Finally, saracatinib significantly decreased TGF-β gene expression. Our results indicate that treatment responsiveness varies between IPF patients in relation to their oxidative and inflammatory status. Interestingly, saracatinib tends to be more effective in IPF than standard antifibrotic drugs.Idiopathic pulmonary fibrosis (IPF) has a detrimental prognosis despite antifibrotic therapies to which individual responses vary. IPF pathology is associated with oxidative stress, inflammation and increased activation of SRC family kinases (SFK). This pilot study evaluates individual responses to pirfenidone, nintedanib and SFK inhibitor saracatinib, markers of redox homeostasis, fibrosis and inflammation, in IPF-derived human bronchial epithelial (HBE) cells. Differentiated HBE cells from patients with and without IPF were analyzed for potential alterations in redox and profibrotic genes and pro-inflammatory cytokine secretion. Additionally, the effects of pirfenidone, nintedanib and saracatinib on these markers were determined. HBE cells were differentiated into a bronchial epithelium containing ciliated epithelial, basal, goblet and club cells. NOX4 expression was increased in IPF-derived HBE cells but differed on an individual level. In patients with higher NOX4 expression, pirfenidone induced antioxidant gene expression. All drugs significantly decreased NOX4 expression. IL-6 ( Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible lung disease of unknown etiology, characterized by scarring of the lung tissue associated with a high burden of disease and a detrimental survival rate ,2 In addRecently, two clinically approved antifibrotic drugs, pirfenidone and nintedanib, were shown to ameliorate the course of the disease by slowing down its progression of , althoughttps://clinicaltrials.gov/ct2/show/NCT04598919), accessed on 11 November 2022.Recently, the non-receptor tyrosine inhibitor saracatinib , which inhibits SRC family kinases (SFK) and BCR-ABL kinases, has been suggested as a potential treatment strategy for IPF ,17,18. SIPF pathology is now commonly considered an epithelial-driven and fibroblast-activated process with mild inflammation , and effPatients were recruited at the Center for Interstitial and Rare Lung Diseases, Thoraxklinik, University of Heidelberg  supplemented with bovine pituitary extract (4 µL/mL), epidermal growth factor (10 ng/mL), insulin (5 µg/mL), hydrocortisone (0.5 µg/mL), triiodo-L-thyronine (6.7 ng/mL) and transferrin (10 µg/mL) , sodium selenite , ethanolamine , phosphorylethanolamine , sodium pyruvate , adenine , Hepes , 1x GlutaMAX (Gibco), and 10 µM Rock-inhibitor , as described previously [Human bronchial epithelial (HBE) cells were obtained from endobronchial lining fluid (ELF) of IPF patients and controls by minimally invasive bronchoscopic microsampling (BMS) during bronchoscopy from subsegmental airways as previously described  After BMeviously .After reaching confluency, cells were transferred to 12-well tissue culture inserts and plated at 90.000 cells/insert . After 2–3 days, cells were subjected to air–liquid interface (ALI) culture by removing the medium from the apical chamber and adding PneumaCult-ALI media  to the basal chamber only. Differentiation into a pseudostratified mucociliary epithelium was achieved after approximately 24–27 days. At day 28, cells were stimulated for 24 h with different concentrations of pirfenidone , nintedanib  or saracatinib   that were added to the basolateral side of the medium to mimic the systemic administration of these drugs to patients. After this incubation, RNA, proteins and supernatants were collected see .To validate differentiation to a pseudostratified mucociliary epithelium, ALI cultures were stained with specific antibodies. The cells were washed with PBS and fixed on slides with 4% paraformaldehyde for 20 min at 4 °C. Afterwards, mounted slides were rinsed with PBS and incubated in PBS containing 0.2% Triton X-100 for 2 min. Slides were then cut in 4 pieces and washed with PBS, blocked in PBS containing 1% bovine serum albumin (BSA) for 30 min, and stained with antibodies against keratin 5 , tubulin-β4 , CC10  and mucin 5AC  overnight at 4 °C. The next day, slides were washed with PBS and blocked for 10 min in PBS/1% BSA. Alexa Fluor secondary antibodies 488 and 594  were added, and slides were incubated for 45 min at 37 °C, followed by Hoechst staining  for 10 min at room temperature. Images were obtained with confocal microscopy (Leica TCS SP5) and Leica Application Suite X software.Secretion of interleukin (IL)-8 and IL-6 in cell culture supernatants was determined using human ELISA DuoSet kits  according to the manufacturer’s instructions.Cells were plated in a 96-well plate, grown to 70% confluency and afterwards stimulated for 24 h. The next day, MTT (Sigma-Aldrich) solution  was added to all wells and incubated for 1 h at 37 °C in the dark. Cells were washed with HBSS, and DMSO was added to solubilize the formed formazan. Afterwards, the plate was incubated for 30 min on a shaker, and absorbance was measured at λ = 540 nm.LDH cell death assay was used according to the instructions of the manufacturer .−∆∆Ct method to relatively quantify the expression of genes of interest. These genes included the ROS-producing enzyme NOX4 as well as selected antioxidant genes  according to the manufacturer’s instructions. The RNA concentration was determined using a NanoDrop spectrophotometer (Thermo Scientific), after which cDNA was synthesized from 500 ng isolated RNA using IScript  according to the manufacturer’s instructions. RT-PCR was performed using SYBR Green PCR Supermix (BioRad) with 4.4 μL of 50 times diluted cDNA and 0.5 μmol/L predesigned primers. PCR amplifications were carried out for up to 55 cycles of denaturation  and annealing/elongation  for selected genes . The gennt genes .p value less than 0.05.All quantitative data were represented as means ± SEM. Statistical differences between groups were evaluated by means of 1-ANOVA analysis corrected with Bonferroni’s post hoc analysis or by means of Student’s t-test in GraphPad Prism software  and considered significant at a Human bronchial epithelial (HBE) cells were isolated from the endobronchial lining fluid (ELF) of patients with IPF and non-IPF controls by BMS and grown in ALI cultures. After 28 days of ALI, a complete bronchial airway epithelium consisting of ciliated epithelial, goblet, club and basal cells has developed. The HBE cells were stained with specific antibodies and confocal microscopy was performed showing the successful differentiation of BMS-derived cultures  from conp = 0.19). Additionally, NOX4 mRNA expression was negatively correlated with the diffusing capacity for carbon monoxide (DLCO) (p = 0.19), as well as CAT (p = 0.23) and SOD2 (p = 0.21), appeared to be slightly downregulated in IPF patients, whereas HO-1 and SOD1 tended to be slightly upregulated  B, althouegulated C.To assess the effects of putative IPF drugs, cells were treated for 24 h at dose ranges similar to those used in previous studies ,17,33,34Next, we examined the effect of the selected concentrations of pirfenidone, nintedanib and saracatinib on mRNA expression of NOX4 and selected antioxidant genes. All drugs significantly reduced NOX4 expression in IPF patients A, but dip < 0.05, IPF development is associated with an increase in fibrotic gene expression, and therefore expression of collagen (COL1A1), fibronectin (FN) and TGF was analyzed. Interestingly, neither pirfenidone nor nintedanib affected COL1A1 A, FN FiB or TGF p = 0.02).As IPF pathology can be accompanied by low-grade chronic inflammation, secretion of pro-inflammatory interleukin (IL)-6 and IL-8 was determined using ELISA. HBE cells isolated from IPF patients did indeed secrete increased levels of IL-6 and IL-8 compared to HBE cells from non-IPF patients A,B. TreaIn this pilot study, we demonstrated that ELF-derived cells from patients with IPF and non-IPF controls can be successfully differentiated into a fully stratified bronchial epithelium, consisting of ciliated epithelial, basal, goblet and club cells. Although our previous study revealed differences in basal airway cell expression between patients with IPF compared to non-IPF controls , this diIPF is a complex heterogeneous disease, and although there are two drugs licensed, they are not fully effective in every patient, as disease progression still occurs during their therapeutic use . Given tOur pilot findings with respect to the effectiveness of saracatinib in IPF are in line with the recent study of Ahangari et al. in which this SFK inhibitor was shown to revert various fibrogenic pathways, including immune responses and extracellular matrix organization, in both animal and human models of pulmonary fibrosis . AdditioOur study is the first study that uses pulmonary bronchial epithelial cells obtained from newly diagnosed and untreated IPF patients to evaluate treatment effects on markers of antioxidant genes and pro-inflammatory cytokine release. Although the current consensus is that alveolar epithelial cells are the key cells driving IPF pathogenesis, there is emerging new evidence that bronchial basal cells are also involved in the development of pulmonary fibrosis. The recent PROFILE study has shown pronounced staining in fibrotic lesions throughout the metaplastic epithelium and enhanced serum levels of three serum proteins, i.e., surfactant protein D, CA19-9 and CA-125 . This fiOur research, being a pilot study, obviously has several limitations. First, the small sample size is likely to have contributed to our inability to detect other potentially biologically relevant differences, and extended studies with larger patient groups would be needed to fully address this. Second, our IPF patients also differed from our control subjects with respect to age and smoking history, which clearly could confound analysis of lung function and redox homeostasis and potentially influenced the results obtained. Additionally, it would be of interest to also measure ROS levels as well as reactive nitrogen species (RNS) levels in the cultures to directly assess the oxidant burden. Lastly, the working mechanisms of pirfenidone and nintedanib in epithelial cells remain to be elucidated to fully understand and clinically translate our findings. The fact that nintedanib also inhibits non-receptor tyrosine kinases including SFK  suggestsWhile designing personalized co-treatment strategies for pulmonary fibrosis, the optimal drug-delivery route should be taken into account. Currently, both pirfenidone and nintedanib are orally administered and associated with various unintended side effects. Direct targeted delivery to the lungs might not only reduce these side effects but might also result in higher local drug concentrations, thereby potentially increasing their efficacy . InteresIn conclusion, there are still many open questions regarding IPF treatment strategies. This pilot study suggests that treatment responsiveness may vary substantially between individual IPF patients, which may partially be due to a differently affected redox status, and further argues that treatment should be personalized to maximize its health benefits .


However, most instruments necessitate the physical presence of a clinician for evaluation, were not designed for PD, nor validated for remote application. (2) Objectives: To develop and validate a self-assessment questionnaire that can be used remotely, and to assess the respondents’ health condition. (3) Methods: A questionnaire, so-called Multidimensional Assessment Questionnaire for Individuals with PD (MAQPD), was developed, administered remotely, and completed by 302 people with PD. MAQPD was validated using factor analysis (FA). The participants’ level of impairment was estimated using factor loadings. The scale’s accuracy was assessed estimating floor and ceiling effects and Cronbach’s alpha. (4) Results: FA suggested classifying the questions into daily activities, cognition, and pain. The respondents did not have extremely severe impairment (most scores ranged from 100 to 180 points), and the factors with the lowest scores were cognition and pain. The instrument had no significant floor or ceiling effects (rates less than 15%), and the Cronbach’s alpha value was larger than 0.90. (5) Conclusion: MAQPD is the only remote self-administered tool found in the literature capable of providing a detailed assessment of the general health status of individuals with PD. Parkinson’s disease (PD) is a complex and progressive neurological disorder that causes tremor, rigidity, bradykinesia, and postural instability . PD has Questionnaires are effective tools for multidimensional evaluation . They ofSeveral instruments are being developed to assess general aspects of health and quality of life in people with PD. However, widely used questionnaires and scales, such as the Movement Disorder Society—Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) , cannot The Parkinson’s Disease Questionnaire-39 (PDQ-39)  is anothThe Barthel Index  and KatzIn summary, most of the instruments used to assess individuals with PD have the following limitations: they require the physical presence of a clinician to perform the assessment, were not designed specifically for patients with PD, and/or have not been validated to be self-administered or applied remotely. As a result, the need for developing a self-applied instrument capable of being used remotely, which can provide a detailed assessment of the overall health status of people with PD, was identified.The goals of this study were to create and validate the structure of a multidimensional self-assessment questionnaire for people with PD that can be used remotely, to propose a model for calculating the final score and level of impairment of the respondents, to assess the scale’s accuracy, and to identify the most common limitations of individuals with PD.The questionnaire’s development and application were approved by the Ethics Committee for Human Research at the Federal University of Uberlandia.Validated questionnaires and assessment scales such as MDS-UPDRS , PDQ-39 Section A—Research presentation. This section presents the aims of the research, describes the risks and benefits, as well as the importance of the participation of people with PD.Section B—Questions about daily activities (Module 1), related to self-care and the capacity of individuals to live independently; and questions concerning cognitive aspects (Module 2), related to the individuals’ feelings about life and society. This section presents 66 questions for evaluation by the respondents, of which 43 are distributed to the subgroups of Module 1 , and 23 distributed to the subgroups of Module 2 .Section C—Multidimensional Pain Inventory. This section presents four subgroups of questions taken from the MPI (Portuguese version) , which rThe “Multidimensional Assessment Questionnaire for Individuals with Parkinson’s Disease” (MAQPD) questionnaire was proposed, with a total of 83 evaluation questions . The lanMAQPD uses two response scales: a LIKERT scale with five classification levels  used in Section B, and a LIKERT scale with seven classification levels ranging from 1 (little pain) to 7 (a lot of pain) used in Section C. In addition, the questionnaire presents optional open-ended questions so that respondents have the opportunity to express in detail complaints or aspects not mentioned in the questionnaire. The average time it took to respond to MAQPD was 18 min.People with PD were invited to participate in the study through a variety of channels, including email lists, discussion forums, PD webinars, interviews for local television stations, and WhatsApp groups. The inclusion criteria for the study were: Having a confirmed diagnosis of PD from a neurologist or geriatrician; and being able to fill out the questionnaire independently. Individuals whose responses to the questionnaire were incomplete were excluded from the analysis. MAQPD was completed by 310 people, but 8 of them did not have PD. As a result, 302 answers were analyzed.Factor analysis (FA) was used to validate the structure of MAQPD. FA is a multivariate statistical method used to define underlying structures in data matrices. FA investigates the interrelationships between multiple variables in order to derive a reduced linear structure from the original data . As a reIn short, FA can be performed in two stages: factor extraction and factor rotation. Factor extraction involves choosing the number of factors to be extracted, and factor rotation aims to obtain a simple structure to improve data interpretability. One approach to factor extraction is principal component analysis (PCA), which explains correlations by explaining (common) variance .PCA analyzes a data set in which observations are described by several quantitative dependent variables that are intercorrelated. The aim of this method is to express important information extracted from the data set as a set of new orthogonal variables. In addition, PCA represents the pattern of similarity of observations and variables .PCA calculates new variables obtained as linear combinations of the original variables, called principal components. The first principal component is required to have the highest possible variance, and in this way, this component explains the largest part of the data set. The second and others components are computed under the restriction to have the biggest variance possible and to be orthogonal to the previous component. The values of these new variables for the observations are called factors scores, which can be interpreted in a geometric way as the observations projections on the principal components. The number of factors retained can be observed using the Scree Plot ,18.X1 represents the normalized variable on a scale of 0 to 4, and X2 represents the variable on a scale of 1 to 7.First, the data were normalized in order to convert the response scale of Section C to the response scale of Section B. The normalization was done to satisfy the following relationship: a low score indicates severe impairment, whereas a high score indicates less impairment. Equation (1) was used to normalize questions 67 to 78 and 81 to 83, as these items have an inverse relationship with the response scale from 0 to 4. Equation (2) was applied to questions 79–80 because they are directly related to the response scale of 0–4. For both equations, The Bartlett sphericity and Kaiser–Meyer–Olkin (KMO) tests were used to determine whether the dataset met the requirements of FA. The Scree Plot was used to determine the number of retained factors that were rotated (oblimin) to improve their interpretation and relationship with the original variables.Variables with factor loadings less than 0.35 were considered insignificant based on the size of a sample between 250 and 349 individuals .Variables with very similar factor loadings, i.e., difference between factor loadings less than 0.10, in more than one factor .Variables with communality under 0.30 ,21. Low To improve the overall solution of FA, the following criteria were used to exclude irrelevant variables that satisfied at least one of the three criteria:After excluding variables that did not meet these criteria, FA was performed again to re-evaluate the need for additional variable exclusion.FA assigns a factor loading to each item of the questionnaire, which reflects the importance of the item in representing a given factor. To compute the final score for each respondent, the factor loadings were multiplied by the score given by the respondent in each question, and the sum of these values was calculated.The Spearman’s correlation coefficient between variables was estimated to determine whether the proposed model is consistent. The correlation coefficient was classified into four categories: very weak (0.00–0.19), weak (0.20–0.39), moderate (0.40–0.59), strong (0.60–0.79), and very strong (0.80–1.00) .The final scores of individuals were analyzed using descriptive statistics such as mean, standard deviation, median, and minimum and maximum values. These measures were computed for the entire instrument as well as for each of the FA-defined factors. The violin plot was shown, which reveals additional details about the data distribution. To gain a better understanding of the distribution of the respondents’ final scores, the histogram and cumulative distribution function of the entire instrument, as well as the measures of skewness and kurtosis, were examined.The floor and ceiling effects, as well as Cronbach’s alpha, were used to assess the scale’s precision. When a proportion of respondents achieves the lowest possible score across a given domain, the floor effect occurs, preventing the identification of changes in situations where the health condition deteriorates. The ceiling effect is interpreted in the opposite way as the floor effect . When thCronbach’s alpha is a coefficient that can be used to determine whether or not the questionnaire items are homogeneous and whether or not the scale consistently measures the characteristic for which it was designed. If it was larger than 0.75 it was deemed adequate . Floor aThe most common answer  for each question was identified in order to find the basic activities of daily living that the respondents had more difficulty performing, as well as the cognitive and pain aspects most affected in these individuals. The responses from the optional open-ended questions were also evaluated.In addition, the main motor, cognitive, and pain limitations were identified and discriminated for each group of respondents according to their diagnosis time, also calculating the most common answer for each question.As shown in 2 = 28247.25; df = 3403 degrees of freedom; p < 0.001) and KMO test  suggested a good adequacy of the data to FA. The adequacy of the data for the application of FA was verified. The results of the Bartlett’s test of sphericity  and the KMO test . FA was performed once more to ensure that the items in the new MAQPD  met any Three factors should be retained because their proportion of variance explained was larger than 60%  and the FACTOR 1: Comprised of 43 variables related to the activities of daily living (ADL).FACTOR 2: Comprised of 14 variables related to pain.FACTOR 3: Comprised of 14 variables related to cognitive aspects.Oblique rotation oblimin was used to improve the interpretation of the factors, and the variables were reorganized into three different groups:No other variables needed to be excluded.The factor loadings of each questionnaire item are shown in The model in Equation (3) was used to compute the sum of each respondent’s final score.Individuals’ final scores could range from 0 to 209.36 points if they answered “0” and “4” to all questions, respectively, according to the model. In addition, the lower the score, the more impaired the individual is.The histogram and cumulative probability distribution function of the individuals’ final scores are depicted in The frequency distribution of the data is shown by the histogram. Most scores ranged from 100 to 180 points, and only a few people had low scores, i.e., less than 50 points. The cumulative distribution function expresses the likelihood that a random observation sampled from the population will be less than or equal to a given value. The probability of an individual’s final score being between 100 and 180 points was approximately 62 percent, and the probability of the score being less than half of the possible score (approximately 105 points) was approximately 30 percent. The skewness was −0.37 and the kurtosis was −0.30.There were no significant floor or ceiling effects found, with values ranging from 0.28% to 10.85%. Cronbach’s alpha was larger than 0.90 for all dimensions, indicating that the proposed questionnaire has strong internal consistency.According to the responses, the activities of daily living that people with PD had more difficulty which were represented by Question 13 with a prevalence of zero, indicated that most people completely disagree with the statement; and Question 16 with a prevalence of one indicated that most people disagree with the statement.The most affected cognitive aspects in patients are represented by the following questions: 48, 49, 50, and 51, which are about living with family and society; 53 and 54, which are about an individual’s personality; 55 and 56, which are about energy and mood; and 63 and 64, which are about emotion and feeling. All of these questions had a score prevalence of one.In terms of pain, the following questions indicate the most impairment of the individual: 67, 68, 72, 76, and 83. All questions had a prevalence of scores equal to one.Additionally, 41.39% of the respondents answered the open-ended questions. Of these, most reported problems in performing household chores, such as washing dishes, cleaning the house and cooking.This work was created in response to the lack of an instrument for remotely assessing the health of people with PD. A study  emphasizMotolese et al.  aimed toFirst, the questionnaires applied by Motolese et al.  were notMAQPD was developed on the basis of existing assessment instruments. It was answered by a large number of people with PD, which contributed to a more robust validation of the instrument structure using FA. This method was used to verify each question’s contribution to the multidimensional evaluation and to group questions into latent dimensions, implying a better distribution and arrangement of the questions ,17. FA sThe estimate of factor loadings from FA enabled weighing the final scores based on the relevance of the question to define the factor. Correlation coefficients ranging from 0.54 to 0.89  were estThe final scores for the entire MAQPD were higher than the measures for the three domains, as expected, because the instrument contains 71 questions in total, while the subgroups of questions related to ADL, cognition, and pain contained 43, 14, and 14 questions, respectively . The relWhen compared to pain, the scores for cognition questions were lower . This meThe data distribution  reveals The skewness and kurtosis coefficients of −0.37 and −0.30, respectively, indicate that the final scores had higher values and were dispersed, indicating a sample with lower impairment but not homogeneous (various diagnosis times).Floor and ceiling effects were not found in statistically significant proportions across the entire instrument or across any domain . As a reQuestion 13—“I drive without difficulty” was the only one in the whole questionnaire that presented a prevalence of answer equal to zero, meaning that most individuals responded to this statement that they totally disagree. In this case, a physiotherapist can direct a rehabilitation session that focuses on specific exercises for alleviating the symptoms that prevent the patient from performing the activity, i.e., driving. Therapies may improve specific functions, allowing the activity to be performed again by the individuals.Furthermore, it was determined that approximately 71.43% of the questions pertaining to cognitive aspects had a prevalence of answers equal to one. This indicates that the majority of respondents had issues with social and family coexistence, lacked the energy and motivation to develop routine activities, and struggled to control their emotions . It is nRegarding pain, 35.71% of the questions presented prevalence of answers equal to one (a lot of pain), one of them being question 76—“How much did your pain affect your ability to do housework?”. This result is associated with that obtained by the open-ended questions, for which most of the respondents answered that they had difficulties in performing housework, suggesting that one of the reasons why individuals feel difficulties in performing domestic chores is pain.Individuals with a diagnosis time of less than 2 years, for example, reported that they had great difficulty in dancing, and that they suffered from a variety of problems related to cognitive issues. For these individuals, doing activities that exercise balance, as well as therapy sessions, may be effective ways to solve these problems. In addition, individuals with a diagnosis time between 2 and 4 years reported that they had difficulty in driving and that they did not meet their friends as much as they would like. Possibly, training the body movements that affect driving—either by using a serious game in which the player is stimulated to drive virtually, or with conventional physiotherapy—may contribute toward the individual performing this activity more easily. Furthermore, promoting support groups with social activities in therapy centers, as well as informing friends and family members about the issue related to social interaction, may help to improve the conviviality of these individuals.Additionally, as individuals with diagnosis time of less than 2 years, between 6 and 8 years and more than 10 years had lack of social interaction, mood changes, and/or lack of disposition, performing a physical activity that requires relating to other people, e.g., dancing, becomes an extremely complex task. These findings agree with those reported by Darweesh et al. . These mA different consideration that also should be highlighted is that all groups presented limitations related to cognitive aspects, which indicates that this domain needs a more attentive and careful look by family members, health professionals, and researchers. In addition, with the exception of the group with diagnosis time of less than 2 years, all the other groups reported problems related to pain. Possibly, improving aspects related to cognition may contribute to reducing the pain experienced by individuals, since one may entail the other.The results of an evaluation based on multidimensional questionnaires contribute to a comprehensive understanding of the individual’s health status, as well as their true limitations and needs ,5. UnderA questionnaire, so-called MAQPD, was developed and its structure was validated by using FA, which allowed for grouping the questions into three domains: ADL, cognition, and pain. The final score for each respondent was calculated using the factor loadings as the weight of the questions. The respondents did not present an extremely severe level of impairment. As the instrument did not present substantial floor or ceiling effects, it can measure both the clinical worsening and improvement of the respondents. The ADL that respondents had the most difficulty in performing was driving. Pain and cognition were also relevant aspects.In conclusion, the MAQPD is the only self-administered questionnaire found in the literature capable of being used remotely, and which can provide a detailed assessment of the general health condition of people with PD.


Staphylococcus aureus ATCC 25293, Escherichia coli ATCC 25922, Candida albicans ATCC 10231. Effective bactericidal activity of PCL with small amount of GNP, especially against C. albicans and S. aureus was confirmed. A decrease in this property or even multiplication of microorganisms was observed in direct proportion to the graphene content in the samples.This work contains an analysis of the impact of modifying a bioresorbable polymer—polycaprolactone (PCL)—with various additives on its antibacterial properties. To this end, samples of PCL filament containing various content levels of graphene (GNP), 0.5%, 5%, 10%, were obtained using injection molding. Polymer samples without additives were used for comparison. The next step was to assess the antimicrobial impact of the preparations under study against the following microorganisms:  Polymers are a very important group of materials used in modern medicine. Polymers are used as scaffolding materials for artificial skin and tissue engineering. Polymers are also used in carrier formulations for transdermal and transdermal drug delivery. Polycaprolactones (PCLs) are used in a wide range of applications due to their properties ,2. Both One of its main advantages is biodegradation ,4, the dSpecific polymers offer a wide variety of possibilities for specific uses in biomedical applications. There are various methods of producing PCL implants, such as solvent casting, foaming, electrospinning, phase separation, or molding ,8. The sPolycaprolactone is a biocompatible, nontoxic aliphatic polyester that can be degraded by hydrolysis of its ester linkages in physiological conditions, and it is approved by the FDA for biomedical applications. It is especially very interesting for the preparation of long-term implantable devices and is widely used in long-term implants and controlled drug release applications.Polycaprolactone possesses some antibacterial properties ,11. ThisMany researchers indicate the need for PCL modifications that increase its bactericidal effectiveness. PCL itself can be a carrier of antibacterial substances ,13. Its Graphene is an advanced material which successfully finds application in medicine. Biomedical applications of graphene and its composites include its use for gene delivery and small drug molecules. In addition, it is used for biofunctionalization of proteins, anticancer therapy, and as an antimicrobial agent for bone and dental implants. One of its characteristics is antibacterial properties . All aspDue to its physical and chemical properties and biocompatibility, graphene and graphene oxide can be used as innovative biomedical materials for biodefense, drug distribution in the body, neo-biotherapy, regenerative medicine, and implant surgery. However, graphene-based nanomaterials can be biocompatible or toxic to living cells. The response of biological cells to these nanomaterials depends largely on the number of layers, lateral size, purity, dosage, surface chemistry, and hydrophilicity.S. aureus, E. coli, and C. albicans were used in the study. The materials intended for research were the filaments sticks manufactured for the 3D printing technique.This work presents a comparative analysis of the bactericidal properties of polycaprolactone samples with the addition of varying content of graphene in the form of nanoparticles (graphene nanoplatelets) (GNP). Typical human pathogens—Graphene nanoplates (GNPs) used as fillers consist of sheets of graphene with a total thickness of several nanometers. SEM images of the introduced GNPs are presented in The lowest graphene concentration selected was 0.5 wt%, an intermediate value of 5 wt%, and the maximum concentration used was 10 wt%.Polycaprolactone blends were prepared containing 0.5, 5, and 10 wt% of graphene content. PCL filaments in the form of composite sticks, for further 3D printing, were made using the injection molding method . Three tAs opposed to pure PCL, the PCL/GNP sticks exhibited a more irregular surface caused by the GNP present on the surface a,c,e. InThe SEM (Scanning Electron Microscope) micrographs of the obtained samples were compared with the same magnification X500, X1000, X3000, and X50000. Small agglomerates of graphene formed by sheets were observed on the surface of all modified filaments .C. albicans.Negative controls—normal . Positive controls—normal . The number of observed microorganisms expressed in the form of the CFU number after 17 h of incubation and the resulting antibacterial efficacy (ABE) are both presented in In the charts , the sumMacro- and microscopic evaluations were performed. The results indicate that graphene nanoparticles were successfully incorporated into the polymer filaments. Higher GNP concentration caused an increase in particle agglomeration. A detailed description of the process parameters and the influence on the properties of the obtained materials was presented in our earlier publication . The resS. aureus and C. albicans. However, this effect appears to decrease with increasing graphene content. We found no antimicrobial effect at all against E. coli.The results show a significant antimicrobial effect against S. aureus, PCL without additives shows only slight antimicrobial activity at the 30% ABE level. Modification with a small amount of graphene (0.5%) significantly increases the antimicrobial efficacy, reaching a level of 90% ABE. Further increasing the graphene content significantly reduces and suppresses the antimicrobial properties of PCL. Several authors emphasized that graphene exhibits antimicrobial properties in the form of oxides [In the case of f oxides . The strDuring sample preparation, polymer granules were dry-mixed with GNP powder. It is highly likely that the carbon particles precipitated during the preparation of the culture suspension served as food for the cultured bacteria. However, we concluded that adding only a small amount of graphene (0.5%) to the PCL samples significantly increased their antimicrobial efficacy compared to pure PCL, and further increasing the graphene content effectively reduced the antimicrobial effect.E. coli. In all cases, the inoculated suspensions induced confluent growth of these bacteria. However, a number of researchers confirmed that PCL shows activity against E. coli, but only if the polymer is modified, for example, by silver nanoparticles [None of the tested samples showed activity against articles ,25.C. albicans: for PCL, the average ABE was about 98%; when GNP was added to PCL, this value dropped to 68%. Increasing the content of graphene in the samples effectively reduced the antimicrobial activity. This may be due to the fact that carbon particles present in the suspension can become food for bacteria, through a similar process as in the case of Staphylococcus aureus.PCL and PCL/GNP_0.5 showed significant antimicrobial activity against This research employed polycaprolactone,  and graphene nanoplatelets . In order to produce filaments for further 3D printing, polymer blends with additives and pure PCL granulate were prepared. The polymer granules were dry mixed with GNP powder to obtain 0.5, 5, and 10% by weight of graphene content in the blend. After combining the ingredients, each of the mixtures was mixed in a mechanical mixer for 20 min. Filaments in the form of modified sticks were manufactured in the process of injection molding using a BabyPlast 6/10P  injection molding machine. The parameters of filament production were described in the work . Microstructural analyses to assess the surface of all samples were carried out in a Nova NanoSEM 200 FEI  scanning electron microscope. All the samples were placed on a stainless-steel plate and covered with a carbon thin film by sputtering to avoid the electrostatic charge accumulation. Filaments in the form of sticks were evaluated using a stereomicroscope from the OPTA-TECH company , equipped with a CMOS 3 camera and OptaViewIS software version 4.3.0.6001. For microbiological tests, filaments were cut and bars with dimensions of 1.75 mm × 6 mm were prepared as shown in (1)Staphylococcus aureus ATCC 25293(2)Escherichia coli ATCC 25922(3)Candida albicans ATCC 10231Polycaprolactone sticks with the addition of 0.5, 5, and 10 wt% graphene were evaluated for bactericidal activity against three selected reference microbial strains:5 CFU / ml in tryptone water . The positive control was the volume 2 mL of a suspension of a given reference strain of microorganism in tryptone water, whereas a negative control was 2 mL of tryptone water. After 17 h of dynamic incubation  at 37 °C (S. aureus and E. coli) or 35 °C  from each test sample and control, 20 μL of the suspension was plated on a solid culture medium: Columbia agar with 5% sheep blood (S. aureus), Mac Conkey agar (E. coli), or Sabouraud agar . After 24 h of incubation at 37 °C (S. aureus and E. coli) or 48 h incubation at 35 °C , the observed microbial colonies were counted using the automatic colony counter “aCOLyte”  to determine bactericidal efficacy  or fungicide  of the tested preparations [All stages of microbiological tests were carried out in accordance with the principles of asepsis and antiseptics. Each preparation was tested in each replicate with four replicates. A single sample of the test preparation was placed in a 2 mL suspension of the standard strain of the microorganism with a final density of 1.5 × 10arations ,27.The use of PCL polymers is widespread in modern medicine. Appropriate modifications of this polymer can enhance its basic properties: in this case, the antimicrobial effect. Adding a small amount of graphene can significantly improve these properties.The results of our research show that increasing the content of graphene in the excipients reduces their bactericidal effect or increases the multiplication of bacteria. It is essential that the newly created biomaterials must always be analyzed in detail. Although the base ingredients separately have good bactericidal properties, their combination may reduce this effect.The bacteria which were selected in our work, constitute a saprophytic flora in humans ,29,30. H


The present study investigates the possibility of obtaining graphene-like phases  as fine suspensions by applying a novel pulsed laser ablation (PLA) approach in flow mode. Two types of suspensions of microcrystalline graphite in aqueous suspensions and two types of microcrystalline graphite in suspensions of 6% hydrogen peroxide solution were irradiated in a quartz tube through which they flow. The third (λ = 355 nm) and fourth harmonics (λ = 266 nm) of an Nd:YAG laser system (15 ns pulse duration and 10 Hz pulse repetition rate) were used. The morphology of the obtained particles was studied by transmission electron microscopy (TEM). Their phase composition and structure were explored by X-ray photoelectron spectroscopy, X-ray diffractometry, and Raman spectroscopy.  Pulsed Laser Ablation in Liquid (PLAL) has been proven as a simple and effective technique to fabricate the different types of carbon-related nanomaterials including nanodiamonds, few-layer to multilayer graphene nanosheets, and graphene oxide nanosheets . SystemiThe use of PLAL for the synthesis of carbon materials is chemically and technically very simple. Almost no byproducts are created and no catalyst is used. Many sources of carbon can be used as the starting material.There are three main types of PLAL techniques used to synthesize carbon nanomaterials.The first experimental design of PLAL included a focused laser beam directed on the surface of a solid target immersed in a liquid for generating the colloidal carbon nanomaterials: nanodiamonds and quantum dots ,9, as weFurther on, the solid target is replaced with a suspension , which significantly increases the useful yield in the form of stable suspensions of nano-sized carbon phases in water . The laAnother implementation of PLAL uses a jet stream of a suspension of carbon particles irradiated by a focused laser beam . The colThe present research examines the opportunity of obtaining graphene-like phases  and studies their modification and oxidation level when applying a pulsed laser ablation in liquid (PLAL) in flow mode.The phase composition of the obtained sedimented suspensions was characterized by different methods: X-ray photoelectron spectroscopy (XPS), grazing incidence X-ray diffractometry (GIXRD), Raman spectroscopy, and transmission electron microscopy (TEM)2O2 in bidistilled water) was used in the other suspension as the liquid medium, maintaining a concentration of 4 mg/mL solid to liquid in all experiments of this study. The suspensions were prepared and placed in an ultrasonic bath 30 min before irradiation.Two different types of suspensions based on two graphites, 99.9995% purity Ultra F , designated as HQ, and low-grade graphite , designated as LQ, which were obtained by mixing 0.4 g of graphite in 100 mL of bidistilled water. Hydrogen peroxide solution  were close to the maximum and minimum absorption values shown; therefore, the radiation was absorbed by all phases of the suspension at λ = 266 nm and mainly by the graphite phase at λ = 355 nm. The irradiation of the entire suspension lasted about 15 min. The obtained graphene-like phases were about 8–11% of the mass of the starting graphites, but we must note that part of the modified particles agglomerated with the main phase and settled. Irradiation took place in an environment isolated from atmospheric air, which, combined with a suitable liquid medium, enabled not only the preparation of different carbon phases but also the realization of their effective functionalization, which is an advantage over other PLAL methods. For the implementation of this method of laser ablation in liquid, no complicated equipment is needed, and there are no moving parts. This makes the PLAL in flow mode method highly reproducible and very easy to apply.−1 or better. The X-ray photoelectron spectroscopy (XPS) analyses were carried out using an ESCALAB MKII spectrometer with a non-monochromatic Al X-ray source under vacuum greater than 10−8 Pa at a 45° take-off angle. The Au4f photoelectron line was used for the calibration of the spectra. The accuracy of the measured BE was 0.2 eV. The photoelectron lines of the constituent elements on the surface were recorded and corrected by subtracting a Shirley-type background and quantified using the peak area and Scofield’s photoionization cross-sections. The C1s, O1s, and Auger lines were recorded for each specimen and further on, the C1s lines were subjected to an additional fitting procedure with XPSPEAK 4.1 software.The Raman measurements were carried out using backscattering geometry with a micro-Raman HORIBA Jobin Yvon Labram HR 800 visible spectrometer equipped with a Peltier-cooled CCD detector with a He-Ne (633 nm wavelength and 0.5 mW) laser excitation. The laser beam was focused on a spot of about 1 μm in diameter, the spectral resolution being 0.5 cmThe structure of the suspensions after drying was characterized by XRD using a PANalytical Empyrean apparatus, whereas the local morphology and structure were revealed by a high-resolution transmission electron microscopy (HR TEM) characterization in both high-resolution (HR) and selected area electron diffraction (SAED) modes with an HR STEM JEOL JEM 2100 microscope.There is always an amount  of unmodified graphite particles in the resulting suspensions, which settle up to 5 min after the laser treatment and are removed from the remaining suspension by decantation. The suspension obtained after decantation was sufficiently stable over time: visible precipitation occurred after more than 24 h. For further research, powders obtained after drying the decanted suspension at 50 °C were used. In a control experiment, suspensions prepared in the manner described were examined without being irradiated and showed results consistent with those of pristine graphite.2 and sp3), structure, and defects  of carbon materials [XPS analysis is useful for identifying the chemical composition, hybridization , sp3-, hydroxyl C–OH/epoxy groups, C–O–C functional groups, and π-π* bond. The C–H bond and oxygen-containing bonds were formed due to the presence of edge and defect sites in the graphite, which provided suitable anchoring sites for the functionalities [2-hybridized C atoms arranged on the corner of a hexagon as in graphite. The peak at ∼285.4 eV corresponds to sp3-hybridized carbon atoms and likely originated from unsaturated carbon atoms reacting with hydrogen during the oxidation process. An increase in the oxygen content on the surface of HQ graphite was observed after laser treatments in H2O, as well as in H2O + 6% H2O2 for both laser wavelengths compared to the pristine one. The calculated sp3/sp2 ratio did not change drastically  [Additionally, in the spectra appeared shake-up satellite peaks in comparison with the pristine one in the high-binding energy range (higher than 292.0 eV). The peaks that appeared at higher-binding energy regions can be associated with the formation of Ces (CNT) .2O2 with 266 nm, seven peaks acted, situated at around 284.8 eV, 286.3 eV, 288.0 eV, 289.5 eV, 291.0 eV, 295.0 eV. In addition, two other peaks appeared at around 282.0 eV and 283.9 eV. The shoulder appeared at around 282.0 eV, most probably due to a charge-neutralized fraction due to the existence of hydrocarbon [2O and H2O2 caused similar changes in the C1s photoelectron line. Only four peaks were used for the fitting of C1s to H2O; however, seven peaks were used for the fitting of C1s to LQ graphite in the H2O2 suspension. Again, the laser treatments of H2O led to the formation of GO, and the same laser treatment of LQ graphite in H2O2 led to the formation of rGO. Furthermore, the peaks that appeared higher than 292.0 eV in the C1s spectra were associated with the formation of C60 and CNT [2 or sp3 features in carbon-like materials [2 bonds, whereas the slopes of the spectra for the other samples gradually changed and tended to that of the sp3 bond, especially for the laser-treated suspensions prepared with LQ graphite , the intensity of the G- and 2D bands decreases. It should also be noted the formation of:-2O2, modified by laser irradiation with a fluence of 0.364 J/cm2;a very fine crystalline (or semi-amorphous) phase of GO  bands , which were associated with the enrichment of graphene/graphite phases with oxygen-containing radicals [−1, whereas the intensity of the 2D band was comparable to that of the G-band. It should be noted that such flakes were observed more frequently in suspensions irradiated with a 355 nm laser wavelength.In contrast to the suspensions discussed above, a large variety of graphene-like phases  were observed in those of LQ graphite in bidistilled water and bidistilled water with 6% Hradicals . SimilarThe XRD patterns obtained from pristine HQ graphite ((002) graphite reflection (COD # 96-101-1061) intensity decreased several times (more than eight times) after the PLA process using 355 nm and fluence of 0.465 J/cm2 in HQ graphite suspensions. Moreover, the addition of 6% H2O2 to the initial suspension of graphite HQ in water further reduced the peak by more than 50%. In parallel, the same peak practically did not change its position but significantly increased its full width at a half maximum (FWHM). The increase in this parameter in those treated with radiation with a wavelength of 266 nm was more than 50% in both suspensions. The same parameter (FWHM of d(002) reflection) determined in the XRD patterns of the suspensions modified with a wavelength of 355 nm increased by 35% in the suspension in bidistilled water and by 63% in that in a solution of hydrogen peroxide in bidistilled water. The above results undoubtedly show that PLA processes cause fragmentation of graphite particles, which most likely takes place by the exfoliation of single- to multilayer graphene and its further agglomeration during sedimentation. The treatment increased the width of all reflections, whereas some reflections disappeared. This allowed us to conclude that a process of amorphization was taking place. The dFor LQ graphite, there was a decrease in the reflection intensity and an increase in the width after the PLA process. The detailed analysis of these results did not differ significantly from that already conducted for the 266 nm- and 355 nm-modified suspensions of the same material in water in a non-flow system . By analThe specimens for the TEM studies were prepared by dropping and drying a drop of any suspension obtained on formvar-covered TEM grids. It should be emphasized that due to the method of obtaining the samples, the visualized nano-sized objects were stuck to each other despite the sonication of the suspension prior to the immobilization on the grid Thus, agglomerates of a much larger size were formed, and individual single-layer graphene flakes were very rarely observed. 2O2 in water, which probably caused a decrease in the crystals after treatment with the H2O2 solution. Amorphous material was detected in the LQ graphite sample even before the H2O2 treatment. Additionally, the TEM characterization of all the suspensions studied showed the formation of different phases after the PLA processes depending on the initial graphite and the composition of the liquid phase of the suspensions:(i)2O2 in water solutions. The graphene/graphene-like flakes were usually stuck to each other.Graphene and defected graphene flakes, 2O2 in water solutions, as well as more frequently in suspensions of LQ graphite. Aggregates of C60 were observed in all suspensions.Bundles of 2D (mainly graphene and graphene-like flakes stuck together with fullerene C60), as shown in (iii)Sedimented agglomerations of amorphized carbon phases surrounded by 2D (graphene and graphene-like flakes or C60 fullerenes arranged in 2–3 layered nanostructures), as well as 3D (carbon nanoparticles up to 10 nm in size or fullerenes C60), nano-sized particles see .60 fullerene (COD # 96-800-0216)  see a. The di216) see b corresp-60 (COD # 96-800-0216) (see 60 (COD # 96-800-0216)  3D particles sedimented on the amorphous particles. These 3D particles were almost spherically shaped a and pos216) see b, with H216) see c. Spheri-60 (COD # 96-101-1061 and COD # 96-800-0216). These agglomerations were observed in HQ graphite suspensions in H2O irradiated with a λ = 266 nm wavelength only.Relatively large formations (size larger than 500 nm) with an irregular form and amorphous structure Practically completely amorphized graphitic carbon see . Such pa2O2 solution as a model system. It has been shown that irradiated suspensions containing mainly nanoscale carbon phases  and bundles of graphene-like flakes) can be produced by this method using laser-beam wavelengths of λ = 266 nm or 355 nm. The new approach for PLAL enables us to effectively achieve the desired degree of functionalization of the processed carbon materials with different types of atoms since the irradiation is carried out inside a quartz tube, isolated from the environment. The ability to control the medium, flow rate, laser wavelength, and irradiation energy flux enables the wide use of this technique. Finally, established flow-mode PLAL procedures have been found to be reproducible, and the main advantages of the proposed method are its simplicity, ease of application, and ability to synthesize large amounts of nano-sized carbon phases, as well as the opportunities it provides for effective functionalization.This work investigates a proposed new method for PLAL in flow mode of an aqueous suspension of microcrystalline graphite, as well as in a flow of microcrystalline graphite, in a 6% H


As deformation and defects are inevitable during the manufacture and service of graphene resonators, comprehensive molecular dynamic (MD) simulations are performed to investigate the vibrational properties of the defective single-layer graphene sheets (SLGSs) during tension. Perfect SLGSs, SLGSs with single vacancy, SLGSs with low-concentration vacancies, and SLGSs with high-concentration vacancies are considered, respectively. The frequencies of the perfect and defective SLGSs at different stretching stages are investigated in detail. The effects of different external forces are also taken into account to study the vibration properties of the defective SLGSs. Results show that the perfect and defective SLGSs both successively perform four stages, i.e., the elastic stage, the yield stage, the hardening stage, and the fracture stage during stretching, and the elastic properties of the SLGSs are insensitive to the vacancy defects, while the ultimate strain is noticeably reduced by the vacancies. The single vacancy has no effect on the vibration properties of SLGS, while the frequency decreases with the increasing vacancy concentration for SLGS at the elastic stage. The frequency of yielded SLGS with a certain vacancy concentration is almost constant even with a varying external force. The two-dimensional 2D) nanomaterial with a monatomic layer, graphene, has attracted significant attention from the scientific and industrial communities since its first isolation in 2004. Due to its extremely high mechanical, electrical, optical, and thermal properties, graphene has been considered an ideal material for use in nanoelectromechanical systems (NEMS) D nanomat,4,5,6, wSimilar to other known materials, perfect graphene sheets are almost impossible to manufacture, even when fabricating experimentally. It is quite well known that defects in materials have a great influence on the properties of crystals and nanostructures ,16,17,18In order to understand the vibrational properties of graphene nanoribbons at different tensile conditions, this paper treats the graphene nanoribbon as a finite-length pre-strained SLGS with clamped ends. The originality of the present work includes: (1) The tensile performances of SLGSs with different vacancy concentrations are studied, (2) the SLGSs with varying pre-strained degrees are taken into consideration to analyze their vibrational properties. We, first, perform extensive MD simulations to analyze the tensile characteristics of the finite-length SLGSs with different vacancy defects, and then study their natural frequencies with varying pre-strained degrees. The effect of different external loads on the resonant response of the pre-strained SLGS with clamped ends is also analyzed to ensure the safety of the graphene nanoribbons. All the calculations related to this work were done by the large-scale atomic/molecular massively parallel simulator (LAMMPS) an open-source molecular dynamic software on Windows .L = 10.365 nm, thickness h = 0.335 nm, and width W = 3.24 nm, including (a) perfect sheet, (b) single vacancy, (c) low-concentration vacancies, and (d) high-concentration vacancies SLGSs. Three layers at both ends, colored in red, of all the SLGSs are fixed, while the rest are free parts that are colored in blue. A periodic boundary is used in the X and Y directions, and a shrink-wrapped boundary is used in the Z direction.In the MD simulations, the adaptive intermolecular reactive empirical bond order potential (AIREBO) is adopted. As given in detail in Stuart et al. , the AIRractions .−1 is implemented along the positive X-axial until the graphene nanoribbons collapse. The strain increment is applied after every 1000-time steps. The Velocity–Verlet algorithm and Nosé–Hoover thermostat are used.Each model is initially relaxed using canonical ensemble (NVT) simulation until the energy of the system is fully minimized for a specified temperature of 100 K, then axial strain applied. The deformation-control method with a strain rate of 0.0005 psAfter computing and exporting the stress and geometrical characteristics of the defective SLGSs during tension through the LAMMPS codes, the tensile stress-strain curves of the SLGSs with different vacancy concentrations are obtained, as shown in To explain the vacancy sensibility, f  per atom  by selecting the kinetic energy of the systems as time-domain signals. In summary, extensive MD simulations are performed to understand the tensile strength of finite-length graphene nanoribbons with varying vacancy concentrations and their vibration properties at different stretching states. The graphene nanoribbon is simulated by SLGS. Perfect SLGSs, SLGSs with a single vacancy, SLSGs with low-concentration vacancies, and SLSGs with high-concentration vacancies were considered. Through the tensile stress-strain curves, all the SLGSs with different vacancy concentrations went through the four stages, i.e., elastic stage, yield stage, hardening stage, and fracture during the tensile process. The vacancies have a greater influence on the ultimate strain of the SLGSs, than the elastic limit and yield limit. As for the vibrations of the defective SLGSs, different external forces are respectively applied to investigate their resonance frequencies at different stretching stages. Even the single vacancy had no effect on the vibrational properties of the SLGSs, the increasing vacancy concentration decreases the resonance frequencies of the SLGSs at the elastic stage. The frequency of yielded SLGS with a certain vacancy concentration is almost constant even with a varying external force.


Graphene, an extraordinary tow-dimensional carbon nanostructure, has attracted global attention due to its electronic, mechanical, and chemical properties; therefore, there is a need to find out an economical mass production method to produce graphene. In the present research, the aim is to find out optimal conditions for exfoliation of few-layers graphene (FLG) in a water–ethanol green solution. We varied different parameters of the ultrasonic probe like power quantity and time duration of sonication to investigate the effects on the number of graphene layers and density of graphene in the solution. Also, an attempt has been made to predict the acoustic pressure distribution by solving the wave equation in various output powers of the ultrasonic probe (sonotrode) using numerical simulations. The simulations and experimentations verify each other. Concluding that modifying the output power at the same condition will significantly alter the acoustic pressure inside the sonoreactor. The difference in acoustic pressure at 90% output power of our experimentations is much higher than in other conditions. Experimentation results utilizing UV–visible spectra, SEM (Scanning electron microscope), TEM (Transmission electron microscope) images and Raman spectrum indicate that the minimum thickness and maximum exfoliation for these samples are acquired for sonication at 90% of the maximum effective output power of the sonicator being 264 W for 55 min. The firstly pioneering experiments have been carried out on micro mechanically cleaved monolayers, but micro mechanical method has the disadvantages of low production yield and poor throughput. In order to utilize graphene for future industrial applications requiring large-scale and high-output processing methods are required6. At present, the reduction of graphene oxide is the preferred scalable method for the preparation of graphene. In this method, the oxidization of graphite by following Hummers modified method, followed by exfoliation of graphite oxide in water, to give aqueous dispersions of graphene oxide (GO) and further the oxidation can be removed by thermal or chemical reduction8. However, the reduction of graphene oxide still retains a high defect density, which degrades their properties.Recently, graphene as a single atomic layer of carbon has attracted significant attention for research due to its unique nanostructure, mechanical, electric and novel thermal properties10. Graphene sheets can be extracted through sonication11 or shear exfoliation12 in an aqueous surfactant solution and certain organic solvents. Several groups have shown that graphene can be prepared in N-methyl-2-pyrrolidone13 (NMP), and dimethylformamide14 (DMF), to produce graphene nano-sheets. These solvents have surface tension close to 40 mJ m−2 and are suitable for direct exfoliation of graphite into FLG15. However, these solvents have high boiling points that limit their viability for real use. Thus, dispersion of graphene in low boiling point solvents is useful in many applications, such as nanocomposites16 nanoplasmonics19, smart coating20, and high-frequency devices23. Many reports present the effect of sonication parameters on produced MoS2 nano-flakes and graphene in DI water-ethanol26. A simple, low-cost, and energy-effective sonication to exfoliate natural graphite flakes in ethanol in a short time sonication is reported27. Studies are aimed at determining the rate of exfoliation of various forms of graphite and its oxide using the top-down method and using UV–Vis spectroscopy for a deeper understanding of the electronic transition are presented29. In another article, one can find out UV–Vis. method to measure the percentage of few-layer sheets in GO dispersions quickly and cheaply30.Most researchers have focused on the direct solution-phase exfoliation of natural graphite flaks to overcome the limitation of graphene oxide. A few layers of graphene were synthesized by evaporating polystyrene at atmospheric pressure by the chemical vapor deposition (APCVD) methodIn this research study, an attempt has been made to predict the acoustic pressure distribution by solving the wave equation in various output power of the ultrasonic probe. Simulations have been carried out by using a numerical model. Then Graphene is prepared via the LPE (liquid phase exfoliation) method utilizing the ultrasonic probe. Then we investigated the various effects of the output power, sonication time duration for the same concentration to find the optimal condition to achieve more graphene with thinner layer from graphite powder. Then simulations are compared with experimental results.In this section material method and the samples preparation are presented.All the chemicals used in this project were research-grade and deionized water (DI) throughout the experiments. The used liquid medium is a green solution prepared by a mixture of 65% DI Water and 35% Ethanol. The sonication is carried out using the ultrasonic processor with a titanium probe (Tip) of 22 mm in diameter (FAPAN-1200UPS model made in Iran by FAPAN Co. Ltd.), operation frequency 20 kHz, and nominal power of 1200 W (RMS power of 300 W).31. But water has a surface tension of about 72 mJ m−2. The range of solvent surface tension that can better exfoliate graphene32 is 40–50 mJ m−2. Optimal surface tension to minimize interfacial tension between graphene and solvent is a mixture of water and a solvent with lower surface tension. Ethanol is a good choice because it has a low boiling point with surface tension close to 22.1 mJ m−2. The 65/35 volume ratio for DI water/ethanol is calculated using the Connors-Wright equation with the surface tension of the solution to be 46 m J m−2 for making binary aqueous organic solutions27. The beaker is kept in an ice-water bath to reduce evaporation of the ethanol due to an increase in the temperature of the solution during the sonication process. Furthermore, the sonicator is adjusted to pulse mode of 50% to prevent overheating. So, the total effective sonication time for preparing the samples is 55 min. The thick flakes are eliminated by centrifugation using a bench top centrifuge: the first step is done for 30 min at 3000 rpm, and then the top 3/4th of the dispersion is retained for the next step, then the rest of the liquid is discarded. Afterward, the second step is carried out again for 15 min at 3600 rpm, and finally, the top 3/4th of the supernatant is collected for the following analyses.Experimentation for all samples is carried by immersing 250 mg of graphite powder added into 125 mL of water–ethanol in a 150 mL glass beaker. We aim to utilize a “green solvent” in order to minimize the environmental impacts. As expected, water is the greenest solventIn this section, an attempt has been made to predict the acoustic pressure distribution by solving the wave equation in various output power of the ultrasonic probe. Simulations have been carried out by using a numerical model. The Computational Fluid Dynamics is a powerful tool that can be used to optimize the characteristic of the sonoreactor. The present numerical study aims to deliver a detailed explanation and instructions to model the sonoreactor under different operating conditions. In the following section, the computational domain, the physical models, and the boundary conditions will be presented. For such an arrangement, it is usually assumed that the highest local pressure value is reached in the close vicinity of the probe. The intensity 34.As the distance from the probe increases, the intensity decrease according to an area into which it is spread in a conical shape. For calculation of the intensity distribution, the distribution of ultrasonic pressure amplitude The acoustic pressure can be obtained by solving wave equation. If linear wave propagation is assumed and the shear stress is neglected which is corrected for liquids and gases, the wave equation has the form:ω is the angular frequency.The pressure p is considered time harmonic i.e.,An axisymmetric 2D section of the ultrasonic processing cell with an ultrasonic probe is used as the modeling geometry. Figure 35.The pressure at the probe tip is calculated from the equation The acoustic pressure distribution zone in the solution is quantified by estimating the area of the regions within the ultrasonic processing cell. The output power is identified to be influential in determining the size of the resultant acoustic pressure distribution zone. The output power for the validation study is listed in Table It is predicted that this output power would be the most effective among the considered range of parameter for the performance of acoustic pressure distribution for ultrasonic liquid processing. For the acoustic pressure analysis, various output powers were selected and the maximum and minimum acoustic pressure and pressure difference i.e. entclass1pt{minima6 Pa and the minimum pressure is − 0.43 × 106 Pa. While, at 90% of output power (~ 264 W) the maximum pressure is 1.4  × 106 Pa and the minimum pressure is − 1.3 × 106 Pa which is supported by the earlier report32. The difference between the maximum and the minimum pressures  in the optimum sonication time of 55 min. From the previous results of the experimentation in this research, the best condition is observed to use a solution with a volume of 125 CC. The effect of changing the output power of sonication in detail by measuring their UV–Vis absorption spectra and SEM are recorded.* transitions of graphene and graphite34. Figure The optical characterization of the graphene sheets is performed with a Perkin-Elmer model lambda 25 spectrometer. Using the UV–Vis spectrometer absorption spectrum one can estimate the approximate thickness of the layers or equally the approximate number of layers. The UV–Vis absorption spectra of graphene samples for 40–90% of the maximum power of the continuous radiation mode are shown in Fig. This result is also confirmed by SEM (ESEM Philips XL30) images, as shown in Fig. 32, the change of output power alters the acoustic pressure range , multi-layer (4–10 layers), and thick layer (> 10 layers) graphene by previous report35.From simulation of acoustic pressure distribution, we found that at the same conditionPa Table  reveals Influence of the effective time duration of the sonication for graphene dispersions in the water–ethanol solutions at 264 W power is studied to find out the range of graphene flakes production in different sonication time duration of 25, 35, 45, 55, and 65 min, which the results are shown in Fig. The SEM micrographs shown in Fig. All findings in this research result in the fact that our most delicate graphene samples in these experimentations are acquired at; power of 264 W, pulse duration of 50% and 55 min irradiation duration which is the “optimum situation” for producing graphene flakes.The structural characterization of the graphene nano-sheets is further examined by TEM Zeiss EM10C, 100kv). Figure C, 100kv.−1, the G peak at ~ 1586 cm−1, and the 2D peak at ~ 2655 cm−1 are existing. Therefore, Raman spectrum of the sample as shown in Fig. For more reassurance, the analysis of Raman spectroscopy is used. Figure s report , the numWe studied the effects of the critical ultrasonic LPE parameters, i.e., power and time duration of sonication, on the quality and yield of FLG flakes produced in DI Water–Ethanol in a vessel with 150 mL of water/ethanol-powder solution. In conclusion, graphene produced by sonication is pure graphene and does not need reduction from GO or purification by washing out chemicals. A distribution of acoustic pressure events throughout the treated volume assisted by acoustic pressure difference is a critical factor in producing more quantity and high-quality FLG flakes. The numerical simulations approve the experimental results, which is an increase in the sonicator radiation power increases the conversion of graphite to graphene due to an increase in pressure differences. In addition, for 264 W of sonicator power, the shift in absorption spectra from 266 to 270 nm occurs, which reveals graphene exfoliation with few-layer (1–3 layers). The UV–visible technique is proven to be very effective in studying the number of layers exfoliated and detecting the difference in conjugated polyenes that affect π to π* plasmon peak.Characterization by UV–visible, SEM, TEM, and Raman spectrum shows that the few-layer graphene for sonication time of 55 min is the optimum for high-quality pure graphene exfoliated from graphite powder for this setup. Graphene with a sonication time of 55 min has a thinner and larger sheet compared to sonication for less or more time duration. Therefore, our findings from the analyses presents the optimum operating parameters for sonicator with the probe of 22 mm in diameter and pulse mode being 50% with 250 mg of graphite in the 125 ml of water–ethanol solution and 150 mL beaker to achieve few-layer graphene flakes with good yield are obtained at 264 W of power, and 55 min of sonication time duration.


B = 30 T), graphene can support the QH effect up to room temperature (RT), concomitant with a non-negligible population of acoustic phonons with a wave-vector commensurate to the inverse electronic magnetic length. Here, we demonstrate that graphene encapsulated in hexagonal boron nitride (hBN) realizes a novel transport regime, where dissipation in the QH phase is governed predominantly by electron-phonon scattering. Investigating thermally-activated transport at filling factor 2 up to RT in an ensemble of back-gated devices, we show that the high B-field behaviour correlates with their zero B-field transport mobility. By this means, we extend the well-accepted notion of phonon-limited resistivity in ultra-clean graphene to a hitherto unexplored high-field realm.The quantum Hall (QH) effect in two-dimensional electron systems (2DESs) is conventionally observed at liquid-helium temperatures, where lattice vibrations are strongly suppressed and bulk carrier scattering is dominated by disorder. However, due to large Landau level (LL) separation (~2000 K at  Monolayer graphene can support the quantum Hall effect up to room temperature. Here, the authors provide evidence that graphene encapsulated in hexagonal boron nitride realizes a novel transport regime where dissipation in the quantum Hall phase is mediated predominantly by electron-phonon scattering rather than disorder scattering. The use of hBN as atomically-flat encapsulating dielectric, in particular, permits a drastic reduction of extrinsic disorder in graphene devices2, leading to the observation of zero-field transport regimes dominated by either electron-electron3, electron-hole4 or electron-phonon (e-ph) interaction5, which manifest over different carrier density and temperature ranges. Toward RT (T ~ 300 K), the scattering of electrons with acoustic phonons was theoretically identified as the main intrinsic contribution to the electrical resistivity in graphene8, implying a carrier mobility exceeding 105 cm2V−1s−1 at low carrier concentration (n < 1012 cm−2). While such figures could already be inferred from early data on disordered SiO2-supported graphene (~104 cm2V−1s−1 mobility)10, at present, the reach of the zero-field acoustic-phonon-limit is firmly established as a generic property of high-quality graphene devices5, also when encapsulated in hBN crystals from different sources11 or engineered to high doping levels (n > 1013 cm−2)12. Notable exceptions to the cleanness-implies-high-RT-mobility scenario are suspended graphene samples, where flexural phonons dramatically contribute to carrier scattering leading to a T2 behaviour of the resistivity13, and rotationally faulted graphene bilayers close to magic-angle, showing strong phonon-driven T-linear resistivity14. The difference between freely suspended graphene and graphene encapsulated in hBN is due to the fact that in the latter case van der Waals interaction between graphene and substrate makes flexural phonons harder, suppressing an intrinsic rippling instability15.Van der Waals heterostructures of graphene and hBN have recently granted experimental access to novel phenomena in condensed matter16 at temperatures close to RT. In this sense, we note that previous literature on the RT-QH effect in graphene20 exclusively includes experiments on SiO2-supported devices, precluding such investigation.In this work, we address the fundamental question whether the e-ph mechanism in clean graphene could also govern the electrical transport in the QH regimeEF) lies on the localised states between two LLs, formed in a perpendicular magnetic field and separated by an energy gap ΔLL. The interplay between this energy scale and the thermal energy kT governs the basic phenomenology of the electrical transport in the QH regime. When ρxx) when measured in four-probe configuration . The pre-factor to the exponential term, ρ0, which is often not considered explicitly, determines the magnitude of the T-activated resistivity . In conventional 2DESs, the small ΔLL leads to a complete suppression of the QH effect within a few K25, where the Eph-controlled phonon population can be considered negligible. Although the low electronic mass in 2DESs such as InSb26 and HgTe29 enables the observation of the QHE up to liquid-nitrogen temperature, this is insufficient to ensure Eph and the corresponding T-dependent excitation probability for acoustic phonons in graphene at B = 30 T are shown in Fig. T-activated resistivity . The Hall conductivity (σxy) presents weak slope changes around filling factors ν = ±2 (Vg ~±20 V), while the shelves-like features at low carrier concentration originate from the onset of electron-hole coexistence in the highly-degenerate N = 0 LL30. ρxx, in addition to the pronounced maximum around the charge neutrality point (CNP), shows two sizable minima  over the whole n range. Importantly, sample D3 shows a μ(n) dependence comparable to the data of ref. 5 (dash-dotted line), demonstrating the standard fingerprint of phonon-limited RT mobility in zero magnetic field12 . The overall high quality of the samples is also supported by the observation of fractional QH states at liquid-helium temperature . In Fig. 32) and the charge inhomogeneity in the CNP region, estimated as the usual n* parameter33 . In addition, as for refs. 34, the linear μ−1(n*) dependence  span a low-disorder range unexplored in previous RT-QH experiments, and (ii) present a well-defined disorder type, with increasing impact along the D4-to-D1 sequence.In Fig. T on the Landau-quantized electrons in graphene at B = 30 T. Toward RT, the broadening of the Fermi-Dirac distribution around EF  ensures excited charge carriers from both the N = 0 and N = 1 LLs, across the giant gap ΔLL. Accordingly, the local resistivity minimum at filling factor ν = 2 leaves zero and displays increasing finite values, as shown in the experimental curves of Fig. T-dependence of ρxx (ν = 2) for samples D1-4, at selected magnetic fields  in graphene on SiO2), and two theoretical calculations defining different dissipation limits (continuous lines). In both cases we take an activation energy equal to ΔLL/2: this was shown to be accurate for high B-fields in ref. 20 and should hold true for clean graphene with reduced LL broadening. The upper line (yellow) assumes the universal conductivity pre-factor due to long-range disorder (2e2/h)23, multiplied by a factor 4 to take into account the LL degeneracy of graphene. The lower line (dark cyan) is based on the work by Alexeev et al.24, who calculated the conductivity mediated by two-phonon scattering for graphene in the RT-QH regime. The relevant e-ph process conserves the LL number, but modifies the in-plane electronic momentum. We note that this phenomenology is fundamentally different from that of magnetophonon oscillations, recently discovered in extra-wide graphene devices36, which rely on resonant inter-LL scattering at T < 200 K. Here, two-phonon scattering within each LL contributes with a conductivity pre-factor σ0 = σN(T/300 K)(B/10 T)1/2, which depends both on temperature and magnetic field (in contrast to the constant pre-factor commonly assumed in QH studies). In the ν = 2 state, the predominant contribution to the σN terms comes from the N = 0 LL 24. Strikingly, the resulting activated behaviour, not including any free parameter, is well approximated by our devices, while the reference data from graphene on SiO2 follow the long-range disorder limit. The qualitative agreement between theoretical calculations and experimental data, together with the contrasting behaviour with respect to previous reports20, indicate that graphene/hBN heterostructures support an e-ph-dominated transport in the RT-QH regime. Arrhenius-type fits to the conductivity37, shown in Fig.We then employ the sample temperature as an experimental knob to control the excitation of both phonons see Fig.  and bulkT curves are acquired for D4 due to experimental limitations) with a generalized relation . This term is intended to account for the effect of residual disorder, and it is the only free parameter in the fits. In Fig. ρD for the three samples at different magnetic fields, as a function of the n* parameter (averaged between the electron and hole-side). The linear ρD(n*) behaviour observed here , is also the one to show a Drude mobility comparable to the zero-field e-ph limit5. Taking into account the sample-dependent correction due to residual disorder, in SI , suggesting a temperature-driven crossover between regimes dominated by either disorder or e-ph interaction . While it is not surprising that the e-ph limit works as a lower bound to the activated resistivity of real samples, the non-universality  of the disorder contribution deserves particular attention in future theoretical treatments of the RT-QH in graphene.Despite the presence of long-range potentials Fig. , our dat SI Fig.  we proceSI Figs.  and S8 w215. In both cases, ripples induce inhomogeneity of electron density with electron and hole puddles in the vicinity of the CNP39. In particular, for the case of graphene on SiO2 the amplitude of induced inhomogeneity of charge-carrier density is estimated as 3 × 1011 cm–239, in agreement with the above cited experimental values of n*. This makes the system strongly disordered, and any intrinsic scattering mechanisms become irrelevant. Oppositely, the hBN substrate is atomically flat1 and at the same time suppresses intrinsic ripples which increases the RT carrier mobility by an order of magnitude and makes intrinsic scattering mechanisms dominant15. Indeed, experimentally measured n* for our samples is an order-of-magnitude smaller than what is supposed to be induced by ripples at RT. This results in an essentially different picture of QH physics at high enough temperatures.The physics of graphene is essentially determined by its deviations from flatness , due to either thermal fluctuations associated to flexural phonons for freely suspended samples or to roughness of substrate like for graphene on SiOIn conclusion, we showed experimental evidence of predominant e-ph scattering in the QH regime. This is realized by uniquely combining strong magnetic fields, high temperatures and hBN-encapsulation of graphene. Although the RT-QH in graphene has long been known, we showed that mitigation of disorder via van der Waals engineering provides novel insights on the transport mechanisms in this phenomenon.5, starting from micromechanically exfoliated graphene flakes previously identified by optical and Raman microscopy. Sample D4 is obtained by CVD growth on Cu foil and direct hBN-mediated pick-up after controlled decoupling via Cu surface oxidation31. All the devices are fabricated making use of electron beam lithography, reactive ion etching and e-beam evaporation of Cr/Au 1D edge contacts5.hBN/graphene/hBN samples D1-3 are assembled using the standard van der Waals dry pick-upρxx and ρxy measurements in four-probe configuration, either under a constant current excitation  or a constant voltage bias . The devices are mounted in a VTI system with low-pressure 4He serving as exchange gas, coupling the samples to a liquid-N2 reservoir. The cryogenic system is accommodated in the access bore of a resistive Bitter magnet at HFML-EMFL, with a maximum field of 33 T.We use standard lock-in acquisition at low frequency (13 Hz), with simultaneous Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.Supplementary Information


A polarization-insensitive broadband terahertz absorber with a sandwich structure of metal–dielectric-graphene is designed and simulated. The graphene is patterned as an array of graphene square blocks with circular apertures. The results of the simulations and theoretical analysis show that the absorption exceeds  The terahertz frequency band located in the transition region from electronics to photonics in an electromagnetic wave is generally regarded as eristics . In receeristics ,4. There0/21 [Graphene, a single layer of carbon atoms, was the first two-dimensional material discovered  and has  TM mode . The proee space . The abs.165 THz . Althougectively . The proof 62.8% . HoweverIn this study, a polarization-insensitive broadband tunable terahertz absorber is designed and simulated. The absorption acquired exceeds The schematic view of the designed absorber is displayed in The structural parameters of the proposed absorber are listed in In the calculations, graphene was considered a very thin dielectric film with a thickness of  formula :(1)σ, there are two factors for the surface conductivity of graphene. The former originates from interband transition and is represented by the symbol  formula :(2)σg≈σiAccording to Formula (2),  spectra . Based oThe properties of the designed absorber are analyzed with a commercial simulation software named FDTD solutions . S21|2 and 11|2, respectively, where S11|2. Considering the impedance matching principle, when the equivalent input impedance matches the free space impedance, the absorption achieves the maximum yield, and the normalized equivalent input impedance can be described as follows [The absorption can be expressed as  follows :(3)Z(ω)=According to Formula (3), when xP = yP = esonance . When thesonance .The influence of different geometrical parameters on absorption properties are illustrated in The effect of the parameter The influence of the middle dielectric layer with different thicknesses is depicted in Whether the absorber has dynamic adjustability was one of the important criteria to judge its performance. As an excellent two-dimensional material, the conductivity of graphene has proven to be connected with its chemical potential An important index parameter to evaluate the performance of an absorber is to see whether it can always retain good absorption performance under the conditions of different polarization modes, different polarization angles, and different incident angles ,22. The The influence of incident angles was also discussed. The related results are illustrated in Finally, comparisons of our designed absorber to other previously reported absorbers were carried out, and the results are shown in In conclusion, a simple structure of a graphene-based terahertz absorber is designed to achieve high absorption, broadband, and polarization insensitivity. The proposed device consists of a periodical array of graphene square blocks with circular apertures, a polyimide dielectric layer as a spacer, and a gold plate as a substrate. Terahertz absorption is more than