Despite the many attractive potential uses of ceramic materials as humidity sensors, some unavoidable drawbacks, including toxicity, poor biocompatibility, long response and recovery times, low sensitivity and high hysteresis have stymied the use of these materials in advanced applications. Therefore, in present investigation, we developed a capacitive humidity sensor using lead-free Ca,Mg,Fe,Ti-Oxide (CMFTO)-based electro-ceramics with perovskite structures synthesized by solid-state step-sintering. This technique helps maintain the submicron size porous morphology of the developed lead-free CMFTO electro-ceramics while providing enhanced water physisorption behaviour. In comparison with conventional capacitive humidity sensors, the presented CMFTO-based humidity sensor shows a high sensitivity of up to 3000% compared to other materials, even at lower signal frequency. The best also shows a rapid response (14.5 s) and recovery (34.27 s), and very low hysteresis (3.2%) in a 33%–95% relative humidity range which are much lower values than those of existing conventional sensors. Therefore, CMFTO nano-electro-ceramics appear to be very promising materials for fabricating high-performance capacitive humidity sensors. It is an extremely important factor in many sectors, including hospitals, textile industries, laboratories, storage rooms for computers, food processing industries, art museums, shopping malls, libraries, exhibition centres, and so on. It is thus necessary to develop highly sensitive humidity sensor materials having good reliability, good linearity, long-term stability, rapid response and recovery, and small hysteresis . Recent 2 , ZnO [112 , Al2O3 [2 [2O3  and  oxide materials [Perovskite-structured , lead titanate (PbTiO3) and lead zirconate titanate (PbZr1−xTixO3) [3, CaxPb1−xTiO3, Li-Ca0.3Pb0.65TiO3 have been used as humidity sensors in many studies [3)-based materials or calcium titanate . CTO is now a common humidity sensing ceramic widely used in the form of porous sintered bodies [1−xLaxTi1−xCrxO3, and Ca1−xBixTi1−xCrxO3 have also been studied [Most of these materials are lead (Pb)-based compounds such as lead magnesium niobate (PbMg−xTixO3) ,30,31 an studies ,33, but  studies ,35. Emphd bodies ,37. To i studied ,39,40. H2+, Fe2+ doped CTO electro-ceramic  (CMFTO)-based humidity sensor nanomaterials with rapid responses and recovery times. The CMFTO electro-ceramics were prepared by an innovative solid-state step-sintering process to get the desired morphology with lower density and high porosity. This facile synthesis process is convenient, eco-friendly and easily controlled, compared to other synthesis techniques [2) and other metal ions from magnesium carbonate (MgCO3), hematite (Fe2O3), and calcium oxide (CaO) have been taken in solution form. This offers an advantage over other expensive synthesis techniques where the prominent sources of TiO2 are titanium alkoxide, oxynitrate, or chloride. This route is also very useful to obtain a homogeneous and fine precursor powder. In this unique synthesis technique, a rapid heating rate, proper isothermal holding method, and considerably reduced processing time and temperature are the main advantageous features compared to conventional ceramic processing methods. This technique can also be implemented in industrial production to reduce production costs and eliminate hazardous by-products. Additionally, TiO2, MgCO3, Fe2O3, and CaO nanopowders are generally nontoxic, and possibly biocompatible, and thus, they have been used in many different biomedical applications. We therefore aimed to design and develop an improved sensitive humidity sensor using a high pressure treated solid-state step-sintering technique and starting from inexpensive Pb-free nanomaterials.The present article therefore aimed to synthesize Mgchniques ,31,32,333, Fe2O3, TiO2 and CaO  in a desired stoichiometry. The dry mixed oxide powders of all the ingredients  were milled with 70% alcohol in a ball-mill  with a sample to ball ratio of 1:200 (w/w) for 72 h. After ball-milling, the milled CMTFO slurry was dried at 105 °C in a convection oven  for 6 h to remove the excess moisture, water and alcohol. The dry-powder was cold pressed into pellets of 10 mm diameter and 1.2 mm of thickness under uniaxial high pressure (450 MPa) at 25 °C into a cylindrical mould using a hydraulic press . Polyvinyl alcohol  was used as binder to reduce the brittleness of the pellets during pelletization. The binder was burnt out at high temperature during sintering. It also helped the sintered pellets to achieve porous morphology [A perovskite-structured polycrystalline sample of CMFTO was prepared by our solid-state step-sintering technique using high-purity ingredients such as MgCOrphology . The steA schematic flow chart of the synthesis processes is depicted in The uniaxially compressed sintered and unsintered pellets were polished with emery paper and cleaned with absolute alcohol, followed by drying at 105 °C before using them as sensors. Thereafter, silver (Ag) electrodes were screen printed on both sides of the each pellet followed by drying at 80 °C for 30 min. Then, copper (Cu) wire was connected to the Ag-electrodes as conducting wire. Finally, the sensor was aged at 150 °C in air for 60 min.ρ, g/cc), open porosity (%), and water absoption (%) ability of electro-ceramics were measured following Equations (1)–(3), respectively, with the help of a modified Archimedes’ principle as highlighted in our previous studies [Wbi, Wdi and Wai represent the initial dry weights of the samples in air before immersion in water, in water during immersion in water, and in air after immersion in water, respectively. The resolution of the weighing machine was ±0.0005 g. At least five identical specimens were prepared to calculate the standard deviation (SD) of all the sintered samples, where The microstructural analysis was carried out by field emission scanning electron microscopy . Pore size distribution (PSD) and relative cumulative frequency (RCF) of the pores were evaluated from the corresponding FESEM images and analyzed with the ImageJ software. An X-ray diffractometer  was used to obtain the different X-ray diffraction (XRD) patterns of the developed electro-ceramics using Cu-Kα radiation in the 2θ range of 20°–50°. Density ρ2, Mg(NO3)2, NaCl, KCl and KNO3) in conical flasks with stoppers, to produce environments with relative humidity (RH) values of 33%, 55%, 75%, 85% and 95% at 25 °C, respectively as described in our previous report [2 Hz–106 Hz. All the humidity sensing measurements were carried out under normal atmospheric pressure. The measurement setup is depicted in The RH-dependent capacitive response of the CMFTO electro-ceramic was determined at 25 °C while the RH was changed from 33% to 95%. First, the final aged sensors were stabilized at 1 V voltage in 95% RH for 24 h. Ambiences with different humidity levels were then created by silting a series of standard saturated salt solutions  along with Fe2O3 (PDF: 01-084-0308), MgCO3 (PDF: 00-002-0905) and CaCO3 (PDF: 01-072-1650), which was converted from CaO during the wet-ball-milling, in the unsintered nanomaterial has already been reported in our previous study [2MgTi3O10, as PDF: 00-013-0353) was formed after sintering at 450 °C. Then, another phase of perovskite CaTiO3 (PDF: 00-008-0092) with sharp small (400) at 2θ = 23.43° and strong (440) at 2θ = 33.29° crystalline peaks was developed after increasing the temperature above 650 °C. The more intense crystalline peaks (230) at 2θ = 32.71° of Fe2MgTi3O10 and (440) of CaTiO3 become stronger after sintering at 1050 °C. A significant shift of a (110) peak of Fe2O3 (2θ = 35.83°) strongly indicates the formation of magnetic Fe3O4 (2θ = 35.70°), which may further stimulate the use of this material in remote sensing and humidity sensor applications [Pore size and pore size distribution (PSD) are two of the most important parameters for all humidity sensor nanomaterials. The PSD and the relative cumilative frequency (RCF) of all the CMFTO ceramics are illustrated in f 4.5 μm . The preus study . XRD pat3 particles was noticed in On the other hand, FESEM images of the materials depicted in 3 . This suggests that the capacitance is an effective physical parameter in order to evaluate the sensor response.All the aforementioned physical properties favour considering the porous CMFTO electro-ceramic sintered at 1050 °C to be the best hydrophilic material among the all ceramics developed in the present study. The hydrophilic characteristics of its surface with meso- and submicropores should be helpful for improving the water molecule adsorption/desorption process and hence, enhance the sensitivity of humidity sensors. To investigate the effect of frequency on sensing characteristics of the CMFTO electro-ceramic based sensor, its capacitance values have been calculated under different humidity environments with various test frequencies such as 104 Hz) but in a higher frequency range, the capacitance value is small and hardly changes with humidity. Generally in the ideal capacitive sensor, the C-value is independent of applied frequency. However, in low humidity environments, the sensing material absorbs a small amount of water, which could be considered as an ideal situation. Due to the absorption of water molecules, the sensing material possesses a leak conduction (γ) [є*, C0 and єr are the complex dielectric constant, capacitance and relative dielectric constant of an ideal capacitor, respectively; ω is the angular frequency, γ is the conductance and є0 is the permittivity at free space. From Equation (4), it can be noticed that the capacitance of the sensing material is inversely proportional to frequency ω and directly proportional to γ. Hence, the C-value decreases with increasing frequency and this relation becomes more prominent under higher RH conditions. In addition, γ is attributed to the physisorption of water molecules on the surface of porous CMFTO electro-ceramic. Here, γ increases with increasing of % RH and as a result, C-value increases with rising RH with respect to ω.The relations between capacitance and frequency at 25 °C of the present capacitive-type sensor at different RH values are depicted in tion (γ) . The caption (γ) ,54: (4)CS) has been calculated by using Equation (5) [C33 and CRH stands for the capacitances measured at 33% RH and at a certain RH level, respectively. C (S (6 to 102 Hz). The C-value increased from 3.2183 × 10−11 F to 9.9741 × 10−10 F with a “S” of ~3000% at the signal frequency of 102 Hz but when the test frequency is 105 Hz, the capacitance varied from 1.2832 × 10−11 to 2.8174 × 10−11 F with “S” of ~120% across the RH range of 33%–95%, respectively. The sensitivity of our developed sensor is higher than that of some other ceramic based humidity sensors reported elsewhere  and so on based humidity sensors) [2 Hz would be considered as best test frequency for the analysis of sensor characteristics.To explain the humidity-dependent capacitive characteristics of CMFTO electro-ceramics, the device sensitivity (tion (5) : (5)S=CRively. C  and S (Fly. C (S  with % Rsensors) ,58,59,603O+) as charge carriers due to the application of external electric field. In very high humidity conditions, the amount of water content increases and the physisorbed water layers gradually behave like normal condensed liquid. In this condition, the protons require very low energy for hopping between adjacent water molecules; as a result the ionic conductivity increases. This charge transport mechanism can be explained by a Grotthuss chain reaction (H2O + H3O+ → H3O+ + H2O) conductivity [The humidity sensing mechanism of the CMFTO electro-ceramic could be explained by adsorption phenomena of water molecules and their effect on the capacitance variation of the system. The mechanism for humidity-dependent electrical characteristics of the oxide-based nanomaterial is not so clear to date. Therefore, a possible physical adsorption mechanism of water molecules for the humidity dependent electrical characteristics of the oxide based electro-ceramic nanomaterials is explained here. The relationship between the capacitance and the RH, can be interpreted by using Equation (4). On the other hand, the ionic conductivity increases with increasing RH , and as uctivity . In addiuctivity . In addi2) values of both the fitted curves were very close to 1 (more than 0.97), representing the best fit of the curves to improve linearity. The two different slopes of logC vs. RH response plot might be contributed to the change of the adsorption mode of water molecules from a monolayer chemisorption and the multilayer physisorption at low and high RH, respectively [The nonlinear characteristics on RH dependent capacitive response  might beectively ,65. ThisC-value between the humidification and desiccation curve is known as hysteresis. High hysteresis values have long been a major drawback in practical humidity sensor applications. The maximum hysteresis rate (Emax) of our developed sensor has been calculated by using Equation (6) [m stands for the maximum hysteresis and YFS is the full scale output.The maximum difference in tion (6) : (6)Emax2O)-based and so on) developed by other studies reported elsewhere [The CMFTO electro-ceramic based humidity sensor showed a maximum hysteresis of about 3.2% corresponding to 85% RH see . This relsewhere ,59,60,66lsewhere ,55.3 based nanomaterials  [The response and recovery time have a significant effect on the performance of humidity sensors. Time taken by a sensor to achieve ~90% of the total capacitance change is defined as the response time in case of adsorption or the recovery time in case of desorption of the water vapors. For a good sensor, the response and recovery time of humidity sensors must be very small. The response see A and rec–98% RH) .2 Hz under fixed humidity levels  for a period of 30 days and its humidity sensing parameters were measured repeatedly every 2 days. The characteristics of the present CMFTO sensor depicted in Stability is one of the most important parameter for any sensor application. The present CMFTO electro-ceramic-based humidity sensor was tested repeatedly at 102 Hz to 106 Hz with 1 V AC signal at 25 °C. The variation in impedance spectra suggests different water absorption mechanisms related to electrical conductivity and polarization occur in the CMFTO electro-ceramic. When the RH is low (33%–75% RH), a half semicircle is observed in the complex impedance plot as depicted in f stands for the resistance of the CMFTO electro-ceramic, which decreases as RH increases, Cf is the capacitance of the CMFTO electro-ceramic and Zi is the interfacial impedance at the electrodes/CMFTO electro-ceramic [3O+ ions. When the RH increases gradually, a huge number of water molecules participate in the physisorbed process on the large specific surface area of the CMFTO electro-ceramic. Thus, proton hopping between the adjacent water molecules occurs easily in CMFTO electro-ceramic. This causes a decrease in the intrinsic resistance of the CMFTO electro-ceramic corresponding to the more depressed semicircles and longer straight lines in Complex impedance spectroscopy is a powerful tool to understand the polarization processes and that take place in a humidity sensor due to absorption of water particles and the conductivity mechanism. The Nyquist/complex impedance plot of the CMFTO-based humidity sensor at different RH (33%–95% RH) was measured over a frequency range of 10-ceramic ,74. At v−11 F to 9.9741 × 10−10 F as the RH changed from 33% to 95% at a testing frequency of 102 Hz. It has shown a high sensitivity (3000%), rapid response (14.5 s) and recovery (34.27), which is much lower than the values of other conventional capacitive humidity sensors. The present capacitive sensor showed a very low hysteresis of 3.2%. Therefore, all the above improved sensing characteristics, together with the good linearity and stability of the CMFTO electro-ceramic indicate that it can be used as a potential humidity sensing material for advanced applications.A novel submicroporous Ca,Mg,Fe,Ti-Oxides electro-ceramic-based capacitive humidity sensor has been fabricated from some inexpensive oxide nanomaterials using a solid-state step-sintering process. The presented sintering technique helped get nanomaterials with the desired morphology, lower density and high porosity. This newly developed Pb-free CMFTO electro-ceramic has shown improved capacitive humidity sensing properties via physisorption. It has shown two distinct conduction mechanisms by displaying a critical RH at 75% RH. The capacitance of the sensor of the CMFTO electro-ceramic increased from 3.2183 × 10


The electrochemical performances of the G/PANI nanocomposites in a three-electrode system using an aqueous sulfuric acid as an electrolyte exhibited a specific capacitance of 436 F g−1 at 1 A g−1, which is much higher than the specific capacitance of pure PANI (367 F g−1). Such a higher specific capacitance of the G/PANI nanocomposite inferred an excellent synergistic effect of respective pseudocapacitance and electrical double-layer capacitance of PANI and graphene.Surfactant-treated graphene/polyaniline (G/PANI) nanocomposites were prepared by the MnO To develop an advanced supercapacitor, low cost, eco-benignity, and high efficiency are indispensable. Based on these needs, much interest has been focused on the electrode materials for supercapacitor to improve both the power and energy density -3. RecenConducting polymers such as polyaniline (PANI), polypyrrole, and polythiophene have been extensively studied for pseudocapacitive materials . Higher The OH, COOH, and epoxy functional groups of graphene oxide (GO) easily interact with polymer backbones to improve the properties of the nanocomposites ,11-13. H2 between the surfactant-treated graphene sheets and followed by oxidative polymerization of aniline (ANI). Sodium lauryl sulfate (SLS) used as the surfactant prevents the aggregation of graphene sheets and spacer for accommodating the PANI between the graphene sheets. By varying the ratio of graphene and PANI, the effect of specific capacitance was studied.Herein, we describe the preparation of graphene/polyaniline (G/PANI) nanocomposites by inserting MnO2SO4), potassium permanganate (KMnO4), hydrochloric acid (HCl), hydrogen peroxide (H2O2), and manganese (II) sulfate monohydrate were received from Samchun Chemical Co., Ltd. . Hydrazine monohydrate (N2H4-H2O), potassium dichromate, SLS, and ANI were provided by Sigma-Aldrich . All the chemicals were used as received without further purification.Expandable graphite (Grade 1721) was obtained from Asbury Carbon . Concentrated sulfuric acid  were added, and the reaction was continued for approximately 3 h. A viscous black-colored solution of GO-SLS/PANI was obtained at the end of the reaction.MnOrocedure , and GO rocedure . GO  was added to that solution, and the reaction was continued for 1 h. The reduced GO-SLS/PANI was washed with 1 M HCl and ethanol to completely remove SLS from the nanocomposite solution. Finally, the G/PANI nanocomposites were dried at 60 C for 24 h to obtain the nanocomposite powders. The blank graphene sample was also prepared by the reduction of the surfactant-treated GO without using PANI. The G/PANI nanocomposites were labeled as G/PANI (GP) 46 and GP 64 according to the weight percent of graphene in the final product.All the electrochemical measurements were performed in a three-electrode cell assembly equipped with a reference electrode (Ag/AgCl), a platinum wire counter electrode, and a glassy carbon working electrode. The electrode was fabricated by sonication in 85% of active material, 10% super-P carbon black, and 5% polytetrafluoroethylene in 2 mL of ethanol solution. The electrochemical performances were investigated using a 2 M aqueous sulfuric acid as the electrolyte. The mass of active material for the electrochemical measurement was about 1.5 mg. The electrochemical charge/discharge and cyclic voltammetry (CV) were performed in the potential window range of −0.2 to 0.8 V. The electrochemical impedance spectra (EIS) were measured at frequencies between 100 kHz and 0.01 Hz.ID and IG values of the graphene were determined using a Raman . The charge/discharge and CV of the G/PANI electrode were performed by using a battery tester . EIS was measured using Bio Logic Science Instruments .Morphologies of the G/PANI nanocomposites were characterized via field-emission scanning electron microscopy . Thermal properties were characterized using a thermogravimetric analysis . Functional group of the nanocomposites was determined using a Nicolet IR 200 FTIR spectrometer  and the 2 on the surfactant-treated graphene significantly prevents the aggregation of the nanocomposites. The MnO2 obtained by the hydrothermal method produces a nanowire of a needle-like morphology , dv/dt is the slope of discharge curve, and m is the mass of the active materials (g) [−1 for the first 5 cycles. As shown in Figure −1. The higher specific capacitance was attributed to the stronger doping effect of sulfuric acid on the PANI nanowires. The surfactant-treated graphene showed a specific capacitance of 220 F g−1 at 0.2 A g−1. Such a high specific capacitance of graphene can be attributed to the pseudocapacitive effect of the residual oxygen groups of graphene sheets [−1, respectively at 0.2 A g−1. Interestingly, the IR drop of the nanocomposites decreased with increasing graphene loadings, indicating the decreasing resistance of the nanocomposites electrode.where ials (g) ,26. The −1. The discharge capacitance of GP 46 was 436 F g−1 at 1 A g−1, which is slightly lower than 0.2 A g−1, indicating a good synergistic effect of the PANI and surfactant-treated graphene sheets. As compared to graphene, the discharge curves of the G/PANI show the deviation from the straight line, attributed to the pseudocapacitance contribution over the electrical double-layer capacitor (EDLC) behavior. The detailed investigation of the specific capacitance of graphene, PANI, GP 64, and GP 46 is listed in Table −1 is larger than those recently reported for G/PANI nanocomposites (Table −1 inferred an efficient ionic transport of PANI and G/PANI electrode during the electrochemical reactions. Figure Figure −1 (Figure The CV of the G, PANI, and G/PANI nanocomposites were performed at 10 mV s1 Figure a. The CV1 Figure b. The se−1 was as high as 436 F g−1 and obtained good cycle stability for 500 cycles. The excellent electrochemical performance of GP 46 at higher current density was attributed to the good synergistic effect of the PANI nanowires and graphene sheets.The intercalation of PANI nanowires and surfactant-treated graphene allows the faster electrolyte diffusion and easy accessibility of the electrolyte at higher current densities. The electrochemical characterizations show the superior performances of GP 46 at higher current densities. For example, the specific capacitance of GP 46 at 1 A g


Several showed good ability to refold after heat denaturation. These L1-binding single domain antibodies, however, failed to recognize the killed vaccinia antigen. Useful vaccinia binding single domain antibodies were isolated by a second selection using the killed virus as the target. The virus binding single domain antibodies were incorporated in sandwich assays as both capture and tracer using the MAGPIX system yielding limits of detection down to 4×105 pfu/ml, a four-fold improvement over the limit obtained using conventional antibodies. This work demonstrates the development of anti-vaccinia single domain antibodies and their incorporation into sandwich assays for viral detection. It also highlights the properties of high affinity and thermal stability that are hallmarks of single domain antibodies.There is ongoing interest to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, single domain antibodies that target vaccinia virus were developed. Two llamas were immunized with killed viral particles followed by boosts with the recombinant membrane protein, L1, to stimulate the immune response for envelope and membrane proteins of the virus. The variable domains of the induced heavy chain antibodies were selected from M13 phage display libraries developed from isolated RNA. Selection via biopanning on the L1 antigen produced single domain antibodies that were specific and had affinities ranging from 4×10 Poxviridae family. It is a complex, enveloped virus that is characterized by a large dsDNA genome (∼190 kbp) that encodes approximately 250 genes Vaccinia virus is the quintessential member of the The L1 protein is a transmembrane, myristoylated protein that is located on the surface of the intact mature virion (IMV) Single domain antibodies (sdAbs) are the recombinantly produced variable domains of heavy chain only antibodies (HcAb) found in members of the family Camelidae There is ongoing interest by the US Department of Defense to develop high affinity, thermal stable recognition elements to replace conventional antibodies in biothreat detection assays. As part of this effort, two llamas were immunized with a combination of killed vaccinia virus material and purified recombinant L1 antigen. It was hypothesized that this immunization protocol would lead to a diverse library that would allow for the isolation of sdAbs capable of binding the L1 antigen as displayed on the virus. In addition, as the animals were immunized with virus, the library would also yield sdAbs capable of recognizing a variety of viral antigens including envelope and membrane proteins of the viral particles. The isolated sdAbs could potentially find application in downstream assays and therapeutics.Vaccinia virus Lister strain, sucrose gradient purified and certified killed by the UV-psoralen method Escherichia coli strain (Agilent Technologies) was used for library construction and throughout the biopanning selection process. The E. coli strain DH5α was used for routine cloning experiments; E. coli Rosetta and Rosetta (DE3) strains (Merck Millipore) served as the bacterial strains for sdAb production.The XL1-Blue Taq DNA polymerase, and T4 DNA ligase for library construction and cloning were purchased from New England Biolabs (NEB). Mutagenesis was performed using the QuikChange II site-directed mutagenesis kit (Agilent Technologies) following the manufacturer instructions. All oligonucleotides were ordered through MWG Operon; DNA sequencing performed by MWG Operon. Anti-camel HRP was from Bethyl Laboratories. Chemical reagents were acquired primarily from Sigma Aldrich, Fisher Scientific, or VWR unless otherwise noted.Restriction enzymes, Two llamas maintained by Triple J Farms  were subjected to a series of identical immunizations with both killed vaccinia virus  and recombinant L1 antigen. The immunization cycle consisted of three rounds with killed virus material  and two additional rounds with the L1 proteins (100 µg each time). The immunization protocol used in this work was specifically approved by the Triple J Farms Institutional Animal Care and Use Committee (IACUC). Triple J Farms has an active IACUC that reviews and approves all immunization protocols. Plasma was titered to assess binding to both the killed vaccinia reagent and L1 following the third and fifth immunization.Serum was separated from whole blood samples via low speed centrifugation (1000 x g) and the use of Uni-Sep Maxi+ conical tubes (Novamed). The buffy coat was transferred to new tubes and peripheral blood lymphocytes (PBLs) pelleted at 2500 x g in a Beckman clinical centrifuge. Total RNA was extracted from PBLs using the QIAGEN RNA Blood Mini Kit and manufacturer's protocol. Extracted total RNA was converted to a cDNA library using the Superscript II First Strand synthesis kit (Invitrogen) and manufacturer's protocol. The cDNA was subjected to two rounds of PCR, the first using degenerate primers for variable domain amplification and the second to incorporate directional SfiI restriction sites. To facilitate phage display, the sdAb library and pECAN 21 vector The sdAb library was interrogated against both recombinant L1 antigen and intact killed vaccinia virus. Antigen was immobilized to microtiter plates either passively or, in the case of the L1 antigen, captured by antibodies that were themselves passively immobilized (sandwich-style). Target antigens and antibodies were diluted in phosphate buffered saline  and aliquoted to individual wells of Nunclon MaxiSorp microtiter plates overnight at 4°C. The viral material was immobilized at a concentration of 10 µg/ml. For the sandwich-style selection, each monoclonal antibody (mAb417 and mAb419) was immobilized individually to separate wells at a concentration of 3 µg/ml. L1 antigen concentrations of 3–5 µg/ml were utilized for both the direct immobilization and sandwich-style selections. After overnight incubation, wells were washed twice with PBS containing 0.05% Tween 20 (PBST) and then blocked with PBST plus 5% milk proteins (PBSTM).12 pfu/ml. Phage were incubated with antigen for 1 hour with agitation. Wells were washed with PBST, the number of washes increasing with each round of biopanning. Bound phage were eluted with 100 mM triethylamine then neutralized with an equivalent volume of 1 M Tris-HCl (pH 8.0). Approximately half of the eluted phage volume was then used to infect an early-log phase XL1-Blue culture. This culture was incubated for one hour at 37°C then plated to antibiotic containing media. Following an overnight incubation, colonies were dislodged from the solid media, a portion of which was used for phage amplification and the remainder diluted in sterile glycerol to a final concentration of 40% v/v for storage at −80°C. Amplified phage was used for additional rounds of selection.Individual rounds of selection were carried out as previously described Following three rounds of selection individual clones were chosen from output plates and assayed for target binding capabilities using a monoclonal phage ELISA E. coli for protein expression. The sdAbs in the pECAN45 expression vector were transformed to Rosetta E. coli for protein expression. Expression and purification from 500 ml shake flasks was performed as previously described, with the exception that protein was eluted from the nickel sepharose resin using 0.5 M imidazole The coding sequence of unique binders was mobilized to pECAN45 Both direct and sandwich assays were performed to characterize the expressed sdAbs and conventional antibodies similar to assays described previously Purified sdAbs and conventional antibodies were biotinylated to serve as tracers in both direct and sandwich assays using a sulfo NHS –LC-LC- biotin (Pierce) reagent as previously described In the MAGPIX experiments, binding is measured from greater than 50 beads; the error of the mean fluorescence intensity (MFI) was found to be 5% or less. Each of the direct binding and sandwich assays were performed on at least two different days. In each case, the experimental data replicated. For some clones, protein from duplicate protein productions was both biotinylated and immobilized on bead sets. Detection trends observed in MAGPIX assays were also consistent across protein preparations; minor variations observed were likely due to variability in the biotinylation and immobilization of the sdAbs.Sandwich assays were also used to screen phage-displayed sdAbs for antigen binding. The anti-L1 monoclonal antibodies 417 and 419 Table 1 Sandwich ELISAs were performed as described previously Direct binding ELISAs were performed essentially as described previously A BioRad ProteOn XPR36 system and standard GLC sensor chip was used to assess binding kinetics The fluorescence based melting assay was conducted using an applied Biosystems Step One Real Time PCR system and Sypro Orange dye (Sigma) m determinations is within ±1°C. Most samples were analyzed by two independent measurements. For three of the samples  replicate protein preparations were analyzed by CD. Replicate measurements showed essentially the same melting and refolding behavior.Refolding and melting temperatures were assessed using a Jasco J-815 CD spectrometer SdAbs and mAbs were diluted to 1.0 mg/ml concentration in PBS (pH 7.4). The samples were heated for 1 hour at 65°C, 70°C, 75°C, 80°C and 85°C in a Tetrad2 thermocycler (MJ Research). All samples were allowed to cool to room temperature on the bench top. Samples were centrifuged to pellet aggregated material then optical density was measured at 280 nm to determine protein concentration. Error on the solubility measurements, determined from duplicate experiments was ±5%. Remaining soluble protein was assayed for binding activity using SPR monitoring binding of the samples to four L1-coated rows. Binding activity was based on the SPR signal generated by 50 nM of sdAb or mAb, measured at 30 seconds, which is still near 1st order binding. Results were reported as percent activity; the error on this value was less than 10%.Two llamas (Whisper and Centavo) were subjected to five rounds of immunization with killed vaccinia virus (3 immunizations) and recombinant L1 antigen (2 immunizations). This immunization strategy was based on our previous work in developing sdAbs towards the MS2 phage Figure S1 in File S1). After the vaccinia immunizations the animals showed a robust response against the killed viral material, but not the L1 antigen. The llama Centavo showed a higher titer than Whisper. After the full immunization course, both animals had improved recognition of L1; however the titer from Centavo was still much better than from Whisper.Whole blood samples were collected from each animal after the three vaccinia immunizations and again after the complete series. The plasma was titered to examine binding towards both the killed vaccinia and L1 antigen  for library construction. Total RNA was extracted, converted to cDNA, and subjected to a two stage PCR to amplify the variable domain genes as described elsewhere Figure S2 in File S1). Given that the animals were immunized with the recombinant protein as well as killed viral material it was anticipated that selection against this protein would have been more successful. It was therefore hypothesized that the L1 antigen may not be amenable to passive immobilization as was used during the initial rounds of selection. An alternate selection protocol was pursued in which the recombinant L1 antigen was captured by conventional antibodies for the L1 antigen. It was believed that this method would ensure that L1 antigen remained in its native conformation and enable the isolation of sdAbs better able to recognize the target antigen. Positive clones were sequenced then representative sdAb were cloned into an expression vector for subsequent purification, biotinylation, and characterization as previously described Initial selection was conducted with passively immobilized recombinant L1 antigen. After three rounds of biopanning, individual clones were assayed for binding to target antigen using a monoclonal phage ELISA protocol. Although binding clones were identified by monoclonal phage ELISA, few of these showed strong binding to the recombinant L1 antigen in MAGPIX assays using the phage-displayed sdAbs , did contain some members with amino acid differences that appeared to directly contribute to the binding kinetics and thermal stability as seen with sdAb L1-C5. The sdAb L1-C5 showed an increase in the off-rate by two orders of magnitude compared to other members of the family; this clone has a single N to Y substitution in the framework 3 region that is not observed in any other members of the L1-H7 family. This shows how minimal changes in the framework can have a significant impact on binding affinity and stability.The thermal stability of individual sdAbs was assayed using a combination of techniques. Initially, the melting temperature for each sdAb was determined using a Sypro Orange fluorescent based melting assay. Sypro Orange is quenched in an aqueous environment but fluoresces as it binds hydrophobic patches that are exposed as the protein unfolds. The L1 binders had a range of melting temperatures from 56–62°C, Figure S4 inFile S1. Both L1-H2 and L1-E2 showed a dramatic decrease in ellipticity after the first round of heating and cooling. This decrease occurred only in the first cycle indicative of a failure to refold, however subsequent rounds showed no additional decrease in ellipticity suggesting that the portion of the sdAb preparation which refolded initially continued to refold successfully.Most sdAbs demonstrate a propensity to refold following denaturation, one of the benefits of the low complexity structure As was seen with the binding kinetics, the amino acid variations in the L1-H7 family contributed to distinct differences in the refolding properties of individual members. The sdAbs L1-H7 and L1-G2 were able to refold to near completion through several cycles of heating and cooling. In contrast, the L1-C5 and L1-E6 sdAbs exhibited a loss in ellipticity after the first heating cycle, an indication of failure to recover secondary structure during the cooling cycle. Subtle variations in amino acids sequences seem to have negative effects on the overall stability of these L1 binders.While CD is able to show the proteins losing and recovering secondary structure when heated at low concentration, it is important to assess the activity of sdAbs after heating to correlate refolding with antigen recognition. SdAbs and mAbs were heated, at a concentration of 1 mg/ml, to defined temperatures and incubated for 1 hour. The samples were allowed to cool to room temperature, and centrifuged to pellet precipitated protein prior to measuring the optical density. Although unfolded material and smaller aggregates may stay in solution, optical density measurements provide information on which samples contain a percentage of soluble protein. If the majority of protein precipitated then the sample would not be expected to show much in terms of binding activity. Samples that retained some soluble antibody were assayed for binding capabilities via SPR, The ability to refold and regain binding activity is a significant advantage of sdAbs compared to their conventional antibody counterparts. However, as seen in this work and others, not all sdAbs refold with the same efficiency Figure S4 inFile S1. Also, in contrast to the parental sdAb, which retained approximately 20% of its binding ability after heating to 85°C, the disulfide mutant shows a complete loss of activity after identical heating. Other researchers have also observed that although addition of a non-canonical disulfide can lead to a substantial increase in Tm, it does not necessarily correlate with an increase in functional material when the protein is heated for an extended time above its Tm The increased melting temperature of the L1-G2+ translated into improved activity at temperatures up to 75°C compared to the parental sdAb as seen in D. These changes in on and off rates may be due to a reduction in the flexibility of the molecule. Other researchers have reported mutants with additional disulfides achieved an increase of ∼10°C in stability The additional disulfide bond also altered the binding kinetics of the L1-G2+ sdAb; however the slightly higher off rate appears to have been balanced by a faster on rate to result in effectively an unchanged KDirect binding assays utilizing the Luminex MAGPIX were employed to determine the specificity of the anti-L1 sdAb as the multiplexed format enabled the simultaneous assessment of binding of each sdAb to L1, four additional recombinant vaccinia proteins, and killed virus preparations. Each of the sdAbs isolated against L1 showed specificity for only the L1 antigen In an effort to isolate sdAbs that recognize the killed vaccinia, the library was panned against the viral target. Twenty six binding clones were sequenced after two rounds of selection against killed vaccinia virus and unique families were identified based on amino acid composition of the complementarity determining regions (CDRs). Sequences of the clones that were expressed for characterization are reported in The ability of select anti-vaccinia sdAbs, anti-L1 sdAbs and mAbs (mAb417 and mAb419) to recognize the vaccinia antigen was assessed via a direct binding ELISA that measured binding to passively immobilized virus Figure 8Fluorescent based melting assays were employed to determine the melting temperatures of the anti-vaccinia sdAbs. The sdAbs showed a range of melting temperatures ranging from 57 to 79°C as reported in Figure S5 in File S1. Limits of detection varied depending on the capture tracer combination used. A limit of detection (LOD) of 4×105 pfu/ml was achieved with the best three combinations . This LOD compares favorably with our results using conventional antibodies (1×106 pfu/ml) (Bottom panel6 pfu/ml) The anti-vaccinia sdAbs were assayed as both captures and tracers in MAGPIX bead-based sandwich assays for the detection of vaccinia, None of the selected vaccinia binders were able to bind the recombinant L1 antigen in either a sandwich or direct binding format (data not shown). Given the complexity of the vaccinia virus, it was not surprising that the sdAbs selected on the viral target did not recognize the L1 antigen. The outer envelope and membranes on vaccinia virus are comprised of no fewer than 25 proteins with significant variability in protein content observed in the various infectious forms of the virus. Four additional recombinant vaccinia proteins  were obtained from BEI Resources and included in the bead based assays; as with L1, none were recognized by the sdAbs isolated against the killed virus. Interestingly, the sdAbs selected against vaccinia bound well to the virus inactivated by the UV-psoralen method, but showed reduced binding to the gamma irradiated vaccinia.We successfully isolated sdAbs from an immunized llama library capable of binding either recombinant L1 protein or killed vaccinia virus Lister strain. Characterization of the L1 binding sdAbs showed nanomolar binding kinetics. A simple modification to the amino acid composition, the addition of cysteines to facilitate disulfide bond formation, had a dramatic effect on the thermal stability of a representative L1-binding sdAb, raising its melting temperature nearly 20°C.Unfortunately, the L1 binding sdAbs were not able to bind the killed vaccinia antigen. The initial immunizations with the viral particle would have produced antibodies against many of the viral proteins. The inability of the L1 binding sdAbs to bind the viral target was unexpected. It was anticipated that immunizing with both the viral particle and boosting with the recombinant antigen would have generated a strong immune response for the L1 antigen both as a viral envelope protein and as a free antigen. The poor virus recognition of the conventional antibody mAb417 and lack of recognition by mAb419, which share binding epitopes with our sdAbs, suggests some deficiency in the sample used for these studies. The vaccinia virus replication cycle and diversity of strains could contribute to variability in virus samples. Future studies assaying live virus as well as killed material from several sources and strains may show better binding capabilities of the anti-L1 sdAbs for the intact viral particle.Selections on vaccinia provided useful reagents that showed improved limits of detection versus conventional antibodies. SdAbs continue to gain momentum for use as both diagnostics and therapeutics. Not only do these molecules demonstrate excellent affinities, specificities, and thermal stability but also characteristics such as ease of production and engineering making sdAb attractive alternatives to conventional mono or polyclonal antibodies.File S1: Titer of plasma on killed vaccinia and recombinant L1 antigen; Phage from individual clones from the initial L1 selection binding to bead-immobilized L1; Surface plasmon resonance data for the L1 binding sdAb; Refolding of the L1 sdAb assessed by circular dichroism; Magplex sandwich assays for limit of detection (vaccinia) using different capture and tracer pairs.(DOC)Click here for additional data file.


We analyzed femoral nerve regeneration in C57BL/6J mice that received this antibody in a hydrogel filled conduit connecting the cut and sutured nerve before its bifurcation, leading to short-term release of antibody by diffusion. Video-based quantitative analysis of motor functions showed improved recovery when compared to mice treated with conduits containing PBS in the hydrogel scaffold, as a vehicle control. This improved recovery was associated with attenuated motoneuron loss, remyelination and improved precision of preferential motor reinnervation. We suggest that function-triggering L1 antibodies applied to the lesion site at the time of injury over a limited time period will not only be beneficial in peripheral, but also central nervous system regeneration.L1 is among the few adhesion molecules that favors repair after trauma in the adult central nervous system of vertebrates by promoting neuritogenesis and neuronal survival, among other beneficial features. In the peripheral nervous system, L1 is up-regulated in Schwann cells and regrowing axons after nerve damage, but the functional consequences of this expression remain unclear. Our previous study of L1-deficient mice in a femoral nerve injury model showed an unexpected improved functional recovery, attenuated motoneuronal cell death, and enhanced Schwann cell proliferation, being attributed to the persistent synthesis of neurotrophic factors. On the other hand, transgenic mice over-expressing L1 in neurons led to improved remyelination, but not improved functional recovery. The present study was undertaken to investigate whether the monoclonal L1 antibody 557 that triggers beneficial L1 functions  The neural cell adhesion molecule L1 is a glycoprotein of the immunoglobulin superfamily expressed in most, if not all, neurons in the central and peripheral nervous systems of mammals. During development, L1 is targeted to the surface of developing neurites and growth cones and mediates axonal outgrowth, fasciculation and guidance as well as neuronal migration and survival in vitroIn vitro, application of recombinant L1 to Schwann cells increased their motility, and, in vivo, virally L1-transduced Schwann cells myelinated regrown/sprouted axons after spinal cord injury, while retaining their motility In the peripheral nervous system, L1 is expressed in axons and Schwann cells during embryonic and early postnatal development, and remains expressed by non-myelinating Schwann cells in the adult L1 monoclonal antibody 557 (L1 Ab 557) was prepared against L1 from adult mouse brain in Lou×Sprague-Dawley F1 hybrid rats as described Application of peptides and proteins via filling of a conduit is sufficient to deliver a protein or peptide of interest to the injury site and has been shown in several studies performed in our group All experiments were conducted in accordance with the German and European Community laws on protection of experimental animals, and all procedures used were approved by the responsible committee of The State of Hamburg (TvG 05/65). Experiments were carried out and the manuscript prepared following the ARRIVE guidelines for animal research.ad libitum, and an artificial 12 h light/dark cycle.Before and after the experiments, three-month-old C57BL/6J mice were kept under standard laboratory conditions, with food and tap water Surgery was performed as described pre, X den and X reinn are values prior to surgery, during the state of denervation (7 days after injury), and at any given time-point during reinnervation, respectively.Analysis was performed over a time-period of 12 weeks using a quantitative video-based approach  A third parameter, the limb protraction length ratio PLR, , was evaTwelve weeks after nerve transection the animals were anaesthetized with fentanyl, droperidol and diazepam for retrograde labeling of regenerated motoneurons Seven days after retrograde labeling the mice were anesthetized by intraperitoneal injection of 16% sodium pentobarbital solution  and transcardially perfused with 4% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.3. The lumbar spinal cords were removed, post-fixed overnight at 4°C in the perfusion solution and then immersed in 15% sucrose solution in 0.1 M cacodylate buffer, pH 7.3, for one day at 4°C. Thereafter the tissue was frozen for 2 min in 2-methyl-butane  pre-cooled to −30°C. For sectioning, spinal cord segments were attached to a cryostat specimen holder using TissueTek . Serial cross-sections of 25 µm thickness were cut on a cryostat  and attached to SuperFrostPlus glass slides (Carl Roth). The sections were air-dried for at least 1 h at room temperature and mounted in anti-fading medium .For the analysis of re-myelination, femoral nerves were dissected from the animals fixed by perfusion and post-fixed in 1% osmium tetroxide  in 0.1 M sodium cacodylate buffer, pH 7.3, for 1 h at room temperature, dehydrated and embedded in resin according to standard protocols. One µm-thick cross-sections from the motor and sensory nerve branches were cut at a distance of approximately 3 mm distal to the bifurcation and stained with 1% toluidine blue/1% borax in distilled water for analysis of axons in the regenerated nerve branches.Serial cross-sections of spinal cords were examined under a fluorescence microscope  with appropriate filter sets. Each section was examined using a 40× objective by focusing through the section thickness starting from the top surface. All profiles, except those visible at the top surfaces of sections, were counted Immunofluorescence staining was performed as described For analyses of perisomatic terminals, spinal cord sections containing retrogradely labeled motoneurons (see above) were freed from the coverslips and mounting medium by soaking in PBS. Antigen retrieval was performed by immersion into 0.01 M sodium citrate solution, pH 9.0, heated to 70°C in a water bath for 30 min. Blocking of non-specific binding sites was then performed using PBS containing 0.2% Triton X-100 , 0.02% sodium azide  and 5% normal goat or normal donkey serum  for 1 h at room temperature. Incubation with primary antibodies against VGAT or ChAT , dilutedhttp://fiji.sc/Fiji) and the LSM image browser (Zeiss) was used to select the image with the largest cell body cross section.Sections containing retrogradely labeled motoneurons and additionally stained for ChAT and VGAT were used to estimate soma areas and motoneuron perisomatic coverage Total numbers of myelinated axons per nerve were estimated in semithin cross-sections on an Axioskop microscope (Zeiss) equipped with a motorized stage and Neurolucida software-controlled computer system  using a 100× oil objective All numerical data are presented as group mean values with SEM. Parametric or nonparametric tests were used for comparisons, as indicated in the text and figure legends. For parametric tests, the mean was used as a representative value if two or more measurements per parameter and animal were performed. Thus, for all these comparisons the degree of freedom was determined by the number of animals. Analyses were performed using the SYSTAT 9 software package . The threshold value for acceptance of differences was 5%. The size of each sample  is indicated in the figures or their captions.t test, Transection of the femoral nerve induces changes in the heels-tail angle (HTA) and foot-base angle (FBA)  as well In addition to ground locomotion, we also evaluated the animals' ability to extend the knee joint during movements without body weight support using the “pencil” test versus 5.4±2.5) . Thus, motoneuron loss and/or failure of axonal regrowth are reduced and precision of preferential motor reinnervation is improved in mice treated with L1 Ab 557.To analyze the numbers of neurons having regrown into the appropriate branch, retrograde labeling of motoneurons was performed at 12 weeks after injury. The total number of motoneurons retrogradely labeled through the motor or the sensory branches, or through both branches of the femoral nerve, was reduced after treatment with PBS compared with non-injured mice and mice treated with L1 Ab 557. The total number of motoneurons in L1 Ab 557 treated mice was similar to that in non-injured mice and displayed preferential motor reinnervation (PMR) when compared with the PBS treated mice . In everWe next analyzed non-injured and regenerated nerves histologically to assess axonal numbers, axonal diameters and degree of myelination 12 weeks after injury. In both motor and sensory femoral nerve branches, the total number of myelinated axons was similar in both experimental groups compared with non-injured mice . However+) terminals and inhibitory  terminals at motoneuron perikarya were measured as indicators for successful recovery at 12 weeks after injury. Somata of motoneurons were larger in L1 Ab 557 treated mice or non-injured mice compared with PBS treated mice (Soma sizes of motoneurons and levels of cholinergic (ChATted mice .+) terminals at motoneuron perikarya were increased in mice treated with L1 Ab 557 compared with the PBS group or mice without injury (+) terminals were decreased in the PBS group as compared with mice treated with L1 Ab 557 and the non-injured group, which were similar  terminals and modulatory cholinergic (ChAT+) terminals at motoneuron perikarya. Cholinergic synaptic input to motoneuron perikarya modulates the excitability of motoneurons and plays an important role in the control of locomotion +) boutons correlate with maintenance of excitatory synapses which contribute to improved recovery after injury in vitro+) terminals, similar in extent to the non-injured situation. Interestingly, L1 activation is necessary for its interaction with the cytoskeletal linker molecule ankyrin With reinnervation, motoneuron cell bodies recover synaptic input In vivo, L1 overexpressing embryonic stem cells, neural stem cells or radial glial cells not only enhanced locomotor recovery after spinal cord injury, but also rescued imperiled host motoneurons and parvalbumin-positive interneurons Our findings also agree with observations in a central nervous system injury paradigm, where treatment of the severed spinal cord with Fab fragments from L1 Ab 557 enhanced regeneration th immunoglobulin domain is deleted, indicate that interactions of L1 on axons with integrins on Schwann cells may be essential for ensheathment and may affect myelination in vitroIn the PNS, L1 is detected on both non-myelinating Schwann cells and non-myelinated sensory axons of adult mice. Non-myelinating Schwann cells show a reduced association with axons in L1 knock-out mice, and the non-myelinated sensory axons undergo degeneration In the present study we observed better myelination in the sensory branch of the femoral nerve of mice treated with L1 Ab 557, while the myelination in the motor branch did not differ from the PBS treated group. This finding might be related to a more pronounced expression of L1 by sensory but not motor axons 2+ or inositol-1,4,5-trisphosphate in vitro paradigm 2+-channel blockers and pertussis toxin. Partial inhibition of neurite outgrowth in these experiments suggests that different molecular mechanisms are involved 2+ and G-protein independent second messenger cascades or even modes of signal transduction independent of second messenger systems. For instance, application of L1 Ab 557 leads to sumoylation and proteolytic processing of L1 in vitroin vivo.The mechanisms by which L1 Ab 557 triggers neurite outgrowth are noteworthy in that the epitope recognized by this antibody recruits second messenger systems in neurons and Schwann cells, such as CaStudies related to regeneration in the presence of L1 antibody have to be taken with caution with regard to systemic application, since L1 is not only expressed in the nervous system but also, for instance, by cells of the immune system, skin, kidney and intestine and application of function triggering L1 antibodies could lead to stimulation of L1-dependent functions in these cells, thereby inducing side effects, which could be avoided by local antibody application. Our study with direct application of beneficial function-triggering L1 antibody indicates favorable effects on recovery and may encourage designing novel, clinically feasible strategies to improve functional outcomes after nervous system injury, for instance by construction of biodegradable conduits and particles that allow controlled release of L1 function triggering reagents, not only the peripheral but also central nervous system.


The crystal structure of the TDP-43 RRM1 domain containing the D169G mutation in complex with DNA along with molecular dynamics simulations reveal that the D169G mutation induces a local conformational change in a β turn and increases the hydrophobic interactions in the RRM1 core, thus enhancing the thermal stability of the RRM1 domain. Our results provide the first crystal structure of TDP-43 containing a disease-linked D169G mutation and a disease-related mechanism showing that D169G mutant is more susceptible to proteolytic cleavage by caspase 3 into the pathogenic C-terminal 35-kD fragments due to its increased stability in the RRM1 domain. Modulation of TDP-43 stability and caspase cleavage efficiency could present an avenue for prevention and treatment of TDP-43-linked neurodegeneration.The RNA-binding protein TDP-43 forms intracellular inclusions in amyotrophic lateral sclerosis (ALS). While TDP-43 mutations have been identified in ALS patients, how these mutations are linked to ALS remains unclear. Here we examined the biophysical properties of six ALS-linked TDP-43 mutants and found that one of the mutants, D169G, had higher thermal stability than wild-type TDP-43 and that it was cleaved by caspase 3 more efficiently, producing increased levels of the C-terminal 35 kD fragments (TDP-35)  Protein misfolding can lead to the formation of intra or extracellular accumulation of aggregated protein and is frequently associated with various pathological conditions such as neurodegeneration. One example is TDP-43, which binds RNA and DNA and functions in mRNA splicing, translation regulation and transport2146TARDBP, the gene encoding TDP-4312TARDBP have been identified in familial and sporadic ALS, most of which are located in the C-terminal G-rich region with only two exceptions to-date, A90V in the NTD and D169G in the RRM11314151617181920212223242526272829303132333435368TDP-43 consists of an N-terminal domain (NTD) and two tandem RNA recognition motifs, RRM1 and RRM2, followed by a C-terminal glycine-rich region (G) . It form8910ALS-linked mutants have been suggested to be more prone to aggregation and more potent in inducing cell death than wild-type TDP-43: Compared to the latter, Q331K and M337V accelerate spontaneous TDP-43 aggregation4045However, it is still unknown why some ALS-linked mutants exhibit increased stability with longer half-lives and how these properties contribute to protein accumulation and disease onset. Progranulin-mediated cleavage of TDP-43 by caspases 3 and 7 may generate the 25 kD (TDP-25) and 35 kD (TDP-35) C-terminal fragments4950515249545556Here, we provide biochemical, structural, simulation, and cellular data to show that compared to wild-type TDP-43, the ALS-linked D169G mutation in TDP-43 generates a more stable protein that is cleaved by caspase 3 more efficiently, producing increased levels of TDP-35, but not TDP-25. The crystal structure of the RRM1 domain containing the D169G mutation, along with molecular dynamics simulations, provides a molecular basis as to how this mutation increases protein stability. We suggest that TDP-35 bearing the RRM1 domain is stabilized by the D169G mutation and is thus the favored product in caspase digestion of TDP-43. This result provides the first crystal structure of TDP-43 containing a disease-associated mutation and the working mechanism showing that the D169G mutation renders TDP-43 more susceptible to production of the pathogenic C-terminal fragment TDP-35, thus linking it to ALS.E. coli. However, the recombinant full-length TDP-43 was highly unstable and degraded within hours of its expression, suggesting that the C-terminal G-rich tail destabilizes TDP-43. We further expressed the His-tagged truncated TDP-43 with a shorter C-terminal G-rich tail and identified a more stable truncated form of TDP-43 (residues 1–330), which could be analyzed in biochemical assays within 24 hours after its expression and purification. This TDP-43 (residues 1–330) is referred to as N-RRM12-G, as it had complete NTD, RRM1 and RRM2 domains, and a C-terminal glycine-rich tail of about 65 residues  and the corresponding D169G mutant with caspases 3, 4, 6 and 7, since it has been suggested that TDP-35 is generated mainly through caspase 3/7-mediated cleavage at D89–A90in vitro digestion experiments  of the wild-type protein  by caspase 3 than wild-type TDP-43 (~20%), likely because A90 is an important determinant for caspase 3 cleavage being right next to the cleavage site of D89–A90 . LikewisTo reveal the molecular basis for the increased stability rendered by the D169G mutation, we co-crystallized RRM1 with DNA and determined the crystal structures of the wild-type RRM1 and RRM1-D169G mutant in complex with a single-stranded DNA, 5′-GTTGAGCGTT-3′ (see RRM1 sequence in 815 DNA (Kd of 20.6 ± 1.8 nM vs. 14.2 ± 1.7 nM)The overall structure of RRM1-D169G mutant is similar to that of wild-type RRM1, nevertheless, a small local difference was observed in the β turn (Turn6) containing the D169G mutation. Superposition of the two proteins yielded an average root-mean-square deviation (RMSD) of 0.39 Å for 71 Cα atoms. Both wild-type RRM1 and D169G mutant exhibit similar affinity for single-stranded (TG)To understand why a local conformational change in Turn6 in the TDP-43 RRM1 is linked to the increased stability, the crystal structures of the RRM1 domain of the wild-type and D169G mutant were subjected to multiple molecular dynamics simulations at a mean temperature of 300 K and pH 7 for a total sampling period of at least 200 ns (see Methods). The simulation protocol adopted maintained the overall protein structure, as evidenced by an average RMSD of 1.0 Å (wild-type) and 0.9 Å (D169G mutant) for the protein backbone atoms from those in the initial structures. To detect subtle conformational changes, the Cα–Cα and SC–SC (where SC denotes the center of mass of the side chain) distances between wild type RRM1 and RRM1-D169G mutant were compared. Furthermore, the pairwise interactions between all residues were decomposed into van der Waals and electrostatic contributions.The simulation analyses suggest that the D169G mutation results in enhanced packing  interactions between Ile168 and several residues involved in the hydrophobic folding of the domain: The D169G mutation enabled the Turn6 backbone to move closer to Loop1: compared to wild-type D169, the Cα–Cα distance between G169 and the residues located on Loop1 and on the N-terminal part of Helix 1 was reduced by roughly 1 Å on average as compared to D169 . This inTo further confirm that Ile168 is a key residue in the inner hydrophobic core of RRM1 domain, we mutated Ile168 into alanine and generated two mutants, RRM1-I168A and RRM1-I168A/D169G. However, these two mutants could not be expressed, indicating that Ile168 is indeed crucial for protein folding. To assess the importance of the hydrogen-bonding interactions between D169G and Thr115 for protein stability, we also mutated Thr115 to alanine in RRM1. Thr115 is located in Loop1 whereas D169G is located in Turn6 in RRM1. The RRM1-T115A mutant could be purified and its secondary structure was similar to that of wild-type RRM1 as revealed by CD . Howeverin vivo, we generated the mouse neuroblastoma (Neuro2a) stable cell lines constitutively expressing the wild-type TDP-43 and the D169G mutant, using the N390D mutant as the positive control. The ALS-associated N390D mutant is more stable and has a higher steady-state level in Neuro2a cells, and it causes higher apoptotic death rate than wild-type TDP-43in vivo result shows that TDP-43 with the D169G mutation was cleaved more efficiently into TDP-35, but not TDP-25. This result is consistent with a recent studyTo further examine the stability and cleavage pattern of TDP-43 in vivo study also shows that expression of the D169G TDP-43 mutant generates a higher level of accumulated TDP-35 than wild type and TDP-35 with the D169G mutation is more stable than wild-type TDP-35.Several TDP-43 mutants have been shown to exhibit an increased protein half-life in different cell models434744In this study we tested six ALS-linked TDP-43 mutants and found one mutant D169G with significantly higher thermal stability than the wild-type protein. The crystal structure and MD simulations further reveal that the higher stability of D169G compared to wild-type TDP-43 is due to a local conformational change in the β turn region containing the D169G mutation and a reinforcement of the hydrophobic interactions in the core of RRM1. Although mutation of D169 to G in TDP-43 has been reported to result in enhanced protein stability44in vitro, and the expression of the D169G TDP-43 mutant in mouse neuroblastoma cells consistently generated higher levels of TDP-35 than that of the wild-type TDP-43  in Alzheimer’s disease, as both proteins are cleaved before aggregation. APP is endoproteolytically cleaved by β and γ secretases to generate Aβ peptides that are the primary components of amyloid deposits in the brain48249TARDBP gene and inserted into the BamHI/HindIII sites in the pQE30 vector (Qiagen) for the expression of the N-terminal His-tagged recombinant proteins. The constructs for the single-point mutants of A90V, D169G, G287S, G294A, G298S and A315T and other mutations for structural study  were generated by QuikChange® Site-Directed Mutagenesis Kit (Agilent Stratagene). All of the plasmids were transformed into Escherichia coli M15 strain for overexpression. Small cell culture of ~25 ml was incubated overnight and then added into 1 liter of LB medium with 50 μg ampicillin for cell growth for about 3–4 hours. The cell culture was cooled down to 18 °C and then induced by adding 0.8 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) and 50 μg ampicillin for 20 hours at 18 °C. The cells were suspended in 50 mM phosphate buffer (pH7.5), 100 mM NaCl and 10 mM β-mercaptoethanol (βME) and lysed by microfluidizer  and centrifuged at 12,000 rpm for 30 minutes. The supernatant of cell extracts were applied onto an equilibrated nickel-nitrilotriacetic acid (Ni-NTA) affinity column (Qiagen) with 50 mM phosphate buffer (pH7.5), 100 mM NaCl and 10 mM βME. The N-terminal His-tagged proteins were eluted by a gradient buffer containing 50 mM sodium phosphate buffer (pH 7.5), 100 mM NaCl, 0–1.0 M imidazole and 10 mM βME. The eluted proteins were then purified by the HiTrap heparin column  and eluted by a gradient buffer containing 50 mM phosphate buffer (pH 7.5), 0.1–1.0 M NaCl and 10 mM βME. The purified proteins (or mutants) were dialyzed against the buffer containing 50 mM phosphate buffer (pH 7.5), 100 mM NaCl and 10 mM βME for further biochemical assays, or the buffer containing 50 mM HEPES buffer (pH7.5), 100 mM NaCl and 10 mM βME for crystallization and caspase digestion assays.The cDNA of RRM1 (residues 101–191), N-RRM1 (1–191), N-RRM12 (1–265) and N-RRM12-G (1–330) were amplified from the human 2 . dmol−1. Thermal denaturation measurements were performed by monitoring the change in CD signals at wavelength 218 nm from 20 °C to 85 °C in 1 degree intervals with 0.5 minute equilibration time.All far-UV CD spectra were recorded on the Aviv Circular Dichroism Spectrometer Model 400. Protein samples were diluted with 50 mM sodium phosphate buffer (pH 7.5), 100 mM NaCl and 10 mM βME to give the appropriate protein concentrations (~20 μM) and a total volume of 300 μl for the measurement in a quartz cell with 1-mm path length. The CD spectra were scanned ranging from 260 to 200 nm three times at 25  °C. The final spectra were represented by mean residue ellipticity [θ] (MRE) in deg . cm® 480 system (Roche) in triplicate using the protocols described previously4 buffer, pH7.5, 100 mM NaCl and 10 mM βME) was incubated at room temperature for 10 minutes. The 125X SYPRO Orange (Invitrogen) dye (1 μl) was then added to the protein and loaded on a multiwell plate (MultiWell Plate 96 White (Roche)) and sealed with LightCycler® 480 Sealing Foil (Roche). The temperature was increased from 20 to 85 °C at a heating rate of 0.012 °C/second and the emitted fluorescence at 568 nm (excited at 483 nm) was recorded every 0.02 °C. The melting temperature (Tm) was calculated by the LightCycler® Protein Melting Analysis.The differential scanning fluorimetry experiments were carried out by the LightCyclert-test which associated comparisons between the wild-type and mutant proteins.The TDP-43 (N-RRM12) (5 μg) and its mutants were incubated respectively with human active recombinant caspase 3 (BD Phamingen), caspase 4 (BioVision), caspase 6 (BioVision), and caspase 7 (BioVision) in a buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 10% glycerol and 0.1% CHAPS at 37 °C for 2 hours . The reactions were stopped by heating the mixture at 95 °C for 5 minutes, and the digested protein samples were resolved in 15% Tricine-SDS-PAGE with Coomassie blue staining. The data obtained from three independent experiments were shown as the mean standard deviation (±S.D.). The differences between the variants were appraised by the ANOVA test. Statistical significance P values were determined by an unpaired two-tailed Student’s 3COONH4, pH 5.0 and 15% v/v Jeffamine ED-2001, pH 7.0.The purified human RRM1 and RRM1-D169G were mixed with the single-stranded DNA with a sequence of 5′-GTTGAGCGTT-3′ in a molar ratio of 1:1.2. The crystals of RRM1-DNA complex were grown by hanging drop vapor-diffusion method at room temperature by mixing 1 μl of protein-DNA solution with 1 μl of reservoir solution containing 0.12 ammonium acetate, 20% PEG 3350 and 0.08 BIS-TRIS pH 5.5. The crystals of RRM1-D169G/DNA complex were grown in the reservoir solution containing 0.05 M CH522 with two complexes per asymmetric unit, whereas the RRM1-D169G/DNA complex was crystallized in the trigonal space group P3221 with four complexes per asymmetric unit. The two complex structures were solved by molecular replacement using the wild-type RRM1 structure (PDB entry code: 4IUF) as the search model. The crystal structures were refined by PHENIX and the data collection and refinement statistics are summarized in The X-ray diffraction data of hRRM1/DNA were collected by Rigaku FR-E + SuperBright X-ray generator with Saturn 944 + CCD detector. The diffraction data of RRM1-D169G/DNA were collected at SPXF beamline BL15A1 at NSRRC (Taiwan). The diffraction data were processed and scaled by HKL2000. The RRM1/DNA complex was crystallized in the hexagonal space group P6The crystal structure of the wild-type RRM1 reported previously (PDB entry code: 4IUF) and RRM1-D169G determined in this study (PDB entry code: 4Y00) were subjected to molecular dynamics simulation. All simulations were performed using the OpenMM5.2 CUDA version of CHARMM version 37, and the CHARMM36 all-atom parameter set. The systems were first submitted to a 30-ps heating phase (from 0 to 300 K), followed by a 500-ps equilibration and a 20-ns production phase in the NPT ensemble. Simulation analysis was performed at room temperature (300 K) with an integration step set to 2 fs, except for the heating phase for which a 1-fs time step was used. Constant pressure was ensured by the use of a Langevin piston with a friction coefficient of 10 ps-1 at a reference pressure of 1 bar. Coordinates were saved every picosecond. All non-bonded interactions were truncated at 16 Å, van der Waals interactions were switched at 12 Å and electrostatic interactions were treated via the particle mesh Ewald summation method. To increase the sampling, several replicates of 20 ns each were performed, rather than a single long-trajectory. As four copies of RRM1-D169G were present in the crystal structure, 3 replicates of each copy were simulated. As for the wild-type RRM1, only one copy was found in the crystal structure, 10 replicates were performed. Therefore, in total, at least 200 ns of sampling was obtained for each form.The cDNA of the wild-type TDP-43, D169G and N390D mutants were cloned into the XhoI/ SacII sites of a pcDNA3.1-Myc/His (B) vector (Invitrogen) with a C-terminal Myc/ His tag. Mouse neuroblastoma (Neuro2a) cells were cultured in Modified Eagle Medium with 10% heat-inactivated fetal bovine serum (Invitrogen), 1% penicillin-streptomycin (Invitrogen) and 1% sodium pyrubate (Invitrogen). For generating stable cell lines, cells were subcultured and transfected in 6-well plates with TDP-43 constructs by selection in 500 mg/ml G418 (Promega) for two weeks and limiting dilution was used to generate monoclonal cell lines.For detecting the TDP-35 protein degradation, stable cells expressing wild-type TDP-43 and mutant (D169G and N390D) constructs were treated with cycloheximide . At various time points thereafter, the transfected cells were lysed and the amounts of the TDP-43 proteins were measured by Western blot analysis using the anti-His antibody (Proteintech). Cells were lysed in RIPA buffer  supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Sigma). The protein concentrations of the lysates were measured using the Bio-Rad protein assay reagent on a Beckman Coulter DU-800 machine. The lysates were then resolved by 12% SDS–PAGE and immunoblotted with the anti-His antibodies. Quantification of the immunoblot band intensities was performed with use of the Image J software (NIH) as described previouslyHow to cite this article: Chiang, C.-H. et al. Structural analysis of disease-related TDP-43 D169G mutation: linking enhanced stability and caspase cleavage efficiency to protein accumulation. Sci. Rep.6, 21581; doi: 10.1038/srep21581 (2016).


We evaluate the damage level and the influence of the HfO2 thickness on graphene. The results indicate that in the case of Al2O3/graphene, whether 1L or FL graphene is strongly damaged under our process conditions. For the case of HfO2/graphene, μRS analysis clearly shows that FL graphene is less disordered than 1L graphene. In addition, the damage levels in FL graphene decrease with the number of layers. Moreover, the FL graphene damage is inversely proportional to the thickness of HfO2 film. Particularly, the bottom layer of twisted bilayer (t-2L) has the salient features of 1L graphene. Therefore, FL graphene allows for controlling/limiting the degree of defect during the PE-ALD HfO2 of dielectrics and could be a good starting material for building field effect transistors, sensors, touch screens and solar cells. Besides, the formation of Hf-C bonds may favor growing high-quality and uniform-coverage dielectric. HfO2 could be a suitable high-K gate dielectric with a scaling capability down to sub-5-nm for graphene-based transistors.Based on micro-Raman spectroscopy (μRS) and X-ray photoelectron spectroscopy (XPS), we study the structural damage incurred in monolayer (1L) and few-layer (FL) graphene subjected to atomic-layer deposition of HfO Most of the reports are related to the latter method, because plasma is rather aggressive  and generally etches graphene11151823Although physical vapor deposition (such as magnetron sputtering), widely used in the semiconductor industry, can provide high deposition rates and preserve film stoichiometry, it generates extensive damage in graphene from high energy sputtered atoms62) and Al2O3 dielectrics on graphene by PE-ALD. In our process, the graphene samples are placed away from the plasma source for outside of the glow discharge. The remote oxygen plasma with low ion bombardment avoids fast etching of graphene. Simultaneously, the reaction between graphene and the precursor still guarantees physical/chemical modification of graphene. Based on micro-Raman spectroscopy (μRS) and X-ray photoelectron spectroscopy (XPS), we study the structural damage induced in 1L graphene underlying HfO2 and Al2O3 upon different oxygen plasma power levels. We evaluate the damage levels in AB-stacked bilayer (AB-2L), twisted bilayer (t-BL) and trilayer (3L) graphene (hereafter they are collectively referred to as FL) underlying HfO2 and Al2O3 for a fixed oxygen plasma power. We also investigate the influence of the HfO2 thickness on graphene with various layer numbers. The results indicate that in the case of Al2O3/graphene, both 1L and FL graphene are strongly damaged under the present process conditions. In the case of HfO2/graphene, μRS analysis clearly shows that FL graphene presents much less disorder than 1L graphene. Moreover, the FL graphene damage decreases with the number of layers. Our results also reveal that an inverse dependence of FL graphene damage with increasing the thickness of HfO2 film. FL graphene allows for controlling/limiting the defect formation during the PE-ALD HfO2 process. Therefore, it could be a good starting material for applications such as graphene-based transistors and sensing devices26In this work, we use mild plasma conditions to directly grow hafnium oxide  is 1.9, and the full width at half maximum (FWHM) of the 2D peak is 31 cm−1. These figures of merit first confirm the presence of as-transferred 1L graphene at the probed locations. At the exact same positions, two new peaks (defect-activated peaks) appear after PE-ALD HfO2 and PE-ALD Al2O3. Namely the D peak located at ~1356 cm−1 and the D’ peak (~1620 cm−1) at the right shoulder of the G mode. The D peak in sp2 graphene is activated by a double resonance Raman process in the presence of disorder and defects and is related to the breathing modes of carbon atoms in the vicinity of the K point in the Brillouin zone  to make the measurement inside the crystalline region (in the center of hexagon) with a Raman laser spot diameter of about 1 μm, the boundary of the hexagons does not contribute to the spectrum here. After the dielectric depositions, the intensity of D peaks becomes very strong. This indicates that the dielectric depositions break the symmetry of the graphene lattice and induce structural defects in graphene. Moreover, the positions of the G peaks are slightly shifted, the FWHM of 2D peaks are broadened and the D’ peaks are separated from the G peaks. These characteristics indicate that graphene is disordered, but it is not completely etched and then still optically visible. We therefore use the area ratio AD/AG between the integrated intensities of the D and G peaks to quantify the amount of disorder (i.e. the AD/AG increases with increasing amount of disorder at low defect concentration range). The area ratio is preferred over the individual intensities since it accounts for variations in peak position, intensity and linewidthμRS is a nondestructive method and is employed here to assess the structural damage in graphene underlying dielectric films. It is worth noting that a laser wave length of 514 nm was used in all the measurements except for the case by special description. 2 and PE-ALD Al2O3, respectively. The corresponding Raman spectra of 1L graphene underlying the two dielectrics are shown in AD/AG ratios are very similar for the different power levels , implying that the amount of generated disorders in graphene is not correlated to the plasma power levels in this range of powers. For ALD Al2O3 on graphene, Lim et al. used nitrogen plasma to pretreat grapheneTo investigate the damage in graphene subjected to different oxygen plasma power levels, we reduce the oxygen plasma power from 300 to 200 W and 150 W for PE-ALD HfO2 and Al2O3 with a thickness of about 5 nm, respectively. The Raman spectra of as-transferred graphene with various layer thicknesses (black curves) are also shown at the bottom of the figure as references. In the case of Al2O3/graphene (red curves), all the AD/AG ratios are large and all the D’ peaks clearly separate from G peaks. These features indicate that all kinds of graphene are damaged during PE-ALD Al2O3. In the case of HfO2/3L graphene, the intensity of the D peak becomes very weak and the D’ peak even almost disappears  is similar to that of the Al2O3/graphene case. These results point out that in PE-ALD HfO2, FL graphene is less damaged than 1L graphene and the damage level of graphene decreases with the number of layers.The layer number is first identified by the color contrast of graphene under optical microscopy, followed by μRS measurements. 2 thickness on graphene. 2 with different thicknesses , respectively. It can be seen from 2 thicknesses, the AD/AG ratio of 1L graphene dramatically increases compared with that of as-transferred graphene. However, the changes in the Raman spectra of FL graphene are less drastic. Surprisingly, all the AD/AG ratios decrease with increasing the thickness of HfO2 film  gratings and three excitation laser energies in the visible range, namely 514.5 nm (2.41 eV), 488 nm (2.54 eV) and 633 nm (1.92 eV). We do not find any peak related to HfO2 or Hf-C in the measurement range from 0 to 4000 cm−1. This implies that the HfO2 film is amorphous due to the low growth temperature.Finally, to identify if the HfOex situ XPS measurements to evaluate the impact of PE-ALD dielectrics on graphene. Four samples are sputtered with an Ar+ gun to perform a depth profile: 5.5-nm-thick Al2O3 and 5.9-nm-thick HfO2 are deposited either on silicon (as references) or on graphene/SiO2/silicon stacks . Core level spectra are recorded from carbon (C 1s), oxygen (O 1s), hafnium (Hf 4f), aluminum (Al 2p), and silicon (Si 2p). The elemental composition of the dielectrics obtained from 20-nm-thick reference layers can be estimated from the ratios of the integrated intensities of the XPS spectra: [O/Hf] = 2.15 ± 0.1 and [O/Al] = 1.47 ± 0.05. These results testify to the good quality of the dielectrics. We now focus on the C 1s atomic concentration profile and spectra of each sample. 2O3/silicon and HfO2/silicon samples, respectively. In both cases, a small amount of carbon (2% on average) is found in the profiles (except for a ~15% concentration corresponding to adventitious carbon on top of the dielectric layers). Moreover, a slight increase of the carbon concentration is observed when approaching the interface between the dielectric and silicon, most likely originating from residual contamination on silicon before PE-ALD. 2O3/graphene and HfO2/graphene samples, respectively. We can clearly identify the presence of graphene between the dielectric and the SiO2/silicon substrate. 2O3/graphene and HfO2/graphene samples at the maximum of the carbon profiles, respectively. The main peak at 284.5 eV in both spectra corresponds to graphene. Strikingly, in contrast to the Al2O3/graphene sample, the HfO2/graphene sample displays an additional peak at 281.5 eV. This peak can be attributed to the formation of the metallic carbide Hf-C. Consequently, the HfO2/graphene profile in et al.+ sputtering of ALD HfO2 induces the formation of Hf-C (at an ion-gun energy of 2000 eV), amounting to 1% of the total composition. To ascertain that the observed Hf-C peak is not related to sputtering (the Ar+ gun is operated on purpose at the very low energy of 200 eV in the hope of preventing Hf-C formation), we have performed an additional experiment. A 1L graphene is transferred onto an HfO2/SiO2/Si substrate and next subjected to sputtering in the same conditions as before. In the corresponding C 1 s spectrum, the carbide-related peak occurs as well, most likely due to intermixing between Hf and C during the erosion, resulting in Hf-C bonding. This means that, from the ex situ XPS analysis only, we cannot conclude if Hf-C forms during the PE-ALD process or during the erosion process or both. Nonetheless, as opposed to Al-C, this illustrates how easy it is to form Hf-C, since the Ar+ sputtering is operated at a very low energy of 200 eV. To further elucidate where Hf-C originates from requires an ALD apparatus fitted with an in situ XPS analyzer.We carry out 2O3/graphene/SiO2/Si , the Al2O3 film starts to nucleate and then grow. Unfortunately, 1L graphene, and even FL graphene, have been degraded during the pretreatment. On the contrary, the XPS results have demonstrated that the Hf-C bonds are quite easily formed, whatever the origin of the bonding. We hypothesize that, during the initial TDMA-Hf precursor pulse, a chemical reaction occurs between the Hf atoms and the C atoms constituting graphene to form Hf-C (its formation temperature is 25 °C)2 growth. Subsequently, the coverage of the first HfO2 layer protects the underlying graphene layer from damage during the following deposition cycles. This may also explain why HfO2 rather than Al2O3 can be directly grown on graphene without out-of-plane covalent functional groups, in low-temperature thermal ALD process41Although the optical image of the Al2/Si see  looks si2/Si see , all theAD/AG ratio on the number of graphene layers is not clear. It may be due to the interlayer interactions between the HfO2 layer and graphene or a direct consequence of the HfO2 deposition process or both. Previous works about the impact of the graphene thickness on its physical properties suggest that graphene’s rigidity may increase with increasing number of layers2 substrate, it conforms more easily to the surface morphology of the underlying substrate, thereby more prone to deform compared with FL graphene. Oxygen plasma modification of 2L and FL graphene has been studied in the literature. Calculation resultset al.462/graphene  made from 2L graphene is strongly suppressed compared with 1L graphene transistors532 and Al2O3) deposited by remote PE-ALD. Our results show that FL graphene is less damaged than 1L graphene; the damage level of FL graphene decreases not only with the number of graphene layers but also with the thickness of HfO2. Interestingly, the Raman spectrum of t-2L graphene underlying HfO2 presents the features of that of as-transferred 1L graphene. XPS measurements indicate that Hf-C is easily formed. After coverage by the first HfO2, the bottom graphene layer has an additional protection. The oxygen plasma pulse in PE-ALD is less reactive to FL graphene than 1L. Graphene rigidity increases with the number of graphene layers. Moreover, it also increases with the thickness of HfO2. These may be the reasons of FL graphene less damaged in PE-ALD HfO2. Therefore, FL graphene, more particularly, t-2L graphene allows for controlling/limiting the defect formation during the PE-ALD HfO2 process and might be a prospective material for applications such as graphene-based transistors and sensing devices. It appears that the thickness of PE-ALD HfO2 can be arbitrarily scaled down to 5 nm. Our results open up direct perspectives for FL graphene and HfO2 gate dielectric in graphene-based transistor applications.We have investigated the structural damage in graphene underlying dielectrics  by the usual method based on PMMA, after etching the copper foil in ammonium persulfate.Graphene is synthesized by APCVD with dilute methane (5% in argon) as hydrocarbon precursor on copper foils (Alfa Aesar #13382). The samples are grown at 1000 °C for 1 h under flows of 500 sccm of argon, 20 sccm of hydrogen, and 0.2 sccm of dilute methane. Graphene is then transferred onto 300-nm-thick SiO2 and Al2O3 films are deposited on graphene/SiO2/Si stacks, by PE-ALD  at 250 °C. The plasma source, inductively coupled at 13.56 MHz, is far away from the samples. It is very important to note that the distance between the plasma source and sample location is larger than 40 cm since the type and concentration of the reactive species  strongly depend on this distance. Outside of the glow discharge, only long lifetime radicals are present while ions and electrons recombine quickly.HfOin situ ellipsometry from reference films directly deposited on silicon substrates. The composition of the dielectric films is characterized by XPS. In order to investigate the damage level of graphene upon different oxygen plasma power levels, HfO2 and Al2O3 films are deposited on graphene/SiO2/Si stacks with nominal 300 W and with reduced oxygen plasma power of 200 and 150 W, respectively.In order to remove the native stress and polymethyl methacrylate (PMMA) residues from growth and transfer, the graphene samples on the SiO2/Si substrate first are annealed at 250 °C for 2 h in the deposition chamber under pressure of 80 mtorr (argon gas). During both dielectric film depositions, the pulse duration of the oxygen plasma (oxidant precursor) is 10 s for each cycle. The flows of the oxygen plasma and the argon carrier gas are 20 and 200 sccm, respectively. The metallic and oxidant precursor pulses are separated by a short argon purge of 5 s. To obtain uniform dielectric films and avoid graphene etching, the metallic precursors are first pulsed on the graphene surface. The metallic precursors can adsorb or react with carbon to form the related metal oxides following the first oxidant precursor pulse. In contrast, if the first pulse is a single cycle of oxygen plasma, the graphene surface is possibly damaged or non-uniform dielectric films are formed. The other parameters related to the metallic precursors and the final thickness of both dielectric films are listed in A LabRam HR 800 confocal laser system from Horiba Jobin Yvon was used for the acquisition of the Raman spectra. The measurements are performed at room temperature with a laser wavelength λ = 514 nm in backscattering geometry. The laser beam is focused on the center of hexagons and a 100 × objective (NA = 0.95) is used to collect the signal. The incident power is kept below 1 mW. Low resolution (150 g/mm) and high resolution (1800 g/mm) gratings are used for the measurements.Kα1,2 x-ray source and a hemispherical deflector analyzer. The spectra are recorded at constant pass energy . A flood gun (low energy electrons and Ar ions) is used during all the measurements. During the sputtering, the Ar+ ion gun is operated at a low energy (200 eV), with an erosion time of 5 s per cycle, and the analysis is done in snapshot mode. The XPS data are treated with the Avantage software. High resolution spectra are fitted by Gaussian-Lorentzian lineshapes with an Avantage “smart” background .A ThermoFisher Scientific K-alpha spectrometer is employed. It is equipped with a monochromatized Al How to cite this article: Tang, X. et al. Damage evaluation in graphene underlying atomic layer deposition dielectrics. Sci. Rep.5, 13523; doi: 10.1038/srep13523 (2015).


Carriers with energy higher than the Schottky barrier between graphene and WSe2 can be emitted over the barrier, thus creating photocurrent. We experimentally demonstrate that the photo-thermionic effect enables detection of sub-bandgap photons, while being size-scalable, electrically tunable, broadband and ultrafast.Finding alternative optoelectronic mechanisms that overcome the limitations of conventional semiconductor devices is paramount for detecting and harvesting low-energy photons. A highly promising approach is to drive a current from the thermal energy added to the free-electron bath as a result of light absorption. Successful implementation of this strategy requires a broadband absorber where carriers interact among themselves more strongly than with phonons, as well as energy-selective contacts to extract the excess electronic heat. Here we show that graphene-WSe The detection of low-energy photons may be enabled by devices that make use of the excess thermal energy from photoexcited carriers as a result of light absorption. Here the authors demonstrate a vertical graphene-WSe2-graphene heterostructure that takes advantage of the photo-thermionic effect. ΦB  even under continuous-wave (CW) excitation1819Graphene, which has recently emerged as an excellent platform for converting photons into hot carriers2/graphene van der Waals heterostructures to detect low-energy photons (with a wavelength up to 1.5 μm) through photo-thermionic (PTI) emission. Te compared with the temperature of the lattice Tph and the environment T0; carriers within this distribution with an energy larger than the Schottky barrier height ΦB at the graphene/WSe2 interface can be injected into the WSe2 and travel to the other graphene layer. The number of carriers with sufficient energy scales with kB is the Boltzmann constant.Here, we use graphene/WSe2—a transition metal dichalcogenide with a bandgap Eg∼1.3 eV—provides an energy barrier between the two graphene sheets with low interfacial defects and reduced Fermi-level pinning. The active device  that enables control of the Fermi energy μ and thereby ΦB of (mainly) the bottom graphene. All measurements presented in the main text are obtained from one particular device comprising a 28-nm-thick WSe2 flake (see 2 flakes of various thicknesses (L=2.2–40 nm) and obtained similar results, consistent with the PTI effect . Signatures of this mechanism are readily visible in the data presented in 2 (Ephoton<Eg). That is expected from a thermally driven photocurrent, given the uniform absorption of graphene in the visible, near-infrared range and the fact that kBTe is independent of Ephoton for constant power15P (P (see Methods). In contrast, the photocurrent in the above-bandgap regime (Ephoton>Eg) varies strongly with Ephoton and scales linearly with P. This photoresponse is characteristic of light absorption in WSe2 and transfer of photoexcited carriers to the graphene electrodes, a process driven by the potential drop across the WSe2 layer2324The PTI process is driven by the light-induced increase of the thermal energy of the electron gas  devices. We note that multi-photon absorption followed by charge transfer could also lead to a superlinear power dependence, but the laser intensity required to induce significant two-photon absorption in either graphene or WSe2 is at least 1–2 orders of magnitude higher than the one used in our experiment   refs , 27. Simt between two sub-picosecond laser pulses generated by a Ti:sapphire laser  and gate voltage (VG). Applying these voltages is necessary in order to generate a finite photocurrent, as it breaks the symmetry of our device, which is composed of two G/WSe2 Schottky barriers with opposite polarity. As the infrared photocurrent maps (Ephoton=0.8 eV) in VB essentially controls over which of the two Schottky barriers hot carriers are injected: for VB=−0.6 V (2 layer (GT/WSe2), whereas the interface with the bottom graphene (GB/WSe2) is photoactive for VB=+0.6 V , the device reaches a responsivity of up to 0.12 mAW−1 at wavelength λ=1,500 nm, which, for 0.5% light absorption in grapheneOne of the hallmarks of thermionic emission is its exponential dependence on the Schottky barrier height. In our device, the gate voltage Te and ΦB, and on the carrier injection time τinj. The values for ΦB(VG) are extracted from temperature-dependent dark current measurements  and laser power. This two-dimensional fit yields a carrier injection time τinj=47±10 ps and a thermal conductance Γ=0.5±0.3 MWm−2 K−1. This value of τinj is almost identical to the one found for ideal G/Si Schottky barriersΓ matches the predicted thermal conductance of G/hBN interfaces due to electron coupling with SPP phononsT0=230 and 330 K) and the analysis yields very similar results  metal filmsΦB. Indeed, we find that the PTI efficiency increases by one order of magnitude (up to 20%) by extrapolating the IQE to higher Te  or lower ΦB , with the important differences that for PETE the photoexcited carriers are in thermal equilibrium with the lattice of a hot semiconductor and are emitted over a vacuum energy barrier3411f=40 MHz and pulse duration dt=100 ps) with a microscope objective (Olympus LCPlanN × 50) on the device. The photocurrent is measured using a preamplifier and a lock-in amplifier synchronized with a mechanical chopper at 117 Hz.The heterostructures are fabricated the same way as described in ref. J thermionically emitted over a Schottky barrier of height ΦB at temperature T isIn the reverse-bias regime, the Schottky barrier model based on the Landauer transport formalisme0 is the elementary charge, kB is the Boltzmann's constant, ħ is the reduced Planck's constant, vF is the graphene Fermi velocity, Φ0 is the band offset at the G/WSe2 interface (0.54 eV) and τinj is the charge injection time. In our experiment, the thermionic photocurrent (PC) we measure is produced by the increase of electronic temperature ΔT=Te−T0 upon illumination of the device with a quasi-CW laser at λ=1,500 nm. Hence, it follows that the photocurrent is A is the area of the laser beam (laser spot size of 1.75 μm), D is the duty cycle (D=dt·f=0.04%) and T0 is the ambient temperature. Using these equations and assuming T0≫ΔT, one can show that α is the light absorption in graphene (0.5%), ηheat is the fraction of absorbed energy that is transferred to the electron bath (∼70%)  ref. , P is thstep see .The data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Massicotte, M. et al. Photo-thermionic effect in vertical graphene heterostructures. Nat. Commun. 7:12174 doi: 10.1038/ncomms12174 (2016).Supplementary Figures 1-7, Supplementary Notes 1-7 and Supplementary References