Interaction of Bovine Serum Albumin and Lysozyme with StainlessSteel Studied by Time-of-Flight Secondary Ion Mass Spectrometryand X‑ray Photoelectron SpectroscopyYolanda 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 ofErlangen−Nuremberg, Martensstrasse 7, 91058 Erlangen, Germany‡Division of Surface and Corrosion Science, Department of Chemistry, School of Chemical Science and Engineering, KTH RoyalInstitute of Technology, Drottning Kristinas vag 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 betweenstainless steel surfaces and proteins is essential from a corrosion and protein-inducedmetal release perspective when stainless steel is used in surgical implants and in foodapplications. The interaction between lysozyme (LSZ) from chicken egg white andbovine serum albumin (BSA) and AISI 316L stainless steel surfaces was studied exsitu by means of X-ray photoelectron spectroscopy (XPS) and time-of-flightsecondary ion mass spectrometry (ToF-SIMS) after different adsorption time periods(0.5, 24, and 168 h). The effect of XPS measurements, storage (aging), sodiumdodecyl sulfate (SDS), and elevated temperature (up to 200 °C) on the proteinlayers, as well as changes in surface oxide composition, were investigated. Both BSAand LSZ adsorption induced an enrichment of chromium in the oxide layer. BSAinduced significant changes to the entire oxide, while LSZ only induced a depletion ofiron at the utmost layer. SDS was not able to remove preadsorbed proteinscompletely, despite its high concentration and relatively long treatment time (up to 36.5 h), but induced partial denaturation ofthe 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 adsorptiontime, between the proteins, and between freshly adsorbed and aged samples. Stainless steel−protein interactions were shown tobe strong and protein-dependent. The findings assist in the understanding of previous studies of metal release and surfacechanges upon exposure to similar protein solutions.

■ INTRODUCTION

Mechanistic understanding of the interaction of stainless steeland proteins is important for many applications, includingcorrosion in protein environments, such as biofouling inseawater,1 and metal release upon protein-induced corrosionfor food-related applications2 and surgical implants.3,4 Gen-erally, proteins are reported to enhance both the extent ofmetal release and the rate of corrosion for stainless steels.4−8

The reverse situation (reduced corrosion or metal release) hasbeen reported in the case of a certain mussel protein5 and forpitting corrosion resistance in serum.9

Time-of-flight secondary ion mass spectrometry (ToF-SIMS)has been reported as a more capable method for detailedsurface analysis of adsorbed proteins on metal substratescompared with X-ray photoelectron spectroscopy (XPS), dueto its higher extent of information on chemical states and aneven higher surface sensitivity.10 Though not quantitative, ToF-SIMS can provide relative semiquantitative information, and

matrix effects are minor in the case of protein investigations.10

ToF-SIMS, often in combination with XPS and multivariateanalysis such as principal component analysis (PCA), has beenused to distinguish between different proteins,11,12 proteinconformation,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 stainlesssteel grade AISI 316L has previously been investigated by ToF-SIMS and dynamic contact angle measurements.17 Many othertechniques have been employed to investigate the interactionbetween BSA and stainless steel surfaces in terms of adsorption(kinetics),6,18−23 binding mechanisms,6,18,20,24,25 and corro-sion,6,18,26 by using electrochemical techniques,18,26 XPS,4,6,22

quartz-crystal microbalance (QCM),6,19,23 solution analy-

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

Article

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© 2012 American Chemical Society 16306 dx.doi.org/10.1021/la3039279 | Langmuir 2012, 28, 16306−16317

sis,4,6,8,19 and vibrational techniques such as infrared spectros-copy,20,22 as well as theoretical considerations.24,25 Bothchemisorption6,8,18,22,24,25 and physisorption6,20 were proposedinvolving interactions with carboxylate groups18,20,24 or aminegroups24 at neutral pH conditions. The surface charge of thestainless steel is a key factor for any interaction. However,scarce information on the surface charge of stainless steels isavailable in the literature, with reported assumptions on, ormeasurements of, an isoelectric point (iep) ranging from pH 3to 8.5,6,18,23 a variation that indicates the importance ofdifferences in surface finish and material properties. Recentmeasurements by some of the authors of this study revealed aniep of pH 3−4 for stainless steel 316 in 1 mM KCl solution andan 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 gradesin phosphate-buffered saline (PBS, pH 7.4) was investigated bysome of the authors of this study, using solution analysis, XPS,electrochemical techniques, QCM, and ζ potential measure-ments, 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 7−13mg/L in serum and about 1.2 g/L in tear fluid.28 The highabundance of albumin in human blood was the reason for itschoice, while lysozyme was chosen on the basis of its oppositenet charge. Both proteins were previously investigated in termsof 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 BSAresulted in the enrichment of surface oxide chromium contentdue to preferential iron complexation and/or release. Incontrast with BSA, LSZ only slightly enhanced the metalrelease and did not induce such strong chromium enrichmentin the surface oxide. This occurred in the cases of both the samemolar and mass concentration of proteins in solution.6 BSAadsorbed at monolayer coverage at 1 g/L BSA in PBS at pH 7.4on stainless steel AISI 316.6 No further increase in thickness

was observed with time.6 It has previously been shown, inprotein concentration-dependent QCM measurements onchromium metal, that the proteins adsorb in monolayercoverage at and above a concentration of 0.5 g/L BSA inPBS at pH 7.4.19 This is in general agreement with manyinvestigations.13,17,23,30,31 Hence, monolayer coverage in thecase of BSA is expected also in this study for 100 g/L BSA inPBS. In contrast, the adsorbed LSZ layer thickness has beenshown to continuously increase with time and correspond toseveral layers on stainless steel 316 after 1 h of exposure at aconcentration of 1 g/L in PBS.6 LSZ has also been reported toadsorb on mica and silica surfaces with an inner, relativelystrongly bound layer and an outer, more weakly adsorbed layerat concentrations above 0.02 g/L.32,33 Hence, LSZ wasexpected to adsorb in several layers also in this study (2.2 g/L in PBS), as previously confirmed by means of XPS.6 Severalpossible binding and metal release mechanisms were identified.Both (a) complexation between BSA or LSZ and the stainlesssteel surface and (b) a local lowering of pH and enrichment ofanions upon charge regulation between the adsorbed proteinlayer and the stainless steel surface were suggested as possiblemechanisms for the observed protein-induced enhancement ofreleased metals from stainless steel.6

The aim of this study was to use the combination of ToF-SIMS and XPS to investige the same system as investigatedpreviously,6 to gain additional information on the interactionbetween the stainless steel AISI 316L surface and the adsorbedprotein layer (LSZ or BSA). This was conducted for differentadsorption time periods up to 1 week. In addition, effects ofsample aging (storage) and X-ray irradiation during XPSmeasurements, sodium dodecyl sulfate (SDS) treatment, andheat 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 toremove proteins from surfaces when the SDS concentration ishigh enough.34,35 The temperature treatment was reported tobe sufficient to remove physisorbed species.36

Figure 1. Overview on previously obtained6 results by means of solution studies, quartz-crystal microbalance, X-ray photoelectron spectroscopy, andζ 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 ofthe 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

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Table

2.AdsorptionandAnalyticalCon

dition

sof

theDifferent316L

Samples

Investigated

andCorrespon

ding

Denotations

sampledenotatio

ngrinding/polishing

procedure

proteinsolutio

n

adsorp-

tion

time

adsorp-

tion

temp

treatm

ent

measuredby

316L

3μm

,cleaned,d

ried,

24haged

atRT

ToF

-SIM

Sfollowed

byXPS

fresh0.5h(BSA

orLS

Z)

1200

grit,

cleaned,

dried,

24haged

atRT

100g/LBSA

or2.2g/L

LSZin

PBS(pH

7.4)

0.5h

RT

ToF

-SIM

Sfollowed

byXPS

fresh24

h(BSA

orLS

Z)

1200

grit,

cleaned,

dried,

24haged

atRT

100g/LBSA

or2.2g/L

LSZin

PBS(pH

7.4)

24h

RT

ToF

-SIM

Sfollowed

byXPS

fresh168h(BSA

orLS

Z)

1200

grit,

cleaned,

dried,

24haged

atRT

100g/LBSA

or2.2g/L

LSZin

PBS(pH

7.4)

168h

4°C

ToF

-SIM

S

SDS0.5h(BSA

orLS

Z)a

immersedin

0.035M

SDSfor0.5hat

RT

ToF

-SIM

S

SDS36

h(BSA

orLS

Z)b

immersedin

0.035M

SDSfor36

hRT

ToF

-SIM

S,followed

byXPS

,followed

byToF

-SIMS

RT,50°C

,100

°C,150

°C,200

°C,and

200°C

+1h(BSA

orLS

Z)

1200

grit,

cleaned,

dried,

24haged

atRT

100g/LBSA

or2.2g/L

LSZin

PBS(pH

7.4)

168h

4°C

samesamplemeasuredconsecutivelyat

RT,5

0°C

,etc.

ToF

-SIM

S,followed

byXPS

(after200°C+1

h)

aged

168h(BSA

orLS

Z)c

-ToF

-SIM

S,followed

byXPS

H2O

rinsed,

SDS0.5h,

SDS24

h,RT,

75°C

,100

°C,150

°C(BSA

orLS

Z)d

consecutivelyrin

sedby

water,immersedin

0.035M

SDSfor0.5

and24

h,andmeasuredat

RT,75°C

,100

°C,and

150°C

ToF

-SIM

S,followed

byXPS

(betweeneach

treat-

ment);for

temperaturetreatm

ent,onlyToF

-SIM

SaConsecutivetreatm

entof

thesample“fresh

168h”

(BSA

orLS

Z).bConsecutivetreatm

entof

thesample“SDS0.5h”

(BSA

orLS

Z).c Sam

esample(BSA

orLS

Z)as

investigated

previously6(sam

econditionsas

“fresh

168h”

butadsorbed

at37

°C).dConsecutivetreatm

entsof

thesample“aged168h”

(BSA

orLS

Z);onlyBSA

samples

inthecase

oftemperature

treatm

ent.

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■ EXPERIMENTAL SECTIONSample Preparation and Protein Adsorption. Stainless steel

(grade AISI 316L) was used as substrate for protein adsorption. Thecomposition is given in Table 1. As a reference, this material waspolished (using 3 μm diamond paste), ultrasonically cleaned (ethanolfor 10 min), dried, aged (stored at ambient conditions) for 1 day, andthen analyzed by means of ToF-SIMS and XPS. This referencematerial is denoted “316L”.Protein adsorption measurements on similarly prepared (but

ground to 1200 grit, as previously6) 316L sheets in PBS (8.77 g/LNaCl, 1.28 g/L Na2HPO4, 1.36 g/L KH2PO4, and 370 μL/L50% NaOH, pH 7.2−7.4) were performed for two different proteins:2.2 g/L LSZ (lysozyme from chicken egg white, Sigma Aldrich) and100 g/L BSA (bovine serum albumin, Sigma Aldrich) in PBS, the samesolutions 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 Fromthese mass concentrations, BSA has a 10 times higher molarconcentration compared with LSZ but is expected to adsorb in asignificantly thinner layer (monolayer) compared to LSZ (severallayers).6 After exposure for 0.5, 24, and 168 h, the sample was rinsedwith 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 168h”, respectively. The “fresh 168 h” samples were not directly measuredby XPS but were first treated in 0.035 M SDS (approximately 4 timesthe critical micelle concentration, cmc35) for 0.5 and 36 h,consecutively, denoted “SDS 0.5 h” and “SDS 36 h”, and finallyanalyzed by means of XPS. An additional set of freshly preparedsamples was heat-treated (within the ToF-SIMS chamber, at roomtemperature and 50, 100, 150, and 200 °C, denoted “RT”, “50 °C”,“100 °C”, “150 °C”, “200 °C”, and “200 °C + 1 h”) for evaluation ofthe interaction strength with the substrate. After each step, and alsoafter XPS investigation, ToF-SIMS measurements were performed. Asimilar SDS treatment (0.5 and 24 h, consecutively) and subsequenttemperature treatment (up to 150 °C, only BSA samples) wereperformed for the aged 168 h samples (cf. below), as well as atemperature treatment (BSA samples, up to 150 °C) for the fresh 24 hsamples.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 1week exposure time, were analyzed by ToF-SIMS and XPS, afterprevious 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 “H2Orinsed”). The adsorption temperature for those samples was 37 °C,while it was room temperature (20 °C) for the fresh 0.5 and 24 hsamples, and 4 °C (to avoid any protein denaturation in solution overthe longer time period) for the fresh 168 h samples. Table 2summarizes the different investigated samples, together with theirdenotations 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 ofthe surface oxide characteristics.Time-of-Flight Secondary Ion Mass Spectrometry. Positive

and negative static SIMS measurements were performed on a ToF-SIMS 5 spectrometer (ION-TOF, Munster, Germany) on at leastthree different spots on each sample. Detailed information is givenelsewhere.16 For the temperature treatment, the temperature wasramped at 0.1 °C/s and then kept constant at the target temperaturefor 30 min. This procedure is reported to be sufficient to removephysisorbed species.36 The measured area was decreased to 250 × 250μm 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 duringheat treatment, making multiple measurements on the same areainevitable). 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

(1486.6 eV, 300 W) as excitation source and a takeoff angle of 45° wasused for the XPS analysis. Binding energies of the target elements (O1s, C 1s, N 1s, S 1s, Fe 2p, and Cr 2p) were determined from detailedspectra by use of pass energy of 23.5 eV with resolution of 0.2 eV. Thebinding energy of the C 1s (C−C, C−H) signal (285.0 eV) was usedas internal reference. Measurements were conducted on areas with adiameter of 800 μm. Background was subtracted via the linear methodin all spectra, and data was evaluated with CasaXPS version 2.3.16Prerel 1.4, Casa Software Ltd. The C 1s peak was resolved into threepeaks; 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 ± 0.1eV), assigned to CC−O and OC−N groups, as describedelsewhere.6,38 Sulfur was present at 164.0 ± 0.4 eV (assigned to S−Sand 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 wasseparated 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 ofprotein spectra for C 1s, S 2p, N 1s, Fe 2p3/2, and Cr 2p3/2 are shownin Figure S2 (Supporting Information).

Multivariate Analysis and Calculations. Spectragui (NBtoolbox v.2.5b) using Matlab was used for multivariate analysis ofthe ToF-SIMS spectra. Principal component analysis (PCA) was usedto identify the peaks that showed the most significant differencesbetween the proteins and different parameters investigated. Allpresented ToF-SIMS spectra are normalized to the total intensityand/or as relative ratios between two signals of the same spectrum.For XPS data, all data are presented in terms of atomic percentage andas relative ratios.

■ RESULTS AND DISCUSSION

Protein-Induced Changes in the Surface Oxide.Combined XPS and ToF-SIMS studies were conducted onsome samples of the previous study,6 that is, on 316L stainlesssteel 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 forapproximately 2 months after exposure). Figure 2 shows similarfindings on chromium enrichment as previously reported whenmeasured directly after exposure.6 The same samples wererinsed with ultrapure water and reanalyzed (“H2O rinsed”).Additional measurements were performed on freshly preparedprotein-adsorbed and water-rinsed samples exposed for 0.5, 24,and 168 h and a reference (316L) without any adsorbedproteins (polished 24 h preanalysis). In agreement withprevious studies,6 Figure 2a clearly reveals a chromiumenrichment (increased oxidized Cr to oxidized Cr+Fe XPSratio) with adsorption time, significant only in the case of BSA,and only after 24 h. The oxidized and metallic signals weredifficult to measure by XPS in the case of LSZ, due to a verythick LSZ layer, in agreement with previous XPS studies.6 Incontrast to XPS (Figure 2a), ToF-SIMS revealed lessdifferences in surface oxide composition between BSA andLSZ. ToF-SIMS showed a significant enhancement of oxidizedchromium in the surface oxide already after 30 min ofadsorption (for both BSA and LSZ, compared to the reference)and a further enhancement with increased adsorption time inthe case of BSA (Figure 2b). This might be due to the highersurface sensitivity of ToF-SIMS43 and be explained by the factthat the outermost surface of the oxide of unexposed samples isricher in iron compared with the inner oxide44 and that proteininteraction, which results in a depletion of iron6 from theoutermost surface, can be detected at an earlier stage. Highersimilarity of BSA and LSZ samples analyzed by means of ToF-SIMS (Figure 2b), compared with XPS, can hence be explainedby a similar outermost surface reaction (depletion of iron),

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while the inner oxide is only, or is more strongly, affected byBSA.Protein Adsorption and Layer Properties. The substrate

(metal or oxide) signals can also be compared with signals fromthe proteins, providing information on the protein layerthickness or density. In Figures 3 and 4, the influence ofsuccessive SDS treatment of the fresh 168 h samples is shownin addition to the untreated samples. Both XPS (Figure 3) andToF-SIMS (Figure 4) revealed surfaces of high proteincoverage compared with the references. With XPS, thedifference between BSA and LSZ in terms of protein layerthickness was evident (Figure 3). For LSZ adsorption, thesurface oxide signal decreased rapidly with adsorption time butincreased with increasing time of SDS treatment. Theseobservations indicate a partial removal of proteins by SDS(concentration 4 times the cmc35), which was stronger for freshcompared with aged samples (Figure 4). Already after 30 minprotein adsorption time, LSZ adsorbed samples revealed alower oxide signal compared with the BSA samples, indicativeof a thicker LSZ layer (Figure 3a). The metallic iron to proteinsignal (oxidized carbon) ratio (Figure 3b), was in generalagreement 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 indicatesa similar BSA layer thickness among the different adsorptionconditions (Figure 3). The same trends were observed whenthe metallic signal was normalized to nitrogen (data notshown). The freshly adsorbed BSA sample showed after 30 minan enhanced oxide signal compared with the other adsorptiontime periods (Figure 3a), in contrast to the metal signal (Figure3b). This is due to initial changes in the oxide to metal ratio(data not shown) in the case of BSA adsorption. In contrast toXPS, ToF-SIMS resulted in similar oxide/protein (CNO−)ratios when BSA and LSZ adsorption were compared (Figure

4a). Even though ToF-SIMS has a lower detection limitcompared to XPS, it is more surface-sensitive, which means thatthe major signal originates from the outermost 2 nm of thesample surface.10 This is reflected by the low ratios (especiallyin Figure 4a) with the signal intensity predominantly derivingfrom the protein fragments (in contrast to XPS, Figure 3a).LSZ, which forms a relatively strongly bound inner layer and aweakly adsorbed outer layer,32 was shown to form a less denselayer compared to BSA on chromium and stainless steel, judgedfrom the dissipation mode of QCM.19 This is in particularimportant at the outermost surface. Any protein signals fromthe outermost surface of BSA and LSZ may therefore be similarin intensity.The metallic substrate signal (Fe+ fragment) per distinct

amino acid signals (sum of C5H8N3+, C8H10N

+, and C9H8N+,

assigned to histidine/arginine, phenylalanine, and tryptophan,respectively) (Figure 4b) showed in general similar trends asthe oxide to CNO− ratio (Figure 4a). The SDS treatmentpartially removed proteins from the surfaces, earlier in the caseof fresh samples compared to aged samples (Figure 4). Foraged samples, the substrate signal was enhanced only for BSAafter 24 h SDS treatment (Figures 3 and 4). For fresh samples,LSZ samples showed a stronger removal of proteins upon SDStreatment compared to BSA, the opposite case from agedsamples. SDS was reported to remove proteins from surfaceswhen the SDS concentration is high enough (>cmc, sometimeseven lower), as reported for LSZ on negatively charged silicasurfaces34 and on negatively charged mica surfaces.35 In thecase of preadsorbed LSZ (as in this study) of relatively highprotein solution concentration (1 g/L; this study, 2.2 g/L) andionic strength (0.16 M; this study, 0.15 M) and SDS atconcentrations well above cmc (as in this study), only a partialremoval of LSZ was observed on neutrally charged chromiumoxide surfaces at pH 7.0.45 Consequently, the protein removalrate is larger, that is, the interaction strength is lower, for fresh

Figure 2. Relative amount of oxidized chromium per oxidizedchromium and oxidized iron, measured by means of (a) XPS and(b) ToF-SIMS. <LOD = below limit of detection.

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

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compared to aged samples, although both are preadsorbed(studied ex situ). This can either be caused by changes in theprotein layer during storage or by the difference in adsorptiontemperature (4 vs 37 °C) resulting in different layer stability.Both water rinsing of the aged sample and 30 min SDStreatment resulted in a thicker LSZ layer compared with thenontreated aged sample (Figure 4). This may be due to anexpansion of the LSZ layer upon short SDS treatment,previously observed for LSZ on negatively charged micasurfaces35 and BSA on silica,46 prior to any desorption. For thefreshly preadsorbed samples, any expansion of the LSZ layer issupposed to occur prior to 30 min of SDS treatment, as thesubstrate to protein signals were already higher after 30 minSDS treatment compared with no treatment (fresh 168 h)(Figure 4). SDS was clearly adsorbed and interacted with theproteins, as a significant increase in fragments corresponding toSDS [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 isinteresting from a surface chemistry perspective since differentmodels were earlier suggested for protein−SDS interaction andany subsequent removal of proteins, including binding to theprotein (in different ways) and binding to the surface.45 Sincethe surfaces were rinsed with water after the SDS treatment, theincrease of SDS fragments must be due to interacting SDSmolecules. 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 reportSDS treatment to denature adsorbed LSZ on negatively

charged mica at SDS concentrations at, or above, cmc.35 Sulfurspecies information provided by XPS and ToF-SIMS wasevaluated to gain an improved understanding of the SDS−protein interaction and the low extent of protein removal. ForXPS (Figure S3, Supporting Information), the sulfur speciesintensity 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 thepeak at 164.0 eV to disulfide bonds or thiolate within

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.

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.

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proteins.40,41 No peak assigned to sulfite, at approximately 166eV,41 was observed. XPS (Figure S3, Supporting Information)showed that the disulfide/thiolate species increased in intensitywith increasing adsorption time (with the exception of BSAafter 0.5 h adsorption). The SDS treatment resulted in a sulfatepeak, probably due to binding or adsorption of SDS to theprotein layer (Figure S3, Supporting Information). At the sametime, the sulfide/thiolate signal decreased with longer time ofSDS treatment of the aged samples, indicative of (partial)protein removal to a higher extent for BSA, in agreement withthe previous discussion (Figures 3 and 4). ToF-SIMS is able todetect the signal produced by disulfide bonds and haspreviously been used to determine protein denaturation.16

The signal decay was investigated as a function of temperaturetreatment, SDS treatments, and XPS measurements (Figures 6and 7). For that purpose, the fresh 168 h samples wereconsecutively treated in SDS for 0.5 and 36 h, and a second setof fresh 168 h samples was exposed to successively increasingtemperature within the ToF-SIMS analysis chamber. XPSmeasurements were not performed in between, but after thecompletion of both experiments. After XPS measurement, thesamples were again investigated by means of ToF-SIMS. Figure6 shows the substrate peaks compared to the protein peaks, as afunction of temperature and SDS treatments. The effect oftemperature treatment during the ToF-SIMS measurementrevealed a slight but significant removal of adsorbed proteinsthat increased with increasing temperature up to 200 °C(Figure 6 and Figure S4 in Supporting Information). For theaged sample after SDS treatment (Figure S4, SupportingInformation), significantly more BSA was removed uponthermal treatment (at even 150 °C), compared with freshlyadsorbed samples.

It seems that long positively charged fragments, assigned tohistidine/arginine, phenylalanine, and tryptophan, decrease to alarger extent compared to the negatively charged amidefragment CNO− with increasing temperature, also whendifferences in Fe+ and FeO− + CrO− signals are considered.One possible explanation is a temperature-induced weakeningof the bonds in the protein, resulting in higher fragmentation ofthe molecules and consequently favored emission of smallerfragments. Also, the increased fragmentation can be caused byrepeated measurements on the same area (cf. ExperimentalSection), even though the primary ion dose density was keptwell below the static limit for each measured spot. Toinvestigate whether ToF-SIMS measurements of the samesample area can cause a protein denaturation or change proteinsignals, the same spot of a sample (1 h BSA adorption in PBS)was analyzed five times consecutively, with a total primary iondose density (PIDD) of 5 × 1011 ions/cm2 (to be comparedwith a maximum exposure of 3 × 1011 ions/cm2 for all otherinvestigated samples). No significant difference in proteinsignals 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 ToF-SIMS (Figure 6). However, the disulfide per CNO− signal wassignificantly reduced after XPS investigation, indicative ofprotein denaturation, cf. Figure 7b. Ultrahigh vacuum (UHV)has previously been shown to induce significant differencescompared to ambient conditions for antiferritin and anti-IgM.47

The influence of UHV has also been highlighted forconformational studies of human serum albumin; however, aneffect was not found to be very significant.13 UHV alone,

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.

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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 indicatethat 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) fragment

correspondingamino acid ref(s)

significant increased signal forprotein (BSA; LSZ)

difference between fresh andaged samples

significant trend withadsorption timeb

30.03 CH4N+ Gly, Lys, Leu, and

others30, 51, 53,54

(LSZ) fresh > aged D

44.05 C2H6N+ Ala, Cys 30, 51, 53,

54(I)

58.07 C3H8N+ Glu 13, 51 BSA D59.05 CH5N3

+ Arg 30, 51, 53 LSZ fresh > aged60.06 C2H6NO

+ Ser 53, 54 LSZ fresh > aged70.03 C3H4NO

+ Asn 30, 51, 53,54

LSZ (fresh > aged)

70.07 C4H8N+ Pro, Arg, Val, Leu 30, 51, 53,

54BSA

72.08 C4H10N+ Val 30, 51, 53,

54BSA fresh > aged (I/D)

73.06 C2H7N3+ Arg 30, 51, 54 LSZ (fresh > aged) (I/D)

74.06 C3H8NO+ Thr 30, 51, 53,

54(LSZ) fresh > aged

81.04 C4H5N2+ His 30, 53, 54 BSA

88.04 C3H6NO2+ Asn, Asp 30, 53, 54 LSZ fresh > aged

91.05 C7H7+ Phe 30 BSA (fresh > aged)

100.08 C4H10N3+ Arg 30, 51,53,

54(LSZ) (I/D)

110.07 C5H8N3+ His, Arg 30, 51, 53,

54BSA fresh > aged (I/D)

120.08 C8H10N+ Phe 30, 51, 53,

54BSA fresh > aged (I/D)

130.06 C9H8N+ Trp 30, 51, 53 LSZ fresh > aged (D)

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

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however, cannot be responsible for any protein denaturation(that is, disulfide signal intensity) observed here, since it is alsopresent during the ToF-SIMS measurements. Instead, the X-rayirradiation or the photoemitted electrons may denature theproteins. This would not be surprising as X-ray irradiation iscommonly used for sterilization of medical equipment.48 InFigure 7a, the aged 168 h samples and consecutive samples allwere measured with XPS before the ToF-SIMS measurementand in between. The observed decrease in disulfide signal mayhence be caused by the XPS measurement, instead of, or inaddition to, the SDS treatment. In Figure 7b, it is shown for thefresh 168 h samples that the disulfide ratio was fairly constantupon short SDS treatment (4 times cmc, 0.5h) for LSZ andtemperature treatment up to 150 °C for both proteins. Even ashort SDS treatment for BSA, longer SDS treatment, andtemperatures of 200 °C induced a significant decrease of thedisulfide ratio, which was even more significant for XPSexposure. This is also illustrated in Figure S1 (SupportingInformation), where the detailed ToF-SIMS spectra are shownfor the disulfide signal and other characteristic signals as afunction of the different treatments.On hydrophilic surfaces (such as stainless steel17), LSZ has

been reported to retain most of its ordered structure,49,50 whileα-lactalbumin50 and BSA49 have lost their ordered structuresalmost completely when investigated on hematite particles.49,50

This effect has been referred to the structural stability of theproteins, which is higher for LSZ compared with BSA.49 Inaddition, less structurally stable proteins adsorb with higheraffinity to surfaces.49 It is therefore expected that BSA shows alower stability (reflected by the disulfide signal), generally andupon SDS treatment, compared with LSZ. Despite the highercontent of disulfide bonds in BSA compared with LSZ perprotein mass unit, the disulfide signal was only slightlyenhanced or similar for BSA compared with LSZ for thefreshly adsorbed samples, and significantly lower in the case ofaged and SDS-treated samples (Figure 7a).The SDS and temperature treatment findings indicate that

aging induces denaturation of proteins and a weaker bonding tothe substrate compared with fresh samples, reflected in higherremoval upon temperature treatment and lower (slower) SDS-induced removal of proteins.Intensities of Amino Acid Fragments. Multivariate

analysis, for example, PCA, has been reported to be usefulfor protein distinction and detection of denaturation11,12,51,52

and to be applicable to identify any conformational changes ofthe protein layer.13−15 PCA was used in this study to identifyamino acids that differed the most between the two proteinsstudied, between adsorption times, and/or between fresh andaged samples. Observed amino acid fragments and theirassignments are compiled in Table 3, together with observeddifferences. In almost all cases, the fragments showed highersignals for the protein that contains more of a given amino acidper 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 exceptionwas 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 resultedin an enhanced amino acid fragment intensity compared withthe aged samples. This finding is in contrast to the similarlythick or even thicker protein layer for the aged 168 h samplecompared with the fresh 168 h sample, deduced from Figures 3

and 4. This effect may be explained by conformational changesthat result in a lower ionization ability of the amino acidfragments and/or by their partial destruction upon aging.Another reason could be a higher amount of surfacecontamination 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, slightlyincreased (nonsignificantly), or did not change significantly,with the exception of signals at 30.03 and 58.07 m/z, discussedbelow. An increase over time would be expected in case of adenser outermost protein layer and no protein orientation orconformation changes. Conformational changes of the proteinand/or speciation (chemical form) changes of some individualamino acids could result in changed ionization properties andhence a decreased signal. Larger amino acid fragments (m/z110.07, 120.08, and 130.06) decreased after longer adsorptiontime (>24 h, Table 3). However, some smaller amino acidfragments constantly decreased throughout the entire screenedadsorption time range, that is, the amine signal (CH4N

+, m/z30.03) and the glutamic acid signal (C3H8N

+, m/z 58.07)(which excludes a single effect of the lower synthesistemperature for the longest adsorption time). The discussedsignals are displayed as a function of adsorption time (for freshsamples only), together with the histidine (arginine) signal(C5H8N3

+, m/z 110.07) for comparison, in Figure 8. The amine

fraction signal corresponds to several different amino acids(Table 3). Its constant decreased intensity with adsorptiontime, in contrast to most other fragments, can be explained byseveral possibilities. First, with increasing and denser proteinlayer over time, the hydrophobicity within the protein layerincreases and the dielectric constant decreases. This maychange the speciation of the otherwise positively charged aminegroup (-NH3

+) to the uncharged -NH2 group within theprotein layer. Second, the positively charged amine group mayinteract with the negatively charged stainless steel surface,either by electrostatic interaction with, for instance, OH− orO2− groups of the oxide or by covalent binding to a metalsurface atom. It can also interact as an uncharged -NH2 groupforming hydrogen bonds to an OH group of the oxide. All these

Figure 8. Relative intensity (normalized to total intensity of eachspectrum) of the positively charged fragments CH4N

+ (amine group),C3H8N

+ (glutamic acid), and C5H8N3+ (histidine, arginine), analyzed

by means of ToF-SIMS.

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interactions could result in a reduced CH4N+ signal. The same

may be true for other amino acid fragments, such as thatderived from glutamic acid. Further studies of single aminoacids or small peptide interactions with stainless steel andadditional time points are, however, needed in order toinvestigate stainless steel binding mechanisms by means ofToF-SIMS. Recently it has been shown that the ratio of thesum of hydrophobic to the sum of hydrophilic amino acidfragments was changed for BSA depending on the hydro-phobicity of the substrate surface.43 No significant trends ordifferences were observed in this study between differentadsorption time periods, different proteins, or upon heating orSDS treatment, when the same ratio was used (data notshown). A theoretical study on Cr2O3 surfaces (as models forstainless steel) and their interaction with glycine, with theassumption that Cr2O3 is uncharged at neutral pH, showed thatseveral types of interactions are possible, including hydrogenbonding and covalent bonding to the amine and carboxylategroup.25 However, as recent findings by the authors showedthat massive stainless steel AISI 316 is negatively charged atneutral 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 isheterogeneous and undergoes continuous changes in, forexample, its surface oxide composition (chromium enrichment,Figure 2).6 Even the stainless steel grade investigated in thisstudy, which contains very low amounts of impurities, possessesmore noble and less noble areas depending on the grainorientations55 as well as grain boundaries of different surfacechemistry at the nanoscale.56 Another indication that severaldifferent interactions may be possible with stainless steelsurfaces is the observation that BSA adsorbed in a similar wayon both positively charged stainless steel nanoparticles and onnegatively charged massive stainless steel.23 Several literatureinvestigations assume stainless steel surfaces to be positivelycharged at neutral pH or pH 7.4 and suggest hence interactionswith negatively charged carboxylate groups.5,18,20

■ SUMMARYThe interaction of LSZ and BSA (in PBS) with AISI 316Lstainless steel surfaces was studied ex situ by means of XPS andToF-SIMS after different adsorption times, and parallelinvestigations of the effect of storage (aging), SDS treatments,XPS measurements, and temperature (up to 200 °C) werecarried out.XPS and ToF-SIMS were found to be highly complementary

techniques for protein−metal interaction studies due todifferences in information depth, chemical information, andsensitivity. XPS measurements (X-ray irradiation and/orphotoelectron emission) most probably induced proteindenaturation, observed in terms of a decreased disulfide signalmeasured by means of ToF-SIMS subsequently conducted afterthe XPS measurements. No denaturation effects were inducedby the use of ToF-SIMS.Both BSA and LSZ adsorption induced an enrichment of

chromium in the surface oxide. LSZ formed a thicker layercompared with BSA. BSA induced significant changes to theentire oxide, whereas LSZ only induced a depletion of iron inthe outermost layer.SDS was unable to completely remove preadsorbed proteins

from either fresh or aged samples, despite its highconcentration (0.035 M; 4 times cmc) and relatively longtreatment time (up to 36.5 h). For the aged samples, BSA was

removed to a larger extent and after shorter time periodscompared with LSZ. The LSZ layer thickness expanded uponSDS treatment prior to any protein removal. Heat treatment upto 200 °C only partially removed adsorbed proteins. Bothtreatments indicate strong protein−surface interactions.In the case of freshly preadsorbed proteins, SDS induced an

enhanced and earlier partial protein removal compared withaged samples for both BSA and LSZ and partially destroyeddisulfide bridges of the proteins. The high-temperaturetreatment (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 weresignificantly enhanced in the case of freshly adsorbed samplescompared with aged samples, despite similar protein surfacecoverage. Reduced intensities of some amino acid fragmentswith adsorption time (especially amine, CH4N

+, and glutamicacid, C3H8N

+) or after longer time periods (especially largeramino acid fragments related to histidine, phenylalanine, andtryptophan) were attributed to changes in speciation (chemicalform) and/or changes in orientation upon, for example, metal(oxide)−protein binding. These strong surface−protein inter-actions confirm and complement earlier studies6 and resulted inan enhanced metal release and enrichment of chromium in thesurface oxide in the case of BSA compared with LSZ.

■ ASSOCIATED CONTENT*S Supporting InformationFive figures as described in the text. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone +46 8 7906670, e-mail yolanda@kth.se.Present Address∥Division of Surface and Corrosion Science, Department ofChemistry, KTH Royal Institute of Technology, DrottningKristinas vag 51, SE-10044 Stockholm, Sweden.Author ContributionsThe manuscript was written through contributions of allauthors. All authors have given approval to the final version ofthe manuscript.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Ulrike Marten-Jahns for XPS measurements and theSwedish Research Council (VR), travel grants from theSwedish Steel Association (Jernkontoret) and the BjornFoundation at KTH, Royal Institute of Technology, Sweden,the German Research Council (DFG), and Cusanuswerk,Germany, and the BMBF for financial support. We thank DanGraham, Ph.D., for developing the NESAC/BIO Toolbox usedin this study and NIH Grant EB-002027 for supporting thetoolbox development.

■ ABBREVIATIONSBSA, bovine serum albumin; LSZ, lysozyme; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; cmc, criticalmicelle concentration; 316L, stainless steel grade AISI 316L;XPS, X-ray photoelectron spectroscopy; ToF-SIMS, time-of-flight secondary ion mass spectrometry; PCA, principal

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

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