{"paper_id":"03152f4f-e81e-40cc-bc95-0ed6bc4f8026","body_text":"License and Terms: This document is copyright 2025 the Author(s); licensee Beilstein-Institut.\nThis is an open access work under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0). Please note that the reuse,\nredistribution and reproduction in particular requires that the author(s) and source are credited and that individual graphics may be subject to special legal provisions.\nThe license is subject to the Beilstein Archives terms and conditions: https://www.beilstein-archives.org/xiv/terms.\nThe definitive version of this work can be found at https://doi.org/10.3762/bxiv.2025.26.v1\nThis open access document is posted as a preprint in the Beilstein Archives at https://doi.org/10.3762/bxiv.2025.26.v1 and is\nconsidered to be an early communication for feedback before peer review. Before citing this document, please check if a final,\npeer-reviewed version has been published.\nThis document is not formatted, has not undergone copyediting or typesetting, and may contain errors, unsubstantiated scientific\nclaims or preliminary data.\nPreprint Title Ultrathin water layers on mannosylated gold nanoparticles\nAuthors Maiara A. Iriarte Alonso, Jorge H. Melillo, Silvina Cerveny, Yujin\nTong and Alexander M. Bittner\nPublication Date 23 Apr. 2025\nArticle Type Full Research Paper\nSupporting Information File 1 250422supp_Iriarte.doc; 868.0 KB\nORCID® iDs Jorge H. Melillo - https://orcid.org/0000-0001-7642-0368; Silvina\nCerveny - https://orcid.org/0000-0001-7727-8156; Alexander M.\nBittner - https://orcid.org/0000-0003-4815-2444\n\n1 \nUltrathin water layers on mannosylated gold nanoparticles \n \nMaiara A. Iriarte Alonso 1, Jorge H. Melillo 2,3, Silvina Cerveny 2,3, Yujin Tong 4,5, and \nAlexander M. Bittner*1,6 \n \n1 CIC nanoGUNE (BRTA), Donostia, Spain \n2 Centro de Fisica de Materiales (CFM-MPC, CSIC-UPV/EHU) Donostia, Spain \n3 Donostia International Physics Center (DIPC), Donostia, Spain \n4 Fritz Haber Inst. der Max Planck Ges., Berlin, Germany \n5 Abt. Physik, Univ. Duisburg, Germany \n6 Ikerbasque Basque Foundation for Science, Bilbao, Spain \n \nEmail: Alexander M. Bittner, a.bittner@nanogune.eu \n \n \n \n \n  \n\n2 \nAbstract \n \nWe investigated t he effect of air humidity on two gold nanoparticle systems, one \nfunctionalized by an oligo ethylene glycol ligand, and one functionalized by a mixture \nof the same with a dimannoside ligand. The dimannoside on a gold particle was chosen \nto mimic the shape and surface chemistry of viral “spike” proteins . We characterized \nthe particles by  electron microscopy, dynamic light scattering, and infrared \nspectroscopy. We probed particles adsorbed on hydrophilic and hydrophobic surfaces \nwith atomic force microscopy (AFM) and vibrational sum frequency generation (VSFG) \nspectroscopy, both operated u nder variable air humidity. For AFM, we additionally \ntested hydrophilic and hydrophobic tips. While VSFG indicated preferential hydration \nof the dimannoside and proved conformational changes in the organic ligands, AFM \nprovided sub-nm changes in particle t opography due to water adsorption. In general, \nthe dimannoside nanoparticles condense ultrathin water layers upon humidity \nincrease. In contrast, we found that the water adsorption on the oligo ethylene glycol \nparticles depends little on humidity. Our insi ghts into structural changes on \nglyconanoparticles and the  hydration properties of glycosylated particles are of \napplication value for biosensors and help model the transmission of airborne viruses , \nsuch as influenza. \n \nKeywords \n \nWater; wetting; AFM; sum frequency generation spectroscopy; nanoparticles; viruses; \nhydrophobicity; hydrophilicity; humidity  \n \nIntroduction \n \nGold nanoparticles (AuNPs) are a staple in biomedical and biophysical research [1,2] \nfor almost a century [3]. They are investigated, e.g., for drug delivery [4], but they are \nalso parts of  actual products , e.g. of sensors [5]. All t his is based on the  ease of \nsynthesis, chemical stability, size tuneability, and unique optical properties [ 6]. The \nextreme dependence of the properties on particl e size and shape has been \ndemonstrated for particle sizes in the 1-100 nm range and on biological interfaces [7]. \nLimited biocompatibility and high tendency to aggregate in solution inspired new \nmechanisms of particle biofunctionalization with proteins, li pids, or carbohydrates. \nCoupling carbohydrates to AuNPs provides particle stability and biocompatibility and \nallows for studying carbohydrate -mediated interactions and designing novel \ncarbohydrate-based antiviral agents [ 8,9]. From a molecular point of vie w, \nglyconanoparticles are water soluble gold nanoclusters with a three -dimensional \ncarbohydrate display, which defines their biological function [10], with a small core size, \nof globular shape, and chemically well -defined composition. Proof of concept stud ies \nhave demonstrated the vast potential of glyconanoparticles for glyconanotechnology \nin solution. However, potential changes of \"glycoclusters\" [2] in dry or humid \nenvironments are not well known, although there are many examples, e.g. sensors that \nuse a ntibodies (glycosylated proteins) linked to AuNPs, such as the now very \n\n3 \nestablished SARS-CoV-2 antigen tests [5]. While practical questions around storage \nconditions and lifetimes call for tests in a realistic environment, the scientific bases are \nassumptions and analogies to chemically similar systems, rather than data. \nSeveral authors have synthesized and investigated (di)mannoside -coated AuNPs. \nWhile there are multiple applications [ 4], such NPs can also be seen as very crude \nmodels of viral “spikes”, wh ich are crucial for virus “survival” during transmission  \n[11,12]. For many, especially mammalian viruses, transmission is in an aqueous \nenvironment (e.g. Dengue [ 13] and Ebola [ 14]), and air humidity does not play any \ndirect role. In contrast, it strongly affects the transmission of influenza [ 11,12,15] or \nSARS-CoV-2 [14] via aerosols. These viruses are enveloped (by glycosylated lipid \nbilayers) and display very large multimers of nanoscale glycoproteins (“spikes”), which \ncontrol virus attachment and fusion  to the host cells [ 16]. Glycosylation, often with \nmannosides [17], is essential to infection. Complete dryness is certainly detrimental \n(lipid bilayers ultimately collapse). Still, how such virus surfaces are preserved in low \nhumidity conditions  is unknow n, as is typical for airborne transmission in Northern \nHemisphere winters [18]. We believe that a glycosylated AuNP can provide a simplified \nmodel of a viral “spike”, whenever the virus is very densely coated, e.g. influenza by \nhemagglutinin [ 18,19]. Although the shape cannot be identical (hemagglutinin is \nroughly a triangular 7 nm prism of 15 nm length), the size is in the typical NP range, \nand a dense coating with oligomannoside should mimic surface physics. In the same \narguments, we note that the adsorption of AuNPs on surfaces would mimic the survival \nof adsorbed viruses, which can either be transmitted mechanically or again become \nairborne. In any case, the role of air humidity for adsorbed viruses is poorly \ndocumented, and its influence on transmission physics is not known. \n \n \nFigure 1. Structures of gold nanoparticles used in this work. (A) PEG AuNPs were obtained in the presence of \ncarboxyl PEG thiol. (B) dimanno-AuNPs were covered with 50 % of dimannoside and 50 % of carboxyl. Note that \nin (A) a nd (B) the linear structure of the particles is displayed below the structural scheme. In (B) only the \ndimannoside is displayed. The yellow circles represent the gold core, the light blue lines the PEG ligand; the thin \nlines the PEGylated ligand terminated by dimannose residues (two hexagons).  \n \nWe chose as models dimannoside gold nanoparticles (dimanno-AuNPs) [6] linked to a \nthiourea PEG thiol chain; the particles also feature COOH -terminated PEG chains \n(Figure 1). We used a typical standard PEG coating on  AuNPs, again with COOH \ntermini (PEG AuNPs) for comparison. Here and in the following, we use the established \nterm “polyethylene glycol” (PEG) to designate our relatively short oligo ethylene glycol \n\n\n4 \nchains. The particles were first characterized by dynamic  light scattering (DLS) and \nzeta potential (ZP) measurements in solution, and by scanning electron microscopy \n(SEM) and scanning transmission electron microscopy (STEM) in vacuum. Samples \nwere adsorbed on flat inorganic surfaces, usually modified with orga nic layers, and \nprobed by Fourier-transform infrared spectroscopy (FTIR), vibrational sum frequency \ngeneration (VSFG), and atomic force microscopy (AFM). For VSFG and AFM, we \nsystematically varied the relative air humidity (RH). \nDLS and ZP yield particle s ize and stability in solution , regarding hydrodynamic \ndiameter and NP surface charge, respectively. Spectroscopy techniques were used to \nanalyze the chemical composition of the organic ligands locally. We used FTIR for the \nmolecular fingerprint infrared re gion to find the characteristic peaks of the organic \nlayers. In contrast, VSFG was applied to obtain interface -sensitive information on CH \nand OH bonds at the AuNP/air interface, under hydration and dehydration. We also \nused a deuterated water (D 2O) atmosphere to distinguish the mannosyl hydroxyl \ngroups from adsorbed and absorbed water.  \nWe achieved detailed spatial characterization by SEM, to measure size and shape, \nand to detect aggregation upon adsorption on surfaces of different hydrophilicity  (see \nSuppl. Info. S1). We used heavy metal staining in STEM on the nm scale, to distinguish \nthe organic ligand shell from the gold core. The main method, however, was \n“noncontact” (AC mode) AFM. Its advantage lies in obtaining a very detailed surface \ntopography, i.e., that is a height image. This also includes adsorbed water layers on \nthe sample, which differ from the adsorption on the surface, such that height variations \ntaking place only on the sample are correlated with air humidity [ 20]. These ideas are \ninspired by previous works of Verdaguer et al. [20,21] and Chiantia et al. [18]. \nUltrathin water layers are extremely delicate, and there is a risk of over -interpreting \nheight information. Indeed, experiments with soft matter in ambient humidity usually \ncreate a thin film of water covering the tip and the sample. A mechanical capillary water \nneck is formed in hydrophilic systems when the AFM tip comes close to the sample \nsurface [22]. In unfavorable cases, this can result in apparent heights up to four times \nlarger than the actual values [ 23]. Experimentally, this issue can be reversed by \ndifferent approaches. First, the height measurements can be achieved at set point \nranges and working distances where only attractive regimes in the amplitude-distance \ncurves are accessed. Second, tips with different hydrophilicity complement \ntopographic information with accurate height determination. Third, the same is true for \nsurfaces; self-assembled monolayers (SAMs) of silanes are well suited to assess the \nwater layer contribution in AFM measurements [24]. They form stable and well-defined \norganic layers on oxides, e.g. on oxidized silicon wafers or glass, where surface charge \nand hydrophilicity are controlled by selecting the appropriate end groups. We \ncombined all the methods mentioned to carry out AFM at variable relative humidity \n(RH) levels in our AuNP systems. Specific care was taken for height measurements, \nas demonstrated by amplitude -distance curves and statistical analysis (t -tests). We \nadded experiments with deuterated water analogous to the mentioned VSFG tests. \nResults \n \nThe dimanno-AuNPs consist of functionalized AuNPs with a mixture of dimannoside \nligand and a PEGylated ligand. The PEG AuNPs were functionalized only with th e \n\n5 \nPEGylated component; they can be seen as a precursor, and we used them as \nnegative controls. \n \nParticle size and shape \n \nParticle size and stability in an aqueous solution were characterized by DLS and ZP, \nwhile size and morphology in completely dry condit ions were measured by electron \nmicroscopy.  \nDLS yields a hydrodynamic diameter of the PEG AuNPs of 67.7 ± 9.4 nm and a \npolydispersity index (PDI) of 0.35. However, the size distribution (Figure 2) reaches a \nmaximum at (41.1 ± 4.2 ) nm. These results indicat e that the sample was slightly \npolydisperse. Hence, a small number of multimers of the NPs is present in the solution. \nIn contrast, the size average of the dimanno-AuNPs was (30.4 ± 1.1 ) nm (PDI 0.29) \nwith a peak at (16.8 ± 0.9) nm. This indicates that the  dimannoside coating results in \nreduced nanoparticle aggregation.  \nIn both cases, the NP surface charge in water at pH ~7 was ~20 mV. The isoelectric \npoint of carboxylate PEG capped particles is ~2.5, so around pH 7 they should exhibit \na negative ZP. This is also compatible with a low carboxylate content, as citrate capped \nAuNPs (with a higher concentration of carboxylate) exhibit a lower ZP (~ 45 mV) at the \nsame pH [1].  \n \nFigure 2: Distribution of hydrodynamic diameters of dimanno-AuNP (black) and PEG AuNP (red) solutions, \nobtained by DLS. \nTo obtain a clearer view of the size distribution, we used SEM to evaluate particle sizes \nand morphologies in high vacuum (i.e. for completely dried samples). By adjusting a \nGaussian fitting to the histograms  (Figure 3), the diameter of the particles adsorbed \non APDMES silicon was estimated. The dimanno-AuNPs exhibited a mean size of \n(14.8 ± 1.6) nm (Figure 3-A and 3-C), and the size of the PEG AuNPs was (14.3 ± 1.5) \nnm (Figure 3-B and 3-D). \n\n\n6 \n \nFigure 3: SEM images of dimanno-AuNPs and PEG AuNPs adsorbed to a hydrophilic surface . (A) Dimanno-\nAuNPs, (B) PEG AuNPs  (scale bars 100 nm) . (C and D) Particle size histograms (diameter) from (A and B) , \nrespectively. \n \nFigure 4: STEM image of negatively stained dimanno -AuNPs (scale bar 100 nm). The gold  cores appear bright, \nthe carbon grid background is dark, and the organic layer appears as grey halo. \n \nThe organic layers from PEG and dimannoside are transparent to electrons in S EM \n(only the gold cores were observed as bright features). We recorded STEM images of \ndimanno-AuNPs deposited on a carbon coated TEM grid to visualize the layer. We \nemployed uranyl as a stain, a soluble heavy metal cation, which attaches to the \nhydrophilic parts of the organic coating , providing good contrast [2 5]. The STEM \nimages in dark field mode (imaging scattered electrons) show the gold cores as bright \nstructures, whereas the thin organic layers are dark. The carbon grid yields practically \nno scattering and appears essentially black, such that both the core and shell of the \nparticles can be distinguished (Figure 4). The estimated organic layer thickness is 1.5 \n\n\n7 \nnm, as obtained by subtraction of the total radius of the negatively stained particles \nand the radius of the gold cores. \n \nAnalysis of the chemical groups \n \nThe nature of the coating layer of (PEG AuNPs and dimanno-AuNPs) was \ncharacterized by FTIR (only in dry nitrogen) and VSFG  at variable humidity . We \ndiscuss details of t he FTIR spectra in the Suppl. Info. S2, including the origin of the \ndifferent bands. Here, we mention that the PEG AuNPs spectra exhibit only a trace of \nO-H stretching bands, in stark contrast to dimanno-AuNPs. It is well known that sugars \nare very difficult to dry completely, so related bands are  easily found in many \ncarbohydrates (see Suppl. Info. S2).  \nFor the VSFG, the ssp polarized spectra were obtained by adding a droplet of H 2O or \nD2O on the dried samples and subsequent evaporation ( G-A and G -B). The VSFG \nspectra focus only on the high frequency region. The vibrational bands are \nsuperimposed on a very broad signal from bulk gold, quasi-constant after normalization \n(like FTIR). In contrast to FTIR, the bands stem only from vibrations at interfaces, \nhence from the NP surface in contact with air. Additionally, highly symmetric vibrations \nare forbidden (with very low or zero intensities) [26].  \nThe PEG signals correspond, as expected, quite well to the infrared spectrum, with a \nstrong peak at 2880 cm-1. This feature points to a lack of local order, which should be \nbased on disorder of the PEG chain (ordered alkyl chains, for example, yield no signal \nfrom CH2). Moreover, the peak is unusually broad, possibly due to the complex shape \nof the disordered PEG alkyl chain. However, the disorder is only present in each chain; \naltogether, the C H stretching vibrations must have a preferred orientation with respect \nto the gold surface. In other words, the nanoparticle has a non -centrosymmetric \nenvironment, which can simply be the presence of the gold surface. The noise -like \nfeature at ~3400 cm-1 is assigned to hydrogen-bonded water, as features above 3600 \ncm-1 should indicate non-hydrogen-bonded water. This is proof that water is adsorbed \non the PEG AuNPs. A quantification is not possible; however, there cannot be more \nthan a few monolayers, otherwise the band would completely dominate the spectrum. \nThis is also in agreement with the absence of the band in FTIR. Hence, the hydrophilic \nPEG chain binds water, but can easily be dried, while the last traces remain. This is \nsimilar to a strongly bound water (mono)layer on biomolecules. \nThe dimanno-AuNPs, under the same conditions, exhibit a very different spectrum: \nThe C H stretching bands at 2850 cm-1 and 2920 cm-1 (see also FTIR) are \"negative\", \ni.e. the background VSFG signal is diminished. The frequency only slightly shifts from \nthat of CH2 groups in alkyl and PEG chains.  The \"dip\"-like features should be due to \nthe interference effect between the resonant and the non-resonant signal. The different \nfeatures of PEG-Au and dimanno-Au may be due either to the other dipole orientation \nof the CH group between the two samples [ 27], or to the charges of the molecules \nbeing opposite [ 28]. The latter seems unlikely since the isoelectric points of the two \nsamples are similar.  \nBy the symmetry rules and the ssp -polarization, one can deduce that the CH 2 groups \nare again oriented, but different from those in the PEG AuNPs. This shows directly that \nthe dimannoside strongly affects the PEG and alkyl chain conformation. We can \n\n8 \nexclude disorder and propose a stretched-out geometry based on the relatively narrow \nbands. The dimannoside must be covered by water, and indeed , a peak assigned to \nliquid water is present. Water on the dimannoside can adsorb in a multitude of \norientations. This averaging results in a low signal.  Figure 5 provides a sketch of the \nprincipal ideas. \nVSFG experiments were repeated after exposure to D 2O. The original idea of using \nD2O was to distinguish the hydroxyl groups of the sugar molecules from those of \nadsorbed/absorbed water. However, the surface hydroxyl groups of amorphous sugar \nmay also be subject to H-D exchange [29]. Nevertheless, Figure 5-B indicates that the \nH2O molecules of the top water layer can exchange back under the experimental \nconditions. The time for the isotope exchange is extremely long: days or even months \n[29]. Deuteration usually assesses the exchange capability of the hydroxyl groups in \ninfrared frequencies between 3100 and 3700 cm-1. Also, the slower structural dynamics \nat the air /D2O interface allow better visualization of  signals related to the air/H 2O \ninterface on the particles  and enable find ing whether there are ordered water \nmolecules at the sample surface.  \nIn this experiment, the D 2O exposure time of 30 minutes is not sufficient to exchange \nthe H in the dimannose OH groups [2 9]. However, due to its large excess, it should \nreplace the thin layers of adsorbed water. Figure 5-B shows that this is indeed the \ncase, the CH region is practically unchanged, while the 3400 cm-1 signals are barely \npresent. Note that the OD stretch in liquid D2O is shifted to 2500 cm-1. In this way, the \nmodel of thin adsorbed water layers is bolstered, also showing the strongly hydrophilic \nnature of both PEG AuNPs and dimanno-AuNPs.  \n \n \n \n \n \n\n\n9 \n \n \nFigure 5: Normalized VSFG spectra in CH and OH region of dimanno-AuNPs and PEG AuNPs on gold surface, \nssp (s VSFG, s vis, p IR) polarization combinations. (A) VSFG spectra in H2O. (B) VSFG spectra in D2O. The black \nline represents the VSFG spectra of dimanno-AuNPs; the red line represents the VSFG spectra of PEG AuNPs. \nNote the different y-axis scale. (C) Model of the proposed conformations of dimanno-AuNPs and PEG AuNPs. PEG \nAuNPs (left) show little order in the PEG chains. Dimanno-AuNPs (right) show a stretched-out geometry provided \nby the dimannose residues (organic layers not drawn to sc ale). The yellow circles represent the gold core; the \nflexible light blue line represent the PEG ligand; the rigid line represent the PEGylated ligand ordered by the \npresence of dimannose residues (two hexagons), surrounded by water (droplet-like blue areas). \n \nAdsorption of the particles on hydrophilic and hydrophobic surfaces \n \nWe assessed the aggregation tendency of the particles after adsorption with SEM and \nAFM. To this end, we determined water contact angles. The hydrophilic APDMES \nsilicon gave 66 ± 3º . In contrast,  on the hydrophobic OTS silicon it was 92 ± 2º . \nComparison of the two surfaces shows characteristic differences (Figure 6), as known \nfrom analogous investigations with tobacco mosaic virus [30] and with surface-layered \nproteins [24].  \nThe dimanno-AuNPs are homogenously distributedon APDMES. Consequently, SEM \nimages have low contrast ( Figure 6-A). Zooming to the nanoscale, we found a \nhomogenous dispersion of the particles ( Figure 6-B). This is supported by AFM \ntopography images at the nanosc ale (Figure 6-C). In contrast, PEG AuNPs are not \nwell dispersed, with more agglomerates (see the analogous Figure 6-G, 6-H, 6-J), and \nparticle clusters form microscale islands.  \nWe analyzed adsorption on the hydrophobic OTS with the same procedure (Figure 6-\nD, 6-E, 6-F and 6-J, 6-K, 6-L). Here, the required drop -casting method resulted in a \ncoffee ring effect [31], as nicely seen in Figures 6-D and 6-J. Local in-homogeneities \nprevail on areas inside and outside the ring. Generally, we observed here a very small \nnumber of NPs and higher agglomeration than on APDMES ( Figure 6-E. 6-F, 6-K, 6-\nL). Like the case on APDMES, PEG AuNPs are less uniformly distributed than \ndimanno-AuNPs. \n\n10 \n \nFigure 6: Representative SEM and AFM images of dimanno-AuNPs and PEG AuNPs on hydrophilic and \nhydrophobic surfaces. (A, B, D, E, G, H, J and K) SEM images. (C, F, I and L) AFM images. (A C) Dimanno-AuNPs \nadsorbed on hydrophilic APDMES modified wafer. (D F) Dimanno-AuNPs adsorbed on hydrophobic OTS modified \nwafer. (G I) PEG AuNPs adsorbed on the hydrophilic surface. (J L). PEG AuNPs adsorbed on the hydrophobic \nsurface. Scale bars 400 µm (A, D, G and J), 500 nm (B and H), 100 nm (E and K). The AFM height scale is adjusted \nto each particle diameter. \n \n \nDetailed height measurements of adsorbed particles in water vapour \n \nAFM under variable RH was based on three distinct hydration conditions, which we \ndefine as 1) “low humidity” , (15 ± 5) % RH, corresponding to desert climate (or very \nlow temperatures); 2) “medium humidit y”, (50 ± 5) % RH, which is slightly above the \nstandard comfort value, and 3) “high humidity”, which required overnight  sample \nincubation to reach (90 ± 5) % RH, corresponding to very wet climate or air during \nprecipitation. \nIn addition, we refer to “hydro philic conditions” in AFM for hydrophilic surfaces \n(APDMES silicon) scanned by a silicon tip. We found a vertical resolution (∆z) of 0.04 \nnm and a surface roughness of 0.16 nm (for scans in the 100 to 1000 nm range). \n\n\n11 \nAmplitude–distance curves (see Suppl. Info. S3) suggest working regimes controlled \nby long-range attractive forces, both at low and high humidity. Under these conditions, \nwe minimize sample damage, and capillary forces, albeit present, play no significant \nrole. \nWe imaged randomly selected NPs under the three humidity conditions and analyzed \ntheir height profiles. In the following, we focus on selected single particles. For the \ndimanno-AuNPs, the maximum height, z max, of the NP in Figure 7-A was 15.6 nm at \n~15 % RH). We found 15.8 nm, hence no measurable increase, at ~50 % RH (Figure \n7-C), but 17.7 nm at~95 % RH (Figure 7-E), a 2.1 nm increase. For PEG AuNPs, we \nchose two particles of 13.3 nm (left) and 12.9 nm (right) ( Figure 7-B). Here, the z max \ndecreased upon humidity increment, to 12.4 nm an d 11.9 nm (Figure 7D), and finally \nat high humidity 12.0 nm and 11.9 nm, respectively ( Figure 7-F). The Suppl. Info. S4 \nprovides additional details. \nTo increase the statistical accuracy of the experiment, we tested ~500 randomly \nselected particles under the same humidity conditions (Figure 8-A and 8-C, Table 1). \nThe resulting box plots for the hydrophilic conditions correlate well with the analysis on \nthe selected single particles (Figure 7, see also related scans in the Suppl. Info. S4): \nThe particle height of (16.7+-1.6) nm increases by 0.3 nm under medium, and by 1.6 \nnm under high humidity. The PEG AuNPs of (13.3 ± 1.0) nm show a slight reduction \nby 0.3 nm, but only at high humidity, which is within the uncertainty of the experiments. \nTo prove that the increase in zmax is solely due to the adsorbed water, we repeated the \nexperiments under hydrophobic conditions:  We employed OTS silicon surfaces and  \nhydrophobic carbon AFM tips. From this, we exclude that the observed phenomena is \nspecific for the type of su rface [32] or based on mechanical contact between tip and \nsurface [22,23,33]. The ∆z in ambient conditions was 0.03 nm and the roughness of \nthe OTS on silicon was 0.55 nm for scans between 100 and 1000 nm. In this case, it \nwas not possible to follow the same particles along the different humidity steps due to \nthe instability of the measurement, which did not allow locating the scanned area after \nchanging the humidity level. We recorded ~250 measurements of single particles on \nrandom positions ( Figure 8-F and 8-D, Table 1). The data at variable humidity for \ndimanno-AuNPs fit well with the observations under hydrophilic conditions, we found \nan increase in z max of 1.7 nm from low to high humidity (Figure 8). In contrast, the \nresults of the PEG AuNPs under hydro phobic conditions differed slightly from those \nunder hydrophilic conditions, we found an increase of 0.8 nm (Figure 8). \n \n\n12 \n  \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \n \nFigure 7: AFM topography of dimanno-AuNPs and PEG AuNPs under hydrophilic conditions (silicon AFM tip, \nAPDMES silicon surface) (A and B) Low humidity (~15 % RH), (C and D) medium (~50 % RH), (E and F) high (~95 \n% RH). (A,C,E) Dimanno-AuNPs; (B,D,F) PEG AuNPs. Lower panel: Overlay of the three profiles shown in (A,C,E) \n\n\n13 \n \nFigure 8: Box plots of AFM heights of dimanno-AuNPs and PEG AuNPs obtained in different humidity levels. AFM \ndata of dimanno-AuNPs under (A) hydrophilic conditions (APDMES surface, silicon tip, ~500 experiments), and (B) \nhydrophobic conditions (OTS surface, carbon tip, ~250 experiments), and of PEG AuNPs under (C) hydrophilic and \n(D) hydrophobic conditions, respectively. Magenta boxes refer to low humidity, blue boxes to medium humidity and \ndark cyan boxes to high humidity. Each box plot shows the interquartile range (from 25 % to 75 %). The extreme of \nthe box represents the 5 % and 95 % quartile, respectively. Middle lines represent the median and the squares of \nthe mean value. Black circles are the 1 % and 99 % quartile, and black stars are the minimum and maximum value. \n \nIn addition, in AFM experiments, we used air humidified with D2O instead of H2O again \non dimanno-AuNPs adsorbed on gold and scanned with a silicon tip (Suppl. Info. S5). \nAlthough we expected no difference, this test was required to compare the conditions \nto those used in the VSFG experiments. Working with D 2O can allow for exploring \nmolecular hydrate structure s [29]. Here, we aimed  to identify if the increase in the \nmaximum height of single particles was merely driven by the adsorption of water layers \non top of the dimannose residues, or by other mechanisms such as swelling, i.e. \nincorporation of water into the NP shell. As in the previously in troduced cases, the \nparticles increased their maximum height when the humidity of the system  increased. \nThis was influenced neither by the surface nor by the tip. So, our D2O data compares \nwell with those under hydrophilic and hydrophobic conditions (Table 1). We evaluated \n300 experiments. \n  \n\n\n14 \nTable 1: Maximum heights (zmax) of individual dimanno- and PEG-AuNPs measured by AFM at different hydration \nconditions. Results expressed as the mean ± SD. Uncertainties were obtained from statistical errors. The number  \nn refers to the evaluated AFM scans. We tested the reliability of our zmax values with a nonparametric Mann-Whitney \ntest (Suppl. Info. S6). For the dimanno-AuNPs, under hydrophilic and hydrophobic conditions and for experiments \nwith deuterated water, we f ound no significant differences in sample distribution at both low (p = 0.46) and high \nhumidity (p = 0.06). PEG AuNPs depend on the scanning conditions, but only when comparing hydrophilic and \nhydrophobic conditions at high humidity (p < 0.0001).  \n \nParticle \n \nSurface \n \n \nwater \nvapor \nzmax \nlow humidity \n(nm) \nzmax \nmedium \nhumidity (nm) \nz max \nhigh \nhumidity \n(nm) \nDimanno-\nAuNPs \nAPDMES/Si, \nhphilic \nH2O  16.7 ± 1.6 \n(n=594) \n17.0 ± 1.4 \n(n=532) \n18.3 ± 2.1 \n(n=492) \nDimanno-\nAuNPs \nOTS/Si, \nhphobic \nH2O  16.7 ± 2.0 \n(n=594) \n17.7 ± 2.3 \n(n=532) \n18.4 ± 2.2 \n(n=492) \nDimanno-\nAuNPs \nGold,hphilic \n \nD2O  16.6 ± 1.6 \n(n=300) \n17.2 ± 1.5 \n(n=300) \n17.9 ± 1.9 \n(n=300) \nPEG-Au\nNPs \n \nAPDMES/Si, \nhphilic \nH2O  13.3 ± 1.0 \n(n=505) \n13.2 ± 1.1 \n(n=495) \n13.0 ± 1.0 \n(n=499) \nPEG-Au\nNPs \nOTS/Si, \nhphobic \nH2O  13.2 ± 2.1 \n(n=505) \n13.7 ± 2.0 \n(n=495) \n14.0 ± 2.2 \n(n=499) \n \nDiscussion \n \nWe first discuss the spectroscopic results. FTIR and VSFG confirm the chemical \ncomposition of the organic layers (Figure 1 and 5). The VSFG results additionally give \nsensitive information on the local ordering of the molecular chains in the ligands. We \nconclude that the dimannose residues are coupled to the PEG chains, and provide \nspecific ordering properties: Under dry conditions, both PEG and dimannoside bind \nwater. However, the higher hydro philicity of dimannoside enables a better interaction \nwith water molecules, which remain bound even after dehydration. This  is also \nsupported by research on SAMs on gold surfaces, which reveal a more hydrophilic \ntendency of surfaces functionalized with man nose terminal groups (C 6H12O6C5S, \ncontact angle < 5°), compared with carboxylic acids (COOHC 11S, contact angle 44° ± \n1º) [3 4]. Whenever NPs cluster, the steric hindrance effects of the bulky dimanno-\nAuNPs should additionally create nanoscale \"traps\" betwee n the NPs. Both effects \nresult in water adsorption that persists even in dry environments. \nFocusing now on particle size and assembly, we found no difference between the \nhydrodynamic size of 16.8 nm (obtained in suspension by DLS) ( Figure 2) and the \nAFM height of 16.7 nm (which we here limit to the case of complete dehydration) (Table \n1). DLS usually gives higher particle sizes due to the hydration layer [ 35,36], which \nslows the diffusion of NPs in aqueous solutions, especially our hydrophilic NPs. We \nsuggest two possible explanations, first, the dimannose residues could be very \nefficiently packed (with PEG ligands acting as spacers), second, the adsorption of the \nNPs distorts the ligands between the NP core and the solid surface. The latter effect is \nwell known from adsorbed NPs, where one should consider up to three ligand spaces, \nligands between NP and surface, ligands that stretch out parallel to the surface, in \ncontact with it, and free ligands, which are merely attached to the NP. \n\n15 \nFor the PEG AuNPs, DLS gave an extreme value of 41.1 nm, correspon ding to three \nAFM height values (13.3 nm). We conclude that we here observe particle \nagglomeration in solution. Previous studies have shown such clusters for PEG AuNPs \nin solution [ 37]; their size is strongly reduced by adding albumin. In our case, the \naddition of dimannose would lead to the case of the dimanno-AuNPs, for which we do \nfind much decreased agglomeration. Generally, nanoparticles produced inside a \nconcentrated carbohydrate solution (more than 40 % of the total aqueous volume) \nprovide a macromolecular crowding environment which diminishes particle interaction \n[38]. The dimanno-AuNPs used in this research were produced in a solution of ~50 % \ndimannoside ligand sufficient to decrease the agglomeration tendency. The observed \nexcellent stability of dimanno-AuNPs may be associated with the physical constraint of \nthe dimannoside being present during synthesis, and the strong intra NP hydrogen \nbonding interactions.  \nThese properties are also reflected in the  macroscale structures formed upon \nadsorption. However, the surface is crucial  here, and our hydrophilic APDMES and \nhydrophobic OTS silanized wafer surfaces (see Suppl. Info. S1) provide two extremes \n(Figure 6). We observed a “coffee ring” drying phenomeno n only on OTS. This is \nrelated to the competition between the time scales of the liquid evaporation and the \nparticle movement: The solvent evaporates so slowly that particles move over at least \nmesoscale distances, thus allowing aggregation in the ring [ 31]. In our research, the \ndeposition protocol may have influenced the observed phenomena. The hydrophilic \nAPDMES favors NP surface interactions with subsequent immobilization. This can be \nbased on multiple hydrogen bonds between the NH3+ groups in APDMES and the OH \ngroups in the dimannoside, which immobilizes the NPs long before solvent \nevaporation. Consequently, in this case, we observed no “coffee ring” effect. The drop-\ncasting method used on the OTS allowed complete evaporation of the droplet. The \nrelatively weak NP surface interactions are now dominated by hydrophobic effects, and \ninter-NP interactions are favored, while NP movement was faster than evaporation, \nresulting in the observed “coffee ring” drying. \nMesoscale observations of NP assembly by AFM and  SEM complement the results \ndiscussed above. The use of hydrophilic and hydrophobic surfaces and also AFM tips \nallows to distinguish ubiquitous water layers, present on all surfaces, from specific \nwater adsorption on the AU NPs. For the APDMES surface at neutral pH, we expect \nelectrostatic interactions between the NH 3+ groups and the carboxylate residues on \nthe NPs, and hydrogen bonds. Hence, there are relatively few differences between \ndimanno-AuNPs and PEG AuNPs, which are related to the particle dispers ion in \nsolution. The clustered PEG AuNPs cannot disperse well on a surface, while the single \ndimanno-AuNPs interact efficiently, indicating that NP-surface interactions dominate in \nthis case over NP -NP interactions. We ascribe this to hydrogen bonds betwee n the \n(multiple) OH groups in the dimannoside and the amine. This finding compares well \nwith TEM on carbon grids: PEG AuNPs with short or long ethylene glycol chains adsorb \nin clusters [39], while glycosaylated AuNPs are homogeneously dispersed [10]. \nOur observations of nanoscale hydration by AFM show a general trend to stronger \nwater adsorption on dimanno-AuNPs, as compared to the PEG AuNPs (Figure 7). This \nagrees with AFM hydration studies of sucrose particles, which become liquid above \n60% RH [ 41], hence , the more hydrophilic nature of the dimannose residues  is \nexpected. Statistical analysis demonstrates that only the water adsorption on dimanno-\nAuNPs is independent from the scanning conditions (Table 1  and Suppl. Info.  S6). \nBetween hydrophilic and hydrophobic conditions we find only small differences, which \n\n16 \nwe interpret as quasi -identical ubiquitous water layers on our silanes: Although \nAPDMES would be expected to bind more water than OTS, this increase is smaller \nthan our experimental error. \nConsidering the size of a water molecule (~0.28 nm) and the typical thickness of \nhydration layers (~0.6 nm) [ 21,22,40,42,43], the absolute values of water adsorption \non dimanno-AuNPs fit to three to four layers of water (~1.5 nm). STEM found that the \norganic layer on dimanno-AuNPs is ~1.5 nm ( Figure 4). Hence, we propose a 100% \nthickness increase due to water adsorption or absorption, i.e., the water can be present \nin a layer, adsorbed, but also in between the various dimannoside groups, absorbed. \nThe latte r case would correspond to a \"swelling\" of the AuNP coating , which is \nagreement with microbalance observations of highly hydrated cellulose films [ 44], \nwhich find 3.6 mol water per cellulose subunit at 97% RH. \nUnder hydrophobic conditions, PEG AuNPs show a moderate increase in AFM height, \ncorresponding to one water layer, not observed under hydrophilic conditions. It is \npossible that this is based on reproducibility issues, which are well known for \nhydrophobic samples. These can be based on irreversible modification of tip or sample, \nor even both [33]. In this regard, Wet STEM and SEM investigations on PEG AuNPs \ndiffer from our data, indicating that particle hydration is independent of the selected \nsurface [21,22,32,45]. However, these studies were performed  on particle clusters in \ncrowded environments, which induce collective phenomena [32,45] that are not in our \nscope.  \n \nConclusions  \n \nGold nanoparticles (AuNPs) have been widely investigated for biomedical applications, \nlike biochemical sensing, imaging or drug delivery systems. The success of these \nplatforms stems from their dispersion in water, stability and biocompatibility in fully \nhydrated states, as well as  in biological fluids. Ou r investigation shows a novel \napproach to these particles by testing the hydration properties at different humidity \nconditions in the air. We characterized AuNPs coated by oligo -ethylene glycol (PEG) \nand dimannoside with a multi-method approach (VSFG, FTIR, DLS, ZP, SEM, STEM, \nAFM). We proved that various properties, mainly those of the adsorbed dimanno-\nAuNPs, depend on environmental conditions, specifically humidity and the \nhydrophilicity of the adsorption surface. \nOur spectroscopic investigations verify t he known chemical properties. With VSFG \nunder controlled humidity, we found ordering phenomena in ligand chains, which we \nattribute to forces exerted by hydrated dimannoside. Such an ordering is absent in \nconventional PEG ligands. Our observations of nanos cale hydration by AFM in a \nhumidity chamber show a general trend of stronger water adsorption on dimanno-\nAuNPs than the PEG AuNPs. Statistical analysis and detailed tests of hydrophilic and \nhydrophobic surfaces, with both hydrophilic and hydrophobic AFM ti ps, helped us to \nexclude the role  of water adsorbed on the surfaces. We found a 100% thickness \nincrease of the 1.5 nm thick dimannoside ligand shell, hence 1.5 nm of water, \ncorresponding to about four layers of water. The water can be absorbed in a layer, and \nbetween the various dimannose groups, absorbed.  \n\n17 \nThe increased water adsorption of dimanno-AuNPs, compared to the well-studied PEG \nAuNPs, makes them candidates for gas sensing applications. Sensors are usually kept \ndry and then become hydrated in standard environments. Whenever dry conditions are \nproblematic, our study suggests testing carbohydrate coatings. \nFinally, revealing the water adsorption of dimannoside from dryness to high humidity \noffers new insights into the molecular role of these surface r esidues also from a \nbiophysical perspective, which is especially valuable when the biological objects in \nquestion are highly pathogenic. Size, surface composition, and controlled orientation \nof the dimanno-AuNPs suggest suitable candidates for emulating vi ral surface \nglycoproteins -like influenza hemagglutinin- under hydration-dehydration cycles in air, \nwhich correspond to the conditions of viral transmission. Indeed, several viral \npathogens display mannose residues on their surface, particularly surface \nglycoproteins. These are responsible for infection, but might also provide adaptability \nto harsh environments, such as dry conditions. Our approach is yet focused exclusively \non water; future investigations should include the effects of adsorbed salts and \nproteins, and the temperature. \n \nExperimental Part \n \nMaterials \nAcetone (99.5 %), 3 -(ethoxy-dimethylsilyl) propylamine (APDMES, 97 %), and \nanhydrous solutions of tolu ene, chloroform, octadecyl trichlorosilane (OTS), and \ndecalin cis+trans were purchased from Sigma Aldrich (Germany). 2-propanol (99.5 %) \nwas obtained from Acros (Belgium) and absolute ethanol (99 %) from Panreac (Spain). \nDimanno-AuNPs and PEG AuNPs aqueous solutions were kindly provided by the Bio \nNano Plasmonics Lab at CIC Biomagune (San Sebasti án, Spain).  Water was of \nultrapure quality (Milli-Q, 18.2 Mcm, <10 ppb total organic content). \n \nSynthesis of gold nanoparticles \nGold nanoparticles were synthesized  by the Bio Nano Plasmonics Lab at CIC \nbiomaGUNE according to [10]. Both PEG AuNPs and dimanno-AuNPs were obtained \nby reduction of AuCl4- with BH4- in the presence of the corresponding thiol ligands. For \nthe PEG AuNPs, a PEG thiol with carboxylic acid termination was used. To obtain the \ndimanno-AuNPs, 50 mol% of the same carboxylic acid ligand was mixed with 50 mol% \nof a dimannoside (Manα1 -2Man) thiourea PEG thiol. After purification and \nlyophilization, the nanoparticles were obtained as brown powders.  \n \nDynamic light scattering (DLS) and zeta potential (ZP) measurements \nDLS was used to determine the hydrodynamic diameter, and ZP was used to estimate \nthe NP surface charge of both particles in solution. We employed Zetasizer Nano ZS \n(Malvern Panalytical, UK) equipment. For DLS measurements, 70 µL of the sample \n(1.0 × 1012 particles/mL) was loaded in a previously Milli Q washed micro-cuvette at 25 \n°C and a detection angle of 173°. For ZP, 7 00 µL of the sample was loaded in a dip -\ncell cuvette. Three runs were performed in three replicates, resulting in nine \n\n18 \nmeasurements per sample. The hydrodynamic diameter (z average) and the NP \nsurface charge (ZP) were calculated with the equipment software (v.7.12) without any \nfurther data processing, hence assuming spherical shapes. For ZP, we set the factor \n𝑓(𝐾𝑎)=1.5 and used the Smoluchowski approximation. \n \nPreparation of hydrophilic and hydrophobic surfaces \nDimanno-AuNPs and PEG AuNPs were adsorbed o n silicon wafers which we \nfunctionalized with APDMES or with OTS  (see Supp. Info.  S1), referred to as \n\"hydrophilic\" or \"hydrophobic”, respectively. The silicon wafers were initially chemically \ncleaned by successive serial sonication steps of 5 minutes each  using acetone, 2 -\npropanol and absolute ethanol and then dried with a nitrogen stream. Oxygen plasma \netching (Diener, DE) was carried out for 8 minutes with 300 W of nominal power (100 \n%), at mbar oxygen pressure, which creates an OH -terminated layer of hy drophilic \nsilicon oxide. \nFor the hydrophilic functionalization, the surfaces were subsequently immersed in 2 \nmL of APDMES in toluene (1:100 v/v) and heated to 60 °C for 30 minutes. To avoid \nthe hydrolyzation of the APDMES, the procedure was achieved in a g love box. After \nfunctionalization, the surfaces were rinsed in toluene and dried in flowing nitrogen.  \nThe hydrophobic functionalization was performed as reported in [ 24]. Silicon wafers \nwere washed first in methanol, then in a 1:1 v/v mixture of methanol and chloroform, \nand finally in chloroform under 5 min sonication at every step. Afterwards, the surfaces \nwere immersed in a 7:2:1 v/v mixture of decalin cis+trans/toluene/chloroform and 0.1 \n% OTS was added. The procedure was done at ambient temperature inside a nitrogen \nglove box. After 12 hours of incubation, the surfaces were rinsed with chloroform, then \nwith a 1:1 v/v mixture of chloroform and methanol, and finally with methanol. The \nprocess was finished by drying the surfaces with a stream of nitrogen. \n \nContact angle measurements \nFor static contact angle measurements, sessile drop experiments were performed in \ntriplicates at ambient temperature (23 -25 °C) with a standard contact -angle \nmeasurement system (G10 goniometer, Krüss). A droplet of ultrapure water of volume \n4 µL was placed onto the functionalized silicon surfaces and contact angles from the \ndrop profile were measured. \n \n \nAdsorption of nanoparticles \nDimanno-AuNPs or PEG AuNPs solutions were incubated for 5 min (1.0 × 10 12 \nparticles/mL) on the hydrophilic functionalized silicon surfaces. The samples were \nwashed with Milli Q water and dried in flowing nitrogen. The adsorption on the \nhydrophobic surfaces was achieved by drop casting. A droplet of dimanno-AuNPs or \nPEG AuNPs was deposited on the surface and dried in air. \n \nInfrared spectroscopy \n\n19 \nFTIR measurements were carried out with a grazing incidence objective lens/mirror \nsystem in a Hyperion 2000 mic roscope with a Vertex 70 spectrometer (Bruker). For \nthis, a concentrated solution of particles (1.9 × 10 13 particles/mL) was cast on  a \ncarefully cleaned gold surface and evaporated under ambient conditions. The \nmeasurements were achieved by recording 2000 scans, from 650 to 4000 cm-1 and a \nresolution of 4 cm-1, in triplicate. Background spectra were recorded on gold areas \noutside the evaporated droplet. The FTIR spectra were obtained with the equipment \nsoftware OPUS v.6.5 without further data processing. \n \nSum frequency generation spectroscopy \nVSFG spectra of dimanno-AuNPs and PEG AuNPs was recorded in a broad -band \nVSFG system at Fritz Haber Institute. Gold thin films (200 nm on 10 nm Cr on glass) \nwere used as surfaces. Previously to sample deposition, the su rfaces were cleaned \nwith ethanol, and Milli Q water under sonication for 10 minutes. UV/ozone cleaning \nwas applied to remove any possible hydrocarbon contamination, and the VSFG \nspectra of the washed gold was used as reference to normalize the non -flat infrared \npower distribution, similar to FTIR recording. About 10 µL of the sample was drop cast \non the surface and evaporated under nitrogen flow. The VSFG experiments were \nachieved under dry air flushing at room temperature (24 °C) at 4 different center \nfrequencies to measure the complete CH and OH stretching region in the broad band \ninfrared. The tunable infrared laser produced femtosecond pulses of ~5.5 mW (at 3300 \ncm-1). Their focus length was 30 cm and the incident angle 45°. The visible picosecond \npulses were at a fixed frequency of 800 nm (12500 cm-1), and of 10 mW power. Their \nfocus length was 100 cm and the incident angle 65°. The sum frequency signal was \ncollected in reflection, accumulated for 1 min for ppp polarization (all beams polarized \nnormal to the surface) and 2 min for ssp (laser beams polarized parallel to the surface, \nsum frequency emission and up -conversion polarized perpendicular and infrared \nparallel to the plane of incidence). \n \nElectron microscope imaging \nSEM and STEM images were obtaine d in a Helios NanoLab 450S dual beam \nmicroscope (FEI  NL / Thermo Fisher Scientific). SEM images were obtained of \ndimanno-AuNPs and PE G AuNPs at 5 kV with 50 pA current in high vacuum mode. \nThe core diameter of single particles was manually counted with ImageJ (NIH, \nhttps://imagej.nih.gov/ij/). The number of particles was expressed as \"n\" and the data \nwere displayed as the mean ± SD. A t otal of 378 particles were counted for the \ndimanno-AuNPs and 491 for the PEG AuNPs.  \nSTEM acquisition was achieved in high vacuum at 30 keV, 50 pA current on negatively \nstained dimanno-AuNPs samples. For the negative staining, 0.35 µL of dimanno-\nAuNPs (1.9 × 1013 particles/mL) were deposited on glow discharged carbon TEM grids \n(air flow, 20 mA, 4 minutes). Then, 0.35 µL uranyl acetate (0.5 % w/w) was deposited \nand dried on the sample. \n \nAFM imaging \n\n20 \nAFM topography images were recorded with an Agilent 5500 AFM (Keysight, Scientec, \nFR), in air in AC “noncontact” mode at 24 °C, at speed between 0.8 and 1.2 lines/s. \nThe scanning was performed in at least six replicates per condition for the PEG AuNPs \nand nine replicates for the dimanno-AuNPs.  \nSilicon cantilevers (Multi 75, Budget Sensors) with a force constant (k) of 3 N/m, a \nresonance frequency (f) of 75 kHz and a nominal radius of <10 nm were used for the \nexperiments on the hydrophilic surfaces. These conditions are referred to as \n\"hydrophilic conditions\".  \nThe visualization of particles on hydrophobic surfaces was achieved by employing \ngold-coated silicon probes with a 1 nm carbon spike (k 5 N/m, f 160 kHz, Hi’Res C14 \n/Cr Au, MikroMasch). These conditions are referred to as \"hydrophobic conditions\". \nThe samples were incubated on the hydrophilic or hydrophobic surfaces, and AFM \ntopography images were taken under humidity-controlled atmospheres at (15 ± 5) %, \n(50 ± 5) % and (90 ± 5) % of RH. These humidity conditions are referred to as \"low \nhumidity\", \"medium humid ity\" and \"high humidity\", respectively. For this, an \nenvironmental isolation chamber (Keysight) was mounted to the AFM, thus providing \na completely sealed and isolated environment. The RH of (15 ± 5) % was achieved by \npumping nitrogen to the isolation cham ber. The RH of (50 ± 5) % was reached by \ncontrolling the mixing ratio between a dry and a wet stream of nitrogen, produced in a \nbubble flask. To reach RH of (90 ± 5) %, the sample was allowed to equilibrate \novernight in the saturated chamber. The images we re examined by using picoView \n1.14 software (Keysight). AFM images were analyzed after scanning with WSxM 5.0 \nDevelop software version 8.0 [ 46] for obtaining the maximum height of the particles \nand height profiles. For this, the images were processed by le velling the plane of the \nimage and applying parabolic flattening. Around 500 individual particles were \nprocessed in the hydrophilic conditions and 250 in the hydrophobic conditions. The \nparticles were manually selected by using the criterion of single part icle observation \nwith a FWHM below 30 nm and 40 nm for the hydrophilic and hydrophobic conditions, \nrespectively. Gwyddion software version 2.47 was used for displaying the topography \nimages. The images were first levelled by mean plane subtraction, aligned  by height \nmedian and occasionally, the z excursions outliers were manually removed by, e.g. \ncorrection of the scan line artefacts in the x axis or misaligned segments within a single \nrow. The height distribution histograms were calculated with Origin 8.0 software and \nheight profiles were obtained with GraphPad Prism version 8.0.1. The number of \nparticles was expressed as \"n\" and the data were displayed as the mean ± SD. \nStatistical Analysis  \nStatistical analysis was performed using GraphPad Prism version 8 .0.1. Data sets \nwere analyzed using unpaired t-tests (Mann -Whitney test) to compare identical \nparticles on different surfaces at the same humidity. Two-tailed P values were reported \nin all the cases, and the alpha level was kept at 0.05. \n \nAcknowledgements  \n \nThe presented results are partially based on Maiara Iriarte -Alonso’s PhD thesis \n“Surface models of Influenza virus envelope: Biophysical studies under various \n\n21 \nhydration scenarios”, University of the Basque Country (2022). We thank Isabel Garcia \n(CIC biomaGUNE, Donostia-San Sebastián, Spain) for the synthesis of the particles. \nAMB acknowledges the Erwin Schrödinger Institute (ESI) for Mathematics and Physics \nin Wien , Austria . We are grateful for funding by grants EU -MSCA 101072645 \nnanoremedi, EIC -pathfinder 101115292 (“textadna”), the Spanish M INECO for the \nMaria de Maeztu Units of Excell ence Program (CEX2020 -001038-\nM/MCIN/AEI/10.13039/501100011033), and for the projects PID2019-104650GB-C22 \n(Bridge), PID2023-147987OB-C32, PID2023-146348NB-I00; the Basque Government \nfor the Elkartek ng ; the Diputacion Foral Gipuzkoa  for the project  “shifte”. MAIA \nreceived an EMBO and a DAAD travel grant that allowed her to work at FHI Berlin. We \nthank the electron microscopy group at CIC nanoGUNE for advice and help.  \n\n22 \nReferences \n \n[1] S. Balci, K. Noda, A.M. Bittner, A. Kadri, C. Wege, H. Jeske, K. Kern, Self -\nAssembly of Metal -Virus Nanodumbbells. Angew. Chemie Int. 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