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[Chiho Kataoka-Hamai](https://orcid.org/0000-0002-4068-0405)

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

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[Quantifying the Thresholds of the Phospholipid Surface Density for Nonspecific Protein Adsorption and Desorption at the Triacylglycerol/Water Interface](https://mdr.nims.go.jp/datasets/f82a615b-792b-4b66-bdb5-76048fc52aa6)

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Quantifying the Thresholds of the Phospholipid Surface Density for Nonspecific Protein Adsorption and Desorption at the Triacylglycerol/Water InterfaceQuantifying the Thresholds of the Phospholipid Surface Density forNonspecific Protein Adsorption and Desorption at theTriacylglycerol/Water InterfaceChiho Kataoka-Hamai*Cite This: Langmuir 2025, 41, 25431−25438 Read OnlineACCESS Metrics & More Article Recommendations *sı Supporting InformationABSTRACT: Quantitative investigation of protein adsorption and desorption at triacylglycerol/water interfaces is of great importance for understanding the properties and functions ofintracellular lipid droplets. In this study, we investigated the adsorption and desorption ofcytochrome c, lysozyme, and bovine serum albumin at the tricaprylin/water interface coveredwith 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) using a surface pressure measurementsystem and a pendant drop tensiometer. The aim of this study was to understand the quantitativerelationships between the area per DOPC molecule and the protein adsorption and desorption.We found that the area per DOPC molecule required to preclude bovine serum albumin adsorption was much smaller than thatrequired to preclude the adsorption of cytochrome c or lysozyme. However, the area per DOPC molecule required for all of thebound protein molecules to desorb from the interface was independent of the type of protein, and this threshold area per DOPCmolecule was in good agreement with an area per lipid value previously reported for fully hydrated DOPC bilayers.■ INTRODUCTIONLipid droplets (LDs) are intracellular organelles that storeneutral lipids, mainly triacylglycerols (TAGs) and cholesterylesters, within their core. This core is surrounded by aphospholipid monolayer decorated with various proteins.1Previous studies have identified 100−150 distinct proteinslocated on the LD surface.2−4 These proteins attach to the LDsurface through at least two pathways. Depending on thepathway, the proteins are grouped into class I and class IIproteins.1 Class I proteins migrate from the endoplasmicreticulum membrane to the LDs through endoplasmicreticulum−LD bridges.5,6 In contrast, class II proteins, whichgenerally contain amphipathic helices, migrate to the LDs fromthe cytosol. Computational studies have shown that theamphipathic helices recognize and preferentially associate withthe phospholipid packing defects where neutral lipids areexposed to the cytosol.7,8The size of LDs dynamically changes through lipolysis andlipogenesis in response to the metabolic conditions.9 The sizechange is thought to regulate the protein composition on the LDsurface. For instance, when a LD shrinks, weekly associatedproteins may dissociate from the LD owing to a decrease in thenumber of packing defects and enhanced molecular crowding.1Considering this regulation mechanism of the proteincomposition, as well as the binding mechanism of class IIproteins, it is clear that phospholipid packing defects play acrucial role in the LD functions. The surface area of defectsvaries with the phospholipid surface density.10 Thus, in thepresent study, we focused on the effect of the phospholipidsurface density on protein adsorption and desorption.A recent study investigated the influence of the phospholipidsurface density on the binding of perilipins, the most abundantLD proteins.11 The results indicated that the binding of differentperilipins to LDs is differently affected by the phospholipidsurface density. However, the quantitative correlation of proteinbinding with the phospholipid surface density remains poorlyunderstood. In this study, therefore, we investigated therelationship between the phospholipid surface density at aTAG/water interface and protein adsorption and desorptionusing a quantitative approach.The phospholipid monolayer surrounding the LD core acts asa barrier to non-LD-binding proteins. However, the minimumphospholipid surface density required to prevent the adsorptionof nonspecific proteins is not well understood. Thus, in thisstudy, we investigated the nonspecific adsorption anddesorption of three globular proteins, lysozyme, cytochrome c,and bovine serum albumin (BSA), at a TAG/water interface.The main phospholipids on LDs are phosphatidylcholines(PCs).12,13 Therefore, we studied the interface covered with aPC monolayer.We determined two thresholds of the PC surface density. Atlow PC surface densities (Figure 1A(i)), many packing defectsare available for protein adsorption. As the PC surface densityincreases, the number of these defects progressively decreasesReceived: June 24, 2025Revised: August 31, 2025Accepted: September 5, 2025Published: September 12, 2025Articlepubs.acs.org/Langmuir© 2025 The Author. Published byAmerican Chemical Society25431https://doi.org/10.1021/acs.langmuir.5c03216Langmuir 2025, 41, 25431−25438This article is licensed under CC-BY 4.0Downloaded via NATL INST FOR MATLS SCIENCE (NIMS) on September 23, 2025 at 23:47:14 (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="Chiho+Kataoka-Hamai"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://pubs.acs.org/action/showCitFormats?doi=10.1021/acs.langmuir.5c03216&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?goto=articleMetrics&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?goto=recommendations&?ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?goto=supporting-info&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=tgr1&ref=pdfhttps://pubs.acs.org/toc/langd5/41/37?ref=pdfhttps://pubs.acs.org/toc/langd5/41/37?ref=pdfhttps://pubs.acs.org/toc/langd5/41/37?ref=pdfhttps://pubs.acs.org/toc/langd5/41/37?ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://pubs.acs.org?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c03216?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/and protein adsorption is eliminated (Figure 1A(ii)). We definethis critical area per PC molecule as Aa. We also determinedanother threshold by considering the case in which proteinmolecules are already bound to the interface (Figure 1B(i)). Inthis case, as the PC surface density increases, protein moleculesbegin to dissociate from the interface, and, eventually, all of theprotein molecules are displaced into the aqueous phase (Figure1B(ii)). We define this critical area per PC molecule as Ad. Aaand Ad can be different values because they are determined fordifferent protein conformations. Aa is determined for undena-tured protein in the aqueous phase whereas Ad is determined fordenatured protein at the interface.We demonstrate a method to determine Aa and Ad usingpendant drop tensiometry. Using this method, we studied theadsorption and desorption of lysozyme, cytochrome c, and BSAat a tricaprylin/water interface covered with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The results showed that theAa value for BSA was much smaller than those for lysozyme andcytochrome c, whereas the Ad values for the three proteins werealmost the same.■ EXPERIMENTAL SECTIONMaterials. DOPC (>99%) was purchased from Avanti Polar Lipids(Alabaster, AL, USA). The DOPC stock solutions were prepared inchloroform, and their concentrations were determined by phosphorusassay.14 Tricaprylin (≥99%) and cytochrome c from equine heart(≥95%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).BSA (≥98%) and lysozyme from egg white were purchased fromFUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). All ofthe chemicals were used as received. Phosphate buffered saline (PBS)buffer (137mMNaCl, 2.7mMKCl, 10mMNa2HPO4, 2mMKH2PO4,pH 7.4) was used for all of the experiments.Vesicle Preparation. The vesicles were prepared from a DOPCstock solution. Bulk chloroform was removed under a nitrogen stream.Any remaining solvent was evaporated under vacuum. PBS buffer wasadded to give a DOPC concentration of 5 mM. The samples weresubjected to 10 freeze−thaw cycles (liquid nitrogen/room temper-ature) and subsequently extruded 11 times through a polycarbonatemembrane filter (100 nm pores) using a mini-extruder (Avanti PolarLipids).Surface PressureMeasurements.The surface pressure at the air/buffer interface was measured using a platinum Wilhelmy plate(perimeter 20 mm) with a KSV NIMA system (Biolin Scientific,Gothenburg, Sweden) equipped with a Langmuir trough (35 cm × 7.5cm) and two barriers. Lipid in chloroform (∼1 mM) was spread ontothe buffer surface (23 °C). The buffer temperature was kept constant byusing a water circulator. After solvent evaporation for 2 min, DOPCmonolayers containing 0−40% tricaprylin and tricaprylin monolayerswere compressed at a rate of 15mm/min. The compression rates for theDOPC monolayers containing 50 and 60% tricaprylin were 45 and 75mm/min, respectively, because these monolayers were unstable underslow compression (Figure S1). The DOPC/tricaprylin ratios areexpressed as molar percentages.Pendant Drop Tensiometry. The interfacial tension at thetricaprylin/buffer interface was measured with a DSA25 drop shapeanalyzer (Krüss GmbH, Hamburg, Germany). A tricaprylin drop wasformed at the end of a J-shaped needle immersed in buffer in a quartzcell (base size 10 mm × 20 mm, height 45 mm). Images of the dropwere recorded under light-emitting diode lamp illumination. The dropshapes were fit to the Young−Laplace equation using ADVANCEsoftware (Krüss GmbH). For the Aa and Ad measurements, theunbound lipids and proteins were removed by flowing buffer throughthe quartz cell at a rate of 2 mL/min using a peristaltic pump and asuction pump (Figure S2). During this washing process, the continuousphase was stirred using a small magnetic stirrer (length 5 mm). Whenprotein was added to the cell for protein adsorption, the solution wasstirred with a magnetic stirrer to give a homogeneous proteinconcentration (Figure S2).Experimental Values. The reported values are the mean(±standard error) of N determinations. The definitions of the symbolsused for the measured values (Aa, Ad, Π, Π0, γ, γ0, Δγpads, γi, Δγ, γi′, Δγ′,Δγb, γi,0, and γbp) are summarized in Table S1.■ RESULTS AND DISCUSSIONDependence of the Interfacial Tension on theArea perDOPC Molecule at the Tricaprylin/Buffer Interface. Todetermine the threshold areas per DOPC molecule (Aa and Ad)from the interfacial tension data, we first determined therelationship between the area per DOPC molecule and theinterfacial tension (Figure 2) using our previously reportedmethod.15,16 First, wemeasured the surface pressures of DOPC/tricaprylin monolayers at the air/buffer interface using aLangmuir trough (Figure 2A). When the DOPC concentrationwas 40% (purple), the surface pressure smoothly increased fromnearly zero upon compression. In this region, all of thetricaprylin molecules were within the DOPC monolayer.However, when the surface pressure reached ∼22 mN/m, theslope of the curve abruptly changed. At this shoulder, tricaprylinmolecules seemed to start to move out of the monolayer to forma new bulk phase.16,17 The surface pressure data after theshoulder overlapped with other data obtained at differentDOPC concentrations. This is because the tricaprylinconcentration in the DOPC monolayer was determined by thearea per DOPC molecule independently of the DOPC/tricaprylin ratio in the lipid mixture initially spread on thebuffer surface.16,17 This one-to-one correspondence between theFigure 1. Measurement of protein adsorption and desorption at atriacylglycerol (TAG)/water interface using pendant drop tensiometry.A TAG drop is pinned at the end of a needle immersed in the aqueousphase. For detecting (A) the inhibition of protein adsorption and (B)the complete desorption of bound protein molecules, the area perphospholipidmolecule is decreased by decreasing the drop volume. (A)Measurement of the threshold area per phospholipid molecule thatprevents protein adsorption (Aa). (i) An interface with lowphospholipid surface density allows protein adsorption. (ii) Once thearea per phospholipid molecule reaches Aa, protein molecules cannotbind. (B) Measurement of the threshold area per phospholipidmolecule at which adsorbed protein molecules are completely displacedinto the aqueous phase (Ad). (i) The interface is initially covered withphospholipid and protein molecules. (ii) Once the area perphospholipid molecule reaches Ad, all of the protein molecules areeliminated.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c03216Langmuir 2025, 41, 25431−2543825432https://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c03216/suppl_file/la5c03216_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c03216/suppl_file/la5c03216_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c03216/suppl_file/la5c03216_si_001.pdfhttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c03216/suppl_file/la5c03216_si_001.pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig1&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig1&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c03216?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-astricaprylin concentration in the monolayer and the area perDOPC molecule resulted from the removal of excess tricaprylinmolecules from the monolayer to the bulk phase. Theoverlapped surface pressure data (Π) were used to correlatethe interfacial tension at the tricaprylin/buffer interface (γ) withthe area per DOPC molecule using the following equation:16= +0 0 (1)where γ0 is the interfacial tension at the pure tricaprylin/bufferinterface and Π0 is the collapse pressure of the pure tricaprylinmonolayer at the air/buffer interface. γ0 was determined to be24.3 (±0.1) mN/m (N = 7) by pendant drop tensiometry. Π0was determined to be 20.0 (±0.1) mN/m (N = 4) using theLangmuir trough (Figure 2B). Using eq 1, we converted thesurface pressure data for the air/buffer interface (Figure 2A) tothe interfacial tension data for the tricaprylin/buffer interface(Figure 2C).Protein Adsorption at the Pure Tricaprylin/BufferInterface. We investigated the adsorption of the proteins (2μM) at the clean tricaprylin/buffer interface using pendant droptensiometry (Figure 3) by measuring the interfacial tensiondifference between the clean interface and the interfaceincubated with the protein for 30 min (Δγpads). BSA showedthe largest Δγpads value, whereas lysozyme and cytochrome cshowed similar but lower Δγpads values (Table 1). These resultsindicate that BSA most strongly bound to the interface andlysozyme and cytochrome c more weakly bound to the interfacewith a similar strength.Determination of the Threshold Area Per DOPCMolecule (Aa). We determined the Aa values at which proteinadsorption was completely prevented (Figure 1A). We firstprepared DOPC-bound interfaces with different DOPC surfacedensities and then added the protein to measure the interfacialtension change due to the protein adsorption. These measure-ments were performed by two methods. In the first method(Figure 4A), a tricaprylin drop was exposed to a vesicle solution(0.07 mM lipid). After the DOPCmonolayer formed, the excessvesicles were removed by flowing buffer into the measurementcell for 10 min with gentle stirring using a magnetic stirrer(Figure 4A, wash). After the washing step, the buffer flow andstirring were stopped to measure the interfacial tension underthe static condition (γi, Figure 4A). Different γi values wereobtained by changing the vesicle adsorption time. Wesubsequently added protein to give a concentration of 2 μMwith gentle stirring for the first 3 min (Figure 4A, proteinadsorption). The protein adsorption was monitored for 30 min.We then calculated the interfacial tension difference between theγi value and the value recorded 30 min after the addition of theprotein (Δγ).The above method was used for γi > 16 mN/m (Figure 4A).For ≤16 mN/m, we used the following method (Figure 4B)because the vesicle adsorption was too slow to achieve γi ≤ 16mN/m. After DOPC monolayer formation, the drop volumewas decreased to reduce the interfacial tension to γi′ (Figure 4B,area decrease). The values of γi′ were recorded withoutremoving free DOPC vesicles. The protein was then added togive a concentration of 2 μM. The protein adsorption wasperformed for 30 min with gentle stirring for the first 3 min(Figure 4B, protein adsorption). For the data analysis, wecalculated the interfacial tension difference between the γi′ valueand the value recorded 30 min after the addition of the protein(Δγ′, Figure 4B). We did not remove the free vesicles during themeasurements because the adsorption of DOPC at the interfaceFigure 2.Determination of the dependence of the interfacial tension onthe area per 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) mole-cule at the tricaprylin/buffer interface. (A) Surface pressure data for theair/buffer interfaces covered with DOPC monolayers containing 0−60% tricaprylin. (B) Surface pressure data for tricaprylin at the air/buffer interface. (C) Dependence of the interfacial tension at thetricaprylin/buffer interface on the area per DOPC molecule. For eachDOPC concentration, we obtained three to four sets of data (differentcolors).Figure 3. Interfacial tension data for tricaprylin drops exposed to purebuffer (black), 2 μM lysozyme (red), 2 μM cytochrome c (blue), and 2μM bovine serum albumin (BSA) (green).Table 1. Δγpads, Aa, and Ad for Lysozyme, Cytochrome c, andBSA Adsorption at the Tricaprylin/Buffer Interface (N = 3−8)protein Δγpads (mN/m) Aa (Å2) Ad (Å2)lysozyme −4.7 (±0.3) 88.3 (±1.4) 66.9 (±0.3)cytochrome c −4.6 (±0.1) 87.4 (±0.04) 66.7 (±0.6)BSA −10.1 (±0.1) 69.8 (±0.3) 68.1 (±0.5)Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c03216Langmuir 2025, 41, 25431−2543825433https://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig2&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig3&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig3&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c03216?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-aswith interfacial tension of ≤16 mN/m was relatively slow. Toextract the interfacial tension change caused solely by proteinadsorption (Δγ), we subtracted the interfacial tension changedue to DOPC adsorption from Δγ′ as follows:= b (2)where Δγb is the interfacial tension change predicted to occurowing to the adsorption of DOPC. Δγb was measured withoutprotein after the drop volume decrease. The Δγb − γi′relationship was fitted to a linear function (Figure 4C, line).This linear approximation was used to estimate the Δγb value ata given γi′ value.Using the results obtained using the above two methods(Figure 4A−C), we determined the Δγ values at different γi (orγi′) values for the three proteins (Figure 4D, squares). The datawere well fitted to linear relationships (Figure 4D, lines), whichwere used to calculate the interfacial tension values at Δγ = 0(γi,0, Figure 4D). γi,0 is the interfacial tension at an area perDOPC molecule of Aa (Figure 1A). Therefore, we determinedthe Aa values from the γi,0 data by using the interfacial tensiondependence on the area per DOPC molecule in Figure 2C. Theresults showed that Aa was the smallest for BSA, and the Aavalues for lysozyme and cytochrome c were larger but similar(Table 1). The results suggest that BSA is the most capable ofadsorbing to tricaprylin exposed to the aqueous phase throughDOPC packing defects, whereas lysozyme and cytochrome c areless capable of adsorbing to tricaprylin. These results wereconsistent with the Δγpads data (Table 1), which showed thatBSA was the most strongly bound to the pure interface.Determination of the Threshold Area per DOPCMolecule (Ad) for BSA. We investigated the threshold areaper DOPC molecule at which the bound protein completelydesorbed from the interface (Ad, Figure 1B). We first studiedBSA (Figure 5A). The experiments consisted of the followingtwo processes: the formation of the interface that bound DOPCand BSA (Figure 1B(i)) and decreasing the surface area todesorb BSA (Figure 1B(ii)). In the first process (Figure 5A),vesicle adsorption (0.07 mM DOPC) was carried out, followedFigure 4. Determination of the threshold area per DOPC molecule(Aa). Twomethods were used to measure the interfacial tension change(Δγ) caused by the adsorption of the protein (2 μM) at the DOPC-covered tricaprylin/buffer interface for 30 min. (A) Method used forobtaining an interfacial tension of >16 mN/m before proteinadsorption. The data for lysozyme are shown. After the adsorption ofDOPC vesicles (0.07 mM lipid), the excess lipids were removed tomeasure γi (wash), and the protein was adsorbed for 30 min (proteinadsorption). The interfacial tension decrease after the addition of theprotein is denoted as Δγ. (B, C) Method used for obtaining aninterfacial tension of ≤16 mN/m before protein adsorption. The datafor BSA are shown. (B) After DOPC adsorption (0.07 mM lipid), thesurface area of the tricaprylin drop was decreased to obtain the γi′ value(area decrease). Without removing the unbound vesicles, the protein (2μM) was adsorbed for 30 min (protein adsorption). The interfacialtension decrease after the addition of the protein is denoted as Δγ′. (C)Interfacial tension change (Δγb) in the absence of the protein afterdecreasing the drop volume in method B. The data (dots) are fitted to alinear equation (line). The Δγb value at a given γi′ value was estimatedfrom this linear relationship. Δγ was obtained by subtracting Δγb fromΔγ′ (eq 2). (D) Determination of the interfacial tension before proteinaddition that gives Δγ = 0 (γi,0). The arrow indicates γi,0 for the BSAdata. The area per DOPC molecule at an interfacial tension of γi,0 is Aa.Figure 5. Determination of the threshold area per DOPC molecule(Ad) for BSA. (A) Formation of a tricaprylin/buffer interface adsorbingDOPC and BSA. After DOPC adsorption (0.07 mM lipid), theinterfacial tension was decreased to γbp by decreasing the volume of thetricaprylin drop (area decrease). The interface was incubated with BSA(2 μM) for 10 min (BSA adsorption), followed by washing with bufferfor 10 min (wash). (B) Measurement of the interfacial tension (solidcircles) with decreasing drop volume (open circles) for the sample in(A). The measurement was continued until the tricaprylin dropdetached from the needle tip. (C)Dependence of the interfacial tensionon the area per DOPC molecule (red) obtained from the data in (B).The data for the protein-free interface in Figure 2C (gray data points)and their fitting curve (blue) are also given. The Ad value is the area perDOPC molecule at which the red and blue curves start to overlap(insert).Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c03216Langmuir 2025, 41, 25431−2543825434https://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig4&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig5&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig5&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c03216?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asby decreasing the drop volume (Figure 5A, area decrease) toachieve a desired interfacial tension value (γbp). This interfacewas incubated with BSA (2 μM) for 10 min (Figure 5A, BSAadsorption) with gentle stirring for the first 3 min. The numberof adsorbed DOPC molecules on the interface was estimatedfrom the γbp value using the interfacial tension data in Figure 2C.This estimated number of adsorbed DOPC molecules wasassumed to be constant throughout the experiment. During theadsorption of BSA at the interface, the adsorption of DOPC wasnegligible because the γbp values were small (<10 mN/m). Afterthe adsorption of BSA, the unbound BSA and vesicles wereremoved by flowing buffer through the measurement cell for 10min with gentle stirring (Figure 5A, wash).For this interface, we measured the interfacial tension (Figure5B, solid circles) while decreasing the drop volume (Figure 5B,open circles) at a rate of 0.25 or 0.5 μL/s. We simultaneouslyrecorded the drop surface area, which was used to calculate thearea per DOPC molecule. Specifically, we divided the dropsurface area by the number of bound DOPC moleculesestimated from the γbp value. The interfacial tension data(Figure 5B, solid circles) were then plotted against the area perDOPC molecule (Figure 5C, red). The results showed that forarea per DOPCmolecule of >∼68 Å2, the interfacial tension wassmaller than that obtained for the protein-free interface (Figure5C, gray data points, blue fitting curve) owing to adsorbed BSA.For area per DOPC molecule of ≤∼68 Å2, however, the twocurves recorded with (red) and without protein (gray datapoints, blue fitting curve) overlapped. The data indicated thatthe bound protein molecules progressively desorbed withdecreasing area per DOPC molecule, and the protein was finallycompletely removed from the interface at an area per DOPCmolecule of ∼68 Å2. This value is Ad (Table 1).Dependence of the Interfacial Tension on theArea PerDOPC Molecule after BSA Desorption. The above datasuggested that BSA completely desorbed from the interface bydecreasing the drop surface area. We confirmed this notion byexperiments consisting of the following three processes. In thefirst process, we formed the interface that adsorbed DOPC andBSA, as described for the data in Figure 5A. In the secondprocess, the drop volume was decreased (Figure 6A, step 1, red)until complete BSA desorption appeared to occur (Figure 6B,red). The desorbed BSA molecules were removed by flowingbuffer through the measurement cell for 5 min with gentlestirring. In the final process, the interface was subjected to a dropvolume change at a rate of 1 μL/s (Figure 6A, steps 2−7). Theinterfacial tension data obtained during these steps (Figure 6B,black, pink, light blue, orange, purple, and green) were in goodagreement with the curve obtained for the protein-free DOPCmonolayer (Figure 6B, gray data points, blue fitting curve).These results verified the complete removal of BSA from theinterface during step 1.Determination of the Threshold Area per DOPCMolecule (Ad) Values for Lysozyme and Cytochrome C.We next determined the Ad value for lysozyme. The experimentsconsisted of the following three processes: the formation of alysozyme-bound DOPC monolayer, shrinking the drop todesorb lysozyme, and drop expansion and shrinkage cycles tomeasure the dependence of the interfacial tension on the areaper DOPC molecule. The first process (monolayer formation)was performed by one of the following methods. To obtain γbp <18 mN/m, we used the method used for BSA (Figure 5A). Toobtain γbp > 18 mN/m, we used the following method (Figure7A). After vesicle adsorption, the unbound lipids were removedby flowing buffer through the measurement cell for 10 min withstirring (Figure 7A, 1st wash). The interface was subsequentlyincubated with the protein (2 μM) for 10 min with stirring(Figure 7A, lysozyme adsorption), followed by the removal ofthe free protein molecules by flowing buffer through themeasurement cell for 10 min with stirring (Figure 7A, 2ndwash).The second process (drop shrinkage) and final process (dropshrinkage/expansion cycles) were performed in similar ways tothose for BSA (Figures 5B,C and 6). For lysozyme desorption(Figure 7B, red, step 1), the volume of the drop to which DOPCand lysozyme adsorbed was decreased at a rate of 0.25 or 0.5 μL/s. The desorbed protein molecules were removed by flowingbuffer for 5 min with gentle stirring. The interfacial tensionduring step 1 (Figure 7C, red) matched the data obtainedwithout protein (Figure 7C, gray data points, blue fitting curve)at an area per DOPC molecule of ∼67 Å2. This area per DOPCmolecule is considered to be the Ad value of lysozyme. After step1, the drop volume was repeatedly increased and decreased at arate of 1 μL/s (Figure 7B, blue, steps 2−5). The interfacialtension data during these steps (Figure 7C, black, pink, lightblue, and orange) were in good agreement with those obtainedfor the pureDOPCmonolayer (Figure 7C, gray data points, bluefitting curve). The results demonstrated that lysozyme waseliminated from the interface during step 1. We also determinedthe Ad value for cytochrome c in a similar manner (Figure 7D).The results showed that the Ad values for the three differentproteins were similar despite the large differences in the Aavalues (Figure 7E and Table 1).Discussion. The three proteins have different properties(Table 2), and they therefore have different interactions withtricaprylin and DOPC. We expect that these differentinteractions are the cause of the different Δγpads and Aa values(Table 1). However, detailed analyses are difficult because of theFigure 6. Interfacial tension data obtained for a tricaprylin/bufferinterface that adsorbed DOPC and BSA. (A) Decreasing the dropvolume to desorb BSA (red, step 1), followed by changing the volumeto investigate the dependence of the interfacial tension on the area perDOPC molecule (blue, steps 2−7). The drop volume during thesesuccessive processes is plotted against the relative time. (B)Dependence of the interfacial tension on the area per DOPC moleculeobtained during step 1 (red) and the subsequent steps 2−7 (black, pink,light blue, orange, purple, and green). The data for the protein-freeinterface in Figure 2C (gray data points) and their fitting curve (blue)are also shown.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c03216Langmuir 2025, 41, 25431−2543825435https://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig6&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig6&ref=pdfpubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c03216?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asfollowing reason. The total energy of the adsorption of proteinsat oil/water interfaces is mainly affected by the contributionsfrom electrostatic interactions, solvation forces, van der Waalsinteractions, and conformational changes.18,19 The first term(electrostatic interactions) needs to be considered because oil/water interfaces are generally negatively charged.20−23 Thesecond term (solvation forces) is repulsive or attractivedepending on the interacting surfaces.19 For example, whenhydrophilic surfaces distributed on the protein interact with thehydrophilic DOPC headgroups, the protein experiences arepulsive force called the hydration force.19 However, whenhydrophobic surfaces distributed locally on the protein interactwith the hydrophobic tricaprylin surfaces exposed throughDOPC packing defects, the protein experiences an attractiveforce.19 The final contribution (conformational changes) arisesfrom the conformational rearrangement within the protein at theinterface. After the conformational changes, the othercontributions from the electrostatic interactions, solvationforces, and van der Waals interactions also change. Because ofthis complexity, it is not possible to infer the factors thatsignificantly influence the protein adsorption for the pure andthe DOPC-covered interfaces.As described above, the Aa values for BSA and the otherproteins were different, probably because of the differentprotein−tricaprylin and protein−DOPC interactions. However,the Ad value was independent of the type of protein (Table 1).Interestingly, the Ad values were very close to the area per lipidvalue previously reported for fully hydrated DOPC bilayers(67.4 Å2 at 30 °C).24 This suggests that the three proteins lostsurface activity when the area per DOPC molecule decreased tothe value for fully hydrated bilayers.■ CONCLUSIONSWe have reported a quantitative method to measure thedependence of protein adsorption and desorption at thetricaprylin/buffer interface on the area per DOPC molecule.We showed that the area per DOPC molecule required toprevent BSA adsorption was much smaller than that required toprevent lysozyme or cytochrome c adsorption. However, thearea per DOPC molecule values required to remove all of theadsorbed protein molecules from the interface were almost thesame, and these values were in good agreement with the area perlipid value reported for fully hydrated DOPC bilayers.Figure 7. Determination of the threshold areas per DOPC molecule (Ad) for (A−C) lysozyme and (D) cytochrome c, and (E) comparison of theresults for the different proteins. (A) Formation of a tricaprylin/buffer interface covered with DOPC and lysozyme. The process consisted of DOPCadsorption (0.07 mM lipid), DOPC removal (first wash), lysozyme adsorption, and lysozyme removal (second wash). (B, C) Interfacial tensionmeasurements for the interface in (A). First, the drop volume (B, red, step 1) was decreased to measure the interfacial tension change caused by thedecrease in the area per DOPC molecule (C, red). Second, the drop volume was varied (B, blue, steps 2−5) to measure the interfacial tension changecaused by cycles of an increase and a decrease in the area per DOPC molecule (C, black, pink, light blue, and orange). (D) Dependence of theinterfacial tension on the area per DOPCmolecule obtained for cytochrome c. Steps 1−5 (red, black, pink, light blue, and orange) were similar to thosefor the lysozyme data in B. (E) Comparison of the results for the different proteins. The BSA data in Figure 5C are overlaid with the lysozyme (C) andthe cytochrome c (D) data. (C−E)Data in the absence of protein (Figure 2C, gray data points) and their fitting curve (Figure 2C, blue) are also shown.Table 2. Properties of the Proteinslysozyme cytochrome c BSAmolecular weight (kDa) 14.3 12.4 66.5molecular size (nm3) 3 × 3 × 4.525 2.5 × 2.5 × 3.726 4 × 4 × 1427isoelectric point 1128 1029 530net charge at pH 731 +7 +6 −18surface hydrophobicity31 7.49 10.33total hydrophobicity31 970 1110 1120instability index 16.932 15.56a 40.1132aCalculated by ProtParam software (https://web.expasy.org/protparam). Proteins with instability indexes of <40 are predictedto be stable.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c03216Langmuir 2025, 41, 25431−2543825436https://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig7&ref=pdfhttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?fig=fig7&ref=pdfhttps://web.expasy.org/protparamhttps://web.expasy.org/protparampubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c03216?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-as■ ASSOCIATED CONTENT*sı Supporting InformationThe Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216.Surface pressure measurements of DOPC monolayerswith high tricaprylin concentrations at the air/bufferinterface, experimental setup for pendant drop tensiom-etry, and summary of the symbols used for the measuredvalues (PDF)■ AUTHOR INFORMATIONCorresponding AuthorChiho Kataoka-Hamai − Research Center for Macromoleculesand Biomaterials, National Institute for Materials Science,Tsukuba, Ibaraki 305-0044, Japan; orcid.org/0000-0002-4068-0405; Email: kataoka.chiho@nims.go.jpComplete contact information is available at:https://pubs.acs.org/10.1021/acs.langmuir.5c03216NotesThe author declares no competing financial interest.■ ACKNOWLEDGMENTSThis work was supported by a KAKENHI grant (22K05179)from the Japan Society for the Promotion of Science. We thankEdanz (https://jp.edanz.com/ac) for editing a draft of thismanuscript.■ REFERENCES(1) Olarte, M. J.; Swanson, J. M. J.; Walther, T. C.; Farese, R. V. TheCYTOLD and ERTOLD Pathways for Lipid Droplet−ProteinTargeting. Trends Biochem. Sci. 2022, 47, 39−51.(2) Bersuker, K.; Peterson, C. W. H.; To, M.; Sahl, S. J.; Savikhin, V.;Grossman, E. A.; Nomura, D. K.; Olzmann, J. A. A Proximity LabelingStrategy Provides Insights into the Composition andDynamics of LipidDroplet Proteomes. Dev. Cell 2018, 44, 97−112.(3) Olzmann, J. A.; Carvalho, P. Dynamics and Functions of LipidDroplets. Nat. Rev. Mol. Cell Biol. 2019, 20, 137−155.(4) Rogers, S.; Gui, L.; Kovalenko, A.; Zoni, V.; Carpentier, M.; Ramji,K.; Ben Mbarek, K.; Bacle, A.; Fuchs, P.; Campomanes, P.; et al.Triglyceride Lipolysis Triggers Liquid Crystalline Phases in LipidDroplets and Alters the LD Proteome. J. Cell Biol. 2022, 221,No. e202205053.(5) Olarte, M. J.; Kim, S.; Sharp, M. E.; Swanson, J. M. J.; Farese, R. V.;Walther, T. C. Determinants of Endoplasmic Reticulum-to-LipidDroplet Protein Targeting. Dev. Cell 2020, 54, 471−487.(6) Wilfling, F.; Wang, H. J.; Haas, J. T.; Krahmer, N.; Gould, T. J.;Uchida, A.; Cheng, J. X.; Graham, M.; Christiano, R.; Fröhlich, F.; et al.Triacylglycerol Synthesis Enzymes Mediate Lipid Droplet Growth byRelocalizing from the ER to Lipid Droplets. Dev. Cell 2013, 24, 384−399.(7) Prévost, C.; Sharp, M. E.; Kory, N.; Lin, Q. Q.; Voth, G. A.; Farese,R. V.; Walther, T. C. Mechanism and Determinants of AmphipathicHelix-Containing Protein Targeting to Lipid Droplets. Dev. Cell 2018,44, 73−86.(8) Rowe, E. R.; Mimmack, M. L.; Barbosa, A. D.; Haider, A.; Isaac, I.;Ouberai, M. M.; Thiam, A. R.; Patel, S.; Saudek, V.; Siniossoglou, S.;et al. Conserved Amphipathic Helices Mediate Lipid Droplet Targetingof Perilipins 1−3. J. Biol. Chem. 2016, 291, 6664−6678.(9) Paar, M.; Jüngst, C.; Steiner, N. A.; Magnes, C.; Sinner, F.; Kolb,D.; Lass, A.; Zimmermann, R.; Zumbusch, A.; Kohlwein, S. D.; et al.Remodeling of Lipid Droplets during Lipolysis and Growth inAdipocytes. J. Biol. Chem. 2012, 287, 11164−11173.(10) Bacle, A.; Gautier, R.; Jackson, C. L.; Fuchs, P. F. J.; Vanni, S.Interdigitation Between Triglycerides and Lipids Modulates SurfaceProperties of Lipid Droplets. Biophys. J. 2017, 112, 1417−1430.(11) Dias Araujo, A. R.; Bello, A. A.; Bigay, J.; Franckhauser, C.;Gautier, R.; Cazareth, J.; Kovács, D.; Brau, F.; Fuggetta, N.; Čopic,̌ A.;et al. Surface Tension-Driven Sorting of Human Perilipins on LipidDroplets. J. Cell Biol. 2024, 223, No. e202403064.(12) Tauchi-Sato, K.; Ozeki, S.; Houjou, T.; Taguchi, R.; Fujimoto, T.The Surface of Lipid Droplets is a Phospholipid Monolayer with aUnique Fatty Acid Composition. J. Biol. Chem. 2002, 277, 44507−44512.(13) Blouin, C.M.; Le Lay, S.; Eberl, A.; Köfeler, H. C.; Guerrera, I. C.;Klein, C.; Le Liepvre, X.; Lasnier, F.; Bourron, O.; Gautier, J. F.; et al.Lipid Droplet Analysis in Caveolin-Deficient Adipocytes: Alterations inSurface Phospholipid Composition and Maturation Defects. J. LipidRes. 2010, 51, 945−956.(14) Chen, P. S.; Toribara, T. Y.; Warner, H. Microdetermination ofPhosphorus. Anal. Chem. 1956, 28, 1756−1758.(15) Kataoka-Hamai, C.; Kawakami, K. Determination of theCoverage of Phosphatidylcholine Monolayers Formed at SiliconeOil−Water Interfaces by Vesicle Fusion. J. Phys. Chem. B 2020, 124,8719−8727.(16) Kataoka-Hamai, C.; Kawakami, K. Determining the Dependenceof Interfacial Tension on Molecular Area for Phospholipid MonolayersFormed at Silicone Oil−Water and Tricaprylin−Water Interfaces byVesicle Fusion. Langmuir 2021, 37, 7527−7535.(17) Mitsche, M. A.; Wang, L. B.; Small, D. M. Adsorption of EggPhosphatidylcholine to an Air/Water and Triolein/Water BubbleInterface: Use of the 2-Dimensional Phase Rule To Estimate theSurface Composition of a Phospholipid/Triolein/Water Surface as aFunction of Surface Pressure. J. Phys. Chem. B 2010, 114, 3276−3284.(18) Sengupta, T.; Razumovsky, L.; Damodaran, S. Energetics ofProtein−Interface Interactions and Its Effect on Protein Adsorption.Langmuir 1999, 15, 6991−7001.(19) Israelachvili, J. N.; Mcguiggan, P. M. Forces between Surfaces inLiquids. Science 1988, 241, 795−800.(20) Marinova, K. G.; Alargova, R. G.; Denkov, N. D.; Velev, O. D.;Petsev, D. N.; Ivanov, I. B.; Borwankar, R. P. Charging of Oil−WaterInterfaces due to Spontaneous Adsorption of Hydroxyl Ions. Langmuir1996, 12, 2045−2051.(21) Roger, K.; Cabane, B. Why Are Hydrophobic/Water InterfacesNegatively Charged? Angew. Chem., Int. Ed. 2012, 51, 5625−5628.(22) Samson, J.-S.; Scheu, R.; Smolentsev, N.; Rick, S. W.; Roke, S.Sum Frequency Spectroscopy of the Hydrophobic Nanodroplet/WaterInterface: Absence of Hydroxyl Ion and Dangling OH Bond Signatures.Chem. Phys. Lett. 2014, 615, 124−131.(23) Vácha, R.; Rick, S. W.; Jungwirth, P.; de Beer, A. G. F.; de Aguiar,H. B.; Samson, J. S.; Roke, S. The Orientation and Charge of Water atthe Hydrophobic Oil Droplet−Water Interface. J. Am. Chem. Soc. 2011,133, 10204−10210.(24) Kucěrka, N.; Nagle, J. F.; Sachs, J. N.; Feller, S. E.; Pencer, J.;Jackson, A.; Katsaras, J. Lipid Bilayer Structure Determined by theSimultaneous Analysis of Neutron and X-Ray Scattering Data. Biophys.J. 2008, 95, 2356−2367.(25) Czeslik, C.; Winter, R. Effect of Temperature on theConformation of Lysozyme Adsorbed to Silica Particles. Phys. Chem.Chem. Phys. 2001, 3, 235−239.(26) Kao, K. C.; Lee, C. H.; Lin, T. S.; Mou, C. Y. Cytochrome CCovalently Immobilized on Mesoporous Silicas as a Peroxidase:Orientation Effect. J. Mater. Chem. 2010, 20, 4653−4662.(27) Peters, T. J. All About Albumin: Biochemistry, Genetics, andMedical Applications; Academic Press, 1996.(28) Alderton, G.; Ward, W. H.; Fevold, H. L. Isolation of Lysozymefrom Egg White. J. Biol. Chem. 1945, 157, 43−58.(29) Hristova, S. H.; Zhivkov, A. M. Isoelectric Point of Free andAdsorbed Cytochrome C Determined by Various Methods. ColloidsSurf. B: Biointerfaces 2019, 174, 87−94.(30) Salis, A.; Boström, M.; Medda, L.; Cugia, F.; Barse, B.; Parsons,D. F.; Ninham, B. W.; Monduzzi, M. Measurements and TheoreticalLangmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c03216Langmuir 2025, 41, 25431−2543825437https://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?goto=supporting-infohttps://pubs.acs.org/doi/suppl/10.1021/acs.langmuir.5c03216/suppl_file/la5c03216_si_001.pdfhttps://pubs.acs.org/action/doSearch?field1=Contrib&text1="Chiho+Kataoka-Hamai"&field2=AllField&text2=&publication=&accessType=allContent&Earliest=&ref=pdfhttps://orcid.org/0000-0002-4068-0405https://orcid.org/0000-0002-4068-0405mailto:kataoka.chiho@nims.go.jphttps://pubs.acs.org/doi/10.1021/acs.langmuir.5c03216?ref=pdfhttps://jp.edanz.com/achttps://doi.org/10.1016/j.tibs.2021.08.007https://doi.org/10.1016/j.tibs.2021.08.007https://doi.org/10.1016/j.tibs.2021.08.007https://doi.org/10.1016/j.devcel.2017.11.020https://doi.org/10.1016/j.devcel.2017.11.020https://doi.org/10.1016/j.devcel.2017.11.020https://doi.org/10.1038/s41580-018-0085-zhttps://doi.org/10.1038/s41580-018-0085-zhttps://doi.org/10.1083/jcb.202205053https://doi.org/10.1083/jcb.202205053https://doi.org/10.1016/j.devcel.2020.07.001https://doi.org/10.1016/j.devcel.2020.07.001https://doi.org/10.1016/j.devcel.2013.01.013https://doi.org/10.1016/j.devcel.2013.01.013https://doi.org/10.1016/j.devcel.2017.12.011https://doi.org/10.1016/j.devcel.2017.12.011https://doi.org/10.1074/jbc.M115.691048https://doi.org/10.1074/jbc.M115.691048https://doi.org/10.1074/jbc.M111.316794https://doi.org/10.1074/jbc.M111.316794https://doi.org/10.1016/j.bpj.2017.02.032https://doi.org/10.1016/j.bpj.2017.02.032https://doi.org/10.1083/jcb.202403064https://doi.org/10.1083/jcb.202403064https://doi.org/10.1074/jbc.M207712200https://doi.org/10.1074/jbc.M207712200https://doi.org/10.1194/jlr.M001016https://doi.org/10.1194/jlr.M001016https://doi.org/10.1021/ac60119a033?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/ac60119a033?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.jpcb.0c06310?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.jpcb.0c06310?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.jpcb.0c06310?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.langmuir.1c01021?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.langmuir.1c01021?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.langmuir.1c01021?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/acs.langmuir.1c01021?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/jp908730t?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/jp908730t?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/jp908730t?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/jp908730t?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/jp908730t?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/la990235s?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/la990235s?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1126/science.241.4867.795https://doi.org/10.1126/science.241.4867.795https://doi.org/10.1021/la950928i?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/la950928i?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1002/anie.201108228https://doi.org/10.1002/anie.201108228https://doi.org/10.1016/j.cplett.2014.09.034https://doi.org/10.1016/j.cplett.2014.09.034https://doi.org/10.1021/ja202081x?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1021/ja202081x?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1529/biophysj.108.132662https://doi.org/10.1529/biophysj.108.132662https://doi.org/10.1039/b005900phttps://doi.org/10.1039/b005900phttps://doi.org/10.1039/b925331ahttps://doi.org/10.1039/b925331ahttps://doi.org/10.1039/b925331ahttps://doi.org/10.1016/S0021-9258(17)41625-5https://doi.org/10.1016/S0021-9258(17)41625-5https://doi.org/10.1016/j.colsurfb.2018.10.080https://doi.org/10.1016/j.colsurfb.2018.10.080https://doi.org/10.1021/la2024605?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-aspubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c03216?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-asInterpretation of Points of Zero Charge/Potential of BSA Protein.Langmuir 2011, 27, 11597−11604.(31) Wahlgren, M. C.; Paulsson, M. A.; Arnebrant, T. Adsorption ofGlobular Model Proteins to Silica and Methylated Silica Surfaces andTheir Elutability by Dodecyltrimethylammonium Bromide. ColloidsSurf. A: Physicochem. Eng. Asp. 1993, 70, 139−149.(32) Beverung, C. J.; Radke, C. J.; Blanch, H.W. Protein Adsorption atthe Oil/Water Interface: Characterization of Adsorption Kinetics byDynamic Interfacial Tension Measurements. Biophys. Chem. 1999, 81,59−80.Langmuir pubs.acs.org/Langmuir Articlehttps://doi.org/10.1021/acs.langmuir.5c03216Langmuir 2025, 41, 25431−2543825438https://doi.org/10.1021/la2024605?urlappend=%3Fref%3DPDF&jav=VoR&rel=cite-ashttps://doi.org/10.1016/0927-7757(93)80282-Jhttps://doi.org/10.1016/0927-7757(93)80282-Jhttps://doi.org/10.1016/0927-7757(93)80282-Jhttps://doi.org/10.1016/S0301-4622(99)00082-4https://doi.org/10.1016/S0301-4622(99)00082-4https://doi.org/10.1016/S0301-4622(99)00082-4pubs.acs.org/Langmuir?ref=pdfhttps://doi.org/10.1021/acs.langmuir.5c03216?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