Hexagonal boron nitride (hBN) is drawing increasing attention as an insulator and substrate material to develop next generation graphene-based electronic devices. In this paper, we investigate the quantum transport in heterostructures consisting of a few atomic layers thick hBN film sandwiched between graphene nanoribbon electrodes. We show a gate-controllable vertical transistor exhibiting strong negative differential resistance (NDR) effect with multiple resonant peaks, which stay pronounced for various device dimensions. We find two distinct mechanisms that are responsible for NDR, depending on the gate and applied biases, in the same device. The origin of first mechanism is a Fabry-Pérot like interference and that of the second mechanism is an in-plane wave vector matching when the Dirac points of the electrodes align. The hBN layers can induce an asymmetry in the current-voltage characteristics which can be further modulated by an applied bias. We find that the electron-phonon scattering suppresses the first mechanism whereas the second mechanism remains relatively unaffected. We also show that the NDR features are tunable by varying device dimensions. The NDR feature with multiple resonant peaks, combined with ultrafast tunneling speed provides prospect for the graphene-hBN-graphene heterostructure in the high-performance electronics. Graphene, a two-dimensional material with unique mechanical, thermal and electronic transport properties3567891011121314NDR in double barrier resonant tunneling diodes (DB-RTD), appears when the quasi-bound levels can no longer enhance the tunneling resonantly19202122242728313334The NDR features in multilayer based devices are to be investigated by exploring current-voltage characteristics as a function of (i) number of hBN layers, (ii) lateral dimensions in determining both the voltage location of NDR peaks and the peak-to-valley ratio, which are essential in the device design, (iii) the role of the asymmetric band offset between hBN and graphene, and (iv) defects and scattering. This, however, necessitates further research to rationalize the underlying physics of the NDR effect and gain insight on how to control its critical properties mentioned above.In this work, we focus on a prototypical multilayer device shown in Next, section 2 defines our method by discussing the underlying Hamiltonians and the methodology for the computation of quantum transport. Section 3 discusses the results including the two underlying mechanisms with the role of electron-phonon scattering included. Section 4 presents the size scaling analysis followed by section 5 concluding the work.heterostructure/multilayer region stacked in AB order . The lattice constant mismatch between hBN and graphene is negligibly small, 1.8%, therefore, we build the device structure with the uniform lattice constant of graphene (2.46Å) only. The system Hamiltonian is constructed using the nearest neighbor tight binding approximation, with the parameters11pz orbitals are considered here; so that the Hamiltonian has the same dimension as the total number of atoms simulated. The effect of number of tunneling hBN layers (Nz), the system width (Nx) and the length of the multilayer stacking region (Ny), where units of Nx and Ny are number of atoms, on the device performance is investigated. The nanostructure thickness is (Nz+2) in units of atomic layers, which includes the two monolayer graphene sheets at the ends.A prototypical heterostructure consists of two semi-infinitely long monolayer armchair-edged graphene nanoribbon (AGNR) electrodes sandwiching an ultra-thin hBN film, with a vertically applied external gate electric field as shown in U = −eVb, where Vb is the applied bias, and the electrostatic potential of the top graphene layer remains zero. The electrostatic potential at each of the sandwiched hBN layers are determined by linearly increasing/decreasing the potential from top to the bottom graphene layer, because the c-axis (out of plane) conductivity of hBN is orders of magnitude smaller than the in-plane conductivity of graphene. The chemical potential of contacts are controlled by bias voltage, namely μB = −eVb and μT = 0 for bottom and top graphene leads respectively. The gate voltage is modeled by shifting the electrostatic potential at the bottom electrode by ΔU = −0.01 eVg, where Vg is the gate voltage. We choose Nx equal to 3n+2, where n is an arbitrary positive integer37The bias across the heterostructure is applied by rigidly shifting the electrostatic potential of the bottom graphene electrode by the amount equal to the applied bias as the metallic graphene layers have much higher conductivity than hBN. The electrostatic potential energy of the bottom layer is E is the electrons energy, I denotes the identity matrix, H is the system Hamiltonian matrix, and i can be calculated subsequently byWe simulate the transport properties of the device by using the state of the art Green’s function (NEGF) formalismf(E) is the Fermi function and μL/R represent the Fermi level of the left (top) and right (bottom) graphene monolayer electrode. In Nx = 200, Ny = 32, and with three hBN layers Nz = 3, at zero bias. The bandgap of hBN is found to be around 4.72 eV, and the valence band offset between hBN and graphene is around 1.38 eV, which is consistent with the prior workThe transmission coefficient is then calculated using the expression,Traditionally, the recursive Green’s function (RGF) approach is applied to solve for the retarded Green’s function. Here, an algorithm named HSC-extension is developed, which is more efficient and is based on the nested dissection method, so that the requisite large scale systems can be simulatedNx = 62 (7.6 nm), stacking length Ny = 32 (6.8 nm) and three hBN layers (1.4 nm) serving as the tunneling barrier. First we consider the highlighted curve with Vg = 0, in which case two NDR peaks emerge at Vb = 0.3 V and 0.66 V, respectively. We attribute the formation of these multiple NDR peaks to the Fabry-Pérot like interference in the multilayer region (mechanism 1), as rationalized below.g and DOShBN) at Vb = 0.3 V, 0.46 V and 0.66 V, corresponding to the first peak, the first valley and the second peak in the current-voltage characteristics. In the DOSg curves . The transmission and DOSg show a strong Fabry-Pérot like resonant feature in the low energy window. The semi-infinite top and bottom AGNRs couple with hBN at the central heterostructure (multilayer) region. The potential discontinuity caused by the interaction between the hBN cut-ends and the graphene layers create a resonant cavity in the overlapping region at both the top and bottom graphene layer. When electrons transport across the boundaries between graphene monolayer and hBN multilayer regions, partial reflections occur at the interfaces. As a result, the transmission is oscillatory with peaks and valleys corresponding to constructive and destructive interferences.To understand the multiple NDR peaks, we calculate the transmission and the average density of states  at E = −0.3 eV in Vb < 0.18 V), we find that this transmission peak P is enhanced, resulting in the increase of current with applied bias. The resonant tunneling occurs when the constructive quantum interference assists the tunneling of electrons from the top to bottom electrodes at specific energies. When the bias is further increased (until 0.46 V), the transmission peak P is reduced due to destructive interference despite the fact that the energy window for carrying current enlarges. This transmission reduction begins to dominate after Vb = 0.3 V, which induces a drop in current. At Vb = 0.46 V, there is a large suppression of transmission within the bias window, which creates a large tunneling gap, leading to the current valley as reflected in the highlighted curve of Vb = 0.66 V due to the constructive interference.The current is determined from the area enveloped by the transmission curve in the energy window bounded by the Fermi levels of two electrodes, E = −0.3 eV (μT), while a symmetric peak at E = 0 eV (μB) is clearly absent. This is due to the fact that the presence of hBN layers break the symmetry. We could understand this from DOShBN plot at Vb = 0.3 V (hBN curve), which shows a peak near E = −0.3 eV but a valley at E = 0 eV. This means that electrons at E = 0 eV see a stronger barrier when tunneling between AGNR layers, and suggests the breaking of the π−π* symmetry. This argument is tested by considering a symmetric tunnel barrier, where such an asymmetry in transmission does not exist. In T) are observed. This significant tunneling enhancement results from the energy level alignment between the subbands of top and bottom graphene electrodes, as reflected in the corresponding DOSg features.An interesting feature of Vg). Vg ranging from −45 V to + 45 V for two devices with different sizes. Take the current-voltage curve at Vg = −45V as an example; At Vb = 0, the negative gate voltage shifts the energy of Dirac point in the bottom AGNR electrode to U = 0.45 eV at equilibrium, while preserving the chemical potentials from two contacts at μB = μT = 0. At Vb = 0.45 V (see mechanism 2) induces the resonant transmission and results in the large current peaks marked by solid arrows in Vb = −0.01Vg) based on mechanism 2 only. This occurs when the strength of mechanism 2 is comparable to that of mechanism 1, when the voltage at which the peak current occurs is influenced by the Fabry-Pérot like interference. The superposition of mechanisms 1 and 2 leads to the current peak displacement, which is larger at low gate voltage .We next discuss the second mechanism that leads to single intense NDR peak by investigating the operational behavior of the heterostructure in the presence of an external gate voltage  dimensions which defines the overlap area between hBN and graphene layers and the z-direction (number of hBN layers), is performed in this section. However, we do not consider the electron-phonon scattering effects during this analysis as they do not significantly alter the outcomes of the analysis.System dimensions are the key ingredients in engineering the device performance. In this particular multilayer heterostructure, for instance, the device width determines the number of subbands in ANGR electrodes and the heterostructure length determines the length of interference region. Based on the two distinct mechanisms responsible for the multiple NDR peaks, it is intuitive that the device dimensions have significant and non-trivial influence on the NDR features rather than simply tuning the current magnitude by following quantum mechanical rules or Ohm’s law. In order to comprehend such influences, the scaling analysis of the device dimensions, namely the lateral  is varied from 1 layer (0.6 nm) to 5 layers (2 nm), while both Nx and Ny are fixed. The magnitudes of current are scaled by a multiplicative factor to present results on the same plot for different values of Nz. The transmission versus energy , the density of subbands for the monolayer AGNR electrodes and the heterostructure region depends on the graphene nanoribbon width, i.e. the energy intervals between subbands for the structure with Nx = 200 are about three times smaller than that for the structure with Nx = 62. Therefore, a larger number of subbands contribute to current under lower biases, resulting in initial increase in current with Nx, as seen in Vb = 0.45 V for different Nx in μB and μT grows with the width of the AGNR electrode  to 64 (13.6 nm), the number of transmission peaks increase as shown in Vb =  0.38 V, 0.8 V for Ny = 32, which shifts to lower Vb i.e. at 0.2 V, 0.4 V respectively, when Ny = 64. This is because the resonant transmission appears at various energies, which vary inversely proportionally with the length of the interference cavity Ny. Experiments where the overlap between two graphene nanoribbons are altered should be able to reveal the differences in oscillations of I-V characteristics as a function of Ny. We note that experiments with changing overlap dimensions have been performed in carbon nanotubes before44The length of the central multilayer region determines the number of incident carriers, and also characterizes the size of the Fabry-Pérot like interference cavity. For mechanism 1, when the heterostructure length Nz = 1), electrons have high tunneling probabilities and thus the current is mainly limited by the number of modes incident within the energy window. Graphene layer has low DOS near Dirac point, yielding the saturation of peak current at large. It is also observed that for larger Nz , the peak current increases with Ny rapidly without saturation. This is consistent with our previous discussion since a thicker hBN tunneling barrier greatly suppresses the overall tunneling transport probability and therefore the peak current magnitude is a strong function of the number of carriers in graphene.With an ultrathin tunneling barrier  of the top and bottom graphene electrodes align, which leads to a second mechanism for resonant tunneling. Both mechanisms respond to gate voltage distinctly. Gate voltage only controls the locations of NDR peaks from mechanism 1 while can tune both PVR and locations of NDR peaks due to mechanism 2. In the presence of electron-phonon scattering decoherence, mechanism 1 is more intensively suppressed compared to mechanism 2, which may be relevant to the disappearance of mechanism 1 in the experiments.Nx), the multiple current peaks preserve but with decreasing PVR values for both mechanisms. (3) For mechanism 1, the bias voltages at which multiple current peaks, occur depend on the length, and the number of peaks increase with length. In contrast to this, the location of the single peak from mechanism 2 is independent of the length. We believe that the negative differential resistance’s sensitivity to the system dimensions will provide additional insights for future theoretical and experimental investigations.Size scaling analysis provides insight into the device physics that determines the number of NDR peaks, the variation of peak current and PVR value with change in device dimensions: (1) The hBN thickness exponentially controls the magnitude of current without significantly affecting the NDR features. (2) For devices with larger widths .


The growth scheme offers metal-free synthesis at low temperature, grain-size customization, large-area uniformity of electrical properties, single-step preparation of graphene/dielectric structures, and readily detachable graphene. We quantify based on thermodynamic principles the activation energies associated with graphene nucleation/growth on γ-Al2O3, verifying the low physical and chemical barriers. Importantly, we derive a universal equation governing the adsorption-based synthesis of graphene over a wide range of temperatures in both catalytic and spontaneous growth regimes. Experimental results support the equation, highlighting the catalytic function of γ-Al2O3 at low temperatures. The synthesized graphene is manually incorporated as a ‘graphene sticker’ into an ultrafast mode-locked laser.The possibilities offered by catalytic γ-Al Owing to its excellent electrical and optical characteristics, graphene has attracted much research attention over the past decade122141517182 substrate.) Recently, the use of catalytic MgO202Transfer-free processes have drawn particular interest because the transfer of large graphene sample synthesized on a metal catalyst generally leads to contamination or the formation of defects8910122O3 catalyst as a novel substrate for the metal-free direct growth of graphene layers at low temperatures. We elucidate the formation mechanism of graphene on γ-Al2O3 with both numerical simulations and experiments, with concordant results obtained. We derive an equation applicable universally for the adsorption-based synthesis of graphene over a wide range of temperatures, which covers both low-temperature catalytic growth and high-temperature spontaneous growth on diverse substrates, including metal catalysts. We show herein that this equation accurately reproduces experimental results. To verify the successful formation of graphene crystals2In this report, we explore γ-Al2O3 substrates is advantageous in a number of regards over conventionally prepared graphene; these include (i) the low-temperature synthesis afforded by γ-Al2O3 catalysis, (ii) the excellent and very uniform electrical properties provided by the ultrahigh-quality crystals that form because of the negligible activation energies associated with carbon diffusion on the substrate and with the surface-regulated crystal nucleation and growth, (iii) the control offered over the thickness of the graphene layer and of the grain size therein—from nano- to micrograins, which is closely related to band gap opening, (iv) the fact that the formation mechanism is demonstrated here both numerically and experimentally, and (v) the ready detachability of the graphene produced and its very low adsorption energy with respect to the substrate, which guarantee the complete transfer of intact layers. Furthermore, the graphene grown can be used with the γ-Al2O3 substrate—an ideal gate dielectric with a high dielectric constant (κ) of 7.920220−4A·cm−2)25Graphene synthesized on γ-Al2O3 from the additional sacrificial layer deposited on the arbitrary substrate to form a graphene/high-κ dielectric. The graphene can be submitted to additional treatments with the assistance of supporting substructures to obtain ultrathin, transparent sheets and a highly bendable Al2O3 substrate. In particular, graphene stickers, as shown in 2O3 as a substrate for growing graphene, we analyzed the growth mechanism theoretically and validated the resulting equation experimentally. An initially amorphous Al2O3 layer was prepared by atomic layer deposition (ALD). This spontaneously transforms into γ-phase during the first stage of graphene growth, and this phase is retained as the substrate is recycled after growing and detaching the graphene. (The specific conditions used for these steps are described in the methods section)To evaluate the versatility of γ-Al2O3 substrate formed was used for the growth of graphene using CVD at different CH4 partial pressures (pCH4), growth temperatures (T), and durations (t), as shown in The γ-Al2O3 substrate and the growth characteristics of graphene, graphene layers were grown at temperatures ranging from 600 °C to 1050 °C. Regardless of the activity of the substrate, graphene always grows under CVD conditions at temperatures above 950 °C via the spontaneous dehydrogenation of CH42O3 can therefore be proved experimentally by demonstrating graphene growth at temperatures below 950 °C. According to previous reports284 is only adsorbed at tricoordinated AlIII sites on the surface of γ-Al2O3. Furthermore, these sites show catalytic activity for dehydrogenation at temperatures as low as 100 °C282O3 substrates. This scheme contains a number of activation barriers with many factors, which are described below.To confirm the catalytic activity of the γ-AlIII sites by dissociative adsorption of a CH4 molecule. The overall activation energy barrier from the CH4 molecule to the generation of a C adatom involves the dissociative adsorption energy, Ead. The generated adatom then diffuses and becomes anchored onto sites that are more or less favorable according to their associated surface diffusion barrier, Ed. Additional C adatoms may come into contact with the anchored C adatom to form linear and ring shaped carbon clusters, as described by the attachment barrier, Eatt. Alternatively, the C adatom may desorb from the γ-Al2O3 substrate immediately after its generation or as it diffuses across the surface according to the desorption energy, Edes. The activation energy for the detachment of a C atom from the perimeter of the graphene nucleus is the detachment barrier, Edet.A carbon adatom can be generated at Ala) via Arrhenius plots, as shown in a includes all contributions from Ead, Ed, Eatt, and Edes, and the complex relationship between these factors. Measuring Ea provides an experimental parameter against which to validate our theoretical model, and provides insights into the formation and growth of the graphene nuclei.After growing graphene at various temperatures, we measured the overall activation barrier for growth  reveals two distinct growth regimes. The gentle slope in c = 0.21 eV, represents Ea in the catalytic growth regime, while the steeper slope reflects the non-catalytic regime with Es = 2.10 eV, in which growth occurs through site independent spontaneous dehydrogenation of CH4. The specific procedure used to obtain these values is described in To sketch out the overall growth of graphene nuclei on the γ-Alc and Es should be given by Equations Based on these theoretical considerations, Ead = 0.88 eV, Eatt = 0.21 eV, Ed = ~0 eV, and ln(q0/N0) = 1.87 eV, for which N0 = 26 / 0.76 nm−2 and q0 = 4 / 0.76 nm−2 represent the total density of adsorption sites respectively on the surface of the substrate and for CH4 on γ-Al2O3; i.e., there are 26 atoms in total and 4 AlIII atoms on each γ-Al2O3 unit cell (with an area of 0.76 nm2)In the above equation, Ec ~ 0.22 eV and Es ~ 2.09 eV, as shown in c = 0.21 eV and Es = 2.10 eV) shown in Inserting these values into the above equations leads to E2O3, Al4C3 forms very slowly and only above 1600 °C, while Al2OC, which is the first metastable product in the formation of Al4C3, forms only at temperatures above 1427 °C2O3 is therefore negligible under the CVD conditions employed here, such that the formation and growth of graphene nuclei on γ-Al2O3 should follow a surface adsorption route similar to that which occurs on Cu. Two-dimensional crystal growth is similar to the nucleation and enlargement of crystals synthesized by physical deposition. We therefore adapted the model for the latter, derived by Robinson and RobinsRuoff and coworkers have shown that graphene synthesis on transition metals involves surface adsorption on Cu but surface segregation or precipitation on Ni due to the differing solubility of C in the two 31des and Ed, at a given growth temperature. The saturation densities in the high-temperature and catalytic growth regimes are derived from the Equation In the above equation, η is used as a determinant for the growth regime, β is an undetermined dimensionless coefficient, C is the number of effective pair-formation sites neighboring each adatom, and the other factors are described in the SI. The determinant is highly dependent on the validity of the values used for the two activation barriers, Ea in the high temperature growth regime does not match the one measured experimentally. We therefore obtained a rough estimate of the transition temperature (Tc) from the intersection of the lines corresponding to Equations c ≈ 4800 K  is given by the following equation,s) are defined as follows:and the activation barriers in the steeper regime governed by spontaneous dehydrogenation  model37d ≈ 0 eV). This contrasts with the values obtained for Ag and Ni,  but is comparable to the barrier on Cu (0.07 eV)a site (C-C-Oa) is the most stable reaction, with a relative energy of 6.06 eV per C atom. No surface diffusion barriers were evidenced in this process, and since the formation of a bridging-metal linear Cn-Oa structure (n is the number of C atoms) is similar to the formation of graphene nuclei on α-Al2O3d for the latter is negligible.To obtain Eatt. A value of 0.21 eV was obtained using a simple model in which the growth of graphene islands is governed by the edge controlled kinetics of the attachment and detachment of C adatoms, as described on page S26 in the The enlargement of graphene nuclei proceeds according to Ed and C solubility, Ni appears at first glance to be the most favorable substrate. However, these two terms can lead to the growth of graphene on Ni substrates both by segregation and precipitation.ad, while as mentioned above, Eatt governs the enlargement of the graphene nuclei. Therefore, these two activation barriers determine the lowest possible growth temperature. For the theoretical calculation of Ec, Kim et al.att of 2 eV for Cu, based on growth experiments performed on Ruatt att of 0.2 eV yields Ec = 1.1 eV, demonstrating that this equation, originally derived for metal oxides, is also valid for metal catalysts. One also notes that the DFT-calculated value for Eatt  is similar to the one measured here (0.21 eV). In this context, Ead has a significant impact on the catalytic performance of the substrates. Since Eads is similar for γ-Al2O3 and Ni, the growth temperature of graphene on γ-Al2O3 should be comparable to that on Nid determines the homogeneity of the graphene layer grown on the substrate, and since Ed is the lowest in γ-Al2O3, this should be the most favorable of these three substrates for the formation of homogeneous graphene. A further discussion of C solubilities and a quantitative comparison of the different substrates may be found on page S28 of the The supply of C adatoms is governed by EIG/ISi), and of the 2D peak to the G peak (I2D/IG), as a function of the growth temperature. The latter ratio reports on the number of graphene layers formed, with a values of ~0.5 at an excitation wavelength of 532 nm indicating the presence of bilayers. IG/ISi reports on the average area of the graphene grains. The variation of this intensity ratio with the growth temperature follows two distinct slopes in Atomic force microscopy (AFM) images of graphene synthesized for 1 h at 600 °C, 800 °C, and 1050 °C, are respectively shown in 2O3 substrate and evaluate the quality of the graphene. High-resolution XPS profiles were acquired in the C 1s We also analyzed our graphene samples by using X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) to identify the crystalline phase of the AlC 1s see  regions s) of the graphene samples as a function of the H2 flow rate used during CVD (under a constant 850 sccm flow of CH4). The linear increase of Rs with the H2 flow observed here can be expressed by the following equation.2 is the flow from the mass flow controller (sccm).H2 flow rate was also investigated by Raman spectroscopy, by looking at its influence on the ID/IG ratio , a strong argument in favor of the use of γ-Al2O3 as a growth substrate. It is noteworthy moreover that the Rs standard deviation is less than 1% in all the samples prepared with H2 flows up to 200 sccm  and n is the refractive index of the Al2O3 substrate (1.77 at 550 nm). According to this equation, a single layer of graphene grown on Al2O3 absorbs ~1.66% of the incident light. As shown in opt at 550 nm is 93.8%, which matches the value (93.76%) predicted using Equation 2O3/Quartz wafer/Al2O3 substratein which ZAs a practical application of our readily detachable graphene layers, we incorporated one of these into an ultrafast mode-locked laser22O3 as a substrate for the highly efficient formation of graphene has been explored and the entire formation mechanism has been elucidated using both numerical simulations and experiments. This synthesis route is effective at low temperatures and without metal, and allows grain size customization and the preparation in a single step of graphene/dielectric layer structures. Moreover, the graphene produced in this way is uniform over large areas in terms of its electrical properties and is readily detachable.In summary, the use of catalytic γ-Al2O3 substrate ensure the synthesis of high quality graphene. Finally, we derived a universal equation, which describes the adsorption-based synthesis of graphene over a wide temperature range  and on a number of different substrates, including metal catalysts. The experimental results obtained in this study support the proposed equation.We evaluated theoretically the activation energies associated with all the stages of graphene nucleation and growth, confirming that the low energy barriers for the surface diffusion of C adatoms and for graphene nucleation/growth on the γ-AlTo demonstrate the potential practical applications of this synthesis route, we manually transferred one of the ‘graphene stickers’ prepared in this way to fabricate an ultrafast mode-locked laser.2O3 substrates (50 nm thick) were synthesized via CVD for the growth of graphene on amorphous ALD Al2O3 substrate with 850 sccm of CH4 and 50 sccm of H2 at 1050 °C. In order to get the γ-Al2O3 substrate, the graphene grown was detached by using a polyimide filmThe γ-Al4 and 50 sccm of H2, with different growth temperatures and durations.Except for the optimization of the gas flow rates, all the graphene samples were synthesized using 850 sccm of CH2O3 (50 nm thick) was synthesized via ALD onto a quartz wafer coated on its backside with a polymer film. The backside was also coated with Al2O3 using the same method. Graphene was then synthesized on both sides of the Al2O3/wafer quartz/Al2O3 substrate for 15 min at 1050 °C.Al2O3 substrates. A D-shaped optical fiber was fabricated by polishing one side of a conventional single-mode fiber (SMF) fixed with epoxy on a slide glassGraphene stickers were prepared by detaching spin-coated polyimide together with the graphene from γ-Al−1. The XPS profiles were acquired using a PHI 5000 VersaProbe (Ulvac-PHI) system at a base pressure of 6.7 × 10−8 Pa using a monochromated Al Ka (1486.6 eV) anode  with a spot size of 100 μm × 100 μm. The transmittance data were recorded on a Cary 5000 (Varian) spectrometer from 175 nm to 3300 nm at a scan rate of 600 nm·min−1 at a resolution of 1.0 nm. The optical spectrum, its characteristics, and those of the pulse waveform, were respectively measured using an autocorrelator , an optical spectrum analyzer , and an oscilloscope .Atomic force micrographs were acquired in non-contact mode with a ppp-NCHR 5M probe (Nanosensors) attached to a XE-100 microscope (Park Systems). The Raman spectra were acquired using a Renishaw In-Via system with a 532 nm excitation laser. The XRD profiles were obtained using a Dmax2500/PC (Rigaku) spectrometer operated at 40 kV, 200 mA, and 8 kW using a Cu target (1.5406 Å) at a scan rate of 2° min2O3 was carried out following the method proposed by Digne, et al.et al.2O3 slab with eight layers was reconstructed after having been cut to a size of Calculations were performed using the Vienna ab initio Simulation Package2O3 surface was defined as follows.The adsorption energy of a C atom on the AlThe energy of one C atom was calculated in a 20 Å × 20 Å × 20 Å vacuum space. A CI-NEB calculation was performed to calculate the migration barrier energy of the attached carbon37III, Oa, Ob, and Oc sites. The AlIII site, which has three O neighbors, is reported to act as a catalytic site for the dissociation of CH428a, Ob, and Oc sites are defined as O atoms having bonds with AlIII and AlV, with AlIII and left-AlIV (AlIV (l)), and with AlIII and right-AlIV (AlIV (r)), respectively.C atom adsorption was tested on top of the AlHow to cite this article: Park, J. et al. Growth, Quantitative Growth Analysis, and Applications of Graphene on γ-Al2O3 catalyst. Sci. Rep. 5, 11839; doi: 10.1038/srep11839 (2015).


Ralstonia solanacearum in microcosms and in tomato plant rhizosphere. We find that bipartite resource competition networks are better predictors of invasion resistance compared with resident community diversity. Specifically, communities with a combination of stabilizing configurations (low nestedness and high connectance), and a clear niche overlap with the pathogen, reduce pathogen invasion success, constrain pathogen growth within invaded communities and have lower levels of diseased plants in greenhouse experiments. Bacterial resource competition network characteristics can thus be important in explaining positive diversity–invasion resistance relationships in bacterial rhizosphere communities.Host-associated bacterial communities can function as an important line of defence against pathogens in animals and plants. Empirical evidence and theoretical predictions suggest that species-rich communities are more resistant to pathogen invasions. Yet, the underlying mechanisms are unclear. Here, we experimentally test how the underlying resource competition networks of resident bacterial communities affect invasion resistance to the plant pathogen  Species-rich communities are thought to be more resistant to pathogen invasion. Here, Wei et al. show that the resource competition network architecture of root-associated bacterial communities are better predictors of plant pathogen invasion resistance than measures of community diversity. Bacterial communities cover virtually all host surfaces from skin, gut and mucosa of animals1567989913141518High diversity might increase community invasion resistance due to interactive effects on community stability2223222627Ralstonia solanacearum, the causative agent of bacterial wilt capable of infecting over 200 different plant species across 50 different familiesR. solanacearum pathogen must first invade the resident bacterial communities and reach a threshold density to become virulentHere we directly examined the distribution of trophic links as underlying mechanisms to predict invasion resistance of plant root-associated bacterial communities against an invading pathogen and the subsequent reduction of disease incidence. We focused on the important soil-borne bacterial pathogen Ralstonia spp. speciesResident bacterial communities were constructed by using five, phylogenetically related but non-virulent, Three different network variables were used to describe (1) similarity in resource consumption between the resident community and the invader (niche overlap) and (2) the intensity of resource competition within resident communities (connectance and nestedness). In our study, niche overlap describes the ecological similarity between the invader and the resident community respective to their resource use. High niche overlap between the resident community and the invader is thus likely to be a decisive factor affecting invasion success3422To test these hypotheses, we conducted replicated invasion experiments in laboratory microcosms (in 96-well microtiter plates) and in the tomato plant soil (rhizosphere) to link resident community composition, invasion resistance and spread of bacterial wilt plant disease. Our results show that bacterial resource competition networks are better at predicting invasion resistance than community diversity. Specifically, resident communities with stabilizing configurations (low nestedness and high connectance), and a clear niche overlap with the pathogen, reduce pathogen invasion success very efficiently leading to lowest levels of bacterial wilt plant disease. Bacterial resource competition network characteristics can thus be important in explaining positive diversity–invasion resistance relationships in bacterial rhizosphere communities.R2=0.11, the effect of niche overlap non-significant, R2=0.46, Increasing resident community diversity reduced pathogen invasion success, defined as the portability of successful invasion . At the Together these results suggest that the characteristics of bacterial resource competition networks could partially explain why more diverse communities were often resistant to pathogen invasions: while nestedness and connectance were important for predicting pathogen invasion probability, high niche overlap was the best predictor for subsequent pathogen growth within successfully invaded resident communities.Ralstonia infection, which correlates with pathogen density in the rhizosphere and thus with invasion successR2=0.43, R2=0.75, R2=0.73, R2=0.75, The proportion of wilted plants  to describe the directed dependencies among diversity and different network variables. We first concentrated on pathogen invasion success in microtitre plate invasions assays. Only high community connectance had direct negative effect on pathogen invasion success . The effIn conclusion, network variables were better predictors of resident community invasion resistance in microcosm experiments compared with diversity. However, this pattern was altered in the rhizosphere invasion experiments, where the mix of both indirect and direct effects of diversity and network variables best explained the pathogen invasion success and subsequent spread of tomato bacterial wilt disease.R. solanacearum. Biodiversity of microbial communities is increasingly recognized to constrain invasion by pathogens9Here we show that simple bipartite resource competition networksWe hypothesized that pathogen invasion success should decrease with increasing niche overlap between the resident communities and invading pathogen due to intensified resource competition. While niche overlap had no effect on pathogen invasion probability in microcosm experiments, it constrained subsequent pathogen multiplication in successfully invaded resident communities. This result is in line with previous studies reporting that high catabolic similarity between the rhizosphere community and the invading pathogen reduces the community invasibilityHigh connectance within rhizosphere communities could decrease pathogen invasion success if it leads to generally more efficient consumption of resources, and hence increased competition91137R. solanacearum densities in rhizosphereLastly, we predicted that low nestedness could increase resident community invasion resistance due to less intense resource competition among generalist and specialist members of resident communities. We found that highly nested resident communities were more susceptible to pathogen invasion and subsequent spread of bacterial wilt plant disease in the tomato plant rhizosphere. Nested resource competition patterns could destabilize rhizosphere communities because specialist species need to compete for a subset of the resources used by generalists2542Interestingly, diversity had direct positive effects on rhizosphere community invasion resistance in the tomato plant soil, which was not explained by resource competition network characteristics. There are multiple non-exclusive explanations for this. First, it is likely that some other factors in addition to resource competition patterns were important for resident community and pathogen fitness in the soil. For example, differences in species ability to colonize plant roots could have played significant role for the establishment of protective rhizosphere communitiesRalstonia species that were closely related with the pathogen. As a result, resident community bacterial diversity could have different effect on invasion resistance depending on the genetic distance between the resident bacterial community and the invading pathogen. Further, our model focused only on resource-mediated competition. In a natural system, increasing bacterial diversity could also increase the strength of antagonistic interactions, which could potentially make communities more susceptible to pathogen invasionR. solanacearum pathogen can rapidly evolve respective to its resource consumption patterns53Albeit being effective in demonstrating causal relationships between resident community interaction network structure, invasion resistance and subsequent disease spread in the soil, our simplified approach has some caveats that might lead to different invasion resistance patterns in more complex natural systems. Firstly, our diversity manipulation included only Soil-borne plant pathogens remain difficult to control because their populations are variable in time and space, and because they can often overcome plant resistance via evolution5R. solanacearum QL-Rs1115 (GenBank accession GU390462) tagged with the pYC12-mCherry plasmid as an invading bacterial pathogenR. solanacearum is a soil-borne, Gram-negative bacterium that invades plant roots and multiplies in the cortical tissue before invading the xylemWe used Ralstonia spp. strains to construct model resident bacterial communities that were exposed to pathogen invasion. All resident community bacterial strains were isolated from the tomato rhizosphere and first grown on South Africa semi selective medium (SMSA-E)Ralstonia colonies were identified on the basis of 16S rRNA gene sequence  of 0.1 and grown in microtitre plates in 150 μl OS minimal medium600>0.05 were scored as positive growth on given substrate. We then used these single-species and one-resource interactions to determine resource consumption patterns (1) between the different members of resident communities and (2) between different resident communities and the invading pathogen. We first defined niche overlap between resident communities and the invading pathogen as the proportion of resources used by the pathogen and by at least one member of the given resident community2627C=L/S2, where L denotes for links between species  and S denotes for species richness of the community. Nestedness describes the trophic interactions between generalist and specialist resource consumers. In highly nested networks, generalist species have many links, which are shared by specialist species that have only few links. Mathematically, nestedness was estimated by the system's ‘temperature' via binary matrixes in three steps by computing first an isocline of perfect order, reorganizing the matrix by permuting rows and columns in the way that maximizes its nestedness and comparing its normalized distance to the isoclineWe used bacterial resource consumption patterns to determine indirect resource competition networks (1) between different members of resident communities and (2) between different resident communities and the invading pathogen. To this end, we first characterized the resource consumption pattern of each bacterial strain individually in single-species monocultures on 48 different single carbon resources representative of tomato root exudates. Briefly, overnight cultures were adjusted to an optical density OD0 of 0.1 −1, peptone 5.0 g l−1, yeast extract 0.5 g l−1, beef extract 3.0 g l−1, pH 7.0) on a shaker at 170 r.p.m., 30 °C for 12 h, after which 31 resident communities were constructed with a starting density of 107 cells per ml. All resident communities were inoculated to 20 different resources environments , each of which were composed of OS minimal medium supplemented with a random mixture of 20 carbons selected from the total pool of 48 resources (R. solanacearum QL-Rs1115 strain (105 cells per ml) or grown alone in the absence of pathogen in 96 wells microtitre plates (200 μl per well) at 30 °C with agitation. Resident community growth was recorded after 48 h on the basis of optical density (OD600) and pathogen growth on the basis of the mCherry fluorescence signal . The invasion process was broken into two steps. First, the probability of successful invasion was measured as a binary variable for each resident community resource environment combination: invasion was scored as successful if the fluorescence signal was higher, and unsuccessful if it was lower, than the background signal of the corresponding control treatment. Second, we quantified the pathogen invasion success within successfully invaded resident communities as pathogen density based on the level of fluorescence signal  were germinated on water-agar plates for three days before sowing into seedling plates containing Cobalt -60-sterilized seedling substrate . Tomato plants at the three-leaf stage (11 days after sowing) were transplanted to seedling trays (8 CFU per g soil)6 CFU per g soil). Tomato plants were grown for 42 days after transplantation in a greenhouse with natural temperature variation ranging from 25 to 35 °C and watered with sterile water regularly. Note that the plants from individual single seedling plates were not considered as independent replicates, but instead the number of wilted plants per seedling plate was used as a disease index. Seedling plates were arranged in randomized order and rearranged randomly every 2 days. The number of wilted plants per seedling plate was recorded at daily basis after 17th day post transplantation (no symptoms were observed before day 17). As we were unable to quantify available carbon resources in the rhizosphere directly, we made the assumption that all 48 resources used to characterize bacterial resource consumption patterns were available in the rhizosphere.The spread of periment . As diseng trays  containi−1) and the asymptotic disease prevalence (% of wilted plants); later referred to as early, intermediate and late stages of infection, respectively. Curves were fitted individually for each tray—an approach very similar to survival analysis—and the resulting variables used as dependent variables in independent GLMs, weighted by –log10(SE) of the parameter estimates to account for the quality of the curve fitting. Due to potential correlation between connectance and nestedness, sequential analysis was used to uncover the most parsimonious GLM's. To this end, we used stepwise model selection using the Akaike information criteria (AIC) to select the model with best explanatory power (step function in R). We used both a backward elimination  and forward-selection model (from simple to more complex) to avoid selecting a local AIC minimumPathogen invasion pattern in microcosm experiment was analysed with GLM assuming binomial data distribution for invasion success analysis and lognormal data distribution for invader density increase analysis. For greenhouse invasions experiments, disease spread over time was assessed as a logistic growth curve spanning the percentage of wilted plants as a function of time during whole duration of the experiment. The curves were fitted individually for each tray, in an approach very similar to mortality curves. This fit produced three variables: the lag phase (days after pathogen addition) before disease onset, the intrinsic increase of wilted plants .Supplementary Figure 1, Supplementary Table 1-4 and Supplementary References


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.