Both graphene samples were functionalised using a pulsed laser deposited (PLD) thin V2O5 layer of average thickness ≈ 0.6 nm. According to the Raman spectra, the SiC/EG has a remarkable resistance against structural damage under the laser deposition conditions. By contrast, the PLD process readily induces defects in CVD graphene. Both sensors showed remarkable and selective sensing of NH3 gas in terms of response amplitude and speed, as well as recovery rate. SiC/EG showed a response that was an order of magnitude larger as compared to similarly functionalised CVDG sensor (295% vs. 31% for 100 ppm NH3). The adsorption site properties are assigned to deposited V2O5 nanophase, being similar for both sensors, rather than (defect) graphene itself. The substantially larger response of SiC/EG sensor is probably the result of the smaller initial free charge carrier doping in EG. Graphene in its pristine form has demonstrated a gas detection ability in an inert carrier gas. For practical use in ambient atmosphere, its sensor properties should be enhanced with functionalisation by defects and dopants, or by decoration with nanophases of metals or/and metal oxides. Excellent sensor behaviour was found for two types of single layer graphenes: grown by chemical vapour deposition (CVD) and transferred onto oxidized silicon (Si/SiO The extraordinary sensitivity of graphene conductance down to a single adsorbed molecule has been demonstrated in the inert gas atmosphere [Graphene, as an atomically thin (semi)conducting material, is a promising sensor platform for monitoring key environmental pollutants, such as NOmosphere . Graphenmosphere ,5,6 and mosphere ,8,9 in tth of a monolayer per laser pulse, is also worth mentioning as a benefit of PLD.For graphene functionalisation, pulsed laser deposition (PLD) is recommended as a highly versatile thin-film deposition technique with a relatively well-controlled synthesis process. It is possible to evaporate practically any solid with a focused laser pulse, thus providing the broadest possible selection of materials for various applications . By chan2 and Ag nanostructured coatings shows substantially improved sensing of NO2 [2 gas is nearly universal. Both CVD graphene transferred to insulating substrate [2 gas down to a few ppb in synthetic air, and thus applicable as NO2 environmental sensors.A single-layer, chemical vapour deposited (CVD) graphene, modified with PLD of ZrOg of NO2 . High seubstrate  and epitubstrate ,13 are c3 adsorber, widely used for catalytic removal of NOx from exhaust gases in the reactions with sacrificial NH3 [2O5, deposited using PLD, renders the CVD graphene a sensitive and selective ammonia gas sensor [Proper functionalisation is needed for selectivity towards less reactive polluting gases. Vanadium (V) oxide is known to be an efficient NHcial NH3 . Indeed,s sensor .3 sensing. Bearing in mind commercially applicable sensors, the properties of graphene, their substrates, and connecting electrode materials, among other features, should be considered. Miniature sensors can be made by transferring the CVD graphene sheet onto functional sensor platforms, equipped with a built-in microheater or embedded light source for better control of operating conditions. Epitaxial graphene on SiC could also be valuable due to a simpler manufacturing process. Moreover, SiC/EG has a higher sensitivity to chemical doping because the intrinsic carrier concentration is less [2O5 is carried out. Raman spectroscopy is applied to reveal the effect of PLD on defect creation in graphene. The influence of functionalisation on NH3 gas sensing properties is studied by comparing pristine and V2O5 deposited graphenes. Second, the gas response measurements are performed in the dark and under ultraviolet light exposure. Also, the dependence of sensor response characteristics on varying humidity level is investigated. Responses of both types of graphene sensors to other polluting gases NO2, CO, SO2 and O3 were tested for comparison in order to assess selectivity. Sensor response and recovery curves are approximated to double exponential functions. Tentative mechanisms of elementary processes are proposed based on phenomenological, biphasic kinetics.The present study will address further important aspects of NH is less . In thisA short version of this paper has appeared in conference proceedings .2 substrate carrying Au (60 nm) electrodes, deposited through a shadow mask via magnetron sputtering  graphene (CVDG) was grown on a commercial, 25-µm-thick polycrystalline copper foil in a home-built CVD reactor. The graphene film was transferred onto a Si/SiOuttering a. The grLarge area epitaxial graphene (EG) was grown using a sublimation method on Si-terminated 4H-SiC (0001) substrates at 2000 °C in argon gas at a pressure of 1 bar . Au  measurement.In the PLD process, which is specified in , a ceram2O5 decorated epitaxial graphene sample, the laser power was reduced to 1 mW in order to diminish the risk of surface damage. The Raman spectrum of graphene on SiC was obtained after subtracting a reference Raman spectrum of pure carrier material 4H-SiC (0001) from the graphene spectrum.The CVD graphene was probed using a micro-Raman spectroscopic system  at the excitation wavelength of 514 nm, with a spot diameter of ≈1 μm and incident radiant power of 1 mW. In case of epitaxial SiC graphene 17 mW of 532 nm laser light was focussed to a spot with diameter of ≈0.9 μm. For the VThe amount of vanadium in the deposited layer was assessed with X-ray fluorescence method, and the oxidation state of deposited vanadium was determined by X-ray photoelectron spectroscopy, as described earlier .3 stainless steel sample chamber, equipped with a gas mixing system, which is more thoroughly described in [2, O2, CO/N2, NO2/N2, SO2/N2, O2/N2 and NH3/N2, all 99.999% pure. All used gases and mixtures were of certified composition from AGA . A gas mixture of 10% O2 + 90% N2 was flown through a UV lamp ozoniser for O3 production, and the resulting O3 concentration was monitored using an ozone analyser . The gas flow through the sample chamber was kept constant at 200 sccm. The nominal relative humidity of the testing gas was regulated between 0 and 50% during the measurements. During the experiments with UV light excitation, the 365 nm light intensity on the sample was ≈15 mW/cm2.Gas sensitivity was measured at room temperature in a 7 cmribed in . The vol2O5. The features appearing darker in the SEM of CVD graphene (Scanning electron microscope (SEM) images are displayed in graphene a,b are tgraphene b and epigraphene d graphen2 contributed surprisingly little to scattering intensity, so that the graphene spectrum could easily be separated. −1 and ≈2690 cm−1, and have full-width at half-maximum (FWHM) of 11 cm−1 and 29 cm−1, respectively. These parameters, together with the 2D to G peak intensity ratio of approximately one to three, corresponded to a single layer of graphene [−1, is an indication of low defect density in the pristine CVD graphene sample [2O5 onto graphene, prominent defect-related D and D´ bands emerged in the Raman spectrum, and, at the same time, the G and 2D bands decreased in height  . As a cospectrum a. HoweveID/IG characterizes disorder induced by point defects [ID/IG and average distance between the point defects LD is non-monotonic and switches from a I(D)/I(G) ∝ LD2 type of dependence to a I(D)/I(G) ∝ 1/LD2 type at around LD ≈ 4 nm [LD < 4 nm. From the ratio ID/IG = 2.88, obtained from LD ≈ 2.3 nm, and the defect density nD of graphene as ≈6.1 × 1012 cm−2. Therefore, the initially pristine CVD graphene with very low defect density suffers damage during the PLD process, acquiring a high level of structural disorder. By contrast, according to Raman analysis, the PLD process did not noticeably increase the defectiveness of the epitaxial graphene sample. The conclusions about the influence of PLD reached on the basis of Raman spectra were supported by the electrical conductivity of samples. The conductivity of the CVD graphene sample, which had the Raman spectrum depicted in The Raman spectrum of graphene and graphene related systems was fairly sensitive to structural disorder. The ratio of the D and G peaks  defects ,29,30. H2O5 with an average thickness of about 0.6 nm.The amount and oxidation state of vanadium deposited with exactly the same PLD procedure was investigated in our previous work by means of X-ray fluorescence (XRF) and X-ray photoelectron spectroscopy (XPS) . In acco3 gas is illustrated for CVD and epitaxial graphene sensors in G: S = (G − G0)/G0, where G0 is the conductance in synthetic air.All sensor measurements in this work were done at room temperature (293–298 K), mainly under continuous excitation with ultraviolet (UV) light (λ = 365 nm), except for several experiments performed without UV exposure for comparison. The influence of graphene functionalisation on the response to NH3 into the test chamber. This was in accordance with the most prominent difference between Si/SiO2/CVDG and SiC/EG given their doping pattern in terms of majority charge carriers, p and n, respectively. In general, CVD graphene exhibits p-type conductivity and gas response at normal atmospheric conditions, primarily because of chemical doping by adsorbed oxygen and water molecules [3 acts as a hole acceptor (electron donor) with respect to pristine graphene [The conductivity of CVD graphene and epitaxial graphene sensors changes to the opposite direction after introducing NHolecules ,32, and olecules ,34. Beargraphene , the con3 and the subsequent recovery curves can be suitably fitted with double exponential functions that give much better approximation than single exponentials:G0 is the initial conductance and G(t) is the conductance at time t; and t0 is the initial moment of time, when the respective stepwise change in gas composition is introduced. The time-independent coefficients A1 and A2 are amplitudes of conductance change. Characteristic times t1 and t2 are inverse first-order rate constants for adsorption and desorption kinetics of gas molecules for pristine or V2O5 functionalised graphene [The electrical response dynamics of a 2D chemiresistor follows the kinetics of adsorption and chemical processes taking place on the surface. In general, the time dependent gas response to NHgraphene .3. For instance, H2O and O2 are known to readily adsorb on graphene surface and influence the electrical properties of graphene [In principle, a single exponential response corresponds to a single type of adsorption site available for molecules in the surface of graphene sensor. In contrast, the double exponential response (Equation (1)) would describe the situation where two sites with different adsorption and desorption rate constants exist . In addigraphene ,32.3 gas injection. This was because the signal relaxation was not exponential but tended to be double-exponential, whereas one of the exponents had quite a large time constant. The reasons behind this slow recovery can be several and depend on the exact mechanism behind the sensor response: high desorption energy barrier of NH3 molecule, or slow re-oxidation of V2O4 to V2O5, or some slow charge balancing processes between graphene and substrate etc.Baseline of the sensor signal  is extremely stable. However, after exposing the sensor to some analyte gas, signal recovery to the baseline can take quite a long time, even when UV light excitation is used. This can be seen in A slow response and recovery of the graphene-based gas senor signal is quite common in the literature ,3,4,8. I3 was considerably improved after functionalisation using PLD. The relative response amplitudes to 8 ppm NH3, under UV light excitation, increased from 5 to 22%, and from 50 to 160% for CVDG and EG samples, respectively. Further, much faster characteristic response times were measured on both types of sensors , whilst it changed to the n-type response under the UV light. Previously, a switch from an n- to p-type response has been observed for epitaxial graphene gas sensors under increasing concentrations of NO2 [2, which results in a lower concentration of free electrons, making the holes a major charge carrier. Besides water vapour and O2, which act as p-type dopants when adsorbing onto graphene in the ambient air [2 and other molecules, which can chemically p-dope graphene and cause n- to p-type switching of pristine graphene, as well as the EG/SiC/V2O5 system. As discussed before, the EG functionalisation with V2O5 did not influence the conductivity and defectiveness of epitaxial graphene samples, and is, therefore, probably not accompanied by a major change in electronic properties (mobility and concentration of charge carriers) of epitaxial graphene. Under the UV light exposure, the photo-activated desorption of molecules responsible for p-doping results in epitaxial graphene exhibiting n-type conductivity.In case of the EG sensor, the effect of UV light was particularly drastic, as the response to NHs of NO2 . The effient air ,32, therient air ,42. Ther3 gas sensing properties, the measurements were carried out under variable relative humidity (RH%) levels of RH0, RH20 and RH50. The results are depicted in t2 at 8 ppm NH3 decreased by 2.1 and 2.6 times for CVDG and EG sensors, respectively. At the same time, the response amplitude increased from 16% to 25% for CVDG, and from 131% to 216% for EG sensor. This clearly implies an important role of H2O in the NH3 adsorption mechanism on the surface of sensing layer.In order to clarify the possible effect of varying humidity level on NH2O5 has been thoroughly investigated to improve the catalytic reduction of NO2 in the presence of NH3, an important technology for detoxification of internal combustion engines exhausts. Two strongly bound species are typically observed as a result of the reaction of NH3 with V2O5 adsorption sites: one with the surface OH group (Brønsted acid site), forming a positively charged NH4+, and the other with the oxygen vacancy (Lewis acid site), forming a species, which is denoted as “coordinated NH3” [3 takes place at both sites, adsorption at the Brønsted site is probably energetically more favourable than adsorption at Lewis site. According to Lin et al. [2O5 is directly related to the presence of water vapor. As a consequence of increasing H2O concentration in the system, a number of Lewis adsorption sites are converted to Brønsted sites (surface OH groups) by H2O adsorption on Lewis sites [2O5.The adsorption of ammonia on Vted NH3” . Althougted NH3”  indicaten et al. , the numis sites . Therefo2O5 is plotted against NH3 concentration between 0.1 and 100 ppm. The response to NH3 gas in this concentration range was between 0.14 and 0.43 for the CVDG sensor and between 0.80 and 3.63 for the EG sensor. The dependence of responses on NH3 concentration followed a Langmuir-type ratiometric function. However, it turned out that stationary responses could not be fitted with a single-site Langmuir adsorption model, but closely followed a two-site Langmuir model [b1 and b2 are the affinity constants, and α1 and α2 are transduction coefficients, according to the simplified transducer function model for CVD graphene oxygen sensor in [1 = 0.26 (±0.023), α2 = 0.19 (±0.024), b1 = 9.8 (±3.0) 1/ppm, b2 = 0.071 (±0.037) 1/ppm; and the following parameters for the EG sensor: α1 = 1.5 (±0.15), α2 = 2.6 (±0.21), b1 = 9.9 (±3.6) 1/ppm, b2 = 0.041 (±0.013) 1/ppm.In ir model :(2)Sx=Gensor in . Of courensor in . The Lan(a)1 and α2 were both much smaller in case of the CVD graphene sensor, which means either a smaller influence of gas adsorption on the conductivity, or a lower concentration of total active adsorption sites.The transduction coefficients α(b)b1 and b2 were very close for both types of graphene sensors, which mean that rate constants for adsorption and desorption were similar for both types of functionalised graphene.The values of corresponding affinity constants Here, two important aspects are to be noted:1/α2 amounted to 1.4 and 0.58 for CVDG and EG sensors, respectively, which means that the proportion of high affinity sites was considerably higher in the case of the CVD graphene or that their influence on the sensor’s conductivity was proportionally lower in the case of the EG sensor.The point (a) is discussed further below. The last deduction is also clearly evident from data in 2 gas is generally much higher than that to other gases from the list of important gaseous pollutants. Strong binding and large charge transfer undoubtedly accompany NO2 molecule adsorption on either pristine, highly defective, or doped graphene [3 molecule can also interact strongly with defects or dopants in graphene, and this is confirmed to a certain extent by experimental evidence [In sensors built on a single layer graphene and other graphene-related materials, the sensitivity to strongly oxidizing NOgraphene . Still, 2O5 was considerably more selective to NH3 gas, rather than to NO2. PLD functionalisation of CVD graphene with Ag or ZrO2, in the contrary, resulted in a higher selective response towards NO2 gas [3 gas increased considerably through functionalisation, and at the same time, the response to NO2 gas diminished, as in the case of EG, or increased only slightly, as in the case of CVDG. The responses of our CVD and epitaxial graphene sensor devices to several polluting gases are compared in  NO2 gas . The sam2O5 also enhanced the response to CO and SO2 gases for both types of sensors. Reactions to O3 and NO2 gases were quite similar, which was expected, as both molecules are strongly oxidizing, with electron affinity (EA) of 2.1028 eV and 2.273 eV [The functionalisation with V2.273 eV , respect2O5 layer on graphene samples, either synthesized using chemical vapour deposition (CVD graphene) and transferred to Si/SiO2, or epitaxial graphene on a SiC substrate. Raman analysis showed that the defectiveness of CVD graphene increased drastically following PLD, which is an expected result of bombardment with a high energy PLD plasma species. By contrast, the defect concentration of epitaxial graphene is not affected by an identical PLD process, which implies considerable resilience against defect creation. This finding is in accordance with the fact that the electrical conductivity of the SiC/EG sample did not change after the PLD of V2O5, while the conductivity of CVD graphene was substantially reduced. Pulsed laser deposition (PLD) was used to create a sub-nanometer V2 substrate subject to PLD plasma.As a possible explanation, we refer to self-healing phenomenon, i.e., reknitting of holes in the graphene during annealing at elevated temperatures , or evenSince the self-healing does not occur at 295 K, the negligible deterioration of structure and conductivity in the epitaxial graphene could rather mean considerably higher energy threshold for defect creation. The substrate supporting graphene can play a major role under a flux of highly energetic particles (≈1 MeV), in which case the majority of defects found in graphene are created using the backscattered ions or sputtered atoms from the substrate . For imp2 graphene and the sp3 bulk material in SiC/EG is covalently bound to SiC substrate and serves as a template during epitaxial growth of graphene on the Si face of SiC [2 surface can be quasi-suspended, with the van der Waals contact area probably much less than 100%. In other words, the potential for C atoms in epi-graphene has a much deeper minimum. Thus, in qualitative terms, SiC/EG is more durable under the bombardment with high velocity particles, owing to the stronger bond of the graphene sheet with the underling substrate.Bearing in mind a much tighter graphene-to-support contact, resilience to plasma treatment of SiC/EG can be understood. The transition layer between the spe of SiC . Thus, e3 gas was achieved for both types of graphene sensors as a result of laser-deposited, thin V2O5 functionalising layer. The magnitude of the relative response of SiC/EG/V2O5 sensor was particularly noteworthy, for instance, amounting to 310% for 40 ppm NH3 at 50% relative humidity. The stationary gas response to the lowest NH3 concentration that could be tested, 0.1 ppm, was 14% and 80% at 20% relative humidity for CVDG and SiC/EG, respectively. A large enhancement of response to the reducing NHPerfect graphene is relatively inert with respect to chemisorption due to the negligible amount of dangling bonds and a lack of charged atoms on the surface ,3. Prist3 concentration is well approximated using a two-site Langmuir model, which yielded two affinity constants b1 and b2, with a difference by two orders of magnitude in both graphenes. However, the corresponding affinities were similar in both instances, showing that CVDG/V2O5 and SiC/EG/V2O5 sensors have sites with similar adsorption energies, i.e., the adsorption sites belonged to the functionalising V2O5 layer, and did not depend on the type of the graphene. The comparable NH3 adsorption affinities of both types of graphene would produce similar responses and recovery times of sensors . Amoussed in . Broadly3 gas than the corresponding CVD graphene sensors. This was reflected in the transduction coefficients α1 and α2 in the Langmuir model (Equation (2)), which were both much smaller in the case of CVD graphene. The major part of the more sensitive nature of EG was most probably related to the particular feature of gas sensing mechanism of the graphene. It was found by Schedin et al. [2 substrate, e.g., the gas adsorption solely modified the concentration of free charge carriers in graphene without changing their mobility. If we assume the same for our sensors, then the change of carrier mobility, caused by the NH3 adsorption was smaller than the effect of carrier concentration, and the gas response amplitude S is determined by a relative change of charge carrier numbers Δn/n0 [n0 is the carrier concentration in the absence of adsorbate gas, and the constant As can be observed form the measurement results in rs Δn/n0 :(3)S=G−n0 of the graphene would correspond to a stronger gas response, and vice versa. In sensors used in this work, the conductivity of pristine CVD graphene was about 15 times higher than that for epitaxial graphene. Given that typical carrier mobility is not very different for pristine samples of CVDG and EG, 1000–3000 and ≈1000 cm2·V−1·s−1, respectively, the charge carrier density in CVDG should be about one order of magnitude higher than that in EG. This conclusion is well supported by the literature data on the typical electronic doping levels of SiC/EG and Si/SiO2/CVDG.The amount of charge transferred per unit change of coverage  was similar for both types of graphene sensors. It follows that the lower initial concentration of carriers 2 are typically highly doped, owing to interactions with the underlying substrate or surface adsorbates, i.e., water, oxygen and other adsorbate molecules [12 cm−2 in the case of SiC/EG [13 cm−2 in the case of Si/SiO2/CVDG [3 (by an order of magnitude) probably arose as a result of the lower initial doping level of SiC/EG. In other words, the Fermi energy level being closer to the Dirac point allowed for higher sensitivity to chemical doping, induced by adsorbing NH3 molecules. The proximity of the Fermi level to a Dirac point in SiC/EG was supported by the fact that under UV light, which is capable of removing the major atmospheric dopants, the response of EG-based sensors switched from p- to n-type.Both epitaxial graphene grown on Si-terminated 4H-SiC (0001) and CVD graphene transferred to Si/SiOolecules ,42. Roomf SiC/EG ,52,53 an2O5 enhanced the response and improved the selectivity of chemiresistive sensors with respect to ammonia. The analysis of sensor kinetics implied the presence of similar adsorption sites in both kinds on functionalised graphenes, indicating that the deposited V2O5 nanophase was a predominating NH3 receptor. The difference in the magnitudes of relative gas response was a result of different charge carrier type and concentration of graphene samples. Epitaxial graphene on SiC was remarkably resistant to PLD plasma. The signals of sensors on SiC/EG platform were larger, faster and more reversible than those for CVD graphene. However, the transferability of CVD graphene onto almost any surface could be of great value.The functionalisation of CVD-grown and epitaxial graphene with VGraphene is almost ideally suited as a signal transducer material in conductive sensors. Since pristine graphene is a relatively weak and non-specific adsorbent, the progress will essentially depend on the quality of receptor function created by means of functionalisation. Only time will tell whether graphene is superior to other categories of thin conductors, such as nanoSMOX  and TMD  ,55,56.


We demonstrate that the threaded macrocycles in interlocked porphyrin–corrole conjugates provide a mechanical “picket fence” without affecting their electronic properties. Building on recent progress in the synthesis of functional porphyrins for a range of applications using the Cu-mediated azide–alkyne cycloaddition (CuAAC) reaction, we describe the active template CuAAC synthesis of interlocked triazole functionalised porphyrinoids in excellent yield. By synthesising interlocked analogues of previously studied porphyrin–corrole conjugates, we demonstrate that this approach gives access to rotaxanes in which the detailed electronic properties of the axle component are unchanged but whose steric properties are transformed by the mechanical “picket fence” provided by the threaded rings. Our results suggest that interlocked functionalised porphyrins, readily available using the AT-CuAAC approach, are sterically hindered scaffolds for the development of new catalysts and materials. The copper-mediated alkyne–azide cycloaddition (CuAAC)The CuAAC reaction has also been widely applied in the synthesis of interlocked molecules,The active template (AT) approach to interlocked molecules,Here we demonstrate the utility of the AT-CuAAC reaction in the synthesis of mechanically interlocked analogues of previously studied triazole-linked porphyrin–corrole conjugates,4⊂3 was synthesised in excellent yield (98% after size exclusion chromatography) by reaction of azide 1, alkyne 2 and macrocycle 3 in the presence of a CuI salt .Porphyrin–corrole diad CuI salt . The ext4⊂3 shows a molecular ion at m/z = 1123.5 consistent with [M + H]2+. Comparison of the 1H NMR spectra , which is in keeping with their location away from the threaded region of the axle, triazole proton Hk is shifted considerably to lower field (Δδ ∼ 1.8 ppm). This is consistent with previous observations of C–H·N hydrogen bonding between the polarised triazole-Hk and the Lewis basic pyridine nitrogen donorsj appear at higher field in the interlocked structure (Δδ ∼ 1.2 ppm) due to the close proximity of the induced magnetic field of the electron rich aromatic units of the macrocycle.The mass spectrum (MS) of [2]rotaxane dyad  spectra  of [2]roA, HB, HC, shielding of protons HF, and HG of the flanking aromatic units and the splitting of HD and HE into diastereotopic pairs due to the non-centrosymmetric axle desymmetrising the faces of the macrocycle on mechanical bond formation. Analysis of triad [3]rotaxane 5⊂23 and pentad [5]rotaxane 6⊂43 by MS also confirmed the presence of the corresponding molecular ions . Their 1H NMR spectra .21Femtosecond transient absorption spectroscopy of These results clearly demonstrate that, as proposed, threading of the macrocycles around the arms of the porphyrin core does not significantly affect the electronic properties of the system.4⊂3, 5⊂23 and 6⊂43 clearly have very different steric properties; encircling the triazole moieties with macrocycle 3 significantly increases the steric demand of the linker units. This difference is particularly striking in the case of pentad 6⊂43 which lacks sterically bulky aryl groups on the central porphyrin unit; the space filling model of 6⊂43 (II-porphyrin unit is significantly encumbered by the threaded macrocycles. This steric hindrance was quantified by determining the % buried volume (% Vbur) of spheres centered on the central ZnII ion (r < 3 Å), the values of % Vbur for 6 and 6⊂43 are identical suggesting that the ZnII center is still accessible to small molecules, as required for catalysis or ligand binding. However, as r increases, the values diverge as the threaded macrocycles lead to a higher excluded volume. The comparison between the variation in % Vbur of 6⊂43 and 6 suggests that although the interlocked macrocycles do not affect the accessible volume immediately around the ZnII center, they provide a steric wall at higher radii similar to covalent picket fence porphyrinoids that have been developed for catalytic and photochemical applications.Although the electronic properties of the interlocked products are unchanged compared with the axle moiety, rotaxanes  of 6⊂34  shows thZnII ion .24 At lo6⊂43 compared with 6 can be found in their 1H NMR spectra; non-interlocked axle 6 displays resonances that are broadened considerably compared with that of [5]rotaxane 6⊂43 under the same experimental conditions  to direct the formation of [(ZnII-porphyrin)2L] dimers.1H NMR spectra  at L : 6 ratios up to 0.5 : 1 a signal was observed at –4.92 ppm in the 1H NMR spectrum, along with a new signal corresponding to Hn′ which is consistent with the formation of a [62L] dimer in slow exchange on the NMR timescale; (ii) once the ratio of L : 6 exceeded 0.5 : 1 the signal at –4.92 ppm disappeared and Hn′ moved to progressively lower field as further ligand was added, stabilising once L : 6 = 1 : 1, consistent with fast ligand exchange once excess L is present and the formation of monomeric complex [6L] in competition with [62L]. This was further confirmed by cooling the equimolar solution of 6 and L; at 273 K a broad signal was observed at –2.96 ppm alongside the reappearance of the signal at –4.92 ppm, consistent with the monomeric species [6L] in equilibrium with [62L] at low temperature. Thus, the speciation of 6 2L] but instead the resonances corresponding to triazole proton Hk and porphyrin β-protons Hn underwent monotonic changes that continued until 1 equivalent of L had been added. Alongside these changes, a new broad signal appeared at –2.94 ppm (L had been added at which point it disappeared. These changes are consistent with the formation of [(6⊂43)L] that undergoes slow exchange with free porphyrin 6⊂43 on the 1H NMR timescale, progressing to fast exchange once excess L is present. Consistent with this, cooling an equimolar mixture of 6⊂43 and L to 223 K (6⊂43)L]. Thus, the speciation diagram of 6⊂43 with respect to equivalents of L L]. However, surprisingly, a small signal was also observed at –4.62 ppm. Reducing the temperature further to 248 K led to a number of significant changes consistent with the formation of [(6⊂43)2L] alongside [(6⊂43)L] and unbound 6⊂43, including the appearance of resonances at –4.62 and 8.6 ppm attributed respectively to L and the porphyrin β protons Hn of the dimeric complex. Reducing the temperature further to 223 K (6⊂43)2L] at the expense of [(6⊂43)L]. Thus, as the temperature is decreased, the mixture of [(6⊂43)L] and 6⊂43 present at 298 K is converted to a mixture of [(6⊂43)2L], [(6⊂43)L] and 6⊂43 until an approximately equimolar mixture of [(6⊂43)2L] and [(6⊂43)L] is produced at 223 K 2L] complex forms at all is remarkable given the sterically hindered environment provided by the macrocycles. The effect of temperature suggests that the balance between negative steric interactions and the positive binding interaction between the ZnII center and the N donors of the DABCO ligand are finely balanced. Ultimately, the entropic cost of forming the ternary complex, along with the restrictions to conformational freedom associated with forming such a crowded structure, appear finely balanced against the enthalpic benefit of maximising Zn–N interactions, leading to a strongly temperature dependent self-assembly process.That the  (Fig. S38L, 6 and 6⊂43 as the temperature is varied; 6 is selectively consumed in formation of [62L] in keeping with the higher stability constant for dimerisation of the non-interlocked axle and we did not observe any evidence of hetero-complex formation. Thus, the mechanical picket fence provided by the macrocycles leads to self-sorting in a mixture of ZnII-porphyrin hosts.Finally, we examined the speciation of mixtures of  Fig. S38. FurtherIn conclusion, we have demonstrated that the ease and utility of the CuAAC reaction, which has led to its widespread use in the design of functional porphyrinoids for various applications,Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.Supplementary informationClick here for additional data file.


Idiopathic pulmonary fibrosis is a rare, progressive and fatal lung disease which affects approximately 5 million persons worldwide. Although pirfenidone and/or nintedanib treatment improves patients’ wellbeing, the prognosis of IPF remains poor with 5-year mortality rates still ranging from 70 to 80%. The promise of the anti-cancer agent nintedanib in IPF, in combination with the recent notion that IPF shares several pathogenic pathways with cancer, raised hope that immune checkpoint inhibitors, the novel revolutionary anticancer agents, could also be the eagerly awaited ground-breaking and unconventional novel treatment modality limiting IPF-related morbidity/mortality. In the current review, we analyse the available literature on immune checkpoint proteins in IPF to explore whether immune checkpoint inhibition may be as promising in IPF as it is in cancer. We conclude that despite several promising papers showing that inhibiting specific immune checkpoint proteins limits pulmonary fibrosis, overall the data seem to argue against a general role of immune checkpoint inhibition in IPF and suggest that only PD-1/PD-L1 inhibition may be beneficial. Idiopathic pulmonary fibrosis (IPF) is the most frequent interstitial lung disease with a devastating prognosis for which there is currently no treatment that cures or reverses the disease. IPF is characterized by excessive accumulation of extracellular matrix in the interstitial and alveolar spaces leading to scarring and the destruction of the normal pulmonary epithelium with subsequent breathing difficulties and diminished oxygen uptake . The preThe identification of novel therapeutic targets critically depends on a better understanding of the pathogenesis of IPF and a plethora of studies have consequently been performed to identify pathogenic pathways that may hold promise as targets for therapy. As outlined below, it emerged from these studies that IPF has several pathogenic pathways in common with cancer based on which it has been proposed to treat IPF patients with drugs known to effectively limit cancer progression . The curAccording to the current paradigm, IPF result from an aberrant wound healing response following repetitive epithelial injury. As cancer may be considered a wound that does not heal , it has Despite the ongoing debate whether IPF should be considered a cancer-like disease, several anticancer agents have already successfully been employed in the setting of IPF. The Food and Drug Administration approval of nintedanib, a tyrosine kinase inhibitor used in the management of NSCLC, is obviously the best example of the applicability of cancer drugs in IPF. However, several other drugs developed as anticancer agents, like GSK2126458  and UCN-01 , show great promise in pre-clinical models of IPF ,23. The Immunotherapy has emerged as a new treatment option for patients with cancer and checkpoint inhibitors are the most commonly used immunotherapy agents . These a+ and type 1 CD4+ helper cells) and subsequent secretion of inflammatory cytokines like interferon gamma (IFNy), PD-L1 is upregulated in peripheral tissues [PD-1 is in normal physiological conditions involved in regulating T cell function . Upon ac tissues ,28. Subs tissues . This pr+ T cells that are again able to engage in cytotoxic killing [In the tumour immune microenvironment (TIME), T cells need to be primed against tumour antigens . Two sig killing . Immune  killing . PD-L1 a killing . BlockinCTLA-4 is involved in attenuating T cell activity . Upon biIn cancer CTLA-4 is upregulated when T cells are activated in the TIME or in lymphoid organs . BlockinBlockade of CTLA-4 also has distinct effects on the differentiation and content of T cells in the TIME . It seemThere are several other checkpoints both on tumour cells, APCs and T cells which can either provide co-stimulatory or co-inhibitory signals to T cells . The folCheckpoint inhibitors are now frequently employed across different types of cancer including lung cancer, melanoma and bladder cancer ,49,50. IThe most commonly used marker to predict clinical benefit of checkpoint inhibitor therapy is PD-L1 expression, measured by immunohistochemistry . In sevePatients with a high tumour mutational burden, reflected by the amount of non-synonymous single nucleotide variants or microsatellite instable tumours, also have a higher chance of responding to checkpoint inhibitors . AlthougAnother promising biomarker is the microbiome. Indeed, the diversity and composition of the gut microbiota has been shown to modify checkpoint inhibitor efficacy in pre-clinical models ,58. MoreOverall, immune checkpoint inhibition truly seems to have revolutionized cancer treatment although its efficacy is highly variable among patients. Unfortunately, there is still no robust biomarker predicting immunotherapy efficacy in cancer and it is envisioned that single biomarkers will never accurately identify patients who will likely benefit from checkpoint therapy . InsteadConsidering the overlap in pathogenic mechanisms between IPF and cancer and the success of anticancer agents in the treatment of IPF, it is tempting to speculate that immune checkpoint inhibition may also hold promise in the setting of IPF. To prove or refute this hypothesis, we here review available literature on immune checkpoints in IPF. To this end, studies focusing on immune checkpoints in IPF were retrieved from PubMed using the key words listed in + T cells and in IPF lung tissue was increased in IPF patients compared to age-matched healthy controls [+ T cells lead to STAT3 upregulation and subsequent IL-17A and TGF-β expression. Indeed, ex vivo blocking the PD-1/PD-L1 axis resulted in a reduction of STAT3 mediated IL-17A and TGF-β production by CD4+ T cells. Interestingly, another study shows that PD-L1 expression on invasive fibroblasts also contributes to pulmonary fibrosis [+ T cells and fibroblasts (see The potential role of the PD-1/PD-L1 checkpoint in IPF has recently been established by showing that the PD-1/PD-L1 axis is induced in IPF patients. In a comprehensive characterization of morphologic and molecular features of pulmonary fibrosis associated cancers, PD-L1 expression was observed in 62% of the patients although the expression analysis in this study was mainly focused on tumour cells . In a picontrols . Irrespefibrosis . Geneticasts see . Overallasts see  instead Another checkpoint inhibitor that has been successfully targeted in NSCLC, especially in combination therapy, is CTLA-4 . Few stuThe potential role of TIM-3, a novel candidate immune checkpoint protein, on IPF progression was recently studied in the preclinical bleomycin model of pulmonary fibrosis . Anti-TIBased on increased expression levels of immune checkpoint inhibitors in IPF patients, these novel anticancer drugs may seem a promising treatment option to pursue in IPF and indeed several studies have hinted upon this approach. The experimental data however argue against a general role of immune checkpoint inhibition in IPF and suggest that only PD-1/PD-L1 inhibition could be beneficial. The underlying preclinical studies do however not address resolution of fibrosis and/or the treatment in established fibrosis and future clinical studies should therefore elucidate whether PD-1/PD-L1 inhibition also improves patient outcome. Such clinical studies should be carefully designed and controlled as anti-PD-1/PD-L1 therapy increases the risk for severe and potentially life-threatening adverse effects. Specifically, checkpoint inhibitors may induce auto-immune related adverse events like dermatitis, pneumonitis, hypothyroidism and colitis ,72. EspeAlthough current data do not provide a strong case for immune checkpoint inhibition in IPF at this particular moment, it should be realised that the number of publications focussing on immune checkpoint inhibition in IPF is rather limited and show conflicting results. Studies that aim to clarify the conflicting results, especially with respect to PD-1 and TIM-3, should shed light on the real future of immune checkpoint inhibition in IPF preventing that the baby is thrown out with the bathwater.In case checkpoint inhibitors will move forward in IPF, it will be important to take into account the heterogeneity of clinical responses observed in cancer patients. Of note, the efficacy of immune therapy is reduced in NSCLC patients with activating mutations in receptor tyrosine kinases . Indeed,In addition to mutations in receptor tyrosine kinases, the gut microbiome also determines the clinical efficacy of immune checkpoint inhibition in NSCLC patients. Indeed, based on the diversity and composition of the intestinal microbiota patients can be stratified in responders and non-responders to PD-1 based immunotherapy ,91. AlthOverall, it is well conceivable that responses to immune checkpoint inhibition will also highly vary in IPF patients and the identification of biomarkers predicting treatment response is of utmost importance. Although no data are currently available, several patient related characteristics may aid in identifying responders from non-responders. Most obvious, expression levels of checkpoint inhibitors on relevant cell types are likely candidate biomarkers. Alternatively, biomarkers predictive of response in cancer patients may be useful in IPF to select appropriate patients. + T cells and fibroblasts resulting in diminished fibrogenesis. The success of the anti-cancer agent nintedanib in IPF, in combination with the recent notion of shared pathogenic mechanisms between IPF and cancer, raised enthusiasm that alternative anti-cancer agents could also benefit IPF patients. Based on its revolutionary success in cancer therapy, several investigators addressed the role of immune checkpoints in IPF. The positive results of some landmark studies lead to several advertorials ,95 claim


The authors of “The Health Buddies App as a Novel Tool to Improve Adherence and Knowledge in Atrial Fibrillation Patients: A Pilot Study” :e98) require corrections in relationship to the use of the Morisky Medication Adherence Scale (MMAS-8).The following text was omitted from the Acknowledgements and should be added:Use of the MMAS is protected by US copyright laws. Permission for use is required. A license agreement is available from: Donald E Morisky, ScD, ScM, MSPH, Professor, Department of Community Health Sciences, UCLA School of Public Health, 650 Charles E Young Drive South, Los Angeles, CA 90095-1772.This statement under the section “Feasibility, Data Collection, and Outcome Measures” requires an advisory warning:A MMAS-8 score of 0-5 indicates a low adherence, 6-7 is medium adherence, and a score of 8 represents a highly adherent patient.The new text, which includes the warning, reads as follows:A MMAS-8 score of 0-5 indicates a low adherence, 6-7 is medium adherence, and a score of 8 represents a highly adherent patient. This MMAS-8 scoring and coding criteria is incorrect, and, if used, would invalidate the MMAS-8 results and potentially put patients at risk of harm.Two additional references should be cited after reference 17 in the following sentence:First, the self-reported 8-item Morisky medication adherence scale (MMAS-8) was used to get an idea about the adherence level from the viewpoint of the patient [17].Krousel-Wood M, Islam T, Webber LS, Re RN, Morisky DE, Muntner P. New medication adherence scale versus pharmacy fill rates in seniors with hypertension. Am J Manag Care 2009 Jan;15(1):59-66.Morisky DE, DiMatteo MR. Improving the measurement of self-reported medication nonadherence: response to authors. J Clin Epidemiol 2011 Mar;64(3):255-7; discussion 258.These will become references 18 and 19, respectively, and all subsequent references will be renumbered accordingly.The correction will appear in the online version of the paper on the JMIR website on November 30, 2018, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article also has been resubmitted to those repositories.Authors and journal were forced to publish this correction due to legal threats by Steven Trubow and Donald Morisky from the company MMAS Research LLC, the copyright holder of the instrument. This is unfortunately not an isolated case, as the developers of this scale are known to comb the literature and ask those who used the scale for research to pay for a retroactive license which may cost thousands or tens of thousands of dollars, and to add references to their work .The Committee on Publication Ethics (COPE) has recently discussed the ethics of this type of behavior by copyright holders of scales ("holding authors to ransom in this way") and recommends to emphasize "the fact that this is not good for the advancement of scientific knowledge or in the public interest" . As open


In this paper, a novel method to realize a dynamically tunable analogue of EIT for the resonance strength rather than the resonance frequency is proposed in the terahertz spectrum. The introduced method is composed of a metal EIT-like structure, in which a distinct EIT phenomenon resulting from the near field coupling between bright and dark mode resonators can be obtained, as well as an integrated monolayer graphene ribbon under the dark mode resonator that can continuously adjust the resonance strength of transparency peak by changing the Fermi level of the graphene. Comparing structures that need to be modulated individually for each unit cell of the metamaterials, the proposed modulation mechanism was convenient for achieving synchronous operations for all unit cells. This work demonstrates a new platform of modulating the EIT analogue and paves the way to design terahertz functional devices which meet the needs of optical networks and terahertz communications. EIT is a concept describing a sharp transmission window generated in a broad absorption profile, and was first discovered in the process of atomic system experiments ,2,3. DueMore recently, graphene, composed of carbon atoms and sp2 hybrid orbital hexagonal honeycomb crystal lattice, have attracted a lot of attention due to their high MRS on the order of picoseconds, adjustable conductivity and other excellent optical, electrical and magnetic properties compared to conventional noble metal materials ,13. In vIn this paper, an innovative method to realize dynamically tunable EIT for the resonance strength rather than the resonance frequency of the transmission windows is proposed at the terahertz spectrum. Apart from the demonstration of the EIT analogue phenomenon by an all-new structure, in which the dark and bright mode resonator are represented by a metal split ring and a cut wire respectively, we integrated monolayer graphene ribbons under the dark mode resonator and then realized the complete modulation for the resonant intensity of the EIT-like transmission window by manipulating the Fermi level of the graphene. It is notable that in the manipulation process there is no need to change the structure parameters and the position of resonant frequency remains unchangeable. Comparing previous structures with discrete modulations for each unit cell, the proposed modulation mechanism is convenient and fast for it can achieve synchronous operations for all unit cells of the metamaterials, which makes the design more accessible for practical applications. In addition, the corresponding group index changed during the modulation process, which proves that the method can obtain a better controllable slow light effect. This work contributes a strategy to increase the diversity and functionality of controllable EIT analogue effects and opens a new platform of modulating the EIT analogue with higher modulation speed and easier fabrication tech for designing novel terahertz functional devices.The thickness of Al and substrate are 200 nm and 400 nm, respectively. The optical properties of metallic Al can be expressed using the Drude model [p is the plasma frequency, and the values are 1.22 × 1014 rad/s and 2.24 × 1016 rad/s respectively.In the formula, γ is the damping coefficient and ωB is the Boltzmann constant, T is the temperature of the environment (T = 300 K), ħ is the simplified Planck constant, and ω is the incident plane wave angle frequency. In particular, the Fermi level of graphene is represented by EF in the formula, and the average relaxation time is represented by Γ.For the graphene material to be added, its conductivity is a major factor affecting its properties. According to the Kubo equation , the conF ≫ KBT and EF ≫ ћω is satisfied to obtain a simplified Drude formula [In the terahertz band, the in-band partial conductivity of graphene is negligible for the electrical conductivity of the total graphene. When graphene is highly doped, condition E formula .(3)σg=(F)/ [The carrier relaxation time define as Γ = (μEm2/(V·s) .Then, as shown in These simulation experiments used FDTD Solution. Precious metal structures at sub-wavelength scale can excite local surface plasmons (LSP) under certain conditions. The electric field distribution of the bright mode structure shown by F in the graphene ribbon increased from 0.2 eV to 0.8 eV, the summit of the spectrum starts to disappear gradually. The transmission spectrum changes from a symmetrical Lorentzian line shape to an asymmetrical Fano line shape. Consequently, complete modulation of the EIT analogue is obtained.Subsequently, in order to conveniently obtain and feasibly control the transmission window of the EIT analogue, the controllable material graphene is added between the dark mode resonator and the substrate. In addition, the graphene ribbon can be connected to all subunit structures on the metamaterial to allow the device to be adjusted simultaneously. Therefore, as displayed in Since graphene is only added to the dark mode resonator, a dark mode resonator is needed to cause a change in the EIT-like transmission spectrum. Next, the coupled harmonic oscillator model will be combined, and then the electric field distribution of the structure during the adjustment process will be analyzed to analyze which properties of the dark mode resonator specifically affect by the graphene . The coub and γb, respectively, while the resonant amplitude and damping coefficient of the dark mode harmonic oscillator are χd and γd. The resonant frequency of bright mode resonator under incident plane wave illumination is denoted by ω0, and its value is 2π × 1.6 THz. δ is the detuning frequency between the two oscillators, and k is the coupling coefficient between the monopole mode and the dipole mode. By way of solving the Equations (4) and (5), the susceptibility χ can be gained by γb, γd, δ and k with the approximate formula ω−ω0 ≪ ω0 [The resonant amplitude and damping coefficient of the bright mode resonator are χ−ω0 ≪ ω0 ,27.(6)χThe loss of energy in the metamaterial device is mainly determined by the imaginary part of the susceptibility. The transmittance of a metamaterial device under plane wave illumination can be expressed by the following Equation (7).In the above formula, the intensity coefficient of the coupling between the incident plane wave and the mode harmonic oscillator is denoted by s. The red dashed line in b, δ and k hardly changed and γd gradually increased during the process of increasing EF. Among them, the values of k, γb and δ were maintained at around 150 rad/ps, 2.5 rad/ps and 0 rad/ps before and after the addition of graphene, respectively. The value of γd is gradually increased from 0.5 rad/ps at the beginning to 5 rad/ps at the end. γb remains unchanged, indicating that graphene does not affect the damping coefficient of the bright mode resonator, and cannot change the inherent energy loss of the bright mode resonator. The constant value of K means that there is no change in the coupling between the dipole mode and the monopole mode. The value of δ close to zero remains unchanged, which indicates that there is no detuning between the bright mode resonator and the dark mode resonator. The damping coefficient of the dark mode resonator has a large variation in the process of adjusting the graphene Fermi level, which is the main factor affecting the transmission spectrum of the metamaterial. When the graphene ribbon is placed under a dark mode resonator and the Fermi level of graphene is gradually increased, the loss of the dark mode resonator is also increased. When the damping coefficient of the dark mode resonator is too large, the dark mode resonator will not maintain the resonance state due to excessive loss. Therefore, when the Fermi level modulation of graphene is 0.8 eV, the analogue of EIT phenomenon on the transmission spectrum of the metamaterial substantially disappears.Combined with the electric field distribution diagram, the electric field distributions at the transmission windows of EIT analogue during the process of modulating the Fermi level of graphene is plotted. As shown in g is employed to represent the capability of slow light, which can be expressed as [In the transparency window of EIT analogue, there is strong dispersion, which means it can slow down the speed of light . The groessed as :(8)ng=nee of the metamaterial can be obtained [By extracting the S parameters, the effective refractive index nobtained . Figure In summary, we have proposed and demonstrated a method to realize a dynamically tunable EIT phenomenon for the resonance strength instead of the resonance frequency of the transmission windows. The transmission spectrum of the metamaterial shows a pronounced transmission peak at 1.6 THz, which is caused by the destructive interference resulting from near field coupling of two resonance modes. Therefore, the complete modulation of the EIT analogue at the specific frequency can be realized without affecting adjacent spectrum by changing the Fermi level of graphene. Furthermore, the coupled harmonic oscillator model is adopted to describe the near field coupling effect and analyze the physical mechanism by the fitting parameter. In the meanwhile, the control of the group index is also calculated for the applications of slowing light. This work offers a new perspective on the design of dynamically adjustable EIT-like devices. Moreover, by changing the size of the device, the design scheme can be easily scalable to other terahertz, infrared or visible regimes for various promising applications in optical networks and terahertz communications.


Background and objectives: In the last few years, graphene oxide has attracted much attention in biomedical applications due to its unique physico-chemical properties and can be used as a carrier for both hydrophilic and/or hydrophobic biomolecules. The purpose of this paper was to synthesize graphene oxide and to obtain multifunctional platforms based on graphene oxide as a nanocarrier loaded with few biologically active substances with anticancer, antimicrobial or anti-inflammatory properties such as gallic acid, caffeic acid, limonene and nutmeg and cembra pine essential oils. Materials and Methods: Graphene oxide was obtained according to the method developed by Hummers and further loaded with biologically active agents. The obtained platforms were characterized using FTIR, HPLC, TGA, SEM, TEM and Raman spectroscopy. Results: Gallic acid released 80% within 10 days but all the other biologically active agents did not release because their affinity for the graphene oxide support was higher than that of the phosphate buffer solution. SEM characterization showed the formation of nanosheets and a slight increase in the degree of agglomeration of the particles. The ratio I2D/IG for all samples was between 0.18 for GO-cembra pine and 0.27 for GO-limonene, indicating that the GO materials were in the form of multilayers. The individual GO sheets were found to have less than 20 µm, the thickness of GO was estimated to be ~4 nm and an interlayer spacing of about 2.12 Å. Raman spectroscopy indicated that the bioactive substances were adsorbed on the surface and no degradation occurred during loading. Conclusions: These findings encourage this research to further explore, both in vitro and in vivo, the biological activities of bioactive agents for their use in medicine. Carbon is an extraordinary element which can exist in different allotropic forms. It is found in large quantities in nature as coal or as natural graphite and in fewer quantities as diamond. The crystal structure of diamond has a cubic arrangement of the atoms compared to graphite which has a special kind of structure containing flat sheets of carbon atoms bonded into hexagonal structures. The later, has remarkable properties, with good thermal and electrical conductivity  and starCarbon nanomaterials contain many different allotropic forms of carbon, the most studied and used ones being carbon nanotubes and graphene. The remarkable properties of graphite a are eveInorganic nanomaterials have made significant progress in the medical field, including cancer therapy, imaging, drug delivery and soft tissue repair and regeneration. Inorganic nanomaterials have been used for improving stem cell engraftment in cellular therapy, the mechanical stability of materials in tissue repair, electrical conductivity in nerve, cardiac regeneration and antibacterial activity in wound dressings. These nanomaterials have also been used to improve or replace common surgical materials and restore functionality to damaged tissue .They have a great potential in biomedical applications including molecular imaging, cancer and gene therapy, drug delivery, biosensors and tissue engineering applications. In medical applications, carbon nanotubes have the ability to penetrate different body tissues and to carry large doses of agents, providing therapeutic effects. Contrast agents associated with carbon nanotubes improve cell and tissue visualization and so new pathological development patterns can be detected. However, various factors and parameters of carbon nanotubes (CNT) including the size, length, shape, surface, agglomeration, doping or functionalization can cause toxicity through oxidative stress  or contrDue to their nanometric structure and remarkable properties, carbon-based materials are studied for biomedical applications. Significant progress has been made to overcome some of the major obstacles in biomedical applications of nanomaterials, especially when it comes to water, namely dispensability and safety issues. A great disadvantage is their insolubility in many solvents, especially in water, carbon nanotubes, as well as graphene sheets, being incompatible with biological systems, but their hydrophilic/hydrophobic behavior can be easily controlled by oxidation. Their toxicity is also a major drawback due to water insolubility and potential metallic impurities . Thus, the functionalization of carbon-based materials is essential in order to become soluble and to have minimal toxicity for biomedical applications ,11,12. UGraphene oxide b is consGraphene oxide and its derivatives have applications in various fields summarized in four main categories, controlled release and cancer treatment, biosensors, bio-imaging, and antibacterial activity ,25,26. TAs the side effects of synthetic substances on the body are well known, lately there has been a great interest in using natural substances and in recent years great emphasis has been placed on the development of nanomaterials loaded with natural extracts . There iGraphene oxide has an easily modified surface with a variety of biocompatible polymers such as chitosan, polyethylene glycol, poly-L-lysine and polyvinyl alcohol. Graphene oxide contains a large amount of marginal hydrophilic groups. These moieties make graphene oxide more attractive for researchers with applications in controlled release, parasitology, tissue engineering, antibacterial activity, cancer treatment, imaging, sensors and diagnostics. The most important applications of graphene oxide are shown in Despite advances in technology in biomedical science, cancer still remains one of the greatest challenges for humanity. In this paper, we wanted to obtain a nanostructured platform for controlled release system using graphene oxide as a nanocarrier for several active agents with anticancer properties as well as antimicrobial and anti-inflammatory properties. The active compounds used were gallic acid, caffeic acid, limonene and two essential oils: nutmeg and cembra pine oil.Graphite powder (99.99%), potassium permanganate (99.22%) , sulphuric acid (95–97%) , hydrochloric acid (36.5–38%) , phosphorus pentoxide (≥98%) , potassium peroxodisulfate (≥98%) (Sigma-Aldrich), hydrogen peroxide (35%) , gallic acid (≥98%) (Merck), caffeic acid (≥98%) (Sigma-Aldrich), limonene (≥95%), cembra pine and nutmeg essential oils by Carl Roth, Karlsruhe, Germany and ethyl alcohol (96%)  were used in this paper. 2SO4 (60 mL), K2S2O8 (10 g) and P2O5 (10 g) was heated at 80 °C. Then, 20 g of graphite was added under stirring condition. The solution was left to cool down to room temperature. Then, a large amount of deionized water was added, and the solution was filtered, and the filtrate was rewashed until reached neutral pH. The washed product was dried for 24 h at 80 °C. Concentrated H2SO4 (460 mL) solution and 20 g of peroxidized graphite were added under stirring condition in a Berzelius flask in an ice bath (temperature below 5 °C). Sixty gram of KMnO4 was then added slowly into the solution and stirred until the solution became dark green. The solution was then transferred to a beaker, where it was stirred for 2 h at 35 °C. About 920 mL of distilled water was added to the mixture and stirred for 15 min, after which 2.8 L of distilled water and 50 mL of 30% H2O2 were added to eliminate the excess of KMnO4. The supernatant was decanted, and the residuals were then rewashed with 5% HCl and distilled H2O until a neutral pH was obtained. The washed GO suspension was dried at 60 °C for 24 h.Graphene oxide was synthesized from graphite powder using a modified Hummers method presented in In order to obtain graphene oxide—gallic acid (GOGA), a 1% solution of gallic acid was prepared. From this solution, three other solutions were made in a 10 mL volumetric flask, adding 0.1 g graphene oxide in each solution. The mixtures were stirred for 16 h and the GOGA nanomaterial was collected via filtration and dried in a vacuum oven at 40 °C for 24 h. The same method was used to load graphene oxide with caffeic acid, limonene, cembra pine and nutmeg essential oils  onto graphene oxide, a phosphate buffer solution with a pH of 7.4 was used. Various studies showed that anions such as of drugs ,55. The 18 . Two-gradient elution system was used: mobile phase A contained water and 0.1% formic acid and mobile phase B was acetonitrile with 0.1% formic acid. The mobile phase composition was 50:50. Elution was performed at a solvent flow rate of 1 mL/min. The sample injection volume was 15 µL and peak detection was at 220 nm. The retention time was 0.85 min. HPLC analysis was conducted on Agilent 1260 Infinity liquid chromatograph  equipped with a DAD detector. The analytical column used was Eclipse Pluse C−1 and 100 cm−1 at a resolution of 4 cm−1.The synthesized products were characterized by FTIR using a Nicolet iS50FT-IR  spectrometer equipped with a DTGS detector which provides information with a high sensitivity in the range of 4000 cmRaman spectroscopy analyses were performed using a Horiba equipment  with an excitation wavelength of 514 nm and a 50X objective with a 10 s acquisition time. 2O3).Thermogravimetric analysis was performed using an STA TG/DSC Netzsch Jupiter 449 °C equipment , with the temperature ranging between 25 and 900 °C in a dynamic atmosphere of 50 mL/min air with a heating rate of 10 K/min in an alumina crucible  with a resolution of 1.2 nm at 30 kV and 3 nm at 1 kV (BSE). 2 F30 S-TWIN high-resolution transmission electron microscope  equipped with STEM with HAADF detector, EDX, PEELS, energy filter, GIF operated at 300 kV. The sample preparation was done as follows: a small amount of GO was placed into water in a centrifuge tube and sonicated for 15 min. After that, 10 microliters of dispersed sample were placed onto 400 mesh holey carbon-coated Cu grid and left to dry prior to the transmission electron microscopy (TEM) analysis.The transmission electron micrographs were obtained by using a Tecnai GZeta Potential was obtained using dynamic light scattering technique . The nanoparticle suspensions in ultrapure water were homogenized at room temperature using an ultrasonication probe for a period of 10 min.3 and 0.1M Na2CO3 separately. The base mixtures containing GO were magnetically stirred for 24 h and then filtered to separate GO sheets from solution. From the obtained extracts, 5 mL was titrated with 0.05 M HCl solution using phenolphthalein as an indicator. For proper interpretation of the results, blank samples were also performed without GO, having the same concentrations [The Boehm method was used to analyze the content of functional groups in GO. Graphene oxide (0.1 g) was immersed into 100 mL of NaOH, NaHCOtrations ,57. 3 neutralized only the carboxylic groups, Na2CO3 neutralizes the carboxylic groups and lactones and NaOH neutralizes all the carboxylic, phenolic and lactonic groups [The concentration of various types of functional groups was calculated assuming that NaHCOc groups .This report presents the analyses of the five nanostructured platforms obtained by loading graphene oxide with biologically active agents: gallic acid, caffeic acid, limonene, nutmeg and cembra pine essential oils. The content of carboxyl group was 0.45 meq/g, of phenolic groups 0.57 meq/g and of lactones 0.15 meq/g, the total number of functional groups was 1.17 meq/g. The content of phenolic groups was higher as they play an important role in the structure of the graphene oxide .Zeta potential, an indicator of the stability of the sample, is around −37.36 mV, which shows a good stability of the suspension .Raman spectroscopy was used to analyze the disorder and defects in the structure of graphene oxide and functionalized graphene oxide .The release behavior of gallic acid loaded onto graphene oxide is presented in In several papers, these biological substances were studied for their benefits in the human body. Gallic acid has many biological applications such as antibacterial, antimutagenic, antiviral and antitumoral . Based o−1 is assigned to C=O stretching vibration present in the carbonyl and carboxyl groups of GO. The presence of the spectral band from 1600 cm−1 is attributed to C=C stretching vibration and the signals at 1224 cm−1 and 1029 cm−1 are corresponding to COC (epoxy) and COH  stretching vibration groups. The broad peak at 3122 cm−1 is corresponding to the associated OH (hydroxyl) group from GO but also due to the adsorbed water [The resulting graphene oxide is water dispersible, the brownish aspect of the suspension is maintained even after 1 day, with only limited deposition after 1 day (~1g/L). As shown in ed water ,31.−1 indicates the presence of the phenolic group, and the signal at 1608 cm−1 is assigned to stretching vibration of C=C bonds of the aromatic ring. The stretching vibration of the carboxyl groups occurs at 1217 cm−1 and the signal at 1021 cm−1 is attributed to C-O stretching vibration of the carboxyl group. The peak at 734 cm−1 corresponds to the δ CC benzene ring vibrations. The signal at 3269 cm−1 corresponds to the OH stretching vibration.−1 and 1580 cm−1 are characteristic to GO, in −1 corresponds to the OH group from carboxylic and phenolic groups. By subtracting between the GOGA nanocomposite spectrum and the pure graphene oxide spectrum a visible shifting and broadening of the band around 1850 cm−1 can be observed, but also an increase in the relative intensity of the peak, also highlighting the interaction of the acid gallic with the graphene oxide, which act as a promising nanocarrier for this BAA. The same can be observed for the peak from 1030 cm−1 [After the loading of graphene oxide with gallic acid, the characteristic bands of the components can be highlighted. The infrared spectrum of graphene oxide loaded with gallic acid (GOGA) shows the characteristic signals of both graphene oxide and gallic acid, suggesting the successful loading. Certainly, some of these bands being overlapped, instead of individual peaks a composed peak can be observed, the intensity is proportional with the sum of the individual peaks. As the peaks from 1715 cm030 cm−1 ,60.−1. The strong intensity band from 1600 cm−1 is attributed to the stretching vibration of C=C bonds present in the carboxyl groups and the signal at 1448 cm−1 is attributed to C-C stretching vibration of the aromatic ring. The signals at 1214 cm−1 and 1071 cm−1 are corresponding to the stretching vibration of the C-OH bonds attached to the aromatic ring. The broad band between 3200 cm−1 and 3500 cm−1 corresponds to the vibration of the associated hydroxyl groups [−1 and 1041 cm−1 indicating interactions between CA and GO. In l groups . The two−1 are assigned to the stretching vibration of C=C bonds of the aromatic ring in the vinyl group. The signals between 3010 cm−1 and 3081 cm−1 are assigned to the stretching vibration of the C-H bonds. The signals below 3000 cm−1 are generally associated with unsaturated groups. The two peaks at 1377 cm−1 and 1437 cm−1 are assigned to the bending CH3/CH2 bonds, while the intense signal at 885 cm−1 may correspond to the CH=CH bending. −1) due to the interaction between the support and the limonene. Also, a difference in relative intensity between the two peaks from 1583 cm−1 and 1721 cm−1 can be observed because of the condition of the characteristics peaks of limonene and GO [e and GO .−1 (corresponding to the CH stretching vibration) and at 1197 cm−1 (corresponding to the CO stretching vibration). The bending vibration of CH3/CH2 groups occurs at 1433 cm−1. In addition, the signal at 3511 cm−1 corresponds to the associated hydroxyl group, and the signal at 2919 cm−1 is attributed to the stretching vibration of the C-H bonds. In Based on the literature data  the main−1, while the G band is noticed at 1592 cm−1 [The Raman spectrum  for the 592 cm−1 ,71.2 carbon forms, is observed at 1589 cm−1 for graphene oxide loaded with GA. The D band, specific for the sp3 carbon forms can be seen at ~1350 cm−1. The 2D band appears in the 2800 cm−1 and 3200 cm−1 range. Raman spectra did not show important differences in the D and G band between GO and GO loaded with bioactive compounds spectra, [D/IG) for GO (0.97) is slightly larger than GO loaded with bioactive substances starting from 0.87 (GO-limonene) to 0.92  which suggests that the active compounds can repair the defects of graphene during functionalization [Comparing the spectra, structural changes occurred during the loading of GO with the active compounds. The G band, specific for the spspectra, ,72,73 th2D/IG for all samples is between 0.18 for GO-cembra pine and 0.27 for GO-limonene, these results indicating that GO materials are in the form of multilayers [In addition, from the Raman spectroscopy, the ratio between the 2D and G band is a method for determining the number of layers presented in the samples. The shape, position and intensity of the 2D band show the difference between single and multilayer graphene oxide. For single layer GO, the 2D band occurs as a single peak and the ratio will be seen to be equal to 2. An increase in the number of layers reduces the intensities of the 2D peaks. In the spectra of the analyzed materials, the ratio Itilayers ,75.All samples show a loss of mass at the beginning around 12%–13%, except for samples with gallic acid and limonene which have a mass loss of about 16%. This may be due to the fact that graphene oxide can easily absorb water and therefore when heated will lose it, together with other volatile compounds that are eliminated in this phase (RT-145 °C).For graphene oxide  the massThermal analysis for gallic acid sample  indicateThe thermal analysis for limonene sample  indicateThermal analysis for essential nutmeg oil sample  shows a The sample loaded with cembra pine essential oil has a slow weight loss (12.35%) between RT and 180 °C, the process is accompanied by a weak endothermic effect, as shown in As a conclusion, all samples present a first mass loss up to 145–180 °C due to the elimination of adsorbed water and some volatile molecules. A second mass loss occurs up to 165 °C for GO sample or up to 215–250 °C for the other samples and represents the loss of labile oxygen groups. The GO and limonene-GO sample undergo an energic oxidation process in this region, with a mass loss of at least 60%, indicating little chemical modification by limonene. Nevertheless, the limonene load on GO has the effect of increasing the thermal stability, the violent oxidation process taking place at 155 °C in the case of simple GO sample, but at 213 °C in the case of the limonene-GO sample. We can also observe that the limonene sample has no mass loss till 400 °C while GO sample presents a slow mass loss after 165 °C, up to 460 °C. Therefore, we can state that while limonene stabilizes the GO up to 213 °C, it will also generate the elimination of all GO oxygen groups in only one process. The gallic acid, caffeic acid, nutmeg and cembra pine essential oil samples contain molecules with -OH moiety and therefore can form hydrogen bonds and can condensate with oxygen groups from GO. The second mass loss process for these samples only represent around 20%–23%, and the exothermic effect is broader and less intense. It can be attributed to a slow oxidation process rather than an explosive one.The last mass loss for all samples is the burning of the carbon skeleton of GO and oxidation of carbonaceous residue from the acids and oils loaded on GO. The processes take place over 400 °C usually and can be overlapped partially or totally giving a broad, intense, sometimes split exothermic effect. For the samples GO and limonene GO where large quantities of carbon were oxidized at a lower temperature, the thermal effect generated by burning of carbon skeleton is less intense. In comparison to gallic acid release behavior, the release rate of the other biologically active agents  loaded onto graphene oxide is immaterial due to very low solubility and the content of the active substance released in the solution is below the limit of detection of used method ,83.The delivery of the gallic acid is quite fast on the first day (a burst delivery) followed by a strong decrease in the delivery for the next days. The cumulative release of gallic acid is about 80% after 10 days and practically is not changed in the next 20 days. These results have potential for GOGA platforms to be used as a drug delivery system having the ability to assure sustained release for over 10 days, as shown in The structure of the graphene oxide appeared in very thin agglomerates sheets, as can be seen at 1000X magnification in As GO has a lamellar layer structure it is also possible to measure the width of the GO sample using SEM analysis. The individual GO sheets were found to have less than 20 µm being in good agreement with the literature .The morphology of graphene oxide loaded with active compounds  does notTEM images showed GO flakes quite flat because of the suspension in a liquid. However, on the TEM grid were still found agglomeration of layers of different sizes, the thickness of the graphene oxide flakes being estimated to be ~4 nm. These findings are in accordance with the literature data ,87. The 2D/IG for all samples was between 0.18 for GO-cembra pine and 0.27 for GO-limonene; these results indicating that GO materials are in the form of multilayers. The individual GO sheets were found to have less than 20 µm. The thickness of GO flakes was estimated to be ~4 nm with an interlayer spacing of about 2.12 Å. The aim of the present work was to obtain graphene oxide-based drug delivery systems by loading caffeic acid, gallic acid, limonene, nutmeg and cembra pin essential oils starting from the corresponding solution. The SEM characterization showed the morphology of the graphene oxide support, which, compared to the loaded samples, a slight increase in the degree of agglomeration of the particles was observed due to the presence of the active compounds onto the graphene oxide samples. Raman spectra showed that no degradation occurred during loading. The ratio between IAccording to the solubility of loaded biologically active agents, the release behavior of the selected biologically active agents was as follows: gallic acid release within 10 days can reach 80% but all the other biologically active agents are practically not released because their affinity for the graphene oxide support is higher than that to phosphate buffer solution. Further analyses are necessary to make biological assessments, both in vitro and in vivo. This results can be exploited accordingly, the systems which are not able to release the biologically active agents being suitable for certain applications  while the systems releasing biologically active agents can be exploited in a classical way, as a platform for delivery of biologically active agents . All these results encourage research towards the next level by carrying out in vitro and in vivo studies to demonstrate the ability of these platforms in the treatment of cancer and severe infections and in development of drug delivery systems and optical biosensing.


These hits mainly bind to the RRM2 domain, which suggests the druggability of the RRM2 domain of TDP-43. These hits also facilitate further studies regarding the hit-to-lead evolution against the TDP-43 RRM domain.The TDP-43 is originally a nuclear protein but translocates to the cytoplasm in the pathological condition. TDP-43, as an RNA-binding protein, consists of two RNA Recognition Motifs (RRM1 and RRM2). RRMs are known to involve both protein-nucleotide and protein-protein interactions and mediate the formation of stress granules. Thus, they assist the entire TDP-43 protein with participating in neurodegenerative and cancer diseases. Consequently, they are potential therapeutic targets. Protein-observed and ligand-observed nuclear magnetic resonance (NMR) spectroscopy were used to uncover the small molecule inhibitors against the tandem RRM of TDP-43. We identified three hits weakly binding the tandem RRMs using the ligand-observed NMR fragment-based screening. The binding topology of these hits is then depicted by chemical shift perturbations (CSP) of the  RNA recognition motifs (RRMs) play diverse roles in post-transcriptional gene expression events such as RNA transport, localization, stability, and mRNA and rRNA processing. RRM is also known as the ribonucleoproteins (RNP) domain, as it contains the short and conserved elements RNP1 and RNP2, or RNA binding domain (RBD), that are abundantly distributed in higher vertebrates . RRMs arThe transactive response DNA-binding Protein 43kDa (TDP-43) is a RRM-containing protein, which plays important functions in mRNA metabolism regulation, including transcription repression, exon skipping, and RNA splicing ,6. TDP-4The dysregulation of TDP-43 is hence associated with a variety of human diseases, especially neurodegenerative diseases, e.g., frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), brain ischemia, aging, and Alzheimer’s disease ,12,13. FApart from the TDP-43 involvement in neurodegenerative diseases, an accumulating amount of evidence suggests that TDP-43 is a cancer responsive factor. TDP-43 positively contributes to the anticancer activity for curcumin in MCF-7 cells  and as a®, technology was used to characterize DNA and RNA oligonucleotides  binding to TDP-43 and their interaction inhibition was assessed [Although more is known about the TDP-43 biology and its association with neurodegenerative and cancer diseases, the development of treatments toward TDP-43 is mostly lagging behind those targeting other proteins involved in such diseases . RRM andassessed . Later, assessed , but infassessed . HoweverNMR spectroscopy is a powerful approach which has been extensively used by the pioneers in fragment-based drug discovery for detecting molecular interactions between the target and the fragment libraries ,38,39 an15N labeled TDP-43 tandem RRMs demonstrate that these hits bind to the same site, mainly on the RRM2 domain. It has also been validated by the chemical shift perturbation experiments for TDP-43 RRM2 alone. The CSP-driven HADDOCK was used to generate the protein-hits binding mode. Collectively, our work provides a class of compounds for further hit-to-lead evolution of the TDP-43 RRM domain and paves the path for targeting protein-RNA interactions using the fragment-based approach.Here, we carried out automated NMR fragment-based screening  to identStructurally, TDP-43 tandem RRMs are approximately 160 amino acids long and display a β1α1β2β3α2β4 arrangement of secondary structure, with an additional β-hairpin named β3’β3’’  or β5 4,49 whichNMR ligand-observed methods detect the weak protein-ligand binding by detecting changes in the characteristics of the ligand spectrum that occur upon binding to the protein. Using the ligand-based experiments, i.e., saturation transfer difference (STD) , water l15N-labeled tandem RRMs of TDP-43 and 3 of them induced significant chemical shift changes of the tandem RRM  of the ndem RRM . This apndem RRM , which sndem RRM . Howeverndem RRM c.Consistently, hits 2 and 3 titrations also point to the same binding topology in the tandem RRM of TDP-43 . For exa15N-labeled RRM2 domain of TDP-43  nucleotides are accommodated on the RRM1 β-sheet and the following two nucleotides (A6A7) act as a connector between two RRMs, while the next three nucleotides (U8G9A10) lie on the RRM2. The U8 nucleotide of RNA is recognized on S258 (β4) through hydrogen bonds, on the backbone carbonyl oxygen of N259 (β4), and the backbone amide of E261 from the C-terminus [8 nucleotide in tandem RRM  interact with RRM1 and RRM2 (PDB code: 4BS2) . Among tterminus . Comparandem RRM a. Furthendem RRM c,d.Accordingly, the crystal structure of TDP-43 RRM2 in complex with ss-DNA 5′-GTTGAGCGTT-3′ (PDB entry: 3D2W) reveals that only three 5′ end nucleotides  make extensive contacts with β-sheet residues of RRM2, whereby T3 particularly contacts with S258, Asn259, and Glu261 through hydrogen bonds , while iTo further characterize the binding mode, a data-driven approach, HADDOCK , was useProteins containing RRM domains function in important aspects of the posttranscriptional regulation of gene expression, mRNA maturation, and other RNA processing machinery. These proteins perform their diverse roles depending on the dual ability to recognize RNA and to interact with other proteins by using their RRM domain . As TDP-We uncovered three small molecules binding to the tandem RRM domain of TDP-43 by using NMR fragment-based screening techniques. The NMR spectroscopy, one of a plethora of biophysical methods, is particularly powerful to detect even ultra-weak protein-ligand interactions. Accordingly, chemical shift perturbations observed in the heteronuclear single-quantum coherence (HSQC) spectra or the linewidth analysis of the small molecules allow the determination of binding affinity ,59. ThisNMR is extensively applied in fragment-based lead discovery . The cenAlthough the 4-aminoquinolines molecules have been discovered through high throughput screening against the full-length TDP-43 , the enl6 tag. The RRM2 domain was amplified from the tandem RRM construct and then sub-cloned into the pET22b vector  with the His6 tag. The constructs were transformed into Escherichia coli BL21 and cultivated in 1 L LB media, incubated at 37 °C. The proteins were expressed at 16 °C after induction by 0.5 mM isopropyl β-D-thiogalactosidase (IPTG) for 20 h. The bacteria were harvested by centrifugation , resuspended in lysis buffer , and then lysed by sonication. The cell lysates were centrifuged . The collected supernatant was purified on a column filled with Nickel-chelated resin . The impurities were washed out using a buffer  containing a linear gradient of 20–40 mM imidazole, then the same buffer containing 500 mM imidazole was used to elute out the target proteins. All proteins were further purified by size exclusion chromatography using a HiLoad 16/600 Superdex 75 column . The target proteins were confirmed by SDS-PAGE.The tandem RRM domain of TDP-43 (residues 101–269) was synthesized by GENEWIZ  and sub-cloned into the pET22b vector  with the His15N-labeled proteins, the cells were first cultured in 1 L LB media, harvested when A600 reached 1.0 and then transferred to 1 L M9 media containing 15NH4Cl. The cells were induced by 0.4 mM IPTG to express the proteins (tandem RRMs and RRM2 domains). The purified proteins were concentrated in PBS buffer plus the 5 mM DTT at pH 7.5.For 1H) were acquired against the 890 fragments library  as described previously in detail [2O (50%). To further confirm the identified primary hits, secondary screening was individually carried out for single hits using the same buffer and NMR experimental settings. We then automatically processed and visualized the primary and secondary data with our ACD/Labs scripts, as previously described [All NMR fragment screening experiments were carried out at 25 °C using an Agilent 700 MHZ spectrometer equipped with a 96 well auto-sampler and a 5 mm cryoprobe. During the primary screening, the ligand-based NMR spectra , in PBS buffer , containing NaCl (200 mM), dithiothreitol (5 mM), and D2O (10%) were titrated by small molecules stocked in DMSO at a concentration of 200 mM, using a series of hit/protein molar ratios of 0.0, 0.5, 1.0, 2.0, 4.0, and 8.0 for TDP-43 tandem RRMs and 0.0, 0.5, 1.0, 2.0, and 4.0 for RRM2, respectively. Spectra were processed in NMRpipe and analyzed with Sparky. The chemical shift changes (∆δ) relative to the free form of protein were defined as follows:δ1H and δ15N are the chemical shift differences of the 1H and 15N dimensions, respectively. We referred to the following chemical shift assignments previously deposited in the Biological Magnetic Resonance Data Bank: RRM1 (BMRB Entry 18765), RRM2 (BMRB Entry 19922), and tandem RRM (BMRB Entry 19290). All structures figures were prepared by Pymol .NMR HSQC spectra were acquired at 25 °C on either an Agilent 700MHZ spectrometer equipped with a cryoprobe or an Agilent 500MHz spectrometer equipped with a room temperature probe. The 1 PDB file was generated by the PRODRG [HADDOCK is an information-driven docking technique used for modeling biomolecule structures by using experimental or predictive restraints ,62. The e PRODRG . The docThe NMR HSQC spectra at molar ratios of 0:1 and 8:1 (hit/protein) were processed using the same NMRpipe script, e.g., 2-fold zero-filling, Fourier transformation, and phase corrections. The spectra were then analyzed, with randomly selected peaks, using Sparky. After peak integration, the linewidth, i.e., the full width at half the peak height, was automatically estimated by Sparky.