Influence of membrane organization on the interactions between persistent pollutants and model membranes

Bioelectrochemistry 87 (2012) 192–198

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Influence of membrane organization on the interactions between persistent pollutants and model membranes Dorota Matyszewska, Ewa Wypijewska, Renata Bilewicz ⁎ Faculty of Chemistry, University of Warsaw, ul. Pasteura 1, 02093 Warsaw, Poland

a r t i c l e

i n f o

Article history: Received 28 June 2011 Received in revised form 17 November 2011 Accepted 28 November 2011 Available online 7 December 2011 Keywords: Perfluorooctanesulphonic acid (PFOS) 1,2-dimyristoyl-sn-glycero-3phosphoethanoloamine (DMPE) menadione Cyclic voltammetry Langmuir monolayer

a b s t r a c t Langmuir monolayer studies and electrochemical methods were employed to investigate the effect of model membrane organization on the interactions with persistent pollutants such as perfluorinated carboxylic acids (PFCAs). 1,2-dimyristoyl-sn-glycero-3-phosphoethanoloamine (DMPE) was employed to construct the model lipid membrane and perfluorooctanesulphonic acid (PFOS) was chosen as the representative of perfluorinated pollutants. We demonstrate that perfluorinated compounds penetrate a model membrane only when it is less condensed. Such liquid-expanded phase was achieved by compressing the Langmuir monolayer to lower surface pressures. PFOS incorporation into model DMPE membrane during membrane formation was observed in liquid-expanded region, while at higher surface pressures, in the well-organized monolayer the expulsion of perfluorinated compound occurred as the result of a strong attraction between the DMPE molecules. The DMPE monolayers prepared by the Langmuir–Blodgett/Langmuir–Schaefer method were transferred onto gold electrode under surface pressure of 3 mN/m and 20 mN/m. The model membrane organization depends on surface pressure during transfer and time of exposure to PFOS solution and is shown to affect the electrode accessibility by three electroactive compounds used as the probes of the blocking properties of the monolayer: menadione, potassium ferricyanide and hexaamineruthenium chloride, differing in the properties and kinetics of electron transfer. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Model biological membranes prepared at the air–water interface by Langmuir technique have been successfully used in the studies of the interactions with numerous drugs, enzymes and toxins [1–3]. This technique allows one to observe the influence of those substances on the model membrane at different stages of membrane formation as well as to study possible interactions with already preformed monolayers compressed to various surface pressures under which the monolayer can exist in different phases of molecular organization. Additionally, it is also possible to transfer such model membranes onto solid support to further characterize model membranes by means of electrochemical, microscopic or spectroscopic techniques. Perfluorinated carboxylic acids (PFCAs) are among the persistent pollutants found in numerous locations across the world [4–7]. These are fully fluorinated analogues of carboxylic acids, which have numerous applications in the industry, including production of paints, waxes, adhesives, fire-fighting foams, paper products and water-proof coatings [8–10]. Among this group of chemicals perfluorooctanesulphonic acid (PFOS) is one of the most commonly occurring

⁎ Corresponding author. Tel.: + 48 22 8220211x345; fax: + 48 22 8224889. E-mail address: [email protected] (R. Bilewicz). 1567-5394/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2011.11.014

(Scheme 1) and therefore is used as a model compound to study the effect of perfluorinated compounds on the environment and living organisms. The presence of PFOS has been detected in samples of human blood, plasma, liver, breast milk and umbilical cord blood collected in different countries [11–14]. The effect of this compound on animals and potentially humans is extensively studied. So far it has been established that it may cause developmental toxicity, loss of body weight or pulmonary deficits [15,16]. Moreover, this chemical can affect the properties of cell membranes [17]. However, to fully understand this problem more studies are still needed. We have recently shown that PFCAs are incorporated from the subphase into DPPC and DMPC monolayers during their formation at the air–water interface [18]. The presence of perfluorinated compounds in the subphase caused changes in the structure and organization of phospholipid molecules in the monolayer, which was proved by the changes in the surface pressure-area per molecule isotherms and in the compressibility of the layers. The character of the changes observed upon the incorporation of PFCAs depended on the type of phospholipid, especially on the length of the acyl chain. Moreover, PMIRRAS studies showed that perfluorinated compounds present in DMPC bilayer induce changes in the orientation and conformation of the acyl chains of DMPC molecules [19]. The observed decrease in the tilt angles led to the increase in membrane fluidity and thickness. We also investigated the blocking properties of DMPC bilayers transferred onto gold electrodes by means of

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193

Scheme 1. The structures of perfluorooctanesulphonic acid (PFOS) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanoloamine (DMPE).

electrochemical methods [20,21]. Changes in the electrode accessibility by selected electroactive species after the incubation in PFOS solutions proved the possibility of incorporation of PFOS into supported model DMPC membranes. In the present study we investigated the effect of organization of model membranes on the ease of penetration of persistent pollutants such as perfluorooctanesulphonic acid (PFOS) through the layer. Model membranes composed of 1,2-dimyristoyl-sn-glycero-3phosphoethanoloamine (DMPE) were formed using Langmuir technique. DMPE is a representative of phosphatidylethanoloamines, which have ethanoloamine as a polar head group (Scheme 1) and are the main components of the inner region of human erythrocytes [22]. Moreover, this phospholipid exhibits interesting surface properties due to the presence of two phase transitions [22]. Therefore, it is possible to follow the incorporation of persistent pollutants into a phospholipid monolayer at different phases of the organization and to observe the influence of model membrane structure and organization on the interactions with such substances. Since the selected perfluorinated compound exhibits some surface activity but does not form stable monolayers at the air–water interface itself, it was dissolved in the subphase on which the monolayer was formed. Changes in the properties of DMPE monolayers upon the incorporation of PFOS dissolved in various concentrations in the subphase were observed. The possibility of PFOS to penetrate the phospholipid monolayers precompressed to selected surface pressures corresponding to different organization of model membranes was also evaluated. In the second part of this study the incorporation of perfluorinated compound into supported DMPE bilayers was verified by electrochemical techniques. The DMPE bilayers were transferred onto gold electrodes by two selected surface pressures: 3 mN/m and 20 mN/m in order to compare the blocking properties of model membranes characterized by different organization of molecules in the membrane. The differences in the electrode access for the selected electroactive probes: menadione, potassium ferricyanide and hexaamineruthenium complex were observed for gold electrodes modified with DMPE bilayers and after the incubation of modified electrodes in PFOS solution.

2. Experimental 2.1. Monolayer experiments 1,2-dimyristoyl-sn-glycero-3-phosphoethanoloamine (DMPE) (Avanti Polar Lipids) was dissolved in chloroform/methanol mixture

(90:10 v/v) to give 1 mg/ml stock solution. DMPE monolayers at the airsolution interface were formed using Langmuir trough KSV LB Trough 5000 (KSV Ltd., Finland) equipped with two movable barriers and a Wilhelmy balance with a paper Wilhelmy plate (paper plate was changed after each experiment). The trough was controlled by a computer using KSV-5000 software version. The monolayers were formed on pure water and subphases containing different concentrations of PFOS (Sigma-Aldrich) solutions in water. After cleaning the subphase and spreading a few microliters of DMPE solution at the interface, the solvent was allowed to evaporate for approximately 15 min. Compression of the film was performed at a speed of 7.5 cm2/min at a constant temperature of 21± 1 °C.

2.2. Electrochemical experiments DMPE bilayers were used as supported model membranes in the voltammetric studies. They were deposited on gold electrodes (11 × 11 mm slides, Arrandee), which were 200–300 nm thick gold films evaporated onto borosilicate glass precoated with an underlayer of chromium. Prior to the deposition, the gold substrates were flame annealed and cleaned in the mixture of H2O2:NH3:H2O with 1:1:5 ratio at 70 0 C for approximately 5 min and rinsed with Milli-Q water. The DMPE layers were transferred from the air–liquid interface onto gold electrode at two selected surface pressures: 3 mN/m and 20 mN/m. The first layer was deposited on the gold surface by vertical withdrawal of the electrode at the speed of 35 mm/min to give a transfer ratio of 1.0 ± 0.1. The electrode was allowed to dry for approximately 1.5 h and the second layer was deposited at a surface pressure of 3 mN/m and 20 mN/m, respectively by employing the horizontal touch method (Langmuir–Schaefer technique). Such a combination of Langmuir–Blodgett/Langmuir–Schaefer techniques has been successfully used to prepare supported phospholipid bilayers of different compositions as reported by other authors [24,25]. Electrochemical experiments were performed by means of AutoLab AUT 71819 with the GPES 4.9 software in three electrode cell with Ag/AgCl as a reference electrode and platinum foil as a counter electrode. The supporting electrolyte was 50 mM phosphate buffer, pH = 6.9 (sodium phosphates from POCh Gliwice, Poland). Menadione (Sigma-Aldrich), potassium ferrocyanide (POCh Gliwice, Poland) and hexaammineruthenium (III) chloride (Sigma-Aldrich) were used as electroactive probes in cyclic voltammetry experiments. Distilled water used throughout all Langmuir–Blodgett and electrochemical experiments was passed through a Milli-Q® water purification system (resistivity 18.2 MΩ/cm).

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3. Results and discussion It is known that DMPE forms stable monolayers at the air–water interface and undergoes two phase transitions [23]. A typical isotherm of DMPE on water subphase is presented in Fig. 1A (black curve). The lift-up of the isotherm starts at area per molecule corresponding to approximately 100 Å 2. Then the first phase transition observed as plateau region at 5 mN/m is associated with the transition from liquid-expanded phase (LE) to liquid-condensed phase (LC). The second transition is attributed to the transition from the condensed phase with tilted chains to a condensed phase with erected chains and is observed at surface pressure of approximately 30 mN/ m. These phase transitions can be easily noticed by following the changes in the reciprocal of compression modulus value, which can be calculated according to the following formula [26]: Cs

−1

¼ −Aðdπ=dAÞ

ð1Þ

where A is area per molecule, and π is surface pressure. The phase transitions are manifested as minima in compression modulus versus surface pressure plot (Fig. 1B). The maximum value of the reciprocal of compression modulus for DMPE monolayers (215 mN/m) is in the range of 100–250 mN/m corresponding to the liquid-condensed phase [27]. The presence of the second phase transition, when the acyl chains change their orientation from tilted to erected, is typical for DMPE and is not observed for other phospholipids (such as DPPC or DMPC) commonly used for the preparation of model cell membranes by means of Langmuir technique. This phenomenon might be explained by differences in the polar head groups of those phospholipids. Contrary to DMPC and DPPC, which have choline group in polar head group region, DMPE molecule contains smaller amine group. Moreover, the interactions between polar heads of DMPE are stronger than those occurring between phosphocholines because hydrogen bonds are formed between the amino and phosphate group of neighboring molecules of DMPE [28]. Therefore, DMPE molecules can pack more tightly in a monolayer and due to those stronger interactions and tighter packing, the second phase transition from the condensed phase with tilted chains to a condensed phase with erected chains occurs. 3.1. Influence of PFOS on DMPE monolayers at the air–water interface The isotherms of DMPE monolayers formed on subphases containing different concentrations of PFOS were compared with an isotherm

Surface pressure / (mN/m)

70

A)

B)

60 50 40

of DMPE obtained on pure water (Fig. 1A). The observed changes in surface properties were of complex nature and strongly depended on the PFOS concentration in the subphase. For smaller concentrations of PFOS the value of mean area per molecule (A0) slightly decreased (Table 1). Additionally, the influence on monolayer shape, particularly on the surface pressure of liquid-expanded (LE)–liquidcondensed (LC) phase transition, was not that significant. The presence of small concentrations of PFOS in the subphase caused only a small shift of the minimum corresponding to LE–LC transition towards higher surface pressures and the maximum value of reciprocal of compression modulus increased in comparison to DMPE monolayer formed on pure water. Increasing PFOS concentration in the subphase to 5 ∙ 10 − 5 M caused not only a significant shift of LE–LC phase transition towards higher surface pressure and an increase in mean area per molecule, but also the maximum value of reciprocal of compression modulus decreased (Fig. 1). It may be explained by the fact that probably PFOS molecules easily incorporate into DMPE monolayer when phospholipid molecules are less organized, i.e. in liquid-expanded state. It is visualized by the observed changes in the surface pressure at which phase transition occurs. However, in case of lower PFOS concentrations in the subphase, PFOS molecules were expelled from the monolayer during its further compression, which resulted in a slight decrease in the value of mean area per molecule (Table 1). The observed decrease might be related to the “memory effect”—the presence of PFOS in the monolayer leaves a permanent change in the monolayer organization. Therefore, even after the repulsion of the perfluorinated compound from the phospholipid monolayer during the compression, the structure of the monolayer remains disturbed and it results in the decrease in the value of area per molecule. Additionally, the maximum value of reciprocal of compression modulus increased when lower concentrations of PFOS in subphase were present, which suggest the slight ordering of the monolayer after the expulsion of PFOS molecules (Table 1). Only in the case of the highest PFOS concentration employed mean area per molecule increased and reciprocal of compression modulus decreased significantly. It might be explained by the fact that probably so many PFOS molecules partitioned into the monolayer, that not all of them were squeezed out from the monolayer during its further compression and as a net result mean area per molecule increased and the monolayer lost its condensed character in the presence of the highest investigated PFOS concentration in the subphase. This situation has not been observed during previous studies of the influence of perfluorinated compounds on DMPC and DPPC monolayers, when even for small concentrations of perfluorinated compound in the subphase an increase in the mean area per molecule was observed. DMPC or DPPC molecules are packed more loosely in a monolayer and may accommodate a larger amount of other small molecules such as PFOS in a monolayer, which leads to the observed changes in characteristic parameters of the monolayers. In case of DMPE monolayers, further compression of the monolayer caused stronger interactions between polar head groups and resulted in the expulsion of PFCAs molecules from DMPE monolayer at higher surface pressures (Fig. 1). Only in case of the highest PFOS concentration no expulsion was detected. A similar observation was made by Hąc-

30 20

Table 1 Characteristic parameters of DMPE Langmuir monolayers formed on subphases containing different concentrations of perfluorinated compounds.

10 0 20

40

60

80

100

120

140

160

Area per molecule / (Å2) Fig. 1. A) Surface pressure—area per molecule (π–A) isotherms B) reciprocal of compression modulus vs. surface pressure plot of DMPE monolayers on pure water (black), 10− 6 M PFOS (red), 5 ∙ 10− 6 M PFOS (green), 10− 5 M PFOS (blue), 5 ∙ 10− 5 M PFOS (cyan) and 10− 4 M PFOS (magenta) subphases.

Subphase

A0 [Å2]

Cs− 1max value [mN/m]

πLE-LC [mN/m]

Water 10− 6 M PFOS 5 ∙ 10− 6 M PFOS 10− 5 M PFOS 5 ∙ 10− 5 M PFOS 10− 4 M PFOS

44.3 42.0 42.2 43.5 44.5 46.0

212 236 225 216 167 151

5.1 8.1 9.3 10.5 15.3 19.1

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3.2. Interactions with precompressed DMPE monolayers

21 18 15

Δπ / (mN/m)

Wydro et al. who investigated surface behavior of mixed monolayers of an antibiotic nystatin and different phospholipids at the air–water interface [29]. For mixed monolayers of nystatin and phospholipids differing in polar head group region they noticed that phosphatidylethanoloamines due to their closer packing in the monolayer are able to accommodate smaller amount of nystatin compared to phosphocholines.

195

12 9 6 3 0

The “squeezing out” effect was observed only at higher surface pressures and for smaller concentrations of PFOS. At lower values of surface pressure when the monolayer is not that tightly packed, PFOS molecules incorporated into the monolayer (Fig. 1). Therefore, the possibility of PFOS to incorporate into already existing model DMPE membranes compressed to lower surface pressures was evaluated. DMPE monolayers were formed on pure water subphase and initially compressed to two different values of surface pressures: 3 mN/m and 20 mN/m corresponding to liquid-expanded and liquid-condensed phase of the monolayer. After reaching the target surface pressure, the barriers were stopped to keep constant area and PFOS solutions were injected into the subphase to obtain different final concentrations. Afterwards, the changes of surface pressure in time were recorded (Fig. 2A and B). For both investigated initial surface pressures the increase in surface pressure after injecting PFOS was observed. The value of surface pressure increase (Δπ) defined as the difference between surface pressure obtained for DMPE monolayer formed on pure water and surface pressure for DMPE monolayer after injecting PFOS solution was calculated after

-3 -6 0

20

40

60

80

100

[PFOS] / (μmol / l) Fig. 3. Difference in surface pressure (Δπ) after injection of PFOS versus PFOS concentration for monolayers initially compressed to (■) π = 3 mN/m; (□) π = 20 mN/m.

140 min (Fig. 3). For the lowest final PFOS concentration in the subphase after injection the Δπ value decreased slightly in case of both initial surface pressures. This observation stays in good agreement with the results of monolayer studies presented in Section 3.1 because it was shown that the presence of lower concentration of PFOS in the subphase may cause some disordering in DMPE monolayers. It is clearly visible especially in case of higher surface pressure investigated in this type of experiments (20 mN/m). However, increasing the final concentration of PFOS in the subphase led to the increase in Δπ value. It suggests that PFOS molecules partition into DMPE monolayer which is not tightly packed at the two investigated surface pressures. Therefore, PFOS molecules may slowly incorporate into precompressed DMPE monolayer and partition into it, which is manifested by the increase in surface pressure in time (Fig. 2A and B). However, the incorporation of PFOS has not been observed for DMPE monolayers initially compressed to 35 mN/m (data not shown). At such high surface pressure DMPE molecules in the monolayer are closely packed and there are strong interactions between polar head groups, which prevent from PFOS incorporation into the monolayer. A similar observation has been made by Bordi et al. who investigated the penetration of two different anthracyclines into DMPE monolayer [30]. The drugs were able to adsorb and penetrate only the monolayers compressed to relatively low surface pressures. 3.3. Transport of menadione through DMPE bilayer in the absence and presence of PFOS Menadione (2-methyl-1,4-naphthoquinone) is an electroactive compound from a group of K vitamins, which are quinones involved in many biological and physiological systems such as energy transduction [31]. Its reduction is described by the following equation [32]:

Fig. 2. Changes of surface pressure in time of DMPE monolayers formed on water (black) and after injecting PFOS solutions to obtain final concentrations of 10− 6 M PFOS (red), 10− 5 M PFOS (green), 5 ∙ 10− 5 M PFOS (blue) and 10− 4 M PFOS (magenta). The monolayers were initially compressed to A) π = 3 mN/m; B) π = 20 mN/m.

This hydrophobic and lipid soluble species, contrary to other commonly used electroactive probes such as ferricyanides (Fe(CN)63 −/4 −), can permeate organic layers assembled on electrode surface [32]. Therefore menadione, along with negatively charged ferricyanides and positively charged hexaamineruthenium complex, which are

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transported to electrode surface through the defects (pinholes) in phospholipid bilayer [33,34], was used to study the changes in the blocking properties of DMPE bilayers transferred onto Au(111) electrodes at two surface pressures: 3 mN/m and 20 mN/m. The preparation of DMPE bilayer supported on gold electrode is described in detail in Section 2.2. The values of surface pressures were selected according to the results of monolayer studies presented in Section 3.3 and correspond to liquid-expanded and liquid-condensed phase, respectively. Transport of menadione through the blocking phospholipid layer was compared for the two types of modified electrodes. Apparently, it takes place both through the defects and by means of permeation through the model membrane. For electrodes modified with DMPE bilayers transferred at 3 mN/m the electron transfer rate constant (ks) for menadione was found to be higher than that for the electrode modified with DMPE bilayer transferred at 20 mN/m (Fig. 4 and Table 2). The values of electron transfer rate constant (for menadione as well as for potassium ferricyanide described in the next section) were calculated by fitting the curves using the GPES 4.9 software. It suggests that the contribution of the transport through the defects in the bilayer deposited at lower surface pressure is greater than that for the bilayer deposited at 20 mN/m. This explanation takes into account the fact that at a surface pressure of 3 mN/m corresponding to liquid-expanded phase the DMPE molecules are loosely distributed in the electrode surface and not well organized, which leads to the presence of relatively great amount of defects in the modifying layer and enables relatively easy access to electrode surface for menadione. However, after the incubation in 10 mM PFOS solution for 100 min the electron transfer rate constant decreases and its value was similar for the both types of electrodes modified with DMPE

-150

A)

j / (μA / cm2)

-100 -50 0 50 100 0.0

-0.1

-0.2

-0.3

-0.4

E / V vs Ag/AgCl

-150

B)

j / (μA / cm2)

-100 -50

0 50 100 0.0

-0.1

-0.2

-0.3

-0.4

E / V vs Ag/AgCl Fig. 4. Cyclic voltammograms in the presence of 0.2 mM menadione in supporting electrolyte (50 mM phosphate buffer pH = 6.9; scan rate 50 mV/s) recorded for bare Au(111) electrode (black), electrode modified with DMPE bilayer (red) and after incubation of modified electrode for 100 min in 10 mM PFOS solution (green); A) DMPE bilayer transferred at 3 mN/m; B) DMPE bilayer transferred at 20 mN/m.

Table 2 Electron transfer rate constants (ks) for menadione and ferricyanides. Electrode modified with DMPE bilayer Menadione (ks/cm s− 1) 3 mN/m 2.58 × 10− 3 20 mN/m 2.01 × 10− 3 Fe(CN)63 −/4 − (ks/cm s− 1) 3 mN/m 5.50 × 10− 5 20 mN/m 5.17 × 10− 5 Ru(NH3)63+/2+ (Ip/μA cm− 2) 3 mN/m − 110 20 mN/m − 75

After 100 min incubation in 10 mM PFOS 1.20 × 10− 3 1.15 × 10− 3 4.49 × 10− 5 7.11 × 10− 5 − 225 − 125

bilayer transferred at the two surface pressures (Table 2). It implies that PFOS blocks the defects in phospholipid bilayer and as a result contribution of menadione transport to the electrode through the defects is significantly reduced. Interestingly, PFOS incorporation into the layer does not influence menadione transport taking place by means of permeation through phospholipid bilayer and therefore the observed values of ks constant for both types of modified electrodes are similar (Fig. 4).

3.4. Potassium ferricyanide and hexaamineruthenium chloride penetration through the DMPE layer in the absence and presence of PFOS Blocking properties of DMPE bilayers transferred at two selected surface pressures were also evaluated by cyclic voltammetry in the presence of potassium ferricyanide and hexaamineruthnium chloride. Ferricyanide is negatively charged electroactive probe which is often used to monitor the changes in electrochemical characteristics of the electrode caused by its modification [35,36]. Its transport to the surface of a modified electrode takes place through the defects in the blocking layer, which is described by a “pinhole model” [32]. Modification of electrode surface with DMPE bilayer resulted in a decrease in ks value, which was greater for the electrodes with DMPE bilayer transferred at 20 mN/m (Table 2). It implies that the model membrane transferred at lower surface pressure is characterized by more defects than better organized DMPE bilayer deposited at 20 mN/m. However, after the incubation in 10 mM PFOS solution the electron transfer rate constant decreased for electrodes modified with DMPE bilayer at 3 mN/m and increased for electrodes modified with DMPE bilayer at 20 mN/m (Table 2). The decrease in the ks value for electrodes modified with DMPE bilayer at 3 mN/m can be explained by the fact that PFOS easily incorporates into the modifying layer, since there is a significant amount of defects and DMPE molecules are not well organized. As a result, there are many PFOS molecules located in the defects in DMPE bilayer, through which the transport of ferricyanides takes place. Apparently, the amount of incorporated PFOS molecules is that big that the layer becomes negatively charged and the negatively charged ferricyanides are repulsed from the electrode. Therefore, the decrease in the electron transfer rate constant is observed. In case of electrodes modified with DMPE bilayer transferred at 20 mN/m situation is slightly different. The bilayer is better organized and fewer defects are present. Incubation in the solution of perfluorinated compound led to the incorporation of PFOS into the layer. In the first place the defects in the bilayer were occupied. However, it is also possible that PFOS penetrated those regions of DMPE membrane that were free of pinholes. As a result, the structure became less compact and the access to the electrode surface for the electroactive probe became easier. Despite electrostatic repulsions between negatively charged PFOS anions in the bilayer and negatively charged ferricyanides, the net effect observed is the increase in electron transfer rate constant. This observation is consistent with previous reports

D. Matyszewska et al. / Bioelectrochemistry 87 (2012) 192–198

on the interactions of ferricyanides with model membranes with incorporated PFOS molecules [20,21]. Hexaamineruthenium ion, positively charged probe undergoing simple 1e outer-sphere electrode process, is also commonly used as a measure of the extent of electrode surface blocking by thin films [33,37]. For electrodes modified with DMPE bilayers the decrease in peak current corresponding to ruthenium reduction was observed (Table 2 and Fig. 5). However, the value obtained for the electrode modified with DMPE bilayer transferred at 3 mN/m was higher than the value for the electrode modified with 20 mN/m, which means that the bilayer transferred at lower surface pressures has worse blocking properties. This observation is consistent with the results obtained in electrochemical experiments in the presence of ferricyanides and menadione (Section 3.4). After incubation in PFOS solutions the peak current increased for both types of modified electrodes (Table 2 and Fig. 5). However, the increase was much more pronounced for the electrode modified with DPME bilayer transferred at 3 mN/m. As it was mentioned above, PFOS molecules incorporate much more easily into DMPE monolayers transferred at lower pressures and due to the relatively high concentration of PFOS in the model membrane, the layer becomes negatively charged. However, this time the electroactive probe used is positively charged and therefore the electrostatic attraction between the probe and PFOS anions present in the bilayer was observed. As a result, the peak current increased significantly. Similar electrostatic attractions occurred in case of electrodes modified with DMPE bilayers transferred at

-300

A)

j / (μA cm-2)

-200

-100

0

100

200 0.0

-0.1

-0.2

-0.3

-0.4

E / V vs Ag/AgCl -300

B) j / (μA cm-2)

-200

-100

0 100

197

20 mN/m but in this case there were fewer PFOS molecules incorporated in the layer and the observed increase in peak current was lower (Table 2). Additionally, control experiments when modified electrodes were incubated for 100 min in pure water (without PFOS) were also carried out to prove that the incubation period has no effect on the organization of the supported phospholipid layers transferred at different surface pressures (Fig. 5). 4. Conclusions Langmuir monolayer technique allowed us to follow the incorporation of perfluorooctanesulphonic acid dissolved in the subphase into DMPE monolayer during its formation. We found that the ease of penetration of the pollutant depended on the integrity of the monolayer. The effect was dependent on PFOS concentration in the subphase and the strongest interactions between DMPE and PFOS molecules were observed in liquid-expanded region when the monolayer was not tightly packed. In the presence of perfluorinated compound the LE-LC phase transition was shifted towards higher surface pressures, which could be easily noticed on the compression modulus versus surface pressure dependence. Further compression of the monolayer led to expulsion of incorporated perfluorinated compound from the monolayer in case of smaller concentrations of PFOS in the subphase. This squeezing out effect was probably caused by strong attractive interactions existing between the DMPE molecules in a well-organized monolayer. Due to those cohesive interactions, DMPE monolayers can accommodate smaller amount of other species when compared to less tightly packed DMPC or DPPC monolayers [18]. PFOS was able to penetrate DMPE monolayer, which was initially compressed to relatively low surface pressures corresponding to liquid-expanded or liquid-condensed phase. PFOS incorporation into supported DMPE bilayers and the influence of model membrane organization on the interactions of this pollutant with the membrane was verified using voltammetry. The selected probes: menadione, potassium ferricyanide and hexaamineruthenium chloride accessed the electrode either by means of permeation through the blocking phospholipid bilayer (menadione) or through the defects in the model membrane (potassium ferricyanide and hexaamineruthenium chloride). It was shown that less organized DMPE bilayers transferred at lower surface pressure (3 mN/m) are characterized by worse blocking properties for the investigated probes than the layers transferred at higher surface pressure (20 mN/m). However, the incorporation of PFOS was facilitated for more loosely organized model membranes with greater amount of defects, which were occupied by PFOS molecules in the first place. Apparently, the presence of perfluorinated compound in a model membrane did not influence the transport of menadione taking place by means of permeation though the phospholipid bilayer but it did influence the electrode accessibility by the other two electroactive species (negatively charged potassium ferricyanide and positively charged hexaamineruthenium chloride), which access the electrode through the defects in the bilayer. The electrostatic interactions between incorporated PFOS anions and the two probes also affect the accessibility of the bilayer covered electrode surface. Acknowledgement

200 0.0

-0.1

-0.2

-0.3

-0.4

This work has been supported by the grant Iuventus Plus IP2010 025370 from the Polish Ministry of Sciences and Higher Education.

E / V vs Ag/AgCl Fig. 5. Cyclic voltammograms in the presence of 1 mM Ru(NH3)63+/2+ in supporting electrolyte (50 mM phosphate buffer pH = 6.9; scan rate 50 mV/s) recorded for bare Au(111) electrode (black), electrode modified with DMPE bilayer (red) and after incubation of modified electrode for 100 min in 10 mM PFOS solution (green); control experiments after incubation of modified electrode for 100 min in water (blue); A) DMPE bilayer transferred at 3 mN/m; B) DMPE bilayer transferred at 20 mN/m.

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Dr. Dorota Matyszewska is currently research associate at the Faculty of Chemistry, University of Warsaw. She received MsC in analytical chemistry and PhD in inorganic chemistry from the University of Warsaw. During her PhD studies she focused on the influence of perfluorinated compounds on model cell membranes. Her research interests also include drug delivery systems.

Renata Bilewicz is Full Professor in Warsaw University, Faculty of Chemistry. She received her PhD in 1984 in the group of Prof. Zenon Kublik, University of Warsaw and in 1996 was appointed as Professor in Warsaw University. In 2000 she became Full Professor in the same university. In 1990 she was awarded a Fellowship for Young Professors of Eastern Europe by the Swiss Government, a PECO fellowship from Brussels and a grant from the Swiss National Foundation. Visiting Professor at University of Basel and University of North Carolina, Raleigh. She was member of the International Advisory Board of Electrochimica Acta and currently is a member of Bioelectrochemical Society Council (2011–2015). Her primary interests include bio- and supramolecular electrochemistry. The research of her team is concerned with electron transfer processes in supramolecular and biomolecular systems, properties of self-assembled mono- and multilayers, molecular recognition processes at interfaces, electrochemically triggered molecular machines and macrocyclic receptors, enzyme-based fuel cells. She is author or co-author of over 150 publications, 5 book chapters and several review papers.