An environmentally friendly nanocomposite polypyrrole@silver/reduced graphene oxide with high catalytic activity for bacteria and antibiotics

In this study, PPy@Ag/rGO nanocomposites were successfully synthesized via the one-pot hydrothermal method using graphene oxide, pyrrole monomer and silver nitrate. The structures and morphologies of as-obtained PPy@Ag/rGO ternary nanocomposites were systematically investigated by scanning electron microscopy (SEM) and transmission electron microscope (TEM). It was found that the PPy@Ag NPs were well distributed on the reduction graphene oxide nanoflakes. The minimum inhibitory concentration (MIC) demonstrated that the PPy@Ag/rGO had enhanced antimicrobial efficiency with Gram-negative (Escherichia coli) bacteria compared with that at the same concentration of silver. From liquid antibacterial cycle experiments, the addition of polypyrrole contributes to the stability of nanosilver and reducing the loss of nanosilver. After several cycles, the antibacterial rate of PPy@Ag/rGO nanomaterials can still be maintained above 90%. In addition, the photocatalytic degradation of tetracycline (TC) under visible light displayed that the composite had good photocatalytic activity and catalytic stability.


Introduction
Nanosilver and silver compounds have potential antibacterial activity against a far-ranging of microorganisms. And these antibacterial materials have been widely used in many sterilization applications [1,2]. Nanosilver is an intolerant disinfectant, which can significantly reduce the use of many bacterial infections, such as benzyl penicillin and tetra, as compared with the general presence. Some bacteria in the environment are resistant to antibiotics. For instance, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are resistant to penicillin benzyl. Therefore, Ag NPs have attracted much attention when nanosilver is found to be effective in killing a many kind of viruses, eumycetes and bacteria. The principium of Ag NPs on bacteria sterilization is not completely clarity. It has been suggested that the antibacterial activity is founded on the electrostatic force between the negatively charged pericellular membrane of the germ and the positively charged Ag ions. As a result, Ag ions can lead to bacterial death by destroying the pericellular membrane, intensely interacting with the sulfhydryl radicals of active enzymes, and even damaging DNA replication capabilities [3,4]. Moreover, the localized surface plasmon effect of precious metal can generate high-energy hot electrons, which can participate in the reaction [5,6]. This phenomenon will help to improve the photocatalytic ability [7,8]. This will effectively reduce the emergence of super bacteria in the environment. It was apparent to all that the antibacterial activity of silver nanoparticles is closely connected to their particle size and spatial distribution [9]. The smaller the particle size of nanosilver is, the better the antibacterial effect is. However, small size silver nanoparticles tend to gather in the preparation process to minimize the surface energy, which results in a significant abasement of their antibacterial performance. To overcome this challenge, it is important to discover and find a suitable carrier material to effectively disperse silver nanoparticles [10,11]. Ali et al. and Tripathi et al. have reported the Ag-doped TiO 2 nanocomposites showed high catalytic activity under visible light and strong inhibition of various bacterial strains [12]. Liu et al. [13] have reported that Ag/ZnO composites have enhanced photocatalytic activity for organic contaminants driven by visible light and synergistically enhanced antibacterial activity against gram bacteria.
Reduced graphene oxide (rGO) is acknowledged as a single layer of carbon atoms that forms a serried honeycomb structure with oxhydryl and epoxy functional groups on two approachable sides and carboxyl groups on the brinks [14][15][16]. These functional groups can better support other materials such as nanometals on the surface of rGO. In 2010, the research first reported the antibacterial activity of graphene oxide (GO) and rGO, where graphenebased nanomaterials have been shown to be effective in choking back the propagation of E. coli [17]. Since then many studies of graphene-based materials have shown the physical interaction between bacterial pericellular membrane and rGO. The sharp edges of reduced graphene oxide can destroy bacterial membrane fracture by puncturing [18]. This local disturbance affects the film potential of phospholipid bilayer of bacterial membrane, interferes with cell metabolic process, and ultimately destroys the pericellular membrane, leading to the cracking and death of bacterial cells [19]. The oxidative stress caused by rGO nanoparticles may participate in irreparable impairment to the bacterial cells that lead to the destruction of cell completeness [20,21].The damage of graphene to pericellular membrane may also be due to the electric-charge transfer mutual effect caused by direct contact between microbiological membranes and graphene [22]. In addition, rGO has inimitable characteristics, including comparatively large specific surface area, lower cytotoxicity and favorable water stability [23,24]. Therefore, it can be used as a plain stage for growth of metal nanoparticles. This form of composite material has been widely used in the field of photocatalysis. The carboxyl group on surface of rGO base allows Ag NPs to interact with rGO nanoparticles through physical adsorption. Specially, the silver positively charged ion can be straightway connected to the carboxy group on the superficies of rGO through electrostatic interactions. Therefore, deoxidization occurs on superficies of GO nano-films, forming a steady rGO-Ag nano-complex. By adding rGO as the carrier, Ag NPs aggregation problems can be minimized or even prevented. Huo et al. [25] have reported Ag/Ag 2 S/ rGO displayed enhanced photocatalytic and antibacterial activity.
However, the Ag nanoparticles supported on the surface of the reduced graphene oxide are easily peeled off during use. So how to avoid the loss of nanosilver during use is an urgent problem to be solved. In the past decade, antimicrobial conductive polymerized substance shows well foreground for development due to their favorable biocompatibility and chemical stability, non-volatility, lack of spread on the skin, low poisonous character, biological activity and long retention time [26][27][28]. Polypyrrole (PPy) is shaped from plenty of linked pyrrole ring architectures and is one of the representative antimicrobial polymerized substances. And PPy has been displayed to possess well antibacterial activity against E. coli [29]. The antibacterial principium of polypyrrole is probably ascribed to seepage of cytoplasm since the mutual effect of bacteria cell membrane and the positive electrical charges in PPy chains. In addition, polypyrrole (PPy) is an organic semiconductor with a narrow band gap, strong electron transport capability and redox properties [30][31][32][33]. Therefore, PPy is widely used in the field of photocatalysis. Polypyrrole is usually prepared by oxidation of pyrrole by chemical or electrochemical means [34][35][36]. Wan et al. and Li et al. have reported the inosculation of cellulose hydrogel and Ag nanoparticles to PPy contribute to the stabilization of the nanomaterial [37]. The addition of polypyrrole can improve the disadvantage of poor stability of binary Ag/rGO nanocomposite during antimicrobial activity. Therefore, it is of great significance to design a simple system to develop nanocomposites with excellent antibacterial properties.
In this work, the PPy@Ag/rGO nano-antibacterial material was synthesized by hydrothermal method. The photocatalytic activities of PPy@Ag/rGO were researched by degrading TC under visible light irradiation. The prepared PPy@Ag/rGO nanomaterial was also used as an antibacterial agent to inactivate E. coli for 12 h at 37°C. At the same time, we further studied the antibacterial effect of PPy on the liquid antibacterial cycle experiment.

Experimental section 2.1 Materials
Silver nitrate (AgNO 3 ), ferric chloride (FeCl 3 ), polyvinylpyrrolidone (PVP) and ethylene glycol ((CH 2 OH) 2 ) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Pyrrole was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. All of the chemical reagents used in the experiments were analytical reagents and did not require further purification. The deionized water used in this experiment also did not require any purification treatment.

Synthesis of PPy@Ag/rGO nanocomposite
GO was prepared by the modified Hummers method [38]. The pyrrole monomer was stored in a freezer and distilled under high-purity N 2 prior to use. A 1.5 g polyvinylpyrrolidone (PVP) was added into 75 mL ethylene glycol (EG) to stir well, and then, 0.05 g AgNO 3 was added to the above solution. The above mixed solution was thoroughly stirred at room temperature for 24 h to prepare a silver colloid. Then, 35 lL pyrrole monomer (Py) and 0.082 g FeCl 3 were sequentially added and continuously stirred at room temperature for 24 h to obtain PPy@Ag. In this experiment, 0.2 g GO was sonicated in ethylene glycol (20 mL) for 100 min to obtain a peeled GO suspension. Afterward, 20 mL GO suspension was added dropwise to the above reaction system and the solution was continuously stirred for 24 h. Lastly, the obtained solution was moved to a 50-mL Teflon lined stainless steel autoclave and then warmed to 200°C in an oven for 24 h. The obtained PPy@Ag/rGO nano-antibacterial material was separated through centrifugation by being washed alternately three times with absolute ethanol and deionized water, respectively. The resulting sample was dried in a vacuum oven at 60°C for 12 h.

Characterization
Field emission scanning electron microscope (FESEM, JSM 7800F) and transmission electron microscope (TEM, JEM-2100) were used to observe the morphologies. The composition and crystal state of the crystals were determined using an X-ray Diffractometer (XRD-6100Lab, Japan), used Cu-Ka, define a scanning speed of 5°/min, and scanning angles ranging from 5 to 80°. The Fourier transform infrared (FTIR) spectra were recorded in transmission mode from 4000 to 500 cm -1 on a FTIR spectrometer (Nicolet iS50). The X-ray photoelectron spectroscopy (XPS) measurements were performed on an X-ray photoelectron spectrometer system (Escalab 250Xi) with Al Ka radiation as the excitation source. The binding energies were calibrated by referencing the C 1s peak (284.8 eV) to reduce the sample charge effect.
In order to study the active substances such as superoxide radicals (ÁO 2 -) and hydroxyl radicals (ÁOH) which may be generated in the catalytic process, electron spin resonance (ESR, Bruker A300) spectroscopy was introduced for analysis and the radical scavenger was 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The electrochemical impedance (EIS) and transient photocurrent response tests were performed on a CHI 760E electrochemical workstation with 0.5 mol/L Na 2 SO 4 solution as the electrolyte. The concentration of silver ions immersed in LB medium was detected by inductively coupled plasma (VISTA-MPX, Australia). The Zeta potential of aqueous colloidal particles was measured using a Malvern Zetasizer Nano ZS90.

Antibacterial tests with PPy@Ag/rGO nanocomposite
The antibacterial activity of composite materials was researched using Gram-negative bacteria Escherichia coli (E. coli) as a model. Before each antibacterial experiment, all glassware and culture medium must be sterilized in a sterilizer at a temperature of 120°C for 20 min.  ). Afterward, the dehydrated bacteria suspension was dropped onto a silicon wafer and allowed to air dry. After spraying gold, it was observed by SEM at an accelerating voltage of 15 kV. At the same time, the dehydrated bacteria suspension was dropped onto a 200-mesh copper mesh and allowed to air dry, and then observed by TEM. The LB liquid culture medium and the E. coli suspension were placed in a refrigerator and stored at 4°C. Then, 200 lL of the E. coli suspension was put in 50 mL LB liquid culture medium, and 3 mg of the antibacterial material was scattered into the above solution. Afterward, it was swayed at 37°C for 12 h. The detection limit for this method of quantification is 10 cfu/mL as follows [40]: When the A 0 is the cfu of control, A t is the cfu of experimental groups.

Photocatalytic activity evaluation
Under the irradiation of visible light, the broadspectrum antibiotic tetracycline (TC) was selected as 3 Results and discussion 3.1 XRD analysis Figure 1 shows that GO has a sharper peak at 2h = 11.2°, which is related to the graphene oxide (001) diffraction layer. Hydroxyl, epoxy, carbonyl and carboxyl functional groups are introduced on the base and edges of the graphite sheet during the deep oxidation of graphite. Therefore, the interlayer spacing (layer spacing: 0.79 nm) of graphene oxide increases to be much higher than that of graphite (layer spacing: 0.34 nm) [41,42]. The diffraction peaks of GO in the nanocomposites PPy@Ag/rGO and Ag/rGO are weakened or almost disappeared, owing to the GO layer is ultrasonically stripped and hydrothermally reduced [43]. The angles of diffraction peaks of Ag/rGO and PPy@Ag/rGO nanocomposites are at 2h = 37.9°, 44.1°, 64.3°and 77.3°a ssigned to Bragg's reflections from the (111), (200), (220) and (311) planes of nano-silver, which is in agreement (JCPDS No. 00-001-1164) [44,45]. Crystallite size of nanoparticles was calculated by Scherer's formula as shown by equation 2 as follows: where D is the crystallite size (Å ), k (Å ) = 1.54 be the wavelength of Cu Ka radiation, and b is the corrected half width of the diffraction peak. For (111) peak of Ag phase, grain size is found to be approximately 75 nm for the PPy@Ag/rGO nanoparticles [46,47]. It proves that the PPy@Ag/rGO, PPy/rGO and Ag/ rGO nanocomposites have been successfully prepared. Figure 2 shows the FT-IR spectrum of PPy@Ag/rGO nanocomposites. The peaks appearing at 1037 cm -1 and 1091 cm -1 can be attributed to C-C and C-H outof-plane deformation stretching and in-plane vibration. The bands at 1187 cm -1 and 1474 cm -1 are related to stretching of C-N. The band at 1553 cm -1 corresponds to C=C stretching of pyrrole ring. The peak appearing at 1712 cm -1 is ascribed to C=O stretching of the overoxidation of PPy [44,48]. The PPy/rGO nanocomposite shows similar peaks as PPy@Ag nanocomposite to some extent. The changes in peak width and displacement of the bands appearing at 668 cm -1 and 1037 cm -1 are related to the doping state of PPy. This indicates that the PPy@Ag/rGO nanocomposite is successfully synthesized.

Morphology analysis
The morphologies of the Ag/rGO, PPy/rGO and PPy@Ag/rGO nanocomposite are studied by FESEM (field emission scanning electron microscopy) analysis (Fig. 3). Figure 3a shows the FESEM image of PPy/rGO nanocomposite obtained from the proposed hydrothermal method. Both graphene oxide and polypyrrole are in the form of flakes and are linked to each other. The agglomeration phenomenon occurs in the nano-silver spheres on the graphene oxide (Fig. 3b). The nano-silver spheres are deposited on the reduced graphene oxide by PPy overlay as shown in Fig. 3c and d. As can be analyzed from

XPS analysis
XPS was applied to investigate the chemical composition of PPy@Ag/rGO nanocomposite. The XPS spectra clearly reveal that the existence of N, C, O, Ag in PPy@Ag/rGO nanocomposite (Fig. 4a). Figure 4d shows the peaks present at a binding energy of 398.4 eV for neutral benzenoid amine nitrogen (-NH-), 398.9 eV for protonated benzenoid amine nitrogen (N ? ), 396.5 eV for quinonoid imine nitrogen (-N=), and 405.7 eV for nitrate ion (NO 3 1-), respectively [49]. The C 1s can be deconvoluted into 283.3 eV, 284.5 eV and 285.9 eV assigned to C-C, C-N and =C-NH ? , respectively (Fig. 4c). In Fig. 4d, XPS spectra show the peak of Ag and a doublet peak of Ag (3d) arising from spin-orbit coupling (Ag 3d 5/2 at 366.5 eV and Ag 3d 3/2 at 372.5 eV).

Antibacterial activities
The antibacterial activities of these prepared nanocomposites were evaluated by bacterium colony computation in the experiment (Fig. 5). The medium without antibacterial material was regarded as a blank control sample (Fig. 5a). For blank control sample, E. coli bacteria show very good in stark contrast to the experimental groups. In the experimental groups with nanomaterials, fewer E. coli colonies (Fig. 5b-d) grow on medium, showing superior antibacterial activity against E. coli colonies. Among them, PPy@Ag/rGO nanomaterials exhibited the best antibacterial properties, killing almost 99% of E. coli.
In order to study the bacterial morphology changes of the nanocomposite during the antibacterial process, the E. coli affected by the PPy@Ag/rGO nanocomposite was observed by TEM. As shown in Fig. 6a and b, during the sterilization process, a large indicating that the bacterial cell membrane has been destroyed and lost its original barrier function (Fig. 6c, d). Figure 7 shows the experimental results of liquid antibacterial. The liquid antibacterial effect of different antibacterial materials is seen in Fig. 7a. The Ag/ rGO and PPy@Ag/rGO nanocomposites exhibit high antibacterial activity. To explore the effect of PPy on antimicrobial activity, we performed a cycling experiment on antimicrobial materials. From the analysis in Fig. 7b, Ag/rGO and PPy@Ag/rGO nanocomposites show higher antibacterial activity when they are initially used. However, as the number of cycles increases, the antibacterial activity of Ag/ rGO nanomaterials significantly decreases due to the loss of nanosilver. PPy has a stabilizing effect on nano-silver in PPy@Ag/rGO nanocomposites, which  can reduce the loss of nano-silver and thus maintain high antibacterial activity for a long time (Fig. 7c).
In order to study the antibacterial mechanism of the nanocomposite PPy@Ag/rGO, we first used ICP to detect the concentration of silver ions immersed in LB medium during liquid antibacterial processes. As a result, as shown in Table 1, the leakage of silver ions is small. This indicates that the nanocomposite does not inhibit and kill bacteria by leakage of precious metals. In addition, we also performed zeta potential testing on nanocomposites. The experimental results show that the polymer PPy and the nanocomposite PPy@Ag/rGO are positively charged in physiological saline (Fig. 8). At the same time, we performed electron paramagnetic resonance analysis on nanocomposites. The results show that nanomaterials do not produce hydroxyl radicals and superoxide radicals under dark conditions (Fig. 11). Based on the comprehensive test and TEM results analysis, we propose a possible antibacterial mechanism. Polypyrrole in nanocomposites exhibits a positive affinity for electronegative E. coli due to its electropositive properties. Therefore, E. coli will accumulate on polypyrrole. The nano-silver particles dispersed on polypyrrole

Photocatalytic performances
The photocatalytic activity of the nanocomposite was evaluated under visible light by photocatalytic degradation of TC. In this way, the application prospect of composite photocatalyst in environmental water treatment was evaluated (Fig. 9a). The adsorption-desorption balance between the degraded liquid and the sample is achieved prior to the start of the reaction. In these experiments, the photodegradation efficiency of TC on the PPy/rGO nanocomposite is only 37.91%, while the TC removal ratios of Ag/rGO and PPy@Ag/rGO are 73.72% and 80.88%, respectively, which is attributed to the surface plasmon resonance effect of nano-silver. Through the analysis of the results of the cycle experiment, the addition of polymer PPy helps to increase the photocatalytic stabilizing ability of nanomaterials (Fig. 9b).
The transient photocurrent response (Fig. 10a) is used to manifest the exciton separation rates of the as-prepared samples [50]. The photocurrent intensity is significantly enhanced from PPy/rGO to PPy@Ag/ rGO, which verifies the most efficient separation of excitons in PPy@Ag/rGO. Electrochemical impedance spectra (EIS) (Fig. 10b) are also collected to  disclose the charge carriers transfer resistance of the as-prepared samples. It is well known that the smaller radius of EIS plot suggests the lower resistance in charge transport or, in other words, the higher charge transfer rate on the interfaces [51]. PPy@Ag/rGO shows the highest the transport ability of photoinduced charge carriers from inside to surface, consistent with the photocurrent result.
In order to understand the active substances generated during photocatalysis and the mechanism of degradation, the DMPO spin-trapping ESR technique is employed. As seen in Fig. 11, a significant characteristic peak of DMPO-ÁO 2 appears of the PPy@Ag/ rGO sample under visible light irradiation. In contrast, no peaks were produced under dark conditions. Similarly, the four characteristic peaks of DMPO-ÁOH also only show under visible light. Secondly, the  Fig. 13 Schematic diagram of the reaction mechanism of photocatalytic degradation of TC active substance capture experiment was carried out to determine the main active substances in the photocatalytic procedure. The superoxide radicals (ÁO 2 -), holes (h ? ) and hydroxyl radicals (ÁOH) were captured by the addition of the capture scavenger ascorbic acid (VC), triethanolamine (IPA) and isopropanol (TEOA), respectively. The results are shown in Fig. 12, indicating that the three actives ÁO 2 -, h ? and ÁOH play an important role in the photodegradation of TC (Fig. 13).

Conclusion
In a word, an environmentally friendly, simple and easy method has been developed to synthesize PPy@Ag/rGO nanocomposites. PPy is Ag NPs as a stabilizer to prevent loss. At the same time, PPy has a strong affinity for E. coli. PPy@Ag/rGO nanocomposite shows extremely low cytotoxicity and longterm stable and effective antibacterial activity against E. coli. The PPy@Ag/rGO nanocomposite also has a good degradation effect on the antibiotic tetracycline. In short, PPy@Ag/rGO is a promising nanocomposite material.