This article demonstrates a dispersed-monolayer graphene-doped polymer/silica hybrid Mach–Zehnder interferometer (MZI) thermal optical switch with low-power consumption and fast response. The polymer/silica hybrid MZI structure reduces the power consumption of the device as a result of the large thermal optical coefficient of the polymer material. To further decrease the response time of the thermal optical switch device, a polymethyl methacrylate, doped with monolayer graphene as a cladding material, has been synthesized. Our study theoretically analyzed the thermal conductivity of composites using the Lewis–Nielsen model. The predicted thermal conductivity of the composites increased by 133.16% at a graphene volume fraction of 0.263 vol %, due to the large thermal conductivity of graphene. Measurements taken of the fabricated thermal optical switch exhibited a power consumption of 7.68 mW, a rise time of 40 μs, and a fall time of 80 μs at a wavelength of 1550 nm.  High-speed optical switch chips are core components in optical switching systems, meeting the requirements of being high speed, large capacity, and low delay ,2,3. Opt2−TiO2 waveguide [According to the structure of the device, TO switches can be categorized into X-junction TO switches  and Machaveguide . A switcaveguide ,17,18. HIn this research, to solve the problems of the graphene film being uneven and fragile during the fabrication of the TO switch, a graphene-dispersed N-Methyl pyrrolidone (NMP) material was synthesized and doped into polymethyl methacrylate (PMMA). This composite material not only guaranteed the large thermal optical coefficient of the polymer, but also an improved thermal conductivity, due to the large thermal conductivity of graphene . TherefoKp and Kg are the thermal conductivities of the PMMA matrix and graphene filler [Kp = 0.19 W/mk; Kg = 5300 W/mk). However, the limitation of the model is that the interfacial thermal resistance between the fillers and the polymer is not considered.The thermal conductivity of the material significantly affects the performance of TO switches. The thermal conductivity of the composite material has been analyzed and calculated using the Bruggeman  and Lewie filler ,22, respD and L represent the diameter and thickness of the filler, respectively . A and B depend on the filler dimensions and the thermal conductivity of each component, respectively, and V is the volume fraction of filler. Vm is the maximum packing fraction of the filler (Vm = 0.673).In the Lewis–Nielsen model, the effects of particle morphology, aggregation method, and orientation on the thermal conductivity of the composites are considered, and the thermal conductivity is calculated by the expression:The thermal conductivity of composites as a function of the volume fraction of filler, calculated using MATLAB software , is shown in a = 5.3 μm and b = 3 μm, respectively. The distance between the electrode and the top of the core was h1 = 1.2 μm, and the thickness of the cladding was h2 = 2 μm. The refractive indexes of the imaginary and real parts of the graphene material are 2.79 and 2.97, respectively [ectively ,24. The ectively . Figure ectively ,24,26. AThe calculated response times and the thermal field distribution of the arc cladding structure for the GD-TO and ND-TO switches are shown in 2, using spin-coating and photolithography processes . The width of the core was 5.3 μm. Next, a graphene-dispersed NMP material was synthesized and doped with 0.263 vol % into PMMA . The composites were spin-coated on the core layer as a waveguide cladding. The arc cladding structure was realized by optimizing the viscosity of the cladding material and the spin rate of the spin coater . Lastly, a 0.1 μm thick Al electrode was fabricated with a width and length of 6 μm and 1.5 cm, respectively. According to the Lewis–Nielsen model, the calculated thermal conductivity of the composites was increased by 133.16%.Following the previous simulation, the polymer/silica hybrid MZI TO switch was fabricated using doped dispersed-monolayer graphene NMP material . In conclusion, we proposed a polymer/silica hybrid MZI TO switch. The predicted thermal conductivity of the composites was then increased by 133.16% for the composites doped with graphene (0.263 vol %). The fabricated TO switch exhibited a power consumption of 7.68 mW, a rise time of 40 μs, and a fall time of 80 μs at a wavelength of 1550 nm. The doped graphene material was not sensitive to the absorption of various polarized modes. In future research, the application of graphene in the all-optical switch field will be studied according to the photothermal efficiency characteristics of graphene at different wavelengths. Moreover, graphene-doped materials have potential applications in both flexible thermal optical switches and wearable fields.


A chemical-sensitive semiconductor film based on graphene has been achieved, being decorated with CH3NH3PbBr3 perovskite (MAPbBr3) nanocrystals (NCs) synthesized, and characterized by several techniques, such as field emission scanning electron microscopy, transmission electron microscopy and X-ray photoelectron spectroscopy. Reversible responses were obtained towards NO2 and NH3 at room temperature, demonstrating an enhanced sensitivity when the graphene is decorated by MAPbBr3 NCs. Furthermore, the effect of ambient moisture was extensively studied, showing that the use of perovskite NCs in gas sensors can become a promising alternative to other gas sensitive materials, due to the protective character of graphene, resulting from its high hydrophobicity. Besides, a gas sensing mechanism is proposed to understand the effects of MAPbBr3 sensing properties.This paper explores the gas sensing properties of graphene nanolayers decorated with lead halide perovskite (CH Nitrogen dioxide (NO2) and ammonia (NH3) are two examples of these harmful species. For these reasons, the in-field monitoring of harmful gases is a mandatory goal to control dangerous levels of contaminants. Above the threshold limits, when human health is at risk, gas sensors can be the last wall to start actions like reducing the access of cars to city centers, to mitigate the impact on the public health.Atmospheric contamination is one of the most important environmental issues in current societies. According to the World Health Organization (WHO), air pollution is linked to 7 million premature deaths in 2012 [Among the different types of gas sensors, chemoresistive sensors have been attracting great interest due to their high sensitivity, low cost, reproducibility and simplicity . Other tMany authors have developed chemoresistive gas sensors employing graphene semiconductors, however the use of pristine graphene for developing gas sensors still presents many intrinsic problems, including low gas sensitivity and complicated processability into devices . On the 3 [2 [Unlike metal oxides based sensors, which usually require high working temperatures to activate the oxidation-reduction processes and achieve the required electron mobility involved in gas sensing , graphen3 , acetone3 , oxygen 3 , and NO23 [2 . However3 [2 .3NH3PbI3 (MAPbI3) usually shows a fast degradation, in most cases attributed to their hygroscopic properties [3), which has been proved to be more resilient to the presence of ambient moisture [3 stability and increasing the sensor time-life, via limiting perovskite degradation. Moreover, the nanocomposite was characterized by several techniques, such as X-Ray photoelectron spectroscopy (XPS), high-resolution transmission electron microscopy (HRTEM), Field Emission Scanning Electron Microscope (FESEM), and Raman spectroscopy. In consequence, this paper shows the results with graphene decorated by MAPbBr3 NCs, registering higher sensitivities and detecting lower concentrations than other gas sensors based on perovskite thin films or similar approaches  and TWA (Time-Weighted Average) by the European Agency for Safety and Health at Work (EU-OSHA) [3 limits. Thus, the development of sensor networks able to detect these trace concentration levels in a wide range of locations would be highly advisable. In consequence, sensors should offer reversible interaction with gas molecules for allowing the continuous monitoring of these species at an affordable cost.Threshold limit values (TLV) to 1-h exposure to NOan Union  and the an Union , respectEU-OSHA)  and by t3) nanocrystals, the method proposed by Schmidt, et al. [2) were mixed in the same solvent. Subsequently, these two solutions were added to the main solution and was cooled to 60 °C. Finally, 5 mL of acetone was added to the final solution to create precipitates of perovskite nanocrystals, which were separated from the unreactive solution by using a centrifugation technique at 6000 rpm during 10 min. Afterwards, the nanocrystals obtained were suspended in toluene.To synthesize lead halide perovskite  dispersed in 1 mL of toluene. Then, graphene nanoplatelets were sonicated employing a Sonic Tip (FisherbrandTM Model 705) at 40% of 700W for 1 h and 30 min using a 1 s on −2 s off pulsed sonication.The exfoliated graphene and the lead halide perovskite were characterized by several techniques such as XPS, HRTEM, FESEM and Raman spectroscopy.The chemical composition of the nanomaterials obtained was studied via XPS, using a SPECS spectrometer  equipped with a Phoibos 150 MCD detector, using a non-monochromatic X-ray source (Al) operating at 200 W. The intensity ratios of the different components were calculated from the area peak after a correction by the transition function of the spectrometer and a non-linear Shirley-type background subtraction. Additionally, graphene crystallinity was evaluated employing a Raman spectrometer from Renishaw, plc. , coupled to a confocal Leica DM2500 microscope. The laser used had a wavelength of 514 nm.The morphology was studied employing a JEOL JEM 2100F  HRTEM, at an operating voltage of 200 kV. Nanomaterials were ultrasonically dispersed in toluene and a drop of dispersion was deposited onto a carbon-coated copper grid, drying it at room temperature. Graphene porosity and perovskites NCs distribution were analyzed by Carl Zeiss AG - ULTRA 55  Field Emission Scanning Electron Microscope.2 quartz substrates previously cleaned in an ultrasonic bath, using an anionic detergent (Alconox) and miliQ water. Afterwards, a 1M solution of hydrochloric acid was employed in order to increase the surface hydrophilicity. Finally, the substrates were cleaned and dried several times with ethanol. Then, a film of graphene nanoplatelets was deposited onto quartz substrates by drop casting method, and subsequently, perovskite nanocrystals (5% wt solution) were deposited by spin coating at 1000 rpm for 120 s. The materials obtained were deposited onto 1.5 cmOnce the hybrid material film was deposited onto quartz substrate, its backside was glued with a thermally conducting epoxy from Heraeus, Inc.  to an alumina hotplate that comprised a screen-printed platinum heater. Then, the samples were placed on a 20 × 30 mm printed circuit board (PCB) to be connected to an airtight test chamber. Finally, two-wire contacts were made on the sensor surface employing a conductive silver paste and platinum wires  mass-flows controllers. The sensors were stabilized under synthetic dry air for 60 min before the application of the desired gas concentration during 30 min of exposure. Besides, a controller evaporator mixer (CEM) from Bronkhorst High-Tech B.V.  was used to humidify the gas mixture and thus, analyze the effect of ambient moisture on the sensing properties  and different dilutions of gases tested to achieve the target concentration. In order to reproduce real atmospheric conditions, NOties see . The relA high-resolution transmission electron microscopy (HR-TEM) analysis was conducted to characterize graphene and perovskite nanocrystals independently. 3 NCs. Additionally, FESEM images were obtained for both, bare and graphene decorated with MAPbBr2 carbon systems in the “graphite-like” [2 carbon systems, but in this case due to the π-π* interactions and π plasmon, which is derived from the energy loss due to the excitation of π electrons [3 configuration appear at 285.0 eV [According to the XPS elemental quantification, the graphene used presents a 7.8% content of oxygen and 92.2% of carbon. The results of the XPS fitting analysis are represented in te-like” . The pealectrons . Compone285.0 eV , and def285.0 eV . Meanwhi285.0 eV . Peak quThe O 1s spectrum is reproduced employing three components centered at different binding energies, which are 531.3 eV, 532.6 eV and 533.9 eV b. The fiTwo different gases, nitrogen dioxide and ammonia, with high interest to be monitored due to the danger to human health derived from their exposure at certain levels were measured. Several dilutions were performed in order to apply different concentrations, especially under the threshold limits. Due to the different baseline levels of the two films studied, normalized responses are show for better comparison in 2. Nitrogen dioxide detection was performed by applying repeated exposure and recovery cycles to increasing concentrations of the analyte considered. 0)/R0, Where R0 is the resistance value immediately before gas exposure , and R is the resistance immediately before the start of a new cleaning step . 2 concentrations measured. Graphene doped with perovskite nanocrystals presents a higher response (up to 3-fold) than bare graphene, even under a hundred ppb of NO2 exposure. Additionally, bare graphene presents a slight saturation above 500 ppb, decreasing the slope obtained and, in consequence, reducing sensitivity. 2 after six months of sensor operation. This result confirms the high stability of MAPbBr3 under ambient moisture and reactive gases, probably due to the nanocrystal structure of the perovskite. Zhu et al. [A stability study of graphene loaded with perovskite nanocrystals was conducted b, revealu et al.  report t2 detection, were applied as well for NH3 detection at ppm range  when moisture is present. However, graphene decorated with perovskite nanocrystals shows a very stable response with only a slight decrease (about 5%) when under humid conditions.Additionally, the sensitivity under dry and humid conditions was evaluated to both, bare and perovskite loaded graphene. 2 leads to a lower sensor resistance. Conversely, an electron-donating gas, such as NH3, decreases the hole concentration resulting in a higher sensor resistance. In films decorated with perovskite nanocrystals, the p-type behavior of the sensor is defined as well by the graphene nanolayers , meanwhile the MAPbBr3 NCs are ambipolar charge transporters [Graphene with oxygen-containing functional groups such as carbonyl, hydroxyl, epoxy, and ether groups exhibited enhanced hole transport characteristics due to its suitable work function ,49. In asporters . In othe2 and NH3, due to the interaction of these gases with the oxygenated defects and functional groups located on the graphene nanosheets. However, all the measurements were done at room temperature, obtaining limited sensitivity derived from the small transfer of electronic charge between graphene and adsorbed gas molecules. Nevertheless, from the gas sensing tests, it was derived that the presence of MAPbBr3 NCs decorating the graphene nanolayers was advantageous for detecting NO2 and NH3 at room temperature. In addition, surface trap sites at the perovskite have been demonstrated to act as active sites for the gas sensing process. It is reported that a net positive charge is formed at the surface of the perovskite due to the loss of bromine and undercoordination of the Pb atom, favouring the perovskite to be sensitive to the environmental gases [3 NCs. Upon the formation of the hybrid perovskite the overall resistance of hybrid films is higher than that of bare graphene due to the intrinsic electrical properties of the perovskite nanocrystals.Bare graphene samples offer a response to both, NOal gases . Regardi3 NCs is associated to the creation of electron-hole pairs by the perovskites, once these are exposed to the gases  generated at perovskite NCs are transferred from the perovskite to the graphene sheet [3, an excess of negative charges (electrons) are generated. As a consequence, perovskite NCs transfer electrons to graphene (and this is associated to an increase in resistivity), enhancing the response of the film to NH3 due to the higher concentration of electrons. This enhancement in the response when graphene is decorated with MAPbBrhe gases . On the ne sheet . Therefo3 NCs and slow down their degradation, due to the limited interaction with ambient moisture [One of the main drawbacks of the sensors based on perovskites films is the fast degradation derived from the exposure to ambient moisture. For that reason, a lead halide perovskite with bromide was chosen, due to the well-known stability over other perovskites based in halides like iodine . Additiomoisture . 2 in humid conditions can be favored by a water-mediated adsorption, as reported in chemoresistive sensors [Considering the effect of ambient moisture in the sensing mechanism, higher sensitivity under humid conditions can be observed for bare graphene. This is in agreement with other works that report the use of graphene as humidity sensors . Normall sensors .2 in the presence of ambient moisture for MAPbBr3 decorated graphene shows interesting characteristics. 2 is achieved for hybrid sensors under dry and humid conditions. The response is only slightly lower in the presence of ambient moisture. Probably water molecules partly passivate the charge transport and the creation of electron-hole pairs of perovskite nanocrystals [3 under dry conditions. The hydrophilic properties of perovskite decorated graphene were further studied by contact angle measurements (see Nevertheless, the detection of NOcrystals , slightlents see .The gas-sensitive layer developed shows its potential for detecting concentrations of nitrogen dioxide and ammonia at trace levels, even below their threshold limit values. These results are a promising beginning for the use of perovskite nanocrystal decorated graphene in gas sensing applications. However, selectivity should be further studied before their use under real conditions. One reasonable approach could be the development of multi-sensor arrays employing decorated graphene with different types of perovskite nanocrystals.2 and NH3 was achieved at room temperature, enabling the integration of these hybrid films in low-power gas sensing devices. In summary, perovskites can constitute an alternative to metals, metal oxides, polymers or other molecules commonly used in the modification of the surface of carbon nanomaterials such as graphene in view of tuning sensitivity and selectivity. This work reports the first use of graphene loaded with perovskite nanocrystals as a chemoresistive sensor. This nanocomposite material has been shown to be a good option to obtain gas sensors with high stability over time, avoiding the main problem associated with perovskites, which is their degradation in contact with ambient moisture. Decorated graphene nanolayers show enhanced nitrogen dioxide and ammonia sensitivity in comparison to their bare graphene counterparts and much reduced cross-sensitivity to the ambient moisture. Furthermore, a reversible response to NO


The transition was accompanied by an electron density redistribution within the molecule, sensed by atomic force microscopy with CO-functionalized tip. This demonstrates the unique capability of the high-resolution imaging technique to discriminate between different spin states of single molecules. Moreover, we present a method for triggering spin state transitions and tuning the electronic properties of molecules through weak non-covalent interaction with suitably functionalized graphene.Nitrogen doping of graphene significantly affects its chemical properties, which is particularly important in molecular sensing and electrocatalysis applications. However, detailed insight into interaction between N-dopant and molecules at the atomic scale is currently lacking. Here we demonstrate control over the spin state of a single iron(II) phthalocyanine molecule by its positioning on N-doped graphene. The spin transition was driven by weak intermixing between orbitals with  Molecules can change their electronic properties when they are adsorbed on substrates, which can be useful for sensing and catalysis. Here, the authors use atomic force microscopy to show that the spin state of an iron complex can be changed upon displacing the molecule to different sites of a nitrogen-doped graphene It shows remarkable physical characteristics, such as large values of intrinsic mobility, Young’s modulus, surface area, thermal and electric conductivity, and optical transmittance6. Hence, both pristine and chemically functionalized graphene have been found to be effective in a broad portfolio of applications, including electronics/optoelectronics, energy generation and storage, and various medical, chemical, catalytic, and environmental processes8.Graphene, a two-dimensional nanoallotrope of carbon, is currently at the forefront of scientific interest owing to its unique electronic, mechanical, optical, and transport properties13. Importantly, the performance of graphene in these technologies is governed by the nitrogen concentration and its local environment in the graphene lattice , which determine its electronic features, including the closed or open electron shell arrangement.Doping of the graphene with foreign elements has been identified as an emerging strategy to open the zero band gap and tune its electronic, magnetic, and optical properties. Nitrogen in various structural configurations is the most frequently studied n-type dopant for improving the conductivity, transport features and magnetic properties of graphene, offering a multitude of applications in related fields like spintronics, energy generation/storage and nanoelectronics16. Thus, graphene and its derivatives are attractive materials for sensing a vast variety of chemicals, (bio)molecules, and gases19. It is well accepted that adsorption of gas molecules onto the surface of graphene can be enhanced by nitrogen doping, causing changes in the local carrier concentration. Such graphene-based sensors can detect even a single gas molecule attached to or detached from the surface of graphene20. Similarly, N-doped carbon allotropes, including graphene, have been shown to act as superior electrocatalysts, establishing the emerging field of metal-free catalysis, with enormous prospects for fuel cells, metal-air batteries and oxygen-reduction reactions21.In addition to allowing control over the intrinsic physicochemical properties of graphene, nitrogen doping can significantly affect its interaction with molecular species, as well as phenomena at the phase boundary of the graphene-molecule, which are particularly important in sensing and electrocatalysis applications22. This work indicated direct relevance of the graphene-molecule interaction for advanced organic electronics22. However, owing to the absence of a central metal atom, the possibility of discriminating or even controlling the spin state of a metal ion has remained challenging.Moreover, very recent work using scanning probe microscopy (SPM) imaging techniques has demonstrated that N-doping of graphene can even tune the physicochemical properties of molecules adsorbed on its surface via non-covalent interactions. In particular, it was shown that 5,10,15,20-tetraphenyl-21H,23H-porphyrin molecules adsorbed on N-doped graphene undergo a downshift of their highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) states if these molecules are located near to nitrogen defects25 and atomic clusters26, bond order analysis27, molecular electrostatic field28 or identification of different products of on-surface synthesis29. Here, we demonstrate that high-resolution images can also be used to discriminate different spin states of iron(II) phthalocyanine (FePc) molecules immobilized on N-doped graphene. The spin crossover was confirmed independently by inelastic spin excitation spectroscopy and theoretical quantum calculations. Importantly, we were able to control the spin state by changing the molecular positioning on N-doped graphene. This is the first example, to the best of our knowledge, of a spin transition induced by non-covalent interaction of a molecule with doped graphene caused by reordering of iron d-orbitals without the use of an external stimulus, such as an external magnetic field, electric field, light, pressure, or temperature. Hence, we show the unique capability of doped graphene to behave as a non-covalent tuner of the electronic and spin properties of molecules.In the present work, we used high-resolution atomic force microscopy (AFM) with a non-magnetic CO-functionalized tip and inelastic spin excitation spectroscopy to characterize molecular electronic states. This state-of-the-art AFM technique has been shown to offer unprecedented spatial resolution, allowing investigation of the chemical structure of molecules30 to prepare N-doped graphene grown on a SiC(0001) surface with the majority of nitrogen defects in the substitutional positions. Hence, the term N-dopant refers to the substitutional (graphitic) variant in the following discussion, unless stated otherwise.We investigated the possibility of tuning the electronic and magnetic properties of iron(II) phthalocyanine molecules via non-covalent interaction with single nitrogen dopants in graphene. We used ion implantation followed by a thermal annealing procedure31 and G/Pt(111)32. On the other hand, deposition of FePc onto N-doped graphene resulted in strikingly different features. The large-scale STM image obtained at 5 K in Fig. We found that the behavior of FePc was very specific depending on whether it was adsorbed on pristine or N-doped graphene. Submonolayer amounts of FePc deposited onto pristine graphene formed large and well-ordered flat islands, which were stabilized by intermolecular non-covalent interactions, as shown in the detailed scanning tunneling microscopy (STM) image in Fig. 34. By comparing the pairs of images acquired before and after the FePc lateral movement, we could precisely determine the register of the FePc molecules with respect to the N-dopants beneath  spectra. Figure I/dV spectra acquired from over the centers of two FePc molecules, one located on graphene and the other above an N-dopant. The FePc adsorbed on graphene exhibited two resonances, at –1.9 and 0.5 V, whereas the spectrum of the molecule adsorbed above the N-dopant showed shifts of the resonances to –1.2 and 0.3 V, effectively lowering the molecular gap by ~0.9 eV. This trend was very reproducible, as we observed it repeatedly during more than ten sessions with different tips on several molecules  and the electrostatic Hartree potential obtained from DFT calculations. Indeed, the electrostatic potential over the N-dopant obtained from DFT calculations . Figure dz2 Fe orbital, as shown in Fig. 42. Indeed, simulated AFM images with the electron density of the triplet and singlet states of FePc reproduced the experimental images obtained for molecules on graphene and the N-dopant, respectively  and E is in-plane magnetic anisotropy, which for planar molecules typically vanishes. Consequently, the Hamiltonian reduces to S = 1 state should show a single inelastic signal symmetric in bias voltage of the magnitude D. Indeed, we observe a symmetric step-wise increase of the conductance at bias voltages approximately ±7 meV. Consequently, we interpret this feature as inelastic spin excitations of the S = 1 spin multiplet (ms = 0 and ms = ±1) due to the zero field splitting driven spin–orbit interaction44. We would like to stress, that very similar spectra were also obtained for Fe-tetraphenyl-porphyrin molecule52, which were also attributed to inelastic spin excitation the S = 1 spin state. Therefore, we believe that the additional inelastic spin excitation measurements fully support the scenario of the spin transition of FePc from triplet (pristine graphene) to singlet (on N-dopant) state.To reinforce our interpretation of the spin transition, we carried out inelastic spin excitation spectroscopysee Fig.  acquiredz-component, i.e., pz of the N-dopant points outward from the surface and the  and dz2-like molecular orbitals of Fe. This causes an upward energy shift of the dz2-orbital with respect to , which promotes electron transfer from dz2 to , leaving the dz2 orbital empty with a closed shell electron configuration, as shown in Fig. z-component d-orbitals is facilitated by the fact they are very close in energies  state. Strikingly, in the proximity of the N-dopant, the low-spin (singlet) state was found to be about 5 kcal/mol more stable than the triplet state. We attributed this effect to weak intermixing between orbitals with para configuration, which has an even stronger local electrostatic field. Supplementary Figure para N-defect. When the molecule was located near the para N-defect, it exhibited a similar STM contrast and dI/dV spectrum to those of molecules located on pristine graphene. However, the contrast and dI/dV spectrum significantly changed when the molecule was manipulated onto a single N-defect due to the spin crossover. Again, this scenario was supported by our DFT calculations, which predicted that the molecule would be in the triplet state when located over the para N-defect. These results also strongly support the role of an open-shell like character of the single substitutional N-defect. The para N-defect has a closed-shell electronic structure which is more stable than the biradical one. The unique chemical activity of graphitic N-defects for spin crossover was further supported by theoretical analysis with different model cases, including a closed-shell pyridinic defect , for which proximity to the molecule again stabilized FePc in the triplet state.The possibility that the spin crossover was due to the local electrostatic field of the N-dopant was ruled out by analysis of a double substitutional nitrogen defect in the 54 in classical coordination chemistry. In our case, we demonstrated that such a transition can be achieved just by sliding the molecule over the surface of N-doped graphene. Thus, the electronic states of FePc molecules on graphene can be tuned locally by means of weak non-covalent interaction with N-dopants causing reordering of the selected iron d-orbitals driven by their weak hybridization with the pz orbital of a single graphitic nitrogen defect. This offers a way for controlling the spin state of a molecular system by simple positioning of the molecule onto a suitably functionalized graphene substrate. We also showed that high-resolution AFM using a non-magnetic CO-functionalized tip can distinguish between different spin states of molecules on a surface. This extends further the outstanding capabilities of the technique and opens new possibilities for studying magnetic properties at the single molecule level.These results demonstrate the diverse chemical activity of nitrogen impurities incorporated into graphene, opening a new way for not only tuning the electronic properties of molecules but also chemical functionalization of graphene. The unique character of the substitutional N-defect allows not only stabilization of the FePc molecule in its vicinity but is also responsible for the spin crossover of FePc. For comparison, such spin crossover of FePc is usually triggered by strong ligands or ligands with unpaired electrons forming covalent dative bondsI/dV measurements were acquired with the conventional lock-in technique with an amplitude modulation of 20 mV at 960 Hz. The metallic tip for dI/dV spectroscopy was formed by repeated indentations into a Au(111) surface until the surface state was clearly visible. Inelastic spin excitation spectroscopies were acquired at 500 MΩ of tunnel resistance with voltage modulation of 0.5 mV at 170 Hz. A graphene sample was prepared on a SiC(0001) substrate by annealing in Ar and was degassed in a UHV system at ≈800 °C55. N atom implantation was achieved by sputtering the graphene sample with N atoms accelerated at 120 eV and subsequent annealing to ≈800 °C. Iron(II) phthalocyanine (FePc) was thermally evaporated from a Ta pocket in UHV onto the graphene surface, which was kept at RT. After deposition, the sample was briefly annealed at temperatures below 200 °C to increase the surface mobility. STM/AFM images were analyzed using WSxM and WSPA software56.STM/AFM measurements were carried out in a UHV chamber equipped with a low-temperature STM/AFM with qPlus tuning fork sensor operated at 5 K (Createc GmbH). During the AFM measurements, a Pt/Ir tip mounted onto the sensor  was oscillated with a constant amplitude of 50 pm. To obtain high-resolution AFM/STM images, prior to the experiment, the tip was functionalized with a CO molecule gathered from a Au(111) surface. dOptimized structures of FePc molecules on N-doped and pristine graphene were calculated using both the cluster and slab models in the Turbomole, Gaussian, and VASP program packages based on ab initio density functional theory (DFT) .Theoretical AFM images were calculated using a probe particle AFM simulator . Further details about the Methods can be found in the Supplementary Methods.The data that support the findings of this study are available from the corresponding authors on request.Supplementary InformationPeer Review File


These two terminal electronic devices can be easily fabricated by forming a Van der Waals contact between large area chemical vapor deposited graphene and CdTe substrates in air and at room temperature. This approach significantly reduces the fabrication cost and improves the reproducibility and stability of electrical properties. A detailed analysis of their AC and DC electrical properties was carried out in order to determine the width of the space charge region and dominant charge transport mechanisms at reverse bias. The unoptimized graphene/CdTe heterojunction detectors exhibited a promising spectral resolution of  Owing to large atomic numbers of the compound components (48 for Cd and 52 for Te), the operation range of CdTe detectors extends to a higher photon energy region (up to 1 MeV) in comparison to Si detectors, and its wider band gap (1.46 eV at 300 K) results in a broad temperature operating range of CdTe detectors without intentional cooling5. Despite significant technical successes, some problems concerning physical properties of Schottky diodes based on high-resistivity materials have not yet been solved. High-resistivity CdTe single crystals suffer from significant temperature instabilities and uncontrollable time fluctuations of electrical characteristics due to polarization and compensation processes12.Graphene is a promising candidate for applications in the form of the front electrical contact for radiation detectors widely employed in science, engineering, medicine, and other fields. An atomically thin layer of graphene would not disturb low energy X-rays on their way to the active layer. Moreover, the relatively high sheet resistance of pristine single-layer graphene will not be a drawback due to the low electrical signal currents generated in radiation detectors or photodiodes13. However, best to our knowledge, there are no reports on two-terminal graphene/single crystal CdTe Schottky-type heterojunction X/γ-ray detectors.Recently, gated graphene field effect transistors, fabricated on intrinsic CdTe were proposed as sensors of ionizing radiationThis study aims to prepare novel graphene/single crystal CdTe heterojunction radiation detectors excluding undesirable vacuum deposition processes and technological heat treatments that may induce surface defects and modify the compensation level in the bulk of the CdTe active layer. Features of their impedance spectra and DC current-voltage characteristics were analyzed in details. Also, their spectroscopic detecting characteristics were measured at different reverse bias and room temperature.The schematic representation and AC equivalent circuit of the graphene/CdTe/Au Schottky-type heterojunction are shown in Fig. Ci is the insulator capacitance, Cb is the barrier capacitance of the graphene/CdTe region, Cb′ is the barrier capacitance of the graphene/insulator/CdTe region.εі = 3 is the dielectric constant of the organic dielectric layer; di = 20 μm is the thickness of the insulator layer; Ас = 0.14 cm2 is the area of the insulator layer under graphene.εCdTe = 10.2 is the dielectric constant of CdTe, W is the width of the space charge region that is one of the key parameters of the Schottky-type semiconductor detectors.Ab = 0.09 cm2 is the area of the graphene/CdTe region.Taking into account the device architecture of the detectors the following capacitances should be considered: Cm = Cb + C′  and imaginary (reactive) parts of the impedance, respectively:Therefore, the measured equivalent capacitance Rs is not included in equation 11) the mRd = 2|Z″min| = 94 ΜΩ and the barrier capacitance Cm = 7.3 × 10−12 F were determined from the analysis of the spectral distribution of the imaginary part of the measured impedance of the graphene/CdTe/Au devices.Values of the depletion region resistance A = Ab + Ac = 0.25 cm2. Equation , , 5), theW can be found as:The physically based solution for WV=0 = 0.18 mm.The calculated width of the space charge region at zero bias W is governed by equation  a small leakage current determines the I–V curve. This is clearly demonstrated by the reverse I–V characteristic presented in double logarithmic (log-log) coordinates in Fig. At small bias (I ~ V1/2) that is determined by the voltage dependence of the width of the depletion region (W ~ V1/2). This type of I–V characteristics is described by the generation-recombination mechanism in the scope of the Sah-Noyce-Shockley model, adapted for a metal-semiconductor contact21. It should be noted that the square root I–V dependence dominates only up to 7 V. This feature of the I–V characteristic well correlates with the previously analyzed AC characteristic which predicts that the CdTe substrate is wholly depleted at reverse bias ≥5.4 V.With increasing reverse bias, the current transport is determined by the generation current within the depletion region in the CdTe substrate. The reverse current exhibits a square root dependence on the applied bias 22. We have already shown that the graphene/CdTe interface is of high quality in comparison to other interfaces formed by the vacuum deposition of metal oxide or nitride films on CdTe substrates . However, the signal from 137Cs isotope (662 keV) is measured with a very promising resolution of 6.1% at the same reverse bias  of detectors strongly depends on the applied voltage. The voltage dependences of FWHM slightly vary for different detectors. Many parameters determine the energy resolution of X- and γ-ray semiconductor detectors: the attenuation length of X-rays with a given energy, the background electrical noise (dark background current) and the charge collecting efficiency. In their turn, the dominant charge transport mechanisms and charge collection efficiency depend on active material, electrical contacts (surface recombination), recombination lifetime, electron and hole mobility, and strength of the internal electrical field (applied bias divided by the active layer thickness)27. The thermoelectric cooler cools both the CdTe detector and the input field effect transistor (FET) of the charge sensitive preamplifier improving its noise-to-signal ratio. All measurements in our study were carried out at 300 K. Our detectors were not optimized, and we did not use a photolithography prepared guard ring protection against the surface leakage. Therefore, the measured relatively low energy resolution is quite reasonable and almost identical to that of wider band gap CdZnTe-based detectors, operated at the same temperature, but much higher DC bias of 450 V28.State-of-the-art commercial CdTe X-ray detectors (Ametek) with an energy resolution in the range of FWHM = 1% operate under a two-stage thermoelectric cooling that significantly reduces electrical noise and allows to increase operating voltages up to 500 VWe proposed a new concept of a Schottky-type semiconductor heterojunction detectors based on a Van der Waals contact between large area CVD-grown graphene and semi-insulating single crystal CdTe. The proposed low-cost and vacuum-free device fabrication process was tailored to skip the undesirable technological heat, which may detrimentally affect the electrical properties and level of compensation in semi-insulating CdTe single crystals leading to uncontrollable and irreproducible characteristics of the detectors based on them.WV=0 = 0.18 mm and the concentration of uncompensated acceptors NA−ND = 2.89 × 1010 cm−3 in the semi-insulating CdTe active material. Also, the dominant charge transport mechanisms through the detectors were determined at a wide range of reverse bias, which are in good correlation with the previously determined AC characteristics.There was developed a quantitative model for the AC characteristics of proposed graphene/CdTe/Au detectors in the scope of the equivalent circuit, which takes into account the features of their device architecture. The developed model was successfully employed to determine the real values of the crucial parameters relevant to Schottky diode-type semiconductor detectors: the width of the space charge region at zero bias 137Cs (662 keV) isotope with the FWHM of about 6% at the reverse bias of 100 V and room temperature.Even the unoptimized graphene/CdTe detectors exhibited a promising spectral resolution, specifically, for a 4) was added to the hydrogen gas flow to start growing graphene. The graphene was deposited at a temperature of 1050 °C. After cooling down, the graphene layers were transferred to the prepared CdTe substrates. For this purpose, the graphene layer was protected with a thin polymer layer, which was spin-coated on top of the graphene-Cu stack. Then, the Cu substrate was etched off using a concentrated ammonium persulfate ((NH4)2S2O8) water solution. The remaining polymer-graphene layer stack was rinsed several times using deionized water to remove residual impurities resulting from the etching procedure. Afterword, the polymer protected graphene layer was transferred onto the CdTe substrate, and the polymer layer was dissolved by rinsing in ethyl acetate  × 109 Ω cm at room temperature that is close to the resistivity of the intrinsic CdTe ρi = 4 × 109 Ω cm, i.e., the semi-insulating CdTe material under investigation can be considered as an almost intrinsic semiconductor.High quality and large area graphene was grown by the chemical vapor deposition (CVD) technique on copper foil. After mechanical polishing, the Cu foil was cleaned in an ultrasonic bath of acetone, and isopropyl alcohol etched in acetic acid and rinsed in deionized water. Then, the Cu foil was transferred to a growth furnace. The native oxide layer of the Cu foil was removed by annealing the substrates to high temperatures and exposing the surface to a flow of ultra-pure hydrogen gas. In a second step, methane  and 241Am (59 keV) X-ray sources by a charge-sensitive amplifier AMPTEK A250 in combination with a multichannel analyzer MCA8000A. The measurements were carried out at different biases and with the shaping time of 1 μs.The detecting properties of the graphene/7CdTe heterojunctions were measured at room temperature using Supporting Information-R1


Methods: MMP-mediated activation of PA-L1 was correlated to anthrax receptor expression and MMP activity in a panel of cancer cells . Uptake of 111In-radiolabeled PA-L1, 111In-PA-WTK563C, or 111In-LFE687A  in tumor and normal tissues was measured using SPECT/CT imaging in vivo. Results: Activation of PA-L1 in vitro correlated with anthrax receptor expression and MMP activity (HT1080 > MDA-MB-231 > B8484 > MCF7). PA-L1–mediated delivery of 111In-LFE687A was demonstrated and was corroborated using confocal microscopy with fluorescently labeled LFE687A. Uptake was blocked by the broad-spectrum MMP inhibitor GM6001. In vivo imaging showed selective accumulation of 111In-PA-L1 in MDA-MB-231 tumor xenografts (5.7 ± 0.9 percentage injected dose [%ID]/g) at 3 h after intravenous administration. 111In-LFE687A was selectively delivered to MMP-positive MDA-MB-231 tumor tissue by MMP-activatable PA-L1 (5.98 ± 0.62 %ID/g) but not by furin-cleavable PA-WT (1.05 ± 0.21 %ID/g) or a noncleavable PA variant control, PA-U7 (2.74 ± 0.24 %ID/g). Conclusion: Taken together, our results indicate that radiolabeled forms of mutated anthrax lethal toxin hold promise for noninvasive imaging of MMP activity in tumor tissue.Increased activity of matrix metalloproteinases (MMPs) is associated with worse prognosis in different cancer types. The wild-type protective antigen (PA-WT) of the binary anthrax lethal toxin was modified to form a pore in cell membranes only when cleaved by MMPs (to form PA-L1). Anthrax lethal factor (LF) is then able to translocate through these pores. Here, we used a  Matrix metalloproteinases (MMPs) are a family of zinc‐dependent proteases that together can degrade most components of the extracellular matrix. MMP activity is essential for tumor growth, allowing cancer cells to invade surrounding tissues and metastasize, as well as interact with the local tumor immune environment . The rolA molecular imaging agent that allows noninvasive imaging of MMP activity and related proteins would allow visualization of these disease processes in vivo, permitting early detection, prognosis, and measurement of therapy efficacy. A diverse range of molecular imaging agents has been developed over the past few years, expertly summarized in a previous publication . This raBacillus anthracis is a spore-forming bacterium that causes anthrax. As a means of suppressing its host’s immune response, the bacterium produces a set of toxins to promote its own survival: protective antigen (PA), lethal factor (LF), and edema factor. After binding to the ubiquitous anthrax receptors (CMG2 and TEM8), full-length PA (83 kDa) is cleaved by furin or furinlike proteases to a 63-kDa isoform  system to visualize MMP activity in tumor tissue using an adapted pretargeting system  9). Bac. Bac9). 168–P176 of the original PA . PA-L1 allows MMP-selective intoxication of tumor cells in vitro, as well as tumor xenografts grown in mice, with a modified LF fusion toxin, incorporating the adenosine diphosphate ribosylation domain of Pseudomonas exotoxin A (FP59)  .E687A, in combination with MMP-activatable PA-L1. In this report, we show that this novel pretargeted imaging system is selective for MMP-expressing cancer cell lines in vitro, and we show selective, noninvasive, in vivo imaging in MMP-expressing tumor xenografts grown in mice.Here, we expand the use of this MMP-activatable system to allow molecular imaging of MMP activity in mouse models of cancer, by SPECT imaging, using a radiolabeled catalytically inactive version of LF, LFB. anthracis LT were expressed and purified as previously described  whenever radiolabeled PA-WT is used  (E687A (90 kDa) containing a defective catalytic domain .PA-WT (83 kDa) is cleavable by furin and other furinlike enzymes . Here, w is used . PA-L1 h is used , whereas is used . LF vari 53 kDa) , LFn mod 53 kDa) , and fulc domain . CleavagK563C were site-specifically conjugated to the metal ion chelator diethylenetriamine pentaacetic acid (DTPA), using the bifunctional agent maleimide-DTPA -13--10-oxo-2,5,8,11-tetraazatridecane-5,8-diyl)diacetic acid) to allow radiolabeling with 111In, whereas PA-L1 or LFE687A was conjugated to p-SCN-benzyl-DTPA (S-2-(4-isothiocyanatobenzyl)-DTPA) before radiolabeling with 111In, using previously described methods -2,5-diphenyltetrazolium bromide) after exposure of cells to PA-WT, PA-WTith FP59 . To inveE687A was added to HT1080 or MCF7 cells growing on glass cover slides, in combination with PA-WT, PA-L1, or a vehicle control. After 3 h, cells were washed, fixed, and mounted with 4′,6-diamidino-2-phenylindole, and images were acquired using a confocal microscope . Full experimental details are laid out in the supplemental materials.Cy3-LFD) and number of binding sites for 111In-PA-WTK563C or 111In-PA-L1. In vitro assays were performed to evaluate the capacity of 111In-LFE687A or 111In-LFn to interact with PA pores and to be delivered into cells after radiolabeling. Full experimental details are laid out in the supplemental materials.Saturation cell binding experiments were performed to determine the apparent dissociation constant  Act of 1986 and with local ethical committee approval. Animals were housed in individually ventilated cages in sex-matched groups of up to 6 per cage in an artificial day–night cycle facility with ad libitum access to food and water. No animals were euthanized for welfare reasons. All analyses were performed masked to experimental group assignment.111In-LFn  or 111In-LFE687E  through a cannula inserted in the tail vein. SPECT/CT images were acquired for up to 3 h in list mode using a VECTor4CT scanner (MILabs). Three mice were used per group. Additional experimental detail is provided in the supplemental materials.To measure plasma clearance, dynamic SPECT/CT imaging of tumor-naïve SCID mice was performed after intravenous administration of 111In-PA-L1 or 111In-PA-WTK563C . Three mice were used per group.To determine the pharmacokinetics of PA-L1 in vivo, dynamic SPECT images were acquired in MDA-MB-231 tumor–bearing mice over 3 h after intravenous injection of 111In-LFE687E  by intravenous injection, together with PA-L1 . To determine the PA-L1 selectivity of tumor uptake, and thus of MMP-mediated uptake, groups of control animals were administered 111In-LFE687A alone or in combination with PA-WT , PA-U7 , or PA-L1 and an excess of unlabeled LFE687A . SPECT imaging was performed 24 h after administration of the 111In-LF/PA combinations. Three mice were used per group.MMP-activatable, PA-L1–mediated uptake of radiolabeled LF was evaluated in a pretargeting-inspired setup. MDA-MB-231 tumor xenograft–bearing mice were administered 111In was determined per gram of tissue, normalized by the administered amount of 111In (expressed as percentage injected dose per gram [%ID/g]). Tumor tissues were stored in 30% sucrose and, after 24 h, flash-frozen with dry ice and stored at −80°C until required for further processing.After imaging, the mice were euthanized by cervical dislocation, and selected organs, tissues, and blood were removed. The amount of t test with the Welch correction for nonequal variances to compare 2 groups. One-way ANOVA followed by Dunnett posttests were used to compare multiple groups. Two-way ANOVA was used to analyze grouped data. All results are reported as mean ± SD of at least 3 independent replicates, unless otherwise indicated.All statistical and regression analyses were performed using GraphPad Prism, version 7 (GraphPad Software). Linear regression was used to test for correlations between measurements. After testing for normality using a Shapiro–Wilk test, means were compared using a In a cell-free setting, recombinant human MMP2 and furin-activated MMP14, but not furin alone, were able to cleave PA-L1. Furin, but not MMP2, was able to cleave PA-WT . Cleavage of PA-L1 by MMP2 and MMP14 at the intended site was further confirmed by mass spectrometry .Bacillus anthracis MMP-activatable LT system. All cell lines, except for HT1080, expressed relatively high levels of furin  was determined for the PA-WT/FP59 and PA-L1/FP59 combinations. Whereas HT1080, MDA-MB-231, and B8484 cells were sensitive to both PA-WT/FP59  and PA-L1/FP59 , MCF7 cells were not . Quality control using instant thin-layer chromatography showed incorporation yields and radiochemical purities of more than 95% . The molar activity of the radiolabeled compounds was 8–15, 8–15, 10, and 5 MBq/nmol for 111In-PA-WTK563C, 111In-PA-L1, 111In-LFE687A, and 111In-LFn, respectively, for in vitro studies. The molar activity of all compounds for in vivo studies is as indicated. All labeled compounds demonstrated satisfactory stability in mouse serum, although a small degree of transchelation could be observed , attributable to transferrin or albumin.The PA/LF variants were conjugated to bifunctional chelator agents, which allowed radiolabeling using 111In-labeled PA-WTK563C showed various levels of binding sites . Affinity of 111In-PA-WTK563C for CMG2 and TEM8 was confirmed by a saturation binding assay on CHO cells that were stably transfected to express CMG2 or TEM8 . The overall affinity of 111In-PA-WTK563C was measured as KD = 4.0 ± 1.2 nM and was not significantly different in any of the cell lines (P = 0.09) , confirming specific binding  . Similar31 cells . Using t111In-LFE678E, demonstrated the selectivity and specificity of the LT system (111In-LFE678E with MDA-MB-231 cells after treatment with the broad-spectrum MMP inhibitor GM6001 demonstrated MMP-mediated PA-L1 activation (P = 0.0003). 111In-LFE678E was also taken up by MDA-MB-231 cells when administered in combination with furin-cleavable PA-WT or MMP-cleavable PA-L1 but not in combination with vehicle control or nonactivatable PA-U7 (P < 0.0001) . Similar results were obtained using a labeled LFn, 111In-LFn, or using HT1080 and B8484 cells . Saturation binding studies of 111In-LFE687A using fixed prepores in MDA-MB-231 cells revealed a binding constant of KD = 0.9 ± 0.09 nM .In a separate series of measurements, exposure of the panel of cancer cells to a radiolabeled full-length catalytically inactive variant of LF, T system . A signi 0.0001) . Additio111In-LFn and 111In-LFE687A was compared in vivo. Dynamic SPECT scans were performed after intravenous bolus injection of either 111In-LFn or 111In-LFE687A in naïve SCID mice . In contrast, the radiolabeled full-length protein, 111In-LFE687A, presented a significantly slower plasma clearance (P < 0.0001) than the truncated protein, maintaining a significant amount in the blood circulation 3 h after administration (35 ± 2.6 %ID/mL). In this case, a monoexponential decay described the radiotracer pharmacokinetic, with a blood half-life of 33 min. Ex vivo biodistribution studies confirmed the in vivo imaging results, revealing low blood signal, yet high kidney uptake (29 ± 1.0 %ID/g), after administration of 111In-LFn, possibly because of glomerular filtration .The pharmacokinetic behavior of CID mice . ImagingK563C and MMP-cleavable PA-L1 were also compared, with a view to optimizing the time of injection in a 111In-LF pretargeting system. Dynamic SPECT imaging after intravenous injection of 111In-PA-L1 or 111In-PA-WTK563C in MDA-MB-231 tumor–bearing mice revealed a distinct pharmacokinetic profile for 111In-PA-WTK563C compared with 111In-PA-L1 (111In-PA-L1 (5.7 ± 1.7 %ID/g) in MDA-MB-231 tumor tissue, compared with 111In-PA-WTK563C , at 3 h after administration of the labeled compound (111In-PA-WTK563C than for 111In-PA-L1 (P < 0.05). All in vivo SPECT and ex vivo biodistribution results were confirmed by autoradiography .The pharmacokinetics of radiolabeled furin–cleavable PA-WTIn-PA-L1 . Both pr111In-LFE687A was considered to be the more appropriate variant to image MMP-mediated PA-L1 pore formation in tumor tissue in vivo, given the extremely fast clearance of 111In-LFn. In addition, the slow but steady increase in tumor uptake of PA-L1 over time indicated that a coinjection of PA-L1 with 111In-LFE687A would be advantageous. The delivery of 111In-LFE678E by PA-L1 was evaluated in mice bearing MDA-MB-231 xenografts (111In-LFE678E to tumor tissue (5.98 ± 0.62 %ID/g), compared with control animals that were not coinjected with PA-L1 , indicating PA-L1–specific delivery. Imaging data at 3 h after injection showed similar results . When animals were also administered noncleavable PA-U7, instead of PA-L1, tumor uptake was significantly reduced , indicating MMP activity–specific targeting of PA-L1 in vivo. Additionally, when the interaction between PA-L1 pores or prepores and 111In-LFE678E was blocked by coadministration of an excess of unlabeled LFE678E, tumor uptake was significantly reduced to nonspecific uptake levels . 111In-LFE678E delivery to tumor tissue was significantly lower when coadministered with furin-cleavable PA-WT . This finding was in line with the rapid plasma clearance, activation by proteases in plasma (P < 0.0001). Contrastingly, PA-WT–mediated delivery of 111In-LFE678E resulted in significantly higher delivery to spleen , again consistent with PA-WT cleavage and activation in blood plasma.On the basis of its pharmacokinetic profile, nografts . Full ex111In-LFE678E by PA-L1 via binding to anthrax receptors, MMP-activation of PA-L1, and LF binding to its de novo receptor site on PA pores.Taken together, our results indicate the selective delivery to tumor tissue of B. anthracis LT system. Previous work by Liu et al. showed in detail that PA-L1, an MMP-cleavable PA variant, allows for selective delivery of LF and related fusion toxins such as FP59 to tumor tissue in vivo for therapeutic gain  did not show the necessary tumor-to-background ratios compared with the longer interval. In contrast, the smaller size of 111In-LFn resulted in renal clearance and therefore negligible liver uptake. 111In-LFn performed equally well in in vitro assays but, in vivo, was cleared from the blood so fast that PA-L1–mediated tumor uptake could not be observed . It may be possible that variants of intermediate size may be cleared more slowly yet are still renally cleared, in analogy with radiolabeled antibody fragments and derivatives, and thus will result in more optimal imaging characteristics  grant. Julia Baguña Torres is funded through a grant from the Pancreatic Cancer Research Fund. Thomas Bugge is funded by the NIDCR, NIH Intramural Research Program, and Samantha Hopkins is funded by the NIAID, NIH Intramural Research Program. Mass spectrometry analysis was performed at the Discovery Proteomics Facility (headed by Roman Fischer), which is part of the TDI MS Laboratory led by Professor Benedikt Kessler. No other potential conflict of interest relevant to this article was reported.Click here for additional data file.


Graphene, a two-dimensional carbon sheet with single-atom thickness, shows immense promise in several nanoscientific and nanotechnological applications, including in sensors, catalysis, and biomedicine. Although several studies have shown the cytotoxicity of graphene oxide in different cell types, there are no comprehensive studies on human embryonic kidney (HEK293) cells that include transcriptomic analysis and an in vitro investigation into the mechanisms of cytotoxicity following exposure to graphene oxide. Therefore, we exposed HEK293 cells to different concentrations of graphene oxide for 24 h and performed several cellular assays. Cell viability and proliferation assays revealed a significant dose-dependent cytotoxic effect on HEK293 cells. Cytotoxicity assays showed increased lactate dehydrogenase (LDH) leakage and reactive oxygen species (ROS) generation, and decreased levels of reduced glutathione (GSH) and increased level of oxidized glutathione indicative of oxidative stress. This detailed mechanistic approach showed that graphene oxide exposure elicits significant decreases in mitochondrial membrane potential and ATP synthesis, as well as in DNA damage and caspase 3 activity. Furthermore, our RNA-Seq analysis revealed that HEK293 cells exposed to graphene oxide significantly altered the expression of genes involved in multiple apoptosis-related biological pathways. Moreover, graphene oxide exposure perturbed the expression of key transcription factors, promoting these apoptosis-related pathways by regulating their downstream genes. Our analysis provides mechanistic insights into how exposure to graphene oxide induces changes in cellular responses and massive cell death in HEK293 cells. To our knowledge, this is the first study describing a combination of cellular responses and transcriptome in HEK293 cells exposed to graphene oxide nanoparticles, providing a foundation for understanding the molecular mechanisms of graphene oxide-induced cytotoxicity and for the development of new therapeutic strategies.  Graphene oxide is a two-dimensional monolayer composed primarily of carbon atoms. It is the oxidized form of graphene and contains various oxygen-containing functionalities, such as epoxide, carbonyl, carboxyl, and hydroxyl groups. Graphene oxide has a large surface area and is cheaper and easier to manufacture than graphene ,2. Due tIn vitro assays are effective strategies as a first approach for determining the cytotoxicity of nanomaterials. Several studies have been conducted to estimate the level of toxicity in different cell types, including pheochromocytoma-derived PC12 [Due to the extensive range of applications for graphene oxide, cytotoxicity studies are essential for the safe and sustainable development of graphene oxide-based nanotechnologies . In vitrved PC12 , HeLa, Mved PC12 ,12,13,14ved PC12 . Oxidatived PC12 .COX2 via epigenetic mechanisms in HEK293T cells.Graphene oxide biocompatibility with several cell lines is dependent on the size of the particles. Graphene oxide was found to elicit toxicity only at high concentrations in human fibroblast cells (HDF); moreover, Gurunathan et al.  reportedCell survival and death are two major toxicity endpoints that can potentially be affected by any nanoparticle treatment. Carbon nanoparticles, in particular, evoke severe toxicity by inducing apoptosis and mitochondrial dysfunction. Due to the extensive use of graphene oxide, it is necessary to minimize its cytotoxicity and determine the associated regulatory molecular mechanisms. Recent findings suggest that the graphene oxide treatment can impair the general cellular priming state, including eliciting disorders of the plasma membrane and cytoskeleton construction . GraphenIn this study, we selected HEK293 cells as a model system to investigate the effects of graphene oxide. These cells are widely used in cell biology and biotechnology to study signal transduction and protein interactions. Non- or low tumorigenic HEK293 cells are frequently used to test the oncogenic properties of cancer-associated genes. Furthermore, these cells most closely resemble adrenal cells, which possess numerous neuronal properties. Given the broad use of HEK293 cells for biomedical research, we used HEK293 cells to investigate the comprehensive cellular effects and molecular mechanisms associated with graphene oxide toxicity.4, NaNO3 anhydrous ethanol, H2SO4, 36% HCl, and 30% H2O2 aqueous solution were purchased from Sigma-Aldrich . The penicillin-streptomycin solution, trypsin-ethylenediaminetetraacetic acid solution, Dulbecco’s modified Eagle’s medium (DMEM), and 1% antibiotic-antimycotic solution were obtained from Gibco . Fetal bovine serum (FBS) and the in vitro toxicology assay kit were purchased from Sigma-Aldrich. The kits for measuring MDA and antioxidants levels were also purchased from Sigma-Aldrich, as were all other chemicals unless otherwise stated. HEK293 cell lines were purchased from the American Type Culture Collection . Graphite powder, NaOH, KMnO2SO4 at 0 °C; then, 8 g of KMnO4 and 1 g of NaNO3 were added gradually while stirring. The mixture was transferred to a 40 °C water bath and stirred for 60 min. Deionized water (250 mL) was slowly added, and the temperature was increased to 98 °C. The mixture was maintained at 98 °C for 30 min and the reaction was then terminated by adding 500 mL of deionized water and 40 mL of a 30% H2O2 solution. The color of the mixture changed to a brilliant yellow, indicating oxidation of pristine graphite to graphite oxide. The mixture was filtered and washed with dilute HCl to remove metal ions. The product was washed repeatedly with distilled water until a pH of 7.0 was achieved and then further sonicated for 15 min. To prepare smaller sized graphene oxide particles, the prepared, brownish mixture of graphene oxide was centrifuged and washed several times with deionized water. The unexfoliated graphene oxide was removed by centrifugation at 12,000 rpm for 5 min, and the as-prepared graphene oxide was dried at 40 °C. To obtain a uniform distribution of smaller sized graphene oxide sheets, a low-speed centrifugation was performed at 5000 rpm for 15 min to remove thick multilayer flakes until all the visible particles had been removed. The supernatant was then further subjected to ultrasonication for 1 h. The resulting graphene oxide samples were used for subsequent analyses.Graphene oxide was synthesized as described previously, with suitable modifications ,27. For 2 at 37 °C. Cells were routinely grown in 100-mm plastic tissue culture dishes  and harvested at the logarithmic phase of growth using a trypsin-EDTA solution. Experiments were performed in 96-, 24-, and 12-well plates and 100-mm cell culture dishes, as indicated. Cells were treated with various concentrations of graphene oxide or the required dose of graphene oxide. Silver nanoparticles (25 μg/mL) were used as a positive control and media without cells used as a negative control. In order to verify the performance of graphene oxide, all the cellular assays experiments, we used media without cells as a negative control (NC) and silver nanoparticles (AgNPs) as a positive control. HEK293 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator in 5% CO2 in a humidified incubator, 10 μL of a CCK-8 solution was added to each well and the plate was incubated for an additional 2 h at 37 °C. The absorbance was measured at 450 nm using a microplate reader . Cell proliferation was determined according to the manufacturer’s instructions (Roche).Cell viability and cell proliferation were measured using a Cell Counting Kit-8 (CCK-8) and a BrdU assay kit. Briefly, HEK293 cells were plated in 96-well flat-bottom culture plates containing various concentrations of graphene oxide. After 24 h of culture at 37 °C and 5% COHEK293 cell membrane integrity was evaluated using an LDH Cytotoxicity Detection Kit according to the manufacturer’s instructions. Briefly, cells were exposed to various concentrations of graphene oxide for 24 h, following which dead-cell protease activity was assessed using a previously described method . The cytHEK293 cells were treated with various concentrations of graphene oxide for 24 h. ROS levels were measured according to a previous method based on the intracellular peroxide-dependent oxidation of 2′,7′-dichlorodihydrofluorescein diacetate  to form the fluorescent compound, 2′,7′-dichlorofluorescein (DCF) [Antioxidant enzyme activities and MDA content were measured using a previously described method . HEK293 HEK293 cells were treated with various concentrations of graphene oxide for 24 h. Changes in MMP were determined using the cationic fluorescent dye, JC-1 . Fluorescence of JC-1 aggregates and JC-1 monomers was measured at an excitation wavelength of 488 nm and emission wavelengths of 583 and 525 nm, respectively, using a Gemini EM fluorescence microplate reader .ATP levels were measured in HEK293 cells according to the manufacturer’s instructions . The cells were exposed to various concentrations of graphene oxide for 24 h and the ATP level was then measured.8-oxo-dG and its analogs were used as biomarkers of oxidative DNA damage and oxidative stress. To evaluate graphene oxide-induced oxidative stress in HEK293 cells, an 8-oxo-dG ELISA assay was utilized as previously described  and follCaspase 3 activity was measured according to previously described methods . Cells wTotal RNA was isolated from both untreated and AdNP-treated HEK293 cells using an RNeasy kit (Qiagen). Before the RNA extraction, the cells were thoroughly washed with ice-cold PBS to remove dead cells. The RNA concentration and quality were measured on a bioanalyzer using Bioanalyzer RNA chips . Total RNAs with ˃7 RNA integrity number (RIN) were used for library preparation. An RNA-Seq library was prepared using an Illumina TruSeq Stranded total RNA sample preparation kit following the manufacturer’s instructions. Approximately 1 µg of total RNA was reverse transcribed. The synthesized cDNA was then subjected to fragmentation, adaptor ligation, PCR amplification, and gel size selection. Sequencing was performed using a NextSeq500 sequencer . After sequencing, the reads were assessed for quality and mapped to the National Center for Biotechnology Information (NCBI) Human Genome Assembly (hg19) using FastQC and the STAR pipeline, respectively. Using the Cufflink tool, differentially expressed genes (DEGs) were determined using a cutoff value (FPKM) >3 and a fold change (FC) >3. The DEGs were then subjected to gene ontology (GO) term analysis using DAVID, and the results were plotted using GOplot . Scatter plot and GOplot images were generated in R (v3.3.2).p-value was calculated by right-sided hypergeometric tests and Benjamin–Hochberg adjustment was used for multiple test correction. KEGG pathways with a p-value < 0.05 were considered significant. Coexpression and physical interactions (protein–protein) of the DEGs were determined using GeneMANIA plug-in  for Cytoscape. A query-dependent weighting method for the selected genes was used in the analysis.GSEA  was used to determine the biological enrichment in the DEGs of the treatment group. For this, gene expression data in GCT format, molecular signature database (MsigDB) in GMT format, and phenotype labels in CLS file format were used. KEGG pathway enrichment analysis for the DEGs from the graphene oxide-treated HEK293 cells was performed using the ClueGO plug-in  for Cytoscape . ClueGO generates functionally clustered KEGG annotation networks with the DEGs. The t-test to determine the one sample to control and significant differences denoted by an asterisk. The results are presented as mean ± standard deviation of three experiments. *p < 0.05 was considered significant, **p < 0.01 was considered highly significant, and ***p < 0.001 was considered very highly significant.Independent experiments were repeated at least three times with three replicates for each concentration. Data were analyzed by a Student’s . Graphene oxide displayed two different characteristic features: A shoulder at approximately 300 nm, corresponding to an n-π* plasmon peak, and a separate shoulder at 230 nm, corresponding to a π-π* plasmon peak [Graphene oxide materials were synthesized as described in our recent study , and themon peak .−1 was assigned to O–H group stretching vibrations. The absorption peaks at 1736 and 1625 cm−1 were assigned to C=O stretching of carboxylic and/or carbonyl functional groups [−1 were assigned to C–O stretching vibrations (The FTIR spectra showed characteristic oxygen-containing groups in which the main absorption band at 3356 cmbrations B. The FTX-ray diffraction (XRD) was performed to determine the magnitude and location of peaks in the XRD curves. In general, natural graphite shows a sharp and highly intense reflection peak at 2θ = 26°. After oxidation, the sharp and intensive graphene oxide peak reflected at 2θ = 10.2° was due to the introduction of functional groups such as hydroxyl and carboxyl during oxidation and ultrasonic treatment C. This iDynamic light scattering is a simple technique that can be used to determine the size distribution of particles in solution. Although the prepared sample had a wide particle size distribution (between 10 and 300 nm), most of the particles were dispersed within 50 nm D in wateThe surface morphology of graphene oxide was confirmed by SEM. The SEM images indicate the formation of graphene oxide sheets with a closely packed lamellar structure, composed of waves that look like fine, alternating layers E. GrapheNext, we examined the surface morphology of graphene oxide by TEM. The TEM images of graphene oxide exhibited a closely packed, lamellar, swollen structure, with a clean and transparent surface. The TEM images also showed a typical flaky structure, which had the wrinkly/wavy characteristic of transparent pure graphene oxide F. Our re−1, which can be attributed to sp3-dependent defects on the breathing modes of the sp2 rings. These defects indicate flaws in the graphene structure and the G band at approximately 1600 cm−1, owing to the vibration of the sp2-bonded carbon atoms were observed in graphene oxide with the ID/IG value of 1.0. The D and G bands were attributed to disorders in the C–C bonds and in-plane vibrations of the C–C bonds, respectively, which is in agreement with previous reports [Raman spectroscopy is used to observe the atomic vibrational, rotational, and other low-frequency dynamic modes in carbon materials. It is a very well designed technique to identify the molecular or atomic composition of samples quickly and reliably. As depicted in  reports ,36,37,38The effects of graphene oxide on the viability and proliferation of HEK293 cells were determined after 24 h of exposure to different concentrations of graphene oxide (0−50 μg/mL) using the CCK-8 and BrdU assays, respectively. The results revealed that graphene oxide has a significant and dose-dependent toxic effect, even at 10 μg/mL A,B. p < 0.05) viability and proliferation with increasing concentrations of graphene oxide. We have previously shown the dose-dependent toxic effects of graphene oxide in a variety of cells, including human breast cancer, ovarian cancer, neuroblastoma, and human lung cancer cells [HEK293 cells show reduced  leakage and dead-cell protease activity appear to be sensitive and reliable. Moreover, both assays have been used previously to measure plasma membrane integrity. Therefore, we measured LDH leakage and dead-cell protease activity to determine the cytotoxicity of graphene oxide. Dead-cell protease activity is represented as the percentage of viable cells. When the cell membrane is damaged, LDH is released into the medium, while dead-cell protease activity is quantified as the level of proteases released from membrane-compromised cells. HEK293 cells were treated with graphene oxide for 24 h and LDH and the dead-cell protease were dose-dependently released into the media A,B.Graphene oxide induces cytotoxicity by increasing LDH leakage in a variety of cancer cells, including ovarian cancer, neuroblastoma, and human lung cancer cells ,28,31. COxidative stress is one of the major mechanisms involved in graphene oxide-induced oxidative stress in a variety of cell types, including bacterial and human cells ,32,43. OGraphene oxide-treated HEK293 cells showed a high rate of ROS formation with increasing doses of graphene oxide (10–50 μg/mL). ROS combine with hydrogen atoms, generating water and a fatty acid radical that can cause oxidative degradation of lipids and, subsequently, cell membrane damage ,44. We eSince thiol metabolism seems to play an important role in defense against exposure to toxicants such as nanoparticles, the concentration of reduced glutathione (GSH) was determined in HEK293 cells after incubation with graphene oxide. The level of GSH was significantly and dose-dependently decreased in graphene oxide-treated cells A. Glutathione is considered one of the most important ROS scavengers and is an important and ubiquitous component of the cellular defense mechanism against oxidative stress . A rapidIncreased levels of oxidative stress disrupts homeostasis, and the enzymes responsible for reducing ROS levels, such as glutathione peroxidase (GPx), cannot compensate for the damage to macromolecules like proteins, DNA, and lipids, which greatly influences cell metabolism and signaling . To bett2+ homeostasis [Mitochondrial function is essential for cellular energy balance, metabolism, modulation of calcium signaling, and cell death. Since mitochondria are the main source of ROS in the cell, we hypothesized that a relationship exists between the mitochondrial membrane potential (MMP) and the rate of ROS production. Therefore, we investigated the effects of graphene oxide on the MMP by measuring the JC-1 aggregate to monomer ratio, and found that it was significantly and dose-dependently lower in graphene oxide-treated HEK293 cells than in the control condition A. The fieostasis . Loss ofeostasis ,48.Since mitochondria are a major source of intracellular ATP generation, and the MMP is vital for ATP production, we measured the ATP levels in graphene oxide-treated HEK293 cells. As shown in Overproduction of ROS can induce oxidative stress, which can subject cells to DNA damage, unregulated signaling, changes in motility, cytotoxicity, apoptosis, and cancer initiation . DNA damC. elegans germ cells and mammalian cells [PUMA and NOXA. Graphene nanoparticles induce cytotoxicity in rat pheochromocytoma cells (PC12 cells) through mitochondrial injury-dependent release of LDH, increased activation of caspase 3, and ROS generation [Overall, caspases play significant roles in cell survival and cell death, since most apoptotic pathways are activated by caspase-dependent cleavage of numerous cytoplasmic and nuclear proteins. Apoptotic signaling by executioner caspases, including caspases 3, 6, and 7, is activated by initiator caspases  ,60. To ian cells . The p53neration  demonstrneration  observedLRP1 and CD24) and upregulated genes (HSPA1A) are illustrated using the IGV genome browser . In the DEG analysis, 119 and 148 genes were down- and upregulated, respectively, in response to graphene oxide treatment A. We nex browser .DDR2, a gene found in the GO term of “cell adhesion” , KEGG, and GeneMANIA was performed to know the mechanism by which graphene oxide promotes apoptosis. In the GSEA, 33 out of 50 and 17 out of 50 gene sets were respectively up- and downregulated in the graphene oxide-treated group, with an enrichment score of 0.4. Among them, the analysis revealed that groups of DEGs were enriched in the apoptosis and G2M checkpoint pathways A. KEGG aFOSB, JUNB, FOS, and FOSL1. Irradiating mammalian cells with ultraviolet (UV) light is known to rapidly induce c-Jun, JUNB, and c-Fos expression [HES5 and RARB  were queried among the DEGs. As shown in pression . In contand RARB B. Importand RARB C. CollecUnderstanding the molecular mechanism of graphene oxide-induced cytotoxicity and cellular responses in HEK293 cells is necessary. We therefore proposed an integrative analytical approach, using various cellular assays and transcriptome analysis, to identify the associated molecular and cellular pathways. We prepared smaller graphene nanoparticles with an average size of 50 nm and then characterized them using various analytical techniques. Furthermore, we also performed a series of assays that measured cell viability, proliferation, cytotoxicity, oxidative stress, antioxidant levels, mitochondrial dysfunction, DNA damage, caspase 3 activity, and the transcriptional profiles of genes involved in various cellular pathways. The CCK-8 and BrdU assays revealed a significant, dose-dependent decrease in cell viability and proliferation in response to graphene oxide. Our findings also demonstrated increased LDH leakage, ROS generation, and decreased levels of glutathione and glutathione peroxidase. This detailed, mechanistic approach shows that graphene oxide exposure results in decreased mitochondrial membrane potential and ATP synthesis, as well as increased DNA damage and caspase 3 activity. Our analysis revealed that graphene oxide directly alters the expression of genes involved in various apoptosis-related biological pathways. The RNA-Seq analysis of graphene oxide-treated HEK293 cells not only establishes mechanistic insights into how graphene oxide evokes extensive cell death in HEK293 cells, but also provides a basis for the development of therapeutic strategies to alleviate harmful effects during use. This study further provides a transcriptomic insight into the role of surface-mediated cellular signaling triggered by graphene oxide and will enable the development of nanomaterial-based therapeutics for regenerative medicine. This approach in understanding nanomaterial–cell interactions illustrates how changes in the transcriptomic profile can predict downstream effects following nanomaterial treatment.


The electrochemical decoration of CVD monolayer graphene with MoO2 is shown to originate from the edge plane like- sites/defects. Edge plane decoration of MoO2 nanowires upon monolayer graphene is observed via electrochemical deposition over short time periods only (ca. −0.6 V for 1 second (vs. Ag/AgCl)). At more electrochemically negative potentials (ca. −1.0 V) or longer deposition times, a large MoO2 film is created/deposited on the graphene sheet, originating and expanding from the original nucleation points at edge plane like- sites/defects/wrinkles. Nanowire fabrication along the edge plane like- sites/defects of graphene is confirmed with Cyclic Voltammetry, Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM) and Raman Spectroscopy. Monitoring the electrochemical response towards [Ru(NH3)6]3+/2+ and comparing the heterogeneous electron transfer (HET) kinetics at CVD grown monolayer graphene prior and post nanowire fabrication reveals key understandings into the fundamental electrochemical properties of carbon materials. The HET kinetics  grown monolayer graphene using a polymer-free transfer methodology is explored through the selective electrodeposition of Molybdenum (di)oxide (MoO Typically, graphene is fabricated via one of two routes; a bottom-up (BU) or a top-down (TD) approach. TD methods, such as chemical/thermal reduction of graphene oxide or using physical/chemical exfoliation, can give rise to a large quantity of graphene sheets; however, the fabricated graphene is generally highly defective and abundant with residual C/O groups or other contamination such as surfactants or metals24. Bottom-up (BU) fabrication routes typically lead to higher quality graphene, but in smaller quantities25. Chemical Vapour Deposition (CVD) growth of graphene onto a copper catalyst is a BU route that facilitates the study of high quality monolayer graphene sheets/films28, which can then either be transferred (commonly using Poly(methyl methacrylate), PMMA) onto a suitable substrate31. PMMA has previously been reported to affect the physical and electrical properties of CVD grown graphene samples transferred via this route38, therefore, in this work we address the fundamental properties and conduct performance studies on CVD grown monolayer graphene transferred via a polymer-free transfer method39.Graphene is extensively studied due to its reported unique electronic, mechanical and optical propertiesvia a BU approach), the reactivity of such electrodes is commonly assigned to the graphene edge planes, which are reported to exhibit ca. 4 orders of magnitude greater specific capacitance, faster electron transfer rates and higher electrocatalytic activity when compared to the graphene basal planes40. Recent work involving the electrochemical behaviour of CVD grown pristine graphene has shown a correlation in the structure of graphene, in terms of a dependence upon its number of layers and the macroscopic electrochemical response/performance42. Moreover, an identical relationship in terms of graphene’s geometric structure  has recently been reported with respect to the lateral flake size, in which smaller graphitic flake sizes  exhibited improved electrochemical properties when compared to the inverse43. Indeed, there have been numerous other studies reporting that, in comparison to the edge plane like- sites/defects45, the basal planes of graphitic and carbon-based materials are effectively inert47. However, regardless of the vast number of recent reports concerning graphene-based electrodes, researchers still debate the real contributions of edge and basal plane like- sites/defects at the macroscopic scale, in future experiments researchers should clarify the experimental configuration of their studies, ie. macro- vs microscopic voltammetry48 with respect to their observed heterogeneous electron transfer (HET) kinetics52; further work is required to explore this edge vs basal argument for carbon surfaces48. It is these aforementioned research considerations that we investigate herein, through the utilisation of a MoO2 deposition technique to decorate CVD grown monolayer graphene (transferred using a polymer-free method) so that one can determine the true electrochemical contributions arising from its structure.In terms of understanding the electrochemical response of CVD grown monolayer graphene  are responsible for the voltammetric signal, and Rowley-Neale et al.59 extended these insights with screen-printed graphitic electrodes (SPEs) with similar results. In these approaches, the selective deposition of metals and metal oxides onto graphite is called ‘step edge decoration’56, and has been utilised to demonstrate that electrochemical reactions at edge plane graphite are “anomalously fast” in comparison to the basal plane, which is effectively inert in comparison60. Through the selective coating and blockage method of the edge plane like- sites/defects with MoO2 nanowires , a reduction in the electrochemical response towards [Ru(NH3)6]3+/2+ 47 was reported; clearly demonstrating that the electrochemical reactivity of the HOPG is due to edge plane like- sites/defects. To the best of our knowledge, the above elegant approach has not previously been applied towards CVD grown monolayer graphene sheets (in addition to the polymer-free transfer method utilised herein) contributing to the understanding of graphene and all akin carbon-based electrode materials.Molybdenum oxide(s) and other metal oxides have been shown to electrochemically nucleate specifically onto the edge plane like- sites/defects of highly oriented pyrolytic graphite (HOPG)2 onto HOPG and SPEs59, where MoO2 selectively deposits upon the available edge plane like- site/defects, allowing their electrochemical contributions to be deduced. In consideration of these approaches, herein we adapt this method towards CVD grown monolayer graphene sheets. Presented within the Experimental Section and ESI (1.1–1.4) are details of the fabrication process, optimisation and physicochemical characterisation of the CVD grown monolayer graphene sheets and their MoO2 decorated counterparts. Briefly, Figure 2 decorated monolayer graphene, displaying the typical monolayer graphene D (1350 cm−1), G (1580 cm−1), 2D (2700 cm−1) and 2D’ (3250 cm−1) peaks, the presence of MoO2 (308 cm−1)61 and the presence of the underlying Si (514 and 985 cm−1) wafer . Additionally, AFM images were collected in order to characterise the MoO2 nucleation upon the edge plane like- sites/defects as depicted in Figure selective nucleation characterised by Rowley-Neale et al.59.Previously reported methodologies have utilised the electrochemical decoration of MoO3)6]3+/2+, which exhibits a peak-to-peak separation (ΔEp) of 160 mV (at 50 mV s−1vs. Ag/AgCl) and a HET . Note that this value is similar but slightly faster than other CVD monolayer graphene sheets previously reported by our research group41, which is due to a higher exposure of the edge plane like- sites/defects upon the graphene sheet. Furthermore, it is worth noting at this stage that each individual graphene sheet is unique in terms of its level of edge plane like- site/defects, so there is natural variation in the reported HET rates throughout the literature; thus, it is important to characterise each monolayer graphene sample62 prior to modification with MoO2. Figure 2 electrochemically decorated monolayer graphene sheet following applying chronoamperometry at a set voltage (−0.6 V vs. Ag/AgCl) for 1 second, where it is readily observed that the MoO2 nucleates upon the edge plane like- sites/defects/wrinkles of the monolayer graphene surface. Figure 3)6]3+/2+, which exhibits a ΔEp of 200 mV (at 50 mV s−1vs. Ag/AgCl) and a −4 cm s−1. Such decreased voltammetric behaviour implies that the insulating MoO2 nanowires have coated some of the available edge plane like- sites/defects, with some edge sites remaining unmodified. Figure θedge) values) the changes observed as one electrodeposits MoO2 onto the graphene electrode’s surface. Such voltammetric profiles show that the decorated electrode (dashed line) still exhibits relatively fast HET kinetics when compared to the unmodified electrode (solid line), but presents a lower current intensity due to the limited availability of edge plane like- sites/defects on the electrode surface. This is corroborated with the SEM presented within Fig. Figure vs. Ag/AgCl). Presented within Fig. 2 electrochemically decorated monolayer graphene sheet that has been applied at −0.6 V (vs. Ag/AgCl) for 10 seconds via chronoamperometry, where it is clear that electrodeposition is no longer isolated upon the edge plane like- sites/defects. Instead, the MoO2 has coated/blocked the entire electroactive surface. Upon electrodeposition at more electrochemically negative potentials (ca. −1.0 V) or longer deposition times, a large MoO2 film is created/deposited on the graphene sheet, originating and expanding from the original edge plane like- sites/defects (non-selective deposition), as shown in Figures 3)6]3+/2+, exhibits a ΔEp of 820 mV and a −6 cm s−1. As expected, the monolayer graphene sheets coated with insulating MoO2 (upon the edge plane like- sites/defects), give rise to an electrochemical response with a larger ΔEp (slower electron transfer) than the unmodified monolayer graphene. The aforementioned MoO2 coverage dependant electrochemical behaviour has been illustrated within the schematic presented within Fig. Next we investigate the effect of increased deposition times at −0.6 V (2 wires that nucleate onto the graphene’s edge plane (and or defect/wrinkle) sites when a electrodeposition for 1 second at −0.6 V is performed, resulting in a decrease in the reversibility of the [Ru(NH3)6]3+/2+ redox probe. Longer deposition times will increase the nucleation of the MoO2 onto remaining edge sites and towards the basal plane of the graphene sheet (deposition of 5 seconds at −0.6 V). Eventually, a complete coverage of the surface of the graphene is confirmed by the absence of cyclic voltammetric redox peaks of [Ru(NH3)6]3+/2+, for example, when a electrodeposition of 10 total seconds at −0.6 V is performed.Figure 2 wires are indeed being electrodeposited selectively onto the graphene edge plane like- sites/defects, at a set potential of −0.6 V (vs. Ag/AgCl) for 1 second and this results in a decrease in the 2 selectively electrodeposits upon the edge plane like- sites/defects, which is confirmed by physicochemical characterisation, indicating that these are the active sites for electron transfer. If no changes in the voltammetry were observed, this would indicate that basal planes sites are the origin of electron transfer, as this is not the case, our work provides convincing evidence that it is the edge plane like- sites/defects of the CVD grown monolayer graphene that are the origin. When utilising an electrodeposition of 10 seconds, an insulating film is created, which largely decreases the amount of available edge plane like- sites/defects by up to 99.9%. Note that in the case of sample B, however, due to the initial low θedge and hence 2 has fewer edge plane like- sites/defects to deposit upon. This demonstrates again that each individually grown CVD graphene sheet is unique in terms of edge plane like- sites/defects and i.e. too long deposition times and high deposition potentials, physicochemical characterisation would indicate a large film is created, which would give the false impression that the basal plane of monolayer graphene is as equally electrochemically active as the edge plane sites, which is not the case. However, there has been a great deal of elegant work published on the micro scale response of electron transfer at carbon materials using novel nanoscale techniques64, therefore it is intended that the data reported herein be considered in-conjunction with such other studies for the electrochemical community to better understand this phenomena as a whole.The experiments performed above confirm that the MoO2 nanowire arrays have been electrodeposited onto the edge plane like- sites/defects of CVD grown monolayer graphene using a polymer-free transfer method, which have been confirmed via physicochemical characterisation. The electrochemical activity pre and post MoO2 electrochemical deposition has been assessed using a near-ideal redox probe and in the latter case indicates that the HET kinetics (MoO3)6]3+/2+ (in 0.1 M KCl), 0.5 M H2SO4 and 1 mM Na2MoO4 (in 1 M NaCl).All chemicals used were of analytical grade and were used as received from the supplier  without any further purification. All solutions were prepared with deionised water of resistivity no less than 18.2 MΩ cm and were vigorously degassed prior to electrochemical measurements with high purity, oxygen free nitrogen. Test solutions were: 1 mM 3+/2+ (in 0.1 M KCl) using the well-known66 and utilised Nicholson method67, for quasi-reversible electrochemical reactions via the following formula59:φ is a kinetic parameter, D is the diffusion coefficient for [Ru(NH3)6]3+/2+ (D = 9.1 × 10−6 cm2 s−1)66, n is the number of electrons that are taking part in the process, F is the faraday constant, vis the scan rate, R is the gas constant and T is the temperature in Kelvin. In order to calculate the HET rate constant, we use the peak to peak separation (ΔEp) to deduce φ, where ΔEp is obtained at various voltammetric scan rates52. The standard heterogeneous constant 6]3+/2+, has been widely determined with a commercial simulation package providing a value of 0.4 cm s−1 for HOPG electrodes58.allowing the coverage of edge plane like- sites/defects upon the graphite electrode to be deduce; we use this and adapt this to the voltammetry observed at the CVD grown monolayer graphene sheets. %θ−1 . Scans were carried out at 40 µm/s with a force set point of 6 nN. Samples were attached to glass microscope slides using double sided tape.Raman Spectroscopy was performed using a DXR Raman Microscope  fitted with a 532 nm excitation laser at a low power of 3 mW to avoid any heating effects. Spectra were recorded using a 3 second exposure time for 3 accumulations at each point. Scanning electron microscope (SEM) images were obtained using a JSM-5600LV  model SEM equipped with an energy-dispersive X-ray microanalysis (EDS) package. Atomic force microscopy (AFM) images were obtained using a DualScope C26 , carried out using AC mode using DS 95 AC probes with a spring constant of 42 N mNote that the area studied is in the macroscale range which is not considered too large because indeed it averages the actual phenomena occurring locally . Such that this makes this study applicable for real applications. We are reporting on the average response of the macro electrode with varying contributions of the two micro features , which is relevant because the likely end use/application of such systems will be on that of the macro scale.Supplementary information