# Graphene Oxide Sheets Decorated with Octahedral Molybdenum Cluster Complexes for Enhanced Photoinactivation of Staphylococcus aureus

Régis Guégan, Xiaoxue Cheng, Xiang Huang, Zuzana Nemeckova, Michaela Kubáňová, Jaroslav Zelenka, Tomáš Ruml, Fabien Grasset, Yoshiyuki Sugahara, Kamil Lang, et al.

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Régis Guégan, Xiaoxue Cheng, Xiang Huang, Zuzana Nemeckova, Michaela Kubáňová, et al. Graphene Oxide Sheets Decorated with Octahedral Molybdenum Cluster Complexes for Enhanced Photoinactivation of Staphylococcus aureus. Inorganic Chemistry, 2023, 62 (35), pp.14243-14251. 10.1021/acs.inorgchem.3c01502. [hal-04196513](https://hal.science/hal-04196513v1)

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Graphene oxide sheets decorated with octahedral molybdenum cluster complexes for enhanced photoinactivation of Staphylococcus aureus

<table><tr><td>Journal:</td><td>Inorganic Chemistry</td></tr><tr><td>Manuscript ID</td><td>ic-2023-015028.R2</td></tr><tr><td>Manuscript Type:</td><td>Article</td></tr><tr><td>Date Submitted by the Author:</td><td>01-Aug-2023</td></tr><tr><td>Complete List of Authors:</td><td>Guegan, Regis; Waseda University; Interfaces Confinement Matériaux et Nanostructures<br>Cheng, Xiaoxue; Waseda University Faculty of Science and Engineering<br>Huang, Xiang; Waseda University Faculty of Science and Engineering<br>Nemeckova, Zuzana; Ustav anorganicke chemie Akademie ved Ceske republiky, Materials Chemistry<br>Kubanova, Michaela; University of Chemistry and Technology Prague<br>Department of Biochemistry and Microbiology<br>Zelenka, Jaroslav; Vysoka skola chemicko-technologicka v Praze,<br>Department of Biochemistry and Microbiology<br>Ruml, Tomáš; Vysoka skola chemicko-technologicka v Praze, Dpt. of<br>Biochemistry and Microbiology<br>Grasset, Fabien; CNRS, UMI3629; ISCR<br>Sugahara, Yoshiyuki; Waseda University Faculty of Science and<br>Engineering<br>Lang, Kamil; Institute of Inorganic Chemistry of the Czech Academy of<br>Sciences,<br>Kirakci, Kaplan; Akademie Ved Ceske Republiky Ustav anorganicke<br>chemie,</td></tr></table>
Graphene oxide sheets decorated with octahedral molybdenum cluster complexes for
enhanced photoinactivation of *Staphylococcus aureus*

Régis Guégan¹,²*, Xiaoxue Cheng³, Xiang Huang³, Zuzana Němečková,⁴ Michaela Kubanová⁵, Jaroslav Zelenka⁵, Tomas Ruml⁵, Fabien Grasset⁶,⁷, Yoshiyuki Sugahara³,⁸, Kamil Lang⁴, Kaplan Kirakci⁴*

¹Global Center for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan

²Interfaces, Confinement, Matériaux et Nanostructures ICMN-UMR 737, CNRS-Université d'Orléans, 1 Rue de la Férollerie, 45100 Orléans, France

³Department of Applied Chemistry, Waseda University, Faculty of Science and Engineering, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan

⁴Institute of Inorganic Chemistry of the Czech Academy of Sciences, 250 68 Husinec-Řež, Czech Republic

⁵Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, 166 28 Praha, Czech Republic

⁶Institut des Sciences Chimiques de Rennes, ISC-UMR 6226, CNRS-Université de Rennes 1, 263 avenue du Général Leclerc, 35700 Rennes, France

⁷CNRS-Saint-Gobain-NIMS, IRL3629, Laboratory for Innovative Key Materials and Structures (LINK), National Institute for Materials Science, 1-1 Namiki, Tsukuba 305-0044, Japan

⁸Kagami Memorial Institute for Materials Science and Technology, Waseda University, 2-8-26 Nishiwaseda, Shinjuku-ku, Tokyo 169-0051, Japan

*email: regis.guegan@univ-orleans.fr, kaplan@iic.cas.cz

Abstract

The emergence of multidrug-resistant microbial pathogens poses a significant threat, severely limiting the options for effective antibiotic therapy. This challenge can be overcome through the photoinactivation of pathogenic bacteria using materials generating reactive oxygen species upon exposure to visible light. These species target vital components of living cells, significantly reducing the likelihood of resistance development by the targeted pathogens. In our research, we have developed a nanocomposite material consisting of an aqueous colloidal suspension of graphene oxide sheets adorned with nanoaggregates of octahedral molybdenum cluster complexes. The negative charge of the graphene oxide and the positive charge of the nanoaggregates promoted their electrostatic interaction in aqueous medium, and
close cohesion between the colloids. Upon illumination with blue light, the colloidal system exerted a potent antibacterial effect against planktonic cultures of *Staphylococcus aureus* largely surpassing the individual contributions of the components. The underlying mechanism behind this phenomenon lies in the photoinduced electron transfer from the nanoaggregates of the cluster complexes to the graphene oxide sheets, which triggers the generation of reactive oxygen species. Thus, leveraging the unique properties of graphene oxide and light-harvesting octahedral molybdenum cluster complexes can open more effective and resilient antibacterial strategies.

**Keywords:** photosensitizer, bactericide properties, graphene oxide, molybdenum cluster, nanocomposite, bacteria

## Introduction

Microbial pathogens exhibiting multidrug-resistance constitute a serious hazard, especially to cancer and immunocompromised patients, and restricts the choices for adequate antibiotic therapy.¹,² Among the many possible ways to inactivate pathogens, their photoinactivation by materials able to produce reactive oxygen species (ROS) upon visible-light irradiation represents an elegant way to achieve this goal, as ROS attack several critical components of living cells thus limiting the emergence of resistance to the treatment.

In this respect, octahedral molybdenum cluster complexes are efficient singlet oxygen photosensitizers and have recently demonstrated major potential as molecular antibacterial agents or for the design of light-triggered disinfecting surfaces. ³,⁴,⁵,⁶,⁷,⁸,⁹ These clusters are stabilized by eight inner ligands, usually iodine atoms and six labile apical ligands of inorganic or organic nature. Upon excitation from the UV up to the green light, the resulting complexes exhibit long lived triplet states that relax via a red-NIR phosphorescence or by energy transfer to molecular oxygen to form the highly reactive singlet oxygen O₂(¹Δg).¹⁰,¹¹,¹² Several studies have reported the effect of these complexes for the photoinactivation of planktonic cultures and biofilms of both gram-negative and gram-positive bacteria.³,⁴,⁵,⁶,⁷,⁸,⁹ When compared to organic photosensitizers, these cluster complexes are less prone to self-quenching of their luminescent properties due to aggregation at high concentration or photobleaching which can limit the efficiency and robustness of their derived materials.⁷

Graphene oxide (GO) sheets, prepared from the etching and oxidation of graphite, exhibit outstanding properties: large specific surface area, electrical and mechanical properties,
that tackle the interests of both the scientific and industrial communities.<sup>13,14,15,16,17</sup> While the inclusion of oxygen atoms within carbon sheets modulates their electrical properties, it also confers a hydrophilic behaviour to GO, making them dispersible in aqueous media as a supporting phase for, e.g., complexes or nanoparticles to prepare nanocomposite materials.<sup>18,19,20,21,22</sup> In addition to the hydrophilic moieties, GO display unaltered hydrophobic graphene like patches which represent many reaction/adsorption sites for the association with other colloids. As in the case of other carbon-based nanomaterials (carbon nanotubes, etc.), GO sheets exhibit antibacterial properties, of which origin comes from the physical presence of the sheets as well as their chemical activities.<sup>13,16,17</sup> Indeed, it has been recognized that GO sheets wrap around bacteria causing irreversible physical damage to the cell membranes while chemically increasing the cellular oxidative stress leading to the end of the bacterial development.<sup>13</sup>

The association of photosensitizing organic compounds such as hypocrellin A to GO sheets lead to nanocomposites showing long-time effects for eradication of cancer cells.<sup>23</sup> In this case, GO sheets act as a preservation matrix for hypocrellin A that could generate O<sub>2</sub>(<sup>1</sup>&Delta;<sub>g</sub>) on a long-time scale. In another study, the association of porphyrins with GO imparts the nanocomposites with antibacterial properties.<sup>24</sup> The mechanism behind the antibacterial activity was assigned to fast electron transfer from the excited singlet states of porphyrin to the conduction band of GO resulting in the production of ROS by the sheets.<sup>24</sup> Thus, we surmise that the association of such cluster complexes with GO could lead to enhancement of photoinactivation of bacteria. In fact, various composites of these cluster complexes with GO have already been utilized with promising results for applications such as photocatalytic water reduction, photodegradation of organics, or gas sensing, but so far, no such composite material has been studied for its antibacterial properties.<sup>25,26,27,28,29</sup>

This research work describes the association of the cluster compound, [Mo<sub>6</sub>I<sub>8</sub>(OCOC<sub>4</sub>H<sub>8</sub>PPh<sub>3</sub>)<sub>6</sub>]Br<sub>4</sub> (Mo<sub>6</sub>), with GO sheets (Fig. 1) for the preparation of nanocomposites with antibacterial properties. Dispersed in dimethyl sulfoxide to provide a homogeneous dispersion of the components and long-term durability of Mo<sub>6</sub> complexes, and then added to aqueous medium, GO sheets act as a template or supporting phase of Mo<sub>6</sub> in the form of nanoaggregates interacting mainly via electrostatic forces. After a careful characterization of the morphology, cohesion, and structure of the nanocomposites as well as of their photophysical properties by various complementary techniques, their bactericide
properties against *Staphylococcus aureus* are demonstrated through a comparative study of the
efficiency of the whole individual materials.

<figure><img src="image_2.png" /><figcaption>**Figure 1.** Schematic representation of (A) GO sheets and (B) positively charged Mo<sub>6</sub> complex [Mo<sub>6</sub>I<sub>8</sub>(OCOC<sub>4</sub>H<sub>8</sub>PPh<sub>3</sub>)<sub>6</sub>]<sup>4+</sup>. Color codes: carbon (grey), oxygen (red), phosphorus (yellow), molybdenum (blue), iodine (magenta).</figcaption></figure>

Results and discussion

Preparation and characterization of GO

GO sheets were prepared using the modified Hummer's method based on oxidation and
exfoliation of graphite.³⁰ As demonstrated by several studies, the diversity of the chemical
landscape of GO sheets and their 2D-surfactant character (Pickering emulsion) allow for
dispersion in many polar and non-polar organic solvents.¹⁴,³¹ However, the use of dimethyl
sulfoxide (DMSO), a polar solvent often used in biological experiments, failed so far to give
stable GO dispersions.³¹ In the aforementioned study, the sheets were dried in a vacuum oven
which favoured the re-stacking of the sheets into the initial graphite-like layered structure,
slowing down their subsequent exfoliation and leading to the formation of GO agglomerates
and sedimentation of the graphite oxide. In the present study, the prepared GO sheets,
displaying a similar oxidation rate, were lyophilized which allowed for an easy dispersion in
DMSO and water at a concentration of 1 mg mL⁻¹, higher than that previously tested using a
wide range of solvents (0.5 mg mL⁻¹) (Fig. S1 in the Supporting Information).³¹ This feature
was confirmed by transmission electron microscopy (TEM) and atomic force microscopy
(AFM) observations, indicating the absence of re-stacking and aggregation of the sheets as the
images showed a majority of individual sheets with a lateral size in the 2-20 µm range and a
thickness close to 1 nm (Figs. 2 and S2 in the Supporting Information), an important point to consider for future applications based on single carbon nanomaterials.

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Characterization of the GO sheets by TEM (A) and AFM with the corresponding section analysis (B).</figcaption></figure>

The GO sheets, after being dispersed in water or DMSO, were collected and characterized by Raman scattering and XPS spectroscopy (Fig. 3), to assess the possible chemical changes after their immersion in DMSO. As expected, the Raman spectra of the GO showed two characteristic bands of graphitized materials, i.e., the D band reflecting structural defects due to inclusion of oxygen atoms in the carbon structure or holes, and the G band representative of the original graphite organization.³² The intensity ratio *I*<sub>D</sub>/*I*<sub>G</sub> is an indicator of the degree of graphitization of the GO sheets and remained the same for the GO sheets (0.87) before and after immersion in water or DMSO (Table 1). It underlines the absence of any modification, nor physical structural alteration of the sheets after the freeze-drying operation. However, the XPS spectrum of the DMSO immersed GO seemed to display a higher proportion of C=O double bonds at the expense of a lowering of C-C bonds (graphite areas), which is contradictory with the Raman data that suggested the same proportion of graphitic domains. Since DMSO is a rather viscous solvent with a high boiling point, it is likely that some DMSO molecules remain on the surface of the GO sheets and the C-S band could overlap with the C 1s spectrum of the GO as well as the resulting distribution of the various bonds. Nevertheless, the characterization by Raman scattering clearly evidences that the GO sheets were not altered neither physically nor chemically after their lyophilization and dispersion in DMSO.
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Raman scattering (A) and XPS (B) of the GO sheets after being dispersed in water (black) and in DMSO (red).</figcaption></figure>

<table><caption>**Table 1.** Component area percentages of the GO sheets dispersed in H₂O and DMSO and corresponding ratios of the G band over the D band.</caption><thead><tr><th>Sample</th><th>C-C</th><th>C-OC, C-OH</th><th>C=O</th><th>I<sub>D</sub>/I<sub>G</sub> (Raman)</th></tr></thead><tbody><tr><td>GO (H<sub>2</sub>O)</td><td>50.2%</td><td>42.6%</td><td>7.2%</td><td>0.87</td></tr><tr><td>GO (DMSO)</td><td>44.6%</td><td>32.8%</td><td>22.6%</td><td>0.87</td></tr></tbody></table>

## Preparation and characterization of GO/Mo₆ nanocomposites

The dispersibility of the GO sheets in DMSO after a freeze-drying operation, with the formation of stable dispersions, opens new routes for the preparation of nanocomposites. Leaning against this strategy, the nanocomposites were prepared by mixing the lyophilized GO and [Mo₆I₈(OCOC₄H₈PPh₃)₆]Br₄ in DMSO for 24 hours under magnetic stirring. Mo₆ complex suffers from a lack of stability and poor solubility in aqueous media where its apical ligands are replaced within days, but it is well soluble in DMSO where the hydrolytic process is greatly slowed down.³ Thus, DMSO solutions of Mo₆ complex did not exhibit any significant aggregation by showing an average size of 3.1 ± 1 nm (Z-average = 6.0 nm, PDI = 0.20) (Fig. S3 in the Supporting Information), which is consistent to the size of isolated Mo₆ complex. Due to the targeted antibacterial application, the GO/Mo₆ nanocomposite was studied in the form of an aqueous colloidal dispersion which was obtained by adding the aliquots of the DMSO dispersion (1 mg mL⁻¹) to deionized water (DMSO/H₂O, 1 % v/v).
Transmission electron microscopy of a drop of the resulting aqueous dispersion of GO/Mo<sub>6</sub> deposited on a TEM grid documented GO sheets decorated by nanoaggregates with an average size of 19 ± 8 nm and made of Mo<sub>6</sub> as demonstrated by the HAADF elemental mapping of Mo and I, (Figs. 4, S4 in the Supporting Information). The Mo<sub>6</sub> nanoaggregates immobilized on the surface of GO are apparently formed during the addition of the DMSO dispersion to H<sub>2</sub>O. Indeed, dynamic light scattering of pure Mo<sub>6</sub> in water evidenced nanoaggregates with an average size by number of 31 ± 10 nm (Z-average = 110 nm, PDI = 0.37) (Fig. S3 in the Supporting Information), reminiscent of the nanoparticles of other pure cluster compounds obtained via the solvent displacement method.<sup>33,34,35</sup> X-ray powder diffraction pattern of GO/Mo<sub>6</sub> featured a very broad peak at approximately 7° demonstrating the amorphous character of the nanocomposite material (Figure S5 in the Supporting Information). ICP-MS analysis of dried GO/Mo<sub>6</sub>, isolated by centrifugation of the water dispersion, revealed a molybdenum content of 68.1 mg g<sup>-1</sup> slightly lower than the theoretical content of 70.5 mg g<sup>-1</sup>, indicating that the majority of the nanoaggregates of Mo<sub>6</sub> are immobilized at the surface of GO. This feature is in agreement with the absorption spectra of the supernatant after centrifugation of the GO/Mo<sub>6</sub> water dispersion, which evidenced only minute amount of free Mo<sub>6</sub> (Fig. S6 in the Supporting Information). The zeta potentials of the aqueous dispersions of GO and Mo<sub>6</sub> amounted to -34 ± 13 and 8 ± 6 mV, respectively, in accordance with the ionization of the hydroxyl and carboxylic groups of GO and the presence of positive charges endowed by the apical ligands of Mo<sub>6</sub> at the surface of the nanoaggregates (Fig. S7 in the Supporting Information). The aqueous dispersion of GO/Mo<sub>6</sub> displayed a negative zeta potential of -19 ± 8 mV and the zeta potential distribution was characterized by a single peak located in the negative range of the zeta potential values. In accordance with the above-mentioned characterizations, these results confirm that, in aqueous medium, the majority of the positively charged Mo<sub>6</sub> nanoaggregates is immobilized at the GO surface, where they partially compensate for its negative charge.

Despite the mild conditions used for the preparation of the nanocomposite in DMSO and the bulkiness of the apical ligands making them poorly labile, it is still possible that an apical ligand exchange could occur in this solvent resulting in bonding of cluster complexes to the surface of GO. In this case, we should expect a homogeneous coverage of the GO surface by Mo₆ considering the high degree of structural defects due to inclusion of oxygen atoms evidenced by Raman spectroscopy. However, TEM images of the nanocomposite clearly show the non-continuous distribution of nanoaggregates of Mo₆ at the surface of GO (Figs. 4 and S4). Thus, the mechanism of the formation of the GO/Mo₆ nanocomposite can be tentatively ascribed to
the precipitation of positively charged nanoaggregates of hydrophobic Mo₆ after mixing the DMSO dispersions of GO and Mo₆ in water, followed by the immobilization of the nanoaggregates at the surface of GO mainly via electrostatic interactions. Indeed, it is unlikely that a significant ligand exchange with hydroxyl or carboxylates function of the GO could occur during the short period (minutes to hours) between the transfer of the DMSO dispersion to water and the measurement of the nanocomposite characteristics.

<figure><img src="image_5.png" /><figcaption>**Figure 4.** TEM images of GO/Mo₆ in the bright field (A, B) with the corresponding particle size distribution of Mo₆ nanoaggregates (C), and in the dark field (D) with the C, Mo (E) and C, I (F) HAADF elemental mapping.</figcaption></figure>

The luminescence of Mo₆ complexes is influenced by their ligands as well as their immediate environment and can be taken as a sensitive indicator of the extent of interaction between Mo₆ and GO. Therefore, the photophysical properties of water dispersions of the nanocomposites and their individual components, freshly prepared in the same way as for DLS experiments, were investigated and are summarized in Table 2. The absorption spectrum of GO/Mo₆ was essentially a composite of its individual components GO and Mo₆ showing typical absorption bands of Mo₆ complexes in the UV/blue region and the featureless UV-vis absorption of GO extending to the red region (Fig. S8 in the Supporting Information). When
compared to free Mo₆ complex, the luminescence band of GO/Mo₆ was broader and the maximum was red-shifted from 706 nm for Mo₆ to approximately 722 nm for GO/Mo₆ (Fig. 5A). A drastic decrease in the luminescence intensity was also noticed, as also evidenced by a significant drop of luminescence quantum yields from 0.24 for Mo₆ to 0.01 for GO/Mo₆. Similarly, the average luminescence lifetime in argon-saturated dispersions decreased from 88 µs for Mo₆ to 2.4 µs for GO/Mo₆ (Fig. 5B). It is worth noting that as-prepared DMSO dispersions of Mo₆ and GO/Mo₆ displayed comparable luminescence spectra indicating that the cluster complex was chemically unchanged prior to addition to aqueous medium as hydrolysis would be characterized by a red-shift of the emission maximum (Fig. S9 in the Supporting Information). In addition, the kinetics of hydrolysis of Mo₆ in aqueous medium were already reported showing that the wide majority of the cluster complexes remained intact after 24 h.³ Thus, hydrolysis of Mo₆ complex in DMSO or immediately after addition to aqueous medium was excluded to account for the strong decrease in emission quantum yield and lifetime of GO/Mo₆ in water relative to pure Mo₆. Instead, the presented results point out at electronic interactions between the immobilized nanoaggregates of Mo₆ and GO, leading to the quenching of their excited states.

<table><caption>**Table 2.** Photophysical parameters of water dispersions of GO, Mo₆, and GO/Mo₆ at room temperature.ª</caption><thead><tr><th>Sample</th><th>λ<sub>L</sub> / nm</th><th>τ<sub>T</sub> / µs</th><th>τ<sub>air</sub> / µs</th><th>Φ<sub>L</sub></th></tr></thead><tbody><tr><td>GO</td><td>670</td><td>0.7 ns<sup>b</sup></td><td>0.7 ns<sup>b</sup></td><td>&lt;0.01</td></tr><tr><td>Mo<sub>6</sub></td><td>706</td><td>88<sup>b</sup></td><td>29<sup>b</sup></td><td>0.24</td></tr><tr><td>GO/Mo<sub>6</sub></td><td>722</td><td>2.4<sup>b</sup></td><td>2.3<sup>b</sup></td><td>0.01</td></tr></tbody></table>

ª λ<sub>L</sub> is the maximum of luminescence emission bands (excited at 400 nm); τ<sub>T</sub> and τ<sub>air</sub> are the average luminescence lifetimes in argon- (oxygen-free) and air-saturated water, respectively (recorded at 700 nm, excited at 405 nm); Φ<sub>L</sub> is the luminescence quantum yield in argon-saturated solutions (excitation at 400 nm, experimental error of Φ<sub>L</sub> is ± 0.01); b The amplitude average lifetimes obtained by the biexponential analysis of corresponding kinetic curves.

Contrasting with free Mo₆ nanoaggregates (Fig. S10 in the Supporting Information), there was negligible quenching of the triplet states by oxygen after their deposition onto the GO sheets as evidenced by the average luminescence lifetimes of GO/Mo₆ in air- and argon-saturated water (2.4 and 2.3 µs, respectively, Table 2) (Fig. 5C). This result also indicates fast
competitive quenching of the triplet states of deposited Mo₆, probably associated with photoinduced electron transfer between Mo₆ and the GO sheets and suggest a poor production of O₂(^1Δg) by deposited Mo₆ upon irradiation. Note that a very weak red emission from a GO water dispersion was observed when exciting at 400 nm. It was characterized by a broad band with a maximum at approximately 670 nm and an emission lifetime of 0.7 ns, which is consistent with the reports on GO (Table 2, Fig. S11 in the Supporting Information).¹⁶

<figure><img src="image_6.png" /><figcaption>**Figure 5.** (A) Normalized luminescence spectra of Mo₆ (black) and GO/Mo₆ (red) in argon-saturated water, excitation was at 400 nm. (B) Luminescence decay kinetics of Mo₆ (black) and GO/Mo₆ (red) in argon-saturated water. Excitation was at 405 nm and emission at 700 nm. (C) Luminescence decay kinetics of GO/Mo₆ in air- (black) and argon-(red) saturated water dispersions. Excitation was at 405 nm and emission at 700 nm.</figcaption></figure>

The inhibition of O₂(^1Δg) production by deposited nanoaggregates of Mo₆ in GO/Mo₆ was confirmed by measuring the weak phosphorescent signal of O₂(^1Δg) centred at approximately 1270 nm. Indeed, while Mo₆ showed a typical phosphorescence band of O₂(^1Δg), as previously reported for this complex,³ both GO and GO/Mo₆ did not display any detectable signal in the same spectral region confirming that GO and GO/Mo₆ did not produce appreciable amount of O₂(^1Δg) under these conditions, as previously reported for GO (Fig. 6A).³⁶ In order to evaluate the capacity of the nanocomposite to generate other reactive oxygen species, such as superoxide, hydrogen peroxide, or hydroxyl radical, we employed an oxidation probe, namely 2',7'-dichlorofluorescein diacetate (DCF-DA). It is a chemically reduced form of fluorescein commonly used as an indicator for ROS. DCF-DA was added to water dispersions containing various concentrations of GO/Mo₆ (0, 0.25, 0.5, 1 and 2 w/w%) followed by irradiation with 460 nm light. The probe was oxidized thus becoming fluorescent, and the amount of oxidation was dose- and time-dependent evidencing the capacity of GO/Mo₆ to photogenerate reactive oxygen species (Fig. 6B).
<figure><img src="image_7.png" /><figcaption>**Figure 6.** (A) Singlet oxygen phosphorescence band of Mo<sub>6</sub> (red), GO (blue) and GO/Mo<sub>6</sub> (black) in oxygen-saturated water. (B) Evaluation of the oxidation of the DCF-DA probe by various concentrations of GO/Mo<sub>6</sub> in water (0, 0.25, 0.5, 1, 2 %) under various blue-light irradiation time (460 nm). Bars labelled 0 % are control experiments in the absence of GO/Mo<sub>6</sub>.</figcaption></figure>

Overall, the observed changes in the zeta potentials and luminescence parameters when comparing Mo₆ and GO/Mo₆ point out at strong electrostatic interactions between the GO sheets and the nanoaggregates of Mo₆ in aqueous medium. The quenching of the luminescence of Mo₆ by GO inhibits the formation of O₂(¹Δg). This feature appears to be related to a photoinduced electron transfer from the excited states of the Mo₆ complex to the conduction band of GO and leads to the formation of ROS, probably superoxide, hydrogen peroxide, or hydroxyl radical, as previously reported in the literature.<sup>37,38,39</sup>

*Bactericide properties of the GO/Mo<sub>6</sub> nanocomposites*

The photoinactivation properties of GO/Mo₆ and its individual components were evaluated against planktonic culture of *S. aureus*, a Gram-positive spherically shaped bacterium. Despite acting as a commensal of the human microbiota, it can be the cause of skin and respiratory infections as well as food poisoning. *S. aureus* constitutes one of the leading lethal pathogens and is associated with antimicrobial resistance, making it an ideal target for photodynamic inactivation.<sup>1,2</sup> The cultures of *S. aureus* were incubated as planktonic cells with GO, Mo₆ and GO/Mo₆ dispersions and illuminated with 460 nm light.
<figure><img src="image_8.png" /><figcaption>**Figure 7.** Photoinactivation of *Staphylococcus aureus* planktonic cultures upon 460 nm irradiation in the presence of (A) GO, Mo<sub>6</sub> or GO/Mo<sub>6</sub> (0.01 g L<sup>-1</sup>) and (B) aged Mo<sub>6</sub> and GO/Mo<sub>6</sub>. Dark controls are represented with black bars and bars labelled Control are experiments in the absence of GO, Mo<sub>6</sub> or GO/Mo<sub>6</sub>.</figcaption></figure>

In the absence of light, GO, Mo<sub>6</sub> and GO/Mo<sub>6</sub> were non-toxic against *S. aureus* at the used concentration of 0.01 mg mL<sup>-1</sup> (Fig. 7A). GO sheets were previously reported to exert an antibacterial effect on *S. aureus*, however in our case we did not observe such effect possibly due to lower concentration and bigger lateral size for the GO used in our experiments.<sup>40</sup> Upon irradiation at 460 nm, GO remained non-toxic, while Mo<sub>6</sub> displayed a mild photoinactivation with a relative decrease of the CFU to 29 ± 12% compared to the control, an effect comparable to previous investigations on the antibacterial activity of this complex.<sup>3</sup> Under the same conditions the photoinactivation ability of GO/Mo<sub>6</sub> was remarkable with a relative decrease of the CFU to 0.06 ± 0.01 % compared to the irradiated control, i. e., the killing was almost three orders of magnitude more efficient than for Mo<sub>6</sub> only (Fig. 7A). Photoinduced intracellular oxidative stress was evaluated by incubating *S. aureus* with 0.01 mg/mL of GO/Mo<sub>6</sub> or its individual components in the presence of DCF-DA as an oxidation probe (Fig. S12 in the Supporting Information). While GO/Mo<sub>6</sub> clearly showed increased photoinduced ROS production, when compared to pure GO, the oxidative ability of Mo<sub>6</sub> alone was higher than that of GO/Mo<sub>6</sub>. Possibly, this feature which is in contradiction with the photoinactivation experiments, arises from different subcellular localization of the materials. Indeed, DCF-DA and Mo<sub>6</sub> should be internalized while GO/Mo<sub>6</sub> should be located at the surface of the bacteria as previously described for GO.<sup>13</sup> Aging of the DMSO solutions of Mo<sub>6</sub> for 6 months led to a total loss of the photoinactivation ability due to hydrolysis of the complex evidently triggered by water traces in DMSO, as previously reported for this complex.<sup>3</sup> On the other hand, aged GO/Mo<sub>6</sub> nanocomposites maintained a high photoinactivation efficiency with a CFU of 0.37 ±
0.26 % (Fig. 7B). The robustness of the system that allows for long-term storage in DMSO suggests a protective of the GO sheets against the hydrolysis of the clusters. Overall, the observed enhancement of the efficiency of the photoinactivation of *S. aureus* was clearly related to a synergistic effect of both components, GO and Mo<sub>6</sub>, probably originating from photoinduced electron transfer from the triplet states of excited Mo<sub>6</sub> to GO leading to the formation of reactive oxygen species.

Experimental

*Reagents and general procedures.* Compound [{Mo<sub>6</sub>I<sub>8</sub>}(OCOC<sub>4</sub>H<sub>8</sub>PPh<sub>3</sub>)<sub>6</sub>]Br<sub>4</sub> (Mo<sub>6</sub>) was prepared according to a previously published procedure.<sup>3</sup> Molybdenum, iodine, sodium methanolate and (4-carboxybutyl)triphenylphosphonium bromide were obtained from Sigma Aldrich and used as received. Solvents for synthesis were purchased from Penta (Czech Republic) and dried over molecular sieves (3 Å). The following reagents were used for the synthesis of graphene oxide without purification: graphite powder (Wako, 98.0%), sulfuric acid (Wako, 95%), sodium nitrate (Wako, 98.0%), potassium permanganate (Kanto, 99.3%), hydrogen peroxide (Kanto, 30.0%), hydrochloric acid (Wako, 35.0%-37.0%).

Raman measurements were recorded with an inVia Reflex spectrometer operating at 732 nm. X-ray photoelectron spectroscopy (XPS) measurements were performed with a JEOL JPS-9010TR spectrometer. Atomic force microscope (AFM) images were obtained using the AFM multimode function on a Digital Instruments AFM. Images of the nanoparticles were acquired by a JEOL 2100 transmission electron microscope (JEOL, Japan). Size distributions and corresponding zeta potentials were determined by dynamic light scattering (DLS) on a particle size analyzer Zetasizer Nano ZS (Malvern, UK). Powder X-ray diffraction (XRD) patterns were recorded using a PANalytical X'Pert PRO diffractometer in the transmission setup equipped with a conventional Cu X-ray tube (40 kV, 30 mA). The molybdenum content of GO/Mo<sub>6</sub> was measured by inductively coupled plasma mass spectrometry (ICP-MS, PerkinElmer, Concord, ON, Canada). The sample was isolated by centrifugation of the GO/Mo<sub>6</sub> water dispersion (10 000 rpm, 5 min), and drying of the solid under reduced pressure for 24 hours. Quantification was carried out via external calibration. The ICP-MS measurement conditions were as follows: RF power 1.1 kW, nebulizer gas flow rate 0.76 L min<sup>-1</sup>, auxiliary gas flow rate 1 L min<sup>-1</sup>, plasma gas flow rate 11 L min<sup>-1</sup>, measured isotope <sup>98</sup>Mo as an analyte and <sup>115</sup>In as an internal standard. UV-vis absorption spectra and luminescence properties were measured on an FLS1000 spectrometer (Edinburgh Instruments, UK) using a cooled PMT-900 photon detection module (Edinburgh Instruments, UK). Aqueous dispersions (0.1 mg mL<sup>-1</sup>
GO/Mo₆) were saturated with air or argon to assure different oxygen concentrations for phosphorescence analyses. The FLS1000 spectrometer was also used for time-resolved phosphorescence measurements ($λ_{exc}$ = 405 nm, VPLED Series) and the recorded decay curves were fitted to exponential functions by the Fluoracle software (v. 2.13.2, Edinburgh Instruments, UK). Phosphorescence quantum yields of the samples were recorded using a Quantaurus QY C11347-1 spectrometer (Hamamatsu, Japan). Singlet oxygen phosphorescence was measured on a Fluorolog 3 spectrometer using a Hamamatsu H10330-45 photomultiplier. In this case, aqueous dispersions of similar absorbance were saturated with oxygen to magnify phosphorescence signals of O₂(^1Δg). Evaluation of the photoinduced oxidation of DCF-DA was performed by adding 10 uM of DCF-DA to the GO/Mo₆ water dispersion (0.01 mg mL⁻¹), illuminating with a 12x10 W LED source (Cameo) (460 nm, 18 mW cm⁻²) for 0, 2, 4, and 6 min and measuring the fluorescence of the whole suspension at 488/525 nm.

*Preparation of graphene oxide.* Graphene oxide (GO) was synthesized based on the Hummer's method. Sulfuric acid (92 mL), sodium nitrate (2 g) and graphite (2 g) were added to a 500 mL beaker and stirred for 30 minutes in an ice bath. After adding potassium permanganate (10 g), the mixture was stirred at 35°C for 40 min. After adding pure water (92 mL) again in the ice bath, the mixture was stirred at 70°C for 20 min. Then, pure water (200 mL) was added, and 30% hydrogen peroxide was added little by little until the absence of formation of bubbles. The precipitate obtained by centrifugation (3000 rpm, 10 min) was washed with 5% hydrochloric acid and pure water. Centrifugation (15000 rpm, 40 min) was performed to remove ions such as sulphate ions that cause a decrease in pH, and the supernatant was discarded. The obtained precipitate was dispersed in pure water and centrifuged to collect the supernatant (8000 rpm, 30 min). Finally, the GO dispersion was concentrated by centrifugation (15000 rpm, 40 min) to reach a concentration of 1 g L⁻¹.

*Preparation of GO/Mo₆ dispersions.* The colloidal dispersions of GO were freeze dried to collect dehydrated GO. The freeze-drying method prevents any restacking of the sheets and facilitates their dispersion in other solvents. Then, colloidal dispersions of GO in DMSO were prepared at a concentration of 1 g L⁻¹. [{Mo₆I₈}(OCOC₄H₈PPh₃)₆]Br₄, dissolved in DMSO solvent at a concentration of 1 g L⁻¹, was added to the GO dispersions. The resulting dispersions were stirred at 200 rpm for 1 day and directly used for formation of water dispersions (DMSO:H₂O v:v=0.01).

*Photoinactivation of bacteria and ROS determination.* Bacterial samples of *Staphylococcus aureus* were cultivated at 37 °C and stored at 4 °C either in the liquid Luria-Bertani (LB)
medium or on LB agar. The stock solutions of Mo₆ were prepared in DMSO. All experiments were performed in triplicates. The stock inoculum of *Staphylococcus aureus* was prepared by diluting bacteria in water and standardizing suspension to 1 McF. A 100 µL aliquot of the inoculum was taken and mixed with GO, Mo₆ or GO/Mo₆ to a final concentration of 0.01 mg mL⁻¹. The samples were incubated for 2 h in the dark at laboratory temperature and afterwards irradiated with a 12x10 W LED source (Cameo) (460 nm, 18 mW cm⁻², 15 min). For quantification of inactivated bacteria, the Miles and Misra method on LB agar was used. Evaluation of the photoinduced oxidation of DCF-DA was performed by treating inoculum of *S. aureus* at 1 McF in water with 0.01 mg mL⁻¹ of GO, Mo₆ or GO/Mo₆ (1% DMSO) for 2 hours, adding DCF-DA in DMSO to a final concentration 10 uM, illuminating with a 12x10 W LED source (Cameo) (460 nm, 18 mW cm⁻²) for 0, 5, and 10 min and measuring the fluorescence of the whole suspension at 488/525 nm. The control experiments were performed using the same concentration of DMSO (1% v/v in final suspension) introduced by the addition of GO, Mo₆ or GO/Mo₆ in the dark or upon irradiation. The effect of aging of dispersions on the antibacterial activity was performed using 6 months-old DMSO dispersions of Mo₆ and GO/Mo₆.

## Conclusion

We have designed a nanocomposite material based on graphene oxide sheets and a Mo₆ compound. Lyophilization of the GO sheets after preparation allowed for the formation of stable colloidal dispersions of GO. Upon the addition of the DMSO colloidal dispersions of GO and Mo₆ to water, the formation of positively charged nanoaggregates of Mo₆ occurred, followed by their immobilization on the surface of the GO sheets *via* electrostatic interaction as demonstrated by DLS, TEM, ICP-MS, and absorption and luminescence spectroscopy. In agreement with electronic properties of GO when combined with photoactive sensitizers, photoinduced electron transfer from excited states of Mo₆ to GO was suggested for explaining the dramatic quenching of the triplet states when Mo₆ is associated with GO. This phenomenon also led to the inhibition of singlet oxygen formation by Mo₆. Injection of electrons from Mo₆ into the conduction band of GO led to the formation of reactive oxygen species, possibly superoxide, hydrogen peroxide or hydroxyl radicals, providing a robust antibacterial activity against *S. aureus* far stronger than that of the isolated components of the nanocomposite. The observation of the synergistic effect paves the way for the design of nanomaterials with enhanced photooxidative properties which potential applications in life and environmental sciences. The easy preparation of the described material as well as the use of inexpensive
components suggest relevant potential for scaling up the production of this nanocomposite material. From a prospective point of view, we believe that the properties of such nanocomposite can be further tuned and optimized by the careful choice of apical ligands of cluster complex and by varying the size, oxidation rate or chemically modifying the surface of the GO sheets. Also, incorporation of the nanocomposite into functional materials such as surfaces and membranes could expand the potential for antibacterial and environmental applications.

## Supporting Information

Photographs of GO dispersions, TEM and AFM of GO, DLS of Mo<sub>6</sub>, Zeta potentials of GO, Mo<sub>6</sub> and GO/Mo<sub>6</sub>, powder XRD of GO/Mo<sub>6</sub>, absorption spectra, luminescence spectra, luminescence decay kinetics, and photoinduced ROS production of GO, Mo<sub>6</sub> and GO/Mo<sub>6</sub> (.PDF).

## Acknowledgments

This research work was supported by the Czech Science Foundation (21-16084J) and the Japan Society for Promotion of Science Kakenhi C project (20K03887). We acknowledge the assistance provided by the Research Infrastructure NanoEnviCz, supported by the Ministry of Education, Youth and Sports of the Czech Republic under Project LM2018124. We are grateful to Petr Bezdička for the measurement of powder XRD pattern and to Antonin Kaňa for the analysis of the molybdenum content by ICP-MS.
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<figure><img src="image_9.png" /></figure>

Photoinduced electron transfer in a graphene oxide/molybdenum cluster nanocomposite material leads to
enhanced Staphylococcus aureus photoinactivation.

329x178mm (600 x 600 DPI)