Liquid Scintillation Spectrometry of Tritium in Studying Lysozyme

Feb 10, 2011 - Liquid scintillation spectrometry of tritium in the application of the scintillation phase method was used for studying the adsorption ...
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Liquid Scintillation Spectrometry of Tritium in Studying Lysozyme Behavior in Aqueous/Organic Liquid Systems. The Influence of the Organic Phase Maria G. Chernysheva* and Gennadii A. Badun Division of Radiochemistry, Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia 119991 ABSTRACT: Liquid scintillation spectrometry of tritium in the application of the scintillation phase method was used for studying the adsorption of lysozyme at the liquid/liquid interface and its distribution in the bulk of the system. The goal of this research was to reveal the influence of the nature of the organic phase on the distribution and adsorption ability of the protein when it is placed in a system containing two immiscible liquids. Based on the radiochemical assay distribution coefficients and adsorption isotherms obtained for aqueous/octane, aqueous/p-xylene and aqueous/octanol systems, it was concluded that the interaction of the protein with the interface plays a dominant role in protein behavior in aqueous/organic liquid systems.

’ INTRODUCTION The experimental study of protein adsorption and its distribution in systems of two immiscible liquids is extremely difficult because only a limited group of methods is appropriate for experimental research. The most prevalent experiments are conducted using the pendant-drop technique,1 Fourier transform infrared spectroscopy,2 total internal reflection fluorescence microscopy3 or the radiotracer method with 14C-labeled proteins, which was first introduced by Graham and Phillips.4-6 In this technique, an excess in concentration is determined by the surface radioactivity method for a water/ air interface and by liquid scintillation spectrometry for a water/ toluene interface. In later research, this technique was successfully used in the investigation of the binding affinity of proteins to a planar triolein/water interface either as individual compounds or in combination with another protein.7,8 Now, it is evident that the rate of protein adsorption at the interface is not simply diffusion-controlled but is governed by the interaction forces that occur between the protein and the interface.9 Other research focuses on the distribution coefficient (KOW), which is determined as a ratio of the compound concentration in an organic liquid (particularly octanol) to its concentration in water. KOW is a biologically important physical parameter, which correlates with the ability of biologically active molecules penetrating the cellular membrane.10 The distribution data are usually obtained by simply shaking a solute with two immiscible solvents, followed by the determination of the solute concentration in one or in both liquids.11 Unfortunately, this technique does not incorporate the possibility of the adsorption of the compound at the liquid/liquid interface. To determine both the distribution coefficient of the protein and its adsorption at the liquid/liquid interface, we propose the r 2011 American Chemical Society

use of tritium-labeled protein to determine, or “trace”, the concentration and liquid scintillation spectrometry as an analytical method. An aqueous solution of a 3H-compound was added to an organic scintillation cocktail practically immiscible with water. Because the path length of tritium β-particles in condensed media is ca. 1.6 μm, the registered radioactivity of the whole system results from the distribution of the labeled compound in the bulk organic phase and its concentration at the liquid/liquid interface. For the first time, this technique was applied to studying the behavior of bovine serum albumin in a water/toluene system.12 Based on the theory briefly described above, this technique was called “scintillation phase method”. Utilization of scintillation additives (naphthalene and 2,5-diphenyloxazole) allowed the application of aliphatic liquids and alcohols equally with aromatic hydrocarbons.13 In this research, we applied the tritium-labeled lysozyme and the scintillation phase approach to the study of the behavior of proteins in aqueous/organic liquid systems. The aim of this work was to reveal the influence of the nature of the organic phase on the protein adsorption at the aqueous/organic interface and its ability to penetrate into the bulk of an organic liquid. To this end, we compared the behavior of lysozyme in several two-phase systems, which contained either p-xylene, octane, or octanol. Lysozyme was chosen as a typical globular protein, which consists of 129 amino acid residues and has a molecular weight of 14.3 kDa and a pI of 11. This protein is highly stabile during adsorption onto hydrophobic surfaces.14 Lysozyme particles in Received: February 6, 2010 Revised: January 14, 2011 Published: February 10, 2011 2188

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each phase were analyzed by the dynamic light scattering (DLS) method.15 The adsorption isotherms obtained were described by the theoretical model proposed by Fainerman for the adsorption of individual proteins.16

’ THEORETICAL SECTION Scintillation Phase Method. The theoretical background of this method is completely described in previous studies.13,17 Thus, here we briefly discuss the merits and demerits of this method. First, the method involves the direct determination of both the bulk concentration of the compound in the organic liquid and its excess at the liquid/liquid interface. Second, the experiment can continue for several months without any destruction.18 Third, it makes it possible to follow the behavior of one component in the mixture in a background of the other.19 As mentioned above, the counting rate of tritium β-radiation of the two-phase system (I) has two components. They are “volumetric” (IV), which results from the 3H-compound in the bulk of the organic phase, and “superficial” (IS), which results from the 3H-compound excess at the aqueous/organic interface.

I ¼ IV þ IS

ð1Þ

IV ¼ εAsp corg V

ð2Þ

Is ¼ εAsp ð0:5Γ þ 0:27cw hÞS

ð3Þ

Here, corg and cW are concentrations of the labeled compound in the organic and aqueous phases, respectively; Γ is the superficial excess of the compound; ε is the registration efficiency of the tritium β-radiation in the organic phase, which is used to convert counts per minute (cpm) readings into decays per minute (dpm); V is the volume of the organic phase, and Asp is the specific radioactivity of the labeled compound (dpm/mol) determined when the tritiated compound is synthesized. h is the zone thickness of the aqueous phase, by which the registration of tritium radiation is possible. The value of h corresponds to the path length of the tritium βparticles in condensed media. Notably, 50% of the particles were calculated to be absorbed by a layer about 0.3-0.4 μm thick, 80% of the particles are calculated to be adsorbed within 1 μm from the source, and the maximum range of the particles in the material of unit density cannot be higher than about 1.5-1.7 μm.20 Thus, the superficial counting rate of tritium β-radiation includes the registration of labeled molecules in both the adsorption layer (registration efficiency is two times less than that for the bulk of organic phase (coefficient equal to 0.5 in eq 3)) and the subsurface aqueous phase, in which the concentration of the compound exceeds its bulk concentration. The bulk concentration of the compound in the subsurface region can be incorporated based on the assumption that for equal distribution of emitters in the subsurface region, the registration efficiency decreases with the coefficient 0.27 in eq 3. The reliability of the coefficient 0.27 was confirmed in the experiments with tritiated water and [3H]alanine.17 The value of registration efficiency of tritium β-radiation in the bulk of the scintillation cocktail was determined with the help of quenching curves that were obtained for each organic scintillation cocktail, according to the procedure previously described.20 Quenching curves were also previously presented.21 In brief, tritiated 2,5-diphenyloxazole was used as the labeled material

Figure 1. Dependence of registration efficiency on the ratio of counting in channels for various scintillation liquids: (1) alkanes; (2) arenes; (3) octanol. “A” corresponds to the number of counts registered in the energy range of 0.02-18.6 keV; “B” corresponds to the number of counts registered in the energy range of (1, 2) 2.0-18.6 keV or (3) 1.018.6 keV.21.

with a high solubility in organic solvents and nitrobenzene was used as a quencher. Quenching was detected by measuring the ratio (B/A) of the counting rates within two pulse-height channels, where A corresponds to the number of impulses registered in the energy range of 0.02-18.6 keV and B corresponds to the number of impulses registered in the energy range of 2.0-18.6 keV (for p-xylene and octane) or 1.0-18.6 keV (octanol). The ratio decreased with an increase in quencher concentration. The value of registration efficiency (ε) was calculated as follows: 1r ð4Þ ε¼ A Here, Ir is the counting rate corresponding to the certain value of the ratio, and A is the radioactivity of the solution. Thus, quenching curves that show the dependence of ε vs the ratio counts in channels B/A were obtained (Figure 1). The value of the ratio of the counting rates was obtained for each sample in the experiment, and the value of registration efficiency was calculated. Because the specific radioactivity of each labeled compound (dpm/mol) is known from the labeled compound characterization and solution concentration, if nonlabeled compound is added for acceptability of the system for measuring, the compound concentration in the organic phase and at the interface can be determined at any point by measuring I and IV and extracted from eq 1, 2, and 3: ð5Þ Is ¼ I - IV IV εAsp V

ð6Þ

Is 2Is - 2  0:27cw h ¼ - 0:54cw h 0:5εAsp S εAsp S

ð7Þ

corg ¼ Γ¼

It has to be emphasized that for compounds that possess high surface activity, the contribution of the component 0.54cWh is not higher than 5%. 2189

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The value of S is under a large amount of influence from interfacial tension. The deformation interface near walls is about capillary length: !1=2 2σwo ð8Þ a¼ ΔFg Here, ΔF is the difference between water and organic liquid densities; σWO is the interfacial tension at the aqueous/organic liquid interface, and g is the free fall acceleration. The interfacial area was calculated as S ¼ πða2 þ r 2 Þ

ð9Þ

Here, r is the inner radius of the vial (0.75 cm). Despite the short path length of tritium β-particles, the data obtained by the scintillation phase technique required detailed analysis. On the one hand, the method is limited by the molar radioactivity of the 3H-compound. Considering the specific radioactivity single-labeled tritiated compound is 29 Ci/mmol, it becomes possible to determine surface concentration to be about 2  10-11 mol/m2. On the other hand, it is very sensitive to the formation of emulsions at the interface.22 The high value of IV (or high value of the distribution coefficient) also limited the application of this technique. The fourth advantage of the scintillation phase method deals directly with tritium as a radionuclide. The nuclear, physical properties of tritium (half-life is 12.4 years and the maximum energy of β-radiation is 18.6 keV) provide a high specific radioactivity of a singly labeled substance and short path lengths of tritium β-particles in the condensed media, while for 14C it reaches ca. 30 μm. It has to be noted that the specific radioactivity of 14C-labeled compound which contains one 14C-atom is 0.062 Ci/mmol. Such a value significantly decreases the sensitivity of the assay. Furthermore, a 14 C-label is usually introduced into the protein via an acylation reaction that results in the labeled analogue.4-6 Tritium can be introduced practically into any organic compound by the thermal activation method, 23-25 which is based on the bombardment of a solid organic target with tritium atoms. 3 H-compounds obtained in such a way are identical to the initial material after chromatographic purification.26 Calculations of Protein Adsorption by the Fainerman Model. This model assumes that protein molecules can exist in a number of states of different molar areas, varying from a maximum value (ωmax) at a very low surface coverage (low surface pressure) to a minimum value (ωmin) at a very high surface coverage.16,27-30 The molar areas of two “neighboring” conformations differ from each other by the molar area increment ω0, chosen to be equal to the molar area of the solvent. The equation of state accounting for the nonideal entropy and enthalpy of mixing for the surface layer reads   πω0 ω0 ¼ lnð1 - θÞ þ θ 1 ð10Þ þ aθ2 RT ω where π is surface pressure, R is the gas law constant, T is the temperature, and a is the intermolecular interaction parameter, and index “p” presented in the cited paper was omitted. Γ¼

n X i¼1

Γi

ð11Þ

is the total adsorption of proteins in all n states, and θ ¼ ωΓ ¼

n X

Γi ωi

ð12Þ

i¼1

is the total surface coverage by protein. Here, ω is the average molar area of protein, where ωi = ωmin þ (i - 1)ω0. (1 e i e n) is the molar area of protein in state i, assuming the molar area increment ω0 at ωmax = ωmin þ (n - 1)ω0. The equation for the adsorption isotherm for each state i of the adsorbed protein molecule reads   ωΓi ωi θ ð13Þ exp -2a cb ¼ ω ð1 - θÞωi =ω where c is the protein bulk concentration and b is the equilibrium adsorption constant for the protein in the ith state. Thus, Pthe adsorption constant for the protein molecule as a whole is b = nb. The distribution function of adsorptions over various states of the protein molecule is ð1 - θÞðωj - ωmin Þ=ω exp½2aθðωj - ωmin Þ=ω  Γj ¼ Γ P n ð1 - θÞðωj - ωmin Þ=ω exp½2aθðωj - ωmin Þ=ω 

ð14Þ

i¼1

The isotherm equation for a bilayer adsorption was derived previously30 assuming that the coverage of the second layer ΓIIωmin is proportional to the adsorption equilibrium constant b2 for the second layer, and to coverage of the first layer as well: ΓII ωmin ¼ θ

b2 c 1 þ b2 c

ð15Þ

’ MATERIALS AND METHODS Lysozyme isolated from egg white was purchased from MP Biomedicals and used without additional purification. The tritium label was introduced into lysozyme by the thermal activation method described previously.31 In brief, 0.8 mL of an 1.25 mg/mL aqueous solution of protein was uniformly distributed on the wall of a glass reaction vessel and lyophilized. The reaction vessel was then connected to the device designed for gaseous tritium and filled with tritium gas after evacuation. The reaction with atomic tritium was performed for 10 s at the target temperature of 298 K.25 The formation of tritium atoms was on the surface of the tungsten filament at 1800 K. After the reaction, the lysozyme-target was dissolved in 2 mL of phosphate saline buffer (PBS, pH 7.2 ( 0.1). To purify the labeled material from labile tritium (tritium in the functional groups), a seven-day dialysis through a 12-kDa cutoff membrane against PBS was conducted. For further purification, sizeexclusion chromatography (SEC) on a 1.5  35 cm column, packed with Fractogel TSK HW-40(F) gel (Merck), was performed. SEC was controlled by measuring the radioactivity of the collected fractions and the UV-absorbance at 280 nm. Such a purification technique provides the further radioactivity of labeled product with tritium in CH-bonds.31 The specific radioactivity of the labeled product was 0.43 mCi/mg. The behavior of lysozyme in the aqueous/organic liquid system was studied using the scintillation phase assay. Scintillation cocktails based on p-xylene, octane, and octanol were prepared as described previously.21 To provide p-xylene, octane and octanol scintillation properties with high registration efficiency, 2,5-diphenyloxazole (0.4 vol. %) was added. Naphthalene (4 vol. %) was also added to the octane and octanol. Scintillation phase experiments were performed according to a procedure described previously.13 A 1-mL aliquot of [3H]lysozyme 2190

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solution in PBS was placed in a polyethylene scintillation vial (PerkinElmer) followed by the addition of 3 mL of corresponding scintillator. Then, the counting rate was measured using a RackBeta 1215 liquid scintillation spectrometer (Finland). The system was incubated at 22 ( 1 °C until equilibrium was achieved. When the system had equilibrated, the organic phase (Vbit) was sampled and its counting rate (Ibit) was measured. IV and IS were calculated as: IV ¼

Ibit V Vbit

ð16Þ

Here, V is the volume of the organic phase. The protein concentration in the organic phase and its excess at the interface were calculated according to eq 6 and 7, respectively. The registration efficiency (ε) of tritium β-radiation in the bulk of the scintillation cocktail was 45 ( 3% for octane and 55 ( 5% for p-xylene. Much stronger quenching was observed for octanol because alcohols are quenchers. In this case, the registration efficiency ranged from 5 to 10%, but these values of ε were sufficient for scintillation phase experiments. The equilibrium concentration in the aqueous phase was calculated from the specific radioactivity of the protein and the value of radioactivity sampled from the aqueous phase (eq 6). For aqueous solutions, the OptiPhase Hi Safe 3 (PerkinElmer) scintillation cocktail was applied. The two-phase systems, and each phase separately, were also analyzed by dynamic light scattering (DLS). Measurements were conducted on a Malvern Zetasizer Nano S (Malvern Instruments Ltd., U.K.) with a detection angle of 173°.15 All measurements in this study were taken at a temperature of 25 °C in a quartz cuvette with a polyethylene cap. When the two-phase system was analyzed, it was prepared directly in the cuvette. At least three repeat measurements of each sample were taken to check for result repeatability. The intensity size distributions were obtained from analysis of the correlation functions using the multiple narrow modes algorithm in the instrument software.

’ RESULTS AND DISCUSSIONS Figure 2 shows the rate of lysozyme distribution in the system, i.e., the aqueous/interface/bulk of the organic liquid. For all tested organic liquids, equilibrium was achieved in 40 h. Equilibration time was largely influenced by the molecular weight of the distributed compound; changes ranged from 5 h for tritium water17 to 40 h for proteins (results of present research and ref 31). Figure 3 shows the correlation between lysozyme concentration in the aqueous phase (cW) and its concentration in the organic phase. In previous work,22 we have shown that appearance of the inflection on the curve corg vs cW corresponds to the formation of micelles in the aqueous phase. Linearity of the correlation indicated the absence of micelle-like formations in the aqueous phase in the presence of the organic phase. The distribution coefficient was calculated as KOW = corg/cW. The values obtained were (3.3 ( 0.2)  10-4, (1.0 ( 0.2)  10-3, (2.6 ( 0.2)  10-3 for aqueous/octane, aqueous/p-xylene, aqueous/octanol systems, respectively. The purification procedure of 3H-labeled material allowed the referring radioactivity of the organic phase provided only by 3H-labeled compound instead of by exchangeable tritium. To confirm this observation, 0.5 mL of the organic phase after the scintillation phase experiment was dried and then dissolved in 0.5 mL of scintillation cocktail. The radioactivity values of the sample before and after drying coincided. The value of the free energy of lysozyme transfer from the aqueous phase to the organic liquid was calculated as ΔGtransfer ¼ - RT ln KOW

ð17Þ

Here, T is 295 K. The values obtained were 4.7, 4.0, and

Figure 2. Variation of the counting rate of tritium-labeled lysozyme, I, in a two-phase system with time, during the protein distribution in the bulk of the system and its adsorption at the aqueous/organic liquid interface. The organic liquids are p-xylene (1), octane (2), and octanol (3).

Figure 3. Correlation between lysozyme concentration in the aqueous phase (cW) and in the organic phase (cO). The organic liquids are octanol (1), p-xylene (2), and octane (3).

3.5 kcal/mol for aqueous/octane, aqueous/p-xylene, and aqueous/octanol systems, respectively. The free energy from transferring 1 mol of protein from an aqueous solution to a nonpolar liquid characterizes its total hydrophobicity. The values for the hydrophobicity of lysozyme obtained in the different systems were similar. The rather high value of hydrophobicity and, as a result, the higher the possibility of a protein molecule interacting with the organic phase, explains the fact that proteins can be dissolved in nonpolar solvents. Furthermore, several typical hydrophilic enzymes were shown to possess catalytic activity in the homogeneous solutions of organic liquids.32 Unfortunately, the structural form that a protein possesses in the organic phase has not yet been determined. However, the influence of the organic phase is manifested in the volume distribution of particles (Figure 4). Indeed, the interaction between positively charged lysozyme and the benzyl-ring of p-xylene could result in the formation of large aggregates, including both protein and solvent molecules. The possibility of the formation of H-bonds with octanol could also results in the corresponding volume distribution of particles (Figure 4 d). 2191

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Figure 5. Volume-particle size distribution of lysozyme in a two-phase system (a) and variation of intensity-particle size distributions of lysozyme with time obtained for a two-phase system obtained at 10, 25, 35, and 45 min after the addition of the organic phase (b).

Figure 4. Volume-particle size distributions of lysozyme obtained for the aqueous phase (a), octane phase (b), p-xylene phase (c), and octanol phase (d).

Despite the fact that the volume distribution of a two-phase system and the aqueous phase are the same (Figure 5 a) in the presence of the organic-phase, the polydispersity index of a two-phase system was increased from 0.5 to 0.8 for 45 min postinjection of the organic phase. The intensity of the second peak that corresponded to large particles, which were observed in the organic phase, increased during the existence of the two-phase system (Figure 5 b). This interaction also should be considered when the adsorption process is discussed. Figure 6 shows the adsorption isotherms of lysozyme at the aqueous/organic liquid interfaces. The experimental data are represented by dots. These data are a more complete presentation of the protein absorption behavior at the aqueous/organic liquid interface than provided elsewhere. The method we used is not limited by interfacial tension that fluctuates with protein adsorption at the interface. The onset of the surface tension decrease for protein solutions corresponds to adsorption values of about 1.0 mg/m2 for globular proteins.16 Furthermore, we described 2-day-old adsorption layers, when the majority of the processes are completed and while the other techniques describe adsorption layers of several hours.29,35 The values of the lysozyme surface concentration for all tested interfaces were similar at concentrations bellow 1  10-5 mol/L.

Figure 6. Adsorption isotherms of lysozyme at the aqueous/octanol (1), aqueous/octane (2), and aqueous/p-xylene (3) interfaces. Dots represent experimental data from the scintillation phase; solid line represents theoretical calculations on the bilayer model with the set of parameters presented in Table 1.

The lysozyme surface excess increased upon bulk concentration growth. For higher concentrations (above 2  10-5 mol/L), the surface concentration of lysozyme at the aqueous/octanol interface was higher than that for interfaces with p-xylene and octane. The values obtained for interfaces with p-xylene and octane were rather similar particularly at concentrations bellow 8  10-5 mol/ L. Surface excess slowly increased upon an increase in concentration, indicating the presence of relaxation processes such as reorientation, denaturation or the formation of a multilayer. At high concentrations the reproducibility of the experimental results is decrease because of the possibility of the formation of microemmulsion phase in the subsurface region.22,34 These results can be explained by the strong affinity of the hydrophobic residues of the protein for the organic phase.35 2192

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Table 1. Parameters Obtained for Lysozyme at Aqueous/ p-Xylene, Aqueous/Octane, and Aqueous/Octanol Interfaces organic phase

a,a m2/mol

ω0, m2/mol

b2,a m3/mol

p-xylene

0.6 ( 0.1

4.95  105

7.5 ( 0.5

octane octanol

0.15 ( 0.05 0.8 ( 0.1

6.35  105 3.15  105

15 ( 2 9.5 ( 0.5

a

Confidence intervals were determined taken into account the spread of the adsorption data.

The experimental data were described by the model proposed by Fainerman and co-workers16,27-30,35 for the adsorption of individual proteins, as described above in the theoretical part of this paper. The theoretical curves shown in Figure 6 (solid lines) were calculated for the following values of lysozyme parameters: ωmin = 7.72  106 m2/mol and ωmax = 2.54  107 m2/mol.30 Other parameters were determined for each two-phase system, according to eqs 10-15, and are summarized in Table 1. The positive values of parameter a (Table 1) at the aqueous/ organic liquid interfaces are the consequence of interactions between the adsorbed protein molecules. According the data obtained for the aqueous/air interface in ref 35, this value obviously contributes in minimizing the contact with the gas phase. However, at the aqueous/organic liquid interfaces, this behavior is generally not observed because of the interactions of the hydrophobic parts of the adsorbed protein molecule with the solvent. Thus, a preferable orientation has to favor both these interactions, which as a consequence, also leads to the high concentration of protein observed at the interface. The values of adsorption at aqueous/organic liquid interfaces were compared with those presented for the aqueous/air interface previously found in ref 30. At higher concentrations, the surface excess of lysozyme at the aqueous/organic liquid interface is higher than at the aqueous/air interface. Indeed, the formation of highly saturated adsorption monolayers at the aqueous/p-xylene interface occurs at concentrations of 1.4  10-5 mol/L. However, at the aqueous/air interface, the more highly saturated adsorption layer consists of longways-on oriented molecules formed at concentrations of 7  10-5 mol/L.33 Neglecting the interfacial protein unfolding, it can be estimated that at concentrations of 7  10-5 mol/L, the lysozyme forms two layers at the aqueous/ p-xylene and aqueous/octane interfaces and five layers at the aqueous/octanol interface. Therefore, liquid scintillation spectrometry and dynamic light scattering measurements indicated a high affinity of the protein for organic liquids. The superficial concentrations of protein at the aqueous/organic liquid interfaces exceeded the value at the aqueous/air interface, which indicates the presence of strong hydrophobic interactions between the hydrophobic amino acid residues of the protein with the organic phase. The values of the distribution coefficients of lysozyme in systems of two immiscible liquids, their adsorption at aqueous/organic liquid interfaces and the adsorption parameters calculated according to the Fainerman model will be used in future research, specifically, the investigation of the interaction of lysozyme with low-molecular-weight ionic surfactants in aqueous/organic liquid systems.

’ ACKNOWLEDGMENT Authors would like to acknowledge Dr. Kalmykov (Lomonosov Moscow State University) for providing the hardware equipment used in this study, and Dr. Filatov for his kind attention to this

study. This work was supported by the Russian Foundation of Basic Research (Grant No. 09-03-00819) and by the Federal Targeted Program, “Scientific and scientific-pedagogical personnel of innovative Russia” from 2009 to 2013 (Project No. 2351P).

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dx.doi.org/10.1021/la1037712 |Langmuir 2011, 27, 2188–2194