A high-quality monolayer graphene with 97% transparency and 350 Ω/□ sheet resistance grown by atmospheric pressure chemical vapor deposition method was transferred onto planar Si cells. An increase in efficiency from 5.38% to 7.85% was observed upon deposition of graphene onto Si cells, which further increases to 8.94% upon SiO2 deposition onto the graphene/Si structure. A large increase in photon conversion efficiency as a result of graphene deposition shows that the electronic interaction and the presence of an electric field at the graphene/Si interface together play an important role in this improvement and additionally lead to a reduction in series resistance due to the conducting nature of graphene.This work presents an experimental and finite difference time domain (FDTD) simulation-based study on the application of graphene as a transparent conducting layer on a planar and untextured crystalline  Graphene has been considered as one of the promising materials for photovoltaic device applications due to its two-dimensional nature with extraordinary optical (transmittance ~98%), electronic , and mechanical properties (Young's modulus 1.0 TPa) . Many atIn silicon solar cell, the power conversion efficiency is limited by many fundamental losses such as incomplete absorption of the solar spectrum, recombination of the photo-generated charge carriers, shading losses, and series resistance losses ,17. AntiIn this paper, we investigate the transparent conducting and surface field properties of graphene layers onto planar and untextured crystalline Si surface by carrying out experimental investigations and finite difference time domain (FDTD) calculations. In addition, the effect of graphene layer on the photovoltaic parameters and spectral responses of planar and untextured Si solar cell has also been investigated.2 (30 sccm) was then introduced into the reaction tube for degassing the air inside. The flow rate of Ar was kept constant (500 sccm) for all the experiments mentioned in this manuscript. The reactor was heated up to 1,030°C in 30 min, and this temperature was kept constant for the next 30 min to anneal the Cu foil. Then, CH4 (3 sccm) was fed into the reactor. After 30 min, the feeding of CH4 was cut off and the reactor was cooled down to room temperature naturally in an Ar and H2 environment. The flow of all the gases was stopped as the temperature reached close to the room temperature.The growth of graphene has been carried out on a 25-μm-thick Cu foil  using an atmospheric pressure chemical vapor deposition (APCVD) system at a temperature of 1,030°C. A split-type furnace with a quartz tube reactor was used for graphene growth. Before loading into the reaction tube, the Cu foil was cleaned in acetic acid followed by acetone, deionized water, and isopropyl alcohol to remove the copper oxide present at the surface. A mixture of Ar (500 sccm) and HMW ~996,000, item no. 182265, 10 mg/ml in anisole) was used for the transfer of graphene onto different substrates like quartz, Si, SiO2-sputtered Si, and solar cells to study graphene quality and its electronic and optical properties. In the first step, the graphene-deposited Cu foil was attached to a glass slide with the help of a scotch tape and then PMMA was spin coated on one side of the Cu foil. The other side of the foil was immersed into 10% HNO3 solution for 2 min to etch out the graphene from that side. Subsequently, the Cu foil was etched using FeCl3 (10% wt./vol.) for 3–4 h. The PMMA coated graphene film was transferred to the desired substrate  on several dips in deionized (DI) water as a cleaning step. In the final step, PMMA was etched out using acetone at 80°C for a duration of 2 h. Some residual PMMA was further removed by annealing in a H2 (500 sccm) and Ar (500 sccm) environment at a temperature of 450°C for 2 h.On successful growth of graphene on Cu foil, polymethyl methacrylate (PMMA)  liquid dopant was used, and the wafers were subjected to elevated temperature in a furnace resulting in the formation of a thin layer of n-doped region (~0.5 μm). The wafers were etched using freon-oxygen (CF4) gas mixture in dry plasma etch machine to remove the junction regions created on the edge. These wafers were then chemically etched to remove the oxides and phosphorous glass formed on their surfaces. The entire backside was metallized with Ag-Al paste. Front contacts on the wafer surface were formed by screen printing the required pattern with a suitable metallic paste on them. The metal paste was dried and sintered in an infrared sintering belt furnace where temperature and belt speed were optimized to achieve a sharp temperature profile. The printed cells were then cut into smaller cells of dimension 10 mm × 10 mm for deposition of graphene. A similar printed cell is kept for comparative studies.In order to study the effect of graphene on photon absorption and carrier collection, we first fabricated Si solar cells with planar and untextured surfaces. A 156-mm monocrystalline silicon wafer was dipped in high-concentration alkali solution at 80°C for 1 to 2 min to remove the roughness of the wafer. A 2 layer was deposited over mc-Si solar cell after the deposition of graphene layer using radio frequency (RF)-magnetron sputtering from a Si target (<111>) with 99.99% purity. The sputtering was carried out for 22 min by introducing Ar (15.8 sccm) and O2 (2.8 sccm) gases at room temperature with an applied RF power of 100 W.A 100-nm-thin film of SiO+ laser for excitation. The scattered light was analyzed with a Renishaw spectrometer having a charged couple device for detection. All the optical measurements were carried out on a Lambda 35 UV/Vis spectrophotometer . The photovoltaic characterization of the solar cell was carried out by measuring the I-V behavior using a 2400 SourceMeter  under simulated AM 1.5 solar illumination at 100 mW/cm2 from a xenon arc lamp in ambient atmosphere.Raman spectroscopic measurements were carried out in backscattering geometry using the 514.5-nm line of Ar4/H2 flow rate. The quality of graphene was analyzed by Raman spectroscopy of the as-deposited graphene on the Cu foil. It is well known that graphene has three most prominent Raman features at ~1,350 cm-1 (D band), ~1,580 cm-1 (G band), and ~2,700 cm-1 (2D band). The D peak is related to the presence of defects  in graphene. The G peak denotes the symmetry-allowed graphite band corresponding to the in-plane vibration of sp2-hybridized carbon atoms, which constitute the graphene sheets. The 2D peak originates from the second-order double resonant Raman scattering from the zone boundary. It is quite established that Raman scattering can be used as a fingerprint for the quality and number of graphene layers. The ratio of the intensity of 2D and G peaks (I2D/IG) and full width at half maximum (FWHM) of the 2D peak are important parameters to evaluate the quality of graphene [-1 and the G peak at 1,580 cm-1, are observed. It is worth noting that the defect-related D  peak decreases with increase in temperature and finally disappears at a temperature of 1,030°C, indicating the improved quality of graphene deposited at higher temperatures. The improved quality of graphene is also confirmed by the I2D/IG ratio and FWHM (2D) plots in Figure I2D/IG ratio increases and FWHM (2D) decreases with increase in temperature.The APCVD conditions have been optimized to synthesize a single-layer graphene by tailoring the growth temperature and CHgraphene ,27. Figu4/H2 flow rate for growing good-quality single-layer graphene, five flow rates of CH4/H2 content were chosen, i.e., 01/10, 03/30, 05/50, 10/100, and 20/200 sccm, while keeping the CH4:H2 flow rate ratio (1:10) constant. The growth temperature was set at the optimized value of 1,030°C with a deposition time of 30 min to ensure complete coverage of graphene. Raman spectra of graphene samples grown at different CH4/H2 flow rates are shown in Figure I2D/IG ratio and FWHM data are shown in Figure -1) and large and symmetrical graphene G  and 2D  peaks. The D peak is negligible in all the cases, indicating a defect-free graphene growth. Furthermore, the FWHM of the 2D peak increases gradually from 30 to 65 cm-2  and FWHM (≈32 cm-1) in graphene grown at 1,030°C and 03/30-sccm CH4/H2 flow rate match well with the previously reported values for monolayer graphene [4 and 30 sccm of H2 flow rates was used for investigating the effect of graphene and G/SiO2 layers on Si solar cell as a transparent conducting and antireflection layer.In order to optimize the CHgraphene ,28-30. B2) graphene transferred onto a SiO2 (300 nm thick)/Si substrate. In order to measure the transmittance values, graphene layer was transferred to a quartz substrate and an average value of transmittance of 97% -coated Si substrate. A comparison of sheet resistance and transmittance of graphene layer used in studies involving G/Si cells is given in Table 3N4 antireflection layer along with a textured surface. It is difficult to deposit/transfer graphene layer on a textured surface. In order to study the transparent conducting properties of graphene layer, it is necessary to remove the Si3N4 layer and texturing of these cells. Therefore, the silicon solar cells with these properties, i.e., with planar Si surface, were fabricated for carrying out these experiments. The procedure followed for fabricating Si (p-n) solar cells with planar top surface (without texturing and AR coating) is described in the ‘Methods’ section. For applying graphene as a transparent conducting and surface field layer on Si solar cells, we chose SiO2 as the antireflection layer. Experimental and simulation studies were performed on the planar Si solar cell to investigate the reflectance properties of monolayer graphene on Si surface. Subsequently, the thickness of SiO2 layer as an antireflection coating for G/Si solar cell was optimized. It was observed that a 100-nm-thick SiO2 layer was sufficient to work as an antireflection layer over the graphene-Si interface. SiO2 (refractive index 1.45) was chosen due to its well-known antireflection properties [Figure operties .2/G overlayers with 100 nm SiO2 thickness were then applied onto the fabricated crystalline Si solar cell having a planar and untextured Si surface  a bare Si solar cell having a planar surface, (ii) graphene on the planar Si solar cell (G/Si), and (iii) 100-nm-thick SiO2 coating on graphene/Si solar cell (SiO2/G/Si). The solar cell performance parameters of open circuit voltage (VOC), short circuit current density (JSC), maximum voltage (VM), maximum current (IM), series resistance (RS), shunt resistance (RSH), fill factor (FF), and the energy conversion efficiency (Eff.) are shown in Table VOC by 20 mV and in JSC by 10.5 mA/cm2. It is important to note that the graphene overlayer on planar Si solar cell (G/Si) has higher conversion efficiency (7.85%) in comparison to the bare Si cell (5.38%) without graphene layer. This conversion efficiency is further increased to 8.94% on introduction of the antireflection SiO2 layer. Improvement in the I-V properties on deposition of graphene onto Si cells may be due to changes in the reflection properties of planar Si cells, improvements in the photovoltaic parameters due to transparent conducting properties of graphene, and the formation of surface electric field layer at the G/Si surface. To separate theses effects, reflectance and junction properties of the G/Si junctions were evaluated.Graphene and SiOe Figure a to expeFigure I-V behavior of graphene/Si (G/n-Si) structure was obtained to study the nature of G/n-Si junction. Figure I-V characteristics of the G/n-Si in dark and light. The forward bias condition was observed with graphene connected to the negative terminal with respect to n-Si. This shows that the interface between the graphene and n-Si behaves like a n+-n junction. The favorable direction of the electric field formed at the interface helps in the reduction of the effective recombination at the front surface and enhances the collection of light-generated free carriers and thus improves the efficiency of solar cell. The n-type or p-type nature of graphene is very sensitive to the synthesis method, adsorbed molecules, nature of the substrate underneath, etc. [n-type) in G/Si cells in the present study acts like an n-type layer.The th, etc. -45. It cJSC and conversion efficiency values as shown in Table n-Si interface and reduction in the associated series resistance. The J-V curve  in G/Si cell (6.2 Ω) in comparison to pristine cell (11.4 Ω). It is important to note that the improvement in efficiency (2.47%) for Si solar cell by using graphene as a surface field layer is larger than or similar to the efficiency improvement (2.38%) obtained by using the n+ doping (thickness ≈ 2 × 1020 cm-3 and 0.07 μm) on the front surface [J-V characteristics are further validated by the incident photon conversion efficiency (IPCE) measurements shown in Figure 2/G layers improve the photon to electron conversion ratio considerably compared to the bare planar Si solar cell. The decrease in the reflectance (∆R) of graphene-deposited Si  is much larger than the decrease in reflectance (∆R) as one goes from Si to G/Si structure. This confirms that the electric field formed at the G/n-Si interface is aiding carrier collection. Thus, the deposition of graphene onto polished n-Si surface is aiding carrier collection or photon absorption in addition to lowering its reflectance. A slight increase in VOC from 573 to 582 mV also indicates the active participation of graphene in the solar cell device. Earlier, a number of studies have reported the effect of graphene quality, number of graphene layers, and adsorbed molecules on the electronic properties of graphene-Si interface. Li et al. reported that the incorporation of graphene introduced a built-in electric field near the interface between the graphene and silicon (n-type) to help in the collection of photo-generated carriers [2 (on going from G/Si to SiO2/G/Si cell), the increase in IPCE is much smaller than the decrease in the reflectance value  is primarily due to the antireflection properties of the 100-nm-thick SiO2 layer. Consequently, the large improvement in JSC and small increase in VOC indicate that graphene behaves like an n+ layer which intrudes a surface field at the interface to enhance the collection of light-generated carriers thereby improving the efficiency of the p-n Si solar cell. Further, a decrease in the series resistance value and a small increase in VOC on deposition of SiO2 layer on the G/Si cell are due to modification in the electronic properties of the G-Si interface during SiO2 deposition process. By modifying the electronic properties of graphene layer, the photovoltaic properties of silicon solar cell can be improved further.A large increase in the short circuit current on graphene deposition onto planar Si solar cell is very interesting on various accounts. As mentioned earlier, there are two important contributions that might result in the enhancement in e Figure b shows avement 2.8% obtainvement 2.8% obtain/Si cell .2 Ω in ccarriers . Attenticarriers ,25,46. Fp-n Si solar cell; (ii) as an antireflection layer, which reduces the reflectance of the planar p-n Si solar cell due to the presence of wrinkles; and (iii) as a surface field layer onto n-type Si due to n+-n nature of the interface, which provides a favorable electric field for reducing the carrier recombination. Due to these effects, an increase in efficiency from 5.38% to 7.85% is observed. Deposition of a layer of SiO2 of an optimized thickness value leads to a further increase in the short circuit current density due to its antireflection properties.The present study is a clear demonstration of the useful combination of the properties of graphene: (i) as a transparent conducting layer, which provides high transmittance (97%) and reduces the series resistance of planar The authors declare that they have no competing interests.2 deposition. SR, SM, SS, and PJ jointly fabricated the p-n Si solar cell. BRM supervised the overall study, analyzed the results, and finalized the manuscript. All authors read and approved the final manuscript.RK carried out all the experiments in this study, analyzed and interpreted the data, and drafted the manuscript. MB was involved in SiORK and MB are PhD students in the Department of Physics, IIT Delhi, India. BRM is a professor (Schlumberger Chair) in the Department of Physics, IIT Delhi, India. SM, SS, and PJ are photovoltaics engineers at BHEL, India.


Carrier mobility and chemical doping level are essential figures of merit for graphene, and large-scale characterization of these properties and their uniformity is a prerequisite for commercialization of graphene for electronics and electrodes. However, existing mapping techniques cannot directly assess these vital parameters in a non-destructive way. By deconvoluting carrier mobility and density from non-contact terahertz spectroscopic measurements of conductance in graphene samples with terahertz-transparent backgates, we are able to present maps of the spatial variation of both quantities over large areas. The demonstrated non-contact approach provides a drastically more efficient alternative to measurements in contacted devices, with potential for aggressive scaling towards wafers/minute. The observed linear relation between conductance and carrier density in chemical vapour deposition graphene indicates dominance by charged scatterers. Unexpectedly, significant variations in mobility rather than doping are the cause of large conductance inhomogeneities, highlighting the importance of statistical approaches when assessing large-area graphene transport properties. Graphene23456781011131416171920232527in-situ electrically gated THz-TDS imaging. In contrast to prior electrically gated THz spectroscopy experiments performed on graphene293031+ boron-doped, nano-crystalline silicon, to ensure a negligible THz response from free carriers injected into the gate-electrode. This allows for isolation of the graphene sheet conductance from non-contact THz-TDS transmission measurements and thus a quantitative extraction of the graphene field-effect mobility.Here we demonstrate quantitative mapping of the field-effect carrier mobility in a large-area monolayer CVD graphene film based on 3N4). The THz response of the poly-Si/Si3N4 stack is found to be negligible with a frequency-independent transmission of 100% ± 1% and a diminishing phase-shift of 2 ° , 50 nm boron-doped poly-crystalline Si (poly-Si), and 165 nm Si nitride 2S2O8. A large-area graphene film is grown on commercially available Cu foil by means of a standard catalytic chemical vapour deposition (CVD) and is subsequently transferred onto the layered substrate, as shown in 3N4/poly-Si/Si were recorded at 21 different gate voltages between 0V and 40V, a selection of which are shown in Vg =24 V THz map. The complex, frequency-dependent sheet conductance 2πf≪1/τ), the Drude-Smith and Drude models, expected for the THz response of pristine graphene, dictates that the real part of THz maps of the CVD graphene on Siσs. The evolution of σs with varying Vg in the series of images in In s as a function of Vg for three distinct areas in the map with highly conducting graphene, less conducting graphene, and no graphene coverage. The measured of σs shows a linear dependence on the applied gate voltage in the range 0V < Vg < 18 V with different slopes throughout the extent of the graphene film, reflecting the local field-effect mobility for hole carriers. This observation is found to be representative throughout the graphene film area. For gate voltages 24 V < Vg < 40 V a plateau with slowly increasing σs is found. This observation indicates that electron-conduction is greatly impaired in this particular graphene film with electron mobilities that are more than an order of magnitude lower than the observed hole mobilities. Application of a gate voltage, Vg, between the poly-Si thin film and the graphene film induces a proportional change in the free carrier density, Δns, in the poly-Si and graphene films, as Vg = 24 V. This minimum represents the charge-neutrality-point (CNP), indicating a constant, and homogeneous presence of residual absorbates with a density of approximately 5 × 1012 cm−2, which p-dopes the entire graphene film. Within the regime of long-range, charged impurity scattering, the added free graphene carriers result in a roughly linear change in the graphene sheet conductance, σs, with the field effect mobility being a proportionality factor. The sheet conductance, σs, of the graphene film is thus described by the relationsCg is the gate capacitance, e is the electronic charge, ε0 is the vacuum permittivity, εSiN = 7.5 is the relative permittivity of Si nitride, t is the thickness of Si nitride gate dielectric, and μFE is the field-effect mobility of the graphene film.where μpoly-Si < 10 cm2/Vs) of our poly-Si thin film relative to the hole mobility of the investigated graphene film, which is approximately two orders of magnitude higher. Because of the choice of a low-mobility, high carrier concentration gate material, the experiments presented here provide a direct and reliable measure of the gate-induced conductance evolution of the graphene film with an insignificant contribution from gate-induced carriers in the substrate. Following |Δσs/ΔVg| in the high density-region well below the CNP. Based on this central result we are able to show a spatially resolved field-effect hole mobility map for the large-area CVD graphene film in Δσs/ΔVg| was found in each pixel by a linear fitting routine to σs vs. Vg from 0 V to +18 V. Similarly, the carrier density due to chemical doping of the graphene film at zero gate bias, can be evaluated across the map from the determined hole mobilities and the sheet conductance at Vg = 0, by utilizing the relation σs= ensμhole. ns (ns is set to 0 in pixels where μhole < 100 cm2/Vs). We note that carrier mobility measured using this technique is affected by the coverage of graphene, because The gate-induced conductance in the gate electrode can be neglected because of the insignificant carrier mobility  from the central region of the graphene film, marked by dashed, black rectangles in μhole = 1174 cm2/Vs and ns(0 V) = 6.2 × 1012 cm−2, and relative standard deviations of std(μhole)/avg(μhole) = 14.0% and std(ns)/avg(ns) = 4.8%.Unexpectedly, We find that the procedure presented here gives far more comprehensive information than the typical convention of relying on a few representative electrical measurements from random or selected areas of a large graphene film. We also note that the features and inhomogeneities in the field effect mobility in We anticipate that quantitative, non-contact mapping of graphene carrier mobility as presented in this work will have a significant technological importance for the advancement of production and implementations of large-area graphene in commercial electronic applications and that it can contribute to the fundamental insight into charge carrier dynamics of the graphene solid-state-system by paving the way for investigations of transport dynamics with unprecedented statistical basis.2 large-area CVD film by in-situ electrically gated THz-TDS measurements. While THz-TDS is non-contact, optical characterization, the frequency range is well below the inverse scattering time of the graphene charge carriers, making the obtained conductance directly comparable the DC value. The technique thus opens up the possibility for assessment of fundamental electrical transport properties such as carrier mobility and chemical doping level on basis of large statistical ensembles, or large-area spatial mapping. Far from the charge-neutrality-point, our measurements show a linear dependence of the THz sheet conductance on carrier density, which is interpreted as a signature of electrical transport limited by long-range, charged impurity scattering, also observed in most DC transport measurements on graphene. Unexpectedly, we find that the mobility varies by up to a factor of 2 on a scale of just few mm, and that significant conductance variations in the investigated CVD graphene are due to mobility rather doping variations, which highlights the importance of techniques that facilitate highly statistical or spatially resolved approaches for assessing transport properties in large-area graphene.In conclusion, we have presented non-contact, quantitative mapping of graphene field effect mobility in a 10 × 10 mm© T-ray™ 4000 fiber-coupled THz time-domain spectrometer relying on photoconductive switches for THz generation and detection, which is detailed elsewhere2 graphene film. Through utilization of the electric field effect in graphene, THz maps are recorded at different carrier densities by applying voltages, Vg, in the range from 0 V to 40 V between poly-Si thin film and graphene film. The full acquisition time for each map is 16 minutes, and Vg is increased in discrete steps of 2 V between each map.The experiments are carried out using a PicometrixSi = 3.4173N4/poly-Si/Si) to that of a reference waveform, 3N4/poly-Si/Si-substrate), and can be related to real and imaginary parts of the sheet conductance asThe sheet conductance of the graphene film was extracted in each pixel of the THz maps based on an approach described previously0 = 377 Ω is the vacuum impedance.where ZHow to cite this article: Buron, J. D. et al. Graphene mobility mapping. Sci. Rep.5, 12305; doi: 10.1038/srep12305 (2015).


Moreover, at low carrier density transport measurements allow probing the density of localized states at edges, thus offering a unique handle on edge physics in graphene devices.Quantum point contacts are cornerstones of mesoscopic physics and central building blocks for quantum electronics. Although the Fermi wavelength in high-quality bulk graphene can be tuned up to hundreds of nanometres, the observation of quantum confinement of Dirac electrons in nanostructured graphene has proven surprisingly challenging. Here we show ballistic transport and quantized conductance of size-confined Dirac fermions in lithographically defined graphene constrictions. At high carrier densities, the observed conductance agrees excellently with the Landauer theory of ballistic transport without any adjustable parameter. Experimental data and simulations for the evolution of the conductance with magnetic field unambiguously confirm the identification of size quantization in the constriction. Close to the charge neutrality point, bias voltage spectroscopy reveals a renormalized Fermi velocity of ∼1.5 × 10 Quantum point contacts are narrow electrical connections of a width comparable to the wavelength of the conducting electrons. Here, the authors create such contacts in graphene by etching constrictions and use them to identify ballistic transport and quantized conductance of size-confined Dirac fermions. The observation of unique transport phenomena in graphene, such as Klein tunnelling35712131416In this work we report on the observation of size quantization and localized trap states in ballistic transport through graphene constrictions approximating quantum point contacts. Away from the Dirac point, the current features evenly spaced, reproducible kinks superposed on a linear background, in agreement with transport simulations. Scattering at the rough constriction edges reduces quantization steps to kinks in both experiment and theory. The kink spacing, and their evolution with magnetic field, allows us to unambiguously identify them as signatures of size quantization. Close to the Dirac point, deviations from ballistic behaviour allow for probing the density of localized trap states.2/Si substrates and use reactive ion etching to pattern narrow constrictions (see Methods) with widths ranging from W≈230 to 850 nm, connecting wide leads  sandwiches on SiOde leads . The grar device . From th V−1 s−1 , resultiW , j>0. In the presence of edge roughness, c0 is reduced to a value below 1 due to limited average transmission, and the higher Fourier components are expected to decay in magnitude and acquire random scattering phases φj≠0. Consequently, the sharp quantization steps turn into periodic modulations as will be shown below. Averaged over these modulations only the zeroth-order term in the expansion (G(0) of a constriction of width W thus features a linear dependenc on kF, or, equivalently, a square-root dependence as a function of back-gate voltage assuming an energy-independent transmission c0 of all modes, in accord with For an ideal constriction xpansion  survivesα for each device (Vg and study both the electron and hole conductance as function of kF (G(kF), the product c0W can be extracted for each device and compared with its width W (c0W extracted from G(0) lie only slightly below the width W, where c0 decreases for decreasing width. This suggests that for the narrower devices reflections, most likely due to device geometry and edge roughness, are playing a more important role. From the data in c0≈0.56 for our smallest constriction. Below we will show that, indeed, reflections at the rough edges of the constriction and not a reduction in active channel width is responsible for the deviation of the experimentally extracted c0W from the SEM width W.By measuring the carrier-density-dependent quantum Hall effect at high magnetic fields4h device . We can on of kF . From th width W  determin width W . The estkF<50 × 106 m−1  the measured conductances systematically deviate from the expected linear behaviour  becomes more apparent when focusing on G around the charge neutrality point (CNP). The conductance as function of n for two different cool-downs of the same graphene constriction  an identical c0W and (ii) almost identical, regularly spaced kink structures  from, for example, localized trap states modifies the relation between n and kF toFor small ehaviour . This deows in n . These oαΔVg will be strongly modified by deviations nT from the linear density of states of ideal Dirac fermions and approaches nT(ΔVg) near the CNP. The trap states do not contribute to transport, yet they contribute to the charging characteristicsG from the nT from experimental conductance data  and the corresponding linear extrapolation to very low values of kF  is probably due to microcracks at the edges of the device.Away from the CNP our data agrees remarkably well with ballistic transport simulations through the device geometry using a modular Green's function approachG(kF), we find reproducible modulations (kinks) in the conductance (G varying in the range of (2−4)e2/h (see arrows in kF), as well as on the constriction width and is strongly influenced by the overall transmission c0 (G of the steps close to 4e2/h for one of our wide samples (W≈310 nm) at elevated conductance values on both the electron and hole sides =G−G(0) provide direct information on the quantized conductance through the constriction (δG(kF) offers an alternative route towards the determination of the constriction width complementary to that from the mean conductance G(0). For example, the pronounced peak of the first harmonic at 230 nm  is more pronounced on the electron side (By subtracting the zeroth-order Fourier component∝triction . One keyron side  than on W (c0W (first term in c0W) from the effective width of the constriction, and proves the relation between the observed Fourier periodicity and the device geometry.Performing such a Fourier analysis for several devices  yields mW  than an E=13.5±2 meV near the CNP (vF=2WΔE/h=(1.5±0.2) × 106 m s−1. This is a clear signature of a substantially renormalized Fermi velocity in nanostructured graphene, possibly enhanced by electron–electron interactionBias voltage spectroscopy measurements yield an estimate for the energy scale of the size quantization steps11 the CNP . With th the CNP  we can eB. The transition from size quantization at zero B-field to Landau quantization at high magnetic fields occurs when the cyclotron radius lC becomes smaller than half the constriction width W. For the Landau level m the transition should occur at lB the magnetic length. This transition line in the B−n plane (see black dashed curve in B=0 T) to the corresponding lowest Landau levels at low temperatures (T=1.7 K) can be easily tracked (T=6 K) the evolution of quantized sub-bands to Landau levels is observed even for higher conductance plateaus  to the Landau levels for large fields (lB<W) remarkably well, further supporting the notion that they are, indeed, a signature of size quantization.Additional clear fingerprints of size quantization appear in the parametric evolution of the conductance steps tracked . At highplateaus . For a cWe have shown ballistic conductance of confined Dirac fermions in high-mobility graphene nanoconstrictions sandwiched by hBN. Away from the Dirac point, we observe a linear increase in conductance as function of Fermi wave vector with a slope proportional to constriction width. Close to the Dirac point, the charging of localized edge states distorts this linear relation. Superimposed on the linear conductance, we observe reproducible, evenly spaced modulations (kinks). Tight-binding simulations for the device reproduce these structures related to size quantization at the constriction. We can unambiguously identify these ‘kinks' as size quantization signatures by both Fourier spectroscopy at zero magnetic field and their evolution with magnetic field, finding good agreement between theory and experiment.6 atmosphere, prior deposition of a ∼10-nm-thick Cr etching mask. Residues of Cr oxide are removed by immersing the samples in a tetramethylammonium hydroxide solution for about 30–35 s. All transport measurements are performed in a four-probe configuration using standard lock-in techniques. Since the distances between the contacted current-carrying electrodes and the voltage probes are small compared with the other length scales of the system, we have an effective two-probe configuration. Importantly, this way we exclude the one-dimensional contact resistances.The hBN–graphene–hBN sandwich structuresWe simulate the experimental device geometry using a third-nearest neighbour tight-binding ansatz. We rescale our device by a factor of four compared with experiment, to arrive at a numerically feasible geometry. We determine the Green's function using the modular recursive Green's function method30How to cite this article: Terrés, B. et al. Size quantization of Dirac fermions in graphene constrictions. Nat. Commun. 7:11528 doi: 10.1038/ncomms11528 (2016).Supplementary Figures 1-15, Supplementary Table 1, Supplementary Notes 1-9 and Supplementary References


Two different polarization configurations are used. In an in-plane configuration, the polarization direction of both incident and scattered light is parallel to the graphene plane. In an out-of-plane configuration, the angle between the polarization vector and the graphene plane is the same as the angle of laser incidence (θ). The normalized Raman intensity of the G-band measured in the out-of-plane configuration, with respect to that in the in-plane configuration, was analyzed as a function of θ. The normalized Raman intensity showed approximately cos2θ-dependence up to θ = 70°, which can be explained by the fact that only the electric field component of the incident and the scattered photon in the out-of-plane configuration projected onto the graphene plane can contribute to the Raman scattering process because of the perfect confinement of the electrons to the graphene plane.Chemical vapor deposition (CVD)-grown single-layer graphene samples, transferred onto a transmission electron microscope (TEM) grid and onto a quartz plate, were studied using polarized Raman spectroscopy with differing angles of laser incidence ( Graphene, an almost perfect two-dimensional crystal, has been intensively investigated for the last decade, since its discovery in 2004 ,2, due t−1, the G-band near 1,585 cm−1, and the 2D-band at approximately 2,675 cm−1. By analyzing both the normalized Raman intensity of the 2D-band with respect to that of the G-band and the line-shape of the 2D-band, the number of graphene layers can be accurately determined [Raman spectroscopy has played an important role in studying graphene ,13. A tytermined .cos2α-dependence in which α is the angle between the CNT axis and the polarization direction of the incident light [Polarizations of the incident laser and the scattered light are important in the Raman scattering on low-dimensional crystals. For a carbon nanotube (CNT), which is a quasi-one-dimensional crystal, the Raman intensities of CNT vibrational modes are maximum when the polarization directions of both the incident and the scattered light are parallel to the CNT direction whereas the Raman scattering is forbidden when the polarization directions of both the incident and the scattered light are perpendicular to the CNT direction . In factnt light . Similarnt light -19.θ, the electric field vector of the incident light can be fully contained in the graphene plane (in-plane configuration) or it can make an angle θ (out-of-plane configuration), depending on the polarization direction of the incident light, as shown in Figure For a two-dimensional crystal, polarized Raman spectroscopy on single-layer graphene revealed strong polarization anisotropy for the Raman intensity of the double-resonant 2D-band, whereas that of the G-band is isotropic, under the normal laser incidence in the backscattering geometry . Polarizout-of-plane configuration, with respect to that in the in-plane configuration, was analyzed as a function of θ. The normalized Raman intensity showed approximately cos2θ-dependence up to θ = 70°.In this work, we investigated polarization anisotropy of the G-band and the 2D-band of single-layer graphene for oblique laser incidence with differing angles of laser incidence for the first time. The normalized Raman intensity of the 2D-band measured in the Single-layer graphene films were grown on 25-μm thick Cu foils  by a commercial plasma-enhanced chemical vapor deposition (CVD) system  at 830°C using methane. Details can be found in ref. .3. Prior to wet-etching, the surface of graphene/Cu was spin-coated with poly-methyl methacrylate (PMMA: 950 A2), followed by baking at 150°C for 3 min. Once Cu foil was dissolved completely, the PMMA/graphene membrane was washed with deionized water and placed on a TEM grid or a quartz substrate. Finally the PMMA film was removed by acetone.For Raman spectroscopy, the synthesized graphene films were transferred either onto transmission electron microscope (TEM) grids or onto quartz substrates by etching the Cu foil in an aqueous solution of FeCl−1.Raman spectra were measured at room temperature by using a 532-nm line from a frequency-doubled Nd:YAG laser  as the excitation light source. Scattered light from the samples was analyzed through a single grating spectrometer  with a focal length of 50 cm  and detected with a liquid-nitrogen-cooled silicon CCD detector . The laser spot size on the sample was approximately 100 μm, and the spectral resolution was approximately 1 cmθ with the graphene plane as the laser incident angle θ. Thus, the in-plane configuration and the out-of-plane configuration can be denoted by (VV) and (HH), respectively. Polarized Raman spectra were measured in both (VV) and (HH) configurations for each laser incident angle θ ranging from −70° to 70°.Two different Raman scattering geometries were used in this work. The backscattering geometry was used for the single-layer graphene on a TEM grid but the forward-scattering geometry for that on a quartz plate. For both scattering geometries, the vertical direction is assigned to the polarization direction of the incident light when its electric field is fully contained in the graphene plane whereas the horizontal direction is assigned to that when its electric field made the same angle Raman spectrum of the CVD-grown graphene sample used in this work is shown in Figure 2α-dependence in which α is the angle between the CNT axis and the polarization direction of the incident light [out-of-plane configuration with respect to that measured in in-plane configuration, 2θ-dependence. Experimental data, shown in Figure θ, agreed quite well with cos2θ-dependence (the black solid line) as expected.Figure nt light . Thus, t2θ-dependence as shown in Figure Figure In order to double-check that the observed polarization anisotropy in Figures out-of-plane configuration with respect to that in the in-plane configuration was analyzed as a function of the laser incident angle θ. The normalized Raman intensity 2θ-dependence up to θ = 70°, which is the direct consequence of the two-dimensional nature of graphene.We investigated polarization anisotropy of the G-band and the 2D-band of single-layer graphene for oblique laser incidence with differing angles of laser incidence for the first time. The normalized Raman intensity of the 2D-band measured in the


Despite the wealth of mental-health information available online to consumers, research has shown that the mental-health information needs of consumers are not being met. This study contributes to that research by soliciting consumer questions directly, categorizing them, analyzing their form, and assessing the extent to which they can be answered from a trusted and vetted source of online information, namely the website of the US National Institute of Mental Health (NIMH). As an alternative to surveys and analyses of online activity, this study shows how consumer questions provide new insight into what consumers do not know and how they express their information needs. The study crowdsourced 100 consumer questions through Amazon Inc.’s Mechanical Turk. Categorization of the questions shows broad agreement with earlier studies in terms of the content of consumer questions. It also suggests that consumers’ grasp of mental health issues may be low compared to other health topics. The majority of the questions (74%) were simple in form, with the remainder being multi-part, multifaceted or narrative. Even simple-form questions could, however, have complex interpretations. Fifty four questions were submitted to the search box at the NIMH website. For 32 questions, no answer could be found in the top one to three documents returned. Inadequacies in the search and retrieval technology deployed at websites account for some of the failure to find answers. The nature of consumer questions in mental health also plays a role. A question that has a false presupposition is less likely to have an answer in trusted and vetted sources of information. Consumer questions are also expressed with a degree of specificity that makes the retrieval of relevant information difficult. The significance of this study is that it shows what an analysis of consumer mental-health questions can tell us about consumer information needs and it provides new insight into the difficulties facing consumers looking for answers to their questions in online resources. Trusted community organizations such as NAMI  also provide comprehensive vetted information on mental health for consumers. Many other countries have similar online resources—the UK National Health Service (NHS) and Mental Health Foundation and the Mental Health Foundation of New Zealand, for example. With such rich resources, it might be expected that consumer mental-health information needs are being taken care of. But research shows otherwise. This article adds to that research, asking what can be learned about mental-health information needs by directly soliciting questions on mental health from consumers and assessing the extent to which those questions can be answered using a trusted and vetted source of online information. Included in our study is an analysis of the questions in terms of their surface form or structure and the categories they fall into.There is a wealth of vetted mental-health information specifically for consumers It has long been known that the Internet plays an important role in mental-health information seeking, with the lived experience of mental illness being of particular interest . KhazaalDespite the rich resources of the Internet, and other sources of patient information such as mental-health professionals, the mental-health information needs of patients and the larger community are not being met. In the Unmet information needs in mental health have been identified in two recent studies focused specifically on depression. In a large-scale survey, twenty one specific topics—organized under general information, lived experience, research and policies, and specific treatments—were confirmed as areas of need in the general population . In postAs an alternative to surveys that assess consumers’ knowledge about specific topics in mental health and as an alternative to analyses of online activity such as in mental-health forums, this study proposes that consumers be prompted to ask questions about mental health and that those questions be studied to better understand unmet information needs. No one has investigated what people want to know about mental health by asking them directly. The hypothesis behind this study is that a question-based analysis can provide important insight into what consumers do not know and, by trying to answer those questions using an existing trusted and vetted source of online information, provide an understanding of the extent to which the information needs represented by these questions can or cannot be met online. Directly solicited questions can also be categorized in terms of content and form to provide insight into the information needs they represent.Clinical information needs have been extensively studied by examining real clinical questions collected from physicians . The queA large-scale source of general consumer health questions is the US National Library of Medicine (NLM). Its customer services receive about 90,000 requests a year from around the world. To provide help in responding to these requests, it recently launched the Consumer Health Information and Question Answering project . The proWhy do people who use drugs experience more mental illnesses?  The general assumption behind this question is that drug use and mental illness commonly occur together, a fact research shows to be the case  What kinds of mental-health questions do people have? (2) What form do the questions take? and (3) How easily are answers to those questions found in trusted and vetted online sources of information? The aim is to develop new insight into what consumers do not know, how they express their information needs in language, and the extent to which those expressed needs can be met using trusted online information.To obtain consumer questions on mental health and illness, we used crowdsourcing; in particular, the platform provided by Amazon Inc.’s Mechanical Turk (AMT). AMT is a digital marketplace that allows workers to perform tasks online in exchange for specified fees. Workers are anonymous to the person requesting the task, as is the requester to the workers. A worker’s unique identifier allows the requester to control the number of times any worker responds to the task request. Results are provided in an Excel spreadsheet that can be downloaded for analysis. AMT has been shown to be a reliable source of participants for clinical studies in consumer health informatics . StudiesWhat do you want to know about depression, anxiety, schizophrenia, bi-polar disorder, suicide or ANY mental-health topic? Enter a full question, not just a phrase. For example: “Do people ever recover from schizophrenia?” NOT ”Schizophrenia recovery.” We do NOT want simple questions of the form “What is X?” where X is a commonly known mental illness. Your question does not have to have perfect grammar but it must be perfectly clear. For example: “Do viruses cause mental illness?” is acceptable. “Is there cause for mental illness?” is not. Spelling mistakes are alright as long as the question is still clear.One hundred questions were collected from 100 unique workers between April 26, 2014 5:47 PM PDT and April 26, 2014 9:46 PM PDT for a total cost of $40 and an effective hourly rate for workers of $16. Each worker was allowed to ask only one question to ensure our data represented the information needs of more than those few people who might respond to the AMT task first and provide many questions each, thereby excluding others from offering their questions.The questions were analyzed three different ways:1.Consumer health category analysis: Using categories adopted from the NLM consumer health question answering project, each question was labelled as to the kind of question it was.2.Analysis of question form: The structure or surface form of each question was investigated.3.Analysis of answer accessibility: An assessment was made as to whether or not each question had an answer that was accessible through the search box at the NIMH site http://www.nimh.nih.gov/.The questions were analyzed as asked, that is, without correction or editing. The number was limited to 100 to permit a thorough manual search of the trusted and vetted information resources of the NIMH. It is especially time-consuming to establish a negative, namely that a given question cannot be answered from a given document. We discuss at the end of the paper the kinds of automated text analysis that will make further studies of this kind possible with a larger collection of questions. The full set of 100 questions is given in Twenty question categories were defined, adapted from the 14 categories of the NLM study of consumer health questions. Adaptions were as follows. In the NLM study, the greatest number of questions (23%) fell in the Management category. This category was replaced by the four NLM subcategories of TREATMENT, CURE, PREVENTION, and OTHER MANAGEMENT to better understand the questions being asked. The Information category had the next highest number of questions (18.1%) in the NLM study. For our study on mental health, any question that appeared to be seeking information to understand mental illness in general or a specific disorder fell into this category. And if a question would require as part of an adequate answer some explanation of mental illness in general or of a specific disorder, it was considered an INFORMATION question. In the NLM study a combined category called Susceptibility had the next highest concentration of questions (14.4%). It was replaced by the three NLM subcategories of DISTRIBUTION of a DISEASE in a POPULATION, INHERITANCE PATTERNS, and TRANSMISSION PATTERNS for infectious diseases. Although the transmission category might not seem justified for mental health, consumers could ask questions that fit the category. Finally, one category was added that is important for mental-health, namely PSYCHOSOCIAL, which covers questions about the social-emotional ramifications of a mental-health disorder. Is Bi-Polar disorder curable at all or will people always be dependent on their medication? falls into four categories; namely, CURE, PROGNOSIS, TREATMENT and INFORMATION, the last because the question suggests the need for general information on bipolar disorder.A question could receive more than one category label. For example, the question All 100 questions were categorized by the first author (CEC) and a randomly selected subset of 20 questions was additionally categorized by the second author (JBK). Those 20 questions are indicated by starred rows in Is schizophrenia genetic? The mother of an adult child similarly diagnosed may say: My father had schizophrenia. Is that why my child has it? Different acts and different words used to express the question but, in important ways, the same question with the same content, namely an inquiry into the heritability of schizophrenia. However, the first question act may have behind it a concern over whether or not the questioner is at risk. The second may have behind it a concern that she or her family heritage are to “blame” for her child’s illness. The reason for or motivation behind a question is an important part of understanding what is being asked and determining the most appropriate answer.It is a fundamental tenet of semantics that meaning does not exist solely within words and sentences but resides in the situations in which the words are uttered and the attitudes of those speaking and hearing the words . To undeIs X true? (Are anti-depressants dangerous?), or What causes X to occur? (What causes PTSD?). This simplifying assumption may have been necessary for early work on question taxonomy development, but questions real people pose are seldom that simple. The NLM questions project recognizes this reality in its work on decomposing complex questions into their simpler parts  was chosen to conduct a review of the extent to which answers are accessible to consumers. Questions were entered into the search box at the NIMH site as they were asked, without any editing or rephrasing. To make the assessment as reasonable as possible, long questions were omitted, thereby precluding many questions of complex form which are naturally more difficult to find answers for. There were 54 short questions (fewer than 255 characters) and these were taken as the test questions. The NIMH website was accessed by CEC on October 17, 2014 and the top three URLs returned for each search were retrieved. A URL (Uniform Resource Locator) is an Internet address that points to a document and it was these documents that were examined for answers to the given questions.It is important not only to know what questions consumers have but also whether or not they can find answers to those questions at comprehensive trusted and vetted sources of online information for consumers. The NIMH site  examined the documents that the top three URLs pointed to and made one of the following three judgments for each question:An answer could not be found in the top three documents returned, or no results were returned.An answer could be found but it was in a long or complex document that required extensive reading and some interpretation.An answer could be found quite easily in the documents returned, starting with the first, moving on to the second or third as needed.In the second assessment, AMT workers were used. For each question an AMT task was set in which the question was presented along with the top URL retrieved by CEC earlier. The worker was asked to indicate if an answer could be found in the document the URL pointed to by following the link and copying and pasting an excerpt containing the answer in a text box or, if no answer could be found, to enter NO ANSWER, or if no document was returned to enter NO DOC. Each task was performed by two workers on February 5, 2015, for a total cost of $76.20. Workers spent longer than expected on each task—on average 13 minutes compared to the 4 minutes expected—giving a lower than intended effective hourly rate of $3.79.Given this average time of 13 minutes, we limited the AMT worker assessment to just the top search result. The length of time it took to ascertain whether or not there was an answer in a document is itself a result that establishes the difficulty of the task for the consumer. We discuss this further in the Results section.For the categorization of all 100 questions by CEC, k = (Na − Nc)/(Np − Nc), where Na = number of agreements overall, Nc = number of agreements that could be expected due to chance, and Np = maximum number of agreements possible. An agreement for a question is an instance in which a given category is either assigned by both raters to that question or by neither. A disagreement for a question is an instance in which a given category is assigned by one rater to the question but not by the other. The number of agreements overall is the sum of the agreements for all 20 questions. With 20 questions and 20 categories, the maximum number of agreements possible is 20 × 20. Chance assignment of a category to a given question is 0.5 for each of the 20 categories, which sums to 0.5 × 20 = 10 for each question. For 20 questions, the number of agreements due to chance is therefore 10 × 20 = 200.For the 20 randomly selected questions that were additionally categorized by JBK, we computed inter-rater agreement using the chance-adjusted measure according to the formula The agreements and disagreements are shown in k = 0.9. If we omit the INFORMATION category, which had a disproportionally high number of agreements, the score stays the same. If we omit the categories shown by shaded row labels in the Table that were not relevant to the subset of 20 questions and so also contributed a high number of agreements, we again get a score of 0.9.Taking all 20 questions and categories, we obtained Is a predisposition for bi-polar disorder hereditary or can it be caused by environmental factors?—may have an exclusive-or or an inclusive-or interpretation and so is not merely two simple questions. That is, the question may have any of the following three summarized interpretations: Is there an inherited predisposition and no environmental effect? Are environmental factors the cause and a disposition is not inherited? Do inherited and environmental factors both play a causal role?The majority of the questions (74 out of 100) are simple in form. Twenty six are complex, with 11 being multi-part, 9 multifaceted and 6 narrative, as shown in Complex questions present another challenge, not so much for their interpretation but for finding answers. Complex questions often include information that is very specific, such as being unfaithful or disappointed, experiencing failure, experiencing a two-week episode, or having a timid father. Although it is often possible to formulate general answers that take specifics into account, search and retrieval technology behind search boxes at websites cannot make the leap from the specific to the general. Nor can the consumer always make the necessary generalization; to know that timidity, for instance, is not a personality trait that generally shows up in discussions of mental health.The first assessment of answer accessibility (by CEC) had the results shown in http://www.nimh.nih.gov/health/topics/depression/index.shtml (accessed 6 February 2015). Some but not all yielded answers to the specific question posed. Sixteen URLs pointed to news items on the NIMH site, such as the following: http://www.nimh.nih.gov/news/science-news/science-news-about-depression.shtml (accessed 6 February 2015). Although an answer might lie within such a document, it was typically difficult to find among all the news article links given, which had to be followed individually. Some URLs pointed to documents such as the following 2011 Director’s Report, which is not consumer-oriented and did not contain an answer: http://www.nimh.nih.gov/about/advisory-boards-and-groups/namhc/2011/september/directors-report.shtml (accessed 6 February 2015). Some URLs pointed to a long pdf (portable document format) document, such as the following budget report, which did not provide an answer: http://www.nimh.nih.gov/about/budget/cj2007_33873.pdf (accessed 6 February 2015).Twenty of the fifty five top URLs that were returned pointed to consumer-oriented material organized under Health Topics at the NIMH website—such as the following, for example: On average, how much time does a person suffering from bi-polar disorder spend in a manic state compared to a depressive state? The underlying assumption is that it is possible to predict the relative lengths of manic and depressive states in general. The question Suicide is most common with which mental illnesses? assumes enough is known about suicide to associate its occurrence with specific mental illnesses in a quantifiable way. Such presuppositions can be identified for 11 of the 32 questions without an answer and these are given at the top of What is the relationship between circadian rhythms and Bipolar Disorder? assumes there is a relation between circadian rhythms and mental illness and that it has been studied. In fact, the NIMH search returns a report on the topic from an NIMH workshop that does provide an answer to the question.A number of the questions entailed presuppositions that are not necessarily true and which may have precluded an answer being found at any trusted and vetted information site. Take, for example, the question The second assessment of answer accessibility, conducted by AMT workers, had similar results, with answers found by both or one of the AMT workers for 20 of the questions. However, the AMT workers failed to find answers to questions numbered 36, 38, 39 and 53 in Question 7: Stress management techniques and meditation can help people with anxiety disorders …, aerobic exercise may have a calming effect. …, certain illicit drugs, and even some over-the-counter cold medications can aggravate ….Question 11: Children who develop depression often continue to have episodes as they enter adulthood. Children who have depression also are more likely to have other more severe illnesses in adulthood.In two instances of answers not being found, the URL pointed to a pdf file which produced the following standard warning before it was opened:Opening <filename>.pdf. Some files can contain viruses or be otherwise harmful to your computer. It is important to be certain that this file is from a trustworthy source. Would you like to open this file?The AMT workers most likely declined to open such a file, which accounts for their reporting the result of NO DOC (no document returned).k = 0.8 between the first and second assessment was calculated using Cohen’s kappa, computed as k = (Pa − Pe)/(1 − Pe) on the results in Inter-rater agreement of The length of time it took for the AMT workers on average to ascertain whether or not there was an answer in a document—13 minutes—made us decide to restrict the second assessment to just the top document. If it takes that long to check one document it seems unlikely that consumers would be prepared to search one document after another for an answer. Despite the difference in the number of documents used in the first and second assessments, there was still high inter-rater agreement. This suggests that much of the time if an answer exists, it can be found in the first, second and/or third documents, just in different forms.http://www.nimh.nih.gov/health/statistics/index.shtml, accessed 20 January 2015). Nonetheless, any follow-up study collecting questions should mention all disorders that are of interest to the study or none, and should explicitly include or exclude substance abuse disorders. Other disorders specifically named in the collected questions were ADD, DID , narcissistic personality disorder, “Neurosis and Psychosis,” borderline personality disorder, drug use, OCD, Erotomania, and PTSD (twice).The high percentage of mental-health questions categorized under INFORMATION—81% compared to 18.1% for the general consumer health questions from the NLM—suggests that consumer grasp of mental illness is low compared to other health topics. Questions sought both general information and information on specific disorders, notably depression (29 questions), bipolar disorder (16), anxiety (14) and schizophrenia (12). These four disorders were named in the AMT task description, which may have increased the likelihood of their being specifically asked about. However, in the US anxiety and depression have the highest 12-month prevalence figures for adults and the four named disorders are among the top six contributors to disability-adjusted life years for mental and behavioral disorders, with drug and alcohol use disorders being the other two  in our study were questions about the lived experience of mental illness for the patient and other affected individuals. The category MANIFESTATION (first cluster) is largely about symptoms and these questions were common, as were questions labeled TREATMENT (first cluster). There were some but not many (nine) questions labeled DIAGNOSIS, which was not in the top two category clusters. There were many questions labeled CAUSE (first cluster). The prevalence of these questions suggests a strong need to understand the origins of mental illness. The CAUSE questions overlap with those categorized under INHERITANCE PATTERNS (second cluster). Taken together the questions in these two categories were twice as frequent as those in any other category besides INFORMATION. There were seven questions about stigma, four asking if something is “normal,” one specifically mentioning stigma, one asking about violence, and one questioning the safety of marriage to a person with a mental illness. There weren’t any questions on disclosure in our study. The many questions combined under PROGNOSIS (first cluster), COMPLICATION (second cluster), and CURE (second cluster) would be aligned with the topics of coping and comorbid health problems identified in earlier studies. Despite the fact that the topics of earlier studies and the NLM categories are not fully aligned, there seems to be broad agreement on the content of consumer questions, but with a lesser emphasis on diagnosis and disclosure in our question collection.What internal coping mechanisms can you use to help deal with bi-polar disorder?, CEC assumed that by “internal coping mechanisms” the questioner meant things she could do herself without help from health-care professionals and so categorized it as a PSYCHOSOCIAL question whereas JBK linked “internal coping mechanisms” with cognitive behavioral therapy and so categorized the question as a TREATMENT question. For question 22, How does poor nutrition during childhood affect one’s susceptibility to develop bipolar or some other mood disorder like depression?, JKB assumed the questioner had in mind that good nutrition might confer a preventive advantage and so assigned the category PREVENTION. For question 5, What causes mental illness to occur in people? JKB determined that an adequate answer would entail some discussion of genetic factors and so assigned the category INHERITANCE PATTERNS.There was strong agreement between the two raters on the categorization of the questions. Differences that did exist revealed something about the process of categorization. Interpreting a question to determine which categories applied to it entailed making assumptions about what the questioner may have had in mind and what an adequate answer to the question would entail. For example, for the question numbered 89 in Why is there a stigma if you have Bipolar Disorder that all of those who have it must be crazy? A grammatically correct version of this question would be Why do people hold the stigmatizing belief that a person with bipolar disorder is “crazy”? This is in the first place a question about stigma, about trying to understand the origins of or reasons behind the stigma of mental illness. But it is also about particular attitudes towards a particular disorder, namely that the person with bipolar disorder is a different, stranger, lesser person, as suggested by the colloquial term crazy.In this initial study, question form analysis was restricted to the top-level characterization of simple, multi-part, multifaceted or narrative. Despite the fact that the majority of questions were simple in form, categorization was not simple and finding answers was not simple. Questions that are simple in form can still have complex meaning. An example is question number 1 in Are X and Y related? implies a different level of knowledge from How are X and Y related? The first question shows uncertainty as to whether a relation exists while the second presumes a relation and seeks to know more about it. The form of the question therefore constrains the kind of answer that is appropriate and relevant to the information need. For these reasons, further study of consumer questions on mental health needs to include a deeper analysis of the question form, one that can profit from the large body of research in education.The form and content of questions have long been studied from the perspective of education. The aim has been to understand what questions to ask students that will best guide their learning. A further aim has been to glean from student questions what they need to learn. An early and oft-cited work is that by http://www.nimh.nih.gov/health/topics/index.shtml, accessed 20 January 2015). However, answers to more than half of the questions were not readily available. Unanswered questions came from any category and included questions that were both simple and complex in form. Admittedly, answers satisfactory to the AMT workers may have been found in the second or third document returned, which research shows is examined around 24–32% of the time. If answers were to be found in the fourth and later documents not checked by CEC, it is less likely these would be found by consumers. There is the possibility that by adding the online resources of trusted consumer organizations such as NAMI and The Mental Health Foundation of New Zealand, more answers would be found. But at the same time a higher number of irrelevant documents would likely be returned as well, the core problem of the online searching experience. Nevertheless, the restriction to the top documents is an acknowledged limitation of the current study.The NIMH provides extensive patient-oriented material and supports research on almost all the topics addressed by the collected questions , that are not used in professional medicine. Here however, we have the problem of precision. The distinction between susceptibility and prevalence important to question 33 is not one that can be elided and carefully written material will not confuse the two. But if the act of asking the question is properly understood, that is, if the questioner’s purpose in asking the question is taken into account, the conceptual overlap between susceptibility and prevalence might be acknowledged and a suitable answer given about the prevalence of anxiety in different age groups.The second reason a question may not find an answer is related to language. There is information at the NIMH site about the prevalence of anxiety in different age groups and it may very well be that the questioner would be satisfied with information about prevalence. It was the word What is the difference between bi-polar 1 and 2? Taking the three top documents returned at the NIMH site for this question, two were about an NIMH Strategic Plan and one was a report from a workshop on mental health and mass violence. The phrase difference between and the word bi-polar were matched to the content in these documents and nowhere, it appears, did the words bi-polar 1 and 2 appear in a document that used the word difference.A third reason for answers not being found has to do with the surface form of questions and the sensitivity of the search process to the surface form. Take the question At what age do symptoms of schizophrenia usually begin to show in those affected? But the second question did not: How early do individuals with schizophrenia begin to manifest symptoms? The most likely reason for the difference is that there are direct discussions of age of onset but these do not use the word early.To appreciate how important the actual phrasing of the question is to the likelihood of an answer being found through the search box, consider the following two questions, both asking about the onset of schizophrenia symptoms. The first question did have an easily found answer in the top document: fourth reason consumer questions may not readily find answers lies in their specificity. “Being more positive,” “children under 10,” “detoxing heavy metals”—these specific references are unlikely to find an exact match in consumer information materials even if there are discussions of patient attitudes and children and toxic metals and illness. The narrative questions all include details that are specific to someone’s circumstances. Such questions will require individualized answers. Is it worth the effort to provide them? That remains to be seen but it is likely that the stories they tell will provide insight for others in similar circumstances. The studies by A In looking at the high number of questions for which answers could not be found, it might be argued that consumers ask the “wrong” questions. That view, however, does not respect the right of consumers to have information needs of any nature and to express those needs in whatever way they are able.This study aimed to contribute to the research on consumer mental-health information by soliciting consumer questions directly, categorizing them, analyzing their form, and assessing the extent to which they can be answered from a trusted and vetted source of online information. The Internet is a much-used resource for health information by consumers. According to a recent Pew report, 59% of U.S. adults, and 72% of Internet users, looked online for health information in the previous year . The dowGiven the need for more research, what is the significance of the study reported in this paper? What do we know about mental-health information and consumers that we did not know before?http://trec.nist.gov). Second, the nature of consumer questions in mental health mitigates against the easy provision of answers. There are presuppositions that consumer questions harbor; a question that has a false presupposition is less likely to have an answer in trusted and vetted sources of information. Consumer questions are also expressed with a degree of specificity that makes the retrieval of relevant information difficult.We have shown that even highly trusted and well-organized sources of information such as the NIMH may not readily provide answers to consumer questions online. Two kinds of explanations presented themselves. First, the clear inadequacies in the search and information retrieval technology deployed at Internet resources such as those of the NIMH. There is no easy fix for this. The problem of matching a question expressed in ordinary language to the text in documents is a long-standing and active area of research on a non-trivial problem, as demonstrated by the 24-year history of the Text Retrieval Conference (TREC) workshop series, which encourages research in information retrieval by providing large test collections for tasks such as the retrieval of documents on the Internet and question answering  words that occur together more often than would be expected by chance. The only four-word n-gram of note in our 100 questions is what is the best and, apart from the phrases mental illness(es), bi(-)polar disorder, and the brain, the other noteworthy bi-grams are the best, symptoms of, and recover from. It appears to be a common theme that consumers want answers that provide comparative results, as indicated by the best. An interest in diagnosis may be suggested by symptoms of, but it may also suggest attempts to understand the experience of someone with a mental illness, wanting to know if observed behavior is a manifestation of a disorder or not. An intuitive reading of the questions as a whole supports the idea that the bi-gram recover from gives voice to a hope that mental illness can be recovered from, as opposed to being managed as a chronic condition. These observations are tentative given the relatively small collection of questions but they point to the potential of large-scale question collection for robust quantitative analysis. Question should be collected in a number of different contexts, including online at a consumer organization such as NAMI, through crowdsourcing, and at locations used in other studies—psychiatrists’ offices and hospitals, for example.With a large enough collection of consumer questions, quantitative analytic methods can be deployed. We end with a brief discussion of what these methods offer. Quantitative text analysis has been used to do text data mining in the scientific literature  and deteHow does depression harm the brain? Because the presupposition—that depression does indeed harm the brain—does not have a consensus evidence-based opinion, an answer will not be found for consumers in trusted and vetted information sources. But using a general-purpose search engine, a consumer will find many “answers” to the question at less trusted and non-vetted online information sources. It is imperative that the most trusted online sources of mental-health information be the ones that consumers go to, because it is at those sources that their questions can be answered.With sufficient consumer questions, quantitative methods can also be deployed to identify common presuppositions in questions. Understanding common presuppositions could lead to a better understanding of the knowledge consumers may need but may not realize they need. Understanding common presuppositions also reveals the questions that must be addressed at trusted and vetted online sites, even if just to point out there is no definitive answer. Take the question 10.7717/peerj.867/supp-1Data S1Click here for additional data file.


Nicotiana benthamiana) leaves transfected with the magnICON deconstructed viral vector expression system. L1 proteins were extracted from agro-infiltrated leaves using a series of pH and salt mediated buffers. Expression levels of L1 proteins and VLPs were verified by immunoblot and ELISA, which confirmed the presence of sequential and conformational epitopes, respectively. Among three constructs tested , TPL1F, containing a full-length L1 and chloroplast transit peptide, was best. Extraction of HPV16 L1 from leaf tissue was most efficient  with a low-salt phosphate buffer. VLPs were purified using both cesium chloride (CsCl) density gradient and size exclusion chromatography. Electron microscopy studies confirmed the presence of assembled forms of HPV16 L1 VLPs. Collectively; our results indicated that chloroplast-targeted transient expression in tobacco plants is promising for the production of a cheap, efficacious HPV16 L1 VLP vaccine. Studies are underway to develop plant VLPs for the production of a cervical cancer vaccine.Cervical cancer is the most common malignancy among women particularly in developing countries, with human papillomavirus (HPV) 16 causing 50% of invasive cervical cancers. A plant-based HPV vaccine is an alternative to the currently available virus-like particle (VLP) vaccines, and would be much less expensive. We optimized methods to express HPV16 L1 protein and purify VLPs from tobacco ( Over 95% of cervical cancers are caused by human papillomaviruses (HPVs). Cervical cancer is the second most common HPV-associated cancer in women, with approximately one-half million new cervical cancer cases and almost 250,000 deaths each year worldwide . The grein vitro and in vivo with either T = 7 symmetry, identical to native virions, or T = 1 comprised of 12 capsomers [The major recombinant HPV vaccines, Gardasil and Cervarix, are composed of major capsid proteins of HPV which self-assemble into highly immunogenic VLPs apsomers –5. Threeapsomers . AlthougPlants have emerged as an alternative biotechnological means of producing pharmaceutical  and therAnother challenge is the chromatographic purification of plant-based proteins. It has been demonstrated that different physiological conditions, together with extraction and purification processes, play a crucial role in denaturation of recombinant protein and determination of yield, stability, and biological activity . CsCl anNicotiana benthamiana) use a high-efficiency binary vector system with a chloroplast signal sequence to assemble the HPV L1 proteins in chloroplasts. The yield of purified plant VLPs was high and antigenically similar to VLPs produced in the baculovirus expression system (Cervarix). Using simple purification steps, this is possibly the first report to present high yield of intact HPV16 L1 VLPs from tobacco plants. We anticipate that our isolated VLPs are safe and can be used as an affordable vaccine worldwide.In this study, to express and purify HPV16 L1 VLPs from tobacco plants, we re-designed our expression construct and addressed major shortcomings from previous plant research, including the low yield and less than optimized purification procedures of earlier plant-based technologies. Our improved conditions for HPV16 L1 production in tobacco plants (BamHI site was obtained by PCR with the end-tailoring primer 16L1d22-Bam-R (5’–GGGGATCCTTATCCTAATGTGAACTTTGGCTT). For chloroplast targeting, a 59 amino acid plastid peptide (TP) coding sequence from rbcS was fused to the initiation codon of the L1 gene using overlap extension PCR. The plastid targeting sequence was amplified from pICH12190  using the primers TP-Xho-F (5’-ccctcgagaacaatggcttcttctatgctttcttc) and TPL1-OL-R (5’-GAAGGGAGCCAAAGAGACATGCATTGGACTCTTCCTCCGT), while the 5’ end of the L1 gene was amplified using primers TPL1-OL-F (5’- acggaggaagagtccaatgcatgtctctttggctcccttc) and 16L1-447-R (5’- cacaactgtgtttgcttgtaatcc). The two overlapping fragments were mixed and amplified using the terminal primers TP-Xho-F and 16L1-447-R, and the resulting product was digested with XhoI and NcoI and ligated with pIBT210.1 [NcoI site. The L1 genes were end-tailored by PCR with BsaI sites to allow insertion into pICH26212 (Icon Genetics), yielding p26212-16L1d22 , p26212-TPL1d22 , and p26212-TPL1F .We designed a plant codon-optimized coding sequence for HPV 16 L1 (505 amino acids) based on a strain isolated at the University of Rochester Medical Center (Robert Rose), which is nearly identical to Genbank accession ACV53978.1, except for the occurrence of Ser in place of Thr at position 389. Codons were chosen to optimize expression in tobacco, and avoiding spurious mRNA processing signals . The C-tIBT210.1 , digesteE. coli and Agrobacterium transformed plasmid DNA was confirmed for integration using BamHI and XhoI restriction digestion analysis and the PCR amplification method. The set of primers used were MZ-1L (5'-GCAGGTGTGGTTGACGAAT-3') and MZ-1R (5'-CAAACGTGCGTTAACAGGTG-3')  for amplification of N- and C- termini of the target gene, respectively.The A. tumefaciens (GV3101) cells were made electro-competent [-1) and incubated with shaking for 1 h and then plated onto YenB media containing appropriate antibiotics.Initially, ompetent , and 50 Agrobacterium cultures containing integrated HPV16 L1 genes. Cultures were grown from starter cultures with shaking at 28°C to attain the exponential phase  in YenB broth containing the appropriate antibiotics. The bacteria were pelleted down by centrifugation at 3000×g for 10 min, suspended in infiltration buffer . Final OD600 after centrifugation and resuspension was 0.1. The vacuum infiltration technique was employed to infiltrate 2–4 week old leaves of N. benthamiana plants. Following infiltration, the plants were grown for 1–9 days post infiltration (DPI) under conditions of 16 h light, 8 h dark at 23°C.Agro-infiltration of tobacco leaves was completed with transformed g) for 10 min at 4°C. The supernatant collected after clarification was used for further analysis.In the preliminary analysis, 200 mg of tobacco leaves were harvested at 6 DPI and proteins were extracted using HEPES buffer as described . For timThe extracted soluble protein samples (supernatant) collected from homogenized and clarified tobacco leaves were mixed with sodium dodecyl sulfate (SDS) loading buffer , and heated at 95°C for 5 min. These proteins were separated on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gels . Gels were either stained with coomassie blue  or transferred to a PVDF membrane  and saturated with 5% milk in Tris-buffered saline and Tween (TBST) for 1 h. The membrane was then incubated overnight at 4°C with HPV16 L1 specific monoclonal  or polyclonal  antibodies with 3% milk/TBST (Tris and NaCl containing 0.1% Tween 20). The membrane was washed three times with TBST and finally incubated with 1:5000 diluted secondary antibody . Proteins were detected by chemiluminescence  and bands were visualized on x-ray film  using an SRX-101A processor . Baculovirus-infected insect cell VLPs made in-house was used as a positive control. The purified fractions from CsCl, ammonium sulfate (AMS), size exclusion chromatography (SEC), and ion-exchange chromatography (IEC) fractions were also analyzed for protein expression, as described above.16A and C are murine monoclonal antibodies made in the laboratory against intact virus-like particles and characterized  whereas The ELISA was performed on all homogenized, purified leaf extracts and sera collected from immunizing mice as described in sections 2.4, 2.5 and 2.14. The 96-well ELISA plates  were coated overnight at 4°C with 100 μL of plant extracts or 500 ng per well of purified plant VLPs re-suspended in 1X Dulbecco's phosphate-buffered saline (DPBS). 500 ng of baculovirus-infected insect cell VLPs were loaded as a positive control. The plates were washed three times with washing buffer (PBS-T) and blocked with 5% BSA in 1XPBS (PBSA) for 1 h at 37°C. After washing three times, plant extracts coated plates were incubated with monoclonal or polyclonal antibodies against HPV16 L1 conformational or sequential epitopes, at a dilution of 1:1000 in 1% PBSA for 1 h at 37°C. Similarly, the purified plant VLPs coated plates were reacted with collected mouse sera, at a dilution of 1:100 in 1% PBSA for 1 h at 37°C. Moreover, to test the immunogenicity of plant derived VLPs, pooled mice sera were diluted 4-fold in 1% PBSA (1:50–1:51200) and titrated against 500 ng of insect cell derived VLPs. Thereafter, plates were washed thrice with PBS-T, and incubated with respective alkaline phosphatase-conjugated goat anti-mouse or anti-rabbit IgG (H + L)  at a dilution of 1:5000 in 1% PBSA for 1 h at 37°C. Unbound secondary antibodies were removed by washing and bound antibodies were stained with an alkaline phosphatase chromogenic substrate , and absorption was measured at 405 nm. One percent PBSA and sera collected from unimmunized mice were used as negative controls for the background reaction.2 and homogenized with extraction buffer . The lysate was centrifuged at  for 30 min at 4°C. The cleared supernatant was collected and mixed with cesium chloride (CsCl) solution to a concentration of 1.31 g mL-1 as determined using a refractometer  and centrifuged at  for 20 h. The bands on CsCl gradients were collected, their densities measured, and the CsCl solution was replaced with 1XDPBS containing CaCl2 and MgCl2 (Life Technologies) by dialysis. The dialyzed sample was again centrifuged for 2 h at the same speed to pellet down the VLPs, which were then suspended in a minimal amount of 1XDPBS for storage at -80°C until analysis.Freshly harvested tobacco leaves (5 g) were grounded in liquid Ng) for 10 min at 4°C. The pelleted protein was re-suspended with PBS+ 0.01% Tween 20, and the re-suspended solution was stored at 4°C.To remove contaminants from L1 proteins, ammonium sulfate precipitation was conducted as previously described . The preg) for 10 min. The obtained supernatant was used for the purification process.To remove further contaminants, the ammonium sulfate precipitate was dialyzed against PBS + 0.01% Tween 20 . Using t-1, followed by the elution buffer PBS containing 2.0 M NaCl  and 0.01% Tween 20. The total run lasted 1.5 h. The fractions corresponding to peaks were pooled and dialyzed against binding buffer and concentrated with an additional 2-h centrifugation step .The supernatant obtained after ammonium sulfate precipitation was dialyzed against the binding buffer  for 3 h at 4°C. The 5 mL cation exchange column  was used to purify the L1 protein. The column was equilibrated with binding buffer, and the dialyzed sample (5 mL) was loaded onto the column. The column was then washed with five column volumes of binding buffer with a flow rate of 0.4 mL min-1 for 4 h; their absorbance at 280 nm was measured with a spectrophotometer, and HPV16 L1 protein was analyzed by SDS–PAGE and immunoblotting, as described above.SEC was performed on dialyzed plant VLPs purified by the CsCl density gradient ultracentrifugation method and ammonium sulfate precipitation. Dialyzed samples were loaded on (16/600) Superdex column . The running buffer for this column was PBS, pH 7.2, 0.01% Tween 20. Fractions were collected from the column at 0.5 mL minTotal protein concentration of each VLP preparation and ammonium sulfate precipitated samples was determined using a Nanodrop ND-8000  at 280 nm and the Bio-Rad Bradford protein assay reagent  with bovine serum albumin  as a standard. The yield of L1 protein was quantified using SDS-PAGE analysis. Serially diluted HPV16 L1 VLPs from leaves and insect cells were loaded on an SDS-PAGE gel, and their band densities were measured by Quantity one image analyzer .The dialyzed, concentrated and purified HPV16 L1 VLPs were absorbed onto carbon-coated grids  and negatively stained with 2% phosphotungstic acid (pH 6.8). The quality of VLPs was examined at a final magnification of 41,000× by transmission electron microscopy (Phillips CM-12).C57BL/6J mice were obtained from the Jackson Laboratory .2 floor area) at a maximum capacity of four mice per pen. Mice were allowed free access to autoclaved food  and acidified water (pH 2.8–3.2). Mice were euthanized using CO2 in a chamber fitted with an appropriate pressure-reducing regulator and flow meter or equivalent equipment to ensure a gradual displacement of 10–30% volume/minute. All studies were done with the University of Louisville, Institutional Animal Care and Use Committee  and Biosafety Committee  approvals.Only female mice were used to minimize fighting. Mice were maintained in humidity-, temperature-, and light cycle (12:12) controlled vivarium under specific pathogen-free conditions. Mice were housed in double-pen polycarbonate cages . Statistical differences were determined by Student's t-test and one was ANOVA. Differences were considered to be statistically significant if the p-value was 0.05.Three constructed vectors p26212-16L1d22, p26212-TPL1d22, and p26212-TPL1F for protein expression in plants were designated as V1, V2, and V3, respectively . RestricThree steps were followed to optimize the expression level of the L1 protein. At first, the expression level of the L1 protein in plants by different expression vectors was confirmed at 6 DPI. Extracted L1 proteins  were detected at target size of 51 or 56 kDa when compared with baculovirus-infected insect cells used as a positive control . AlthougSecondly, to optimize and characterize the purification condition for the L1 protein extracted from infiltrated plant leaves, different physiological buffers with various pH (2–8), ascorbic acid supplement, and salt (0 and 0.1 M) were used. Proteins were successfully detected in crude extracts homogenized with pH 4 to 8 buffers, and higher concentrations were found in the presence (0.1 M) rather than the absence of salt containing buffers . OverallThirdly, a time point study was conducted to determine the optimal duration of protein expression in V3 transformed tobacco plants. Interestingly, the expression levels of the L1s were high at 5 to 9 DPIs, whereas non-infiltrated control leaves were negative for all the tested antibodies . The proBased on obtained results, 6 DPI leaves transformed with V3 vector and extracted with neutral PBS buffer was chosen for further purification and quantification studies. Compared with purified VLPs from insect cells, >2.5% of TSP for L1 proteins (2.5 μg L1 / 100 μg TSP) was calculated and overall, 250 mg of L1 yield was obtained per kg of plant biomass.3 density in centrifuge tubes and were referred to as the top and lower bands. L1 proteins were detected in both the collected bands, however, a relatively stronger signal was identified from the top collected band than the lower one  column. Differently assembled forms of L1 VLPs were eluted in a peak with an elution volume of approximately 42 mL of running buffer between 2000 and 75 kDa when compared with different molecular weight protein standards . L1 protIn order to optimize the conditions and strategy for collecting the accumulated VLPs, alternative purification methods were tested. Using forty-five percent of AMS, most of the L1 proteins and VLPs were successfully recovered. Following each step of the AMS treatment, TSPs were precipitated and clarified, which drastically reduced the TSPs to one-fifth of 760 mg (152 mg).These results indicated that more than 75% of TSPs were effectively precipitated; most of the recovered L1 proteins were soluble in PBS buffer, as shown in Based on the isoelectric point (pI) of 8.5 for the L1 protein, crude extracts, CsCl, and AMS precipitated protein samples were also subjected to CM Sepharose Fast Flow, a weak cation exchanger. We found that plant protein samples showed low binding affinity with the cation column and started eluting early with only 0.3 M of NaCl. Unlike the plant protein, the insect cell-expressed L1 protein was eluted at ≥ 0.7 M NaCl (data not shown). These results were also verified by immunoblot assay, which showed that most of the plant L1 protein was eluted without being bound and lost during the wash .Our results confirmed that purified HPV16 L1 protein self-assembled into stable VLPs in plants. As shown in To test the immunogenicity of plant VLPs, antibody titers were evaluated in immunized mice , where 1The aim of our study is to successfully express and purify HPV16 L1 VLPs from tobacco plants using MagnICON expression system. The L1 protein was designed to be accumulated in chloroplasts, which lead to the assembly of predominantly 35–55 nm VLPs with maximal protein level (>2.5% of TSP).Low levels of recombinant HPV L1 protein expression lead to decreased formation of VLPs, which in turn reflects the total outcome yield of purified VLPs ,24. TherCompared to nuclear transformation, it has been documented that genetic engineering of plastids offers many advantages, which make the system ideal for applications involving plant-made pharmaceuticals . These iExpression of L1 proteins was shown in plants transfected with all three constructs upon extraction with an HEPES buffer , and theLow yield of L1 in extracts with HEPES buffer suggested that L1 protein might be degraded during the extraction and homogenization process. To overcome the problem of low yield, we optimized our extraction protocol using different buffers and salts, and an antioxidant supplement was added to reduce L1 oxidation. Our results indicated that extraction of HPV16 L1 from leaf tissue was most efficient (>2.5% of TSP) with a low-salt phosphate buffer (pH 7.0). Using this neutral pH phosphate buffer, we found that L1 expression reached the maximum between 6 and 7 DPI. A study had previously shown that neutral or lower pH aided in the assembly of T = 1 or T = 7 L1 VLPs . SimilarNotably, our improved purification protocol gave higher L1 VLP's recovery with the successful removal of major plant proteins such as Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) from the purified product. After that, different purification strategies to intensively recover highly stable and purified VLPs were tested in this study. Therefore, combinations of CsCl density gradient analysis, ammonium sulfate precipitation, and different chromatographic purification techniques were employed to remove contaminated proteins and recover purified ones and eventually to standardize the best conditions for purification. Since VLPs were only partially purified when a single purification method was used, we used a combination of steps to remove contaminated proteins. The VLPs recovered after the purification steps were abundant and highly antigenic as evident by electron microscopic and ELISA analysis. Although the HPV-neutralization activity of plant VLP-induced antibodies was not assessed in the present study, their high binding titers to insect cell-produced VLPs, as shown in Another problem in previous studies was the aggregation of proteins. Therefore, purification conditions that maintain the intact conformation of VLPs are important because recombinant VLPs are inherently unstable and tend to denature and aggregate in solution , 28. To AMS was used as an alternative method to purify L1 proteins. In agreement with a previous study , our resIn conclusion, we demonstrated that a magnlCON plant virus vector with full-length L1 and a chloroplast transit peptide successfully produced HPV16 L1 VLPs in tobacco plants. Extraction of HPV16 L1 from leaf tissue was most efficient (>2.5% of TSP) with a low-salt phosphate buffer in the presence of HPV16 L1 VLPs, predominantly 35–55 nm in size, evident from electron microscopic studies. Although we used CsCl density gradient centrifugation as the preliminary step to remove plant proteins and collect VLPs, these data may be useful for the development of chloroplast-based vaccines. We anticipate that our new purification methods described herein will aid in recovering highly antigenic HPV16 VLPs from tobacco plants towards the goal of reducing time, cost, and labor while increasing the capacity for industrial production.S1 FigImmunoblot represents the detection profile of HPV16 L1 protein where NC; negative control corresponds to non-infiltrated tobacco leaves, V3; PBS extracted 6 DPI tobacco leaves with V3 construct and PC; positive control, VLPs derived from insect cells. MoAbs (16E and 1H8) and PoAb (disCPV2) were used to detect sequential epitopes of HPV16 L1 protein.(TIF)Click here for additional data file.