Charge transport at the Dirac point in bilayer graphene exhibits two dramatically different transport states, insulating and metallic, that occur in apparently otherwise indistinguishable experimental samples. We demonstrate that the existence of these two transport states has its origin in an interplay between evanescent modes, that dominate charge transport near the Dirac point, and disordered configurations of extended defects in the form of partial dislocations. In a large ensemble of bilayer systems with randomly positioned partial dislocations, the distribution of conductivities is found to be strongly peaked at both the insulating and metallic limits. We argue that this distribution form, that occurs only at the Dirac point, lies at the heart of the observation of both metallic and insulating states in bilayer graphene. In seemingly indistinguishable bilayer graphene samples, two distinct transport regimes, insulating and metallic, have been identified experimentally. Here, the authors demonstrate that these two states originate from the interplay between extended defects and evanescent modes at the Dirac point. Contradicting this expectation, recent transport experiments on ultra-clean bilayer graphene report the existence, near charge neutrality, of an insulating phase. Curiously, this occurs only in about 50% of an apparently identical set of ultra-clean high-mobility samples, with the remainder showing the expected minimal metallic conductivity8. Two different explanations for the existence of the insulating state have been proposed: that the eight-fold degeneracy of the bilayer electron fluid at the Dirac point is responsible for an interaction-driven symmetry breaking to an insulating phase7 or, alternatively, that the insulating state is a manifestation of charge blocking by partial dislocations8, a structural defect recently observed in many bilayer graphene samples12.Transport at the Dirac point in structurally perfect bilayer graphene is expected to exhibit a minimal metallic conductivity, very similar to that found in single layer grapheneIn both these models the occurrence in experiment of metallic and insulating states with approximately equal probability is difficult to explain. In the interaction model we have the difficulty of apparently identical ultra-clean samples resulting only sometimes in the transition to an insulating phase; similarly, in the blocking model the metallic state requires for its existence samples that are entirely free of partial dislocations, a situation that would appear unlikely.Here we resolve this conundrum by identifying an unusual transport physics that arises from the interplay of extended defects and evanescent Dirac point transport in bilayer graphene. We find that some positions of partial dislocations entirely block transport while others appear not to impede charge transport at all. In particular, in an ensemble of systems with randomly placed partials, the distribution function of conductivities has pronounced—and approximately equal—maxima at both the insulating and the metallic limits. This behaviour, which sharply deviates from the well-known paradigm that disorder should generally act to suppress transport, we argue that this underpins the observation of both insulating and metallic transport states in experiment.12, described by one of the three partial Burgers vectors: a is the graphene lattice parameter, and di the nearest neighbour vectors of the honeycomb lattice). Note that these stacking fault partial dislocations are very different in character from the dislocations found in single-layer graphene14. Indeed, as they require for their existence only local strain within the constituent layers of the bilayer, they may occur between graphene sheets that are essentially structurally perfect. The weak interlayer bonding of bilayer graphene then implies a rather low energy cost of stacking fault partial dislocations and they are, for example, found to form a dense network in epitaxial graphene grown on SiC (0001)10, where they arise due to strain induced by the lattice mismatch between graphene and the SiC substrate.The microscopic origin of partial dislocations arises from the fact that Bernal stacking may be achieved in two equivalent ways, typically referred to as AB and AC stacking, as shown in Fig. 8. We will return to this point subsequently. Even with this simplifying assumption, however, modelling the transport experiments remains a challenge due to the presence of two very different length scales: the partial dislocation which occurs on a scale of ≈5 nm, and the length of the suspended bilayer graphene sample which is typically of the order of 1 μm. This is therefore a multi-scale problem, and evidently precludes the use of an atomistic approach such as the tight-binding method. To circumvent this difficulty we will make use of a recently developed effective Hamiltonian theory capable of treating large-scale structural deformations in low-dimensional systems15, and shown recently to provide a very good description of the electronic structure both for partial dislocations15 as well as twist faults in bilayer graphene18. In this scheme the individual layers of the bilayer are modelled by the Dirac–Weyl Hamiltonian H0 = vFσ.p , while the spatially dependent stacking order is represented by an interlayer stacking field that, importantly, treats all stacking types on an equal footing . The transport problem is then addressed within the framework of Landauer theory with the leads modelled as highly doped bilayer graphene2. Further details may be found in the Methods section.In order to understand the impact of such stacking defects on charge transport, we will consider a model of partial dislocations that are parallel with the left and right electrodes contacting the bilayer sample Fig. . This isG shows the expected L−1  or x1/L = 1/2 and x1/L = 2/10, respectively  . As may be seen, this function has pronounced peaks at both the insulating and minimally metallic transport states. These results, it should be stressed, are rather independent of the lead structure, the strain state of the partial, and indeed the partial Burgers vector—as may be seen in Fig. σ(x1) dependence is found for all three possible partial Burgers vectors of bilayer graphene . Furthermore, the window for which a terrace is open to charge transport depends significantly on its length, with the resonance centre given byl⊥ ≈ 15 Å. To bring this single terrace physics to the problem of a dislocation threaded sample, we simply insert an ideal  lead between the partial dislocation and the second terrace such that the transport problem is then recast as a multiple scattering problem involving single terrace transmission functions19, with the total transmission given by t = tAC(1 − rABrAC)−1tAB. In this expression |tAB/AC|2 are the transmission functions of the individual terraces, with (1 − rABrAC)−1 encoding multiple scattering between them. From this we see that transport will be profoundly influenced by the resonance centres tAB/AC|2 have common evanescent momenta for which they are themselves non-zero. For a symmetric partial position, entailing AB and AC segments with similar L, |tAB|2 and |tAC|2 do have coinciding ranges of their open values of Imkx and so a conducting state is found  of Fig. lead| < 1 at x = L giving us the expression L, can be recast as x1/L = 0.75—panel (c) of the same figure—the terraces are of different length and the matching condition cannot be satisfied with the same Imkx in each terrace. As a consequence of these incompatible matching conditions, the wavefunction in the second terrace collapses, and a blocking of charge transport ensues. While two-state transport for a single partial configuration can thus be clearly motivated in terms of the interplay between evanescent transport and partial dislocations, it generates the expectation that only high symmetry  configurations of partials will conduct. Interestingly, as we now show, this is not the case.This physics may also be understood in an intuitive way from the point of view of the evanescent transport wavefunction. The dominant component of such an exponential wavefunction has the form entclass1pt{minimaxi} which, as each partial is defined by one coordinate, may be characterised by a single number—the RMS of the terrace length distribution Lrms. Three distinct regions of transport vs. Lrms may be seen. For small Lrms < 0.1, corresponding to ordered configurations in which the partials are approximately uniformly spaced, the transport resembles that of the high symmetry single partial configuration: a few percent resonant enhancement over the minimal metallic conductivity of pristine bilayer graphene. This is the multi-partial geometry directly analogous to the central position in the case of a single partial, and conducts for a similar reason: each terrace shares similar values of evanescent momentum for which transport is allowed. Contrasting this, in the limit of large Lrms > 0.65 the transport is seen to be completely blocked. This Lrms regime corresponds to case of a partial pairing geometry in which two of the three partials have come close to their minimal allowed separation of 125 Å. This is analogous to the unsymmetrical partial position, as the very different terrace lengths lead to non-coinciding transmission windows and thus to transport blocking. Most interesting, however, is the intermediate no-pairing but disordered regime. In this region the same value of Lrms is found to yield almost all transport states from quite strong resonant enhancement  to complete suppression of transport. The probability density function P(σ)  at the Dirac point and at a finite energy of 10 meV). Finally, to make a direct comparison with experiment we take the data from ref. 4 which, after rescaling, is presented by the grey bars in the right hand panel of Fig. bar represents a transport measurement in a single suspended bilayer graphene sample, and the experimental distribution of conductivities is seen to match very well the theoretical probability density function.For more than one partial, the generic case is disordered among the partial coordinates {(σ) Fig.  again shLrms ≈ 0.4 but very different transport states. In a similar way to the analysis for the single partial configuration, we break each bilayer sample into two sub-systems: the first three terraces and partial dislocations and the last terrace −1t119, where |t|2 is the transmission probability through the whole system, |t1/2|2 the sub-system transmission probabilities and |r1|2 (|r2|2) the probability of a left (right) moving wave reflecting from sub-system 1 (2). As may be seen in Fig. r1r2)−1| and t1 coincide, resulting in a pronounced enhancement of the system transmission function t. Given that the term (1 − r1r2)−1 = 1 + r1r2 + (r1r2)2 +… encodes multiple back and forth scattering at the boundary between the two sub-systems, this enhancement corresponds physically to a multiple scattering resonance between bilayer terraces (as opposed to the evanescent resonance intrinsic to a single terrace). It is the presence of such resonances, absent in system (b) for which the maxima of |(1 − r1r2)−1| evidently does not coincide with the maxima of either t1/2, that is responsible for the existence of both insulating as well as conducting samples in the intermediate disorder regime.To probe the reason behind the occurrence of insulating as well as conducting samples in the intermediate disorder regime, we perform a scattering analysis for two configurations having nearly identical L−1 length dependence characteristic of the minimal metallic state  and so will reopen propagating channels in the transport.Application of a layer perpendicular field ky) from, at the Dirac point, imaginary transport momenta (kx), which in an irregular partial geometry will mix. At the Dirac point, however, states of real momenta are apparently blocked by partials8 and so we expect our Dirac point results to be robust to this more complex mode matching. On the other hand at finite doping the re-established propagating channel is expected to generate a higher conductivity in an irregular partial geometry than in a straight partial geometry, precisely due to this mixing of momenta. We thus expect the weaker establishment of the metallic state at finite doping and finite symmetry breaking field to be the most significant impact of the straight partial approximation.In both Fig. 24. In the light of the role that structural disorder plays in explaining the physics at zero field, it will be of interest to examine the impact of structural disorder at finite fields.We have shown that the approximately equal occurrence of both conducting and insulating transport states in experimental samples of bilayer graphene may be explained by an anomalous Dirac point transport involving partial dislocations and transport by evanescent states. This is underpinned by the fact that random arrangements of partial dislocations can support charge transport, leading to minimal metallic conductivity for arrangements of partials that one might expect to block transport. For an ensemble of systems this leads to an unusual two-state distribution function of conductivities in which minimal metallic and insulating states occur with approximately equal probability at the Dirac point. The dramatically different transport states found in experiment are thus simply a manifestation of this hidden structural degree of freedom, which will naturally change from sample to sample as well as be changed by annealing the same sample. Finally, we note that ultra-clean suspended bilayer graphene has, in addition to interesting zero field transport physics, interesting physics at finite magnetic fields, in particular possible indications of a fractional quantum Hall effect4 in which two distinct types of bilayer graphene are reported: those having a very high mobility, for which two-state transport is observed, and those with a substantially lower mobility that were found always to be conducting. As the high mobility samples presumably exclude the kinds of single layer disorder likely to significantly disrupt the terrace wavefunction, we infer that bilayer terraces free from disorder are required for the two-state mechanism to hold.For the transport calculation we use the Landauer formalism, appropriate as the experimental system is mesoscopic in dimensions, ultra-clean, and at low temperatures. Transport is thus likely to be well described by the phase coherent approach. In this context it is worth nothing the recent work of Bao et al.In what follows we will describe the three distinct aspects of this calculation: obtaining the system Hamiltonian; determination of the transfer matrices of the bilayer sample; and finally deriving the overall scattering matrix and conductance from these transfer matrices.H Hamiltonian15, and we will employ this method here . For our tight-binding method we employ two hopping functions δ the length of the hopping vector. The in-plane constants vF = 106 ms−1 and the known in-plane nearest neighbour hopping strength of 2.8 eV, while the interlayer constants A⊥ and B⊥ are similarly fixed to reproduce the energy separation of the bonding and anti-bonding states in AB stacked bilayer graphene (0.78 eV) while maintaining a nearest neighbour hopping strength of 0.38 eV.The multi-scale nature of the transport problem mandates the use of a continuum approach in which the dislocation threaded bilayer is treated via some generalisation of the well-known Dirac–Weyl equation of graphene. Recently, a general and exact scheme has been presented by which the tight-binding Hamiltonian of any system can be directly mapped to a effective continuum u(x) that describes the local relative displacement of the two layers of bilayer graphene from some initial reference stacking due to, for example, a series of partial dislocations. For the case of a single type, three partial dislocation such a stacking field is shown in Fig. u(x) only to the first layer. In this case the effective Hamiltonian for a graphene bilayer system with a general u(x) field is given byvF the Fermi velocity of graphene. The diagonal blocks of this Hamiltonian are the Dirac–Weyl operators for each layer, with the first layer operator augmented by fields arising from strain due to the application the displacement field u(x) to this layer, while the off diagonal blocks encode the complex spatially dependent interlayer stacking. The strain induced layer–diagonal fields are given by the standard results25 with the effective scalar field given byCV and CA are the coupling strengths (that depend on the in-plane hopping constants 15) and uij = ∂jui the components of the strain tensor with ui the ith component of the displacement field u(x). The strain associated with a type three partial is shown in Fig. 15AUC is area of the bilayer unit cell. Note that this field—and all subsequent objects related to it—is a 2 × 2 matrices in interlayer sublattice space, i.e., each element represents an electron hopping amplitude between the sublattices of different layers. In this expression the sum i is over the translation group of the high symmetry point K = 2π/a: Ki = K + Gi where Gi are the reciprocal space lattice vectors of bilayer graphene. The matrices Mi are given by να (νβ) the basis vectors of layer one (two) of the bilayer. Finally, the function t⊥(q) is the Fourier transform of the interlayer hopping function t⊥(δ) where δ is the separation vector of the two sites (in different layers) that the electron hops between: δ = |Rj − Ri|. As this function, and hence its Fourier transform t⊥(q), are exponentially decaying a first star approximation in which the sum is restricted to include only K, and the two equivalent high symmetry K points of the bilayer Brillouin zone already constitutes a good approximation for S(x). Assuming the AB structure as a reference state, and taking a standard choice of basis vectors ν1 = 0 and ν2 = a is that it treats AB, AC, and any intermediate stacking on exactly the same footing. To see this note that insertion of u = 0 into Eq.  shown in panel of Fig. a is chosen to reproduce as closely as possible the calculated partial structure described in ref. 10. As may be seen—see panel (b) of Fig. S(x) can be conveniently expressed by projection onto a complete set of four stacking matrices which, with a c-number coefficient, are sufficient to describe all possible interlayer coupling fields: S(x) = cAB(x)τAB + cAC(x)τAC + cAA(x)τAA + cz(x)τz. These matrices are given byτ matrices evidently inhabit the interlayer and sublattice space of the S(x) field, is directly informative of the local stacking of the bilayer. This projection is exhibited in panel (c) of Fig. τz type of stacking is zero throughout the partial dislocation.The partial dislocation, which interpolates between these two limits, we model using the displacement field H(x), we proceed by dividing the bilayer sample into a series of small strips  within which H(x) can be considered constant. By expressing the strip Hamiltonian as H = H0 + Hxkx + Hyky we may rewrite the eigenvalue problem HΨ = EΨ asE and ky, the constants of motion of transport problem . These u(x) is presented in panel (a). From these, and the corresponding eigenfunctions Ψi, we can easily construct the transfer matrix of the strip. Denoting as W that matrix in which each column is one of the eigenfunctions Ψi, and as B the diagonal matrix with elements L the strip width, then the transfer matrix is simply WBW−1.Having established the system Hamiltonian 2. The leads are encoded in a WL matrix in which each column consists of a highly doped bilayer graphene wavefunction (two right moving and two left moving states), for an explicit form of WL see the appendix of ref. 2. We have checked that the qualitative nature of the insulating and conducting partial dislocation configurations is independent of the doping of the lead  yields a series of strip transfer matrices Mstrip having exactly the form of Eq. (19, combined to give the overall system scattering matrix Ssystem. From this the conductance G is then obtained via the Landauer formula.The remainder of the calculation follows the standard methodology of the scattering theory Landauer approach, see refs. The data that support the findings of this study are available from the corresponding author upon request.Peer Review File


Moreover, this designed architecture shows excellent stability in the battery operation. After 100 charging–discharging cycles, the electrode not only shows no observable morphology change, it can also be reused in another battery and practical with the same performance. It is believed that this novel structure including the synthesis procedure will provide a new developing direction for the VRFB electrode.3D graphene‐nanowall‐decorated carbon felts (CF) are synthesized via an in situ microwave plasma enhanced chemical vapor deposition method and used as positive electrode for vanadium redox flow battery (VRFB). The carbon fibers in CF are successfully wrapped by vertically grown graphene nanowalls, which not only increase the electrode specific area, but also expose a high density of sharp graphene edges with good catalytic activities to the vanadium ions. As a result, the VRFB with this novel electrode shows three times higher reaction rate toward VO However, in addition to the high cost of these metals, they are also active toward water splitting. As a result, electrical energy will be wasted on the undesirable water splitting process instead of the redox reactions of vanadium ions. This not only decreases the energy efficiency (EE), but also leads to oxygen evolutions which in turn causes stability and safe problem in the battery.Over the past few years, intensive efforts have been devoted to address the above‐mentioned challenge by modifying the electrode surface to introduce more active sites or to enhance their catalytic activities. For example, noble metals such as Au, Pt, Ru, and Ir have been deposited on the carbon felt (CF) electrodes and exhibited improved catalytic activity toward VO2+/VO2+ couple, these thin films show good electrochemical activity ascribing to their high specific area and oxygen containing groups on them.2+/VO2+ couple.On the other hand, graphene materials have also been found to have promising applications on electrodes for VRFB.2+/VO2+ redox couple and 11% increased EE over VRFB with an unmodified CF electrode. It was also found that the graphene‐coated CF electrode show excellent stability in the battery operation. After 100 charging–discharging cycles, the electrode not only shows no observable morphology changes, it can also be reused in another battery and practically with the same performance.To achieve these improvements, we designed and prepared a unique hierarchical structure by growing a uniform and dense layer of 3D graphene on CF via a simple microwave plasma enhanced chemical vapor deposition (MPCVD) process. It was observed that the carbon fibers in CF were successfully wrapped by vertically grown graphene nanowalls, which not only increase the electrode specific area, but also exposing a high density of sharp graphene edges with good catalytic activities to the vanadium ions. We applied the electrode in VRFB and observed about three times higher reaction rate toward VO2FigureFigure2 g−1 while that of CF is only about 1.9 m2 g−1 . In addition, compared with bare CF sample, all CF‐G samples show significantly decreased area electric resistance after the 3D graphene in situ growth process , implying smaller physical polarization for CF‐G electrodes during the VRFB operation.A typical photograph of an as‐prepared electrode material (CF‐G‐1) is shown in The CF‐G‐1 material was grinded into powder, dispersed in alcohol solution and dropped onto a porous carbon film for transmission electron microscopy (TEM) observation. Figure Figure−1 (G band) and the appearance of shape peak at 2668 cm−1 (2D band) confirming the TEM observation that the vertically nanowalls are several layer graphene materials.2+/VO2+ redox reactions.Crystal structures of the CF and the CF‐G‐1 samples were characterized with X‐ray diffraction (XRD). As shown in Figure−1 . The peaks at about 1.1 V correspond to the oxidation process of VO2+ → VO2+, with the oxygen transferring from water to VO2+ and electron output .FigureElectrochemical impendence spectroscopy (EIS) was used to measure the charge transfer resistance (Rct) during the redox reactions. As shown in −1 discharge capacity for CF‐G‐1 and 14.3 Ah L−1 discharge capacity for CF. For VRFB, the EE value is a key performance indicator for evaluating the energy conversion efficiency between chemical and electric energies. The energy efficiency (EE) within 100 cycles at the current density of 25 mA cm−2 for CF‐G‐1 and CF are shown in Figure −2 is obtained, respectively  and the Rct  can be respectively estimated from the starting point and the diameter of the semicircles. The resistance of diffusion (Zw) during the reaction process can be determined from the slope of the straight line.−2, the capacity decreases from 20.5 Ah L−1 for the initial value to 9.7 Ah L−1 after 100 cycles . As explain, degradation caused by the vanadium ion counter diffusion through the membrane is well‐known main cause for capacity fading in VRFB.−1 which is larger than its initial value of 20.5 Ah L−1, indicating the present CF‐G‐1 electrode practically not only having no electrochemical degradation after 100 cycles, but also completing an activation process. The surface chemical status of CF‐G‐1 after 100 cycles was further analyzed by XPS method. As shown in Figure S9 of the Supporting Information, the surface oxygen content increases from 7.13% to 12.3%, suggesting some of the graphene (probably the edge part with more defects) has been oxidized in the strong oxidation electrolyte during the charge–discharge processes.2+/VO2+ redox reactions. Inspired by this interesting phenomenon and previous reported activation method,−1 of initial discharge capacity can be obtained and the Coulombic efficiency keep above 96% at 50th cycle, further demonstrating that introducing of surface oxygen functional groups can improve the electrochemical activity of graphene toward VO2+/VO2+ redox reactions. The proposed catalytic mechanism is schemed in Figure S11 of the Supporting Information. For the oxidation process, VO2+ ions connect with the surface oxygen containing groups first, then the oxygen atom transfer from the H2O molecule to the VO2+ with the assistance of surface oxygen containing groups. Finally, the reaction product (VO2+) diffuses from the electrode surface into the electrolyte. For the reduction process, the reaction is opposite and can also be facilitated by the surface oxygen functional groups.To further test operation stability of the VRFB with a CF‐G‐1 electrode, we measured its capacity changes for 100 cycles at a current density of 25 mA cmThe structural stability is also crucial for stable cycling. The battery was disassembled and CF‐G‐1 electrode was washed and dried after battery test. SEM images for this used CF‐G‐1 electrode are shown in Figure S12a of the Supporting Information; the vertically standing characteristic of the graphene nanowall is well maintained without any sign of peeling off. Other than the electrode, operation stability of VRFB is also limited by the membrane which makes the battery couldn't operate for a long time. To eliminate the effects from this reason, we have specially designed a testing setup as shown in Figure S12b of the Supporting Information, the CF‐G‐1 electrode was putted into a glass container with flowing electrolyte (no membrane and current were applied during this adhesion strength test). After 10 d scouring test in the electrolyte, the CF‐G‐1 electrode was taken out and characterized by SEM . No obvious change can be observed and the graphene nanowalls still possess 3D structure and are well connect with the CF substrate, which further confirm that the bonding between carbon fibers and graphene nanowalls are strong enough to withstand the battery's mechanical operation.3In summary, graphene nanowall decorated CF was successfully fabricated via a simple MPCVD method and used as positive electrode for VRFB for the first time. Compared with previous reported graphene based catalysts, this unique structure can provide more active sites for vanadium redox reactions. Excellent cycle stability  and rate capability can be obtained when employing the composite materials as positive electrode during VRFB test. Moreover, this in situ growth process ensures good bonding between the graphene nanowalls and the carbon fibers. The electrode show no observable degradation in long time scouring test and 200 charge–discharge cycles. We believe this fabrication method and this novel structure will have a very good promise in VRFB electrode application. Moreover, this all carbon based material may also have applications in catalyst substrate and other battery electrodes.4Preparation of 3D Graphene Modified Carbon Felt Electrode: The graphene nanowall decorated carbon felt was prepared by a simple MPCVD process, as reported elsewhere with modification.4/H2 gas mixture at a constant flow rate ratio at 30 Torr. The microwave power was kept at 1200 W, and the substrate was heated to about 800 °C as monitored by an infrared pyrometer and maintained for 2 h. After the plasma was switched off, the sample was cooled down naturally. Details on the growth conditions are listed in Table S1 of the Supporting Information. Samples synthesized under different conditions are marked as CF‐G‐1, CF‐G‐2, CF‐G‐3, and CF‐G‐4, respectively. CF‐G‐1 sample was further modified in concentrated nitric acid at 80 °C for 6 h, and then washed by deionized water and alcohol for several times and dried in vacuum.Characterization: Structures of the materials were characterized with an X‐ray powder diffractometer . Morphologies of the carbon felt and graphene‐modified carbon felts were observed with a transmission electron microscope  at 200 kV and a scanning electron microscope . Raman spectroscopy was carried out with a Renishaw 2000 Raman microscope with 633 nm excitation source. Surface areas of the samples were measured with nitrogen sorption isotherms 77 K on a Micromeritics ASAP 2020 apparatus. The surface chemical status for CF‐G materials was analyzed by X‐ray photoelectron spectroscopy . Electric conductivity of all samples is tested by the equipment shown in Figure S3 and the test method is also described in detail in the Supporting Information.Electrochemical Measurement: Electrochemical measurements were carried out using a three‐electrode cell on an electrochemical workstation (Autolab 302N). A working electrode with a geometric area of 1 cm2 was clamped by an electrode holder where only a small part of the carbon felt was contacted with the platinum current collector was equipped with a platinum plate counter electrode and Ag/AgCl reference electrode in 0.1 m VOSO4  in 2 m H2SO4 solution. A salt bridge was used to eliminate the liquid junction potential. Cyclic voltammetry (CV) was recorded over a fixed voltage window between 0 and 1.4 V versus Ag/AgCl under different scan rates at room temperature. Electrochemical impendence spectroscopy (EIS) was recorded over a frequency range from 100 kHz to 10 mHz at open‐circuit voltage. For battery test, thickness of ≈4 mm graphene modified carbon felt electrode and pristine carbon felt  with an active area of 6.25 cm2 (2.5 × 2.5 cm) were used as the positive and negative electrode, respectively. The batteries were fabricated by sandwiching the proton exchange membrane (Nafion 212) between two pieces of electrodes placed in polytetrafluoroethylene frames. Copper sheet and conductive plastic were served as current collector and bipolar plate, respectively. Flexible graphite was place between the current collector and bipolar plate which was used to reduce the contact resistance during the battery operation due to the full contact between them. The carbon felt compression ratio in the battery apparatus is about 75%. The starting electrolytes for both the positive and the negative part were 2.5 m VOSO4+ 2.5 m H2SO4 (15 mL). The test batteries were charged and discharged over the operating potential range of 0.7–1.7 V.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Here we report a transfer method using rosin as a support layer, whose weak interaction with graphene, good solubility and sufficient strength enable ultraclean and damage-free transfer. The transferred graphene has a low surface roughness with an occasional maximum residue height of about 15 nm and a uniform sheet resistance of 560 Ω per square with about 1% deviation over a large area. Such clean, damage-free graphene has produced the four-inch monolithic flexible graphene-based organic light-emitting diode with a high brightness of about 10,000 cd m−2 that can already satisfy the requirements for lighting sources and displays.The large polymer particle residue generated during the transfer process of graphene grown by chemical vapour deposition is a critical issue that limits its use in large-area thin-film devices such as organic light-emitting diodes. The available lighting areas of the graphene-based organic light-emitting diodes reported so far are usually <1 cm Ultraclean and damage-free transfer of graphene over large areas is crucial for the future development of flexible electronics and optoelectronics. Using a rosin-assisted method, the authors transfer graphene with an ultraclean surface and uniform small sheet resistance—a 4-inch monolithic organic light-emitting diode is demonstrated. Different from previously-used support materials, rosin has a super solubility in organic solvents, a weak interaction with graphene and sufficient strength, all of which enable clean and damage-free transfer. The transferred monolayer graphene films have a very low surface roughness with a maximum height of about 15 nm and a uniform sheet resistance of 560 Ω per square with about 1% deviation over a large area. Such clean and damage-free graphene greatly improves the current efficiency (CE) and power efficiency (PE) of OLEDs, with maxima as high as 89.7 cd A−1 and 102.6 lm W−1, respectively, even without doping. More importantly, we have fabricated the four-inch monolithic flexible graphene-based OLEDs with a uniform lighting area of 56 cm2 and a high brightness of about 10,000 cd m−2 that can already satisfy the requirements for lighting sources and displays.Here, we have found that rosin  sufficient support strength. Good solubility allows the support layer to be easily dissolved in the commonly used chemical solvents. A low Ead. is beneficial for the separation of the polymeric support layer from the graphene surface. Sufficient support strength can effectively prevent fragmentation or tearing of the graphene film during transfer.Based on the understanding of graphene transfer by using macromolecular polymers and small organic molecules, an ideal polymeric support layer should satisfy all the following three requirements in order to obtain residue- and damage-free graphene films of a large area: (i) a good solubility in solvents, (ii) a low ca. 302) mainly consisting of resin acids (primarily abietic acid)44Ead. of rosin, pentacene, SPPO1 and HMMA  molecules on a graphene surface (Ead. (1.04 eV) of rosin on graphene with the most stable adsorption configuration , scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), and atomic force microscopy (AFM) to characterize the surface of rosin-transferred graphene films. For comparison, the surface structure of PMMA-transferred graphene films was also studied. The individual dark dots shown in 343536372) were measured for each sample. Consistent with OM observations, large residue particles are frequently observed on the PMMA-transferred sample (Rmax) of the large residue particles is a more important parameter for large-area thin-film device applications, because this determines whether the devices are likely to be short-circuited. A typical statistical histogram of Rmax collected from 100 randomly selected areas (5 × 5 μm2) for a rosin-transferred graphene film on a SiO2/Si substrate was shown in Rmax of about 15 nm  is surface-sensitive quantitative spectroscopic technique that can measure the elemental composition of a surface in the parts-per-thousand range, and Raman spectroscopy provides a high-resolution characterization tool to give both the atomic structure and electronic properties of graphene such as the number and orientation of layers, doping, disorder and functional groupsne on Cu . No rosi2, which was divided into 100 equal areas for electrical property measurements. All these areas show a very uniform sheet resistance of 560 Ω per square with a standard deviation of about 1%  and ITO as the anode.We first used rosin-transferred three-layer graphene as an anode to fabricate phosphorescent green OLEDs with a lighting area of 0.4 × 0.4 cm−1 and 102.6 lm W−1, respectively, which are comparable to the best values of graphene-based OLEDs reported in the literature without any outcoupling structure and cavity resonance enhancement design  of the large PMMA residue particles far exceeds the thickness of the active layer (about 140 nm), resulting in electrical micro-short circuits. Therefore, many dark spots are observed and these grow quickly with increasing operating time and applied voltage, and spark discharge can be found at some spots of the lighted area, which lead to quick burn-out of the device.In contrast, for the PMMA-transferred graphene TCEs, no four inch OLED devices can be lighted. As shown in 2 OLEDs can light more than two times longer than four-inch ones (8 × 7 cm2) in air at same luminance. The main reason is that the large-area devices easily exist further hole defects that cause short circuit, thus reducing stability. Another important problem is that the anode resistance has much larger influence on the large-area devices than the small-area ones. It can cause a big voltage drop and current distribution non-uniformity in large-area devices, which will greatly reduce device stability. Therefore, for our large-area OLED devices using rosin-transferred graphene as anode, much effort should be made to further reduce its resistance to enhance device lifetime in the future.We also compared the stability of large-area and small-area OLED devices. The unencapsulated 0.4 × 0.4 cm−2. This rosin-based transfer method provides a universal approach for the ultraclean and damage-free transfer of graphene and other 2D materials grown by CVD on metals, which paves the way for electronic and optoelectronic applications, in particular, large-area thin-film devices of 2D materials.Ultraclean and damage-free transfer of large-area CVD-grown graphene films has been achieved using the small organic molecule rosin as a support layer based on its good solubility, weak interaction with graphene and adequate support strength. The transferred graphene films have a very low surface roughness with a maximum height of residue particles up to 15 nm and an extremely uniform sheet resistance of 560 Ω per square with about 1% deviation over a large area. Such clean and damage-free graphene greatly improves the CE and PE of OLEDs, and more importantly, it has enabled to production of the 4-inch monolithic flexible graphene-based OLEDs exhibiting uniform light emission and a high brightness of about 10,000 cd mab-initio simulation package3) was used to calculate the adsorption energies of different organic molecules on the graphene surface. Only the Γ point was used to sample the first Brillouin zone for all calculations due to the large size of the graphene supercell. For the geometry relaxations and energy calculations, van der Waals interactions were incorporated by the optB88 exchange functional603 supercell until the residual force per atom decreases to <0.01 eV Å−1.DFT calculations were performed using the projector augmented wave method56wca. 302 by gel permeation chromatogram, Alfa-Aesar CAS no. 8050-09-7, dissolved in ethyl lactate with a concentration of 50 wt.%) was first spin-coated on the CVD-grown graphene film at 500 r.p.m. for 10 s and then at 1,200 r.p.m. for 60 s. The rosin layer was then cured at room temperature, followed by etching the Cu foil in an aqueous solution of FeCl3 (0.03 g ml−1) to obtain a rosin/graphene stack floating on the solution. After washing with deionized water to remove residual etchant, the rosin/graphene stack was collected on the target substrate, and then taken out from the solution. To ensure the rosin/graphene film stack remained intact and was fully in contact with the target substrate, the rosin/graphene/target substrate was first treated at 40 °C for 1 h, and the temperature then was slowly increased to 120 °C for 20 min to evaporate the residual water. Subsequently, the rosin layer was dissolved by acetone  and banana oil solutions  in sequence. Finally, the graphene film was blow dried using high-purity nitrogen.Monolayer graphene films were grown by CVD on copper foils as reported previously23243 HIL layer. After that, phosphorescent green OLEDs were fabricated by subsequently depositing a 60 nm di-[4--phenyl] cyclohexane (TAPC) hole transportation layer (HTL), two 8 nm layers of bis(2-phenylpyridine) (acetylacetonate)iridium(III) [Ir(ppy)2(acac)] doped with 1,1-bisphenyl] cyclohexane (TCTA) and a bathophenanthroline (Bphen) light emission layer, a 60 nm Bphen electron transportation layer (ETL) and a 0.5 nm Li/130 nm Al cathode. The active area defined by the cathode is 0.4 × 0.4 cm2. For comparison, PMMA-transferred 3-layer graphene films were also prepared, selectively oxidized and patterned by the same methods, and fabricated into OLEDs with the same device structure. For the fabrication of large-area devices, rosin- and PMMA-transferred 5-layer graphene films were used to further reduce the sheet resistance, while the other fabrication procedures and device structures remained the same.First, three-layer graphene films were transferred layer-by-layer onto a PET substrate using rosin as the support layer. Ozone treatment was carried out at 120 °C for 5 min to obtain a GO/G/G heterostructure to simultaneously increase the work function and compatibility with HIL as described in our earlier work2 (300 nm thick)/Si substrate and a TEM grid. AFM  was used to characterize the surface roughness of the graphene transferred onto the SiO2/Si substrate with a tapping mode. Particle analysis function in NanoScope Analysis 1.40, a software package for analysing scanning probe microscopy data, was used to analyse the height of the residue particles to obtain the Rmax of each measured area. Rmax of a transferred graphene sample is the maximum of the Rmax values of all measured areas. XPS was used to characterize the surface chemical composition on an ESCALAB 250 instrument with Al Kα and He I radiation sources. The XPS spectra were fitted using the XPS peak 4.1 software in which a Shirley background was assumed. Raman spectra were measured using a Jobin Yvon LabRam HR800, excited by a 532 nm laser. The laser spot size was about 1 μm with the laser power below 2 mW to avoid laser-heating-induced sample damage.OM (Nikon Eclipse LV100), SEM  and HRTEM  were used to characterize the morphology and structure of the graphene films transferred onto a SiOThe sheet resistance and transmittance of graphene films with different numbers of layers transferred onto PET were measured by a 4-probe resistivity measurement system  and UV–vis-NIR spectrometer (Agilent Model Cary 5E), respectively. Current-brightness-voltage characteristics of the unencapsulated OLEDs were characterized by Keithley source measurement units (Keithley 2400 and Keithley 2000) with a calibrated silicon photodiode in air. Note that our OLED devices were stable enough when measuring their basic optical and electric properties although they were not encapsulated.The data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Zhang, Z. et al. Rosin-enabled ultraclean and damage-free transfer of graphene for large-area flexible organic light-emitting diodes. Nat. Commun.8, 14560 doi: 10.1038/ncomms14560 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Notes and Supplementary References2 fabricated with a rosin-transferred graphene anode.A 4-inch monolithic flexible green OLED with a lighting area of 8 × 7 cmA 3-inch monolithic flexible green OLED fabricated with a PMMA-transferred graphene anode.


And the graphene/PANI aerogel presents a high recoverable compressive strain of 90% due to the strong interaction between PANI and graphene. The all-solid-state supercapacitors were assembled to demonstrate the compression-tolerant ability of graphene/PANI electrodes. The gravimetric capacitance of graphene/PANI electrodes reaches 424 F g−1 and retains 96% even at 90% compressive strain. And a volumetric capacitance of 65.5 F cm−3 is achieved, which is much higher than that of other compressible composite electrodes. Furthermore, several compressible supercapacitors can be integrated and connected in series to enhance the overall output voltage, suggesting the potential to meet the practical application.Superelastic graphene aerogel with ultra-high compressibility shows promising potential for compression-tolerant supercapacitor electrode. However, its specific capacitance is too low to meet the practical application. Herein, we deposited polyaniline (PANI) into the superelastic graphene aerogel to improve the capacitance while maintaining the superelasticity. Graphene/PANI aerogel with optimized PANI mass content of 63 wt% shows the improved specific capacitance of 713 F gThe online version of this article (10.1186/s11671-017-2395-z) contains supplementary material, which is available to authorized users. Rapid development of portable and wearable electronic devices not only enriches our daily lives, but also requires matchable energy storage devices, which should have the capability to endure high-level strains –3. Among−1 [−1 [Superelastic graphene aerogels with ordered porous structure  are sti3O4 [2 [2 aerogel as the electrodes of SCs [−1, strain = 50%). It is attributed to that the attachment of MnO2 particles on graphene/CNT scaffold is relative weak, and the mass content of MnO2 and compressive strains must be kept at low-level to avoid the peel-off of MnO2 from the scaffold.To improve the specific capacitance of the graphene aerogel, an effect method is to combine the graphene aerogel with pseudocapacitive materials to form a composite aerogel electrode , 22. For3O4 , MnO2 [23O4 [2 , 25, pol3O4 [2 , and pol3O4 [2 , have bes of SCs . However−1 is obtained and retains 96% even under 90% compression strain, allowing us achieve a high volumetric capacitance of 65.5 F cm−3.Conducting polymer of PANI has been studied extensively as the electrode material due to its high conductivity, electroactivity, and specific pseudocapacitance . And PAN−1, 10 mL) was first mixed with L-ascorbic acid (100 mg) by stirring for 30 min. Then the mixture solution was poured into the glass vials and heated for 30 min at 90 °C for the synthesis of partially reduced graphene hydrogel. The obtained hydrogel was treated by the freeze-thaw process in the refrigerator (− 20 °C) and room temperature. Subsequently, further reduction process for the freeze-recast hydrogel was carried out for 5 h at 90 °C by the initial reductant (L-ascorbic acid) to get completely reduced graphene hydrogel. Finally, the graphene hydrogel was subjected to dialysis in deionized water and dried at 60 °C for 48 h to obtain the superelastic graphene aerogel.Graphene oxide (GO) was prepared by the oxidation of flake graphite according to the modified Hummers’ method , 31. The−1 for 100, 200, 300, and 400 cycles in 1 M H2SO4 and 0.05 M aniline aqueous solution. Subsequent to the electrochemical deposition, the samples were washed with deionized water and then dried at 60 °C for 24 h. The mass contents of PANI in graphene/PANI aerogels were calculated from the mass changes of the aerogels before and after electrochemical deposition. The graphene/PANI aerogels were defined based on the deposition period. For example, the graphene/PANI-2 aerogel was prepared by 200 CV sweeping cycles.The electrochemical deposition of PANI into the superelastic graphene aerogel was carried out by cyclic voltammetry (CV) method using a three-electrode electrochemical workstation (CHI660E), where the superelastic graphene aerogel was used as the working electrode, a platinum electrode as the counter electrode, and a Ag/AgCl electrode as the reference electrode. The deposition process was performed in the potential range from − 0.2 to 0.8 V at a sweep rate of 50 mV s2SO4 gel electrolyte was first prepared through mixing H2SO4, PVA powder, and deionized water according to the mass ratio of 4:5:50. Subsequently, the mixture was stirred for 30 min at 80 °C to form a clear electrolyte. After that, the graphene/PANI aerogels were immersed into the PVA/H2SO4 gel electrolyte for 30 min and were curdled in the air. Then two pieces of the aerogels were placed onto two poly (PET) substrates with Au (~ 100 nm), respectively. One piece of porous separator (Celgard 3501) was also infiltrated with PVA/H2SO4 gel electrolyte. The compressible all-solid-state SCs were obtained by assembling the as-prepared two electrodes sandwiched with the separator under pressure. Finally, the devices were kept at 45 °C for 24 h to remove excess water in the electrolyte.The compressible all-solid-state SCs were assembled to investigate the electrochemical performances of the graphene/PANI electrodes under various compressive strains. The assembly procedure has been mentioned in previous literatures , 32–34. −1.Micro-Raman spectroscopy  was performed using a laser excitation wavelength of 514.5 nm. The microstructure of the graphene/PANI aerogels was observed using a Hatchi S-4800 scanning electron spectroscopy (SEM) equipped with energy dispersive spectroscopy (EDS). The chemical structure of the aerogels was investigated by Fourier transform infrared spectroscopy  and X-ray photoelectron spectroscopy . Compression tests were carried out on an Instron-5566 with a strain rate of 100 mm min2SO4 aqueous electrolyte. The graphene/PANI aerogel, Pt wire, and Ag/AgCl were used as working electrode, counter electrode, and reference electrode, respectively. The specific capacitance (Cs) was calculated from the GCD curves according to the following equation:I is the constant discharge current, ∆t is the discharging time, m is the mass of the working electrode, ∆V is the voltage drop upon discharging.Electrochemical characterizations including CV, galvanostatic charge–discharge (GCD) and electrochemical impedance spectroscopy (EIS) were carried out by the CHI660E electrochemical workstation. Electrochemical measurements of the individual electrode were performed in a three-electrode system with 1 M HCg) and volumetric capacitance (CVol) of the graphene/PANI electrodes in the SCs were calculated from the GCD curves according to the following formulas:I is the constant discharge current, ∆t is the discharging time, m is the total mass of two electrodes, ∆V is the voltage drop upon discharging, ρ is the density of the graphene/PANI aerogel under various compressive strains.The electrochemical measurements of the electrodes under various compressive strains were performed in the all-solid-state SCs in the original state or at certain compressive strains. The gravimetric capacitance (E) and power density (P) of the SCs were calculated from the GCD curves using the following equations.The energy density  at 398.8, 399.3, and 401.1 eV, respectively [+ illustrates a high proton doping level for the deposited PANI on graphene cell walls, leading to the enhancement of electron conductivity and pseudocapacitive performance. Additional file In order to reveal the chemical structure of the graphene/PANI aerogels, the FTIR spectrum of graphene/PANI-2 aerogel is shown in Fig. n of C─H , 40, 41.─H [42− , 38. The─H [42− . The decectively , 43. TheAs mentioned in the previous reports, graphene aerogels with honeycomb-like and oriented cellular structure can present superelasticity , 46. The2SO4 aqueous solution. As shown in Fig. −1 are shown in Fig. −1). This value of specific capacitance of the graphene/PANI-2 aerogel in this work is located at a moderate level among other 3D graphene/PANI composites in the previous reports . The gravimetric capacitance values of graphene/PANI-2 electrodes with/without compression are higher than that of other compressible composite electrodes listed in Table −3 at 90% strain  condition and dynamic (repeated compression/release) condition, there is only slight fluctuation of capacitances (Fig. −1 at a power density of 0.4 kW kg−1. The maximum power density is 2.1 kW kg−1 at an energy density of 6.4 W h kg−1. The obtained energy density and power density are located at a moderate level among other similar all-solid-state symmetric SCs [The EIS of the SCs based on graphene/PANI-2 electrodes was also characterized Fig. . The Nyqces Fig. . For lonces Fig. . The oritric SCs , 34, 51.The output voltage and product current of a single SC based on graphene/PANI-2 electrodes is too low to power the practical electron devices. Thus, we connected several compressible SCs either in parallel or in series to improve the output voltage or product current. As illustrated in Fig. −1 for graphene/PANI-2 aerogel, is obtained by the study in three-electrode system. The compression-tolerant ability of the graphene/PANI electrodes was demonstrated in the all-solid-state SCs. The gravimetric capacitance of the compressible graphene/PANI-2 electrodes reaches 424 F g−1 and retains 96% under 90% compressive strain. Resulting from the invariant of gravimetric capacitance and significant increase of density of the graphene/PANI-2 electrodes under high compression, the volumetric capacitance reaches 85.5 F cm−3 at 90% strain, which is far higher than other compressible composite electrodes. Furthermore, several SCs based on the graphene/PANI electrodes can be integrated and interconnected together on one chip to power the electronic devices. This work paves the way for advanced applications of SCs in the area of compressible energy-storage devices.For acquiring the compressible electrodes with both high compression-tolerant ability and high capacitances, PANI was deposited into superelastic graphene aerogel by electrochemical deposition method. Different contents and uniform distribution of PANI are obtained by controlling the deposition period. Compression tests show that the recoverable compressive strain of graphene/PANI aerogels reaches 90%, indicating that the superelasticity is preserved well after the deposition of PANI. And the optimized PANI content of 63 wt%, corresponding to the specific capacitance of 713 F g


A high-quality programmed cell-death ligand 1 (PD-L1) diagnostic assay may help predict which patients are more likely to respond to anti-programmed cell death-1 (PD-1)/PD-L1 antibody-based cancer therapy. Here we describe a PD-L1 immunohistochemical (IHC) staining protocol developed by Ventana Medical Systems Inc. and key analytical parameters of its use in formalin-fixed, paraffin-embedded (FFPE) samples of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC).An anti-human PD-L1 rabbit monoclonal antibody (SP263) was optimized for use with the VENTANA OptiView DAB IHC Detection Kit on the automated VENTANA BenchMark ULTRA platform. The VENTANA PD-L1 (SP263) Assay was validated for use with FFPE NSCLC and HNSCC tissue samples in a series of studies addressing sensitivity, specificity, robustness, and precision. Samples from a subset of 181 patients from a Phase 1/2 study of durvalumab (NCT01693562) were analyzed to determine the optimal PD-L1 staining cut-off for enriching the probability of responses to treatment. The scoring algorithm was defined using statistical analysis of clinical response data from this clinical trial and PD-L1 staining parameters in HNSCC and NSCLC tissue. Inter-reader agreement was established by three pathologists who evaluated 81 NSCLC and 100 HNSCC samples across the range of PD-L1 expression levels.The VENTANA PD-L1 (SP263) Assay met all pre-defined acceptance criteria. For both cancer types, a cut-off of 25 % of tumor cells with PD-L1 membrane staining of any intensity best discriminated responders from nonresponders. Samples with staining above this value were deemed to have high PD-L1 expression, and those with staining below it were deemed to have low or no PD-L1 expression. Inter-reader agreement on PD-L1 status was 97 and 92 % for NSCLC and HNSCC, respectively.These results highlight the robustness and reproducibility of the VENTANA PD-L1 (SP263) Assay and support its suitability for use in the evaluation of NSCLC and HNSCC FFPE tumor samples using the devised ≥25 % tumor cell staining cut-off in a clinical setting. The clinical utility of the PD-L1 diagnostic assay as a predictive biomarker will be further validated in ongoing durvalumab studies.NCT01693562ClinicalTrials.gov:  This article provides the full results of two abstracts that have previously been presented and published , 2.Patients with recurrent and metastatic non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) have considerable unmet medical needs and require improved treatments with the potential to provide prolonged survival and reduced exposure to toxic chemotherapies –5.It has long been recognized that the immune system is capable of recognizing cancer as foreign and developing a specific immune response. A number of stimulatory and opposing inhibitory proteins have been shown to regulate the quality and magnitude of the antitumor response –9. For ePD-L1  is part of a complex system of receptors and ligands that are involved in regulating T-cell activation. Its main function is to regulate the balance between T-cell activation and tolerance through interaction with its two receptors, PD-1 CD279) and CD80 (B7-1). In normal tissue, PD-L1 has been reported to be expressed on a subset of T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells, and bone marrow-derived mast cells, as well as various non-hematopoietic cells , 17. PD- and CD80PD-L1 biology is further complicated in additional ways. For example, full-length PD-L1 is composed of an extracellular domain that contains the PD-1 binding domain, a shorter intracellular domain, and a short transmembrane region. A number of antibodies to the extracellular and intracellular domains have been generated and several of those appear useful as IHC reagents –24. It iNot surprisingly then, given the various complexities noted, substantial discordance is seen among published studies relying on PD-L1 IHC –24. NoneDurvalumab (MEDI4736) is a selective, high-affinity human IgG1 monoclonal antibody that blocks PD-L1 binding to PD-1 and CD80 but does not bind to programmed cell death ligand-2 (PD-L2) . PD-L2 pMost NSCLC tumors do not express PD-L1 at high levels, although reported levels vary; , 36, 37 The development and application of diagnostic tests in clinical practice to identify patients most likely to benefit from anti-PD-1/PD-L1 therapy could improve patient outcomes and decrease healthcare costs, while directing patients that are less likely to respond towards other alternative treatment options. Research into the relative performance of different PD-L1 assays and their reliability for detecting PD-L1 is ongoing . These dIn this paper, we describe the development and validation of the VENTANA PD-L1 (SP263) Assay (PD-L1 [SP263]) , which has been designed for the detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) NSCLC and HNSCC tumor samples. In addition, we describe the identification and validation of scoring criteria  that can be used to classify samples as PD-L1 high expression or PD-L1 low/no expression in NSCLC and HNSCC tissue.The PD-L1 (SP263) assay uses an anti-human PD-L1 rabbit monoclonal antibody (SP263) directed against the cytoplasmic region of human PD-L1. This peptide was synthesized and conjugated to the carrier protein keyhole limpet hemocyanin. New Zealand White rabbits were immunized with keyhole limpet hemocyanin conjugated peptide emulsified with complete Freund’s adjuvant followed by a series of booster doses of immunogen emulsified with incomplete Freund’s adjuvant. The rabbit that generated IHC-positive polyclonal antibody was then selected for monoclonal antibody development using Spring Bioscience’s proprietary technology.Briefly, antibody-expressing cells were isolated and screened by enzyme-linked immunoabsorbent assay for reactivity to the immunogen and by IHC on control tissue blocks. Complementary DNA coding for antibody heavy chains and light chains was isolated from IHC-positive clones. Monoclonal antibodies were produced by co-transfection of heavy- and light-chain complementary DNA and tested again by IHC. The clone producing antibodies with the best specificity was selected. A larger transfected production lot was produced and the monoclonal antibody was purified through a Protein A column.The antibody was designed to be used in conjunction with the VENTANA OptiView diaminobenzidine tetrahydrochloride (DAB) IHC Detection Kit (P/N 760-700) and the staining protocol for the assay is provided in Table Normal-term placenta can be used as a positive and negative tissue control for the assay. Tissue controls were used to monitor the correct performance of processed tissues, test reagents, and instruments. One placenta control was included on each staining run.The SP263 antibody was tested by immunocytochemistry on the following cell lines: Calu-3, KARPASS 299, H820, H1975, MDA-MB231, T-47D, LOX, ACHN, MCF-7, and HCT-116. In addition, HEK293 cell lines transfected to express varying levels of PD-L1 were prepared to test PD-L1 expression across the dynamic range and were also transfected to express PD-L2 to demonstrate antibody specificity.Tumor cell lines  were evaluated for surface PD-L1 expression and the number of receptors per cell was estimated using flow cytometry. Briefly, tumor cell suspensions were incubated with 100 μl of anti-human PD-L1 antibody  diluted in flow cytometry analysis (FACS) buffer  for 45 min at 4 °C. After primary monoclonal antibody incubation, cells were washed with cold FACS buffer and resuspended in 100 μl QIFI Kit FITC secondary antibody diluted 1:50 with FACS buffer . Secondary detection antibody incubation was conducted for 45 min at 4 °C, protected from light. After secondary incubation, cells were washed once with cold FACS buffer and resuspended in FACS buffer for flow cytometric analysis performed on a BD LSR II Flow Cytometer . Using the setup provided in the QIFI kit, a standard curve was plotted using the mean fluorescent intensity values and calculated using GraphPad Prism 6 software. The x values were determined, which correlated to the number of receptors per cell.Western blot studies were conducted by SDS-PAGE. Cell lysates were prepared from four different cell lines that demonstrated varying IHC protein expression . A recombinant human PD-L1 protein served as a positive control and a recombinant BCL-2 protein served as a negative control for the study. An anti-actin antibody (8H10D10)  was used to detect a ~42 kD protein actin. This constitutively expressed reference protein ensured equivalent loading of samples onto the gel.Normal and tumor tissue array samples  were stained with the PD-L1 (SP263) rabbit monoclonal antibody using the final optimized protocol on the BenchMark ULTRA. A rabbit monoclonal negative-control Ig was also analyzed for the array staining run.All tumor sample evaluations were conducted by board-certified pathologists at Ventana’s College of American Pathologists accredited and Clinical Laboratory Improvement Act certified laboratory. Upon receipt of each sample, hematoxylin and eosin staining was performed to determine the number of tumor cells. The sample was considered acceptable for further analysis if it contained ≥100 viable tumor cells. The PD-L1 (SP263) assay was carried out on 4–6 μm sample sections mounted onto positively charged slides. PD-L1 staining was evaluated by estimating the percentage of cells stained at different intensities, from 0 to 3, with 0 representing no staining and 3 representing strong staining. This percentage was determined for staining localized in the cytoplasm and membrane of tumor cells. In addition, PD-L1 staining in tumor-associated immune cells  was evaluated as a percentage of immune cells present in the tumor area.In NSCLC and HNSCC samples, PD-L1 was generally expressed as a continuum, ranging from a few tumor cells and/or immune cells expressing PD-L1 to the majority of tumor cells and immune cells expressing the target. Intensity of staining also varied, with mixtures of weak, moderate and intensely-stained tumor, and immune cells sometimes seen in single tumor specimens. To understand the association of the biomarker with clinical response, patient samples need to be classified into categories. For these purposes, samples were classified into two categories: high and low/no expression. The optimal scoring criteria were determined by Maximal Chi-square  and receThe endpoints used for the actual cut-off determination were the confirmed objective response (Y) per RECIST v1.1 based on investigator assessment and the percentage tumor cell membrane positivity for PD-L1 (X).This scoring algorithm and the selected cut-off were utilized in the definitions of PD-L1 high and PD-L1 low/negative in the subsequent verification studies.Cut-slide stability was evaluated on four tissue samples stained with the recommended assay protocol after storage under two different conditions (2–8 and 30 °C). Cut-slide stability was also assessed on slides stained at various timepoints after sectioning. Slides sectioned and stained at the Day 0 timepoint served as the comparator for the remainder of the tested timepoints .Tissues were sectioned at various thicknesses (3–7 μm) to assess the effect of thickness on the staining performance of the PD-L1 (SP263) assay. The placenta tissue positive-control slides included in the staining run were sectioned at 4 μm. Slides were required to stain within 1.0 intensity point of each other and to maintain the same PD-L1 status (high or low/no expression).The effects of fixative type and fixation time were evaluated in tonsil-positive control tissue. Tonsil cases were processed in six fixatives , Z5 and Prefer) with multiple timepoints  in each fixative. The effects of ischemia were also investigated using KARPASS 299 xenograph tissues with ischemic times varying between 0, 0.5, 1, 2, 6, and 24 h. The immunostained slides were read and scored by a board-certified pathologist on a 0–3 intensity scale for specific PD-L1 staining and background. Data were then compared to the reference standard (10 % NBF for 24 h).This design verification study was conducted to assess the PD-L1 (SP263) assay’s staining precision in NSCLC and HNSCC. Samples included both PD-L1 high and PD-L1 low/negative tissues across i) three different PD-L1 (SP263) antibody lots; ii) three OptiView DAB IHC Detection Kit lots; and iii) three BenchMark ULTRA instruments. This study was designed to verify the intermediate staining precision  of the PD-L1 (SP263) assay. A total of nine PD-L1 high and nine PD-L1 low/negative samples for each tumor type, spanning the range of PD-L1 expression, were used in the study.A total of 10 NSCLC and 10 HNSCC whole tissue samples (comprising five PD-L1 high and five PD-L1 low/negative samples for each tumor type) spanning the range of PD-L1 staining were evaluated across a cohort, which represented the dynamic range of PD-L1 expression. For intra-day repeatability, five replicate slides from each of the NSCLC or HNSCC specimens were stained on a single BenchMark ULTRA instrument  across two staining runs for NSCLC and HNSCC, respectively.A total of 10 NSCLC and 10 HNSCC samples (comprising five PD-L1 high and five PD-L1 low/negative samples for each tumor type) spanning the range of PD-L1 expression were evaluated across three BenchMark ULTRA instruments. Three staining runs were conducted for NSCLC and HNSCC, respectively.A total of 10 NSCLC and 10 HNSCC whole tissue samples (comprising five PD-L1 high and five PD-L1 low/negative cases for each tumor type) spanning the range of PD-L1 expression were evaluated. Two replicate slides were stained for each sample on three BenchMark ULTRA instruments on five non-consecutive days over a minimum span of 20 days.A total of 14 NSCLC and 14 HNSCC whole tissue samples (comprising seven PD-L1 high and seven PD-L1 low/negative cases for each tumor type) were evaluated. Cases were selected to cover the range of expression and included borderline cases around the cut-off. Each case was stained five different times in each laboratory. Three different laboratories were assessed. Two pathologists per laboratory conducted the scoring.Between-reader (inter-reader) agreement with the PD-L1 (SP263) assay scoring algorithm was determined using a cohort of NSCLC tissue samples (previously screened for PD-L1 status) obtained from commercial sources and stained using the PD-L1 (SP263) assay. A total of 81 NSCLC samples (40 PD-L1 high and 41 PD-L1 low/negative) together with 100 HNSCC samples (50 PD-L1 high and 50 PD-L1 low/negative) were evaluated across the dynamic range of staining. Three readers were trained on the scoring algorithm and provided with the blinded and randomized study samples for scoring. Readers completed the initial scoring within 2 weeks and the scores were analyzed. There was a mandatory 2-week break period prior to the intra-reader precision scoring. Slides were then re-randomized and provided to the readers for intra-reader scoring.The SP263 antibody showed a high degree of sensitivity and specificity for PD-L1 in cell lines with various levels of PD-L1 expression, as determined by flow cytometry, immunocytochemistry  was detected by Western blot in the H820, MDA-MB231, and H1975 cell lines using the PD-L1 (SP263) antibody  assay for the PD-L1 analyte. In cell lines with differential native PD-L1-specific expression, PD-L1 staining intensity with the IHC assay corresponded to the relative levels of PD-L1 expression (LOX > ~MDA-MB231 > ACHN > HCT116 > MCF-7) , and placenta. As tonsil and lymph node are known to contain immune cells expressing PD-L1, staining in these tissues was expected. Trophoblast-lineage cell staining in placenta was also anticipated. No specific PD-L1 staining was observed in the parenchyma of a single normal lung core.The following tumor tissue samples stained using the SP263 assay demonstrated PD-L1 immunostaining of tumor cells: squamous cell carcinoma of the skin, infiltrating ductal carcinoma of the breast, cholangiocarcinoma of the liver, Hodgkin lymphoma, squamous cell carcinoma of the lung, gastric adenocarcinoma, diffuse large B-cell lymphoma (in stomach), squamous cell carcinoma of the uterine cervix, endometrial adenocarcinoma, and thyroid papillary carcinoma. Most specimens also demonstrated immunostaining of tumor-infiltrating immune cells. The degree of apparent background staining was reported as ≤0.5 (faint) intensity on all evaluated cores, a level that generally did not interfere with specific PD-L1 labeling and therefore was judged to be acceptable. Additional commercial samples of NSCLC and HNSCC tissue showed similar PD-L1 labeling in a number of tumors. Figure p-value = 0.0003, Fig. Clinical response to durvalumab and PD-L1 expression in tumor biopsies correlated best with PD-L1 expression in tumor cells rather than immune cells. In a subset of 155 patients with NSCLC in the Phase 1/2 trial, the optimal cut-off identified statistically Fig.  was 38 %The staining performance on NSCLC and HNSCC tissues stored at 2–8 and 30 °C passed the acceptance criteria. This test also demonstrated that staining at the Day 3 and 14, and Months 1–13 timepoints was consistent with results obtained on Day 0 for NSCLC tissues and staining at the Day 3 and 14, and Months 1–10 timepoints was consistent with results obtained on Day 0 for HNSCC tissues.Appropriate antibody staining was achieved across all tissue section thicknesses tested  (Table The intra-day reproducibility of the PD-L1 (SP263) assay within a single staining run exceeded the 90 % pass criterion set for this study; overall percentage agreement (OPA) for PD-L1 high vs. low/no expression was 100.0 % with a 95 % confidence interval (CI) of 92.9–100.0. The positive percentage agreement was 100.0 % (95 % CI: 88.6–100.0) and the negative percentage agreement was 100.0 % (95 % CI: 83.9–100.0). The assay also exceeded the 90 % pass criteria set for intra-platform precision (performance across three BenchMark ULTRA instruments) and inter-day precision (performance on the same BenchMark ULTRA instrument over 5 non-consecutive days for a minimum of 20 days) Table . All theThe intermediate precision design verification study was conducted to assess PD-L1 (SP263) assay staining precision on NSCLC tissues representing the clinical status range. The antibody exceeded the 90 % pass criteria set across the three antibody lots, three detection kit lots and three BenchMark ULTRA platforms Table . All theFor NSCLC, the OPA between the three readers was 96.7 %. The average positive agreement (APA) and average negative agreement (ANA) between the three readers was 96.6 and 96.8 %, respectively Table . The intFor NSCLC, the average positive and average negative inter-laboratory reproducibility was 93.3 % and 79.5 %, respectively. The OPA was 86.4 %. For HNSCC, the average positive and average negative inter-laboratory reproducibility was 89.4 and 99.5 %, respectively. The OPA was 94.5 %  assay showed the required level of analytical specificity and sensitivity for detecting engineered and endogenous PD-L1 protein in cell lines across a wide dynamic range of expression. Additionally, it was shown to detect PD-L1 expression levels as low as ~1,600 receptors per cell. The rabbit monoclonal antibody used in the assay was raised against the cytoplasmic region of human PD-L1 and produced a single clear band of the appropriate molecular weight in Western blot analysis. Target specificity was also demonstrated by transfecting PD-L1 into cell lines with no endogenous PD-L1 expression, producing clear staining with the PD-L1 (SP263) assay, and no staining in cells transfected with PD-L2. Antibodies raised against the cytoplasmic domain of PD-L1 have been reported to offer better visualization of membrane PD-L1 compared with those raised against the extracellular domain .The assay detected PD-L1 immunoreactivity with the anticipated staining patterns in a panel of normal tissue samples with known PD-L1 expression  and in tumor cells from NSCLC and HNSCC tissue samples with a range of PD-L1 staining intensities and frequencies representing the heterogeneity of tissue expression. No unexpected cross-reactivity was observed with the antibody.Numerous analytical variables have the potential to affect the reliability of assay results and the impact of these variables was extensively evaluated during assay development. In terms of preanalytical variables, the antibody demonstrated appropriate staining across all tissue section thicknesses tested and also accurately represented the PD-L1 high or PD-L1 low/negative staining status in each case. Changes in the recommended tissue fixative used  over time periods ranging from 6 to 72 h and cold tissue ischemia for periods ranging from 0 to 24 h had a negligible effect on staining performance, as did cut-slide storage conditions of 2 − 8 and 30 °C for varying time periods of up to 12 months.For assay performance over repeated measurements, the assay exceeded the 90 % pass criterion for intra-day reproducibility within a single staining run, inter-day precision (performance on the same BenchMark ULTRA instrument over 5 non-consecutive days for a minimum of 20 days) and intra-platform precision (performance across three BenchMark ULTRA instruments). The assay also exceeded the 90 % pass criteria for determination of intermediate precision across three antibody lots, three detection kit lots, and three BenchMark ULTRA platforms. Assay performance test scores  were in the same range as those reported for the FDA-approved Dako PD-L1 IHC 28-8 pharmDx and PD-L1 IHC 22C3 pharmDx assays , 15. IntA scoring algorithm for PD-L1 staining with the PD-L1 (SP263) assay was defined on the basis of an analysis of PD-L1 staining parameters observed from NSCLC and HNSCC samples from the NCT01693562 clinical trial of durvalumab, together with clinical response data. Statistical analysis of PD-L1 staining parameters in this study indicated that the parameter that correlated best with clinical response to durvalumab was PD-L1 expression in the membrane of tumor cells, regardless of staining intensity. Other PD-L1 parameters, however, remain under investigation. Samples were considered to have high expression when ≥25 % of viable tumor cells demonstrated membrane staining for PD-L1 at any intensity [Although several different assays to determine PD-L1 positivity in tumor tissue have been developed, few have been performance tested using comparable assay conditions. There may be differences in assay performance owing to the diversity of antibody clones with different affinities, raised against different areas (epitopes) on the PD-L1 molecule . Furthermore, a number of associated IHC protocols have been developed, relying on differing antigen retrieval conditions and staining platforms in particular. These differences are likely to generate different staining patterns . Tissue The heterogeneity of PD-L1 expression also poses a challenge for pathologists , 29. UnlThis analysis demonstrated the reliability of the PD-L1 (SP263) assay to determine the PD-L1 expression of NSCLC and HNSCC tumor samples obtained in a clinical setting based on a ≥25 % cut-off value for positively stained tumor cells. Preliminary data from retrospective analyses of tumor samples obtained from a clinical trial of the anti-PD-L1 agent durvalumab, demonstrated that PD-L1 high expression, identified by the PD-L1 (SP263) assay and scoring algorithm, was a useful predictor of treatment response [The VENTANA PD-L1 (SP263) Assay met all of the predefined acceptance criteria , showing analytical specificity, sensitivity, robustness and precision, and obtaining the required performance scores for day-to-day, site-to site and observer-to-observer repeatability and reproducibility, confirming its reliability for staining of FFPE NSCLC and HNSCC samples across different testing environments.The PD-L1 (SP263) assay is currently in use in ongoing clinical trials of durvalumab, as monotherapy and in combination with tremelimumab, as part of a comprehensive clinical development program in NSCLC and HNSCC. These studies support the use of the PD-L1 (SP263) assay for testing on clinical samples and form the basis for confirming clinical utility.


Whales were subject to one no-sonar control session and either two successive ramp-up sessions  or a ramp-up session (RampUp1) and a full-power session (FullPower). Full-power sessions were conducted only twice; for other whales we used acoustic modelling that assumed transmission of the full-power sequence during their no-sonar control. Averaged over all whales, risk indicators in RampUp1 (n=11) differed significantly from those in FullPower (n=12) by −3.0 dB (SPLmax), −2.0 dB (SELcum) and +168 m (Rmin), but not significantly from those in RampUp2 (n=9). Only five whales in RampUp1, four whales in RampUp2 and none in FullPower or control sessions avoided the sound source. For RampUp1, we found statistically significant differences in risk indicators between whales that avoided the sonar and whales that did not: −4.7 dB (SPLmax), −3.4 dB (SELcum) and +291 m (Rmin). In contrast, for RampUp2, these differences were smaller and not significant. This study suggests that sonar ramp-up has a positive but limited mitigative effect for humpback whales overall, but that ramp-up can reduce the risk of harm more effectively in situations when animals are more responsive and likely to avoid the sonar, e.g. owing to novelty of the stimulus, when they are in the path of an approaching sonar ship.Exposure to underwater sound can cause permanent hearing loss and other physiological effects in marine animals. To reduce this risk, naval sonars are sometimes gradually increased in intensity at the start of transmission (‘ramp-up’). Here, we conducted experiments in which tagged humpback whales were approached with a ship to test whether a sonar operation preceded by ramp-up reduced three risk indicators – maximum sound pressure level (SPL Highlighted Article: Variation in behavioural responses of humpback whales limits the effectiveness of a ramp-up procedure used to mitigate physiological effects of naval sonar on marine animals. Noise-induced hearing loss may affect individual survival, as acoustic communication in marine animals facilitates vital behaviours such as feeding and resting, and decisions about habitat selection via social information . GuideliTesting the effectiveness of ramp-up and other noise mitigation measures has been highlighted as a high priority for research in the scientific, conservation and regulatory communities , indicatList of symbols and abbreviationsagamma shapebgamma scaleEsingle-pulse sound exposureGEEgeneralized estimating equationPLpropagation lossPTSpermanent hearing threshold shiftrPearson's correlation coefficientRminminimum source–whale rangeSELsingle-pulse sound exposure levelcumSELcumulative sound exposure level of the sessionSLsource level based on mean-square sound pressureESLenergy source levelSPLsound pressure levelmaxSPLmaximum sound pressure level of the sessionTeffective pulse durationt0reference time=1 sTTStemporary hearing threshold shiftZtest statistic for Wald test and Barnard's unconditional testκconcentrationµmeanσstandard deviationUsing theoretical modelling, Megaptera novaeangliae Borowski 1781). After being exploited during the whaling era, most humpback whale populations have been recovering relatively quickly compared with several other baleen whale sub-populations, many of which remain endangered or critically endangered  active sonar systems for longer-range detections of submarines . These tmax), cumulative sound exposure level (SELcum) and minimum source–whale range (Rmin). In addition, effectiveness of ramp-up was assessed by identifying avoidance responses to the sound source and determining their influence on the risk indicators. We tested the specific scenario in which a whale was located directly in front of an approaching source ship and the sonar operation commenced relatively close to the whale, as that scenario likely poses the greatest risk of harm to whales in their natural environment.Effectiveness of ramp-up of 1.3–2.0 kHz sonar was assessed in terms of whether risk of harm was less in whales presented with a full-power sequence preceded by a ramp-up sequence, i.e. the RampUp session, compared with whales presented with a full-power sequence preceded by a period without sonar, i.e. the FullPower session. In addition, the potential for short-term habituation or sensitization to the sonar exposure was investigated by presenting whales with two identical RampUp sessions. We analysed three risk indicators: maximum sound pressure level  or 8 h (2011) of baseline data were collected per whale. Each tagged whale was subject to three experimental sessions: first a no-sonar control, then two sonar sessions. Sessions had a 10-min duration each and were separated by 1 h or more. The no-sonar control was always conducted first to test how whales responded to the ship alone, before they heard sonar transmitted from the vessel. During no-sonar control the ship approached the tagged whale in the same way as during sonar sessions, but without transmitting sonar.−1 (8 knots) and on a predetermined straight intercept course during no-sonar control and sonar sessions , developed for the Norwegian Navy, and RU-tool (custom-built in MATLAB by P.J.W.). The surface positions of the whale, relayed via VHF radio by the observers on the tracking boat, and the GPS positions of the source ship were fed into the tools, which then calculated the time and position of the intercept and the best sailing path. The two tools used somewhat different logic: MARIA primarily used the automatic identification system signal from the tracking boat, which was usually around 100 m from the whale; and RU-tool predicted the future movements of the whale from the last sightings by applying different rules based on the whale's general movement pattern. The final course correction and start time of the session were determined only minutes before the start. Ultimate decisions on course changes and start of transmission were always made by the experiment coordinator based on all available information.The source ship approached the whale at a speed of 4.1 m sThe two sonar sessions were either two identical ramp-up sessions (‘RampUp1’ and ‘RampUp2’) or a ramp-up session (‘RampUp1’) and a full-power session (‘FullPower’) without ramp-up . Ramp-upThe research vessel towed the sonar source  at 50 m depth using 250–300 m of cable. The source transmitted a 1.3–2.0 kHz upsweep pulse at 20 s interval. The single-pulse source level  was gradually increased from 152 to 214 dB re. 1 µPa m in the first 5 min and then kept at 214 dB re. 1 µPa m during ramp-up sessions . Full-po−1) and sonar source used , in compliance with the ethical use of animals in experimentation. The research protocol was approved by the University of St Andrews Animal Welfare and Ethics Committee and WHOI's Institutional Animal Care and Use Committee.t=5 min, in combination with the full-power transmission sequence, with first the sonar transmission at t=5 min . In this simulation, avoidance behavior was modelled by randomly drawing samples from empirical distributions  derived from all observations collected during experimental sessions. The simulation showed that there was a negligible chance that an avoidance response would substantially reduce SPLmax (Appendix A).Full-power sessions were only conducted twice  and always as the third session . Records t=5 min , was detUse of full-power outcomes in this way was considered the most appropriate approach from an animal-welfare perspective because it (1) reduced the number of times the source suddenly was switched on at maximum level at relatively close range from the tagged animal and (2) allowed us to obtain information about habituation and sensitization by exposing animals to two identical subsequent ramp-up sessions instead of to one ramp-up session and one full-power session. The two experimental full-power sessions were conducted to provide information about potential effects of the sudden nearby onset of sonar on the magnitude of the behavioural response .max), cumulative sound exposure level (SELcum) and minimum source–whale range (Rmin) – which we could estimate from our at-sea treatments. Single-pulse sound pressure level (SPL) and sound exposure level (SEL) for sonar transmissions received on the DTAG were measured following the procedures in https://www.iso.org/standard/62406.html). SELcum is the logarithm of the sum of the single-pulse sound exposures over all pulses in the session, and SPLmax is the maximum SPL of all pulses in the session. We combined SL with modelled acoustic propagation loss (PL) using ray-trace software . To avoid misinterpretation of normal and coincidental changes in behaviour, the turn angle of each potential avoidance was compared with the empirical cumulative distribution of turn angles for all clockwise or counter-clockwise turns during 4 h of baseline data preceding the session. This baseline data set included turns during the period before the no-sonar control and, for sonar sessions, periods between experimental sessions. The duration of 4 h was chosen to reduce differences in the amount of data available per whale and variation in turn angles owing to whales switching behavioural state. Only turns in one direction were used to reduce autocorrelation in the baseline data set. Turns with a P-value of less than a Bonferroni-corrected significance level (0.05 divided by the number of tests on the experiment) were classified as avoidance.For each session, a quantitative analysis to identify avoidance responses in the whale's horizontal track was conducted. This analysis aimed to replicate the definition of avoidance used by We also assigned presence/absence of feeding just before or during the experimental session based upon the detection of lunges D–F, i.e.We compared the presence/absence of avoidance between no-sonar control sessions and ramp-up sessions (RampUp1 and RampUp2 combined) using a two-sided Barnard's unconditional test of superiority on a contingency table with avoidance  and a null hypothesis on no effect of ramp-up session, relative to no-sonar control.max, SELcum and Rmin) changed across the three types of sonar session and across whales that avoided the source versus whales that did not. We used generalized estimating equations (GEEs), which are an extension of generalized linear models that accounts for correlated and clustered data by appropriately inflating the standard errors . All tagged whales were used in the analysis, resembling the real-world situation where animals encountering a sonar ship may or may not exhibit avoidance. We also fitted models with avoidance, ramp-up session  and their interaction as explanatory variables to examine the effects of avoidance and presentation order. Only ramp-up sessions were used in this analysis because avoidance responses were not observed in full-power sessions.All explanatory variables were factor covariates. We fitted models with session  as the explanatory variable to examine the effects of transmission scheme and presentation order. As described above, we pooled 10 full-power outcomes that were based on no-sonar control sessions with two observed full-power sessions to create the FullPower dataset. This pooling assumed that the behaviour of the two whales during full-power sessions was unaffected by the previous two sessions. This assumption appears to be reasonable based on the movement tracks of the two exposed animals  animal. The jackknife variance estimator was applied because the sandwich variance estimator can be biased for small sample sizes . An indeFor inference purposes, we used prediction plots generated from the GEE models. The 95% confidence intervals (CIs) for the predictions were calculated using a parametric bootstrap on the GEE covariance matrix. The 2.5th and 97.5th percentiles were calculated from 5000 bootstrap iterations. We used two-sided Wald tests to assess differences between factor levels in models with session. For models with avoidance and ramp-up session, we used the output from the 5000 bootstraps to make comparisons between specific factor level combinations of interest, as the interaction term precluded the use of the standard Walt tests. We calculated the differences between predictions for different factor levels across all bootstraps as well as a 95% CI for these differences. We concluded that there was a significant increase or decrease in the risk indicator in cases where the upper and lower confidence bounds for the differences were exclusively positive or negative.Table S1.All statistical analyses were performed in R version 3.0.2 using R packages geepack , MuMIn  and BarnWe successfully tagged 13 humpback whales, five in 2011 and eight in 2012 . One whamax=173 dB re. 1 µPa (s.d. 4 dB) and SELcum=177 dB re. 1 µPa2 s (s.d. 3 dB) over all ramp-up and full-power sessions. The source passed at relatively close range to the whale: mean Rmin was 299 m (s.d. 209 m) over all sessions. The experimental design, with the whale in front of an approaching source, resulted in strong correlations among the three risk indicators: SPLmax and SELcum , SPLmax and Rmin , and SELcum and Rmin .Whales were exposed to mean SPLFig. S2). The presence of avoidance thus differed between stimulus presentations , suggesting that responses were caused by the sonar exposure and not the approaching ship. Between-whale and within-whale variations in the presence of avoidance were large; avoidance occurred during five of 10 RampUp1 sessions  and three of eight RampUp2 sessions . Only one whale (mn12_180b) avoided the source during both RampUp1 and RampUp2 .Avoidance responses were identified in eight ramp-up sessions  but never in the 11 no-sonar control sessions or two full-power sessions , −2.0 dB for SELcum  and +168 m for Rmin  as explanatory variable. FullPower consisted of observed sessions  and modelled outcomes . The average differences in risk indicators between RampUp1 and FullPower were small but statistically significant ; Table 2max, −3.4 dB for SELcum and +291 m for Rmin (max and +4.1 dB for SELcum) and their minimum distance to the source averaged 209 m lower , we also compared whales that avoided the sound source with whales that did not avoid. For RampUp1, we found statistically significant differences between these two categories: −4.7 dB for SPLfor Rmin , Table 3for Rmin , Table 3 m lower , Table 3max=167 dB re. 1 µPa (s.d. 5 dB), which was ∼6 dB lower than other whales that avoided . Similar trends were observed for SELcum and Rmin: whales that initiated avoidance from a non-feeding state in RampUp1 generally had a lower SELcum and a greater Rmin than other whales that avoided.There were indications that a combination of behavioural state  and order of presentation influenced avoidance behaviour. The three whales in RampUp1 that initiated avoidance from a non-feeding state received a mean SPLmax and SELcum and increase in minimum distance to the whale, of exposure to humpback whales, but only in the first sonar exposure session (RampUp1). The average reductions in risk for RampUp1 compared with FullPower were statistically significant, but relatively small  have been reported in parallel studies of the same experiments , 2016. TThe only tagged whale that avoided during both sonar sessions was a mother (mn12_180b) with a small calf. Whale mn12_180b responded during both ramp-up sessions with an unusual three-dimensional avoidance response, which included a descent to >100 m depth E,F. The cum was 183 dB re. 1 µPa2 s . Direct measurements of temporary (TTS) or permanent hearing threshold shift (PTS) for humpback whales, or any other baleen whale, do not currently exist. The National Marine Fisheries Service has recently published updated risk thresholds, including an estimate of TTS onset (defined as a TTS of 6 dB) for low-frequency hearing specialist cetaceans, which includes humpback whales, at frequency-weighted SELcum=179 dB re. 1 µPa2 s  can be directly compared with the risk thresholds of cum is still far (16 dB) below the PTS risk threshold of 199 dB re. 1 µPa2 s  and the receiver (∼1.3 m s−1). All simulated whales avoided instantaneously and in a direction perpendicular to the source track . Simulations were informed by parameters derived from avoidance responses during ramp-up sessions, and it is possible that the full-power protocol would, on average, induce stronger responses that would reduce risk of hearing loss more effectively. However, no avoidance responses were observed in the experimental two full-power sessions conducted, but both these whales were previously exposed.One reason for this result was likely the large difference in speed between the source  was implemented. The range of beam take-off angles in the vertical plane was ±89 deg. The number of traced Gaussian beams was 3200 . The modelled source was horizontally omnidirectional and was placed at the mean tow depth of the real source calculated over all pulses in the experimental session.E) was derived from the SL based on mean-square sound pressure via SLE=SL+10log10(T/t0) dB, where t0 is 1 s. Because of a gradual onset and offset in the waveform, the effective duration T of a transmitted sonar pulse was 0.47 s and 0.93 s for the time intervals 0≤t<5 min and 5≤t≤10 min, respectively. The received single-pulse SEL and single-pulse SPL were derived from the modelled PL as SEL=SLE−PL and SPL=SL−PL, respectively. SELcum of the experimental session was then calculated by taking 10 times the 10-base log of the sum of the single-pulse sound exposures E received in the session, where E=10SEL/10 dB µPa2 s. SPLmax was taken as the maximum over all SPL in the session.The energy source level (SLmax and SELcum). For whale depth we used a normal distribution centered at the depth measured by the DTAG at the time of the sonar transmission and s.d.=1 m reflecting the precision of the pressure sensor . This mean difference based on all sessions measured was added to the modelled received level, reflecting our higher confidence in the accuracy of the measurements. This correction only affected absolute values and did not affect the main results of this study, as they are based on differences in received level. Potential reasons for the discrepancy between the modelled and measured levels included the simplified assumptions of the acoustic modelling, limited environmental information and the difference in root mean square averaging time for the SPL calculations. In addition, the total SLE calculated over all pulses in the ramp-up session was 0.43 dB higher than the total SLE calculated over all pulses in the full-power session (cum for ramp-up sessions so that any potential statistical differences between RampUp and FullPower in this metric were caused by differences in behavior and not by differences in transmission protocols.For all humpback whales exposed to sonar,  session . Therefo