Interaction of Bovine Serum Albumin and Lysozyme with Stainless

Article pubs.acs.org/Langmuir

Interaction of Bovine Serum Albumin and Lysozyme with Stainless Steel Studied by Time-of-Flight Secondary Ion Mass Spectrometry and X‑ray Photoelectron Spectroscopy Yolanda S. Hedberg,*,†,‡,∥ Manuela S. Killian,† Eva Blomberg,‡,§ Sannakaisa Virtanen,† Patrik Schmuki,† and Inger Odnevall Wallinder‡ †

Institute for Surface Science and Corrosion, Department of Materials Science and Engineering 4, Friedrich-Alexander-University of Erlangen−Nuremberg, Martensstrasse 7, 91058 Erlangen, Germany ‡ Division of Surface and Corrosion Science, Department of Chemistry, School of Chemical Science and Engineering, KTH Royal Institute of Technology, Drottning Kristinas väg 51, SE-10044 Stockholm, Sweden § Institute for Surface Chemistry, YKI, Post Office Box 5607, SE-114 86 Stockholm, Sweden S Supporting Information *

ABSTRACT: An in-depth mechanistic understanding of the interaction between stainless steel surfaces and proteins is essential from a corrosion and protein-induced metal release perspective when stainless steel is used in surgical implants and in food applications. The interaction between lysozyme (LSZ) from chicken egg white and bovine serum albumin (BSA) and AISI 316L stainless steel surfaces was studied ex situ by means of X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) after different adsorption time periods (0.5, 24, and 168 h). The effect of XPS measurements, storage (aging), sodium dodecyl sulfate (SDS), and elevated temperature (up to 200 °C) on the protein layers, as well as changes in surface oxide composition, were investigated. Both BSA and LSZ adsorption induced an enrichment of chromium in the oxide layer. BSA induced significant changes to the entire oxide, while LSZ only induced a depletion of iron at the utmost layer. SDS was not able to remove preadsorbed proteins completely, despite its high concentration and relatively long treatment time (up to 36.5 h), but induced partial denaturation of the protein coatings. High-temperature treatment (200 °C) and XPS exposure (X-ray irradiation and/or photoelectron emission) induced significant denaturation of both proteins. The heating treatment up to 200 °C removed some proteins, far from all. Amino acid fragment intensities determined from ToF-SIMS are discussed in terms of significant differences with adsorption time, between the proteins, and between freshly adsorbed and aged samples. Stainless steel−protein interactions were shown to be strong and protein-dependent. The findings assist in the understanding of previous studies of metal release and surface changes upon exposure to similar protein solutions.

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matrix effects are minor in the case of protein investigations.10 ToF-SIMS, often in combination with XPS and multivariate analysis such as principal component analysis (PCA), has been used to distinguish between different proteins,11,12 protein conformation,13 polar and nonpolar amino acid groups,13−15 denaturation states of proteins,11,16 and layer thicknesses.13,15 The adsorption of bovine serum albumin (BSA) on stainless steel grade AISI 316L has previously been investigated by ToFSIMS and dynamic contact angle measurements.17 Many other techniques have been employed to investigate the interaction between BSA and stainless steel surfaces in terms of adsorption (kinetics),6,18−23 binding mechanisms,6,18,20,24,25 and corrosion,6,18,26 by using electrochemical techniques,18,26 XPS,4,6,22 quartz-crystal microbalance (QCM),6,19,23 solution analy-

INTRODUCTION Mechanistic understanding of the interaction of stainless steel and proteins is important for many applications, including corrosion in protein environments, such as biofouling in seawater,1 and metal release upon protein-induced corrosion for food-related applications2 and surgical implants.3,4 Generally, proteins are reported to enhance both the extent of metal release and the rate of corrosion for stainless steels.4−8 The reverse situation (reduced corrosion or metal release) has been reported in the case of a certain mussel protein5 and for pitting corrosion resistance in serum.9 Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been reported as a more capable method for detailed surface analysis of adsorbed proteins on metal substrates compared with X-ray photoelectron spectroscopy (XPS), due to its higher extent of information on chemical states and an even higher surface sensitivity.10 Though not quantitative, ToFSIMS can provide relative semiquantitative information, and © 2012 American Chemical Society

Received: October 2, 2012 Revised: November 1, 2012 Published: November 1, 2012 16306

dx.doi.org/10.1021/la3039279 | Langmuir 2012, 28, 16306−16317

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Figure 1. Overview on previously obtained6 results by means of solution studies, quartz-crystal microbalance, X-ray photoelectron spectroscopy, and ζ potential measurements, on different stainless steel grades, for example, 316 L sheets exposed to LSZ or BSA in PBS at pH 7.4. Local lowering of the surface pH and enrichment of chlorides are caused by charge regulation.

Table 1. Nominal Bulk Composition of the 316L Sheet Based on Supplier Information composition (wt %) Fe

Cr

Ni

Mo

Mn

Si

C

S

P

Cu

68.9

16.6

10.6

2.1

1.0

0.4

0.03

0.001

0.02

0.3

sis,4,6,8,19 and vibrational techniques such as infrared spectroscopy,20,22 as well as theoretical considerations.24,25 Both chemisorption6,8,18,22,24,25 and physisorption6,20 were proposed involving interactions with carboxylate groups18,20,24 or amine groups24 at neutral pH conditions. The surface charge of the stainless steel is a key factor for any interaction. However, scarce information on the surface charge of stainless steels is available in the literature, with reported assumptions on, or measurements of, an isoelectric point (iep) ranging from pH 3 to 8.5,6,18,23 a variation that indicates the importance of differences in surface finish and material properties. Recent measurements by some of the authors of this study revealed an iep of pH 3−4 for stainless steel 316 in 1 mM KCl solution and an approximate ζ potential of −100 mV at pH 7.4.6 Recently, the influence of net positively charged lysozyme (LSZ) and net negatively charged BSA on corrosion, adsorption, and metal release of different stainless steel grades in phosphate-buffered saline (PBS, pH 7.4) was investigated by some of the authors of this study, using solution analysis, XPS, electrochemical techniques, QCM, and ζ potential measurements, summarized in Figure 1.6 The normal albumin concentration in human plasma is 42 ± 3.5 g/L,27 while the human lysozyme concentration is 7−13 mg/L in serum and about 1.2 g/L in tear fluid.28 The high abundance of albumin in human blood was the reason for its choice, while lysozyme was chosen on the basis of its opposite net charge. Both proteins were previously investigated in terms of adsorption on AISI 316,19 complexation with chromium,29 and induced metal release for different stainless steel grades.6,19 The strong enhancement of metal release induced by BSA resulted in the enrichment of surface oxide chromium content due to preferential iron complexation and/or release. In contrast with BSA, LSZ only slightly enhanced the metal release and did not induce such strong chromium enrichment in the surface oxide. This occurred in the cases of both the same molar and mass concentration of proteins in solution.6 BSA adsorbed at monolayer coverage at 1 g/L BSA in PBS at pH 7.4 on stainless steel AISI 316.6 No further increase in thickness

was observed with time.6 It has previously been shown, in protein concentration-dependent QCM measurements on chromium metal, that the proteins adsorb in monolayer coverage at and above a concentration of 0.5 g/L BSA in PBS at pH 7.4.19 This is in general agreement with many investigations.13,17,23,30,31 Hence, monolayer coverage in the case of BSA is expected also in this study for 100 g/L BSA in PBS. In contrast, the adsorbed LSZ layer thickness has been shown to continuously increase with time and correspond to several layers on stainless steel 316 after 1 h of exposure at a concentration of 1 g/L in PBS.6 LSZ has also been reported to adsorb on mica and silica surfaces with an inner, relatively strongly bound layer and an outer, more weakly adsorbed layer at concentrations above 0.02 g/L.32,33 Hence, LSZ was expected to adsorb in several layers also in this study (2.2 g/ L in PBS), as previously confirmed by means of XPS.6 Several possible binding and metal release mechanisms were identified. Both (a) complexation between BSA or LSZ and the stainless steel surface and (b) a local lowering of pH and enrichment of anions upon charge regulation between the adsorbed protein layer and the stainless steel surface were suggested as possible mechanisms for the observed protein-induced enhancement of released metals from stainless steel.6 The aim of this study was to use the combination of ToFSIMS and XPS to investige the same system as investigated previously,6 to gain additional information on the interaction between the stainless steel AISI 316L surface and the adsorbed protein layer (LSZ or BSA). This was conducted for different adsorption time periods up to 1 week. In addition, effects of sample aging (storage) and X-ray irradiation during XPS measurements, sodium dodecyl sulfate (SDS) treatment, and heat treatment (up to 200 °C) on protein coverage, denaturation, and surface oxide composition were examined. SDS is known for protein denaturation and to be able to remove proteins from surfaces when the SDS concentration is high enough.34,35 The temperature treatment was reported to be sufficient to remove physisorbed species.36 16307


16308

consecutively rinsed by water, immersed in 0.035 M SDS for 0.5 and 24 h, and measured at RT, 75 °C, 100 °C, and 150 °C

H2O rinsed, SDS 0.5 h, SDS 24 h, RT, 75 °C, 100 °C, 150 °C (BSA or LSZ)d

ToF-SIMS, followed by XPS (between each treatment); for temperature treatment, only ToF-SIMS

ToF-SIMS, followed by XPS

a

Consecutive treatment of the sample “fresh 168 h” (BSA or LSZ). bConsecutive treatment of the sample “SDS 0.5 h” (BSA or LSZ). cSame sample (BSA or LSZ) as investigated previously6 (same conditions as “fresh 168 h” but adsorbed at 37 °C). dConsecutive treatments of the sample “aged 168 h” (BSA or LSZ); only BSA samples in the case of temperature treatment.

-

4 °C

aged 168 h (BSA or LSZ)c

168 h

ToF-SIMS ToF-SIMS, followed by XPS, followed by ToFSIMS ToF-SIMS, followed by XPS (after 200°C+1h)

ToF-SIMS

4 °C

ToF-SIMS followed by XPS

ToF-SIMS followed by XPS

same sample measured consecutively at RT, 50 °C, etc.

100 g/L BSA or 2.2 g/L LSZ in PBS (pH 7.4)

measured by ToF-SIMS followed by XPS

RT

RT

treatment

RT, 50 °C, 100 °C, 150 °C, 200 °C, and 200 °C + 1 h (BSA or LSZ)

168 h

24 h

0.5 h

adsorption temp

immersed in 0.035 M SDS for 0.5 h at RT immersed in 0.035 M SDS for 36 h RT




protein solution

adsorption time

SDS 0.5 h (BSA or LSZ)a SDS 36 h (BSA or LSZ)b

fresh 168 h (BSA or LSZ)

1200 grit, cleaned, dried, 24 h aged at RT

1200 grit, cleaned, dried, 24 h aged at RT 1200 grit, cleaned, dried, 24 h aged at RT 1200 grit, cleaned, dried, 24 h aged at RT

fresh 0.5 h (BSA or LSZ)

fresh 24 h (BSA or LSZ)

3 μm, cleaned, dried, 24 h aged at RT

sample denotation

316L

grinding/polishing procedure

Table 2. Adsorption and Analytical Conditions of the Different 316L Samples Investigated and Corresponding Denotations

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(1486.6 eV, 300 W) as excitation source and a takeoff angle of 45° was used for the XPS analysis. Binding energies of the target elements (O 1s, C 1s, N 1s, S 1s, Fe 2p, and Cr 2p) were determined from detailed spectra by use of pass energy of 23.5 eV with resolution of 0.2 eV. The binding energy of the C 1s (C−C, C−H) signal (285.0 eV) was used as internal reference. Measurements were conducted on areas with a diameter of 800 μm. Background was subtracted via the linear method in all spectra, and data was evaluated with CasaXPS version 2.3.16 Prerel 1.4, Casa Software Ltd. The C 1s peak was resolved into three peaks; C1 (285.0 eV), assigned to C−C and C−H groups; C2 (286.5 ± 0.2 eV), assigned to C−N and C−O groups; and C3 (288.1 ± 0.1 eV), assigned to CC−O and OC−N groups, as described elsewhere.6,38 Sulfur was present at 164.0 ± 0.4 eV (assigned to S−S and S−H)39−41 and/or 168.7 eV ± 0.2 eV (assigned to sulfate).41,42 No signal assigned to sulfite41 was observed. The Fe 2p peak was separated according to its metallic (707.2 ± 0.1 eV) and oxidized (711.4 ± 0.4 eV) states. A similar deconvolution was made for Cr 2p (metallic, 573.9 ± 0.2 eV; oxidized, 577.2 ± 0.2 eV). Typical peaks of protein spectra for C 1s, S 2p, N 1s, Fe 2p3/2, and Cr 2p3/2 are shown in Figure S2 (Supporting Information). Multivariate Analysis and Calculations. Spectragui (NB toolbox v.2.5b) using Matlab was used for multivariate analysis of the ToF-SIMS spectra. Principal component analysis (PCA) was used to identify the peaks that showed the most significant differences between the proteins and different parameters investigated. All presented ToF-SIMS spectra are normalized to the total intensity and/or as relative ratios between two signals of the same spectrum. For XPS data, all data are presented in terms of atomic percentage and as relative ratios.

EXPERIMENTAL SECTION

Sample Preparation and Protein Adsorption. Stainless steel (grade AISI 316L) was used as substrate for protein adsorption. The composition is given in Table 1. As a reference, this material was polished (using 3 μm diamond paste), ultrasonically cleaned (ethanol for 10 min), dried, aged (stored at ambient conditions) for 1 day, and then analyzed by means of ToF-SIMS and XPS. This reference material is denoted “316L”. Protein adsorption measurements on similarly prepared (but ground to 1200 grit, as previously6) 316L sheets in PBS (8.77 g/L NaCl, 1.28 g/L Na2HPO4, 1.36 g/L KH2PO4, and 370 μL/L 50% NaOH, pH 7.2−7.4) were performed for two different proteins: 2.2 g/L LSZ (lysozyme from chicken egg white, Sigma Aldrich) and 100 g/L BSA (bovine serum albumin, Sigma Aldrich) in PBS, the same solutions used as previously.6 BSA is net negatively charged (iep 4.7− 5.2)27 and LSZ is net positively charged (iep 11) at pH 7.4.37 From these mass concentrations, BSA has a 10 times higher molar concentration compared with LSZ but is expected to adsorb in a significantly thinner layer (monolayer) compared to LSZ (several layers).6 After exposure for 0.5, 24, and 168 h, the sample was rinsed with ultrapure water (>18 MΩ·cm), dried, and analyzed by ToF-SIMS (the same day) and XPS (not in the case of 168 h) consecutively. Those samples are denoted “fresh 0.5 h”, “fresh 24 h”, and “fresh 168 h”, respectively. The “fresh 168 h” samples were not directly measured by XPS but were first treated in 0.035 M SDS (approximately 4 times the critical micelle concentration, cmc35) for 0.5 and 36 h, consecutively, denoted “SDS 0.5 h” and “SDS 36 h”, and finally analyzed by means of XPS. An additional set of freshly prepared samples was heat-treated (within the ToF-SIMS chamber, at room temperature and 50, 100, 150, and 200 °C, denoted “RT”, “50 °C”, “100 °C”, “150 °C”, “200 °C”, and “200 °C + 1 h”) for evaluation of the interaction strength with the substrate. After each step, and also after XPS investigation, ToF-SIMS measurements were performed. A similar SDS treatment (0.5 and 24 h, consecutively) and subsequent temperature treatment (up to 150 °C, only BSA samples) were performed for the aged 168 h samples (cf. below), as well as a temperature treatment (BSA samples, up to 150 °C) for the fresh 24 h samples. In addition, the same samples as studied previously,6 that is, 316L sheets in LSZ (2.2 g/L) and BSA (100 g/L) solutions in PBS after 1 week exposure time, were analyzed by ToF-SIMS and XPS, after previous XPS measurements and about 2 months of storage (desiccator and successive transportation at ambient conditions), denoted “aged 168 h”, before and after water rinsing (denoted “H2O rinsed”). The adsorption temperature for those samples was 37 °C, while it was room temperature (20 °C) for the fresh 0.5 and 24 h samples, and 4 °C (to avoid any protein denaturation in solution over the longer time period) for the fresh 168 h samples. Table 2 summarizes the different investigated samples, together with their denotations and adsorption and analytical conditions. The SDS and heat treatments were selected to remove physisorbed proteins,34−36 and to minimize any interaction resulting in a change of the surface oxide characteristics. Time-of-Flight Secondary Ion Mass Spectrometry. Positive and negative static SIMS measurements were performed on a ToFSIMS 5 spectrometer (ION-TOF, Münster, Germany) on at least three different spots on each sample. Detailed information is given elsewhere.16 For the temperature treatment, the temperature was ramped at 0.1 °C/s and then kept constant at the target temperature for 30 min. This procedure is reported to be sufficient to remove physisorbed species.36 The measured area was decreased to 250 × 250 μm and the primary ion dose density (PIDD) was kept at 1011 ions/ cm2 for each measured spectrum, with a maximal exposure to 3 × 1011 ions/cm2 for each measured area (the sample cannot be moved during heat treatment, making multiple measurements on the same area inevitable). Typical ToF-SIMS spectra are shown in Figure S1 (Supporting Information). X-ray Photoelectron Spectroscopy. A Perkin-Elmer Physical Electronics 5600 spectrometer using monochromated Al Kα radiation

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RESULTS AND DISCUSSION Protein-Induced Changes in the Surface Oxide. Combined XPS and ToF-SIMS studies were conducted on some samples of the previous study,6 that is, on 316L stainless steel exposed for 1 week (168 h) to BSA (100 g/L) and LSZ (2.2 g/L) in PBS, referred to as “aged 168 h” (stored for approximately 2 months after exposure). Figure 2 shows similar findings on chromium enrichment as previously reported when measured directly after exposure.6 The same samples were rinsed with ultrapure water and reanalyzed (“H2O rinsed”). Additional measurements were performed on freshly prepared protein-adsorbed and water-rinsed samples exposed for 0.5, 24, and 168 h and a reference (316L) without any adsorbed proteins (polished 24 h preanalysis). In agreement with previous studies,6 Figure 2a clearly reveals a chromium enrichment (increased oxidized Cr to oxidized Cr+Fe XPS ratio) with adsorption time, significant only in the case of BSA, and only after 24 h. The oxidized and metallic signals were difficult to measure by XPS in the case of LSZ, due to a very thick LSZ layer, in agreement with previous XPS studies.6 In contrast to XPS (Figure 2a), ToF-SIMS revealed less differences in surface oxide composition between BSA and LSZ. ToF-SIMS showed a significant enhancement of oxidized chromium in the surface oxide already after 30 min of adsorption (for both BSA and LSZ, compared to the reference) and a further enhancement with increased adsorption time in the case of BSA (Figure 2b). This might be due to the higher surface sensitivity of ToF-SIMS43 and be explained by the fact that the outermost surface of the oxide of unexposed samples is richer in iron compared with the inner oxide44 and that protein interaction, which results in a depletion of iron6 from the outermost surface, can be detected at an earlier stage. Higher similarity of BSA and LSZ samples analyzed by means of ToFSIMS (Figure 2b), compared with XPS, can hence be explained by a similar outermost surface reaction (depletion of iron), 16309


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Figure 3. (a) Oxidized iron and chromium per nitrogen, and (b) metallic iron signal per oxidized carbon, measured by means of XPS.

Figure 2. Relative amount of oxidized chromium per oxidized chromium and oxidized iron, measured by means of (a) XPS and (b) ToF-SIMS. cmc, sometimes even lower), as reported for LSZ on negatively charged silica surfaces34 and on negatively charged mica surfaces.35 In the case of preadsorbed LSZ (as in this study) of relatively high protein solution concentration (1 g/L; this study, 2.2 g/L) and ionic strength (0.16 M; this study, 0.15 M) and SDS at concentrations well above cmc (as in this study), only a partial removal of LSZ was observed on neutrally charged chromium oxide surfaces at pH 7.0.45 Consequently, the protein removal rate is larger, that is, the interaction strength is lower, for fresh

while the inner oxide is only, or is more strongly, affected by BSA. Protein Adsorption and Layer Properties. The substrate (metal or oxide) signals can also be compared with signals from the proteins, providing information on the protein layer thickness or density. In Figures 3 and 4, the influence of successive SDS treatment of the fresh 168 h samples is shown in addition to the untreated samples. Both XPS (Figure 3) and ToF-SIMS (Figure 4) revealed surfaces of high protein coverage compared with the references. With XPS, the difference between BSA and LSZ in terms of protein layer thickness was evident (Figure 3). For LSZ adsorption, the surface oxide signal decreased rapidly with adsorption time but increased with increasing time of SDS treatment. These observations indicate a partial removal of proteins by SDS (concentration 4 times the cmc35), which was stronger for fresh compared with aged samples (Figure 4). Already after 30 min protein adsorption time, LSZ adsorbed samples revealed a lower oxide signal compared with the BSA samples, indicative of a thicker LSZ layer (Figure 3a). The metallic iron to protein signal (oxidized carbon) ratio (Figure 3b), was in general agreement with the oxide to nitrogen ratio (Figure 3a), showing barely detectable substrate signals in the case of LSZ, and constant substrate signals in the case of BSA. This indicates a similar BSA layer thickness among the different adsorption conditions (Figure 3). The same trends were observed when the metallic signal was normalized to nitrogen (data not shown). The freshly adsorbed BSA sample showed after 30 min an enhanced oxide signal compared with the other adsorption time periods (Figure 3a), in contrast to the metal signal (Figure 3b). This is due to initial changes in the oxide to metal ratio (data not shown) in the case of BSA adsorption. In contrast to XPS, ToF-SIMS resulted in similar oxide/protein (CNO−) ratios when BSA and LSZ adsorption were compared (Figure 16310


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Figure 4. Oxidized iron and chromium, (a) per CNO− and (b) per the sum of distinct ToF-SIMS amino acid fragments for histidine/arginine, phenylalanine, and tryptophan, measured by means of ToF-SIMS.

compared to aged samples, although both are preadsorbed (studied ex situ). This can either be caused by changes in the protein layer during storage or by the difference in adsorption temperature (4 vs 37 °C) resulting in different layer stability. Both water rinsing of the aged sample and 30 min SDS treatment resulted in a thicker LSZ layer compared with the nontreated aged sample (Figure 4). This may be due to an expansion of the LSZ layer upon short SDS treatment, previously observed for LSZ on negatively charged mica surfaces35 and BSA on silica,46 prior to any desorption. For the freshly preadsorbed samples, any expansion of the LSZ layer is supposed to occur prior to 30 min of SDS treatment, as the substrate to protein signals were already higher after 30 min SDS treatment compared with no treatment (fresh 168 h) (Figure 4). SDS was clearly adsorbed and interacted with the proteins, as a significant increase in fragments corresponding to SDS [CH2SO4− (m/z 109.97), C3H5SO4− (m/z 137.00), and C12H25SO4− (m/z 265.177)] were detected for the SDS-treated samples compared with the fresh samples (Figure 5). This is interesting from a surface chemistry perspective since different models were earlier suggested for protein−SDS interaction and any subsequent removal of proteins, including binding to the protein (in different ways) and binding to the surface.45 Since the surfaces were rinsed with water after the SDS treatment, the increase of SDS fragments must be due to interacting SDS molecules. The signal corresponding to the total SDS molecule, C12H25SO4−, increased most strongly upon SDS treatment. Denaturation upon SDS or Temperature Treatment and Effect of XPS Measurements. Literature findings report SDS treatment to denature adsorbed LSZ on negatively

Figure 5. ToF-SIMS negative fragments at mass numbers 109.97, 137.00, and 265.18 (corresponding to CH2SO4−, C3H5SO4−, and C12H25SO4−, respectively) for BSA and LSZ fresh 168 h samples and successive SDS treatment.

charged mica at SDS concentrations at, or above, cmc.35 Sulfur species information provided by XPS and ToF-SIMS was evaluated to gain an improved understanding of the SDS− protein interaction and the low extent of protein removal. For XPS (Figure S3, Supporting Information), the sulfur species intensity is displayed for the peaks at 164.0 ± 0.4 and 168.7 ± 0.2 eV. The peak at 168.7 eV is assigned to sulfate41,42 and the peak at 164.0 eV to disulfide bonds or thiolate within 16311


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Figure 6. (a) Oxidized iron and chromium per CNO− signal and (b) metallic iron per sum of the distinct amino acid fragments for histidine (arginine), phenylalanine, and tryptophan, determined by means of ToF-SIMS as a function of SDS and temperature treatment.

proteins.40,41 No peak assigned to sulfite, at approximately 166 eV,41 was observed. XPS (Figure S3, Supporting Information) showed that the disulfide/thiolate species increased in intensity with increasing adsorption time (with the exception of BSA after 0.5 h adsorption). The SDS treatment resulted in a sulfate peak, probably due to binding or adsorption of SDS to the protein layer (Figure S3, Supporting Information). At the same time, the sulfide/thiolate signal decreased with longer time of SDS treatment of the aged samples, indicative of (partial) protein removal to a higher extent for BSA, in agreement with the previous discussion (Figures 3 and 4). ToF-SIMS is able to detect the signal produced by disulfide bonds and has previously been used to determine protein denaturation.16 The signal decay was investigated as a function of temperature treatment, SDS treatments, and XPS measurements (Figures 6 and 7). For that purpose, the fresh 168 h samples were consecutively treated in SDS for 0.5 and 36 h, and a second set of fresh 168 h samples was exposed to successively increasing temperature within the ToF-SIMS analysis chamber. XPS measurements were not performed in between, but after the completion of both experiments. After XPS measurement, the samples were again investigated by means of ToF-SIMS. Figure 6 shows the substrate peaks compared to the protein peaks, as a function of temperature and SDS treatments. The effect of temperature treatment during the ToF-SIMS measurement revealed a slight but significant removal of adsorbed proteins that increased with increasing temperature up to 200 °C (Figure 6 and Figure S4 in Supporting Information). For the aged sample after SDS treatment (Figure S4, Supporting Information), significantly more BSA was removed upon thermal treatment (at even 150 °C), compared with freshly adsorbed samples.

It seems that long positively charged fragments, assigned to histidine/arginine, phenylalanine, and tryptophan, decrease to a larger extent compared to the negatively charged amide fragment CNO− with increasing temperature, also when differences in Fe+ and FeO− + CrO− signals are considered. One possible explanation is a temperature-induced weakening of the bonds in the protein, resulting in higher fragmentation of the molecules and consequently favored emission of smaller fragments. Also, the increased fragmentation can be caused by repeated measurements on the same area (cf. Experimental Section), even though the primary ion dose density was kept well below the static limit for each measured spot. To investigate whether ToF-SIMS measurements of the same sample area can cause a protein denaturation or change protein signals, the same spot of a sample (1 h BSA adorption in PBS) was analyzed five times consecutively, with a total primary ion dose density (PIDD) of 5 × 1011 ions/cm2 (to be compared with a maximum exposure of 3 × 1011 ions/cm2 for all other investigated samples). No significant difference in protein signals or any denaturation were observed (Figure S5, Supporting Information). The XPS measurement after the temperature treatment did not significantly change the amount of protein adsorbed, judged from the substrate to protein signals measured by ToFSIMS (Figure 6). However, the disulfide per CNO− signal was significantly reduced after XPS investigation, indicative of protein denaturation, cf. Figure 7b. Ultrahigh vacuum (UHV) has previously been shown to induce significant differences compared to ambient conditions for antiferritin and anti-IgM.47 The influence of UHV has also been highlighted for conformational studies of human serum albumin; however, an effect was not found to be very significant.13 UHV alone, 16312


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Figure 7. Disulfide signal per CNO−, determined by means of ToF-SIMS, as a function of (a) SDS and (b) temperature treatment. Asterisks indicate that the samples were measured by XPS prior to the ToF-SIMS measurements.

Table 3. Selected Positive ToF-SIMS Fragments, Their Corresponding Amino Acids, and Observed Significant Differencesa mass (m/z) 30.03

fragment CH4N+ +

corresponding amino acid Gly, Lys, Leu, and others Ala, Cys

44.05

C2H6N

58.07 59.05 60.06 70.03

C3H8N+ CH5N3+ C2H6NO+ C3H4NO+

Glu Arg Ser Asn

70.07

C4H8N+

Pro, Arg, Val, Leu

72.08

C4H10N+

Val

73.06 74.06

C2H7N3+ C3H8NO+

Arg Thr

81.04 88.04 91.05 100.08

C4H5N2+ C3H6NO2+ C7H7+ C4H10N3+

His Asn, Asp Phe Arg

110.07

C5H8N3+

His, Arg

120.08

C8H10N+

Phe

130.06

C9H8N

+

Trp

ref(s) 30, 51, 53, 54 30, 51, 53, 54 13, 51 30, 51, 53 53, 54 30, 51, 53, 54 30, 51, 53, 54 30, 51, 53, 54 30, 51, 54 30, 51, 53, 54 30, 53, 54 30, 53, 54 30 30, 51,53, 54 30, 51, 53, 54 30, 51, 53, 54 30, 51, 53

significant increased signal for protein (BSA; LSZ) (LSZ)

difference between fresh and aged samples fresh > aged

significant trend with adsorption timeb D (I)

BSA LSZ LSZ LSZ

D fresh > aged fresh > aged (fresh > aged)

BSA BSA

fresh > aged

(I/D)

LSZ (LSZ)

(fresh > aged) fresh > aged

(I/D)

BSA LSZ BSA (LSZ)

fresh > aged (fresh > aged)

BSA

fresh > aged

(I/D)

BSA

fresh > aged

(I/D)

LSZ

fresh > aged

(D)

(I/D)

a

Differences between BSA and LSZ (where parentheses indicate a slight difference), fresh and aged samples, and different adsorption times are shown. The selection was based on multivariate analysis. bD, decreasing, or I, increasing. 16313


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and 4. This effect may be explained by conformational changes that result in a lower ionization ability of the amino acid fragments and/or by their partial destruction upon aging. Another reason could be a higher amount of surface contamination compared to freshly adsorbed samples. The amino acid intensities increased (to 24 h, 20 °C) and decreased (to 168 h, 4 °C) with adsorption time, slightly increased (nonsignificantly), or did not change significantly, with the exception of signals at 30.03 and 58.07 m/z, discussed below. An increase over time would be expected in case of a denser outermost protein layer and no protein orientation or conformation changes. Conformational changes of the protein and/or speciation (chemical form) changes of some individual amino acids could result in changed ionization properties and hence a decreased signal. Larger amino acid fragments (m/z 110.07, 120.08, and 130.06) decreased after longer adsorption time (>24 h, Table 3). However, some smaller amino acid fragments constantly decreased throughout the entire screened adsorption time range, that is, the amine signal (CH4N+, m/z 30.03) and the glutamic acid signal (C3H8N+, m/z 58.07) (which excludes a single effect of the lower synthesis temperature for the longest adsorption time). The discussed signals are displayed as a function of adsorption time (for fresh samples only), together with the histidine (arginine) signal (C5H8N3+, m/z 110.07) for comparison, in Figure 8. The amine

however, cannot be responsible for any protein denaturation (that is, disulfide signal intensity) observed here, since it is also present during the ToF-SIMS measurements. Instead, the X-ray irradiation or the photoemitted electrons may denature the proteins. This would not be surprising as X-ray irradiation is commonly used for sterilization of medical equipment.48 In Figure 7a, the aged 168 h samples and consecutive samples all were measured with XPS before the ToF-SIMS measurement and in between. The observed decrease in disulfide signal may hence be caused by the XPS measurement, instead of, or in addition to, the SDS treatment. In Figure 7b, it is shown for the fresh 168 h samples that the disulfide ratio was fairly constant upon short SDS treatment (4 times cmc, 0.5h) for LSZ and temperature treatment up to 150 °C for both proteins. Even a short SDS treatment for BSA, longer SDS treatment, and temperatures of 200 °C induced a significant decrease of the disulfide ratio, which was even more significant for XPS exposure. This is also illustrated in Figure S1 (Supporting Information), where the detailed ToF-SIMS spectra are shown for the disulfide signal and other characteristic signals as a function of the different treatments. On hydrophilic surfaces (such as stainless steel17), LSZ has been reported to retain most of its ordered structure,49,50 while α-lactalbumin50 and BSA49 have lost their ordered structures almost completely when investigated on hematite particles.49,50 This effect has been referred to the structural stability of the proteins, which is higher for LSZ compared with BSA.49 In addition, less structurally stable proteins adsorb with higher affinity to surfaces.49 It is therefore expected that BSA shows a lower stability (reflected by the disulfide signal), generally and upon SDS treatment, compared with LSZ. Despite the higher content of disulfide bonds in BSA compared with LSZ per protein mass unit, the disulfide signal was only slightly enhanced or similar for BSA compared with LSZ for the freshly adsorbed samples, and significantly lower in the case of aged and SDS-treated samples (Figure 7a). The SDS and temperature treatment findings indicate that aging induces denaturation of proteins and a weaker bonding to the substrate compared with fresh samples, reflected in higher removal upon temperature treatment and lower (slower) SDSinduced removal of proteins. Intensities of Amino Acid Fragments. Multivariate analysis, for example, PCA, has been reported to be useful for protein distinction and detection of denaturation11,12,51,52 and to be applicable to identify any conformational changes of the protein layer.13−15 PCA was used in this study to identify amino acids that differed the most between the two proteins studied, between adsorption times, and/or between fresh and aged samples. Observed amino acid fragments and their assignments are compiled in Table 3, together with observed differences. In almost all cases, the fragments showed higher signals for the protein that contains more of a given amino acid per mass. For example, fragment intensities of valine (C4H10N+), histidine (C4H5N2+, C5H8N3+), and phenylalanine (C7H7+, C8H10N+) were all enhanced for BSA compared with LSZ, as expected from the protein sequences. One exception was aspartic acid (C3H6NO2+), which was increased for LSZ, however, theoretically with a higher content per mass in BSA. In nearly all cases (Table 3), freshly adsorbed proteins resulted in an enhanced amino acid fragment intensity compared with the aged samples. This finding is in contrast to the similarly thick or even thicker protein layer for the aged 168 h sample compared with the fresh 168 h sample, deduced from Figures 3

Figure 8. Relative intensity (normalized to total intensity of each spectrum) of the positively charged fragments CH4N+ (amine group), C3H8N+ (glutamic acid), and C5H8N3+ (histidine, arginine), analyzed by means of ToF-SIMS.

fraction signal corresponds to several different amino acids (Table 3). Its constant decreased intensity with adsorption time, in contrast to most other fragments, can be explained by several possibilities. First, with increasing and denser protein layer over time, the hydrophobicity within the protein layer increases and the dielectric constant decreases. This may change the speciation of the otherwise positively charged amine group (-NH3+) to the uncharged -NH2 group within the protein layer. Second, the positively charged amine group may interact with the negatively charged stainless steel surface, either by electrostatic interaction with, for instance, OH− or O2− groups of the oxide or by covalent binding to a metal surface atom. It can also interact as an uncharged -NH2 group forming hydrogen bonds to an OH group of the oxide. All these 16314


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interactions could result in a reduced CH4N+ signal. The same may be true for other amino acid fragments, such as that derived from glutamic acid. Further studies of single amino acids or small peptide interactions with stainless steel and additional time points are, however, needed in order to investigate stainless steel binding mechanisms by means of ToF-SIMS. Recently it has been shown that the ratio of the sum of hydrophobic to the sum of hydrophilic amino acid fragments was changed for BSA depending on the hydrophobicity of the substrate surface.43 No significant trends or differences were observed in this study between different adsorption time periods, different proteins, or upon heating or SDS treatment, when the same ratio was used (data not shown). A theoretical study on Cr2O3 surfaces (as models for stainless steel) and their interaction with glycine, with the assumption that Cr2O3 is uncharged at neutral pH, showed that several types of interactions are possible, including hydrogen bonding and covalent bonding to the amine and carboxylate group.25 However, as recent findings by the authors showed that massive stainless steel AISI 316 is negatively charged at neutral pH,6 an interaction with the positively charged -NH3+ group is more probable compared with the negatively charged -COO− group. On the other hand, the stainless steel surface is heterogeneous and undergoes continuous changes in, for example, its surface oxide composition (chromium enrichment, Figure 2).6 Even the stainless steel grade investigated in this study, which contains very low amounts of impurities, possesses more noble and less noble areas depending on the grain orientations55 as well as grain boundaries of different surface chemistry at the nanoscale.56 Another indication that several different interactions may be possible with stainless steel surfaces is the observation that BSA adsorbed in a similar way on both positively charged stainless steel nanoparticles and on negatively charged massive stainless steel.23 Several literature investigations assume stainless steel surfaces to be positively charged at neutral pH or pH 7.4 and suggest hence interactions with negatively charged carboxylate groups.5,18,20

removed to a larger extent and after shorter time periods compared with LSZ. The LSZ layer thickness expanded upon SDS treatment prior to any protein removal. Heat treatment up to 200 °C only partially removed adsorbed proteins. Both treatments indicate strong protein−surface interactions. In the case of freshly preadsorbed proteins, SDS induced an enhanced and earlier partial protein removal compared with aged samples for both BSA and LSZ and partially destroyed disulfide bridges of the proteins. The high-temperature treatment (200 °C), and in particular the XPS exposure, induced an even more significant denaturation of both proteins. ToF-SIMS intensities of amino acid fragments correlated (in most cases) with their natural protein content and were significantly enhanced in the case of freshly adsorbed samples compared with aged samples, despite similar protein surface coverage. Reduced intensities of some amino acid fragments with adsorption time (especially amine, CH4N+, and glutamic acid, C3H8N+) or after longer time periods (especially larger amino acid fragments related to histidine, phenylalanine, and tryptophan) were attributed to changes in speciation (chemical form) and/or changes in orientation upon, for example, metal (oxide)−protein binding. These strong surface−protein interactions confirm and complement earlier studies6 and resulted in an enhanced metal release and enrichment of chromium in the surface oxide in the case of BSA compared with LSZ.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Five figures as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*Phone +46 8 7906670, e-mail [email protected]. Present Address ∥

Division of Surface and Corrosion Science, Department of Chemistry, KTH Royal Institute of Technology, Drottning Kristinas väg 51, SE-10044 Stockholm, Sweden.

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SUMMARY The interaction of LSZ and BSA (in PBS) with AISI 316L stainless steel surfaces was studied ex situ by means of XPS and ToF-SIMS after different adsorption times, and parallel investigations of the effect of storage (aging), SDS treatments, XPS measurements, and temperature (up to 200 °C) were carried out. XPS and ToF-SIMS were found to be highly complementary techniques for protein−metal interaction studies due to differences in information depth, chemical information, and sensitivity. XPS measurements (X-ray irradiation and/or photoelectron emission) most probably induced protein denaturation, observed in terms of a decreased disulfide signal measured by means of ToF-SIMS subsequently conducted after the XPS measurements. No denaturation effects were induced by the use of ToF-SIMS. Both BSA and LSZ adsorption induced an enrichment of chromium in the surface oxide. LSZ formed a thicker layer compared with BSA. BSA induced significant changes to the entire oxide, whereas LSZ only induced a depletion of iron in the outermost layer. SDS was unable to completely remove preadsorbed proteins from either fresh or aged samples, despite its high concentration (0.035 M; 4 times cmc) and relatively long treatment time (up to 36.5 h). For the aged samples, BSA was

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 We thank Ulrike Marten-Jahns for XPS measurements and the Swedish Research Council (VR), travel grants from the Swedish Steel Association (Jernkontoret) and the Björn Foundation at KTH, Royal Institute of Technology, Sweden, the German Research Council (DFG), and Cusanuswerk, Germany, and the BMBF for financial support. We thank Dan Graham, Ph.D., for developing the NESAC/BIO Toolbox used in this study and NIH Grant EB-002027 for supporting the toolbox development.

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ABBREVIATIONS BSA, bovine serum albumin; LSZ, lysozyme; PBS, phosphatebuffered saline; SDS, sodium dodecyl sulfate; cmc, critical micelle concentration; 316L, stainless steel grade AISI 316L; XPS, X-ray photoelectron spectroscopy; ToF-SIMS, time-offlight secondary ion mass spectrometry; PCA, principal 16315


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component analysis; PIDD, primary ion dose density; UHV, ultrahigh vacuum; QCM, quartz crystal microbalance

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Interaction of Bovine Serum Albumin and Lysozyme with Stainless

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