Wildlife has been traditionally used by forest communities as a source of protein, and the Peruvian Amazon is no exception. The articulation of colonist and indigenous communities to urban centers and markets results in changes in livelihood strategies and impacts on wildlife populations. To address the threat of overhunting and forest conversion, we provide a generalized characterization of colonist and indigenous communities and their hunting activities near Pucallpa, Ucayali, Peru.H).A semi-structured household survey was conducted to characterize hunters and describe their prey collections. The data were analyzed by conducting a Kruskal-Wallis test, a multiple regression analysis, and by estimating the harvest rate (Cuniculus paca) were the most frequently hunted prey, followed by red brocket deer (Mazama americana) and primates. While hunting intensity was not significantly different between indigenous and colonist communities, hunting rate disparities suggest there are different types of hunters  and that prey composition differs between communities.Less wealthy households were more actively engaged in hunting for food security and as a livelihood strategy. Additionally, older hunters were associated with higher hunting rates. Although the percentage of hunters was relatively low, estimated hunting rates suggest overharvesting of wildlife. Lowland pacas  contains supplementary material, which is available to authorized users. Subsistence hunting has traditionally been an essential livelihood strategy for food security and nutritional health in Amazonian communities. However, economic development, population growth, and the construction of roads in the Amazon have transformed the landscape and the livelihoods of native inhabitants and incoming settlers , 41. TheAmazon deforestation figures in Peru indicate that almost eight million hectares of primary forest have been converted to agriculture and other land uses. This represents approximately 10% of the original forest cover . SeekingLocal extinctions of wildlife species have been reported due to overharvesting , 10. TheA better understanding of hunter characteristics and hunting activity within a given socioeconomic and cultural context is critical to address the threat of overharvesting, to support knowledge-based decision making, and to formulate responsible wildlife management policies. At the forest-agriculture interface near Pucallpa in the Amazon Department of Ucayali in Peru, we hypothesize that indigenous communities remain more heavily dependent on hunting activities as a livelihood strategy compared to colonist communities. Furthermore, we hypothesize that hunting activities among indigenous communities are more sustainable than those of colonist communities. Motivated by current knowledge gaps regarding hunting activity in Ucayali, we aimed to (1) compare hunting intensity and (2) characterize hunters in colonist and indigenous communities near the city of Pucallpa in the Department of Ucayali in the Central Peruvian Amazon and (3) describe hunting activity and its potential implications on prey viability. While most ethnozoological studies on hunting in the Peruvian Amazon have been carried out in heavily forested or protected areas , 26, 38,This study is part of a larger research project called “Attaining Sustainable Services from Ecosystems through Trade-Off Scenarios,” or ASSETS , which a−1. The rainy season usually takes place between the months of February and May [As part of a larger and multicountry research project, in Peru, ASSETS was carried out in communities located in the lowland Amazon rainforests in the provinces of Coronel Portillo and Padre Abad within the Department of Ucayali. The region has a typical tropical climate, with monthly temperatures ranging between 20 and 36 °C and precipitation between 1535 and 2100 mm year and May .The Department of Ucayali has an estimated urban population of 40%, with approximately 210,000 people living in the capital city Pucallpa . The remThe communities included in this study Fig. ; Table 1Among the ASSETS project data collection activities, a comprehensive household survey questionnaire composed of 30 modules was developed by an interdisciplinary team of scientists, which was led by the University of Southampton. The survey instrument included multiple choice and open-ended questions  regarding household characteristics, use of natural resources, livelihood strategies, and dietary habits . From thα = 0.05). In addition, a multiple regression analysis (backwards elimination method) was conducted in SPSS version 20 to determine the characteristics which collectively best reflect hunters in the Ucayali region. The estimated total number of animals hunted per month was set as the response variable, and seven objective and subjective hunter characteristics were used as explanatory variables: age, educational level, ethnicity, wealth , number of heads of cattle owned, frequency of fishing, and personal perception of household food security. The variable for wealth was used instead of income as a long-term economic indicator and as a way to estimate the level of comfort in a less biased manner given that income wages may be variable among indigenous communities. Yet, the variable was estimated on a per capita basis for each household. Gender was not considered in the analysis as 95% of the hunters surveyed were male. Categorical explanatory variables were converted to continuous data, and all were ln-transformed to improve the linear fit to the response variable and to ensure normality of the residuals. Given that the number of hunters in the sample was relatively low, the regression model was run to determine predictor variable significance and the type of relation (negative or positive) with the response variable. Standard statistical test procedures (not shown) were carried out to assess the performance of the regression model.Data analysis focused on (1) the relation between community ethnicity and hunting intensity , (2) hunter characterization, and (3) hunting activity data inferences . To address our first hypothesis, the monthly average number of prey animals reported by hunters was compared based on community type  using a Kruskal-Wallis test conducted in R version 3.2.3 (2008). Significant differences in the average number of animals hunted were determined at the 95% confidence interval (H) concept proposed by Robinson and Redford [2). The estimated values for commonly harvested species/taxa were compared to the sustainable harvest values modeled by Robinson and Redford [To address our second hypothesis, hunting activity among communities and community types was explored by comparing the types of animals harvested, their relative reproductive strategies, and the potential impacts of hunting in the region. From the interview data, it was not possible to estimate the maximum sustainable yield of commonly hunted species or groups. Instead, we used the harvest rate ( Redford . Harvest Redford  for the n = 42) of these households were involved in hunting, who collected at least one prey item per month. The number of hunters per community was as follows: Caco Macaya 10, Puerto Belén 7, La Unión 7, Naranjal 6, Cunchuri 3, Junín Pablo 3, Monte de los Olivos 3, Pueblo Libre 2, and Yerbas Buenas 1 (Table p = 0.675). Most indigenous and colonist hunters collected small quantities of animals . At the same time, both indigenous and colonist communities include individuals who hunted much higher numbers of animals . In the indigenous community of Caco Macaya for instance, most hunters collected between one and five animals per month. Yet, there were two hunters who estimated harvesting between 18 and 30 animals per month each.A total of 230 heads of household were interviewed in the nine communities. Only 18% (R2 = 0.288). Wealth, or the number of durable items owned by indigenous and colonist hunters, ranged from 1 to 12 , and their age varied from 16 to 70 years . By examining the demographic information collected from the household survey , and a positive relation between age and hunting success (regression coefficient β = 0.374). These results suggest that hunting activities decreased with wealth and increased with age.According to the multiple regression analysis, only the variables for wealth and age were significant in predicting the number of preyed animals per month, explaining 29% of the variation (adjusted Cuniculus paca) and the armadillo (Dasypus sp.) were the most commonly hunted species across all communities. In terms of overall numbers, the lowland paca was also the most harvested game animal, followed by spider monkeys (Ateles sp.), armadillos, and brocket deer (Mazama Americana). During the rainy season when the data were collected, mammals were the main group hunted in the region. These were followed by birds, while the collection of reptiles was less common. Harvested animals were classified according to their general reproductive productivity (K or r selection strategies). Species vulnerable to overhunting are typically K-selected, which are those characterized by relatively low reproductive cycles and large body mass [Table ody mass . In indiH) would be 4.42 animals/km2/year. By the same standards, the harvest rate for spider monkeys, armadillos, and red brocket deer would be 1.26, 1.09, and 1.03 animals/km2/year, respectively. At the community type level and within their respective territories, if we compare the estimated harvest rates for each species, we find that indigenous communities reported higher takeoff values  may be displacing hunting as a primary food procurement activity, which agrees with findings elsewhere . While While most hunters reported to consume the harvested prey, the disparity in hunting intensity among hunters suggests there are two types of engagement in this activity by both indigenous and colonist communities: a more common occasional and opportunistic subsistence hunting, and what could be perceived as commercial hunting. The low number of animals harvested by most hunters suggests a sporadic and opportunistic approach to hunting. For these hunters, prey items may be occasionally harvested while carrying out other activities such as tending to agricultural plots, fishing, and/or collecting other forest products. In contrast, a smaller group of frequent or specialized hunters tends to harvest larger quantities of game. Similar findings were reported by van Vliet et al. , who claWhile the relation between household socioeconomics and hunting is not yet fully understood , 24, 60,Age was also significant in characterizing hunters, showing that hunters tend to be older rather than young. The relationship between age and hunting success has been previously examined by Walker et al. . Their rAnimals with both low and high reproductive rates were similarly hunted in indigenous and riverine colonist communities, while mainly high reproductive species/taxa were hunted by land-based communities. This reflects a lower availability of K-selected species in areas that have undergone greater land cover changes. Among land-based colonist communities, La Unión had the highest hunting rates. The higher hunting activity reported for this community may be related to a greater presence of forest cover in the village surroundings compared to the other land-based communities surveyed  and white-lipped peccary (Tayassu pecari) have relatively medium to fast reproductive characteristics, making them potentially abundant in the landscape and thus readily harvested. Yet, they are known to be susceptible to anthropogenic pressures such as human-induced forest cover change, road density, and the expansion of settlements [According to Bodmer , riverintlements . Populattlements . Forest/tlements . Hence, 2/year) than that reported at the forest/agriculture interface in Ucayali. Given that the actual hunting territories utilized by the communities and hunters were difficult to estimate due to the varying degree of forest cover and fragmentation , in turd Thoisy , we could Thoisy , sustainIn further examining harvest rates by community type for frequently hunted species, we can observe that the values reported by indigenous communities are higher compared to riverine or land-based colonist communities. While challenging our second hypothesis, indigenous communities collected a greater variety of species, which reduces the overall hunting pressure exerted on any single population. Yet, according to our results, indigenous communities seem to harvest unsustainable numbers of individuals, in particular slow-reproducing species Table . While tLivelihoods strategies of colonist and indigenous communities near Pucallpa in Ucayali depend directly and indirectly on the services provided by natural and anthropogenic ecosystems. At the forest/agricultural interface, these services include wildlife as a protein food source and/or as a trading commodity. Given that indigenous communities have traditionally hunted for subsistence purposes, significant differences in hunting intensity between indigenous and colonist communities were expected. Yet, changes in land cover due to logging, agriculture, and the commercialization of non-timber forest products may be shifting livelihood strategies and displacing hunting as a primary food procurement activity among indigenous communities. Hunting as a frequent livelihood strategy was practiced by a few individuals , while hunting as a complementary (opportunistic) livelihood strategy was more common. Furthermore, higher hunting activity was associated with less wealthy households and with older rather than young individuals across the studied communities.Irrespective of the lack of difference in hunting intensity between indigenous and colonist communities, the composition of game species varied between communities. Villages with medium to high forest cover, which included indigenous and riverine colonist communities, reported greater game diversity, as well as a greater number of game species with slow reproductive strategies. Estimations on hunting rates indicate that the lowland paca is susceptible to local extinction, despite of its relatively rapid reproductive strategy. Other commonly hunted species such as primates may also be vulnerable to overharvesting given their slower population turnover reproductive characteristics.As the agriculture frontier expands into forested areas in response to the continuous influx of migrants, population growth, and extractive economies, food procurement and the livelihood strategies of indigenous and colonist communities are expected to adapt. Hunting activities for subsistence purposes may not be attractive to younger generations, nor feasible as the distribution and abundance of game populations declines. As the potential demand for wildlife increases due to the development of market economies in the region, further research on hunter-prey dynamics is necessary to target effective conservation strategies aimed at protecting healthy faunal populations, conserving valuable cultural practices and traditional resource-use knowledge, along with developing inclusive and sustainable livelihoods for future generations.Additional file 1:Appendix S1. Selection of questions extracted from household survey to be used in regression analysis. (DOCX 60 kb)Additional file 2:Appendix S2. ASSETS study Peru village characterization. (DOCX 81 kb)


Communities juxtaposed to protected areas (PAs) often disproportionally accrue the costs of conservation, but they can also receive benefits from the existence of a PA. The extent to which local communities benefit or incur costs as a result of residing next to PAs is of interest to conservationists and policy-makers. This study sought to understand the costs, benefits, and attitudes of local people living adjacent to Save Valley Conservancy (SVC), Zimbabwe. The purpose was to determine whether benefit and loss accrual has a bearing on the levels of illicit wildlife-based activities experienced in the SVC. Data were collected through a household questionnaire survey and key informant interviews from April to July 2014. A three-stage sampling was adopted: firstly, purposive sampling was employed to select wards adjacent to the SVC; secondly, random sampling was used to select villages within the selected wards; and thirdly, systematic sampling was used to select 71 household questionnaire respondents. Snowball sampling was used to select 9 key informants. The study results show that the majority of locals living close to SVC are not deriving discernable benefits and the costs of conservation are escalating influencing negative attitudes towards wildlife conservation, thus causing them to view wildlife as a nuisance. Overall, our results indicate that conservation losses and benefit accrual by local communities influence their attitudes toward SVC and conservation in general. We conclude that costs incurred outweighed the benefits accrued, a situation that triggers a more negative form of reciprocity towards SVC and wildlife conservation. It is recommended that a more socially and economically inclusive management approach based on a stakeholder-driven access and benefit sharing (ABS) framework be instituted to promote a more positive form of reciprocity towards SVC and nature conservation. The advent of colonialism in the 18th and 19th centuries in Africa saw the twin processes of land and wildlife alienation creating hostility to wildlife among the affected local people. Colonialism was the entry point through which the fortress conservation doctrine slithered its way into Africa . This moIn recent years, after a period of strictly centralized wildlife management and exclusive wildlife conservation, there has been a commendable attempt to balance the needs for conservation with those for rural development . In an eLocal communities seek benefits and avoid costsLocal communities are rationale beingsThe standards that local communities use to evaluate costs and benefits vary over time from person to personRelationships are interdependent and dynamicThe conceptual framework  explainsThe proposed framework examines the relationship between the PA management and the local communities who are either positively or negatively affected by the PA. Two factors that influence PA-community relationships are identified and each of these factors affects the way PA management and communities relate with each other. Costs and benefits as outcomes of conservation affect the way PA management and communities relate with each other. If communities do not receive benefits and bear costs, they are likely to have a negative relationship with the PA , 23. If 2. Up until April 2014, it was a cooperatively managed private wildlife area, but in the month of May 2014, it was placed under the custodianship of the Zimbabwe Parks and Wildlife Management Authority (ZPWMA). The conservancy is located in agroecological region V which is a semiarid area in the South East Lowveld of Zimbabwe. Its southern boundary is approximately 45 km northeast of Chiredzi town, whereas the Save River and Sangwe communal lands mark its eastern boundary. Its northern boundary lies not far from Birchenough Bridge, and its western boundary is formed by a resettlement scheme on land of the former Devuli Ranch and to the South by Matsai Communal area. It is found in the province of Masvingo and covers two districts, which are Chiredzi and Bikita. It is surrounded by three other districts, which are Zaka, Buhera, and Chipinge. The conservancy is bordered primarily by high-density communal land (of between 11 and 82 people per km2) [The study was conducted in two wards: Ward (3) and Ward 26) in Bikita district adjacent to the southwestern border of SVC, southeastern Zimbabwe . SVC spa in BikitA mixed-methods approach was used in data collection; it comprised structured household questionnaires which were administered via in-person interviews; the questionnaires included both open-ended and fixed response questions. Fixed response questions were used to ensure precision of responses, whilst open-ended questions were also included to obtain more detailed information on the nature of costs and benefits of conservation. The researchers also used participatory rural appraisal techniques particularly making use of semistructured interviews with key informants. The semistructured interviews were used to solicit more information. Schensul et al.  noted thIn addition, in order to achieve successful field data collection, a three-stage sampling design was adopted due to the nature of the sampling frame, to select the sampling units. The first stage was purposive sampling, where two wards (3 and 26) were selected from a total of eight wards which were under consideration for sampling. Purposive sampling was found suitable as these study sites were under the PA-community outreach initiative and also due to their geographical proximity to the SVC boundary. The second sampling stage used was simple random sampling for village selection where five villages from eleven villages constituting the two wards were selected , Village 2 Angus (65), twenty-six (73), twenty-seven (65), and thirty-one (69) with a total of 350 households). The third sampling technique, systematic sampling, was done to select 71 households where questionnaires were administered and collected in the 5 randomly selected villages representing 20% of the targeted population; this was ensured by walking through the village interviewing every second household. In addition, key informants interview questionnaire guides were administered to nine community leaders including traditional leaders, village elders, and ward councilors, all from the abovementioned wards through snowball sampling.χ2)) test was also used to determine whether given responses on costs, benefit from and attitudes towards the SVC differ across the five villages. Quantitative analyses were conducted using Statistical Package for Social Sciences version 19 for Windows . Qualitative data from semistructured interviews with key informants were summarized into percentages, and a qualitative content analysis technique was used.Descriptive statistics were used to summarize quantitative data sets from household questionnaires. A nonparametric  claimed that they incurred costs from wildlife whilst a minor proportion of the respondents  claimed no such costs  in respondents claims for incurring costs from wildlife across villages.In village twenty-six, a high proportion of respondents  of the respondents in village thirty-one reported livestock depredation to be the most prevalent cost incurred whilst a few  of the respondents in village (2) Angus mentioned restricted access to natural resources as a cost incurred  on the nature of costs incurred by the local communities across the villages.Respondents incur different kinds of costs from conservation in the SVC. Most  mentioned that they do not get benefits, whereas 45% (n=32) of the respondents indicated that they benefit from the SVC. There was no significant difference  on local communities' views in regard to deriving benefits from SVC across the villages.Most respondents (n=16) of the respondents across the sampled villages claimed that the renovation of local schools is the major benefit derived from SVC, with few respondents  pointing to borehole drilling as the least derived benefit from the SVC  on the nature of benefits derived from SVC across the villages.On the nature of benefits derived from the SVC, fifty percent  of the respondents in village thirty-one strongly agreed that SVC is more of a liability, whilst a few  of the respondents in Matsai village strongly disagreed that SVC is more of a liability. There was no significant difference  on the ratings of respondents attitudes based on (a) (“SVC is more of a liability”), across the villages.Most  of the respondents in village (2) Angus strongly agreed that SVC is for foreign interests, whereas a few  of the respondents in Matsai village strongly disagreed that SVC is for foreign interests. There was no significant difference  on the ratings of respondents' attitudes based on (b) (“SVC is for foreign interests”), across the villages.Many  of the respondents in village twenty-six strongly agreed that the relationship between SVC and the communities is bad, whereas a minor proportion  of the respondents agreed that the relationship between SVC and the communities is bad. There was no significant difference  on the ratings of respondents' attitudes across the villages based on (c) (“the relationship between SVC and the communities is bad”), across the villages, respectively (Majority (ectively .Respondents' attitudes toward SVC based on the following response options: (a) “SVC is more of a liability,” (b) “SVC is for foreign interests,” and (c) “the relationship between SVC and the communities is bad.”n=4; 44%) of the key informants had some knowledge of various laws and policy frameworks that could be used to help locals derive conservation benefits from PAs. For instance, they had basic knowledge of the CAMPFIRE-based projects and the wildlife-based land reform policy. The majority  of the informants had a little idea of any policy-related instruments; the informants reported that the SVC is not conducting awareness campaigns so as to equip local communities with the basic fundamental conservation policy issues. Additionally, all the traditional leaders, villages elders, and ward councilors  stated that their knowledge of how the SVC community trust a community-based conservation intervention functions is poor.Few  reported that the devolution of benefits is unclear, whereas the distribution channels are poorly defined. In addition, most of the interviewees, particularly traditional leaders and village elders , highlighted that the SVC community trust is no longer functional since there are no follow-ups being made to ensure that communities are still benefiting. Furthermore, councilors  associate the current challenges with the failure of communities to know any policies addressing the roles PAs should play in enhancing local communities well-being. Additionally, all the interviewees  mentioned that the aspect of resource nationalism through the indigenization of the SVC has resulted in the individual landowners in the SVC to be less concerned with meeting local communities aspirations and needs. The informants believe it is a challenge for their welfare to be addressed as the individual landholders in the SVC are highly antagonistic.All traditional leaders, villages elders, and ward councilors , spotted hyena (Crocuta crocuta), and lions (Panthera leo). This can be a major factor as wild game numbers in the SVC are anecdotal. This phenomenon has been also identified by Gandiwa et al. [In addition, the rate of occurrence of wildlife-induced damage can be due to the fact that the study sites are close to the SVC boundary. Results agree with findings by Salerno et al.  and Macka et al.  in GonarFurthermore, results on benefits show that a higher proportion of the respondents from the household survey are disgruntled with the nature of benefits derived from the SVC. On a preliminary basis, the dissatisfaction expressed in regard with benefits derived can be due to the awarding of benefits at community level not household level. Other studies have demonstrated that local people hold favourable attitudes toward wildlife conservation when personal benefits are derived from PAs –37. SongMoreover, this study recorded that the majority of the respondents across the villages hold negative attitudes toward SVC. Negative attitudes were significantly more common across households that had been affected by wildlife problems. Shibia  and GandBased on our results, it can be concluded that costs outweigh benefits in local communities living adjacent to SVC and that there are no formal benefit sharing mechanisms (BMSs), leading to local communities having negative attitudes towards SVC and wildlife conservation in general. It is recommended that costs incurred by local communities can be offset by a number of actions such as (i) putting in place formalized BSMs to ensure a more consistent flow of benefits to local people living on the edge. This is critical as Jensen  argues t


Here we show with experimental data that a laser-driven proton beam focused onto a target heated it in a localized spot with size strongly dependent upon material and as small as 35 μm radius. Simulations indicate that cold stopping power values cannot model the intense proton beam transport in solid targets well enough to match the large differences observed. In the experiment a 74 J, 670 fs laser drove a focusing proton beam that transported through different thicknesses of solid Mylar, Al, Cu or Au, eventually heating a rear, thin, Au witness layer. The XUV emission seen from the rear of the Au indicated a clear dependence of proton beam transport upon atomic number, Z, of the transport layer: a larger and brighter emission spot was measured after proton transport through the lower Z foils even with equal mass density for supposed equivalent proton stopping range. Beam transport dynamics pertaining to the observed heated spot were investigated numerically with a particle-in-cell (PIC) code. In simulations protons moving through an Al transport layer result in higher Au temperature responsible for higher Au radiant emittance compared to a Cu transport case. The inferred finding that proton stopping varies with temperature in different materials, considerably changing the beam heating profile, can guide applications seeking to controllably heat targets with intense proton beams.Intense lasers can accelerate protons in sufficient numbers and energy that the resulting beam can heat materials to exotic warm (10  Other potential applications of laser-driven beams include generation of neutrons or exotic isotopes5 and the ion fast ignition inertial fusion concept7. It has been demonstrated that protons can be accelerated to 10 s of MeV via short-pulse lasers interacting with solid targets. Several different methods have been investigated for ion acceleration including the most studied mechanism target normal sheath acceleration (TNSA)9, radiation pressure acceleration (RPA)11, breakout after burner (BOA) 13 and collisionless shock acceleration14. While proton acceleration with various mechanisms has been intensively studied, relatively few works focus on the physics arising during intense proton beam transport in various states of materials.Creation of intense proton beams and their transport are active research topics due to the unique ability of such beams to volumetrically and isochorically heat matter for fundamental studies of non-equilibrium states16. For these studies, proton beams were generated through the TNSA mechanism utilizing curved foil targets which provide the beam focusing effect as protons accelerate normal to the target toward the geometric center. When focused, these proton beams became intense enough to heat a matter sample to 10 s of eV. For instance, (>1011) protons generated from a 10 J laser were focused to a small spot (<50 μm) resulting in isochoric heating of Al foil to over 20 eV temperature15. Using a higher energy (170 J) laser, the brightest emission on Al foil corresponding to temperature of ~80 eV was measured16.Early studies on proton beam transport focus on demonstration of isochoric heating of a single solid materialMeV in a metal, the stopping power and total range of the proton are dominated by electronic stopping power caused by frequent inelastic collisions with electrons. For cold materials, probabilistic yet accurate stopping power values exist for charged ions including protons, drawing from broad experimental measurements and theoretical values18. However, model predictions for ion stopping in more complex states such as warm dense matter (WDM) or dense plasma, being neither solid nor ideal plasma, are uncertain and unverified. Major efforts to describe stopping of charged particles in this regime, a partially ionized medium, have been made via theoretical approaches and numerical calculations. One well-known approach is separately treating bound and free electrons’ contributions to electronic stopping power (B + F model)20, where fractions of these two populations are determined by plasma state conditions. A more self-consistent standard approach for charged particle stopping, the BPS model21, includes perturbative approaches to interactions and dynamic screening instead of simplified methods for the Coulomb energy exchange. With this model, alpha particle stopping range is predicted to be about 20–30% longer compared to most models for deuterium-tritium fusion23. Applying the local density approximation is another well-known method24. Recent calculation (SCAALP)25 uses the local-density and average-atom approximations where the inhomogeneous total electron density is taken into account. Simulations using molecular dynamics26 have shown that they are reasonably applicable to calculate stopping of ion transport in strongly coupled plasmas. While these approaches can calculate stopping power values, each has limitations and none is valid over a wide range of density and temperature.In proton beam transport, accurate accounting of how protons transfer their energy to matter as they slow down, known as proton stopping power, is one of the requirements to estimate beam projected range and heating profiles in a transport medium. For a non-relativistic proton with initial energy >3He reaction to probe a laser-heated sample. The precise energy of these protons allowed for reliable comparison of the stopping calculated by various means but only for a single, high projectile velocity27. The experiment was modeled with various models in a recent stopping study and found to be reproduced well using a time-dependent orbital-free density functional theory (TD-OF-DFT) method28. A different experimental method was applied to make ion stopping power measurements in laser-heated warm-dense matter, but for a projectile much closer to the expected thermal velocity of the sample, where stopping power is highest and most subject to disagreement between models29. The results of that experiment promote advanced stopping models including BPS and T-matrix formulation30.Few experiments have attempted to measure proton stopping power in the WDM range because of the paradoxical situation that measurable samples are difficult to create. One measurement of stopping power was made using lower flux protons born from the D, 32 and PIC with Monte Carlo stopping33. However, these transport studies with simple stopping models are limited to a few materials and would benefit from experimental findings in relevant conditions for various materials.Drivers powerful enough to reach WDM conditions may cause the thermodynamic state of the sample to change rapidly, causing difficulties in predicting the behavior of the beam transport. In this case, responses of the matter and the beam to one another need to be taken into account simultaneously. Self-consistent description of intense proton beam dynamics with collective effects has been studied using PIC simulations with a B + F stopping model by this groupIn this paper, we present experimental and simulation results of proton beam transport in different solid materials with a proton beam with a fixed source. Presented first are measurements of thermal emissions due to focused beam heating. To address the experimental questions such as the cause of dependence of the emission characteristics upon transport materials and heated temperature at the rear of the target, simulations using a particle-in-cell code were carried out. Simulations of the time-dependent source are described in the second Results subsection, followed by transport simulations using the same code. Details of the experimental and computational setups are described in Methods.rc = 300 μm was focused as discussed below. At a distance ~500 μm, ~1.7 × rc, the focused proton beams entered then transported through solid foils having a broad range of atomic number, Z, including Mylar, Al, Cu or Au with different thicknesses and a thin Au witness layer.A schematic diagram of the experiment and example data types are shown in Fig. 18. Two series of foils were chosen. First, Mylar (CH), Al, Cu and Au had a thickness of 50 μm, 30 μm, 15 μm and 12.5 μm, respectively, all chosen to range out 1.7 ± 0.1 MeV protons. Second 13 μm Mylar and 12.5 μm Al were chosen to range out 0.9 ± 0.1 MeV protons. For all materials, 0.5 μm Au layer was deposited on the rear of the transport foil to be a consistent surface for thermal emission measurements. An aluminum cylinder was fabricated to fix the distance between the Au hemi and target rear for all target types. Hot electrons generated by the laser interacting with the Au hemi can spread through the structure and reach the rear, potentially affecting the measurements. To reduce this effect, the structure was made wide (R = 550 μm) and the Au hemi was connected to it with thin support strips.The transport foil thickness for the different materials was chosen to have equivalent stopping range in cold matter using the NIST data of proton stoppingAs a main measurement of beam transport, an extreme ultraviolet (XUV) imaging system imaged thermal emission from the back side of the Au layer in two dimensions. Since Planckian radiation intensity sensitively varies depending on the temperature of a material, the time integrated brightness of radiation is an indicator of the surface temperature of a target with caveats discussed later.μm Au hemi rear. The proton source was also measured in these shots. Second, a target consisting of a flat 10 μm Au target foil and Au transport foil was irradiated with the laser fully focused on the front foil to achieve highest possible intensity. The XUV emission from the rear Au foil in this case was undetectable. This indicates that the heating from an unfocused proton beam and from any electrons that may have transited the Al structure were insufficient to heat the rear Au surface to the point of considerable 68 eV emission. Note that a typical unfocused TNSA proton beam with 50 μm source size and expansion cone angle of 20 degrees would overfill the 500 μm diameter transport foil.There are two important reference cases in the XUV dataset to consider. First, targets with no foils behind the Au hemi were shot, and bright emission was observed spread over the 300 For all other shots the laser irradiated target was a Au hemi. Contrary to the flat target case the XUV emitted from the surface behind the transport foil was confined locally, even smaller than the laser focus in some cases, and filters were necessary as the emission brightness was at least 3 orders of magnitude greater. This is due to the focused proton heating. Collected XUV images from different targets are shown in Fig. ρτ. Interestingly, all the targets with Al had a much wider emission size than targets with any other transport layer material or thickness, including those with higher atomic number and higher areal density. The largest emission size (~85 μm radius enclosing 50% XUV energy) from a target with Al is more than twice the size (~35 μm) of the Au foil cases. In contrast to this strong material dependence, emission size is seen to be weakly sensitive to transport layer thickness comparing data points with the same material, but different thicknesses such as Al (open vs. closed green squares) and CH (open vs. closed blue circles). XUV peak brightness versus target ρτ is plotted in Fig. Z or ρτ) is not straightforward, but low Z materials (CH and Al) tend to show brighter signal than Cu and Au. A clear trend is that targets with the thin layer have much brighter emission (~4×) than their thicker counterparts having the same material.Details of emission results for all transport foils are shown in Fig. 34 predict lateral straggling that increases with thickness but has extent of only several μm for the thicknesses investigated with thick Au being the most extreme at ~12 μm. As seen in the data above, the heated sizes vary by a much wider range, from 30 μm to 80 μm. Also, wider emission size for low ρτ material is contradictory to Moliere nuclear scattering formulas35 where protons traveling in high ρτ material have wider scattering angle. Further, one might speculate that Cu and Au, having higher room temperature thermal conductivity and more free electrons than Al and CH, would experience greater lateral spreading of the heat deposited via proton stopping, heating a wider portion of the Au emission surface. However, the data oppose all these trends with Cu and Au cases having the smallest emission regions. For these results, it can be concluded that transport for dense proton beams cannot be explained by only cold stopping and scattering models. Particle-in-cell (PIC) computational modeling of the proton beam transport with fields and an advanced model for proton electronic stopping power32 was conducted to aid in the interpretation of experimental observation as described in the following section. It will be shown that the nonlinear dependence of emissivity versus temperature must also be taken into account in understanding these experimental results.Discrepancies exist between these experimental results and predictions of simple existing models. For example, Monte Carlo calculations36. Simulating the configuration at full scale would require ~40 ps simulation run time at great computational expense so that the entire beam including low energy ~1 MeV protons can traverse the transport and witness foils positioned 450 μm behind the hemi. Moreover, this simulation would need to be repeated for different transport materials. Thus, to efficiently use computational resources, the problem was divided into two steps. First, proton beam generation and drift was modeled and compared to the measured spectrum. The resulting proton beam source was then used in a second series of transport simulations. Details of the simulations are given in the Methods section.Simulation of the experiment was conducted using particle-in-cell code LSPTV/m) to accelerate protons to MeV energy. Those protons initially accelerated normal to the target (moving toward the target geometric center) then expanded transversely by radial fields generated in the beam. Figure The source simulation was performed by injecting a population of electrons with parameters based on the measured laser qualities at the laser interaction location on the hemi. The target geometry was reconstructed in 2D Cartesian coordinates and a kinetic hydrogen species was initialized on the hemi rear. Electrons quickly spread out from the front side of the hemi target and expanded into the vacuum away from surfaces, driving a typical TNSA sheath field. At the rear side of the hemi, this field was strong enough . Fast moving protons  present a higher slope temperature, while those arriving later (10−20 ps) had a lower slope temperature. The second characteristic of the proton beam is that higher energy protons are focused to a small waist when they reach the extraction plane, whereas lower energy protons are uniformly spread over a larger radial area. For these reasons, it was decided that the beam entering the transport layers could not be represented by a beam with a steady-state, single characteristic spectrum, nor single size. Instead, the early and late protons were fit to two beams with duration, total energy, size and characteristic slope temperature given in Fig. Critically, it was observed from the proton generation simulation that the proton source could not be well parameterized with single fixed values. Two main characteristics of the proton beam are (i) proton energy distribution varied with time and (ii) the beam spatial profile at the extraction plane was different for differing proton energies. Figure 31 that updates according to the thermodynamic state of the material locally. Cases were also run with cold stopping18 for comparison. Two materials were chosen, Al and Cu, since in the experiment these transport materials exhibited dramatically different features of XUV emissions. Two proton beams were injected sequentially into the transport material with beam input parameters according to the representative fits discussed in the previous section. The total energy in both beams is 1.2 J, which is about 1.5% of the laser energy used in the experiment. Details can again be found in Methods.To study transport in the foil layers, one key factor is particle stopping power, which determines how much energy particles lose and ultimately where they stop in the medium. The same PIC code was used in a different mode to study transport in the secondary target. While the PIC algorithm precludes explicit binary collisions, quantum and atomic-scale effects cannot directly be included. However, this code can allow new understandings into intense beam transport due to an included dynamic proton stopping power moduleμm and 15 μm) of the two layers were determined by cold stopping range (for a proton of 1.7 MeV), similar energy fluences are expected. Varied energy fluence in the dynamic stopping simulation indicates that protons traveling in Al lose proportionally less energy than the Cu case as proton stopping power changes with the thermodynamic states of the transport layer. This result informs us that choice of a dynamic, or at least non-room-temperature stopping power model is necessary to reproduce the differences in proton transport observed in the experiment.Figure Total proton energy fluences deposited in the Au layer are found by the energy lost by particles between the Au entrance and exit extraction planes. Particles that were counted in the entrance extraction plane but not the exit plane were assumed to deposit all their energy in the entrance bin. The deposited fluence curves are shown in Fig. 6 MeV/μm2 at the first bin (0–1 μm) in x-axis corresponding to a volume of 0.5 × 1 × 1 μm3. Using the volumetric heat capacity of Au, 2.43 × 10−12J/Kμm3, the calculated Au temperature is 40 eV. This estimation is similar to the simulation result as shown in Fig. Here, the heated temperature of Au can be estimated using the deposited energy fluence. For instance, energy fluence through the Al layer is ~7.5 × 10eV has a peak in spectral radiance at 68 eV. This means that the response of the imaging system is not linear and can exaggerate the cooler regions further out from the spot. Additionally, emittance is strongly dependent on temperature. The Stefan-Boltzmann law describes the total power of black-body radiation depending on the object’s temperature with the relation j = σT4, where j, σ and T are respectively the radiated emittance, Stefan-Boltzmann constant and temperature. While this does not hold true for a single photon band, its scaling shows how a temperature difference of just 5 eV can lead to a large difference of radiated emittance. As shown in Fig. Using XUV emission brightness as a temperature diagnostic has caveats. A radiating blackbody with temperature 14 37 predicted that the XUV brightness corresponding to a Cu target temperature below ~20 eV is hardly detectable. Considering this limit, it can be inferred that the Al case has a broader area over which XUV emission can be detected. For Al and Cu layers, ranges (X-axis) for temperature above 30 eV are 130 μm and 70 μm, respectively; for temperature above 25 eV they are 200 μm and 120 μm respectively. This difference in size of the signal in simulations has the same trend observed in the experiment where emission size (R ~ 80 μm) for Al target is about twice that from a Cu target. Note that the beam central area is heated by two characterized beams, but only the second beam (low energy protons) is involved in heating of the wings. Thus, an inference can be drawn that low energy protons are responsible for the clear distinction in heating profile at the wings for the two different material cases.An important feature is the relatively gradual lateral temperature gradient in the Al case compared to the Cu case. This trend is clearly seen particularly for the outer part in the transverse direction (x-axis), leading to >2× higher emission brightness for the Al case. The XUV imaging has a limitation for measuring emission brightness because the target emitting XUV expands rapidly, resulting in the reduction of absolute brightness. For example, previous work using LASNEX (radiative-hydrodynamics code) simulationAdditionally, the induced magnetic field during proton beam transport was measured in the simulation to see if the magnetic field had any influence on beam profiles in different materials. However, the proton beam was not intense enough to produce high magnetic fields, (the maximum magnetic field is below 10 Tesla for a given proton beam) and it cannot affect the beam transport in the low thicknesses used.In this work, the experiments showed that the size and brightness of XUV emissions caused by proton beams vary by a factor of >2 with the target material, demonstrating an unexpectedly strong dependence of proton beam transport on materials.Simulation modeling of the experiments showed that protons moving through an Al transport layer heat the rear of Au to higher temperature resulting in higher radiated emittance compared to a Cu transport case. This result is consistent with the experimental measurement showing the different brightness of XUV between two material cases. Higher temperature over an entire area of Au for the Al case helps explain the brighter and wider XUV emission size seen from the experiment. These results provide an understanding that the variation of stopping power with temperature in different materials can considerably change the beam heating profile in targets. This effect turned out to be dominant rather than other effects such as beam scattering and induced weak magnetic field for the pertinent proton beam density.In the range of target temperature (<40 eV) encountered in this work, change of stopping power is relatively considerable only for protons having energies below 1 MeV. Thus it can be inferred that low energy protons played an important role in varying heating profiles in different materials with given thicknesses. Currently, stopping power models for low energy protons in CH or Au materials are limited and unvalidated, inhibiting the range of our investigation. More accurate stopping models in this regime would help us carry out further investigations of the physics of intense beam transport in solids.J on a target in a duration of 670 ± 50 fs with an incidence angle of 22.5°. The spectral phase and pulse shape of the laser have been carefully characterized by the facility38 through frequency resolved optical gating (FROG). Measurements were taken with a spectrometer and a FROG on most shots in this experiment of the pulse leaving the compressor as well as the pulse reflected from the target. The laser spot was characterized by relaying an image onto a CCD at low laser power. The spot at the target apex plane contained 50% of the laser energy within 65 μm, achieved by deliberately defocusing to uniformly cover a large area of a target with the desired outcome that doing so can aid beam focusing by the TNSA mechanism with a curved target40. The intensity was 3 ± 0.5 × 1018 W/cm2. No attempt was made to measure or mitigate spatio-temporal coupling effects41 which, if present, would equally affect all shots. Similarly, non-uniformities in the defocused laser spot, which are known to manifest in the proton beam42, are presumed to affect the proton source equally for all shots. The 10 μm Au hemi was chosen as the proton source target because experience has shown it to be reliable and relatively free from instability-induced structures in the proton beam profile.The experiments were conducted using the Trident laser facility at Los Alamos National Laboratory. Chirped pulse amplified laser pulses delivered an energy of 74 ± 7 37, the first optic was a spherical mirror with 50 cm radius of curvature, rcurv, consisting of 21 pairs of Mo2C and Si layers. It was placed 20 degrees off the target rear normal at a distance of 27.6 cm, where it reflected 68 eV light to a second, flat mirror in a turning chamber and imaged it onto a CCD filtered with Al.To measure the energy spectrum of generated protons, a stack of radiochromic films (RCF) and Thomson Parabola (TP) were used on each shot; for the electron spectrum, a magnetic spectrometer was fielded just below the TP line of sight. In the XUV imaging system36. The simulation method used here to model the TNSA mechanism is to inject relativistic electrons that are representing those accelerated by laser interaction with a target43. For this injection, electrons of Te = 0.5 MeV distribution with 650 fs duration were chosen, based on the fast electron spectrum measured with an electron spectrometer during the experiment as well as a reasonable agreement to the Beg and Ponderomotive scalings45 for the Trident laser parameters. Energy coupling from laser to electron was assumed to be 20%46. In a 2D cartesian simulation, both Au ions and electrons in a partial hemi with the same size as the real Au target are treated as a fluid, and a kinetic hydrogen layer is positioned at the rear of the hemi target.Modeling was conducted using the implicit, hybrid, particle-in-cell code LSP47. Although PIC simulations have limits to provide accurate interpretation of quantum phenomenon for warm dense matter, many effects for proton beam transport are self-consistently modeled with dynamic stopping calculation which is based on matter’s local thermodynamic states  Also fields evolution is determined by both beam current and matter’s local resistivity change. Electrons co-moving with protons in the TNSA beam have an energy of only several keV, stopping at the target front surface. As their impact on the transport is negligible, only protons were sent to the target in these simulations. Once protons travel through a transport layer (Al or Cu) up to distance d = 30 μm and 15 μm respectively, the proton data including particle energy (3 dimensions in velocity space) and positions in 2D are recorded in the extraction plane and re-injected from that plane into an Au target. The last extraction plane is positioned at 0.5 μm inside the Au as following the experiment where 0.5 μm Au layer was coated at back surface of transport foil. In these simulations, dynamic proton stopping power31 that is updated based on its thermodynamic state at each time step was applied for Al and Cu. For Au medium, only cold stopping was applied due to a limit of using an equation of state that is required for dynamic stopping calculation.In the proton transport simulations, both ions and electrons for materials are set up as fluid where their ionization and heating capacity are updated from the equation of state tables produced by PROPACEOS


The fast development of high-resolution electron microscopy (EM) demands a background-noise-free substrate to support the specimens, where atomically thin graphene membranes can serve as an ideal candidate. Yet the preparation of robust and ultraclean graphene EM grids remains challenging. Here we present a polymer- and transfer-free direct-etching method for batch fabrication of robust ultraclean graphene grids through membrane tension modulation. Loading samples on such graphene grids enables the detection of single metal atoms and atomic-resolution imaging of the iron core of ferritin molecules at both room- and cryo-temperature. The same kind of hydrophilic graphene grid allows the formation of ultrathin vitrified ice layer embedded most protein particles at the graphene-water interface, which facilitates cryo-EM 3D reconstruction of archaea 20S proteasomes at a record high resolution of ~2.36 Å. Our results demonstrate the significant improvements in image quality using the graphene grids and expand the scope of EM imaging. High-resolution electron microscopy requires robust and noise-free substrates to support the specimens. Here, the authors present a polymer- and transfer-free direct-etching method for fabrication of graphene grids with ultraclean surfaces and demonstrate cryo-EM at record high resolution. However, atomic-resolution cryo-EM is still challenging when biomolecules are embedded in unsupported vitrified ice layer at cryogenic temperature. During the cryo-EM specimen preparation, unsupported protein particles tend to adsorb at the air–water interfaces, which might cause preferential orientation and denaturation of proteins, thus limited the attainable resolution of the reconstructions5. To address this issue, conventional amorphous carbon films were often used to support the proteins. However, their non-negligible background noise and radiation-induced specimen motion hamper the further improvement of image resolution6.Technological breakthroughs in electron microscopy (EM) have enabled the imaging of individual atoms at the sub-angstrom resolution for inorganic materials9, reduce radiation-induced sample motion10, and especially avoid air–water interface adsorption of proteins during cryo-EM12. An ideal graphene grid as specimen support should exhibit several crucial characteristics: robust enough to avoid breakage14; clean surface to provide high contrast16; and tunable hydrophilicity for efficient specimens loading10. Although large-area high-quality graphene films can be grown on a metal substrate by chemical vapor deposition (CVD) and allow for the manufacturing of EM grids18, scalable fabrication of robust and ultraclean graphene EM grids is still a great challenge. Current preparation methods mainly rely on the transfer of graphene onto the perforated EM grids21, which always result in the low efficiency and low coverage of suspended graphene membrane. Furthermore, polymer films have often been coated on the graphene surface as supports during previous fabrication techniques, so that polymer residues on the graphene membranes would inevitably degrade the intrinsic performances of graphene22 and introduce extra background noises in EM21. It is therefore critical to develop a robust and scalable method to produce ultraclean and hydrophilic graphene grids for more general application in high-resolution EM imaging.Graphene, an atomically thin carbon two-dimensional crystal with high conductivity and mechanical strength, is an ideal specimen support to substantially minimize background noise28.Herein, we exploit a polymer- and transfer-free method for scalable fabrication of robust and ultraclean graphene grids from large-area CVD-grown graphene films on metal foil. Graphene grids show tunable surface hydrophilicity, which enables efficient specimens loading for atomic-resolution EM at both room- and cryo-temperature. Such grids have also been proved to achieve ultrathin vitrified ice layer (~30 nm in thickness) with most protein particles surrounded at the graphene–water interface, achieving 3D reconstruction of archaea 20S proteasome at a record high resolution of 2.36 Å by single-particle cryo-EMFigure 32. The graphene membrane bent down due to the capillary pressure Pcap induced by the liquid on the grid holes. Pcap can be estimated according to Young-Laplace equation Pcap = 2γl-g/Rl, where γl-g is the surface tension of the liquid and Rl is the curvature radius of the liquid surface. Since the grid hole has an inclined wall from fabrication, Rl decreases during the drying process, leading to an increasing Pcap accordingly. Thus, the deformation of the graphene membrane will increase gradually  of the graphene membrane is much lower than that of amorphous carbon film  can be modulated via adjusting the condition of plasma treatments. As shown in Fig. ID/IG) of Raman spectroscopy for graphene membrane maintained suppressed (~0.05) within 10 s of plasma treatments, and then ID/IG distinctly increased to ~2 with a corresponding LD of ~10 nm as the treatment prolonged  and C (Z = 6)36. The oxygen-containing function groups on graphene membranes not only improved their hydrophilicity, but also can efficiently anchor and stabilize the single atoms, such as Cu on graphene basal plane  of the graphene grids from 90 ± 2° to 50 ± 3° under controllable oxygen-plasma treatments within 40 s Fig. . Meanwhiane Fig. .Fig. 3St−2. The radiation robustness of graphene grid allows it to be operated in a range of operating voltage of 60–300 kV at variable temperatures. We utilized the graphene grids to load and observe biological specimens such as ferritin molecules (comprising of protein shells and iron-containing cores) at 300 kV. As shown in Fig. 3O4  plane spacing of 1.48 Å can also be clearly resolved at cryogenic temperature  under the same H2 and CH4 flow. Then, the supply of both H2 and CH4 was stopped. For the super-clean graphene preparation, CO2 gas (500 sccm) was pumped into the LPCVD system to selectively remove the amorphous carbon on graphene surface at ~500 °C for 3 h, as previously reported by our group39.The clean single-crystal graphene films were grown on commercial copper foils  in a low-pressure CVD (LPCVD) tube furnace system. Firstly, the copper foil was electrochemically polished in the electrolyte solution composed of ethylene glycol and phosphoric acid (v/v = 1:3) with a voltage of 2–3 V for 10–30 min. The copper substrate was loaded into the tube furnace and heated to 1030 °C under a flow of 100 sccm H2. Subsequently, 1000 sccm H2 and 1 sccm CH4 were introduced into the LPCVD system for ~1.5 h with a pressure of ~2000 Pa. Note that the high pressure of ~2000 Pa in the CVD system helps to increase the growth rate and the coverage of bilayer graphene. After growth, the bilayer graphene films were treated with CO2 etching at 500 °C as mentioned above.For the growth of bilayer graphene films, a piece of electrochemically polished copper foil (Alfa-Aesar #46365) was placed on a quartz substrate before loaded into the tube furnace. The copper foil was heated to 1030 °C in one hour and annealed for 30 min under a flow of 100 sccm H2, the copper foil was firstly heated to 1030 °C for 1 h and then annealed for 30 min. Subsequently, the graphene film was grown on the copper foil for 2 h under the flow of 2000 sccm H2 and 2 sccm CH4 with a pressure of ~2000 Pa. After that, the same CO2 treatment was used to clean the few-layer graphene surface.For the growth of few-layer graphene films, electrochemically polished Cu foil was placed on the quartz substrate and loaded into the LPCVD system. Under a flow of 100 sccm HPlasma treatment to remove the undesired graphene: To remove the graphene on one side of copper foil, we placed the copper foil with graphene on a smooth hard substrate such as clean glass slide or polyethylene glycol terephthalate (PET) film. Then the scotch tape was used to seal the edges of copper foil on the substrate. In this way, the graphene on the other side of copper foil was protected from being damaged by the following plasma treatment. After placing the hard substrate in the reactive ion etcher , a flow of 10 sccm air was introduced, then the air plasma was generated at a power of 150 W for 3 min to remove the undesired graphene.Photolithography: The positive photoresist  was spun onto the Cu foil at 4000 rpm and baked at 110 °C for 3 min. Subsequently, the positive photoresist was exposed to 365 nm ultraviolet for 6–10 s with the designed mask. After being post-baked at 110 °C for 1 min, the foil was developed in dilute developer  for 6–10 s. In this way, the patterned photoresist was fabricated on the copper foil.2S2O8 aqueous solution. Note that a stir bar could be used for controlling shapes of the holes.Selective etching: The exposed area of the patterned copper foil was selectively etched with 0.1 M NaN-methyl pyrrolidone and acetone; then the grids were rinsed in the isopropyl alcohol.Rinsing: The floating graphene grids were washed gently with deionized water to remove etchant traces, and the photoresist was carefully removed with Drying: The bilayer and few-layer graphene grids were normally dried in the super-clean room to avoid extra contaminants after being took out from the isopropyl alcohol. The monolayer graphene grids were dried in the critical point drier (samdri-795) to protect the suspended graphene membranes from rupturing by surface tension.A reactive ion etcher  was used to modulate the species of source gas, volume flow rate of gas, treatment time and power  , scanning electron microscopy (SEM)  and Raman Spectrometer . The WCAs of graphene grids were measured using contact angle measurement (Dataphysics OCA 20). The selected area electron diffraction (SAED) patterns of graphene were collected by transmission electron microscopy . And the aberration-corrected STEM images of graphene were performed using a Nion U-HERMS200 microscope at 60 kV.The aberration-corrected STEM images and electron energy loss spectrum (EELS) data were performed using a monochromatic Nion U-HERMS200 electron microscope which is operated at 60 kV. Both aberration-corrected STEM images and EEL spectra were acquired with a 35-mrad convergence semi-angle. The images were acquired using a HAADF detector with an 80–210 mrad collection semi-angle and the EEL spectra data were acquired with a 24.9-mrad collection semi-angle. EEL spectra were recorded by the EELS CCD with 2048 channels, the dispersion of which is 0.41 eV/channel, hence we could detect the loss range of 200–1050 eV which contains the K edge of carbon (284 eV) and L edge of copper (931 eV). The diameter of the focused electron beam is at the angstrom level. To minimize the damage of such a focused beam and acquire signals from multi atoms, we made the beam slightly defocused of 50 nm and set the exposure time as 5 s for each spectrum.The ferritins from equine spleen (Sigma-Aldrich) were used for TEM characterization. Specifically, 1 μL of ferritin solution diluted 100 times from the original solution was loaded onto the membrane surface at room temperature, followed by drying naturally in the air. At room temperature, TEM characterizations and the EDX mapping of ferritins were carried out using a FEI 80–300 Environmental Titan operated in monochromated mode at 300 kV.2. The cryo-holder is then inserted into the TEM column (~1 s), and during the whole process the sample is kept at −178 °C. TEM characterizations were carried out using a FEI Titan 80-300 environmental (scanning) transmission electron microscope (E(S)TEM) operated at 300 kV. The microscope was equipped with an aberration corrector in the image-forming (objective) lens, which was tuned before each sample analysis.For the characterization at cryogenic temperature, the as-prepared graphene grid with ferritin was plunged-frozen into liquid nitrogen and then carefully mounted onto a TEM cryo-holder (Gatan 626 cryo-holder) using a cryo-transfer station to ensure the entire process under liquid NT. acidophilum 20S proteasome was provided by the Xueming Li lab in Tsinghua University24. Shortly, His-tagged 20S plasmids were transformed into Escherichia coli cells and induced for protein expression. The protein complex was purified by nickel affinity columns as previously reported. For cryo-EM specimen preparation, graphene grids were glow-discharged for 10 s, and then ~3 μL of 20S proteasome solution at a concentration of ~0.2 mg mL−1 was loaded onto the grids. After incubation for 2 s in a cryo-plunger at 8 °C and 100% relative humidity , the grids were blotted for 5 s with filter paper and plunge-frozen into liquid ethane cooled at liquid nitrogen temperature. The grids were stored in liquid nitrogen until examined by cryo-EM.The −2 and these frames were motion-corrected by MotionCor2 (REF). CTF estimation was performed by CTFFIND4 and 156667 particles were auto-picked by Relion (REF). After 2D and 3D classification, 124,212 particles were finally used for further 3D auto-refine with D7 symmetry. The resolution of the final 3D reconstruction was reported as 2.3(6) Å estimated by the FSC 0.143 criterion .The as-prepared frozen specimen grids were loaded into a FEI Titan Krios (300 kV) TEM equipped with an X-FEG and Cs-corrector, and the dataset was collected by Gatan K2 direct electron detector camera (Gatan Company). A total of 935 micrographs were recorded by AutoEMation2 , with a pixel size of 0.67 Å and defocus range of 0.5–1.9 μm. Each micrograph was fractionated to 32 frames with a collective dose of 50 e Åion Fig. . The cry−2), and each micrograph was dose-dependently fractionated to 32 frames. Particles were auto-picked and their coordinates in each frame were determined by Relion3.0. These coordinates were then applied for calculating the displacements of 20S proteasome particles on individual supporting membrane versus dose accumulation.To measure the displacement of particles on various supporting membranes, we collected movies of specimens under Titan Krios (300 kV) with constant dose rate (with accumulated dose of ~50 e Å−2 s−1 from 0 min to 70 min. The relative integrated intensity variation of diffractograms (the first-order integrated intensity was divided by the third-order integrated intensity) versus dose was calculated to evaluate the stability of the graphene membranes.The stability of graphene membranes at liquid nitrogen temperature was investigated by Tecnai F20 (200 kV). Selected area electron-diffraction patterns of graphene were continuously collected at the same position on a CCD camera (Gatan US4000) with a dose rate of ~15 e ÅSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3


Thermomyces Lanuginosus (TLL).Biographene was successfully produced in water from graphite flakes by a simple, rapid, and efficient methodology based on a bioexfoliation technology. The methodology consisted in the application of a lipase, with a unique mechanism of interaction with hydrophobic surfaces, combined with a previous mechanical sonication, to selectively generate lipase-graphene sheets conjugates in water at room temperature. The adsorption of the lipase on the graphene sheets permits to keep the sheets separated in comparison with other methods. It was possible to obtain more than 80% of graphene (in the form of multi-layer graphene) from low-cost graphite and with less damage compared to commercial graphene oxide (GO) or reduced GO. Experimental analysis demonstrated the formation of multi-layer graphene (MLG) mainly using lipase from  Graphene is a material with excellent electrical and thermal properties, very high mechanical strength and elasticity which make it ideal for application in areas such as electronics, materials, biomedicine, biotechnology, etc. ,11,12,13Production of graphene has been performed by scotch tape peeling , differeIn particular, the synthesis of graphene by the chemical reduction of graphene oxide (GO) by the Hummers method represents at present the most scalable method for the production of few-layer graphene ,17. HoweIn this way, in recent years, strategies to synthesize graphene by a more sustainable way have been reported. The direct exfoliation of graphite to graphene in water seems to have the best reported outcome ,26,27,28In particular, the application of proteins for the exfoliation of graphite is an interesting alternative. The application of a mechanical step to create enough space between graphene layers in graphite (interlayer distance 0.33 nm) is needed for the intercalation of proteins (protein size 5–10 nm). A few examples have proven to be successful in the application of proteins ,28,29. HAt present, the most specific methodology is represented by using hydrophobins, which present a hydrophobic part and another hydrophilic . It is dTherefore, the development of a highly selective methodology to produce a completely homogeneous modification of the graphene protein surface is mandatory. Moreover, from an economical point of view, the availability of the enzyme is critical.This way, the use of lipases can solve these drawbacks. Lipases are enzymes with a particular structure based on their natural function, fat hydrolysis. In nature, they work at the interface between oil and water . They prThis capacity has been demonstrated to work in the presence of different hydrophobic interfaces  ,33,34,35Recently a lipase interacting with graphene has been described although in all cases as a covalent immobilization technique on graphene oxide using crosslinkers ,37.In this work, we applied for the first time the special selective capacity of these lipases for their selective adsorption on graphene from graphite flakes. These represent a very fast, cost-efficient and sustainable methodology for the bioexfoliation of graphite to graphene. These enzymes can be homogeneously located on the graphene surface, permitting their direct production from the graphite present in a water suspension at room temperature.This also represents a method for the specific functionalization of graphene, because we can selectively control the enzyme location, thus controlling the number of groups  for the modification of graphene.Furthermore, we demonstrate how the capacity of the lid has an influence on the final results and how it is possible to obtain biographene very easily on a large scale.Graphite flakes and p-nitrophenilpropionate (pNPP) were purchased from Sigma-Aldrich Co . Lipase B from Candida antarctica (CAL-B) solution and lipase from Thermomyces Lanuginosus (TLL) were obtained from Novozymes . Aqueous solutions were prepared using UltraPure™ DNase/RNase-Free Distilled Water (Thermo Fisher Scientific). To recover the biohybrids, a Biocen 22 R  refrigerated centrifuge was used. −1 band of a pure silicon sample. Two 60-second scans were performed for each spectrum. The obtained spectra were baseline corrected in order to remove any background fluorescence. Spectrophotometric analyses were run on a V-730 spectrophotometer . Atomic force microscopy images were obtained using an NTEGRA PRIMA NT-MDT microscope. The obtained images were analyzed using the built-in software provided by the manufacturer.Inductively coupled plasma-optical emission spectrometry (ICP-OES) was performed in an OPTIMA 2100 DV instrument . X-Ray diffraction (XRD) patterns were obtained using a Texture Analysis D8 Advance Diffractometer  with Cu Kα radiation. Transmission electron microscopy (TEM) and high-resolution TEM microscopy (HRTEM) images were obtained on a 2100F microscope . Interplanar spacing in the nanostructures was calculated by using the inversed Fourier transform with the GATAN digital micrograph program . Scanning electron microscopy (SEM) imaging was performed on a TM-1000 microscope . Vibra-Cell™ Ultrasonic Liquid Processors VCX130 (Sonics) was used for graphite sonication process. Raman spectra were collected at room temperature using a dispersive JASCO NRS-5100 equipment with a focal distance of 300 mm and a maximum resolution of 1 cm-1. The laser used in the experiments was a solid-state laser (532 nm) with a sample output power of 4 mW. The equipment is coupled to an MPLFLN 20 × UV objective. Prior to analysis, the Raman shift was calibrated using the 520 cmThe graphite flakes (1 g) are added together with 20 mL of distilled water to a 50 mL centrifuge tube . Then, the graphite is exfoliated by alternating cycles of 5 min sonication/rest, for 30 min at an amplitude of 80%. To avoid excessive heating of the mixture, the 50 mL Falcon tube is introduced into a mixture of ice and cold acetone. This exfoliation allows the weakening of the bonds between sheets. The non-exfoliated graphite is separated from the mixture by centrifugation. The mixture was transferred to 15 mL centrifuge tubes  and centrifuged at 400 rpm for 1 min. The supernatant is carefully separated from the unfolded graphite solid. Finally, the supernatant was placed in a Petri dish and dried in the stove at 50 °C. The method allows obtaining 100 mg of Sgraphene-1, final yield of 10%.The graphite flakes (1 g) are added together with 20 mL of distilled water to a 50 mL centrifuge tube . Once this is done, the graphite is exfoliated by alternating cycles of 5 min sonication/rest, for 1 h at an amplitude of 80%. To avoid excessive heating of the mixture, the 50 mL Falcon tube is introduced into a mixture of ice and cold acetone. This exfoliation allows the weakening of the bonds between sheets. The non-exfoliated graphite is separated from the mixture by centrifugation. The mixture was transferred to 15 mL centrifuge tubes  and centrifuged at 400 rpm for 1 min. The black supernatant was carefully separated from the graphite solid. Finally, it was placed in a Petri dish and dried in the stove at 50 °C. The method allows obtaining 300 mg of Sgraphene-2, final yield of 30%.The graphite flakes (1 g) in 20 mL of water were sonicated as described in Method 2. After that, commercial lipase solution of TLL (0.43 mL) or commercial lipase solution of CAL-B (1 mL), in both cases offering 5 mg lipase, were directly added to that and allowed to stir for a period of time between 30 min and 1 h. The selective adsorption of the lipase was followed by measuring the supernatant using the pNPP activity assay (described below). After lipase immobilization, the suspension turned cloudy black. Then, the mixture was transferred to 15 mL Falcon centrifuge tubes and centrifuged at 400 rpm for 1 min to remove some non-exfoliated graphite. After that, the black suspension was centrifuged at 8000 rpm for 10 min and then the water was removed. One mL of acetone was added to dissolve the black powder and then this mixture was transferred to a Petri dish and dried at 50 °C for 4 h. The method allowed obtaining 800 mg of biographene-1, final yield of 80% using TLL and 600 mg, 60% yield using CAL-B.The graphite flakes (1 g) were added to a solution of 20 mL of water to which 0.43 mL of commercial lipase was previously added in the case of TLL or 1 mL in the case of CAL-B, (enzymatic loading: 5 mgenzyme/gsupport). Then this mixture was sonicated using the aforementioned Method 2. After that, the mixture was transferred to 15 mL Falcon centrifuge tubes and centrifuged at 400 rpm for 1 min. The supernatant was carefully separated from the unfolded graphite solid. After that, the black suspension was centrifuged at 8000 rpm for 10 min and then the water was removed. One mL of acetone was added to dissolve the black powder and then this mixture was transferred to a Petri dish and dried at 50 °C for 4 h. The method allowed obtaining 500 mg of biographene-2, final yield of 50%.This assay was performed by measuring the increase in the absorbance at 348 nm produced by the release of p-nitrophenol in the hydrolysis of 0.4 mM pNPP in 25 mM sodium phosphate buffer at pH 7 and 25 °C. To initialize the reaction, 20 μL of lipase solution or suspension was added to 2.5 mL of substrate solution. One international unit of pNPP activity was defined as the amount of enzyme that is necessary to hydrolyze 1 μmol of pNPP per minute (IU) under the conditions described previously. Four different methods have been performed in order to produce graphene from cheap graphite flakes. Sgraphene-1 and Sgraphene-2 were obtained. These strategies gave moderate to low yields of graphene (black suspension) of 10% to 30%, respectively. First, two different methods based on a mechanical force through ultrasound to the flakes of graphite were tested, one with a simple mechanical sonication (Method 1) and another with double sonication (Method 2) of graphite flakes in distilled water. After that, two materials, Sgraphene-1 (Simple Sonication) and Sgraphene-2 (Double sonication), for example in the SEM images, that material presented a larger flake size by using Method 1  or T. lanuginosus (TLL) on the Sgraphene-2  was fully adsorbed to the solid material at room temperature. The spacing obtained previously between the graphite sheets allows the lipase to be placed between them, giving rise to hydrophobic sheet–lipase interactions  that permit separating said sheets obtaining graphene . biographene-1, the yield was higher using TLL than CAL-B, 80% against 60%. These phenomena could be explained considering the aminoacid composition and tridimensional structure of both lipases. Although both enzymes have similar molecular weight, TLL presents a much higher hydrophobic area than CAL-B , the method using CALB seems to generate a material with a larger flake size when compared with the method using TLL . The TEMd manner B. HoweveConsidering these promising results, a fourth method adding the enzyme directly from the beginning (sonication plus lipase immobilization in one-pot) was attempted .The lipase must be introduced between the sheets of graphene while the ultrasound weakens this interaction between sheets separating them completely. The strategy was applied using shorter and longer sonication times. In this case, also both lipases were tested.The best results were obtained using the longest strategy for mechanical sonication described in Method 2 but in the presence of the enzyme. However, in both cases, yields of black solid around 50% were obtained. TEM analysis demonstrated differences between using one lipase or the other . In the However, when comparing this method with the aforementioned Method 3, the sizes of the obtained aggregates were much larger than in Method 3, together with a higher density of sheets.biographene-1-TLL). It should also be noted that the treatments applied to the different samples did not create more defects on the graphene layers, as evidenced by the comparable D band between the graphene samples and the parent graphite  is obtained, because peak at 2710 cm−1 is mainly present [−1  and a function of the number of graphene layers, the results have focused on samples with a number of layers <6 and thus a comparison with our results may not be applicable.The  present . In the  present  have stuIn order to corroborate the promising results hinted at by the Raman spectra, AFM images were collected see  and analWe have developed for the first time a very simple, rapid, low-cost, and environmentally safe process to produce graphene sheets from very cheap graphite flakes in water and room temperature. Themomyces lanuginosus lipase (with a very high hydrophobic area) gave the best results. It was possible to produce multi-layer graphene (MLG) at 80% yield, obtaining 800 mg of graphene per gram of graphite flakes by a very simple way. Additionally, this produced better quality  biographene than commercial GO or rGO tested.The novelty consists in the application of a biomolecule, a lipase, an enzyme with a very high selectivity to interact with hydrophobic surfaces, fixing the open conformation which permits the location of lipases in open conformation uniformly distributed on the graphene sheets surface, avoiding the interactions between sheets, generating an exquisite bioexfoliation of graphite. Two different enzymes with different methods were employed and the use of This is very important from an industrial point of view, because economically the advantage is clear, 1 g of graphite flakes costs 3.36 Eurocents , whereas 1 g of graphene oxide costs €524 (SIGMA), that means it is 15,000 times cheaper. Another advantage of this produced biographene is that it is already functionalized, because the enzyme presents a high number of carboxylic groups in the area exposed to the solvent. Therefore, this strategy could have a high potential in the application of graphene in different areas .


TS) of the film with 74% graphene increased by 27.22% and the total soluble matter (TSM) of the film with 93% graphene decreased by 11.30%.This study applied a facile and green approach to synthesize a stable graphene aqueous dispersion, and the graphene aqueous dispersion was employed to modify the renewable, compatible and biodegradable soy-protein-isolated (SPI) films to enhance their thermal stability, mechanical properties and water resistance. Atomic force microscopy (AFM) images confirmed the monolayer structure of graphene. The hydrogen bonds and π–π interactions between graphene and the SPI molecules were showed with the attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy, and X-ray diffraction (XRD). As expected, compared to the pure SPI film, the tensile strength ( Nowadays, with the increased awareness of sustainable development and environmental protection, interest has risen in developing biopolymer films as an environment friendly and renewable alternative to petroleum-based materials . These fAmong the biopolymer studies, soy protein isolate (SPI), a common by-product of the edible oil industry, showed a wide range of potential applications in drug delivery, packaging and mulching fields due to its low cost, abundance, biocompatibility, sustainability and film-forming capacity ,4,5. How2-hybridized carbon atoms arranged in a honeycomb lattice [Recently, graphene has attracted increasing interest as an emerging functional material. Graphene is a two-dimensional (2D) crystalline monolayer sheet, with sp lattice ,10,11. T lattice ,10,12,13 lattice ,15,16. HInspired by the excellent hydrophobic and mechanical properties of graphene, Wang et al., synthesized graphene-reinforced poly (PVA) composite films . The resIn this study, we compounded series of graphene with different ultrasonic time via a facile method, and prepared renewable and environment friendly SPI–graphene composite films. The hydrogen bonds and π–π interactions between the SPI and graphene molecules improved the mechanical properties, thermal stability and water resistance of the resultant composite films. This advanced performance provides SPI–graphene films wide potential application in drug delivery, packaging and the food industry.SPI with a protein content of 95% was purchased from Yuwang Ecological Food Industry Co., Ltd. . Graphite powder was provided by Sinopharm Chemical Reagent Co., Ltd. . Bovine serum albumin of biotechnology grade was obtained from Beijing Labest Bio Technology Co., Ltd. . Glycerol (99% purity) of biotechnology grade, sodium hydroxide of analytical grade and other chemical reactants of analytical grade were purchased from Beijing Chemical Reagents .−1·m−1) previously determined [50.0 mg of bovine serum albumin (BSA) powder was mixed with 900 mL of deionized water at 50 °C and stirred for 12 h. The pH of the aqueous BSA solution was adjusted to 3.6 with dilute HCl solution. Then 1 g graphite power were dispersed in 600 mL of the aqueous BSA solution. The mixture was probe-sonicated and stirred for 6, 12, 18, 24, 30 and 36 h. The product was allowed to stand for 24 h at room temperature to settle some of the unreacted graphite particles and large graphene aggregates. It was then centrifuged at 3000 rpm for 30 min to separate the supernatant from the sediment. The concentration of aqueous graphene dispersion was calculated from the Beer–Lambert law with the extinction coefficient of graphene (α = 1390 mL·mgtermined  at the wThe SPI film-forming solutions were prepared by SPI (4.0 g), glycerol (2.0 g), graphene aqueous dispersion and deionized water. The pH of the mixture was adjusted to 9.0 ± 0.1 with sodium hydroxide solution, then heated in a water bath at 85 °C for 30 min. The solution (75.0 g) was poured onto a Teflon-coated plate after removing bubbles by ultrasound treatment. The films were dried in drying oven at 45 °C for 24 h, and preconditioned at the relative humidity of 50 ± 2% and temperature of 25 ± 2 °C for 48 h before testing. The composition parts of the films were presented in The TU-1901 ultraviolet-visible spectrophotometer  was used to record the absorption spectra of aqueous graphene within the range of 250–400 nm.The morphology of graphene was observed by atomic force microscopy . The topographic (height) and phase images were collected in the tapping mode using a monolithic Si tip with a resonance frequency between 250 and 300 kHz.The surface morphology of the graphene–SPI films was observed using the SU8010 field emission scanning electron microscopy  with an acceleration voltage of 5 kV.−1. A total of 32 scans were performed at 4 cm−1 resolution.The chemical structure of the graphene–SPI films was examined according to the Nicolet 6700 attenuated total reflectance Fourier transform infrared spectroscopy  with a wave number range from 650 to 4000 cm−1. The temperature ranged from 30 to 600 °C under a nitrogen atmosphere (100 mL·min−1) to avoid thermo-oxidative reactions.Samples were dried at 45 °C for 24 h before testing. The thermal stability of the graphene–SPI films was measured using the Q50 thermo gravimetric analysis (TGA) analyzer  with a rate of 10 °C·minX-ray diffraction (XRD) patterns were observed by the D8 advance diffractometer  equipped with a Cu Kα radiation source. The 2θ value was ranged from 5° to 60° in continuous scanning mode with an increment of 0.02°.The surface hydrophilicity of the graphene–SPI films was investigated using the water contact angles . A sessile droplet  of distilled water was dropped onto the surface of the films. The angles of both sides were recorded at an interval from 0.1 to 180 s. Five replicates were conducted for each specimen.TS), elongation at break (EB) and Young’s modulus (E) were determined by five replicates of each film.Each sample was cut into 10 × 80 mm pieces and the thickness of the film was measured with a digital micrometer (0–25 ± 0.001 mm) before testing. The mechanical properties were determined on the tensile testing machine  according to ISO527-3:1995 (E). The tensile strength (2) of each film were tested to determine the moisture content (MC), total solution matter (TSM) and water uptake (WU).Three specimens (20 × 20 mmma) was determined. The specimens were dried in an air-circulating oven at 103 ± 2 °C for 24 h and weighed again (mb). MC was calculated as follows: The initial mass  for 48 h, and the initial mass was weighed as md. The films were immersed in a breaker with 30 mL of deionized water at room temperature for 24 h. Then the water was moved and the mass of specimens were weighted as me. WU was calculated as follows:The specimens were firstly stored in P−1 [UV–vis spectroscopy was used to confirm the presence of graphene. −1 ,23. The The stripped graphene aqueous dispersion (1 mL) was obtained, then diluted five times directly before investigated the surface morphology of the graphene by AFM. The 2D morphologies and height images are shown in −1. The increasing intensity of the broad absorption band at 3274 cm−1 was attributed to the O–H stretching in the SPI films [−1 were the stretching vibrations of the methylene groups [−1 were the main characteristic amide bonds of amide І (C=O stretching), amide ІІ (N–H bending), and amide ІІІ (C–N and N–H stretching), respectively [−1 corresponded to the hydrogen bond between SPI and the graphene molecules. Based on the above analysis, we concluded that the interactions between SPI and the graphene molecules were mainly due to the hydrogen bonds, and the π–π interactions also provided a possibility for the modification of SPI molecules.ATR FT-IR was used to investigate the structural characteristics of the SPI-based films . These sPI films . The peae groups . Among aectively ,27. The The structure of SPI-based films was observed by XRD . ClearlyThe surface morphology of graphene–SPI films was observed by SEM . ObviousTmax1,2) of sample B (with 74% graphene content) increased from 151 to 195 °C, and from 293 to 309 °C. Overall, the improved thermal stability was attributed to the hydrogen bonds and π–π interactions between graphene and the SPI matrix. However, the excessive content of graphene may have strengthened the π–π interactions between graphene molecules, thereby weakening the interactions between the graphene and the SPI molecules.The thermal properties of these SPI-based films were investigated by thermos-gravimetric (TG) and derivative TG (DTG) analysis, as shown in TS), Young’s modulus (E) and elongation at break (EB) of SPI-based films are shown in TS and E, while reducing the EB. Compared to the pure SPI film, the TS of sample B (with 74% graphene content) increased from 4.74 to 6.03 Mpa, accounting for the increase of 27.22%. The results may be attributed to the deformation of SPI molecule chains. Graphene was distributed in the SPI matrix in the form of a single layer, which enhanced interaction and prevented slipping [The results of tensile strength , total soluble matter (TSM) and water uptake (WU) of the six groups of SPI-based film samples. The introduction of graphene did not result in significant changes in the MC result. The addition of graphene decreased the TSM and WU, but the WU trend was not obvious. Compared to the pure SPI film, the TSM of sample A (with 93% graphene content) decreased by 11.30%. The results may be accounted as the outstanding compatibility and strong interactions between graphene and the SPI matrix, which exposed more hydrophobic groups, thereby exhibiting improved surface hydrophobic properties [operties ,37,38.TS of sample B (with 74% graphene content) increased by 27.22%. Water resistance and thermal abilities were also improved due to the addition of graphene. This improved performance may be due to the hydrogen bonds and π–π interactions between graphene and the SPI matrix, which were confirmed by the ATR FT-IR and XRD. Overall, this research indicated that SPI–graphene composite films have potential applications in drug delivery, packaging and mulching.In the study, we synthesized graphene aqueous dispersion via a simple and green method successfully. Composite SPI–graphene films with enhanced thermal stability, mechanical properties and water resistance were prepared. When preparing the graphene aqueous dispersion, the best ultrasonic time was 24 h. Compared to the pure SPI film, the