barrier layers based on nanostructured fluorocarbon … · 2. materials and methods as a model...

Barrier layers based on nanostructured fluorocarbon films: structure, interaction with microorganisms, mechanical properties

V. Elinson1, S. Andreevskaya2, A. Lyamin1, N. Shevlyagina2, V. Zhukhovitsky2, 3, P.A. Shur1 1Moscow Aviation Institute (National Research University), 125993, Volokolamskoe Shosse, 4, Moscow, Russia; 2Gamaleya Research Center for Epidemiology and Microbiology, Ministry of Public Health, 123098, Gamaleya str, 18,

Moscow, Russia; 3Sechenov The First Moscow Medical University, Ministry of Public Health, 119991, Bol’shaya Pirogovskaya str., 8,

Moscow, Russia

Nowadays the problem of the polymeric materials destruction under the influence of microorganisms (biodestruction) is one of the most important problems restricting the use of polymers and products from them. One of the approaches to solve this problem is to create barrier layers on the polymers’ surface that impede the adhesion of microbial cells, what prevents a biofilm formation and the subsequent biodegradation of polymeric materials.

It is well known that polymeric materials are used in various fields of science and technology: in micro- and nanoelectronics (in particular in the polytronics), in medicine, in the food industry, etc., therefore barrier layers for polymeric materials and products, in addition to resistance to biodedestraction, should have a number of properties that would allow them to be used for appropriate purposes. Mechanical characteristics, such as nanohardness and Young's modulus, belong to the most common properties of materials.

The use of fluorocarbon films obtained by ion-plasma technology methods, especially using a two-component plasma-forming mixture C6H12 + CF4, which contains a component for the film deposition and component for film etching, allows the formation of barrier layers that prevent the biofilms formation and subsequent biodestruction of materials.

The present chapter is devoted to the study, based on scanning electron microscopy and X-ray microanalysis (SEM-EDAX), of the structure and relief of the obtained layers, as well as the processes of biofouling by Staphylococcus aureus cells of the barrier layers surface formed with a different content of CF4 in the plasma-forming mixture.

The obtained results demonstrated the presence of transient processes area from the films deposition to their etching. Barrier layers formed in this area have a specific relief that prevents the adhesion of microbial cells.

In addition, it was shown that for a small number of microbial cells adhered on the surface of layers formed outside the transient area, there are no signs of cell division and biofilm formation. The study of the nanohardness and Young's modulus demonstrated interesting features of the mechanical characteristics of these barrier layers.

Keywords barrier layers; nanostructured fluorocarbon films; biofilms; biodestruction; adhesion of bacteria; transient processes; specific relief; mechanical characteristics

1. Introduction

Nowadays the problem of the polymeric materials destruction under the influence of microorganisms (biodestruction) is one of the most important problems constraining the use of polymers and products from them [1-3]. One of the approaches to solve this problem is to create barrier layers on the polymers’ surface that impede the adhesion of microbial cells, what prevents a biofilm formation and the subsequent biodegradation of polymeric materials [1,4,5]. It is well known that polymeric materials are used in various fields of science and technology: in micro- and nanoelectronics (in particular in the polytronics), in medicine, in the food industry, etc., therefore barrier layers for polymeric materials and products, in addition to resistance to biodedestraction, should have a number of properties that would allow them to be used for appropriate purposes. Mechanical characteristics, such as nanohardness and Young's modulus, belong to the most common properties of materials. It was shown in papers [6, 7] that using a plasma-forming mixture CF4 + C6H12 in ion-stimulated film deposition from the gas phase, an area of transient processes was observed (transition from film deposition to etching). Nanostructured fluorocarbon films formed in conditions of transient areas have antiadhesive properties with respect to S. aureus. However, in order to understand the mechanism of this phenomenon and for the further development of proposed approach for the formation of nanostructured fluorocarbon films, it is necessary to expand the technological range and investigate the antiadhesive properties of the obtained nanostructures, paying special attention to the process parameters that affect the nature of the relief change. Thus, the aim of this work was to study the features of the interaction between Staphylococcus aureus and nanostructured barrier layers formed by methods of ion-plasma technology under various conditions, based on fluorocarbon films, and the general mechanical characteristics of the obtained materials.

Antimicrobial research: Novel bioknowledge and educational programs (A. Méndez-Vilas, Ed.)

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2. Materials and methods

As a model material polytetrafluoroethylene (PTFE) was chosen as a polymeric material widely used in polytronics, in medicine, in the food industry, etc. S. aureus, a widespread microorganism known for its ability to adhere to various surfaces, form biofilms on them, and its’ destructive potential, was used for the study [1-4]. Samples were formed with the help of an ion source operating in crossed electric and magnetic fields. The plasma-forming mixture CF4 + C6H12 containing a film deposition component (C6H12) and a component for film etching (CF4) was used. The samples were chosen on the basis of the data of [6,7], in which it was shown that at the content of CF4 in the plasma-forming mixture from 40 to 60 vol.% the area of transient processes was observed (from film deposition to etching with the creation of specific relief). Samples formed in the area of transient processes have antiadhesive properties with respect to S. aureus. In this work, the conditions for the formation of samples were extended. The list of samples is presented in Table 1.

Table 1. List of samples for the investigation of the adhesive properties of PTFE

№ Sample formation conditions

0 Glass (control 1) 1 PTFE (control 2) 2 PTFE, CF4 treatment, 30 min

3 PTFE, CF4 treatment, 30 min Deposition (С6Н12 + CF4) (90% + 10%), 20 min



6 PTFE, CF4 treatment, 20 min




2.1. Scanning electron microscopy and X-ray microanalysis of samples’ surfaces

Intact samples were attached to aluminum tables using carbon scotch and a 5 nm thick gold layer was deposited onto their surface using the SPI-MODULE Sputter Coater (SPI Supplies, USA). The surface structure was studied with the help of dual ion-electron scanning microscope Quanta 200 3D (FEI Company, USA). For elemental analysis the X-ray microanalyzer Genesis XM2 (EDAX, USA) was used. The set of spectra was carried out under conditions of high vacuum with an accelerating voltage of 10 kV for 50 seconds, with a counting rate per second of 1500-2000 and dead time of 20-40%. Using the mapping function for PMA, a visual assessment of the distribution of elements in the samples was carried out.

2.2. The interaction of S. aureus with the obtained materials.

The samples of PTFE with different treatment were placed in well plates containing the daily culture of S. aureus 29213 ATCC in liquid nutrient medium NB, in an amount of 9.5x108 CFU/ml. The microorganisms were incubated in the presence of the test materials without the addition of a nutrient medium for 5 days at room temperature. In the same conditions, control samples of glass and PTFE samples without treatment were incubated. After incubation, the samples were washed with sterile water for three times and fixed in a 10% neutral aqueous formalin solution for 24 hours. Preparations for the scanning electron microscope investigation consisted in drying samples under natural conditions with further deposition of a 5 nm thick gold layer.

2.3. The investigation of the mechanical properties of the obtained materials

Nanohardness and Young's modulus was measured by durometer Nanovea («Micro Photonics Inc.», USA)by the Oliver-Pharr method. The Berkovich indenter was used.


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Indentation load to the indenter is 2.5 mN, the holding time at the maximum load of the indenter is 20 seconds, loading and unloading speed at the load 5 mN per minute. Each sample was tested at least 12 times. During measurement a loading and unloading diagrams were constructed as a function of the force applied to the indenter from indentation depth. Nanohardness defined as the ratio of maximum load to the area of the indentation H=Pmax/A. All calculations with some refinement coefficients were made automatically with the help of the instrument software. Measurement of mechanical characteristics was carried out on fluorocarbon films formed on the polystyrene (PS) surface.

3. Results and discussion

3.1 Structure and elemental analysis of materials.

The control sample of PTFE according to the results of SEM had the smoothest surface. With the help of X-ray microanalysis of surfaces it was shown that the average weight content (WT%) of fluorine in its composition was the largest among all presented samples and equal to 62.92%. (Fig. 1 and 3)

Fig. 1 Surface of PTFE control sample without pretreatment

Preliminary CF4 ions treatment of the material surface for 30 min resulted in the formation of a specific relief on the surface with parallel crimped crests to each other. The further deposition of the film from the gas mixture of CF4 + C6H12 with the content ofCF4from 10% to 60% was manifested on the surface in the relief enhancement and change in the shape of the crests (Fig. 2 a, b, c, d). The relative content of fluorine varied downward to 57% and 46% with pretreatment for 30 min and applying the film with a content of 10% CF4 in the mixture, respectively (Fig. 2 and 3). In a sample with a deposited film with a CF4 content of 40%, the ratio of fluorine and carbon in the sample was approaching of 49.90 and 50.10, respectively. (Figure 3). The film deposition using the plasma-forming mixture containing 60% CF4 and 40% C6H12 again resulted in an increase in the fluorine content in the sample. (Fig. 2 and 3)

(а) (b) (c) (d)

(e) (f) (g) (h)

Fig.2 Change in surface relief after 30 min pretreatment. (a) and with deposited film with a CF4 content of 10% (b), 40% (c), and 60% (d) in the plasma-forming mixture. Change in the surface relief after 20 min pretreatment. (e) and with deposited film with a CF4 content of 10% (f), 40% (g), and 60% (h) in the plasma-forming mixture.

A similar pattern of changes in the surface relief was achieved after pretreatment of PTFE with CF4 ions for 20 min, although the height of the crests formed after film deposition from the plasma-forming mixture is not so visible (Fig. 2 e, f, g, h). The relative content of fluorine in the sample composition decreased compared to the control sample after CF4 ions treatment 20 minutes, and when the film was applied from a mixture containing 10% CF4 and 90% C6H12, the ratio was equal to the carbon in the sample obtained from the mixture, Containing 40% CF4 and 60% C6H12, and again increased in the sample obtained from a mixture containing 60% CF4 and 40% C6H12 (Fig. 3).

1 mkm 1 mkm 1 mkm 1 mkm



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It must be noted that irrespective of the options of preliminary CF4 ion treatment of the PTFE for 30 or 20 minutes, the ratio of the weight content of fluorine and carbon to the sample approaching to the sample was equal to that of the plasma-forming mixture containing 40% CF4 and 60% C6H12 (Fig.3).

Fig. 3. Ratio of the fluorine and carbon content in the composition of the obtained materials according to X-ray microanalysis.

3.2. The interaction of S. aureus with the investigated surfaces.

On the surface of the glass, which was chosen as a control material, S. aureus formed massive clusters, sometimes multilayered, partially covered with exomatrix. This indicates the ability of this type of microorganism to adhere to abiotic surfaces, active reproduction and the formation of biofilm on them. On the PTFE surface without preliminary treatment such massive accumulations of bacterial cells was not found, they were located at a distance from each other. The chains of fissile bacteria were identified. (Figure 4)

(a) (b)

Fig.4. Adhesion of microorganisms on the control surfaces of materials: a) cover glass (sample № 0); b) PTFE (sample № 1) The pattern observed in the interaction of S. aureus with the experimental samples was significantly different. After preliminary CF4 ions treatment of materials for 30 min, single bacterial cells without signs of division were detected on the surfaces of samples № 2 and № 3 (only pre-treatment and film deposition from a mixture containing 10% CF4, respectively). (Figure 5 a, b). Surfaces of samples No. 4 and No. 5 remained free of bacterial cells, what indicates the presence of antiadhesive properties with respect to S. aureus to modified surfaces. (Figure 5c, d)

(a) (b) (c) (d)

Fig.5. Adhesion of microorganisms to the surface of PTFE samples after 30 min preliminary treatment: a) sample № 2; b) sample № 3; c) sample № 4; d) sample № 5

37,07 42,7153,74 50,10 44,44 45,20

55,11 50,60 45,52

62,92 57,2946,26 49,90 55,56 54,78

44,87 49,40 54,48

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After 20 minutes preliminary treatment of the PTFE surface on all fluorocarbon films’ surfaces the adhesion of bacteria was detected of a different degree of severity. After only surface treatment (sample № 6) (Fig. 6a), from 8 to 15 bacterial cells were detected in each field of view, also dividing cells and small clusters of them. On the surface of sample № 7 single cells of microorganisms with no signs of division were detected in each field of view. (Fig. 6b). On samples № 8 and № 9 single bacteria were found in rare fields of view. (Fig.6 c and d).

(а) (b) (c) (d)

Fig.6. Adhesion of microorganisms on the surface of PTFE samples after 20 minutes preliminary treatment of: a) sample № 6; B) sample № 7; C) sample № 8; D) sample № 9

3.3. Mechanical properties.

The average values of nanosurface and Young's modulus of the samples are presented in Table 2 and in Fig.7. The data shows that samples with treatment with CF4 ions for 10 minutes have the smallest values of nanohardness and Young's modulus of elasticity. All values of the Young's modulus after the deposition of the fluorocarbon film significantly exceed the values for the untreated polystyrene surface (by 3 times) and for the surface after treatment with CF4 ions (by 200 times). The values of nanohardness after CF4 ions treatment for 10 minutes also sharply decrease, but after further deposition of the fluorocarbon film, they practically do not differ from the values of the untreated polystyrene, especially after deposition of fluorocarbon film from the gas mixture containing 20%, 30% and 40% CF4. Samples with treatment with CF4 ions for 10 minutes have the smallest value of hardness and Young's modulus of elasticity. After the deposition of the fluorocarbon film, the nanohardness of the samples increase significantly from 0.01 GPa (sample 0) and reach a maximum of 0.61 GPa after deposition of fluorocarbon film with 30% CF4 content (sample 3). The values of Young's modulus of elasticity also vary from 0.02 GPa (sample 0) to 4.09 GPa (sample 3).

Table 2. The values of the nanohardness and the Young's modulus of elasticity of deposited coatings on polystyrene.

Sample parameters Nanohardness Н, GPa

Young's modulus Е, GPa

Untreated PS 0,68±0,06 1,34±0,06 CF4-10 min 0,01±0,001 0,02±0,001 CF4-20 min 0,322±0.09 4,53±0,16 CF4-30 min 0,287±0,04 3,43±0,13

CF4-10 min+ CF4(10%)/С6Н12(90%) 0,27±0,03 1,58±0,08 CF4-10 min+ CF4(20%)/С6Н12(80%) 0,58±0,05 3,98±0,12 CF4-10 min+ CF4(30%)/С6Н12(70%) 0,61±0,04 4,09±0,17 CF4-10 min+ CF4(40%)/С6Н12(60%) 0,58±0,03 3,85±0,12 CF4-10 min+ CF4(50%)/С6Н12(50%) 0,56±0,05 3,35±0,15

It was found that pretreatment with CF4 ions plays a significant role in the formation of barrier layers. The PS was treated for 10, 20 and 30 minutes. The highest values of mechanical characteristics are achieved after 20 minutes of preliminary treatment, which is possibly related to the maximum values of root- mean-square roughness deviation Rq for this relief (Nanohardness-0.32 GPa, Young's modulus - 4.53 GPa) (Fig.7)



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a) b)

Fig. 7. Histograms of the dependence of the mechanical characteristics of fluorocarbon coatings from the CF4 content in the plasma-forming mixture CF4 + C6H12 and from the preliminary treatment time: a) nanohardness; B) Young's modulus of elasticity.

Conclusion

The data obtained as a result of the research allows to draw the following conclusions: 1. The structure and microelement composition of the studied materials based on PTFE differ depending on the type of surface modification. Both the relief and the ratio of the fluorine and carbon content in the surface composition depend on the time of preliminary treatment of the initial material. After 30-minute preliminary treatment, the relative fluorine content of the materials is generally higher than that of the 20-minute preliminary treatment. 2. The antiadhesion properties of the materials also depend on the deposition conditions of the fluorocarbon coating. All materials have antiadhesive properties to some extent, in comparison with the control glass. Nevertheless, the smallest adhesion of microorganisms (S. aureus 29213 ATCC) is observed after 30-minute preliminary treatment of the initial material and further deposition of the fluorocarbon film with 40% and 60% CF4 content in the gas mixture. Thus, it can be assumed that most important factors determining the antiadhesive properties of the obtained samples are the specific relief of the surface and the fluorine content in the surface layer. 3. It was found that preliminary treatment with CF4 ions plays a significant role in the formation of barrier layers. All values of the Young's modulus after preliminary treatment with CF4 ions and deposition of the fluorocarbon coating significantly exceed the values for the untreated polystyrene surface. The values of nanohardness after 10 minutes treatment with CF4 decrease dramatically, but after further deposition of the fluorocarbon film they practically do not differ from the values of the untreated polystyrene. The highest values of mechanical characteristics are achieved with 20 minutes of preliminary treatment, what is possibly related to the maximum values of root-mean-square roughness deviation Rq for a given relief.

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00,10,20,30,40,50,60,70,8

Nanohardness, GPa

00,5

11,5

22,5

33,5

44,5

5

Young's modulus, GPa


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[7] Elinson V.M., Didenko L.V., Shevlyagina N.V., Avtandilov G.A., Lyamin A.N., O.A.Silnitskaya. The chapter “Nanostructured fluorine-containing surfaces: physicochemical properties and resistance to biodestruction” in the book “Polymer science: research advances, practical applications and educational asppects”, Formatex Research Center, Madrid, Spain, 2016, pp. 342-347 (A.Mendez-Vilas, A.Solano Eds). ISBN-13:978-84-942134-8-9


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barrier layers based on nanostructured fluorocarbon … · 2. materials and methods as a model...

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