2011-1

Leading Opinion

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Keywords:PeptideStainless steelPilin receptor binding domain

t bind

peptides [4], although recently a reasonably high afnity (116 nM)binding peptide for titaniumwas obtained by phage display [5]. TheRBD binds directly to steel and this interaction does not appear tobe mediated by hydrophobic interactions [2]. However, the RBDinteraction with epithelial cells is of considerably lower afnity(w200,000 times lower) and the interaction with the minimalreceptor of GalNAc-Gal appears to be mediated largely throughhydrophobic interactions [6].

q Editors Note: This paper is one of a newly instituted series of scientic articlesthat provide evidence-based scientic opinions on topical and important issues inbiomaterials science. They have some features of an invited editorial but are basedon scientic facts, and some features of a review paper, without attempting to becomprehensive. These papers have been commissioned by the Editor-in-Chief andreviewed for factual, scientic content by referees.* Corresponding author. Tel.: 1 780 492 5374; fax: 1 780 492 7521.

Contents lists availab

Biomat

journal homepage: www.elsev

Biomaterials 32 (2011) 5311e5319E-mail address: [email protected] (R.T. Irvin).a vast array of surfaces, from human cells to stainless steel sinks andtubs, and surprisingly Pseudomonas aeruginosa (PA) utilizesa receptor binding domain (RBD) displayed at the tip of its type IVpili (T4P) to bind to both. T4P are powerful nanomotors that pullthe bacteria along surfaces by retracting the pilus once it hasbounddgenerating large forces on the RBDesurface interface. ThePA RBD has an incredibly high apparent afnity for stainless steel[1,2] which cannot be readily understood given the high entropicpenalty associated with a exible peptide interacting with an

PA is a human opportunistic pathogen that utilizes its T4P toadhere to and colonize a wide variety of biotic and abiotic surfaces.Binding is mediated by the RBD, a semi conserved 17-amino acidregion that includes an intra-chain disulde loop encoded withinthe C-terminal region of the pilin structural protein, termed PilA, inPA T4P. The RBD has an extremely high afnity for stainless steel,exemplied by a Kiapparent ofw0.2 nM for the inhibition of puriedpili (thought to display 3 RBDs at the tip of the ber) and viable PAbacteria (each cell has a number of T4P located at the poles of therod shaped cells) binding to steel, particularly when compared tothe mM range of afnities previously reported for metal bindingElectron work functionXPSAdhesive forceCorrosion

1. Introduction

Bacteria are amazingly efcient a0142-9612/$ e see front matter 2011 Elsevier Ltd.doi:10.1016/j.biomaterials.2011.04.027has a signicantly increased electron work function (4.9 0.05 eV compared to 4.79 0.07 eV), decreasedmaterial adhesive force (19.4 8.8 nN compared to 56.7 10.5 nN), and is signicantly harder than regular304 stainless steel (w40% harder). A formal or semi-formal organoemetallic covalent bond is generatedbetween a pilin receptor binding domain and stainless steel based on XPS analysis which indicates that theelectronic state of the surface is altered. Further, we establish that the peptideesteel reaction demonstratesa degree of stereospecicity as the reaction of native L-peptide, D-peptide and a retro-inverso-D-peptideyields bioorganic steel products that can be differentiated via the resulting EWF (4.867 0.008 eV,4.651 0.008 eV, and 4.919 0.007 eV, respectively). We conclude that electron sharing between thepeptide and steel surface results in the stabilization of surface electrons to generate bioorganic steel thatdisplays altered properties relative to the initial starting material. The bioorganic steel generated from theretro-inverso-D-peptide yields a protease stable product that is harder (41%harder at a 400 mN load), andhasa 50% lower corrosion rate compared with regular stainless steel (0.11 0.03 mpy and 0.22 0.04 mpy,respectively). Bioorganic steel is readily fabricated.

2011 Elsevier Ltd. All rights reserved.

ing to and growing on

immobile surface and yet direct measurement of RBDesteel inter-action with atomic force microscopy has conrmed a very strongmolecular interaction [2,3].Accepted 8 April 2011Available online 7 May 2011generate an altered formof stainless steelwhichwe termbioorganic stainless steel. Bioorganic stainless steelArticle history:Received 29 March 2011

A synthetic peptide derived from thenativeprotein sequence of ametal binding bacterial piluswas observedto spontaneously react with stainless steel via a previously unreported type of chemical interaction toA peptide e stainless steel reaction thatof matterq

Elisabeth M. Davis a, Dong-yang Li b, Randall T. IrviaDepartment of Medical Microbiology and Immunology, University of Alberta, EdmontobDepartment of Chemical and Material Engineering, University of Alberta, Edmonton, A

a r t i c l e i n f o a b s t r a c tAll rights reserved.ields a new bioorganic e metal state

,*

lberta T6G 2H7, Canadata T6G 2H7, Canada

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[1,10] and the force required to break the RBD-steel interaction is

teriaconsiderably higher (w2.2 fold) at grain boundaries thanwithin thegrains [3] suggesting that surface electron activity or the differen-tial material composition of the grain associated with the grainboundary contributed signicantly to the interaction. Generally,bacterial binding to abiotic surfaces is thought to involve theinteraction of the cells with a conditioning lm of adsorbed organicmaterial bound to the surface. Binding is mediated throughhydrophobic interactions due to energy contributions from thebulk solvent being excluded from the site of interaction [11,12].However, the RBDesteel interaction did not require bulk solvent,and did not appear to involve a conditioning lm [2,3,13]. Thesurface free energy of stainless steel can be signicantly altered byregulating the grain size [3]. Nano-structured (steel with small butdened grain sizes) and of specic surface electron activities orsurface free energies as determined by EWF and adhesive forcemeasurements, was employed to demonstrate that the strength ofthe RBDesteel interaction is directly proportional to the surfaceelectron activity [13]. Thus the mechanism of interaction(s) thatmediate RBD binding to biotic surfaces differs substantially fromthat which mediates binding to stainless steel surfaces, eventhough the RBD is the same in both cases.

While our observations concerning the RBDesteel interactionappeared to have a sound materials basis and reected the mate-rials property of the steel surface, the molecular basis for the RBDinteractionwith stainless steel was puzzling from a biological pointof view as most biologically relevant ligandereceptor interactionsare driven by the energetics of the hydrophobic effect. Given thatthe afnity of the RBD for steel was extremely high (beyond whatwould be anticipated for a exible peptide binding to a solid surfacewhere an entropic penalty would be encountered), that the inter-action with steel did not require a conditioning lm, that hydro-phobic interactions did not appear to contribute to the interaction,and that the strength of interaction was directly proportional tosurface electron activity or surface free energy, we hypothesizedthat the RBDmay form a semi-formal or formal covalent bond withthe stainless steel surface to delocalize surface electrons throughthe RBD and thus investigated the nature of the RBDesteel inter-action. We report that the PA RBD forms a previously unreportedchemical interaction with steel that alters surface electron orbitalsof the surface layer of stainless steel to form a new material whichwe term bioorganic K122-4 stainless steel (borg-K122SS).

2. Materials and methods

K122-4(128-144)ox peptide was synthesized by solid phase peptide synthesisand puried by reversed-phase high-performance liquid chromatography (HPLC) asdescribed by Wong et al. [14,15]. The disulphide bridge form of the peptide used inthis study was generated by air-oxidization [16]. Biotinylation of the peptide wasperformed as previously described by Yu et al. [17]. Peptides were synthesized bythe Peptide and Protein Chemistry Core Facility of the University of Colorado HealthSciences Center at Fitzsimons on a fee for service basis. The ability of biotinylated piliand peptide to bind to stainless steel was demonstrated by Giltner et al. [1].

Grade 304 stainless steel 2B nish plates (1 mm thick, 20 gauge steel) were cutinto 1 cm 1 cm coupons and annealed at 1040 C for 1 h. The surface was polishedStainless steel is polycrystalline in nature, with individualcrystals being termed grains while the interfacial region locatedbetween adjoining crystals is termed the grain boundary. Grainboundaries are regions of the metal where there are signicantcrystal defects and various types of dislocations. Electrons in theseregions of imperfections are at higher energy levels, rendering theregions more reactive and more capable of interacting with othermaterials beyond the metal [7]. The grain boundary materialsurface properties differ from the surface properties observedwithin a grain [8,9]. PA binds preferentially to grain boundaries

E.M. Davis et al. / Bioma5312to uniformity using sandpaper of increasing grit size (MetTech Inc, Calgary), from120# to 1200# grit and an aqueous slurry of 0.05 mm colloidal silica. Coupons werewashed with dish detergent, thoroughly rinsed with distilled water, and immersedin 95% (v/v) ethanol for 15minwith gentle agitation. Coupons were then rinsed withdistilled water, washed with 15 mL of acetone for 1 min, and rinsed with distilledwater. Theywere placed in 6-well (1 coupon per well) tissue culture plates (Corning)and covered with 3 mL of sterile phosphate buffered saline (PBS)(pH 7.4) containing10 mg/mL of K122-4(128-144)ox peptide and were incubated at room temperature(RT) for 1 h with gentle agitation. Samples were washed 6 times with distilled waterand allowed to air dry.

Aluminum (5 mm thick) was cut into 1 cm by 1 cm samples. Aluminum sampleswere cleaned, polished, and coated with K122-4(128-148)ox peptide. Aluminumwas chosen for a control surface as the RBD did not appear to interact with thealuminum and hence was used as control for what the materials property of RBDdeposited on a surface.

For corrosion, steel coupons were annealed as described and encased in epoxy(LECO Corporation, Mississauga) prior to polishing. Once polished, samples werecleaned and coated as described.

The EWF was determined using a scanning Kelvin probe [18] and three areasmeasuring 0.5 mm 0.5 mmwere scanned per sample. Data was plotted as meansin electron volts (eV) per area scanned. The electron work function (EWF) wasmeasured using a scanning Kelvin probe (KP Techonology Ltd, UK) tted with an Autip with a 1 mm diameter [18]. The probe was composed of three sub-systemscontrolled by a computer, including a digital oscillator, data acquisition, andsample translation. A three-axis microstep positioner allowed for precise position ofthe tip on the sample and controlled scanning steps of 0.4 mM per step. The oscil-lation frequency of the Kelvin probe was 173 Hz. Three separate areas of 0.5 mm by0.5 mmwere scanned on each sample to determine the electron activity, with a totalof 100 reading per area scan.

An AFM equipped with a silicon nitride tip was used to measure the adhesiveforce [3]. Fifty adhesive force measurements were obtained per sample. The adhe-sive force between a standard AFM silicon nitride tip (Veeco, CA) and a peptide-coated surface was measured using an atomic force microscope (AFM) (Hysitron,Minneapolis, USA). The AFMwas used in contactmode. The tip was approached tothe surface, allowed to made contact, and the total amount of deection of thecantilever when the tip is pulled away from the surface, which reects the adhesiveforce, was detected by laser beam. The related force can be quantitatively deter-mined if the spring constant is known. The spring constant for the nitride tip was0.06 N/m.

Nanoindentation measurements were performed using a triboscope equippedwith a diamond indenter. Samples were tested using loads of 50e800 mN and totaldisplacement (in nm) of the tip into the sample was measured. A triboscope(Hysitron, Minneapolis, USA), a combination of a nanomechanical probe attachmentand an AFM, was used to examine the changes in the mechanical properties ofpeptide-coated samples. The probe, a diamond pyramidal Vickers indenter, hada nominal radius of 150 nm with a force sensitivity of 100 nN and a displacementresolution of 0.2 nm. During nanoindentation a force-depth curve was obtained foreach indentation and the total depth displacement of the tip into the surface of thesample was obtained from this curve. Five force-depth curves were obtained foreach force load.

Some samples were tested as to their corrosion properties. A minimum of threelinear polarization and Tafel plot corrosion tests using a computerized scanningpotentiostat were performed per sample to determine the polarization resistanceand corrosion rate of peptide-coated samples. Electrochemical tests were performedusing a computerized scanning potentiostat (Model PC4-750, Gamry). A saturatedcalomel electrode (SCE) and a platinum (Pt) foil were used as the counter andreference electrodes for all corrosion experiments. The electrolyte solution used wastap water and all experiments were performed at room temperature. At least threeseparate linear polarization corrosion tests were performed to measure the polari-zation resistance Rp, starting at 0.02 V below the open circuit potential (OCP) andending 0.02 V above the OCP and using a scanning rate of 0.125 mV/s. A minimum ofthree Tafel plot corrosion tests were performed. A scanning rate of 1 mV/s was usedand scans began from 0.25 V below the OCP and ended 0.25 V above the OCP. Thecorrosion rate and the polarization resistance were obtained using the SterneGearyequation.

Surface electron activity was examined via XPS analysis. XPS analysis was per-formed using an AXIS-165 spectrometer (Kratos Analytical). The base pressure in theanalytical chamber was lower than 3 108 Pa. Monochromatic Al Ka source(hn 1486.6 eV) was used at a power of 210W. The analysis spot was 400 700 um.The resolution of the instrument is 0.55 eV for Ag 3d and 0.70 eV for Au 4f peaks.Survey scans were collected for binding energy from 1100 eV to 0 with analyzerpass-energy of 160 eV and a step of 0.35 eV. For the high-resolution spectra the pass-energy was 20 eV with a step between 0.1 and 0.15 eV. No charge neutralizationwasrequired. In order to increase the surface sensitivity the samples were tilted so thatthe angle between the sample surface and the analyzer was 30 . No sample etchingwas performed prior to analysis. Data analysis was done using Casa XPS software,version 2.3.13.

We examined the surface morphology samples by standard SEM and AUGERanalysis methods. SEM and Auger data were collected with a eld-emission Augermicroprobe JAMP-9500F (JEOL). The primary electron beam energy was 10 kV, and

ls 32 (2011) 5311e5319the probing beam current was 7 nA. The lateral resolutions for SEM and AES are 3.0

Fig. 1. Changes in surface electron reactivity, adhesive force and hardness of borg-K122SS steel compared with 304 stainless steel and aluminum with and without K122-4(128-144)ox peptide. A) Electron work function measurements of borg-K122SS and 304 steel performed using a Kelvin probe. Three areas (1 mm 1 mm) were scanned. A totalof 100 measurements were taken per area scanned and means were plotted. B) Electron work function measurements of aluminumwith and without exposure to K122-4 (128-144)ox peptide. C) Adhesive force measurements on borg-K122SS and 304 stainless steel performed with an atomic force microscope (AFM) equipped with a non-conductive siliconnitride cantilever. Fifty force measurements were taken per sample. D) Adhesive force measurements on aluminum with and without exposure to K122-4 (128-144)ox peptide. E)Nanoindentation measurements of borg-K122SS and 304 steel. Five displacement measurements were taken per load. F) Nanoindentation measurements of aluminum with andwithout exposure to K122-4 (128-144)ox peptide.

E.M. Davis et al. / Biomaterials 32 (2011) 5311e5319 5313

and 8 nm, respectively. The sample was tilted 30 away from the primary beamtoward the axis of the electron analyzer. The SEM images were used to locatepositions for collecting the Auger energy spectra, line proles, and images. Autoprobe tracking was in effect during the imaging to eliminate the drifting due toinstabilities in power, temperature, etc. The intensity in the image and line proledistributions was calculated as (P B)/B to remove edge effects, P being the peakintensity and B being the background intensity. The images are presented in

3. Results and discussion

E.M. Davis et al. / Biomaterials 32 (2011) 5311e53195314To elucidate the nature of the interaction between stainless steeland the T4P of P. aeruginosa, a synthetic peptide of the RBD, termedK122-4(128-144)ox, was applied to stainless steel surfaces (notethat we are reporting on a single P. aeruginosa type IV pilin RBDsequence but our data indicates that all tested RBD sequences todate interact in a similar manner). Derived from the sequence ofthe RBD of P. aeruginosa strain K122-4 PilA protein, the peptide isN-a-acetylated, has a C-terminal amide, and contains a disuldeloop (Fig. S1). This new bioorganic steel, borg-K122SS, was exam-ined for changes in the physical and mechanical properties of itssurface, including the reactivity of surface electrons, as a result ofpeptide binding. We speculated that any interactions between thesteel surface electrons and the peptide would result in measurablechanges in these surface properties.

Metal electron activity is described by the electron work func-tion (EWF). EWF measures the minimum amount of energyrequired to move an electron from the Fermi level to just beyondthe surface of the metal. Gold, considered the standard fromwhichall other EWFs are compared to, has an EWF of 5.1 eV [19,20].Regions of higher electron activity, such as the grain boundary, havecorrespondingly lower EWFs because less energy is required forremoving electrons from the regions. Conversely, surfaces withhigher EWF values are more stable and inert because they possess

Fig. 2. XPS spectra analysis of elemental composition of borg-K122SS and 304 stain-less steel. Overlays of borg-K122SS and 304 steel XPS spectra of A) O 1S orbitals,B) sulfur 1S orbital region with an unidentied peak associated with the borg-K122SSsurface, and C) iron 3S orbital region with an unidentied peak associated with theborg-K122SS surface. XPS spectra of borg-K122SS is plotted in green while the XPSa thermal scale, the brighter area corresponding to the highest intensity.As receptor binding domain contains potential trypsin cleave sites, we examined

the ability of trypsin to modify steel surfaces. Grade 304 stainless steel 2B nishplates (20 gauge, 1 mm thick) were cleaned as described previously [1] andassembled into a Schleicher and Schuell MinifoldTM System (Mandel Scientic).Synthetic biotinylated PAK(128-144)ox peptide were added at a concentration of10 mg/mL (50 mL per well in replicates of ve) to the stainless steel manifold andincubated at RT for 1 h with gentle agitation. The manifold was washed six timeswith 250 mL per well of phosphate buffered saline (PBS, pH 7.4). To test whether thepeptide was sensitive to trypsin, 1 mg/mL of trypsin (100 mL per well, 5 wells) wasadded and the manifold was incubated for 1 h at 37 C with gentle agitation. Themanifold was washed six times with PBS and remaining bound peptidewas assessedusing streptavidin-horseradish peroxidase (HRP, Sigma) diluted 1:5000 in PBS.Following a 1 h incubation at RT, the manifold was washed with PBS and substratebuffer (0.01 M sodium citrate, pH 4.2 containing 1 M 2,20-Azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) diammonium salt (ABTS) (Sigma) and 0.03% (v/v)hydrogen peroxide) was added (125 mL per well). The manifold was incubated for10 min at RT with gentle agitation. One hundred mL per well of the reaction solutionwas transferred to a 96-well at bottom microtiter plate (Corning) and absorbanceat 405 nm was determined using a FluoStar Optima plate reader.

To measure the EWF of the samples following trypsin treatment, steel couponswere polished, cleaned, and coated with K122-4(128-144)ox peptide as describedpreviously andwere in 1 mg/mL trypsin for 45min at 37 C. Samples werewashed sixtimes with distilled water and allowed to air dry before testing the EWF.

Data were analyzed using the statistics program PRISM 5.0 (GraphPad Sofware)utilizing ManneWhitney analysis, as the data distribution was non-parametric innature. Nanoindentation data were analyzed using a linear regression model togenerate a best-t line through the data points. Signicant differences between datapoints were determined using 2-way non-parametric ANOVA analysis followed byBonferroni posttests. A signicance cut-off of 0.05 was employed for all tests.spectra of 304 stainless steel is plotted in red. (For interpretation of the references tocolour in this gure legend, the reader is referred to the web version of this article.)

less reactive surface electrons. Borg-K122SS had a signicantlyhigher EWF compared to unmodied 304 steel (Fig. 1A). Thepresence of bound peptide increased the EWF from 4.5 tow5.0 eV(P < 0.001), suggesting that binding of the peptide stabilizes steelsurface electrons. The EWF of a second metal aluminum, did notincrease when peptide was added to the surface or dried on thesurface, as the EWF was not signicantly different from uncoatedaluminum (Fig. 1B). Aluminum is utilized as a control surface onwhich peptide could be deposited where the peptide would notinteract and thus allows for the assessment of the materials prop-erties of the peptide rather than a peptide coupled to a surface.Aluminum may have fairly unique properties as the type IV pilinRBD appears to be designed to interact with most solid phasesurfaces. The ability of the peptide to stabilize steel but notaluminum suggests that the peptideesteel interaction occursbetween the peptide and one or more specic components withinthe steel.

The adhesive force, a second physical property of metalsurfaces, describes the ability of a metal surface to stick andinteract with other surfaces and is increased at grain boundaries[21,22]. Higher adhesive forces correspond to areas where the EWFis lower [23] and the RBD has been shown to bind with higheradhesive force to grain boundaries compared with regions withingrains [3]. The adhesive force of borg-K122SS and 304 steel wasmeasured using atomic force microscopy (AFM). The AFM canti-lever, possessing a silicon nitride tip, is lowered until the tipinteracts with the surface. The cantilever is then raised and theamount of force necessary to break the interaction between the tipand the surface is measured (Fig. S2). Surfaces with reactivesurface electrons interact more readily with the tip and greaterforce is necessary to break the interaction. The AFM silicon nitridetip did not interact as strongly with borg-K122SS (P < 0.001), asthe adhesive force of borg-K122SS was 19.4 8.8 nN compared toan observed adhesive force of 56.7 10.5 nN for untreated 304

entaical

E.M. Davis et al. / Biomaterials 32 (2011) 5311e5319 5315Fig. 3. Auger-SEM scan of distribution of elements on the surface of borg-K122SS. ElemB) sulfur, note the presence of nano-structured material (see arrowheads). C) Topograph

from the borg-K122SS surface plotted as the frequency of electrons at a given energy (in eV)N peaks (originating solely from the peptide) are low but the energies of the backscatteredl scan of A) nitrogen, note the presence of nanostructed material (see arrowheads) andscan of the surface of borg-K122SS. D) Analysis of the backscattered electrons obtained

and labeled as to the element generating electrons of that energy value. Note the S andelectrons are observed at previously reported energy levels for these elements.

E.M. Davis et al. / Biomaterials 32 (2011) 5311e53195316

steel (Fig. 1C). These results suggest that the surface of borg-K122SS is less reactive than 304 steel and that the surface elec-trons of borg-K122SS interact less strongly with the AFM tip. Theadhesive force of peptide dried on aluminum did not differ fromunaltered aluminum (Fig. 1D).

Another important property of surfaces is their hardness. To

no classical sulfur electron orbitals were detected by XPS (Fig. 2B).However, the peptide contains two sulfur atoms within the disul-de loop that would be visible by XPS. A signicant peak wasobserved near the location of unbonded sulfur 1S orbital (Fig. 2B)for borg-K122SS. This peak did notmatch any known shifts in sulfurpeaks and could not be positively identied as sulfur based on XPS

304tainl)oxsequ

E.M. Davis et al. / Biomaterials 32 (2011) 5311e5319 5317compare the hardness of borg-K122SS to 304 steel, an AFMequipped with a triboscope was used to obtain nanoindentationloadedisplacement plots, where total displacement of a diamondindenter tip into the surface of borg-K122SS and 304 steel wasmeasured for loads of 50e400 mN (Fig. S3A,B). At all loads, greaterdisplacement was observed for 304 steel compared to borg-K122SS(Fig. 1E). Even at the highest load of 400 mN, total displacement intoborg-K122SS was 20 nm compared to 35 nm for 304 steel(P< 0.0001). This differential hardness was maintained in the samefashion even with a load of 800 mN, the limits for this apparatus(Fig. S3C). No marked difference in displacement was observedbetween aluminumwith applied peptide and untreated aluminum(Fig. 1F), further suggesting that the peptide interacts with speciccomponents in steel and that, in the presence of bound peptide,a harder material is created as a result of changes in the physicaland mechanical properties of the steel when it interacts with theorganic peptide. The changes in thematerials properties of the steelsuggested that there was a chemical interaction of the peptide withthe steel surface and that the peptide was not simply binding to thesurface of the steel. To conrm that the peptide was not simplybound to the surface of the steel we determined whether one coulddisplace a limited amount of bound biotinylated-peptide (the RBDwas synthesized with a N-terminal biotin and tetra-glycine linkerand approximately 50% of maximal peptide loading of the surfaceas determined by probing with a streptavidin-horseradish peroxi-dase probe) with a large excess of unlabeled free peptide. Boundbiotinylated K122-4(128-144)ox could not be displaced from thesteel surface by exogenous peptide (Fig. S4) indicating that thebound peptide is not in dynamic equilibrium.

We hypothesized that the peptide was chemically reacting withthe surface electrons of steel to alter the properties of the steelsurface by undergoing a previously unreported form of chemicalinteraction. Such an interaction that generates a new materialwould result in changes in the electronic state of the surface, withmeasurable changes in the electron orbitals of the metal. To furthercharacterize the chemical properties of borg-K122SS, as well as todetermine whether bonding was occurring and to identify whichelements were involved in the interaction, X-ray photoelectronspectroscopy (XPS) analysis was used to examine the electronicstate of the elements on the surface of borg-K122SS in comparisonto 304 steel. Spectra analysis revealed that the iron and chromium2p 1/2 and 3/2 orbitals (Fig. S5A,B) did not appear to play animportant role in bond formation and electron stabilization as noshifts were observed in the borg-K122SS spectrawhen compared to304 steel. An increase in the peak of the oxygen 1S orbital of borg-K122SS was observed compared to 304 steel (Fig. 2A), with theborg-K122SS O1S peak peaking at 50 counts per second (CPS)compared to 40 CPS for 304 steel, suggesting a role for oxygen inbond formation. No signicant changes were observed in thespectra of the nitrogen 1S orbital and the carbon 1S orbital(Fig. S5C,D). The 304 steel contains negligible amounts of sulfur and

Fig. 4. Evidence that stereospecic interactions/products are differentially produced onA) Tryspin sensitivity biotinylated K122-4(128-144)ox peptide on the surface of 304 sC) EWF of borg-K122Ss, borg-K122SS D-amino (generated by utilizing K122-4(128-144using a synthetic peptide synthesized with all D amino acid residues but in the inverted

and a C-terminal amide). D) EWF analysis of borg-retro-inverso-K122SS and 304 stainlessinverso-K122SS and 304 stainless steel with a load of 200 mN and a load of 400 mN. F) Cordata alone. However, since sulfur must be present and was notidentied anywhere else, this peak strongly suggests involvementof sulfur in this bonding event which has not been previouslydescribed. Electrons can ow through disulde bonds, making itfeasible that the disulde bond could interact with and stabilizesurface electrons by chemically binding with them. Further exam-ination of the XPS spectra revealed the presence of a strong peaknear the location of the iron 3S orbital (Fig. 2C) in borg-K122SS thatwas absent from the spectra of 304 steel. The differences betweenthe electronic states of oxygen, iron, and sulfur on the surface ofborg-K122SS and 304 steel conrm that borg-K122SS is a newmaterial that is chemically different from 304 steel. The XPS spectradata support the involvement of several elements in the formationof borg-K122SS.

To supplement the XPS data, Auger scanning electron micros-copy (SEM) scans of the surface of borg-K122SS were indepen-dently performed to look at the surface distribution of the elementsnitrogen, oxygen, sulfur and carbon by determining the numberand source of origin of backscattered secondary electrons of theappropriate energy levels for the specic elements on the borg-K122SS surface. Carbon, and oxygen were distributed across thesurface in a mostly homogenous pattern, with a few areas of higherelement concentration. Given the high concentration of carbon andoxygen in stainless steel, the lack of any specic distribution patternwas anticipated and no nanostructural pattern of distribution wasobserved for either carbon or oxygen (Fig S6A,B). Nitrogen andsulfur (which are not normally components of steel surfaces andthe presence of these elements is due to presence of the peptide)were clearly seen to be distributed across the surface in discretenanostructures (see arrowheads in Fig. 3A,B) which suggests thatthe peptideesteel interface was in fact imaged. These nano-structures were not discernable in a standard SEM surface scan(Fig. 3C) and one would not anticipate imaging the peptide giventhat this was an uncoated specimen and that the acceleratingpotential used was 10.0 keV (Fig. 3C). The auger-SEM energyspectra of the backscattered electrons indicated the presence of lowamounts of sulfur on the surface (Fig. 3D). These results suggestthat at least one of the unidentied electron orbitals observed inthe XPS spectra of borg-K122SS is likely a far red-shifted-sulfurelectron orbital, perhaps suggesting a conjugated electron. It islikely that electrons are delocalized throughout the disulde loopof the peptide and shared through multiple contact points with thesteel surface. This hypothesis is supported by the observation thattrypsin treatment of borg-K122SS releases the peptide from thesteel surface (the RBD has two lysine residues, see Fig. S1) witha resulting change in the surface EWF (Fig. 4A,B). It is worth notingthat a formed disulde bridge has previously been noted asa requirement for RBD functionality [14]. As protease sensitivity ofbioorganic steel would severely limit potential applications, weexamined the ability of synthetic D-K122-4(128-144)ox (an enan-tiomeric form of the native peptide, see Fig. S1) and retro-inverso

stainless steel dependent upon the stereochemistry of the synthetic peptide utilized.ess steel. B) Trypsin sensitivity of borg-K122SS as determined by measuring the EWF.consisting of all D amino acid residues), and borg-retro-inverso-K122SS (generated byence or what is termed a retro-inverso peptide with a standard acetylated N-terminus

steel with and without trypsin digestion. E) Nanoindentation analysis of borg-retro-rosion rates of borg-retro-inverso-K122SS and 304 stainless steel.

59:1083e96.[2] Yu B, Giltner CL, van Schaik EJ, Bautista DL, Hodges RS, Audette GF, et al.

phage display. Applied Microbiology and Biotechnology 2005;68:505e9.

[14] Wong WY, Campbell AP, McInnes C, Sykes BD, Paranchych W, Irvin RT, et al.Structure-function analysis of the adherence-binding domain on the pilin of

teriaD-K122-4(128-144)ox (a peptide of inverted sequence and syn-thesized with D-amino acid residues which will position the aminoacid side chains in the correct relative positions, see Fig. S1) to alterthe properties of steel as both the all D- and the retro-inversopeptide would be protease resistant. As peptides composed ofD-enantiomeric amino acid residues are protease resistant, theseforms of the peptide sequence would increase the potential utilityof these peptides. The all D- and the retro-inverso forms of the RBDinteracted with the steel surface and physical properties of the steelsurface. Strikingly, the D-K122-4(128-144)ox peptide interactedwith the steel but did not signicantly increase the EWF of the steelwhile the retro-inverso D-K122-4(128-144) signicantly increasedthe EWF of the steel beyond that observed for the native L-form ofthe peptide (Fig. 4C). The retro-inverso D-K122-4(128-144)ox was,as anticipated, resistant to trypsinization (Fig. 4D) and also signif-icantly increased the hardness of the steel (Fig. 4E). As the EWF isa measure of the energy level of the surface electrons and theability of those electrons to react with other materials, a high EWFgenerally reects good resistance to corrosion and the high EWF ofthe borg-retro-inverso-K122SS suggested that its corrosion ratewould be low. We thus tested the corrosion rate of borg-retro-inverso-K122SS. Borg-retro-inverso-K122SS is signicantly morecorrosion resistant than regular stainless steel, having a corrosionrate w50% of normal steel (Fig. 4F). Our results strongly suggestthat the peptideesteel reaction is a stereospecic process thatresults in different forms of bioorganic steel that can be differen-tiated based on their biophysical properties. Themolecular basis forthe observed stereospecicity in the products is unclear at thecurrent time.

The high strength of the RBDestainless steel interaction, whichanchors the pilus to the surface and enables the progressivemovement of the cell along that surface when the pilus retracts,appears reasonable given the formation of a chemical interactionwith the surface. The extremely high apparent afnity of the RBDfor the steel surface is also reasonable given that a bond is formed.The observation that the strength of the RBDesteel interactionvaries as a function of the surface electron activity of stainless steel[13] suggests that the bond formed with the RBD requires surfaceelectrons that can be delocalized through the peptide. The PA RBDhas been demonstrated to interact with a wide range of surfacesandmetals including titanium (aluminum being an interesting casewhere the RBD does not interact with the metal) suggesting thatthe nature of the surface electrons may be more important forbinding than the elemental or material composition.

4. Conclusions

In summary, we have characterized a new material, which wehave termed borg-K122SS, which is readily and spontaneouslyformed from stainless steel with exposure to a synthetic peptidethat is the functional adhesin of the T4P. Borg-K122SS has signi-cantly different physical and chemical properties compared withregular 304 stainless steel. Borg-K122SS had a higher EWF and wasless adhesive than unmodied 304 steel. Borg-K122SS surfaceswere signicantly harder and corroded signicantly more slowlythan the parental 304 steel. XPS spectra data of borg-K122SSsuggests that multiple contact points exists between the peptideand the steel, with two currently unassigned electron orbitals (onelikely due to a far red-shifted-sulfur electron binding energy) whichsupports the formation of a chemical interaction, possibly withextensive electron de-localization, that results in chemical andphysical stabilization of the surface.

The observation that the RBD bonds with stainless steel to forma previously unreportedmaterial that has altered physical/chemical

E.M. Davis et al. / Bioma5318attributes has signicant implications for both the facile design,Pseudomonas aeruginosa strains PAK and KB7. Biochemistry 1995;34:12963e72.

[15] Wong WY, Irvin RT, Paranchych W, Hodges RS. Antigeneantibody interac-tions: elucidation of the epitope and strain-specicity of a monoclonal anti-[5] Khoo X, OToole GA, Nair SA, Snyder BD, Kenan DJ, Grinstaff MW. Staphylococcusaureus resistance on titanium coated with multivalent PEGylated-peptides.Biomaterials 2010;31:9285e92.

[6] Schweizer HP, Po C. Regulation of glycerol metabolism in Pseudomonas aer-uginosa: characterization of the glpR repressor gene. Journal of Bacteriology1996;178:5215e21.

[7] Tao S, Li DY. Nanocrystallization effect on the surface electron work functionof copper and its corrosion behaviour. Philosophical Magazine Letters 2008;88:505e9.

[8] Song S. In situ high resolution eletronc microscopy of grain-boundarymigration through ledge motion in an AleMg alloy. Philosophical MagazineLetters 1990;79:3125e7.

[9] Artemyev AV, Fionova LK. Grain-boundary morphology change in aluminumduring heating. Fizika Metallov I Metallovedenie 1988;66:132e6.

[10] Stanley PM. Factors affecting the irreversible attachment of Pseudomonasaeruginosa to stainless steel. Canadian Journal of Microbiology 1983;29:1493e9.

[11] Li X, Logan BE. Analysis of bacterial adhesion using a gradient force analysismethod and colloid probe atomic force microscopy. Langmuir 2004;20:8817e22.

[12] Pereni CI, Zhao Q, Liu Y, Abel E. Surface free energy effect on bacterialretention. Colloids and Surfaces B: Biointerfaces 2006;48:143e7.

[13] Yu B, Lesiuk A, Davis EM, Irvin RT, Li DY. Surface nanocrystallization forbacterial control. Langmuir 2010;26:10930e4.A novel biometallic interface: high afnity tip-associated binding by pilin-derived protein nanotubes. Journal of Bionanoscience 2007;1:73e83.

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[4] Zuo R, Ornek D, Wood TK. Aluminum- and mild steel-binding peptides frommodication and engineering of a common, widely used industrialmaterial based on a natural process and our understanding oftwitching motility and T4P structure and function.

Acknowledgments

The authors gratefully acknowledge the nancial support of theNatural Sciences and Engineering Research Council of Canadathrough Discovery operating grants to DYL and RTI, and through anAlexander Graham Bell Canada Graduate Scholarship to EMD. EMDwas also the recipient of a University of Alberta PhD scholarship.The technical assistance and helpful suggestions of Bart Hazes,particularly in the generation the enantiomeric structure of thetruncated PilA pilin protein and receptor binding domain is grate-fully acknowledged. We gratefully acknowledge the technicalassistance of D. Karpuzov, A. He, and S. Xu in relation to the XPS andSEM Auger analysis. We thank B. Yu, R. Chung, and X. Tang fortechnical assistance and helpful suggestions. The authors disclosethat a patent application based on this technology has been ledand assigned to Arch Biopartners Inc., and that the authors and theUniversity of Alberta hold an equity position in Arch BiopartnersInc.

Appendix. Supplementary material

Supplementary data related to this article can be found online atdoi:10.1016/j.biomaterials.2011.04.027.

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A peptide stainless steel reaction that yields a new bioorganic metal state of matter1 Introduction2 Materials and methods3 Results and discussion4 Conclusions Acknowledgments Appendix. Supplementary material References