Covalent Attachment of Poly(ethylene glycol) to Surfaces, Critical for

Also, preventing the adsorption of conditioning film components is in itself not a trivial exercise and needs to be confirmed using very surface-sensi...
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Covalent Attachment of Poly(ethylene glycol) to Surfaces, Critical for Reducing Bacterial Adhesion Peter Kingshott,*,† Jiang Wei,† Dorthe Bagge-Ravn,‡ Nikolaj Gadegaard,† and Lone Gram‡ Danish Polymer Centre, Risø National Laboratory, Building 124, P.O. Box 49, DK-4000 Roskilde, Denmark, and Department of Seafood Research, Danish Institute for Fisheries Research, Søltofts Plads, c/o Technical University of Denmark, Building 221, DK-2800 Kgs. Lyngby, Denmark Received January 8, 2003. In Final Form: May 28, 2003 The effects of different poly(ethylene glycol) (PEG) attachment strategies upon the adhesion of a Gramnegative bacteria (Pseudomonas sp.) was tested. PEG was covalently immobilized, at the lower critical solution temperature of PEG, to a layer of branched poly(ethylenimine) (PEI). PEI was both physically adsorbed to a stainless-steel (SS) substrate and covalently immobilized to a carboxylated poly(ethylene terephthalate) (PET-COOH) surface. On both substrates, the PEI and PEG grafting conditions were optimized so that the levels of surface coverage after each step were maximized and were the same on both substrates, as judged by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Also, ToF-SIMS imaging showed that both substrates were chemically uniform after each surface modification step. Thus, the two surfaces differ only in the mode of attachment of PEI to the substrate. In bacterial adhesion experiments, the optimal SS-PEG surface was not capable of reducing the number of adherent Pseudomonas sp. when compared to the controls. However, the PET-PEG surface reduced the level of adhesion by between 2 and 4 orders of magnitude for up to 5 h. ToF-SIMS analysis showed that both PEG surfaces adsorbed low but comparable levels of proteinaceous growth medium components (tryptic soy broth), as indicated by the addition of unique amino acid fragment ions in the spectra, most likely small peptides. Thus, bacterial adhesion was strongly dependent on the PEG immobilization strategy and not on the extent of peptide/protein adsorption. However, for the best PEG surfaces the residual bacterial adhesion is most likely from recognition of the small amount of adsorbed peptides. This highlights the necessity for preventing the adsorption of small biological species that can even penetrate PEG layers of high graft density, in the quest for the ultimate “nonfouling” surface.

Introduction Bacterial colonization of surfaces has a major impact in a broad range of technologically significant areas. Examples where detrimental consequences arise include: (1) the medical device industry where implants with adherent bacteria cause infections,1 (2) the food processing industry where lack of hygiene can cause food spoilage or pose a serious risk to public health,2 and (3) industries where degradation of materials by adherent bacteria is very costly,3 to name a few. Pseudomonas sp. is one bacterium that is ubiquitous in the environment that tenaciously and rapidly colonizes surfaces. The colonies eventually develop into mature biofilms and drastically increase the risk of severe infections with improper hygiene.4 The initial attachment of bacteria is speculated to be mediated by either the specific recognition of elements on the surface, such as * Corresponding author. Telephone ++ 45 4677 5480. Fax: ++ 45 4677 4971. E-mail: [email protected]. † Risø National Laboratory. ‡ Danish Institute for Fisheries Research. (1) Costerton, J. W.; Lewandowski, Z.; Caldwell, D. E.; Korber, D. R.; Lappin-Scott, H. M. Annu. Rev. Microbiol. 1995, 49, 711-745. Bryers, J. D. Colloids Surf., B 1994, 2, 9-23. (2) Storgårds, E.; Simola, H.; Sjoberg, A.-M.; Wirtanen, G. Chem Eng. Res. Des. 1999, 77, 137-145. Boulange-Petermann, L. Biofouling 1996, 10, 275-300. (3) Dawood, Z.; Brozel, V. S. J. Appl. Microbiol. 1998, 84, 929-936. Bode, H. B.; Kerkhoff, K.; Jendrossek, D. Biomacromolecules 2001, 2, 295-303. (4) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Science 1999, 284, 1318-1322.

components of a protein film,5 or specific binding to sugar receptors.6 Also, nonspecific, physicochemical interactions such as electrostatic or hydrophobic interactions between the bacteria and the surface can play a role in adhesion.7 Once the bacteria are attached, the transcription of specific genes is activated, resulting in the synthesis of exopolysaccharides (EPSs; or slime) that encase the bacteria and enable them to form a fully established biofilm.8 Biofilms are exceedingly resistant to antibiotics and biocides as a result of the impenetrable slime protecting the bacteria.9 Therefore, preventing biofilm formation requires the development of surfaces that could ideally fully resist the initial attachment of the bacteria, or at least discourage them from “switching on” the genes that enable them to produce EPSs and develop microcolonies that eventually lead to the formation of a mature biofilm.4 To date no surface exists that can fully prevent bacteria adhesion, and this remains a major challenge to the field of biological surface science, defined as the interdisciplinary area where properties and processes at interfaces between synthetic materials and biological environments are investigated and new materials are designed.10 From (5) An, Y. H.; Friedman, R. J. J. Biomed. Mater. Res. 1998, 43, 338348. (6) Scharfman, A.; Degroote, S.; Beau, J.; Lamblin, G.; Roussel, P.; Mazurier, J. Glycobiology 1999, 9 (8), 757-764. (7) Poortinga, A. T.; Bos, R.; Norde, W.; Busscher, H. J. Surf. Sci. Rep. 2002, 47, 1-32 and references therein. (8) Davies, D. G.; Geesey, G. G. Appl. Environ. Microbiol. 1995, 61, 860-867. Sutherland, I. W. Microbiology 2001, 147, 3-9. (9) Stoodley, P.; Sauer, K.; Davies, D. G.; Costerton, J. W. Annu. Rev. Microbiol. 2002, 56, 187-209 (10) Kasemo, B. Surf. Sci. 2002, 500, 656-677.

10.1021/la034032m CCC: $25.00 © 2003 American Chemical Society Published on Web 07/23/2003

Grafting of PEG To Reduce Bacterial Adhesion

the literature, poly(ethylene glycol) (PEG) as a surface modifying agent is clearly the most effective molecule at reducing bioadhesion (protein adsorption, bacterial and cell adhesion).11-16,54 The best reports have shown that protein adsorption can be suppressed to a fraction of the uncoated surface or even remain undetected when analyzed by the most sensitive analytical methods.12-15,17,18 However, the most promising reports investigating bacterial adhesion to PEG surfaces have so far only been marginally successful, by suppression of at best up to 1-2 orders of magnitude.19-23 Why is this the case? Theoretical considerations predict that PEG can fully prevent adhesion;24-26 however, the question remains whether these predictions can ever be put into practice by experiments: in particular, the generation of stable PEG layers with a sufficiently high graft density and surface uniformity to provide the optimal steric repulsive barrier against bioadhesion. Furthermore, investigations into the specific structural properties and physicochemical forces involved in the interaction of proteins and biological material with PEG layers27,28 would greatly assist in the design of the ultimate surface. Finally, the design of the ultimate “nonfouling” surface is dependent on being able to detect protein adsorption below the threshold where no subsequent events can occur (such as bacterial adhesion). This requires extremely sensitive surface analytical tools capable of detecting fractions of a monolayer. In addition, many studies have demonstrated that adsorption from single solutions of model proteins can be prevented by PEG. However, recent work has shown that low-molecularweight proteins and peptides can adsorb from complex biological media, such as human tears, onto surfaces such as contact lenses.29 Preventing the adsorption of lowmolecular-weight proteins (5000 in positive mode. Spectra were calibrated using the known masses of hydrocarbon ions (H+, C+, CH+, CH2+, CH3+, C3H2+, C4H6+, C5H9+, C6H5+, and C7H7+). SIMS imaging was obtained by selecting masses of interest and recording their intensities with respect to the position of the primary ion. The analysis conditions were the same as those used for spectral acquisition. Atomic force microscopy (AFM) measurements were performed in air on a Digital Instruments Dimension 3000 in tapping mode using standard Si3N4 tips. Bacterial Adhesion. A Gram-negative bacterium (Pseudomonas sp.) was isolated from the processing equipment in a processing plant producing cold-smoked salmon after cleaning and disinfection.30,31 The adhesion of Pseudomonas sp. to the different SS surfaces was tested according to the method of BaggeRavn et al.37 The surfaces investigated were sterilized by immersion in 97% ethanol for 10 min. The Pseudomonas sp. was precultured in tryptic soy broth (TSB; Oxoid CM129) with agitation at 25 °C. After 24 h, the bacteria were harvested (3000× g for 10 min), resuspended, and diluted in PBS (0.8% NaCl, 0.02% KCl, 0.144% Na2HPO4, and 0.0024% NaH2PO4). The assay for studying bacterial adhesion is deliberately carried out in PBS so the bacteria will not grow. Bacterial growth is avoided to create a system where we can study the immediate interaction between the bacterial cell and the surface. The bacterial density in PBS at every sampling-time point is monitored by the total aerobic plate counts in the beginning and in the end of each experiment, and there is no killing of bacteria throughout the experiment. The number of bacteria remains constant within the first 8 h; thereafter, there is a slight increase in the numbers.37 Sterile disks and holders were conditioned in dilute (1:7) TSB for 30 min with agitation; thereafter, the holders were transferred to a new sterile beaker containing the investigated bacteria suspended in PBS at approximately 106 cfu (colony-forming units)/ mL. Adhesion was allowed to take place on both sides of the disks under slow stirring at room temperature. At different time points, disks were sampled and the number of adhered microorganisms was quantified by indirect conductometry.37,38 The disks were transferred to a test tube containing a TSB growth medium. The growth of the adherent bacteria developed CO2, which diffused to an inner tube containing sterile NaOH. Electrodes measured the change of conductance in NaOH as CO2 dissolved in the alkali. The detection time, that is, the time point at which a significant change in conductance occurs, is inversely related to the initial number of bacteria. By use of a calibration curve constructed by a 10-fold dilution series of each of the bacteria, the initial number of bacteria on the surfaces was calculated. The conductometric detection system will detect as low as one bacterial cell per vial. Two batches of PEG-coated PET surfaces were tested. Protein Adsorption. Surfaces that were preconditioned with the TSB growth medium were also subjected to XPS and ToF-SIMS analysis to elucidate whether any of the proteinaceous components adsorbed to the PET-PEG or SS-PEG surfaces. (37) Bagge-Ravn, D.; Hjelm, M.; Johansen, C.; Huber, I.; Gram, L. Appl. Environ. Microbiol. 2001, 67, 2319-2325. (38) Johansen, C.; Falholt, P.; Gram, L. Appl. Environ. Microbiol. 1997, 63, 3724-3728.

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Table 1. XPS Elemental Composition for the Derivatization of the Modified PET Samples with either TFAA or PFP sample

%C

%O

%F

F/C

PET PET (theory) PET-OH PET-TFAA PET-OH-TFAA PET-COOH PET-PFP PET-OH-PFP PET-COOH-PFP

70.8 71.4 69.5 72.7 67.7 68.8 68.6 68.9 65.8

29.2 28.6 30.5 26.8 26.2 31.3 29.5 30.1 28.1

0.0 0.0 0.0 0.5 6.0 0.0 1.8 1.0 6.1

0 0 0 0.007 0.09 0 0.03 0.01 0.09

After rinsing with PBS, the surfaces were rinsed with water to remove any buffer salts that could interfere with the analysis.

Results Functionalization of PET. The two-step process to functionalize PET with reactive carboxyl groups was confirmed by derivatization with fluorinated probes that could be detected by XPS, and the results are shown in Table 1. Before derivatization, there is little difference between the PET, PET-OH, and PET-COOH surfaces with only small changes to the elemental composition after either the hydroxylation or the carboxylation steps. This is also shown in Table 2, which summarizes the data from the curve-fitted high-resolution C(1s) spectra of the PET, PET-OH, and PET-COOH surfaces. Each of the three spectra contains three main components at 285.0 eV (CdC/CsC/CsH), 286.5-286.7 eV (peak B; ether), and 288.9-289.1 eV (peak C, acid or ester), and there is little change to the concentration of each species evident. Also present are the π-π* shake-up satellite peaks. Therefore, derivatization was necessary to provide evidence of the presence of the functional groups. First, the hydroxyl groups on the PET-OH substrates were derivatized in a TFAA vapor, as depicted in Figure 1C. The increase in the F/C ratio from 0.007 for the untreated PET to 0.09 for PET-OH indicates the introduction of hydroxyl groups on the PET surface. PET-COOH was derivatized with PFP in ethanol, and the F/C ratio increased from 0.01 for the PET-OH surface to 0.09 for the PET-COOH surface. The presence of F on the PET and PET-OH surfaces after each type of derivatization is indicative of either nonspecific binding of the probe or reaction with PET endgroups. For the PET-OH surface, the theoretical F/C ratio for 100% conversion is 0.23; therefore, a hydroxyl group is introduced to only one in every two or three PET monomer units. For the PET-COOH surface, the theoretical F/C ratio is 0.26 for 100% conversion; therefore, one in three PET monomer units gets functionalized with carboxyl groups. The results are in good agreement with the previous reports.33,34 It would appear that the derivatization reactions confirm that the hydroxyl to carboxylation step proceeds to almost 100% conversion; however, AFM measurements (Figure 2) suggest that significant surface erosion occurs and a significant number of carboxyl groups are most likely generated from hydrolysis of PET. The root-mean-square (RMS) roughness increases from 3.1 nm for the PET surface (Figure 3A) to 12 nm for the PETOH surface (Figure 3B) and 11.6 nm for the PET-COOH surface (Figure 3C). Despite these findings, a significant number of reactive groups are present on PET-COOH for further functionalization. Grafting of PEI to the PET and SS Surfaces. Both the SS and the PET-COOH surfaces were modified with a branched PEI linker layer to provide a high concentration of reactive amine groups that would sub-

Figure 2. Positive-ion ToF-SIMS mass spectra for (A) the SSNH2 surface and (B) the PET-COO-NH2 surface. The concentration of PEI used for grafting was 30 mg/mL.

sequently produce a PEG surface of high graft density. PEI is a highly branched polymer with about 25% primary amine groups. The remaining amine groups are secondary (50%) and tertiary (25%). Only a small fraction of the groups react with the surface; thus, a high concentration of free amino groups are available for PEG grafting. We have recently shown that the level of PEI coverage on the SS surface is optimal when adsorption takes place from a concentrated solution (30 mg/mL).36 For PEI grafting to the PET-COOH surface (Figure 1F), EDAC/NHS was used for covalent immobilization, and a 30 mg/mL buffered (pH 7.4) solution was used to provide a direct comparison to the SS surface. For SS, surface modification occurs by adsorption of positively charged PEI to the negatively charged SS surface. The XPS elemental composition results for both the SS-NH2 and PET-COO-NH2 surfaces are shown in Table 2. The successful grafting of PEI to both surfaces is clearly shown from the substantial increase in nitrogen content to >11%. On SS-NH2, the N/C ratio increases to 0.282 compared to the PET-COONH2 surface (0.166). However, some nitrogen is present on the control SS surface (1.8%). Also detected on the SS surface were metals (Cr, Fe, Mo, and Mn) that are present in the native SS as metal oxides and hydroxides and S introduced during the Piranha cleaning step.36 Despite the optimal level of PEI achieved on both surfaces, from XPS overlayer calculations the dry thickness for the PEI layer is only 0.7 and 0.8 nm on the SS and PET-COOH surfaces, respectively.39 (39) The thickness calculation (d) assumes an electron takeoff angle of 55° and an inelastic mean free path (λ) for nitrogen photoelectrons of 3 nm.40 I ) I∞ exp[-d/(λ cos θ)]. In the equation, I is the measured intensity of the overlayer (in %) and I∞ is the intensity of an infinitely thick PEI layer ()33%). (40) Blomberg, E.; Claesson, P. M.; Fro¨berg, J. C. Biomaterials 1998, 19, 371-386.

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Table 2. Summary of the XPS High-Resolution C(1s) Data Derived from Curve-Fitting C1

C2

C3

C4a

C5a

sample

BE (eV)

%

BE (eV)

%

BE (eV)

%

BE (eV)

BE (eV)

PET PET-OH PET-COOH PET-COOH-PEI

285.0 285.0 285.0 285.0

53.5 53.2 58.8 59.9

286.6 286.5 286.7 286.5

22.7 28.8 16.1 24.4

289.0 289.0 289.1 288.9

23.8 18.1 25.1 15.7

291.1 290.9 291.0 291.3

292.8 292.2 293.4 292.4

a

π-π* shake-up satellite peaks.

Figure 3. AFM images for the (A) PET, (B) PET-COOH, (C) PET-COO-NH2, and (D) PET-PEG surfaces. The concentration of PEI used for grafting was 30 mg/mL.

ToF-SIMS analysis was also performed on both the PEImodified surfaces, and the positive-ion spectra (m/z 0-100) are shown in Figure 2. For the SS-NH2 surface (Figure 2A), three types of ions were observed: N-containing ions, hydrocarbon ions, and metal ions. The most abundant peaks are N-containing ions, which mainly arise from the fragments of repeat unit: Rn ( H (m/z 42 and 44) and Rn ( CH2 ( H (m/z 30, 55, 56, 57, 68, and 70). The hydrocarbon peaks (m/z 27, 39, 41, 43, 55, 57, and 69) arise from the fragments, which are due to the C-N cleavage in the backbone, loss of N, and most likely hydrocarbon contamination. The most intense peaks in the spectrum are the Cr and Fe ions, which come from the substrate SS. The high intensity of these ions is most likely due to their higher ionization probability and greater emission depth of small atomic species. The latter phenomenon has been observed for Si+ ion emission through polyelectrolyte

multilayers.41 For the PET-COO-NH2 surface (Figure 2B) the most intense peaks generated are also those that arise from the fragmentation of the PEI backbone. The only striking difference between the two surfaces is that the most intense ion is the C3H8N+ ion (m/z 58), which has a very low abundance in the spectrum for SS-NH2. We speculate that a high concentration of protonated amine groups exist in the PEI layer, thereby increasing the ionization efficiency of the PEI endgroup. The covalent immobilization of PEI with EDAC/NHS to the PETCOOH surface occurs at pH 7.4, and the isoelectric point of PEI is >10. On the other hand, the pH of the PEI solution during physical adsorption to the SS surface was 11.2, where very little protonation of PEI is expected.42 (41) Delcorte, A.; Bertrand, P.; Jonas, A.; Wischerhoff, E.; Mayer, B.; Laschewsky, A. Surf. Sci. 1996, 366, 149.

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Table 3. XPS Elemental Compositions (Atomic % and Ratios) for PEI- and PEG-Modified SS and PET Surfaces sample

%C

%O

%N

N/C

O/C

% Cr

% Fe

%S

other

PET-COOH PET-COO-NH2 PET-PEG PET-PEG + TSB SS (Piranha cleaned) SS-NH2 SS-PEG SS-PEG + TSB

68.8 66.9 68.8 67.6 24.8 43.2 60.8 60.9

31.3 22.0 28.9 29.5 55.2 37.1 36.7 34.4

0 11.1 2.4 2.9 1.8 12.1 1.4 2.9

0 0.166 0.035 0.043 0.072 0.282 0.023 0.048

0.455 0.329 0.420 0.436 2.225 0.858 0.606 0.565

8.9 4.5 0.9 1.8

2.5 2.8

2.7

4.1

Figure 4. Surface analysis of the SS-PEG and PET-PEG surfaces: (A) high-resolution C(1s) spectrum of the PET-PEG surface; (B) corresponding positive-ion ToF-SIMS spectrum of the PET-PEG surface; (C) high-resolution C(1s) spectrum of the SS-PEG surface; and (D) corresponding positive-ion ToF-SIMS spectrum of the SS-PEG surface. The concentration of PEI used for grafting was 30 mg/mL.

Grafting of PEG to the SS-NH2 and PET-COONH2 Surfaces. Linear PEG chains (methoxy-terminted PEG-aldehyde, MW 5000) were chemically grafted directly onto the SS-NH2 and PET-COO-NH2 surfaces by reductive amination in an attempt to generate a surface that was resistant to protein adsorption and bacterial adhesion. The grafting conditions were chosen to optimize the graft density. This was performed at the LCST (60 °C in 0.6 M K2SO4) of PEG.13,14 Under these conditions, PEG in the solution starts to phase separate as a result of the disruption of the hydration shell around the individual polymer chains. Subsequently, when an individual molecule first couples to the surface it exists in a collapsed state and does not repel the next molecular chain to reach (42) The pH of the PEI solution at a concentration of 30.0 mg/mL was measured to be 11.2. It has been shown that the number of charged amine groups decreases with increasing pH of the PEI solution (Smits, R. G.; Koper, G. J. M.; Mandel, M. J. Phys. Chem. 1993, 97, 57545751), and as a result, very few charges exist in the PEI backbone. However, we expect that the principle interfacial driving force for PEI self-assembly on SS is still electrostatic in nature because it has also been shown that only a small number of charged groups are needed to neutralize the opposite changes on the planar substrate (Claessen, P. M.; Paulson, O. E. H.; Blomberg, E.; Burns, N. L. Colloid Surf., A 1997, 123-124, 341-353).

the surface away from neighboring reactive sites. because this process is reversible, upon rehydration a PEG layer is regenerated with the maximum possible graft density necessary for optimal steric repulsion properties.24,25 The final degree of PEG coverage is, thus, controlled by the initial density of reactive groups on the surface. Both XPS and ToF-SIMS was used to evaluate the success of the PEG grafting to the SS-NH2 and PETCOO-NH2 surfaces. From the XPS elemental composition data in Table 3 and high-resolution C(1s) spectra in Figure 4, there is a clear indication that a high level of PEG grafting is achieved on both surfaces. That is, for the PETPEG surface the O/C ratio increases from 0.329 to 0.420, and the N/C ratio decreases from 0.166 to 0.035. For the SS-PEG surface, the O/C ratio decreases from 0.858 to 0.606, and the N/C ratio decreases from 0.282 to 0.023. The O/C ratios never reach the theoretical value of 0.5 for a pure PEG layer because an individual PEG chain of MW 5000 (radius of gyration: Rg ) 3 nm)43 is smaller than the sampling depth of XPS (6 nm). In the case of PET-PEG, the O/C is less than 0.5 as a result of a lower (43) Malmsten, M.; Van Alstine, L. M. J. Colloid Interface Sci. 1996, 68, 3751.

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oxygen content in the substrate, and for the SS-PEG surface, the higher O/C ratio arises from residual metal oxides that are not fully attenuated to the PEG overlayer. Finally, the large attenuation of the nitrogen signal from the underlying PEI layer also indicates that the surface has a high degree of PEG grafting. The high-resolution C(1s) XPS spectra of the PET-PEG (Figure 4A) and SSPEG (Figure 4C) surfaces also show that the surface consists mainly of PEG. Both spectra are dominated by the ether component (CsO, 286.5 eV), which contributes up to 86.5 and 84.1% for the total carbon species on the PET-PEG and SS-PEG surfaces, respectively. The C(1s) spectra for both surfaces show small levels of neutral carbon (CsH/CsC, 285.0 eV) and amide functionality (CdOsN, 288.2 eV). The latter component arises from the amide bond formation between PEG and PEI. Figure 4B,D shows the positive-ion ToF-SIMS spectra (0-100 m/z) for the PET-PEG (B) and SS-PEG (D) surfaces. The most intense ions generated from both surfaces come from the fragmentation of PEG, producing the characteristic fingerprint, as shown for ions of Rn ( H+ (m/z 43, 45, 87, and 89) and Rn ( CH2 ( H+ (m/z 31, 59, 71, and 73) structure. The most intense fragment ion is C2H5O+ (m/z 45), which is expected from PEG analysis by SIMS. The characteristic ions of PEI were still detected with very low intensities, suggesting that the PEI layer was fully covered by a substantial PEG layer. The only major difference between the two surfaces is the presence of Cr+ (m/z 51.9) and Fe+ (m/z 55.9) ions that are still detected even though the overlayer is approaching the thickness (6 nm) of the XPS sampling depth. However, they are of lower intensity than the SS-PEI surface (Figure 2A). It has been estimated from XPS and X-ray reflectivity that the SIMS emission depth of Si+ ions is greater than 10 nm, when polyelectrolyte multilayers uniformly cover a Si substrate.41 A decrease in emission depth with increasing ion size was also observed. Therefore, we speculate that the same phenomenon occurs with Fe+ and Cr+ ion emission through the PEI and PEG layers. Finally, to evaluate the homogeneity of the PEI and PEG layers, ToF-SIMS imaging was performed. Under the analysis conditions chosen (3-pA current, 600-ps pulse width), the lateral resolution of the ToF-SIMS images is 6-10 µm. The data are presented in Figure 5 (100 × 100 µm images). Parts A and B of Figure 5 show the image of the sum of several of the major nitrogen fragment ions and the total ion image for the PET-COOH-HN2 surface, respectively. After PEG grafting, the image generated from the sum of several of oxygen fragment ions (Figure 5C) and the total ion image (Figure 5D) are shown. Similarly, the images for the SS-NH2 and SS-PEG surfaces are shown in Figures 5E-G. All images on both substrates demonstrate that both steps in the modification on the two substrates are chemically uniform (on the micrometer scale). For the PET-PEG surface, the homogeneous nature of the surface is also confirmed by AFM measurements, as shown in Figure 3D, where the RMS value is significantly reduced compared to those of the other steps in the surface modification procedure (Figure 3A-C). From the AFM pictures of the SS-PEG surface (data not shown), the RMS values were of a magnitude similar to those achieved on PET-PEG, which is well below the reported values where topography has an influence on bacterial adhesion.44 Bacterial Adhesion to the Modified Surfaces. The initial bacterial adhesion experiments were performed on the SS-NH2(1)-PEG surface, and untreated SS, SS-clean (44) Verran, J.; Boyd, R. D. Biofouling 2001, 17, 59-71.

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Figure 5. ToF-SIMS chemical images for the (A) PET-COONH2, the sum of the nitrogen fragments from the PEI layer; (B) PET-COO-NH2, the total ion image; (C) PET-PEG, the sum of the major oxygen fragments from the PEG layer; (D) PETPEG, the total ion image; (E) SS-NH2, the sum of the nitrogen fragments from the PEI layer; (F) SS-NH2, the total ion image; (G) SS-PEG, the sum of the major oxygen fragments from the PEG layer; and (H) SS-PEG, the total ion image. The concentration of PEI used for grafting was 30 mg/mL.

(cleaned in the Piranha solution), and SS-NH2(1) were used as comparison surfaces. Our previous results have demonstrated that there is a significant difference in the adsorbed amount of PEI on SS when a 1 mg/mL solution concentration is used compared to 30 mg/mL. Consequently, the resulting grafted PEG surface is lower in surface coverage and was demonstrated to be less effective at reducing protein adsorption.36 The number of bacteria adhering was not different on the four surfaces, and within 2 h of exposure to the bacterial suspension, the final saturation level was reached. Similarly, the SS-NH2(30)PEG surface was also exposed to a suspension of Pseudomonas sp., and again similar numbers adhered on this surface as compared to those on the SS surface.36 The concentration of PEI used for surface modification was

Grafting of PEG To Reduce Bacterial Adhesion

Figure 6. Bacterial adhesion to the modified PET and SS surfaces. (A) Adhesion of Pseudomonas sp. to PET-PEI(1)PEG, PET-OH, PET-PEI, and untreated SS; number of cfu/ mL ) 2.0 × 107. (B) Adhesion of Pseudomonas sp. to PETcontrol, PET-PEI(30)-PEG, and untreated SS; number of cfu/ mL ) 2.7 × 106. (C) Adhesion of Pseudomonas sp. to PETcontrol, PET-PEI(30)-PEG, and untreated SS; number of cfu/ mL ) 1.6 × 107.

30 mg/mL, and the surface analysis is described above. Therefore, the optimal surface layer of PEG on SS was incapable of preventing bacterial adhesion even though there is a very high graft density, and a uniform layer of PEG is presented to the bacteria. Figure 6A-C shows the results for the adhesion of Pseudomonas sp. to the modified PET surfaces used in the study. In contrast to that of the modified SS substrate, the level of adherent bacteria was markedly reduced by

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grafting PEG to the PET surface. In Figure 6A, results are shown for adhesion to the PET-NH2(1)-PEG surface, with covalent grafting of the PEI from a 1 mg/mL solution. The data are presented as log cfu/cm2 versus time. The initial level of Pseudomonas sp. adhesion to the PETNH2(1)-PEG surface after 1 h was approximately log 3 cfu/cm2, or 1000 bacteria/cm2, and this value increased to log 4 cfu/cm2, or 10 000/cm2, after 5 h. In contrast, a level of log 5 to log 6 cfu/cm2 adhered to the untreated SS and PET-NH2(1) controls. Both the PET-OH and PET-NH2(1)-PEG surfaces were not significantly different after 5 h, and this could be due to variability in the experiment. The surface analysis results for this particular surface (data not shown) suggest that a very uniform but thinner layer of PEG is present on the surface, compared to the PET-NH2(30)-PEG surface (30 mg/mL PEI used). In this case, less PEI was coupled to the surface, and, therefore, fewer reactive groups were available for achieving the higher level of PEG grafting. In Figure 6B,C, the results are presented for the Pseudomonas sp. adhesion to the PET-NH2(30)-PEG surface in two separate experiments, where the PEI was grafted from a 30 mg/mL solution and a very high level of PEG grafting was achieved, comparable to the SSNH2(30)-PEG, as shown from XPS and ToF-SIMS measurements. Adhesion was compared to that of the PET and SS controls. In the first experiment (Figure 6B), the level of adhesion varied from log 4 to log 5 cfu, or 104-105 bacteria/cm2, for the entire time course of the experiment (5 h), compared to log 8 (108 bacteria/cm2) for the controls. This represents up to 4 orders of magnitude reduction in bacterial adhesion. In the second experiment (Figure 6C), the PEG coating also reduced the number of adhering Pseudomonas sp. very significantly (up to 2 orders of magnitude). Surface analysis revealed that chemically the two batches were identical; hence, the difference in adhesion could be associated with a difference in the peptide composition of the TSB used to precondition the surfaces. More peptide adsorption would allow for higher numbers of adherent bacteria. Protein Adsorption to the PEG Surfaces. Both XPS and ToF-SIMS analysis were performed on SS-PEG and PET-PEG (with 30 mg/mL PEI as the linker layer) after exposure to the TSB growth medium. The adsorption experiments were identical to that for the preconditioning of the surfaces prior to the bacterial adhesion experiments, that is, for 30 min in 1:7 TSB solution followed by PBS rinsing. The elemental compositions for the SS-PEG and PET-PEG after TSB adsorption are shown in Table 3. For both surfaces, there is a slight increase in the N/C ratio after exposure to the TSB, which may be associated with the adsorption of peptides. However, the greater error when measuring low surface concentrations with XPS places uncertainty in this assumption. In addition, the level of protein adsorption may be below that detectable by XPS when nitrogen is present, as previously demonstrated.14,45 To determine whether protein adsorption actually occurs to these “low-fouling surfaces”, ToF-SIMS analysis was performed before and after exposure of the SS-PEI(30)-PEG and PET-PEI(30)-PEG surfaces to the TSB media. ToF-SIMS has previously been shown to be capable of detecting very low levels of protein adsorption to PEG surfaces when XPS measurements showed that no protein adsorbed.45 This is due to the inherently higher surface sensitivity and specificity of ToF-SIMS. In the SIMS (45) Kingshott, P.; McArthur, S.; Thissen, H.; Castner, D. G.; Griesser, H. J. Biomaterials 2002, 23 (24), 4775-4785.

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Figure 7. ToF-SIMS data for the PET-PEI(30)-PEG and SS-PEI(30)-PEG surfaces after adsorption experiments with the TSB growth medium. The sum of the intensities of the amino acid fragment ions, C2H5S+ (m/z 61), C3H4NO+ (m/z 70), C3H8NO+ (m/z 74), and C4H6NO+ (m/z 84), is normalized against the sum of the two characteristic PEG fragment ions, C2H3O+ (m/z 43) and C2H5O+ (m/z 45). For the control samples, the amino acid fragment ions were not detected; hence, the data are not included in the figure.

ionization of proteins, characteristic immonium ions and their fragment ions are generated from the amino acids that constitute the backbone of the proteins.46,47 The ToF-SIMS data for the two surfaces before and after TSB adsorption are shown in Figure 7. The data show the sum of the intensity of selected nitrogen-containing ions of amino acids normalized against two PEG fragment ions. That is, the sum of the intensity of the amino acid fragment ions, C2H5S+ (m/z 61), C3H4NO+ (m/z 70), C3H8NO+ (m/z 74), and C4H6NO+ (m/z 84), is normalized against the sum of the PEG fragment ions, C2H3O+ (m/z 43) and C2H5O+ (m/z 45). These nitrogen fragments were chosen because they appeared in the spectrum acquired from a bulk film of TSB deposited on a silicon wafer,48 and they are absent in the spectrum for PEI. They originate from the fragmentation of methionine (C2H5S+), asparagine and proline (C3H4NO+), and threonine (C3H8NO+).47 The increase in intensity compared to that of the controls indicates the adsorption of some peptide or protein from the TSB solution, confirming speculations from XPS measurements. The similarity in the intensities for both PEG surfaces suggests that the level of adsorption is quite comparable, and, hence, the profound difference in bacterial adhesion to the two surfaces cannot be correlated with differences in peptide or protein adsorption. Discussion An optimal PEG (MW 5000) surface in terms of surface coverage has been created on two different substrates (SS and PET) in a two-step process. The common features of the attachment were (1) the use of a PEI linker layer to provide reactive amino groups for the PEG grafting and (2) grafting of PEG at the LCST. The only difference in the procedure was that the PEI layer was physically adsorbed to the SS surface but covalently attached to the PET substrate. This combination of immobilization methodology offers the best opportunity of creating a steric repulsive barrier against bacterial adhesion using linear (46) Mantus, D. S.; Ratner, B. D.; Carlson, B. A.; Moulder, J. F. Anal. Chem. 1993, 65, 1431-1438. (47) Wagner, M. S.; Castner, D. G. Langmuir 2001, 17, 4649-4660. (48) Wei, J.; Bagge-Ravn, D.; Gram, L.; Kingshott, P. Manuscript in preparation.

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PEG chains of MW 5000. Characterization of both surfaces by high-sensitivity chemical surface analysis (XPS and ToF-SIMS) after PEG grafting reveals that they are identical in terms of both surface uniformity and surface coverage, consisting of a very high graft density in both cases. For both surfaces, a thickness of 4-4.5 nm for the PEG layer is estimated from the attenuation of the N signal using XPS overlayer calculations, which is slightly more than the Rg (3 nm) of the PEG chains (MW 5000). This suggests that the attached PEG chains exist in a brush conformation, which is a necessary requirement for good nonfouling behavior.24,25 Both the SS-PEG and PET-PEG surfaces were tested for the adhesion of Pseudomonas sp. that was isolated from a fish processing plant. However, contrary to the expectations from surface analysis the adhesion results revealed that the two substrates differed markedly in their ability to prevent colonization. The SS-PEG surface was incapable of having any effect in the level of Pseudomonas sp. up to 5 h compared to controls, whereas the PETPEG surface showed up to a fourfold or 10 000 times reduction in bacterial adhesion. ToF-SIMS analysis showed that proteinaceous species adsorbed from the TSB solution in comparable levels. The results clearly demonstrate the importance of the covalent attachment of surface modifying agents if bacteria adhesion is to be prevented. The bacterial adhesion results raise the question of why one surface (the SS-PEG) cannot stop bacteria attachment whereas another is extremely effective, despite the surface chemistries of the final PEG layers being almost identical. We thought that the sterilization procedure (rinsing in 97% ethanol) prior to adhesion experiments might have a profound effect on the stability of the two different PEG layers. However, because the surface composition remained unchanged after sterilization we ruled this out as a possible explanation. Also, at the quite harsh conditions of the PEG grafting step (high salt concentration and temperature) no PEI delamination occurs. The profound difference in bacterial adhesion between the SS-PEG and PET-PEG surfaces, in the absence of a conditioning film effect, clearly points to additional mechanisms that may be used by bacteria to colonize the surfaces. The tenacity that bacteria show in adhering to surfaces appears significantly greater than that shown by mammalian cells. For example, it has been shown49 that the attachment and growth of cornea epithelial cells, which synthesize their own extracellular matrix proteins to help them attach to surfaces, can be prevented by PEG (MW 5000) layers grafted to plasma polymer layers at the LCST, even in the presence of serum proteins. This raises the question about possible explanations. It is known from the literature that Pseudomonas sp. produce biosurfactants, which are used to solubilize hydrocarbon material by micellization and increase biodegradation rates.50 We speculate that such mechanisms could be used by bacteria to desorb material and assist in their colonization of surfaces. In such a case, the SS-PEG system may not be stable enough in a bacterial environment compared to the covalent PET-PEG system. From the literature, it has been recently demonstrated that densely packed, physically adsorbed PEG-containing (49) Thissen, H.; Hayes, J. P.; Kingshott, P.; Johnson, G.; Harvey, E. C.; Griesser, H. J. Smart Mater. Struct. 2002, 11, 792-799. (50) Al-Tahhan, R. A.; Sandrin, T. R.; Bodour, A. A.; Maier, R. M. Appl. Environ. Microbiol. 2002, 66 (8), 3262-3268.

Grafting of PEG To Reduce Bacterial Adhesion

layers such as those based on Pluronics51 or hydrophobic surfactants21 can reduce the attachment of certain bacteria up to 1 order of magnitude. Maybe these surfaces would be more effective if covalently attached. However, from a speculative point of view there could be a number of other possible interrelated mechanisms that may explain our findings, including: (1) On the PET-PEG surface, apart from protein adsorption, quite possibly a small population of the PEI-PEG molecules are not covalently attached. These could be desorbed by bacteria and attachment using the mechanisms described above. (2) PEG may be incapable of repelling the EPS produced by the bacteria. Also, it is known that both Gram-positive52 bacteria and Gramnegative53 bacteria release natural virulent factors such as lipopolysaccharides and potent hydrolytic enzymes, which may also adsorb or even destroy the PEG layer over time. These hypotheses regarding the coating stability and bacterial attachment mechanisms need further elucidation. In addition, further investigations into the biochemical pathways used by bacteria to attach to surfaces in the absence of a conditioning film are clearly needed. Also, preventing the adsorption of conditioning film components is in itself not a trivial exercise and needs to be confirmed using very surface-sensitive methods such as ToF-SIMS.36,45 Our results are extremely promising, but some bacteria (1000/cm2) still adsorb to the best PET-PEG despite the high graft density. From ToF-SIMS analysis, peptide or protein adsorption is detected from the TSB solution in comparable levels on both the SS-PEG and PET-PEG surfaces. We have analyzed TSB by matrix-assisted laser desorption/ionization mass spectrometry (data not shown) and found that it consists of a number of low-molecularweight proteins (ca.