Impact of the Degree of Ethoxylation of the Ethoxylated Polysorbate

Jul 21, 2014 - The results presented show that varying the degree of ethoxylation of the polysorbate surfactant changes the interaction between the su...
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Impact of the Degree of Ethoxylation of the Ethoxylated Polysorbate Nonionic Surfactant on the Surface Self-Assembly of HydrophobinEthoxylated Polysorbate Surfactant Mixtures Jeffrey Penfold,*,†,‡ Robert K. Thomas,† Peixun Li,† Jordan T. Petkov,§ Ian Tucker,§ Andrew R. Cox,§ Nick Hedges,§ John R. P. Webster,‡ and Maximilian W. A. Skoda‡ †

Physical and Theoretical Chemistry Laboratory, Oxford University, South Parks Road, Oxford OX1 2JD, United Kingdom STFC, Rutherford Appleton Laboratory, Chilton, Didcot OX11 0QX, United Kingdom § Unilever Research and Development Laboratory, Port Sunlight, Quarry Road East, Bebington, Wirral CH62 4ZD, United Kingdom ‡

S Supporting Information *

ABSTRACT: Neutron reflectivity measurements have been used to study the surface adsorption of the polyethylene sorbitan monostearate surfactant, with degrees of ethoxylation varying from 3 to 20 ethylene oxide groups, with the globular protein hydrophobin. The surface interaction between the ethoxylated polysorbate nonionic surfactants and the hydrophobin results in self-assembly at the air−solution interface in the form of a well-defined layered surface structure. The surface interaction arises from a combination of the hydrophobic interaction between the surfactant alkyl chain and the hydrophobic patch on the surface of the hydrophobin, and the hydrophilic interaction between the ethoxylated sorbitan headgroup and the hydrophilic regions on the surface of the hydrophobin. The results presented show that varying the degree of ethoxylation of the polysorbate surfactant changes the interaction between the surfactant and the hydrophobin and the packing, and hence the evolution in the resulting surface structure. The optimal degree of ethoxylation for multilayer formation is over a broad range, from of order 6 to 17 ethylene oxide groups, and for degrees of ethoxylation of 3 and 20 only monolayer adsorption of either the surfactant or the hydrophobin is observed.



benefit agents, including perfumes, flavors, and antibacterial/ antimicrobial agents. In this Article, we explore the extent of surface ordering in some specific protein−surfactant mixtures. Protein−surfactant interactions are of central importance in many biomedical and biotechnological applications,17 and are important in personal care products, cosmetics, and food formulations. Protein−surfactant interactions18 and especially the surface properties of protein−surfactant mixtures19−22 have been extensively studied. Protein−surfactant surface adsorption properties resemble in many cases those encountered in polyelectrolyte−surfactant mixtures,4 resulting in enhanced adsorption due to surface interactions, and in competitive

INTRODUCTION The spontaneous self-assembly in dilute surfactant solutions at interfaces to form highly ordered layered structures has been recently reported for a range of different systems. It has been demonstrated in ionic/nonionic surfactant mixtures1,2 and in anionic surfactants3 by the addition of multivalent counterions, such as Ca2+ or Al3+, and in different polyelectrolyte/ionic surfactant mixtures.4 It has also been reported in other dilute mixed surfactant systems at the air−water interface.5,6 Surface ordering in surfactant-based systems is more widely observed in lung surfactants7−10 and in more concentrated surfactant systems.11−16 The surface multilayer structures can exhibit quite unusual and extreme wetting properties, and have potential for applications in soft or biolubrication. The enhanced adsorption associated with the surface multilayer structures has potential for enhanced delivery and retention of a range of © 2014 American Chemical Society

Received: June 10, 2014 Revised: July 21, 2014 Published: July 21, 2014 9741

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adsorption at higher surfactant concentrations.21,23 The changes in the patterns of adsorption broadly correlate with surface tension23 and surface rheology.19 Gunning et al.22 described protein−surfactant competitive adsorption as an orogenic mechanism associated with the disruption of protein networks at the surface. In this general context, the globular protein hydrophobin has attracted much recent interest,24−26 and its surface interaction with surfactants is the subject of this study. Hydrophobin is a small (∼7−10 kDa) globular protein, which is compact and robust due to four intramolecular disulfide bridges;24,25 see Figure 1a. It has a well-defined surface patch

of this Article is to explore in more detail how the structure of the ethoxylated polysorbate surfactant, through varying the degree of ethoxylation, affects the conditions for surface ordering. This has potentially important implications in the context of the application of protein−surfactant mixtures in foods, cosmetics, and personal care products. The ethoxylated polysorbate surfactants, more commonly encountered as the Tween series of nonionic surfactants, are an important class of surfactant. The Tween surfactants are ethoxylated sorbitan esters of different fatty acids. The Tween 20, 40, 60, and 80 all have 20 ethylene oxide groups attached to the sorbitan headgroup and a single alkyl chain, lauric, palmitic, stearic, or oleic; see Figure 1b. The specific interaction between the Tween surfactants and a range of proteins has led to the exploitation of Tween−protein mixtures in foam and emulsion stabilization,28−33 in the development of protein resistant surfaces and soft lubrication,34,35 and in the manipulation of protein folding and membrane protein extraction.36−40 The specific interaction between the Tween surfactants and proteins has also given rise to competitive and synergistic interactions at interfaces and in self-assembly.41−46 Here, we explore a different but related aspect associated with self-assembly at the interface. This study extends the earlier reported observation of surface self-assembly in ethoxylated polysorbate surfactant−hydrophobin mixtures at the air−water interface.27 It reports the impact of varying the degree of ethoxylation of the ethoxylated polysorbate surfactant on the surface self-assembly in ethoxylated polysorbate surfactant−hydrophobin mixtures, obtained from a series of neutron reflectivity, NR, measurements. Results are presented for mixtures of hydrophobin and a range of ethoxylated sorbitan monostearate surfactants, with degrees of ethoxylation that vary from 3 to 20. Results are also presented for a fixed hydrophobin concentration and variable surfactant concentrations and vice versa.



Figure 1. Schematic representation of (a) hydrophobin and (b) Tween 60.

EXPERIMENTAL DETAILS

a. Materials. The hydrophobin, class II hydrophobin, HFBII, was produced by yeast fermentation at an external fermentation company (BAC), and purified by a two-phase extraction, as described in more detail elsewhere.23 The purified material was freeze dried before preparation in aqueous solution. The solutions were prepared in degassed water to minimize disadvantageous bubble formation and the formation of large aggregates. A range of different ethoxylated polysorbate surfactants were synthesized by ethoxylation, using deuterium labeled ethylene oxide, of the sorbitan ester, sorbitan monostearate.27,49 The sorbitan monostearate was supplied by SigmaAldrich, and the synthesis and characterization are described in detail elsewhere.27,47 For consistency with the Tween nomenclature (the standard Tween surfactants have 20 ethylene oxide groups attached to the sorbitan headgroup), the surfactants synthesized here are abbreviated as PESn60, where n is the degree of ethoxylation. The data presented here are for n = 3, 6, 7, 9, 12, 13, 16.5, and 20. High-purity water (Elga Ultrapure) was used, and the D2O was obtained from SigmaAldrich. All of the glassware and Teflon troughs were cleaned in alkali detergent (Decon90) and rinsed thoroughly in high-purity water. b. Neutron Reflectivity. The neutron reflectivity, NR, measurements were made at the air−water interface on the INTER and SURF reflectometers at the ISIS neutron source.48 On INTER the reflectivity R(Q) was measured over a Q range of 0.03−0.3 Å−1, which was covered using a grazing angle of incidence of 2.3° and neutron wavelengths from ∼1 to 15 Å. On SURF the measurements were made over a slightly narrower Q range, 0.045−0.5 Å−1, using neutron wavelengths from ∼1 to 7 Å and an angle of incidence of 1.5°. The reflectivity, R(Q), was calibrated with respect to the direct beam intensity and the reflection from a D2O surface. The measurements were made in sealed Teflon troughs at 25 °C with sample volumes ∼25 mL. Each neutron reflectivity

and is hence highly surface active, resulting in surface layers that are highly adhesive and have unusually high surface elasticity values.26 Recent studies on the coadsorption of hydrophobin with a range of conventional surfactants23 investigated the competitive adsorption between the two. At low surfactant concentrations, the surface adsorption is dominated by the hydrophobin, whereas for concentrations ≫ critical micelle concentration, cmc, the hydrophobin is displaced from the surface by the surfactant. In relationship to this study on surface self-assembly, it has been shown that at low pH the interaction between the hydrophobin and the anionic surfactant SDS, and the nonionic surfactant C12E6, was altered, and the pattern of adsorption changed. In the case of SDS, this resulted in some surface ordering of SDS/hydrophobin mixtures in the form of a trilayer. Tucker et al.27 have demonstrated more extensive surface ordering at the air−water interface in the form of a well-defined layered structure at the surface corresponding to three bilayers of hydrophobin and surfactant, due to the specific interaction between ethoxylated polysorbate surfactants and hydrophobin. This is driven by the hydrophobic interaction between the hydrophobin and the surfactant alkyl chain and the hydrophilic interaction between the ethoxylated sorbitan headgroup and the hydrophilic regions on the surface of the hydrophobin. The focus 9742

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Figure 2. NR data for (a) PES1260/0.025 mg/mL hydrophobin in nrw, for PES1260 concentrations from 3.9 to 500 μM (see legend for more details), and (b) 125 μM PES1360/hydrophobin, for hydrophobin concentrations from 0.003 to 1.0 mg/mL (see legend for details). The solid lines are model calculations as described in the text and for the model parameters summarized in Table 1. The different curves are shifted vertically by a factor of 2 for clarity. profile took ∼20−30 min on INTER and ∼60 min on SURF. The measurements were made sequentially in a five or seven position sample changer, and repeated at least once until the reflectivity no longer changed with time. Hence, the structures presented represent equilibrium or at least steady-state structures, and were established after a total lapse time ≥2−3 h. In the kinematic approximation,49 the reflectivity is related to the square of the Fourier transform of the scattering length density profile, ρ(z), normal to the surface (ρ(z) = ∑ini(z)bi, ni(z) and bi are the number density and neutron scattering length of the ith component, and ρ(z) is related to the neutron refractive index, n(z), and n(z) = 1 − λ2ρ(z)/2π), such that: R(Q ) =

16π 2 | ρ(z) e−iQz dz|2 Q2



D), the neutron reflectivity profile can directly provide information about the amount adsorbed at the air−water interface, and the structure of the adsorbed layer. This has been extensively demonstrated and exploited for a range of surfactant, mixed surfactant, polymer/surfactant, and protein/surfactant systems.4,49 This approach is used here to characterize the hydrophobin-ethoxylated polysorbate surfactant adsorption at the air−water interface. The NR data reported here correspond to surface structures that range from a simple monolayer to a more complex layered structure comprising up to six layers (see later for details). The specular reflectivity can be equally well described using the kinematic approximation description outlined above or the more exact optical matrix method.49 The reflectivity data here are fitted by comparing them with a profile calculated using the optical matrix method for the simplest structural model that is consistent (evaluated on a least-squares criterion) with the data and is physically consistent with the dimensions and densities of the different components. Each layer in the model is characterized by a thickness, d, and a scattering length density, ρ. A flat

(1)

By manipulation of ρ(z) through deuterium labeling (H, D have different scattering lengths, −3.7 × 10−6 Å for H, and 6.67 × 10−5 Å for 9743

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a

9744

34 29 17 16 17 18 18 17 14 16

5.0 1.0 0.4 0.2 0.1 0.05 0.025 0.0125 0.0063 0.0031

−6

0.9 1.3 0.8 1.0 1.7 1.6 1.6 1.7 1.6 1.7

ρ1 (×10

2.5 2.2 2.4 2.2 2.1 1.6 1.5 1.2 1.3

Å )

−2

Å )

−2

29 24 21 23 28 39

d2 (Å)

19 28 31 28 26 27 32 30

d2 (Å)

−6

Å )

−2

0.6 0.6 0.9 0.8 0.9 1.1 0.9 1.0

ρ2 (×10

0.3 0.6 0.6 0.6 0.4 0.6

ρ2 (×10

−6

14 16 16 16 16 15

9 7 14 13 12 15 15 15

d3 (Å)

d3 (Å)

Å )

−2

The errors in d and ρ for each layer are ±2 Å and ±0.2 × 10−6 Å−2, respectively.

d1 (Å)

25 16 16 20 16 14 26 31 30

500 250 125 62.5 31.3 15.6 7.8 3.9 1.98

ρ1 (×10

hydrophobin conc (mg/mL)

d1 (Å)

Tween conc (μM)

−6

0.8 1.4 2.0 2.3 2.3 2.5 2.0 1.7

ρ3 (×10−6 Å−2)

(b)

1.6 2.2 2.8 2.8 1.0 0.5

ρ3 (×10−6 Å−2)

(a)

46 38 28 30 34 30 29 30

d4 (Å)

23 27 24 26 22 34

d4 (Å)

0.6 0.6 0.6 0.5 0.5 0.9 0.5 0.3

ρ4 (×10−6 Å−2)

1.0 1.0 0.8 0.9 0.9 0.6

ρ4 (×10−6 Å−2)

17 14 11 13 12 16 15 23

d5 (Å)

18 13 15 16 18

d5 (Å)

2.2 2.2 2.8 2.6 2.6 2.7 2.9 1.8

ρ5(×10−6 Å−2)

2.1 3.0 2.6 2.5 1.8

ρ5(×10−6 Å−2)

48 42 36 32 33 24 28 23

d6 (Å)

34 37 25 41 46

d6 (Å)

Table 1. Key Model Parameters from Analysis of NR data for (a) PES1260/0.025 mg/mL Hydrophobin in nrw, and (b) 125 μM PES1360/Hydrophobin in nrwa

0.6 0.4 0.6 0.6 0.6 1.0 0.9 0.6

ρ6 (×10−6 Å−2)

0.5 0.8 0.6 0.5 0.5

ρ6 (×10−6 Å−2)

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surfactant concentration range ∼30−500 μM six separate layers are required. Taking into account the surfactant and hydrophobin dimensions and scattering lengths, the structure is best described as an initial monolayer (∼15−20 Å) of deuterated ethoxylated polysorbate surfactant followed by layers of hydrophobin (∼30 Å) separated by thin layers (∼10−15 Å) of the deuterated ethoxylated polysorbate surfactant headgroups. This is then represented by the three bilayer structure at the interface, as illustrated in Figure 3, where the molecular model

background is visible in the data at high Q values. It is included in the modeling and does not significantly affect the data interpretation. The reflectivity in the regions of monolayer adsorption was analyzed as a single layer of uniform composition, characterized by a thickness and scattering length density, d and ρ. The area/molecule and adsorbed amount can then be evaluated in the usual way48 using:

A=

∑b dρ

(2)

c. Measurements Made. The NR measurements presented here were all made in null reflecting water, nrw, for PESn60/hydrophobin mixtures for n = 3, 6, 9, 12, 16.5, and 20 at a hydrophobin concentration of 0.025 mg/mL, and for n = 3, 7, 9, 13, 16.5, and 20 at a hydrophobin concentration of 0.05 mg/mL, and for PESn60 concentrations in the range ∼2.0−1000.0 μM. Further measurements were made for 125 μM PESn60, for n = 3, 7, 13, 16.5, and 20.0 and hydrophobin concentrations from 0.001 to 5 mg/mL.



RESULTS Figure 2a shows a series of NR measurements made in nrw for the combination of hydrophobin and the ethoxylated polysorbate surfactant PES1260, with the ethylene oxide groups deuterium labeled. The NR data presented in Figure 2a were measured at PES1260 concentrations from 2.0 to 500 μM (shown in Figure 2a from 3.9 to 500 μM). At the extremes of surfactant concentration, at 2.0, 3.9, and 500 μM, the NR data are monotonically decreasing curves, consistent with a monolayer adsorbed at the interface. At PES1260 concentrations of 2.0 and 3.9 μM, the adsorbed layer thickness, d, is ∼30 Å, and the scattering length density, ρ (see Table 1a for values), is consistent with a close-packed layer of hydrophobin adsorbed at the interface. These values are similar to those previously reported for hydrophobin adsorption at the air−water interface.23 At a PES1260 concentration of 500 μM, the adsorbed layer thickness is ∼25 Å, and the scattering length density is ∼2.5 × 10−6 Å−2 (see Table 1a). This corresponds to an adsorbed monolayer of PES1260 surfactant with an area/molecule ∼110 Å2. The area/ molecule, A, is determined using eq 2 and with ∑b = 5.72 × 10−3 Å, as described elsewhere.49 This is close to the area/molecule at saturation adsorption, ∼90 Å2, obtained for PES126050 in the absence of hydrophobin. At PES1260 concentrations from 7.8 to 250 μM, the NR data have a distinctly different form. It corresponds to a thicker layer overall, but with a structure more complex than a single layer. The reflectivity is now dominated by two principal interference features, which become more pronounced as the surfactant concentration increases. They are most pronounced at surfactant concentrations from ∼60 to 120 μM, and become less distinct again at even higher surfactant concentrations. The peak centered at a Q value ∼0.15 Å−1 is a broad Bragg peak arising from a layered structure at the interface. The structure corresponds to a multilayer with three bilayers. The fringe at the lower Q value, ∼0.08 Å−1, is a “total thickness” fringe and arises from the average of overall layer of adsorbed material at the interface. The data are modeled using up to six discrete layers. The refractive index contrast between the deuterated ethoxylated surfactant headgroups and the hydrophobin enhances the sensitivity to the structure of the adsorbed layer. The modeling is achieved by using the least number of layers required to obtain a good representation of the data, and in which the d and ρ values can be physically related to the adsorbed species. The different d and ρ values are refined separately, but are constrained by the principal interference features of the reflectivity profiles. Where the interference features are most well developed, in the

Figure 3. Schematic representation of the hydrophobin/ethoxylated polysorbate surfactant surface multilayer structure (reproduced from ref 27).

takes into account the dimensions and structure of the surfactant and hydrophobin. At the more extremes of the surfactant concentration (in the low and highs) and depending upon the degree of ethoxylation, the structures are less well defined and can be described by a smaller number of layers consistent with a less well-defined structure. The key model parameters are summarized in Table 1a. Where the surface structure is less well developed, at a surfactant concentration of 7.8 μM, the surface structure is described by only four layers (or in some cases two bilayers). The key model parameters for PES960 and PES16.560 with 0.025 mg/mL are summarized in Table S1 in the Supporting Information. This variation in the surface structure was further discussed in more detail where the variation in the surface structure was encountered in the ethoxylated polysorbate surfactant/hydrophobin mixtures at a fixed degree of ethoxylation and due to changes in the surfactant alkyl chain length.27 A broadly similar sequence of NR data was obtained at a fixed surfactant concentration and variable hydrophobin concentrations. The profiles were measured for 125 μM PES1360 and hydrophobin in the concentration range 0.003−5.0 mg/mL, and are shown in Figure 2b for hydrophobin concentrations from 0.003 to 1.0 mg/mL. The key model parameters are listed in Table 1b. The surface structures are similar to those summarized in Figure 2a and Table 1a, and illustrated in Figure 3. The surface multilayer structure persists over most of the hydrophobin concentration range measured, and notably down to relatively low hydrophobin concentrations. The structure is still relatively well-defined at a hydrophobin concentration ∼0.003 mg/mL. At the higher hydrophobin concentrations ≥1.0 mg/mL, the surface adsorbed layer is in the form of a monolayer. The corresponding values for d and ρ (see Table 1b) are consistent with a monolayer of hydrophobin at the interface. Patterns of behavior similar to those shown in Figure 2a were observed for 0.025 and 0.05 mg/ 9745

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Table 2. Key Model Parameters for (a) PES2060/0.025 mg/mL Hydrophobin, and (b) PES360/0.05 mg/mL Hydrophobin (a) ρ1 (±0.2 × 10−6 Å−2)

d1 (±1.0 Å)

Tween conc (μM) 1000 500 250 125 62.5 31.3 15.8 7.9 3.9

27 31 28 30 31 31 30 30 31 (b)

2.0 1.7 1.6 1.5 1.5 1.4 1.5 1.4 1.3

Tween conc (μM)

d1 (±1.0 Å)

ρ1 (±0.2 × 10−6 Å−2)

1000 500 250 125 62.5 31.3 15.8 7.9 3.9

21 27 24 25 28 32 32 31 31

2.3 1.6 1.7 1.6 1.5 1.2 1.2 1.2 1.2

d2 (±2.0 Å)

ρ2 (±0.2 × 10−6 Å−2)

67 67

0.4 0.3

mixtures. Furthermore, this tendency was enhanced at low pH, due to a greater interaction between the two components. The variation in surface adsorption behavior obtained from the NR data for the different ethoxylated polysorbate surfactants mixed with hydrophobin can be represented as approximate surface phase diagrams. These are shown in Figure 4 for the variation in surfactant concentration of the different ethoxylated polysorbate surfactants at the fixed hydrophobin concentrations of 0.025 mg/mL (Figure 4a) and at 0.05 mg/mL (Figure 4b) and at a fixed surfactant concentration of 125 μM and variable hydrophobin concentrations (Figure 4c). The approximate surface phase diagrams in Figure 4 show the extent over which surface multilayer formation occurs between the differently ethoxylated polysorbate surfactants and hydrophobin. Figure 4 a and b shows that at the fixed hydrophobin concentration of 0.025 and 0.05 mg/mL, multilayer formation occurs over a wide range of surfactant concentraions, for degrees of ethoxylation from 6.0 to 16.5. In that range of ethoxylation, the surfactant concentration range over which multilayer formation occurs is almost independent of the degree of ethoxylation. At the higher hydrophobin concentration measured, 0.05 mg/mL, the region of surfactant concentration over which multilayer formation occurs is more extended and shifted to higher surfactant concentrations. At the extremes of surfactant concentration, the transition from multilayer to monolayer formation results in surface structures that are less well-defined and can be described with a smaller number of layers with less contrast in the density between the layers (as indicated in Figure 2 and Table 1). This is especially pronounced in the variations shown in Figure 4a, at the lower hydrophobin concentration of 0.025 mg/mL. For the degrees of ethoxylation of 3 and 20 in Figure 4a and b, there is an almost complete absence of multilayer formation, and only a narrow region with a weakly defined multilayer structure exists. In the surface phase diagram shown in Figure 4c, where the variation in structure with hydrophobin concentration at a fixed surfactant concentration is presented, the multilayer structures with surfactants with a degree of ethoxylation from 7 to 16.5 persist over a wide range of

mL hydrophobin and the ethoxylated polysorbate surfactants PES660, PES760, PES960, PES1360, and PES16.560, although the extent of the surfactant concentration range over which ordering exists differs. Furthermore, the range of ordering observed for PES1360 at a fixed surfactant concentration of 125 μM and variable hydrophobin concentrations, as shown in Figure 2b, is replicated for PES760 and PES16.560. However, the NR data for PES360 and PES2060 with hydrophobin, at hydrophobin concentrations of 0.025 and 0.05 mg/mL and at a fixed surfactant concentration of 125 μM and variable hydrophobin concentrations, have a distinctly different form. Over most of the surfactant concentration range and at the different hydrophobin concentrations, the adsorption is in the form of a monolayer. However, at the higher surfactant and hydrophobin concentrations, there is a narrow range of concentrations where the reflectivity shows a weak interference fringe and the data are consistent with a monolayer and an adjacent dilute thicker layer. Some typical parameters are summarized in Table 2a and b for PES2060/0.025 mg/mL hydrophobin and for PES360/0.05 mg/ mL hydrophobin. As discussed earlier in the context of the NR data in Figure 2, the adsorption at the higher surfactant concentrations is dominated by the surfactant adsorption and represents an adsorbed layer of surfactant close to its saturation adsorption value. At the lower surfactant concentrations, the adsorption is dominated by the hydrophobin adsorption. It was previously shown for hydrophobin adsorption with the surfactants sodium dodecyl sulfate, SDS, hexadecyltrimethylammonium bromide, CTAB, and monododecyl hexaethylene glycol, C12E6, at the air− water interface that a sharp transition from a surface dominated by hydrophobin to one dominated by the surfactant occurred at the surfactant cmc.23 This transition is not so clear in the data presented in Table 2a and b. This arises from a stronger surface interaction between the ethoxylated polysorbate surfactant and hydrophobin, resulting in a broad region where a mixed monolayer exists at the interface. Zhang et al.23 reported a greater tendency to form mixed monolayers in hydrophobin/ C12E6 mixtures as compared to hydrophobin/ionic surfactant 9746

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Figure 4. Approximate surface phase diagrams for PESn60/hydrophobin, (a) 0.025 mg/mL hydrophobin, (b) 0.05 mg/mL hydrophobin, and (c) 125 μM PESn60. See legend for details of the differently colored regions.

hydrophobin concentrations. Notably the multilayer structures persist down to extremely low hydrophobin concentrations. Consistent with the data presented in Figure 4 a and b, there is an almost complete absence of multilayer formation for surfactants with degrees of ethoxylation of 3 and 20.

Information), the general structure that describes the surface layering in the ethoxylated polysorbate/hydrophobin mixtures is remarkably consistent and robust over the concentration ranges and variations in surfactant structure studied. However, there are regions at the extremities of the surfactant or hydrophobin concentration ranges where the structures are less well-defined. In some cases, this results in a wider variation in the structural parameters, and in other cases a lower number of layers is required to describe the data. However, the overriding structure



DISCUSSION Although there are some variations in the model parameters derived (see Table 1a and b and Table S1 in the Supporting 9747

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3.5 5.0 4.8

ratio surfactant/hydrophobin

0.7 0.8 0.6 49 37 25

645 770 967

mean hydrophobin a/m

2.8 3.0 2.6 14 13 14

199 153 199

mean surfactant a/m

0.9 1.0 0.6 24 27 29

472 550 1080

(b)

a/m6

1.5 2.2 3.1

d6 (Å) ρ5(×10−6 Å−2) d5 (Å) ρ4 (×10−6 Å−2) d4 (Å) ρ3 (×10−6 Å−2)

a

The equivalent saturated area/molecules are ∼450, 70, 90, and 110 Å2, respectively.

109 147 213 794 600 931 330 163 185 650 1125 890 167 150 200 9.0 12.0 16. 5

a/m2 a/m1 degree of ethoxylation, n

20 16 17 9.0 12.0 16.5

1.3 2.4 2.3

a/m3

30 24 26

a/m4

0.8 0.6 0.7

a/m5

9 16 14

(a) d3 (Å) ρ2 (×10−6 Å−2) d2 (Å) ρ1 (±0.1 × 10−6 Å−2) d1 (±1.0 Å) degree of ethoxylation, n

Table 3. (a) Key Model Parameters for 125 μM PESn60/0.025 mg/mL Hydrophobin, for n = 9.0, 12.0, and 16.5; and (b) Area/Molecule and Mean Values per Structure, for ∑b for Hydrophobin, PES960, PES1260, and PES16.560 of 16.2, 4.34, 5.72, and 7.78 × 10−3 A−2a

is one that is well described by 6 layers, or effectively 3 bilayers, as shown schematically in Figure 3 and summarized in Table 1. This is interpreted as an initial layer of surfactant with a thickness ∼15−20 Å, and alternate layers of predominantly hydrophobin or surfactant. The hydrophobin layers are typically ∼25−35 Å thick, and the subsequent surfactant layers (which contain predominantly the ethoxylated headgroups) are ∼10−15 Å thick. As illustrated in the schematic representation in Figure 3, it is assumed that the layers of hydrophobin correspond to layers of dimers, and that the surfactant alkyl chains interleave between the hydrophobin molecules. The subsequent surfactant layers, apart from the initial layer, are then comprised of the ethoxylated headgroups and solvent. Hence, it is likely that the structure arises from two different types of interaction between the hydrophobin and the surfactant. The first of these is the hydrophobic interaction between the surfactant alkyl chain and the hydrophobic patch on the surface of the hydrophobin. The initial results on ethoxylated polysorbate/hydrophobin interactions27 showed that in terms of the formation of the layered structure this interaction was optimal for the stearic alkyl chain length, with a fully extended length of ∼22 Å, and less so for the unsaturated oleic chain and the shorter lauric and palmitic chains. The second interaction is between the ethoxylated surfactant headgroup and the hydrophilic regions of the hydrophobin. In this study, we have investigated specifically how the surface ordering varies with the degree of ethoxylation of the surfactant headgroup, where the number of ethylene oxide group was varied from 3 to 20. We have observed (see Figure 4) that the surface ordering persists over a wide range of different degrees of ethoxylation, from 7 to 16.5. For degrees of ethoxylation of 3 and 20, there is essentially no surface ordering and monolayer adsorption is observed. In Table 3, the detailed structural parameters for 125 μM PESn60/0.025 mg/mL hydrophobin mixtures for n = 9, 12, and 16.5 are presented. In addition to the thickness and scattering length densities summarized in Table 3a, in Table 3b we have included the calculated amounts (expressed as area/molecule) of surfactant and hydrophobin in their respective layers; using eq 2 and the ∑b parameters listed in the table caption. In this calculation, it is assumed that the alternate layers contain only surfactant or hydrophobin. In both cases, the contribution of the alkyl chain is considered to be negligible, and there is no solvent contribution to the scattering as the measurements were made in nrw. Furthermore, the values of ρ obtained indicate that the layered structures observed cover the entire surface, and are not within error fragmented. From the average area/molecule values of surfactant and hydrophobin in each of the layers, the ratio of surfactant/hydrophobin in the structure is approximately estimated (see Table 3b). As the degree of ethoxylation increases from 9 to 16.5, the mean areas/molecule of the surfactant and hydrophobin both increase. For the surfactant layers, the average area/molecule corresponds to a packing ∼0.55 of saturated surfactant adsorption. For the hydrophobin layers, the packing of the hydrophobin for degrees of ethoxylation of 9 and 12 is ∼0.7 of a closed-packed layer of hydrophobin, but for the higher degree of ethoxylation of 16.5 it has dropped to ∼0.45. However, the average ratio of surfactant/hydrophobin across the overall structure varies between 3.5 and 5.0. A more complete summary of the area/molecule in each layer for PES960, PES1260, and PES16.560 at the concentrations where the surface structures are broadly similar is presented in Table S2 in the Supporting Information. Hence, over a broad range of ethoxylation of the polysorbate headgroup, the packing can be adjusted to an

ρ6 (×10−6 Å−2)

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nonionic surfactants result in denaturation. For example, the Tween surfactants can be the most efficient for the extraction of some proteins and yet the least efficient for others. Bam et al.36−38 showed that the hydrophobic interaction between the Tween surfactant and recombinant human growth hormone reduces aggregation during refolding. The range of ethoxylated polysorbate surfactants available from the different degree of ethoxylation and alkyl chain length offer the potential for a wide range of molecular chaperoning function in protein folding and related mechanisms. The wetting properties associated with the ethoxylated polysorbate surfactant/hydrophobin mixtures were reported more fully elsewhere.27 However, in summary, the adsorption of hydrophobin or hydrophobin/surfactant mixtures for which the adsorption is predominantly monolayer strongly wet hydrophobic surfaces, whereas the adsorption of ethoxylated polysorbate surfactant/hydrophobin mixtures that form multilayer structures result in hydrophobic surfaces that remain highly hydrophobic. This is in marked contrast to the multilayer structures that form for polyelectrolyte/ionic surfactant4 or ionic surfactants in the presence of multivalent counterions,1−3 which persistently wet hydrophobic surfaces. Related to this, Graca et al.35 have demonstrated that the adsorption of Tween surfactants onto hydrophobic surfaces forms robust layers with potentially attractive lubrication properties. Prime et al.52 have demonstrated the role of ethoxylated surfaces in inhibiting nonspecific adsorption of a range of proteins. Hence, the ethoxylated polysorbate/hydrophobin mixtures present a different range of opportunities for modifying the macroscopic surface properties.

optimal value to maximize the hydrophobic and hydrophilic interactions between the surfactant and hydrophobin that stabilize the observed surface structures. For the smaller and larger degrees of ethoxylation of 3 and 20, the corresponding surfactant areas/molecule (the saturated adsorptions for PES360 and PES2060 have area/molecule of 37 and 125 Å2) are too small or too large to accommodate the packing required to optimize both the hydrophobic and the hydrophilic interactions. The area/molecule of the nonionic surfactant is not the only requirement associated with optimizing the interactions, but the structure of the ethoxylated polysorbate headgroup and the way in which the ethylene oxide groups are distributed are clearly important. This was demonstrated with some NR measurements on decaethylene monostearyl ether (C18E10)/hydrophobin mixture, where the nonionic surfactant has a stearic alkyl chain length and an area/molecule in the range studied, but a different headgroup structure. In this case, only monolayer adsorption of either the surfactant or the hydrophobin was observed. As discussed in the Introduction, the specific interaction between ethoxylated polysorbate Tween surfactants and different proteins has led to a number of applications associated with their diverse properties, ranging from foam and emulsion stabilization to the manipulation of protein folding and enhanced surface delivery of bioactive components.28−40,51 Here, the specific nature of the interaction between the ethoxylated polysorbate surfactants is tuned by the alkyl chain length and the degree of ethoxylation. The combination of the hydrophobic and hydrophilic interactions between the two species gives rise to some unusual and spectacular surface ordering. This has not been observed in any other surfactant−protein interaction at surfaces. This was further confirmed here with some preliminary NR measurements using a mixture of 125 μM PES1360 surfactant and 0.05 mg/mL β-lactoglobulin, β-casein, and lysozyme. For each of the three mixtures, the NR measurements showed only monolayer adsorption, and in which the reflectivity was largely independent of protein type. The data were consistent with a layer of surfactant with an area/molecule ∼100 Å2. Some more limited surface ordering, in the form of a single trilayer, was previously reported by Zhang et al.20 for SDS/hydrophobin mixtures at pH 3. However, this results from quite a different interaction between the surfactant and protein. It arises from a charge attraction between the two species, similar to that encountered in polyelectrolyte/ionic surfactant mixtures.4 Surface multilayer formation has been extensively demonstrated in anionic and anionic/nonionic surfactant mixtures arising from the addition of multivalent ions1−3 and in a range of polyelectrolyte or oligomer/surfactant mixtures.4 The nature of protein/surfactant interactions has wider implications, as outlined in the Introduction. For example, it is generally assumed that the interaction of polyoxyethylene-based nonionic surfactants with proteins results in a reduced tendency toward protein denaturation as compared to ionic surfactants. This can be an important factor in terms of membrane protein extraction, solubilization, and stabilization. However, Le Marie et al.39 remarked that the associated solubilization rates were slower for the nonionic surfactants, and that in the case of the Tween surfactants this was associated with the bulky headgroup and related curvature. The ethoxylated polysorbate surfactants with the reduced degree of ethoxylation show a pronounced synergy with hydrophobin, and this may also offer a distinct advantage with other proteins. Arachea et al.40 showed that the extraction efficiency and stability after extraction varied substantially for different membrane proteins and surfactants, and that even some



SUMMARY The interaction between the ethoxylated polysorbate surfactant, with degrees of ethoxylation ranging from ∼6 to 17, with the protein hydrophobin results in the formation of a well-defined layered structure, comprising three bilayers, at the air−water interface. The structures form spontaneously over this wide range of variations in the degree of ethoxylation of the surfactant, and for a wide range of surfactant and hydrophobin concentrations. Although the layered surface structures are observed over a wide range in the degree of ethoxylation of the surfactant, they do not form using the standard Tween polysorbate surfactants where the degree of ethoxylation is on average 20. In particular, the results show that the protein− surfactant interaction can be manipulated by varying the degree of ethoxylation of the ethoxylated polysorbate surfactant. The multilayer structures formed offer great potential for the manipulation of surface adsorption properties and for the control of wetting properties. More broadly, the ethoxylated polysorbate surfactants provide a greater range of opportunities for manipulating the interaction of surfactants with proteins and other biomolecules, in applications such as membrane protein extraction, drug delivery, and emulsion/foam stabilization.



ASSOCIATED CONTENT

S Supporting Information *

Additional key model parameters and derived area/molecule values from the NR data. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*E-mail: jeff[email protected]. 9749

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Notes

(17) Otzen, D. Protein-surfactant interactions: a tale of many states. Biochim. Biophys. Acta 2011, 1814, 562−592. (18) Goddard, E. D.; Ananthananpadmanbhan, K. P. Interaction of Surfactants with Polymers and Proteins; CRC Press: Boca Raton, FL, 1993. (19) Kotsmar, C. S.; Pradines, V.; Alahrerdjieva, V. S.; Absenenko, E. V.; Fainerman, V. B.; Kovalchuk, V. I.; Kragel, J.; Leser, M. E.; Noskov, B. A.; Miller, R. Thermodynamics, adsorption kinetics and rheology in mixed protein-surfactant interfacial layers. Adv. Colloid Interface Sci. 2009, 50, 41−54. (20) Maldonado-Valderrama, J.; Rodriguez-Patino, J. M. Interfacial rheology of protein-surfactant mixtures. Curr. Opin. Colloid Interface Sci. 2010, 15, 271−282. (21) Green, R. J.; Su, T. J.; Joy, H.; Lu, J. R. Interaction of lysozyme and SDS at the air-liquid interface. Langmuir 2000, 16, 5797−5805. (22) Gunning, P. A.; Mackie, A. R.; Gunning, P.; Woodward, N. C.; Wilde, P. J.; Morris, V. J. Effect of surfactant type on surfactant-protein interactions at the air-water interface. Biomacromolecules 2004, 5, 984− 991. (23) Zhang, X. L.; Penfold, J.; Thomas, R. K.; Tucker, I. M.; Petkov, J. T.; Bent, J.; Cox, A.; Campbell, R. A. Adsorption behavior of hydrophobin and hydrophobin/surfactant mixtures at the air-water interface. Langmuir 2011, 27, 11316−11323. (24) Hakanpaa, J.; Szilvray, G. R.; Kalijunen, H.; Maksimainen, M.; Linder, M. B.; Rouvinen, J. Two crystal structures of Trichodema reesi hydrophobin HFB1- the structure of the protein- amphiphile with and without detergent interactions. J. Protein Sci. 2006, 15, 2129−2140. (25) Kallio, J. M.; Linder, M. B.; Rouvinen, J. Crystal structure of hydrophobin hfbII in the presence of surfactant implicate the formation of fibrils and monolayer films. J. Biol. Chem. 2007, 282, 287333−28739. (26) Stanimirova, R. D.; Gurkov, T. D.; Kralchevsky, P. A.; Balasheva, K. T.; Stoyanov, S. D.; Pelan, E. G. Surface pressure and elasticity of hydrophobin layers at the air-water interface; rheology versus structure detected by AFM. Langmuir 2013, 29, 6053−6067. (27) Tucker, I. M.; Petkov, J. T.; Penfold, J.; Thomas, R. K.; Li, P. X.; Cox, A. R.; Hedges, N.; Webster, J. R. P. Spontaneous surface selfassembly in protein-surfactant mixtures: interactions between hydrophobin and ethoxylated polysorbate surfactants. J. Phys. Chem. B 2014, 118, 4867−4875. (28) Ruiz-Henestrosa, V. P.; Sanchez, C. C.; Patino, J. M. R. Adsorption and foaming characteristics of Soy Globulins and Tween20 mixed systems. Ind. Eng. Chem. Res. 2008, 47, 2876−2885. (29) Yang, Y.; Leser, M. E.; Sher, A. A.; McClements, D. J. Formation and stability of emulsions using a natural small molecule surfactant: Quilaja Saponin. Food Hydrocolloids 2013, 30, 589−596. (30) Davies, R.; Graham, D. E.; Vincent, B. Water-cyclohexaneSpan80-Tween80 systems: solution properties and water/oil emulsion formation. J. Colloid Interface Sci. 1987, 116, 88−99. (31) Weiss, J.; McClements, D. J. Mass transport phenomena in oil-inwater emulsions containing surfactant micelles: solubilisation. Langmuir 2000, 16, 5879−5883. (32) Schmidts, T.; Dobler, D.; Wissing, C.; Dunkel, F. Influence of hydrophilic surfactants on the properties of multiple w/o/w emulsions. J. Colloid Interface Sci. 2009, 338, 184−192. (33) Sharma, S. C.; Warr, G. C. Phase phenomena, self-assembly and emulsification of Tween80/water mixtures with Limonene and perfluoromethyldecalin. Langmuir 2012, 28, 11707−11713. (34) Shen, L.; Guo, A.; Zhu, X. Tween surfactants: adsorption, selforganisation and protein resistance. Surf. Sci. 2011, 605, 494−499. (35) Graca, M.; Bongaerts, J. H. H.; Stokes, J. R.; Granick, S. Friction and adsorption of aqueous polyoxyethylene (Tween) surfactants at hydrophobic surfaces. J. Colloid Interface Sci. 2007, 315, 662−670. (36) Webb, S. D.; Cleland, J. L.; Carpenter, J. F.; Randolph, T. W. A new mechanism for decreasing aggregation of recombinant Human Interferon-γ by a surfactant: slow dissolution of lypophilised formulations in a solution containing 0.03% Polysorbate20. J. Pharm. Sci. 2002, 91, 543−558. (37) Bam, N. B.; Cleland, J. L.; Yang, J.; Manning, M. C.; Carpenter, J. F.; Kelley, R. F.; Randolph, T. W. Tween protects recombinant human

The authors declare no competing financial interest.



ACKNOWLEDGMENTS The provision of beam time on the INTER and SURF instruments at ISIS is acknowledged. The invaluable scientific and technical assistance of the Instrument Scientists and support staff is gratefully recognized.



REFERENCES

(1) Penfold, J.; Thomas, R. K.; Dong, C. C.; Tucker, I. M.; Metcalfe, K.; Golding, S.; Grillo, I. Equilibrium surface adsorption behavior in complex anionic/nonionic surfactant mixtures. Langmuir 2007, 23, 10140−10149. (2) Petkov, J. T.; Tucker, I. M.; Penfold, J.; Thomas, R. K.; Petsev, D. N.; Dong, C. C.; Golding, S.; Grillo, I. The impact of multivalent counterions, Al3+, on the surface adsorption of the anionic surfactant, alkyloxyethylene sulfate and anionic/nonionic surfactant mixtures. Langmuir 2010, 26, 16669−16709. (3) Xu, H.; Penfold, J.; Thomas, R. K.; Petkov, J. T.; Tucker, I. M.; Webster, J. P. R. The formation of surface multilayers at the air-water interface from sodium polyethylene glycol monoalkyl ether sulfate/ AlCl3 solutions: the role of the size of the polyethylene oxide group. Langmuir 2013, 29, 11656−11666. (4) Taylor, D. J. F.; Thomas, R. K.; Penfold, J. Polymer-surfactant interactions at the air-water interface. Adv. Colloid Interface Sci. 2007, 132, 69−110. (5) Takumi, H.; Noda, M.; Matsubara, T.; Takuie, T.; Aratono, M. Dynamics of condensed monolayer and multilayer formation of hexadecylpyridinium chloride - sodium dodecyl sulfate mixed systems at the air-water interface. Chem. Lett. 2012, 14, 1218−1220. (6) Kawai, T.; Yamada, Y.; Kondo, T. Adsorbed monolayers of mixed surfactant solutions of sodium dodecyl sulfate and cetylpyridinium chloride studied by Infrared external reflection spectroscopy. J. Phys. Chem. C 2008, 112, 2040−2044. (7) Wang, L.; Cai, P.; Galla, H. J.; He, H.; Fluch, C. R.; Mendelsohn, R. Monolayer-multilayer transition in a lung surfactant model: IR reflection and adsorption spectroscopy. Eur. Biophys. J. 2005, 234, 243−254. (8) Alonso, C.; Alig, T.; Yoon, J.; Bringezu, F.; Warriner, H.; Zasadzinski, J. A. More than a monolayer: relating lung surfactant structure and mechanics to composition. Biophys. J. 2004, 87, 4188− 4202. (9) Malcharek, S.; Hinz, A.; Hilterhaus, L.; Galla, H. J. Multilayer structures in lipid monolayer films containing surfactant protein C: effects of cholesterol and DOPE. Biophys. J. 2006, 88, 2638−2649. (10) Ravsio, A.; Cruz, A.; Perez-Gil, J.; Haller, T. High throughput evaluation of pulmonary surfactant adsorption and surface film formation. J. Lipid Res. 2008, 49, 2479−2488. (11) Gerstenberg, M. C.; Pedersen, J. S.; Majewski, J.; Smith, G. S. Surface induced ordering in triblock micelles at the solid-liquid interface. Langmuir 2002, 18, 4933−4943. (12) Hamilton, W. A.; Butler, P. D.; Baker, S. M.; Smith, G. S.; Hayter, J. B.; Magid, L. J.; Pynn, R. Shear induced hexagonal ordering observed in an ionic viscoelastic fluid flowing past a surface. Phys. Rev. Lett. 1974, 72, 2219−2223. (13) McGillivray, D. J.; Thomas, R. K.; Rennie, A. R.; Penfold, J.; Sivia, D. S. Ordered structures of di-chain cationic surfactants at interfaces. Langmuir 2003, 19, 7719−7726. (14) Smit, B.; Hilbers, P. A.; Esselink, K.; Rupert, L. A. M.; va Os, N. M.; Schlijper, A. G. Structure of an oil-water interface in the presence of micelles: a computer simulation study. J. Phys. Chem. 1991, 95, 6361− 6368. (15) Wolff, M.; Seitz, R.; Gutfreund, P.; Voss, N.; Gerth, S.; Walz, M.; Magerl, A.; Zabel, H. Shear induced relaxation of polymeric micelles at the solid-liquid interface. Langmuir 2008, 24, 11331−11333. (16) Hellsing, M. S.; Rennie, A. R.; Hughes, A. V. Adsorption of aerosol-OT to sapphire: lamellar structures studied with neutrons. Langmuir 2011, 27, 4669−4678. 9750

dx.doi.org/10.1021/la5022627 | Langmuir 2014, 30, 9741−9751

Langmuir

Article

growth hormone against agitation-induced damage via hydrophobic interactions. J. Pharm. Sci. 1998, 87, 1554−1559. (38) Bam, N. B.; Cleland, J. L.; Randolph, T. W. Molten globule intermediate recombinant human growth hormone: Stabilisation with surfactants. Biotechnol. Prog. 1996, 12, 801−809. (39) Le Maire, M.; Champeil, P.; Møller, J. V. Interaction of membrane proteins and lipids with solubilized detergents. Biochim. Biophys. Acta 2000, 1508, 86−111. (40) Arachea, B. T.; Sun, Z.; Potente, N.; Malik, R.; Isailovic, D.; Viola, R. E. Detergent selection for enhanced extraction of membrane proteins. Protein Expression Purif. 2012, 86, 12−20. (41) Kragel, J.; Wustneck, R.; Husband, F.; Wilde, P. J.; Makievski, A. V.; Grigoriev, D. O.; Li, J. B. Properties of mixed protein/surfactant adsorption layers. Colloids Surf., B 1999, 12, 399−407. (42) Seo, D. J.; Fujita, H.; Sakoda, A. Effects of a nonionic surfactant, Tween20, on adsorption/desorption of saccharificated enzymes onto/ from lignocelluloses and saccharification rate. Adsorption 2011, 17, 813−822. (43) Joshi, O.; McGuire, J. Adsorption behaviour of lysozyme and Tween20 at hydrophilic and hydrophobic silica-water interfaces. Appl. Biochem. Biotechnol. 2009, 152, 235−248. (44) Lu, D.; Rhodes, D. G. Mixed composition films of Spans and Tween80 at the air-water interface. Langmuir 2000, 16, 8107−8112. (45) Di Marzio, L.; Marianecci, C.; Petrine, M.; Rinaldi, F.; Carafa, M. Novel pH-sensitive non-ionic surfactant vesicles: comparison between Tween21 and Tween20. Colloids Surf., B 2011, 82, 18−24. (46) Singhal, S.; Moser, C. C.; Wheatley, M. A. Surfactant stabilized micro-bubbles as ultrasound contrast agents: stability study of Span60 and Tween80 mixtures using a Langmuir trough. Langmuir 1993, 9, 2426−2429. (47) Oxford Isotope Facility, www.isis.stfc.ac.uk/apply-for-beamtime/ Oxford-isotope-facility8717.html. (48) INTER and SURF reflectometers at the ISIS facility, http://www. isis.stfc.ac.uk/instruments/. (49) Lu, J. R.; Thomas, R. K.; Penfold, J. Surfactant layers at the airwater interface: structure and composition. Adv. Colloid Interface Sci. 2000, 84, 143−304. (50) Penfold, J.; Thomas, R. K.; Li, P. X.; Petkov, J. T.; Tucker, I.; Webster, J. R. P.; Terry, A. Adsorption at the air-water and oil-water interfaces and self-assembly in aqueous solution of the ethoxylated polysorbate nonionic surfactant. Langmuir 2014. (51) O’Sullivan, S. M.; Woods, J. A.; O’Brien, N. M. Use of Tween40 and Tween80 to deliver a mixture of phytochemicals to human colonic adenocarcinoma cell (CaCO-2) monolayers. Br. J. Nutr. 2004, 91, 757− 764. (52) Prime, K. L.; Whitesides, G. M. Adsorption of proteins onto surfaces containing end-attached oligo(ethylene oxide): a model system using self-assembled monolayers. J. Am. Chem. Soc. 1993, 115, 10714− 10721.

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