Graphene Antibacterial Agents Comprising of mono

Subscriber access provided by UNIV OF DURHAM

Biological and Environmental Phenomena at the Interface

Ion-based Metal/Graphene Antibacterial Agents Comprising of mono-Ionic and bi-Ionic Silver and Copper Species Anna Perdikaki, Angeliki Galeou, George Pilatos, Anastasia Prombona, and Georgios N. Karanikolos Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01880 • Publication Date (Web): 26 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Ion-based Metal/Graphene Antibacterial Agents Comprising of mono-Ionic and bi-Ionic Silver and Copper Species Anna Perdikaki1, Angeliki Galeou2, George Pilatos1, Anastasia Prombona2, Georgios N. Karanikolos3,4* 1

Institute of Nanoscience and Nanotechnology (INN), Demokritos National Research Center, Athens, 153 10, Greece 2

Institute of Biosciences and Applications, Demokritos National Research Center, Athens, 153 10, Greece

3

Department of Chemical Engineering, The Petroleum Institute, Khalifa University of Science & Technology, P.O. Box 2533, Abu Dhabi, UAE 4

Center for Membranes and Advanced Water Technology, Khalifa University of Science & Technology, P.O. Box 127788, Abu Dhabi, UAE *

Corresponding author email: [email protected]

Keywords: ions, silver, copper, bi-metallic, graphene, antibacterial, E.coli

Abstract Design of novel and more efficient antibacterial agents is a continuous and dynamic process due to the appearance of new pathogenic strains and inherent resistance development to existing antimicrobial treatments. Metallic nanoparticles (NPs) are highly investigated, yet the role of released ions is crucial in the antibacterial activity of the NP-based systems. We developed herein ion-based, metal/graphene hybrid structures comprising of surface-bound Ag, Cu mono-ionic and Ag/Cu bi-ionic species on functionalized graphene, without involvement of NPs. The antibacterial performance of the resulting systems was evaluated against 1

ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 36

Escherichia coli cells using a series of parametrization experiments of varying metal ion type and concentration, and compared with the respective NP-based systems. It was found that the bi-ionic Ag/Cu-graphene materials exhibited superior performance compared to the mono-ionic analogues owing to the synergistic action of the combination of the two different metal ions on the surface and the enhancing role of the graphene support, while all ionic-based systems performed superiorly compared to their NP-based counterparts of same metal type and concentration. In addition, the materials exhibited sustained action, as their activity was maintained after reuse in repeated cycles employing fresh bacteria in each cycle. The systems developed herein may open new prospects towards development of novel, efficient, and tunable antibacterial agents by properly supporting and configuring metals in ionic form.

Introduction Development of new generations of antimicrobial agents is crucial as pathogenic microorganisms severely impact current health, environmental, and technological activities. Consequently, the importance of metallic species in battling bacteria has been recognized long ago with research efforts mainly focusing on nanoparticle (NP)-based formulations.1-7 However, ions released from the NP surfaces play a key role in the antibacterial activity through direct interaction with the microbial membranes.8-9 The metallic species react with proteins by combining with the -SH groups of enzymes leading to inactivation of the cell proteins and disruption of DNA replication.10 Raffi et al.11 attributed the antibacterial activity of copper NPs to the adhesion of released Cu2+ ions to bacteria because of their opposite electrical charges, eventually resulting in a copper reduction at the bacterial cell wall, while Gunawan et al.12 showed that soluble copper species are responsible for the 2


Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

antimicrobial action, and their efficacy mainly depends on the speciation of such soluble species. The proposed mechanism of action was the cytolysis of cells due to high oxidative stress and the release of cellular materials, resulting in the aggregation of the ruptured cells. Silver and copper ions have also been shown to be efficient disinfectants for treatment of wastewater containing infectious microorganisms.13-21 Other metals such as platinum, gold, nickel, iron oxide, and silicon and its oxides, have also been investigated yet often with less significant bactericidal effect in studies with Escherichia coli (E. coli).15, 22-23 Systems comprising of more than one type of metal NPs are also under investigation, as they often exhibit enhanced performance compared to monometallic analogues. For instance, the enhancing effect of bimetallic species has been reported in Cu-Ag bimetal nanotube-based copper silicates,24 and bimetals and bimetal oxides for removal of contaminants from water.25 Ion release quantification studies have also revealed that Cu/Ag alloys formed on stainless steel by laser cladding exhibited an up to 28-fold increased release of Cu1+ ions compared to the pure elements, resulting in enhanced killing of E.coli bacteria.26 Various materials, such as inorganic (TiO2, SiO2),

27-30

carbon-based,

zeolites and metal organic frameworks (MOFs),33-35 and macromolecules

36-37

31-32

have

been used as supports for metallic species. The combined nanocomposites exhibit enhanced performance in bio-imaging, cancer detection and therapy, and as antibacterial agents.38 MOFs for instance could be used as reservoirs of metal ions, such as silver (Ag+), zinc (Zn2+), copper (Cu+ or Cu2+), or nickel (Ni2+), where the gradual release of the metal ions by biodegradation of the framework structures can provide a sustained antibacterial action.33-35 Graphene is also under intense investigation for development of graphene-based metal composites for various applications.7, 31, 39 Its oxidized form, which is obtained by chemical modification to 3


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 36

introduce hydroxyl, carboxyl, and epoxy functional groups, is more preferable for growth of composites for biological applications, such as antibacterial, drug delivery, and cancer treatment and diagnostics,

40-46

since it is hydrophilic and constitutes an

excellent building block for preparing hybrid materials by nanoparticle attachment on the graphene surface functionalities. Several studies have demonstrated the beneficial effect of graphene when used as support for metallic NPs compared to unsupported NPs.47-48 This effect originates from the fine dispersion of the NPs on the surface49-51 and the graphene/NP interactions that may alter the activity of the involved metallic species. The graphene support can also contribute by enhancing the oxidative stress to the cell membranes.52 In addition, when graphene exists in a tortuous/porous morphology, it can facilitate trapping of the bacteria cells in its framework and enable direct contact with the deposited metallic particles ensuring a locally high concentration of released ions able to induce significant inhibition of bacterial growth.7, 38, 53 In this work, we prepared ionic-based graphene composites comprised of Ag, Cu and bimetallic Ag/Cu ions anchored on the active surface sites of porous oxidized graphene, without the involvement of NPs, towards development of more active yet robust antibacterial systems. The porous graphene support was produced by chemical vapor deposition (CVD) using ferrocene as precursor and subsequently was oxidized to introduce oxygen functional groups as to enable anchoring of the ionic species. We evaluated the antibacterial performance of the resulting systems against E. coli cultures by varying ion type and concentration in both the mono-ionic and bi-ionic forms, and compared them with their NP-based analogues of same metal concentration. We also evaluated the stability of the materials by testing their performance after repeated washing cycles following preparation and before the 4


Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

antibacterial testing. Furthermore, experiments were performed by utilizing the same material in sequential cycles of bacterial exposure as to examine the reusability potential of the developed hybrids.

Experimental Section Graphene growth and functionalization. Sonochemical oxidation was applied to functionalize the graphene support following its growth. Growth was realized by CVD and the procedure is analytically described in our previous report.54 Briefly, it involves sublimation of ferrocene powder at 250 °C in a preheated zone and transfer of the generated vapors into the main CVD zone to enable graphene growth. No other precursor was involved. Reaction pressure and temperature were maintained at 1 bar and 800 °C, respectively, and the duration of growth was 1h. Following growth, oxidation was performed using an acidic solution of 8M HNO3 (65%) and 8M H2SO4 (98%) under sonication and heating at 60 °C for 3h.55 After treatment, the resulting graphene oxide (GO) was recovered by repeated cycles of washing/centrifugation until neutral pH, followed by drying at 80°C. Synthesis of metal ion-graphene hybrids. The ionic silver graphene hybrids were prepared using silver nitrate (AgNO3, 99.8%, Panreac) as metal ion precursor. Firstly, 0.03 g of graphene oxide was dispersed in 11 ml of deionized water by sonication for 10 min. Subsequently, 9 ml of aqueous AgNO3 solution of various concentrations were added and the mixture was kept sonicated for 30 min in order to better disperse the graphene particles and metal ions and enhance their mixing in water. According to literature, reduction is not expected to take place by such mild procedure without additional reducing agents, except if prolonged sonication is applied, e.g. for 48 hours as reported by Li et al.

56

. The resulting ionic Ag - graphene hybrids (AgIONs5


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 36

graphene) were recovered after washing with deionized water. To ensure that only the solvents were removed without any loss of graphitic or metallic species, a rotary evaporator was used in all drying steps. The ionic copper graphene hybrids were prepared using copper (II) chloride (CuCl2, 99%, Sigma-Aldrich) as ion precursor. 0.03 g of graphene oxide, desired amounts of CuCl2, and 20 ml deionized water were mixed together under sonication for 1h. The resulting ionic Cu - graphene hybrids (CuIONs-graphene) were collected after washing with water and drying in a rotary evaporator. The bi-ionic silver/copper graphene hybrids (Ag/CuIONs-graphene) were equimolar with respect to each metal, and were prepared by a two-step procedure, according to which Cu and Ag ions were sequentially formed and attached on the graphene surface based on the aforementioned procedures. To prepare the final hybrid suspensions used in the antibacterial experiments, specific amounts of the dried hybrids were re-dispersed in 2 ml of H2O as to achieve the desired metal concentration. Regarding amounts of added ions, during preparation of the bi-ionic Ag/Cu IONs-graphene hybrids for example, the following amounts of each metal ion were used: For a total metal concentration of the final hybrid suspension that was used in the antibacterial tests of 5x10-3M, 0.54 mg of Ag ions and 0.32 mg of Cu ions were used. For a total metal concentration of 10-4M, 0.01 mg of Ag ions and 0.006 mg of Cu ions were used. 1 ml from each of the above suspensions was added in the bacterial culture to conduct the antibacterial experiments. Synthesis of metal NP-graphene hybrids. For comparison purposes, nanoparticlebased graphene hybrids analogous to the ionic ones were also prepared and tested. AgNP-graphene hybrids were synthesized by reducing the ionic suspension formed as shown above using 4 ml of N,N-dimethylformamide (DMF, 99%, Sigma-Aldrich). The reaction and metal NP growth took place at 60 °C for 6h under stirring.57 The 6


Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

CuNP-grahene hybrids were prepared by exposing the Cu ionic suspension prepared as shown above to 1 ml of potassium borohydride (KBH4, 99.9%, Sigma-Aldrich) aqueous solution at 100°C for 24h.58 Finally, the bimetallic Ag/Cu NP graphene hybrids were prepared in a two-step sequential procedure, according to which Cu and Ag NPs were sequentially formed and attached on the graphene surface based on the aforementioned procedures. The resulting NP hybrid powders were collected after washing with water and drying in a rotary evaporator. Characterization. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were carried out using a FEI Quanta Inspect SEM, and a FEI CM20 TEM operating at 200 kV, respectively. Samples for TEM analysis were prepared by depositing a drop of the graphene suspension on a carbon-coated copper TEM grid. Inductively coupled plasma mass spectrometry (ICP-MS) was performed on an Agilent 7700 ICP-MS spectrometer. Antibacterial activity/Growth inhibition assay. The activity of the developed materials against bacteria was evaluated by a growth inhibition assay, using E. coli (strain DH5α) cells. Liquid bacteria cultures were grown in Luria-Bertani (LB) growth medium (1% Tryptone, 0.5% yeast extract, 1% sodium chloride, pH adjusted to 7.0 with 5 N NaOH). For the solid bacterial growth medium, LB was supplemented with 15.0 g/l agar. The growth inhibition assay was performed as follows: A starting bacterial culture (5 ml liquid culture, grown overnight from a single colony) was diluted 50 times with LB and distributed in glass Erlenmeyer flasks (10 ml/sample). Subsequently, 1 ml of the various sample metal hybrid suspensions was added, and the cultures were allowed to grow for 6h at 37 °C under constant agitation (220 rpm). Every 2h, 1 ml from each sample was used in serial dilutions from 10-1 to 10-6. Subsequently, 100 µl of the last two dilutions were plated on LB-agar plates and 7


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 36

incubated at 37 °C for 16h. The agar plates were then inspected for bacterial growth and the number of colonies was recorded. The CFU/ml (Colony Forming Units/ml) for each sample was plotted vs. time as to depict the bacterial growth. Digital images of all the plates were also captured. A control culture, without any antibacterial hybrid, was tested in every run by the same method for comparison purposes. Two or three independent experiments using the same starting culture were carried out for each data set as to confirm reproducibility. The different data sets were extracted independently using their own respective starting cultures. Finally, the half maximum effective concentration, EC50, which is referred as the metal concentration required in a specific material to achieve a 50% reduction in bacterial growth compared to control sample after a specified exposure time, was also determined. The concentration-effect curves were used for the EC50 calculations through a four-parameter logistic equation using the SigmaPlot Curve Analysis software.59-60

Results and Discussion Morphological and structural characteristics of the produced graphene material used to anchoring the metal ions are shown in Fig. 1. The SEM image of Fig. 1a reveals that the graphene support exhibits a porous morphology formed by the deposition of irregularly shaped and sized particles upon CVD growth, which took place at 800 °C for 1 h using exclusively ferrocene vapors as dual-action carbon and catalyst precursor. The formed tortuous network is preferred as to provide a high surface area and enable fine dispersion of the metallic ions, high ion concentration per unit mass of the material, and enhanced contact with the bacteria cells. The specific surface area of the produced graphene-based support is around 60 m2/g, as calculated by the Brunauer Emmett Teller (BET) method. 54, 61 Fig. 1b is a high-resolution TEM 8


Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

image indicating an average of ~3 layers in the generated graphene, a fact that is further confirmed by Raman analysis (inset in Fig. 1a) that shows a 2D band with a full width at half maximum (FWHM) of 50.7 cm-1 indicating a material that consists of 2-3 graphitic layers.62-63 The good graphitic quality of the support is also revealed by the high intensity and symmetry of the 2D band,54, 64 and the high relative intensity ratios of the main bands, i.e. 1.1 and 2.5 for I2D/IG and IG/ID, respectively. Introduction of surface polar groups on the graphene in high density is crucial as to achieve a high concentration of attached metal ions on the surface. The versatility of graphene to functionalization that can enable introduction of a variety of oxygen functionalities (epoxy, carboxyl, hydroxyl) on the surface renders it a unique material for attachment of NPs and ions compared to other potential solids that could be used as support. We applied sonochemical oxidation using an acidic solution of nitric and sulfuric acids, as described in the experimental section, in order to introduce oxygen

functionalities

on

the

surface.

This

treatment,

as

revealed

by

thermogravimetric analysis (TGA, Fig. S1 of the Supporting Information), introduces a high number of oxygen functionalities onto the surface given the significantly higher weight loss upon TGA thermal treatment for the oxidized graphene compared to the non-oxidized one. The ion-graphene hybrids were resulted by attraction and attachment of the metal ions dispersed in solution on the surface due to electrostatic interactions with the epoxy, hydroxyl, and carboxyl functional groups50,

65

on the

graphene oxide (Fig 1c). A weight loss of around 4 wt%, as per the TGA profile shown in Fig. S1 of the Supporting Information, is attributed to the oxygen functional groups on the surface of the graphene oxide. Taking a surface area of 60 m2/g, as determined by BET analysis, we can see that 1 g of the graphene support contains 0.04 g of oxygen functionalities on a 60 m2 area. This yields a surface density of these 9


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 36

functionalities of around 0.0007 g/m2. Taking an average molecular weight of the three main graphene oxide functionalities (carboxylic (-COOH), carbonyl (-CO), and hydroxylic (-COH)) of 34, then the surface concentration of the functionalities is 2*10-5 mole/m2 or 2*10-23 mole/nm2 or 12 functionalities/nm2. Assuming that each functionality binds one metal ion, then the ion concentration on the surface is approximately 12 metal ions/nm2. It should be noted that without attachment of the metal ions, both the as-grown and the oxidized graphene do not exhibit considerable antibacterial activity. This was revealed by control bacterial experiments without metal loading, which were performed similarly to the experiments with the metal hybrids, namely, by using a constant quantity of 1 ml of aqueous as-grown or oxidized graphene suspension with 5x10-4 M graphene concentration injected into 10 ml of the bacterial culture (Fig. S2 in the Supporting Information). The insignificant antibacterial effect of the graphene support allows us to attribute the observed activity of the developed systems exclusively to the function of the surface-bound metal ionic species. Following attachment of the ions from solution to the active sites of the graphene oxide surface, we proceeded to evaluate the performance of the resulting ion-based composites. The antibacterial effect of the mono-ionic systems was evaluated firstly by inspecting bacterial growth for an incubation period of up to 6 h. Three different metal concentrations of the graphene-based suspensions added to the culture medium were used to test the activity, namely 10−4, 10−3, and 5x10−3 M. Fig. 2a shows the bacterial growth in the presence of AgIONs-graphene and CuIONsgraphene. It is evident from the growth profiles that both Ag- and Cu-based samples suppressed bacterial growth, with the effect being more intense as metal ion concentration in the hybrids increases. It should be noted however that the Ag-based 10


Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

samples exhibited significantly higher activity compared to the Cu-based ones in all metal concentrations tested. Indeed, as revealed by Fig. 2a, while all Cu-based samples tested inhibited bacterial proliferation, thus acting as bacteriostatic agents, the AgIONs-graphene composites with a metal ion concentration of 10-3 M or higher could also be characterized as bactericidal, as they killed all the existing bacteria. Subsequently, the activity of the bi-ionic Ag/Cu hybrids was evaluated and compared with that of the mono-ionic systems of same metal concentration. Three different metal concentrations of the suspensions added to the culture medium were used, comparable to the ones used in the monocationic systems, namely 10−4, 10−3, and 5x10−3 M. The bi-ionic samples consisted of equimolar concentrations of each of the two metallic cations. To confirm that both metal ions are in almost equal concentration on the surface of graphene, we performed a control experiment by involving strong reducing agents as to completely convert all metal ions into particles, i.e. DMF and KBH4 for the reduction of Ag and Cu ions, respectively, and then we analyzed the elemental composition of the resulting particles-graphene structures. EDS analysis corresponding to bimetallic Ag/Cu-graphene indicated a metal content of 3.64 wt% for Ag and 2.25 wt% for Cu, which corresponds to atomic content of 0.45% and 0.47%, respectively, confirming the equimolar metallic composition on the surface. Fig. 2b compares the activity of the mono-ionic and bi-ionic systems with a 10-4 M metal concentration. Interestingly, the antibacterial activity of the bi-ionic hybrid was superior compared to the mono-ionic counterparts of same total metal concentration, an outcome that demonstrates the beneficial effect of bimetallic formulations as compared to monometallic ones. The bi-cationic materials at the 10−3 and 5x10−3 M metal concentration not only resulted in complete bacterial growth inhibition but they were also able to kill the initial E.coli colonies thus performing 11


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 36

bactericidal action. It should be highlighted that the enhanced activity of the bi-ionic hybrids versus the mono-ionic ones exists despite the fact that the bi-ionic materials consist of equimolar amounts of each of the two metals and therefore contain significantly lower Ag concentration compared to the pure Ag-based mono-ionic systems, which were significantly more active than the Cu-based ones. In this set of comparable experiments, the antibacterial activity and the stability of the hybrids for a longer incubation period was also examined in order to investigate whether their activity is reduced over time. For this purpose, the bacterial growth profiles of the mono- and bi-ionic systems were recorded for double the incubation time as compared to the typical duration used throughout this work, i.e. from 6 h to 12 h. Evidently, as shown in Fig. 2b, all metal-containing samples kept the bacterial growth suppressed up to the extended exposure duration indicating that the generated composites can be suitable for applications that require prolonged stability and sustained activity. If we compare the growth curve of the control and the treated samples, it is obvious that, with the same growth medium, the control sample has reached a significantly higher number of colonies than the treated samples. This shows that the treated cultures have in fact the potential for further growth. It should be noted that our data are extracted from measurements of living bacteria (number of colonies) and not from optical density measurements that may include non-living bacteria. Therefore, we can conclude that the growth inhibition of the treated samples is not a result of nutrient deficiency or population pressure, but of the generated hybrids effect. It should also be clarified that each set of experiments, distinguished by presenting them in different plots, has its own starting control sample. That is why for each set of experiments, the control that corresponds to that set is always measured and reported. As one can observe, the controls have also different values 12


Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

from plot to plot depending on initial bacterial colonies. Therefore one cannot compare curves from one plot to the other in absolute values. What is important to compare is the degree of bacterial growth inhibition. For instance, by comparing the 10-4 M CuIONS-graphene sample between Fig 2a and 2b, we observe that at 6 hours, the inhibition in comparison to their respective control sample is 28 3 % in both plots. Taking a second sample, e.g. 10-4 M AgIONS-graphene, the percentage inhibition value is 70 4 % in both plots. This shows that although the experiments have been performed independently using different starting control samples, the antibacterial effect for each ion-based hybrid is reproducible. Nanoparticle-based systems analogous to the ionic ones were subsequently prepared so as to compare the activity of the ionic- versus NP-based graphene composites of same metal concentration, in both monometallic and bimetallic forms. The metal NP-graphene hybrids were realized by in-situ reduction of the graphenestabilized ions converting the surface-bound ions into surface-bound NPs, as described in the experimental section. Characterization data of the bimetallic Ag/CuNP-graphene systems are shown in Fig. S3 of the Supporting Information. Fig. 3a compares the antibacterial effect of the monocationic Ag-, Cu- and bi-cationic Ag/Cu-graphene samples with the analogous monometallic AgNP-, CuNP- and bimetallic Ag/CuNP ones for a 5×10−3M total metal concentration of the hybrid suspension added to the culture medium. It is evident that the order of activity follows the profile of Ag/Cu>Ag>Cu in both NP and ionic hybrids, yet the ionic systems exhibit superior performance compared to their NP-based counterparts in both monometallic and bi-metallic types. It is known that ions play a crucial role in bacterial destruction as they interact directly with the cell membrane and can induce loss of replication abilities to the cells by interaction with thiol groups causing 13


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

inactivation of the bacterial proteins.2,

6, 12, 66

Page 14 of 36

To this extent, the ionic systems

developed here provide well-stabilized ions in high local concentrations that are available to interact with the bacterial cells directly from their surface sites providing immediate and enhanced action. In nanoparticles however, the antibacterial action mainly relies on release of ions from the surface, which typically depends on limitations due to diffusion and ion formation/detachment from the particle surfaces. Indeed, given that ions are responsible for the antibacterial effect through interaction with cell membrane, for same metal amount per unit of support surface, a system consisting of supported ions is anticipated to exhibit faster and higher activity compared to a NP-based system, since in the former case all metal content can be active provided that the ions are well dispersed, while in the latter case, NP metal core is inactive and inaccessible by the bacteria, and only the external surface metal can participate in the antibacterial action. Of course if the NP size becomes very small, then the antibacterial activity of such NP-based system is significantly enhanced, due to high external surface to volume ratio. The enhanced kinetics of antibacterial action of the ionic- versus the NP-based hybrids is also revealed by comparing the half maximum effective concentration, EC50, of the materials, i.e. the metal concentration required to achieve a 50% reduction in bacterial growth. Taking an incubation time of 4 h as a basis, the EC50 values for the CuIONs-graphene and AgIONs-graphene hybrids are 2x10-3M and 0.55x 10-3M, respectively, which are significantly lower than the values of the analogous NP-based systems, i.e. 4.2x10-3 Μ and 1.6x10-3 M for the CuNPs-graphene and AgNPs-graphene hybrids, respectively. This confirms the superior performance of the ion-based systems, since in order to achieve the same antibacterial effect, lower metal concentration is required compared to that required in the NP-based analogues. 14


Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

To examine whether the ionic hybrids contain any free-standing ions that may have remained after the drying procedure used to recover the material, we exposed the as-prepared powder in washing/centrifugation as to remove any free-standing ions and keep only the surface-bound ones. Specifically, the as-prepared material was dispersed in deionized water and washed under sonication. Centrifugation was employed next to recover the powder, and the supernatant containing any freestanding ions was discarded. The above procedure was repeated twice. Subsequently, we evaluated the antibacterial performance of the washed samples. As shown in Fig. 3b, the activity of the washed samples is similar to that of the as-produced ones. In particular, the washed Ag-based hybrid achieves greater cell growth inhibition than the Cu-based one, while the washed bi-ionic Ag/Cu material performs superiorly compared to the mono-ionic ones by not only suppressing cell proliferation but also killing the initial colony cells, i.e. performing bactericidal action. The EC50 concentrations further confirm the above performance. Indeed, for the washed CuIONs-graphene and AgIONs-graphene hybrids, the EC50 values are 2.7x10-3M and 0.59x10-3M, respectively, which are close to those of the as-produced hybrids reported above. The EC50 of the bi-ionic Ag/CuIONs-graphene material is 0.55x10-3M and that of the washed bi-ionic one is 0.56x10-3M. Fig. 3c shows visually the effect of the hybrids by inspecting and comparing the respective photographs from the agar plates containing the cell colonies after a 6-h incubation period for a total metal concentration added to the culture medium of 5x10-3 M. Evidently, the ionic Ag/Cu material performs superiorly compared to all other materials tested, including the NPbased ones, by completely eliminating the bacterial colonies, while its washed analogue performs similar action, confirming that the ions are well-bound to the surface and do not easily migrate to solution after exposure to the culture medium. It 15


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 36

can be therefore emphasized that the oxidized graphene exhibiting high concentration of active anchoring surface sites provides an ideal medium for attachment of a high number of metal ions per unit of surface area yielding a material with a high local concentration of surface-bound ions. Their action is also facilitated by the nanostructured morphology of the used graphene, which consists of aggregated particles forming an extended macro-porosity with high surface area that facilitates contact with the bacterial cells upon mixing. To study the release of ions from the graphene surface, we performed ICP-MS as to quantify the amount of ions released into the medium. The 5x10-3M Ag/CuIONs hybrid was dispersed in the LB medium following the same conditions as in the antibacterial tests (1 ml of the final hybrid dispersion added in 10 ml of LB medium, 37 °C, 220 rpm) for 4 hours. After centrifugation, the supernatant was diluted at a ratio of 1:1000 in deionized water, and 1.3 g of the resulting liquid underwent microwave digestion with 4 ml HNO3 (65%), 1 ml H2O2, and 5 ml H2O. A final dilution to 50 ml H2O took place before the ICP-MS analysis. According to the analysis, the LB sample had a concentration of 2.35 ppm of Ag ions and nondetectable Cu ions concentration. Considering the fact that in an actual antibacterial test involving the above mentioned bi-ionic hybrid the total concentration of each of the two ions after dispersion of the hybrid in the LB medium is 24.5 ppm for Ag and 14.4 ppm for Cu, the amount of Ag ions released after the above 4-h treatment was approximately 10% of the total Ag ions, with non-detectable existence of Cu ions in the medium. This indicates that the majority of ions remain attached on the graphene surface and their release from the surface towards the liquid medium is rather slow and gradual. Release of Cu ions could only take place after extensive washing of the sample. Indeed, ICP analysis of the washed sample prepared according to the washing 16


Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

cycles described above indicated a 4.2 ppm Cu ions concentration in the supernatant. The ICP analysis in the washed sample was performed as follows: The washed 5x103

M Ag/CuIONs hybrid was diluted at a ratio of 1:500 to deionized water. After

centrifugation, 1.53 g of the supernatant underwent analogous microwave digestion and further dilution as for the as-prepared sample. It is evident from the ICP results that Ag ions are more prone to migrating to the liquid medium compared to their Cu counterparts implying a stronger interaction of Cu ions with the graphene surface. For practical applications, in addition to high activity, antibacterial agents should also exhibit durability and sustained action. To examine whether the developed ionic hybrids meet this target, we repeatedly exposed the materials in fresh bacterial cultures as to explore their reusability. Specifically, after a first cycle of antibacterial action using a 6-hour incubation period, the hybrids were exposed to a second cycle, and then to yet another one, each time using fresh bacterial colonies and keeping the incubation time constant at 6 hours for every cycle. After each bacterial exposure cycle and before the next one, the liquid containing the graphene composite was centrifuged for 5 min at 5000 rpm and the supernatant, mainly containing the LB, was discarded as to recover the solid material. Subsequently, 1 ml of sterilized water was added followed by homogenization at a vortex apparatus. In order to ensure that no live bacteria remained after exposure to a given bacterial cycle, a thermal treatment was also performed as to kill any remaining bacteria.67 By executing blank parametric experiments, the optimum conditions for this treatment were found to be 80 °C for 15 min. After executing the thermal treatment, the material was kept under refrigeration overnight in order to be re-used in the next day for a new 6-hour incubation test using fresh bacterial colonies. The results of the reusability experiments are shown in Fig. 4. As revealed by Fig 4a, which corresponds to the bi-ionic Ag/Cu graphene hybrid with 17


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 36

a 5x10-3 M total metal concentration, the material keeps its superior antibacterial activity even after three cycles of repeated exposure to fresh bacteria, achieving not only bacteriostatic but also bactericidal action killing all existing E. coli cells after a 6-hour incubation period in each cycle. Fig. 4b shows a close view of only the profiles corresponding to the material, after removing the curve of the control sample. Indicatively, the performance of the hybrid is almost identical for the first two cycles, and only a minor delay in the bactericidal action at the third cycle is apparent for the first 4 hours of exposure. Following this time, the material recovers its complete bactericidal effect for up to 6 hours of incubation. The inset of Fig. 4b shows corresponding photographs of the agar plates containing the hybrid ionic material after the completion of the 6-hour incubation period for each of the three cycles of exposure. The control sample containing no active material after the same period of incubation is also appended for comparison. Evidently, almost all bacterial colonies are being exterminated in all three cycles, in agreement to the activity curves, confirming that in addition to high activity, fast kinetics of action, and stability, the developed ionic hybrids exhibit also reusability.

Conclusions Metal-graphene composites comprising of metallic ions attached on functionalized porous graphene were prepared and their antibacterial behavior was investigated. The graphene support was produced by CVD using ferrocene as a dualaction catalyst and carbon precursor, exhibited a particulate, tortuous morphology, and was further oxidized to introduce abundant functionalities on the surface in order to enable attachment of the introduced metal ions in high densities. Three types of ions were employed, namely Ag, Cu, and bi-ionic Ag/Cu using various 18


Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

concentrations, and the antibacterial performance of the resulting hybrids was evaluated against E. coli bacterial cultures. It was found that both Ag- and Cu-based ionic systems suppressed bacterial growth, yet the Ag-based ones exhibited significantly higher activity compared to their Cu-based counterparts. Notably, the biionic Ag/CuION-graphene hybrids exhibited superior performance compared to the mono-ionic ones in all metal concentrations tested achieving in many cases also bactericidal action, owing to the synergistic effect of both ionic types on the surface as well as their coordination with the functionalized graphene. Ion release (ICP) studies indicated that the release of ions from the graphene surface is slow and gradual taking place over long exposure to liquid media owing to strong anchoring of the ions with the surface functionalities, while Ag was found to be more prone to migrating to the liquid than Cu. By comparing the developed ionic systems with nanoparticle-based ones of same metal concentration, it was found that the former performed superiorly compared to the latter, in both activity and kinetics of action. In addition, the developed ionic systems were stable, as their performance remained almost unchanged even after washing the material repeatedly before executing the antibacterial testing, indicating that the ions are well attached on the surface functional groups and do not migrate into the liquid phase. Furthermore, the hybrids are reusable as their high activity was maintained after exposing the same material to repeated cycles of bacterial action using fresh bacteria each time. These structures can open new prospects toward development of efficient, robust, and tunable antibacterial agents that are based on ionic species properly configured on engineered nanostructured surfaces.

Acknowledgments 19


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 36

Anna Perdikaki acknowledges the National Center for Scientific Research Demokritos for a Ph.D. Scholarship. We thank Drs. Ιoannis Karatasios, Nikos Boukos, Sergios Papageorgiou, and George Pappas for assistance with material characterization.

References (1)

Bragg, P. D.; Rainnie, D. J. The effect of silver ions on the respiratory chain of

Escherichia coli. Can. J. Microbiol. 1974, 20, 883. (2)

Feng, Q. L.; Wu, J.; Chen, G. Q.; Cui, F. Z.; Kim, T. N.; Kim, J. O. A

mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. Journal of Biomedical Materials Research 2000, 52, 662. (3)

Gogoi, S. K.; Gopinath, P.; Paul, A.; Ramesh, A.; Ghosh, S. S.;

Chattopadhyay, A. Green Fluorescent Protein-Expressing Escherichia coli as a Model System for Investigating the Antimicrobial Activities of Silver Nanoparticles. Langmuir 2006, 22, 9322. (4)

Liau, S. Y.; Read, D. C.; Pugh, W. J.; Furr, J. R.; Russell, A. D. Interaction of

silver nitrate with readily identifiable groups: relationship to the antibacterial action of silver ions. Lett Appl Microbiol 1997, 25, 279. (5)

Choi, O.; Deng, K. K.; Kim, N.-J.; Ross Jr, L.; Surampalli, R. Y.; Hu, Z. The

inhibitory effects of silver nanoparticles, silver ions, and silver chloride colloids on microbial growth. Water Res. 2008, 42, 3066. (6)

Perdikaki, A. V.; Tsitoura, P.; Vermisoglou, E. C.; Kanellopoulos, N. K.;

Karanikolos, G. N. Poly(ethylene oxide)-b-Poly(propylene oxide) Amphiphilic Block

20


Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Copolymer-Mediated Growth of Silver Nanoparticles and Their Antibacterial Behavior. Langmuir 2013, 29, 11479. (7)

Perdikaki, A.; Galeou, A.; Pilatos, G.; Karatasios, I.; Kanellopoulos, N. K.;

Prombona, A.; Karanikolos, G. N. Ag and Cu Monometallic and Ag/Cu Bimetallic Nanoparticle-Graphene Composites with Enhanced Antibacterial Performance. ACS Applied Materials & Interfaces 2016, 8, 27498. (8)

Jose Ruben, M.; Jose Luis, E.; Alejandra, C.; Katherine, H.; Juan, B. K.; Jose

Tapia, R.; Miguel Jose, Y. The Bactericidal Effect of Silver Nanoparticles. Nanotechnology 2005, 16, 2346. (9)

Sondi, I.; Salopek-Sondi, B. Silver Nanoparticles as Antimicrobial Agent: a

Case Study on E. coli as a Β Model for Gram-Negative Bacteria. J. Colloid Interface Sci. 2004, 275, 177. (10)

Jeon, H.-J.; Yi, S.-C.; Oh, S.-G. Preparation and Antibacterial Effects of Ag-

SiO2 Thin Films by Sol-Gel Method. Biomaterials 2003, 24, 4921. (11)

Raffi, M.; Mehrwan, S.; Bhatti, T. M.; Akhter, J. I.; Hameed, A.; Yawar, W.;

ul Hasan, M. M. Investigations into the antibacterial behavior of copper nanoparticles against Escherichia coli. Annals of Microbiology 60, 75. (12)

Gunawan, C.; Teoh, W. Y.; Marquis, C. P.; Amal, R. Cytotoxic Origin of

Copper(II) Oxide Nanoparticles: Comparative Studies with Micron-Sized Particles, Leachate, and Metal Salts. ACS Nano 5, 7214. (13)

Jiang, B.; Tian, C.; Song, G.; Chang, W.; Wang, G.; Wu, Q.; Fu, H. A Novel

Ag/Graphene Composite: Facile Fabrication and Enhanced Antibacterial Properties. J. Mater. Sci. 2013, 48, 1980. (14)

Chernousova, S.; Epple, M. Silver as Antibacterial Agent: Ion, Nanoparticle,

and Metal. Angew. Chem. Int. Ed. 2013, 52, 1636. 21


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(15)

Page 22 of 36

Ruparelia, J. P.; Chatterjee, A. K.; Duttagupta, S. P.; Mukherji, S. Strain

Specificity in Antimicrobial Activity of Silver and Copper Nanoparticles. Acta Biomaterialia 2008, 4, 707. (16)

Rispoli, F.; Angelov, A.; Badia, D.; Kumar, A.; Seal, S.; Shah, V.

Understanding the Toxicity of Aggregated Zero Valent Copper Nanoparticles against Escherichia coli. J. Hazard. Mater. 2010, 180, 212. (17)

Chatterjee, A. K.; Sarkar, R. K.; Chattopadhyay, A. P.; Aich, P.; Chakraborty,

R.; Basu, T. A Simple Robust Method for Synthesis of Metallic Copper Nanoparticles of High Antibacterial Potency against E. coli. Nanotechnology 2012, 23, 085103. (18)

Cioffi, N.; Torsi, L.; Ditaranto, N.; Tantillo, G.; Ghibelli, L.; Sabbatini, L.;

Bleve-Zacheo, T.; D'Alessio, M.; Zambonin, P. G.; Traversa, E. Copper Nanoparticle/Polymer Composites with Antifungal and Bacteriostatic Properties. Chem. Mater. 2005, 17, 5255. (19)

Chaloupka, K.; Malam, Y.; Seifalian, A. M. Nanosilver as a New Generation

of Nanoproduct in Biomedical Applications. Trends Biotechnol. 2010, 28, 580. (20)

Xiu, Z.-m.; Zhang, Q.-b.; Puppala, H. L.; Colvin, V. L.; Alvarez, P. J. J.

Negligible Particle-Specific Antibacterial Activity of Silver Nanoparticles. Nano Lett. 2012, 12, 4271. (21)

Pal, S.; Yoon, E. J.; Tak, Y. K.; Choi, E. C.; Song, J. M. Synthesis of Highly

Antibacterial Nanocrystalline Trivalent Silver Polydiguanide. J. Am. Chem. Soc. 2009, 131, 16147. (22)

Cho, K.-H.; Park, J.-E.; Osaka, T.; Park, S.-G. The study of antimicrobial

activity and preservative effects of nanosilver ingredient. Electrochim. Acta 2005, 51, 956.

22


Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(23)

Williams, D. N.; Ehrman, S. H.; Pulliam Holoman, T. R. Evaluation of the

microbial growth response to inorganic nanoparticles. Journal of Nanobiotechnology 2006, 4, 3. (24)

Fang, W.; Chaofa, X.; Zheng, J.; Chen, G.; Jiang, K. Fabrication of Cu-Ag

Bimetal Nanotube-Based Copper Silicates for Enhancement of Antibacterial Activities. RSC Advances 2015, 5, 39612. (25)

Fu, F.; Cheng, Z.; Lu, J. Synthesis and Use of Bimetals and Bimetal Oxides in

Contaminants Removal from Water: a Review. RSC Advances 2015, 5, 85395. (26)

Hans, M.; Támara, J. C.; Mathews, S.; Bax, B.; Hegetschweiler, A.;

Kautenburger, R.; Solioz, M.; Mücklich, F. Laser Cladding of Stainless Steel with a Copper–Silver Alloy to Generate Surfaces of High Antimicrobial Activity. Appl. Surf. Sci. 2014, 320, 195. (27)

Sotiriou, G. A.; Meyer, A.; Knijnenburg, J. T. N.; Panke, S.; Pratsinis, S. E.

Quantifying the Origin of Released Ag+ Ions from Nanosilver. Langmuir 28, 15929. (28)

Sotiriou, G. A.; Pratsinis, S. E. Antibacterial Activity of Nanosilver Ions and

Particles. Environmental Science & Technology 44, 5649. (29)

Cai, Y.; Tan, F.; Qiao, X.; Wang, W.; Chen, J.; Qiu, X. Room-temperature

synthesis of silica supported silver nanoparticles in basic ethanol solution and their antibacterial activity. RSC Advances 6, 18407. (30)

Hou, X.; Ma, H.; Liu, F.; Deng, J.; Ai, Y.; Zhao, X.; Mao, D.; Li, D.; Liao, B.

Synthesis of Ag ion-implanted TiO2 thin films for antibacterial application and photocatalytic performance. J Hazard Mater 2016, 299, 59. (31)

Xu, Z.; Gao, H.; Guoxin, H. Solution-based Synthesis and Characterization of

a Silver Nanoparticle-Graphene Hybrid Film. Carbon 2011, 49, 4731.

23


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(32)

Page 24 of 36

Xu, S.; Man, B.; Jiang, S.; Wang, J.; Wei, J.; Xu, S.; Liu, H.; Gao, S.; Liu, H.;

Li, Z., et al. Graphene/Cu Nanoparticle Hybrids Fabricated by Chemical Vapor Deposition As Surface-Enhanced Raman Scattering Substrate for Label-Free Detection of Adenosine. ACS Appl. Mater. Interfaces 2015, 7, 10977. (33)

Berchel, M.; Gall, T. L.; Denis, C.; Hir, S. L.; Quentel, F.; Elleouet, C.;

Montier, T.; Rueff, J.-M.; Salaun, J.-Y.; Haelters, J.-P., et al. A silver-based metalorganic framework material as a 'reservoir' of bactericidal metal ions. New J. Chem. 35, 1000. (34)

Lu, X.; Ye, J.; Zhang, D.; Xie, R.; Bogale, R. F.; Sun, Y.; Zhao, L.; Zhao, Q.;

Ning, G. Silver carboxylate metal-organic frameworks with highly antibacterial activity and biocompatibility. J. Inorg. Biochem. 138, 114. (35)

Aguado, S.; Quiros, J.; Canivet, J.; Farrusseng, D.; Boltes, K.; Rosal, R.

Antimicrobial activity of cobalt imidazolate metal-organic frameworks. Chemosphere 113, 188. (36)

Li, W.; Zhou, S.; Gao, S.; Chen, S.; Huang, M.; Cao, R. C. Spatioselective

Fabrication of Highly Effective Antibacterial Layer by Surface-Anchored Discrete Metal–Organic Frameworks. Advanced Materials Interfaces 2, 1400405. (37)

Wyszogrodzka, G.; MarszaΕ‚ek, B.; Gil, B.; DoroΕΌyΕ„ski, P. a. Metal-

organic frameworks: mechanisms of antibacterial action and potential applications. Drug Discovery Today 21, 1009. (38)

Ji, H.; Sun, H.; Qu, X. Antibacterial applications of graphene-based

nanomaterials: Recent achievements and challenges. Advanced Drug Delivery Reviews 105, Part B, 176.

24


Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(39)

Wang, C.; Li, J.; Amatore, C.; Chen, Y.; Jiang, H.; Wang, X.-M. Gold

Nanoclusters and Graphene Nanocomposites for Drug Delivery and Imaging of Cancer Cells. Angew. Chem. Int. Ed. 2011, 50, 11644. (40)

Park, S.; Ruoff, R. S. Chemical Methods for the Production of Graphenes. Nat

Nano 2009, 4, 217. (41)

Hu, W.; Peng, C.; Luo, W.; Lv, M.; Li, X.; Li, D.; Huang, Q.; Fan, C.

Graphene-Based Antibacterial Paper. ACS Nano 2010, 4, 4317. (42)

Akhavan, O.; Ghaderi, E. Toxicity of Graphene and Graphene Oxide

Nanowalls against Bacteria. ACS Nano 2010, 4, 5731. (43)

Vermisoglou, E. C.; Pilatos, G.; Romanos, G. E.; Devlin, E.; Kanellopoulos,

N. K.; Karanikolos, G. N. Magnetic carbon nanotubes with particle-free surfaces and high drug loading capacity. Nanotechnology 2011, 22. (44)

Geetha Bai, R.; Muthoosamy, K.; Shipton, F. N.; Pandikumar, A.;

Rameshkumar, P.; Huang, N. M.; Manickam, S. The biogenic synthesis of a reduced graphene oxide-silver (RGO-Ag) nanocomposite and its dual applications as an antibacterial agent and cancer biomarker sensor. RSC Advances 2016, 6, 36576. (45)

Muthoosamy, K.; G. Bai, R.; Manickam, S. Graphene and Graphene Oxide as

a Docking Station for Modern Drug Delivery System. Current Drug Delivery 2014, 11, 701. (46)

Geetha Bai, R.; Muthoosamy, K.; Zhou, M.; Ashokkumar, M.; Huang, N. M.;

Manickam, S. Sonochemical and sustainable synthesis of graphene-gold (G-Au) nanocomposites for enzymeless and selective electrochemical detection of nitric oxide. Biosens. Bioelectron. 2017, 87, 622.

25


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(47)

Page 26 of 36

Shao, W.; Liu, X.; Min, H.; Dong, G.; Feng, Q.; Zuo, S. Preparation,

Characterization, and Antibacterial Activity of Silver Nanoparticle-Decorated Graphene Oxide Nanocomposite. ACS Applied Materials & Interfaces 2015, 7, 6966. (48)

Zhang, P.; Wang, H.; Zhang, X.; Xu, W.; Li, Y.; Li, Q.; Wei, G.; Su, Z.

Graphene film doped with silver nanoparticles: self-assembly formation, structural characterizations, antibacterial ability, and biocompatibility. Biomaterials Science 3, 852. (49)

Ma, J.; Zhang, J.; Xiong, Z.; Yong, Y.; Zhao, X. S. Preparation,

Characterization and Antibacterial Properties of Silver-Modified Graphene Oxide. J. Mater. Chem. 2011, 21, 3350. (50)

Shen, J.; Shi, M.; Li, N.; Yan, B.; Ma, H.; Hu, Y.; Ye, M. Facile Synthesis and

Application of Ag-Chemically Converted Graphene Nanocomposite. Nano Research 2010, 3, 339. (51)

Xu, W.-P.; Zhang, L.-C.; Li, J.-P.; Lu, Y.; Li, H.-H.; Ma, Y.-N.; Wang, W.-D.;

Yu, S.-H. Facile Synthesis of Silver@Graphene Oxide Nanocomposites and Their Enhanced Antibacterial Properties. J. Mater. Chem. 2011, 21, 4593. (52)

Liu, S.; Zeng, T. H.; Hofmann, M.; Burcombe, E.; Wei, J.; Jiang, R.; Kong, J.;

Chen, Y. Antibacterial Activity of Graphite, Graphite Oxide, Graphene Oxide, and Reduced Graphene Oxide: Membrane and Oxidative Stress. ACS Nano 2011, 5, 6971. (53)

Chen, J.; Peng, H.; Wang, X.; Shao, F.; Yuan, Z.; Han, H. Graphene oxide

exhibits broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal conidia by intertwining and membrane perturbation. Nanoscale 6, 1879. (54)

Pilatos, G.; Perdikaki, A. V.; Sapalidis, A.; Pappas, G. S.; Giannakopoulou,

T.; Tsoutsou, D.; Xenogiannopoulou, E.; Boukos, N.; Dimoulas, A.; Trapalis, C., et

26


Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

al. Graphene by One-Step Chemical Vapor Deposition from Ferrocene Vapors: Properties and Electrochemical Evaluation. J. Appl. Phys. 2016, 119, 064303. (55)

Xing, Y. Synthesis and Electrochemical Characterization of Uniformly-

Dispersed High Loading Pt Nanoparticles on Sonochemically-Treated Carbon Nanotubes. J. Phys. Chem. B 2004, 108, 19255. (56)

Li, C.; Wang, X.; Chen, F.; Zhang, C.; Zhi, X.; Wang, K.; Cui, D. The

antifungal activity of graphene oxide–silver nanocomposites. Biomaterials 2013, 34, 3882. (57)

Yang, Y.-K.; He, C.-E.; He, W.-J.; Yu, L.-J.; Peng, R.-G.; Xie, X.-L.; Wang,

X.-B.; Mai, Y.-W. Reduction of Silver Nanoparticles onto Graphene Oxide Nanosheets with N,N-Dimethylformamide and SERS Activities of GO/Ag Composites. J. Nanopart. Res. 2011, 13, 5571. (58)

Zhang, K. Fabrication of Copper Nanoparticles/Graphene Oxide Composites

for Surface-Enhanced Raman Scattering. Appl. Surf. Sci. 2012, 258, 7327. (59)

Navarro, E.; Piccapietra, F.; Wagner, B.; Marconi, F.; Kaegi, R.; Odzak, N.;

Sigg, L.; Behra, R. Toxicity of Silver Nanoparticles to Chlamydomonas reinhardtii. Environmental Science & Technology 2008, 42, 8959. (60)

Mortimer, M.; Kasemets, K.; Kahru, A. Toxicity of ZnO and CuO

Nanoparticles to Ciliated Protozoa Tetrahymena thermophila. Toxicology 2010, 269, 182. (61)

Pilatos, G.; Vermisoglou, E. C.; Perdikaki, A.; Devlin, E.; Pappas, G. S.;

Romanos, G. E.; Boukos, N.; Giannakopoulou, T.; Trapalis, C.; Kanellopoulos, N. K., et al. One-Step, in Situ Growth of Unmodified Graphene - Magnetic Nanostructured Composites. Carbon 2014, 66, 467.

27


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(62)

Page 28 of 36

Hao, Y.; Wang, Y.; Wang, L.; Ni, Z.; Wang, Z.; Wang, R.; Koo, C. K.; Shen,

Z.; Thong, J. T. L. Probing Layer Number and Stacking Order of Few-Layer Graphene by Raman Spectroscopy. Small 2010, 6, 195. (63)

Graf, D.; Molitor, F.; Ensslin, K.; Stampfer, C.; Jungen, A.; Hierold, C.;

Wirtz, L. Spatially Resolved Raman Spectroscopy of Single- and Few-Layer Graphene. Nano Lett. 2007, 7, 238. (64)

Pimenta, M. A.; Dresselhaus, G.; Dresselhaus, M. S.; Cancado, L. G.; Jorio,

A.; Saito, R. Studying Disorder in Graphite-Based Systems by Raman Spectroscopy. PCCP 2007, 9, 1276. (65)

Goncalves, G.; Marques, P. A. A. P.; Granadeiro, C. M.; Nogueira, H. I. S.;

Singh, M. K.; Gracio, J. Surface Modification of Graphene Nanosheets with Gold Nanoparticles: The Role of Oxygen Moieties at Graphene Surface on Gold Nucleation and Growth. Chem. Mater. 2009, 21, 4796. (66)

Greulich, C.; Braun, D.; Peetsch, A.; Diendorf, J.; Siebers, B.; Epple, M.;

Köller, M. The toxic effect of silver ions and silver nanoparticles towards bacteria and human cells occurs in the same concentration range. RSC Advances 2012, 2, 6981. (67)

Stringer, S. C.; George, S. M.; Peck, M. W. Thermal inactivation of

Escherichia coli O157:H7. Journal of Applied Microbiology 2000, 88, 79S.

28


Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure Captions: Figure 1. (a) SEM image of the ferrocene-derived graphene support exhibiting a porous-like morphology. Inset is the Raman spectrum of the as-grown graphene after CVD. (b) High-resolution TEM image showing 3-4 graphene layers. (c) Schematic representation of the production of bi-ionic graphene hybrids comprising of Ag and Cu ions attached on the graphene oxide anchoring surface functionalities. Figure 2. (a) Antibacterial activity of the mono-ionic AgIONs-graphene and CuIONsgraphene hybrids using different metal concentrations.

(b) Comparison of the

bacteriostatic activity of the bi-ionic Ag/CuIONs-graphene hybrids versus the monoionic ones for a total metal ion concentration of 10−4 M and for an extended incubation period of 12 hours. Figure 3. (a) Comparison of the antibacterial activity of the ion-based graphene hybrids with that of nanoparticle-based analogues of same metal concentration (5x10-3 M) in both monometallic and bimetallic formulations. (b) Antibacterial activity of the mono-ionic and bi-ionic systems following repeated washing employed to remove any free-standing ions revealing the stability of the hybrids. (c) Photographs of the agar plates showing the bacterial colonies after a 6-hour incubation period in the presence of both NP- and ion-based materials. The superior antibacterial action of the bi-ionic Ag/Cu hybrid is evident even after exposing it to extensive washing prior to the bacterial testing. Figure 4. (a) Bactericidal action of the bio-ionic Ag/CuION-graphene hybrid with a total metal concentration of 5x10-3 M after three cycles of exposure to fresh bacteria cultures revealing constantly high activity. (b) Close-view of the antibacterial profiles corresponding to the Ag/CuION-graphene hybrid. The inset shows corresponding

29


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 36

photographs of the agar plates containing the hybrid ionic material after the completion of the 6-hour incubation period for each of the three cycles of exposure.

30


Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figures:

(a)

(b)

10 nm

(c)

Figure 1.

31


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

(a)

(b)

Figure 2.

32


Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(a)

(b)

33


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 36

(c)

control

5x10-3 Μ, Ag/CuNPs-graphene, equimolar

5x10-3 Μ, CuNPs-graphene

5x10-3 Μ, ΑgNPs-graphene

5x10-3 Μ, Ag/CuIONs-graphene, equimolar

5x10-3 Μ, Ag/CuIONs-graphene, equimolar, washed

Figure 3.

34


Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(a)

(b)

control

Ag/CuIONsgraphene 1st cycle

Ag/CuIONsgraphene 2nd cycle

Ag/CuIONsgraphene 3rd cycle

Figure 4.

35


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 36

TOC Graphic:

36


Graphene Antibacterial Agents Comprising of mono

Recommend Documents