Relative Contribution of Lateral Packing Density to Albumin

Jul 27, 2016 - †Department of Chemical and Materials Engineering, and §Department of Electrical and Computer Engineering, University of Kentucky, L...
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Relative Contribution of Lateral Packing Density to Albumin Adsorption on Monolayers Leila Safazadeh,† Victor E. F. Zehuri,† Samuel P. Pautler,‡ J. Todd Hastings,*,§ and Brad J. Berron*,† †

Department of Chemical and Materials Engineering, and §Department of Electrical and Computer Engineering, University of Kentucky, Lexington, Kentucky 40506, United States ‡ Department of Bioengineering, University of Missouri, Columbia, Missouri 65211, United States S Supporting Information *

ABSTRACT: The effect of functional group density on protein adsorption is systematically studied to support ongoing efforts in molecular imprinting of surfaces and bulk materials. In these applications, functional commodity chemicals are molded to complement the shape and chemistry of the target molecule. Here, we study the relationship between bovine serum albumin adsorption and ligand density for carboxylate, alcohol, and alkyl terminal groups. Control surfaces consisting of densely packed self-assembled monolayers (SAMs) are contrasted with low-density SAMs formed through thiol−yne chemistry. Direct comparison consistently yielded greater protein adsorption on low-density SAMs than conventional pure component SAMs of the same functional group. Critically, the carboxylate and alcohol low-density SAMS are more hydrophobic than their analogous dense SAMs. Mixed functional group, dense SAMs were formed with alkyl diluents to match the hydrophobicity of the low-density SAMs. Once hydrophobicity is matched, the dense carboxylate and alcohol SAMs have higher adsorption than the low-density SAMs. We conclude (1) surface charge and hydrophobicity trends dominate over surface density contributions; (2) when hydrophobicity is matched, greater adsorption occurs on dense hydrophilic groups than on lower density hydrophilic groups; (3) when hydrophobicity is matched, greater adsorption occurs on lower density hydrophobic groups than on higher density hydrophobic groups.

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INTRODUCTION The nature of protein adsorption onto solid surfaces is critically important across all areas of biotechnology, biomaterial science, and medicine. While much protein adsorption research is singularly focused on nonfouling surfaces,1−4 there are many applications where the balance between specific and nonspecific adsorption is the primary metric. In particular, sensing systems often use proteins, oligonucleotides, or synthetic analogues to create a complex multifunctional surface to promote only specific interactions. As more researchers seek to mimic the specificity of biologically complementary molecules, there is increasing interest in heterogeneous surface interactions with biomolecules. In many early studies, self-assembled monolayers (SAMs) were frequently used to investigate the mechanistic aspects of interfacial protein adsorption.5 SAMs of long alkanethiols on gold are convenient, as they form stable and well-defined organic layers of diverse terminal functionalities (Figure 1a).6 Some heterogeneity in chemical functionality has been achieved by coadsorption of two or more distinct alkanethiols (Figure 1c).5,7 In these pure component and mixed component SAMS, the effects of composition on protein attachment are well established.7−10 The adsorption of albumin is preferred on © 2016 American Chemical Society

more hydrophobic surfaces (e.g., alkyl-terminated SAMs) over hydrophilic surfaces (e.g., alcohol- or carboxylate-terminated SAMs).11−15 This general tendency of low adsorption on hydrophilic SAMs concurs with the recent focus on zwitterionic16−18 and PEG based coatings for antifouling surfaces.19−21 Conversely, low-energy surfaces favor strong interactions with proteins over interactions with water. In mixed monolayer studies, an analogous trend is clearly observed through a gradual protein adsorption decrease as the surface energy increases.3 Even with the extensive literature on albumins and SAMs, little is understood of the effect of lateral packing density of ligands on protein adsorption. This is significant, as most biological binding occurs with functional groups spaced along a polypeptide, oligonucleotide, or a polysaccharide backbone. The available research on lateral chain spacing investigates a single functional group at a time. One study indicates that human serum albumin protein (HSA) binds more tenaciously on an alkyl terminated low-density SAM (LD-SAM) than on a Received: May 17, 2016 Revised: July 19, 2016 Published: July 27, 2016 8034

DOI: 10.1021/acs.langmuir.6b01885 Langmuir 2016, 32, 8034−8041

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chemistry. Our synthetic approach is adaptable to a wide range of functional groups and yields a y-shaped molecule (Figure 1b), where the 2:1 ratio of substrate interfacing groups to environment interfacing groups gives a predicable 50% chain density.26,27 These LD-SAMs are even more electrochemically stable than conventional SAMs by virtue of a doubly bound adsorbate and significant van der Waals interactions in the region near the gold substrate.26 This predictable, stable, lowdensity structure provides an ideal platform chemistry to isolate packing density influences on protein adsorption. We use the functional diversity of click LD-SAMs26 to prepare surfaces with 50% lateral density of carboxylate (COO−), hydroxyl (OH), and alkyl (CH3) terminal groups (Figure 1b). This work is the first report of LD-SAMs prepared by thiol−yne chemistry with hydroxyl and alkyl terminal groups. The LD-SAM adsorption data is contrasted against those obtained for analogous well-packed SAMs with the same terminal functional groups (Figure 1a). While previous adsorption studies on alkyl chains do not measure or account for changes in surface energy and charge with varying chain density, the alteration of carboxylate and hydroxyl based SAMs will dramatically alter these parameters. In this work, we also evaluate protein adsorption on wettability-matched surfaces made from mixed SAMS of carboxyl/alkyl and hydroxyl/alkyl (Figure 1c).

Figure 1. Three classes of monolayers studied: (a) traditional SAMs of high packing density and homogeneous adsorbate composition (from left to right: carboxylate, hydroxyl, and methyl terminated SAMs). (b) LD-SAMs with 50% lateral packing density of traditional SAMs and homogeneous adsorbate composition (from left to right: carboxylate, hydroxyl, and methyl terminated LD-SAMs). (c) Mixed SAMs of high packing density and binary adsorbate composition (from left to right: carboxylate/methyl mixed SAMs and hydroxyl/methyl mixed SAMs).

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EXPERIMENTAL SECTION

1,10-Decanedithiol (98%) was obtained from TCI America. n-Hexane (>95%), acetone (>99.9%), 10-undecynoic acid (95%), 1-hexyne (97%), 5-hexyn-1-ol (96%), ethanol (>99.5%), dichloromethane (>99.8%), 11-mercaptoundecanoic acid (MUA; 95%), 11-mercapto1-undecanol (%97), 1-dodecanethiol (%96), glycerol (>99.5%), and silica gel (35−60 mesh particle size) were purchased from SigmaAldrich (St. Louis, MO) and were used as received. 3-Mercaptopropyl trimethoxysilane (95%) was purchased from Gelest and used as received. Irgacure-184 was used as received from Ciba Specialty Chemicals. Deionized water was produced using an 18 MΩ Millipore water purification system. Silicon wafers (P/Boron ⟨1-0-0⟩), 150 ± 0.2 mm diameter, with thickness of 600−650 μm, were obtained from WRS Materials. Glass microscope slides were purchased from VWR. Gold (99.9%) and chromium (99.9%) targets used in sputtering were purchased from Kurt J. Lesker, Inc. BK7 specific index matching fluid was purchased from Cargille, Inc. Preparation of SPR Sensors. The experimental SPR sensor consists of a gold-coated glass substrate with a 3-mercaptopropyl trimethoxysilane (MPTS) adhesion layer. Microscope slides (VWR Micro slides, 48382-171, 1 mm thick) were cut to 1.5 cm square pieces and rinsed sequentially with acetone, ethanol, and deionized water for 5 min each in an ultrasonic bath. After being blown dry with a stream of nitrogen, slides were spin coated with a solution of MTPS:ethanol:water with a weight ratio of (1:95:5) at 2000 rpm for 30 s. Samples were then placed on a hot plate at 110 °C for 10 min to remove residual solvent. Slides were immediately transferred to a Hummer 8.1 DC sputter system chamber where gold (50−55 nm) was deposited onto the MPTS layer. Slides were rinsed with ethanol and deionized water and blown dry with a stream of nitrogen prior to use. Gold Substrate Preparation. Gold-coated silicon wafers with chromium adhesion layers were used for all non-SPR studies. Silicon wafers were plasma cleaned prior to sputtering. Then, chromium (10 nm) and gold (50 nm) were sequentially deposited onto the wafers. Substrates were typically cut to 1 × 3 cm samples, cleaned with piranha solution (3:1 H2SO4:30% H2O2) for 1 min, rinsed with deionized water, and dried under a stream of nitrogen prior to use. Preparation of Low-Density Monolayers. Complete molecular synthesis procedures are provided as Supporting Information. The 10,11-bis(10-mercaptodecylthio)undecanoic acid was synthesized according to our previously described methods.26 5,6-Bis(10-

densely packed SAM. The more tenacious binding of HSA to LD-SAMs was attributed to the specific interactions between the alkyl chains on the surface and the HSA binding pockets.22 This argues in favor of the important role that specific binding and intercalation potential play in binding of proteins to LDSAMs. While alkyl terminated monolayers appear to have an increase in protein binding with increasing chain spacing, the opposite trend exists in hydrophilic, oligo(ethylene glycol) (OEG) chains.23−25 While OEG chains are promising for nonfouling surfaces, they are not similar to any of the hydrophilic functional groups currently studied for specific binding. Most researchers studying imprinted recognition focus on commercially available precursors which resemble existing biological moieties. Terminal carboxylate, alcohol, and alkyl groups are frequently used to mimic peptide functional groups of isoleucine, aspartic acid, glutamic acid, and serine. The structure of hydrophilic groups used for imprinting has a significant distinction from the OEG groups: the impact of chain spacing on surface hydrophobicity. In the OEG system, the spacing of the terminal groups does not change the hydrophobicity of the surface. In contrast, the lateral spacing of alcohol or carboxylate terminated chains will expose underlying methylene chains, increasing the hydrophobicity over a densely packed surface of these same chains. As a result, the role of chain spacing on adsorption is fundamentally different. Here, we seek to address the role of chain spacing in terminal motifs which are common in both commercially available precursors and biological recognition molecules. This knowledge is a first step in our long-term goal of spatially arranging these functional groups to design artificial biorecognition sites through surface imprinting. To study the role of surface density on carboxylate, alcohol, and alkyl terminated chains, we leverage recently developed low-density monolayers composed of a dithiol adsorbate synthesized through thiol−yne click 8035

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theoretical fits to the spectrum were based on a data set for the dielectric constant of gold provided by Johnson and Christy.33 In a typical adsorption experiment, deionized water was injected for 4 min, phosphate buffer saline (pH = 7.4) was injected for 4 min and 45 s, and 0.38 μM BSA in phosphate buffered saline was injected for 10 min ± 15 s. To calibrate the system, additional trials included an injection of aqueous glycerol solution (1%) to impart a known bulk refractive index change of 0.001 13 from that of deionized water.34 Calculation of the Adsorbed BSA Concentration on Surface. The mass of adsorbed BSA is determined from the change in SPR resonance wavelength. The resonance wavelength corresponds to a local minimum in a plot of reflected light intensity versus wavenumber.30 Using the theoretical Fresnel multilayer model (parameters shown in Table 1), changes in SPR resonance wavelength

mercaptodecylthio)hexan-1-ol and 5,6-bis(10-mercaptodecylthio)hexane adsorbates were prepared in an analogous synthetic approach using 5-hexyn-1-ol and 1-hexyne in place of 10-undecynoic acid, respectively. The product adsorbates were then reconstituted in pure hexane to a concentration of 1 mM. Gold substrates were immersed in this solution for 24 h at room temperature to form SAMs. The substrates were sequentially rinsed with ethanol and deionized water and then blown dry with a stream of nitrogen. Preparation of Pure and Mixed Monolayers. Pure solutions of 11-mercaptoundecanoic acid (HS(CH2)10CO2H), 1-dodecanethiol (HS(CH2)11CH3), and 11-mercapto-1-undecanol (HS(CH2)11OH) were prepared in ethanol at a concentration of 1 mM. Mixtures of two thiols (CH3(CH2)11SH/HS(CH2)10CO2H) and (CH3(CH2)11SH/ HS(CH2)11OH) were obtained by mixing the pure 1 mM thiol solutions in different ratios. Gold substrates were immersed in alkanethiol solutions at room temperature for 24 h. The substrates were sequentially rinsed with ethanol and deionized water and then blown dry with a stream of nitrogen. Spectroscopic Ellipsometry. SAM thicknesses were measured with a spectroscopic ellipsometer (M-2000, J.A. Woollam Co. Inc.). The complex refractive index of the bare gold substrate was measured over a wavelength range of 375−1000 nm and incident angles between 65° and 75° using 2° increments. Scans were then taken of monolayer coated substrates, and the monolayer coating thickness and optical constants were fit to experimental data using a standard Cauchy model. For albumin adsorption studies, the samples were initially immersed in deionized water for 4 min, followed by immersion in phosphate buffer saline (pH = 7.4) for 4 min and 45 s and then in BSA solution in phosphate buffer saline (0.38 μM)15,28 for 10 min ± 15 s. To remove any weakly adsorbed BSA from surface, samples were immersed again in phosphate buffered saline for 4 min and 45 s and deionized water fo 4 min. Samples were then dried in a stream of nitrogen. The mass of adsorbed BSA was determined by measuring the ellipsometric thickness of the BSA layer. To calculate the surface concentration (mass per area) of BSA, the ellipsometric thickness was divided by the density of BSA (1330 g/L).29 Results shown are means from at least three measurements on each of at least four samples. The reported values are the mean ± standard deviation. Statistically significant differences are determined by a p-value less than 0.05 using a two-tailed t-test. Contact Angle Goniometry. Advancing and receding contact angles of deionized water on monolayer surfaces were determined with a manual contact angle goniometer (Rame-Hart model 100). Measurements were taken for one side of a drop volume of approximately 5 μL. Results shown are means from at least three measurements on each of at least four samples. The reported values are the mean ± standard deviation. Statistically significant differences are determined by a p-value less than 0.05 using a two-tailed t-test. Surface Plasmon Resonance. SPR measurements for the BSA adsorption study on monolayers were performed on a homemade SPR system based on the traditional Kretschmann configuration.30 The SPR experimental system and measurement principle are described extensively in literature.31,32 The sensor substrate was placed in contact with a BK7 equilateral prism (Esco Products, Inc.) using an index matching fluid (Cargille Laboratories). The prism and sensor were clamped into a custom-made acrylic cassette with fluorinated ethylene propylene coated channels. Liquids were introduced to the sensor surface using poly(tetrafluoroethylene) tubing and a low-pulsation peristaltic pump (Isamatec). The sensor was illuminated with light from a halogen lamp (Model DH-2000, Ocean Optics, Inc.) using a 200 μm core, multimode, optical fiber, and a collimating lens. The polarization of the incident light was controlled by a calcite GlanTaylor polarizer (ThorLabs, Inc.), and the angle of incidence was set at 65.5° (inside BK7 prism). The reflected light was collected by another lens and coupled to a multimode fiber, which routed it to a CCD based spectrometer (OceanOptics model HR-4000). Surface plasmon resonance shifts and spectra were recorded and analyzed using custom software developed in LabView (National Instruments). The

Table 1. Parameters Used for the Correlation of SPR Data with Mass of Adsorbed Albumin layer

media

n

k

ref

1 2 3

glass prism and substrate gold film on the SPR sensor SAM layer on gold film and adsorbed albumin phosphate buffered saline

1.52 0.17 1.45

0.0 4.86 0

15,38 15 13, 15

1.33

0

15, 39

4

are attributed to changes in the binding layer thickness.30,35−37 This model gives a linear dependence between the wavelength change and thickness, where a 1 nm shift in SPR wavelength corresponds to 0.63 Å in thickness. The value of surface concentration of BSA (M) in units of mass per area is calculated by the following equation: M=

d p(np − nb) (dn/dc)

(1)

where nb and np are the refractive indices of the PBS buffer and BSA solution, dp is the protein layer effective thickness, and dn/dc = 0.182 mL/mg. The adsorbed protein and monolayer contributions are approximated as a single layer, as the optical constant values are indistinguishable. Results shown are means from at least four samples. The reported values are the mean ± standard deviation. Statistically significant differences are determined by a p-value less than 0.05 using a two-tailed t-test.

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RESULTS AND DISCUSSION The focus of this paper is to elucidate the differences in albumin adsorption between densely packed and loosely packed molecules. Our investigation centers on the carboxylate, alcohol, and alkyl ligands which are common in both natural biorecognition and synthetic biorecognition. Our study design first evaluates the spacing of these chains versus a densely packed monolayer of identical ligand composition. Then, we design densely packed, mixed monolayer surfaces with the same surface energy as that of the low-density monolayers. With lowdensity and high-density surfaces of matched hydrophobicity, we then study the role of chain spacing in these systems. This study design directly contrasts the role of chain packing from hydrophobicity for the first time on functional group chemistries commonly used in natural and synthetic biorecognition. Differences in Magnitude of BSA Adsorption between SAMs and LD-SAMs. Here, we compare the magnitude of BSA adsorption on traditional, pure component SAMs (Figure 1a) to BSA adsorption on pure component LD-SAMs (Figure 1b) of the same functional group. We measured adsorption differences by analogous studies using both SPR and ellipsometry. Gold coated SPR sensors were functionalized with the appropriate monolayer and then contacted sequen8036

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the thickness change caused by BSA adsorption (Figure SI-4). Ellipsometry shows a qualitative trend for increasing adsorption with decreasing surface energy when considering the SAMs and LD-SAMs (COOH ∼ OH < CH3). This roughly agrees with the trend from SPR analysis (COOH < OH < CH3). SPR measures the in situ solvated coupling of proteins to the surface, while ellipsometry measurement is the ex situ analysis of dehydrated residual protein on the surface. The aggregate SPR measurement approach is able to provide a statistically significant difference in adsorption on hydroxyl versus carboxylate surfaces (OH SAM vs COOH SAM: p = 1.× 10−5, OH LDM vs COOH LDM: p = 9 × 10−11), while our ellipsometric approach is unable to determine differences between these surfaces (OH SAM vs COOH SAM: p = 0.28, OH LDM vs COOH LDM: p = 0.08). As a result the subsequent studies are performed exclusively with SPR. The agreement of measured trends within the SPR data set, the ellipsometric data set, and prior publications supports the use of the SPR analysis to establish new trends in protein adsorption relating to chain density.41−44 When contrasting BSA adsorption on SAMs and LD-SAMs for each functional group (Figure 2), the amount adsorbed on LD-SAMs is consistently higher than that on SAMs of the same terminal functionality. When considering the root cause of these differences, it is important to revisit the magnitude of intermolecular interactions. The strongest driving forces considered here are electrostatic interactions. BSA has an isoelectric point of 5.4, supporting a net negative charge at our experimental pH of 7.4.45 The carboxylate terminated adsorbates in SAMs and LD-SAMs are expected to be deprotonated at pH 7.4.46,47 Lower adsorption of BSA on the acid terminated SAMs and LD-SAMs is reasonably attributed to electrostatic repulsions. As a result, the reduced surface density of the charge in the LD-SAM directly results in a decreased electrostatic repulsion and therefore higher BSA adsorption. This change in surface charge and hydrophobicity between the SAMs and the LD-SAMs prevents a direct, conclusive determination of the influence of chain packing based on this data set alone. For the hydroxyl terminated SAMs, the relatively weaker polar interactions promote the formation of a hydration layer at the polar, hydroxyl terminus of the adsorbate. As a result, proteins are discouraged from interacting with the surface. Similar to the carboxylate terminated system, the changing lateral density of the polar groups between the SAMs and the LD-SAMs alters the hydrophilicity of these surfaces. The additional exposure of the underlying methylene chains increases the hydrophobicity of the surface. As such, we see a corresponding increase in protein adsorption for the LD-SAMs over the SAMs (Figure 2). This findingtogether with the increased adsorption on carboxylate LD-SAMs over carboxylate SAMsis in direct contrast with prior studies varying hydrophilic chain density. The critical difference between the present data and prior studies is the agreement with the hydrophobicity between the LD-SAM and SAM systems. In Figure 2, the change in both hydrophobicity and chain density makes it challenging to distinguish the role of chain density on protein adsorption with the ligands used in protein imprinted materials. For the nonpolar alkyl SAMs and LD-SAMs, the higher adsorption on LD-SAMs is largely driven by van der Waals interactions. In this case, the lateral spacing of the alkyl groups has a negligible effect on surface charge, polarity, and

tially with water, phosphate buffer, BSA in phosphate buffer. The net changes in resonance wavelength caused by BSA adsorption on monolayers were measured over time (Figure 2a,

Figure 2. SPR analysis of BSA adsorption on monolayers. (a) Representative SPR data for SAMs. (b) Representative SPR data for LD-SAMs. (c) Calculated adsorbed mass per area on indicated monolayers. Data points and error bars represent mean and standard deviation of the measurement.

2b), and the mass of adsorbed BSA on each surface was calculated (Figure 2c). This SPR data does not distinguish between strongly or loosely adsorbed BSA. If there is a significant difference in interaction strength between the SAMs and the LD-SAMs, the trends in the amount of specifically adsorbed BSA may be different upon rinsing or under different fluidic shear conditions. For pure component SAMs, the magnitude of adsorbed protein increases with decreasing surface energy. This trend qualitatively agrees with previous observations in adsorption onto pure component SAMs.3,40 As expected, this same qualitative trend of increasing adsorption by SPR with decreasing surface energy also holds true in the LDSAM surfaces. These trends were further supported by ex-situ ellipsometry. The ellipsometric thicknesses of each monolayer before/after exposure to BSA protein solution were measured, and the concentration of BSA adsorbed on surfaces was calculated from 8037

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Langmuir hydrophilicity. This is further supported by comparable contact angles for alkyl (∼107°)3 and methylene surfaces (∼101°).48 As a result, the adsorption differences observed for this system are likely driven by changes to the surface conformation. This system also closely mirrors the previous work by Choi and Foster on the adsorption of HSA on laterally spaced alkyl chains. They observed a similar increase in adsorption with increasing chain spacing, and they hypothesized the major driving force for the increase in adsorption was intercalation of the chains into hydrophobic pockets in the HSA. For our data, we also see an increase in adsorption. This is potentially attributed to the void spaces between the chains of the adsorbates, allowing for intercalation of the monolayer into hydrophobic albumin domains. The intercalated BSA molecules would increase the van der Waals interactions between the BSA molecules and the surface.49,50 While we cannot verify the specific site of intercalation, the absence of charge density or hydrophobicity effects supports intercalation as a likely mechanism for increasing protein adsorption on laterally spaced alkyl terminated chains in protein recognition applications. Decoupling Chain Density from Surface Energy and Charge Contributions. In order to isolate the effect of chain packing density on protein adsorption, we studied protein adsorption on well-packed SAMs with similar wettability and functionality as the carboxylate and hydroxyl LD-SAMs. Selfassembled monolayers containing binary mixtures of carboxylate/alkyl or hydroxyl/alkyl terminated alkanethiols with similar chain length were prepared on gold to systematically vary surface charge and hydrophobicity. The advancing and receding values of contact angle of water on these surfaces were measured and plotted against the solution phase molar percentages of the HS(CH2)10CO2H or HS(CH2)11OH in the solution relative to the HS(CH2)11CH3 (Figure 3). The pure component advancing and receding angle for carboxylate,51,52 hydroxyl,53 and alkyl54 terminated monolayers agree well with other literature reports on these surfaces. The general nonlinear trend qualitatively agrees with previous reports of carboxylate/alkyl5 and hydroxyl/alkyl54 mixed monolayer systems.3 The advancing and receding values of water for the prepared LD-SAMs are presented in Table 2, and lines corresponding to the advancing contact angle of each LDSAM are also provided in Figure 3. For the carboxylate terminated monolayers, there is good agreement between both the advancing (69°) and receding (34°) water contact angles for the LD-SAM and a mixed SAM with a solution phase molar composition of 70% HS(CH2)11COOH. This agreement in wettability supports at least a moderate agreement in carboxylate surface density by virtue of the Cassie Equation,27 and an agreement of carboxylate density is expected to result in a matched density of charge on the surface equal to half the charge density of a well-packed carboxylate surface. We also find a solution phase molar fraction of 60% HS(CH2)10OH agrees well with the advancing water contact angle of a hydroxyl terminated LD-SAM (74°), while there is only moderate agreement in receding contact angles (LD-SAM ∼ 36°, mixed SAM ∼ 47°). We then used these charge and energy matched surface to specifically investigate the influence of chain density on the magnitude of protein binding on hydroxyl and carboxylate terminated surfaces. For carboxylate and hydroxyl terminated monolayers, adsorption on LD-SAMs is lower than on the hydrophobicity matched, dense SAMs (Figure 4, p = 2 × 10−14

Figure 3. Water contact angle dependence on solution phase composition of thiols in mixed self-assembled monolayer systems; 1 mM total thiol content in ethanol with overnight incubation. Horizontal dashed line indicates advancing water contact angle of the corresponding LD-SAM. Vertical dashed line indicates mixed SAM composition to give matched advancing water contact angle. (a) Mixed SAMs of HS(CH2)10COOH and HS(CH2)11CH3. (b) Mixed SAMs of HS(CH2)11OH and HS(CH2)11CH3. Each data point is the mean ± standard deviation of four samples.

Table 2. Advancing and Receding Water Contact Angle on Functional Loosely-Packed Monolayers LD-SAM

θA (deg)

θR (deg)

COOH OH CH3

69 ± 2 74 ± 2 104 ± 2

34 ± 3 36 ± 1 93 ± 2

and p = 2 × 10−9, respectively). This is now similar to the trend observed with OEG monolayers, where the loosely packed forms of OEG SAM are more resistant to protein adsorption than their analogous dense forms of PEG monolayers.23 By matching charge and energy (Figure 4), the adsorption trend is reversed from that seen on unmatched surfaces (Figure 2). Based on this dramatic change, we conclude that charge and hydrophobicity have a greater influence on protein adsorption than chain density for hydrophilic ligands. Taken together, the matched hydrophobicity studies show a different influence of chain density on protein adsorption for carboxylate and hydroxyl terminated chains when compared to alkyl terminated chains. The carboxylate and hydroxyl LD8038

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chains when compared with the hydrophilic carboxylate and hydroxyl end groups. Intercalation is only likely to drive adsorption phenomena within the first monolayer of BSA. Beyond the 200 ng/cm2 of adsorbed mass attributed to this first layer, other factors will dominate adsorption. This threshold is only exceeded in the hydroxyl and alkyl terminated LD-SAMs (Figure 2C) and the hydroxyl mixed SAM (Figure 4B). In each of these samples, the major differences in adsorption rate are observed in the initial adsorption phase, while the adsorption rates are nearly identical beyond 7 min (Figures 2B and 4B).

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SUMMARY AND CONCLUSIONS Adsorption of BSA was studied on pure LD-SAMs of different terminal functionalities, and the results were contrasted against those for traditional well-packed SAMs. When LD-SAMs are compared to pure component SAMs of the same functional groups, the adsorption of BSA onto LD-SAMs is significantly greater than on SAMs for all functional groups. When LDSAMs are compared to mixed SAMs of matched charge density and hydrophobicity, the adsorption of BSA onto LD-SAMs is significantly less than on SAMs for hydrophilic groups. The change in adsorption from altering the hydrophobicity of the SAM was greater than the change in adsorption due to changes in packing density. This trend is opposite that associated with hydrophilic tail groups, and these differences are attributed to the favorability of functional group intercalation into hydrophobic domains of BSA.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b01885. Details of synthesis and characterization of LD-SAM adsorbates; adsorbate chemical structures; ellipsometric determination of BSA adsorption on pure component SAMs and LD-SAMs (PDF)

Figure 4. SPR analysis of adsorbed BSA on monolayers with matched hydrophobicity to that of LD-SAMs. (a) Representative SPR data for hydrophobicity matched SAMs. (b) Calculated adsorbed mass per area on indicated monolayers. Data points and error bars represent mean and standard deviation of the measurement.

SAMs are more resistant to BSA adsorption than their wellpacked analogue, while the alkyl LD-SAMs encourage more adsorption of BSA than a densely packed SAM. For monolayer/protein interactions, the differences in chain density and functionality play a significant role, but this role is less prominent than hydrophobic effects. The structuring of pure component and mixed SAMs prohibits the response of the SAMs to even large electrochemical potential driving forces on charged SAMs.26,55 In contrast, LD-SAMs are capable of molecular level rearrangement in response to changes in applied electrochemical potential or in the presence of an intercalating solvent.56 These rearrangements may alter the nature of protein−monolayer interactions through intercalation,57 but this intercalation is only favored if the chains favor interaction with hydrophobic protein domains. This likely drives the major adsorption differences we observe in the role of chain density between hydrophilic and hydrophobic functional end groups. The hydrophilic groups at the end of chains lack favorable interactions with the hydrophobic core of the protein. This scenario provides little benefit for the loosely packed chains to penetrate and interact strongly with the protein when compared to a densely packed surface. For hydrophobic end groups, the alkyl chain greatly prefers interaction with the hydrophobic protein core over interaction with an aqueous environment. As a result, the driving force for intercalation is significantly greater for the hydrophobic alkyl

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.T.H.). *E-mail: [email protected] (B.J.B.). Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported partially by the National Science Foundation under Award EEC-0851716. Acknowledgment is made to the Donors of the American Chemical Society Petroleum Research Fund (52743-DNI5) and the University of Kentucky Reese S. Terry professorship (J.T.H.) for partial support of this research. Funding agencies had no involvement in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication. The authors also acknowledge U.K. Center for Nanoscale Science and Engineering for 8039

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use of a sputtering machine, a spectroscopic ellipsometer, and spin coater.

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ABBREVIATIONS BSA, bovine serum albumin; HSA, human serum albumin; LDSAM, low-density self-assembled monolayer; MPTS, 3mercaptopropyl trimethoxysilane; OEG, oligo(ethylene glycol); SAM, self-assembled monolayer; SPR, surface plasmon resonance

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DOI: 10.1021/acs.langmuir.6b01885 Langmuir 2016, 32, 8034−8041