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.


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.


Parkinson’s disease (PD) is the second most common neurodegenerative disease in the United States after Alzheimer’s disease (AD). To help understand the electrophysiology of these diseases, N27 neuronal cells have been used as an in vitro model. In this study, a flexible graphene-based biosensor design is presented. Biocompatible graphene was manufactured using a liquid-phase exfoliation method and bovine serum albumin (BSA) for further exfoliation. Raman spectroscopy results indicated that the graphene produced was indeed few-layer graphene (FLG) with  Parkinson’s disease (PD) is a neuronal disease that is caused by the death of dopaminergic neurons in the substantia nigra pars compacta region of the brain . It is c2-bonded carbon atoms 3−/4− and [Ru(NH3)6]3+/2+ redox peaks have been measured, a study on the bioelectrical signaling of neuronal cells using graphene-based sensors is lacking [There have been many previous studies on constructing biosensors using graphene-based materials . One suc lacking ,31,32,33 lacking . Since g lacking ,35,36,37In this study, we describe a prototype graphene biosensor to sense electrical signaling in N27 cells. The biosensor was produced with an inkjet printer to create conductive, biocompatible, and defect-free graphene. The graphene was exfoliated from graphite using the LPE method. This design presents a facile technique that can be used to manufacture biosensors for a variety of applications. In a previous study, bovine serum albumin (BSA) was used to exfoliate graphene in an aqueous state and resulted in graphene that exhibited strong biocompatibility. Since this technique was easily implemented, it resulted suitable for large-scale graphene production and, hence, was adopted in this study. The graphene and printed graphene chips were characterized using Raman spectroscopy, Atomic force microscopy (AFM), and scanning electron microscopy (SEM). To characterize cell viability within the biosensor, multiple live–dead cell assays were performed.w/v, Sigma-Aldrich, St. Louis, MO, USA); sodium chloride (NaCl) purchased from Sigma-Aldrich, St. Louis, MO, USA; Kapton polyimide (PI) . RPMI medium 1640 (1X) , L-glutamine 200 mM(100X) , pen/strep solution  was purchased from Gibco Life Technologies. Fetal bovine serum (FBS)  was purchased from ThermoFisher Scientific, Waltham, MA. Polyurethane ether tubes  were purchased from Superthane®, 3 mL Luer-Lok syringes were purchased from Allegro Medical. Inc.  Six-well cell culture clusters (Lot# 23314037) were purchased from Costar®. A GenieTouch™, syringe pump was purchased from Kent Scientific Corporation. A 4-axis CNC USB controller Mk3/4 for mini CNC mill was purchased and controlled by a PlanetCNC® . A sinometer digital multimeter (MS8261) was used to measure print conductivity. A JEOL FESM JCM-6000 scanning electron microscope and a Zeiss Axio Observer Z1 inverted microscope were used for SEM imaging of the graphene prints and the live–dead cell assays, respectively. A Raman spectrometer  with a CW laser  was used for Raman spectroscopy measurements. A PerkinElmer UV–Vis spectrophotometer (Lambda 750) at λ = 660 nm at room temperature was used to provide absorption spectra and consequently the concentration of graphene ink.The materials and equipment used in this study were: graphite ; Albumin, from bovine serum ; poly(sodium 4-styrenesulfonate) ; poly(ethyleneimine), (PEI) solution (50% 2O. The Vibro-Energy mill (shaker mill) was run for 90 h at 300 rpm , and finally submerged into a PEI solution (30 mg/mL) of DI-H2O and an NaCl solution (0.5 mol L−1) for additional 20 min. The substrate was then allowed to air-dry for 12 h.Because of its great thermal resistance, Kapton PI was chosen as the polymer substrate. It can withstand temperatures from −269 to 400 °C. Due to the hydrophilicity and negative charge of the FLG ink, it was necessary to perform a hydrophilicity treatment on the Kapton PI substrate. To treat Kapton PI, PI was first washed in acetone. The substrate was next washed with PSS (3.5 mg/mL), submerged in a solution of 50 mL DI-H−1. The ink was injected onto the PI substrate, which was fixed on a 22 × 22 mm glass cover chip. The chip was covered with two pieces of copper tape, with a 0.50 mm gap between the two pieces RBased on Equation (1), the conductivity measurements were performed by serval trails with different annealing temperatures and different time periods. 2 substrate and air-dried. A Raman spectrometer with a CW laser provided a laser beam at wavelength 532 nm to the graphene on the Si/SiO2 sample. The samples obtained from the shaker mill and kitchen blender had similar cartelization when Raman spectroscopy was performed A. A thinImaging of the printed graphene patterns on PI was performed using a SEM with a 2–5 kV accelerating voltage. The thickness is shown in 6 cells/vial were added to the chips using a micro pipette; 10 µL of cell suspension was introduced in each of the prints. The cells were left to grow in an incubator maintained at 37 °C with 5% CO2. The cells were checked under an inverted microscope at 72 h, as shown in A flask of eight-time-passaged N27 cells was chosen for testing the biocompatibility of the graphene sensor. The graphene chips were placed in six-well cell culture plates. The chips were then placed under a biological safety cabinet and exposed to UV radiation for 12 h. The UV exposure ensured that the chips were sterile and biocompatibility would be maximized. In this study, N27s cells were cultured in 3 mL of maintenance medium (MM) that included RPMI medium 1640 (1X), 10% FBS, 1% penicillin, and 1% L-glutamine. Then, 3 mL medium was introduced in each well which was covered with the graphene chip. N27 cells with a cell density ≥1 × 102 atmosphere. The dye was then removed, and FBS-free RPMI medium was added to maintain moisture for sample imaging. N27 cells were imaged after 72 h of incubation. As shown in the control well (Live–dead cell assays were performed using a 70 µM CellTracker™ CMFDA solution combined with an 8 µM propidium iodide (PI) solution in FBS-free RPMI medium. The CellTracker™ CMFDA solution was prepared by dissolving 50 µg of CellTracker™ in 10.8 µL of dimethyl sulfoxide (DMSO). The PI was diluted from a stock provided by Invitrogen. The 70 µM CellTracker™ CMFDA solution and 80 µM PI solution were dissolved in FBS-free RPMI medium to reach the correct concentrations. Then, 1 mL of solution was prepared to facilitate the calculations and minimize the error. MM was carefully removed from the desired well of the 6-well plate. After removal, the well was rinsed with FBS-free RPMI medium (500 µL), 500 µL of dye was added to the well, and the cells were then incubated for 30 min at 37 °C in a 5% COrol well A, live cA Zeiss Axio Observer Z1 inverted microscope was used to gather the live–dead cell assay results. The cells were carefully shielded from light after performing the live–dead cell assay so as to not affect the results. CellTracker™ CMFDA has an excitation of 492 nm and an emission of 517 nm. Propidium iodide has an excitation of 535 nm and an emission of 617 nm. The microscope was set to capture these wavelengths and image the fluorescence resulting from the live–dead cell assay. The results can be seen in 2 with a Si/285 nm laser (34). Another fabrication method uses surface plasmon resonance to fabricate graphene on a gold sensor to achieve higher sensitivity than that of a traditional gold thin film SPR sensor [Traditional graphene-based biosensors are expensive and are still in a conceptual stage. One fabrication method consists in synthesizing graphene on a glass slip and etching SiOR sensor .In our design, the graphene biosensor chip included three components: a glass coverslip, a copper tape, and a polyimide (Kapton) polymer substrate . The copAqueous graphene was pre-prepared using the wet-ball milling technology as described previously . This pa2 was performed using a laser with a wavelength of 532 nm  of live cells. This design could maximize printed FLG conductivity up to 6800


Annulohypoxylon stygium (Lév.) were assembled from raw reads from PacBio and Illumina sequencing. The diversity of genomic structures, conserved genes, intergenic regions and introns were analyzed and compared. Genome sizes ranged from 132 to 147 kb and contained the same sets of conserved protein-coding, tRNA and rRNA genes and shared the same gene arrangements and orientation. In addition, most intergenic regions were homogeneous and had similar sizes except for the region between cytochrome b (cob) and cytochrome c oxidase I (cox1) genes which ranged from 2,998 to 8,039 bp among the six isolates. Sixty-five intron insertion sites and 99 different introns were detected in these genomes. Each genome contained 45 or more introns, which varied in distribution and content. Introns from homologous insertion sites also showed high diversity in size, type and content. Comparison of introns at the same loci showed some complex introns, such as twintrons and ORF-less introns. There were 44 short fragment insertions detected within introns, intergenic regions, or as introns, some of them located at conserved domain regions of homing endonuclease genes. Insertions of short fragments such as small inverted repeats might affect or hinder the movement of introns, and these allowed for intron accumulation in the mitochondrial genomes analyzed, and enlarged their size. This study showed that the evolution of fungal mitochondrial introns is complex, and the results suggest short fragment insertions as a potential factor leading to larger mitochondrial genomes in A. stygium.Mitochondrial DNA (mtDNA) is a core non-nuclear genetic material found in all eukaryotic organisms, the size of which varies extensively in the eumycota, even within species. In this study, mitochondrial genomes of six isolates of  The mitochondrial organelle is found in most eukaryotic organisms , a set of tRNAs, and small and large ribosomal RNA (rns and rnl). However, the size of fungal mtDNAs varies greatly. The smallest mitochondrial genomes, such as those of Harpochytrium sp. and Candida orthopsilosis, are around 20 kb in size with few or no introns and few or no non-conserved genes. Several large mitochondrial genomes in fungi (more than 150 kb) have been released in the organelle genome database in NCBI recently, including those of Rhizoctonia solani AG-1 and AG3  , A. stygium 90081906 (AY951667 and AY951775), A. stygium 90041409 (AY951666 and AY951776), A. stygium var. annulatum 3 (AY951669 and AY951777) and A. stygium var. annulatum 91042205 (AY951668 and AY951778) were employed as controls. Sequences were aligned using Clustal W with gap opening penalty of 5 and gap extension penalty of 2. The distances were computed using the Neighbor-joining method. All positions containing gaps and missing data were eliminated from phylogenetic analysis. Evolutionary analyses were conducted in Mega 7 . Borders between exons and introns were confirmed manually using BLASTP against NCBI protein databases and codon usage table 4  following Haridas and Gantt  , the cytochrome oxidase subunits 1, 2, and 3 , NADH dehydrogenase subunits 1, 2, 3, 4, 5, 6, and 4L , apocytochrome b (cob), and one ribosomal protein (rps3 in rnl). Twenty-six tRNA genes were comprised of three copies of tRNA-met encoding genes, two for each of tRNA-ser, tRNA-arg, tRNA-leu, and tRNA-lys and the other 15 standard tRNAs, most of which were located between rnl-nad2 (11), rns-nad6 (8), and cox3-rnl (3). All of these genes were predicted from the same forward strand  and cytochrome c oxidase I (cox1) genes which ranged from 2,998 to 8,039 bp among the six isolates.From the sequence comparisons, the same sets of 15 conserved protein-coding genes, 26 tRNAs, and small and large subunit rRNAs were found in mitochondrial DNAs of all six isolates . An aminotransferase class-III like gene (containing two short introns) was in the region between atp9 and cox2, and a fragment encoding AAT_I super family domain was located within the area between atp6 and rns. These genes and fragments were identified in mtDNAs of all six sequenced isolates.Three transferase genes as well as a transferase-related fragment were predicted from mtDNA of cox1 was found to have 17 sites; rnl had eight; nad5 had seven each; cob and cox3 had six; rns and nad2 had five each; nad1 had four; atp6 had three; and single sites were also found in nad4, nad6, atp9, and cox2. According to the length and similarity (92% identity), nad1-i4 had five different intron types; cox1-i2 and cox1-i11 contained four types; five introns had three types; 14 introns had two types; and the other 43 introns were singletons. As a result a total of 99 different introns were identified from the six isolates. They varied greatly in size, ranging from 12 bp for rns-i2 (As03) to 6,259 bp for cob-i3 (As31).Different from the conserved genes and transferase genes, the content and distribution of introns within conserved genes varied greatly among isolates Table . Intronscox1-i2 of As23. Among nine group II introns, three carried genes encoding RNA-dependent-DNA polymerase. Interestingly, the group II intron rns-i1 only had an orf366 encoding double LAGLIDADG_1 domain. In addition to group I and group II introns, the other five introns were not typical mitochondrial introns. These included rnl-i6 (containing the rps3 gene), rnl-i4 (type II) and three short introns  with 12 bp, and rns-i2 (type II) with 50 bp).Most introns (86%) belonged to group I or contained group I-characterized conserved domains, which included LAGLIDADG_1, double LAGLIDADG_1, LAGLIDADG_2, GIY-YIG motifs, or mixtures (Table rnl-i6 (containing conserved gene rps3) and nad5-i5 (containing two IDs), other “common introns” were group I with the length ranging from 900 to 2 kb. Each contained one conserved domain-encoded ORF or fragment. Different introns from the same insertion sites at 15 positions had different sizes, but shared high sequence similarity (>90%) among partial fragments. Different introns from the same insertion sites of cox1-i9, atp6-i3, and atp9-i1 shared less similarity (80–90% identity) among partial fragments. Two completely different introns were also detected from each insertion site of cox1-i14, rns-i2, and rnl-i1, and rnl-i7 , group II (As23 and As24), and group III (As03). An exception was As28:124339-124385, which was found in mtDNAs of As03, As23, As24, and As28. Twenty seven short fragments had a single SIR , three had two SIRs, and one had three SIRs. No typical SIR structures were found in 14 other short fragments.Some short fragments were multi-copy in individual mtDNAs. They included As03:124881-124916 (8 copies in As03), As23:118651-118685 (7 copies in As23 and As24), As23:118686-118723 (5 copies in As23 and As24), As23:7552-7599 (3 copies in As23 and 2 copies in As24), As24:16469-16531(3 copies in As23 and As24), As24:37117-37158 (3 copies in As23 and As24), As28:35488-35535 . In addition, there were two copies of As23:137527-137604, As23:140496-140543, As23:47023-47067, and As23:80096-80143 in the corresponding mitochondrial genomes. The copy fragments showed more than 90% identity with their corresponding insertions.nad1-i4, nad5-i3, cox1-i2, and cox1-i11. Five different introns found in nad1-i4 (Table nad1-i4 of As31 (and As15) was the smallest one , and contained a GIY-YIG domain encoded sequence, which was interrupted, forming three segments. There were two more fragments by comparing nad1-i4 of As28 with that of As31: a 46-bp SIR and a 1329-bp parasitic intron encoding a double LAGLIDADG_1 domain. Parasitic introns are self-splicing introns which insert into another host introns , 2,561 bp (As24), 2,693 bp (As31), and 4,039 bp (As23)  Table . A membecox1-i11, which were 1,576 bp (As15), 3,086 bp , 3,212 bp (As23), and 4,817 bp (As03) in length ; loss of the fragment from 717 to 1,938 bp and short fragment from 20 to 64 bp to form a 1,022-bp intron of As03 (without double LAGLIDADG_1). A similar case was found in rnl-i3 of As23. A fragment with orf253 encoding a GIY-YIG domain was lost from rnl-i3 of As28 to form a 521-bp one of As23.Comparison of introns at the same loci also showed the formation of three ORF-less introns Figure . The rnlA. stygium. Sixty-five insertion sites and 99 different introns were identified. Introns from each of 16 insertion sites varied greatly in size, but shared high similarity among partial fragments. The similar sequences were inferred to be shared in the common ancestor, and each intron represented one of its evolutionary descendants. Intron comparisons may demonstrate their possible evolution. For cox1-i2, as an example, the intron of As28 was a typical mitochondrial intron, with a group IB RNA structure and a LAGLIDAGE_1-like gene. It was inferred to be the ancestor of all introns in cox1-i2, since it carried the least content. An additional parasitic IC2 intron was detected in the intron of As31. A 52-bp SIR-like fragment was detected within double LAGLIDADG_1 region of the parasitic IC2 intron of As23, indicating that the intron of As23 may have originated from the ancestral intron currently conserved in As31. Similar to the intron of As23, that of As24 contained two additional fragments at the 5′ end, but lacked the parasitic IC2 intron. The intron of As24 was a presumed descendant of the one from As23, but with the loss of the IC2 intron. As a result, four introns in cox1-i2 may have derived from the ancestral homolog from the As28 form, to the As31 form, to the As23 form, and then to the As24 form , a hypothetical protein of Pestalotiopsis fici (YP_009317063.1), and an N-acetyl-transferase of P. fici (YP_009317072). However, there were three transferase genes as well as a transferase-related fragment in all six mtDNAs of A. stygium. They included two copies of putative N-acetyl-transferase genes and an aminotransferase class-III-like gene. Corresponding transferase genes from different mtDNAs shared high similarity. P. fici and A. stygium belong to same order of Xylariales, and these transferase genes likely evolved via vertical gene transfer from their ancestor, rather than horizontal gene transfer.The presence of transferase encoding genes was another feature of mtDNA in O. sinensis (Kang et al., cox1-i2 in A. stygium was a large intron of 4,039 bp, which harbored three ORFs with LAGLIDAGE HE genes. Altogether, introns accounted for the range from 59.6 to 62.2% of the full mitochondrial genome sizes among the size isolates. Our findings as well as the previous studies demonstrated that the number and length of introns are two key factors that contribute to the size of large fungal mtDNAs.The six mtDNAs were 131,996–147,325 bp in length. To our knowledge, only eight completely sequenced and well annotated fungal mtDNAs are larger than 150 kb (Losada et al., Aspergillus and Penicillium species (Joardar et al., Cordyceps militaris (Zhang et al., Rhizophagus irregularis (Formey et al., Rhynchosporium species (Torriani et al., Fusarium species (Al-Reedy et al., Chrysoporthe species (Kanzi et al., cob and cox1.Comparative mitochondrial genomic analyses among isolates within intraspecies or closely related species might enhance our understanding of recent fungal mitochondrial evolution. Related researche on several genera has been performed in the past few years. Nine mitochondrial genomes from Aspergillus and Penicillium species have zero common introns (Joardar et al., Cordyceps militaris strains have four common introns (Zhang et al., Rhizophagus irregularis isolates have five common introns (Formey et al., A. stygium was much greater than those species with small mtDNA size, indicating that there might be one or more mechanisms in A. stygium which accelerate degradation, and hinder the mobility of their mitochondrial introns.Common introns were found in all tested mtDNAs, and they could be derived from their ancestors. These introns should be stable in their host genomes and have little or no activity of mobility. Mitochondrial genomes of nine Small inverted repeats are short elements <100 bp that can fold into hairpin-like structures. These hairpin-like elements have been widely found in the organellar genomes of plants, aquatic animals (Lukić-Bilela et al., A. stygium is the presence of short fragment insertions in introns. Insertion of short fragments may result in the change of secondary structure of RNA, altered structure of the conserved domain of HE genes, or interruption of ORF integrity in introns, thus reducing or hindering the activity of RNA or/and HE. Broken introns may increase the frequency of different types of mutations, including insertions/deletions of parasitic introns, loss of HEGs, single base mutation and insertions of other short fragments. All these mutations can give rise to a great number of high-diversity introns, which may contribute to the large genomic size of A. stygium mitochondria.A possible factor for so many introns in mtDNA of A. stygium ranged from 132 to 147 kb in size and contained the same sets of conserved protein-coding, tRNA and rRNA genes, and shared the same gene arrangement and orientation. Those six genomes varied greatly in intron distribution and content. Comparison of introns within the same insertion site demonstrated formation of some complex introns, such as twintrons and ORF-less introns. Complex introns underwent not only insertion/deletion of introns, but also insertion/deletion of short fragments such as small inverted repeats. Movements of short fragment within an intron might affect or hinder the activity of introns, which could cause intron accumulation in mitochondrial genomes, and enlarge their size.MtDNAs of six isolates from BX and RM conceived this study. YD analyzed the data, prepared figures and drafted the manuscript. TH participated in early analysis of preliminary data and manuscript writing and revision. SL provided suggestion for the research, contributed to the data interpretation, writing, and revising the manuscript critically. QC participated in the data analysis. LL and QW sequenced and analyzed the mitochondrial genome. All authors have read and approved the final version of 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.