Journal of Colloid and Interface Science 349 (2010) 1318

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Journal of Colloid and Interface Science

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Bioengineering of stainless steel surface by covalent immobilization of enzymes. Physical characterization and interfacial enzymatic activity
Anne Caro a, Vincent Humblot a, Christophe Mthivier a, Michel Minier b, Lucica Barbes b, Joachim Li b, Michle Salmain b,*, Claire-Marie Pradier a
a b

Universit Pierre et Marie Curie, Laboratoire de Ractivit de Surface (UMR CNRS 7197), 4 place Jussieu, case 178, 75252 Paris Cedex 05, France Chimie Paristech (Ecole Nationale Suprieure de Chimie de Paris), Laboratoire Charles Friedel, CNRS UMR 7223, 11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France

a r t i c l e

i n f o

a b s t r a c t
Two hydrolytic enzymes, namely lysozyme and trypsin, were covalently immobilized onto stainless steel surfaces using wet chemistry processes. The immobilization strategy took advantage of the spontaneous physisorption of the polymer poly(ethylene imine) (PEI) onto stainless steel to yield a rmly attached, thin organic layer containing a high density of primary amine functions. Both enzymes were then covalently grafted to the surface via a glutaraldehyde cross-linker. Alternatively, a thicker underlayer of PEI was chemisorbed by cross-linking two PEI layers by glutaraldehyde. The effective presence of both enzymes on the stainless steel surfaces and their relative amount were assessed by immunochemical assays employing specic anti-enzyme antibodies. Eventually, the hydrolytic activity of the immobilized enzymes was evaluated by local enzymatic tests with suitable substrates. This work demonstrates that, although the amount of enzymes did not vary signicantly with the underlayer thickness, their hydrolytic activity could be much improved by increasing the distance from the oxide surface and, likely, by favoring their accessibility. Our data suggest that the immobilization of enzymes on solid oxide surfaces is feasible and efcient, and that the enzymes retain catalytic activity. It may thus provide a promising route towards biolm-resistant materials. 2009 Published by Elsevier Inc.

Article history: Received 10 September 2009 Accepted 1 December 2009 Available online 4 December 2009 Keywords: Stainless steel Poly(ethylene imine) Lysozyme Trypsin Anti-biolm surfaces

1. Introduction All surfaces in contact with a liquid medium are likely to get covered with a biolm, resulting from the adhesion of microorganisms [1]. Most of the time, this phenomenon is detrimental for example colonization of marine structures, clogging of water pipes or contamination of food-processing equipments. In the marine environment, biofouling can induce many damages, such as corrosion of structures or modication of ow dynamics. These damages lead in turn to substantial economical issues. Paints containing metal compounds, like tributyl tin or copper complexes have been used up to now to protect marine structures against biofouling [2]. However, because these highly toxic and harmful species are eventually released in the environment, they are now subject to international rules, aiming at prohibiting their use in 2008 in Europe [3]. There is thus an urgent need to propose environmentally friendly alternatives to these additives [4]. Hydrolytic enzymes (esterases, proteases and glycosidases) from various sources have been investigated as ecologically compatible antifouling agents. When included (formulated) within
* Corresponding author. Fax: +33 1 43 25 79 75. E-mail address: michele-salmain@chimie-paristech.fr (M. Salmain). 0021-9797/$ - see front matter 2009 Published by Elsevier Inc. doi:10.1016/j.jcis.2009.12.001

appropriate paints, they are slowly released in the surrounding medium where they can exert their biocide properties [58]. An alternative to this strategy would be to permanently attach the antifouling agents to the material surface so that their anti-adhesion/anti-bacterial activity is exerted at the solidliquid interface [912]. Several pathways can be envisaged to immobilize enzymes onto the widely used stainless steel material. The most straightforward method that would consist in their direct physisorption was immediately set aside as this may induce protein conformation changes [13], possibly resulting in loss of catalytic activity. Another pathway, using genetically engineered enzymes including a biomimetic short aminoacid sequence binding selectively to inorganic materials [14,15], may also be employed but require to produce high quantities of recombinant proteins, which may be time-consuming. A more accessible method would be to introduce reactive organic functions onto the surface of stainless steel from which the enzymes could be readily linked by covalent means. The surface of stainless steel being mostly composed of chromium oxide/ hydroxide (see the results part) [16], we and others employed the trialkoxysilane APTES to functionalize the surface with amine groups [1719], by analogy with the classical surface chemistry on silica [20]. Unfortunately, the chemical bonds created by

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reaction of APTES and stainless steel were found rather labile when exposed to aqueous medium as deduced from surface infrared spectroscopic measurements [21]. A more efcient approach to introduce amine groups onto stainless steel takes advantage of the spontaneous physisorption of branched poly(ethylene imine) (PEI) to yield a thin organic layer including a high density of primary amine groups [22,23]. This approach recently allowed us to covalently immobilize the glycosidase hen egg white lysozyme (HEWL) to stainless steel surfaces via cross-linking with glutaraldehyde (GA) as depicted in Fig. 1. The resulting modied surfaces displayed anti-adhesion properties toward Listeria ivanovii and anti-bacterial properties toward Micrococcus luteus [24]. In this paper, we describe the immobilization onto stainless steel of another hydrolase, namely trypsin. This proteolytic enzyme could be useful to hydrolyse proteins involved in the adhesion of other micro-organisms. Moreover, when incorporated with water-based paints, some proteases were able to inhibit attachment of barnacle and bryozoan larves onto plastic panels, thus presenting potentially interesting antifouling properties in the marine environment [5,1012]. The surface of stainless steel samples coated with trypsin or lysozyme was characterized by X-ray photoelectron spectroscopy (XPS) to gain access to the elemental composition of the solid interface after each step of functionalization. The presence of trypsin and lysozyme at the surface was semi-quantitatively assessed by immunochemical tests employing antibodies against both enzymes. Eventually, the enzymatic activity of lysozyme- and trypsin-coated substrates was measured with appropriate substrates under standard assay conditions. Both enzymes were shown to retain satisfactory catalytic activity upon binding to stainless steel, thus conrming the pertinence of the immobilization method. The main input of this study is the importance of the thickness of the binding layer to maintain good enzymatic activity.

0.21 g NaHCO3 in 100 ml H2O and adjusting the pH to 10 with 1 M NaOH. Phosphate buffered saline PBS (pH 7.4) was prepared by dissolving 0.34 g KH2PO4, 1.42 g Na2HPO4, 0.2 g KCl and 8 g NaCl in 1 l H2O. Sulfochromic acid was prepared by dissolving 6 g of K2CrO4 in 100 ml H2SO4 96%. Caution: Manipulation of sulfochromic acid must be performed in a fume hood with appropriate protection. AISI 316L stainless steel coupons (10 10 2 mm) mechanically polished to 0.5 lm were purchased from Goodfellow (Lille, France). 2.2. Experimental procedures 2.2.1. Cleaning and conditioning of stainless steel substrates Coupons were cleaned and pre-conditioned according to literature methods [16,25]. Briey, substrates were successively polished onto 0.25 lm (abrasive/polishing disk and diamond suspension), rinsed with ethanol, then ultrasonically washed 15 min in cyclohexane (three times), 10 min in water then 20 min in acetone. They were etched by sulfochromic acid at 60 C for 10 min to generate a reactive oxide/hydroxide layer. Caution: This step must be performed under appropriate ventilating conditions wearing gloves and goggles. They were extensively washed with water and dried under a ow of N2. At this stage the coupons were named SS-SC. 2.2.2. Physisorption of PEI Substrates were covered with 1.5 ml of a 3% PEI solution in water (w/v) and gently stirred for 2 h on a rocking table. They were washed with water to yield SS-SC-PEI coupons. 2.2.3. Grafting of enzymes The coupons were covered with 1.5 ml of a solution of GA obtained by diluting one part of the stock solution with four parts of ethanol. After 2 h, they were rinsed with ethanol to yield SSSC-PEI-GA. Some of these coupons were again submitted to the PEIGA sequence to yield SS-SC-(PEI-GA)2. The GA activated coupons placed in individual glass reactors were covered with 1.5 ml of a 1 mg/ml enzyme solution in carbonate buffer and left overnight at 4 C under gently shaking. For some samples, solid NaCNBH3 was added to the enzyme solution at a nal concentration of 0.1 M. Final washings consisted in three bathes of PBS for 40 min. Enzymatic assays were performed on the washing solutions to ensure that complete desorption of loosely bound enzymes occurred. The nal samples are named SS-SC-PEI-GA-ENZ, SS-SCPEI-GA-ENZ-red, SS-SC-(PEI-GA)2-ENZ and SS-SC-(PEI-GA)2-ENZred where ENZ is HEWL or TRYP and red means imine reduction step by treatment with NaCNBH3. 2.2.4. Immunochemical detection of immobilized enzymes Substrates coated with HEWL and placed in a 24-well plate (IWAKI) were covered with 0.5 ml of a 2 lg/ml solution of antilysozyme antibody in incubation buffer (PBS containing 0.1% Tween 20 v/v and 1% BSA w/v) for 2 h at room temperature. After extensive rinsing with washing buffer (PBS containing 2 M NaCl and 0.05% Tween 20 v/v), coupons were covered with 500 ll of a solution of anti-rabbit IgG peroxidase conjugate (1:8000) in incubation buffer. After extensive rinsing with washing buffer, coupons were covered with 0.5 ml of a 0.7 g/l solution of OPD and 0.04% H2O2 in citratephosphate buffer pH 5.0. After 10 min, the enzymatic reaction was stopped by addition of 0.3 ml of 2.5 M H2SO4. After another 5 min in the dark, 0.15 ml of each solution was transferred into a 96-well plate and the absorbance at 490 nm measured with a microtitre plate reader (Biorad Model 550). The same procedure was applied for the stainless steel coupons covered with trypsin using anti-trypsin antibody at 1 lg/ml.

2. Materials and methods 2.1. Materials Poly(ethylene imine) PEI (branched, average MW = 25,000), glutaraldehyde GA (25% solution in water, w/v) and sodium cyanoborohydride were purchased from Aldrich (Saint Quentin Fallavier, France). Hen egg white lysozyme HEWL (EC 3.2.1.17, 3 times crystallized, 23,000 units/mg protein), bovine pancreas trypsin TRYP (EC 3.4.21.4, TPCK-treated, 12,400 units/mg protein), Nabenzoyl L-arginine ethyl ester BAEE, goat anti-rabbit IgG peroxidase conjugate, lyophilized Micrococcus lysodeikticus, o-phenylene diamine OPD and 30% H2O2 were purchased from Sigma (Saint Quentin Fallavier, France). Polyclonal rabbit anti-lysozyme and anti-trypsin antibodies were purchased from Chemicon (Millipore, Molsheim, France). Carbonate buffer was prepared by dissolving

Fig. 1. Schematic representation of functionalization steps of stainless steel surface by HEWL or TRYP.

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2.2.5. Enzymatic activity of lysozyme coated substrates The enzymatic activity of the stainless steel coupons coated by HEWL was measured on M. lysodeikticus using a procedure adapted from the classical lysozyme assay [26]. Substrates coated by HEWL were placed in separate glass reactors and covered with 3 ml of a 0.015% (w/v) suspension of M. lysodeikticus reconstituted from lyophilized bacteria in 66 mM phosphate buffer pH 6.24. The reactors were placed on a rocking table. Monitoring of enzymatic activity was carried out over a period of 250 min in the following manner. One ml of each suspension was withdrawn every 15 min and the turbidity measured at 450 nm after which the suspension was put back into the reactor. Two control experiments were carried out to measure non enzymatic bacterial lysis (= autolysis), whereby the turbidity of a stirred bacterial suspension alone or in the presence of an untreated SS-SC coupon was monitored. Plots of optical density versus time were generated and slopes measured. By denition, one unit produces a DA450nm of 0.001 per min at pH 6.24 and 25 C using a 0.015% (w/v) suspension of M. lysodeikticus in a 2.6 ml reaction mixture. The calculated enzymatic activity of the coupons was corrected by a volume correction factor fv of 3 ml/2.6 ml = 1.15 and a substrate concentration correction factor fc of 1.25. This latter term comes from the nonlinear dependence of A450nm versus M. lysodeikticus concentration and was measured independently [16]. 2.2.6. Enzymatic activity of trypsin-coated substrates The enzymatic activity of the stainless steel coupons coated with trypsin was measured by monitoring the hydrolysis of BAEE by UV spectrometry [27]. Coupons coated by trypsin were placed in separate glass reactors and covered with 3 ml of 0.25 mM BAEE in 67 mM phosphate buffer pH 7.6. The reactors were placed on a rocking table and the suspensions gently stirred. Monitoring of enzymatic activity was carried out over a period of 250350 min in the following manner. One ml of each suspension was withdrawn every 15 min and the absorbance of the solutions at 253 nm was measured after which the solutions were put back into the reactors. Two control experiments were carried out to measure non enzymatic hydrolysis whereby the absorbance of a stirred BAEE solution alone or in the presence of an untreated SS-SC coupon was monitored. Plots of optical density versus time were generated and slopes measured. By denition, one unit produces a DA253nm of 0.001 per min at pH 7.6 and 25 C using a 0.25 mM solution of BAEE in a 3.2 ml reaction mixture. The calculated enzymatic activity of the coupons was corrected by a volume correction factor fv of 3 ml/3.2 ml = 0.94. 2.2.7. XPS analysis XPS analyses were performed on a PHOIBOS 100 X-ray photoelectron spectrometer (SPECS) with a Mg Ka X-ray source (hm = 1253.6 eV) at 1010 Torr pressure. Spectra were carried out with a 10 eV pass, a 7 2 mm spot size and 150 W electron beam power. A take-off angle of 90 with respect to the surface was employed. Because some samples did not show the current CC/CH C 1s peak, the binding energies were calibrated against the Cr 2p3/2 peak of Cr2O3 set to 576.2 eV. Elemental peak intensities were corrected by Scoeld factors [28]. The high resolution C 1s, N 1s, and Cr 2p spectra were tted using the Casa XPS software and applying a GaussianLorentzian function 70/30. 3. Results The surface analysis data presented here have been collected after each step of functionalization, until the chemical grafting of HEWL or TRYP; XPS data are useful to check the binding of the enzymes and compare their relative amounts on the surface. Results

of the immunochemical reactions and enzymatic assays are presented after these surface characterizations. 3.1. XPS analysis of surfaces The survey spectrum of the stainless steel sample showed intense iron, chromium and oxygen peaks (not shown) and their high resolution spectra suggested that the surface was mostly covered by metallic chromium, chromium oxides and hydroxides, with the Cr 2p3/2 peak decomposed into three contributions at 574.2, 576.1 and 577.6 0.2 eV, respectively (spectra not shown). Treatment of the stainless steel samples by sulfochromic acid resulted in an increase of the chromium hydroxide contribution together with a slight decrease of the oxide one [16,25]. The O 1s spectrum could be tted with three components at 530.1, 531.6 and 532.7 0.2 eV, assigned to oxygen in metal oxides, in metal hydroxides and in organic oxygen and water, respectively (spectra not shown). Furthermore this treatment enabled to strongly reduce carbon contamination (the C 1s peak area decreased by more than 60%). Thereby, the sulfochromic acid treatment led both to a segregation of reactive chromium oxides and hydroxides at the surface layer, as well as a reduction of surface impurities. Note eventually that a weak peak of sulfur was sometimes detected, owing to sulfuric acid contained in the sulfochromic acid mixture. PEI-treated surfaces (SS-SC-PEI) displayed marked changes in the chromium, oxygen, carbon and nitrogen composition compared to the SS-SC sample. The C 1s and N 1s peaks, as well as their decomposition, are shown in Fig. 2, together with those recorded after surface exposure to GA and, HEWL or TRYP. Physisorption of PEI resulted in a strong attenuation of the Cr 2p peak while the carbon and nitrogen peaks increased and changed in shape (see lower spectra of Fig. 2). The C 1s spectrum was tted with a major component, at 286.3 eV, assigned to carbon bound to nitrogen (CN), and a very weak one at 288.2 eV, attributed to residual contamination. The absence of any CC/CH components is in agreement with the PEI atomic composition. Accordingly, an intense N 1s peak now appeared on the survey spectrum. The high resolution N 1s spectrum was decomposed into two contributions, at 400.0 and 402.1 0.2 eV characteristic of non-protonated nitrogen in NH2 (89%), and protonated nitrogen in NH amino groups 3 (11%), respectively. The N/C ratio was equal to 0.5, i.e. in line with the theoretical N/C ratio for the pure PEI polymer. After treatment of the metallic substrates with the ethanolic glutaraldehyde solution, the N 1s peak area, and in particular that of the NH2 contribution was shown to decrease, while that at

Fig. 2. High resolution C 1s, and N 1s spectra of the stainless steel surfaces after the successive functionalization steps, PEI, PEI-GA, PEI-GA + TRYP and PEI-GA + HEWL.

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higher binding energy slightly increased. The clearest evidence of GA grafting was the change in the C 1s peak prole; the latter was now dominated by a new contribution at 285 eV, attributed to the CC/CH bonds of the GA molecules. The carbon involved in the C@N imine bond is expected to give a contribution at ca. 287 eV, which is too small and too close to appear separately from the intense peak centered at 286.3 eV. A new contribution at 287.8 eV was assigned to the carbon in the terminal aldehyde group of GA. The N/C ratio decreased to 0.3 as a result of GA binding and N 1s photoelectrons screening by the upper organic layer. Prior to enzyme grafting, some surfaces were submitted twice to the PEIGA treatment in order to search for a possible optimization of the enzyme activity. This induced no change in the respective C 1s and O 1s peak proles (spectra not shown) but a marked decrease of the Cr 2p peak intensity (13,800 vs. 8000 cts for the PEIGA and (PEIGA)2 surfaces, respectively), conrming that a thicker polymeric layer had been formed. After treatment of the GA surfaces with HEWL or TRYP, a further decrease of the Cr 2p contributions and a change in the C 1s and N 1s peak proles (see the two upper spectra of Fig. 2). The high resolution C 1s spectrum was tted with 3 contributions at 285, 286.3 and 288.1 0.2 eV. The latter, assigned to C(@O)O and C(@O)N groups, is characteristic of amide and carboxylic acid bonds present in proteins. The N 1s spectra were now dominated by the NH2 contributions of the enzymes at 400.0 eV; in addition, an intense and new peak showed up at 400.9 0.2 eV, that can be attributed to nitrogen in the amide bonds of the proteins. An increase of the N/C to 0.37 originating both by an increase of C and N brought by the proteins was observed at this stage.

binding in the presence of reductant. The addition of NaCNBH3 at the HEWL chemisorption step resulted in an amount of enzyme bound the surface higher by 30% (compare sample D to sample F and E to G in Fig. 3). Not surprisingly, a thicker organic adlayer prepared with two alternating treatments with PEI and GA allowed the same amount of lysozyme to be grafted to the surface. Finally, the reproducibility of the immobilization procedure was studied on 6 SS-SC-PEI-GA-HEWL samples. This test yielded a coefcient of variation of 10% for the amount of HEWL on the samples. The sample type of experiment was repeated for the substrates covered with trypsin (Fig. 4, bar chart). This time, very weak amounts of trypsin were immobilized by direct adsorption to the SS-SC and SS-SC-PEI samples. The covalent immobilization procedures allowed comparable amounts of enzyme to be bound to the surface and addition of reductant did not increase the amount of surface-bound trypsin.

3.3. Enzymatic assays The enzymatic activity of lysozyme is typically measured spectrophotometrically by monitoring the decrease of turbidity of a cell suspension of Micrococcus lysodeikticus [26]. The enzymatic activity of the stainless steel samples was measured during 250 min using a related procedure whereby the coupons were dipped into a suspension of bacteria. This suspension was retrieved from time to time and its turbidity measured at 450 nm. To correct from nonspecic effects, the turbidity of a suspension alone or in the presence of a bare stainless steel sample was monitored in parallel. The enzymatic activity expressed in units is reported as a point graph in Fig. 3. Weak activities were measured for samples where lysozyme had been adsorbed noncovalently. Conversely, signicant activities were measured for the samples where lysozyme had been chemisorbed via the PEI + GA procedure. The reproducibility of the immobilization procedure was studied on three SSSC-PEI-GA-HEWL samples. This test yielded a coefcient of variation of 25% for the enzymatic activity. The measurements highlight the benet of the reductant addition during the enzyme immobilization step (activity multiplied by 4 or 2 for the single and double layers, respectively). They also highlight the benet of building a thicker adlayer with the two alternating treatments with PEI and GA (activity multiplied by 2 for both the unreduced and reduced samples).

3.2. Immunochemical detection of enzymes The actual presence of lysozyme on several stainless steel samples and its relative amount were assessed by an immunochemical assay using a rabbit anti-lysozyme antibody, followed by a revelation step with anti-rabbit IgG peroxidase conjugate. Detection of surface-bound lysozyme was eventually achieved by addition of the chromogenic substrate OPD and H2O2. In the presence of peroxidase and hydrogen peroxide, OPD is converted into a yellow-orange dye that can be conveniently measured colorimetrically (kmax = 492 nm). Results are displayed as a bar chart in Fig. 3. The immobilization methods could be ranked in the following order: physisorption/chemisorption < covalent binding < covalent

Fig. 3. Immunochemical detection of lysozyme coated on stainless steel coupons (bar chart). Samples were successively exposed to anti-lysozyme antibody and antirabbit IgG HRP conjugate. Enzymatic activity of lysozyme coated stainless steel coupons (curve). Each point represents the means standard deviation on two samples. A: SS-SC; B: SS-SC-HEWL; C: SS-SC-PEI-HEWL; D: SS-SC-PEI-GA-HEWL; E: SS-SC-(PEI-GA)2-HEWL; F: SS-SC-PEI-GA-HEWL-red; G: SS-SC-(PEI-GA)2-HEWL-red. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

Fig. 4. Immunochemical detection of trypsin-coated on stainless steel coupons (bar chart). Samples were successively exposed to anti-trypsin antibody and anti-rabbit IgG HRP conjugate. Enzymatic activity of trypsin-coated stainless steel coupons (curve). Each point represents the means standard deviation of two samples. A: SS-SC; B: SS-SC-TRYP; C: SS-SC-PEI-TRYP; D: SS-SC-PEI-GA-TRYP; E: SS-SC-(PEIGA)2-TRYP; F: SS-SC-PEI-GA-TRYP-red; G: SS-SC-(PEI-GA)2-TRYP-red. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

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The esterase activity of trypsin was used to evaluate the catalytic activity of the trypsin-coated stainless steel substrates. This activity was measured by a spectrophotometric monitoring of the hydrolysis of the substrate BAEE at 253 nm (Fig. 4, curve). Samples resulting from the direct physisorption of trypsin displayed no signicant enzymatic activity. Conversely, all the stainless steel samples resulting from the covalent binding procedures displayed a signicant esterolytic activity. The reproducibility of the immobilization procedure was studied on 3 SS-SC-PEI-GA-TRYP samples. This test yielded a coefcient of variation of 30% for the enzymatic activity. Compared to the SS-SC-PEI-GA-TRYP sample, the addition of reductant resulted in a twice higher enzymatic activity. Building of the thicker adlayer also resulted in a twice higher enzymatic activity. Similarly to what had been observed for lysozyme, the sample with the highest enzymatic activity was obtained by combining a thick organic adlayer and reduction of imine bonds with NaCNBH3. To assess whether BAEE hydrolysis originated from immobilized or released enzyme, a simple test was performed where spectrophotometric monitoring was carried on after the coupons were removed from the substrate solution (Fig. 5). The absorbance of the solutions at 253 nm that linearly increased with time when the stainless steel coupons were in contact them was shown to level off as soon as they were removed. This clearly indicated that the measured enzymatic activity was exclusively related to the immobilized enzyme. 4. Discussion The purpose of our work was to prepare and characterize functional stainless steel surfaces covered with a thin lm of two strongly attached hydrolytic enzymes, namely hen egg white lysozyme and bovine trypsin. Lysozyme is a glycosidase that catalyzes the hydrolysis of the b(1?4) linkage between N-acetylglucosamine and N-acetylmuramic acid. As these two sugars constitute the building blocks of the polysaccharidic component of certain bacterial cell walls, this hydrolysis causes cell lysis, hence the origin of its anti-bacterial activity. Trypsin is a serine endoproteinase that selectively cleaves peptide bonds at the carboxyl side of basic aminoacids. Both enzymes may actively protect surfaces against biolm formation by degrading the molecules biofouling species use to colonize surfaces [7]. XPS analysis rst conrmed the presence of PEI on the surfaces, with both NH2 and NH functionalities; let us remember that PEI is 3

Fig. 5. Hydrolysis of BAEE catalyzed by stainless steel samples coated with trypsin monitored by spectrophotometry at 253 nm. Arrows indicate time at which samples were removed from substrate solutions.

a weak polybase whose degree of protonation depends upon the pH and the ionic strength [29]. As the pH of the 3% solution of PEI is 11.2 [22], the polymer is expected to be weakly charged. So the presence of cationic nitrogen atoms on the surface after adsorption might give an evidence that adsorption of PEI occured via electrostatic interactions as on mica [30]. Then, upon grafting of glutaraldehyde, the change in the N 1s peak may be explained by rst, the covalent binding of GA chains onto the PEI layer, that screened part of N 1s photoelectrons from the NH2 groups, and second, by the detection of nitrogen from the new formed imine bonds [31]. A further change observed in the C 1s and N 1s peaks after treatment with the protein solutions led us to conclude that protein molecules were indeed bound to the surface. At this point, one may rst wonder whether the surface amounts of HEWL and TRYP were similar for the SS-SC-PEI-GA-enzyme samples. The area of the C 1s288eV peak contribution (corresponding to the carbons in the C(@O)N/C(@O)O bonds) ratioed against the total C 1s area was considered as a good indication of the amount of enzyme on the surfaces; it was equal to 15% and 13% of the total C 1s peak area after binding of HEWL and TRYP, respectively. When considering the sequence of both enzymes taken from the Expasy database, this percentage should be equal to 25% and 28% for HEWL and TRYP, respectively. These discrepancies tend to suggest that C atoms belonging to the lower layer(s) participate to the total C 1s signal so that the thickness of the enzyme layers is not sufcient to screen them. The fact that the actual percentage is closer to the theoretical percentage in the case of HEWL might indicate that the density of HEWL molecules at the surface of the sample is larger than that of TRYP. This is conrmed by the higher intensity of the N 1s contribution at ca 400.9 eV on the HEWL layer (see Fig. 2). Second, one may wonder whether the immobilization procedure had any inuence on the quantity and activity of the immobilized enzymes. Samples for which lysozyme or trypsin was grafted via glutaraldehyde cross-linking gave higher responses (in terms of OD492) than samples for which the enzymes were simply chemisorbed to the PEI or directly physisorbed to the stainless steel surface. This indicated that the covalent binding strategy was a more efcient method than simple physisorption to immobilize the enzymes to stainless steel. The benecial effect of the reduction step on the attachment of lysozyme may be possibly explained by the stronger bonds created between the protein and the organic adlayer. Surprisingly, a detectable amount of lysozyme was adsorbed to bare stainless steel and to stainless steel covered with a lm of PEI; this latter nding was unexpected as lysozyme is positively charged at the pH of the immobilization step that is lower than its isoelectric point (pI = 11). A similar nding was reported for sulfonated membrane coated with PEI [32]. Lysozyme was also found to adsorb to titanium alloy coated with a thin lm of poly(allylamine) obtained by plasma polymerization [33]. However, although simple physisorption of either lysozyme or trypsin did lead to a number of strongly bound (resisting to severe rinsing procedures) molecules, it resulted into surfaces having no or very little catalytic activity towards the selected substrates. The construction of an attachment layer, both to strongly bind the proteins and to anchor them away from the surface, was thus compulsory to get an active surface. Note however that the fractions of carbon involved in C@O bonds with respect the total carbon content measured at the surface by XPS (see Fig. 2) were far below those calculated from the proteins sequence, suggesting that the surface was not fully covered with enzymes. Catalytic activity measurements revealed the importance of both the reduction step that likely strengthens the binding of enzymes to the surface and the thickness of the binding layer formed here by PEI in combination with GA. Although the amount of each protein was comparable whatever the covalent immobilization procedure used, the catalytic activity was always

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A. Caro et al. / Journal of Colloid and Interface Science 349 (2010) 1318 [7] S.M. Olsen, L.T. Pedersen, M.H. Laursen, S. Kiil, K. Dam-Johansen, Biofouling 23 (2007) 369383. [8] M.E. Pettitt, S.L. Henry, M.E. Callow, J.A. Callow, A.S. Clare, Biofouling 20 (2004) 299311. [9] C.K. Bower, M.H. Daeschel, J. McGuire, J. Dairy Sci. 81 (1998) 27712778. [10] Y. Duk Kim, J.S. Dordick, D.S. Clark, Biotechnol. Bioeng. 72 (2001) 475482. [11] M. Tasso, M.E. Pettitt, A.L. Cordeiro, M.E. Callow, J.A. Callow, C. Werner, Biofouling 25 (2009) 505516. [12] M. Tasso, A.L. Cordeiro, K. Salchert, C. Werner, Macromol. Biosci. 9 (2009) 922 929. [13] K. Nakanishi, T. Sakiyama, K. Imamura, J. Biosci. Bioeng. 91 (2001) 233244. [14] A.R. Statz, R.J. Meagher, A.E. Barron, P.B. Messersmith, J. Am. Chem. Soc. 127 (2005) 79727973. [15] C. Tamerler, S. Dincer, D. Heidel, M. Hadi Zareie, M. Sarikaya, Prog. Org. Coat. 47 (2003) 267274. [16] M. Minier, M. Salmain, N. Yacoubi, L. Barbes, C. Mthivier, S. Zanna, C.-M. Pradier, Langmuir 21 (2005) 59575965. [17] T.-W. Chuang, M.H. Chen, F.H. Lin, J. Biomed. Mater. Res. A 85 (2008) 722730. [18] H.P. Jennissen, T. Zumbrink, M. Chatzinikolaidou, J. Steppuhn, Materialwiss. Werkstofftech. 30 (1999) 838845. [19] T. Yoshioka, K. Tsuru, S. Hayakawa, A. Osaka, Biomaterials 24 (2003) 2889 2894. [20] H.H. Weetall, Appl. Biochem. Biotechnol. 41 (1993) 157188. [21] A. Caro. Fonctionnalisation de surfaces dacier inoxydable an dinhiber les premires tapes de formation dun biolm. Universit Pierre et Marie Curie, Paris, 2008. [22] P. Kingshott, J. Wei, D. Bagge Ravn, N. Gadegaard, L. Gram, Langmuir 19 (2003) 69126921. [23] J. Wei, D. Bagge Ravn, L. Gram, P. Kingshott, Colloids Surf. B 32 (2003) 275291. [24] A. Caro, V. Humblot, M. Minier, M. Salmain, C.-M. Pradier, J. Phys. Chem. B 113 (2009) 21012109. [25] J.M. Chovelon, L. El Aarch, M. Charbonnier, M. Romand, J. Adhes. 50 (1995) 43 58. [26] D. Shugar, Biochim. Biophys. Acta 8 (1952) 302308. [27] H.U. Bergmeyer, K. Gawehn, M. Grassl, in: H.U. Bergmeyer (Ed.), Methods in Enzymatic Analysis, second ed., vol. I, Academic Press Inc., New York, 1974, pp. 515516. [28] J.H. Scoeld, J. Electron, Spectrosc. Relat. Phenom. 8 (1976) 129137. [29] G.M. Lindquist, R.A. Stratton, J. Colloid Interface Sci. 55 (1976) 4559. [30] P.M. Claesson, O.E.H. Paulson, E. Blomberg, N.L. Burns, Colloids Surf. A 123124 (1997) 341353. [31] C. Jones, E. Sammann, Carbon 28 (1990) 509514. [32] J. Etheve, P. Dejardin, M. Boissire, Colloids Surf. B 28 (2003) 285293. [33] D.A. Puleo, R.A. Kissling, M.-S. Sheu, Biomaterials 23 (2002) 20792087.

improved when the reduction step was carried out and when a thicker organic layer was built. The double-layer effect was particularly noticeable in the case of HEWL. This feature may be related to the very large size of the substrate used for the enzymatic tests, namely the bacterium M. lysodeikticus that required a good accessibility of the enzyme to be lyzed. Conversely, the small substrate BAEE (MW = 343) used for assaying the TRYP-coated surfaces may be able to freely diffuse within the organic layer and be hydrolyzed. In conclusion, we showed that the covalent immobilization processes led to rmly bound and active enzyme layers and that their hydrolytic activity originated exclusively from surface-bound molecules. The thickening of the attachment layer contributed signicantly to the increase of surface enzymatic activity by moving away the proteins from the surface. This last point is very important as regards their possible application as antifouling agents. Indeed, we showed that the lysozyme coated surface displayed antiadhesion and anti-bacterial activity against live bacteria [24]. Acknowledgments The Centre National de le Recherche Scientique (CNRS) and the French Ministry of Research are gratefully acknowledged for nancial support. A.C. acknowledges the French Dlgation Gnrale lArmement (DGA) for her PhD grant. References
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