Botrytis cinerea is a ubiquitous necrotrophic filamentous fungal phytopathogen that lacks host specificity and can affect more than 1000 different plant species. In this work, we explored L1 [(E)-2-{[(2-aminopyridin-2-yl)imino]-methyl}-4,6-di-tert-butylphenol], a pyridine Schiff base harboring an intramolecular bond (IHB), regarding their antifungal activity against Botrytis cinerea. Moreover, we present a full characterization of the L1 by NMR and powder diffraction, as well as UV–vis, in the presence of previously untested different organic solvents. Complementary time-dependent density functional theory (TD-DFT) calculations were performed, and the noncovalent interaction (NCI) index was determined. Moreover, we obtained a scan-rate study on cyclic voltammetry of L1. Finally, we tested the antifungal activity of L1 against two strains of Botrytis cinerea . We found that L1 acts as an efficient antifungal agent against Botrytis cinerea at 26 °C, even better than the commercial antifungal agent fenhexamid. Although the antifungal activity was also observed at 4 °C, the effect was less pronounced. These results show the high versatility of this kind of pyridine Schiff bases in biological applications. Schiff base compounds are organic compounds containing an azomethine C=N group ,2,3,4. PBotrytis cinerea is a ubiquitous necrotrophic filamentous fungal phytopathogen, virtually lacking host specificity, able to infect more than 1000 different plant species -methyl}-4,6-di-tert-butylphenol]  calculations to corroborate the experimental UV–vis data, as well as the noncovalent interaction (NCI) index of L1, in order to better understand the nature of the IHB. Moreover, we performed a scan-rate study on cyclic voltammetry of L1 to determine whether these processes are controlled by diffusion of species. Finally, we tested the antifungal activity of L1 against two strains of Botrytis cinerea   , a pyridassessed . In addiberries) , and othL1 synthesis and some characterizations have already been reported -methyl}-4,6-di-tert-butyl-phenol), L1, exerted a cytotoxic effect against yeasts (Cryptococcus spp. MIC: 4.468 ppm) -methyl}-4,6-di-tert-butyl-phenol (L1) compound was prepared by direct interaction between 1,2-diaminopyridine and 3,5-di-tert-butyl-2-ol-benzaldehyde (1:1) in methanol, at room temperature, according to a previously described non-template method [(E)-2-{[(2-aminopyridin-2-yl)imino]-methyl}-4,6-di-tert-butyl-phenol (L1) yellow powder, 0.68 g ; FTIR : 3487 and 3255 (νOH), 2947 (νOH), 2947 and 2905 (νNH2), 2863 (νCH), 1607 (νHC=N), 1589 (νC=C). UV–Vis:  λ (nm)(ε) = 239 (25.63 × 103 mol-1 dm3 cm-1), 277 (18.07 × 103 mol−1 dm3 cm-1), 375 (15.26 × 103 mol−1 dm3 cm−1);  λ (nm)(ε)= 274 (32.78 × 103 mol−1 dm3 cm−1), 372 (15.80 × 103 mol−1 dm3 cm−1);  λ (nm)(ε)= 236 (23.77 × 103 mol−1 dm3 cm−1), 274 (16.15 × 103 mol−1 dm3 cm−1), 368 (13.69 × 103 mol−1 dm3 cm−1);  λ (nm)(ε) = 378 (7.24 × 103 mol−1 dm3 cm−1). 1H-NMR at 25 °C : δ =1.35 , 1.48 , 5.51 , 6.71 , 7.52–7.43 , 7.53 , 7.95 , 8.88 , 13.29 ; : δ =1.25 , 1.40 , 4.93 , 6.65 , 7.31-712 , 7.41 , 7.87 , 8.57 , 12.94 ; : δ =1.11 , 1.24 ,6.60-6.40 , 7.18 , 7.28 , 7.24 , 7.28 , 7.63 , 8.57 ; 1H-NMR at 4 °C : δ = 1.25 , 1.40 , 4.89 , 6.70 , 7.27–7.25 , 7.43 , 7.94 , 8.59 , 12.93 ; 13C-NMR at 25 °C : δ = 28, 113, 119, 125, 127, 130, 136, 140, 146, 154, 158, 165; : δ = 29, 30, 31, 34, 114, 118, 125, 127, 128, 130, 136, 141, 145, 153, 157, 165; : δ = 28, 29, 30, 100, 113, 125, 127, 128, 144, 165. DEPT at 25 °C : δ = 31, 113, 125, 127, 128, 146, 165; : δ = 29, 30, 31, 114, 125, 127, 128, 145, 165; : δ = 28, 29, 30, 114, 125, 127, 128, 144, 165.The (e method . (E)-2-{L1. We assessed the purity and corroborated the compound structure by a combination of techniques, including , and complemented with DFT and TD-DFT using standard CAM-B3LYP/TZ2P level of theory. Our analysis showed the high stability and strength of the intramolecular hydrogen bond in different solvents by performing noncovalent interactions index (NCI) studies. Furthermore, we determined the electrochemical profile of L1 (and compared it with L2), providing a scan-rate study, demonstrating that the electrochemical calculated processes are controlled by mass transfer (diffusion) only for L1. These results show that small structural changes in these compounds could lead to changes in their properties. Finally, we showed that L1 exerts an antifungal effect against two strains of Botrytis cinerea (mold), including a strain isolated directly from the field, primarily at 26 °C, remarking the potential of these king of Schiff bases as modulators of biological activities.In this manuscript, we provided a further characterization of


Spelling is a fundamental literacy skill facilitating word recognition and thus higher-level reading abilities via its support for efficient text processing . However Literacy is widely recognized as a foundational educational skill e.g., . AlthougIn contrast, spelling ability has received relatively little attention in second language (L2) learners. Existing research shows evidence both for overlap with L1 spelling  and L1-bTo address these gaps, the current study analyzed English spelling data from adult ESL users in three L1 groups , as well as a native speaker comparison group. Data were additionally collected on phonological awareness ability to facilitate cross-linguistic comparisons of not only spelling ability, but also how spelling is related to other key literacy skills.Although spelling ability frequently gets only secondary attention in literacy research , it is iSpelling is also a key component of writing. The Simple View of Writing e.g.,  describeFurther evidence for the interconnectedness of spelling with word recognition, reading comprehension, and writing comes from research examining the impact of instructional interventions. Targeted spelling instruction improves not only spelling and phonological awareness e.g.,  but alsoOne perennial point of contention in literacy research is whether word recognition is best modeled via a single-route, connectionist approach e.g.,  or a duaCross-linguistic research on spelling development suggests that the degree to which individuals rely on these two major sources of knowledge – phonological and visual/orthographic – varies across languages, and in particular across writing systems. As has been widely discussed in the literature on cross-linguistic literacy, the varying phonological and morphological characteristics of different languages are typically associated with particular approaches to encoding the language in writing, such that “every language gets the writing system it deserves” , p. 266.optional in abjads, and in fact are frequently omitted in texts written for fluent adult readers   . Howeverhope, and rimes such as -ough)  .Research has also indicated that individual spellers may take somewhat different approaches to spelling – either lexical (whole-word) or sub-lexical (based on phoneme-grapheme correspondences) e.g., . These iGiven the importance of high-quality lexical representations to support word recognition and overall reading skill, surprisingly few studies have included a detailed consideration of L2 spelling skills. This is true even for English as a second/foreign language (ESL/EFL), despite the international prevalence of this second language e.g., . HoweverA number of studies on ESL spelling have found substantial overlap between L1 (English) spelling development and L2 (ESL/EFL) spelling development. For example, ESL spelling ability relies on largely the same component literacy skills  as does monolingual L1 English spelling ability . In addiThese general tendencies also influence the types of spelling errors or misidentifications that are made most frequently by learners from different L1 backgrounds. For example, Wang and colleagues  examinedAnother well-established domain of L1 influence on L2 spelling is L1 phonology: when examining the specific misspellings produced by L2 spellers, errors frequently appear for the spellings of sounds that do not exist or are not contrastive in learners’ L1. For example, very) and < s > and < z > , as well as the spellings of /i/ and /I/ . Similarly, in L1 Arabic speakers, spelling errors frequently involve confusion between graphemes representing sounds present in Arabic  and their voicing pairs, representing sounds absent from Modern Standard Arabic ; examples include < cabable > for capable and < habet > for habit. Similar evidence of L1 phonological influence on L2 spelling has been documented for L1 German  show less awareness of, lower sensitivity to, or less robust representations of vowels compared to consonants – “vowel blindness” . Early sIn sum, the limited research on ESL spelling abilities indicates that there is substantial overlap with L1 spelling development, including the reliance on phonological versus orthographic information. However, ESL spelling is also influenced by both L1 phonology (often leading to difficulties spelling non-native phonemes or distinguishing spellings for non-native phonemic contrasts) and the L1 writing system .As detailed above, despite the centrality of spelling ability to overall literacy skill, relatively little research has examined ESL spelling abilities. Most of the research that has been conducted – like L1 spelling studies  – has foThe current study was conducted in an effort to begin filling in these gaps. In contrast to much of the existing research, the target participants were adult learners of ESL at the university level (rather than children in elementary or middle school). Data were collected from learners with a range of L1 backgrounds; these were grouped into three main categories on the basis of L1 writing system: alphabet, abjad, or morphosyllabary. Collecting data from this range of L1 backgrounds facilitates cross-linguistic comparisons using uniform task items and procedures. In addition, data were collected from a comparison group of L1 English speakers, so that ESL performance (across L1 groups) could be understood in the context of native-level performance. Finally, participants also completed a phonological awareness task, so that spelling ability could be examined in relation to this key literacy skill. Phonological awareness was chosen because it is a critical component skill for literacy, particularly in the opaque orthography employed in English e.g., . It alsoData were collected from 70 participants: 47 ESL learners and 23 L1 English speakers (serving as a native speaker reference group). All participants were adult (18 years or older) students recruited from the intensive English program, undergraduate, and graduate student populations at a mid-sized regional university in the midwestern United States. The 47 ESL learners  were either preparing for or enrolled in undergraduate or graduate studies. They were classified into three groups based on the type of writing system employed in their L1: 27 alphabetic L1 participants ; 7 abjad L1 participants ; and 13 L1 morphosyllabary participants . Specific age data were not available for the 23 L1 English speakers , though all participants fell within the typical undergraduate student age range of 18–25 years old.For the ESL learners, L2 proficiency information was available in the form of a score from one or both of two different standardized English proficiency tests: the Test of English as a Foreign Language (TOEFL) or the Oxford Online Placement Test (OOPT). Because there is no established, validated method of converting scores from one of these two assessments to an equivalent score on the other, the scores on each assessment were instead converted to the corresponding level of proficiency as described in the Common European Framework of Reference : A1, A2,1. The 16 pseudoword items were taken from a variety of published studies examining pseudoword spelling in ESL/EFL learners  were resolved through discussion between a trained research assistant and the first author. A strict scoring procedure (implemented via an Excel formula) was used to determine accuracy: if the participant’s production was spelled exactly correct it was marked as correct, and any deviations from the correct spelling resulted in an incorrect marking.Responses to the Elision items were also transcribed into a digital spreadsheet for scoring and analysis. All responses to the Elision task were transcribed by a minimum of two trained research assistants; inter-rater reliability was initially 89% and items for which there was disagreement were resolved after consulting a third coder.r = 0.17, p = 0.30 with word accuracy, r = 0.24, p = 0.15 with pseudoword accuracy, r = 0.21, p = 0.23 with elision accuracy; gender: r = 0.09, p = 0.47 with word accuracy, r = 0.18, p = 0.15 with pseudoword accuracy, r < 0.01, p = 0.98 with elision accuracy) and were thus not included in any of the mixed effects models.Pearson correlations and generalized linear mixed effects models with random intercepts for participants and items , a number of item characteristics were significantly associated with real word spelling accuracy . Thus, length and number of orthographic and phonological neighbors were considered in model building. Log frequency  was also considered due to its substantial influence in general lexical processing; however, age of acquisition was not included in models because of its high correlation with length  and concerns regarding issues with multicollinearity.Correlations were also used to guide the selection of item characteristics to consider in modeling word and pseudoword spelling accuracy. Although no item characteristics were significantly associated with pseudoword spelling accuracy  were used to calculate semi-partial correlation coefficients between real word spelling accuracy, pseudoword spelling accuracy, and elision accuracy, controlling for L2 English proficiency.Pearson correlations  among real word spelling accuracy, pseudoword spelling accuracy, and elision accuracy are in 2(df = 3) = 35.86, p < 0.001, and the L1 English speakers were significantly more accurate than all three ESL groups: alphabetic L1 speakers , abjad L1 speakers , and morphosyllabic L1 speakers . Next, the ESL speaker groups were examined in more detail to explore the influence of both L1 writing system and L2 English proficiency on Elision accuracy. The overall effect of L1 writing system was significant, χ2(df = 2) = 8.11, p = 0.02. Specifically, the abjad L1 and morphosyllabic L1 speakers were both significantly more accurate than the alphabetic L1 speakers . However, the difference between the abjad L1 and morphosyllabic L1 speakers was not significant, β = 0.04, z = 0.07, p = 0.94. The effect of L2 English proficiency was also significant, β = 1.60, z = 3.97, p < 0.001, such that higher proficiency participants were significantly more accurate than lower proficiency participants. However, the interaction between L1 writing system and proficiency was not significant, χ2(df = 2) = 2.21, p = 0.33, suggesting that the effect of proficiency is the same regardless of L1 writing system.The means and standard deviations for Elision accuracy are given in z = 6.01, p < 0.001; the effect of length was marginally significant, β = −0.24, z = −1.80, p = 0.07; and the effect of orthographic neighborhood size was not significant, β = −0.004, z = −0.08, p = 0.93. The effect of elision accuracy was significant, β = 2.77, z = 5.71, p < 0.001, as was the overall effect of L1 writing system, χ2(df = 12) = 69.51, p < 0.001. Critically, L1 writing system also interacted significantly with lexicality, χ2(df = 3) = 13.50, p = 0.004. Therefore, further analyses were conducted for real words and pseudowords separately.The means and standard deviations for real word and pseudoword spelling accuracy are given in 2(df = 3) = 25.11, p < 0.001, as were the effects of elision accuracy  and word length . In addition, there were two significant interactions involving comparisons between the L1 English speakers and the alphabetic L1 speakers  as well as two marginally significant interactions involving comparisons between the L1 English speakers and the abjad L1 speakers . Thus, the final models of real word spelling accuracy examined L1 English speakers and the ESL speakers separately, and L2 English proficiency was also added as a factor for the ESL model.For real word spelling, model building initially considered all participant characteristics  and item characteristics  that correlations had suggested may be relevant, as well as log frequency. The overall effect of L1 writing system was significant, χ2(df = 2) = 0.20, p = 0.91 (though it was maintained in the model due to significant interactions with other variables). There were significant effects of proficiency and elision accuracy, such that participants with higher L2 English proficiency and with higher phonological awareness had significantly higher spelling accuracy than participants with lower L2 English proficiency and with lower phonological awareness. There were also significant effects of orthographic and phonological neighborhood size, with lower spelling accuracy for words with larger orthographic or phonological neighborhoods.These final models are given in 2(df = 2) = 7.82, p = 0.02; follow-up analyses within each L1 writing system group were used to examine this further. For the alphabetic and morphosyllabic L1 participants, the effect of phonological awareness was significant and positive: participants with higher elision accuracy had higher real word spelling accuracy . In contrast, for the abjad L1 participants, the effect of phonological awareness was marginally significant, but in the opposite direction: abjad speakers with higher elision accuracy had lower real word spelling accuracy, β = −4.60, z = −1.89, p = 0.06.There were also a number of significant interactions that qualified these overall effects. The interaction between L1 writing system and elision accuracy was significant, χThe interactions between elision accuracy and orthographic neighborhood size and phonological neighborhood size were also significant. In each case, participants who had higher phonological awareness experienced a larger effect of both orthographic neighborhood size and phonological neighborhood size. Finally, the interactions between word frequency and orthographic neighborhood size and phonological neighborhood size were significant. These interactions indicate that higher-frequency words were associated with smaller effects of both orthographic neighborhood size and phonological neighborhood size.2(df = 3) = 50.97, p < 0.001, as was the main effect of elision accuracy, β = 2.22, z = 4.36, p < 0.001. In addition, there were two significant interactions involving comparisons between the L1 English speakers and the abjad L1 speakers  as well as two significant interactions involving comparisons between the L1 English speakers and the morphosyllabic L1 speakers . Thus, the final models of pseudoword spelling accuracy again considered L1 English speakers and the ESL speakers separately, and L2 English proficiency was added as a factor for the ESL model.For pseudoword spelling, model building considered the same participant characteristics  and applicable item characteristics  that were considered with real word accuracy so that the results could be directly compared. The overall effect of L1 writing system was significant, χ2(df = 2) = 2.91, p = 0.23 (though it was again maintained in the model due to significant interactions with other variables). There was also a significant effect of proficiency, such that participants with higher L2 English proficiency had significantly higher pseudoword spelling accuracy than participants with lower L2 English proficiency.These final models are given in 2(df = 2) = 12.09, p = 0.002; and between L1 writing system and orthographic neighborhood size, χ2(df = 2) = 9.58, p = 0.01. Follow-up analyses within each L1 writing system group were used to examine these interactions further. For the alphabetic L1 participants, neither the effect of length  nor the effect of orthographic neighborhood size  were significant. However, within this alphabetic L1 model, the effect of proficiency was significant, β = 1.50, z = 1.98, p = 0.048, with higher pseudoword spelling accuracy among higher proficiency participants. For the abjad L1 participants, the effect of length was significant , with longer pseudowords being spelled more accurately than shorter pseudowords. The effect of orthographic neighborhood size was marginally significant , with larger orthographic neighborhoods associated with somewhat lower pseudoword spelling accuracy, but the effect of proficiency was not significant and was dropped from the final abjad L1 model. Finally, for the morphosyllabic L1 participants, the effect of length was significant , again with longer pseudowords being spelled more accurately than shorter pseudowords. The effect of orthographic neighborhood size was also significant , again with larger orthographic neighborhoods associated with lower pseudoword spelling accuracy, though the effect of proficiency was not significant and also dropped from the final morphosyllabic L1 model.There were two significant interactions that qualified these overall effects: between L1 writing system and length, χtypes and quantities of spelling errors committed by participants was conducted. First, the spelling of each grapheme in each item was examined to determine whether it was correct or incorrect. Generally following dress was divided into the following graphemes corresponding to phonemes: d| r| e| ss; separate was divided into s| e| p| a| r| aCe| t (with aCe representing the non-linear sequence of vowel + consonant + < e > indicating long vowel); and knowledge was divided into kn| ow| l| e| dge.Following the quantitative analysis of overall (strict) accuracy described above, a more in-depth investigation into the napkin); incorrect vowel grapheme (the wrong letter(s) representing a vowel phoneme in the word, e.g., < seperate > for separate); missing consonant grapheme (no letter(s) present to represent a consonant phoneme in the word, e.g., < resonsible > for responsible); missing vowel grapheme (no letter(s) present to represent a vowel phoneme in the word, e.g., < decsion > for decision); extra consonant grapheme  representing a consonant phoneme not present in the word, e.g., < grownd > for grown); or extra vowel grapheme  representing a vowel phoneme not present in the word, e.g., < recoganize > for recognize). Inter-rater reliability was initially 91% for real words and 63% for pseudowords; items for which there was disagreement were resolved through discussion among coders.Incorrect spellings of graphemes were further categorized in terms of whether the particular sound was misspelled or missing or whether additional sounds were represented in the spelling that were not present in the dictated word. More specifically, the categories were as follows (with examples drawn directly from the data): incorrect consonant grapheme (the wrong letter(s) representing a consonant phoneme in the word, e.g., < napcen > for rate of errors per item (rather than strict accuracy) as a function of L1 background, L2 English proficiency, and phonological awareness. The average number of errors per item of different types are given in Similar to the main analyses examining overall accuracy, linear mixed effects analyses were used, this time to examine the t = 4.40, p < 0.001, with significantly fewer errors made to real words than pseudowords. The overall effect of L1 writing system was also significant, χ2(df = 3) = 34.50, p < 0.001, as was the effect of phonological awareness, β = −0.44, t = −3.17, p = 0.002, with higher phonological awareness associated with fewer errors. The interaction between L1 writing system and item type was also significant, χ2(df = 3) = 9.66, p = 0.02. Thus, L1 English speakers and ESL speakers were further examined separately, with L2 English proficiency also added as a factor for the ESL model.The first error analysis examined the errors made to real words versus pseudowords . The effect of lexicality was significant β = −0.36, t = −0.19, p < 0.001, with higher phonological awareness associated with fewer errors. The effect of lexicality was also significant, with fewer errors made to real words than pseudowords, β = −0.61, t = 4.07, p < 0.001. The interaction between phonological awareness and lexicality was only marginally significant, β = 0.31, t = 1.70, p = 0.096; the trend was such that the effect of phonological awareness was slightly stronger for real words than it was for pseudowords.For the L1 English speakers, the effect of phonological awareness was significant, β = −54, 2(df = 2) = 3.28, p = 0.19. In addition, the effect of proficiency was not significant, β = −0.03, t = −0.30, p = 0.77. However, the effect of lexicality was significant, β = −0.74, t = −6.59, p < 0.001, with fewer errors made to real words than to pseudowords. The interaction between lexicality and proficiency was also significant, β = 0.49, t = 3.55, p < 0.001, with more errors made to real words than pseudowords among lower proficiency speakers, but more errors made to pseudowords than to real words among higher proficiency speakers.For the ESL speakers, the overall effect of L1 writing system was not significant, χFor the second error analysis, the incorrect, missing, and additional consonant errors and vowel errors were summed together by phoneme type to calculate the total number of consonant-related errors and vowel-related errors for each participant. These were then examined across L1 groups and L2 English proficiency to determine whether there were any differential accuracies by segment type (consonant versus vowel sounds), as has been demonstrated in previous research with abjad L1 speakers e.g., .2(df = 3) = 27.58, p < 0.001. The main effect of lexicality was not significant, β = 0.10, t = 1.42, p = 0.16, though the main effect of segment type was marginally significant, β = 0.05, t = 1.66, p = 0.099, with a slight trend toward more vowel errors than consonant errors. However, these main effects were qualified by a number of significant interactions, including between L1 writing system and lexicality, χ2(df = 3) = 18.38, p < 0.001 and between L1 writing system and segment type, χ2(df = 3) = 15.44, p = 0.001. Due to these significant interactions with L1 writing system, as with previous analyses, the L1 English speakers and ESL speakers were examined in more detail separately.There was a significant overall effect of L1 writing system, χt = −3.08, p = 0.01; lexicality, β = −0.15, t = −9.30, p < 0.001. Specifically, higher phonological awareness and real words were associated with fewer errors than were lower phonological awareness and pseudowords. Notably, the effect of segment type was not significant.For the L1 English speakers, only phonological awareness and lexicality were significant predictors: phonological awareness, β = −0.12, 2(df = 2) = 7.26, p = 0.03. However, a number of main effects were not significant: proficiency, β = −0.02, t = −0.33, p = 0.75; phonological awareness, β = 0.07, t = 0.52, p = 0.60; lexicality, β = 0.01, t = 0.10, p = 0.92; and segment type, β = 0.04, t = 1.11, p = 0.27. Importantly, these main effects were qualified by a significant interaction between L1 writing system and segment type, χ2(df = 2) = 14.44, p < 0.001. Thus, each L1 writing system group was examined separately.For the ESL speakers, the overall effect of L1 writing system was again significant, χt = −0.53, p = 0.60; phonological awareness, β = 0.04, t = 0.23, p = 0.82; and lexicality, β = −0.04, t = −0.40, p = 0.69. The main effect of segment type was marginally significant, β = 0.11, t = 1.97, p = 0.05, with somewhat more errors made involving vowels than consonants. However, there were a number of significant interactions among these variables. Proficiency interacted with lexicality, β = 0.20, t = 2.63, p = 0.01, such that there was no lexicality effect in higher proficiency participants, but there were more errors per item to real words than to pseudowords in lower proficiency participants. Proficiency also interacted with segment type, β = −0.15, t = −2.39, p = 0.02, such that higher proficiency participants made somewhat more errors involving vowels than consonants, but there was essentially no difference in lower proficiency participants. Lastly, phonological awareness interacted with lexicality, β = −0.35, t = −2.55, p = 0.01, such that phonological awareness was not related to the error rate among pseudowords, but higher phonological awareness was associated with a lower error rate among real words.For alphabetic L1 speakers, most main effects were not significant: proficiency, β = −0.05, t = −1.88, p = 0.08, with a lower error rate among real words than pseudowords. The effects of proficiency and phonological awareness were not significant and thus removed from the final model. However, the effect of segment type was significant, β = 0.16, t = 2.45, p = 0.02: abjad L1 speakers made significantly more errors involving vowels than consonants.For abjad L1 speakers, the effect of lexicality was marginally significant, β = −0.13, t = −2.66, p = 0.01, with a lower error rate among real words than pseudowords. The main effect of segment type was also significant, β = 0.24, t = 3.72, p < 0.001, with more errors involving vowels than consonants. As with the abjad L1 speakers, the effects of proficiency and phonological awareness were not significant and thus removed from the final model. However, the interaction between lexicality and segment type was also significant, β = −0.18, t = −2.05, p = 0.047; there were substantially more errors involving vowels than consonants among pseudoword items, but this difference was minimal among real words.For morphosyllabic L1 speakers, the effect of lexicality was significant, β = −0.17, The correlations among real word spelling accuracy, pseudoword spelling accuracy, and phonological awareness accuracy showed different patterns in each L1 group. For the L1 English speakers, all three tasks were significantly positively correlated with one another, whereas for the morphosyllabic L1 speakers, there were no significant correlations among these three tasks. The alphabetic and abjad L1 speakers fell somewhere in the middle: phonological awareness accuracy was significantly correlated with real word spelling accuracy for the alphabetic L1 speakers and was significantly correlated with pseudoword spelling accuracy for the abjad L1 speakers, though these were the only significant correlations in these groups.Regarding spelling accuracy, the native speaker comparison group (L1 English speakers) had substantially higher real word and pseudoword spelling accuracy than the three ESL groups. Interestingly, the L1 English speakers had relatively few predictors of spelling accuracy: only phonological awareness  and length . No other lexical characteristics influenced L1 English speaker spelling accuracy.longer pseudowords and for those with smaller orthographic neighborhoods.The picture was much more complex among the ESL participants. L2 English proficiency was consistently related to both real word and pseudoword spelling accuracy, such that spelling accuracy increased along with increasing L2 proficiency. Similarly, higher phonological awareness scores were associated with higher real word spelling accuracy in the alphabetic and morphosyllabic L1 speakers, but the opposite pattern was found for the abjad L1 speakers: higher phonological awareness was associated with lower real word spelling accuracy. In contrast, higher phonological awareness scores were associated with higher pseudoword spelling accuracy across L1 groups. Lexical characteristics also influenced ESL spelling accuracy: higher phonological awareness scores were associated with larger effects of orthographic and phonological neighborhood size in real words, whereas higher word frequency was associated with smaller effects of orthographic and phonological neighborhood size in real words. Among pseudowords, the abjad and morphosyllabic L1 speakers had more accurate pseudoword spelling for Considering the phonological awareness scores themselves, the L1 English speakers had higher accuracy than all ESL groups on the elision task. Among the ESL participants phonological awareness accuracy increased along with higher L2 English proficiency across L1s. However, the alphabetic L1 speakers were significantly less accurate in their elision performance than the abjad and morphosyllabic L1 speakers, who did not differ from one another.Finally, the L1 English speakers and ESL speakers also showed different patterns regarding the rates at which they made specific types of spelling errors. Among L1 English speakers, the only two patterns that emerged were that higher phonological awareness was related to a significantly lower error rate, and that the error rate was lower to real words than pseudowords. Once again, the pattern of results was more complex when considering the ESL speakers. Considering error rates to real words versus pseudowords, there was no overall effect of L1 writing system or L2 English proficiency level, but there was an unexpected interaction between lexicality and proficiency, such that lower proficiency ESL speakers had a higher error rate to real words than pseudowords, whereas higher proficiency ESL speakers had a higher error rate to pseudowords than real words. Considering error rates involving vowels versus consonants, there was a general tendency for a higher error rate to vowels than consonants. This was found among higher proficiency alphabetic L1 speakers, with pseudowords in morphosyllabic L1 speakers, and in abjad L1 speakers regardless of proficiency level or item type.The current study examined real word and pseudoword spelling accuracy in adult learners of ESL from a variety of L1 backgrounds. To facilitate cross-linguistic comparisons, these ESL speakers were grouped by L1 writing system: alphabetic L1s, abjad L1s, and morphosyllabic L1s. Participants also completed the Elision subtest from the CTOPP to measure their phonological awareness. Analyses focused on the relationships among real word and pseudoword spelling and phonological awareness skills, detailed analyses of the types of errors made by participants, and the variations in these patterns across L1 groups.As a whole, the results were largely consistent with the findings from previous research on ESL spelling abilities. The comparison group of L1 English speakers consistently had higher spelling accuracy , fewer errors per item, and higher phonological awareness accuracy than all three ESL groups. Considering the three ESL groups, previous research has generally found that ESL speakers from a morphosyllabic L1 typically have relatively strong real word spelling skills, particularly for irregular/exception words, but have substantial difficulties reading and spelling pseudowords, whereas ESL speakers from an alphabetic L1 typically have stronger phonological skills and rely on them for word reading and spelling, resulting in a substantial number of overregularization errors e.g., . The resGiven the relatively strong phonological skills that are typically found in learners with an alphabetic L1, it may initially seem surprising that the alphabetic L1 speakers in this study had such low pseudoword spelling and phonological awareness performance. However, there are a number of factors that can help explain this result. First, as discussed further below, the distribution of participants with different L1 writing systems was not even across proficiency levels, and there were more alphabetic L1 speakers at the lowest levels of proficiency  than in the other ESL groups. Given that spelling accuracy was also significantly related to L2 English proficiency, it is likely that the overall lower proficiency level of the alphabetic L1 speakers contributed to their lower raw scores. This interpretation is supported by the fact that, when L2 English proficiency was considered, the differences among the ESL groups were much smaller and real word spelling accuracy did not differ between the alphabetic and morphosyllabic L1 speakers.Another contributing factor is likely the nature of phonological skill development in L1 Spanish . Due in part to the highly consistent nature of the Spanish orthography, fine-grained phonological awareness skills  are not needed for Spanish literacy and thus may not develop readily e.g., , 2011. IIt must also be noted that, although the alphabetic L1 speakers had low strict spelling accuracy, a different picture emerges if error rates per item are considered. In word spelling, the alphabetic L1 speakers frequently had low overall rates of errors per word, particularly on pseudowords. Thus, although their spellings may not have been accurate in terms of strict coding, the spellings produced by the alphabetic L1 speakers were relatively more target-like than those produced by the other ESL groups. This finding also highlights another important point: the conclusion that is reached from research results may be heavily dependent on the format of the task e.g., , 2011, tless consistent than pseudowords. If these items are not well-known words for participants (as may be the case for those with lower levels of proficiency), they are likely to be more difficult to spell and to result in an increased error rate. However, with increased proficiency (and increased vocabulary knowledge), this effect should diminish or disappear – as is attested in the current data.Examination of the results for error rates also highlights another initially unexpected pattern in the findings: a higher error rate to real words than to pseudowords, particularly in lower proficiency learners. Given that real words may be known, but pseudowords by definition are unknown, it might be expected that spellings, regardless of scoring approach, would be more accurate for real words than pseudowords. However, a closer examination of the characteristics of English words and pseudowords themselves helps illustrate why this may not always be the case. English spelling is notoriously opaque , meaningThe differential relationships between spelling and phonological skills found in the current study are also consistent with previous research. In the L1 English speakers, both real word and pseudoword spelling accuracy were significantly related to phonological awareness skills. This is consistent with the continuing importance of phonological skills for literacy in the opaque English orthography, compared to other languages in which the importance of phonological awareness decreases rapidly with increasing reading proficiency e.g., . Consistnegative beta weight for elision accuracy as a predictor of real word spelling accuracy among the abjad L1 speakers, in contrast to the positive beta weights for the alphabetic and morphosyllabic L1 speakers. Based on a reviewer recommendation, we examined whether proficiency was a significant predictor of elision accuracy, and whether this relationship was different among the ESL groups. This is in fact what we found: there was a significant positive relationship between proficiency and elision accuracy for the alphabetic L1  and morphosyllabic L1 speakers  but not for the abjad L1 speakers . Thus, larger and more detailed datasets would be beneficial in future research to more directly explore these interactions and what they can tell us about L1 differences in literacy skills.Examining the varying relationships between spelling and phonological skills is also an area where additional research with a larger number of participants at both lower and higher proficiency levels would be beneficial. In the current results there was an unexpected finding of a The results from the current study do provide new data to contribute to an unresolved issue in L2 spelling research: whether (L1-specific) errors decrease with increasing L2 proficiency, or whether they persist over continued L2 development. Some previous studies have found that such errors do decrease with increasing L2 proficiency e.g., , whereasAnother question addressed by the current study was whether there would be significant L1 differences in accuracy spelling consonant versus vowel phonemes, as has been found for both L1 Arabic and L1 Hebrew speakers in prior research e.g., . BroadlyAlthough this study makes important contributions toward our understanding of adult ESL spelling abilities, there are still a number of limitations that must be acknowledged. First, although data were collected from a wide range of L2 English proficiency levels and three representative L1 writing systems, there were relatively small sample sizes in some combinations of L2 English proficiency and L1 writing system. In addition, the number of participants from each L1 writing system was not consistent across all levels of proficiency, with somewhat more alphabetic L1 speakers at lower levels of proficiency compared to the abjad L1 and morphosyllabic L1 groups. Future research would benefit from a larger sample size, including targeted recruitment of a balanced sample of participants across L1 writing systems and proficiency levels; this was unfortunately not possible given the current context of data collection. Such research will be useful for confirming the specific findings from this study.Another improvement that could be made by future research would be to include a wider variety of target items, both real words and pseudowords, and to specifically consider lexical characteristics during item selection. The items chosen for the current study were based on items previously used successfully in published research on ESL spelling abilities, but were not chosen with regard to their frequency, orthographic neighborhood size, or phonological neighborhood size. However, modeling the results for spelling accuracy suggested that these three lexical characteristics in particular may influence ESL spelling abilities, and are thus deserving of dedicated attention in future research.A strength of the current study was the use of a dictation task to assess spelling ability. Although this type of assessment is common in L1- and child-focused literacy research, it is much lesson common in adult L2 research, and using it in this study eliminated the influence of learner avoidance found in studies of spelling errors from naturalistic writing samples . HoweverAlthough spelling is a critical literacy skill it has received relatively little attention, especially in the domain of cross-linguistic L2 literacy research. The current study sought to address a number of gaps left by existing research, by focusing on adult ESL users (rather than children), considering not only real word spelling ability but also pseudoword spelling ability and phonological awareness, and using the same materials and procedure with participants from a variety of L1 backgrounds (thus facilitating cross-linguistic comparisons). In many ways the results were consistent with previous research, demonstrating relatively high  spelling accuracy yet somewhat weak phonological skills in morphosyllabic L1 speakers; more reliance on phonological skills in alphabetic L1 speakers; and significantly more errors involving vowels than consonants in abjad L1 speakers. However, there were also some unexpected patterns in the results, particularly the relatively low spelling accuracy and phonological awareness performance by the alphabetic L1 speakers. To some degree, these unexpected findings can be accounted for when considering another way to analyze the data: in terms of error rates, rather than strict accuracy. This highlights perhaps the most important individual finding from the study: the substantially different patterns of results, and conclusions, that may be reached when the data are scored differently. The field of L2 literacy research has much important work to do to document such variations and establish best practices for task choice, item selection, and particularly scoring procedures to improve reliability and validity.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Human Subjects Committee at Southern Illinois University Carbondale. The patients/participants provided their written informed consent to participate in this study.KM contributed to study design, item selection, data collection, data analysis, and writing. EL contributed to data analysis and writing. KC contributed to study design, item selection, data collection, and writing. EH contributed to study design, item selection, data collection, and writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The development of polymer-based devices has attracted much attention due to their miniaturization, flexibility, lightweight and sustainable power sources with high efficiency in the field of wearable/portable electronics, and energy system. In this work, we proposed a polyvinylidene fluoride (PVDF)-based composite matrix for both energy harvesting and energy storage applications. The physicochemical characterizations, such as X-ray diffraction, laser Raman, and field-emission scanning electron microscopy (FE-SEM) analyses, were performed for the electrospun PVDF/sodium niobate and PVDF/reduced graphene oxide composite film. The electrospun PVDF/sodium niobate nanofibrous mat has been utilized for the energy harvester which shows an open circuit voltage of 40 V (peak to peak) at an applied compressive force of 40 N. The PVDF/reduced graphene oxide composite film acts as the electrode for the symmetric supercapacitor (SSC) device fabrication and investigated for their supercapacitive properties. Finally, the self-charging system has been assembled using PVDF/sodium niobate (energy harvester), and PVDF/reduced graphene oxide SSC (energy storage) and the self-charging capability is investigated. The proposed self-charging system can create a pathway for the all-polymer based composite high-performance self-charging system.  The extensive use of renewable energy/power sources paves the way for the growing energy crisis and the environmental pollution issues, which creates the demand towards the need for energy sources ,2. To ov2O5), potassium permanganate (KMnO4), sulfuric acid (H2SO4), hydrogen peroxide (H2O2), hydrochloric acid (HCl), sodium hydroxide (NaOH), hydrazine hydrate (assay 50%), polyvinyl alcohol (PVA), phosphoric acid (H3PO4), N,N-dimethylacetamide and acetone were obtained from Dae Jung Chemicals and Metal Co., Ltd. . All the chemicals used in this research were research-grade, and double distilled water was used throughout the experiments.Graphite powder and polyvinylidene fluoride (PVDF) were procured from Sigma Aldrich Ltd. Niobium oxide  and galvanostatic charge-discharge (CD) measurements using an Autolab PGSTAT302N electrochemical workstation.The supercapacitor device was fabricated using the prepared PVDF/reduced graphene oxide composite film as the electrode for the device with PVA/H+ ion laser. The data were collected using a 10 s data point acquisition time. The PVDF/sodium niobate nanofibers were prepared to utilize the electrospinning/spray system . The surface morphology of the PVDF/reduced graphene oxide composite film and electrospun PVDF/sodium niobate mat were analyzed using a field-emission scanning electron microscope . The external force applied to the nanogenerator device using a linear motor  and the open-circuit voltage and the short-circuit current were recorded using nano voltmeter  and a picoammeter (Keithley 6485), respectively.The phase formation and the crystallinity of the graphene nanosheets and sodium niobate nanostructures were determined using the Rigaku X-ray diffractometer operated at 40 keV and 40 mA Cu Kα radiation. The ultrasound irradiation used for the dissolution of PVDF polymer solution was carried out on a SONIC  VCX 500 system  with the aid of direct immersion titanium (Ti-6Al-4V) horn. The Raman spectra of the PVDF/reduced graphene oxide composite film and sodium niobate were obtained using Lab Ram HR Evolution Raman spectrometer , operated at 10 mW laser power with an excitation wavelength of 514 nm with an ArP21ma. −1 [−1 all correspond to the ferroelectric phase of sodium niobite [−1 (ν1) accompanied by a shoulder at around 571 cm−1 (ν2) confirms the orthorhombic perovskite structure of the sodium niobate in the PVDF matrix [The physicochemical characterization of the sodium niobate and PVDF/sodium niobate nanofibers are provided in 21ma. −1  whereas  niobite . The preF matrix . FurtherF matrix . The surThe piezoelectric characterization of the bare PVDF and electrospun PVDF/sodium niobate nanofibrous mat was investigated by fabricating the nanogenerator device . Figure From the obtained results, it is evident that the output of the PVDF/sodium niobate nanogenerator is highly dependent on the applied compressive force and directly proportional to its piezo-voltage. The electrospun PVDF/sodium niobate mat as the active piezoelectric layer shows enhanced output compared with the other reported piezoelectric nanogenerators and the improved performance is due to the following reasons: (i) high ferroelectric property of the PVDF and sodium niobate results in the enhancement of the piezoelectric property of the composite, (ii) high voltage applied in the fabrication methodology, which enhances the arrangement of dipoles in the prepared composite mat ,39. Thes−1 corresponds to the D and G band of the graphene in the PVDF matrix [−1 corresponds to the characteristic band of PVDF [In the view of energy storage application, PVDF/reduced graphene oxide has been chosen as the electrode material. F matrix , and the of PVDF . The sur of PVDF . The FES of PVDF . Besides3PO4 as the electrolyte. Initially, the cyclic voltammetric analysis for the fabricated PVDF/reduced graphene oxide SSC was recorded over the voltage window of 0–0.8 V measured at various scan rates ranging from 1 to 10 V s−1 as shown in −1) symbolizing the excellent capacitive nature of the PVDF/reduced graphene oxide SSC device. The current of the CV profiles is increasing with the increase in the scan rate, suggesting the better capacitive nature of the device [C) of the PVDF/reduced graphene oxide SSC device was calculated using the relation [The electrochemical properties of the prepared PVDF/reduced graphene oxide film were investigated by fabricating a symmetric solid-state supercapacitor device using PVDF/reduced graphene oxide as the electrode and PVA/He device . The gale device . The CD e device . From Fie device . The sperelation : C = I ×I” is the discharge current (A), “Δt” is the discharge time (s), “ΔV” is the voltage window (V), and “A” is the area of the PVDF/reduced graphene oxide SSC device the electrode (cm−2). The PVDF/reduced graphene oxide SSC device delivered a specific capacitance of 683 nF was calculated from the CD profile measured at a constant discharge current of 10 nA. The change in the specific capacitance of the PVDF/reduced graphene oxide SSC device with respect to the current is provided in −2 was obtained at a higher current density of 50 nA cm−2, which almost retained 69.69 % of its initial capacitance with an increase of 5-fold in the current suggesting the better rate capability of the PVDF/reduced graphene oxide SSC device [Here “C device . Figure C device . Figure To investigate the capability of the self-charging system as a practical power source for electronic devices, the PVDF/sodium niobate nanogenerator was integrated with the PVDF/reduced graphene oxide SSC device to form the self-charging power system as shown in the scheme provided in The charging behavior of the PVDF/reduced graphene oxide SSC connected with the PVDF/sodium niobate nanogenerator under various applied compressive force is provided in In this work, we successfully fabricated the PVDF/reduced graphene oxide electrode and electrospun PVDF/sodium niobate nanofibrous mat as the active materials for the supercapacitor and nanogenerator device. The individual performance has been examined by fabricating the PVDF/reduced graphene oxide supercapacitor device and PVDF/sodium niobate nanogenerator device, which shows an excellent performance as the energy storage and harvester, respectively. The integrated self-charging power system can charge the supercapacitor device to 0.8 V in 190 s. Collectively, from the obtained results, the novel self-charging power system will paves the way towards the development of polymer-based self-charging system for wearable/portable electronics applications.


Forkhead box C2 (FOXC2) is a crucial factor involving in various cancers. However, its functions in hepatocellular carcinoma (HCC) is unknown. Here, we explored the role of FOXC2 in the progression of HCC and its potential mechanisms.FOXC2 expression in HCC tissue and cells were detected by immunohistochemistry or western blot and real-time PCR. CCK8, wound healing and transwell assay were used to measure cell growth and invasion. Tumor formation experiment was carried out to assess the tumorigenicity of HCC cells. Regulation of FOXC2 on Ang-2 was validated by luciferase assay and complementary experiments.Increased FOXC2 expression was found to be associated positively with more aggressive clinicopathologic features. HCC patients with higher FOXC2 expression had significantly shorter overall survival. FOXC2 expression was indentified as an independent risk factor for resectable HCC. Increased FOXC2 expression accelerated the migration and invasion of HCC cells, accompanied by enhanced Ang-2 expression. Likewise, FOXC2 knockdown yielded opposite results. Moreover, FOXC2 stimulated the activation of the Ang-2 promoter. Suppression of Ang-2 expression hindered the FOXC2-mediated EMT processs, cell migration and invasion of HCC.FOXC2 is a novel prognostic predictor for HCC and may facilitate the growth and invasion through Ang-2. As a fatal cancer with poor prognosis, hepatocellular carcinoma (HCC) causes the third-highest number of cancer-related mortality in sub-Saharan Africa, East Asia and the second-highest number deaths for males in China, with an increasing number of cases in Europe and the United States –3. In thA range of processes occurring in the cell, like metabolism, stress resistance, cell cycle arrest, apoptosis, and aging are regulated by the FOX (Forkhead box) family of proteins, which characteristically possess a DNA binding domain, that is evolutionarily conserved –7. The FAbnormal angiogenesis is one of the hallmarks of malignancy and a research hotspot for the development of novel targeted regimen . The A nTherefore, the expression levels and prognostic significance of FOXC2 in HCC tissues was determined in the present study. Additionally,we explored potential FOXC2 target genes involved in cell invasion and migration. Luciferase assays and western blot analysis were used to identify Ang-2 as a target gene of FOXC2. Ultimately, we knocked down endogenous Ang-2, using its shRNA, to elucidate the role of FOXC2 in tumor development.Paraffin-embedded HCC samples were obtained from the Nanfang hospital biobank. 280 HCC patients treated at Nanfang hospital between January 2009 and December 2014 were included. The inclusion criteria were (1) histologically confirmed the diagnosis and (2) no previous treatment. The exclusion criteria were (1) serious complications, (2) presence of other malignant diseases, or (3) incomplete follow-up data. For tumor staging, the Cancer staging system defined by the 7th Edition of the American Joint Committee was used. Besides, 40 pairs of HCC and respective adjacent non-tumorous samples were collected to analyze the relationship between FOXC2 and Ang-2.Nude mice were purchased from Southern Medical University, Laboratory Animal Center. Normal hepatic cell line L02 and HCC cell lines including sk-hep-1, hep3B, SMMC-7721, Huh-7 and MHCC-97H were purchased from the Chinese Academy of Sciences, Shanghai Cell Bank (China). 293T cells were purchased from ATCC (American Tissue culture collection). All cells were maintained in RPMI1640  containing 10% fetal bovine serum at 37 °C.For the gene knockdown (KD) studies, shRNA containing lentiviral particles for either FOXC2 or Ang-2 were purchased from Santa Cruz Biotechnology (Cat. No: sc-43767-V and sc-39305-V). The virus was collected after transfection for 60 h into 293T cells with all vectors and then infected target cells along with 8 mg/mL polybrene (Sigma-Aldrich). Western blotting was performed for selection of stable and independent clones.Lentiviral expression clones LV-pGV208-FOXC2, lentiviral packaging plasmids (pHelper 1.0 and 2.0) and control vector LV-pGV208 were purchased from Genechem . The supernatant containing lentivirus particles were harvested after transfection for 48 h in 293T cells and then were used to infect Huh-7 and MHCC-97H cells. Based on EGFP assay, lentiviral-infected cells were sorted with flow cytometry to achieve 100% cell infection. Overexpression of FOXC2 was confirmed through western blot.The protein of each group was collected after cell lysis with RIPA buffer and quantified using a BCA assay kit from Abcam . 20 µg of protein was separated on 10% SDS-PAGE gel, electrotransferred to nitrocellulose membranes , blocked with 1% BSA for 1 h and incubated with rabbit primary antibodies in 1:1000 dilution, including anti-Ang-2(#2948S), anti-FOXC2(#12974S),anti-E-cadherin(#14472S), anti-vimentin(#5741S), anti-fibronectin(#4705S) and anti-β-actin(#4970S) . Subsequently, the membranes were washed extensively with PBST  followed by incubation of secondary antibody, HRP-conjugated anti-rabbit IgG incubation for 1 h at room temperature. The ECL method was then used to visualize protein bands.® III First-Strand Synthesis System (Invitrogen). Real-time PCR was performed with a CFX96 Real-Time System (Bio-Rad) in the presence of 2X SYBR green master mixture (Invitrogen) in a final reaction volume of 10 μL. The primer sequences were: FOXC2 sense 5′-CCTACCTGAGCGAGCAGAAT-3′, antisense 5′-ACCTTGACGAAGCACTCGTT-3′, GAPDH sense 5′-TGTTGCCATCAATGACCCCTT-3′, and antisense 5′-CTCCACGACGTACTCAGCG-3′. The internal control was GAPDH. All experiments were run in triplicates in three independent experiments.Total RNA was extracted from HCC cells according to manufacturer’s instructions using Trizol (Invitrogen). 1 μg of RNA was used to synthesize cDNA with the SuperScriptCCK-8  was used to assess the cell proliferation as manufacturer’s instructions recommend. Briefly, cells were inoculated in 96-well plates at the density of 1.5 × 103 cell/well, and cultured in incubator for 24 h before evaluated at day 1, 2, 3, 4, 5, respectively. CCK-8 solution was then dripped into each well, and the plate was transferred to the incubator for 4 h. Finally, OD value at 450 nm was detected by HBS-1096B microplate reader .4 cells were inoculated in each well of a 6-well plate and maintained for 24 h in an incubator. Next day, a 1 mL pipette tip was used to scratch the cells at a width of 500 μm and then washed with RPMI-1640. At 24 and 48 h after scratch, cells were observed with an inverted microscope and photograph. Data summarize three independent experiments.The migration of cells was determined through wound healing tests following a standardized protocol. Briefly, 2 × 10Cells were incubated in PBS  and the upper chambers coating with 50% Matrigel (BD Biosciences) of 24-well transwell plates . After incubating for 24 h, invaded cells were examined and photographed through bright field microscopy  after staining with 0.5% crystal violet. The invasion rate was determined by counting the invaded cells in five randomly selected fields of each chamber under a fluorescence microscope . Data summarize three independent experiments.Tissue sections were boiled in citrate-hydrochloric acid (pH 6.0) for 30 min for epitope retrieval and incubated at 4 °C overnight with anti-FOXC2  and anti-Ang-2  primary antibodies. Immunostaining was performed using the Envision System with diaminobenzidine .The stained sections were scored independently by two pathologists who were blinded to the clinical parameters. FOXC2 expression was evaluated using the extent and intensity of staining. The staining intensity was scored as 0-no staining, 1-weak staining(seen as light yellow), 2-moderate staining(seen as yellow–brown),or 3-strong staining(seen as brown).The extent of staining was scored as 0(0%), 1(1 to 25%), 3(51 to 75%) and 4(76 to 100%), based on the percentage of positive stain relative to the entire cancerous area or the entire section for normal samples. The final staining score (0 to 7) was defined through the sums of the intensity and extent scores. Tumors with a final staining score < 3 were considered as low expression, while those with high expression had scores > 3 to 7.The binding regions of FOXC2 on Ang-2 promoter were predicted on the high-quality transcription factor binding profile database JASPAR . Three s6 HCC cells in 100 µl 1:1 of PBS and matrigel on both sides of each mouse. The growth rate of the tumor was monitored using bioluminescent imaging. Mice were anesthetized with 0.7% pentobarbital sodium (10 μl/g) and injected intraperitoneally with 150 mg/kg luciferin . Tumors were imaged through the IVIS Lumina II platform and analyzed with Live Image software . Each group had six mice.FOXC2-OE Huh-7 and FOXC2-OE-Ang-2 KD Huh-7 cells were transfected to express luciferase. 6 to 7-week-old male NOD/SCID mice  were injected were with 2*10t test or ANOVA; The differences were assessed using SPSS 19.0 software . P ≤ 0.05 was considered as statistically significant.All data are presented as mean ± SEM unless stated otherwise. Statistical significance (P < 0.05) was determined by two-side 280 paraffin-embedded HCC samples and 40 normal (non-cancer) samples were immunohistochemically analyzed for FOXC2 expression. According to the intensity of IHC staining, no or weak staining of FOXC2 protein was seen in 115 of 280 (41.1%) paraffin-embedded HCC tissues, while moderate staining (in the membrane and cytoplasm of cancer cells) was observed in 104 of 280 (37.1%) samples and strong staining was observed in 61 of 280 (21.8%) samples. In 40 non-cancerous control tissues, no FOXC2 staining was seen in 32 cases and weak expression was seen in two cases  and late stage (stage III-IV). In the early stage group, 69 patients showed high FOXC2 expression in tumor cells. Patients with high FOXC2 expression suffered from worse OS as compared to those with low FOXC2 expression  and overexpression (OE) HCC cell lines using MTT assay. As shown in Fig. To analyze the impact of FOXC2 on the migration of both HCC cell lines, we performed wound-healing assays. As illustrated in Fig. 2 = 0.709, p < 0.0001). Thus, FOXC2 may impact the production of Ang-2 to induce metastasis and invasion in HCC.According to the above results, we further analyzed FOXC2 and Ang-2 expression in 40 HCC tissues, of which 60.2% showed high FOXC2 expression. The result showed that tissues with high FOXC2 expression also exhibited high Ang-2 expression. As shown in Fig. Since Ang-2 also plays a crucial role in metastasis and invasion of malignancy, we further investigated whether FOXC2 modulates Ang-2 expression in MHCC-97H and Huh-7 cells via western blot. As shown in Fig. To determine whether Ang-2 is a direct target of FOXC2, three Ang-2 promoter fragments  were cloned into a pGL3-control vector for dual luciferase reporter assay. As observed in Fig. As expected, Ang-2 knockdown (KD) abated the growth of HCC cells with FOXC2 overexpression (OE) Fig. a, b. As The function of Ang-2 knockdown on the FOXC2-induced EMT process in HCC was assessed by measuring the expression of E-cadherin, vimentin, and fibronectin in FOXC2 OE and FOXC2 OE Ang-2 KD cells through western blot. Reduced Ang-2 expression in FOXC2 OE cells led to the enhanced expression of epithelial marker E-cadherin and weakened the expression of mesenchymal markers fibronectin and vimentin Fig. g. These The aforementioned in vitro findings strongly indicated that FOXC2 promoted the proliferation and invasion of HCC cells by modulating Ang-2 expression. We next evaluated the effect of FOXC2 on tumor growth by in vivo optical imaging in NOD/SCID mice.FOXC2 OE and FOXC2 OE Ang-2 KD Huh-7 cells with luciferase activity were injected into the flanks and shoulders of NOD/SCID mice Fig. a and e. By far, recurrence and metastasis remain the major cause of HCC-related mortality, and the underlying molecular mechanisms are still to be clarified.As a focus of current research field, FOXC2 has been reported as an oncogene in a range of cancers. It was well accepted that FOXC2 was a metastasis-related gene and might consequently serve as a predictor for the treatment response or prognosis of cancer patients in clinic use . In lineFurther analysis revealed a positive correlation between the expression of FOXC2 and Ang-2, led to our assumption that FOXC2 promotes Ang-2 expression for the invasion and metastasis of HCC. Luciferase assay showed that FOXC2 noticeably augmented the transcriptional activity of Ang-2, together with the result that Ang-2 expression was decreased in FOXC2-knockdown MHCC-97H and Huh-7 cell lines, suggesting that Ang-2 was a direct target of FOXC2.Hypervascularity is one of the remarkable features of HCC. In the past decades, the development of neovascular targeting therapy has dawned on HCC patients. The current therapeutic target of the angiogenic pathway includes VEGF signaling, angiopoietin 1(Ang-1), Ang-2 and tyrosine-protein kinase receptor Tie2. Agents targeting VEGF axis like bevacizumab, axitinib, lenvatinib, pazopanib, ramucirumab, sunitinib and vandetanib has been approved for the treatment of various cancers including colorectal cancer, non-small cell lung cancer, cervical cancer,ovarian cancer. Angiogenesis inhibitor targeting both VEGFR and Tie2 like cabozantinib, regorafenib and sorafenib have also shown efficacy in the treatment of renal cancer, thyroid cancer, gastrointestinal stromal tumor and liver cancer . Among tTo sum up, this study indicated that FOXC2 promote HCC migration and invasion, and the regulation of Ang-2 exacerbates these effects. Thus, FOXC2 and Ang-2 might be potent and promising targets for the prevention and treatment of metastatic HCC.Additional file 1: Figure S1. Fibronectin, vimentin and E-cadherin expression in FOXC2 OE and FOXC2 OE Ang-2 KD tumors were analyzed through western blot.


As a derivative material of graphene, graphene oxide films hold great promise in thermal management devices. Based on the theory of Fourier formula, we deduce the analytical formula of the thermal conductivity of graphene oxide films. The interlaminar thermal property of graphene oxide films is studied using molecular dynamics simulation. The effect of vacancy defect on the thermal conductance of the interface is considered. The interfacial heat transfer efficiency of graphene oxide films strengthens with the increasing ratio of the vacancy defect. Based on the theoretical model and simulation results, we put forward an optimization model of the graphene oxide film. The optimal structure has the minimum overlap length and the maximum thermal conductivity. An estimated optimal overlap length for the GO (graphene-oxide) films with degree of oxidation 10% and density of vacancy defect 2% is 0.33 μm. Our results can provide effective guidance to the rationally designed defective microstructures on engineering thermal transport processes. Graphene-oxide (GO) films show fascinating performance and have been widely applied in various fields ,3,4,5,6.The peculiarity and complexity of the flux transfer are a bottleneck for the thermal conductivity thin-film material at atomic scale. Efforts can be made to research the influence factors and thereby improve the quality of such heat transfer efficiency. A multilayered film can be fabricated by a single sheet in a layer-by-layer assembly approach See in a. This no ~ 100) . The vaco ~ 100) ,32. Meano ~ 100) ,33. Someo ~ 100) ,35 and to ~ 100) , could bDue to the limitation of experimental methods and techniques, the structure with a continuous large individual GO sheet is difficult to synthesize. The GO film structure with gaps is more easily formed See in b 37]. I. I37]. IIn this article, we highlighted a theoretic formula framework based on Fourier law to decouple the effects of in-plane and out-of-plane on thermal transfer in overlap GO films model. The in-plane thermal property of GO has been studied in our previous work . In thisy-axis is set to 1, and the value of In this work, a novel effective theoretical formula of GO films thermal conductivity is deduced based on the Fourier law, systematically considering the heat transfer both in-plane and out-of-plane directions. The derivation of the formula is similar to the previous work on the thermal properties of graphene-based layered materials . Herein,Bring Equation (2) into Equation  and eliminate A general solution of Equation  isAs shown in Using Equation (7) and a few mathematical techniques to substitute for From the above relations, the effective thermal conductivity From Equation (9) it can be found that uctivity ,42. λ caThe existence of the constant 2 is due to the periodic conditions of RVE.In this work, we perform classical molecular dynamics (MD) simulations to study the t groups . The hydt groups  simulatit groups ,44 impleMD simulations are performed using the large-scale atomic/molecular massively parallel simulator (LAMMPS) ,46. The i-th slab and The system is equally divided into 100 thin slabs along the heat transfer direction. The heat source and sink slabs are located at the middle and the two ends of the model, respectively. The time step of 0.1 fs is selected for integration on the equations of atomic motion in the simulations. The system reaches the equilibrium state at 300 K in the Nosé–Hoover thermal bath for 0.2 ns. Then, the system exchanges the kinetic energies every 100 time steps between the coldest atom in the heated slab and the hottest atom in the sink source slab for 0.8 ns. The total heat flux 8 Wm−2 K−1), the corresponding model is the graphene overlap structure. The effect of thermal transfer between GO strengthened gradually with an increasing degree of oxidation. When 8 Wm−2 K−1. 8 Wm−2 K−1, whereas both the degree of oxidation and ratio of the vacancy defect are the maximum  to the out-of-plane direction, which results in the increment of the thermal conductance. The vector arrows among the overlap structure are intensive as the increasing ratio of single vacancy and a hydroxyl group with heat flux see a. Especi6 GWm−3 K−1, 6 GWm−3 K−1, −3 K−1, 6 GWm−3 K−1 by fitting the curves in To obtain the Another key point in this work is to find the optimal overlap length From the above discussion, us works , and the−1 K−1, 6 GWm−3 K−1, When the ratio of vacancy defect is constant, the overlap length corresponding to In summary, an analytical model is developed to investigate the thermal properties of overlap GO films. The influence of coupled hydroxyl group (the ratio


In the current research, the biodiesel was prepared from feedstocks of Neem oil and Karanja oil employing a single step direct transesterification method using acid-base catalysts simultaneously. The fuel properties of both Neem and Karanja biodiesel along with different biodiesel-diesel blends were studied and compared. Biodiesel produced from Neem oil was found better in terms of kinematic viscosity, calorific value and cloud point for all its blends with diesel compared to Karanja biodiesel-diesel blends. Experiments were conducted to study the effects of addition of graphene nano particles on fuel properties of biodiesel-diesel blends. The B20 biodiesel-diesel blend was selected, which was blended with graphene nano particles in different proportions  to get different stable and symmetric B20-nano blends. The fuel properties except kinematic viscosity were further improved with higher dosages of nano particles with the biodiesel-diesel blend. The performance and emissions tests were conducted on 4-stroke variable compression ratio diesel engine. Higher concentrated B20-nano blends of Neem (NOME20GO105) and Karanja (KOME20GO105) resulted in 31 and 30.9% of brake thermal efficiency, respectively, compared with diesel of 32.5%. The brake-specific fuel consumption (BSFC) was reduced by 10 and 11% for NOME20GO105 and KOME20GO105, respectively, compared to their respective B20 blends. Similarly, carbon monoxide (CO) was reduced significantly by 27 and 29% for NOME20GO105 and KOME20GO105, respectively.  The mixture was shaken well for proper mixing. Later boron triflouride (BF3), which acts as an acid catalyst, was added to the reactor containing methanol and sodium methoxide mixture. Finally, crude oil was added to the reaction mixture. The crude oil, methanol, sodium methoxide and boron triflouride were taken in the ratio 1:4:0.1:0.1, respectively. The reaction was carried out for just two hours at 50 °C. After 2 hours of direct transesterification, the reaction was stopped. The author used petroleum ether to separate biodiesel from the reaction mixture in his previous published research. The method described was not economical and was consuming more time when performing evaporation and condensation of petroleum ether . TherefoIn this study, Graphene oxide (GO) has been used as a fuel additive synthesized using chemical vapor deposition (CVD). GO possess excellent thermal and electrical conductivities, higher magnetism besides high surface area to volume ratio. It improves the convective heat transfer coefficient, calorific value, cetane number of fuel if it is blended with the fuel. Furthermore, it reduces ignition delay and enhances micro-explosion. 2 v−1 s−1, surface area 2630 m2g−1, thermal conductivity 3300 Wm−1K−1 and purity of 95–98.5%The size of GO NPs is 23–40 nm, Young’s modulus 1.0 TPa, an inherent mobility of 200,000 cmThe physico-chemical properties of Neem and Karanja and their respective methyl esters along with their blends have been found in accordance with the ASTM D6751 standards. Experiments were conducted on single cylinder four stroke variable compression ratio engine with electric starter connected to eddy current type dynamometer for loading as shown in Throughout the experiments, the compression ratio, fuel injection pressure and ignition timing were kept constant at 17.5: 1, 205 bar and 23° before top dead center, respectively. To record different emissions such as HC and CO, an AVL DiGas444G gas analyzer  was used, which works on a non-dispersive infra-red technology (NDIR). Smoke in the exhaust gas was measured by the Smoke Meter AVL . All the readings were noted after the engine is reached steady state. The engine was loaded with the help of an eddy current dynamometer. The uncertainties and accuracies of the measured parameters in the current investigations are given in R in Equation (3) reveals the function of X1, X2,……nX, and X1, X2,……nX indicates the no. of obtained readings. Therefore, The variance of the measured factors estimated utilizing Gaussian distribution as described in Equation (1), under the of limits confidence of ±2σ. The limit 2σ reflects the average bound of 95% of the calculated values.e limits .By utilizing Equation (4), the uncertainties in various quantified and specific variables were analyzed. The errors bars were by averaging 3 readings and included in all the engine characteristics using NOME, KOME and graphene oxide fuel blends.2/s) biodiesel is less by 4.97% compared to Karanja (5.0901 mm2/s) biodiesel at 40 °C as shown in The calorific values of diesel, Neem biodiesel and Karanja biodiesel are 45,369, 40,050 and 39,538 kJ/kg, respectively, and are shown in The nano-additives are used to improve some of the biodiesel fuel properties, which further improve the performance of the engine besides reduced emissions with no or little engine modifications. Nano particles (NPs), which possess enhanced surface area/volume ratio, also act as a catalyst. The higher surface area/volume ratio improves the quality of air and fuel mixture and, hence, increases the rate of chemical reaction during combustion . Higher In the current investigation, an ultrasonication technique is used to stably blend the nanoparticles in the fuel blends. Initially, different concentrations of nanofluids are prepared using bath and probe sonication method. The nanofluids are blended with a diesel–biodiesel (B20) blend, initially the nanofluids  are transferred to diesel–B20 fuel blend, to remove excess moisture, the blends are heated at 60 °C using magnetic stirrer; later, it is sonicated for one hour using bath sonication and, finally, the nano fuel blends are stably dispersed using probe sonication at a frequency of 15–20 Hz for 20 min for each fuel blend. These steps enable stable dispersion of nanoparticles in fuel blends, thus preventing the nanoparticles from agglomerating. Ultrasonication technique avoids the agglomeration to the larger extent and, hence, is used in the present research for the preparation of NPs dispersed in base biodiesel fuel. Metal based nano additives results in agglomeration, clustering and settling due to their short-term stability. Hence, in the present research, the author has used graphene oxide, a carbon-based NP, to make the Biodiesel-Diesel-Nano blends. Nano additives are blended with a B20 biodiesel-diesel blend in the three different dosage levels of 35, 70 and 105 ppm. They are denoted as NOME20GO35, NOME20GO70 and NOME20GO105 for Neem Biodiesel-Diesel-Nano blends. Similarly, KOME20GO35, KOME20GO70 and KOME20GO105 are for Karanja biodiesel-diesel–graphene oxide nano blends.Higher kinematic viscosity of the fuel affects the fuel supply system in the engine. The fuel injection system and charge preparation are reliant on density, volatility and viscosity of fuel, which are often interdependent. The kinematic viscosity of biodiesel-diesel blends increase with the addition of nano additives as shown in The variation of brake thermal efficiency (BTE) with load for diesel, NOME20, KOME20, along with different dosage levels  of graphene oxide NPs has shown in The brake-specific fuel consumption (BSFC) is an important performance parameter of an IC engine. Hydrocarbons are the result of incomplete combustion of the fuel. Diesel engines are the major source of formation of smoke and are more spontaneous at maximum engine load conditions, which can be observed from Unavailability of sufficient quantity of oxygen for combustion inside the combustion chamber is the main reason for incomplete combustion and, hence, formation of CO. The formation of CO with brake power for different fuel blends has been shown in The current research is a comparison of fuel properties biodiesel and their blends prepared by using the non-edible feedstocks of Neem and Karanja by employing direct transesterification process. Furthermore, B20 biodiesel-diesel blends were blended with graphene oxide NPs in different proportions. The following points are drawn from the results.Biodiesel prepared by direct transesterification reduced the reaction timing.Neem biodiesel is slightly better compared to Karanja in terms of calorific value, kinematic viscosity. There is a marginal difference in cloud point and flash point.The fuel properties of both blends improved with addition of higher dosages of graphene oxide nano particles.Maximum BTE of diesel, NOME20GO105 and KOMEGO105 is 32.5, 31 and 30.9%, respectively, at full load.BSFC for NOME20GO105 and KOME20GO105 was reduced by 10 and 11%, respectively, compared to NOME20 and KOME20.CO for NOME20GO105 and KOME20GO105 was reduced by 27 and 29%, respectively, compared to NOME20 and KOME20.BTE, BSFC, CO, HC and smoke were slightly better for the diesel-fueled engine compared to NOME20GO105 and KOME20GO10 run engines.


Lactate is an important organic molecule that is produced in excess during anaerobic metabolism when oxygen is absent in the human organism. The concentration of this substance in the body can be related to several medical conditions, such as hemorrhage, respiratory failure, and ischemia. Herein, we describe a graphene-based lactate biosensor to detect the concentrations of L-lactic acid in different fluids (buffer solution and plasma). The active surface (graphene) of the device was functionalized with lactate dehydrogenase enzyme using different substances  to guarantee stability and increase selectivity. The devices presented linear responses for the concentration ranges tested in the different fluids. An interference study was performed using ascorbic acid, uric acid, and glucose, and there was a minimum variation in the Dirac point voltage during detection of lactate in any of the samples. The stability of the devices was verified at up to 50 days while kept in a dry box at room temperature, and device operation was stable until 12 days. This study demonstrated graphene performance to monitor L-lactic acid production in human samples, indicating that this material can be implemented in more simple and low-cost devices, such as flexible sensors, for point-of-care applications. Lactate is present in various biological processes and medical conditions, and it has an important role in the anaerobic metabolic pathway ,2. UsualMost of the reported biosensors used to detect lactate concentration depend on a specific enzyme to catalyze the reaction and enhance the selectivity; the product of the reaction is usually proportional to the analyte concentration ,9,10,11.In the clinical environment, lactate concentration is commonly measured using a blood gas analyzer that is large bench-top point-of-care (POC) equipment and it has certain limitations regarding processing time and sample preparation ,8,24. ThGraphene is a single-layer material with a large surface area and paramount electronic and chemical properties that can increase the sensitivity and specificity of a biosensor when applied as an active layer ,35,36,37In this study, we present a multiplexed solution-gated graphene-based field-effect transistor sensor to determine the concentration of lactate in different fluids. The lactate was detected using the LDH enzyme to catalyze the chemical reaction in the biological fluid. The enzyme was immobilized using different substances to guarantee stability and selectivity over the active layer. The transfer characteristics of the common-gate GFET were measured for each concentration in different electrolytes (buffer solution and blood plasma). The selectivity was verified using interference substances , and the stability of the GFET devices was monitored for up to 50 days.AZ-1512, SU-8 2075, hexamethyldisilazane (HMDS), and (1-methoxy-2-propyl) acetate (SU-8 developer) were purchased from MicroChem Corp. . Polydimethylsiloxane (PDMS) (Sylgard 184 A/B) was obtained from Dow Corning . Graphene layers grown by chemical vapor deposition (CVD) were acquired from Graphenea . Human plasma, uric acid, ascorbic acid, iron (III) chloride powder, and phosphate-buffered saline were supplied by Sigma-Aldrich Corp. . Illustrations were created with BioRender.com.The fabrication of the coplanar electrodes on a glass substrate was reported in our previous works . Briefly, the glass substrate was spin-coated with the adhesion promoter (HMDS) and with the positive photoresist (AZ-1512). The electrodes were patterned using a photomask and ultraviolet radiation over the glass by a mask aligner . The titanium and the gold layers were deposited over the glass using a vacuum thermal evaporator system (Evaporation System SHE-6T-350D). The CVD-grown graphene coated with a PDMS layer was transferred onto the region between the eight pairs of electrodes (source and drain) after the copper layer was etched away, and the supportive layer (PDMS) was detached from the graphene during an acetone bath. The PDMS microfluidic channels containing eight channels were bonded onto the substrate by UV–ozone treatment. In The electrode array was assembled with PDMS channels and the active layer surface was treated with different substances to guarantee stability and selectivity of the device. The enzymatic matrix was fabricated using Nafion, chitosan, lactate dehydrogenase enzyme, and glutaraldehyde. First, 3 µL of Nafion (NA) was drop-casted over the graphene for 1 h, after which the channels were washed with phosphate-buffered saline (PBS). Nafion is a sulfonate fluoropolymer–copolymer and is used as a membrane to block anionic interference species, such as ascorbic acid ,44,45,46Dirac) of the bare graphene was determined considering the lowest drain current (ID) measured while applying a constant bias (VD) on the drain terminal of 0.4 V and varying the bias (VG) applied to the gate. The microchannels were cleaned with deionized water and dried in an oven after the characterization of the bare devices. Then, the enzyme was immobilized following the steps mentioned in the previous subsection to produce the immobilization matrix. The transfer characteristics of the graphene layer were verified using an HP Agilent 4145B Semiconductor Parameter Analyzer  while using the buffer solution (PBS) as an analyte. The Dirac point voltage  to focus on clinical applications. A schematic of the experiment is illustrated in Dirac of 1 V (standard deviation (SD) of 0.03 V). The same was reproduced for the individual gate structure, with a mean VDirac of 1.03 V (SD of 0.06 V). The Dirac point voltage values of each structure are presented in The measured transfer characteristics of the graphene-based devices were used to define reference points before the immobilization matrix formation and the lactate assays. In our previous work, a solution-gated GFET with a coplanar electrode array was developed for cancer diagnostics . The intAfter electrical characterization, all the microchannels were cleaned with deionized water and the lactate detection assay was performed. First, the enzyme was injected over the graphene surface into the eight microchannels. The device was placed in the probe station, and different concentrations of L-lactic acid (0.25 to 10 mM) were mixed with the LDH enzyme, considering that the reference line for bare graphene was measured with the buffer solution only. The concentration range was defined based on the reference values used to detect sepsis shock in blood samples, since lactate is a prognosis marker among septic shock patients ,7. Dirac are shown in Dirac values demonstrate a linear relationship with lactate concentration in 2 = 0.8549). Adding high concentrations of L-lactic acid in the channel with the presence of the LDH enzyme causes the production of more positive and neutral charges (NADH and H+), then the neutrality point is higher when the concentration increases [The Dirac point voltages were measured after the LDH enzyme catalyzed L-lactic acid, resulting in a product that doped the graphene surface with negative charges, as shown in Dirac obtained with bare graphene. Based on the results presented in Dirac only shifted without the presence of chitosan. However, the matrix was still too thick for the microchannel; some devices experienced issues regarding the flow of analyte in the channel, even after drying them for more than 60 min. To overcome this clogging issue, a cleaning procedure with deionized water was repeated more than three times after the deposition of each component in the matrix. New devices were fabricated to evaluate the proposed immobilization matrix. It is necessary to reduce the number of layers over the active layer to avoid clogging the microchannels. Since Nafion and glutaraldehyde are essential to blocking the determined substances, we verified the performance of the enzyme with and without chitosan, since this material is an electron mediator that can improve the LDH enzyme immobilization capacity ,19,48. ADirac values of devices measured during the 50 days are shown in Dirac increased greatly after 12 days of measurement, as did the number of failed devices. The instability of the enzyme can be considered a drawback of the method presented in this study, and it is necessary to evaluate a new storage approach to guarantee the enzyme function for at least 6 months, since the device will be adapted as a point-of-care sensor in future study.Considering the final immobilization matrix composed of Nafion, chitosan, LDH, and glutaraldehyde, stabilization of the enzyme was verified for up to 50 days while the devices were kept in a dry box at room temperature. In this study, we tested the storage of the devices in a refrigerator and a dry box, although the devices maintained in a refrigerator had no electrical response even after 2 days of storage. We decided to proceed only with the devices stored in the dry box. For each day measured, 5 mM of L-lactic acid were injected into the microchannel, and the signal response was recorded. The VDirac increased from the lowest to the highest concentration in blood plasma samples. For each concentration, three channels were used to obtain the error bars. The variation of the VDirac for the selected biological fluid is presented in 2 = 0.9787, n = 3). Compared with the works in the literature that also used human plasma samples, the linear response observed with the graphene-based device was similar or superior, and we also investigated a wider range of concentrations [Previously, the performance of common-gate GFET devices was verified using the proposed immobilization in buffer solutions with different concentrations of L-lactic acid. Then, blood plasma was used as an electrolyte to confirm the capability of the device to detect lactate in real human samples. First, blood plasma was injected (10 µL) into the channels after being mixed with different concentrations of lactate, from 0.25 to 7.5 mM, based on the reference values in the literature considering clinical emergencies where the level of lactate is abnormal . As obsetrations ,51. BesiDirac of the buffer solution was used as a reference point. The plasma sample containing 7.5 mM of L-lactic acid was injected into the same microchannel as when the transfer characteristics were measured; this procedure was repeated for each interference species with concentrations that were considered for a normal blood sample from a healthy human. New experiments were performed to evaluate the interference from electroactive species in the human samples ,21. The Dirac values are presented in The GFET devices were measured, and the output signals were analyzed, and the VDirac, indicating a limitation of this enzyme with the respective immobilization matrix. Additionally, different interference substances  were injected into the GFET devices during the lactate assays to verify the selectivity of the sensor. The variation of VDirac was significant after injection of the plasma sample with 7.5 mM of lactate considering the reference point (PBS solution), and the maximum shift observed was 0.09 V among interference substances. Considering the results obtained in this study, graphene demonstrated the potential to detect L-lactic acid in human fluids without the interference of other biochemicals. To the best of our knowledge, a GFET device to detect the concentrations of lactate in human samples had not been reported, also considering the combination of elements in the immobilization matrix. With the implementation of low-cost materials to our proposed method, it is possible to detect lactate in a fast and precise diagnostic method for clinical applications taking advantage of the properties of the graphene-related materials, e.g., graphene oxide and reduced graphene oxide. The next step of this study is to include two new analytes to increase the prediction of a sepsis shock by developing a flexible, low-cost, and disposable chip for a point-of-care device with a rapid and accurate response.We demonstrated in this study a graphene-based biosensor to provide rapid quantitative measurement of lactate in the buffer solution and blood plasma. Determination of L-lactic acid was performed by measuring the transfer characteristics of graphene in a field-effect transistor structure with different concentrations of the analyte. Different materials were tested for the immobilization matrix to guarantee the stabilization and selectivity of the device; the best combination includes Nafion, chitosan, lactate dehydrogenase, and glutaraldehyde. The proposed immobilization matrix was structured considering the role of each biochemical to improve the performance of the graphene-based sensor. After the measurements, a linear response was observed for all the samples, including the blood plasma, with different concentrations (from 0 mM to 7.5 mM). The capability of detection of lactate at high concentrations (up to 7.5 mM) indicates that the proposed device can be implemented for POC applications for different disorders, such as septic shock and left ventricular failure. The presence of chitosan was essential to maintain the stability of the LDH enzyme, which was evaluated through the electrical measurements after storing the GFET devices at room temperature in a dry box for up to 50 days. After 12 days, there was a significant shift of V