Based on the necessity for enclosure protection of temperature and relative humidity sensors installed in a hostile environment, a wind tunnel was used to quantify the time that the sensors take to reach equilibrium in the environmental conditions to which they are exposed. Two treatments were used: (1) sensors with polyvinyl chloride (PVC) enclosure protection, and (2) sensors with no enclosure protection. The primary objective of this study was to develop and validate a 3-D computational fluid dynamics (CFD) model for analyzing the temperature and relative humidity distribution in a wind tunnel using sensors with PVC enclosure protection and sensors with no enclosure protection. A CFD simulation model was developed to describe the temperature distribution and the physics of mass transfer related to the airflow relative humidity. The first results demonstrate the applicability of the simulation. For verification, a sensor device was successfully assembled and tested in an environment that was optimized to ensure fast change conditions. The quantification setup presented in this paper is thus considered to be adequate for testing different materials and morphologies for enclosure protection. The results show that the boundary layer flow regime has a significant impact on the heat flux distribution. The results indicate that the CFD technique is a powerful tool which provides a detailed description of the flow and temperature fields as well as the time that the relative humidity takes to reach equilibrium with the environment in which the sensors are inserted. Technological development with respect to instrumentation systems and measures increasingly demands the means to provide greater reliability of the data collected.Computational fluid dynamics (CFD) simulations are increasingly used to predict various issues in agriculture and food processing Protective housing for temperature and relative humidity sensors is normally required to prevent damage due to the physical and environmental influences on agricultural processes. A major concern about protected temperature and relative humidity sensors is the response time delay in a process reaching equilibrium with its environment, which may affect its accuracy if the measurement is taken at an inadequate time.The main objective of this work was to identify how much time it takes for temperature and relative humidity sensors to respond properly in a dynamic varying process, under certain specified operational conditions.As a first step, experiments were performed in a wind tunnel, and CFD simulations, to predict the distributions of temperature and relative humidity in a non-isothermal flow, were accomplished.A small wind tunnel, 200 mm in diameter and 500 mm high, was designed to provide a laminar flow with a speed between 0.5 and 1 The investigation of the heat exchange and mass transfer between the environment and the housing was divided into two parts: (1) a study of the interface between the fluid medium (air) outside and inside the housing, (2) analysis of the fluid flow (air) at constant speed for high temperature (77.5°C), external to the housing.Measurements were taken at the point closest to the center outside the housing and at the center point inside. The Reynolds number Results were obtained from the standard In order to gain further insight into the heat transfer and mass structures of PVC (polyvinyl chloride) materials and to analyze the behavior of the temperature and relative humidity sensors more deeply, CFD model simulations were performed to investigate the distribution of temperature and relative humidity in the transient regime at the observation points, and the findings were compared with results obtained in the wind tunnel.A data acquisition system, based on 1-Wire technology, with real-time monitoring was implemented using addressable digital devices The 1-Wire network uses only a conductor for data transmission, in common with all instruments or devices (slaves) connected to the network, and a computer or microprocessor (master), which manages the whole system through an appropriate computer program, and is technically and economically feasible The temperature and relative humidity in the wind tunnel were measured at two central points. A sensor without protective housing was placed close to the wind tunnel center and exposed to the air stream. Another sensor with protective housing was installed at the center of the wind tunnel and received the airflow through the perforations made in the housing wall. The average airflow rate was held constant through the wind tunnel. External environmental variables, such as temperature and relative humidity, were also monitored with a sensor without housing.The housing for the circuit with a temperature sensor (DS2438) and a humidity sensor (HIH4000) with 1-Wire technology inside the wind tunnel was constructed using a PVC tube 100 mm long with a diameter of 32 mm. Perforations of 5 mm diameter were drilled into the housing wall to allow for airflow around the sensors, connected by shielded wire pairs, as illustrated in A data acquisition system was programmed using the 1-Wire protocol and based on a system called STRADA As previously discussed, one sensor with protective housing and one without were installed inside the wind tunnel. A third sensor was installed outside the tunnel to monitor the external environment conditions. The sensors were interconnected by a four-way telephone wire to acquire and transmit data The temperature and relative humidity data were recorded with a resolution of The airflow speed in the wind tunnel was set at Due to the complexity of the housing geometry, the ANSYS ICEM CFD software was used to construct a tetrahedral computational mesh of the object under study.The tetrahedral volume element is very useful for maintaining the high quality of the mesh while articulating different forms of complex geometry to be modeled in the region of interest, even though it requires more nodes than the mesh elements and results in a hexahedral file size and increased computation time The results obtained by simulation using CFD were verified and compared with the corresponding data obtained experimentally in the wind tunnel.The measured values and those obtained experimentally in the wind tunnel were used to assign the boundary conditions of the model. Thus, it was necessary to provide the initial values of variables such as relative humidity and air temperature inside and outside the enclosure, calculate the humidity and temperature for each step in time, and solve the equations of conservation of energy, mass and momentum.The CFD technique was used to solve the Navier–Stokes equations written for the velocity and energy fields, quantifying the fields of temperature and velocity by the finite volume method h - Convective heat transfer coefficient m - Mass transfer coefficient hP – Pressure (Pa)T – Temperature (K)T – Time (s) - Dissipation rate of speed fluctuations k - Turbulent kinetic energy coefficient The Reynolds tensor was modeled using the standard turbulence After exposure of the sensors to the airflow for a specific period of time for a given level of humidity, a relative humidity value is reached, which depends on the temperature and the amount of water contained in the air at the equilibrium state.Relative humidity is a measure of the amount of water contained in the air, defined by the ratio between the actual pressure of water vapor in the atmosphere and the pressure of saturated water vapor in the atmosphere at the same temperature, usually expressed as a percentage.The empirical relationship between the saturation vapor pressure and the temperature is given by the following equation The ANSYS CFX computer program was used to implement the proposed model with the following assumptions:Transportation in transient state.Incompressible flow.A residue with a value less than The variables were tested for normality using the Shapiro–Wilk test and subsequently an analysis of variance was carried out. Comparisons between independent replications were performed by analysis of variance for data with a normal distribution and by the nonparametric Kruskal–Wallis test for data without a normal distribution.After the appropriate transformations, data were submitted for nonparametric testing using the SigmaPlot, version 11.0, computer program, from Systat Software Inc. This work was developed at the Departamento de Engenharia Agrícola, Universidade Federal de Viçosa, Viçosa, State of Minas Gerais, Brazil.To solve the problem of heat and mass transfer using CFD, first the behavior of the fluid (air) under two experimental conditions  was analyzed. The behavior of the temperature variation is shown in After the experimental behavior of the fluid (air) in the proposed scenarios was analyzed, the process was modeled. The boundary conditions took into account the temperature at the entrance of the control volume.The temperature of the fluid stream outside the protective sensor's housing was measured, starting at instant zero corresponding to the ambient air temperature until a maximum steady temperature was reached, according to the power and airflow rate of the fan used. A mathematical model was fitted to the experimental data by nonlinear regression analysis. The model that best fitted to the experimental data was an exponential type equation, expressed by y - Temperature (t - Time (s)a, b - Regression coefficientsThe coefficient estimates of the fitted model, along with their standard error, t-statistics values, and probability significance level, are presented in The adjusted coefficient of determination  for predicting the response of temperature and relative humidity sensors inside the protective housing. This is very important to determine the exact instant at which the measurements should be taken to obtain the correct, or at least, a satisfactory value of the measure of interest, principally if the process is taking place in a hostile or dirty environment.The model previously discussed was used to estimate the boundary conditions (input conditions) of the simulation model.In order to accomplish the simulation, ICEM ANSYSTM software was used to generate meshes for the system. A tetrahedral mesh was created with a total of 356,654 finite volumes comprising two distinct domains: (1) formed by the fluid confined in the protective housing consisting of 217,069 finite volumes, and (2) formed by the unconfined fluid consisting of 139,585 finite volumes. Both domains were interconnected by a fluid-type interface at the openings in the PVC tube. Boundary conditions were established for each face of each domain, as shown in Using During the study period , it was observed that the time for temperature and relative humidity to reach the equilibrium conditions for sensors with and without protective housing differed by 1,020 s and 540 s, respectively, with a variation of It should be pointed out that these results are for a particular case and, certainly, may vary for other conditions or processes. However, they give an indication of how to proceed when measuring temperature and relative humidity, for example, in the mass of a biological product, such as cereal grains.A high correlation was found between the measured and simulated temperature inside the sensor's housing, as well as between the measured and simulated relative humidity inside the sensor's housing, as shown in It can be seen in As a material of high resistance and low cost, PVC tubes are appropriate for housing sensors exposed to hostile environments.The results obtained from simulations of temperature and relative humidity show a good correlation with experimental data .Video S1Temperature and relative humidity variation.(AVI)Click here for additional data file.


We developed a simple, scalable and high-throughput method for fabrication of large-area three-dimensional rose-like microflowers with controlled size, shape and density on graphene films by femtosecond laser micromachining. The novel biomimetic microflower that composed of numerous turnup graphene nanoflakes can be fabricated by only a single femtosecond laser pulse, which is efficient enough for large-area patterning. The graphene films were composed of layer-by-layer graphene nanosheets separated by nanogaps (~10–50 nm), and graphene monolayers with an interlayer spacing of ~0.37 nm constituted each of the graphene nanosheets. This unique hierarchical layering structure of graphene films provides great possibilities for generation of tensile stress during femtosecond laser ablation to roll up the nanoflakes, which contributes to the formation of microflowers. By a simple scanning technique, patterned surfaces with controllable densities of flower patterns were obtained, which can exhibit adhesive superhydrophobicity. More importantly, this technique enables fabrication of the large-area patterned surfaces at centimeter scales in a simple and efficient way. This study not only presents new insights of ultrafast laser processing of novel graphene-based materials but also shows great promise of designing new materials combined with ultrafast laser surface patterning for future applications in functional coatings, sensors, actuators and microfluidics. Surface micro/nano patterning is a highly promising approach to control the optical, electrical, chemical, biological, mechanical and other properties of a solid surface1246781313 w/cm2) and ultrashort irradiation periods, fs laser fabrication presents unique advantages of nonlinear nonequilibrium processing212225In general, scalable methods used to create surface patterns can be divided into two categories: bottom-up methods such as chemical synthesis, self-assembly and dip-pen nanolithography1551819In this study, large-area uniform 3D rose-like microstructures were fabricated using fs laser pulses. The 3D rose-like structure can be obtained by only one laser pulse, which offers great potential for the high-throughput large-scale manufacturing. The home-made freestanding graphene films, prepared from direct filtration of the aqueous reduced graphene oxide colloidal suspensions, were composed of both nanoscale graphene nanosheets separated by nanogaps and subnanoscle graphene monolayers with a large interlayer spacing of ~0.37 nm. The unique hierarchical structure offers great potential for the generation of tensile stress during fs laser ablation, which acted as a driven force for rolling up the graphene nanoflakes. Our approach shows potential application in fabrication of centimeter-scale 3D micro/nanostructures in a controllable and highly-efficient manner, which is distinct from earlier reports in that the graphene film itself served as an important precursor for the formation of graphene microflowers when it was processed with fs laser pulses.v), and scanning pitch (2) shows superhydrophobility with a large water contact angle of 150°, whereas the original graphene film only exhibits hydrophilicity with a low water contact angle of 78°  to 4 J/cm2 (more than 20 times larger than the threshold), the ablated structures on HOPG films changed from smooth craters to nanoripples to through holes with the increase of the pulse number  of 1.1 J/cm2. At N = 1, several intact and flat graphene flakes were peeled off from the center of the ablated region after a single pulse irradiation, leaving a relative clean and flat surface in the ablation crater (The size and shape of the graphene flower can be further controlled by tuning the laser fluence (F) and pulse number (N), respectively. n crater . The foln crater . In thisn crater . Hence, v), and scanning pitch. The details of the flower density (D) calculation, and the relation between scanning parameters and flower densities were described in 2. It is shown that the graphene microflowers within the scanning area have almost the same geometrical feature, indicating the excellent repeatability. The density of graphene microflowers increases gradually from Large-area micropatterning of graphene films with controllable densities of flower patterns was realized by a simple scanning technique with coordinated adjustments of laser repetition rate, scanning speed , high-density (>1021 cm−3) electrons3434It is worth noting that the microflowers can be obtained on graphene films that have unique hierarchical layering structures, while they cannot be fabricated on HOPG films with a uniform interlayer spacing. On the other hand, the microflowers consist of graphene nanoflakes that were exfoliated from the surface by fs laser pulse, which are geometrically similar to the fragmentation of thin spalls induced by tensile stressIn summary, a simple and efficient method for large-area fabrication of 3D biomimetic microflowers on the graphene films with hierarchical layering structure have been developed using femtosecond laser pulses. The unique structure of the graphene films offers great potential for the generation of tensile stress during femtosecond laser ablation to roll up the graphene nanoflakes for the formation of a complex microflower by only a single laser pulse. Moreover, the size and shape of the microflowers can be precisely controlled by tuning the laser fluence and pulse number, respectively. After large-scale fabrication, the surface of graphene film was covered with uniform arrays of flower patterns with controllable density, exhibiting superhydrophobility and a high retention force to water. The fabricated biomimetic microflowers on the freestanding and flexible graphene films can find broad applications in bionics, microfluidics, sensors, micro/nano-droplets manipulation, single droplet analysis, Janus interface materials for actuators, and in fabrication of lab on a chip that need to retain tiny quantities of liquid without leaking.The graphene films were prepared by direct filtration of aqueous reduced graphene oxide colloidal suspensions through a filter membrane as detailed in our previous reportThe setup of the fabrication system was described in The pump-probe experiments were conducted with the same fs laser. A laser beam was divided into a pump and a probe beam by a beam splitter. The pump beam was focused onto the target surface by a 5 × objective lens. The probe beam was frequency doubled to 400 nm by a beta barium borate crystal. Using a CCD camera, the shadowgraphs of the early plasma were detected.The surface morphology was investigated by a scanning electron microscope . The average interlayer spacing was evaluated by a parallel beam Bruker D8 Advance X-ray diffractometer (XRD). The electrical conductivity was measured using a four-probe conductivity test meter  at room temperature. Raman spectra were obtained using a Renishaw InVia Reflex spectrometer with a 532 nm light source at a low power. The transmission electron microscopy (TEM) was performed using an FEI Tecnai G2 F30 transmission electron microscope. Roughness measurements were carried out using an atomic force microscope . The water contact angles were measured using a K12 tensiometer . X-ray photoelectron spectroscopy (XPS) was carried out using a PHI Quantera X-ray photoelectron spectrometer.How to cite this article: Shi, X. et al. Femtosecond laser rapid fabrication of large-area rose-like micropatterns on freestanding flexible graphene films. Sci. Rep.5, 17557; doi: 10.1038/srep17557 (2015).


Sarcoidosis involves abnormal collections of inflammatory cells (granulomas) which may form as nodules in multiple organs. 90% of affected patients have respiratory tract abnormalities. We present a 61-year-old male with sarcoidosis who was admitted for respiratory distress. Fibrosing mediastinitis was seen in the chest computograph. Management was conservative and included steroids, antibiotics, and oxygen therapy. Sarcoidosis and fibrosing mediastinitis are rare. Fibrosing mediastinitis is more commonly seen with histoplasmosis. We explore the clinical similarities between histoplasmosis and sarcoidosis. We also explore the potential cause and effect relationship and workup for each disease entity. H. capsulatum and causes symptoms that are almost identical to those present with sarcoidosis. Clinicians rely on patient history, lab values, and radiological and pathological studies to distinguish between them. However, there are false negative tests and atypical findings which may complicate the diagnosis. Misdiagnosing one for the other and starting the incorrect treatment plan could cause significant morbidity. In this case, we present a male patient diagnosed with sarcoidosis. History was obtained by personal interview and clinic and hospital medical records. His radiographic finding of fibrosing mediastinitis is rare in sarcoidosis and more commonly seen with histoplasmosis. We review this case to make clinicians aware of the striking resemblance between these two entities and the workup that is recommended to accurately diagnose each disease.Sarcoidosis involves abnormal collections of inflammatory cells (granulomas), which may form as nodules in multiple organs. It is considered a diagnosis of exclusion and most commonly presents with respiratory symptoms. Histoplasmosis is caused by the fungus2 of 57 (on 50% FiO2). Due to his level of respiratory distress, he was intubated with ventilator support. He was treated with antibiotics, steroids, and airway clearance measures. Chest X-ray (CXR) showed extensive fibrocalcific scarring in the upper lobes and compensatory emphysema of the lower lobes  at 18%. Arterial blood gas analysis revealed respiratory acidosis with a pH of 7.27 and COer lobes . Culture Aspergillus. For this reason, oral antifungal therapy was instituted.Our patient first presented with respiratory symptoms at 28 years of age. His symptoms at that time included intermittent fatigue, shortness of breath, a “sore” tongue, and “facial swelling.” While spirometry was normal, his CXR revealed left hilar adenopathy and calcification. Mediastinoscopy was performed, including a mediastinal lymph node biopsy where he was found to have noncaseating granulomas. The fungal cultures and AFB (acid fast bacilli) stains by bronchoscopy were negative. The patient was diagnosed with sarcoidosis and started on prednisone for several months, to which his symptoms responded favorably. For 2 decades, he was treated with inhaled steroids and intermittent courses of prednisone. In his 40s, he presented to an ENT  specialist with uveitis and a hoarse voice. The ENT physician felt that the cause of the hoarse voice was iatrogenic . No biopsy was performed. At the age of 55, a cavitary lesion was detected on chest CT, and sputum cultures were positive forInitial CXRs at the age of 28 showed left hilar and left paratracheal adenopathy. There were also calcified nodules in right and left lung fields. CXRs from the age of 45 through 52 showed progressive upper lobe fibrotic changes. From the age of 52 to 62, the images were stable. The CT scan during the most recent admission was stable as compared to the one done 3 years prior. It showed extensive calcification and soft tissue within the mediastinum with retraction of the mediastinum consistent with fibrosing mediastinitis. Both upper lobes had extensive calcifications and scarring. The esophagus was dilated above the fibrosing mediastinum  associated with sarcoidosis. Devaraj et al. reported two cases in a series of 12 patients with CT images and histology of fibrosing mediastinitis (16% of patients) . There w Histoplasma grew from the liver biopsy specimens [There are other clinical markers which can be used to diagnose sarcoidosis. However, these also can be misleading. ACE (angiotensin converting enzyme) levels are usually only elevated in sarcoidosis but there was a reported case where a patient had elevated ACE levels andpecimens . ACE levpecimens . Noncasepecimens . Histoplpecimens . A positpecimens . However Histoplasma organisms are difficult to visualize in tissues and have often been initially overlooked [ Histoplasma capsulatum has been identified in clinical samples by a highly sensitive molecular marker, a fragment of the Hcp 100 gene. PCR assays for the Hcp 100 gene have been validated in several studies and have shown high specificities [Laboratory methods to diagnose histoplasmosis include cultures, histology, molecular techniques, serology, and antigen detection.erlooked . In addierlooked , 24. Hiserlooked . In receficities –29.In summary, chronic disseminated histoplasmosis and sarcoidosis have strikingly similar features. Diagnostic tests including radiography also have similarities. Unfortunately, applying loose criteria for the diagnosis of sarcoidosis may result in overlooking specific infectious diseases including histoplasmosis. Cultures alone cannot exclude histoplasmosis due to a significant rate of false negatives. As a result, the recommended workup to accurately differentiate between sarcoidosis and histoplasmosis should include radiography, tissue biopsies, cultures, ACE level, serology, PCR assay, and urine antigen. Future studies will be needed to investigate the link between these two diseases.


Graphene has drawn considerable attention due to its intriguing properties in photonics and optoelectronics. However, its interaction with light is normally rather weak. Meta-surfaces, artificial structures with single planar function-layers, have demonstrated exotic performances in boosting light-matter interactions, e.g., for absorption enhancement. Graphene based high efficiency absorber is desirable for its potential applications in optical detections and signal modulations. Here we exploit graphene nanoribbons based meta-surface to realize coherent perfect absorption (CPA) in the mid-infrared regime. It was shown that quasi-CPA frequencies, at which CPA can be demonstrated with proper phase modulations, exist for the grapheme meta-surface with strong resonant behaviors. The CPA can be tuned substantially by merging the geometric design of the meta-surface and the electrical tunability of graphene. Furthermore, we found that the graphene nanoribbon meta-surface based CPA is realizable with experimentally achievable graphene sample. Recently, graphene is been widely investigated for potential applications in photonics and optoelectronics123471113η0 where η0 represents the characteristic impedance of vacuum434445464748495051Artificially engineered photonic structures have been employed as a platform for boosting light-matter interactions for decades15182022232425293031323334353637383940In this paper, we propose a tunable mid-infrared coherent perfect absorber with a graphene nanoribbon based meta-surface. It is found that quasi-CPA frequencies, which is the necessary formation condition of coherent absorption, does exist in the mid-infrared regime for properly designed graphene nanoribbon arrays. The scattering of coherent beams can be perfectly suppressed at the quasi-CPA frequencies with proper phase modulations on the input beams. For the case with two crosses on the transmission and reflection spectra, we can achieve coherent perfect absorption at the two quasi-CPA frequencies, simultaneously. The flexible tunabilities of the graphene meta-surface based CPA are of interests for tunable infrared detections and signal modulations.I+ and I−), O+ and O− are the respective output magnitudes, the graphene sheet is illuminated with perpendicularly polarized light . The meta-atoms, i.e., graphene nanoribbons, are periodically arranged in x-y plane with the lattice  constant and ribbon’s width being P and w, respectively. Both lattice constant and width of the graphene nanoribbon meta-atoms play important roles in determining the optical resonant behaviors of graphene meta-surface, the lattice constant is set to be P = 0.7 μm for the study of CPA in the mid-infrared regime.σg) since a one-atom-thick graphene sheet is sufficiently thin compared with the concerned wavelength. In the theoretical perspective based on random-phase-approximation (RPA)5455EF represents the Fermi energy, μ = 104 cm2V−1s−1 and Fermi velocity vF ≈ 106 m/s.The graphene layer is considered as a sheet material modeled with complex surface conductivity (EF = 0.5 eV. r) and transmission coefficients (t) for the case of graphene nanoribbon meta-surface with width of w = 0.33 μm. We can see an obvious resonance around 23.0 THz. The resonance was confirmed to be electric dipolar mode (which will be verified from the effective surface conductivity below), similar to the low-frequency resonance of split-ring-resonators as that inWe first took O±) of the graphene nanoribbon meta-surface can be related to the two input beams  through a scattering matrix, Sg, defined as:The complex scattering coefficients (t/r)+ and (t/r)− are scattering elements of forward  and backward  beams, since the linear monolayer graphene under investigation is of reciprocity and spatial symmetry, the scattering matrix can be simplified with t± = t and r± = r. Then the amplitude of the scattering coefficients would bewhere  of the input coherent beams leads to significant suppression of the scattering outputs at the quasi-CPA frequencies. The normalized total absorption as a function of frequency for the phase modulation ϕ0 = 1.03π is plotted in To implement the perfect absorption with the graphene meta-surface, we set a chirped phase modulation r and t) of the graphene meta-surface on the widths of nanoribbons. We can see that the resonant frequency of the electric dipolar mode shows a monotonous red-shift with the increase of w, which is similar the cut-wire casex axis. Increasing of the width w or the graphene’ filling factor in the unit cell of the meta-surface leads to stronger light-graphene interaction, i.e. high resonant strength of the electric dipolar resonance, and thus higher r and lower t around the resonance that introduce a regime where w = 0.138 μm, which has its boundary (as the solid line indicated) being the quasi-CPA points. The discrete spheres on top of the solid curve, representing the extracted surface conductivities with The meta-surface structures together with the electrically-controlled graphene will provide more wider tunable space for the design of mid-infrared CPA, we first consider the geometric tunability of the graphene nanoribbon based CPA. μm, the left edge of the solid boundary in On the other hand, the graphene meta-surface is also expected to have higher resonant strength for graphene with larger Fermi level3P = 0.22 μm, w = 0.20 μm) made from experimentally practical graphene, i.e., the Yan et al. graphene with Drude weight τ−1 = 9.8 THz2Here, we also consider a graphene nanoribbon array . The periodic boundary conditions were set for the simulation of periodic graphene nanoribbon structures. The effective surface conductivities of the graphene nanoribbon based meta-surfaces were carried out using a recently proposed sheet retrieval methodHow to cite this article: Fan, Y. et al. Tunable mid-infrared coherent perfect absorption in a graphene meta-surface. Sci. Rep.5, 13956; doi: 10.1038/srep13956 (2015).


The devices also showed excellent selectivity over other metal cations such as, Na+, K+, Mg2+, and Ca2+, suggesting the capability of working in a complex sample matrix. We further successfully demonstrated the detection of Pb2+ ions in real blood samples from children by using our aptasensors, and explored their potential applications for quantification. Our results underscore such graphene FET aptasensors for future applications on fast detection of heavy metal ions for health monitoring and disease diagnostics.Lead is a cumulative toxicant, which can induce severe health issues, especially in children’s case due to their immature nervous system. While realizing large-scale monitoring of children blood lead remains challenging by utilizing traditional methods, it is highly desirable to search for alternative techniques or novel sensing materials. Here we report a label-free and portable aptasensor based on graphene field effect transistor (FET) for effective children blood lead detection. With standard solutions of different Pb When BLL is below 75 μg/L, the reduction of children’s intelligence quotient (IQ) was reported to be associated with as wellLead (Pb) is a widely distributed contaminant due to industrial and mining activities, coal combustion, and use of leaded paints/gasoline12Some basic criteria to realize large-scale monitoring of blood Pb include good selectivity, high sensitivity, superior portability, fast response and low cost. However, due to the complicated matrix of blood, it is highly challenging to develop a detection technique, which combines all these required features. For example, some widely-used instruments in laboratories such as atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS) have shown good selectivity and high sensitivity, but they are quite expensive and cumbersome67891011121315161718192021Graphene, the world’s thinnest conductive and elastic material, emerges as an ideal candidate. As a prototype two-dimensional (2D) material, graphene is easy to be functionalized and highly sensitive to environment, making it suitable to detect various objects, such as proteins, DNA, gas atoms and heavy metal ions192324252627282+concentrations, we obtained a detection limitation below 37.5 ng/L. Excellent selectivity to was also demonstrated over other metal cations Na+, K+, Mg2+, and Ca2+, suggesting the devices’ capability of working in a complex sample matrix. Furthermore, we successfully demonstrated the detection of Pb2+ ions in real blood samples from children by using our aptasensors, and explored their potential applications for quantification.In this work, we report a label-free and portable aptasensor based on graphene FET for effective children blood Pb detection. With Tris-HCl solutions of different Pb2+ detection, including G-quadruplex15172019212+ binding affinity and selectivity. The original 8–17 DNAzyme comprises of an enzyme strand (17E), which cleaves the RNA base on the substrate (17S) strand at the cleavage site upon binding to Pb2+. Typically, 8–17 DNAzyme based Pb2+ sensors relied on the release of cleaved 17S strand for signaling19212+ binding alone (by replacing the cleavable site rebonucleotide “A” to uncleavable deoxyribonucleotide “A” of the 8–17 DNAzyme) could give rise to significant signal changes with no need for the dissociation of the cleaved 17S strand. This could potentially improve the response speed of the 8–17 DNAzyme based Pb2+ sensors2+ binding capability of the DNAzyme. To ensure strong and specific binding of the aptamers to the graphene surface, we introduced a pyrene group to the 5′-end of 17E. This allows the aptamers to be anchored to the graphene surface of the devices through π-π stacking interactions. Moreover, this could successfully avoid the non-specific adsorption and denaturation of 8–17 DNAzyme on the graphene surface, which was reported to be a problem for many graphene based sensors2+ sensing. Such approaches have also been demonstrated to be valid to detect other heavy metal ions, such as Hg2+  2) were chosen for our studies due to higher quality, while CVD  grown graphene is believed to behave similarly. Shadow mask alignment or electron beam lithography followed by electron beam evaporation was applied to pattern the electrodes, which consist of 5 nm Ti and 50 nm Au, covered by an extra layer of 15 nm SiO2 to protect electrodes from electrochemical reactions . The atomic force microscopy (AFM) image of a single layer graphene flake is presented in 2 250 sccm, 300K) before electrical measurements were performed.The optical microscopic image of a typical graphene device is shown in back gate)-dependent resistance measurement results of a typical graphene FET device, showing the ambipolar field-effect characteristics. Both the carrier type and density of graphene can be modulated by Vback gate. In cnp (or the Dirac point in the case of monolayer graphene), indicating how the graphene flake is intrinsically doped, i.e. p/n doped when Vcnp is positive/negative or non-doped when Vcnp is equal to zero. For this particular device, Vcnp is around 18V, suggesting it is intrinsically p-doped. The feature of being sensitive to surrounding environment renders graphene an ideal sensing material for detecting charged objects. With the presence of certain charged environment, the charge doping level (or Fermi level) of graphene will be changed, resulting in a shift of Vcnp, or the resistance-Vback gate curve.We first confirmed the successful linking of pyrene to the 5′ of 17E, as supported by the UV-Vis spectrum shown in liquid gate curves within 2 minutes, which minimized the effect of solution evaporation. The pH of standard solutions was kept at neutral to avoid false response. At least two replicates were conducted for each concentration and the relative standard deviations between replicates were 2.4–15.7%, indicating good reproducibility of our measurement. The test results of standard solutions with four concentrations  are shown in cnp was around 0.2 V, indicating the p-doping nature of the device in Tris-HCl environment. After adding standard solution of 37.5 ng/L, we clearly observed left shifts of the curve, i.e. smaller Vcnp. For higher concentrations, the resistance-Vliquid gate shifted further left and Vcnp became smaller.To examine the performance of our graphene aptasensors for detection, we used 10 mg/L solution diluted with Tris-HCl to obtain standard solutions with different concentrations. The devices were immersed in the 0.5 M/L Tris-HCl solution during test to simulate human body fluid environment and build the ion liquid gate type of FETs . After adding standard solutions with different concentrations, we measured the resistance-Vcnp. For solutions with higher concentration, larger doping of electrons induces larger shift of Vcnp, which can be defined by ∆Vcnp2628cnp versus concentration  of the tested standard solutions from two representative devices  by approaching its surface, which leads to a left shift of the transfer curves or a smaller V2+, we measured the response of other commonly found ions in blood, including Na+, K+, Mg2+ and Ca2+ at a concentration of 0.1 M/L. As shown in cnp was much smaller than that from Pb2+ at a significantly lower concentration (0.5 nM/L). Therefore, our device can be used to measure real blood samples with no interference from other metal cations in blood. Such a great selectivity can be attributed to the preferential binding of 8–17 DNAzyme to Pb2+ over other metal ions. These results also suggested that functionalization of 8–17 DNAzyme to the graphene surface did not affect its Pb2+ binding affinity and selectivity.To study the selectivity of the aptasensor toward Pbcnp increased with Pb2+ concentration. In order to rule out possible background disturbance in real samples, a parallel test was performed with another non-aptamer device, with results shown in cnp on the bare graphene.To the best of our knowledge, although a few graphene FET aptasensor has been reported, none of them was used for the measurement of Pb level in real blood sample. To address this challenging issue, we tested the feasibility of our device to measure BLL in real children blood samples. Three children blood samples were collected from town of Zhuhang in Nantong , which is an industrial town with intensive wire rope production. Emission of Pb-containing smoke and dust during the wire rope manufacturing process result in elevated levels of Pb in various environmental matrix of this area. Several children living in a nearby residential district were diagnosed with hyperactivity and emotional problems owing to their elevated BLL higher than 100 μg/Lcnp. We defined these concentrations as aptasensor concentrations, which were plotted together with ICP-MS measured concentrations for comparison in We further explored the potential application of our aptasensor devices on Pb quantification in real blood samples. The results from the standard solutions . 17E and 17S was separately synthesized by Genscript Biotechnology Co.  and dissolved in deionized water (4 μM/L). Our real samples of children blood were collected from Zhuhang, Nantong, Jiangsu Province, China. The collection of blood samples and the related experiments performed in this study were approved by the center for disease control and prevention of Changshu, China, and the methods were carried out in accordance with the guidelines established by the center. The informed consent was obtained from parents of these children. A conventional electron-beam lithography process followed by standard electron-beam evaporation of metal electrodes  was used to fabricate graphene FET devices.17E and 17S were synthesized by Genscript Biotechnology Co. . The functionalization consists of two steps: firstly, we dropped the 17E solution (4 μM/L) on the devices, and waited for 30 minutes to ensure good attachment; Secondly, we dropped the 17S to form 8–17 DNAzyme on the surface of graphene. The devices were washed with deionized water to remove the aptamer residue after each step.3 in a graphite digestion system at 105 °C for 5 h, and followed by the addition of 1 mL of 30% H2O2. The digestion was continued to reach near dryness, which was diluted to 10 mL with Milli-Q water. Lead concentration in the digestion solution was measured using ICP-MS. We measured it with ICP-MS . During ICP-MS analysis, indium isotope (114In) was used as internal standard to test the instrument performance. Before tested with our biosensor, the digested samples were neutralized by NaOH (1M/L).The children blood was digested according to U.S. EPA Method 3050B through a Hot Block digestion system . Briefly, 0.5 mL of blood was initially digested with 10 mL of concentrated HNO2 layer of the wafers was used as the back gate. For the ion liquid gate type of FET devices, a gold wire was inserted into the solution as the liquid gate electrode. We applied a constant bias = 0.1 V while sweeping the back/liquid gate voltage forward at a speed of 0.01 V/s. There was a one-minute internal between each gate voltage sweep to ensure a stable state. For measurements of real samples, an initial signal offset was adjusted by adding a highly-diluted pretreated sample with concentration (~5 ng/L). The error bars arose from the upper and lower limit of multi-measurements operated in a certain concentration.NI USB-6251  was used as a voltage source and DL 1211  was used as a current amplifier. The doped silicon layer underneath the SiOHow to cite this article: Wang, C. et al. A label-free and portable graphene FET aptasensor for children blood lead detection. Sci. Rep.6, 21711; doi: 10.1038/srep21711 (2016).


There is an error in the Results subsection of the Abstract as well as third paragraph of the Results section. In both of these instances, 43.9% should be 43.1%.There is an error in the caption of


A foundational library called MORT (Molecular Objects and Relevant Templates) for the development of new software packages and tools employed in computational biology and computer-aided drug design (CADD) is described here.MORT contains several advantages compared with the other libraries. Firstly, MORT written in C++ natively supports the paradigm of object-oriented design, and thus it can be understood and extended easily. Secondly, MORT employs the relational model to represent a molecule, and it is more convenient and flexible than the traditional hierarchical model employed by many other libraries. Thirdly, a lot of functions have been included in this library, and a molecule can be manipulated easily at different levels. For example, it can parse a variety of popular molecular formats , create the topology and coordinate files for the simulations supported by AMBER, calculate the energy of a specific molecule based on the AMBER force fields, etc.http://cadd.suda.edu.cn/MORT/index.htm.We believe that MORT can be used as a foundational library for programmers to develop new programs and applications for computational biology and CADD. Source code of MORT is available at Molecular modeling techniques have been widely used in the fields of chemistry, biology, drug design, and materials science for studying molecular systems ranging from small molecules to large biological molecules and even material assemblies. A lot of molecular simulation and visualization tools or packages have been developed-9. AmberMORT is written in C++ that is a native OOP language, and a relational model and other well-designed patterns have been applied to this library, which makes it very flexible. Furthermore, many commands/functions supported by XLEAP and AmberTools have been merged into this library. Based oAn advantage of MORT is that it employs the relational model rather than the traditional hierarchical model to store all the information of a molecule. In the hierarchical model, a molecule owns some residues, and a residue owns some atoms. Therefore, a molecule does not own atoms directly. In the relational model, a molecule owns residues and atoms directly, while a residue does not own atoms, and they just have relations between each other.The hierarchical model is used by XLEAP and some other software packages . But itHowever, all these problems do not exist or can be easily fixed for the relational model employed in MORT. Firstly, the problem of iterating on atoms does not exist because a molecule owns atoms directly, and then users can perform the iteration on a molecule directly disregarding residues. Secondly, the relational model does not have any problem dealing with bonds, angles and torsions, because in the relational model a molecule directly owns bonds, angles (if any) and torsions (if any). Finally, compared with the hierarchical model, the relational model is extremely easy to be extended. Taking the strand as an example, to introduce a strand by employing the relational model, users do not need to change the old code. Strand can only appear in DNA-related program and all users need to do is to make sure that the relations between strand and residues have been created correctly.atom, bond, resd, angl, tors, oops, tor2 and ptor.The names of these object types are encoded into HASH values in order to save the time of comparing characters. Each HASH is 10 letters long and only composed of letters, digital characters and underlines. Some HASH values are predefined, and the others can be generated by using a function with a string as the input. The flowchart for the generation of a HASH value is illustrated in Figure It has been mentioned that in the relational model, atoms, bonds and residues are owned directly by a molecule, so are angles, torsions and any other possible components such as strands and improper torsions. In MORT, these objects  are called molecular objects (referred as MOs). Currently, MORT supports eight types of MOs: atom, bond, residue, angle, torsion, out-of-plane stretch , torsion-torsion interaction, and pi-torsion (the latter two are used exclusively by the AMOEBA force field). A four-character-long-code is assigned to each MO type, and they are molecule_t) owns several MOs , and each MO has its own properties and there are relations between them. In order to save the properties and relations, two variables are created. The properties are stored in components (represented by m_components), and the relations are stored in adjacencies (represented by m_adjacencys). Both of m_components and m_adjacencys are the member variables of class molecule_t. A molecule usually has several components  and has several adjacencies . Descriptions of the important classes of variables are listed in Table The basic idea of MORT is: a molecule  but in a 1D array of floating numbers. The component is implemented in this way to achieve the best space efficiency.An adjacency records the relations between two components. For each pair of connected molecular objects, there is a record in the adjacency. More specifically, it uses a 2D array to store connections.morf_t, which is the base class for all MO classes. In brief, class molecule_t is designed for the storage of data, whereas class morf_t is created for the modification, creation or deletion of objects, and it does not contain any data information.The composition of a molecule is shown in Figure molecule_t, another class (represented by class database_t) is created to store the information of a molecule: the molecule’s pointers and their corresponding names. Therefore, if a molecule needs to be modified, its corresponding pointers will be returned from the database by using the function get_mol with its name as the parameter. The structure of class database_t is shown in Figure database_t and molecule_t are both inherited from class entity_t, which is the base class for the storage of data.Except for class morf_t is created for the modification, creation or deletion of objects. In order to achieve the above goals, some functions are necessary to access the properties of MOs, and therefore several member functions have been designed as follows:As has been mentioned before, class void set_x;value_type get_x( const string& pname ) const;bool get_x;x’ in the names of these functions can be any of the following five characters: ‘i’ (for integer), ‘d’ (for double precision), ‘s’ (for string), ‘v’ (for numeric vector) and ‘a’ (for any other data type), while the value_type can be int, double, string, numvec and boost::any depending on the ‘x’. In order to accelerate the process, string can be replaced by HASH values, and these functions can be transformed into the following ones:The character ‘void set_x;value_type get_x( const long long& pid ) const;bool get_x;iter_T) or MORANGE .Two methods can be used to iterate on the MOs of a molecule: MOITER ;iter_t operator--(int);iter_t& operator + =(ptrdiff_t pdif);iter_t& operator- = (ptrdiff_t pdif);morf_t. The following two member functions of class molecule_t return the starting and ending iterators of a certain type of MO:With all these member functions implemented, MOITER works just like a pointer to class iter_t xxxx_begin ;iter_t xxxx_end;xxxx could be any of the eight 4 character ID of a molecular object type.Here, range_T). Class range_T has the following member functions:Another way to enumerate MOs is to use MORANGE  const;morf_t at(int id) const;molecule_t has the functions xxxxs that return the MORANGE of a certain type of MOs, while class morf_t has similar member functions related_xxxxs that returns the MORANGE of related MOs.MORANGE in a sense works just like an array of MOs. Class molecule_t), and are composed of MOs . Molecules are stored in database (represented by class database_t). Both of molecule_t and database_t are inherited from class entity_t. Class morf_t and its child classes are used to modify, add and delete MOs. The functions in MORT can be roughly divided into two categories: object-related and property-related.A lot of functions supported by Antechamber and XLEAP have been developed in MORT, and therefore based on MORT it is very easy for users to develop new applications for computational biology and CADD. As shown in Figure Functions in this category can handle the MOs defined in MORT. These functions have various usages, and they are distributed in different directories.OBJFUN: a lot of functions are defined in this directory, and they can be used to modify objects. For example, the function fixbond can be used to fix the bond order of a molecule based on several rules, such as hard rule, length rule and conjugation rule, as illustrated in our previous work" may represent a carbon isotope. After the judgment, the corresponding functions will be used to analyze the character: parse_charge can be used to get the charge information from "+" and "-"; parse_ring can be used to recognize the digit outside a bracket as a starting/ending point of a ring; parse_weight can be used to parse the element’s weight that is listed in a bracket; parse_alpha can be used to interpret the element’s type, etc. Finally, all the information will be recorded into a molecule.(3). d SMARTS. SMILES CAPBOX: functions in this directory are mainly designed to add the solvent environment to an object. Four functions were designed to build different kinds of solvation environments: solvatecap can be used to add a solvate cap around the specified position of a solute; solvatebox can be used to add a solvate box to the solute in a cuboid way; solvateoct can be used to add the solvate box to a solute in a truncated octahedron way, which can reduce the number of the added solvent molecules; solvateshl can be used to add a solvate shell around the whole solute. The algorithms of these four functions are very similar. The process of solvateshl is illustrated in Figure (4). addions can be used to add positive/negative ions to the whole molecule.Apart from the functions mentioned above, the function ATMASK: sometimes, users may want to get the selected partition of a molecule, such as the atoms within 5Å from the 4th atom of a molecule. To achieve this goal, the function mask_atom was designed. This function can be employed to the specified atoms and residues. The argument of this function is a little complicated, and it is composed of some figures and symbols. For example, ":1-10" means to get the residues 1 to 10 from a molecule; "@4 < @5" means to return all the atoms within 5Å from the atom 4.(5). atom_node will then be invoked, and the following letters or numbers will be parsed as a MO in a specified molecule (here "4" represents the 4th atom). The symbol "<" means to get the atoms/residues in a certain distance, and then it will invoke the function parse of class dist_node to interpret the next section. The previous MO will be regarded as a core. The second "@" indicates that the queried objects are atoms, and "5" equals to the threshold of the distance. Finally, the function match checks each atom in the molecule that satisfies the condition, and returns the qualified ones.Following is the steps to interpret "@4 < @5". The first "@" indicates that this is something about atoms ("@" represents atoms and ":" stands for residues). The functions of class FORMAT: functions defined here can be used to handle different kinds of formatted files. For example, the function read_sdf can be used to parse the molecule from a MOL/SDF formatted file, and the function load_mdb can be used to load the molecules from a database file, which contains a lot of molecules with the MOL2, MOL/SDF or OFF format. Once a molecule has been loaded, it will be stored in a database (represented by class database_t) with its name as the identity.(6). The functions in this category can add, modify, save and delete different properties of a molecule, and all these functions are distributed in their corresponding directories.ENEFRC: functions in this directory are energy-related. The function eval_bond, eval_angl or eval_tors can be used to calculate the energy of bonds, angles or torsions. The non-bond energy can be calculated in two ways. The function get_dir can be used to calculate the energy based on periodic boundary condition (PBC) by using Ewald summation[nonbond_egb can be used to calculate the polar contribution of desolvation for a non-periodic model by using the Generalized Born (GB) model[(1). ummation,16. And Ke,C and NA are electrostatic constant, Coulomb constant and Avogadro’s contant, respectively, ε is the dielectric constant of water (78.5), INVCHG2 is a constant value of 332.05 kcal/mol, rij is the distance between two atoms, αi and αj are equal to the Born radii of atoms i and j. More detailed descriptions of the energy-related functions can be found in Additional filewhere AMBFMT: In this part of MORT, some functions were designed to read the AMBER and AMOEBA force fields and save the properties of a molecule into topology files. For example, write_amber_prmtop can be used to generate two files: topology  and coordinate files (space information), which can be used as the input files for MD simulations. read_frc/read_amoeba_frc can be used to get the AMBER/AMOEBA force field parameters from an AMBER/AMOEBA parameter file and then store them into molecules.(2). For the better use of MORT, several commands have been defined in directory "plugin", which makes it very convenient to develop one serviceable application with just a few or sometimes just one command for the developers.PLUGIN: commands in this directory can be regarded as the MORT’s interface, and users can call these commands to accomplish many kinds of tasks with less effort than using functions. They can help the developers do more work in less time.(3). In each command class, apart from its constructor and destructor, one function is necessary:virtual bool exec;command_i, which is the base class of all the command classes. exec performs variously in command_i’s child classes, reflecting the polymorphism of C++ language. Once one command class is declared, exec should be used to execute the corresponding commands at the end.This function is a virtual function inherited from class source can be used to interpret the files displayed in directory "dat/cmd", which contains the commands used for loading the force field parameters for the preparation of other operations ; merge can be used to put the objects listed in its arguments together; solvate can be used to add the solvents around a molecule; moloper can be used to add the missing information to a molecule (mentioned in directory OBJFUN part), etc. These commands are all defined in their own classes, and users need to declare the class first before doing any operation. The details of the important functions and classes in MORT and the related information of important commands can be found in Additional fileA lot of commands have be defined, for example, fixbond, addHs and other functions can work together to add the missing information.MORT can be used as a foundational library to develop new programs or software packages for computational biology and CADD. Usually, based on MORT, only a few codes are needed to solve a problem. For example, if users want to calculate the energy of a protein, the energy-related functions can be used conveniently. If the information of a molecule is incomplete, test directory.MORT can serve as the core of one program, what users need to do is to package this core with some necessary codes. They can be compiled as a standalone program to solve different kinds of problems. Figure A C++ based library MORT is developed as a new foundation library for computational biology and CADD. This new library has many advantages, especially for its data structure and powerful functions. By employing the relational model instead of the hierarchical model to store data, less time is taken while iterating on the atoms and it solves the annoying problems such as determining the belongings of inter-residue bonds. In this model, all bonds and atoms are independent and extra entities are created to store the connection information and other relations between these entities. A lot of functions have been developed in this library, and Figure setpchg can be used to call the standalone program in Antechamber[parmchk can be used to call Parmchk to add missing force field parameters. With all these commands implemented, it is easy to create the topology file for a protein-inhibitor complex inside MORT without calling any outside programs.Moreover, many commands in AmberTools have been integrated into MORT. For example, echamber to assighttp://cadd.suda.edu.cn/MORT/index.htm, and it can be compiled into a static library on Linux platform. It’s written in C++ and the boost library (version 1.46.1 or newer) is needed.MORT is available atThe authors declare that they have no competing interests.TH, QZ and WZ instigated the project, designed and implemented the algorithms and developed the MORT library. TH, QZ, YL, JW and JZ drafted the manuscript. All authors read and approved the final manuscript.The code organization, naming rules, and the detailed descriptions of the important classes and functions of MORT.Click here for fileSource code of MORT.Click here for file


TDP-43 is the major disease-associated protein involved in the pathogenesis and progression of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions linked to TDP-43 pathology (FTLD-TDP). Abnormal phosphorylation, truncation and cytoplasmic mis-localization are known to be the characteristics for the aggregated forms of TDP-43, and gain of toxic abnormal TDP-43 or loss of function of physiological TDP-43 have been suggested as the cause of neurodegeneration. However, most of the post-translational modifications or truncation sites in the abnormal TDP-43 in brains of patients remain to be identified by protein chemical analysis. In this study, we carried out a highly sensitive liquid chromatography-mass spectrometry analysis of Sarkosyl-insoluble pathological TDP-43 from brains of ALS patients and identified several novel phosphorylation sites, deamidation sites, and cleavage sites. Almost all modifications were localized in the Gly-rich C-terminal half. Most of the cleavage sites identified in this study are novel and are located in N-terminal half, suggesting that these sites may be more accessible to proteolytic enzymes. The data obtained in this study provide a foundation for the molecular mechanisms of TDP-43 aggregation and ALS pathogenesis. TARDBP gene, is a highly conserved, ubiquitously expressed nuclear protein. It has conserved RNA recognition motifs (RRM1/RRM2) flanked on either side by N-terminal and glycine-rich C-terminal domains that mediate protein-protein interactions345678910TARDBP gene causes early embryonic lethality12Transactivation response (TAR) DNA-binding protein 43 (TDP-43), encoded by the otifs RRM/RRM2 flaTDP-43 is the major disease-associated protein involved in the pathogenesis of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD) linked to TDP-43 pathology (FTLD-TDP)1416171819212223TDP-43 is predominantly localized in the nucleus, but under pathological conditions it is translocated to the cytosol14152627282914Immunoblotting of the Sarkosyl-insoluble fractions from FTLD and ALS cases using phosphospecific antibodies clearly demonstrated that hyperphosphorylated full-length TDP-43 of 45 kDa, smearing substances, and fragments at 18–25 and 35kDa are the major species of TDP-43 26312627TDP-43 phosphorylation, cleavage and cytoplasmic mislocalization are all associated with TDP-43 aggregation. However it remains to be clarified whether these modifications are the cause of the disease. Recent reports on post-translational modifications, including fragmentation, were based on studies of cellular or animal models overexpressing TDP-43 or its derivatives343536Immunoblot analysis revealed that abundant abnormal TDP-43 was recovered in the Sarkosyl-insoluble fractions of brains from both ALS cases as shown in After SDS-PAGE, the gel portions corresponding to these bands were cut out as shown in We identified at least four intrinsically cleaved TDP-43 peptides in the pathological TDP-43, i.e., three N-terminal peptides  and causes mislocalization of TDP-43 in motor neuronsAccumulation of filamentous inclusions composed of abnormally phosphorylated full-length TDP-43 (45 kDa) and its fragments (35 and 17–27 kDa) is a defining feature of TDP-43 proteinopathies3034However, no such TDP-43 peptides cleaved by caspase or calpain reported were not detected in the pathological TDP-43 in the brains of patients in this study. On the other hand, uncleaved peptides by caspase or calpain were detected as the major molecules in the digests. Furthermore, several novel intrinsically cleaved TDP-43 peptides were identified in the pathological TDP-43, i.e., three and five N-terminal peptides   and two Cellular expression of TDP-43 is tightly regulated at the transcriptional and post-translational levels. Under pathological conditions, however, formation of TDP-43 aggregates within the cell nucleus or cytoplasm results in reduced free nuclear TDP-43, and therefore the TDP-43 binding-receptor sensor detects a fall in protein levels and responds with increased TDP-43 production; the produced TDP-43 is then sequestered by TDP-43 aggregatesin vitro and in vivo4344It has been recently reported that C9ORF72 repeat expansions in mice cause TDP-43 pathology. In these mice, insoluble phosphorylated TDP-43 was predominantly present in monomeric form and high-molecular-weight or truncated pTDP-43 species were not detectedWe identified some intrinsically cleaved TDP-43 peptides at the N-terminus and C-terminus of Sarkosyl-insoluble TDP-43 in this study. These cleavage sites were neither the caspase cleavage sites nor the calpain cleavage sites of TDP-43 as reported previously46in vitro experimentIn our previous studies, we identified phosphorylation sites by immunochemical methodsIt has known that non-phosphorylated intact TDP-43 forms fibrous aggregateFurthermore, it seems reasonable to speculate that heavily phosphorylated peptides may not be detected because of low recovery from the column and low ionization efficiency in mass analysis. S409/S410-phosphorylated TDP-43 was immunochemically included in almost all fractions as shown in 2627Indeed, there were individual differences in other modifications, as shown in Interestingly, inclusions within the brain of ALS and FTLD-TDP patients are readily labelled with antibodies that recognize the C-terminus of TDP-43, but not with N-terminal TDP-43 antibodies. In contrast, spinal cord inclusions are labelled with both N- and C-terminal TDP-43 antibodies, suggesting that they are composed of full-length TDP-43 It has already reported that a Gly-rich region (287–322), which contains multiple glycine repeats, may contribute significantly to fiber formation as well as aggregation propensity5033Sarkosyl-insoluble abnormal TDP-43 was prepared from brains of two ALS cases with abundant and widespread TDP-43 pathologies. Brain samples (0.5 g) from patients with ALS  and from vascular dementia  were homogenized in 10 ml of homogenization buffer . Sarkosyl was added to the homogenates , which were then incubated for 30 min at 37 °C and centrifuged at 20,000 g for 10 min at 25 °C. The supernatants were centrifuged at 100,000 g for 20 min at 25 °C. The pellets were further washed with sterile saline and centrifuged at 100,000 g for 20 min. The resulting pellets were used as Sarkosyl-insoluble fraction. This study was approved by the research ethics committee of Tokyo Metropolitan Institute of Medical Science (number: 15–5), and carried out in accordance with the approved guidelines. Informed consent about the brain donation was obtained from all subjects.The Sarkosyl-insoluble, SDS/urea-soluble fractions were separated on 4 ~ 20% polyacrylamide gradient gels by SDS-PAGE and immunochemically detected as described-123  was used at a flow rate of 300 nl / min with a 2–80% linear gradient of acetonitrile for 60 min. Eluted peptides were directly detected with an ion trap mass spectrometer, Velos Pro  at a spray voltage of 1.9 kV and a collision energy of 35%. The mass acquisition method consisted of one full MS survey scan followed by an MS/MS scan of the most abundant precursor ions from the survey scan. Dynamic exclusion for the MS/MS was set to 30 seconds. An MS scan range of 400–2000 m/z was employed in the positive ion mode, followed by data-dependent MS/MS using the CID or HCD operating mode on the top 10 ions in order of abundance. The data were analyzed with Proteome Discoverer , Mascot software  and Scaffold software . Swiss prot and GenBank databases were used.The sample was resuspended in 0.1% formic acid containing 2% acetonitrile and introduced into a nano-flow HPLC system, DiNa fitted with an automatic sampler . A packed nano-capillary column NTCC-360/75-3How to cite this article: Kametani, F. et al. Mass spectrometric analysis of accumulated TDP-43 in amyotrophic lateral sclerosis brains. Sci. Rep.6, 23281; doi: 10.1038/srep23281 (2016).


The graphene FETs fabricated on poly (PEN) are found to have a lower density of charging sites than those on SiO2/Si substrate, mainly due to reduced interfacial interaction between the graphene and the PEN. This method can be an indispensable means to improve the stability of devices using a graphene as it provides an accurate and quantitative way to define the quality of graphene after the device fabrication.Defects of graphene are the most important concern for the successful applications of graphene since they affect device performance significantly. However, once the graphene is integrated in the device structures, the quality of graphene and surrounding environment could only be assessed using indirect information such as hysteresis, mobility and drive current. Here we develop a discharge current analysis method to measure the quality of graphene integrated in a field effect transistor structure by analyzing the discharge current and examine its validity using various device structures. The density of charging sites affecting the performance of graphene field effect transistor obtained using the discharge current analysis method was on the order of 10 The electrical and physical properties of graphene have been extensively investigated for diverse applications such as electronic switch, sensors, transparent electrodes, fuel cells, and catalysts1234568910111314For example, the conductivity of graphene is found to be greatly affected by many factors such as metallic residues originating from the growth processes, substrate materials, capping dielectrics, device fabrication processes such as patterning and cleaning, initial defect density, contact metals, operation ambient, temperature, and more. In addition, the operation history-dependent device drifts such as hysteresis, charge trapping, and interfacial reactions also affect the characteristics of a device using a graphene.3 type defects can be measured using the shift in the relative position and width of the G-peak, D-peak, and 2D-peak of the Raman spectrum. Various TEM and STM analyses of grain size, defects, and contaminants have been reported in the literature19Since the defects in graphene and the influences of the environmental factors play a critical role in device-level operation1718While Raman spectroscopy, AFM, TEM, and STM are useful means in the analysis of the initial quality of graphene, these methods are not easy to use when examining graphene integrated in a device structure2122Thus, the characteristics of graphene integrated in a device structure have been primarily investigated indirectly using the electrical characteristics of devices such as current-voltage (I-V) or capacitance-voltage (C-V) characteristics222327In this work, a new electrical characterization method to obtain quantitative information on defects and other environmental factors representing the quality of graphene FETs has been proposed and its validity has been examined using devices fabricated on a large area monolayer graphene sheets with different levels of physical defect density (see Methods). This method, called the discharge current analysis (DCA) method, modulates the carrier concentration of graphene periodically using an external pulse bias and translates the frequency dependence of the charges discharged from the graphene channel into a density of charging sites. The validity of this DCA method is examined by correlating the electrically measured density of charging sites with the initial physical defect density of graphene measured with Raman spectroscopy.For silicon MOSFETs, a charge pumping (CP) method has been used to accurately analyze the interfacial defect density, which affects the device performance. The CP method fills up defect sites at the silicon-dielectric interface using minority carriers supplied from the source and drain at an inversion state, as shown in δI, highlighted in red in oI, does not change as a function of frequency because it is proportional to the total width of the pulse peak as shown in δI, discharged from the charging sites in each cycle increases as a function of frequency. Thus, the discharge current can be approximately represented using the following equation, cI is the measured discharge current, oI is the current due to the charges accumulated in the graphene itself, k is a frequency-dependent loss factor to account for the charge loss to the source side due to asymmetric metal contacts and other factors, δI is the additional discharge current from charging sites, and f is the measurement frequency.The baseline current, cI, is linearly proportional to the frequency in the 10 KHz to 100 KHz range. As mentioned above, the tail current, δI, consists of charges released from defects sites or other surface states. The slope slightly increases above 100 KHz and then saturates at a higher frequency.Typical discharge current measured at 10 KHz to 1 MHz from CVD-grown graphene FETs is shown in cI curve, the charge trapping and detrapping mechanism in the graphene should be explained first. Recently, our group suggested that the hysteresis of a graphene I-V curve is primarily caused by two representative mechanisms, tunneling and a surface redox reaction16To understand the frequency response of the cI primarily monitors the discharge current due to the tunneling component, and it is proportional to the initial defect density as shown below. At this frequency range, cI linearly increases in proportion to the frequency. Thus, the slope of the linear portion can be used to extract the density of charging sites. On the other hand, at a frequency above 100 KHz, charge traps that cannot release charges within 5 μs start to continue holding the charges and those traps no longer contribute to cI. As a result, the slope of cI increases and eventually becomes saturated as shown in steps 5 and 6 of In the 10 KHz to 100 KHz range, c-f curves for four representative devices with different initial defect densities. All four curves show two regions of different slopes as a function of frequency at 20 to 100 KHz and 100 to 150 KHz, respectively. The cI curves are saturated at ~200 KHz and above. The linear slope at 20 to 100 KHz is attributed to the charge exchange between the charge sites and the graphene through the tunneling mechanism explained above. To investigate the influence of initial defect density (defined as I(D)/I(G) ratio) of graphene, we conducted Raman spectroscopy on each graphene channel after the graphene channel patterning /I(G) as shown in δI value representing the density of charging sites originating from both the graphene bulk channel and various charging sites near the graphene can be used to extract the density of graphene defect sites even after device fabrication.tterning , and theoI, due to the Dirac point shift during the measurement. Steps 5 and 6 of oI. The second slope showed a strong temperature dependence, indicating that it is related to the thermally activated mechanism such as chemical reaction./I(G) /I(G) . Nchargic-f measurement method can be instrumental in analyzing graphene quality, especially after device fabrication, because this method can be used to compare the impact of processes on the same graphene even without calibration.There is appreciable data scattering in c-f) curve is used to analyze the density of the charging sites while the electron-hole pair recombination at the interfacial charging sites is measured at a fixed frequency in the charge pumping method.The DCA method may look similar to the charge pumping method, which measures the interface state density of silicon MOSFETs, but the measurement principle is quite different31c-f curves measured in bottom gate graphene FETs with/without a 30 nm Al2O3 passivation layer. Since the Al2O3 passivation decreases contamination from the graphene surface, the density of the charging sites decreased to 2.47 × 1014/cm3 from 3.19 × 1014/cm3 after passivation. A more prominent slope increase before the passivation in the 100 to 180 KHz region indicates that the graphene surface is more prone to the water redox-induced charge generation, as expected2536372/silicon substrate with a poly (PEN) substrate  process was transferred to 90 nm SiO2O3 using atomic layer deposition (ALD) process at 130°C and annealed in N2 ambient at 200°C for 30 min to minimize the influence of ambient.For the device fabrication, 100 nm Au electrode patterns were formed on a graphene sheet using i-line contact photolithography and a lift off process. Then, graphene channels between source and drain electrode were patterned using i-line photolithography and oxygen plasma ashing process . The surface of graphene channel was passivated with 30 nm AlAfter the graphene channel patterning, the quality of graphene channel defined as I(D)/I(G) ratio was measured for all devices using Raman spectroscopy . 2 gate dielectric, using a pulse generator (Agilent 81110) as shown in For discharge current analysis, square pulse biases in 10 KHz to 500 KHz range were applied to the substrate, which works as a bottom gate electrode with 90 nm SiOU.J. performed electrical measurement and data analysis and worth the main manuscript with M.-H.H. and B.H.L., J.J.K. and H.J.H. performed the electrical measurement of devices and data analysis. Y.G.L. programed lab view software to collect the data presented in Fig. 2 and Fig. 3. C.G.K. and S.L. fabricated graphene FET on oxide and PEN substrate used in Fig. 3 respectively. S.K.L. developed the graphene process and fabricated the graphene sheet used in this work. M.-H.H. and B.H.L. wrote the main manuscript and jointly developed a model for data analysis. All authors reviewed the manuscript.Supplemetal data


We find that the substrate is, in fact, of great importance in the defect formation process, as the defects in graphene are mostly generated through an indirect process by the sputtered atoms from the substrate.Although ion beam technology has frequently been used for introducing defects in graphene, the associated key mechanism of the defect formation under ion irradiation is still largely unclear. We report a systematic study of the ion irradiation experiments on SiO Graphene has recently attracted great attention for a variety of novel applications, such as in nanoelectronics, gas detectors, solar cells, and DNA sequencing123468In ion-matter interaction theory, an energetic ion transfers its energy to the target system by both the elastic collisions with the target nuclei and the inelastic interactions with the target electrons (e.g. causing electronic excitations and/or ionization). In most of the previous reports112 substrate. Contrarily, Mathew et al.2 substrate under 35 keV C ion irradiation had negligible effect on the graphene. As described above, many apparently contradictory claims have been made, and the key mechanism of the defect formation under ion irradiation is still a mystery.On the other hand, the effect from the substrate underneath the graphene was also found playing an important role in the defect formation process. Compagnini et al.Y) in graphene under energetic ion irradiation. A variety of ion species with a large range of the ion energy were used. We further performed MD simulations to compare the experimental results in order to understand the underlying mechanism. We found that the defect formation only depends on the nuclear collision until eS reaches a threshold of ~ 2.2 keV/nm, above which the electronic energy loss becomes important; moreover, the majority of the defects are actually created by the sputtered atoms from the substrate rather than the direct ion-graphene collisions. Therefore, the interaction between the energetic ions and the substrate should be paid great attention to, if one would like to use the ion beam technique for engineering graphene.In this work, we report a systematic experimental study of the defect yield  monotonously increases with nS, following almost a linear relation in the log-log plot. But no such positive correlation was found with respect to eS, although the electronic energy loss is in general the major energy loss pathway for MeV heavy ions . Nevertheless, the indirect process also includes the defects generated by the recoiled incident ions16; however, they are very rare as found in our simulation, so they are safely ignored in the present discussion. Note that, by this indirect process, the defects can be generated fairly far from the incident spots. Statistically, we choose the defect-to-incident-spot distance of 5 Å as the dividing point to separately count for the defect yields by the direct process (dirY) and the indirect process (indY). As the results shown in nS used in this work: the defects generated by the direct process are consistently at least one order of magnitude less.As for the nuclear collisions, one would expect that the defects are generated by direct collision process (direct process) where incident ions directly knock out the graphene carbon atoms and leave the vacancy-type defects in graphene. However, by carefully analyzing the simulation results, we found that the majority of the defect sites were laterally quite far (a few nanometers) from the incident spots of the impacting ions, thus they were unlikely generated by the direct process. In fact, they were generated by the sputtered atoms from the SiOdT) for a carbon atom to be knocked out from a freestanding graphene sheet is about 25 eV using the AIREBO potential2, the displacement energy depends on the direction of the atoms being knocked out. We previously show that2 substrate, while it still remains around 25 eV for being knocked out into the vacuum. The former situation corresponds to the direct nuclear collision process with the impacting ions, thus the associated defect generation is greatly suppressed due to the high dT. The latter situation (towards vacuum) corresponds to the collisions initiated by the sputtered atoms from the substrate. Generally, the sputtered atoms carry energies spreading in a fairly wide range from several eV to several tens of eV following Thompson distribution2 as an example  is fairly close to 1, so the defect formation cross-section follows a simple linear relationship with respect to the nuclear stopping power. This simple linear relationship suggests that the simple binary collision model can still be used to describe the defect formation by the direct process with only a slight modification on the displacement energy (resulting in the decreased prefactor). On the other hand, for the indirect process, the defect formation is rather complicated; the cross-section depends on many factors, such as the interaction between the impacting ions and the substrate, the sputtering yield of the substrate, the types of the sputtered species, and their kinetic energy distributions. Nevertheless, the defect yield still follows reasonably well with the power law of nSends. The fitted parameters are practically useful, as they represent some universal features of the defect formation process, and can be used as a first-hand estimation for choosing the ion irradiation conditions in future.Based on the above analysis, we separately fit the direct and indirect defect yields by the power laws with respect to the nuclear stopping power in graphene  and 6 MeV Si (3.01 keV/nm) ions used in this work. On the other hand, we noticed that the substrate SiO2 has a considerably lower thermal conductance of only 1.44 W/mK2. In fact, we previously showed that the sputtering yield of bare SiO2 started to increase when eS of the incident ions exceeds 2.2 keV/nm30. Thus, the increased defect formation was most likely to be formed by an indirect process in which the sputtering yield of SiO2 was enhanced by the high electronically lost energy of the incident ions. Accordingly, for this graphene-on-SiO2 system, the threshold of eS should be determined by the substrate, i.e., 2.2 keV/nm. In addition, it is worth mentioning that, via the strong electronic excitations, part of the sputtered particles might be ionized, like oxygen radicals, which are known to chemically react with graphene . As reported before31eSshould be largely reduced. In fact, we found that, the experimental defect yield of graphene on copper was much reduced compared to that of SiO2 supported graphene (see 12 ions/cm2).For the ions in Group B, the experimental defect yields were clearly higher than the MD simulation results, as in which only nuclear collisions were considered. The experimental yields for 3 MeV and 6 MeV Si ions were roughly two times higher than the simulated counterparts, and the discrepancy became much larger when 250 MeV Sn ions were used (at least 10 times higher). This is because the electronic energy losses of these ions are so high see  that thehene see  online, 2-supported graphene were further analyzed from the MD simulation results, and compared with the suspended graphene. Taking 3 MeV Si ion irradiation as an example, typical simulation snapshots are shown in 2-supported graphene, whereas, in the suspended graphene, large complex defects (pores) were the majority. Histograms comparing the size distributions of the defects are shown in 8, and molecular adsorptionThe defect patterns in the SiO2-supported graphene by systematic ion irradiation experiments and MD simulations. Our results clearly demonstrated the great importance of the substrate during the defect formation process. The direct nuclear collision process between the impacting ions and graphene carbon atoms can also generate defects, but in much less yield (at least one order of magnitude less) than the indirect process. Simple power-law fittings of the defect yields with respect to the nuclear energy losses were given for both the direct and the indirect processes, which can be, in future, used as a first-hand estimation for choosing the ion irradiation conditions. As for the electronic energy loss of the impacting ions, this portion of energy usually does not generate defects in graphene, unless the electronic energy loss of the ions is above a threshold of 2.2 keV/nm. We further reasoned that this threshold is actually set by the SiO2 substrate, as this value is the turning point for the sputtering yield of bare SiO2 to increase, which again high lights the importance of the substrate effect. In addition, our MD simulation also showed that the small-size point defects are preferentially formed in the supported graphene. Lastly, we believe that, by choosing the right substrate along with the right irradiation conditions, we can create defects in graphene with a highly controllable manner by the ion beam technique.In summary, we have carefully investigated the mechanism of the ion-irradiation induced defect formation in the SiO2 substrate were used in the ion irradiation experiments. The monolayer graphene was grown by CVD on a copper foil, and was then transferred onto a SiO2(300nm)/Si substrate by standard transfer processY) in graphene, i.e., the average number of the defect sites produced by one single incident ion. This defect yield was derived, based on a phenomenological model19D band and the G band (D/IGI) in the Raman spectrum of the irradiated graphene. The G band (~1580 cm−1) is associated with the in-plane stretching motions of the sp2-bonded carbon atoms, whereas the D band (~1345 cm−1) corresponds to the defects in graphene36Samples of monolayer graphene on SiO2 substrate and graphene, were listed in 11 to 3 × 1014 ions/cm2.C, Si ions irradiations were carried out on the 2 × 1.7 MV tandem accelerator at Peking university in Beijing, and Xe ions irradiations were carried out on the 320 kV ECR platform at IMP (Lanzhou). Ion energies, together with their stopping powers in SiO2 slab  was 196.52 × 204.23 Å2, and the thickness was chosen to be around 60 Å. The adaptive intermolecular reactive empirical bond order (AIREBO) potential function2 slab was assumed to be van der Waals (vdW) type, and was modeled by the Lennard-Jones potential with the parameters taken from Ref. For the simulation of the ion-solid interaction process, we used MD simulation to simulate this process in order to carefully investigate the mechanism of the defect formation in graphene under ion irradiation. In our MD simulation, the lateral size of the graphene/SiOW.S.L. did the irradiations and Raman spectrum analysis of all the samples. W.S.L., X.T.Z., and S.J.Z did the MD simulations and defect statistics. W.S.L., X.W.W., H.L.D. and J.M.X. have written the paper. All authors have reviewed the manuscript.Supporting information