Aphasia in bilingual patients is a therapeutic challenge since both languages can be impacted by the same lesion. Language control has been suggested to play an important role in the recovery of first (L1) and second (L2) language in bilingual aphasia following stroke. To test this hypothesis, we collected behavioral measures of language production  in each language and language control (linguistic and nonlinguistic switching tasks), as well as fMRI during a naming task at one and four months following stroke in five bilingual patients suffering from poststroke aphasia. We further applied dynamic causal modelling (DCM) analyses to the connections between language and control brain areas. Three patients showed parallel recovery in language production, one patient improved in L1, and one improved in L2 only. Language-control functions improved in two patients. Consistent with the dynamic view of language recovery, DCM analyses showed a higher connectedness between language and control areas in the language with the better recovery. Moreover, similar degrees of connectedness between language and control areas were found in the patients who recovered in both languages. Our data suggest that engagement of the interconnected language-control network is crucial in the recovery of languages.  Due to the increasing number of multilinguals in modern society, the incidence of language impairments induced by brain lesions (aphasia) in this population is growing rapidly , 2. The Most of the current literature indicates that language recovery in bilingual aphasic patients depends on the degree of language mastery or language-specific factors –7. For eAbutalebi and Green , for insWhile this theory accounts for the large variability in recovery patterns of multilingual aphasia, there is only sparse evidence for any association between control function and language recovery since control functions are rarely specifically assessed in aphasic patients. Aglioti et al.  reportedThe network underlying language control described by Abutalebi and Green  and AbutAs a descriptive marker of behavioral improvement/changes in language and control functions, the patients were behaviorally evaluated for their pattern of recovery of language and executive functions using general aphasia evaluation (GAE), picture naming and executive tasks (linguistic and nonlinguistic switching).In order to investigate the connections within the language-control network, we used fMRI analyses and applied dynamic causal modelling in the fMRI picture naming task in L1 and L2 to examine whole brain activation patterns and the effective connectivity between the control areas  and the regions involved in language production . We further examined whether global changes in connectedness within language-control network are associated with the recovery of languages.To directly assess the hypothesis advanced in the language-control model , we examBased on the hypothesis of a key role for cognitive control in bilingual language production and in the recovery of bilingual aphasia, our study aims to test whether among the different control areas proposed by Abutalebi et al. , changes We chose to evaluate bilingual aphasic patients during the subacute phase since this population has rarely been studied in the acute and subacute phases. This will allow us to better understand the contribution of the control system in the recovery of language in bilingual aphasia, especially during the period when spontaneous recovery process mainly takes place , 23. In We recruited right-handed late (age of acquisition (AoA) of L2 after 6 y/o) bilingual patients aged between 18 and 85 years old, who suffered from aphasia following a focal left-sided ischemic or hemorrhagic stroke. The following languages were included in the study: French (in each case as subjects' L1 or L2) and English, German, Spanish, or Italian. During the recruitment procedure, we excluded patients with a history of premorbid language impairment, several brain lesions, or severe aphasia.A total of eleven patients were recruited for this project. However, only six patients completed all the steps of the study and, among them, five subjects fulfilled our criteria of the selection of regions of interest (ROIs) for the DCM analyses and therefore are reported in this paper. Five more patients performed the first session of the study and then declined to participate in the second session see . The stuCase DescriptionPatient 1. YL is a 61-year-old man who is a French (L1)-English (L2) bilingual. Mr. YL was born in French-speaking part of Switzerland. The language of teaching at school was French. Mr. YL estimates an advanced level of English for reading, speaking, and comprehension . Before the stroke, his language usage was mainly in French; he spoke 100% French with his family and 80% with his friends. He followed TV and radio programs only in French. However, his reading was 50% in French and 50% in English (readings in English are mainly work-related books and documents), and he used mainly English at his workplace (80%).Mr. YL was admitted to Geneva University Hospital (HUG) with right sensorimotor hemiparesis, right facial palsy, and impaired comprehension and language production mainly manifested in L2 following a left frontotemporal ischemic stroke. A secondary hemorrhagic event in the ischemic area was seen three days after the ischemic event . A firstPatient 2. MR is a 65-year-old Italian (L1)-French (L2) bilingual woman. She was born in Italy to Italian parents and followed primary school in Italy. She moved to the French-speaking part of Switzerland at the age of 24, and then she has taken some courses to learn French. Before the stroke, she used Italian and French quite equally; she used French at work (100%), and Italian for TV or radio programs (100%). She used 50% in Italian and 50% in French to speak with her family and friends and to read books and journals.MR was admitted to HUG for resection of a meningioma on the left greater wing of the sphenoid bone. Two days after the resection of the meningioma, she presented a right sensorimotor hemiparesis and a severe language production problem plus a lesser degree of comprehension problems in both languages, caused by an epidural hematoma with pressure over the operation site and ischemic changes in the left frontobasal area . The iniPatient 3. CA is a 63-year-old woman who is an Italian (L1)-French (L2) bilingual. She was born in Italy to Italian parents and followed primary school in Italy. She moved to the French-speaking part of Switzerland at the age of 10; thereafter she started to learn French. Mrs. CA followed secondary school in Switzerland where the teaching language was French. She has also basic knowledge in English and Spanish, which she has learned at school. Before the stroke, the main language of conversation was French with her husband and children (90%) and at work (75%) and she spoke Italian with her parents (100%).She was admitted to HUG because of right hemiparesis and severe global aphasia due to a left basal ganglia hemorrhagic stroke with no evidence of midline shift . Within Patient 4. RG is a 49-year-old bilingual French (L1)-English (L2) male patient. He finished primary and secondary schools in the French-speaking part of Switzerland. He started to learn English at school at the age of 14. He used English quite frequently in his daily life; he used French and English equally at work (50% French and 50% English for teaching and customer care). He followed TV and radio programs and also read books and journals 50% in French and 50% in English. However, he spoke only in French with his friends and family. According to the self-evaluation questionnaire filled in by his wife, his language abilities were estimated as follows: speaking 50%, comprehension 70%, reading 85%, and writing 30%.He was admitted to Fribourg Cantonal Hospital with a sudden right hemiparesis and anomia and no other language symptoms due to a left sylvian ischemic stroke .Patient 5. GH is a 79-year-old bilingual French (L1)-English (L2) male patient. He has learned English around the age of 18 when he first travelled to the US and England. He has then moved to Sweden and started to learn Swedish too. He has been working in Sweden for about 18 years teaching guitar in both English and Swedish. He then moved back to Switzerland at the age of 66. He then continued to teach playing guitar to children. He used both French and English in his teachings (50% French and 50% English). With his family he spoke only in French; however with his friends he spoke 50% in French and 50% in English. He followed TV and radio program mostly in French (75% in French and 25% in English) and he read books and journals only in French.He was admitted to HUG with a paresthesia in his left arm and global aphasia. GH was a known case of auricular fibrillation before this acute event. The cerebral CT scan after the acute event confirmed an ischemic lesion in the left frontal, insula, and sylvian areas . Within http://dx.doi.org/10.1155/2016/8797086.More details of patients can be found in Tables The data and results on the control subjects are presented in Supplementary Data 2 and 3.Subjects were assessed using a questionnaire on their immersion in both L1 and L2, AoA, how long they had lived in a region where predominantly the second language was spoken, which language they spoke with their family members, in school, and in present activities , and if the language was acquired in school or out of school only. In the self-evaluation part, subjects (or their family members) had to indicate in percentages how well they would estimate their reading, speaking, comprehension, and writing skills.Patients were assessed at subacute  and chronic  phases. In both sessions we used the same procedures, listed as follows: (1) behavioral assessment of the severity of aphasia as well as a combination of language-control function evaluations; (2) in an fMRI recording session, the patients performed a language production task (picture naming) in each language : global severity of the aphasia and language capacities was assessed using a separate evaluation of language capacities in each language . This evaluation consisted of a brief test of object naming (ten objects to name), automatic speech , word and phrase repetition, yes/no questions, object recognition, following oral and written instructions , description, and verbal fluency. All these tests were extracted from the Bilingual Aphasia Test (BAT)  except fA linguistic switching task : forty A nonlinguistic switching task: four images (a red or blue circle or square) were presented on the upper or the lower part of the screen. Subjects were instructed to name, as quickly as possible, the color of the image when the image was presented on the upper part of the screen and to say the shape of the image when it was presented on the lower part of the screen. After a fixation cross of 500 ms, the images were presented on the screen for 5,000 ms and were followed by a blank screen of variable duration of 3,000–7,000 ms . Therefore, the subjects had at most between 8,000 and 12,000 ms to respond. However, only first-attempt correct responses within five seconds of the presentation of the image were scored as correct. Each trial was started manually by the experimenter when the word “ready?” was presented on the screen. The first six trials of the task were cued with the category in which the image should be named (color or shape) written on the left of the image . The tasLanguage-control functions were evaluated using the following: For all the tasks, instructions were given both written on the screen and orally, and the subjects performed a short training session just before starting the task. The evaluation of the language-control function was performed in the more proficient language . Moreover, because of slowness of patients and fatigability, for all the tasks we did not record reaction times, and the analyses were focused on response accuracy.Because of the limited number of patients, differences in lesion size and site, and variability of symptoms, we used primarily a multiple single-case approach for our analyses between T1 and T2. For comparison of the patients' scores in the two sessions, a McNemar Chi-squared test is used for each case. GAE, picture naming, and “combined production score”  are assessed as language performances. Specifically, we focused on the “combined production score” which could better represent language production performance.Stimuli. Five lists (one list per language) of 40 noncognate words (black and white line drawing pictures) were selected from Snodgrass and Vanderwart [nderwart . The worProcedure. Each fMRI session started with a picture naming in L1; in this part, the subjects were instructed to name the pictures that appeared on the screen in their L1. After a fixation cross of 500 ms, the images were presented on the screen for 5,000 ms and were followed by a blank screen of variable duration of 4,100–6,100 ms . Therefore, the subjects had at most between 9,100 and 11,100 ms to respond. However, only first-attempt correct responses within five seconds of the presentation of the image were scored as correct. Each task lasted around 7-8 minutes . After about 30 seconds of rest, the subjects started their second task in which they had to name the pictures in their second language. The first six trials of the task were cued with the language in which the image should be named (L1 or L2) written on the left of the image. For the fMRI tasks, a short training was performed before the subjects entered the scanner. In this training, which contained 10 trials, the subjects were presented with black and white line drawing pictures selected from Snodgrass list and were asked to name the pictures in their L1 or L2 to become familiar with the task.Data of the aphasic patients were acquired using three different 3T scanners on two different sites; Site 1: Fribourg Cantonal Hospital (HFr) and Site 2: University Hospital of Geneva (HUG). The scanners which were used were (1) Discovery MR750; GE Healthcare, Waukesha, Wisconsin, with a 32-channel receive head coil (Site 1), (2) Magneton Trio, Siemens Medical Solutions, Erlangen, Germany, with a 12-channel receive head coil (Site 2), and (3) Magneton Prisma, Siemens Medical Solutions, Erlangen, Germany, with a 20-channel receive head (Site 2). Subjects were in a supine position with their heads stabilized by foam to reduce head movements. They wore headphones  coupled with an MRI-compatible microphone  to record oral response during the experiment. In the first scanner, visual stimuli were presented on an LCD screen . In the other two scanners, the stimuli were displayed on a screen by a video projector  through a mirror system. In all three cases, the stimuli resolution was 1024 × 768 with a refresh rate of 60 Hz. The E-Prime 2 software  was used to show stimuli and record behavioral data.∗weighted echo planar images (EPI) with blood oxygenation level-dependent (BOLD) contrast were acquired with voxel size: 2.3 × 2.3 × 3 mm, FOV = 220 mm, 37 ascending axial slices, interslice spacing = 0.2 mm, TR/TE = 2000/30 ms, flip angle = 85, and PIAF: 2. In addition, a B0 field inhomogeneity mapping sequence was acquired to correct for geometrical distortion that occurred along the phase-encoding direction (using a Gradient Echo protocol) with the same scan coverage as the functional scan: number of slices = 37, FOV = 220 mm, TR/TE1/TE2 = 50/4.9/7.3 ms [∗weighted EPI with BOLD contrast were acquired with voxel size: 2 × 2 × 3.5 mm, FOV = 240 mm, 29 ascending axial slices, interslice spacing = 0.35 mm, TR/TE = 2000/30 ms, flip angle = 85, and PIAF: 2. A B0 field inhomogeneity mapping sequence was also acquired with the same scan coverage as the functional MRI sequences: number of slices = 29, FOV = 240 mm, and TR/TE1/TE2 = 400/5.19/7.65 ms. On average, a total of 248 volumes were acquired during the picture naming in L1 and picture naming in L2. Each fMRI acquisition session started with six seconds of dummy scans to ensure a steady-state magnetization of the tissues.MRI acquisition parameters were optimized for each site. From the first site in Fribourg (Scanner 1), T1-weighted images were acquired with a FSPGR BRAVO sequence, voxel size: 0.86 × 0.86 × 1 mm, field of view (FOV) = 220 mm, number of coronal slices: 276, TR/TE = 7300/2.8 ms, flip angle = 9, phase acceleration factor (PAF) = 1.5, and intensity correction (SCIC). Functional T29/7.3 ms . From th3 voxel size, and smoothing with a Gaussian kernel of 8 mm full width at half maximum (FWHM). In order to exclude the brain lesion from the analyses, a mask file of the brain lesion of each subject was manually drawn on axial slices of the standard Montreal Neurological Institute's (MNI) brain template using the MRIcron software (https://www.nitrc.org/projects/mricron) and used during the preprocessing of data on SPM. The preprocessed volumes were submitted to fixed effects analyses at the subject level by applying the general linear model to each voxel [We used the SPM8 software , running on MATLAB 2012b , to analyze functional MRI data (fMRI). FMRI images were preprocessed following the standard procedure proposed by Friston . Preprocch voxel . Each stDCM is a widely used method for investigating context-dependent causal interactions between brain regions and it describes the architecture of the network . In DCM, the brain is treated as a dynamic input-state-output system. A given experiment is considered as a designed perturbation of neuronal dynamics that is propagated throughout a network of interconnected nodes. Three sets of parameters are estimated in DCM: the direct influence of stimuli on regional activity (driving input), the intrinsic connections between regions, and the changes in the intrinsic connectivity between regions induced by adding or removing a modulatory influence (modulatory effect). a priori hypotheses already tested by previous works. Therefore, the selection of the ROIs and the intrinsic connections was based exactly on the work by this group. Accordingly, the following five ROIs were selected for the network: BTLA, LIFGTri (areas related to language processing), head of left caudate, ACC (areas involved in cognitive control function), and LIFGOrb as a part of both language and cognitive control systems. As per Abutalebi et al. [p < 0.05 uncorrected were extracted from each 7 mm spherical ROI centered at the subject's local maximum inside the ROIs. For the patients, we have verified visually whether the ROIs were affected by the lesion. When the patients or control subjects did not fulfill our criteria  they were removed from the analyses. This way, we have removed one patient (as one of the ROIs was inside the lesion) and one control participant (as he did not show activation in one of the ROIs in the desired threshold) [We based our analyses on this model, which has been defined by Abutalebi et al. . Becausei et al. , we alsoreshold) .However, in order to take into account the modulatory effect of the language task on the network , we inserted the modulatory effect of the task over BTLA (as the sensory input of the network)  and LIFGConsidering the limited number of patients and the effect of the different scanners used in this study, we primarily performed the analyses at a single-case level. Patterns of brain activation in the four different conditions (picture naming in L1 and L2 at T1: subacute phase and T2: chronic phase) for each patient are shown in the figure representing the data related to the patient Figures .Regarding the DCM analyses, in order to investigate how connection strength changes over time for single intrinsic connections within the network, the differences in the strength of connection between L1 and L2 (L2 − L1) at each session are presented in a graph for each patient. These graphs represent the pattern of difference in connection strength in language-control network while performing picture naming in L1 and L2 across time  and the changes in combined production scores for each language separately.(A) We first conducted McNemar Chi-squared tests comparing language performance (GAE and picture naming scores) in L1 and L2 and control function (linguistic and nonlinguistic switch task scores) across time; (B) using DCM on fMRI, we compared the strength of connectivity within the language-control network between L1 and L2 across time at single subject level; (C) at the group level, we then performed a correlation analysis between the recovery of language production scores and the changes in the strength of connection between language and control areas. A description of the main results of the analyses is provided here, and a complete reporting of the scores and results is provided in (A) Behavioral Scores. At T2 (chronic phase), the combined production score showed improvement in L1  but no changes in L2. In addition, no improvement was found in linguistic and nonlinguistic switching tasks accuracy (accuracy .(B) Changes in Connectivity in the Language-Control Network. For each single intrinsic connection within the network, the differences in connection strengths between L1 and L2 (L2 − L1) at each session are shown in (A) Behavioral Scores. At T2 (chronic phase), the combined production score improved in both L1  and L2 . However, no significant improvement was found in linguistic  and nonlinguistic switching tasks  (p: 0.47)  (see Tab(B) Changes in Connectivity in the Language-Control Network. Regarding the DCM analyses for each single intrinsic connection within the network, the same approach was used as for patient 1 (atient 1 . Importa(A) Behavioral Scores. At T2 (chronic phase), the combined production score showed improvement in both L1  and L2  at T2 (chronic phase). The patient also showed a significant improvement in both linguistic and nonlinguistic switching tasks across time    (see Tab(B) Changes in Connectivity in the Language-Control Network. The differences between L1 and L2 (L2 − L1) in the strength of single intrinsic connections within the network are shown in (A) Behavioral Scores. At T2 (chronic phase), the combined production score improved in L2  and no improvement was seen in L1 ). His performance in the linguistic switching task improved significantly  and his nonlinguistic switching performance was spared at T1 (subacute phase) (e phase) .(B) Changes in Connectivity in the Language-Control Network. At the single intrinsic connection level, the differences between L1 and L2 (L2 − L1) in strength of single intrinsic connections within the network for each session are shown in (A) Behavioral Scores. The combined production score improved in both L1  and L2  at T2 (chronic phase), although the patient still made several language switching errors. However, no improvement was seen in the linguistic and nonlinguistic switching task performances ((B) Changes in Connectivity in the Language-Control Network. At the single intrinsic connection level, the differences between L1 and L2 (L2 − L1) in the strength of single intrinsic connections within the network for each session are shown in p < 0.001 was selected to visualize the main effects  Correlation Analysis between Language Production Recovery and Changes in the Strength of Connection. At the group level, in the aphasic patients, the changes in the strength of intrinsic connections between language and control areas  were implemented to correlate with the changes in combined production scores.p: 0.026). Moreover, changes in the combined productions score in L2 were negatively correlated to the changes in the strength of connections from LIFGTri to LIFGOrb .In the aphasic patients, we found a significant correlation between changes in the combined production scores in L1 and changes in the strength of connection from ACC to LIFGTri (while performing picture naming in L1) .To better compare the changes in the number of connections with higher strength values between the improved versus unimproved language across time, we concatenated the data of patients 1 and 4 (who improved language production in only one language). This combined analysis showed a higher number of connections in the improved language at T2 (chronic phase)  we monitored modifications in language and control performance to identify whether language recovery was linked with the recovery of control functions. Moreover, (B) using a DCM approach, we examined how the interconnections between language and control areas changed with the recovery of language production, and (C) we then investigated the possible correlation between changes in language production performances and changes in the strength of each single connection within language-control network across time.Considering the changes in the combined production scores, three of our five patients recovered in both L1 and L2 , one patient recovered in L1 (patient 1), and one (patient 4) in L2 only . Two patients (patient 3 with recovery in both languages and patient 4 with recovery in L2) showed improvement in language-control functions . No decrDescriptive analyses of the DCM suggested a relationship between the pattern of recovery of language production and changes in the strength of connections across time. In patient 1, who recovered only L1 production score across time, the majority of connections within language-control network (10 out of 15 connections) had higher connection strength values at the chronic phase, indicating a higher connectedness within the language-control network while picture naming in L1. The similar pattern of changes in the connectedness within language-control network took place in patient 4 who recovered only L2 . In these two patients with recovery of only one language, combined analyses revealed that improvement in production score in one language was associated with an increase in the number of connections with higher strength values at T2 (chronic phase) while performing the task in that specific language. In addition, showing a similar pattern of changes in the language-control network connectedness, patients 2 and 5 at T1 (subacute phase) in the majority of connections had higher coupling values for picture naming in L2, while at T2, the coupling values of 7 connections were higher in L1 and 8 connections had higher coupling values in L2 task. Also, patient 3 showed higher coupling values for the majority of connections for L1 picture naming at T1 (subacute phase), while at T2, the coupling values of 7 connections were higher in L1 and 8 connections had higher coupling values in L2 task. Taken together, in patients 2, 3, and 5 who recovered both L1 and L2, a redistribution of the connection strength occurred across time; the strength of the connections between language and control areas was similarly distributed at T2 (chronic phase) over the network during picture naming in L1 and L2. In the control group with main L2 exposure and usage in daily life, a higher connectedness was seen within the network for L1 compared to L2 (see Supplementary Data 2 and 3). We will discuss each of these results in turn.Although the role of control functions in the recovery of bilingual aphasia has been suggested in several studies , 9, in oOur findings on the changes in the differences in connectedness between L1 and L2 within the language-control network are in line with the results of Abutalebi et al. , in the Furthermore, our results suggest that a change in connection strength from ACC to LIFGTri during picture naming in L1 was associated with L1 recovery; the coupling between these two areas became stronger when L1 recovered. LIFGTri, along with the LIFG pars opercularis (BA44), is known to be involved in different steps of language production , namely,Analyses of the changes in connectivity strengths across time suggest that, in patients with L2 recovery (four out of five patients), the connection from LIFGTri to LIFGOrb becomes weaker for L2 compared to L1. This finding is also supported by the result of a correlation analysis showing that the recovery of combined production scores in L2 negatively correlates with changes in the strength of connection from LIFGTri to LIFGOrb. In other words, when L2 recovers, the coupling from LIFGTri to LIFGOrb decreases. These two regions are strongly anatomofunctionally interconnected as subregions of the inferior frontal gyrus . LIFGTriThe present study suffers from several limitations. First, we could not implement certain language and control tasks since they were too demanding for aphasic patients, especially in the acute and subacute phases. A comprehensive evaluation of language and control would have helped better understand the possible correlation between the recovery of control functions and the recovery of language performance following a stroke. Even though we narrowed our selection of tasks to a limited series of evaluation materials, half of the initially recruited patients dropped out of the second session of the study. Another limitation of this study was the small number of included patients; their results may not be easily applicable to all bilingual aphasic patients. In addition, we could not control the age of the patients in the study (the age of patients ranged from 49 to 79 years old). It is known that the age factor can affect behavioral performance, functional brain activity, and connectivity within brain areas as a result of alteration in neuronal activity and connectivity in aging brain , 49. HowIt worth noting that, in the present study, only three patients followed language therapy sessions and the therapy was computer assisted to improve lexical access and in turn improve naming performances. One patient (patient 1) received therapy in his L1 (French) and he then improved in L1 production. This lack of improvement in L2 could be explained by the very low L2 usage and immersion by this subject. It has been previously suggested by Edmonds and Kiran  that theTaken together, our findings supply additional evidence that the engagement of the interconnected language-control network is crucial for the recovery of languages. Furthermore, we suggest that L1 recovery is improved by increased connectivity between ACC and LIFGTri, which prevents conflicts from the second language. However, L2 recovery requires a decrease in connectivity from LIFGTri to LIFGOrb in order to decrease the automatic activation of the L1 lexical system, which, according to the Revised Hierarchical Model, has stronger links with the conceptual system.In order to support data included in the main text, additional data is provided in Supplementary Material in three parts. Supplementary data 1 includes detailed information on patients' scores in General Aphasia Evaluation and Picture naming task. Supplementary data 2 includes information on control subjects as well as the results of DCM analysis on this subjects. Supplementary data 3 shows the pattern of brain activation whilst picture naming in L1 and L2 in control subjects.


The NH3 molecule is found to play a dominant role in enhancing the strength of defective graphene by up to ~15.6%, while other gas molecules decrease the strength of graphene with varying degrees. The remarkable strength enhancement can be interpreted by the decomposition of NH3, which saturates the dangling bond and leads to charge redistribution at the defect site. The present work provides basic information for the mechanical failure of gas-adsorbed graphene and guidance for manufacturing graphene-based electromechanical devices.Pure graphene is known as the strongest material ever discovered. However, the unavoidable defect formation in the fabrication process renders the strength of defective graphene much lower (~14%) than that of its perfect counterpart. By means of density functional theory computations, we systematically explored the effect of gas molecules (H So far, a considerable number of theoretical and experimental studies have been performed to investigate the effect of adsorbates on the structural and electronic properties of host materials. However, the effect of gas adsorption on the mechanical properties has been rarely studied. Unveiling the effect of gas molecule adsorption on the mechanical properties of the graphene layer would help us understand this issue, which is important for its applications in flexible electronic nanodevices and other related fields.Noticeably gas adsorption on nanomaterials202122232425262, N2, NH3, CO, CO2 and O2, have been considered due to their importance to environmental and industrial applications. We first investigate the structural and mechanical properties of the perfect graphene (P-graphene) with adsorbed gas molecules. Then, the change of ideal strain for the D-graphene upon gas molecule adsorption is investigated. Finally, the mechanisms behind the strength change are analysed to gain more insights into the effects of adsorbed molecules on the mechanical properties of D-graphene.In this paper, we performed a systematic study of gas adsorption on the perfect/defective graphene layer to evaluate the mechanical strength change. Many common gases, namely Hab intio simulation package (VASP) code3032−5 eV and 0.005 eV/Å, respectively. A 5 × 5 × 1 and 17 × 17 × 1 Monkhorst–Pack k-point sampling was used for geometry optimizations and static electronic structure calculations, respectively. The adsorption energy (Ead) of a gas molecule on graphene layer was obtained by the following equation: Ead = E(gas+G) − (EG + Egas), where E(gas+G), EG and Egas are the total energies of the gas adsorbed graphene system, graphene and the adsorbed gas molecule, respectively. According to this definition, a more negative Ead value indicates a more favourable adsorption.Our spin polarized DFT computations were performed by utilizing Viena a, which is determined by the expression a = a0 (1 + ε). Here, a0 is the equilibrium lattice constants of graphene at 0% strain. The values of ε increased in steps of 0.4% until the graphene sheet fractured and the strain-stress relation was obtained. To eliminate the artificial effect of the out-of-plane thickness of the simulation box on the stress, we employed the second Piola–Kirchhoff stress37The strain36First, we search for the lowest-energy configuration when a gas molecule is adsorbed on P-graphene. To find the most favourable position for gas adsorption, we place the single gas molecule above the graphene layer at different distances and orientations. After full optimization, the obtained configurations are compared, and the energetically most favourable states are selected for further discussions.3 is presented in 3 molecule prefers to locate in the hexagon center  are given in the 2, NH3 and O2 is exothermic while others are not favoured energetically, and only O2 adsorbed system is magnetic.As a representative, the case for NHn center , and then center  between Before studying the strain effect on the gas-adsorbed graphene, we investigated the strain-stress relationship in the P-graphene as shown in the 3, where the fracture point drops to only 15.6% relative to that of P-graphene (>30% of the fracture point). These results well explain why the synthesized graphene layers exhibit relatively low intrinsic strength (~15% of the fracture point) under realistic experimental conditions, which include gas exposure.However, the adsorption of a single gas molecule on the top of graphene sheet can significantly decrease its mechanical strength . The larNormally, a material’s ideal (intrinsic) strength can be traced down to its bond strength2, N2, CO, CO2 or O2 molecules makes the critical strain for D-graphene decrease by 0.4~2.0%  for NH3 also indicates that it dissociates on the D-graphene sheet. Furthermore, all of the six gas-adsorbed systems become magnetic.Our computations show that the adsorption of H0.4~2.0% . The mosne sheet , and the3 adsorption on defective graphene, experimentally it has been demonstrated that the NH3 molecule can easily dissociate on this host layer3 + D-graphene system at different strains, as shown in 3 decomposition needs to overcome an energy barrier (Ea) of 0.5 eV for the strain-free D-graphene, while the Ea value decreases as the strain increases. As a 0.5 eV energy barrier is not sufficient to hinder the NH3 decomposition, it is reasonable to expect the NH3 decomposition to occur without stain, while the finite strains can accelerate this process.With regard to the NH3 decomposition on defective graphene, as revealed by our NEB calculations, is further verified by the optimized structural configuration of NH3 + D-graphene system  (3 adsorption induces obvious corrugation on the D-graphene layer (The high possibility of NHe system . The NH3m (-c-H) . This dine layer  due to t2 is further dissolved into NH and -c-H .


Antibodies targeting the PD-1/PD-L1 immune checkpoint achieved spectacular success in anticancer therapy in the recent years. In contrast, no small molecules with cellular activity have been reported so far. Here we provide evidence that small molecules are capable of alleviating the PD-1/PD-L1 immune checkpoint-mediated exhaustion of Jurkat T-lymphocytes. The two optimized small-molecule inhibitors of the PD-1/PD-L1 interaction, BMS-1001 and BMS-1166, developed by Bristol-Myers Squibb, bind to human PD-L1 and block its interaction with PD-1, when tested on isolated proteins. The compounds present low toxicity towards tested cell lines and block the interaction of soluble PD-L1 with the cell surface-expressed PD-1. As a result, BMS-1001 and BMS-1166 alleviate the inhibitory effect of the soluble PD-L1 on the T-cell receptor-mediated activation of T-lymphocytes. Moreover, the compounds were effective in attenuating the inhibitory effect of the cell surface-associated PD-L1. We also determined the X-ray structures of the complexes of BMS-1001 and BMS-1166 with PD-L1, which revealed features that may be responsible for increased potency of the compounds compared to their predecessors. Further development may lead to the design of an anticancer therapy based on the orally delivered immune checkpoint inhibition. Anticancer therapies targeting immune checkpoint receptors have witnessed a spectacular success in the last years. Antibodies blocking CTLA4 interaction with CD80, and, to even more extent, PD-1/PD-L1 interaction, provided unprecedented results. This merited accelerated approvals by regulatory agencies, offering a real cure to certain formerly lethal cancers .PD-1 is expressed on activated T cells  and tranRecent data demonstrate that PD-L1 not only is overexpressed on the surface of cancer cells, but the level of soluble PD-L1 (sPD-L1) is elevated in the plasma of some cancer patients , 16. It Antagonizing the PD-1/PD-L1 interaction reverts the exhausted phenotype of T cells and allows efficient killing of cancer cells , 21. TheThe development of small-molecular weight inhibitors is expected to bring a number of profits in the field of the PD-1/PD-L1 immune checkpoint blockade (ICB). This is due to several advantages of small molecules over therapeutic antibodies, which are: lower production costs, higher stability, improved tumor penetration, amenability for oral administration and elimination of immunogenicity issues . However1H-15N HMQC NMR titration experiment with positive results, as reported elsewhere [1H-15N resonance signals of the 15N labeled PD-1 is observed due to an increased transverse relaxation rate of the higher molecular weight entities [1H-15N signals of PD-1  showed moderate toxicity  used in all further assays were exposed to the increasing concentration of the tested compounds for 48 h. Considerable differences in the toxicity of the tested compounds were noted. The most toxic compounds (BMS-37 and −242) showed ECy Figure . Low toxy Figure . These t50 values in range of 0.333–1.15 nM  modified to constitutively express PD-1 and carrying a luciferase reporter gene driven by TCR-inducible NFAT response element  was used in T cell activation experiments. When antigen-presenting surrogate CHO cells, constitutively expressing the TCR agonist and PD-L1 (aAPCs) are presented to these ECs in co-culture, TCR signaling is repressed by PD-1 and the reporter remains silenced. Agents efficiently interfering with the PD-1/PD-L1 interaction activate the expression of the reporter gene resulting in the increase in luminescence intensity. Clinically relevant PD-1-antagonizing antibodies (nivolumab and pembrolizumab) and the PD-L1-blocking antibodies  release TCR signaling with ECM Figure . Both BMM Figure . Neverths Figure . BMS-200Increased level of sPD-L1 is thought to negatively affect the T cell anticancer response –19. ThisTo evaluate the potential of BMS compounds in abrogating the inhibitory effect of sPD-L1 on the activation of T cells, sPD-L1 was pre-incubated with tested compounds and presented to ECs together with the anti-CD3-activating antibody. BMS-1001 and −1166 dose-dependently abolished the inhibition of ECs stimulation by sPD-L1 Figure . ImportaTo test if the observed effect of BMS compounds was directly associated with the decreased sPD-L1 recruitment to the cell surface of the PD-1-expressing cells, a His-tagged sPD-L1 was labeled with the Ni-NTA-conjugated fluorescent dye and flow cytometry analysis was performed. When ECs were contacted with labeled sPD-L1, a clear staining was observed Figure . Pre-incATyr56 sidechain  by 40 degrees -4-hydroxypyrrolidine-2-carboxylic acid substituent is stabilized through hydrogen bond with NH2 group of BLys124 side chain -2-amino-3-hydroxypropanoic acid moiety forms hydrogen bonds with the carbonyl of AAsp122 sidechain and additional water-mediated hydrogen bond with the NH2 group of ALys124 and mainchain carbonyl of ATyr123. The 3-cyanobenzyl substituent provides additional hydrophobic contacts with ATyr123 aromatic ring and AArg125 sidechain  in vitro binding assay [Several classes of non-antibody molecules have been patented in the recent years, including one group of small-molecule inhibitors, disclosed by Bristol-Myers Squibb in the two consecutive patents , 25, butng assay , 25. Ourin vitro [We have shown previously that the compounds BMS-8, BMS-37 and BMS-242 bind to PD-L1 and efficiently dissociate the human PD-1/PD-L1 complex in vitro . Here weBoth structural and biochemical data suggest the formation of dimers of sPD-L1 in the presence of BMS compounds. This unique formation of PD-L1 dimers may facilitate the inhibition of PD-1/PD-L1 interaction, as the dimerization engages PD-1-binding surfaces of both PD-L1 protomers. Since the level of soluble PD-L1 is increased in the serum of cancer patients and correlates with poor prognosis , 16, andOur structural results show that the tested BMS compounds force the conformational changes at the surface of PD-L1 molecule upon binding. Both conformational changes and additional interaction sub-sites absent in previously published structures result in improved interaction between the characterized compounds and the target protein Figure . These rCollectively, our results advocate for PD-1/PD-L1-blocking potential of the evaluated BMS compounds and present a likely mode of this inhibition by forcing the dimerization of PD-L1 molecules.Following antibodies were used: anti-PD-1 antibody nivolumab , anti-PD-1 antibody pembrolizumab , anti-PD-L1 antibody atezolizumab  and anti-PD-L1 antibody durvalumab .1H NMR, 13C NMR, high-resolution mass spectrometry (HRMS) and UPLC/MS.Six small-molecule compounds disclosed by Bristol-Myers Squibb were tested in respect to their biological activities in disrupting of the PD-1/PD-L1 interaction. The compounds were synthesized according to procedures described in BMS patents , 25 withExpression and purification of human PD-L1  and human PD-1  was carried out as described previously . A singl15N labelling was obtained by expressing proteins in the M9 minimal medium containing 15NH4Cl as the sole nitrogen source. For NMR measurements the buffer was exchanged by gel filtration to PBS pH 7.4 (PD-L1) or 25 mM sodium phosphate containing 100 mM NaCl pH 6.4 (PD-1). 10% (v/v) of D2O was added to the samples to provide the lock signal. All spectra were recorded at 300K using a Bruker Avance III 600 MHz spectrometer. The interaction of the compounds with PD-L1 was evaluated by monitoring the perturbations in chemical shifts of NMR resonances in the 1H-15N 2D HMQC upon titration with the compound. The ability of tested compounds to dissociate PD-L1/PD-1 was evaluated using the Antagonist Induced Dissociation Assay (AIDA) [15N-labeled PD-1 (0.2 mM) was slightly overtitrated with the unlabeled PD-L1. The compounds were aliquoted into the resulting mixture. During the experiment the 1H-15N signals were monitored by the HMQC.Uniform y (AIDA) , 28. In + aAPC/CHO-K1 cells, called aAPCs) overexpressing TCR ligand and PD-L1, and T cell surrogate, a modified Jurkat T cell line overexpressing PD-1 and carrying a luciferase reporter under the control of NFAT promoter  [Mycoplasma contamination using PCR-based method [To verify the potency of BMS compounds in the inhibition of the PD-1/PD-L1 interactions, a cell-based model of the PD-1/PD-L1 immune checkpoint blockade was used. In the assay, two model cell lines are utilized: the artificial Antigen-Presenting Cells (PD-L1led ECs) . The celd method .5 000 ECs were seeded on transparent 96-well plates and cultured for 48 h in the presence of increasing concentrations of the BMS compounds or DMSO as a control . Following the treatment, a metabolic activity test was performed with the use of Biolog Redox Dye Mix MB (BioLog), according to the manufacturer's instructions.50) and maximal luminescence values (RLUmax) were determined by fitting the Hill equation to the experimental data.The aAPCs were seeded in white 96-well plates at the density of 10 000 per well in the culture medium 24 h prior to the assay. On the day of the assay, 3.5x serial dilutions of the antibodies were prepared in the RPMI 1640-containing 1% FBS. Serial dilutions of BMS compounds were prepared in DMSO and formulated in RPMI 1640-containing 1% FBS. By this, the concentration of DMSO was kept constant in all samples. 95 μl of the medium was removed from the wells and the cells were overlaid with 40 μl of the compound dilutions. 20 000 of ECs were added to each well in 40 μl RPMI 1640 containing 1% FBS. Following 6 h incubation at 37°C, the plates were equilibrated at room temperature for 10 min and 80 μl of the Bio-Glo reagent (Promega) was added to each well. After incubation for 10 min, luminescence was quantified using FlexStation 3 (Molecular Devices). Half maximal effective concentrations  was diluted in PBS supplemented with the penicillin/streptomycin solution  in the presence of the BMS compounds or a corresponding volume of DMSO. Then, 15 μl of the solution was added to each well of the antibody-coated plate. ECs were centrifuged and diluted to 50 000 per ml, and 60 μl of the cell solution was added to each well. The final concentration of sPD-L1 was 10 μg/ml (0.6 μM). The final concentrations of the BMS compounds were: 0.12, 0.3, 1.2 and 3 μM, giving the following BMS:sPD-L1 molar ratios: 1:5, 1:2, 2:1 and 5:1. The cells were cultured for 24 h and the luciferase activity assay was performed using the Bio-Glo Luciferase Assay System (Promega) according to the manufacturer's instructions.6 cells per ml. 50 μl of ECs was added to each sample and incubated on ice for additional 60 min. The final concentrations of the components were: 25 μg/ml of PD-L1 (1.5 μM), 125 μg/ml the anti-PD-L1 antibodies and control antibodies and 1 μM of the BMS compounds. The samples were analyzed using the BD FACS Verse flow cytometer and BD FACSuite v1.0.6 software.Binding of sPD-L1 (aa 18–134) to ECs was evaluated by flow cytometry. The His-tagged PD-L1 protein or its mutants were stained with NTA-Atto 647 N fluorescent dye (Sigma Aldrich) for 2 h at 22°C, at 8:1 molar ratio (protein:dye). PD-L1-Atto was formulated in 150 μl PBS with the tested compounds or antibodies. The samples were incubated for 30 min at 4°C in the dark. Meanwhile, ECs were centrifuged, washed with PBS and suspended in fresh PBS at concentration of 1 × 10Purified proteins in 10 mM Tris (pH 8.0) containing 20 mM NaCl, were concentrated to 5 mg/ml, mixed with the inhibitor in 1:3 molar ratio (protein:compound) and clarified by centrifugation at 15 000 × g for 10 min. Supernatant was used for screening using a sitting-drop vapor diffusion method. Diffraction-quality crystals were obtained at room temperature from 0.1 M Tris pH 8.5 containing 0.2 M magnesium chloride and 30% (w/v) PEG 4000 for the hPD-L1/BMS-1001 complex and from 0.01 M Tris pH 8.4 containing 0.28 M sodium chloride and 27% (w/v) PEG 4000 for hPD-L1/BMS-1166 complex.The X-ray diffraction data were collected at the PETRA III P11 beamline at DESY  and on Superdex S75 10/30 HR column was equilibrated with 10 mM Tris pH 8.0 containing 20 mM NaCl. The column was calibrated with globular protein standards. The retention time of hPD-L1 (4 mg/ml) was determined. hPD-L1/inhibitor complexes were formed at 3:1 (inhibitor:protein) molar ratio, incubated for 15 min at room temperature and clarified by centrifugation for 10 min at 15 000 × g. The samples were analyzed in the same manner as the apo-hPD-L1.hPD-L1 was concentrated to 10 μM in PBS pH 7.4 and mixed with BMS-1001 or BMS-1166 in the 1:1 molar ratio, followed by the addition of the BS3 cross-linking reagent at 0.25, 0.5 or 1mM final concentration (Thermo Scientific). After 30-min incubation at room temperature, the reaction was quenched by the addition of Tris pH 7.5 to the final concentration of 25 mM. Control sample contained hPD-L1 and 1 mM BS3 reagent. The samples were analyzed by SDS-PAGE.APD-L1 and BPD-L1 protomers were extracted from co-crystal structures with BMS-1001 or −1166, described in this manuscript, and used as receptors. BMS compounds were also extracted from the co-crystal structures, served as ligands and treated as rigid.Docking of BMS compounds at the surface of PD-L1 was performed using Hex 8.0.0 with default parameters . APD-L1 Coordinates and structure factors were deposited in the Protein Data Bank with accession numbers 5NIU (BMS-1001) and 5NIX (BMS-1166).


The G-FET transfer characteristics showed repeatable and reliable responses in all surface modifying steps using a direct current (DC) readout system. The hCG concentration gradient showed a detection limit of ~1 pg·mL−1. The proposed method facilitates the cost-effective and viable production of graphene point-of-care devices for clinical diagnosis.We report on a label-free immunosensor based on graphene field effect transistors (G-FETs) for the ultrasensitive detection of Human Chorionic Gonadotrophin (hCG), as an indicator of pregnancy and related disorders, such as actopic pregnancy, choriocarcinoma and orchic teratoma. Pyrene based bioactive ester was non-covalently anchored onto the graphene channel in order to retain the sp Human Chorionic Gonadotrophin (hCG) is a glycosylated protein 37.5 kDa) secreted by placenta during pregnancy or gestational trophoblastic diseases ,2. The f7.5 kDa s2 nanobelt, ZnO nanofilm, Au nanoparticles, molybdenum disulphide (MoS2), cerium oxide (CeOx) and single-walled carbon nanotubes (SWCNT) to name a few [2 to tetrahedral sp3 carbons leading to charge impurities and vacancy defects. To the best of our knowledge, there is only one report on BG-gFET based immunoassay for hCG detection, using multilayer epitaxial graphene grown on silicon carbide and a covalent functionalization method was used to anchor the necessary biomolecules. Other BG-gFET based immunoassays adopted intermediary modification of graphene either by hexamethyldisilazane (HMDS) or reduced to graphene oxide targeting rotavirus, DNA or cancer biomarkers [Since the first report by Bergveld et al. on FET based immunosensor (immunoFET), numerous studies have been conducted on immunoFET based on silicon nanowires (SiNW), SiOme a few ,9. Recenme a few ,11,12,13me a few ,17,18,19omarkers ,16,17.2/Si substrate. A non-covalent bond between monolayer graphene and antibody was facilitated by applying a linker molecule known as 1-pyrenebutyric acid–N-hydroxysuccinimide ester (Pyr-NHS) via π-π bonding. The performance of the device was assessed under ambient conditions for over 10 devices to establish higher stability and repeatability. Changes in resistance difference between consecutive surface modifications displayed a distinctive pattern not commonly seen in relation to the usual increase in resistance owing to biomolecular absorption. The sensor exhibited an ultralow label-free detection limit of 1 pg·mL−1 with high sensitivity (0.30 Ω·ng−1·mL−1) and reproducibility (>87% yield). An in-depth analysis on carrier transfer for each surface modification was carried out for the first time for non-covalently functionalized BG-gFET illustrating the importance of net charge, amino acid content and the isoelectric point of biomolecules. These key factors are vital for bioelectronic sensing mechanism and device performance. The rapid, sensitive, selective, stable, repeatable and reliable detection suggests promising applications of bgFET structure for multiple marker detection on a point-of-care device.In the present study, we demonstrate a scalable CVD grown monolayer graphene based FET immunosensor on a SiO−1 complementary anti-hCG (ab8466) in PBS buffer were purchased from Abcam  and the prepared aliquots were stored at −20 °C.Monolayer CVD graphene on Cu foil was purchased from Graphene Supermarket . Photoresist 1805 G2, lift-off resist 3B (LoR) and their corresponding developer and remover were purchased from A-Gas Electronic Materials . All the other chemicals, such as Pyr-NHS, iron nitride, nitric acid, PBS, Poly (methyl methacrylate) (PMMA), Ethanolamine etc. at biochemical grade were purchased from Sigma Aldrich . Bovine serum albumin (BSA) at biochemical grade was purchased from Sigma Aldrich . 100 µg·hCG (ab126652) lyophilized powder and 1 mL of 0.02 mg·mL−1 to 3000 cm−1 range with five accumulations each lasting 15 s.Raman spectra was collected in an XploRA Raman system  running a LabSpec 6.4.2.5 and equipped with a 532 nm HeNe laser delivering 20 mW of laser power at the sample. An Olympus BX41 microscope  with a 100 µm slit width, a 300 µm confocal hole, 1200 T grating and 1000× magnification was used. An MPlan N 100× microscope objective  focused the laser on the sample into a spot of ~7.45 µm diameter. A thermoelectrically cooled charge coupled device (CCD) camera was used for detection. Single spectra were collected for bare, coated and non-coated dentine by graphene from 1100 cm−1.X-ray photoelectron spectroscopy (XPS) was performed with Kratos AXISULTRA with a mono-chromated Al kα X-ray source (1486.6 eV) using an emission current of 10 mA and an anode potential of 12 kV (120 W). The ULTRA was used in fixed analyzer transmission (FAT) mode, with pass energy of 80 eV for wide scans and pass energy 20 eV for high resolution scans. Cyclic Voltammetry (CV) measurements were carried out in 10 mM ferricyanide aqueous solution (1 M KCl solution) at room temperature with a scan potential range from 0.8 V to −0.4 V and a scan rate of 100 mV·sA Keysight B1500A semiconductor device parameter analyzer with an MPS150 probe station  was employed to investigate the electrical characteristics and sensing performance of the FET device at room temperature. The transistor measurement on the sensor was carried out by measuring the drain current (Ids) as a function of the gate voltage (Vg) (−100 V to +100 V) with a fixed drain-source voltage (Vds = 100 mV). The sensing signal of the device was recorded by monitoring the Ids as a function of drain-source voltage (Vd) (−1 V to +1 V) with a fixed gate voltage (Vds = 0 V). The data were analysed using SigmaPlot 13 software .2/Si substrates as follows. Firstly, PMMA was dissolved in the chlorobenzene with a 10 mg·mL−1 concentration and spin-coated on the one side of the graphene film at a spin speed of 4000 rpm for 30 s followed by baking at 180 °C for 1 min. To etch the Cu substrate, 10% HNO3 was used for 2 min followed by etching in 0.1 M ammonium persulfate for approximately 3 h with the endpoint determined when Cu was no longer visible. The resulted PMMA/graphene membrane was transferred to a rinse bath of deionized (DI)water for 10 min. Subsequently, monolayer graphene was directly transferred onto SiO2/Si substrates. Finally, PMMA layer was removed by acetone treatment at 50 °C for five minutes following by cleaning with Isopropyl alcohol (IPA) and DI water. Graphene on SiO2/Si substrates obtained as such was dried in vacuum before characterization and measurements.The monolayer graphene was obtained from Graphene Supermarket , and it was synthesized on both sides of 25 µm thick copper foil through chemical vapour deposition (CVD) method . The wet2/Si substrate was obtained by wet-transfer method as mentioned above [2/Si at 50 W and 6 × 10−7 Torr for 2 min [−7 Torr and 4 mTorr base and Ar pressure [2 is 300 nm and the width and length of each FET unit is 90 µm and 80 µm respectively.A schematic illustration of the fabrication of hCG biosensor is shown in ed above ,22. In oed above . We usedor 2 min . We depopressure . The thi−1 anti-hCG solution and incubated at 4 °C for 4 h. The surface of graphene channel was rinsed with PBS buffer and immersed in BSA solution with a concentration of 0.5 mg·mL−1 to block the free amino groups to prevent the non-specific binding of hCG. Finally, hCG solution with concentrations ranging from 1 pg·mL−1 to 100 ng·mL−1 were prepared by diluting 1 µg·mL−1 stock solution with PBS buffer and then incubated on graphene channel at 4 °C for 4 h to ensure the strong antibody/antigen binding. Finally, the samples were treated with 100 mM ethnolamine for 30 min and washed with DIW for 5 min to remove non-specific passivation followed by vacuum dry at 30 Torr for an hour before characterization [Pyr-NHS was used as the linker between graphene channel and anti-hCG via π-π interaction. Consisting of hydrophobic pyrenyl moiety base and a bio-active ester head, Pyr-NHS does not require any pre-treatment of antigen with EDAC/NHS chemistry is required . 2 mM Pyrization .2 before and after adding linker and others. The intensity ratios of 2D-band to G-band are higher than 5. These data confirm that the transferred graphene before and after treating with linker and others is high-quality and continuous. For effective removal of photoresist during fabrication, deep UV (DUV) technique was used within the fabrication process to reduce photoresist contamination. The details of our work to obtain clean, uniform, and continuous graphene, with a typical low sheet resistance along with XPS and Cyclic voltammetry analysis have been elaborated in our recent works in detail [The morphology of the samples and surface modification was characterized by Raman spectroscopy, XPS and cyclic voltammetry. n detail ,23. The −1) and 2D-peak (~2700 cm−1) are clearly resolved with strong intensity and sharp shape of 2D peak, indicating that the graphene film is monolayer. To avoid possible surface contaminations, all devices were thermal-annealed  before electrical measurements were performed. back·gate)-dependent resistance measurement results of a typical graphene FET device, showing the ambipolar field-effect characteristics. Both the carrier type and density of graphene can be modulated by Vback·gate. The maximum resistance  point corresponds to the charge neutrality point Vcnp (or the Dirac point in the case of monolayer graphene), indicating how the graphene film is intrinsically doped, i.e., p/n doped when Vcnp is positive/negative or non-doped when Vcnp is equal to zero. For this particular device, Vcnp is around 12 V, suggesting it is intrinsically p-doped. The p-doping characteristics of graphene are usually caused by exposure to an oxygen-containing atmosphere, as oxygen molecules adsorbed onto defect or oxidized sites on a graphene surface will function as p-type dopants by trapping electrons during device fabrication process [cnp or the resistance-Vback·gate curve.Raman spectroscopy was used to confirm the layer number of graphene film. In the Raman spectrum shown in  process . Several2D-peak ~00 cm−1 a process . The sli process ,30.The f−1 to 100 ng·mL−1. All the devices were formed by modification processes under controlled physiochemical conditions. We used 100 µg·L−1 PBS to obtain standard solutions with different concentrations. After adding standard solutions with different concentrations for four hours at 4 °C followed by washing with PBS and N2 drying, we measured the resistance-Vback·gate curves within 2 min. The pH of standard solutions was kept at neutral to avoid false response. At least three replicates were conducted for each concentration and the relative standard deviations between replicates were 5%–24%, indicating good reproducibility of our measurement. The test results of standard solutions with five concentrations  are shown in cnp was around 12 V, indicating the p-doping nature of the device in dry state. After adding standard solution of 1 pg·mL−1, we clearly observed right shifts of the curve, i.e., higher Vcnp. For higher concentrations, the resistance-Vback·gate shifted further right and Vcnp became higher.To examine the performance of bgFET immunosensor for detection, we performed a quantitative analysis on multiple BG-gFET devices to examine the change in resistance of the graphene channel after Ab-hCG binding ranging from 1 pg·mLcnp [cnp versus concentration  of the tested standard solutions from three representative devices. A lognormal fitting was performed to obtain a calibration curve for quantification. Based on our measurement, the lowest concentration our graphene aptasensor detected was 1 pg·mL−1, and a concentration-response calibration curve showed a good fitting (R2 = 0.97) in the range of concentration evaluated, which is advantageous for the detection of hCG having wide-ranging expression profiles in the different stages of diagnosis i.e., pregnancy or cancer development. The obtained limit of detection (LOD) was 600 times more sensitive than the one achieved with the previously published hCG biosensor on BG-gFET [−1 directly without pre- or post-amplification process. This demonstrated that our aptasensor can work well for samples much lower than the safe level.Such results indicate that our bgFET immunosensors are effective for detecting at different concentrations. The detection mechanism can be explained by a simple picture that, in dry condition, higher concentrations of hCG cause electron doping of the graphene  by approaching its surface, which leads to a right shift of the transfer curves or a higher Vcnp  and pote BG-gFET  and twen BG-gFET . In addiSelectivity tests were carried out by using interfering species, such as BSA, glucose, uric acid (UA), ascorbic acid (AA) and 1 pg/mL of hCG a. It can−1, which is much lower than the clinical limit (5 ng·mL−1) with a broad dynamic range in concentration (1 pg·mL−1 to 100 ng·mL−1) [In summary, we demonstrated a BG-gFET based immunosensor functionalized with a non-covalent linker for high sensitivity and label-free detection of hCG. The sensors showed the detection of hCG at concentration levels as low as 1 pg·mLng·mL−1) . Further


For non–small cell lung cancer (NSCLC), treatment with pembrolizumab is limited to patients with tumours expressing PD-L1 assessed by immunohistochemistry (IHC) using the PD-L1 IHC 22C3 pharmDx  companion diagnostic test, on the Dako Autostainer Link 48 (ASL48) platform. Optimised protocols are urgently needed for use of the 22C3 antibody concentrate to test PD-L1 expression on more widely available IHC autostainers.We evaluated PD-L1 expression using the 22C3 antibody concentrate in the three main commercially available autostainers Dako ASL48, BenchMark ULTRA , and Bond-III (Leica Biosystems) and compared the staining results with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Several technical conditions for laboratory-developed tests (LDTs) were evaluated in tonsil specimens and a training set of three NSCLC samples. Optimised protocols were then validated in 120 NSCLC specimens.Optimised protocols were obtained on both the VENTANA BenchMark ULTRA and Dako ASL48 platforms. Significant expression of PD-L1 was obtained on tissue controls with the Leica Bond-III autostainer when high concentrations of the 22C3 antibody were used. It therefore was not tested on the 120 NSCLC specimens. An almost 100% concordance rate for dichotomized tumour proportion score (TPS) results was observed between TPS ratings using the 22C3 antibody concentrate on the Dako ASL48 and VENTANA BenchMark ULTRA platforms relative to the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Interpathologist agreement was high on both LDTs and the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform.Availability of standardized protocols for determining PD-L1 expression using the 22C3 antibody concentrate on the widely available Dako ASL48 and VENTANA BenchMark ULTRA IHC platforms will expand the number of laboratories able to determine eligibility of patients with NSCLC for treatment with pembrolizumab in a reliable and concordant manner. Pembrolizumab is a humanised monoclonal antibody that targets programmed death 1 (PD-1), a negative co-stimulatory receptor expressed on the surface of activated T cells that can be exploited by tumours to evade T-cell–induced anti-tumour activity . It has in-vitro diagnostic (IVD) antibody, the use of which in combination with laboratory-developed tests is considered to be the most financially sustainable. Thus, there is an urgent need for a reliable protocol that can allow PD-L1 IHC testing using the 22C3 antibody on widely available IHC platforms worldwide.Pembrolizumab is currently approved as a monotherapy in >60 countries for one or more of the following indications: unresectable or metastatic melanoma; metastatic NSCLC with PD-L1 expression and disease progression on or after platinum-containing chemotherapy; metastatic NSCLC with high PD-L1 expression and no prior systemic chemotherapy; recurrent or metastatic head and neck squamous cell carcinoma with disease progression on or after platinum-containing chemotherapy; adult and paediatric refractory classical non-Hodgkin’s lymphoma; cisplatin-ineligible metastatic urothelial carcinoma; and microsatellite-instability–high solid tumours. It is also approved in combination with pemetrexed and carboplatin as a first-line treatment for patients with metastatic non-squamous NSCLC ,6. CurreOur study investigated the 22C3 antibody concentrate , in the three main commercially available autostainers, Autostainer Link , BenchMark ULTRA , and Bond-III  and compared the results with the ‘gold standard’ PD-L1 IHC 22C3 pharmDx kit  on the Dako ASL48 platform, in order to develop standardized protocols for PD-L1 IHC using the 22C3 antibody concentrate.Archival formalin-fixed paraffin-embedded (FFPE) tumour samples from 120 patients with stage I–IV NSCLC were retrospectively selected from the Laboratory of Clinical and Experimental Pathology and the Nice Hospital-Integrated Biobank (BB-0033-00025) . All tumour specimens were collected, stored, and used with informed written consent from the patients. The study was approved by the local ethics committee  and was performed in accordance with the guidelines of the Declaration of Helsinki.Tumour samples were obtained from patients who underwent surgical resection or biopsy in the departments of pulmonary medicine and thoracic surgery at Hôpital Pasteur between March 2007 and March 2016. Of the 120 tumour specimens evaluated, 95% consisted of surgical resection specimens and the remaining 5% were bronchial biopsies and transbronchial mediastinal lymph node biopsies. A sample was considered eligible for the study if the tumour morphology was preserved and if a minimum of 100 cancer cells were present in the tissue section. Morphologic classification was assigned according to the last World Health Organization/International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society criteria, and confirmed by the immunohistochemical phenotype  by the same three senior lung pathologists who reviewed the NSCLC IHC . The 120 NSCLC samples were to include 80 adenocarcinomas  and 40 squamous cell carcinomas . Sources of tumour samples included patients who may have had common genetic alterations such as epidermal growth factor receptor mutations or anaplastic lymphoma kinase translocations.PD-L1 IHC using the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform was performed according to manufacturer recommendations . The 22CTM FLEX Wash Buffer 20x). Each IHC run contained a positive control (on-slide tonsil tissue) and a negative antibody control  (http://dx.doi.org/10.17504/protocols.io.iw9cfh6).Specimens were sectioned at a thickness of 3 μm and stained on positively charged glass slides stored at 4°C within 3 days after sectioning. Deparaffinization, rehydration, and antigen retrieval were performed on PT Link (Dako PT100) using the EnVision™ FLEX Target Retrieval Solution, at low pH 6.0 for 53 minutes at room temperature. Following FLEX peroxidase block for 5 minutes, specimens were incubated with primary mouse anti–PD-L1 monoclonal antibody , using a concentration of 1:50 for 60 minutes at room temperature. Specimens were then incubated with the EnVision™ FLEX+ Mouse LINKER for 30 minutes at room temperature, followed by incubation with the EnVision™ FLEX HRP visualization reagent for 30 minutes at room temperature. The enzymatic conversion of the subsequently added 3,3’-diaminobenzidine tetrahydrochloride (DAB) chromogen for 10 minutes at room temperature followed by DAB enhancer for 5 minutes at room temperature resulted in precipitation of a visible reaction product at the site of antigen. The specimens were then counterstained with haematoxylin and coverslipped. The entire described steps are followed by buffer washes for 5 minutes (EnVisionhttp://dx.doi.org/10.17504/protocols.io.ixacfie).Specimens were sectioned at a thickness of 3 μm and stained on positively charged glass slides stored at 4°C within 3 days after sectioning. Deparaffinization, rehydration, and antigen retrieval were performed by CC1  antigen retrieval solution , performed on the VENTANA BenchMark ULTRA automated slide stainer for 64 minutes at 100°C. Specimens were incubated with primary mouse anti–PD-L1 monoclonal antibody  using a concentration of 1:50 for 32 minutes at 37°C, followed by visualization with the OptiView DAB IHC Detection Kit (Ventana) and OptiView Amplification Kit (Ventana) for 12 minutes. The specimens were then counterstained with haematoxylin II and bluing reagent (Ventana) and coverslipped. Each IHC run contained a positive control (on-slide tonsil tissue) and a negative antibody control   antigen retrieval solution (ref. AR 9640) performed on the Bond-III Leica automated slide stainer for 20 minutes at 100°C. Specimens were incubated with primary mouse anti–PD-L1 monoclonal antibody  using a concentration of 1:10 for 2×60 minutes at room temperature, followed by visualisation with the Leica Bond detection kit for 20 minutes at room temperature (Ref. DS 9800). The specimens were then counterstained with haematoxylin and coverslipped. Each IHC run contained a positive control (tonsil tissue)Tumour proportion score (TPS) was evaluated on samples with at least 100 viable tumour cells in the specimen. TPS was calculated as the percentage of PD-L1–positive cells  relative to all viable tumour cells (positive or negative) present in the sample.Scoring was assessed by three trained pathologists ; pathologists B and C were trained by pathologist A, who underwent a live course specially designed to train pathologists for scoring and interpretation of PD-L1 IHC results that are generated using the PD-L1 IHC 22C3 pharmDx kit (Dako) on the Dako ASL48 platform. Each pathologist scored the same stained glass slides independently and was masked as to the staining protocol used on a particular slide. All slides generated from staining on one platform (with no knowledge of which platform) were read at the same time. Slides stained from each platform were read independently at different times. Moreover, between each analysis for a given platform the order of samples was randomly rearranged by the technicians.Micrographs from the slides used for assay development  were created by whole-slide scanning, using a Nanozoomer HT 2.0 Scanner . Automated analysis was used to complement the manual analysis made by pathologists, allowing differences between tests, such as variation in intensity and number of cells for the development of LDTs to be seen. While intensity is not used in determining the TPS, the automatic analysis may provide clues on small variations, approximately 1%, which is an important score in certain clinical scenarios, especially in second-line treatment.To complement the IHC manual scoring, automated densitometry measurements of immunoprecipitates in scanned whole slides were made by using the Interactive Learning and Segmentation Toolkit that allows for tissue recognition within the Calopix software  in order to analyse only the epithelial component (membranous/cytoplasmic). The Calopix IHC algorithm was then applied to each scanned whole-slide.TPS scores using optimised protocols for two LDTs using the 22C3 antibody concentrate on the Dako ASL48 and VENTANA BenchMark ULTRA platforms were compared with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform (gold standard). The sample size of 120 NSCLC samples for evaluating the concordance of LDTs using the 22C3 antibody and the PD-L1 IHC 22C3 pharmDx kit was based on precision of estimates of positive and negative percentage agreement to ensure that the margin of error was near or below 5%.Statistical analyses assessing agreement were performed on TPS scores as continuous variables as well as on dichotomized variables using two positivity cut points: TPS positive (≥1%) and TPS strongly positive (≥50%). Positive percentage agreement (PPA) and negative percentage agreement (NPA) were calculated for the comparison between the dichotomized TPS results using each of the 22C3 concentrate LDTs relative to the TPS results observed with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. 95% confidence intervals were calculated using 2-sided Wilson score confidence limits. Intraclass correlation coefficients were calculated for the comparison of TPS raw scores as continuous variables from each of the 22C3 concentrate LDTs, relative to the TPS score observed with the 22C3 pharmDx kit. κ scores were calculated for dichotomized TPS results at the 1% or 50% level to assess the interpathologist agreement for each of the LDTs and for the PD-L1 IHC 22C3 pharmDx kit.A total of 10 protocols were evaluated on the Dako ASL48, 15 protocols on the VENTANA BenchMark ULTRA, and 11 protocols on the Bond-III platforms in serial sections from a tonsil specimen see . Based oThe optimised protocol for the PD-L1 IHC assay using the 22C3 antibody concentrate on the VENTANA BenchMark ULTRA platform is described in the Methods section . StaininFor each of the three pathologists, a 100% concordance rate for dichotomized TPS results at <1% versus ≥1% was observed between TPS ratings from each of the 22C3 antibody concentrate LDTs relative to the gold standard observed with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform . SimilarInterpathologist agreement was high on both LDTs and the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform (gold standard). Using a TPS cut point of ≥1%, κ scores for interpathologist agreement were 1 for both LDTs and the gold standard. Using a TPS cut point of ≥50%, κ scores were 1 for interpathologist agreement for VENTANA BenchMark ULTRA, and 0.99 for the gold standard and Dako ASL48.The PD-L1 IHC 22C3 pharmDx kit was recently approved by the US Food and Drug Administration as a companion diagnostic assay to identify patients with metastatic NSCLC who are most likely to respond to pembrolizumab ,9. The PWe have developed optimised protocols for determining PD-L1 expression by IHC using the 22C3 antibody concentrate  on both the VENTANA BenchMark ULTRA and Dako ASL48 platforms. The 22C3 antibody concentrate may be compatible with the Leica Bond-III autostainer provided that high concentration (1:10) is used. In addition, send-out testing may be considered on national validated and accredited platforms for those laboratories that carry a Leica Bond III. TPS ratings demonstrated high concordance compared with the gold standard 22C3 pharmDx kit on the Dako ASL48 for both LDTs.In our protocol development experiments, we first used tonsil specimens as widely recommended positive controls, with the highest expression of PD-L1 in crypt epithelium, macrophages homing into the germinal centres, and interfollicular mononuclear leucocytes. However, when several protocols at 1:50 dilution, different by only subtle modifications of the technical conditions  NSCLC sample should be used instead to provide a dynamic range of PD-L1 staining for intensity and spatial distribution. The ideal control would be weakly positive staining NSCLC tissue, as this would provide greater sensitivity for detecting degradation of reagents as well as intratumour spatial heterogeneity. Additionally, it is important to note that pathologist training is necessary for correct interpretation of PD-L1 staining on IHC ,15. In tOur study has a number of limitations, including its retrospective nature, the fact that it was conducted at a single institution, and the fact that no patient was treated with pembrolizumab. To overcome these limitations, national efforts are currently ongoing to validate LDTs in conjunction with pembrolizumab treatment in prospective clinical trials .To date, only one study has investigated whether it is possible to adapt Dako’s PD-L1 IHC 22C3 pharmDx assay to the BenchMark XT platform (Ventana) . Howeverhttp://www.nordiqc.org/downloads/assessments/96_102.pdf) [Another harmonization effort for PD-L1 testing was recently made by the Nordic immunohistochemical Quality Control organization  . While o102.pdf) ,14. As nAvailability of standardised protocols for determining PD-L1 expression on the widely available Dako ASL48 and VENTANA BenchMark ULTRA IHC platforms will expand the number of laboratories able to determine eligibility of patients with NSCLC for treatment with pembrolizumab in a reliable and concordant manner. Moreover, using the 22C3 antibody concentrate with the specific reagents for each IHC platform provides greater practicality for pathology laboratories worldwide. Development of protocols for PD-L1 IHC using the 22C3 antibody concentrate on other commercially available IHC platforms such as VENTANA BenchMark XT and Dako Omnis should also be urgently considered.S1 TableShaded rows represent conditions used in the optimised protocols. LDT, laboratory-developed test; PD-L1, programmed death ligand 1; IHC, immunohistochemistry; ASL48, Autostainer Link 48; na, not applicable.(DOCX)Click here for additional data file.S1 FigTechnical conditions used are indicated. Original magnification 5×. ×. PD-L1, programmed death ligand 1; IHC, immunohistochemistry; ASL48, Autostainer Link 48; LDT, laboratory-developed test; Ab, antibody.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigTechnical conditions used are indicated. Original magnification 5×. PD-L1, programmed death ligand 1; IHC, immunohistochemistry; ASL48, Autostainer Link 48; LDT, laboratory-developed test; Ab, antibody.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 FigTechnical conditions used are indicated. Original magnification 5×. PD-L1, programmed death ligand 1; IHC, immunohistochemistry; ASL48, Autostainer Link 48; LDT, laboratory-developed test; Ab, antibody.(TIF)Click here for additional data file.S6 FigA-C) and VENTANA BenchMark ULTRA platforms (D-F) (LDTs) relative to the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform (gold standard).Pathologist ratings of TPS on each of the PD-L1 IHC assays using the 22C3 antibody concentrate on the Dako ASL48 Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.


We performed first-principles calculations to reveal the possibility of applying pristine, defective, and B-doped graphene in feasible negative electrode materials of ion batteries. It is found that the barriers for ions are too high to diffuse through the original graphene, however the reduced barriers are obtained by introducing defects  in the graphene. Among the three types of defects, the systems with a double vacancy could provide the lowest barriers of 1.49 and 6.08 eV for Li and Na, respectively. Furthermore, for all kinds of B-doped graphene with the vacancy, the systems with a double vacancy could also provide the lowest adsorption energies and diffusion barriers. Therefore, undoped and B-doped graphene with a double vacancy turn out to be the most promising candidates that can replace pristine graphene for anode materials in ion batteries. At present, although the rechargeable lithium ion battery (LIB) has been widely used in various types of electronic equipment, it still faces the potential risk of gradually rising cost of lithium . TherefoRecently, some results of experiments show that it is possible to use graphene ,18 and iBesides the high capacity, a promising anode material should also be corrosion resistant in organic electrolytes and form a stable solid electrolyte interphase (SEI). In experiment, graphene was prepared to protect the silicon anode in LIBs as a coating, which acted as an artificial SEI . It prevt, L and D are the ion diffusion time, the diffusion length and the diffusion coefficient, respectively [On the one hand, for the practical purpose of using graphene as a protective coating for the anode material in LIBs and SIBs, the barriers should be low enough for Li and Na diffusing through the graphene. On the other hand, according to the following equation, ectively . The curThere are more and more computational studies focusing on the interaction between ions and graphene now. Some theoretical research investigated the bonding and charge transfer between the adsorbed Li and pristine graphene ,28,29,30et al. examined mechanisms of charge transfer upon interaction of hydrated protons with a variety of doped graphene nanoribbons, and found that the B, N, and O doped edges all show active proton affinity. In addition, their theory study of charge carrier adsorption onto zigzag edge-shaped graphene nanoribbons (ZGNRs) indicates that a maximum charge loading is achieved with incorporating edge substitution with boron.Furthermore, it is said that chemical modification is also an effective way to adjust the performance of graphene ,44,45,46Based the above summary of studying on the electrochemical properties of pristine, defective, and chemically modified graphene, two outstanding issues still exist: how defects and chemical doping affect the diffusion of Li and Na on graphene, and what are the differences of the diffusion between the two of them. To answer these questions in detail, in the present study, the first-principles calculations have been executed to thoroughly explore the Li and Na diffusion through the defected graphene including vacancies and Stone–Wales defects. Furthermore, because the boron doping keeps the planar structure of graphene without too much deformation , boron-dIn this work, first-principles calculations were implemented in CASTEP  code , which eThe primitive unit cell of graphene with the experimental lattice constant of 2.46 Å was built by placing the two C atoms on two-dimensional honeycomb lattice with a hexagonal structure. Based on the primitive cell, the supercell consists of 4 × 4 primitive cells, as shown in adE) have been calculated by the following equationsy + ionE, syE and ionE are the total energies of the graphene system with a ion(Li or Na), the graphene system and a ion, respectively. For both Li and Na, the results of the three sites after relaxation show that the H site is the most stable position with the lowest value of +0.49 and +0.61 eV separately. The positive adsorption energies indicate that the adsorption of ions on pristine graphene is unstable energetically. Therefore, the clustering of ions is finite and the diffusion of ions is difficult on the graphene. Furthermore, after relaxation, the most stable position for Li is 1.71 Å away from the plane of the graphene above the center of the hexagon, as well as 2.30 Å for Na.Li and Na are located initially at the position 2.0 Å and 2.5 Å from the graphene plane, respectively. To find the most stable position, three adsorption sites with high symmetry are considered by optimizing the structures: in the center of a carbon hexagon (H), on the top of a carbon atom (T), at the midpoint of a C-C bond (M), as shown in Next, we calculate the energy barrier for ions to diffuse through the graphene plane. In order to save computational time, the barriers were calculated without relaxing the intermediate structures. This simplified method turns out to be a good approximation as relaxation plays a very small role in bringing down the barrier height. We validate our approach for a few representative systems against a complete linear and quadratic synchronous transit (LST/QST) transition state (TS) search algorithm in CASTEP . The barWe consider the possibility of the ion diffusion through the defected graphene, including single vacancy (SV) , double 1(BC31) is about 70 mAh/g [It is said that chemical doping is an effective way to adjust the performance of graphene ,55. Rece70 mAh/g . We also70 mAh/g ,58. Howexy plane)), the blue and red filled areas separately means gaining and losing electrons. It demonstrates that there is no charge around Li and B. Because of the electro-positive nature of B, graphene layer receives all its charge. Both Li and Na transfer one electron to the layer as well. The diffusion barriers of Li and Na are 8.80 and 31.37 eV, respectively, as given in Therefore, the effect of B-doping on the barrier for ions to diffuse through the graphene is explored. The adsorption energies of Li and Na for B-doped graphene are −0.95 and −0.74 eV, respectively, as shown in Next, to further discuss the effect of doping on the electronic structure, the calculation of the density of states (DOS) were carried out, as shown in Boron was doped in the graphene with a single vacancy (SV) and a double vacancy (DV). For the SV defect case, three types of configurations are formed by doping with 1, 2 and 3 boron, as shown in Furthermore, we estimate the diffusion barriers of ions through the B-doped graphene with DV. There are six kinds of models for Li and Na diffusion. The structures doped by one to four boron atoms and their corresponding diffusion barriers are shown in We implemented first-principles calculations to clarify the impact of defects and B-doping on the graphene as a possible negative electrode material. Though the barriers for ions are too high to diffuse in pristine graphene, the barriers reduce significantly because of the presence of defects . It can also be found that the barriers for ions diffusion are determined by the sizes of the open space formed by the defects. Therefore, there are the lowest barriers of 1.49 and 6.08 eV for Li and Na, respectively, in the graphene with DV. The impact of B-doping on the diffusion barriers was also considered. Though the adsorptions of Li and Na on the B-doped graphene are more stable, they are not low enough barriers for fast ion diffusion. Due to the B-doping in the graphene with SV, the high diffusion barriers for Li and Na are observed, which rise gradually with the increase of the concentrations of B in the configurations. However, the B-doped systems with DV provide significantly low adsorption energies and diffusion barriers. Our results show the advantages and disadvantages of various types of graphene, and thus we believe that undoped and B-doped graphene with DV will be better negative electrode and synthetic SEI materials for ion batteries.