There are two critical challenges which determine the array density of 3D RRAM: 1) the scaling limit in both horizontal and vertical directions; 2) the integration of selector devices in 3D structure. In this work, we present a novel 3D RRAM structure using low-dimensional materials, including 2D graphene and 1D carbon nanotube (CNT), as the edge electrodes. A two-layer 3D RRAM with monolayer graphene as edge electrode is demonstrated. The electrical results reveal that the RRAM devices could switch normally with this very thin edge electrode at nanometer scale. Meanwhile, benefited from the asymmetric carrier transport induced by Schottky barrier at metal/CNT and oxide/CNT interfaces, a selector built-in 3D RRAM structure using CNT as edge electrode is successfully fabricated and characterized. Furthermore, the discussion of high array density potential is presented. As one of the most promising emerging non-volatile memory (NVM) devices, oxide-based resistive switching memory (RRAM) has attracted significant interests due to the super endurance, fast switching speed, low power consumption and good CMOS compatibility12345681011y and Leffective represent the thicknesses of the metal plane and the effective perimeter which is in touch with resistive switching layer at horizontal direction. For traditional metal plane electrode based 3D RRAM, since it has cylindrical shape with surrounding electrode, the Leffective could be calculated as:Where Lx,pillar, Lx,RRAM, Lx,S.L. represent thicknesses of pillar electrode, resistive switching layer and selector layer respectively. Previous studies on planar RRAM structure have already shown that 3 nm RRAM device could deliver normal resistive switching performances14y? 2) How to find an efficient method to integrate a selector device? Similar to 2D crossbar structure, 3D RRAM array also has sneak path current issue, as shown in 10x,S.L.) in those approaches are even larger than that of switching layer , which limits the size scaling in horizontal directionWhere LRecently, carbon based low dimensional materials, including carbon nanotube(CNT) and graphene, have drawn significant scientific and technological interests as the emerging interconnect solution18212O5-x/TaOy RRAM cells are fabricated using monolayer graphene as the edge electrode. 2 substrate by an electrochemical approach2O5-x/TaOy RRAM devices using monolayer graphene as the edge electrode. Two-layer 3D Ta2O5-x/TaOy RRAM with graphene edge electrode is shown in y oxide layer contacts with graphene layer during deposition process, the edge of graphene could be oxidized. The previous studies have shown that, in the graphene/Ta2O5-x/TaOy/graphene system, a certain concentration of epoxide groups were grafted onto the basal plane of grapheney and graphene. Serving as an internal resistor, this high resistance region helps control the overshoot current and achieves self-compliance property during SET and forming operations. 4 s at 85 °C.The electrical performance of two-layer 3D Ta2O5-x interface, other than graphene/TaOy interface. During forming process, the oxygen vacancies in TaOy layer move to Ta2O5-x layer, at the Pt/Ta2O5-x the conductive filaments are formed. On the graphene/TaOy side, there are a lot of oxygen vacancies in TaOy which has good conductivity. Electrons could transport from graphene to TaOy easily and enough current could be supplied. Since the graphene electrode has little effects on this process, it is possible that the filament size is still in several-nanometers order, which could be larger than graphene electrode.Furthermore, It is necessary to investigate the mechanism when the edge electrode scaling to sub-nanometer. Therefore, The temperature dependent transport characteristics in HRS and LRS are studied to understand the conduction mechanism. The above physical characterizations and electrical results prove the proposed 3D RRAM cell could switch successfully with monolayer graphene as edge electrode. This ultra-small feature size (~0.3 nm) confirms the 3D RRAM could scale down to sub-nanometer in vertical direction.2x103D RRAM has more challenges of scaling down in horizontal direction. Due to the sneak path current issue, as shown in 2O5-x/TaOy RRAM using CNT as the edge electrode. The effective area Seffective in this case is further reduced, which closes to the magnitude of CNT cross-section area. The fabrication process is similar to that of 3D RRAM with graphene edge electrode, as shown in 2 substrate directly by CVD method2 insulator layer using HF chemistry which wouldn’t damage the CNT, as shown in A novel approach of using CNT as the edge electrode and a self-integrated selector is proposed and devices are fabricated. 2O5-x/TaOy RRAM with CNT edge electrode. Both metallic and semiconducting CNTs were investigated. The metallic CNT with TaOy and Sc electrodes shows a near-ohmic behaviour as shown in 4 s at 85 °C, as shown in y. At reverse cut-off state with positive voltage on edge electrode and negative voltage on pillar electrode, the Schottky barriers block the hole transport at Sc/semiconducting CNT end and the electron transport at TaOy/semiconducting CNT end, as shown in the inset of y could serve as the built-in bi-directional selector for 3D RRAM without inserting any additional selector layer.For semiconducting CNT as edge electrode case, a totally different transport property was observed, as shown in 4 s at 85 °C at 10 GΩ and 100 MΩ with 5 V read voltage, as shown in By choosing the metal contact Sc and TMO deposition sequence, the built-in selector could reduce the over-shoot current and sneak-path current simultaneously: During SET process with negative voltage on pillar electrode, the build-in selector is under reverse bias, which reduces the over-shoot current and achieve better self-compliance property. While in RESET process, the selector under forward bias supplies enough driving current to assist the resistive switching process. Since the 3D RRAM has been experimentally demonstrated at single cell level, it is important to evaluate the performance potential at the array level. Next, a simulation study is conducted to evaluate the 3D RRAM architecture with metal plane/2D/1D nano-materials. A resistor network model is constructed using SPICE method assisted by MATLAB. Three types of 3D RRAM cells, using Pt edge electrode, graphene edge electrode and semiconducting CNT edge electrode, have been compared based on measured data. Moreover, the interconnect resistance between neighboring cells is taken into account, following previous literature4 bits for device with Pt edge electrode. In contrast, for graphene and CNT edge electrode devices, the voltage degrade to 2 V when the array sizes increase to 108 bits and 1010 bits. The decrease of write access voltage mainly comes from the interconnect resistance. With the array size increase, the equivalent resistance of interconnect is comparable with RRAM cells, which causes the divided voltage on selected cell decrease. Therefore the 3D RRAM with Pt edge electrode has the limited array size due to small HRS/LRS resistances. For 3D RRAM cells with graphene and CNT edge electrodes, the HRS/LRS resistances are 1000X larger. This property offers much large array size without write access voltage degradation.m = 80 mV is set as the criterion6 bits which is still not good enough for high density applications. With semiconducting CNT edge electrode case, owing to the built-in selector benefit, the sneak path currents could be reduced efficiently and less voltage drops on other unselected cells. As a result, more than 108 bits array size could be achieved.The simulation results show graphene/CNT edge electrode based 3D RRAM have better performance at array level, comparing with metal edge electrode based 3D RRAM. In addition, some non-ideal effects, such as non-uniformity, device imperfection, RTN noise, etc. are not included in the simulation. Although these effects will not affect the comparison results, they still need to be investigated in the future. The excellent vertical and horizontal scaling limits give 3D RRAM technology great potential in high density memory applications.2O5-x/TaOy RRAM cells with monolayer graphene as edge electrode are fabricated. 3D RRAM cells could switch normally with sub-nanometer electrode thickness. In horizontal direction, selector-layer free is realized by using 3D Ta2O5-x/TaOy RRAM with semiconducting CNT edge electrode. In such case, the Schottky barriers formed at CNT/Sc and CNT/TaOy contacts are served as the built-in selector. Based on the experimental and simulation results, different edge electrode material options are evaluated for high density application potential.In this work, 3D RRAMs with graphene or CNT edge electrodes are demonstrated, to explore the vertical and horizontal scalability. In vertical direction, two-layer 3D Ta2O5-x/TaOy RRAM with graphene edge electrode: 1) Synthesize and transfer the first layer graphene to SiO2 substrate. 2) Deposit 100 nm SiO2 insulating layer by RF sputtering. 3) Synthesize and transfer the second layer graphene to SiO2 substrate. 4) Deposit second SiO2 insulating layer. 5) Pattern by lithography and ICP dry etch with C4F8/Ar to form the drilled holes. 6) Deposit the Ta2O5-x/TaOy TMO layer by RF reactive sputtering. 7) Pattern by lithography and deposit 30 nm Pt as the pillar electrode. 8) Pattern by lithography and deposit the 40 nm Pd as the contact metal on graphene.3D Ta2O5-x/TaOy RRAM with CNT edge electrode: 1) Synthesize CNT on SiO2 substrate using CVD method. 2) Deposit 100 nm SiO2 insulating layer by sputtering. 3) Pattern by lithography and wet etch the drilled hole without damaging the CNT. 4) Etch the CNT using oxygen plasma in the drilled hole. 5) Deposit the Ta2O5-x/TaOy TMO layer by reactive sputtering. 6) Pattern by lithography and deposit 30 nm Pt as the pillar electrode. 7) Pattern by lithography and deposit the 40 nm Sc as the contact metal on CNT.3D Ta2 ambience. Then the reaction gases composed by CH4 and H2 were introduced into the growth quartz chamber with a total gas flow rate of 800sccm. The ratio of CH4:H2 is 0.0078:1 and the substrates were soaked in the reaction gas mixture for 60 minutes before cooling down step. The synthesized graphene films were transferred to SiO2 substrates by an electrochemical methodSynthesize and transfer the graphene edge electrode. Pt foils were heated to 1050 °C in H2 substrates. After the growth of CNTs, the samples were annealed at 80 °C in a low pressure environment for 5 min to make the substrates more hydrophobic.Synthesize the CNT edge electrode: The CNTs were synthesized by a CVD methodThe quality of graphene was characterized by Raman spectra (RENISHAW RM2000). The CNTs were observed using TEM (FEI Tecnai TF20) and SEM (QUANTA FEG450). The 3D RRAM cross-section structures were investigated by TEM, EELS (FEI Tecnai G2 F20) and FIB (FEI Nova600). The electrical characteristics were tested by Agilent B1500A semiconductor parameter analyzer and Agilent 81110A pulse generator with Cascade Summit 11000 probe station.How to cite this article: Bai, Y. et al. Stacked 3D RRAM Array with Graphene/CNT as Edge Electrodes. Sci. Rep.5, 13785; doi: 10.1038/srep13785 (2015).


We design a dual-band absorber formed by combining two cross-shaped metallic resonators of different sizes within a super-unit-cell arranged in mirror symmetry. Simulations indicate that absorption efficiencies greater than 99% can be achieved at two different frequencies under normal incidence. We employ a design scheme with graphene integration, which allows independent tuning of individual absorption frequencies by electrostatically changing the Fermi energy of the graphene layer. High absorbance is maintained over a wide incident angle range up to 50 degrees for both TE and TM polarizations. It thus enables a promising way to design electrically tunable absorbers, which may contribute toward the realization of frequency selective detectors for sensing applications. Metamaterial absorbers356791011141516171819202123252931323436363839404142In this paper, we numerically demonstrate, via highly confident numerical simulations, a dual-band metamaterial absorber where the two absorption bands can be independently tuned. This can be accomplished through the application of a voltage bias to modify the conductivity of the graphene layer that has been integrated into the metamaterial absorber super-unit-cell. The metamaterial device offers high absorbance over a wide range of incidence angles, and the independently tunable absorption bands might find applications in fields like tunable sensors and selective thermal emitters.ts = 0.285 μm, and the thickness of gold is tm = 0.1 μm. The periods along the x and y directions are Px = 14.4 μm and Py = 3.6 μm, the lengths of the bigger and smaller gold crosses are S1 = 3.0 μm and S2 = 2.0 μm, and their widths are W1 = 0.72 μm and W2 = 0.6 μm, respectively. The permittivity of the PTFE spacer is 2. The graphene layer is structured into interdigitated fingers with width Wg = 3.4 μm and spacing d = 0.2 μm. Two gate voltages, Vg1 and Vg2, are applied to the two graphene interdigitated finger sets in order to tune their Fermi energies independently.The schematic design of the proposed tunable metamaterial absorber is illustrated in S21 for transmission and S11 for reflection) and the absorbance was derived using The simulations were carried out using the well-established three-dimensional full wave electromagnetic solver, CST Microwave Studio 2012. We obtained the S parameters (ωp) and the scattering rate (Γ) for various Fermi energy as explained in Methods. By optimizing the thickness of the spacer ts = 0.285 μm, we obtained two absorption bands with peaks absorbance greater than 99%. When Ef = 0.2 eV, the two absorption peaks are positioned at wavelengths of 5.88 μm and 8.46 μm. Varying Ef from 0.2 eV to 0.8 eV, which could be accomplished by applying a voltage bias between the ground plane and graphene layer, both absorption peaks show blue-shift while the high absorbance is remained. The longer wavelength absorption peak shifts by ΔλS1 = 0.46 μm (5.4% shift), and the shorter wavelength absorption peak shifts by ΔλS2 = 0.15 μm (2.6% shift). The difference in the absorption wavelength shift is mostly due to the wavelength dependent change of the imaginary conductivity We first investigated a dual-band metamaterial absorber shown in H1 and H2, which apply to the corresponding two resonant structures. The wave reflected from the metamaterial absorber is quantified byIn order to further understand the blue-shift, we describe the metamaterial absorber as a combination of two independent transfer functions, E0 is the normally incident electric field. The absorbance is then given by ZG across the capacitor C. The entire circuit is backed by a transmission line of impedance ZL, representing the gold ground plane. The length of transmission line ZD is equal to the dielectric spacer thickness employed in the simulations and has the same effect of creating a resonant cavity between ZL and the RLC circuitwhere Zi to the wave incident from a transmission line of impedance Z0 = 377 Ω. The transfer functions are equivalent to field reflection coefficientsThe circuit presents a complex, frequency-dependent impedance ZL = 2 Ω was calculated using tabulated bulk gold conductivity valuesd~100 nm). The graphene impedance consists of real and imaginary parts,The load impedance Lk = 1/α is the kinetic inductance of the graphene and, being variable with  Ef, is the tuning mechanism of the absorber. By the equation for ZG it should be hypothesized that because ZG should play a minor role. However, this should not hold true at longer wavelengths and GR should begin to have a noticeable effect.The term Ri, Li, and Ci were determined empirically by matching the resonance frequency, Q-factor, and peak absorbance qualities of both resonances against simulations not involving any graphene layer. It was found that these factors were highly constrained if all three qualities were to be simultaneously matched. Numerical values for ZGi were calculated asThe values for Bi is a constant fitting parameter that is easily obtained by matching the magnitude of the frequency shift of the absorption peak over Ef = [0.2 : 0.8] eV. After ZGi was determined, the factors Ri, Li, and Ci were fine-tuned to give the final model parameters. These are summarized in where ZG shunted the entire RLC circuit or the LC sub-circuit, were also studied. In these cases, the absorbance bandwidth and/or the peak absorbance could not be kept constant in tuning over the entire Fermi energy range. By restricting the graphene effect to the capacitor alone, the net inductive reactance of Zi is largely maintained during tuning, being dominated by Li. Though the effective capacitive reactance changes, it has very little effect on the Q-factor. Thus, absorption bandwidth is almost completely unaffected during tuning. The net resistive impedance of Zi is also maintained, being dominated almost entirely by Ri for all values of Ef. This prevents any change in the peak absorbance during tuning. The third implication is the validation of the hypothesis that RG plays a required role in the tuning behavior at long wavelengths. By artificially zeroing RG in the circuit model, a negligible effect was observed on the CR2 resonant absorbance, whereas the CR1 resonant absorbance peak continually decayed during tuning.The equivalent circuit model results are summarized as the circles in Vg1 and Vg2, respectively. First, we calculated the absorbance spectrum of our absorber by changing the Fermi energy of graphene under the small resonators (CR2) via tuning bias voltage Vg2, while keeping the Fermi energy of graphene under the bigger resonators (CR1) fixed to be 0.5 eV. The simulated absorbance spectra are shown as the solid curves in λ2 = 5.88 μm to λ2 = 5.73 μm, a wavelength shift of 2.6%. An alternative expression of modulation is obtained by fixing the observation wavelength to λ2 = 5.88 μm. In this case one would observe an absorbance change λ1 = 8.23 μm arising from the bigger resonators remains nearly invariant. This implies that the two resonances are spectrally separated enough to prevent any noticeable mutual coupling. Thus we can independently tune the resonant frequency of the CR2 resonator by changing the Fermi energy of graphene under the small crosses. The same results can be obtained from the circuit model by fixing the Fermi energy of H1 to be 0.5 eV, which are shown as the circles in To investigate the independent tuning of each resonance, we adopt the device design shown in λ1 blue-shifts from 8.45 μm to 8.0 μm, a wavelength shift of 5.3%. An alternative expression of modulation is obtained by fixing the observation wavelength to λ1 = 8.45 μm. In this case one would observe an absorbance change Next, we calculated the absorption while varying the Fermi energy of the graphene layer under the bigger resonators (CR1) while fixing the Fermi energy of graphene under the smaller resonators (CR2) to 0.5 eV. The simulated absorbance spectra for various Fermi energies are shown as the solid curves in We have conducted additional simulations to investigate the behavioral dependence on angle of incidence. For these simulations we fix the Fermi energy of graphene under both resonators to be 0.4 eV, while maintaining all other aforementioned parameters. Absorbance as a function of wavelength and incident angle is shown in It should be noted that polarization-insensitive absorbers are highly desirable47484950We also note that the scattering rate of graphene may vary due to the impurities, defects, and interfacesIn conclusion, we showed that a dual-band absorber can be formed by using horizontally cascaded cross-shaped resonant structures at mid-infrared frequencies. Absorption peaks of greater than 99% were obtained under normal incidence, and high absorbance remained at large incident angles up to 50 degrees for both of the bands under TE and TM polarizations. Moreover, our metamaterial absorber structure enabled independent tunability of the two absorption bands by electrically controlling the Fermi energy of corresponding graphene layers. With its excellent performance, our design should be applicable in many fields such as tunable sensors and selective thermal emitters.tg = 0.5nm for all of our simulations. The effective dielectric constant of graphene can be calculated by tg is the thickness of graphene.The simulations were carried out using commercial Finite Element full wave electromagnetic solver CST Microwave Studio 2012. We employed a time-domain solver to obtain frequency-dependent absorption spectra in The complex surface conductivity of graphene can be calculated from the well-known Kubo formula and is described with interband and intraband contributions as32whereω is the angular frequency, Ef is the Fermi energy of graphene, Γ is the collision angular frequency, T is the temperature, kB is the Boltzmann constant, e is the elementary charge, and Ef, the intraband contribution dominates the graphene conductivity as the interband transitions are negligible due to Pauli blockingEf >0.2 eV for wavelengths of interest ranging from 5.5 μm to 10 μm. Then, graphene can be described by taking only the intraband part of the Drude-like conductivity as described in Eq. tg = 0.5 nm is the thickness of graphene layer. In our simulations, we set T = 300 K and scattering where The relationship of the Fermi energy 6 m/s), 2 and Kapton which require lower voltage bias while offering higher dielectric breakdown for our experimental demonstration. A recent demonstration shows that the gate voltage bias can be significantly reduced by applying a layer of ionic gel over graphene film with top gating architecturewhere How to cite this article: Zhang, Y. et al. Independently tunable dual-band perfect absorber based on graphene at mid-infrared frequencies. Sci. Rep.5, 18463; doi: 10.1038/srep18463 (2015).


The growth of graphene using resistive‐heating cold‐wall chemical vapor deposition (CVD) is demonstrated. This technique is 100 times faster and 99% lower cost than standard CVD. A study of Raman spectroscopy, atomic force microscopy, scanning electron microscopy, and electrical magneto‐transport measurements shows that cold‐wall CVD graphene is of comparable quality to natural graphene. Finally, the first transparent flexible graphene capacitive touch‐sensor is demonstrated. Therefore, a way forward to increase the throughput and reduce the production cost is to grow graphene in a cold wall CVD system which heats selectively only the Cu foils. Few types of cold wall CVD have been investigated so far for the growth of grapheneChemical vapor deposition (CVD) of monolayer graphene on copper2 fabricated on standard SiO2 substrates we measure a charge carrier mobility of 3300 cm2 V−1 s−1 and the quantum Hall Effect typical of single layer graphene. In contrast, the quality of graphene grown by hot‐wall CVD is often gauged only by carrier mobility,Here we report a completely new mechanism for the growth of graphene by resistively heated stage cold‐wall CVD which is markedly different from the growth mechanism of graphene in a hot‐wall CVD. Through a combined study of Raman spectroscopy, atomic force microscopy (AFM), and scanning electron microscopy (SEM) we elucidate the early stage formation of graphene by monitoring the transition from disordered carbon adsorbed on Cu to graphene. We also demonstrate for the first time 1) high‐throughput production, 2) ultralow cost, and (3) high quality monolayer graphene grown on Cu foils by resistively heated stage cold‐wall CVD. Our technique merges short deposition time (approximately few minutes) with high‐efficiency heating of a cold‐wall CVD system, resulting in ≈99% reduction in graphene production cost. The Raman spectra of our graphene films shows a low defect related peak and in devices with an area of 5600 μm high‐thr ultralowStudies of the growth mechanism of graphene on copper (using methane) in a hot‐wall CVD have thus far suggested the direct growth of 2D films involving several steps. The first step is the direct formation of 2D nuclei of grapheneFigure2/Si substrates using a wet transfer method.Our experiments show that the growth mechanism of graphene in cold‐wall CVD is markedly different from that of the hot‐wall CVD described above. Specifically, over a range of growth temperatures that we have investigated, we always observed a thick carbon film 100 nm), which forms in the early stages of the growth  ranging from 1 to 6 min. For all the samples we observe the characteristic peaks of sp2 bonded carbon atoms: the D‐peak at ≈1340 cm−1, the G‐peak around 1600 cm−1, the D′‐peak around 1620 cm−1, and the 2D‐peak at ≈2700 cm−1. For short tG  the D‐ and G‐peaks have considerable higher intensities than the 2D‐peak, which is typical of disordered carbon films.tG increases we observe changes in intensities, sharpness, and positions of the D and G peaks, and for tG > 4 min a well‐defined 2D‐band emerges. At the same time, AFM measurements show a reduction in the film thickness from 116 to 2.7 nm with increasing tG from 1 to 6 min , the full width at half maximum ) and the peak positions ). According to the three stage model for classification of disorder,ID/IG, FWHM and Pos(G) allow us to assess the ordering/amorphization in carbon materials ranging from graphite and amorphous carbontG = 1 min, the presence of a 2D peak with Pos(2D) = 2683 cm−1 and FWHM(2D) = 88 cm−1, the absence of a doublet in the D and 2D peaks, together with the overlap of G and D′ peaks indicate the formation of nanocrystalline graphite with no 3D ordering. Figure I2D and IG increase with increasing tG, whereas the ratio ID/IG decreases from ≈3.9 to 0.2 , and FWHM with increasing tG is consistent with the stage 1 ordering trajectory leading from nanocrystalline graphite to graphite. In this regime the size of sp2 clusters (La) increases with increasing ordering and can be estimated using the Tuinstra–Koenig relation ID/IG = C(λ)/La where C(532 nm) ≈ 4.96 nm.a ≈ 2 nm for tG = 1 min, which increases to La ≈ 25 nm for tG = 6 min as shown in Figure Lorentzian fitting of the D‐, G‐, and 2D‐peaks allows us to ascertain the structural ordering within the films by analyzing the band intensities  = 2678 cm−1 and FWHM(2D) = 30 cm−1 indicating the formation of monolayer graphene.For Figure2. Raman measurements confirm that these islands are composed of graphene. SEM analysis of films grown for 1000 and 1035 °C reveals a similar behavior of the saturation in island separation and a maxima in island size (see the Supporting Information). We observe that an increase in growth temperature leads to a reduction in the time required to achieve the maximum island size and to form a monolayer graphene as shown in the inset of Figure Ea = (Edes + Eatt – Ed – Ead)/2 and with the density of graphene islands Ni ≈ PCH4· exp(2Ea/KT), with Edes the desorption energy of a carbon monomer on the Cu surface, Eatt the barrier of attachment for the capture of a monomer by supercritical nucleus, Ed the activation energy of surface diffusion of a monomer, Ead the activation energy for dissociative adsorption of CH4 on Cu, PCH4 the methane partial pressure, K the Boltzmann constant, and T the growth temperature. Figure i has a dependence on growth temperature which is typical of the desorption controlled regime with an activation energy of 1.66 eV. The desorption model is also consistent with the formation of holes inside the islands at 8 min of growth , and FWHM for a 1 × 1 cm graphene film. Raman mapping measurements shown in the Supporting Information demonstrate the uniformity and high‐quality of the continuous films. The total processing time of this procedure is about 20 min (see the Supporting Information); this includes (1) heating up time for the CVD system from room temperature to the growth temperature, (2) Cu foil annealing time, (3) graphene nucleation and growth time, (4) cooling down time for the system to room temperature. The demonstrated processing time is significantly shorter than the processing time needed by hot‐wall CVD .−2 (see the Supporting Information). Compared with other CVD studies and neglecting the base cost of copper we see a reduction in the production costs of 98.83%–99.89%. This extraordinary reduction in the production cost, together with the possibility of reconstitution of high purity copper from etchant solutions by electrolysis that can yield up to 99% of the original foil,Having established the initial stages of graphene formation, we investigate the transition from graphene islands to a continuous film. Figure th time,  cooling 2/Si substrates. Using the parallel plate capacitor model we estimate the field effect mobility to be 3300 cm2 V−1 s−1 at 1.4 K and 2773 cm2 V−1 s−1 at room temperature. This mobility, measured across a large area device (≈0.05 mm2), is comparable to the mobility measured in smaller area devices of graphene either grown by CVD (50 μm222) and deposited on oxidized silicon substrates.2 V−1 s−1) multiplied by the area of the device (μm2). As shown in the Supporting Information, graphene grown by resistive heating cold‐wall CVD has Q ranging from 4 × 106 to 7.2 × 106, whereas most reports of monolayer graphene grown by hot‐wall CVD have Q ranging from 103 to 7 × 106. Hence cold‐wall CVD grown graphene has a narrow spread of Q stemming from a reproducible high quality growth process. This is in stark contrast to the spread of Q over three orders of magnitude reported for hot‐wall CVD grown graphene.To ascertain the quality of the electronic properties of graphene produced by cold‐wall CVD we characterized the charge carrier mobility in transistor devices fabricated on SiOvice ≈0.0 mm2, is Figure2, see inset) fabricated on standard SiO2/p‐Si substrates. The Si substrates are heavily doped and act as the gate electrode. By applying a voltage to the gate (Vg) we tune the carrier concentration from 3 × 1011 to 6 × 1012 cm−2. The charge neutrality point is at 0 V, indicating low residual doping levels in our samples. Figure σxy) against the applied Vg, taken at 13 T and at a temperature of 250 mK. Clearly developed conductance plateaus are visible when the Fermi energy is within a Landau level (N) that correspond to the conductance relationship σxy = (N + 1/2)4e2/h, typical of the half‐integer quantum Hall effect (QHE) of monolayer graphene with two‐fold spin and valley degeneracy.ρxx = 0 Ω where the Fermi energy is within the N = 0 Landau level for both electrons and holes, indicating that the graphene quality is high enough to observe the localization in the QH regime. At the same time, σxx shows well‐defined Shubnikov–de Haas oscillations which are periodic with the applied gate voltage. A color map of the differential Hall conductance plotted against perpendicular applied field and charge carrier concentration shows that Landau levels up to N = 6 are visible at high fields , therefore allowing us to demonstrate for the first time a flexible and transparent capacitive touch sensor using graphene for both the top and bottom electrodes.ρ ≈ 1.3 kΩ and the contact resistance <68 Ω. A detailed description of the fabrication procedure and electrical characterization is provided in the Supporting Information. The individual elements of the touch sensors are formed at the intersection between the graphene strips and each element represents a parallel plate capacitor. As pressure is applied to an element of the array, the dielectric elastically deforms reducing the spacing between the graphene electrodes resulting in an increase in capacitance. Figure Figure C = 6 pF was observed with a return to the original state after unloading. The sharp change in capacitance demonstrated indicates a fast responsivity to loading and unloading of the element. Finally we tested the flexibility and durability of the devices by bending the substrate and systematically measuring the resistance of the graphene strips across the device. Figure To test the responsivity of the device we periodically loaded and unloaded an element with a human finger and measured the change in capacitance shown in Figure In summary, we have shown a new growth mechanism of graphene by cold‐wall CVD, which starts with the formation of a thick carbon film in the early stages of the growth, that becomes progressively thinner with increasing the growth time and finally evolves into graphene islands. At the same time we demonstrate an extremely high‐throughput and cost efficient growth procedure for preparing high quality monolayer graphene using cold‐wall CVD. Finally, we use graphene as electrode material and demonstrate the first flexible and transparent graphene capacitive touch sensor using processing techniques that are compatible with existing transparent and flexible electronic technologies. Besides its importance for the quick industrial exploitation of graphene since cold‐wall CVD systems are found in semiconductor industries manufacturing plants, our work could lead to new generations of flexible electronics and offers exciting new opportunities for the realization of graphene‐based disruptive technologies.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


This functional contrast agent is intended for the specific and non-invasive detection of prostate cancer cells that are PSMA positive, a marker implicated in prostate tumor progression and metastasis. The two-step carbodiimide reaction used to conjugate the antibody to the nanoparticle was efficient and we obtained an elemental iron content of 1958±611 per antibody. Immunofluorescence microscopy and flow cytometry showed that the conjugated muJ591:MIRB complex specifically binds to PSMA-positive (LNCaP) cells. The muJ591:MIRB complex reduced cell adhesion and cell proliferation on LNCaP cells and caused apoptosis as tested by Annexin V assay, suggesting anti-tumorigenic characteristics. Measurements of the T2 relaxation time of the muJ591:MIRB complex using a 400 MHz Innova NMR and a multi-echo spin-echo sequence on a 3T MRI  showed a significant T2 relaxation time reduction for the muJ591:MIRB complex, with a reduced T2 relaxation time as a function of the iron concentration. PSMA-positive cells treated with muJ591:MIRB showed a significantly shorter T2 relaxation time as obtained using a 3T MRI scanner. The reduction in T2 relaxation time for muJ591:MIRB, combined with its specificity against PSMA+LNCaP cells, suggest its potential as a biologically-specific MR contrast agent.In this study we developed, characterized and validated  In the last 10 years, the incidence of prostate cancer has steadily risen, remaining the second most common cancer diagnosed in men worldwide. In Canada alone, it accounts for roughly 27% of newly diagnosed cancers in 2012 in vivo prostate cancer specific targeting strategies Studies have suggested that, by specifically targeting prostate specific membrane antigen (PSMA) expressing cells, both localized and aggressive conditions can be treated PSMA receptors are localized primarily to the apical plasma membrane of prostate epithelium and play an integral role in the progression of prostate malignancies, probably by promoting anti-apoptotic signaling, ensuring cell resilience and promoting cell proliferation Endorectal magnetic resonance imaging (MRI) is becoming common part of the local work-up for prostate cancer. Dynamic studies using gadolinium-DTPA contrast agents have demonstrated useful enhancement of tumor images In this study, we developed a functional super-paramagnetic iron oxide (SPIO)-based MR contrast agent that specifically attaches to PSMA receptors for MR imaging of prostate cancer cells. We present a method to develop this functional MR contrast agent using a commercially available SPIO, Molday ION Rhodamine-B Carboxyl (MIRB), conjugated to the anti-PSMA receptor antibody J591. We characterized and validated this antibody:SPIO-nanoparticle complex for specific detection of PSMA positive prostate cancer tumor cells using immunofluorescence, flow cytometry and 3T clinical MRI. Our new contrast agent can be used for the specific and non-invasive detection of prostate cancer cells with PSMA, a marker implicated in prostate tumor progression and metastasis Murine J591 mAb, at a concentration of 5 mg/mL and with known specificity for PSMA, was procured from Dr. Neil H Bander's laboratory  through an institutional material transfer agreement. The isotype control antibody for synthesis and cell culture experiments was a murine IgG1  at a concentration of 5 mg/mL in 1× phosphate buffered saline without calcium or magnesium ions , as per manufacturer's recommendation.N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-(4-Morpholino)ethanesulphonic acid hydrate (MES), sodium bicarbonate, HPLC-grade water (ChromoSolv Water), chloroform, acetonitrile, and deuterium oxide, were purchased from Sigma Aldrich, Oakville, ON, Canada, unless otherwise stated.Molday ION Rhodamine Carboxyl  was used to prepare the antibody:SPIO complexes. All the reagents required for the conjugation reactions and analysis, including Antibodies were conjugated to MIRB based on a modification of the manufacturer's method . Briefly, MIRB (0.2 mg of iron) was added to 100 µL of muJ591 or muIgG at 1 mg/mL in 1× PBS buffer (pH 7.65) in sterile 1.5 mL microfuge tubes. The antibody with MIRB solution was lightly vortexed prior to the addition of 100 µL of 0.1 M sodium bicarbonate buffer (pH 8.0), ice incubated for 15 min with occasional vortexing, then kept 24 h at 4°C in a rotator mixer. The overnight incubated antibody:MIRB were mixed with 50 µL of 2 mg/mL EDC solution in deionized water plus 100 µL of MES buffer (pH 5.6) and kept at 37°C for 5 min with occasional vortexing, prior to addition of 50 µL of 2 mg/mL NHS and incubation at 37°C for further 15 min, with occasional mixing. The solution was then cooled on ice for 15 min and kept at 4°C for 48 h in a rotator mixer. After 48 h the mixture was transferred to a sterile 15 mL conical tube and rinsed twice with 500 µL 1× PBS buffer to extract out the antibody:MIRB conjugates attached to the tube wall. In order to remove any unbound antibodies, the reaction mixture was transferred to a fresh sterile 15 mL conical tube containing 50 µL of 2 mg/mL NHS plus 150 µL of 0.25 M MES buffer and vortexed. This tube was kept overnight at 4°C in a rotator mixer, washed with an equal volume of 0.1 M Sodium bicarbonate buffer (pH 8.0) and centrifuged using 10 K Amicon Ultra Centrifugal Filters , based on the manufacturer's protocol for ultrafiltration. The solution retained in the filtration column, containing the remaining antibody:MIRB conjugates, was washed with 0.01 M sodium bicarbonate (pH 7.65) using the buffer exchange steps described in the manufacturer's protocol. Each of the eluants was analyzed for both antibody and iron contents using the Bradford protein assay kit and inductive coupled plasma atomic emission spectroscopy (ICP-AES) based assay, respectively. The total protein concentration of the conjugates was determined by Bradford protein assay using gradient concentration of BSA and human γ-globulin as standards for indirect estimation of antibody content.ICP-AES was used to determine the elemental iron contents of the antibody:MIRB conjugates. Antibody:MIRB conjugates were acid digested using an equal volume of 70% nitric acid in deionized LC-MS grade water, in a final sample volume of 6 mL; a buffer only (negative control) was similarly digested. All the samples were run on the Varian Vista Pro CCD simultaneous ICP-OES instrument .The complex muJ591:MIRB was immobilized on a Nucleopore membrane  and heated at 57°C in a hot air oven for 25 min to remove moisture. The immoblized complex was then mounted and sputter-coated with carbon. SEM images were acquired at 5000 and 10,000× magnification using a Hitachi Su-70 Schotty Field Emission SEM  with a resolution of 1.5 nm at 1 kV. The same system was used to perform Energy Dispersive X-ray (EDX) spectroscopy at 5000× magnification to confirm the presence of organic matter and SPIO nanoparticles.Particle size of muJ591:MIRB conjugate was determined by dynamic light scattering. The muJ591:MIRB conjugate was dispersed in 0.9% saline at a protein concentration of 0.01% w/v. Measurements were carried out using a Zetasizer Nano ZS instrument . The particle size results were reported as the harmonic intensity averaged particle diameter (or Z-average) and they are expressed in nanometers.To determine whether the muJ591:MIRB conjugate generated from the labelling reactions retained the super-paramagnetic characteristics required of an MR contrast agent, we measured the T2 relaxation time in NMR. Samples with equal total protein concentration (0.04 µg/µL) of muIgG:MIRB and muJ591:MIRB complexes were separately prepared in 600 µL of deuterium oxide and transferred into Wilmad NMR tubes . T2 relaxation times were obtained after optimizing the magnetic field of the Innova Unity 500 NMR machine and samples were run as per the manufacturer's recommendation.max) using a Synergy4 fluorimetric plate reader . The peak λmax and λmax shift were determined by plotting the fluorescence intensity values at different excitation and emission wavelengths.To test whether the conjugation reaction caused fluorescence enhancement or quenching of the Rhodamine-B fluorophore on the MIRB conjugates we performed a fluorimetric analysis. MIRB nanoparticles and muJ591:MIRB complexes samples with an equal iron concentration were analyzed for fluorescence intensity and wavelength at peak intensity  and 1% antibiotics/antimycotics. The cells were grown to 80% confluence in 75 cm2 vented cap, canted neck falcon flasks  with media replenished every three days.PSMA-positive LNCaP cells (CRL-1740) and PSMA-negative DU145 cells (HTB-81) were procured from American Type Cell Culture, Manassas, VA, USA. All cells were grown in a 37°C, 5% COCells were seeded at an initial concentration of 1000 (for DU145) and 2000 cells (for LNCaP) per well in 96 well plates and grown for 3 days before performing the experiments. Immunofluorescence studies were then conducted on both LNCaP and DU145 after treatment with 0.1 µg/µL muJ591:MIRB for 1 h with or without addition of secondary antibody conjugated to AlexaFluor-488 fluorophore. Then, cells were incubated with warm 0.5% BSA in RPMI media to encourage cells to internalize the antibody. The cells were then visualized at different time points  under a standard inverted fluorescence microscope  attached to a closed circuit camera .A flow cytometric assay was performed based on a modification of the method described by Peldschus K et al (2010) 2 for 40 minutes. Cells were finally washed with serum-free RPMI-1640 until cells in the uncoated wells stopped detaching. All cells were then fixed using ice-cold methanol and stained with 0.1% crystal violet. Filtered 2% SDS was added and the plates were incubated with agitation at room temperature for 30 minutes, after which time, absorbance readings at 550 nm were taken using the PowerWave XS plate reader . We compared the absorbance readings from the muJ591- and muJ591:MIRB-coated plates with respect to the untreated control wells to analyze adhesion.Cell adhesion after treatment of plates with muJ591 and muJ591:MIRB was evaluated. The muJ591:MIRB complex and muJ591 mAb was prepared in 1× phosphate buffered saline (PBS) at a concentration of 0.01 µg/mL. We then precoated 96-well plates with 100 µL of muJ591:MIRB complex and muJ591 mAb in quadruplicates, and incubated overnight at 4 °C for immobilization of the complex on the plate surface. After overnight incubation, we aspirated out the solution and blocked the wells with 1% BSA in 1× PBS for at least 10 minutes and aspirated out again. Non-treated wells were used as control. About 100,000 LNCaP or DU145 cells were then seeded in the precoated and control wells and incubated at 37°C, 5% CO2 for 2 hours followed by 20 min incubation with agitation in the dark at room temperature. The fluorescence intensities were then measured at 2, 6, 12 and 24 h post muJ591:MIRB treatment using the FLx800 plate reader at excitation of 540 nm and emission of 590 nm.For cell proliferation assessment, a resazurin-based fluorimetric assay  was performed. After the 96 well plates were precoated with muJ591:MIRB or 1× phosphate buffered saline (control) and blocked with 1% BSA as described above, 1000 DU145 or 2000 LNCaP cells were seeded. The cells were grown until control wells reached 80% confluence. The LNCaP and DU145 cells were further treated with muJ591:MIRB complex (0.01 µg/mL) or serum free media (control) for 1 h, after which media was replaced with serum free media. The Resazurin solution was added, as recommended by the manufacturer's protocol, and plates were further incubated at 37°C, 5% COFor determining cell apoptosis and death caused by the muJ591:MIRB complexes on LNCaP cells, an Annexin V-FITC assay was performed . Cells were dislodged from the flasks using 2 mM EDTA in 1× phosphate buffer saline, pelleted and resuspended in sterile, supplemented media, and then seeded in 6 well culture plates at a concentration of 10,000 cells per well. Once cells reached 80–90% confluence, muJ591:MIRB or control  treatment was added to the wells for 1 h followed by 3 washes in phosphate-buffered saline (PBS). Flow cytometric analysis was then performed based on method described by Richard et al (2010) To validate we could detect changes on the T2 relaxation time with a clinical MR scanner we performed MR imaging of the conjugates first and then conjugate-treated cells. The muJ591:MIRB and muIgG:MIRB complexes, as well as muJ591, muIgG and MIRB alone were suspended on PBS at different concentrations . Then, 100 µL of serum free media were added to 96-well plates (n = 3) and placed on the MR scanner.in vitro cell MR imaging, both LNCaP and DU145 cells were plated, in triplicate, at concentrations of 105 cells/well in 96 well plates and grown for 36–48 h. The cells were then treated with 100 µL of antibody:MIRB conjugates or non-conjugated antibodies for 1 h, rinsed 2× times with serum free media, 1× time with 1× Tris-EDTA buffered saline, and 1× times with 0.01 M Sodium bicarbonate buffer to remove unbound conjugates, and then resuspended in 100 µL serum free media and placed on the MR scanner. Different concentrations of muJ591:MIRB, muJ591, muIgG:MIRB and muIgG  were used.For 5, 7.55, 55, 2.55 and 1.255 cells/well and grown for 48 h. The cells were then treated with 100 µL of muJ591:MIRB for 1 h, rinsed 2× times with serum free media, 1× time with 1× Tris-EDTA buffered saline, and 1× times with 0.01 M Sodium bicarbonate buffer to remove unbound conjugates, resuspended in 100 µL serum free media and placed on the MR scanner.To determine the limit of detection LNCaP cells were plated in 96 well plates, in triplicate, at concentrations of 10A clinical 3T MRI scanner  with a small surface Flex coil was used. Images were acquired with a multi-echo spin echo sequence with 64 echo times spaced by 5.8 ms . The intensity as a function of echo time was fitted to:Z is the intensity at echo time TE; A, B and T2 are the fitting parameters Where MAll statistical analyses were performed using Graphpad Prism software . All data were tested for normality and homogeneity of variances using a Shapiro-Wilks test and a Bartlett's test, respectively, before choosing a suitable parametric or non-parametric statistical test. A resulting p-value of less than 0.05 was considered to be significant. Parametric data sets were analysed using Student's t-tests or one-way ANOVAs, followed by an appropriate post-hoc such as a Tukey HSD. Non-parametric data sets were analysed using one-way Kruskal Wallis ANOVA followed by a Nemenyi's post hoc, and Friedman's tests followed by a pair-wise comparison using a Wilcoxon signed rank test with a Bonferroni adjustment.ICP-AES was used to estimate the amount of elemental iron present in each of the conjugated muIgG:MIRB and muJ591:MIRB complexes. We observed that muJ591:MIRB complexes had 1958±611 (n = 8) elemental iron per antibody while muIgG:MIRB complexes had 2906±631 (n = 8) elemental iron per antibody. The iron nanoparticles presence on the muJ591:MIRB complex was further confirmed by Energy Dispersive X-ray spectroscopy . The preThe presence of the muJ591:MIRB complex immobilized on salt crystals from the buffer can be observed in the superposed EDX-SEM images . EDX mapThe average particle sizes for the muJ591:MIRB conjugates were 34.75±14.41 nm with a polydispersity index (PdI) value of 0.23±0.01. For comparison, we performed particle size measurements for the MIRB nanoparticles alone and obtained 34.64±10.83 nm with a PdI of 0.25±0.02 which are within the values reported by the manufacturer (35 nm).Immunofluorescence microscopy was used to determine whether PSMA positive LNCaP cells were specifically detected by the muJ591:MIRB complex. max of MIRB alone and muJ591:MIRB complex  treated LNCaP cells. The complex antibody:MIRB demonstrated faster T2 relaxation time with increasing iron concentration . For nonLive PSMA-positive (LNCaP) prostate cancer cells showed a reduced T2 relaxation time when treated with the muJ591:MIRB complex , and T2 5 or lower seeded cell counts, showing there is a limit on the detection provided by the MRI that depends on the cell population.For live PSMA-positive (LNCaP) prostate cancer cells treated with muJ591:MIRB the T2 relaxation time became faster for higher seeded cell counts . No chanLNCaP and DU145 prostate cancer cells also showed a reduced T2 relaxation time when treated with the MIRB nanoparticle alone, with a faster T2 for higher iron concentrations. LNCaP cells treated with MIRB alone at an iron concentration of 0.125 µg/µL showed a T2 of 385±33 ms, whereas for muJ591:MIRB-treatment at 0.1 µg/µL, the T2 was 272±18 ms, showing that at a similar iron concentration, we can still differentiate between MIRB uptake and muJ591:MIRB binding by the T2 relaxation time values. DU145 cells treated with MIRB alone at an iron concentration of 0.125 µg/µL had a T2 of 161±18 ms, and this value was consistently faster than DU145 cells treated with muJ591:MIRB. This showed that both cell lines were taking up MIRB when it was not conjugated, and once MIRB was conjugated to antibody, only the PSMA-positive LNCaP cells were detected as changing their T2 relaxation time values. This confirmed that muJ591:MIRB complex detection of PSMA-positive LNCaP cells using a clinical 3T MRI was feasible and sensitive for iron concentrations as low as 0.02 µg/µL (0.035 µg/µL protein concentration).in vitro, flow cytometry, and immunofluorescence based methods.We were able to stably conjugate the prostate cancer-detecting muJ591 antibody and MIRB to generate antibody:SPIO complexes with an iron to antibody concentration that is sufficient to serve as a functional MR contrast agent detectable by clinical MRI Several studies have shown how antibodies conjugated to super-paramagnetic iron oxide particles were used for specific MR imaging of human lymphocytes, the Her-2/neu receptor in breast cancer cells, and apoptotic cells When 1 µg/µL of muIgG antibody or muJ591 antibody were conjugated to MIRB using our two-step carboiimide reaction, we recovered in average 0.45±0.03 µg/µL of muIgG:MIRB and 0.56±0.05 µg/µL of muJ591 antibody:MIRB complex. This protein recovery after the conjugation was estimated by Bradford protein assays performed before and after the reaction.The MIRB cluster-conjugated muJ591 antibody had similar values of elemental iron per antibody to the muIgG antibody. It is therefore likely that we have similar nanoparticle per antibody ratio and the muIgG is therefore an acceptable control. However, when the T2 relaxation time was obtained, the muIgG:MIRB had a slower T2 which was very closer to the value obtained for the MIRB nanoparticle alone. The conjugation with the muJ591 antibody seems to have an effect on the relaxation time in a favorable direction to its detection since the T2 relaxation time becomes faster.Note that, due to limitations in sample preparation and the resolution of the SEM instrument which we used, we were not able to accurately estimate the particle size of the conjugates on the SEM images. The drying step performed during the sample preparation step to immobilize the muJ591:MIRB conjugates on to the Nucleopore filters resulted in formation of crystalline buffer salts which are prominently seen in the SEM images as large micrometer-size structures. However, by performing the EDX elemental mapping we were able to confirm the presence of the muJ591:MIRB conjugates immobilized within these structures. We therefore used dynamic light scattering to obtain particle sizes of the conjugates.When equal protein concentrations of antibody:MIRB conjugates were subjected to NMR analysis, we found that the T2 relaxation time of the muJ591:MIRB was similar to the one for the muIgG:MIRB complex. This could be attributed to the similar amount of elemental iron contents and molecular size of the conjugates. The T2 relaxation time measurements with the MRI showed differences between the muIgG:MIRB and the muJ591:MIRB complexes. The difference between NMR and MRI results can be explained by the use of a standard multi echo spin echo sequence on the MRI and calculations based on processed MR images intensity changes. Nevertheless, our findings showed that the antibody:MIRB complex presents a significantly faster T2 relaxation time that makes it a good candidate as MR contrast agent.max values as well as the fluorescence intensity of Rhodamine-B fluorophore in the MIRB nanoparticles. The observed Rhodamine-B quenching was exploited to indirectly determine the success of antibody conjugation to MIRB nanoparticles. Fluorescence intensity measurements were performed routinely as quality control assessment measure following each synthesis experiments and to determine the overall loading concentration. Our results agree with previous reports that indirectly suggest that the solvent polarity and proximity of quenching species to the metal-fluorophore conjugation system may play a role in the fluorescence quenching The success of our two-step carboiimide conjugation could be confirmed by determining the changes in peak λin vitro experiments, the muIgG:MIRB conjugates were easily cleared following the washes during the immunostaining steps while the muJ591:MIRB conjugates were found to remain adherent to the PSMA expressing LNCaP cells. Thus by using muIgG antibody as a control, we ensured the overall efficacy and functionality of the complexes.Our findings suggest that the binding of the conjugate muJ591:MIRB to the PSMA-positive cells is highly specific. The control muIgG:MIRB conjugate could not be detected in the MRI images or T2 relaxation time maps of the cells. We have used a muIgG antibody as an experimental control antibody for both synthesis and validation studies. Several studies used IgG antibody to demonstrate the specificity of their functional MR contrast agents in vivo studies. We first determined the best buffer in which the antibody:MIRB conjugates would maintain stable surface charge and remain in solution without flocculation or aggregation during storage. We performed a zeta potential analysis (data not shown) and observed that the 0.01 M sodium bicarbonate buffer could maintain uniform surface charge of antibody:MIRB complex. Preliminary in vivo tests were performed  and confirmed 0.01 M sodium bicarbonate as the best buffer for in vivo use.We additionally determined potential stability issues while formulating the conjugates in different buffers in order to maximize the functionality and specificity of the antibody:MIRB conjugates for future in vitro. Flow cytometry-based detection showed as well that the muJ591:MIRB was binding to LNCaP cells.Androgen-dependent LNCaP, which specifically express PSMA receptors when compared to other prostate cancer cell-lines Flow cytometric and immunofluorescence approaches were both successful in discerning the binding capacity and also the relative uptake capacity of muJ591:MIRB conjugates in both DU145 and LNCaP cells. Flow cytometric-based detection was more accurate in determining the extent of binding, which is especially useful when picogram level of contrast agents are used. A future line of experiments will be to determine the antibody:MIRB uptake kinetics using confocal microscopy.We could detect the presence of muJ591:MIRB on live PSMA positive cells using a clinical 3T MR scanner. The change in T2 relaxation time was significant and standard multi-echo, spin-echo sequences allowed us to quantify it. It should therefore be possible to obtain images using standard T2-weighted imaging at echo times selected by the T2 relaxation time value of the complex, as well as T2-mapping techniques. There is however a limit on the detection capabilities of the technique related to the amount of cells and therefore SPIO signal.Because prostate cancer cell lines take up non-conjugated MIRB and this also modifies the T2 relaxation time, special caution should be taken when comparing non-conjugated SPIO-based contrast agents to the antibody:SPIO agents. However, the fact that non-conjugated MIRB is taken up by multiple cells and the antibody:MIRB only by specific cell types should give the necessary specificity to detect PSMA-positive cells only.in vitro detection using the MR scanner we suspended cells on serum-free media during the imaging. Other more tissue-like experimental settings such as agar or agarose were preliminary investigated but they were found to strongly modify the T2 relaxation time and reduce the sensitivity of the detection.For live cell Our findings showed that the muJ591:MIRB treated LNCaP cells showed significantly reduced cell adhesion and increased Annexin V expression, along with significant reduction in cell proliferation. Tumor cells that are less adherent may be more likely to cause metastasis as there is enhanced migratory and invasive potential. At the same time, some studies also show that certain tumor cells that become less adherent may undergo anoikis, a form of apoptosis induced by anchorage-dependent cells detaching from extracellular matrix (ECM) in vivo images should be done at different time points. A longer time course study would be necessary for long term in vivo effects targeting PSMA-expressing cells.Knowing that muJ591:MIRB likely has pro-apoptotic effects on prostate cancer cells, care should be taken with the imaging timing to strike a balance between detection of treated cells and their death due to the muJ591. The Recent years have given rise to the field of theranostics, whereby non-invasive detection and treatment are possible due to advances in nanoparticle synthesis and advanced imaging methods. Theranostic nanomedicines, mostly nanoparticles carrying therapeutic or diagnostic markers in form of targeted small interference RNA or antibodies, have been designed to improve current cancer therapies thus addressing specific limitations of current treatment and diagnostics approaches in vivo prostate cancer MRI studies.In conclusion, we demonstrated the generation and characterization of muJ591:MIRB nanoparticle conjugates, which can specifically detect PSMA expressing LNCaP cells using 3T Clinical MRI. Our findings suggest that muJ591:MIRB could be a promising diagnostic agent that shows therapeutic effects —a possible prostate cancer theranostic agent. This study also paves the way for our next goal, to appropriately validate the MR contrast agent for


Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease of unknown origin. Recent findings suggest that IPF results from multiple factors that eventually lead to interstitial lung injury. In the pathogenesis it is likely that complex relationships between genetic predispositions, environmental exposures, and lung infections promote the fibrotic processes causing IPF; it is this complexity and the multiplicity of causes that make the population and clinical course of IPF so heterogeneous. Thus, it is clear that one common factor driving IPF pathogenesis in all patients would be far too simplified of an understanding. In recent years, efforts have been made in finding therapeutic strategies that target disease progression rather than disease onset. The biochemical composition and abnormal stiffness of the matrix might be crucial in controlling the cellular phenotype in fibrotic lungs that promotes disease progression and persistence. Though there has been substantial progress in the IPF field in recent years, much more work is required in order to improve the prognosis associated with this disease. Idiopathic pulmonary fibrosis (IPF) is one of the most common diseases classified as a rare disease. The prognosis of IPF is poor, with most patients succumbing to their illness at a rate comparable to aggressive cancers . The cauUnlike fish or amphibians, evolution has favoured fibrogenesis over regeneration in complex organisms such as mammals , 6. Evenin vivo, that interleukin-1β induces an early inflammation promoting the activation of pro-fibrotic pathways through transforming growth factor (TGF)-β1, able to self-sustain up to day 60 independently of any signs of residual inflammation and trigger clustering of myofibroblasts and collagen similar to myofibroblastic foci observed in humans [Exposure to inhaled environmental agents, most of all cigarette smoke, represents an important risk factor for IPF. The increased risk of developing IPF remains even after smoke cessation, suggesting the establishment of self-sustained (or autocrine) mechanisms after the initial injury , 10. Cign humans . This hiIn vitro, the differentiation of myofibroblasts is strongly correlated with substrate stiffness; it is clear that stiff substrates promote the production of profibrotic mediators and ECM deposition [in vitro approaches strongly suggest that stiffness alone can drive myofibroblast activation and subsequent ECM deposition. However, they lack the spatial cues that ECM and growth factors encounter in the 3D fibrotic lung in vivo. Booth et al. [. [Myofibroblasts are the major producers of the fibrotic extracellular matrix (ECM) which results in the characteristic stiffness of a fibrotic lung, decreased lung volumes, and shortness of breath in patients. position , 18, wheposition . Activatposition –22. The position . These ih et al.  showed, h et al. . These nh et al. . Moreovet al. [.  demonstrt al. [. . FurtherIPF is a complex disease involving multiple steps which eventually overcome physiological repair mechanisms and lead to fibrosis. Even if the etiologic events causing the onset of IPF remain unknown, decades of research have highlighted the fact that fibrogenesis requires a combination of several factors which cause both epithelial injury and impaired wound healing. It is this complexity and the multiplicity of causes that make the population and clinical course of IPF so heterogeneous. For the time being, it seems more realistic to continue investigating therapeutic strategies that limit disease progression rather that prevent its development. Due to the multiple pathways involved in abnormal fibrogenesis, multi-target therapies appear essential.


In metastatic breast cancers, the acquisition of metastatic ability, which leads to clinically incurable disease and poor survival, has been associated with acquisition of epithelial-mesenchymal transition (EMT) program and self-renewing trait (CSCs) via activation of PI3K/AKT and IL6/JAK2/STAT3 signaling pathways. We found that TrkB is a key regulator of PI3K/AKT and JAK/STAT signal pathway-mediated tumor metastasis and EMT program. Here, we demonstrated that TrkB activates AKT by directly binding to c-Src, leading to increased proliferation. Also, TrkB increases Twist-1 and Twist-2 expression through activation of JAK2/STAT3 by inducing c-Src-JAK2 complex formation. Furthermore, TrkB in the absence of c-Src binds directly to JAK2 and inhibits SOCS3-mediated JAK2 degradation, resulting in increased total JAK2 and STAT3 levels, which subsequently leads to JAK2/STAT3 activation and Twist-1 upregulation. Additionally, activation of the JAK2/STAT3 pathway via induction of IL-6 secretion by TrkB enables induction of activation of the EMT program via induction of STAT3 nuclear translocation. These observations suggest that TrkB is a promising target for future intervention strategies to prevent tumor metastasis, EMT program and self-renewing trait in breast cancer. There are several discrete steps in the biological cascade of metastasis, loss of cellular adhesion, increased motility and invasiveness, entry and survival into circulation, exit into new tissue, and eventual colonization of a distant site , 2. Durihigh/CD24low antigenic phenotype, which results in their having stem cell-like characteristics (CSCs). There is accumulating evidences of a role of PI3K/AKT and JAK/STAT3 signaling pathways in tumor metastasis and EMT. Activation of the PI3K/AKT pathway, IL-6/JAK2/STAT3 pathway, and stem cell-like characteristics contribute to the poor outcomes of metastatic breast cancers [Metastatic breast cancers are more chemoresistant forms of breast cancer with expression of high levels of EMT markers and exhibit a CD44 cancers , 9. More cancers , while i cancers , 12.Independent of these findings, TrkB, a member of the tropomyosin-related kinase (Trk) family of neurotrophin receptors, is critical to biological processes in the developing and mature nervous systems, including neuronal growth, differentiation, and survival . Also, TIt is reported that ectopic TrkB expression in RIE-1 cells suppresses anoikis through AKT activation . HoweverWe initially examined whether the elevated levels of TrkB associated with the increase in phosphorylation of TrkB. As shown in Figure These observations led us to speculate that TrkB might modulate expression or activation of c-Src. To determine whether TrkB contributes to regulation of c-Src in MAD-MB-231 cells, we selected highly metastatic MDA-MB-231 and Hs578T cells stably expressing the TrkB-shRNAs. TrkB-shRNA partially suppressed the expression of endogenous TrkB  in MDA-MB-231 and Hs578T cells . We nextTo further investigate the induction of c-Src phosphorylation by TrkB, we examined whether TrkB directly interacts with c-Src in MDA-MB-231 cells. Interestingly, endogenous TrkB was able to interact with endogenous c-Src under physiological conditions Figure . To confTherefore, we examined whether c-Src activation is involved in the TrkB-induced Mek1 and AKT activation as well as cyclin D1 expression. To test this notion, we selected MDA-MB-231 cells stably expressing the c-Src-shRNAs. As shown in Activation of STAT3 is required for c-Src-mediated proliferation, survival and transformation –31, and Given-mentioned role of TrkB as c-Src activator via direct interaction, it is possible that the TrkB/c-Src complex could activate JAK2 via interaction. Indeed, we found that both TrkB and c-Src interacted with JAK2 after transient transfection . MoreoveInterestingly, MDA-MB-231 TrkB-shRNA cells demonstrated much lower Twist-1 and Twist-2 mRNA levels than MDA-MB-231 cells treated with SU6656 Figure . AdditioWe next attempted to investigate whether TrkB knockdown, which was identified above as being activated by the JAK2/STAT3/Twist-1 axis without c-Src, affects activation of the JAK2/STAT3/Twist-1 axis in Hs578T and MDA-MB-231 cells. Immunoblotting analysis revealed that JAK2 and STAT3 phosphorylation levels were markedly reduced in Hs578T and MDA-MB-231 TrkB-shRNA cells relative to its control-shRNA cells. Interestingly, the JAK2, STAT3, and Twist expression levels were also significantly reduced in Hs578T and MDA-MB-231 TrkB-shRNA cells Figure . AdditioBecause TrkB knockdown reduced the JAK2 protein levels, we initially focused on JAK2 expression. TrkB knockdown significantly reduced the JAK2 protein levels, but had no effect on JAK2 mRNA levels Figure , suggest+CD24+ and CD44+CD24− breast cancer cells have high phospho-STAT3 via their expression of genes such as IL6, PTGIS, and HAS1, which activate an autocrine loop [STAT3 phosphorylation by IL-6 promotes its nuclear translocation and DNA binding followed by the transactivation of genes involved in a number of cellular functions, including proliferation, differentiation, and survival . Moreover, the IL-6/STAT3/JAK2 pathway is involved in the maintenance of stem cell–like cancer cells  because ine loop . Furtherine loop . We specTo further examine the TrkB-mediated regulation of the JAK2/STAT3 pathway, we assessed IL-6-induced JAK2 and STAT3 activation in MBA-MB-231 and Hs578T control-shRNA or TrkB-shRNA cells. Relative to the MDA-MB-231 and Hs578T control-shRNA cells, TrkB-shRNA cells showed significantly reduced activation and expression of JAK2/STAT3, as well as Twist-1 expression after stimulation with or without IL-6 Figure . FurtherThe results of a recent study suggests that Twist-1 and Twist-2 induce activation of the EMT program to maintain the resulting mesenchymal state –40. MoreDuring the process of tumor metastasis, which is often enabled by EMT program, pleiotropically acting transcription factors, such as E12, Goosecoid, SIP1, Snail, Twist-1, and Twist-2, are induced that orchestrate EMT programs , 41. TheAlso, relative to the parental MDA-MB-231 control-shRNA cells, TrkB-shRNA cells significantly downregulated the expression of mesenchymal markers such as N-cadherin and fibronectin, whereas they upregulated the expression of epithelial markers (such as E-cadherin and α-catenin). Moreover, the levels of Twist-1 was significantly downregulated in MDA-MB-231 TrkB-shRNA cells relative to MDA-MB-231 control-shRNA cells . FurtherTo determine the contribution of TrkB to primary tumor formation, we injected MDA-MB-231 cells expressing either TrkB-shRNA or control-shRNA into the mouse mammary fat pads of BALB/c Nu/Nu mice and examined the resulting primary tumors 30 days later. Control MDA-MB-231 cells formed primary mammary tumors at identical rates, whereas primary tumor formation by MDA-MB-231 TrkB-shRNA cells was markedly decreased Figure . These rTo determine if the loss of TrkB expression affected the ability of MDA-MB-231 cells to metastasize, MDA-MB-231 cells expressing either TrkB-shRNA or the control shRNA were injected into the tail veins of BALB/c Nu/Nu mice, and their lungs were examined for metastases 35 days after injection. Suppression of TrkB expression strongly reduced the number of metastatic nodules relative to MDA-MB-231 control-shRNA cells Figure . Additiohigh/CD24low cells, which show a stem cell-like phenotype, are enriched in highly metastatic breast cancer cells  have been proposed to be resistant to cancer therapies through activation of the PI3K/AKT pathway, IL-6/JAK2/STAT3 pathway, and EMT [TrkB is overexpressed in several human cancers, ranging from neuroblastomas to pancreatic ductal adenocarcinomas, and its overexpression suppresses anoikis as an EMT inducer by regulation of Zeb1 , 42. Ind and EMT , 43–47. In the present study, we found that activation of the MEK and PI3K/AKT pathway through TrkB-mediated c-Src activation after TrkB-c-Src complex formation induced tumorigenicity and metastatic potential of breast cancer. These our results are supported by those of a previous study in which the phosphatidylinositol-3-OH kinase/protein kinase B signaling pathways required for EMT, anokis suppression, c-Src activated PI3K/AKT and Ras/MAPK cascades , 48, 49.in vitro and was required for cell transformation of a number of oncogenes and activation of STAT3 by interleukin-6 or expression of activated c-Src induced Twist expression at the protein and mRNA levels [We further dissected the role of TrkB in tumorigenicity and metastasis. Activated STAT3 was able to transform cells A levels , 50–52. A levels , 53–57. Recent studies showed that the IL-6 inflammatory feedback loop leads to CSC self-renewal and induction of EMT, both of which are implicated in tumor metastasis and poor outcomes by therapeutic resistance , 37, 58.Recent evidence indicates transcription factors Twist-1 and Twist-2, which are master regulators of embryonic morphogenesis, play an essential role in metastasis, CSCs and EMT of breast cancer , 60–66. Human breast cancer , SYF, 293T, and MDCK cell lines were maintained as previously described , 69, 70.Each of the two shRNA-encoding oligonucleotides against mouse and human TrkB was designed and verified to be specific to TrkB through BLAST searches against the mouse and human genomes, respectively. The primers corresponding to TrkB were cloned into the pLKO lentiviral vector to generate the TrkB-shRNA expression plasmid . shRNA tAssays were performed as previously described, with modification , 69. TheAll assays were performed as previously described , 71. TheThe primer sequences are listed in the supplemental experimental procedures . Total RE-cadherin, Twist-1, Twist-2, or STAT3 reporter gene constructs and 0.5 μg of pCMV-β-gal were cotransfected per well. Cell extracts were prepared 48 hrs after transfection, and the luciferase activity was quantified using an Enhanced Luciferase Assay Kit (Promega). All experiments were performed in triplicate.Cells that were 50% confluent in 12-well dishes were transfected using Lipofectamine 2000 (Invitrogen). A total of 0.5 μg 5 cells suspended in 50 μl PBS/Matrigel (BD Biosciences) were injected subcutaneously into the left and right hind flank regions under anesthesia. Mice were euthanized at 5 weeks and primary tumors were excised for analysis. For tail-vein injection, 1 × 105 cells suspended in 50 μl PBS were injected into the tail vein of 7-week-old BALB/c Nu/Nu mice.BALB/c Nu/Nu mice were purchased from the Korea Research Institute of Bioscience and Biotechnology  and handled in compliance with the IACUC. For tumorigenicity studies, 1 × 10Mice were given intraperitoneal injections of 150 ng/g D-Luciferin  and anesthetized with 2.5% isoflurane. At 7–8 min after injection, animals were imaged using the Xenogen Spectrum (IVIS-200) imaging system.The equal numbers of MDA-MB-231 control-shRNA or TrkB-shRNA cells were plated and cultured for 3 days. Subsequently, conditioned media from these cell cultures were collected and analyzed by the Human IL-6 Quantikine ELIZA kit (R&D systems) according to the manufacturer's instructions.t test (two-tailed).Data are expressed as the means ± SEM. Statistical analyses of these data were conducted via a Student's


We investigate optical field enhancement for a wide mid-infrared range, originating from the excitation of graphene plasmons, by introducing a graded dielectric grating of varying period underneath a graphene monolayer. Excitation of the plasmonic mode can be achieved by illuminating a normal-incidence plane wave on the gratings due to guided-mode resonance. The gratings of varying period enable the excitation of the plasmonic mode with a very high field enhancement factor (to the order of magnitude of 1000) within a wide spectral band, which leads to the frequency-dependent spatially separated localization of the infrared spectrum modes. We also demonstrate that the excitation position of the plasmonic mode can be freely tuned by varying the thickness of the interlayer as well as the chemical potential of the graphene monolayer. This structure enables the design of two-dimensional plasmonic photonic circuits and metamaterials targeted towards numerous potential applications including optoelectronic detectors, light-harvest devices, on-chip optical interconnects, biosensors, and light-matter interactions. The efficient concentration of optical energy into nanoscale objects and structures and the consequent enhancement of the optical field by optical antennas224It would be hugely advantageous to extend the concept of energy concentration to the infrared and THz regime, which would enable significantly increased applications including probingRecent concerns regarding plasmonics have been partly shifted to graphene plasmons (GPs)1415142210d1, is assumed to be 500 nm. In this case, the grating layer can be treated as infinitely thick since the silicon substrate below the grating does not affect the field distribution of the GPs in the grating/silica/graphene/air system. As the grating period is much smaller than the wavelength of the incident wave, the grating can be approximately modeled as an effective medium with the equivalent permittivity24εsilica = 3.9 and εsilicon = 11.9 are the respective permittivities of silica and silicon at the mid-infrared rangef = w2/Λ is the filling ratio of the silica.We first consider the dispersive relationship of the plasmonic mode supported by a graphene monolayer on a uniform diffractive grating substrate with a silica layer as the interlayer 1/2  are the permittivities and wavevectors of the grating layer (i = 1), silica (i = 2), graphene (i = g) and air (i = 0), respectively, while β is the propagation constant of the GPs along the x axis. The dispersion relation of the GPs by equation μc = 0.65 eV is represented by solid lines in j is the diffraction order and θ is the incident angle. To excite the plasmonic waves with wavelength λ and the fundamental order j = 1 under the condition of normal incidence θ = 0, the following expression should be satisfiedFor the four-layer grating/silica/graphene/air system, the dispersion relation of the GPs can be derived by combining the Maxwell equations and continuous boundary conditions26d2, with μc = 0.65 eV and w1 = 62 nm. It can be clearly seen from d2 = 10 nm, which can be attributed to the excitation of the fundamental (j = 1) and third-order (j = 3) plasmonic modes on the graphene surface. The missing second-order (j = 2) mode is here caused by the vanishing dipole moment. The scaling of the resonant wavelength, associated with the grating period, is consistent with equation d2 increases due to the fact that the overlap between the GPs and the grating reduces as d2 increases and hence reduces the excitation efficiency. The scaling of the fundamental-mode resonant wavelength with respect to the thickness of the silica layer, d2, by finite element method (FEM) simulation agrees with the theoretical results of equation d2. This is because the effective index of the plasmonic mode in the grating/silica/graphene/air system reduces with the increased thickness of the silica layer, d2. From 2) and light source (|E0|2). The enhancement level presented here is quantitatively comparable to that reported in plasmonic nanocavity gratings made of nanogrooves in a gold filmw1, along the x direction, while keeping the width of the silica layer, w2, constant. Wide-band plasmonic field enhancement can be anticipated under the illumination of a normal-incidence plane wave. The simulation result shown in 102/|E0|2) is independent of the power enhancement on the graphene surface, which is proportional to the power of incident light. The average electric field enhancement for the first 3 um in the left region is approximately 1000, but this does not mean the power enhancement is larger than 1 as the power of the incident light source is unchanged. The GPs excited by the silicon grating propagate along the graphene surface and hence suffer an absorption loss from the graphene monolayer. This is why the electric field intensity reaches its maximum value at the center position for each excitation wavelength . The calculated results show that the propagation loss by a UDGS is almost equal for the three wavelengths. For example, the values of Im(β) with λ = 7, 8 and 9 μm at w1 = 20 nm are 1.81 × 105, 1.62 × 105 and 1.46 × 105 m−1, respectively. Additionally, the left-going plasmon for a longer wavelength undergoes a much larger propagation distance between the excitation position and the 0 ~ 2 μm region. As a result, the field enhancement for λ = 7 μm is higher than that for a longer wavelength in the first 0 ~ 2 μm region.The UDGS can only excite the GPs on a graphene monolayer within a very narrow bandwidth around the resonant frequency. Bearing in mind that the resonant frequency is dependent on the grating period , we furtd2. According to equation λ = 8 μm in our case), a much wider w1 is required to excite the GPs as d2 increases due to the gradual reduction of the value of n ) is the Fermi-Dirac distribution, ε is the energy, μc is the chemical potential, T is the temperature, e is the electron charge, kB is the Boltzmann constant, ω is the radian frequency, and τ is the momentum relaxation time representing the loss mechanism due to the carrier intraband scattering.The optical properties of graphene are typically described by its surface conductivity. The Kubo formula has been widely employed to retrieve its surface conductivity in a series of theoretical and experimental studies on graphene313233μc| ≫ kBT, the surface conductivity of graphene could be approximated as32In the mid-infrared range with |T = 300 K and τ = 1 ps. Considering that in high-quality suspended graphene a DC mobility of μ > 100000 cm2V−1s−1 has been experimentally achievedτ > 1.5 ps, our setting of τ = 1 ps can be said to conservatively reflect the practical transport loss of graphene. However, as a graphene monolayer with a carrier mobility of more than 100000 cm2V−1s−1 is rarely seen in graphene samples in practice, we have also studied the electric field enhancement utilizing a much more realistic scattering time of graphene [see In our study, hene see .η0 (≈377 Ω) is the impedance of air, k0 is the light wavevector in free space, and dg is the thickness of the graphene monolayer (assumed to be 0.5 nm in our work).By treating the graphene monolayer as an ultra-thin metal film, graphene’s equivalent permittivity can be written as14To calculate the dispersion relation of the GPs, two-dimensional FEM simulation based on the mode solver of COMSOL is employed. In the simulation, 5-layer meshes are employed to denote the graphene layer while non-uniform meshes with a maximum element size of 500 nm are adopted to represent the other regions besides graphene.How to cite this article: Zhang, T. et al. Tunable broadband plasmonic field enhancement on a graphene surface using a normal-incidence plane wave at mid-infrared frequencies. Sci. Rep.5, 11195; doi: 10.1038/srep11195 (2015).


AoB PLANTS was conceived. I hope that the range of papers included in the special issue will reveal some of the complexities in soil community-mediated plant invasion.Soil microbial communities are one of the multiple factors that facilitate or resist plant invasion. Regional and biogeographic studies help to determine how soil communities and the processes mediated by soil microbes are linked to other mechanisms of invasion. Both the success of plant invasions and their impacts are profoundly influenced by a wide range of soil communities and the soil processes mediated by them. With an aim to better understand the mechanisms responsible for the soil community-driven routes, a special issue of  Soil microbial communities play an important role in explaining why some exotic species become aggressive invaders and form mono-dominant communities in non-native ranges but coexist with their neighbours in species-diverse native communities. Research on invasion ecology during last two decades has made a significant contribution in understanding how soil communities can either resist or facilitate invasions a. van deIn the boom phase, the positive benefits of introduction phase increase to facilitate invasive species by evolving increased competitive ability , invasivAlliaria petiolata (M. Bieb.) Cavara & Grande) suppress mycorrhizae and disrupt mutualistic associations between mycorrhizae and native tree seedlings  by disrupting mutualistic root-fungal symbioses in native plant species. Similar results were obtained in a long-term field experiment: where garlic mustard was present, vital rates of native perennials were suppressed relative to weeded plots, suggesting a general link between the chemically driven disruption of the mutualism and plant physiological declines, carbon stress and demographic declines.One of the hypotheses that links novel chemicals and suppression of root-fungal symbioses is disruption of mutualistic hypothesis  and the University of Delhi supported this work.The None declared.


The effect of hygrothermal conditions (air temperature and relative humidity) on the dehydration of theophylline monohydrate was investigated. Firstly, the equilibrium states of theophylline were investigated. The data from gravimetric analysis at constant temperature and humidity were reported as desorption isotherms. The PXRD analysis was used to identify the different polymorphic forms of theophylline: the monohydrate, the metastable anhydrate, and the stable anhydrate. Solid-solid phase diagrams for two processing times were proposed. Secondly, the dehydration kinetics were studied. The water content evolutions with time were recorded at several temperatures from 20°C to 80°C and several relative humidities from 4% to 50%. Different mathematical models were used to fit the experimental data. The spatially averaged solution of 2D Fickian transient diffusion equation best represented the water mass loss versus time experimental relationship. The dehydration rate constant was found to increase exponentially with air temperature and to decrease exponentially with air relative humidity. Its hydration behaviour and solid-state transitions have been extensively studied and presently the compound is known to exist as a monohydrate (M) and four anhydrous polymorphs  [Theophylline (CThe M form has been shown to lose water in low relative humidity (or water activity for solvents) to produce form II which is the most prevalent form and has been long considered as the only stable form at room temperature , 5. FormThe dehydration kinetics of theophylline monohydrate has been the subject of several investigations. Airaksinen et al.  investign of 0.33, respectively. Lower temperatures resulted in higher concentration of form III in the dehydrated product.Nunes et al.  investign = 0.25 and that at 40°C the dehydration of theophylline was a two-step process. Each step could be linearized using the Avrami-Erofeev's relationship with n = 0.25. The same conclusion was given by Agbada and York [Suzuki et al.  reportedand York .Matsuo and Matsuoka  have fouIt follows from the above short literature review, that the dehydration rates of theophylline were studied mainly with temperature as the controlling parameter and the influence of relative humidity has not been much investigated. The aim of this work was to experimentally study the influence of both parameters, varying in a broad range, on the solid phase transition pathways and kinetics during dehydration of theophylline. From equilibrium data, a phase diagram was determined and from kinetic data a simple predictive model for dehydration progress with time was derived. The model proposed here was compared to the common ones from the literature concerning the dehydration of theophylline and was proven to interpret better the experimental curves. Unlike the others, this model included explicitly the relative humidity and was based on water diffusion as rate controlling transition mechanism.2. 6H2O, Mg(NO3)2, K2CO3, NaBr, NaCl and KCl salts used for hygrothermal conditioning of hydrated theophylline samples were also supplied by Sigma-Aldrich.Anhydrous theophylline was purchased from Sigma-Aldrich . Hydrated theophylline was recrystallised from a saturated solution of anhydrous theophylline. Anhydrous theophylline was first dissolved in hot (60°C) distilled water. The solution was then filtered and allowed to cool overnight. Hydrated theophylline crystals were harvested and dried at room temperature. CsF, LiCl, MgClA differential scanning calorimeter  was used. The instrument was calibrated with pure indium. About 4-5 mg theophylline sample was packed in aluminum pans, crimped with lids having several pinholes. Tests were run from room temperature to 300°C, at a heating rate of 5°C/min under dry nitrogen purge.The thermogravimetric analyser (TGA Netzsch TG 209 F1 Iris ASC) was used. The sample was heated in an open aluminum pan from room temperature to 200°C, under nitrogen purge, at a rate of 1°C/min.α radiation (λ = 1.54 Å) using Göbel mirror bent gradient multilayer optics. The scattered intensities were measured with a scintillation counter. The angular range was 5–50° with a scan speed of 5° 2θ min−1.X-Ray powder (XRPD) theta-theta diffractometer  was used. The XRPD experiments were performed in symmetrical reflection mode with CuK2·6H2O, Mg(NO3)2, K2CO3, NaBr, NaCl, and KCl were prepared and stored at 10, 20, 30, 40, 50, and 80°C providing the different required relative humidity conditions. The relative humidity values were verified with an Activity-meter  and are detailed in Saturated solutions of CsF, LiCl, MgClHitachi S-800 Field Emission Gun (FEG) Scanning Electron Microscope (SEM) was used. All samples were coated with gold for increased signal/noise ratio.In order to interpret and fit the experimental dehydration curves, several equations were proposed in the literature. Among them, the models A and B are the most widely used for solid-state transitions see . ConcernDSC and TGA curves for the recrystallized and commercial theophylline are shown in Figures The DSC curve of commercial theophylline showed only one endothermic peak at 272°C due to melting and no weight loss was observed on the TGA curve. Therefore, the commercial product appeared effectively as the anhydrous form of theophylline. Similar results were reported by Suzuki et al. .The powder X-ray diffraction patterns of the recrystallized and commercial theophylline are shown in X versus air relative humidity RH) in The final equilibrium contents obtained from gravimetric experiments at constant temperature and constant relative humidity were used to plot the desorption isotherms of theophylline. The plot of desorption equilibriums  and low temperatures. The three forms of theophylline coexisted for moderate temperatures and humidities.θ).The evolution of PXRD patterns of theophylline during processing at 3.8% and 20°C (conditions which favor the occurrence of the anhydrous metastable form) is presented in The transition between the monohydrate and the metastable form corresponds to the reversible loss of water through channels in the crystal lattice according to Phadnis and Suryanarayanan . The cryA second solid-solid phase diagram corresponding to equilibrium states (after one month) of theophylline monohydrate processing at different relative humidities and different temperatures is presented on Our results did not agree closely with the above cited literature data. Ticehurst et al.  used aceX  versus time plots for the dehydration of theophylline monohydrate at 11.61% relative humidity and at different temperatures  is shown in The water content X  versus time plots for the dehydration of theophylline monohydrate at 40°C, and at different relative humidities  is presented in The water content These experiments showed that both temperature and humidity affected the dehydration rate of theophylline monohydrate. The same kinds of curves were also obtained for all temperatures and relative humidities considered, but these results are not shown here.X/X0 ≤ 0.8) with a slow quasilinear trend and a second stage (0.8 ≤ X/X0 ≤ 0) with a fast quasiexponential trend. The first stage was practically neither observed for the highest applied temperatures when probably form II was exclusively produced nor for the lowest relative humidity when probably the form III was exclusively produced. According to the literature [Most of the experimental curves exhibited two dehydration stages: the first stage  and a linear expression for lnk0 could be a possible mathematical representation of the theophylline's dehydration curves during the second stage. This representation would include the influence of both temperature and relative humidity in a quite simple way. The predicted dehydration curves of the theophylline monohydrate, according to different models are shown in Thus, the Fickian diffusion-based model (C) coupled with the Arrhenius expression for Except for the lowest relative humidity (2.4%) and the highest temperature (80°C), the points are rather narrowly distributed along the bisecting line and thus the correlation between the two sets of data can be considered satisfactorily.The capability of the model C to interpret correctly the experimental data may indicate that the dehydration rate of theophylline in the second  stage of the process is controlled by water transfer within the crystal rather than the nucleation and growth of the new crystal phase or the progression of the interface between the new and old phases. This corroborates the hypothesis of a “WET4” dehydration mechanism with a process rate controlled by diffusion suggested at the end of Section 3.3.Theophylline is classified as a lattice channel (or tunnel) hydrate, which class is known for releasing water rather easily. However, unlike in many others channel hydrates, theophylline's molecules are very tightly packed and its channels are particularly narrow (roughly of the size of the water molecule) according to Ahlqvist and Taylor . Water mThe effect of hygrothermal conditions (air temperature and relative humidity) on the dehydration of theophylline monohydrate was investigated. The results of gravimetric experiments provided the conditions where the monohydrate transforms to the anhydrate. The analysis of PXRD patterns allowed the determination of the conditions where only one of the anhydrate forms was obtained or where the two forms could coexist. The metastable form appeared preferably for low temperatures and low humidities, while the stable form was preferred for high temperatures. The coexistence occurred only for short processing times (few days). The proposed solid-solid phase diagram can be very useful for predicting the state of theophylline during drying or during storage.The dehydration rate was found to increase strongly with air temperature and to decrease with air relative humidity. Three mathematical models: Avrami-Erofeev's solid-state transition, first order chemical kinetic, and Fick's 2-D diffusion were used to fit the experimental data. The measured evolutions of water mass with time during dehydration of theophylline for all considered conditions were found to be best represented by the spatially averaged solution of 2-D Fickian transient diffusion equation. The parameters of this equation were correlated to temperature and relative humidity by exponential expressions, and finally a simple but comprehensive model for prediction of the dehydration progress with time was obtained. This kind of procedure of fitting semi-empirical equations to dehydration curves can provide a fast way for drying process modeling and optimal control in pharmaceutical industry.