Dextran- and Chitosan-Based Antifouling, Antimicrobial Adhesion, and

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Dextran and Chitosan-based Antifouling, Antimicrobial Adhesion and Self-Polishing Multilayer Coatings from pHResponsive Linkages-Enabled Layer-by-Layer Assembly Gang Xu, Peng Liu, Dicky Pranantyo, Koon-Gee Neoh, and En-Tang Kang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04286 • Publication Date (Web): 22 Jan 2018 Downloaded from http://pubs.acs.org on January 29, 2018

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ACS Sustainable Chemistry & Engineering

Dextran and Chitosan-based Antifouling, Antimicrobial Adhesion and Self-Polishing Multilayer Coatings from pH-Responsive Linkages-Enabled Layer-by-Layer Assembly

Gang Xu, Peng Liu, Dicky Pranantyo, Koon-Gee Neoh and En-Tang Kang*

Department of Chemical & Biomolecular Engineering National University of Singapore 4 Engineering Drive 4, Kent Ridge, Singapore 117585

* To whom correspondence should be addressed E-mail: [email protected] (E.T.K)

1 ACS Paragon Plus Environment

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Abstract To meet the demand for more environmentally-friendly antifouling coatings, and to improve the

fouling-resistant

coatings

with

both

‘offense’

and

‘defense’

functionalities,

polysaccharides (PSa)-based self-polishing multilayer coatings were developed for combating biofouling. Dextran aldehyde (Dex-CHO) and carboxymethyl chitosan (CMCS) were synthesized and alternatively incorporated via imine linkage into the multilayer coating in layer-by-layer (LbL) deposition. Surface plasmon resonance (SPR) technique was utilized to monitor the LbL assembly process. With increasing number of assembled bilayers, the antifouling performances against bovine serum albumin (BSA) adsorption, bacterial (S. aureus and E. coli) adhesion, and alga (Amphora coffeaeformis) attachment improved steadily. The self-polishing ability of the multilayer coatings was achieved via cleavage of pH-responsive imine linkage under acidic environments. As such, dense bacterial adhesion induced detachment of the outmost layer of the coatings. The efficacies of antifouling and antimicrobial adhesion were thus enhanced by the self-polishing ability of the multilayer coatings. Therefore, the LbL-deposited self-polishing dextran/chitosan multilayer coatings offer an environmentally-friendly and sustainable alternative for combating biofouling in aquatic environments.

Keywords: Dextran aldehyde, carboxymethyl chitosan, pH-cleavable, self-polishing, antifouling, layer-by-layer deposition, and surface coatings


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Introduction Polysaccharides (PSa) are carbohydrate macromolecules comprising of monosaccharides joined together by glycosidic linkages. They exist in abundance in nature, such as in plants, microorganism (fungi and bacteria), algae, and animals.1-3 Recently, naturally occurring PSa has attracted much attentions in biomedical and biomolecular fields4-6 due to their abundance, biocompatibility7 and biodegradability.8,9 Among the various PSa, dextran, a branched glucan, is commonly employed as an antithrombotic agent10 and as a volume expander in the case of hypovolaemia.11 In comparison to other PSa, dextran is soluble in polar solvents and dextranfunctionalized substrates are hydrophilic. However, up to now, the fouling-resistant efficacy of dextran and its derivatives, are rarely reported.12 On the other hand, chitosan, derived from the alkaline deacetylation of chitin, is known for its biocompatibility, biodegradability, and hemostatic and antimicrobial properties.13 Carboxymethyl chitosan (CMCS), one of the water-soluble chitosan derivatives, is known to have antimicrobial properties and is applicable in a wide pH range, allowing its development as a functional biopolymer of the future.14

Biofouling, the accumulation of micro- and macro- organisms on artificial surfaces, has been intensively addressed due to its economic and ecologic consequences.15-17 Biofouling is problematic, for instance, to marine structures arising from the bio-diversity and intricate ecosystem of the marine environment. The drag force is significantly increased once the hulls of marine vessels are infested with organisms, and more fuels have to be consumed to overcome the increased drag force. Furthermore, the by-products from increased fuel combustion, acidic CO2 and SO2 in particular, could critically impact the marine ecosystem by promoting ocean acidification.18 As for the economic impact, it is estimated that more than



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US$ 5.7 billion was spent annually to combat marine biofouling.19 As such, it is of urgency to develop an efficient and sustainable antifouling strategy for the marine environment.

During the past decade, various antifouling strategies have been developed to overcome the biofouling-induced

environmental

issues.20-24

Notably,

surface

modification

and

functionalization via molecular design are employed to impart substrates with anti-adhesion and antimicrobial efficacies.22,25-27 For example, poly(ethylene glycol) (PEG) was employed to resist protein adsorption and cell adhesion, although the stability and sustainability of PEG-based coatings are yet to be improved.28 Subsequently, zwitterionic polymers were designed to combat marine biofouling, since the electrostatically induced hydration in zwitterionic polymers is more pronounced.29 These surface coatings belong to foulingresistant polymer coatings, which act as a deterrent to foulants and fouling organisms. Alternatively, the self-polishing coatings were designed not only to resist the initial adhesion of micro- and macro- organisms, but also to refresh the antifouling capability or efficacy by detachment of the heavily fouled outermost layer.30-32 The detached coating layers should also be biocompatible, biodegradable and environmentally-benign, to prevent secondary pollution to aquatic lives and ecosystems.

Stimuli-responsive linkages have been widely applied in drug delivery systems as the release of pro-drugs can be controlled by regulating, for example, the pH values.33-35 pH-sensitive linkages, such as imine, hydrazide, oxime and β-thiopropionate bonds undergo cleavage at specific pH range from 5.0 to 6.5,36-38 while they are stable slightly above the neutral pH. The pH value of seawater is typically basic (pH 7.5-8.4),39 in which these linkages undergo minimal or slow cleavage. Consequently, it is desirable to develop pH-cleavable linkages in


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self-polishing multilayer coating via layer-by-layer (LbL) deposition,40 to mitigate biofouling in the aggressive marine environment.

In this study, two polysaccharides (PSa), dextran aldehyde (Dex-CHO) and carboxymethyl chitosan (CMCS), were synthesized and employed to construct a self-polishing antifouling and antimicrobial multilayer coating via LbL deposition in aldehyde-amine reactions. As shown in Scheme 1a, a mussel adhesive-inspired polydopamine (PDA) layer was first deposited on the stainless steel (SS) surfaces (denoted as the SS-PDA surface). Subsequently, Dex-CHO was deposited on the SS-PDA surface via aldehyde-amine reaction,41 to form the SS-DEX surface. The first Dex-CHO/CMCS bilayer coating was completed by functionalization with CMCS (referring to as the SS-FF1 surface). The multilayer-coated SSDEXCHTn surface was subsequently prepared by alternative deposition of the hydrophilic Dex-CHO and the antimicrobial CMCS in aqueous solutions. Besides the SS surface, glass slides (GS), silicon wafer (Si) and Au-chips were also utilized as the substrates. Notably, surface plasmon resonance (SPR) technique was used to characterize the fabrication process of LbL-deposited multilayer coatings.42,43 The antifouling and antimicrobial adhesion efficacies of the as-prepared multilayer coatings were evaluated against protein adsorption (bovine serum albumin, BSA), bacterial adhesion (gram-positive bacteria S. aureus and gram-negative bacteria E. coli), and microalgal attachment (Amphora coffeaeformis). The stability and sustainability of the coatings were ascertained after 6-time injection of BSA protein in SPR measurements. Cytotoxicity of the coatings was assayed with 3T3 fibroblasts. In addition, the self-polishing ability of the as-prepared multilayer DEXCHTn coating was evaluated under slightly acidic condition of heavily fouled surfaces from bacterial adhesion (Scheme 1b).



Experimental Section Materials AISI-type 304 stainless steel (SS) foils of 0.05 mm in thickness were purchased from Goodfellow Ltd. of Cambridge, U.K. Medical grade silicone sheets with thickness of 1 mm were obtained from BioPlexus Inc., Los Angeles, USA. SPR Au chips (BR100405) were purchased from GE Healthcare Pte Ltd., Boston, USA. Dopamine hydrochloride (98%), tris(hydroxymethyl) aminomethane hydrochloride (Tris-HCl), sodium periodate, dextran (DEX, Mw 6000), chitosan (CHT, deacetylation degree ≥75%) and albumin-fluorescein isothiocyanate conjugate (BSA-FITC, product no. A9771) were purchased from SigmaAldrich Chem. Co. All other reagents and solvents (reagent-grade) were purchased from either Sigma-Aldrich or Merck Chem. Co. and were used without further purification. Dextran aldehyde (Dex-CHO)44 and carboxymethyl chitosan (CMCS)45 were synthesized according to the methods reported previously (Supporting Information). Gram-negative bacteria strain of Escherichia coli (E. coli, ATCC, 14948) and Gram-positive bacteria strain of Staphylococcus aureus (S. aureus, ATCC 12228) were obtained from American Type Culture Collection, Manassas, VA. The Live/Dead BacLight Bacterial Viability Kit L131152 was purchased from Molecular Probes Inc., Eugene, Oregon, USA. The seawater was drawn from 20 m depth and 50 m off the southern shoreline of St John’s Island in the Singapore Strait, and was filtered (0.2 µm membrane) prior to use.

Fabrication of layer-by-layer (LbL)-deposited multilayer coatings Stainless steel (SS) substrates of 2 × 2 cm2 in area were activated in piranha solution, and then ultrasonically washed with deionized water, acetone and ethanol. The resulting hydroxyl-enriched SS surfaces were denoted as the pristine SS surfaces. Polydopamine was used as the anchor layer on the pristine SS surfaces (SS-PDA), according to the procedures


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reported previously.46 The SS-PDA substrates were immersed in the aqueous solution of Dex-CHO (4 mg/mL) for 30 min, and then rinsed with deionized water. The obtained SSDEX surfaces were then immersed in the aqueous solution of CMCS (4 mg/mL) for another 30 min, and rinsed with deionized water, to obtain the first Dex-CHO/CMCS bilayerdeposition on SS surface (SS-DEXCHT1). Subsequently, substrates functionalized with 5 bilayers (SS-DEXCHT5 surface), 8 bilayers (SS-DEXCHT8 surface) and 11 bilayers (SSDEXCHT11 surface) were prepared by alternative deposition in Dex-CHO and CMCS aqueous solutions. As a control experiment to investigate the self-polishing ability, SSDEXCHT11 surfaces were immersed in 10 mM NaBH4 solution for 30 min,47 and denoted as SS-DEXCHT11(Reduced) surfaces, to reduce the imine linkages to secondary amines. In addition, the layer-by-layer (LbL) assembled multilayer coatings were also deposited on other substrates, including the glass slides (GS), silicon wafer (Si) and Au chips, using the same procedures as that for the SS substrates.

Antifouling and antimicrobial performances Protein Adsorption Assays The custom-built SPR sensor was used to measure the real-time protein adsorption on multilayer-coated Au chips. For the adsorption assays, the pristine and bilayer-functionalized Au SPR chips were exposed to a flow (50 µL/min) of HEPES buffer (pH 7.4) for 100 s. Subsequently, 0.1 mg/mL BSA protein in HEPES was injected at the same flow rate (50 µL/min) for 10 min. After which, the sensor chips were rinsed with HEPES for 5 min. SPR signals were recorded during the three processes, HEPES pre-rinsing, BSA protein injection, and HEPES post-rinsing. The shifts in SPR signal (∆RU) were used to quantify the amount of adsorbed protein.



Bacterial Adhesion Assays The antimicrobial and antifouling performance of the pristine and functionalized SS surfaces were evaluated using gram-negative bacteria E. coli and gram-positive bacteria S. aureus. One mL of bacteria with a concentration of 107 cells/mL was cultured according to the method reported previously.48 ImageJ software (http://imagej.nih.gov.libproxy1.nus.edu.sg/ij/) was used to quantify the number of adhered bacteria from fluorescence microscopy images.49 The fraction of bacterial adhesion was expressed as percentages relative to the cell number (both live and dead cells) observed on the pristine SS surface (100%). ANOVA analysis between the pristine and bilayer-coated surfaces was used for pairwise comparison.

Microalgal Attachment Assays The anti-adhesion performance against the microalgal Amphora coffeaeformis (UTEX B2080) was evaluated following the procedures described in the literature.50 ImageJ software was also used to obtain the statistics of adhered Amphora cells. The fraction of attached Amphora was expressed as percentages relative to the cell number obtained from the pristine SS surface (100%). ANOVA analysis between the pristine and bilayer-coated surfaces was used for pairwise comparison.

Cytotoxicity Performance Evaluation To assess the cytotoxicity of the multilayer coatings, standard 3T3 fibroblast cells were cultured following the procedures reported previously.51 The multilayer-coated substrates were immersed into 1 mL of cell suspension (50,000 cells/mL) and incubated at 37 °C for 24 h. The pure growth medium without the substrate was assayed as the non-toxic control. The results were expressed as percentages relative to the absorbance obtained from the control experiment.


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Self-Polishing Performance Evaluation Three immersion experiments were conducted and compared in the investigation of the selfpolishing ability of the multilayer coatings. The GS-DEXCHT11 substrates were immersed in an acidic aqueous solution (pH 5.5) to allow the hydrolysis of imide linkages.36,37 As the first control, the GS-DEXCHT11(Reduced) substrate was immersed in the acidic solution. In addition, the GS-DEXCHT11 substrate was immersed in seawater as the second control. The coating thicknesses of these immersed GS substrates were continuously determined at 1-h interval for the first 6 h and then at 6-h interval for 24 h, using the ellipsometry. In addition, SPR was also employed to evaluate the self-polishing capability of the multilayer coatings. The Au-DEXCHT11 and Au-DEXCHT11(Reduced) surfaces were pre-rinsed at a flow (10 µL/min) of ultra-pure water for 400 s. Subsequently, the acidic solution (pH 5.5) was injected at the same flow rate for 4000 s. After which, the sensor chips were rinsed with ultra-pure water. SPR signals were recorded for the three runs to evaluate the detachment of the multilayer coating. The relationship between the self-polishing property and the antifouling performance was further investigated. S. aureus and E. coli bacteria with a high concentration of 108 cells/mL were culture using the similar protocol, and then incubated with the SSDEXCHT11 and SS-DEXCHT11(Reduced) surfaces for 4 h and 8 h. The respective fluorescence images of adhered bacteria were obtained. In addition, the thickness of multilayer coating as a function of incubation time in 108 cells/mL of E. coli bacterial suspensions were also determined by ellipsometry.

Materials characterization Chemical structures of dextran and Dex-CHO were characterized by proton nuclear magnetic resonance (1H NMR) spectroscopy. Chitosan and CMCS were characterized by Fourier transform infrared (FT-IR) spectroscopy. The chemical composition of the polymer-



functionalized GS surfaces was determined by X-ray photoelectron spectroscopy (XPS). The growth of the multilayer coating on Au surface was monitored using a custom-built SPR and a variable angle spectroscopic ellipsometer. The surface wettability of the multilayer-coated surface was assessed using contact angle goniometry. Atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM) were utilized to evaluate surface morphologies of the functionalized surfaces. Details of all these characterization techniques are available in Supporting Information.

Results and Discussion Synthesis of dextran aldehyde (Dex-CHO) and carboxymethyl chitosan (CMCS) To confirm the successful oxidization of dextran to dextran aldehyde (Dex-CHO), the structural variances of dextran were evaluated using 1H NMR spectroscopy measurements. In Figure S1a (Supporting Information), the chemical shift at 4.95 ppm and the multiplet at 3.44.1 ppm in the 1H NMR spectrum of dextran are assigned to the anomeric proton 1 and protons at positions 2-6, respectively.52 Upon NaIO4 treatment, the six-carbon rings in dextran molecules are partially oxidized to two aldehyde groups at the ends.53 Correspondingly, several characteristic chemical shifts at 5.7-4.7 ppm have appeared in the 1

H NMR spectrum of Dex-CHO in Figure S1b, which are assigned to protons of the oxidized

saccharides (positions 1’, 2’, 5’ and 6’).54

Chitosan and carboxymethyl chitosan (CMCS) were characterized by Fourier transform infrared (FT-IR) spectroscopy, as shown in Figure S2. The main characteristic peaks of chitosan are discernible at the wavenumber of 1029 cm-1 and 1655 cm-1 (Figure S2a), attributable to the C-O stretch at C6 and the -NH- bend, respectively.55 After


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carboxymethlylation, two additional characteristic peaks are present at the wavenumber of 1409 cm-1 and 1600 cm-1 in Figure S2b. Both peaks are ascribed to the stretching vibration of -COOH groups,55 indicating the successful functionalization of carboxymethyl groups. Moreover, the maintained characteristic -NH2 peak at 1655 cm-1 indicates the feasibility of subsequent functionalization via amine-aldehyde reaction.

Layer-by-layer (LbL)-deposited multilayer coatings X-ray photoelectron spectroscopy (XPS) characterization X-ray photoelectron spectroscopy (XPS) was used to study the chemical compositions of surface coatings (Figure 1). In the C 1s core-level spectrum of the SS-PDA surface (Figure 1a), the five curve-fitted peak components with binding energies (BEs) at 284.6, 285.6, 286.2, 287.8 and 288.7 eV are attributable to the C−H, C−N, C−O, C=O, and O-C=O species, respectively.48,50 After deposition of a Dex-CHO layer, a new characteristic peak with BE at 287 eV, ascribed to C=N species in imine linkage,56,57 appears in the C 1s core-level spectrum of the SS-DEX surface (Figure 1b). Moreover, the intensity of C-O species increases significantly due to the coupling of dextran. Both the appearance of C=N species and the increased intensity of C-O species indicate the successful functionalization of DexCHO via aldehyde-amine condensation reaction. Subsequently, the intensity of C-O species in the C 1s core-level spectrum of the SS-DEXCHT1 surface increases further, resulting from the functionalization of CMCS layer (Figure 1c). Similar peak component contributions are observed in the C 1s core-level spectrum of the SS-DEXCHT11 surface (Figure 1d). In particular, the intensity of C=O species decreases while those of C=N and O-C=O species increase slightly, indicating that as more CMCS are deposited, more aldehyde groups are converted to imine linkages in the assembled bilayers. The [C]/[N] elemental ratio also decreases progressively from 13.0:1.0 (SS-DEXCHT1), 12.1:1.0 (SS-DEXCHT5), 10.8:1.0



(SS-DEXCHT8) to 9.8:1.0 (SS-DEXCHT11) (Figure 1e). The increasing nitrogen concentration is consistent with the presence of more amine/imine groups, as more CMCS molecules are deposited with increasing number of the assembled bilayers.

Growth of the multilayer coatings To characterize the layer-by-layer (LbL)-deposited multilayer coatings, surface plasmon resonance (SPR) technique was used to monitor the real-time thickness growth on Au-chip surface. The SPR signal shifts to higher response as more materials are bonded or attached on Au chip surface.58 In Figure 2a, the SPR signal increases significantly in the first 7 min, resulting from the formation of polydopamine layer on Au surface. The inset of Figure 2a reveals a complete cycle of the alternative injection of Dex-CHO (blue section) and CMCS (red section) aqueous solutions, in which the SPR signal spikes (initial attachment), levels off (association process) and drops sharply (buffer rinsing) for 2 times. As the time progresses, the number of deposited bilayer increases from 1 (Au-DEXCHT1 at 24 min) to 11 (AuDEXCHT11 at 190 min). Correspondingly, the SPR sensorgram increases from 24.8×103 RU to 30.1×103 RU with a homogeneous upward trend (~250 RU/bilayer), implying that the LBL-deposited multilayer coating is evenly fabricated on the Au chip surface.

Besides the measurement by SPR technique, the thickness of the multilayer coating on Si surface was also determined using an ellipsometer. As shown in Figure 2b, the thickness increases continuously with increasing number of assembled bilayers. The coating thickness of the Si-DEXCHT1 surface was about 42 nm. It increases to ~80 nm when 5 bilayers are deposited, and a final thickness of ~140 nm for the Si-DEXCHT11 surface, suggesting an average thickness of ~10 nm for each Dex-CHO/CMCS bilayer. The thickness monitored by SPR and the thickness determined by ellipsometry, vs. number of assembled bilayers are


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plotted in Figure 2b. In both cases, an approximately linear increase in thickness is observed during LbL assembly of the bilayers on the substrate surface. In addition, as more bilayers are deposited, there is a good correlation between coating thickness monitored by SPR technique and the thickness determined by ellipsometer, indicating the uniform and homogenous growth of the Dex-CHO/CMCS bilayers.

Surface properties of the layer-by-layer (LbL)-deposited multilayer coatings Surface wettability Static water contact angle (SWCA) goniometry was used to quantify the hydrophilicities of pristine and multilayer-functionalized glass slides (GS) surfaces. Figure S3 shows the water droplet images and the corresponding SWCA. The SWCA of the pristine GS surface is 42° and it decreases to 30° as the first dextran/chitosan bilayer is deposited. In comparison to the SS-DEXCHT5 surface (SWCA of 24°), the SWCA of SS-DEXCHT8 surface decreases to 14°. The lowest SWCA of 9° is observed for the GS-DEXCHT11 surface, indicating superhydrophilicity of the final multilayer coating. Thus, the hydrophilicity of the multilayer coating increases continually as more bilayers are assembled. In addition, the DEXCHT11coated stainless steel (SS), silicon wafer (Si) and Au-chip surfaces also exhibit comparable small SWCAs (Figure S4).

Surface morphology Figure 3 shows the atomic force microscopy (AFM) images of the Si substrates functionalized with different numbers of Dex-CHO/CMCS bilayers. The root mean square roughness (Rq) over an area of 5 × 5 µm2 of the Si-PDA surface is 16.8 nm (Figure S5a). It decreases to 6.7 nm after functionalization of the first bilayer (Figure 3a). With the deposition of more bilayers, the morphologies of functionalized surface become smoother. For example,



the respective Rq values of the Si-DEXCHT5 and Si-DEXCHT8 surfaces are 3.1 nm (Figure 3b) and 1.6 nm (Figure S5b). Finally, after deposition of 11 bilayers, a relatively smooth surface is obtained with a Rq value as small as 1.1 nm (Figure 3c). In addition to AFM images, FESEM images of the bilayer-coated surfaces were also captured to evaluate their surface morphologies (Figure S6). As shown in Figure S6a, a number of clusters are observed on the Si-DEXCHT1 surface. After deposition of 5 bilayers, the Si-DEXCHT5 surface becomes smoother with fewer aggregates. Finally, a rather uniform coating without sporadic spots is observed on the Si-DEXCHT11 surface. These phenomena are consistent with the LbL mechanism,46,48,59 in which the new layer fills in the uneven spaces of the outermost layer, as the deposition of bilayers progresses.

Antifouling and antimicrobial adhesion performances of the layer-by-layer (LbL)deposited multilayer coatings Protein adsorption assays Since protein adsorption is the initial stage of biofilm formation, which also facilitates the subsequent micro- and macro- adhesion, SPR technique was employed to quantitatively assess the anti-adsorption performances of the multilayer-coated surfaces. Figure 4a shows the SPR signal curves of Au-chips, functionalized with different numbers of bilayers, upon injection of bovine serum albumin (BSA) solution. After the association process and rinsing with buffer solution, the pristine Au surface registers the highest response unit (RU), reaching 3.5×103 RU. In contrast, SPR sensorgrams of the Au-DEXCHT1 and Au-DEXCHT5 surfaces are shifted to lower responses, with the lowest protein adsorption (only 1.7×102 RU) being observed on the Au-DEXCHT11 surface. The significant reduction in SPR responses suggests that protein adsorption on the multilayer-coated Au surface is substantially reduced. A better antifouling capability is achieved once more Dex-CHO/CMCS bilayers are assembled.


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In addition, the injection of BSA protein on the Au-DEXCHT11 surface was repeated 6 times to evaluate the sustainability and stability of its resistance to protein adsorption. As shown in Figure 4b, the SPR sensorgram shows minimal and uniform increases after each injection, and the total increase in SPR signal is only 3.2×102 RU after 6 cycles. The maintenance of antifouling capacity upon cyclic loading suggests stability and durability of the multilayer coatings on the surface.

Bacterial adhesion assays Bacterial adhesion on artificial surfaces is a critical step during the biofouling process,60 as it enables the subsequent biofilm formation. In this study, two bacteria strains, Staphylococcus aureus (S. aureus, gram-positive) and Escherichia coli (E. coli, gram-negative) were employed to study the antifouling and antimicrobial activities of the Dex-CHO/CMCSincorporated multilayer coatings. Live/Dead BacLight Kit-based method, in which the live and dead cells are distinguished with different colors, is used to observe the distribution of adhered bacteria. After incubation in the respective S. aureus and E. coli bacterial suspensions (107 cells/mL) for 4 h, a large number of viable cells (green spots) and few dead cells (red spots) are observed in Figure 5a, indicating the negligible antifouling and antimicrobial ability of the pristine SS surface. For the SS-DEXCHT1 surface, the number of adhered viable bacteria is markedly reduced, while more dead cells are observed (Figure 5b), indicating that the assembled Dex-CHO/CMCS bilayer has inhibited the bacterial adhesion and killed some of the adhered bacteria. After functionalization of 5 bilayers, the amount of viable cells is further reduced (Figure 5c), while the number of dead cells remains almost constant. Notably, the SS-DEXCHT11 surface exhibits only a small number of adhered bacteria (both viable and dead cells, Figure 5d). The marked reduction in adhered bacteria



can be ascribed to the enhanced antifouling and antimicrobial efficacies. As revealed by the SWCA and AFM measurements, the surface hydrophilicity increases progressively while the surface roughness decreases continually, as more bilayers are coated. The synergistic effect of the two surface properties contributes to a better antifouling performance.

For quantitative evaluation of the antifouling and antimicrobial performances of the multilayer-functionalized surfaces, the adhered bacteria were counted using the ImageJ software.49 Figure 6 shows the fractions of adhered S. aureus and E. coli on the pristine and functionalized SS surfaces. All results are normalized by defining the fraction of adhered bacteria (both live and dead cells) on the pristine SS surface as 100%. With increasing number of the assembled bilayers from 1 to 5, 8 and 11, the fraction of viable cells decreases correspondingly from 34% to 26%, 11% and only 4%. For the adhered dead bacteria, the fraction is 17% on the first bilayer-coated surface, attributable to the antimicrobial efficiency of the CMCS compounds. The dead fraction decreases to 12% on the SS-DEXCHT5 surface, and only 6% on the SS-DEXCHT11 surface. These quantitative results are consistent with the fluorescence microscopy images (Figure 5), confirming the antifouling and antimicrobial efficacies of the multilayer coatings. In addition, similar conclusions can be drawn from both qualitative and quantitative analysis of adhered E. coli bacteria, as shown in Figure 5e,f,g,h, and Figure 6, confirming the combined antifouling and antimicrobial efficacies of the surfaces against both gram-positive and gram-negative bacteria strains.

Microalgal attachment assays The antifouling capability of the pristine SS and functionalized SS substrates were also investigated by the attachment assay of Amphora coffeaeformis, a widespread micro-fouler in the marine environment.61 Figure 7 shows the florescence images of the substrate surfaces


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after exposure to Amphora suspension (105 cells/mL) for 24 h. A large number of adhered Amphora cells is captured on the pristine SS surface (Figure 7a). After functionalization of the Dex-CHO/CMCS bilayers, the number of adhered Amphora cells decreases significantly as the number of the bilayer increases (Figure 7b to 7d), indicating increasing resistance of the surface to microalgal attachment. The observed Amphora attachment on the pristine and bilayer-coated SS surfaces are in good agreement with the bacterial adhesion results (Figure 5), confirming that antifouling performance improves with increasing number of surfacecoated bilayers.

Figure 7e shows the quantitative assay of Amphora on the pristine and the functionalized SS surfaces. The attachment on pristine SS surface is set as the reference (100%), since the surface is most susceptible to Amphora attachment. In conformity with the observed fluorescence images, the fraction of Amphora attachment decreases progressively with increasing number of bilayers in the multilayer coating. The respective fractions of attached Amphora on the SS-DEXCHT1, SS-DEXCHT5 and SS-DEXCHT8 surfaces are 56%, 29% and 21%. The best antifouling performance against Amphora attachment is observed for the SS-DEXCHT11 surface, with the lowest fraction of attachment of only 7%.

Self-Polishing performance of the layer-by-layer (LbL)-deposited multilayer coatings Characterization of the SS-DEXCHT11(Reduced) surfaces The LbL-deposited multilayer coating can undergo self-polishing since the imine linkages between Dex-CHO and CMCS layers are cleavable under acidic conditions.33,36 To investigate the self-polishing performance of the SS-DEXCHT11 surfaces, the multilayer coating was treated with NaBH4 to reduce the imine linkages to secondary amines, denoted as the SS-DEXCHT11(Reduced) surfaces. As shown in Figure S7a, the SWCA of the GS-



DEXCHT11(Reduced) surface is found to be 12°, indicating the retention of hydrophilicity after the reduction reaction. In comparison to the Si-DEXCHT11 surface, the roughness (Rq) of the Si-DEXCHT11(Reduced) surface was maintained at the small value of 1.3 nm (Figure S7b).

Bacterial adhesion and microalgal attachment assays were conducted to assess the antifouling performance of the SS-DEXCHT11(Reduced) surface. In comparison to the SS-DEXCHT11 surface (Figure 5d, h), the number of adhered bacteria (for both S. aureus and E. coli) or attached Amphora on the SS-DEXCHT11(Reduced) surface increase slightly, as shown in Figure S8. These phenomena suggest that NaBH4-reduced multilayer coating has similar surface properties as the original SS-DEXCHT11 coating, including the surface wettability, surface morphology, and the antifouling and antimicrobial efficacies.

Evaluation of the self-polishing performance of the SS-DEXCHT11 surfaces To assess the self-detachability of the fouled outerlayer from the SS-DEXCHT11 surface, thicknesses of the coatings on the GS-DEXCHT11 and GS-DEXCHT11(Reduced) substrates immersed in an acidic solution (pH 5.5), as well as that of the GS-DEXCHT11 substrate immersed in seawater, were determined by ellipsometry as a function of exposure time. In Figure 8a, the thickness of the GS-DEXCHT11 surface decreases rapidly from 143 to 79 nm in the first 6 h of exposure to an acidic solution (pH 5.5), followed by a more gradual decrease until a final thickness of 39 nm was obtained after 24 h of exposure. Since the average thickness of one Dex-CHO/CMCS bilayer is ~10 nm (Figure 2), it is estimated that the outermost layer takes approximately one hour to detach from the multilayer coating. On the contrary, the GS-DEXCHT11(Reduced) substrate exhibits minimal decrease in thickness from 145 nm to 130 nm over a 24-h period in the acidic solution. The GS-DEXCHT11 surface


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exhibits an almost constant thickness of ~140 nm over the 24-h period of immersion in seawater, indicating that cleavage of the multilayer coating is negligible in seawater.

In addition, SPR technique was employed to ascertain the self-polishing performance of the resulting multilayer coating. As shown in Figure 8b, after injection of an acidic solution (pH 5.5) for 4000 s and rinsing with ultra-pure water, the SPR sensorgram of Au-DEXCHT11 surface is lower than the initial baseline, with a reduction of ~320 RU. In contrast, the AuDEXCHT11(Reduced) surface exhibits only a slight reduction in SPR response upon acidic injection. The SPR and ellipsometry results readily suggest that the LbL-deposited multilayer coating is self-polishing through sequential detachment of its outmost layers via cleavage of the imine linkages under an acidic environment (pH 5.5), while reduction of the imine linkages by NaBH4 treatment disables the self-polishing ability.

Enhanced antifouling performance of the self-polishing SS-DEXCHT11 surfaces To further evaluate the self-polishing effect of the surface in enhancing antifouling and antimicrobial adhesion performances, the pristine SS, SS-DEXCHT11(Reduced) and SSDEXCHT11 surfaces were incubated in S. aureus and E. coli bacterial suspensions of a higher concentration (108 cells/mL) for 4 h to 8 h. The high concentration of bacteria stimulates the production of an acidic environment, as bacteria decompose organics during their growth and metabolism.62,63 After incubation in bacteria suspension (108 cells/mL) for 4 h, both the SSDEXCHT11(Reduced) (Figure 9b) and SS-DEXCHT11 surfaces (Figure 9c) outperform the pristine SS surface (Figure 9a) in antifouling performance. In particular, some clusters of live bacteria are observed on the SS-DEXCHT11(Reduced) surface. The aggregation of live bacteria on the SS-DEXCHT11(Reduced) surface indicates a lower anti-adhesion ability of the surface as compared to that of the SS-DEXCHT11 surface. Furthermore, with the increase



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in incubation time to 8 h, the number of adhered bacteria on the SS-DEXCHT11(Reduced) surface (Figure 9b’) is increased substantially, while that on the SS-DEXCHT11 (Figure 9c’) surface remains almost constant. Similar results are observed for the E. coli adhesion assays (Figure 9 d,e,e’,f,f’). In the absence of self-polishing functionality, a larger number of adhered

bacteria

and

aggregation

of

live

bacteria

are

observed

on

the

SS-

DEXCHT11(Reduced) surface than that on the SS-DEXCHT11 surface.

In addition, the thickness profile of multilayer coating as a function of incubation time in bacterial suspension was determined by ellipsometry. As shown in Figure 10, the coating thickness of the GS-DEXCHT11 surface decreases continuously as a function of incubation time in high concentration of E. coli bacterial suspension (108 cell/mL). The coating thickness decreases slightly from 145 nm to 133 nm after 4 h, and decreases further to 100 nm after 12 h of incubation. With the incubation time extended to 48 h, the coating thickness decreases to 55 nm, indicating that about 9 Dex-CHO/CMCS bilayers have detached from the multilayer coating surface. In contrast, only a slight reduction in coating thickness is observed on the GS-DEXCHT11(Reduced) surface after incubation in the dense bacteria suspension for 48 h. The results of thickness measurements after incubation in the bacterial suspension are consistence with that observed in Figure 8, confirming the bacterial aggregation-induced self-polishing capability of the PSa-based multilayer coatings in the presence of dense bacterial fouling or adhesion.

Cytotoxicity test 3T3 fibroblasts were cultured to assess the cytotoxicity effect of the self-polishing multilayer coating via MTT assays. In Figure S9, the viability of 3T3 cells incubated in the control medium is normalized as 100%. The pristine SS surface exhibits minimal toxicity to the


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growth of 3T3 cells, with a high cell viability of 95%. After deposition of the DexCHO/CMCS bilayers on the SS surface, the cell viabilities decrease correspondingly. For example, 86%, 78% and 82% of 3T3 cells survived after 24-h of incubation with the SSDEXCHT1, SS-DEXCHT5 and SS-DEXCHT8 surface, respectively. The lowest viability of 74% is observed on the SS-DEXCHT11 surface, which contains the highest amount of CMCS. Nevertheless, this cell viability of more than 70%, relative to that of the pure medium, still indicates the biocompatibility and environmental-friendliness of the self-polishing multilayer coatings.



Conclusion A self-polishing multilayer coating, fabricated by alternative deposition of dextran aldehyde (Dex-CHO) and carboxymethyl chitosan (CMCS) in imine-linkage induced layer-by-layer (LbL) deposition, was developed for antifouling and antimicrobial adhesion applications. Surface plasmon resonance (SPR) was employed to characterize the LbL deposition process, as well as the stability of the multilayer coatings. The natural polysaccharide (PSa)-based multilayer coatings exhibited good performance against adsorption of bovine serum albumin (BSA), adhesion of both S. aureus and E. coli, and attachment of Amphora coffeaeformis. Notably, the antifouling and antimicrobial performances improved with increasing number of Dex-CHO/CMCS bilayers, attributable to the progressive increase in surface hydrophilicity and continuous decrease in surface roughness. The stability and biocompatibility of the multilayer coatings were ascertained by the cyclic protein loading test and MTT assays. The self-polishing effect of the multilayer coatings was evaluated by changes in coating thickness, as revealed by ellipsometry and SPR, under acidic condition. In comparison to the traditional multilayer coatings, the self-polishing multilayer coating exhibited enhanced antifouling and antimicrobial efficacies upon exposure to high concentrations of bacteria. The anti-adhesion performance of the self-polishing multilayer coating refreshed by detaching its outmost layer, while NaBH4 reduction of the inter-layer imine linkages disables the self-polishing capability. Thus, the PSa-based self-polishing multilayer coating provides an environmentally-friendly alternative to combat biofouling in aggressive aquatic environments, as well as mitigating biofouling-induced environmental problems.


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Associated Contents Supporting Information Supporting Information is available free of charge on the ACS Publications website. Synthesis of dextran aldehyde (Dex-CHO) and carboxymethyl chitosan (CMCS) (Section 1), materials characterization (Section 2), 1H NMR spectra of dextran and Dex-CHO (Figure S1), FT-IR spectra of chitosan and CMCS (Figure S2), static water contact angle of the pristine GS and GS-DEXCHTn surfaces, n=1, 5, 8 and 11 (Figure S3), and DEXCHT11-functionalized SS, Si and Au substrates (Figure S4), AFM images of the Si-PDA and Si-DEXCHT8 surfaces (Figure S5), FESEM images of the multilayer-coated Si surfaces (Figure S6), static water contact angle and AFM image of the GS/Si-DEXCHT11(Reduced) surfaces (Figure S7), fluorescence microscopy images of adhered S. aureus, E. coli and attached Amphora on the SS-DEXCHT11(Reduced) surface (Figure S8) and relative 3T3 cell viability via MTT assays (Figure S9).

Acknowledgement The authors would like to acknowledge the financial support for this study from Singapore Millennium Foundation under Grant No. 1123004048 (NUS WBS No. R279-000-428-592).



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Captions for Scheme and Figures Scheme 1. (a) Preparation of multilayer coatings via layer-by-layer deposition on SS surface, and (b) the self-polishing mechanism. Figure 1. XPS C 1s core-level spectra of the (a) SS-PDA, (b) SS-DEX, (c) SS-DEXCHT1 and (d) SS-DEXCHT11 surfaces, and (e) XPS wide-scan spectra of the SS-DEXCHTn surfaces (n=1, 5, 8 and 11). Figure 2. Characterization of the LbL-deposited multilayer coatings. (a) SPR time traces and a complete injection cycle (inset) of alternative deposition of Dex-CHO and CMCS, and (b) Thickness (determined by SPR and ellipsometry) vs. the number of assembled bilayers. Figure 3. AFM images of the (a) Si-DEXCHT1, (b) Si-DEXCHT5 and (c) Si-DEXCHT11 surfaces (Rq, rms roughness). Figure 4. (a) SPR sensorgrams of the pristine Au and Au-DEXCHTn surfaces (n=1, 5 and 11) against BSA injection and HEPES rinsing, and (b) SPR sensorgrams of the Au-DEXCHT11 surface against 6-time injection of BSA solution. Figure 5. Fluorescence microscopy images of the (a, e) pristine SS, (b, f) SS-DEXCHT1, (c, g) SS- DEXCHT5 and (d, h) SS- DEXCHT11 surfaces after exposure to (a-d) S. aureus and (e-h) E. coli (107 cells/mL) at 37 °C for 4 h. Scale bar: 50 µm. Figure 6. Fractions of S. aureus and E. coli adhered on the pristine SS and SS-DEXCHTn surfaces (n=1, 5, 8 and 11) after immersion in the bacterial suspension (107 cells/mL) at 37 °C for 4 h. (Three replicates for each surface and error bar represents standard deviation; *denotes significant difference (P < 0.05) as compared to the pristine SS surface using ANOVA analysis). Figure 7. Fluorescent microscopy images of attached Amphora on the (a) pristine GS, (b) SSDEXCHT1, (c) SS-DEXCHT5, (d) SS-DEXCHT11 surfaces, and (e) the respective fraction of attached Amphora after exposure to the Amphora coffeaeformis medium at 25 °C for 24 h. Scale bar: 50 µm. (Three replicates for each surface and error bar represents standard deviation; *denotes significant difference (P < 0.05) as compared to the pristine SS surface using ANOVA analysis). Figure 8. Characterization of the self-polishing property. (a) Ellipsometric thickness and (b) SPR sensorgrams versus time of the GS-DEXCHT11 (red line) and GS-DEXCHT11(Reduced) (black line) surfaces incubated in acidic solution (pH 5.5), and the GS-DEXCHT11 surface incubated in seawater (blue line in Figure 8a). Figure 9. Fluorescence microscopy images of the (a, d) pristine SS, (b, e) SSDEXCHT11(Reduced) and (c, f) SS-DEXCHT11 surfaces after exposure to (a-c) S. aureus and (d-f) E. coli (108 cells/mL) at 37 °C for (a-f) 4 h and (b’, c’, e’ and f’) 8 h. Scale bar: 50 µm. Figure 10. Thickness profile (determined by ellipsometry) of the multilayer coatings on the GS-DEXCHT11 and GS-DEXCHT11(Reduced) surfaces, as a function of incubation time in high concentration (108 cells/mL) of E. coli bacterial suspension.


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Scheme 1. (a) Preparation of multilayer coatings via layer-by-layer deposition on SS surface, and (b) the self-polishing mechanism.



Figure 1. XPS C 1s core-level spectra of the (a) SS-PDA, (b) SS-DEX, (c) SS-DEXCHT1 and (d) SS-DEXCHT11 surfaces, and (e) XPS wide-scan spectra of the SS-DEXCHTn surfaces (n=1, 5, 8 and 11).


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Figure 2. Characterization of the LbL-deposited multilayer coatings. (a) SPR time traces and a complete injection cycle (inset) of alternative deposition of Dex-CHO and CMCS, and (b) Thickness (determined by SPR and ellipsometry) vs. the number of assembled bilayers.



Figure 3. AFM images of the (a) Si-DEXCHT1, (b) Si-DEXCHT5 and (c) Si-DEXCHT11 surfaces (Rq, rms roughness).


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Figure 4. (a) SPR sensorgrams of the pristine Au and Au-DEXCHTn surfaces (n=1, 5 and 11) against BSA injection and HEPES rinsing, and (b) SPR sensorgrams of the Au-DEXCHT11 surface against 6-time injection of BSA solution.



Figure 5. Fluorescence microscopy images of the (a, e) pristine SS, (b, f) SS-DEXCHT1, (c, g) SS- DEXCHT5 and (d, h) SS- DEXCHT11 surfaces after exposure to (a-d) S. aureus and (e-h) E. coli (107 cells/mL) at 37 °C for 4 h. Scale bar: 50 µm.


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Figure 6. Fractions of S. aureus and E. coli adhered on the pristine SS and SS-DEXCHTn surfaces (n=1, 5, 8 and 11) after immersion in the bacterial suspension (107 cells/mL) at 37 °C for 4 h. (Three replicates for each surface and error bar represents standard deviation; *denotes significant difference (P < 0.05) as compared to the pristine SS surface using ANOVA analysis).



Figure 7. Fluorescent microscopy images of attached Amphora on the (a) pristine GS, (b) SSDEXCHT1, (c) SS-DEXCHT5, (d) SS-DEXCHT11 surfaces, and (e) the respective fraction of attached Amphora after exposure to the Amphora coffeaeformis medium at 25 °C for 24 h. Scale bar: 50 µm. (Three replicates for each surface and error bar represents standard deviation; *denotes significant difference (P < 0.05) as compared to the pristine SS surface using ANOVA analysis).


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Figure 8. Characterization of the self-polishing property. (a) Ellipsometric thickness and (b) SPR sensorgrams versus time of the GS-DEXCHT11 (red line) and GS-DEXCHT11(Reduced) (black line) surfaces incubated in acidic solution (pH 5.5), and the GS-DEXCHT11 surface incubated in seawater (blue line in Figure 8a).



Figure 9. Fluorescence microscopy images of the (a, d) pristine SS, (b, e) SSDEXCHT11(Reduced) and (c, f) SS- DEXCHT11 surfaces after exposure to (a-c) S. aureus and (d-f) E. coli (108 cells/mL) at 37 °C for (a-f) 4 h and (b’, c’, e’ and f’) 8 h. Scale bar: 50 µm.


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Figure 10. Thickness profile (determined by ellipsometry) of the multilayer coatings on the GS-DEXCHT11 and GS-DEXCHT11(Reduced) surfaces, as a function of incubation time in high concentration (108 cells/mL) of E. coli bacterial suspension.



For Table of Contents Use Only

Synopsis: Self-polishing multilayer coatings from layer-by-layer deposition of dextran aldehyde and carboxymethyl chitosan as an environmentally-friendly and sustainable alternative for combating biofouling.


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Dextran- and Chitosan-Based Antifouling, Antimicrobial Adhesion, and

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