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Transparent Copper-loaded Chitosan/Silica Antibacterial Coatings with Long-term Efficacy Debirupa Mitra, Min Li, En-Tang Kang, and Koon-Gee Neoh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b07700 • Publication Date (Web): 09 Aug 2017 Downloaded from http://pubs.acs.org on August 12, 2017

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ACS Applied Materials & Interfaces

Transparent

Copper-loaded

Chitosan/Silica

Antibacterial

Coatings with Long-term Efficacy

Debirupa Mitra, Min Li, En-Tang Kang, Koon Gee Neoh* Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 117576

* Corresponding author: Tel: +65 65162176; Fax: +65 67791936; Email: [email protected]

1 ACS Paragon Plus Environment


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Abstract Bacteria-contaminated inanimate surfaces within hospitals and clinics result in transmission of pathogens via direct or indirect contact, leading to increased risk of healthcare-associated infections (HAI). The use of antibacterial coatings is a potential way of reducing the bacterial burden, but many surfaces like instrument panels and monitors necessitate the coatings to be transparent while being highly antibacterial. In this work, silica nanoparticles (SiO2 NPs) were first grown over a layer of acrylated quaternized chitosan (AQCS) covalently immobilized on commercially available transparent polyvinyl fluoride (PVF) films. The SiO2 NPs then served as nano-reservoirs for adsorption of copper ions. The coated PVF films were transparent and reduced viable bacterial count by ~ 99 % and 100 %, when incubated with a bacteria-loaded droplet for 60 min and 120 min respectively. The killing efficacy of these coatings, after wiping 100 times, with a deionized water-wetted cloth was reduced slightly to 97-98 %. The stability of these coatings can be further improved with the deposition of another layer of cationic quaternized chitosan (QCS) over the negatively charged SiO2 NP layer, wherein the coatings maintained ~ 99 % killing efficacy even after 100 wipes. These coatings showed no significant toxicity to mammalian cells, and hence can potentially be used in a clinical setting for reducing HAI.

Keywords: antibacterial, copper, quaternized chitosan, silica nanoparticle, coating

Introduction Contamination of inanimate surfaces with bacteria has long been a cause of concern, and its consequences range from spoilage of food1 to occurrence of infection in implanted medical devices.2 There is now significant evidence that contaminated surfaces in clinics or hospitals contribute to the transmission of pathogens, resulting in the spread of healthcare-associated


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infections (HAI).3 Although regular cleaning of environmental surfaces in hospitals with appropriate detergent or disinfectant is commonly used to reduce the spread of HAI, recent reports suggest that bacterial population reverts back to its pre-cleaning level within just a few hours of cleaning, irrespective of the disinfectant used.4 Thus, there is a pressing need for strategies that can effectively inhibit bacterial colonization on surfaces. A potential approach is the use of antibacterial coatings that can substantially reduce the surface-attached bacterial burden to break the “nosocomial infection loop” and prevent transmission while preserving the properties of the bulk material.5

Strategies

employed

in

endowing

surfaces

with

antibacterial properties

include

immobilization of bactericidal polymers by “grafting to” or “grafting from” methods,6,7 layerby-layer (LbL) assembly of bactericidal polymers,8 polymeric coating with eluting antibiotics or metals or antimicrobial peptides,9 nanoparticle-based coating10 or a combination of these methods.11 Metal ions like Ag, Cu and Zn possess intrinsic broad-spectrum antibacterial activity at concentrations that are non-cytotoxic to mammalian cells.12 As such, metal-based antibacterial coatings (with Ag and Cu in particular) have been widely investigated because of their high antibacterial efficacy.10,13 While Ag has been the preferred choice in most cases,14 it has been shown that Cu surfaces exhibit higher efficacy, compared to Ag surfaces, at temperatures and humidity that are typical of indoor environments like hospitals.15 The potential of Cu surfaces in reducing transmission of pathogens in clinical environment has also been demonstrated.16-18 However, the use of metallic Cu or alloyed Cu surfaces on medical instruments (like syringe pump drivers, ventilator panels, etc.), switchboards, monitors and keyboards within the hospital wards is not practical due to the opacity of Cu metal or alloy. For these applications, transparent and long-lasting Cu-containing antibacterial coatings can be an attractive alternative.



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Cu-based coatings have been mostly fabricated by magnetron sputtering,19 cold-spraying,20 aerosol-assisted chemical vapour deposition,21 and electroless plating.22 Although these methods are effective in depositing Cu, the prepared surfaces are generally coloured and opaque. We propose to address this issue by loading Cu ions onto transparent coating materials such as silica nanoparticles (SiO2 NPs). SiO2 can be easily fabricated as transparent NPs, and SiO2 NP-coated glass has demonstrated better optical transmission properties than uncoated glass.23 Mesoporous SiO2 NPs have been widely investigated for a variety of biomedical applications,24,25 and recent studies have shown Cu-containing bioactive glass NPs and Cu-doped mesoporous SiO2 NPs to be potential agents for therapeutic Cu ion release.26,27

Herein we report the fabrication of transparent Cu-containing coatings with a high degree of antibacterial efficacy and durability on polymeric films via the LbL method. Since direct immobilization of SiO2 NPs on inert polymeric films can be difficult, the NPs were grown in situ after deposition of a polycation layer. Quaternized chitosan (QCS) was selected as the base polycation due to its antibacterial properties and non-cytotoxic nature.28,29 It was immobilized on transparent polyvinyl fluoride (PVF) films by UV-induced grafting of acrylated quaternized chitosan (AQCS). SiO2 NPs were then grown over the AQCS coating, followed by adsorption of Cu (II) ions. Subsequently, another layer of QCS was deposited over the SiO2 NP layer to enhance the stability of the nanoparticulate coating. The antibacterial efficacy, durability, transparency and cytotoxicity of the Cu-loaded coatings were evaluated.

Experimental section


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Materials Low molecular weight chitosan (CS, ≥ 75 % deacetylation, viscosity 0.02-0.3 kg/m-s for 1 wt.% solution in 1 wt.% acetic acid), hexyl bromide, methanesulfonic acid, acryloyl chloride, tetraethylorthosilicate (TEOS), tryptic soy broth, Luria-Bertani broth (Miller) and agar were purchased from Sigma-Aldrich, USA. Potassium iodide, sodium hydroxide, ammonium hydroxide and copper (II) chloride were procured from Merck, Germany. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Alfa Aesar, USA. PVF film (thickness 0.05 mm) was purchased from Goodfellow, UK. Staphylococcus aureus (S. aureus, ATCC 25923), Pseudomonas aeruginosa (P. aeruginosa, ATCC 15692), 3T3 mouse fibroblast cells (ATCC CRL-1658) and adult human dermal fibroblasts (HDF, ATCC PCS-201-012) were obtained from American Type Culture Collection (ATCC, USA). All other solvents used were of analytical reagent grade.

Preparation of AQCS grafted PVF film QCS was synthesized as described in our previous work.30 Briefly, 2 g of CS was dispersed in 100 mL of N-methyl-2-pyrrolidone under stirring. Then, the temperature was increased to 60 °C and 30 mL of 1.5 N NaOH was added. After overnight stirring, the temperature was increased to 80 °C and 2.4 g of KI and 26 g of hexyl bromide were added. The reaction was carried out at 80 °C for 48 h. The reaction mixture was then precipitated in acetone and the precipitate, obtained after centrifugation, was dissolved in deionized (DI) water. This aqueous solution was dialysed against DI water and freeze-dried to obtain QCS. QCS was then acrylated using a procedure similar to that reported earlier.31 Seventy-five milligrams of QCS was dissolved in 20 mL of methanesulfonic acid under stirring for 1 h at room temperature. The solution was then stirred in an ice-bath to lower its temperature to 0 °C. Ninety-five microliters of acryloyl chloride was added dropwise and the reaction was allowed to proceed



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for 1 h at 0 °C and for another 5 h at 25 °C. The reaction was then terminated by adding 30 g of crushed ice. The solution was dialysed against DI water for 3 days and freeze-dried to obtain AQCS.

To prepare AQCS-grafted PVF films, PVF was first cut into 15 × 8 cm2 pieces and cleaned by ultrasonication in ethanol followed by DI water for 10 min each. The clean and dry PVF film was then subjected to plasma treatment (Model ATTO, Diener electronic, Germany) in the presence of oxygen gas for 10 min at 150 W power. Then, 2.5 mL of 5 wt.% AQCS aqueous solution was poured over the plasma-treated PVF film and spread with the help of a spatula to completely coat the film with the solution. The film was placed inside a glass container, degassed with argon for 1 h and irradiated with 365 nm UV light inside a crosslinker chamber (Model CL-1000L, UVP, USA) for 2 h to obtain AQCS-grafted PVF. The film was then washed thoroughly with DI water to remove any physically adsorbed polymer, dried under nitrogen flow and stored in a dry-box for further use (denoted as Film A).

Preparation of SiO2 NP coatings SiO2 NPs were grown in situ on Film A via Stöber method of SiO2 condensation and growth.32-34 Film A, measuring 2 × 2 cm2 (cut from the 15 × 8 cm2 film) was placed in a 25 mL glass beaker and 5 mL of ethanol, 1 mL of DI water and 100 µL of 25 % NH4OH (in water) were added to it. This was placed in a shaker-water bath maintained at 30 °C and 100 rpm. Fifty microliters of TEOS was added to the solution and the reaction was allowed to proceed for 1 to 5 h to prepare Films S1-S5 (Table 1). The film was removed from the SiO2 sol, washed with copious amounts of DI water and dried in an oven at 70 °C for 1 h. The Si content of Films S1-S5 was determined by subjecting the films to hot alkali digestion (boiling in 4 M NaOH for 20 min) followed by overnight incubation at 25 °C, prior to analysis using


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inductively coupled plasma optical emission spectrometry (ICP-OES, iCAP 6200 duo, Thermo Scientific, USA) analysis. For preparation of films with a second layer of QCS (Films Q1 or Q3), 50 µL of 5 wt.% QCS aqueous solution was spread over Films S1 or S3, respectively, and kept at 25 °C for 5 h. The films were then washed with DI water and dried in an oven at 70 °C for 1 h.

Cu loading on coated PVF films and release profile of Cu and Si Cu was loaded on Films A, S1-S5 and Q1, Q3 by immersing the films in copper salt solution. Film A, S1, S2, S3, S4, S5, Q1 or Q3 measuring 2 × 2 cm2, was immersed in 6 wt.% CuCl2 aqueous solution and placed in a shaking water bath kept at 30 °C and 50 rpm for 3-15 h to prepare Cu-loaded films. The films were removed from the CuCl2 solution after the treatment period, washed with DI water and dried in an oven at 70 °C for 1 h. From these preliminary experiments, it was established that the optimal treatment time was 6 h (see Results and Discussion), and Films A-Cu, S1-Cu, S2-Cu, S3-Cu, S4-Cu, S5-Cu, Q1-Cu or Q3-Cu (Table 1) were prepared using this treatment time. The Cu content of the Cu-loaded films was determined by subjecting the films to hot acid digestion (in 65 % HNO3 at 70 °C for 2 h) followed by ICP-OES analysis.

Cu release from Films A-Cu, S1-Cu and Q1-Cu was investigated by immersing the respective film (measuring 1 × 1 cm2) in 1 mL of DI water at 25 °C for different time periods and subjecting the release medium to ICP-OES analysis. The film size and aqueous volume for this experiment were chosen to simulate release (of Cu or Si) in the cytotoxicity experiment (described below). Si release from Films S1, S1-Cu, Q1 and Q1-Cu was similarly measured, except that in this case the release medium was further digested in 4 M NaOH by overnight incubation at 25 °C, prior to ICP-OES analysis.



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Characterization AQCS was analysed using Fourier Transform Infrared spectroscopy (FTIR) (Vertex 70, Bruker, USA). For electron microscopy observation, the modified surfaces of Films A, S1, S3 and S5 (cut into 5 × 5 mm2 pieces) were coated with platinum in a sputter-coater (20 mA for 100 s, Model JFC 1300, JEOL, USA) and observed under a field emission scanning electron microscope (FESEM) (Model JSM-6700, JEOL, USA) at an acceleration voltage of 5 kV. Water contact angle of Films A, S1, S3, Q1 and Q3 was measured using a telescopic goniometer (Model 1 00-00-230, Ramé-Hart, USA). Surface composition of the films was analysed by X-ray photoelectron spectroscopy (XPS) (AXIS UltraDLD spectrometer, Kratos Analytical, UK) using monochromatic Al Kα as the X-ray source (1468.6 eV photons). Peak fitting was carried out using XPSPEAK41 software, with the C 1s peak at 284.6 eV as reference, and the full width at half-maximum for all peaks within a core level was kept constant. Zeta potential of Films A, S1, S3, Q1 and Q3 was measured in 0.1 M NaCl using an electrokinetic analyzer (Model SurPASS, Anton Paar, Austria). Optical transmission of coated films (2 × 2 cm2) in the visible region was measured using UV-Vis spectroscopy (UV 3600, Shimadzu, Japan) with pristine PVF film as reference.

Assays for determining antibacterial efficacy The ability of the coated PVF films to resist bacterial colonization when challenged with bacteria-loaded droplets (subsequently referred to as droplet assay) was assessed as described in our earlier publication.30 Briefly, S. aureus (or P. aeruginosa) was cultured in tryptic soy broth (or Luria-Bertani broth) for 18-24 h at 37 °C in an orbital shaker-incubator (Model ES20, Grant Instruments, UK). The growth medium was removed by centrifugation for 8 min at 2700 rpm and the bacteria were re-suspended in sterile phosphate-buffered saline (PBS) at a


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concentration of either ~ 5 × 106 cells/mL or ~ 108 cells/mL. Pristine or coated PVF films, measuring 1 × 1 cm2, were first sterilized inside a biological safety cabinet fitted with a 30 W UV lamp for 20 min. One microliter of the prepared bacterial suspension (containing ~ 5 × 103 cells or ~ 105 cells ) was carefully pipetted on each of the films and allowed to incubate at 25 °C for a pre-determined time (30, 60, 90 or 120 min). One set of experiments was carried to evaluate the effect of droplet size by using 10 µL droplet (containing ~ 5 × 103 P. aeruginosa) and an incubation time of 120 min at 25 °C. After incubation, each film was immersed in a tube containing 2 mL of PBS and subjected to 7 min ultrasonication followed by 10 s vortexing to release the surface-attached bacteria into the medium. One hundred microliters of this PBS suspension was spread on agar-plates and the number of colony forming units (CFU) was counted after 24 h incubation at 37 °C. The agar plates were prepared from nutrient-agar solution obtained by dissolving 30 g/L of tryptic soy broth (or 25 g/L of Luria-Bertani broth) and 30 g/L of agar in DI water at 100 °C followed by autoclaving at 120 °C for 20 min. The sterilized solution was then poured in to sterile 94 mm Petri dishes (~ 10 mL solution in to each Petri dish) and allowed to cool to room temperature prior to use.

Another assay was carried out to assess the antibacterial efficacy of the coated films when contacted with a bacteria-loaded dry surface (subsequently referred to as contact assay).30 S. aureus was suspended in sterile PBS at a concentration of ~ 107 cells/mL and 1 µL of this suspension containing ~ 104 cells was pipetted onto the surface of a sterile, hydrophilic 0.2 µm polyester track-etch membrane. After the water droplet was absorbed by the membrane, it was placed over the pristine or coated PVF film (1 × 1 cm2) such that the contaminated side of the membrane was in contact with the film surface. A 30 g weight was immediately placed on top of the membrane and after 5 s, the weight and the membrane were removed. The film



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was then incubated at 25 °C for 60 min followed by quantitative counting of bacteria by spread-plate method as described above.

Durability assay The stability of the deposited SiO2 NP layer when subjected to friction was assessed as follows: Films S1, S2, S3, S4 and S5 were wiped 30 times (in one direction) with a piece of cloth moistened with DI water and the films were allowed to air-dry after each wipe. The Si content of the dried films was then analysed by ICP-OES analysis as described above. All Cu-loaded films were subjected to the durability test for 30, 60 and/or 100 wipes with the DI water-wetted cloth. The stability of the coating was assessed from the Cu or Si content of these wiped and dried films as determined by ICP-OES analysis or from the antibacterial efficacy by droplet assay.

Cytotoxicity assay The possible cytotoxicity of the coated films with or without Cu was investigated using the standard MTT assay. 3T3 mouse fibroblast cells (or HDF) were cultured in growth medium comprising Dulbecco’s Modified Eagle Medium, 10 % fetal bovine serum, 1 mM Lglutamine and 100 IU/mL penicillin. One milliliter of 3T3 cell (or HDF) suspension in the growth medium was seeded into each well of a 24-well plate at a density of 40,000 cells per well and incubated at 37 °C, 5 % CO2 for 24 h. The medium was removed and pristine PVF or coated PVF films measuring 1 × 1 cm2 were then gently placed in the wells with the coated surface touching the cell layer. One milliliter of fresh medium was added to each well and incubated for another 24 h at 37 °C, 5 % CO2. Wells containing cells in growth medium without films served as the negative control. The films and medium were then removed from each well and 100 µL of MTT reagent (5 mg/mL in sterile PBS) and 400 µL of fresh medium


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were added to each well. After incubation for 4 h, the solution containing medium and MTT reagent was removed from the wells and 750 µL dimethyl sulfoxide was added to dissolve the formazan crystals. Absorbance was measured at 570 nm using a microplate reader (Multiskan GO, Thermo Scientific, USA) and the viability of the cells was expressed as a percentage relative to the negative control.

Statistical analysis Experiments were carried out in triplicate unless otherwise stated. All experimental values were reported as mean ± standard deviation and statistical analysis was carried out using oneway analysis of variance (ANOVA) with Tukey post hoc test. Statistical significance was accepted at p < 0.05.

Results and Discussion Characterization of AQCS and AQCS/SiO2 NP coating CS was quaternized via an alkylation reaction with hexyl bromide,29 and the QCS was further modified to AQCS by the introduction of acrylate groups at C-6 position by O-acylation under acidic conditions.31 Successful preparation of AQCS was confirmed by FTIR spectroscopy (Figure S1). The absorption band at 1527 cm-1 in the AQCS spectrum corresponds to the N+-C bond formed from quaternization of the amine groups at C-2 position of CS.30 The bands at 777 cm-1 and 1630 cm-1 are likely due to C=C out of plane deformation35 and C=C stretching36 respectively, of the acrylate group in AQCS. The strong absorption at 1158 cm-1 can be attributed to C-O stretch of the ester group introduced by acylation.36 The characteristic absorption of CS at 1070 cm-1 corresponding to bending of CO-C groups is preserved in the AQCS spectrum.29



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The coating procedure is shown schematically in Scheme 1. PVF was chosen as the substrate in this work due to its transparency, durability and its use as a protective film in industrial applications. In the first step, PVF film was subjected to plasma treatment (Step (1)) in presence of oxygen to generate an oxygen-rich surface comprising hydroxyl and peroxyl groups.37 AQCS was immobilized on the surface-activated PVF by UV-induced covalent grafting and cross-linking via the acrylate groups of AQCS to prepare Film A, as shown in Step (2).38 In the next step (Step (3)), SiO2 NPs were deposited on Film A to prepare Film S1 (or S2-S5, depending on the SiO2 deposition time). Growth of SiO2 on the AQCS layer was carried out via Stöber method, where TEOS is hydrolysed in presence of ammonia and ethanol resulting in the condensation of SiO2 on the surface.34 Ammonia helps to generate SiO2 NPs with a negatively charged surface, which in turn promotes electrostatic interaction with the positively charged AQCS surface.33 This electrostatic interaction provides the driving force for the simultaneous nucleation and growth of SiO2 NPs on the polymer surface. An additional layer of QCS was deposited on Film S1 (or S3) by electrostatic assembly of the positively charged QCS over the negatively charged SiO2 NPs to prepare Film Q1 (or Q3) as shown in Step (4). As a final step, Cu was loaded on Films A, S1 (or S2S5) and Q1 (or Q3) by the adsorption of Cu ions from copper salt (CuCl2) solution.

XPS spectra of Films A-Cu and S1-Cu are shown in Figure 1. The wide-scan spectrum of Film A-Cu (Figure 1(a)) shows substantial reduction in the F 1s peak at ~ 685 eV as compared to that of pristine PVF (Figure S2), indicating that the surface of the PVF film has been modified. The appearance of an N 1s peak at ~ 400 eV in the spectrum Film A-Cu is attributed to the surface-immobilized AQCS, since this peak is absent in the PVF spectrum. The N 1s core-level spectrum (inset of Figure 1(a’)) can be fitted with two peaks at 399.7 eV and 402 eV corresponding to N-H and N+ of AQCS, respectively, further confirming the


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successful immobilization of AQCS on the PVF surface.29 The intensity of O 1s peak at ~ 530 eV in the spectrum of Film A-Cu (Figure 1(a)) also increased compared to that in the PVF spectrum, and this is consistent with the presence of oxygen in AQCS. Although pristine PVF and Film A-Cu did not have any deposited SiO2 NP, a very small Si 2p peak was observed at 102 eV (Figures S2 and 1(a’)), which may be due to possible contamination from the double-sided tape used for fixing the samples on the XPS sample stub or from within the XPS analysis chamber. The presence of Cu in Film A-Cu was confirmed from the Cu 2p core-level spectrum (Figure 1(a”)), which showed a doublet at ~ 935 eV and ~ 955 eV, corresponding to Cu 2p3/2 and Cu 2p1/2 respectively. The Cu 2p3/2 peak could be fitted with a major peak at 935.2 eV, which can be attributed to Cu (II), confirming the presence of adsorbed Cu (II) on the AQCS layer.39 Shake-up satellites accompanying the doublet, which are a characteristic of Cu (II) species, were also observed.40 A relatively small peak at 933.1 eV was also present which is attributed to a small amount of Cu (I) species, that may have been formed either from the interaction of Cu (II) with AQCS since CS is known to reduce some metals or the reduction of Cu (II) by X-rays during the XPS analysis.40

The wide-scan spectrum of the SiO2–containing film, S1-Cu, (Figure 1(b)) shows strong Si 2s and Si 2p peaks at ~ 154 eV and ~ 103 eV respectively, along with an increased O 1s intensity as compared to that of A-Cu. The Si 2p core-level spectrum (Figure 1(b’)) can be fitted with a major peak at 102.9 eV, which is attributed to amorphous SiO2,41 indicating successful deposition of SiO2 NPs. A smaller peak at 101.7 eV was also observed, which could be due to the presence of either sub-oxides of silicon42 or a silyl-alkyl moiety, possibly TEOS.43 The N 1s spectrum of S1-Cu (inset of Figure 1(b’)) can be deconvoluted in the same manner as Film A-Cu with peaks for N-H and N+. The Cu 2p core-level spectrum of



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S1-Cu (Figure 1(b”)) shows the characteristic Cu (II) spectrum as mentioned above, with two Cu 2p3/2 peaks at 935.1 and 933 eV, corresponding to Cu (II) and Cu (I), respectively.

Effect of SiO2 NP deposition time on Cu content and durability The SiO2 NPs act as “nano-reservoirs” for Cu ions that endow the coating with antibacterial property. While a larger amount of deposited NPs would constitute a larger reservoir for Cu ions, the electrostatic interaction between individual NPs and the positively charged AQCS layer may be weakened. Strong electrostatic interaction between SiO2 NPs and AQCS is essential to prevent leaching of the NPs from the coating. The condensation of SiO2 NPs by Stöber’s method is known to be dependent on environmental conditions like solution pH, composition of reactants, amount of NH3 catalyst, time and temperature;33,44 and the size of NPs formed by this method may vary from 20-800 nm.45 In our work, the reaction mixture was fixed at 0.04 mol/L TEOS, 0.1 mol/L NH3 and 9.8 mol/L H2O in ethanol and the reaction temperature was kept at 30 °C. The reaction time was varied from 1 h to 5 h to investigate the time-dependent growth of SiO2 NPs on the AQCS surface. FESEM images of Films A, S1, S3 and S5 are shown in Figure 2. The AQCS layer is relatively featureless (Figure 2(a)), and after a reaction time of 1 h, it was uniformly covered with NPs of size 50-150 nm (Figure 2(b)). When the reaction time was increased to 3 h and 5 h, the NPs became larger (50-300 nm) with a higher degree of polydispersity and aggregation (Figure 2(c,d)). Our observations are in accordance with a previous study, which reported that with increasing time, SiO2 NPs grow by aggregation of smaller particles.44 The growth of SiO2 NPs was further investigated in a quantitative manner by determining the Si content of Films S1-S5 by ICP-OES analysis and the results are shown in Figure S3(a). The amount of Si increased with increase in reaction time from ~ 300 µg/cm2 after 1 h to ~ 480 µg/cm2 after 4 h. The increase in Si content is expected due to the growth of the SiO2 NPs. However, there was no significant


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change (p > 0.05) in the Si content after a reaction time of 4 h (comparing Films S4 and S5) possibly due to exhaustion of the reactants.

The Cu loading on the SiO2 NP-modified films is expected to be governed by the amount of SiO2 NPs and the duration the films were exposed to the Cu salt solution. To investigate the latter, the duration of exposure of Film S5 to the Cu salt solution was varied from 3 to 15 h in a preliminary set of experiments and it was observed that the Cu content in the film increased with increase in loading time from 3 h to 6 h but no further increase was observed when the time was increased to 15 h (data not shown). Thus, the Cu loading time was fixed at 6 h thereafter and the effect of SiO2 deposition time on Cu loading was investigated (Figure 3(a)). The Cu content increased with increase in SiO2 NP deposition time from 1 h to 4 h (Films S1 to S4), correlating well with the increase in Si content (Figure S3(a)) on the surface of these films. However, no further significant (p > 0.05) increase in Cu content was observed when the SiO2 NP deposition time was increased from 4 h to 5 h, which is due to the absence of SiO2 NP growth after 4 h, as discussed earlier. The direct correlation between the Si and Cu contents highlights the importance of surface area provided by the microporous Stöber’s SiO246 for Cu adsorption. The surface of SiO2 has abundant silanol groups (-SiOH)24 and more so in Stöber’s SiO2 due to the presence of NH4OH in the reaction mixture.33 These surface –OH groups are often ionized depending on pH and composition of the aqueous environment, and serve as sites for metal ion adsorption by electrostatic interaction and in some cases, coordinative interaction with the metal ion. The metal cation may either adsorb on the ionized site in a monodentate or a bidentate fashion or the adsorption of the monohydroxometal (II) may also take place in the case of Cu (II) which can be hydrolysed at the interface.47,48 To compare the Cu loading due to AQCS versus that due to the SiO2 NPs, the AQCS-modified surface without SiO2 NPs (Film A) was also treated with Cu salt solution



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for 6 h. The Cu content of Film A-Cu was measured to be ~ 0.9 µg/cm2 (Figure 3(b)), which is significantly (p < 0.05) lower than that obtained when Films S1-S5 were used (Figure 3(a)). Although Cu can form complexes with CS and its derivatives,49,50 the number of sites available on the AQCS layer is expected to be limited as compared to the that provided by the large surface area of the SiO2 NPs.

The stability of the deposited SiO2 NP layer after being subjected to wiping 30 times with a DI water-wetted cloth was assessed by measuring the Si content. Figure S3(a) shows that there was no significant decrease (p > 0.05) in the Si content of Films S1 and S2 after wiping 30 times with DI water. The Si content of Film S3 decreased by ~ 10 % (p = 0.045) while that of Films S4 and S5 decreased by ~ 20 % (p = 0.002 and 0.0004 for S4 and S5, respectively) after 30 wipes. The decrease in the Si content can be attributed to the loss of loosely bound SiO2 NPs upon wiping. Thus, it can be inferred that SiO2 deposition time > 3 h resulted in a less stable SiO2 NP layer possibly due to a decrease in electrostatic interaction between the AQCS layer and the SiO2 NPs which were deposited further away from it. Therefore, Films S1 and S3 were chosen for further tests.

The Cu content of Films A-Cu, S1-Cu and S3-Cu after 60 and 100 times wipes is shown in Figure 3(b). For Film A-Cu subjected to 60 and 100 wipes, the Cu content reduced by ~ 67 % and ~ 89 %, respectively, as compared to as-prepared A-Cu, indicating that the surfaceadsorbed Cu was easily wiped away. For Film S1-Cu, there was no significant decrease in the Cu content after 60 wipes as compared to the as-prepared film. However, the Cu content decreased by ~ 68 % (from 1.9 µg/cm2 for the as-prepared film to 0.6 µg/cm2) after 100 wipes. On the other hand, the Cu content of Film S3-Cu decreased by ~ 61 and ~ 90 % after 60 and 100 wipes respectively, with respect to the as-prepared film. Since the Si content of


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Film S1-Cu decreased by ~ 42 % (Figure S3(b)) after 100 wipes, the corresponding decrease in Cu loading of Film S1-Cu after 100 wipes is due largely to the loss of SiO2 NPs, which served as reservoirs of Cu ions. It is likely that the loss of Cu from Film S3-Cu after 100 wipes is due to the same reason. Since a loss of the Cu-containing SiO2 NPs would diminish the long-term antibacterial efficacy, improvement in the durability of the coating is essential for practical applications. We hypothesized that an additional layer of positively charged QCS deposited over the negatively charged SiO2 NPs would mitigate the loss of these NPs upon wiping, and this strategy is discussed below.

Effect of additional QCS layer on durability QCS was deposited on to Films S1 and S3 to prepare Films Q1 and Q3 respectively. The deposition of QCS was driven by electrostatic interaction with the SiO2 NPs on the surface. The water contact angle changed from 33 ± 2 ° for Film S1 to 45 ± 2 ° for Film Q1 and from 24 ± 2 ° for Film S3 to 42 ± 2 ° for Film Q3 (Table S1). The zeta potential changed from -35 mV for Film S1 to 25 mV for Film Q1 and from -65 mV for Film S3 to 23 mV for Film Q3. The increase in water contact angle, and change in the zeta potential from negative to positive indicated that QCS was successfully deposited on the SiO2 NP-modified surface. The effect of this additional QCS layer on the stability of the coating was investigated. From Figure 3(b), it can be seen that there is no significant difference (p > 0.05) in Cu content between Films Q1-Cu and S1-Cu, and between Films Q3-Cu and S3-Cu Although QCS can provide additional sites for Cu complexation, the number of such sites in the QCS layer would be low as compared to those provided by the NPs. The improvement in the durability of the coating with the deposition of the additional QCS layer is evident from Figure 3(b). For Film Q1-Cu, the Cu content did not decrease by a significant amount (p > 0.05) after 60 wipes but decreased by ~ 39 % (p < 0.05) after 100 wipes. Nevertheless, after 100 wipes, the Cu



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content of Film Q1-Cu was still double that of Film S1-Cu subjected to similar treatment. Similarly, the Cu content of Film Q3-Cu did not change significantly after 60 wipes as compared to the as-prepared film. After 100 wipes, the Cu content of Film Q3-Cu decreased by ~ 53 % as compared to the as-prepared film, but it was still six times more than the Cu content of Film S3-Cu subjected to similar treatment. The Si content of Q1-Cu is shown in Figure S3(b), and it can be seen that the amount of Si decreased by only ~ 14 % after wiping 100 times with DI water for Film Q1-Cu, whereas for Film S1-Cu, the Si content decreased by ~ 42 %. These findings show that the additional QCS layer greatly improved the stability of the SiO2 NP coating.

Antibacterial efficacy of coatings The primary aim of this work was to design antibacterial coatings with high efficacy and durability, intended for use in clinical or other bacteria-burdened public environment. The antibacterial efficacy of the prepared coatings was first evaluated using the droplet assay, in which the coated surfaces were exposed to a 1 µL droplet of bacterial suspension for a definite period of time, and the number of viable bacteria on the surface was counted thereafter.30 The droplet assay simulates the settling of infectious droplets such as those from sneezing or coughing of patients or splashes from a nearby contaminated water source like sink or shower on inanimate surfaces.51 S. aureus was chosen as the model bacteria for most of the experiments since it is one of the most commonly occurring and virulent pathogens in clinical settings and is also known to survive up to 7 months on dry surfaces.52

In the first set of experiments, Films S1-Cu, S3-Cu, Q1-Cu, and Q3-Cu before and after wiping 30, 60 or 100 times with a DI water-wetted cloth were subjected to the droplet assay to assess their long-term antibacterial efficacy. The results presented in Figure 4(a) show


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that as-prepared Films S1-Cu and S3-Cu reduced CFU count by ~ 99 %, as compared to pristine PVF, after incubation with S. aureus for 60 min. There was no significant increase (p > 0.05) in the CFU count after 30 (data not shown) and 60 wipes, for both S1-Cu and S3-Cu. However, after 100 wipes, the CFU count increased by 12 and 14 % for Films S1-Cu and S3Cu respectively. Although these films showed significantly higher (p < 0.05) CFU count as compared to their respective as-prepared coated films, the CFU counts on S1-Cu and S3-Cu after 100 wipes were still 98 and 97 %, respectively, lower than that on pristine PVF film. The as-prepared Films Q1-Cu and Q3-Cu also resulted in ~ 99 % reduction in CFU as compared to pristine PVF film, similar to S1-Cu and S3-Cu, and this efficacy was maintained for both Films Q1-Cu and Q3-Cu even after 100 wipes with a DI water-wetted cloth. It is interesting to note that the antibacterial efficacy of as-prepared Film Q1-Cu (or S1-Cu) is similar to that of Film Q3-Cu (or S3-Cu), even though the Cu content of Q3-Cu (or S3-Cu) was 5 times higher than that of Q1-Cu (or S1-Cu). This may be related to the mechanism by which Cu exerts antibacterial activity. Bacterial killing upon contact with Cu surfaces has been attributed to the intracellular uptake of Cu ions, which in turn leads to membrane damage, generation of reactive oxygen species, inhibition of enzymes and possible degradation of DNA.53,54 Since the uptake of Cu ions by bacteria is the key event for Cucontaining surfaces to exhibit “contact-killing”,53 it follows that the killing efficacy depends on the amount and rate of Cu ions released into the aqueous phase surrounding the bacteria as well as the time of contact. This leads to a concentration-dependent55 and time-dependent56 reduction in bacteria, when other factors (like bacteria type, bacteria plating method, temperature, etc.) are kept constant. For our case (Figure 4(a)), the effect of Cu content in the reduction of CFU was not observed, likely because the Cu concentration of all the asprepared films was high enough to kill ~ 99 % of the bacteria within 60 min of incubation.



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However, the concentration-dependent effect can be observed at lower Cu content, as discussed in the next paragraph.

The importance of SiO2 NPs in maintaining the antibacterial efficacy of the coated films is demonstrated by comparing the performance of Film A-Cu with that of Film Q1-Cu before and after wiping. As-prepared A-Cu showed ~ 99 % reduction in CFU (Figure 4(a)), as compared to pristine PVF, similar to the other Cu-loaded films containing SiO2 NPs like S1Cu and Q1-Cu (Figure 4(a)). However, after 60 and 100 wipes with DI water-wetted cloth, Film A-Cu reduced CFU count by only 92 % and 88 %, respectively, unlike Q1-Cu whose efficacy remained unchanged at 99 %. The Cu content in A-Cu after 100 wipes was ~ 0.1 µg/cm2, which is about 1 order of magnitude lower than that of the as-prepared A-Cu (~ 0.9 µg/cm2) or Q1-Cu after 100 wipes (1.3 µg/cm2). Since the rate of Cu release into the aqueous phase in the vicinity of the bacteria is dependent on the Cu content of surfaces,55 a lower surface Cu content may result in a decrease in Cu release to such an extent that an incubation time of 60 min was not sufficient to exert high contact-killing.

In the above experiment, the bacteria loading was ~ 5 × 103 cells/droplet on 1 cm2 sample, which was chosen based on the average CFU counts (1-103 CFU/cm2) on hospital surfaces reported in earlier studies.17,57 However, on certain occasions, the bacterial concentration may be even higher since aerosols generated from cough of some patients were found to contain > 104 CFU.58 Therefore, a second set of droplet assay experiments was carried out with bacterial concentration of ~ 105 cells/droplet on 1 cm2 sample and the results, obtained after 60 min incubation with S. aureus, are shown in Figure S4. It was found that Film A with only a coating of AQCS reduced CFU by ~ 83 %, while A-Cu reduced CFU by 97 % as compared to pristine PVF film. Both Films S1-Cu and Q1-Cu exhibited > 98 % CFU


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reduction as compared to pristine PVF, indicating that these coatings are able to inhibit bacterial colonization even at high bacterial loading.

In both sets of experiment described above, the bacteria-contaminated droplet was placed on the films for 60 min and the highest kill rate achieved was ~ 99 %. A third set of droplet assay was carried out by varying the S. aureus incubation time on the surface from 30-120 min, to study the time-dependent killing efficacy. It can be seen from Figure 4(b) that the CFU count on pristine PVF after 120 min decreased slightly compared to the count obtained after 30 min. Since PVF is not known to possess any antibacterial activity, the decrease in number of bacteria over 120 min can be attributed to natural death of S. aureus in the absence of nutrients.59 Film A, coated with only AQCS, showed ~ 55 % and ~ 90 % decrease in CFU, as compared to corresponding pristine PVF, after 30 and 120 min of incubation respectively, indicating that antibacterial activity exerted by AQCS is highly time-dependent. The activity of AQCS is expected to be similar to that of QCS, which exerts bactericidal effects as a result of membrane damage induced by electrostatic interaction of the positively charged polymer with the negatively charged bacterial membrane as well as hydrophobic interaction between the alkyl substituent and the lipophilic membrane.28 Similar to Film A, a steady increase in kill rate with incubation time was also observed with the Cu-containing films (Films A-Cu, S1-Cu and Q1-Cu), and 100 % kill was achieved after 90 to 120 min. The time-dependent killing efficacy of Cu surfaces is related to the time required for intracellular accumulation of Cu ions, and the results shown in Figure 4(b) are consistent with an earlier report, where intracellular Cu concentration of bacteria incubated with moist metallic Cu coupons increased steadily over a period of 3 h and no live bacteria could be detected at the end of 3 h.36



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The droplet assay was also carried out with P. aeruginosa on Films PVF, S1-Cu and Q1-Cu. P. aeruginosa is a Gram-negative bacterium which can survive on dry surfaces for up to 16 months and is known to be a frequent isolate from patients with HAI.52 When 1 µL of bacterial suspension was incubated on Films S1-Cu and Q1-Cu for 120 min at 25 °C, 100 % kill was achieved (Figure S5), which is the same as that obtained using Gram-positive S. aureus (Figure 4(b)). Another set of experiment was carried out with 10 µL instead of 1 µL droplet to evaluate whether evaporative stress contributed significantly to the high kill rate. The results showed that 100 % kill was again achieved for Films S1-Cu and Q1-Cu (Figure S5). Thus, the efficacy of the Cu-containing films in killing both Gram-positive and Gramnegative bacteria can be attributed to the bactericidal activity of copper.

Another major route of bacterial transmission is via direct physical contact with contaminated surfaces,51 such as gloves of healthcare workers.60 To mimic the transmission of bacteria under “dry” conditions, the contact assay was carried out.30 The CFU count on the film surface obtained after 60 min incubation with S. aureus is shown in Figure 5. For the contact assay, all Cu-containing films showed 100 % kill after 60 min, which is a higher kill rate compared to the droplet assay. A similar observation was made by Espírito et al., where bacteria incubated on a dry Cu surface were killed more rapidly than those on a moist surface. This was attributed to the absence of a buffering medium on dry surfaces which promoted a more rapid uptake and accumulation of Cu within the bacteria.56 The results of the contact and droplet assays indicate that Films S1-Cu and Q1-Cu have good potential for use as effective antibacterial coatings in moist and dry environments, with Film Q1-Cu being preferred for longer term applications considering its higher durability.

Cytotoxicity of coated films


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In view of the potential application of the Cu-loaded films in clinical settings, it is of utmost importance that the coatings possess minimal or no significant cytotoxicity. Hence, the possible cytotoxicity of selected coated films was evaluated using the standard MTT assay. As shown in Figure 6, Film A did not exhibit significant toxicity (p > 0.05) after 24 h of incubation with 3T3 mouse fibroblasts. The lack of cytotoxicity of Film A can be expected as QCS has been previously shown to be non-cytotoxic and the biocompatibility of CS derivatives is well established.28 SiO2 NP-containing Films S1, S3, Q1 and Q3 also did not show any cytotoxicity to 3T3 cells. There have been conflicting reports on the potential cytotoxicity of SiO2 NPs. These NPs have been previously reported to be well-suited for biomedical applications due to their biocompatibility.25 However, studies that reported on the possible cytotoxic effects of SiO2 NPs have shown the cytotoxicity to be dependent on various factors like size of NPs, crystallinity, surface charge, concentration of NPs and the cell type.61-63 The differences in the type of SiO2 NPs and experimental design among the different studies may have contributed to the conflicting cytotoxicity results reported in the literature. Amorphous SiO2 is reported to be less cytotoxic than crystalline SiO2.61 For amorphous SiO2 NPs of size ranging from 20-200 nm, no cytotoxicity was reported at SiO2 concentration of ≤ 50 ppm (≈ 23 ppm Si) for most cases62-64 although one report showed reduced cell viability at 10 ppm SiO2 (≈ 4.6 ppm Si). Another report demonstrated that amorphous SiO2 NPs of sizes ranging from 15-300 nm did not show cytotoxicity to 3T3 cells, even at a concentration of 100 ppm when incubated up to 72 h.65 SiO2 NPs are also listed under “Generally Accepted as Safe” by the US Food and Drug Administration and used in many commercially available cosmetics and food additives.24 In the present work, the lack of cytotoxicity of SiO2 NP containing films towards 3T3 cells as shown in Figure 6, is likely due to the very low concentration of released Si (< 0.4 ppm) after 24 h (Figure S6(a)). Although the release studies were carried out in absence of cell growth medium and cells, the



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release profile is expected to be similar when the film is incubated with cells. None of the Culoaded films, namely, Films A-Cu, S1-Cu, S3-Cu, Q1-Cu and Q3-Cu showed significant toxicity (p > 0.05) towards the 3T3 cells after 24 h of incubation. Additional cytotoxicity assays of Films S1-Cu and Q1-Cu were carried out with adult HDF, and Figure S7 shows that no significant difference (p > 0.05) in cell viability was observed after 24 h incubation, as compared to HDF incubated only in growth medium. Although cupric ions have been reported earlier to be cytotoxic to different cell lines, the concentrations of Cu that resulted in significantly reduced cell viability in these cases were > 25 ppm.66-68 Cu ions in the concentration range of 0.01-0.1 mM (0.64-6.4 ppm) have been shown to possess high antibacterial activity without any cytotoxic effect toward mouse fibroblast cells.12 Films ACu, S1-Cu and Q1-Cu resulted in < 0.8 ppm (< 0.012 mM) of released Cu after 24 h of incubation in water (Figure S6(b)), and thus, the lack of cytotoxicity as indicated by the results in Figures 6 and S7 can be expected. Therefore, the modified PVF films not only showed high antibacterial efficacy, but they also did not significantly affect mammalian cell viability, demonstrating their potential for clinical applications.

Optical properties of coated films The application of antibacterial coatings on instrument panels, monitors, keyboards, necessitates a high degree of transparency in order to maintain the functionality of these devices. The coated films prepared in this study are all clear and transparent as shown by the photographs of Films A-Cu, S1-Cu and Q1-Cu in Figure 7(a). The optical transmission of Cu-loaded films was also characterized by UV-Vis spectroscopy, and their transmittance in the visible region (350-750 nm) is shown in Figure 7. Film A-Cu showed 65-85 % transmission across the visible region, with reference to pristine PVF film. On the other hand, the transmission of Films S1-Cu and S3-Cu was improved to 95-99 % and 90-96 %


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respectively, across the visible spectrum. This increase in the transparency over that of Film A-Cu is attributed to the presence of SiO2 NPs which have been widely used for making optically transparent and anti-reflective coatings.23,69 Pure SiO2 is an inherently transparent compound, and the deposition of SiO2 NPs result in the creation of voids and nanoporosity that lowers the apparent refractive index of the material, which in turn reduces reflective losses and increases light transmission.69 In addition, SiO2 NPs can also enhance transparency by reducing light scattering. Scattering is largely dependent on the size of SiO2 NPs used and NPs with size of ~ 100 nm or less are considered optimal for higher transparency.70 As such, Film S1-Cu which was prepared with smaller SiO2 NPs as compared to Film S3-Cu (comparing Films S3 and S1 in Figure 2), showed increased transmission due to reduced scattering. With the addition of another QCS layer, i.e. Films Q1-Cu and Q3-Cu, the transmission reduced to 76-90 % and 74-88 % respectively. This decrease in transparency can be due to either increased scattering, absorption or reflectance or a combination of these by the polymer layer. Nevertheless, the transparency of Films Q1-Cu and Q3-Cu is still higher than that of Film A-Cu. Thus, the incorporation of SiO2 NPs in the coating not only enhances the durability of the Cu-containing antibacterial coatings, but increases their transparency as well.

Conclusion In this work, SiO2 NPs were grown over a layer of covalently immobilized AQCS followed by adsorption of Cu to prepare antibacterial coatings. The SiO2 NPs served as nano-reservoirs for the bactericidal Cu ions and also increased coating transparency. The amount of Cu that could be deposited on the coating can be controlled by the amount of SiO2 NPs, but the antibacterial efficacy of the Cu-containing SiO2 NP films is independent of the Cu content as long as it is sufficiently high. A 100 % killing efficacy of S. aureus and P. aeruginosa in



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contact with the Cu-containing SiO2 NP films can be achieved after 120 min. The deposition of another QCS layer on the SiO2 NP containing films enhanced the durability of the coating and 99 % killing efficacy was retained even after 100 wipes with DI water. In spite of its high antibacterial efficacy, the Cu-containing SiO2 NP films did not exhibit significant cytotoxicity towards mammalian fibroblasts. Thus, these coatings show good potential for use as transparent and highly efficacious antibacterial coatings for overcoming the transmission of bacteria within the clinical environment.

ACKNOWLEDGMENT This work was financially supported by the National University of Singapore (Grant number: R279000416112). We acknowledge Ms. Steffie Mano for her assistance in culturing HDF.

ASSOCIATED CONTENT Supporting Information. FTIR characterization of AQCS, XPS spectra of pristine PVF, Si content of modified films, results of droplet assay using ~ 105 cells/sample (S. aureus), and using ~ 5 × 103 cells/sample (P. aeruginosa), Cu and Si release from coated films, cytotoxicity assay with HDF, and water contact angle and zeta potential of Films A, S1, S3, Q1 and Q3 are provided in the Supporting Information.

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Scheme

Scheme 1 Schematic showing surface modification of PVF substrate via plasma treatment (1), AQCS grafting (2) followed by in situ growth of SiO2 NPs (3) and deposition of a second QCS layer (4). The different samples were treated with CuCl2 solution to prepare Cu-loaded coatings. Chemical structures of QCS and AQCS are also depicted.



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Figures

Figure 1 XPS wide-scan (a,b), Si 2p (a’,b’), N 1s (inset of a’,b’) and Cu 2p (a”,b”) spectra of (a,a’,a”) Film A-Cu and (b,b’,b”) Film S1-Cu.

Figure 2 FESEM images of Films (a) A, (b) S1, (c) S3 and (d) S5 showing the deposited SiO2 NPs. Scale bar represents 1 µm. 36 ACS Paragon Plus Environment

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Figure 3 Cu content of (a) Cu-containing SiO2 NP films prepared with different SiO2 deposition time (time in h is indicated by numeral in sample name) and (b) Films A-Cu, S1Cu, Q1-Cu, S3-Cu and Q3-Cu after durability assay. * Significant difference (p < 0.05) between the samples indicated.



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Figure 4 Quantitative count of viable S. aureus from droplet assay on pristine and coated PVF films (a) before and after the durability assay (incubation time = 60 min), and (b) after different incubation time. * Significant difference (p < 0.05) as compared to pristine PVF. # Significant difference between the samples indicated. Droplet assay was carried out with ~ 5 × 103 bacterial cells/sample.


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Figure 5 Quantitative count of viable S. aureus from contact assay on pristine and coated PVF films after 60 min of incubation. * Significant difference (p < 0.05) as compared to pristine PVF.

Figure 6 Viability of 3T3 fibroblast cells incubated with pristine and coated PVF films for 24 h, expressed as a percentage relative to control (cells incubated in medium without any film).



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Figure 7 (a) Photographs of pristine PVF and Films A-Cu, S1-Cu and Q1-Cu placed their labels on a sheet of paper, and (b) transmittance of Films S1-Cu, S3-Cu, Q1-Cu, Q3-Cu and A-Cu (from top to bottom), with pristine PVF as the reference.


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Table Table 1 Film notation and description Film

Steps* involved in preparation

Silica deposition duration (h)

Copper loading duration (h)

PVF

No modification

-

-

A

PVF + steps (1) and (2)

-

-

A-Cu

Cu adsorbed on A

-

6

S1

PVF + Steps (1), (2) and (3)

1

-

S2

PVF + Steps (1), (2) and (3)

2

-

S3

PVF + Steps (1), (2) and (3)

3

-

S4

PVF + Steps (1), (2) and (3)

4

-

S5

PVF + Steps (1), (2) and (3)

5

-

S1-Cu

Cu adsorbed on S1

1

6

S2-Cu

Cu adsorbed on S2

2

6

S3-Cu

Cu adsorbed on S3

3

6

S4-Cu

Cu adsorbed on S4

4

6

S5-Cu

Cu adsorbed on S5

5

6

Q1

PVF + Steps (1), (2), (3) and (4)

1

-

Q3

PVF + Steps (1), (2), (3) and (4)

3

-

Q1-Cu

Cu adsorbed on Q1

1

6

Q3-Cu

Cu adsorbed on Q3

3

6

* Step numbers corresponding to Scheme 1



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TOC GRAPHIC


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