Directed Assembly of Binary Monolayers with a High Protein Affinity

J. Phys. Chem. B 2007, 111, 2347-2356

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Directed Assembly of Binary Monolayers with a High Protein Affinity: Infrared Reflection Absorption Spectroscopy (IRRAS) and Surface Plasmon Resonance (SPR) Xuezhong Du* and Yuchun Wang Key Laboratory of Mesoscopic Chemistry (Ministry of Education), State Key Laboratory of Coordination Chemistry, and School of Chemistry and Chemical Engineering, Nanjing UniVersity, Nanjing 210093, People’s Republic of China ReceiVed: August 17, 2006; In Final Form: January 10, 2007

Infrared reflection absorption spectroscopy (IRRAS) and surface plasmon resonance (SPR) techniques have been employed to investigate human serum albumin (HSA) binding to binary monolayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and cationic dioctadecyldimethylammonium bromide (DOMA). At the air-water interface, the favorable electrostatic interaction between DPPC and DOMA leads to a dense chain packing. The tilt angle of the hydrocarbon chains decreases with increasing mole fraction of DOMA (XDOMA) in the monolayers at the surface pressure 30 mN/m: DPPC (∼30°), XDOMA ) 0.1 (∼15°), and XDOMA ) 0.3 (∼0°). Negligible protein binding to the DPPC monolayer is observed in contrast to a significant binding to the binary monolayers. After HSA binding, the hydrocarbon chains at XDOMA ) 0.1 undergo an increase in tilt angle from 15° to 25∼30°, and the chains at XDOMA ) 0.3 remain almost unchanged. The two components in the monolayers deliver through lateral reorganization, induced by the protein in the subphase, to form multiple interaction sites favorable for protein binding. The surfaces with a high protein affinity are created through the directed assembly of binary monolayers for use in biosensing.

Introduction Numerous biochemical processes, such as cellular signaling, molecular recognition, and immunoreaction are directly related to protein adsorption.1 The adsorption of proteins and other biological molecules at the interfaces is of critical importance in bioprocessing, biomaterials, and biosensing.2 The surfaces terminated with protein-repelling groups can be used in clinical implants and contact lenses,3 and those terminated with functional groups can be used for biosensors and bioseparation.4 In a variety of biological processes, protein-membrane interactions are often multivalent, and their properties can differ markedly from the corresponding monovalent constituents.5 Multivalent interactions are crucial for discovering a broad range of biosensors with various functions for use in medicine, environment, fermentation, and food processing. A few model surfaces offer platforms for studying proteinmembrane interactions. Protein adsorption at the self-assembled monolayers (SAMs) consisting of binary lipids has been studied,6-8 but the multivalent interactions between the proteins and functional lipids are obviously depressed due to the limitation of lateral mobility through the immobilization of covalent attachment. On the other hand, the molar ratios of different components in the SAMs cannot be precisely controlled, because they are generally different from the original molar ratios in the solutions, from which the SAMs are formed.9 However, the multivalent protein binding can be realized through the interactions with unilamellar vesicles or fluid-supported lipid bilayers (SLBs) rearranged from the unilamellar vesicles.10,11 Phospholipids are well-known to form the framework of cellular membranes, and the distribution of the phospholipids with different headgroups varies depending on the membrane leaflet. * Corresponding author. E-mail: [email protected]. Fax: 86-2583317761.

The asymmetric distribution of the lipids in the SLBs is more recently demonstrated by Rossetti et al.12 It is obvious that the mole fractions of the functional lipids on the surfaces of the multicomponent vesicles or fluid-SLBs cannot be precisely controlled. Langmuir monolayers at the air-water interface are known to be two-dimensionally fluid, which enables them to mimic cell surface rearrangement. Lateral mobility is expected to play a key role in free rearrangement of ligand molecules in membranes,13-15 because a ligand for the second and subsequent binding events can be delivered through the lateral rearrangement of monolayer components.11,16 In comparison with the fluid-SLBs (a thin layer water of about 1 nm thick between the bilayer and an underlying support17), the molar ratios of different components in the Langmuir monolayers can be precisely controlled. Besides, the lipid components in the monolayers are capable of lateral mobility. This study is motivated by the possibility of directed protein binding to the fluid monolayers to generate tailor-made surfaces with a high protein affinity for biosensing and high-density information storage. The zwitterionic phospholipids with the headgroups phosphatidylcholine (PC), such as dipalmitoylphosphatidylcholine (DPPC), are one of the major components of cellular membranes. The zwitterionic headgroups can bind significant amounts of water and possess good biocompatibility to resist protein adsorption.18-20 In addition, changes in temperature, pH, and added electrolyte have been found to have minimal effect on the zwitterionic headgroups.21 Dioctadecyldimethylammonium bromide (DOMA) is a synthetic double-chain cationic surfactant. The first synthetic bilayer membrane, similar to the cellular membrane in structure, was prepared with DOMA.22 In addition, both DPPC and DOMA can form stable Langmuir monolayers at the air-water interface.23,24 Human serum albumin (HSA, M ) 68 kDa, pI 4.8) is the most abundant protein in the circulatory system. Acting as a multifunctional

10.1021/jp0653196 CCC: $37.00 © 2007 American Chemical Society Published on Web 02/08/2007

2348 J. Phys. Chem. B, Vol. 111, No. 9, 2007 transporter protein, it has a concentration of approximately 50 mg/mL plasma. Here, the DPPC lipids are used to constitute a protein-resistant matrix monolayer in which the DOMA surfactants as ligands are distributed. The binding of the receptor HSA then proceeds to achieve multiple interactions between them before the binary monolayers at the air-water interface are immobilized using the Langmuir-Blodgett (LB) technique. A variety of techniques has been used to study the interaction of proteins with monolayers. Infrared reflection absorption spectroscopy (IRRAS) has emerged as one of the leading methods for structural analyses of monolayers at the air-water interface and can provide abundant information concerning lipid conformation, tilt, and headgroup structure.25 This technique is currently the only physical method able to directly monitor protein secondary structure in situ in Langmuir monolayers.26 Surface plasmon resonance (SPR) is an excellent method for the investigation of protein adsorption in real time without labeling analytes. The observation of a SPR shift, during the adsorption of protein on the surface, will give rich information about protein binding kinetics and surface density.27 In this paper, the IRRAS technique is used to investigate the interactions of HSA with the binary monolayers of DPPC and DOMA at the air-water interface to obtain information on chain orientation before and after protein binding and secondary structure of the protein adsorbed at the monolayers. The SPR technique is employed to study directed assembly of the binary monolayers with a high protein affinity. Experimental Section Chemicals. Synthetic L-R-DPPC (∼99%) and 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. HSA (99%) was supplied by Sigma and used as received. All experiments were carried out at a final albumin concentration of 1.0 µg/mL. Isotherm Measurements. The surface pressure (π)-area (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 paper Wihelmy plate 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 then 15 min was allowed for solvent evaporation. Two barriers compressed symmetrically at the same rate (5 mm/min), corresponding to 1.36 nm2/molecule min. The subphase temperature was kept at 18 °C. Each sample was run at least three times to ensure reproducibility. IRRAS Spectrum Measurements. IRRAS spectra of the monolayers at the air-water interface were recorded on a Bruker Equinox 55 FTIR spectrometer connected to an XA-511 external reflection attachment (Bruker, Karlsruhe, Germany) with a shuttle trough system and a narrow band mercury-cadmiumtelluride (MCT) detector. Sample (film-covered surface) and reference (film-free surface) troughs were fixed on a shuttle device driven by a computer-controlled stepper motor for

Du and Wang allowing spectral collections from the two troughs in an alternating fashion. A KRS-5 polarizer was used to generate perpendicularly polarized lights, and the efficiency of the polarizer was determined to be about 99.2%. The polarizer efficiency is an important parameter for accurate orientation estimation. The IRRAS experiments were carried out at 22 °C. The film-forming molecules were spread from chloroform solution of desired volumes, and 20 min was allowed for solvent evaporation. The measurement system was then enclosed for humidity equilibrium and monolayer relaxation for 4 h prior to compression. The monolayers were compressed discontinuously to the desired surface pressure (30 mN/m) from ∼0 mN/m. After 30 min of relaxation, the two moving barriers were stopped and the monolayer areas were kept constant. For the experiments of protein binding, concentrated albumin solutions were injected into the unstirred subphase beneath the compressed monolayers to achieve a final protein concentration of 1.0 µg/mL. The external reflection absorption spectrum of pure water was used as a reference. The spectra were recorded with a resolution of 8 cm-1 by coaddition of 1024 scans. A time delay of 30 s was allowed for film equilibrium between trough movement and data collection. Spectra were acquired using p-polarized radiation followed by data collection using s-polarized light. During the data collection at different angles of incidence, surface pressure changed slightly for the monolayers (e2.1 mN/m). The IRRAS spectra were used without any processing or baseline correction. The attenuated total reflection FTIR (ATR-FTIR) spectrum of aqueous albumin solution (10 mg/mL) was measured on the Bruker Equinox 55 FTIR spectrometer with a DTGS detector at the resolution 8 cm-1. The ATR accessory contained a ZnSe crystal at a nominal angle of incidence 45°. The final spectrum of albumin was obtained after the spectrum of water was subtracted. SPR Measurements. A Teflon trough with a dimension of 4 cm × 2 cm × 1 cm was home-built and modified from the microtrough used before.16,28 The trough walls were undercut by 45° to eliminate the formation of a meniscus presenting a planar interface,16 on which an integrated optics SPR sensor (Spreeta, Texas Instruments) was used to measure protein adsorption kinetics for each monolayer.16,28 The Spreeta sensors combine the sensor surfaces with all optic and electronic components required for SPR experiments in a compact and lightweight assembly.27 The SPR sensor was first cleaned using an aqueous solution of 1% Triton X-100 and 0.1 M NaOH followed by double-distilled water. Its sensing gold surface was made hydrophobic by immersion in a 2 mM ODT solution in absolute ethanol for 20 min followed by rinsing with doubledistilled water. The SPR sensor was then dried and positioned above the monolayer free air-water interface. The SPR sensor was initialized in air and calibrated in double-distilled water, and a SPR baseline was obtained in water. A binary monolayer of DPPC and DOMA was spread until the desired surface pressure of 30 mN/m was reached, and then it was allowed for relax for 1 h. In the case of the control experiment (a monolayer at the solid-water interface), the ODT-modified SPR sensor was slowly lowered into contact with the monolayer and brought to a depth of ca. 2 mm using a micromanipulator. This procedure was equivalent to a horizontal monolayer transfer to a solid substrate but without ever removing the substrate (i.e., the sensor) carrying the transferred monolayer from the trough.16 Upon contact of the SPR sensor with the monolayer, a step increase of the SPR signal was recorded and a new SPR baseline was established for a period of 10 min to ensure the integrity of the transferred monolayer prior to injecting protein or

Assembly of Monolayes with a High Protein Affinity

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exchanging the subphase to cause protein desorption. The protein solution of desired volumes was injected into the subphase to reach a final albumin concentration of 1.0 µg/mL. The protein binding was allowed to proceed for at least 2 h; the subphase was then exchanged with acidic water of pH 3.0 to remove protein from the monolayer and finally with pure water of pH 5.6 prior to reintroducing protein for binding. In the case of the fluid monolayer (a monolayer at the air-water interface), protein was injected beneath the compressed monolayer after 1 h of relaxation and allowed to bind for at least 2 h. An ODT-modified SPR sensor was brought into contact with the protein-bound monolayer, and SPR signals were recorded. The following procedures were the same as those in the case of the control experiment. For the initial protein adsorption on the fluid monolayers, the binding kinetics were not attained as the SPR sensors could not be in contact with the monolayers if they were to remain fluid. However, the final adsorption values were obtained by placing the SPR sensors in contact with the monolayers after 2 h of binding. At a given molar ratio, the two types of monolayers had the same molecular densities because the identical amounts of the samples were spread at the fixed trough area. This meant that the SPR angle shifts, contributing from the lipid monolayers and pre-modified ODT monolayers on the SPR sensors, were practically identical in the two cases. The SPR angle shifts due to the protein adsorption were only presented after the subtraction of the contributions from the lipid and ODT monolayers. Results and Discussion Monolayers at the Air-Water Interface before Protein Binding. The π-A isotherms of DPPC, DOMA, and their mixtures at the mole fractions of DOMA (XDOMA) 0.1 and 0.3 are shown in Figure 1a. The miscibility and stability of the binary monolayers of DPPC and DOMA (XDOMA ) 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, and 0.5) on pure water have been studied recently.28 All of the mixed monolayers show the condensed features in comparison with the monolayers of the two individual components. The liquid-expanded phase and mean limiting molecular area of the mixed monolayers gradually diminish with increasing XDOMA. The interaction between the two components is obviously strengthened with the increase of XDOMA. The liquidexpanded phase virtually disappears up to XDOMA ) 0.25.28 When XDOMA reaches 0.3, the most condensed monolayer is obtained,28 indicating the strongest attractive interaction between the two individual components in this case. An increase in limiting molecular area is followed for XDOMA ) 0.4 and 0.5.28 Figure 1b shows IRRAS spectra of the monolayers of DPPC, DOMA, and their mixtures (XDOMA ) 0.1 and 0.3) on pure water at a surface pressure of 30 mN/m. In the DOMA monolayer, the bands at 2924 and 2854 cm-1 are assigned to the antisymmetric and symmetric methylene stretching vibrations [νa(CH2) and νs(CH2)], respectively. The νa(CH2) and νs(CH2) frequencies are known to be sensitive to the conformation order of hydrocarbon chains.29 Lower wavenumbers are characteristic of preferential all-trans conformers in highly ordered chains, and the number of gauche conformers increases with frequency and width of the bands. The two band positions indicate that the alkyl chains are in the gauche conformation. A weak band around 1466 cm-1, due to the methylene scissoring mode [δ(CH2)], indicates an amorphous packing of alkyl chains. A peak at 988 cm-1 is attributed to the antisymmetric C-N+-C stretching vibration. In the DPPC monolayer, the νa(CH2) and νs(CH2) bands appear at 2920 and 2851 cm-1, which indicates that the acyl chains are almost in the trans conformation with

Figure 1. (a) Surface pressure-area isotherms of the monolayers of DPPC, DOMA, and their mixtures (XDOMA ) 0.1 and 0.3) on pure water at 18 °C. (b) IRRAS spectra of the monolayers of DPPC, DOMA, and the mixtures of DPPC and DOMA (XDOMA ) 0.1 and 0.3) on pure water at the surface pressure 30 mN/m at 22 °C: p-polarization; angle of incidence 30°.

a small amount of gauche kink. A strong band at 1735 cm-1 is attributed to the carbonyl stretching vibration [ν(CdO)]. A singlet δ(CH2) band at 1470 cm-1 indicates that the acyl chains in the monolayers are packed in a hexagonal subcell structure where each chain can freely rotate around its axis.30 The bands at 1230 and 1088 cm-1 are assigned to the antisymmetric and symmetric phosphate stretching vibrations [νa(PO2-) and νs(PO2-)],31,32 and the two weak peaks around 1172 and 1057 cm-1 are due to the antisymmetric and symmetric C-O-C stretching vibrations, respectively.33 The band at 972 cm-1 is owing to the antisymmetric C-N+-C stretching vibration.33 In the mixed monolayers, the νa(CH2) band frequencies shift slightly to 2919 cm-1, and the νs(CH2) band frequencies remain unchanged. However, their intensities are increased with XDOMA, which results from an increase of chain packing and/or a decrease of chain tilt. This suggests that the two components are miscible. The IRRAS results are in agreement with those obtained from the corresponding isotherms. The ν(CdO) band shifts up to 1738 cm-1 at XDOMA ) 0.3 from 1735 cm-1 in the pure DPPC monolayer. An increase in the ν(CdO) frequency indicates the partial dehydration of the hydrated carbonyl groups,34,35 which reflects a strong interaction between the zwitterionic DPPC and cationic DOMA, forcing the hydrated water away from the carbonyl groups. The δ(CH2) band becomes sharper. The νs(PO2-) band frequency remains almost unaltered, and the νa(PO2-) band increases in width. This is because the partial constituents shift to higher frequency due to the dehydration of the phosphate groups,36,37 which means

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Figure 2. IRRAS spectra of a DPPC monolayer on pure water at the surface pressure 30 mN/m at different angles of incidence: (a) p-polarization; (b) s-polarization.

that there is a strong interaction between the phosphate groups of PC and the cationic headgroups of DOMA. The close packing requires the water in the hydration shell to be squeezed out.38 Figure 2 shows IRRAS spectra of a DPPC monolayer on pure water at different angles of incidence for p- and s-polarization. For p-polarized radiation, which is polarized parallel to the plane of incidence, the bands are initially negative and their intensities increase with increasing angle of incidence and reach a maximum; then a minimum in the reflectivity is found at the Brewster angle (Figure 2a). The exact position of the Brewster angle φ depends on the wavelength of light and the optical properties of the substrate. For the air-water interface, the Brewster angle can be estimated by calculating tan φ ) n2/n1 (n1 ) 1 for air), where n2 is the real part of the H2O refractive index at a given wavenumber [φ ≈ 54° for the νa(CH2) and νs(CH2) bands]. Beyond the Brewster angle, the bands become positive and their intensities decrease upon further increase of incidence angle. For s-polarized radiation, which is polarized perpendicular to the plane of incidence, the bands are always negative and their intensities decrease with increasing angle of incidence (Figure 2b). It is known that the IRRAS data are defined as plots of reflectance-absorbance (RA) versus wavenumber. RA is defined as -log(R/R0), where R and R0 are the reflectivities of the film-covered and film-free surfaces, respectively. Herein, the Kuzmin and Michailov’s model,39,40 which was further developed by Gericke et al.,25,41,42 is used to describe IRRAS band intensities. The following parameters are required to calculate a RA value using Kuzmin and Michailov’s formulation:39,40 refractive index and extinction coefficient of air, n1 )

Figure 3. Comparison of the simulated (lines) and measured (symbols) RA values of νa(CH2) bands for the monolayers on pure water at 30 mN/m. The surface film parameters for the simulation are L ) 2.05 nm, R ) 90°, and degree of polarization 99.2%: (a) θ ≈ 30° and kmax ) 0.60 for the DPPC monolayer; (b) θ ≈ 15° and kmax ) 0.60 at XDOMA ) 0.1; (c) θ ≈ 0° and kmax ) 0.65 at XDOMA ) 0.3.

1 and k1 ) 0; refractive index and extinction coefficient of H2O, n2 and k2, obtained from the literature;43 angle between dipole moment vector and chain axis, R ) 90° for the νa(CH2) vibration; film thickness, d, obtained by taking into account the tilt angle of hydrocarbon chains θ and extended chain length L; ordinary and extraordinary refractive indices of hydrocarbon chains in the mid-IR region, nord ) 1.46 and next ) 1.57,25 and corresponding directional refractive indices of the film, nx ) ny and nz, obtained from nord, next, and θ; and directional extinction coefficients of the film, kx ) ky and kz, obtained for the given tilt angle and transition moment direction when the film extinction coefficient, kmax, is known. Here, only two unknowns remain, kmax and θ. A set of different combinations of kmax and



Figure 4. Time-dependent p-polarized IRRAS spectra of a DPPC monolayer at the surface pressure 30 mN/m upon injecting HSA with a final concentration of 1.0 µg/mL at the angle of incidence 30°.

θ are used by computer simulation to calculate RA values to fit the measured data. Figure 3a shows the theoretical RA values (lines) of the νa(CH2) bands for the DPPC monolayer on pure water against angle of incidence as well as the experimental data (symbols). The tilt angle of the acyl chains is evaluated to be ∼30°, and the kmax value is 0.60 by the best fit. Previous neutron and X-ray reflection measurements yielded 33 ( 3° for the tilt angle of acyl chains in the perdeuterated DPPC monolayers at a surface pressure of 42 mN/m.44 A synchrotron X-ray diffraction determined a tilt angle of 25° or 30°, depending on surface pressure for the DPPC acyl chains in a condensed phase.45 Previous IRRAS results gave a tilt angle of 26 ( 2° of acyl chains for a DPPC monolayer at 28 ( 2 mN/m on pure water,46 and recent IRRAS results showed a dependence of tilt angle of acyl chains on surface pressure, 35 ( 3° at 10 mN/m, 32 ( 2° at 25 mN/m, and 29 ( 1° at 40 mN/m.47 More recently, sum frequency generation (SFG) estimated a tilt angle of 25° for the acyl chains of DPPC monolayer at 42 mN/m.38 The tilt angle obtained here is in agreement with those derived from the IRRAS and other techniques taking into account the discrepancy in conformation order (2920 vs 2918 cm-1), subphase temperature (22 vs 19 ( 0.5 °C), and spectral resolution (8 vs 4 cm-1).46 The fact that the DPPC acyl chains must tilt in the condensed phase has been established.48,49 In the DPPC molecule, the size of the headgroup is relatively large, and the chain must be tilted to some extent to compensate for the headtail mismatch to form a stable monolayer at the air-water interface. The kmax value is related to the extinction coefficient for the vibrational mode and the density of the film-forming molecules at the air-water interface.47 The relative magnitude of the molecular density can be obtained from the appropriate π-A isotherms. The kmax values for the νa(CH2) bands in the mixed monolayers at XDOMA ) 0.1 and 0.3 are thus estimated to be 0.60 and 0.65, respectively, taking into account their molecular areas at 30 mN/m and the kmax value obtained in the case of pure DPPC monolayer. From the p- and s-polarized IRRAS spectra of the binary monolayers of DPPC and DOMA (data not shown), the tilt angles of hydrocarbon chains are readily evaluated by the fit of theoretical calculations to the experimental data (Figure 3b, c). The tilt angles are about 15° and 0° with respect to the normal of the monolayer surface at XDOMA ) 0.1 and 0.3, respectively. Obviously, the tilt angle of the tail chains decreases with the increase of XDOMA, and the chains

Figure 5. Time-dependent p-polarized IRRAS spectra of the binary monolayers of DPPC and DOMA at the surface pressure 30 mN/m upon injecting HSA with a final concentration of 1.0 µg/mL at the angle of incidence 30°: (a) XDOMA ) 0.1; (b) XDOMA ) 0.3, (c) XDOMA ) 0.3 in the region 3900-2700 cm-1. The difference spectra before and after albumin adsorption and the ATR-FTIR spectrum of aqueous albumin solution are presented for comparison.

are almost oriented perpendicular to the water surface at XDOMA ) 0.3, where the most condensed monolayer is formed.28 Monolayers at the Air-Water Interface after Protein Binding. Figure 4 shows time-dependent IRRAS spectra of a DPPC monolayer at 30 mN/m upon injection of albumin. The spectrum after 5 h is practically identical to that before protein injection, which indicates that HSA does not adsorb at the condensed zwitterionic monolayer. It is known that the zwitterionic headgroups are protein-resistant;18-20 no protein adsorption at the zwitterionic monolayers on water is observed at surface pressures above 30 mN/m.47,50-53 However, for the mixed monolayers of DPPC and DOMA (XDOMA ) 0.1 and 0.3),


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Figure 6. IRRAS spectra of the binary monolayers at the surface pressure 30 mN/m after albumin adsorption saturation at different angles of incidence: (a) XDOMA ) 0.1, p-polarization; (b) XDOMA ) 0.1, s-polarization; (c) XDOMA ) 0.3, p-polarization; (d) XDOMA ) 0.3, s-polarization.

albumin adsorption is clearly observed (Figure 5a, b), which indicates that the protein binding is primarily driven by the electrostatic interaction (net negative charges for HSA at pH 5.6). During the interaction of the protein with the monolayers, surface pressures remain almost constant, i.e., a decrease of 0.2 mN/m for DPPC, unchanged at XDOMA ) 0.1, and an increase of 0.3 mN/m at XDOMA ) 0.3 after 3 h of protein adsorption. Thus, HSA molecules hardly penetrate into the compressed monolayers at 30 mN/m but bind to the hydrophilic headgroup regions of the monolayers at XDOMA ) 0.1 and 0.3. Protein injection results in the appearance of amide bands and an increase in the intensities of the water bands (Figure 5c). The latter indicates an increasing surface layer thickness due to protein adsorption.50 Blume and co-workers investigated the relation between the intensity of the ν(OH) band at approximately 3600 cm-1 and the film thickness at the air-water interface.54 They could correlate the increase in the intensity of the band directly with the increase of film thickness.54 From the difference spectra before and after protein adsorption, the two strong bands at 1657 and 1548 cm-1 are clearly due to the amide I and II bands, respectively. The amide I band is primarily from the peptide bond CdO stretching mode, and the amide II band results from C-N stretching and N-H bending vibrations. Previous studies have shown that a direct correlation exists between amide I band frequencies and protein secondary structures.55,56 The position of the amide I band at 1657 cm-1 suggests the presence of a great amount of R-helical conformation.55,57 The maximum peak positions of the amide I and II bands for the adsorbed HSA are almost identical to those for the aqueous protein solution. The above spectral features indicate

that the secondary structures of the adsorbed protein at the hydrophilic headgroup regions of the mixed monolayers are mostly maintained as in bulk solution. The weak bands around 1400 cm-1 are likely due to the symmetric stretching mode of carboxylate groups [νs(COO-)] from some of amino acid residues, and the corresponding antisymmetric carboxylate stretching band [νa(COO-)] may be overlapped with the amide II band. At XDOMA ) 0.1, a weak band near 1258 cm-1 is attributed to the amide III band of the protein. The presence of two weak positive peaks at 2919 and 2851 cm-1 indicates that the hydrocarbon chains increase in tilt angle after protein adsorption. At XDOMA ) 0.3, neither positive peak nor negative peak appears in this region, which indicates that the chain orientation remains unchanged even after 5 h protein adsorption. The monolayers are first compressed to 30 mN/m, and barrier movement is stopped after monolayer relaxation (at the fixed trough area); then the protein is introduced. Thus, the number of lipid molecules on the surface does not vary, and the frequencies of the vibrational modes do not change; i.e., the chain conformation is not altered as the protein is introduced. The same kmax values are used for fitting the experimental data before and after protein injection. From the p- and s-polarized IRRAS spectra of the binary monolayers after protein adsorption (Figure 6), the tilt angles of the hydrocarbon chains are estimated by the fit of theoretical calculations to the experimental data (Figure 7). At XDOMA ) 0.1, the tilt angle of the chains increases from 15° before protein binding to 25∼30° after protein binding. At XDOMA ) 0.3, the chains almost remain oriented perpendicular to the water surface (a tilt angle of ∼0°) before and after protein adsorption. The use of D2O as subphase is avoided


Figure 7. Comparison of the simulated (lines) and measured (symbols) RA values of νa(CH2) bands for the monolayers at the surface pressure 30 mN/m after albumin adsorption saturation. The surface film parameters for the simulation are L ) 2.05 nm, R ) 90°, and degree of polarization 99.2%: (a) θ ) 25∼30° and kmax ) 0.60 at XDOMA ) 0.1; (b) θ ≈ 0° and kmax ) 0.65 at XDOMA ) 0.3.

here, both because of the easier use of H2O and because increasing amounts of HOD in the subphase during long-term measurements, such as adsorption measurements, sometimes causes baseline problems.50 Directed Assembly of Binary Monolayers. The monolayers are prepared at the surface pressure 30 mN/m to prevent albumin in the subphase from penetrating. Figure 8a shows SPR sensorgrams of protein binding to the monolayers of the DPPC, DOMA, and their mixtures on pure water followed by rising with acidic water adjusted with HCl. During the SPR monitoring, the monolayers are immobilized with the hydrophobically modified SPR gold surfaces, which is different from the binary SAMs to some degree because the mixed monolayers are already miscible prior to immobilization. The amounts of adsorbed protein on the monolayer surfaces at saturation estimated on the basis of the relationship,58 an angle shift of 0.1° ∼ a protein surface density of 1 ng/mm2, as a function of XDOMA are presented in Figure 8b. Negligible albumin binding to the zwitterionic DPPC monolayer is observed in contrast to a significant binding to the cationic DOMA monolayer, which indicates that the protein binding is primarily driven by the electrostatic interaction. With increasing XDOMA, the amount of bound albumin on the mixed monolayers is much higher than the values estimated only from XDOMA on the basis of the additive rule (the dashed line in Figure 8b). The binary monolayers with different XDOMA at the surface pressure 30 mN/m are miscible due to the negative deviation of the excess molecular areas and the negative Gibbs free energy of mixing.28


Figure 8. (a) SPR sensorgrams of HSA binding to the monolayers of DPPC, DOMA, and their mixtures at different XDOMA at the surface pressure 30 mN/m with a final concentration of 1.0 µg/mL. (b) Amount of bound albumin on the monolayers at saturation as a function of XDOMA.

The binding behaviors indicate that the amount of adsorbed protein is related not only to the number of positive charges (XDOMA) but also to the distribution of positive charges (distribution fashion of DOMA in the DPPC matrix monolayers) on the surface. This suggests that the functional ligands with favorable distribution in the matrix monolayers could match well with the residues on the surface of protein to improve protein adsorption even at a given molar ratio of the two components. Figure 9 shows SPR sensorgrams of albumin adsorption/ desorption processes on the surfaces of initially fluid and immobilized monolayers of DPPC and DOMA at different XDOMA, respectively. The amounts of bound albumin on the two types of monolayer surfaces are considerably different. In the case of XDOMA ) 0.1, the adsorbed amount at the initially fluid monolayer after 2 h (Γ ) 0.132 µg/cm2) is 78% greater than that at the initially immobilized monolayer (0.074 µg/cm2) during the first binding stage. It is most likely that the enhanced protein affinity results from the redistribution of the binary components delivered through the lateral reorganization of cationic DOMA in the monolayer at the air-water interface, which is induced by the directed binding of negative-charged HSA in the subphase. At the surface pressure 30 mN/m, the binary monolayers are more or less in a liquid-condensed phase, and the hydrocarbon chains are almost in the trans conformation with a small amount of gauche kink, which means that the chains at the air-water interface are capable of lateral mobility to some extent. The above SPR results also demonstrate the occurrence of lateral mobility of the chains. The directed assembly of the binary monolayer in the presence of protein gives rise to a positive charge pattern in the monolayer matching with the distribution of negative charges on the surface of albumin. This is different from the original charge pattern in the binary


Du and Wang

Figure 9. SPR sensorgrams of HSA adsorption/desorption processes on the binary monolayers of DPPC and DOMA (XDOMA ) 0.1, 0.25, 0.3, and 0.5) for initially fluid monolayers (solid line) and initially immobilized monolayers (dashed line): arrow a, injecting albumin with a final concentration of 1.0 µg/mL; arrow b, rinsing with acidic water (pH 3.0); arrow c, introducing pure water (pH 5.6).

monolayer only determined by the favorable electrostatic interaction between the two components on pure water free of protein. The new charge pattern in the monolayer creates more multiple binding sites available for protein. It is clear that protein binding is related not only to the mole fraction of the functional lipids but also to the distribution of the functional lipids in the matrix monolayers. The increased amount of the adsorbed HSA results from the directed assembly of the fluid monolayer induced by the protein in the subphase and the creation of multiple interaction sites in the monolayers favorable for protein binding. As XDOMA increases, the amounts of bound albumin on the monolayers increase in the two cases; however, the bound amount on the initially fluid monolayer is greater than that on the initially immobilized one at a given XDOMA. The relative amount of bound albumin against XDOMA is shown in Figure 10. The protein affinity is enhanced more at the low XDOMA than that at the high XDOMA. The favorable reorganization of DOMA in the DPPC monolayers gives rise to an increase in the number of multiple binding sites for protein binding at the low XDOMA. At the high XDOMA, the lateral reorganization of DOMA cannot cause a significant change in the number of multiple binding sites due to the excess DOMA. The directed assembly of the binary monolayers with a high protein affinity is schematically represented in Figure 11, together with protein binding to a control monolayer for comparison. The created multiple binding sites may have a similar effect of antibodies to recognize the original protein (antigens). On the other hand, real antibodies may be immobilized through the directed

Figure 10. Relative surface density of bound albumin on the binary monolayers (initially fluid monolayer vs initially immobilized monolayer) after 2 h adsorption for the first binding stage as a function of XDOMA.

assembly for biosensing. The high amount of bound antibodies will lead to a significant increase in sensing efficiency. When attempting to remove the adsorbed protein from the two types of monolayer surfaces by exchanging the albumincontaining subphase with pure water, no desorption is observed due to the strong electrostatic binding. However, the adsorbed protein can be removed by lowering pH of rinsing solutions with the addition of HCl. Substantial but not complete desorption is observed for most of the monolayers up to pH 3.0, at which


J. Phys. Chem. B, Vol. 111, No. 9, 2007 2355 binding times are enhanced in comparison with those at the initially immobilized monolayers. The high protein affinity results from the multiple interactions of protein with the monolayers through the lateral reorganization of binary components at the air-water interface, induced by the surface charges of protein in the subphase. The created multiple interaction sites on the monolayer surfaces are preserved for subsequent protein binding. Acknowledgment. The work was supported by the National Natural Science Foundation of China (Grant No. 20303008) and the Fund of Nanjing University. References and Notes

Figure 11. Schematic illustration of directed assembly of a fluid binary monolayer induced by protein in the subphase in comparison with an immobilized monolayer. The gold surfaces of the SPR sensors are hydrophobically modified with ODT so as to interact favorably with the hydrophobic chains of the monolayers. The diagram is not drawn to scale, but the shapes of the SPR sensors are trapezoid-like, similar to their sketches.

the PC headgroups still remain zwitterionic.59,60 Albumin is known to exist in aqueous solutions under different wellcharacterized structural conformations: the most compact structure (N form) with 55% helical content is found around the isoelectric point and up to neutral pH; upon a decrease in pH to 2.7, the albumin undergoes an unfolding to the expanded state (E form) with 35% helical content.61 All the structural transitions have been shown to be reversible.62 Prior to reintroducing HSA into the subphase, the rinsing solution of pH 3.0 is exchanged with pure water of pH 5.6; the remaining albumin with the E form after desorption can recover back to the N form as existed during the first binding stage. Upon reintroducing albumin into the subphase, the total amounts of adsorbed proteins are a little reduced in the two cases. Obviously, the already adsorbed proteins have an influence on the subsequent binding, which reflects the existence of the protein-protein interactions during protein binding and desorption.63 The above protein adsorption kinetics indicates that protein binding is determined not only by the proteinmonolayer interaction but also by the protein-protein interaction. However, the total amount of the adsorbed protein at the initially fluid monolayer (0.118 µg/cm2 at XDOMA ) 0.1) at saturation is greater than that at the control monolayer (0.066 µg/cm2) during the second binding stage (except XDOMA ) 0.5). This indicates that the multiple interaction sites formed in the initially fluid monolayers are preserved for the subsequent protein binding. The surfaces with a high protein affinity are created through the directed assembly for use in biosensing. Conclusions In the DPPC monolayer at the air-water interface, the acyl chains at the surface pressure 30 mN/m adopt a tilt angle of about 30° with respect to the normal of the surface, and no albumin binding to the zwitterionic monolayer is observed. In the binary monolayers of DPPC and DOMA at the interface, a significant protein binding is observed due to the electrostatic interaction. At XDOMA ) 0.1, the hydrocarbon chains undergo a change in tilt angle from 15° before protein binding to 25∼30° after protein binding, and at XDOMA ) 0.3, the chains almost remain oriented perpendicular to the water surface (a tilt angle of ∼0°) before and after protein binding. The amounts of the adsorbed albumin at the initially fluid monolayers after identical

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Directed Assembly of Binary Monolayers with a High Protein Affinity

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