Wrapping Bacteria by Graphene Nanosheets for Isolation from ...

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Wrapping Bacteria by Graphene Nanosheets for Isolation from Environment, Reactivation by Sonication, and Inactivation by Near-Infrared Irradiation O. Akhavan,*,†,‡ E. Ghaderi,† and A. Esfandiar‡ † ‡

Department of Physics, Sharif University of Technology, P. O. Box 111559161, Tehran, Iran Institute for Nanoscience and Nanotechnology, Sharif University of Technology, P.O. Box 1458889694, Tehran, Iran ABSTRACT: Bioactivity of Escherichia coli bacteria (as a simple model for microorganisms) and interaction of them with the environment were controlled by their capturing within aggregated graphene nanosheets. The oxygen-containing functional groups of chemically exfoliated single-layer graphene oxide nanosheets were reduced by melatonin as a biocompatible antioxidant. While each one of the graphene (oxide) suspension and melatonin solution did not separately show any considerable inactivation effects on the bacteria, aggregation of the sheets in the melatoninbacterial suspension resulted in trapping the bacteria within the aggregated sheets, i.e., a kind of inactivation. The bacteria trapped within the aggregated sheets were biologically disconnected from their environment, because they could not proliferate in a culture medium and consume the glucose of their environment. However, after removing the sheets from the surface of the microorganisms by using sonication, they could again interact with their environment. The reactivated bacteria consumed glucose and could be proliferated; i.e., they were alive within the aggregated graphene sheets (here, at least for 24 h). The trapped alive bacteria could be photothermally inactivated forever by near-infrared irradiation at 808 nm. These results suggest that graphene nanosheets may potentially serve as an encapsulating material for delivery of such microorganisms and as an effective photothermal agent for inactivation of the graphenewrapped microorganisms.

1. INTRODUCTION Graphene as a one atom thick sheet constructed by sp2bonded carbon atoms in a closely packed honeycomb lattice with unique and promising characteristics has attracted much attention, particularly after its realization in 2004.1 Indeed, it has been used as a fascinating nanomaterial in a variety of fields such as condensed-matter and high-energy physics,24 electronics,58 material science,912 and a broad range of technological applications.1319 There is also an increasing attention to application of graphene in the fields of bioelectronics, biosensing, and biology. For example, it was applied in biosensors,2023 glucose sensors,2429 single bacterium sensor and DNA transistor,30 sensitive immunosensor for cancer biomarker,31 and antimicrobial purposes.3234 So far, the following current methods have been proposed for preparation of graphene nanosheets: micromechanical cleavage of graphite,13,35,36 chemical vapor deposition,37,38 epitaxial growth,3941 cutting carbon nanotubes,42,43 direct sonication44,45 and chemical exfoliation from bulk graphite.4656 Among them, the chemical exfoliation method has been widely utilized as a favorable, effective, reliable, large-scale production and low-cost method. In addition, the chemically exfoliated graphene oxide nanosheets are versatile in producing functionalized graphene nanosheets.57,58 However, the chemically exfoliated graphene oxide nanosheets require a subsequent reduction to convert into graphene r 2011 American Chemical Society

nanosheets. So far, the graphene oxide nanosheets have been reduced by chemical reductants such as hydrazine and sodium borohydride (see, for example, refs 59 and 60), heat treatment in a reducing environment,61,62 microwave irradiation,63 flash photoreduction,64 catalytic,65 and photocatalytic32,6669 processes. However, because of the high toxicity of chemical reductants such as hydrazine and the high temperatures required in the thermal reductions (>500 °C), the most current chemical and thermal reducing processes cannot be extensively used in chemistry, biology, and electronic applications. There are attempts to find environmentally friendly reduction methods to produce graphene nanosheets. For instance, Zhou et al. studied a “green” reduction of graphite oxide into graphene nanosheets using hydrothermal dehydration.70 Recently, reduction of graphene oxide nanosheets by vitamin C as a biocompatible and strong antioxidant60,71 and also by bacterial respiration72 was reported. In 1993, it was discovered that melatonin can act as a strong antioxidant,73 in addition to its function as synchronizer of the biological clock. It is known as an antioxidant that can easily cross cell membranes and the bloodbrain barrier.74 Melatonin is a Received: January 22, 2011 Revised: March 14, 2011 Published: April 22, 2011 6279

dx.doi.org/10.1021/jp200686k | J. Phys. Chem. B 2011, 115, 6279–6288

The Journal of Physical Chemistry B scavenger of reactive oxygen/nitrogen species, due to its capacity to act as an electron donor.7578 Unlike conventional antioxidants, melatonin has been referred to as a terminal antioxidant, because it does not undergo a redox cycling.79 In fact, redox cycling in conventional antioxidants (e.g., vitamin C) can result in a prooxidation. But, once melatonin has oxidized, it cannot be reduced to its initial state because it forms some ireversible end products under reaction with free radicals. Graphene nanaosheets tend to irreversibly aggregate or even restack to form graphite through strong ππ stacking and van der Waals interaction.80 On the other hand, it was recently demonstrated that a monolayer graphene sheets is impermeable to even He.81 In fact, graphene can act as the world’s thinnest membrane to provide a unique separation barrier between two distinct regions with only one atom thick separation.81 Therefore, a graphene sheet, as a biocompatible material,82,83 may be also applied in wrapping microorganisms to separate and/or protect them from the environment or vice versa. Melatonin as a powerful antioxidant can be utilized for biocompatible reduction of graphene oxide sheets and, so, their aggregation in a suspension containing microorganisms. The aggregating graphene sheets can trap the microorganisms within them to isolate them from the suspension. On the other hand, biological systems such as microorganisms are known to be highly transparent to 7001100 nm nearinfrared (near-IR) light irradiation.84 It was previously shown that the strong near-IR absorbance of single-wall carbon nanotubes (SWCNTs)85 can cause cell death due to excessive local heating of the stimulated SWCNTs in vitro. This intrinsic property of SWNTs resulted in application of functionalized SWCNTs in selective and photothermal destructions of cancer cells.86,87 Graphene sheets can also absorb the near-IR light irradiation (see, for example, refs 69 and 88). Hence, the microorganisms wrapped by graphene nanosheets may loss their high transparency to the near-IR irradiation and, so, may be photothermally killed within the closed and the absorbent environment caused by the aggregated graphene sheets. In this work, the effect of melatonin on reduction of the oxygen-containing functional groups of the chemically exfoliated graphene oxide nanosheets was investigated by X-ray photoelectron spectroscopy (XPS). Trapping Escherichia coli (E. coli) bacteria, as a simple model for microorganisms, within the aggregated graphene sheets was examined by atomic force microscopy (AFM) and Raman spectroscopy. Two kinds of the as-prepared graphene oxide suspension with the oxygen-containing functional groups and homogeneous graphene sheetmelatonin suspension with the reduced functional groups were applied to study the trapping the bacteria within the aggregated reduced sheets at different concentrations of graphene (oxide) sheets in the bacterial suspension. To study the biological disconnection of the bacteria trapped within the aggregated graphene sheets from the environment, proliferation and glucose consumption of the trapped bacteria were investigated. Furthermore, the possibility of the bacteria surviving within the aggregated graphene sheets and their reactivation after removing the graphene sheets was examined. The influence of nearIR irradiation on the bioactivity of the trapped bacteria was also investigated.

2. EXPERIMENTAL SECTION Preparation of Graphene Oxide Nanosheets. The modified Hummers procedure89,90 was used to oxidize natural graphite powders (particle diameter of 45 μm, Sigma-Aldrich). At first,

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50 mL of H2SO4 was added into a beaker including 2 g of graphite at room temperature. Then, the temperature of the beaker decreased to 0 °C by using an ice bath. After that, 6 g of potassium permanganate (KMnO4) was slowly added to the above mixture while it gradually warmed to room temperature. The suspension was stirred for 2 h at 35 °C. After cooling the suspension in an ice bath, it was diluted by 350 mL of deionized (DI) water. Then, H2O2 (30%) was added until the gas evolution ceased in order to be sure about reduction of residual permanganate to soluble manganese ions. Finally, the resulting suspension was filtered, washed with 1 M HCl and several times with DI water, and dried at 60 °C for 24 h to obtain brownish graphite oxide powders. Different amounts of the obtained graphite oxide powder were dispersed in DI water and sonicated for 30 min to obtain graphene oxide nanosheet (GOS) suspensions with the different concentrations (from 0.05 to 5 mg/mL). Reduction of the GOSs in MelatoninBacterial Suspension. To study the interaction between the reduced GOSs and a microorganism, E. coli (ATCC 25922) bacterium was selected as a model of the Gram-negative bacteria. Before the microbiological test, all glassware and samples were sterilized by autoclaving at 120 °C for 15 min. The prepared GOS suspension (20 mL) was added to 20 mL nutrient broth inoculated by about 106 colony forming units (CFU)/(mL of the bacteria). The medium used for bacterial activity was a LuriaBertani (LB) culture medium containing (amounts in milligrams per milliliter) the following: peptone, 10; NaCl, 10; and yeast extract, 5 (developed by glucose (D-glucose monohydrated), 20; KH2PO4, 7; K2HPO4, 3; (NH4)2SO4, 1; sodium citrate, 0.5; and MgSO4 3 7 H2O, 0.1). The pH of the medium was also adjusted in the range of pH 6.97.1 using NaOH. Then, 150 mg of N-acetyl-5-methoxytryptamine (melatonin (C13H16N2O2), Merck, with purity of g98%) in 10 mL of DI water was added to the GOSbacterial suspension to reduce the graphene oxide sheets. After that, the suspension was incubated at 37 °C and irradiated by a 100 W tungsten lamp for 96 h. The temperature of the suspension was controlled through contacting the beaker of the suspension to a water bath. The reduction process resulted in aggregation of the reduced GOSs in the melatoninbacterial (GOSmelatonin bacterial) suspension. pH of the GOSmelatoninbacterial suspensions decreased from 6.5 to 5.3 by decreasing the concentration of the GOSs from 5 to 0.01 mg/mL. In addition, we found that by increasing the pH of the GOSmelatonin suspension to about pH 9, a homogeneous graphene sheet melatonin (GSmelatonin) suspension (without aggregation of the reduced sheets) can be obtained. A portion (20 mL) of the prepared bacterial suspension was also added to 20 mL of the GSmelatonin suspension. By adding 10 mL of the melatonin solution to the GSmelatonin suspension, the pH of the suspensions decreased to about pH 6.3, which resulted in aggregation of the GSs in the initially homogeneous suspension. After elapsing 24 h in the dark, a portion of all of the reduced suspensions was sonicated for 5 min in each 12 h interval to detach the aggregated graphene sheets from the bacteria. Longer sonications were not used to avoid the probable denaturing of the enzyme of the bacteria. After 48 h, the bacterial activity and glucose concentration of the sonicated suspensions were measured and compared with those of the unsonicated ones. To study the bacterial activity of the various prepared suspensions, 100 μL of each suspension was spread on a LB agar plate and incubated at 37 °C for 24 h for counting the active bacterial colonies using an optical microscope. The total number of the 6280

dx.doi.org/10.1021/jp200686k |J. Phys. Chem. B 2011, 115, 6279–6288

The Journal of Physical Chemistry B cells forming unit were determined by area-based estimation. The reported data were the average value of three separate similar runs. Glucose consumption of the bacteria (through the glycolysis process of the bacteria in the culture medium) was also measured by a glucose meter (ACCU-CHEK Aviva). Effect of Near-IR Irradiation on the GrapheneMelatonin Bacterial Suspensions. After the 96 h reduction process mentioned above, the GOSmelatoninbacterial and GSmelatoninbacterial suspensions with GOS concentration of 5 mg/mL were irradiated by an 808 nm diode laser (JENOPTIK uniquemode GmbH, Germany) with the beam diameter of about 1 cm at 7.5 W/cm2 for various irradiation times. During the near-IR irradiation, the temperature of the suspensions was controlled around 39 °C (with (2 °C precision) through contacting the beaker of the suspensions to a water bath. Similar to the procedure described above, the irradiated suspensions were sonicated and their bacterial activity was measured. Material Characterization. AFM images of the GOSs and the reduced GOSs containing the trapped bacteria were obtained by a Nanoscope III Multimode (VEECO) in tapping mode. The graphene thin films for AFM imaging were prepared by dropcasting the GOS suspension with a concentration of 0.5 mg/mL onto cleaned SiO2/Si substrates which were then allowed to dry in air. XPS was employed to investigate the changes that occurred in the chemical states of the GOSs by melatonin. The graphene thin films for this purpose were prepared by the suspensions with 5 mg/mL GOS concentration. The data were acquired by using a hemispherical analyzer equipped with a monochromatic Al KR X-ray source (hν = 1486.6 eV) operating at a vacuum better than 107 Pa. The XPS peaks were deconvoluted by using Gaussian components after a Shirley background subtraction. Raman spectroscopy of the latter thin films (prepared by the suspensions with 5 mg/mL GOSs) was obtained at room temperature using a Raman Microprobe (HR-800 Jobin-Yvon) with 532 nm Nd YAG excitation source to examine the carbon structure and the single-layer and/or multilayer characteristics of the as-prepared GOSs and the GOSs reduced in the various suspensions. A Jasco V530 UVvisiblenear-IR spectrophotometer was utilized to determine the optical absorption of the suspensions in the wavelength range of 3001100 nm.

3. RESULTS AND DISCUSSION To characterize the topography of the graphene oxide nanosheets and the reduced graphene sheets which captured the bacteria, AFM was utilized as an effective method, as shown in Figure 1. An AFM image of the graphene oxide nanosheets deposited on the surface of the SiO2/Si(100) substrate has been presented in Figure 1a. The surface of the SiO2/Si(100) substrate was smooth with root-mean-square surface roughness of about 0.5 nm. On such a smooth substrate, the surface of the sheets also showed a relatively smooth planar structure. The height profile diagram of the AFM image showed that the thickness of the nanosheets was about 0.8 nm, which is in good consistency with the typical thickness of the single-layer graphene oxide sheets.48,49 The overlapping edges of the nanosheets can be also seen in the image. Figure 1b shows the bacteria trapped among the aggregated graphene oxide nanosheets after reduction by the melatonin solution. The agglomerated graphene sheets are clearly seen in the image. To better confirm trapping of the bacteria within the aggregated graphene oxide sheets reduced by the melatonin solution, a 1 μm 1 μm close-up

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Figure 1. AFM images of (a) the graphene oxide sheets on the SiO2/ Si(100) substrate and (b) the bacteria trapped between the graphene oxide sheets aggregated together after reduction by melatonin. Covering one of the bacteria by the aggregated graphene sheets can be seen in c. The height profile diagram of the line depicted in each AFM image is also shown beside the image.

AFM image was taken from the top surface of one of the bacteria, as shown in Figure 1c. The aggregated graphene sheets with typical thicknesses of about 48 nm are well-distinguishable on the top surface of the bacterium. This indicated that the graphene oxide nanosheets could wrap the bacteria (as a model for microorganisms) by the reduced graphene sheets during their aggregation in the melatonin solution. In general, the exfoliated graphene oxide nanosheets contain a diversity of functional groups such as hydroxyl (COH) and epoxide (COC) groups in addition to carbonyl (>CdO) and carboxyl (COOH) groups usually present at the defects and/or edges of the sheets. Hence, to analytically study the chemical state of the graphene oxide nanosheets in the various suspensions, the C(1s) XPS core levels of the graphene thin films prepared from the various graphene suspensions were deconvoluted, as shown in Figure 2. The deconvoluted peak centered at the binding energy of 285.0 eV was assigned to the CC, CdC, and CH bonds. The deconvoluted peaks located at the binding energies of 286.0, 287.7, and 289.2 eV were attributed to hydroxyl, carbonyl, and carboxyl functional groups, respectively.32,9193 It should be noted that, in the most structural models of graphite oxides, the hydroxyl and epoxide groups exhibit similar C(1s) 6281


The Journal of Physical Chemistry B

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Table 1. Peak Area (A) Ratios of the Oxygen-Containing Bonds to the CC Bonds (by XPS) and the Peak Intensity Ratios of ID/IG (by Raman) of the Graphene Thin Films Prepared from the Various Suspensions XPS suspensions used for preparing the thin films

Figure 2. Peak deconvolution of the C(1s) XPS core level of the graphene thin films prepared from suspensions of (a) the as-prepared GOS, (b) the GOSmelatonin, (c) the GOSmelatoninbacteria, (d) the GSmelatonin, and (e) the GSmelatoninbacteria.

binding energies.94 Thus, the contribution of hydroxyl and epoxide functional groups was not separated in the deconvoluted C(1s) peak. For the as-prepared GOSs (Figure 2a), large amounts of the functional groups were found in the deconvoluted carbon peak, as expected. To quantitatively study variations of the functional groups on the surface of the nanosheets, the peak area ratios of the COH, CdO, and OdCOH bonds to the CC bonds (containing CC, CdC, and CH bonds) were evaluated and compared. The evaluated values were listed in Table 1. For the asprepared GOSs, the peak area ratios of the COH, CdO, and OdCOH bonds to the CC bonds were evaluated to be 0.71, 1.18, and 0.19, respectively. After chemical reduction of the GOSs by melatonin, the XPS analysis (Figure 2b) indicated that the peak area ratios of the COH, CdO, and OdCOH functional groups to the area of the CC bonds were reduced 46, 61, and 47% relative to the corresponding bonds on the as-prepared GOSs, respectively (see Table 1). The deconvoluted C(1s) peak of the reduced graphene nanosheets that trapped the bacteria has been also presented in Figure 2c. It was found that the melatonin solution in the bacterial suspension could also well-reduce the graphene oxide nanosheets. In fact, the peak area ratios of the COH, CdO, and OdCOH functional groups to the area of the CC bonds decreased 56, 68, and 57% relative to the corresponding bonds on the as-prepared GOSs, respectively, indicating a slightly (∼18%) better reduction of the GOSs in the bacterial melatonin suspension than their reduction in the pure melatonin solution. This slight development in the reduction of the GOSs can be assigned to production of hydrogen and ethanol by the bacteria in mixed-acid fermentation with anaerobic conditions through a glycolysis process.95 For example, it was previously reported that E. coli bacteria can reduce some oxide materials, such as reduction of CuO to Cu2O after an immediate contact with the bacteria during the bacterial inactivation process.96 Furthermore, two peaks of K(2p3/2) and K(2p1/2) were observed at binding energies of 292.7 and 295.6, respectively. The presence of the K element in the XPS spectrum originated from the buffer solution used to prepare the bacterial suspensions. Figure 2d shows the deconvoluted C(1s) core level of the graphene thin films prepared from the

ACOH/ACC ACO/ACC AOCOH/ACC

Raman ID/IG

as-prepared GOS

0.71

1.18

0.19

GOSmelatonin

0.38

0.45

0.10

0.92 0.85

GOSmelatoninbacteria

0.31

0.37

0.08

0.82

GSmelatonin

0.50

0.48

0.11

0.86

GSmelatoninbacteria

0.39

0.37

0.09

0.83

GSmelatonin suspension. It was seen that the reduced GOSs in both thin films obtained from the GOSmelatonin and the GSmelatonin suspensions contained similar amounts of carbonyl and carboxyl functional groups (see also Table 1). The higher amount (31% increase) of the hydroxyl functional groups in the graphene thin films obtained from the GSmelatonin suspension than the corresponding amount of those obtained from the GOS melatonin suspension was assigned to the presence of further OH bonds on the surface of the prepared GSs. In fact, the OH bonds originating from the NaOH of the suspension prevented aggregation of the reduced GOSs in the suspension, due to electrostatic repulsion. Figure 2e also shows further (∼22%) reduction of the functional groups on the surface of the graphene thin films obtained from the GSmelatoninbacterial suspension as compared to the graphene thin films obtained from the GSmelatonin suspension (Figure 2e), due to production of hydrogen and ethanol by the bacteria through the glycolysis process. It should be noted that, in this work, to provide the conditions compatible with the bacteria, the temperature of reduction of the GOSs in the GOSmelatonin and GOSmelatoninbacterial suspensions was limited to 37 °C. However, on the basis of the XPS analysis, we also found that increasing the temperature of the GOSmelatonin suspension to 80 °C resulted in further deoxygenation of the GOSs (73, 78, and 68% reduction of the COH, CdO, and OdCOH functional groups relative to the corresponding bonds of the as-prepared GOSs). Moreover, use of hydrazine (as a common and powerful reducing agent) at a temperature of 80 °C and pH ∼ 10 (adjusted by ammonia solution) resulted in 71, 83, and 66% reduction of the COH, CdO, and OdCOH functional groups of the GOS suspension relative to the corresponding bonds of the as-prepared GOSs, respectively. These results showed that melatonin can be used as an efficient reducing agent at 80 °C and its reduction efficiency is comparable with the hydrazine’s efficiency. To investigate about the effect of the melatonin solution and the melatoninbacterial suspension on the carbon structure, reduction, and aggregation of the GOSs, Raman spectroscopy was employed, with its spectra shown in Figure 3. Indeed, Raman spectroscopy is known as a useful technique to study the ordered/ disordered crystal structures of carbonaceous materials, such as graphene nanosheets. The well-known characteristics of Raman spectra of carbon materials are D and G bands (typically located at ∼1350 and 1580 cm1), which are usually assigned to the local defects/disorders (particularly located at the edges of graphitic and graphene platelets) and the sp2 graphitized structure, 6282



Figure 3. Raman spectra of the thin films prepared from the suspensions of (a) the as-prepared GOS, (b) the GOSmelatonin, (c) the GOSmelatoninbacteria, (d) the GSmelatonin, and (e) the GS melatoninbacteria.

respectively.97,98 This means that smaller ID/IG peak intensity ratios of Raman spectra correspond to lower defects/disorders in a graphitized structure such as graphene. Figure 3 shows that, after reduction of the GOSs by the melatonin solution, the ID/IG ratio decreased from 0.92 for the as-prepared GOSs to 0.85 for the reduced sheets. In addition, the melatoninbacterial suspension resulted in more reduction of the ID/IG ratio to 0.82. The decreases in the ID/IG ratio were assigned to improvement in the sp2 graphitized structure of the sheets due to the reduction process. Similar decreases in the ID/IG ratio of the graphene thin films obtained from the GSmelatonin and GSmelatonin bacterial suspension were also found, as mentioned in Table 1. Using Raman spectra analysis, it is also possible to study the single-layer, bilayer, and multilayer characteristics of graphene layers. In fact, the G peak position of the single-layer graphenes (typically centered at 1585 cm1) shifts into lower wavenumbers after stacking further graphene layers.99101 For example, the G band shifts 6 cm1 into lower wavenumbers for the sheets constituted by 26 layers. In this work, the peak positions of the G bands of the as-prepared GOSs and the GOSs reduced by the melatonin solution and the melatoninbacterial suspension were found to be centered at about 1584, 1575, and 1579 cm1, respectively. In addition to the G band, shape and position of the 2D band effectively help to distinguish the layer numbers of graphene sheets.99102 For instance, the 2D peak position of the single-layer graphenes (typically centered at 2679 cm1) shifts to higher wavenumbers by 19 cm1 for the multilayer graphenes with 24 layers.98 Figure 3a shows that the 2D band of the asprepared GOSs was centered at a wavenumber of 2680 cm1 with a low-intensity shoulder at the higher wavenumbers. Thus, the single-layer structure was the predominant structure of the asprepared GOSs. However, the 2D band of the Raman spectrum of the GOSs reduced by the melatonin solution (Figure 3b) exhibited a peak position shift into the higher wavenumber of ∼2760 cm1. This shift was assigned to substantial aggregation of the reduced GOSs in the melatonin solution, consistent with our observation. For the GOSs reduced in the melatoninbacterial suspension (Figure 3c), the 2D peak showed a shift to the lower wavenumbers (as compared to the 2D peak shown in Figure 3b) with a peak position centered at about 2730 cm1. Approaching the 2D peak into the typical wavenumber of the single-layer structure (2679 cm1) indicated a smaller amount of aggregation of the GOSs reduced in the melatoninbacterial suspension than that of the sheets reduced

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Figure 4. Ratio of the number of the active bacteria obtained from the as-prepared GOSbacterial, the GOSmelatoninbacterial, and the GSmelatoninbacterial suspensions.

in the pure melatonin solution. Furthermore, the asymmetric 2D peak presented in Figure 3c shows a significant shoulder at wavenumber of about 2680 cm1, relating to the presence of the singlelayer structure in the sheets reduced in the melatoninbacterial suspension. Therefore, the Raman analysis indicated that the bacteria could moderate aggregation of the GOSs in the melatonin solution. This can be assigned to trapping the bacteria within the aggregated graphene sheets, which is also consistent with the AFM observations (see Figure 1b,c). A trace of the glucose of the GOS melatoninbacteria suspension was also observed in the Raman spectrum shown in Figure 3c. The 2D band of Figure 3d confirmed the presence of single-layer graphene sheets in the GSmelatonin suspension. This indicated that the pH adjustment of the GOS melatonin solution could well-keep the homogeneity and separateness of the graphene sheets in the GSmelatonin suspension, the same as the homogeneity and separateness of the GOSs in the asprepared suspension (compare Figure 3a,d). A weak shoulder in the higher wavenumbers was assigned to a slight aggregation that occurred during the reduction of the GOSs in the pH-adjusted melatonin solution. By removing the hydroxyl functional groups from the reduced graphene sheets in the GSmelatoninbacterial suspension using the excess melatonin, aggregation of the graphene sheets occurred, as can be seen from the 2D peak of the Raman spectrum shown in Figure 3e. A weak shoulder at the wavenumber of ∼2680 cm1 indicated the presence of a single-layer graphene in the GSmelatoninbacterial suspension. But, its lower intensity as compared to the intensity of the 2D shoulder of the GOS melatoninbacterial suspension at such a wavenumber indicated that the latter suspension included further reduced graphene sheets with single-layer characteristics. This can be assigned to lower trapping of the bacteria within the aggregated sheets of the GS melatoninbacterial suspension as compared to the GOS melatoninbacterial one, as we discuss in the following. To further investigate trapping of E. coli bacteria within the GOSs reduced by melatonin, the bacterial activity of the GOS melatoninbacterial and GSmelatoninbacterial suspensions with the various graphene oxide concentrations was examined. Figure 4 shows ratios of the number of the active bacteria obtained from the graphenemelatoninbacterial suspensions with the different graphene oxide concentrations to the number of the active bacteria obtained from the bacterial suspension with no GOSs. To have a benchmark, ratios of the active bacteria 6283


The Journal of Physical Chemistry B obtained from the bacterial suspension (without the melatonin solution applied for reducing the GOSs) with the different graphene oxide concentrations were also presented in Figure 4. It was found that increasing the graphene oxide concentration could slightly decrease the activity of the bacteria in the bacterial suspension. In fact, the GOS suspensions could not present a significant bactericidal property. However, in the GOS melatoninbacterial suspension, the activity of the bacteria was substantially decreased with increasing graphene concentration so that, for a concentration of 5 mg/mL, no active bacterium was observed. For the suspensions with no graphene sheets, further bacterial activity was observed in melatoninbacterial suspension as compared to the activity in the bacterial suspension without the melatonin, as can be seen in Figure 4. Therefore, a substantial fraction of the bacteria in the GOSmelatoninbacterial suspension was trapped within the aggregated graphene sheets, while only a small fraction of them might be inactivated through probable other mechanism(s) in the suspension. Figure 4 also shows that although aggregation of the GSs in the GSmelatoninbacterial suspensions could slightly inactivate and capture the bacteria, the aggregated GSs could not effectively trap the bacteria as they were trapped within the reduced and aggregated GOSs in the GOSmelatoninbacterial suspensions. The better capturing of the bacteria by the GOSs was attributed to better attachment of the bacteria on the surface of the GOSs with the oxygen-containing functional groups as compared to the GSs with the reduced functional groups. It is worthy to note that, although there are some reports about biocompatibility of the graphene,82,83 it was also reported that graphene-based materials can show antibacterial property.32,103 Such discrepancies were also frequently reported for cytotoxicity of one of the other carbon nanostructures, i.e., carbon nanotubes, due to their various structures, sizes, amounts of defects, and types of functionalizations.104 Similar physicochemical characteristics can also affect the cytotoxicity of graphene-based materials prepared by different research groups at various experimental conditions. In this work, although the GOSs did not behave as fully biocompatible nanomaterials, they did not exhibit a strong antibacterial activity. Besides probable differences in the physicochemical characteristics of the graphene oxides, the smaller antibacterial activity of the GOSs than the activity of graphene oxide reported by Hu et al.103 can be assigned to the characteristics of the bacterial suspension which for the former was the nutrient LB culture medium (as a suitable medium for proliferation of bacteria) and for the latter was a simple saline solution. In fact, Hu et al.103 showed that graphene oxide sheets could prevent the proliferation of E. coli bacteria in the saline solution while they did not exhibit a considerable cytotoxicity. Elsewhere, we showed that cytotoxicity of the graphene can be highly increased by providing direct interaction between the cell wall membrane of bacteria and the edge of the graphene sheets synthesized on a substrate as graphene nanowalls.32 In fact, without a considerable physical interaction between the membrane of bacteria and the edge of the graphene sheets, no considerable cytotoxicity can be expected for graphene sheets, as was not observed in this work. Figure 5 shows glucose concentrations of the bacterial, the GOSbacterial, and the GSmelatoninbacterial suspensions with the different graphene oxide concentrations. It can be seen that glucose concentration slightly increased in the bacterial suspension by increasing the concentration of the as-prepared GOSs in the suspension. This is consistent with the slight inactivation of the bacteria by increasing the concentration of the

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Figure 5. Glucose concentrations of the GOSbacterial, GOS melatoninbacterial, and GSmelatoninbacterial suspensions at the various concentrations of the graphene sheets.

GOSs in the suspension. In the GOSmelatoninbacterial suspension, however, the glucose concentration considerably increased by raising the concentration of the GOSs in the suspension. Since E. coli bacteria consume glucose through a glycolysis process, the increase in glucose concentration of each suspension was assigned to decrease of the number of the active bacteria of that suspension. Furthermore, the decrease in the number of the active bacteria can corresponded to an increase of the number of bacteria trapped within the aggregated graphene sheets. It was found that the glucose concentration of the melatoninbacterial suspension with 5 mg/mL graphene sheets was nearly unchanged relative to the initial glucose concentration used in the suspension (20 mg/mL). This is completely consistent with observation of no active bacteria at such conditions (see Figure 4). Therefore, when all of the bacteria were trapped within the aggregated graphene sheets, they were also isolated from their environment. Also, the lower glucose concentrations in the GSmelatoninbacterial suspensions than the glucose concentrations in the GOSmelatoninbacterial suspensions confirmed that the aggregated GSs could not effectively capture the bacteria of the suspension, unlike their effective capturing in GOSmelatoninbacterial suspension (particularly at the graphene oxide concentration of 5 mg/mL which resulted in a complete capturing). To release the bacteria trapped within the aggregated graphene sheets, the melatoninbacterial suspensions irradiated for 96 h were sonicated. Figure 6 shows the relative difference of the active bacteria and the glucose concentration of the sonicated GOSmelatoninbacterial suspensions. The relative difference of the active bacteria was defined as (Ns(C) Nu(C))/Nu(C=0) in which Ns(C) and Nu(C) are the number of active bacteria obtained from the sonicated and the unsonicated suspensions at a graphene oxide concentration (C), respectively. Figure 6 indicates that the sonication resulted in a slight inactivation of the bacteria in the melatoninbacterial suspension with no GOSs, due to its probable effect on the nature of the enzyme of the bacteria. However, by increasing the concentrations of the graphene oxide sheets from 0.05 to 1 mg/mL, the activity of the bacteria obtained from the sonicated suspensions significantly increased from about 4 to 33%. However, after increasing the graphene oxide concentration to 5 mg/mL, the bacterial activity slightly decreased. This change can be assigned to high 6284



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Figure 6. Variations of the number of the active bacteria and glucose concentrations of the sonicated GOSmelatoninbacterial suspensions relative to those of the unsonicated ones.

aggregation of the reduced graphene sheets in the high concentration of 5 mg/mL of the graphene sheets, which resulted in harder separation of the highly aggregated sheets from the bacteria by using the sonication. These results showed that after 48 h inactivation of the bacteria within the aggregated graphene sheets in the GOSmelatoninbacterial suspension, maximally about one-third of the bacteria could be reactivated in the suspension by applying the sonication method. For the GSmelatoninbacterial suspension, the maximum ratio of the reactivated bacteria was found to be only about 16% at 1 mg/mL of the GOS concentration. The reactivated bacteria should also consume the glucose of the melatoninbacterial suspensions. Figure 6 also presents the relative difference of the glucose concentration of the sonicated GOSmelatoninbacterial suspensions as (Gu(C) Gs(C))/Gu(C) in which Gu(C) and Gs(C) are the glucose concentrations of the unsonicated and the sonicated suspensions at a graphene oxide concentration (C), respectively. Corresponding to the bacterial activity, it was found that the sonication of the GOSmelatoninbacterial suspensions resulted in increasing the glucose consumption of the suspension by the reactivated bacteria. Indeed, the glucose consumption increased by increasing the concentration of the graphene oxide sheets in the suspension from 0.05 to 1 mg/mL, but it exhibited a slight decrease when the graphene concentration increased to 5 mg/mL, in a complete consistency with the bacterial activity. The effect of the near-IR irradiation at wavelength of 808 nm on the activity of the bacteria in the various graphene suspensions was also studied. Figure 7a shows the ratios of the number of the active bacteria obtained from the irradiated and then sonicated GOSbacterial, GOSmelatoninbacterial, and GSmelatonin bacterial suspensions with GOS concentration of 5 mg/mL to the number of the active bacteria obtained from the unsonicated bacterial suspension with no GOSs. The near-IR irradiation for 120 s resulted in a complete inactivation of the bacteria in the GOS melatoninbacterial suspension, while it could inactivate only 45 and 58% of the bacteria in the GOSbacterial and GSmelatonin bacterial suspensions, respectively. Since it was shown that graphene layers can absorb infrared irradiation (see, for example, refs 69 and 88), the optical absorption characteristic of the GOS and the GOSmelatonin suspensions with GOS concentration of

Figure 7. (a) Ratio of the number of the active bacteria obtained from the sonicated GOSbacterial, GOSmelatoninbacterial, and GS melatoninbacterial suspensions with 5 mg/mL GOS concentration, after exposure to near-IR irradiation for different periods of time. The inset shows the ratio of the active bacteria obtained from the GOS melatoninbacterial suspension in a closed up window. (b) UV visnear-IR spectra of the GOS and the GOSmelatonin suspensions with GOS concentration of 1 mg/mL.

1 mg/mL were studied, as presented in Figure 7b. Compared with the GOS suspension, the GOSmelatonin suspension showed an extension of the absorption to the higher wavelengths, a considerable extension to the visible region (400700 nm) and a slighter extension to the near-IR region (>700 nm). This extension in the absorption could be also distinguished by a color change of the suspension from light brown for the GOS suspension to black for the GOSmelatonin suspension. This kind of color change was previously attributed to partial restoration of the π network within the carbon structure as a witness for chemical reduction of the graphene oxide sheets.105,106 Since aggregated graphene sheets can further absorb near-IR irradiation, the temperature of the aggregated sheets (as the local temperature of the suspension, not the temperature of the whole suspension) increased relative to the constant temperature of the suspension at 39 °C. Therefore, the increase in inactivation of the bacteria in the graphene suspensions exposed to near-IR irradiation was assigned to excessive local heating of the graphene sheets, and consequently, photothermal inactivation of the bacteria. Since the active bacteria in the GOSmelatoninbacterial suspension were completely trapped within the aggregated graphene sheets, the near-IR irradiation could successfully inactivate all of the bacteria of the suspension in 120 s. 6285



4. CONCLUSIONS Single-layer GOSs synthesized by the chemical exfoliation method were chemically reduced by melatonin solution. By variation of pH of the GOSmelatonin suspension, both homogeneous and inhomogeneous GSmelatonin suspensions could be obtained. In the melatoninbacterial suspension, aggregation of the sheets resulted in trapping of the E. coli bacteria within the aggregated sheets (a kind of bacterial inactivation), while the graphene (oxide) suspension and melatonin solution did not individually exhibit any considerable inactivation effects on the bacteria. The GOSs with the oxygen-containing functional groups could better trap the bacteria than the GSs with reduced functional groups. On the basis of measuring the glucose consumption of the bacteria, it was found that the bacteria trapped within the aggregated graphene sheets were biologically disconnected from their environment. In addition, the bacteria trapped within the graphene sheets were inactive without any chance for proliferation in a culture medium. But, after removing the aggregated graphene sheets from the surface of the bacteria by using sonication, they could be reactivated. The reactivated bacteria consumed the glucose of the suspension and also could proliferate in a culture medium; i.e., they were alive while they were temporarily inactivated inside the graphene sheets (here, at least for 24 h). The near-IR irradiation could inactivate the bacteria forever by excessive local heating of the bacteria trapped within the aggregated graphene sheets. Since E. coli was used as a simple model for microorganisms, the results of this work can be utilized in various biological applications in which photothermal inactivation of microorganisms and/or reactivation of them after a protected delivery is required. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Tel.: þ982166164566. Fax: þ982166022711.

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