Antibacterial Activity of In Situ Generated Silver Nanoparticles in Hybrid Silica Films

Abstract

Herein we present silver nanoparticles (AgNPs)-doped inorganic–organic hybrid silica films on glass and polypropylene substrates. A hybrid inorganic–organic silica sol in alcoholic medium was prepared at room temperature using TEOS, GLYMO, and APTES. Silver nanoparticles were generated in situ within the hybrid silica sol. AgNPs-SiO₂ film was obtained by dip coating method following drying at 80 °C. FTIR spectra shows several vibrational bands of the hybrid silica network and amine functionalization. AgNPs formation was observed from the XRD spectra of the dried film. UV–Visible spectra show sharp surface plasmon resonance (SPR) band centered at 412 nm arising from the evenly distributed silver nanoparticle inside the silica film that was supported by morphological characterization. Both the coated films showed good antibacterial activity against E. coli bacterial strain by forming a zone of inhibition in the agar diffusion test. The antibacterial efficiency for coated glass and polypropylene was 72.5% and 83.75%. This coating approach provides a straight-forward solution to prepare antibacterial coatings on various substrates especially on plastics, where low temperature processing is necessary.

1. Introduction

The outbreak due to the super spreader and highly infectious coronavirus (SARS-CoV-2) has revealed the need to develop antibacterial and antiviral coating materials [1]. In particular, high-touch surfaces such as public transport, door handles, elevator keys, and other common areas in hospitals and offices are at high risk of spreading the airborne pathogens [2]. Silver has been used as a potent antimicrobial agent since ancient times [3,4]. Silver nanoparticles (AgNPs) are considered very potent antimicrobial/antifungal agents due to their strong cytotoxic effect toward a broad range of microorganism and remarkably low human toxicity compared to other heavy metals [5]. Although the actual reason for the antibacterial activity of AgNPs is not clear, many reports propose that Ag ions are released from the AgNPs, which strongly binds to the thiol groups present in the bacterial/microbial cell membrane and thus destroy the cell by puncturing the wall leading to cell death [5,6]. Many researchers have proposed several synthesis procedures to prepare highly antibacterial silver nanomaterials [5,7,8,9,10,11,12]. AgNPs have been successfully loaded on various porous supports to enhance the antimicrobial efficiency; Yang et al. described E. coli inactivation by AgNPs anchored on titania nanotubes [13], Joarder et al. reported enhanced antibacterial activity of AgNPs deposited on MCM-41 type mesoporous silica [14], Guo et al. described sustained release behavior of halloysite nanotubes loaded with the AgNPs [15], Dung et al. fabricated silver-doped ceramic filter for antimicrobial water purification [16], and Chen et al. reviewed the antibacterial activity of silver doped polymeric nanostructures [17]. However, it is necessary to achieve a proper antimicrobial coating that can be applied to a variety of substrates since bare or supported AgNPs could exhibit some adverse effects due to excessive leaching in moist environments that could lead to diminished antibacterial activity and make the environment toxic as well [18,19,20]. Antimicrobial coatings based on AgNPs have been used in medical implants, biomedical devices, and in packaging material to inhibit the bacterial infection [3,21,22,23,24,25]. Most of the reported works describe either high temperature sintering or complex multistep procedures that might show difficulties in obtaining antibacterial coatings on a large scale [3,22,25].

In this work, we report one-pot synthesis of in situ generated AgNPs in organically modified hybrid silica films coated on glass and polypropylene substrates without using any additional reducing agents. Hybrid silica films show many advantageous properties compared to their inorganic counterpart due to their flexibility and film adherence on a range of substrates without crack formation when comparatively thicker coating is required [26,27,28]. They serve as excellent host for trapping and uniformly dispersing the metal and semiconductor nanoparticles because of their ability to act as stabilizers that terminates further growth of the nanoparticles by controlling the nucleation process [29,30]. AgNPs were generated inside the hybrid silica film during the synthesis process and coatings were deposited on glass slide and polypropylene sheets by the dip coating method. The films were characterized by UV–Visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) analysis, and scanning electron microscopy (SEM). The antibacterial activity of the coated films was tested against Gram negative Escherichia coli (E. coli) bacterial strain.

2. Materials and Methods

2.1. Chemicals and Biological Reagents

All the chemicals in the synthesis procedure were used without any modification. Tetraethylorthisilicate (TEOS, Sigma-Aldrich, St. Luis, MO, USA, Reagent grade, 98%) was used as inorganic precursor, and (3-Glycidyloxypropyl)triethoxysilane (GLYMO, Sigma-Aldrich, ≥98%) and (3-Aminopropyl)triethoxysilane (APTES, Sigma-Aldrich, ≥98%) were used as the organic precursors of silica. Silver nitrate (AgNO₃, Alfa Aesar Gmbh & Co KG, Karlsruhe, Germany, 99+%) was used as the silver source. Agar powder was purchased from A.C.E.F., Luria-Bertani (LB) broth (Miller) and E coli culture BL21 (DE3) strain were purchased from Merck KGaA, Germany. Milli Q ultrapure water (Resistivity 18.2 MΩ cm) was used throughout the experimental procedure.

2.2. Hybrid AgNPs-SiO₂ Film Preparation

Inorganic–organic hybrid coating approach was followed to prepare the AgNPs-SiO₂ coatings on glass slides and PP sheets. Ag nanoparticles were generated inside the SiO₂ film matrix at sol processing stage. This inorganic–organic hybrid coating approach not only helps to induce the formation of AgNPs inside the SiO₂ film, but also increases the adherence to the polypropylene sheets, which was not achieved from conventional silica sol. In addition, 10 equivalent wt% of SiO₂ was maintained to produce a thicker coating without suffering the cracking and 5 mol% of Ag with respect to SiO₂ was used as the dopant. The molar composition of TEOS: GLYMO: APTES was maintained at 6:3:1 and 1.5 mole of H₂O and 10⁻² mole of HNO₃ per mole of the alkoxide group was used in the sol to initiate the hydrolysis-condensation reaction, while 2-propanol was used as the solvent. First, the hybrid silica sol was prepared and kept in a closed container for one day. Then, the required amount of AgNO₃ dissolved in a little amount of water was mixed with stirring. After 10 min of stirring, the color of the sol changed gradually to dark brown indicating the formation of AgNPs in the sol. Cleaned glass slides and polypropylene sheets were coated with the above sol by dip coating method using the lifting speed of 20 cm/min. After coating deposition, the coated substrates were kept in an oven at 80 °C overnight to densify the coating. The coated films showed uniform bright yellow color, which indicates that AgNPs have been formed inside the silica film. The schematic diagram of the film forming process is presented in Figure 1.

2.3. Characterizations

Nanocrystalline nature of the AgNPs in silica film on glass and polypropylene substrates was characterized by X-ray diffraction (XRD) measurement, performed on a Rigaku Ultima X-ray diffractometer using CuK_α radiation (λ = 1.5406 Å) operating at 40 kV/30 mA with the step size of 0.02°. FTIR spectra of the dried film were recorded with a JASCO FTIR-6300 instrument over the range 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹ by accumulating 256 scans for each measurement, adopting the KBr disc method. A little amount of the film was scrapped off from the coated glass substrate and mixed with the KBr (powder: KBr weight ratio of 1:10) to prepare the IR transparent disk. UV–Visible absorption spectra of the films coated on glass and polypropylene were measured with Agilent Cary 5000 UV–Visible–NIR spectrophotometer. The coated glass slides and PP sheets were fixed to the solid sample holder accessory and mounted into the sample compartment in transmission mode. Uncoated glass slide and PP sheet were used as reference material while performing baseline correction. The absorption spectra were measured directly from the drop-down measurement command (Absorbance) in the software. Morphology of the AgNPs-SiO₂ film was observed with a Zeiss (model Sigma VP) field emission scanning electron microscope (FESEM).

2.4. Assay of Antimicrobial Activity

The antimicrobial activity of AgNPs-SiO₂ coated glass slide and polypropylene sheet was investigated against the common pathogenic bacteria E. coli as the model microorganism, which was pre-cultured at 37 °C to reach a concentration above 10⁸ colony forming units (CFU/mL). The turbidity assay was performed to quantitatively evaluate the antimicrobial activity by observing the optical density of the strain at 600 nm. Each sample was added into 25 mL of E. coli. nutrient broth, seeded with 0.05 mL of test strains (1.6–1.9 × 10⁸ CFU of total bacteria number), and incubated at 37 °C for 18 h. Different dilutions of the bacterial suspensions were then transferred onto nutrient agar plates and incubated at 37 °C for 18 h. After that, the viable bacteria were monitored by counting the number of colony forming units from the appropriate dilution on nutrient agar plates and expressed as CFU. The same procedure was performed on bare substrates as control.

3. Results and Discussion

Silver nanoparticles were generated in the inorganic–organic hybrid silica matrix without using any additional reducing agent. Here, APTES played a crucial role to reduce Ag⁺ to Ag⁰. Choi et al. reported that aminosilane could be utilized to produce metallic silver nanoparticles embedded in sol-gel silica matrix without using any external reducing precursor [30]. Therefore, we can assume that the amine functional groups originating from APTES could reduce AgNO₃ to form Ag nanoparticles in the hybrid silica matrix in presence of water. The reduction process was monitored by observing the sol color change from colorless sol to light brownish to deep brown. The amine functionalization to the silica matrix and the formation of SiO₂ network was investigated by FTIR measurements, which is presented in Figure 2. The most informative region is situated in the wavenumber range between 946 to 1250 cm⁻¹ due to the Si−O−Si network, where the most intense band is observed that consists of a group of bands. The characteristics of this band are quite different than those reported for inorganic–organic hybrid silica film, where a relatively sharp band has been shown [26,31]. The vibrational bands centered at 1092 and 805 cm⁻¹ are assigned to the asymmetric and symmetric stretching of Si−O−Si, respectively [26,32]. The Si−O−Si asymmetric stretching band is overlapped with the Si−O−C vibrational band. A peak due to the NO⁻³ group at 1387 cm⁻¹ is also observed indicating the presence of nitrates inside the film originating from silver nitrate [31]. Absence of the epoxide bands at 1260–1240 cm⁻¹ (epoxy ring breathing) and 950–810 cm⁻¹ (asymmetrical ring stretching) indicate the polymerization of epoxide groups [31]. The broad band around 3300–3600 cm⁻¹ and relatively low intense band at 976 cm⁻¹ are attributed to the stretching vibration of H-bonded silanols (Si−OH) with hydroxyl groups of the adsorbed water molecules. Interestingly, there are new bands centered at 1035 and 1150 cm⁻¹, which are assigned to Si−O−Si transverse optical (TO) and Si−O−Si longitudinal optical (LO) stretching modes, respectively (Figure 2), which strongly suggest the functionalization of APTES with the silica network [33,34]. This functionalization is further confirmed by the appearance of CH₃ asymmetric mode at 2975 cm⁻¹ and CH₂ asymmetric and symmetric stretching modes at 2931 and 2865 cm⁻¹ [33]. There are also some low intense peaks between 1500 to 1600 cm⁻¹ that could be assigned to the −NH₂ scissor vibration and asymmetric NH⁺₃ deformation mode, both originating from APTES, which also support the APTES functionalization with the Si−O−Si.

Figure 3 shows the XRD pattern of the AgNPs/SiO₂ film coated on glass and polypropylene substrates dried at 80 °C. In both cases, low intensity and broad diffraction peaks appeared around 38.18° 2θ that correspond to the <111> plane of the face centered cubic crystalline phase of metallic silver [26]. The appearance of broader diffraction peaks indicates the formation of evenly distributed small sized-silver nanoclusters inside the silica film that is supported by the morphological investigation discussed later on. We did not observe any additional peaks due to the silver oxides [35].

UV–Visible absorption spectra of the AgNPs/SiO₂ coated films on glass slide and polypropylene sheet are presented in Figure 4. Both the films show a strong absorption band centered at 412 nm wavelength that can be explained by the surface plasmon resonance (SPR) band of the AgNPs. The appearance of sharp and intense SPR band without having any additional shoulder peaks at higher wavelength region strongly suggests the uniformly dispersed AgNPs throughout the silica film having narrow size distribution without forming any agglomeration [36]. This is also supported by the appearance of the bright yellow color of the coated films (inset of Figure 4).

The electron micrographs (FESEM) images of the AgNPs in silica film is shown in Figure 5 with different magnification (Figure 5a–c). It is clearly evidenced that the AgNPs are evenly distributed inside the silica film matrix. No distinguishable agglomeration was observed. The average size of the AgNPs was found to be about 20 nm (Figure 5d), which is in good agreement with the optical absorption spectra [36].

Biocidal activity of the AgNPs-SiO₂ films coated on glass slides and polypropylene sheets were evaluated qualitatively and quantitatively against E. coli, which is the most commonly used Gram-negative food borne pathogen that is also present in lower intestinal tract of warm-blooded animals, including humans [37]. E. coli is often responsible for bacterial infection of the environment through fecal discharge and wastewater from sewage [38]. It is also easy to grow the E. coli very fast with diverse species in laboratory conditions (~37 °C) that is also well understood so far by several studies [37,38,39]. The qualitative evaluation was examined by observing the inhibition zone formation around the coated film using the disk diffusion method (Figure 6a,b). It is clearly visible from the figure that AgNPs/SiO₂ coated films inhibited the bacterial growth by forming a well resolved inhibition zone around the coated film that confirm no bacterial growth around the coating, whereas there was no evidence of inhibition zone formation in case of uncoated substrates that reveal no antibacterial activity of the uncoated substrates. It is also noticeable that the diameter of the inhibition zone is higher for polypropylene that the glass slides which suggests strong adherence of the AgNPs/SiO₂ film with the glassy surface that might control the release of Ag⁺ ions, which are responsible for the killing of the bacteria. Since the glass surface has many hydroxyl groups (Si-OH) favoring the surface wettability, and AgNPs containing hybrid silica sol also have silanol groups, a strong chemical bonding could occur between the glass slide and the hybrid silica sol. On the other hand, polypropylene, having a chemical formula of (C₃H₆)_n, where the methyl groups (CH₃) are arranged on the carbon chain, shows strong hydrophobicity that makes it difficult to coat with only TEOS-derived silica sol. Although the inorganic–organic hybrid sol favored the film formation on PP substrate, it may lack the strong adherence achieved in case of the glass slides. The qualitative antibacterial efficiency was obtained by the turbidity assay of the growth solution, where the optical absorption at 600 nm (OD₆₀₀) was recorded and expressed as colony forming unit (CFU). The antibacterial efficiency was calculated by the following equation [6]

Efficiency = [1 − CFU_Ag/CFU₀]

(1)

where CFU_Ag and CFU₀ represent the bacterial concentration with AgNPs/SiO₂ film and with uncoated substrates, respectively. Figure 6c shows the bacterial growth solution with AgNPs/SiO₂ coated glass and polypropylene substrates and a control solution after 18 h of incubation. The growth solution containing coated films show optically clear appearance compared to turbid appearance of the control solution indicating the coated film inhibited the E. coli growth. The antibacterial efficiency is presented in Figure 6d, where it is observed that 83.75% efficiency was obtained for the coated polypropylene substrate, whereas for the coated glass substrate it was 72.5%.

Comparatively lower antibacterial efficiency of the glass slides is in agreement with the lower inhibition zone formation compared to the coated polypropylene sheets (Figure 6a,b). Similar antibacterial activities of the AgNPs dispersed in silica coating have been also reported [40,41,42].

4. Conclusions

In this work, we have reported a simple coating approach based on AgNPs in hybrid silica sol to effectively obtain biocide film on various substrates. Since the AgNPs are produced in situ inside the silica sol, it excludes the second step of reducing the Ag⁺ to Ag⁰, either by using a reducing agent or by thermal curing. The process could be interesting for low temperature curing substrate such as plastics and/or where a reduction step is difficult to carry out, e.g., common touch surfaces. AgNPs containing silica sol can also be sprayed or rolled to coat larger substrates. Since the coated films showed moderate antibacterial activity (72.5% and 83.75% for glass and polypropylene), and the AgNPs are protected inside the glassy SiO₂ film, they could be potential candidate in food packaging industries, where there are limited chances of contamination due to silver leaching. We will investigate in detail how Ag⁺ releases from the films on various substrates and its effect on the antibacterial activity.