# Fileset

[[Langmuir]Potential-switchable viscoelasticity of protein nanolayers at a liquid liquid interface.pdf](https://mdr.nims.go.jp/filesets/15ad5176-6629-46d7-af46-607b4b904861/download)

## Creator

Kosuke Ishii, [Takeshi Ueki](https://orcid.org/0000-0001-9317-6280), [Jun Nakanishi](https://orcid.org/0000-0003-4457-6581), [Kazuhiro Akutsu-Suyama](https://orcid.org/0000-0002-4797-6604), [Norifumi L. Yamada](https://orcid.org/0000-0002-8370-4526), [Yuko Yokoyama](https://orcid.org/0000-0002-3943-0978), [Tetsuo Sakka](https://orcid.org/0000-0002-1892-8056), [Naoya Nishi](https://orcid.org/0000-0002-5654-5603)

## Rights

[Creative Commons BY Attribution 4.0 International](https://creativecommons.org/licenses/by/4.0/)

## Other metadata

[Potential-Switchable Viscoelasticity of Protein Nanolayers at a Liquid/Liquid Interface](https://mdr.nims.go.jp/datasets/be3eb78a-d5b8-4887-86dd-393bca897f7c)

## Fulltext

Potential-Switchable Viscoelasticity of Protein Nanolayers at a Liquid/Liquid InterfacePotential-Switchable Viscoelasticity of Protein Nanolayers at aLiquid/Liquid InterfaceKosuke Ishii, Takeshi Ueki,* Jun Nakanishi, Kazuhiro Akutsu-Suyama, Norifumi L. Yamada,Yuko Yokoyama, Tetsuo Sakka, and Naoya Nishi*Cite This: Langmuir 2025, 41, 17973−17981 Read OnlineACCESS Metrics & More Article Recommendations *sı Supporting InformationABSTRACT: Protein nanolayers (PNLs) formed at an electrochemical liquid|liquid interface between water (W) and a fluoroussolvent (F) were examined by using interfacial rheological measurement (IRM) and neutron reflectometry (NR) under theexternally controlled condition of the phase boundary potential differences EFW(= φW − φF + const.), where F contained ahydrophobic ionic liquid (IL) as a supporting electrolyte and W, whose pH was 7.4, contained a protein, bovine serum albumin(BSA). The IRM and NR results illuminated that both static and dynamic properties of the PNL at the electrochemical F|Winterface were varied by applying EFW. NR found minimal EFW dependence on the adsorption amount of BSA in the PNL. In contrast,IRM revealed that although the interfacial shear loss moduli G″ of the PNL was constant regardless of EFW, the interfacial shearstorage G′ of the PNL increased dramatically at more negative EFW, showing a more elastic response. This difference between staticand dynamic properties results from the increase in intermolecular and intramolecular interactions between BSA molecules in thePNL at more negative EFW due to the accelerated denaturation of negatively charged BSA that formed complexes with IL cationsaccumulated on the F side of the F|W interface. The G′ and G″ reversibly responded to switching between different potentials (apositive and a negative EFW). These IRM results unveiled that the viscoelasticity of the PNL at the electrochemical F|W interface isreversibly potential-switchable. The present interface-specific method using the potential control is a new promising method todiversify and switch the PNL structure reversibly. The reversible structural control of the PNL would enable us to perform real-timeobservation of cells reacting to environmental changes at liquid|liquid interfaces.1. INTRODUCTIONMechanobiology is a research field that focuses on how cellssense and respond to mechanical cues such as substrateviscoelasticity. In this context, cell culture platforms with well-defined and tunable mechanical properties have becomeindispensable tools for studying cellular phenotypes such asadhesion, migration, and differentiation. Against this back-ground, hydrophobic liquid interfaces have recently emergedas a novel platform for mechanobiology. The liquid phase usedas a cell scaffold must form a clear biphasic system with water,be noncytotoxic, and have a density higher than water. SinceRosenberg reported in 1964,1 that cells adhere to and spreadon fluorinated liquids such as FC-70, numerous studies haveexplored cell dynamics at the interfaces of various molecularliquids, including silicone oils2,3 and fluorinated liquids.4−8 Inrecent years, notable biological phenomena have been reportedat liquid interfaces, including selective neuronal differentiation9of human mesenchymal stem cells (hMSCs) and themaintenance of their undifferentiated state.10 At these liquidinterfaces, proteins from the culture medium spontaneouslyaccumulate to form a protein layer with a thickness of severalnm,11,12 so-called protein nanolayer (PNL), which acts as amechanically robust, solid-like scaffold for cell adhesion andspreading. However, PNLs formed via spontaneous proteinself-assembly are often mechanically fragile, sometimes failingReceived: April 12, 2025Revised: June 15, 2025Accepted: June 20, 2025Published: July 1, 2025Articlepubs.acs.org/Langmuir© 2025 American Chemical Society17973https://doi.org/10.1021/acs.langmuir.5c01819Langmuir 2025, 41, 17973−17981This article is licensed under CC-BY 4.0Downloaded via NATL INST FOR MATLS SCIENCE (NIMS) on September 2, 2025 at 04:08:32 (UTC).See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Kosuke+Ishii"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Takeshi+Ueki"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Jun+Nakanishi"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Kazuhiro+Akutsu-Suyama"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Norifumi+L.+Yamada"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Yuko+Yokoyama"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Yuko+Yokoyama"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Tetsuo+Sakka"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Naoya+Nishi"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/showCitFormats?doi=10.1021/acs.langmuir.5c01819&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?goto=articleMetrics&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?goto=recommendations&?ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?goto=supporting-info&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=tgr1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=tgr1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=tgr1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=tgr1&ref=pdfhttps://pubs.acs.org/toc/langd5/41/27?ref=pdfhttps://pubs.acs.org/toc/langd5/41/27?ref=pdfhttps://pubs.acs.org/toc/langd5/41/27?ref=pdfhttps://pubs.acs.org/toc/langd5/41/27?ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c01819?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://pubs.acs.org/Langmuir?ref=pdfhttps://pubs.acs.org/Langmuir?ref=pdfhttps://acsopenscience.org/researchers/open-access/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/to adequately support cell adhesion. To address this problem,Keese and Geaver reported a method to enhance themechanical robustness of PNLs by anchoring them tofluorinated liquid interfaces using reactive surfactants.13−15Other strategies have also been proposed, including pretreat-ment with certain proteins that support cell adhesion16 andactively denaturing proteins at the interface17 to reinforce themechanical properties of PNLs.Recently, we introduced hydrophobic ionic liquid (IL)interfaces as a new class of liquid scaffolds for cell culture.18We demonstrated that certain alkylphosphonium-based ILs(and some alkylammonium-based ILs19) exhibit low cytotox-icity and can support cell culture at their interfaces. Similar toconventional liquid scaffolds, PNLs also form at IL interfaces,and their mechanical robustness plays a key role in celladhesion. The apparent Young’s modulus of PNLs in thevertical direction at IL interfaces is lower than that atfluorinated liquid interfaces. Nevertheless, cell adhesionoccurred at the IL interface, and it was found that the degreeof cell spreading at the interface varied depending on subtledifferences in the IL chemical structure.18 Furthermore, byleveraging the high miscibility of ILs with various (macro)molecules, we successfully modified IL-based PNLs byincorporating a cross-linked polymer to enhance the bulkmechanical properties, thereby modulating cell spreading andmorphology.In the present study, to diversify cell culture on liquid|liquidinterfaces, the PNL structure at the electrochemical fluorinatedliquid|water (F|W) interface was controlled by modulating theinterfacial ionic composition through phase boundary potentialdifference switching. Given the high solubility of ILs with afluorinated anion in fluorinated liquids,20 we employed an ILas a supporting electrolyte in the subphase. Interfacialrheological measurements (IRM), which have been extensivelyused to investigate the rheological properties of PNLs atnonelectrochemical liquid|liquid interfaces,8,21−27 were appliedhere to the electrochemical liquid|liquid interface. Neutronreflectometry (NR) was utilized to characterize the PNL at theelectrochemical F|W interface. While NR has been previouslyused to investigate PNLs at electrode|W interfaces28,29 andthose at nonelectrochemical oil (O)|W interfaces,8,26,30,31 aswell as electric double layers at electrochemical O|W32 and F|W33 interfaces, to the best of our knowledge, NR has neverbeen applied to examine the potential-dependent structure ofPNLs at electrochemical liquid|liquid interfaces. In this study,we demonstrate that IRM and NR are powerful tools forprobing the static and dynamic properties of PNLs underelectrochemical conditions. Unlike chemical modificationsused in previous studies3,13,14,34 to enhance the mechanicalproperties of PNLs, our approach enables interfacial-specificreinforcement of PNLs via physical (electrochemical)methods. Because IL interfaces have higher polarity comparedto conventional subphases such as silicone oils and somefluorinated liquids, PNLs formed via interfacial tension-drivenassembly tend to be mechanically weaker. However, we showthat electrochemical modulation can improve the mechanicalrobustness of PNLs, potentially overcoming this limitation.Furthermore, by utilizing a highly switchable electrochemicalstimulus, we achieve a reversible modulation of interfacialelasticity across an order of magnitude, with high temporalresolution. The creation of cell scaffolds capable of deliveringreversible mechanical stimuli and enabling real-time observa-tion of cell dynamics at interfaces represents a growing trend inmechanobiology.35−38 Our system offers a qualitatively newapproach to liquid-based cell scaffolds, providing a means toapply localized mechanical stimuli to cells at liquid interfaces ina manner distinct from conventional methods.2. EXPERIMENTAL SECTION2.1. Materials. The fluorinated liquid (F) used was1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-(trifluoromethyl)pentane(DMTMP, TCI, Figure 1). As the supporting electrolyte in F,trihexyltetradecylphosphonium bis(nonafluorobutanesulfonyl)amide([THTDP+][C4C4N−]), which is a hydrophobic IL, was dissolvedat 2.5 mM, as was in our previous study on the electric double layer atthe F|W interface.33 [THTDP+][C4C4N−] (Figure 1) was preparedfrom [THTDP+]Cl− (Sigma-Aldrich) and Li+[C4C4N−] (MitsubishiMaterials Electronic Chemicals) and purified by using the samemethods as those described elsewhere.39For IRM, a phosphate buffer (PB, pH 7.4) was prepared bydissolving 0.2 mM Na2HPO4·2H2O and 0.2 mM NaH2PO4·12H2O(Wako) in H2O (Milli-Q). With this PB, a bovine serum albumin(BSA) solution for IRM was prepared which contained 1 mg/mL BSA(Wako, first grade, pH 5.2) and 1 mM NaCl (Kishida). The finalconcentration of BSA in W7.4 (the pH 7.4 buffered W phase) for IRMwas 0.5 mg/mL, half of that in the above BSA solution because thetwo-phase system was constructed with a 1 mM NaCl solutionwithout BSA first and then an equal amount of the BSA solution wasadded to start the BSA adsorption (see Section 2.3 for the detail).For NR, a PB (pH 7.4) was prepared by dissolving 0.2 mMNa2HPO4 and 0.2 mM NaH2PO4 (Wako) in D2O (Silanes, >99.9%).A tartaric acid buffer (pH 2.6) was also prepared by dissolving 0.8mM C4H4Na2O6 (Wako) and 1 mM NaCl in D2O. With the PB andthe tartaric acid buffer, two BSA solutions with pH 7.4 and 2.6, wereprepared, both containing 1 mg/mL BSA (Wako, Crystallized) and 1mM NaCl (Kishida). The final concentration of BSA both in W7.4 andW2.6 was 0.5 mg/mL, because of the dilution similar to the IRM case(see above and Section 2.2). It is noted that the isoelectric point ofBSA in a 1 mM NaCl solution is pH 4.8,40 indicating that BSA isnegatively and positively charged at pH > 4.8 and pH < 4.8,respectively.2.2. Neutron Reflectometry. NR was performed using ahorizontal-type neutron reflectometer, SOFIA, at BL16 of theMaterials and Life Science Experimental Facility (MLF) of theJapan Proton Accelerator Research Complex (J-PARC).41,42 The qrange was 0.02−0.08 Å−1 (the incident angles were 0.4/1.0°). TheNR cell used was the same as our previous NR study for theelectrochemical F|W interface.33 The F|W interface was formed in thefollowing way. First, 15 mL of 1 mM NaCl D2O solution (upperphase) was gently placed in a quartz cell, and then 8 mL of F (lowerphase) was slowly injected into the cell from the bottom using asyringe pump to form the F|W interface. Then, 15 mL of the BSAFigure 1. Structures of DMTMP and [THTDP+][C4C4N−].Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c01819Langmuir 2025, 41, 17973−1798117974https://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig1&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c01819?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-assolution (pH 7.4 or 2.6) was added to W to start the BSA adsorptionat the F|W interface. NR was performed 1 h after the addition of theBSA solution when the BSA adsorption was saturated, judging frominterfacial tension measurements (Figure S1-2). The bulk concen-tration of BSA in the W was 0.5 mg/mL, which is among theconcentration range used in previous studies on the PNL formation ofBSA at liquid/liquid interfaces.12,43The reflectivity data were analyzed using a one-slab model takinginto account the interface layer (L) on the W side of the F|Winterface, which corresponds to the PNL on the interface and doesnot take into account the molecules/ions in F: THTDP+, C4C4N−,and DMTMP. The scattering length density (SLD) changes on the Fside of the interface are regarded as negligible, from the fact that thenumber density of THTDP+, C4C4N−, and DMTMP at the interfacewas not so high (<3 × 10−3 Å−2 from our previous NR study on theelectric double layer at the F|W interface)33 compared with that ofBSA (2 × 10−4 Å−2 roughly estimated from a reported adsorptionamount of BSA at the O|W interface, 2 mg/m2)30 while theirscattering lengths (THTDP+: −36.5, C4C4N−: 193.2, and DMTMP:114.6 fm)33 are 2 orders of magnitude smaller than that of BSA (bBSA= 26000 fm, calculated from the structure of BSA taken from NCBIdata sets44 by using a calculator provided by MULCh45). Thisindicates that THTDP+, C4C4N−, and DMTMP do not substantiallyaffect the SLD in the PNL. The following conditions were used forfitting. The SLD of F and W (ρF = 3.13, ρW = 6.16 × 10−6 Å−2) wereset to the values obtained from the NR at the F|air and W|airinterfaces (see S2 in Supporting Information). The surface roughnessbetween L-F (namely, the F|W interface), σL-F was fixed to the valuesof σA estimated from the capillary wave theory46,47 (see S3). Todetermine the surface roughness between W-L σW-L, we evaluated twomodels: one with σW-L fixed at the same value as σL-F and the otherwith nonfixed σW-L. The corrected Akaike Information Criterion(AICc),48 a measure of the model likelihood, showed that the fixedσL-F model was more likely (Table S4−1). In the following, we discussthe NR results obtained employing a one-slab model with the fixedσW-L even though both fitted results were similar. The fitted resultswithout fixed σW-L are shown in Figure S4−2 and Table S4−2.The value of ABSA (= d(bBSA × nBSA−ρF)), with the number densityof BSA in L nBSA and thickness of L d, reflects the adsorption amountof BSA and was extracted by using a code made by ourselves used inour previous papers.33,49,50 In the following section, ABSA was used asa parameter of the accumulated BSA to the interface.2.3. Interfacial Rheological Measurements. The viscoelasticityof the PNL at the electrochemical F|W interface was measured byusing a rheometer (HR20, TA Instruments) equipped with a Pt-Irring wire in a double wall-ring geometry (Figure 2). The cross-sectionof the ring wire was square with a diagonal of 1 mm. A Pt coil and Ag/AgCl wire were placed in F as the counter and quasi-referenceelectrode, respectively, which were covered with PTFE tubes not tocontact with W. An Ag/AgCl coil was placed in W as the counter/reference electrode. The potential difference between the Ag/AgClwire in F and the Ag/AgCl coil in W EFW(= φW − φF + const.) wascontrolled by using a potentiostat (CompactStat, Ivium). The PNL atthe F|W interface was prepared as follows. The F (15 mL) wasinjected into the cell. The Pt-Ir ring was slowly lowered until rippleswere observed on the surface of F, which indicated that the loweredge of the ring had touched the F surface. The ring was lowered afurther 460−500 μm to position the height of the midplane of the ringdiagonal to the surface. Then, 1 mM NaCl H2O solution (15 mL) wasgently added on F to form the F|W interface. In the followingexperiments, the interfacial shear storage moduli G′ and loss moduliG″ did not change when the ring height was shifted by ± 300 μm.After the F|W interface was formed, EFW = −0.6, −0.3, 0, +0.3, and+0.6 V were applied and then the BSA solution (pH 7.4, 15 mL) wasslowly added to the W to start the formation of the PNL on theinterface. The bulk concentration of BSA in the W7.4 was 0.5 mg/mL.The time dependence of G′, G″, and tan δ ( = G″/G′) of the PNL atthe F|W interface was measured at a strain of γ = 1% and an angularfrequency of ω = 1 Hz for 1 h, which is the saturation time of the BSAadsorption estimated from the interfacial tension measurements (SeeS1, Figure S1−2). 100% strain corresponds to the ring rotation overthe same distance as that between the ring and outer wall in the radialdirection.51 After 1 h, an amplitude sweep at 1 Hz was performed witha strain γ range of 0.1−200% (at EFW = 0, +0.3, +0.6 V) and0.1−1000% (EFW = −0.3 V) to verify the strain resistance of the PNLat each EFW.3. RESULTS AND DISCUSSION3.1. Neutron Reflectometry. To analyze the amount ofBSA adsorbed on the electrochemical F|W7.4 interface, ABSA atthe F|W7.4 interface was measured at each EFW (Figure 3, redsolid circles). The reflectivity data are shown in Figure S4−1.EFW = 0 V should be close to the uncharged condition judgingfrom the potential of zero charge (pzc) for the case withoutBSA, +0.05 V.33 The ABSA at EFW = 0 V was the same within theerrors as the case using F without IL (Figure 3, a black plotshown at EFW = 0 V). This illustrates that ABSA is not affectedwith or without IL on the F side of the interface when theinterface is not charged. With some degree of variabilitypresent in the data, the ABSA with W7.4 slightly increased withdecreasing EFW, suggesting that the BSA amount at the F|Winterface increases at more negative potentials. This tendencywas also observed in the interfacial tension measurementsshown in Figure S1−2. At the BSA-free F|W interface, EFW =0.05 V is the pzc.33 Considering the composition of the electricFigure 2. Schematic cross-section of IRM cell in a double wall-ringgeometry. The electrodes for the F side are covered with PTFE tubesin W. The Pt-Ir ring wire was placed at the F|W interface. The cross-section of the ring was square with a vertical diagonal length of 1 mm.Figure 3. Potential dependence of ABSA of the PNL at the interfacebetween W7.4 and F with IL (red). The data for the neat F casewithout IL, and therefore without external potential control, is shownat EFW = 0 V (black) for comparison. The error bars are the standarderrors of fitting results. The red dotted line is from the least-squaresfitting for the data with IL.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c01819Langmuir 2025, 41, 17973−1798117975https://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig3&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c01819?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asdouble layer at the F|W interface,33 THTDP+ (C4C4N−) ionsare more accumulated on the F side of the interface at EFW < 0V (EFW > 0 V). At EFW < 0 V, the electrostatic interactionbetween THTDP+ and BSA, which is negatively charged at pH7.4, could increase the adsorption of BSA at the interface. Thisis similar to the electrodeposition of lysozyme at an O|Winterface accelerated by the electrostatic interaction ofpositively charged lysozyme and anions in O.52 At EFW > 0 V,although the electrostatic repulsion between C4C4N− and BSAcould decrease the adsorption of BSA at the interface, BSA wasstill adsorbed to the negatively charged interface to form thePNL as in a previous study where the positively charged part ofBSA was adsorbed on a negatively charged silica surface.53 Tofurther investigate the electrostatic interaction between BSAand the IL ions on the F side of the interface, we alsoperformed NR at the F|W2.6 interface, using a pH 2.6 bufferwhere BSA is positively charged. The NR results at the F|W2.6interface (Figure S5−3) were analyzed similarly to those at pH7.4. The ABSA of the PNL at the F (with IL)|W2.6 interface isshown in Figure S5−4. Opposite to the W7.4 case, withincreasing EFW, the ABSA values weakly increased, and thereforethe BSA amount at the interface increased. This means that theCoulombic interaction behavior between IL ions and BSA atthe F|W interface observed at pH 7.4 still holds at pH 2.6; theelectrostatic repulsion (attraction) from THTDP+ (C4C4N−)at EFW < 0 V (> 0 V) could decrease (increase) the adsorptionof positively charged BSA at the interface. The slope signchange in the ABSA vs EFW plots at pH 2.6 indicates that the BSAamount in the PNL is controllable either by applying EFW or bychanging pH. It should be noted, however, that the EFWdependence of the BSA adsorption amount, revealed usingNR and interfacial tension measurements, was minimal, whichis in stark contrast to a dramatic change in the viscoelasticityshown below.3.2. Interfacial Rheological Measurements. To analyzehow the phase boundary potential affects the viscoelasticity ofthe PNL, we performed the IRM at the electrochemical F|W7.4interface. The G′ (storage modulus) and G″ (loss modulus)obtained from IRM provide insight into the mechanicalcharacteristics of the PNL at the interface. G′ reflects theelastic nature, likely originating from reversible intermolecularinteractions between BSA molecules, such as hydrogenbonding, hydrophobic association, or electrostatically mediatedclustering. G″, in contrast, is associated with dissipativeprocesses, including rearrangement of protein molecules orpartial unfolding of loosely bound protein dangling ends orinterfacial viscosity. These mechanical parameters are highlyrelevant to cell culture applications. Recent studies inmechanobiology have revealed that not only G′ but also G″plays a critical role in regulating cell behavior on viscoelasticsubstrates. For instance, the “molecular clutch model” suggeststhat viscous dissipation influences focal adhesion dynamics andactin flow in addition to stiffness.54,55 Cooper et al. alsoreported that changes in G″, with constant G′, modulate cellspreading and cytoskeletal organization. Therefore, the abilityto electrochemically and reversibly tune both G′ and G″ at aliquid|liquid interface offers a promising approach toinvestigating dynamic mechanosensing in cells.56,57We first examined the effect of IL addition to F on theviscoelasticity of PNL at the F|W interface. The time evolutionof interfacial shear storage and loss moduli, G′ and G″,respectively, for 1 h is shown in Figure S5−1. For both thecases in the presence and absence of IL, one can see that G′and G″ increase with increasing time, keeping G′ > G″, whichmeans the formed PNL has a gel-like nature. The differencebetween the two cases is clearly discernible in Figure S5−1.The two moduli steeply rise for the case without IL on theorder of 100 s, contrasting with a 10 times slower PNLformation when F contained IL. The slowdown in the presenceof IL can be explained by the fact that the F side of the F|Winterface is covered by an IL ion-rich layer when F contains IL,even without applying external potential, which was unveiled inour previous NR study.33 Such accumulation of IL ions leadsto the slow formation of the interfacial structure,58 resulting inthe slow rise in the two moduli in the presence of IL, as shownin Figure S5−1. This indicates that the IL, even though it isseemingly an additive in F with a low concentration (2.5 mM),has a strong impact not only on the interfacial structure butalso on the viscoelasticity of PNL on the interface. Then weinvestigated the time evolution of the two moduli in the IL-added case for a longer time (>10 h), the results of which areshown in Figure S5−2. Note that in this case, we applied EFW =0 V, which is close to the uncharged condition withoutpotential control. Figure S5−2 shows both G′ and G″ evolveon the order of 103 with a steeper increase in the G′, indicatingthat the PNL gradually exhibits a more elastic-dominantresponse on this time scale (11 h). In the following, weexamine the effect of the phase boundary potential on thePNL. It is noted that G′ and G″ did not reach equilibrium at 1h. Figure 4 shows the time evolution of G′ and G″ during 1 hafter the BSA solution was injected at EFW = −0.6, 0, +0.6 V. AsEFW decreased, G′ and G″ increased more steeply. Consideringthat the time evolution of the surface pressure (Figure S1−2)was not significantly different regardless of EFW, IRM indicatedthat at lower EFW BSA was more denatured and formed moreintermolecular bonds. Figure 5a shows the G′ and G″ 1 h afterthe injection of the BSA solution at EFW = −0.6, −0.3, 0, +0.3,+0.6 V. As EFW decreased, G′ significantly increased, whereasG″ slightly increased, which shows that the PNL was moreelastic (lower tan δ) at EFW = −0.6 V (on the positively chargedF surface), and was more viscoelastic (higher tan δ) at EFW = 0.6V (on the negatively charged F surface). Controlling EFW had asignificant effect on the viscoelasticity of the PNL at the F|W7.4interface (Figure 5a) unlike that in the amount of BSA in theFigure 4. Time evolution of interfacial shear storage and loss modulusG′ (solid lines) and G″ (dashed lines) at EFW = − 0.6 (blue), 0 (black),+ 0.6 V (red) at the F|W interface after t = 0 when the BSA solutionwas injected. G′ and G″ were measured with a strain of γ = 1% and anangular frequency of ω = 1 Hz. Those at EFW = −0.3 and +0.3 V areshown in Figure S5−4 and 6, respectively.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c01819Langmuir 2025, 41, 17973−1798117976https://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig4&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c01819?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asPNL (Figures 3, S1−2b, S4−4). At EFW < 0 V, IL cations aremore accumulated at the F side of the interface.33 Chargedproteins at the electrochemical O|W interface were reported toform complexes with organic ions in O that have the countercharge.52,59−62 Similarly, in the present study, the complexformation of BSA, which is negatively charged, with IL cationsis likely to accelerate the denaturation of BSA depending onEFW, which strengthened the intermolecular and intramolecularbonds of BSA in the PNL. In addition to the denaturation ofBSA, the viscosity of ILs at the interface,63−74 including at theliquid|liquid interface,58,70,75 was reported to be much higherthan that in the IL bulk because of spontaneously formed well-ordered ionic multilayers at the interface. Our previous studyon the ionic compositions at the F|W interface revealed that ILions are accumulated at the interface, especially at EFW < 0 V upto 400 times higher concentrations than those in the bulk,33forming an IL-like environment at the interface. This impliesthat at EFW < 0 V, well-ordered IL-rich layers are formed at theinterface, contributing to increased stiffness and more elasticinterfacial behavior of the PNL.At EFW > 0 V, PNL showed less elasticity than that at EFW < 0V. BSA at the interface is likely to be less denatured becausethe F side of the interface was covered by relatively hydrophilicsulfonyl groups of C4C4N− that orient their perfluorobutylgroups to the F bulk, according to the compositional analysisof the F|W interface33 and the orientational one of the IL ionsat the IL|W interface.76 These factors could inhibit theformation of intermolecular bonds between neighboring BSAand make the PNL less elastic at EFW > 0 V. Figure 5b showsthe loss tangent of the PNL, tan δ (= G″/G′), at each EFW. AsEFW increased, tan δ increased from 0.4 at EFW = −0.6 V to 0.8 atEFW = 0.6 V, which also indicated that the PNL is more elastic(lower tan δ) at EFW = −0.6 V and more viscous (higher tan δ)at EFW = 0.6 V. These results show that EFW can switch theviscoelasticity of the PNL at the F|W interface. Figure 6 showsG′ and G″ profiles as a function of strain γ which weremeasured at 1 h after the BSA solution was injected. Thepoints in Figure 6 are the yield points where G′ and G″ profilescross over (G′ = G″), γYP, which is a measure of how resistantthe PNL is to strain. As EFW decreased from EFW = +0.6 to −0.6V, the γYP increased, indicating that the structure of PNL at EFW< 0 is much more strain-resistant. Figure 7 shows the EFWdependence of γYP. In Figure 7, γYP at EFW = −0.6 V reached347%, which came from a peculiar behavior in the decayingparts of G′ and G″ profiles, where the former showed plateausaround 100% strain and then crossed over at greater strain(Figure S5−8a,b). These peculiar behaviors in the decayingparts were observed in all experiments (3 out of 3 times) at EFW= −0.6 V (Figure S5−8), sometimes observed (2 out of 5times) at EFW = −0.3 V (Figure S5−9b). Similar behavior wasalso observed in Figure S5−9c, with the decay in G′ beingsmaller at 100%. In contrast, those behaviors were notobserved (0 out of 3 times) at EFW = 0, +0.3, and +0.6 V(Figures S5−10−12). In Figure 7, the γYP at EFW = −0.3 V wasnot taken into account when the peculiar behavior wasFigure 5. Potential dependence of (a) interfacial shear storage andloss modulus, G′ (red) and G″ (blue), and (b) tan δ (= G″/G′, black)at the F|W interface 1 h after the BSA solution was injected. G′ andG″ were measured with a strain of γ = 1% and an angular frequency ofω = 1 Hz. The error bars are the standard errors from threeexperiments at each EFW shown in Figure S5−3−7.Figure 6. Strain γ sweep of interfacial shear storage G′ (solid lines)and loss modulus G″ (dashed lines) at EFW = − 0.3 (blue), 0 (black),+0.6 V (red) at the F|W interface at 1 h after the BSA solution wasinjected. The points are the yield point where G′ and G″ profiles crossover (G′ = G″), γYP. These profiles are the ones whose γYP are theclosest to the average of γYP out of the three profiles at EFW = −0.3, 0,and +0.6 V shown in Figure S5−9, 10, and 12, respectively. Those atEFW = −0.6 and +0.3 V are shown in Figure S5−8 and 11, respectively.Figure 7. γYP of the PNL at the F|W interface.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c01819Langmuir 2025, 41, 17973−1798117977https://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig7&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c01819?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asobserved. The peculiar behavior of the G′ and G″ at EFW = −0.6and −0.3 V might be because the reduction in G′ decreaseddue to the reformation of the PNL structure and newinteractions between BSA molecules at large strains around γ =100%. At EFW = 0, +0.3, and +0.6 V, the plateau was notobserved probably because the number of BSA in PNL wassmaller and BSA was less denatured, making it harder toreform the PNL structure and new interactions between BSAmolecules. The results in Figures 5−7 indicate that PNLs at EFW< 0 V have both the high elasticity and the high strainresistance without any chemical treatments to PNL.The PNL at the liquid|liquid interface was reported torecover after cracking the PNL.18 The following experimentswere carried out to investigate the potential dependence of therecovery speed of the cracked PNL. After the PNL had beenformed for 1 h, it was cracked by applying a 1000%deformation at EFW = −0.6, 0, +0.6 V and then the timesweep was performed to measure the recovery time forreaching the values of G′ and G″ before cracking (FigureS5−13). The recovery time was 10 s at EFW = −0.6 V, 350 s atEFW = 0 V, and 600 s at EFW = 0.6 V; as EFW decreased, therecovery time was shortened. The electrostatic attraction islikely to accelerate the adsorption of BSA and recovery of thePNL at the F|W interface. This tendency agrees with the caseat the silica|W interface where the adsorption of positivelycharged BSA at pH > 5 is faster than that of negatively chargedBSA at pH < 5.77 The recovery time at EFW = 0 V, 350 s, iscomparable to that at the IL|W interface measured by usinghigh-speed AFM, 300−500 s.18 Although F is a dilutedsolution of IL, the recovery time at the F|W and IL|Winterfaces without externally controlling the phase boundarypotential difference was close. This also supports that thecomposition of the F side of the F|W interface is IL-like.33The switching effect of EFW on G′ and G″ of the PNL wasinvestigated to control the structure and mechanical propertyof PNL. Figure 8 shows the profiles of G′ and G″ when the EFWwas switched between EFW = +0.3 V and EFW = −0.6 V. G′ andG″ were reversible against the EFW switch, implying that themechanical interaction of PNL and cells on the electrochemicalF|W interface is actively switchable. The transition of G′ andG″ is different between EFW = +0.3 to −0.6 V and EFW = −0.6 to+0.3 V. This indicates that the structural changes of the PNLinduced by switching EFW have various processes such as theadsorption and desorption of BSA, and the rearrangement ofthe adsorption part and the inter and intramolecular bonds ofBSA. The fact that G″ responds more rapidly to potentialswitching than G′ may suggest that dissipative processes suchas rearrangement of protein molecules or partial unfolding ofloosely bound protein dangling ends occur quickly, whereasthe development of a more elastic network (reflected in G′)involves slower maturation of intermolecular interactions. Thisdifference implies that multiple, time scale-dependentprocesses contribute to the viscoelastic modulation of thePNL.4. CONCLUSIONSWe successfully analyzed the PNL structure at the F|Winterface under the condition of EFW by using NR and IRM.Although the NR results showed that EFW has minimal effect onthe adsorption amount of BSA in the PNL at the electro-chemical F|W interface, IRM unveiled that the in-planestructure was strengthened at EFW < 0 V, and was reversiblyswitchable by applying EFW. The present proof-of-concept studydemonstrates that the potential control is an interface-specificmethod to diversify and switch the PNL structure reversibly.This method with interface-specificity and reversibility is instark contrast to previously reported ones that change thehydrophobic liquids4,14,18,26,43,78,79 or add reagents inW.5,13,14,26,34 The reversible structural control of the PNLwould enable us to perform real-time observation of cellsreacting to environmental changes at liquid|liquid interfaces.■ ASSOCIATED CONTENT*sı Supporting InformationThe Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819.Interfacial tension measurements at the F|W interface;NR at the liquid|air interface; interfacial roughness of theF|W interface evaluated using the capillary wave theory;and NR and interfacial rheological measurements at theF|W interface (PDF)■ AUTHOR INFORMATIONCorresponding AuthorsTakeshi Ueki − Research Center for Macromolecules &Biomaterials, National Institute for Materials Science(NIMS), Tsukuba, Ibaraki 305-0044, Japan; GraduateSchool of Life Science, Hokkaido University, Sapporo 060-0810, Japan; orcid.org/0000-0001-9317-6280;Email: UEKI.Takeshi@nims.go.jpNaoya Nishi − Department of Energy and HydrocarbonChemistry, Kyoto University, Kyoto 615-8510, Japan;orcid.org/0000-0002-5654-5603;Email: nishi.naoya.7e@kyoto-u.ac.jpAuthorsKosuke Ishii − Department of Energy and HydrocarbonChemistry, Kyoto University, Kyoto 615-8510, JapanJun Nakanishi − Research Center for Macromolecules &Biomaterials, National Institute for Materials Science(NIMS), Tsukuba, Ibaraki 305-0044, Japan; GraduateSchool of Advanced Science and Engineering, WasedaFigure 8. Time courses of G′ (blue) and G″ (green) of the PNLagainst multiple potential switches between at EFW = +0.3 (gray) and−0.6 V (yellow) at every 3600 s.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c01819Langmuir 2025, 41, 17973−1798117978https://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?goto=supporting-infohttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c01819/suppl_file/la5c01819_si_001.pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Takeshi+Ueki"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0001-9317-6280mailto:UEKI.Takeshi@nims.go.jphttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Naoya+Nishi"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0002-5654-5603https://orcid.org/0000-0002-5654-5603mailto:nishi.naoya.7e@kyoto-u.ac.jphttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Kosuke+Ishii"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Jun+Nakanishi"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig8&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?fig=fig8&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c01819?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asUniversity, Tokyo 169-8555, Japan; Graduate School ofAdvanced Engineering, Tokyo University of Science, Tokyo125-8585, Japan; orcid.org/0000-0003-4457-6581Kazuhiro Akutsu-Suyama − Neutron Science and TechnologyCenter, Comprehensive Research Organization for Scienceand Society (CROSS), Naka 319-1106 Ibaraki, Japan;orcid.org/0000-0002-4797-6604Norifumi L. Yamada − Neutron Science Laboratory, Centerfor Integrative Quantum Beam Science, High EnergyAccelerator Research Organization, Naka 319-1106, Japan;orcid.org/0000-0002-8370-4526Yuko Yokoyama − Department of Energy and HydrocarbonChemistry, Kyoto University, Kyoto 615-8510, Japan;orcid.org/0000-0002-3943-0978Tetsuo Sakka − Department of Energy and HydrocarbonChemistry, Kyoto University, Kyoto 615-8510, Japan;orcid.org/0000-0002-1892-8056Complete contact information is available at:https://pubs.acs.org/10.1021/acs.langmuir.5c01819NotesThe authors declare no competing financial interest.■ ACKNOWLEDGMENTSWe thank Dr. Jun-ichi Horinaka for the fruitful discussion onrheological measurements. This work was partly supported bythe Japan Society for the Promotion of Science (JSPS)KAKENHI Grants (23H03829 for N.N. and 23H02030 forT.U.). The neutron reflectivity experiments were performed atthe Materials and Life Science Experimental Facility in J-PARC(Proposal Nos. 2023A0070 and 2024A0237).■ REFERENCES(1) Rosenberg, M. D. Cell Surface Interactions and InterfacialDynamics. In Cellular Control Mechanisms and Cancer; Emmelot, P.;Muhlbock, O., Eds.; Elsevier: Amsterdam, 1964; pp 146−164.(2) Kong, D.; Nguyen, K. D. Q.; Megone, W.; Peng, L.; Gautrot, J.E. The culture of HaCaT cells on liquid substrates is mediated by amechanically strong liquid-liquid interface. Faraday Discuss. 2017,204, 367−381.(3) Kong, D.; Megone, W.; Nguyen, K. D. Q.; Cio, S. D.; Ramstedt,M.; Gautrot, J. E. Protein Nanosheet Mechanics Controls CellAdhesion and Expansion on Low-Viscosity Liquids. Nano Lett. 2018,18, 1946−1951.(4) Minami, K.; Mori, T.; Nakanishi, W.; Shigi, N.; Nakanishi, J.;Hill, J. P.; Komiyama, M.; Ariga, K. Suppression of MyogenicDifferentiation of Mammalian Cells Caused by Fluidity of a Liquid-Liquid Interface. ACS Appl. Mater. Interfaces 2017, 9, 30553−30560.(5) Kong, D.; Peng, L.; Di Cio, S.; Novak, P.; Gautrot, J. E. StemCell Expansion and Fate Decision on Liquid Substrates Are Regulatedby Self-Assembled Nanosheets. ACS Nano 2018, 12, 9206−9213.(6) Jia, X.; Minami, K.; Uto, K.; Chang, A. C.; Hill, J. P.; Ueki, T.;Nakanishi, J.; Ariga, K. Modulation of Mesenchymal Stem CellsMechanosensing at Fluid Interfaces by Tailored Self-AssembledProtein Monolayers. Small 2019, 15, No. e1804640.(7) Peng, L.; Gautrot, J. E. Long term expansion profile ofmesenchymal stromal cells at protein nanosheet-stabilised bioemul-sions for next generation cell culture microcarriers. Mater. Today Bio2021, 12, No. 100159.(8) Kong, D.; Peng, L.; Bosch-Fortea, M.; Chrysanthou, A.; Alexis,C. V. J. M.; Matellan, C.; Zarbakhsh, A.; Mastroianni, G.; del RioHernandez, A.; Gautrot, J. E. Impact of the multiscale viscoelasticityof quasi-2D self-assembled protein networks on stem cell expansion atliquid interfaces. Biomaterials 2022, 284, No. 121494.(9) Jia, X.; Minami, K.; Uto, K.; Chang, A. C.; Hill, J. P.; Nakanishi,J.; Ariga, K. Adaptive Liquid Interfacially Assembled ProteinNanosheets for Guiding Mesenchymal Stem Cell Fate. Adv. Mater.2020, 32, No. 1905942.(10) Jia, X.; Song, J.; Lv, W.; Hill, J. P.; Nakanishi, J.; Ariga, K.Adaptive liquid interfaces induce neuronal differentiation ofmesenchymal stem cells through lipid raft assembly. Nat. Commun.2022, 13, No. 3110.(11) Jia, X.; Minami, K.; Uto, K.; Chang, A. C.; Hill, J. P.; Ueki, T.;Nakanishi, J.; Ariga, K. Modulation of Mesenchymal Stem CellsMechanosensing at Fluid Interfaces by Tailored Self-AssembledProtein Monolayers. Small 2019, 15, No. 1804640.(12) Ueki, T.; Uto, K.; Yamamoto, S.; Tamate, R.; Kamiyama, Y.;Jia, X.; Noguchi, H.; Minami, K.; Ariga, K.; Wang, H.; Nakanishi, J.Ionic Liquid Interface as a Cell Scaffold. Adv. Mater. 2024, 36,No. 2310105.(13) Keese, C. R.; Giaever, I. Cell Growth on Liquid Microcarriers.Science 1983, 219, 1448−1449.(14) Giaever, I.; Keese, C. R. Behavior of cells at fluid interfaces.Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 219−222.(15) Keese, C. R.; Giaever, I. Substrate mechanics and cellspreading. Exp. Cell Res. 1991, 195, 528−532.(16) Ando, J.; Albelda, S. M.; Levine, E. M. Culture of human adultendothelial cells on liquid-liquid interfaces: A new approach to thestudy of cell-matrix interactions. In Vitro Cell. Dev. Biol.: Anim. 1991,27, 525−532.(17) Shiba, Y.; Ohshima, T.; Sato, M. Growth and morphology ofanchorage-dependent animal cells in a liquid/liquid interface system.Biotechnol. Bioeng. 1998, 57, 583−589.(18) Ueki, T.; Uto, K.; Yamamoto, S.; Tamate, R.; Kamiyama, Y.;Jia, X.; Noguchi, H.; Minami, K.; Ariga, K.; Wang, H.; Nakanishi, J.Ionic Liquid Interface as a Cell Scaffold. Adv. Mater. 2024, 36,No. 2310105.(19) Nakanishi, J.; Ueki, T.; Dieb, S.; Noguchi, H.; Yamamoto, S.;Sodeyama, K. Data-driven optimization of the in silico design of ionicliquids as interfacial cell culture fluids. Sci. Technol. Adv. Mater. 2024,25, No. 2418287.(20) Katano, H.; Kuroda, Y.; Uematsu, K. Applicability of a fluoroussolvent 1,1,1,2,3,4,4,5,5,5-decafluoropentane for the non-aqueousmedium in liquid-liquid electrochemistry. J. Electroanal. Chem. 2017,788, 232−234.(21) Roth, S.; Murray, B. S.; Dickinson, E. Interfacial shear rheologyof aged and heat-treated β-lactoglobulin films: Displacement bynonionic surfactant. J. Agric. Food Chem. 2000, 48, 1491−1497.(22) Benjamins, J.; Lyklema, J.; Lucassen-Reynders, E. H.Compression/expansion rheology of oil/water interfaces withadsorbed proteins. Comparison with the air/water surface. Langmuir2006, 22, 6181−6188.(23) Grigoriev, D. O.; Derkatch, S.; Krägel, J.; Miller, R.Relationship between structure and rheological properties of mixedBSA/Tween 80 adsorption layers at the air/water interface. FoodHydrocolloids 2007, 21, 823−830.(24) Maldonado-Valderrama, J.; Miller, R.; Fainerman, V. B.; Wilde,P. J.; Morris, V. J. Effect of gastric conditions on β-lactoglobulininterfacial networks: Influence of the oil phase on protein structure.Langmuir 2010, 26, 15901−15908.(25) Mitropoulos, V.; Mütze, A.; Fischer, P. Mechanical propertiesof protein adsorption layers at the air/water and oil/water interface: Acomparison in light of the thermodynamical stability of proteins. Adv.Colloid Interface Sci. 2014, 206, 195−206.(26) Bergfreund, J.; Diener, M.; Geue, T.; Nussbaum, N.; Kummer,N.; Bertsch, P.; Nyström, G.; Fischer, P. Globular protein assemblyand network formation at fluid interfaces: effect of oil. Soft Matter2021, 17, 1692−1700.(27) Bergfreund, J.; Bertsch, P.; Fischer, P. Adsorption of proteins tofluid interfaces: Role of the hydrophobic subphase. J. Colloid InterfaceSci. 2021, 584, 411−417.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c01819Langmuir 2025, 41, 17973−1798117979https://orcid.org/0000-0003-4457-6581https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Kazuhiro+Akutsu-Suyama"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0002-4797-6604https://orcid.org/0000-0002-4797-6604https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Norifumi+L.+Yamada"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0002-8370-4526https://orcid.org/0000-0002-8370-4526https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Yuko+Yokoyama"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0002-3943-0978https://orcid.org/0000-0002-3943-0978https://pubs.acs.org/action/doSearch?field1=Contrib&text1="Tetsuo+Sakka"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0002-1892-8056https://orcid.org/0000-0002-1892-8056https://pubs.acs.org/doi/10.1021/acs.langmuir.5c01819?ref=pdfhttps://doi.org/10.1039/C7FD00091Jhttps://doi.org/10.1039/C7FD00091Jhttps://doi.org/10.1021/acs.nanolett.7b05339?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.nanolett.7b05339?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acsami.7b11445?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acsami.7b11445?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acsami.7b11445?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acsnano.8b03865?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acsnano.8b03865?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acsnano.8b03865?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1002/smll.201804640https://doi.org/10.1002/smll.201804640https://doi.org/10.1002/smll.201804640https://doi.org/10.1016/j.mtbio.2021.100159https://doi.org/10.1016/j.mtbio.2021.100159https://doi.org/10.1016/j.mtbio.2021.100159https://doi.org/10.1016/j.biomaterials.2022.121494https://doi.org/10.1016/j.biomaterials.2022.121494https://doi.org/10.1016/j.biomaterials.2022.121494https://doi.org/10.1002/adma.201905942https://doi.org/10.1002/adma.201905942https://doi.org/10.1038/s41467-022-30622-yhttps://doi.org/10.1038/s41467-022-30622-yhttps://doi.org/10.1002/smll.201804640https://doi.org/10.1002/smll.201804640https://doi.org/10.1002/smll.201804640https://doi.org/10.1002/adma.202310105https://doi.org/10.1126/science.6828872https://doi.org/10.1073/pnas.80.1.219https://doi.org/10.1016/0014-4827(91)90406-Khttps://doi.org/10.1016/0014-4827(91)90406-Khttps://doi.org/10.1007/BF02631282https://doi.org/10.1007/BF02631282https://doi.org/10.1007/BF02631282https://doi.org/10.1002/(SICI)1097-0290(19980305)57:5<583::AID-BIT10>3.0.CO;2-Dhttps://doi.org/10.1002/(SICI)1097-0290(19980305)57:5<583::AID-BIT10>3.0.CO;2-Dhttps://doi.org/10.1002/adma.202310105https://doi.org/10.1080/14686996.2024.2418287https://doi.org/10.1080/14686996.2024.2418287https://doi.org/10.1016/j.jelechem.2017.01.060https://doi.org/10.1016/j.jelechem.2017.01.060https://doi.org/10.1016/j.jelechem.2017.01.060https://doi.org/10.1021/jf990976z?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/jf990976z?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/jf990976z?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/la060441h?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/la060441h?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1016/j.foodhyd.2006.08.018https://doi.org/10.1016/j.foodhyd.2006.08.018https://doi.org/10.1021/la102294u?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/la102294u?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1016/j.cis.2013.11.004https://doi.org/10.1016/j.cis.2013.11.004https://doi.org/10.1016/j.cis.2013.11.004https://doi.org/10.1039/D0SM01870Hhttps://doi.org/10.1039/D0SM01870Hhttps://doi.org/10.1016/j.jcis.2020.09.118https://doi.org/10.1016/j.jcis.2020.09.118pubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c01819?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as(28) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. Theconformational structure of bovine serum albumin layers adsorbed atthe silica-water interface. J. Phys. Chem. B 1998, 102, 8100−8108.(29) Koutsioubas, A.; Lairez, D.; Zalczer, G.; Cousin, F. Slow andremanent electric polarization of adsorbed BSA layer evidenced byneutron reflection. Soft Matter 2012, 8, 2638−2643.(30) Campana, M.; Hosking, S. L.; Petkov, J. T.; Tucker, I. M.;Webster, J. R. P.; Zarbakhsh, A.; Lu, J. R. Adsorption of bovine serumalbumin (BSA) at the oil/water interface: A neutron reflection study.Langmuir 2015, 31, 5614−5622.(31) Cheung, D. L. Adsorption and conformations of lysozyme andα -lactalbumin at a water-octane interface. J. Chem. Phys. 2017, 147,No. 195101.(32) Tummino, A.; Scoppola, E.; Fragneto, G.; Gutfreund, P.;Maestro, A.; Dryfe, R. A. W. Neutron reflectometry study of theinterface between two immiscible electrolyte solutions: Effects ofelectrolyte concentration, applied electric field, and lipid adsorption.Electrochim. Acta 2021, 384, No. 138336.(33) Ishii, K.; Akutsu-Suyama, K.; Yamada, N. L.; Yokoyama, Y.;Sakka, T.; Nishi, N. Accumulation of ionic-liquid ions at theelectrochemical liquid/liquid interface between water and a fluoroussolvent studied using neutron reflectometry. Electrochim. Acta 2025,513, No. 145563.(34) Chrysanthou, A.; Kanso, H.; Zhong, W.; Shang, L.; Gautrot, J.E. Supercharged Protein Nanosheets for Cell Expansion onBioemulsions. ACS Appl. Mater. Interfaces 2023, 15, 2760−2770.(35) Rosales, A. M.; Vega, S. L.; DelRio, F. W.; Burdick, J. A.;Anseth, K. S. Hydrogels with Reversible Mechanics to Probe DynamicCell Microenvironments. Angew. Chem., Int. Ed. 2017, 56, 12132−12136.(36) Grim, J. C.; Brown, T. E.; Aguado, B. A.; Chapnick, D. A.;Viert, A. L.; Liu, X.; Anseth, K. S. A Reversible and Repeatable Thiol-Ene Bioconjugation for Dynamic Patterning of Signaling Proteins inHydrogels. ACS Cent. Sci. 2018, 4, 909−916.(37) Homma, K.; Chang, A. C.; Yamamoto, S.; Tamate, R.; Ueki, T.;Nakanishi, J. Design of azobenzene-bearing hydrogel with photo-switchable mechanics driven by photo-induced phase transition for invitro disease modeling. Acta Biomater. 2021, 132, 103−113.(38) Ueki, T.; Osaka, Y.; Homma, K.; Yamamoto, S.; Saruwatari, A.;Wang, H.; Kamimura, M.; Nakanishi, J. Reversible SolubilitySwitching of a Polymer Triggered by Visible-Light ResponsiveAzobenzene Photochromism with Negligible Thermal Relaxation.Macromol. Rapid Commun. 2024, 45, No. 2400419.(39) Nishi, N.; Minami, K.; Motobayashi, K.; Osawa, M.; Sakka, T.Interfacial Structure at the quaternary ammonium-based ionic liquids|gold electrode interface probed by surface-enhanced infraredabsorption spectroscopy: Anion dependence of the cationic behavior.J. Phys. Chem. C 2017, 121, 1658−1666.(40) Salgın, S.; Salgin, U.; Bahadir, S. Zeta potentials and isoelectricpoints of biomolecules: The effects of ion types and ionic strengths.Int. J. Electrochem. Sci. 2012, 7, 12404−12414.(41) Micheau, C.; Ueda, Y.; Motokawa, R.; Akutsu-Suyama, K.;Yamada, N. L.; Yamada, M.; Moussaoui, S. A.; Makombe, E.; Meyer,D.; Berthon, L.; Bourgeois, D. Organization of malonamides from theinterface to the organic bulk phase. J. Mol. Liq. 2024, 401,No. 124372.(42) Mitamura, K.; Yamada, N. L.; Sagehashi, H.; Torikai, N.; Arita,H.; Terada, M.; Kobayashi, M.; Sato, S.; Seto, H.; Goko, S.; Furusaka,M.; Oda, T.; Hino, M.; Jinnai, H.; Takahara, A. Novel neutronreflectometer SOFIA at J-PARC/MLF for in-situ soft-interfacecharacterization. Polym. J. 2013, 45, 100−108.(43) Jia, X.; Minami, K.; Uto, K.; Chang, A. C.; Hill, J. P.; Ueki, T.;Nakanishi, J.; Ariga, K. Modulation of Mesenchymal Stem CellsMechanosensing at Fluid Interfaces by Tailored Self-AssembledProtein Monolayers. Small 2019, 15, No. 1804640.(44) https://www.ncbi.nlm.nih.gov/gene/280717 (accessed Jun 15,2025)(45) https://smb-research.smb.usyd.edu.au/NCVWeb/input.jsp(accessed Jun 15, 2025).(46) Braslau, A.; Pershan, P. S.; Swislow, G.; Ocko, B. M.; Als-Nielsen, J. Capillary waves on the surface of simple liquids measuredby x-ray reflectivity. Phys. Rev. A 1988, 38, No. 2457.(47) Buff, F. P.; Lovett, R. A.; Stillinger, F. H. Interfacial DensityProfile for Fluids in the Critical Region. Phys. Rev. Lett. 1965, 15,No. 621.(48) Sugiura, N. Further analysis of the data by Akaike’s informationcriterion and the finite corrections. Commun. Stat.-Theory Methods1978, 7, 13−26.(49) Nishi, N.; Uchiyashiki, J.; Ikeda, Y.; Katakura, S.; Oda, T.;Hino, M.; Yamada, N. L. Potential-Dependent Structure of the IonicLayer at the Electrode Interface of an Ionic Liquid Probed UsingNeutron Reflectometry. J. Phys. Chem. C 2019, 123, 9223−9230.(50) Nishi, N.; Uchiyashiki, J.; Oda, T.; Hino, M.; Yamada, N. L.Overscreening Induced by Ionic Adsorption at the Ionic Liquid/Electrode Interface Detected Using Neutron Reflectometry with aRational Material Design. Bull. Chem. Soc. Jpn. 2021, 94, 2914−2918.(51) Vandebril, S.; Franck, A.; Fuller, G.; Moldenaers, J.; Vermant, J.Double Wall Ring Geometry to Measure Interfacial RheologicalProperties. Rheol. Acta 2009, 49, 131−144.(52) Hartvig, R. A.; Méndez, M. A.; Van De Weert, M.; Jorgensen,L.; Østergaard, J.; Girault, H. H.; Jensen, H. Interfacial complexesbetween a protein and lipophilic ions at an oil-water interface. Anal.Chem. 2010, 82, 7699−7705.(53) Kubiak-Ossowska, K.; Jachimska, B.; Mulheran, P. A. HowNegatively Charged Proteins Adsorb to Negatively Charged Surfaces:A Molecular Dynamics Study of BSA Adsorption on Silica. J. Phys.Chem. B 2016, 120, 10463−10468.(54) Gong, Z.; Szczesny, S. E.; Caliari, S. R.; Charrier, E. E.;Chaudhuri, O.; Cao, X.; Lin, Y.; Mauck, R. L.; Janmey, P. A.; Burdick,J. A.; Shenoy, V. B. Matching material and cellular timescalesmaximizes cell spreading on viscoelastic substrates. Proc. Natl. Acad.Sci. U.S.A. 2018, 115, E2686−E2695.(55) Chaudhuri, O.; Cooper-White, J.; Janmey, P. A.; Mooney, D. J.;Shenoy, V. B. Effects of extracellular matrix viscoelasticity on cellularbehaviour. Nature 2020, 584, 535−546.(56) Cameron, A. R.; Frith, J. E.; Cooper-White, J. J. The influenceof substrate creep on mesenchymal stem cell behaviour andphenotype. Biomaterials 2011, 32, 5979−5993.(57) Cameron, A. R.; Frith, J. E.; Gomez, G. A.; Yap, A. S.; Cooper-White, J. J. The effect of time-dependent deformation of viscoelastichydrogels on myogenic induction and Rac1 activity in mesenchymalstem cells. Biomaterials 2014, 35, 1857−1868.(58) Yasui, Y.; Kitazumi, Y.; Ishimatsu, R.; Nishi, N.; Kakiuchi, T.Ultraslow response of interfacial tension to the change in the phase-boundary potential at the interface between water and a room-temperature ionic liquid, trioctylmethylammonium bis-(nonafluorobutanesulfonyl)amide. J. Phys. Chem. B 2009, 113,3273−3276.(59) Scanlon, M. D.; Jennings, E.; Arrigan, D. W. M. Electro-chemical behaviour of hen-egg-white lysozyme at the polarised water/1, 2-dichloroethane interface. Phys. Chem. Chem. Phys. 2009, 11,2272−2280.(60) Arooj, M.; Gandhi, N. S.; Kreck, C. A.; Arrigan, D. W. M.;Mancera, R. L. Adsorption and Unfolding of Lysozyme at a PolarizedAqueous-Organic Liquid Interface. J. Phys. Chem. B 2016, 120, 3100−3112.(61) Alvarez de Eulate, E.; O’Sullivan, S.; Arrigan, D. W. M.Electrochemically Induced Formation of Cytochrome c Oligomers atSoft Interfaces. ChemElectroChem 2017, 4, 898−904.(62) Booth, S. G.; Felisilda, B. M. B.; De Eulate, E. A.; Gustafsson,O. J. R.; Arooj, M.; Mancera, R. L.; Dryfe, R. A. W.; Hackett, M. J.;Arrigan, D. W. M. Secondary Structural Changes in Proteins as aResult of Electroadsorption at Aqueous-Organogel Interfaces.Langmuir 2019, 35, 5821−5829.(63) Yokota, Y.; Harada, T.; Fukui, K. I. Direct observation oflayered structures at ionic liquid/solid interfaces by using frequency-modulation atomic force microscopy. Chem. Commun. 2010, 46,8627−8629.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c01819Langmuir 2025, 41, 17973−1798117980https://doi.org/10.1021/jp981239t?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/jp981239t?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/jp981239t?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1039/c2sm07265chttps://doi.org/10.1039/c2sm07265chttps://doi.org/10.1039/c2sm07265chttps://doi.org/10.1021/acs.langmuir.5b00646?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.langmuir.5b00646?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1063/1.4994561https://doi.org/10.1063/1.4994561https://doi.org/10.1016/j.electacta.2021.138336https://doi.org/10.1016/j.electacta.2021.138336https://doi.org/10.1016/j.electacta.2021.138336https://doi.org/10.1016/j.electacta.2024.145563https://doi.org/10.1016/j.electacta.2024.145563https://doi.org/10.1016/j.electacta.2024.145563https://doi.org/10.1021/acsami.2c20188?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acsami.2c20188?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1002/anie.201705684https://doi.org/10.1002/anie.201705684https://doi.org/10.1021/acscentsci.8b00325?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acscentsci.8b00325?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acscentsci.8b00325?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1016/j.actbio.2021.03.028https://doi.org/10.1016/j.actbio.2021.03.028https://doi.org/10.1016/j.actbio.2021.03.028https://doi.org/10.1002/marc.202400419https://doi.org/10.1002/marc.202400419https://doi.org/10.1002/marc.202400419https://doi.org/10.1021/acs.jpcc.6b10826?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.jpcc.6b10826?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.jpcc.6b10826?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1016/S1452-3981(23)16554-0https://doi.org/10.1016/S1452-3981(23)16554-0https://doi.org/10.1016/j.molliq.2024.124372https://doi.org/10.1016/j.molliq.2024.124372https://doi.org/10.1038/pj.2012.156https://doi.org/10.1038/pj.2012.156https://doi.org/10.1038/pj.2012.156https://doi.org/10.1002/smll.201804640https://doi.org/10.1002/smll.201804640https://doi.org/10.1002/smll.201804640https://www.ncbi.nlm.nih.gov/gene/280717https://smb-research.smb.usyd.edu.au/NCVWeb/input.jsphttps://doi.org/10.1103/PhysRevA.38.2457https://doi.org/10.1103/PhysRevA.38.2457https://doi.org/10.1103/PhysRevLett.15.621https://doi.org/10.1103/PhysRevLett.15.621https://doi.org/10.1080/03610927808827599https://doi.org/10.1080/03610927808827599https://doi.org/10.1021/acs.jpcc.9b01151?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.jpcc.9b01151?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.jpcc.9b01151?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1246/bcsj.20210328https://doi.org/10.1246/bcsj.20210328https://doi.org/10.1246/bcsj.20210328https://doi.org/10.1007/s00397-009-0407-3https://doi.org/10.1007/s00397-009-0407-3https://doi.org/10.1021/ac101528r?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/ac101528r?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.jpcb.6b07646?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.jpcb.6b07646?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.jpcb.6b07646?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1073/pnas.1716620115https://doi.org/10.1073/pnas.1716620115https://doi.org/10.1038/s41586-020-2612-2https://doi.org/10.1038/s41586-020-2612-2https://doi.org/10.1016/j.biomaterials.2011.04.003https://doi.org/10.1016/j.biomaterials.2011.04.003https://doi.org/10.1016/j.biomaterials.2011.04.003https://doi.org/10.1016/j.biomaterials.2013.11.023https://doi.org/10.1016/j.biomaterials.2013.11.023https://doi.org/10.1016/j.biomaterials.2013.11.023https://doi.org/10.1021/jp9006312?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/jp9006312?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/jp9006312?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/jp9006312?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1039/b815589ehttps://doi.org/10.1039/b815589ehttps://doi.org/10.1039/b815589ehttps://doi.org/10.1021/acs.jpcb.6b00536?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.jpcb.6b00536?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1002/celc.201600851https://doi.org/10.1002/celc.201600851https://doi.org/10.1021/acs.langmuir.8b04227?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.langmuir.8b04227?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1039/c0cc02643chttps://doi.org/10.1039/c0cc02643chttps://doi.org/10.1039/c0cc02643cpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c01819?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as(64) Ueno, K.; Kasuya, M.; Watanabe, M.; Mizukami, M.; Kurihara,K. Resonance shear measurement of nanoconfined ionic liquids. Phys.Chem. Chem. Phys. 2010, 12, 4066−4071.(65) Nishi, N.; Yamazawa, T.; Sakka, T.; Hotta, H.; Ikeno, T.;Hanaoka, K.; Takahashi, H. How Viscous Is the Solidlike Structure atthe Interface of Ionic Liquids? A Study Using Total InternalReflection Fluorescence Spectroscopy with a Fluorescent MolecularProbe Sensitive to High Viscosity. Langmuir 2020, 36, 10397−10403.(66) Zhang, S.; Nishi, N.; Sakka, T. Electrochemical surface plasmonresonance measurements of camel-shaped static capacitance and slowdynamics of electric double layer structure at the ionic liquid/electrode interface. J. Chem. Phys. 2020, 153, No. 044707.(67) Bou-Malham, I.; Bureau, L. Nanoconfined ionic liquids: Effectof surface charges on flow and molecular layering. Soft Matter 2010, 6,4062−4065.(68) Sha, M.; Wu, G.; Dou, Q.; Tang, Z.; Fang, H. Double-layerformation of [Bmim][PF6] Ionic liquid triggered by surface negativecharge. Langmuir 2010, 26, 12667−12672.(69) Kislenko, S. A.; Amirov, R. H.; Samoylov, I. S. Influence oftemperature on the structure and dynamics of the [BMIM][PF6]ionic liquid/graphite interface. Phys. Chem. Chem. Phys. 2010, 12,11245−11250.(70) Nishi, N.; Hirano, Y.; Motokawa, T.; Kakiuchi, T. Ultraslowrelaxation of the structure at the ionic liquid-gold electrode interfaceto a potential step probed by electrochemical surface plasmonresonance measurements: Asymmetry of the relaxation time to thepotential-step direction. Phys. Chem. Chem. Phys. 2013, 15, 11615−11619.(71) Comtet, J.; Nigues̀, A.; Kaiser, V.; Coasne, B.; Bocquet, L.;Siria, A. Nanoscale capillary freezing of ionic liquids confined betweenmetallic interfaces and the role of electronic screening. Nat. Mater.2017, 16, 634−639.(72) Lhermerout, R.; Perkin, S. Nanoconfined ionic liquids:Disentangling electrostatic and viscous forces. Phys. Rev. Fluids2018, 3, No. 014201.(73) Gil, P. S.; Jorgenson, S. J.; Riet, A. R.; Lacks, D. J.; Comtet, J.;Nigues̀, A.; Kaiser, V.; Coasne, B.; Bocquet, L.; Siria, A.; Lhermerout,R.; Perkin, S.; Dou, Q.; Sha, M.; Fu, H.; Wu, G. Nanoscale capillaryfreezing of ionic liquids confined between metallic interfaces and therole of electronic screening. ChemPhysChem 2018, 122, 2438−2443.(74) Ntim, S.; Sulpizi, M. Role of image charges in ionic liquidconfined between metallic interfaces. Phys. Chem. Chem. Phys. 2020,22, 10786−10791.(75) Nishi, N.; Yasui, Y.; Uruga, T.; Tanida, H.; Yamada, T.;Nakayama, S. I.; Matsuoka, H.; Kakiuchi, T. Ionic multilayers at thefree surface of an ionic liquid, trioctylmethylammonium bis-(nonafluorobutanesulfonyl)amide, probed by x-ray reflectivity meas-urements. J. Chem. Phys. 2010, 132, No. 164705.(76) Ishii, K.; Sakka, T.; Nishi, N. Potential dependence of the ionicstructure at the ionic liquid/water interface studied using MDsimulation. Phys. Chem. Chem. Phys. 2021, 23, 22367−22374.(77) McUmber, A. C.; Randolph, T. W.; Schwartz, D. K.Electrostatic Interactions Influence Protein Adsorption (but NotDesorption) at the Silica-Aqueous Interface. J. Phys. Chem. Lett. 2015,6, 2583−2587.(78) Zare, D.; Allison, J. R.; McGrath, K. M. Molecular DynamicsSimulation of β-Lactoglobulin at Different Oil/Water Interfaces.Biomacromolecules 2016, 17, 1572−1581.(79) Bergfreund, J.; Bertsch, P.; Kuster, S.; Fischer, P. Effect of OilHydrophobicity on the Adsorption and Rheology of β-Lactoglobulinat Oil-Water Interfaces. Langmuir 2018, 34, 4929−4936.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c01819Langmuir 2025, 41, 17973−1798117981https://doi.org/10.1039/b923571jhttps://doi.org/10.1021/acs.langmuir.0c01528?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.langmuir.0c01528?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.langmuir.0c01528?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.langmuir.0c01528?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1063/5.0011671https://doi.org/10.1063/5.0011671https://doi.org/10.1063/5.0011671https://doi.org/10.1063/5.0011671https://doi.org/10.1039/c0sm00377hhttps://doi.org/10.1039/c0sm00377hhttps://doi.org/10.1021/la101107b?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/la101107b?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/la101107b?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1039/c0cp00220hhttps://doi.org/10.1039/c0cp00220hhttps://doi.org/10.1039/c0cp00220hhttps://doi.org/10.1039/c3cp51463chttps://doi.org/10.1039/c3cp51463chttps://doi.org/10.1039/c3cp51463chttps://doi.org/10.1039/c3cp51463chttps://doi.org/10.1039/c3cp51463chttps://doi.org/10.1038/nmat4880https://doi.org/10.1038/nmat4880https://doi.org/10.1103/PhysRevFluids.3.014201https://doi.org/10.1103/PhysRevFluids.3.014201https://doi.org/10.1002/cphc.201000198https://doi.org/10.1002/cphc.201000198https://doi.org/10.1002/cphc.201000198https://doi.org/10.1039/D0CP00409Jhttps://doi.org/10.1039/D0CP00409Jhttps://doi.org/10.1063/1.3398029https://doi.org/10.1063/1.3398029https://doi.org/10.1063/1.3398029https://doi.org/10.1063/1.3398029https://doi.org/10.1039/D1CP02484Ahttps://doi.org/10.1039/D1CP02484Ahttps://doi.org/10.1039/D1CP02484Ahttps://doi.org/10.1021/acs.jpclett.5b00933?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.jpclett.5b00933?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.biomac.5b01709?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.biomac.5b01709?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.langmuir.8b00458?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.langmuir.8b00458?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.langmuir.8b00458?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-aspubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c01819?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://www.cas.org/solutions/biofinder-discovery-platform?utm_campaign=GLO_ACD_STH_BDP_AWS&utm_medium=DSP_CAS_PAD&utm_source=Publication_ACSPubs