Silver nanoparticles grafted onto PET: Effect of preparation method on antibacterial activity

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Silver nanoparticles grafted onto PET: Effect of preparation method on antibacterial activity H.Y. Nguyenovaa, , B. Vokatab, K. Zarubac, J. Siegela, Z. Kolskad, V. Svorcika, P. Slepickaa, A. Reznickovaa ⁎

a

Department of Solid State Engineering, University of Chemistry and Technology, 166 28 Prague 6, Czech Republic Department of Biochemistry and Microbiology, University of Chemistry and Technology, 166 28 Prague 6, Czech Republic c Department of Analytical Chemistry, University of Chemistry and Technology, 166 28 Prague 6, Czech Republic d Faculty of Science, J.E. Purkyne University, 400 96 Usti nad Labem, Czech Republic b

ARTICLE INFO

ABSTRACT

Keywords: Nanoparticle Grafting Plasma Polymer Antibacterial activity

Incorporating silver nanoparticles (Ag NPs) into surface structure is one of the way to prepare antibacterial surfaces. This study focuses on preparation of antibacterial polymer surface by grafting polyethylene terephthalate (PET) with Ag NPs differing by method of preparation. Ag NP dispersions were synthesized by chemical (Ag NPCH), electrochemical (Ag NPE) and physical (Ag NPP) methods. They were characterized by transmission electron microscopy and UV–Vis spectroscopy. Ag NPs were grafted onto plasma treated PET using dithiol interlayer because of Ag high affinity to thiol groups. Success of grafting was determined by X–ray photoelectron and energy dispersive X-ray spectroscopies. Atomic force and scanning electron microscopes also showed presence of both thiol and Ag NPs on plasma treated PET. Prepared samples were subjected to antibacterial tests against Escherichia coli and Staphylococcus epidermidis. Ag NPE were the smallest and their amount grafted onto PET surface was the highest. Therefore, PET with Ag NPE would be expected to have the best antibacterial effect. However, the highest antibacterial activity (for both strains) turned out to be on PET grafted with Ag NPP because far greater NP amount was situated more in the volume of grafted layer than on PET surface itself.

1. Introduction

polymers is however hindered by their susceptibility to bacterial attachment and consequent biofilm formation, especially if they are used for longer period. This problem can lead to nosocomial infections that are difficult to treat. On top of that, higher bacterial resistance is a result of the frequent use of antibiotics (resistance acquired from evolutionary processes) and biofilm formation, which is onerous to remove. Bacteria within biofilms are surrounded by extracellular matrix, which add to their resistivity [7–10]. Effective prevention of bacterial contamination is to use antibacterial polymers. Some polymers have antibacterial properties on their own but antimicrobial polymers are also fabricated via proper modification - introducing bactericidal agents into their structure or in form of surface coatings [7,11,12]. Bactericidal polymers usually work on the principle of releasing antimicrobial substances from their surface or kill bacteria in contact with the surface. They are prepared by incorporating antibacterial agents into their structure, such as cationic biocides, antibacterial peptides, antibiotics etc. [8,9]. Prospective approach is polymers with metallic particles. Metals,

Polymer materials are irreplaceable part of our life for a long time owing to their physical-chemical properties, diverse chemical composition and easy processing. Despite that, pristine polymers usually do not possess characteristics required for applications and have to be subjected to surface treatment. Diverse methods of surface modification are employed (e.g. UV-irradiation, plasma treatment, grafting) [1,2]. Among them, plasma treatment is one of the most popular. This technique is easy to execute and do not produce toxic waste. Plasma discharge cause cleavage of polymer chains in thin surface layer of material. During the process, radicals and oxygen functional groups are formed, too. On top of that, physico-chemical characteristics like surface roughness and wettability can be easily adjusted by using different type of plasma [1,3,4]. Polymer materials became important materials utilised in medical field, particularly biocompatible polymers, as medical devices used outside, but also inside the body [1,5,6]. The application of these

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Corresponding author. E-mail address: [email protected] (H.Y. Nguyenova).

https://doi.org/10.1016/j.reactfunctpolym.2019.104376 Received 30 July 2019; Received in revised form 30 September 2019; Accepted 2 October 2019 1381-5148/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: H.Y. Nguyenova, et al., Reactive and Functional Polymers, https://doi.org/10.1016/j.reactfunctpolym.2019.104376

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like Ag or Cu, are known and used for their antibacterial activity since ancient times [12,13]. In nanosized form, their antibacterial properties are enhanced thanks to higher surface-volume ratio and small size enabling easier penetration through cell membrane. NPs have effect against broad bacterial spectrum (even resistant strains) in low dosage, which present low to none toxicity to human [7,14–16]. However, mechanism of Ag NPs action against microorganisms is, despite the great interest, still not fully known [15]. Various theories how NPs can affect bacteria are described. Ag NPs are able to accumulate onto bacterial cell membrane and cause disruption in its structure. High level of reactive oxygen species (ROS) was also observed in cells after exposure to Ag NPs. ROS inflict oxidative stress on bacteria and damage DNA thus inhibiting some enzymes essential for bacterial growth. Also, Ag NPs affinity toward –SH groups can affect biological system of bacteria as thiols are apart of enzymes important for cell metabolism. Ag NPs form bonds with them and disturb metabolism pathways. NPs toxicity can be result of combination of above mentioned theories, however it is influenced by NPs size, shape or surface charge as well [12–14,16]. This work object was to study the effect of preparation method, stabilization medium and size of Ag NPs grafted onto plasma treated PET on antibacterial activity. NPs were synthesized by chemical (Ag NPCH), electrochemical (Ag NPE) and physical (Ag NPP) methods. For grafting of Ag NPs onto plasma activated PET, biphenyl-4,4′-dithiol (BPD) was selected due to high affinity of silver to sulphur [17,18]. Among main factors impacting antibacterial activity of Ag NPs are their concentration and size [19]. These parameters were determined using X-ray photoelectron (XPS) and EDS energy dispersive spectroscopy (EDS) and by transmission electron microscopy (TEM). Surface morphology changes and presence of BPD and Ag NPs on PET were investigated by AFM and SEM. TEM was also used for capturing NPs interaction with cell membrane of Escherichia coli (E. coli) and Staphylococcus epidermidis (S. epidermidis). Ag NPs has been chosen in this study because of their high bactericidal activity, which make them attractive in medical applications (wound dressing, surgical meshes, dentistry) or environmental applications (water purifying, air disinfection) [20,21].

(ii) electrochemical Ag NPE Electrochemical Ag NPs were prepared using two silver plates (size 50 × 12 × 2 mm3; purity 99.95%) connected to the DC voltage source (PS 305D) in parallel as electrodes in the 100 ml glass beaker placed on the magnetic stirrer. The electrodes were immersed in 100 ml of trisodium citrate dihydrate solution (4∙10−4 mol/l). The solution was stirred for 30 min at 300 rpm [23]. The solution of colloidal NPs had rich yellow colour. (iii) physical Ag NPP Ag sputtering (Ag target of purity of 99.98%, Safina a.s., CZ) was performed by Sputter Coater SCD 050 (Baltec). Sputtering of Ag into 2 ml of polyethylene glycol (PEG; Mw = 600 g/mol, Sigma-Aldrich Corp., US) was carried out at room temperature (25 °C), the argon pressure of 8 Pa (gas purity 99.997%, Siad a.s., CZ), current 30 mA and electrode distance 4.5 cm. The deposition time was 300 s. Immediately after the sputtering of Ag, PEG with Ag nanoparticles was mixed with distilled water in volume ratio 1:8 (PEG:water) [24]. The colour of nanoparticle dispersions was deep yellow. 2.2. Analytical methods Samples for transmission electron microscopy (TEM) were prepared by the deposition of a drop of Ag NP dispersion on carbon-coated copper grid and excess of dispersion on the grid was dried by Whatman filtration paper. The grid was next put onto drop of distilled water for rinsing twice and then dried again. The samples were studied by transmission electron microscope JEOL JEM-1010 (JEOL Ltd., JPN) operated at the accelerating voltage of 80 kV. Images were taken by SIS Mega View III camera (Soft Imaging Systems, DEU) and analysed by AnalySIS v. 2.0 software (Münster, DEU). Average size of the prepared NPs was determined based on AnalySIS v. 2.0 software calculation of 300 particles. For pictures of bacteria, a concentrated bacteria suspension was deposited onto a carbon grid and left to dry. The suspension was previously exposed for two hours to Ag NPs grafted onto PET surface. The grids were then dyed by solution of silicotungstic acid (4%) and left to dry on a filtration paper. Ultraviolet-visible (UV–Vis) spectroscopy was used to study optical properties of colloidal dispersions of Ag NPs. Absorption spectra were recorded on Lambda 25 spectrophotometer (PerkinElmer Inc., US) in spectral range 300–700 nm with a 1 nm data step and scan speed 240 nm/min. The colloidal dispersions were kept in 1 cm polystyrene cuvette. Reference spectrum of solvent (trisodium citrate dihydrate or PEG) was subtracted from the measured spectrum of Ag NP colloids. Concentration of Ag in colloidal dispersions was determined by atomic absorption spectroscopy using AAS spectrometer Varian AA 880 (Varian Inc., US). Flame atomization and absorption at 242.8 nm was used for determination. Wettability of samples was examined by contact angle measurement and surface free energy calculation on DSA 100 device (KRÜSS GmbH, DEU). A drop of liquid of (2.0 ± 0.2) μl was deposited by automatic pipette onto the surface of sample and evaluated by Advance software. Contact angles of distilled water were measured at 5 to 6 different sample's positions. Surface energy was calculated from contact angles of distilled water and diiodomethane (Sigma Aldrich, US) using OwensWendt-Rabel-Keable (OWRK) model. Atomic concentrations of elements on surface of samples were determined by X-ray photoelectron spectroscopy (XPS). The measurement was carried out on Omicron Nanotechnology ESCAProbe P spectrometer (Omicron Nanotechnology GmbH, DEU) at pressure of 2∙10−8 Pa. A source of radiation was monochromatic X-ray beam of 1486.7 eV with step size 0.05 eV. Samples were investigated under two take off angles – 0 and 81° from the surface normal. Scan size was 2 × 3 mm2 Spectra were processed by CasaXPS software.

2. Experimental part 2.1. Materials and sample preparation Polyethylene terephthalate in the form of 23 μm thick foil (PET, density 1.3 g/cm3, Goodfellow Ltd., UK) was used in this experiment. At first, samples were treated in Ar+ plasma on Balzers SCD 050 device: exposure time 120 s, discharge power 8.3 W, pressure 10 Pa, electrode distance 50 mm. The samples were then inserted into methanol solution of biphenyl-4,4´-dithiol (1∙10−3 mol/l; BPD, Sigma-Aldrich Corp., US) for 24 h and subsequently moved into dispersions of Ag NPs for 24 h. After removing from the solution, samples were rinsed off in water and nitrogen-dried. The sample modification and storage was accomplished at laboratory temperature. 2.1.1. Preparation of silver nanoparticles Three types of Ag NPs were used in this study: (i) chemical – Ag NPCH, (ii) electrochemical – Ag NPE and (iii) physical – Ag NPP; according to the preparation method. (i) chemical Ag NPCH Chemical Ag NPs were synthesized by AgNO3 reduction in a water solution of trisodium citrate dihydrate [22]. A mixture of water (98 ml) and AgNO3 (1 ml; 0.106 mol/l) was heated up to boiling point and subsequently trisodium citrate (1 ml; 3.8∙10−2 mol/l) was added. The mixture was then kept boiling for 1 h. The final colloidal dispersion was of grey colour with yellow tint. 2

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Surface morphology and roughness was examined by atomic force microscopy (AFM, Dimension ICON microscope, Bruker Corp., US). Measurements were carried out in ScanAsyst mode in Air. Silicon tip on nitride SCANASYST-AIR with a spring constant 0.4 N/m was used. Data were evaluated by NanoScope Analyssis software. Average roughness (Ra) represents arithmetic mean of deviations from the centre plane of sample. Scan size was 3 × 3 μm2. Morphology of sample's surface was also studied using scanning electron microscopy (SEM, Tescan Lyra microscope, Tescan, CZ) at the accelerating voltage of 2 kV. Elemental mapping was performed using an energy dispersive X-ray spectroscopy (EDS, analyser X-MaxN, 20 mm2 SDD detector, Oxford Instrumets plc, UK). The accelerating voltage of EDS measurement was 10 kV. Size of scan was 10 × 10 μm2. The samples were sputtered by thin layer of gold (20 nm). Electrokinetic potential was measured by electrokinetic analyser SurPASS (Anton Paar GmbH, AT). Samples were attached to holder of size of 2 × 1 cm2. Solution of KCl (10−3 mol/dm3) flew through the holder as an electrolyte. Each sample was measured 8 times at the constant pH of 6.3 and room temperature. Zeta potential was calculated according to streaming current method, which use HelmholtzSmoluchowski equation. 2.3. Antibacterial tests Antibacterial properties of Ag NPs were tested using “drop test” against two bacterial strains, gram-positive strain S. epidermidis (DBM 2124) and gram-negative strain E. coli (DBM 3138). Inoculum of bacteria was prepared by sterile transferring of one colony of bacteria into liquid medium Lura-Bertani (LB; 4 ml of LB for S. epidermidis and 25 ml for E. coli). Then the mixture was incubated over the night in orbital shaker at 37 °C. Growth of bacteria was confirmed by measurement of optical density (ideal OD = 1). Inocula were then serially diluted using sterile phosphate buffered solution (PBS). Resultant mixtures of suspension of bacteria and PBS were of concentration 5500 bacteria/1 ml (S. epidermidis) and 2750 bacteria/1 ml (E. coli). 100 μl of bacterial suspension with PBS was put onto sample surface. They were then kept under laboratory conditions on table for two hours. Three drops of 25 μl of the suspension from each sample were then deposited on Petri dishes with cultivation media and cultivated overnight. S. epidermidis was cultivated on Plate Count Agar (PCA) at the temperature of 37 °C and E. coli on LB at room temperature (24 °C). The tests were performed on five samples from each preparation step meaning that there were 15 drops for one preparation stage. Simultaneously with tests on samples, control test (CTRL) was carried out by depositing 15 drops of diluted bacterial suspension onto cultivation dish. Images of dishes were captured by UVIdoc HD instrument (UVITEC Ltd., UK).

Fig. 1.. TEM images of chemically (AgNPCH), electrochemically (AgNPE) and physically (AgNPP) synthesized Ag NPs.

images (Fig. 1). Ag NPCH were the largest with the average size of (59.6 ± 7.1) nm. NPs were mainly spherically shaped, however small amount of rods was also formed in the Ag NPCH dispersion, possibly due to lower concentration of trisodium citrate acting as a reducing agent as well as stabilizer. The distribution of NPs size is the widest out of the used Ag colloidal dispersions. The poorer long time stability of dispersion also caused aggregation of Ag NPs. Average diameter of Ag NPP used in this study was approximately (19.7 ± 2.9) nm. Ag NPP also aggregated (see Fig. 1) but did not clump together as much as in the case of Ag NPCH. Size distribution in this dispersion is wide, too. TEM image of Ag NPP shows nearly spherical NPs (black spots) forming large group, but there are tiny spots of several shade of grey spread all over image as well. These are presumably disintegrated PEG used for stabilization of dispersion (they seem to encapsulate aggregating NPs) but the darker ones can very much be very small Ag NPs. Ag NPE were the smallest with average diameter of (9.8 ± 1.5) nm. Ag NPE aggregated only a bit. Unlike other types of NPs, they formed smaller groups that are spread all over the image. NPs size seems to be more uniform in Ag NPE dispersion. Their shape is that of spheres, too. The shape is more homogeneous compared to other two Ag NPs types. Colloidal dispersions of Ag NPs were further characterized using UV–Vis spectroscopy. UV–Vis spectra of each Ag NPs are shown in Fig. 2. Samples were diluted by distilled water to approx same concentration of Ag (i.e. 5 mg/l) to evaluate shifts of surface plasmon resonance (SPR) band in all colloidal dispersion. The SPR peak in the range of 400 to 430 nm confirms the presence of spherical Ag NPs in all studied dispersions [25,26]. According to TEM image of Ag NPCH (Fig. 1), there were nanospheres and nanorods in the dispersion. However, concentration of nanospheres significantly predominate nanorods, therefore there is only single peak in Ag NPCH dispersion absorption spectra. SPR band of Ag NPCH was, in comparison to others, the highest and the widest. This suggests the highest concentration out of

3. Results and discussion Three types of Ag NPs, differing by the preparation method, stabilizing agents and size were used in this study. The goal was to appoint which Ag NPs grafted onto plasma treated PET may have the greatest antibacterial performance. Surface and antibacterial properties were investigated on pristine PET and after each preparation step. 3.1. Ag NPs characterization Ag NP dispersions were examined by several analyses determining their characteristic like concentration or size and shape of NPs. The concentration of metal in dispersion was determined using AAS analysis. The highest concentration of silver in dispersion (105 mg/l) was detected in chemically (Ag NPCH) synthesized NPs. Ag concentration in electrochemically (Ag NPE) and physically (Ag NPP) prepared dispersions was about half the amount in Ag NPCH (55.4 mg/l and 43.4 mg/l, respectively). Size and shape of Ag NPs were determined from TEM 3

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measured on PET grafted with BPD (97.9°). WCA of PET with grafted Ag NPP was lower than those with the other type of Ag NPs, the contact angle value is 46.0°. WCA was influenced by the sample surface but also by the chemistry of substances grafted onto PET. The difference between PET with Ag NPP and the others is caused most likely by their stabilizing agents. Since PEG (used for stabilization of Ag NPP) is well miscible in water, the sample's surface was more hydrophilic. Ag NPCH and Ag NPE were both stabilized by weak water solution of sodium citrate, resulting in their contact angles being comparable (69.3° and 64.6°, resp.). SFE is responsible for interaction between a polymer surface and other participating phases, which determine wettability of polymer. A molecule in volume of every material is exposed to interaction with other molecules of its kind. However, molecules located in the surface layer interact less with molecules in the material volume and experience interaction with outer phases, too [29]. The Young equation describes force balance in a place of surface where three phases are in contact (in the place of liquid drop). The equation serves as mean to calculate surface energy of material, employing contact angle of liquid drop on the surface and surface energy of solid-liquid and liquid-gas interfaces [29,30]. A high SFE enable liquid to spread over a material surface. Whereas, the lower the value of SFE is, the worse are wettability properties of material [3,29]. As we can see in Table 1, surface with the highest energy was PET modified by Ar+ discharge (67.8 mJ/ m2), which was as well the most hydrophilic surface. Contrariwise, PET grafted with BPD had the lowest surface energy (37.0 mJ/m2). These outcomes supported results of WCA measurement. Surface chemical composition of samples was determined by X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray spectroscopy (EDS). Samples of untreated PET and after each step of sample preparation (plasma treatment for 120 s – PET/120; grafting with BPD – PET/120/BPD; anchoring of Ag NPs – PET/120/BPD/AgNP) were investigated under take off angle of 0°. Samples were also examined under take off angle 81° to determine whether the grafted substance (BPD, Ag NP) is more on the sample surface (within atomic layers) or underneath the PET surface (2–3 nm) [31]. Table 2 summarizes an elemental composition of the samples surface. Plasma treatment of polymer inflicts breakage of polymer chains in the thin layer of surface and formation of radicals and oxygen functional groups, which explains change of C concentration (from 73.7 to 67.8 at. %) and increase of atomic concentration of oxygen (from 26.3 to 32.2 at. %) [3]. Presence of S in the spectra confirms successful grafting of BPD onto plasma treated PET [32,33]. C concentration of sample grafted with BPD rose (69.1 at. %), under take off angle 81° was even higher (72.5 at. %), which molecules of BPD are responsible for (C content in its molecule). From Table 2, decrease of S concentration can be observed after grafting of Ag NPCH and Ag NPP. The concentration drop is caused by bonding of NPs to –SH groups [33,34]. PET with Ag NPE was the only

Fig. 2.. UV-Vis absorption spectra chemically (AgNPCH), electrochemically (AgNPE) and physically (Ag NPP) synthesized Ag NPs.

used Ag NPs and a wide size distribution. The peak maximum was also shifted to longer wavelength, confirming the Ag NPCH as the largest out of Ag NPs used in this study [27]. SPR band of Ag NPE is rather wide inclining a wider NP size distribution. The absorption of dispersion is lower than those of other NPs and signifies the lowest concentration of NPs. Ag NPP absorbed nearly as much radiation as Ag NPCH which would suggest similar concentration of NPs in colloid solution. Ag NPP were stabilized by PEG that have formed thin layer around NPs, so they absorbed more radiation. The SPR band of this colloidal solution is thin, which would mean small size distribution. This outcome is in disagreement with TEM image (Fig. 1) in which the wide distribution of NPs size can be observed. The reason might be (i) instability of Ag NPP dispersion and (ii) different refractive index of surrounding media. Among size and type of metal, SPR position is known to be affected by surrounding environment, too [28]. Wavelength of the absorption maxima of Ag NPE and Ag NPP is comparable (408 and 405 nm, respectively) inclining there is not big difference regarding their size. Results from UV–Vis analysis are in good agreement with TEM measurement concerning size of NPs. 3.2. Surface properties Water contact angle (WCA) and surface free energy (SFE) values are summed up in Table 1. Plasma treatment causes not only perturbation of macromolecular chains in the top layer of PET sample, but also polar groups are introduced to the sample surface. Surface polarity of PET thus dramatically increases. The most hydrophilic surface was determined on plasma treated PET, with the contact angle value around 35.1°. On contrary to that, the highest WCA (hydrophobic surface) was

Table 2 Concentration of C, O, S and Ag elements on samples surface determined by XPS on pristine PET, PET treated by plasma for 120 s (PET/120), plasma treated and grafted by BPD (PET/120/BPD) and subsequently grafted with chemically (AgNPCH), electrochemically (AgNPE) and physically (AgNPP) synthesized Ag NPs.

Table 1 Values of water contact angle (WCA) and surface free energy (SFE) determined using goniometric measurement on unmodified PET, plasma treated PET for 120 s, after grafting of BPD and afterward grafting with chemically (AgNPCH), electrochemically (AgNPE) and physically (AgNPP) synthesized Ag NPs. Sample

WCA (°)

PET PET/120 PET/120/BPD PET/120/BPD/AgNPCH PET/120/BPD/AgNPE PET/120/BPD/AgNPP

83.9 35.1 97.9 69.3 64.6 46.0

± ± ± ± ± ±

−2

SFE (mJ·m 0.8 2.4 1.9 1.6 0.9 2.0

45.6 67.8 37.0 48.0 49.5 66.7

± ± ± ± ± ±

Sample

Concentration of elements (at. %) C (1 s)

)

0.3 1.9 1.8 0.8 0.6 1.0

PET PET/120 PET/120/BPD PET/120/BPD/AgNPCH PET/120/BPD/AgNPE PET/120/BPD/AgNPP

4

O (1 s)

S (2p)

Ag (3d)

0°

81 °

0°

81 °

0°

81 °

0°

81 °

73.7 67.8 69.1 64.8 71.3 67.9

– – 72.5 65.8 69.4 80.3

26.3 32.2 28.6 33.3 21.3 31.3

– – 24.4 32.2 21.2 19.3

– – 2.4 1.1 4.4 TA

– – 3.1 0.7 6.2 –

– – – 0.8 3.0 0.8

– – – 1.3 3.3 0.4

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sample that did not record concentration drop of S. On the contrary, amount of S was even twofold for an incomprehensible reason. This phenomenon will be subject to further testing in the future. Merely trace amount (TA) of S was observed on the sample with Ag NPP. Owing to PEG having more viscous character, “thick” PEG layer created on the sample. Thereafter monochromatic beam was unable to reach the surface through the layer well. A penetration depth of beam under the take off angle of 81° is smaller than under 0°, thus resulting in none amount of S detected [31]. Under take off angle 81°, concentration of C on PET with Ag NPP was detected up to 80.3 at. % on account of the PEG layer on the sample surface. The highest concentration was, under both take off angles, found on the PET with Ag NPE, whereas the least amount was observed on the sample with Ag NPP. Large amount of Ag NPE grafted onto PET is probably caused by high S concentration on the surface constituting to larger number of binding sites for Ag NPs. Ag NPCH and Ag NPE concentrations were both determined higher under take off angle 81°. Amount of Ag NPP on the other hand was lower, which incline that Ag NPP was more in the volume of sample (in the layer of PEG) than on the surface. EDS analysis was employed to determine distribution of grafted elements on plasma activated PET surface and their concentration in greater depth of sample surface. EDS maps of sulphur and silver distribution on PET surface are presented in Fig. 3. Concentration of elements (at. %) of C, O, S and Ag on the plasma activated PET (for 120 s) grafted with BPD and then with Ag NPs are summarized in Table 3 together with measurement error (acquired area 10 × 10 μm2). Occurrence of S attests successful grafting of BPD molecules onto plasma treated PET. From Table 3 can be seen, that concentration of S decreased after Ag NPs grafting (from 0.4 to 0.3 at. %). The only exception is PET sample grafted by Ag NPP, where no S was found. These results correspond well with XPS analysis. Like it was mentioned above, no S was detected due to the “thick” PEG layer created on the PET sample, through which the detection beam was unable to reach surface. On the other hand, Ag on this sample was over double the amount of Ag on PET grafted with other Ag NPs (Ag NPP – 2.0 at. % compared with Ag NPCH - 0.8 at. %). The lowest Ag concentration was detected on sample with Ag NPE (0.1 at. %). Results of EDS measurement diverge from XPS results for electron beam used in EDS (depending on accelerating voltage) penetrates deeper into the sample surface than X-ray beam of XPS

Table 3 Elemental concentration (at. %) on grafted PET samples (with BPD and chemically (AgNPCH), electrochemically (AgNPE) and physically (AgNPP) synthesized AgNPs) determined by EDS method. Sample

PET/120/BPD PET/120/BPD/AgNPCH PET/120/BPD/AgNPE PET/120/BPD/AgNPP

Concentration of elements (at. %) C

O

S

Ag

83.9 82.7 83.1 83.7

15.7 16.2 16.5 14.3

0.4 0.3 0.3 –

– 0.8 0.1 2.0

method [35]. The scan area is also different. Scan size measured by XPS is 2 × 3 mm2, while EDS measured surface of 10 × 10 μm2. As can be seen in EDS maps, sulphur is homogenously distributed over the surface and its concentration is nearly the same in all studied samples. There might have been faint diminish after grafting with Ag NPs as data from Table 3 suggest. S was not found on the sample with Ag NPP, therefore a map for this sample is not included in the Fig. 3. EDS maps of Ag showed significant differences in amount of grafted silver between the samples. Ag NPP grafted on PET was visible the most. The Ag quantity was clearly few times the amount of Ag NPCH. The lowest amount of Ag was found on sample grafted with Ag NPE (see the image PET/120/ BPD/AgNPE). Surface morphology and roughness was examined by atomic force microscopy (AFM) (Fig. 4). Plasma treatment causes disruption of macromolecular chains in upper layer of PET, which results in increase of surface roughness Ra from 0.5 to 1.4 nm. Ra further rose after grafting. While Ra of samples grafted with BPD (2.8 nm) and subsequently by Ag NPCH (2.4 nm) and Ag NPE (2.6 nm) are similar, roughness of polymer with Ag NPP was nearly twofold (4.6 nm). The reason lies in PEG layer created on PET surface. Since PEG molecules are larger than BPD or Ag NPs, the roughness of surface was higher. Successful grafting by BPD confirms presence of BPD displayed in AFM image (PET/120/BPD). As can be seen in the image, BPD is spread all over the surface of PET sample. BPD seems to start crystallise, therefore there are few large light clumps detected on the surface as well. BPD then decreased after grafting with Ag NPs (see Fig. 4 - PET/120/BPD/AgNP). The BPD amount dropped dramatically with grafting of polymer with

Fig. 3.. Elemental distribution maps of sulphur and silver on plasma treated PET grafted with BPD (PET/120/BPD) and subsequently with chemically (AgNPCH), electrochemically (AgNPE) and physically (AgNPP) synthesized Ag NPs determined by EDS analysis. 5

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Fig. 4.. AFM image of pristine PET, plasma treated PET for 120 s, plasma treated PET grafted with BPD and subsequently with chemically (AgNPCH), electrochemically (AgNPE) and physically (AgNPP) synthesized Ag NPs. Ra represents surface roughness of samples in nm.

Ag NPCH. After grafting of Ag NPE onto PET surface, S quantum appears to be greater. On the other hand, AFM image (PET/120/BPD/AgNPP) shows that BPD was not even found because of the “thick” PEG film. These results correspond well with elemental concentrations of S determined by XPS. Ag NPCH and Ag NPE on PET sample surface for the most part presumably aggregated and created clusters. Ag NPs are thus seen as light sizeable spots in the AFM images. Number of Ag NPs is similar on these two samples. Nevertheless, it is not certain if those are Ag NPs or crystallized BPD. Ag NPP did not aggregate as much as the other two Ag NP types; while there are few clumps, a good deal of small dots can be observed in (PET/120/BPD/AgNPP) image. Compared to the other two grafted samples, there were detected far more NPs on Ag NPP grafted PET. Changes in morphology of larger surface area have been also supported by SEM measurement (see Fig. 5). Only samples grafted with BPD and Ag NPs were investigated by this method to evaluate distribution of grafted substances on samples. The analysis confirmed presence of grafted BPD on plasma treated PET, too. BPD can be seen as elongated forms of lighter grey colour in the Fig. 5 (PET/120/BPD). Like in AFM, few light clumps are seen on this sample surface, which are presumably crystallized form of BPD. Slight decrease in the BPD amount can be observed on samples with Ag NPs, with the exception of the sample with Ag NPP. BPD was, as in the previous analyses, not found on the surface of this sample. However, macromolecular chains of PEG, alongside the NPs, can be clearly seen in the image PET/120/ BPD/AgNPP. As mentioned above, PEG creates “thick” layer on the PET surface. Ag NPs seem to aggregate on this sample thus large clusters are displayed in the image. From electron image of PET/120/BPD/AgNPCH sample, quite number of NPs can be distinguished as well as their shape (i.e. spheres and few rods, see Fig. 5). Moreover, Ag NPCH can be seen homogeneously distributed over the sample surface. Amount of grafted Ag NPCH onto PET seems to be nearly the same as on the Ag NPP sample. SEM image of sample with Ag NPE shows, compared to other two Ag NPs types, the lowest number of grafted NPs. Similarly, Ag NPE aggregated on sample surface, too. Ag NPs clusters of similar size as in Ag NPCH case can be seen in image PET/120/BPD/AgNPE sample. NPs are evenly distributed all over the surface of samples. Zeta potential values of tested samples are documented in Fig. 6. Only slight changes in surface chemistry and charge on PET surface after plasma treatment can be clearly seen. This corresponds well with

Fig. 5.. SEM images of plasma treated PET grafted with BPD (PET/120/BPD) and subsequently with chemically (AgNPCH), electrochemically (AgNPE) and physically (AgNPP) synthesized Ag NPs.

the slight increase of oxygen groups (see Table 2, XPS results). More pronounced changes were observed after grafting surface with BPD and afterwards with Ag NPs. BPD grafting lead to the zeta potential of more negative value due to negative surface charge caused by –SH groups on the surface. Grafting with Ag NPCH significantly changes surface chemistry and charge due to increasing presence of oxygen groups again to the less negative value. Ag NPE grafting resulted in dramatic zeta potential decrease which can be explain by three effects: (i) the smallest presence of oxygen, (ii) the most S amount, which causes strongly negative charge on surface [32] and also (iii) the most amount of Ag which results in accumulation of electrons in the case of metalsolution interface leading again in negative surface charge [33,36]. Due to presence of PEG molecules, no amount of S and minimum of Ag NPP grafted onto surface, zeta potential increases to less negative values. All 6

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Fig. 6.. Zeta potential determined on pristine PET, plasma activated PET for 120 s, plasma treated and grafted with BPD and subsequently with chemically (AgNPCH), electrochemically (AgNPE) and physically (AgNPP) synthesized Ag NPs.

of these results correlated very well with XPS results. 3.3. Bactericidal effect Antibacterial activity was tested against E. coli and S. epidermidis. Tests were performed on pristine PET and after each preparation step. Fig. 7 summarizes average count of surviving bacterial colony producing units (CPU) of both bacteria on each sample. Surviving units of E. coli from CTRL test add up to 53 CPU. The number of CPU on untreated PET was slighter than in CTRL, nevertheless it was still within error range. CPU further dropped on plasma treated PET sample. The cause is most likely the presence of radicals and oxygen species. Number of surviving E. coli bacteria deposited on BPD grafted PET increased in comparison to plasma treated sample. It was nearly the same as the count of CPU in CTRL. E. coli also seems to be not much affected by Ag NPCH. Number of surviving bacteria was just a bit lower than on PET treated by plasma (plasma treated PET – 43 CPU; PET grafted by Ag NPCH – 41 CPU). The other two Ag NP types had better antibacterial effect. Mortality of bacteria subjected to contact with Ag NPE was 50%. In the case of Ag NPP, antibacterial effect was almost 100%. Only 2 CPU survived in just one drop out of 15 drops. The rest 14 drops were free of any living bacteria. Surviving S. epidermidis CPU on the first three samples (PET, PET/120 and PET/120/BPD) were about same as in the CTRL. The number of CPU is, in the case of these three samples and S.

Fig. 8.. Images of cultivation dishes with grown bacterial colonies E. coli and S. epidermidis. Images are those of control test (CTRL) and bacteria exposed to chemically (AgNPCH), electrochemically (AgNPE) and physically (AgNPP) synthesized Ag NP grafted onto PET.

epidermidis CTRL, about 34 units. Ag NPCH had far greater effect this time than on E. coli. Average CPU count in this case is around 3. In the Fig. 7, enlarged columns of surviving CPU after exposition to Ag NPE and Ag NPP seems to be same. The only difference is lack of error line in the case of Ag NPP because there were no surviving S. epidermidis. Ag NPE was nearly 100% effective. There was only one surviving colony producing unit. Fig. 8 documents images of Petri dishes with overnight cultivation of E. coli, which were exposed to samples with grafted Ag NPs. No significant changes from CTRL can be observed on the Ag NPCH since there was only slight to none fluctuation in amount of surviving CPU. Visible CPU decrease is on the sample with Ag NPE. According to count, around 50% bacteria survived. Petri dish with E. coli which were in contact with Ag NPP grafted PET (2 CPU) seems to be empty. S. epidermidis was much more sensitive to Ag NPCH sample. Few drops of bacterial suspension were completely free of any surviving bacteria, which also show in the image of Petri dish (Fig. 8– PET/120/BPD/ AgNPCH) where there are some empty spots. Ag NPE sample was nearly 100% effective. Only one CPU survived. It was however hardly visible on the Petri dish, therefore the image (PET/120/BPD/AgNPE) appear to be completely empty. Petri dish of Ag NPP sample is clear of any CPU

Fig. 7.. Count of CPU on PET samples from drop antibacterial test against E. coli and S. epidermidis. Samples were tested after each stage of preparation: pristine PET, plasma treated PET (120 s), after grafting with BPD (PET/120/BPD) and then grafted with chemically (AgNPCH), electrochemically (AgNPE) and physically (AgNPP) synthesized Ag NPs. 7

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Fig. 9.. TEM images of E. coli and S. epidermidis exposed to PET samples grafted with BPD and subsequently with chemically (AgNPCH), electrochemically (AgNPE) and physically (AgNPP) synthesized Ag NPs.

since no bacteria survived. Fig. 9 documents TEM images of E. coli and S. epidermidis exposed to Ag NPs grafted PET samples. While bacteria can be seen quite clearly, blurry substances around them are lysing bacteria or their part. Fig. 9 confirmed that Ag NPCH seems to not affect E. coli much; the other two are more effective. Antibacterial performance of Ag NPCH against S. epidermidis improved greatly as can be seen in the image PET/120/ BPD/Ag NPCH. TEM image of S. epidermidis treated with Ag NPP shows besides lysing bacteria freed Ag NPs. The reason why Ag NPE seems to be more effective than Ag NPCH may lie in their size since Ag NPE were determined as the smallest out of the three. It would also incline to the best bactericidal effect [14]. However, the results of antibacterial test determined Ag NPP as the most effective. There are many factors that could have led to this outcome. First of all, the surface of PET sample with Ag NPP was more hydrophilic than the rest, enabling bacterial suspension to spread over the sample surface well. The suspension was then in contact with wider area than in the other samples case and therefore with greater number of Ag NPs. Another reason might be “thick” layer of PEG formed on the surface of sample. From XPS and EDS analyses commented above, Ag NPP appear to be in the volume of PEG layer rather than on the surface. By adding solution onto it, the topmost layer of PEG chains might have started to shift their conformation or simply loosen up. Therefore, great deal of NPs gets to resurface from the volume. Bacteria were also reported to be capable of degradation of PEG with lower molecular weight [37]. That could have helped uncover Ag NPs in the volume. On top of that, some Ag NPP seems to liberate from surface to the solution (see Fig. 9). Freed Ag NPs might have adhered onto bacteria surface leading to easier disruption of their cell wall.

which confirmed similar chemical composition of both samples surfaces (stabilization by sodium citrate). XPS and EDS confirmed successful grafting with both BPD and NPs. Sulphur was not detected on the surface with Ag NPP, though, because of PEG film formation on the sample. The highest concentration of Ag NPs grafted onto plasma treated PET was determined on Ag NPE sample. On contrary to that, the lowest NPs concentration was found on the Ag NPP sample. Nevertheless, examination under take off angle of 81°, disclosed that Ag NPP are located more in the volume of sample than on the surface, most likely in the PEG layer. EDS detected the highest concentration of grafted Ag NPs to be on Ag NPP sample, which confirms NPs to be more in volume of the sample surface. SEM and AFM affirmed predominating amount of Ag NPP grafted onto PET, as well as creation of PEG layer on the surface of Ag NPP sample. Electrokinetic analysis confirmed both BPD and Ag NPs grafting and correlates well with XPS determination. Although Ag NPE were determined to be smallest and their concentration on the topmost layer of PET was highest, the most effective against bacteria was Ag NPP sample. Bacterial suspension deposited onto this sample came to contact with larger area. The great amount of Ag NPP was enclosed in PEG layer immobilised on the PET surface, too. Upon contact with suspension, PEG macromolecular chains gained greater mobility and so the Ag NPs could resurface from the layer volume. Data availability The raw/processed date required to reproduce these findings cannot be shared at this time due to technical or time limitations. Declaration of Competing Interest The authors declare no competing financial interest.

4. Conclusion

Acknowledgements

The main object of this study was to compare antibacterial effect of PET samples grafted with Ag NPs synthesized by different methods. PET surface was treated by plasma discharge for 120 s, subsequently grafted with biphenyl-4,4′-dithiol and afterward with Ag NPs. With the help of TEM and UV–Vis spectroscopy, Ag NPs shape was revealed to be mainly that of spheres. Average size of Ag NPs was determined from TEM images: Ag NPE (9.8 ± 1.5) nm, Ag NPP (19.7 ± 2.9) nm and Ag NPCH (59.6 ± 7.1) nm. Because of PEG good solubility in water, the sample surface grafted with Ag NPP was more hydrophilic. On contrary to that, Ag NPCH and Ag NPE WCA were comparable (69.3° and 64.6°, resp.),

The authors gratefully acknowledge the Czech Science Foundation (GA CR) (Grant No. 17-00939S) and specific university research (MSMT No 21-SVV/2019) for the financial support of this research work. References [1] O. Nedela, P. Slepicka, V. Svorcik, Surface modification of polymer substrates for biomedical applications, Materials 10 (2017) 1115, https://doi.org/10.3390/ ma10101115.

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