump distances and the measured ones,as shown in s + d for most of the binary mixture concentrationsreported for large spacing above s ≥ 40 μmeither considering preferential ethanol evaporation in phase I (E)or both phases considered together (M).As the spacing between pillars increases, θ, for the different pillar spacing and binary mixtureconcentrations is presented in s ≤ 20 μm during most of the phases reported, i.e., independently of the assumption whether pure ethanol(phase I) preferentially evaporates or taking into consideration theevaporation of both water and ethanol (M). The measured and reportedchanges in contact angle, δθ, reportedin At the same time, followingvolume conservation, the depinningof the contact line causes an increase in the contact angle duringeach stick–slip occurrence. The change in contact angle, δ43 By establishing a 2D pinning force balance at the contact line basedon the change in contact angle δθ duringthe pinning stage, the pinning/depinning force, δF, displayed in 43γ is the liquid–gas surface tension and θ0 is the initial apparent intrinsic contact angle.Themagnitude of the contact line jumps and/or changes in contact angleare attributed to the pinning/depinning events as a consequence ofthe pinning force imposed by the structures at the contact line, whichin turn increases the excess surface free energy of the droplet asits shape deviates from spherical cap during evaporation.θ0 is approximated as the average of the initial contactangles during evaporation in the stick–slip period. This isvalid since most of the stick–slip events occur within thesame range of contact angles, except for the cases where the dropletsevaporate in the increasing or decreasing contact angle mixed stick–slipmode. We also note here that the average pinning/depinning force,δF, assuming that only water evaporates afteralmost all ethanol has evaporated, i.e., phase III,and those for the total mixture (M) evaporation are in good quantitativeagreement. Hence, for simplicity, we only represent results for ethanoland for the mixture within Supporting Information, S3: Pinning/depinning force δF in Figure S9.The 2D force balance established in F , calculated and reported in et al.9 In the present case,pinning/depinning force, δF, is attributedto the presence of structures. Despite the different number and densityof microstructures , as well as the larger jumps of the contactline for high ethanol concentrations when compared to high water concentrationsfor large pillar spacing (s ≥ 40 μm).For the same micropillared configuration, pure water and high surfacetension binary mixtures require short jumps of the contact line toovercome similar pinning/depinning forces. The short magnitude ofthe jumps, in turn, gives rise to a larger number of discrete jumps,with the maximum number of these discrete jumps taking place for purewater and high water concentration fluids on the shortests micropillarspacing of s = 5 μm.Meanwhile, the different type of fluid does impactthe magnitudeof the pinning/depinning forces, δF, as per the different wettingregimes and evaporation behaviors reported. The rather uniform pinning/depinningforces, δsion see 1, conseqBasedon the different results and analysis presented above, we proposethe following unified typical evaporation mechanism function of thefluid surface tension and the pillar spacing, as shown in the schematicsin s + d during the stick–slip and/or the mixed stick–slipmodes. On one hand, for high surface tension fluids, i.e., pure water and its high water concentration mixtures, on shortmicropillar spacing, droplets typically sit in the Cassie–Baxterregime, and the jumps of the contact line ensue from one micropillarstructure top to the next micropillar structure top, i.e., δD ≈ s + d. In comparison, for larger pillar spacing, there is nodirect correlation between the magnitude of the jumps and the expectedjump, although δD is typically smaller than s + d and the droplets evaporate in a similarmanner to the smooth counterpart with δD < s + d.i.e., δD > s + d. We note here that in these particular cases, the occurrence ofasymetric droplets may lead to further deviation from the sphericalcap with the consequent higher energy gained by the droplet and thereported greater jumps of the contact line. However, for these lowsurface tension fluids on large spacing, there is also no direct correlationbetween the magnitude of the jumps and the expected jump δD < s + d, similarto the behavior reported for higher surface tension fluids.On the other hand, as theethanol concentration increases on shortpillar spacing, droplets display a larger droplet footprint and lowercontact angles in the partial non-wetting Wenzel regime with contactline jumps typically larger than the expected ones, 45 However, while this phenomena may occur on smooth surfaces, in thecase of our structured surfaces, the presence of finite pinning siteshinders any further spreading or retracting dynamics of the contactline that may occur as a consequence of any surface tension gradient.This is further supported by the absence of any initial contact linespreading or retracting during the droplet evolution over time representedin We would like to further note here that in the case of binary mixtures,spreading or contraction of the droplet contact line may ensue dependingon the volatility and the mixture as well as depending on the differencein surface tensions between the two miscible fluids, which generatesa surface tension gradient locally near the contact line. In the presenceof binary mixtures, solutal Marangoni induces further droplet spreadingthan their respective pure fluids, whereas if the most volatile componenthas the highest surface tension, then the retraction of the contactline may ensue.Local and overall pinning force and free energy analyses appliedto the different evaporating phases, fluid concentration, and pillarstructure, are proposed as further research aiming to reach a moreholistic understanding of the discrete pinning and depinning mechanismsand energy barriers present during binary mixture sessile dropletevaporation on structured surfaces.13Last, we anticipate thatthe different new mixed stick–slipmodes reported here are to be found for other evaporating fluids.More in particular, on one hand, the decreasing contact angle mixedstick–slip mode is to be found at the end of evaporation notonly in the case of this or other binary mixture fluids but also inthe case of pure fluids such as pure water at the end of the evaporation3b,c and i.e., ethanol, evaporates preferentially,and the water concentration within the droplet increases, which eventuallyincreases locally the droplet surface tension leading to the contactangle increase upon a depinning event. This regime was also noticedfor pure ethanol as water present in a humid environment adsorbs-absorbsand/or condenses on the droplet as ethanol evaporates. The decreasingcontact angle mixed stick–slip mode occurs at the end of thedroplet evaporation when the pillars affect considerably the contactline motion and the shape displayed by small volume droplets. Theextent and duration of these evaporation modes are dependent on thefluid surface tension and on the pillar spacing, as reported here.Moreover, the magnitude of the stick–slip contact line jumpsalong with the changes in contact angle was studied. It was foundthat the contact line movement is dependent on the spacing betweenpillars and self-contained within the spacing and diameter of thepillared configuration (s + d) inthe case of high surface tension fluids and short pillar spacing.For larger pillar spacing, the magnitude of the jumps is below theexpected value s + d. In addition,all fluids used on structured surfaces exhibit similar values of pinning/depinningforce except for short spacing where this force increases as the ethanolconcentration increases. The increase in the pinning force for mediumand high ethanol concentrations lies in the different wetting behaviorsas partial non-wetting Wenzel and the consequent greater droplet–surfaceinteractions when compared to Cassie–Baxter droplets, thislatter regime ensuing in the case of low ethanol concentration andpure water. In comparison for large spacing, most/all average valuesare within the standard deviations. It is concluded that by choosingthe appropriate surface structure and fluid surface tension, the wettingregime, droplet shape, initial pinning time, evaporation mode, andits duration can be tailored, which can prove to be beneficial tomany engineering, biological, and/or medical applications.The evaporation behavior of pure waterand pure ethanol and theirbinary mixtures on hydrophobic structured surfaces has been experimentallyinvestigated. Three different evaporative behaviors have been noticed:pinning, stick–slip, and mixed mode, with the absence of aconstant contact angle mode, which are consistent with the literature.In addition, two further mixed stick–slip modes are reportedhere for the first time, namely, the increasing contact angle mixedstick–slip and the decreasing contact angle mixed stick–slipmodes. The increasing contact angle mixed stick–slip mode occursrelatively near the beginning of the evaporation as the more volatilefluid,


Soil moisture content (SWC) can change the diversity and composition of soil fungal communities by affecting soil texture and soil nutrients. To explore the response of soil fungal communities to moisture in the grassland ecosystem on the south shore of Hulun Lake, we set up a natural moisture gradient that was subdivided into high (HW), medium (MW), and low (LW) water contents. Vegetation was investigated by quadrat method, and aboveground biomass was collected by the mowing method. Soil physicochemical properties were obtained by internal experiments. The composition of the soil fungal community was determined using high-throughput sequencing technology. The results showed significant differences in soil texture, nutrients, and fungal species diversity under the moisture gradients. Although there was significant clustering of fungal communities in different treatments, the fungal community composition was not significantly different. According to the phylogenetic tree, the Ascomycota and Basidiomycota were the most important branches. The fungal species diversity was smaller when SWC was higher, and in this environment (HW), the fungal-dominant species were significantly related to SWC and soil nutrients. At this time, soil clay formed a protective barrier for the survival of the dominant classes Sordariomycetes and Dothideomycetes and increased their relative abundance. In summary, the fungal community responded significantly to SWC on the southern shore of the Hulun Lake ecosystem in Inner Mongolia, China, and the fungal community composition of the HW group was stable and easier to survive. Soil microorganisms are the most active components of soil ecosystems and are involved in many key soil processes, such as organic matter formation and transformation, and elemental biogeochemical cycling ,2. In a Soil water content (SWC) is one of the important factors that maintains soil microbial activity and affects ecosystem function . DirectlHulun Lake is a large lake in the high-latitude, semi-arid region of northern China, a unique natural lake ecosystem with diversity and ecological functions similar to those of arid regions . HoweverHulun Lake is the largest lake in the Inner Mongolia Autonomous Region, located at 117°00′10″–117°41′40″ E and 48°30′40″–49°20′40″ N. The lake surface is irregularly rectangular. This study was carried out in the grassland ecosystem of the south shore of Hulun Lake, characterized by low anthropogenic disturbance and single grazing. The area is a temperate grassland, and the establishment species are mainly Stipa krylovii Roshev and Leymus chinensis (Trin.) Tzvel. The climate is characterized by a semi-arid continental climate, with an annual precipitation of about 280 mm and an average annual evaporation of 5–6 times the annual precipitation. In August 2019, we established a natural moisture gradient sample strip on the south shore of Hulun Lake, extending about 25 km landward along the lake, with a total of nine sampling sites, and three 10 m × 10 m replicate plots were randomly established in each sample site. Using a handheld GPS, each sample site was located at a distance of about 2–5 km. The sample site closest to the lake was sample point 1, and each sample point was further away from the lake in increasing order. We divided the natural moisture gradient sample strip into different groups of high water content (HW), medium water content (MW), and low water content (LW) based on distance from the lake and SWC .We randomly selected three 1 m × 1 m quadrats in each sample plot, and the aboveground biomass (AGB) within each quadrat was mowed, and brought back to the laboratory to dry to a constant weight in a desiccator, and weighed. Using a soil auger boring of 8 cm in diameter and 10 cm in height, a 0–10 cm soil layer was drilled in each of the mowed sample quadrat, three soil samples were mixed into a soil sample in each plot, three soil samples were collected for each site, and a total of 27 soil samples were collected in the natural moisture gradient. The soil was sieved at 2 mm to remove obvious roots. The sieved fresh soil samples were divided into two parts, and one part was stored at 4 °C for the determination of basic physical and chemical properties of the soil. Soil bulk density (BD) was measured by the cutting ring method and SWC was measured by the drying method. Soil pH was measured using a combination glass electrode. SOC and TN were measured with the elemental analyzer FLASH 2000. Soil texture  was analyzed with a laser particle analyzer Mastersizer 2000. The other part of fresh soil samples was put into sterile centrifuge tubes and stored in a refrigerator at −80℃ and sent to the Research Center for Eco-Environment Sciences, Chinese Academy of Sciences for high-throughput sequencing of soil microorganisms.Genomic DNA was extracted from 0.5 g of fresh soil using a PowerSoil DNA Isolation Kit (MO BIO). DNA quality was determined using a NanoDrop spectrophotometer . For fungi, PCR was performed to amplify the ITS2 region using primers ITS3 (5′-GCATCGGAAGAACGCAGC-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) .The amplhttp://unite.ut.ee, 27 May 2020) was used with USEARCH10 [ITS gene sequences were processed with USEARCH10 . First, SEARCH10 ,34 for fhttp://itol.embl.de/ (2 November 2022) for beautification. The Spearman correlation was calculated using the "psych" package in R, and corrected for p-values using the “fdr” method, followed by a correlation heat map of environmental factors and fungal strains using “pheatmap”.The AGB, soil physicochemical properties, and relative abundance of fungi under different soil water contents were estimated by one-way ANOVA with SPSS 26 and Duncan’s post-hoc test. The “hclust” package was used in R to group the raw data after hierarchical clustering. Soil microbial diversity was computed using the “Vegan” package in R 3.6.0 software. Box line plots of fungal alpha diversity and stacked histograms of species composition were plotted using the “Hmisc” and “ggplot2” packages. Fungal β-diversity was analyzed in principal coordinates using the “Euclidean” distance matrix and visualized through the ImageGP website. The fungal-specific ASVs and shared ASVs sequences in different soil water contents were visualized using the “VennDiagram” package in R to obtain Venn diagrams. The sequence alignment was performed using Muscle software, followed by the rapid construction of maximum likelihood method (ML) evolutionary trees using IQ-TREE, and the obtained evolutionary tree data were imported into SWC, soil clay, and sand content, SOC and TN were significantly different in the different moisture gradients. Among them, SOC and TN showed a trend of first increasing and then decreasing with the decrease in SWC . AbovegrThe fungi were divided into three clades, and Ascomycota and Basidiomycota were the dominant clades . The ASVThere was no significant difference between the dominant phyla Ascomycota and Basidiomycota . At the The fungal species diversity  was significantly different under soil water contents. Additionally, there was an increasing trend with the decrease in SWC . HoweverBased on Euclidean distance, PcoA showed that the fungal communities were clearly separated under a natural moisture gradient, and the first and second axes explained 14.13% and 7.08%, respectively . HW, MW,The fungal communities were significantly correlated with soil physicochemical properties . The domSoil fungi have improved stress and drought resistance for better adaptation to soil nutrient restriction and drought conditions . Soil phWe established a phylogenetic tree by maximum likelihood method, and the fungi were divided into three main clades (phylum); with Ascomycota and Basidiomycota being the two largest clades . It waspThe species diversity  of the fungal communities showed an increasing trend from the near lake to far lake . Some stThe natural moisture gradient in the grassland ecosystem on the south shore of Hulun Lake caused a significant variation in microbial taxonomic. Horton et al.  also obsUnder HW, MW, and LW, 116, 90, and 86 unique ASVs, respectively, were observed in our study , showingp < 0.05). The relative abundance of Sordariomycetes decreased when soil nutrient content was higher (Soil physicochemical properties are important factors in the variation of fungal communities . Soil nus higher B. Furthes higher B. In thes higher . Secondls higher B, indicaA natural moisture gradient in the grassland ecosystem on the south shore of Hulun Lake caused differences in the soil fungal community diversity and structural composition. SWC, as the most significant factor, altered the soil texture and nutrients, and thus affected the fungal communities. The fungal species diversity tended to decrease with increasing SWC. Furthermore, although the fungal β-diversity clustered significantly, their differences were not significant under different soil water contents. Ascomycota and Basidiomycota were the main fungal phyla when SWC was high. Additionally, Sordariomycetes and Dothideomycetes as the main dominant fungal classes had high relative abundance. Moreover, the unique ASVs of fungi were significantly affected by SWC and their numbers increased with increasing SWC. In general, the fungal community structural composition was stable in the high water environment, indicating that the fungal communities in the grassland ecosystem on the south shore of Hulun Lake are more likely to survive in the near-lake environment.