N

LD

ARRAA

KBNESA

1

aeidpibsstw

c[eftoecbmc

0h

Colloids and Surfaces B: Biointerfaces 111 (2013) 252– 256

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

jou rn al hom epage: www.elsev ier .com/ locate /co lsur fb

afion coated stainless steel for anti-biofilm application

i Juan Zhong, Li Qing Pang, Li Ming Che, Xue E. Wu ∗, Xiao Dong Chen ∗

epartment of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China

a r t i c l e i n f o

rticle history:eceived 1 April 2013eceived in revised form 20 May 2013ccepted 25 May 2013vailable online 2 June 2013

a b s t r a c t

Biofilms can adhere to most surfaces and have caused a wide range of problems in various industrialprocesses as well as daily life activities. In this work, the anti-biofilm ability of Nafion-coated stainlesssteel surface was investigated and our results showed that stainless steel discs coated with 1% Nafioncan significantly reduce E. coli adhesion. Nafion has a large amount of negatively charged sulphonategroups, and the findings of this study suggest that the negative surface charge can greatly reduce bacterial

eywords:iofilmafion. colitainless steel

adhesion through increasing the electrostatic repulsion between negatively charged bacterial cells andNafion coated stainless steel surface. The roughness of coated and uncoated stainless steel discs made nosignificant differences while the hydrophobic of the discs increased after coated with Nafion.

© 2013 Elsevier B.V. All rights reserved.

nti-biofilm

. Introduction

Biofilms have been defined as a group of microorganismsttached to a surface which are embedded in a matrix of hydratedxtracellular polymeric substances (EPS) produced by microorgan-sms [1]. Bacteria attached to the surfaces are more resistant toisinfectants than free-living cells and biofilm has been a seriousroblem in many areas, including food, environmental and biomed-

cal, etc., and has posed many problems [2–6]. The adhesion ofacteria to a solid surface is a complex process [7,8], and manytudies have investigated the correlations between bacterial adhe-ion and surface properties, such as the surface roughness [9–13],he surface charge [9,14–23], the surface free energies [24–31], asell as the hydrophobic/hydrophilic properties [14,31–38].

It is known that electrostatic interactions contribute to biofilmohesion [9,14], and are likely to enhance the stability of biofilms15]. A number of studies have tried to evaluate the influence oflectrostatic interactions on bacteria adhesion, and most studiesocused on the influence of cations in solution (e.g. Ca2+, Mg2+) onhe formation of biofilms [16–20]. On the other hand, as a resultf the basic nature of most of the surface features, bacteria usuallyxhibit a negative net charge at neutral pH [39–41]. The surfaceharge of the substrate might affect the electrostatic interactionetween bacterial and substrate surfaces and the attachment of

icrobial cells on the surface could be reduced by a negatively

harged surface [21–23].

∗ Corresponding authors. Tel.: +86 592 2189073; fax: +86 592 2188855.E-mail addresses: xewu@xmu.edu.cn (X.E. Wu), xdc@xmu.edu.cn (X.D. Chen).

927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfb.2013.05.039

Due to its stability of physical and chemical properties at a var-ious processing temperature, stainless steel is widely used in thefood and medical industries as well as daily life. However, becauseof the cracks and crevices on its surface, bacterial attachment andsubsequent biofilm formation can occur on stainless steel surface.Therefore, effective surface modification strategies are urgentlyneeded in practice. There are various methods to develop anti-biofilm surfaces on stainless steels, such as: heating, abrading,chemical treatment, ion bombardment, surface coating [42,43].Compared with other complex processes, surface coating is thesimplest method of preventing the growth of bacteria and biofilmformation [44,45], and the key to this method is to find effectivecoating reagents which can prevent bacteria adhesion or biofilmformation.

Nafion polymers are a family of non-cross-linked, perfluoro-sulphonate cation-exchange polymers which is super selective,thermal stable and biocompatible [46–48]. The sulfonic acidgroups at the side chains of Nafion would dissociate the hydro-gen ion in solution, and the remaining polymers are negativelycharged (Fig. 1(b)) [49]. Nafion membrane has been widely usedto prevent electrode fouling by organic, surface-active compounds[50–52]. Recently, Pontie et al. deposited poly (diallyl-dimethyl-ammonium) chloride (PDADMAC) and poly (styrene sulfonic) acidon a Nafion membrane to improve its anti-biofouling ability [53].However, to our knowledge, the anti-biofilm effect of Nafion coat-ing on stainless steel has not yet been reported.

In this paper, Nafion was used as stailess steel coating material

and the ability of Nafion coating to reduce the biofilm formationby E. coli on stainless steel discs was studied and the roles ofsubstratum properties on the biofilms formation by E. coli wereinvestigated.

L.J. Zhong et al. / Colloids and Surfaces B: B

Fe

2

2

Gbgrp

2

pawtppwfi

2

1t5sld

2

w(ta

Ho

ig. 1. Structure of Nafion: (a) chemical structure of Nafion; (b) clusters of sulfonate-nded perfluoroalkyl ether groups of Nafion.

. Materials and methods

.1. Biological materials

Bacteria strain Escherichia coli DH5� was purchased from Chinaeneral Microbiological Culture Collection Center (CGMCC). Theacterial strain was stored in a refrigerator (Thermo) at −60 ◦C, andrown on LB medium (pH 7.0) for 12 h at 37 ◦C to active the bacte-ia before use. The LB medium contains 0.5% (w/v) yeast extractowder, 1% (w/v) peptone, and 1% (w/v) sodium chloride.

.2. Chemicals

Nafion solution (DE520CS 5%) was purchased from Dupont Com-any. During the experiment, 5% Nafion was diluted 5 times bybsolute ethanol (99%) to get 1% Nafion solution. Ultrapure wateras obtained directly from a Milli-Q academic water purifica-

ion system (Millipore Corporation, USA), fitted with an organicurification column to remove organic matter. All solutions wererepared by dilution of the above stock solutions with ultrapureater immediately prior to determination unless otherwise speci-ed.

.3. Treatment and Nafion coating of stainless steel discs

Stainless steel (304) discs (8 mm diameter and approximately mm thick) were treated with 0.5% (wt%) sodium hydroxide solu-ion for 30 min, and sonificated twice with distilled water set at3 kHz for 5 min (KUDOS). These stainless steel discs were thenterilized by autoclave (Zealway G154DW) at 121 ◦C for 20 min fol-owed by dipping these discs in 1% Nafion solution and then airried.

.4. Contact angle and roughness measurement

The contact angle of stainless steel discs before and after coatingith Nafion was measured by contact angle measuring instrument

KRÜSS, DSA 30) to get the information of the physical properties ofhe discs; distilled water was used as a probe for measuring contact

ngles. Each disc was measured 6 times.

A surface measuring instrument (Hommel Tester T8000,ommel-Etamic GmbH) was used to characterize the roughnessf stainless steel discs.

iointerfaces 111 (2013) 252– 256 253

2.5. Biofilm formation

Commercially available 24-well-microtiter plates (Costar) wereused for biofilm formation. During the experiments, stainless steeldiscs were placed in the wells of microtiter plate.

A single E. coli DH5� colony was chosen to inoculate in 100 mlLB medium in a 250 ml flask, shaken, cultured at 37 ◦C at a rotat-ing speed of 150 rpm for 16 h. 100 �l of the inoculum was thentransferred into a new 250 ml flask containing 100 ml LB medium,and incubated at 37 ◦C, 150 rpm for 12 h. 2 ml of the inoculum wasadded into each well of the microtiter plate. Unless other stated,the stainless steel discs with or without coating by 1% Nafion wereplaced in the wells respectively, and cultured at the same conditionas above for 96 h to form biofilms.

2.6. Bacterial adhesion assay

2.6.1. Plate counting methodThe number of the bacteria colonies in biofilm was determined

by plate counting method. Six pieces of stainless steel discs werewashed and sterilized, as described above. Three of them werecoated with 1% Nafion while the other three were not. All the sixdiscs were put into the wells of 24-microtiter plate individually, andthen 2 ml E. coli suspension was added to each well before incuba-tion. After incubating for 24 h, Stainless steel discs were taken outfrom the microtiter plates and washed three times with phosphatebuffer solution (PBS) containing 0.027% (w/v) KH2PO4, 0.142% (w/v)Na2HPO4, 0.8% (w/v) NaCl, and 0.02% (w/v) KCl (pH 7.0) to removeloosely adherent planktonic bacteria. The discs were then put into1.5 ml centrifuge tubes containing 1 ml PBS respectively and soni-ficated for 5 min with an ultrasonic cleaner set at 20 kHz for 5 minto release the embeded bacteria into PBS solution. The suspensionwas diluted 100 times with PBS after sonification, and 20 �l of thediluent was transfered onto LB agar medium plates, and culturedat 37 ◦C in an incubator for 24 h to count the number of bacteria indifferent discs. Each sample was cultured parallelly in three plates.

2.6.2. MicroscopeMicroscope (OLYPUS, BX41) was used to primarily check the

biofilms on discs.

2.6.3. Phase shiftThe surface morphology of stainless steel discs were measured

by the phase shift (MicroXAM-3D). Stainless steel discs were halfcoated with Nafion, and the discs without biofilm were used ascontrol.

2.6.4. SEMThe biofilms formed on stainless steel discs were washed three

times with phosphate buffer solution (PBS), and once with distilledwater to remove the remaining ions. The discs underwent a dryingprocess and sputter-coated with gold for 60 s at 20 mA. SEM wasperformed using a Quanta 200 environmental scanning electronmicroscope.

3. Results and discussion

3.1. Surface fabrication and their characterization of Nafioncoated stainless steel

Although most bacteria have a negative and hydrophobic sur-

face at neutral pH, the degree of cell membrane hydrophobicitychanges during growing [39–41]. As mentioned previously, theproperties (e.g. roughness, surface charge, hydrophobicity) of sub-stratum contributed to the formation of biofilm and understanding

254 L.J. Zhong et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 252– 256

Table 1Water contact angles (by degree) of stainless steel discs before and after coated with1% Nafion.

No. Contact angles (◦)

Before modification Coated by 1% Nafion

1 87.02 99.272 86.02 96.683 85.93 98.084 85.33 100.60

tersrea

hsdh1ccnt

3s

occac

ba

Fig. 2. CFU of the discs with or without coating with 1% Nafion solution: (a) the

TR

5 82.15 102.376 83.78 100.32

he interactions between the bacteria and the substratum prop-rties may help to find anti-biofilm surface. Up to now, mostesearchers agree that bacteria tend to adhere on hydrophobicurfaces rather than hydrophilic surfaces [9,32,33]. As for theoughness, most reports revealed that the surface roughness influ-nces bacterial adhesion and the extent of adhesion will increases the surface roughness increases [9,11,12].

In this study, the effects of Nafion coating on the roughness andydrophobicity of stainless steel discs have been investigated. Ashown in Table 1, the water contact angles of the stainless steeliscs increased after modification with Nafion, indicating that theydrophobicity of stainless steel discs increased after coated with% Nafion. The roughness of three stainless steel discs samplesoated with 1% Nafion and three discs without Nafion coating asontrol were measured and the results showed that there wereo significant differences in the roughness of control samples andesting samples (Table 2).

.2. Anti-bacterial adhesion of Nafion coated stainless steelurfaces

The plate counting method is used here to count the E. coli cellsn the stainless steel discs. The CFU of stainless steel discs withoutoating or coated by 1% Nafion is shown in Fig. 2. As can be seen,ompared with the stainless steel discs without Nafion coating,n obviously decrease in the adherence of the bacteria on Nafion

oated stainless steel discs was observed.

The microscopic image of a half coated stainless steel disc beforeiofilm forming is shown in Fig. 3(a), while the microscopic imagefter biofilm forming steps is presented in Fig. 3(b). Compared with

able 2oughness (�m) of stainless steel discs coated with 1% Nafion and discs without coating.

Control(1) Control(2) Control(3

Ra (�m) 0.030 0.026 0.027

Rs (�m) 0.291 0.255 0.255

Fig. 3. (a and b) Microscopic images of half-Nafion-co

mean value of CFU and standard deviation of 3 discs without Nafion coating; (b) themean value of CFU and standard deviation of 3 discs coated with Nafion.

Fig. 3(a), Fig. 3(b) shows the Nafion coated half disc had less sub-stance than the half without Nafion coating. Fig. 4 were the resultsof Phase Shift MicroXAM-3D, two discs were half coated with 1%Nafion, one was used to cultivate biofilm, named testing disc, andthe other was used as control. Fig. 4(a) shows the morphology ofthe control disc while the morphology of biofilm on the half coateddiscs is revealed in Fig. 4(b). According to Fig. 4(a), there were no sig-nificant differences between the Nafion-coated half and uncoatedhalf of the control disc before biofilm forming. Fig. 4(b) reveals thatthe substance attached on the Nafion-coated half of testing discwas much less than the half disc without Nafion coating, whichindicates that stainless steel coated with Nafion can inhibit bacte-ria adhesion. The SEM images (Fig. 5) also shows that there weremuch more bacteria on the half disc without Nafion coating thanthe half which had been coated with 1% Nafion.

It has been suggested that electrostatic interactions were themost important factor for biofilm formation [15]. Most bacteria are

negatively charged and making the surface of negatively chargedwill decrease the abundance of attached bacteria by enhancing therepulsive forces between negatively charged surfaces and bacteria.

) Testing(1) Testing(2) Testing(3)

0.027 0.030 0.0250.239 0.260 0.216

ated stainless steel disc of E. coli biofilm formed.

L.J. Zhong et al. / Colloids and Surfaces B: Biointerfaces 111 (2013) 252– 256 255

Fig. 4. Phase shift MicroXAM-3D of half-Nafion-coated stainless steel disc: (a) the control disc; (b) the testing disc.

iscs: (

AwNbbpaotc

d

Fig. 5. SEM images of biofilms on half-Nafion-coated stainless steel d

s an ion exchange resin, the Nafion molecule has a Teflon backboneith perfluorine side chains containing sulfonic acid groups. Whenafion is exposed to water solutions, the sulfonic acid groups wille dissociated, leaving the surface negatively charged. And it haseen reported that Nafion can completely reject negatively chargedrobes, but attract positively charged species. In the case of E. colidhesion, a significant reduction in adherent bacteria was observedn the Nafion coated stainless steel discs which could be attributed

o these repulsive forces between the negatively charged bacterialells and Nafion coated stainless steel surfaces.

From above results, we can conclude that the adhesion of E. coliecreased on a negative charged and more hydrophobic surface.

a) half disc without Nafion coating; (b) half disc with Nafion coating.

According to most researchers, bacteria are prone to adhere onhydrophobic surfaces rather than hydrophilic surfaces [9,32–34].And we proposed that the electrostatic interaction plays a moreimportant role than hydrophobicity during the adhesion of E. colito Nafion coated stainless steel.

4. Conclusion

In this work, we have developed an environmentaly friendlyand simple procedure for coating stainless steel with 1% Nafionand our results showed that the Nafion coated stainless steel discssignificantly reduced the bacteria adhesion compared with

2 es B: B

utisoiw

A

P

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

56 L.J. Zhong et al. / Colloids and Surfac

ncoated stainless steel discs. In this way, this paper demonstratedhat negative charged surface can reduce E. coli adhesion by increas-ng the electrostatic repulsion between the bacterial cells and thetainless steel surfaces. This work also showed that the differencef roughness between stainless steel with and without Nafion coat-ng was negligible, and the hydrophobicity increased after coating

ith Nafion.

cknowledgment

Thanks to Tsinghua University for their help in taking SEM andhase Shift MicroXAM-3D photos.

eferences

[1] J.W. Costerton, K.J. Cheng, G.G. Geesey, J.C. Nickel, M. Dasugupta, T.J. Mar-rie, Bacterial biofilms in nature and disease, Annu. Rev. Microbiol. 41 (1987)435–464.

[2] S.A. Kirtley, J. McGuire, On differences in surface constitution of dairy productcontact materials, J. Dairy Sci. 72 (1989) 1748–1753.

[3] M.C. Te Giffel, R.R. Beumer, L.P.M. Langeveld, F.M. Rombouts, The role of heatexchangers in the contamination of milk with Bacillus cereus in dairy processingplants, Int. J. Dairy Technol. 30 (1997) 43–47.

[4] W.G. Scott, H.M. Scott, R.J. Lake, M.G. Baker, Economic cost to New Zealand offood borne infectious disease, N Z Med. J. 113 (2000) 281–284.

[5] J.D. Brooks, S.H. Flint, Biofilms in the food industry: problems and potentialsolutions, J. Ind. Microbiol. Biotechnol. 43 (2008) 2163–2176.

[6] X. Shi, X. Zhu, Biofilm formation and food safety in food industries, Trends FoodSci. Technol. 20 (2009) 407–413.

[7] J.W. Costerton, Z. Lewandowski, D.E. Caldwell, D.R. Korber, M. Hilary, L. Scott,Microbial biofilms, Annu. Rev. Microbiol. 49 (1995) 711–745.

[8] B.E. Christensen, W.G. Characklis, Physical and chemical properties of biofilms,in: Biofilms, John Wiley & Sons, New York, 1990, pp. 93–103.

[9] M. Fletcher, G.I. Loeb, Influence of substratum characteristics on the attachmentof a marine pseudomonad to solid surfaces, Appl. Environ. Microbiol. 37 (1979)67–72.

10] P.J. Eginton, H. Gibson, J. Holah, P.S. Handley, P. Gilbert, The influence ofsubstratum properties on the attachment of bacterial cells, Colloids Surf. B:Biointerfaces 5 (1995) 153–159.

11] K. Pedersen, Biofilm development on stainless steel and PVC surfaces in drink-ing water, Water Res. 24 (1990) 239–243.

12] G. Wirtanen, H. Ahola, T. Mattila-Sandholm, Evaluation of cleaning proceduresin elimination of biofilm from stainless steel surfaces in open process equip-ment, Trans. Int. Chem. Eng. 73 (1995) 1–9.

13] S.H. Flint, J.D. Brooks, P.J. Bremer, Properties of the stainless steel substrate,influencing the adhesion of thermo-resistant streptococci, J. Food Eng. 43(2000) 235–242.

14] K.C. Marshall, R. Stout, R. Mitchell, Mechanism of the initial events inthe sorption of marine bacteria to surfaces, J. Gen. Microbiol. 68 (1971)337–348.

15] H.C. Flemming, J. Wingender, M.C. Griegbe, C. Mayer, Physico-chemical prop-erties of biofilms: recent advances in their study and control, Biofilms (2000)19–33.

16] M. Fletcher, Attachment of Pseudomonas fluorescens to glass and influence ofelectrolytes on bacterium–substratum separation distance, J. Bacteriol. 170(1988) 2027–2030.

17] D. Lattner, H.C. Flemming, C. Mayer, 13C NMR study of the interaction ofbacterial alginate with bivalent cations, Int. J. Biol. Macromol. 33 (2003)81–88.

18] H.C. Flemming, P.S. Murthy, R. Venkatesan, K.E. Cooksey, Marine and IndustrialBiofouling, Springer, Berlin, 2009.

19] L. Tian, X.D. Chen, Q.P. Yang, J.C. Chen, L. Shi, Q. Li, Effect of calcium ions on theevolution of biofouling by Bacillus subtilis in plate heat exchangers simulatingthe heat pump system used with treated sewage in the 2008 Olympic Village,Colloid Surf. B: Biointerfaces 94 (2012) 309–316.

20] B. Song, L.G. Leff, Influence of magnesium ions on biofilm formation by Pseu-domonas fluorescens, Microbiol. Res. 161 (2006) 355–361.

21] K. Hibiya, S. Tsuneda, A. Hirata, Formation and characteristics of nitrifyingbiofilm on a membrane modified with positively-charged polymer chains, Col-loids Surf. B: Biointerfaces 18 (2000) 105–112.

22] A. Terada, K. Okuyama, M. Nishikawa, S. Tsuneda, M. Hosomi, The effect of sur-face charge property on Escherichia coli initial adhesion and subsequent biofilmformation, Biotechnol. Bioeng. 109 (2012) 1745–1754.

23] B. Gottenbos, H.C. Vandermei, H.J. Busscher, Initial adhesion and surfacegrowth of Pseudomonas aeruginosa on negatively and positively charged poly(methacrylates), J. Mater. Sci.: Mater. Med. 10 (1999) 853–855.

24] C.I. Pereni, Q. Zhao, Y. Liu, E. Abel, Surface free energy effect on bacterial reten-tion, Colloids Surf. B: Biointerfaces 48 (2006) 143–147.

[

[

iointerfaces 111 (2013) 252– 256

25] R.E. Baier, Substratum influences on the adhesion of microorganisms and theirresultant new surface properties, in: Adsorption of Micro-organisms to Surface,Wiley-Interscience, New York, 1980, pp. 59–104.

26] Q. Zhao, Y. Liu, C. Wang, S. Wang, H. Müller-Steinhagen, Effect of surface freeenergy on the adhesion of biofouling and crystalline fouling, Chem. Eng. Sci. 60(2005) 4858–4865.

27] Y. Liu, Q. Zhao, Influence of surface energy of modified surfaces on bacterialadhesion, Biophys. Chem. 117 (2005) 39–45.

28] A.S. Jonssona, B. Jonsson, The influence of nonionic and ionic surfactants onhydrophobic and hydrophilic ultrafiltration membranes, J. Membr. Sci. 56(1991) 49–76.

29] L.E.S. Brink, S.J.G. Elbers, T. Robbertsen, P. Both, The anti-fouling action of poly-mers preadsorbed on ultrafiltration and microfiltration membranes, J. Membr.Sci. 76 (1993) 281–291.

30] M. Fletcher, K.C. Marshall, Are solid surfaces ecological significance to aquaticbacteria? Adv. Microbial Ecol. 47 (1982) 135–143.

31] L. Boulange-Peterman, B. Baroux, M.N. Bellon-Fontaine, The influence of metal-lic surface wettability on bacterial adhesion, J. Adhes. Sci. Technol. 7 (1993)221–230.

32] U. Husmark, U. Ronner, Forces involved in adhesion of Bacillus cereus spores tosolid surfaces under different environmental conditions, J. Appl. Bacteriol. 69(1990) 557–562.

33] J.S. Peng, W.C. Tsai, C.C. Chou, Surface characteristics of Bacillus cereusand its adhesion to stainless steel, Int. J. Food Microbiol. 65 (2001)105–111.

34] N. Cerca, G.B. Pier, M. Vilanova, R. Oliveira, J. Azeredo, Quantitative analysisof adhesion and biofilm formationon hydrophilic and hydrophobic surfacesof clinical isolates of Staphylococcus epidermidis, Res. Microbiol. 156 (2005)506–514.

35] B. Bendinger, H.H.M. Rijnaarts, K. Altendorf, A.J.B. Zehnder, Physico-chemicalcell surface and adhesive properties of coryneform bacteria related to the pres-ence and chain length of mycolic acids, Appl. Environ. Microbiol. 59 (1993)3973–3977.

36] S. Mceldowney, M. Fletcher, Variability of the influence of physico-chemicalfactors affecting bacterial adhesion to polystyrene substratum, Appl. Environ.Microbiol. 52 (1986) 460–465.

37] R.J. Doyle, Contribution of the hydrophobic effect to microbial infection,Microbes Infect. 2 (2000) 391–400.

38] H.C. Flemming, A. Tamachkiarowa, J. Klahre, J. Schmitt, Monitoring systems forthe detection of biofouling in technical systems, Water Sci. Technol. 38 (1998)299–307.

39] V.P. Harden, J.O. Harris, The isoelectric point of bacterial cells, J. Bacteriol. 65(1953) 198–202.

40] M. Rosenberg, S. Kjelleberg, Hydrophobic interactions: role in bacterial adhe-sion, Adv. Microbial Ecol. 9 (1986) 353–393.

41] L. Boulange-Peterman, Processes of bioadhesion on stainless steel surfaces andcleanability: a review with special reference to the food industry, Biofouling:J. Bioadhes. Biofilm Res. 10 (1996) 275–300.

42] R.O. Adams, A review of the stainless steel surface, J. Vac. Sci. Technol. A: Vac.Surf. Films 1 (1983) 12–18.

43] N. Shaigan, W. Qu, D.G. lvey, W. Chen, A review of recent progress incoatings, surface modifications and alloy developments for solid oxidefuel cell ferritic stainless steel interconnects, J. Power Sources 6 (2010)1529–1542.

44] A. Héquet, V. Humblota, J.M. Berjeaud, C.M. Pradier, Optimized grafting ofantimicrobial peptides on stainless steel surface and biofilm resistance tests,Colloids Surf. B: Biointerfaces 84 (2011) 301–309.

45] X.L. Liu, Z. Wu, F. Zhou, D. Li, H. Chen, Poly(vinylpyrrolidone-b-styrene) blockcopolymers tethered surfaces for protein adsorption and cell adhesion regula-tion, Colloids Surf. B: Biointerfaces 79 (2010) 452–459.

46] H.L. Yeager, A. Steck, Ion-exchange selectivity and metal ion separationswith a perfluorinated cation-exchange polymer, Anal. Chem. 51 (1979)862–865.

47] R.F.B. Turner, D.J. Harrison, R.V. Rojotte, Preliminary in vivo biocompatibilitystudies on perfluorosulphonic acid polymer membranes for biosensor applica-tions, Biomaterials 12 (1991) 361–368.

48] Z. Dai, H. Mohwald, Highly stable and biocompatible Nafion-based capsuleswith controlled permeability for low-molecular-weight species, Chemistry:Eur. J. (2002) 4751–4755.

49] W.Y. Hsu, T.D. Gierke, Ion transport and clustering in Nafion perfluorinatedmembranes, J. Membr. Sci. 13 (1983) 307–326.

50] B. Hoyer, T.M. Florence, G.E. Batley, Application of polymer-coated glassycarbon electrodes in anodic stripping voltammetry, Anal. Chem. 59 (1987)1608–1614.

51] R.C. Mercado, F. Moussy, In vitro and in vivo mineralization of Nafion mem-brane used for implantable glucose sensors, Biosens. Bioelectron. 13 (1998)133–145.

52] L.B. Blom, G.M. Morrison, M.S. Roux, G. Mills, R. Greenwood, Metal diffusionproperties of a Nafion-coated porous membrane in an aquatic passive samplersystem, J. Environ. Monitor. 5 (2003) 404–409.

53] M. Pontie, S. Ben Rejeb, J. Legrand, Anti-microbial approach onto cationic-exchange membranes, Sep. Purif. Technol. 101 (2012) 91–97.