Surface plasmons, collective electromagnetic excitations coupled to conduction electron oscillations, enable the manipulation of light–matter interactions at the nanoscale. Plasmon dispersion of metallic structures depends sensitively on their dimensionality and has been intensively studied for fundamental physics as well as applied technologies. Here, we report possible evidence for gate-tunable hybrid plasmons from the dimensionally mixed coupling between one-dimensional (1D) carbon nanotubes and two-dimensional (2D) graphene. In contrast to the carrier density-independent 1D Luttinger liquid plasmons in bare metallic carbon nanotubes, plasmon wavelengths in the 1D-2D heterostructure are modulated by 75% via electrostatic gating while retaining the high figures of merit of 1D plasmons. We propose a theoretical model to describe the electromagnetic interaction between plasmons in nanotubes and graphene, suggesting plasmon hybridization as a possible origin for the observed large plasmon modulation. The mixed-dimensional plasmonic heterostructures may enable diverse designs of tunable plasmonic nanodevices. Surface plasmons have unique physical properties that make them also interesting for technology. Here, the authors observe plasmons in mixed-dimensional heterostructures that can be highly modulated with electrostatic gating, which may be explained by plasmon hybridization Dimensionality has profound effects on the plasmon properties. Plasmons in low dimensions have been intensely studied for their potential in manipulating light–matter interactions far below the diffraction limit5. Notably, in one-dimensional (1D) materials such as single-walled carbon nanotubes (SWNTs), electrons are strongly correlated with each other as a Luttinger liquid, resulting in peculiar properties of 1D Luttinger plasmons8. The 1D Luttinger plasmon in metallic SWNTs combines a nondispersive plasmon velocity, deep subwavelength confinement, and low loss, but it cannot be actively modulated by gating due to its intrinsic independence on carrier density9. Two-dimensional (2D) graphene sheets encapsulated between hexagonal boron nitride (h-BN) layers host highly confined and low-loss 2D plasmons with gate tunability16. Despite extensive efforts to investigate surface plasmons in each dimension as well as in coupled plasmonic systems within the same dimension , the exploration of plasmonic modes in systems of mixed dimensionality has been surprisingly limited21. Recently, mixed-dimensional van der Waals (vdW) heterostructures have emerged as a promising platform to explore intriguing physical phenomena and new device functionalities25. Theoretical studies further suggest that the coupling between plasmonic materials of mixed dimensionality can drastically change the plasmon dispersion27. However, an experimental realization of the collective plasmon modes of a coupled mixed-dimensional heterostructure has yet to be demonstrated.Surface plasmons are collective charge oscillations coupled to electromagnetic wavesIn this article, we report experimental and theoretical studies of plasmons in mixed-dimensional SWNT/h-BN/graphene heterostructures, which serve as an exemplary 1D-2D hybrid plasmonic system. The results indicate that the experimentally observed tunable plasmon modes are most likely to be interpreted as hybrid plasmons due to the coupling between the 1D SWNT plasmons and the 2D graphene plasmons. This coupling is theoretically expected to lead to efficient electrical control of the highly localized plasmon excitations along the nanotube. Our findings demonstrate that dimensionally mixed coupled plasmonic systems may host plasmons with on-demand properties that cannot be attained in the constituents alone. The approach can be used to design hybrid plasmonic devices with varying functionalities.2/Si. A thin layer of top h-BN (~2 nm) is inserted between SWNT and graphene to avoid direct charge transfer while maintaining efficient electromagnetic coupling. Ultraclean SWNTs are directly grown onto SiO2/Si substrates by chemical vapor deposition (CVD)9. Graphene and h-BN flakes are mechanically exfoliated onto SiO2 (285 nm)/Si substrates. SWNT/h-BN/Graphene heterostructures are assembled by the standard polymer stamp dry-transfer technique28 (see Methods for details). The top h-BN flake does not fully cover the graphene to allow a direct metal contact with the exposed graphene region. The optical image of an as-prepared heterostructure is shown in Fig. As schematically shown in Fig. 11. This infrared nano-imaging technique is based on a tapping mode atomic force microscopy (AFM). The metallic AFM tip is illuminated with a focused infrared beam of wavelength 10.6 μm (28.3 THz). The sharp tip (~20 nm radius at the apex) acts as an optical nanoantenna and concentrates the incident light into a near-field nanoscale spot, which launches the plasmons in the sample. The launched plasmon wave propagates towards and reflects off the sample edges or other scatterers, forming a standing wave with a period of half plasmon wavelength, λp/2. The signal backscattered by the tip apex contains essential plasmon information and is captured by a mercury cadmium telluride (MCT) detector in the far field. By a raster scan of the sample surface, near-field images are obtained simultaneously with topography. Figure 8.We probe the plasmon modes in the heterostructure using infrared scanning near-field optical microscopy (IR-SNOM) as illustrated in Fig. 1aλ0 of 10.6 μm. The topography and corresponding near-field images of a long SWNT M1 are shown in Fig. Vg is varied away from the charge neutral point Vcnp, the graphene carrier density n and associated Fermi energy EF are tuned continuously as Cg is the capacitance density, ℏ is the reduced Planck constant, and 15. We study the heterostructure for a wide range of gate voltage with a maximum at − 120 V, corresponding to a hole density maximum of 22. The evolution of the twin fringes with gate voltage indicates that the graphene carrier density and associated graphene plasmons are effectively tuned by the applied gate voltage Vg in our measurement. More importantly, we clearly observe that prominent near-field oscillations emerge near the nanotube ends and that they depend sensitively on the gate voltage. The oscillation peaks near the right end correspond to the constructive interference between the plasmon wave excited by the tip and that reflected by the right end7. Note that the plasmon wave reflected by the left end experiences substantial damping during the long-distance roundtrip propagation and therefore has negligible contribution to the signal near the right end. The white double arrow  decreases from 7 to 4. The achieved plasmon wavelength modulation can be estimated to be ~(1/4 − 1/7)/(1/7) ~75%. The gate-tunable plasmons in SWNT M1 and M2 at the positive gate voltage side are shown in Supplementary Fig. The modulation of plasmon excitations is also evident in a short SWNT M2. Fig. ity Fig. , we cleaide Fig. , the cavVg | , the intensity maxima move from the nanotube axis to the top and bottom edges , the graphene plasmons suffer from severe Landau damping by exciting interband electron-hole pairs. When the intraband conductivity dominates at substantially high Fermi energy, the graphene plasmon wavelength EF-dependence is also clearly observed in the infrared nano-imaging of the gate-dependent graphene plasmon interference patterns near the edge of graphene in the same heterostructure  is the gate-tunable Fresnel-like graphene response function for the incoming evanescent waves of an arbitrary momentum k larger than kmed. To solve Eq. 1 analytically, we use the permittivity ratio εin/εmed = −210 + 23.52i to include the substrate effect. It yields λp ~80 nm and Q ~15 at EF = 0 eV, which are consistent with the experimental observations in Figs. A theoretical model is established to better understand the large plasmon modulation in this mixed-dimensional heterostructure, suggesting the likely origin to be the plasmon hybridization. SWNTs can be modeled as ultrathin cylindrical waveguides with radius entclass1pt{minimakp at a given Fermi energy EF, we can obtain the plasmon wavelength Q of the hybrid plasmon modes using the relation r = 0 reduces to that of freestanding SWNTs providing a single solution. On the contrary, Eq. 1 with r ≠ 0 yields double solutions exhibiting the plasmon hybridization in the mixed-dimensional heterostructure as shown in Fig. Vg, but progressively becomes graphene-like (SWNT-like) at higher Vg. The experimentally obtained plasmon wavelength as a function of gate voltage from infrared nano-imaging data of both SWNT M1 and M2 are displayed in black and blue squares, respectively as illustrated in Fig. Solving Eq. 1 for the complex-valued plasmon momentum 29. Second, the quality factor of the lower plasmon branch is smaller than the higher branch. As shown in the simulation results in Supplementary Fig. We hypothesize that the absence of the clear lower plasmon mode in the experimental data is due to a combination of two effects. First, for the lower plasmon mode, the out-of-phase charge oscillations in SWNT and graphene leads to a shorter plasmon wavelength and concentration of the electric field at the gap between SWNT and graphene, resulting in the weaker coupling of this mode to the metallic AFM tip. The gap localization of the lower mode 2 and its consequence are demonstrated in Supplementary Fig. Ez) at SWNT’s bottom surface when an oscillating point dipole source mimicking the tip is used to excite the surface plasmons in the heterostructure. Two dispersion branches in the numerical simulation can be resolved and match well with the theoretical results indicated by the white dashed lines  (see Methods for details). In the numerical simulation, the thin h-BN flake between SWNT and graphene is replaced by air as an approximation to avoid the huge numerical cost, while the dielectric screening effect can be included in the effective permittivity of SWNT. Figure ω(k), will be highly desirable. Systematic study of the hybrid plasmon dispersion in heterostructures with different nanotube diameters and h-BN thicknesses can further elucidate the evolution of the 1D-2D hybrid plasmon as a function of the coupling strength. Experimental realization of these measurements, however, can be challenging. For example, measurements of the plasmon dispersion require a tunable laser over a very broad spectral range away from the phonon bands of both SiO2 and h-BN layers . Such a broadly tunable infrared laser is not available in our current IR-SNOM setup. In addition, the fabrication of clean and gate-tunable SWNT/h-BN/graphene heterostructures with high-quality plasmon modes is quite challenging experimentally, which makes it very difficult to systematically examine the hybridized plasmon oscillations in heterostructures with varying nanotube diameters and h-BN thicknesses. Future improvements on the IR-SNOM apparatus and 1D-2D heterostructure fabrication technique will be needed to realize these measurements.Further experimental studies are needed to establish conclusively the 1D-2D hybrid plasmons in the SWNT/h-BN/graphene heterostructure. A full characterization of the plasmon dispersion as a function of frequency and momentum, that is 21. Optical responses of other metal/polar crystal heterostructures are constrained to the Reststrahlen bands of the polar crystals and cannot achieve broadband spectral coverage16. Although we have focused on a single infrared frequency (28.3 THz) in our study, the coupling effect and plasmon modulation are valid for a broad infrared spectrum where bare SWNT and graphene plasmon momentum can match. The coupled SWNT/h-BN/graphene heterostructure exhibits the combination of broadband response, ultimate confinement limit, and gate tunability, which is superior to other coupled plasmonic systems reported in the literature.It should be noted that some coupled systems have been studied before. For instance, graphene was shown to affect the plasmon resonance in a silver nanowire/graphene structure, but only a few percent modulation has been achieved due to the large mode mismatch between graphene plasmons and silver plasmons33.To conclude, we have experimentally realized a gate-tunable plasmonic system composed of low-dimensional vdW materials with mixed dimensionality in a SWNT/h-BN/graphene heterostructure. We observed gate-tunable plasmons in the heterostructure with a 75% plasmon wavelength modulation. Assisted by theoretical modeling and numerical simulations, we suggest that the plasmon modulation may be possible evidence for the hybridization between 1D plasmons in SWNTs and 2D plasmons in graphene. The coupled plasmonic systems with materials of different dimensionality may enable diverse applications in plasmonic nanodevices and light–matter interactions2/Si substrates by CVD. Graphene and h-BN flakes are mechanically exfoliated onto SiO2/Si substrates. SWNT/h-BN/Graphene heterostructures are assembled by the standard polymer stamp dry-transfer technique. A polydimethylsiloxane (PDMS) stamp coated with a thin polypropylene carbonate (PPC) film is used to sequentially pick up a thick h-BN flake, a graphene flake, a thin h-BN flake, and SWNTs. Graphene is partially covered by the top thin h-BN flake to allow direct contact of a metal electrode on the exposed area. The PPC thin film together with the heterostructure (SWNT/thin h-BN/graphene/thick h-BN) is peeled from the PDMS stamp, flipped over, and transferred onto a clean SiO2 (285 nm)/Si substrate. The sample is annealed in ultrahigh vacuum . At the excitation wavelength of 10.6 µm, the plasmonic responses of both SWNT and graphene are prominent and the effects from the phonon bands of h-BN and SiO2 substrates are negligible. The metallic tip is tapped at a frequency of Ω ~240 kHz, with an amplitude of ~80 nm. The signal backscattered from the tip apex carries local optical information of the sample and is captured by an MCT detector (Kolmar Technologies KLD-0.1-J1/11/DC) in the far field. To suppress the background scattering from the tip shaft and sample, the detector signal is demodulated at a frequency of 3 Ω by a lock-in amplifier (Zurich Instruments HF2LI). By recording the demodulated signal while scanning the sample, near-field images are obtained simultaneously with the topography.The infrared nano-imaging measurement in this work is accomplished using a home-built scattering-type scanning near-field optical microscope (s-SNOM). To perform infrared nano-imaging, the metallic tip  of a tapping mode AFM (Bruker Innova) is illuminated from the side with a p-polarized infrared beam  and the wavelength of the excitation light (10.6 μm), the thin h-BN spacer is replaced by an air gap of 2 nm. A point electric dipole mimicking the tip normal to the graphene surface (that is z-direction) excites the heterostructure at an oscillating frequency of 28.3 THz (~10.6 μm). The radial electric field, Re(Ez), is taken at the bottom surface of SWNT to avoid dipolar source fields and their diffraction, which can complicate the plasmon dispersion in Fourier transformed profiles.We use COMSOL Multiphysics to simulate the plasmon dispersion and electric field distribution in the heterostructure. SWNTs are modeled by a metallic cylinder with a radius Supplementary Information for Gate-tunable plasmons in mixed-dimensional van der Waals heterostructuresPeer Review File


Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) that exist on a spectrum of neurodegenerative disease. A hallmark of pathology is cytoplasmic TDP-43 aggregates within neurons, observed in 97% of ALS cases and ~ 50% of FTLD cases. This mislocalisation from the nucleus into the cytoplasm and TDP-43 cleavage are associated with pathology, however, the drivers of these changes are unknown. p62 is invariably also present within these aggregates. We show that p62 overexpression causes TDP-43 mislocalisation into cytoplasmic aggregates, and aberrant TDP-43 cleavage that was dependent on both the PB1 and ubiquitin-associated (UBA) domains of p62. We further show that p62 overexpression induces neuron death. We found that stressors (proteasome inhibition and arsenic) increased p62 expression and that this shifted the nuclear:cytoplasmic TDP-43 ratio. Overall, our study suggests that environmental factors that increase p62 may thereby contribute to TDP-43 pathology in ALS and FTLD. ALS, the most common form of motor neuron disease, is a progressive and ultimately lethal disorder with an average life expectancy of 2–3 years post diagnosis2. FTLD is a leading cause of early onset dementia. Although most cases (~ 90%) of both diseases are sporadic , the pathology and phenotypic presentation of sporadic cases are, for the most part, indistinguishable to inherited forms. The exact aetiology of ALS and FTLD remains unclear, however, research over the past few decades indicates that several mechanisms may play a role in pathobiology. These include dysfunctional protein homeostasis through changes to autophagy or lysosomal function or the ubiquitin–proteasome system, endoplasmic reticulum stress, dysfunctional mitochondria and/or energy metabolism, increased oxidative stress, impaired axonal transport and trafficking defects and dysfunctional RNA-binding proteins3.Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are genotypically, phenotypically and pathologically related neurodegenerative diseases that are now referred to as part of a disease spectrum4. TDP-43 aggregates are also found in other neurodegenerative and neuromuscular conditions (e.g. Alzheimer’s disease and inclusion body myositis). Mutations in TARDBP, coding for TDP-43, are a rare cause of ALS and FTLD6. In familial and sporadic cases, the TDP-43 observed in affected tissue is found in cytoplasmic aggregates and is aberrantly phosphorylated, ubiquitinated and cleaved7. TDP-43 is normally a primarily nuclear protein, with roles in mRNA translation, splicing and cryptic exon retention, nuclear export, and axon outgrowth4. The cytoplasmic mislocalisation of TDP-43 is considered an important pathogenic mechanism in ALS and FTLD that is likely to impact diverse biological processes that reduce neuronal viability4. TDP-43 pathology may be due to both loss and gain of function10. Abnormal nuclear depletion of TDP-43 causes a loss of splicing function in neurons leading to widespread transcriptome changes, including altered splicing11 and also leads to defects in DNA double-strand repair12. TDP-43 cytoplasmic aggregation and cleavage leads to toxic gain of function phenotypes13.A key characteristic of ALS and FTLD is the presence of cytoplasmic aggregates containing TDP-43 in neurons, present in 97% of ALS cases, familial and sporadic, and ~ 50% of FTLD cases, indicating a common final pathway17. The consistent presence of p62 in pathological inclusions and the identification of Sequestosome1/p62 mutations as a rare cause of ALS and FTLD suggest a role in pathogenesis18. p62 shuttles ubiquitinated proteins to the proteasome or to the autophagy-lysosomal system for degradation and regulation of signalling pathways that are induced by oxidative stress (Nrf2) and nerve growth factor . p62 facilitates the degradation of proteins implicated in neurodegenerative diseases, including mutant superoxide dismutase 1 (SOD1) in ALS19 and tau in Alzheimer’s, and FTLD20. Further to roles in autophagy and the ubiquitin proteasome system the adaptor protein regulates transcription factors. p62 is a scaffold protein in NF-κB signalling pathways important for neuronal differentiation and survival21. Oxidative damage to the p62 promoter is associated with neurodegenerative diseases, p62 knockout mice develop dementia-like symptoms, and loss of p62 caused a syndrome that includes childhood neurodegeneration23. Together, these studies suggest that increasing p62 levels may be of therapeutic benefit. However, previous studies highlight that p62 knockout can enhance neuron survival or that p62 expression can be detrimental25. It remains unclear whether p62 expression is harmful or helpful in the context of ALS and FTLD. Here, we describe that overexpression of p62 leads to TDP-43 cellular mislocalisation, cleavage and aggregation and leads to neuronal death.TDP-43 positive protein aggregates in ALS and FTLD often also contain p62 protein17. Thus, we wondered if p62 expression or its aggregation within the cytoplasm might affect TDP-43 localisation. We observed that overexpression of EGFP-p62WT with TDP-43-tdTomatoWT in motor neuron-like NSC-34 cells causes TDP-43 nuclear depletion with localisation shifting into cytoplasmic aggregates. Co-localisation of TDP-43 with the nuclear stain was significantly reduced in cells expressing EGFP-p62WT compared with those expressing EGFP alone (p =  < 0.001) with Pearson’s co-efficient of 0.508 ± 0.047 versus 0.786 ± 0.021.  and have previously been shown to reduce ubiquitin-binding capacity26. We found that co-expression of EGFP-p62ΔUBA with TDP-43-tdTomato did not lead to mislocalisation and nuclear depletion of TDP-43, as co-localisation with the nucleus was unaffected by EGFP-p62ΔUBA over-expression (Pearson’s co-efficient of 0.651 ± 0.036). Of note, we observed that TDP-43 in the cytoplasm formed a distinct shell surrounding EGFP-p62WT  domain, we investigated whether nuclear mislocalisation of TDP-43 was dependent on the UBA domain. Of note, several We observed that TDP-43 mislocalisation from the nucleus seemed to be associated with p62 foci, as TDP-43 visually formed a shell around p62 puncta. Thus, we performed fractionation of cellular proteins into a soluble and an insoluble (containing protein aggregates) fraction to determine the effects of p62 on TDP-43 aggregation ; which appears to be due to the decreased soluble TDP-43 in cells, EGFP-p62WTp = 0.080  and without p62 (p62−/−). The amount of nuclear TDP-43 was increased in p62−/− cells compared with p62+/+, thus increasing the nuclear:cytoplasmic ratio , sodium arsenate (NaAsO2) or heat shock and observed that MG132 and arsenic both increased p62 expression in p62+/+ cells  with , in cells overexpressing EGFP-p62WT compared with cells overexpressing EGFP. Thus, in the time frame of our experiment 48 h post-transfection EGFP-p62WT overexpression, and the subsequent cleavage of TDP-43 to TDP-35, did not enhance TDP-43 degradation  of TDP-43, indicating that the N-terminal region of TDP-43 was presumably cleaved , 58.27% (p = 0.0004) and 48.3% (p = 0.0018) with EGFP-p62WT expression alone at 48, 72 and 96 h post transfection, respectively. In cells expressing both TDP-43WT and EGFP-p62WT this was further reduced to 42.73% (p < 0.0001) and 32.97% (p = 0.0004) at 72 and 96 h post transfection, respectively.While previous studies have shown that p62 knockdown causes neurodegenerationNext we investigated whether the effects of p62 overexpression on cell viability were dependent on TDP-43. To this end, we transfected cells with phosphorodiamidate morpholino oligomers (PMOs) used routinely in our laboratory to knock-down expression of TDP-43, or a control PMO Fig. . We founStag2, Poldip3, and Madd transcripts was carried out on RNA from NSC-34 cells transfected with the p62 overexpression plasmid or an empty vector control, following a 1, 3 or 5-day incubation  in the p62 transfected cells after 3 and 5 days compared with empty vector transfected cells, with 39% of + 30b transcripts compared to 33% within the empty vector transfected cells  transcript was observed and deemed to be a PCR artefact. Following p62 overexpression, we observed up to a 10% decrease in the full-length Madd transcript after 5 days, when normalised to Smn loading , the UBA domain (1–385) or the nuclear export sequence (Δ303–320) did not induce TDP-43 cleavage to a TDP-35 C-terminal fragment  or expression of a mutant protein incapable of self-dimerisation (K7A/D69A) induced TDP-43 cleavage to a similar extent as expression of EGFP-p62WT Fig. . However6 or expr∆PB1 or EGFP-p62∆UBA failed to mislocalise TDP-43 or cause nuclear depletion, however overexpression of the PB1 and UBA domains alone (EGFP-p62Δ123–385) was not sufficient to cause significant TDP-43 nuclear depletion  providing further evidence that p62 expression depletes nuclear TDP-43 by shifting TDP-43 into the cytoplasm. It is not clear exactly how this occurs however, p62 may interact with TDP-43 via its PB1 domain, and/or recognize ubiquitinated TDP-43 via its UBA domain, thereby inducing mislocalisation and aggregation.The mislocalisation of TDP-43 away from the nucleus and into cytoplasmic aggregates in neuronal cells is a hallmark of ALS and non-tau related FTLD. These protein aggregates invariably also contain p62, which is thought to be a marker of dysregulated proteostasis. The cause of protein aggregation and its potential role as either a cytoprotective or cytotoxic process has long been debated and is still unclearSTAG2 mature RNA35, exon 3 skipping from POLDIP336 and regulation of MADD35 in human neuronal cells. We amplified Stag2, Poldip3, and Madd transcripts from RNA extracted from NSC-34 cells transfected with the p62 overexpression plasmid or an empty vector control, at 1, 3 or 5-days post transfection. Consistent with TDP-43 knockdown in previous reports, there was an increase in Stag2 transcripts containing exon 30b in the p62 transfected cells after 3 and 5 days, compared with the empty vector transfected cells. Additionally, we observed a decrease in the full-length Madd transcript after 5 days. The moderate effect size that we observed may be due to low transfection efficiency in NSC-34 cells (~ 20–30%), such that only a subset of the total RNA extracted is from cells overexpressing p62. We found, in contrast to previous reports, a decrease in the number of transcripts skipping exon 3 (Δ3) in Poldip3 following p62 overexpression compared with empty vector transfected cells at 5 days. Nonetheless, our findings are interesting as while changes in regulation of Stag2 and Madd transcripts following p62 overexpression supports a loss of functional nuclear TDP-43, the splicing of Poldip3 in mouse neurons was inconsistent with a previous report36. This may be due to differential splicing patterns and regulation between species. In support of this, TDP-43 regulation of STMN2 cryptic splicing in ALS patient neurons was not observed in the mouse37.It is unclear whether the ALS and FTLD pathology associated with TDP-43 is due to loss of nuclear function or gain of toxic cytoplasmic functions. We hypothesised that p62 overexpression, leading to TDP-43 nuclear depletion, may cause changes in TDP-43 mediated splicing events. We investigated the effects of we p62 overexpression in neuronal cells on TDP-43-mediated RNA regulation. Loss of functional TDP-43 within the nucleus has previously been reported to induce the retention of exon 30b within the 33. It may be that the time frame of our experiments monitoring TDP-43 turnover was insufficient to observe the effects of p62 expression on TDP-43 degradation.In order to assess the effects of p62 overexpression on TDP-43 aggregation and possible toxic cytoplasmic gain of function we performed cell fractionation studies after co-expressing p62  with TDP-43 and found that p62 wild type overexpression reduced the amount of soluble TDP-43 and increased the soluble:insoluble ratio. This suggests that expression of p62 may either induce TDP-43 degradation, thereby decreasing the soluble TDP-43 observed, or p62 may shift TDP-43 expression into insoluble protein aggregates. It appears to be the latter, as the total amount of wild type TDP-43  was unchanged in cells overexpressing EGFP-p62 compared with cells expressing EGFP. Taken together, our experiments suggest that p62 is shifting TDP-43 localisation from a soluble nuclear fraction into an insoluble cytoplasmic fraction, rather than inducing degradation of TDP-43. To investigate this further we performed experiments to determine the effects of p62 overexpression on TDP-43 turnover. We treated cells with a proteasome inhibitor (MG132), serum starvation (to induce autophagy) or serum starvation with Bafilomycin treatment  followed by cell fractionation into soluble and insoluble fractions. In keeping with our previous observations, these studies did not reveal significant changes in the amount of total TDP-43 (soluble and insoluble combined) in cells over-expressing EGFP-p62 compared with EGFP expressing cells. However, a previous report has shown that p62 expression induced TDP-43 degradation38. We observed that p62 overexpression induced TDP-43-tdTomato cleavage into a 90-kDa fragment. Given that the tdTomato tag is C-terminal and is ~ 55-kDa, this means the fragment corresponds to a C-terminal TDP-43 protein of ~ 35-kDa. Our TDP-43 turnover experiments revealed that this cleaved TDP-43 fragment was also produced in control EGFP-expressing cells, but only following treatment with a proteasome inhibitor, whereas this fragment was observed in lysates from cells expressing EGFP-p62 under all treatment conditions including in the lysates from untreated cells. Thus, proteasome inhibition and overexpression of EGFP-p62 lead to TDP-43 cleavage. Importantly a C-terminal 35-kDa fragment has been identified in patients and is associated with aggregation and loss of function38. Thus p62-induced cleavage of TDP-43 may be important in ALS and FTLD pathology. We excised the bands containing cleaved TDP-43 from our control, MG132 treated cells and the bands from untreated cells expressing p62 and subjected them to tryptic digestion and mass spectrometry. We determined that the fragment corresponds to amino acids 85–414 of TDP-43.In addition to cytoplasmic mislocalisation and aggregation, TDP-43 cleavage is associated with ALS and FTLD pathology40. The cleavage sites for calpain is in the C-terminus of TDP-43, and we determined by mass spectrometry that our cleavage site is in the N-terminus, thus it seemed unlikely that the TDP-35 fragment observed is mediated by calpain cleavage. TDP-43 contains a Caspase-3 dependent “DETD” recognition site, with cleavage occurring between amino acids 84–8541. Thus, we investigated whether p62-mediated cleavage of TDP-43 was dependent on calpain or caspases, using specific inhibitors. The amount of the cleavage product, TDP-35-tdTomato, in cells expressing EGFP-p62 was not significantly different in lysates from caspase inhibitor treated cells compared with untreated cells. However, in EGFP-p62 expressing cells treated with a calpain inhibitor the amount of TDP-35-tdTomato observed in the insoluble fraction was reduced compared to non-treated cells expressing EGFP-p62. This could suggest that the TDP-43 cleavage that is induced by p62 overexpression is partly dependent on calpains, but not caspases, however it is more likely a reflection of the reduced total TDP-43 that we observed in calpain-treated cells. It is possible that pre-treatment with the calpain inhibitor prior to transfections reduced the transfection efficiency thereby reducing the total TDP-43 and the TDP-35 fragment observed. Thus, we interpret our results to be consistent with the notion that p62-mediated TDP-43 cleavage occurs via another mechanism that is independent of both caspases and calpains.We performed a literature search of TDP-43 cleavage and found that both calpain and caspase-3 can lead to TDP-43 cleavage to a 35-kDa fragment−/− MEFs, which showed significantly increased nuclear TDP-43 when compared with p62+/+ MEFs. As p62 is a stress-induced gene we hypothesise that stressors associated ALS, such as cigarette smoking or exposure to pesticides42, or proteasomal or oxidative stress44 may induce p62 expression and thereby lead to TDP-43 pathology. Here, we show that proteasome inhibition and exposure to the heavy metal arsenite induced p62 expression and this was associated with increased levels of cytoplasmic TDP-43. We also show that p62 overexpression induced neuronal death. However, we found that this cell death was independent of TDP-43 expression as knock-down of TDP-43 did not significantly reduce the cell death observed. Thus, while increased levels of p62 leads to TDP-43 mislocalisation and aggregation and misregulation of RNA transcripts in neurons, p62 appears to induce cell death via additional TDP-43 independent pathways.Our results show that p62 overexpression induced signs of TDP-43 pathology and we confirmed an important role for p62 in TDP-43 localisation using p62Our results show that p62-induced cleavage of TDP-43 requires the PB1 domain, the UBA domain and the nuclear export sequence, whereas mislocalisation required the PB1 and UBA domains only. Thus, cytoplasmic mislocalisation was not necessarily required for TDP-43 cleavage to occur. Importantly, our results show that overexpression of p62 that is lacking either the PB1 or the UBA domains does not induce TDP-43 cleavage or mislocalisation. Of note, p62 overexpression exacerbates disease in a SOD1 mouse model of ALS and here we have shown that p62 overexpression leads to altered TDP-43-mediated RNA regulation consistent with reduced TDP-43 nuclear function. Thus, modulating p62 expression may present a potential therapeutic target for ALS and FTD-TDP. We are currently investigating the use of phosphorodiamidate morpholino oligomers to regulate the expression of p62. Determination of the effects of p62 modulation on TDP-43 pathology in ALS patient-derived neuronal cells is the subject of our future research.+/+) and p62 knockout (p62−/−) MEFs were a generous gift from Professors Yu-shin Sou and Masaaki Komatsu, described previously45. NSC-34 cells were maintained in Dulbecco’s modified essential medium (DMEM)/F12-Ham supplemented with 10% foetal calf serum in a 37 °C incubator with 5% CO2. MEF cells were maintained in DMEM with 10% foetal calf serum. Chemical treatments were purchased from Sigma-Aldrich. Cells were incubated overnight with 2 μM MG132, 10 μM sodium arsenite or were heat shocked at 42 °C for 1 h, followed by 2 h recovery at 37 °C. For inhibitor assays, cells were pre-treated for 1 h with either 20 μM caspase inhibitor (Promega) or 5 calpain inhibitor (Sigma-Aldrich) or left untreated prior to transfection. Cells were maintained in media containing the inhibitors for 48 h post-transfection. Cells were then fractionated into soluble and insoluble fractions and subjected to SDS polyacrylamide electrophoresis and western blotting.All experiments described were carried out in NSC-34 motor neuron-like cells or mouse embryonic fibroblasts (MEFs). Wild type , 1 × 106 cells were resuspended in Buffer R. PMO was added at 100 μM. Cells were electroporated using the neon transfection tips as per manufacturer’s instructions (Invitrogen). The following day cells were transfected with 2 μg expression plasmids for either EGFP or EGFP-p62 and 48 h later were assessed for cell viability with Sytox Red by flow cytometry.Cells were transfected with constructs as indicated for 6–24 h. The pcDNA3.1, pcDNA3.1/EGFP, pcDNA3.1/EGFP-p62 (wild type and UBA deficient) were described previously+/+ or p62−/− MEFs were fractionated following cell treatments. Briefly, cells were lysed with 500 µL fractionation buffer (10 mM HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), 1.5 mM MgCl2, 10 mM KCl, 0.5 μM dithiothreitol, 0.05% Tergitol, pH 7.9) on ice for 10 min. Lysates were clarified by centrifugation at 3000 rpm at 4 °C for 10 min. Cytosolic supernatants were collected and frozen. The pellet was resuspended in 374 μL Buffer B , pH 7.9) and 26 μL 4.6 M NaCl then passed through a 25-guage needle 10 times and then incubated on ice for 30 min. The lysates were centrifuged at 15,000 rpm at 4℃ for 30 min. Nuclear supernatants were collected.p622 flasks. The following day cells were transfected with an expression plasmid for TDP-43-tdTomato and either pcDNA3.1/EGFP, pcDNA3.1/EGFP-p62 wild type [WT] or deletion constructs as indicated. Forty-eight hours post-tranfection cells were lysed on ice with 1% Triton X-100 in phosphate buffered saline (PBS) supplemented with complete EDTA-free protease and phosphatase inhibitor cocktails. Lysates were freeze/thawed and then clarified by centrifugation at 15,000g for 30 min at 4 °C and the 1% Triton X-100 soluble fractions were collected. The pellets were subsequently washed four times with 1% Triton X-100 in PBS and Triton-X insoluble proteins were solubilized with the addition of 1% SDS and incubated for 1 h at 60 °C. Triton X-100-insoluble fractions were collected following centrifugation at 15,000g for 30 min at 4 °C.NSC-34 cells were seeded into T25 cmProtein samples were electrophoresed through SDS polyacrylamide gels and transferred to nitrocellulose membranes. Western blot analyses were performed as follows: membranes were blocked with 5% skim milk powder (SMP) in Tris-Buffered Saline (TBS) for 1 h at room temperature, membranes were then incubated at room temperature with primary antibody as follows: anti-RFP (ThermoFisher) at a dilution of 1:1000 in 5% BSA in TBS-T for 2 h, anti-GFP (ThermoFisher) at a dilution of 1:5000 in 5% SMP in TBS with 0.05% Tween-20 (TBS-T) for 1 h, or anti-α-tubulin (Sigma-Aldrich) at a dilution of 1:10,000 in 5% SMP in TBS-T for 1 h. Membranes were washed 3× with TBS-T and then incubated with a species appropriate horseradish peroxidase conjugated secondary antibody for 1 h at room temperature in 3% SMP in TBS-T. Membranes were washed 3× with TBS-T and protein bands visualised with enhanced Chemi-luminescence Plus reagent (Perkin Elmer). Membranes were stripped between western blots using mild stripping buffer  and checked for residual signal with Chemiluminescence reagent (Perkin Elmer).Densitometry of bands from Western blot analyses was performed using the in-built analysis function in the BioRad ImageLab software. Data presented has been corrected to α-tubulin expression within samples and depicts the mean of ≥ 3 independent experiments +/− SEM.2 flasks. Cells were transfected at various time points to allow for simultaneous assessment of viability at the following times post-transfection; 96, 72, and 48 h. Cells were transfected with pcDNA3.1/EGFP-p62 (wild-type) and TDP-43-tdTomato or suitable tagged-empty vector control. Cells were washed twice with 1 mL ice-cold PBS and transferred to 1.5 mL microfuge tubes and centrifuged at 1000 rpm for 2 min. Cells were resuspended in 200 µL ice-cold PBS + 2% FBS. A positive control sample was prepared by taking half of an untransfected sample and heating it at 65 °C for 1 min, followed by a 1 min incubation on ice. Half of the “killed” sample was resuspended in an equivalent volume of the remaining untransfected sample.For assessment of cell viability, NSC-34 cells were seeded into T25 cmSamples were transferred to tubes and 0.2 µL SYTOX Red stain added to each sample that were then gently mixed and incubated at room temperature, shielded from light, for a minimum of 15 min. Samples were then analysed on a Fortessa flow cytometer, with 50,000 events collected per sample. A population of cells co-expressing EGFP-p62 and TDP-43-tdTomato was created, and viability was assessed by measuring the amount of fluorescence in the SYTOX Red channel. SYTOX Red is an exclusion dye and only permeates the membrane of dead cells, therefore populations with higher fluorescence were considered non-viable. The kill control sample was used to define the gating parameters for the viable vs dead/non-viable cell population. For assessment of the effect of TDP-43 knockdown on p62 overexpression induced cytotoxicity, cells were electroporated with PMOs to knockdown TDP-43 expression or a PMO control and plated in a 6-well plate. The following day cells were transfected with 2 μg EGFP or EGFP-p62. Forty-eight hours later cells were collected and stained with Sytox Red then assessed by flow cytometry as described.WTtransfected (cells were left untreated) cells were separated in duplicate through 10% SDS polyacrylamide gels. One gel was Coomassie stained and destained in 7% acetic acid, 10% methanol while the second gel was transferred to nitrocellulose membrane and western blot analysis for TDP-43-tdTomato  was performed. Bands of ~ 90 kDa in the Coomassie stained gel were identified by comparison with the molecular weight of the TDP-43-tdTomato from the western blot results. The bands in the gel were excised and cut into 1–2 mm pieces and destained in 50 mM ammonium bicarbonate pH 8, followed by 50 mM ammonium bicarbonate/50% acetonitrile pH 8. The destained gel pieces were dehydrated in 100% acetonitrile and the solution was removed to allow gel pieces to air dry. The proteins were reduced and alkylated with 10 mM DTT and 20 mM iodoacetamide (IAA) respectively and digested with trypsin (12.5 ng/µL) overnight at 37 °C as described48. Following overnight digestion, the supernatant was transferred to a fresh tube and the tryptic peptides from the gel pieces were extracted twice with 50% acetonitrile/2% formic acid and combined with the supernatants. The tryptic peptides were vacuum centrifuged to remove acetonitrile and desalted on a pre-equilibrated C18 Omix tip. The eluted peptides were further dried under vacuum centrifugation until ~ 10 µL remained for LC–MS/MS analysis.Triton-X insoluble fractions from cells transfected with TDP-43-tdTomato and either (i) pcDNA3.1 empty vector (cells were treated overnight with 10 μM MG132) or (ii) EGFP-p6218 column  employing a 60 min gradient  with a flow rate of 300 nL/min. The peptides were eluted and ionized into Q-Exactive Plus mass spectrometer (ThermoFisher). The electrospray source was fitted with an emitter tip 10 μm (ThermoFisher) and maintained at 1.6 kV electrospray voltage. FTMS analysis was carried out with a 70,000 resolution and an AGC target of 1 × 106 ions in full MS (m/z range 400–2000); and MS/MS scans were carried out at 17,500 resolution with an AGC target of 2 × 104 ions. Maximum injection times were set to 30 and 50 ms respectively. A top-10 method was employed for MS/MS selection, ion selection threshold for triggering MS/MS fragmentation was set to 1 × 104 counts, isolation width of 2.0 Da, and dynamic exclusion for 20 s was used to perform HCD fragmentation with normalised collision energy of 27.Tryptic peptides were separated on a Ultimate 3000 (ThermoFisher) equipped with a Thermo Acclaim™ PepMap™ 100 CRaw spectra files were processed using the Proteome Discoverer 2.2 software (ThermoFisher) incorporating the Sequest search algorithm. Peptide identifications were determined using a 20-ppm precursor ion tolerance and a 0.1 Da MS/MS fragment ion tolerance for FTMS and HCD fragmentation. Carbamidomethylation modification of cysteines was considered a static modification while oxidation of methionine, and acetyl modification on N-terminal residues were set as variable modifications allowing for maximum two missed cleavages. The data was processed through Percolator for estimation of false discovery rates. Protein identifications were validated employing a q-value of 0.01.To enable separation of single cells for better imaging, cells were transfected and then reseeded onto coverslips the following day. Briefly, NSC-34 cells were transfected in 6-well plates with expression vectors for TDP-43-tdTomato (wild type [WT] or mutant [p.M337V] and either pcDNA3.1-EGFP (empty vector [EV]), pcDNA3.1-EGFP p62 (WT or deletion constructs as indicated). The following day cells were trypsinised and reseeded onto Poly-L-Lysine coated coverslips in 6-well plates. The following day 48 h post-transfection cells were washed 2 times with PBS and then fixed with 4% paraformaldehyde in PBS for 20 min, followed by washing 3 times with PBS. Coverslips were incubated with Hoechst 33342 (ThermoFisher) dye diluted 1:10,000 in PBS for 10 min at room temperature. Cells were washed 3 times with PBS and the coverslips mounted onto slides using Prolong Gold antifade mounting media (ThermoFisher). Images were taken using a Nikon A1 confocal microscope using a 40× oil objective. Pearson’s co-efficient to determine the co-localisation of TDP-43-tdTomato with Hoechst nuclear stain was performed using the co-loc 2 plug in for ImageJ on cell images taken from 3 independent replicate experiments (minimum 32 cells per transfection). A Pearson’s co-efficient of > 0.6 indicates co-localisation.NSC-34 cells were transfected with p62 overexpression plasmids or an empty vector, as above, and incubated for 1, 3 or 5 days prior to harvesting for transcript analysis. RNA was isolated from cells using a MagMAX total RNA isolation kit, including a DNase treatment (Life Technologies), performed using a Kingfisher RNA extractor, according to the manufacturer’s protocol. RT-PCRs were performed using a One-step SuperScript III RT-PCR kit with platinum Taq polymerase (Life Technologies), according the manufacturer’s instructions. Each reaction contained 50 ng of RNA and 25 ng of each forward and reverse primer, with the primer sequences and PCR cycling conditions listed in Table p ≤ 0.05.All statistical analyses were performed in SPSS Version 24.0.0.0. The significance of differences between means was determined using the One-way analysis of variance with post-hoc Bonferroni test with significance set at Supplementary Figures.


Drosophila model of ALS based on TDP-43 expression in motor neurons that recapitulates hallmark features of motor neuron disease including TDP-43 aggregation, locomotor dysfunction, and reduced lifespan. To gain insights into metabolic changes caused by TDP-43, we performed global metabolomic profiling in larvae expressing TDP-43  and identified significant alterations in several metabolic pathways. Here, we report alterations in multiple metabolic pathways and highlight upregulation of Tricarboxylic acid (TCA) cycle metabolites and defects in neurotransmitter levels. We also show that modulating TCA cycle flux either genetically or by dietary intervention mitigates TDP-43-dependent locomotor defects. In addition, dopamine levels are significantly reduced in the context of TDP-43G298S, and we find that treatment with pramipexole, a dopamine agonist, improves locomotor function in vivo in Drosophila models of TDP-43 proteinopathy.Amyotrophic lateral sclerosis (ALS) is a fatal, complex neurodegenerative disorder that causes selective degeneration of motor neurons. ALS patients exhibit symptoms consistent with altered cellular energetics such as hypermetabolism, weight loss, dyslipidemia, insulin resistance, and altered glucose tolerance. Although evidence supports metabolic changes in ALS patients, metabolic alterations at a cellular level remain poorly understood. Here, we used a  Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that affects both upper and lower motor neurons (MNs). It leads to the loss of motor coordination, paralysis, respiratory insufficiency, and eventual death within 30 months of symptom onset in most patients. Although several risk factors have been identified, the disease etiology remains poorly understood ,2. Several clinical studies have reported that ALS patients exhibit hypermetabolism, altered energy and lipid metabolism, weight loss, dyslipidemia, insulin resistance, and altered glucose tolerance, as reviewed in ,4. MetabDrosophila model of ALS based on the over-expression of TDP-43 in MNs. Notably, these models recapitulate several features of motor neuron (MN) disease including locomotor dysfunction, TDP-43 containing cytoplasmic aggregates, and reduced lifespan [Drosophila expressing TDP-43 in MNs exhibit alterations in glycolysis, the carnitine shuttle, and lipid beta oxidation [Drosophila or human mitochondrial isocitrate dehydrogenase 3 (IDH3), or by feeding pyruvate, mitigates TDP-43 dependent locomotor defects. In addition, levels of dopamine are reduced, consistent with reports in ALS patients. We find that the administration of pramipexole, a dopamine agonist, improves locomotor function in Drosophila models of TDP-43 proteinopathy. These results show that dietary intervention or pharmacological and genetic manipulations mitigate TDP-43 induced locomotor dysfunction in vivo and identify broad ranging metabolic deficits that may inform therapeutic strategies based on improving cellular energetics.One of the pathological hallmarks of ALS is the accumulation of TAR DNA binding protein (TDP-43) into irreversible cytoplasmic aggregates . These clifespan ,9. Additxidation ,11,12,13Drosophila models of TDP-43 proteinopathy based on overexpression of wildtype TDP-43 (TDP-43WT) or an ALS-associated mutant variant, G298S (TDP-43G298S), in motor neurons, specifically. Metabolomic profiling was conducted on whole third instar larvae over-expressing TDP-43 in MNs (D42 > TDP-43WT or D42 > TDP-43G298S) and genetic background controls (D42 > w1118) . In brief, five replicates of 100 larvae each were submitted to Metabolon, Inc where they were analyzed for the presence of 572 compounds of known identity (metabolites). Biochemical profiling identified 106 significantly altered metabolites in TDP-43WT compared to controls  and 217 significantly altered metabolites in TDP-43G298S compared to controls   see . These a.05) see .G298S exhibit a significant increase in several TCA cycle intermediates compared to controls  [WT or TDP-43G298S significantly increased the larval turning times  compared to w1118 controls (8.19 ± 0.09 s). Interestingly, overexpression of either hIDH3a or dIDH3a in motor neurons on their own caused a significant increase in larval turning time   subunit a, the catalytic domain of IDH3  , with TD dIDH3a) ,9, expreH3a) see . HoweverWT . These results indicate that high levels of pyruvate rescue TDP-43-induced locomotor deficits and are consistent with increased flux through the TCA cycle as a compensatory mechanism.We have previously shown that the end product of glycolysis, pyruvate, is upregulated in TDP-43-expressing larvae, and genetic interaction experiments with phosphofructokinase  showed that this is a compensatory mechanism , in TDP-43WT, and a trend towards a decrease in TDP-43G298S larvae . These results indicate that a dopamine agonist rescues TDP-43 induced locomotor deficits in vivo.Our data showing that dopamine levels are decreased in the context of mutant TDP-43 overexpression led us to hypothesize that pramipexole, a dopamine agonist that has been shown to reverse motor deficits caused by dopamine depletion in Parkinson’s disease patients, may also be protective in the context of TDP-43 proteinopathy. To investigate if pramipexole can rescue locomotor deficits in fect see , 10 μM pDrosophila ALS models based on the overexpression of TDP-43 in MNs. Notably, our model has previously identified metabolic and molecular alterations that were subsequently validated in ALS spinal cords and induced pluripotent stem cell (iPSC)-derived MNs [Clinical observations identified metabolic alterations in ALS patients that are consistent with altered cellular energetics reviewed in . Howeverived MNs ,18,19. SDrosophila and human metabolism and/or due to limitations in our experimental approach . Alternatively, it is possible that our Drosophila model of ALS does not recapitulate all the aspects of motor neuron disease in humans.In this study, we used global metabolomics to gain insight into altered metabolic pathways in ALS, which could uncover potential therapeutic strategies. Here we report several altered metabolites including amino acids, peptides, lipids, and purine metabolism see , severalDrosophila IDH3a, the catalytic subunit of NAD+-dependent mitochondrial isocitrate dehydrogenase, although toxic on their own, mitigate locomotor deficits in the context of TDP-43. Similarly, dietary supplementation with pyruvate, which is predicted to fuel the TCA cycle, also mitigates TDP-43-induced locomotor deficits. Alternatively, pyruvate could be taken up into glia, where it is converted into lactate and then taken back up into neurons for fueling ATP production  [To further understand the contribution of the TCA cycle defects to TDP-43-dependent toxicity, we used genetic and dietary interventions. IDH3a catalyzes the production of α-ketoglutarate, which can also be replenished by glutaminolysis. In the absence of glutaminolysis, we tested whether IDH3a overexpression in the context of TDP-43 can rescue TDP-43-induced locomotor deficits. Indeed, we found that the overexpression of either human or shuttle) . These rshuttle) . TCA cycshuttle) . While tDrosophila expressing TDP-43 in MNs [At this time, we cannot distinguish whether pyruvate supplementation is protective due to increasing the TCA cycle or due to acting as free radical scavenger, as reported in tumor cells . It will3 in MNs   was added to reach a final concentration of 250 μM. The food was then dispensed into vials and allowed to solidify. For pramipexole supplementation, pramipexole  was dissolved in DMSO and was added to the food to reach a final concentration of 10 μM.Metabolite analyses were performed by Metabolon. Inc as previously described ,13,32. FLarval turning assays were performed as previously described ,9. BriefLarval turning data were analyzed using Kruskal–Wallis tests using GraphPad Prism v9.0.


The photoconductive gain was improved by two orders of magnitude compared to the gain of a photodetector using a graphene/Bi2Te3 nanoplate junction. Additionally, the position of photocurrent generation was investigated at the graphene/Bi2Te3 NWs junction. Eventually, with low bandgap Bi2Te3 NWs and a graphene junction, the photoresponsivity improved by 200% at 2200 nm (~0.09 mA/W).A graphene photodetector decorated with Bi Graphene has attracted much interest for optoelectronic applications due to its unique photonic properties, such as its broad bandwidth absorption, short carrier lifetime, and high gain by the carrier multiplication process ,3,4,5,6.7 A/W photoresponsivity at 895 nm  and exp[−(t − toff)/τoff], respectively, as shown in 2Te3 NW decorated devices, the photocurrent generation time is increased to 8 ms, which represents a two-fold increase compared to the control group without Bi2Te3 NWs. In the case of the falling time, Bi2Te3 NW decorated devices show a longer time of 1.2 ms, compared to 0.8 ms for the control group.A transient photocurrent measurement with/without Bitr, where τ is the lifetime; ttr is the transit time, which is equal to L2/μVd; L is the channel length of the device; μ is the field-effect mobility; and Vd is the voltage applied to the drain. The photoconductive gain of the Bi2Te3 NWs/graphene photodetector was 3 × 104 at 1100 nm, which is two-fold larger than that of the only graphene sample.In addition, the photoconductive gain of each device was calculated using the result of the rising and falling time. The following equation can be employed to calculate the photoconductive gain: G = τ/t2Te3 NWs decoration show a longer rising/falling time compared to the control group due to the charge exchange between Bi2Te3 NW and graphene during the light illumination. In the graphene photodetector, the photocarriers are generated in the whole graphene channel, but the photocarriers generated near the graphene/metal junction can only be collected due to the short recombination time [The devices with Biion time . 2Te3 NWs/graphene photodetector. For the graphene channel device, the photocurrent was generated in the graphene/metal junction area [2Te3 NWs/graphene photodetector, the photocurrent was primarily generated through the whole channel, as shown in 2Te3 NWs decorated the graphene channel, the recombination time seemed to be increased, and the whole channel could be attributed to photocurrent generation. As a result, the absorption was improved for the Bi2Te3 NWs decorated graphene photodetector. This observation confirms that the Bi2Te3 NWs improved the photocurrent generation across the graphene channel. The maximum photocurrent increased five times using Bi2Te3 NWs, from 80 to 400 nA.Scanning photocurrent mapping (SPCM) measurement was carried out with a 530 nm single-mode fiber laser to clarify the exact location of the photocarrier generation in our devices and compare the photocurrent generation mechanism of both the graphene photodetector and Biion area . In the 2Te3 NWs by measuring the photoresponse under an infrared illumination condition  [2Te3 nanoplates/graphene junction (around 83).Overall, the performance of the Bits (103)  and dras2Te3 NWs/graphene junction using the drop-casting method. Graphene was n-doped after making contact with Bi2Te3 NWs, and the photoconductive gain was enhanced further, to 3 × 104, being 1.61 times larger than that of the only graphene photodetector. Additionally, the photoresponsivity under near-infrared was improved by 200% compared to the graphene devices. Moreover, the large area solution was processible and broadband absorption was possible using this structure.In this work, we investigated the Bi


Although the linguistic properties of polar questions have been extensively studied, comparatively little is known about how polar questions are processed in real time. In this paper, we report on three eye-tracking experiments on the processing of positive and negative polar questions in English and French. Our results show that in the early stages, participants pay attention to both positive and negative states of affairs for both positive and negative questions. In the late stages, positive and certain negative polar questions were associated with a bias for the positive state, and this bias appears to be pragmatic in nature. We suggest that different biases in mental representations reflect the hearer’s reasoning about the speaker’s purposes of enquiry. In everyday conversation, we frequently ask and answer polar questions. Indeed, in the Switchboard Speech Act Corpus, polar questions and their answers were among the most frequent speech acts, occurring more often than all wh-questions taken together .Polar questions can be either high negative) and negative polar questions where the negation follows the subject (low negative) as illustrated in (2).Certain languages make a further distinction within the class of negative polar questions depending on the surface position of the negation. For example, English distinguishes negative questions where the negation follows the auxiliary verb . In particular, we were interested in seeing whether positive and negative questions make different propositions cognitively salient, as several of the aforementioned semantic theories suggest.In order to answer this research question, we carried out a series of visual-world eye-tracking tasks. In these tasks, participants were presented with visual scenes containing relevant objects while listening to linguistic stimuli  and an open window in the case of (2b); another image‒the competitor‒mismatched with the content of the sentence, e.g., it showed an open window in the case of (2a) and a closed window in the case of (2a). The remaining two images were distractors, showing, e.g., a sliced pie and an unsliced pie.While listening to these assertions, participants were presented with four images. One of these images‒the Tian and colleagues found that when processing simple positive assertions, participants’ attention was shifted to the target image as soon as the verb was encountered. When processing simple negative assertions, however, participants’ attention was drawn toward both the target and the competitor image for a few hundred milliseconds before being shifted to the target. No difference was found between processing cleft positives and negatives.Tian and colleagues argue that processing assertions out of the blue triggers the representation of different pragmatic contexts. The sentences in their experiment, when uttered out of the blue, evoke different QUDs. In particular, the sentences in (2a-b) tend to be construed as answers to a positive polar question such as ‘Has Matt shut his dad’s window?’, while the sentences in (2c-d) tend to be construed as answers to a wh-question such as ‘Who has shut his dad’s window?’ for (2c), and ‘Who hasn’t shut his dad’s window?’ for (2d).In our study, participants listened to polar questions and responses, such as:While listening to these question–answer pairs, participants saw four images: one image showed an ironed shirt (the p image), one image showed a crumpled shirt (the ~p image), and the other two images were distractors that were included to ensure that participants were unable to predict the upcoming content of the question.We wanted to determine whether, upon hearing a polar question, participants fixate on both the p and ~p image, or whether they fixate primarily on only one of these images. The former is more in line with the partition view; while the latter is more consistent with the proposition–extraction view, as well as the proposals of Roelofsen and Farkas , Krifka In addition, we wanted to see how quickly participants would fixate on either the p or ~p image. For positive assertions, participants fixate on the correct image from the verb onwards. Hence, if polar questions are essentially assertions, as the proposition–extraction view proposes, the same should hold for polar questions. However, if the association between positive polar questions and the p image, and between negative polar questions and the ~p image is pragmatic in nature, we might expect a delay, as is generally found for pragmatic inferences  followed by answers.40 native English speakers were recruited from University College London via an online pool of participants. They all had uncorrected or corrected to normal vision. They were paid four pounds for their participation.The materials consisted of 42 question–answer pairs, such as the ones shown in (3). The questions always contained binary predicates, i.e., predicates whose negation corresponds to a well-defined situation, such as ‘ironed the shirt’, ‘opened the window’, and ‘broke the eggs’. See the “Each question had three forms: positive, high-neg, and low-neg. There were 8 lists containing 1 form of each of the 42 questions. Each list consisted of 14 positive questions, 14 high negative questions, and 14 low negative questions. These lists were supplemented with another 14 positive questions, thus ensuring an equal number of positive and negative questions. Since the latter 14 questions were only tested in one condition, they will not be included in the analyses below. Half of the questions in each condition were confirmed ; the other half rejected . The order of presentation was randomised across the eight lists.target images representing p and ~p , and two distractors, which were images of an item in two states . These distractors were included so that the participants could not predict the verb before hearing it. The critical images and the distractors all measured 250 × 250 pixels. The locations of the target and distractor images were randomised between the eight lists.Each question–answer pair was associated with a visual scene consisting of five images see Fig. : in the t-tests indicated that there were no significant differences in word duration across conditions .Sentences were recorded by a male speaker of South-East British English. There was a pause of 1.5 s between the question and the answer. The speaker was instructed to read all sentences with natural intonation. The average duration of the individual word regions that were identical across the three question forms  never differed by more than 20 ms. Paired The experiment was conducted using the E-Prime software and a Tobii T300 eye-tracker. Participants were calibrated at the beginning of the experiment using a nigh-point display. Before each trial, there was a fixation cross in the centre of the screen, and participants’ eye gaze had to be fixed on this point for a continuous 1.5 s before the trial started. Then a scene of five images (as described above) appeared for one second before the audio started. Participants were instructed to listen and look at the scene. A third of the trials were followed by a comprehension question to ensure that participants paid attention to the audio.We analysed fixations on the p and ~p images against the unfolding audio stimuli. For the analysis of eye movements, word region boundaries were offset by 200 ms to allow for the time it takes to launch eye movements Hallett, . FixatioFigure The results show that participants, upon hearing a positive or negative polar question, fixate on both the p and ~p images from the verb region  onwards. In this respect, polar questions differ from positive assertions. Recall that Tian et al.  observedIn the positive condition, a bias towards the p image emerged, starting in the genitive region  and persisting into the gap region. In the high-neg condition, the bias was similar but less prominent. In the low-neg condition, there was also a mitigated and less persistent bias towards the p image, occurring in the noun region .In order to test whether these biases were statistically significant, we calculated the natural log ratio of the probability of looks to the p image over looks to the ~p image. This measure is symmetrical around 0, meaning that a positive log ratio corresponds to a bias towards the p image and a negative log ratio to a bias towards the ~p image. We conducted mixed linear regression analyses to determine if the log ratios differed reliably from 0. The results of these analyses are shown in Table In the positive condition, there was a significant bias towards the p image from the genitive onwards. In the high-neg condition, the bias emerged in the noun region. In the low-neg condition, the bias also emerged in the noun region, but disappeared immediately afterwards.t(34) = 2.1, p = 0.04, model = intercepts only; positive vs. low-neg: t(36) = 3.2, p = 0.00, model = intercepts for subjects and items; slopes for items; high-neg vs. low-neg: t(36) = 2.8, p = 0.01, model = intercepts for subjects and items; slopes for items). The positive bias was thus more persistent in the positive condition and in the high-neg condition compared to the low-neg condition; there was no significant difference between the positive and high-neg condition.In order to determine if the log ratio differed across conditions, we conducted, for each pair of conditions, mixed linear regression analyses predicting log ratio based on condition. In these analyses, we included random intercepts and slopes for participants and items  had to select the image showing an ironed shirt.40 native English speakers were recruited from University College London via an online pool of participants. All participants had normal or corrected to normal vision and were paid four pounds for their participation.The materials were the same as in Exp. 1.Participants were instructed to identify the image that corresponded to the answer in the question–answer pair. To this end, the four images were geographically mapped to the keys ‘e’, ‘c’, ‘i’, and ‘m’ on the keyboard. These buttons were marked with coloured stickers. Participants were asked to place four fingers on these keys and not look down during the experiment. This did not pose any difficulties to the participants. Participants were familiarised with the procedure by means of four practice trials that included feedback on their performance. Other than this, the procedure was the same as for Exp. 1.All participants achieved an accuracy of over 85% and data from all participants were included. Filtering of eye-tracking data followed the same procedure as Exp. 1.First, we briefly consider the behavioural data to validate the experimental task. Overall, accuracy was close to the ceiling at 94.4% correct responses. Participants were most accurate for positive questions (96.9% correct for both ‘yes’ and ‘no’ responses) and least accurate for low-neg questions (90.3% correct for ‘yes’ responses and 93.4% for ‘no’ responses). Accuracy for high-neg questions fell in between (95.8% correct for ‘yes’ responses and 93.4% correct for ‘no’ responses).Response times, measured from onset of the answer particle, were fastest for positive questions (1282 ms for ‘yes’ answers and 1230 ms for ‘no’ answers). Response times for high-neg questions were substantially slower (1412 ms for ‘yes’ answers and 1514 ms for ‘no’ answers). Response times for low-neg questions were the slowest . All other comparisons were not significant.In order to determine if the log ratio differed across conditions, we conducted, for each pair of conditions, mixed linear regression analyses predicting log ratio based on condition, with random intercepts and slopes for participants and items. Again, we stepwise dropped the random slopes if the models failed to converge. The alpha value was corrected for multiple comparisons and set at 0.016. This analysis indicated that the log ratio in the positive condition is numerically but not significantly higher than the low-neg condition during the noun region and during the first half of the gap  bias for the p image in the positive and high-neg conditions.The first two experiments tested the interpretation of polar questions in British-English, even though the conclusions that we would like to draw are much wider in scope. To address this, we replicated Exp. 2 in a different language: Belgian-French. Of course, even if we confirm the findings of Exps. 1 and 2 in one different language, this would not guarantee that the pattern of results is universal. It would, however, increase the experimental base on which to build more wide-ranging conclusions.There are a couple of differences between polar questions in English and French that warrant discussion up front. First, French has three constructions that can be used to ask questions. To illustrate, the question ‘Do you dance? can be translated into French as:In order to facilitate the comparison with English, and because it is the most natural form with which to ask negative questions, we tested questions that are marked by means of subject-verb inversion, as in (5b).Second, French has no distinction between high-neg and low-neg questions . Hence, we only tested two conditions: positive polar questions  and negative polar questions .Third, the response particle system in French is less ambiguous than in English. In particular, when responding to a negative question, ‘Yes’ and ‘No’ in English are variously used to confirm or reject the corresponding question. In our experiment, however, we always used ‘Yes’ to confirm and ‘No’ to reject questions, and the intended meaning was disambiguated in the continuation . In French, by contrast, ‘Si’ unambiguously confirms and ‘Non’ unambiguously rejects negative questions , we instead placed a temporal adverb between the verb and the object. Participants thus listened to sequences such as:t-tests indicated that there were no significant differences in word duration across conditions .The average duration of the individual word regions that were identical across the three question forms  never differed by more than 30 ms. Paired The procedure was the same as in Exp. 2.First, we briefly consider the behavioural data to validate the experimental task. Accuracy was close to ceiling at 96.3% correct responses. Participants were more accurate for negative questions (98.4% correct for ‘yes’ responses and 95.0% for ‘no’ responses) than for positive questions (98.2% correct for ‘yes’ responses and 93.5% for ‘no’ responses). Recall that, for English, we observed the lowest accuracy for ‘yes’ responses to negative questions; for French, accuracy was the highest in this condition. This difference is likely caused by the presence of French ‘si’ as an unambiguous way of referring to a positive state of affairs, whereas in English ‘yes’ is ambiguous between referring to a positive or negative state of affairs.Response times were also numerically faster for negative questions (1425 ms for ‘yes’ answers and 1582 ms for ‘no’ answers) compared to positive ones (1418 ms for ‘yes’ answers and 1603 ms for ‘no’ answers). However, the difference between positive and negative questions was extremely small. Again, these observations accord with our prior expectations. Hence, we now turn to the eye-tracking data.Figure To test whether the biases were statistically significant, we calculated the natural log ratio of the probability of looks to the p image over looks to the ~p image. We conducted mixed linear regression analyses to determine if the log ratios differed reliably from 0. In these analyses, we included random intercepts and slopes for participants and items. If this analysis did not converge, we stepwise dropped random slopes for items and participants. The results of these analyses are shown in Table Again, participants initially paid attention to both p and ~p images in both positive and negative conditions. In the positive condition, there was a significant bias towards the p image in the noun region and during the second half of the gap between question and answer. In the negative condition, there was a marginally significant positive bias in the verb region and during the first half of the gap between question and answer.t(79) = 2.3, p = 0.03, model = intercepts only).In order to determine if the log ratio differed across conditions, we conducted mixed linear regression analyses predicting log ratio based on condition (positive or negative), with random intercepts and slopes for participants and items. Again, we stepwise dropped the random slopes if the models failed to converge. The alpha value was corrected for multiple comparisons and set at 0.016. This analysis indicated that, in the noun region, the log ratio in the positive condition was numerically but not significantly higher than in the negative condition  and an image of a crumpled shirt (the ~p image).In Exp. 1, participants merely listened to these dialogues while watching visual scenes, occasionally answering comprehension questions to determine whether they were paying attention. In Exps. 2 and 3, participants were instructed to indicate which image corresponded to the answer in the question–answer pair. Exps. 1 and 2 tested polar questions in English ; Exp. 3 tested polar questions in French (positive and negative).The results of the three experiments were largely concurrent. In all conditions, participants paid increased attention to both p and ~p starting soon after hearing the verb, suggesting that participants consider both alternative states of affairs when processing a polar question. In this respect, polar questions differ from positive assertions, as Tian et al.  found thIn later regions, positive questions were associated with a bias towards the p image from the noun region  onwards. A smaller and less persistent bias towards the p image was observed for high-neg questions in English and for negative questions in French. Low-neg questions did not have a clear bias pattern. Although Exp. 1 observed a marginally significant bias towards the p image during the noun region, this effect was not replicated in Exp. 2. A negative bias  was never observed.Assuming that semantic representations straightforwardly map onto cognitive representations . A negative question in French can have the function of a high-neg or a low-neg English question, depending on the context and the question prosody. Given such states of enquiry, we might expect to observe a negative bias, but that was never observed. Why? One possibility is that some low-neg questions, especially in British English, can carry an epistemic bias for p. For example, ‘Is there not some call on judges to be just a little more respectful perhaps when they’re dealing with cases like this?’ . This question implies that the speaker expects p. The other possibility is that the optional positive epistemic bias counter-balanced the negative evidential bias Sudo, ; just liOverall, we argue that gaze biases in the late regions represent pragmatic processing: we represent the questioner’s ‘state of enquiry’. This conclusion rests on the assumption that semantic processing occurs relatively early, whereas the preferences that we observed often emerged more than a second after the question type was disambiguated. We should acknowledge, however, that, while there is evidence in favour of mapping early and late preferences to semantic and pragmatic processing (e.g., Bott & Noveck, This paper investigated the online processing of polar questions, and the properties that may influence the mental representation of polar questions. Through three visual world eye-tracking experiments, we found that in both English and French, the mental representations of polar questions involve representing both p and ~p, which is different from what Tian et al.  observed


This could be attributing to the lubrication effect of the water molecules, which sped up the migration of charged groups in the polyelectrolytes. Moreover, molecular dynamics simulations confirmed that a microphase separation occurred when the fraction reached an extreme value owing to the dominated interaction between PSS and PAH. These results provide support for the structural stability of this composite material and its applications in devices.Graphene/poly-(sodium-4-styrene sulfonate)(PSS)/poly- (PAH) composite is a frequently adopted system for fabricating polyelectrolyte multilayer films. Swelling is the bottleneck limiting its applications, and its effects on the conductivity is still controversial. Herein, we report successful swelling of a graphene/PSS/PAH composite in a vapor atmosphere, and the relation with the mass fraction of water is uncovered. The composite was prepared via a layer-by-layer assembly technique and systematically characterized. The results indicated that the average thickness for each bilayer was about 0.95 nm. The hardness and modulus were 2.5 ± 0.2 and 68 ± 5 GPa, respectively, and both were independent of thickness. The sheet resistance decreased slightly with the prolongation of immersion time, but was distinct from that of the water mass fraction. It reduced from 2.44 × 10 This because the swelling of poly/poly-(sodium-4-styrene sulfonate) (PAH/PSS) multilayers may open cation-exchange sites that preferentially bind K+ to enable highly selective transport [3− molecules exhibit interesting optoelectronic properties, making graphene-based materials potentially useful in a variety of optoelectronic device applications [Polyelectrolyte multilayer (PEM) films have been extensively explored in the last decade due to their high chemical and physical stability and their high permeability and selectivity. They have shown great promise in applications such as fabricating ultrathin conducting layers, permeation-selective membranes, protective coatings, sensors, and materials for nonlinear optics . For exa4 was about −10 kJ mol−1 for PAH/PSS, indicating the relatively strong association between the polymer segments [−1 [The PAH/PSS was the first system where the stratified nature of linearly growing multilayers was established, and now, it is a widely used polyelectrolyte combination for the buildup of PEM films . PSS andsegments . Varyingents [−1 . Furtherents [−1 ,10 and hents [−1 ,12.3Na and graphene multilayer films formed via π–π interactions not only dispersed well in water owing to the ionic side chains of -SO3Na, but also prevented the aggregation of graphene by electrostatic repulsion [Despite extensive investigations that revealed the factors affecting the building of PEM films, relatively little is known about the humidity that exists within these applications and the methods that control their swelling. For PAH/PSS assembly films, swelling in water was deteriorative for the stability of the nanostructures. Graphene oxide and its derivatives were composited to overcome this obstacle. For example, homogeneous and controllable -SOepulsion . In thisepulsion . The priw = 70,000), poly-(ethylenimine) , and poly-  were all purchased from Sigma-Aldrich . Concentrated sulfuric acid , sodium nitrate (NaNO3), hydrochloric acid (HCl), hydrazine monohydrate (N2H4·H2O), hydrogen peroxide (H2O2), potassium permanganate (KMnO4), and other chemicals were supplied by Sinopharm Chemical Reagent CO., Ltd. . All chemicals and reagents were used directly as received. Deionized water  was produced with a Milli-Q Plus system (Millipore) .Natural graphene flake (Grade-300), poly-(sodium-4-styrene sulfonate)  solution. The π-like stacking of benzene rings of PSS benefits the binding of PSS and graphene sheets, leading to a high fraction of individualized graphene after dispersion and reduction. Details: Graphene oxide was synthesized by Hummer’s method [2SO4/H2O2 (v/v: 3/1) at 80 °C for 1 h [Reduced graphene oxide composite was assembled on glass following the schematic shown in  for 1 h . General for 1 h . Hence, 2O mass fraction). After that, the sheet resistance was measured using a four-probe method.The traditional method, which involves immersing the assembled film in electrolyte solution for a certain amount of time, followed by washing and drying, leads, no doubt, to mass loss, especially for the polyelectrolytes in the film. Disruption of inter-polyelectrolyte binding also lies in the heart of salt-induced and pH-induced dissolution of multilayers. So as to avoid an erosion phenomenon in pure water, we swelled the composite film with water vapor at room temperature. The prepared composite films were immersed in water vapor for 24 h to complete the swelling, and then were dried in the air to reach a target mass  and FT-IR spectra (Fourier-Transform Infrared Spectrometer 3100) were assessed under a laser wavelength of 532 nm and an intensity of 1.5 mW. The wave number range was 4000–400 cm−1. A minimum of 16 scans signal-averaged with a resolution of 2 cm−1 was employed.The spectroscopic properties were investigated using the UV-Vis spectra  with a scanning range of 200–800 nm and a scanning speed of 300 nm·minThe morphologies were investigated with an atomic force microscope  on a Veeco Icon in the taping mode, and scanning electron microscopy  was performed on a HITACHI S4300 system at the accelerated voltage of 20 kV. Transmission electron microscopy measurements were performed on a Tecnai G2 F30 microscope  at 80 kV.The mechanical properties were tested by using nano-indentation experiments on a Nano Indenter XP system after a standard calibration. The Poisson ratio was set to 0.25, the surface-approach velocity was 10 nm/s, the strain rate target was 0.05/s, and the frequency target was 45 Hz. Keithley S4200  was used to measure the sheet resistances with a four-probe method under different levels of humidity at room temperature (30 °C). 3− groups showed the strongest inter-polyelectrolyte binding, resulting in inhibited chain exchange within PEMs. Prior to the assembly, PEI was first absorbed on the substrate to enhance the binding strength between the film and substrate. The growth of the multilayers was monitored using the UV-Vis spectrum. The UV-Vis curves are shown in −2 . Note that PAH and PSS had the same linear charge density; thus, the surface coverage was the same, and each adsorption step was accompanied by charge overcompensation [− ions dissolved from the multilayer, which was now positively charged. The surface charge density was again unusually low at 0.62 ± 0.22 mC m−2. Actually, the uniformity of the composite was key in obtaining excellent mechanical properties.Polycations with a high density of primary amino groups and polyanions with SOensation . During −1. The decrease in the peak intensity indicated that most of the –OH groups in graphene oxide were removed after the reduction by N2H4·H2O. The peaks of the C=O group were located between 1650 and 1750 cm−1 owing to asymmetric stretching. The peaks at 1540 and 1182 cm−1 were assigned to C=C bonds and C–O–C groups. The peak at 800 cm−1 was attributed to para-disubstituted benzene rings. The Raman peaks located at 1580 and 1380 cm−1 were G and D bands, respectively [3 to sp2.ectively ,20. The 2/Si substrate (A typical AFM test in the taping mode was performed, which indicated an average film thickness of 95 nm on a SiOubstrate a,b. The The mechanical properties, e.g., elastic modulus and hardness, were characterized by a nano−indentation method owing to its excellent performance on at the nano−scale. The variations in the hardness and modulus with the indentation depth are shown in 5 to 2.38 × 105 ohm/sq after immersion for 24 h. The sheet resistance decreased slightly with the prolongation of immersion time; however, the modified value was less than only 10% and was eventually saturated, which should not affect the fundamental properties. The influence of the H2O mass fraction, however, was different. It was reduced from 2.44 × 105 to 2.34 × 105 ohm/sq, and the rate of change increased with the increase in the H2O mass fraction, especially when the mass fraction was larger than 5%. This can be attributed to the lubrication effect of the water overflow in the film, which accelerated the migration of charged groups into the polyelectrolytes. However, when the mass fraction of H2O continued to increase, the interaction between PSS and PAH dominated and a microphase separation occurred, which led to restacking of the graphene sheets and, thus, a decrease in sheet resistance. Furthermore, graphene sheets could help to stabilize the polyelectrolyte films and greatly reduce the inner resistance by changing the transport of the electrons from a “point-to-point” mode to a more effective “plane-to-point’’ mode [The effect of the swelling of thin films on the sheet resistance is the key for further consideration. t’’ mode . 2O mass fraction, however, was different. It was reduced from 2.44 × 105 to 2.34 × 105 ohm/sq, and the rate of change increased with the increase in the H2O mass fraction, especially when the mass fraction was larger than 5%. This could be attributed to the lubrication effect of the water overflow in the film, which accelerated the migration of charged groups into the polyelectrolytes. When the mass fraction of H2O continued to increase, the interaction between PSS and PAH dominated and a microphase separation occurred. These results provide support for applications of the graphene/PSS/PAH thin films. In summary, we report the successful fabrication of a graphene/PSS/PAH composite for swelling studies. The composite was prepared via a layer-by-layer assembly technique and systematically characterized by UV-Vis, Raman, and FT-IR spectra, atomic force microscopy, a nano-indentation procedure, a four-probe method, and a theoretical calculation. An average film thickness of 0.95 nm was found for each bilayer. The average hardness and modulus were about 2.5 ± 0.2 and 68 ± 5 GPa, and both were independent of the thickness, which made the composite film easy to peel off and transfer to an arbitrary target. The sheet resistance decreased slightly with the prolongation of the immersion time. The influence of the H