Complex three-dimensional structures inspired by origami, kirigami have promise as routes for two-dimensional to three-dimensional transformation, but current examples lack the necessary combination of functional materials, mechanics designs, system-level architectures, and integration capabilities for practical devices with unique operational features. Here, we show that two-dimensional semiconductor/semi-metal materials can play critical roles in this context, through demonstrations of complex, mechanically assembled three-dimensional systems for light-imaging capabilities that can encompass measurements of the direction, intensity and angular divergence properties of incident light. Specifically, the mechanics of graphene and MoS 2 engineered in 3D Kirigami geometries.The strain tolerance and promising optoelectronic properties of 2D materials can be leveraged to design functional optical sensing devices. Here, the authors provide a demonstration of arrays of independently addressable photodetectors constructed from graphene and MoS Progress in this area is often limited by the relatively small range of choices in controlled, reliable, reproducible strategies for producing 3D geometrical forms in advanced functional materials11. Among the most recently introduced methods is a scheme in which compressive buckling associated with a stretched elastomeric substrate guides the mechanical assembly of elaborate 3D mesostructures, some with designs reminiscent of those achieved in macroscale structures by origami/kirigami, with specified shapes and with sizes that can span several orders of magnitude in characteristic dimensions, down to the submicron regime in lateral features and to a few tens of nanometers in thickness15. These ideas leverage an intimate interplay between materials and microstructural mechanics, with diverse examples of use with silicon membranes, metallic electrodes, and polymer films in hundreds of different 3D geometries16. Full, quantitative modeling of the mechanics forms an essential aspect of design in all such cases; without such theoretical guidance, the buckling process itself can lead to cracking and/or defect formation in the constituent materials in ways that can deteriorate their properties. Physical toughness and materials structure geometries are, therefore, critically important in maximizing the diversity of realizable 3D structures.Electronic and optoelectronic materials deployed in complex, three-dimensional (3D) structures can offer qualitatively expanded levels of functionality compared to those in their corresponding two-dimensional (2D) planar counterparts. Many examples demonstrate clearly the value of 3D structures in achieving unique properties with simple constituent materials19. In fact, recent work demonstrates that graphene can be built into common, kirigami-type structures and in mechanical metamaterials such as stretchable electrodes, springs, and hinges21. The assembly approaches in these cases, however, rely on externally imposed forces, with limited ability to address complex, 3D architectures and/or functional systems that form naturally in a parallel fashion with the requisite heterogeneous collection of patterned materials.Atomically thin, 2D materials have a well-established set of excellent mechanical properties, some of which, for graphene, are unmatched; these characteristics have direct and essential relevance in the context of 3D assembly2 and graphene, each of which offers an extraordinary combination of electrical, mechanical and optical properties relevant for present purposes. Specifically, graphene offers the best set of parameters for flexible, transparent conductors, with potential to replace indium–tin–oxide in flat panel displays and touch screens23. Additionally, MoS2 is of great interest for its excellent semiconducting properties at atomic thicknesses26. Owing to a direct band gap of 1.9 eV in monolayer MoS2, this material is well-suited for applications in the unique photodetecting systems32. Realizing 3D arrays of MoS2/graphene photodetectors involves first optimizing the parameters for assembly of origami-inspired 3D shapes  using finite-element analysis (FEA). The set of assembly parameters defined in this way serves as starting points for experimentally constructing the targeted 3D shapes, with integrated devices, by compressive buckling. Here, MoS2 and graphene serve as the channel and electrodes, respectively, in ultrathin semiconductor photoresistor supported by a layer of polymer. The resulting system forms spontaneously, and without external application of targeted forces, from a 2D planar geometry into a 3D configuration that allows tracking of both the direction and intensity of incident light. As an additional feature, the atomically thin MoS2 and graphene yield optically transparent devices, such that light passing through the device can be detected at two sensing locations (the entry and exit sites), thereby providing further information of relevance to divergence angle.Here, we explore the use of 2D materials in functional, 3D systems formed via geometry transformation guided by compressive buckling, with a focus examples in constructs that provide 3D photodetection/imaging capabilities by use of light sensing elements that incorporate monolayer MoS2 on an SiO2/Si wafer. Patterned encapsulation using SU-8 yields thick and thin regions designed specifically to aid in guiding the assembly process. Finally, a layer of photoresist (PR) serves to pattern the exposure of selected regions to ultraviolet (UV) ozone. After removing the SiO2 by immersion in an etchant, transfer printing with a flat slab of polydimethylsiloxane (PDMS) delivers the planar device assembly onto a film of polyvinyl alcohol (PVA) such that the topside can be exposed to UV ozone. Subsequent lamination onto a pre-stretched elastomeric substrate, pre-exposed to UV ozone, leads to strong bonding at the regions of SU-8 previously treated with UV ozone. Removing the PR layer leaves a slight physical separation between the device platform and the elastomeric substrate at corresponding regions. Releasing the substrate 37. The maximum strain in the MoS2 is ≤0.15%, indicating that MoS2 occupies a relatively flat region of the structure and that the strain is bending dominated. It is noteworthy that the maximum strain of MoS2 is much higher in the hemisphere than the other two configurations, due to the non-negligible bending deformations. The resistance of the graphene under repeated buckling and release appears in Fig. As the 3D shape evolves, the strains induced by bending and twisting must remain below the maximum endurance limit for each layer in the construct, to avoid cracking or other forms of mechanical degradation of the materials. Optimization in this context, with a goal toward realizing the desired geometry , involves analytic modeling and full 3D FEA techniques that capture all details of the mechanics. The analysis starts with a precursor device design on the 2D planar surface, which subsequently morphs into a 3D structure via the process of controlled compressive buckling. Precursor designs for the octagonal prism, octagonal prismoid, and hemisphere at the 2D planar surface appear on the left side of panels b, c, and d, respectively, in Fig. els Fig.  and 2. Aain Fig. . The max35. The areal proportion of the regions that undergo significant strain (>2%) is less than 0.2% .System-level operation requires electrical interconnects to external data acquisition equipment, with designs capable of accommodating the 3D transformation and associated buckling processes. Figure ces Fig. . Such slI–V) characteristics for different illumination intensities  increases with intensity and with bias voltage, in a manner that is symmetric around 0 V. The linear behavior and symmetric I–V characteristics are consistent with approximately ohmic contacts between the graphene and MoS2  through multiple cycles at each bias voltage32. The estimated rise (τrise) and decay (τdecay) times of the Iph are 290 ± 130 ms and 420 ± 210 ms, respectively,31. The time-resolved photoresponses are lower than those of similar devices fabricated on SiO2 substrates  can be reversibly stretched and deformed from 2D to 3D states; the corresponding stability behaviors, as in Fig. Visible light 532 nm) passed over a representative photodetector induces a photoresponse that can be captured as current–voltage  exhibit high optical transmittance (~87%) at 550 nm  in addition to an entry (P1) . The direction of the incident beam (P1 → P2) follows from the spherical locations of the P1 and P2 vectors with respect to the hemisphere center. The P1 → P2 direction can be defined in terms of the azimuthal angle θ in the x–y plane and the polar angle φ between the x–y plane and z direction  increasing θ from −45.0° to 45.0° while fixing φ at 90.0°, (2) decreasing φ from 90.0° to 67.5° while fixing θ at 0.0°, and (3) increasing θ from 45.0° to 135° and φ from 67.5° to 112.5°. In each, three combinations of θ and φ are tested, as shown in Fig. 1 → P2) can be expressed in spherical coordinates θ and φ. In all combinations in the three scenarios , the θ and φ coordinates calculated from the measured photoresponses match the coordinates directly measured with a protractor scale  Fig. . Figure ion Fig. . This si2 and graphene in complex 3D system architectures allows their unique electronic, mechanical, and optical properties to be leveraged in a photodetection and imaging device that can sense both the direction and intensity of illumination, in real time. Similar capabilities cannot be achieved easily, with solid state, integrated operation, by using conventional designs or materials. Key enabling properties of 2D materials in this context include high threshold strains, to avoid fracture even in extreme cases of 2D to 3D transformation, ultrathin geometries, to minimize bending-induced strains that follow from this transformation and optical transparency, to allow simultaneous illumination on the front and backsides of the system. The example presented here might foreshadow other opportunities to engineer unique system-level function by deploying 2D materials in 3D designs.Exploiting MoS2 were grown on a Cu foil and a SiO2/Si wafer, respectively, by the low pressure chemical vapor deposition and the metalorganic chemical vapor deposition (MOCVD). The Cu foils (16 × 8 cm2) were inserted in a circular quartz tube and thermally annealed up to 1000 °C in the presence of H2 gas (8 sccm) at 80 mTorr for 2 h. The chamber was then injected with CH4 precursor gas (20 sccm) at 1.6 Torr for 1 h. Subsequently, the quartz tube (heated zone) was allowed to naturally cool at an initially rapid rate, before being gradually raised to room temperature in the presence of H2 gas (8 sccm) at 80 mTorr. Chemical precursors to Mo and S for growth of MoS2 were molybdenum hexacarbonyl (MHC) and dimethyl sulfide (DMS), respectively. The gas phase MHC (0.5 sccm) and DMS (1 sccm) were inserted into the MOCVD quartz tube along with H2 (10 sccm) and Ar (300 sccm), and the tube was heated to 550 °C at 7.5 Torr for 20 h. Raman and photoluminescence (PL) results from MoS2 and graphene are shown in Supplementary Figs. 2 is monolayer and graphene is double-layer.Monolayer graphene and MoS2 plasma) and on the MoS2 monolayers to provide support during the transfer process. The Cu foil was etched by floating the PMMA-coated graphene/Cu foil on ammonium persulfate (APS) solution (20 g ℓ−1) for 5 h. After etching the Cu foil, the PMMA/graphene film was floated multiple times on deionized (DI) water to completely wash away the APS residue. Finally, the graphene was transferred to the desired wafer and the PMMA was removed by acetone. The SiO2 on the MoS2 was etched by floating the PMMA-coated MoS2 SiO2/Si wafer on diluted (1%) hydrogen fluoride (HF) solution. Afterward, the HF residues were washed away by floating the PMAA/MoS2 film on DI water in a manner similar to that for the PMMA/graphene film.Polymethyl methacrylate (PMMA) was spin-coated on top of the graphene of one side of the Cu foil . After transferring the graphene, the electrodes were defined in an interdigitated geometry by photolithography and reactive ion etching (RIE) with O2 plasma . MoS2 was transferred onto the interdigitated pattern of graphene electrodes and the channel regions were defined by photolithography and RIE with CHF3/O2 plasma (35/10 sccm) at 100 W for 5 s. Another SU-8 (5 µm) layer was spin-coated and patterned in a manner similar to that for the first SU-8 layer, but with openings in regions designated for creases. PR was spin-coated and patterned to expose and cover the bonding and non-bonding regions, respectively. Assisted by HF treatment, this fabricated structure was transferred with a slab of PDMS to a tape of PVA. Next, an elastomer substrate  was bi-axially pre-stretched to the optimized strain determined by FEA simulations. The structure on the PVA tape and the pre-stretched substrate were exposed to UV ozone, laminated together and then baked in an oven at 70 °C for 5 min. The PVA was then dissolved in DI water. Finally, the PR was dissolved in acetone, which loosened and/or slightly delaminated the non-bonding regions of the devices from the pre-stretched substrate, facilitating the 3D assembly process upon release of the pre-stretching strain. The sizes of MoS2 photoresistor and the 3D pop-up hemisphere structure are 80 × 80 µm and 3.5 × 1.4 mm (diameter × height), respectively. Detailed descriptions of the 3D hemispherical structure and diagrams of the operating principles are in Supplementary Figs. First, SU-8 (2 µm) was spin-coated on an SiO2/Graphene/SU-8 or SU-8/Graphene / SU-8) were used to model the silicone substrate and 2D precursors, respectively. Refined meshes of those elements ensured the computational accuracy. The critical buckling strains and corresponding buckling modes determined from linear buckling analyses were implemented as initial imperfections in the postbuckling calculations to obtain the deformed configurations and strain distributions during the pre-strain release. The simulations of postbuckling process were performed using conventional static analysis in the commercial software ABAQUS. The elastic modulus (E) and Poisson’s ratio (ν) are Esubstrate = 166 kPa and νsubstrate = 0.49 for substrate; Egraphene = 500 GPa and νgraphene = 0.15 for graphene; 2; and Three-dimensional FEA allowed prediction of the mechanical deformations and strain distributions of photodetector structures and the entire circuit system enabled by controlled buckling. Eight-node 3D solid elements and four-node shell elements with a multiple stack design . The photoresponse was recorded in real time by using a data acquisition system (DAQ) (Keithley 3706A) and a source meter (Keithley 2612). The raw photoresponse data from the DAQ were input into a program (encoded in MATLAB) to allow visualization of the photocurrent mapping. The program represents the intensity of the photocurrent mapping by a color scale and calculates the direction based on Eq. (Data supporting the findings of this study are available within the article and its Supplementary Information file, and from the corresponding authors upon reasonable request.Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3


Long interspersed nuclear element-1 (LINE-1 or L1) is a type of retrotransposons comprising 17% of the human and mouse genome, and has been found to be associated with several types of neurological disorders. Previous post-mortem brain studies reveal increased L1 copy number in the prefrontal cortex from schizophrenia patients. However, whether L1 retrotransposition occurs similarly in major depressive disorder (MDD) is unknown. Here, L1 copy number was measured by quantitative PCR analysis in peripheral blood of MDD patients (n = 105) and healthy controls (n = 105). The results showed that L1 copy number was increased in MDD patients possibly due to its hypomethylation. Furthermore, L1 copy number in peripheral blood and five brain regions  was measured in the chronic unpredictable mild stress (CUMS) model of depression in mice. Intriguingly, increased L1 copy number in blood and the decreased L1 copy number in the prefrontal cortex were observed in stressed mice, while no change was found in other brain regions. Our results suggest that the changes of L1 may be associated with the pathophysiology of MDD, but the biological mechanism behind dysfunction of L1 retrotransposition in MDD remains to be further investigated. Major depressive disorder (MDD) is a common psychiatric disorder that is defined by episodes of depressed mood lasting for more than 2 weeks with the symptoms of disturbed sleep and appetite, excessive guilt, reduced concentration, slowed movements and suicidal thoughts1Increasing evidence suggests that mobile elements are regarded as sensors of environmental stress in the brain4464In this study, we quantified L1 copy number in peripheral blood genomic DNA and found that patients with MDD had a significant increase in L1 copy number. We also measured the methylation levels of L1 5′-UTR and found that MDD patients had less methylation levels in peripheral blood than healthy controls. Next, we quantified L1 copy number in the mouse model of depression , and found that CUMS mice had higher L1 copy number in peripheral blood than controls, consistent with results from MDD patients. Chronic unpredictable stress causes major structural and functional maladaptations in multiple brain regions10et al.Quantitative multiplex polymerase chain reaction (PCR) to detect L1 copy number in the human brain regions and other tissues was first developed by Coufal Methylation sensitive restriction enzymes (MSREs) with RT-PCR were used to measure L1 5′UTR DNA methylation. A 315 bp fragment rich in CpG sites was amplified and four CpG restriction enzyme sites that can be cleaved by the methylation sensitive restriction enzymes . The schematic diagram is shown in et al.14p = 0.0003, p < 0.0001, To further examine the changes of L1 copy number in MDD, we established the depression model of mice via chronic unpredictable mild stress (CUMS) paradigm, which is the widely applied and most accepted animal model of depression. CUMS was firstly reported by Katz and Hersh, and further improved by Willner et al. were the first to develope an approach based on single cell genomic qPCR to detect the activity of endogenous L1 retrotransposition within the mouse genomeet al. quantified brain L1 content in a schizophrenia animal model that showed increased L1 content as well as in schizophrenia patientsMuotri  0.05,It is well known that a long-term exposure to stress can trigger deleterious effects on the structure and function of the brain. Animals exposed to chronic stress have morphological and functional damages in various brain regions, including PFC and hippocampuset al. reported that voluntary exercise could increase L1 insertions in matured neurons in the hippocampus21231227in vivo1212133132et al. revealed that the activation of L1 retrotransposition caused an age-dependent neuronal declineThe occurrence of many disorders is related to genomic instability. L1 retrotransposition, as one of the DNA damaging agents, can lead to various types of genomic instability17The present results suggested that the changes of L1 copy number might be related to MDD. We observed increased L1 copy number in peripheral blood of MDD patients and the animal model. We also found that L1 was robustly hypomethylated in peripheral blood of MDD patients compared to healthy controls. Possibly, increased L1 copy number in blood may be closely correlated with DNA demethylation in MDD. Hypomethylation in L1 may increase their activity, resulting in genomic instability and leading to many diseases, such as cancer36et al. found that there was a tendency towards increase in L1 copy number in the prefrontal cortex of MDD patients. The differences in biological background of study samples may be the major reasons for the different results between our work and the study reported by Bundo et al. In addition, the extents of L1 changes were obviously smaller in CUMS mice than MDD patients in our study. This may be due to the small sample size in study of mouse model; mice exposed to CUMS for 35 days failed to completely express the symptoms observed in MDD patients.Furthermore, our results showed that L1 copy number was decreased in the prefrontal cortex of the animal model of depression. We speculate that there may be different mechanisms of regulating L1 retrotransposition between peripheral blood and the prefrontal cortex of depressive mice. Bundo et al. reported that small interfering RNAs could inhibit L1 retrotransposition at a post-transcriptional level through induction of mRNA degradation41et al. proposed a model that microprocessor complex could bind to L1 mRNA and cleave hairpin structures, resulting in a decrease in L1 mRNA and retrotransposition rates ultimately44There are several molecular mechanisms behind L1 retrotransposition repression. DNA methylation is the most common one to repress L1 retrotranspositionA number of brain regions have been reported to mediate the various symptoms of depression. Both human brain imaging and postmortem studies supported that depression involved structural and functional alterations in many brain regions including the hippocampus, PFC, amygdala, NAC and thalamus523053To our knowledge, this is the first study to explore L1 copy number and its methylation status in peripheral blood of MDD patients. It is also the first time to investigate L1 copy number in five brain regions and in blood in the animal depression model. The results should be repeated in a larger sample of depression patients and animals, and additional animal models of depression is also needed to clarify the role of L1 in the MDD. In conclusion, L1 misregulation may be associated with MDD although L1 activity may be variable between peripheral blood and brain tissues. Possibly, there are different mechanisms that control L1 activity in both the central nervous system and peripheral blood.The complete details of the entire study design and procedures involved were in accordance with the Declaration of Helsinki. All patients and control subjects were of the Chinese Han origin from the northern area of China. They all gave written informed consent to attending this study as approved by the Ethics Committee of Chinese Academy of Medical Sciences and Peking Union Medical College. All animal work was approved by the Experimental Animal Center of Chinese Academy of Medical Sciences and Peking Union Medical College and in accordance with the institutional guidelines of the Beijing Administration Office of Laboratory Animals. This study was approved by the Institutional Review Board of Chinese Academy of Medical Sciences and Peking Union Medical College.A total of 105 unrelated patients with MDD  and 105 healthy controls  were recruited for analysis of L1 copy numbers. Among them, 60 MDD patients  and 41 healthy controls  were used for analysis of L1 5′-UTR DNA methylation. All the patients were recruited as inpatients by First Hospital of Shanxi Medical University. Clinical diagnosis of MDD was made by at least two experienced psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders Fourth Edition (DSM-IV) criteria. Patients were interviewed with the same psychiatrists using the 17-item Hamilton Rating Scale for Depression (HAMD-17) during sampling. None of these patients received psychotropic medication within 4 weeks. Control subjects were recruited from local communities. The eligible controls were in physical health and did not have a history of psychiatric illness, serious somatic disease, alcoholism or drug abuse.Male C57BL/6 J mice at 8–9 weeks of age  were housed in groups of 2 per cage under a 12-h light/dark cycle at constant temperature (25 °C) with ad libitum access to food and water. All C57BL/6 J mice were allowed 1 week of habituation to the housing conditions before the start of experiments.QuickGene DNA whole blood kit  was used with Automatic DNA/RNA Extraction System  to isolate genomic DNA from human blood, which enables automatic extraction of about 50 μg of DNA from 2 ml of whole blood. Quantitative PCR experiments were performed using a CFX96 Real-Time PCR Detection System  according to standard protocol. The quantitative multiplexing PCR assay of human sample was performed using 100 pg DNA and SYBR-Green assay of mouse sample was performed using 500 pg DNA. Standard curves of genomic DNA ranging from 2.5 ng to 4 pg were performed with 5 times dilution. The slope of linear regression to the standard curve was nearly −3.32, which means that the primer efficiency and multiplexing effectiveness were acceptable. All RT-PCR reactions were performed in triplicates.et al. describedHuman L1 copy number was measured with ORF2, HERVH or SATA as internal controls. The quantitative multiplexing PCR strategy to investigate endogenous L1 activity in humans was done as Gage Human L1 5′-UTR DNA methylation was measured by methylation sensitive restriction enzymes (MSREs) with quantitative RT-PCR (MSREs RT-PCR). The OneStep qMethyl™ kit  was used to measure the level of DNA methylation. Primers used for L1 amplifications in 5′-UTR of human DNA sample were as follows: hL1-methF 5′-ACAGCTCCGGTCTACAGCTC-3′ and hL1-methR 5′-GTGTGGGATATAGTCTCGTGGTG-3′. The experimental procedure was based on manufacturer’s standard protocol. For each DNA sample to be analyzed, both a Test Reaction and a Reference Reaction mixture were set up properly. PCR was performed in a 20-μl reaction volume with 10 μl of 2 × Test Reaction PreMix or Reference Reaction PreMix, 5 μl of DNA (100 pg/μl) and 1 μl of mixed primers (5 μmol/L). The detailed quantitative PCR conditions were as follows: 37 °C MSREs digestion for 2 h, 95 °C for 10 min, followed by 45 cycles of amplification and quantification , 72 °C final extension for 7 min.−ΔΔCt) method was employed to evaluate the relative quantity of L1 5′-UTR DNA methylation status. ΔCt = the Ct value from the Test Reaction minus the Ct values from the Reference Reaction. The large ΔCt represents the high status of non-methylated DNA.The comparative Ct  for 6 min. The procedure was recorded by video and the immobility time in the last 4 min was scored by an experienced observer who is blind to the mouse identities. Immobility was defined as floating or remaining motionless without leaning against the wall of the beakerTo determine whether mice acquired anhedonic phenotype induced by CUMS, a standard sucrose consumption assay was performed. Mice were habituated to drink 1% of sucrose solution  in a 50 ml conical tube for 48 h, followed by 12 h fluid deprivation. On the test day, mice were presented with 1% of sucrose solution for 1 h and the consumption was determined by the difference after weighing the conical tube pre- and post-test.After mice were deeply anesthetized with 10% chloral hydrate, 1 ml of eyeball blood was obtained and all mice were killed by cervical dislocation. Genomic DNA in blood was extracted by TIANamp Blood DNA Kit  from 500 μl of whole blood. The brains were rapidly extracted and frozen in the cold isopentane for 30 sec, then stored at −80 °C until processed. Brains were embedded in the optimal cutting temperature compound  for 3 h at −22 °C in a cryostat  to get equilibration of temperature before sampling. Tissues from different brain regions were sampled using a punch  with a diameter of 1.25 mm . The hipA 20-μl reaction volume containing 10 μl of 2 × FastStart Essential DNA Green Master Mix , 1 μl of mixed primers (8 μmol/L for 5srRNA and 2.5 μmol/L for others), and 1 μl of DNA (500 pg/μl) was used for quantitative RT-PCR amplification . The detailed RT-PCR conditions were as follows: 95 °C for 10 min, followed by 36 cycles of amplification and quantification . The blood and brains from twenty-one CUMS and twelve control mouse were quantified except that one sample of PVN did not meet our quality and was discarded.−ΔΔCt) method was used to quantify expression of L1 copy number and fold change (FC) was used to present data. Binary logistic regression was used to examine the difference in L1 levels and L1 5′-UTR DNA methylation between the patient group and control group with adjustment for age and sex. Pearson correlation was applied to analyze the correlation between the total score of HAMD-17 and either L1 levels or L1 5′-UTR DNA methylation. Mann-Whitney U test was used to examine the differences in L1 copy number, sucrose consumption and immobility time between CUMS mice and controls.The ratio of L1 copy number was calculated and then normalized relative to the average value of control samples. The comparative Ct .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Influence of the ionic strength on the transistor performance is also investigated. Double contacts are found to decrease the effective resistance and the transfer length, but do not improve the transconductance. An electrochemical annealing/cleaning effect is investigated and proposed to originate from the out-of-plane gate leakage current. The devices are used as a proof-of-concept for bioelectronic sensors, recording external potentials from both: ex vivo heart tissue and in vitro cardiomyocyte-like HL-1 cells. The recordings show distinguishable action potentials with a signal to noise ratio over 14 from ex vivo tissue and over 6 from the cardiac-like cell line in vitro. Furthermore, in vitro neuronal signals are recorded by the graphene transistors with distinguishable bursting for the first time.This work is focused on the fabrication and analysis of graphene-based, solution-gated field effect transistor arrays (GFETs) on a large scale for bioelectronic measurements. The GFETs fabricated on different substrates, with a variety of gate geometries (width/length) of the graphene channel, reveal a linear relation between the transconductance and the width/length ratio. The area normalised electrolyte-gated transconductance is in the range of 1–2 mS·V Graphene field effect transistors (GFETs) are the main focus of the work. In general, transistors are very interesting for bioelectronics, since when compared to microelectrode arrays (MEAs)4 they are active elements and are therefore more functional and tunable. Graphene transistors have already been shown to be extremely sensitive to changes in the gate potential in a liquid environment5. Moreover, it is possible to decrease the device’s size without impairing its performance (if the W/L ratio is preserved), which is a great advantage when compared to classical microelectrode arrays (MEAs)4. Additionally, even large areas of graphene have been proven to be both biocompatible and cytocompatible7.In the field of bioelectronics, graphene is a promising candidate for very efficient, flexible, biocompatible and implantable sensors9.Additionally, in order to conduct good quality extracellular measurements reproduciblythe devices need to be identical or close to identical. However, up to now most fabrication routes for graphene-based bioelectronics are at an early development stage where devices are processed individually or in small arrays comprising only of a few devices and fabricated on a chip-scale11, while some have attempted using chemical vapor deposition (CVD) graphene for the wafer scale fabrication of devices14. One of the main problems in this regard is the quality of CVD-grown graphene15. However, up to now, CVD graphene can be grown on Cu or Cu-Ni foils with grain sizes up to the centimeter scale17, and recent advances in graphene growth show that even in cold wall CVD reactors it is possible to fabricate high quality monolayers of graphene18. However, the graphene still needs to be transferred to device-compatible substrates and the transfer process can introduce defects and consequently a low yield in functional devices20.In recent years there have been many attempts to scale up the single-device processing to wafer-scale fabrication; some are still focused on epitaxially grown graphene2 of the graphene-on-copper for fabrication of one 4-inch wafer with 52 devices per wafer21. This high-throughput transfer and large scale fabrication approach, combined with the cm-scale sizes of graphene domains17, will result in more reproducible performance of the GFETs.In our previous work we demonstrated efficient transfer of graphene, which requires only 4 cm2, HfO2, polyimide), passivation, geometric considerations and graphene channel size; (b) measurement conditions, including ionic strength of the gating solution used and applied potentials. Bio-experiments, consisting of ex vivo (heart tissue) and in vitro  recordings, prove the applicability of such graphene transistors for bioelectronics. A new passivation type, the feedline follower is introduced and argued to be better for neuronal interfacing. An electrochemical, gate leakage current induced cleaning of graphene and consecutive improvement of the GFETs performance is also investigated in the scope of this work.In this work, we present a large scale fabrication of GFETs aimed for bioelectronics applications. Fabricated on a 4-inch scale, the process can be further adjusted to 6- and 8-inch processes with similar yield. Altogether, we evaluate the performance of the solution-gated GFETs  depending on: (a) processing parameters, including substrate type (SiO2/Si (further denoted as Si-I and Si-II), one HfO2/Si (Hf) and one polyimide/Si (PI)  is far within the p-doping regime  is reached . The characterisation of a GFET consists of two steps: current annealing and measurement. The annealing step, was discovered during initial measurements, and is required to bring the transistor into its operative state. Failure to perform this step prevents further accurate analysis of the devices. During the annealing step, the drain-source current, Isee Fig. . Howeversee Fig. . Once anGS potentials  increases the gate leakage current up to tens of nA  via an electrochemical reaction driven by the out-of-plane leakage current. Application of high Vsee Fig. . We obsesee Fig. . HoweverThe removal of contaminants can result in an intermediate step, when a local doping creates a second conduction minima  and analysed for their performance, including transconductance, mobility, Dirac resistance, contact resistance and sheet resistance . The first two parameters  are usually taken into consideration for comparison with other materials and works25. Remarkably, some of the values, like sheet resistance, RS, can be used for the rough evaluation of the GFET’s quality, while values of mobility have to be carefully examined in order to provide a correct and valuable comparison (see Supplementary Information for details).Following the annealing steps, the GFETs are characterised  and high ionic concentrations of the solution gate, when the usual approach would end up in an unrealistic case of ions coming too close to the graphene surface31. Finally, we combine all three capacitances, with the calculation details given in the Supplementary Information. In order to provide a comprehensive analysis, we measured six GFETs in a PBS titration series PBS from 10x down to 0.001x. 10x PBS is a solution has an ionic strength of 1.62 M, which would theoretically result in a Debye length of 0.24 nm, however, such values are not physically possible31. Therefore, for the highly concentrated salt solutions, the air-gap simplified model is used for the capacitance calculations. In the model, the diffuse layer breaks down for high concentrations and steric repulsion has to be considered. Nevertheless, the overall capacitance is dominated by the air gap (which takes the place of the Stern layer in our model)31. Therefore we use the simpler model, taking into account only the quantum and air gap capacitances  ratio of the device, as previously reported33. In Fig. 33. Such normalisation brings more comparable values important to describe and compare intrinsic properties of GFETs. The other wafers were analysed in a similar way and the averaged fitting curves, which show the slope of the transconductance are given in Supplementary Fig. −1·□, and plot in statistical details in Fig. DS and number of squares (□), for all five wafers are in the range of 1-2 mS·V−1·□.Further analysis of a device’s performance is done regarding the processing parameters, i.e. substrate, width and length of the graphene area, and the type of contacts. For this purpose, over 500 GFETs from four different wafers  with different lengths and widths were measured and analysed. The ionic solution and V2·V−1·s−1). However, one can see that there are rare occurrences of extra-large mobility values especially for the HfO2 and polyimide substrates . One of the possible explanations of this phenomena is a finite crystallinity of the graphene. Regardless of the grain sizes of the CVD grown graphene, in smaller devices there are fewer grain boundaries, reaching a situation when a GFET consists of a single graphene crystal. In this case we see the drastic increase in that transistor’s charge carrier mobility. While for the GFETs with channels above 100 μm2 in area, the chance of grain boundaries increases, limiting electrical performance of the GFETs35. The principle was also shown when boundaries were specifically introduced for ion channel sensing applications36. Another explanation for the observed mobility values is substrate-induced scattering, which constricts the electrical properties of even single crystalline devices, as visible on the example of SiO2-based devices. Although the devices on SiO2 substrates have the largest average mobility (750 ± 350 cm2·V−1·s−1), the SiO2 substrate results in a suppression of the performance37 compared to HfO2 and polyimide substrates, where the value can reach up to 4.9 × 103 cm2·V−1·s−1.In order to understand the field effect mobility values , the values are averaged for each wafer see Fig. . Since ttes Fig. . In ordetes Fig. . From thC, and transfer length, LT. The transmission line measurement technique (TLMT) has been used to determine the parameters38. Details of the calculations are given in the Supplementary Information. We analysed the difference in the contact resistance for wafer Si-I (top contacted) and Si-II (double contacted) and the results can be seen in Supplementary Fig. S, Si-I = 1550 ± 820 Ω and RS, Si-II = 1210 ± 1040 Ω. Clearly double side contacting reduces the contact resistance and therefore the overall sheet resistance of the GFETs. The computed transfer length for the wafers are: LT, Si-I = 8.5 ± 2.2 µm and LT, Si-II = 3.6 ± 2.2 µm. The transfer length is the path a charge carrier has to travel underneath the contact area before transfer to the feedline. Decreasing transfer length is important for further miniaturisation of the devices. This shows that the transfer length could be reduced by more than a factor of 2 using double contacted graphene.The last two important parameters for characterisation of the GFETs are the contact resistance, R41 when the geometrical and environmental factors are taken into account. Interesting to see is that first of all just voltage normalised transconductance values can be misleading, and the values are extremely over/underestimated. When the values are further normalised per number of squares, the final performance shows a similar trend across publications.Table in, to the gate electrode, while source-drain signal amplitude, Vout, is monitored and the overall amplification factor DS is simply measured, and the changes (∆VDS) are transformed into ∆VGS by dividing by AV .In order to show the applicability of the devices for bioelectronics applications, the devices were encapsulated and prepared for cell culture (see Supplementary Information). The devices were later measured at the custom-built multi-channel measurement set-up, the principle of which is schematically shown in Fig. see Fig. . Every fex vivo bioelectronics. The overall signal-to-noise ratio (SNR) of the measurements is up to 17, while considering the noise as twice the median absolute distribution .Afterwards, an embryonic heart tissue is placed on the chip’s surface see Fig. . A Ag/Ag42. The shape of the APs is shown in Fig. 44.Cardiomyocyte-like cells (HL-1 cell line) are further cultured on the chips’ surface see Fig. . A typic2. The neuronal network was cultured for at least 14 days in vitro (DIV14) until mature, when the cultured neurons produce spontaneous APs that can propagate through the network. As expected, the extracellular neuronal APs are one or two orders of magnitude smaller than those of heart tissue45. Nevertheless, we were able to record the APs and even bursting activity of neuronal networks with the GFETs  is presented in Fig. Further, cortical neurons were cultured on top of the chips (details are given in Methods) with an approximate density of 1500 cells per mmETs Fig. . More tiImportant to consider in further comparison is that in all our SNR measurements we use 2 × MAD instead of root mean square (RMS) for the noise value and only a 50 Hz filter applied to the recordings to remove the power line hum and to keep the signal as undisturbed as possible. In addition to that, there is a 3 kHz anti-aliasing low-pass filter installed in the amplifier system. However, an appropriate filter and use of the RMS value would result in the SNR estimation up to 5 for neuronal recordings, 10 for HL-1s and 25 for heart tissue (see Supplementary Information for details).46. Moreover, the final signal shape depends on the signal transport from a cellular membrane to a transistor. Here the important factor is the so-called cell-chip coupling47. Since the cells are alive and dynamically growing, the coupling might be different from culture to culture and from chip to chip49. Nevertheless, our neuronal recordings from graphene transistors are in good accordance with previously published neuronal AP shape data8.Understanding the shape and amplitude of the recorded neuronal action potentials is a complex task, considering that different parts of a neuronal network produce different kinds of potentials50, which form a large confluent layer all-over the surface, create a sealed environment and therefore increase the signal51. Neuronal networks, in contrast, do not form confluent layers, but rather grow a large network of neurites. The neurites could be as small as 1 µm in diameter. Therefore, neurons usually do not cover the entire passivation opening, and the large gap distance is compounded by unsealed areas at the cell edges  was then spin-coated (3000 rpm), soft baked (110 °C for one minute), exposed to i-line UV light with a dose of 55 mJ/cm2, developed in 0.26% TMAH solution, and was used to protect the graphene active areas during exposure to oxygen plasma . The stack of 10 nm Ti and 90 nm Au was e-beam assisted evaporated on the wafer through a pre-defined structure of lift-off resists. For this purpose, LOR-3B  was spin-coated (3000 rpm) and soft baked (150 °C for 5 minutes) on top of another resist, nLOF-2020 , spin-coated (3000 rpm) and soft baked (100 °C for 2 minutes) in advance. The resists were exposed to i-line UV light at a dose of 40 mJ/cm2, post-exposure baked (110 °C for 1 minute) and developed in 0.26% TMAH. Photostructurable polyimide, HD8820 (HD Microsystems) was used in the last step to form the passivation. Spin-coated at 5000 rpm, soft-baked at 120 °C (with a slow ramp), exposed to 250 mJ/cm2 i-line UV light, developed in 0.26% TMAH for 80 seconds and hard baked , the polyimide formed a pinhole-free, 3 µm thick passivation. Post-cleanroom treatments, such as device encapsulation protocols are given in the Supplementary Information.The graphene was CVD grown on 25 µm thick copper foils in an Ar/H51. Prior to the seeding, the chips were cleaned with 70% ethanol and coated with fibronectin/gelatin solution . When reaching 100% confluency, the cells were passaged and seeded on top of the GFETs at 200 cells/mm2. The chips were then placed in an incubator (37 °C and 5% CO2) for the cells to mature. Claycomb medium, supplemented with 10% fetal bovine serum, 100U/ml-100µg/ml penicillin-streptomycin, 0.1 mM norepinephrine and 2 mM L-glutamine was exchanged every day (100%) and two hours before the measurements.The cardiomyocyte-like cell line HL-1 was cultured in T25 flasksThe heart tissue was prepared by dissecting embryonic tissue from an E18 Wistar rat. The heart of an embryo is quickly isolated, washed in Hank’s balanced salt solution (HBSS), then stored and measured in supplemented Claycomb medium (see HL-1 cell culture).52. Prior to culturing the cells on a chip, a glass ring was mounted onto the chip to form a culture container and the surface of the chip was coated with GpECM  for improved cellular adhesion. The cells were diluted in Neurobasal medium (Life Technologies), supplemented with B27 (1% (v/v), Gibco), L-glutamine  and Gentamicin (0.05 mg/mL), and plated on top of the chip with an approximate density of 1500 cells per mm2. Chips were kept in an incubator (37 °C and 5% CO2) and the supplemented Neurobasal medium was exchanged with 100% fresh supplemented medium two hours after the plating then with 50% fresh supplemented medium twice per week and one day before the measurements. The neurons were grown until at least DIV14. Measurements were performed on DIV 14–26.The primary cortical neurons were isolated from E18 Wistar ratsLive-Dead staining was performed using 1 µg/ml Calcein-AM and 2 µM Ethidium Homodimer (both Life Technologies) in supplemented cell growth medium to stain live and dead cells in green and red, respectively. Cells and dyes were incubated for 15 minutes in a 37 °C incubator or on a 37 °C hot plate (if performed after the measurements). The samples were observed using an Axio Imager Z1 microscope (Carl Zeiss).53. After the culture, the samples are chemically fixed using a 3.2% glutaraldehyde solution in PBS for 15 minutes at room temperature. Following that dehydration is performed in a series of ethanol washing steps (from 10% to 100% of ethanol concentration). The samples are then stored in 100% ethanol until they are dried via the CO2 critical point drying (CPD) technique. In order to improve the charge flow, the samples were sputtered with approximately 10 nm of platinum prior to the SEM imaging. Figure In order to see the topography of the neuronal network, a high-resolution image is required. We used scanning electron microscopy (SEM) to observe the outgrowth of neurons on the chip surface. It is important to use a special preparation technique in order to image biological specimens due to their normal existence in an aqueous environment and lack of conductivityIf not stated otherwise, all chemicals were purchased from Sigma Aldrich. The experiments were done with the approval of the Landesumweltamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Recklinghausen, Germany, number 84–02.04.2015.A173.The data that support the findings of this study are available from the corresponding author on request.Supplementary


Coccinella septempunctata) at different humidities by horizontal traction experiments. We found that both low (15%) and high (99%) relative humidities lead to a decrease of attachment ability. The significantly highest attachment forces were revealed at 60% humidity. This relationship was found both in female and male beetles, despite of a deviating structure of adhesive setae and a significant difference in forces between sexes. These findings demonstrate that not only dry adhesive setae are affected by ambient humidity, but also setae that stick due to the capillarity of an oily secretion.Many insects possess adhesive foot pads, which enable them to scale smooth vertical surfaces. The function of these organs may be highly affected by environmental conditions. Ladybird beetles (Coccinellidae) possess dense tarsal soles of tenent setae, supplemented with an adhesive fluid. We studied the attachment ability of the seven-spotted ladybird beetle ( Substrate attachment plays an important role in the niche occupation of plant-dwelling insects, since it is substantial for resting and locomotion in a complex environment. Consequently, a high diversity of friction and adhesion enhancing structures has evolved among insects –2. SeverCoccinella septempunctata) were carried out in an environmental chamber at various controlled humidities. Experiments were carried out with female and male beetles, which differ in the structure of their adhesive pads [C. septempunctata? (2) Is the attachment ability in both sexes likewise affected by ambient humidity?In the present study, force measurements with tethered seven-spotted ladybirds  was chosen (Pteridium aquilinum) in the New Botanical Garden at Kiel University, Schleswig-Holstein, Germany. They were kept individually in plastic tubes at a temperature of 22–24 °C and relative humidity of 40–50%. These conditions were consistent with those at which the beetles were captured. The beetles were fed with honey. C. septempunctata. The tarsus is composed of three tarsomeres and two ventrally curved claws  seted claws , (3) seted claws , and (4)ed claws . All foued claws  for all ed claws –J, exceped claws .Traction force experiments were performed in a polymethylmethacrylate (PMMA) chamber 30 × 14 × 14 cm) in which relative humidity could be manipulated by the controlled mixture of dry and wetted air  were used: 15%, 60% and 99%. The RH was monitored with a P330 digital hygrometer . To generate humid conditions, air was passed through a water bottle, so that the vapour concentration in the air increased. To reach 99% of relative humidity, slight heating was used to increase the amount of water vapour. This did not affect the temperature within the experimentation chamber. To generate a dry environment, dry air from a pressurized air pipe was pumped into the experimentation box. To stabilize ambient humidity and the thickness of adsorbed water films, the procedure was started about one hour before the experiments.A strain-gauge force transducer  connected to a Biopac MP100 amplifier  was used to measure forces generated by the beetles. Force data were recorded with the AcqKnowledge 3.7.0 software (Biopac Systems Inc.). A half Plexiglas tube was installed inside the chamber, in order to guide beetle locomotion perpendicular to the force sensor and prevent it from climbing onto the lateral walls.Prior to experiments the beetles were anesthetized with carbon dioxide and the elytra were glued together with a droplet of molten wax, to prevent them from flying (for details see ). IndiviC. septempunctata, a Kruskal–Wallis One Way ANOVA on Ranks followed by an all pairwise multiple comparison procedure  has been performed. In order to evaluate the effect of sex on the attachment force, a Mann–Whitney Rank Sum Test has been applied to the pooled data. Therefore, the data of the different relative humidity has been pooled individually for both experiments and sexes.The experiment was carried out in two different runs. The first run was done with 15 beetles . All beetles were tested with one single level of humidity per day, further called Experiment 1. To exclude an influence of physiological conditions of individual animals on different experimental days, a second run was executed. In this run, 9 beetles  were tested. Individual beetles were tested on one day at all three levels of relative humidity in a randomized fashion, further called Experiment 2. To evaluate the effect of the relative humidity on attachment force in both sexes of A Plexiglas sheet was fixed with double side adhesive tape at three different positions: horizontal (0°), vertical (90°) and upside-down (180°) inside the experimentation chamber. We observed the locomotion behaviour of individual beetles on these surfaces. Additionally, to the three previously used humidity adjustments , we made some observations at RH higher than 99%, when water began to condense on the substrate.We observed that the beetle’s attachment is affected, as the water begins to condense on the substrate, when the relative humidity exceeds 99%. In this case, beetles were not able to hold on the vertical Plexiglas slide and slid along its surface.N = 40; H = 30.035; df = 2; P ≤ 0.001). An all pairwise comparison . An all pairwise comparison . An all pairwise comparison . An all pairwise comparison  adhesive pad is influenced by ambient humidity. Our results indicate an optimal range of relative humidity with maximal traction forces in ladybird beetles. Similar observations have been made in dry adhesive pads of spiders, using a similar setup and method as in the present study , and gecFor the dry adhesive pads of spiders and geckos the humidity-related effects on adhesion can be explained by three different mechanisms: (1) capillary forces due to the formation of liquid bridges; (2) changes in the effective short-range interactions due to adsorbed monolayers of water on the substrate ,14; (3) C. septempunctata it was recently shown that the setal tips contain high amounts of the soft rubber-like protein resilin [For  resilin , which i resilin . Peisker resilin  showed aLeptinotarsa decemlineata) on a dry surface, which were kept prior the experiment under humid conditions if compared to beetles kept prior the experiment under dry conditions. In the present study we observed a distinct difference between both types of experiments (Experiment 1 and 2). The differences between the measured traction forces, although significant, are smaller in case of all humidity levels measured on one day (Experiment 2), than in the case of one humidity level measured per day (Experiment 1). Since in Experiment 2 the humidity levels were randomized for individual runs, there were some beetles that were measured first at higher humidity and then at lower humidity. In combination with relatively short durations between individual runs, those individuals, according to the idea of changing material properties, should then show a better performance also at the lower humidity level if compared to Experiment 1, thus, reducing the observable differences between different humidity levels.Two other observations may further support the hypothesis that a change in material properties with increasing humidity is responsible for at least the increase in traction forces from 15% RH to 60% RH. Voigt et al.  observedAlthough the hypothesis of changing material properties seems to be quite plausible, there are also several aspects speaking against it. The tarsal secretions in hairy attachment pads of beetles were found to be mainly lipid-based –2749–50,27C. septempunctata, viscosity of the tarsal secretion was found to be of order 20 mPa·s [Coming back to the abovementioned mechanisms of capillary forces and/or changes in the effective short-range van der Waals interaction, which might influence the humidity dependent adhesion in geckos , it need20 mPa·s  and drop20 mPa·s ). Moreov20 mPa·s , which m20 mPa·s .The reduction in traction forces observed in the present study at 99% RH (without visible water condensation) may be explained by a sufficient amount of adsorbed water on the substrate. It has been shown that the friction behaviour between two solids crucially depends on the water layer thickness between them . At critIn nature, animals are constantly exposed to certain humidity. Most of insects live on plants containing a humid boundary layer in the vicinity of the plant surface. The humidity close to leaf surfaces ranges between 30% and 75% . It is pC. septempunctata beetles, we found evidence for a sexual dimorphism. Male beetles generated higher adhesion than females, which was previously reported and explained by the presence of specialized setae with discoidal tips that can generate higher adhesion on smooth substrates due to their advantageous, geometry-induced homogenous stress distribution [Finally, comparing absolute values in the attachment forces of ribution –60. Simiribution ,61. The The present study demonstrates, for the first time, the effect of ambient humidity on a wet adhesive pad that is supplemented by a lipid-based secretion. This shows that humidity similarly affects the function of both dry and wet adhesive pads. However, the underlying mechanisms leading to this effect remain unclear. To be able to distinguish between different mechanisms , experiments with substrates of different surface free energy should be performed in the future.


Mutagenesis study revealed that two hot-spot residues  of PD-L1 contribute significantly to the binding of atezolizumab. The structure also shows that atezolizumab binds PD-L1 with a distinct heavy and light chain orientation and it blocks PD-1/PD-L1 interaction through competing with PD-1 for the same PD-L1 surface area. Taken together, the complex structure of PD-L1/atezolizumab solved here revealed the molecular mechanism of atezolizumab in immunotherapy and provides basis for future monoclonal antibody optimization and rational design of small chemical compounds targeting PD-L1 surface.Monoclonal antibodies targeting PD-1/PD-L1 signaling pathway have achieved unprecedented success in cancer treatment over the last few years. Atezolizumab is the first PD-L1 monoclonal antibody approved by US FDA for cancer therapy; however the molecular basis of atezolizumab in blocking PD-1/PD-L1 interaction is not fully understood. Here we have solved the crystal structure of PD-L1/atezolizumab complex at 2.9 angstrom resolution. The structure shows that atezolizumab binds the front beta-sheet of PD-L1 through three CDR loops from the heavy chain and one CDR loop from the light chain. The binding involves extensive hydrogen-bonding and hydrophobic interactions. Notably there are multiple aromatic residues from the CDR loops forming Pi-Pi stacking or cation-Pi interactions within the center of the binding interface and the buried surface area is more than 2000 Å There are more than forty ongoing trials with atezolizumab either as monotherapy or in combination with other agents . Clinical indications under investigation include renal cell carcinoma, hepatocellular carcinoma, triple negative breast cancer, colorectal cancer, hematologic malignancies in addition to other tumor types as well. Several other anti-PD-L1 antibodies including avelumab, durvalumab and KN035 are also in intensive clinical trials. Crystal structures of these PD-L1 antibodies such as avelumab, durvalumab, BMS936559 and KN035 have been reported recently [Atezolizumab (TECENTRIQ) developed by Genetech is the first therapeutic anti-PD-L1 antibody approved in the United States for the treatment of patients with metastatic urothelial carcinoma or non-small cell lung cancer who have progressed during or following platinum-containing chemotherapy [recently –19, howeE.coli as inclusion body and purified after refolding as previously described [Atezolizumab Fab fragment was expressed in HEK293 expression system and purified from the culture medium. The IgV domain of PD-L1 was expressed in escribed . Crystalescribed , 20.2 in PD-L1/atezolizumab binding interface, which is the largest among the known PD-L1/antibodies structures. The buried surface area of atezolizumab is 1029 Å2 with 784 Å2 from the heavy chain and 245 Å2 from the light chain in the binding interface, and the buried surface area of PD-L1 is 971 Å2  shows that there are 13 hydrogen bonds  and abou2 Figure . Althoug2 for PD-1, 644.4 Å2 for KN035, 875.4 Å2 for avelumab, 742.4 Å2 for BMS-936559 and 971 Å2 for atezolizumab respectively. These binding areas largely overlap each other  or IgG4 isoform (BMS-936559) . These dIt is also somewhat surprising that all these PD-L1 blocking antibodies bind PD-L1 on the flat front beta-sheet as generally targeting a relatively flat protein surface is very challenging. When compared with other immune checkpoint blocking antibodies, we have found that CTLA-4 blocking antibodies such as ipilimumab (PDB:5TRU) and tremelimumab (PDB:5GGV) similarly target the front surface of IgV domain of CTLA-4, while PD-1 blocking antibodies such as pembrolizumab (PDB:5GGS) and nivolumab (PDB:5GGR) mainly bind PD-1 on the connecting loops of the front beta-sheet , 22–24. It has been shown that many residues are often involved in a protein binding interface, but only a few of them make critical contributions towards complex formation. These key residues are often called the hot-spot residues of a given protein surface and they are general targets for rational drug design in blocking protein interactions , 26. We In conclusion, the crystal structure of therapeutic antibody atezolizumab complexed with PD-L1 solved here shows that the antibody blocks PD-1/PD-L1 pathway through competing with PD-1 for the binding surface on PD-L1 and this provides basis for further antibody optimization and for the development of novel chemical PD-L1 inhibitors for immunotherapy.*106/ml. Culture medium were collected after 3 days through centrifugation. The assembled Fab fragment of atezolizumab with a his-tag at the C-terminus of the heavy chain was purified by Ni column  from the medium after dialysis against 10 mM Tris-HCl, pH 7.4, 150 mM NaCl. IgV domain of PD-L1 is prepared as previously described [E.coli BL21 (DE3) as inclusion bodies. Cells were cultured at 37°C in LB and induced with 0.5 mM IPTG  once the optical density at 600 nm reached 1.0. After a further 16-hour incubation at 37°C, the cells were collected by centrifugation, resuspended in lysis buffer  and lyzed by sonication. Inclusion bodies were recovered by centrifugation at 15,000 g for 10 minutes and were then washed 3 times with lysis buffer, followed by washing with buffer without Triton X-100. The inclusion bodies were dissolved in 6 M GuHCl, 0.5 mM EDTA, and 10 mM DTT, 20 mMTris, pH 7.4 and added drop-wise into a refolding buffer consisting of 1 M Arg hydrochloride, 0.1 M Tris, pH 8.0, 2mM EDTA, 0.25 mM oxidized glutathione, 0.25 mM reduced glutathione and 0.1 mg/ml of atezolizumab. The PD-L1 IgV domain/atezolizumab complexes were subsequently purified using ion exchange and gel filtration columns .The Fab fragment of atezolizumab was expressed in the transient HEK293 expression system. Mixture of PEI (Polyscience) and expression plasmids coding the heavy chain and light chain of the Fab fragment of atezolizumab (w/w 3:1) was transfect into HEK293 suspension cell at a density of 4escribed . BrieflyPD-L1/atezolizumab complex was concentrated to ~10 mg/ml and screened for crystallization conditions using commercially available buffers with sitting-drop vapor diffusion method where 0.2 μl of the protein solution was mixed with 0.2 μl of reservoir solution. Diffraction-quality crystals of PD-L1/atezolizumab complex were obtained at room temperature from 2 M ammonium sulfate and 0.1 M Tris pH7.0 after optimization.Crystals were cryoprotected in 20% glycerol in the mother liquor and flash-cooled in liquid nitrogen. Diffraction data were collected on beamlines BL17U at SSRF, Shanghai, China. The data were indexed and processed with iMosflm and scaled with Aimless from the CCP4 suite . The strThe binding kinetics of PD-L1 variants to atezolizumab was measured as previously described . Briefly


Finally, we present the solution NMR structure of a head‐to‐tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher‐order polymerization. These data provide structural detail for the established mechanistic role of the well‐folded TDP‐43 NTD in splicing and link this function to LLPS. In addition, the fusion‐tag solubilized, recombinant form of TDP‐43 full‐length protein developed here will enable future phase separation and in vitro biochemical assays on TDP‐43 function and interactions that have been hampered in the past by TDP‐43 aggregation.TDP‐43 is an RNA‐binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C‐terminal domain which mediates ALS inclusions, TDP‐43 has a globular N‐terminal domain (NTD). Here, we show that TDP‐43 NTD assembles into head‐to‐tail linear chains and that phosphomimetic substitution at S48 disrupts TDP‐43 polymeric assembly, discourages liquid–liquid phase separation (LLPS)  Therefore, these data are consistent with the region near S48 playing a role in assembly.Inspired by the observation that pentamerization of nucleophosmin is regulated by phosphorylation  and sedimentation equilibrium analytical ultracentrifugation (AUC). The results from both methods support a model of weak infinite linear self‐association, specifically isodesmic assembly resulting in unbranched linear chains. CG‐MALS data were collected for wild‐type TDP‐43 NTD in a wide range of concentrations up to more than 300 μM. The mass average molecular weight increases continuously as a function of the concentration of the wild‐type NTD Fig E. ModelsKD ~ 40 μM (We observe evidence of the same self‐association behavior for TDP‐43 NTD in AUC experiments. In sedimentation velocity (SV) experiments, at least three species are observed when the data are modeled to determine the sedimentation coefficient distribution of the present species, and their fractional populations shift to larger species in a concentration‐dependent manner . However ~ 40 μM . The diset al, in vitro  cells, TDP‐43RRM‐GFP localizes to the nucleus and concentrates into a single large liquid–liquid phase separated assembly per cell over time . These findings suggest that the details of the NTD structure and interactions in the region surrounding S48, not just phosphorylation at S48, are critical for mechanisms that mediate TDP‐43 control of splicing. To further determine the requirement for NTD oligomerization in TDP‐43 splicing regulatory function, we studied NTD Y4R and E17R, two other self‐assembly deficient variants (see below), in our cellular assays. We observed a dramatic decrease in TDP‐43 activity upon introduction of both mutations  TDP‐43 NTD wild‐type titrated with increasing concentrations of NMR‐invisible  S48E TDP‐43 NTD would show chemical shift deviations only near S48. Indeed, chemical shift deviations are only observed in the C‐terminal half (residues 40–80) of wild‐type  TDP‐43 NTD S48E shows chemical shift deviations at residues in the N‐terminal half (residues 1–35) of the S48E protein  may be the consequence of the conformational change caused by the interaction occurring in the N‐terminal interface. These residues are the only outliers from the single interface behavior observed for other residues in the domain. Collectively, these data immediately point to a head‐to‐tail assembly  that are high for positions in close proximity to a stabilized nitroxide spin label, even in transiently populated states. Assuming a two‐state monomer–dimer equilibrium, the relative orientation of two NTD monomers in the assembled dimer state can be discerned given a sufficient number of samples with conjugated nitroxide radical spin labels at single positions spatially separated across the monomer surface. Therefore, MTSL spin labels were conjugated to the NTD at engineered cysteine variants at positions 2 or 29 or at native cysteine positions 39 or 50 and mixed with 15N isotopically enriched NTD C39S/C50S in order to measure 1HN transverse paramagnetic relaxation enhancements, Γ2, arising only from intermolecular contacts  S48E resulted first in disappearance of peaks due to the kinetics of exchange between monomer and complex  would inhibit self‐assembly as potently as S48E. In order to test this hypothesis, we generated NTD variants E17R and Y4R and examined their effects on self‐assembly. When the NMR chemical shift deviations for increasing concentrations of each mutant alone were compared to wild‐type, we found that E17R and Y4R NTD variants do not disrupt folding  but do d–30 would of S48E . The res13C/15N‐edited and 13C/15N‐filtered/edited NOESY measurements using the 13C/15N‐labeled S48E (or Y4R) complexed with natural abundance Y4R (or S48E)  . The filor S48E) . With thet al, et al, The structure of each subunit is largely consistent with the previously published NTD monomer structure of 2N4P  alter the interface dramatically, it is also likely that dimer (and higher‐order structures) is not saturated in their samples of polymerization competent wild‐type, as evidenced by the smaller chemical shift differences compared to our NMR titration, complicating interpretation of filtered NOESY experiments which can contain spectral artifacts from incomplete filtering  that the NTD oligomerization is important for function, (ii) that phosphosite S48  tag, and TEV cleavage site  fusion tag (designated pJ4M) was constructed using overlap extension PCR (Horton The TDP‐43‐MBP expression construct (designated pJ4M/TDP‐43) was created by subcloning the DNA sequence for TDP‐43, which was amplified from pJ411/TDP‐43 by PCR using primers 5‐TAATACGACTCACTATAGGG‐3 and 5‐TCATCACTCGAGCATGCCCC‐3, into pJ4M via restriction sites NdeI and XhoI.Escherichia coli BL21 Star DE3 (Life Technologies). Cells were grown in LB media for natural abundance isotope labeling. For expression of  isotope labeling, cells were grown in M9 minimal media supplemented with 13C‐glucose and/or 15NH4Cl (Cambridge Isotope Laboratories). In order to purify the TDP‐43 NTD, cell cultures were grown at 37°C to an OD600 of 0.6–0.9. Protein expression was induced by addition of isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) (GoldBio) to 1 mM, followed by incubation at 37°C for 4 h. Cells were harvested by centrifugation, resuspended in NTD binding buffer  supplemented with complete EDTA‐free protease inhibitor cocktail (Roche), and lysed using an EmulsiFlex C3 (Avestin). Cell lysates were clarified by centrifugation and applied to a 5 ml Histrap HP column . The column was washed with five volumes of binding buffer, and TDP‐43 NTD was eluted with a linear gradient of NTD elution buffer . Cleavage and removal of the N‐terminal Thio‐His tag was achieved by incubation with 0.03 mg/ml TEV protease concomitant with dialysis into NTD dialysis buffer , followed by a subsequent purification with the 5 ml Histrap HP column. TDP‐43 NTD was further purified by SEC over a Superdex 75 26/60 column  with NTD SEC buffer . Purified TDP‐43 NTD was concentrated to ~500 μM, flash frozen, and stored at −80°C.Point mutations were introduced either by Quikchange mutagenesis or by overlap extension PCR with mutagenic primers. Proteins were overexpressed in For purification of full‐length TDP‐43‐MBP fusion protein, cell cultures were grown at 37°C to an OD600 of 0.6–0.9. Cell cultures were then cooled to 16°C, after which protein expression was induced with addition of IPTG to 1 mM, followed by incubation at 16°C overnight. Cells were harvested by centrifugation, resuspended in TDP‐43 binding buffer  glycerol, 1 mM DTT) supplemented with complete EDTA‐free protease inhibitor cocktail, and lysed with the EmulsiFlex C3. Cell lysates were clarified by centrifugation, applied to the 5 ml Histrap HP column, and washed with five volumes of TDP‐43 binding buffer. TDP‐43‐MBP was eluted with a linear gradient of TDP‐43 elution buffer  glycerol, 1 mM DTT).Eluant was further purified by SEC over a Superdex 200 26/60 column  with TDP‐43 SEC buffer . Purified TDP‐43‐MBP was concentrated to 250 μM, flash frozen, and stored at −80°C.et al, NMR spectra were processed with NMRPipe CO, CBCA(CO)NH, and HNCACB.15N and 1H chemical shifts were measured from the crosspeaks on the HSQCs of 5, 20, 100, 200, 350 and 500 μM WT and 5, 100 and 200 μM variants in the condition of 20 mM HEPES pH 6.8, 1 mM DTT, and 10% D2O. Chemical shift deviations for 1H and 15N were quantified by subtracting the chemical shift of the 5 μM sample from the respective value of each other samples. The average chemical shift deviations (CSDs) were calculated by the equation: 15N S48E was titrated by 50 μM, 100 μM, 400 μM, 1 mM, 1.6 mM, and 2 mM of natural isotopic abundance Y4R. 15N Y4R was titrated by 50 μM, 100 μM, 400 μM, 800 μM, 1 mM, and 2 mM of natural isotopic abundance S48E. The CSDs for 15N and 1H were quantified by subtracting the chemical shifts of 100 μM 15N S48E (or 15N Y4R) from the respective value of each mixture samples and then calculated by the equation above.For the titrations of S48E and Y4R, the 100 μM of either 1H‐15N HSQCs were acquired at 500 MHz 1H Larmor frequency with 32 transients per data point with 64* and 1,536* complex pairs in the indirect 15N and direct 1H dimensions, with corresponding acquisition times of 35 and 219 ms, and sweep widths of 36.0 and 14.0 ppm centered at 116.5 and 4.7 ppm, respectively.15N‐labeled C39S/C50S in 20 mM HEPES pH 6.8, 10% D2O.Since TDP‐43 1–80 contains two intrinsic cysteines, C39 and C50, either cysteine was substituted by serine to generate the variants C39S and C50S, respectively. Other PRE variants including single cysteine substitution at S2 and S29 were engineered from the construct of C39S C50S TDP‐43 1–80. One mM MTSL  or 1 mM AcMTSL  was added into 50 μM of each variant protein under the condition of 20 mM Tris–Cl pH 8.0, 150 mM NaCl. After incubating 1 h at room temperature, the excess label was removed by HiPrep desalting column and the protein was exchanged to the buffer of 20 mM HEPES pH 6.8. Finally, the paramagnetic probe MTSL  was conjugated at C2, C29, C39, and C50, respectively. The samples were concentrated and flash frozen. For the intermolecular PRE measurement, 100 μM MTSL (or AcMTSL)‐conjugated PRE variant was mixed with 100 μM 1HNR2, were measured at 500 MHz 1H frequency for paramagnetic and diamagnetic samples, with 192* and 1,536* complex pairs in the 15N indirect and 1H direct dimensions, corresponding acquisition times of 105 and 21.9 ms, and sweep widths of 36 and 14 ppm centered at 116.5 and 4.7 ppm, respectively. Each 1HNR2 experiment comprised three interleaved 1HNR2 relaxation delays: 0.1, 2.1, 5.1 ms. PRE values (Γ2 = 1HNR2para − 1HNR2dia) were obtained from the data collected with 0.1‐ and 2.1‐ms delays using the two time point method as described previously . Distance restraints were derived from sets of 3D‐ NOESY experiments acquired with mixing time of 120 ms. Isotope‐filtered NOESY experiments  were performed to identify intermolecular NOE, which were further classified in strong, medium and weak restrains corresponding to 3.5, 4.5, and 6.0 Å upper limits. Dihedral angle restraints were calculated by using software TALOS+ , which uses a software‐controlled multiple syringe pump to create a concentration gradient, and a DAWN‐HELEOS multi‐angle light‐scattering photometer to collect data from the incoming sample stream. Static light‐scattering data were collected at 14 scattering angles as a function of protein concentration. For each injection, the solution was allowed to come to equilibrium for 60 s. Equilibrium MALS data as a function of concentration were analyzed as described by Attri et al , and datMx is the molar mass of protein X, and [Xi] is the concentration of the species Xi. R is normalized to an optical constant K* defined asin which n0 denotes the refractive index of the solvent, λ0 the vacuum wavelength of incident light (690 nm), NA, Avogadro's number, and dn/dc is the refractive index increment of the sample . The concentrations of each species are related to the equilibrium constants and total protein concentration, resulting in the following equations for typical monomer–dimer, monomer–trimer, monomer–i‐mer equilibria:where i = 1 represents the free monomer, the total molar concentration [X]total is known at each gradient injection, and R(0)/K* is measured. Non‐linear least square optimization is used to obtain a single KA value that fits the data across the entire concentration range of interest.Above, et al, To describe isodesmic self‐association, we used equations previously described  and the molecular weight of the building block, which was in agreement with a TDP‐43 NTD monomer within error.Fitting parameters were The SEC multi‐angle light‐scattering (SEC‐MALS) experiments were carried out using a WTC‐0150S5  size‐exclusion column  with three detectors connected in series: an Agilent 1200 ultraviolet (UV) detector , a Wyatt DAWN‐HELEOS‐II multi‐angle light‐scattering (MALS) detector, and a Wyatt Optilab T‐rEX differential refractive index (RI) detector . The column was equilibrated with 20 mM HEPES pH 6.8, 1 mM DTT, and either 0 or 150 mM NaCl. All data were collected at 25°C. A 100 μl sample was injected into the column using an auto‐sample injection method with a flow rate of 0.5 ml/min. Protein in the eluent was detected via UV absorbance at 280 nm, light‐scattering, and refractive index detectors. The data were recorded and analyzed with the Wyatt Astra software (version 7.0).Protein samples of TDP‐43 NTD WT and S48E were prepared at 400 μM in the following buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, and 1 mM DTT. The amine cross‐linker BS3 (bis(sulfosuccinimidyl)suberate, Fisher Scientific) was added to create a molar ratio of 50. Reactions were incubated at room temperature for 30 min. The reactions were quenched by the addition of 100 mM Tris pH 7.5 and were incubated at room temperature for at least 15 min prior to taking samples for analysis on a gel.et al, https://sedfitsedphat.nibib.nih.gov/software/default.aspx).The cell assembly, containing identical sample and reference buffer volumes of 360 μl, was placed in a rotor and temperature equilibrated at rest at 20°C for 2 h before it was accelerated from 0 to 50,000 rpm. Rayleigh interference optical data were collected continuously for 12 h separately. The velocity data were modeled with diffusion‐deconvoluted sedimentation coefficient distributions c(s) in SEDFIT, using algebraic noise decomposition and with signal‐average frictional ratio and meniscus position refined with non‐linear regression  following standard protocols unless mentioned otherwise  using the reversible one‐step self‐association model. The association scheme used in this analysis was [A] ↔ [A2] ↔ [A3] ↔ [A4] ↔ …. [isodesmic] with A being the TDP‐43 NTD monomer, and KDiso the single dissociation constant for the association steps were obtained from the analysis. Errors of the fits represent the 68.3% confidence interval (CI) using an automated surface projection method , 25,000 (for 24 h), and 43,000 rpm (for 24 h) (Zhao Oligopeptides and phospho‐specific rabbit antibodies against TDP‐43 amino acids 40–53 (GLRYRNPVSQCMRG) were custom produced by GenScript . Confirmation of antibody specificity was performed by serially diluting TDP‐43 oligopeptides in 8 M urea and spotting to nitrocellulose membranes, which were then blocked in 6% milk in Tris‐buffered saline (TBS). Membranes were then probed with polyclonal antibodies specific to the phospho‐peptide (α‐TDP‐43 pSer48) or non‐specific to the phosphorylated state (PAN antibody). Primary antibodies were detected with IRDye conjugated secondary antibodies , and their fluorescence was measured and imaged using the Odyssey LI‐COR Imaging System.HEK293T cells were obtained from ATCC (CRL‐11268) and maintained under standard conditions at 37°C in DMEM‐based media (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum and 1% P/S/G (penicillin–streptomycin–glutamine). Lysates were prepared by suspension and incubation of cells in nondenaturing lysis buffer  100 μl phosphatase inhibitor cocktail (Sigma‐Aldrich P0044), 1,250 units Benzonase nuclease (Sigma‐Aldrich E1014).® Precast AnyKD™ acrylamide gels (Bio‐Rad 456‐9034) and nitrocellulose membranes (Bio‐Rad 162‐0146). Immunoblotting with custom and commercial α‐TDP‐43 antibodies (CST 3449) and detection with the LI‐COR imaging system were performed as described above. Immunoprecipitation of HEK293T cell lysates was performed according to the manufacturer's protocol with Protein G Dynabeads (Invitrogen 10007D) and mouse monoclonal α‐TDP‐43 antibody (Proteintech 60019‐2).Western blotting was performed using standard laboratory procedures with Mini‐PROTEAN2, 1 mM DTT) at 37°C. The membranes were then probed as described above.Phosphatase treatment of nitrocellulose membranes was performed using calf intestinal phosphatase . Following detection of the 43 kDa species by Western blotting, membranes were stripped of primary and secondary antibodies using stripping reagent (Thermo Fisher 46430). Membrane sections containing putative TDP‐43 species were cut and blocked with 5% bovine serum albumin and 0.1% Triton X‐100 in TBS. Identical membrane pieces were treated with and without CIP in reaction buffer . Mammalian expression constructs were cloned into modified pCS‐DEST backbones (Addgene 22423).2 in DMEM high glucose  supplemented with 10% FBS, 2 mM l‐glutamine (Gibco), 1 mM non‐essential amino acids (Gibco), and 1% penicillin/streptomycin (Gibco), unless otherwise noted.All TDP‐43 phase separation reporter assays were performed in HEK‐293T cells obtained from the American Tissue Culture Consortium . Cells were maintained and imaged at 37°C and 5% COMammalian TDP‐43 expression constructs (500 ng) were transfected into HEK‐293T cells grown on tissue culture‐treated μ‐slides (ibidi) with X‐tremeGENE 9 (Roche). To compare expression levels, cells were harvested 24 h after transfection and analyzed by (i) flow cytometry with a BD Accuri C6 instrument  or (ii) immunoblotting using anti‐GFP (Novus NB600‐308) and anti‐alpha‐tubulin (Sigma T6199) primary antibodies. Blots were developed with a LiCor Odyssey imager using appropriate dye‐conjugated secondary antibodies. To follow TDP‐43 reporter phase formation over time, cells were imaged in L15 medium (Gibco) + 10% FBS at 37°C every hour for a total of 24 h after transfection with a Leica DMI‐6000B microscope equipped with an Andor Yokogawa spinning disk confocal unit. A 488‐nm laser was used for GFP excitation and an Andor iXon Ultra EMCCD camera for signal detection.RRM‐GFP nuclear and droplet signals in live cells, each confocal image was taken with identical detector settings. Mathematica (Wolfram Research) was used to outline nuclei based on the total nuclear GFP signal, and to quantify the fluorescence signal of different TDP‐43RRM‐GFP variants in the nucleoplasm versus the droplet phase.A Leica SP8 confocal laser scanning microscope equipped with a 64× glycerol (NA 1.4) objective (Leica) and a temperature‐ and gas‐controlled incubation chamber (Life Imaging Services) was used for image acquisition. The 488‐ and 561‐nm laser lines were used for excitation of GFP (green channel) or mCherry (red channel), respectively. For quantification of TDP‐43t), the fluorescence intensities within the bleached droplet hemisphere were integrated and normalized to the fluorescence intensity of the corresponding unbleached droplet hemisphere. These normalized, time‐dependent fluorescence intensities It were then used to calculate the fluorescence recovery (FR) according to the following formula: FR(t) = (It − It0)/(Ibefore bleaching − It0), with t0 being the first time point observed after photobleaching.Half‐bleach experiments were performed 24 h after transfection of the indicated constructs using the FRAP module of the Leica SP8 microscope. The Leica Application Suite X software was used for quantification and GraphPad Prism to plot and analyze replicate half‐bleach experiments. For each recorded time point . This served as template for site‐directed mutagenesis using the QuikChange Site‐Directed Mutagenesis Kit (Agilent Technologies) to generate TDP‐43 mutants for mammalian expression. Oligonucleotides used for mutagenesis: Y4R_FW, gacgacaagctttctgaacgtattcgggtaaccgaagatg; Y4R_RV, catcttcggttacccgaatacgttcagaaagcttgtcgtc; E17R_FW, agaacgatgagcccattcgaataccatcggaagacgatg; E17R_RV, catcgtcttccgatggtattcgaatgggctcatcgttct; S48A_FW, cgctacaggaatccagtggctcagtgtatgagaggtg tc; S48A_RV, gacacctctcatacactgagccactggattcctgtagcg; S48E_FW, cgctacaggaatccagtggaacagtgtatgagaggtgtc; S48E_RV, gacacctctcatacac tgttccactggattcctgtagcg.l‐glutamine, and sodium bicarbonate and supplemented with filtered fetal bovine serum at 10%. RNAi‐mediated downregulation of TDP‐43 was carried out as previously described (1) and siCONTROL Nontargetting siRNA#1 (Dharmacon) was used as control. TDP‐43 downregulation was confirmed by immunoblotting.Human cell lines were grown in growth media‐Dulbecco's modified Eagle's medium, 4,500 mg/l glucose, et al, Splicing assays were carried out in HeLa cells transiently transfected with Lipofectamine 2000 and Oligofectamine in the case of siRNA (Life Technologies) according to manufacturer protocols. Splicing assays were performed using the CFTR reporter minigene as previously described  was used to detect endogenous and tagged TDP‐43. GAPDH was used as the loading control (Abcam ab181602).NLF, AW, AEC, DRM, MTN, and VHR designed and conducted the gel filtration, protein expression and purification, and structural NMR experiments. AEC developed the C‐terminal MBP vector and phase separation assay. MTN solved the structure of the dimer from samples made by AW and data collected together with AW. NLF, AW, and AEC wrote the manuscript with input from the authors. TM and EWM conceived and analyzed the CG‐MALS and cross‐linking experiments, and EWM carried them out. TM, EWM, and AN conceived the AUC experiments, and AN carried out and analyzed them. HBS conceived, performed, and analyzed the phase separation cell culture experiments, with input and resources provided by RR. YMA and ANR conceived and analyzed the splicing experiments, and ANR carried them out. FS and SNR conceived of, performed, and analyzed the immunoblotting experiments.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Review Process FileClick here for additional data file.


A study on graphene that was synthetized by means of a novel transfer-free fabrication approach and is employed as sensing material is herein presented. Multilayer graphene was deposited by chemical vapour deposition (CVD) mediated by CMOS-compatible Mo. The utilized technique takes advantage of the absence of damage or contamination of the synthesized graphene, because there is no need for the transfer onto a substrate. Moreover, a proper pre-patterning of the Mo catalyst allows one to obtain graphene films with different shapes and dimensions. The sensing properties of the material have been investigated by exposing the devices to NO2, NH3 and CO, which have been selected because they are well-known hazardous substances. The concentration ranges have been chosen according to the conventional monitoring of these gases. The measurements have been carried out in humid N2 environment, setting the flow rate at 500 sccm, the temperature at 25 °C and the relative humidity (RH) at 50%. An increase of the conductance response has been recorded upon exposure towards NO2, whereas a decrease of the signal has been detected towards NH3. The material appears totally insensitive towards CO. Finally, the sensing selectivity has been proven by evaluating and comparing the degree of adsorption and the interaction energies for NO2 and NH3 on graphene. The direct-growth approach for the synthesis of graphene opens a promising path towards diverse applicative scenarios, including the straightforward integration in electronic devices.The sp Due to its extraordinary electronic, chemical, mechanical, thermal and optical properties, graphene has been defined as the “wonder material” of the 21st century with plentiful applications in several fields such as energy storage, biomedical, electronics –6. HowevAnother issue that needs to be addressed is the problem of slow and incomplete recovery in the operative regime of standard temperature and pressure, which can be solved by providing the sensors with appropriate electronics, able to perform data analysis  or a ref2 interface. More recently, Lukosius et al. have shown the successful growth of a graphene layer underneath Ni bars on insulating SiO2 layers, so avoiding the metal contamination problems and complexity associated to graphene transfer [All that said, the development of new approaches enabling the direct growth of large-area graphene layers on arbitrary insulating substrates, compatible with conventional Si technology, is of crucial importance –15. A potransfer .2 substrates by means of a transfer-free CVD method mediated by Mo. The pre-patterning of the Mo layer allows the graphene to be shaped in the desired form by means of standard lithography techniques, following a completely Si-technology-compatible approach. Chemiresistive devices have been realized by shaping the sensing layer in form of rectangular strips.In the present work, we show the results of the sensing performances of conductometric devices in which multilayer graphene films have been directly synthesized on insulating SiO2, NH3 and CO. In particular, NO2 and NH3 are known to exhibit an electron acceptor- and donor-like nature respectively, as extensively shown in the literature [In order to probe the gas-sensing capabilities of the fabricated devices, three standard pollutants have been chosen, namely NOterature ,22–23. T2 and NH3.Finally, the sensing specificity of the material has been probed by evaluating and comparing the degree of adsorption and the interaction energies for NOThe material, prepared according to the procedure discussed in detail in the Experimental section, has been morphologically and structurally characterized in a previous work , from whIn −1) and D′ (ca. 1600 cm−1) bands is related to presence of defects and in particular the D intensity, roughly 5-fold lower than that of the G peak, attests a low density of defects. This evidence confirms the non-degradation of the material even during ageing.Starting from this premise, we have further deepened the analysis of the material by performing a Raman analysis in several different sites of the graphene film. As can be seen in I(D)/I(D′) ratio allows one to discriminate the type of defects in the film. Indeed, as reported by Eckmann et al., the value of the slope of the I(D)/I(G) vs I(D′)/I(G) curve, namely I(D)/I(D′), can be related to the preponderant typology of defects in the different graphene samples [I(D)/I(D′) ratio exhibits its maximum value, around 13, for sp3-like defects, it decreases down to about 7 in the case of vacancies and it is minimum for edge defects (ca. 3.5). In our case the estimated I(D)/I(D′) value is around 4.4, indicating that defects in the material are reasonably ascribed to the boundaries.The analysis of the  samples . In partl = 206 μm, w = 5 μm  was chosen because of the undesired influence of oxygen. The number of active sites on the graphene surface decreases due to binding of O2 molecules via weak physisorption [In order to investigate the reliability of the fabrication process in terms of gas-sensing behaviour, two identical devices have been considered, labelled with A and B. As described below in the Experimental section, the sensing layer has a rectangular geometry: w = 5 μm  and t = sorption . This ph2. As can be observed, the base conductance values are comparable (GA ≈ GB ≈ 22 µS). During exposure, both chemiresistors exhibit an identical percentage variation of the conductance without reaching a plateau. In the figures the asymptotic values of conductance are also displayed. Such quantities have been evaluated by extrapolating the final conductance value by fitting each curve, within the time window of exposure to the analyte, with the exponential functionGMIN/MAX represents the asymptotic value of the conductance after exposure to the analyte. When the gas inlet is stopped, a slow decrease of the device conductance and thus an incomplete recovery follows. This is not surprising and can be explained in terms of interaction energy between graphene and analyte, as will be explained later. Since NO2 acts as an electron acceptor, during interaction the electronic charge is transferred from graphene to the analyte. Thus, the observed increase in the device conductance is naturally expected because of the interaction of the molecule with a p-type material.where 3. Even in this case both chemiresistors exhibit a comparable percentage variation of the conductance, as well as similar kinetics  has been converted into a number of analyte molecules per unit time, , by knowing the incoming concentrations (1 ppm for NO2 and 250 ppm for NH3). The total pressure in the testing chamber (pTOT = 1 atm) has been taken into account. Furthermore, a uniform molecule density dV = Nt/Vc in the chamber (Vc = 0.4 dm3) has been assumed:The absolute number of analyte molecules approaching the devices has been firstly estimated, referring to the responses reported in t = 600 s has finally been evaluated as:The effective number of colliding analyte molecules during the exposure in the time interval Δ2 and NH3 is 250−2/3. This factor will be used later to normalize the number of adsorbed molecules to the same amount of incoming analyte.It is worth noting that the ration of these quantities for NOG0 of the devices  obeys Ohm’s law:The initial conductance value The conductivity can be expressed in terms of volumetric density and mobility of the charge carriers:2 and 103 cm2/V·s), in the calculations, a carrier mobility of μ ≈ 1000 cm2/V·s has been assumed [G/G0 in 10 min. This quantity is linked to the number of adsorbed molecules via the virtual charge-transfer amount resulting from the interaction between graphene and analyte molecules, Δq. Hence, the effective number of analyte-induced charge carriers NS onto the devices surface can be expressed asAccording to the mobility values reported in literature for CVD graphene  >> 1, it is possible to writeAnalogously to the conventional treatment of doped semiconductors, after having estimated the maximum number of adsorbed analyte molecules, they have been assimilated as punctual impurities in the crystal lattice. Herein the Maxwell–Boltzmann approximation for the statistical description of charge carriers can be justified by virtue of the semi-classical regime of the system, with low induced charge density, of the order of 10graphene ) and a t2, both for devices A and B (kBT ≈ 25 meV) is not sufficient to “escape” from the potential well associated to the adsorption.In particular, the estimated characteristic energies for NO A and B , appear G/G0, NS, , α,  and Eint) calculated for devices A and B, for NO2 and NH3, are summarized in The primary quantities  Mo target. The metal film thickness can be made as thin as 50 nm without any segregation at the graphene CVD temperature (≈1000 °C), because of the higher melting point of Mo (2623 °C) compared to that of other conventional catalysts such as Cu (1085 °C) or Ni (1455 °C). Moreover, this allows a pre-patterning the film for the selective growth of graphene down to micrometre-size dimensions. A photo-lithographic process, combined with SF6 dry etching, has been used to shape the Mo in the desired form.4″ Si wafers coated by 90 nm thick thermal SiO2/Si substrate by means of AIXTRON BlackMagic Pro equipment, setting the temperature at 1000 °C, the pressure at 25 mbar and using Ar/H2/CH4 as feedstock. At the end of the CVD growth, the Mo has been selectively etched away underneath graphene employing phosphoric acid. Due to anchoring at the sides of the patterned catalyst, the graphene layer sticks directly to the underlaying SiO2. Cr/Au metal contacts have finally been patterned by a lift-off process to design a four-point probe structure. The final chemiresistive devices consist of a material strip 206 µm long and 5 µm wide, placed between two couples of metal electrodes , placed in a thermostatic box. Precise amounts of analyte were let into the chamber via a system of programmable mass flow controllers (MKS) and electro-pneumatic valves. Relative humidity can be adjusted as required through a water bubbler placed in a thermostatic bath. The bias voltage is provided by TTi QL355T Precision Power Supply.The chemiresistors have been tested in a Gas Sensor Characterization System  consisting of a stainless steel testing chamber  20 min carrier gas flow (baseline), (2) 10 min exposure to the analytes at the chosen concentrations, (3) 20 min carrier gas flow (recovery). The measurements have been carried out in inert NG0 is the unperturbed conductance value and Gf is the conductance value at the end of the exposure.where