Miscibility of Binary Monolayers at the Air−Water Interface and

Langmuir 2006, 22, 6195-6202

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Miscibility of Binary Monolayers at the Air-Water Interface and Interaction of Protein with Immobilized Monolayers by Surface Plasmon Resonance Technique Yuchun Wang and Xuezhong Du* Key Laboratory of Mesoscopic Chemistry, State Key Laboratory of Coordination Chemistry, and School of Chemistry and Chemical Engineering, Nanjing UniVersity, Nanjing 210093, P. R. China ReceiVed February 28, 2006. In Final Form: April 22, 2006 The miscibility and stability of the binary monolayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and cationic dioctadecyldimethylammonium bromide (DOMA) at the air-water interface and the interaction of ferritin with the immobilized monolayers have been studied in detail using surface pressure-area isotherms and surface plasmon resonance technique, respectively. The surface pressure-area isotherms indicated that the binary monolayers of DPPC and DOMA at the air-water interface were miscible and more stable than the monolayers of the two individual components. The surface plasmon resonance studies indicated that ferritin binding to the immobilized monolayers was primarily driven by the electrostatic interaction and that the amount of adsorbed protein at saturation was closely related not only to the number of positive charges in the monolayers but also to the pattern of positive charges at a given mole fraction of DOMA. The protein adsorption kinetics was determined by the properties of the monolayers (i.e., the protein-monolayer interaction) and the structure of preadsorbed protein molecules (i.e., the protein-protein interaction).

Introduction Numerous biochemical processes, such as, cell signaling, molecular recognition, and immunoreaction are directly related to protein adsorption.1 The adsorption of proteins and other biological molecules at the liquid-solid interfaces is of critical importance in bioprocessing, biomaterials, and biosensing.2 The solid surfaces investigated are varied from native surfaces3,4 to immobilized layers on surfaces such as self-assembled monolayers (SAMs).5-7 The SAMs terminated with protein-repelling groups can be used in clinical implants and contact lenses,8 and those terminated with functional ligands can be used for biosensors and bioseparation.9 However, the protein-surface interactions that govern the behavior of immobilized protein molecules are not well-established.10 The molar ratios of binary components in the SAMs cannot be precisely controlled, since they are generally different from the molar ratios of the two components in the SAM-forming organic solutions; on the other hand, phase separation readily takes place in the binary SAMs probably due to the limitation of lateral mobility by the immobilization of covalent attachment. Langmuir monolayers at the air-water interface are known to be two-dimensionally fluid, which is favorable to the mixing of binary components to constitute * To whom correspondence should be addressed. E-mail: xzdu@ nju.edu.cn. Fax: 86-25-83317761. (1) Diretrich, C.; Boscheinen, O.; Scharf, K.-D.; Schmitt, L.; Tampe´, R. Biochemistry 1996, 35, 100. (2) Ihalainen, P.; Peltonen, J. Langmuir 2002, 18, 4953. (3) Brusatori, M. A.; Tie, Y.; Van Tassel, P. R. Langmuir 2003, 19, 5089. (4) Marchin, K. L.; Berrie, C. L. Langmuir 2003, 19, 9883. (5) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Angew. Chem. Int. Ed. 2001, 40, 163. (6) Ostuni, E.; Grzybowski, B. A.; Mrksich, M.; Roberts, C. S.; Whitesides, G. M. Langmuir 2003, 19, 1861. (7) Metallo, S. J.; Kane, R. S.; Holmlin, R. E.; Whitesides, G. M. J. Am. Chem. Soc. 2003, 125, 4534. (8) Silin, V.; Weetall, H.; Vanderah, D. J. J. Colloid Interface Sci. 1997, 185, 94. (9) Lahiri, J.; Isaacs, L.; Tien, J.; Whitesides, G. M. Anal. Chem. 1999, 71, 777. (10) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2006, 128, 3939.

different monolayer surfaces. The miscible monolayers are immobilized through Langmuir-Blodgett (LB) technique for protein binding. On the other hand, the molar ratios of binary components in the monolayers can be precisely controlled in principle. Phospholipids form the framework of cellular membranes. The lipids containing the polar groups phosphatidylcholine such as dipalmitoylphosphatidylcholine (DPPC) are one of the most major components of cellular membranes. The zwitterionic headgroups can bind significant amounts of water and possess good biocompatibility to resist protein adsorption.11 Dioctadecyldimethylammonium bromide (DOMA) is a synthetic doublechain cationic surfactant. The totally synthetic bilayer membranes, similar to cellular membranes in structure, were first prepared with DOMA;12 thus, DOMA and its homologues are widely used as the model systems for the simulation of various physicochemical processes in biological membranes.13-15 In addition, both DPPC and DOMA can form stable Langmuir monolayers at the air-water interface.15,16 Here, the DPPC lipids were used to act as a protein-resistant matrix monolayer, in which DOMA surfactants as ligands were distributed, and protein binding was proceeded after the mixed monolayers at the airwater interface were immobilized through LB technique. Ferritin is an almost spherical protein with the molecular weight 680 kDa and the diameter 12.5 nm. Ferritin has an isoelectric point (pI) of 4.8, and its net charge is negative at pH > 5.17 Ferritin was selected as a model protein due to its stable, compact, (11) Murphy, E. F.; Lu, J. R.; Brewer, J.; Ressell, J.; Penfold, J. Langmuir 1999, 15, 1313. (12) Kunitake, T.; Okahata, Y. J. Am. Chem. Soc. 1977, 99, 3860. (13) Cavalli, A.; Dynarowicz-Ła¸ tka, P.; Oliveria, O. N., Jr,; Feitosa, E. Chem. Phys. Lett. 2001, 338, 88. (14) Gonc¸ alves da Silva, A. M.; Roma˜o, R. S.; Lucero Caro, A.; Rodrı´guez Patino, J. M. J. Colloid Interface Sci. 2004, 270, 417. (15) Souza, S. M. B.; Chaimovich, H.; Politi, M. J. Langmuir 1995, 11, 1715. (16) Roma˜o, R. I. S.; Gonc¸ alves da Silva, A. M. Chem. Phys. Lipids 2004, 131, 27. (17) Johnson. C. A.; Yuan, Y.; Lenhoff, A. M. J. Colloid Interface Sci. 2000, 223, 261.

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and nearly spherical structure, which could eliminate discrepancy in adsorption orientation and structure deformation upon binding. A variety of techniques have been used to study protein adsorption.18-26 Among them, surface plasmon resonance (SPR) is a excellent method for the investigation of protein adsorption in real time without labeling analytes. The observation of a SPR shift, during the formation of a protein layer on the surface, will give abundant information about protein adsorption kinetics and surface density.27 The protein binding to the binary monolayers was studied by the SPR technique. During the monitoring, the monolayers were in fact immobilized with the hydrophobically modified surfaces of SPR sensors.18 In this paper, the miscibility and stability of the mixed monolayers of DPPC and DOMA and the interaction of ferritin with the monolayers were systematically investigated using surface pressure-area isotherms and SPR technique, respectively. The protein binding kinetics on surfaces of the mixed monolayers with varied molar ratios and adsorption mechanism were determined not only by the protein-monolayer interaction but also by the protein-protein interaction. Experimental Section Materials. Synthetic L-R-dipalmitoylphosphatidylcholine (DPPC, ∼99%) and dioctadecyldimethylammonium bromide (DOMA, >99%) were purchased from Sigma and Acros Organics, respectively. These chemicals were used without further purification. Their stock solutions were prepared in pretreated chloroform (analytical grade) at a concentration of 1 mM and stored at -20 °C prior to use. The mixtures of DPPC and DOMA were prepared volumetrically from their stock solutions. 1-Octadecanethiol (ODT, 95%) was purchased from Fluka. Triton X-100, ethanol, NaCl, and NaOH were of analytical grade. Water used was double-distilled (pH 5.6, resistivity 18.2 MΩ cm) after a deionized Milli-Q exchange. Horse spleen ferritin (76 mg/mL, Type 1) in 0.15 M NaCl was supplied by Sigma and used as received. Ferritin solution was prepared by diluting the stock solution with double-distilled water to the desired concentrations. All experiments were carried out at a final ferritin concentration of 1.0 µg/mL. Monolayer Spreading and Isotherm Measurements. The surface pressure-area (π-A) isotherms were recorded on a Nima 611 Langmuir trough (Nima Technology, England) equipped with computer controls. The maximum available surface area was 30 cm × 10 cm and could be varied continuously by moving two Teflon barriers. A Wihelmy plate (filter paper) was used as the surface pressure sensor with an accuracy of (0.1 mN/m and situated in the middle of the trough. Chloroform solutions of DPPC, DOMA, and their mixtures at various mole fractions were spread on pure water and aqueous subphase containing 100 mM NaCl, respectively, and then 15 min was allowed for solvent evaporation. Two barriers compressed symmetrically at the same rate of 5 mm/min. The subphase temperature was kept at 18 °C. Each sample was run at least three times to ensure reproducibility. Protein Penetration into/Binding to Mixed Monolayers at the Air-Water Interface. The mixed monolayers of DPPC and DOMA were spread on pure water as described in the isotherm measurements. The two barriers moved at a rate of 2 mm/min. After the surface (18) Du, X.; Hlady, V.; Britt, D. Biosens. Bioelectron. 2005, 20, 2053. (19) Cornec, M.; Narsimhan, G. Langmuir 2000, 16, 1216. (20) Polverini, E.; Arisi, S.; Cavatorta, P.; Berzina, T.; Cristofolini, L.; Fasano, A.; Riccio, P.; Fontana, M. P. Langmuir 2003, 19, 872. (21) Xicohtencatl-Cortes, J.; Mas-Oliva, J.; Castillo, R. J. Phys. Chem. B 2004, 108, 7307. (22) Roach, P.; Farrar, D.; Perry, C. C. J. Am. Chem. Soc. 2005, 127, 8168. (23) Moraille, P.; Badia, A. Angew. Chem. Int. Ed. 2002, 41, 4303. (24) Ngankam, A. P.; Van Tassel, P. R. Langmuir 2005, 21, 5865. (25) Rosilio, V.; Boissonnade, M.-M.; Zhang, J.; Jiang, L.; Baszkin, A. Langmuir 1997, 13, 4669. (26) Kent, M. S.; Him, H.; Sasaki, D. Y.; Satija, S.; Seo, Y.-S.; Majewski, J. Langmuir 2005, 21, 6815. (27) Weimar, T. Angew. Chem. Int. Ed. 2000, 39, 1219.

Wang and Du pressures reached the preset values, the monolayers were held for 5 min for equilibration, then the two barriers were stopped and the monolayer areas were kept constant. The surface pressure as a function of time at the fixed area was recorded. After the 2 h monolayer relaxation, 2 mL of 0.09 mg/mL ferritin solution was injected into the aqueous subphase to achieve a final protein concentration of 1.0 µg/mL. Ferritin was then allowed to adsorb from the unstirred subphase to the monolayers at the air-water interface. The change in surface pressure was recorded for another 2 h. SPR Spectroscopy and Protein Binding. A Teflon trough with a dimension of 4 cm × 2 cm × 0.5 cm and a total subphase volume of 5.6 mL was home-built to minimize the amount of protein needed. The trough walls were undercut by 45° to eliminate the formation of meniscus presenting a planer interface on which an integrated optics SPR sensor (Spreeta, Texas Instruments) was used to measure protein adsorption kinetics for each monolayer.18 The SPR sensor was first cleaned using 1% Triton X-100 and 0.1 M NaOH solution followed by double-distilled water. Its sensing Au surface was made hydrophobic by immersion in a 2 mM ODT solution in absolute ethanol for 10 min. The SPR sensor was initialized in air and calibrated in double-distilled water, and a SPR baseline was obtained in water. The mixed monolayer of DPPC and DOMA was spread until a desired surface pressure of 30 mN/m was achieved. A modified SPR sensor was brought into contact with the mixed monolayer after the monolayer was allowed to relax for 1 h. The monolayer was immobilized on the surface of the modified SPR sensor, and then the SPR signal was recorded and a new SPR baseline was obtained. A total of 5.6 µL of ferritin solution was injected into the subphase to attain a final concentration of 1.0 µg/mL and allowed to adsorb for at least 2 h, followed by exchange of the subphase with water of pH 3.0 to remove ferritin from the monolayer. The subphase was then exchanged with pure water of pH 5.6 prior to reintroducing ferritin into the subphase for binding.

Results and Discussion Isotherms, Miscibility, and Stability of Monolayers at the Air-Water Interface. The π-A isotherms of the monolayers of DPPC, DOMA, and their mixtures at various mole fractions on pure water and aqueous subphase containing 100 mM NaCl were shown in Figure 1. On pure water (part a of Figure 1), the DPPC monolayer underwent a liquid expanded-liquid condensed phase transition around 5 mN/m followed by a liquid condensed phase until a collapse point at about 60 mN/m. The DOMA monolayer showed the feature of expansion with a phase transition around 25 mN/m after which a liquid condensed phase was formed. The corresponding collapse surface pressure was around 47 mN/m. The two isotherms on pure water were in agreement with those in the literature.13,28 All of the mixed monolayers showed the condensed features as compared with the monolayers of the two individual components. The liquid expanded phase and mean limiting molecular area of the mixed monolayers gradually diminished with increasing the mole fraction of DOMA (XDOMA). It was obvious that the interaction between the two individual components was strengthened gradually with the increase of XDOMA. The liquid expanded phase virtually disappeared up to XDOMA ) 0.25. When XDOMA reached 0.3, the smallest limiting molecular area occurred, indicating the strongest attractive interaction between the two individual components in this case. An increase in limiting molecular area was followed for XDOMA ) 0.4 and 0.5 due to the large XDOMA in the binary mixtures. It was clear that the favorable electrostatic interactions occurred between the zwitterionic and cationic headgroups. It was recently reported that the mixing of zwitterionic lipids with cationic ones produced gel-phase supported lipid bilayers free of defects detectable using atomic force microscopy, which (28) Yu, Z.-W.; Jin, J.; Cao, Y. Langmuir 2002, 18, 4530.

Miscibility of Binary Monolayers

Figure 1. Surface pressure-area isotherms of the monolayers of DPPC, DOMA, and their mixtures at various mole fractions of DOMA on different subphases at 18 °C: (a) pure water; (b) aqueous solution containing 100 mM NaCl.

contrasted with the observation of massive defects when anionic or no charged lipids were added.29 It was expected that the cationic lipids mixed well with the zwitterionic ones and “stitched” the bilayers together.29 On the NaCl-containing subphase (part b of Figure 1), the monolayers of the two individual components were considerably condensed in comparison with those on pure water, particularly for the DOMA monolayer. The repulsive interactions between the positively charged headgroups of the DOMA molecules were obviously weakened by the presence of NaCl in the subphase. The collapse surface pressure of the corresponding monolayer was increased to 55 mN/m. In the cases of the mixed monolayers, the limiting molecular areas first reduced with increasing XDOMA from 0 to 0.25, and then reversed upon a further increase in XDOMA up to XDOMA ) 0.5. This result was similar to that on pure water, but the smallest limiting molecular area occurred at XDOMA ) 0.25, due to the salt effect, instead of XDOMA ) 0.3 in the case of pure water. The mixed monolayers on pure water and the NaCl-containing subphase displayed single collapse pressure varying with XDOMA, higher than that of the DOMA monolayer. 30 This suggested that the two components of DPPC and DOMA were miscible on the two subphases at the molar ratios studied. The miscibility of the binary monolayers could be further investigated from the variation of mean molecular area. At a given surface pressure, the excess area can be represented by comparing the mean molecular area of a mixed monolayer (29) Zhang, L.; Spurlin, T. A.; Gewirth, A. A.; Granick, S. J. Phys. Chem. B 2006, 110, 33. (30) Shahgaldian, P.; Coleman, A. W. Langmuir 2003, 19, 5261.

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Figure 2. Excess mean molecular area of the mixed monolayers as a function of mole fraction of DOMA at different surface pressures: (a) pure water; (b) aqueous solution containing 100 mM NaCl.

consisting of components 1 and 2 with that of an ideally mixed monolayer.31-35

Aex(π) ) A12(π) - Aid(π) ) A12(π) - [X1A1(π) + X2A2(π)] (1) where A12 and Aid are the experimental and ideal molecular areas of the mixed monolayers at a given surface pressure, respectively. A1 and A2 stand for the area per molecule of pure components 1 and 2 at the same surface pressure, and X1 and X2 denote the mole fractions in the mixtures. If two components are ideally mixed or totally immiscible, the excess molecular area, Aex, will be zero. Any derivation from ideality would indicate that the two components are miscible. In general, negative derivation implies an attractive interaction between them, whereas a repulsive interaction will lead to positive derivation from an ideal behavior.35 Figure 2 shows the excess molecular area as a function of XDOMA in the mixed monolayers on pure water and the NaCl-containing subphase. Negative derivation for various mole fractions at different surface pressures was observed in the two cases. The (31) Gaines, G. L., Jr. Insoluble Monolayers at Liquid-Gas Interfaces; WileyInterscience: New York, 1966. (32) Chou, T.-H.; Chang, C.-H. Langmuir 2000, 16, 3385. (33) Sennato, S.; Bordi, F.; Cametti, C.; Coluzza, C.; Desideri, A.; Rufini, S. J. Phys. Chem. B 2005, 109, 15950. (34) Sosperdra, P.; Espina, M.; Alsina, M. A.; Haro, M. A. I.; Mestres, C. J. Phys. Chem. B 2003, 107, 203. (35) Rey Go´mez-Serranillos, I.; Min˜ones, J., Jr.; Dynarowicz-Ła¸ tka, P. Minones, J.; Conde, O. Langmuir 2004, 20, 11414.

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Wang and Du

Figure 3. Excess free energy of mixing as a function of mole fraction of DOMA on different subphases at various surface pressures: (a) pure water; (b) aqueous solution containing 100 mM NaCl.

extent of negative derivation diminished with increasing surface pressure, which could be expected since the mixed monolayers at the high surface pressures were more compact and the effect of intermolecular interaction on molecular packing would be less important.32 Moreover the extent of the variation of mean molecular areas on the NaCl-containing subphase was less than that on pure water, especially at the high surface pressures. This indicated that the presence of salt reduced the influence of intermolecular interaction on molecular packing, meaning that the binary components were already close-packed on the NaClcontaining subphases. All of the above results showed that the incorporation of DOMA molecules into the DPPC monolayers might increase the interaction between them and that the mixed monolayers at the molar ratios investigated were miscible through the attractive intermolecular interactions. The stability of a mixed monolayer can be investigated by the Gibbs free energy of mixing, ∆Gmix, and the extent of derivation from an ideal mixing can be represented by the excess free energy of mixing, ∆Gex32,35,36

∆Gex )

∫0π{A12(π) - [X1A1(π) + X2A2(π)]} dπ ∆Gmix ) ∆Gex + ∆Gid

(2) (3)

where ∆Gid is the ideal free energy of mixing, which can be evaluated from

∆Gid ) RT(X1 ln X1 + X2 ln X2)

(4)

where R is the universal gas constant and T is the absolute (36) Pagano, R. E.; Gershfeld, N. L. J. Phys. Chem. 1972, 76, 1238.

Figure 4. Free energy of mixing as a function of mole fraction of DOMA on different subphases at various surface pressures: (a) pure water; (b) aqueous solution containing 100 mM NaCl.

temperature. The ∆Gex of the mixed monolayers versus XDOMA at a fixed surface pressure on the two subphases was shown in Figure 3. The values of ∆Gex were negative at various mole fractions, indicating the mutual attractions of the molecules in the mixed monolayers and the improved stability of the mixed monolayers. Moreover, the negative derivation of ∆Gex was enhanced with increasing surface pressure from 5 to 40 mN/m. Clearly, the more condensed the mixed monolayers, the stronger the intermolecular interactions between the binary components. The occurrence of a minimum suggested that the influence of intermolecular interaction on monolayer stability was more significant for the mixed monolayers at the mole fractions than for those at the other mole fractions. In general, the ∆Gex values were not particularly high (