Solid Interface - The

J. Phys. Chem. C 2009, 113, 2053–2062

2053

Buffer Effect on Protein Adsorption at Liquid/Solid Interface† Tao Wei, Sarawut Kaewtathip, and Katherine Shing* Mork Family Department of Chemical Engineering and Material Science, UniVersity of Southern California, Los Angeles, California 90089 ReceiVed: July 25, 2008; ReVised Manuscript ReceiVed: December 25, 2008

This work shows buffer choice and buffer concentration can drastically affect protein adsorption. We used ATR/FTIR to compare the adsorption kinetics and secondary structural evolution of BSA, IgG, fibrinogen and lysozyme on a Ge surface, buffered in phosphate buffered saline (PBS) and tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) at pH 7.4. The adsorption behaviors of the four proteins are multilayer in nature, and all exhibit a short period of rapid initial adsorption with larger secondary structural changes, followed by a long period of quasi-linear adsorption. For BSA, IgG and fibrinogen, PBS buffer depresses the adsorption in the quasi-linear kinetic region compared with Tris-HCl buffer, whereas lysozyme adsorption is relatively insensitive to buffer choice. Buffer concentration also affects protein adsorption. BSA adsorption increases monotonically with Tris-HCl concentration while the variation with PBS concentration is nonmonotonic. The secondary structure in the adsorbed phase is in general quite different from that in the bulk solution; however, buffer choice does not have a significant effect on secondary structural evolution of adsorbed proteins especially when comparisons are made on the basis of adsorbed amount. Although PBS is the most commonly used buffer at physiological pH, the role of phosphate ions in PBS buffer and their effect on protein adsorption are rather complex because phosphate ions adsorb competitively with protein molecules and more than one type of phosphate ions can exist both in the bulk solution and in the adsorbed phase. The competitive nature of protein and phosphate ion adsorption is demonstrated for IgG adsorption in a sequenced adsorption/flushing/displacement experiment. 1. Introduction Protein adsorption at solid/solution interface has been a research focus for more than three decades due to its importance in the development of biocompatible materials, various biotechnological processes, food and pharmaceutical industries, and promising new areas such as biosensor, gene microarray, biochip, biofuel cell and so on. In order to control and manipulate protein adsorption, the mechanisms which govern the adsorption process need to be well understood. At present, it is generally accepted that the adsorption behavior of proteins at relatively high concentrations often does not follow the true equilibrium isotherm,1,2 because the slow relaxation of nonequilibrium structures1,3,4 leads to multilayer build-up. Such behavior has been monitored by atomic force microscopy (AFM) measurements,5-8 neutron reflection9,10 dual polarization interferometry,11 circular dichroism (CD),12 and Fourier transform infrared attenuated total reflectance (FTIR/ATR),2 among other techniques. The process of macromolecular multilayer adsorption, accompanied by structural rearrangements, is still too complicated to be modeled using macroscopic kinetic models and too computationally expensive to be atomistically simulated. At low concentrations, an interfacial cavity kinetic model has been used to characterize monolayer or submonolayer protein adsorption with surface-induced structural transitions.3,4,13 Atomisticsimulations,suchasMolecularDynamic(MD)simulation14-18 and Monte Carlo (MC) simulation,19-21 are able to model the atom/atom interactions realistically and to elucidate the orientational alignments and conformation evolution of an adsorbed † Part of the special section “Physical Chemistry of Environmental Interfaces”. * To whom correspondence should be addressed. E-mail: [email protected].

protein molecule; however, these techniques are still largely limited to the simulation of a single protein molecule on a surface with nanometer dimensions at nanosecond time scales. Many studies have focused on the effect of various modifications in the adsorption systems, including surface modification,22-24 protein modification,25-27 the use of excipients such as saccharides28 and surfactants,29 and adjustment of solvent conditions such as ionic strength30 and pH,30 for the purpose of either reducing or promoting protein adsorption. Although buffer research is a well-established area for the stabilization of proteins in the bulk solution in the pharmaceutical field,31 it did not seem to have attracted significant interest in adsorption studies and only a small number of studies have been reported. Adsorption capacity of cytochrome c on Cu2+-chelated poly(HEMA-MAH) bead was demonstrated to be dependent on the buffer type with the observed adsorption in the order phosphate > N-(2hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES) > morpholinopropane sulfonic acid (MOPS) > morpholinoethane sulfonic acid (MES) > tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl).32 Vasina and Dejardin reported that the adsorption of R-chymotrypsin on muscovite mica was depressed by increasing the concentration of Tris-HCl buffer at pH 8.6, close to R-chymotrypsin’s isoelectronic point.33 PBS is the most commonly used buffer at the pH range close to 7, since it is reported to be able to stabilize protein structure in bulk solution environment in most cases.31 Phosphate ions are known to adsorb strongly onto most metal oxides such as hematite,34 goethite,35-37 TiO2,38 and some solid surfaces such as Au39 and Pt.40 It was reported that adsorbed IgG on TiO2 surface buffered by PBS can be partially displaced with phosphate ions by washing with higher concentration of proteinfree PBS buffer solution at pH 7.0 and pH 10.38 The adsorption

10.1021/jp806586n CCC: $40.75  2009 American Chemical Society Published on Web 02/05/2009

2054 J. Phys. Chem. C, Vol. 113, No. 6, 2009 of human and bovine serum albumin (HAS and BSA) on silica-titania surfaces was found to be much slower in phosphate buffered saline (PBS) buffer compared with adsorption in HEPES buffer.41 BSA adsorption on hydroxyapatite (HAP) was found to be depressed by the presence of phosphate ions.42 The structural rearrangement of adsorbed cytochrome c on graphite electrodes surface43 and hematite surface6 was found to be efficiently suppressed with the presence of phosphate ions. Due to the great variety of proteins and surfaces studied as well as the complex solvent environments employed, no general conclusions or physical models have been proposed. In order for reported experimental adsorption data to have significance or relevance to actual applications (such as surface fouling due to protein adsorption) and for meaningful comparisons to be made, the role of buffers in the adsorption process must be delineated and selection of buffer type and buffer concentration must be made with care. The objective of this paper is to study the buffer effect on the protein adsorption behavior. In this study, we chose TrisHCl and PBS, two commonly used buffers at physiological pH. The proteins we used include bovine serum albumin (BSA), lysozyme, immunoglobulin G (IgG), and fibrinogen. These proteins have different isoelectric points and are different in size and shape. The temperature was fixed at 25 °C and the pH was kept at 7.4. We chose a simple uniform surface (Ge) so that comparisons of data for different proteins and buffers can be made without complications arising from surface heterogeneity, reproducibility of surface morphology or surface changes as result of adsorption. The technique employed is FTIR/ATR. We report results for total adsorption and secondary structure as a function of time and as a function of adsorbed layers. A sequenced adsorption/flushing/displacement experiment was carried out on ZnSe surface to study the interaction between adsorbed phosphate ions and IgG molecules. 2. Materials and Methods 2.1. Materials. BSA and lysozyme were purchased from Sigma-Aldrich and were used without further purification. Fibrinogen and IgG were purchased from Serologicals Inc. and also were used as received. EM Science OmniPur tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl) was obtained from VWR (purity > 99%), and Dulbecco’s phosphate buffered saline (PBS) buffers were obtained from Irvine Scientific. Proteins were dissolved in the buffers to prepare the solutions and the pH was adjusted to 7.4 by adding either NaOH or HCl. For fibrinogen, 0.9% (w/w) of NaCl was added to facilitate dissolution. Unless noted otherwise, the protein concentration is 0.1 mg/mL and the buffer concentration is 10 mM. 2.2. Experimental Method and Procedure. FTIR/ATR measurements were performed using a Genesis II spectrometer (Mattson Instruments, Madison, WI). The protein spectra were detected with a deuterated triglycine sulfate (DTGS) detector at 25 °C. The spectrometer was equipped with a horizontal attenuated total reflectance (ATR) accessory (Pike Technologies). The ATR flow cell has a cell volume of 0.5 mL. The internal reflection element (IRE) is a Ge crystal surface with a native oxide layers. The phosphate displacement experiment (Section 3.4.2) was carried out on a ZnSe crystal. Ge surfaces with native oxide layers (water advancing contact angle: around 40 °)44 and ZnSe surfaces (water advancing contact angle: 76.3 °)45 are known to be hydrophilic. The interferometer and the sample compartment were purged with CO2 free dry air for at least 24 h before each experiment. The spectra were collected

Wei et al.

Figure 1. Calibration curves for BSA, lysozyme, IgG and fibrinogen adsorption on Ge surface.

at the resolution of 4 cm-1 using a normal Beer-Norton apodization function. Unless stated otherwise, a typical experiment proceeded as follows: First, a blank cell spectrum was recorded at 500 scans. Second, the buffer solution was injected into the ATR cell using a peristaltic pump (Harvard Apparatus) at a rate of 2 mL/min. After 10 min, the background (buffer solution) spectra were taken at 500 scans. The buffer flow was then replaced by protein solution at the same flow rate. A 38-scan spectrum was recorded every minute. During the adsorption experiment, protein concentration in the ATR cell was maintained constant due to continuous injection at a constant flow rate (2 mL/min). A large amount of fresh protein solution was prepared, and the protein solution was not circulated. The adsorption process was typically monitored for 360 min, after which the protein solution flow was replaced by a protein-free buffer solution for 120 min to monitor desorption. 2.3. Data Processing. 2.3.1. Quantification of Adsorption Amount and Number of Adsorbed Layers. In a protein IR spectrum, the amide II band (1480-1590 cm-1) is the least sensitive to structural rearrangement and is the most stable. Thus it is traditionally used to quantify the total amount of protein adsorbed. In this work, the experimentally measured absorbance was converted to surface excess through calibration, using a modification of the procedures described by Vermette et al.46 A fixed volume of protein solution of known concentration was dropped evenly onto the Ge surface using a syringe. The solution volume and concentration were used to calculate the actual amount of protein on the surface. This value was divided by the total area of the Ge surface to obtain the surface density. The Ge crystal with protein solution on top was vaccuum-dried for 24 h. The protein solution was spread as evenly as possible on the entire crystal top surface to ensure that the dried protein film was as uniform as possible. The next step was to obtain the spectra of the dried protein on the crystal. A blank spectrum was first taken at 500 scans after the FTIR spectrometer was well purged. Then the water vapor spectrum was collected at 128 scans. The ATR accessory, containing the ATR crystal with dried protein film on top, was then placed inside the sample compartment, and the spectrum was collected at 128 scans. The protein spectrum was obtained after water vapor subtraction, and the amide II peak was integrated. The procedures were repeated for different protein surface densities which were varied by changing the protein solution concentration. The amide II areas were then plotted against the corresponding surface density to yield the calibration curve. Figure 1 shows the calibration curves of the four proteins on the Ge surface. In visualizing the adsorbed protein phase, it is useful to estimate the approximate number of adsorbed layers in correspondence to various values of the adsorbed amount. In

Buffer Effect on Protein Adsorption

J. Phys. Chem. C, Vol. 113, No. 6, 2009 2055

TABLE 1: Protein Molecular Weight, Size and Estimated Adsorbed Mass per Layer

a

BSA IgG fibrinogen lysozyme a

molecular weight, kDa

size, Å3

estimated adsorbed mass per layer, mg/m2

66.4 150 340 14.3

84 × 84 × 84 × 31.548 235 × 44 × 4447 450 × 90 × 6048 45 × 30 × 3051

2.1 4.9 5.5 1.5 48

BSA is modeled as a triangular prismatic shell, while IgG, fibrinogen and lysozyme are modeled as elongated ellipsoids.

general, the mass per layer depends on molecular packing, which is sensitive to many parameters such as bulk protein concentration, protein molecular shape, molecular size and flexibility as well as protein/surface interactions, among others. Table 1 shows our estimates made by assuming closed pack coverage of the protein molecule and applying the jamming limit (approximately 50% for many common shaped objects47). IgG, fibrinogen, and lysozyme protein molecules are treated as ellipsoids, while BSA is modeled as a triangular prismatic shell48 with molecular dimensions shown in Table 1. In estimating the molecular packing, we assumed half of protein molecules adsorbed are lying flat at the maximum projected area and the other half are standing at the minimum projected area. With these assumptions, the estimated mass per adsorbed layer for BSA is 2.1 mg/m2, which is comparable to the reported experimental saturation adsorption value (1.8-2.4 mg/m2).47,49 The estimated mass per adsorbed layer for IgG is 4.9 mg/m2, which is within the reported experimental range of 2.1-8.4 mg/m2 depending on the experimental conditions.47 The estimated mass per adsorbed layer for fibrinogen is 5.5 mg/m2, which is comparable to the reported experimental saturation adsorption value of 5 mg/m2 on tantalum film.50 The estimated mass per adsorbed layer for lysozyme is 1.5 mg/m2, which is comparable to the experimental range of 0.95-2.1 mg/m2 reported on silica surface11 and 1.7 mg/m2 on SiO2 surface.51 We emphasize that the values shown in Table 1 are approximations and are used only to aid visualization of the adsorbed phase. 2.3.2. Secondary Structure Identification and Quantification. Protein biological function is sensitive to secondary structure. Surface induced denaturing is well-known; therefore it is important to monitor secondary structure in the adsorbed layers. The amide I region (1600-1700 cm-1) is sensitive to the type of secondary structure and has linear relationship with the secondary structural contents. Second derivative deconvolution and Fourier self-deconvolution are typically performed on the amide I band to determine the number and position of the overlapping peaks. Differences in the assignment of deconvoluted absorbance peaks to particular structures are found in the literature.52-55 The assignments used in this work are summarized in Figure 2, which are primarily based on X-ray crystal data and solution data in H2O and in D2O. Intramolecular antiparallel β-sheet has a strong absorbance band at 1630-1640 cm-1 and at higher frequency (1670-1685 cm-1), which overlaps with turn structure.56 The band near 1610-1628 cm-1 is assigned to intermolecular β-sheet (or intermolecular extended chains), particularly at high protein concentrations, or adsorbed in polyelectrolyte polymer multilayer matrix,54,57,58 although no exact assignments for this band have been established.59 Side chains can also contribute to absorbance in this region (1610-1628 cm-1). The other main structural component, R-helix band, is generally assigned to the region of 1649-1658 cm-1 in H2O and D2O, although absorption close to 1641 cm-1 has also been reported.54 The random

Figure 2. Empirical protein structure-frequency relationship in the amide I region from crystal X-ray measurements and from solution measurements in H2O and D2O:52-55 a)intermolecular β-sheet or possible side chain; b)intermolecular β-sheet.

TABLE 2: Protein Isoelectric Point (pI) and Net Charge at pH 7.4 pI net charge at pH7.4

BSA

fibrinogen

IgG

lysozyme

4.9 (4.7) -

5.5 -

6.1 -

11.4 +

coil bands centering at about 1643 cm-1 in D2O shifts to higher frequency in H2O (up to 1657 cm-1) and overlaps with the signal arising from R-helix.53,54 Therefore, the R-helix content in H2O cannot be unambiguously discriminated from random coil in the amide I region.30,56,60 In this paper, we followed the common practice of attributing this peak to a combination of R-helix and random coil in H2O environment.30,60 Specific peak assignments for the four proteins studied in this work are shown later in Table 3. Baseline correction, water subtraction, curve-fitting and smoothing were carried out carefully and consistently. The Levenberg-Marquardt algorithm was used for curve-fitting. The Savitzky-Golay algorithm was applied to smoothing the curves and to suppressing noises, which got amplified during Fourier deconvolution or second derivative analysis. Secondary derivative analysis was used for the estimation and selection of the reasonable range for the peak parameters (height, width and peak position).30 The same numerical processing methods are used for all data obtained in this study with little subjective judgment. Therefore, the small number of arbitrary numerical parameters (such as the range of peak height) should have minimal effect on the observed trends. 3. Results and Discussion It is known that protein size and net charge have significant effects on adsorption. In this study, we selected four proteins with different sizes (Table 1) and isoelectric points (pI) (Table 2.) In this section, we compare the adsorption behavior of negatively charged proteins BSA, IgG and fibrinogen with the positively charged protein, lysozyme, at the same bulk protein concentration of 0.1 mg/mL on a Ge surface in Tris-HCl (10 mM) and PBS (10 mM) buffers. This section is divided into four parts and is organized as follows: in section 3.1, we compare the adsorption kinetics of the four proteins in the two buffers. In section 3.2, we compare the time evolution of the average secondary structure of the adsorbed proteins and the secondary structural content as a function of layer number in the two buffers. In section 3.3, we study the effect of buffer concentration on protein adsorption. Because PBS is the most important and commonly used buffer at physiological pH, and due to the sensitivity of protein adsorption to competition from phosphate ions present in PBS buffer, we examine the role of phosphate ions in section 3.4 by monitoring the adsorption of phosphate ions on the solid surface in the presence of the four proteins. Also shown in section 3.4 is a detailed illustration of the effect of phosphate ions on IgG

2056 J. Phys. Chem. C, Vol. 113, No. 6, 2009

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TABLE 3: Amide I Spectral Band Positions (cm-1) of Adsorbed BSA, IgG, Fibrinogen and Lysozyme on Ge Surface β-sheet side chain

intermolecular

intramolecular

R-helix/ random coil

BSA

1615 ( 1.5

1637 ( 0.3

1651 ( 0.4

IgG

1615 ( 1.5

1626 ( 0.3 1690 ( 1.2 1626 ( 0.3 1690 ( 1.2 1618 ( 0.5 1685 ( 0.3 1622 ( 0.1 1691 ( 0.8

1637 ( 0.3

1651 ( 0.4

1635 ( 0.1

1646 ( 0.8 1653 ( 0.3 1654 ( 0.1

fibrinogen lysozyme

adsorption using a sequenced adsorption/flushing/displacement experiment where the IgG molecules previously adsorbed in Tris-HCl buffer were displaced by PBS buffer. 3.1. Adsorption Kinetics of BSA, IgG, Fibrinogen and Lysozyme in Tris-HCl and PBS Buffers. Unless otherwise marked, adsorption at bulk concentration of 0.1 mg/mL was monitored for 360 min after which the protein solution flow was switched to protein-free buffer flow to monitor the presence (or absence) of desorption. Figure 3 shows the adsorption kinetics curves for BSA. The LHS ordinate shows the adsorbed amount as computed from absorbance and the calibration curve. The RHS ordinate shows the corresponding estimated number of adsorbed layers as described in section 2.3.1. The adsorption kinetic behavior shown in Figure 3 follows the generic trend common to high-affinity multilayer adsorption: a short period of rapid initial rise is followed by a long quasilinear region. It is obvious that no adsorption equilibrium is reached and a large number of adsorbed layers are formed. There is virtually no desorption when the surface was flushed with BSA-free buffer after adsorption for 360 min, indicating that the adsorption was irreversible. Before discussing the kinetic behavior, it is useful to first examine the driving force underlying the adsorption in this system. In high-affinity adsorption with negligible desorption, the driving force within a relatively short fixed distance is controlled by (µib - µis(top layer(s)), where µib is the chemical potential of the protein i in the bulk solution phase and µis(top layer(s)) is the corresponding value on the adsorbed phase (Figure 4). The magnitude of µib depends on the nature of protein i and the bulk conditions (temperature, protein concentration, buffer/solvent type, etc). In a flow experiment, µib is independent of time (t). The magnitude of µis(top layer(s)), on the other hand, hinges on the nature of the protein i, the nature of the solid surface, and the adsorbed phase conditions (adsorbed amount and the composition of the adsorbed layers). Hence, unlike µib, µis(top layer(s)) varies with time. Because the adsorption layers are not a homogeneous

Figure 3. BSA adsorption at pH 7.4 and 0.1 mg/mL bulk concentration in different buffers on Ge surface.

1633 ( 0.2 1642 ( 0.4

turn 1661 ( 1.0 1675 ( 0.7 1661 ( 1.0 1675 ( 0.7 1666 ( 0.4 1673 ( 0.4 1674 ( 0.4 1683 ( 0.5

phase, we expect the chemical potential of the top adsorbed protein molecules, µis(top layer(s)), to vary from layer to layer. µis(top layer(s)) has two asymptotic values: µis0 and µis∞, corresponding to the values at t ) 0 and t f ∞ respectively. µis0 represents the chemical potential of the protein i adsorbed on the bare surface and is dominated by protein-surface interactions, whereas µis∞ stands for the chemical potential of protein i in the topmost adsorbed layer (generally far from the solid surface), and hence is dominated by protein-protein and protein-solvent interactions. It is expected that, in general, the limit t f ∞ is reached after a few layers have been adsorbed. It is also expected that in general µis0 is lower (or more negative) than µis∞, that is, µis0 < µis∞. In the short initial rapid adsorption region, the adsorbed protein chemical potential µis(top layer(s)) changes rapidly from µis0 to µis∞ as the surface is covered and the first few layers are adsorbed. The driving force during this short period varies with time, resulting in the observed rapid and nonlinear kinetics curve. After a few layers have been adsorbed, the driving force reaches the asymptotic and constant value of (µib - µis∞) resulting in the observed quasi-linear region. The slope in the quasi-linear region is smaller than that in the initial region because µis0 < µis∞. With this physical picture in mind, Figure 3 implies that both buffers are able to stabilize BSA in the bulk solution phase (by lowering µib) compared with in pure water solvent case so the initial adsorption is lower in the buffered solutions. The ability of buffers to stabilize protein in bulk solution is well-known. The long-time total adsorption is sensitive to the slope of the quasi-linear region, and Figure 3 shows that the adsorption rate in this region is the highest in Tris-HCl, followed by that in PBS. The unbuffered case has the lowest adsorption rate in this region. This trend is different from that during the initial adsorption where the unbuffered case has the highest adsorption. We believe this is because in the initial region, the buffer affects primarily only one of the chemical potentials in the driving force (µib) whereas both chemical potentials terms in the driving force in the quasi-linear region are affected by buffer choice and the observed behavior depends on the difference between the two chemical potentials in the driving force in the two buffers. BSA adsorption on Ge is lower in PBS than in Tris-HCl both in the initial rapid region and in the quasi-linear region, implying that (µib - µis0)PBS < (µib - µis0)Tris-HCl and (µib - µis∞)PBS < (µib µis∞)Tris-HCl; that is, the driving force in PBS is lower than in both regions. Our results are consistent with reported observation of depressed BSA adsorption on silica-titanium41 and on HAP surface in the presence of phosphate ions. Due to the importance of PBS buffer at physiological pH, we will return to a more detailed examination in section 3.4. The adsorption kinetic curves for IgG, fibrinogen, and lysozyme in Tris-HCl and PBS are shown in Figure 5. They are all qualitatively similar to those for BSA; namely, the adsorption is multilayer with a short period of rapid initial



Figure 4. Driving force during protein multilayer adsorption on solid surface.

Figure 5. Surface excess and estimated layers of adsorbed IgG, fibrinogen and lysozyme as a function of time in PBS and Tris-HCl on a Ge surface at pH 7.4: (a) IgG (0.1 mg/mL); (b) fibrinogen (0.1 mg/ mL); (c) lysozyme (0.1 mg/mL and 1.0 mg/mL).

adsorption followed by a long period of quasi-linear adsorption. The adsorption of lysozyme is slightly more reversible compared with the other proteins. This is probably due to lysozyme’s much smaller size. Some quantitative differences are observed, however. The adsorption amounts of the negatively charged proteins, IgG and fibrinogen, are significantly depressed in PBS buffer compared with in Tris-HCl buffer (as is the case for BSA). This observation is also consistent with published results which show that phosphate ions are able to depress negative charged IgG (at pH 7.0 and 10.0) adsorption and can partially

displace adsorbed negative charged IgG (at pH 7.0 and 10.0) on TiO2 surface, whereas little displacement of adsorbed IgG molecules was observed at pH 4.0 where IgG carries a net positive charge.38 For the positively charged protein in this study, lysozyme, buffer choice has little effect (although a small enhancement in adsorption in PBS buffer can be noted in Figure 5(c)). To further verify the insensitivity of lysozyme adsorption to buffer choice, we repeated the adsorption experiment at a higher bulk concentration of 1.0 mg/mL (Figure 5(c)), and the same trend was observed. The difference observed in behaviors of the two buffers for proteins with positive and negative charges is most likely due to the fact that the negatively charged phosphate ions in PBS buffer have a tendency to adsorb on the solid surface and retard the approach of negatively charged proteins such as BSA, IgG and fibrinogen, resulting in lower adsorption. In addition, the negatively charged phosphate ions are better able to stabilize the positively charged proteins in the bulk solution, hence reducing the driving force, (µib - µis∞). 3.2. Secondary Structural Changes upon Adsorption. Changes in protein secondary structure are frequently monitored as indications of denaturing. Denaturing upon adsorption is important in many applications such as implants, biofouling, etc. In this section we show the time evolution of secondary structure of the four proteins adsorbed on Ge in PBS and TrisHCl buffers as measured by FTIR/ATR. The secondary structural assignments of IgG,53 BSA,53 fibrinogen57,61 and lysozyme62,63 in the bulk solution and adsorbed on the solid surface have been reported in the literature. Quantification of secondary structure is sensitive to the peak assignments. There are no definitive peak assignments, and a range of values is reported in the literature (see Figure 1). To conduct comparative studies and to observe changes over time, it is important to have a set of consistent peak assignments for the various principal secondary structural components for each protein. In this study, we apply the peak assignments shown in Table 3. We assign peaks centering around 1685-1692 cm-1 to intermolecular β-sheet, although some studies have also attributed these peaks to the turn structure. As stated in section 2.3.2, R-helix can be distinguished from random coil structures in D2O, however, in H2O, R-helix overlaps with random coil. In this study, we follow the practice of combining them as a single peak and identify the structure as R-helix/random coil. Intramolecular β-sheet peak usually centers around 1635-1638 cm-1 for BSA, IgG and fibrinogen. For lysozyme, intermolecular β-sheet exhibits two peaks, at 1633 and 1642 cm-1. These are combined and identified as intermolecular β-sheet. In graphical representation, for the sake of clarity, we show only the three principal secondary structural components: R-helix/random coil,


Wei et al.

Figure 6. Secondary structural evolution of adsorbed proteins at 0.1 mg/mL bulk concentration in Tris-HCl and PBS on Ge surface at pH 7.4: (+) R-helix/random-coil (Tris-HCl); (O) R-helix/random-coil (PBS); ([) β-sheet (Tris-HCl); (0) β-sheet (PBS); (1) turn (Tris-HCl); (4) turn (PBS).

TABLE 4: Compositions of Secondary Structurea for Protein in Bulk Solution (D2O) (%) 53

BSA IgGc fibrinogen57 lysozyme62

R-helix/coil

β-sheetb

turn

51-55 7-9 35-37 53

23-27 57-64 49-61 7

18-24 30-32 8-11 40

a

Contribution of side chain is counted but not listed. b β-Sheet includes both intramolecular and intermolecular β-sheet. c Measured in this work using transmission FTIR in D2O.

β-sheet (combination of intermolecular and intramolecular β-sheets) and turns. Side chain structures make a minor contribution and are relatively stable and are, therefore, not shown. 3.2.1. Secondary Structure as a Function of Adsorption Time. Figure 6 shows the time evolution of the three main secondary structural components (R-helix/random-coil, β-sheet and turn) of adsorbed BSA, IgG, fibrinogen, and lysozyme on Ge buffered with PBS and Tris-HCl. Data beyond 360 min (when the protein solution flow was switched to buffer flow) are not shown because no further secondary structure changes are observed. This means also that the adsorbed protein molecules do not exhibit long-time aging on the surface. Of the four proteins in this study, IgG has the most stable secondary structure (Figure 6(b)). The percentages of the three principal secondary structural components show little variation with time, and the magnitudes are similar to those in the bulk solution (Table 4). Buffer choice also has little effect on the secondary structure of IgG. Significant changes in secondary structure are observed upon adsorption for the other three proteins. Figures 6(a), 6(c) and 6(d) indicate that BSA, fibrinogen and lysozyme all show similar qualitative secondary structure changes over an extended period of time: decreases in R-helix/ coil and turn structures, and increase in β-sheet structure. This is consistent with the observation for BSA adsorbed on polyelectrolyte substrate.58 The effect of buffer choice, however,

depends on the protein. For lysozyme, there is little difference between PBS and Tri-HCl. This is perhaps expected because the adsorption curves for lysozyme in the two buffers are very similar (Figure 5(c)). For BSA and fibrinogen, buffer choice results in observable differences in secondary structure changes. More importantly, the average secondary structure at long-time for adsorbed BSA, fibrinogen and lysozyme are quite different from their bulk solution secondary structure. We also expect the secondary structure of the adsorbed phase not to be homogeneous (i.e., the layers near the solid surface are likely to have different secondary structure versus the layers further away from the solid surface); however, the FTIR/ATR data yield the average secondary structure across the whole adsorbed phase. Therefore, it would be meaningful and informative to deduce the secondary structure as a function of layer number. This is shown in the next section. 3.2.2. Secondary Structure of Adsorbed Proteins as a Function of Adsorbed Layer Number. As stated above, FTIR data and Figure 6 yield the average secondary structure of all adsorbed molecules as a function of time. By combining Figure 6 with the adsorption curves (Figure 5), we can deduce the differential structures as a function of incremental adsorbed amount as time increases. This is done as follows: Figure 6 shows secondary structural percentage as a function of time (t), that is, x% vs t. Figure 5 shows adsorbed amount or adsorbed layer number (N) as a function of time, or N vs t. By combining Figure 5 and Figure 6 and eliminating t we obtain (average) secondary structure x% as a function of (total) adsorbed layer N, i.e., x% vs N. By slightly smoothing this curve of x% vs N and taking the ratio of the differences ∆(x% N)/∆(N), we obtain the secondary structural components in each successive adsorbed layer. The results are shown in Figure 7, where each bar shows the average secondary structural content in a particular adsorbed layer. For example, Figure 7(a) shows that when BSA is buffered with PBS, in the second adsorbed layer, the BSA molecules exhibit about 30% β-sheet structure, 34% R-helix/ coil structure and 28% turn structure. Also shown for compari-



Figure 7. The secondary structural content in each successive adsorbed layer: (a) BSA; (b) IgG; (c) fibrinogen;* (d) lysozyme.# *No differential secondary structure is obtained for the first few layers in Tris-HCl because the initial adsorption for fibrinogen is very large and rapid; no results are obtained for larger layer numbers in PBS because the adsorption rate in the quasi-linear region is very low (Figure 5(b)). #The column marked with + is obtained from a separate experiment at a lower bulk concentration of 0.01 mg/mL.

son (as horizontal dashed lines) are the contents in the bulk solution. Because Figure 7 is the result of a number of approximations and smoothing operations, we only make a number of qualitative observations. These observations are, nevertheless, very informative. First, the effect of buffer choice on secondary structure change is not significant. The observed differences in Figure 6 are primarily due to the large differences in the adsorbed amount. Second, the secondary structure in the adsorbed layers is in general quite different from those in the bulk solution. Third, the secondary structure distribution is not homogeneous across the adsorbed phase. The secondary structure in the first few layers can be very different from those in subsequent layers, (see in particular, Figure 7(d) for lysozyme). Finally, adsorbed BSA, fibrinogen and IgG all show relatively stable secondary structures beyond the first few layers. On the other hand, adsorbed lysozyme secondary structure continues to vary from layer to layer. This is most likely related to the fact that of the four proteins studied, lysozyme suffers the largest change in secondary structure as a result of adsorption (for example, the β-sheet content changes from less than 10% to 30-40% while the R-helix/random coil content decreased from over 50% to 30-40%), which implies that lysozyme secondary structure is more easily perturbed under the effects of the substrate surface. 3.3. The Effect of Buffer Concentration on Protein Adsorption. Since the type of buffer significantly affects the adsorption behavior, it is expected that buffer concentration will also have an effect. Here we demonstrate the magnitude of this

effect for BSA adsorption. Figure 8(a) shows BSA adsorption on Ge at three Tris-HCl concentrations (0, 10 and 100 mM). Corresponding results for BSA in PBS buffer are shown in Figure 8(b). The observed effect of buffer concentration is rather complex. Tris-HCl depresses the initial adsorption of BSA on Ge, but enhances the adsorption in the quasi-linear region. The enhancement (slope of the quasi-linear region) increases monotonically with Tris-HCl concentration. In PBS buffer, the initial adsorption is also depressed as in the Tris-HCl buffer; however, in the quasi-linear region, the adsorption rate varies nonmonotonically with the PBS concentration. The adsorption rate was increased when the PBS concentration was changed from 0 mM to 10 mM, but when the PBS concentration was further increased to 100 mM, the adsorption rate was drastically reduced. We expect this to be due to the complex dependence of the driving force (µib - µis∞) on PBS concentration. This complex behavior is perhaps to be expected for a number of reasons: (a) phosphate ions adsorb on solid surfaces and can compete with protein adsorption; (b) phosphoric acid has three states of deprotonation resulting in three types of phosphate ions, with pH dependent relative distribution (Figure 9); (c) phosphate ions can possibly form complexes (especially with metal oxides such as R-Fe2O334). We will briefly examine some of these issues in the following section. 3.4. PBS Detailed Study. Because PBS is the most commonly used buffer at physiological pH, in this section we examine the role of phosphate ions in PBS buffered protein adsorption. We first study the adsorption of the two dominant


Wei et al.

Figure 10. Phosphate IR absorbance spectra at 300-min adsorption in protein solution (fibrinogen, lysozyme, IgG and BSA) buffered by10 mM PBS and in the protein-free PBS buffer (10 mM) on Ge.

Figure 8. BSA adsorption at pH 7.4 and 0.1 mg/mL bulk concentration on Ge surface: (a) at different Tris-HCl concentrations; (b) at different Tris-HCl concentrations.

Figure 9. Mole fraction of phosphate species (H3PO4, H2PO4-, HPO42and PO43-) as a function of pH (the equilibrium constants of the three successive deprotonation steps of H3PO4 are pKa1 ) 2.15; pKa2 ) 7.2; pKa3 ) 12.4 respectively).

phosphate ions at pH 7.4, H2PO4- and H2PO4-, during protein adsorption in section 3.4.1. In section 3.4.2, we show the use of phosphate ions to desorb previously adsorbed IgG molecules in a sequential adsorption/flushing/displacement experiment. 3.4.1. Presence of Phosphate Ions during Protein Adsorption in PBS Buffer. As stated above, some of the complexities observed in PBS buffered systems are due to the tendency of phosphate ions to adsorb on surfaces. When protein solutes are present, competitive adsorption is expected to occur. Since FTIR spectra are sensitive to the phosphate deprotonation states, we monitor the absorption peaks associated with specific types of phosphate ions during protein adsorption. In aqueous environment, phosphate exists in four possible states, depending on the pH (Figure 9). At pH 7.4, the dominant ionic forms are HPO42- and H2PO4-, in the mole ratio of about 1.6 to 1.0. The H2PO4- ion has four IR absorption peaks at 1156-1159, 1077, 940-944, and 875-879 cm-1; and HPO42- has 3 peaks at 1078-1080, 990 and 850-855 cm-1.35,64 Figure 10 compares the phosphate absorption spectra at 300 min of adsorption for BSA, fibrinogen, IgG and lysozyme in

Figure 11. IgG adsorption at 0.1 mg/mL bulk concentration in TrisHCl and in PBS on ZnSe surface: (a) calibration curves for IgG on Ge and ZnSe; (b) adsorption amount and estimated layers as a function of time.

PBS buffer and that for the protein-free PBS buffer on Ge surface. Several interesting observations can be made: first, the protein-free buffer phosphate spectrum shows the broad peak around 1077-1080 cm-1 characteristic of either HPO42- or H2PO4-, but the 1160 cm-1 peak unique to H2PO4- was not apparent, despite the fact that the expected mole ratio of HPO42to H2PO4- is about 1.6 to 1.0 in the bulk. This means that the Ge surface was able to shift the phosphate deprotonation equilibrium so that the phosphate ion relative distribution in the bulk is not the same as that on the surface. The second interesting observation is that the 1160 cm-1 peak unique to H2PO4- is also not observed during adsorption of BSA, IgG and lysozyme, but is very prominent in fibrinogen adsorption.



Figure 12. IgG sequential adsorption/flushing/displacement experiment. (a) IgG adsorbed amount and estimated layers.* (b) Phosphate absorption spectra as a function of time. (c) Integrated phosphate peak area. (d) IgG adsorbed amount as a function of phosphate peak area. *I: Adsorption in Tris-HCl. II: Flushing with Tris-HCl. III: Displacement in PBS.

It is possible that fibrinogen promotes the co-adsorption of the H2PO4- ions. 3.4.2. Displacement of Adsorbed IgG with Phosphate Ions on ZnSe Surface. As shown in section 3.1, the presence of phosphate ions in PBS buffer significantly affected the adsorption kinetics of negatively charged protein such as BSA, IgG and fibrinogen. In this section, we examine the correlation between protein adsorption and phosphate adsorption. For illustration purpose, we selected IgG because it has the most stable secondary structure during adsorption. We performed the experiment on the ZnSe surface because the slope of the IgG calibration curve on ZnSe is significantly larger than on Ge (Figure 11(a)) so the results should be more reliable. We first performed a standard 360-min adsorption experiment on ZnSe using both Tris-HCl and PBS buffers. The qualitative behavior of IgG on ZnSe (Figure 11(b)) is very similar to that on Ge (Figure 5(a)). We see that IgG adsorption is considerably depressed in PBS buffer compared to Tri-HCl buffer. We then conducted a sequential adsorption/flushing/displacement experiment. In this experiment, IgG adsorption at bulk concentration of 0.1 mg/mL in Tris-HCl buffer was first carried out on ZnSe for 360 min. At the end of 360 min, the IgG solution flow was replaced by protein-free Tris-HCl buffer at the same flow rate. This flushing step was carried out for 120 min. The Tris-HCl buffer flow was then switched to protein-free PBS buffer in a displacement step for 360 min. The surface excess/adsorbed layer number as a function of time during this experiment is shown in Figure 12(a). The IgG molecules adsorbed on ZnSe in Tris-HCl buffer were only very slightly desorbed by flushing with protein-free Tris-HCl buffer. However, when PBS buffer was introduced, the adsorbed IgG molecules were rapidly

desorbed. The desorption process was smooth and continuous, reducing the number of adsorbed layers from over 7 to about 2 layers in 360 min. The presence of phosphate ions on the surface during this sequential experiment was monitored and shown in Figure 12(b). From the absorbance spectra in Figure 12(b), the phosphate absorbance peak at around 1078 cm-1 is absent before 480 min but is clearly visible at 600 and 860 min after switching to PBS buffer (at 480 min). The phosphate peaks were integrated and shown as a function of time in Figure 12(c). A comparison of Figure 12(a) region III and Figure 12(c) shows a clear correspondence between the adsorption of phosphate ions and desorption of IgG molecules during the same time period. To further illustrate this correlation, we plotted the IgG surface excess (from Figure 12(a)) against the phosphate absorbance peak area (from Figure 12(c)) in Figure 12(d). The linear relationship confirms that the desorption of IgG molecules was directly related to and caused by the adsorption of phosphate ions. 4. Conclusions Buffer type as well as buffer concentration can have significant effects on protein adsorption. In this work, we showed that PBS significantly depressed BSA, IgG and fibrinogen adsorption on Ge at pH 7.4 compared to Tris-HCl. Lysozyme adsorption is not significantly affected by buffer choice. A possible explanation is that phosphate ions have a tendency to adsorb on the Ge surface making the surface negatively charged, resulting in the suppression of the adsorption of proteins carrying net negative charge (at pH ) 7.4, BSA, IgG and fibrinogen are

2062 J. Phys. Chem. C, Vol. 113, No. 6, 2009 negatively charged, whereas lysozyme is positively charged). This sensitivity to buffer type and buffer concentration means that care must be exercised when selecting the buffer conditions for adsorption studies and when evaluating biomedical implants as well as when comparing adsorption data. Protein secondary structure can change significantly upon adsorption; however, buffer choice seems to have negligible effect on the secondary structure of the adsorbed proteins studied here provided comparisons are based on adsorbed amount rather than on adsorption time. The behavior of PBS buffer, the most commonly used buffer at physiological pH, is particularly complex in adsorption studies due to the various types of phosphate ions present and the tendency of these ions to adsorb competitively and/or to form complexes either with the proteins or with the surfaces. Therefore, unless necessary, it is recommended that PBS should not be used as the routine buffer of choice for adsorption studies. Acknowledgment. The authors wish to acknowledge support of this work from the USC WISE program. References and Notes (1) Heinrich, L.; Mann, E. K.; Voegel, J. C.; M., K. G. J.; Schaaf, P. Langmuir 2996, 12, 4857. (2) Kaewtathip, S. Ph.D Thesis, University of Southern California, 2004. (3) Brusatori, M. A.; Tie, Y.; Van Tassel, P. R. Langmuir 2003, 19, 5089. (4) Tie, Y.; Ngankam, A. P.; Van Tassel, P. R. Langmuir 2004, 20, 10599. (5) Kim, D. T.; Blanch, H. W.; Radke, C. J. Langmuir 2002, 18, 5841. (6) Khare, N.; Eggleston, C. M.; Lovelace, D. M.; Boese, S. W. J. Colloid Interface Sci. 2006, 303, 404. (7) Hong, X. Y.; Christopher, R. L. J. Colloid Interface Sci. 1996, 182, 586. (8) Xu, H.; Zhao, X.; Grant, C.; Lu, J. R. Langmuir 2006, 22, 6313. (9) Su, T. J.; Lu, J. R.; Thomas, R. K.; Cui, Z. F.; Penfold, J. Langmuir 1998, 14, 438. (10) Lu, J. R.; Su, T. J.; Thirtle, P. N.; Thomas, R. K.; Rennie, A. R.; Cubitt, R. J. Colloid Interface Sci. 1998, 206, 212. (11) Lu, J. R.; Swann, M. J.; Peel, L. L.; Freeman, N. J. Langmuir 2004, 20, 1827. (12) Billsten, P.; Wahlgren, M.; Arnebrant, T.; McGuire, J.; Elwing, H. J. Colloid Interface Sci. 1995, 175, 77. (13) Calonder, C.; Van Tassel, P. R. Langmuir 2001, 17, 4392. (14) Zhou, J.; Zheng, J.; Jiang, S. Y. J. Phys. Chem. B 2004, 108, 17418. (15) Zheng, J.; Li, L.; Tsao, H. K.; Sheng, Y. J.; Chen, S.; Jiang, S. Biophys. J. 2005, 89, 158. (16) Agashe, M.; Raut, V.; Stuart, S. J.; Latour, R. A. Langmuir 2005, 21, 1103. (17) Raut, V. P.; Agashe, M. A.; Stuart, S. J.; Latour, R. A. Langmuir 2005, 21, 1629. (18) Pitt., W. G.; Weaver, D. R. J. Colloid Interface Sci. 1997, 185, 258. (19) Mungikar, A. A.; Forciniti, D. Biomacromolecules 2004, 5, 2147. (20) Mungikar, A. A.; Forciniti, D. Biomacromolecules 2006, 7, 239. (21) Song, D.; Forciniti, D. J. Chem. Phys. 2001, 115, 8089. (22) Li, L.; Chen, S.; Zheng, J.; Ratner, B. D.; Jiang, S. J. Phys. Chem. B 2005, 109, 2934. (23) Zhang, Z.; Chen, S.; Jiang, S. Biomacromolecules 2006, 7, 3311. (24) Clare, T. L.; Clare, B. H.; Nichols, B. M.; Abbott, N. L.; Hamers, R. J. Langmuir 2005, 21, 6344. (25) Malmsten, M.; Veide, A. J. Colloid Interface Sci. 1996, 178, 160. (26) Daly, S. M.; Przybycien, T. M.; Tilton, R. D. Langmuir 2005, 21, 1328.

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