Introduction

The discharge of industrial oily wastewater and frequent oil spill accidents are causing increasingly severe water pollution problems, which have a catastrophic impact on the environment, particularly the aquatic ecological environment, and can result in huge economic loss1,2,3,4,5. For example, in July 2020, a Japanese bulk carrier broke off the Mauritius coast after spilling hundreds of tons of fuel oil. The spill included 814 metric tons of oil liquid waste, 318 metric tons of solid waste sludge, and contaminated debris. Accidents can occur at any time, and the cost of managing the after-effects of these accidents is enormous6. Therefore, it is essential to develop effective technologies for managing oil spills and harmful wastewater contaminants.

An increasing amount of oily wastewater is produced by several sources, and the efficient separation of these oil–water wastes has remained a significant challenge. Membrane filtration has attracted attention as an oil–water separation method owing to its convenience, low cost, environmental friendliness, and lack of requirement of chemicals or additives7,8,9,10. Accordingly, various studies have been conducted to functionalize the membrane filtration method to enhance its oil–water separation performance. For example, the use of superhydrophobic/superoleophobic coatings to control the wettability of membrane surface has been employed to enhance the various separation performance of this method11,12,13,14,15,16,17,18,19,20.

However, in real-world environments, such as oil spills, there can be damages caused by external stimuli, as well as oil and water21. First, oily wastewater from industries contains different types of oil, lubricants and cooling agents, metal residues, and other organic and inorganic components22,23. In addition, oily wastewater from domestic sewage is composed of not only organic substances but also a significant number of pathogen microorganisms (e.g., bacteria, virus, and fungi). Second, oily wastewater sometimes contains solid lumps, which affect the flow of fluid through membrane surface; thus, damaging the membrane and reducing its performance24. Therefore, it is essential to develop innovative integrated removal processes based on filtration technologies owing to the varying characteristics and origin of the vast array of pollutants present in oily wastewater. Particularly, an ideal oil–water separation membrane should possess desirable surface wettability to achieve a high separation efficiency and permeability.

Versatile nanofiber fabrication electrospinning technique has been employed to fabricate nonwoven membranes containing continuous sub-micron or nano-sized entangled fibers25,26,27. Electrospun nanofibrous membranes have attracted attention for energy-saving and high-efficiency oil–water separation owing to their high porosity, high surface area-to-volume ratio, controllable pore size, and tunable morphology. However, the poor mechanical property of these membranes has limited their practical application. In addition, the poor long-term durability of current nanofibrous membranes in harsh environments has restricted its application. Therefore, it is essential to develop a novel functional membrane that can selectively and efficiently separate oil–water mixtures under harsh conditions.

Generally, anti-microbial materials and coatings are used in separation membranes to eliminate bacteria and drug release owing to their excellent bactericidal performance28,29; however, their side effects, such as the occurrence of super-bacteria, which can cause defects in the separation membrane, from continuous usage, have limited their further application30. In this study, we developed a highly durable porous fiber for oil–water separation and bacteria penetration blocking using an in-and-out fluorinated polyurethane (F-PU) coating. The F-PU materials was prepared via the urethane reaction of perfluoroalkyl alcohol, ethylene glycol (EG), and isophorone diisocyanate (IPDI). The prepared F-PU coating materials easily permeated and reacted in the inner site and outer surface of porous fibers. The in-and-out F-PU coating was used to fabricate a coated heterogeneous fabric, and the fabric exhibited excellent highly hydrophobic properties, anti-scratch performance, oil–water separation performance, and anti-bacterial effects (Fig. 1). Furthermore, the fabric maintained its bacterial penetration blocking ability even after the membrane was subjected to a scratching test; thereby, verifying the high durability of the fabricated in-and-out F-PU-coated fabric.

Fig. 1: Highly durable and sustainable heterogeneous fabric with in-and-out coatings.
figure 1

Schematic illustration of the in-and-out fluorinated polyurethane (F-PU)-coated fabric for oil–water separation and bacterial penetration blocking.

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Results and discussion

In this study, in-and-out F-PU coating materials were synthesized using urethane reaction. The synthesis pathway of the F-PU coating materials is shown in Fig. 2a. The F-PU coating material was synthesized using IPDI, EG, and TDFO. One molecule of the F-PU coating material consisted of three urethane bonds, one NCO group, and one perfluoroalkyl group. The three urethane bonds reinforced the mechanical properties of the material by bonding hydrogen bonds to each other to form hard segments31,32,33. The NCO group reacted with the –OH group, carboxylic acid, and amine group on the surface of ACF. Owing to its low surface energy, the perfluoroalkyl group enhanced the hydrophobicity of the surface of the ACF. The different roles of the various chemical structures in the F-PU coating material exhibited a harmonious effect in the resulting ACF membrane.

Fig. 2: Synthesis of in-and-out F-PU coating materials.
figure 2

a Schematic illustration of F-PU coating materials with the fluorination and urethane bonding of ethylene glycol (EG), isophorone diisocyanate (IPDI), and 1H,1H,2H,2H-Tridecafluoro-1-n-octanol (TDFO) for the synthesis of the F-PU coating materials (b) Fourier transform-infrared (FT-IR) transmittance of the F-PU coating materials (TDFO-IPDI-EG-IPDI), TDFO, and IPDI.

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The FT-IR spectra of the F-PU coating material, TDFO, and IPDI are shown in Fig. 2b. In the FT-IR spectra of the F-PU coating material, the absorption peak at 2270 cm−1, which corresponded to NCO, decreased, and the absorption peak at 3500 cm−1, which corresponded to the OH of TDFO disappeared. Furthermore, two additional peaks were observed at 1705 and 1535 cm−1, which corresponded to the stretching vibration modes of the C=O and N–H bonds in the urethane groups, respectively. In addition, a broad absorption band was observed at ~3340–3320 cm−1, which corresponded to N–H stretching vibration, indicating the generation of –OCOHN– groups in the F-PU coating material. This could be attributed to the fact that PU is capable of forming several kinds of hydrogen bonds owing to the presence of a N–H donor group and a C=O acceptor group in the urethane linkage.

To verify the successful formation of the F-PU in-and-out coating in the coated fabric, the morphology of the bare ACF and F-PU in-and-out-coated ACF were investigated using FESEM/EDS analysis. The FESEM image revealed that the surface of the bare ACF exhibited a high surface roughness and porosity (Fig. 3a). In contrast, after the addition of the F-PU in-and-out coatings, the F-PU in-and-out-coated ACF exhibited a significantly decreased surface roughness and porous structure, which could be attributed to the inflow of F-PU coating materials into the pores of ACF (Fig. 3b). Furthermore, the BET measurements revealed that the specific surface area of the F-PU in-and-out-coated ACF was 11.13 m2/g, which was 200 times lower than that of the bare ACF. This indicates that the micropore (<2 nm) of the bare ACF was almost completely filled with the in-and-out coating after the F-PU coating treatment (Supplementary Fig. S1).

Fig. 3: Characterization of F-PU in-and-out coated ACF.
figure 3

Field emission scanning electron microscopy (FE-SEM) images of (a) bare activated carbon fabric (ACF) and (b) F-PU in-and-out-coated ACF. c FESEM/energy dispersive spectroscopy (EDS) of the F-PU in-and-out-coated ACF (red_carbon, green_oxygen, blue_fluorine, and purple_nitrogen). Contact angle measurement of the (d) bare ACF I (e) F-PU in-and-out-coated ACF. (f) Photographic image of the F-PU in-and-out-coated ACF after immersion in distilled (DI) water (5 uL).

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Although BET has decreased a lot, it is unlikely that flux will decrease because pores <2 nm are lost. The BET analysis results provided information on nano-sized pores, whereas we used porosimeter to confirm the micro-sized space between fibers and fibers according to F-PU coating. There are spaces between fibers within the ACF fabric, and micro-sized spaces rather than nano-sized spaces in this space are very dominant. As shown in the Supplementary Fig. S2, the pore volume can be checked before and after coating. The pore volume slightly decreased after coating, but there were many spaces large enough, so there is no problem with liquid flux.

In addition, these results demonstrate the entry of the F-PU coating materials into the pores of the ACF and their reaction with the functional groups on the surface of ACF. The elemental distribution of the cross–section of the F-PU in-and-out-coated ACF was investigated using FESEM/EDS analysis and XPS (Fig. 3c and Supplementary Fig. S3). The results revealed the presence of fluorine and nitrogen elements from TDFO and urethane bonds inside and outside the F-PU in-and-out-coated ACF. In contrast, only carbon and oxygen elements were observed in the bare ACF (Supplementary Fig. S3). Next, the effect of the TDFO of the F-PU coating materials on the hydrophobicity of the coated ACF was investigated using contact angle analysis. Generally, bare ACF exhibits highly hydrophilic properties because of its polar functional groups. The results revealed that the contact angle of the bare ACF was 0°, and the bare ACF absorbed the water droplet used for the measurement (Fig. 3d). In contrast, the F-PU in-and-out-coated ACF exhibited highly hydrophobic properties with a water contact angle of 133.3 ± 2.7°, and a uniform hydrophobicity (Fig. 3e and f).

The oleophilic and hydrophobic characteristics of the F-PU in-and-out-coated ACF are shown in Fig. 4. In an aqueous condition, oil droplets rapidly spread and were absorbed by the F-PU in-and-out-coated ACF. In contrast, under oil conditions, water droplets were retained on the F-PU in-and-out-coated ACF, and the water contact angle was higher than that under air condition (Supplementary Fig. S4a). In addition, the coated ACF was wetted with oil to form an oil-containing region, which exhibited superhydrophobicity under oil conditions. In contrast, the bare ACF does not selectively absorb oil and water, and exhibited high absorption capacity for all liquid. The F–PU in-and-out-coated ACF exhibited a significantly reduced water absorption capacity; however, its oil absorption capacity was similar to that of the bare ACF (Fig. 4b and Supplementary Fig. S4b). This indicates that the F–PU in-and-out coated ACF exhibited a high selectivity for oil/water mixtures.

Fig. 4: Wettability and water-oil selective properties of F-PU in-and-out coated ACF.
figure 4

a Photographs of the flow of CF on the F-PU in-and-out-coated ACF in aqueous condition. b Absorption capacity (mg/cm2) of the bare ACF and F-PU in-and-out-coated ACF for DI water, dichloromethane (DCM), toluene, hexane, olive oil, and CF. The error bars represent the standard deviations of the data.

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Coated fabric has a highly hydrophobic properties and oleophilic properties, whereas bare fabric is hydrophilic and oleophilic. So, once the oil–water solution contacts a surface of the coated fabric, the oil droplets would be quickly adsorbed and spread across the coated fabric because of the synergistic effects of gravity force and high affinity. The interconnected pores between the fibers and the pores within the coated ACF serve as a “high pathway” for oil diffusion. However, the adsorption or diffusion of water would be rejected because of the highly hydrophobic property of perfluorocarbon chain on the coated ACF. Water droplets are inferior due to the competitive adsorption with oil droplets on the surface of coated ACF. Therefore, the water droplets were rejected and the oil droplets spread out as an “oil layer” on the surface of ACF surface.

In addition, because the coated fabric is highly hydrophobic, the breakthrough pressure is a positive value (ΔP > 0) following the equation. So the coated ACF can sustain a certain pressure (negative capillary effect). Without an external pressure, water droplet can’t spontaneously penetrate through the coated ACF. On the contrary, when the coated ACF is highly oleophilic (θ < 5°), the breakthrough pressure is a negative value (ΔP > 0), so the coated ACF cannot withstand any pressure (capillary effect). The oil droplet are able to permeate the coated ACF spontaneously34,35,36,37.

Based on the aforementioned high hydrophilicity and high oleophilicity of the F-PU in-and-out-coated ACF, The oil–water mixture separation performance of the F-PU in-and-out-coated ACF was examined using a customized oil/water separation system (Fig. 5a). The separation efficiencies of the F-PU in-and-out-coated ACF for various oil–water mixtures were above 95%, indicating the efficient oil–water mixtures separation performance of the F-PU in-and-out-coated ACF for the six mixtures (Fig. 5b). Furthermore, to investigate the reusability of the F-PU in-and-out-coated ACF, the coated ACF was used to separate CF/water mixture for 20 times successively. The material exhibited a remarkable flux rate of above 2 × 104 L ∙ m−2 ∙ h−1 for the CF/water mixture. In addition, the separation efficiency of the material after each application was in the range of 95%, verifying the good stability of the F-PU in-and-out-coated ACF, and its potential for continuous reusability for oil contamination separation (Fig. 5c). These results indicate that the F-PU in-and-out-coated ACFs were ideal reusable separation membranes for the potential treatment of various oily wastewater discharges.

Fig. 5: Oil/water separation performance of F-PU in-and-out coated ACF.
figure 5

a Photograph images of the oil/water separation system containing the F-PU in-and-out coated ACF. b Separation efficiencies of the F-PU in-and-out coated ACF for CF/water, nitromethane (NM)/water, Trichloroethylene (TCE)/water, dichloromethane (DCM)/water, chlorobenzene (CB)/water, and 1,2-dichloroethane (DCE)/water mixtures. c Separation efficiencies of F-PU in-and-out coated ACF for CF/water mixture as a function of the number of oil/water separation cycles. The error bars represent the standard deviations of the data.

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To demonstrate the resistance of the material to external impact and its continuous reusability after impact, the F-PU in-and-out-coated ACF was subjected to a scratch test, and its maintained wettability was investigated. The results revealed that there was a slight change in the water contact angle of the F-PU in-and-out-coated ACF after scratching the coated fabric for 50 times using sand paper (Fig. 6a). In addition, although the scratch test did not induce any significant damage to the F-PU in-and-out coating, the coated ACF membrane maintained its wettability after the occurrence of damages or crack owing to the presence of F-PU in-and-out coatings inside the ACF pores. Moreover, the F-PU in-and-out coating contained numerous urethane chemical structures, which strengthened the intermolecular hydrogen bonds between the hydrogen of the secondary amine group and the carbonyl oxygen. In addition, the F-PU in-and-out coating acted as a physical crosslinker and enhanced the mechanical properties of the ACF. Figure 6b shows a comparison of the stress–strain curve of the bare ACF fabric and the F-PU in-and-out-coated ACF. The tensile strengths of the bare ACF and F-PU in-and-out coated ACF were 322.41 and 575.2 MPa, respectively. The tensile strength of the F-PU in-and-out coated ACF was 1.78 times higher than that of the bare ACF. This indicates that the F-PU in-and-out-coated ACF can withstand further external pressure.

Fig. 6: Durability and mechanical properties of F-PU in-and-out coated ACF.
figure 6

a Water contact angle of the F-PU in-and-out-coated ACF after the scratch test. b Tensile strength of the bare ACF and F-PU in-and-out-coated ACF. The error bars represent the standard deviations of the data.

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To evaluate the bacterial penetration blocking ability of the F-PU in-and-out-coated ACF, we designed and fabricated a bacterial penetrating system using trans-well plates (Supplementary Fig. S5). To prove that it is not toxic after coating, the C2C12 cell viability test before and after coating was carried out using Cell Counting Kit-8 with a density of 3 × 104 cells/well for 48 h incubation. Although the viability was slightly reduced in samples with ACF, the cell viability is higher after coating than before coating. This means that there is no toxicity by the coating materials (Supplementary Fig. S6). Anti-bacterial property is the ability of a material to destroy bacteria or suppress their growth or their ability to reproduce. Antibiotics exhibit a very powerful anti-microbial effect; however, they are constantly consumed and exhibit fatal side effects, such as super-bacteria generation. Therefore, separation membranes containing antibiotics are unsuitable in an environment where there is a continuous flow of fluid. This indicates that it is essential to develop separation materials that exhibit anti-fouling properties and high durability.

Figure 7a shows the quantity of bacteria that penetrated through or was retained on the bare ACF and the F-PU in-and-out-coated ACF. No bacteria was observed on the bare ACF owing to its non-selectivity, which enabled the penetration of the S. aureus and E. coli. In contrast, numerous light yellow aggregates, which were formed by bacteria colonies, were observed on the surface of the F–PU in-and-out-coated ACF subjected to 30 and 50 scratch cycles. Further experiments were conducted to determine whether the F-PU in-and-out-coated ACF subjected to scratch test can effectively prevent the permeation of bacteria. Bacteria were seeded on the bare ACF and the F–PU in-and-out-coated ACF, cultured for 1 day, after which the OD value of the solution below the inserts of the trans-well plate was measured. The F-PU in-and-out-coated ACF effectively prevented the permeation of S. aureus and E. coli by ≥99% (Fig. 7b). Particularly, after the 30- and 50-cycles scratch test, the F-PU in-and-out-coated ACF maintained its bacteria penetration blocking ability for S. aureus and E. coli. These results of the performances of the F-PU in-and-out-coated ACF demonstrate the promising potential of the coated -ACF for the prevention of bacterial penetration (≥99%), and its high durability external impact.

Fig. 7: Bacteria blocking performance of F-PU in-and-out coated ACF.
figure 7

a Photograph images of the bare ACF and the F-PU in-and-out-coated ACF with 30 and 50 scratch cycles after the bacterial penetration tests. b Bacterial viability of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) for the bare ACF, the F-PU in-and-out coated ACF, and the coated ACF after 30 and 50 scratch cycles (coated_S30 and S50, respectively). The error bars represent the standard deviations of the data.

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In summary, we reported a strategy to modify the surface wettability of porous ACF using F-PU in-and-out coatings. The F-PU coating material was prepared via the reaction of EG, IPDI, and TDFO. The coating of the inner and outer surface of ACF with the F-PU in-and-out coatings was achieved via urethane reactions. The in-and-out coating enabled the formation of a heterogeneous structure in the ACF fabric. The heterogeneous structure of the F-PU in-and-out-coated ACF endowed the fabric with a high durability for scratch resistance. In addition, further investigations revealed that the F-PU in-and-out-coated ACF exhibited goods hydrophobic properties after scratch tests, enhanced tensile strength, oil absorption properties, and excellent oil–water separation performance. Particularly, during the oil–water separation experiments, oils passed through the F-PU in-and-out-coated ACF at a high flux (2 × 104 L · m−2 · h−1) and high efficiency (>95%). Lastly, we confirmed that the F-PU in-and-out-coated ACF effectively blocked bacteria permeation regardless of the bacteria type (gram-negative S. aureus and gram-positive E. coli), and maintained its bacteria blocking performance after the scratch tests. These results indicate that F-PU in-and-out coating is effective for fabricating a heterogeneous-structured membrane with high durability and excellent bacterial blocking performance.

Methods

Materials

Activated carbon fabric (ACF, ACC-5092-20) was purchased from Kynol®. The weight, thickness and specific surface area of the ACF were 135 g/m2, 0.55 mm, and 1800 m2/g, respectively. Isophorone diisocyanate (IPDI), EG, dimethyl sulfoxide, dichloromethane (DCM), toluene, hexane, olive oil, nitromethane (NM), trichloroethylene (TCE), chlorobenzene (CB), 1,2-dichloroethane (DCE), and chloroform (CF) were purchased from Sigma–Aldrich. 1H,1H,2H,2H-Tridecafluoro-1-n-octanol (TDFO) was obtained from BLD Pharmatech Inc.

Synthesis of F-PU coating materials

First, TDFO–IPDI–EG–IPDI coating materials were synthesized with a two-step addition process, in which the prepolymer was prepared by reacting EG with excess IPDI, after which the end isocyanate (NCO) groups were reacted with the hydroxyl group (–OH) perfluoroalkyl chain of TDFO. The successful preparation of TDFO–IPDI–EG–IPDI coating materials was verified using Fourier transform-infrared (FT–IR) analysis (model 4700 device Jasco, Easton, USA). The molar ratio of IPDI, EG, and TDFO in the prepolymer was 2:1:1. To synthesize the TDFO–IPDI–EG–IPDI coating materials, first, IPDI–EG–IPDI was synthesized by mixing 4.32 mL of IPDI and 0.58 mL of EG in 85.22 mL of DMF, after which the mixture was reacted at 80 °C for 12 h under stirring. Subsequently, 4.66 mL of TDFO was added to the prepared IPDI–EG–IPDI mixture and reacted at 80 °C for 12 h under stirring in an oil bath.

Fabrication of F-PU in-and-out-coated fabric

Generally, ACF contains numerous functional groups including –OH group, ketone, and carboxylic acid. To enhance the reactivity of ACF using the prepared TDFO–IPDI–EG–IPDI coating materials, the ketone of ACF was reduced using sodium borohydride to produce an –OH group. Next, the treated ACF was immersed in TDFO–IPDI–EG–IPDI coating materials solution. Subsequently, the –OH group of ACF and the NCO of TDFO–IPDI–EG–IPDI coating materials were reacted at 80 °C for 72 h in an oil bath.

Characterization of TDFO–IPDI–EG–IPDI in-and-out-coated fabric

The surface morphology and elemental analysis of the TDFO-IPDI-EG-IPDI in-and-out coated fabric were analyzed using X-ray photoelectron spectroscopy (XPS; K-alpha, ThermoFisher Scientific, USA), SEM (IT-500, JEOL, Japan) and field emission scanning electron microscopy equipped with an energy dispersive X-ray spectrometer (FE-SEM/EDS; SUPRA 55VP, Carl Zeiss, Germany). Nitrogen adsorption measurements of TDFO–IPDI–EG–IPDI in-and-out coated fabric were performed using a Brunauer–Emmett–Teller (BET) analyzer (ASAP 2020 system, Micromeritics Instrument Corp., USA). The contact angles of various liquids on the TDFO–IPDI–EG–IPDI in-and-out-coated fabric were evaluated from a contact angle goniometer (Smart Drop Standard, Femtobiomed, Korea). A universal testing machine (UTM; Model 3366, Instron, USA) was used to measure tensile strength of bare ACF and coated ACF (tensile rate = 5 mm/min−1).

Oil–water absorption and separation

The water or oil absorption of the coated fabric was investigated using six types of test liquids (distilled (DI) water, DCM, toluene, hexane, olive oil, and CF). Feed solution was prepared at a 1:1(v/v) ratio of water and oil. To measure the absorption capacities of the membrane, 1 cm2 of bare ACF and the in-and-out F-PU-coated ACF was prepared and immersed in the DI water or the oils until the fabrics were totally saturated, after which the samples were removed and immediately weighed. The weight measurement was performed quickly to avoid any absorbate evaporation. The absorption capacity of the bare ACF and in-and-out F-PU-coated ACF was determined in weight:area ratio, as given in Eq. 1:

$${{{\mathrm{Absorption}}}}\;{{{\mathrm{capacity}}}} = \frac{{W_a\;\left( {{{\mathrm{g}}}} \right) - W_0({{{\mathrm{g}}}})}}{{Area\;({{{\mathrm{cm}}}}^2)}}$$
(1)

where Wa is the weight of the bare ACF or in-and-out F-PU-coated ACF in the oil/organic solvents at a saturated state and W0 is the weight the bare ACF or in-and-out F-PU-coated ACF at the initial state. Each test was repeated three times.

For an oil–water separation test driven only by self-gravity, the bare ACF and in-and-out F-PU-coated ACF were placed between two glass containers. Subsequently, oil–water mixtures were poured onto the bare ACF or in-and-out F-PU coated ACF. Each separation experiment was performed three times, and the separation efficiency was determined using Eq. (2):

$${{{\mathrm{Separation}}}}\;{{{\mathrm{efficiency}}}} = \frac{{V_a}}{{V_0}}\,\times\,100\%$$
(2)

Where Va is the volume of solvent that penetrates to the in-and-out F-PU-coated ACF after the oil–water mixture was poured and V0 is the volume of solvent in the oil–water mixture before pouring.

The flux (F) of the bare ACF and in-and-out F-PU-coated ACF was calculated using Eq. (3):

$${{{\mathrm{Flux}}}}\;\left( F \right) = \frac{V}{{St}}$$
(3)

where V is the volume of the liquid permeating through the bare ACF and in-and-out F-PU-coated ACF, S is the effective contact area of the bare ACF and in-and-out F-PU-coated ACF, and t is the permeating time.

Bacteria penetration through the F-PU in-and-out-coated fabric

Staphylococcus aureus (S. aureus; 25923, ATCC, USA) and Escherichia coli (E. coli; 25922, ATCC, USA), which are gram-positive and gram-negative bacteria, respectively, were used in the bacterial penetration blocking experiment. Briefly, both bacteria were cultured in a Tryptic soy broth (soybean–casein digest medium, Becton Dickinson and Company, USA) medium and incubated at 37 °C under aerobic conditions. Subsequently, the bacterial suspension was diluted to an optical density (OD) concentration of 0.1 (measured at 600 nm).

Bare ACF, was used as a control sample, and the coated ACF was used as the experimental sample. All samples used in this experiment were sterilized under ultraviolet-irradiation before culturing with bacterium. The samples were placed in each insert of 24-transwell plates (6.5 mm insert and 8.0 μm diameter pore), after which the inserts were mounted in each well. Subsequently, 0.8 mL of the fresh bacteria culture medium was poured into each well at the bottom of the transwell plate. Thereafter, 80 μL of S. aureus or E. coli suspension was added to each insert containing the samples. To validate the experiment, several wells were filled with a pristine medium without bacteria. After incubation of the well plates for 24 h, all inserts were removed from the transwell plates. The OD value of the culture medium remaining at the bottom of each well was measured to evaluate the degree of contamination of the culture medium by bacterial permeation. The OD of the medium was measured at 600 nm using a plate reader.