Solvent-Evaporation-Induced Synthesis of Graphene Oxide/Peptide Nanofiber (GO/PNF) Hybrid Membranes Doped with Silver Nanoparticles for Antibacterial Application

Abstract: Designing functional membranes through the collaboration of multi-dimensional nanomaterials is of particular interest in environmental and biomedical applications. Herein, we propose a facile and green synthetic strategy by collaborating with graphene oxide (GO), peptides, and silver nanoparticles (AgNPs) to synthesize functional hybrid membranes with favourable antibacterial effects. GO nanosheets are functionalized with self-assembled peptide nanofifibers (PNFs) to form GO/PNFs nanohybrids, in which the PNFs not only improve the biocompatibility and dispersity of GO, but also provide more active sites for growing and anchoring AgNPs. As a result, multifunctional GO/PNFs/AgNP hybrid membranes with adjustable thickness and AgNP density are prepared via the solvent evaporation technique. The structural morphology of the as-prepared membranes is characterized using scanning electron microscopy, transmission electron microscopy, and X-ray photoelectron spectroscopy, and their properties are analyzed by spectral methods. The hybrid membranes are then subjected to antibacterial experiments and their excellent antibacterial performances are demonstrated. Keywords: solvent evaporation; graphene membrane; silver nanoparticles; peptide nanofifibers; antibacterial application

1. Introduction Nanomaterial-based functional membranes have attracted great attention owing to their wide applications in fifiltration, dialysis, surface modifification, tissue engineering, antibacterial materials, and many other fifields [1–5]. Typically, various membranes can be prepared through the methods of casting, coating, electrospinning, layer-by-layer selfassembly, evaporation, and vacuum fifiltration [6–8], by which the thickness, pore size, and surface functionality of the fabricated membranes can be tailored effectively. According to different applications, the precursor materials for the fabrication of membranes can range from polymers [9] to biomass nanofifibers [10], inorganic nanowires [11], carbon nanofifibers [12], carbon nanotubes [13,14], two-dimensional (2D) materials [15–18], and others. As a single-layer, biocompatible 2D nanomaterial, graphene, especially in the form of graphene oxide (GO) and reduced graphene oxide (RGO) nanosheets, exhibits high potential for the preparation of functional membranes with excellent electrical, thermal, and mechanical properties [15,19,20]. GO also has good antibacterial activity and the sharp edges of the nanosheets are in direct contact with bacterial cell membranes, thus effectively disrupting the cell membranes and exerting antibacterial effects [21]. Besides these unique structures and properties, GO nanosheets have extra advantages for the preparation of functional membranes. For instance, there are abundant functional groups like -COOH, -OH, and C=O on the surface and edges of GO nanosheets, which provide the possibility for the modifification of GO with other nanomaterials to enhance the properties of created GO hybrid membranes [22,23]. In addition, the 2D nanosheet structure makes GO a good support material for conjugation with various nanoparticles, polymers, biomolecules, and biomass nanofifibers with high density [24–26]. Previously, studies have indicated that GObased membranes can be utilized for water purifification, gas fifiltration, electronic devices, flflexible sensors, battery separators, and antibacterial materials [7,27,28]. Li et al. developed the method of directly mixing acetone and GO aqueous dispersion to prepare GO fifilms with adjustable thickness [29]. Jang et al. studied the adhesion of GO fifilms to several different bacteria, and found that the ability of GO to obtain electrons from bacteria led to the adhesion effect of GO to bacteria. In addition, the adhesion effect of different bacteria is different, and the quality of the GO fifilm also inflfluences the adhesion effect of bacteria [30]. AgNP is a kind of metal nanomaterial that is widely used in medicine, catalysis, food, environment, biosensors, and many others. AgNP, as an excellent broad-spectrum antibacterial material, has a strong antibacterial effect on a variety of bacteria, pathogens, fungi, and so on. The damage mechanism of AgNP to bacteria is that it binds with the bacterial cell membrane to cause membrane damage, interacts with DNA and proteins in bacteria to cause molecular damage, and produces reactive oxygen species, among others. It has also been reported that AgNP can release Ag+ to bind with proteins in bacteria to change its active site and three-dimensional structure to provide an additional bactericidal effect [31]. The size of AgNPs is also a key factor affecting their antibacterial properties. Morones et al. explored the antibacterial effect of AgNPs in the range of 1–100 nm [32]. Experiments show that small-sized AgNPs (1–10 nm) can then bind to the cell membrane of bacteria and enter the bacterial interior to play an antibacterial role. This is because the small-sized AgNPs have a larger specifific surface area and are more likely to bind to bacteria and enter the bacterial interior. In addition, the surface charge of AgNP also has an impact on its antibacterial activity. Badawyetal et al. evaluated the antibacterial performance of AgNPs with different surface charge intensities, such as the strong electrostatic attraction between negatively charged Bacillus (−37 mV) and positively charged AgNPs. This results in a stronger interaction between AgNPs and bacteria, which improves antibacterial performance. On the other hand, Bacillus will form a certain degree of electrostatic shielding effect on negatively charged AgNPs, which limits the interaction between the two, and thus reduces the antibacterial performance of AgNPs. This study provides ideas for the rational design of AgNPs as antibacterial agents [33]. When applying GO membrane materials for the applications of wound repair, wearable biosensors, water disinfection, and food packaging, one of the key issues that should be considered is the antibacterial performance of the GO membrane used [34,35]. Previous studies have proved that GO nanosheets contributed to the antibacterial effects of related materials through disrupting the cell membrane [36]. However, the antibacterial activity of graphene materials is limited compared with traditional nanomaterials such as silver, copper, and titanium oxide nanoparticles [24,37,38]. In addition, the aggregation and oxide status of GO nanosheets have potential effects on the antibacterial effificiency of GO materials [39]. Therefore, the fabrication of hybrid membranes of GO and highly effective antibacterial nanoparticles is popular for utilizing the combined properties and functions of 2D GO and other materials via synergistic effects. For example, by modifying silver nanoparticles (AgNPs) and copper oxide nanoparticles (CuONPs) onto the surface of GO nanosheets, Menazea et al. chemically prepared functional GO/AgNPs and GO/CuONPs hybrids, respectively. Both GO-based nanohybrids were biocompatible and revealed good inhibitory effects on both S. aureus and Gram-negative bacteria [24]. In recent work, Wang et al. designed a simple method to modify GO with CuS nanoparticles to form CuS@GO nanocomposites, and then CuS@GO electrostatically interacted with chitosan (CS) hydrogel to produce CuS@GO-CS antibacterial hydrogel. The antibacterial hydrogel has a large antibacterial effect on Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) [40]. In another work, Liang et al. used electrostatic interactions to load zinc oxide quantum dots (ZnO QDs) onto GO nanosheets to synthesize ZnO QDs@GO hybrids, which were further introduced into the as-prepared hydrogel to prepare a hybrid hydrogel with antibacterial activity and wound-healing ability [41]. In this work, we propose a facile solvent evaporation method for the fabrication of GO-based functional hybrid membranes. GO nanosheets are fifirstly functionalized with self-assembled peptide nanofifibers (PNFs), which show high affifinity for binding GO to form GO/PNF nanohybrids. The introduction of PNFs onto GO nanosheets not only improves the biocompatibility and dispersity of GO, but also provides more active sites for binding AgNPs via the biomimetic synthesis. By the solvent evaporation, GO/PNF/AgNP hybrid membranes with adjustable thickness and AgNP density are prepared. The created hybrid membranes that exhibit excellent antibacterial activity towards E. coli and S. aureus owing to the synergistic antibacterial effects of both GO and AgNPs. This facile, green, economic strategy for the fabrication of large-scale GO-based membranes will inspire the design and synthesis of 2D material-based functional membranes for advanced applications.

2. Materials and Methods 2.1. Materials All chemicals used in this work are of analytical level. Peptide with the sequence of KIIIIKYWYAF was bought from the SynPeptide Biotechnology Co., Ltd. (Nanjing, China). Monolayered GO aqueous dispersions (GO-1, 10 mg/g) were provided by the Hangzhou Gaoxi Technology Co., Ltd. (Hangzhou, China). D-anhydrous glucose (AR), phosphoric acid, ethanol, glycerol, glutaraldehyde, NaCl, tryptone, and agar powder were purchased from the Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China). 2.2. Preparation of PNFs via Self-Assembly The formation of PNFs is achieved through the peptide self-assembly method. In brief, 1 mg of KIIIIKYWYAF peptide monomer powder was dissolved in 10 mL of water and well dispersed by sonication to confifigure a 0.1 mg/mL peptide solution. Ultrasound can ensure the full dispersion of peptides. The peptide solution was then incubated in a 37 ◦C water bath and sampled every 24 h for 3 days to observe the self-assembly status using atomic force microscopy. 2.3. Fabrication of GO/PNF/AgNP Hybrid Membrane by Solvent Evaporation The preparation of GO/PNF/AgNP hybrid membranes at the air–liquid interface was carried out via the solvent evaporation method, as shown in Scheme 1. Specififically, 2.5 g GO dispersion was diluted to 50 mL to obtain 0.5 mg/mL of GO dispersion. Then, 1 mL of peptide solution and 25 µL of AgNO3 solution were mixed into the GO dispersion. Then, the peptide solution incubated with 1 mL for 3 days and 25 µL AgNO3 (0.5 mol/L) solution was mixed into GO dispersion. After the reaction, the mixed solution was placed in a water bath at 80 ◦C and evaporated for 40 min to obtain GO/PNF/AgNP heterogeneous membranes. The GO/PNF/AgNP hybrid membrane is formed at the water–air interface, the thickness of the hybrid fifilm can be adjusted by changing the evaporation time, and the shape of the hybrid fifilm can be adjusted by changing the loading substrate of the hybrid fifilm. 2.4. Characterization Techniques All AFM samples were characterized by dropping 10 µL of sample onto a newly dissociated mica substrate and drying in air. AFM measurements were performed in air in tap mode using‍ an FM-Nanoview 6800 AFM (FSM-Precision, Feisman Precision Instruments, Suzhou, China)‍. A Tap300Al-G silicon probe (300 kHz, 40 Nm−1 ) was used for AFM image capture. Tap mode images were recorded and analyzed using Gwyddion software (version 2.57). Transmission electron microscopy (Tecnai G2 F20, FEI Co. Hillsborough, OR, USA) was used to observe the structure and morphology of GO/PNF/AgNP suspensions. Scanning electron microscopy (SEM, Regulus 8100, Hitachi Co.Tokyo, Japan) was used to observe the microstructure of GO/PNF/AgNP hybrid membranes. XPS characteriza-tion of the samples was performed on a PHI 5000 VersaProbe III spectrometer (PHI-5000 Versaprobe III, UlVAC-PHI Co., Kanagawa, Japan).