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Easy-cleaning surfaces functionalized with an interface-binding recombinant lipase
Colloids and Surfaces B: Biointerfaces 184 (2019) 110433
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Easy-cleaning surfaces functionalized with an interface-binding recombinant lipase
T ⁎
Liting Zhanga,b, Yaofei Sunb, Yibing Wangb, Xiaoli Wangb, Haifeng Zhuanga, Chao Chenb, , ⁎ Ping Wangc, a
Key Laboratory of Recycling and Eco-Treatment of Waste Biomass of Zhejiang Province, Zhejiang University of Science and Technology, Hangzhou, 310023, PR China State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology, East China University of Science and Technology, Shanghai, 200237, PR China c Department of Bioproducts and Biosystems Engineering, University of Minnesota-Twins City, St Paul, MN, 55127, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Surface fabrication Self-assembly Cellulose-binding domain Functional durability Easy-cleaning surfaces
Surface fabrication is an effective method for functional materials development. This work investigated the use of cellulose-binding domain (CBD) fused detergent lipase for fabrication of easy-cleaning surfaces. As a result, the CBD conjugated Lipase-A (LipA-CBD) demonstrated a multi-layer self-assemble on cotton fabric surface and enhanced hydrophobicity as detected by both scanning electron microscope and water contact angle measurement. Compared to the normal cotton surfaces, such self-assembly bioactive surfaces afforded effective easycleaning functionality against both water and lipids based stains with the most significant stain removing ratio as examined through the simulated laundering test. Additionally, this surface assembled LipA-CBD presented good thermal stability with 15 days of half-life detected for 70°C and over 60 days for room temperature. Although there is a gradual decrease in sun irradiation stability and laundering durability, the functionality could be quickly recovered by re-applying the LipA-CBD as the self-assembly coating in the rinsing process. These results presented a green, simple and timesaving method to develop new easy-cleaning cotton fabrics via interfacial selfassembly of biomacromolecules.
1. Introduction Over the past decades, surface functionalization has been proven particularly effective in realizing unique smart materials with desired functions and properties. In particular, the functional textiles with waterproofing, anti-bacterial, flame retardancy and anti-ultraviolet properties have been widely explored and in some case have led to commercialized products [1–5]. In view of the close relationship with our daily life, the self-cleaning and easy-cleaning textiles are especially attractive. They have so far developed primarily based on the principal of surface hydrophobization. The most wildly used approach to fabricate hydrophobic surfaces is the use of low surface energy chemical coatings including fluorine or silicon containing chemicals in combination with proper surface roughness [6–8]. For example, Deng et al. reported recently a durable superhydrophobic cotton fabric prepared by the γ-rays radiation induced graft polymerization of a commercially available fluorinated acrylate monomer (1H, 1H, 2H, 2H-nonafluorohexyl-1acrylate) onto the cotton fabric [9]. Alternatively, Zimmermann et al. developed a robust superhydrophobic textiles through
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the gas phase coating procedure by which a layer of polymethylsilsesquioxane nanofilaments was grown onto the individual textile fibers [10]. Both treatments were efficient to generate fluorocarbon or silico finishing on cotton fabrics to realize the water resistant properties. However, such chemical fabrications are associated with time-consuming, environmental safety risks to human, and the resultant cotton fabrics become not biodegradable, also at a cost of losing some original physical properties at different extents [11–13]. As a result, development of “simple” and “green” fabrication methods with less influences on cotton natural characteristics are highly desired. Different from surface physicochemical property manipulations as have done before [14], and inspired by the novel proteinaceous coating for functional regulations on solid surfaces [15,16], we explored a new biocatalysis based surface fabrication protocol utilizing the green biocatalysts to realize functional fabrics that can selectively decompose contaminants, thus avoid surface contamination. And to achieve that, we adapted a fusion protein strategy to afford the biocatalyst stable adsorption, while maintains a favorable biocompatible environment to ensure original activity on the fabric surface. Cellulose-binding domains
Corresponding authors. E-mail addresses: [email protected] (C. Chen), [email protected] (P. Wang).
https://doi.org/10.1016/j.colsurfb.2019.110433 Received 10 April 2019; Received in revised form 23 July 2019; Accepted 7 August 2019 Available online 10 August 2019 0927-7765/ © 2019 Published by Elsevier B.V.
Colloids and Surfaces B: Biointerfaces 184 (2019) 110433
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energy-dispersive X-ray spectroscopy (EDX) (PhenomProX G5, Netherland) to reveal the elemental changes before and after LipA-CBD modification.
(CBDs), a protein structure evolved in native biological world, offer excellent affinitive effect on cellulosic materials, and can be conjugated with proteins, peptides, and antibodies as immobilization anchors [17–19]. Some previous researches also demonstrated CBD adsorption could adjust the surface characteristics of the cellulosic materials and make them hydrophobic [20,21]. Cotton fabric is generally composited by more than 90% of cellulose, as a result, provides ideal surface properties for CBD affinitive adsorption through the aromatic polarization and hydrophobic interactions [22]. In this work, a hydrolysis enzyme, Lipase-A (LipA), as a lipids digestive functional domain was fused with CBD monomer. We expect such a unique CBD-LipA recombinant biomacromolecule can spontaneously and quickly self-assemble on cotton matrix, then effectively generate the easy-cleaning capabilities through the hydrolysis process, at the same time, enhance the surface hydrophobic property to alleviate the stain contaminations on cotton fabric surfaces.
2.4. Functionality demonstrations The blood and lipstick were selected as the model water based and lipids based stains to demonstrate the easy-cleaning functionality by monitoring the stain removing efficiency. One drop of human blood 20.0 μL was gently loaded onto the cotton fabric samples with bounded LipA-CBD 0.6 μmol/g cotton fabric or without LipA-CBD adsorption. The samples were left under ambient condition for overnight or days of air-drying, and then each sample was washed by 100 mL of tap water for 10 min with 200 rpm mechanical stirring to simulate the laundering process. Finally, the tested samples were collected by filtration and airdrying, and the stain removing efficiency was approximately estimated according to the residual stain changes on cotton fabric surfaces. For lipstick stain removing test, a cross symbol was gently painted onto the different pretreated cotton fabric samples by stamp using lipstick as the inkpad, then each sample was immersed into 1.0 mL sodium alcohol ether sulphate (AES) solution (1.0%) at 30 °C for 0.5 h, after that the samples and solutions were transferred into 100 mL tap water in presence of 0.5% AES in glass flask with mechanical stirring at 200 rpm. The stain removing process was recorded by camera and the stain removing efficiency was estimated as mentioned above.
2. Experimental Section 2.1. Materials The primers used in plasmid (pET28a-LipA-CBD) construction and DNA sequencing services were provided by Ruidi Biotech (Shanghai, China); Restriction endonucleases (NcoI and XhoI), T4 ligase, Premix Taq DNA polymerase, DNA marker, and Loading buffer were purchased from TaKaRa (Dalian, China). The Ni-NTA agarose resin and plasmid extraction kit was purchased from Qiagen (Shanghai, China). The Promega Wizard SV gel purification kit was purchased from Promega, (Fitchburg, USA). Cotton fabric was provided by Jintai (Hangzhou, China), and the free fatty acids analysis kit was from Jincheng (Nanjing, China). The E.coli DH5α from Invitrogen and E.coli BL21 (DE3) from Novagen were used to LipA-CBD cloning and expression. All other chemicals were purchased from Sangon (Shanghai, China).
2.5. Functional durability For the laundering durability test, it was basically carried out according to the American Association of Textile Chemists and Colorists test method 61–2006 with certain modifications. In each test cycle, 10 pieces of samples with bounded LipA-CBD 0.6 μmol/g cotton fabric were laundered in a plastic beaker containing 50 mL aqueous solution of AES (0.1%, w/w) and 50 stainless steel balls on a 25 °C shaker with 40 rpm for 10 min. After filtration and centrifugation, the recovered samples were rinsed in 50% ethanol solution for 3 times to remove the adsorbed AES. Finally, the WCAs of the cotton fabric samples were recorded after the oven drying. The effect of temperature and UV irradiation on surface bound LipA-CBD stability was investigated and described as the relative activities between the activities after and before treatments. For UV irradiation stability testing, the samples were directly exposed under the sunshine at strong irradiation time for 2 h (generally at 12:00–14:00 p.m.), and repeat this irradiation for 6 times in the following 6 days with average intensity at 2000–2800 μW/cm2 under 30–40 °C. The activities of the LipA-CBD were measured by a colorimetric method, which was modified from Guo’s protocol using pnitrophenyl palmitate (p-NPP) as chromogen [23].
2.2. Fabrication of LipA-CBD modified cotton fabric The cloning and preparation of LipA-CBD processes were specified in supplemental information. Based on this, 1.0 × 1.0 cm2 cotton fabric samples were pre-cleaned by an alternate cycle of shaking in 95% ethanol for 1 h to remove the lipids contaminations, which was followed by rinsing with deionized water, and then the clean cotton fabrics were dried at 60 °C in oven for 24 h. The pre-cleaned samples were directly immersed into LipA-CBD containing solutions for 0.5 h, which could ensure the Lip-CBD reach the adsorption equilibrium. Finally, the cotton fabric samples were collected from solutions by filtration and centrifugation, and then dried at 40 °C in oven for 24 h before further tests. For the adsorption characteristics investigation, 10.0 mg of cotton fabric with buffer saturated was immersed into 1.0 mL solution with different concentrations of LipA-CBD. After 0.5 h of incubation at 20 °C, the cotton fabrics were collected and repetitively washed by 1.0 mL of buffer for three times. The amount of bound LipA-CBD was calculated from the change between the original and remaining amounts of the LipA-CBD in supernatant and washing solutions. Each point was measured in triplicate.
3. Results and discussion 3.1. LipA-CBD preparation and adsorption Bio-based stains such as polysaccharides, lipids and proteins could be digested by hydrolases mostly in aqueous environments, and the reaction products are small molecule residues, which could be easily removed from solid surfaces in presence of surfactants [24]. Targeting the lipids based stain in this study, LipA was selected to afford the easycleaning functionality. LipA could efficiently decompose triglyceride to water soluble units including free fatty acids and glycerol in neutral, slightly acidic or alkane solutions [25]. However, LipA is highly watersoluble, which makes it difficult to retain the enzyme on cotton fabric surface. Accordingly, CBD fusion with LipA protein was designed and prepared by over expression in E.coil BL21(DE3) with a 6 His-tag. The recombinant fusion protein was recovered by using Ni-NTA affinity chromatography. As shown in Fig. 1A, the purified CBD-LipA product has a molecular weight (MW) of ˜36.7 kDa, which agrees well with the
2.3. Surface characteristics of cotton fabric For changes in surface hydrophobicity induced by LipA-CBD adsorption, it was characterized by water contact angle (WCA) analysis under ambient conditions. Typically, one drop 5.0 μL of purified water was gently loaded onto fabric samples with different amount of preadsorbed LipA-CBD by a syringe and the contact angles were then measured within 5 s. The morphology changes of the fabrics before and after LipA-CBD adsorption was visualized by SEM (Hitachi S3500 N, Tokyo, Japan) with a 10 Å thickness of platinum sputter-coating, and the elemental composition of the surfaces were further inspected by the 2
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Fig. 1. SDS-PAGE analysis of the LipA-CBD preparation (A) (Lane-1: Markers, Lane-2: LipA-CBD product, Lane-3: LipA-CBD product after cellulose particles pretreatment); Adsorption isotherm of LipA-CBD on cotton fabric (B).
surface before and after LipA-CBD adsorption with a bounded LipA-CBD 0.6 μmol/g cotton fabric. As shown in Fig. 2, the sample modified with the LipA-CBD has no obvious changes in fiber appearance under 500X magnifications (A and C of Fig. 2). The crevice between fibers were well retained to keep air permeable, at the same time, the other characteristics of the cotton fabric including tensile strength, dry weight, and thickness have not been obviously influenced by the LipA-CBD coating formulation. However, the fiber surface revealed a pronounced change when it was examined under 20,000X magnification (B and D of Fig. 2). The wrinkles on surfaces of original fibers disappeared and the modified surface became much rougher and more irregular. The EDX qualitative analysis presented new peaks for nitrogen and sulphur elements, but no obvious nitrogen or sulphur component was detected on blank cotton fabric surface. As a result, it is reasonable to conclude that the newly generated irregular layer is an indication of ununiformed distribution of LipA-CBD on cotton fiber surface. According to the reported crystal structure of the LipA and CBD, both are spherical approximate proteins with average diameter of 6.0 nm and 5.9 nm [27,28]. Considering the total amount of the bounded LipA-CBD and the specific surface area of the cotton fabric, it is sufficient to form 20 layers of coverage on cotton fabric surface. Water contact angle (WCA) measurements were also conducted to investigate changes in surface properties. The cotton fabric has hydrophilic surface and it is quite water wettable that water droplets could hardly formed (Fig. 2A); After LipA-CBD adsorption, the water wettability significantly decreased. It appears that the hydrophobicity is dependent on the LipA-CBD loading. When the bounded LipA-CBD is higher than 0.22 μmol/g cotton fabric, the water droplet could stabilize for more than 1 h with WCA between 135° and 143° (Fig. 2C). While the hydrophobicity became unstable that the water droplet spread out within 5 min and the WCA decreased dramatically (Fig. 2E), when the bounded LipA-CBD is lower than 0.22 μmol/g cotton fabric. Although this hydrophobic modification did not generate super hydrophobic cotton materials that the WCA is higher than 150°, it still provided a green and simple method for moderate hydrophobization of the cellulosic materials.
theoretical calculation according to the amino acid sequence of the protein. Furthermore, the band significantly receded when the protein solution was pretreated with microcrystalline cellulose Avicel PH101, which means the resultant protein has specific adsorption on cellulosic matrix. Both results demonstrated the over expression of LipA-CBD fusion protein was successful and the cellulose affinitive functionality of the CBD subunit was well preserved. The adsorption isotherm at 20 °C was obtained using LipA-CBD solutions with initial concentrations between 0 and 58.5 μM, which was quite next to the solubility of the fusion protein under the experimental condition. According to Fig. 1B, it appears that a multi-layer adsorption happened, and the binding capacity increased gradually with the raise of LipA-CBD concentration in solution phase without obvious saturation. Meanwhile, the partition coefficient of 0.05 L/g was calculated from the initial slope of the isotherm. Both cases indicated a more significant adsorption than CBD monomer under the same conditions that the maximum binding capacity was 0.67 μmol/g cotton fabric and the partition coefficient was 0.0072 L/g [26]. The enzyme kinetic parameters Km, kcat and Vmax of cotton adsorbed LipA-CBD and free LipA-CBD were also evaluated through the Lineweaver-Burk plotting, as shown in Fig. S1, and they were specified in Table 1. The Km of immobilized LipA-CBD was 4.68 mM, which is higher than that of free LipA-CBD (2.85 mM), demonstrating lower affinity of immobilized lipase to substrate in comparison with free LipA-CBD. Furthermore, Vmax value was apparently decreased for immobilized LipA-CBD with respect to equivalent free counterpart, and the decreased Vmax values of immobilized LipA-CBD was probably owing to steric hindrance of catalytic site by cotton matrix and multi-layer adsorption structure. Meanwhile, the kcat and kcat/ Km values of immobilized were also sharply reduced. These changes in the kinetic parameters may be due not only to structural changes occurring in the enzyme upon immobilization, but also to the reduced accessibility of the substrate to the active sites of the immobilized LipACBD. 3.2. Characterizations of LipA-CBD modified surfaces SEM images revealed the morphology changes on cotton fabric fiber
3.3. Surface functionality tests
Table 1 The kinetics of free and immobilized LipA-CBD. Catalyst
Km (mM)
Vmax (mM/min)
kcat (min−1)
kcat/Km (mM−1 min−1)
Free LipA-CBD Immobilized LipA-CBD
2.85 4.68
0.07 0.04
575.34 332.55
201.92 71.07
As the self-assembly LipA-CBD coating is successfully designed and prepared, the easy-cleaning functionality of such prepared cotton fabrics against lipids based stain was tested. Lipstick was selected as the model stain, because it is mainly composed by triglycerides, natural vegetable oils, waxes, and certain portion of pigments, which is less than 10% of the mass fraction. As shown in Fig. 3, the reference cotton fabric pre-adsorbed CBD did not promote any removing of the stain, in 3
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Fig. 2. Effects of LipA-CBD adsorption on surface characteristics of cotton fabrics (cotton fabrics modified with (C and D) and without LipA-CBD (A and B); The relationship between the LipA-CBD loading and WCA (E)).
presented perfect blood un-wettability and the contact angle was even higher than 150°, while the blood drop quickly penetrated into the blank cotton fabric (Fig. 4A-0 and B-0). The blood contact angle became lower with the oxidation degree increase and the water evaporation of the blood drop, after 24 h, the air dried blood residual still stayed on the surface of the cotton fabric (Fig. 4B-1 and B-2), which is beneficial to the removing of the blood stain, and the bloodstain retained on LipACBD treated fabric was much less than that without LipA-CBD treatment (Fig. 4A-3 and B-3). When the bloodstain was not cleaned in time, and kept for days or one week after contamination, the blood became more stubborn than that blood stained within 24 h (Fig. 4A-3 and A-4). However, the retained bloodstain on LipA-CBD modified samples was much less than that on blank samples (Fig. 4A-4 and B-4), and the residual bloodstain could be further removed by gently hand rubbing. This easy cleaning functionality should be attributed to the surface hydrophobization induced by the LipA-CBD adsorption, which caused bloodstain unwettability and less dispersion that further makes the removing efficiency for surface aggregated bloodstain much higher than that for blank fabrics.
contrast, the stain removing ratio was even lower than blank cotton fabric. It indicates that the CBD adsorption hindered the lipstick removing, and this negative effect could be induced by the hydrophobization that makes the cotton surface more compatible with the lipstick stain. The cotton fabric samples pre-adsorbed LipA-CBD presented the expected functionality with the most significant stain removing ratio. The stain removing ratio increased with the washing time extension, while it was no obvious increase for the blank and reference cotton fabric samples. Therefore, it is reasonable to attribute the easycleaning functionality to the LipA produced lipids hydrolysis bioactivity, which effectively hydrolyzed the triglycerides in the stain and promoted the pigment and fatty acids released from the stain body. This assumption was further certified by the simultaneously fatty acids analysis, and the results were shown in Fig. 3D. Additional fatty acids continuously released into the washing solution within the 1.5 h of the washing time and the free fatty acid concentration became stable between 1.5 h and 3.0 h, which is in good corresponding with the stain washing process as observed in Fig. 3C. The release rate and total amount of fatty acids in solution phase are also more obvious than that obtained from blank samples and CBD modified samples. Another easy functionality for water based stain contamination was demonstrated in Fig. 4. Cotton fabric samples modified with LipA-CBD
Fig. 3. Effects of LipA-CBD modification on easy-cleaning functionality against lipstick stains (A: blank sample, B: CBD modified sample, C: LipA-CBD modified sample; 0: before washing, 1: after washing 30 min, 2: after washing 60 min); Free fatty acids released from different cotton fabric samples (D). 4
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Fig. 4. Effects of LipA-CBD modification on easy-cleaning functionality against human blood stain (A: blank sample, B: Lip-CBD modified sample; blood stained on cotton fabric before air dying (0), after air drying for 1 h (1), and 24 h (2); the blood easy-cleaning results for 24 h (3) and 168 h (4) contaminated samples.
which should effectively resist the infrared heat effect induced denature, the bioactivity decrease should be attributed to the ultraviolet light radiation induced aromatic residuals excitation and disulfide bond disassociation, then protein chain unfolding [30]. Finally, the laundering durability was tested to investigate the surface hydrophobicity change and evaluate the functional stability of the modified cotton fabrics. As shown in Fig. 6B, The WCA was approximately 140° for the sample before the laundering test and it decreased slowly with increasing of laundering cycles, while the WCA decreased significantly and became unstable after 5 laundering cycles. Considering the dependence between WCAs and adsorbed amount of LipA-CBD in Fig. 2E, and the non-covalent binding in the coating layer, the degradation of surface hydrophobicity might be caused by the LipA-CBD desorption in the laundering test. This was further verified by the protein determination of the laundering solution using the Bradford method (Fig. S2). However, it is interesting to see that this defect can be remedied by reapplying the bio-agent as the coating material in rinsing process, as demonstrated in the 7th laundering cycle. Therefore, this Lip-CBD protein could also be popularized as a separated stabilizing agent with this easy-cleaning cotton fabric. In summary, the durability performances, especially under the strong UV irradiation and laundering, for such LipA-CBD coated cotton seems not as good as we expected, however, we have to note the harsh testing conditions, and this is rarely the case in practical applications. As we can see in the Fig. 5A, the bioactive stability could retain well in room temperature indoor environment. Even in laundering process, there is a slow protein desorption, however, the LipA-CBD coating
3.4. Functional durability tests Based on these good easy-cleaning performances, it is believed that the functionality against lipids based stains is closely related with the stability of the immobilized LipA, and the functionality against water based stains is induced by the surface hydrophobization. However, the enzyme stability and the surface hydrophobicity might be declined in practical processes including laundering, drying, and dressing. As a result, the influences of thermal denaturation, sun-irradiated denaturation, and laundering durability were systematically investigated. The LipA-CBD pre-adsorbed on cotton fabric is quite stable under room temperature environment that no obvious activity loss observed in the 15 days of incubation. Even under 70 °C, which is the general temperature for dryer, the residual activity of LipA-CBD could retain 97% or higher in the beginning 48 h and the half-life time could reach half month (Fig. 5A). In contrast, the relative activity obtained from the natural LipA directly adsorbed cotton fabric was less than 10% after 24 h of incubation, and completely denatured after 72 h in the 70 °C oven environment, as shown in Fig. 5B. These results indicated that the fusion of CBD significantly improved the thermal stability of LipA, which is well consistent with the results reported by Thongekkaew et al [29]. Moreover, the effect of sun irradiation on stability of the preadsorbed LipA-CBD was further investigated. The residual activity was measured as function of sun irradiation time in Fig. 6A. Although there is an obvious activity loss, the residual activity is still retained about 63% after total 12 h of intermittent insolation with the strong UV index between 6 to 7. In view of the good thermal stability of LipA-CBD,
Fig. 5. Thermal stability of LipA-CBD (A) and LipA (B) modified cotton fabrics (square: 25 °C, cycle: 70 °C). 5
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Fig. 6. The sun irradiation (A) and laundering stability (B) of LipA-CBD modified cotton fabrics.
induced functionalities could be retained at least 5 laundering cycles, and the function decline could be easily recovered in rinsing process in any laundering cycle. As a result, this LipA-CBD coated cotton material should meet the requirement for our daily life.
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4. Conclusions CBD fusion mediated a significant self-assembly that successfully promoted the bioactive enzyme adsorption on cotton fabric surface and afforded easy-cleaning functional surfaces. The adsorbed LipA-CBD fusion protein well retained original bioactivity, which allows the fabric selectively hydrolyzed the lipids stain-forming biomolecules, avoiding formation of stubborn stains on cotton fabric surfaces. Meanwhile, the LipA-CBD adsorption induced an effective surface hydrophobization affording excellent easy-cleaning functionality against water-rich stains such as human blood. As a result, this water and lipids based stains easy-cleaning cotton materials could help to reduce water, surfactant and energy consumption by shortening the washing time and decreasing rinsing cycles. Such a recombinant biomacromolecule strategy also promised the convenience for regeneration of the functional surface by re-applying the bio-agents as the self-assembly coating. Combined with the enhanced thermo-stability and recoverable durability, a variety of functional fabric materials including anti-bacteria and anti-fouling depending on the bioactive molecules applied, could be ultimately developed. Acknowledgment This research was financially supported by National Natural Science Foundation of China (21636003 & 51708505), and the Science and Technology Department of Zhejiang Province (2017C03010). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110433. References [1] K. Shen, M. Yu, Q. Li, W. Sun, X. Zhang, M. Quan, Z. Liu, S. Shi, Y. Gong, Synthesis of a fluorine-free polymeric water-repellent agent for creation of superhydrophobic fabrics, Appl. Surf. Sci. 426 (2017) 694. [2] M. Wu, B. Ma, T. Pan, S. Chen, J. Sun, Silver nanoparticle colored cotton fabrics with tunable colors and durable antibacterial and self-healing superhydrophobic properties, Adv. Funct. Mater. 26 (2016) 569. [3] M. Yu, Z. Wang, M. Lv, R. Hao, R. Zhao, L. Qi, S. Liu, C. Yu, B. Zhang, C. Fan, J. Li, Antisuperbug cotton fabric with excellent laundering durability, ACS Appl. Mater. Interface 8 (2016) 19866. [4] C. Xue, L. Zhang, P. Wei, S. Jia, Fabrication of superhydrophobic cotton textiles with flame retardancy, Cellulose 23 (2016) 1471.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Easy-cleaning surfaces functionalized with an interface-binding recombinant lipase
T ⁎
Liting Zhanga,b, Yaofei Sunb, Yibing Wangb, Xiaoli Wangb, Haifeng Zhuanga, Chao Chenb, , ⁎ Ping Wangc, a
Key Laboratory of Recycling and Eco-Treatment of Waste Biomass of Zhejiang Province, Zhejiang University of Science and Technology, Hangzhou, 310023, PR China State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology, East China University of Science and Technology, Shanghai, 200237, PR China c Department of Bioproducts and Biosystems Engineering, University of Minnesota-Twins City, St Paul, MN, 55127, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Surface fabrication Self-assembly Cellulose-binding domain Functional durability Easy-cleaning surfaces
Surface fabrication is an effective method for functional materials development. This work investigated the use of cellulose-binding domain (CBD) fused detergent lipase for fabrication of easy-cleaning surfaces. As a result, the CBD conjugated Lipase-A (LipA-CBD) demonstrated a multi-layer self-assemble on cotton fabric surface and enhanced hydrophobicity as detected by both scanning electron microscope and water contact angle measurement. Compared to the normal cotton surfaces, such self-assembly bioactive surfaces afforded effective easycleaning functionality against both water and lipids based stains with the most significant stain removing ratio as examined through the simulated laundering test. Additionally, this surface assembled LipA-CBD presented good thermal stability with 15 days of half-life detected for 70°C and over 60 days for room temperature. Although there is a gradual decrease in sun irradiation stability and laundering durability, the functionality could be quickly recovered by re-applying the LipA-CBD as the self-assembly coating in the rinsing process. These results presented a green, simple and timesaving method to develop new easy-cleaning cotton fabrics via interfacial selfassembly of biomacromolecules.
1. Introduction Over the past decades, surface functionalization has been proven particularly effective in realizing unique smart materials with desired functions and properties. In particular, the functional textiles with waterproofing, anti-bacterial, flame retardancy and anti-ultraviolet properties have been widely explored and in some case have led to commercialized products [1–5]. In view of the close relationship with our daily life, the self-cleaning and easy-cleaning textiles are especially attractive. They have so far developed primarily based on the principal of surface hydrophobization. The most wildly used approach to fabricate hydrophobic surfaces is the use of low surface energy chemical coatings including fluorine or silicon containing chemicals in combination with proper surface roughness [6–8]. For example, Deng et al. reported recently a durable superhydrophobic cotton fabric prepared by the γ-rays radiation induced graft polymerization of a commercially available fluorinated acrylate monomer (1H, 1H, 2H, 2H-nonafluorohexyl-1acrylate) onto the cotton fabric [9]. Alternatively, Zimmermann et al. developed a robust superhydrophobic textiles through
⁎
the gas phase coating procedure by which a layer of polymethylsilsesquioxane nanofilaments was grown onto the individual textile fibers [10]. Both treatments were efficient to generate fluorocarbon or silico finishing on cotton fabrics to realize the water resistant properties. However, such chemical fabrications are associated with time-consuming, environmental safety risks to human, and the resultant cotton fabrics become not biodegradable, also at a cost of losing some original physical properties at different extents [11–13]. As a result, development of “simple” and “green” fabrication methods with less influences on cotton natural characteristics are highly desired. Different from surface physicochemical property manipulations as have done before [14], and inspired by the novel proteinaceous coating for functional regulations on solid surfaces [15,16], we explored a new biocatalysis based surface fabrication protocol utilizing the green biocatalysts to realize functional fabrics that can selectively decompose contaminants, thus avoid surface contamination. And to achieve that, we adapted a fusion protein strategy to afford the biocatalyst stable adsorption, while maintains a favorable biocompatible environment to ensure original activity on the fabric surface. Cellulose-binding domains
Corresponding authors. E-mail addresses: [email protected] (C. Chen), [email protected] (P. Wang).
https://doi.org/10.1016/j.colsurfb.2019.110433 Received 10 April 2019; Received in revised form 23 July 2019; Accepted 7 August 2019 Available online 10 August 2019 0927-7765/ © 2019 Published by Elsevier B.V.
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energy-dispersive X-ray spectroscopy (EDX) (PhenomProX G5, Netherland) to reveal the elemental changes before and after LipA-CBD modification.
(CBDs), a protein structure evolved in native biological world, offer excellent affinitive effect on cellulosic materials, and can be conjugated with proteins, peptides, and antibodies as immobilization anchors [17–19]. Some previous researches also demonstrated CBD adsorption could adjust the surface characteristics of the cellulosic materials and make them hydrophobic [20,21]. Cotton fabric is generally composited by more than 90% of cellulose, as a result, provides ideal surface properties for CBD affinitive adsorption through the aromatic polarization and hydrophobic interactions [22]. In this work, a hydrolysis enzyme, Lipase-A (LipA), as a lipids digestive functional domain was fused with CBD monomer. We expect such a unique CBD-LipA recombinant biomacromolecule can spontaneously and quickly self-assemble on cotton matrix, then effectively generate the easy-cleaning capabilities through the hydrolysis process, at the same time, enhance the surface hydrophobic property to alleviate the stain contaminations on cotton fabric surfaces.
2.4. Functionality demonstrations The blood and lipstick were selected as the model water based and lipids based stains to demonstrate the easy-cleaning functionality by monitoring the stain removing efficiency. One drop of human blood 20.0 μL was gently loaded onto the cotton fabric samples with bounded LipA-CBD 0.6 μmol/g cotton fabric or without LipA-CBD adsorption. The samples were left under ambient condition for overnight or days of air-drying, and then each sample was washed by 100 mL of tap water for 10 min with 200 rpm mechanical stirring to simulate the laundering process. Finally, the tested samples were collected by filtration and airdrying, and the stain removing efficiency was approximately estimated according to the residual stain changes on cotton fabric surfaces. For lipstick stain removing test, a cross symbol was gently painted onto the different pretreated cotton fabric samples by stamp using lipstick as the inkpad, then each sample was immersed into 1.0 mL sodium alcohol ether sulphate (AES) solution (1.0%) at 30 °C for 0.5 h, after that the samples and solutions were transferred into 100 mL tap water in presence of 0.5% AES in glass flask with mechanical stirring at 200 rpm. The stain removing process was recorded by camera and the stain removing efficiency was estimated as mentioned above.
2. Experimental Section 2.1. Materials The primers used in plasmid (pET28a-LipA-CBD) construction and DNA sequencing services were provided by Ruidi Biotech (Shanghai, China); Restriction endonucleases (NcoI and XhoI), T4 ligase, Premix Taq DNA polymerase, DNA marker, and Loading buffer were purchased from TaKaRa (Dalian, China). The Ni-NTA agarose resin and plasmid extraction kit was purchased from Qiagen (Shanghai, China). The Promega Wizard SV gel purification kit was purchased from Promega, (Fitchburg, USA). Cotton fabric was provided by Jintai (Hangzhou, China), and the free fatty acids analysis kit was from Jincheng (Nanjing, China). The E.coli DH5α from Invitrogen and E.coli BL21 (DE3) from Novagen were used to LipA-CBD cloning and expression. All other chemicals were purchased from Sangon (Shanghai, China).
2.5. Functional durability For the laundering durability test, it was basically carried out according to the American Association of Textile Chemists and Colorists test method 61–2006 with certain modifications. In each test cycle, 10 pieces of samples with bounded LipA-CBD 0.6 μmol/g cotton fabric were laundered in a plastic beaker containing 50 mL aqueous solution of AES (0.1%, w/w) and 50 stainless steel balls on a 25 °C shaker with 40 rpm for 10 min. After filtration and centrifugation, the recovered samples were rinsed in 50% ethanol solution for 3 times to remove the adsorbed AES. Finally, the WCAs of the cotton fabric samples were recorded after the oven drying. The effect of temperature and UV irradiation on surface bound LipA-CBD stability was investigated and described as the relative activities between the activities after and before treatments. For UV irradiation stability testing, the samples were directly exposed under the sunshine at strong irradiation time for 2 h (generally at 12:00–14:00 p.m.), and repeat this irradiation for 6 times in the following 6 days with average intensity at 2000–2800 μW/cm2 under 30–40 °C. The activities of the LipA-CBD were measured by a colorimetric method, which was modified from Guo’s protocol using pnitrophenyl palmitate (p-NPP) as chromogen [23].
2.2. Fabrication of LipA-CBD modified cotton fabric The cloning and preparation of LipA-CBD processes were specified in supplemental information. Based on this, 1.0 × 1.0 cm2 cotton fabric samples were pre-cleaned by an alternate cycle of shaking in 95% ethanol for 1 h to remove the lipids contaminations, which was followed by rinsing with deionized water, and then the clean cotton fabrics were dried at 60 °C in oven for 24 h. The pre-cleaned samples were directly immersed into LipA-CBD containing solutions for 0.5 h, which could ensure the Lip-CBD reach the adsorption equilibrium. Finally, the cotton fabric samples were collected from solutions by filtration and centrifugation, and then dried at 40 °C in oven for 24 h before further tests. For the adsorption characteristics investigation, 10.0 mg of cotton fabric with buffer saturated was immersed into 1.0 mL solution with different concentrations of LipA-CBD. After 0.5 h of incubation at 20 °C, the cotton fabrics were collected and repetitively washed by 1.0 mL of buffer for three times. The amount of bound LipA-CBD was calculated from the change between the original and remaining amounts of the LipA-CBD in supernatant and washing solutions. Each point was measured in triplicate.
3. Results and discussion 3.1. LipA-CBD preparation and adsorption Bio-based stains such as polysaccharides, lipids and proteins could be digested by hydrolases mostly in aqueous environments, and the reaction products are small molecule residues, which could be easily removed from solid surfaces in presence of surfactants [24]. Targeting the lipids based stain in this study, LipA was selected to afford the easycleaning functionality. LipA could efficiently decompose triglyceride to water soluble units including free fatty acids and glycerol in neutral, slightly acidic or alkane solutions [25]. However, LipA is highly watersoluble, which makes it difficult to retain the enzyme on cotton fabric surface. Accordingly, CBD fusion with LipA protein was designed and prepared by over expression in E.coil BL21(DE3) with a 6 His-tag. The recombinant fusion protein was recovered by using Ni-NTA affinity chromatography. As shown in Fig. 1A, the purified CBD-LipA product has a molecular weight (MW) of ˜36.7 kDa, which agrees well with the
2.3. Surface characteristics of cotton fabric For changes in surface hydrophobicity induced by LipA-CBD adsorption, it was characterized by water contact angle (WCA) analysis under ambient conditions. Typically, one drop 5.0 μL of purified water was gently loaded onto fabric samples with different amount of preadsorbed LipA-CBD by a syringe and the contact angles were then measured within 5 s. The morphology changes of the fabrics before and after LipA-CBD adsorption was visualized by SEM (Hitachi S3500 N, Tokyo, Japan) with a 10 Å thickness of platinum sputter-coating, and the elemental composition of the surfaces were further inspected by the 2
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Fig. 1. SDS-PAGE analysis of the LipA-CBD preparation (A) (Lane-1: Markers, Lane-2: LipA-CBD product, Lane-3: LipA-CBD product after cellulose particles pretreatment); Adsorption isotherm of LipA-CBD on cotton fabric (B).
surface before and after LipA-CBD adsorption with a bounded LipA-CBD 0.6 μmol/g cotton fabric. As shown in Fig. 2, the sample modified with the LipA-CBD has no obvious changes in fiber appearance under 500X magnifications (A and C of Fig. 2). The crevice between fibers were well retained to keep air permeable, at the same time, the other characteristics of the cotton fabric including tensile strength, dry weight, and thickness have not been obviously influenced by the LipA-CBD coating formulation. However, the fiber surface revealed a pronounced change when it was examined under 20,000X magnification (B and D of Fig. 2). The wrinkles on surfaces of original fibers disappeared and the modified surface became much rougher and more irregular. The EDX qualitative analysis presented new peaks for nitrogen and sulphur elements, but no obvious nitrogen or sulphur component was detected on blank cotton fabric surface. As a result, it is reasonable to conclude that the newly generated irregular layer is an indication of ununiformed distribution of LipA-CBD on cotton fiber surface. According to the reported crystal structure of the LipA and CBD, both are spherical approximate proteins with average diameter of 6.0 nm and 5.9 nm [27,28]. Considering the total amount of the bounded LipA-CBD and the specific surface area of the cotton fabric, it is sufficient to form 20 layers of coverage on cotton fabric surface. Water contact angle (WCA) measurements were also conducted to investigate changes in surface properties. The cotton fabric has hydrophilic surface and it is quite water wettable that water droplets could hardly formed (Fig. 2A); After LipA-CBD adsorption, the water wettability significantly decreased. It appears that the hydrophobicity is dependent on the LipA-CBD loading. When the bounded LipA-CBD is higher than 0.22 μmol/g cotton fabric, the water droplet could stabilize for more than 1 h with WCA between 135° and 143° (Fig. 2C). While the hydrophobicity became unstable that the water droplet spread out within 5 min and the WCA decreased dramatically (Fig. 2E), when the bounded LipA-CBD is lower than 0.22 μmol/g cotton fabric. Although this hydrophobic modification did not generate super hydrophobic cotton materials that the WCA is higher than 150°, it still provided a green and simple method for moderate hydrophobization of the cellulosic materials.
theoretical calculation according to the amino acid sequence of the protein. Furthermore, the band significantly receded when the protein solution was pretreated with microcrystalline cellulose Avicel PH101, which means the resultant protein has specific adsorption on cellulosic matrix. Both results demonstrated the over expression of LipA-CBD fusion protein was successful and the cellulose affinitive functionality of the CBD subunit was well preserved. The adsorption isotherm at 20 °C was obtained using LipA-CBD solutions with initial concentrations between 0 and 58.5 μM, which was quite next to the solubility of the fusion protein under the experimental condition. According to Fig. 1B, it appears that a multi-layer adsorption happened, and the binding capacity increased gradually with the raise of LipA-CBD concentration in solution phase without obvious saturation. Meanwhile, the partition coefficient of 0.05 L/g was calculated from the initial slope of the isotherm. Both cases indicated a more significant adsorption than CBD monomer under the same conditions that the maximum binding capacity was 0.67 μmol/g cotton fabric and the partition coefficient was 0.0072 L/g [26]. The enzyme kinetic parameters Km, kcat and Vmax of cotton adsorbed LipA-CBD and free LipA-CBD were also evaluated through the Lineweaver-Burk plotting, as shown in Fig. S1, and they were specified in Table 1. The Km of immobilized LipA-CBD was 4.68 mM, which is higher than that of free LipA-CBD (2.85 mM), demonstrating lower affinity of immobilized lipase to substrate in comparison with free LipA-CBD. Furthermore, Vmax value was apparently decreased for immobilized LipA-CBD with respect to equivalent free counterpart, and the decreased Vmax values of immobilized LipA-CBD was probably owing to steric hindrance of catalytic site by cotton matrix and multi-layer adsorption structure. Meanwhile, the kcat and kcat/ Km values of immobilized were also sharply reduced. These changes in the kinetic parameters may be due not only to structural changes occurring in the enzyme upon immobilization, but also to the reduced accessibility of the substrate to the active sites of the immobilized LipACBD. 3.2. Characterizations of LipA-CBD modified surfaces SEM images revealed the morphology changes on cotton fabric fiber
3.3. Surface functionality tests
Table 1 The kinetics of free and immobilized LipA-CBD. Catalyst
Km (mM)
Vmax (mM/min)
kcat (min−1)
kcat/Km (mM−1 min−1)
Free LipA-CBD Immobilized LipA-CBD
2.85 4.68
0.07 0.04
575.34 332.55
201.92 71.07
As the self-assembly LipA-CBD coating is successfully designed and prepared, the easy-cleaning functionality of such prepared cotton fabrics against lipids based stain was tested. Lipstick was selected as the model stain, because it is mainly composed by triglycerides, natural vegetable oils, waxes, and certain portion of pigments, which is less than 10% of the mass fraction. As shown in Fig. 3, the reference cotton fabric pre-adsorbed CBD did not promote any removing of the stain, in 3
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Fig. 2. Effects of LipA-CBD adsorption on surface characteristics of cotton fabrics (cotton fabrics modified with (C and D) and without LipA-CBD (A and B); The relationship between the LipA-CBD loading and WCA (E)).
presented perfect blood un-wettability and the contact angle was even higher than 150°, while the blood drop quickly penetrated into the blank cotton fabric (Fig. 4A-0 and B-0). The blood contact angle became lower with the oxidation degree increase and the water evaporation of the blood drop, after 24 h, the air dried blood residual still stayed on the surface of the cotton fabric (Fig. 4B-1 and B-2), which is beneficial to the removing of the blood stain, and the bloodstain retained on LipACBD treated fabric was much less than that without LipA-CBD treatment (Fig. 4A-3 and B-3). When the bloodstain was not cleaned in time, and kept for days or one week after contamination, the blood became more stubborn than that blood stained within 24 h (Fig. 4A-3 and A-4). However, the retained bloodstain on LipA-CBD modified samples was much less than that on blank samples (Fig. 4A-4 and B-4), and the residual bloodstain could be further removed by gently hand rubbing. This easy cleaning functionality should be attributed to the surface hydrophobization induced by the LipA-CBD adsorption, which caused bloodstain unwettability and less dispersion that further makes the removing efficiency for surface aggregated bloodstain much higher than that for blank fabrics.
contrast, the stain removing ratio was even lower than blank cotton fabric. It indicates that the CBD adsorption hindered the lipstick removing, and this negative effect could be induced by the hydrophobization that makes the cotton surface more compatible with the lipstick stain. The cotton fabric samples pre-adsorbed LipA-CBD presented the expected functionality with the most significant stain removing ratio. The stain removing ratio increased with the washing time extension, while it was no obvious increase for the blank and reference cotton fabric samples. Therefore, it is reasonable to attribute the easycleaning functionality to the LipA produced lipids hydrolysis bioactivity, which effectively hydrolyzed the triglycerides in the stain and promoted the pigment and fatty acids released from the stain body. This assumption was further certified by the simultaneously fatty acids analysis, and the results were shown in Fig. 3D. Additional fatty acids continuously released into the washing solution within the 1.5 h of the washing time and the free fatty acid concentration became stable between 1.5 h and 3.0 h, which is in good corresponding with the stain washing process as observed in Fig. 3C. The release rate and total amount of fatty acids in solution phase are also more obvious than that obtained from blank samples and CBD modified samples. Another easy functionality for water based stain contamination was demonstrated in Fig. 4. Cotton fabric samples modified with LipA-CBD
Fig. 3. Effects of LipA-CBD modification on easy-cleaning functionality against lipstick stains (A: blank sample, B: CBD modified sample, C: LipA-CBD modified sample; 0: before washing, 1: after washing 30 min, 2: after washing 60 min); Free fatty acids released from different cotton fabric samples (D). 4
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Fig. 4. Effects of LipA-CBD modification on easy-cleaning functionality against human blood stain (A: blank sample, B: Lip-CBD modified sample; blood stained on cotton fabric before air dying (0), after air drying for 1 h (1), and 24 h (2); the blood easy-cleaning results for 24 h (3) and 168 h (4) contaminated samples.
which should effectively resist the infrared heat effect induced denature, the bioactivity decrease should be attributed to the ultraviolet light radiation induced aromatic residuals excitation and disulfide bond disassociation, then protein chain unfolding [30]. Finally, the laundering durability was tested to investigate the surface hydrophobicity change and evaluate the functional stability of the modified cotton fabrics. As shown in Fig. 6B, The WCA was approximately 140° for the sample before the laundering test and it decreased slowly with increasing of laundering cycles, while the WCA decreased significantly and became unstable after 5 laundering cycles. Considering the dependence between WCAs and adsorbed amount of LipA-CBD in Fig. 2E, and the non-covalent binding in the coating layer, the degradation of surface hydrophobicity might be caused by the LipA-CBD desorption in the laundering test. This was further verified by the protein determination of the laundering solution using the Bradford method (Fig. S2). However, it is interesting to see that this defect can be remedied by reapplying the bio-agent as the coating material in rinsing process, as demonstrated in the 7th laundering cycle. Therefore, this Lip-CBD protein could also be popularized as a separated stabilizing agent with this easy-cleaning cotton fabric. In summary, the durability performances, especially under the strong UV irradiation and laundering, for such LipA-CBD coated cotton seems not as good as we expected, however, we have to note the harsh testing conditions, and this is rarely the case in practical applications. As we can see in the Fig. 5A, the bioactive stability could retain well in room temperature indoor environment. Even in laundering process, there is a slow protein desorption, however, the LipA-CBD coating
3.4. Functional durability tests Based on these good easy-cleaning performances, it is believed that the functionality against lipids based stains is closely related with the stability of the immobilized LipA, and the functionality against water based stains is induced by the surface hydrophobization. However, the enzyme stability and the surface hydrophobicity might be declined in practical processes including laundering, drying, and dressing. As a result, the influences of thermal denaturation, sun-irradiated denaturation, and laundering durability were systematically investigated. The LipA-CBD pre-adsorbed on cotton fabric is quite stable under room temperature environment that no obvious activity loss observed in the 15 days of incubation. Even under 70 °C, which is the general temperature for dryer, the residual activity of LipA-CBD could retain 97% or higher in the beginning 48 h and the half-life time could reach half month (Fig. 5A). In contrast, the relative activity obtained from the natural LipA directly adsorbed cotton fabric was less than 10% after 24 h of incubation, and completely denatured after 72 h in the 70 °C oven environment, as shown in Fig. 5B. These results indicated that the fusion of CBD significantly improved the thermal stability of LipA, which is well consistent with the results reported by Thongekkaew et al [29]. Moreover, the effect of sun irradiation on stability of the preadsorbed LipA-CBD was further investigated. The residual activity was measured as function of sun irradiation time in Fig. 6A. Although there is an obvious activity loss, the residual activity is still retained about 63% after total 12 h of intermittent insolation with the strong UV index between 6 to 7. In view of the good thermal stability of LipA-CBD,
Fig. 5. Thermal stability of LipA-CBD (A) and LipA (B) modified cotton fabrics (square: 25 °C, cycle: 70 °C). 5
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Fig. 6. The sun irradiation (A) and laundering stability (B) of LipA-CBD modified cotton fabrics.
induced functionalities could be retained at least 5 laundering cycles, and the function decline could be easily recovered in rinsing process in any laundering cycle. As a result, this LipA-CBD coated cotton material should meet the requirement for our daily life.
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4. Conclusions CBD fusion mediated a significant self-assembly that successfully promoted the bioactive enzyme adsorption on cotton fabric surface and afforded easy-cleaning functional surfaces. The adsorbed LipA-CBD fusion protein well retained original bioactivity, which allows the fabric selectively hydrolyzed the lipids stain-forming biomolecules, avoiding formation of stubborn stains on cotton fabric surfaces. Meanwhile, the LipA-CBD adsorption induced an effective surface hydrophobization affording excellent easy-cleaning functionality against water-rich stains such as human blood. As a result, this water and lipids based stains easy-cleaning cotton materials could help to reduce water, surfactant and energy consumption by shortening the washing time and decreasing rinsing cycles. Such a recombinant biomacromolecule strategy also promised the convenience for regeneration of the functional surface by re-applying the bio-agents as the self-assembly coating. Combined with the enhanced thermo-stability and recoverable durability, a variety of functional fabric materials including anti-bacteria and anti-fouling depending on the bioactive molecules applied, could be ultimately developed. Acknowledgment This research was financially supported by National Natural Science Foundation of China (21636003 & 51708505), and the Science and Technology Department of Zhejiang Province (2017C03010). Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110433. References [1] K. Shen, M. Yu, Q. Li, W. Sun, X. Zhang, M. Quan, Z. Liu, S. Shi, Y. Gong, Synthesis of a fluorine-free polymeric water-repellent agent for creation of superhydrophobic fabrics, Appl. Surf. Sci. 426 (2017) 694. [2] M. Wu, B. Ma, T. Pan, S. Chen, J. Sun, Silver nanoparticle colored cotton fabrics with tunable colors and durable antibacterial and self-healing superhydrophobic properties, Adv. Funct. Mater. 26 (2016) 569. [3] M. Yu, Z. Wang, M. Lv, R. Hao, R. Zhao, L. Qi, S. Liu, C. Yu, B. Zhang, C. Fan, J. Li, Antisuperbug cotton fabric with excellent laundering durability, ACS Appl. Mater. Interface 8 (2016) 19866. [4] C. Xue, L. Zhang, P. Wei, S. Jia, Fabrication of superhydrophobic cotton textiles with flame retardancy, Cellulose 23 (2016) 1471.
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