Shigella. As its molecular mechanism of action is still poorly understood, we used its structure to guide site-directed mutagenesis and to dissect its function. We identified mutants predominantly affecting all known features of MxiC regulation as follows: secretion of translocators, MxiC and/or effectors. Using molecular genetics, we then mapped at which point in the regulatory cascade the mutants were affected. Analysis of some of these mutants led us to a set of electron paramagnetic resonance experiments that provide evidence that MxiC interacts directly with IpaD. We suggest how this interaction regulates a switch in its conformation that is key to its functions.Type III secretion systems are complex nanomachines used for injection of proteins from Gram-negative bacteria into eukaryotic cells. Although they are assembled when the environmental conditions are appropriate, they only start secreting upon contact with a host cell. Secretion is hierarchical. First, the pore-forming translocators are released. Second, effector proteins are injected. Hierarchy between these protein classes is mediated by a conserved gatekeeper protein, MxiC, in  Escherichia coli, Salmonella, and Shigella species.Type III secretion systems (T3SSs)Shigella species are the etiological agent of bacillary dysentery in humans  form a flat, elongated structure   .Yersinia YopN .We generated an x et al. . In our degraded B.A) , indicating deletion of the N terminus affects its ability to become secreted. However, secretion of IpaB, IpaC, and IpaD was unaffected in mxiCΔNterm; small differences between ΔmxiC/mxiC+ and mxiCΔNterm were not statistically significant (A and C). Thus, the ability of MxiC to prevent effector secretion and to promote inducible translocator secretion are not coupled, and only the former requires its secretion signal.As described previously, MxiCΔNterm is not secreted after Congo red (CR) induction A 23) an anA) , which are equivalent to the last 11 residues of SepL  ; translocators were only weakly induced, and effector proteins were leaked. In addition, MxiCΔCterm itself was only poorly secreted  even in ΔipaB . Therefore, unlike for SepL, MxiC's C terminus is essential.The 11 C-terminal residues of the EPEC MxiC homolog, SepL, are required for regulation of effector secretion but not for translocator secretion . As a coFig. S3) . The restop) to generate mxiCΔCBD, lacking residues 32–72. This construct was stably expressed, but it was leaked and only weakly secreted upon CR induction (A). Interestingly, secretion of MxiCΔCBD was enhanced in a ΔipaB background . This indicates the chaperone-binding domain influences the regulation of MxiC secretion rather than the intrinsic ability of the protein to become secreted. Furthermore, although mxiCΔCBD could not block effector secretion, it was able to promote translocator secretion (A).The chaperone-binding domain of YopN has a chaperone-independent role in secretion regulation . We thusnduction A. Intereecretion A.supplemental Fig. S5), we made several single and combined charge-swap mutations. mxiCK66E was unable to complement ΔmxiC. Paradoxically, the mutant protein was detected in culture supernatants and, to a lesser degree, in the supernatants after CR induction, but only low levels were detected in whole culture lysates (C). mxiC had a wild-type-like phenotype, except for some premature MxiC secretions. mxiC leaked MxiC at high levels and was unable to prevent effector secretion (data not shown). A similar phenotype was observed in mxiCK68E . Translocator IpaD and early effector IpgD were secreted at intermediate levels of ∼40–50%. Thus, either only a small proportion of intracellular MxiC is involved in blocking secretion and/or its secretion per se is not required to release this block.MxiC lacking its secretion signal is not secreted and is also unable to prevent effector secretion . In an mreleased , 16. ThuYersinia YopN needs its heterodimeric chaperone SycN/YscB for efficient secretion and regulation  and a triple deletion (ΔipgEΔipgAΔspa15). However, even the deletion of multiple chaperones had no effect on MxiC . This suggests that none of these chaperones influences MxiC function. Therefore, either there are no such chaperones binding to the CBD of MxiC, or we have not identified them because of low sequence similarities. However, we think the latter is unlikely because MxiC is primarily monomeric in cytosol, and no known class 1 chaperone was identified, even using mass spectrometry after cross-linking , suggesting it as a site for protein-protein interactions. Cherradi et al.  do not effect its function. We mutated residues Leu-222 and Leu-242 into serines and found that neither mxiCL222S and mxiCL242S nor the double mutant mxiC had any effect on MxiC stability or function (data not shown).Deane et al.  noticed i et al.  changed mxiC mutant was stably expressed (C) and able to induce translocator secretion after induction (A). However, mxiC was unable to prevent effector secretion (A and B). Thus, the hydrophobic patch is involved in preventing effector secretion.As change to a polar side chain may not have been enough to disrupt interactions in this area, we introduced two charges. The xpressed C and ablnduction A. Howeveecretion , A and Btop), is not conserved. However, it contains multiple serines, aspartates, valines, and threonines that are not classically helix favoring . We generated a model of the “bent” wild-type protein using the YopN/TyeA structure . In comparison with the crystal structure, the bent MxiC model is not only folded at the hinge region but undergoes a slight twisting motion. We then made three mutants as follows: one mutant introducing a proline  that would likely cause a break in helix 9 and thus a conformation similar to that of YopN/TyeA; a second mutant introducing three glycines  in the area to favor switching between both putative forms; and a third mutant that might stabilize a straight helix . MxiC Ile-251 is structurally equivalent to Phe-268 of YopN, which is sandwiched between hydrophobic residues in the core of TyeA, thus likely stabilizing the bent conformation.The sequence in MxiC helix 9, its putative “hinge” region , top, isfavoring . Multiplode 1XL3 ) as tempC). The triple glycine mutant mxiC behaved like ΔmxiC. MxiC was not secreted efficiently, and translocator secretion was reduced (43 ± 23% of the complemented strain), and effector proteins were leaked. Under the straight mutant mxiC, induced secretion of both IpaB and IpaC was reduced, whereas that of IpaD was as efficient as in the complemented strain or even increased (D). This mutant did not affect secretion of MxiC or effector proteins. The bent mutant mxiCV256P leaked high levels of a low molecular weight protein. We identified this band by mass spectrometry using a sample equivalent to the “−CR” sample in Fig. 5A. The top T3S-related protein identified was effector IpgB1 . The similarly sized effector/anti-activator OspD1 was not detected in this band. The equivalent band from a ΔmxiC “+CR” sample was analyzed in parallel; again, IpgB1 was the top T3S-related hit . These results suggest that a “straightened” MxiC favors the earliest step in the induction hierarchy, i.e. IpaD secretion, whereas a bent MxiC cannot prevent or favors, independently of its own secretion, the final one, i.e. effector release. In the wobbly mutant, three residues are altered that are initially identical to those changed in the straight one, but its phenotype is opposite, i.e. more like the bent one. This indicates that it is not the chemical nature of the amino acids in this surface patch that leads to these phenotypes but rather the stability of the secondary structure they form.All mutant proteins were expressed at wild-type levels or better C. The trncreased D. This mated hit . The mxiThe secretion patterns observed in the helix 9 mutants suggested movement in this area of MxiC is required for its function. We decided to examine the molecular conformation(s) of MxiC using double electron-electron resonance (DEER), also known as pulsed electron double resonance (PELDOR). This is an increasingly popular electron paramagnetic resonance (EPR) technique used to measure distances between paramagnetic spin labels on a nanometer scale .2VJ4 , we chose residues Ala-247 and Ser-290 to introduce paramagnetic centers into the molecule as the distance between these residues was predicted to be in the measurable range for both forms, and the difference in distance between both forms was predicted as significant enough to be detectable . To covalently couple the paramagnetic nitroxide label MTSL to these residues, Ala-247 and Ser-290 were mutated into cysteines. At the same time, the two endogenous cysteines Cys-184 and Cys-233 were mutated into alanine and serine, respectively, to finally generate a quadruple mutant mxiC or mxiC(Cys). Alternatively, complementary mutant MxiCs, with cysteines for labeling inserted at two sets of different but equally suitable locations, could not be purified in sufficient amounts (data not shown).Based on the MxiC structure (chain A of PDB entry 2VJ4 ) and themxiC(Cys) behaved like the complemented strain in a CR secretion assay . A His-tagged version of the cysteine mutant (His-MxiC(Cys)) was soluble when expressed in E. coli . We also combined the quadruple cysteine mutant with the previously generated bent (mxiCV256P) and straight ) mutants. However, neither His-MxiC nor His-MxiC were solubly expressed in E. coli (data not shown). Thus, we could only analyze the “wild-type” His-MxiC(Cys).The m sample (A). Using a previously obtained standard curve, the spin concentration was calculated to be 66 μm. The amount of residual unbound label was calculated to <1%; thus a spin labeling efficiency of close to 100% could be extracted. The spectrum also indicated the protein was folded; because of increased mobility in a disordered structure, an unfolded protein would result in a spectrum similar to that of the free label.MTSL was covalently coupled to His-MxiC(Cys) at residues 247 and 290. The protein was concentrated and mixed with deuterated glycerol for EPR/DEER experiments . The labeling efficiency was assessed using continuous wave room temperature EPR on a 35 μm sample A. Using B and C). Reliability of the two peaks comes from the high signal-to-noise F(t) trace. We simulated the expected distance distribution for the bent MxiC model (C) and the MxiC structure  using MMM . Although the modeled form factors all differ in their overall shapes, they all show distances in the 1.5–3-nm range, in line with the experimental setup. However, the simulated peak at 3 nm was not as prominent in the experimental data. We thus conclude that the experimental data are not consistent with the structures nor with the bent model. When modeling the possible observable distances in the structure and the bent model using the “all rotamers” function in MMM  , a protein that is not known to interact with MxiC (A), indicating Spa15 does not influence the rotational freedom of the labels in MxiC. Consistently, the DEER form factor and resulting distance distribution were also unchanged (data not shown). Thus, Spa15 does not affect MxiC's conformation in vitro.As EPR only detects signals from paramagnetic centers, one can add potential binding partners to the sample without affecting the signal as long as they are diamagnetic. To determine whether other T3S-related proteins influence MxiC's conformation, we mixed labeled His-MxiC(Cys) with a 10-fold molar excess of the proposed interaction partner IpaD  or Spa15ith MxiC . Spa15 aith MxiC A, indicaCsupplemental Fig. S11) changed the continuous wave (cw) spectrum obtained (C), indicating the conformational freedom of the spin-labeled side chains has been affected by IpaD-MxiC complex formation, either due to direct interaction with IpaD residues or to subsequent conformational changes in MxiC. To confirm IpaD was responsible for signal modification, we mixed MxiC with different concentrations of IpaD (D). We found that the larger the stoichiometric fold of IpaD versus MxiC, the bigger was the spectral change detected. This shows the signal modification is specific to IpaD addition and that, in the concentration range used, we can modify the ratio of the MxiC-IpaD complex versus MxiC alone in the equilibrium ensemble. We also diluted the 10:1 IpaD/MxiC mix 1:4 (E). This confirmed that in preventing the interaction between MxiC and IpaD by dilution, the MxiC alone signal is recovered, meaning the interaction is reversible.In contrast, addition of a 10-fold molar excess of purified His-IpaD  (B) or when added in the presence of a 10-fold molar excess of IpaD (C). Taken together, these data indicate that in solution only IpaD displays an affinity for MxiC. However, we found no evidence of a distance change between the two spin-labeled side chains by DEER upon addition of any protein partners (data not shown).Given the evidence that MxiC family proteins can interact directly with translocator chaperones , 36–38, 36–g. S11D) . Neitherg. S11D) B or when of IpaD C. Taken mxiC mutants that leak effectors do so because MxiC's function(s) in blocking effector secretion is compromised or because they cannot retain MxiC intracellularly. We examined this by combining them with a mutation that prevents MxiC secretion, mxiHK69A.To dissect at which point they are deregulated, we wanted to understand whether our mxiHK69A mutant in the needle protein does not secrete effector proteins (mxiHK69A mutant lacking MxiC (ΔmxiHΔmxiC/mxiHK69A) can secrete effectors  is affecting function should continue to leak effectors even if MxiC is retained inside bacteria. For instance, the hydrophobic core mxiCF206S mutant . We focused solely on MxiC mutants displaying premature secretion of effectors and MxiC leakage. For completeness, we also included mxiC, which carries mutations in a negatively charged surface patch (top), secretes translocators and MxiC prematurely, and is defective in IpaD secretion upon activation , and its inducible secretion was also slightly reduced compared with wild type but of a similar level to that of ΔmxiCΔmxiH/mxiCmxiHK69A (top panels). Indeed, slightly fewer functionally mature T3SSs are assembled in ΔmxiH/mxiHK69A relative to wild type (mxiCΔmxiH/mxiCmxiHK69A could not leak or induce secretion of the effectors IpaA or IpgD nor of MxiC (bottom panels), respectively. However, it secreted slightly reduced amounts of the translocators inducibly, as observed previously . We then examined how each mxiC mutant behaved in an mxiHK69A background. We focused on leakage in the absence of induction because activation of MxiC secretion is blocked in an mxiHK69A background but effector secretion might be allowed by a functionally defective MxiC.We were unable to detect leakage from Δild type . Howevereviously . In ΔmximxiCK68E was unable to leak or induce effector secretion in the presence of mxiHK69A but not of mxiH. This indicates it is the only mutant where MxiC would be functional in blocking effector secretion, if it were not secreted prematurely. All other mutants leak effectors similarly whether mxiH or mxiHK69A is present, indicating that in these mutants MxiC's function(s) in blocking effector secretion are affected, either directly as in mxiC or indirectly, via premature secretion of translocators as in mxiC.Only i.e. regulation of translocator, MxiC, or effector secretion. This indicates the three regulatory functions of MxiC are interlinked. Therefore, the mutants were classified according to where their main initial defect lies, assuming the regulatory steps occur in this order.Our mutants can be organized into classes according to their phenotypes . HowevermxiC, mxiCΔCterm, which we therefore placed in class 0. However, unlike mxiCΔCterm, mxiCΔNterm and mxiCΔCBD were still largely able to induce translocator, but not MxiC, secretion, suggesting they are not full loss-of-function mutants (class 1a). Mutant mxiCV256P also could not secrete MxiC well inducibly or in a ΔipaB background, suggesting it has some defect in MxiC secretion. This mutant leaks effectors, but as it can still stimulate secretion of translocators, it is also not a complete loss-of-function mutant (class 1a). Both classes of mutants readily by-passed the block on effector secretion imposed by mxiHK69A, although none secreted MxiC efficiently in this background, indicating they are unable to establish all or some of MxiC's regulatory functions.Only one mutant showed such severe loss-of-function as to resemble ΔmxiCK66E and mxiCK68E are phenotypically similar in that they secrete MxiC prematurely and hence show reduced translocator induction and increased effector leakage. However, MxiCK68E is secreted much more efficiently than MxiCK66E. Furthermore, among all mutants, only MxiCK68E does not leak effectors in an mxiHK69A background. This indicates that the mutations in MxiCK68E and MxiCK66E, despite their proximity and chemical similarity, lead to different defects. K68E (class 1c) primarily deregulates MxiC secretion, leading it and effectors to be secreted prematurely. In addition to some premature secretion, K66E (class 1b) seems to confer more fundamental defect(s) leading to effector leakage. Finally, the only defect seen in mxiC is effector leakage (class 1d).Class 2 mutants are primarily, if oppositely, affected in translocator release. MxiC cannot induce IpaD secretion but leaks IpaB, IpaC, itself, and effectors , althougi.e. mxiCΔCterm and mxiCΔNterm, but still lead to full or partial null phenotypes, respectively.Using the mutant classes, we conclude that several physiological features of MxiC are regulated as follows: 1) its secretion, because several mutants up- or down-regulate it; 2) hierarchical secretion of translocators and effectors, and even of proteins within those two categories; and 3) the ability to switch from translocator to effector secretion. There are also uncharacterized fundamental properties of MxiC as indicated by mutations that most likely do not affect its overall structure, et al.  might localize the YopN-TyeA complex to the secretion apparatus. Ferracci et al. , these regions may also be involved in binding it to and releasing it from that site.MxiC's secretion signal and putative CBD are dispensable for stimulation of translocator secretion, which indicate domains 1–3 alone mediate this function. Multiple residues in the C-terminal helix of MxiC are highly conserved. Schubot et al.  suggestei et al.  found Tyi et al.  of MxiC i et al. . In contecretion , 33. Howi et al.  were oveV mutant  also led ) or not at all ). Thus, the action of MxiC on translocators and effectors can be uncoupled. MxiC acts as if it had already received the activation signal  leaks IpaB and IpaC and is unable to induce IpaD secretion  instead displays reduced induction of IpaB and IpaC, although IpaD secretion is slightly increased. These opposite effects correlate with the mutations being on opposite faces of MxiC (top). These faces are also the ones that may undergo a conformational change from flat to bent . Furthermore, the predicted bent mutant (mxiCV256P) and the predicted straight mutant show opposite phenotypes; although the former leaks effectors and is only weakly affected in translocator secretion, the latter shows differential induction of translocators. Thus, reduced secretion of the repressor IpaD could affect secretion of the hydrophobic translocators. Alternatively or additionally, MxiCV256P was only poorly secretable and could be affected in more fundamental functions. Although we cannot exclude that these mutations mainly affect MxiC interactions by altering its surface, the type of mutations, the fact that the mutations are directly adjacent, and their opposite phenotypes suggest that we have modified MxiC's ability to undergo conformational changes.Even hydrophilic h et al. ). Intere of MxiC , top. Th to bent . In factmxiC lies on the face of the molecule that is flat in MxiC but concave in YopN/TyeA and the E. coli homolog SepL , or both. The latter is supported by the work of Archuleta and Spiller  and IpaD were mixed have two possible explanations as follows: (i) the increased molecular weight of the complex(es), which is reflected in slower overall rotational correlation times; (ii) a direct interaction between the spin-labeled probes and residues in IpaD. Although the reason of the spectral effects observed cannot be clarified, it corroborates the notion that complex formation between MxiC and IpaD occurs. From Fig. 7ipaDL99P and mxiC (A), with the negatively charged patch of MxiC  at their interface, yielding an overall elongated form. The interacting surface is relatively flat and contains several charged residues on the edges and stacked tyrosine residues in the center giving an interface formed by two charge-complementary surfaces (B). This model is consistent with the data of Lee et al.  ), we buiK,E293K) A, with tsurfaces B. This me et al. , who fouiCE201K,E6K,E293K mxiCΔNterm mutation in the constitutive secretor background ΔipaB, we confirmed that this mutant is unsecretable. Similar to the N-terminal secretion signal, the putative chaperone-binding domain is required for efficient MxiC secretion. However, this domain did not affect secretability. Thus, MxiC secretion is merely deregulated in mxiCΔCBD. Interestingly, two different mutations in the CBD of MxiC led to increased MxiC leakage, mxiCK68E and mxiCK66E. Taken together, these data suggest that the putative chaperone-binding domain positively regulates MxiC secretion.By analyzing the ipaB background. This is reminiscent of the lack of requirement of MxiC for translocator secretion in the same background. Similar to the repression of translocator secretion  has gained the ability to activate its own secretion  is transmitted to the cytoplasm, most likely through the needle.The mechanism preventing premature translocator secretion is counteracted by MxiC. Secretion of IpaD requires the negatively charged patch and probably a flat conformation of the molecule. Removal of IpaD may initiate a conformational change in MxiC, which bends to release the IpgC chaperone and its bound translocators allowing these to become secreted in turn. IpaB and secreted IpaC form a pore, connected to IpaD at the needle tip and also inserted in the host cell membrane, which transmits “Signal 2” to the cytoplasm. Reception of this signal may allow finalization of a conformational change in MxiC that releases it for secretion. Then, early effectors are secreted. Moreover, as both negative regulators are now removed, the secretion rate is enhanced , possiblmxiC, we dissected at which stages of the regulatory cascade MxiC acts, including which of its functional steps can be uncoupled. This also allowed us to determine how the different steps are connected. Finally, we provide the first evidence that a directionally defined conformational change in MxiC is involved in controlling the secretion hierarchy. The conservation of the components involved indicates the importance of this regulatory pathway to T3SS-carrying bacterial pathogens of animals.By using structure- and sequence-based mutagenesis of supplemental Tables S1 and S2 list the strains and plasmids used. S. flexneri strains were grown in Trypticase Soy Broth (BD Biosciences) at 37 °C with appropriate antibiotics the following final concentrations: ampicillin 100 μg ml−1, kanamycin 50 μg ml−1, tetracycline 5 μg ml−1, and chloramphenicol 10 μg ml−1. IPTG was used at the final concentrations indicated in the figure legends. The supplemental Table S3 lists the primers used.The Plasmids were generated as described below and verified by sequencing.mxiC) was generated by amplifying mxiC from the Shigella virulence plasmid pWR100 using primers mxiC_SacI and mxiC_BamHI. The purified PCR product was digested with SacI and BamHI and cloned into pWSK29* . The resulting PCR products were digested with SacI and BamHI. Fragments mxiCΔNterm and mxiC(1–341) were cloned into pACT3 digested with SacI/BamHI yielding pDR60 and pDR73, respectively.To express the terminal mxiCΔCBD, an internal deletion mutant that was created by two-step PCR and lacks residues 32–72. The mxiC gene was amplified from pIMA208 using primer pairs mxiC_SacI/mxiC_D32–72_R and mxiC_D32–72_F/mxiC_BamHI. The obtained PCR fragments were used as template for the second PCR step using primers mxiC_SacI and mxiC_BamHI. The PCR product was purified, digested with SacI/BamHI, and ligated into pACT3 digested with the same enzymes.pDR80 contains mxiCK66E was generated by two-step PCR. First, mxiC was amplified from pIMA208 using primers mxiC_SacI and mxiC_K66E_R and/or mxiC_K66E_F and mxiC_BamHI, respectively. These fragments were combined and reamplified using mxiC_SacI and mxiC_BamHI. The PCR products were purified, digested with SacI and BamHI, and cloned into pACT3 digested with the same enzymes yielding pDR96. pDR100 contains mxiCK68E cloned via SacI/BamHI, equivalent to amplification of mutant mxiC using primers mxiC_SacI and mxiC_BamHI. This mutant was obtained by chance and retained as the mutation was in a relevant area of the molecule.Mutant mxiC was generated by two-step PCR. pIMA212 was used as template for reactions with primer pairs mxiC_SacI/mxiC_M226K_R and mxiC_L242D_F/mxiC_BamHI. The obtained fragments were combined and reamplified using primers mxiC_SacI and mxiC_BamHI. The product was purified, digested with SacI and BamHI, and cloned into pACT3 digested with the same enzymes, yielding pDR72.Double mutant mxiC(Cys) (C184A (TGT to GCA), C233S (TGT to TCT), A247C (GCA to TGT), and S290C (AGT to TGT)) required for EPR experiments was synthesized by MWG Eurofins and supplied as pEXA-mxiC_EPR2. For pDR104 (pET28bmxiC(Cys)), mxiC(Cys) was amplified from the pEX-A-mxiC_EPR2 using primers mxiC_NdeI_F/mxiC_EcoRI_R. The PCR product was purified and digested with NdeI/EcoRI before cloning into pET28b (Novagen).Mutants in helix 9 were generated by two-step PCR; first, mxiC was amplified from pIMA208 using primer pairs mxiC_SacI/mxiC_V256P_R, mxiC_V256P_F/mxiC_BamHI, mxiC_SacI/mxiC_wobble_R, mxiC_wobble_F/mxiC_BamHI, mxiC_SacI/mxiC_straight_R2, and mxiC_straight_F2/mxiC_BamHI. The obtained PCR fragments were used as template for the second PCR using primers mxiC_SacI and mxiC_BamHI. The products were purified and digested with SacI/BamHI before ligation into pACT3 digested with the same enzymes. The resulting plasmids were named pDR91 (pACT3mxiCV256P), pDR92), and pDR93), respectively. The background quadruple cysteine mutant gene Shigella wild type yielding ΔipgA, ΔipgE, and Δspa15, respectively. For ΔipgE Δspa15 (abbreviation of ΔipgE::FRT Δspa15::kan) and ΔipgE ΔipgA (abbreviation of ΔipgE::FRT ΔipgA::kan), the kanamycin cassette was removed in ΔipgE by FLP-mediated recombination using the introduced FRT sites yielding ΔipgE::FRT. The same fragments used for the deletion of spa15 and ipgA in the wild-type background were now used for λ Red recombination in ΔipgE::FRT. This step was of low efficiency as the recombination preferentially occurred at the FRT scar at the ΔipgE site and not up- and downstream of spa15 and ipgA, respectively. Colonies were prescreened by colony PCR using primers annealing ∼200–300 bp upstream and downstream of the original chaperone gene, seeking to obtain a PCR fragment with a size compatible with the insertion of the kanamycin cassette versus the chaperone gene. ΔipgE ΔipgA Δspa15 (abbreviation of ΔipgE::FRT ΔipgA::FRT Δspa15::kan) was generated by removing the kanamycin cassette in ΔipgE ΔipgA by FLP recombination yielding ΔipgE::FRT ΔipgA::FRT and subsequent λ Red recombination using the ipgA::kan PCR fragment. Again, this step was of low efficiency as now two FRT sites were available on the virulence plasmid and colonies were prescreened by colony PCR as described for the double knockouts. All insertions and cassette removals were verified by sequencing.Deletions of the chaperone genes were performed using the method of Datsenko and Wanner . A kanammxiC plasmids detailed above were then combined with plasmids carrying mxiHK69A or wild-type mxiH as control in ΔmxiCΔmxiH  were mixed with 4× Laemmli sample buffer (“whole culture lysates”). Samples from equivalent cell numbers (∼3 × 106 cfu) were separated by 10% SDS-PAGE and immunoblotted.Whole cultures of S. flexneri strains grown to A600 ∼1 were collected by centrifugation at 15,900 × g for 10 min at 4 °C. The supernatants were mixed with 4× Laemmli sample buffer, normalized for equivalent cell numbers, separated on 10% SDS-PAGE, and visualized by silver staining or immunoblotting. On silver-stained SDS-PAGE, labels indicate the position of proteins as determined by mass spectrometry or from deletion strains.Shigella culture leads to a burst of Ipa protein secretion called “induction”  was added to a final concentration of 200 μg ml−1. After incubation at 37 °C for 15 min, samples were centrifuged at 15,900 × g for 10 min at 4 °C. 20 μl of the supernatants denatured in Laemmli sample buffer were subjected to 10% SDS-PAGE and visualized by silver staining or immunoblotting.CR, a small amphipathic dye molecule, is an artificial inducer of T3S. Its addition to a duction” , 48. S. et al. (Proteins were transferred onto Immobilon FL (Millipore) membrane using a semi-dry method. Primary antibodies used were as follows: anti-IpaA mouse monoclonal, gift from Kirsten Niebuhr ; anti-Ipet al. ; anti-Ipet al. ; and antet al. . Near-inShigella were grown to A600 ∼1 and resuspended in PBS to an A600 of 15. The suspension was brought to 37 °C in a water bath before CR was added at a final concentration of 200 μg ml−1, and the cultures were incubated for 8 min. For secreted proteins, samples were centrifuged at 15,000 × g for 10 min at 4 °C, and supernatants were denatured in Laemmli sample buffer. For whole cultures, the bacterial suspension was directly denatured in sample buffer. To calculate the secreted percentage of each protein, we compared protein amounts in supernatants and whole cultures by Western blotting. Undiluted and 1:4 diluted supernatant was compared with a dilution series of the whole cultures. Near-infrared fluorescent secondary antibodies were quantified on a LI-COR Odyssey imaging system. A linear fit of the dilution series allowed us to determine the concentration of protein in the supernatant in comparison with the whole culture.Wild-type 2VJ4  and thet et al. ) were uset al.  were extracted so that only a single polypeptide chain was present in each PDB file. This was necessary as the labeled residues (247 and 290) are in close proximity to the other polypeptide chains in the original PDB files. The neighboring chains thus also influence the rotamer modeling; however, MxiC is most likely monomeric in solution C322S was amplified from pUC18 ipaDC322S , and cloned into pET15b (Novagen) via NdeI/BamHI. The changes to the protocol for each protein are detailed in supplemental Table S4.Each protein was purified first by nickel affinity chromatography and then by size-exclusion chromatography. The protocols for these purifications were based on articles herewith: Spa15 ; IpgC/IppaDC322S , using psupplemental Table S4). Bacteria were grown to an A600 of ∼0.6 before cooling to 20 °C and induction with a final concentration of 1 mm IPTG. Cultures were then left shaking at 20 °C overnight.All protein concentrations, measured with a Nanodrop Lite spectrophotometer (Thermo Scientific), were adjusted using molecular weight, extinction coefficient (as obtained from the ExPASy server) of the protein, and consideration of the path length. Prior to each protein purification, the appropriate bacterial strain was streaked out on an Lysogeny Broth (LB) agar plate containing the appropriate antibiotics and grown overnight at 37 °C. The next day small LB cultures were made overnight. In the morning, cultures were made with overnight cultures (g for 15 min), washed in 10 ml of PBS (3500 × g for 5 min), and resuspended with ∼30 ml of Binding buffer with protease inhibitors . Bacteria were lysed by sonication (Sonics Vibra CellTM) at amplitude 60%, pulse 1-s on and 1-s off, time of 30 s, and variable cycle numbers .Cells were harvested by centrifugation (5000 × g at 4 °C. Supernatants were filtered through a 0.45-μm and then 0.22-μm syringe filter (Sartorius) and applied to a 5-ml HisTrap FF column  equilibrated in binding buffer  using a peristaltic pump . The entire supernatant was passed over the column three times at 2.5–5 ml/min at 4 °C. The column was then connected to an ÄKTA  and washed with 10–15 column volumes of Binding buffer, followed by elution buffer . Elution of bound proteins was carried out in the presence of protease inhibitors, with an imidazole gradient from 20 mm to 1 m  to 3 ml/min for 25 min. 5-ml fractions were collected. Peak fractions potentially containing proteins of interest had their concentration determined at A280 and were examined using Coomassie-stained SDS-polyacrylamide gels. The appropriate fractions were pooled and concentrated using Amicon Ultra spin concentrator .Lysates were clarified by centrifugation for 30 min at 20,000 × m Tris-HCl, pH 7.5, 100 mm NaCl, and 100–250-μl samples were applied, and the column was run at 0.5 ml/min collecting 1-ml fractions. Location and purity of proteins of interest were verified by Coomassie-stained SDS-PAGE. After the final gel filtration step, all samples were concentrated as above  and flash-frozen in aliquots in liquid nitrogen and stored at −80 °C until use.These concentrated fractions were run on a Superdex75 10/30 size exclusion chromatography column  equilibrated in 20 mm freshly made DTT (Sigma) was added to MxiC(C184A/C233S/A247C/S290C) to reduce the SH of the cysteines before concentration and application on the gel filtration column. The fractions containing His-MxiC(C184A/C233S/A247C/S290C) (∼2.12 mg/ml) were pooled, and the spin label MTSL  was added at 2-fold molar excess (∼121 μm). This means one molecule of MTSL was added per cysteine in the sample. After incubation for overnight at 4 °C in the dark, the sample was concentrated to ∼1.842 mm (∼70 mg/ml) as determined by the A280 using Amicon Ultra-4 spin concentrators .1 mQ cavity. The samples were thawed on ice, and 20 μl were transferred into a 1.5-mm outer diameter glass capillary. A 14-mT field sweep was performed, with 0.15 mT modulation amplitude, 7.96 milliwatt incident microwave power, and ∼9.38 GHz frequency. As the measured signal is the first derivative, the resulting curve has to be integrated to obtain the absorbance spectrum. Double integration yields the spin concentration as horizontal asymptote. The area under the absorbance spectrum is proportional to the spin concentration. The correlation factor was experimentally determined with a solution of known concentration of tempol in water.To determine the labeling efficiency of His-MxiC(Cys) with MTSL, continuous wave EPR spectra were detected at room temperature on an E500 Elexsys Bruker spectrometer equipped with a super high Q-band DEER experiments were performed as described in Polyhach et al. (Q-band DEER experiments are described in Bordignon and Polyhach (V(t) = V(0)) are background corrected to obtain the DEER form factor (F(t) = F(0)) by division by the background function (which is mainly due to intermolecular interactions). The form factor oscillates around a horizontal line at (1 − Δ) (Δ is the modulation depth) after background correction. The distance distribution is extracted from the form factor using DeerAnalysis2013 , and contained a final concentration of ∼75 μm MxiC and 10-fold more of the other proteins (∼750 μm).To analyze the interaction between MxiC and other proteins, we performed continuous wave X-band EPR spectroscopy at room temperature to detect eventual changes in the dynamic properties of the two spin labels on MxiC upon complex formation. Each EPR sample was prepared in gel filtration buffer (20 mA. D. R., E. B., R. B. S., I. M. A., and A. J. B. conceived and designed the experiments; A. D. R., E. B., S. M., D. K. S., X. L., M. P., I. M., R. B. S., and A. J. B. performed the experiments; A. D. R., E. B., I. M. A., R. B. S., and A. J. B. analyzed the data; X. L. and M. P. generated reagents and materials; and A. D. R., E. B., I. M. A., R. B. S., and A. J. B. contributed to the writing of the manuscript. All authors approved the final version of the manuscript.


Both theoretical and experimental researches on gas response of graphene-based sensors to these typical gases are summarized. Finally, the future research trend about graphene synthesis technique and relevant perspective are also given.Graphene-based materials have aroused enormous focus on a wide range of engineering fields because of their unique structure. One of the most promising applications is gas adsorption and sensing. In electrical engineering, graphene-based sensors are also employed as detecting devices to estimate the operation status of gas insulated switchgear (GIS). This paper reviews the main synthesis methods of graphene, gas adsorption, and sensing mechanism of its based sensors, as well as their applications in detecting SF The groundbreaking fabrication of graphene [2/g, graphene displays a wonderful performance for surface chemistry [The past several decades have witnessed increasing attention by researchers to carbon-based materials, especially those formed by spgraphene  has arougraphene ,4. Givenhemistry ,6,7. Thehemistry ,10,11,12hemistry ,16,17,186) gas, a most commonly utilized kind of insulation gas in high voltage equipment of power systems, especially gas-insulated switchgear (GIS) due to its excellent chemically inert and wonder dielectric strength [2, H2S, SO2F2, CF4 and SOF2 [6 in order to estimate the operation status of GIS, guarding against more serious insulated faults.It has been well-known that sulfur hexafluoride . Results indicated that, although some twisted parts at the graphene do exist, the thick places account for the most proportions.Stankovich  prepared2, NH3, H2O, and CO and proved that these gaseous chemicals can be detected individually, since graphene-based sensors have such required sensitivity.For carbon materials, gas adsorption is primarily depended on physisorption on their surfaces, and sometimes the electrostatic and dispersion reactions that can be tuned by chemical substitution can happen as well. Two factors, namely the surface characteristic of the adsorbent and the properties of the target gas molecules, can influence the interaction strength and further determine the adsorbing rate . Specifi2/graphene oxide sheet was employed to study its sensing property to H2, and they found that high sensitivity (92%) and fast response (no more than 20 s) could be obtained at 180 °C when the concentration of hydrogen reaches 500 ppm. In another research [2 nanorods arrays on graphene sheets enhanced sensitivity to H2S, which is mainly attributed to the outstanding electronic characteristics of graphene and the synergism of the large surface area of SnO2 nanorods arrays. Based on the first principles theory, Congcong et al. [2 and NO.In addition, the flexibility of modification and functionalization in graphene surfaces has opened up many possibilities for the development of tailored functional materials, so that they possess varied physicochemical properties to be employed as gas adsorbent, gas sensors, or some other electrochemical devices. Liscio et al. , with grresearch , it was g et al.  investig2 significantly improved compared with the unmodified one, as Pd has a good affinity toward H2 detection. In the research by Fowler et al. [2 and NH3. Results showed that NO2 receives electrons from graphene, while NH3 donates electrons to graphene, resulting in different sensing mechanisms of these two gases. That is, holes induced conduction and electrons induced conduction for NO2 and NH3, respectively.In terms of the application of graphene-based materials for gas sensors, its fundamental principle is based on the conductivity change after the adsorption of gas molecules . In detar et al. , the graTherefore, the nature physical property of a high specific surface area allows graphene to be prepared as gas sensors for detecting individual gaseous chemicals. More importantly, the chemical modification by functional groups, metal or nonmetal atoms, as well as nanoparticle decoration can enhance the sensitivity and selectivity of graphene-based sensors to one kind of gas in the environment with mixed gases, and this improves its availability for gas sensing in related industrial applications.6 decomposed gases. Much research has been conducted both theoretically and experimentally to investigate the adsorption mechanism and response characteristics. This section is divided into two parts to separately summarize theoretical calculations and experimental analyses.In view of excellent physicochemical properties of graphene-based materials discussed above, their application for preparing sensing devices have never failed to be emphasized, without the exception of fabricating related sensors employed in electrical engineering to detect SF6 decomposed gases. Based on first principles theory, relevant adsorbing parameters including total adsorption energies (Ead), charge transfer (Qt) value, interaction distances (D), energy gap (Eg), and total or local density of state [2F2, SOF2, H2S, CF4, and SO2, and the graphene model should be set up and then optimized to obtain their most stable geometric structures. Then, targeted gas molecules enable us to approach the surface of graphene so as to proceed with the adsorption process. After optimizing whole system, the related adsorbing configurations and parameters mentioned above can be gained. One thing should be mentioned that the adsorbing energy of each system could not be obtained directly. Instead, it is calculated by the following equation:Egraphene/molecule, Egraphene, and Emolecule, respectively, represent the systemic energy after reaction, and the total energies of the graphene and the gas molecule before the reaction. A negative Ead indicates spontaneity of the corresponding reaction. Similarly, Eg of each reaction is calculated asELUMO and EHOMO represent the Lowest Unoccupied Molecular Orbital and the Highest Occupied Molecular Orbital, respectively. 6 decomposed gases, which are all geometrically optimized.As a complete modeling and simulation software, a materials studio was designed to help predict and understand the relationship of a material’s atomic and molecular structure with its properties and behavior ,46. Hereof state ,47 can bof state ,49. Firs6 decompositions. In the theoretical research [2S on intrinsic graphene is proven to be quite weak as a consequence of the small binding energy, the large bonding length, and the small net charge transfer; while for Pt-doped graphene, it has the ability to adsorb up to seven H2S molecules on its surface according to larger binding energy and a shorter Pt H2S bonding distance. Such results provide powerful proofs for the conclusion that metal-doped graphene has better gas response to gaseous chemicals analyzed before. In fact, Pt that naturally has a strong catalytic activity undoubtedly helps its doped graphene with good gas sorption [In terms of theoretical calculations, plenty of research has been conducted for the purpose of discovering a novel kind of graphene-supported material that has a good gas response to SFresearch , adsorptsorption  if it ac2S sensing, and they found that, compared with the film sensors prepared by intrinsic graphene, those decorated with copper (Cu) manifested better sensing behavior. Moreover, the adsorbing ability of Cu-graphene to H2S depends on the concentration of decorated Cu. Too much or too little Cu concentration would deteriorate the gas response of graphene-based sensors to H2S. Another interesting phenomenon is that the Cu decoration perfectly changes the type of the semiconductor, i.e., the intrinsic graphene-based sensors present a p-type semiconductor, while the dopant of Cu present an n-type one. Generally speaking, the doping of metal that has redundant electrons enables electrons to pad the holes and form a duplet. Once all the holes are full of electrons and certain free electrons still exist, it shows an n-type conductor; conversely, if the holes fail to be filled, it remains a p-type conductor. It is no wonder that, in this study, the Cu atom in a proper concentration provide enough electrons, leading to the transformation of the conductor type. However, too little Cu only reduces the concentration of carriers, lowering the conductivity of graphene-based sensors, while too much Cu improves the number of electrons, enhancing dramatically the conductivity of the prepared sensors. Such changes are, on the contrary, detrimental to the gas response as the conductivity changes inconspicuously after the gas adsorbing reactions. That is, they present an insensitivity to their environment. However, it is not contradictive to our previous analysis that the modification of metal can enhance sensing performance. If the dopant concentration is proper, the modified complex presents a further improved response to the target gas.Berahman et al.  designed2 onto light metal-doped (Li and Al) graphene oxide (GO) was investigated by Chi Chen et al. [2 adsorption performance, with a binding energy of up to 1.08 eV, as the light metals that stably anchor on the GO via hydroxyl and epoxy groups provide many active adsorption sites for gas molecules. Rad et al. investigated the SO2 adsorption on pristine graphene, N-doped graphene [Ead comes to the adsorption when N-graphene acts as adsorbent (−19.6/mol), followed by B-graphene (−11.2 kJ/mol) and finally pristine graphene (−9.1 kJ/mol), which suggests that N-doped graphene has the best adsorbing ability to SO2. B-graphene shows as an n-type semiconductor because of a highly negative charge of C-atoms neighboring the boron dopant. When the SO2 molecule is adsorbed on the B-graphene surface, the energy gap reduces by 0.60 eV from 2.90 eV for insolated B-graphene, implying a sharp increase for conductivity of the B-graphene-based sensor. Even so, the adsorption of SO2 on B-graphene or intrinsic is physisorption, on N-graphene is chemisorption, as their different adsorbing cases in these two systems. The inter-molecule bond of SO2 witnesses little change after adsorption in SO2/B-graphene system due to weak sorption; while deforms destructively in SO2/N-graphene system because of their strong binding force.Based on first principles theory, adsorption of SOn et al. , and thegraphene , and B-dgraphene   and d2 (1.467 Å) present no significant change after adsorption compared with that before reaction , the M5 site for H2S adsorbed on intrinsic graphene is the most negative (−0.617 eV), indicating that the adsorption in this site occurs most easily; upon Au-graphene, the reaction for H2S is the most likely to take place when the S atom is oriented to the Au-graphene . In terms of the other gases, the lowest Ead among several adsorbing sites are −0.467 eV for SOF2/graphene with the B site, −0.961 eV for SOF2/Au-graphene with 2F- and S-oriented site, −0.472 eV for SO2F2/graphene with 2O- and F-oriented site, −1.790 eV for SO2F2/Au-graphene with 2F- and O-oriented site, −0.302 eV for SO2/graphene with the T site, and −0.587 eV for SO2/Au-graphene with O-oriented site, respectively. These results reveal that a much stronger adsorption reaction happens in the Au-graphene system than that of the intrinsic one. Moreover, the adsorptions between the Au-doped graphene and gas molecules are proven to be chemisorption since their Ead are far negative than the critical value (−0.8 eV) of the chemical adsorption reported elsewhere [6 decomposition gases, coming up with a new method to research the adsorption process and a new type of possibly workable sensing material.It can be seen that, although all of the 2F2, SOF2, and SO2 were set up, to study the adsorption behaviors and gas response of Ag modified graphene to these gases. To attain a better understanding of these adsorbing reactions and adsorbing capacity of Ag-doped complex to an individual gas, both single gas molecules and double ones were investigated in this research, with optimized geometrical structures shown in In other representative research  based on6 decompositions. Among three systems, SO2F2 shows the most active effect to Ag-doped complex with the lowest adsorption energy (−1.448 eV) and the shortest adsorption distance (2.013 Å), which means that Ag-doped graphene sensors have the best sensitivity to SO2F2 compared with the other two types of gases. Furthermore, all negative Qt values indicate that the electrons transfer from the Ag-graphene to the gas molecules; the largest number of charges that transfer from the adsorbent to the SO2F2 molecule further verifies their strong reaction, proving that SO2F2 can be easily adsorbed on the Ag-doped complex.In terms of one gas molecule adsorption, it can be seen that new bonds are formed in every system between certain atoms of gas molecules and Au atom on the surface of graphene, suggesting the strong binding forces of Ag-graphene to SF2F2 and SOF2 molecules adsorption, the processes appear similar to single gas adsorption, namely that only one in two molecules interacts with Ag-graphene and adsorbed on its surface, and the other one have little interaction with it. While for double SO2 molecule systems, the adsorption seems to be much stronger as the Ead become more negative and the Qt doubled. Such conditions imply that Ag-graphene has an adsorbing capacity to SO2 that is superior to SO2F2 and SOF2. The adsorption to double SO2 molecules can be regarded as chemisorption, and that to SO2F2 or SOF2, physisorption. Even so, the Ead and D in the SO2F2 system still indicate their relatively strong reaction with Ag-graphene.Taking two gas molecules’ adsorption into consideration and comparing their adsorbing parameters with those of single gas molecule adsorption systems, it can be concluded that, for doubled SOEg) about different systems can be obtained to clearly understand the influence of the adsorbing gas molecules on the conductivity of Ag-graphene prepared sensors. Through analyses, it can be obtained that Eg of the SO2F2/Ag-graphene and Ag-graphene/SO2 systems increases sharply when the number of adsorbed gas molecules increases from 1 to 2, revealing a decreased conductivity of the graphene sensors; as for the Ag-graphene/SOF2 system, the number of adsorbed gas molecules may have little impact on the conductivity of graphene sensors. Overall, it was eventually concluded that the sensitivity of Ag-graphene to diverse gases agrees with the following order: SO2F2 > SO2 > SOF2, since the energy gap has little change with respect to the SOF2/Ag-graphene system.Based on further studies on frontier molecular orbital theory, the HOMO, LUMO, and energy gap . The adsorptions were performed at different temperatures in order to study its influence on the adsorption process of SO2 on GO. Near ambient pressure, the adsorption abilities of GO to SO2 are 156 mg/g (15 °C), 153 mg/g (25 °C), and 135 mg/g (35 °C), respectively. Given the decreased adsorbing ability with the increasing temperature, the predominate adsorption of SO2 on GO gives rise to physisorption, which depends on a relatively strong van der Waals interaction due to the large dipole moment of SO2. Upon N2 plasma treatment on GO, a certain amount of N functional groups for ~3.5 atom % indeed is gained; however, it fails to noticeably facilitate the SO2 adsorption on treated GO.Babu et al.  prepared6 decomposition components, H2S, via an electrochemical workstation. The sensors were prepared by repeatedly dropping the graphene suspension onto the film with concomitant drying treatment, for the sake of obtaining a dense, uniform, and smooth graphene film on the substrate. Afterward, the sensor was exposed to environments in a sealed steal tank with different concentrates of H2S to determine its change in resistance.Yu lei et al.  introduc2S against the resistance measured without the presence of sulfuretted hydrogen, expressed as [R represents the real-time resistance of the sensors immersed in H2S, and R0 represents resistance measured in the initial vacuum environment. In this study, three concentrations, namely 300 ppm, 50 ppm, and 100 ppm, of H2S were detected to obtain the sensitivity–time curves for further analysis, expressed in The response of sensors is characterized as the percentage of the resistance change due to the exposure to a gas containing Hessed as (3)S=R−R2S environment; (ii) their sensitivities to H2S reach as high as −7.93%, −10.63%, and −15.78%, respectively, despite their poor performances in terms of long response time to H2S (about 10 min); (iii) the higher the concentration of H2S is, the higher the sensitivity of graphene-based sensors is. Through detection of H2S by graphene-based sensors, it can be proved that graphene-based sensors have a relatively high sensitivity to H2S, and the concentration of such decomposition can be approximately estimated.From 2S and SOF2 adsorption performances. Two concentrations of each gas  are taken into consideration, with the sensitivity–time curves obtained under ambient condition. For H2S adsorption, an increased resistance of 28.15% and 18.73% are obtained for 100 ppm and 50 ppm, respectively; while upon SOF2 adsorption, 100 ppm and 50 ppm result in 23.83% and 15.36% decrease in resistance, respectively. These results manifest that Au-graphene can realize the selectively detection for SOF2 and H2S. Moreover, the probable concentration of individual gas can be estimated, given the dependence between resistance change and gas concentration. In comparison with the above sensitivity of graphene to H2S, Au-modified graphene obviously presents superior sensing behavior, leading to a better response.In , Au-dope2, as shown in 2, the existed target gas is expelled until its inexistence and subsequently begins the nest round of detection. From 2S and SOF2. Overall, it can be concluded that Au-graphene is a promising sensing material for detecting H2S and SOF2 according to its remarkable sensitivity, selectivity, and reusability. It is hoped that Au-doped graphene sensors can selectively detect these two gases in GIS.To investigate the repeatability of such material, the recover lines that used to explore its recovery property are obtained by injecting N6 decomposition components in order to estimate the insulation state of the power system has also been reviewed.In this paper, the main approaches for the synthesis of graphene, its composites, and its based materials have been summarized; simultaneously, the application of such materials in electrical engineering for the detection of SFThe technique of exfoliation, showing good promise to industrially produce graphene, TCVD, which can reproduce graphene on a centimeter scale substrate, and PECVD, which exhibits a fine feasibility to synthesize graphene on any substrate together, constitute the three main synthesis means for graphene preparation.6 decomposed products for use in power systems, which, to a certain degree, broadens their applications, contributing to the further exploitation of their properties.Based on previously theoretical and experimental researches, it can be confirmed that graphene-based materials might be a novel category of sensors because of their exceptional properties such as large specific surface area, high sensitivity, and selectivity to gases. Their outstanding sensing performances enable SF6 decomposition component detection, more high-accuracy experiments that lower the minimum response concentration as much as possible to meet industrial demands are still required. Not only do repeatable experiments need to be performed, but experiments about gas response to SO2F2 and SOF2 also need to be implemented, improving the probability of comprehensively understanding this issue.However, essential work focused on the effective preparation of graphene and relevant experiments aimed at repeatedly verifying the feasibility of graphene-based sensors should be conducted in order to realize its application on the diagnosis of GIS. First, in terms of synthesizing graphene, the inadequate ability for controlling the layer of graphene and realizing large-scale production ought to be solved. Besides, though the theoretical results prove their suitability for SF6 decomposition detection can be built up, detection precision and accuracy can be greatly developed. That is, the individual gases of SF6 decomposed products could be qualitatively and quantitatively detected and, with a short response time, a high sensitivity, environmental friendliness, and chemical stability can to a large extent substitute current electrical devices.For all of these efforts, the ultimate aim is to find a series of graphene-based sensors in which each sensor is quite sensitivity to a specific gas. It is expected that, if the sensor array prepared by graphene-based materials applied for SF


Graphene functionalization with organics is expected to be an important step for the development of graphene-based materials with tailored electronic properties. However, its high chemical inertness makes difficult a controlled and selective covalent functionalization, and most of the works performed up to the date report electrostatic molecular adsorption or unruly functionalization. We show hereafter a mechanism for promoting highly specific covalent bonding of any amino-terminated molecule and a description of the operating processes. We show, by different experimental techniques and theoretical methods, that the excess of charge at carbon dangling-bonds formed on single-atomic vacancies at the graphene surface induces enhanced reactivity towards a selective oxidation of the amino group and subsequent integration of the nitrogen within the graphene network. Remarkably, functionalized surfaces retain the electronic properties of pristine graphene. This study opens the door for development of graphene-based interfaces, as nano-bio-hybrid composites, fabrication of dielectrics, plasmonics or spintronics. Organic functionalization is key to the development of graphene-based functional composites, yet selective covalent functionalization is hindered by graphene chemical inertness. Here, the authors demonstrate a versatile route to graphene covalent bonding with amino-terminated organic molecules. Graphene, one of the most relevant materials of the decade, has to strengthen several drawbacks before it may step from fundamental physics to technology. Particularly, the absence of an electronic band-gap and its extreme chemical inertness significantly compromise their use as an active element in electronic devices. Development of highly precise and selective chemical functionalization strategies is understood as a breakthrough to circumvent this issue. A ruled covalent functionalization will provide added value to pristine graphene to develop applications in domains as important as band-gap engineering, creation of magnetism in graphene for applications in spintronics, fabrication of dielectrics, formation of nano-bio hybrid composites for improved biosensors or tailoring optical-plasmonic properties. To this aim, wet chemistry protocols  have been settled12345678910111281415-SiC(0001) by used chemical vapour deposition at 1,600 °C under an argon (Ar) laminar flow in a hot-wall Aixtron VP508 reactor182 V−1 s−1. The present work has been performed using both, SLG and QFSMG. Both surfaces exhibit similar results, and therefore, we will refer to them indistinctly.For our highly selective covalent organic functionalization we used high quality grown epitaxial graphene on 4HTo validate our mechanism, we have chosen p-aminophenol (p-AP) as a model molecule as it incorporates an aromatic ring and two different ending groups. The only requirement for our methodology is to use a molecule terminated in an amino group, as the mechanism we will describe hereafter is selective to this particular group. The terminal group is the one that has to be chosen paying attention to the possible applications. In our case the phenol-linked molecule can be considered as a chemical spacer or linker for anchoring more complex molecular nanoarchitectures in the search of designing functional structures2361020In this work we show an unprecedented chemical mechanism, described at an atomic level, by which the excess of charge located in unsaturated dangling bonds of C atoms around single-atomic vacancies (SAV) induces a selective oxidation reaction of any molecule containing an amino group, in which the N gets incorporated into de graphene lattice followed by a redistribution of the charge of the molecule in the neighbourhood of the vacancy. This mechanism can be used as a rational functionalization strategy to covalently couple amino-terminated organic molecules, as p-AP, to the surface of graphene epitaxially grown on SiC without disrupting its outstanding electrical properties over mm distances. Our method is based on making use of the localized electrons appearing around atomic vacancies. The physics of induced point defects on graphene have emerged in the last years as a source of new controlled properties, giving rise to magnetism27+ ion irradiation. These vacancies have been previously observed and isolated by transmission electron microscope (TEM) and STM, and their formation protocol is well established29303132333435We have performed First-principles density functional theory (DFT)-based calculations on a fully relaxed model of 4H-SiC(0001) SLG/(6✓3 × 6✓3)R30° with a unit cell with 13 × 13 SLG on topRegarding the SAV structural morphology, it has been recently shown that two distinct configurations of a SAV can be found in graphene32We observe that the apparent height of the SAV in STM images is about 1±0.2 Å and its width about 1±0.2 nm. These values are unaltered for the case of the QFSMG surface and are in good agreement with previously published valuesOnce SAVs are created they appear stable and spread all over the graphene surface. The next step is dosing about four Langmuirs of p-AP. STM images recorded after this process show that the surface topography has importantly changed. All the localized threefold radiating 1 Å high bumps become larger and higher, losing their starring appearance. The average apparent height is now 1.3±0.6 Å . Upon fus core-level peak derived from N in a graphitic network has been reported to appear at 400.5 eV  spectra recorded on the surface after dosing are indicative of the existence of N and carbonaceous species on the surface, suggesting that the bumps depicted in STM images corresponds to p-AP molecules anchored to the SAVs with an unique adsorption site for N atoms . The bin eV ref. , howeverRemarkably, if p-AP is dosed on pristine graphene (no SAVs) no absorption takes place, as depicted by STM and XPS measurements . Thus, wWe have checked the thermal stability of the anchored molecules, and no significant changes were found neither in the line-shape and intensity of the XPS spectra nor in the STM images appearance up to temperatures above 250 °C, indicating the covalent nature of the bonding, and allowing us to disregard any unspecific molecular adsorption . Moreove9Interestingly, a recent work explores the catalytic activities promoted by the unsaturated dangling bonds that may remain on a surface after SAV formation2 groups. We have computed the height of the barrier, ΔE, at the transition state (TS) in the double (−NH2) dehydrogenation process of the p-AF molecule. This TS has been investigated within the climbing-image nudge elastic band approach (see full details in the 2) dehydrogenation (around 1.5 eV), besides the fact that the remaining dehydrogenated oxygen would not be able to perfectly integrate within the SAV into the graphene lattice, only getting attached to one (of three) of the C atoms of the SAV. All calculated energy barriers are deeply discussed in the With all this chemical information in mind we have carried out a large battery of DFT-based calculations to confirm the above-mentioned scenario  molecule transfers 0.56e− towards the graphene lattice, while the N atom (considered as belonging to the molecule) accumulates an extra electronic charge of around 0.82e− with respect to its neutral state, coming from a charge rearrangement in the molecule, leaving the rest of the molecule with a net loss of almost 1.4e−. This is related to the fact that the N atom integrates into the graphene lattice, stabilizing its charge state by adsorbing and accumulating charge on the N atom from the rest of the molecule, and acting as a drain-pipe towards the C atoms at the neighbourhood.Interestingly, the N atom in the proposed structure has the charge unbalanced, since it is surrounded by four C atoms . To evalThe left panel of N region—within the solid line–, the resembling of pristine graphene DOS is quite poor, and some induced features arises just above and below associated to a reflection of the electronic properties of the positively charged molecule. In particular, the one arising between −1 and 0 eV—labelled as (*) in right panel of N on their closest C atoms. On the other hand, the highly hybridized molecular HOMO and LUMO positions are indicated as blue and red shadowed region, respectively, and they have visibly spread out by effect of the chemical interaction. The tunnelling region, where experimental images were recorded (−0.7/−0.3 eV) is also indicated in the right panel of To evaluate the spatial extension of the electronic charge transferred from the molecule towards the graphene lattice, right panel of Nevertheless, as we move away from the N atom, the C-atoms set (crown)—between the dot and solid lines in 12 cm−2 but it can be controlled by changing the duration of the ion irradiationThe density of immobilized groups is an important point when focus on applications12 molecules per cm2. For doses leading to higher coverage many events overlap and the characteristic electronic properties of the system collapse.This mechanism can be theoretical described with a simple phenomenological calculation based on random numbers. We have computed the density for different fluencies removing from the counting all overlapping events. The overlapping criterion has been taken in such a way that isolated events are considered if a minimum lateral distance larger than 1 nm separates them. This simple computational model reproduces quite perfectly our experimental data, and let us to conclude, both from calculation and experiments, that the maximum number of independent molecules that can be covalently linked is 8 × 1013 molecules per cm2. The statistical nature of our methodology only allows us to have a density four times lower.It is important to note that assuming a purely geometrical model of hard spheres with the experimental radius of 1nm, the maximum number of immobilized molecules is about 3.2 × 10Moreover, from R) versus inverse temperature measurements at zero magnetic field recorded for a QSG sample. Of paramount importance is the fact that the functionalized system does not exhibit a measurable gap at low temperatures (below 34 K); the thermally activated regimes at intermediate (between 34 and 140 K) and high temperature (above 140 K) regimes exhibit low-activation energies  curves evidence the presence of Landau Levels in p-AP functionalized samples. Shubnikov–de Haas oscillations are evident in Upon cooling below 34 K, Moreover, from a fundamental point of view, the electronic band structure of the sample does not seem to be altered (at most slightly modified) after p-AP functionalization. This is shown in 38Atomic force microscopy (AFM) measurements further prove that this functionalization process did not either alters the morphology of the graphene samples. Topographic and surface potential images of the functionalized samples look very much alike to pristine graphene on SiC, with no evidences of large-scale damage .2-terminating molecules, with the doubly dehydrogenated N atom incorporated within the graphene lattice (as in the present case): 4-aminothiophenol, 4-aminobenzaldehyde, 4-(oxiran-2-yl)aniline, among many others, where the same previous behaviour has been also observed. For all these cases the molecules transfer a net electronic charge of around 0.5–0.6e− to the graphene, while N atom accumulates an extra electronic charge of around 0.9–1e−. These calculations support the experimental observed generality of the process.To validate our mechanism, we have chosen p-AP as a model molecule as it incorporates an aromatic ring and two different ending groups. The only requirement for this methodology is to use an amino-terminated molecule, as the mechanism is highly selective and efficient to this particular group. The other ending group has to be chosen towards potential applications. Several variations of this molecule are nowadays commercially available or possible to synthetize by several laboratory routes. We have experimentally confirmed that our protocol perfectly works also with aminothiophenol and p-Phenylenediamine molecules , and the2 per molecule  during 45 s at normal incidence angle. The acceleration energy of the electrons inside the ion gun was 140 eV, the current sample was 1 μA and the gas pressure during this process was kept at 1 × 10−7 mbar. Aminophenol was dosed from a crucible hold at room temperature inside the UHV chamber.Growth, STM, XPS and ARPES experiments were performed XPS spectra were recorded using a Phoibos 150 electron analyser equipped with a 2D-DLD detector and an Al-Kα monochromatic X-Ray source. ARPES spectra have been acquired at RT at BaDElPh beamline located at ELETTRA Synchroton in Trieste using a SPECS Phoibos 150 with a 2D-CCD detector system2 square samples using the Van der Pauw configuration in 9 T and 14 T Quantum Design PPMS, using Wimbush press-on contacts on Cu-sheet pads placed at the sample corners. The magnetic field was applied perpendicular to the graphene sheet. Resistance measurements were taken in three configurations: two of them optimized for resistivity measurements along nominally orthogonal directions in the sample while the remaining configuration maximized the Hall resistance. Further information is given in the Experimental Section of the Resistance as a function of magnetic field and temperature (1.8 K–360 K) was measured on 1 × 1 cmFirst-principles computational simulations have been carried out within the basis of different DFT-based implementations. In particular, we have combined the localized-basis-set and plane-wave DFT-schemes as implemented in the FIREBALL50The data that support the findings of this study are available from the corresponding author on request.How to cite this article: Bueno, R. A. et al. Highly selective covalent organic functionalization of epitaxial graphene. Nat. Commun.8, 15306 doi: 10.1038/ncomms15306 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Notes, Supplementary Methods and Supplementary References


The co-doping of graphene with nitrogen and sulfur was investigated aiming at understanding their interactions with the presence of oxygen in graphene. The co-doped graphene (NS-G) was synthesized via a one-pot hydrothermal route using graphene oxide as starting material and L-cysteine, an amino acid containing both N and S, as the doping agent. The obtained NS-G with a three-dimensional hierarchical structure containing both macropores and mesopores exhibited excellent mechanical stabilities under both wet and dry conditions. As compared to N or S singly doped graphene, the co-doped sample contains significantly higher concentrations of N and S species especially pyrollic N groups. The co-doped sample considerably outperformed the singly doped samples when used as free-standing electrode in supercapacitors due to enhanced pseudocapacitance. The simultaneous incorporation of S and N species with the presence of oxygen significantly modified the surface chemistry of carbon leading to considerably higher doping levels, although directly bonding between N and S is neither likely nor detected. Hence, the synergetic effect between N and S occurred through carbon atoms in neighboring hexagonal rings in a graphene sheet. The incorporation of heteroatoms in carbon materials can significantly modify their surface and bulk properties and considerably broaden their applications in various fields. Among others, nitrogen doping of graphene has been intensively investigated in the last few years. Sulfur and boron are among other commonly used heteroatoms for the doping1−1, which are far below the theoretical electrochemical double-layer capacitance of 520 F g−19Recently, graphene has been widely explored as electrode materials in supercapacitors due to its large specific surface area and high electronic conductivity3456Much progress has been made in the synthesis of graphene with different morphologies so far. For example, two-dimensional graphene nanosheets, one-dimensional graphene nanoribbons, and zero-dimensional graphene quantum dots have been obtained91112182045141516171819Other than the morphology control, another effective way in improving the capacitive performance of graphene is the introduction of heteroatoms222425It has been reported that nitrogen doping can improve the specific capacitance and cyclability of graphene in supercapacitors142728Although co-doped carbon materials potentially possess promising properties for various applications, there are only a few reports about graphene that are simultaneously doped with sulfur and nitrogen31323334353632Recently, we used GO as precursor and nine different amino acids as environment-friendly nitrogen sources for the synthesis of nitrogen-doped graphene via hydrothermal method, where the influence of acidity of amino acid on the capacitive performance was investigated. The charged amino acid, and the ensuing electrostatic interactions between amino acid and GO affect the morphology and specific surface area of nitrogen doped graphene (N-G), and ultimately affect its electrochemical performance. The specific capacitance for the obtained N-G samples are following the order of acidic amino acids > neutral amino acids > basic amino acids−1, an energy density of 29.4 Wh kg−1 and a power density of 10.0 kW kg−1, which significantly outperformed the nitrogen doped graphene (N-G) and sulfur doped graphene (S-G). The location, interaction and synergetic effect of nitrogen and sulfur on carbon surface were discussed.The present study aims at the understanding of the interaction between nitrogen and sulfur as dopants in graphene. Graphene oxide and L-cysteine were used as starting material in the hydrothermal synthesis. The obtained NS-G exhibits three-dimensional hierarchical structure containing macropores. The NS-G hydrogel with high mechanical stabilities was used as an additive/binder-free electrode and showed an excellent electrochemical performance with a high specific capacitance of 566 F g−1, indicating a predominantly elastic response. The G″ of NS-G hydrogel is slightly sensitive to the frequency and does not cross over G′, implying a high degree of covalent or non-covalent cross-linking in the hydrogel13The co-doped NS-G, singly doped N-G and S-G hydrogels were successfully synthesized via the one-pot hydrothermal method. When preparing using the same reactor, the dimension of the hydrogel with different doping elements appears to be rather similar . We alsoThe morphology of the NS-G, S-G and N-G aerogel samples was investigated by SEM. As displayed in The results from XRD measurements are shown in −1 corresponding to the D and G band, respectively , S-G (1.03) and N-G (1.00) are essentially equal, which means the defect levels of these samples are similar. In the doped samples, the weak and broad peaks at around 2800 cm−1 can be assigned to the combination of 2D-band and D+G-band. The peaks at around 2680 cm−1 are related to the second-order two phonon mode 2D-band and the weak contribution at around 2920 cm−1 is a combination of the D-and G-band, which are related to the disorder-induced feature of carbon material−1 is also observed. The presence of the broad peak in RGO, NS-G, N-G and S-G confirms the presence of defects.ectively . The relectively  and d, i0 = 0.4-1). The co-doped samples prepared from different cysteine to GO ratios show similar pore structure but with less pronounced hysteresis in isotherms (2 g−1) is slightly lower than those of S-G and N-G. It is also lower than the nitrogen-doped graphene prepared using acidic amino acid in our previously study (367.1 m2 g−1)2 g−129332 g−1 for NS-G005 and 156.9 m2 g−1 for NS-G1).The BET surface area and pore structure of the freeze-dried samples were investigated by nitrogen physisorption, and the obtained isotherms are shown in sotherms . As a wh3 g−1. This is considered as a positive feature in this work, since mesoporous structure with large pore size facilitates electrolyte diffusion and therefore can potentially enhance the capacitive performance when used as electrode.The pore size distributions are listed in The doped samples were further investigated by high resolution XPS. As shown in 4138n–C (n = 1 or 2) and conjugated –C = S– bonds respectively3031n– species, which usually form at the edges of grapheneThe surface atomic concentrations of C, O, S and N were derived from the corresponding peak areas of the XP spectra and the results are summarized in Above-mentioned results demonstrate the unique features of the co-doped NS-G, including the high specific surface areas and pore volume, 3D porous structure, and high nitrogen and sulfur contents. These features strongly suggest that NS-G could have a better electrochemical performance than the singly doped samples.−1. The voltammograms of the three samples show approximately rectangular shape, which are typical for double layer capacitors. Obviously, the NS-G has the highest capacitance in the three samples. −1. The curves show an approximately triangular form for the three samples, indicating the electric double-layer performance. The time of accomplishing a charge/discharge cycle is clearly longer for NS-G than that for N-G and S-G. The mass specific capacitances (Csc) calculated by using the equation Csc = (IΔt)/(mΔV) were 566, 282 and 371 F g−1 for NS-G, S-G and N-G, respectively. The Csc value of NS-G is higher than that of single doped graphene, RGO  is higher than the theoretical electrochemical double-layer capacitance of 520 F g−1The doped graphene hydrogels were used as free-standing electrodes, and tested using cyclic voltammetry (CV) and galvanostatic charge-discharge (GCD) techniques with a three-electrode system. It has been reported that pyridinic N in graphene layers results in the attraction of ions, and pyrrolic N has an enhancing effects on the capacitance due to its pseudocapacitive contributions46−1 is shown in −1 for 2000 cycles (−1) was obtained after 2000 cycles, which demonstrates the excellent cycling performance of the NS-G electrode.The calculated specific capacitance (Csc) at different current densities from 0.5 to 10 A g0 cycles . The capElectrochemical impedance spectroscopy (EIS) provides information on the internal resistance of the electrode material and the resistance between the electrode and electrolyte. The real part of resistance (Z′) measured is the ohmic resistance derived from the electrolyte and the contact between the electrode and the current collector. As shown in 30−1 was determined to be 385 and 263 F g−1 for NS-G005 and NS-G1, respectively  was obtained after 2000 cycles.Finally, the power density and energy density of NS-G was investigated by a symmetrical two-electrode system in 6 M KOH. The CV and GCD curves of NS-G at different scan rates and current densities are shown in 0 cycles  in a sym−1 at 0.5 A g−1. The enhanced pseudocapacitance in the co-doped sample can be assigned to higher contents of pyrrolic N groups and S species.In summary, nitrogen and sulfur co-doped hydrogels and aerogels were successfully synthesized through a hydrothermal method by using amino acid and GO as precursor. The obtained NS-G with a three-dimensional hierarchical structure containing both macropores and mesopores exhibited excellent mechanical stabilities. The simultaneous incorporation of S and N species with the presence of oxygen significantly modified the surface chemistry of carbon leading to considerably higher doping levels, although directly bonding between N and S is neither likely nor detected. Hence, the synergetic effect between N and S occurred through carbon atoms in neighboring hexagonal rings in a graphene sheet. When used as free-standing electrode in supercapacitors, the co-doped sample considerably outperformed the singly doped samples due to an enhanced pseudocapacitance and a lower charge transfer resistance. The specific capacitance of NS-G reached 566 F g−1, 30 ml) containing 0.05 mmol L-cysteine (C3H7NO2S the mass ratio of L-cysteine to GO is 0.1:1) was first treated by sonication for 10 min. The resulting stable suspension, sealed in a Telfon-lined autoclave, was hydrothermally treated at 160°C for 4 h. After that, the autoclave was naturally cooled to room temperature and the as-prepared NS-G hydrogel was removed from the autoclave and immersed in deionized water for seven days, in which deionized water was renewed once a day to remove any unreacted reagents. Finally, the NS-G hydrogel was freeze-dried overnight to obtain the aerogel. In addition to the L-cysteine to GO ratio of 0.1:1, a lower mass ratio of 0.05:1 and a higher ratio of 1:1 was investigated and the obtained samples are designated as NS-G005 and NS-G1, respectively. For comparison, N and S singly doped hydrogels (N-G and S-G) were prepared using the same procedure. L-alanine  and 3-mercaptopropionic acid  with 30 ml GO suspension (2 mg/ml) were employed for N-G and S-G, respectively. The hydrothermal synthesis was also performed without doping agent and the obtained sample is designated as RGO. The water content was determined by weighing the samples before and after drying.Graphene oxide was prepared from graphite powder using a modified Hummers methodα, (λ = 1.5418 Å) radiation. Raman spectra were recorded with a Bruker spectrometer with 532 nm laser, and X-ray photoelectron spectroscopy (XPS) measurements were carried out using a ESCALab220i-XL electron spectrometer (VG Scientific) using a 300W Al Kα radiation. Nitrogen physisorption was carried out at 77 K with an ASAP 2020 Physisorption Analyzer. The mechanical properties of the prepared hydrogels were investigated using a dynamic mechanical analyzer (DMA Q800).All of the samples were characterized by scanning electron microscopy (SEM) using a Hitachi S-4800 system and transmission electron microscopy . X-ray diffraction (XRD) measurements were performed with a D8 Bruker diffractormeter with a Cu K2 and the area of the prepared electrode is 1 cm2. In the three-electrode system, the graphene hydrogel samples were used as the working electrode, platinum foil as the counter electrode and standard Hg electrode as reference one, and 6.0 M KOH aqueous solution as electrolyte. CV and GCD test in the potential range of −1.2 to −0.2V was performed at different scan rate and current densities. For the electrochemical test in the two-electrode system, two slices of hydrogel with the same mass were pressed onto a nickel foam sheet (3 × 1 cm) at 10 MPa for 30 s and used as electrodes. The electrode contained approximately 2.0 mg/cm2 of active material. The two electrodes were separated by a filter paper, which was fully soaked with electrolyte. The potential range for CV and GCD tests was 0–1.0 V. Electrochemical impedance spectroscopy (EIS) measurements were carried out by applying an alternating current with the voltage of 5 mV over a frequency range from 0.01 Hz to 100 kHz. The gravimetric capacitance, CCV (F g−1), was calculated from CVs using the following equation: CCV = Σ|I|/2νmΔV, where Σ|I| is the area of the current (A) profile, m is the mass of active material in the electrode (g), ν is the scan rate (V/s) and ΔV is the potential window (V). The specific discharging capacitances were determined using the equation Csc = (IΔt)/(mΔV) from three-electrode measurement, and in the two-electrode system the energy density and power density were calculated using the equation E = 1/2 Csc ΔV2 and P = E/Δt, respectivelyCyclic voltammetry and galvanostatic charge/discharge of doped graphene electrodes were performed with a CHI660D electrochemical workstation with both two-electrode and three-electrode systems. The working electrodes were fabricated by the prepared hydrogels in the wet state without using any binder or conducting additives. A slice of the wet hydrogel was cut and blotted with a filter paper to remove excess water; then it was pressed onto a nickel foam sheet (3 × 1 cm) at 10 MPa for 30 s. The mass loading of active material on each current collector was about 2 mg/cmSupplementary Information for Interaction between Nitrogen and Sulfur in Co-Doped Graphene and Synergetic Effect in Supercapacitor


Moreover, performance on word order and social-pragmatic scores independently predicted both groups' later performance on wh-question comprehension. Our findings indicate that both grammar and social-pragmatics are implicated in the comprehension of wh-questions. The “grammatical-origins” argument is supported because the ASD group did not reveal earlier and stable comprehension of wh-questions; furthermore, their performance on SVO word order predicted their later success in linguistic processing of wh-questions. The “pragmatic-origins” argument is also supported because children's earlier socialization and communication scores strongly predicted their successful performance on wh-question comprehension.Compared to typically developing children, children with autism (ASD) show delayed production of wh-questions. It is currently controversial the degree to which such deficits derive from social-pragmatic requirements and/or because these are complex grammatical structures. The current study employed the intermodal preferential looking (IPL) paradigm, which reduces social-pragmatic demands. The IPL paradigm can help distinguish these proposals, as successful comprehension promotes the “pragmatics-origins” argument whereas comprehension difficulties would implicate a “grammatical-origins” argument. Additionally, we tested both the linguistic and social explanations by assessing the contributions of children's early grammatical knowledge  and their social-pragmatic scores on the Vineland to their later wh-question comprehension. Fourteen children with ASD and 17 TD children, matched on language level, were visited in their homes at 4-month intervals. Comprehension of wh-questions and SVO word order were tested via IPL: the wh-question video showed a costumed horse and bird serving as agents or patients of familiar transitive actions. During the test trials, they were displayed side by side with directing audios . Children's eye movements were coded offline; the DV was their percent looking to the named item during test. To show comprehension, children should look longer at the named item during a where-question than during a subject-wh or object-wh question. Results indicated that TD children comprehended both subject and object wh-questions at 32 months of age. Comprehension of object-wh questions emerged  According to the DSM-V , usually occurring in the beginning of the sentence (in English). Syntactically, these wh-words stand for information that is missing in the sentence. Wh-questions probe for missing arguments  or adjuncts . Furthermore, argument wh-questions can ask for the grammatical subject of a sentence  Who __ likes Mary?) or the grammatical object of the sentence  Who does Mary like __?). Notice that both subject and object wh-questions involve wh-movement from the original argument location; however, the movement for subject wh-questions does not change the canonical word order of English sentences  above), whereas the movement for object wh-questions changes the word order of the sentence to OSV , indicating that children's proportion of looking to the named object was different for the “what” and “where” questions. There was no main effect of visit , nor a significant group × trial interaction . A significant group effect emerged , with greater overall looking to the named object by the TD group than by the ASD group. Given these significant trial and group effects, the next set of analyses investigated each group's looking patterns separately for subject- and object-what questions.A repeated-measures analysis of variance (2 × 4 × 2) was conducted with group (ASD or TD) as the between-subjects variable, and visit  and trial type  as within-subjects variables. The results showed a main effect of trial  but no main effect of visit . The visit × trial interaction trended toward significance . The second ANOVA compared the object wh-questions and “where” questions, and revealed a main effect of trial , and no main effect of visit . This visit × trial interaction also trended toward significance .For the TD group, the first repeated-measures analysis of variance (4 × 2) was conducted with visits  and trial type (subject-what questions and where questions), revealed a main effect of trial  but no main effect of visit  and no visit × trial interaction . Similarly, the repeated-measures analysis of variance (4 × 2) for the object wh-questions with visit  and trial type (object-what questions and where questions) revealed a main effect of trial  but no main effect of visit  nor a significant visit × trial interaction .For the ASD group, the first repeated-measures analysis of variance (4 × 2), conducted by visit  and trial type  = 1.90, p = 0.038; visit 4: t(16) = 3.68, p = 0.001; visit 5: t(16) = 4.09, p < 0.001; visit 6: t(15) = 6.26, p < 0.001; see Figure t(16) = 1.27, p = 0.111; visit 4: t(16) = 3.75, p < 0.001; visit 5: t(16) = 3.57, p = 0.001; visit 6: t(15) = 8.52, p < 0.001; see Figure Overall, then, both groups demonstrated wh-question comprehension—they correctly looked t(13) = 3.39, p = 0.002, this effect disappeared at visit 4, t(13)= 0.998, p = 0.168 and visit 5, t(11) = 1.05, p = 0.157, then re-emerged at visit 6, t(11) = 2.07, p = 0.031; see Figure t(13) = 2.30, p = 0.019; visit 4: t(13) = 0.807, p = 0.217; visit 5: t(11) = 1.58, p = 0.07; visit 6: t(10) = 0.857, p = 0.206; see Figure The ASD group's performance was less consistent for object-what questions: while they appeared to show comprehension at visit 3, visit4: M = 43.31, SD = 11.95; ASDvisit6: M = 48.28, SD = 19.35; t(28) = −0.859, p > 0.05] or their expressive (EOWPVT) vocabulary  Thus, it appears that the TD and ASD groups achieved comprehension of wh-questions at similar language levels.In sum, TD children displayed evidence of wh-question comprehension by 32 months of age . The ASD group demonstrated significant comprehension at visit 3; however, the ASD group was unable to maintain this level of comprehension consistently for the rest of the visits (with re-emerging significant comprehension for object wh-questions at visit 6). When the two groups are compared by age and/or visit, there is a discrepancy in the point of wh-question comprehension attainment; however, it is important to compare the groups by language level as well. As Table 2 = 3.76, p = 0.05]; visit 4: , visit 5: ; and visit 6: }, indicate that in all the visits there were more Comprehenders than Non-comprehenders in the TD group. Within the ASD group, there were more Comprehenders than Non-comprehenders at visit 3 .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.