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Jun 8, 2007 - Templating Polypeptides on Self-Assembled Hemicylindrical Surface Micelles. Julio Martinez,Raisa Talroze,Erik Watkins,Jaroslaw P. Majews...
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J. Phys. Chem. C 2007, 111, 9211-9220

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Templating Polypeptides on Self-Assembled Hemicylindrical Surface Micelles Julio Martinez,† Raisa Talroze,†,‡ Erik Watkins,§ Jaroslaw P. Majewski,§ and Pieter Stroeve*,† Department of Chemical Engineering and Materials Science, UniVersity of CaliforniasDaVis, DaVis, California 95616, Los Alamos National Laboratory, Manuel Lujan Neutron Scattering Center, Los Alamos, New Mexico 87545, and Department of Pharmaceutical Chemistry, UniVersity of CaliforniasSan Francisco, San Francisco, California 94143 ReceiVed: December 20, 2006; In Final Form: March 6, 2007

The adsorption of both random coil and R-helical poly L-arginine (PLA) on a surface templated by sodium dodecyl sulfate (SDS) hemicylindrical micelles is investigated and compared. Both secondary structures of PLA irreversibly adsorb on the SDS hemimicelles preserving the secondary conformation present in solution. However, the hemicylindrical SDS surface micelles induce aligned ordering of the R-helical PLA molecules by the adsorption of them into the grooves and at the top of the hemimicelles. For random coil PLA, ordering is not promoted by the surface hemimicelles. The surface concentration of the R-helical PLA is approximately 2 times larger than that for the random coil PLA although electrostatic attraction to the surface is largely screened by the presence of NaClO4 for the R-helical case. The results indicate that the adsorption process for R-helical PLA case is governed by a combination of electrostatic and van der Waals forces. Neutron reflectivity data show that the templated surface of surface micelles is not appreciably distorted by PLA adsorption. The nanostructure of surface micelles can induce order of a subsequent layer of adsorbed molecules allowing construction of macromolecular assemblies.

Introduction Biosurfaces made of electrostatically adsorbed biopolymers that produce layers of different functionalities have been applied in fields such as biosensors, biochips, and biomaterials.1-4 Layer by layer (LbL) deposition is perhaps the method of choice to produce such bio-surfaces due to its simplicity and flexibility of biopolymer and surface. One of the characteristics of LbL films is that they give unordered structures.5 Recent developments are focused on the production of LbL films that presents locally organized structures by the adsorption of biomolecules onto texturized surfaces, which induces biopolymer ordering and alignment.6-8 One of the existing approaches employs stiffrod like biopolymers for the production of anisotropic LbL films.6-7 In this case, surface patterning consisting of parallel channels was obtained by polishing a silicon surface in one direction with diamond paste. Then, the texturized surface was exposed to a solution of R-helical poly L-lysine (PLL). Experimental observations showed that PLL preferentially adsorbed into the channels due to the larger contact to charge area between biopolymer and solid substrate.6-7 Kumar and Hahm (2005) proposed a different approach to achieve the controlled positioning of biopolymers using the self-assembly of diblock copolymer. By this approach, surface patterning with regular nanometric dimensions can be achieved, making it superior to mechanical surface patterning approaches.8 Surfactants can also self-assemble at hydrophilic and hydrophobic surfaces to form cylindrical or hemicylindrical surface micelles when conditions such as molecular structure, ionic * Corresponding author. E-mail: [email protected]. Address: Department of Chemical Engineering and Materials Science, University of CaliforniasDavis, 1 Shields Ave, Davis, CA 95616. † University of CaliforniasDavis. ‡ University of CaliforniasSan Francisco. § Los Alamos National Laboratory.

strength, surface characteristics, and surfactant concentration are appropriate.9-12 The presence of charged head-groups can provide an electrostatic driving force for the adsorption onto oppositely charged surfaces. For hydrophobic surfaces, van der Waals forces between the alkyl chain of surfactant molecules and the surface are responsible for the adsorption process. Hemicylindrical micelles of sodium dodecyl sulfate (SDS) have been observed by atomic force microscopy (AFM) on hydrophobic surfaces at concentration lower than the critical micelle concentration (8.1 mM).13 These types of structures turn out to be very uniform at the nanometric scale. However, once the surface micelles meet different crystal planes of the surface, a change of micelle orientation has been observed.13 Overcoming this obstacle, Levchenko et al.14 observed by AFM that hemicylindrical aggregates of SDS on a self-assembled monolayer (SAM) of undecanethiol on gold did keep its parallelism even across different crystal planes. Our goal is to investigate the adsorption of a model polypeptide, poly L-arginine (PLA), on a surface texturized by SDS hemimicelles adsorbed on a hydrophobic SAM, as an approach for the production of anisotropic and organized nanofilms. The PLA was selected due to its known chemistry for the modification of the lateral chain group guanidino, making it a good candidate for LbL deposition with applications for biosensors and drug delivery.15-16 The adsorption of surfactants onto SAMs of different types has been studied for many years,14,17,18 but the sequential adsorption of polymer on adsorbed surfactant micelles was not reported until recently.19 In this work, we study the adsorption and stability of the SAM/ SDS/PLA systems by surface plasmon resonance (SPR). Assuming that the gaps between surface micelles resemble channels, charged stiff rod like molecules should have a preference to align in the channels.6,7 On the other hand, molecules in random coil conformation would adsorb onto the

10.1021/jp0687798 CCC: $37.00 © 2007 American Chemical Society Published on Web 06/08/2007

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surface without any preferential orientation. We examine the conformation of adsorbed PLA onto the SAM/SDS by attenuated total reflection Fourier transform infrared (ATR-FTIR) from a solution presenting R-helical or random coil conformation. Furthermore, the ordering capability of the templated surface is assessed by neutron reflection (NR) studies. Conceptually, the ordering of stiff rod like molecules is possible by the adsorption of molecules into the SDS hemimicelle’s grooves. Neutron reflection is capable to resolve the isotopic density distribution normal to the surface by the use of isotopic substitution, so that the degree of penetration of PLA molecules into the SDS layer and the consequent ordering can be determined by this experimental technique. Material and Methods Sodium perchlorate, undecanethiol, PLA hydrochloride (degree of polymerization 73%, with 3% or less ornithine residue), hydrogenated SDS (H-SDS) and D2O were purchased from Sigma-Aldrich. Water of 18 MΩ-cm or higher was used in this research. Perdeuterated SDS (98% isotopic enriched, D-SDS) from Cambridge Isotopes Laboratories was used. The H-SDS and D-SDS were crystallized 3 times from ethanol before use. Ethanol (200 proof) from Gold Shield Chemical was used in this research. Hydrophobic SAMs were formed by exposure of gold covered surfaces to a 5 mM undecanethiol/ethanol solution for 18 h.14 After SAM formation, the surfaces were rinsed with large amounts of ethanol and dried with nitrogen. The solution concentrations were 16.2 mM SDS and 150 µM PLA and these concentrations were employed throughout this research. The research was divided into two experimental situations. One situation is called the “helical case” where the solutions contained 250 mM NaClO4. The other situation is called the “random case”, in which the SDS and PLA solutions contained no NaClO4. The reason for this division is based on the fact that PLA in water is in random coil state, while in 250 mM NaClO4 solution the PLA is R-helical.20 Surface Plasmon Resonance (SPR). Gold (99.999%) was deposited on clean LaSFN9 glass slides (Schott, Germany) by thermal evaporation in an Edwards AUTO 306 at a vacuum of 1 × 10-5 Torr or lower. The rate of gold deposition on the LaSFN9 glass slides was between 0.2 and 0.4 Å/s. The SPR apparatus has been described by Levchenko et al.14 A SPR flow cell, identical to the one described in Artyukhin et al.,19 was employed in this research. The SDS and PLA solutions were introduced into the cell at a rate of 5 mL/min with a peristaltic pump. After the SDS solution flowed into the cell, the PLA solution was introduced into the cell with a 0.3 mL air bubble separating the two solutions. The modeling of reflectivity versus angle data was carried out with WINSPALL 2.0 (Max-Planck Institute for Polymer Research, Mainz, Germany), where the refractive index of each layer was fixed and the thickness was selected as the estimated parameter. The refractive indexes for SDS and PLA solutions as well as water were measured with an Abbe 60 refractometer using a sodium lamp as light source and are reported in Table 1. Nonequilibrium data for the adsorption or desorption process were obtained 1° lower than the plasmon resonance angle for the SAM/solvent. Thicknesses equal to 473 ( 31 Å for Au and 16 ( 1 Å for SAM were measured. The surface concentrations for SDS and PLA were determined following Feijter et al:21

Γ)

t(nf - ns) dn/dc

(1)

TABLE 1: Refractive Index and Scattering Length Density of Materials material D-SDSa H-SDS 16.2 mM SDS random case helical case PLA 150 µM PLA random case helical case SAM H2O D2O

refractive index

scattering length density (10-6 Å-2)

1.46b

6.6 0.4

1.334 1.335 1.5b

2.7

1.334 1.335 1.44 1.333

-0.3 -0.56 6.4

a D-SDS stands for 98% isotopic enrichment. b Values with no water content.

where Γ is the surface concentration, t is the film thickness, nf and ns are the refractive index of the adsorbed film and solution, respectively, and dn/dc is the change of refractive index due to change of solute concentration. Equation 1 assumes that the refractive index changes linearly as function of solute concentration.21 The dn/dc was measured as 3.7 × 10-5 and 4.1 × 10-5 mM-1 for 16.2 mM SDS for the random and helical cases respectively. The dn/dc for PLA was the same for both cases and equal to 3.9 × 10-3 mM-1. The dn/dc values were obtained from data that showed a high degree of linearity, so we can assume the applicability of eq 1. Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR). The ATR-FTIR experiments were carried out using a Nicolet, Prote´ge´ 460 spectrometer with an MCT detector. Five hundred scans with a resolution of 4 cm-1 were performed. Apodization Happ-Genzel, none zero filling and ATR correction were employed. A KRS-5 crystal (50 mm × 10 mm × 3 mm, 45° SPP) was used for the ATR-FTIR experiments. Five hundred angstroms of thermally evaporated gold was deposited onto one of the 50 mm by 10 mm sides, while all the other sides of the crystal were masked with tape. After SAM formation on the gold, the crystal was placed in a twin parallel mirror ATR holder (Harrick Scientific Products, Inc) inside a flow cell that provided the fluid/SAM contact. Due to the fact that the specimen chamber was vented with dry air, IR measurements were carried out 2 h after placing the ATR cell into the chamber. The different solutions were introduced to the ATR-FTIR flow cell following the steps mentioned in the SPR section. All ATRFTIR measurements were performed at an angle of 45°. Second derivates of FTIR spectra were obtained after smoothing with a nine-point function. FTIR data processing was performed with OMNIC version 3.1. Neutron Reflection. The NR experiments were carried out at the Manuel Lujan Neutron Scattering Center, Los Alamos National Laboratory, on a time-of-flight reflectometer. The momentum transfer vector, Qz, was measured from 0.008 to 0.25 Å-1 and reasonable statistics were obtained up to R ≈ 10-6. Neutron reflection data collection times were between 6 and 8 h. The uncertainty of Qz, defined as dQz/Qz, was approximately equal to 3%. Single side polished, N-type silicon wafers of 5 mm thickness and a diameter of 76.2 mm were used for the NR studies. A chromium layer, for adhesion purposes, followed by a gold layer, was deposited on the clean silicon wafers (polished side) by sputtering. After metal deposition, SAM formation was carried out as previously mentioned. NR experiments were carried out in the solid-liquid flow cell described in Doshi et al.22 Data were analyzed using a model dependent

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approach, based on a number of boxes that can physically represent the adsorbed layers. The calculations were done with PARRATT32 version 1.6 (HMI-Berlin, Germany) using a fitting procedure that minimized the sample error weighted sum square differences, χ2. Only the fitting results that yielded meaningful physical parameters are considered. The scattering length density for H2O and D2O were considered constant and the values are reported in Table 1. The scattering length densities (F) from NR data modeling for Cr, Au, SiO2, and silicon were equal to 3.6 × 10-6, 4.6 × 10-6, 3.1 × 10-6, and 2.09 × 10-6 Å-2, respectively, which are close to published data for similar system.23,24 The resulting thicknesses were 25 ( 7 Å for Cr, 114 ( 11 Å for Au, and 5 ( 2 Å for SiO2. Scattering length density for the SAM was calculated as proposed by Schwendel et al.25 and taken as being constant. Due to the low molecular weight PLA employed in this research, we assume that the proton is fully exchange by a deuterium ion when D2O was present in the solvent media (assumption later tested by ATRFTIR). The resulting FPLA is shown in Table 1, which was calculated from the contributions of all atoms in the PLA, assuming a volume 188.2 Å3 per arginine residue from consensus volume.26 The scattering length densities for each atom were obtained from the National Institute of Standards and Technology, www.ncnr.nist.gov/resources/n-lengths. The FD-SDS for 98% isotopic enrichment perdeuterated SDS was calculated by atomic contribution to be equal to 6.6 × 10-6 Å-2. The interfacial roughness between consecutives layers cannot be precisely estimated due to the fact that Qz was limited to 0.25 Å-1, so it was arbitrarily chosen and not allowed to vary during the data fitting procedure. The interfacial roughness between Si/SiO2, SiO2/Cr, Cr/Au, and Au/SAM layers was set equal to 2 Å, which is the measured roughness for a native SiO2 film on a Si substrate.27 Interfacial roughness between organic layers and organic layer/solvent were set equal to 3 Å, which has been used to describe adsorbed organic films.28 Results and Discussion Adsorption and System Stability. With SPR, we studied the adsorption of SDS on the hydrophobic SAM, followed by PLA adsorption on the adsorbed SDS. Figure 1A shows the change of the normalized reflectivity at a fixed angle as a function of time. Changes in the normalized reflectivity during the injection steps are attributed to adsorption since the refractive indices of the bulk solution before and after the injection are practically identical.19 Adsorption of SDS onto the SAM reached equilibrium soon after injection for the random case, as well as the helical case where large electrostatic screening by the high salt concentration is present. The adsorption process for SDS is governed by forces other than those of electrostatic character due to the hydrophobic nature of the SAM. Once SDS adsorption reached its equilibrium reflectivity value, PLA was introduced into the system and equilibrium conditions were reached approximately 5 min after injection (Figure 1A). Adsorption of biopolymers onto hydrophobic and hydrophilic surfaces can be driven by a combination of electrostatic, van der Waals, hydrophobic and hydrogen bonding interactions.29,30 At the conditions of the experiments (pH 6.5), PLA has an overall positive charge. On the other hand, stable R-helical conformation for PLA occurs due to charge cancellation induced by the binding of the ClO4- anions to positive charged sites on the PLA leaving it without charge.20,31 Direct exposure of PLA in water to hydrophobic SAM did not yield any change of reflectivity (data not shown), so PLA adsorption for the random case results from the presence of adsorbed SDS and may be

Figure 1. Change of normalized reflectivity during the adsorption (A) and desorption process (B).

driven by electrostatic forces. However, R-helical PLA adsorption onto the bare hydrophobic SAM did occur and reached equilibrium conditions approximately 1 h after injection. Shortrange van der Waals forces are considered responsible for such behavior.32 This may imply that the adsorption of R-helical PLA onto SAM/SDS is driven by van der Waals forces. On the other hand, the adsorption of R-helical PLL (induced by ClO4- anions) onto silicon oxide6-7 indicates electrostatic attraction. Therefore, R-helical PLA adsorption onto SAM/SDS is governed by a combination of short-range van der Waals and electrostatic attraction forces. The residual normalized reflectivities for the cases after solvent rinsing are shown in Figure 1B. We can observe that material is irreversibly adsorbed on the surface since the residual reflectivity values are larger than 1. Among all the cases, the SAM/SDS/PLA for the helical case has the largest residual reflectivity, which indicates that this case presents the largest concentration of irreversible adsorbed species. The change of reflectivity as function of the laser incident angle, once adsorption process reached equilibrium, was also studied and it is shown in Figure 2. Inserts in Figure 2 show details of the change of the minimum plasmon angle. Sarkar and Somasundaran33 observed an increase of reflectivity value at the minimum plasmon angle during the adsorption of a cationic surfactant (dodecyltrimethylammonium chloride) to a

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Figure 2. Reflectivity change as a function of angle for (A) random and (B) helical cases. Inserts show a representation around the minimum plasmon angle.

previously adsorbed layer of anionic polymer (poly(acrylic acid)). They attributed this behavior to an increase of the hydrophobic nature of adsorbed polymer due to the binding and intermixing of surfactant at a molecular level, which increases the dielectric constant of the adsorbed layer, leading to the evanescent wave being pushed back to the gold surface. The reflectivity of the plasmon angle is the same for SDS and PLA curves as shown in Figure 2A,B, and the results lead us to conclude that PLA and SDS are not appreciable intermixed. Furthermore, the injection of the PLA solution, which contains

no surfactant, can result in desorption of the SDS layer exposed to the solvent.14,18 On the basis of the observations that PLA for the random case adsorbed on the surface only if SDS is previously adsorbed, and that there is negligible molecular intermixing between PLA and SDS, we conclude that the SDS layer is confined between the SAM and the PLA. On the other hand, the increase in the plasmon angle for the helical case could be associated with the adsorption of PLA onto the SAM with displacement of SDS from the surface. However, we have previously observed that the largest amount of material remain-

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TABLE 2: Thicknesses and Surface Concentrations Measured by SPRa random

b

helical

material

thickness (Å)

Γ (µmol/m2)

thickness (Å)

Γ (µmol/m2)

SDS PLA PLA rinseb

10.3 ( 0.4 10.6 ( 0.6 10.2 ( 0.8

3.5 ( 0.1 0.045 ( 0.003 0.043 ( 0.003

11.9 ( 0.3 19.9 ( 0.2 21 ( 0.4

3.7 ( 0.1 0.085 ( 0.001 0.089 ( 0.002

a Mean and standard deviations obtained from at least 3 samples. For PLA parameters after solvent rinse.

ing on the surface after solvent rinse is for the SAM/SDS/PLA helical case. In principle, this can only be possible if the SDS remains on the surface isolated from the solvent, so the assumption that SDS is confined between the SAM and the PLA for helical case is reasonable. The thicknesses and surface concentrations of each layer at equilibrium conditions are shown in Table 2. The thicknesses of SDS for the random and the helical cases are equal to 10.3 ( 0.4 and 11.9 ( 0.3 Å, respectively. Screening of the repulsive electrostatic forces among adsorbed SDS molecules due to the high salt concentration is assumed to be responsible for the increment in the thickness.13 The thicknesses of adsorbed PLA layer is obtained by assuming no appreciable desorption of SDS from the surface due to the confinement effect. Values of 10.6 ( 0.6 and 19.9 ( 0.2 Å are determined for the random and helical cases, respectively. The R-helical diameter for PLA with extended side chains is 24 Å.34 On the other hand, the a-axis of the hexagonal unit cell with 1/2 water molecule per residue was 14 Å measured by X-ray diffraction, and this 10 Å difference is attributed to the folding of side chains around the helical axis.34 The value of 19.9 Å “dry layer” of adsorbed PLA for R-helical case turns out to be between those limits and significantly larger compared to 10.6 Å measured for random case. For comparison purpose, the thickness and surface concentration of adsorbed R-helical PLA with no SDS were 18 Å and 0.077 µmol/m2. Rinsing the SPR cell with the respective solvents did not substantially modify the surface thicknesses indicating irreversible adsorption. Secondary Conformation of Adsorbed PLA. We discussed in the previous section the adsorption and stability of the SAM/ SDS/PLA systems under consideration. In this section, we estimate the conformation of PLA to assess the possibility of conformational changes following adsorption to the surface. ATR-FTIR is used for the study of secondary conformation of proteins adsorbed on gold/thiol-modified surface since this approach allows in situ studies.35 The ATR-FTIR spectra in this section were measured after rinsing the ATR cell with the respective solvents in order to avoid any contribution from nonadsorbed PLA to the FTIR signal. Figure 3 shows the respective absorption spectra for the random and the helical cases in D2O. The symmetric stretching of the SO3 group from SDS is observed at about 1080 cm-1 for the random case,15,36 which supports the assumption that SDS remains on the surface. Unfortunately, the asymmetric stretching for ClO4- is between 1070 and 1100 cm-1,37 so the symmetric stretching of SO3 cannot be observed for the helical case. Asymmetric stretching of SO3 should be between 1200 and 1300 cm-1 and is represented by a doublet.15,36 Instead, a large asymmetrical peak centered on 1210 cm-1 is observed for both situations. The asymmetry is attributed to the contribution of D2O absorption, which is around 1209 cm-1.38 Inserts in Figure 3 show the IR absorption spectra from 1200-1300 cm-1 for the same cases in H2O where the doublet for asymmetric stretching of SO3 is observed. Therefore, SDS remains on the surface after PLA

Figure 3. ATR-FTIR spectra in D2O for (A) helical and (B) random cases. Inserts are same experimental conditions with H2O as solvent.

adsorption. The amide I’ region peak presents a shoulder located at 1580 cm-1 that is assigned to the asymmetric stretching of free COO- at the end of peptide chain.39,40 The absence of the amide II band (∼1540 cm-1) indicates a large degree of H to D exchange.40 This point is particularly useful for the estimation of the scattering length density of PLA for the NR experiments. The large band from 1410 to 1480 cm-1 contains the methylene scissor (∼1450 cm-1) and wagging (∼1410 cm-1) deformations15 and the amide II′ band (∼1430 cm-1) due to the H to D exchange.40 The second derivates of amide I′ region for both cases are represented in Figure 4. Peaks for the different secondary structure have been assigned as follow: R-helical 1650-1659 cm-1, β-sheet 1666-1680 cm-1, unordered 1640-1648 cm-1, and the extended chains and β-sheet 1621-1637 cm-1.41-43 The large intensity of a few distinct second derivate peaks indicates that a particular conformation dominates in each case. For instance, adsorbed PLA for the random case shows a large contribution of unordered structure. Furthermore, the R-helix is the main peak for the helical case. Adsorbed PLA for the helical case presents about 70% of the helical structure, which is smaller than the reported helicity obtained for the same conditions in solution.20 The relative amount of the R-helix on the surface is underestimated by FTIR since the guanidine shows a strong absorption which overlaps with the amide I′ band.44 The absorption of guanidine group in D2O solution has been

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Figure 4. Second derivate of absorbance: amide I′ region.

TABLE 3: NR Thickness and Volume Fraction Model Parameters assignment SDS hemimicelles volume fraction thickness SDS/PLA for SDS matching unperturbed thickness PLA thickness PLA volume fraction

random case

helical case

0.37 ( 0.06 16 ( 0.5 Å

0.51 ( 0.02 15.5 ( 0.5 Å

4Å 25 Å 0.38

0Å 34 Å 0.44

reported at 1586 and 1608 cm-1 for symmetrical and antisymmetrical vibrations, respectively.44 In this work, we observed the shift of guanidine maximum absorption to ∼1595 and ∼1615 cm-1 assumed to be caused by PLA-SDS and PLA-ClO4 electrostatic interactions at guanidine site.44 We conclude from this set of data that the adsorption process does not induce appreciable conformational change of PLA with respect to the conformation in solution. Similar observations have been reported for LbL systems for polypeptides and proteins.38,45 PLA Organization on the SDS Surface. The large amount of stiff-rod-like conformation found on the surface indicates there is no conformational restriction for PLA to adsorb in the channels between the surface hemimicelles. If this is the case, some degree of interpenetration of the PLA layer into the SDS layer could occur. NR has been used for studying LbL deposition and penetration of biomolecules into surface adsorbed layers28,46 because isotopic substitution provides contrast between adsorbed layers. First, we characterize surface SDS hemimicelles formed on the SAM before PLA adsorption step following Schulz et al.47,48 with the thickness and the volume fraction of surface hemimicelles as estimable parameters in order to minimize χ2. The thickness of the hemimicelles is modeled as a multiple of layers with a thickness equal to 1 Å with each layer having a volume fraction resulting from geometrical considerations.47 In order to have good contrast between the SAM and SDS layers, we selected the following isotopic substitution: H-SAM/D-SDS/ bulk solution. Bulk solutions with different scattering length densities, H2O and contrast matching 5 (CM5), and a mixture of H2O and D2O with a scattering length density of 4.9 × 10-6 Å-2 were used for hemimicelles characterization. Table 3 summarizes the results of the fitting approach. A micellar thickness of 16 ( 0.5 Å, volume fraction of 0.37 ( 0.06, and calculated periodicity of 67 Å are obtained for the random case, which are similar to reported values for SDS on hydrophobic surfaces obtained by AFM.13,14 For the helical case, a thickness

Figure 5. Reflectivity curves for LA adsorption onto SAM/D-SDS for D-SDS matching bulk solution. (A) Helical case. (B) Random case. Error bars are the standard deviations of NR data. Inserts show the resulting scattering length density profiles. Length ) 0 Å represents the Si/SiO2 interface.

Figure 6. Distribution of PLA onto surface hemimicelles. (A) Random case. (B) Helical case (not draw to scale).

of 15.5 ( 0.5 Å and a volume fraction of 0.51 ( 0.02 are obtained. The resulting periodicity is equal to 49 Å. The increase of ionic strength by the presence of NaClO4 yielded a smaller SDS periodicity with no significant change of micelle dimensions. The result agrees with previous observation for SDS

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Figure 7. PLA surface distribution for helical case at CM5 condition. (A) NR data and best fit before (dashed line) and calculated after (solid red line) helical PLA adsorption. Error bars are the standard deviations of NR data. (B) Resulting scattering length density profiles. Length ) 0 Å, representing the Si/SiO2 interface. The schematic superimposed on the graph shows the layer of the SAM, hemicylindrical SDS micelle, and helical PLA.

surface micelles on graphite, and it was associated with the screening of repulsive electrostatic forces.13 A common assumption used in the modeling of NR data is that the thickness and scattering length density of the previously adsorbed layer is not affected by the adsorption of a new layer. However, this is not the case for the present research due to possible penetration of PLA into the SDS layer. Penetration (or the lack of it) for biomolecules into monolayers has been described using the box model by allowing the scattering length density to vary.28 Unfortunately, we cannot rely on such approach because the surface hemimicelles are modeled by a stack of layers of 1 Å thickness with different scattering length

densities. For such a model, we would be forced to assume the penetration depth of PLA molecules in order to know the number of layers that are affected. To overcome such constraints and study the adsorption of PLA with the simplest possible box model, we propose to match the D-SDS and bulk solution scattering length densities. The idea was originally proposed by Lu et al.,49 but our work employs it in a conceptually different way. By using D2O as solvent, an error of about 3% would be produced to match the D-SDS scattering length density. This difference is even smaller when the hydrated hemimicellar layer is taking into consideration, so it is considered negligible for the purpose of this research. The benefits of the proposed

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Figure 8. PLA surface distribution for random case at CM5 condition. (A) NR data and best fit before (dashed line) and calculated after (solid line) random PLA adsorption. Error bars are the standard deviations of NR data (B) resulting scattering length density profiles. Length ) 0 Å, representing the Si/SiO2 interface. The schematic superimposed on the graph shows the layer of the SAM, hemicylindrical SDS micelle, and random-coil PLA.

approach can be contemplated in eq 2. Essentially, the portion of hemimicelle that is not affected by PLA can be modeled by a single layer of variable thickness with a scattering length density of 6.4 × 10-6 Å-2. In this way we can assess the thickness of the unperturbed hemimicellar layer. Selecting a number of boxes and allowing the thickness and scattering length density of the boxes to vary is the method we use to obtain the distribution of PLA normal to the surface. In order to know if the box model for adsorbed PLA substantially improve the description of the system, this research will relay not only on reducing the sum square error but also on

using Hamilton’s table to determine whether the new model is statistically different:25,50

F ) FD-SDS φD-SDS + FPLA φPLA + Fsolv(1 - φD-SDS - φPLA) FD-SDS ) FsolV F ) FPLAφPLA + Fsolv(1 - φPLA)

(2)

The resulting parameters from the proposed approach indicate that the thickness of unperturbed micellar layer is equal to 0 and 4 Å for the helical and the random cases, respectively (inserts in Figure 5A,B). The thicknesses of unperturbed D-SDS

Self-Assembled Hemicylindrical Surface Micelles layer for random case can be appreciated in the Figure 5B insert by the small peak in the scattering length density (SLD) profile at about 170 Å. This set of data indicates that the layer of R-helical PLA is in contact with the SAM, while the layer of random coil PLA is away from it by a distance equal to 4 Å. A total PLA thickness of 25 Å for the random case is observed, about 1/4 of the expected size of the random coil in a good solvent. Surprisingly, the adsorbed PLA layer turns out to be 34 Å thick for the helical case, which is larger than the maximum PLA helical diameter reported.34 The volume fractions of PLA for the random and the helical cases are equal to 0.38 and 0.44, respectively. Govardhan et al.16 observed a monolayer of electrostatically adsorbed PLA on human growth hormone crystal in a low strength solution. This is likely to occur for random case, so PLA molecules followed the contour of patterned surface as shown by the proposed schematic in Figure 6A. The NR data for the SAM/PLA system yield a layer of 21 Å thick and a volume fraction of 0.81, which is close to 0.79 for a monolayer of tightly packed, parallel cylinders on a surface. By comparison with the SPR results, we conclude that R-helical PLA adsorbs on the SAM with a resulting helical diameter equal to 21 Å. Furthermore, the calculated surface concentration for R-helical PLA in such distribution would be 0.072 µmol/m2 (assuming a diameter of 21 Å and a length of 110 Å per PLA molecule), which is close to the estimated 0.077 µmol/m2 by SPR. We have previously observed that the surface concentration for the R-helical PLA is not substantially different whether or not SDS is previously adsorbed on the SAM. This indicates a full coverage by adsorbed R-helical PLA independent of the presence of SDS at the surface. For the helical case, PLA molecules should preferentially adsorb in the intermicellar space due to a larger density of surface charge6,7 and in contact with the SAM favored by short-range van der Waals forces.32 This surface distribution is partially satisfied by the NR data reported in Table 3, but it does not correlate with the thickness of PLA layer being larger than the R-helical diameter (21 Å) and the full surface coverage observed by SPR. Furthermore, R-helical PLA adsorbs as a monolayer as previously observed for the SAM/PLA system, so the thickness of the PLA layer observed by NR for SAM/SDS/PLA system cannot be explained by a the adsorption of PLA molecules at the top of others PLA. These experimental observations suggest that PLA can also adsorb on the top of the SDS hemimicelles as shown by the proposed surface distribution in Figure 6B. The NR studies for SAM/DSDS/PLA with CM5 as background were also performed in order to test the proposed surface distributions assuming that surface hemimicelles were not distorted by the adsorption of PLA. A large number of parameters could be estimated from the adsorption of PLA, so NR data were not subjected to a fitting algorithm. Calculated R versus Qz data points are obtained from the dimensions for the surface hemimicelle at CM5 solvent condition and from the PLA volume fractions reported in Table 3. The PLA thickness measured for the random case was divided into layers of 1 Å thickness with the same scattering length densities. The helical case was approached by assuming cylinders of 21 Å in diameter arranged as shown in Figure 6B. These cylinders were divided into layers of 1 Å thickness with their resulting scattering length densities based on geometrical considerations. Figures 7 and 8A show the NR reflectivity data with their respective fitting for SAM/D-SDS and calculated SAM/D-SDS/PLA curves for helical and random cases. We can observe that the adsorption of PLA onto SAM/D-SDS induces changes on the NR data profile specially observed at Qz ≈ 0.05

J. Phys. Chem. C, Vol. 111, No. 26, 2007 9219 Å-1. Simulated data for the helical case based on the proposed distribution in Figure 6B can closely represent the experimental data in Figure 7A, except for Qz from ∼0.06 to 0.12 Å-1. For random coil case, the proposed distribution in Figure 6B shows better agreement with some deviation for Qz larger than 0.16 Å-1. Among all the possible reasons behind the small deviations observed between the calculated and experimental data, the assumption that surface micelles are not deformed by the presence of adsorbed PLA may not be strictly correct especially for the R-helical PLA case due to the rigidity of helical conformation. Conclusion PLA adsorption on SDS surface hemimicelles is described in this paper. The PLA/SDS does not desorb after solvent rinsing which indicates stable LbL deposition. No appreciable intermixing of the PLA/SDS/SAM was observed by SPR measurements. Adsorbed PLA for the random and the helical cases show no appreciable change in the secondary conformation when it is compared with its conformation in solution. Adsorption of PLA for the random case is governed by electrostatic attraction, while for the helical case van der Waals forces also contribute to attraction of PLA on the SAM/SDS surface. The NR data suggest that, for the random case, PLA adsorbed on the surface and mimics the contour of the surface hemimicelles while helical PLA adsorbs in the channels and at the top of the SDS hemimicelles to achieve larger surface coverage than in the random coil case. NR studies based on the contrast matching approach were essential to quantify the degree of penetration of adsorbates on the texturized surface by SDS hemimicelles. Acknowledgment. We gratefully acknowledge the financial support from the Manuel Lujan Jr., Neutron Scattering Center through the CARE program. The Manuel Lujan Jr., Neutron Scattering Center is a national user facility funded by the United States Department of Energy, Office of Basic Energy SciencesMaterials Science. At the time that this research was completed, the Manuel Lujan Jr., Neutron Scattering Center was managed by the University of California under Contract No. W-7405ENG-36. Supporting Information Available: NR reflectivity experimental data and simulated curves for SAM/PLA/CM5 for helical case and SAM/D-SDS/H2O for helical and random cases. This information is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Mu¨ller M.; Meier-Haack, J.; Schwarz, S.; Buchhammer, H. M.; Eichhorn, K. J.; Janke, A.; Kebler, B.; Nagel, J.; Oelmann, M.; Reihs, T.; Lunkwitz, K. Polyelectrolyte Multilayers and Their Interactions. J. Adhes. 2004, 80, 521-547. (2) Ngankam, A. P.; Mao, G.; Van Tassel, P. R. Fibronectin Adsorp. Polyelectrolyte Multilayer Films 2004, 20, 3362-3370. (3) Trubeskoy, V. S.; Loomis, A. L.; Hagstrom, J. E.; Budker, V. G.; Wolff, J. A. Layer by Layer desorption of Oppositely Charged Polyelectrolytes on the Surface of Condensed DNA particles. Nucleic Acid Res. 1999, 27, 3090-3095. (4) Caruso, F; Niikura, K.; Furlong, D. N.; Okahata, Y. Assembly of Alternating Polyelectrolyte and Protein Multilayer Films for Immunosensing. Langmuir 1997, 13, 3427-3433. (5) Decher, G. Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites. Science 1997, 277, 1232-1237. (6) Mu¨ller, M.; Kessler, B.; Lunkwitz, K. Induced Orientation of R-Helical Polypeptides in Polyelectrolytes Multilayers. J. Phys. Chem. B 2003, 107, 8189-8197. (7) Mu¨ller, M. Orientation of R-Helical Poly(L-Lysine) in Consecutively Adsorbed Polyelectrolyte Multilayers on Texturized Silicon Substrates. Biomacromolecules 2001, 2, 262-269.

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