Social media is used increasingly by the general public to access health information. However, a lack of models for health information distribution limits the presence of publicly funded services on social media sites.The goal of the study was to present a model for delivering child health information to parents through a social media site.A Facebook site was launched for 11 months based on a question-and-answer service produced by a pediatrician and open to Facebook users over 18 years old. If the answer did not include a further referral to a health care service provider, the question was considered comprehensively answered. The site was funded by a pharmaceutical company, and it included an advertisement of a pharmaceutical product for children’s fever and pain.P=.003).During the study, 768 questions were submitted: an average of 69.8 (SD 31.7) per month. There were 245,533 independent Facebook users on the site, with an average of 727.0 (SD 2280.6) per day. Infections were the most common theme in questions . Questions were more likely to be comprehensively answered if they were related to infections  than questions related to non-infectious symptoms (265/423, 64.2%, On this site aimed at parents of small children, personalized answers were an effective way of delivering information. The service is likely to have reduced the need for further contacts with a health care service provider in more than half of the cases. The site could serve as a model for publicly funded health information distribution. Social media has changed the way the general public accesses health information . With thHowever, user-generated health content on social media is generally inconsistent with clinical guidelines and professional knowledge, and non-biased information can be difficult to obtain . PubliclWhile social media can be used to distribute specific information on issues addressed by individual Internet users, these discussions can be accessed by countless others on an open site, making the information available to a larger audience. Depicted in our study is a social media site for distributing child health information to parents. The information was distributed by a pediatrician. The aim was to determine the number of questions generated by the site, the profile of parents’ concerns, and the number of visitors on the site. Also, the parental contact was assessed by evaluating whether or not the answers to questions included further referral to a health care provider.A Facebook site, Kysy lastenlääkäriltä . The results from these 11 months are used in this analysis.The service was intended to answer questions on children’s fever and pain, but parental postings included questions on all subjects. A non-medically trained moderator evaluated all postings and messages left on the site twice daily, and postings that included product labels were removed according to Finnish law on pharmaceutical product promotion. However, the sender was given the opportunity to re-send the posting if the product label was changed to a generic name. All questions were answered by the pediatrician (the author), even if they were outside the focus of the service. The pediatrician was employed on an hourly basis to the site, and at the time was also a research coordinator at a clinical postgraduate school at the University of Helsinki, Finland. The pediatrician answered questions once daily on weekdays. In addition, the moderator contacted the pediatrician regarding possible emergency-related questions potentially requiring an urgent response, in which case the parent received a notification to seek immediate treatment.Typically, each posting included several questions. Postings were first analyzed for the number of questions, and then the questions were categorized according to the symptoms presented . Most of the questions did not include sufficient information to set a specific diagnosis. Therefore, categorization was performed in relation to the clinical entity the question belonged to. Infections were further categorized into subgroups. Otitis media (a middle ear infection) was the largest subgroup and as such was included in the analysis. Each of the other infection subgroups included only 1-30 questions, and thus these subgroups were omitted from further analyses, although included in the infection category.Answers were evaluated by the author by reviewing the questions in each posting separately. If the answer did not include a referral to a health care service provider, the question was considered comprehensively answered. If the answer included a referral at a later time should certain criteria be met (such as in the case of new symptoms), the question was also considered comprehensively answered.Analyses were performed using GraphPad Prism 6.0. Question data are presented as mean (SD). Proportions were compared using the chi-square test. User statistics were gathered from information in the Facebook user database, Page insights.During the study, 245,533 independent Facebook users visited the site, an average of 727.0 (SD 2280.6) per day. During closure of the service (April to August 2012), there were an average of 17.4 (SD 48.8) daily visitors. In the average month, 75.0% of the visitors were women (SD 13.7). The largest visitor age group was 25-34 years in both women and men: 39.6% (SD 2.9) and 11.5% (SD 2.4) of all monthly visitors, respectively. Most of the visitors were inhabitants of the five largest cities in Finland (data not shown). By December 31, 2012, there were 12,328 Facebook likes since the beginning of the service.There were an average of 42.6 (SD 27.6) monthly postings from parents, 478 in total. Men sent a total of 9 postings. Each posting contained a mean 1.6 (SD 0.6) questions (N=768), 69.8 (SD 31.7) questions per month. Of all postings, 63.4% (303/478) were comprehensively answered.Infections were the most common theme in questions , with otitis media–related questions being the most common subgroup of infections . There were 413 non–infection-related questions during the study period. The other symptom groups with more than 30 questions in the study period were non-infectious rash , allergies , fever , neurological symptoms , and nutrition . Within the otitis media category, 85.5% of questions (59/69) were considered comprehensively answered.In general, only one exchange of postings occurred for each question. However, the need for referral was evaluated from each posting by the pediatrician. Questions relating to fever were most likely to be comprehensively answered  while questions relating to neurological symptoms were least likely (19/34, 55.9%; Large seasonal variation in question topics was present. Infection-related questions dominated in the fall , tracingThis study provides an overview of a social media site for distributing child health information, a profile of the questions asked by parents, the proportions of questions comprehensively answered, and the volume of visitors on the site. On this site, which was aimed at parents of small children, personalized answers were an effective way of delivering information. Based on the number of questions considered comprehensively answered, the service may have reduced the need for further contacts with a health care service provider in more than half of the cases.The service was directed to questions on child health. According to the site visitor profile, mostly women aged 29-34 years, it is likely that the audience was reached. The number of individual visits was almost 250,000. There was a great variation in the amount of daily visitors, but no trend could be seen to explain this. The number of postings generated by this audience was 478, so it is likely that the answer given to one posting was read by multiple users and information spread was greater than just one person, which is typical for this kind of media . EvaluatInfections were the most common topic for parental questions. These are also the most typical cause for ambulatory pediatric doctor’s visit. Pediatric infectious diseases is a subspecialty within pediatrics, and this finding supports the fact that a specialist of this field would be the optimal person to work on a site with such a focus. In addition, the seasonal variation in questions on infections followed the general trend of pediatric infections in Finland. This underlines the fact that the questions presented were not just sporadically written, but followed general trends.Social media is an interactive and sometimes face-to-face information distribution channel, and it is therefore considered a more reliable media than passive Internet pages . It is uThe proportion of questions comprehensively answered was over 70%. Without this social media contact, many of these questions would likely have been presented to a health care service provider, possibly by making an appointment to see a doctor. Although it was not possible to evaluate in the study, the service may well have reduced the pressure on “well-baby” clinic personnel. Questions about infections were the most likely to be comprehensively answered, while questions concerning neurological symptoms were least likely to be comprehensively answered, demonstrating the difficulty in assessing neurological symptoms in general. In health information technology, effectiveness of the service is rarely evaluated . On the When planning a site for health information, the scope has to be defined. If the scope is comprehensive and aimed at, for example, a wide array of child health issues, the expertise also needs to be more comprehensive. This is of course more costly. On the other hand, a narrow scope can also be recommended. The experience of this site suggests that even a narrow focus on infection-related questions only would have been functional especially because these questions were more likely to be comprehensively answered than non–infection-related questions. Here, the focus of the questions changed over time and the number of infection-related questions increased towards winter. Therefore, the expertise needed to answer the questions may vary over time. Another issue to be decided on is the lapse between questions and answers. It is recommended here that the lapse be fairly short. On this site, the lapse was 24 hours on weekdays and more than 2 days at weekends. This may have resulted in a lower number of visits and postings. The method here would serve its purpose best if it were focused narrowly and manned in such a way that the delay in answers was shorter.One of the crucial concerns when launching a site is the possible discrepancy between the demand and the number of personnel answering to the demand, that is, the possibility of an overflow of questions. According to the data from this site, the number of questions is likely to be rather limited in comparison with the number of visitors. Hence, one question and answer may be viewed a multitude of times and the information delivered efficiently. While active social media participants are usually a minority , it is pAnother possible limitation to the study is the fact that having a pharmaceutical product advertisement on the site may have skewed the focus of the questions presented on the site. As the product was an antipyretic drug, it is likely the possible effect was an increase in the number of questions on infections.There is a constant demand for social media–based child health information services. A social media health information site is likely to be well accepted if the consultant is a specialist and, when delivering child health information, possibly a pediatrician. On this site aimed at parents of children in Finland, the greatest number of questions was presented between September and January, and most of these concerned infections. Over half of the questions were estimated to be comprehensively answered, and most likely did not require further contact with a health care service provider. These factors should be taken into account when planning such a service.


Furthermore, we examined how empirical relationships between aboveground production and plant species richness are modified when standing biomass is expressed per unit volume rather than surface. We propose that biomass packing emphasizes species coexistence mechanisms and may be an indicator of resource use efficiency in plant communities.Aboveground production in terrestrial plant communities is commonly expressed in amount of carbon, or biomass, per unit surface. Alternatively, expressing production per unit volume allows the comparison of communities by their fundamental capacities in packing carbon. In this work we reanalyzed published data from more than 900 plant communities across nine ecosystems to show that standing dry biomass per unit volume (biomass packing) consistently averages around 1 kg/m When expressed per unit volume (g/m3), however, his model also predicted that standing biomass should be independent of stem density across the plant kingdom  scaled per unit of sampled volume.The present study addresses two questions: Are there fundamental limits to the amount of standing dry biomass that is packed per unit volume in terrestrial plant communities? Is biomass packing affected by community composition and species richness? To investigate these questions, we reviewed the scientific literature and compiled data for more than 900 plant communities. This synthesis highlights broad patterns in biomass packing across contrasted ecosystem types. It is not a meta-analysis of the reviewed literature, which would require the inclusion of climatic or edaphic moderator variables as well as accounting for spatial auto-correlation and random (data source) effects.2 and height values to m  and standing dry biomass (g/m2) to evaluate their association.To determine whether standing dry biomass approximates the volume occupied by plants, we compiled a dataset on 42 wetland plant communities from the Lac St-Pierre ecosystem . In August 2014, we sampled vegetation quadrats of 0.5 × 0.5 m covering a broad range of species assemblages and vegetation types. In each quadrat we clipped all the vegetation standing above 2 cm from the ground. We then evaluated plant volume using the water displacement method in a bucket of 12.6 cm radius. We expressed the amount of water displaced by the submersed vegetation in cubic meters. We then oven dried the vegetation at 70 °C for 48 h and weighed the dry biomass. Finally, we report the relationship between plant volume .From our analysis of the reviewed data, we found that standing dry biomass per unit surface varied by over three orders of magnitude across the plant kingdom . In fore3 (the average 50th percentile being 0.88 kg/m3) and by less than an order of magnitude across ecosystems , to nonexistent when expressed per unit volume .The positive relationship between plant species richness and biomass production among experimental meadow communities vanished when standing dry biomass was expressed per unit volume rather than surface , indicating that standing biomass is a good proxy for the volume occupied by plant modules, at least for wetland communities  in the first dataset and 5.3 kg/m3 (median of 0.6 kg/m3) in the second. We feel confident that the inclusion of more plant communities would not alter these general results.The literature review highlights that if plant community biomass is represented as dry biomass per unit surface area, major ecosystem types are drastically different. However, when accounting for height and expressing standing dry biomass per unit volume, plant communities from different ecosystems appear remarkably similar in their capacity to pack biomass. Biomass packing across plant communities peaked just below 5 kg/mOur results revealed that cropland and meadow communities were equally capable of packing biomass per unit volume. However, the caveat to this result is that it is true only when plant communities are compared at their peak of biomass production. For instance, cornfields sown in widely spaced rows attain their maximum of biomass packing only towards the end of the growing season. Likewise, meadows are repeatedly harvested within a year, after which a regrowth period of sparse vegetation and low biomass packing initiates. Moreover, while tropical and temperate forests may show similar packing values at their peak of production, air temperature and sunlight duration impose stronger seasonal variations in the biomass packing of temperate ecosystems. Such examples illustrate the importance of accounting for changes in biomass packing over time and, perhaps, the inaccuracy of treating it as a static metric.3 were placed in 107 woody stands, such that stems would fill the frames from top to bottom. Overall, the maximum biomass packing measured across stands and vegetation types varied from 3 kg/m3 in poplar and birch forests to 6 kg/m3 in pine and spruce forests  forest stands . In this forests . Since sGarnering much attention in the literature, positive relationships between species richness and biomass production have been repeatedly reported in experimentally manipulated plant communities e.g., . FurtherDivergent from those aforementioned biodiversity experiments, a negative relationship between plant species richness and biomass production can be observed in freely assembled herbaceous communities, wherein the productive stands are dominated by fewer species . The negTo understand why the richness-biomass relationship flattens when expressing biomass per unit volume, we examined how much of the variation in standing biomass per unit surface is explained by community height. Among our 971 plant communities, height explains 88% of the variation in standing biomass when the axes are on a logarithmic scale . This stOur reanalysis of the literature casts a new light on how plant biomass accumulates in terrestrial ecosystems. Specifically, while the widespread metric of biomass per unit surface highlights differences between plant communities, expressing their standing dry biomass per unit of volume reveals their striking similarity. Expressing biomasses per unit volume goes against a longstanding tradition of measuring primary production per unit surface in terrestrial ecosystems, probably reflecting our utilitarian view of ecosystems conditioned by questions such as: how much crop  can be produced and harvested per hectare of land?3 can be exceeded in communities grown under controlled conditions, although croplands did not reach higher biomass packing values over that of other ecosystems in our dataset. Biomass packing may thus represent a simple and generic indicator of resource use efficiency in plant communities.Our findings suggest that biomass packing is influenced less by baseline climatic and edaphic conditions, but rather emphasizes local plant–plant interactions. We propose that a fundamental limit might exist to the amount of dry biomass that natural plant communities pack per unit volume in terrestrial ecosystems; since the long-term maintenance of standing dry biomass beyond this point may be biologically unsustainable . It is l10.7717/peerj.849/supp-1Table S1Click here for additional data file.


Active manipulation of light in optical fibres has been extensively studied with great interest because of its compatibility with diverse fibre-optic systems. While graphene exhibits a strong electro-optic effect originating from its gapless Dirac-fermionic band structure, electric control of all-fibre graphene devices remains still highly challenging. Here we report electrically manipulable in-line graphene devices by integrating graphene-based field effect transistors on a side-polished fibre. Ion liquid used in the present work critically acts both as an efficient gating medium with wide electrochemical windows and transparent over-cladding facilitating light–matter interaction. Combined study of unique features in gate-variable electrical transport and optical transition at monolayer and randomly stacked multilayer graphene reveals that the device exhibits significant optical transmission change (>90%) with high efficiency-loss figure of merit. This subsequently modifies nonlinear saturable absorption characteristics of the device, enabling electrically tunable fibre laser at various operational regimes. The proposed device will open promising way for actively controlled optoelectronic and nonlinear photonic devices in all-fibre platform with greatly enhanced graphene–light interaction. Active control of light in optical fibres is of great interest, to this end, electric control of all-fibre graphene devices is desirable but highly challenging. Here, Lee et al. demonstrate electric control of the optical properties of a graphene sheet deposited on a side-polished fibre mediated by an ion liquid. The extraordinary electrical and optical properties of graphene have attracted strong interest in the fields of optoelectronics, plasmonics and photonics applications125710111348In this work, we demonstrate electrically controllable all-fibre graphene devices for the first time. The graphene-based field effect transistor (FET) was built on a side-polished fibre (SPF) with an ion-liquid dielectric. Here the ion-liquid dielectric plays a key role as an extremely efficient electrical gating medium2 graphene layer covers the side-polished section. A magnified image of the interaction region is shown in the inset of All-fibre graphene devices were fabricated on the SPF as shown schematically in VG), the TE mode suffers strong absorption of 3.47 dB (orange solid line at zero VG), whereas transmission of the TM mode increases by 0.09 dB (blue solid line at zero VG). This is because the ion-liquid over-cladding alleviates the scattering loss caused by the graphene and the polished surface. More importantly, optical transmission of the device was actively tuned with an applied gate voltage. As shown in VG ranging from 0.7 V to −1.8 V. The TE mode shows a more drastic transmission change from 39.2 to 83.4% for the same VG range. This indicates that 44.2% of total incident light recoverably interacts with the monolayer graphene for the TE mode. It is worth noting that the total insertion loss (<1 dB) in our device is much smaller than that of previously reported silicon waveguide-based device (25 dB)4VD) of 2 mV. The drain current (ID) varies with VG where the on–off current ratio was measured as 16.7 within the applied VG of ±2 V. The voltage at charge neutral point (VCNP) appears to be 1.16 V , which indicates that the graphene on the quartz substrate is initially p-doped. The inset of T) of the TE mode with respect to the drain current variation (ΔID). The experimental results (black solid square) and the fitted data (red solid line) clearly reveal that the derivative has a maximum at ID=6.55 μA (black dotted line), which corresponds to an applied gate voltage of −0.49 V . The maximum change in optical transmission will occur when the Fermi level approaches half the photon energy A shift of Fermi level in graphene is responsible for this optical transmission change. Limited density of state of electrons confined in a two-dimensional graphene leads to a significant change of the Fermi level near the Dirac point with the applied gate bias, resulting in strong electro-optic absorption10112) of the graphene samples (see I2D/G) obtained from Raman images are depicted for mono-, bi- and quad-layer graphenes, respectively, from top to bottom (left). Shown together are the corresponding Raman spectra at several sampling points (right). The monolayer graphene has a nearly constant I2D/G of around 2.9–3.6 over the most area (>95%). For multilayer graphene samples, the histograms extracted from the Raman images indicate that only small portion of films (6.3% for bilayer and 10.1% for quad-layer) demonstrates the similar I2D/G to that of the monolayer graphene. In these areas, electrical properties of each graphene layer are expected to be preserved owing to the weak layer-to-layer interactionI2D/G less than that of monolayer graphene, which indicates that there is a significant interlayer coupling even in our randomly stacked multilayers similar to Bernal-stacked grapheneThe simple structure of the proposed device readily allows it to be extended to multilayer graphene-based systems, providing an opportunity to enhance device efficiency and to explore the electro-absorption features of multilayer graphene. Large-area bi- and quad-layer graphenes prepared with a multiple stacking methodples see . Figure VG of −1.05 V where the measured pulse duration was 423 fs at a repetition rate of 30.9 MHz as shown in VG, the linear optical transmission decreases, while both modulation depth and saturation fluence of the SA increase. This significantly modifies the Q-switching instability condition26VG of −0.18 V. The repetition rate of the laser was measured as 25.4 kHz  at a low operation voltage (<3 V). Furthermore, electrically manipulated nonlinear absorption properties of our devices enable it to apply as a tunable in-line graphene SA, which can electrically control the fibre laser operation at different regimes as follows: continuous wave, Q-switching and passive mode-locking. Although its gating speed is currently limited by the ionic mobility in the ion liquid56780% at a l302 size, cleaned using nitric acid for few seconds, rinsed off with deionized water to remove any unwanted particles on the both surfaces of the foil and rolled into the fused silica chamber of the CVD furnace. Then the copper foil was heated at 1,000 °C under the flow rate of 20 sccm of H2 for 30 min, and CH4 gas was allowed to flow into the chamber with 50 sccm for 30 min. After completing the thermal process, the furnace was cooled down to room temperature. The poly(methyl methacrylate) (PMMA) was spin-coated on the as-grown graphene with a thickness of 300 nm as a supporting layer, which is then cut to desired sizes after removing the graphene layer grown on the other side of the copper foil by a reactive-ion etching method. The copper foils were etched using ammonium persulphate aqueous solution. For the randomly stacked few-layer graphene films, the PMMA/graphene films were directly transferred on to the monolayer graphene-grown copper foil without additional PMMA film and the etching process was repeated. The PMMA-coated graphene films were then transferred onto the SPF and the PMMA support layer was dissolved in an acetone bath for an hour and rinsed with isopropyl alcohol.A conventional single-mode fibre (SMF-28e) was embedded into the quartz block with a radius curvature of 250 mm and side-polished until the insertion loss increased to −60 dB in the presence of the index oil with a refractive index of 1.458. The insertion loss of the SPF was 0.5 dB without index oil. Cr/Au electrodes with a thickness of 50 nm (Cr: 5 nm and Au: 45 nm) were deposited using an e-beam evaporator and a metal mask. Graphene monolayer films were grown on 25-μm-thick copper foils via low-pressure thermal CVD . The copper foil was cut into 10 × 10-cmA ring cavity fibre laser was built as shown in D.-I.Y. conceived and designed the experiment. S.Y.C., M.H.K., S.S., S.B., S.J.K., F.R. and B.H.H. prepared the graphene samples. E.J.L., S.Y.C., H.J. and N.H.P. fabricated the graphene devices. E.J.L., H.J., N.H.P., W.Y., J.-Y.P. and D.-I.Y. tested the performance of the devices. J.-K.P. and Y.H.A. measured the Raman signal. K.L., Y.H.A., K.J.A., J.-Y.P., F.R. and D.-I.Y. prepared the manuscript.et al. Active control of all-fibre graphene devices with electrical gating. Nat. Commun. 6:6851 doi: 10.1038/ncomms7851 (2015).How to cite this article: Lee E. J. Supplementary Figures 1-4, Supplementary Note 1 and Supplementary Reference


Upon intercalation, a charge density per graphene layer of 5 ⋅ 1013 ± 5 ⋅ 1012 cm−2 is attained, making this material an attractive platform for the study of plasmonic excitations in the infrared wavelength spectrum of interest to the telecommunication industry. Finally, we demonstrate the potential of this material for flexible electronics in a transparent circuit on a polyethylene naphthalate substrate.The efficiency of flexible photovoltaic and organic light emitting devices is heavily dependent on the availability of flexible and transparent conductors with at least a similar workfunction to that of Indium Tin Oxide. Here we present the first study of the work function of large area (up to 9 cm The development of flexible photovoltaic and light emitting devices depend on the availability and compatibility between flexible and transparent electrodes and photoactive materials. The most widely used transparent conductors nowadays -i.e. Indium-Tin-Oxide (ITO) and Fluorine doped Tin Oxide (FTO)- are brittle and their electrical properties degrade significantly under small applied strains12Meshes of metallic nano-wires are emerging as an attractive alternative, since they have high optical transmission ≈80% at 550 nm) and low sheet resistance (38Ω/◻)50 nm andThe sheet resistance of functionalized CVD graphene reported to date still greatly exceeds that of ITO ≈8Ω/◻ causing excessive dissipation in electrical devices910112 on glass and up to 1 cm2 on Si) FeCl3-FLG obtained from Ni-grown graphene. We find values as high as 5.1 eV, that is 0.3 eV larger than the work function of ITO, making this material an ideal replacement for ITO in PV and OLED since it would not require the additional charge blocking layer typically used with ITO electrodes. With the aid of Raman spectroscopy, we estimate a charge density per graphene layer of FeCl3-FLG up to 5 ⋅1013 ± 5⋅1012 cm−2, making this material an attractive platform for studying the physics of plasmons in the infrared region. Finally we demonstrate the potential of this material for flexible electronics in a transparent circuit on an insulating, transparent and flexible sheet of Polyethylene naphthalate (PEN) with a room-temperature resistivity of 20.52Ω/◻ and high optical transmission of 77% in the visible wavelength range.Here we present the first experimental study of the work function (WF) of large area  was transferred to glass substrates by using PMMA as a support during the wet-etching of nickel in a FeClCPD) measured in static Kelvin probe measurements. The accuracy with which the absolute work function of a material using Kelvin probe methods can be calculated depends on the accuracy with which the work function of the probe can be determined. For these measurements we employed an NT-MDT NTEGRA Aura system using frequency-modulated SKPM mode and conductive Bruker PFQNE tips in ambient conditions.Scanning Kelvin probe microscopy (SKPM) is used to determine the absolute work function (Φ) and its spatial distribution over the surface of intercalated graphene. SKPM maps the electrostatic forces between the surface of the sample and a conductive atomic force microscopy (AFM) probe which is oscillated above the sample surface. The electrostatic force between the cantilever and the sample is nullified by applying a backing dc bias to a conductive AFM probe. The applied bias which is equal and opposite to the electrostatic potential is termed the surface potential (SP) and is equivalent to the contact potential difference (Vprobe = ΦAu + VCPD) where ΦAu is assumed to be 5.1 eVAu with an average SP of −0.43 V and a full width at half maximum (FWHM) of 0.014 eV giving a Φprobe of 4.67 eV ± 8 meV, see μm2 areas of the intercalated sample highlighted in the micrograph picture of sample. The most commonly occurring Φsample were recorded at 5.1, 5 and, 4.9 eV with the majority of the surface area of the sample ≈60% showing a work function value as large as 5.1 eV  graphene sandwiched between FeCl3, (2) graphene with FeCl3 only on one side and (3) graphene with no direct contact to FeCl3. While the SP was observed to vary on the nanoscale between 4.8–5.2 eV, the average Φsample value of 5 eV was uniform over the scale of the sample -that is 1 cm2.Gold electrodes were deposited onto the intercalated samples serving both as a grounding contact and a calibration standard for the measurements of Φ, see 1 eV see . The obs2 multilayer graphene on a Si/SiO2 substrate intercalated for 36 h. We find that the G-band (1580–1630 cm−1) is composed by four Lorentzian peaks labelled as G0 (1580 cm−1), −1), G1 (1611 cm−1) and G2 (1622 cm−1). Each G-peak corresponds to a different level of charge transfer1419−1) is upshifted and its shape becomes the convolution of a smaller number of Lorentzians as compared to the pristine multilayer graphene−1 (2D0), 2684 cm−1−1 (2D1) and 2721 cm−1 (2D2).Raman spectroscopy is another valuable tool for the characterization of this material. 0 and 2D0 are characteristic of un-doped (pristine) graphene layers. G1 and 2D1 indicate that graphene is in direct contact to only one adjacent FeCl3 layer (low doping), whereas G2 and 2D2 appear when graphene is sandwiched between two FeCl3 layers (high doping)3 layer but with a lower density of FeCl3 molecules, thus giving lower charge transfer.The positions of G- and 2D- peaks are unique identifiers of the doping level in this intercalated compound. More specifically, G3-FLG from the Raman spectra we use the model developed by Lazzeri et al.g2 phonon mode of the Raman G-peak due to the non-adiabatic removal of the Kohn anomaly at Γn and the upshift of the G-peak position was obtained and this can efficiently be used to determine n with an accuracy better than 10% from the values of charge density obtained with complementary methods such as quantum oscillations of magneto-conductance, see n over a representative surface area of 104 μm2 in our material show three different doping levels, see n0 = 0.3 ⋅ 1013 ± 1 ⋅ 1012 cm−2, −2 and n1 = 4.6⋅1013 ± 5 ⋅ 1012 cm−2, see 3-intercalated multilayer graphene system, such as Ni-grown CVD graphene, the total charge density can reach values as high as 1015 cm−2 for a sequence of 15 graphene layers.To characterize the Fermi energy in FeCl3-intercalation of multilayer graphene provides an attractive platform for pioneering studies of surface plasmons at wavelengths of interest to the telecommunication industry. More specifically, the plasmon frequency in graphene scales as ħω ∝ (EF/D)1/2 where EF is the Fermi level and D is the size of the resonant plasmonic structureEF ≈ 1 eV, corresponding to a total charge density of ≈7 ⋅ 1014 cm−2, would give a plasmon resonance at a photon wavelegth of ~1.4μm3-intercalated multilayer graphene.Since the exploitation of collective charge oscillations in graphene (i.e. plasmons) for light manipulation strongly relies on doping, FeClμm film of ITO of 80%The room temperature resistivity of the Ni-grown intercalated multilayer graphene was characterized by measuring the four-terminal resistance in devices with different aspect ratios of the conductive channel, that is device length -ranging from 0.15 cm to 0.7 cm- divided by the device width (1 cm), see 3-FLG and using a thermal release tape to mechanically peal the functionalized graphene off the glass substrate see 3-FLG of 1 cm2 was dry transferred to a PEN substrate, while the tape and PMMA are removed by a heat process and rinsing in acetone, respectively  large work function, (2) low electrical resistivity and (3) high optical transmittance make this material an attractive system for flexible electronics and PV applications. To demonstrate further its potential, we have transferred the intercalated Ni-graphene to a standard flexible PEN substrate. This is readily done by spin coating PMMA onto FeClvely see . We then3-FLG obtained from Ni-grown graphene (up to 9 cm2 on glass and 1 cm2 on Si and PEN), with values as large as 5.1 eV, which qualify this material as a replacement for ITO for example in PV and OLED. Upon intercalation, a charge density per graphene layer of ≈5 ⋅ 1013 cm−2 is attained, making this material an attractive platform for the study of plasmons in the infrared region potentially of interest to the telecommunication industry. Finally, we demonstrate the potential of this material for flexible electronics in a transparent circuit on a PEN substrate with a room temperature resistivity of 20.52Ω/◻ and optical transmission of 77% in the visible wavelength range.In conclusion we have presented the first systematic study of the work function of large area FeClHow to cite this article: Bointon, T. H. et al. Large-area functionalized CVD graphene for work function matched transparent electrodes. Sci. Rep.5, 16464; doi: 10.1038/srep16464 (2015).


Due to their stability, easy chemical modifications and proneness to nanostructured device construction, aptamer-based sensors have been incorporated in a variety of applications including electrochemical sensing devices. In recent years, the performance of aptasensors has been augmented by incorporating novel nanomaterials in the preparation of better electrochemical sensors. In this review, we summarize the recent trends in the use of nanomaterials for developing electrochemical aptasensors.Electrochemical aptasensors, which are based on the specificity of aptamer-target recognition, with electrochemical transduction for analytical purposes have received particular attention due to their high sensitivity and selectivity, simple instrumentation, as well as low production cost. Aptamers are functional nucleic acids with specific and high affinity to their targets, similar to antibodies. However, they are completely selected  Electrochemical aptasensors (aptamer-based sensors) facilitate simple, effective and rapid detection of biomolecules which are important in medicine, environment and food applications. Aptasensors are getting considerable interest due to their unique capabilities compared to other biorecognition elements. The artificial aptamer selection procedures allow the delivery of custom-made biorecognition for any kind of target. In recent years, numerous electrochemical biosensors have been reported using a variety of nanomaterials such as quantum dots, gold nanoparticles, and carbon nanotubes. Among novel nanomaterials, carbon nanostructures provide the basis for emerging technologies in the field of molecular electronics devices. A number of recent reports specifically introduced graphene as an emerging nanomaterial for improved electrochemical sensors ,2. Carbon materials have been widely employed in analytical electrochemistry, outperforming the traditional electrodes. Their performance originates largely from structural polymorphism and chemical stability. Micro-structured carbon materials, such as carbon nanotubes (CNT), improved electrochemical applications by enabling novel designs in sensing, electrocatalysis and electronics, compared to traditional carbon materials such as glassy carbon, diamond or carbon black. Most recently, graphene emerged as a material with future potential for improved applications. These expectations are based on the more advanced properties of graphene compared to CNTs . Graphene is a two-dimensional single atom thick nanomaterial, which has recently been at the scientific focus due to its remarkable mechanical and electronic properties. In comparison to CNTs, graphene has the advantages of high thermal and electrical conductivity due to small thickness and large surface area. Integration of graphene with metal nanoparticles is usually necessary for enhancing biosensing properties. The attachment of graphene on electrodes and biorecognition elements was achieved through a plethora of nanocomposites. Major additional premises of graphene compared to other carbon based nanomaterials are low cost and large production scale in biosensor development.Electrochemical aptasensors integrate the aptamer functionality with the sensitivity of electrochemical technologies for detecting a variety of targets ,5. In thin vitro selection procedure, SELEX , is a powerful technique which can identify functional nucleic acid sequences with specific affinity to desired target molecules [Aptamers are target affinity nucleic acids selected artificially via a combinatorial procedure. They have frequently been proposed as alternatives to antibodies with additional desirable properties. In the last decades, due to a variety of materials with unique properties for sensor development, aptasensors have been given more attention for developing novel analytical tools ,7,8,9,10olecules ,12. The olecules ,14. Moreolecules ,16,17. Polecules . Interactions of nucleic acids with various nanomaterials allowed improved designs of biosensors . For exain situ measurements. Limitations for this signal transduction mechanism include the potential interferences to the response, if several electroactive compounds generate false current values. These effects have been eliminated for clinical applications through the use of selective membranes, which carefully control the molecular weight or the charge of compounds that have access to the electrode. Amperometric sensors determine the presence of target by detecting the changes in current resulting from the electrochemical oxidation or reduction of an electroactive species. It is usually performed by maintaining a constant potential at working electrode with respect to a reference electrode. The resulting current is directly correlated to the bulk concentration of the electroactive compound. This signal-transduction mechanism is frequently used for enzymatic and catalytic biosensors. The main advantage of this class of transducer is the low cost, therefore disposable electrodes are often used with this technique. The high degree of reproducibility that is possible for these (one time use) electrodes eliminates the cumbersome requirement for repeated calibration. The type of instrument used for these measurements is also very easy to obtain and can be inexpensive and compact, this allowing for the possibility of −6 to 10−1 mol/L. Their continuous measurement capability is also an interesting advantage for many applications. The apparatus is inexpensive, portable, and it is well suited for in situ measurements. The main disadvantage is that the limit of detection for some samples can be high (10−5 mol/L or 1 ppm) and the selectivity can be poor. One of the attractive characteristics of potentiometric measurements with ISE is the relative independence of signal from sample volume [Potentiometric sensors measure the potential difference between a working and a reference electrode or two reference electrodes separated by a selective membrane, when there is no significant current flowing between them. The transducer is usually an ion-selective electrode (ISE). The main advantage of such devices is the wide concentration range for which ions can be detected, generally between 10e volume . 2+ ion selective microelectrode  measures the response (current and its phase) of an electrochemical system to an applied oscillating potential as a function of the frequency. EIS is a rapidly developing electrochemical technique for the investigation of bulk and interfacial electrical properties of any kind of solid or liquid material which is connected to an appropriate electrochemical transducer. Moreover, the EIS method is label-free, simple, and requires no external modification of biomolecules. In the field of biosensors, it is particularly well-suited to the detection of binding events on the transducer surface. The large applicability of conductimetric detection is due to the observation that almost all enzymatic reactions involve either consumption or production of charged species. The electric field is generated using a sinusoidal voltage (AC) which helps in minimizing undesirable effects such as Faradic processes, double layer charging and concentration polarization. The primary advantage of this technique is the use of inexpensive, reproducible and disposable sensors. The main disadvantage is that the ionic species produced must significantly change the total ionic strength to obtain a reliable measurement. This requirement increases the detection limit to unacceptable levels and results in potential interferences from variability in the ionic strength of the sample. High sensitivity of the method is highly advantageous, but also can be associated with nonspecific impedance changes that could be easily mistaken for specific interactions. Aptamers have been used in conductimetric/impedimetric sensors in a variety of designs in order to exploit direct biomolecule measurements ,34. ExamSensors based on field effect transistors (FET) rely on electrical response modulation of conductors. FET consists of a source, a drain and a gate. The current in gate voltage can be regulated by the voltage between source and drain. The gate terminal can be modified with a molecular receptor or ion-selective membrane for establishing a biosensor. The binding of target analyte on molecular receptors is detected as an electrical signal based on the dependence of gate voltage and channel conductance . AptamerGraphene is an excellent conductor material and thus, graphene modified electrodes exhibit fine electrochemical response . The outThe strong interaction between graphene and single stranded nucleic acids is another advantage that has been used to develop simple and effective electrochemical aptasensors. Covalent modifications on the graphene surfaces have also been exploited for aptamer immobilization. Researchers have usually combined graphene with various nanomaterials in order to facilitate development of electrochemical sensors.Aptamers can be immobilized on graphene nanocomposites via covalent bonds for developing reusable sensors or via physical adsorption of aptamers on graphene to obtain one time use sensors. Both strategies have been demonstrated to be useful in aptasensor development. 3,4,9,10-perylene tetracarboxylic acid (PTCA) is an archetypal π-stacking organic perylene dye with favorable photo and chemical stability. PTCA strongly adsorbs on graphene through π-π stacking and thus prevents graphene aggregation. Another advantage of PTCA composites is the added carboxyl groups which can be exploited for covalent attachment of aptamers. Graphene-promoted PTCA (GPD) nanocomposites has been synthesized as redox probe for developing an electrochemical thrombin aptasensor . The autStructure-switching of aptamers has been fruitful in designing electrochemical biosensors. A sensitive aptasensor was reported based on gold nanoparticles-graphene nanosheets using structure-switching histidine aptamers . Gold naA couple of graphene based formats were reported by using aptamer adsorption on the graphene and then electrochemical signal was recorded by target induced release of the aptamer molecules . Wang anA small molecule electrochemical sensor was designed by using target-induced release of ferrocene-labeled aptamers from a magneto-controlled graphene surface . In magnFinally a complex of silica-graphene-gold based system was developed as ATP sensor. Guo and co-workers reported a triplex-DNA design of electrochemical aptasensor by using silver microspheres (SMS) as separation element and graphene-mesoporous silica-gold nanoparticle nanocomposite (GSGH) as sensing platform . In thisCarbon nanotubes (CNT) and their hybrids have attracted great interest in biosensor development due to their unique properties. Large surface to volume ratio is the major characteristic of CNTs. Nucleic acids can readily be adsorbed on the surfaces of carbon nanotubes through the π-π stacking. Various sensing formats have been created by using displacement of adsorbed aptamers from the nanotube surfaces in the presence of complementary DNA, aptamer target, exonucleases or some dye molecules such as methylene blue. Reusable sensors have been designed through covalent attachment of aptamers to hybrid components of graphene containing composites. Two groups of CNTs can be synthesized; single-walled carbon nanotubes (SWCNT) or multi-walled carbon nanotubes (MWCNT). SWCNTs are formed when a graphene sheet is rolled to a tube and two or more concentric tubes form MWCNTs. Detailed information on synthesis of carbon nanotubes and their electrochemical applications can be found in some recent reviews ,61,62,63SWCNTs have abundant accessible surface area for biological immobilization and their high electron mobility, low electrical resistance, favorable biocompatibility make them excellent candidates for biosensing. They can promote the electron transfer and improve the catalytic activity of nanoparticles. Bai and co-workers functionalized platinum-gold nanoparticles and horseradish peroxidase with SWCNTs to label thrombin aptamer for developing an ultrasensitive electrochemical biosensor . An aptaKara and co-workers developed a thrombin aptasensor based on impedance spectroscopy. MWCNTs were attached on the surface of screen-printed carbon electro-transducers. Aptamers were attached on the carbon nanotubes through amine bond and the thrombin binding was monitored by resistance to charge transfer for obtaining a limit of detection of 105 pM. In a similar report, MWCNTs were modified with thrombin aptamers hybridized to ferrocene labeled complementary DNA . Target 32+) based platforms have been known for high electrochemical stability and reversible behavior. An ultrasensitive thrombin aptasensor was designed based on solid state Ru(bpy)32+ ECL [co-pyrrole propylic acid) nanoparticles loaded with aptamer and ruthenium complex [−16 M.Trisruthenium(II) (Ru(bpy))32+ ECL . A compo complex . The nanLi and co-workers developed a novel biosensor by immobilizing thrombin binding aptamer molecular beacon on soluble CdSe quantum dots modified on top of the glassy carbon electrode . MethyleAnother interesting study described an electrochemical aptasensor, based on disposable screen-printed electrodes for the detection of the mycotoxin ochratoxin A (OTA) . Two str3−/4− redox couple, which can be measured by electrochemical impedance spectroscopy. The linear range of detection for the sensor was demonstrated to be 1–30 nM thrombin.To improve the sensitivity of an impedimetric sensor, 15 nm gold nanoparticles (AuNP) bearing thrombin-binding aptamers were immobilized on the surface of gold electrodes via hexanedithiol . The traThe exceptional properties of carbon-based nanomaterials make them compelling for electrochemical biosensor development. Aptamers have already proven to be one of the potentially best suited biorecognition elements in nanomaterial-based sensing platforms. The complex nanocomposites consisting of carbon nanomaterials, various nanoparticles and aptamers promise label-free, ultrasensitive biosensors at reasonable costs for any analyte of interest. The graphene-nanoparticle combinations provide improved electrochemical platforms and aptamer incorporation serves as a unique element in achieving a universal biosensor for any desired target. Recently reported applications, as proof-of-principle studies, have been confined to a couple of aptamer sequences (mostly thrombin or ATP binding aptamers). Future trends in electrochemical aptasensor research should broaden the spectrum of applications in medical diagnosis, environmental monitoring and other biosensing fields.


The GSH-coated Au29–43(SG)27–37 nanoclusters can escape the RES absorption, leading to a good tumor uptake (~8.1% ID/g at 24 h post injection). As a result, the as-designed Au nanoclusters led to a strong enhancement for radiotherapy, as well as a negligible damage to normal tissues. After the treatment, the ultrasmall Au29–43(SG)27–37 nanoclusters can be efficiently cleared by the kidney, thereby avoiding potential long-term side-effects caused by the accumulation of gold atoms in the body. Our data suggest that the ultrasmall peptide-protected Au nanoclusters are a promising radiosensitizer for cancer radiotherapy.Radiotherapy is often the most straightforward first line cancer treatment for solid tumors. While it is highly effective against tumors, there is also collateral damage to healthy proximal tissues especially with high doses. The use of radiosensitizers is an effective way to boost the killing efficacy of radiotherapy against the tumor while drastically limiting the received dose and reducing the possible damage to normal tissues. Here, we report the design and application of a good radiosensitizer by using ultrasmall Au Cancer remains one of the world's most devastating diseases with more than 10 million new cases each year, and radiotherapy is a leading cancer treatment approach that addresses the needs of more than 50% cancer patients234In general, the radiosensitizing agents can be classified into two major categories according to their mechanisms of action: (type-1) chemotherapeutics that modulate the cell response to enhance the radiation damage, and type-2) materials that interact directly with the radiation and generate additional damages to the cell material36336101213in vivo biodistribution. For example, the naked Au NPs of particle sizes of 1.9 and 4.8 nm, while small, have low colloidal stability due to the protein corona acquired in blood. These Au NPs eventually formed large aggregates of ~20–100 nm, which could not be rapidly metabolized and certainly unable to escape the RES18Type-2 radiosensitizers are mostly metal-based materials that can strongly absorb, scatter, and reemit radiation energy, resulting in a local radiation dose increase when they are accumulated in tumors151819202222242627282930333435in vivo pharmacokinetics, and enhance their deposition in tumors29–43(SG)27–3729–43(SG)27–37 NCs have attractive features of high tumor uptake, strong sensitizing enhancement for radiation, and low toxicity, and they could be a good candidate for next generation radiosensitizers for clinical use. This study has therefore enriched the family of Au NPs and NCs that could show good performance for cancer radiotherapy33Taken together of the two key attributes (size and surface) for NPs-based radiosensitizers, we hypothesized that: 1) small naturally-occurring peptides, such as glutathione or GSH, could be a good surface ligand for Au NPs by helping them escape the RES absorption and improving their deposition in tumors; and 2) ultrasmall Au NPs with core sizes below 2 nm  in combination with the GSH ligands can ensure a small hydrodynamic diameter (HD), which could provide good interface with the biological system, improve their 29–43(SG)27–37 NCs were prepared by a reported procedure404142434429–43(SG)27–37 NCs was determined to be ~2.8 nm by using dynamic light scattering 27–37 NCs showed strong orange luminescence with an emission peak at ~610 nm 27–37 NCs and the extent of plasma protein that binds to the NCs by missing Au29–43(SG)27–37 NCs  with blood plasma (0.5 mL). The photoluminescence of the mixture of Au29–43(SG)27–37 NCs and blood plasma (at 24 h after mixing) was not decreased significantly as compared with the aqueous solution of the NCs (29–43(SG)27–37 NCs were sufficiently stable in blood. The unbound Au29–43(SG)27–37 NCs were separated from the protein-bound Au NCs by filtering the mixture of Au29–43(SG)27–37 NCs and blood plasma (at 24 h after mixing) using ultrafiltration with a molecular weight cut-off, MWCO of 50 kDa. About 40% of Au29–43(SG)27–37 NCs were recovered from the filtrate as determined by their photoluminescence intensity 27–37 NCs. The mice were intraperitoneally injected with the Au29–43(SG)27–37 NCs (~5.9 mg-Au/kg-body). As shown in 1/2α) of Au29–43(SG)27–37 NCs in blood was determined to be 6.5 h. As compared with the reported Au10–12(SG)10–12 and Au25(SG)18 NCs, the longer distribution half-life of the Au29–43(SG)27–37 NCs could be attributed to their larger hydrodynamic diameters3329–43(SG)27–37 NCs in blood was gradually stabilized after ~12 h (29–43(SG)27–37 NCs in blood may lead to high tumor uptake of the NCs.We further performed er ~12 h . The hig29–43(SG)27–37 NCs was measured using inductively coupled plasma mass spectrometry 18 and Au10–12(SG)10–12, and their tumor uptake were determined to be 13% and 50% ID/g, respectively3325(SG)18 and Au10–12(SG)10–12, the tumor uptake of Au29–43(SG)27–37 is relatively lower. However, one salient point of Au29–43(SG)27–37 is its strong orange emission at 610 nm with a high quantum yield of 15%; such strong emission could be advantageous for some biomedical applications. The ratios of the concentration of Au in tumor relative to that in other tissues and organs are important parameters to evaluate the specificity of the NCs. The tumor/kidney, tumor/blood, and tumor/liver ratios were determined to be 2.1/1.0, 4.5/1.0, and 14.2/1.0, respectively.The tumor uptake of the Au ICP-MS, . The tum29–43(SG)27–37 NCs were further investigated. 29–43(SG)27–37 NCs at 24 h and 28 days p.i. Tumor and kidney possessed predominant distributions relative to spleen, liver, heart, and lung at 24 h p.i., which supports that Au29–43(SG)27–37 NCs could escape RES absorption and achieve efficient targeting. The majority of Au were cleared at 28 days p.i. because only 0.2% ID/g Au in liver, ~0.4% ID/g Au in kidney, and <0.1% ID/g in tumor were found, suggesting a high efficacy of renal clearance of Au NCs4828375029–43(SG)27–37 NCs with GSH ligands on the NC surface featured with a different biodistribution from that of the Cy5-labeled Au25(SG)1825(SG)1829–43(SG)27–37 NCs, the GSH ligand on the NC surface may help mitigate the serum protein adsorptionDetailed biodistribution and clearance of Au29–43(SG)27–37 NCs by the X-ray computed tomography in vivo imaging 27–37 NCs  were injected into mice via tail vein, and two-and three-dimensional X-ray CT images were recorded. We measured the tumor uptake of Au29–43(SG)27–37 NCs using U14 tumor bearing mice. As shown in 29–43(SG)27–37 NCs at the time points of 1 and 24 h p.i., measured using nude mice without tumor. The bladder 27–37 NCs by kidneyWe also confirmed the tumor uptake and efficient renal clearance of Au imaging . X-ray C imaging , which i29–43(SG)27–37 NCs by using U14 tumor bearing nude mice as the animal model. The mice were intraperitoneally injected with Au29–43(SG)27–37 NCs of a concentration of 5.9 mg-Au/kg-body. As a maximum tumor uptake of Au29–43(SG)27–37 NCs was reached at 24 h p.i. 27–37 NCs plus radiation (p < 0.05). In addition, compared with the mice treated by radiation only, the tumor volume decreased to ~66% in mice treated with Au29–43(SG)27–37 NCs plus radiation (p < 0.05). 29–43(SG)27–37 NCs plus radiation. Similarly, a significant tumor weight decrease was seen in mice treated with Au29–43(SG)27–37 NCs plus radiation relative to that in mice treated with radiation only, suggesting that the Au29–43(SG)27–37 NCs can enhance the radiation therapy.We also examined the cancer radiation treatment of Au4 h p.i. , the micmeasured . Compare29–43(SG)27–37 NCs did not induce a significant liver and kidney toxicity. In contrast, the naked Au NPs, PEG-coated Au NPs, and BSA-protected Au NCs with the hydrodynamic diameter of ~6–100 nm have been found with acute liver toxicity, such as the increase of alanine aminotransferase (ALT) and aspartate aminotransferase (AST)3750545529–43(SG)27–37 NCs developed in this study could emerge as an attractive radiosensitizing agent with its low toxicity and high tumor uptake.We finally checked the toxicological responses by examining blood biochemistry  and path29–43(SG)27–37 NCs covered by GSH can escape the RES absorption and showed high tumor accumulation via the improved EPR effect. The hydrodynamically ultrasmall Au29–43(SG)27–37 NCs showed very efficient renal clearance, and no obvious toxicity was observed in the body. The as-designed Au NCs can also significantly enhance the efficacy of the cancer radiotherapy. These advantageous features allow the Au29–43(SG)27–37 NCs to be attractive radiosensitizer materials for further testing.In summary, the Au29–43(SG)27–37 NCs followed the published procedures404  and GSH  were mixed with 4.35 mL of ultrapure water at 25°C. The reaction mixture was heated to 70°C under gentle stirring (500 rpm) for 24 h. An aqueous solution of intensely orange-emitting Au29–43(SG)27–37 NCs was formed. The orange-emitting Au29–43(SG)27–37 NC solution could be stored at 4°C for 6 months with negligible changes in their optical properties. The as-prepared Au29–43(SG)27–37 NCs were purified through ultrafiltration (3 kDa membrane).The synthesis and purification of Au6 cells suspended in 50 μL of PBS into the right shoulder of male nude mice. The mice treated with Au29–43(SG)27–37 NCs were sacrificed at 0.5, 1, 2, 6, 12, 24, 48, and 72 h post injection (p.i.). The main organs, such as tumor, liver, kidney, spleen, heart, lung, brain were collected. The organs of Au29–43(SG)27–37 NCs treated mice were digested using a microwave system CEM Mars 5  to determine their Au content, which was determined by an inductively coupled plasma mass spectrometer .The studies were approved by the Institute of Radiation Medicine, Chinese Academy of Medical Sciences and Animal Care Research Advisory Committee of Institute of Radiation Medicine, Chinese Academy of Medical Sciences, while experiments conducted following the guidelines of the Animal Research Ethics Board of Institute of Radiation Medicine, Chinese Academy of Medical Sciences. Forty-eight mice were purchased, maintained, and handled using protocols approved by the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (CAMS). The U14 tumor models were generated by subcutaneous injection of 2 × 106 cells suspended in 50 μL of PBS into the right shoulder of male nude mice. Before the experiments, the mice were anesthetized by chloral hydrate. For CT imaging, 200 μL of GSH-protected Au29–43(SG)27–37 NCs  were injected through the intraperitoneal routes into mice. Each mouse was imaged on a small-animal scanner . The mice were exposed to a 10-min CT scan and the images were reconstructed using the filtered back-projection algorithm with CT-based photon-attenuation correction. CT data were analyzed for regions of interest, including tumor, bladder, and spleen.Eighteen mice were purchased, maintained, and handled using protocols approved by the Institute of Radiation Medicine, Chinese Academy of Medical Sciences (CAMS). The U14 tumor models were generated by subcutaneous injection of 2 × 106 cells suspended in 50 μL of PBS into the right shoulder of BALB/c mice. The male mice were intraperitoneally treated with the Au29–43(SG)27–37 NCs when the tumor volume reached 100–120 mm3 (7 days after tumor inoculation). For each treatment, Au29–43(SG)27–37 NCs (0.59 mg-Au/mL) were intraperitoneally injected at a dosage of 5.9 mg/kg in the mice. As the control, 200 μL of saline was intraperitoneally injected into each mouse in the control group. Subsequently, the mice were irradiated by 5 Gy gamma-rays from 137Cs (photon energy 662 keV) with an activity of 3600 Ci at 24 h p.i. for Au29–43(SG)27–37 NCs injections. Thirty two male mice were assigned to the following four groups (eight mice per group): control, Au29–43(SG)27–37, radiation alone, and Au29–43(SG)27–37 + radiation. The tumor size was measured every two or three days, and calculated using the equation: tumor volume = (tumor length) × (tumor width)2/2.All animals were purchased, maintained, and handled using protocols approved by the Institute of Radiation Medicine, CAMS. The U14 tumor models were generated by subcutaneous injection of 2 × 10The treated mice were weighed and assessed for behavioral changes. All mice were sacrificed at 28 days p.i., and their blood and organs were collected for hematology, biochemistry and toxicological investigation. The blood was drawn for hematology analysis (potassium EDTA collection tube) and serum biochemistry analysis (lithium heparin collection tube) using a standard saphenous vein blood collection technique. During necropsy, liver, kidney, spleen, heart, lung, brain, genitals, tumor, and thyroid were collected and weighed. Major organs from these mice were then fixed in 4% neutral buffered formalin, processed into paraffin, and stained with hematoxylin and eosin (H&E). Pathology was examined using a digital light microscope.in vivo experiment. X.Z., Z.L., S.F., D.T.L. and J.X. analyzed the data and co-wrote the paper. All authors discussed the results and commented on the manuscript.X.Z., Z.L., J.X. and M.G. conceived the project and designed the experiments. J.C., Z.L., X.S., S.S., X.Y. and X.Z. performed the experiments. Z.L., H.W. and X.Y. synthesized the materials and J.C., X.S., L.Z., K.G., Y.S. and S.S. performed the SI


There were 8,385 cases with a diagnosis of sarcoidosis representing 0.054%  of all hospitalizations in Switzerland. These cases were compared with age- and sex-matched controls without the diagnosis of sarcoidosis. Hospitalization and mortality rates in Switzerland remained stable over the observed time period. Comorbidity analysis revealed that sarcoidosis patients had significantly higher medication-related comorbidities compared to matched controls, probably due to systemic corticosteroids and immunosuppressive therapy. Sarcoidosis patients were also more frequently re-hospitalized , had a longer hospital stay , had more comorbidities , and had a significantly higher in-hospital mortality (2.6% [95% CI 2.3%-2.9%] vs. 1.8% [95% CI 1.5%-2.1%] (p < 0.001). A worse outcome was observed among sarcoidosis patients having co-occurrence of associated respiratory diseases. Moreover, age was an important risk factor for re-hospitalization.Sarcoidosis is a multisystem disease with an unpredictable and sometimes fatal course while the underlying pathomechanism is still unclear. Reasons of the increasing hospitalization rate and mortality in the United States remain in dispute but incriminated are a number of distinct comorbidities and risk factors as well as the application of more aggressive therapeutic agents. Studies reflecting the recent development in central Europe are lacking. Our aim was to investigate the recent mortality and hospitalization rates as well as the underlying comorbidities of hospitalized sarcoidosis patients in Switzerland. In this longitudinal, nested case-control study, a nation-wide database provided by the Swiss Federal Office for Statistics enclosing every hospital entry covering the years 2002–2012 ( Sarcoidosis is a systemic disease of unknown cause resolving in complete remission in many cases but sometimes leading to substantial and even fatal organic dysfunctions . NowadayThe aim of this study was to investigate the hospitalization rate and in-hospital mortality among sarcoidosis patients over the last years in Switzerland, as well as the associated comorbidities and risk factors of this disease.http://www.who.int/classifications/icd/en/). The coding version ICD-10 was uniformly used all along the study time period.All our observations were derived from a dataset provided by the Swiss Federal Office for Statistics, which offers a nation-wide coverage of all hospitalized patients since 1998. In this database, the patient information is fully anonymized. No written informed consent was given to the patients who were unidentifiable due to the anonymization. The data belong to the Swiss Federal Office for Statistics  who provides regulated access to the data for research purpose. In analogy to the study of Deubelbeiss and colleagues  or Baty For the current study, sarcoidosis was defined by the ICD-10 code D86*. The cases were defined as any hospitalizations with the diagnosis of sarcoidosis either as main or concomitant diagnosis. Geriatric, obstetric, psychiatric hospitalization and cases of rehabilitation were excluded from the study. The controls consisted of hospitalizations not including sarcoidosis in the main or concomitant diagnoses. Controls were extracted from the hospitalization database using a random procedure and matching the cases 1:1 for age, gender, and month of hospitalization.Sarcoidosis-comorbidities were defined as any additional diagnosis coded by the ICD-10 nomenclature. The Charlson’s comorbidity index  was usedp-values from likelihood ratio tests. Time to first re-hospitalization was defined as the time elapsed between the first and second hospitalization record of a given sarcoidosis patient in the database. Data from patients without re-hospitalization, i.e. recorded only once in the database, were censored (lost to follow-up censoring). Time—to—event data were analyzed using Kaplan-Meier estimates, log-rank tests and Cox-proportional hazards regression. Results were reported as hazard ratio (HR) with 95% confidence intervals (CI) together with the Wald test statistic. Hazard ratios provide the percentage of increase in the hazard rates for an increase of one unit in the explanatory variable. Generalized estimating equations (using working independence correlation structure) was used to test associations with one or several covariates while accounting for within-patient variability.Descriptive statistics were reported as median and inter-quartile-range (IQR). The enrichment of sarcoidosis-associated comorbidities was tested using conditional logistic regression in order to account for the way the controls were sampled in our nested case-control design. Results were reported as odds-ratio (OR) with 95% confidence intervals (CI) together with Principal component analysis (PCA) was used to explore correlations among sarcoidosis-associated comorbidities. PCA depicts how comorbidities are related to each other and how sarcoidosis cases are grouped according to comorbidities. A data-matrix summarizing the presence/absence (1/0) of comorbidities in sarcoidosis cases was created. Scaled PCA was applied to the comorbidity matrix. The role played by external explanatory variables, including age, length-of-hospital stay, gender, in-hospital death, hospitalization rate, and number of comorbidities, was further explored using vector fitting procedure to identify the directions of maximal correlation with the external variables in the PCA space . PCA ressurvival, geepack, Epi, multipleNCC, ade4 and vegan.All analyses were done at the hospitalization level, except for the re-hospitalization analysis which was done at the patient level. All statistical computations were done using the R statistical software (version 3.1.2) . The folThe number of hospitalized cases with a diagnosis of sarcoidosis either as main or secondary diagnosis between 2002 and 2012 was 8,385 corresponding to 0.054%  of all hospitalizations in Switzerland, and representing 4,664 unique patients . OverallThe proportion of hospitalizations due to sarcoidosis was age-dependent. A higher percentage of hospitalization was found among patients aged 40–50 years (1%) and elderly patients defined as more than 70 year old (6.3%). Re-hospitalizations occurred 1.80 times per patient on average .In 30% of cases, sarcoidosis was the main reason for hospitalization (primary diagnosis).p < 0.001)  .p < 0.001) and (70–96] (p < 0.001). The in-hospital mortality rate (lower panel) did not increase significantly in any age category over the observed time period (2002–2012), although there was a trend towards an increase of cases in the older category (age >70) (p = 0.085) (= 0.085) .p < 0.001), had a longer hospital stay , had more comorbidities , and had a significantly higher in-hospital mortality (2.6% [95% CI 2.3%-2.9%] vs. 1.8% [95% CI 1.5%-2.1%] (p < 0.001).The characteristics of sarcoidosis cases and matched controls are summarized in p < 0.05). Of these, 57 with the lowest p-values (p < 1 × 10−10) were kept in the analysis  and a lower rate of cancer disease . Under-represented comorbidities among sarcoidosis cases consisted of orthopedical, gestational, geriatric, and mental disorders.Sarcoidosis cases had a higher rate of pulmonary embolism . Cases with the combined diagnosis were older  and tended to be more frequent among females (sex ratio: 0.42). They had a worse outcome with more comorbidities , a longer in-hospital stay  and a higher chance of in-hospital mortality  in comparison to sarcoidosis cases without pulmonary embolism.Overall, there were 119 hospitalized cases with sarcoidosis and pulmonary embolism (ICD-10 code: I26*), corresponding to 1.4% of all sarcoidosis hospitalizations. Compared to the control group, sarcoidosis cases had a higher rate of pulmonary embolism . The risk of re-hospitalization was also significantly higher in patients having a larger amount of comorbidities with an increase of 5.4% in the hazard rates for each additional comorbidity . No statistically significant difference was found between females and males . In this longitudinal, nested case-control study, we investigated the recent mortality and hospitalization rates as well as the underlying comorbidities of hospitalized sarcoidosis patients in Switzerland from 2002 to 2012. Furthermore, we analyzed possible risk factors and predictors of outcome compared to matched controls.Overall, our analysis showed stable mortality and hospitalization rates among sarcoidosis patients in Switzerland over the observed time period. This is in contrast to recent findings of a significant mortality increase, at least for Caucasians, in the United States –6. ReasoBy clearly displaying the burden of the disease, our data reveal that sarcoidosis patients showed a significantly worse outcome compared to matched controls regarding re-hospitalization, in-hospital mortality and length of hospital stay. Similar results were recently found when comparing sarcoidosis patients with pedestrian traffic accident patients . In the In our study, we identified possible predictors and risk factors associated with a worse outcome. We found that age was a significant prognostic factor of the disease, which is supported by other previous studies . There wSpecific comorbidities possibly contributing to a worse outcome were also detected. For instance, associated respiratory diseases were significantly correlated to a longer hospital stay and a higher number of comorbidities. This defines a phenotype of sarcoidosis patients with a higher morbidity. Among these associated respiratory diseases, there was a higher risk of pulmonary embolism. A link between sarcoidosis and pulmonary embolism was postulated in previous studies  while thMedication-related events seem to substantially affect sarcoidosis patients leading to a significantly higher hospitalization rate as well as to a shorter time to re-hospitalization. Long-term benefits of the corticosteroidal and immunosuppressive therapy are often discussed controversially . Our finOne of the strength of our study is the comprehensive number of patients registered by the Swiss coding system, providing good data quality. In addition, this study is one of a few European studies investigating hospitalizations and mortality rates among sarcoidosis patients and the only one displaying risk factors for re-hospitalization. As a limitation, our analysis is based on a database which is essentially hospitalization-oriented. Our cohort only included hospitalized sarcoidosis cases while a high fraction of patients is typically treated in an out-patient setting. Further information such as medication or laboratory data were not available nor was it possible to contact patients to complete or confirm data.Overall, the current study is showing stable mortality and hospitalization rates among sarcoidosis patients over the observed time period. However, the burden of the disease becomes apparent, as patients with sarcoidosis show a significantly worse outcome compared to matched controls. This could be due to the disease itself as well as to a cluster of specific comorbidities and complications namely associated respiratory diseases. On the other hand, there is also evidence that side effects of more aggressive treatment approaches could explain this poorer outcome, probably especially affecting patients at older age.A better knowledge of these prognostic factors could lead to a better understanding of the disease and guide to a more specific monitoring as well as more individualized therapeutic approaches. Further research should be done to evaluate this and maybe contribute to a better outcome.


As a result, thin magnesium hydroxide layers spontaneously grew, giving rise to characteristic volume expansion effects, which depended on the size of the particles. Rounding of the originally sharp cube edges leads to a significant loss of the morphological definition specific to the MgO cubes. Comparison of different regions within one sample before and after exposure to liquid water reveals different transformation processes, such as the formation of Mg(OH)2 shells that act as diffusion barriers for MgO dissolution or the evolution of brucite nanosheets organized in characteristic flower-like microstructures. The findings underline the significant metastability of nanomaterials under both ambient and high-vacuum conditions and show the dramatic effect of ubiquitous water films during storage and characterization of oxide nanomaterials.Physisorbed water originating from exposure to the ambient can have a strong impact on the structure and chemistry of oxide nanomaterials. The effect can be particularly pronounced when these oxides are in physical contact with a solid substrate such as the ones used for immobilization to perform electron or ion microscopy imaging. We used helium ion microscopy (HIM) and investigated morphological changes of vapor-phase-grown MgO cubes after vacuum annealing and pressing into foils of soft and high purity indium. The indium foils were either used as obtained or, for reference, subjected to vacuum drying. After four days of storage in the vacuum chamber of the microscope and at a base pressure of  Knowledge about the stability of engineered nanomaterials in aqueous systems is critical for predicting their functionality under environmental conditions. For example, for transient electronics it has become a major challenge to understand and control the factors of materials transformation in aqueous systems –2. UnderAmong the many microscopic techniques available –6 scanniAdditionally, metal oxides can also be damaged by an electron beam . Indeed,2 (from ρmagnesia = 3.5 g·cm−3 to ρbrucite = 2.3–2.4 g·cm−3) generates a significant volume expansion. For refractory materials, related phenomena can have desired [The density mismatch that follows the conversion from MgO to Mg(OH) desired  as well  desired . In the There is growing awareness in the scientific community  that a vT = 1173 K for a minimum duration of one hour in order to eliminate surface adsorbates including residual hydroxyls of high thermal stability [−7 mbar) at room temperature for four days. The latter procedure was applied in order to remove physisorbed species such as weakly bound water that forms during exposure of the foil to the ambient. The shape change of the cubes was quantified by image analysis, using the edge length increase factor, defined as L2/L1, where L1 and L2 are the edge lengths before and after high-vacuum treatment in the HIM chamber, respectively. Since it is not possible to simultaneously measure the three edge lengths of the cubes, we calculated the corresponding volume increase factor, defined as (L2/L1)3. More than 45 edge lengths were measured before and after high-vacuum treatment, for both types of samples . The cubes selected for the measurements had an estimated maximum tilt angle of 30° relative to the incident ion beam. This does not change the edge length increase factor (L2/L1), but induces a maximum error of less than 15% in edge length (L1). To verify that the tilt did not, indeed, affect our measurements, we measured cubes that were lying exactly flat on the surface (tilt = 0°) and found essentially the same dependence of L2/L1 on L1 . The microscope is equipped with an Everhardt–Thornley (ET) detector to record secondary electron images. The acceleration voltage of the ions and the beam current were 30 kV and approx. 0.25 pA, respectively. The MgO cube powders were vacuum annealed at −7 mbar, HIM imaging reveals that important morphological changes of the MgO cubes have occurred 2 layer coats the MgO cubes, leading to a partial fusion of the particles.After four days under high-vacuum conditions at a base pressure lower than 3·10occurred . As showL2/L1 of 1.14 ± 0.10 (standard deviation of the measurement) when the indium foil was used as received, where L1 and L2 are the edge lengths before and after high vacuum treatment, respectively. This corresponds to a volume increase factor (L2/L1)3 of 1.52 ± 0.44 . In the case of a complete transformation of MgO cubes into Mg(OH)2 cubes, a similar volume increase factor, i.e., 1.52, would be expected. The high value of the volume expansion measured is attributed to the Kirkendall effect, which has been observed many times in the case of solid-to-solid transformations [2+, and H2O molecules/hydroxide ions) are thus expected to lead to the growth of the Mg(OH)2 layer outwards the cubes, explaining the large increase in volume measured here. The proposed transformation process, which is schematically shown in Figure S2 of As shown in rmations –28. SuchFurthermore, a clear size dependence is observed as shown in Interestingly, contrast changes between the different imaging sessions are quite evident . In this15 cm−2. Because the defects mainly occur close to the stopping range of the ions, which is ca. 160 nm according to the SRIM software [The build-up of carbon on the sample areas during imaging may appear similar. As illustrated in , the secsoftware , it is c2+ and OH− ions and lamellar Mg(OH)2 structures crystallize and give rise to flower-like morphologies  [We performed exploratory experiments to address the impact of liquid water on thermally activated MgO cubes. A comparison was performed on sample spots having cubes of different sizes put in contact with a water droplet  , as a mi (BaSO4) –38. Inde (BaSO4) ,39. The 2 core–shell structures as a result of the Kirkendall effect.This work underlines the metastability of metal oxide nanostructures and reports drastic material changes during storage under high vacuum conditions. We observed significant and size-dependent volume expansion effects of the MgO cubes that were grown and processed in dry vacuum environments prior to imaging with helium ion microscopy. The volume expansions observed are attributed to surface reactions between the MgO cubes and water molecules originating as physisorbed species from an indium foil that was employed as a conductive substrate for imaging. Depending on their size the MgO-based cubes  become sThese results are significant and should be relevant for everyone working with nanomaterials under ambient and high-vacuum conditions. Conductive substrates require appropriate drying and surface cleaning prior to sample immobilization and measurement. Apart from the transformation behavior of cubic oxides such as MgO, NiO and CdO and its relevance for the synthesis of hydroxides with controlled morphologies –41, the File 1Additional experimental data.


The exposure to graphene oxide did not induce significant changes in the body weights, organ weights, and food consumption during the 14 days of recovery time. The microalbumin and lactate dehydrogenase levels in the bronchoalveolar lavage (BAL) fluid were not significantly changed due to the exposure. Similarly, total cell count, macrophages, polymorphonuclear leukocytes, and lymphocytes were not significantly altered in the BAL fluid. Plus, the histopathological examination of the rat lungs only showed an uptake of graphene oxide in the alveolar macrophages of the high-concentration group. Therefore, these results demonstrate that the single inhalation exposure to graphene oxide induce minimal toxic responses in rat lungs at the concentrations and time points used in the present study.Graphene is receiving increased attention due to its potential widespread applications in future. However, the health effects of graphene have not yet been well studied. Therefore, this study examined the pulmonary effects of graphene oxide using male Sprague-Dawley rats and a single 6-hour nose-only inhalation technique. Following the exposure, the rats were allowed to recover for 1 day, 7 days, or 14 days. A total of three groups were compared: control (fresh air), low concentration (0.46 ± 0.06 mg/m Graphene is a two-dimensional graphitic carbon nanomaterial with unique physical and chemical properties that enable a wide variety of applications . Plus, gα (TNF-α), tumor growth factor-β (TGF-β), and interleukin-1β (IL-1β) and IL-6, were significantly elevated in the bronchoalveolar lavage (BAL) fluid during the recovery period of 28 days. In another study, graphene exposure increased the polymorphonuclear neutrophil (PMN) levels in the BAL fluid from male Wistar rats following exposure to graphene at target concentrations of 2.5 or 10.0 mg/m3 for 6 hours per day for 5 consecutive days with 7 or 28 days of recovery [β were also increased at day 7 in a dose-dependent manner, while graphene-loaded alveolar macrophages were observed in the lungs of all the treated animals in a concentration-dependent manner. Meanwhile, when using a pharyngeal aspiration technique with female C57BL/6 mice, graphene nanoplatelets (50 μg) were found to increase the pulmonary PMN number and proinflammatory cytokines, such as monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and IL-1β, in the BAL fluid [As a result, several toxicological studies have recently been performed to determine the toxicity of graphene nanomaterials. For example, graphene nanoplatelets were found to accumulate in mouse lungs for up to 28 days after a single intratracheal instillation at a dose of 2.5 or 5.0 mg/kg . Plus, aAL fluid , 6. in vivo and in vitro studies, the inhalation of graphene nanomaterials would be expected to exert toxicity in humans, as in experimental animals. However, since only a small amount of data with limited experimental designs has so far been reported, more data obtained from a variety of experimental settings needs to be accumulated. We also hypothesized that relatively lower concentration of graphene exposure would represent more valuable outcome in order to translate into a human exposure scenario. Also, the selected concentrations were based on the previous experimental setting employed in inhalation toxicity studies for graphene nanomaterials [3 or 3 mg/m3) for 6 hours using a nose-only inhalation chamber and then allowed to recover for 1 day, 7 days, or 14 days. The results showed that the graphene oxide nanomaterials produced minimal toxic effects in the male rats for the experimental settings used in this study.Thus, according to these findings obtained fromaterials . In addiThe graphene oxide nanomaterials were kindly provided by Dr. Heon Sang Lee of Dong-A University . A transmission electron microscope equipped with an energy-dispersive X-ray analyzer (TEM-EDX) was used to measure the graphene oxide nanomaterials based on National Institute for Occupational Safety and Health (NIOSH) analytical method 7402 [3 and 3 mg/m3 for the low- and high-concentration chamber, respectively.Male Sprague-Dawley (SD) rats were exposed to the graphene oxide nanomaterials using a nose-only exposure system . The nose-only exposure system provided reliable exposure condition with accuracy and consistency. Thus, this exposure system was extensively used for inhalation of carbon-based nanomaterials including graphene in our laboratory and by others –10. The  ad libitum. During the acclimation period, the animals were trained to adapt to the nose-only inhalation chamber. The rats were divided into 3 groups: control , low-concentration group (n = 12), and high-concentration group (n = 12). The low- and high-concentration groups were exposed to the graphene oxide for a single period of 6 hours, while the control group received filtered fresh air. The animals were examined daily for any evidence of exposure-related toxic responses. The body weights were measured at the time of purchase, at the time of grouping, after the 6 h inhalation exposure, and before necropsy. The food consumption (g/rat/day) was measured once a week. After the single 6 h inhalation exposure to graphene oxide, the rats were allowed to recover for 1 day, 7 days, or 14 days (n = 4 per treatment group for each time period) to investigate the toxic responses. At sacrifice, gross observations of the organs were recorded, and the testes, kidneys, spleen, liver, lungs, and brain were all carefully removed and weighed. All the animal protocols were approved by Hanyang University Institutional Animal Care and Use Committee.The six-week-old male specific-pathogen free SD rats were purchased from OrientBio  and acclimated for 2 weeks before initiating the inhalation exposure. During the acclimation and inhalation exposure, the rats were housed in polycarbonate cages (maximum of 3 rats per cage) installed in individually ventilated cage racks. The rats were kept under a controlled temperature (21.8 ± 0.17°C) and humidity (49.16 ± 2.37%) and a 12 h light/dark cycle. The rats were fed a rodent diet  and filtered waterThe concentrations of graphene oxide in the chambers were measured using polycarbon filters connected to a MAS Escort ELF sampling pump  at a flow rate of 1.0 L/min. The weight difference of the polycarbon filter before and after sampling was calculated. The size distribution of the graphene oxide was then measured directly using a scanning nanoparticle spectrometer  connected to a condensation particle counter  and dust monitor . The volume of sheath air and polydispersed aerosol air used in the SNPS and CPC was 15 and 1.5 L/min, respectively.α (TNF-α), interleukin- (IL-) 1β, IL-18, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), and vascular endothelial growth factor (VEGF) in the BAL fluid was measured using a Bio-Plex Rat 23-Plex assay  according to the manufacturer's protocol. The level of tumor growth factor β1 (TGF-β1), matrix metallopeptidase-9 (MMP-9), and tissue inhibitor of metalloproteinase-1 (TIMP-1) was measured using Quantikine ELISA kit  according to the manufacturer's instructions.At sacrifice, the right lungs were 4 times with 3 mL aliguots of cold 0.9% NaCl solution . The BAL fluid samples were then centrifuged for 7 min at 500 ×g, and the BAL cells were collected and resuspended in 1 mL of 0.9% NaCl solution for evaluation. The total cells number was determined using a hemocytometer. The cells were first smeared and then stained with Wright Giemsa Sure Stain for counting the total number of cells, macrophages, polymorphonuclear leukocytes (PMNs), and lymphocytes. The BAL levels of microalbumin and lactate dehydrogenase (LDH) were also measured using reagents obtained from Randox Laboratories Ltd.  and Waco Pure Chemical Industries, Ltd. , respectively. The level of inflammatory cytokines such as tumor necrosis factor-At sacrifice, the left lungs were removed and fixed in a 10% formalin solution containing neutral PBS under 25 cm water pressure followed by embedding in paraffin and staining with hematoxylin and eosin. The stained lung tissue sections were examined under a light microscope for histopathological evaluation.P < 0.05 and P < 0.01.The statistical analysis was performed using SPSS (Version 19) and the statistical evaluation performed using an analysis of variance (ANOVA) following multiple comparison tests using Duncan's method. The level of statistical significance was set at θ = 26.4°, corresponding to the interlayer spacing (d = 0.34 nm). The peak in the XRD pattern for the dried graphene oxide particles showed broadening, as well as a shift to a lower angle (2θ = 9.5°), indicating that the interlayers were 0.93 nm apart due to intercalation by the hydroxyl, carbonyl, and epoxide groups and moisture. The DLS measurement showed the average flake size of the graphene oxide in a solution. Plus, the equivalent hydrodynamic diameter of the graphene oxide was estimated using Stokes-Einstein equation to be 150–250 nm (The field emission TEM (FE-TEM) analysis characterized the graphene oxide as a folded and stacked platelet structure Figures . The TEM0–250 nm .3) measured based on the weight of the polycarbon filter before and after sampling were 0.46 ± 0.06 and 3.76 ± 0.24 for the low- and high-concentration groups, respectively (3) in the chambers was measured directly using SNPS and was 3.33 × 106 ± 2.95 × 105 and 6.17 × 106 ± 4.13 × 105 for the low- and high-concentration groups of graphene oxide, respectively (2/cm3) of the graphene oxide were 50.6 ± 1.82 nm and 6.45 × 1010 ± 7.04 × 109 nm2/cm3, respectively, for the low-concentration chamber and 72.9 ± 2.02 nm and 2.72 × 1011 ± 3.70 × 1010 nm2/cm3, respectively, for the high-concentration chamber . No significant body weight changes were observed for the low- and high-concentration groups during the exposure and recovery periods (Supplement 2). The examination of the rat organs, including the testes, kidneys, spleen, liver, lungs, and brain, also revealed no significant clinical signs or organ weight changes during the observation period (Supplements 3–11).No significant gross effects were observed during the exposure and recovery periods. Also, there were no significant differences in food intake between the control and graphene oxide-treated groups  in order to observe the causality and dose-dependent toxic responses in animals. Concentrations of graphene oxide were chosen based on preliminary data which presented dose-dependent toxic responses after 5-day repeated inhalation of graphene nanomaterial. Moreover, our concentration is more relevant to worker exposure levels compared to other previous study which used up to 10 mg/m3 [μg/m3 in the work area sampling [Since graphene nanomaterials are experiencing rapid development with predicted widespread application, concerns about human exposure have also been recently increasing. However, safety information, particularly the inhalation toxicity of graphene-based nanomaterials, remains limited . Therefosampling .3, 3.05 mg/m3, and 10.1 mg/m3) and a longer period of inhalation (6 hours per day for 5 consecutive days) and similar concentration (0.68 and 3.86 3.05 mg/m3) and exposure period (6 hours per day for 5 consecutive days and 28 days after exposure) [Overall, no significant clinical changes due to graphene oxide exposure were observed during the 6-hour inhalation and 14-day recovery period, unlike other carbon nanomaterial toxicity studies such as MWCNTs and SWCNTs , 15, 16.α, IL-1β, IL-18, G-CSF, M-CSF, VEGF, TGF-β1, MMP-9, and TIMP-1) demonstrated that only MMP-9 (day 1) and IL-18 and TGF-β1 (day 7) were significantly elevated due to exposure of graphene oxide at high concentrations. These elevated parameters returned to baseline level after 14-day recovery period. This temporary elevation of MMP-9, IL-18, and TGF-β1 may be due to phagocytosis of graphene oxide by alveolar macrophage and other types of lung cells [μg of unoxidized graphene platelets using a pharyngeal aspiration technique and the results demonstrated significant increases in LDH and the total cells and PMNs numbers at 24 h after exposure [μg graphene (approximately 2.5 mg/kg mouse) used in the previous study was significantly higher than the doses used in the present study, where the approximate deposited doses were 0.026 mg (0.087 mg/kg rat) for the high-concentration group and 0.003 mg (0.01 mg/kg) for the low-concentration group assuming a deposition rate of 10% and a minute ventilation of 0.19L/min in a 300 g rat [3 × (0.19 L/min) × 6 h × 0.1 = 0.003 mg deposited a day (0.46/1000 × 0.19 × 360 min × 0.1 = 0.003); high-concentration group, 3.76 mg/m3 × (0.19 L/min) × 6 h × 0.1 = 0.026 mg deposited a day (3.76/1000 × 0.19 × 360 min × 0.1 = 0.026). Another previous study also showed markedly elevated damage parameters in the BAL fluid [3 for 6 h/d, for 5 days with 7 or 28 days of recovery) showed an elevated number of total cells and lymphocytes at 10.01 mg/m3 and PMNs at 3.05 mg/m3. Yet, the particle concentrations were significantly higher than the concentrations used in the present study. Interestingly, the lowest concentration  in the previous study did not increase the lung toxicity parameters, and this concentration is similar to the high concentration  used in the present study in terms of the approximate total deposited dose in the rat lungs. The previous study also found a dose-dependent increase in the LDH levels in the BAL fluid after exposure to graphene. Thus, when taken collectively, while the experimental designs differed significantly between the two studies, the dose would seem to be the major factor determining the toxicity of graphene.The present study also observed several toxicological indicators to determine the toxicity of graphene oxide in the exposed rats. However, the results showed limited level of toxicity in the lungs of the exposed rats. BAL fluid evaluation of nine inflammatory parameters . Therefore, this data demonstrated that the exposure concentration was an important factor determining the amount of graphene oxide accumulated in the alveolar region of the rat lungs, and the graphene oxide used in this study persisted in the lungs up to 14 days after exposure. However, no other histopathological changes were found in the lungs of the exposed rats. Similarly, Ma-Hock et al.  also fouIn conclusion, the results of this study suggest that the single inhalation of graphene oxide in male Sprague-Dawley rats exerted minimal toxic responses, indicating that the selected graphene oxide was relatively inert at the concentrations used. However, further studies with varying experimental settings are still needed to determine the toxicity of graphene oxide nanomaterials.Supplement 1: shows food intake of rats.Supplement 2: shows body weight of rats before and after the exposure.Supplement 3-5: shows gross findings of rats during the recovery period.Supplement 6-11: shows absolute and relative organ weight of rats during the recovery period.