bk-2012-1120.ch032

Chapter 32

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High-Throughput Study of Protein–Surface Interactions Using a Surface Plasmon Resonance Imaging Apparatus Yusuke Arima,1 Rika Ishii,1 Isao Hirata,2 and Hiroo Iwata*,1 1Institute for Frontier Medical Sciences,

Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan 2Department of Biomaterials Science, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8553, Japan *E-mail: [email protected]

Protein adsorption at the liquid–solid interface is critically important for the development of biomedical materials. Surface plasmon resonance (SPR) has been widely used in real-time monitoring of protein adsorption behavior on surfaces. However, the number of combinations of protein–surface interactions that can be measured in a single experiment is limited by a conventional SPR apparatus. We assembled an SPR imaging apparatus for high-throughput studies of many sets of protein–surface interactions. In addition, SPR sensor surfaces with arrayed spots carrying different surface properties were prepared by taking advantage of self-assembled monolayers of alkanethiols. The combination of the SPR imaging apparatus and the arrayed sensor surfaces allows for multiple and real-time monitoring of protein–surface interactions.

Introduction Protein adsorption at the liquid–solid interface is of critical importance in a number of fundamental fields and applications, such as development of biomaterials, medical devices, and biosensors, and thus has been extensively studied. However, many questions remain to be addressed to better understand the phenomena of protein adsorption on artificial surfaces. © 2012 American Chemical Society In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Various surface-sensitive methods have been introduced to study protein adsorption on artificial surfaces, including ellipsometry (1–3), Fourier transform infrared reflection adsorption spectroscopy (FTIR-RAS) (4, 5), sum frequency generation spectroscopy (6, 7), atomic force microscopy (8, 9), quartz crystal microbalance (5, 10), optical waveguide light mode spectroscopy (11), and surface plasmon resonance (SPR) (12–15). SPR can offer real-time and label-free analysis of the interfacial events that occur on the surface of thin films formed on a metal (usually gold or silver) layer under physiological conditions (16). Thus, it coordinates well with self-assembled monolayers (SAMs) of alkanethiols easily formed on metal surfaces. We have examined protein–surface interactions including non-specific protein adsorption and complement activation using SPR (17–19). However, the number of studies of protein–surface interaction that can be performed by a conventional SPR apparatus is limited, leading to time-consuming series of analyses for comprehensive study of these interactions. Therefore, a high-throughput method is required. Recently, a technique of SPR imaging (also designated as SPR microscopy) has been developed and applied to monitor adsorbing organic materials and biomolecules in a spatially resolved manner (20, 21). The combination of SAMs of alkanethiols and the SPR imaging technique is expected to result in a high-throughput, real-time, label-free, and highly sensitive method to study protein–surface interactions. In this study, SPR sensors with arrays of spots carrying SAMs of alkanethiols with different terminal functional groups were made on a gold thin film. We examined the efficacy of the SPR imaging technique using an SPR imaging apparatus that we developed for quantitative analyses of protein adsorption on the functionalized SAM surfaces.

Surface Plasmon Resonance (SPR) Imaging Apparatus Principle of SPR A surface plasmon is a longitudinal charge density wave that is propagated in a parallel manner along the interface of two media, where one surface is a metal and the other is a dielectric layer (22). A metal with a free electron is an essential component, as described by the free electron model of the SPR phenomenon. Most experimental work has been performed using gold and silver thin layers. The surface plasmon is excited by a light wave in SPR sensors. The Kretchmann configuration based on the total internal reflection of light has been employed for development of an SPR optical unit (23). The evanescent wave of the incident light can couple with a surface plasmon at a specific incident angle, θSPR; that is, the wave vector ksp for the propagating surface plasmon is coupled with the wave vector of the evanescent field kev, resulting in energy loss of the incident light to the metal film, observed as a minimum in the reflected light intensity (Figure 1 (b)). The electromagnetic field of a surface plasmon is confined at the metal–dielectric boundary and decays exponentially with ~200 nm of a typical penetration depth in common (24). The SPR angle (θSPR) thus sensitively depends on the refractive index of the medium in the vicinity of the metal film. Changes in the refractive index above the metal 696 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


surface caused by various biological processes, such as adsorption of proteins, result in a change in θSPR.

Figure 1. (a) Schematic illustration of the Kretschmann configuration for SPR. The surface plasmon is excited at the metal–ambient interface when the evanescent component of a wave vector of incoming light (kev) is equal to the wave vector of the propagating surface plasmon (ksp). (b) Reflectance as a function of incident angle before (black) and after (red) adsorption of substances. (c) Schematic representation of an SPR apparatus. Reprinted with permission from reference (25). SPR Imaging Apparatus The optical construction of our SPR imaging apparatus is simple, as shown schematically in Figure 1 (c) (25). The SPR sensor is a glass plate coated with gold (~44 nm), with an underlayer of chromium (~1 nm) as an adhesive layer. The plate is optically coupled to a glass prism using an index-matching fluid. A collimated and p-polarized laser beam is directed to the back side of a sample plate through a glass prism, and reflected light is captured by a CCD camera. Near-infrared light (λ = 905 ± 5 nm) was used to improve sensitivity for the SPR imaging experiment (26). By changing the incident angle of the collimated light, we can determine the SPR angle (θSPR) as the minimum in reflectance (Figure 1 (b)). When the angle of incident light is fixed, the intensity of the reflected light changes due to a shift in θSPR that occurs (ΔθSPR) in a manner dependent on the amount of the 697 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

deposited substances. Detection of light intensity allows for monitoring of protein adsorption behavior on the sensor surface in real time. The light intensity change can be converted to the amount of protein adsorbed using Fresnel fits for the system glass/Cr/Au/protein/water (27, 28).

Model Surfaces


Self-Assembled Monolayers of Alkanethiols We employed alkanethiols, HS(CH2)nX, which form SAMs on the SPR sensor surface, to prepare model surfaces presenting functional group “X.” Alkanethiols chemisorb from a solution onto a metal surface such as gold, silver, or platinum. A gold thin layer on a glass plate is commonly used to form SAMs because it is easy to prepare and is stable in the ambient environment. The gold–sulfur bond is relatively stable with ΔH° ≈ 28 kcal/mol (29, 30). In addition, van der Waals interactions between each long alkyl chain lead to self-assembly of the molecules. Alkanethiols carrying a long alkyl chain (n > 11) form closely packed SAMs with approximately 21.4 Å2 of occupied area per molecule (31, 32). Due to anchoring of the thiol to gold and the close packing of the alkyl chain, a functional group, X, at another terminal end is effectively displayed at the surface of the SAM. Alkanethiols with various functional groups, X, are commercially available. It is easy to prepare SAMs displaying various functional groups. In addition, the surface properties of SAMs can be finely controlled by coadsorption from a mixture of alkanethiols with different functional groups. The composition of alkanethiols in SAMs reflects the mole fraction of alkanethiols in solution but is not the same as their composition in the solution. The composition of SAMs can be determined by spectroscopic methods such as FTIR-RAS and X-ray photoelectron spectroscopy. Figure 2 summarizes mixed SAMs, which are prepared from a mixture of hydroxyl-terminated alkanethiols (11-mercapto-1-undecanol) and methyl-terminated alkanethiols with different alkyl chain lengths (33). The surface composition of the alkanethiol in the mixed SAM was determined from the FTIR-RAS spectra using an absorption band assigned to the asymmetric stretching mode of the methyl groups. The surface fraction of the alkanethiols in the mixed SAM does not linearly reflect the mole fraction in the original solution (Figure 2 (a)). Rather, it is highly dependent on the alkyl chain length of alkanethiols and the terminal functional groups (18, 34). The water contact angle reflects hydroxyl content in the mixed SAMs (Figure 2 (b)). Thus, the preparation of mixed SAMs from a mixture of different alkanethiols allows us to systematically change surface properties and provides different kinds of model surfaces for studies of protein adsorption on artificial materials. Arrays for SPR Imaging Experiment Protein adsorption onto surfaces with different surface properties can be simultaneously monitored using the SPR imaging apparatus and a sensor chip with arrayed spots with different SAMs. We prepared the sensor chip using 698 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


photo-patterning of SAMs. Other techniques such as microcontact printing (35) and inkjet printing (36) are also available for preparing the arrays. Preparation of the array is schematically depicted in Figure 3. A gold-coated glass substrate was immersed in an ethanol solution of methyl-terminated alkanethiol to form a hydrophobic SAM. The SAM was photo-oxidized by UV irradiation through a photomask, and then the oxidized SAM was removed by rinsing with ethanol. Droplets of ethanol solutions of alkanethiols (300 nL) were subsequently placed on the oxidized spots to form the SAM. A hydrophobic SAM of the methyl-terminated alkanethiol was employed as a base SAM to prevent spreading of ethanol solution placed on each spot. The surface properties of the spots were controlled by varying the terminal functional group of the alkanethiol or by mixing two kinds of alkanethiols.

High-Throughput Protein Adsorption Studies Using the SPR Imaging Apparatus Non-Specific Protein Adsorption When artificial materials are brought into contact with biological fluids, non-specific protein adsorption occurs at the initial phase and affects subsequent biological responses, including blood coagulation, complement activation, and cell adhesion. Therefore, it is of critical importance to understand and control non-specific protein adsorption to develop materials for biomedical uses. We employed the SPR imaging apparatus to examine the effect of surface functional groups on non-specific protein adsorption.

Figure 2. Composition (a) and water contact angles (b) of SAMs formed from various reaction mixtures of 11-mercapto-1-undecanol and several methyl-terminated alkanethiols with different alkyl chain lengths. The composition of SAMs was determined by FTIR-RAS spectra. Reprinted with permission from reference (33). Copyright 2003 John Wiley & Sons. 699 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 3. Scheme for fabrication of an array of SAMs carrying different functional groups. Spots carrying alkanethiols with different functional groups, e.g., methyl (CH3), hydroxyl (OH), carboxylic acid (COOH), amine (NH2), or poly(ethylene glycol) (PEG)-coupled amine (PEG) groups, were formed in an array format on the SPR sensor as depicted in Figure 4 (c) (25). PEG-tethered spots were prepared by reacting the N-hydroxysuccinimidyl ester of methoxy–PEG propionic acid (PEG-NHS; MW of PEG: 2,000) with spots of an amine-terminated alkanethiol. In our SPR imaging apparatus, spots appeared as ellipses because images of the arrayed spots were observed at an oblique angle as schematically shown in Figure 1(c). Spots except the PEG-tethered spots were darker than background (Figure 4(a)). The number of repeating methylene units in an alkanethiol molecule on spots (n = 11) was smaller than that on the surrounding area (n = 15). Supporting the notion that refractive indices of all alkanethiols are the same (i.e., 1.45) (37), the brightness in the CCD image reflects the thickness of the monolayer. The difference in thickness between spots and background is expected to be 0.5 nm. This caused darkness in the SPR images for spots with alkanethiols with methyl (CH3), hydroxyl (OH), carboxylic acid (COOH), and amine (NH2) groups. These results demonstrated the high vertical resolution of the SPR imaging apparatus. The PEG-tethered spots, where PEG-NHS (MW = 2,000) was reacted with NH2 spots, appear to be the same brightness as the background. The brightness in the SPR image depends on the effective refractive index near the metal surface. This association might be due to hydration of the surface-bound PEG chains. The effective refractive index of hydrated chains is much lower than that of ethylene glycol (n = 1.4306) and dehydrated PEG film (high molecular weight poly(oxyethylene), n = 1.4563) (38). 700 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 4. SPR image of the array in buffer (a) before and (b) after BSA adsorption. The surface functional group of each spot is shown in (c). Image (b) was acquired by subtraction of the image from image (a). Scale bar, 1 mm. Reprinted with permission from reference (25).

We examined adsorption of bovine serum albumin (BSA) on the SAM array. Figure 4(b) shows a CCD image acquired by subtraction of the two images collected before and after exposure of the array to a BSA solution for 2 hours. Brightness of spots is related to the amount of adsorbed protein. It is noted that BSA adsorption also occurs onto the background surface, which carries a methyl-terminated alkanethiol. Therefore, contrasted in Figure 4 (b) are results from the difference in the adsorbed amount of BSA between spots and the background. The observed pattern allows for qualitative analysis of protein adsorption. The intensities of reflected light averaged for 7 × 7 pixels in each spot were continuously monitored during the entire period of the flowing solutions. Figure 5 shows the time course of BSA adsorption for multiple regions of the array. First, Dulbecco’s phosphate buffered saline (DPBS) was introduced in a flow cell to establish baselines, and then a BSA solution in DPBS (1 mg/mL) was introduced. The SPR signal sharply increased upon introduction of the BSA solution. Change in reflected light intensity was caused by the combination effects of the refractive index increase of the solution containing BSA and BSA adsorption onto the surface. Net SPR signal increase due to the BSA adsorption could be seen after replacement with DPBS. After a 2-hour exposure, the BSA solution was replaced with DPBS. For protein adsorption studies, the change in the intensity of reflected light (ΔI) should be converted to the adsorbed amount of proteins (Γ) by the following equation according to a calibration curve of the SPR signal (25),

The adsorbed amounts of proteins are depicted at the right vertical line. 701 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 5. Parallel monitoring of BSA adsorption onto an array carrying different functional groups. BSA concentration: 1 mg/mL. Reprinted with permission from reference (25).

We further examined the effect of protein type and the pH of buffers on protein adsorption (Figure 6). We employed BSA, γ-globulin, and lysozyme for model proteins and buffers of pH 3–11 (McIlvaine buffer for pH 3, 5, and 7, and 0.2 M carbonate buffer for pH 9 and 11). As expected, coupling of PEG reduced non-specific adsorption for all proteins and at all pH values examined. For BSA and γ-globulin, the amounts of adsorbed proteins showed the maximum at pH 5 regardless of the surface functional group of SAM spots. This pH value is close to the isoelectric points (IEPs) of the proteins (BSA: 4.8, γ-globulin: ~6) (39). The maximum in adsorbed protein around its IEP is in agreement with previous studies on pH-dependent adsorption (40–42). Intermolecular and intramolecular electrostatic repulsions of proteins are minimized at this pH value. These results suggest that the protein property resulting from the electrostatic interaction plays important roles in protein adsorption. According to the dimensions of BSA (14 × 4 × 4 nm), the amounts of adsorbed proteins are estimated to be 250 ng/cm2 for side-on and 600 ng/cm2 for end-on orientations (39). Our results suggest that BSA adsorbs onto the surfaces in side-on orientation even at pH 5. For γglobulin (24 × 4.4 × 4.4 nm), the adsorbed amounts are calculated to be 270 ng/cm2 for side-on and 1480 ng/cm2 for end-on orientations (39), suggesting that γ-globulin adsorbs onto SAM surfaces partially in end-on orientation. As the solution pH became alkali, the amount of adsorbed proteins decreased. Especially, the amounts of adsorbed proteins drastically decreased at pH 9 and 11 for COOHSAM. At this pH, both COOH-SAM surface and proteins are negatively charged. Therefore, electrostatic repulsion plays an important role in reducing the nonspecific adsorption to the COOH-SAM surface at this pH range.

702 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 6. Effect of solution pH on the amount of BSA (a), γ-globulin (b), or lysozyme (c) adsorbed onto SAMs carrying different terminal groups. Protein concentration: 1 mg/mL. Data shown are means ± SD (n = 5).

These results demonstrate the usefulness of the SPR imaging apparatus for real-time and parallel analysis (high-throughput) of protein adsorption on many spots presenting different surface functional groups. Ostuni et al (43) examined the relationship between characteristics of functional groups and the ability to resist the non-specific adsorption of proteins on 58 kinds of surfaces on gold by SPR analysis in a one-spot format. However, their method requires large numbers of measurements, one for each sample surface. In our system, protein adsorption on 25 different surfaces can be simultaneously examined. Thus, parallel sensing using the SPR imaging apparatus has great advantages in its ability to allow label-free, high-sensitivity, and real-time detection of interfacial events.



Complement Activation The complement system is composed of approximately 30 fluid-phase and cell-membrane–bound proteins. It is activated through a cascade of enzyme reactions and plays an important role in the body’s defense systems against pathogenic xenobiotics (44, 45). It is also non-specifically activated by artificial polymeric materials. For example, hemodialysis membranes made of cellulose or its derivatives strongly activate the complement system, and this process has been extensively studied (46, 47). The complement activation by artificial materials also has been seen in various clinical settings, such as open heart surgery, blood transfusion medicine, and extracorporeal immunotherapies (48–50). Understanding the complement activation by artificial materials is important to the rational design of biocompatible surfaces of synthetic materials. We examined the effect of surface functional groups on complement activation using the SPR imaging apparatus. Human serum (10%) was exposed to the array with the pattern shown in Figure 4, and deposition of serum proteins was monitored in real time. Deposition of serum proteins occurred for all spots. The largest amount of deposited proteins was observed for the spots carrying hydroxyls and PEG (Figure 7(a)). This behavior greatly differs from that in a single protein solution of BSA, γ-globulin, or lysozyme, as shown in Figure 5. After the array was rinsed with buffer, the surfaces carrying layers of adsorbed serum protein were further characterized by exposure to an anti-human C3b antiserum solution. Anti-C3b antibody was expected to be bound to C3b or C3 convertase (C3bBb) deposited on the SAM surfaces. A much larger amount of anti-C3b antibody was bound to the protein layers deposited on hydroxyl and PEG spots (Figure 7(b)), indicating that a major component in the protein adsorbed layer is C3b or C3bBb. These results demonstrated that the surface carrying the hydroxyl groups and PEG strongly activated the complement system, resulting in C3b or C3bBb deposition. When C3b is deposited on the material surface, it forms C3bBb, which cleaves C3 in the vicinity of the surface into C3a and C3b. The rate of C3b deposition onto the surface is accelerated by an autocatalytic positive feedback mechanism after C3 convertase has been generated once. The above inference was supported by results from enzyme-linked immunosorbent assay (ELISA) of the fragments of complement proteins produced during the complement activation. Human serum was incubated with surfaces, and concentrations of complement fragments or complexes (C3a, Bb, or SC5b-9) in the serum samples were then determined by ELISA. Surfaces on which a large amount of anti-C3b antibody was deposited also produced fragments of complement proteins, such as Bb, C3a, and SC5b-9, in serum (51–53). The combination of SPR and ELISA thus provides coordinated information about complement activation behavior on material surfaces, but SPR is superior for rapid and real-time monitoring of complement activation compared to ELISA. Strong complement activation by a SAM carrying a hydroxyl group is consistent with our previous work (51). It has also been reported that materials surfaces carrying hydroxyl groups, such as a hemodialysis membrane made from cellulose, strongly activate the complement system (46, 47). Of interest, a large amount of C3b was deposited on a PEG-tethered amine surface. Based 704 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


on our previous studies, this result suggests that the complement system is strongly activated by a PEG-tethered amine surface even though we did not quantify concentrations of complement fragments generated in human serum after incubation with the surface. We reported that methoxy-terminated PEG surfaces do not activate the complement system although long-term storage makes the surface a strong activator for the complement system (54, 55). PEG, which was used in this study, carries a methoxy group at its terminus. The PEG might be degraded during storage, or the mixed surface of PEG and amine groups might affect triggers for complement activation. Further study is needed to understand the mechanism in detail. We also examined the effect on complement activation of the density of hydroxyl groups. Spots carrying different hydroxyl contents were prepared by mixing two kinds of alkanethiols: 1-nonanethiol (CH3(CH2)8SH) and 11-mercapto-1-undecanol (HO(CH2)11SH). Alkanethiol solutions (mole percent of HO(CH2)11SH: 0, 25, 50, 75, 100%; total thiol concentration: 1 mM) were placed on a photo-patterned SAM surface for 1 hour. Methyl-terminated alkanethiols with shorter alkyl chains were used so that the hydroxyl contents on the surface became nearly identical to those in solution (see Figure 2(a)) (33). The amount of deposited proteins increased with an increase in hydroxyl content (Figure 8a). The amount of bound anti-C3b antibody also increased with increasing hydroxyl content (Figure 8b), consistent with our previous report (33). These results clearly demonstrate that the surface density of hydroxyl groups greatly modulates the complement behavior. To further investigate complement activation behavior on material surfaces, a combination study of SPR and ELISA is needed. The SPR imaging apparatus is still useful for screening complement behavior on materials surfaces.

Figure 7. Time course of protein adsorption from 10% human serum (a) and subsequent binding of anti-C3b antiserum onto SAMs carrying different functional groups.



Figure 8. Relationship between hydroxyl content on SAM surface and the amount of adsorbed serum proteins (a) and bound anti-C3b antiserum (b). Data shown are means ± SD (n = 5).

Summary We described a high-throughput method using an SPR imaging apparatus and a sensor with arrayed spots for quantitative and parallel analysis of protein–surface interactions, including non-specific protein adsorption and complement activation. The array presenting spots with different surface characteristics was fabricated by photo-patterning of SAMs, which has the great advantage of stable and well-defined surfaces. A large number of spots with various surface properties is displayed on a sensor chip. Thus, our method is a powerful tool for screening of protein–surface interactions in designing biomaterials with desired functions.

Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Nanomedicine Molecular Science” (No. 2306) and a Grants-in-Aid for Young Scientists (B) (No. 23700559) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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