We tested specificity of [111In]PD-L1-mAb and NIR-PD-L1-mAb in cell lines and in tumors with varying levels of PD-L1 expression. We performed SPECT/CT imaging, biodistribution and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-PDL1) and in controls (CHO). Results were confirmed in triple negative breast cancer (TNBC) (MDAMB231 and SUM149) and non-small cell lung cancer (NSCLC) (H2444 and H1155) xenografts with varying levels of PD-L1 expression. There was specific binding of [111In]PD-L1-mAb and NIR-PD-L1-mAb to tumor cells in vitro, correlating with PD-L1 expression levels. In mice bearing subcutaneous and orthotopic tumors, there was specific and persistent high accumulation of signal intensity in PD-L1 positive tumors  but not in controls. These results demonstrate that [111In]PD-L1-mAb and NIR-PD-L1-mAb can detect graded levels of PD-L1 expression in human tumor xenografts in vivo. As a humanized antibody, these findings suggest clinical translation of radiolabeled versions of MPDL3280A for imaging. Specificity of NIR-PD-L1-mAb indicates the potential for optical imaging of PD-L1 expression in tumors in relevant pre-clinical as well as clinical settings.Antibodies targeting the PD-1/PD-L1 immune checkpoint lead to tumor regression and improved survival in several cancers. PD-L1 expression in tumors may be predictive of response to checkpoint blockade therapy. Because tissue samples might not always be available to guide therapy, we developed and evaluated a humanized antibody for non-invasive imaging of PD-L1 expression in tumors. Radiolabeled [ Tumor cells avoid the immune response by exploiting immune checkpoints through expression of immunosuppressive molecules, recruitment of suppressive immune cell populations and secretion of soluble suppressive factors . Immune PD-L1, a 290 amino acid type I transmembrane glycoprotein, is the primary ligand of programmed death-1 (PD-1). Binding of PD-L1 to PD-1 suppresses T-cell immune activity and restricts tumor cell killing . PD-L1 eAntibodies targeting PD-L1  have demonstrated anti-tumor activity in diverse tumor types, including renal cell carcinoma (RCC) , advanceKd) of 0.43 nM and 0.13 nM, respectively PD-L1-mAb was produced with a specific activity of 4.8±0.65 μCi/μg with > 98% and ∼75% radiochemical purity and immunoreactive fraction, respectively , H2444 (95%), MDAMB231 (27%), SUM149 (0.1%), H1155 (0%) and CHO cells (0%). Furthermore, PD-L1 expression levels, as shown by mean fluorescence intensity (MFI), also varied between cell types. The graded expression profile was: CHO-PDL1 > H2444 > MDAMB231 > SUM149 > H1155 > CHO Figure . The uptO Figure .111In]PD-L1-mAb binding to CHO-PDL1 cells, by addition of 10-fold molar equivalent excess of unlabeled antibody, reduced radioactivity uptake by 75%, indicating that [111In]PD-L1 mAb binding is specific. A similar uptake profile was observed with NIR-PD-L1-mAb  was in spleen (23.5±8.2), followed by CHO-PDL1 tumor (4.7±0.7) and liver (8.2±4.5)  biodistribution in the CHO-PDL1 tumor model at 120 h  in CHO-PDL1 tumor radioactivity uptake  in vivo specificity and dose dependent distribution of the anti-PD-L1 antibody.These data demonstrate: (i) [111In]PD-L1-mAb showed high radioactivity accumulation in the MDAMB231 tumors compared to SUM149 , Macrocyclics Inc.  and Nordion , respectively. All cell culture related reagents were purchased from Invitrogen, unless otherwise specified.The published sequence for anti-hPD-L1 monoclonal antibody  was used to guide the synthesis, performed by Evitria  , 18. Allhigh), NCI-H1155 , MDAMB231  and CHO-K1, , were purchased from the American Type Culture Collection (ATCC) and passaged for fewer than 3 months after which new cultures were initiated from vials of frozen cells. The SUM149  cell line was kindly provided by Dr. Stephen P. Ethier, Medical University of South Carolina, and authenticated by STR profiling at the Johns Hopkins genetic resources facility. SUM149 cells were maintained in Ham's F-12 medium with 5% FBS, 1% P/S and 5 μg/mL insulin, and 0.5 μg/mL hydrocortisone. All other cell lines were cultured in ATCC recommended media in an incubator at 37°C in an atmosphere containing 5% CO2. The CHO-PDL1 cell line generation and maintenance are described in Four cell lines NCI-H2444 . The harvested cells were washed twice with flow cytometry buffer (1xPBS with 2 mM EDTA and 0.5% FBS). Cells were stained with anti-human PD-L1 antibody conjugated with phycoerythrin (PE)  according to the manufacturer's protocol and were analyzed on a FACSCalibur flow cytometer (Becton Dickinson). At least 20,000 events were recorded and analyzed using FlowJo software (Tree Star).6), CHO (10×106), H2444 (10×106), or H1155 (2×106) cells and orthotopically in the upper mammary fat pads with MDAMB231 (2×106), and SUM149 (2×106) cells in 100 μL of HBSS containing 50% matrigel (Corning). Mice were used for imaging or biodistribution experiments when the tumors reached a volume of 200-300 mm3.Animal studies were performed according to the protocols approved by the JHU Animal Care and Use Committee (ACUC). Six-to-eight weeks old, female, non-obese diabetic severe-combined immunodeficient gamma (NSG) mice were obtained from the JHU Immune Compromised Animal Core. Mice were implanted subcutaneously in the upper flanks with CHO-PDL1 (10×106 in 30 μL HBSS containing 50% matrigel), were injected directly though the intercostal space into the left lung, by using a 29G needle on a 0.5 mL syringe. Skin incisions were closed by sutures and antibiotics were applied topically. Growth of the orthotopic lung tumor was monitored by CT imaging.Orthotopic mouse models of lung tumors were generated by injecting lung cancer cells into the left lung of NSG mice. A 1 cm skin incision was made on the left scapula of the anesthetized mouse and the thoracic muscles were separated to expose the costal layer. The H2444 cells, (1×10111In was carried out in 0.3 M ammonium acetate (pH 5.5) for 1 h at 37°C, under metal free conditions. The resulting [111In]PD-L1-mAb was incubated with ethylenediaminetetraacetic acid (EDTA) at a final concentration of 5 mM for 5 min to chelate unbound 111In3+, and further purified on a desalting column. Radiochemical purity was tested by instant thin-layer chromatography (ITLC)  using citrate-phosphate-dextrose solution as a mobile phase (The PD-L1 mAb was reacted with N-[(R)-2-Amino-3-(p-isothiocyanato-phenyl) propyl]-trans--cyclohexane-1,2-diamine-N,N,N',N”,N”-pentaacetic acid  in 0.1 M sodium bicarbonate buffer (pH = 9.5) at 1:50 mAb:CHX-A”-DTPA molar ratio, for 1 h at room temperature. Unconjugated CHX-A”-DTPA was removed using a Zeba™ spin desalting column, pre-equilibrated with 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer. The DTPA-conjugated mAb was further purified on an Amicon ultra-4 centrifugal filter with 10k Da MWCO , using 0.1 M ammonium acetate (pH 5.5). Conjugation of CHX-A”-DTPA to antibody was confirmed by matrix assisted laser desorption ionization mass spectrometry. Radiolabeling of mAb-DTPA with le phase .® 800CW NHS ester. The reaction mixture was gently stirred for 2 h at room temperature, followed by purification using PBS pre-equilibrated Zeba™ spin desalting columns, 7K MWCO, 0.5 mL . Analysis of the NIR-PD-L1-mAb was carried out using a Nanodrop 2000 UV-vis spectrophotometer (Thermo Fisher Scientific), which indicated 1:2 mAb:IR800 molar ratio (Synthesis of PD-L1 mAb conjugated with Licor800 dye (NIR-PD-L1-mAb) was performed according to manufacturer's protocol. Briefly, 3 mg of mAb dissolved in 400 μL of 1xPBS were mixed with 4 μL of DMSO containing 0.09 mg of IRDyear ratio .In vitro binding of [111In]PD-L1-mAb to CHO-PDL1, CHO, MDAMB231, SUM149, H2444, and H1155 cells was determined by incubating 1 μCi of [111In]PD-L1-mAb with 1×106cells (in triplicate for each cell line) for 1h at 37°C. PD-L1 blocking was performed by adding a 10-fold molar equivalent excess of the non-labeled mAb. After incubation, cells were washed three times with cold PBS prior to counting on an automated gamma counter .111In]PD-L1-mAb to 4, 2, 1, 0.5 and 0.25 million CHO-PDL1 cells was carried out as described above. IF was calculated using the Lindmo assay PD-L1-mAb , images were acquired at the specified time points in mice. The tomographic data were acquired in 64 projections over 360o, at 45 s per projection, using medium energy pinhole collimators. CT images were acquired in 512 projections to allow anatomic co-registration. Images were reconstructed using the ordered subsets-expectation maximization algorithm, and 3D volume rendered decay corrected images were generated using Amira 5.5.0 software (Visage Imaging Inc.).Whole-body SPECT-CT images were acquired on an X-SPECT small animal SPECT/CT system  as described previously . Briefly111In]PD-L1-mAb was optimized for in vivo distribution and tumor uptake in mice harboring CHO-PDL1 and CHO xenografts. Mice (n = 3-5/group) were injected intravenously with 40 μCi of [111In]PD-L1-mAb alone (approximately ∼ 8.5 μg of the protein), or in combination with 10, 30, and 90 μg of unmodified PD-L1-mAb. For the blocking study, mice were pre-injected with 1.5 mg of unmodified mAb 30 min before injection of [111In]PD-L1-mAb. Blood, tumors, and selected tissues were harvested, weighed and counted in an automated gamma counter  at 48 h after the [111In]PD-L1-mAb injection. Following this optimization, all other biodistribution studies were performed with 40μCi [111In]PD-L1 mAb combined with 30 μg of unmodified mAb.Specific activity of [111In]PD-L1-mAb injection. The percentage of injected dose per gram of tissue (%ID/g) values were calculated based on signal decay correction and normalization to external 111In standards, which were measured in triplicate. Biodistribution data shown is mean ± the standard error of the mean (SEM).Biodistribution studies were also carried out to confirm the imaging study results in CHO/CHO-PDL1, SUM149/MDAMB231 and H1155/H2444 xenograft models with low or high expression of PD-L1 respectively, at various time points after [n = 3-5) bearing tumors with low and high expression of PD-L1. Mice were anaesthetized with isoflurane and serial images of the dorsal, left lateral, ventral and right lateral surfaces were captured using the Pearl Impulse Imager in white light and 800 nm channels  at 24, 48, 72, 96 and 120 h post injection. On day 5 after the injection of NIR-PD-L1-mAb, mice were euthanized and tumors and selected tissues were dissected and imaged ex vivo. To quantify the signal, equal sized regions of interest (ROIs) were drawn on tumors and tissues and on an area outside the mouse and representative of background. Mean signal intensity in each ROI was normalized by subtracting the background signal, and used for statistical analysis. Data shown is mean fluorescence intensity values ± SEM.PD-L1 expression in different tumor models was assessed by optical imaging using NIR-PD-L1. The NIR-PD-L1 (22 μg) was injected into the tail vein of the mice  at 1:500 dilution at 4°C overnight. Subsequently, using Dako CSAII Biotin-free Tyramide Signal Amplification System kit, slides were incubated with secondary antibody, amplification reagent, and with anti-fluorecein-HRP. Finally, staining was carried out by adding DAB chromogen. Sections were counterstained with hematoxylin, followed by dehydration with alcohol gradients, xylene washes and mounted with a cover slip.Tumor sections were evaluated for PD-L1 expression by immunohistochemistry (IHC). Harvested tumors were fixed in 10% neutral buffered formalin, embedded in paraffin, and 4 μm thick sections were obtained on slides. After deparaffinizing with xylene and alcohol gradients, antigen retrieval was done using 10 mM citrate buffer, pH 6.0 . Tumor sections were then treated with 3% Hin vitro receptor binding assay data and ex vivo biodistribution data were performed with Graphpad Prism 6 software using an unpaired two-tailed t test. When P < 0.05, the difference between the compared groups was considered to be statistically significant.Statistical analysis of


We discuss the range of application of this technique, which is currently limited by oligomer contaminants, and possible means to expand it by eliminating the contamination problem.Graphene has recently attracted much interest as a material for flexible, transparent electrodes or active layers in electronic and photonic devices. However, realization of such graphene-based devices is limited due to difficulties in obtaining patterned graphene monolayers on top of materials that are degraded when exposed to a high-temperature or wet process. We demonstrate a low-temperature, dry process capable of transfer-printing a patterned graphene monolayer grown on Cu foil onto a target substrate using an elastomeric stamp. A challenge in realizing this is to obtain a high-quality graphene layer on a hydrophobic stamp made of poly(dimethylsiloxane), which is overcome by introducing two crucial modifications to the conventional wet-transfer method – the use of a support layer composed of Au and the decrease in surface tension of the liquid bath. Using this technique, patterns of a graphene monolayer were transfer-printed on poly:polystyrene sulfonate and MoO Graphene, a one-atom-thick layer of carbon atoms arranged in a hexagonal lattice, has outstanding electrical136813141619et al. demonstrated a technique capable of transferring graphene monolayers without an adhesion or a self-release layerAttention, therefore, has been focused on development of dry-transfer techniques3, which are representative examples of organic electronic materials and practically important metal oxidesHere, we demonstrate a low-temperature, dry transfer process capable of transfer-printing a patterned graphene monolayer onto a target substrate that can be damaged or degraded by a wet, plasma or high-temperature process. In this process, a graphene monolayer on Cu foil, which is grown by chemical vapor deposition (CVD) and then patterned using a conventional lithographic process, is transferred onto a stamp made of poly(dimethylsiloxane) (PDMS), and subsequently transfer-printed from the stamp onto the target substrate. The graphene transfer from Cu foil to PDMS is achieved using the conventional wet-transfer processTo transfer a graphene monolayer onto a target substrate that can be damaged or degraded by a wet or high-temperature process , we firsFirst, as a support layer material, we use thermally deposited Au, instead of PMMA, which is most widely used for this purpose in the wet-transfer method2 gun, the graphene and substrate form a conformal contact without wrinkles throughout the substrate, as the water is laterally displaced [Second, for the liquid on which the graphene–Au bilayer floats and from which it is scooped with a PDMS stamp , we use isplaced . Since tisplaced .2 RIE of graphene using photoresist patterned by photolithography as an etch mask191919When patterning of graphene is required, a conventional patterning process, such as O2 layer following a process described in –2, which was obtained by counting the number of defects distributed over the whole sample area using an optical microscope. When the PDMS stamp was observed by an optical microscope after Step (f) in 2 gun, these locally conformal contacts laterally expand, generating narrow wrinkles with water droplets trapped inside, as shown in the right image of 2 pressure causes the water droplets to burst, resulting into defects such as that shown in To show that the surface tension of a liquid used in Step (d) in Rsh) was measured for graphene monolayers transfer-printed on glass substrates, using the van der Pauw methodRsh values were obtained with an injected current of 1 mA. In the following, a graphene monolayer transfer-printed onto a final substrate from a PDMS stamp onto which a graphene–Au bilayer was scooped from a bath of water and the ethanol–water mixture are referred to as −1, 2D: 2686 cm−1, G: 1588 cm−1), (ii) the height of the D peaks is negligible, and (iii) the 2D/G peak ratios are larger than 2.7, confirming that the transfer-printed graphene is indeed a monolayerRsh for Rsh, which has been ruled out by an experiment described in Note 1 in The sheet resistance (T), plotted on the y-axis, is the intensity of the optical beam transmitted through a glass/graphene sample normalized to that transmitted through a glass substrate. The size of the optical beam at the sample location was approximately 2 mm by 8 mm. For both T are consistent with what was previously measured for a graphene monolayer on a quartz substrateT for α) can be estimated as α calculated at each wavelength in 2 blow-dry and transfer-printing tend to cause defects in the graphene layer, as shown in Rsh of a transfer-printed graphene monolayer by this approach was 4 kΩ/sq, even with a self-release layer inserted for reliable graphene transferAs expected, successful transfer-printing of graphene requires a defect-free graphene monolayer that is globally conformal to a PDMS stamp. Our proposed technique achieves this with the water–ethanol mixture, which provides a continuous lubrication layer, and with a Au support layer, which allows for its removal without swelling PDMS. Alternatively, one may attempt to obtain a defect-free graphene monolayer on a PDMS stamp by pressing the stamp onto a graphene layer grown on Cu foil and then etching away the Cu foil by floating the Cu/graphene/PDMS on a bath of a Cu etchant. Since the surface of Cu foil commonly used in CVD growth of graphene typically has corrugations on the micron scale2 RIE using a photoresist etch mask patterned by photolithography. Next, the patterned graphene was transfer-printed on a Si/SiO2 substrate coated with MoO3 or PEDOT:PSS, both of which are susceptible to degradation when exposed to an aqueous condition or aggressive patterning process. μm and 15 μm, respectively, which are identical, within the resolution of the optical imaging system used (~0.5 μm), to those of the corresponding features on the photomask. A closer observation of the pattern edge using a field emission scanning electron microscope (FE-SEM) revealed that it is not straight on the nanoscale, with an “edge resolution” of 50 nm, which is probably attributed to the edge resolution of the photomask patterns and/or limitation of the photolithography process . As shown in 2 substrate coated with a PEDOT:PSS separation layer, and then transferred onto a lacey carbon TEM grid using the conventional wet-transfer method . An EDS spectrum obtained from a region shown in 252 and Ar, as shown in Fig. 4(b)Next, we observed the surface of the transfer-printed graphene on a Si/SiOThe morphological and elemental characterizations of the surface of transfer-printed graphene discussed above help determine the range of application of our technique in its current form. Since the oligomer residues are likely to be present only on the top surface, that is, the graphene surface that used to be in contact with the PDMS, our technique can be applied to fabrication of (i) devices where only the bottom surface of the graphene electrode is involved in injection or collection of charge carriers, such as LEDs and solar cells, made of organic semiconductors1314452, whose cost may not be of a concern for many device applications. Another concern may be effects of the Au etchant on the electrical quality of transfer-printed graphene. In our experiments, exposure of graphene to the Au etchant is rather brief (<5 min), after which samples are thoroughly rinsed with clean water. As a result, the elemental characterization of the transfer-printed graphene layer in Fig. S8 does not show any peaks associated with the Au etchant, such as those of iodine. Furthermore, through a simple experiment described in Note 2 in Rsh due to the Au etchant is negligible. Finally, we note that Au in our demonstration can be considered as a representative material for the support layer satisfying the following conditions — it must not be etched away by the Cu etchant, and can be removed without swelling PDMS and damaging graphene — and hence can be replaced by other material, not necessarily metallic, that is more cost-effective and/or easily removed.A possible concern with choosing Au as a material for the support layer is its high cost. Although Au is an expensive material, the thinness of the support layer translates to a small amount of Au used per area: the 200-nm-thick Au support layer corresponds to 0.386 mg of Au per cm3 and PEDOT:PSS, with the smallest feature size and edge resolution of ≃10 μm and 50 nm, respectively. Immediate application areas of this technique include organic electronic devices whose top electrodes are composed of graphene. Moreover, by eliminating siloxane oligomer residues on graphene using alternate stamp material, the technique can be further applied to devices whose graphene electrodes are in their interiors, such as tandem LEDs and solar cells. Finally, with possible appropriate modification, it may also be applied to dry-transfer of other two-dimensional materials, including boron nitrideIn summary, we have developed a low temperature, dry process capable of transfer-printing a patterned graphene monolayer grown on Cu foil on a target substrate. Two features distinct from the conventional wet-transfer method2 at 20 mTorr, and during growth, the reactor was filled with a mixture of CH4 and H2 at a total pressure of 150 mTorr, whose flow rates are 35 and 5 sccm, respectively.A graphene monolayer on Cu foil was grown in a CVD system consisting of a tubular quartz reactor and a furnace. Experimental details described in ref.−7 Torr) (b). To etch away the Cu foil, the Cu/graphene/Au multilayer was floated on an ammonium persulfate solution, prepared by dissolving 10 g of ammonium persulfate (Sigma Aldrich) in 500 ml of water (c). After the etch was completed, the graphene–Au bilayer was scooped with a glass slide, and then transferred on a bath of water to remove residual ammonium persulfate. Next, the graphene–Au bilayer was moved onto a bath composed of an ethanol–water mixture (70 vol % ethanol and 30 vol % water), from which the bilayer was scooped by and transferred onto a PDMS stamp (d). The sample was then blow-dried using a N2 gun (e), and was further dried on a hot plate at 40 °C for more than 4 h. The Au support layer was etched using an ammonium iodide solution  (f), after which the PDMS/graphene sample was rinsed with water. After water droplets on the sample were blown away using a N2 gun, the graphene-coated PDMS stamp was gently pressed onto a target substrate, inducing intimate contact throughout the substrate area (g). Before separation of the stamp from the substrate, the sample was stored at room temperature for 1 h under a pressure of 9.9 kPa, and then placed on a hot plate at 70 °C for 10 min without application of pressure. Finally, the stamp was carefully peeled off from the substrate (h), resulting in the transfer-printed graphene monolayer on the target substrate.Low-temperature, dry transfer of graphene monolayers was carried out by following processes described in μm-thick photoresist  was spin-coated on a Cu/graphene sample, and then patterned by photolithography. The patterned graphene on Cu foil was obtained, when the graphene in the areas not covered by the photoresist was etched by reactive ion etch in O2 . Performing the processes described in 3 layer. The target substrate was a 500-μm-thick Si substrate pre-coated with a 285-nm-thick thermal SiO2 layer, and the PEDOT:PSS (Heraeus) and MoO3  layers were deposited by spin-coating  and thermal evaporation in high vacuum , respectively.In this process, a graphene monolayer on Cu foil was first patterned using conventional photolithography and reactive-ion etch, as described in 2 substrate as the PEDOT:PSS layer was dissolved (c). Next, the bilayer was transferred to another water bath and kept floating on it for more than 24 h to ensure that PEDOT:PSS remaining on the graphene surface was removed. Then, the bilayer was scooped with a lacey carbon TEM grid  (d), after which the grid was placed on a hot plate at 40 °C for more than 2 h. Finally, the PMMA layer was removed by acetone (e), resulting in the graphene monolayer on the TEM grid (f).Samples for the elemental analysis were prepared following the processes described in The surface morphology of transfer-printed graphene layers was characterized using a FE-SEM  and an AFM . The sheet resistance was measured using a source meter  and a multimeter . The Raman spectra were obtained using a confocal Raman microscope  with an excitation wavelength of 514.5 nm emitted from an Ar laser. An ultraviolet–visible spectrophotometer  was used to measure the optical transmittance spectra. The elemental analysis was carried out using a STEM equipped with an EDS .How to cite this article: Cha, S. et al. Low-Temperature, Dry Transfer-Printing of a Patterned Graphene Monolayer. Sci. Rep. 5, 17877; doi: 10.1038/srep17877 (2015).


In this study, we examined the expression of PD-L1, CD25, PD-1, FoxP3, CD4, CD8, and EpCAM in 42 peripheral blood samples and fresh tumor specimens using multiparametric flow cytometry. Our results showed that the percentages of PD-L1-expressing CD25+ CD4+ T cells were significantly higher in TILs than in peripheral blood lymphocytes . The density of CD25+ PD-L1+ CD4+ TILs positively correlated with that of PD-1+ CD8+ TILs but negatively correlated with interferon (IFN)-γ+ and tumor necrosis factor (TNF)-β+ CD8+ TILs. However, the high ratio of CD8+ TILs to that of CD25+ PD-L1+ CD4+ TILs or to EpCAM+ tumor cells were associated with high percentages of IFN-γ+ and TNF-β+ CD8+ TILs. Moreover, inhibition of PD-L1 and PD-1 decreased the density of CD25+ PD-L1+ CD4+ cells and PD-1+ CD8+ TILs but increased the percentage of IFN-γ+ and TNF-β+ CD8+ cells. High ratios of CD8+ TILs to CD25+ PD-L1+ CD4+ TILs or to EpCAM+ tumor cells enhanced the activity of tumor-specific CD8+ T cells after PD-1/PD-L1 blockade therapy. Taken together, our results highlighted the importance of CD25+ PD-L1+ CD4+ TILs in mediating the tumor microenvironment immune response. Our findings also indicated that high ratios of CD8+ TILs to CD25+ PD-L1+ CD4+ TILs or to EpCAM+ tumor cells in patients may be more effective after PD-1/PD-L1 blockade therapy. Thus, the variable density of CD8+ and CD25+ PD-L1+ CD4+ T cells within the tumor microenvironment caused differential responses to PD-1/PD-L1 blockade.The programmed cell death-1 (PD-1)/programmed cell death-1 ligand (PD-L1) pathway has been shown to play a pivotal role in tumor evasion. Inhibition of PD-1 and its ligand PD-L1 using an immune checkpoint inhibitor has emerged as a promising immunotherapy for the treatment of various types of cancer. The expression of PD-L1 in tumor cells and tumor-infiltrating lymphocytes (TILs) has been shown to be correlated with improved efficacy of antibodies against PD-1 or PD-L1. Moreover, the density of CD8


Graphene and other two dimensional (2D) materials are currently integrated into nanoscaled devices that may – one day – sequence genomes. Graphene and other two dimensional (2D) materials are currently integrated into nanoscaled devices that may – one day – sequence genomes. The challenge to solve is conceptually straightforward: cut a sheet out of a 2D material and use the edge of the sheet to scan an unfolded biomolecule from head to tail. As the scan proceeds – and because 2D materials are atomically thin – the information provided by the edge might be used to identify different segments – ideally single nucleotides – in the biomolecular strand. So far, the most efficient approach was to drill a nano-sized pore in the sheet and use this pore as a channel to guide and detect individual molecules by measuring the electrochemical ionic current. Nanoscaled gaps between two electrodes in 2D materials recently emerged as powerful alternatives to nanopores. This article reviews the current status and prospects of integrating 2D materials in nanopores, nanogaps and similar devices for single molecule biosensing applications. We discuss the pros and cons, the challenges, and the latest achievements in the field. To achieve high-throughput sequencing with 2D materials, interdisciplinary research is essential. Key learning points(1) [Mechanism] To learn the working principle of nanopores for biomolecule detection and sequencing.(2) [General] To learn the advantages and the challenges of the implementation of 2D materials in nanopore schemes.(3) [Mechanism] To learn about nanogap and the other proposals as the next generations of biomolecular sensors.1Nanopore sensors have emerged as powerful devices for probing biomolecules. Head-to-tail sequencing is the ultimate goal of nanopore research. In comparison to existing sequencing technologies, nanopores potentially provide a single molecule platform for sequencing unlimited DNA lengths without chemical modifications or labeling. The working concept is straightforward: a DNA molecule is electrophoretically driven through a nanoscale pore in a membrane separating two aqueous environments. The signal (ionic current) corresponding to a nucleotide – momentarily present at the narrowest constriction of the nanopore – provides the information essential to identifying that specific nucleotide, primarily its molecular size or charge depending on the background ionic strength.e.g. α-hemolysin and MspA, embedded in a lipid bilayer membrane were the first type of the nanopores experimentally probed, with now the proof that sequence information can be obtained.1 Solid-state nanopores, however, are more stable alternatives.2 The tunability of their physical and chemical properties, and their compatibility for mass-production are significant advantages.2Ionic channels, naturally occurring in biological nanopores 6 As the thinnest possible materials with thicknesses comparable to the spacing between the nucleotides, monolayers of 2D materials promise very high spatial resolution, potentially eligible to DNA sequencing.7The channel of a nanopore in a freestanding silicon-based membrane typically contains hundreds of bases simultaneously. Consequently such long channel nanopores are not capable of addressing and detecting single nucleotides composing a DNA strand. The integration of two-dimensional (2D) materials, especially graphene in nanopore systems, has drawn a lot of interest in recent years.11 Some of the 2D materials, particularly graphene, are electronically conductive with highly mobile and gate tunable charge carriers. Such properties open up new modalities for single molecule detection, e.g. by probing the doping effect of a translocating DNA molecule on the conductivity of graphene.12 Hexagonal boron nitride (h-BN) and molybdenum disulfide (MoS2) are the other 2D materials with demonstrated capabilities in nanopore applications.14Large, continuous and stable films of 2D materials can now be grown chemically to form free-standing membranes.5 and single-stranded6 DNA molecules.The far-too-rapid translocation of DNA molecules through nanopores, however, makes the detection of the individual nucleotides very challenging, both for 2D and long channel nanopores. Additionally, the low mechanical stability of atomically thin membranes is the Achilles' heel of 2D nanopores, adding considerable noise to the measured signal and reducing the achievable resolution. Due to such limitations, attempts to sequence DNA with 2D nanopores have not yet met the initial hopes. Instead, the platform has been extensively used to successfully detect the translocation of individual double-strandedConsidering the rapidly growing interest in the application of 2D nanopores and nanogaps, this article serves as a comprehensive tutorial, reviewing the latest progress in the field. Background literature, fabrication methods and important achievements are presented in each section. We explain the remaining challenges and, importantly, offer appropriate solutions. This review bridges chemistry and physics and suggests new development routes in this still young and emerging research field.22D materials and particularly graphene were recently integrated in nanopore sensors. Their mono-atomic thickness is at the center of the research interest. Although 2D materials conceptually promise single nucleotide resolution, they did not yet prove themselves as an alternative to commercially available Oxford Nanopore Technology sequencing platforms (www.nanoporetech.com).2.1L and separates two adjacent fluidic cells (cis and trans) containing a buffer solution. By applying a transmembrane potential (hundreds of mV), a constant ionic current is established as a result of the migration of ions between the cells. Upon the addition of single stranded or double stranded DNA molecules in the cis reservoir, the negatively charged DNA molecules are electrophoretically driven towards the trans reservoir. As single DNA molecules pass (i.e. translocate) through the nanopore, they partially block the flow of ions in the buffer, resulting in a series of sharp dips in the measured current  is the driving force and the ultimate goal of nanopore analysis: theoretical calculations confirmed the possibility of distinguishing base pairs composing a double-stranded DNA (dsDNA) in graphene nanopore systems,15 as the ionic current largely depends on the conformation (stretching) of the translocating molecule. While the externally applied electrostatic field tends to stretch and deform base pairs occupying the nanopore, intermolecular hydrogen bonding in A–T and G–C base pairs  resists against the deformation to some extent. Additionally, harsh electrostatic force can even break the hydrogen bonds and locally deform the molecules.15 The G–C base pair, however, is more robust against stretching than the A–T pair, as the former possesses an extra hydrogen bond. It was shown by computer simulation that an optimal driving force (∼1 V in this simulation) can sufficiently deform the A–T base pair so that the resulting ionic current can be distinguished from the G–C pair signal.Identifying the building blocks of biomolecules  can lead to the reduction of the signal to noise ratio.5 (x) membranes with few tens of nanometers thicknesses are fabricated and used. Focused electron or ion beams are standard techniques to sculpt microscale openings in such membranes. In a next step, a 2D material is transferred onto the substrate covering the opening. The interface between the substrate and the 2D material should be well sealed against any ionic leakage to maximize sensitivity, for example using an elaborate transfer process.16 As the final step, a nanopore is drilled in the 2D material. Few techniques have been successfully used for drilling such nanopores.Exfoliated or chemically grown, the flakes have to be transferred onto a substrate with a previously drilled opening as shown in the bottom inset of 5 . The sup2.2.112 The bonds between the atoms in the 2D crystal are mechanically broken by collisions of highly focused and accelerated electrons (inset of x membrane can be done simultaneously.12 The just-sculpted structures can be readily imaged in the scanning mode of the TEM. The formation of defects and amorphization close to the nanopore in monolayer graphene membrane (less likely to happen in few layer membranes17) is a common problem in the normal application of the technique. Additionally it is shown that organic process residuals and contaminations can migrate to the beam spot and accumulate, locally raising the effective thickness of the edge far beyond the atomic thickness of the membrane.183Transmission electron microscopy (TEM) is the most used technique for sculpting nanopores and arrays of nanopores in graphene and other 2D materials.inset of . Further2.2.218in situ annealing (T ≥ 600 °C) during electron beam sculpting in TEM can effectively prevent contamination and amorphization of graphene. Remarkably, high-resolution TEM images confirmed that the crystallinity of graphene is preserved up to the edges (10 very fine nanostructures (e.g. nanopores of d ≈ 2 nm) were sculpted with predefined positions and sizes  to break three carbon–carbon bonds in the graphene lattice to nucleate a defect  Ar+ ion beam nucleates arrays of vacancy defects which are subsequently grown with unfocused subthreshold energy electron beams. Nanopores with diameter as small as 6 Å were obtained with this approach , the nanorods deform into nano-hemispheres which are mobile under the light source. The temperature in the vicinity of the nano-hemispheres largely exceeds the oxidation temperature of graphene, finally yielding nanopores. Photothermal nanopore formation is another scalable technique yielding arrays of nanopores at room temperature.2.2.513 and MoS214).2.3So far 2D nanopores have been extensively used for probing the translocation of single and double stranded DNA. No successful implementation of 2D nanopores for DNA sequencing has been reported yet. Several parameters affect the performance of a nanopore, which will be discussed in this section and some are summarized in 2.3.16An example of the electrical signal measured from a nanopore is shown in 2.3.2d = 2r in a membrane of thickness L can be regarded as a cylinder:23 The conductance increases with increasing nanopore diameter and decreases with increasing channel length, which can be obtained by selecting thicker membranes. This model is mainly valid when L > d (thick nanopores). For nanopores in thin materials e.g. 2D nanopores, the convergence of the electric field lines at the “mouth” of the nanopores  is proposed as:24σ refers to the ionic conductivity of the buffer solution. The first and the second terms show the nanopore channel resistance and the access resistance respectively. For ultra-thin membranes (L ≪ d), the first term approaches zero and the access resistance dominates; hence the conductance increases linearly with the nanopore diameter: G ∝ d. The coefficient of proportionality depends on the concentration of the ions and their mobilities. In contrast, when the thickness of the membrane is comparable to or larger than the nanopore size, the ionic conductance scales quadratically with the nanopore size: G ∝ d2.A general model for the conductance of a nanopore (d = 8 nm) and from a nanopore with a similar diameter (d = 6 nm) in a SiNx membrane of L = 40 nm.4 The baseline current in the 2D nanopore is several times larger than the one in the SiNx membrane. This observation is consistent with eqn (1) since the reduction of the membrane thickness yields a higher conductance. Indeed, ions feel much less resistivity passing through nanopores in 2D materials, compared to long channel nanopores.25 Molecular dynamic simulations showed that once a negatively charged ssDNA enters the nanopore, the change of the ion distribution results in an electric double layer near the surface of the DNA strand, reducing the sensitivity of the ionic current to the size of the bases. The effect is stronger in thin (monolayer) graphene membrane; hence the relatively thick membranes (few layer membranes) are claimed to be more efficient for sequencing ssDNA.Although achieving higher resolution is an important motivation for using 2D nanopores, some reports argue about the efficiency of using monolayer membranes.2.3.315 Induction of low frequency noise on the measured signal is another drawback of the instabilities.6The mechanical instability of the membrane in which the nanopore is sculpted greatly affects the spatial resolution. As freestanding 2D materials are mechanically less stable than thick membranes, their mechanical instability is more of a concern. In a 2D nanopore, the amplitude of the membrane vibrations may reach a level comparable to the thickness of the membrane.19 Increasing the working temperature also has similar consequences.26Several approaches can reduce the mechanical vibration of 2D nanopores in buffer solutions. The process for the fabrication of free standing membranes may induce some mechanical stress in the final membrane which limits the mechanical fluctuations. Additionally, the concentration of salt in the buffer solution also affect the mechanical vibrations: the more concentrated the salts, the more ions colliding the membrane and the less mechanical stability.i.e. the relative size of the nanopore versus the size of the DNA molecule) is another parameter affecting the sensitivity of nanopores. Simulations demonstrated that in long channel nanopores, the ionic current density is uniformly distributed throughout the channel width as the edges of 2D nanopores strongly localize the ionic current6  on the membrane and may finally clog the nanopore . AFM map4 did not show enough wettability to translocate DNA. Instead, depositing a thin layer of hydrophilic TiO2 films on both sides of the graphene membrane further improved the wettability of the devices.A rapid UV/ozone treatment can turn graphene into graphene oxide with hydrophilic properties. The oxidation by itself may not be enough as 30 percent of the devices measured after the treatment6 Note that a further increase in the pH (up to ∼12) may denature dsDNA into ssDNA. In a separate study, 3M of KCl did not prevent DNA from interacting with graphitic surfaces:19 AFM mappings showed that ssDNA still sticks rather strongly to the surface of HOPG  and high pH of the buffer solution (up to 10) minimize the interaction of DNA with the surface, leading to faster DNA translocation. of HOPG . Instead2.3.54 Experimental results showed that even coating graphene with 5 nm thick TiO2 film could not block such parallel paths effectively.4 Minimizing the suspended graphene area could be an effective method to minimize the contribution of the pin-holes to the total ionic current.Pin-holes, nanometer scale defects in 2D crystals, create parallel and unfavorable paths for the migration of the ions (rather than through the nanopore), and thus reduce the sensitivity of the measurement. Such pin-holes may form during the growth or transfer of the 2D materials or because of the post treatment used to modify the hydrophobicity of the surface. As a result, the blockade current in the nanopore could be one order of magnitude less in defected CVD graphene compared to nanopores with the same diameter in silicon nitride membranes.2.4It is now well established that the detection of the translocation of entire DNA molecules in solid-state nanopores is possible. Attempts, however, to identify individual nucleotides within a DNA strand have not yet succeeded. The rise of 2D materials in nanopore technology raised hopes for achieving single base resolution. Despite the considerable efforts over the last five years, high throughput sequencing has not been realized yet with 2D materials. This section reviews the drawbacks and challenges.2.4.1versus nanopore diameter for 2D nanopores, early experimental results3 depicted a quadratic relation which is the expected behavior for a cylindrical and long channel nanopore  predicts a linear dependency of the conductivity inset of . Indeed 0.1 nm19 .2.4.227 a nucleotide unbinds (hydrophobic interaction) from one side of the membrane and binds to the other side after a small sliding of the strand along the surface. Simulations predict a time span of 16 ns for each nucleotide (nt) to pass through a graphene nanopore .27 Experimentally, translocation times ranging between 10 ns6 to ∼60 ns3 per base (pair) have been reported in different conditions and for single and double stranded DNA (To be able to differentiate between nucleotides composing single-stranded DNA (ssDNA), the translocation must be performed in single-nucleotide steps:nded DNA .28 Using such a filter sets a maximum temporal resolution of ∼50 μs. The achieved resolution is therefore much inferior to the required resolution to resolve single nucleotides.The bandwidth of the measurement electronics sets an upper limit to the achievable temporal resolution. The combination of the membrane with the ions at its both sides can be regarded as a parallel plate capacitor which adds high frequency noise to the measured signal. To attenuate this noise, it is inevitable to use a low-pass filter of ∼10 kHz as a part of the electronic detection scheme.Slowing down the translocation speed of the DNA is an important objective followed by many researchers, particularly when using biological nanopores. In fact, a DNA translocating through a nanopore experiences three different forces, namely the electrophoretic driving force, the nanopore-molecule interaction force, and the drag (viscosity) force from the buffer solution. While the electrophoretic driving force accelerates the translocation of DNA, the two others impede against the translocation. Several parameters modulate those forces and therefore the translocation speed of DNA; they will be discussed in the following sections.2.4.2.16 the smaller the nanopore, the faster the translocation. The observation was explained in terms of the decreased drag on the translocating molecule in smaller nanopores. However it seems this effect is not universal since a contradictory trend was also reported elsewhere for 2D nanopores19  is another confirmation that in ssDNA purines and pyrimidines are more accessible for pi-stacking interaction with graphene.A more detailed study about the DNA–membrane hydrophobic interaction was performed by probing the translocation of single and double stranded DNA through nanopores in graphene–Alr = 1.2 nm) in a positively charged membrane (+3.6e) can happen almost twice faster than a similar nanopore in a negatively charged membrane (–3.6e).15 Indeed the repulsive interaction between the negatively charged DNA molecule and nanopore rim reduces the effective nanopore diameter.In addition to the naturally occurring hydrophobic interaction between the DNA and 2D materials, electrostatic interaction also can be induced. Chemically functionalizing the nanopore with positively and negatively charged groups is a way to achieve nanopores with different charge states: as DNA is also charged , the DNA–membrane electrostatic interaction can affect the translocation speed. Simulations illustrated that the translocation through a nanopore  an electrostatic interaction. Stacked membranes in which an electrically connected graphene membrane is sandwiched between two dielectric materials is a model systems for this purpose.2.4.2.4F) felt by the DNA molecule translocating through the nanopore depends on the externally applied electrical field (E) and the electrical charge of the molecule inside the nanopore (q): F ∝ Eq. While a larger electric field  is established across thinner membranes, the total negative electrical charge on the portion of the DNA residing inside the thinner nanopore is smaller. Consequently, in theory, no strong effect of the channel length on the total electrical force and hence on the translocation speed is predicted.The effects of the fluidic channel length – set by the thickness of the membrane – on the translocation speed have been studied both theoretically and experimentally. The total electrical force  membranes13 reveals an opposite trend i.e. faster translocation through h-BN nanopores in comparison than in SiNx ones. Such contradictionary observations may also point out the influence of chemical composition of the membrane material on the strength of the DNA–membrane interactions.Experimentally, nanopores drilled in few layer graphene yielded slower translocations (more than twice as slow) compared to similar nanopores drilled in 20 nm-thick SiN2.4.3Nucleotides can take diverse molecular conformations while passing through nanopores. Examples of such conformations are depicted in the snapshots in the top panels of 27 Hence even in presence of conformational instabilities, single nucleotide differentiation is possible.The relatively uncontrollable and arbitrary conformations of the nucleotides affect the ionic current blockade. Nevertheless simulations showed that the dependency of the signal to the nucleotide type is stronger than to its conformation.2.4.4x nanopores  can efficiently minimize the high frequency noise.17The noise characteristics in 2D and long channel nanopores are different. The difference in the noise levels can be readily observed by comparing the time traces measured in graphene and in SiNanopores . The powns cells . The dief noise – in nanopores in graphene and other 2D materials is normally much larger (up to 100 times) than in long channel nanopores.32 As a source of a considerable reduction in the signal-to-noise ratio, the high amplitude of 1/f noise is an important challenge in 2D nanopores. It was demonstrated that for nanopores in SiNx membranes the amplitude of the low frequency noise is inversely dependent on the number of ionic charge carriers ∝ 1/N:33 a principle known as Hooge's relation. However experimental results do not show such a dependency in monolayer graphene nanopores.32 The reasons behind this discrepancy are not yet understood.The magnitude of the low frequency noise – known as 1/32 We note that those experiments were performed at relatively high KCl concentration (1 M); more decisive conclusions could be drawn by lowering the ionic strength of the buffer solution.The origin of the high-amplitude low-frequency noise in 2D nanopores is under debate. Charge fluctuations in the membrane are potentially capable of inducing noise in the ionic current. Protonation and de-protonation of the carboxyl groups formed at the nanopore rim may cause such fluctuations and can be triggered by varying the pH of the environment. However experimental results did not confirm any strong dependency of the noise to the pH of the buffer solution.4 To cope with this effect, the hydrophobicity of the graphene membrane was modified by a rapid UV/ozone exposure, yielding graphene oxide. The improved wetting of the membrane on the cost of inducing defects, however, did not lead to a remarkable reduction of the noise level. Importantly, the evaporation of a hydrophilic TiO2 layer on graphene considerably reduced the noise. The idea of covering the membrane with hydrophilic materials was followed later by20 in which relatively thick membranes of multi-stacked graphene and Al2O3 layers were made. The top hydrophilic Al2O3 layer was supposed to provide adequate wetting during the experiments. Indeed very low 1/f noise, comparable with long channel nanopores and considerably less than in pure 2D nanopores was measured.The incomplete wetting of the hydrophobic graphene has been hypothetized as the source of the measured noise.f noise level while increasing the thickness (i.e. the number of layers) of graphene and h-BN membranes were measured.32 Such results point out the effect of the mechanical fluctuations as an important source of low frequency noise in 2D nanopore devices. In fact conventional solid state membranes  are mechanically more rigid and stable compared to few layer 2D materials. We note that unlike the interpretation of early reports,4 the reduced noise level measured by covering graphene with other materials4 or in stacked systems20 can be explained in terms of the increased mechanical stability, rather than the modification of the hydrophobicity of the graphene membrane. Minimizing the area of the free-stranding membrane is an approach to improve the stability of the membrane without affecting the surface properties. In fact reduced noise levels have been reported in two samples with very small free standing graphene areas.6Mechanical instability of the membranes is another parameter which is suspected to cause the noise: the fluctuation of the membrane may lead to the fluctuation of the ion flux. Increasing the thickness of the membrane is an approach to improve its mechanical stability. Indeed prominent trends in lowering the 1/x opening blocks a large amount of the pin-holes in graphene which would result in an enhanced signal. The important disadvantage of using thick graphene layers or of depositing other materials is that the increased thickness of the membrane eludes the mono-atomic nature of graphene which potentially provides single base resolution.Comparing the approaches to improve the mechanical stability of the membrane we note that minimizing the SiN2.5The challenges to improve the resolution of DNA detection have been discussed in the previous section. Fast translocation speed and high noise levels are among the challenges directly linked to the conventional measurement scheme of the nanopores. Hence researchers developed alternative detection methods to avoid these limitations.22 Fluorescently-tagged DNA molecules were electrostatically driven through a nanopore in a graphene membrane made by photothermal sculpting  is another 2D material with appealing properties in the field. Contrarily to graphene, MoS2 has a band gap of 1.8 eV which is demanded for sensing applications. The mobility of MoS2 is yet inferior to graphene: this limitation can be overcome by engineering the environment of the device.Most of the research on 2D nanopores so far has been performed using graphene membranes. Molybdenum disulfide (MoS37 A monolayer MoS2 is ∼1 nm thick and is relatively (three times) thicker than a monolayer graphene. Hence on a cost of the slight reduction of the resolution of the device, higher signal to noise ratio is predicted in the 2D nanopores in MoS2 membranes. In an early work, a signal to noise ratio of 10 has been measured for 2D nanopores in MoS2 which is almost three times higher than what can be achieved in graphene 2D nanopores.14The considerably large noise in graphene nanopores has been attributed to the low mechanical instability of the membrane as a result of its atomic thickness. Indeed simulations predict that the noise level reduces by using trilayer graphene membrane of ∼1 nm thickness.2, even without any additional surface treatments.26 This result is in agreement with experiments.14 Indeed, the presence of the hydrophilic Mo sites in the structure of the MoS2 membranes reduces the DNA–membrane interaction. In contrary to MoS2, experimentations with h-BN membranes demonstrate remarkably higher interaction with DNA molecules (in comparison to SiNx and graphene6) even at strong alkaline and salty condition .13DNA–membrane interaction is another important parameter to select the right material. Molecular dynamics simulations show that in contrary to the graphene, DNA does not stick significantly to the surface of MoS2, its low interaction with DNA and the high enough mechanical stability which a free standing MoS2 membrane promise a good replacement for graphene in nanopore applications.The electrically conducting nature of MoS2.7in situ heating. For large-scale production of sensors, however, significant research efforts are still needed: although TEM is great for proof-of-concept experiments, it is not viable for scalable applications.Nanopore technology offers a fast, cheap and easy solution for biomolecule sensing. The implementation of 2D materials can potentially improve the resolution of the devices to reach single nucleotide resolution. The possibility of chemically and electrically functionalizing the 2D membranes opens new routes for the customization of the sensors. Only a few methods have been utilized to drill nanopores in 2D materials. Among them, the most robust is to use a beam of energetic electrons combined with f ionic current noise which severely affects the signal to noise ratio, is another major challenge, very particular to 2D nanopores. The origin of this noise is not entirely clear yet, although the existing reports point the mechanical fluctuations of the membrane as the most probable source. Improving the mechanical stability of the membrane by reducing the area of freestanding part seems to be the most efficient technique to overcome the limitation. Utilizing thicker 2D materials (e.g. MoS2) is another potential approach.DNA sequencing with 2D nanopores met important challenges. The very rapid translocation of the nucleotides through the nanopore is far beyond the sensitivity of the ionic current. Several approaches can be taken to slow down DNA. Separately, the large 1/Alternative detection schemes – not necessarily based on the ionic conduction – have been proposed to overcome the existing difficulties. The conducting nature of 2D materials with externally tunable conductivity is the important supplement of graphene and some other 2D materials to develop such methods. In principle, schemes based on the electronic conduction could be faster than the existing measurement approach based on the ionic current. Theoretical propositions and experimental results of such schemes will be discussed in details in the next section.3E would be unable to overcome the potential barriers with a higher energy U (U > E). In quantum mechanics, however, electrons can tunnel through the barrier with a non-zero probability, yielding a measurable tunneling current38  current between two electrodes – separated by a nanogap – as the biomolecule slides through is a remarkable proposition. In classical mechanics, an electron with energy urrent38 . Increas40 Separately, the method potentially provides temporal resolution that would allow recording several data points per nucleotide. As the nucleotides pass one-by-one across the gap, recording the tunneling current over time should, in principle, resolve the particular sequence of the nucleotides in the strand.The detection and sequencing of single molecules could be implemented in a simple device composed of two electrodes separated by a small gap through which a biomolecule would translocate . Upon ap3.141 The modulations of the tunneling current between the STM tip and the substrate allowed visualizing the DNA molecules (Tunneling current for the detection of DNA molecules was for the first time exploited in scanning tunneling microscopy (STM). There, a sub-nanometer distance between the tip and the conducting substrate can be realized and maintained using a ‘feed-back loop’. In an early experiment, DNA molecules were imaged individually with STM.olecules .e.g. a thin wire) yields, in time, a thinner and thinner wire, a few atoms wide junction, a single atom junction, and finally a gap , guanosine 5′-monophosphate (GMP) and cytidine 5′-monophosphate (CMP), were probed.43 In each case, unique tunneling current signatures were detected  and ultrahigh vacuum which is not adequate for biological specimens; break junctions do not provide 3.25 As opposed to metallic break junctions, in an ultimate nanogap design, the surface of graphene could be covered with insulating materials, blocking any path for parallel conduction. We note that unlike the 2D nanopore systems, the capacitance between the cis and trans cells does not influence the tunneling current, hence a lower high-frequency noise is expected.The application of graphene for DNA sequencing with tunneling electrons has been theoretically studied. The presence of highly mobile charge carriers is the most distinctive property of graphene over the other 2D materials. Additionally, freestanding graphene membranes are of relatively high mechanical strength and as opposed to metals, each carbon atom is covalently bonded and conjugated to each other. 2D materials can also stand against large transmembrane pressures in liquid environments.39 while a nanogap width of less than 1.5 nm  would yield specific tunneling current signatures for each nucleotide, small metric variations in the width of the nanogap drastically impact the measured current. Important parameters that influence the detected signal and are crucial for achieving the single nucleotide resolution will be discussed in details in the next sections.The concept of using in-plane graphene electrodes for DNA sequencing  was inve3.2.1N bases in a DNA strand  translocating through a graphene nanogap, the measured current can be expressed as function of the position of the center of the DNA molecule (x0) with respect to the gap, and the applied bias voltage (V):39d is the width of the gap, xi is the position of the base i along the backbone,  is a base type-dependent coefficient  and k is the decay constant which depends on the work function of graphene. The results of the simulation for a specific sequence of the bases and for two different gap sizes are shown in Considering a set of 3.2.240 By means of the density functional theory (DFT), the tunneling currents associated to the four nucleotides, namely deoxyadenosine monophosphate (dAMP), deoxythymidine monophosphate (dTMP), deoxyguanosine monophosphate (dGMP), and deoxycytidine monophosphate (dCMP) in a nanogap between graphene electrodes were simulated.40 Size-wise, the nucleotides can be classified in two categories: purine-based  and pyrimidine-based  nucleotides. The pyrimidines have a smaller size than purines hence the distance between the nucleotides and the graphene edge is larger for pyrimidine-containing nucleotides. The discrepancy of the sizes affects the transmission  spectra. The transmission spectra associated to the dAMP, oriented in several angles with respect to the edge of graphene is plotted in –20 to 10–6G0 which is different from pyrimidine-based nucleotides, ranging from 0 to 10–8G0. The transmission peaks also provide distinctions between the nucleotides: the first peak below the Fermi energy  of the electrodes is found to be in resonance with the HOMO  of the nucleotide and is therefore dependent on the type of nucleotide. For all the studied orientations, the width of the resonance peak ranges between ∼0.05–0.10 eV for pyrimidines and between ∼0.1–0.2 eV for purines: the size of the nucleotides (at least the graphene–nucleotide coupling) provides means for distinguishing the two categories of nucleotides. The next challenge would therefore be distinguish between the nucleotides within each group . The position of the resonance peak is, however, different for dGMP and dAMP. For dGMP, the peak is located close to the Fermi energy of the electrodes (E – Ef ≤ 0.5 eV) while it is far away for the dAMP (E – Ef ≥ 0.5 eV). All the discussion so far was based on the zero biased transmission function. The results are summarized in IdGMP > IdAMP > IdCMP > IdTMP with very small overlaps.The nucleotides passing through a nanogap may take many different orientations and arbitrary distance to the graphene electrodes.45 The results showed a giant improvement in the sensitivity of the device: more than one order of magnitude difference in tunneling current between the nucleotides. Furthermore, the stabilization of the DNA bases against any possible thermal fluctuations dramatically reduced the electrical noise. Slowing down the translocation of DNA is another benefit of generating hydrogen-bonding interactions between the translocating nucleotides and the nanogap electrodes. Applying a large bias voltage can lead to similar results. Indeed, the transverse electric field established between the biased electrodes can be very large and stronger than the driving transmembrane field.46 The resultant stabilization of the translocating DNA preserves the sensitivity of the readout for different bases (distinguishability).Functionalization of the electrodes is an effective approach to minimize the conformational uncertainty. The effect of the hydrogen bonds formed in between single nucleotide from a single-stranded DNA and the individual gold atoms at the electrodes  were investigated theoretically.3.2.339 which is yet smaller than the separation between the bases. In other words, even though the mechanical fluctuations add some noise to the measured signal, high enough sensitivity for sequencing can still be achieved. We note that the thermal vibration of a monolayer graphene in water has not been studied yet.Thermal vibration of the graphene membrane influences the measured signal. The amplitude of the vibrations in few-layer graphene membrane in vacuum may reach 0.16 nm (for a membrane with thickness of 0.6 nm and length of 500 nm)In the most simple design of a nanogap , the sur3.347 . The current tunneling between the edges of each graphene layer (at the nanopore) would allow identifying the nucleotides. Simulations demonstrated that, indeed, the different nucleotides can be distinguished  protocol for the fabrication of well-defined and tunable twisted bilayer graphene. Deposition of the electrodes on one layer without electrically touching the other one is another fabrication challenge.In a real device the current in between the large layers of graphene can be several orders of magnitude larger than the conductance through the edges of the nanopore. In theory, the interlayer conduction depends on the relative orientation angle in between the layers. Therefore, the two layers can be electrically decoupled at some certain twist angles.49 It is postulated that in the presence of the molecule in the nanopore, the transmitted current through the two possible paths (i) via the molecules, and (ii) via the h-BN layer, may show quantum interference which could further improve the sensitivity of the device.49 The relative orientation (stacking) of the graphene/h-BN/graphene crystals affects the electrical isolation of the graphene layers. We note that the separation of the two graphene electrodes by the h-BN layer increases the tunneling length, decreasing the tunneling current. Separately, increasing the tunneling channel length also implies that more nucleotides may contribute to the tunneling current (at least if more than one layer of h-BN are used to separate the two graphene electrodes) which decreases the resolution of the measurement.Using the insulating few- (mono-) layer hexagonal boron nitride (h-BN) in-between graphene layers reduces the interlayer conductance.3.4Several studies  showed that tunneling current is sensitive to the molecular composition of translocating biomolecular strands. In comparison to the ionic current, the tunneling current could be potentially more sensitive to alternations in the chemical structure of the nucleotides. Unlike the nanopore scheme, the transmembrane capacitance does not directly affect the tunneling current; hence low amplitude of high-frequency noise is predicted. Indeed, the theoretical work and recent experiments with metallic break junctions show that it is possible to detect subtle differences in chemical compositions using tunneling current (nucleotides and amino acids).The complex fabrication of the electrodes with a narrow gap (<1.5 nm) is certainly the most important challenge which has prevented the realization of graphene based in-plane electrodes sensors so far. Additionally, theoretical works confirmed the efficiency of the detection scheme for single stranded DNA. As such, ssDNA must first translocate through the nanogap in an unfolded conformation, for example in the presence of unfolding agents such as urea. Separately, a nanopore in a bilayer graphene intrinsically has a physical gap in between the two layers, which could be used also to distinguish nucleotides in a DNA strand.4f noise always circumvent the data acquisition: the low mechanical stability of 2D membranes seems to be the origin of this noise. And importantly, to reach potential applications of 2D nanopores for DNA or other biomolecular sequencing applications, the poor scalability of the techniques developed so far to fabricate 2D nanopores have to be thought through.Nanopore technologies in solid-state materials – particularly graphene and other 2D materials – are still far from reaching the goal of sequencing long stretches of single-stranded DNA molecules primarily because the conventional measurement scheme based on ionic conduction has not much more advantages over what is currently pursued with biological nanopores. Additionally, the transmembrane capacitance produces a high frequency noise in the same frequency regime as the sequence information. Using low-pass filters to cut-off the noise also cuts-off the sequencing information, unfortunately. The situation is even worse in 2D graphene nanopores since a high level of low frequency 1/Nanogap in 2D materials is a proposition which potentially can overcome some of the sequencing challenges. Their fabrication is, however, even more complicated than nanopores. While the tunneling current has been predicted to be very sensitive to the available electronic states in nucleotides and therefore may provide more accurate readings, all predictions are based on theoretical work . Certainly, the transmembrane potential used to drive the molecules could be isolated further from the measurement circuit to limit the high frequency noise. The electrodes could also be supported on relatively thick membranes providing higher mechanical stability.Very importantly, small nanogaps with a width of less than ∼1.5 nm are also required to establish a measureable tunneling current. While high temperature STEM techniques showed the feasibility for the fabrication, tunneling has not been measured so far with graphene for example. Additionally, tunneling currents are very sensitive to the gap width and only a few percent increase in the gap width dramatically reduce the signal by orders of magnitude. Additionally, conventional nanofabrication methods are hardly capable to produce such short nanogaps, explaining why the idea has not been realized yet experimentally.An alternative to longitudinal nanogaps was proposed theoretically: the distance between the layers in a bilayer graphene can be regarded as a natural gap between the two basal plane graphene electrodes. This proposition may ease the fabrication in the sense that the gap can be achieved naturally and reproducibly. However the isolation of the two conducting layers is hard to achieve, even in twisted graphene bilayers.To conclude, the approaches to sequence DNA with 2D materials are still at a very early stage. The existing nanofabrication methods failed in realizing sequencing. But the principle is still viable: an atomic layer of a 2D material – particularly a graphene edge – can scan a genome and record its sequence as DNA is scanned. It is now clear that developing unconventional fabrication methods are vital .It is hard to predict now whether 2D crystals will ever meet the goal of DNA sequencing one day. Certainly, however, in the quest of this goal, new science will be discovered setting out even more horizons for the research.


We report a one-step polymer-free approach to patterning graphene using a stencil mask and oxygen plasma reactive-ion etching, with a subsequent polymer-free direct transfer for flexible graphene devices. Our stencil mask is fabricated via a subtractive, laser cutting manufacturing technique, followed by lamination of stencil mask onto graphene grown on Cu foil for patterning. Subsequently, micro-sized graphene features of various shapes are patterned via reactive-ion etching. The integrity of our graphene after patterning is confirmed by Raman spectroscopy. We further demonstrate the rapid prototyping capability of a stretchable, crumpled graphene strain sensor and patterned graphene condensation channels for potential applications in sensing and heat transfer, respectively. We further demonstrate that the polymer-free approach for both patterning and transfer to flexible substrates allows the realization of cleaner graphene features as confirmed by water contact angle measurements. We believe that our new method promotes rapid, facile fabrication of cleaner graphene devices, and can be extended to other two dimensional materials in the future. Graphene, a two-dimensional carbon allotrope, has received immense scientific and technological interest since its discovery789e.g., patterning and transfer) for creating interconnects, transistor channels, or device terminals have slowed the implementation of graphene in a wider range of applications14171423242526Significant progress has been made in the direct synthesis of large-area, uniform, high quality graphene films using chemical vapor deposition (CVD) with various precursors and catalyst substrates1112Conventional microfabrication-based photolithography requires extensive process steps, and yet suffers from polymeric residue171423242526Here we report a one-step facile method to pattern graphene by using stencil mask and oxygen plasma reactive-ion etching (RIE), and subsequent polymer-free direct transfer to flexible substrates. In conjunction with the recent evolution of additive and subtractive manufacturing techniques such as three-dimensional (3D) printing and computer numerical control (CNC) milling, we developed a simple and scalable graphene patterning technique using a stencil mask fabricated via a laser cutter. Our approach to patterning graphene using a stencil mask is based upon the shadow mask technique that has been employed for contact metal deposition292 plasma etching. While oxygen gas is commonly used for plasma etching of graphene313233In 2/Si substrate with 300 nm thermal oxide for better visual contrast. We demonstrate the potential of our method for effective pattern replication and parallel processing in device fabrication. To show such potential, we produced micron-sized features with a variety of repeated shapes etched on the graphene sheet grown on copper, and then successfully transferred the patterned graphene onto rigid and flexible substrates in a conformal fashion. Optical microscope images , and S2 To characterize the pattern replication capability and resolution of our stencil-based patterning, we determine the ratio of feature size of patterned graphene to that of stencil mask for various design feature sizes . Unity . This mi−1)2 plasma (for d = 1 to 9 μm)3237To substantiate the quality and integrity of our patterned graphene, we carried out Raman analysis at different distances from the etched feature edge. εpre,x ~ 250% and εpre,y ~ 150% before the patterned graphene transfer (See Methods). Scanning electron microscope (SEM) images  images  show theεtensile,x = 0% was plotted over applied strain (R/R0) versus increasing tensile strains  exhibited two different regimesεtensile,x = 0 to 40%), while at higher strain, the strain sensitivity is ~2.52   . This cost cycle (R/Rcycle 1) over one thousand cyclical strains of εtensile,x ~ 50%  with hydrophobic characteristics can enable dropwise condensation and therefore improve heat transfer characteristics. Recently, researchers have shown that hydrophobic and hydrophilic channels have improved condensation heat transfer performance compared to only hydrophobic surfaces based on the size and shape of hydrophilic region47Graphene patterning can also have significant impact in areas that require selective hydrophobicity or combined hydrophilic-hydrophobic patterns2 substrate, as clearly shown by water film having the shape of letter “E” and “C” . The E-SEM allows real-time image acquisition during water vapor condensation on a cooled sample. Our capability to selectively pattern graphene resulted in filmwise condensation on the bare SiO and “C” , in cont and “C”  50e.g., laser cutter and 3D printer) rather than the process itself. Furthermore, previous studies have shown enhanced oxidation of graphene by O2 plasma in a slightly reducing environment of ammonia and argon (or hydrogen) for controlled etch rate predominantly at the graphene nanoribbon edgesOur approach demonstrates a new possibility to overcome limitations imposed by existing post-synthesis processes to achieve graphene micro-patterning. Our method to pattern graphene with a stencil mask followed by direct lamination transfer has several key advantages over existing techniques. First, the simple patterning process circumvents the costly lithographic patterning and post processing steps required in traditional patterning techniques. Furthermore, our method allows rapid design iterations and pattern replications. Second, our proposed polymer-free patterning technique promotes graphene of cleaner quality than fabrication techniques that necessitate graphene exposure to resist or PMMA. Third, the ubiquity, scalability, and low cost of stencil mask fabrication by additive and subtractive manufacturing techniques such as laser cutter and 3D printing offers a pathway that is conducive to discovering potential applications. Lastly, our patterning technique provides a simple and robust platform that can be applicable to patterning of a wide range of other novel two-dimensional materials of recent interest , hydrogen (H2), and argon (Ar). Subsequent graphene patterning on as grown CVD-grown graphene was achieved after by aligning stencil mask first, then by oxygen plasma reactive-ion etching. The stencil masks shown in Graphene was grown on a copper foil by low-pressure CVD using a mixture of methane . E-SEM images  were obtained using FEI quanta 450 SEM  by increasing water vapor pressure to or above saturation pressure during E-SEM imaging. Raman spectra were obtained with the laser excitation wavelength of 633 nm, 1800 l/mm grating, 10 seconds of accumulation . Water contact angles were measured by a goniometer KSV CAM200 .εpre,x ~ 250% and εpre,y ~ 150%. Patterned, serpentine-shaped graphene on a Cu foil was then transferred onto a pre-strained VHB film. The Cu foil was then chemically etched with sodium persulfate solution. Thin gold film (40 nm) was deposited by thermal evaporator  to create contact pads. The device was biaxially re-stretched during the thermal deposition to create corrugated gold contact electrodes serving the device stretchability. The crumpling of graphene serpentine structures was accomplished by subsequent releasing of the elastomeric substrate.A VHB tape , a highly stretchable acrylic film, was biaxially pre-strained by x-direction as illustrated in Two terminal resistance measurements were performed with a probe station  and a sourcemeter . The serpentine-shape crumpled graphene strain sensor device was uniaxially stretched along the direction with higher pre-strain .


In situ SR-XPS during the graphitization annealing clarified that graphene is formed during the cooling stage. We conclude that Ni silicide and Ni carbide formation play an essential role in the formation of graphene.Low-temperature (~1073 K) formation of graphene was performed on Si substrates by using an ultrathin (2 nm) Ni layer deposited on a 3C-SiC thin film heteroepitaxially grown on a Si substrate. Angle-resolved, synchrotron-radiation X-ray photoemission spectroscopy (SR-XPS) results show that the stacking order is, from the surface to the bulk, Ni carbides(Ni This is mainly due to its giant carrier mobility  [Graphene, a single layer of sp cm2/Vs) , high th cm2/Vs) , as well cm2/Vs) , CVD on  cm2/Vs) –8, and e cm2/Vs) –11. AmonIn this context, Suemitsu et al. developed a so-called graphene-on-silicon (GOS) technology, in which EG is formed on a 3C-SiC thin film heteroepitaxially grown on a Si wafer –15. The A graphitization temperature of ~1473 K or higher, however, is still too high to be fully compatible with Si technology. Several attempts have been made to reduce the graphitization temperature of GOS, including the addition of a small amount of oxygen  or monos2. The sample fabrication consists of four stages: (a) growth of 3C-SiC thin (~100 nm) films using gas-source molecular-beam epitaxy (GSMBE), (b) ultrathin (~2-3 nm) Ni film deposition using electron beam, (c) annealing at temperatures 973–1173 K for 30 min, and (d) cooling down to 373 K at a rate of 12.5 K/min. In GSMBE, monomethylsilane (MMS: CH3SiH3) with a pressure of 10−2 Pa was used as the single source of silicon and carbon with a typical substrate temperature of 1323 K [An n-type Si(111) wafer was cut into pieces to form specimens with a size of 7 × 7 mmf 1323 K . Graphit−1), G’(~2700 cm−1), and D(~1360 cm−1) appear, which proves the graphene formation at these low temperatures. The grain size of graphene (La) is obtained from the intensity ratio between the D and G bands asFigure Elaser is the energy of the excitation laser in electron volt, and ID and IG are the intensities of the D and G bands, respectively [where ectively . At 11733C (~284 eV) and NiCx  [λ of the photoelectrons from Ni-C, Ni, and Ni-Si layers are in the order of 1–1.7 nm, which decreases down to 0.5–0.8 nm in the surface-sensitive measurement at θ = 60°. Although this value is less than the thickness of the Ni layer (~2 nm), we here observe a strong SiC component in the C 1s spectrum. This result can be related to the fact (a) that XPS measurement substantially detects photoelectrons from the surface up to 3λ in depth and (b) that our XPS setup integrally collects the photoelectrons that leave the surface at angles in the range of ±8° around the line-of-sight direction from the surface to the analyzer. The inset shows the detection-angle dependence of the peak intensities normalized by that of the SiC peak. The graphene and the Ni-carbide peaks show higher intensities in the surface-sensitive observations, indicating the formation of these layers on top of the SiC film. Figure 3C and NiCx. They are higher in the bulk-sensitive observations, and this tendency is pronounced in the IG/INi3C ratio. It is therefore concluded that the graphene layer is located beneath the Ni carbide layers, and the Ni3C layer is located above the C-rich NiCx (x > 0.33) layer   appear iyer Fig. .Fig. 2C 3/2 core-level spectrum obtained in a surface-sensitive mode with the detection angle of 60° from the surface normal. The Ni silicide components such as Ni2Si (~854 eV) and NiSi (~854.9 eV) and the Ni carbide component Ni3C (852.4 eV) [2Si to NiSi increases in the surface-sensitive observations, indicating that the Ni-rich Ni2Si silicide is on top of the Si-rich NiSi silicide. We therefore conclude that the Ni and Ni-silicide layers are stacked, from the surface to the bulk, in the order of Ni/Ni2Si/NiSi   appear iiSi Fig. .Fig. 3NiFigure 3C and NiCx as well as that of 3C-SiC, appear in the spectra in the early stage of the heating. Graphene component, on the other hand, appears only in the cooling stage , and cooling of the sample Fig. . The Ni age Fig. . In the tra Fig. , the pea2Si as a major reaction path for the graphite layer formation. As for graphene, Cao et al. [From these time evolutions of both C 1s and Ni 2p core-level peaks, we conclude that part of the 3C-SiC film is decomposed due to reactions with Ni to form Ni silicides and Ni carbides, and the excess C atoms created by this decomposition are used to form graphene. Of the Ni silicide and carbide formations, the former should be by far the major reaction path because the formation enthalpy for Ni silicide is negative (−80 kJ/mol) while that for Ni carbide is positive (30 kJ/mol) –37. Kurio et al.  have repo et al. , 38, theo et al. , 40. In o et al.  either t3C/NiCx)/graphene/Ni/Ni silicides (Ni2Si/NiSi)/SiC/Si. In situ SR-XPS during heating/annealing/cooling procedures has proved that Ni silicides and Ni carbides form during heating/annealing periods while graphene forms only in the cooling period. It is suggested that excess C atoms are liberated during heating/annealing along with the formation of Ni silicides, and part of these excess C atoms form into graphene. Graphene is formed during cooling because the C atom solubility in Ni decreases with decreasing temperature. The Ni carbide layers are suggested to act as a good capping layer for graphene formation as well as a C source through its decomposition during cooling, which is favored because of its exothermic nature of the reaction.In order to verify the Ni-assisted graphene formation on the SiC thin films on Si substrates and to clarify its growth mechanism, we conducted Raman and in situ SR-XPS analysis. We have successfully demonstrated that graphene actually forms on a 3C-SiC thin film at a temperature as low as 1073 K with the aid of the Ni layer. Angle-resolved SR-XPS proved that the stacking order, from the top to the bottom, is Ni carbides , and there is a lot of room for improvement toward low-temperature formation of graphene. Moreover, graphene produced by this method grows on a non-crystalline Ni layer, not directly on the SiC crystal, which cannot be called “epitaxial graphene” in its true meaning of the term. Despite these limitations, the present findings, especially that on the role of Ni carbide formation, will greatly contribute to the improvement of Ni-assisted graphene formation on Ni/SiC as well as ohmic-contact formation for both graphene and SiC devices.


Staphylococcus aureus (MSSA). This study aimed to assess the surrogate predictive value (SPV) of oxacillin and cefoxitin for the susceptibility of commonly utilized parenteral β-lactams against MSSA as well as to evaluate the concordance between predictive susceptibility testing and the in vivo exposures for ceftriaxone. Broth microdilution MICs were determined for cefazolin, cefoxitin, ceftaroline, ceftriaxone, nafcillin, and oxacillin against a national collection of 1238 MSSA from US hospitals. Pharmacodynamic profiling was utilized to establish a clinical breakpoint for commonly utilized doses of ceftriaxone. Oxacillin had good SPVs for all the β-lactams tested, whereas cefoxitin produced unacceptable major errors for all four agents and thus appears to be an unacceptable susceptibility surrogate. While oxacillin is an adequate surrogate based on the currently defined laboratory criteria, our data also suggest that caution should be exercised when incorporating this testing approach in the clinical setting in view of the fact that the MIC distribution of MSSA coupled with the commonly utilized low doses of ceftriaxone may result in inadequate in vivo exposures against this pathogen.Susceptibility testing with the use of surrogate agents is common among clinical microbiology laboratories. One such example is oxacillin and cefoxitin for β-lactams against methicillin-susceptible  Staphylococcus aureus (MSSA) infections [For decades, the parenteral antistaphylococcal penicillins, oxacillin and nafcillin, have been utilized for the treatment of methicillin-susceptible fections . Alternafections . Based ofections . Since cS. aureus were ≤4 μg/mL, 8 μg/mL and ≥16 μg/mL, for susceptible, intermediate, and resistant, respectively assuming the utilization of 2 grams intravenously every 12–24 h [fT > MIC) was insufficient to achieve >90% probability of target attainment (PTA) for MSSA with MICs of ≤8 μg/mL [At the time of this analysis, the FDA breakpoints for ceftriaxone against  12–24 h . This su≤8 μg/mL . OverallMore recently the CLSI eliminated the MIC based breakpoints for many β-lactams for MSSA and now suggests the use of oxacillin or cefoxitin as a surrogate agent for predicting the susceptibility of MSSA to many antimicrobials including ceftriaxone ,9. Althoin vivo exposures for ceftriaxone.Herein, we aimed to (1) assess the predictive value of surrogate testing of oxacillin and cefoxitin for commonly utilized parenteral antibiotics for MSSA; (2) to evaluate the concordance between predictive susceptibility testing and the MSSA were collected from U.S. medical centers over the period of 2011 to 2013. Isolates were obtained from all anatomical sites with the exception of the urinary tract. Once collected, the organisms were transferred to trypticase soy agar slants for shipment to the central laboratory  for MIC determinations.S. aureus 29,213 was used on all MIC trays prior to and during the susceptibility testing. Results were interpreted based on the current CLSI or FDA breakpoint criteria listed in MIC testing was conducted for cefazolin, ceftaroline, cefoxitin, ceftriaxone, nafcillin, and oxacillin using broth microdilution according to CLSI recommendations. For quality control purposes, the ATCC Given the multitude of ceftriaxone doses used clinically, a pharmacodynamic breakpoint of 2 μg/mL was used for ceftriaxone 1 gram every 24 h regimen and the 4 μg/mL value was employed when considering the higher 2 gram every 12–24 h dose of ceftriaxone [n = 196) of isolates had a ceftriaxone MIC of ≤2 μg/mL and remaining 980 (79%) had a ceftriaxone MIC of 4 μg/mL. According to the FDA breakpoint of ≤4 μg/mL, oxacillin’s SPV for ceftriaxone produced 5% minor error. However, when considering the resulting in vivo exposures obtained when administering the 1 gram every 24 h dose and the overall percentage of isolates that had MICs >2 μg/mL, the discordance between the oxacillin SPV and the pharmacodynamic breakpoint for this dose is 84%  isolates that displayed MICs of ≤2 and 4 μg/mL were resistant to cefoxitin, thus major error was observed . In contThe use of ceftriaxone to treat MSSA infections has increased due to its once-daily dosing regimen, absence of dose adjustments in patients with renal dysfunction, and reduced cost . Currentin vivo potency of ceftriaxone when considering the MIC distribution of MSSA as well as the variety of doses used in clinical practice. To add additional confusion for the clinician utilizing ceftriaxone for MSSA infections, both the FDA and the CLSI have now abandoned the use of ceftriaxone specific susceptibility criteria for MSSA, thus the prescriber has no potency reference on which to base decisions regarding the appropriateness of dose. To further emphasize the clinical challenges associates with the appropriate use of ceftriaxone for MSSA, although the package insert advocates a dose of 2 to 4 gm per day for MSSA infections [While oxacillin appears to have acceptable SPVs for the β-lactams tested, careful consideration should be taken with the interpretation of these results as this approach predicts the likelihood of susceptibility based on laboratory criteria, but does not describe the reduced fections , the 1 gfections ,13,14.in vitro pharmacodynamic model [in vitro pharmacodynamic assessment appears to suggest the inadequacy of low dose ceftriaxone regimens for MSSA infection, additional clinical data are required to determine if the poor outcomes are associated with these doses for the management of MSSA in the setting of CABP.In this study, we further highlight the importance of dose selection in order to optimize the pharmacodynamic profile of ceftriaxone for the management of MSSA based infection. As a result of the current MIC distribution of ceftriaxone against this national collection of MSSA, low dosing regimens appear insufficient to drive the most favorable outcomes for this pathogen. To test this hypothesis, Iacovides and colleagues assessed the bactericidal activity of ceftriaxone against MSSA using an ic model . These iin vivo exposures of ceftriaxone.In summary, cefoxitin is frequently associated with major errors and is therefore not a reliable surrogate for the susceptibility of the β-lactams tested. While cefoxitin was a poor surrogate, our data indicate that oxacillin has excellent surrogate predictive value for cefazolin, ceftriaxone, ceftaroline and nafcillin. While oxacillin is an adequate surrogate based on the defined laboratory criteria, our data also suggest that caution should be exercised when utilizing this testing approach because the MIC distribution of MSSA as well as the variety of doses used in clinical practice may result in inadequate