And we replaced the substrate with Ni/SiO2/Si and measured the thickness of Ni film by Rutherford backscattering spectrometry (RBS). Combined with suitable anneal conditions, these samples implanted by various small carbon clusters were made to grow graphene. Results from Raman spectrum reveal that few-layer graphene were obtained and discuss whether IG/I2D can contribute to explain the relationship between the number of graphene layers and cluster implantation dosage.Based on the extensive application of 2 × 1.7MV Tandetron accelerator, a low-energy cluster chamber has been built to explore for synthesizing graphene. Raman spectrum and atomic force microscopy (AFM) show that an amorphous carbon film in nanometer was deposited on the silicon by C For instance, the energetic ion-solid interaction was taken as a classic model to characterize some structure information of superconductor at room temperature or high K by projecting MeV ions to impact on superconductive targets [in situ characterization of these transients which can exhibit a clear physical image on changeable process of the structure was performed by the accelerator-transmission electron microscopy (TEM) interface system [In the past of several decades, ion beam analysis (IBA) based on low-energy accelerator has developed to be a comprehensive particle analytical discipline system -4. A fur targets . In ordee system ,8. For pe system ,10. In ae system .In Wuhan University, the double 1.7 MV Tandetron accelerator was inherited from Physical Institution of Chinese Academy of Sciences in 2004. After several important maintenances and upgrades of facility, some primary ion beam analysis with terminal voltage at 1.2 MV can be performed in a good state, such as Rutherford backscattering spectrometry (RBS), elastic recoil detection analysis (ERDA), and nuclear reaction analysis (NRA). Besides, we have developed some extensive applications, including accelerator-TEM interface system [As another kind of ultra-thin carbon film, graphene is a promising material which is probable to replace silicon integration technique due to its advanced and novel physical properties ,13. But −, C−, Si−, P−, Fe−, Cu−, and Au−[A source of negative ion by cesium sputtering (SNICS) can produce various negative ions from solid targets, such as B, and Au−,17, whic−) from SNICS at an energy of 20 keV are chosen for desirable targets by mass analyzer, then are decelerated to a few hundred electron volt or below 3 keV by the deceleration field after voltage scanner mounted on two aligned directions of X and Y-axis, finally to soft-land to the substrate. The current integrator is used for monitoring implantation dose simultaneously. To eliminate some impacts on the current integrator from high voltage at decelerated filed, an isolation transformer was introduced to guarantee safety. In addition, a rotated target holder  is about 5.10 nm for thin film, which indicates a great promise of preparing ultra-thin film under much lower energy ion implantation.Figure  edition . About 12/Si with about 300 nm Ni film deposited Ni atoms onto silica by e-beam evaporating.It is an essential purpose that we designed this low-energy cluster chamber for graphene preparation. In the process of exploring some effective methods, after depositing carbon films with the scale of several nanometers on the silicon, we selected suitable substrates to succeed in achieving few-layer graphene. Uninstalling the decelerated field, we selected small carbon cluster ions to inject to the substrate below 30 keV. The substrate Ni/SiO2+ which was produced by the double 1.7 MV tandem accelerator was collimated to the target with ion current of 5 nA and the round beam spot of 1.5 mm. The backscattered ions were detected by passivated implanted planar silicon (PIPS) detector with the resolution of 14 keV for α particle at 165°. The abscissa of spectra stands for channel numbers of multi-channel analyzer (MCA), which is proportional to the energy of scattered ions. A broad peak indicates that the surface edge of Ni is about channel 269 and the back edge is about channel 195. The channel difference of both edges is corresponding to the energy loss of projectile Li ions in Ni in correlation with the thickness of thin film. A straightforward route is simulating the trajectories of incident ions in matter. The red curve of this graph is simulation result from SIMNRA6.05 code, which is in coincidence with experimental data absolutely. The simulated results reveal that the areal density of Ni film is 2.1 × 1018 atoms/cm2, and a corresponding thickness is 227.3 nm in the case of the volume density at 8.9 g/cm3, which is thinner than the estimated value.The thickness of Ni film has influence on carbon segregation from inside up to the surface, so it is significant to evaluate the thickness of the substrate, and RBS spectra of the sample was carried out, as shown in Figure 15 cm−2 by ion implantation consisting of different cluster sizes at 20 keV. After implantation, these samples were annealed from room temperature to 900°C and dwell time was 60 min, then cooled down to room temperature naturally at 2.0 torr.With regard to depositing Ni film onto silica but not silicon substrate, it was reported that the silicon oxide at a thickness of 300 nm can enhance scattered signals of Raman resonance spectrum drastically because photon can evoke continuous interferences at the interface between Ni and silica . All the−1 is relative to the disorder and defects in the structures performing sp3 hybridization of carbon atoms, while sp2 hybridization induced by the in-plan optical phonon E2g near the first Brillouin Zone center is characterized as G peak at 1,580 cm−1[−1 of graphene is single and symmetrical to characterize monolayer. These samples were implanted with the same dosage of 8 × 1015 carbon atoms/cm2 at 20 keV by the different small carbon cluster sizes . Almost the three characteristic peak positions appear, and every peak position for different cluster sizes has also negligible shifts, as shown in Figure −1 and IG/I2D (the intensity ratio of G peak and 2D peak), which is the smaller and thinner film that can be obtained, were also evaluated to differentiate graphite and confirm the layers of graphene sheets [−1 in the spectra, corresponding to three and more layers. A visualized trend is observed that IG/I2D decreases as carbon cluster size increases, described in Figure 1 to C4, meanwhile larger clusters C4, C6, C8 are presenting a relatively gradual shrink. In the case of such low-energy ion implantation, light cluster can penetrate into deeper sites than heavy cluster in the substrate, which is dependent on the energy distribution of cascade collision between cluster and matter.Raman spectroscopy is always employed as one of the powerful non-destructive methods to identify graphene and determine the layer of graphene ,21. In t,580 cm−1. The 2D C1, C2, C, C6, C8.1 implantation instead of saving time. For example, implantation time is about 8.5 h for cluster C8 at 20 keV in this work, but the IG/I2D ratio is the smallest which indicates that the graphene quality is better than that in the other smaller cluster sizes.In previous studies, we have achieved some carbon cluster mass spectra at the different extractor voltages of sputtering ion source in this machine and paid much attention to investigate Raman characterizations of graphene by means of the carbon cluster ion implantation ,23. BecaE0 is cluster energy, and every atom of Cn cluster can be allocated as homogeneous energy of E0/n. Therefore, in comparison with C1, Cn (n > 1) has more sophisticated interactions with the substrate, involving in non-linear damage effect and atomic self-sputtering effect [15 cm−2 and 8 × 1015 cm−2 of carbon concentration on the surface are in agreement with monolayer and bilayer graphene [n (n > 10) under suitable energy is more likely to develop this process. But we have to take the atomic self-sputtering effect and more sophisticated cluster-matter interaction into consideration. More investigations are probable to promote the nucleation mechanism of graphene including ion-matter interaction, crystal quality of the substrate, anneal temperature, and other details about growth conditions.Simply, g effect ,25. Durig effect , no mattg effect ,15,20,214 cluster ions to the silicon at 14 keV, and the RMS is about 5.10 nm. Another substrates Ni/SiO2/Si whose thickness was measured at 227.3 nm by RBS were implanted with the same carbon atoms at 8 × 1015 cm−2 by several kinds of small clusters Cn ; after annealing, Raman spectra indicate that few-layer graphene was prepared successfully. And the ratio IG/I2D shows that the number of graphene layers cannot be controlled by implantation dosage purely but are associated with carbon atoms precipitation and segregation from inside to the surface grain boundaries of the substrate during thermal treatment. From ultra-thin carbon film to graphene by means of the similar cluster ion implantation technique, it is conductive for cluster implantation of light elements to develop low-energy shallow ion implantation in semiconductor industry.We have developed a low-energy cluster chamber on the base of extensive application for the double 1.7 MV Tandetron accelerator, which was used to explore for graphene synthesis. In our previous work, a kind of amorphous ultra-thin carbon film was fabricated by projecting CAFM: atomic force microscopy; ERDA: elastic recoil detection analysis; IBA: ion beam analysis; MCA: multi-channel analyzer; NRA: nuclear reaction analysis; RBS: Rutherford backscattering spectrometry; RMS: root mean square roughness; TEM: transmission electron microscopy.The authors declare that they have no competing interests.ZW designed parts of the experiments and sample preparations and drafted the manuscript. DF is the corresponding author and provided a great help for experimental designs. Other co-authors took part in sample preparation and characterizations and discussed the results. All authors have read and approved the final manuscript.


Tξ in the graphene layer.We report on gate-tuned locality of superconductivity-induced phase-coherent magnetoconductance oscillations in a graphene-based Andreev interferometer, consisting of a T-shaped graphene bar in contact with a superconducting Al loop. The conductance oscillations arose from the flux change through the superconducting Al loop, with gate-dependent Fraunhofer-type modulation of the envelope. We confirm a transitional change in the character of the pair coherence, between local and nonlocal, in the same device as the effective length-to-width ratio of the device was modulated by tuning the pair-coherence length  W) superconductor–normal-conductor–superconductor (SNS) proximity junction1x1 on the interface N/S1 is governed by sum of phase-coherent contributions from positions x2 along the opposite interface N/S2, located within the pair coherence length Tξ from x1. For an S1-to-S2 spacing L1 sufficiently shorter than Tξ, however, the pair transport depends on the phase difference between the same lateral positions (x1 = x2) only, revealing the local nature of pair coherenceCarrier transport in a mesoscopic system is governed by quantum mechanical interference over the spatial phase-coherence range where the interfering phase of carrier waves is determined by the nonlocal response to electromagnetic fields. This nonlocal nature also emerges for the transport of superconducting pair carriers in a laterally extended (width h/2e). The critical current periodicity of h/e was observed, employing separate extended Josephson junctions of channel length different L1/W ratiosdifferent physical geometryL1 and W of a junction changeFully nonlocal Josephson pair coupling is predicted to show twice the magnetic flux periodicity of the junction critical current for local pair interference4φl (> L1 > Tξ). In contrast to studies in the Josephson configuration, in existing pair-coherence studies using Andreev interferometry, the nonlocal character has been commonly observed1213Tξ marginally close to L1/2. To that end, we adopted an extended S-graphene-S junction consisting of a graphene normal-conductor (N) layer16Tξ as adopted in studies of graphene-based Josephson phenomena192021222324In an SNS proximity junction, the pair coherence can be examined more closely by employing the Andreev interferometry67891011121314Tξ in highly doped range (near the CNP) in graphene. We continuously adjusted the character of the superconductivity-induced pair coherence in a GAI by changing the effective aspect ratio of the junction along with gate-tuning Tξ in the graphene layer. All three GAI devices that we examined exhibited similar features and we present below the data for one particular device. Accurate tracing of the pair-coherence characteristics, in this study, was possible by utilising the highly stable gate tunability of both the carrier density and the corresponding Tξ in graphene. The Andreev interferometry adopting graphene offers a convenient and unique platform to investigate the locality of the pair coherence effect.Andreev interferometers adopted in this work consisted of an extended SNS junction with the graphene N layer. We observed Fraunhofer-like magnetoconductance (MC) modulation; with the local  character for a long (short) h/2e threading the area of loop and (ii) Fraunhofer-type envelop variations of the rapid conductance oscillations, attributed to the magnetic flux threading the closed carrier path in the phase-coherent region of the TGBh/2e  and h/e  along with the gate-dependent variation of Tξ. This cannot be due to a change in the flux focusing because the physical geometry of the junction did not vary with gating. The gradual shift of the field periodicity is a consequence of tuning the locality of the superconductivity-induced phase coherence represented by Tξ in a GAI.The GAI consisted of a T-shaped graphene bar (TGB) and an open Al superconducting loop, with the two ends of the Al loop in direct contact with the two arms of the TGB see . In the 2/Vs for a backgate voltage bgV of 50 V at a base temperature of 50 mK. The carrier motion in the TGB was diffusive  of the left (right) G/Al interface, while each attains the phase of the superconducting order at x1(2). These Andreev-reflected partial waves propagate back to P in a diffusive way to be recombined. The phase value at x1(2) can be expressed as ϕ10(20) is the phase at the center of the left (right) G/Al interface and x-axis direction.The superconductivity-mediated phase coherence of the carriers was established in the TGB between the two G/Al lateral interfaces. Suppose that an electron is scattered at position P in graphene into two electron-like partial waves. These waves are then Andreev-reflected as hole-like partial waves at different points ϕ = ϕ1 − ϕ2 = 2πΦ/Φ0, where Φ0 = h/2e is the flux quantum and Φ is the magnetic flux threading the area, A1 (≈13.3 μm2), enclosed by the centers of the Al-loop wires connected to the left and right G/Al lateral interfaces .The phase difference between the ends of the superconducting Al loop was controlled by an external magnetic field with fluxes threading loops (i) and (ii); this induced the above conductance oscillations with a modulated envelope. The total phase difference over loop (i) was Δterfaces . The areG as a function of the magnetic field B for bgV = 50 V at T = 50 mK, which resulted from combined contributions of the superconductivity-induced conductance enhancement and the conductance oscillation ΔG below the critical field of Al, cB = 10.5 mT. The conductance enhancement defined by (SG − NG)/NG was a consequence of the penetration of the Al superconducting order into the graphene at the G/Al interfaces. Here, SG and NG represent the differential conductance of the graphene in contact with the Al ring in the superconducting and normal states, respectively. The maximum of (SG − NG)/NG was ~ 0.18, where G (B = 0 mT) and G (B = 12 mT) were used as SG and NG, respectively. The phase coherence of the GAI over the length L2 was represented by the conductance oscillation of ΔG(B) . ΔG(B) consisted of fast h/2e oscillations in B in loop (i) was 0.158 mT, which is in good agreement with the value of 0.155 mT estimated from the GAI geometry  is about an order of magnitude larger than a previous report for a GAIG (~ 0.3e2/h), we obtained similar Fraunhofer behaviour (not shown) as the device with the large value of ΔG.etry see . In Fig.G oscillation in G(B) in h/2e, h/4e, and h/6e oscillations, which led to the field dependence of ΔG as G(B) [solid curve in i.e., the constructive interference of hole (electron) carriers implying the diffusion  of carriers with an increase (a decrease) of the conductance.The ΔR (≡ 1/G) as a function of bgV, where bgV = −19 V is the CNP. ΔG in graphene increased with the electron- or hole-like carrier density by positive or negative gating away from the CNP. The oscillation periods (ΔB) of the ΔG envelope for bgV = 50, −10, −19, −30, and −50 V were 2.85, ~5, ~6, ~5, and 2.95 mT, respectively. The significant variation in ΔB with the carrier density was caused by the change in the spatial distribution of the e–h phase coherence  at the G/Al lateral interfaces (described below), rather than by the change in A2. The weak gate dependence of the phase-coherent TGB area did not induce a significant change in A2  is the effective temperature of the carriers, and Fv is the Fermi velocity in graphene. Tξ was estimated using the relationship ΔG (B = 0) ~ exp(−L2/Tξ); ~320 nm for bgV = 50 V and decreased as bgV approached the CNP [see G (B = 0) remained finite at ~0.1e2/h at the CNP due to e–h puddle-induced fluctuations of the carrier density, which kept Tξ finite on the scale of the e–h puddle-induced density fluctuations . Assuming processes involving only a single Andreev reflection from each G/Al interface, the conductance oscillation is given byf can be approximated by a Gaussian-type weighting factor as x1 in a G/Al interface and x2 in the opposite-side interface. The parameter α simply controls the range of interference. For 2Tξ ≈ L1, the overlap of Tξ between x1 and x2 is weighted at x1 ≈ x2, thus, the magnitude of the e–h phase coherence at the overlapped region of Tξ is much larger than that at the other region and the corresponding effective range of interference is also localised , f ≈ δ(x1 − x2), which is 1 for x1 = x2 and 0 otherwise. For this local pair coherence, ΔG is simplified  becomes almost constant for all combinations of , leading to nonlocal pair coherence. In this case, an Andreev-reflected hole at a point x1 of the left G/Al interface interferes, with equal probability, with an Andreev-reflected hole at any value of point x2 of the right interface, and vice versaG is given by . Here, as ΔG is proportional to the square of the sine function, ΔG has a zero phase value for any magnetic field B  modulation has recently been observed in S-graphene-S-type Andreev interferometersB-field-dependent phase relationship. It was accomplished by varying the effective L1/W ratio continuously with fixed physical geometry of the devices along with changing the backgate voltages and the resulting Tξ in a given device. The local behaviour was confirmed by fine tuning Tξ marginally close to L1/2 in the highly doped range of graphene, while the nonlocal behaviour was obtained for Tξ much shorter than L1 close to the CNP of graphene. The close examination of the pair coherence characteristics was made possible using the highly stable and effective gate tunability of both the carrier concentration and the corresponding pair coherence length in graphene. GAI provides a convenient and unique platform to investigate the locality of the pair coherence.The locality of the pair coherence in a normal conductor in contact with superconductors is at the core of studies on the Josephson coupling in mesoscopic scales. We report the first Andreev-interferometry observation of the local behaviour of the pair coherence as well as the common nonlocal behaviour and a continuous tuning between them using GAIs, which was confirmed by the flux period of the Fraunhofer-type conductance variations and the contrasting L1 between the two G/Al lateral interfaces was 550 nm. The spacing L2 between the voltage leads B and C was ~0.8 μm. The Al loop was prepared by in-situ sequential deposition of a Ti/Al/Au (7 nm/70 nm/5 nm thick) tri-layer onto the prepatterned e-beam resist. The Ti and Au layers improved the contact at the G/Al interfaces and protected the Al surface from oxidation, respectively. The high transparency of the G/Al interfaces was confirmed by the almost vanishing (<0.1 Ω) contact resistance. With the interferometer mounted in a dilution fridge , a bias current I was injected into the interferometer in a symmetrical manner between leads A and D. The voltage difference V was monitored between leads B and C [A monolayer graphene sheet was exfoliated from a thin graphite flake onto a heavily electron-doped Si substrate capped with a 300-nm-thick oxidation layer. The Si substrate was used as a backgate to modulate the carrier density and types in the graphene. An open-square Al loop of 400 nm in width was directly coupled to the graphene by electron (e)-beam patterning, e-gun deposition, and the lift-off technique. The graphene layer was patterned using e-beam lithography and oxygen-plasma etching into a T-shaped structure having a linewidth of 500 nm. The spacing  B and C  using a M.K., D.J. and H.-J.L. designed the experiments. M.K. prepared the samples and performed measurements. D.J. and G.-H.L. assisted in measurements. M.K., G.-H.L., Y.-S.S. and H.-J.L. analysed the data, H.-W.L. provided the theoretical consultation and H.-J.L. supervised the project. M.K., Y.-S.S. and H.-J.L. wrote the manuscript. All authors contributed to the discussion and reviewed the manuscript.Tuning Locality of Pair Coherence in Graphene-based Andreev Interferometers


Contextual elements have significant impact on uptake of health care innovations. While existing conceptual frameworks in implementation science suggest contextual elements interact with each other, little research has described how this might look in practice. To bridge this gap, this study identifies the interconnected patterns among contextual elements that influence uptake of an anticoagulation clinic improvement initiative.a priori approach based on the Promoting Action on Research Implementation in Health Services (PARIHS) model and an emergent thematic analysis.We completed 51 semi-structured interviews and ethnographic observations across five case study sites involved in an evidence-based practice (EBP) quality improvement initiative. We analyzed data in NVivo 10 using an Key contextual elements, such as leadership, teamwork, and communication, interacted with each other in contributing to site-level uptake of the EBP, often yielding results that could not be predicted by looking at just one of these elements alone. Sites with context conducive to change in these areas predictably had high uptake, while sites with uniformly weak contextual elements had low uptake. Most sites presented a mixed picture, with contextual elements being strongly supportive of change in some areas and weak or moderate in others. In some cases, we found that sites with strong context in at least one area only needed to have adequate context in other areas to yield high uptake. At other sites, weak context in just one area had the potential to contribute to low uptake, despite countervailing strengths. Even a site with positive views of EBPs could not succeed when context was weak.Interrelationships among different contextual elements can act as barriers to uptake at some sites and as facilitators at others. Accounting for interconnections among elements enables PARIHS to more fully describe the determinants of successful implementation as they operate in real-world settings. The uptake of evidence-based practices (EBPs) and quality improvement (QI) interventions can be notoriously challenging. The effort to implement EBPs entails attention not only to designing an appropriate intervention, but also to the dynamic organizational context in which the intervention is to be implemented. Established implementation science (IS) work on barriers and facilitators to uptake has proven useful in real-world implementation efforts, especially in regard to identifying individual contextual elements and noting ways these singular contextual components influence the implementation of health care innovations -6.Implementation researchers anticipate that when a majority of contextual elements are conducive to change, implementation is usually successful. In contrast, when a majority of contextual elements are not conducive to change, implementation is not successful. However, in many cases where EBPs or QI projects are implemented, there is a more mixed, intermediate picture that can produce interesting, yet unpredictable, patterns of uptake. Our study identifies and describes a range of such situations at five sites with a “mixed” picture of contextual elements. Therefore in this study we discuss more broadly how interactions of contextual elements impact QI projects. Since little is known about these interactions , the preWe used the Promoting Action on Research Implementation in Health Services (PARIHS) model prospectively to implement a large anticoagulation improvement project. PARIHS states that successful implementation (SI) is a function (f) of the nature and type of evidence (E), the qualities of the context (C) where the evidence is being employed and the way the process is facilitated (F) or as Kitson et al. represent it, SI = f  . EvidencContext is dynamic, multivalent, and highly variable in organizational life. Key work on PARIHS recognizes the dynamic relationship among elements and sub-elements and therefore PARIHS contains within it an understanding that the same intervention will likely vary widely in different settings ,10. HowePARIHS elements are often described on a continuum which ranks their “strength” in regard to support for the intervention from low to high . This asEvidence is the stakeholder’s perceptions of the quality and validity of the evidence supporting the intervention, in this case the ACC treatment algorithm, the dashboard that measures TTR and work processes that increase standardization. In this paper, a leader refers to those in positions of management such as Pharmacy Service Line Chiefs and middle managers . Leadership is an element of Context in PARIHS. In this study, leadership has three dimensions, where it: (1) describes the activities (such as supporting effective teamwork) and attitudes taken by leaders (those in management positions) to respond to the ACCII as documented by the research and implementation teams, (2) includes the perceptions staff have of their leadership’s actions and behaviors in response to the ACCII, and (3) refers to the way leaders represent themselves and their responses to the ACCII in interviews. Teamwork is a contextual factor that is embedded within the PARIHS constructs of culture and leadership. In this study, teamwork has two dimensions: (1) the ethnographically observed behaviors and actions that indicate whether a cooperative, coordinated effort by the ACC team is present and the extent of its presence in regard to the ACCII, and (2) the perception ACC staff have of the working relationships among their fellow ACC staff and the general staff attitude toward the ACCII. Communication is a contextual factor that is embedded in the PARIHS contextual constructs of Receptive Context, Leadership and Evaluation and Feedback. For this project, communication is understood as the imparting of information and meaning through speech, technology, body language, and signs. Communication also has several dimensions, referring to (1) communication from leadership to ACC team about the ACCII, and (2) the quality and strength of intra-ACC team communication about ACCII and about ACC care.Recognizing that contextual elements are multidimensional, we characterize each element here. interaction to be a relationship in which two or more contextual elements may affect one another, as opposed to referring to an interaction among people. These effects are not unidirectional but rather multi-directional. Interplay can be understood as reciprocal action, interaction and the mutual influencing that occurs among contextual elements. The present article is an attempt to capture and understand these additional dimensions of how contextual features may work to promote SI.However, characterizing contextual elements only in terms of their relative strength (conducive or not conducive to change) relative to SI, while useful, is not sufficient. In fact, contextual elements can have mixed effects, or their effects can vary over time. In this study we do rank the strength of contextual elements  was introduced in a Veterans Integrated Service Network (VISN), based in the northeastern United States. The ACCII’s focus is all pharmacy-run anticoagulation clinics and all anticoagulation clinical staff in the VISN, encompassing eight medical centers and their affiliated outpatient clinics.The most commonly used anticoagulant in the United States is warfarin, also known by the brand name Coumadin . While wTo improve anticoagulation care, clinical pharmacists in the VISN have been asked to adopt a dosing algorithm, an EBP, which has been shown to improve anticoagulation control and reduce rates of patient complications ,17. SecoThe ACCII is delivered by two overlapping teams. The core facilitation team at the VISN level consists of a clinical pharmacist with training in facilitation, anticoagulation care and QI, an industrial engineer, and an executive level leader, who is a QI specialist. This team is delivering and facilitating the intervention to each medical center and all satellite ACCs. The research team is studying the entire process of implementation, aggregating data on the implementation, and feeding back information to the core team to assist in tailoring and facilitating uptake. After each site visit, the research team quickly processed interim data to identify any barriers to SI which the research team then fed back to the core implementation team for tailoring their facilitation efforts. The extended team involves both the core facilitation team and research team.The present study focused on five ACC sites. Our cross-site comparison combined a case-oriented approach and a variable-oriented approach  to betteData was collected in the latter half of 2012 and early part of 2013 (October 2012 – January 2013) and analyzed in 2013 (February – May 2013). All frontline anticoagulation pharmacy staff in the VISN was eligible to participate in the study of the ACCII by the research team. We specifically requested participation from clinical pharmacists, pharmacy technicians and medical clerks who worked a majority and/or a regular amount of time in the anticoagulation clinic. The purposive sample included pharmacy administrators, clinical pharmacists, nurses, as well as support staff . Fifty-oTwo trained qualitative observers shadowed clinic staff as they engaged in delivery of care including but not limited to, work processes, job tasks and roles, and social dynamics at each ACCII site for at least an entire day and often two days. Observations were collected in the form of field notes. Field notes are written records made during site visits or immediately thereafter. The Institutional Review Board (IRB) of the Bedford VA Medical Center approved the study.a priori coding scheme guided by PARIHS to identify information on evidence, context and facilitation was developed. We also used an iterative thematic analysis so that we could capture any interesting relationships, patterns, surprises, and inconsistencies among people and within and across sites  for data management, coding and analysis. An ss sites . Two resiability . ObservaMembers of the qualitative team met regularly to review coded texts and resolve discrepancies through consensus as well as identify and resolve within site and cross-site variation in themes. The qualitative team also used the same process to resolve any discrepancies in our definitions and characterizations contextual elements at each site and our characterizations of the strength of contextual elements in regard to the ACCII Table  and our The degree of evidence-based practice (EBP) uptake varied across sites and we found interaction among contextual elements was a major factor influencing uptake. In this section, we provide site summaries which describe the state of contextual elements in site-specific case study tables Tables  and 10. Our findings demonstrate how interaction among contextual elements, like teamwork, communication and leadership, led to what seemed to us to be relatively consistent patterns with regard to the impact on uptake. In the context of our implementation effort, we ranked the contextual elements as strong, medium and weak Table  in termsWe observed three general patterns of interaction among contextual elements and evidence. First, belief in the evidence, while necessary for uptake, was not sufficient. Second, we observed that while belief in evidence was important, a site needed at least one other contextual element to be strongly favorable, be it leadership, teamwork, or communication, for uptake to occur. In contrast, if a site had unfavorable context in all three of areas, successful uptake was unlikely to occur. Third, we observed that when belief in the evidence was limited, even noteworthy strengths in other areas only served to reinforce the determination to resist any change to embrace the EBP. Details about these interaction patterns are found below.“We have very strong communication…We try to be very thorough in our notes so that the next person picking it up will know exactly where it was left off, what the story is…. We use (Microsoft) Communicator. We use the phone and we use the spreadsheet to share info…and I think that we’re really good about talking to each other about difficult cases and that kind of helps us to make the best clinical decisions that we can. …we work really well as a team…we talk a lot and we communicate about what’s going on”.One of our most striking findings was that belief in and comfort with the evidence, while important, was not by itself a determining factor in ACCII uptake. Sites A Table  and B T perceive“We are looking to optimize the process that we use… I think it’s a fabulous idea. I’m looking for ways that we can improve… so you are less likely to make mistakes or worry about making mistakes … what would be wonderful is if we could figure out who’s doing a better job with that kind of thing and how can we implement it here”.Site A already had a culture of improvement, as seen in the following quote:Having a new leader who needed time to adjust and learn on the job was not a barrier to change for Site A, because of their strong teamwork and communication.“…It’s really individual specific…For some folks it’s…really good. Others, there’s a chronic lack of communication and lack of…intuition or empathy, you know, as far as what other people’s schedules are and… [this] impacts other people’s function”.Site B had generally adequate teamwork and communication, which was not without its weaknesses however. One staff member remarked of team work and communication at Site B,“[The middle manager] is so organized… [The manger] is wonderful”.It was through the enthusiastic leadership of a new middle manager that Site B had high levels of uptake in the ACCII. One staff person remarked,“[MM] is just so helpful and…she really understands the issues that we face and, I mean, she is obviously on board with anticoag project…”Another stated:Teamwork and communication was mediocre at Site B, but in this case it was strong leadership, and a team willing to be led, that changed the contextual interplay dynamics and enabled the site to become a site with high uptake. In this case, the interaction among contextual elements that varied in strength nevertheless led to high uptake. Here again, we see that at least a moderate belief in the evidence, combined with at least one strong contextual factor  were sufficient to enable successful implementation to occur.“…I’m kind of willing to give it a shot. But …I think we might have some other pharmacists here that might want to use the clinical judgment card pretty frequently on that one”.At Site C Table , many el“…We’re such a good team, we really work together to help everybody and I think when you respect the person that you’re working with and that feeling is mutual I think it makes the clinic run a lot smoother. You know, I think patients are satisfied and… We really do work together as a team”.They were referring to their clinical leader. Teamwork and communication were strong at this Site C. One pharmacist stated:“… if I have a question…Like, procedural stuff a lot of times I’ll ask…. If I have a quick question about…dosing or something like that, a lot of times I’ll ask [the middle manager]”.Staff made it clear that they felt their leader was very much a member of the ACC team. A newer staff member notes that intra-team communication was great and that communication with their leader was excellent as well. In fact this staff member noted,Staff felt comfort with and respect for their middle manager. However, this site remained at a mid-level of uptake initially in part because of this leader’s lukewarm response to the EBP, resulting in only partial fidelity to the ACCII. Site C illustrates the pattern where if belief in the evidence is lukewarm, even strengths in other areas cannot entirely compensate enough to improve uptake.At other sites, there was an open distrust of EBPs; we observed that uptake was limited in proportion to the extent of the distrust. At Site D Table , distrus“…to me you can’t categorize every patient into that algorithm. They don’t all fit and a lot of times it’s a gut feeling that you need to change a dose”. A participant at Site D referred to anticoagulation care as an “art” more than a “science,” indicating a discomfort with the EBP. This pharmacist explains,“… [We] know that we can depend on each other and anyone’ll back you up and help you out”. Another noted“We all [have]… a similar philosophy, a similar training and a similar mindset, so I think our team play [emphasis in interview] works well…”In the context of such dismissal of evidence, the strong teamwork and communication present at Site D only served to reinforce resistance to the effort to implement ACCII. In regard to teamwork, one team member stated,“the most resistant to change”. In discussing why there was resistance to change, there was a celebration of this team’s closeness. For example, in the past, there had been an effort to standardize one aspect of ACC practice at this site. This local QI effort met with no success at Site D as seen in almost identical quotes by two Site D team members,“Well we didn’t want to use that [the proposed QI tool]…and I don’t remember actually my own feelings about it but I remember that you know as a team we kind of shut it down…we decided to just stay with our way….”And another staff member noted that they anticipated that their site would be, “We didn’t want to use it [the proposed QI tool]…as a team we… said no”.Another staff member said,“I don’t keep my ear that close to the ground so, I just sit back and wait for official things to come…Just [heard from leadership] that it’s… [ACCII] coming…It’s [ACCI] in the works…”Communication and teamwork worked together to prevent uptake of a local QI project. Leadership engagement and communication about the fact that the ACCII was being implemented had not even reached all staff when the research team began interviewing. A staff person said,Since leadership had a “hands-off” approach to the running of the ACC at Site D, staff felt that participation in the ACCII was not required and therefore they either did not participate or participated reluctantly and infrequently. At Site D Table , the res3“…I have seen a change in people’s attitudes towards…using the algorithm. I like it. [It] takes a little bit more time when we’re using [the algorithm] because we’re kind of dosing a little bit differently than we had in the past…and you kinda have to think things through a little bit more which isn’t a bad thing”.At Site E Table , we foun“It [teamwork] is awful …There are certain people that will not be in a team, no matter how hard you try…I would love to have a team atmosphere and be able to count on everybody and feel like there’s trust and that we’re providing the best care, but we do not have that here”.However, other contextual factors were notably weak. Teamwork at Site E is described by a staff member as,“…the other thing is communication…we don’t have staff meetings. We…just get emails…but nothing is really discussed among the group”.The feeling was that poor intra-team relations and communication impacts work performance. Furthermore communication between leadership and the ACC staff also impacted staff attitude toward their teamwork, their work and their attitude toward change. A Site E pharmacist reported,;“I think the hardest part of our job is communication…. the volume of our workload is very high so that’s challenging. We have so many patients that walk right in our door. …I think another challenge is the lack of communication among the pharmacists. With the clinical staff [some] have meetings with our [leadership] …they knew about all these…changes, however it doesn’t get trickled down to the rest of the clinic… we’re not [the entire staff] informed as to what’s happening”.This was reinforced by another staff member“…looking at what can we do better. But anyone above him, we don’t see, we don’t hear from”. The following quote illustrated general staff attitude in this area:“…People here want to always make an impression, okay? Especially the people in management… They come around when there’s the photo op, I always like to say… On a daily basis, forget it, okay? That’s the way it is, you know”.As to leadership, the picture is generally one of frustration. Some staff note that that the middle manager is, Here, we see that even a site with a positive view of the evidence cannot succeed when context was very weak.Evidence alone was not sole determining factor in uptake.A site needed at least one and possibly two contextual elements to be strong  for uptake to occur. If a site was relatively weak in all three of these, uptake would not occur, despite strong belief in the evidence.If belief in the evidence was limited, strengths in other areas often did not work in favor of uptake, but rather served to reinforce the status quo (i.e. resistance to change).Theoretical frameworks in implementation science and related fields generally state that several contextual elements contribute to successful implementation, defined as full and high-fidelity uptake of an EBP. While interactions among these contextual elements have been tacitly assumed to occur, they have rarely been described in detail. Our study examined five sites as examples of how contextual elements may interact with each other and with perceptions of the evidence to promote or inhibit adoption of an evidence-based practice. We observed that no single shortcoming served as a “fatal flaw,” but that three general interaction patterns emerged from our data that mapped with successful or unsuccessful uptake of the ACCII.With the recent emphasis on theory-driven implementation studies, theory has seemed to diverge regarding whether to focus on the key role of individuals in uptake of interventions, or whether to shift attention to the roles of teamwork and social networks . In partIf there is a contextual element that is particularly strong, this strength may compensate for weaker areas, as was seen with Site A Table . Site A Many conceptual frameworks present taxonomies of contextual elements which can be used prospectively to guide a theory-driven implementation, to tailor interventions to address perceived needs on an ongoing basis, and to analyze implementation retrospectively. In our study, PARIHS was used prospectively as part of the overall study design which mapped out how the research team would study the implementation as it rolled out, developed and spread. Specifically, the research team drew on PARIHS to construct the interview guide and to develop the code book for the interviews and ethnographic observations. However, in the course of collecting and analyzing our data we realized that we were encountering a limitation in our conceptual model. PARIHS did not provide us a way to describe and the interplay among contextual elements, nor did it represent how contextual interrelationships influenced initial rates of uptake. In this study, we learned that interactions among contextual elements can produce a synergy which magnifies contextual effects and moves them in certain patterns, some of which we were able to characterize here.We operationalized the patterns we observed in the qualitative data by feeding the information back to the ACCII facilitation team and working with them to tailor the intervention to be site specific. Some of this tailoring took the form of doing some targeted capacity building and development of contextual elements at sites to enhance uptake. For example, there were acts of facilitation such as teamwork development, encouraging and facilitating better communication within and across sites about the ACCII, and reaching out to leadership to enlist support for the intervention that came directly out of the research team’s rapid site assessment. Additionally there were increased efforts in algorithm presentations, journal clubs and other venues to educate ACC staff about the scientific basis of the algorithm and the importance of its use, especially where belief in the evidence seemed to be the weakest link.As a concrete example of the value added by our approach to analyzing not only contextual elements, but also their interaction, the research team observed that teamwork was likely to be the “weak link” at Sites D and E. The team was “weak” at Site D – with regard to our intervention – because they worked so well together and mutually reinforced each other’s views of the limited value placed ACCII. In this case, team cohesion created resistance that negatively impacted implementation of the intervention. The team at site E was weak because they did not know how to work toward a common goal, they did not support one another and they did not have established means of communicating about common goals because they did not hold regular meetings. Our assessment of teamwork as the key factor driving successful implementation at these sites was in part predicated on our understanding that “unlocking” teamwork would also enable other aspects of context to improve. Informed by this observation, both the research team and the facilitation-implementation team concentrated on team-building activities at Site E, and on trying to convince team members at Site D regarding the evidence for the algorithm, as well as reaching out to the Site D leadership to educate them. Based on our interim findings, our efforts seem to be making a difference at both Sites D and E, and indeed focusing on teamwork first at both sites  did enable other aspects of context to improve subsequently. This serves as a concrete example of how our interactional approach to data analysis also helped us tailor the intervention more effectively.Quality improvement (QI) does not rest on the intervention itself but often on the context in which the intervention takes place. For example, one recent study examined how contextual elements interacted in the setting of a single-site intervention to reduce surgery cancellations among 20 staff . In thatOur study’s cross-site analysis suggests that the relationship between evidence, contextual factors and uptake is a very mixed picture but that there are common patterns among interactional data that influence uptake Tables  and 3. FThis study has some limitations. Most importantly, interactional data can be challenging to identify and discuss as they are based on social actions and actors who are always changing. We added ethnographic observations, which allowed for a more detailed understanding of interactional data, to ameliorate this limitation.Interrelationships among different contextual elements can act as barriers to uptake at some sites and as facilitators at others. Accounting for interconnections among elements enables PARIHS to more fully describe the determinants of successful implementation as they operate in real-world settings. Our study is an important start to better understand these interactions, which have been proposed and assumed, but rarely described in detail. This approach to understanding the interaction of contextual elements also has the potential to help inform a more effective approach to tailoring the intervention to promote successful implementation.


PGK1 promoter (PPGK1) from the methylotrophic yeast Pichia pastoris. The L1 gene was cloned under regulation of PPGK1 into pPGKΔ3 expression vector to achieve intracellular expression. In parallel, secretion of the L1 protein was obtained through the use of an alternative vector called pPGKΔ3α, in which a codon optimized α-factor signal sequence was inserted. We devised a work-flow based on the detection of the L1 protein by dot blot, colony blot, and western blot to classify the positive clones. Finally, intracellular HPV VLPs assembly was demonstrated for the first time in yeast cells. This study opens up perspectives for the establishment of an innovative platform for the production of HPV VLPs or other viral antigens for vaccination purposes, based on constitutive expression in P. pastoris.The human papillomavirus (HPV) L1 major capsid protein, which forms the basis of the currently available vaccines against cervical cancer, self-assembles into virus-like particles (VLPs) when expressed heterologously. We report the development of a biotechnology platform for HPV16 L1 protein expression based on the constitutive  HPVs are a large family of dsDNA viruses that cause benign warts and malignant tumors. Persistent infection by HPV imposes a huge burden on health services worldwide owing to its links with cancer of the vagina, vulva, penis, anus, tongue, and, in particular, uterine cervix, which is the most serious outcome . About 7 Saccharomyces cerevisiae and baculovirus-infected cells, respectively. Although these vaccines are highly effective and safe  was routinely used as a host for cloning and plasmid manipulations. This strain was cultured at 37°C in LB medium  supplied with appropriate antibiotics. P. pastoris X-33 strain (wild-type) used in this study was purchased from Invitrogen. The yeast cells were grown at 30°C on YPD medium  and YPDS  supplemented with 100 μg/mL zeocin (Invitrogen) when necessary.TheAll the molecular cloning techniques were carried out as previously described . Restric P. pastoris. Restriction sites for XhoI and NotI were added to the flanking regions of the L1 gene so that the cloning could be directed into the expression vector. The gene was synthesized by Epoch Biosciences , and cloned into pBSK plasmid. The resulting construct called pBSK/L1 was amplified in E. coli DH5α. L1 gene was released from pBSK after double-digestion with XhoI and NotI and employed for creating the expression vectors.A DNA sequence encoding the HPV16 L1 protein (Gen Bank access number GI: 27752860) was designed with codons optimized for expression inGK1 promoter from P. pastoris for constitutive expression of the L1 protein. The original 2 kb PPGK1 sequence described by de Almeida et al. [PGK1 sequence was used to generate the pPGKΔ3 expression vector for intracellular expression. Additionally, a pPGKΔ3α expression vector carries a codon-optimized S. cerevisiaeα-factor signal sequence (α-MF) cloned downstream to PPGK1 to drive recombinant protein secretion. Both PPGK1-based vectors contain the zeocin resistance gene Sh ble for positive selection of E. coli and P. pastoris recombinants, as well as a C-terminal polyhistidine (6xHis) tag for detection of the fusion protein by immunoblot assays. The L1 gene previously digested with XhoI and NotI was cloned into pPGKΔ3α and pPGKΔ3 expression vectors digested with the same enzymes, and the resulting vectors were called pPGKΔ3α/L1 and pPGKΔ3/L1, respectively. The construction of the vectors was confirmed by restriction digestion, PCR and DNA sequencing with specific primers for both L1  and PPGK1 (5′ TCATAGTTCATCCCTCTCTCC 3′) sequences.Two expression vectors were constructed with the Pa et al.  was redua et al. . The res PGK1 locus of P. pastoris was achieved after linearization of the 5 μg PPGK1-based expression vectors with SacI [ P. pastoris electrocompetent cells were prepared and transformed, as described elsewhere [ P. pastoris/pPGKΔ3α/L1 or P. pastoris/pPGKΔ3/L1, depending on the vector that was used.Stable integration into theith SacI . P. pastlsewhere . In thisμg/mL zeocin, the transformants were subjected to a general procedure for the selection of clones containing multiple copies of the expression cassette, called Posttransformational Vector Amplification (PTVA) process [μg/mL).After selection on agar plates containing YPD supplemented with 100  process . For thiμg/mL zeocin were subjected to a general screening for selection of recombinant strains expressing high levels of HPV L1 protein. The screening was carried out as follows: (i) P. pastoris/pPGKΔ3α/L1 clones were cultivated in agar plates and subsequently subjected to colony blot for detection of HPV L1 protein with the CamVir anti-HPV16 L1 monoclonal antibody ; (ii) In contrast, P. pastoris/pPGKΔ3/L1 clones were subsequently grown in deep-well plates to detect HPV L1 protein by dot blot with the anti-L1 monoclonal antibody.Multicopy clones resistant to 1000  P. pastoris/pPGKΔ3α/L1 multicopy clones in YPD agar plates for 3 days at 30°C, the PVDF membranes were cut as discs and left standing with the surface colonies for 3 hours at 28°C. Colonies on the master plate could be replicated by placing the PVDF discs on a fresh agar plate when desired. The PVDF discs were washed with tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST) to remove adhering cells. The membranes were blocked by incubation in TBST, supplemented with 5% nonfat milk for 1 hour at room temperature, and then incubated overnight at 4°C with anti-L1 antibody properly diluted (1 : 1000) in the blocking solution. PVDF discs were washed three times with TBST for 10 min and then incubated for 1 hour at room temperature with peroxidase-conjugated goat anti-mouse immunoglobulin  diluted 1 : 3000 in the blocking solution. Chemiluminescence reaction was detected with an ECL kit .In the case of colony blot, the protocol described by Goodnough et al.  was modi P. pastoris/pPGKΔ3/L1 multicopy clones were first grown in a deep-well plate containing YPD medium for 3 days at 30°C. The plate was shaken at 300 rpm and P. pastoris cells were harvested by centrifugation at 3000 rpm for recovery of the pellets. Preparation of yeast extracts was performed with an “alkaline lysis” procedure as previously described [β-mercaptoethanol) and heated to 90°C for 10 min and the lysate was brought to neutral pH. To improve solubilization, the lysate was heated again for 10 min and mixed with loading buffer . The PVDF membranes were cut in a proper way and inserted into a dot blotter apparatus. Protein transfer to the membranes was carried out with 100 μL of the lysate for 1 hour and, thereafter, the immunoblot proceeded as already described for the colony blot. The HPV16 L1 protein that was episomally expressed in P. pastoris [For the dot blot assay,escribed . Brieflypastoris  was usedP. pastoris multicopy clones were selected for baffled-flask cultivation in accordance with the highest detected level for the HPV L1 protein. P. pastoris/pPGKΔ3α/L1 clones were first preinoculated in 5 mL of YPD to achieve the secretion of the L1 protein and grown at 28–30°C in a shaking incubator (250–300 rpm) until the cultures reached an OD600 of ~10 (24 hours). Following this, the cells were inoculated in 20 mL of YPD for 3 days and aliquots were taken for analysis at periodic intervals. These samples were centrifuged at 12000 g for 2 min and the supernatants were stored for further analysis. For the intracellular expression of the L1 protein, P. pastoris/pPGKΔ3/L1 clones were subjected to the same protocol but the cell pellets were stored instead of the supernatants.β-mercaptoethanol, 0.1% bromophenol blue) was then added to the washed pellet (corresponding to 100x concentration) and this mix was resolved on 15% SDS PAGE after the samples had been heated for 10 min at 75°C. Polyacrylamide gel was either stained with Coomassie brilliant blue  or transferred to PVDF membranes using a V20-SDB semidry protein transfer apparatus . Immunoblot was performed as described in Aliquots of supernatants were submitted to precipitation with trichloroacetic acid (TCA) (Sigma-Aldrich) to a final concentration of 10% to allow the secretory production of the HPV L1 protein to be analyzed. After concentration, the supernatant was discarded, and the protein pellet was resuspended again in 100% acetone to remove residual TCA. A final volume of SDS-loading buffer , according to Cregg et al. . This crAt the end of the induction course, both the cells and culture supernatants were subjected to absorption into carbon-coated grids, as recommended by Falcón et al. . The gri P. pastoris PGK1 promoter which was originally described and employed for secretion of α-amylase from Bacillus subtilis [ PGK1 promoter sequence containing ~400 bp was used to allow integration of the expression vector via homologous recombination, as well as a codon-optimized α-factor from S. cerevisiae to drive the secretion of recombinant proteins  together with the detection of the L1 protein expressed episomally in P. pastoris cytosol ensured the specificity of the reaction.Compared withanalyzed . Multicoanalyzed . Hence,  Figures . The lac P. pastoris secretes few proteins into the medium [It is worth noting that this practical and rapid workflow provides a strategy to screen well-expressing clones through cultivation under nonoptimal conditions—with regard to culture volume, shaking, and aeration, as is the case for deep-well and agar plate cultures—among a considerable high number of colonies. The colony blot assay has an important feature, particularly in the case of PGK-based clones that secrete L1 protein, which is the dispensable use of liquid media. Sincee medium , 30, 43, P. pastoris was first reported by Bazan et al. [ S. cerevisiae. Despite being innovative and important means of demonstrating the feasibility of Pichia in producing HPV VLPs, the work carried out by Bazan and colleagues, employed a nonintegrative system. As the authors (and other studies) point out, the use of episomal plasmids is not recommended for industrial purposes, since they have been associated with the generation of recombinant strains that are genetically unstable and have two undesirable outcomes: (i) instability in the heterologous expression levels; (ii) continuous antibiotic selection which is needed to maintain the expression plasmids [ P. pastoris by employing AOX1-based integrative vectors [Expression of the major capsid protein of HPV16 inn et al. . Accordiplasmids , 26, 44. vectors , 23, 24. P. pastoris strains present three distinct phenotypes: (i) the wild-type phenotype designated as methanol utilization plus (Mut+) and characterized by the presence of a functional copy of the alcohol oxidase 1 gene (AOX1) which is responsible for 85% of the utilization of methanol by the alcohol oxidase enzyme; (ii) methanol utilization slow (Muts) phenotype, characterized by the absence of the AOX1 gene and presence of the AOX2 gene, which is about 97% homologous to AOX1 but much less expressed; (iii) methanol utilization minus (Mut−) phenotype, where both AOX genes are absent. Hence, while Muts strains show a poor growth on methanol medium, the Mut+ strains have a greater growth rate and Mut− strains, in contrast, are unable to grow on methanol as the sole carbon source. Interestingly, Cregg et al. [ P. pastoris Mut+strains expressed 10-fold less HBsAg than Muts strains, which suggests that consumption of methanol at high levels, coupled with a rapid growth rate, may not lead to efficient HBsAg assembly [AOX1 induction are nonoptimal for the production of HBV and HPV VLPs. Finally, even though recombinant protein expression in Pichia Muts strain could circumvent the deficiencies observed for the generation of VLPs in Mut+ strains, the slower methanol utilization phenotype requires long fermentation times to reach peak product concentrations. Once induced, the entire culture cannot be used to start a new culture [With regard to the metabolization of methanol,g et al.  showed tassembly . In addiassembly , althoug culture .AOX1-based system, we constitutively expressed the L1 protein through PPGK1 in the presence of glucose and by employing an easier cultivation/expression schedule in shake flasks, since L1 expression occurred together with the cell growth and the laborious control of the methanol/inducer levels was dispensable  status by the FDA (Food and Drug Administration) and has been recognized as an efficient vehicle for the delivery of viral antigens when administered by an oral and intramuscular route [The assembly of HBV and HCV core proteins into VLPs has been previously reported in yeast , 51. Howar route . Furtherar route . P. pastoris recombinant strains described here provides evidence of electrodense structures with estimated diameters of 55 nm, as expected for the HPV VLPs (Figures  P. pastoris strains that had been transformed by the parental vectors ( P. pastoris through immunoelectron microscopy were hindered by the retention of cell membrane components in the architecture of the HCV core particles which has an envelope-like structure [Electron microscopy analysis of Figures  and in a Figures  and insi Figures  (either  vectors . We alsotructure . Accordi P. pastoris recombinant strains. It has been shown that assembly of HPV VLPs can be achieved with neutral pH, high ionic strength, and relatively low concentrations of reducing agents [ P. pastoris growth progresses in unbuffered medium, the pH drops to 3 or below [We attempted to characterize assembled HPV VLPs in the culture media fromg agents . Conversor below . It can or below , 48–51.  P. pastoris PGK1 promoter for the production of HPV16 VLPs. Constitutive expression of the HPV16 L1 capsid protein was efficiently achieved for the first time through an easier production schedule than is the case when an AOX1 inducible promoter is employed. In addition, an optimized α-MF secretion signal downstream to PPGK1 provided a prompt secretion of L1 protein in the culture media. Although further experiments are needed to determine which production strategy is more effective for HPV L1 protein production, the data outlined here underline the efficiency of a PGK1-based platform as a heterologous expression system and support its employment for the production of HPV VLPs for vaccination purposes. Moreover, our preliminary data showing intracellular VLP assembly open up perspectives for the employment of P. pastoris cells that can express HPV L1 protein as a live attenuated vaccine. To the best of our knowledge, this is the first time that the expression of HPV L1 protein was achieved in P. pastoris through an IP-free biotechnology platform. The discovery of alternatives to biotechnology platforms for producing an efficient and cost-effective vaccine has the potential both to offer greater protection—since not all the HPV genotypes are covered by the current vaccines—and to allow the vaccine to be widely disseminated.The data presented in this work demonstrate the functionality of a biotechnology platform based on


Background: Although poorer cognitive performance has been found to be associated with anxiety, it remains unclear whether neurocognitive function affects biased cognitive processing toward emotional information. We investigated whether general cognitive function evaluated with a standard neuropsychological test predicts biased cognition, focusing on attentional bias toward threat.Methods: One hundred and five healthy young adults completed a dot-probe task measuring attentional bias and the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) measuring general cognitive function, which consists of five domains: immediate memory, visuospatial/constructional, language, attention, and delayed memory. Stepwise multiple regression analysis was performed to examine the relationship between attentional bias and cognitive function.Results: The attentional domain was the best predictor of attentional bias toward threat . Within the attentional domain, digit symbol coding was negatively correlated with attentional bias .Conclusions: The present study provides the first evidence that general attentional ability, which was assessed with a standard neuropsychological test, affects attentional bias toward threatening information. Individual cognitive profiles might be important for the measurement and modification of cognitive biases. Cognitive theories suggest that cognitive processing biased toward affective significance confers an increased risk for the development and exacerbation of emotional disorders Beck, . Such biBiased attention toward threatening information is one of the most widely studied cognitive biases. Attentional bias refers to a tendency to quickly identify and easily dwell on emotional stimuli , chronic subdural hematoma (n = 1), cerebral palsy (n = 1), Wilson disease (n = 1), histories of subarachnoid hemorrhage (n = 1) and hydrocephalus (n = 1), and strabismus (n = 1). Thus, data from 105 participants were included in the analyses .Participants were 113 individuals recruited via advertisements in a local magazine and billboards at Kitasato University. The eligibility criteria were as follows: no Axis-I psychiatric disorders or substance abuse history, which were determined using the Mini-International Neuropsychiatric Interview . BDI-II is a 21-item, self-report questionnaire to assess depressive symptoms experienced during the past 2 weeks  was employed to assess multiple domains of cognitive function. The RBANS is a representative, clinician-administered neuropsychological test for adults aged between 20 and 89 years  Parts A and B Reitan, , 1955 weTo measure attentional bias, we used the dot-probe task (DPT), the most commonly used and innovative program for attentional bias modification . The significance threshold was set at 0.05 (two-tailed).r(99) = −0.27, p = 0.006; r(99) = 0.20, p = 0.049, respectively], indicating that lower performance in attention-related functions was associated with biased attention toward threatening information. Although attentional bias was not significantly correlated with trait anxiety in the current linear model, its relationship with depressive symptoms bordered on statistical significance (p = 0.050).Mean scores and standard deviations (SDs) of the RBANS, TMT, depressive symptoms, trait anxiety, and attentional bias are presented in Table F = 6.06, R2 = 0.20, adjusted R2 = 0.16, p < 0.001; Table p = 0.006).Next, stepwise multiple regression analysis revealed that the attentional domain of the RBANS was the best predictor of attentional bias toward threat , was negatively correlated with attentional bias toward threat .As a reference analysis to specify which component of the attentional domain in the RBANS was more relevant to attentional bias, partial correlation analysis showed that the digit symbol coding of the attentional domain, not digit span [p = 0.041) and delayed memory scores , compared to men. This is partly consistent with a previous study that found a similar gender difference in the RBANS .Additionally, we performed a confirmatory analysis to examine whether there are gender differences in the RBANS. The ANCOVA, in which age and years of education were controlled for, showed that women had higher immediate memory scores  .Some limitations should be noted when interpreting the results. First, we used the RBANS to measure neurocognitive function. More detailed assessment tools for assessing neurocognitive function are necessary. Second, the semantic fluency scores in the present study were lower than the scores in the original study (Randolph, In summary, we revealed that general attentional ability, assessed with a standard neuropsychological test, affects attentional bias toward threat. Compromised neurocognitive function in a specific domain might affect biased cognition toward an emotional stimulus therein. Consideration of individual differences in neurocognitive function might be important for the measurement and modification of cognitive bias.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


With transmission-type graphene SA, the EDFL shortens its pulsewidth from 483 to 441 fs and broadens its spectral linewidth from 4.2 to 6.1 nm with enlarging the pumping current from 200 to 900 mA. In contrast, the reflection-type SA only compresses the pulsewidth from 875 to 796 fs with corresponding spectral linewidth broadened from 2.2 to 3.3 nm. The reflection-type graphene mode-locker increases twice of its equivalent layer number to cause more insertion loss than the transmission-type one. Nevertheless, the reflection-type based saturable absorber system can generate stabilized soliton-like pulse easier than that of transmission-type system, because the nonlinearity induced self-amplitude modulation depth is simultaneously enlarged when passing through the graphene twice under the retro-reflector design.The  Thus, graphene could overcome the non-uniform distribution and self-aggregation problem happened on carbon nanotube. In addition, graphene exhibits lower threshold intensity for saturate absorption than that of carbon nanotubes to take over other saturable absorbers for the passively mode-locked EDFL89To get rid of the complicated synthesizing and transferring process, the hydrogen-free and low-temperature plasma enhanced chemical vapor deposition (PECVD) of graphene has emerged3 and the lifted graphene was transferred to a smooth Si wafer afterwards. The atomic-force microscopy (AFM) top-view image and cross-sectional profile of few-layer graphene on Si wafer shown in in-situ dissolution-and-reduction after the hydrogen-free and low-temperature PECVD growth is roughly estimated as 6 ~ 7 layers. To characterize the saturable absorption feature of the few-layer graphene, the nonlinear transmittance obtained under the pumping with high-peak-power fiber laser  is shown in T of 3.5%. The absorption saturates at a pumping power of more than 3.23 mW due to the Pauli blocking effect, where the photons could pass through the optically bleached graphene. The corresponding modulation depth of the few-layer graphene is about 28%, which is already comparable with those of about 30% as obtained from the seven-layer graphene synthesized under high-temperature and hydrogen-rich environmentIn order to measure the thickness and calculate the layer number of the few-layer graphene, the ultra-thin nickel catalyst film was etched by FeCl2/Si substrate is applied as the catalyst for the dissolution-and-reduction synthesis procedure. Several advantages of nickel which favor the in-situ graphene synthesis are addressed below. First, the carbon atoms can still dissolve into the ultra-thin nickel film at relatively low temperature (<500 °C), which facilitates the reduction of few-layer graphene from cooling the nickel matrix after carbon deposition−1 is induced by the structural defects in graphene, the G peak at 1580 cm−1 certifies the C-C sp2 network, and the unique 2D peak at 2760 cm−1 denotes the existence of single-layer graphene3 carbon bondsD/IG intensity ratio value of about 0.3. The better quality of graphene could be obtained by post annealing on the nickel substrate at 900 °C2D/IG intensity ratio of the graphene after dissolution-and-reduction from ultra-thin nickel film grown by low-temperature hydrogen-free PECVD is about 0.45, indicating that the graphene is not mono-layer (with I2D/IG > 1) and bi-layer (with I2D/IG = 1). For tri- or more-layer graphene synthesized on ultra-thin nickel catalyst film with hydrogen-free and low-temperature PECVD, the layer number should be further defined by measuring its transmittanceTo facilitate the hydrogen-free low-temperature PECVD growth of few-layer graphene, the ultra-thin nickel coated SiOAs pumping current ranged from 400 to 600 mA, the measured TBP is deviated from 0.315 to indicate the incomplete soliton mode-locking phenomenon. Almost the same temporal and spectral shapes are obtained with nearly transform-limited TBP at pumping current of larger than 700 mA.In more detail, the autocorrelation traces obtained at different pumping currents beyond the threshold of 100 mA are shown in In comparison, three major differences between the reflection- and transmission-type graphene based passively mode-locked EDFL systems are addressed, the cavity length (due to the aid of circulator), the doubled linear loss and the twice enlarged saturable absorbance (due to retro-reflection in graphene). As the light passes through the graphene layer and reflects by the gold coated end-face, the equivalent layer number increases twice such that the reflection-type graphene based mode-locker inevitably causes more insertion loss than the transmission-type one. Nevertheless, the reflection-type based saturable absorber system was believed to generate stabilized soliton-like pulse easier than that of transmission-type system. That is because the nonlinearity induced self-amplitude modulation depth is simultaneously enlarged passing through the graphene twice under the retro-reflector design. Unfortunately, the mode-locking performance would be slightly degraded with the circulator induced insertion loss, which needs a detailed analysis for comparing both reflection- and transmission-type graphene mode-locked EDFLs.To perform the retro-reflection type mode-locker, the From the monitored autocorrelation traces shown in et al. have demonstrated a stable mode-locked EDFL with pulsewidth of 415 fs and pulse energy of 7.3 nJ by using a CVD-grown atomic layer grapheneet al. have also utilized the CVD-grown multilayer graphene to produce a stable mode-locked fiber laser with 432-fs pulsewidthGraphene with high crystalline quality can serve as an effective saturable absorber for ultrafast mode-locked lasers due to its excellent optical properties, including ultrafast carrier response time, low saturation intensity, high nonlinear modulation depth and less scattering loss81027209253234363739in-situ dissolution-and-reduction CVD synthesized few-layer graphene.With our approach, the desorbed carbon atoms from the Ni film can self-assemble the few-layer graphene with highly layer uniformity. The PECVD synthesis can lower down the growth temperature to a critical value of 550 °C such that the dissolution rate of the decomposed carbon atoms in the Ni film can be minimized. By precisely controlling the growth temperature near the phase transition temperature of Ni, the minimal layer number of graphene can be obtained due to the smallest quantity of carbon atoms desorbed from Ni film. Without hydrogen passivation, the few-layer graphene with suppressed defects can still be obtained under low-temperature growth. Although there is a small amount of defects existed in few-layer graphene to degrade its mode-locking force, the sub-picosecond ultrafast passively mode-locked EDFL pulse can still be generated with the saturable absorption in the in-situ dissolution-and-reduction synthesis is roughly estimated as 6 ~ 7 by AFM. The few-layer graphene obtained under low-temperature and non-hydrogen environment demonstrates a nonlinear transmittance increased from 87.5% to 91% with a ΔT of 3.5% and a corresponding modulation depth of 28%. At maximal pumping power, the transmission-type graphene mode-locked EDFL shows a central wavelength of 1572 ± 0.5 nm with a pulsewidth of 441 fs and a spectral linewidth of 6.1 nm. The EDFL with a cavity length of 7.43 m reveals the repetition frequency of 28.57 MHz. The soliton mode-locking is induced by observing the significant Kelly sidebands. In contrast, the reflection-type mode-locker can only generate the passively mode-locked EDFL with a broadened pulsewidth of 796 fs and a shrunk spectral linewidth of 3.3 nm. The layer number the reflection-type graphene based mode-locker is doubled such that the insertion loss becomes twice more than the transmission-type one. The repetition frequency concurrently decreases to 16.66 MHz as the cavity lengthens to 12.7 m. Nevertheless, the reflection-type based saturable absorber system can generate stabilized soliton-like pulse easier than that of transmission-type system, because the nonlinearity induced self-amplitude modulation depth is simultaneously enlarged when passing through the graphene twice under the retro-reflector design.For the first time, a low-temperature and hydrogen-free PECVD synthesized graphene is used as a mode locker in transmission- and reflection-types for passively mode-locked EDFL systems. A dissolution-and-reduction synthesis facilitates to dissolve carbon atoms into the ultra-thin nickel film at relatively low temperature (~550 °C). Subsequently, the formation of few-layer graphene is observed from cooling the nickel matrix after carbon deposition. The measured layer number of few-layer graphene synthesized by In experiment, the few-layer graphene at 550 °C is performed with the mixed methane and argon at gas flow rate of 3 and 200 SCCM, respectively. The deposition on 50 nm of Ni substrate by PECVD with the plasma power of 100 W sustains for 100 s. At initial stage, the carbon atoms are gradually dissolved into Ni film at substrate temperature beyond threshold, where the formation of hexagonal carbon ring structure cannot be initiated in the interstices of Ni film. By lowering the substrate temperature, numerous carbon atoms desorb from Ni matrix to form the hexagonal carbon ring structure, and the layer number of graphene on the Ni film is dominated by the amount of desorbed carbon atomsThe advantage of low-temperature growth is to rigidly control the quantity of carbon atoms desorbed from Ni film so as to precisely determine the layer number of self-assembled graphene. Without the need of high-temperature procedure, the precipitation of the dissolved carbon atoms in the Ni film can be reduced. Under the temperature as low as 550 °C , only a small amount of carbon atoms can be dissolved into the Ni film, so as to prevent the precipitation of abundant carbon atoms after cooling21In previous work, the layer number of graphene can be controlled by detuning either the deposition time of argon-diluted methane fluence or the thickness of the evaporated Ni film. For example, when decreasing the deposition time of methane from 600 to 100 s, the graphene layer number can be reduced from 8 to 3 layers3, where the Fe3+ ion can etch the Ni film to make graphene floating upon solution9423 solution is diluted by injecting deionized (DI) water. Eventually, the graphene is attached onto the end-face of a SMF patchcord that is put into the solution of DI water. The transfer procedure is illustrated in After hydrogen-free and low-temperature PECVD growth, the synthesized graphene on Ni film is immersed in an aqueous solution of FeClThese fiber pigtails with few-layer graphene were placed in the passively mode-locked EDFL system, as illustrated in In contrast, the second system with a reflection-type few-layer graphene saturable absorber in the passively mode-locked EDFL system is demonstrated as shown in To perform the passive mode-locking at appropriate pumping condition, the intracavity gain of the EDFL was determined under bi-directional pumping regime. The relationship of the output power and the intracavity gain versus input power is shown in How to cite this article: Peng, K.-J. et al. Dissolution-and-reduction CVD synthesis of few-layer graphene on ultra-thin nickel film lifted off for mode-locking fiber lasers. Sci. Rep.5, 13689; doi: 10.1038/srep13689 (2015).


Moreover, we show that the top hBN layer used in the transfer process acts as an effective top encapsulation resulting in improved stability to ambient exposure. The transfer method is applicable to other CVD-grown 2D materials on copper foils, thereby facilitating the preparation of van der Waals heterostructures with controlled doping.We report a CVD hexagonal boron nitride (hBN-) assisted transfer method that enables a polymer-impurity free transfer process and subsequent top encapsulation of large-area CVD-grown graphene. We demonstrate that the CVD hBN layer that is utilized in this transfer technique acts as a buffer layer between the graphene film and supporting polymer layer. We show that the resulting graphene layers possess lower doping concentration, and improved carrier mobilities compared to graphene films produced by conventional transfer methods onto untreated SiO Owing to its unique mechanical42O3101122 V−1 s−1. Recent attention has been devoted to graphene encapsulation using hexagonal boron nitride (hBN) layers, a lattice-matched insulating 2D material with atomically smooth surfaces that has enabled near phonon-limited room temperature carrier mobilities of 140,000 cm2 V−1 s−1 to be realised in exfoliated hBN/graphene/hBN heterostructures17The robustness of graphene during fabrication processes and the long-term stability of graphene devices are additional key issues that must be resolved in order to make graphene commercially applicable. Improved stability is attainable by encapsulating graphene devices with a protection layer. A number of approaches have been reported employing a variety of encapsulation materials including metal oxides Al2O31011, TiO213,2/Si substrate by a scooping technique (AT-hBN/Gr/SiO2 sample), but the final result can be further improved by dry transfer and full encapsulation with hBN.2 surface passivation techniques such as SAMs or hBN buffer layers significantly improve the electrical properties of CVD graphene2021222 structures by charged surface states and impurity scattering24252628292 sample is compared to three samples prepared using the WT technique differing only in the substrate treatment, including untreated SiO2 (WT-Gr/SiO2), SAM-modified SiO2 (WT-Gr/SAM) and hBN covered SiO2 (WT-Gr/hBN). For the SAM modification, an octadecyltrichlorosilane (ODTS) layer was utilized due to its higher efficiency of screening the charge impurities present on SiO2 layer20The novel transfer method is compared to the conventional, and improved wet transfer (WT) techniques. It has been demonstrated that SiO2, and a topological mismatch of PMMA/hBN and Gr/Cu interfaces is likely to be present because the PMMA film conforms to the morphology of the underlying copper substrate of the first hBN layerOptical microscopy images of the transferred graphene films confirm that regardless of the target substrate, the WT method results in continuous films with low density of defective structures such as tears and cracks . We noteThe crystalline quality of the graphene films was analysed by Raman spectroscopy, as shown in 343536373839404138393839−1 is 50 times weaker than for the G peak of graphene under same measurement conditions2 sample) does not introduce significant defects and results in a defect density even lower than that detected for the WT-Gr/hBN sample.Consistent with optical microscopy, the obtained micro-Raman maps show uniform coverage of the graphene films transferred using both conventional WT and the presented AT methods . Raman s2 where graphene is in contact with the hBN film, a pronounced upshift of the Pos(2D) is observed, indicating increased strain. Sample AT-hBN/Gr/SiO2 exhibits an elongated distribution, providing further evidence of increased strain variation, as expected from the AT technique. In contrast, the conventional WT-Gr/SiO2 sample exhibits correlations between Pos(G) and Pos(2D) that are consistent with doping variations in the sample. This is attributed to the fact that the graphene film is in direct contact with SiO2, which introduces doping variation due to the presence of charged surface states and impurities2425202 substrate. However, since the CVD hBN layer was transferred using the conventional WT method, polymer residues present on the surface introduce additional doping variations in the WT-Gr/hBN sample. Notably, the AT-hBN/Gr/SiO2 sample demonstrates lower doping variations even though the graphene film is in direct contact with SiO2, which is consistent with the absence of surface polymer residues. The correlations between the Pos(G) and I(2D)/I(G) are shown in the 2D histograms of 2 sample makes the doping analysis difficult.The 2D histograms for Pos(G) and Pos(2D) are shown in D), defined as the conductivity minimum, provides direct information about the magnitude of the extrinsic doping and facilitates comparison between the different transfer techniques. Although carrier mobility is another important parameter for the material quality characterization, device length scale is important. In the current study, we have chosen to make the devices using a shadow mask technique. The advantage of this technique is that it avoids exposing the graphene to additional polymer residues for example during a photoresist or ebeam resist step, allowing us to isolate extrinsic doping induced by the transfer method alone. Additionally, shadow masking prevents graphene removal and delamination during lift off processThe material quality was further analysed by examining the electrical properties of graphene FET devices in terms of the charge neutrality point and carrier mobility (see Methods section for more details on the fabrication process). The charge neutrality point, or the Dirac point . For the sample WT-Gr/SAM, the VD is located at lower gate voltages compared to WT-Gr/SiO2 implying reduced p-type doping, consistent with the passivation of the SiO2 surface by the SAM. In contrast, the hBN buffer layer (WT-Gr/hBN) appears to have a negative effect, displaying a VD upshift compared to the WT-Gr/SiO2 sample, indicating an increased p-type doping, consistent with the presence of polymer residues on the underlying hBN surface. The lowest doping is observed for the hBN-assisted transfer (AT-hBN/Gr/SiO2) which exhibits a VD ~ 7 V, significantly downshifted compared to WT-Gr/SiO2. Since the graphene film is still in direct contact with the SiO2 substrate, this result can be attributed directly to the absence of polymer residues on the graphene surface. From analysis of the VD we deduce that the absence of polymer residues results in a 2.7 × 1012 cm−2 reduction in the doping level , ε0 is the permittivity of free space and dg is the thickness of SiO2 (90 nm). This method provides results comparable to those reported previously using a similar calculation of the graphene mobility from two-point FET measurements2031482 V−1 s−1 is obtained for the Gr-SiO2 sample, which is consistent with literature values31μh/μe ~ 3.2), which can be attributed to the presence of charge trap states on the SiO2 substrate2. This is perhaps surprising, since the SAM is expected to passivate reactive sites such as silanol groups at the SiO2 surface20μh/μe ~ 2.2 indicating that the ODTS layer screens the charged impurities from the adsorbates on the SiO2 surface. In the case of the WT-Gr/hBN sample, while the hole mobility remains quite high, the hole/electron mobility imbalance is much stronger with μh/μe ~ 9 providing further evidence of the presence of polymer dopants on the hBN surface. Finally, we find that for the AT-hBN/Gr/SiO2 sample the electron and hole mobilities are increased several-fold (even taking into account reproducibility across devices) compared to the standard WT-Gr/SiO2 sample. Noticeably, the asymmetry of the electron and hole mobilities remains high (μh/μe ~ 3.7) due to direct contact of the graphene film with the SiO2 substrate.where 2, WT-Gr/SAM and WT-Gr/hBN  followed by a thorough rinse in DI water. The floating PMMA/graphene or PMMA/hBN stack was scooped out using the target substrate. After drying, the sample was annealed at 180 °C for 30 mins. Finally, the PMMA was removed in acetone followed by IPA rinse. Three samples were prepared using the conventional wet transfer (WT) technique differing only in the substrate treatment, including untreated SiO2 (WT-Gr/SiO2), SAM-modified SiO2 (WT-Gr/SAM) and hBN covered SiO2 (WT-Gr/hBN). For the SAM modification, an octadecyltrichlorosilane (ODTS) layer was utilized due to its higher efficiency of screening the charge impurities present on SiO2 layer202-plasma activation of the SiO2 and spin casting from toluene solutionMonolayer graphene/Cu (25 μm) and hBN/Cu (25 μm) foils were purchased from Graphenea and Graphene Supermarket, respectively. Graphene/Cu or hBN/Cu layers were first covered with PMMA A4 495 by spin coating at 3500 rpm for 60 s and curing at room temperature overnight. Graphene or hBN present on the bottom side of the foils were removed using an O2 using the WT procedure (AT-hBN/Gr/SiO2 sample).The hBN/copper foil was coated with a relatively thick layer of PMMA (7% at 2500 rpm for 60 s). A frame of kapton tape was then applied to the PMMA/hBN/Cu stack to facilitate the dry transfer. After Cu etching and rinsing, the PMMA/hBN stack was removed from the DI water and dried at 60 °C for 1 hour. The sample was then ready for application directly to the target substrate, which in this case was the graphene/Cu foil. By these means a clean interface between hBN and graphene layer was created. After annealing at 180 °C for 30 mins, Cu etching and rinsing steps were repeated. The number and order of stacked 2D layers is in principle unlimited. To provide comparable results, in this work we limit ourselves to one hBN-Gr stack, which was applied to the SiO2-plasma treatment, while the second step provided the necessary pattern for contact deposition (Cr (5 nm)/Au (40 nm)). Graphene FET channel width and length were 100 and 1000um, respectively. The transfer characteristics of the graphene FETs were analyzed using a Keithley 2634B semiconductor parameter analyser unit under ambient environment with a two-point measurement for source and drain electrodes.The electrical properties of the transferred graphene films were examined in a field-effect transistor (FET) geometry. For the WT samples, top-contact Cr (5 nm)/Au (40 nm) electrodes were thermally evaporated through shadow masks directly onto graphene films to avoid further contact with polymers such as photoresist. For the hBN-assisted sample, photolithography can be used due to the protection provided by the top layer hBN. In this case, the edge-contacts were fabricated using a two-step photolithography process−1 and lateral resolution of 0.3 μm. Areas of 15 × 15 μm were mapped out and analysed to provide a sufficient statistical description of the overall graphene quality, since strong spatial variations in the Raman spectra are usually observed over the transferred graphene filmRaman spectroscopy measurements were performed using a confocal scanning Raman microscope (WiTec Alpha300 system) in backscattering geometry with 532 nm excitation and a 100 × objective (0.9 NA). An incident laser power of 1 mW was used to avoid laser induced heating of graphene samples. Measurements were performed at room temperature under ambient conditions, with an accumulation time of 0.5 s, a spectral resolution of 3 cmThe data that support the findings of this study are available from the corresponding authors on request .How to cite this article: Shautsova, V. et al. Hexagonal Boron Nitride assisted transfer and encapsulation of large area CVD graphene. Sci. Rep.6, 30210; doi: 10.1038/srep30210 (2016).