An Original Route to Immobilize an Organic Biocide onto a

release of an antifouling biocide or by the presence of such compounds on the surface. ... immobilization of an organic biocide (chloramine) on the el...
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Langmuir 2007, 23, 3873-3879

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An Original Route to Immobilize an Organic Biocide onto a Transparent Tin Dioxide Electrode Catherine Debiemme-Chouvy,* Sanae Haskouri, Guy Folcher, and Hubert Cachet Laboratoire Interfaces et Syste` mes Electrochimiques, UPR 15 du CNRS, UPMC, Case Courrier 133, 4 place Jussieu, 75252 Paris Cedex, France ReceiVed December 14, 2006. In Final Form: January 18, 2007 Prevention of biofilm growth on surfaces immersed in an aqueous environment could be obtained either by the release of an antifouling biocide or by the presence of such compounds on the surface. In this paper it is shown, for the first time, that an electrochemical treatment performed in the presence of chlorides and proteins allows the immobilization of an organic biocide (chloramine) on the electrode. This electrode is a stable transparent conductive tin dioxide film coated on glass. It is polarized to oxidize chloride ions into hypochlorous acid, which reacts with the organic matter (bovine serum albumin) present at the electrode/solution interface, leading on one hand to the chlorination of the proteins with in particular the chloramine formation and on the other hand to the protein aggregation on the surface.

Introduction The surfaces immersed in aqueous environments gradually become covered by complex layers of biofouling organisms. To prevent biofilm formation, marine structures are coated with paints containing an antifouling compound: organotin (now prohibited in many countries for small boats and aquaculture), copper, zinc, etc.1 In addition, chlorination is the predominant disinfection method applied in water and wastewater treatment. Chloramine is increasingly being considered as an alternative final disinfectant to chlorine in drinking water treatment even if it is generally not as potent as free chlorine against planktonic organisms. In this way, a relatively stable disinfecting residual is provided that can be maintained over a relatively long period of time during and after chlorination.2-4 Finally, numerous biological studies devoted to the potential role on host tissues of excessive or misplaced generation of hypochlorous acid (HOCl) are reported in the literature. Indeed the heme enzyme myeloperoxidase catalyzes the reaction of hydrogen peroxide (H2O2) with chloride ions generating HOCl, which in excess can damage tissues. This process may be implicated in several human diseases.5-7 Chemical modifications in biological tissues can actually be induced by HOCl, which reacts notably with the protein side chains.5,8 From Table 1, it is clear that the most reactive amino acids with HOCl are the sulfur-containing ones (Met, Cys, and also cystine). These reactions give rise to sulfoxide for Met and disulfides and oxy acids for Cys and cystine. Amine functions like those on His and Lys side chains or R-amino groups react rapidly with HOCl. These reactions result primarily in the formation of chloramines * Corresponding author. E-mail: [email protected]. (1) Omae, I. Chem. ReV. 2003, 103, 3431-3448. (2) Goel, S.; Bouwer, E. J. Water Res. 2004, 38, 301-308. (3) Momba, N. B. M.; Cloete, T. E.; Venter, S. N.; Kfir, R. Water Res. 1999, 33, 2937-2940. (4) Donnermair, M. M.; Blatchley, E. R., III. Water Res. 2003, 37, 15571570. (5) Hawkins, C. L.; Pattison, D. I.; Davies, M. J. Amino Acids 2003, 25, 259274. (6) Senthilmohan, R.; Kettle, A. J. Arch. Biochem. Biophys. 2006, 445, 235244. (7) Pattison, D. I.; Davies, M. J. Curr. Med. Chem. 2006, 13, 3271-3290. (8) Pattison, D. I.; Davies, M. J. Chem. Res. Toxicol. 2001, 14, 1453-1464.

(>N-Cl: RNHCl, RNCl2, and RR′NCl) that retain the oxidizing ability of HOCl.9-13 The aim of the present paper is to show that the electrochemical production of HOCl in the presence of protein can yield the immobilization of chloramines on the electrode surface. This will be demonstrated by promoting the oxidation of chloride ions at conductive tin dioxide (SnO2) films in the presence of bovine serum albumin (BSA). This protein contains numerous His and Lys residus. The primary structure of the BSA is given in ref 14, and its partial composition is reported in Table 1. It has been shown that HOCl can be produced at doped SnO2 electrodes, anodically polarized in seawater, either as transparent conductive films15 or as conductive paint containing SnO2 particles16 for the electrochemical inactivation of marine bacteria. SnO2 is a well-suited electrode material for such an application because of its stability and the large overpotential for oxygen evolution.17 In the following, the adsorption/deposition of BSA and its possible chemical modifications are investigated. First, the protein adsorption on SnO2, at open circuit potential, is monitored in situ using a quartz crystal microbalance. The morphology of the resultant surface is characterized by scanning electron microscopy (SEM), and its chemical composition is determined by X-ray photoelectron spectroscopy (XPS) for surface analysis (N-Cl + H2O f Figure 7. Survey XPS spectra from SnO2 films after polarization at 1.5 V/SCE for 2 h in (A) 0.5 M NaCl without BSA, (B) 1 mg/mL BSA + 0.5 M Na2SO4 solution, and (C) 1 mg/mL BSA + 0.5 M NaCl solution.

electrode mass only slightly increases (Figure 1A). Under polarization, the SnO2 mass increase is high; after one potential scan the deposit mass is about 4 µg cm-2. In potentiostatic conditions, at a potential at which chloride oxidation occurs (here 1.5 V/SCE), after a few tens of minutes, it could increase up to 50 µg cm-2 (see Figure 1B). In addition, the comparison of Figures 5 and 6 clearly shows that, for a given chloride concentration, the presence of BSA does not modify the anodic current, i.e., the electrochemical process. The SEM images reported in Figures 3C, 3D, and 4 show that, after an anodic polarization in the presence of chlorides and BSA, the SnO2 surface is coated with an irregular porous deposit. The EDX spectra depicted in Figure 4 evidence that the Sn signals are attenuated (compare spectra A and C), indicating that the SnO2 film is buried under a deposit composed of C, N, O, and S, elements characteristic of the BSA; notably the C peak is largely increased (Figure 4C). It must be noticed that Cl is detected, whereas no Na is evidenced (1 keV). In order to determine the chemical composition of the SnO2 surfaces, XPS analyses were also performed. Figure 7 shows the survey spectra obtained from electrochemically treated SnO2 films in various conditions. In the case of NaCl solution without BSA, only the photopeaks due to the substrate are detected (see Figure 2A for comparison). In the presence of BSA but without chloride, the XPS survey spectrum is similar to the one obtained at open circuit potential (see Figure 2B). Finally, in the presence of both chlorides and BSA, the substrate is no longer detected; C1s, N1s, and O1s photopeaks are detected and also Cl2p and Cl2s ones. No Na signal (Na1s: 1070 eV) is detected; this means that the Cl atoms are linked to the organic matter likely via chloramine groups. These results were obtained using BSA concentrations of 0.5 to 3 mg/mL. For higher BSA concentrations, for example, 30 mg/mL, the aggregation of the protein was observed, but chlorine was not detected. In fact, the XPS survey spectrum was identical to the BSA one. 3. Chloramine Characterization. After polarization for 2 h in a 0.5 M NaCl solution containing 1 mg/mL BSA, the SnO2 electrode was rinsed with water and then dipped into the yellow TNB solution. It was observed that immediately the solution began to bleach. This is shown in Figure 8, in which absorbance spectra obtained after various dipping times are reported; the same sample was dipped successively for 30 s, 1 min 30 s, and 2 min 30 s. For this example, after 5 min of immersion, the TNB solution was totally bleached. To confirm the specificity of the electrochemically treated SnO2 surface, the same experiments were conducted using an as-prepared SnO2 film. The adding of

R-SO-CH3 + >N-H + Cl- + H+ (5) As one can notice from Figure 9 after the methionine treatment, the absorbance of the TNB solution only slightly decreases (see Figure 9). Remarkably, if after the electrochemical treatment the SnO2 electrode is exposed to air for 1 month, the TNB solution bleaches, too. The organic deposit was mechanically removed from the SnO2 film, and the obtained powder was added (after weighing) to the TNB solution. Several experiments were performed in order to quantify the number of chloramines per adsorbed BSA molecules. Unfortunately, the dissolution of the powder was never complete even if the TNB solution was rapidly partially decolored.

Discussion If the BSA tertiary structure is preserved, the mass of the equivalent of one BSA monolayer is between 0.2 and 0.7 µg‚cm2 depending on the orientation of the molecules on the surface.27 Since on SnO2, at open circuit potential, in the presence of BSA, the substrate mass increase was of 0.4 ( 0.1 µg‚cm-2, we can conclude that no more than the equivalent of one monolayer adsorbs on it. This finding is in good agreement with literature since it has been shown that, on hydrophilic surfaces, BSA adsorbs in a two-step process28 and that the deposited layer prevents further protein adsorption. The as-prepared SnO2 surface is hydrophilic.29 After anodic polarization in BSA-containing chloride solution, the electrode mass increase was up to 50 µg‚cm-2, about 100 times higher than at open circuit potential (Figure 1). This is in agreement with the SEM images (Figures 3 and 4) and XPS and EDX spectra (Figures 7 and 4, respectively). As a result, in order to form an organic film, chloride ions and proteins should be present at the electrode interface. This is well demonstrated by the XPS survey spectra depicted in Figure 7. They were recorded after SnO2 polarization in the presence of only chlorides or only BSA or in the presence of both BSA and chlorides. In the absence of chloride, the XPS spectrum is similar to the one obtained at open circuit potential, i.e., a monolayer of BSA adsorbs on SnO2 (spectrum B in Figure 2). No protein aggregation occurs. The signature of the substrate disappears only after performing the electrochemical treatment in the presence of both BSA and chloride ions (spectrum C in Figure 7), and Cl is detected due to the reaction of HOCl with the BSA. Moreover, the detection of chlorine by XPS (surface sensitive) (23) Connick, R. E.; Chia, Y. J. Am. Chem. Soc. 1959, 81, 1280-1284. (24) Standard Potentials in Aqueous Solution; Bard, A. J., Parsons, R., Jordan, J., Eds.; Dekker: New York, 1985; pp 76-77. (25) Morris, J. C. J. Phys. Chem. 1966, 70, 3798-3805. (26) Peskin, A. V.; Winterbourn, C. C. Free Radical Biol. Med. 2001, 30, 572-579. (27) Bendedouch, D.; Chen, S.-H. J. Phys. Chem. 1983, 87, 1473-1477. (28) Sweryda-Krawiec, B.; Devaraj, H.; Jacob, G.; Hickman, J. J. Langmuir 2004, 20, 2054-2056. (29) Cachet, H.; Folcher, G.; Haskouri, S.; Tribollet, B.; Festy, D. Mater. Techn. 2004, 7-8, 1-7.

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Figure 8. Optical absorbance spectra of (A) (s) DTNB solution; (- - -) TNB solution; and (B) TNB solution after (9) addition of BSA powder; (4) soaking of an untreated SnO2 film; soaking for (0) 30 s, ([) 2 min, and (O) 5 min of a SnO2 film polarized at 1.5 V/SCE during 4 h in 0.5 NaCl solution containing 1 mg/mL BSA. Cuvette length: 1 cm.

Figure 9. Chemical characterization of chloramines by TNB. Left: SnO2 films and optical images of cuvettes (1 cm) containing the TNB solution. (A) Unmodified TNB solution. Top: Before immersion of the SnO2 films coated with a large organic deposit. Bottom: Sample B after a 3 h immersion of the SnO2 films polarized at 1.5 V/SCE in 0.5 M NaCl solution containing 1 mg/mL BSA; sample C as sample B but before immersion the SnO2 film was dipped for 4 h in 0.1 M methionine solution. Right: optical absorbance spectra of TNB solutions (cuvette length: 0.2 cm). Black lines: Reference (A). Blue and red lines correspond to samples B and C, respectively. Full and dotted lines: Spectra recorded after 30 min and 3 h, respectively, of immersion of the SnO2 films.

and by EDX (bulk sensitive) indicates that the organic deposit is chlorinated throughout its thickness. From a detailed XPS study given elsewhere,30 the shape of the C1s level is identical to the one obtained for the BSA powder, indicating that at least the primary structure of the protein is retained in the deposit. The S2p level shows two contributions, one assigned to S(-II) and S(-I), i.e., to thiols and disulfides (at 163.8 eV), and the other one, at the higher binding energy (168 eV), attributed to S(+VI), i.e., to -SO2- groups. No S(+IV) (-SO- group) is detected either because in BSA the number of Met residues is low (see Table 1) or because thioether (RS-CH3) oxidized by HOCl yields sulfone (R-SO2-CH3), as suggested in ref 8. Moreover, two types of Cl are present into the deposit. The first one, detected at the lower binding energy, is attributed to N-Cl bonds. It has been noticed that after contact with a methionine solution this contribution largely decreases. The second Cl2p contribution is assigned to sulfonyl chloride groups (-SO2-Cl) even if in solution they undergo electrolysis. We can assume that on the SnO2 surface these groups are stabilized. In addition, it has been noticed that this XPS contribution is too high to be attributed to 3-Cl-Tyr since BSA contains only 20 Tyr residues. (30) Debiemme-Chouvy, C.; Haskouri, S.; Cachet, H. Appl. Surf. Sci., accepted, doi:10.1016/j.apsusc.2006.12.077.

Thus, during the HOCl production at the SnO2/solution polarized interface, the chlorination of protein could take place following these reactions:

R-SH + 3 HOCl f R-SO2-Cl + 2 Cl- + 2 H+ + H2O (6) R-S-S-R′ + 5 HOCl f R-SO2-Cl + R′-SO2-Cl + 3 Cl- + 3 H+ + H2O (7) R-NH2 + HOCl f R-NHCl + H2O

(8)

yielding sulfonyl chloride and chloramine groups.31 The latter were characterized by reaction with TNB according to reaction 2. For TNB, the molar extinction coefficient at 412 nm was reported to be 14 100 M-1‚cm,21,32 and for DTNB33 at 324 nm it is 17 780 M-1‚cm-1. The presence of chloramines in the organic deposit formed at the SnO2 surface during HOCl production is evidenced on one hand by the decrease of the TNB absorbance peak (proportional to 2 × 14 100) and on the other hand by the (31) Pattison, D. I.; Davies, M. J. Biochemistry 2005, 44, 7378-7387. (32) Eyer, P.; Worek, F.; Kiderlen, D.; Sinko, G.; Stuglin, A.; Simeon-Rudolf, V.; Reiner, E. Anal. Biochem. 2003, 312, 224-227. (33) Riddles, P. W.; Blakeley, R. L.; Zerner, B. Anal. Biochem. 1979, 94, 75-81.

An Original Route to Immobilize an Organic Biocide

increase of the DTNB peak (proportional to 17 780), as shown by Figure 8. The quantification of the chloramine groups was not obvious; on one hand the TNB solution is not stable, and on the other hand it was not easy to determine the endpoint because the powder dissolution rate was low. However, XPS analysis allowed us to estimate that one BSA molecule immobilized at the surface contains about 50 chloramine groups.30 As mentioned before, the oxidizing ability of HOCl is retained by chloramines, which are known to have biocide activity.34 We have recently evidenced the antibacterial property of these surfaces by checking the inhibition of the growth of Escherichia coli on electrochemically modified SnO2 surfaces in the presence of BSA.35 Such a finding has to be connected with observations made during long-term experiments at IFREMER-Centre de Brest in which SnO2-coated glasses were continuously polarized at +1.5 V/SCE in seawater for three weeks. After switching off the anodic polarization, a persistent antifouling effect was observed for about two weeks.36 Finally, aggregation of proteins on the SnO2 surface during HOCl formation is explained by considering the reaction between a sulfonyl chloride group (-SO2-Cl) due to Cys or cystine oxidation (reactions 6 and 7) and an amine function,37,38

R-SO2-Cl + NH2-R′ f R-SO2-NH-R′ + Cl- + H+ (9) resulting in intra- and intermolecular cross-linking sulfonamides. Thus, this process yields stable covalent bonds leading to a very high molecular mass protein. This can explain the low solubility of the organic matter observed during the attempts of chloramine quantification. It was also observed that protein aggregation could lead to sulfinamide formation that occurs via sulfenic and/or sulfinyl chloride intermediates that decompose upon reaction with amine, yielding stable covalent bonds.39,40 However in our case no such groups were detected by XPS30 likely because, at the SnO2/solution interface, the molar ratio of HOCl to BSA was high, allowing oxidation of S(-I) and S(-II) up to S(+VI). Moreover, it has been evidenced that, in solution, protein (34) Carr, A. C.; van den Berg, J. J. M.; Winterbourn, C. C. Biochim. Biophys. Acta 1998, 1392, 254-264. (35) Haskouri, S.; Cachet, H.; Duval, J.-L.; Debiemme-Chouvy, C. Electrochem. Commun. 2006, 8, 1115-1118. (36) Festy, D. Private communication. (37) Winterbourn, C. C.; Brennan, S. O. Biochem. J. 1997, 326, 87-92. (38) Fu, X.; Mueller, D. M.; Heinecke, J. W. Biochemistry 2002, 41, 12931301. (39) Raftery, M. J.; Yang, Z.; Valenzuela, S. M.; Geczy, C. L. J. Biol. Chem. 2001, 276, 33393-33401. (40) Raftery, M. J.; Geczy, C. L. J. Am. Soc. Mass Spectrom. 2002, 13, 709718.

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aggregation takes place when the HOCl concentration is higher than the BSA one.41,42 For the highest BSA concentration tested, no Cl was detected by XPS at the surface of electrochemically treated SnO2 film. Obviously, in this condition the ratio of HOCl to BSA is lower than previously. Since the rate constants are the highest for S-containing residues (see Table 1), we can assume that HOCl reacts mainly with Met, Cys, and cystine,7 leading mainly to the formation of sulfonamide groups; thus, the chloramine formation is low.

Conclusion The immobilization of an organic biocide on a SnO2 surface has been shown to be effective. Indeed, an important deposit of organic matter containing chloramine groups occurs during the anodic polarization of the SnO2 electrode in the presence of chlorides and proteins. Actually, during HOCl formation due to chloride oxidation, two phenomena take place: the aggregation of proteins onto the SnO2 surface and the chlorination of the proteins due to the reaction of HOCl with some protein side chains, leading notably to chloramines, which give the SnO2 surface antibacterial properties. Interestingly, we have also obtained an organic film deposition using bromide solutions instead of chloride ones. In that condition, hypobromous acid (HOBr) is produced, yielding in the presence of BSA the formation of bromamine6,7,43 with a protein aggregation process, too.44 In this context, studies are currently in progress for comparing the biocide properties of the organic film deposited on SnO2 during anodic polarization in the presence of proteins either in artificial solutions (0.5 M NaCl or NaBr) or in natural seawater, which contains 10-3 M bromide and 0.5 M chloride. Moreover in order to specify the reactions that take place at the electrode/solution interface, small model peptides will be used, for example without Tyr. Finally, one can notice that the present biocide immobilization process can be extended to any electrode material for which halogen oxidation occurs at potentials lower than that of oxygen evolution. Acknowledgment. The authors are very grateful to Stephan Borensztajn for performing the SEM and EDX analyses and would like to thank Dr. Mathieu Lazerges for QCM experiments performed at open circuit potential. LA063613J (41) Hawkins, C. L.; Davies, M. J. Chem. Res. Toxicol. 2005, 18, 1600-1610. (42) Chapman, A. L.; Winterbourn, C. C.; Brennan, S. O.; Jordan, T. W.; Kettle, A. J. Biochem. J. 2003, 375, 33-40. (43) Pattison, D. I.; Davies, M. J. Biochemistry 2004, 43, 4799-4809. (44) Haskouri, S. Ph.D. Thesis, University Paris VII, 2006. (45) Armesto, X. L.; Canle L. M.; Fernandez, M. I.; Garcia, M. V.; Santaballa J. A. Tetrahedron 2000, 56, 1103-1109.