Anti-adhesion and antibacterial activity of silver nanoparticles supported on graphene oxide sheets

Highlights

• Graphene oxide (GO) decorated with silver nanoparticles were successfully prepared.

• The AgNPs supported onto GO showed an average size of 7.5 nm.

• The role of oxidation debris on the anchoring of AgNPs on GO was proposed.

• GO–Ag nanocomposite showed high antibacterial activity against Pseudomonas aeruginosa strain.

• GO–Ag also presented biocide activity against adhered cells of P. aeruginosa.

Abstract

This work reports on the preparation, characterization and antibacterial activity of a nanocomposite formed from graphene oxide (GO) sheets decorated with silver nanoparticles (GO–Ag). The GO–Ag nanocomposite was prepared in the presence of AgNO₃ and sodium citrate. The physicochemical characterization was performed by UV–vis spectroscopy, X-ray diffraction (XRD), thermogravimetric analysis (TGA), Raman spectroscopy and transmission electron microscopy (TEM). The average size of the silver nanoparticles anchored on the GO surface was 7.5 nm. Oxidation debris fragments (a byproduct adsorbed on the GO surface) were found to be crucial for the nucleation and growth of the silver nanoparticles. The antibacterial activity of the GO and GO–Ag nanocomposite against the microorganism Pseudomonas aeruginosa was investigated using the standard counting plate methodology. The GO dispersion showed no antibacterial activity against P. aeruginosa over the concentration range investigated. On the other hand, the GO–Ag nanocomposite displayed high biocidal activity with a minimum inhibitory concentration ranging from 2.5 to 5.0 μg/mL. The anti-biofilm activity toward P. aeruginosa adhered on stainless steel surfaces was also investigated. The results showed a 100% inhibition rate of the adhered cells after exposure to the GO–Ag nanocomposite for one hour. To the best of our knowledge, this work provides the first direct evidence that GO–Ag nanocomposites can inhibit the growth of microbial adhered cells, thus preventing the process of biofilm formation. These promising results support the idea that GO–Ag nanocomposites may be applied as antibacterial coatings material to prevent the development of biofilms in food packaging and medical devices.

Introduction

Microorganisms are widespread in nature, and many can adhere to surfaces, forming so-called biofilms [1]. These microorganisms are found in many different environments (ocean, soil, skin, and air), and their adherence to surfaces constitutes a survival strategy [2]. Bacterial communities can anchor on surfaces and produce biofilms, which are commonly surrounded by an extracellular polymeric matrix of exopolysaccharide (EPS) [3]. The dynamic of biofilm formation occurs in different steps; the primary phase corresponds to the adhesion of the microorganisms to the surface, which can in turn act as a support for microbial growth, a source of nutrients or a supplier of the optimal oxygen required by aerobic microorganisms [2]. The presence of biofilms can be harmful to several industrial processes because they cause mechanical blockage in fluid systems, disturbances of heat transfer processes and corrosion in metallic surfaces [4].

In particular, the presence of biofilms in the food industry can lead to severe hygienic problems, economic losses due to food spoilage and even serious diseases when pathogens are involved [5]. In a hospital environment, biofilms may contaminate medical implant devices [6], thus increasing the risk of the transmission of infectious diseases and offering higher microbial resistance to antibiotics [2], [6], as the cells living in the form of biofilms are usually more resistant to antimicrobial compounds than planktonic cells [1].

Pseudomonas aeruginosa is a classic example of a microorganism with the ability to grow and form biofilms on non-sterile solid surfaces [3]. Pseudomonas sp. represent a large and versatile group of bacteria that have adapted to live in soil, seawater and fresh water [7], and due to their excellent adaptability, they are found in food processing environments, including drains, floors, meat surfaces, and dairy products [5]. In hospitals, the presence of P. aeruginosa has been associated with the occurrence of lung infections and cystic fibrosis [6]. The formation of biofilms by P. aeruginosa is related to the presence of both flagellar and pili-mediated motilities [8], the production of large amounts of EPS [4] and the quorum sensing system that controls the cell-cell signaling processes [2].

The removal and eradication of biofilms is generally achieved by mechanical force, acid- or alkaline-based detergents or chemical disinfectants. However, the efficiency of these chemical products is strongly affected by factors such as pH, temperature, solubility, concentration and exposure time [5]. In this context, there is a strong desire to develop new strategies to control biofilm formation on food and medical products. According to Romero and Kolter [2], even after many efforts dedicated to develop new anti-adhesion agents, initiatives to improve existing products and standard protocols are encouraged.

Nanotechnology has emerged as an alternative tool for developing products with new properties to meet the increasing demand of the industrial sectors for advanced functional materials. In particular, nanostructured materials such as carbon nanotubes and graphene have been developed as model systems in nanoscience and nanotechnology. Graphene-based materials are a special class, and they have attracted a large amount of attention because of their large superficial area and exceptional electronic, mechanical and thermal properties [9]. Graphene is a two-dimensional material composed of a hexagonal sp²-hybridized carbon network [10], [11], while graphene oxide (GO) is a chemically modified graphene featuring hydroxyl, carboxyl and epoxy functional groups [12]. This functionalized form of graphene is able to produce stable dispersions in water that consist primarily of single-layer GO sheets [13]. This material has been used as a promising building block for preparing new composites [13]. On the other hand, it is well-known that silver nanoparticles possess antimicrobial activity and have been used as biocide agents in health, food and textile applications [14]. Accordingly, silver nanoparticles assembled on graphene oxide sheets (GO–Ag) have been exploited as novel antibacterial systems [15], [16]. Liu et al. [15] and Das et al. [16] demonstrated antibacterial activity for GO–Ag nanocomposites against Gram-negative Escherichia coli. However, the potential for these nanocomposites to prevent biofilm formation has not been explored. Moreover, the antibacterial activity of GO itself is controversial and demands further investigation. For instance, Liu et al. [17] compared the antibacterial activity of four types of graphene-based materials toward E. coli. GO was reported to exhibit the highest antibacterial activity against E. coli when compared to graphite, graphene and reduced graphene oxide. In contrast, Ruiz et al. [18] conducted a study to characterize the antimicrobial activity of GO, and the results showed that the bacteria grew faster when GO was added to the culture medium. Furthermore, the presence of graphene oxide stimulated the bacterial growth by acting as a surface for their attachment and proliferation. Here, we propose the synthesis and chemical characterization of GO decorated with silver nanoparticles. We also highlight the role of oxidation debris fragments in this process. The GO–Ag nanocomposite showed promising antibacterial activity against P. aeruginosa, and this result raises the possibility of applying GO–Ag nanomaterials as anti-adhesion agents, an application that has not been considered until now.