Although the great potential of droplet based microfluidic technologies for routine applications in industry and academia has been successfully demonstrated over the past years, its inherent potential is not fully exploited till now. Especially regarding to the droplet generation reproducibility and stability, two pivotally important parameters for successful applications, there is still a need for improvement. This is even more considerable when droplets are created to investigate tissue fragments or cell cultures (e.g. suspended cells or 3D cell cultures) over days or even weeks. In this study we present microfluidic chips composed of a plasma coated polymer, which allow surfactants‐free, highly reproducible and stable droplet generation from fluids like cell culture media. We demonstrate how different microfluidic designs and different flow rates (and flow rate ratios) affect the reproducibility of the droplet generation process and display the applicability for a wide variety of bio(techno)logically relevant media. The reThis broad range of biological applications is associated with the use of highly diverse culture media possessing different properties regarding e.g. viscosity and surface energy. As culture medium components like proteins or salts significantly determine the surface energy it is obvious that the droplet generation process as well as the droplet behavior inside the micro channels highly depends on the concentration of these components. In this study we concentrate on the characterization of the droplet generation process depending on the kind of aqueous (culture) medium. The investigations focus on the droplet generation micro‐system (DMS) which is of pivotal importance for a reproducible droplet generation process and thus for future reliable assay results. Two key parameters of the DMS have to be tailored in order to achieve a high quality droplet generation: (i) provide perfect geometrical conditions for the reproducible generation of droplets from the aqueous phase and (ii) reduce the adhesion of the aqueous phase to the channel walls past the droplet generation zone.In the first part of this study the reproducibility of the droplet generation from cell culture medium as the disperse (aqueous) phase as a function of the DMS design and the applied flow rates was assessed 22.1Each DMS was assembled from two polycarbonate (PC) plates , the droplet generation micro‐system DMS Fig. A and 1B,s/s had to be introduced. This factor represents the ratio of the droplet length average sD,SP, calculated from the sample volume delivered by the syringes pump (SP) and divided by the number of generated droplets nmax, and the droplet length average sD,OD determined by the optical detection Ts/ss/s was used to calculate the length sTD,nOD of the non‐deformed droplets:In Eq. TD,nOD the exact droplet volumes were calculated. Subsequently, the volume of each individual droplet was determined employing this correction factor that is specific for each experiment. All processes including droplet generation, droplet detection and all calculations were performed using a home made C++‐procedure.From this corrected s2.22F4 as precursor 4F8, Linde, Pullach, Germany, 4.8) was used as a mixture with argon (5.0) containing 5% (v/v) c‐C4F8. The discharge was powered by a TIGRES V20‐901.Atmospheric‐pressure plasma‐activated chemical vapor deposition (PA‐CVD) of perfluorinated amorphous carbon films, then termed a‐C:F in analogy with hydrogenated “diamond‐like” carbon films, a‐C:H, was achieved using a “corona”‐activated CVD process with tetrafluoroethylene C2.32.3.1a =  0 .79 μm +/− 0.05 μm before and after the plasma coating procedure.The surface free energy (including polar and dispersive components) was determined employing the OWRK approach . Briefly, contact angles for the four fluids deionized water, formamide, ethyleneglycol (predominantly polar) and diiodomethane (disperse) were recorded using the OCA System . The investigations were performed on milled polycarbonate plates with a roughness of S2.3.22, resolution: 512 × 512 pixels).Part of the plasma film was removed from a plasma coated glass slide with a scalpel. The thickness of the film was determined at this artificially created edge employing the NanoWizard AFM  fetal calf serum and 0.01% (w/v) phenol red). All experiments were performed with PFD as continuous hydrophobic phase.Experiments with five DMS designs and various flow rate sets were performed to investigate their influence on both, the droplet volume and the droplet volume reproducibility. All deviations were evaluated using the coefficient of variation (CV). The experiments were performed using Dulbecco's Modified Eagle's Medium  of 10, 5 and 2.5 (Table 2).Prior to the experiment the DMS was intensively rinsed with PFD . The experiments were performed with PFD flow rates QTo investigate the droplet generation reproducibility in terms of the flow rate, various experiments with nine different flow rate combinations were performed. These nine experiments representing the first run started with the flow rate combination 250/25 and ended up with the flow rate combination 1000/400 (Table intra‐run‐deviation (standard deviation for each experiment of a single run),inter‐run‐deviation (standard deviation only for the 5/1 ratio experiments of the first and the second run as well as for the third and the 4th run),total inter‐run‐deviation (standard deviation for Series 1 and Series 2 experiments with the same flow rate combination).To investigate the long term behavior of the DMS the first run and the second run experiments were repeated as third and 4th run using the same DMS. The delay between Series 1 and Series 2 was ∼48 h. To evaluate the droplet generation reproducibility three deviation types were defined (Table 2.5In order to estimate the applicability of the coating for bio(techno)logically and biomedically relevant applications, various challenging aqueous media were used for droplet generation employing the 2D 90° DMS . These media comprise anticoagulated blood products , Iscove's modified dulbecco's medium . Furthermore, the surface energy decreased to 10.6 mN/m . The coated surfaces also displayed oleophobic properties (limited wettability with the not‐fluorinated tetradecane) but they highly supported the adhesion of fluorinated oils like PFD (PFD contact angle of 0°).The uncoated PC surface displayed a water contact angle of 88.3° ± 1.4° and a surface tension of 40.4 mN/m (39.6 mN/m dispersive and 0.7 mN/m polar components). After plasma coating with the precursor c‐CThe film thickness was estimated to be in the range of ∼140 nm, since AFM measurements on the identical coatings deposited on glass revealed a film thickness of 143 nm Fig. .3.23.2.1c/Qd: 500/100 and 1000/200. Being given a constant Qc and Qc/Qd ratio, the volumes of the droplets are highly dependent on the design of the DMS . This leads to the formation of a thin PFD film on the surface of the channel walls, which prevents the droplets from contacting the channel surface In the case of the junction DMS the injected PFD pushed the sample fluid against the PFD film being on the channel surface. The resulting pressure led to interactions between the involved interfaces. In the case of 90° DMS the PFD injection led to the largest pressure whereas in the case of 45° DMS there was a pressure component in z‐direction leading to a faster and more reproducible droplet generation Fig. B. These 3.3The influence of the flow rates on the droplet generation process has been described in the literature 3.3.1d a proportional increase in Qc resulted in a decrease of the droplet volume. Furthermore, a proportional increase of Qd at a constant Qc led to the formation of droplets with an increased volume. Within the investigated Qc/Qd range the droplet volume reduction with a decreasing Qc was logarithmic while there was a linear increase in the droplet volume with rising Qd values. These correlations are known from the literature and could be confirmed Given a constant Q3.3.2A strong correlation of the reproducibility of the droplet generation process with the flow rates is displayed by the experimental results. In the case of increasing the flow rate of the disperse phase the reproducibility increased. In the case of increasing the flow rate of the continuous phase the reproducibility also increased, except for the 3D DMS, which behaved reversely. For the 2D 45° DMS there was no clear correlation regarding the reproducibility of the flow rates of both phases.When the flow rate of the continuous phase was increased, the flow velocity rose and consequently the pressure increased. As a result the shear forces that were imposed onto the disperse phase also increased. As a consequence, there was a stronger constriction of the connection between the developing droplet and the disperse phase. The increased force of the continuous phase on the developing droplet obviously caused a faster  and more reproducible droplet generation.When the flow rate of the disperse phase was increased, the flow velocity rose and consequently the injection pressure, too. This also reduced the influence of the viscosity and the interfacial tension, which significantly affected the droplet generation reproducibility. In general, increasing flow rates of the continuous or the disperse phase increased the dynamics of the droplet generation and thus the reproducibility.c and for 3D DMS at low Qc. The reasons for this unusual behaviour of the 3D DMS shall be discussed following. During the droplet generation with the 3D DMS (notably at the flow rate Qc of 1000 μL/min and the Qc/Qd ratios of 5 and 10) it was observed, that the droplet diameter was smaller than the diameter of the microfluidic channel. Consequently, due to the missing stabilization that is normally exerted to the droplet by the wall of the microfluidic channel the droplet generation was inconstant (higher degree of freedom) causing a decreasing reproducibility.Comparing the results of the 2D 90° DMS Fig. A with thCompared to the 90° DMS in the 3D DMS there is a distinct downstream force component caused by the flow of the continuous phase PFD. The horizontal force component pushes the developing droplet downstream. As the vertical component of the PFD flow is smaller than in the 2D 90° DMS, the squeezing zone will be influenced by the pushed developing droplet whereas the influence of the vertical PFD force component becomes smaller. Thus, the droplet pinching–off will be more influenced by stretching the squeezing zone caused by the pushed droplet than by the squeezing effect caused by the vertical component of the PFD flow.Furthermore, in the case of 2D 90° DMS the PFD is guided from the two side channels directly into the main channel. In the case of the 3D DMS the PFD also comes from two side channels but it will be spatially distributed into an annular gap surrounding the main channel like a funnel. The abrupt junction between the side channels (arranged in 2D) and the annular gap (3D) causes undefined flow conditions leading to flow gradients with irregular flow rates. These irregular flow rates also influence the droplet generation.3.4In addition to the DMEM medium further fluidic samples Table  were subAlmost all of the investigated application relevant media could be segmented over a period of 8 h with a high quality, indicating that the DMS coating was stable over that period. However, whole blood  and human plasma slightly reduced the quality of the droplet generation process. For whole blood a perfect droplet generation was observed initially. After a few minutes the whole blood non‐cyclically interacted with the channel wall downstream the droplet generation zone. This adhesion of droplets was predominantly observed at sites at which the lowest thickness of the PFD film was expected (compare chapter 3.2). At the sites of the reduced PFD film thickness the probability of an adhesion of medium components to the channel surface obviously increases. The adhesion induced changes of the surface properties were irreversible and resulted in a decreased reproducibility of the droplet generation process.Varying the flow rates affected the droplet adhesion significantly. At flow rates of 250/25 the adhesion tendency increased while it was almost completely inhibited when the flow rates were raised to 500/100. This may be attributed to the short time period in which an individual droplet may interact with the surface.The exact reason for the high adhesion potential of blood to the channel walls could not be fully elucidated, yet. Since similar effects were observed for both anticoagulated whole blood and plasma (whole blood without cells) it is highly unlikely, that the blood cells play a major role in the changes at the interface . Consequently, it is more likely that coagulation factors interfere with the channel surface, which mask the surface and thus change its physico‐chemical properties. The adhesion would likely lead to an initiation of the coagulation cascade, which was suppressed by the added citrate in these experiments . The hypothesis that coagulation factors may interfere with the droplet generation is further supported by the observation, that media  containing serum  supported a high quality droplet generation process. However, further research on the adhesion of blood cells and coagulation factors will be performed in order to support this hypothesis.+ and OH− ions. We also suspect that the coating is irreversibly damaged since even after rinsing the system with deionized water and PFD the droplet generation process of DMEM was severely affected. The detailed elucidation of how the high H+ and OH− concentrations interfere with the coating (detachment or modification) is the subject of ongoing research.Even more dramatic, negative effects on the droplet generation process were observed when media with extreme pH values were employed. After the generation of a few initial droplets, media with a pH 10 and pH 2 could not be segmented in the DMS but only at the transition zone from the DMS to the PTFE tubing. Since this is a phenomenon usually only observed for uncoated DMS we hypothesize that the coating is not stable against high concentrations of H44F8 precursor is a perfectly suited technology for equipping the surface of microfluidic systems with the hydrophobicity that supports a stable droplet generation process. Consequently, it is possible to perform the droplet generation process without surfactants, which may negatively interfere with the bio(techno)logical applications. By choosing an appropriate DMS design and adjusting the flow rates, the droplet volumes could be tuned in a range of ∼435 nL to ∼1065 nL.A plasma coating of PC materials with the c‐COne of the most relevant parameters of the DMS was its reproducibility, which was dependent on the DMS design and the flow rates. Independent of the design all DMS revealed an excellent reproducibility for short term applications. Furthermore, a high reproducibility for long term applications was observed for high flow rates. These high flow rates of 1000 μL/min resulted in a higher dynamic of the droplet generation with a low variation in droplet volume and are thus preferable. A higher dimensionality of the PFD injection led to an improved reproducibility of the droplet generation process. Similarly, the 2D injection at an angle of 90° resulted in a superior reproducibility as compared to an injection at 45°. Consequently, the 2D 90°DMS is a promising DMS design for a reproducible droplet generation process. Further research will be focused on the development and characterization of the 3D DMS since it is highly likely, that this combination of the dimensionality and the 90° injection angle may further improve the reproducibility.c flow rate was 2000 μL/min which corresponds at a Qc/Qd ratio of 5 to a droplet generation frequency of 10 s−1. Further increasing of the Qc flow rate caused decreasing distances between the droplets which led to undesired droplet contact and, as we operate without surfactants, to fusion of droplets.The upper limit for the QThe here presented plasma coated DMS were demonstrated to be well applicable for the reproducible generation of droplets from a wide variety of bio(techno)logically relevant media. Even the droplet generation from anticoagulated blood products (like whole blood and plasma) was possible although it was less reproducible due to an increased adhesion of droplets to the channel walls in 2D DMS. However, using the 3D DMS this adhesion should be significantly reduced because of the much more pronounced PFD film between the droplets and the channel surface caused by the 3D flow‐focusing effect. Adhesion phenomena depending on the chip geometry and the used fluids will be investigated further.The results presented in this study are the basis for further development of functional chips and applications with delicate samples like suspensions of (multi)cellular systems or even tissue fragments. Both, polymer as DMS material and DMS preparation by plasma coating are convenient and cost effective. Consequently, it will be possible to offer the systems as single use devices for high‐throughput applications in industry and academia.Droplet based microfluidics has a promising application potential in the field of drug screening for personalized medicine or for pharmaceutical research, to mention only a few. Precondition for all applications is a stable and reproducible droplet generation and manipulation as well as cost‐effective equipment like microfluidic chips. In this study we present microfluidic polymer chips that are capable to generate and manipulate aqueous droplets highly reproducibly and without the use of surfactants. The droplets could be used to cultivate even mammalian cells e.g. as spheroids or tissue fragments. The chips could be manufactured by both precision manufacturing, e.g. milling, and injection molding for use as disposables. Thus, for each application the microfluidic channel structure could be milled and tested before molding a higher number of disposable chips. Each chip consists of two symmetrical chip‐halves that can be coated application‐related before assembly.The authors have declared no conflict of interest.


The triboelectric charges, generated by friction with a Pt-coated atomic force microscope tip and injected through defects, are trapped at the air–SiO2 interface underneath graphene and act as ghost floating gates. Tunnelling triboelectrification uniquely permits to create, modify and destroy p and n regions at will with the spatial resolution of atomic force microscopes. As a proof of concept, we draw rewritable p/n+ and p/p+ junctions with resolutions as small as 200 nm. Our results open the way to time-variant two-dimensional electronics where conductors, p and n regions can be defined on demand.Gates can electrostatically control charges inside two-dimensional materials. However, integrating independent gates typically requires depositing and patterning suitable insulators and conductors. Moreover, after manufacturing, gates are unchangeable. Here we introduce tunnelling triboelectrification for localizing electric charges in very close proximity of two-dimensional materials. As representative materials, we use chemical vapour deposition graphene deposited on a SiO Once fabricated, the gates of conventional electronic devices are spatially fixed. Here, the authors introduce tunnelling triboelectrification to create, modify and destroy on-demand ghost floating gates underneath 2D materials, with the spatial resolution of an atomic force microscope. In conventional field-effect devices1357Triboelectrification is the electrical charging by friction between two materials910122 /Si wafer using a wet transfer method. As schematically shown in 2 area using a grounded Pt-coated AFM tip in contact mode, with a force of 15 nN. We verified by AFM that rubbing does not result in detectable mechanical damages to graphene . 2 area after rubbing the 0.5 × 0.5 μm2 central square; the rubbed region has a ∼50 mV higher surface potential than the unrubbed region. Such potential variation may not be attributed to charges stored in the graphene as electric charges may be localized for long times only in insulating materials. We therefore conclude that some of the charges generated during rubbing tunnel through the monolayer graphene and are locally trapped on the underlying insulator. The trapped charges act as an immaterial , bottom floating gate and locally change the polarity and density of charges in graphene as well as the graphene work-function; for simplicity we will therefore refer to these trapped charges as to ghost floating gates. The ghost floating gates effectively control the carriers within graphene because of the ultimate thinness of graphene as well as of the comparably thin air-gap.We deposited CVD graphene on a SiOgraphene  and alsoVTT  as a function of position . As evident, the potential variation is very well preserved even after 72 h. VTT as a function of time and the best fit function (blue line)τshort∼3 h and 21 min), there is a dominant term with much higher initial amplitude and with an exceptionally long time constant /Si substrates, and measured the surface potential of a 2 × 2 μm2 graphene layer both before and after rubbing the 1 × 1 μm2 central area with a Pt AFM tip. Vafter−before), both in the rubbed (ΔVr) and in the unrubbed (ΔVu) parts of the graphene films. ΔVr  and the triboelectric charges would simply charge the graphene–air–SiO2–silicon capacitor. Except for HOPG, the graphene layers are not equipotential as the trapped charges, similar to the presence of ghost floating gates at the SiO2–air interface, locally increase or decrease, depending on their polarities, the graphene potential. In practice, tunnelling triboelectrification and the spread of charges on the entire graphene layer are two competing processes. The spread of charges involves the entire graphene layer and is global; on the contrary, tunnelling triboelectrification is restricted to an area of graphene comparable with the contact area of the AFM tip and, therefore, is extremely localized. As evident from 2–silicon capacitor. By contrast, when increasing the number of layers, tunnelling becomes more and more unlikely and triboelectric charges tend to spread over the entire graphene layer and, consequently, increase or decrease the potential of the entire graphene layer.To gain further insight on tunnelling triboelectrification, we also studied the cases of monolayer (1L), bilayer (2L), trilayer (3L) CVD graphene and highly ordered pyrolytic graphite (HOPG). Similar to the experiments described in 2/Si substrates. 2 (white box region) with the Pt AFM tip. As shown in 22We also performed similar experiments on mechanically exfoliated graphene on SiOx comparable with the very sharp tip of the AFM. In the simplified, lumped elements, equivalent circuit for tunnelling triboelectrification shown in x2) and the global section . Owing to rubbing, a fraction of the triboelectric charges tunnel through graphene and is injected at the interface between air and SiO2 ; the rest of the triboelectric charges  spreads all over the graphene layer. Immediately after tunnelling, the charges QTT, trapped at the air–SiO2 interface, electrostatically attract charges of the opposite type on the graphene layer and/or on the silicon underneath silicon oxide . However, as graphically illustrated in 2 capacitor  of the triboelectric charges injected into a rubbed insulating material can be controlled by the application of a biasing voltage voltages , negativvoltages , positiv density  can be c2, between the sense electrodes S1 and S2 in R/R0 (where R is the resistance of graphene after rubbing and R0 is the resistance before rubbing) as a function of the tunnelling triboelectric charge density  can be dynamically changed over time, possibly resulting in truly time-variant electronic devices and systems. Since AFMs can be fully integrated on a single chip2526+  and p/n++ . Tunnelling triboelectrification occurs in defective CVD graphene, especially for single-layer samples, but not in high-quality exfoliated graphene or in HOPG. We also found a decay time of several days, which is more than two orders of magnitude longer than for standard triboelectrification. Owing to the ultimate thinness of 2D materials and to the sub-nanometer insulating air-gap, the immaterial ghost gates very effectively control charges inside graphene. In striking contrast with conventional electronic devices which, after manufacturing, may not be changed, the ghost floating gates as well as the resulting p and n regions can be created, enlarged, reduced or destroyed on-demand, with the resolution of AFMs and without the intricate process-compatibility issues for integrating many independent conventional field-effect devices on a single chip. Although these immaterial ghost gates are floating, with obvious restrictions for the design of analogue and digital circuits282930312/Si substrate, which is obviously compatible with the integration of CMOS analogue/digital circuits and/or MEMS, including micromachined AFMs. In fact, since AFMs can be fully integrated on a single chip2526333435363738+ and p/p+ junctions with resolutions as small as 200 nm. Our results may greatly facilitate the very large-scale integration of independent 2D field-effect devices and can open the way to time-variant 2D electronics, where conductors , p and n regions can be defined on demand.In conclusion, we have introduced tunnelling triboelectrification for dynamically localizing charges on immaterial, floating, bottom ghost gates underneath 2D materials. We rubbed graphene with a Pt-coated AFM tip and found that a fraction of the triboelectric charges, generated by friction between graphene and the AFM tip, tunnel through graphene and are trapped at the interface between the air-gap and the underlying insulator (SiO2 (1 Torr). Then, the copper foil was annealed for 30 min to clean its surface. The CVD graphene was synthesized by a mixture of CH4 (20 sccm) and H2 (10 sccm) for 30 min (1 Torr). After the growth was complete, the gas supply was ceased and the chamber was cooled below 100 °C at a cooling rate of 160 °C min−1. The CVD graphene synthesized on copper foil was spin-coated with poly(methyl methacrylate) (PMMA) using spin coater with 1,000 r.p.m., 30 s and it was cured at 120 °C for 10 min. Then, the CVD graphene on copper foil was floated in an etchant  to etch away the copper foil. After the copper foil was completely etched away, the graphene with PMMA was rinsed in deionized water three times to wash away the etchant residues. The CVD graphene was transferred onto SiO2 (300 nm)/Si  substrate using a well-known wet transfer method. Multi-layer CVD graphene was prepared by repeating the same processA 75 μm-thick copper foil (Wacopa) was used for graphene growth. The copper foil was placed in a rapid thermal chemical vapour deposition (CVD) chamber, where the temperature was increased from room temperature to 1,000 °C under a 10 sccm flow of HThe rubbing process between a Pt-coated tip  and graphene was carried out by the contact mode of an AFM system  under a contact force of 15 nN and at a scan rate of 1 Hz, to generate triboelectric charges. In the experiments of The data that support the findings of this study are available from the corresponding authors upon reasonable request.How to cite this article: Kim, S. et al. Rewritable ghost floating gates by tunnelling triboelectrification for two-dimensional electronics. Nat. Commun.8, 15891 doi: 10.1038/ncomms15891 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Programmed cell death ligand 1 (PD-L1) is a cell surface glycoprotein that regulates the cellular immune response and serves as a targetable immune checkpoint molecule. PD-L1 is expressed on tumor cells and the immune microenvironment of several human malignancies, including a subset of aggressive lymphomas. We sought to investigate further the clinical and pathologic features of EBV-negative diffuse large B-cell lymphoma (DLBCL) cases that express PD-L1. Immunohistochemical staining using an anti-PD-L1 monoclonal antibody was performed on DLBCL cases from 86 patients. These patients received standard chemotherapy treatment and were followed for up to 175 months. Overall, 14 cases (16%) were considered positive for PD-L1 in tumor cells. In comparison with PD-L1 negative cases, PD-L1 positive cases had a higher rate of non-GCB type , and higher Ann Arbor stage (II-IV) . No significant differences were seen in the immunohistochemical expression of BCL2, MYC, or Ki67. Patients with tumors expressing PD-L1 demonstrated inferior overall survival (OS) upon long term follow up (P=0.0447). Both age/sex-adjusted and multivariate analyses identified PD-L1 as an independent predictor for OS (P=0.0101 and P=0.0424). There was no significant difference, however, in terms of remission rates after first treatment, relapse rates, and progression free survival between the groups. Identification of DLBCL cases that express PD-L1 may serve to select a subset of patients that could further benefit from targeted immunotherapy. Immune checkpoint blockade strategies have revolutionized the approach to cancer therapy and have provided oncologists and patients with novel therapeutic options for a range of malignancies . ImmunotThese findings have provided a rationale for disrupting the PD-1 axis using antibodies against these antigens with the objective of restoring the anti-tumor activity of suppressed T cells. Clinical trials with humanized monoclonal antibodies against PD-L1 and PD-1 have yielded robust, durable responses in patients with advanced malignancies including metastatic melanoma, tumors with mismatch-repair deficiency, and relapsed or refractory classical Hodgkin lymphoma –12. RespBCL2, BCL6, and/or MYC [DLBCL, not otherwise specified (NOS), is the most common type of non-Hodgkin lymphoma among adults and is both clinically and pathologically heterogeneous. Several pathologic features are known to predict a worse clinical outcome for DLBCL patients, including non-germinal center B-cell (non-GCB) phenotype , increasd/or MYC –20. IdenIn the present study, we sought to investigate further the clinical and pathologic features of EBV-negative DLBCL cases with PD-L1 expression. Using a well-annotated cohort of patients with treatment information and long term clinical follow-up, we examined the immunohistochemical expression of PD-L1 in tumor cells and the microenvironment, and correlated these data with additional histologic parameters and clinical outcome data.PD-L1-positive tumor cells, along with PD-L1-positive non-malignant cells, were recorded as a percentage of the total cellularity within tumor sections . The Ki67 labeling index ranged from 10% to 95% . CD10, BCL6, and MUM1 showed significant differences between the PD-L1 positive and negative groups reflecting the non-GCB vs. GCB phenotype differences  received rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) as standard chemotherapy. Other treatment regimens included dose-adjusted etoposide, vincristine, doxorubicin, cyclophosphamide and prednisone (EPOCH); hyper-fractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone (hyper-CVAD); chlorambucil plus prednisone; rituximab alone; methotrexate alone; and radiation alone. None of the patients received hematopoietic stem cell transplant. All patients were followed by clinical and radiological assessments over a period ranging from 2 days to 175 months (median follow-up 21 months).Of 72 patients who received R-CHOP treatment and had available followup information, the overall response rate  was 78% (56/72). No significant differences were found among the three groups . Within the PD-L1 negative group, there was no significant difference in OS between the mPD-L1+ and mPD-L1- subgroups (data not shown).After initial treatment, the median progression-free survival (PFS) time was 18.5 months  and the median overall survival (OS) time was 21 months . For survival analyses, the 86 cases were divided into 2 groups: PD-L1 positive and PD-L1 negative groups (including mPD-L1 positive and mPD-L1 negative cases). Kaplan-Meier curves of PFS and OS showed apparent decreased survival of the PD-L1 positive group , high stage (II-IV) (P=0.0064), and intermediate-high IPI (2-5) (P=0.0120) were statistically significant predictors of OS Table . Non-GCBImmune checkpoint blockade therapy is a promising chemotherapeutic strategy that will likely lead to novel multimodal and combinatorial approaches to treating hematologic malignancies . In the In this study, we correlated PD-L1 expression in EBV-negative DLBCL cases with clinical and pathologic features. In our cohort, 16% of cases were positive for PD-L1 in tumor cells and an additional 27% had positive microenvironment staining. Several recent studies have examined the incidence of PD-L1 positivity in DLBCL, NOS cases and variants of DLBCL, including T-cell/histiocyte-rich large B-cell lymphoma and EBV-positive DLBCL of the elderly , 23–25. In addition to non-GCB phenotype, we found that patients with PD-L1 positive DLBCL had higher-stage tumors than those with PD-L1 negative DLBCL. We also found a trend towards poorer outcomes in terms of PFS and OS for patients with PD-L1 positive tumors. Decreased OS was found to be statistically significant in our cohort. Two analyses of clinical outcome in PD-L1 positive DLBCL have recently been reported , 25. KiyAmong the studies examining PD-L1 expression in DLBCL, it should be noted that the thresholds for determining PD-L1 positivity have varied widely. In earlier studies of PD-L1 staining in DLBCL, a lower threshold, such as 5% tumor positivity, was employed . In termThe finding that a subset of DLBCL cases had PD-L1 expression in non-malignant cells within the tumor microenvironment supports previous studies that observed a distinct proportion of classical Hodgkin lymphoma and DLBCL with this finding . In our PD-L1 and IGH gene loci [In the case of classical Hodgkin lymphoma, at least two independent genetic mechanisms, 9p24 amplification and EBV infection, are thought to lead to overexpression of PD-L1 in the malignant Reed-Sternberg cells , 7. Simiene loci . These cWith the anticipation of additional clinical trials of immunotherapy directed at the PD-1 axis, treatment challenges will include determining the ideal time to initiate targeted immunotherapy; whether to combine immunotherapy with conventional chemotherapy, other immune checkpoint inhibitors, or hematopoietic stem cell transplant; and whether treatment should be continued following the first remission to prevent recurrence. Identification of DLBCL cases that express PD-L1 may form a rational basis for guiding therapy and can serve to select a subset of patients that could further benefit from targeted immunotherapy. Threshold levels of PD-L1 expression will need to be further examined to determine a biologically relevant level of expression that can predict tumor response to therapy and/or predict patient prognosis.Cases of DLBCL, NOS, diagnosed between 2000 and 2014 were retrieved from the surgical pathology files and medical records of our institution. The study was approved by the University of Massachusetts Medical School Institutional Review Board. All DLBCLs were diagnosed and classified according to 2008 World Health Organization (WHO) criteria. Eighty-six DLBCL cases were included, composed of 46 female and 40 male patients, with a median age of 70 years (range 15-91 years). The primary sites involved by DLBCL in descending order of frequency included lymph node (32 cases), soft tissue (18 cases), spleen (5 cases), bone (4 cases), central nervous system (4 cases), skin (4 cases), salivary gland (3 cases), paranasal sinuses (3 cases), small bowel (3 cases), testis (3 cases), breast (2 cases), liver (2 cases), lung (2 cases), and bladder (1 case). All cases were negative for EBV-encoded RNA (EBER) by in situ hybridization study.Immunohistochemistry using a rabbit anti-PD-L1 monoclonal antibody  was performed on 5 μm-thick, formalin-fixed paraffin embedded (FFPE) tissue sections and tissue microarray sections using a Dako Autostainer  with antigen retrieval methods (0.01M citrate buffer at pH 6.0) as described previously [All IHC-stained sections were evaluated and scored by two hematopathologists independently. Discrepancies in scoring (<10% of cases) were resolved by consensus conference between the two pathologists. Threshold values of 30% for BCL2, BCL6, CD10, and MUM1 staining were chosen reflecting routine clinical practice. MYC and Ki67 expression was recorded using a percentage scale of positive tumor ranging from 0% to 100%. Staining intensity of PD-L1 was scored as follows: 0 (no staining), 1+ (weak), 2+ (moderate), or 3+ (strong). Tumor cells exhibiting 2+ or 3+ membrane staining were recorded as a percentage of total tumor cellularity. PD-L1-positive non-malignant cells as a percentage of total tumor cellularity was also recorded. Appropriate external positive (placenta) and negative (tonsil) controls were included with each staining run.Patient demographics, clinical data, treatment and outcome information were obtained from the medical record. Clinical parameters included: presence of B symptoms, performance status, CBC, kidney and liver function tests, lactate dehydrogenase (LDH), bone marrow involvement, other extranodal sites involvement, bulky disease (>10cm), Ann Arbor stage, International Prognostic Index (IPI), positron emission tomography-computed tomography (PET/CT) scan at diagnosis and after treatment, treatment history, response to first treatment , major complications to treatment , the dates of disease progression, relapse or death and cause of death.Comparisons of clinical data between groups were carried out using the Fisher exact test, Chi-Square test, and survival analysis in SAS v9.3  and R 3.2.1. Progression-free survival (PFS) was measured from the date of original diagnosis to the date of last follow-up or of the first progression, relapse or death, toxicity events being excluded. Overall survival (OS) was measured from the date of original diagnosis to the date of last follow-up or death from any cause. PFS and OS were estimated using the Kaplan-Meier method. Differences of PFS or OS at different PD-L1 expression levels were assessed using the Log-Rank tests at the two-sided significance level of 0.05.Age and sex-adjusted and multivariate analyses were conducted to evaluate the potential association between PD-L1 expression as well as other covariates with clinical outcomes. The stratified Cox proportional hazards model was used to determine hazard ratios and confidence intervals (CI) at the 95th confidence level. The variables included in multivariate analysis for OS were age, sex, expression of PD-L1, GCB/non-GCB type, stage and IPI. All of these variables had a P-value <0.1 in the age- and sex-adjusted analysis. Occasional missing values in the data set did not appear to present significant problems in the modeling strategy. The proportional hazard assumptions were satisfied in both age and sex-adjusted and multivariate analyses based on the log-log plots and Schoenfeld residuals method.


On the other hand, the WF of graphene increases to ~5.38 eV on F-SAM. By tuning the WF of graphene, an upward band bending is found at the ZnPc/graphene interface on F-SAM. At the interface between C60 and NH2-SAM modified graphene, a downward band bending is observed.The impact of graphene work function (WF) on the electronic structure at the graphene/organic interface has been investigated. WF manipulation of graphene is realized using self-assembled monolayers (SAMs) with different end groups. With this method, the upper surface of the functionalized graphene remains intact, and thus precludes changes of molecular orientation and packing structures of subsequently deposited active materials. The WF of NH Graphene is a promising alternative to transparent electrodes in organic electronic devices ,5,6,7,8,3 has been utilized for hole injection enhancement in related devices [3, was employed to decrease the WF of the electrode for better electron injection. In addition to the insertion of an interfacial layer, the manipulation of the graphene WF can also be realized using plasma treatments. O2 or SF6 plasma treatments have been used to increase the WF of graphene, which was attributed to the presence of oxygenated or fluorinated species on the graphene basal plane [The insertion of an interfacial layer is one of the most commonly used surface functionalization methods to manipulate the WF of electrodes. Poly :poly (styrenesulfonate) (PEDOT:PSS) facilitated the extraction of holes at the active layer/anode interface . Moreove devices ,27,28. I devices ,30,31, ial plane ,33,34. Aal plane . It is wal plane ,38,39,402 substrates. After the formation of SAMs, graphene was transferred on the top of SAMs, leaving the upper surface of SAM functionalized graphene intact as pristine graphene. Without the insertion of an interfacial layer or plasma treatments, the molecular orientation and packing structures of subsequently deposited active materials remained unchanged, which helped to separate the impact of the graphene WF from molecular orientation and packing structure changes. Using SAM modified graphene, we have successfully tuned the Fermi level of graphene without disturbing molecular orientations and packing structures of active materials on top. Model systems of graphene/organic molecule interfaces have been constructed, which simplifies factors affecting the interface energy levels compared with previous works.Recently, n-type and p-type doping of graphene using self-assembled monolayers (SAMs) with different end groups has been demonstrated ,42. In g2-SAM, and the WF decreased to ~3.90 eV; in the case of graphene on F-SAM, its Fermi level moved downward, and the graphene WF increased to ~5.38 eV. The influence of the graphene WF on the interfacial energy levels was been investigated. An upward band bending was observed at the interface between ZnPc and F-SAM functionalized graphene, while a downward band bending was found for the growth of C60 on NH2-SAM functionalized graphene.In the present study, we demonstrated the manipulation of the graphene WF using different SAMs. As revealed by ultraviolet photoelectron spectroscopy (UPS) results, the Fermi level of graphene shifted upward on NH2 substrate was treated with O2 plasma for 15 min to generate a hydrophilic surface for the formation of NH2-SAM and F-SAM  water. Finally, they were dried by nitrogen flow. In the case of NH2-SAM, 0.8 mL 3-aminopropyltriethoxysilane  was added into a sealed vial together with clean SiO2 substrates under nitrogen environments. The vial was then heated to 80 °C and kept for 2 h before being thoroughly washed with toluene, methanol  and DI water. The functionalized SiO2 wafer was then immerged in DI water for more than 12 h at room temperature (RT), which is helpful to complete the hydrolysis of residual ethoxy groups. Finally, NH2-SAM functionalized SiO2 substrates were dried with nitrogen flow. For the formation of F-SAM, clean SiO2 substrates were placed in a sealed vial filled with 0.5 mL 1H, 1H, 2H, 2H-perfluorooctyltriethoxysilane (Sigma-Aldrich). The sealed vial was then heated in an oven at 120 °C for 1 h. After, F-SAM functionalized SiO2 wafers were rinsed by toluene, methanol, and DI water to remove the physically adsorbed F-SAM precursor molecules. Finally, they were dried by nitrogen gas. The SiO2, and O2 was introduced into the LPCVD chamber when the pressure reached 0.01 Pa. The Ar and H2 flow rates were kept at 600 sccm and 100 sccm during growth, which was calibrated with flow meters; the O2 flow rate varied from 0 to 0.1 sccm in different growth stages. Thermal treatments at 1050 °C to copper foil lasted for 1 h and kept for 2 h for better crystallinity before 0.5 sccm methane was introduced into the LPCVD chamber. In order to obtain graphene with good quality and large size, the growth of graphene lasted for 1 h, and then the system was cooled to RT. Graphene samples were transferred to SiO2, NH2-SAM/SiO2, and F-SAM/SiO2 substrates by commonly used method using poly(methyl methacrylate) (PMMA). In order to remove organic residues and contaminants during the transfer process, graphene/SiO2, graphene/NH2-SAM, and graphene/F-SAM were thoroughly cleaned by acetone vapor. After, all graphene samples were loaded into a quartz tube with base pressure greater than 5 × 10−2 Pa, and then heated to 150 °C for 15 min under H2 environment.The growth of graphene on copper foil was in a custom designed low-pressure chemical vapor deposition (LPCVD) system . A combi−10 mbar. For UPS measurements, He I (21.2 eV) was the excitation source. A −5 V sample bias was applied during WF measurements. XPS measurements were performed with an Al Kα source. Before deposition, ZnPc and C60 were thoroughly degassed overnight, and then thermally evaporated onto graphene, NH2-SAM modified graphene, and F-SAM modified graphene in the growth chamber. The deposition rate for ZnPc and C60 was 0.1 and 0.2 nm/min, respectively. The nominal thicknesses of ZnPc and C60 were determined with the attenuation of Si 2p peak after deposition. Using a Renishaw inVia Raman Microscope,  Raman spectra were acquired. The wavelength of the excitation laser was 514 nm. During measurements, focused laser spot of ~1 μm, power of ~1 mW and integration time of 10 s were chosen.A multifunctional ultrahigh vacuum (UHV) VT-SPM system  was used for in situ UPS and X-ray photoelectron spectroscopy (XPS) measurements. The base pressure in the analysis chamber was greater than 3 × 102-SAM and F-SAM on SiO2 substrates are shown in 2-SAM and F-SAM are determined by the UPS spectra at the low kinetic energy region (2-SAM is ~3.46 eV, and it is ~5.56 eV for F-SAM on SiO2 substrates, which agrees well with the previously reported values [2-SAM, F 1s and C 1s spectra for F-SAM, respectively. For F-SAM on SiO2 substrates, two peaks can be identified in the C 1s region. The peak located at ~292.8 eV derives from carbon atoms in CF2 and CF3 species; the peak located at ~285.5 eV is attributed to carbon atoms from the alkyl chain of F-SAM. The XPS results shown in 2-SAM and F-SAM on SiO2 substrates in the present study.The UPS spectra for NHy region a; the WFd values . Figure 2-SAM, SiO2, and F-SAM are shown in 2-SAM, the WF decreases to ~3.90 eV, which is evidenced by the shift of secondary electron cutoff toward the lower kinetic energy part. The decrease of the sample WF suggests that electrons transfer from NH2-SAM to graphene and thus it upward shifts its Fermi level. On the other hand, the sample WF change is in the opposite direction for graphene on F-SAM. The secondary electron cutoff shifts to the higher kinetic energy part (3 group, electrons transfer from graphene to F-SAM and thus lowers its Fermi level [2-SAM. These shifts of characteristic Raman peaks suggest that doping of graphene has been successfully realized using SAMs [The UPS spectra for graphene on NHrgy part a, indicami level . The maning SAMs . 60, were chosen to investigate the impact of graphene WF changes. UPS spectra with the increasing thickness of ZnPc up to 5.0 nm on graphene/F-SAM and graphene/NH2-SAM are displayed in 2-SAM differs from the growth of ZnPc on graphene/F-SAM, and no notable vacuum level shift is observed with the increasing coverage of ZnPc, which is evidence for vacuum level alignment at the ZnPc/graphene interface on NH2-SAM. As shown in 2-SAM, we can clearly observe that the HOMO leading edge is at ~1.04 eV. Despite the increasing peak intensity with the increasing coverage, no binding energy shift for the HOMO leading edge of ZnPc is found. After the deposition of 5.0 nm ZnPc, it still locates at ~1.04 eV below the Fermi level. Moreover, similar behavior is observed with the increasing coverage of ZnPc on graphene/SiO2 (2, and graphene/NH2-SAM are shown in 2-SAM modified graphene, the Fermi level locates in the middle of ZnPc band gap, and hence charge transfer is impeded at the interface. As a result, there is little or no shift of vacuum level as well as the HOMO leading edge of ZnPc.As we mentioned in ene/SiO2 ; when th60, a typical n-type organic semiconductor with relatively high ionization potential [60 on graphene/F-SAM and graphene/NH2-SAM are shown in 60, a clear sign of vacuum level alignment at the interface between C60 and F-SAM modified graphene. In addition, no binding energy shift of the C60 HOMO leading edge is found. After the deposition of 5.0 nm C60, we can see that the HOMO leading edge of C60 is ~0.96 eV. The UPS results for the growth of C60 on graphene/SiO2 are similar to that on graphene/F-SAM. There are no vacuum level and HOMO leading edge shifts during the growth of C60 (60 increases to 5.0 nm, it locates at ~1.70 eV below the graphene Fermi level. Unlike the growth of C60 on pristine graphene and graphene/F-SAM, an unambiguous downward band bending can be recognized after the growth of C60 on graphene/NH2-SAM. The HOMO leading edge of C60 is ~2.06 eV when 0.5 nm C60 has been evaporated onto graphene/NH2-SAM, and an upward shift to ~1.78 eV below the Fermi level is observed when the thickness of C60 increases to 5.0 nm. Because of the n-type doping of graphene, the WF of NH2-SAM modified graphene decreases to ~3.90 eV, which is even lower than the EA of C60 (~4.18 eV as shown in 60 is lower than the graphene Fermi level on NH2-SAM. Therefore, spontaneous electron transfer from NH2-SAM modified graphene to C60 takes place upon deposition, resulting in the downward band bending. The schematic energy level diagrams at the interface between C60 and graphene/F-SAM, pristine graphene, and graphene/NH2-SAM are shown in The growth behavior of Cotential , on prish of C60 . When th2-SAM decreases to ~3.90 eV, and increases to ~5.38 eV on F-SAM. Spontaneous electron transfer from ZnPc to F-SAM modified graphene occurs, which is induced by the downward shift of the graphene Fermi level on F-SAM, leading to an upward band bending at the interface. In contrast, we identify a downward band bending at the interface between C60 and NH2-SAM modified graphene, which can be attributed to the upward shift of the graphene Fermi level on NH2-SAM. By using SAMs to manipulate the Fermi level of graphene, the electronic structure at the graphene/organic interface is optimized without disturbing molecular orientation on top, which facilitates the charge injection or collection at the electrode. This can greatly enhance the performance of graphene-based organic electronic devices, and shed light on interfacial engineering approaches for other kinds of transparent electrodes.The impact of graphene WF on interfacial energy levels between graphene and organic molecules has been investigated by tuning its Fermi level using SAMs with different end groups. The WF of graphene on NH


The goal is to evaluate avelumab, an anti-PD-L1 monoclonal immunoglobulin G antibody labeled with zirconium-89 in human PD-L1-expressing cancer cells and mouse xenografts for clinical translation.89Zr]Zr-DFO-PD-L1 monoclonal antibody (mAb) was synthesized using avelumab conjugated to desferrioxamine. In vitro binding studies and biodistribution studies were performed with PD-L1+MDA-MB231 cells and MDA-MB231 xenograft mouse models, respectively. Biodistributions were determined at 1, 2, 3, 5, and 7 days post coinjection of [89Zr]Zr-DFO-PD-L1 mAb without or with unlabeled avelumab .[89Zr]Zr-DFO-PD-L1 mAb exhibited high affinity (Kd ∼ 0.3 nM) and detected moderate PD-L1 expression levels in MDA-MB231 cells. The spleen and lymph nodes exhibited the highest [89Zr]Zr-DFO-PD-L1 mAb uptakes in all time points, while MDA-MB231 tumor uptakes were lower but highly retained. In the unlabeled avelumab dose escalation studies, spleen tissue–muscle ratios decreased in a dose-dependent manner indicating specific [89Zr]Zr-DFO-PD-L1 mAb binding to PD-L1. In contrast, lymph node and tumor tissue–muscle ratios increased 4- to 5-fold at 20 and 40 µg avelumab doses.[89Zr]Zr-DFO-PD-L1 mAb exhibited specific and high affinity for PD-L1 in vitro and had target tissue uptakes correlating with PD-L1 expression levels in vivo. [89Zr]Zr-DFO-PD-L1 mAb uptake in PD-L1+tumors increased with escalating doses of avelumab.[ Overexpression of PD-L1 has been observed across a wide variety of human cancers including skin, blood, lung, breast, ovarian, gastric, and prostate and is linked to a worse prognosis.10 Therapeutics designed to block PD-1 receptor binding to its respective ligand have been shown to be beneficial in reversing the immunosuppressive environment.12The programmed cell death-1 receptor (PD-1)/ programmed cell death ligand 1 (PD-L1) pathway has emerged as an important immunotherapeutic target for cancer.13 In these clinical trials, durable responses were observed but only in a subset of patients who were not all identified as positive for PD-L1 expression using immunohistochemistry (IHC) from tumor biopsy tissue.14 Although tumor expression of PD-L1 determined from IHC methods has shown promise as a predictive biomarker for patient selection and therapeutic responses, these IHC results have not been conclusive. For instance, some responders were scored as PD-L1 negative, and some nonresponders were scored as PD-L1 positive.16 This may relate to differences between in vivo “real-time” expression compared to IHC results from an earlier biopsy specimen. Additional issues include differences in detection levels of the PD-L1 mAb used for the IHC and the inherent sampling limitations of biopsy specimens. Moreover, most studies score tumor PD-L1 expression and may not include the expression on other cells in the tumor microenvironment which may be an important factor in determining therapeutic responsiveness.1719To date, several monoclonal antibodies (mAb) targeting the PD-1/PD-L1 pathway have been approved by the Food and Drug Administration (FDA) including nivolumab and pembrolizumab.1/2 = 12.7 hours) and was shown to have potential for PD-L1 immuno-PET imaging in preclinical human tumor xenograft mouse models in which tumor uptakes of copper-64 labeled atezolizumab were consistent with known tumor PD-L1 expression levels.20 Although these tumors had high uptake levels and were discernible in biodistribution and imaging studies, respectively, some nontarget tissue uptake was seen as well. These studies would suggest that radiolabeling the mAb with a longer-lived radionuclide (>12.7 hours) would make possible imaging at later times, thereby achieving higher tumor to background ratios due to increased clearance.Radiolabeling of these antibodies could provide in vivo expression levels of PD-L1 in tumors which could help select patients for this therapy. While the high affinity and specificity of these therapeutic molecules readily fulfill a primary requirement for a successful positron emission tomography (PET) imaging agent, these large molecular weight molecules require longer lived PET radionuclides to account for the slower clearance from non-target tissue. Atezolizumab is one of the new therapeutic anti-PD-L1 mAb, which has been radiolabeled with copper-64 (t1/2= 78.4 hours) and evaluated in vitro and in vivo in PD-L1 expressing human tumor cells and mouse xenograft models, respectively. In vivo biodistribution evaluations with escalating doses of unlabeled mAb were also included to determine whether tumor uptake may be improved with prior specific mAb dose loading which was previously reported with indium-111 radiolabeled atezolizumab. This fully humanized mAb recognizes both human and murine PD-L1 with high affinity and functions not only as an immune checkpoint inhibitor but also as a mediator of antibody-dependent cellular toxicity (ADCC).21 Avelumab has shown efficacy in preclinical mouse bladder cancer models and is currently in human clinical trials in which positive patient responses were observed but not reliably predicted by PD-L1 IHC.23 This work represents a prelude to potential clinical PET imaging with zirconium-89 avelumab which may establish the relevance of tumor PD-L1 expression as a biomarker for patient selection and identifying early therapeutic responses. The overall goal from these preclinical studies was to collect the in vitro binding data and the human dosimetry estimates of zirconium-89 labeled avelumab required for an IND filing for clinical application.In this report, avelumab, a therapeutic anti-PD-L1 monoclonal immunoglobulin G (IgG) antibody (PD-L1 mAb) was labeled with the longer-lived radionuclide, zirconium-89 (tp-isothiocyanatobenzoyl- desferrioxamine (DFO-Bz-NCS) was purchased from Macrocyclics, Inc. . Sodium acetate and Tris-HCl were purchased from Thermo Fisher Scientific . Whole human serum was obtained from MP Biomedicals, LLC . All other chemicals and solvents were received from Sigma Aldrich  and used without further purification. PD-10 desalting columns were obtained from GE Healthcare Biosciences . Zirconium-89 oxalate was obtained from the National Institutes of Health cyclotron facility . Instant thin-layer chromatography (iTLC) papers were purchased from Biodex Medical Systems, Inc. . The iTLC papers were developed using 20 mmol/L citric buffer (pH 5) and read in an Eckert & Ziegler TLC scanner . Analytical high-performance liquid chromatography (HPLC) analyses were performed on an Agilent 1200 Series instrument equipped with a multi-wavelength UV detector connected in series with a Bioscan flow count radiodetector. The size-exclusion column , TSKgel SuperSW3000, was obtained from Tosoh Bioscience LLC . HPLC condition: eluent, 0.1 mol/L sodium phosphate, 0.1 mol/L sodium sulfate, 0.05% sodium azide, 10% iso-propyl alcohol (pH 6.8), flow rate, 0.3 mL/min. BCA Protein Assay Kit (Thermo Fisher Scientific) with bovine gamma globulin standard was used to determine the conjugate concentrations.PD-L1 mAb  was provided by Dr John Greiner . The 2 in RPMI-1640 ([MDA-MB231 (human breast adenocarcinoma)]), RPMI-1640 + 0.1 mmol/L NEAA + 1 mmol/L sodium pyruvate (MKN-45 [human gastric carcinoma]) and RPMI-1640 ATCC modified with10 mmol/L HEPES, 4500 mg/L glucose . All media were supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine and Pen/Strep/Amphotericin B.Cell lines were grown at 37°C in 5% COp-isothiocyanatobenzyl-desferrioxamine (DFO-Bz-NCS) was conjugated with lysine residues of PD-L1 mAb to produce DFO-PD-L1 mAb following the literature method.24 Briefly, PD-L1 mAb (10 mg/mL) was buffer exchanged into 0.1 mol/L NaHCO3 , concentrated to 5 mg/mL, and treated with a 5-fold molar excess of DFO-Bz-NCS (5 mg/mL in DMSO). The mixture was gently rocked at 37°C for 75 minutes before stopping the conjugation reaction by the addition of 1 mol/L Tris . The DFO-PD-L1 mAb conjugate was purified twice by 2 PD-10 columns (spin protocol) into 0.25 mol/L sodium acetate (pH 5.5). Purity of PD-L1 mAb and DFO-PD-L1 mAb conjugates were determined by HPLC using size exclusion column (SE-HPLC). The concentration was measured using bicinchoninic acid assay (BCA). The number of chelators per PD-L1 mAb was determined using 25 mmol/L nonradioactive zirconium choloride mixed with a trace amount of zirconium-89 following a literature method.25The bifunctional chelating agent 89Zr]Zr-DFO-PD-L1 mAb was prepared according to the method described in the literature with minor modifications.24 Briefly, zirconium-89 (74-111 MBq) in 1 mol/L oxalic acid was neutralized with Na2CO3 (2 mol/L). The reaction was incubated for 3 minutes. HEPES buffer  and gentisic acid solution  were added to the reaction and mixed. The pH of the reaction was adjusted between 7.3 to 7.5 with Na2CO3 (2 mol/L). DFO-PD-L1 mAb in 0.25 mol/L sodium acetate  was added, and the pH was readjusted between 7.3 to 7.5 with Na2CO3 (2 mol/L). The reaction was incubated for 1 hour at room temperature before challenging with DTPA  for an additional 10 minutes. The progress of the reaction was monitored by iTLC. The iTLC papers were developed with 20 mmol/L citric buffer (pH 5) and read in an Eckert & Ziegler TLC scanner . The radiolabeled conjugate was purified by PD-10 column using 0.9% NaCl (pH 7). The molar activity and the purity of the radiolabeled conjugate were determined by HPLC (Rt = 9.5 minutes) using size exclusion column. Human serum albumin (20% w/v solution) was added to the radiolabeled conjugate to obtain a final concentration of 1%. The stability of the radiolabeled conjugate in whole human serum at 37°C was assessed by SE-HPLC up to 7 days .[d and Bmax using plated cells  or cells in tubes  to which increasing concentrations of [89Zr]Zr-DFO-PD-L1 mAb (0.15-52 nmol/L) were added to duplicate tubes; nonspecific binding was determined by adding unlabeled PD-L1 mAb (10−6 mol/L) to another set of duplicates. For competition studies, [89Zr]Zr-DFO-PD-L1 mAb at a single concentration (0.15-2.9 nmol/L) and increasing concentrations (0-1000 nmol/L) of competitors (PD-L1mAb) were added to MDA-MB231 or HCC-827. After incubation , the cell bound radiolabeled PD-L1mAb was separated from free antibody: (1) plated cells were washed with phosphate buffered saline (PBS), treated with trypsin, and collected in vials or (2) cells in tubes were pelleted by centrifugation, washed twice (PBS), and supernatants removed. The cell bound radioactivity for these samples was determined by γ counting . From the saturation studies, the Kd and Bmax were determined from 6 to 8 concentrations of radiolabeled PD-L1 mAb and analyzed using nonlinear regression curve fitting (one-site specific binding); from the competition studies, Ki was determined from 8 competitor concentrations  to which a constant concentration of [89Zr]Zr-DFO-PD-L1 mAb (0.15-0.40 nmol/L) was added. Nonspecific binding was determined by adding unlabeled PD-L1 mAb (10−6 mol/L) to another set of duplicates. All assay tubes were then incubated at 37°C for 1 hour, and the procedures performed to remove the free antibody were as described above. For the other method described by Morris, the percentage of immunoreactivity was determined by a self-displacement method derived from a radiolabeled PD-L1 mAb saturation curve and competition curve using as the competitor, unlabeled PD-L1 mAb as described earlier.28The biological molar activity or immunoreactive fraction (% immunoreactivity) of the [6) in PBS:30% matrigel. All animal studies were performed in accordance with NIH Guidelines for the Care and Use of Laboratory Animals using IACUC-approved protocols.Athymic female nude mice  were injected subcutaneously in the right thigh with either MDA-MB231 (2-5 × 1089Zr]Zr-DFO-PD-L1 mAb  and euthanized  at selected times. Blood samples and tissues were excised from each animal, weighed, and radioactivity content was determined (Perkin Elmer 2480 Wizard3). Radioactivity content in the blood and each tissue was expressed as % injected dose per gram of tissue  and then normalized for body weight to a 20 g mouse (2):Tumor-bearing mice (tumor weights: 0.1-0.8 g) were injected while awake via the tail vein with [tissue) / (tissue weight (g))] / [cpm total injected dose] × 100(1) %ID/g = [(counts per minute (cpm) normalized to a 20 g mouse = (%ID/g) × [(body weight)/(20 g)](2) % ID/g t test with P < .05 as significant (GraphPad In Stat 3 for Windows).Statistical analysis of the differences between the 2 groups was done using the Student 2 (1.5%-3% v/v) and imaged at various times after intravenous injection of [89Zr]Zr-DFO-PD-L1 mAb . Whole-body static PET images were obtained at 2 bed positions  followed by CT images  using the BioPET . The images were reconstructed by a 3-dimensional ordered subsets expectation maximum (3D-OSEM).Tumor-bearing mice were anesthetized with isoflurane/OHuman dosimetry estimates extrapolated from the mouse biodistribution studies were calculated using OLINDA V1.1  with mouse to human fractional organ extrapolation of the mean residence times of the ligand measured by the biodistribution described earlier. The %ID/g values (determined from the biodistribution studies described above) for a set of organs determined over a 7-day time course were used to extrapolate human dosimetry in the same organs. The whole organ was dissected from the carcass and counted to measure the organ radioactive content. For the bone, skin, muscles, and blood samples, a sample was dissected, weighed, and counted in the γ counter. Because of the relatively small uptake in the skin, muscle, and blood, these tissues were not included in the kinetic input form of the OLINDA dosimetry estimation software.29 This calculation gives an estimate bone mass of 1.07 g for a 20 g mouse. The %ID/g at each time point for each mouse was multiplied by the bone mass fractional estimate for each mouse.Biodistribution data showed radioconjugate uptake in the mouse skeleton above background. To account for this, a special case was made for bone dosimetry estimation. Instead of including the bone activity in the body remainder volume, the whole bone activity was estimated and entered into the trabecula bone input field in OLINDA. The bone tissue %ID/g and the %ID/organ were estimated using a murine bone fraction model of 53.3 (g/kg).30 The result was that 75% of the activity was assigned to the small intestine and 25% to the large intestine. To further separate out the large intestine into upper large intestine and lower large intestine, the ICRP 80 standard of 57% contributes to the upper large intestine, and the remaining 43% was assigned to the lower large intestine.From the %ID/organ, time–activity curves (TAC) were generated from PET images, and residence times were calculated in units of hours. Between the time points of the TAC, a trapezoidal model was used to estimate the area under the curve. For the last time point, an exponential decay curve with the half-life of zirconium-89 was used to extrapolate the tail of the TAC. Since the %ID/organ of the whole intestine was measured (including the contents), the absorbed activity between the large and small intestine was determined using the MOBY fractional mass model for a 25 g mouse.All tissues were routinely processed and sectioned at 5 µm for automated hematoxylin and eosin (H&E) staining and digitized with an Aperio ScanScope XT (Leica) at 200× in a single z-plane. Slides and digital images were reviewed by a board-certified veterinary pathologist.H score .Immunohistochemistry (IHC) staining was performed on Leica Biosystems BondMax autostainer with the following conditions: heat-induced epitope retrieval for 20 minutes and human-specific PD-L1  dilution 1:250 for 30 minutes. Positive control tissues included human thymus. Negative controls included isotype control reagents substituted for the primary antibody. For mouse-specific PD-L1, the following conditions were used: heat-induced epitope retrieval with Decloak Citrate for 10 minutes, mouse-specific PD-L1  at 1:500 dilution overnight at 4°C. Positive controls included mouse spleen and negative controls included isotype control reagents substituted for the primary antibody. Slides were digitized with an Aperio ScanScope XT (Leica) at 200X in a single z-plane. Slides and digital images were reviewed by a board-certified veterinary pathologist and staining was quantified using automated image analysis algorithms from which H scores were determined. The intensity of staining was evaluated according to the following scale: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The proportion of all cells  found to express PD-L1 was determined and then multiplied by the staining intensity score to obtain a final semiquantitative 89Zr]Zr-DFO-PD-L1 mAb at 37°C (80% intact after 7 days).The DFO-PD-L1 mAb conjugate was prepared using a 5-fold molar excesses of DFO-BZ-NCS. HPLC chromatogram (size exclusion) at 280 nm indicated no aggregation of protein due to the conjugation reaction. The radiotitration assays revealed an average of 1.4 ± 0.2 DFO chelator per mAb molecule. Radiolabeling reactions with zirconium-89 proceeded at room temperature, and the isolated radiochemical yields were in the range of 80% to 95% (n = 30). The molar activities of the radioimmunoconjugates were 22,200 to 70,300 MBq/µmol (n = 30) with >95% radiochemical purity as confirmed by SE-HPLC . SE-HPLC chromatogram revealed the slow decomposition  of [89Zr]Zr-DFO-PD-L1 mAb exhibited high specific binding and affinity for PD-L1 with a Kd of 0.392 ± 0.0481 nmol/L (n = 7) in both MDA-MB231 (moderate/low PD-L1 expression) and HCC-827 cells  than MDA-MB-231 cells  were 25- to 12-fold lower than HCC-827 and MDA-MB231 cells, respectively, with the majority of the bound [89Zr]Zr-DFO- PD-L1 mAb representing nonspecific binding , 0.43 ± 0.048 nmol/L (n = 7), compared favorably with the Kd of [89Zr]Zr-DFO-PD-L1 mAb denoting that the conjugation with desferrioxamine (DFO) and radiolabeling with zirconium-89 had not altered the high-affinity binding to PD-L1 (89Zr]Zr-DFO-PD-L1 mAb determined by the Lindmo antigen excess method and Morris self-displacement method was 91.0% ± 3.4% (n = 6 batches) and 92.0% ± 3.4% (n = 6 batches), respectively (89Zr]Zr-DFO-PD-L1 mAb was highly retained and reproducible across batches.The Kto PD-L1 . The bioectively . Togethe89Zr]Zr-DFO-PD-L1 mAb would be appropriate for in vivo tumor imaging with low to moderate PD-L1 expression (>20 000 receptors per cell) as was observed with the HCC-827 and MDA-MB231 cells, whereas tumors with lower PD-L1 expression (<2200 sites per cell) as in the case of MKN-45 tumors would not be clearly discernable.These in vitro results indicate that [89Zr]Zr-DFO-PD-L1 mAb was determined in MDA-MB231 xenografts, at 1, 2, 3, 5 and 7 days postinjection (89Zr]Zr-DFO-PD-L1 mAb distributed rapidly and cleared from nontarget tissues except for the femur over the 7-day time course (89Zr]Zr-DFO-PD-L1 mAb was highly retained in MDA-MB231 tumors from day 1 (2.8%ID/g) to day 5 (3.0%ID/g) with a 13% loss occurring at day 7 (2.4%ID/g) compared to day 1. The lower MDA-MB231 tumor uptake indicates lower levels of PD-L1+ compared to the spleen and lymph nodes which agrees with the known modest PD-L1 expression levels in this cell line determined in vitro. Femur uptakes (%ID/g) increased 1.7-fold from day 1 (7.6%ID/g) to day 7 (13.2%ID/g) likely indicating loss of zirconium-89 from the DFO chelate that would be expected to localize in the bone (32 The highest tissue to muscle ratios (T: M) occurred in the spleen at day 2 (48:1) and day 3 (46:1) with decreases in T: Ms observed at day 5 (39:1) and day 7  only group (89Zr]Zr-DFO-PD-L1 mAb (2 µg) only group  co-injected with Zr-DFO-PD-L1 mAb uptakes in these tissues represent specific targeting to PD-L1 , associated mass ∼ 20 µg; 89Zr]Zr-DFO-PD-L1 mAb imaging would be able to detect PD-L1+ lesions with low to moderate PD-L1 concentrations.Small animal PET imaging studies were performed with MDA-MB231 xenograft mice at 1,2, 3, 6, and 7 days postinjection of [89Zr]Zr-DFO-PD-L1 mAb biodistribution results with the MDA-MB231 xenograft mice, the radioactivity content for the organs and tissues over the imaging time course was determined and used for extrapolation for radioconjugate residence times in humans  compared to the dose with no protein loading .37 The increased tumor uptake of the radiolabeled antibodies could be explained by the longer blood circulation times resulting in increased levels of the radiolabeled antibody to be delivered to the tumor mass. The protein loading dose also would block specific uptake in non-tumor binding sites such as the spleen allowing a greater blood concentration of radiolabeled antibody available for tumor uptake. This interaction may explain the increased [89Zr]Zr-DFO-PD-L1 mAb tumor uptake observed with increased loading doses of unlabeled PD-L1 mAb in this study. The implication of this finding is that for translation to human imaging studies, an infusion of unlabeled antibody prior to radiolabeled antibody injection would improve tumor uptakes and diagnostic potential. However, this dose of avelumab would still result in serum levels below that which is expected to elicit a treatment response.These results are similar to previous reports in which tumor targeting and uptake of the radiolabeled antibody were affected by the total antibody protein dose.d∼ 0.4 nmol/L) affinity of [89Zr]Zr-DFO-PD-L1 mAb for human PD-L1, it would be expected to detect lower concentrations of PD-L1 compared to other nanomolar affinity probes such as [89Zr]Zr-atezolizumab , [89Zr]Zr-C4 mAb (IC50= 9.9 nmol/L), zirconium-89 labeled anti-PD-L1 domain Ab, KN035  and the radiolabeled PD-L1 specific peptide, [64Cu]Cu-WL12 (EC50 =2.9 nmol/L WL12D).3741 Using the Kd (0.4 nmol/L) of [89Zr]Zr-DFO-PD-L1 mAb and the concentration of 6 nmol/L for PD-L1 on MDA-MB231 tumor cells  a potential in vivo target to nontarget ratio (T: NT) of 15:1 would be predicted by applying the mathematical binding model, Bmax/Kd. (derived from the equilibrium equation described by Scatchard).43 This predicted T: NT ratio compares favorably with the in vivo MDA-MB231 T: M ratio of 11:1 obtained at 20× molar excess of avelumab  and the Bmax of 6 nmol/L for the PD-L1 concentration in H1975 tumor cells (in vitro), the predicted T: NT ratio is 3:1 which is comparable to the in vivo H1975 T: M ratios ranging from 5:1 and 3:1 . Therefore, these data would suggest that in humans [89Zr]Zr-DFO-PD-L1 mAb would be able to discern lesions with lower PD-L1 expression levels compared to other PD-L1 targeted imaging agents with lower affinity.Due to the lower sub-nanomolar  in MDA-MB231 xenografts compares with the biodistributions of [89Zr]Zr-atezolizumab and [89Zr]Zr-C4 (anti-PD-L1 recombinant IgG1 mAb) using H1975 xenografts. The tissues with the highest uptakes (%ID/g) were spleen, liver, kidney, lungs, and bone for the 3 radiotracers, although [89Zr]Zr-DFO-PD-L1 mAb uptake values were higher in these tissues (2- to 5-fold) at the same times.37 In part, the higher targeted uptakes of [89Zr]Zr-DFO-PD-L1 mAb in these tissues could be related to the differences associated with the radiolabeled mAb such as specific activity and the mass of mAb associated with the injected dose . For example, the highest spleen uptake , whereas a 2-fold decrease in the spleen uptake (15. 6%ID/g) was observed with the addition of 20 µg of unlabeled PD-L1 mAb to the tracer dose for a total mass dose of 20.1 µg , the uptake values for the spleen and lungs are now similar. However the uptake of [89Zr]Zr-DFO-PD-L1 mAb in the kidneys, liver, and bone are still higher (2- to 3-fold). The [89Zr]Zr-DFO-PD-L1 mAb decreased spleen and lung uptakes at the lower molar specific activity might be expected, since increased mass has been shown to alter radiotracer biodistributions in tissues with saturable target sites.42 On the other hand, the higher uptakes in the kidney liver and bone maybe more associated with clearance of the radiolabeled mAb and its metabolites rather than specific targeting. Therapeutic mAbs have been found to have complex pharmacokinetics that are influenced by their target as well as their ability to elicit immune responses. Avelumab has a biologically intact Fc receptor that is able to mediate ADCC, whereas both [89Zr]Zr-atezolizumab and [89Zr]Zr-C4 lack Fc receptor-mediated binding.44 In addition, the immunodeficiency status of the host mouse (strain) and other molecular properties unique to the mAb  have been shown to be a factor in determining their in vivo biodistribution.45 Moreover, in humans avelumab has been shown to have faster clearance (t1/2 = 3.9 − 4.1 days [10 and 20 mg/Kg doses]) than atezolizumab (t1/2 = 27 d) which maybe a consequence of the Fc/γ receptor-mediated binding.47 Clearance of mAb is usually biexponential with a nonlinear phase representing the pharmacokinetics of the specific binding component and a linear phase representing clearance of the nonspecific component. This faster clearance of avelumab may explain the higher kidney, liver, and bone uptake values of [89Zr]Zr-DFO-PD-L1 mAb compared to [89Zr]Zr-atezolizumab in the mouse biodistributions, as the majority of the radioactivity in these tissues would not be expected to represent intact radiolabeled mAb but radiolabeled metabolites (Ab fragments) or in the case of bone free zirconium-89.32 Further, this faster clearance of avelumab would indicate that suitable target to nontarget ratios maybe achieved earlier with [89Zr]Zr-DFO-PD-L1 mAb than with [89Zr]Zr-atezolizumab; thus, PET images maybe acquired sooner after the radiotracer injection.Although the higher affinity of [89Zr]Zr-atezolizumab PET imaging showed high uptakes in tumors, normal tissues known to have PD-L1 expression , and other tissue regions of chronic inflammation.48 In some cases, [89Zr]Zr-atezolizumab PET imaging was able to identify PD-L1+tumors in patients who had been scored previously as negative by IHC. While these first clinical imaging results are encouraging, further PD-L1 immuno-PET imaging studies need to be included in clinical trials to establish the predictive value of PD-L1 expression as a reliable biomarker for distinguishing responders from nonresponders, monitoring therapeutic responses and evaluating efficacy of this new class of immunotherapeutics.49 Likewise the clinical translation of [89Zr]Zr-DFO-PD-L1 mAb would aid in determining the diagnostic and prognostic potential of PD-L1 immunoPET imaging as well as increase our understanding of PD-L1 target engagement and Fc/γ-mediated responses. Moreover, since [89Zr]Zr-DFO-PD-L1 mAb represents the radiolabeled version of avelumab which is currently in clinical trials, this agent could not only serve as a companion diagnostic imaging agent but aid in the drug development process by establishing dosing and then tracing in vivo tissue disposition of avelumab.50 Although a fluorine-18-labeled small molecule radiotracer would be more desirable for clinical applications, these zirconium-89 labeled FDA-approved therapeutic mAbs are more easily translated into the clinic making possible “proof-of-concept” PET imaging studies earlier in human patients. If these studies prove that PD-L1 has value as a predictive biomarker then this would warrant further development of small molecules or Ab fragments targeting PD-L1 labeled with shorter lived radionuclides (i.e fluorine-18) for clinical applications.51In ongoing clinical studies, [In conclusion PET imaging could provide quantitative assessment of PD-L1 tumor expression in the whole tumor and its microenvironment as well as in metastatic disease sites. This noninvasive imaging method could also be useful in overcoming IHC limitations in identifying patients with PD-L1 target levels predictive of treatment response. This imaging could also potentially serve as a biomarker for response monitoring for anti-PD-L1 therapy. Validated PET imaging agents designed to detect and quantitate tumor PD-L1 expression will have clinical diagnostic and prognostic value and aid in drug development by providing an accurate assessment of tumor PD-L1 status.Click here for additional data file.Supplemental Material, PDL1_supportingInfo_ejagoda_(1) for Immuno-PET Imaging of the Programmed Cell Death-1 Ligand (PD-L1) Using a Zirconium-89 Labeled Therapeutic Antibody, Avelumab by Elaine M. Jagoda, Olga Vasalatiy, Falguni Basuli, Ana Christina L. Opina, Mark R. Williams, Karen Wong, Kelly C. Lane, Steve Adler, Anita Thein Ton, Lawrence P. Szajek, Biying Xu, Donna Butcher, Elijah F. Edmondson, Rolf E. Swenson, John Greiner, James Gulley, Janet Eary and Peter L. Choyke in Molecular Imaging


In this paper, owing to the electrostatic interaction between graphene and h-BN, a facile liquid phase exfoliation method was carried out to fabricate h-BN/graphene based van der Waals heterostructure nanocomposites without additional chemical cross-linkers. The physicochemical properties of as-prepared composites were characterized by several electron microscopic and spectroscopic measurements. The h-BN/graphene heterostructure composites were employed to use as the anodes of asymmetric supercapacitor, and exhibited exceptional capacitive performance due to their synergistic effects. It is expected that the as-prepared h-BN/graphene materials can boost scalable heterostructure electrodes in supercapacitors, and our liquid phase exfoliation method can be used for the construction of the other energy storage and electronics. Graphene, as one of the most important representative of 2D nanomaterials, has been a research hotspot in recent years  of the h-BN/graphene heterostructure film prepared by chemical vapor deposition method is 3.5 times higher than that of the single-layer graphene film   is defined as “white graphene” or “graphene-like” Boron Nitride, with an approximate honeycomb lattice formed by sp2 hybridization and a band gap of 5.9 eV  method, and then transfer and mechanical superposition; the other is to directly use CVD method to grow graphene on substrate and then continue to grow h-BN on the surface of graphene, while this method needs to investigate the lattice mismatch problem. It is noted that the CVD method can ensure integrality of h-BN/graphene heterostructure, but the reaction condition is extremely harsh , the heterostructure size is limited and the cost is high, so it's insufficient to meet the needs of practical application. Therefore, an effective preparation method is urgently needed to make up for the shortcomings of the CVD method to prepare the h-BN/graphene heterostructure materials.As our best knowledge, liquid phase exfoliation method has been rarely carried out to prepare h-BN/graphene heterostructure. Nevertheless, some investigators have attempted to use this chemical method to create heterostructures, like graphene-black phosphorous-graphene sandwich heterostructure . Meanwhile, the assembled asymmertic supercapacitor (ASc) exhibits maximum energy density of 2.05 Wh/kg at high power density of 1998.5 W/kg.Graphite powder  or h-BN powder  were dissolved in the urea/glycerol (molar ratio = 2:1) dispersion. After that, 200 mL of the graphite or h-BN dispersion were transferred to a 800 mL flat bottom beaker, under which graphite or h-BN powder were exfoliated and dispersed through mechanical stirring at 800 rpm for 24 h. Then, the obtained products were evenly transferred to a 50 ml centrifuge tube, then centrifuged for 25 min at 5,000 rpm. The top half of the centrifuged graphite or h-BN dispersion were collected and re-dispersed in DMF, followed by filtration and ultrasonic washing with large amounts of DMF and ethanol, and drying in vacuum oven at 60°C. The yield of graphene or h-BN was decided by taking off the mass of the residual solid . The resulting mixed solution was ultrasonic for 30 min, then stirred at room temperature for 24 h and centrifuged at 1,000 rpm for 30 min. The upper liquid was discarded, and the solid precipitation was finally obtained, namely, h-BN/grapheme (BN/G) heterostructure materials.Transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM) images were obtained using a JEM-2001F  equipped with a Gatan CCD camera. The morphology of the samples was observed by using S-4800  cold field emission scanning electron microscope (FESEM). The crystal structure of heterostructure was analyzed by D8 Advance X-ray diffraction systems . Raman spectra of the samples were tested by an InVia  spectrometer to reflect the composition equipped by a 633 nm laser. The thickness of all samples was also analyzed by MFP-3D-SA AFM  using a tapping mode at a scan rate of 1 Hz.2/g, purity>94.2%, XFNANO Materials). The counter electrodes were prepared via the same method as the above working electrodes. The loading mass of each electrode was about 0.15 mg/cm2. Microporous polypropylene and 2 M KOH solution were used as the separator and electrolyte, respectively.The BN/G working electrodes were prepared by mixing 90 wt.% active material  and 10 wt.% polyvinylidene fluoride (PVDF) in NMP solvent to form a slurry. Then the slurry was coated onto a clean Ni mesh (current collector) and dried in a vacuum oven at 60 °C for 24 h. The counter electrode was the activated carbon  mThe electrochemical performance of samples was measured on the CHI660E  electrochemical workstation. Cyclic voltammetry (CV) tests were carried out at the scan rate of 10~200 mV/s. Galvanostatic charge-discharge (GCD) tests were performed at various current densities from 0.5 to 10 A/g. Electrochemical impedance spectroscopy (EIS) was employed in a frequency from 0.01 Hz to 100 kHz at an amplitude of 5 mV. All the related electrochemical tests were performed at room temperature. Specific capacity of the BN/G in two-electrode was calculated from Equations (1) and (2) , s is the scanning rate (V/s), Δt and ΔV are the discharged time (s) and the voltage drop upon discharging, respectively, and m is the mass of the electroactive materials (g). The energy density  and the power density  of the BN/G//AC were calculated based on the following Equations (3) and (4)  calculated according to Equation (2), Δt and ΔV are the discharged time (s) and the voltage drop upon discharging, respectively.Where In order to further observe the graphene prepared by the glycerol/urea system mentioned above, the graphene/DMF dispersion samples were analyzed with the SEM morphology. We also used AFM to conduct random sample analysis of the exfoliated graphene, which was compared with the sampling method of TEM: the sample to be tested was dripped with pipette gun to the newly prepared mica film after vacuum dust removal. It can be observed the 2D morphology of typical graphene in the tapping mode from To test the universality of glycerol/urea in the above system, we used the same process to treat other 2D material, such as h-BN, and also obtained h-BN/DMF dispersion solution. Then the h-BN samples were observed by TEM. As shown in To understand the crystal structure characteristics of the as-prepared BN/G heterostructure samples, XRD analysis was performed in −1) and G peak , which are shown in Graphene and BN/G, respectively, demonstrating the presence of graphene in BN/G heterostructure. Due to the strong photoluminescence background of h-BN, the photoluminescence background shown in the BN/G region can be attributed to the defect states of h-BN, including defects along grain boundaries . It is found that the graphene is inserted into the interlayer space of h-BN to play the role of interval layering, preventing the re-stacking of h-BN nanosheets. Therefore, the charge storage mechanism in BN/G may be caused by the synergistic effect of graphene and h-BN. The variation trend of BN/G samples with different mass ratios is shown in + and OH−  was further taken as a representative to study its cycle stability for charging and discharging 10,000 times under the current density of 10 A/g. Toward studying the electrochemical properties of our assembled BN/G//AC ASc, we further reflected its power density and energy density. In summary, we reported a well-constructed heterostructure materials, in which the h-BN nanosheets were assembled with graphene through electrostatic interaction based on solution method. This scalable stacked heterostructure are well-aligned in vertical, but the layers are randomly stacked in horizontal. The synthesis method adopted in this work has extensive use, low cost and facile process, which can be applied to other 2D materials. Meanwhile, we further developed BN/G heterogstructure electrode material, and explored the feasibility of BN/G heterostructure electrode material in the application of asymmertic supercapacitors. The incorporation of h-BN not only constructs the electrolyte channel for the graphene layer, but also improves the electrochemical performance. Moreover, the assembled BN/G//AC ASc exhibits maximum energy density of 2.05 Wh/kg at high power density of 1998.5 W/kg, and excellent long cycling stability with 96% of initial specific capacitance after 10,000 cycles at 10 A/g, indicating its promising application in energy storage component.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.XZ, GW, FH, HL, and CG were responsible for all the experiments and the analysis of data. FH, SW, YH, GZ, and DC were responsible for the drafting. All authors contributed equally to the final writing of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here, we reported an ingenious fabrication of moisture responsive graphene-based actuator via unilateral two-beam laser interference (TBLI) treatment of graphene oxide (GO) papers. TBLI technique has been recognized as a representative photoreduction and patterning strategy for hierarchical structuring of GO. The GO paper can be reduced and cut into grating-like periodic reduced graphene oxide (RGO) microstructures due to laser ablation effect. However, the lower light transmittance of the thick GO paper and the corresponding thermal relaxation phenomenon make it impossible to trigger complete reduction, leading to the formation of the anisotropic GO/reduced GO (RGO) bilayer structure. Interestingly, the RGO side that feature lower OCGs and higher roughness shows strong water adsorption due to the formation of micronanostructures. Due to the different water adsorption capacities of the two sides, a flower moisture-responsive actuator has been fabricated, which exhibits “opening” and “closing” behavior under different humidity conditions. The traditional actuators, such as energy conversion devices, sensors, robotics, and micro-electromechanical systems, are driving devices which have been extensively studied because of their irreplaceable application in basic scientific research and engineering technologies , as derivatives of graphene materials  treatment by the photothermal effects, causing the removal of most of the OCGs on and between the GO sheets. Simultaneously, abundant RGO micronanostructures including periodic micro-gratings and nanoscale sheets are formed, increasing the richness of the surface morphology, this changes the wettability of the resultant graphene films, causing hydrophobicity. Laser processing technology is an effective means to realize controlling of surface wettability by fabricating micro/nano structures on the surface of materials  treatment, the graphite powder was oxidized into single-layer and few-layer graphene nanosheets containing a large number of hydroxyl and carboxyl groups. Due to the presence of a mass of OCGs, the obtained GO material is easily soluble in water and becomes a water-soluble suspension. In order to remove residual sulfate and chloride ions, GO aqueous solution should be washed several times with deionized water. The synthesized GO solution was collected by centrifugation and then dispersed in distilled water at a concentration of 3 mg/mL under ultrasonic treatment. Subsequently, graphene oxide paper has been fabricated by ultrafiltration of the prepared GO aqueous solution with the help of the filtration membrane with pore diameter of 0.22 μm. And drying the GO/membrane in the ambient air, and then peeling off the GO paper for next treatment.The graphene oxide material we used here was prepared by Hummer's method. After high strong oxidant  was used as the light source for the GO reduction process. The laser beam with the diameter size of 8 mm was split into two beams, which own the same optical path lengths to the sample surface. The RGO with multilevel structures were fabricated by exposing the GO papers to the interfered laser region. Taking advantage of laser holography technique, the periodicity of the grating could be precisely controlled by changing the angle of two laser beams. By adjusting the appropriate laser power (~0.4 mW), exposure time (30 s) and laser light path, here we fabricated the graphene grating with 2 μm. The laser power is 0.4 mw, and the exposure time is 30 s, which is the relatively suitable laser processing parameter. When the laser power is too large and the exposure time is too long, the surface morphology of the processed sample will be ablated seriously. On the contrary, when the laser power is too low and the exposure time is too short, it is difficult to form a complete continuous periodic micro-nano grating structure . Taking where Λ is the period, λF is the laser wavelength and θ is the angle between two beams.3COOK, MgCl2, K2CO3, NaBr, NaCl, and KCl in a closed glass vessel, which yielded ~23, 33, 44, 57, 75, and 86% RH, respectively. All of the measurements were conducted in air at room temperature (25°C).SEM images were obtained using a field-emission scanning electron microscope , which referred to as FE-SEM. X-ray photoelectron spectroscopy (XPS) was performed using an ESCALAB 250 spectrometer for chemical bond analysis. Raman spectroscopy was recorded on a Jobin-Yvon T64000 Raman spectrometer equipped with a liquid-nitrogen-cooled argon ion laser at 514.5 nm (Spectra-Physics Stabilite 2017) as the excitation source; the laser power used was ~10 mW with an average spot size of 1 μm in diameter. The contact angle (CA) measurements were performed using the Contact Angle System OCA 20  at ambient temperature. The CA was measured using a 4 μL water droplet. The humidity response cycle test is carried out in an environment with 100% humidity. The controlled humidity environments were achieved using saturated aqueous solutions of CHAs an efficient tool for large-area micronano-patterning without any shadow masks and chemicals, laser holography technique is undoubtedly a preferred choice for fabrication of micronanostructures  of the two sides of the RGO paper irradiated by TBLI light. As shown in −1 (D band) and 1,588 cm−1 (G band) are observed. The G peak is used to characterize the degree of graphitization of carbon materials, and the D peak represents the defects, caused by dislocation, grain boundary, fold, and so on. In general, after the removal of OCGs by appropriate reduction method, the C-C hybridization on the surface would restore on a certain extent, and ID/IG shows an obvious decreased. However, the ID/IG ratio of RGO in our work slight increased from 0.97 (GO) to 0.98, which means that more new defects have been brought during the laser ablation. To further investigate the uniformity of laser interfered region of RGO, we also measure the Raman maps in an area of 5 × 10 μm2, as shown in Furthermore, another characterization method, Raman spectroscopy has been used to test the degree of hybridization of GO and RGO, as shown in After TBLI treatment, the GO/RGO bilayer structure has been formed, which would curve to the absorbent layer in moisture. The humidity response characteristics of the GO/RGO bilayer structure provide possibilities for the design and fabrication of intelligent graphene actuators. In our work, inspired from opening and closing behaviors of flowers, the unique flower-robot has been demonstrated. We cut the GO/RGO bilayer papers into six pieces to simulate the shape of the petals; and then put them together and glue to a branch directly, as shown in In conclusion, TBLI treatment of GO papers has been evolved for successful fabrication of GO/RGO bilayer structure toward preparation of moisture-responsive graphene-based actuators. TBLI treatment was performed to remove the OCGs on the GO sheets and form graphene micronanostructures. Due to the light gradient transmittance and delayed thermal transmission of GO paper, the gradual reduction along its lateral direction occurred, resulting in differences in the reduction degree as well as surface morphology on both sides. In spite of hydrophilicity of GO, the RGO side has been observed stronger absorption ability of water molecules than GO side because of auxetic material porosity induced under laser ablation, which rendered the anisotropic GO/RGO structure impressible to environmental moisture. Interestingly, the graphene micronanostructures formed during the reduction process simultaneously increased the roughness of the RGO surface, leading to hydrophobicity on the RGO side. A stripe GO/RGO bilayer structure has been fabricated to demonstrate the reversible bending behaviors. A curvature of −0.047 to 0.175 was obtained, when the RH was adjusted between 23 and 86%. Inspired by the floral behavior, the fabricated humidity-driven graphene actuators were assembled to mimic the opening and closing behavior of flower. We deem that this laser lithography technology may enable biomimetic design and fabrication of intelligent graphene-devices.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.H-BJ, YL, and L-QR conceived the idea and designed the experiments. H-BJ, S-YL, and Y-YS made the characterizations. H-BJ and JL performed the detections. H-BJ, D-DH, and YL contributed to data analysis and interpretation. H-BJ and D-DH wrote the paper. All authors discussed the results and commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.