Paper material containing Ag cations immobilised in faujasite: synthesis, characterisation and antibacterial effects

Ageing was carried out in a climatic chamber (MEMMERT HCP246) at 90 °C and with a relative humidity of 59%, for 12 days. All samples were aged under the same conditions.

The pH measurements of the paper samples were carried out according to the TAPPI T 529 om-88 norm using a flat plate electrode (Flatrode Hamilton, 809 Titrando from Metrohm). Samples were cut out from the prepared paper sheets, and a drop of water was placed on their surfaces. The electrode was then applied to each sample, and the pH was measured until a stable value was reached.

The zero-span breaking strength was measured according to the TAPPI T236 norm (TAPPI 1996). Before measurements, samples were conditioned according to the D685 ASTM standard, at 23 °C and 50% RH.

Surface topology was analysed by optical microscopy (Leica DM2000) equipped with a digital camera. Imaging was carried out using reflected and transmitted light modes, respectively. To obtain representative images, imaging was carried out at five different places of the material (test of material homogeneity).

The surface topography of the prepared materials was examined by using scanning electron microscopy (SEM) equipped with a probe for chemical analysis of specimens in microareas with energy dispersive X-ray spectroscopy. A scanning electron microscope (Nova Nano SEM 300 FEI Company) was used for high-quality magnification of the paper morphology.

Measurements were carried out using the ColourQuestXE (HunterLab) colorimeter. Measurement conditions were as follows: measuring surface ∅ = 9.5 mm, observer 2°, illuminant D65, specular reflection excluded.

A micro fading tester was used to assess the lightfastness of the prepared paper samples. The micro fading tester, a relatively novel device which can be used to obtain quick and non-destructive measurements of material colour change caused by incident light, was described in (Lerwill et al. 2008; Łojewski et al. 2011). The instrument was equipped with a high power xenon light source and VIS spectrometer, which allow induced colour changes to be monitored. Three lightfastness measurements were taken for each type of paper.

The weight-averaged degree of polymerisation (DP_w) of cellulose (cellulose triphenylcarbamate, CTC) was determined by size exclusion chromatography (SEC). The details concerning the analytical procedures used can be found elsewhere (Pawcenis et al. 2015). In brief, analysis of molecular mass distributions was carried out in tetrahydrofurane (THF) solvent used as a mobile phase, with double detection: (1) by multiple angle laser light scattering (MALLS, Waters), and (2) refractive index (RI). Both detectors worked at a light wavelength of 658 nm. In the measurements, a value of the specific increment of the refractive index equal to 0.162 cm³/g was used.

The paper samples containing Ag⁰, Ag⁺ and zeolite were first defibred in a beaker in 50 cm³ of water (the process was aided by ultrasound waves). The standard procedure of preparing cellulose material was applied (Lauriol et al. 1987; Stol et al. 2002), using cellulose derivative CTC in THF solution. The AgFAU was removed from the CTC solutions by filtration through a Teflon syringe filter with pore diameter of 0.45 μm. The other additives were removed before derivatisation by washing.

Escherichia coli from the culture collection of the laboratory of the Plant-Microbial Interaction Group of the Jagiellonian University (Kraków, PL) were used in the laboratory tests. A detailed description of the sample preparation and analytical procedure can be found in our previous work (Łojewska et al. 2015). The efficiency of the biocidal process on the prepared paper samples was checked using the test LuciPac Pen equipped with a lumitester (Kikkoman lumitester PD-20) (ATP + AMP, Hygiene Monitoring test kit from Kikkoman Corp., code 60331), in accordance with the manufacturer’s instructions. The test gives relative content of ATP, ADP and AMP (RLU—relative luminescence units).

The progress of ion exchange was monitored by XRF analysis, the results of which are shown in Fig. 1. It can be noted that the time necessary to achieve saturation in ion exchange was around 2 h. For the assumed half exchange, a time of 1 h was chosen. The degree of ion exchange was then measured by the XRF method, by digesting the samples in concentrated HNO₃.

In order to dispose of the external unattached silver moieties, complexation using EDTA sodium salt was employed. The progress of elution with the EDTA solution of a given concentration was monitored by XRF analysis of eluate solutions. To check the efficiency of EDTA washing, the AgFAU samples were also treated with the same quantities of water. After repeated treatment with the same portions of EDTA, the amount of silver in the eluent solution stabilises and reaches a low constant value, which is 0.15% of the initial value. This was assigned as the completion of the removal process. The state and amount of silver on the zeolite surface at different stages of elution were also analysed with XPS, which indicated the presence of silver NPs on the initial sample. Semi-quantitative results are shown in Table 2.

Corresponding with analysis of eluents, the amount of silver at the surface stabilises after treatment with the EDTA solution of higher concentration (> 0.01 M), and is around four times lower than for the initial sample. Neither the 0.01 M EDTA nor the water were able to remove external silver oxide particles from the zeolite samples. The remaining silver will be called stable silver attached to Al–O- groups in zeolite, impossible to remove by treatment with a chelating agent.

The content of silver present in the paper samples is shown in Table 1. For the final PZAg⁺_EDTA, the amount of silver was measured as 1.5 wt%. It can be noted that, due to the washing procedure, the silver content in the PZAg⁺_EDTA sample was 27% lower (1.5 wt% drops to 1.1 wt%) than in the PZAg⁺ untreated sample. In the PAg⁺ and the PAg⁰ control samples, Ag content was from two to three times lower compared to the paper modified with the zeolite. The silver content in the paper was not controlled other than by the affinity of different additives (silver cations, silver NPs or AgFAU particles) to cellulose fibres, which varies from sample to sample.

As can be inferred from the optical images of the final paper sample PZAg⁺_EDTA, the zeolite filler dispersed evenly within the paper pulp (Fig. 2a, b). The zeolite particles can be noted as black spots in the transmission image of the sample (Fig. 2a).

What is more, SEM analysis confirmed that zeolite is presented on the cellulose fibres. The back-scattered electron mode allowed silver-exchanged zeolite to be shown as brighter spots (Fig. 3). In the picture, there are also marked (as numbers) places where EDX analysis was performed. The average values of element content (Table 3) confirm the presence of elements typical for zeolite (Al, Si, O), as well as for silver which was introduced to zeolite.

The pH values (Fig. 4) of the paper samples containing silver in any form were lower than for the reference samples without silver; for P and PZ0, pH amounted to 8.8 ± 0.25, 9.4 ± 0.08, respectively. The lowest value was observed for sample PAg⁺(4.7 ± 0.11). For samples which contained zeolites, both unmodified and modified, the pH values were higher, 5.9 ± 0.03; 7.00 ± 0.04, respectively. Although, the addition of zeolite with trapped silver causes a decrease in the pH value, the decrease is much lower than for materials in which the active agent was presented in unbound form. It seems that the higher acidity of paper material designed for packaging is not an obstacle. In fact, in the long run the acidity of paper is responsible for the loss of the mechanical strength of paper or cellulose-based materials caused by hydrolysis of cellulose fibres (Chu 1981; Łojewski et al. 2010). Low pH of cellulose-based materials may result from, amongst other things, the manufacturing process, the presence of acids in the raw materials, or as products of cellulose degradation. In the presence of water molecules (atmospheric moisture, bound water) an acid hydrolysis reaction occurs. Therefore, hydrolytic cleavage of β(1-4) glycosidic bonds leads to shortening of cellulose chains. This process will be reflected in the consecutive decrease of average DP value, and possibly in loss of mechanical strength of cellulose fibres.

According to the results of zero-span breaking strength of the paper samples, of all tested materials the PZAg⁺_EDTA was around 20% stronger than unmodified paper, and stronger than the rest of the samples (Fig. 5). The presence of silver or silver oxide crystallites in paper (PAg⁺, PAg⁰, PZAg⁺) weakens the strength of cellulose fibres in comparison with the pure paper sample P. This can be explained by the modification of the hydrogen bonding network with silver cations, which interact directly with –OH groups in cellulose chains or are electrostatically attracted by them as high electronic density is located just on the surface of the fibres due to the enrichment with –OH groups (He et al. 2003). Indeed, it has been documented that hydrogen bonding is responsible not only for the crystallinity of cellulose, but also for tensile strength (Chu 1981). Let us note that, even in the sample containing zero valent silver NPs (PZAg⁰), the presence of Ag⁺ cannot be excluded as the sonication process used for silver reduction proceeded with high but limited efficiency. In the paper sample PZAg⁺, which was not eluted, the crystallites of silver oxide are still present. However, it has to be taken into account that the conclusions drawn from the results of the measurements of mechanical properties are not very firm, due to high experimental error.

The results of paper mechanical resistance are consistent with paper acidity (compare Figs. 4, 5). This means that paper acidity can be correlated with cellulose fibre strength and used as a gauge for predicting mechanical properties. In brief, paper sample PAg⁰, containing silver NPs, demonstrated both much lower pH and breaking strength than the final material prepared by us containing silver-exchanged zeolite PZAg⁺_EDTA. It can also be noted from the comparison of the PZAg⁺and PZAg⁺_EDTA samples that washing with EDTA improves both the acidity and mechanical properties of the modified paper material.

The degree of polymerisation (DP_w) of the initial unaged and aged samples was assessed based on the SEC measurements of molecular mass distribution of cellulose. When comparing the unaged samples, all the initial values were similar and fell in the range 1200-1400 DP_w, except the unaged sample containing zeolite PZAg⁺, for which DP_w was about 1070. An important observation is also that the DP_w of the final sample (PZAg⁺_EDTA) is the highest among the studied samples – even up to around 100 units higher than for the reference paper sample (see Fig. 6). The same observation can be made for the initial PAg⁰ paper containing silver NPs. Upon thermal ageing, the decrease in DP_w values can be noted for all samples. The lowest degradation was measured for P samples, and there is almost no degradation at all for the pure zeolite-enriched paper sample PZ0. The highest drop in DP_w values was shown by the PAg⁺ and PAg⁰ samples with silver additives, indicating highly degraded cellulose chains of the 200 DP_w value. There is a noteworthy difference between the PAg⁰ sample with silver NPs and the final sample of PZAg⁺_EDTA, which confirms the conclusion about the positive influence of faujasite exchanged with silver on the condition of both cellulose chains and fibres in the paper material.

The parameter L* from the CIELab colour model was used as the measure of brightness—L* with a range from 0 (black) to 100 (white). The total colour change (∆E) was calculated from the L*, a*, b* values following the equation described in (Gulrajani 2010).

For the initial samples, regardless of the filler composition, no colour differences were noted among any sample containing silver, by either colour change indicator (L* and ∆E) in comparison with the reference paper sample P. The results presenting the differences that occurred after 2 months of storage at 23 °C and 50% RH are shown in Fig. 7. There, for all samples containing silver, an average drop of around 20% in the L* values was observed in comparison with the reference paper sample P. An important observation is that there was almost no difference between paper samples with silver NPs (PAg⁰) and our final material (PZAg⁺_EDTA).

Colour stability (lightfastness) over time was also measured by spot light ageing tests performed with a micro fading tester. The results are expressed by ∆E values in Fig. 8. Again, the highest colour change values were noted for the samples containing silver, reaching 35 units for the PAg⁺ sample. The lightfastness of paper Ag NPs and the final sample PZAg⁺_EDTA is very much alike, with only three ∆E units difference.

The antimicrobial properties of the obtained final material and the reference samples are presented in Fig. 9. The tests were performed with respect to E. coli strains used typically as a representative to assess the biocidal properties of materials. The highest and comparable activity can be noted for both paper samples containing zeolites, as can be judged from the comparison of the ATP/ADP/AMP content in the samples (expressed as counts) in Fig. 9. The content of adenosine phosphates can be used as a direct measure of the bioactivity of the living organisms, especially if the bacterial strain is of the same identity (as discussed by Kwiatkowska et al. (2016) and references therein). The greatest drop in the values was observed for both the samples containing zeolites, PZAg⁺ and the final PZAg⁺_EDTA, and slightly less so for the PAg⁺ sample. This indicates that in fact the active silver species in the mechanism of biocidal activity are silver cations and not silver zero valent NPs (PAg⁰), which respond comparably to the paper reference sample P, indicating no biocidal activity and a low concentration of Ag⁺.

To illustrate the action of materials against E.coli strain, the inhibition zone is presented (Fig. 10). A halo, proving the antimicrobial behaviour of manufactured material, can be seen around the paper disc.