Through in-situ measurements, we directly probe the charge transfer as the interfacial dipoles are formed. It is found that the adsorbed molecules do not affect electron scattering rates in graphene, indicating that charge transfer is the main mechanism governing the level alignment. From the amount of transferred charge and the molecular coverage of the grown films, the amount of charge transferred per adsorbed molecule is estimated, indicating very weak interaction.The unique density of states and exceptionally low electrical noise allow graphene-based field effect devices to be utilized as extremely sensitive potentiometers for probing charge transfer with adsorbed species. On the other hand, molecular level alignment at the interface with electrodes can strongly influence the performance of organic-based devices. For this reason, interfacial band engineering is crucial for potential applications of graphene/organic semiconductor heterostructures. Here, we demonstrate charge transfer between graphene and two molecular semiconductors, parahexaphenyl and buckminsterfullerene C In addition to high transparency, high mechanical strength, flexibility, thermal as well as chemical stability, and ease of functionalization, there are also crucial advantages of graphene as an electrode material in organic electronics. These are mainly based on the favorable band alignment with many organic semiconductors and on their impeccable growth morphologies on graphene, which relies on the van der Waals (vdW) nature of the interface3. As a consequence, these heterostructures exhibit low injection barriers, high charge extraction and injection efficiencies with electronic decoupling and preservation of the intrinsic functionality of the molecular crystals at the interface19. Hence, graphene is employed as a carrier injection layer between organic semiconductors and metallic contacts4 and as vdW electrode7. Efficient charge separation in graphene/organic semiconductors photo-transistors15 as well as organic light emitting diodes23 have been demonstrated. Moreover, heterointerfaces between organic semiconductors and other two-dimensional (2D) materials – like hexagonal boron nitride and MoS2 – have been realized, enhancing existing and enabling new functionalities in organic electronics based devices25.Graphene has a significant potential to be used as a new transparent conductive electrode material in flexible and wearable electronics, optoelectronics, and energy applications10. Using a gated graphene field-effect device enables precise control of graphene’s Fermi level position26, which affects charge transport through the interface23. This allows even tuning the molecular orbitals at the interface27. Furthermore, in-situ sheet resistance measurements combined with ultraviolet photoelectron spectroscopy have been used to investigate charge transfer between graphene and Cs2CO3 in an organic matrix22, where the formation of a large interface dipole and n-type doping of graphene were observed. In general, materials with a work function (Φ) substantially lower than that of graphene are needed to achieve n-type doping, as was also demonstrated by deposition of potassium and ZnO28. Recently, interfaces between graphene and metal oxides have been investigated as effective hole-injection layers in organic light emitting diodes21, exhibiting p-type doping of graphene through charge transfer and formation of an interfacial dipole.Interfacial band engineering–through charge transfer and band alignment–is crucial for potential applications of graphene-organic semiconductor heterostructures. Doping of graphene by charge transfer with organic molecular layers has been investigated using Kelvin probe force microscopy and electrical measurements of graphene field effect devices26 and the exceptionally low electrical noise29, field effect devices can be utilized as extremely sensitive potentiometers for probing charge transfer between graphene and adsorbed species. This was used even to detect individual adsorption/desorption events of gas molecules as NO2 on micrometer-scale devices30. Moreover, in-situ electrical characterization was employed to reveal the relation between SiO2 and the effective p-type doping of graphene under ambient conditions31, showing the necessity of both, water and oxygen in this process.Owing to the unique density of states of graphenein-situ electrical characterization is needed which strictly avoids further sample treatment and exposure to ambient atmosphere, especially in the cases when charge transfer is expected to be small. Yet, a study of charge transfer between molecular crystals and graphene by in-situ electrical measurements with simultaneous capability of tuning graphene’s Fermi level position and type of majority carriers is greatly lacking.Nonetheless, the high sensitivity of graphene devices goes along with a lack of selectivity. The effects of marginal remnants, arising from the device preparation  often mask the charge transfer effect that is actually under investigation. For this reason, 33, a wide HOMO-LUMO gap, intrinsic organic semiconductor with well matching Φ to graphene35; and buckminsterfullerene C60, an n-type semiconductor with Φ over 1 eV larger than that of graphene37. The organic thin films (0.5–10.0 monolayers (ML)) have been prepared using a hot wall epitaxy (HWE) setup38 equipped with electrical contacts to the sample in order to enable in-situ electrical characterization. As substrates, field effect transistors (FETs) with an exfoliated graphene channel have been used.In this study, we examine charge transfer between graphene and two molecular crystals: para-hexaphenyl (6P)in-situ the effect that deposited molecular crystals have on the transfer curves of graphene FETs, and thus probe the formation of the interfacial dipole. The setup allows us to control graphene’s Fermi level position prior (and during) the growth experiments. The estimated charge transfer per adsorbed molecule deduced from the graphene’s Fermi level shift and the molecular coverage indicate a very weak interaction. Furthermore, the same setup has been used for desorption experiments with 6P, showing that the charge transfer process is completely reversible upon 6P desorption.We study in-situ electrical measurement setup. Graphene films exhibit trace amounts of photoresist contaminations from FET fabrication39. These residues were observed to have similar impact on the morphology of the grown films as in the case of wet-transferred chemical vapor deposited graphene40.The custom-built HWE chamber used in this study has three electrical contacts attached to the sample holder, which allow to probe and tune electrical properties of the samples prior and during the growth. A layout of the growth chamber is illustrated in Fig. SD(VSG)) it is possible to recalculate the position of graphene’s Fermi level, both prior and after the growth (EF0 and EF1) as illustrated in Fig. SG = 0 V prior to the growth experiments. This has been attributed to the unintentional doping that results mainly from the trapped interfacial layer between graphene and SiO2, exposure to ambient water vapor, and lithography residues, thus giving a different CNP value for each sample39. For this reason, performing in-situ measurements of the charge transfer during the growth is essential to eliminate all other contributions  which would affect the transfer curves of graphene in a similar way (see supplementary information for the details on the pre-treatment of graphene FETs). Figure From the transfer curves of graphene FETs (I−6 mbar (300 K) in the growth chamber, directly prior and after the growth of ~0.8 ML of 6P. The device was slightly p-doped with a CNP atVCNP = (0.8 ± 0.2) V, giving EF = −(56 ± 8) meV. The Fermi level was kept below the neutrality point (EF0 = −(81 ± 5) meV), within the linear part of the transfer curve by setting VSG = −1V during the growth experiments. The in-situ measurements of ISD during the growth  were detected in all growth experiments. This fact unambiguously proves that the deposited thin layers of the molecular crystals did not affect scattering rates in graphene. Thus, the doping mechanism is mainly based on charge transfer between graphene and the adsorbed molecules.Solid lines in Fig. SG = (1.0 ± 0.1) V. In order to ensure that graphene’s Fermi level is above the CNP at the beginning of the growth experiment, a constant voltage VSG = 2.5 V was applied during the growth, thus giving the initial position of the Fermi level of EF0 = (74 ± 3) meV. The inset in Fig. SG was ramped from 0 V to 2.5 V prior to 6P deposition. As the gate voltage increases, the ISD curve changes from p-type graphene–through CNP–to n-type graphene. Then, the device was exposed to 6P vapor and the current immediately decreased. Since the experiment started from initially n-type graphene, a decrease in current (at constant VSG) indicates again p-type doping by the adsorbed molecules. Interestingly, the minimal value in current which was reached during the growth experiment matches exactly the minimal value reached by sweeping VSG. This indicates that adsorbed molecules lower the Fermi level of graphene from n-type–through the neutrality point and into p-type–where further p-type doping is seen as an increase in the current. This was confirmed by measuring transfer curves after the growth  in high vacuum for an extended period of time, hence releasing the molecules from the surface of the graphene FETs. The same 31. For comparison, we provide in Fig. SD was measured and correlated to sample temperature TD, as shown in Fig. SD indicates n-type doping demonstrating the reversibility of the charge transfer mechanism. The minimal value of the current reached by annealing was slightly higher than for the case of electrostatic gating  K, then, a rapid reduction in the current sets in, which is attributed to 6P desorption. This might be useful to determine the desorption temperature of organic semiconductors from graphene. However, both molecular rearrangement prior to desorption and the heating rate could affect the desorption temperature42.Figure z scale for both images is used to highlight that the majority (over 95%) of the material was successfully removed by annealing for only 80 minutes. Extended annealing times or higher annealing temperatures would entirely remove the 6P, however, this could also affect the serial resistance of the thin gold electrodes and was therefore avoided.AFM was used to monitor the amount of material removed from the surface by annealing. An overview of the device prior to thermal desorption is presented in Fig. 60 was also investigated. Figure in-situ electrical measurements during the growth of C60. Data for two devices are presented in the same manner as for 6P  V. Due to strong initial intrinsic p-doping of the sample, the CNP could not be reached within the accessible range of VSG (without risking electrostatic breakdown of the SiO2). The position of the CNP was estimated considering the value observed in air prior to degassing and annealing of the device. Hence, there is a larger uncertainty of the exact initial position of the Fermi level (EF0 = −(160 ± 20) meV). However, this does not affect the shift of the transfer curves and the amount of transferred charge introduced by the adsorbed molecules, which can still be estimated from Fig. SD was recorded as a function of time 44 and the ionization energy of the molecular crystals is considered. In both cases, the Fermi level in graphene is expected to be at least 1 eV above the highest occupied molecular orbitals. Figure 60 prior to interaction . The data for the ionization energies, Φ, and band gaps of 6P and C60 were taken from ultraviolet photoelectron spectroscopy (UPS) of bulk molecular crystals and thin films deposited on highly oriented pyrolytic graphite45. As the interface is formed, electrons are transferred from graphene into the growing molecular crystals. Considering the low density of states of both, 6P and C60, and the small amount of material in the grown films (less than 10 nm), Fermi level alignment in the ordinary sense (bulk interfaces) is most likely not possible and would require significantly thicker films37.Transfer of electrons from graphene–and consequent p-type doping of graphene by both 6P and C60 , allow an estimation of the number of deposited molecules. From these two values, the average charge transfer per adsorbed molecule can be calculated. A compilation of the estimated charge transfer per adsorbed molecule as function of the CNP position  is provided in Fig. x axis indicate the uncertainty of the initial CNP position, while error bars in the y axis mainly originate from the uncertainty of the total volume of the molecular crystals grown on the device surfaces (measured by AFM). The obtained charge transfer per adsorbed 6P molecule was found to be ~4.7 × 10−4 electrons/6P, and for the case of C60 somewhat higher values of ~1.1 × 10−3 electrons/C60 were deduced. No significant change of the charge transfer was observed within the available range of the applied gate voltage , and the observed differences between individual devices are likely related to sample-to-sample variations.Since the shift of the CNP is known, it is possible to estimate the number of transferred electrons from graphene. The detailed morphology of the grown films revealed by AFM (see supplementary information) together with the most probable crystal phases of both 6P and C6 and for the graphene/C60 interface measured by scanning tunneling spectroscopy and transport characteristics46, revealing weak interaction and small charge transfer from graphene. Vertical heterostructures using a graphene/C60 interface have shown slight downshifts of graphene’s Fermi level upon C60 deposition14. In addition, charge transfers between epitaxial graphene and gold, antimony, and bismuth have somewhat larger values47, in the order of 2–10 × 10−3 electrons taken from graphene per adsorbed metal atom. Very recently, molecules (as acetone and toluene) trapped at the interface between graphene an SiO2, and the influence of arrangement of C60 on graphene and the role of the supporting substrate on the charge transfer have been investigated49, in both cases exhibiting charge transfer in the order of ~10−3 electrons per molecule.Similar results were obtained for the charge transfer at the graphene/pentacene interface measured by UPS51. Fractional charge transfer has been reported for molecules adsorbed on clean metal surfaces, usually through weak chemisorption or covalent bonding52 resulting in an excess charge homogeneously distributed among the molecules, yielding an effective fractional charge per each molecule. As an alternative scenario, the integer charge transfer was reported for weakly interacting interfaces like passivated metal surfaces51. In this case, electrons tunnel from the metal and are localized only on some molecules, while the others remain electrically neutral. In this study, fractional charge transfer is less likely due to the vdW nature of the interface. Moreover, measurements of the source-drain current during the growth did not show significant changes between the first and several subsequent layers, for the case when multi-layers are grown. This observation further indicates that the charge transfer mechanism is more likely an integer than a fractional one.The fact that over thousand molecules are needed to extract only one electron from graphene raises the question of the nature of the charge transfer mechanism. The charge transfer could either be fractional or integer60) was observed. As the interface forms, less than 10−3 electrons per molecule are taken from graphene, consequently lowering graphene’s Fermi level and introducing p-type doping of graphene. This result was found not to be dependent on the initial type of the majority carriers in graphene , confirming that band alignment is more important than the type of doping when considering interfacial band engineering with graphene. Moreover, the fact that the adsorbed molecules have mainly affected the ambipolar transfer curves of graphene field-effect devices through lateral shifts unambiguously proves that the scattering rates in graphene were not affected by the adsorbed molecules. This confirms that the interaction occurs principally through charge transfer and formation of an interfacial dipole, with further indications that integer charge transfer occurs in these systems.In summary, a weak electronic interaction through charge transfer between graphene and molecular semiconductors  and drain (D) top contacts were made by positive mask UV photolithography, with a (20–30) nm thick gold layer. Channel length was ~10 μm, and width (W) was (5–30) μm (depending on the shape of the flakes).Graphene flakes were prepared by micromechanical exfoliation of kish graphite. Flakes were deposited on highly doped Si substrates (less than 0.01 Ω cm), serving as a back-gate electrode (G), with (80 ± 2) nm thin layer of dry thermal SiO−6 mbar) for 12 hours, followed by annealing in high vacuum prior to the growth of molecular crystals. More details on the pre-treatment of the graphene FETs is given in the supporting information, where 6P crystallites are also grown at elevated TD (365 K) in order to accent the impact of the surface contaminations on the growth of the molecules.In order to have stable transfer characteristics of the devices and to ensure reproducibility of the data, all samples were left to degas in high vacuum (≈1 × 10SD) and S and G (VSG) were applied, and the currents between S and D (ISD) and S and G (ISG) were measured. To avoid electrostatic breakdown of the SiO2, ISG was monitored and set not to exceed 1 nA. All transfer curves ISD(VSG) were measured at room temperature in either high vacuum or ambient atmosphere. Five subsequent transfer curves were measured each time to ensure reproducibility, with ΔVSG = 0.01 V and time steps of 200 ms. For in-situ measurements of ISD, the time step was set to 500 ms. The sample temperature (TD) was kept constant at (300 ± 2) K in order to exclude other potential contributions to the charge transfer process. The position of the Fermi level in graphene was calculated considering a parallel plate capacitor between graphene and SiO2 and taking the Fermi velocity of electrons in graphene as 106 m/s (see supplementary information for more details). Field effect mobilities were extracted considering the diffusive regime of charge transport (L = 10 μm). ΔISD/Δ VSG was obtained from the linear parts of the transfer curves. Serial resistances (RS) of the devices (mainly arising from thin Au pads) were measured for each device batch by Kelvin probe force microscopy measurements of the voltage drop between S and D. RS was found to be between 20% and 30% of the total device resistivity. It is worth to mention that only the absolute values for the field-effect mobilities are affected by RS.Electrical measurements were carried out within an HWE chamber, using a Keithley 2636 A SYSTEM SourceMeter. Voltages between S and D  with fix source (TS) and wall (TW) temperatures, for 6P TS = 508 K, TW = 518 K, and for C60 TS = 623 K, TW = 638 K.Thin films of molecular semiconductors were grown using a home-built HWE setup with electrical connections to the sample holder that allow β-phase of 6P crystallites is expected (the Baker structure) and the fcc structure of C60. The morphology of C60 films indicated layered growth (step edge height of ~0.8 nm), while 6P films were found to consist of both, islands of up-right standing molecules (~2.6 nm) and needles with flat-laying molecules .The morphology of the samples was investigated employing an Asylum Research MFP-3D AFM system operating under ambient conditions. Olympus AC160TS probes were used with typical force constants of 20–80 N/m and tip curvature radii of 5–7 nm. AFM topography images of the samples were processed using the open source software Gwyddion (version 2.38). The thickness of the molecular crystals was estimated considering the total volume on the device active area and presented as an equivalent in complete monolayers (ML) of the bulk structure  of the molecular crystals, since an ideal layer-by-layer growth was not observed. In the considered ranges of the growth temperatures, only the monoclinic The datasets generated during and/or analysed during the current study that are not included in this published article (and its supplementary information files) are available from the corresponding author on reasonable request.Supplementary Information


The formation mechanisms of two different types of grain boundaries (GBs), the weakly bound overlapping GB and the covalent bound GB, during graphene domain coalescence are revealed by both theoretical modeling and experimental observations. The formation of grain boundaries (GBs) in graphene films is both fundamentally interesting and practically important for many applications. A GB in graphene is known as a linear defect and is formed during the coalescence of two single crystalline graphene domains. The covalent binding between domains is broadly known as the mechanism of GB formation during graphene chemical vapor deposition (CVD) growth. Here, we demonstrate another GB formation mechanism, where two graphene domains are connected by weak van der Waals interactions between overlapping graphene layers. The formation mechanism of the overlapping GBs (OLGBs) is systematically explored theoretically and the proposed conditions for forming OLGBs are validated by experimental observations. This discovery leads to a deep understanding of the mechanism of graphene CVD growth and reveals potential means for graphene quality control in CVD synthesis. Among these methods, graphene CVD growth on a transition metal surface stands out because of the potential for the massive production of high quality, very large area graphene at a relatively low cost, and the existence of numerous parameters which allow for the precise tuning of the number of graphene layers and/or the achievement of controllable doping.7 In most CVD experiments, a graphene film is formed through the coalescence of a huge number of micro-sized domains. The coalescence of two graphene domains of different orientations will certainly lead to a grain boundary (GB) between them and therefore GBs in a graphene film are considered to be the main type of defects that greatly impede the quality of the graphene.11 Although the structures of GBs in graphene have been broadly studied theoretically,16 their formation mechanism during CVD growth remains unexplored.Various methods for the synthesis of high-quality graphene, including the mechanical exfoliation of graphite,15 Here we term this kind of GB a covalently bonded GB (CBGB). The formation process of CBGBs has been broadly adopted to understand multicrystalline graphene film growth. However, some recent experimental observations showed evidence of another type of GB in graphene. In 2011, Robertson et al. found that graphene grown on Cu substrates by atmospheric pressure CVD (APCVD) with a high H2 partial pressure contains two types of boundaries, CBGBs and boundaries from the overlapping of graphene layers from both domains, which we term overlapping GBs (OLGBs).17 Afterwards, OLGBs were also observed by Tsen et al.18 and Rao et al.19 with both studies showing that OLGB formation has a large impact on the current across the resulting graphene sheet. These experimental evidences clearly indicate that two graphene domains can be connected either by CBGBs or OLGBs which therefore, needless to say, impose distinct differences on graphene's performance. Apart from the effect on conductance, we can anticipate that graphene with CBGBs is mechanically much stronger than graphene with OLGBs, and that the thermal conductivity through an OLGB must be much lower than that through a CBGB. Therefore, it is of great importance to unveil the formation mechanism of these two types of GBs in order to control them during graphene synthesis.In most CVD processes, the nucleation of graphene domains is a random process and therefore there is no control over their locations and crystalline orientations. The coalescence of two graphene domains can be achieved by forming covalent C–C bonds between them, with the disruption of the two periodic hexagonal lattices resulting in a linear defect, which is composed of pentagons and heptagons (5|7s).2 partial pressure in the feedstock is the key experimental parameter for controlling the GB type. Considering the high formation energy of CBGBs with a misorientation angle of 10–25°, OLGBs are expected to be easily formed between graphene domains with crystalline orientation angle differences in the same range. Careful experimental studies and data in the existing literature unambiguously validate both the role of hydrogen in GB formation, and our theoretical predictions.In this manuscript, the formation processes of CBGBs and OLGBs and their competition during graphene CVD growth on Cu substrates are systematically investigated. It is demonstrated that the formation of OLGBs is due to the high stability of the hydrogen passivated graphene edge and therefore the H2.ab initio simulation Package (VASP).21 The exchange correlation potential is described by the Perdew–Burke–Ernzerhof implementation of the Generalized Gradient Approximation (GGA).22 The projector augmented wave (PAW) method is employed to treat the interaction between the valence electrons and the ion cores.23 Due to the unsatisfactory performance of GGA functionals in describing weak van der Waals (VDW) interactions, these calculations are corrected by the more accurate DFT-D2 method.24 An energy cutoff of 400 eV is adopted for the plane wave basis set.All DFT calculations in this study are performed by using the Vienna 3, as shown in Fig. S1 in the ESI.–1. The k-point mesh is sampled as 1 × 5 × 1, which is tested to provide enough accuracy.To investigate the structural stability of the various edges and GBs in graphene grown on the Cu substrates, a slab of 3 atomic layers with the bottom layer fixed is chosen to represent the metal (111) surface. The slabs are separated by over 15 Å to eliminate interactions between the repeated images. To keep the size of the Cu slab commensurate with graphene, the lattice constant of Cu is reduced by less than 4%. The unit cell size of the substrate is 33.95 × 6.53 × 30 Å3.θ, ranging from 0 to π/3, and the angle of alignment, φ, ranging from –π/6 to π/6.15 The combination of these two degrees of freedom  leads to a very large number of potential GB structures15 and therefore it is impractical to study the formation of all possible GBs. Herein, a representative GB, as shown in θ = 21.8° and φ = 0° on a Cu(111) surface is adopted as an example to explore the formation processes of CBGBs and OLGBs.A GB in graphene has two degrees of freedom, the misorientation angle, P(H2).31 As explained in our previous studies,32 the edge of a graphene domain can be either passivated by the transition metal surface at low P(H2) or terminated by hydrogen atoms at high P(H2).Many experiments have shown that graphene growth is highly dependent on the partial pressure of hydrogen in the carrier gas, –1. Then, when the two edges are sufficiently close to each other, C–C bonds form between them spontaneously  at high temperature and with low P(H2),33 so we can anticipate that the experimental condition for forming CBGBs is the same.Firstly, let us consider the coalescence of two metal passivated graphene edges. As shown in aneously  and someubstrate . The fin32 The growth of two H terminated graphene edges on a Cu(111) surface is shown in μ), which is defined as the chemical potential difference between a carbon atom in the feedstock and in graphene . This is expected because the formation of an overlapping GB only involves the variation of the very weak van der Waals interactions and a slight bending of the graphene. So we can conclude that the formation of the OLGB is very possible during graphene CVD growth if the edges of the graphene domains are terminated by H atoms.Next, let us consider the coalescence of two graphene domains with H terminated edges, which is known to be crucial for understanding graphene CVD growth.e see ESI. One canversus that of metal passivated graphene edges is crucial for the formation of OLGBs. As demonstrated in previous studies, the Gibbs free energy difference between an H terminated graphene edge and the corresponding metal passivated graphene edge is32EF = EF(HTGE) – EF(MPGE) is the formation energy difference between an H terminated graphene edge and a metal passivated graphene edge, ΔFV is the vibrational free energy of the H atoms at the H passivated graphene edge, μH is the chemical potential of H2 in the gas phase as a function of temperature, T, and P(H2), and NH is the number of H atoms. The details of the calculation are provided in the ESI.The above analysis shows us that the formation of H terminated graphene edges is a precondition for forming OLGBs, and thus the stability of H terminated graphene edges T = 1300 K) the metal passivated graphene edge is more stable than the H terminated graphene edge even if the hydrogen partial pressure P(H2) < 10–3 Torr and therefore the coalescence of two metal passivated graphene edges would lead to a CBGB. In contrast, the H terminated graphene edge prevails at lower temperatures or higher P(H2) values and the coalescence of the two hydrogen terminated graphene domains may lead to an OLGB.The thermodynamic diagram of the specific H terminated graphene edge and the metal passivated graphene edge on the Cu(111) surface can be obtained by solving eqn (1) and is plotted in –1. As expected, it can be clearly seen that the OLGB is increasingly preferred if the formation energy of the CBGB is higher.It is important to note that two H terminated graphene edges might merge into a CBGB because graphene growth always involves the fracture and reforming of C–C and C–H bonds at the edge. In the case that a CBGB is energetically more favorable than a corresponding OLGB, two H terminated graphene edges certainly have the probability of being welded into a CBGB. To include this probability, let us compare the Gibbs free energy of a CBGB and a corresponding OLGB see ESI. Fig. 3b4.2 rich and H2 poor conditions. Graphene samples were grown on copper foil  using low-pressure CVD (LPCVD). Before growth, the copper foils were electropolished according to the method reported by Han et al.34 Then, the copper foil was loaded into the hot center of the furnace and heated to 1000 °C in a 350 sccm H2 atmosphere with a pressure of 500 Pa within 30 min and annealed under this temperature for 2–4 h to enlarge the domain sizes of the polycrystalline copper foil. The H2 poor CVD experiments were carried at a H2 partial pressure of ∼3.7 × 10–2 Torr and the specific experimental parameters are 5 sccm H2, 1 sccm CH4, and 300 sccm Ar with the system pressure kept at 500 Pa for 20 min. The H2 rich experiments were carried with a H2 partial pressure of ∼10 Torr and the experimental parameters were 1 sccm CH4, and 800–900 sccm H2, and the system pressure was kept at 1200–1500 Pa for 1 h. The graphene samples were transferred onto a 300 nm SiO2/Si substrate using a wet-etching method with the help of polymethyl methacrylate (PMMA) for characterization.We carried out experiments to verify the above predictions. We grew graphene under both H2 partial pressures  and . From an experimental perspective, the measured misorientation angle of a GB is usually folded in the range of  because it is impractical to identify the true misorientation angles θ from 60°–θ without atomic resolution  in the observed misorientation angle range of θ ∈ . To verify this prediction, we counted the numbers of OLGBs and CBGBs in versus the misorientation angle is shown in T is 1300 K, β and λ are then found to be 1.24 × 10–5 and 12.6, respectively. Clearly, the fitting line shown in Recent studies have shown that the misorientation angle dependent formation energy of CBGBs shows an “M” shape, with two peaks appearing in the ranges of  and  respectively, ranging from 0° to 60° .12–15 Thsolution . Therefo33 So, OLGBs were rarely observed for graphene growth on these catalysts because of the high stability of metal passivated graphene edges.It is worth mentioning that the coalescence of two domains with metal passivated graphene edges must lead to a CBGB due to the high activity of the edge C atoms and the high instability of the metal passivated graphene edges. Previous studies have demonstrated that most catalysts, such as Ni, Co, Pt, Pd, Rh, Ir, and Ru, are more active than Cu and the metal passivated graphene edges on these surfaces have much lower formation energies than that on Cu.5.In summary, we performed a systematic theoretical study on the formation mechanism of overlapping grain boundaries (OLGBs) during graphene CVD growth, including the thermodynamic stability and the formation processes. This study reveals that the hydrogen passivation of the graphene edge is a precondition of forming OLGBs, and therefore OLGBs might be formed during graphene CVD growth on a less active catalyst surface, with high hydrogen partial pressure and high temperature. Further analysis reveals that an OLGB can be easily formed during the coalescence of two graphene domains with crystalline orientation differences in the range of 10 to 25 degrees, due to the high instability of the CBGBs. Furthermore, both our experimental study and literature data validated the proposed experimental conditions for OLGB growth and the theoretical prediction of the abundance of OLGBs in the misorientation angle range of 10 to 25 degrees. This study explains well the mystery of forming the OLGBs observed in previous graphene synthesis experiments, and leads to a deeper insight into graphene CVD growth. It also allows us to achieve the desired graphene growth by rational experimental design.


Scanning transmission electron microscopy, x-ray absorption spectroscopy, and theoretical calculations suggest that the Pt2 dimers are likely in the oxidized form of Pt2Ox. In hydrolytic dehydrogenation of ammonia borane, Pt2 dimers exhibit a high specific rate of 2800 molH2 molPt−1 min−1 at room temperature, ~17- and 45-fold higher than graphene supported Pt single atoms and nanoparticles, respectively. These findings open an avenue to bottom–up fabrication of supported atomically precise ultrafine metal clusters for practical applications.Supported metal clusters containing only a few atoms are of great interest. Progress has been made in synthesis of metal single-atom catalysts. However, precise synthesis of metal dimers on high-surface area support remains a grand challenge. Here, we show that Pt Controlled fabrication of few-atoms supported catalysts is a major challenge in the synthesis of nanomaterials. Here, the authors show a bottom-up approach to precisely synthesize platinum dimers supported on graphene, which display higher catalytic activity and stability than single atoms and nanoparticles. Decreasing metal particle size is desirable for improving metal utilization, since catalytic reactions take place on the surface of metal nanoparticles (NPs). When a metal cluster contains only a few metal atoms, it could have a discrete energy band structure, tightly correlated with the number of metal atoms. Changing one atom in the ultrafine cluster might largely alter the electronic structure and drastically change its catalytic properties. Such atom-dependent catalytic behaviors have been successfully demonstrated by the model catalysts of mass-selected metal clusters, which were fabricated by soft landing of mass-selected ions from their physical vapor under ultrahigh vacuum conditions14. However, such complicated approach is only limited to model catalyst studies and is not applicable to high-surface area supports for practical applications.Supported metal catalysts are among the most important categories of heterogeneous catalysts in many reactions including chemical upgrading, automobile exhaust treatment, Fischer-Tropsch synthesis, biomass conversions, and many other processes23. Nonetheless, synthesis of atomically precise ultrafine metal clusters, such as dimers, on high-surface area supports, remains a grand challenge. The decisive limitation is the lack of precise control over the aggregation process, which often causes metal NPs formation with a broad size distribution. Protecting metal clusters with a strong ligand can certainly inhibit metal aggregation to a large extent, such as in the case of thiolate-protected Au magic clusters24. However, these strong protective ligands typically poison the metal clusters, and decrease their catalytic activities considerably30. Alternatively, Gates et al. demonstrated that precisely defined iridium and rhodium clusters were achieved by grafting the corresponding carbonyl complexes with a specific number of metal atoms onto oxide supports33. But the success is limited. As a consequence, a general bottom–up approach to synthesize atomically precise metal clusters on high-surface area supports is still missing. Atomic layer deposition (ALD) relies on two sequential self-limiting surface reactions at the molecular level, which are separated by inert gas purging36. This unique character makes ALD possible to bottom–up construct catalytic materials on a high-surface area substrate uniformly and precisely39.Recently, synthesis of supported metal single-atom catalysts (SACs) has been extensively explored and a number of successful examples have been demonstrated2 dimers can be bottom–up fabricated on a graphene support by depositing Pt on phenol-related oxygen anchor sites atom-by-atom in a sequential manner using Pt ALD. The dominant presence of isolated Pt1 single atoms and Pt2 dimers in the corresponding samples were confirmed by both aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and X-ray absorption fine structure spectroscopy (XAFS). Their structures were determined through a combination of density function theory (DFT) calculations and XAFS spectra simulations. In hydrolysis of ammonia borane (AB) for hydrogen generation, graphene supported Pt2 dimers (Pt2/graphene) exhibited a striking activity, which is ~17- and 45-fold higher than that of graphene supported Pt1 single atoms and Pt NPs, respectively. Compared to Pt1 single atoms and Pt NPs, the decreased adsorption energies of both AB and H2 molecules on Pt2 dimers are likely the major reason for the high activity. More importantly, the Pt2 dimers were stable under the current reaction condition and in the inert environment at below 300 °C.Here, we show that Pt22, the nucleation sites of isolated phenols or phenol–carbonyl pairs suggested by Shenoyl et al40. for Pt ALD were first created on pristine graphene nanosheets through acid oxidation followed by high-temperature thermal reduction, as illustrated in the schematic model in Fig. 2/g. Next, Pt ALD was performed on the graphene support by alternately exposing trimethyl(methylcyclopentadienyl)-platinum(IV) (MeCpPtMe3) and molecular O2 at 250 °C. The self-limiting surface reactions between MeCpPtMe3 and the support ensure nucleation of one MeCpPtMe3 molecule on one phenol-related nucleation site during the saturated MeCpPtMe3 exposure .Based on our recent strategy1 single atoms was further utilized as nucleation sites for anchoring the secondary MeCpPtMe3 molecule in the following cycle. Again, the steric hindrance between MeCpPtMe3 molecules restricts chemisorbing one MeCpPtMe3 molecule only on one isolated Pt1 atom. However, we noticed that a considerable amount of Pt NPs were formed after two successive cycles of Pt ALD at 250 °C , a stronger oxidizing regent was utilized to remove the ligand efficiently to form Pt2 dimers (Pt2/graphene) , Pt2 dimers were dominantly formed along with a certain number of Pt1 single atoms . First, the influence of the Pt precursor ligand on the second Pt ALD cycle was examined. In this case, half cycle of Pt ALD was executed on the graphene support by performing the MeCpPtMe3 pulse step only (denoted as MeCpPtMe/graphene) at 250 °C. After that, the ALD reactor was cooled to near room temperature and half amount of the MeCpPtMe/graphene sample was taken out of reactor for ICP-AES analysis; while the rest of the sample was put back to the ALD reactor quickly to perform the second cycle (Pt-MeCpPtMe/graphene) at 150 °C. In nine independent trials, the ICP-AES results showed that the ratio of the Pt loadings of Pt-MeCpPtMe/graphene to those of the corresponding MeCpPtMe/graphene were all very close to one  located at between Pt foil and PtO2, indicating that the Pt in MeCpPtMe/graphene, Pt1 single atoms, and Pt2 dimers were all in a similar oxidation state between Pt0 and Pt4+. The MeCpPtMe3 reference sample exhibits two well-resolved peaks at 1.62 and 1.99 Å in the Fourier transformed (FT) k3χ(k) curve in the real-space (R-space)  Fig. , assignece) Fig. 47,50. Ap1/graphene spectrum had a higher intensity and slightly shifted to 1.65 Å, while the peak at 1.99 Å disappeared. Clearly, the MeCp ligand was combusted off after the O2 exposure step at 250 °C. The first shell peak is assigned to Pt−O and/or Pt−C coordinations. Similar to MeCpPtMe/graphene, a very weak peak at 2.4 Å was visible in the spectrum of Pt1/graphene. However, this peak is significantly different from the Pt–Pt coordination peak (at ~2.62 Å) in Pt foil, thus assigned to the second nearest C/O neighbors of Pt. This suggests the absence of Pt NPs in Pt1/graphene, consistent with our STEM observation  and two O atoms at the interface (the Pt–O bond distance: 2.02 Å) produces an EXAFS spectrum in good agreement with the experimental result  clusters by Schneider et al. previously54. Interestingly, we also noticed that the tilted angle of the Pt2O6 chain could vary largely from ~8 to ~50°, depending on both the size of carbon vacancies and the configurations of two interfacial O atoms  than that of the Pt atom in Pt1/graphene-R (0.40 eV), which indicates that Pt1/graphene-R is more prone to accept electrons than Pt2/graphene-R  revealed by DFT calculations agrees well with the literature57, where Pt NP catalyst deactivation induced by B poisoning was observed during the AB hydrolysis reaction. Bearing this in mind, we further examined the recycling stabilities of the Pt1/graphene, Pt NP, and PtO2 catalysts and measured the B contents in the used samples using ICP-AES. Indeed, catalyst deactivations and considerable amounts of B adsorption were observed on all the used samples  is remarkably stronger than that on Pt2/graphene-R (−1.29 eV). More interestingly, we found that H2 chemisorbs dissociatively on Pt1/graphene-R, but molecularly on Pt2/graphene-R, indicated by the H–H bond distance of 2.02 and 0.96 Å, respectively. Such molecular adsorption of H2 on the Pt2 dimer with a moderate adsorption energy favors H2 desorption during the AB hydrolysis reaction, thereby further boosting the catalytic activity. Taken together, compared to Pt1 single atom, the higher energy position of the unoccupied state of the Pt 5d orbital of the top Pt in the Pt2 dimer might play an important role in weakening the adsorption of both AB and H2 molecules, thus facilitating the activity remarkably.Hion Fig. . It was 2/graphene catalyst exhibited a very high stability in the AB hydrolysis reaction during the recyclability test for five cycles  and 400 °C (Pt2/graphene-400C), respectively  clusters on high-surface area supports for advanced catalysis.In conclusion, we have successfully demonstrated that Pt3, 98%), chloroplatinic acid , ammonia borane (97%), the commercial PtO2 , and Pt/carbon catalysts  were all purchased from Sigma Aldrich. Silica gel was purchased from Alfa Aesar  surface area 300 m2/g). Pristine graphene nanosheet (99.5%) was bought from Chengdu Organic Chemicals Co. Ltd., Chinese Academy of Sciences. All materials were used as received without further purification.Trimethyl(methylcyclopentadienyl)platinum(IV)  and stirred at room temperature for 22 h. Then the mixture was cooled down to 0 °C to add 1.8 g potassium permanganate (KMnO4). After stirring at room temperature and 35 °C for 2 and 3 h, respectively, the mixture was heated to 98 °C and kept at this temperature for another 30 min. Next, it was cooled down to 40 °C, and 90 mL of water and 7.5 mL of hydrogen peroxide  were slowly added into the mixture. After that the precipitate was filtered out by washing with HCl (5%) and ultrapure water, it was dried in a vacuum oven at 45 °C overnight. The dry material was grinded to obtain graphene oxide powder. Finally, the reduced graphene oxide was obtained by thermal deoxygenation of graphene oxide powder at 1050 °C for 2 min under helium at a flow rate of 50 mL/min.Pristine graphene nanosheet was first oxidized to graphene oxide according to the procedure described previously3 precursor and O2 (99.999%) at 250 oC64. Ultrahigh purity N2 (99.999%) was used as the carrier gas at a flow rate of 200 mL/min. The Pt precursor was heated to 65 °C to get a sufficient vapor pressure. The reactor inlets were held at 110 °C to avoid any precursor condensation. The timing sequence was 90, 120, 60, and 120 sec for the MeCpPtMe3 exposure, N2 purge, O2 exposure, and N2 purge, respectively (90-120-60-120).Pt ALD was carried out on a viscous flow reactor  by alternatively exposing to MeCpPtMe1/graphene SAC at 150 °C. Here, O3 was used as the oxidant to make sure that the precursor ligand can be fully removed65. The timing sequence was 90-120-60-120.The second Pt ALD cycle was performed on the PtTwo consecutive cycles of Pt ALD was also performed on the reduced graphene oxide at 250 °C using the same timing sequence.Pt ALD was performed on the silica gel support for one cycle at 250 °C using the same timing sequence.−2 M H2PtCl6 aqueous solution (0.9 mL). Then, the mixture was stirred for 30 min, and then dried in air at room temperature for 12 h. The dried material was first calcined in air at 120 °C for 12 h, and then reduced in 10% H2 in argon at 300 °C for another 2 h to get the Pt/graphene-WI catalyst.A Pt/graphene NP catalyst was synthesized by the wetness impregnation method (Pt/graphene-WI). In this case, 100 mg graphene support was slowly added into a 1.93 × 10L3-edge  were performed in the transmission mode with the Si (111) monochromator at the BL14W1 beamline of the Shanghai Synchrotron Radiation Facility (SSRF), China. The storage ring of SSRF worked at 3.5 GeV with a maximum current of 210 mA.The Pt loadings in these samples were determined by ICP-AES measurements; therein all samples were dissolved in hot fresh aqua regia. The BET surface area was measured on a Micromeritics ASAP 2020 system. Raman spectra were recorded on a LabRAM HR Raman spectrometer with a 514 nm Ar laser in backscattering geometry. Aberration-corrected HAADF-STEM measurements were taken on a JEM-ARM200F instrument (University of Science and Technology of China) at 200 keV. XAFS measurements at the Pt 61.The EXAFS oscillation functions χ(k) were obtained by subtracting the post-edge background from the overall absorption spectra and then normalized with respect to the edge-jump step. The Rbkg value of 1.0 was used for all samples. Subsequently, k3-weighted χ(k) fucntions in the k range of 2.2–13.5 Å−1 were FT to the R space by using a Hanning window of dk = 3.0 Å−1.The acquired EXAFS data were processed according to the standard procedures using the ATHENA module implemented in the IFEFFIT software packages62 using the structural models suggested by DFT calculations. The simulated EXAFS χ(k) functions were also k3-weighted and FT into the R-space by using the same k range of 2.2–13.5 Å−1 as that in the experimental data. During simulations, the coordination numbers were set to the values of the model structures generated by DFT calculations. The amplitude reduction factor S02 was fixed at the value of 0.86 which was determined by fitting the reference metal Pt foil. The Debye–Waller factors for the nearest Pt−O/C and Pt−Pt pairs were set at the typical values of 0.0030 and 0.0065 Å2 determined from the fittings of PtO2 and Pt foil references, respectively, and they were set at 0.008 Å2 for all the other distant paths which contributed barely discernible signals as seen from the experimental data in the R-space. To further improve the match between the simulation and the experimental data, for the MeCpPtMe/graphene sample, the two nearest Pt–C and Pt–O interatomic distances were optimized to 2.00 and 2.02 Å, respectively, both of which are within ~ 2% error level as compared to the optimized structure by DFT calculations . For the other two samples, the simulated EXAFS spectra based on the DFT-generated structures match well with the experimental data, thus no further structure optimization was performed during EXAFS simulations.EXAFS simulations were performed with the FEFF8.4 code66 with a double numerical basis set together with polarization functions (DNP) were adopted to form the Perdew-Burke-Ernzerhof (PBE) exchange-correlation functional within the generalized gradient approximation67, implemented in DMol3 package 68. A DFT-D semi-empirical correction with Tkatchenko-Scheffler (TS) method is applied with the PBE functional to account for the dispersion interaction. Conductor-like screening model (COSMO) with a dielectric constant of 78.54 is adopted to consider the water solvent effect regarding the adsorption of molecule and fragment in AB hydrolysis. A smearing of 0.001 Ha to the orbital occupation is applied to achieve electronic convergence. The real-space global cutoff radius is set to be 4.5 Å. A hexagonal supercell containing (8 × 8) unit cells of graphene monolayer with about 17 Å vacuum layer was used as a support. The convergence tolerances of energy, force, and displacement for the geometry optimization were 1 × 10−5 Ha, 0.002 Ha/Å, and 0.005 Å, respectively. 1 × 1 × 1 k-points grid is used to describe the Brillouin zone for geometric optimization. The adsorption energy is defined by the formula: Eads(AB) = E– and Eads(H2) = E–, where E, E, and Ecatalyst are the total energies for the optimized equilibrium configurations of catalyst with and without AB or H2, respectively; and EAB  is the energy of the AB (gas phase H2) molecule in its ground state. For Pt(111), the supercell is (4×4).All spin-polarized calculations were performed by using the DFT method. The DFT Semi-core Pseudopotential method2/graphene catalyst was used, while the weight of other catalysts was adjusted to keep the same amount of Pt with Pt2/graphene. Typically, 5 mL aqueous AB solution (6.5 × 10−2 M) were introduced into the glass container via a syringe. For the commercial PtO2 catalyst, the mole ratio of Pt to AB was kept as the same with other samples, and the result was normalized to other samples based the amount of Pt. The AB solution and the catalyst were well-mixed by using a magnetic stirrer at a speed of 800 r/min to eliminate any mass-transfer issue. The generated volume of H2 was measured by a water-filled gas burette, where the volume of water discharged was converted into the volume of hydrogen generated69.As a probe reaction, hydrolytic dehydrogenation of AB was performed in a three-necked flask at 27 °C under atmospheric pressure. The flask was immersed in a water bath to control the reaction temperature. 10 mg of the Ptr) of these catalysts were calculated according to the Eq. (The specific rates ( the Eq. :2\docume2, while t is the reaction time in min.Here All the relevant data are available from the authors upon request.Peer Review FileSupplementary Information


Staphylococcus aureus (S. aureus) is a common and dangerous pathogen that causes various infectious diseases. Skin damage, such as burn wounds, are at high risk of Staphylococcus aureus colonization and infection, which increases morbidity and mortality. The phage lysin LysGH15 exhibits highly efficient lytic activity against methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. Apigenin (api) significantly decreases haemolysis of rabbit erythrocytes caused by S. aureus and shows anti-inflammatory function. LysGH15 and api were added to Aquaphor to form an LysGH15-api-Aquaphor (LAA) ointment. The LAA ointment simultaneously exhibited bactericidal activity against S. aureus and inhibited haemolysis. In an LAA-treated mouse model of an MRSA-infected skin wound, the mean bacterial colony count decreased to approximately 102 CFU/mg at 18 h after treatment (and the bacteria became undetectable at 96 h), whereas the mean count in untreated mice was approximately 105 CFU/mg of tissue. The LAA ointment also reduced the levels of pro-inflammatory cytokines  and accelerated wound healing in the mouse model. These data demonstrate the potential efficacy of a combination of LysGH15 and api for use as a topical antimicrobial agent against S. aureus. Staphylococcus aureus causes an extensive range of infections, from skin to systemic infections, which cause organ failure and death [S. aureus can colonize wounds and cause infection [S. aureus colonization and infection, increasing morbidity and mortality among burn patients [Staphylococcus aureus (MRSA), threaten the efficacy of antimicrobial therapies [nd death ,2,3,4. Wnfection ,6,7,8. Dpatients ,10,11 bepatients ,13. Antipatients ,15,16. Hherapies .S. aureus (MSSA), both in vitro and in vivo [S. aureus was unable to develop resistance against LysGH15 under the conditions tested, and that specific anti-LysGH15 antibodies produced in mice are unable to block the activity of LysGH15 [hla and agrA genes that encode alpha-hemolysin (Hla), which ultimately reduces the production of alpha-hemolysin in S. aureus [S. aureus has not been determined yet. Thus, in this study, api was selected. Endolysins encoded by bacteriophages are capable of lysing host cells by breaking down the cell wall ,18,19. P in vivo ,19. LysG in vivo . We also LysGH15 . Api has LysGH15 ,28,29,30. aureus . In a prIn this study, we analysed the effect of an ointment consisting of a combination of LysGH15 and api on the skin decolonization of MRSA. The ointment could not only remove MRSA from the skin wound but also decrease skin inflammation and accelerate wound healing.ad libitum, and the mice were monitored daily. All the animal experimental procedures were performed in strict accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals, which were approved by the State Council of the People’s Republic of China (1.11.1988) and the Animal Welfare and Research Ethics Committee at Jilin University. The permission code was 201701002 and the permission date was 5, Janaury, 2017.Female specific-pathogen-free BALB/c mice that weighed 20–22 g  were housed in filter-top cages in an air-conditioned animal facility at the National Experimental Teaching Demonstration Centre of Jilin University . Water and normal mouse chow were provided ® BAA-1717™) was obtained from the American Type Culture Collection  and employed throughout this study. USA300 was routinely grown in brain heart infusion (BHI) broth  at 37 °C with shaking at 200 revolutions per minute (rpm). The community-associated MRSA strain USA300-TCH1516 (USA300) (ATCCEscherichia coli BL21 (DE3) strain that expressed the full-length LysGH15 protein was previously constructed in the laboratory, and LysGH15 was expressed and purified as described in a previous report [An s report . Api wass report ,18. LysGs report . 600) of 0.6) in BHI broth at 37 °C, with shaking at 200 rpm. The bacteria were collected and washed three times (5000× g for 1 min at 4 °C) with phosphate-buffered saline (PBS) [7 CFU) was spread onto BHI agar. The following ointment formulations were prepared, using the ULTRA-TURRAX disperser  to mix them. Aquaphor containing LysGH15 (60 μg per 0.1 g ointment) and api (500 μg per 0.1 g ointment), Aquaphor containing LysGH15 (60 μg per 0.1 g ointment), Aquaphor containing api (500 μg per 0.1 g ointment), pure api (100 μg per 100 μL PBS), pure LysGH15 (120 μg per 100 μL PBS), pure Aquaphor (0.1 g), and the commercially available topical ointment mupirocin  [We performed a plate lytic assay to determine the efficacy of the LysGH15-api-Aquaphor ointment. The MRSA strain USA300 was grown to the exponential growth phase  ,19. The 0.05 mg)  were use7 CFU) cultures and incubated at 37 °C [The bactericidal activities of different formulations were determined by using time kill assay in vitro. The LAA ointment consisted of lyophilized LysGH15 (50 µg LysGH15 per 0.1 g ointment) and api (500 μg api per 0.1 g ointment) mixed with Aquaphor. The LysGH15-Aquaphor (LA) ointment consisted of LysGH15 (50 µg LysGH15 per 0.1 g ointment) mixed with Aquaphor. The api-Aquaphor (AA) ointment consisted of api (500 μg api per 0.1 g ointment) mixed with Aquaphor. To determine the activity of different formulations in vitro, time kill assays were performed . For theat 37 °C . The CFU. USA300 was grown to the exponential growth phase (OD600 of 0.6) in BHI broth at 37 °C, with shaking at 200 rpm. The bacteria were collected and washed three times (5000× g for 1 min at 4 °C) with PBS. The different formulations were separately added to the USA300 suspensions (1 × 107 CFU). The bacterial lysates were harvested by centrifugation (1100× g for 1 min) at 0, 1, or 2 min after treatment. Subsequently, the bacterial lysates were fixed with glutaraldehyde and dehydrated in a graded ethanol series for 20 min. The specimens were dried overnight at room temperature. The dried and frozen samples were prepared for SEM analysis  [Scanning electron microscopy (SEM) was performed to assess the activity of the different formulations in vitroGermany) .4 CFU/mL). The turbidity of the bacterial culture solutions were measured at 600 nm at 10 h after culturing (to an OD600 of 2.5), and the supernatants were harvested via centrifugation (5500× g for 5 min at 4 °C) and then filtered. To achieve a final volume of 1 mL, 100 μL supernatant and a 25 μL red blood cell pellet were added to the PBS. The mixture was incubated for 30 min at 37 °C. Subsequently, unlysed blood cells were removed by centrifugation (5500× g for 1 min at 4 °C). After centrifugation, the OD450 of the supernatant was measured. The supernatants of the pure USA300 culture served as the 100% haemolysis control [Haemolytic activity was assessed using rabbit erythrocytes, as previously described . The dif control ,31.2 section of the dorsum of each mouse was shaved and cleansed with 70% alcohol, and the epidermis was then stripped using autoclave tape to induce skin damage [7 CFU) suspended in PBS. All the animals were housed individually to prevent post-wounding traumatic damage to the wounds by other mice.A mouse model of skin damage was established using the female BALB/c mice according to the methods used in a previous study, with some modifications . The micn damage . The damAt 24 h after colonization, the mice were randomly separated into six groups: the PBS, Aquaphor, AA, LA, LAA, and mupirocin groups. Each group contained 6 mice. The 0.1 g dose of ointments and commercial mupirocin were applied to the wounds according to the previous study method, with some modification . The micAfter the different formulations were administered and allowed to remain on the USA300-challenged skin for 18 h, 6 mice from each group were randomly selected for euthanasia with an injection of Fatal Plus   and each wound area was excised and weighed to ensure uniformity among the samples. The skin samples were suspended in filter-sterilized PBS and homogenized with sterile mortars and motor-driven Teflon pestles  on ice. The bacterial loads in the skin sample homogenates were measured by serial dilution and plating on mannitol salt agar plates . The concentrations of cytokines  in the skin samples were quantified using an enzyme-linked immunosorbent assay (ELISA)  according to the manufacturer’s instructions .At 5 d after injury, one mouse from each of the six groups  was randomly selected, and the wound specimens from each group were harvested and fixed in 4% formaldehyde buffered with PBS. The formalin-fixed tissues were then processed and stained with haematoxylin and eosin (H&E) using a routine staining procedure and subsequently analysed using microscopy ,37. It has been reported that the alternatively activated macrophage (AAM) plays an important role in wound healing. Arginase I and YM1 were identified as the markers of AAM activation . Thus, q7 CFU/mL) and further incubated at 37 °C. The CFUs were counted at various time points.BALB/c mice were shaved, tape-stripped, and treated daily with LAA or PBS for 9 d. At 10 d, serum samples from the LAA-treated mice were obtained. Each group contained 6 mice. Blood samples were also obtained from the LAA-treated mice for the detection of antibodies using Western blotting. The titre of antibodies against LysGH15 was determined using an ELISA ,32. To dp-value < 0.05 was considered to be statistically significant. The error bars in the figures represent the standard deviations of the means.GraphPad Prism 5  was utilized to analyse the data from the ELISAs. SPSS version 13.0  was employed for the statistical analysis of other experimental data using a one-way analysis of variance (ANOVA). A To test the bactericidal activity of LAA in vitro, both a plate lytic assay and time kill assay were performed. The plate lytic assay demonstrated that LysGH15-api-Aquaphor and LysGH15-Aquaphor ointment or mupirocin ointment can efficiently inhibit the growth of USA300 and induce distinct halos. AA, Aquaphor, and api exhibited no lytic activity a. When LThe USA300 culture supernatant, which contains haemolysins, induced haemolysis of rabbit erythrocytes. When Aquaphor was added to the USA300 culture, the haemolytic activity was unaffected. However, when api, AA, LAA, LAA with anti-LAA serum, or AA with anti-LAA serum was added to the rabbit erythrocytes, no haemolysis was detected, as shown in 7 CFU USA300, the colony count was 2 × 105 CFU per mg of skin after the challenge, and the skin wound became red and swollen with minimal bleeding and no skin necrosis. Aquaphor, AA, LA, LAA, and mupirocin were used to treat the USA300 infection in the mouse model of skin damage, with PBS being used as the control. As shown in When the shaved and tape-stripped area of the mice was topically colonized with 1 × 10Mouse skin sections stained with hematoxylin and eosin (H&E) were used to evaluate the degree of wound healing. As shown in 5 CFU/mg) at 18 h compared with one dose of AA, PBS, or Aquaphor. The USA300 reduction was lower in the LA group (2.8 log units) than in the LAA group. However, the difference in the efficacy of the dose in the LAA group (3.3 log units reduction) compared with the dose in the mupirocin group (3.1 log units reduction) was not significant. The ability of the different treatments to reduce the bacterial counts in the skin samples was investigated . One dosSince IFN-γ, TNF-α, and IL-1β play a critical role in the pathogenesis of MRSA infections, the levels of these pro-inflammatory cytokines were determined. As shown in −1 of 1.8 (1:64)  a. The efOur study provides the first description of the efficacy of LysGH15-api-Aquaphor (LAA) ointment as a topical treatment in vitro and in vivo. Aquaphor was employed as the ointment base. Aquaphor did not affect the lytic activity of LysGH15 or the haemolysis inhibition of api, and it served as a hydrating agent for the LAA ointment . Thus, tS. aureus strains was observed after repeated in vitro exposure to the agent after only one week [S. aureus cells have been recovered after repeated exposure to LysGH15 under the conditions tested [In vivo, skin wounds were created in mice and subsequently treated with topical applications of different ointments. Both LAA and LA ointments promoted fast re-epithelialization during the nine-day study period, similar to the effect of mupirocin. However, the AA ointment was no better than Aquaphor or PBS. The aetiology of wound healing is complex. According to our results, an important factor in accelerating wound closure and wound healing was the decolonization of the MRSA strain USA300, which was attributed to the lytic activity of LysGH15. Mupirocin is the standard agent for MSSA and MRSA nasal and skin decolonization ,40. Mupione week . Mupirocs tested . Additios tested ,32.S. aureus infection [The healing process for injured tissues generally involves multiple cytokines that are primarily secreted by immune cells and that are important mediators of host defence, post-injury repair, cell growth, and maturation ,38,42. Tnfection . Thus, tApi is a naturally occurring flavonoid that is considered to be nontoxic and non-mutagenic ,45. Api S. aureus in vitro or in vivo. We also discovered that LAA can trigger LysGH15-specific antibody production when used to treat wounded skin. However, the titres of antibodies were very low even when LAA was repeatedly administered, as noted in previous research [As an exogenous protein, LysGH15 has the potential to be immunogenic, stimulating the production of antibodies. Our previous study revealed that LysGH15 triggered the generation of LysGH15-specific antibodies in mice when it was subcutaneously administered . Howeverresearch ,32Moreover, the anti-serum did not affect the lytic activity of LAA ointment. In addition, the anti-serum did not affect the haemolysis inhibition exhibited by LAA. No specific antibody against api was detected in this study. Considering the small size of api , this finding is reasonable. S. aureus [It has been proved that the N-terminal CHAP domain and C-terminal SH3b domain are the active regions of LysGH15, and their grooves involving the key residues play important roles in lysing and binding . aureus . Anti-Ly. aureus . Our results provide information on a topical antimicrobial agent that works against staphylococci. Specifically, this study provides evidence that the topical application of LysGH15 and api has significant potential as an alternative strategy for MRSA infections.


In this work graphene-based aerogel anodes and graphene/stainless steel cathodes have been optimised as platinum-free electrodes in Rhodopseudomonas palustris microbial fuel cells, achieving a maximum power output of ∼3.5 W m–3. Rhodopseudomonas palustris CGA009. We achieve a volumetric peak power output (PV) of up to 3.51 ± 0.50 W m–3 using graphene based aerogel anodes with a surface area of 8.2 m2 g–1. We demonstrate that enhanced MFC output arises from the interplay of the improved surface area, enhanced conductivity, and catalytic surface groups of the graphene based electrode. In addition, we show a 500-fold increase in PV to 1.3 ± 0.23 W m–3 when using a graphene coated stainless steel (SS) air cathode, compared to an uncoated SS cathode, demonstrating the feasibility of a platinum-free, graphene catalysed MFCs. Finally, we show a direct application for microwatt-consuming electronics by connecting several of these coin sized devices in series to power a digital clock.Microbial fuel cells (MFCs) exploit the ability of microorganisms to generate electrical power during metabolism of substrates. However, the low efficiency of extracellular electron transfer from cells to the anode and the use of expensive rare metals as catalysts, such as platinum, limit their application and scalability. In this study we investigate the use of pristine graphene based electrodes at both the anode and the cathode of a MFC for efficient electrical energy production from the metabolically versatile bacterium  Global population expansion and economic development result in increasing demand for energy and clean water, leading to a pressing need for innovative renewable energy sources and more efficient and sustainable waste treatment technologies. Microbial fuel cell (MFC) technology may satisfy both requirements by tapping into the significant chemical energy in wastewater, exploiting the electrogenic nature of various microorganisms that oxidize organic substrates and donate electrons to an external electron acceptor. Although examples of carbon-based anodes and platinum (Pt) cathodes exist, there remains major scope for improving the performance of electrodes for MFCs. Enhanced understanding of the parameters determining electrode performance will help in the development of environmentally-friendly, abundant catalytic cathode materials, and highly electron-accepting anode materials. Here we identify key parameters of a range of MFC electrodes and characterise the performance of a set of novel, environmentally-friendly low-cost graphene-based anodes and cathodes.via direct extracellular electron transfer (DEET) from anodic biofilms2O formation and evaporation via ORR at the air exposed cathode.11Power output from a single chamber MFC is primarily limited by the efficiency of extracellular electron transfer (EET) from the cell to the anode,i.e. their resistance to corrosion in an aqueous environment), surface area (∼0.5 m2 g–1) and electrical conductivity.2 g–1 ,via an electron shuttling processes, and potentially providing an alternative and cheaper cathodic catalyst to commonly used Pt–1). MFC anodes using chemically modified GO have been shown to enhance power densities to 2.67 W m–2 (∼18-fold) for stainless steel mesh,–3 (∼3-fold) from carbon cloth,–3 (∼19-fold) for nickel oxide foams.–3 . Their low density and high surface area, together with high conductivity, establish aerogels based on GO as high performance MFC anodes.2 g–1) structures.e.g. methane) for CVD,2O.4 S m–1), and positively charged backbone that may interact electrostatically with negatively charged cells to facilitate cell–anode interactions and biofilm formation.44Cost-effective carbon-based materials such as carbon felt,Rhodopseudomonas palustris  CGA009  from MFCs using carbon foam anodes was doubled to 265 ± 12.1 mW m–3 by coating with a pristine graphene/PD (Gr–PD) based ink, and the enhanced surface from composite Gr–PD aerogel anodes increased PV 13-fold to 3.51 ± 0.50 W m–3, closer to our benchmark provided by carbon fibre as an anode material (5.37 ± 1.16 W m–3). As a practical application, we show that a circuit of 10 single chamber MFC devices operated with Gr–PD aerogel anodes with a total volume of 1.32 cm3 generated 4.19 μW of power, sufficient to run a digital clock. In addition, we show that a Gr–PD ink coating is able to impart catalytic activity onto a standard marine grade stainless steel mesh (SS), showing feasibility as a cost-effective Pt-free air cathode. Finally, we demonstrate a fully pristine graphene-enabled MFC by integrating our pristine graphene-based anode aerogel and air cathode in a single chamber MFC, paving the way to a cost-effective, environmentally friendly energy source.Pristine graphene can be produced sustainably by liquid phase exfoliation (LPE) or cracking of methane biogas (derived from food waste and other renewable sources) both of which we used to produce low-cost, scalable, and environmentally friendly electrodes for MFCs. In the anodic chamber we use the metabolically diverse, purple non-sulphur α-proteobacterium, ) CGA009 . R. paluWe prepared low density, highly porous aerogel anodes using a biocompatible and biodegradable non-conductive polymer, carboxymethylcellulose sodium salt (CMC) as a scaffold material.–1 (PD1) and ∼1508 cm–1 (PD2), are assigned to the asymmetric Cα = Cβ stretching and symmetric Cα = Cβ (–O) stretching vibrations respectively.2g phonon at the Brillouin zone centre in graphene, while the D peak  is due to the breathing modes of carbon sp2 atoms and requires a defect for its activation.–1 nm–1) for each of the aerogels with graphene flakes  indicates that the D peak originated from defects in the basal plane of the graphene in addition to defects along the flake edges.R. palustris cells were used to determine the charge transfer resistance (Rct) of each aerogel. Nyquist plots are generated by plotting the imaginary impedance Im(Z) versus the real impedance Re(Z) for each aerogel, and show a semicircle at high frequencies, where the system is under kinetic control , followed by a straight line at low frequencies, where the system is under a diffusive controlled regime .Rs) combines the ionic resistance of the electrolyte, the intrinsic substrate resistance and the contact resistance, and it is defined as the value where the semicircle intercepts the real impedance (Re(Z)) axis.Cd), and the charge transfer resistance (Rct). The diffusive regime is modelled by the Warburg resistance (ZW), which describes the frequency dependence of the ion transport to the electrode. In cases where the Rct is sufficiently high and the diffusive regime is not reached, ZW is set to zeroRct is ∼101 kΩ and ∼176 kΩ respectively, with the single semicircle indicating that the electrochemical reaction is kinetically controlled, hindering the process of electron transfer and indicating a resistive behaviour of the materials.Rct for A-CMC–Gr (∼46.2 Ω) (red curve) and A-CMC–Gr–PD (∼21.0 Ω) (blue curve) suggests that the addition of Gr flakes is primarily responsible for decrease in Rct, while PD likely helps to bridge between the conductive graphene flakes, improving Rct further. On the other hand, we noticed the absence of a diffusion element in the A-CMC–PD electrode, which indicates a slower ion transfer process. Therefore we can conclude that the addition of graphene flakes can improve electron transfer in the anode aerogels.The Nyquist plots of the electrochemical impedance spectra (EIS) before the addition of Sa) was 3.9 m2 g–1 for the A-CMC–PD aerogel and 7.1 m2 g–1 for A-CMC, while Sa increased to 20.2 m2 g–1 and 8.2 m2 g–1 for A-CMC–Gr and A-CMC–Gr–PD respectively, with the addition of graphene flakes. We suspect that the surface areas are indeed much higher (i.e. 50–100 m2 g–1) than those calculated. However, a collapse of the aerogels due to increasingly high mercury pressure (∼400 psi) is known to affect soft foams analysed with mercury porosimetryThe pore size distribution of the aerogels was estimated by mercury porosimetry . 2 g–1) carbon foam (CF) coated with a graphene–PD ink (Gr–PD–IPA) formulated by liquid phase exfoliation (LPE) . Isopropyl alcohol (IPA) was used as the solvent for this ink as the low surface tension (∼29 mN m–1) helps to transport the graphene flakes by capillary action around the porous structure. Additionally, to investigate the catalytic activity of a graphene–PD coating as a Pt-free air cathode we coated a standard SS mesh with a graphene–PD ink (Gr–PD–W), by vacuum filtration , to make a graphene coated stainless steel (SS–Gr–PD) cathode. Water was chosen as the solvent for this ink as organic solvents such as IPA will dissolve the nitrocellulose membrane used in the vacuum filtration. The optical absorption spectra of the Gr–PD–W and Gr–PD–IPA inks  statistics showed a thickness of ∼6 nm , surface tension (γ), and density (ρ) for the two inks showed that ηGr–PD–W ∼ 0.89 mPa s, γGr–PD–W ∼ 70 mN m–1, ρGr–PD–W ∼ 1.02 g cm–3; ηGr–PD–IPA ∼ 2.5 mPa s, γGr–PD–IPA ∼ 27 mN m–1, ρGr–PD–IPA ∼ 0.785 g cm–3, consistent with previous reports.In order to establish the effect of enhancing anode conductivity and surface area on MFC performance, we compared the aerogel anodes with a conductive graphene coated carbon foam (CF-Gr–PD) . This was prepared using a low surface area  and CF-Gr–PD anode (red curve) have the typical D, G and 2D peaks of graphene as described in the previous section which are in line with the spectra of Gr–PD–IPA flakes (green curve). The blue curve showed a combination of both Gr–PD and PD spectra, while the red curve brings additional features to the CF anode (black curve) where the absence of a distinct 2D peak and the G peak position Pos(G) ∼ 1600 cm–1 indicated the more defective nature of the CF.–1 nm–1.–1 nm–1) lower than that expected for disordered carbon.49Raman spectroscopy was also used to characterize the quality of the cathode and anodes. Rct of CF-Gr–PD anode decreased with respect to that of the CF anode from 41.4 kΩ to 0.930 kΩ.  and CF-Gr–PD (2 m2 g–1) anodes. SEM of the CF and CF-Gr–PD anodes showed average pore sizes of ∼2 μm  OD660 = 3.0 without stirring, to encourage cells to form an electroactive biofilm on the anode surface  Rsur , and has similar surface resistance (15.3 ± 1.21 Ω m–2), however the higher OCV of CFi (605 ± 70.3 mV) may have facilitated higher current (39.7 ± 4.86 A m–2) due to the unbroken conductive CFi connection, as opposed to discontinuous pristine graphene flakes.We used linear sweep voltammetry (LSV) to calculate polarization and power curves of the aerogel anodes by applying a linear sweep potential from the open circuit voltage (OCV) to 0 V. The OCV , surfaceve) Rsur , maximumRsur .71R. palustris taken at 1 mV s–1 between –900 mV and 900 mV after 72 hours in the devices. A broad oxidation–reduction peak pair at ∼360 mV and ∼–200 mV from A-CMC are most likely a result of the interaction between cells and the stainless steel anode connector,R. palustris on carbon paper,via interaction with excreted mediators.Shewanella oneidensis.R. palustris homologs to OmcA have low genetic and structural similarityR. palustris is known to have other important mechanisms of both oxidising and reducing its surroundings such as the phototrophic iron oxidation (Pio) pathway.R. palustris in a MFC. However, further work is required to elucidate the molecular basis of the precise redox active mechanisms acting in synergy with graphene.Cyclic voltammetry (CV) can be used to gain a qualitative insight into the redox mechanisms used to transfer electrons between the cell and the anode and thus give information on pristine graphene's catalytic properties  and 138 ± 28.2 mW m–3 (p = 0.007) respectively  CF-Gr–PD anodes to 3.51 ± 0.504 W m–3 from the A-CMC–Gr–PD aerogel anodes with a surface area of ∼10 to 20 m2 g–1. This clearly indicates that increasing anodic surface area is a key factor to improve MFC performances.We assessed the influence of surface area on the MFC anodes by acquiring the polarisation and power curves from linear sweep voltammetry from MFC devices equipped with CF anodes and the SS anode connector alone (used as control), as done for the aerogels. The n curves  and powen curves  of the CR. palustris biofilms on the SS, CF, and CF-Gr–PD anodes after operations, the anodes were removed from the MFC and prepared for SEM imaging after performing the bioelectrochemical measurements. The SS anode connector  of pristine graphene with the SS–Gr–PD electrode. ID and PV of 0.172 ± 0.0378 A m–2 and 1.04 ± 0.252 W m–3 respectively, which are 500-fold greater than those achieved with SS (black curve) at the cathode (0.000373 ± 0.000103 A m–2 and 0.00228 ± 0.000552 W m–3) (p = 0.011) and is only slightly lower than what was achieved when using a CP-Pt cathode (red curve) (ID ∼ 0.672 ± 0.152 A m–2 and PV ∼ 5.01 ± 0.302 W m–3). We attribute the improved performance of the SS–Gr–PD to the catalytic properties of pristine graphene flakes on the SS mesh facilitating the ORR.ID of 0.0753 ± 0.739 A m–2  at the cathode.39 A m–2  and PV o90 W m–3 , demonstID and PV decreasing from 8.3 to 6.5 μA and from 4.19 to 2.76 μW, respectively. This application demonstrates how graphene based MFCs are able to produce sufficient power to run commercial electronic devices such as those that may be found in wearable technology or low powered sensors.We tested the feasibility of graphene based MFCs to power small electrical devices, such as a digital clock. A circuit of 10 MFC chambers using A-CMC–Gr–PD anodes and Nafion® carbon–Pt cathodes was connected, with two series of five MFCs in parallel . This coIn this study we demonstrated the three-fold advantage of graphene based aerogels towards enhancing the efficiency of MFC electrodes. First, we demonstrated the effectiveness of unmodified, pristine graphene composite aerogels. Second, we showed that a pristine graphene coating can enhance EET compared to standard carbon anodes. Third, we reveal that pristine graphene can catalyse the cathodic ORR as well as anodic EET. We provide a direct demonstration of graphene enabled MFCs powering a commercial digital clock, illustrating the possibility of MFC in power demanding applications such as micro-electronics, beyond the already existing application ranges of sustainable electricity production, treatment of municipal waste water streams and biosensors.An isopropanol (IPA) based ink (Gr–PD–IPA) was prepared by ultrasonication  of graphite flakes (Sigma Aldrich) for 9 hours in IPA with PD (1% v/v). Additionally, a water based graphene ink (Gr–PD–W) was prepared by ultrasonication of graphite flakes for 9 hours in deionized water with PD (1% v/v) (Sigma 739316) to produce graphene flakes. In both ink preparations, the small amount of PD acts as a stabilisation agent and conductivity enhancer. The stabilization mechanism is due firstly to the π–π interaction between the graphene sheets and the backbone of the PEDOT and secondly the electrostatic repulsion between the negatively charged PSS.via the Beer–Lambert law expressed in the formula A = αcl, where α [L g–1 m–1] is the absorption coefficient, c [g L–1] is the concentration and l [m] is with the beam path length. The Gr–PD–W and Gr–PD–IPA inks were diluted 1 : 10 with water–PD and IPA–PD respectively. Assuming α ∼ 1390 L g–1 m–1 .The surface tension of the inks was measured using the pendent drop method (FTA1000B). The shape of the drop results from the relationship between the surface tension and gravity. The surface tension is then calculated from the shadow image of a pendent drop using drop shape analysis. A parallel plate rotational rheometer (DHR rheometer TA instruments) was used to evaluate the viscosity as a function of shear rate and the infinite-rate viscosities were determined for the Gr–PD–W and Gr–PD–IPA inks. Ink density was evaluated from a (Sartorius ME5) microbalance where the density is the mass per unit volume (–1) of the Gr–PD–IPA ink transported the graphene flakes by capillary action around the porous structure. For the aerogel anode fabrication a CMC/water precursor for each aerogel was created by adding 5 mg ml–1 CMC  to deionised water. Graphene powder (10 g L–1) (Cambridge Nanosystems) produced by cracking methane and carbon dioxide in a plasma torch was then added to the CMC/water solution for the A-CMC–Gr–PD and A-CMC–Gr aerogels while PD (10% v/v) (Sigma 739316) was added to the CMC/water solution for the A-CMC–Gr–PD and A-CMC–PD aerogels. Each sample solution (1 ml) was pipetted into an aluminium container and frozen at –10 °C allowing the growth of ice crystals to shapes the pore geometry of each structure. The samples were then placed into a freeze dryer (Telstar LyoQuest) to remove the ice crystals by sublimation under vacuum.A graphene coated carbon foam CF-Gr–PD anode was created by drop casting 3 ml of Gr–PD–IPA ink onto a conductive carbon foam anode . The low surface tension  of each cathode was determined with a Jandel probe head in a 4-point probe configuration. The Rs of a Gr–PD film transferred onto glass is 3.7 kΩ □–1, significantly higher than the Rs of the SS cathode (<10 Ω □–1).The cathodes were prepared by vacuum filtration transfer. First (∼4 ml) of the Gr–PD–W ink was diluted with deionized water at a ratio of 1 : 9 respectively and passed through a nitrocellulose membrane (100 nm pore size), hastened with the use of a Büchner flask attached to a vacuum pump. The graphene/PD film on the membrane was then transferred onto a stainless steel mesh with 0.2 mm spacing. After oven annealing (∼80 °C) the sample was placed in an acetone bath overnight in order to dissolve the nitrocellulose membrane and leave behind a graphene/PD film of thickness 635 nm (Bruker Dektak Stylus Profilometer) on the metal mesh (cathode SS–Gr–PD). SS–Gr–PD was then cleaned in acetone and isopropanol baths sequentially and then died in a nitrogen flux. The sheet resistance (P) and the pore diameter (D) can be found through the Washburn equation assuming the pores are cylindrical,D = –4γ cos(θ)/P where γ is the surface tension of mercury (485 mN m–1), θ is the contact angle (130°) acting along the parameter of the pore. The pore size diameter is plotted as a function of the differential intrusion (ml g–1 Å–1) which is found by dividing the incremental intrusion (ml g–1) by the difference in pore diameter (Å). The specific pore area (A) is then calculated (A = 4V/D) assuming a cylinder pore volume (V = πD2h/4) and open cylinder pore area (A = πDh). For each specific surface area measurement the contribution from the macro (∼75 μm) and meso (∼1 μm) pores while the contribution from the micropores (∼10 nm) could not be determined as the foams collapsed at the higher pressures (∼400 psia), a known problem when examining soft foams with mercury porosimetry.The pore size distribution and specific surface area of the anodes were determined using a mercury intrusion technique (Micromeritics AutoPore IV 9500). Mercury was pushed into the sample from 6.9 kPa (1 psia) to a maximum pressure of 206 843 kPa (30 000 psia). The relationship between this pressure  and pressed together between PTFE blocks. The cell was immersed in 1.0 M tetraethyl ammonium tetrafluoroborate (TEABF)/propylene carbonate (Sigma-Aldrich) non-aqueous electrolyte. EIS experiments were conducted using a BioLogic VSP-300 potentiostat, using an AC voltage of 0.2 V with 5 mV amplitude over a frequency range of 10 mHz to 10 kHz. All experiments were performed at room temperature.40Ø: 13 mm, height 30 mm), between which were clamped in descending order: a marine grade stainless steel  anodic electrode connector, a dialysis membrane of 10 kDa pore size (Thermo), a Nafion® proton exchange membrane (Dupont) with a conductive and catalytic carbon–Pt coating facing into the air exposed cathodic chamber, and finally a second SS mesh. The top and bottom SS mesh layers served as contacts for the anode and cathode respectively. Carbon fibre and aerogel electrodes were placed over the anodic SS mesh with wet thickness of 4 mm and 1 mm respectively, and total geometrical area of 1.33 cm2. No electron shuttle mediator was used. 4 ml of adjusted cell culture was injected into the top anodic chamber and cells were allowed to settle and form a biofilm on the anode. To characterize the SS–Gr–PD cathodes, the MFC device was modified with a secondary channel (Ø: 6 mm) drilled perpendicular and intersecting midway to the anodic channel. The SS–Gr–PD cathode was placed at the end of this channel, with a total geometrical area of 0.283 cm2, and 2 g of carbon fibre  was used in the anodic chamber. Platinum coated carbon paper (CP-Pt) was used as a benchmark (hydrogen electrode/reformate cathode Alfa Aesar 45452). Bioelectrochemical characterisation was carried out with a PALM-SENS MultiEmstat 8-channel potentiostat. Following stabilisation at the open circuit potential, polarisation and power curves were calculated by Ohm's law via linear voltage sweeps scanning the applied voltage from its open circuit potential to 0 volts with a slow scan rate of 1 mV s–1 to avoid overpotential effects.–1 between –900 mV and 900 mV after incubation for 3 days in the MFC.The MFCs used in this study were based around the design shown in λL, where λL is the laser excitation wavelength. Raman spectra for the Gr–PD–IPA and Gr flakes were acquired on Si/SiO2 substrate while all other spectra were acquired on the anodes and cathodes directly.Raman spectra for the cathode and anodes were acquired with a Renishaw 1000 InVia micro-Raman spectrometer at 457, 514.5, and 633 nm and a ×20 objective, with an incident power of below ∼1 mW.Scanning electron microscopy images were taken with a high resolution Magellan 400L scanning electron microscope (SEM). The field emission gun was operated at an accelerating voltage of 5 keV and gun current of 6.3 pA. Images were obtained in secondary electron detection mode using an immersion lens and TLD detector. The biological samples were taken on a FEI Verios 460 scanning electron microscope. Samples were washed in distilled water, fixed in liquid ethane, and mounted on a liquid nitrogen cooled plate before freeze drying overnight and coating in 14 nm of iridium.R. palustris and prepared cell cultures, T. P. C. and P. B. designed and constructed the MFCs and performed electrochemical measurements. T. P. C. undertook SEM on biological samples while P. H. preformed EIS on the aerogel and carbon foam samples with the assistance of T. C. The manuscript was written by T. C., T. P. C., F. T. and C. J. H., in close consultation with other authors.T. C. and T. P. C. conceived and designed the experiments. T. C., T. P. C., P. B., F. T. and C. J. H. analysed the experimental data and interpreted the results. T. C. developed the inks and composites and performed UV-vis, rheometry, AFM, Raman and pendant drop analysis on the inks and conducted mercury porosimetry, SEM, optical microscopy on the carbon foam and aerogel composites. T. P. C. and D. L. S. curated The authors have no conflict of interest.Supplementary informationClick here for additional data file.