Although immunotherapy has emerged as an attractive therapy for refractory cancers, its limited efficacy in hepatocellular carcinoma (HCC) suggests the need for a combination strategy that can either enhance or complement therapeutic effect. We investigated whether combination of immune checkpoint blockade (ICB) and radiation could enhance antitumor effect in a murine HCC model.Using murine HCC, HCa-1, the effect of radiation on programmed death-ligand1 (PD-L1) expression was determined by real-time PCR, flow cytometry, and western blotting. Signaling pathways involved in altered PD-L1 expression were examined. Tumor growth and survival rate were evaluated for a combination of anti-PD-L1 and radiation. Immunological parameters in the tumor were assessed using flow cytometry and histological study.P<0.01). Survival was significantly improved in the combination group compared to anti-PD-L1 alone or radiation alone group . The underlying mechanism involved increasing apoptosis, decreasing tumor cell proliferation, as well as restoration of CD8+ T cell functions.Radiation upregulated PD-L1 expression in tumor cells through IFN-γ/STAT3 signaling, which could facilitate therapeutic action of anti-PD-L1. Combination of anti-PD-L1 and radiation significantly suppressed tumor growth compared to treatment with anti-PD-L1 alone or radiation alone group (The combination of anti-PD-L1 and radiation significantly improved the antitumor effect shown in tumor growth delay as well as in survival, supporting a novel combination strategy of immunoradiotherapy in HCC. Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with a high mortality rate. Particularly, advanced HCC patients have a poor prognosis with a 5-year survival rate <5% and a high recurrence rate , 2. AlthRecently, immunotherapy has emerged as an effective and promising treatment for cancers that were refractory to conventional treatment. Recent advances in the understanding of tumor biology and immune checkpoint molecules have provided novel therapeutic strategies using immune checkpoint blockade (ICB). Several studies have shown that the blockade of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) or programmed death-1 (PD-1) resulted in significant antitumor effect and prolongation of survival –13. HoweRadiation therapy (RT) has long been utilized as one of major cancer modality. RT results in direct tumor cell death and induces tumor-specific immune response , 16. It Several preclinical studies showed that radiation combined with ICB enhanced local tumor control and survival rates in breast cancer, colon cancer, and melanoma –26, provWe hypothesized that if radiation could induce programmed death-ligand1 (PD-L1) expression in the tumor, the antitumor effect of PD-L1 blockade would be augmented along with the antitumor effect of radiation per se. In this study, we investigated whether radiation could enhance PD-L1 expression in tumor cells, which may provide an effective milieu for anti-PD-L1 binding to tumor cells, as well as its impact on tumor growth suppression and survival, using murine HCC syngeneic to C3H/HeN mice as a preclinical model for HCC.in vitro effect of radiation on PD-L1 expression in murine HCC (HCa-1). The change of PD-L1 mRNA expression after radiation was determined by real-time PCR and PD-L1 protein expression was determined by flow cytometry and western blotting. Figure in vivo effect of radiation on PD-L1 expression by immunohistochemistry (IHC) and western blotting. HCa-1 cells (1 × 106) were inoculated intramuscularly into the right thighs of mice, and tumors were irradiated with a single dose of 10 Gy when the tumor reached to 8 mm in mean diameter. To examine the PD-L1 expression, tumor samples were harvested 7 days after radiation. Tumor sections were stained with PD-L1 antibody for IHC and tumor cell lysate was isolated for western blotting. As shown in Figure We first assessed the In several cancer cells, upregulation of PD-L1 expression is strongly associated with a Toll-like receptor or the IFN-γ signaling pathway , 29. RadThe above results suggest that radiation could upregulate PD-L1 expression via IFN-γ production. To understand whether radiation upregulated the PD-L1 expression in HCa-1 cells via IFN-γ signaling, blocking experiments were performed. We observed the effect of IFN-γ inhibition on the expression of PD-L1. Pre-treatment with the anti-IFN-γ neutralizing antibody resulted in significant inhibition in radiation-induced upregulation of PD-L1 expression, as assessed by flow cytometry and western blotting Figure . These det al. showed that Janus activated kinase/STAT/interferon regulatory factor-1 (IRF-1) signaling pathway plays a critical role in the upregulation of PD-L1 expression in response to IFN-γ in human lung cancer cell lines [Lee ll lines . Therefoin vivo and in vitro. Expression of PD-L1 may allow tumor cells to evade the host immune response and attenuate the efficacy of the antitumor immune response [in vivo. HCa-1 cells (1 × 106) were inoculated intramuscularly into the right thighs of mice, and treatment was started when the tumor reached to 8 mm in mean diameter. Right thighs of mice were irradiated with 10 Gy in 1 fraction. For the PD-L1 blockade experiment, anti-PD-L1 (10 mg/kg) was given a total of 4 injections in 3-day intervals after radiation , but was significant in the radiation alone group compared with the control group (P<0.05). The lack of tumor growth suppression in anti-PD-L1 alone could have resulted from low baseline level of PD-L1 in HCa-1 cells. Combination group significantly suppressed tumor growth compared with anti-PD-L1 or radiation alone group, respectively  .The above data showed that radiation led to enhancement of tumor cell PD-L1 expression response . We nextn Figure . The antP<0.001) . After all mice in the other experimental groups had died, 70% of mice still survived in the combination group  Figure .We next examined the mechanism underlying the significant regression of HCa-1 tumors exhibited in the combination treatment. The rates of cell proliferation and apoptosis in tumor tissues from each group were analyzed by IHC staining for Ki67 and terminal deoxynucleotidyl transfer dUTP nick end labeling (TUNEL), respectively. The combination group exhibited significantly decreased tumor cell proliferation compared with the anti-PD-L1 or with the radiation alone group. The level of apoptosis was increased in the combination group compared with the anti-PD-L1 or with the radiation alone group Figure , which m+ tumor infiltrating lymphocytes (TILs) are known to play a key role in antitumor effect and PD-1-expressing CD8+ TILs are involved in tumor-associated immunosuppression [+ TIL functions, we performed flow cytometry analysis using TILs isolated from HCa-1-bearing mice. The percentage of CD8+ T cells was significantly increased in the combination group, while anti-PD-L1 and radiation alone groups showed a modest increase in CD8+ T cells , and cell proliferation (Ki67). As shown in Figure +CD8+T cells, CD107a+CD8+ T cells, and Ki67+CD8+ T cells, indicating an increased effector cell cytotoxicity and proliferation . These results were confirmed at the protein level by IHC  radiation upregulated PD-L1 expression in tumor cells in a time- and dose-dependent manner, (ii) radiation activated STAT3 through the production of IFN-γ, which led to PD-L1 upregulation in tumor cells, and (iii) the efficacy of radiation could be increased by combination with anti-PD-L1 antibody, leading to the abrogation of exhausted CD8Although immunotherapy has emerged as a novel cancer treatment, its modest response rate particularly in HCC throws an issue of how to escalate its antitumor effect. Tumors with a high mutation burden, such as melanoma and non-small cell lung cancer, showed clinical benefits to immune checkpoint therapy. High frequency of non-synonymous mutation, which results in the formation of neo-antigens that can be recognized by immune cells, has been reported to be successful for cancer immune checkpoint therapy . In addi+ T cells were associated with poor disease progression, such as shorter disease-free survival in HCC. These data suggest that blocking of PD-1/PD-L1 interaction may increase anti-tumor effect in HCC patients. For this reason, we focused on blocking the PD-1/PD-L1 pathway using anti-PD-L1 antibody in treatment of HCC.Radiation induces not only direct tumor cell killing but also phenotype alteration of tumor cells, including MHC-I, carcinoembryonic antigen, mucin-1, Fas, and ICAM-1, which may affect tumor immunogenicity –23. Radiet al [In this study, we also found that radiation increased PD-L1 mRNA and protein expressions in HCa-1 cells in time- and dose- dependent manners Figure  and 2B. et al . These ret al , 46–48. et al , 50. We in vivo. We further demonstrated whether combination of anti-PD-L1 and radiation enhances the antitumor effect in a murine HCC model. Combination of anti-PD-L1 and radiation resulted in reduction of tumor growth and improved the survival rate when compared to treatment with either anti-PD-L1 or radiation . All mice were maintained in a specific pathogen-free facility of the Yonsei University Medical College guidelines, the Guide for the Care and Use of Laboratory Animals and the Guidelines and Policies for Rodent experiment provided by the Assessment and Accreditation of laboratory Animal Care (AAALAC). All mice were raised with free access to food and water under specific pathogen-free conditions in a room maintained on a 12 h light/dark cycle.2 and 95% air.Murine HCC, HCa-1, was maintained in Dulbecco's minimal essential medium  supplemented with 10% (v/v) fetal bovine serum (GIBCO), 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO) at 37°C in a humidified atmosphere of 5% COCell Fixation/Permeabilization Kits for intracellular cytokine analysis was purchased from BD Bioscience . STAT3 inhibitor VI, S31-201 was purchased from Santa Cruz Biotechnology . Recombinant mouse IFN-γ and TNF-α were purchased from Peprotech .Total RNA was extracted from HCa-1 cells using TRIzol reagent . One μg of total RNA was reverse transcribed to cDNA using Omniscript RT Kit  according to the manufacturer's instructions. Real-time PCR was performed on the Step One Plus-Real Time System  using the Power SYBR Green Master Mix (Applied Biosystems). The primers were as follows: mouse PD-L1;5’-AAA TCG TGG TCC CCA AGC-3’ and 5’-TCC TCA TGT TTT GGG AAC TAT CT-3’, mouse INF-γ; 5’-CCT AGC TCT GAG ACA ATG AAC GCT-3’ and 5’-TGC CAG TTC CTC CAG ATA TCC AAG-3’, mouse TNF-α;5’-AAA ATT CGA GTG ACA AGC CTG TAG-3’ and 5’-CCC TTG AAG AGA ACC TGG GAG TAG-3’, mouse STAT1; 5’- TGA GAT GTC CCG GAT AGT GG-3’ and 5’-CGC CAG AGA GAA ATT CGT GT-3’, mouse STAT3; 5’-GTC TGC AGA GTT CAA GCA CCT-3’ and 5’-TCC TCA GTC ACG ATC AAG GAG-3’, mouse IRF-1; 5’-GGA CTC AGC AGC TCT ACC CTA CCT-3’ and 5’-GCT GGA GTT ATG TCC CTT TCC ATA TC-3’, and mouse GAPDH; 5’-CGA CTT CAA CAG CAA CTC CCA CTC TTC C-3’ and 5’-TGG GTG GTC CAG GGT TTC TTA CTC CTT-3’. The relative levels of mRNA were determined by normalization of the data to the expression levels of GAPDH mRNA.Whole cell extracts were prepared using RIPA buffer  with protease inhibitor cocktail . Equal amounts of cell lysates were separated on 10% SDS–polyacrylamide gel, and were then electrotransferred to polyvinylidene difluoride membrane . The membranes were blocked with 5% skim milk in 0.1% TBS-Tween20 for 1 h at 25°C prior to incubation with primary antibodies against PD-L1 , IFN-γ , TNF-α (Santa Cruz Biotechnology), phospho-STAT1 (S727) , STAT1 , phospho-STAT3 (Y705) (Santa Cruz Biotechnology), STAT3 (Santa Cruz Biotechnology), and GAPDH (Santa Cruz Biotechnology). Primary antibodies were incubated for overnight at 4°C after blocking. For antibodies detection, goat-anti-rabbit IgG-HRP antibody (Santa Cruz Biotechnology) was incubated for 1 h at 25°C. The blots were developed using chemiluminescent substrate  and Signals were detected using ImageQuant LAS-4000 . GAPDH was used as a loading control.6) were inoculated intramuscularly into the right thighs of mice, and treatment was started when the tumor reached to 8 mm in mean diameter. Four experimental groups were set; control, anti-PD-L1 alone, radiation alone, and combination of anti-PD-L1 and radiation. Mice were immobilized in specially designed mice jig and lead shield was used to avoid unwanted irradiation to other body parts. The right thighs of the mice were irradiated with 10 Gy in 1 fraction using an X-Rad 320 irradiator . Mice were treated 69 cm from the radiation source (SSD) with a dose rate of 150 cGy/min with 300 kVp X-rays, using 12.5 mA and an X-ray beam filter consisting of 2.0 mm Al. For the PD-L1 blockade experiment, anti-PD-L1  was given as a total of 4 injections in 3-day intervals after radiation. For CD8+ T cell depletion experiment, anti-CD8  was given in a total of 4 injections in 3-day intervals. Antibody treatment started from one day before radiation.HCa-1 cells  guidelines of the Yonsei University Health System. After experiments, the experimental mice were sacrificed before reaching the maximum allowable size using carbon dioxide (CO2).Tumor volume was measured every 3 days with an electronic caliper and calculated as volume = π/6 × ab2 inhalation and the date was recorded to calculate survival rate.Mice were monitored daily to ensure their survival rate for 56 days period. When tumor reached to ≥ 25 mm in mean diameter and/or mice showed signs of poor body condition including respiratory distress, hypoactivity, and failure to respond to stimuli, the mice were sacrificed by COFor the isolation of TILs, solid tumors were excised and chopped by clipper then incubated in 1 mg/ml collagenase type IV  solution containing 0.01 mg/ml DNase I  at 37°C for 30 min. TILs were isolated by density gradient centrifugation on an 80%/40% Percoll (GE Life sciences) gradient after washing the dissociated tissues by chilled complete RPMI medium.Flow cytometry was performed by FACS LSR II . The data was analyzed using the FlowJo software .For staining of surface proteins, single cells were resuspended in cold PBS with 3% BSA and stained with a fluorescent-labeled antibodies against PD-L1  and CD8  at a final concentration of 1 μg/mL for 15 min at 4°C on ice/light protected. Cells were then washed twice and immediately analyzed.For intracellular molecule staining, single cells were stained using Cell Fixation/Permeabilization Kits, according to the manufacturer's instructions. Briefly, surface stained cells were fixed and permeabilized for 30 min in a fixation/permeabilization buffer. After washing with 1× Perm/Wash buffer, cells were stained with fluorescent-labeled antibodies against IFN-γ (ebioscience), TNF-α (BioLegend), p-STAT3 (Y705) (ebioscience), CD107a (ebioscience), and Ki67 (ebioscience) for 30 min on ice/light protected. Cells were then washed twice with 1× Perm/Wash buffer and immediately analyzed.For paraffin sections, tumor samples were fixed in 4% formalin and were embedded in paraffin, which was cut into 5-μm-thick sections. For frozen sections, formalin-fixed tumor samples were snap frozen over liquid nitrogen in a Tissue-Tek OCT compound  and cryosectioned (15 μm). The antibodies used in this study are described in 2O2 in methanol for 30 min in order to reduce endogenous peroxidase activity. They were blocked with 5% normal horse serum (Gibco) for 1 h and then incubated with a primary antibody against PD-L1 (Proteintech). The samples were incubated with biotinylated secondary antibody  and peroxidase-labeled streptavidin (DAKO Corp.). Staining was developed using the 3-3 diaminobenzidine substrate chromogen system (DAKO Corp.). The samples were counterstained with hematoxylin (DAKO Corp.) and imaged by light microscopy.For IHC staining, the deparaffinized sections were treated with 3% HFor immunofluorescence staining, frozen sections were incubated with 5% normal horse serum and 0.3% Triton X-100 (Sigma) in PBS, pH 7.5, and subsequently with primary antibodies against CD8 , IFN-γ (Bioss), CD107a (Abcam), and Ki67 (Abcam) overnight at 4°C. Finally, sections were incubated with alexa Fluor 488 donkey anti-rabbit IgG  and alexa Fluor 594 goat anti-rat IgG (Invitrogen) at 25°C for 1 h. The sections were then counterstained with DAPI and imaged by confocal microscopy .Tumor apoptosis was assessed by TUNEL staining. About 5 μm sections of tumor tissues embedded in paraffin were stained with the DeadEnd™ Colorimetric TUNEL System , according to the manufacturer's instructions.P< 0.05 was regarded as statistically significant. All experiments were conducted at least twice.Data are expressed as means ± standard error (SE). Statistical significance was determined via one-way ANOVA using Prism 5.0 software . A 2-tailed Student's t test was used to compare data between 2 groups. Two-way ANOVA was used to compare tumor size at multiple time points within groups. Differences in mouse survival rate were determined by a log-rank (Mantel-Cox) test of the Kaplan-Meier survival curves. A value of


Van der Waals heterostructures combining hexagonal boron nitride (h-BN) and graphene offer many potential advantages, but remain difficult to produce as continuous films over large areas. In particular, the growth of h-BN on graphene has proven to be challenging due to the inertness of the graphene surface. Here we exploit a scalable molecular beam epitaxy based method to allow both the h-BN and graphene to form in a stacked heterostructure in the favorable growth environment provided by a Ni(111) substrate. This involves first saturating a Ni film on MgO(111) with C, growing h-BN on the exposed metal surface, and precipitating the C back to the h-BN/Ni interface to form graphene. The resulting laterally continuous heterostructure is composed of a top layer of few-layer thick h-BN on an intermediate few-layer thick graphene, lying on top of Ni/MgO(111). Examinations by synchrotron-based grazing incidence diffraction, X-ray photoemission spectroscopy, and UV-Raman spectroscopy reveal that while the h-BN is relaxed, the lattice constant of graphene is significantly reduced, likely due to nitrogen doping. These results illustrate a different pathway for the production of h-BN/graphene heterostructures, and open a new perspective for the large-area preparation of heterosystems combining graphene and other 2D or 3D materials. Despite this inherent structural compatibility, most successful demonstration heterostructures have relied on stacked flakes which have been mechanically exfoliated from bulk samples of the two materials678101112131415161718191727In this manuscript we demonstrate a method for the production of h-BN/graphene heterostructures which allows both materials to form on the surface of the Ni substrate. Recently we have shown that Ni is an effective substrate for the growth of both atomically thin h-BN and graphene via molecular beam epitaxy292 pieces of MgO(111) by electron beam evaporation, and back-coating them with 1 μm of Ti. The Ni/MgO(111) templates were transferred through air to a UHV MBE system (~1 × 10−10 Torr) where they were outgassed at 300 °C for 60 minutes, and sputtered with Ar . The films were then annealed at 850 °C for 20 minutes and cooled to 730 °C for C saturation, which was accomplished by evaporating elemental C from an electron beam heated graphite target. The amount of carbon utilized for the saturation step was chosen to avoid the concomitant formation of graphene at the Ni surface32The main steps involved in the fabrication of the h-BN/graphene heterostacks studied here are illustrated schematically in AFM scans of the h-BN/graphene heterostructure samples show the network of wrinkles typically seen in two-dimensional films grown on metallic substrates  is attributed to a non-resonant second-order Raman scattering in graphene3840−1 range (G1), and a small shoulder centered at 1555 ± 20 cm−1 (G2 - see the following discussion for further details). 1 component, which was taken at the exact same position as the h-BN map  allowed the continuity of the h-BN over the full heterostructure to be verified. h-BN map . As in tults see . In thathing see .1 peak (see −1)4345472 peak is a possible alloying of the h-BN and graphene films close to their interface. In previous work, the observation of a red shift and broadening of the G peak in h-BNC alloys with respect to pure graphene has been reported49et al. recently proposed that the G peak of graphene downshifts (towards the h-BN phonon line), when a homogeneous single phase alloy of h-BNC is formedet al.As already pointed out, our Raman investigations provide evidence for the presence of unintentional modifications in the few-layer graphene film formed at the interface. In fact, for our growth experiments, graphene doping and/or alloying close to the h-BN/graphene interface cannot be ruled out. During the growth of h-BN see , when B peak see  is generet al.545556XPS analyses provided information on the chemical composition and bonding nature of the MBE-grown material. The results obtained from a heterostructure sample exhibit marked differences in the N1s and B1s spectral regions. They are both now composed of a dominant component with binding energies around 398.3 (N1s) and 190.7 eV (B1s). These values are in good agreement with previously published data for h-BN on top of graphene104362sp2 bonded C atoms within graphene10114365665052The existence of graphene in the heterostructure film is confirmed by measuring the C1s spectral region , which iet al.29d states and π states of the 2D layer)686970et al.vs. Frank-van der Merwe (FM) growth)h-BN/Ni), between graphene layers (γGr/Gr), between graphene and h-BN layers (γGr/hBN), and between graphene and Ni (γGr/Ni). Given the similar van der Waals interactions between layers in both graphene and h-BN – and its small absolute magnitude – the strength of the bonding between each material and the Ni surface is likely to dominate. If the energetic driving force to maintain the h-BN/Ni bonding is larger than the graphene/Ni binding energy:Importantly, the transition from two-component to one-component XPS spectra reveals that the heterostructure formation leads to a more homogeneous interaction of the h-BN film with the underlying template, a result of the continuous few-layer graphene coverage formed at the h-BN/Ni interface via C precipitation. It indicates that our synthesis approach results in the growth of graphene underneath the h-BN, even in regions where the first h-BN atomic layer is strongly bound to the Ni surface. This is different from what has recently been reported for the CVD-based synthesis of graphene under h-BN, in which graphene could only be formed under weakly bound h-BN islandsthe graphene will preferentially form thicker multilayer deposits, minimizing the total area of h-BN which is decoupled from the Ni surface upon C intercalation (similar to VW growth mode). However, if the graphene/Ni binding energy is larger:the formation of a continuous graphene film will be more favorable (comparable to FM growth mode). The observation of the continuously detected graphene Raman signal across the sample surface proves that the C precipitated as a continuous graphene film, consistent with the behavior expected from the h-BN/Ni and graphene/Ni interactions−1 results from the (10.0) reflection of the h-BN lattice, and corresponds to an in-plane lattice spacing of 2.502 Å which is very close to its bulk value−1, however, refers to an average real-space in-plane lattice parameter of 2.352 Å, and reveals that the graphene lattice is strongly compressed in about 4% (in comparison to graphite - a = 2.461 Å)4361et alBoth constituent materials of the h-BN/graphene heterostructures could also be detected via GID. This technique has proven to be very powerful in obtaining precise information about the structural properties of single graphene layers2/Si substrate without having its structural properties substantially affected, as verified by Raman spectroscopy. In general, the procedure described here offers an alternative route for the scalable production of h-BN/graphene heterostructures which overcomes many of the drawbacks faced by other methods. It also opens a new pathway for the production of other heterosystems composed of graphene in combination with another material, especially in cases where the formation of a homogeneous graphene underlayer (e.g. as a bottom electrode) might be required.We have demonstrated a novel growth method for the production of layered h-BN/graphene heterostructures based on MBE. The utilization of a Ni film pre-saturated with C as a substrate for h-BN deposition is shown to enable the growth of few-layer graphene at the h-BN/Ni(111) interface by controllably ramping the temperature down to precipitate the C out of the metal. The primary benefit of this technique is that both constituent materials of the heterostructure form in the chemically favorable environment offered by the Ni surface, resulting in continuous graphene and h-BN layers. The top h-BN layer in the stack is strain free, consistent with it being decoupled from the Ni substrate due to the interfacial graphene growth. A careful analysis of the material properties also revealed a significant contraction of the graphene’s crystal lattice due to N-doping. We correlate the N-doping to the possible existence of undissolved C atoms and graphene inclusions present at the Ni surface at the moment when h-BN starts to be grown. Further adjustment of growth parameters shall be implemented in order to mitigate this effect. In terms of thickness homogeneity, it is anticipated that the use of Ni films with smoother surfaces and thus a lower density of step clusters will be beneficial. The h-BN/graphene heterostructure film could be transferred to a SiO2 flow and 350 W power. The h-BN growth duration is 5 hours. More details for the h-BN growth can be found elsewhereFor the pure graphene and h-BN films whose Raman results are depicted in Micro Raman spectroscopy was employed to investigate the structural properties as well as the lateral continuity of the grown material. Excitation wavelengths (λ) in the visible (473 nm) and ultraviolet (244 nm) regions were employed.AFM was used to examine the surface morphology of the resulting heterostructure films.−11 Torr. The chamber is equipped with a monochromatic Al x-ray source providing photons with 1486.6 eV for XPS. The experimental condition was set so that the full width at half maximum (FWHM) of the clean Au 4f7/2 line was 0.65 eV. All spectra were collected at room temperature with a photoelectron takeoff angle of 0°  and without sample pre-annealing.XPS was employed to investigate the chemical composition and binding states of the heterostructures. The samples were transferred in air for the XPS measurements. The measurements were performed using a Scienta ESCA 200 spectrometer with a base pressure of 7.5 × 10Grazing incidence X-ray diffraction (GID) was used to evaluate the crystalline configuration of the surface layers of the heterostructure. These experiments were performed at beamline BM25B (SpLine) at the European Synchrotron Radiation Facility, Grenoble, France. The X-ray energy used was 20 keV, with the beam at a 0.1° angle of incidence to ensure a high degree of surface sensitivity.How to cite this article: Wofford, J. M. et al. A hybrid MBE-based growth method for large-area synthesis of stacked hexagonal boron nitride/graphene heterostructures. Sci. Rep.7, 43644; doi: 10.1038/srep43644 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The Appalachian region of the United States is experiencing a large increase in hepatitis C virus (HCV) infections related to injection drug use (IDU) . Fifth, growth of SSPs and service integration in these states is rapid, and the most recent data on SSPs should be sought through the state or local health department or harm reduction coalition. Finally, these data cannot be used to evaluate quality of service delivery and whether service delivery is adequate to meet the needs of the population.SSPs can be implemented through a variety of models and by a variety of agencies and organizations including those in rural areas. Demand for syringe services is growing rapidly in these three states with underserved populations of persons who inject drugs, representing an opportunity to implement, evaluate, and improve access to evidence-based services known to reduce the considerable morbidity and mortality associated with injection drug use.Opioid overdose, human immunodeficiency virus, and viral hepatitis have increased among persons who inject drugs in the United States. Comprehensive syringe services programs (SSPs) reduce risks associated with injection drug use (IDU); however, access to SSPs has been limited.SSPs have increased dramatically in Kentucky, North Carolina and West Virginia with support from government officials, community advocates, and healthcare providers.Comprehensive SSPs can mitigate the health effects of IDU. With appropriate authorization and support, agencies can successfully implement SSPs in underserved areas.


Mid-infrared spectroscopy is of great importance in many areas and its integration with thin-film technology can economically enrich the functionalities of many existing devices. In this paper we propose a graphene-based ultra-compact spectrometer  that is compatible with complementary metal-oxide-semiconductor (CMOS) processing. The proposed structure uses a monolayer graphene as a mid-infrared surface waveguide, whose optical response is spatially modulated using electric fields to form a Fabry-Pérot cavity. By varying the voltage acting on the cavity, we can control the transmitted wavelength of the spectrometer at room temperature. This design has potential applications in the graphene-silicon-based optoelectronic devices as it offers new possibilities for developing new ultra-compact spectrometers and low-cost hyperspectral imaging sensors in mid-infrared region. Compared to simple photodetection, a spectroscopic study which resolves a spectrum of signal as a function of wavelength adds another dimension into detection and reveals much more information of the object, such as its temperature, motion and composition123567891011121314151819In this paper we report an ultra-compact Fabry-Pérot spectrometer design based on graphene plasmonics and show the response of this newly proposed structure through theoretical and numerical studies. This Fabry-Pérot spectrometer couples propagating beams into a monolayer graphene as surface waves and uses a pair of Bragg reflectors to form an optical cavity of hundreds of nanometers in size. A scanning gate voltage regulates the optical phase shift between the Bragg reflectors to select the transmission wavelength by electrically controlling the optical property of the graphene layer. A high-transmission peak presents when constructive interference occurs between transmitted beams. The proposed structure is ultra-compact in size  and is compatible with those graphene-based photodetectors891011Hgx1−CdxTe in the 1950 s has provided unprecedented degree of freedom to design infrared detectorsCd x, the bandgap of Hgx1−CdxTe increases from a negative value to a positive one, allowing the detection region to cover the whole mid-infrared spectral range. More recently, mid-infrared photodetectors have been made using quantum-well and quantum-dot structures on III-V materials1011101121Detection of mid-infrared light has attracted many research interests since the first observation of infrared radiation from sunlight in the early 1800 scm2V−1s−1 at room temperature, which is among the highest in known materials; graphene-based techniques are compatible with CMOS processing because of graphene’s two dimensional character and its great dynamic tunability, which enables cost-effective integration of electronics and optics onto a single chip; graphene-based detectors also have a low dark current in general and thus low energy consumption, making them an ideal candidate in high-sensitivity and low-noise detection systems. The unique electronic structure and dynamics of graphene can also support prorogation of surface plasmon polaritons (SPPs), which are polariton mode of photon and electron density waves at a conductor and dielectric interface. Propagating light can be efficiently coupled into SPPs, which have many scientific and engineering applications in computing, data storage, communication, nanoelectronic and nanophotonics2627282930313233Graphene, a monolayer of carbon atoms arranged in a honeycomb lattice, offers a promising platform to overcome these obstacles8910111211212489101121Vbias while the bridge is subjected to a gate voltage Vg. The two voltages are electrically isolated using a thin layer of silica (SiO2). When Vbias is applied between graphene (Graphene) and highly-doped silicon (Si) spaced by SiO2, the induced electric field has a periodic modulation due to the grating structure. Since the thickness of SiO2t is typically small in our model compared to the width of trench in the silicon grating, we negelect the edge effect and assume the field is given by the solution in large parallel plate capacitor t(y) indicates thickness is a periodic step function of y and SiO2 layer. Thus, the surface charge density of graphene can be approximated using Gauss’s law ε0 is the vacuum permittivity and e is the elementary charge. The change of surface charge density modifies the electron population in graphene, leading to the modulation of Fermi level EF according to vF is the Fermi velocity of electron, 106 m/s in graphene. The change in Fermi level in turn affects the scattering of electrons, characterized by the relaxation time μ is the carrier mobility in graphene. In the mid-infrared spectral region, the optical properties of a monolayer graphene can be characterized by a complex surface conductivity σG. σG is generally attributed to intraband transitions and interband transitions, which correspond to the first term and the second term of the Kubo formula37The schematic and working principle of our proposed spectrometer are illustrated in 293035kB is the Boltzmann constant, T is the temperature, ħ is the reduced Planck constant and ω is the angular frequency of optical excitation. Note here we have taken the approximation ħω ≫ kBT and EF ≫ kBTσG results in a periodic variation in the effective refractive index neff felt by the propagating SPPs in graphene (see Methods Section for more information). Each boundary causes a partial reflection of the incoming waves due to the mismatch between adjacent effective refractive indices. When the reflected beams add up constructively, high reflection occurs. This happens to a range of wavelengths, known as photonic “stopband”, which is a generic feature when wave propagates in periodically modulating media, as shown in δ introduced by the intermediate bridge, the overall reflection and transmission coefficients for the whole structure are given by Fresnel equations in a three layer systemwhere r is the reflection coefficient defined as the ratio between the incident electric field and the reflected electric field when only one Bragg reflector is present. To get the forms in δ = 2k0neffL = 4πneffL/λ, where k0 is the amplitude of wave vector of wavelength λ in free space, neff is the effective refractive index defined as the real part of the ratio between the wave vector in graphene and k0, L is the physical length of the bridge and the factor 2 comes from the fact that the beam is reflected twice before getting out from the same boundary. As δ → 2mπ , rtot → 0 and ttot → 1 regardless the value of r, implying total transmission T ≡ |ttot|2 = 1, as indicated in where m = 0). By taking two Bragg reflectors as highly reflecting mirrors and writing δ as a pure real number, we have neglected the material losses along propagation in order to emphasize the idea.The selective transmission of light can be resolved across the stopband, which essentially works a spectrometerThe proposed idea is investigated using a commercial software package implementing finite element method–COMSOL Multiphysics. A nominal thickness of graphene is needed to simulate its optical response. This is because the general harmonic response of graphene at the macroscopic scale is described by Maxwell’s equationsεb is the ion background contribution into the relative permittivity of graphene and tG. By comparing the surface current density α = σGE and the bulk current density σG/tG. Definewhere εb and tG are typically chosen to be 2.5 and 1 nm, respectively254041μm to 8 μm), i.e. εAir = 1, εSi = 11.7. Like many others254243μm because light absorption of SiO2 is strong when the wavelength exceeds 8 μmso SiO2t, as shown in t is critical for two reasons: on one hand, t should be small compared to w1 and w2 to give sufficient spatial modulation of applied electric field; on the other hand, t should be large to avoid electric breakdown of SiO2. The lengths of the bridge and the trenches are also important in order to achieve noticeable shift in spectrum by moderate voltage variations. So the values of voltages (including Vbias and Vg), widths (including width of trench in Reflector I w1 and width of trench in Reflector II w2), periods (including period of grating in Reflector I p1 and period of grating in Reflector II p2) and thicknesses  are dependent on each other and should be optimized globally.In order to calculate the transmission of the structure, we use a same boundary mode to excite SPPs in graphene for different wavelengths. This is achieved by inserting a graphene waveguide embedded in air into the beam path before the beam passes through the interested structure. The advantage of doing this is twofold: (1) COMSOL can accurately find the boundary mode near the given effective refractive index so the model is robust when changing excitation wavelength as well as geometrical parameters; (2) the single mode excitation gives good definition of transmission that makes the comparison between different wavelengths meaningful. An important parameter in the simulation is the thickness of Vg as other parameters are set to be constants Vbias = 3.2 V, w1 = w2 = 40 nm, L = 20 nm, p1 = p2 = 80 nm, t1 = t2 = 10 nm and t = 5 nm. The numbers of periods in two reflectors are both chosen to be 3. In this case, the distribution of applied electric field is verified to be close to a periodic step function. As expected, a transmission line is present within a stopband formed by two identical Bragg reflectors. The sidelobes outside the stopband drawn as dotted lines are common in Bragg reflectors, resulting from incomplete constructive interference of transmitted beams. Another prominent feature is that the transmission peak gets red shifted as the gate voltage decreases. This is because effective refractive index neff increases as Vg decreases (see Methods Section). Recalling from neff, so the transmission peak is supposed to have a red-shift. To further confirm that the working principle of this spectrometer is as proposed, we plot the distribution of the normalized amplitude square of electric field in λt, as indicated in the fourth panel of λt, the phase shift introduced by the bridge part is 2m0π, where m0 is a certain integer, the transmitted beams interfere constructively, leading to complete passing-through of the incident beam. And the electric field in the bridge region, i.e., the area between two highly reflective mirrors, gets greatly enhanced, as shown by the normalized quantity |E|2/|E0|2 in E0|2 is the value taken at the leftmost edge of the plotted structure. Note the outline of silicon grating structure in thin solid line in λr, also indicated in E|2/|E0|2 along a cutline for two excitation wavelengths λt and λr are shown in SiO2 side. The normalized value |E|2/|E0|2 in the bridge area at λt is roughly 55 times stronger than that at λr. The electric field inside the bridge region has multiple peaks, this is because L is several times larger than the wavelength of progagating SPPs. In principle, this value can be smaller. So the several micrometer long structure of Fabry-Pérot spectrometer can be further shrinked, making it even more compact in the realm of mid-infrared devices. In both cases in μm to around 7.5 μm only by changing the widths of trenches in two Bragg reflectors. As shown in Vbias offers another knob to dynamically tune the position of stopband. Similar to the trend observed in Vbias gives rise to the red-shift of stopband. This can be understood by the same reasoning: the reduced voltage leads to increased effective refractive index, which in turn requires longer wavelength to meet the condition of Bragg reflection.Another advantage of the proposed structure is its multiple degrees of freedom to engineer the propagation stopband as well as the transmission line. This can be achieved by two ways: one is to change the geometry of the structure and another is to modify the effective rafractive index of graphene, as will be discussed in more details in wave impendance analysis in Methods. We have demonstrated a new concept of Fabry-Pérot spectrometer based on ideal graphene with low losses. By dynamically tuning the gate voltage applied to the bridge area of graphene, the wavelength of the transmitted line can be continuously changed. More tunability of the structure can be achieved by adjusting the geometric parameters and by dynamically changing the biased voltage applied to graphene. Furthermore, the proposed structure inherits many advantages of graphene-based devices: small footprint, fast speed, broad bandwidth and compatibility with CMOS technologies.cm2V−1s−1 which is a moderate value for graphene on SiO2cm2V−1s−1, which is about the best value reported in a suspended graphene monolayercm2V−1s−1 have been reported by placing graphene on hexagonal boron nitride42When material losses are considered, as shown in SiO2 with other low loss materials4950We expect the proposed idea of graphene-based Fabry-Pérot spectrometer can be generalized to guide the design of structures using other materials. One example is to push the upperbound of the operation bandwidth into an even longer wavelength by replacing the limiting material SiO2 we can approximate the structure as a multilayer model, where a thin layer of SiO2 of thickness SiO2. In this waveguide model, graphene is modeled as an infinitely thin surface layer characterized by its complex conductivity σG, whose imaginary part is typically a positive value in the mid-infrared spectral region of our interest, taking the form σG = σG,r + iσG,i with σG,i > 0. In COMSOL, the definition of permittivity is in a different form σG = σG,r − iσG,i, where σG,i > 0. In this paper, we use the first sign convention unless otherwise stated. One consequence of this property is that the graphene layer can support transverse magnetic (TM) modes in the Air − Graphene − SiO2 − Si multilayer structure41Hy; Ez, Ex] if we set the propagation direction of the surface wave to be y direction and plane normal direction along x axis, as indicated in x = 0 plane, the field components have the formThe analytical solution of SPPs in simple cases can provide us good insights into their properties and help us to understand their response in complex structures. By neglecting the variations in thickness of γi  is defined as μi = 1). Here, we have assigned the subscripts Air, SiO2, Si as 1, 2, 3, respectively. εi and μi are relative permittivity and relative permeability of the corresponding material, respectively. In source-free time-harmonic EM fields, electric field E is related to magnetic field bywhere eiωt−. Thus,where we have assumed the time-harmonic term takes the form x = 0 and x = −t2, leading toTo solve the dispersion relationship of propagating surface waves along graphene, we need the boundary conditions at both αz and local electric field αz = σGEz. Substituting In deriving Ha2 and Hb2 givesCombining SiO2, leading to the increase in neff. Note when t2 → ∞, C1,2 → −1, which is equivalent to53η0 is the intrinsic impedance of free space. If ε1 = ε2 = ε, where neff is essentially the real part of effectively refractive index of air-graphene-air surface waveguide, indicated by nm), which is not the actual physical thickness of graphene (about 0.33 nm). It is shown that as long as the mesh gird is sufficiently small, the difference between results obtained for 1 nm and 0.33 nm is negligible4144The numerical simulations using the proposed structures are conducted using a commercial finite element method software package, COMSOL Multiphysics. To ensure the same excitation for SPPs in graphene, we set up port boundary conditions for the left and right boundaries. To be specific, we utilize the numeric type of port and run boundary mode analysis first to find boundary mode near From ω = 3 × 1014rad/s and τ = 6 × 10−13 s. This indicates that the propagation length of SPPs in graphene is much longer than their wavelength, making it reasonable to use wave impedance to analyze the reflection and transmission of SPPs in the structure. In addition, Z = ET/HT at a particular point. The longitudinal components of electric field get transmitted according to the continuity of displacement field. The propagation of wave impedance from Z(l) to Z(0) in one medium is given by57In the infrared region of our interest, the optical excitation of graphene is mostly contributed by intraband transitions3855η is the intrinsic impedance of the medium, defined as δ = k0neffl ∈ [0, 2π) matters in the analysis of impedance propagation. Two particular cases are when the propagation distances  equal to half and quarter of the wavelengthwhere H) and low (L) effective refractive indices, taking Reflector II shown in λFor a Bragg reflector made of alternating layers of high . Similarly, for Reflector Iwhere When the optical lengthη0), continuity of displacement field ganrantees the perfect transmission of longitudinal components. So the total transmission is 1 for the particular wavelength λ. Note from How to cite this article: Wang, X. et al. A graphene-based Fabry-Pérot spectrometer in mid-infrared region.Sci. Rep.6, 32616; doi: 10.1038/srep32616 (2016).


The promise of graphene and its derivatives as next generation sensors for real-time detection of toxic heavy metals (HM) requires a clear understanding of behavior of these metals on the graphene surface and response of the graphene to adsorption events. Our calculations herein were focused on the investigation of the interaction between three HMs, namely Cd, Hg and Pb, with graphene quantum dots (GQDs). We determine binding energies and heights of both neutral and charged HM ions on these GQDs. The results show that the adsorption energy of donor-like physisorbed neutral Pb atoms is larger than that of either Cd or Hg. In contrast to the donor-like behavior of elemental HMs, the chemisorbed charged HM species act as typical acceptors. The energy barriers to migration of the neutral adatoms on GQDs are also estimated. In addition, we show how the substitution of a carbon atom by a HM adatom changes the geometric structure of GQDs and hence their electronic and vibrational properties. UV-visible absorption spectra of HM-adsorbed GQDs vary with the size and shape of the GQD. Based on our results, we suggest a route towards the development of a graphene-based sensing platform for the optical detection of toxic HMs. Three main attributes of graphene make it attractive for such applications: (i) a surface area of 2600 m2·g−1, thereby providing a large platform for surface chemistry2, (ii) its ability to act as either a reducing agent (electron donor) or an oxidizing agent (electron acceptor)3 and (iii) low Johnson noise, even at low charge densities and at room temperature, as a result of its high conductivity and lack of a band gap4. Combined, these properties mean that graphene-based sensors are capable of detecting changes in the local charge concentration of less than single electron4, and thus the presence of small concentrations of surface functional groups and adsorbates.As a two-dimensional material with unique electronic properties, graphene is anticipated to be a key element in novel sensing platforms5, which results in a variable photoluminescence emission6. The adsorption of certain ions or molecules can modify the size of the band gap and thereby cause a photoluminescence quenching or enhancement effect. Such a change in the optical properties can be effectively detected and so harnessed to create sensors.Further functionality can be achieved by considering zero-dimensional graphene quantum dots (GQDs). Due to the quantum confinement effect, GQDs exhibit a size-dependent band gap7, such as Cd, Hg and Pb. Beyond a certain limit, exposure to these elements can cause severe detrimental effects to human health and to the environment in general8. Cd is classified as a human carcinogen, resulting in lung cancer as well as various renal problems. Hg exposure is a major public health concern, causing widespread problems to the nervous, digestive and immune systems as well as to the development a fetus. Although most exposure to Hg occurs by eating contaminated fish and shellfish, elemental mercury vapors can also be inhaled by workers in industrial processes. Lead poisoning affects the central nervous system, and is particularly harmful to children. Its widespread use in paint and water pipes mean that is significant public health concern in many parts of the world. According to the World Health Organization reports9, the maximal permissible limits of Cd, Hg and Pb in drinking water are estimated to be 3, 1 and 10 μg/L, respectively.Driven by environmental and health concerns, there is currently a strong impetus towards the development of fast, inexpensive commercial sensors for the detection of hazardous heavy metals10. This is due to their ability to form salts with different solubilities13. For example, Hg1+ (mercurous) and Hg2+ (mercuric) valence states mercury form different inorganic salts. Hg2+ - containing salts are more soluble and hence more dangerous than Hg1+-containing salts. Thus, the ability to detect very low concentrations of these non-biocompatible heavy metals in different oxidation states is vital.The relative toxicity of these ions also depends strongly on their oxidation state27. The fundamental operational principle of these sensors is the chemical interaction between the adsorbate  and the graphene electrode, which strongly modifies the measurable electrical properties of the graphene-adsorbate system, such as the electric current or potential. The resulting electrochemical signal can then be used to quantitatively determine the concentration of toxic heavy metals on the surface. Several experimental investigations have considered how charged ions of Cd, Hg and Pb interact with carbon-based materials, such as functionalized graphene, graphene oxide and carbon nanotubes41.To date, a number of graphene-based sensors for detection of heavy metals have been proposedGQDs are, to date, not as well investigated for sensing applications as those based on functionalized graphene. Here, we explore the adsorption of neutral, monovalent and divalent ions of Cd, Hg and Pb on both graphene quantum dots and infinite graphene, in order to determine their suitability as sensors for heavy metal detection.54H18 graphene quantum dots. We then discuss the electronic properties of the interacting systems. Next, we consider the nature of the mobility and clustering of heavy metal adatoms on graphene quantum dots and the effects of substitutional impurities on the structural and physical properties of GQDs. Finally, we show that the interaction between the HM atoms and the GQD depends on the size and edge of the latter, and determine how the latter will influence the UV-visible adsorption spectra. Our results confirm a possibility to develop realistic optical sensing applications for HMs detection.The current section is organized as follows. First, we describe the geometric and energetic features associated with the adsorption of heavy metal atoms and ions on C23, here we explore four adsorption sites, namely the hollow site (H), where the adatom sits in the center of the hexagonal ring, the bridge site (B), where atoms are placed at the center of a C-C bond, the top site (T), where the adatom is adsorbed directly on top of a carbon atom and a defect site (D), where the adatom is adsorbed on top of a carbon vacancy in the GQD. These configurations are illustrated in Fig. As the adsorption site of the adatom is known to influence the electronic properties54H18 GQD. Such as large GQD was chosen to minimize the effect on the edge on the adsorption location, and thereby to approximate infinite graphene. The results are shown in Fig. We determine the binding site and binding height of the adatoms by plotting the total energy as a function of their distance from a zigzag-edged C42.We find that the binding height and binding position depend on the adatoms. The most energetically-favorable binding site for the adsorption of Cd° and Hg° on the GQD is the hollow site, while that of the Pb° is the bridge site. These results are summarized in Table 43. The calculated adsorption energies of neutral Cd°, Hg° and Pb° indicate the physisorption nature of the interaction between neutral HMs and hydrogen-terminated GQDs.The smallest binding energies are found for Cd° and Hg° adatoms, at 0.170 eV and 0.161 eV, respectively. The binding energy for Pb° adatoms is found to be larger, at 0.199 eV. Adatoms are generally considered to be physisorbed on a surface when their binding energy is lower than 0.5 eV per adsorbed species44. A small charge transfer of approximately 0.09e occurs from the Cd° and Hg° adatoms to C54H18 quantum dot. In contrast, the Pb° atom donates approximately 0.24e to the carbon atoms. The difference in the charge transfer between GQD and the Cd°, Hg° and Pb° adatoms can be understood in terms of the ionization energy of the metal45. The ionization potential of Pb° is approximately equal to 7.41 eV47, whereas Cd and Hg have larger ionization energies of 8.99 eV and 10.43 eV47, respectively. Thus, the Pb° transfers electrons more easily to the GQD than Cd° and Hg° adatoms.The charge transfer to or from the adatom was calculated using Mulliken charge analysis1+, Cd;2+ Hg1+, Hg;2+ and Pb1+, Pb2+) adsorbed on the C54H18 as a function of the binding height. Unlike the weakly-bound neutral adatoms, the charged ions are now chemisorbed on the GQD. In all cases, the binding position changes for the charged ions compared to their neutral counterparts. Cd1+ and Cd2+ are now preferentially adsorbed on the T and B site, respectively. The Hg ions adsorb on the T-site, compared to the H-site for the neutral adatom. While the monovalent Pb adatom remains on the B-site, as for the neutral adatom, it switches to the H-site for the divalent adatom. As expected, the positively charged ions reverse the direction of charge transfer compared to the charge-donating neutral adatoms. This amounts to approximately 0.9e− for Cd1+, 1.8e− for Cd2+, 0.9e− for Hg1+, 2e− for Hg2+, 0.2e− for Pb1+ and 0.9e− for Pb2+, as shown in Table Figure 1+ > Cd1+ > Pb1+ and Hg2+ > Cd2+ > Pb2+  and a significantly larger energy difference between it and the hollow site (119 meV). The binding height of the Cd adatom is found to be 3.46 Å. This is slightly larger than that found using the GQD approach (3.441 Å). A similar slight increase in binding height occurs for the case of a Hg (Pb) adatom, from 3.322 Å (2.904 Å) to 3.33 Å (2.96 Å). These differences can be due to the influence of the finite size of the GQDs, their hydrogened edgesB when adsorbed on the graphene surface. The non-magnetic solution is 0.385 eV higher in energy.The supercell calculations give increased binding energies for Cd and Hg compared to the calculations for the GQD: 0.218 eV and 0.246 eV for Cd and Hg, respectively, compared to 0.170 eV and 0.161 eV. The binding energy of Pb is also increased to 0.372 eV. Due to the unpaired electrons of the Pb adatom, it exhibits a magnetic moment of 1.78 µn-doping from the adsorbant.In agreement with the GQD calculations, the supercell calculations also find minimal charge transfer between the physisorbed adatoms and the graphene sheet. We find Cd (Hg) transfers 0.02e (0.05e) to the carbon atoms, while the Pb atom transfers 0.27e to the graphene sheet. The effects of this charge transfer can be seen in the shift of the graphene Dirac cone towards higher binding energies after Pb adsorption. This is shown in Fig. 54H18 GQDs after the adsorption of neutral heavy metal adatoms. It is clear that the Cd° and Hg° atoms do not contribute to the formation of both LUMO and HOMO , which also suggests a very weak interaction between the two. In contrast, Pb@C54H18 exhibits qualitatively different behavior , which still retains the attributes of the bigger one. As in the case of the C54H18, the chosen path for total energy calculations on C30H16 GQD passes over all three high-symmetry positions, namely the T, H and B sites  adatoms and a C16H10 GQD. The choice of the small and simplest C16H10 QQD is justified because we are mainly interested in the search of the critical distance when the metal-metal attraction becomes dominating. At this nano-scale, this distance cannot exceed the HM isomer bond lengths in the gas-phase and thus even the simplest GQD can be representative of GQD family to understand the specificity of the metal clustering on the sp2 bonded conjugated hexagonal rings. The effect of the GQD size on the metal-metal interaction is expected to be minor.In the previous sections, we showed how heavy metal adatoms adsorb and diffuse on C2 and Hg2 dimers are in the range of 3.78–4.33 Å55 and 3.605–3.69 Å58, respectively. The bond length of a stable Pb2 dimer is 2.93 Å59. Our calculations find the equilibrium distances for isolated Cd2, Hg2 and Pb2 dimers to be 4.131 Å, 3.968 Å and 2.939 Å, respectively. Apart from the equilibrium distance for Hg, these values are in good agreement with the experimental parameters. To determine the tendency of two heavy metal adatoms adsorbed on graphene to cluster, we consider two initial geometrical configurations where the adatoms have been separated by different distances. After optimization, we find that two adatoms, initially placed far from each other, tend to bind to graphene quantum dot, rather than each other. On the other hand, two adatoms placed close to each other form dimer structures, with equilibrium distances of 3.691 Å, 3.759 Å and 2.917 Å for Cd2, Hg2 and Pb2, respectively. For the case of Cd and Hg, the total energy of the system is lower when the two adatoms are far from each other, by 24 meV per atom. This suggests that despite a low diffusion barrier, these adatoms should not tend to form islands on graphene. On the other hand, the formation of Pb dimers on the graphene surface is highly favourable, with an energy gain of 1.046 eV per Pb atom. The relaxed structures are shown in Fig. First, we explore the formation of heavy metal dimers, i.e. a cluster consisting of two adatoms. There are two competing mechanisms here: (1) the formation of bonds between the metal atoms and graphene and (2) the formation of bonds between the two metal atoms. The relative strength of these interactions will be dependent on the distance between the adatoms on the surface of graphene. The experimental bond lengths for isolated Cd16H10 GQDs vary with the number of atoms in the cluster. The binding energy of an isolated neutral Cd and Hg atoms on the graphene quantum dot is 175 meV and 167 meV, respectively, but decreases upon increasing the number of adsorbed atoms (from 1 atom to 4 atoms) to 102 meV and 76 meV. This decrease is caused by the competition between metal-surface and metal-metal interactions. For the case of shorter metal-metal distances the binding energy becomes lower with an increasing number of adatoms. Increasing the number of Pb atoms adsorbed on the graphene surface results in an even more dramatic decrease in the binding energy. This is driven by the energetic preference of Pb atoms to cluster when adsorbed on graphene.Figure The HOMO-LUMO gap of the combined system is also dependent on the number of adatoms. Figure 24H12 quantum dots. A carbon atom belonging to the central hexagonal ring is replaced with a heavy metal atom. The relaxed structures after geometry optimization are shown in Fig. sp2 plane. The Cd-C, Hg-C and Pb-C bonds are longer than the C-C bond at 2.22 Å, 2.28 Å and 2.26 Å, respectively. As a result, the graphene quantum dot experiences a considerable distortion. We define the out-of-plane distortion of the GQD as:Zmax is the maximum value of the out-of-plane coordinates of the carbon atoms and Zave is the average value. We find that Δh due to the Cd doping (0.912 Å) is much lower than that due to Hg and Pb doping . This difference may be understood in terms of electron affinity: Hg has the lowest value of the electron affinity among the heavy metals considered here. A lower value of electron affinity results in lower values of charge transfer and binding energy, implying that the mercury atom does not accept electrons as easily. For this reason the C-Hg bond length is longer than the C-Cd and C-Pb bond lengths and the remaining carbon atoms are reorganized into a distorted graphene quantum dot. For the case of a substituting Pb atom, the graphene quantum dot is also distorted, but now Pb is bonded to three carbon atoms, due to its high electron affinity.Substitutional and vacancy defects in graphene can occur during the growth stage and may significantly modify the typical electronic and vibrational fingerprints of graphene. Heavy metal atoms may therefore also adsorb on a vacancy site as well as on a defect-free carbon surface and it is important to distinguish these two events. In this section, we investigate how the substitutional replacement of a carbon atom in the GQD with a heavy metal atom affects the local structural disorder and changes the electronic and Raman spectra of CThe charge on the substituting heavy metal atoms is changed significantly compared to the charge on the isolated atoms. The charges on Cd, Hg and Pb atoms are calculated to be + 0.843e, + 0.650e and + 0.544e, respectively.23H12 GQD  is observed for Cd-doped and Hg-doped GQDs due to the strong localization of the LUMO orbitals on the Cd and Hg atoms. The HOMO-LUMO gap of Pb-C23H12 is 2.01 eV due to the weak contribution of the Pb to the total DOS.Features of electron density distribution that depend on the interaction of the coronene with heavy metals are correlated with the values of the HOMO-LUMO gap. The size of the HOMO-LUMO gap follows the sequence C−1 to 1700 cm−1, as it includes the characteristic fingerprint of in-plane C-C movements, that is, the G-mode. It is generally accepted that the relationship between the D and G modes can be regarded as an effective parameter for quantifying disorder in graphene, which is induced by point defects, external doping and edges 60. We do not discuss here the modes up to 1200 cm−1  and hydrogen-related high-frequency vibrations above 1700 cm−1. In addition, it is important to note that the local phonon modes induced by substitution manifest themselves only in low-frequency region of the Raman spectra due to very large value of oscillator mass and the so-called isotopic effect. Due to the D6H symmetry and the structural properties of C24H12 cluster, the irreducible representation and selection rules imply that there are 24 Raman active modes: 6A1g, 6E1g, and 12E2g61. As can be seen in Fig. −1 and 1652 cm−1. These features can be assigned to A1g and E2g phonons, in good agreement with ref. E2g zone center phonon is allowed by the selection rules and therefore only the G mode is usually observed in the Raman spectra of the high-quality graphene. The activation of the Ag1 zone-boundary mode  is associated with the relaxation of the k = 0 Raman selection rule due to disorder in the graphene sheet63. In the case of the graphene quantum dot, the edges are responsible for the presence of this zone-boundary phonon mode. Furthermore, we can see that the intensity of the D band is higher than that of the G band, indicating the important role of the hydrogen termination in the Raman scattering processes in small GQDs.Finally, we discuss the vibrational properties of the defective GQDs. The substitution of a carbon atom with a heavy metal atom will significantly influence the out-of-plane and in-plane movement of the host GQD, leading to a modification of the intensities and frequencies of the allowed phonon modes. The Raman scattering spectra were calculated for pristine coronene and for the systems with heavy metal adsorbants. Due to the hydrogen-terminated edges of the GQDs, there will not only carbon-related phonon modes, but also mixed modes corresponding to C-H stretching and vibrations involving only hydrogen atoms. We consider the Raman spectra located in the frequency region from 1200 cmG mode for defective coronene is reduced compared to pristine coronene. A set of new Raman peaks corresponding to in-plane carbon vibrations can be observed due to the presence of Cd, Hg and Pb  and C54H18 (with zigzag edges) GQDs, Fig. u symmetry). Compared with the armchair GQDs, the absorption peak of zigzag GQDs exhibits a red-shift of 45 nm, while the oscillator strength of dominating transitions are similar in both cases. The adsorption spectra of the armchair GQDs is mainly dominated by two peaks at 371 and 369 nm. The former is associated with H-1- > L + 1 (75%) and HOMO- > LUMO (16%) electronic transitions and the latter is originated from H-1- > LUMO (46%) and HOMO- > L + 1 (48%) transitions. The same electronic transitions contribute to the adsorption spectra of the zigzag-edged GQDs.For this aim, we have explored the edge and size effects by optimizing the structures of zigzag-edged and armchair-edged GQDs with different diameters before and after applying heavy metals (Tables Ag symmetry), namely n-π* transitions can occur. These electronic transitions can be regarded as unique electronic signatures serving for detection of the heavy metals.When adsorbed by nanographene surface, heavy metal species introduce new local states  into the electronic structure of graphene nanomaterials, thereby modifying the total density of states as well as optical properties, which are strongly governed by electronic structure. As a result, a new kind of the electronic transitions  and graphene, the HOMO-LUMO gap of zigzag and armchair Cd@GQDs and Hg@GQDs follows the size dependence of the energy gap of their pristine counterparts. Taking these features into account, it is reasonable to assume that UV-vis adsorption spectra of the GQDs after complexation with Cd and Hg are not strongly affected by incoming adsorbates and no significant red-shift is expected.The adsorption of neutral Cd and Hg adatoms is slightly dependent on the size of the GQD and the type of the edge termination. Analyzing the geometrical configuration of the neutral Cd and Hg at the surface of the varying sized zigzag and armchair edged GQDs, it was revealed that in most cases the hollow site is the most favorable position for Cd and Hg adsorption. Our calculations show that the binding energy increases with increasing diameter of GQD, reaching the saturation level for larger quantum dots  interaction of heavy metals with sensitive GQDs and (ii) subsequent measurements of the optical response of graphene-based solutions to exposure of the heavy metal species. Since the optical properties of graphene nanomaterials are significantly affected by incoming adsorbates, it is expected that each of HMs will lead to unique optical fingerprint. In fact, collecting the information about the peak dispositions, peak quenching and novel electronic singularities gives us a possibility of a deeper understand of the heavy metals effect on the properties of the graphene-related materials. Thus, a comprehensive analysis of the optical response of the graphene-based sensing platform to HMs exposure can lead to an effective tool for a fast detection of the heavy metals in aqueous solutions and even in biological liquids.sp2 surface. We showed that the Raman scattering spectra of the GQDs provides a unique fingerprint of each interacting system. In view of the high sensitivity of the vibrational properties of graphene quantum dots to adsorbates, graphene quantum dots-based Raman sensors can be used to detect toxic heavy metals.DFT calculations were employed to investigate the interaction between neutral and charged heavy metal atoms of cadmium, lead and mercury and different zigzag and armchair-edged GQDs. The binding energy, the charge transfer and the equilibrium distances between the adsorbates and graphene quantum dots are found to be strongly dependent on the adsorption site, the charge state of the metal adsorbate, the size/edge geometry of the GQDs and the concentration of heavy metals on graphene quantum dot. We find that the neutral atom of Pb tends to bind more strongly to GQD than either Cd or Hg, acting an electron donor. This can be explained by differences in their ionization energies. We show that the most favorable adsorption site for Cd and Hg is the high-symmetry hollow site, whereas Pb binds preferentially on the bridge site. The adsorption properties of the charged ions differ significantly from those of the neutral atoms, due to the chemisorption nature of the interaction. The charged heavy metal species were found to act as electron acceptors in the GQDs–HMs systems. A general increase of the binding energy of the neutral and charged heavy metals species, and the corresponding charge transfer increase, with the GQD size was found. The adsorption ability of the armchair GQDs to HMs is stronger compared to zigzag GQDs. The energetic barriers for Cd or Hg motion across the GQDs are very small. The Pb atom migrates mostly along the C-C bond, avoiding the energetically unfavorable hollow site. The binding energy of heavy metals decreases with an increasing a number of metal adatoms. Such behavior is driven by the energy gain originating from the competition between metal-metal interaction and metal-GQD complexation. We find that unlike the formation of a Pb dimer on graphene quantum dots, the formation of Hg and Cd dimers is not energetically favorable. We also show that the substitution of a carbon atom in the GQD by the heavy metal atoms causes a significant distortion of the nanographene plane, accompanied by the protrusion of the metal atoms from the 0 (Hg0)-GQDs interaction. The adsorption spectra of the GQDs after complexation with heavy metal ions are found to be highly sensitive to the size and edge configuration of the GQD. In this case, the doping effect induces a significant red-shift of the absorption spectra of both zigzag and armchair GQDs as well as an appearance of new adsorption peaks in long wavelengths region. Our results show that the effect of the chemisorbed HM ions on UV-vis absorption spectra of GQDs is very complex and the changes in the nature of the excited electronic transitions correlate directly with charge transfer from electron-donating GQDs to acceptor-like heavy metal ions. The obtained results provide insightful nano-engineering approach based on size and edge geometry control toward development of advanced optical graphene-based sensors for detection of the heavy metals in different oxidative states.Finally, using the results of TDDFT calculations we explained in detail the nature of excited states in the zigzag and armchair GQDs before and after complexation with HMs. Based on the results, we discussed how UV-vis adsorption spectra can be used for development of realistic optical sensors. Our study revealed that due to the weak interaction of the neutral Cd and Hg adatoms with GQDs, the adsorption spectroscopy is not appropriate method for detection of these two. On the other hand, a probing of the GQDs with substitutional defects by Raman spectroscopy can provide more valuable information about Cd64, to investigate the interaction of GQDs with three different heavy metal atoms, namely cadmium, mercury and lead. Geometry optimization of the interacting systems was done at the Becke3LYP level of DFT with a 6–31 G basis set for carbon and a basis set developed by the Stuttgart-Dresden-Bonn group for the heavy metal atoms65, using the default convergence criteria. We use C16H10, C24H12, C30H16, and C54H18 as representatives of GQDs.In this paper, DFT calculations were performed using the Gaussian 09 package66 method as implemented in the Vienna ab initio simulation package68 (vasp-5.3), which utilizes periodic boundary conditions. In this case, infinite graphene was described using a 4 × 4 supercell, i.e., with 32 carbon atoms. The Perdew-Burke-Ernzerhof (PBE)69 generalized gradient approximation (GGA) was employed. The plane wave basis set was converged using an 800 eV energy cutoff. Structural relaxations were carried out using a 5 × 5 × 1 k-point Monkhorst-Pack mesh70 to sample the Brillouin zone. A 9 × 9 × 1 mesh was then used to determine the total energies. The DFT-D3 method of Grimme was used to describe van der Waals interactions71. A vacuum layer of at least 20 Å was included in the direction normal to the surface to ensure no spurious interactions between repeating slabs. The positions of the carbon atoms, as well as the adatom, were optimized until all residual forces were less than 0.01 eV·Å−1.We also determined how these heavy metal adsorbants interact with extended graphene. This was done using the PAWn is the total number of interacting heavy metal atoms or ions. In a case of the interaction between graphene quantum dots and metal clusters  we cannot neglect the contribution of the metal-metal attraction to binding energy of the heavy metal and thus the later can be evaluated from the energy difference between the total potential energy of the interacting “GQD-metal cluster” system, The binding energy of a heavy metal adatom to a graphene quantum dot is defined as:72.It should be mentioned that the binding energies have corrected for the basis set superposition error (BSSE) by means of counterpoise methodThe excited electron states – transitions between occupied and unoccupied states – of the zigzag and armchair varying sized GQDs before and after applying the heavy metals species were investigated by means of the time-dependent density functional theory (TD-DFT) approach. 100 single excited states were involved to calculate the ultraviolet-visible adsorption spectra at the TDDFT/B3LYP level of theory using 6–31 basis set, implemented in the GAUSSIAN 98 program. The oscillator strengths for each transitions is proportional to the intensities of absorption peaks. The shift and quenching the adsorption peaks is due to the charge transfer between graphene quantum dots and heavy metal species.Supplementary Materials