Tannic acid anchored layer-by-layer covalent deposition of parasin I peptide for antifouling and antimicrobial coatings

Abstract

Tannic acid can serve as an initiator anchor for surface functionalization. Parasin I is an antimicrobial peptide derived from histone. Multilayer coatings on stainless steel were prepared by alternative deposition of these two materials via the Michael addition/Schiff base reaction-enabled layer-by-layer (LBL) deposition technique. The as-prepared multilayer coating exhibits good resistance to Gram-negative bacteria (Pseudomonas sp. and E. coli), Gram-positive bacteria (S. aureus and S. epidermidis) and microalgae (Amphora coffeaeformis). The antifouling and antimicrobial efficacy increase with an increasing number of the assembled multilayers. The stability and durability of multilayer coatings were also ascertained by prolonged exposure to seawater. The LBL covalently deposited multilayer coatings are thus potentially useful as effective and environmental benign coatings to combat biofouling in marine and aqueous environments.

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1. Introduction

Biofouling, caused by the unwanted adsorption of a variety of microorganisms on artificial surfaces, has become a worldwide problem which costs an estimated 30–50 billion dollars annually.^1–3 In the marine and aquatic environments, once bacteria adhere to material surfaces, the voyage efficiency of boats and service life of submerged structures will be reduced.⁴ In order to resist the accumulation of microorganisms as well as prevent the biofilm formation, surface modification via molecular design is essential. Traditionally, biocidal coatings based on a release mechanism were commonly employed since these coatings can control the biofilm formation via releasing of metallic, organometallic and organic biocides.^5–8 However, these environmentally malign antifouling approaches are under scrutiny because of their detrimental effects on the marine ecosystem.^6,9 Thus, there is an urgent need to develop more environment-friendly antifouling surfaces.

Among the various techniques developed to confer substrate surfaces with antifouling properties,^10–12 tethering of nontoxic functional brushes on substrate surfaces has been suggested as an effective and environmentally benign coating technique.^13–15 For this coating method, a stable and durable initiators layer that anchors readily on the surface is of great importance.¹⁶ To a large extent, the initiator layer determines the type of resultant polymer brushes on surfaces.¹³ Recently, tea stains-inspired plant polyphenolic tannic acid (TA), was modified to form brominated TA (TABr) which was then employed as the primer anchor for surface-initiated atom transfer radical polymerization (SI-ATRP). The enhanced antifouling performance and good durability of the coating demonstrate that TA based anchor is a practical and valid candidate as the initiator layer for surface functionalization.¹⁴ Moreover, besides a stable and durable anchor layer on the surface, the sequential buildup strategy for the brush coating is also of critical importance.

Among developed coating strategies, layer-by-layer (LBL) deposition has been shown as an effective and versatile method for modifying surfaces with functional polymers.^17–22 The LBL deposition technique can involve non-covalent interactions, such as electrostatic forces,²³ halogen bonding,²⁴ hydrogen bonding²⁵ and charge-transfer interaction.²⁶ It can also involve covalent cross-linking within the LBL films.⁶ In contrast to non-covalent interaction, covalently assembled multilayer is expected to provide better stability and durability under harsh environments.^27,28

Michael addition/Schiff base interaction between catechols and thiols/amines under oxidizing conditions provides a simple means for stable covalent coupling.^29,30 On the other hand, parasin I, an amine-rich peptide derived from histone H2A in the catfish, is well known for its excellent antimicrobial property.^31–33 Nevertheless, its anti-adhesion and antifouling efficacy is still under investigation. Thus, it will be meaningful to fabricate parasin I-based coatings and analyze their antifouling efficiency toward various kinds of microfoulers, to further evaluate their potential applications in maritime industries.

In this study, we explore the covalent LBL deposition of tannic acid and amine-rich parasin I peptide, to fabricate low-fouling and antimicrobial polymer coating (Scheme 1). Commercial tannic acid was first anchored on stainless steel (SS) surface (SS-TA) through the formation of tridentate complexes of the trihydroxyphenyl moieties, followed by the covalent deposition of parasin I peptide (SS-TAPEP₁) via Michael addition/Schiff base reaction with the remaining trihydroxyphenyls. The multi-layer SS-TAPEP_n substrates were prepared by LBL alternative deposition of TA and the peptide. The antifouling efficiency of the resulting TAPEP_n coating was assayed by the adhesion of Gram-negative bacteria (Pseudomonas sp. and E. coli), Gram-positive bacteria (S. aureus and S. epidermidis) and microalgae (Amphora coffeaeformis).

Scheme 1 Schematic illustration of preparation of multilayer coatings via LBL deposition on SS surface.

2. Experimental part

2.1 Materials

Stainless steel foils (AISI type 304, Fe/Cr18/Ni10, 0.05 mm thickness) were purchased from Goodfellow Ltd., Cambridge, U.K. Parasin I peptide was purchased from ChinaPeptides Co., Ltd. Shanghai, China. Tannic acid (TA) was purchased from Sigma-Aldrich Chemical Co., St. Louis, MO. Gram-negative bacteria strain of Pseudomonas sp. (NCIMB 2021) was obtained from the National Collection of Industrial Marine Bacteria, Sussex, U.K. Gram-negative bacteria strain of Escherichia coli (E. coli, ATCC, 14948), Gram-positive bacteria strain of Staphylococcus aureus (S. aureus, ATCC 12228) and Staphylococcus epidermidis (S. epidermidis, ATCC 36984) were obtained from American Type Culture Collection, Manassas, VA. The LIVE/DEAD BacLight Bacterial Viability Kit L131152 was purchased from Molecular Probes Inc., Eugene, OR.

2.2 Polymer multilayer coatings prepared by covalent LBL assembly

Tannic acid-coated stainless steel substrates were prepared as reported in the previous literature.¹⁴ Briefly, the SS foils were first cut into 2 cm × 2 cm coupons and immersed in the piranha solution (H₂SO₄ (95–97%) : H₂O₂ (30%) = 3 : 1, v/v) for 30 min to oxidize the organic residues. Then, the SS coupons were subjected to sequential ultrasonic cleansing in deionized water, acetone, and ethanol for 10 min each. Subsequently, TA (2.0 mg mL⁻¹) was dissolved in aqueous NaCl solution (0.6 M, pH 8.5) in a Petri dish (ϕ 90 mm). Four coupons of SS were introduced into the solution and the Petri dish was kept sealed and agitated in an orbital shaker for 24 h to allow homogeneous anchoring of the initiator primer on the SS surfaces. The as-prepared TA-anchored SS substrates, denoted as SS-TA, were immersed in deionized water and ethanol for 5 min each to remove the loosely adsorbed salt and TA primer.

Subsequently, the resulting SS-TA substrates were exposed to an aqueous solution of parasin I peptide (2.0 mg mL⁻¹) for 1 h, then washed with deionized water to obtain the first TA/peptide bilayer coating on the SS surface, denoted as the SS-TAPEP₁ substrates. The multilayer-coated SS substrates were prepared by n-times of alternative immersion into TA and parasin I peptide solutions, and denoted as the SS-TAPEP_n substrates, where n represents the number of bilayer assembled. In addition, glass slides were also used as the substrates for the LBL deposition to facilitate measurement of coating thickness. The process for preparing polymer multilayer coatings on glass surface was the same as that on the SS surface.

2.3 Surface functionalization of Au electrodes

Prior to the modification, the gold electrode (GE) (2 mm diameter) was first pre-treated with freshly made piranha solution (H₂SO₄ : H₂O₂ = 7 : 3, v/v) for 5 min and then rinsed with water. Sequentially, the GE was polished with 0.3 and 0.05 mm alumina powder, cleaned with ethanol and deionized water for 5 min each. After that, the electrode was scanned in 0.1 M H₂SO₄ between 0.2 and 1.55 V at 100 mV s⁻¹ until a steady-state redox wave was observed.³⁴ The procedures for immobilization of the multiple polymer layers on GE surfaces were the same as that on SS and glass surfaces.

2.4 Bacteria adhesion assay

Marine bacteria, Pseudomonas sp. was cultured in nutrient-rich simulated seawater for 3 days according to the method described previously.³⁵ E. coli and S. aureus were cultured in aqueous nutrient broth for 1 day, as described previously.³⁶ S. epidermidis was incubated in tryptic soy broth at 37 °C overnight. After incubation, each bacterial suspension was centrifuged at 2700 rpm for 10 min to remove the supernatant. Subsequently, Pseudomonas sp. was washed twice with simulated seawater, E. coli, S. aureus and S. epidermidis were washed twice with phosphate buffered saline (PBS), and then resuspended at a concentration of 10⁷ cells per mL.

Each sample substrate was cut into 1 cm × 1 cm in size and placed in 24-well plates (Nalge, Nunc Int., Rochester, NY). One mL of each bacterial suspension was added to immerse samples and incubated under static condition at 37 °C for 4 h. After washing thrice with ultra-pure water to remove the non-adhered bacteria, each sample substrate was stained with LIVE/DEAD BacLight solution for 15 min. The live (appearing green) and dead (appearing red) bacterial cells adhered on the sample surfaces were distinguishable under a Nikon ECLIPSE Ti-U fluorescence microscope (Tokyo, Japan), equipped with a green filter (excitation/emission: 450–490 nm/500–550 nm) and a red filter (excitation/emission: 510–560 nm/605–685 nm).

2.5 Amphora coffeaeformis attachment assay

Amphora coffeaeformis (UTEX B2080) was maintained in F/2 medium at 24 °C under a 12 h light : 12 h dark regime for at least a week in tissue culture flasks and cultured according to that described in the previous literature.³⁷ The quantification of adhered Amphora coffeaeformis on substrate surfaces was achieved by the fluorescence microscopy technique and autofluorescence intensity-based procedures, as described in published literatures.^14,29

2.6 Characterization

Surface compositions of the modified SS substrates were determined by X-ray photoelectron spectroscopy (XPS) on a Kratos AXIS Ultra spectrophotometer. The XPSPEAK version 4.1 software was used in peak analysis, using the C 1s hydrocarbon peak at 284.6 eV as a reference for all binding energies (BEs). In spectral deconvolution, the full width at half-maximum (FWHM) for the Gaussian peaks was maintained constant for all components in a particular spectrum. Surface elemental stoichiometries were derived from the peak-area ratios, after correcting with the experimentally determined sensitivity factors.

Static water contact angles of the pristine and functionalized SS substrates were measured and captured at 25 °C using the sessile drop method with 2 μL water droplets on a telescopic goniometer (Model 100-00-(230), Rame-Hart Inc., Mountain Lake, NJ) with a magnification power of 23× and a protector of 1° graduation. Each static water contact angle reported was the average value from three substrates.

The topography of the polymer-modified substrate surfaces was investigated in a dry state by atomic force microscope (AFM), using a Nanoscope IIIa AFM from Digital Instruments, Inc. The root-mean-square (rms) roughness (R_s) of the surfaces was calculated by AFM software. To measure the thickness of multilayer polymer coating, the polymer-functionalized substrates were scratched using a razor blade, as described previously.³⁸

The thickness of multilayer polymer coating was also determined by electrochemical impedance spectroscopy (EIS) measurements on an Autolab PGSTAT 30 electrochemical workstation (Ecochemie Co., Netherlands). The impedance spectra were recorded using a 10 mV amplitude sinusoidal signal in the frequency range of 0.005–100 000 Hz. Data were recorded and processed using the WVASE32 software package.

2.7 Stability and durability of the LBL deposited multilayer

To assess the stability and durability of the LBL assembled multilayer on SS surfaces, the SS-TAPEP₁₁ substrates were exposed to filtered (0.2 μm membrane) natural seawater for 15 days. After the immersion, the substrates were rinsed with deionized water and dried under reduced pressure. The composition of each substrates surface was then determined by XPS analysis, and the antifouling efficacy was assayed by adhesion of bacteria and Amphora.

3. Results and discussion

3.1 Assembly of tannic acid/peptide multilayer coating on stainless steel (SS) surface

Tannic acid (TA) was first coated on the SS substrate surface as the initiator primer for subsequent covalently LBL assembly of parasin I peptide (Scheme 1). The trihydroxyphenyl moieties of TA readily anchor on the SS surface via metal chelation to form a tridentate coordination complexes layer (SS-TA surface).^39,40 By comparing the X-ray photoelectron spectroscopy (XPS) wide-scan spectrum of the SS-TA surface to that of the pristine SS surface (Fig. 1a and b), the characteristic signals of metallic elements, viz., the Cr 2p, Fe 2p and Ni 2p core-level signals with respective binding energies (BEs) at 577, 711 and 860 eV,¹⁴ become weaker after coupling of the TA initiator layer. However, the thickness of coated TA layer is still below the probing depth (8–10 nm) of XPS technique in organic matrices.⁴¹ The XPS C 1s core-level spectrum of SS-TA surface (Fig. 1c) can be curve-fitted with C–H, C–O and O–C=O components with respective BEs at 284.6, 286.2 and 288.4 eV,⁴² consistent with a TA-functionalized SS surface.

Fig. 1 XPS wide-scan and C 1s core-level spectra of the (a) pristine SS and (b and c) SS-TA surfaces.

The trihydroxyphenyl groups in TA are reactive and exhibit inherent affinity toward various polymer and metal substrates.^39,40,43 Consequently, as shown in Scheme 1, the SS-TA surface is a suitable platform for fabricating the antifouling and antibacterial surface with parasin I peptide^44,45 in the subsequent LBL disposition processes. In comparison to the wide scan spectrum of SS-TA surface, a new signal characteristic of the N 1s species appears in the wide scan spectrum of peptide-coated SS-TA substrate (SS-TAPEP₁, Fig. 2a). The respective BEs at 399.7 and 401.5 eV in the XPS N 1s core-level spectra are associated with the amine ((C)–N–) and protonated amine (NH₃⁺) species.⁶ Apart from the new N 1s signal, the XPS C 1s core-level spectra of the SS-TAPEP₁ surface can be curve-fitted into five peak components with BEs at 284.6, 285.6, 286.2, 287.8 and 288.7 eV (Fig. 2b), attributed to the C–H, C–N, C–O, N–C=O and O–C=O species, respectively.¹⁵ Both the appearance of N 1s signal in the wide-scan spectrum and the C–N and N–C=O peak components in the C 1s core-level spectrum confirm the coupling of peptide to SS-TA surface.

Fig. 2 XPS wide-scan, C 1s and N 1s core-level spectra of the (a and b) SS-TAPEP₁, (c and d) SS-TAPEP₅, (e and f) SS-TAPEP₁₁ and (g and h) aged SS-TAPEP₁₁ surfaces (aged substrates: immersion in filtered seawater for 15 days).

Fig. 2 shows the XPS wide-scan, C 1s and N 1s core-level spectra of the (c and d) SS-TAPEP₅ and (e and f) SS-TAPEP₁₁ surfaces. The XPS-derived surface compositions of the LBL functionalized SS surfaces (SS-TAPEP₁, SS-TAPEP₅ and SS-TAPEP₁₁) are summarized in Table 1. The presence of the C–N species in the XPS C 1s core-level spectra (Fig. 2d and f) are consistent with the immobilization of peptide-containing multilayers on the surface via Michael addition/Schiff base reaction. The respective [C]/[N] molar ratios are calculated from the XPS C 1s and N 1s core-level spectral area ratios (Table 1). The [C]/[N] molar ratios of tannic acid and parasin I peptide layers of the SS-TAPEP₁, SS-TAPEP₅ and SS-TAPEP₁₁ surface remain at 0.98, 1.20 and 1.29, respectively. In addition, the stability of LBL-functionalized surfaces was investigated by exposing them to filtered (0.2 μm membrane) seawater for 15 days. XPS analysis of the aged SS-TAPEP₁₁ surface (Fig. 2g–h and Table 1) reveals a slight increase in [C]/[N] molar ratio, in comparison to that of the as-prepared SS-TAPEP₁₁ surfaces, indicating the relative stability and durability of the LBL assembled multilayer coating.

Table 1 Surface composition, elemental analysis, static water contact angle and thickness of pristine and functionalized SS surfaces

Samples	Surface composition (molar ratio)	Static water contact angle (mean ± SD)	Thickness (mean ± SD, nm)
Pristine SS	—	66 ± 3°	—
SS-TAPEP1	[C] : [N] : [O] = 4.6 : 1.0 : 2.1	38 ± 3°	8 ± 4
SS-TAPEP5	[C] : [N] : [O] = 5.1 : 1.0 : 2.3	33 ± 2°	107 ± 3
SS-TAPEP11	[C] : [N] : [O] = 5.3 : 1.0 : 2.0	27 ± 2°	223 ± 2
Aged SS-TAPEP11	[C] : [N] : [O] = 5.5 : 1.0 : 1.8	32 ± 2°	203 ± 2

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The static water contact angles of surface-functionalized substrates were examined and summarized in Fig. S1,† (ESI). After the modification of first TA/peptide bilayer coating, the water contact angle of SS surface decreases from 66° (pristine SS surface) to 38°. The contact angle decreases continuously with increasing number of coated bilayer on the SS surface, and reaches a value of 27° for the SS-TAPEP₁₁ surface, indicative of strong hydration of the multilayer assembled surface.⁴⁶ The aged SS-TAPEP₁₁ surface retains a low water contact angle of 32°, demonstrating the stability of the hydration layer after prolonged exposure in seawater. These observations are consistent with the XPS analysis results, and confirm the successful coupling of stable multilayers.

The topography of the multilayer assembled surfaces was investigated by AFM, as shown in Fig. 3. R_s value of the multilayer-functionalized surfaces over an area of 5 × 5 μm² decreases from 3.5 to 1.5 nm after deposition of 11 bilayers. The reduction in surface roughness suggests the formation of more complex and denser polymer coatings on the SS surface with increasing number of bilayer via the LBL covalently deposition technique. The thickness of the multilayer coatings was analysed from the step-profile in the AFM images of the scratched surface. The coating thickness increases gradually from about 8 to 223 nm (Fig. 3a and c) after 11 polymer bilayers have been LBL covalently assembled onto the surface, indicative of an average bilayer thickness of about 20 nm. The thickness is consistence with that observed from the cross-sectional SEM image of the SS-TAPEP₁₁ surface (Fig. S2, ESI†). These results are also in good agreement with the measurement by electrochemical impedance spectroscopy (EIS, see below), which is an effective method to investigate the interfacial properties of modified electrodes.^47,48

Fig. 3 AFM images of the (a) glass-TAPEP₁, (b) glass-TAPEP₅ and (c) glass-TAPEP₁₁ surfaces. R_s = rms roughness, h = thickness of polymer multilayer coatings.

Significant changes and homogeneous enhancement in the electrochemical impedance spectra of the TA/peptide bilayers modified electrode are observed as a function of the number of assembled bilayer (Fig. 4a). The diameter of the semicircle parts increases to 8134 Ω after 11 bilayers were assembled, confirming that the TA/peptide bilayer could assemble on the surfaces via the layer-by-layer method. The growth of coating thickness alters the coating characteristics, such as ion transfer resistance and the dielectric capacitance.⁴⁹ Fig. 4b compares the coating thickness determined from AFM and EIS analyses. Both plots show that, except for the first bilayer, the thickness of assembled bilayer remains uniform at about 20 nm. The reduced thickness in the first bilayer could be attributed to differences in surface functionalities and morphology of pristine and TA/peptide functionalized SS surfaces.

Fig. 4 (a) EIS data of the TA/peptide bilayer assembled GE as a function of bilayer number. (a–g) n = 1, 2, 3, 4, 5, 8 and 11. (b) Thickness of LBL assembled multilayer coatings determined by EIS and AFM scratch analysis.

3.2 Antibacterial performance of LBL assembled SS surface

The antibacterial efficacy of multilayer assembled SS surfaces was evaluated using four different bacteria, viz., marine Gram-negative Pseudomonas sp., Gram-negative E. coli, Gram-positive S. epidermidis and waterborne Gram-positive Staphylococcus aureus (S. aureus). Live/dead two-color fluorescence method was applied, with viable cells appear green while dead cells appear red after staining under the fluorescence microscope. Fig. 5 shows the fluorescence microscopy images of the pristine and LBL-functionalized SS surfaces were obtained after exposure to the Pseudomonas sp. suspension for 4 h. A large number of viable cells adhered on the pristine SS surface (Fig. 5a), with a very small amount of dead cells (Fig. 5b), which is in agreement with previous reports that pristine SS surface exhibits high susceptibility to bacterial adhesion and colonization.^15,50

Fig. 5 Fluorescence microscopy images of the (a and b) pristine SS, (c and d) SS-TAPEP₁, (e and f) SS-TAPEP₅, (g and h) SS-TAPEP₁₁ and (i and j) aged SS-TAPEP₁₁ surfaces after exposure to Pseudomonas sp. (10⁷ cells per mL) for 4 h. Scale bar: 50 μm (aged substrate: immersion in filtered seawater for 15 days).

For the LBL assembled SS surface, the antibacterial and anti-adhesion performances are dependent on the number of functionalized bilayer on the substrates. For the SS-TAPEP₁ surface (Fig. 5c), an apparent reduction in live bacterial adhesion was observed in comparison to that of the pristine SS surface. The SS-TAPEP₅ surface (Fig. 5e) exhibits improved resistance to bacterial adhesion over that of the SS-TAPEP₁ surface. Only a few viable bacterial cells attach to the SS-TAPEP₁₁ surface (Fig. 5g), indicating a superior antibacterial fouling efficacy of the denser multilayer coating from the assembly of 11 bilayers. As the anti-adhesion property of layer-by-layer functionalized surface is dependent on the thickness of hydration layer which prevents the bacterial attachment, the enhanced antibacterial (adhesion) and antifouling performance of the SS-TAPEP₁₁ surface, evaluated from fluorescence images, is consistent with the results from static water contact angle measurements.

Fig. 5d, f and h show the bactericidal efficacy of functionalized SS surfaces. In comparison to the pristine SS surfaces, the concentration of dead bacterial cells adhered to the SS-TAPEP₁ surfaces (Fig. 5d) has increased substantially. This phenomenon is consistent with the antimicrobial activity of parasin I towards Gram-negative bacteria. However, as an interesting contrast to the viable cells attached, the concentration of dead bacteria changes only slightly with an increasing number of the assembled bilayers (Fig. 5f and h). As the total number of attached bacteria on the functionalized surfaces are decreasing, the similar dispersion of dead bacteria illustrates that the antimicrobial efficacy increases with a number of bilayers. The anti-adhesion and antifouling performance of present tannic acid-peptide multilayer and environmentally-friendly coatings are comparable to those of polydimethyl siloxane nanocomposites,⁵¹ polyethylenimine-scaffold soft material coatings⁵² and bifunctional block copolymer architecture,⁵³ reported in the recent literature.

As an important criteria for long-term antifouling applications, stability and durability of the LBL deposited multilayer coating should be evaluated. After 15 day exposure to filtered (0.2 μm membrane) natural seawater, the fluorescence images of aged SS-TAPEP₁₁ surface (Fig. 5i and j) reveal only a small number of attached bacteria. The number of adhered bacteria increases only slightly when compared to that of the as-prepared SS-TAPEP₁₁ surfaces. In addition, the persistence of antimicrobial ability was demonstrated by the presence of dead bacterial cells on the aged SS-TAPEP₁₁ surface in Fig. 5j. Thus, the stability and durability of the TA/peptide multilayer prepared by LBL covalently deposition has been ascertained. Similar conclusions can be obtained by analysing the fluorescence images captured from Gram-negative E. coli bacteria (Fig. 6), Gram-positive S. aureus bacteria (Fig. 7) and S. epidermidis bacteria (Fig. 8). Thus, the multilayer coated SS surfaces exhibit low susceptibility to both Gram-negative and Gram-positive bacteria. The proposed TA/peptide multilayer coatings prepared by LBL covalently deposition, indeed have good antimicrobial and anti-adhesion efficacy, indicative of their antifouling applications in maritime industries.

Fig. 6 Fluorescence microscopy images of the (a and b) pristine SS, (c and d) SS-TAPEP₁, (e and f) SS-TAPEP₅, (g and h) SS-TAPEP₁₁ and (i and j) aged SS-TAPEP₁₁ surfaces after exposure to E. coli (10⁷ cells per mL) for 4 h. Scale bar: 50 μm (aged substrate: immersion in filtered seawater for 15 days).

Fig. 7 Fluorescence microscopy images of the (a and b) pristine SS, (c and d) SS-TAPEP₁, (e and f) SS-TAPEP₅, (g and h) SS-TAPEP₁₁ and (i and j) aged SS-TAPEP₁₁ surfaces after exposure to S. aureus (10⁷ cells per mL) for 4 h. Scale bar: 50 μm (aged substrate: immersion in filtered seawater for 15 days).

Fig. 8 Fluorescence microscopy images of the (a and b) pristine SS, (c and d) SS-TAPEP₁, (e and f) SS-TAPEP₅, (g and h) SS-TAPEP₁₁ and (i and j) aged SS-TAPEP₁₁ surfaces after exposure to S. epidermidis (10⁷ cells per mL) for 4 h. Scale bar: 50 μm (aged substrate: immersion in filtered seawater for 15 days).

3.3 Resistance of the SS-TAPEP_n surfaces against microalgal attachment

Amphora coffeaeformis, a member of raphid diatoms, is widely used as a microfouler to evaluate the antifouling efficacy in marine environment. The anti-adhesion properties of the pristine and TA/peptide bilayer functionalized SS surfaces were challenged with Amphora cultures. Fig. 9 shows the corresponding fluorescence images of adhered Amphora after 24 h of immersion in the microalgae suspension. A high concentration of Amphora cells was observed on the pristine SS surface, as in the bacterial adhesion. Significantly less Amphora cells were found on the bilayer coated SS surface, especially on the SS-TAPEP₅ and SS-TAPEP₁₁ surfaces. This phenomenon indicates that LBL assembled multilayer coatings indeed have good antifouling efficacy, as is also evidenced by the water contact angle and bacteria attachment results. However, the fluorescence images can only provide a visual comparison between different surfaces on Amphora adhesion, so it is necessary to develop a more quantitative assay.

Fig. 9 Fluorescence microscopy images of the (a) pristine SS, (b) SS-TAPEP₁, (c) SS-TAPEP₅, (d) SS-TAPEP₁₁ and (e) aged SS-TAPEP₁₁ surfaces after immersion in the Amphora coffeaeformis solution at 25 °C for 24 h, and (f) their detachment from the pristine SS after ultrasonic treatment for 10 min. Scale bar: 50 μm (aged substrate: immersion in filtered seawater for 15 days).

According to previous reports,^14,29 the adhered Amphora cells can be thoroughly removed from surfaces by ultrasonic agitation, and their chlorophyll autofluorescence can be detected to quantify the attachment of Amphora cells. Complete detachment of algal cells from the fouled surface was achieved after 10 min of ultrasonic agitation, as no red spot of Amphora remained on surface after ultrasonication (Fig. 9f). Upon excitation at the wavelength (λ_ex) of 440 nm, the autofluorescence intensity of Amphora coffeaeformis cells detected at ∼690 nm exhibits a linear relationship with the Amphora cell concentration (inset of Fig. 10). Fig. 10 shows the results of quantitative assay of pristine and multilayer-coated SS surfaces. In comparison to the pristine SS surface (100%), the anti-adhesion efficacy of SS-TAPEP_n (n = 1, 5, 11) enhances progressively (Fig. 10). The percentage of attached Amphora cells reduces correspondingly to 59%, 14% and 5%. The aged SS-TAPEP₁₁ also retains the antifouling performance with only 18% adhesion. These results are in good agreement with the observations from fluorescence images investigation (Fig. 9).

Fig. 10 Percentage of settled Amphora on the pristine and functionalized SS surfaces after immersion in the 10⁵ cells per mL algal suspension for 24 h. Inset is the calibration curve of the standard algal suspension presented in logarithmic scale. Error bars denote the standard deviation from three replicates.

4. Conclusion

Robust polymer multilayer coatings have been fabricated via the layer-by-layer deposition technique for combating marine biofouling. The dense and durable multilayer coatings were prepared by covalent assembly of alternative layers of tannic acid (also serving as the initiator anchor) and antimicrobial parasin I peptide via Michael addition/Schiff base reaction. The anti-adhesion property of the as-prepared multilayer coatings was challenged with Gram-negative bacteria (Pseudomonas sp. and E. coli), Gram-positive bacteria (S. aureus and S. epidermidis) and microalgae (Amphora coffeaeformis). The multilayer coatings exhibit good resistance to bacterial and microalgal attachment. The antifouling efficacy improves with increasing number of the coated bilayer. Moreover, the antifouling efficiency of the multilayer coatings remains relatively unchanged after 15 days of exposure to seawater. Thus, the proposed multilayer coatings are potentially useful to the maritime industries as effective and environmental benign antifouling coatings.

Acknowledgements

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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Footnote

† Electronic supplementary information (ESI) available: Images of water droplets on pristine SS, SS-TAPEP₁, SS-TAPEP₅, SS-TAPEP₁₁ and aged SS-TAPEP₁₁ surfaces (Fig. S1). Cross-sectional SEM image of SS-TAPEP₁₁ surface (Fig. S2). See DOI: 10.1039/c5ra23374g

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