Enzymatic waterborne polyurethane towards a robust and environmentally friendly anti-biofouling coating

Abstract

A novel anti-biofouling coating was designed and prepared by directly mixing antifouling enzymes (lipase or protease) with a castor oil-based waterborne polyurethane (WPU) dispersion. Covalent incorporation of the enzymes into the WPU coating was realized via the reaction of the excessive NCO groups in WPU and the NH₂ groups in the enzyme molecules as investigated using enzyme leaking and IR analysis. The incorporation of the enzymes had a plasticization effect on the WPU coating, e.g. the tensile strength decreased and elongation at break increased after addition of lipase to the WPU coating. SEM images and contact angles (CAs) analysis showed that the enzymes were evenly distributed in the coating surface. The enzymatic WPU coatings displayed robust catalytic capabilities, e.g. 70% initial activity after ten continuous usages, ca. 50% initial activity after storage at room temperature for 90 days and ca. 20% initial activity after incubating at 60 °C for 90 days were obtained for the WPU coating containing lipase. Additionally, the enzymatic WPU coating afforded a self-cleaning ability against lipid and protein stains. This robust and environmentally friendly enzymatic WPU coating may provide a new strategy for resisting biomolecular contaminations.

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Introduction

Biomolecules such as lipids, proteins and polysaccharides can easily attach to a material surface and cause biocontaminations. The lipid-stained surfaces are inaesthetic and insanitary due to the further adhesion of other pollutants. Reusable medical devices may be contaminated with the fats in adipose tissues and the soil left can pose a risk to patients.¹ Microfluidic channels meet the challenge in the reusability when dealing with oil species in chemical analysis.² Protein adsorption on biological implants will reduce the effect of the equipment and may lead to blood clots.³ Biomolecule-related fouling often results in monetary costs to vessels by increasing fuel costs due to drag.⁴ Various detergents and organic solvents have been developed to clean them, but these chemical substances are not only potentially contaminative to environments but also harmful to health after a long-term accumulation inside the human body.^5,6 Therefore, it is important to develop new methods to solve the above problems. The design of anti-biofouling coatings to eliminate contamination in situ has been of great interest. During the past few decades, many different synthesis strategies have been developed to design and fabricate superhydrophobic, superoleophobic and PEG-modified surfaces to realize anti-biofouling based on lipid or protein-repellent properties.^7–13 However, such surfaces are very difficult to design due to the ability of low surface tension liquids to spread on most of the surfaces.¹¹ Chemical composition and hierarchical microstructures are key elements in the design and the fabrication is often complicated.¹¹

Besides the synthetic approaches, biomimetic strategies have been recently developed to create bio-inspired anti-biofouling surfaces.^8,14 Enzymes are natural catalysts that can lower the activation energy of chemical reactions. Lipases can catalyse the hydrolysis of triglyceride to glycerol and fatty acid and have been widely used in the treatment of oil pollution.^15,16 Protease can catalyse the hydrolysis of the peptide bonds in proteins to form peptide fragments. Lipases and protease can increase the cleaning efficacy in biofouling treatment and help to minimize the need for manual brushing and scrubbing. In addition the enzyme-based anti-biofouling strategy is environmentally friendly and easy to achieve under mild operating conditions. Direct utilization of free enzymes is expensive; thus immobilization of the enzymes on a material surface is an effective way to improve the reusability of the enzymes. Interfacial immobilization of lipase by covalently grafting it on a glass substrate followed by incorporation into a coating has been reported.^17–19 Direct incorporation of enzymes into solutions prior to coating, represents a simple, economical and scalable method of producing enzymatic coatings.¹⁹ However, a large leakage of enzymes from the coating often occurs resulting in a limited lifespan.^20,21 Enzyme–nanotube conjugates have been shown to be highly efficient at retaining enzymes in coatings,^22–24 but this method requires complicated modification of the enzymes and has a potential security issue for the toxicity of carbon nanotubes.

In this study, castor oil-based WPU was used as a coating carrier for its environmental friendliness as well as its highly adjustable properties.²⁵ Furthermore, the WPU coating represents a potentially ideal polymeric matrix for multipoint and covalent immobilization of enzymes. Drevon directly added an enzyme to two-component waterborne polyurethane prior to polymerization and the enzyme was irreversibly attached to the polymeric matrix.²⁶ Different to the previous study, the WPU used in this research was one-component with iron groups in the polyurethane skeleton and the petroleum-based poly component was replaced by bio-renewable castor oil. Polyurethane dispersions (PUDs) with excess isocyanate groups were first prepared and then mixed with enzymes before coating on surfaces. The catalytic activities, reusabilities, stabilities and the anti-lipid/protein abilities of the coatings were investigated.

Experimental section

Materials

Castor oil and olive oil were purchased from Acros. Soybean oil (Arowana) and salad dressing (kewpie) were purchased from the market. Isophorone diisocyanate (IPDI), dibutyltin dilaurate (DBTDL) and triethylamine (TEA) were purchased from TCI. 2,2-Dihydroxy methyl butyric acid (DMBA) was purchased from Aldrich. Acetone, isooctane, copper acetate and 4 A molecular sieves were purchased from Sinopharm. Candida antarctica lipase B (CALB), trypsin from a porcine pancreas and casein were purposed from Sigma. Hexadecanoic acid and 4-nitrophenyl ester were purchased from Alfa Aesar. To remove residual water from the raw materials, castor oil was distilled at 120 °C under vacuum for 2 h, DMBA was dried at 60 °C for 3 h and acetone was incubated with 4 A molecular sieves overnight.

Synthesis of the castor oil-based PUDs

Stoichiometric castor oil, IPDI and DMBA were added to a 250 mL three-necked flask equipped with a mechanical stirrer, thermometer and a condenser with a nitrogen in/out let. A homogeneous mixture was obtained by stirring and then DBTDL was added drop wise. The reaction was carried out at 60 °C for 3 h under a nitrogen atmosphere. During the reaction appropriate acetone was added to reduce the viscosity and prevent gelation. After cooling down to room temperature, the solution was neutralized by the addition of TEA and stirred for 1 h. Then deionized water was slowly added to the solution under vigorous stirring (2000 rpm) to obtain stable dispersions. Finally the acetone was removed under vacuum and dispersions with a solid content of ca. 20 wt% were obtained. By variations of the DMBA content from 5 to 9 wt%, five aqueous PUDs were prepared and they had an identical NCO/OH value of 1.4.

Preparation of the enzymatic WPU coatings

The CALB or trypsin solution was mixed with the PUDs at a proportion of 20 μL, 50 μL and 100 μL per mL of latex. Then 1 mL of the mixtures was poured into a Teflon dish with a bottom diameter of 36 mm. The enzymatic latex was dried at room temperature for 7 days to form WPU coatings containing lipase (WPU-L) or trypsin (WPU-T). The coating could be peeled off from the dish with forceps. The obtained films had a thickness of ca. 20 μm and were identified as WPU-L20, WPU-L50 and WPU-L100 respectively. To determine the amount of enzymes released from these films, the films were immersed in phosphate buffer (20 mM, pH 7.2) for 6 h and the amount of enzymes in the solution was measured using the micro bicinchoninic acid (Micro BCA) assay (Pierce Biotechnology, Rockford, IL).

Characterization of the enzymatic WPU coatings

An infrared spectrophotometer (NICOLET iS50, Thermo Fisher Scientific, USA) equipped with Fourier transform analysis (FTIR) was used to analyse the surface chemical structure of the WPU-L films. Measurements were carried out using attenuated total reflectance (ATR). FTIR spectra were collected in the frequency range of 4000–500 cm⁻¹ using 32 scans and at a resolution of 4 cm⁻¹. Thermal gravimetric analysis (TGA) of the WPU-L films was performed on an SDT Q600 (TA Instruments, USA). Approximately 10 mg of the samples was heated from 25 °C to 600 °C at a heating rate of 10 °C min⁻¹ under a nitrogen atmosphere. Stress–strain measurements of the WPU-L films were carried out using a DMA Q800 (TA Instruments, USA) equipped with a tensile clamp. The specimens had a length of 5 mm, a width of 3 mm, and a thickness of 0.2 mm. A linearly increased force at a speed of 0.5 N min⁻¹ was applied to the films and the stress and strain were recorded instantly. Water contact angles (CAs) were measured using an optical contact angle meter (DSA100, Kruss, Ger) at ambient temperature with 10 μL of distilled water. The average value of three measurements at different positions on the surface was determined as the CA.

Catalytic activity of the enzymatic PUDs and WPU coatings

It was not feasible to directly detect the change of absorbance in the PUDs due to their opacity. Therefore, the hydrolytic activity of the lipase in the PUDs was evaluated using the hydrolysis of olive oil in a two-phase system based on a colorimetric determination of liberated free fatty acids in the organic phase. Typically, 5 mL of olive oil dissolved in isooctane (10%, w/v) was added to 5 mL of phosphate buffer (20 mM, pH 7.2) containing 5 μL of the PUDs–CALB. The reactant mixture was shaken at 200 rpm and 37 °C for 10 min. Then 1 mL of the upper layer was taken and mixed with 0.2 mL of the cupric acetate–pyridine reagent. After violent vibration the mixture was measured at 715 nm using a UV/VIS-Spectrophotometer (Unico 2800, Shanghai Experiment Equipment Co., China). One unit of lipase activity was defined as the amount of the enzyme required to liberate 1 μmol of free oleic acid per minute. The thermo and storage stabilities of the PUDs–CALB were examined by measuring the residual enzyme activities at regular intervals at 25 °C and 60 °C respectively.

Surface activity of the WPU-L coatings was determined using the hydrolysis of hexadecanoic acid and 4-nitrophenyl ester. In a typical process, WPU-L film corresponding to 1 mL of the PUDs mixed with 20 μL of CALB was cut into 1 cm² squares and dipped in 2 mL of phosphate buffer (20 mM, pH 7.2). Then 20 μL of hexadecanoic acid and 4-nitrophenyl ester (0.5 M) dissolved in acetone was added to the above buffer. The reaction system was incubated at 40 °C for 10 min and shaken at 50 rpms. Afterwards, the absorbance of the supernatant was measured at 410 nm. One unit of the WPU-L film was defined as the dimension of the film required to liberate 1 μmol of p-nitrophenol per minute.

Surface activity of the WPU-T coatings was determined using the hydrolysis of casein to tyrosine. WPU-T film (trypsin, 5% w/w) was cut into 1.5 cm × 1.5 cm squares and incubated with 1 mL of casein solution (20%, w/v). After incubation at 40 °C for 10 min, 2 mL of trichloroacetic acid (0.4 M) was added to the above solution and then the mixture was centrifuged at 5000 rpm for 10 min. The supernatant was mixed with 5 mL of Na₂CO₃ solution (0.4 M) and 1 mL of Forint phenol reagent, incubated at 40 °C for 20 min, and then the absorbance at 660 nm was measured. One unit of the WPU-T film was defined as the dimension of the film required to liberate 1 μmol of tyrosine per minute.

The thermo and storage stabilities of the WPU-L and WPU-T films were examined by measuring the residual enzyme activities at regular intervals at 25 °C and 60 °C respectively. The reusability of the films was determined by measuring the residual activity after sequential use, between two use cycles the films were incubated with phosphate buffer at 25 °C.

Anti-biofouling text

A mixture of olive oil and soybean oil (1 : 1, v/v), the common oils used in foods, was dropped and spread uniformly onto the WPU-L coatings. After incubation at 25 or 60 °C for a period of time the CAs were measured. A WPU coating without CALB was used as a control. The degree of changes in the CAs was used to describe the anti-lipid contamination ability. Salad dressing was spread to a word “foul” on the WPU-T coatings. After baking at 60 °C for 1 h, the salad dressing-shrouded coating was rinsed with water in a shaker. The degradation of the salad dressing on the coating was observed at the determined time point. A WPU coating without trypsin was used as a control.

Results and discussion

Physicochemical properties of the enzymatic WPU coatings

Incorporation of CALB or trypsin shows similar effects on the morphology, thermal, mechanical and hydrophilic properties of the WPU coatings. For conciseness, we only show the results related to CALB.

Transparent and smooth enzymatic WPU coating was obtained. Fluorescence microscopy images show that CALB is uniformly distributed in the WPU coating (Fig. 1a and b). In addition, the surface and cross section of the WPU coating had no obvious change after the incorporation of the enzyme and the microscopic structure was flat without large aggregations (Fig. 1c–f). These results suggest that the enzyme molecules had a good compatibility with WPU.

Fig. 1 Fluorescence microscopy images of (a) WPU coating and (b) WPU-L100 coating (CALB was pre-labelled with FITC, excitation light was 450–490 nm); SEM images of the surface of (c) WPU coating and (d) WPU-L100 coating; SEM images of the cross section of (e) WPU coating and (f) WPU-L100 coating.

The FTIR spectra of the synthesized WPU-L films are shown in Fig. 2. The peaks at 3335 cm⁻¹ and 1698 cm⁻¹ correspond to the N–H stretching vibration and C=O stretching vibration respectively, indicating the formation of urethane groups (–NH–COO–). The NH stretching vibration exhibits a strong absorption peak at around 3330–3400 cm⁻¹ arising from the hydrogen bonding between the NH and carbonyl groups, whereas the free NH stretching vibration appears at ca. 3493–3520 cm⁻¹. The band at 1698 cm⁻¹ was assigned to the hydrogen-bonded urethane carbonyl groups.

Fig. 2 IR spectra of the WPU-L20 film prepared by the mixing of the PUDs with CALB after drying at room temperature for 1 (black line) or 7 days (red line).

TGA was used to analyse the thermal resistance of the WPU films with different CALB contents (Fig. 3a). Five degradation stages were found for the WPU-L films. The weight loss of the WPU film before 200 °C was due to the evaporation of residual moisture. The WPU-L films had a degradation of CALB around 190 °C and the weight loss increased with the increase in CALB content. The weight percent of CALB in the films was calculated as 2.1, 4.5, and 8.2% for WPU-L20, WPU-L50 and WPU-L100 respectively using black WPU as a control. Alicyclic diisocyanate which has a limited heat resistance was used in this study. The degradations at around 260 °C and 310 °C were due to the fracturing of the weak urethane bonds. The addition of CALB to the WPU made the decomposition temperature of the urethane bonds decrease from 317 °C to 309 °C. This might result from the steric effect of the CALB in the PU polymer skeleton weakening the interaction between hard segments. The last two degradations at around 355 °C and 430 °C were related to the decomposition of the soft segments of castor oil.

Fig. 3 (a) TGA, (b) stress–strain curve and (c) contact angles of the WPU-L films containing different levels of CALB content.

The mechanical properties of the WPU-L films were measured using a stress–strain test (Fig. 3b) and the tensile strength and elongation at break were obtained. The WPU film showed a tensile strength of 7.2 MPa and an elongation at break of 181%. As the content of CALB increased from 2 to 8 wt%, a decrease of the tensile strength and an increase of the elongation at break of the films were observed. The WPU-L20 film had a tensile strength of 3.9 MPa which was decreased by 45% compared with the WPU film. When the content of CALB continued to increase the tensile strength had a slight decrease to 2.7 MPa while the elongation at break increased to 270%, which was similar to typical elastomeric polymers. Those changes were because the CALB worked as a plasticizer in the polymer. The covalently embedded CALB in the WPU films decreased the crosslinking density of the polymer mainly because the NCO group reacted with the NH₂ groups of CALB could not react with other molecular chains. In addition, the steric hindrance of CALB decreased the interactions between the molecular chains and enhanced the mobility of the polymer segments. Thus the addition of CALB decreased the strength and improved the elasticity of the WPU films.

The hydrophilicity of the WPU-L films was determined using CA measurement (Fig. 3c). With the increase of CALB content, the CAs decreased from 83.8 °C to 75.5 °C. This was due to the existence of hydrophilic CALB on the film surface. Five different places on the films were measured and no obvious difference in the CAs was found, indicating that CALB was distributed on the surfaces uniformly and homogeneous incorporation of CALB in the WPU polymeric matrix was achieved. For mass transfer resistance the catalytic activity of the enzymatic coating resulted primarily from the enzymes on the surfaces of the coating.²⁷ It can be predicted that the CALB on the film surface may have a catalytic activity.

Covalent immobilization of enzymes into the WPU coatings

In the BCA analysis less than 1% (w/w) of the protein loaded to the WPU films was detected in the incubation solution, and no activity was observed in the solution, indicating that almost all of the CALB and trypsin was irreversibly immobilized on the WPU films. IR was used to analyse the changes of the chemical groups of the WPU-L film during drying. After the PUDs with a NCO/OH value of 1.4 were prepared, CALB was immediately mixed with the PUDs and the mixture was dried at room temperature. When the emulsion began to transform to a transparent film, a piece of the film was detached and analysed using ATR-IR. A peak at 2270 cm⁻¹ corresponding to the NCO group was obviously observed suggesting that some isocyanate groups still existed when most of the water was evaporated (Fig. 2). After continual drying for three days the peak disappeared (Fig. 2). Based on the reactivity of the isocyanates with different active hydrogen compounds: aliphatic NH₂ > aromatic NH₂ > primary OH > water > secondary OH > tertiary OH > phenolic OH > COOH.²⁸ When CALB was mixed with the PUDs, the primary amine groups of CALB and water compete to react with the NCO groups on the PUDs. Because of the higher activity of the primary amine groups, some amino groups at the surface of the enzyme molecules can react with isocyanate to generate ureas, resulting in the covalent incorporation of enzymes into the WPU coating.

Catalytic activity of enzymatic WPU coatings

The effects of the hydrophilicity of the WPU films on the catalytic activity of CALB were analysed. DMBA was used as a chain extender in the preparation of WPU, and the carboxyl groups of DMBA endowed the films with a certain hydrophilicity. The hydrophilicity of the WPU films increased as the DMBA content increased. The apparent activity retention of CALB in the WPU films with different contents of DMBA are shown in Fig. 4a. The CALB in the WPU film with 6 wt% DMBA showed the highest activity. A relatively hydrophobic environment might favour the activity of lipase because of the interfacial activation and easy accessibility of the hydrophobic substrates.²⁹ However, the activity of CALB in the most hydrophobic WPU films (DMBA, 5 wt%) was lower, indicating that some other factors might affect the activity, such as conformational inactivation. In contrast to the lipase, trypsin had a favourable catalytic activity when the WPU coating had 8 wt% DMBA. A relatively hydrophilic environment might contribute to the activity of trypsin for the easy accessibility of hydrophilic substrates.

Fig. 4 (a) Effect of the DMBA content on the catalytic activity of the WPU-L20 and WPU-T coatings; (b) reusability of the WPU-L20 and WPU-T coatings; (c) thermostability and storage stability of the WPU-L20 coating at 60 °C and 25 °C, respectively; (d) thermostability and storage stability of the WPU-T coating at 60 °C and 25 °C, respectively.

Reusability

For a consideration of coating applications, reusability and long-term use of the biocatalytic coatings and films would be desirable. Thus, the reusability of the WPU-L20 and WPU-T coatings was tested. After the measurement of initial activity, the film was incubated in aqueous buffer for 2 h and then the activity was tested and this process was repeated. The hydrolytic activity of the films after ten washes retained ca. 70% of the initial activity for both enzymatic coatings (Fig. 4b). Furthermore, no activity was observed in the wash solution, suggesting that there was no loss of CALB or trypsin from the films and the observed activity resulted from enzymes incorporated into the films. A sharp initial loss of activity of the enzymatic coatings is usually found mainly due to the leaching of unfixed enzymes.²⁰ In this study, the film maintained almost unchanged activity in the first five cycles. Covalent incorporation made the enzymes efficiently entangle within the polymer matrix, thereby allowing enzymes to be retained and possess activity after long-term reuse of these materials.

Stability

Thermostability is one of the most concerning properties for enzyme-based materials in practical applications. Thermostability of CALB in the WPU films was investigated using incubation of the WPU-L20 film at 60 °C. The initial rapid deactivation occurred during the first month and then the activity of the films reached a stable stage with ca. 20% residual activity for at least two months (Fig. 4c). The WPU-L20 and WPU-T coatings retained ca. 50 and 70% residual activity respectively after incubation at room temperature for 90 days, while the half lifetime of the free enzymes was less than a week (Fig. 4c and d). The high stability for the enzymatic coating might be attributed to the covalent incorporation of enzymes within the polymeric network, which could help stabilize the conformation of the enzymes to stand against protein chain unfolding, a common mechanism of inactivation for free enzymes.

Anti-biofouling performance

Due to the stable hydrolytic activity of the WPU-L and WPU-T coatings, the surfaces could be considered as a kind of self-cleaning layer for anti-protein and anti-oil applications. The anti-lipid ability of the WPU films with different CALB contents was analysed using CA measurements. The CAs of the samples after different treatments are shown in Fig. 5. The original samples had deceased CAs with an increase in CALB content, which has been discussed above. After coating with a layer of hydrophobic oil the CAs of all the samples had an increase to ca. 84 °C. A decrease of the CAs of the oil-stained WPU-L film surfaces was observed under ambient conditions. For example, the CAs of the WPU-L20 films decreased from 83.74 °C to 82.8 °C, 80.7 °C and 77.6 °C after incubation at 25 °C for 12, 24 and 48 h respectively. The time needed to decrease the CAs to the original level decreased with the increase of CALB content. The films WPU-50 and WPU-L100 had the ability to restore the original CA within 24 and 12 h respectively. In addition, all samples could achieve this change within 1 h when incubation was at 60 °C. The control experiment showed that the CA of the surface coated with the mixture of oleic acid and glycerol had a negligible change (Fig. S1†). Therefore, the decrease of the CAs was attributed to the hydrolysis of the oil to the relatively hydrophilic glycerol and fatty acid catalysed by the CALB fixed on the film surface. Higher enzyme content and temperature facilitated the process, which could account for the previous results. The CAs of the oil-stained WPU films had no obvious change after these treatments indicating that the anti-lipid ability of the WPU-L films solely resulted from the catalysis of CALB. The prepared WPU films contained some residual moisture and have the capability to adsorb water molecules due to the hydrophilic component, 2,2-dihydroxy methyl butyric acid (DMBA), as proven by our previous study.²⁵ Therefore, we suggest that the water molecules used by lipase to catalyze the hydrolysis of the oil come from both the residual moisture and absorbed water molecules from the air.

Fig. 5 Contact angles of the WPU-L coatings containing different levels of CALB content after different treatments.

Proteins are another type of key component of many biofoulings. The anti-protein ability of the WPU-T coating was tested using the hydrolysis of salad dressing on the surface of the coating (Fig. 6). With gradual hydrolysis of the proteins in the sauce dressing to small water soluble molecules, the salad stain on the WPU-T coating disappeared after soaking in the water for 1 h (Fig. 6a). While on the control WPU coating without trypsin, the stain remained (Fig. 6b), indicating that the WPU-T coating can resist protein stains.

Fig. 6 Antifouling ability of (a) WPU-T and (b) blank WPU coatings covered with salad sauce.

Conclusions

In summary, robust and environmentally friendly anti-oil WPU coatings (films) have been designed based on covalent incorporation of antifouling enzymes. The enzymatic WPU coatings are capable of the hydrolysis of lipids and proteins on contact and without the release of enzymes. Covalent binding of the enzymes within a polyurethane network via the NH₂ groups of the enzymes and the NCO groups of the PUDs results in a markedly enhanced reusability and stability of the enzymes. The enzymatic coatings show highly selective surface bioactivity to selectively degrade lipids and proteins, facilitating the elimination of biomolecular stains. Such self-cleaning behaviour may help to reduce the consumption of water and chemical detergents for washing when applied to household or medical facilities. WPU coatings with covalently embedded enzymes represent a facile route to develop highly efficient anti-biofouling coatings. This approach may be extended to other antifouling enzymes and is promising for anti-biofouling applications.

Acknowledgements

The authors are thankful for the support from the National Natural Science Foundation of China (Grant No. 21376249, 21336010, 20976180), and the 973 Program (2013CB733604). National Major Scientific Equipment Development Project (2013YQ14040508).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra04583a

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