Plastocyanin is a copper containing protein that is involved in the electron transfer process in photosynthetic organisms. The active site of plastocyanin is described as an entatic state whereby its structure represents a compromise between the structures favored by the oxidized and reduced forms. In this study, the nature of the entatic state is investigated through density functional theory‐based hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations. The strain energy is computed to be 12.8 kcal/mol and 14.5 kcal/mol for the oxidized and reduced forms of the protein, indicating that the active site has an intermediate structure. It is shown that the energy gap between the oxidized and reduced forms varies significantly with the fluctuations in the structure of the active site at room temperature. An accurate determination of the reorganization energy requires averaging over conformation and a large region of the protein around the active site to be treated at the quantum mechanical level. © 2016 The Authors. Journal of Computational Chemistry Published by Wiley Periodicals, Inc. In the active site of plastocyanin, copper is bound in an approximate trigonal plane to the sulphur of cysteine and two nitrogen atoms of histidine ligands, with a further methionine ligand in an axial position.Copper containing proteins are naturally occurring proteins involved in many biological processes, including respiration and photosynthesis.−1 and 12,800 cm−1, which are assigned to a Cys(π) → SOMO ligand to metal charge‐transfer (LMCT) and d ligand field → SOMO excitations, respectively, the first of which is much more intense than the second.−1) there is an additional band which is assigned to the Cys(σ) → SOMO transition. In plastocyanin this band is very weak, but in the closely related proteins cucumber basic protein and nitrite reductase its intensity increases.During electron transfer copper is reduced from Cu(II) to Cu(I). In its oxidized form, plastocyanin has a dThe structure of the active site plays a key role in the function of the protein. One debated aspect of plastocyanin redox chemistry has been the entatic−1.This concept of an entatic state was questioned following quantum mechanical calculations of the structure of the active site.The studies of the strain energy and the reorganization energy have been largely based on a static view of the structures. It is known that conformational averaging is important in determining the properties of these proteins.The structure of the gas‐phase model active site see Fig.  was opti+ and thirteen Cl− ions were added to neutralize the overall charge of the system, giving a physiologically relevant concentration of 0.15 M. Periodic boundaries were employed, using the Particle‐mesh Ewald summation method for long‐range electrostatics, with a cutoff distance of 12 Å and a pair‐list distance of 16 Å. Minimization was performed using the steepest‐descent algorithm for 10,000 steps. Equilibration was carried out for a total of 1 ns, with a timestep of 2 fs, with rescaling of velocities every 500 steps. All bonds to hydrogen were constrained using the SHAKE algorithm. This gave an equilibrated protein and water molecules system that provides a suitable starting point for further calculations.Initially, reduced and oxidized forms of plastocyanin from the Dryopteris fern −4 kcal mol−1, before a short period of equilibration was performed . Production QM/MM dynamics were performed for a total of 40 ps. The ωB97X‐D functional and the 6‐31G(d) basis set were used throughout. All of the quantum chemical calculations were performed with the Q‐Chem software,Using the equilibrated system, a QM/MM partitioning was performed, using the active site shown in Figure Tables The average structural parameters from the QM/MM simulations are in closer agreement with the crystal structure, particularly considering some variation between the reported crystal structures and the dynamical nature of the protein in solution is to be expected. The predicted Cu—S(cys) bond length for the oxidized form of 2.16 Å is consistent with the value of 2.15 Å derived from analysis of the absorption spectrumτ4 parameter,α and β are the two largest bond angles of the copper coordination site and the value of τ4 varies between 0 for square‐planar and 1 for tetrahedral geometries. For the isolated active site values of τ4 = 0.57 and τ4 = 0.91 are obtained for the oxidized and reduced forms. This shows that the reduced form is close to tetrahedral while the oxidized form is intermediate between square‐planar and tetrahedral; this is typical for 4‐coordinate Cu(II) systems. For the average structures from the QM/MM simulations the value of τ4 for the oxidized form increases to 0.88, while the reduced form has a value close to 1. Consequently, the distortion from tetrahedral evident in the model gas phase structure of the oxidized form is decreased significantly.The reduced form exhibits tetrahedral coordination to the copper center, while the oxidized form shows a more distorted geometry. For the oxidized form, it is useful to calculate the geometrical The strain energy provides a measure of the extent to which the protein holds the active site in an energetically nonoptimum structure, and is calculated from the difference between the energy of the ensemble averaged QM/MM active site structure in the gas‐phase  and the energy of the corresponding optimized active‐site gas‐phase structure. This gives values of 12.8 kcal/mol and 14.5 kcal/mol for the oxidized and reduced forms of the protein, respectively. The value for the oxidized form is close to the value of ∼10 kcal/mol determined from LFMM calculations.E, where ΔE = Eox − Ered, during the QM/MM simulations for the oxidized and reduced forms of the protein. This corresponds to the energy change between the two states without allowing for any relaxation of the structure. For both trajectories there is significant variation in ΔE. For the reduced trajectory, the average value of ΔE is 82.5 kcal/mol with a standard deviation of 11.2 kcal/mol. The oxidized trajectory shows a surprising amount of deviation in ΔE, which is initially positive and then falls rapidly, where it varies predominantly between −10 kcal/mol and −40 kcal/mol. The initial rapid change in ΔΕ at the start of the simulation is likely to reflect that the protein was still equilibrating during this period. Focusing on the simulation from 3 ps onwards gives an average value for ΔΕ of −29.2 kcal/mol with a standard deviation of 9.0 kcal/mol. This variation in ΔΕ suggests that the view of the protein being held in a single entatic state is not the complete picture since an ensemble of structures with significantly different ΔΕ will be sampled.Figure G is often termed the driving force of the reaction and is the change in free energy between the final and initial states and V is the diabatic electron coupling matrix element between the two states. The total reorganization energy, λ, can be calculated from the average of the instantaneous difference in energy between the oxidized and reduced forms (ΔE) from both the oxidized trajectory and the reduced trajectory. Within the linear response approximation the reorganization energy is given by〈ΔE〉red and 〈ΔE〉ox are the average values of ΔE from the reduced and oxidized trajectories, respectively. The total reorganization energy has been measured to lie between 16 kcal/mol and 28 kcal/mol,λ =55.9 kcal/mol, which is significantly larger than the experimental value, predominantly owing to the high value for 〈ΔE〉red. The accuracy of the computed value of λ can depend on a number of factors including applicability of the linear response approximation, the completeness of the sampling and the accuracy of the QM/MM method. The computational cost of QM/MM approaches makes it challenging to achieve a thorough sampling of configurations, and we explore the accuracy of the QM/MM model in more detail later.The reorganization energy is a key component in Marcus theory where the rate of electron transfer is given byt = 0 and the calculated ΔE for the oxidized and reduced trajectories. For the oxidized form, the plot suggests a significant degree of correlation between the structure and the calculated ΔE. There is also a large amount of change in the RMSD for the structures in the early part of the simulation (those with positive ΔE) confirming that the protein was still undergoing some equilibration in this period. For the reduced form there is less variation in the RMSD during the simulation, and correspondingly less variation in ΔE.Figure τ4 = 0.76, and if such a structure was thermally accessible it would result in very fast rates of electron transfer. To explore the energetics of these structures further, additional calculations with a larger QM region describing the active site have been performed for the structures corresponding to the times of 2.6 ps and 24.4 ps. These structures were chosen based on their associated ΔE, with the structure at 2.6 ps corresponding to the minimum value and the structure at 24.4 ps close to the average value during the part of the simulation were the structure had stabilized. The larger QM region is shown in Figure Ε change from −0.5 kcal/mol to −7.0 kcal/mol at 2.6 ps and −25.0 kcal/mol to −17.7 kcal/mol at 24.4 ps. This shows that the smaller QM active site calculations correctly predict that the structure at 2.6 ps has the smaller reorganization energy. However, while the variation in the reorganization energy remains significant, it suggests that the smaller QM region tends to exaggerate these fluctuations. This is likely to be associated with having a larger number of classical point charges very close to the copper active site center.Assuming that the electronic coupling matrix elements do vary significantly with the fluctuations in the structure,QM/MM molecular dynamics simulations have been performed for the oxidized and reduced forms of plastocyanin. The structural parameters describing the active site determined through averaging the geometries through the MD simulation are close to those of the crystal structure and show some significant differences to those of the optimized isolated gas phase active site. The strain energy for the active site is computed to be 12.8 kcal/mol for the oxidized form, in agreement with previous work.λ is higher than values estimated from experiment, although the simulations do sample structures that have a much lower value for λ. This may indicate more extensive sampling is required. The evidence of the calculations presented here is that using a QM region that includes the typical active site treated in QM calculations reproduces the trends in the energy gap between oxidized and reduced forms, but tends to overestimate the fluctuations. This illustrates the challenge of modeling the electron transfer in these systems within a QM/MM framework. Treating a very large region of the protein surrounding the active site at the QM level is desirable for a reliable estimate of the reorganization energy. However, these calculations are very computationally demanding and are not practical for the study of many conformations or the use in molecular dynamics simulations. One solution to this problem might be to use more approximate methods, such as LFMM, provided they give a balanced treatment of both oxidized and reduced forms of the protein.There is considerable variation in the computed instantaneous energy difference between the oxidized and reduced forms of the protein arising from the thermal fluctuations in the structure at room temperature. This necessitates some averaging over conformation in determining the reorganization energy, and means that the view of the protein constraining the active site in a structure that is optimal for electron transfer does not provide the complete picture of the electron transfer process. The computed value for


When irradiated in a transmission electron microscope, plunge-frozen, amorphous water ice specimens accumulate a pattern of static charge that changes dynamically as the specimen is irradiated, and which can deflect the transmitted electrons and blur the resultant micrographs. Here we provide a physical description of this charge accumulation and characterise its dynamic behaviour in the context of low-dose electron cryomicroscopy (cryoEM). We observe the accumulation of positive charge in the primary irradiation area as expected from earlier work. To our surprise, we also observed a build-up of negative charge in nearby unirradiated regions of the specimen. Using a standard carbon support foil containing a pure water ice specimen, we collect a portion of this negative charge in the micrometer sized specimen holes which act as electrostatic lenses. These unusual, diverging micro-lenses are extremely sensitive charge detectors that allow us to directly measure the magnitude and dynamics of charge accumulation and neutralisation that occur during cryoEM imaging. Using these measurements, we find that the build-up of charge on the specimen saturates to a dynamic equilibrium at an electron fluence which is orders of magnitude lower than required for a typical low-dose micrograph. The measurements here will guide the development of optimal imaging conditions for biological specimens and contribute to a complete theory of information loss in electron cryomicroscopy. To conduct an experiment as diagrammed in mode 1 was collected, which becomes the first image in the movie. The microscope was then switched to mode 2 and the centre region of the square was irradiated for the specified time corresponding to a particular fluence of electrons. The microscope was then switched back to mode 1, and a movie was collected by taking a series of 10–300 images (depending on the particular experiment) with a set delay between each image to allow the files to be written out. The delay for the supplementary movie and Frozen specimens were transferred to the load lock system of an electron cryomicroscope (FEI Polara) and loaded into the specimen position. All micrographs were recorded using 300 kV bright field transmission mode, and detected with a 2048  ×  2048 phosphor-coupled CCD camera (Gatan Orius). Micrographs were collected using a pre-specimen beam blanker using the data collection software attached to the camera , which allows capture of a series of micrographs using a set exposure time and a fixed delay between images. A diagram of the typical experiment is presented in This experimental scheme was tested on holey amorphous carbon foils having an additional carbon film on top of the foil, to establish the appropriate conditions, and then conducted on cryo-specimens prepared as described above. To determine the focal length of the microlenses we repeated the experiment several times under the same conditions until the maximum intensity for the focused microlenses was found; in this case - 60 mm. The error in determining this maximum is estimated to be  ± 5 mm. The beam current was measured using the current amplifiers attached to the two phosphor viewing screens, which had been previously calibrated against a picoammeter attached to a Faraday cup in the projection chamber. The total error in the measurement of the beam current, which is the dominant source of error in the reported flux, is estimated to be less than 30%. All other errors are less than 10%.2.3Images were converted to MRC format and cropped, converted to 8 bit depth and saved as TIFF files for creating the panels in 33.1To investigate the kinetics of charge build-up and neutralisation, it was important to use specimens consisting of amorphous ice on a holey carbon film, which is the specimen used in most single particle cryoEM experiments 3.2r, the radius of the hole) in c is the speed of light in vacuum. Thus the lateral velocity, sv required to give a deflection angle of 10 µrad is 2  ×  103 m/s, which is imparted on the electron during its transit through the electric field of the charged specimen. If we take the vertical distance over which the lateral field is experienced by the electron as approximately 5 µm, which is approximately the distance between holes, then to accelerate the electron to this velocity over this distance, a field of 0.6  ×  106 V/m is required. Thus the potential drop from the centre of the suspended ice to the edge of the hole in the foil is For a 300 keV electron to be displaced by 0.6 µm , is likely to be dominated by the charge that builds up on and around the illuminated area. Thus the contrast observed by Longchamp et al. in recent low-energy holography experiments Another interesting question to consider in this context is whether the influence on the quality of TEM images of electrostatic charge build-up on the specimen would be expected to be less if a microscope with a more powerful objective lens was used. In typical electron cryomicroscopes, the focal length of the objective lens is a few millimetres, with Spotscan imaging has been advocated 2 or Si3N4. We have shown that a small disk of insulating material, when appropriately charged, can act as both a converging and diverging electron microlens. We envision that structures such as these could be incorporated in other devices to locally detect secondary electrons in specific regions and even create electron micro-optical devices.Finally, the electrostatic micro-lensing phenomenon that has allowed us to estimate the charge build up due to the inverse Berriman effect may turn out to be useful for other types of investigation on different substrates or devices, such as those fabricated with common insulating materials like SiO52. This means that the positive charge that builds up in the irradiated region of a cryoEM specimen saturates at a fluence that is much less than that incurred even in the first frame  of a low-dose micrograph on a direct electron detector. Slight increases in potential can occur after this but are thought to be small and mostly related to the charge-up of regions within the low intensity tails of the beam. This positive charge is a likely physical explanation for offsets in defocus that are often observed in micrographs of ice imbedded specimens but further work is required to quantify this accurately and compare it to the physical movement of the ice layer. It is still not possible to say unequivocally that specimen charging under these conditions has no effect on image quality; it is possible that there will be a small defocus change during the first 2 of the exposure, but based on the measurements here it is thought to be much less than the movement of the amorphous water at the onset of irradiation. While here we have considered the steady-state charge that accumulates on the specimen, it will be accompanied by a fluctuating charge that occurs due to the discrete nature of the electron scattering events and we consider this aspect of specimen charging in the accompanying paper From these measurements we conclude that charge, in the form of low energy secondary electrons, can accumulate on insulating regions of the specimen in the electron microscope even tens of microns away from the primary beam. This charge can be measured by incorporating insulating structures within the specimen that act as microlenses for a particular region of a low flux, parallel beam. Further, the positive charge left behind after the loss of secondary electrons saturates on a typical cryoEM specimen after between


A new adaptive switching algorithm is presented where two adaptive filters are switched correspondingly for lower and higher noise ratio of the image. An adaptive center weighted vector median filter is used for the lower noise ratio whereas for higher noise ratio the noisy pixels are detected based on the comparison of the difference between the mean of the vector pixels in the window and the approximated variance of the vector pixels in the window. Then the window comprising the detected noisy pixel is further considered where the pixels are given exponential weights according to their similarity to the other neighboring pixels, spatially and radio metrically. The noisy pixels are then replaced by the weighted average of the pixels within the window. The filter is able to preserve higher signal content in the higher noise ratio as compared to other robust filters in comparison. With a little high in computational complexity, this technique performs well both in lower and higher noise ratios. Simulation results on various RGB images show that the proposed algorithm outperforms many other existing nonlinear filters in terms of preservation of edges and fine details. Filtering is one of the most essential steps in the applications of image processing. An image must contain the required data to show the correct information before it is used for any image processing application. But images are usually corrupted with unwanted information that causes hindrance to an efficient image processing operations. These unwanted information which are termed as noise must be removed properly from the image as a preprocessing step. Additive random noise (Gaussian noise) and salt and pepper noise are some of the most common noises found in digital image. Impulse noise which may be fixed valued noise (FVN) or random valued noise (RVN) is one of the most naturally occurring noises in digital images and it is induced in the image during image acquisition by faulty sensors or during transmission through communication channels. Noise removal techniques depends on the type of noises degrading the image and also largely on the percentage of noise corrupting the image. A number of robust filters have been proposed in literature for filtering the color images corrupted with impulse noise. Non-linear filters which actually work in spatial domain suit well for impulse noise removal from color images  angular part from the VDF in calculating the cumulative distances from a vector pixel to the other in the filtering window. The center weighted VMF (CWVMF)  treats the color image channel wise in a scalar form which often leads to color artifacts Pitas . The nonTo overcome this particular issue noise detection schemes are introduced in the rank order static filters Lukac , that chn is the number of vector pixels in the window. If the difference between any two pixels from the peer group is greater than user-specified threshold, then the center pixel is replaced with the output of VMF. And if the difference of the individual pixel, in the peer group m, from the center pixel is less than a user specified threshold, then the center pixel is replaced with the VMF, which results in the fast PGF (FPGF)  the pixels in the window is sorted according to their differences from the center pixel, then a peer group of  T Lukac , 2003.υ , entropy VDF (EVDF) and entropy DDF (EDDF) are the group of entropy vector filters which replace the center pixel with the output of VMF, VDF and DDF respectively, if the local contrast entropy of the center pixel is greater than its local contrast entropic threshold multiplied by a weighting factor l is the number of pixels in the ordered set, then position of the noisy center pixel will be outside the range of l. In the rank-conditioned and threshold VMF (RCTVMF), rank-conditioned and threshold VDF (RCTVDF) and rank-conditioned and threshold DDF (RCTDDF) (Singh and Bora The Neuvo VMF (NVMF) , fuzzy vector directional filter (FVDF) and fuzzy directional distance filter (FDDF) weight the pixels in the filtering window according to their respective cumulative distance before replacing the center pixel with the average of the weighted pixels. These filters use certain parameters Ta, Tb, Tc and Td respectively, Moore et al. , if the difference between the center pixel and the first four pixels of the rank-ordered set of pixels is greater than four separate thresholds e et al.  then theThe non-causal linear predictor based filters use the concept of linear prediction Singh  for estip, which corresponds to the least valued element from the sorted array of cumulative distance of the vector pixels in the window, is compared with a threshold, which is calculated based on the standard deviations of the separate three channels of the image. If the value of k is greater than that of the threshold T, then the center pixel is predicted considering the 2nd order non-causal regions. The non causal vector median filter 2nd order non causal (NCVMF_2nc) considers only the upper four non causal pixels out of the eight, from the 2nd order region excluding the center pixel. The non causal vector median filter 1st order non causal (NCVMF_1nc) considers the four 1st order pixels from the non causal region. The non causal vector median filter 1st order causal (NCVMF_1c) predicts the center pixel using the two pixels of the 1st order causal region.The non-causal vector median filter (NCVMF)  compares the cumulative distance of the center pixel with that of the sorted array of the cumulative distance of all the pixels in the window, so that center pixel lies in the μ. This results to the corresponding non-adaptive sigma vector filters as SVMF_MEAN, SVMF_RANK, SVDF_MEAN, SVDF_RANK, SDDF_MEAN and SDDF_RANK respectively. Then the groups of adaptive sigma vector filters are ASVMF_MEAN, ASVMF_RANK, ASVDF_MEAN, ASVDF_RANK, SDDF_MEAN and ASDDF_RANK correspondingly. Here the center pixel is replaced with output of VMF, VDF and DDF respectively if the difference between the center pixel and the mean vector or the vector median is greater than the product of the corresponding approximated variance and a weighting parameter μ, where the approximated variance is calculated based on the cumulative distance of the vector pixels in the window from that of the mean vector.The vector sigma filters . As usual in the basic filtering approach, a window W of odd size m × n centered at x is considered  from the mean, xmean is compared with the approximated variance, σ2 multiplied by a weighting factor x is considered to be a noisy pixel.The variance Ewmf is an unbiased one, namely: (a) each weight xi is a positive number, yi ≥ 0 and (b) α, η and β are used to tune the amount of weight to be given to the pixels. To obey the above constraints so that the output is unbiased, it is made that d(i) and l(i) will be in the range of k where β is the value of α at which the weighting function wi takes the maximum derivative. The value of η = 1 − α also makes it sure that the two components in Eq. d(i) for a particular pixel xi is larger, than it means that the intensity difference of the particular pixel from its surrounding is very large and has more tendencies to be an impulse noisy pixel. Therefore less weight is given to the pixel and vice versa. Whereas the second component as described by Eqs. xi with a large value of l(i) is far away from the center pixel x and is of less resemblance or importance, to be assigned with less weight. And it can be also seen that the parameters α and η are inversely proportional to the weight wi.This particular detection algorithm based on ASVMF_MEAN is suitable for higher noise region of the image because the approximated variance is calculated based on the cumulative difference of the vector pixels from the mean, which means that all the pixels whether impulse noise or not are considered and are given similar importance. And the vector pixel is treated as impulse noise if it is having a bigger difference from the mean, as compared to average of the difference of vector pixels from the mean as seen in Eqs. mentclasspt{minimad(i) and l(i), the amount of weight assigned is again controlled by the respective values of α and η. If any of α or η is having a value of 1 then the other will be 0 that avoids the condition for both the components to be considered simultaneously. The condition that d(i), more weight is allowed to be assigned to the pixel by the first component of Eq. α, thus highlighting the first component, that subsequently makes the second component which is made of l(i) to be less important with a comparatively higher value of d(i), less weight is made to be assigned by the first component with higher value of α and thus the second component is given more importance with a comparatively lesser value of α and η for fixed valued and random valued noise correspondingly. And the values of α and η at which the value of PSNR and other measuring parameters attain their maximum values is highlighted. The selection of values for the parameters is further discussed in part C: Parameter selection, of section III.Further for a particular value of entclass1pt{minimaL1 and L2 norms. Figure The filters are evaluated in this section on a variety of RGB color images, which includes scientific images, biomedical images, photographic images, synthetic images and a group of images generally used in the color image processing literature, for a better comparison on the performance of the filters, by considering a 3 × 3 window size with b be the probability of corruption of the color image with the impulse noise. A color image has three vector components, where each component has a chance of being corrupted by the impulse noise with a respective corruption probability. Let bR, bG and bB be the probabilities of impulse noise corruption of the three components R, G and B respectively.ηk can be either 0 or 225, whereas it can be any random value ranging from 0 to 255 for the random valued noise.An impulse noise model which is commonly used in the literature of filtering of color images is used in this work   for fixed valued noise and random valued noise respectively (see Tables α plays a bigger role as compared to the second component controlled by parameter η in Eq. β and ν is considered as 3 so that u goes from 3 to 5, for which three different xCWVMF is calculated with three different TL and TH in Eq. The exponential weight is found using Eq. mentclass2pt{minimse ratio 0 and 90%The performance comparison of the various filters is shown in Tables l of the Figs. After working on various numbers of images it can be seen that the proposed filter, Noise percentage based switching filter maintains a good PSNR value at lower noise ratio by outperforming robust filters like ACWVMF, ACWVDF, etc. and also definitely overtakes even the most robust filters, which are very efficient in the higher noise ratios, like ACWVMF, EVMF and some vector sigma filters. This is supported by the experimental results shown in the Tables The proposed filter is able to outperform the other robust filters, in maintaining the signal content and preserving the fine details of the image corrupted with fixed valued and random valued impulse noise at the lower noise ratios as well as at the higher noise ratios. And the filter also maintains the chromaticity of the filtered image at a lower value of NCD and MAE as compared to some of the very efficient filters in literature. The weighted average output of the filter does not belong to the vectors in the window because of which the filter can be extended further for Gaussian noise and mixed Gaussian and impulse noise removal. Other than this, an unwanted commonly occurring issue called smoothing can be still further minimized. Future work will be in introducing a directional distance factor in the exponential weight function so that the chromaticity maintenance of the image is improved further.


Despite the relative large size, both peptides were able to permeate the dense active layer of the AIM membrane and the transport mechanism was determined to be diffusion-based. Interestingly, the membrane permeability increased 3.65 times for the 692 Da peptide (1.39 × 10−12 m2·s−1) compared to the 375 Da peptide (0.38 × 10−12 m2·s−1). This increase thus occurs for an 85% increase in Mw but only for a 34% increase in peptide radius of gyration (gR) as determined from molecular dynamics (MD) simulations. This suggests that gR is a strong influencing factor for membrane permeability. Thus, an increased gR reflects the larger peptide chains ability to sample a larger conformational space when interacting with the nanostructured active layer increasing the likelihood for permeation.Forward osmosis (FO) membranes have gained interest in several disciplines for the rejection and concentration of various molecules. One application area for FO membranes that is becoming increasingly popular is the use of the membranes to concentrate or dilute high value compound solutions such as pharmaceuticals. It is crucial in such settings to control the transport over the membrane to avoid losses of valuable compounds, but little is known about the rejection and transport mechanisms of larger biomolecules with often flexible conformations. In this study, transport of two chemically similar peptides with molecular weight ( Within the membrane community there has been an increasing focus on the filtration technique forward osmosis (FO), where filtration is driven by a concentration gradient as opposed to the traditional pressure gradient known from reverse osmosis (RO). FO membranes have gained interest in several disciplines and are finding applications in different markets such as seawater and brackish water desalination ,2, wasteOne of the key performance indicators for FO membranes is the rejection of organic molecules. In drinking water or wastewater treatment, high rejection is important to ensure that micropollutants such as pesticides, pharmaceuticals, and endocrine disruptors do not end up in the final product. In concentration or dilution applications, such as downstream processing of pharmaceuticals and fertigation, it is also important to control the transport of other molecules than water across the membrane to avoid loss of valuable components.WM < 365 Da) and with a relatively well defined and rigid structure  is the membrane permeability coefficient of the membrane.In FO, water transport is driven by an osmotic pressure gradient, Δπ [bar], between the feed and draw solution. For an ideal membrane, the water flux, C [g·L−1], as described by Equation (2).B [L·m−2·h−1] is the salt permeability coefficient and sJ [g·m−2·h−1] is the salt flux.For the same membrane, salt will diffuse from the draw to the feed solution as a result of the salt concentration gradient, ΔR [%] is the rejection of the specific solute, fc is the average concentration of the solute in the feed during experiments and pc is the solute concentration in the permeate. The average concentration of the solute in the feed during experiments is described by Equation (4).0c is the initial concentration of the solute at the start of the experiment and endc is the concentration at the end of the experiment. Any concentration unit can be used.The rejection of the solutes in an FO process is described by Equation (3).In a previous study with the same membrane, the rejection of three small organic molecules  was modeled with a solution diffusion based model, while a pore flow model could not be fitted . Since t−2·s−1] is the flux of peptides, P [m2·s−1] is the permeability and −3] is the average concentration difference across the membrane.In the solution-diffusion model, the membrane is viewed as a solid barrier, through which the molecules move by first adsorbing to the surface and then diffusing through the membrane matrix until they reach the other side of the membrane from where they desorb and move into the permeate. The flux through the membrane is described by Equation (5).D [m2·s−1] is the diffusion coefficient for the peptide in the membrane matrix and S is the sorption coefficient of the peptide to the membrane material. S is dimensionless, since it is the ratio of the peptide activity in solution relative to the membrane surface. In practice, it is sufficient to determine the permeability coefficient when modeling the rejection.The permeability coefficient can further be described as a function of the diffusion coefficient of the peptide through the membrane and the sorption coefficient of the peptide to the membrane material as shown in Equation (6).Experimentally, the permeability coefficient for each peptide was determined by measuring the flux of peptides across the membrane at different concentration difference values in the absence of water flux. In the assays, the water flux was eliminated by removing the osmotic gradient over the membrane, which in practice was done by using a solution of the same osmolarity on both sides of the membrane. The peptide flux was then determined by measuring the peptide concentration in the permeate after a given amount of time, and the permeability coefficient could be found as the slope of the linear plot.Biomimetic FO membranes from Aquaporin A/S were used to study the diffusion mechanisms through the membrane. The membranes contain functional aquaporin channels embedded in vesicles of 200 nm size which are further incorporated in the membrane through a thin film composite active layer formation . The TFCTwo different peptides were used for the up-concentration assay and transport mechanisms experiments, one larger peptide (GGG SGA GKT) of 692 Da and a second smaller peptide (AGKT) of 375 Da, see From a practical point of view, the two peptides are interesting since they potentially can be used to capture and recover phosphate. The peptide sequence Ser-Gly-Ala-Gly-Lys-Thr (SGA GKT) is a reconstructed P-loop in which proteins are able to bind phosphate groups of GTP and ATP. This hexapeptide has previously been reported to bind to  high pH  and may An FO operating module was designed, built, and used to conduct the experiments, as shown in bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid) consisting of 10 mM TES, 1 mM EDTA and the pH was adjusted to 8 with 1 M NaOH to generate stock solutions of 100 mg/L. From this, peptide solutions of 1 mg/L, 5 mg/L, and 10 mg/L were prepared. 0.5 mL of TES buffer (without peptide) was placed in the reservoir compartment and 100 mL of the buffered peptide solution was re-circulated in the bottom compartment. The system was saturated for five hours or overnight to avoid any start up effects such as the loss of peptide to the chamber walls and the membrane. Before starting the diffusion experiment, the reservoir compartment containing the pure TES buffer was emptied and washed with TES buffer. The experiment was initiated by placing a fresh 0.5 mL aliquot of TES buffer in the reservoir compartment and was then set to run for five hours. At the end of the experiment, the TES buffer solution was removed from the reservoir compartment, weighed, and analyzed with HPLC to quantify potential diffusing peptides. The previously described steps were followed for each of the chosen concentrations for both the peptides.The FO module was used to determine the diffusion of the peptide molecules through the membrane matrix. A vial of each peptide (10 mg) was solubilized in TES buffer , equipped with a ZORBAX Eclipse XDB-C18 5 µm column . For GGG SGA GKT, an eluent mixture of acetonitrile and MilliQ  in the ratio 10/90 was used. The injection volume was 50 µL and the flow rate 1 mL/min. For AGKT, the peptides were eluted with a 67 mM phosphate buffer at pH 7.4. The injection volume was 50 µL and the flow rate 0.5 mL/min. The chosen absorption wavelength was 205 nm for both peptides.−1, and the pressure was set to 1 bar using the Berendsen barostat. Non-bonded interactions were cut off at 10.0 Å, full electrostatics for the periodic system were calculated using the Particle Mesh Ewald approach  from a molecular configuration can be calculated as shown in Equation (7).ith atom with position vector The mass weighted radius of gyration, To compare the influence of a rigid secondary structure on permeability, the radius of gyration was determined for three pesticides from a previous study . The radt is the membrane thickness (m) and wJ the water flux (m3·m−2·s−1). The equation assumes that feed and retentate concentration are equal, appropriate for low recovery values typically used to determine rejection values and that the concentration difference across the membrane can be approximated to be equal to the feed concentration, which is a good approximation for high rejection values (R > 95%).2) to generate the peptide flux in mol·s−1·m−2. The molar flux was then plotted as a function of the concentration difference across the membrane. A linear plot shows a diffusion driven process where the slope is the permeability coefficient, P, from Equation (6).To determine the rejection mechanisms, the permeate concentrations from the HPLC measurements were used to determine the molar flux of the peptides. The concentration of the peptide was multiplied with the volume in the draw chamber (0.5 mL) and divided with the membrane area  [Another factor influencing the permeability of the peptide through the membrane is their polarity and charge. Polarity can influence the sorption of the peptides to the membrane surface, which would be the first step in a solution-diffusion based transport mechanism, and the orientation of the molecule towards the membrane surface . Charge nd 9.60) . The chaIn The finding, that a molecule like the 692 Da peptide is capable of penetrating a selective layer that has been found to show almost complete rejection of molecules that are 4.5 times smaller  is highly interesting. It shows that for even dense and highly rejecting membranes, permeation of significantly larger molecules is still possible. This phenomenon in FO filtration might lead to a slow accumulation of the molecules in the draw solution, especially if the draw solution is recovered with membrane distillation and the molecules have a low volatility, which is the standard for biomolecules. The membrane penetration of the compounds might also ultimately alter the membrane performance. This is especially true for compounds that have a higher affinity for the membrane material and as such move into the structure and accumulate, which may ultimately change membrane characteristics. Also, within the FO community there is a constant search for the holy grail of draw solutes. If potential candidates are toxic even in very low concentrations, it is crucial to know that they might cross the membrane barrier to the feed side even when the size of the draw solute is relatively large.Finally, biomimetic membranes are based on the concept that biologically active molecules are incorporated in the selective layer of the membrane. This study shows that biomolecules not too dissimilar to proteins are capable of diffusing through the membrane matrix. The question is then at which size does it become completely impossible for these molecules to diffuse through the membrane. Biomimetic membranes and in general mixed matrix composite membranes are based on the incorporation of particles in the selective layer. The results of this study indicate that ultimately the structure of a mixed matrix membrane might not be static, leading to a leakage of membrane components to the surrounding medium. One way to overcome this could be to covalently bind the proteins in biomimetic structures to their surrounding environment in the selective layer.In this study, the transport of two peptides of molecular sizes 375 Da and 692 Da with flexible structures across a biomimetic forward osmosis membrane with a dense selective layer was investigated. The membrane exhibited high but incomplete rejection rates where 1% of the peptides were still able to cross the membrane barrier in spite of their relatively large size. The peptide with the smallest molecular mass was found to have the lowest permeability, which might be explained by the radius of gyration. A higher radius of gyration enables the peptide to assume several conformations when in contact with the membrane matrix, which ultimately could increase the probability of the peptide being transported through the membrane. The transport mechanism was found to be diffusion based. The overall finding of this study was that even relatively large molecules can cross an otherwise dense membrane layer. This is significant primarily for choosing the optimal operating conditions and run time of the system in downstream processing and needs to be considered for each and every forward osmosis application. Ultimately, it has a significance in the robustness of the membrane fabrication process where the incorporation of biomolecules in the selective layer is one of the key steps.


Globally, the World Health Organization reports that the chances of a child dying is highest in the first month of life, the neonatal period. The neonatal mortality rate in Cambodia is 18 per 1000 live births. In the province of Kampong Chhnang, that rate is the fifth highest among the 24 provinces of Cambodia at 27 per 1000 live births. We piloted a project to determine the feasibility of using a mHealth intervention  to increase mothers’ awareness about neonatal care and promote the government policy ‘Safe Motherhood Protocols for Health Centres’ which are in line with WHO recommendations for neonatal care.Between September and December 2013, we piloted an Interactive Voice Response system that sent pre-recorded messages to mothers of newborns using the theme ‘It takes a village to raise a baby’. Four hundred fifty-five mothers were registered onto this program and the intervention involved delivering seven periodic 60 to 90 s voice messages directly to the mobile phones of these mothers from day three of their neonate’s life to day 28. An evaluation of the pilot was conducted in December 2013. One hundred twenty-nine mothers were randomly selected from the 455 registered mothers and interviewed using a quantitative questionnaire. We also held two focus group discussions with three mothers and seven health workers.Quantitative and qualitative results of 126 respondents were included for analysis. They indicate that the intervention was well accepted. Seventy-one percent of respondents reported that they would recommend the intervention to other mothers, and 83% reported that they would be willing to pay for the service.This type of mHealth intervention is an acceptable and feasible way of promoting the awareness of newborn care to rural Cambodian mothers. The Intof life. –5. The aof life. , 4, 6. Ioutcomes . It also mHealth . In Camb mHealth . Accordi mHealth . We piloPeople in Need (PIN) – Czech Republic, a locally based International Non Government Organization (INGO), implemented a mHealth pilot in the local health administrative unit called the Kampong Tralach Operational District (OD) in Kampong Chhnang Province called ‘Baby Village Care’. It was designed to create more awareness regarding proper neonatal care in the first month of life based on what is recommended by the Ministry of Health Guideline for Safe Motherhood, issued in 2010 .Between 1 and 19 July 2013, a small formative research study was done in the target area of Kampong Tralach OD in Kampong Chhnang Province. This consisted of three pre-pilot assessments. Firstly, two brief interviews with local health authorities at the OD and Provincial Levels regarding the status of recent neonatal mortality. Secondly, we undertook a pretested quantitative questionnaire on 78 randomly selected mothers with newborns to ask about their attitudes regarding of piloting an mHealth program. Thirdly, midwives in the target health administrative area were also interviewed using another pretested questionnaire at the health centre and referral hospital level regarding their perspectives of a mHealth intervention in this area. The results of the formative study fed into the design of the mHealth pilot.The formative research results were incorporated into the design of the program. The ‘Baby Village Care’ program primarily targets mothers with newborns aged under 28 days who have access to a mobile phone. Our platform, an online cloud program entitled ‘Verboice’, used IVR technology to send seven voice-messages 60 to 90 s long directly to the mothers’ mobile phone starting from Day 3 of the newborn’s life. Verboice would call to the registered mobile phone number every 4 days until the newborn is 28 days old. Each message uses the different voices of several respected village ‘characters’ such as a doctor, a midwife, a village health worker, another mother, the village chief and an elderly grandmother. Given the novelty of mHealth programs, trained midwives at the health facilities were chosen to be the ‘gatekeepers’ of access to the service. Midwives from the target area received a half day interactive, hands-on training on how to register eligible mothers before the pilot was launched. Registration onto the service is done after the participating mother has given birth and before they are discharged from the health facility. We designed the pilot intervention with sustainability in mind and a conceptual framework was used to project future plans for the program that involved a model of self-funding from Public Private Partnerships. This is the unique feature of the pilot as often these are implement without a long-term goal in mind . . In the pilot evaluation, we interviewed 128 of the 455 participants using a pretested and back-translated quantitative questionnaire. These participants were randomly selected from the registered list of phone numbers recorded on the Verboice program and were interviewed over the phone. Two FGDs were conducted with one facilitator and one scribe. One FGD consisted of three mothers and three community health workers, the other consisted of all health workers . The quantitative survey data was entered into SPSS version 20 and the results of the pilot evaluation were performed using SPSS version 22. Frequency analyses were mostly used to yield the results reported here.For the pilot evaluation 128 mothers were interviewed using a quantitative questionnaire. Their general characteristics were an average age of 27 years, all but two were married, 47% were first time mothers, 70% had access to their own mobile phones (mobiles that belonged to the participant and not to their husband or other family members) Table This (initiative) makes mothers more aware of how to take care of (their) babies’. Our quantitative results indicate that 85% of the mothers surveyed listened to some messages not just once but several times, using the option in the IVR to repeat again, allowing the messages to be heard by other members of the household such as their husbands (n = 87), mothers (n = 67), siblings and older children (n = 23). The FGD results also showed that some first-time mothers said they had ‘shared the messages for other members of the household to listen to’.A key aim of the pilot intervention was to assess whether or not this type of intervention would be acceptable to the target audience and thus feasible for potential scale-up. The results indicate that acceptability was high with all mothers listening to at least four of the seven messages. Sixty-one percent mothers surveyed report that they told others about the service, 71% would recommend the service to other mothers and 63% reported sharing the information they heard with others. The results from our FGD with the health workers also responded well to the program. They report that the program was a good way of connecting with mothers, as one midwife said ‘Further, 96% of respondents reported the length of the messages (60 to 90 s) was appropriate and all respondents reported that the intervention was helpful for them when looking after their newborn. Additionally, 45% also reported learning something new from listening to the messages. The majority of respondents (96%) report that they would like to see the service extended, 44% of which suggested a service extension of at least 12 months. Finally, results also showed that 43% of mothers reported that they took their babies to the health centre as a result of listening to the messages.‘At anytime (when I was listening to the message) the line was clear. The timing was convenient for me because normally mothers with new babies stay home after birth and do not go anywhere’.We also assessed a number of technological points to judge user satisfaction. The timing of the automated calls  was one aspect of importance because we wanted to target a time when the mother is at home to receive the message and increase the chances of the messages being heard. Our evaluation results indicate that 36% of respondents received a call during the preferred time slot and the majority received calls at other times of day. Despite this, only 6 of those surveyed reported that they thought the timing of calls was inconvenient. The main reasons for inconvenient call time were that they did not have their phone with them at the time of the call or they were too busy to answer the call. The technical aspects of line rental provided by the private telecommunications company was also assessed . All respondents surveyed reported the line was clear and only 12 out of those surveyed reported their call was cut off while listening to the messages. Discussion during the FGDs included one first-time mother mentioning that n = 75) of respondents said that they would be willing to pay for the service. The fee ranged being between 100 and 3000 Cambodian Riels (4000 Riels = 1 USD) with an average willingness to pay of 577 Riels. One mother from the FDG remarked ‘I would like to pay USD 0.10 for the messages because they are very important to child health’. Despite this willingness to pay reported from the mothers, it should be noted that during the FDG with the midwives and community health workers, they reported that the program should be made free to mothers as they suspected that ‘most mothers do not have enough credit on their phones (to pay for the messages)’.To make this type of mHealth intervention financially sustainable willingness to pay was assessed. A majority . Finally, we did not collect any information on the literacy, education level or social economic factors of the targeted mothers. Such factors may have influenced the outcome of the pilot intervention. Our study focused on the feasibility of a mHealth intervention of this kind, however, we did not have the resources to assess the effects of the intervention on health outcomes. Our messages were also only available to mothers who delivered their children in a health facility, which meant that those who still delivered at home who maybe more likely to be a poorer, more vulnerable group are still at risk.For others considering implementing a similar project in other settings, we recommend that detailed pre-pilot work be considered to assess the community and local authority buy-in to the mHealth idea if it is novel. The results of this can feed into the design of the program and can be of valuable importance. During the development of the program content, based on our experience and results, the more realistic the messages can be, the more likely the target audience will be to responding to them. In our case, we tried as much as possible to use ordinary local wording, spoke clearly and slowly, using ‘trusted’ voices such as a village chief, a midwife, a peer mother etc. Though our pilot targeted mothers with infants, a largely female audience, we did not limit our messages to women only, but also reached out to men as they are a key part of the decision making process. As such, for other programs wanting to use IVR technology, we would suggest considering messages (their content and ‘voice’) to be as broad-reaching as it is possible, as it was in our case, so that those other than the targeted mobile user may also be listening in. However, on the flip side, if the program being implemented is to discuss highly private content (such as issues of domestic violence), consideration will need to be given to the potential that there may be other secondary users of the mobile. Finally, depending on the technology platform being used, try to make the system as user-friendly as possible. Consider putting fail-safes in place wherever possible so that if a mistake were to be made, there would be an option redo for the user.This pilot study was effective in justifying that this type of mHealth intervention, using IVR technology, is an acceptable way of improving the mothers’ awareness of newborn care. The results indicate that while this type of intervention is feasible on technological, cultural and economic grounds, more studies need to be done to show the impact of messages on behaviour change that can lead to improved MCHN outcomes through expansion to over the 1000 days period.


Background: Gas sensors are very important in several fields like gas monitoring, safety and environmental applications. In this approach, a new gas sensing concept is investigated which combines the powerful adsorption probability of metal oxide conductive sensors (MOS) with an optical ellipsometric readout. This concept shows promising results to solve the problems of cross sensitivity of the MOS concept.Results: Undoped tin oxide (SnOx) and iron doped tin oxide (Fe:SnOx) thin add-on films were prepared by magnetron sputtering on the top of the actual surface plasmon resonance (SPR) sensing gold layer. The films were tested for their sensitivity to several gas species in the surface plasmon resonance enhanced (SPREE) gas measurement. It was found that the undoped tin oxide (SnOx) shows higher sensitivities to propane (C3H8) then to carbon monoxide (CO). By using Fe:SnOx, this relation is inverted. This behavior was explained by a change of the amount of binding sites for CO in the layer due to this iron doping. For hydrogen (H2) no such relation was found but the sensing ability was identical for both layer materials. This observation was related to a different sensing mechanism for H2 which is driven by the diffusion into the layer instead of adsorption on the surface.Conclusion: The gas sensing selectivity can be enhanced by tuning the properties of the thin film overcoating. A relation of the binding sites in the doped and undoped SnOx films and the gas sensing abilities for CO and C3H8 was found. This could open the path for optimized gas sensing devices with different coated SPREE sensors. Due to the wide field of possible applications, many different concepts for gas sensing were developed in the past. Typical state-of-the-art concepts for gas measurements are electrochemical and infrared sensors, pellistors and metal oxide conductive sensors (MOS) . All seninterest –10. Thesinterest , sol–gelinterest , spray pinterest , sputterinterest –16 and einterest . In our x add-on layer was then added by using radio frequency (RF) magnetron sputtering  with 13.56 MHz frequency and 200 W power. Here, a commercially available pure SnO2 target (99.9%) obtained from FHR GmbH was used. The target has a diameter of 200 mm and a thickness of 6 mm. For the Fe-doped samples, a home-built RF magnetron sputtering equipment with 13.56 MHz frequency was used with a SnO2 target (99.95%) of 48 mm diameter. In this case, an additional rectangular Fe-strip (99.95%) of size 10 mm × 20 mm and a thickness of 2 mm was attached at the center of the SnO2 target. The deposition was done by applying 50 W of DC pulsed power at a frequency of 50 kHz with a pulse duration of 4 μs. Depending on the intended layer properties, the parameters of the sputtering were changed. These parameters are the partial pressure of argon and (if added) oxygen, bias and deposition time. For the undoped SnOx layers, pure argon with a pressure of 0.3 Pa was used. The Fe-doped SnOx was deposited with a 20%/80% oxygen/argon mixture with a pressure of 0.2 Pa. The thickness of the SnOx and Fe:SnOx overcoatings was 7 nm and 5 nm, respectively. The thickness was determined by using spectroscopic ellipsometry. The layer morphology was also determined by means of atomic force microscopy (AFM) and transimission electron mircoscopy (TEM) measurements. These measurements are reported in [x) was achieved and the surface roughness is low (root mean square average of height deviation Rq < 0.2 nm). Due to the low Rq value, roughness effects were not taken into account for explanation of the results. Furthermore, no indication of changes in the porosity of the layers which probably could effect the gas sensing ability was found.The sensors were prepared by a combination of two different techniques which were already reported –16. The orted in –19. It w2, 80% N2) and one added analyte gas . The analyte gas was mixed with synthetic air by using a home-built mixing setup which is regulated by a MKS 647 C multi gas controller monitor. The flow was kept constant at 100 sccm. The pressure inside the gas tube was manipulated by using a high-precision valve . The unit of sensor and attached gas tube is placed into the measurement spot of a single wavelength laser ellipsometer (Sentech SE 400). The ellipsometer is equipped with a helium–neon laser source (632.8 nm), a polarizer (P), a switchable prism compensator (C) and a rotating analyzer (AR). The angle of incidence (AOI) can be changed by means of a manual goniometer with a precision of 0.05°.The gas sensing technique called surface plasmon resonance enhanced ellipsometry (SPREE) has been described previously –22. The A theoretical description of the SPREE technique is given in detail elsewhere –25. In erp,s as the Fresnel coefficent and δp,s as the phase for the p- and s-polarization state. tanΨ and Δ are the consecutive amplitude ratio and phase difference of ρ. If a thin semitransparent layer like a gold layer is introduced between the two dielectric media, the overall reflection coefficient will change towith r01 as the Fresnel coefficient at the interface of the incident layer (BK7 glass) and the thin gold and r12 as the interface between the gold layer and the dielectric on the bottom side of the setup (air). The β value describes the film phase thickness given by the formulawith N1 as the complex refractive index of medium 1 und Φ1 as the incident light angle in medium 1.with Rp, the SPR effect can be described by the reflectance  = |Rp|2. Close to the surface plasmon resonance, the reflectance can be written asBy knowing that the intermediate layer is gold, the effect of the surface plasmon resonance has to be taken into account for the description of the reflection coefficients. Because this phenomenon effects mostly the reflection coefficient of the p-polarized part kx0 as the component of the wave vector of the incident light in the direction of the surface plasmon propagation and kSP =  + iΓ∞ as the wave vector of a surface plasmon on an interface of two semi-infinite media according to [ + iΓrad. In the ellipsometric measurement the Ψ and Δ values can be interpreted by [25with rding to . Due to reted by –2528.If the angle of incidence is varied close to the resonance condition, the Ψ function shows a minimum at the exact resonance angle, while in the Δ graph a step function can be observed. Both values reach their minimum due to the complete coupling to the plasmon resonance if an ideal system is assumed. If an additional layer is introduced between the gold layer and the ambient dielectric medium (air), the simulation of the system becomes more complex. In this case an extra pertubation has to be taken into account which will disturb the ideal resonance conditions. This case is shown in By changing the dielectric medium (air) with an addition of a certain volume of another gas, a shift in the dependence of Ψ and Δ on the AOI is predicted. The reason of this change can be explainend by different effects. First, a change of the refractive index of the dielectric medium (air) due to addition of another gas can be considered. In this case, the new refractive index of the gas mixture is given by the Lorenz–Lorentz-equation i as the partial density of the ith component, NA as the Avogadros constant, Mi as the molecular weight and αi as the polarizability. By using the changed refractive index of the gas mixture, the values of Ψ and Δ in the SPREE experiment can be calculated using −6 (ppm), this theory does not explain the measured changes in Ψ and Δ as presented in this study because the quantitative change would be immeasurably small. To explain this sensitivity, the adsorption of molecules on the surface of the sensor has to be taken into account. In this case, as known from SPR spectroscopy [with ρtroscopy –31, a neAs described above, the gas experiments show a pressure dependence, which changes the Δ response. By changing the relative pressure inside the gas tube, a signal could be detected with the SPREE experiment. The resulting signal in dependence to the relative pressure is shown in This pressure dependence can be a result of the changed amount of molecules adsorbed to the surface which effect the position of the SPR signal. Another explanation could be a changed refractive index of the gas medium due to the different density by increasing the pressure. This behavior shows a linear dependency in the studied pressure region to the pressure which is in agreement with both explanations. Supposedly, a combination of both mechanisms occurs when the total pressure of the gas phase is changed. To achieve the linearity and sensitivity shown in the following gas measurement section, it is necessary to keep the pressure constant during the experiment. Otherwise this additional degree of freedom will result in unpredictable response of the Δ signal to changes in the gas tube. We solved this by using a high precision pressure valve which is adjusted in dependence of the pressure gauge to keep the pressure constant. The pressure value for all gas experiments was adjusted to be 0.4 bar overpressure compared to ambient.A typical gas sensing experiment is shown in Here, a shift of 0.5° of the Ψ minimum and the Δ phase change can be observed on the AOI axis. The amount of this shift is related to the change in frequency of the surface plasmon resonance due to the adsorption of the gas molecules on the surface as described above. If the response of the Ψ and Δ signal is monitored over a certain time while the gas concentration is changed, a difference of the signal is measured as shown in x overlayer, as shown in We decided to focus on the evaluation of the ΔΔ values instead of analyzing the ΔΨ values because the signal to noise ratio is much better for the ΔΔ measurement. The calculated values of the standard deviation σ for the gas measurement of CO on a Fe:SnO3H8, CO and H2, can be measured down to the low ppm range with different sensitivity. All gases show a fast response to concentration changes. It takes approximately 3–4 measurements to reach the plateau of the signal which represents 9–12 s response time. This time is likely to be dominated by the time needed for the gas mixture to establish in the apparatus. Therefore, we suppose that all surface processes generating the measurement effect are faster than the time resolution of the experiment. All changes are fully reversible and the signal regenerates to the initial value within the same time of 9–12 s. As shown in x add-on layer shown in 3H8 shows the highest signal of 20° in ΔΔ for 100 ppm and is well resolved at 10 ppm with 2.2°. For H2 the maximum signal for 100 ppm reaches ΔΔ = 15° and can only barely identified at 10 ppm with ΔΔ = 1.5°. The lowest response was detected for CO with ΔΔ = 7.5° at 100 ppm and almost no visible signal at 10 ppm.It was found that all three gases, Cx layer as the overcoating for the gold sensing layer, the adsorption probability of the sensor layer system is changed. The undoped SnOx coating causes a stronger response of the sensor signal for C3H8 as for CO. This observation supports the assumption that C3H8 is more likely adsorbed than CO.In contrast, the response to H2 is driven by an increased diffusion into the sensing layer in contrast to an adsorption at the surface due to the small molecule size [x) overcoated sensors with an increased number of binding sites for CO were built [2 with 14.5° for 100 ppm which is comparable with the response of the undoped SnOx layer analyzed in the prior experiment. As the response of H2 is the same for undoped and Fe-doped SnOx, the assumption that the sensing mechanism is not based on adsorption of the molecule on the surface but on a diffusion process into the sensing layer is supported for this gas species. The signal for 10 ppm is still well resolved with ΔΔ = 1.6°. The better resolution in comparison to the undoped sample is a result of the much better signal to noise ratio achieved with the Fe:SnOx add-on layer. This effect is probably due to a changed thickness (5 nm for Fe:SnOx and 7 nm for SnOx) of the layer which also effects the distinct sharp form of the SPR signal. In another study, we have found that the thickness of the overlayer affects the gas sensing sensitivity but not the selectivity to a specific gas species. By tuning the thickness to a certain limit , the ability to measure a lower concentration of the added gas species is improved [3H8 gas with ΔΔ = 6.1° at 100 ppm and ΔΔ = 1.0° at 10 ppm. This extensive decrease in the response for C3H8 in comparison to the undoped SnOx coating corroborates the theory of a reduction of the amount of adsorbed molecules due the raised number of binding sites for CO.All signals respond linearly to concentration changes as plotted on the right graph in ule size . To suppre built . This waimproved . The sec2, CO and C3H8) is fully reversible even after several cycles. Furthermore, a linear response to the concentration of every gas species was found which shows a promising behavior for future applications. Further investigations to optimize the sensitivity for specific gas species have shown that the response of the ellipsometric Δ signal depends on the overcoating of the actual gold sensing layer. Two different add-on layers were tested, undoped SnOx and iron doped SnOx (Fe:SnOx) due to their different amount of binding sites for C3H8 and CO. The assumed effect that an additional amount of binding sites for CO, as present in Fe:SnOx, will lead to an increased sensing ability for CO is supported by the results. In consequence, a decreased signal for C3H8 was found. No effect in dependence of the doped layer was observed for H2. This was explained by a different sensing mechanism which does not rely on the adsorption of the molecules on the surface but on the diffusion into the sensing layer. However, the detailed sensing mechanism could not be determined due to the lack of temperature dependent measurements. An additional linear dependence to the gas pressure was observed which needs to be controlled during the gas measurement to obtain reliable data on the dependence of the measurement signal on the additional layer. The new type of gas sensing with the SPREE method showed promising results by confirming that the sensitivity to a specific gas can be controlled by changing the add-on coating of the sensing gold layer. Additional studies investigating the influence of the add-on layer properties to the gas sensitivity and selectivity will follow.The recently developed gas measurement technique, surface plasmon resonance enhanced ellipsometry (SPREE), was investigated in terms of sensitivity, reversibility and linearity. Additionally, the relation between changes of the dielectric function due to changes of the gas species and concentration was investigated. It was found that the gas measurement with all probed gas species (H


Dynamic wedges have been used in clinical practice for many years. Obvious superiority of dynamic over physical wedges is accompanied by the increased overhead involved in verifying the accuracy and reliability of their use. Contrary to very limited QA required to ensure proper functioning of the physical wedges, dynamic wedges, like any other dynamic treatment, require a robust QA program. This work expands upon previous suggestions and describes a comprehensive QA program for Varian enhanced dynamic wedges (EDWs) and presents the results of an 18‐month evaluation of these wedges. The QA program includes daily, monthly, and yearly tests and individual treatment QA at the onset of use of the EDWs. The results of the 18‐month evaluation show reproducibility in the wedge factors of better than 1% and in dose profiles of better than 2% on a monthly basis. Daily output measurements are generally within 2% of expected values.PACS numbers: 87.53.Xd, 87.56.Fc The enhanced dynamic wedge (EDW) is an option on newer Varian LINACs. In this mode of treatment, one of the upper jaws sweeps across the field from its maximum open position to within 0.5 cm of the opposite jaw, creating a wedged beam profile. In order to deliver a dynamically wedged field, the length of the treatment field is divided into 20 segments, and the speed of the moving jaw and the dose rate within each segment are controlled based on a calculated segmented treatment table (STT) generated by the LINAC computer. The STT is essentially a table of positions of the moving jaw versus the cumulative monitor units delivered at each position. The STT for a particular wedged delivery is a product of weighted averaging between an open‐field STT and a 60° “golden” STT. The details of STT generation and delivery have been explained by Varian.Like any other mode of dynamic treatment, use of EDWs should be accompanied by a rigorous QA program. The QA procedures described here have been implemented to assure proper functioning of the EDWs and can be easily performed in a clinical environment. This work is meant to be an update to previous reports on EDW implementation and QA, such as those by Moeller et al.The proposed QA of EDWs includes daily, monthly, and yearly tasks, as well as individual treatment verifications. The daily QA includes delivery of one EDW field per beam energy as part of the morning QA of the LINAC and recording the value of the central axis reading at a standard depth. The monthly QA includes measuring a sample of wedge factors, obtaining a sample of beam cross profiles, and saving a sample of dynalog files generated by the LINAC. Dynalog files contain information such as date and time of treatment, energy, monitor units, orientation, and type of treatment, as well as the STT calculated for the treatment and the actual delivered STT. The annual QA includes a larger sampling of wedge factors, similar to that for physical or hard wedges, and an analysis of saved dynalog files. The individual treatment verification or “per patient” QA includes visual inspection of the wedge direction at the conclusion of treatment and may only be necessary at the onset of the use of dynamic wedges.±3% must be satisfied pursuant to TG‐40 specificationsTwo EDW fields, one for each photon energy, have been added to the morning photon QA list. The wedge angle of 30° was chosen for this because delivering a 30° wedged field acts as a check of the integrity of the algorithm, since a “weighting” of the open and golden STTs must be performed to deliver this field. The central axis wedge factor can be obtained by dividing the “wedged” reading by the “open” reading. This value is used as a constancy check and is proportional to the clinically used wedge factors measured under standard conditions. Similar to open field constancy measurements, a limit of 20×20cm2 is used for the morning output checks.The morning output check device used here is the VeriDose system  and is calibrated on a bimonthly basis. This device is diode‐based, thus eliminating the need for temperature and pressure corrections. The diodes are beneath 2.3 cm water equivalent buildup; no additional buildup was used for measurements. A field size of Monthly QA consists of wedge factor and profile measurements. In addition, a number of dynalog files are saved in order to create a dataset for future analysis. Wedge factors are measured for a rotating selection of fields, of varying field sizes and energies, month to month. All the wedge factors are measured in a water phantom under standard measurement conditions (100 cm source‐to‐surface distance (SSD), 10 cm depth). A total of 14 wedge factors (one per wedge angle per energy) are measured each month. The wedge factors are then compared to those in the dosimetry tables, as obtained at the time of commissioning. Linear interpolation is used to obtain wedge factors for field sizes not measured at the time of commissioning.20×20cm2. All the Profiler measurements performed here were at 100 cm SSD with a 20×20cm2 field size without any additional buildup added to the device.Profile measurements are performed using the Profiler  for a fixed field size and all the wedge angles and energies. The purpose of this test is to check the constancy of the delivery of EDW fields. The Profiler consists of a linear diode array of 46 detectors with 0.5 cm spacing and with a 1‐cm water equivalent internal buildup. Additional buildup plates can be added to bring the scan plane to the desired depth. Our experiment with the addition of buildup plates showed no improvement in the reproducibility of the profiles for these energies (6 MV and 10 MV); thus, we chose not to add plates for ease of measurement. The Profiler's performance has been evaluated by Zhu et al.30×20cm2 field (largest deliverable field size in the Y‐direction), 30° EDW, for both energies. The saved dynalogs are used for the annual review and may be used for diagnosing problems that may manifest over time.During the delivery of a dynamically wedged field, the LINAC constantly checks the dose delivered against the desired delivery; that is, the delivered  STT is compared to the calculated STT, and an interlock is triggered should a discrepancy greater than 0.5 cm spatially or 0.3 monitor units dosimetrically be observed.10×10cm2 field and compared with commissioning data. Again, all measurements are performed in a water phantom at 100 cm SSD and 10 cm depth. The analysis of the dynalog files saved on a monthly basis is done during the annual QA by plotting the delivered STTs and comparing them.Annual QA is similar to that used for physical wedges, with wedge factors measured for all wedges and both energies for a Patient QA is an essential part of QA at the commencement of EDW implementation, and unfamiliarity of the therapists with the EDW may add to its importance. Normally, the matching of wedge direction between the plan, chart, and record‐and‐verify system is checked by the dosimetrist and physicist. It is, however, good practice to ask the therapists to confirm the orientation due to its importance in proper delivery of wedged fields. In the case of physical or hard wedges, the heel and toe direction of the wedge is usually checked by the therapists before the commencement of the first treatment. For EDWs, the verification of wedge direction prior to treatment can only be done by checking the wedge direction graphics on the collimator head. Posttreatment, the wedge direction can be verified by visually inspecting the location of the “light strip.” The light strip, which indicates the final position of the moving jaw, is the “toe” direction of the wedge that can be checked against the documentation in the chart. The therapists need to be trained to enter the room following the conclusion of treatment of each field to check the light strip on the patient's skin. This is particularly important when beginning to use EDWs, as a secondary check to proper transfer of information from the treatment‐planning system to the record‐and‐verify system, and can be eliminated once it is clear that no problems exist.All the QA tasks performed are summarized in 0.31(±0.29) for the 6‐MV and 0.71(±0.35) for the 10‐MV beam as compared to the values obtained at commissioning.Monthly wedge factor measurements over the 18‐month period show an average percentage difference of dmax. So some difference between the two quantities is expected, mostly due to scattering off the collimator jaws and variations in partial transmission through the moving jaw at various positions; otherwise, they should follow the same trend. As seen in the figures, the maximum difference between STT and dose profiles is about 2% for the 6‐MV beam and 6% for the 10‐MV beam. The error bars in these figures do not represent measurement uncertainty; rather, they are drawn to illustrate the degree of agreement between the sets of data. We have also found that larger field sizes exhibit a greater difference between these two curves. In general, however, the monitor units delivered as a function of jaw position (delivered STT) correlate consistently and reproducibly with the dose profile measured.The reproducibility of the dose profiles is shown in A comprehensive QA procedure for enhanced dynamic wedges has been proposed. Most of these QA routines can also be applied to the dynamic wedges available on other LINACs. An evaluation of the performance of these wedges is also presented. We have found very good reproducibility of these dynamic treatments over an 18‐month period for the 21ex accelerator, as well as accuracy and repeatability of the STT generation and delivery. We have outlined a workable QA procedure for this dynamic modality. The daily and monthly tests discussed here  should be adequate to detect any potential problems in the delivering of an EDW field.


A single-electron transistor is a nano-device with large potential for low-power applications that can be used as logic elements in integrated circuits. In this device, the conductance oscillates with a well-defined period due to the Coulomb blockade effect. By using a unique technique, we explore single-electron transistors based on a single metallic nanoparticle with tunable coupling to electric leads. We demonstrate a unique regime in which the transistor is characterized by multi-periodic oscillations of the conductance with gate voltage where the additional periods are harmonics of the basic periodicity of the Coulomb blockade and their relative strength can be controllably tuned. These harmonics correspond to a charge change on the dot by a fraction of the electron charge. The presence of multiple harmonics makes these transistors potential elements in future miniaturization of nano-sized circuit elements. Single-electron transistors are elements for nanoscale electronics. Employing single-electron transistors based on gold nanoparticles, Bitton et al., report a fabrication technique that allows precise control over the coupling between a nanodot and leads, resulting in new transport characteristics. Hence the device exhibits conductance oscillations as a function of gate voltage, Vg, with a well-defined CB periodicity (PCB = e/CG) equal to the ratio of an electron charge e to a gate capacitance CG. The periodic conductance oscillations make the SET a promising ground for applications such as an electronic switch6, memory device7, extremely sensitive charge and displacement sensor9, logic gates11, voltage amplifier12, and so on.Single-electron transistors (SETs) are based on a nanostructure such as a nanoparticle, molecule, or a quantum dot, which is resistively coupled to the source and drain leads and capacitively coupled to a gate electrode. Electrons are confined to a small volume and their number in the nanostructure is quantized. The current through the nanostructure can be tuned via the gate voltage which controls the number of electrons in the SET. Each time a single electron is added, the current is blocked due to Coulomb blockade (CB) effect13. This requires the fabrication of multi-SETs for each logic circuit. For instance, two multi-gate SETs can produce three current levels. A significant increase in SET functionality may be achieved if a single SET would be able to produce multiple well-distinguished current values between the high state (CB peak) and low state . This would enable the use of a single SET for switching between more than two states and therefore would facilitate going beyond binary logic. In this case, a set of multiple inputs, all connected to the gate electrode of a single SET, would generate an output which can have a few different values. Such a device will not only allow easier access to a multiple-valued logic function but would also be a large step toward further miniaturization of integrated circuit elements.Applications of multi-input gate SETs for multiple-valued logic circuits have also been proposedIn this work, we show that under certain conditions an SET can manifest multiple-periodicity where the relative intensity of each period can be well-controlled. A superposition of multiple periods having different intensities naturally yields different output values. The ability to manipulate the relative intensity of each period enables one to fine-control the output current values of the SET device.e2/h. In this weak coupling regime, the charge confined in such a closed dot is quantized and one observes a well pronounced set of conductance peaks. Each peak is associated with the change of total charge on the dot by one electron. The other limit is the strong coupling regime for which at least one of the leads is open, with the coupling greater than e2/h. In this open dot the charge is not quantized, CB effects are suppressed, and one observes only a weak modulation of the conductance through the dot, with the same period14. The crossover between closed and open dots is seldom investigated. In recent theoretical works16 the crossover regime was approached from an open dot limit  image of Fig. The SETs are prepared utilizing a unique technique that combines nanolithography, atomic force microscope (AFM) nanomanipulation and electrodeposition as illustrated in Fig. G(Vg) of such an SET system taken at T = 4.2 K is shown in Fig. PCB and the other is half this period. We measured CB effects on 21 similar samples. Multiple-periodicity was observed in nine such SET devices. In most cases the SETs exhibit conductance oscillation with the base period PCB and additional oscillation with a period PCB/2. However, some SETs exhibit oscillations that give rise to the third h/e2, controlled by the resistances between the dot and both source (RS) and drain (RD) electrodes. We only observe multiple-periodicity for SETs which are tuned to the appropriate coupling regime. Unfortunately, it is not possible to measure the coupling strength directly from the conductance. Moreover, since our dot is asymmetrically coupled, these two quantities are quite different. While the measured conductance, G, is governed by the weakly connected lead, the coupling strength, η is determined by the well connected one. Nevertheless, it is possible to extract η by fitting I−V curves to the results of ref. 31 as discussed in the 30). Doing so we find that there is a small coupling window where multiple-periodicity can be observed, that lies in the interval 2 < η < 5. This is demonstrated in Fig. G(Vg) for two SETs with two different coupling strengths. In Fig. η = 7.7 ± 0.1) and the conductance oscillates with a base PCB only, as demonstrated in the Fourier transform  and the conductance exhibits additional periods. In this case, the structure is much richer than those of Fig. fCB.It turns out that the intensity of the faster periods is very sensitive to the dot-lead coupling strength orm Fig. . In Fig.fCB. Though the theory predicts  for fabrication of source and drain electrodes separated by a small gap of 10–30 nm and a side gate electrode at a distance of 200 nm.L-Lysine (P.L.L) as an adhesive layer. After the deposition of the adhesive layer the sample is ready for the deposition of the gold colloids. The Au colloid deposition takes place by adding a 10–20 μl drop of the solution on the modified substrate for an hour. This results in 20–30 gold colloids within a 1 μm square area.The nanoparticles we use are gold colloids with diameter of 30 nm. We use colloidal particles in solution which are stabilized by negatively charged ions that prevent agglomeration of the particles in solution. By using an organic layer that is terminated with amino groups, it is possible to adsorb the negative shell of the particles to the substrate by electrostatic interaction. We chose Poly-For placing the nanoparticle in a desired location we utilize AFM nanomanipulation (Nanoman system in DI Veeco 3100 Scanning Probe Microscope). The nanoparticle is moved between the electrodes using the AFM tip, by pushing the particle to the right position.After positioning the colloid in the gap, the distance to each lead is usually smaller than 10 nm; however, the dot is not yet electrically connected to the leads. At this stage, we are not able to monitor current through the device. For minimizing the gap between the nanoparticle and the leads, we use an electrodeposition process by which we grow atoms on the leads. During the deposition process, we measure the conductance between the source and drain and stop the process when current can be measured. For the electrodeposition process, we place the sample on a holder which is made of inert materials (glass and Teflon). This is very important for preventing any metal dissolution that may result from reaction with the solution. The electrodes are connected to the circuit through Cu wires pressed by Indium which are placed out of the solution.2) and a buffer (PH 10) composed of 1 M potassium bicarbonate (KHCO3) and 0.2 M potassium hydroxide (KOH). A 25 mm diameter Au wire (99.9985% purity) is used as the counter electrode and the reference electrode. The two separated evaporated gold electrodes are the working electrodes. When applying electrochemical DC voltage between the working electrodes and the counter electrode, the cyanurate ion accepts an electron from the working electrodes and liberates the cyanide ligands. Hence, neutral gold atoms collect on the surface of the two gold electrodes and close the gap between the leads and the dot.Our electrodeposition set-up consists of solution, counter electrode, reference electrode, and working electrodes Fig. . The eleThe deposition occurs only on the electrodes and not on the colloid as it is not connected to the circuit. Deposition occurs on the colloid only after it becomes electrically connected to one of the electrodes and becomes part of it.−1. Under these conditions the deposition is very uniform and smooth, thus the possibility of large gold clusters forming during the electrodeposition process becomes improbable. In addition, the slow rate enables to stop the process at different degrees of couplings.The value of the DC voltage between the counter electrode and the source and drain electrodes determines the deposition rate. The slower the deposition, the higher is the quality of the film. We found that a value of 1 V yields a slow enough deposition rate of 0.3 nm minThe deposition is isotropic, hence the gold is deposited uniformly on the surface of the electrodes with no preference to a certain direction. Therefore, bridging the dot-leads distance causing the widening of the electrodes tips. It is for this reason that the coupling between the dot to the leads and hence the dot-lead conductance is believed to be achieved by a large number of channels.For measuring the conductance between the working electrodes during the electrodeposition process, we use AC conductivity measurement which ensures equal deposition on both the electrodes. An AC voltage of 2 mV is applied between the two working gold electrodes. We are able to monitor the separation between the electrodes once the distance becomes very small. The oscillation frequency should be very low in order to reduce the ion conductivity of the solution which appears as a background measurement. Applying frequency of 2 Hz reduces the measured conductivity of the solution to 5 MΩ. This value is always measured at the background. Therefore it is extremely difficult to stop the process at the stage of a very weakly connected barrier. Figure After electric contact is achieved the sample is taken out of the aqueous solution and transferred to a measurement probe where it is cooled down to 4.2 K for electrical measurements. High resolution scanning electron microscope (SEM) images of different samples taken after different fabrication stages are shown in Fig. G ≤ e2/h while still being able to observe CB effects. Twenty-five percent of our electrodeposited samples showed CB effects while the rest showed ohmic conductance curves, apparently due to strong antisymmetric geometry. An example of such asymmetric device is shown in Fig. The success of the electrodeposition process crucially depends on the symmetry of the distance between the dot and the leads. If the metallic colloid is not positioned at the center of the gap, there is a very small chance to stop the electrodeposition process at measured conductance of Finally, it is important to note that the same fabrication technique, including the adsorption of the adhesive layer, P.L.L, was applied to several devices without placing a nanoparticle in the gap. All these devices did not show any signature of CB effects in the conductance curves . This asThe data that support the findings of this study are available from the corresponding author on reasonable request.Supplementary InformationPeer Review File