Structure and Electrostatic Interaction Properties of Monolayers of

Jul 29, 2000 - Monolayers at the air/water interface of a new amphiphilic molecule derived from a C60-fullerene were studied at different lateral pres...
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Structure and Electrostatic Interaction Properties of Monolayers of Amphiphilic Molecules Derived from C60-Fullerenes: A Film Balance, Neutron-, and Infrared Reflection Study† Andreas P. Maierhofer,‡ Michael Brettreich,§ Stephan Burghardt,§ Otto Vostrowsky,§ Andreas Hirsch,§ Sean Langridge,| and Thomas M. Bayerl*,‡ Universita¨ t Wu¨ rzburg, Physikalisches Institut EP5, D-97074 Wu¨ rzburg, Germany, Universita¨ t Erlangen-Nu¨ rnberg, Institut fu¨ r Organische Chemie, D-91054 Erlangen, Germany, and Rutherford Appleton Laboratory, ISIS, Chilton, U.K. Received March 1, 2000. In Final Form: May 25, 2000 Monolayers at the air/water interface of a new amphiphilic molecule derived from a C60-fullerene were studied at different lateral pressures by a combination of film balance techniques, neutron reflection (NR), and infrared reflection-absorption spectroscopy (IRRAS). The amphiphilic fullerene derivative (AF) consisted of a dendrimeric hydrophilic region, and 10 alkyl chains covalently attached to the fullerene cage formed the hydrophobic part. The AF monolayers could be compressed and expanded without significant hysteresis and the alkyl chains remained fluid at all pressures. By a titration series, the pK value of the AF monolayer was determined as 7.5 and pH dependent measurements allowed a variation of the negative AF headgroup charge by about 18 charges. The thickness of the AF monolayer at high lateral pressure was 30 Å, thus similar to that of typical phospholipid monolayers in the condensed state. In contrast, the AF molecular area was about 6-fold higher than that of phospholipids at high pressure. Moreover, the hydration capacity of the AF headgroup is significantly higher than that of phospholipids. The negatively charged AF monolayer showed a strong coupling of the water-soluble protein cytochrome c from the subphase, leading to the formation of a 30 Å thick protein layer underneath the AF layer. The protein content of this layer varied drastically with the pH value. The properties of the AF monolayers may be useful in the design of dedicated biomimetic surfaces.

Introduction Optimized membrane mimetics are a prerequisite for many biosensor applications. So far, this is achieved by biofunctionalization of solid surfaces using self-assembled phospholipid monolayers (SAMÅs) or bilayers, where the latter are either separated from the solid surface by an ultrathin water layer or (additionally) by a water-swollen polymer cushion. Often the purpose of the functionalization requires the presence of electrically charged phospholipids in the mono- or bilayer to control and modulate surface charge properties. However, a drawback of the most abundant charged phospholipids is that they feature not more than one charge per molecule at relevant pH values. Hence, the achievable charge density of biofunctionalized surfaces is often limited by the molecular area of the lipids used. Furthermore, since the pK values of the lipids are mostly outside the biologically tolerable pH limits, slight variations in pH around the neutral value (pH ) 7.0) do not result in a modulation of the surface charge density. Finally, in some cases where the functionalized surfaces have to be phospholipase resistant, the use of phospholipids is prohibited. Here we present a new bilayer- and monolayer forming amphiphile which is based on fullerene derivatives where † Part of the Special Issue “Colloid Science Matured, Four Colloid Scientists Turn 60 at the Millenium. * Corresponding author. Address: Universita¨t Wu¨rzburg, Physikalisches Institut EP-5, D-97074 Wu¨rzburg, Germany. Phone: 49-931-8885863. Fax: 49-931-8885851. E-mail: bayerl@ physik.uni-wuerzburg.de. ‡ Universita ¨ t Wu¨rzburg. § Universita ¨ t Erlangen-Nu¨rnberg. | Rutherford Appleton Laboratory.

the hydrophobic part is made of alkyl chains attached to the fullerene cage and the hydrophilic part consists of a dendrimeric structure which can carry up to 18 negative charges at the corresponding pH (Figure 1). While lipophilic derivatized fullerenes and their nanostructure formation in phospholipid bilayers by self-assembly processes has been studied in some detail,1-3 amphiphilic derivatized Fullerenes represent a completely new class of amphiphiles4 with many unknown physical properties. Very recently, we have demonstrated the ability of amphiphilic fullerene derivatives to form spontaneously bilayer vesicles in excess water.5 An interesting aspect of these amphiphiles for biological surface functionalization is the potentially large number of negative charges per molecule. Thus, slight pH variations could be used to achieve drastic changes of the surface charge density and thus of the electrostatic interaction potential of the surface with water-soluble biomolecules. Thin films of derivatized fullerenes (for review see ref 6) have been studied over several years and for some of these molecules stable Langmuir monolayers were pre(1) Hetzer, M.; Bayerl, S.; Camps, X.; Vostrowsky, O.; Hirsch, A.; Bayerl, T. M. Adv. Mater. 1997, 9, 913-917. (2) Hetzer, M.; Gutberlet, T.; Brown, M. F.; Camps, X.; Vostrowsky, O.; Scho¨nberger, H.; Hirsch, A.; Bayerl, T. M. J. Phys. Chem. 1999, 103, 637-642. (3) Hetzer, M.; Clausen-Schaumann, H.; Bayerl, S.; Bayerl, T. M.; Camps, X.; Vostrowsky, O.; Hirsch, A. Angew. Chem., Int. Ed. Engl. 1999, 38, 1962-1965. (4) Cardullo, F.; Diederich, F.; Echegoyen, L.; Habicher, T.; Jayaraman, N.; Leblanc, R. M.; Stoddart, J. F.; Wang, S. Langmuir 1998, 14, 1955-1959. (5) Brettreich, M.; Burghard, S.; Bo¨ttcher, C.; Bayerl, S.; Hirsch, A. Angew. Chem. 2000. In press. (6) Prato, M. Top. Curr. Chem. 1999, 199, 173-187.

10.1021/la000297e CCC: $19.00 © 2000 American Chemical Society Published on Web 07/29/2000

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Figure 1. Structure of the amphiphilic fullerene derivative (AF) studied. For neutron reflection and IRRAS experiments, the C12 alkyl chains were perdeuterated.

pared and studied by different techniques.4,7-13 However, none of these molecules showed the potential to form spontaneously vesicles in a similar way as phospholipids do. The amphiphilic fullerene derivative presented in this work exhibits this feature. In this paper we wish to concentrate on the physical properties of monolayers at the air/water interface formed by this amphiphilic molecule. We have combined the film balance technique with neutron reflection (NR) and infrared reflection-absorption spectroscopy (IRRAS) to obtain complementary information about the lateral (7) Fukuto, M.; Penanen, K.; Heilmann, R. K.; Pershan, P. S.; Vaknin, D. J. Chem. Phys. 1997, 107, 5531-5546. (8) Ravaine, S.; Mingotaud, C.; Delhaes, P. Thin Solid Films 1996, 285, 76-79. (9) Ravaine, S.; Faye, V.; Nguyen, H. T.; Delhaes, P. J. Phys. Chem. Solids 1997, 58, 1753-1756. (10) Vaknin, D. Phys. B 1996, 221, 152-158. (11) Wang, P.; Metzger, R. M.; Chen, B. Thin Solid Films 1998, 329, 96-99. (12) Wang, S. P.; Leblanc, R. M.; Arias, F.; Echegoyen, L. Thin Solid Films 1998, 329, 141-144. (13) Zhang, W.; Shi, Y. R.; Gan, L. B.; Wu, N. Z.; Huang, C. H.; Wu, D. G. Langmuir 1999, 15.

pressure dependence of the monolayer structure and to observe the adsorption of a water-soluble protein (cytochrome c) to the monolayer from the subphase. We were particularly interested in monolayer properties of these “designed” amphiphiles which can help to overcome the above-mentioned shortcomings of phospholipids in biofunctionalization and which may improve the functional and structural properties of advanced surface coatings. Materials and Methods Materials. The synthesis of the amphiphilic hexakisadduct of a C60 fullerene (AF) as shown in Figure 1 and of its alkyl chain perdeuterated analogue (AF-d250) was performed according to procedures described elsewhere.5 The final product was dissolved in chloroform at a concentration of 0.2 nmol/µL. For all monolayer experiments at the air/water interface, the subphase contained 0.25 mM EDTA and was buffered with either 25 mM phosphate or 20 mM HEPES. If not indicated otherwise, the pH was adjusted by NaOH (approximately 13 mM for the phosphate buffer, approximately 4 mM for the HEPES buffer) to pH 7.0 ( 0.1. Buffers were prepared using either Millipore purified water (film balance measurements), or D2O (97.5% purity from

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Deuchem GmbH, Leipzig, Germany) for IRRAS measurements, or D2O (99.9% purity from Fluorchem Ltd., UK) and a mixture of 91.1:8.7 (vol/vol) H2O:D2O for NR experiments. The latter mixture gives a neutron scattering length identical to that of air and will be denoted as contrast matched to air (CMA) water in the following. Cytochrome c was obtained from Fluka (Deisenhofen, Germany) and was dissolved in an aliquot of the buffer at a concentration of 2 mg/mL. The dissolution in D2O containing buffer was done at least 2 h in advance of the experiments to allow labile protons to exchange with deuterons from the buffer. AF monolayers were spread from the organic solution using a microsyringe and afterward compressed to the desired surface pressure. For NR and IRRAS experiments, Langmuir troughs were used which allowed the pressure tight sealing of an inner “protein compartment” after compression by closing a 2 mm wide channel link with a Teflon plate.14 This procedure avoided film leakage and allowed a better control of the protein concentration in the subphase. Only the sample in the separated compartment was illuminated by the neutron or IR beam and contributed to the recorded signal. The maximum subphase surface area of the Langmuir troughs was 289 and 223 cm2 for the NR and IRRAS experiments, respectively. The inner “protein compartments” had areas and volumes of 64.5 cm2 and (38.5 ( 2.5) mL for NR and of 49.7 cm2 and (27.5 ( 2.5) mL for IRRAS. Film balance experiments at different pH values were carried out on a Langmuir trough with a maximum surface area of 422 cm2 and a subphase temperature of 20.0 ( 0.2 °C for all experiments. The pK value of the AF monolayer was determined employing a dedicated film balance equipped with an inner “titration compartment” as follows: An AF monolayer was first compressed to 4.5 mN/m on a subphase of pH 4.0. After this, the channel link between the trough and the titration compartment was pressure tightly closed and the pH of the compartment subphase was successively increased by injecting a total of 500 µL of 1.00 M NaOH into the subphase through the injection hole and the change of π was recorded after appropriate equilibration. Methods. Neutron reflection (NR) was measured at the CRISP spectrometer of the ISIS spallation source (Rutherford-Appleton Laboratory, Chilton, U.K.) according to procedures previously described in detail.14-17 After compression of an AF-d250 monolayer to approximately 35 mN/m, the channel link of the protein compartment was closed and reflectivity curves were recorded. After this, 100 µL of cytochrome c solution was injected into the subphase through submersed injection holes located in the edge of the inner protein compartment, giving a final cytochrome c concentration of c0 ) 420 ( 30 nM in all experiments. Subsequently, a series of NR measurements (duration 15 min each) were performed for about 2 h and the lateral pressure π was recorded. Only those data recorded after equilibration of the surface pressure were considered for data analysis. To aid data analysis of NR measurements on a D2O subphase, samples of the subphase were collected during the experiments and their isotopic purity was checked by proton NMR, which accurately quantified any changes in subphase neutron scattering length due to unavoidable atmospheric H-D exchange. To facilitate the data analysis in terms of multilayer model fits, the measurements were performed on D2O and CMA subphase (contrast variation). IRRAS measurements were performed using a Perkin-Elmer Spectrum 2000 spectrometer with a liquid N2 cooled MCT detector. For each spectrum 512 interferograms were acquired at a resolution of 4 cm-1 (acquisition time ca. 9 min). The system was equipped with a user-modified Specac (LOT, Langenberg, Germany) external reflection unit and a home-built film balance. The angle of incidence was 28° with respect to the surface normal. (14) Naumann, C.; Dietrich, C.; Behrisch, A.; Bayerl, T.; Schleicher, M.; Bucknall, D.; Sackmann, E. Biophys. J. 1996, 71, 811-823. (15) Naumann, C.; Dietrich, C.; Lu, J. R.; Thomas, R. K.; Rennie, A. R.; Penfold, J.; Bayerl, T. M. Langmuir 1994, 10, 1919-1925. (16) Naumann, C.; Brumm, T.; Rennie, A. R.; Penfold, J.; Bayerl, T. M. Langmuir 1995, 11, 3948-3952. (17) Maierhofer, A. P.; Bayerl, T. M. In Modern optics, Electronics and High Precision Techniques in Cell Biology; Isenberg, G., Ed.; Springer-Verlag: Berlin, 1998; p 139-157.

Maierhofer et al. To maintain a constant water vapor content and temperature, the setup was placed in a hermetically sealed and thermally insulated sample container. The change of water level in the trough due to evaporation during the experiment was found to be negligible. Spreading of the lipid monolayers and injection of the protein solution were achieved without opening the sample container by operating through small holes. Measurements were done by switching between two troughs at regular intervals (every 10 min) at the beam position using a home-built trough shuttle system controlled by the acquisition computer. One trough equipped with the inner “protein compartment” contained the monolayer system under study (sample) while the other (reference) was filled with the pure subphase. The shuttle motion did not cause any additional changes of the lateral pressure in the sample trough during the experiment compared to a trough kept fixed for the same time. Reflectionabsorbance (RA) spectra were generated from subsequent sample and reference measurements using GRAMS Version 3.01 software (Galactic Industries Corp., Salem, NH). Here RA is defined as RA ) -log(RS/RR), where RS and RR represent the reflectance of the sample and of the reference compartment, respectively. In a first step several RA spectra of the bare subphase surface were recorded as “reference spectra”. After the water vapor bands had reached a constant level, AF or AF-d250 monolayers were spread and RA spectra were recorded (denoted “AF spectra”). Since H2O exhibits a strong absorbance in the amide I region (1700-1600 cm-1), it is unsuitable for quantitative measurements in this region. Therefore, all IRRAS experiments on CdO stretching or amide I absorption were conducted using a D2O subphase, which shows only very weak absorption in this region. After the injection of 80 µL of cytochrome c solution into the subphase, resulting in a concentration of c0 ) 420 ( 30 nM, spectral features of the amide I bands became visible in the RA spectra. “Protein spectra” were recorded over a total time of up to 5 h. Only those data recorded after equilibration of the surface pressure were considered for data analysis.

Data Analysis NR Data. NR data were analyzed by least-squares fitting of multilayer models to the reflectivity curves using the program MULF which implements the optical matrix method.18 Each layer in the fit is characterized by its thickness dj, its scattering length density (SLD) Fj, and a Gaussian roughness σj. In our case, all fits were performed with σj ) 0. Errors of the fits were calculated from the variance/covariance matrix generated during the fitting procedure. For pure AF-d250 monolayers those solutions with comparable fit quality parameters χ2 15,16 were selected which gave consistent results for both D2O and CMA as the subphase. Using a two-layer model significantly improved the fit quality in comparison to a one-layer model. There remained multiple possible solutions with equal χ2 values which were characterized by a common total monolayer layer thickness but different single layer thicknesses and SLDs. To interpret the two-layer fits by a model which represents the hydrophobic part of the molecule (deuterated chains and fullerene body) and the hydrophilic part (dendrimer + water), additional constraints were applied: In the CMA contrast the water molecules around the dendrimer do not contribute to the SLD. For this reason it is possible to calculate the molecular area

Amol,j ) bj/Fjdj

(1)

separately from both layers of the model. Here bj, Fj, and dj are the scattering lengths calculated from the molecular composition and the fitted SLD and thickness of the layers. (18) Born, M.; Wolf, E. Principles of Optics, 6th ed.; Pergamon Press: Oxford, 1993.

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Only those fits were selected for which Amol,1 and Amol,2 agreed within the error limits. Another constraint was a reasonable agreement between Amol,j and Amol,FB obtained from the film balance measurement. The protein coupled to the monolayer was considered as a third layer14,19 in the NR data analysis. In the fitting process of this three-layer system, the values obtained for the pure AF-d250 layer were kept constant and the values d3 and F3 (thickness and SLD of the protein layer) were allowed to vary (first iteration step). In a second iteration step the thickness of the hydrophobic region only was kept constant while all other parameters of the AF layer were allowed to vary close to their initial values. The latter step considers that changes in hydration and orientation of the headgroup as well as a slight shift of layer boundaries may occur as a result of the protein adsorption. This resulted in a significant improvement of χ2 even though the changes in the AF layer in the second iteration step were very small and remained always within the limits known from several previous experiments. 15,19,20 From the fitting results the value ηPr, the fraction of protein in the “protein layer”

ηPr ) (FW - FPr)/(FW - FPr,0)

Figure 2. Pressure-area isotherms of AF monolayers at T ) 20.0 °C at different subphase pH values.

(2)

was calculated. Here FW is the SLD of the subphase and FPr,0 is the SLD of the pure protein, considering that FPr,0 is a function of FW owing to the exchange of labile protons in the protein. IRRAS Data. To improve the compensation of water vapor bands, difference spectra were calculated from the “AF-spectra” and the reference spectra. For the determination of the height of the amide I absorbance of cytochrome c, difference spectra of the “protein spectra” and the “AF-spectra” were calculated. Results The dendrimer-like hydrophilic part of AF comprises 18 carboxyl groups which release their protons under appropriate pH conditions. This allows the variation of the surface charge density of the monolayer and the electrostatic repulsion between the molecules over a wide range. Pressure-area isotherms of AF were recorded on subphases with pH 2.1, pH 3.9, pH 7.0, and pH 12.1 (Figure 2). From these π(A)-isotherms the increasing dominance of electrostatic repulsion with increasing pH values becomes quite obvious: For low pH (2.1 and 3.9), the lowpressure regime (π < 5 mN/m) at a molecular area A beyond 350 Å2 is characterized by a pressure-area behavior typical for a lipid monolayer in the gas phase. For A e 350 Å2 the slope of the isotherm changed rather abruptly, indicating a diminished compressibility of the layer. In contrast, at pH 7.1 and even more at pH 12.1, this transition becomes increasingly diffuse and π remained remained above zero pressure even at high molecular area. In the high-pressure regime (π > 25 mN/m) the compressibility is similar at all pH values but A increases by about 30% from pH 2.1 to pH 12.1. For the case of monovalent ions in the subphase the electrostatically induced lateral pressure πel follows a (19) Johnson, S. J.; Bayerl, T. M.; Weihan, W.; Noack, H.; Penfold, J.; Thomas, R. K.; Kanellas, D.; Rennie, A. R.; Sackmann, E. Biophys. J. 1991, 60, 1017-1025. (20) Brumm, T.; Naumann, C.; Sackmann, E.; Rennie, A. R.; Thomas, R. K.; Kanellas, D.; Penfold, J.; Bayerl, T. M. Eur. Biophys. J. 1994, 23, 289-296.

Figure 3. Titration isotherm of an AF monolayer. The solid line represents the number of negative charges n as calculated by eq 3.

[cosh(eΨ0/(2kT)) - 1] dependence.21 Here e is the elementary charge, Ψ0 the surface potential, k the Boltzmann constant, and T the temperature. According to the Grahame equation the surface charge density σ is proportional to sinh(eΨ0/(2kT)).22 Thus, for a high potential (Ψ0 < -100 mV) which is the case at low ionic strength, πel increases linearly with σ and, because of σ ) ne/A, with n (n, number of negative charges per AF molecule; A, molecular area). Therefore, the surface pK value of the monolayer can be determined from a titration series. The titration isotherm is shown in Figure 3. The solid line represents a calculation of the negative charges per molecule as a function of the pH value according to

n ) nmax/(1 + 10(pK-pH))

(3)

The steep increase of π from 6.0 mN/m at pH 6.0 to 37 mN/m at pH 9 is clearly a result of increasing electrostatic repulsion upon dissociation of the carboxyl groups. From the turning point of the titration curve the average pK value was obtained as pK ) 7.5 ( 0.1. The molecular area at 35 mN/m and pH < 6 was A ) 247 ( 3 Å2. Because there are 10 C-12-chains attached to the fullerene cage, the area per C-12 chain at this pressure was still well above the lower packing limit of alkyl chains (g20 Å2). Hence, no squeeze-out of molecules can be (21) Payens, T. A. Philips Res. Rep. 1955, 10, 425-432. (22) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1992.

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Maierhofer et al. Table 1. Structural Parameters of the AF-d250 Monolayera D2O

CMA

n AFB/Å2 π/[mN/m] b1/10-5 Å b2/10-5 Å

8.0 276 ( 9 34.0 ( 0.2 3081 ( 20 578 ( 13

4.3 283 ( 9 32.0 ( 0.2 3081 ( 20 512 ( 6

d1/Å F1/10-6 Å-2 d2/Å F2/10-6 Å-2

18.0 ( 0.5 5.96 ( 0.3 11.8 ( 0.5 4.77 ( 0.3

18.5 ( 0.5 5.53 ( 0.02 11.8 ( 0.5 1.43 ( 0.04

ANR/Å2 ANR,2/Å2 nw

287 ( 5

301 ( 5 305 ( 12

55 ( 3

a

Figure 4. IRRAS spectrum of AF-d250 monolayers at π ) 33 mN/m and 20 °C on a buffered D2O subphase (HEPES buffer, pD ) 7.4). The peaks were assigned to the symmetric and asymmetric C-D stretching mode (2199 and 2097 cm-1), the CdO stretching mode of the acid moiety (1749-1704 cm-1), the amide I band (1637 cm-1), and the amide II band (1561 cm-1). The insert shows the C-H stretching region of AF at 33 mN/m on buffered H2O subphase, pH 7.0.

expected at this pressure. This is consistent with the fact that we observed no significant hysteresis upon multiple cycles of compression and expansion. No indication of a phase transition to a more condensed phase comparable to the LC or S phase in lipid monolayers was found by film balance measurements for any of the pH values studied. To study possible phase transitions in more detail, IRRAS measurements were performed on both AF and AF-d250 monolayers. A typical IRRAS spectrum obtained at high lateral pressure of the AF monolayer is shown in Figure 4. The peak frequencies of the asymmetric and symmetric methylene stretching vibrations at 35 mN/m were measured as νasym ) 2926 cm-1 and νsym ) 2855 cm-1. The chain perdeuterated AF-d250 monolayer exhibited at the same pressure values for the C-D stretching vibration of νasym ) 2199 cm-1 and νsym ) 2097 cm-1. No change of frequency for both monolayers was observed for measurements at low pressure (π < 6 mN/m). Together with the film balance data this strongly indicates that the alkyl chains are in a disordered, fluidlike state at all pressures. To obtain structural information, neutron reflection (NR) experiments using monolayers of the chain perdeuterated AF-d250 were performed. Analysis of the NR curves in terms of a two-layer model (hydrophilic dendrimer region and hydrophobic fullerene cage and alkyl chain region) constrained by the film balance (area/ molecule) and IRRAS (fluid phase state at all pressures) results (cf. methods section) gave for both D2O and CMA contrast a total monolayer thickness of dtot ) 29.5 ( 1.0 Å. The results of NR data analysis are summarized in Table 1. Since the protons of the carboxyl groups can readily exchange with deuterons from the D2O subphase, the measured scattering length of the hydrophilic headgroup must be different for D2O and CMA subphases and will vary for different pH values. The number n of bound protons/deuterons can be calculated using eq 3. For the measurements on D2O, a

Top part: data obtained from film balance measurements (n, number of negative charges per molecule; AFB, molecular area; π, lateral pressure) or directly from the chemical composition (b1, b2: scattering lengths of the hydrophobic and hydrophilic part of the molecule). Middle part: parameters obtained from two-layer fits to the NR data (d1, d2, F1, F2: thicknesses and SLDs of the hydrophobic and hydrophilic part of the molecule, respectively). Bottom part: parameters calculated from the NR data (ANR, molecular area calculated from the hydrophobic part; ANR,2, molecular area calculated from the dendrimer part; nw, number of water molecules per AF headgroup).

correction to the pH value measured by a glass electrode must be applied according to pD ) pH meter reading +0.4.23 Moreover, for the case of D2O contrast there is an additional SLD contribution by all the D2O molecules present in the dendrimer region. Hence, the hydration number of the headgroup is given by

nw ) (F2d2Amol - b2)/bw

(4)

Here F2 and d2 are the fitted SLD and the thickness of the headgroup region, b2 is the calculated scattering length of the dendrimer, and Amol is the molecular area obtained from the film balance and NR experiments. Using nw, the volume fraction occupied by water molecules for a single AF-dendrimer headgroup can be calculated as

x ) nwVw/V2

(5)

Here Vw ) 30.3 Å3 is the volume occupied by one water molecule24 and V2 ) d2Amol is the volume occupied by the AF headgroup and the surrounding water molecules. We obtained nw ) 55 at π ) 34 mN/m, thus x ≈ 0.5, indicating that even at high lateral pressure there is still about 50% of the headgroup volume occupied by water. In a second series of experiments, the adsorption of a water-soluble protein which exhibits 9 cationic excess charges at neutral pH (cytochrome c) to the AF monolayer was studied. The motivation of this study was the fact that the AF molecule shows approximately the same number of anionic charges at pH 7.5 and thus a very strong Coulomb coupling can be expected. Protein adsorption experiments were carried out on subphases of different ionic strength and pH. The injection of cytochrome c to the subphase at π ) 33 ( 1 mN/m resulted in a pressure increase which depended on the subphase ionic strength and pH value (Table 2). Figure 5 shows the reflectivity curve of the AF monolayer at 34 mN/m before and after the coupling of cytochrome c from (23) Glasoe, P. K.; Long, F. A. J. Phys. Chem. 1960, 64, 188-190. (24) Nagle, J. F.; Wiener, M. C. Biochim. Biophys. Acta 1988, 942, 1-10.

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Table 2. Pressure Increase ∆π Due to Cytochrome c Adsorption to a AF-d250 Monolayer at π ) 33 mN/m under Different Buffer Conditions

a

pD

[Na+]

∆π/[mN/m]

teq/ha

7.4 7.4 7.4 4.4

5 38 58 0

11 11 7 6

1.5 2 3 0.7

teq: time for complete equilibration of the surface pressure

Table 3. Results of Three-Layer Data Fits (Thickness and SLD for Each Layer) of the AFul-d250 Monolayer (d1, d2, G1, G2) and of the Adsorbed Cytochrome c Monolayer (d3, G3) d1/Å F1/10-6 Å-2 d2/Å F2/10-6 Å-2 d3/Å F3/10-6 Å-2 ηa

D2 O

CMA

18.0 5.56 ( 0.03 11.8 4.77 29.3 ( 0.4 5.10 ( 0.02 0.34 ( 0.02

18.5 5.20 ( 0.2 11.8 1.45 ( 0.04 32.1 ( 0.5 0.64 ( 0.02 0.35 ( 0.02

a η: volume fraction of cytochrome c in the “protein layer” (third layer).

Table 4. Intensities of the Amide I Band of Cytochrome-c Obtained by IRRAS upon Adsorption of the Protein to AF-d250 Monolayers under Different Subphase PD Conditions pD

[Na+]/mM

I/mAUa

7.4 7.4 4.4 7.4b

5 58 0 5

6.1 2.4 2.0 5.9

a mAU: milli absorbance units. b The bottom row gives the intensity I after the pD was raised from 4.4 to 7.4 by titration of NaOH into the subphase.

Figure 5. Neutron reflectivity curves for aF-d250 monolayers before (circles) and after (squares) the addition of 420 ( 30 nM cytochrome c to the subphase, measured at π ) 33 mN/m and 20 °C on a buffered D2O (pD ) 7.4) (a) subphase and on (b) a subphase of water (pH ) 7.0) contrast matched to air. The solid lines represent the best fits to the data in terms of a two-layer model for the pure monolayer and of a three layer model for the monolayer with cytochrome c adsorbed, respectively. The inserts show the scattering length density (SLD, in 10-6 Å-2.) profiles as calculated from the fits before (solid line) and after (doted line) the adsorption of cytochrome c.

the subphase. The significant changes of the reflectivity profile indicate a strong coupling of the protein with the AF monolayer. Data analysis in terms of a three layer model (two for the AF monolayer as above, one for the protein layer) provided the thickness and SLD of the individual layers as summarized in Table 3. While the thickness values and densities of the AF head and tail regions remain rather unchanged, a third (protein) layer of about 30 Å thickness can be clearly observed after the cytochrome c adsorption. The SLD of this layer allowed the calculation of its protein density at pH 7 and [Na+] ) 38 mM as 0.34 ( 0.02 in both D2O and CMA. The protein adsorption was also observed by IRRAS measurements by analyzing the amide I signal intensity arising from adsorbed cytochrome c. Under the assumption that cytochrome c couples spatially oriented to the

Figure 6. IRRAS amide I band intensity of cytochrome-c adsorbed to AF-d250 monolayers at different D2O subphase buffer conditions: The solid lines show the signal at low ionic strength at pD ) 4.4 (small signal) and after the addition (titration) of NaOH to the subphase, giving pD ) 7.4 (big signal). The dotted line shows the cytochrome-c signal at low ionic strength, pD ) 7.4 and the dashed line in a subphase with of 58 mM Na+, pD ) 7.4 (independent measurements).

monolayer and retains its conformational state, the intensity of this band scales with the amount of cytochrome c adsorbed. This allowed us to explore the variation of protein adsorption with the pH value of the subphase by perfoming experiments at pD 7.4 and pD 4.4 subphases (Table 4 and Figure 6). An IRRAS measurement at pD 4.4 showed a dramatic reduction in the amide I signal compared to that of the experiment at pD 7.4. Increasing the pD from 4.4 to 7.4 by injecting NaOH to the subphase resulted in a recovery of the amid I intensity back to the value which was obtained previously for a pD 7.4 subphase. The fact that at pD 4.4 still some protein adsorbed to the monolayer can be attributed to charge fluctuations at the low ionic strength (0.1 mM, corresponding to a Debye length of ≈30 nm) at which the experiments were performed.

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It is unlikely that conformational changes of the protein contribute significantly to the observed pH dependent absorption since the position of the amide I band maximum was 1646 ( 2 cm-1 under all buffer conditions. This suggests that cytochrome c was not a subject to major conformational changes in the pH range studied, which is in agreement with previous findings by other authors.25 Discussion In this paper we describe a new amphiphilic molecule which can form stable monolayers at the air/water interface. Although there is quite a number of amphiphilic molecules known beside the well established phospholipids which can potentially form monolayers (e.g., peptides, lipopolymers), many of these molecules exhibit unstable monolayers at higher lateral pressure. In most cases, such molecules tend to partition into the aqueous subphase upon increase of π. Even pure monolayers of certain anionic phospholipids such as phosphatidyl-glycerols are unstable at high lateral pressures under low ionic strength conditions. In contrast, the AF monolayer can be compressed with virtually no expulsion of molecules into the subphase. A similar stability of an amphiphilc derivatized Fullerene monolayer was already observed for a molecule which consisted of a hydrophilic dendrimer attached to the C60 (i.e., without the alkyl chains).4 Despite high lateral pressure, the AF monolayer retained the fluidity of the AF alkyl chains and a remarkable high hydration (about 50% of the headgroup volume at 34 mN/m is occupied by water). Furthermore, the pK value of 7.5 obtained for the AF monolayer allowed the control of the monolayer charge state (from 18 negative charges to neutral) by a pH variation around the neutral value. Thus, a maximum charge density of one negative charge per 18 Å2 can be achieved, comparable to that of a condensed (LC) arachidic acid monolayer. The fact that the AF monolayer is still fluid at this charge density suggests a more biocompatible interface than for the fatty acids. Common charged phospholipids require even in the LC phase at least twice the area per charge compared to the AF. This stresses the importance of these new molecules for biomimetic applications. The summarized properties may be important in future applications of AF monolayers for the functionalization of surfaces and for the study of electrostatic coupling and immobilization of biomolecules (enzymes, proteins, peptides). After all, the tremendous charge concentration per AF molecule and their high headgroup hydration may provide an optimized surface for multilayer preparations involving polyelectrolytes. It is interesting to compare our results with those obtained for phospholipid monolayers at the air/water interface. The monolayer thickness of AF of dtot ) 29.5 ( 1.0 Å at 34 mN/m (20 °C, pH 7.0) is similar to that of commonly used phospholipids such as dipalmitoyl-phosphatidyl-choline (DPPC) at comparable conditions (28 Å at 35 mN/m,16) though the DPPC monolayer is in liquidcondensed (LC) phase under this pressure and temperature. Also the headgroup thicknesses of both molecules are comparable at these conditions: 10.9 Å for DPPC16 and 11.8 Å for AF headgroups (Table 1). Finally, also the hydrophobic regions compare well in thickness: 17.0 Å for DPPC16 and 18.5 Å for AF (Table 1). On the other hand, the DPPC monolayer exhibits a hydration of nw ) 4 at this pressure while the AF (25) Goto, Y.; Hagihara, Y.; Hamada, D.; Hoshino, M.; Nishii, I. Biochemistry 1993, 31, 11878-11885.

Maierhofer et al.

monolayer has nw ) 55. This is not just a consequence of the lower molecular area of DPPC: At 35 mN/m and pH 7.0, the molecular area of DPPC is 52 Å2 16 while that of AF is 287 Å2 and thus approximately 5-fold higher while the hydration of the latter is a factor of 14 higher. To relate the hydration with the molecular area rather than the headgroup volume is justified in this special case since the headgroup thicknesses of DPPC and AF are similar. However, one may argue that the hydration comparison at 34 mN/m is hampered by the fact that DPPC is in a much higher ordered state (LC or S phase) at this pressure. Therefore, a value of 9 mN/m (LE phase) might be more appropriate for a comparison. At this pressure the DPPC monolayer exhibits nw ) 10 and AM ) 77 Å.2 16 Comparing this with the AF values for 34 mN/m gives now a considerably better match. The latter shows a 5.5-fold higher hydration at a 4.5-fold higher molecular area. Nevertheless, even in comparison with a fluidlike DPPC does the AF monolayer exhibit a higher hydration per molecular area. The measurements of protein adsorption at pH 7.0 gave a cytochrome-c content of the 30 Å thick adsorbed protein layer of 34%. This is close to the cytrochrome-c coverage of 39% observed previously by NR for a condensed (LC phase) phospholipid monolayer of equimolar DPPC with dimyristoyl-phosphatidyl-glycerol (DMPG) at pH 7.0.17 In contrast, the maximum coverage assuming a twodimensional hexagonal lattice of spherical protein molecules (30 Å diameter) can be estimated as 60%. Calculating the area per single anionic charge in the monolayer gives 37 Å2 for AF and 50 Å2 for the lipid mixture at comparable high pressure π. The reason this higher charge density does not manifest itself in higher protein coverage lies in the different salt conditions. The phospholipid experiments were performed under very low ionic strength while the NR measurements of the AF monolayer were done at [Na+] ) 38 mM. Indeed, a comparison of the amide I intensity of cytochrome c adsorbed to the AF monolayer at high π under conditions of 5 and 58 mM sodium in the subphase (pD 7.4) shows a significant higher intensity for the low salt condition (Table 4). Nevertheless, other factors may contribute to differences in the protein coverage for the AF and phospholipid monolayers: (1) The charges in the AF monolayer may not be equally accessible to the protein due to steric reasons which arise from the effective molecular shape of the dendrimeric structure. (2) The projected area of cytochrome c (≈700 Å2) is roughly twice the molecular area of an AF molecule at high π, thus the protein’s eight positive charges can cover a monolayer area occupied by at least two AF molecules having approximately eight negative charges each. For the equimolar lipid mixture, the ratio is more favorable since an average of seven anionic DMPG molecules will nearly compensate for the protein charge over the same area. This charge/area mismatch of the AF/cytochrome c system is very likely the reason we did not observe in our NR experiment any significant changes in the protein coverage when the D2O subphase was substituted by CMA, which can be expected to reduce the charges per AF molecule by approximately 50%. A theoretical estimate on the basis of the Gouy-Chapman and the Grahame equations confirms this observation. The electrostatic potential at 1 nm mean distance off the charged monolayer surface varies by just 6% when the number of eight charges per AF molecule (D2O subphase) is reduced to four (CMA subphase). However, if the number of charges is further reduced as was the case in our pH variation IRRAS measurements, then the monolayer becomes increasingly undercharged

Monolayers of Amphiphilic Molecules

compared to the protein layer which, in the limit, leads to the detachment of the latter into the subphase. Conclusion We have shown that AF molecules can form stable monolayers at the air/water interface of a layer thickness similar to that of typical phospholipids and exhibit surface properties (high hydration and protein binding capacity, electrostatic interaction potential modulation over a wide range by pH variation around the neutral value) which could be useful in advanced biomimetic surface applica-

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tions. Since the AF can also form stable bilayers, future studies will focus on the modulation of bilayer surface and micromechanical properties by this new class of amphiphiles. Acknowledgment. The technical assistance of Gabriela Cocora during the neutron experiments is gratefully appreciated. This work was supported by research grants from the DFG and the BMBF. LA000297E