Tea Stains-Inspired Antifouling Coatings Based on ... - ACS Publications

Research Article pubs.acs.org/journal/ascecg

Tea Stains-Inspired Antifouling Coatings Based on Tannic AcidFunctionalized Agarose Liqun Xu, Dicky Pranantyo, Koon-Gee Neoh, and En-Tang Kang* Department of Chemical and Biomolecular Engineering National University of Singapore 4 Engineering Drive 4, Kent Ridge, Singapore 117576

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S Supporting Information *

ABSTRACT: It is well-known that tannic acid (TA) and its analogs bind strongly to various substrates to produce, for example, the familiar and unpleasant “tea stains”. Functionalization of a polymer or macromolecule with TA would confer the resulting biomacromolecules with similar binding or anchoring ability on many surfaces. To verify the hypothesis, the naturally occurring polysaccharide agarose (Agr) was functionalized with alkyl bromo moieties, followed by etherification with tannic acid under basic conditions via Williamson ether synthesis. The TA-functionalized Agr (AgrTA) so obtained can be deposited onto titanium (Ti), stainless steel (SS), and silicon surfaces via direct adsorption and intermolecular oxidative cross-linking. The AgrTA-deposited SS surfaces show good stability in flowing electrolytes of varying pH. The AgrTA-deposited SS surfaces can also effectively reduce the adsorption of bovine serum albumin and the adhesion of Escherichia coli and 3T3 fibroblasts. In perhaps what is an ironic twist, through proper molecular design, the undesirable “tea stains” have inspired the production of sustainable antifouling coatings. KEYWORDS: Tannic acid, Agarose, Antifouling, Tannic acid-containing polymer, Tea stains-inspired coating

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interactions,27−30 which are similar to the binding mechanism of the DOPA-enriched adhesive of MFPs.2,31 It has been reported that TA is capable of forming a versatile surface coating on gold, titanium (Ti), stainless steel (SS), and polymeric substrates.32−34 TA has also been utilized as building blocks for the formation of nano-/microparticles,35,36 capsules,37 multilayer films,38,39 mucoadhesives,40 cytoprotective cellin-shell hybrids,41 hydrogels,42 and graphene nanocomposites.43 Ejima et al. reported a one-step approach for the fabrication of a TA-based capsule via the metal-polyphenol complexation between TA and Fe3+.32 Sileika et al. utilized TA as the precursor to form macromolecular coatings on both inorganic and organic solid substrates.33 Zhang et al. deposited a TA coating on the polysulfone ultrafiltration substrate as an interlayer to anchor the thin and smooth polyamide selective layer.44 Although numerous attempts to develop TA-based coatings have been made, the incorporation of TA into polymeric materials and the direct use of TA-containing polymers for the preparation of functional surfaces and coatings have not been previously explored. In this work, TA-functionalized agarose (AgrTA) was prepared via etherification of aromatic hydroxyls in TA with alkyl bromide in bromo-functionalized Agr (AgrBr, Scheme 1). The ability of AgrTA to deposit onto various substrate surfaces

INTRODUCTION Recent developments in surface coating technologies have been widely inspired by the adhesive mussel foot proteins (MFPs).1−4 The unrivaled amino acid, 3,4-dihydroxyphenylalanine (DOPA), has been identified as the essential fraction of these MFPs that contributes to the adhesive properties.5,6 DOPA analogs, especially dopamine, can undergo oxidative self-polymerization under mild alkaline conditions, leading to the formation of adherent polydopamine layers on various substrate surfaces.2,7−9 DOPA and its analogous compounds have also been integrated into small molecules and macromolecules as active adhesive constituents for deposition of functional coatings.10−22 Although the use of MFPs-inspired approaches for the deposition of functional coatings has become commonplace in the past few years, the effort to exploit other nature-inspired materials for preparing universal coatings on a diverse array of substrates remains strong in both academic and industrial communities. People have consumed tea for centuries. Interestingly, tea drinking always leaves stains on the tea cup.23 Inspired by tea stains, a variety of phenolic compounds present in tea, red wine, chocolate, and plants have been identified for application as versatile universal coatings.24−26 Tannic acid (TA), an abundant phenolic dendroid found in plants and fruits,27 consists of a center glucose molecule with all five hydroxyl moieties esterified with two gallic acid (3,4,5-trihydroxybenzoic acid) molecules. The trihydroxyphenyl moieties exhibit strong interfacial binding ability through physical and chemical © 2017 American Chemical Society

Received: November 11, 2016 Revised: February 27, 2017 Published: March 3, 2017 3055

DOI: 10.1021/acssuschemeng.6b02737 ACS Sustainable Chem. Eng. 2017, 5, 3055−3062

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ACS Sustainable Chemistry & Engineering

Scheme 1. (a) Synthesis Routes of Bromo- and Tannic Acid-Functionalized Agarose; (b) Schematic Illustration of the Tea Stains-Inspired Approach To Prepare the AgrTA Coating on the Substrate Surface

of AgrTA2, the procedure was similar to that used for the preparation of AgrTA1, except that AgrBr2 (1.0 g, 0.70 mmol of alkyl bromide), TA (5.95 g, 3.50 mmol), K2CO3 (0.48 g, 3.50 mmol), and 80 mL of DMF were used. Deposition of AgrTA coatings on substrate surfaces. AgrTA was suspended in doubly distilled water (2 mg/mL). The pH of the mixture solution was adjusted to 8.5 using a 2 M NaOH aqueous solution. The mixture solution was stirred at 90 °C until the complete dissolution. After cooling to 37 °C, the pH of the solution was adjusted to 8.5, again using NaOH aqueous solution (2 M). The SS coupons, Ti foils, and silicon wafers were immersed in the AgrTA solution at 37 °C for 48 h. The AgrTA-coated substrate surfaces were washed with copious amounts of doubly distilled water and blown dried with purified argon. The resulting AgrTA1- and AgrTA2-coated SS (Ti or Si) surfaces were referred to as SS (Ti or Si)-AgrTA1 and SS (Ti or Si)-AgrTA2 surfaces. Protein adsorption onto the pristine and AgrTA-coated SS surfaces. Protein adsorption was assayed via measuring the surface compositions of protein-adsorbed SS substrates by X-ray photoelectron spectroscopy (XPS) according to the method reported in the literature.53 The adsorption of fluorescent BSA-FITC was assayed using a fluorescence microscope, according to the procedures described in the literature.51 Bacteria adhesion on the pristine and AgrTA-coated SS surfaces. Escherichia coli (E. coli, DH5α) was used for evaluating the anti-adhesion characteristics of the pristine and AgrTA-coated (before and after aging) SS surfaces to bacteria. The pristine and AgrTAcoated SS coupons, before and after aging in phosphate buffered saline (PBS) for 30 days, of 1 × 1 cm2 in size were placed in a 24-well plate and covered with 1 mL of bacterial suspension (5 × 107 cells/mL) at 37 °C for 4 h. After that, the solution in each well was removed, and the SS substrates were washed with PBS thrice. For scanning electron microscopy (SEM) observations, the substrates were fixed in 4 wt % glutaraldehyde aqueous solution, dehydrated with ethanol−water mixtures, and dried.54 The number of adhered bacteria on the pristine and modified SS substrates were quantified using the spread plate method.55 Cell adhesion on the pristine and AgrTA-coated SS surfaces. 3T3 fibroblast cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (v/v), L-glutamine (1 mM), and penicillin (100 IU/mL) in a humidified 5% CO2 incubator at 37 °C. After harvesting, the cells were diluted in DMEM to a concentration of 2 × 105 cells/mL. The pristine and surface-modified SS coupons of 1 × 1 cm2 in size were placed in a 24well plate and covered with 1 mL of medium with 3T3 fibroblasts at 37 °C for 4 days. After that, the medium in each well was removed,

was examined by static water contact angle measurements and X-ray photoelectron spectroscopy (XPS). The stability of the AgrTA coating on SS surfaces was studied by cyclic surface ζ potential measurements. Agr, a natural polysaccharide, has found biotechnological and biomedical applications in the form of beads for affinity chromatography and gels for electrophoresis and tissue cultures.45−48 It is low-cost, nontoxic, biocompatible, stable at body temperature, and nonimmunogenic. Agr is known to exhibit antifouling properties for proteins, bacteria, algae, and barnacle cyprids.49−52 Thus, conjugation of TA with fouling resistant Agr could confer good antifouling properties to the surface coatings. The antifouling properties of AgrTA coatings were investigated by protein adsorption, as well as bacterial and cell adhesion assays.

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EXPERIMENTAL SECTION

Materials. Type 304 stainless steel (SS) foils (0.05 and 0.5 mm in thickness) and titanium (Ti) foils (0.5 mm in thickness) were purchased from Goodfellow Inc. (Cambridge, UK). Silicon ((100)oriented single crystal silicon, Si) wafers were obtained from Unisil (Santa Clara, CA). 6-Bromohexanoyl chloride (BrHACl, 97%), bovine serum albumin (BSA), fluorescein isothiocyanate-labeled BSA (BSAFITC), and tannic acid (TA, ACS reagent) were purchased from Sigma-Aldrich Chemical Co. Agarose (Agr) was purchased from BioRad Laboratories Inc. Preparation of bromo-containing Agr (AgrBr). AgrBr was prepared via esterification reaction between Agr and BrHACl (Supporting Information). The resulting products, with BrHACl to agarobiose (basic repeating unit of Agr) molar feed ratios of 0.25 and 0.5, were referred to as AgrBr1 and AgrBr2, respectively. Preparation of tannic acid-functionalized Agr (AgrTA). For the etherification reaction, AgrBr1 (1.0 g or 0.33 mmol of alkyl bromide) and anhydrous N,N-dimethylformamide (DMF, 50 mL) were mechanically stirred at 50 °C. After complete dissolution of AgrBr1, the solution was purged with argon for 30 min at room temperature. TA (2.83 g, 1.66 mmol, 500 mol % relative to alkyl bromide) and K2CO3 (0.23 g, 1.66 mmol) were successively added under flowing argon. The reaction was mechanically stirred at 60 °C for 2 days under inert atmosphere. After cooling down to room temperature, the reaction mixture was filtered. The filtrate was poured into 200 mL of doubly distilled water (pH = 3). The cloudy solution was dialyzed with deionized water for 3 days. About 0.84 g of white AgrTA1 powder was obtained after lyophilization. For the preparation 3056


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Figure 1. 1H NMR spectra of (a) Agr, (b) AgrBr2, (c) TA, and (d) AgrTA2 in DMSO-d6 at 25 °C.

with 1H NMR spectrum shown in Figure 1a, was first esterified with 6-bromohexanoyl chloride (BrHACl) to introduce the alkyl bromo side chains. The resulting bromo-functionalized Agr (AgrBr), with BrHACl to agarobiose molar feed ratios of 0.25 and 0.5, were referred to as AgrBr1 and AgrBr2, respectively. The 1H NMR spectra of AgrBr1 and AgrBr2 (Figure S1a of Supporting Information and Figure 1b, respectively) show new chemical shifts at 1.40, 1.56, 1.71− 1.81, and 2.34 ppm which are characteristic proton signals of the 6-bromohexanoyl side chain.51 The degrees of substitution (DS’s) of alkyl bromide per agarobiose in AgrBr1 and AgrBr2 were estimated from the 1H NMR spectra to be about 0.103 and 0.243, respectively. The alkyl bromo groups in AgrBr were then substituted by TA in the presence of K2CO3. An excess amount of TA (5× relative to the alkyl bromide) was added to ensure a high degree of substitution of the alkyl bromo groups and to minimize the intermolecular cross-linking of TA and AgrBr. The obtained AgrTA prepared from AgrBr1 and AgrBr2 were referred to as AgrTA1 and AgrTA2, respectively. Figure S1b (Supporting Information) and Figure 1d show the respective 1H NMR spectra of AgrTA1 and AgrTA2. Besides the chemical shifts associated with the protons in Agr, the aromatic hydroxyl (8.64−10.32 ppm), aromatic (6.71−7.51 ppm), and glucoside (5.43, 6.00, and 6.44 ppm) proton signals in TA (Figure 1c) are also discernible in the 1H NMR spectra of AgrTA1 and AgrTA2. The DS’s of TA per agarobiose in AgrTA1 and AgrTA2 were determined from the respective 1H NMR spectra, using the integrals of the methine peak of the glucoside ring in TA at 6.44 ppm and C1′-H and C2-OH protons in the range of 4.93−5.35 ppm. The molar ratios of TA to agarobiose are about 0.104 and 0.115 (Table 1), respectively,

and the SS substrates were washed one time with PBS. The cells were stained with 4,6-diamidino-2-phenylindole (DAPI) according to the manufacturer’s manual, and observed using a Nikon A1R inverted fluorescence microscope. The number of adhered 3T3 fibroblast cells was counted using NIH ImageJ software (http://imagej.nih.gov/ij/). Statistical analysis of the results over 3 fluorescence microscopy images was carried out using one-way analysis of variance (ANOVA) with a Tukey post hoc test. Characterization. The chemical structures of TA and Agr derivatives were characterized by 1H NMR spectroscopy on a Varian 600 MHz spectrometer. XPS measurements were carried out on a Kratos AXIS Ultra HSA spectrophotometer equipped with a monochromatized Al Kα X-ray source (1468.71 eV photons). SEM (JEOL JSM 5600 LV) was used for imaging the adhered bacteria on the SS substrates. An electrokinetic analyzer (SurPass, Anton Paar) comprising an adjustable-gap cell was used to determine the zeta (ζ) potential of the SS surfaces. The static water contact angle measurements were carried out on an optical video contact angle system (OCA-15-plus, Dataphysics). The thicknesses of AgrTA coatings on silicon wafer surfaces were measured by ellipsometry on a variable angle spectroscopic ellipsometer.56 The topography of the AgrTA coatings on silicon wafers was investigated by atomic force microscopy (AFM) in “tapping mode” in air on a Nanoscope IIIa AFM from Digital Instrument, Inc.

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RESULTS AND DISCUSSION Synthesis of tannic acid-functionalized agarose (AgrTA). Etherification of aromatic hydroxyls with alkyl bromide (Williamson ether synthesis) can be carried out efficiently with a suitable base, NaHCO3 or K2CO3. The abundant trihydroxyphenyl moieties in tannic acid (TA) facilitate the preparation of TA-based conjugates via Williamson ether synthesis. To prepare AgrTA (Scheme 1a), agarose (Agr), 3057


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ACS Sustainable Chemistry & Engineering Table 1. Characteristics of the Bromo- and TA-Functionalized Agr AgrBr1 AgrBr2 AgrTA1 AgrTA2

BrHACl/agarobiosea molar feed ratio

Alkyl bromide per agarobioseb

0.25:1 0.5:1

0.108 0.243

TA/alkyl bromide molar feed ratio

4:1 4:1

TA per agarobiosec

efficiency

0.104 0.115

41.2%d 47.8%d 96.3%e 47.3%e

a

Basic repeating unit of Agr. bDetermined from the 1H NMR spectra: using the integrated area ratios of methylene protons at 1.56 ppm and C1′-H and C2-OH protons in the range of 4.97−5.34 ppm. cDetermined from the 1H NMR spectra: using the integrated area ratios of methine protons in the glucoside ring at 6.44 ppm and C1′-H and C2-OH protons in the range of 4.93−5.35 ppm. dEsterification efficiency (ratio of obtained versus theoretical alkyl bromide per agarobiose). eEtherification efficiency (ratio of obtained TA versus alkyl bromide per agarobiose).

phenomenon probably has resulted from the weaker physical interactions between the AgrTA and Si surfaces. The successful deposition of AgrTA coatings on the substrate surfaces was also confirmed by static water contact angle measurements (Figure S5, Supporting Information). The pristine SS, Ti, and Si surfaces have static water contact angles of about 73°, 60°, and 86°, respectively. The static water contact angles of the corresponding SS, Ti, and Si surfaces decrease to 17°, 15°, and 44° after deposition of AgrTA1, and to 39°, 29°, and 51° after coating of AgrTA2. Since the single crystal silicon wafer has an atomically flat surface, the thicknesses of the AgrTA1 and AgrTA2 coatings on its surface were measured by ellipsometry to be 19.4 ± 5.0 and 5.5 ± 1.9 nm, respectively. The surface topography of the AgrTA-coated silicon wafers was subsequently investigated by AFM. Figure 3 shows the representative AFM images of pristine and AgrTA1- and AgrTA2-coated silicon wafers. The scan sizes of all the AFM images are 5 μm × 5 μm. The pristine silicon wafer shows a smooth surface, with a root-mean-square roughness (Rq) of 0.3 nm. After deposition of the AgrTA coatings, the surface roughness values increase significantly to 2.2 and 1.6 nm, respectively, for the Si-AgrTA1 and Si-AgrTA2 surfaces. The successful deposition of AgrTA coatings on the SS surfaces was further verified by zeta (ζ) potential measurements. As shown in Figure 4A, the ζ potentials of the pristine SS surface increase with the decrease in pH, with an isoelectric point (IEP) at pH of about 5. The AgrTA1- and AgrTA2coated SS surfaces exhibit negative charge from pH 3 to 10. The negatively charged AgrTA coatings over this pH range may be due to the dissociation of hydroxyl groups in AgrTA, or specific ion adsorption of solution to the AgrTA coating surfaces.57 The ζ potential measurements were carried out by placing two AgrTA-coated SS surfaces with a rectangular size of 1 cm × 2 cm parallel to each other and forming a microslit with a separation of about 100 μm. An electrolyte solution (1 mM KCl) was forced through the channel with a flow rate of up to about 600 mL/min. Thus, the ζ potential measurements can also be used to investigate the durability of the AgrTA coatings under varying pH and dynamic conditions. Figure 4B shows the ζ potentials of SS-AgrTA2 surfaces between pH 3 and 10 after 8 test cycles. The cyclic erosion/abrasion by the flowing electrolyte with varying pH does not significantly affect the quality of AgrTA2 coating. Only a slight increase in the ζ potentials was observed between pH 3 and 5. The ζ potentials of the SS-AgrTA2 surface become stable again after cycle 3. These results indicate that the AgrTA coatings are stable and durable after exposure to flowing electrolytes of varying pH. The stability of the AgrTA coatings on the SS surfaces was also assessed by conducting the contact angle measurement after they were aged in PBS at 25 °C for 30 days. As shown in Figure

for AgrTA1 and AgrTA2. Because of the steric hindrance, the extents of TA substitution in AgrTA1 and AgrTA2 are close, even in the presence of two times the alkyl bromo groups in AgrBr2 over that in AgrBr1. Deposition of AgrTA coatings on substrates. Deposition of the AgrTA coatings on a variety of substrates was achieved by immersing the substrates in the aqueous solution of AgrTA at pH 8.5 for 48 h, and by intermolecular oxidative coupling of TA under basic conditions33 between the adhered and free polymer chains in the solution. The presence of AgrTA coatings on the substrate surfaces was revealed by XPS measurements. Figures S2−S4 (Supporting Information) show the XPS wide-scan spectra of stainless steel (SS), Ti, and Si surfaces before and after coating of AgrTA. The enhanced C 1s signal intensities and attenuated underlying substrate signal (Fe 2p, Ti 2p, or Si 2p) intensities indicate the successful deposition of AgrTA coatings on the substrate surfaces. The C to underlying substrate signal (Fe, Ti, or Si) intensity ratios in the XPS wide-scan spectra (Figures S2−S4, Supporting Information) are shown in Figure 2. The [C]/[Fe],

Figure 2. XPS-derived C to underlying substrate signal (Fe, Ti, or Si) intensity ratios of the SS, Ti, and Si surfaces before and after AgrTA coatings.

[C]/[Ti], and [C]/[Si] ratios all increase after the deposition of AgrTA1 and AgrTA2 coatings. The significant increase in [C]/[Fe] ratios on the SS-AgrTA1 and SS-AgrTA2 surfaces indicates the effective coating of AgrTA on the SS surface. The C to underlying substrate signal intensity ratios of the AgrTA2 coated SS and Ti surfaces are higher than those coated with AgrTA1. Since the interactions between metal substrates and TA mainly rely on the formation of coordination complexes,23 the higher TA content in AgrTA2 results in thicker coatings on the SS and Ti surfaces. On the other hand, both AgrTA1 and AgrTA2 give rise to only a thin coating on the Si surface. This 3058


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Figure 3. AFM images of the surface morphology of the (a) pristine Si, (b) Si-AgrTA1, and (c) Si-AgrTA2 surfaces.

Figure 4. (a) Zeta (ζ) potential of the prisitine SS, SS-AgrTA1, and SS-AgrTA2 surfaces as a function of pH; (b) Cyclic ζ potential measurements of the SS-AgrTA2 surfaces.

S6 (Supporting Information), the respective contact angles of the SS-AgrTA1 and SS-AgrTA2 surfaces are 22° and 40° at 30 days, which are comparable to the values before aging (17° and 39°, respectively). Antifouling efficacy of the AgrTA-coated SS substrates. Since SS is of considerable importance arising from its widespread applications in biomedical devices, food industry, and underwater structures,58 the antifouling performance of the AgrTA-coated SS surfaces was investigated in detail. Protein adsorption plays a significant role in the subsequent adhesion of fouling organisms, as the adsorbed proteins may adversely affect the surface functionality.59,60 To qualitatively the examine protein fouling resistance of the AgrTA coatings, the pristine and AgrTA-coated SS surfaces were immersed in the PBS solution containing fluorescent BSA-FITC for 12 h. The insets of Figure 5 show the fluorescence microscope images of the pristine SS, SS-AgrTA1, and SS-AgrTA2 surfaces after exposure to the BSA-FITC solution. A large amount of BSA-FITC covers the pristine SS surface. The SS-AgrTA1 and SS-AgrTA2 surfaces have a comparatively small amount of BSA-FITC adsorption. The protein repellent capacity of AgrTA coatings can be attributed to the repulsive force associated with the presence of a hydration layer on the hydrophilic surface.59,61 The adsorption of BSA on the pristine and AgrTA-coated SS surfaces was also assessed from the XPS C 1s and N 1s corelevel spectral area ratios ([N]/[C] ratios).53 Figure 5 compares the [N]/[C] ratios of the pristine SS, SS-AgrTA1, and SSAgrTA2 surfaces before and after BSA exposure. The [N]/[C] ratios of the pristine SS, SS-AgrTA1, and SS-AgrTA2 surfaces are negligible before BSA exposure, as no nitrogen atom is present in the SS and AgrTA structure. After BSA exposure, the [N]/[C] ratios of the pristine SS, SS-AgrTA1, and SS-AgrTA2

Figure 5. Evaluation of protein adsorption, expressed as XPS-derived surface [N]/[C] ratios on the pristine SS, SS-AgrTA1, and SS-AgrTA2 surfaces after incubation in PBS (10 mM, pH = 7.4) containing 1 mg/ mL of BSA for 12 h. Inset: fluorescence microscopy images of the pristine SS, SS-AgrTA1, and SS-AgrTA2 surfaces after exposure to BSA-FITC PBS (10 mM, pH = 7.4) solution (1 mg/mL) for 12 h. The scale bar is 50 μm.

surfaces are 0.19, 0.08, and 0.05, respectively. As proteins have high [N]/[C] ratios,53 the large [N]/[C] ratio of the pristine SS surface would indicate nonspecific BSA adsorption of the surface. Based on the [N]/[C] ratios of the pristine SS, SSAgrTA1, and SS-AgrTA2 surfaces, BSA adsorption on the respective SS-AgrTA1 and SS-AgrTA2 surfaces is reduced by 58% and 74% in comparison to that on the pristine SS surface. The AgrTA2 coating is less susceptible to protein adsorption than the AgrTA1 coating. This phenomenon can be attributed to the thicker AgrTA2 coating on the SS surface, which 3059


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Figure 6. Representative SEM images of the (a) pristine SS, (b) SS-AgrTA1, and (c) SS-AgrTA2, and (d) the number of adhered E. coli cells per cm2, after exposure to E. coli suspension in PBS (5 × 107 cells/mL) for 4 h. (*) denotes significant difference (p < 0.05) between pristine and AgrTA-coated SS surfaces before and after aging in PBS for 30 days. (n.s.) denotes no significant difference (p > 0.05) betweern AgrTA-coated SS surfaces before and after aging.

(p > 0.05) in inhibition of bacterial adhesion between the AgrTA-coated SS surfaces before and after aging in PBS for 30 days. Thus, the AgrTA coatings on the SS surfaces are stable. Adhesion of 3T3 fibroblasts on the pristine and AgrTAcoated SS surfaces was evaluated after culturing for 4 days. As shown in Figure S7 (Supporting Information), the nuclei were stained with DAPI dye in blue. A large number of 3T3 fibroblasts readily adhere on the pristine SS surface, while the number of adhered cells on the SS-AgrTA1 and SS-AgrTA2 surfaces is significantly (p < 0.05) reduced. Thus, the AgrTA coatings are stable and can effectively inhibit the nonspecific adhesion of 3T3 fibroblast cells.

provides a better surface coverage and more uniform hydration layer to reduce protein adsorption. To evaluate further the antifouling performances of AgrTAcoated SS surfaces, resistance to bacterial adhesion of the AgrTA coatings was investigated. Adhesion of Gram-negative E. coli on the pristine SS, SS-AgrTA1, and SS-AgrTA2 surfaces is shown in Figure 6. Figure 6a−c show the SEM images of the pristine SS, SS-AgrTA1, and SS-AgrTA2 surfaces after exposure to the bacterial suspension for 4 h. A large number of bacterial cells are evident in the SEM image of the pristine SS surface. After deposition of AgrTA coatings, sparsely distributed bacterial cells are observed on the SS-AgrTA1 and SSAgrTA2 surfaces. The resistant to bacterial adhesion of the AgrTA coatings can be attributed to the hydration of the hydrophilic AgrTA layers.61 Quantitative assay of bacteria adhesion on the pristine and AgrTA-coated SS surfaces was performed using the spread plate method. Figure 6d shows the amount of adhered viable E. coli cells on the pristine SS, SSAgrTA1, and SS-AgrTA2 surfaces after immersion in the bacterial suspension for 4 h. The pristine SS surface is most susceptible to bacterial adhesion with an adhered E. coli density of 4.1 × 105 cells/cm2. The AgrTA coatings on the SS surfaces are effective in reducing the number of adhered cells. The number of adhered E. coli on the SS-AgrTA1 and SS-AgrTA2 surfaces decreased significantly (p < 0.05) to 1.3 × 105 and 0.8 × 105 cells/cm2, respectively. These results are consistent with the observed SEM images of Figure 6a−c. The SS-AgrTA2 surface exhibits better antibacterial adhesion (and antiprotein fouling) performance than the SS-AgrTA1 surface. These results indicate that the surface morphology of the AgrTA coating plays an important role in inhibiting bacterial adhesion and protein fouling, as the thicker AgrTA2 coating could provide better surface coverage, and thus a more uniform hydration layer. The numbers of viable bacteria adhered on the aged SS-AgrTA1 and SS-AgrTA2 surfaces are 1.5 × 105 and 0.9 × 105 cells/cm2, respectively. There is no significant difference

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CONCLUSIONS Tannic acid-functionalized agarose (AgrTA) was successfully prepared via Williamson ether synthesis. The obtained AgrTA were deposited onto the titanium, silicon, and stainless steel (SS) surfaces via direct adsorption and intermolecular oxidative cross-linking. The AgrTA-coated SS surfaces exhibited good durability after aging in flowing electrolyte with varied pH. In comparison to the pristine SS surface, the AgrTA-deposited SS surfaces reduced the adsorption of bovine serum albumin. Due to the formation of surface hydration layers on the AgrTA coatings, the AgrTA-coated SS surfaces also reduced the adhesion of Escherichia coli and 3T3 fibroblasts. AgrTA can confer fouling resistance to many other substrate surfaces of interest. The preparation of AgrTA can be extended to the functionalization of other macromolecules with intriguing antifouling, antibacterial, and stimuli-responsive properties.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b02737. 3060


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ACS Sustainable Chemistry & Engineering H NMR of AgrBr1 and AgrTA1 in DMSO-d6 at 25 °C (Figure S1); XPS wide-scan spectra of the SS, Ti, and Si surfaces before and after deposition with AgrTA (Figures S2−4); Static water contact angles of the SS, Ti, and Si surfaces before and after deposition of AgrTA (Figure S5); Static water contact angle evolutions of SS-AgrTA surfaces after aging in PBS (Figure S6); 3T3 fibroblast adhesion on the pristine SS and SS-AgrTA surfaces (Figure S7) (PDF) 1

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Liqun Xu: 0000-0002-6780-114X Koon-Gee Neoh: 0000-0002-2700-1914 En-Tang Kang: 0000-0003-0599-7834 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Singapore Millenium Foundation (SMF) for the financial support under the grant 1123004048.

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REFERENCES

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