Here, we demonstrate a strong isotope effect associated with the VUV photodissociation of CO by measuring the branching ratios of 12C16O and 13C16O in the Rydberg 4p(2), 5p(0) and 5s(0) complex region. The measurements show that the quantum yields of electronically excited C atoms in the photodissociation of 13C16O are dramatically different from those of 12C16O, revealing strong isotope effect. This isotope effect strongly depends on specific quantum states of CO being excited, which implies that such effect must be considered in the photochemical models on a state by state basis.Rare isotope ( Carbon monoxide is the main C and O reservoir in interstellar media, where it undergoes photochemistry. The authors show that isotopic substitution affects not only its absorption. spectrum and photodissociation rates but also the branching ratio, key to understand isotopic. fractionation in the solar system It has long been known that rare isotope  substitutions can substantially change photoabsorption line positions, oscillator strengths and photodissociation rates of CO8, and these are key input parameters for the self-shielding models11. These models have been proposed for explaining the anomalous oxygen isotopic distributions observed among various early Solar System objects which contain both 16O-rich and 16O-poor reservoirs12. Self-shielding process happens when a light beam with broad spectral distribution penetrates a sample of mixed gaseous molecules which have different absorption line positions. Due to the different column densities of the gaseous components, which result in different attenuation speeds according to Beer’s law, the relative amount of light absorption for different molecular species will change along the light propagation direction. To test the self-shielding models, Thiemens and coworkers performed an experiment at the advanced light source in Berkeley, strong and wavelength selective mass-independent oxygen isotope fractionation processes have been observed, from which they concluded that the self-shielding effect is not the major reason for the anomalously enriched atomic oxygen reservoirs observed in the Solar System13. However, their experiment and discussion did not convince most of the other researchers16. Now it is generally agreed that self-shielding effect is the main reason of the massive isotope fractionation observed in the above experiment and also that in the solar system, while Thiemens´ experiment does have the effect of encouraging people to include the different photoabsorption and photodissociation cross sections for different CO isotopologues in their models, which has been proved to make the modeling results in closer agreement with the experimental measurements18. Compared to the absorption line positions, accurate absolute values of photoabsorption and photodissociation cross sections are much more difficult to be measured, thus continued recent efforts are being devoted to obtain more precise values of these for all the CO isotopologues in hope of further improving the photochemical modeling22.Carbon monoxide (CO) is one of the most important molecules in astronomy, not only because it is the second most abundant molecular species in the universe only after the molecular hydrogen  are depleted in the Sun compared with that in the Earth and other terrestrial planetary materials by ∼7%26, and the heavy nitrogen isotope (15N) is depleted even more in the Sun by ∼40%27. The reasons for such big difference between O and N in the degree of heavy isotope depletion in the Sun are still unclear. Clayton proposed that this is caused by the different reactivity of the atomic photodissociation products of CO and N2 when react with H223. He assumed that dissociation of CO only produces O atoms in the ground state O(3P), and that of N2 can produce 50% N atoms in the excited state N(2D). N(2D) can be much more efficiently trapped due to its higher reaction rates with H2 compared with O(3P), and this caused the much higher degree of depletion for 15N compared with 17O and 18O in the Sun as observed by the Genesis mission. To verify this assumption, the photodissociation branching ratios of 12C16O have been measured recently33, which showed that photodissociation of 12C16O not only generates C and O atoms in the ground state, but also produces significant amount of C and O atoms in the excited states, and the ratios between them strongly depend on the specific rovibronic state of 12C16O being excited. These observations clearly showed that the initial assumption by Clayton is oversimplified. By using the measured branching ratios and the photoabsorption cross sections of 12C16O and 14N2 reported in the literature, Shi et al. carefully modeled the possible fractionation processes of O and N isotopes in the framework of self-shielding models by considering the different chemical reactivity between the excited O(1D) [N(2D)] and the ground O(3P) [N(4S)] produced by VUV photodissociation of CO [N2]. The modeling results qualitatively support the observations by the Genesis mission24. Besides O and N, Lyons et al. have recently showed that the heavy C isotope (13C) is also depleted in the Sun compared with that in the Earth and other planetary objects25. In their work, they have also considered the possible effect caused by the production of excited C atoms C(1D) from photodissociation of CO, because C(1D) can react with H2 several orders of magnitude faster than any other reactions in their models.While most of the current photochemical models have considered the rare isotope substitution effects in the photodestruction process of CO due to VUV absorption, only few of them have talked about what chemical processes would happen following the production of C and O atoms, which can play important roles in sequestering the isotope fractionation results generated in the photodissociation process25. In the present work, we measure the photodissociation branching ratios of 13C16O in the Rydberg 4p(2), 5p(0) and 5s(0) complex region and find that the percentages of producing C(1D) and O(1D) for 13C16O can be dramatically different from that for 12C16O in the same absorption band, revealing very strong isotope effect. The underlying mechanism for such isotope effect is general for any indirect predissociation processes34, like that in O2, N2. This could potentially have great impact on the current photochemical models for understanding the various isotope fractionation phenomena observed in the fields of astrochemistry, geochemistry, and planetary atmospheric chemistry36.Even though not mentioned in the above two examples, they actually both assumed that all CO isotopologues produce the same amount of O and C atoms in the excited states, the validity of which has not been experimentally proved yet−1) generated by the two-photon resonance-enhanced four-wave mixing scheme  was crossed by the VUV laser beam in the photodissociation and photoionization (PD/PI) region of the TSVMI setup. CO molecules absorbed a single VUV photon to be excited to a specific rovibronic state, and then predissociated into the three possible channels as follows  Fig.  which eqeme Fig. , as showsee Fig. :1\documeThe TSVMI setup collected the velocity-map ion images of C atoms produced in the above photodissociation processes. The images were then converted to the total kinetic energy release (TKER) spectra, from which the branching ratios of the above three dissociation channels can be deduced (see “Methods” section).13C16O, and compared with those of 12C16O from previous studies in the Rydberg 4p(2), 5p(0) and 5s(0) complex region (92.85–94.00 nm), where complicated photoabsorption structures due to various Rydberg–Rydberg and Rydberg–valence interactions have been observed, and the absorption line positions and oscillator strengths were found to strongly depend on the isotopic substitution20. The measured branching ratios of seven absorption bands in the wavelength range 92.85–94.00 nm are listed in Table 12C16O and 13C16O. The branching ratio data for absorption bands of 1Π(v′ = 2), (4pπ) 1Π(v′ = 2), (5pπ) 1Π(v′ = 0), (5pσ) 1Σ+(v′ = 0) and I 1Π for 12C16O are adopted from our previous study32, and the newly measured branching ratios in the current study for the absorption bands of C′1Σ+(v′ = 7) and I´(5sσ) 1Σ+(v′ = 0) for 12C16O are consistent with the previous measurement in the literature31. In most of the raw TSVMI images as shown in Fig. 3P)+O(3P), C(1D)+O(3P) and C(3P)+O(1D), respectively. The outermost, middle and innermost rings in the TSVMI images are converted to the peaks with largest (2.0–2.3 eV), medium (0.8–1.0 eV) and smallest (0.1–0.3 eV) kinetic energies in the TKER spectra, respectively. After normalization by using the photoionization cross sections of C(3P) and C(1D) (see Methods)39, the relative intensities of the three peaks in the TKER spectra directly reveal the branching ratios of the three dissociation channels.In the present study, we measured the branching ratios of the above three channels for the photodissociation of 12C16O and 13C16O in the absorption bands with upper states 1Π(v′ = 2), (5pπ) 1Π(v′ = 0), C′1Σ+(v′ = 7) and I′(5sσ) 1Σ+(v′ = 0). This is especially prominent for the comparison between the lowest dissociation channel C(3P)+O(3P) and the spin-forbidden channel C(1D)+O(3P), which respectively produce C atoms in the ground and first excited states. The largest variations happen in the absorption bands of 1Π(v′ = 2) and C′1Σ+(v′ = 7), where the relative ratios of C(3P)+O(3P) and C(1D)+O(3P) are completely inverted. For 12C16O, dissociation into the lowest channel dominates the predissociation process with percentages of ~53% and ~77% for the absorption bands of 1Π(v′ = 2) and C′1Σ+(v′ = 7), respectively; while for 13C16O, dissociation into the spin-forbidden channel producing C atoms in the excited state dominates the predissociation process with percentages of ~79% and ~62%, and the corresponding percentages of the lowest dissociation channel decrease to ~9% and ~22%, respectively for the two absorption bands. These huge branching ratio variations (up to 50%) between 12C16O and 13C16O in the above two absorption bands have been directly revealed in Fig. 1Π(v′ = 0), in which case the substitution of 12C by 13C decreases the relative percentage of C(3P)+O(3P) from ~62% to ~45%, and increases that of C(1D)+O(3P) from ~22% to ~48%, as shown in Fig. 1Σ+(v′ = 0), only the outermost ring corresponding to the lowest dissociation channel can be observed for 12C16O, as shown in Fig. 12C16O. While for 13C16O, the two inner rings and the corresponding peaks are clearly observable from the TSVMI image and the TKER spectrum, as shown in Fig. 12C by 13C has also enhanced the generation of excited C atoms in the absorption band of (5pσ) 1Σ+(v′ = 0) at the band head, i.e. from ~ 12% to ~25%. Other than enhancing the generation of C(1D), 13C substitution could also decrease it as in the cases of (4pπ) 1Π(v′ = 2) and I 1Π, while the decreased amount is relatively small, less than 10% for both of the two absorption bands, as shown in Table 1D)+O(3P) caused by the 13C substitution, relatively small changes on the percentages of the channel C(3P)+O(1D) have been observed in the energy range investigated in the current study, which are usually less than 10%. As shown in Fig. 1Π(v′ = 0) and C´1Σ+(v′ = 7), in which the percentages of generating excited O atoms decreases and increases by ~10%, respectively.In Fig. 12C by 13C can greatly change the photodissociation branching ratios of CO, which varies from less than ~10% to ~50%. This isotopic effect strongly depends on the specific electronic and vibrational quantum level of CO being excited. Previous studies have shown that the photodissociation branching ratios of CO also depend on the rotational quantum number of the excited state, which revealed the detailed photodissociation dynamics32. In the current study, we investigate the rotational dependence of the branching ratio of the absorption band with upper state 1Π(v′ = 2) for 13C16O, and compare it with that of 12C16O in Fig. 1Π(v′ = 2). Our previous study revealed that the branching ratios of the lowest dissociation channel C(3P)+O(3P) increase linearly as a function of J′(J′+1) with J′ being the rotational quantum number of the upper state32. The linear dependence is due to the heterogeneous perturbations by the repulsive D′1Σ+ state that is correlating to the dissociation channel C(3P)+O(3P). As shown in Fig. 13C16O is almost the same with that of 12C16O, the substitution of 12C by 13C shifts up the line representing the branching ratios of the channel C(1D)+O(3P), and at the same time shifts down the line for C(3P)+O(3P), without changing the slopes of the lines. This observation implies that the isotopic substitution has negligible effect on the electronic perturbation coefficient between the Rydberg state 1Π(v′ = 2) and the repulsive D′1Σ+ state, while it does greatly enhance the coupling schemes which finally dissociate CO molecules into the spin forbidden (or triplet) channels. This will be discussed furthermore in next section.From the above presentation, we can see that the substitution of 13C16O are dramatically different from that of 12C16O, revealing conspicuous isotope effect which depends on the specific quantum state of CO being excited. To our knowledge, this isotope effect has not been noticed before. From the chemical physics point of view, the substitution of 12C by 13C must have dramatically changed the relative coupling strengths among different rovibronic states which determine the partitions of the CO photodissociation into the three available channels. Due to the complicated potential curves of CO and the mutual interactions among them in this high excited region40, quantitative explanations of the observed isotope effect is obviously out of scope for the current study. Here we employ a simple diabatic coupling model to qualitatively explain the strong isotope effect observed in the current study. This model was recently discussed by Thiemens and coworkers for explaining the variations of absorption line positions and oscillator strengths of N2 due to isotopic substitution, which should also work for the photoabsorption and photodissociation processes of CO as pointed out by those authors41. This is actually similar to the coupled equations method used by Lefebvre-Brion and coworkers for quantitatively predicting the line positions, oscillator strengths, accidental perturbations, and photodissociation branching ratios of CO and their dependences on the isotopic substitution43. In this model, two bound diabatic potential curves with different bonding types, usually one Rydberg and one valence, cross at certain point and interact with each other through an electronic coupling constant H, this is shown schematically in Fig. H/(Ea−Eb), where Ea and Eb are the energy positions of the two interacting quantum states as shown by the double head arrow in Fig. H, but it does modify the reduced masses of diatomic molecules, this will, in turn, change the vibrational and rotational constants of the molecules, and thus the energy positions Ea and Eb. Because the two diabatic potential curves have different bonding characters , variations of the reduced mass change their vibrational and rotational energy ladders in different ways, this will modify the energy gap Ea−Eb between the two interacting levels, and thus the coupling strength between them . This mechanism has been argued to be the origin of the strong isotopic selectivity in absorption line positions and oscillator strengths of N2, especially in the crossing region of the two diabatic potential curves34. This physical picture can also explain the isotopically selective accidental local perturbations which are ubiquitous in the absorption spectra of N2, CO, and their isotopologues44.In the above section, it is shown that the photodissociation branching ratios of −1 for the (5pσ) 1Σ+(v′ = 0) state to ~101 cm−1 for the (4pπ) 1Π(v′ = 2) state, this observation is consistent with the above discussion that isotopic substitution shifts the vibrational energy ladders in different manners for electronic states of different bonding natures. It has long been known that photodissociation of CO is an indirect process with all the possible dissociation pathways summarized before45. There are two different pathways for dissociating into the lowest channel C(3P)+O(3P), Pathways I and II, as shown schematically in Fig. 1Σ+ state (the repulsive black curve), which leads to the linear dependence of the photodissociation rates and branching ratios on J′(J′+1) for states of 1Π symmetry, like the 1Π(v′ = 2) state as shown in Fig. 1Σ+ state is repulsive in the Franck-Condon region, thus the above physical model, which is for interaction between two bound electronic states, does not work34. The coupling strength between a bound electro-vibrational level and a repulsive state will not be significantly changed by the small energy shift of the bound level caused by isotopic substitution if it is well separated from the curve crossing region, this might explain why the substitution of 12C by 13C did not change the way how the branching ratios depend on J′(J′+1) for the 1Π(v′ = 2) state, i.e. the slopes of the linear fitting lines are the same for both 12C16O and 13C16O, as shown in Fig. 1Π or 3Π symmetry (the red curve), then dissociate into the channel C(3P)+O(3P) through a repulsive state of 1Π or 3Π symmetry (the green curve). Even though the detailed dynamics of this pathway has not been clarified yet, its existence has been confirmed in several previous studies41. Because the first step involves coupling between two bound states, its strength could be strongly affected (increased or decreased) by isotopic substitution as discussed above. Reduction of the total contribution from Pathway II could be one of the reasons why the relative percentage into the C(3P)+O(3P) channel is greatly reduced for 13C16O compared with 12C16O in the 1Π(v′ = 2) state. Besides the above discussed reason, it could also be due to the enhancement of the photodissociation rate into the channel C(1D)+O(3P) which, in turn, decreases the relative percentage of the channel C(3P)+O(3P). Since the directly accessible quantum state from the ground electronic state by single VUV photon absorption is singlet, which cannot directly correlate with the channel C(1D)+O(3P), it usually couples with a repulsive state of 3Π symmetry through spin-orbit interaction to dissociate into C(1D)+O(3P). In this process, coupling with an intermediate bound state of 1Π or 3Π symmetry (the red curve) as a first step could be possible41. This mechanism is shown in Fig. 1D)+O(3P) channel for 13C16O compared with 12C16O in the 1Π(v′ = 2) state. Dissociation pathways into the channel C(3P)+O(1D) are similar to that into the C(1D)+O(3P) channel, thus will not be repeated here.Table channel CP+O3P), P, −1 for1Σ+ state, the coupling strengths in the other two pathways as summarized in the previous study30 can be both significantly altered by the isotopic substitution, and this should be the origins of the strong isotope effect of the photodissociation branching ratios of CO observed in the current study. One thing to be noticed is that the magnitude of the isotope effect does not depend on the amount of energy shift in a monotone way. For example, the biggest energy shift (~101 cm−1) is observed for the (4pπ) 1Π(v′ = 2) state, while its branching ratios have only been changed by ~10%; on the other hand, the energy shift for the (5pπ) 1Π(v′ = 0) state is only ~45 cm−1, its branching ratios are varied by ~25% due to the isotopic substitution, as shown in Table Ea−Eb, which not only depends on the energy position of the directly excited state (Ea), but also on the energy position of the state that is being coupled with (Eb). Thus, it is not possible to tell directly from the isotopically selective photoabsorption spectra that how much the branching ratio of a specific absorption band will be changed by the isotopic substitution. Quantitative experimental measurement like that presented in the current study is needed for all the CO absorption bands, and this is being performed in our lab.Thus except for Pathway I which involves the repulsive D′17O and 18O are mostly not unity and strongly wavelength dependent. This contradicts with the prediction by photochemical models which only consider the self-shielding effect13. Being stimulated by this experimental work, Lyons performed a detailed quantitative simulation, and found that pure self-shielding alone can account for the magnitude of the massive isotopic fractionation observed in the experiment, while consideration of other isotopic effects can definitely make the model more realistic, for example, the isotopic differences in dissociation probabilities can cause the slope increase observed in Thiemens’ experiment18. Even though the branching ratios of 12C17O and 12C18O have not been directly measured yet, the present experiment strongly implies that different relative amounts of excited 17,18O atoms [O(1D)] might be possibly produced, especially when considering the fact that 13C16O and 12C18O have very similar reduced masses35. This could affect the subsequent trapping reaction rate between CO and O which forms CO2 for detection in Thiemens’ experiment18. Since CO photodissociation can only generate excited O atoms [O(1D)] at VUV wavelength shorter than 94.94 nm (see reaction (3)), thus the present isotope effect observed for the branching ratio should not affect any absorption bands at longer wavelength in Thiemens’ experiment. Direct measurements of the photodissociation branching ratios for 12C17O and 12C18O have been planned in our lab, since they might be important for any photochemical models based on the self-shielding effect, for example, the recent one by Shi et al. for explaining why the rare isotope 15N is much more enriched (~7 times) than the rare isotopes 17O and 18O in the terrestrial planets relative to the Sun as revealed by the recent NASA’s Genesis mission27.Early photochemical models based on the self-shielding effect only considered the isotopic fractionation effect caused by the shifts of photoabsorption line positions due to isotopic substitutions and assumed that no other isotopic fractionation processes occurred in the photodissociation process. This assumption was directly challenged recently by Thiemens and coworkers who found that the relative degrees of enrichment between 46, Lyons et al. concluded that the primary reservoirs of C on the terrestrial planets are enriched in 13C relative to the Sun, similar to O and N isotopes25. To explain this finding, they have built a photochemical timescale model which included all the relevant chemical reactions between C atoms and other molecular species, like H2, CH, CH2, and also VUV photons, that could be present in the early Solar system or parent molecular cloud. In the model, they have highlighted the potential possible roles played by the excited C(1D) atoms produced from the photodissociation of CO, because the reaction of C(1D) towards H2 is faster by a factor of ~103 than any other photochemical processes in the model, this could sequester the C atoms into large molecules to at least partially preserve the 13C isotope signature formed in the self-shielding process before they are ionized by VUV photons. The photoionization process can erase the 13C isotope signature through the ion exchange reaction between C+ and CO. Our current study showed that at some absorption bands of 13C16O, the production of excited C(1D) atoms can be significantly enhanced compared with that in 12C16O, for example, at the 1Π(v′ = 2) and C´1Σ+(v′ = 7) bands the relative percentages of C(1D) are increased by ~50% as shown in Fig. 13C more preferential than 12C due to the much faster reaction rates of C(1D) than that of the ground state C(3P) when react with H2. Without systematic branching ratio measurements like that in the present study for all the relevant 13C16O absorption bands and quantitative photochemical modeling as that by Lyons and coworkers25, it is not possible to predict how exactly the current observation could affect the modeling outcomes, while we believe that consideration of the isotope dependent branching ratios on a state by state basis could improve any future quantitative photochemical models based on the CO self-shielding effect.Besides the O isotopes, C isotopes are also of central importance for understanding the formation history of the Solar system. By reanalyzing the ATMOS Fourier transform spectrometer data for CO13C16O in the wavelength range 92.85–94.00 nm. The measurements showed that the branching ratio can be strongly altered through the substitution of 12C by 13C. This isotope effect also depends on the specific rovibronic quantum state of CO that is being excited. We employed a simple diabatic coupling model to explain this strong isotope effect, which could potentially work for any other molecules which undergo indirect predissociation process into multiple channels, for example N234. This strong quantum state dependent isotope effect of the photodissociation branching ratio could potentially improve the current photochemical models, thus need to be considered on a state by state basis in any future quantitative modeling.In summary, we have demonstrated a strong isotope effect in the VUV photodissociation of CO by measuring the branching ratios of 37, together with a tunable VUV laser radiation source generated by the two-photon resonance-enhanced four-wave mixing scheme. A schematic diagram of the experiment is shown in Fig. 12C16O, 99.9%; 13C16O, 13C = 99%, 18O < 5%) generated by a pulsed valve (PV) operating at 10 Hz intersects with a VUV laser beam perpendicularly in the photodissociation and photoionization region. CO absorbs a single sum-frequency VUV photon (2ω1+ω2) and is excited to a specific rovibronic state and then undergoes predissociation through various pathways to form the three dissociation channels  = 11.09 eV is the bond dissociation energy47; E(C) and E(O) are the internal energies of the C and O atoms respectively, with E(C(3P)) = E(O(3P)) = 0 eV, E(C(1D)) = 1.2637 eV, E(C(1D)) = 1.9674 eV; ETKER is the total kinetic energy release (TKER), EC is the kinetic energy of the C atoms; MC and MO are the masses of C and O atoms respectively. Since the three dissociation channels as shown in Eqs. ( with E(CP) = E(O(3P) and C(1D) to obtain the branching ratios of the three dissociation channels as listed in Table 12C(3P) was measured to be ~16 Mb with an uncertainty of ~30%38, and that of 12C(1D) was calculated to be ~27 Mb in the VUV range of the present study with no associated uncertainties reported39, thus this could be the biggest potential uncertainty source of the current photodissociation branching ratio measurements. Since there are no relevant results on the photoionization cross sections of 13C, here we used the photoionization cross sections of 12C to also calibrate the TKER spectra of 13C16O.According to eqs.  and 5),,5), TKER3P) and CD to obta1. ω1 is fixed at 222.568 nm (ω1) which is in resonance with the two-photon transition of Xe: (5p)5(2P1/2)6p2[1/2](J = 0) ← (5p)6 1S0 at 89,860.018 cm−1. The dye laser pumped by the second harmonic gives out the tunable visible laser beam ω2, which was scanned from 560 to 610 nm in the present study. The UV (ω1) and visible (ω2) laser beams were combined through a dichroic mirror (DM) and focused into a T-shape channel by two plano-convex lens both with f = 600 mm. The Xe gas is pulsed into the T-shape channel when the two laser beams arrive, and the four-wave mixing process takes place to generate the tunable VUV laser radiation, which has the photon energy of 2ω1+ω2. The time duration of the VUV laser pulse is 5–10 ns, and the predissociation life times of CO are usually in the picosecond range, thus in the present study a single VUV laser pulse is enough to first dissociate CO molecules and then ionize the C atoms for detection in the same laser pulse through a direct photoionization process. The VUV intensity generated this way is generally weak, no saturation of the photoionization has been noticed, thus the absolute VUV intensity should have no effect on the relative branching ratio measurements.The tunable VUV laser radiation source used in the current study is generated by using the two-photon resonance-enhanced four-wave mixing scheme as shown in Fig. Peer Review File


Artificial proteins binding any predefined “target” protein can now be efficiently generated using combinatorial libraries based on robust protein scaffolds. αRep is such a family of artificial proteins, based on an α-solenoid protein repeat scaffold. The low aggregation propensity of the specific “binders” generated from this library opens new protein engineering opportunities such as the creation of biosensors within multidomain constructions. Here, we have explored the properties of two new types of artificial bidomain proteins based on αRep structures. Their structural and functional properties are characterized in detail using biophysical methods. The results clearly show that both bidomain proteins adopt a closed bivalve shell-like conformation, in the ligand free form. However, the presence of ligands induces a conformational transition, and the proteins adopt an open form in which each domain can bind its cognate protein partner. The open/closed equilibria alter the affinities of each domain and induce new cooperative effects. The binding-induced relative domain motion was monitored by FRET. Crystal structures of the chimeric proteins indicate that the conformation of each constituting domain is conserved but that their mutual interactions explain the emergent properties of these artificial bidomain proteins. The ligand-induced structural transition observed in these bidomain proteins should be transferable to other αRep proteins with different specificity and could provide the basis of a new generic biosensor design. Each library variant is characterized by the presence of random amino acid substitutions on its outside surface. New binding properties can emerge from this hypervariable surface, and although very rare in the initial library, specific binders can be selected by genetic sorting methods such as phage, ribosome or yeast display. It is now clear that these strategies, which bypasse immunization, are generally applicable, as illustrated by the fact that a large set of new proteins binding specifically to almost any predefined protein targets were obtained from several published libraries4. Those specific binding proteins based on alternative scaffolds are usually more efficiently produced and engineered than antibody-based chimeric proteins, that suffer from high aggregation propensity. Furthermore, specific binders from disulfide-free protein scaffolds can be efficiently used in reducing environments such as the cytoplasm of living cells.Efficient methods have been recently developed to create artificial proteins binding specifically to almost any desired protein target. The most established strategy is to design and assemble a very large library of protein variants, in which all variants share a same architecture usually based on a natural protein “scaffold”7.Engineering of specific proteins based on robust scaffolds opens a range of new opportunities. Sophisticated multidomain proteins such as artificial receptors, sensors or switches could be designed by integrating tailor-made binding proteins into multi-modular constructions10. Alternatively, the ligand-binding event can induce a structural transition detected in a second “reporter” domain. A simple head to tail fusion of recognition and reporter domains is often not adapted as it usually produces functionally-independent domains. Only few general solutions have been suggested to create such inter-domain coupling. For example, insertion of a peptide within the surface loop of an enzyme11 or inserting a whole protein into another unrelated enzyme were shown to generate new bi-active proteins with unexpected functional properties12. The coupling of the inserted and host domains can be further engineered or evolved to create new ligand responsive enzymes14. Structural transitions were also engineered by designing a bidomain protein in such a way that the folding of the two domains are mutually exclusive15. Specific “affinity clamps” have been further developed into protein16 or peptide17 specific biosensors by fusing each of the two peptide-binding domains to a fluorescent protein giving rise to reconstituted GFP fluorescence16 or to a ligand-dependent FRET signal17. Recently, new biosensors using computationally designed protein-binding domain and ligand-induced protein stabilization have been described21.In order to convert a specific binder into a specific bio-sensor, the target-binding domain must be integrated with a transducing component in such a way that the target-binding event will give rise to a measurable signal. The signal can result from environment-sensitive fluorescent probes chemically grafted near the binding site of the recognition unitIn this paper, we propose a new design of a multi-domain protein switch based on an open/closed bivalve-shell-like protein and we describe the experimental behavior of the resulting bidomain protein based on this design.22 composed of a HEAT-like motif23. Each protein member of the αRep library has the same general architecture but is endowed with a unique binding surface made by the juxtaposition of hypervariable residues24. Specific αRep binders for a wide range of arbitrarily predefined target proteins with unrelated sequences and structures have been selected by phage display or protein complementation assay25. This suggests that the αRep library is a general source of specific reagents. The crystal structures of five αRep-target protein complexes  clearly showed that the targets are bound, as expected, on the hypervariable concave face of the αRep fold. Given the versatile binding surface grafted on a precisely defined and very stable fold, a designed molecular switch based on αRep proteins may later be adaptable to other proteins of this family binding different unrelated targets. This prompted us to explore new molecular devices using αReps as building blocks.We used a recognition unit based on our previous work with αRep proteins. αReps are a family of artificial repeat proteins23. In the dimeric structure, the binding concave surfaces made from the variable residues from each monomer face each other and the dimer resembles a closed bivalve shell. Although relatively tightly associated, we showed that the two monomers can dissociate in presence of another αRep that binds to the concave surface of A3. These results suggest that a synthetic bi-domain protein made by two A3 monomers linked together should fold as a closed bivalve, but could then be opened by a second distinct protein. This structural transition will a priori give rise to a large relative motion of the two linked domains. The opening and closing of a bidomain bivalve shell protein, consecutive to the binding of its ligand appear as a general way to create a conformational switch in this protein family and could later be used to create a range of αRep-based biosensors.The general design described here is based on the quaternary structure of one the previously described αRep proteins, named A3. This protein spontaneously forms a homodimer in solutionDespite this opening and closing possibility, it is difficult to predict precisely how such bidomain proteins will behave. For example, it is expected that covalently linking the two subunits of the A3 homodimer should reinforce the interactions between the two monomers and therefore should stabilize a closed form. But, would this closed bivalve still be able to open in order to bind an external ligand? Would it be possible to observe such a structural transition in αRep with other binding specificities? For example, how a chimeric protein made by an A3-domain and a different unrelated αRep domain would behave in solution? Would it give rise to a closed-bivalve? What will be the behavior of such a bidomain in the presence of ligands specific for each domain? The properties of such proteins are currently unpredictable and a better appreciation of these effects is clearly essential for the conception of multi-modular devices.Our goal was to address experimentally these questions and to establish clear observations on the possible behavior of such proteins.23 and bGFPD26 were connected by a flexible linker (SGGGG)2, creating two bidomains named A3_A3 (homo-bidomain) and A3_bGFPD (hetero-bidomain)  Figure .24. The interaction surface was determined using the crystal structure of the A3/bA3-2 complex (PDB ID 4JW2). bGFPD was selected from the αRep library as a specific binder for eGFP. It also contains 4 internal repeats and 2 caps.αRep A3 contains 4 internal repeats between an N-cap and a C-cap motif and its biophysical properties and crystal structure were previously described. The A3 homodimer dissociates in solution upon interaction with two bA3-2 and bA3–17 αReps that were selected from the library as specific binders for A326. As expected, the bidomain single chain, A3_ A3 is monomeric in solution, as the two linked domains interact together. The hydrodynamic radius of the bidomain A3_A3 (3.0 nm) is similar to the dimeric A3 (2.9 nm) indicating that the chimeric bidomain protein also adopts a closed bivalve conformation. SEC-MALS analysis of the A3_bGFPD construct yielded a broad and dissymmetric peak. Analysis of the higher elution volume fraction of the peak indicates a 44.8 kDa mass, which corresponds to A3_bGFPD monomer mass  A3 and bGFPD domains using ITC: A3 was titrated with bGFPD and the isotherm clearly showed a weak interaction with a KUpon the addition of one molar equivalent of each ligand (bA3-2 or eGFP) to the bidomains, changes in the SEC elution volumes and/or in the peak area were observed, indicating the formation of complexes of higher molecular weight. The stoichiometry of these complexes was determined by using the measured molar masses and molecular extinction coefficients. In both bidomains, each domain binds one molar equivalent of ligand Figure . Taken t26, bA3-2 interacts with A3 in a 1:1 stoichiometric ratio with a KD value of 4.5 ± 7 nM and eGFP interacts with bGFPD with a KD value of 2.7 ± 2.0 nM. The interaction between two A3 monomers resulting in the A3 dimer has been measured using Analytical Ultracentrifugation (AUC) and a KD value of 37 ± 6 nM (KD A3/A3) was determined  is higher than with A3 alone (without linker: 4.5 ± 7 nM), suggesting that the intramolecular link between the two binding sites stabilizes the closed form and induces a lower apparent affinity for an external ligand. The ITC data were reasonably well fitted with a model assuming equivalent and independent sites. It seems however clear that, if the two binding sites of the chimeric protein A3_A3 are nearly identical, the binding of a ligand on the first site will stabilize an open conformation of the bidomain structure and will facilitate the binding on the second site. The KD determined by this experiment should therefore be considered as an apparent value  Table , Fig. 2A24. The A3_A3 bidomain was pre-incubated with one of its ligands and the second ligand was then added as a competitor. In this experiment, bA3–17 was not able to replace pre-bound bA3-2 as illustrated by the lack of signal in ITC. This result is in accordance with the known lower affinity of bA3–17 for A3, relatively to bA3-2 for A3. In the reverse experiments, bA3-2 was able to replace bA3-17 with a KD value 9.7 ± 1.5 nM . We thus measured the affinity of bA3-2 for the monomeric form of A3, which is, as expected, higher than for the A3 dimer.The KWhen the bidomains are immobilized on the chip, the profile of the kinetics of ligand association and dissociation obtained with either bA3-2 Fig.  or eGFP Bidomains A3_A3 and A3_bGFPD were then used as circulating analytes on bA3-2 and eGFP immobilized ligands. The curves could not be fitted using simple models, but, clearly, the creation of chimeric proteins affects both association and dissociation phases. For both complexes bA3-2/A3_A3 and eGFP/A3_bGFPD Fig. , a slowe23. This suggests that this interface between caps of each monomer contributes to the dimer association. As the conformation of the A3_A3 bidomain is fully superimposable to the A3 dimer, the interactions involving the N-cap and C-cap are also present in the bidomain and favor the close conformation.Structural studies of the two bidomains were undertaken in order to have a better understanding of their behavior. The crystal structures of the two bidomains were determined with a resolution of 1.94 and 2.55 Å for A3_A3 and A3_bGFPD respectively. In neither structure electronic density was observed for the linker, which was expected as it was designed to be totally flexible. A3_A3 crystallizes with one A3 subunit in the asymmetric unit. SDS-PAGE of dissolved crystals showed that they contained the intact fusion protein Figure . The cryvia its A3 subunit. This dimer is identical to the A3 dimer and to the A3_A3 bidomain protein. SEC-MALS and SAXS results indicate the presence of an equilibrium between a monomeric and a dimeric form of the bidomain and at the concentration used for crystallization, the dimeric form is favored. For the dimer observed in the crystal structure  is higher than the interface (638 Å2) between the A3 and bGFPD domain of the same molecule. Furthermore, these two interfaces involve different types of residues. The dimeric interface between A3 domains is established by contact between their variable side chains. In the interdomain interface, the variable residues of the bGFPD domain are in contact with a non-variable part of the surface of A3  bidomain crystals have a single A3_bGFPD in the asymmetric unit. The structure shows an open conformation but the two domains are in contact: the loop linking the consecutives repeats of the A3 domain interact with the GFP binding surface of the second bGFPD domain. Importantly, the crystal symmetry generates a dimer with a neighbor molecule ure Fig. , two dis A3 Fig. . Since tg = 26.8 Å, Dmax = 90 Å), and could adopt different possible geometries of the closed forms.A SEC-SAXS experiment has been used to determine the conformation of A3_bGFPD in solution. The successive scattering patterns recorded at the end of the elution peak Figure , corresp27. A unique C-terminal cysteine was covalently grafted with an “ATTO 488 maleimide” dye used as FRET donor and the N-terminal His-tag was coupled with a “NTA-ATTO 647 N” dye used as a FRET acceptor. For both bidomains, . However even this relatively weak affinity between domains is sufficient to take place predominantly within a bidomain protein. This fortuitous and relatively weak interaction between A3 and bGFPD moieties may in fact be due to the interactions between the N-cap of the A3 domain and the C-cap of the bGFPD domain. All αReps share the same C-cap repeat. The A3 dimer structure clearly indicates that the specific sequence of the A3 N-cap can interact with the common C-cap repeat and this could contribute to the observed affinity.The SEC-MALS and SAXS results clearly show that, at low micromolar concentration both bidomains adopt a closed bivalve conformation. This was expected for A3_A3: when not linked together the two A3 monomers dimerize with relatively high affinity  but the underlying principles can be more generally useful. αRep binders for various unrelated proteins have already been generatedAs shown in the FRET assays for these two “model” bidomains, it is clearly possible to monitor the open/closed conformational change into a variation of fluorescence signal correlated to ligand binding. Such a closed/open transition in a bivalve-like protein could later prove useful as a general way to transduce the target-binding event into a measurable quantitative readout. Here, it will be possible to tailor chimeric proteins with domains that bind a target of choice, which is a substantial improvement over existing sensors.Moreover the cooperative effects observed between the binding partners of a chimeric protein made from two different αReps with different specificities could also be a basis to design future molecular switches. The structural transition induced by the ligand of one domain can enhance the binding on the second domain of a different, unrelated partner.a3s) previously described33. The DNA sequence a3s was designed with different codons at equivalent positions in consecutive repeat and therefore could be easily amplified by PCR.The αRep proteins are mainly composed of repeated sequences and therefore molecular biology procedures such as cloning based on PCR can be difficult due to the repeated codon sequences. This is further enhanced in sequences of bidomain proteins based on αRep. In order to simplify the constructions steps, two distinct DNA sequences coding for the same αRep A3 protein were used. The first one is the gene directly obtained from the αRep library (A3). The second one is a synthetic gene (2 were inserted ahead of the multiple cloning site in PQE81L using circular polymerase extension cloning (CPEC). In this step a new pair of unique cloning sites (BamH I/Hind III) was inserted just after the sequence of the linker. These sites were used to insert the a3 sequence using classical restriction sites cloning. The gene coding to bGFPD was cloned using the same cloning site (BamH I/Hind III).The sequence coding for TEV-A3 and a linker (SGGGG)A3_A3 and A3_bGFPD are described more precisely in Supplementary Figure ® constructs (IBA technology) were obtained from PQE80L in which the His-tag was removed.Gene construction of both bidomains 26. E. Coli BL21-Gold (DE3) Competent Cells (Agilent Technologies) were transformed with the plasmid coding for the different proteins. Cells were grown at 37 °C in 2YT medium containing 200 µg.L−1 ampicillin until the OD600nm reached 0.6. Protein expression was induced by addition of 1 mM IPTG and the cells were further incubated for 5 h at 37 °C. The cells were harvested, suspended in 50 mM sodium phosphate pH7.5, 150 mM NaCl (PBS), submitted to three freezing/thawing cycles, treated with DNase I for 30 min and sonicated. The His-tagged proteins were purified using nickel-affinity chromatography . The His-tag A3-A3 and A3-bGFPD was cleaved by TEV protease for some SPR experiments.Expression and purification of His-tagged proteins were performed as describedTwin-Strep®-tagged proteins  were also purified using a StrepTactin Sepharose affinity chromatography ; both samples obtained from this first step purification were then submitted to a size exclusion chromatography.−1) were loaded on a Superdex 200 10/300GL increase  equilibrated in PBS at a 0.5 mL.min−1 flow rate (Shimadzu HPLC system). Detection was performed using a MiniDAWN TREOS multi-angle light scattering detector and an Optilab T-rEX differential refractometer (Wyatt Technology). Absolute molar masses were determined with the Astra 6.1 software, using a differential index of refraction (dn/dc) value of 0.183 mL.g−1. Hydrodynamic radius was determined using the WyattQELS (Quasi-Elastic Light Scattering) module.Purified proteins  or with a VP-ITC . For the measurement monitored with the ITC 200, 2 µL aliquots of the titrant  at 300 µM were injected from a computer-controlled 40 µL microsyringe at intervals of 180 s into the solution of target (25 µM) dissolved in PBS (stirring at 700 rpm). For the measurement monitored with the VP-ITC, 10 µL aliquots of the titrant at 100 µM were injected from a 250 µL microsyringe at intervals of 240 s into the solution of target (8 µM) with or without 40 µM competitor) in PBS (stirring at 350 rpm). The data were integrated and analyzed using the MicroCal Origin software provided by the manufacturer according to the one-binding-site model.−1. ProteOn HTG sensor chip (Bio-Rad) were used to immobilized αRep proteins (bA3-2 and eGFP) at two densities on two of the six channels chip following the Tris-NTA / His-tag coupling protocol. Five concentrations (from 0 to 3 µM) of purified proteins  were tested. Signals were corrected with the uncoated reference channel and fitted with the Proteon Manager software by Langmuir model. The final figures were obtained using RStudio software.Surface plasmon resonance was measured using a ProteonTM XPR36 instrument (Bio-Rad). Immobilized proteins contain a N-terminal His-tag. Circulating proteins have no His-tag . All measurements were performed in PBS containing 0.05% Tween20 at a flow rate of 50 µL·min−1 of bovine serum albumin (BSA). For protein sample from 1 nM to 60 nM, only labeled protein was used; for higher concentrated samples labeled protein (60 nM) was mixed with unlabeled protein. 400 μL of A3 samples were centrifuged at 42,000 rpm . Sedimentation profiles were collected every 5 min and analyzed as described36.All sedimentation velocity experiments were performed on an analytical ultracentrifuge XLI  with an An-50 Ti rotor at 20 °C equipped with a fluorescence detection system . Sedimentation velocity experiments were done in two-channel 12 mm path-length Epon charcoal-filled centerpieces. A3 protein was labeled with Monolith NT.115 Protein Labeling Kit BLUE NHS (Nanotemper Technologies). Samples were prepared by dilution of concentrated stocks in PBS, 0.1 mg.mLSEC-SAXS data were collected with A3_bGFPD samples at the BM29 line at the ESRF, Grenoble, with a size-exclusion HPLC column (Agilent Bio sec-3) online with a SAXS measuring cell (a 1.5 mm diameter quartz capillary in an evacuated sample chamber) and the data analysis is detailed in Note S1.−1 ≤ c ≤ 8.0 mg mL−1) of A3_bGFPD were placed in a quartz capillary thermalized cell inserted into an evacuated sample chamber. SAXS data were analyzed using the program PRIMUS (https://www.embl-hamburg.de/biosaxs/primus.html).Other SAXS experiments were performed on an in-house SAXS instrument . 30 µl of concentrated solutions . The two bidomains crystallized under several different conditions. For A3_A3, the optimized crystallization conditions are 0.1 M Sodium-citrate pH 5.6, 2.4 M Ammonium sulfate. For A3_bGFPD best crystals were obtained in 1.6 M Tri-Sodium citrate pH6.5. Crystals were flash-frozen by soaking into mother liquor supplemented with 30% glycerol as cryoprotectant for A3_A3 and 3.8 M malonate for A3_bGFPD. Diffraction data were collected at 100 K on beamline ID29 at the ESRF synchrotron using a PILATUS 6 M detector. The images were analyzed with the XDS programfree crystallographic factors of 17.48% and 22.21% respectively . A 10 mM solution of ATTO 488 maleimide (Sigma) was prepared in anhydrous DMSO and mixed with the proteins samples in PBS . The mixtures were incubated overnight at 4 °C in the dark. Excess of ATTO 488 maleimide was washed off using Zeba Micro Spin desalting columns. ATTO 488 concentrations were determined by absorbance measurements at 501 nm using a molar absorptivity of 90 000 Mhttps://www.atto-tec.com) via the N-terminal His-tag. 10 molar equivalents of dye were added to 0.1 µM of ATTO 488-bidomain and incubated for 5 min in the dark.Each ATTO 488-labelled bidomain was then conjugated with NTA-ATTO 647 N  flx-Xenius V2 spectrofluorometer. Emission spectra were measured from 500 nm to 800 nm while exciting the samples at 488 nm. Spectra were recorded using a 1 nm step, a 10 nm bandwidth for both channels, an averaging time of 0.1 s and a PMT voltage of 500 V. Successive aliquots of bA3-2, BFP or albumin (without His-tag) were added in the cuvette containing 0.1 µM of labelled bidomain. The solutions were incubated for two minutes at room temperature in the dark before measuring each spectrum. Fluorescence emission intensities at 664 nm plotted as a function of total ligand concentration were fitted according to the following equation using the Origin program:Supplementary Information


A favorable energy band alignment facilitating interfacial photocarrier separation and efficient carrier injection into the MoS2 layer inside the 0D–2D mixed‐dimensional vdWHs are confirmed by a series of optical characterizations. Owing to the synergistic effect of the photogating mechanism and the modulation of Schottky barriers, the corresponding phototransistor exhibits a high photoresponsivity of 7.7 × 104 A W−1, a specific detectivity of ≈5.6 × 1011 Jones, and an external quantum efficiency exceeding 107%. The demonstration of such 0D–2D mixed‐dimensional heterostructures proposed here would open up a wide realm of opportunities for designing low‐cost, flexible transparent, and high‐performance optoelectronics.2D transition metal dichalcogenide (2D‐TMD) materials and their van der Waals heterostructures (vdWHs) have inspired worldwide efforts in the fields of electronics and optoelectronics. However, photodetectors based on 2D/2D vdWHs suffer from performance limitations due to the weak optical absorption of their atomically thin nature. In this work, taking advantage of an excellent light absorption coefficient, low‐temperature solution‐processability, and long charge carrier diffusion length, all‐inorganic halides perovskite CsPbI Great breakthrough has been made in this structure, of which the photoresponsivity was strikingly enhanced by a factor of 7.7 compared with that of pristine MoS2 device.To tackle these problems, several strategies such as integrating with strong light absorption materials,2 MvdWHs was reported to exceed 105 A W−1,[[qv: 13b]] and HgTe QDs/MoS2 structure had a broadband photodetection beyond 2 µm.Semiconducting 0D QDs with superior high‐absorption coefficient, large spectral coverage, tunability bandgaps, and low‐cost solution processing were also considered as an ideal sensitizer to combining with other light‐sensitive materials for improving photoresponse.3−xBrx QDs (PQDs) was integrated with monolayer MoS2 for low‐cost and high performance 0D–2D mixed‐dimensional heterostructured photodetector. A favorable band alignment and efficient carrier extraction by 2D‐MoS2 layer in this 0D–2D MvdWH was demonstrated, which facilitated to the photocarrier generation efficacy and photogating effect. Owing to the synergistic effect of photogating effect and the modulation of Schottky barriers, the optimized device exhibited an extremely high photoresponsivity of 7.7 × 104 A W−1, a specific detectivity of ≈5.6 × 1011 Jones, and an ultrahigh external quantum efficiency exceeding 107%. The demonstration of such 0D–2D mixed‐dimensional structure here would open up a wide opportunity for designing flexible, transparent, and high‐performance optoelectronics.In this work, all‐inorganic cesium lead halides perovskite CsPbI23−xBrx PQDs/MoS2 monolayer mixed‐dimensional phototransistor was shown in Figure2, which served as the channel of the hybrid phototransistor, was synthesized by oxygen‐assisted chemical vapor deposition2/Si substrate by poly(methyl methacrylate) (PMMA)‐assisted transfer method. Then the as‐synthesized colloidal CsPbI3−xBrx QDs dilute solution was uniformly spin‐coated onto the MoS2 monolayer. Before spin‐coating, the density of ligands in PQDs was controlled to improve the conductance by the method reported by Li et al.2 by thermal evaporation. Two characteristic Raman peaks of MoS2 monolayer were observed at 385.8 cm−1 (E12g mode) and 404.8 cm−1 (A1g mode); the difference between E12g and A1g mode peaks was 19 cm−1, well corresponding to the theoretical value of monolayer MoS2  spectra analysis on the pristine MoS2 monolayer, PQDs, and PQDs/MoS2 hybrid system. The UV–vis absorption spectrum of the pristine MoS2 layer presented two excitonic absorption peaks between 600 and 700 nm . In comparison, the PQDs/MoS2 MvdWH showed enhanced absorption below 645 nm wavelengths, which resulted from the synergetic absorption effect of PQDs and MoS2 layer.To investigate the optical characteristics of the PQDs/MoSm Figure d, which WF) of materials,2 MvdWH. Dual‐pass amplitude‐modulated‐KPFM mode on Bruker Dimension Icon with a Pt/Ir‐coated probe  was employed to measure the surface potential at ambient atmosphere. Before measuring, the work function of the tip was calibrated by taking the contact potential difference (CPD) on Cu foil. The CPD was nearly zero which indicated that the work function of the tip was close to that of Cu (≈4.8 eV). The measured CPD between the sample and the tip can be expressed by the following equation[[qv: 24b]]: e × VCPD = WF, tip − WF, sample, where WF, tip and WF, sample are the work functions of the tip and sample, respectively, and e is the elementary charge.Since Kelvin probe force microscopy (KPFM) is sensitive to the surface work function  toward the MoS2 layer  toward the PQDs/MoS2 MvdWH  analysis ,] coinciding with the KPFM results.The topography profile of the pristine MoS2 monolayer and PQDs were reported to be 4.25[[qv: 25a]] and 3.6 eV2 MvdWH was determined to form a type‐II energy band alignment  versus VG (gate voltage)) were investigated both in dark and under illumination at a constant drain voltage of 1 V , the drain current of the hybrid phototransistor was dramatically enhanced by 15.3 times compared with pristine MoS2 device (obtained at VD = 1 V), manifesting the strong absorption characteristics of PQDs and effective separation of photoexcited carriers.To evaluate the improvement in the optoelectronic performance of the PQDs/MoS2 mixed‐dimensional phototransistor under 532 nm illumination with a range of optical powers (from dark to 1.5 µW) are shown in Figure Iph = Ilight − Idark, obtained from FigurePeff) at a fixed gated voltage of 60 V is plotted in Figure Iph ∼ Peffα, where the obtained exponent α via linearly fitting the curve is 0.72, indicating there existed trap‐assisted recombination at the interface of MvdWH.ID–VD curves (obtained at VG = 0 V) both in dark and under illumination with different powers are shown in the inset of Figure 4  and the output characteristics under illumination and dark  both manifest good gate modulation of the hybrid device.The photoinduced transfer curves of the PQDs/MoSR = Iph/Peff) and specific detectivity (D* = RA(1/2)/(2eId)1/2),Peff is the effective incident power (Peff = Pin × Adevice/Alaserspot), A is the effective detection area, Id is the dark current, and e is the unit charge. The curves of calculated R and D* as functions of illumination power were plotted in Figure 2 mixed‐dimensional photodetector reached a photoresponsivity (black line) of 7.7 × 104 A W−1 and a specific detectivity (orange line) of ≈5.6 × 1011 Jones under illumination power in the nanowatt scale, which is quite outstanding performance compared to those previously reported TMD‐based photodetectors.]Then, we analyzed two most important figures of merit for a photodetector, external photoresponsivity  is defined as the number of electrons collected per incident photon bought about and expressed by the equation: EQE(%) =Nel/Nph =IphPeff hcλe× 100,h is the Planck's constant, c is the speed of light in vacuum, and e is the elementary charge. As shown in Figure 2 mixed‐dimensional phototransistor yielded a very high EQE value exceeding 107% at a VG of 60 V, which is several orders of magnitude higher than those of previously reported ones.Vth) from the relatively linear part of the transfer curves in Figure Vth as a function of effective incident power (Peff) was plotted in Figure ΔVth=aPeffb,b value ≈ 0.21, smaller than 1, confirmed the existence of photogating effect in our phototransistor. Specifically, the dominate photocurrent generation mechanism can be determined through a simple power‐law, Iph ∝Peffα,[[qv: 33b]] where we extracted α by linear fitting the log‐plotted of Iph versus Peff . The obtained α ranges from 0.83 to 0.22 as the applied gate voltage increases, manifesting that the dominate mechanism can be tuned from photoconduction to high gain photogating when the applied gate voltage gradually increased.Note that both R and D* decreased exponentially with increasing incident power. This phenomenon was attributed to higher probability of scattering and recombination under stronger illumination,qv: 9,13b]] and higher values of b] and hib] and hiVG < Vt, OFF state) and the accumulation regime .] As is illustrated in Figure Moreover, as shown in Figure  As is  As is  As is 2 MvdWH played an important role for the photocurrent generation, we conducted time‐resolved photoluminescence (TRPL) and fluorescence lifetime imaging microscopy (FLIM) measurements. The transient photoluminescence decay curves were fitted by a two‐component exponential decay model (F(t)=∑aie−(t−t0)/τi, i=1,2) in Figure 1 and slow decay τ2. The fast component related to trap‐assisted recombination at grain boundaries in pure PQDs or carrier extraction by layers in PQDs/MoS2 MvdWH, whereas the slow one related to the radiative recombination inside the grains or at the interface of the MvdWH.To further investigate and verify whether the photoinduced interfacial charge transfer of our PQDs/MoS2 layer.2 layer,2 MvdWH were obtained with the samples excited by a pulsed diode laser  at excitation fluences of 3.5 µJ cm−2, filtered by 690/50 and 620/50 nm bandpass filters, and spatially mapped in Figure 2 layer obtained from 690/50 nm bandpass filters, which was determined to be 0.9 ns and is comparable to previously reported values2 layer, obtained from 620/50 nm bandpass filters, was significantly shortened (dark blue triangle) than that of pure PQDs on glass substrate (bright red dots), which are well consistent with the TRPL results in Figure 2 channel was accelerated, leading to the dramatically increased photocurrent and ultrasensitive photoresponse.We found that the photoluminescence transients of the PQDs in the MvdWH were significantly slowed down compared with that of pure PQDs, from average 26.4 ns in pure PQDs to about 7.8 ns in the MvdWH. This phenomenon indicated the appearance of a strong quenching effect resulting from the effective charge transfer from PQDs to MoS2 hybrid photodetector was investigated under 1.5 µW pulsed illumination power over multiple cycles at VD = 0.1 V  powder (0.01 g) and covered by a cleaned Si/SiO2 substrate was pushed in the center of a tubular furnace. Another ceramic boat filled with sulfur (S) powder was placed at the upstream region of the furnace at a lower temperature zone. The temperature was first raised to 300 °C and held for 30 min to preheat MoO3 powder, then to 850 °C/30 min to grow large monolayer MoS2 crystals. Meanwhile the S powder was heated to 180 °C by a heating belt, and the sulfur vapor flowed into the furnace by the carrier gas of Ar. Particularly, 2‐sccm O2 was used to decrease the nucleation density at the growing stage. Finally, the furnace was gradually cooled down to room temperature. During the whole process, the ultrahigh‐purity Ar was held at 500 sccm under atmospheric pressure.3−xIx Quantum dotsSynthesis of CsPbBr: Colloidal CsPbI3−xBrx perovskite QDs with excitation peak at 646 nm were synthesized as Joseph M. Luther's reported method with some modification.2 (0.03 g) and PbI2 (0.1 g) mixtures along with 10 mL of octadecene (ODE) were loaded into a 50 mL three‐neck flask, and degassed at 120 °C for 30 min under vacuum. The oleylamine and oleic acid  acted as ligands and were injected into the flask at 120 °C under protection of N2 after being preheated to 70 °C. Then the flask was put in vacuum again until the mixtures completely dissolved and no gas released. After that, the Cs‐oleate , prepared by dissolving CsCO3 in ODE and OA at 150 °C, was quickly injected at 170 °C. After 5 s, the reaction mixture was quenched by immediately immersing it in ice bath, and the color turned to dark red. Finally, the colloidal QDs were precipitated by tert‐butanol and separated via repeatedly centrifugation.Device Fabrication: The as‐grown MoS2 films were transferred on Si/SiO2 substrate by PMMA‐assisted method, that is, first spin‐coated a PMMA supporting layer on MoS2 films, then lifted off the PMMA/MoS2 layers by NH4OH solution (25%) and followed by removing the PMMA layer in acetone. Then Au (100 nm) source and drain contacts with a 2 µm in length and 20 µm in width channel, which was defined by standard photolithography and electron‐beam lithography, were fabricated on MoS2 layer by thermal evaporation. The MoS2 samples were annealed at ≈250 °C for 2 h under Ar/H2 (100/10) to remove resist residue and improve contact conductance as previously reported.−1) was uniformly spin‐coated onto MoS2 layer with a speed of 2000 rpm. The surface ligands' density was controlled by ethyl acetate and 1‐octane  to increase conductance before spin‐coating. Finally, the device was annealed at 60 °C for 15 min to evaporate the organic solvent and improve contacts at the interface for electrical measurements.Optical and Electrical Measurements: The TEM images of the perovskite QDs were taken on an FEI‐Tecnai G2 F20 TEM instrument with an accelerating voltage of 200 keV. Raman and PL spectra were measured by a confocal Raman microscopic system (Horiba Jobin Yvon HR800) with a 532 nm laser as the excitation source; the absorption spectra were obtained by UV–visible–NIR spectrometer (Cary 5000). The KPFM measurement was performed on a Bruker Dimension Icon with a Pt/Ir‐coated probe  by amplitude‐modulated‐KPFM mode at ambient atmosphere. Before measuring, the work function of tip was calibrated by Cu foil, which work function is about 4.8 eV. UPS measurements were performed using He I (21.22 eV) excitation lines as excitation sources in an ultrahigh vacuum (2E−8 mbar) chamber (Thermo Scientific ESCALab 250Xi). A confocal two‐photon fluorescence lifetime image microscope system (ARsiMP‐LSM‐Kit‐Legend Elite‐USX) was employed for the FLIM and corresponding TRPL measurements. The electrical measurements were characterized by a semiconductor parameter analyzer (Keithley 4200‐SCS) combined with a probe station at room temperature, and a 532 nm diode laser (spot size ≈1 mm) with a tunable attenuator was used as the illumination source.The authors declare no conflict of interest.SupplementaryClick here for additional data file.


LIM and SH3 protein 1 (LASP1) is a specific focal adhesion protein that promotes metastasis in a variety of tumours. However, its role in head and neck squamous cell carcinoma (HNSCC) has not been fully validated. The purpose of this study was to analyse the interaction of LASP1 and its binding partner in HNSCC. The expression of LASP1 and HSPA1A in HNSCC was analysed by real‐time PCR and Western blot. The effects of LASP1 on the biology behaviour of HNSCC cell lines were observed in vivo and in vitro. Co‐immunoprecipitation analysis was performed to confirm the interaction between LASP1 and HSPA1A. LASP1 was highly expressed in HNSCC and associated with poor prognosis for patients. LASP1 also promoted cell proliferation, colony formation, invasion and cell cycle G2/M phase transition. Heat shock protein family A member 1A (HSPA1A) is identified as a chaperone protein of LASP1 and co‐localized in the cytoplasm. HSPA1A positively regulates the interaction of LASP1 with P‐AKT and enhances the malignant behaviour of HNSCC cells. LASP1 and HSPA1A are both up‐regulated in HNSCC, and directly binds to each other. Double inhibition of LASP1 and HSPA1A expression may be an effective method for the treatment of HNSCC. In conjunction with other heat shock proteins, HSPA1A mediates the folding of newly translated protein and makes existing proteins stable against aggregation in the cytosol and in organelles.In this present study, we found that HSPA1A directly binds with LASP1, co‐located in the cell cytoplasm and plays a synergistic role in promoting tumours in HNSCC by elevating the interaction between LASP1 and P‐AKT. Our results suggested that LASP1 promoted HNSCC proliferation, metastasis and invasion through direct interaction with HSPA1A.22.1The clinical samples used in this study are 40 pairs of HNSCC tissues and adjacent normal tissues. All the samples were collected from the Department of Oral and Maxillofacial‐Head and Neck Oncology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine .2.22 incubator at 37°C.The HNSCC cell lines HN4, HN6 and HN30 were obtained from the University of Maryland Dental School, USA, and CAL‐27 was provided by the American Type Culture Collection, USA. The periodontal ligament cells (PDLC) which are epithelial cells from head and neck in this study were primarily cultured in DMEM supplemented with 10% heat‐inactivated FBS, penicillin (100 units/mL) and streptomycin (100 μg/mL).2.3‐ΔΔCt.Total RNA was isolated from head and neck tumour tissues and cell lines according to the manufacturer's instructions (9108/9109) and measured at optical density measurements at 260 nm and 280 nm for total RNA concentration and purity; the RNA was then converted to cDNA using a reverse transcriptase kit (DRR420A). Experiments were performed using the real‐time PCR instrument (ABI PRISM 7500). The primers were synthesized by Shanghai Sheng Gong Bioengineering Co., Ltd., and the sequence was as follows: LASP1: F: 5′‐CTG TCT CTG CCT TAT AGC AAC AC‐3′, R: 5′‐CAT CTC GAA CCT GGC TGT TTG‐3′. HSPA1A: F: 5′‐CCA GAA CAA GCG AGC CGT GAG‐3′, R: 5′‐TCG GAA CAG GTC GGA GCA CAG3′. GAPDH: F:5′‐CAT CTC TGC CCC CTC TGC TGA‐3′, R: 5′‐GGA TGA CCT TGC CCA CAG CCT‐3′. Three replicate wells were set in each group, GAPDH was used as an internal reference, and the relative expression level of the gene was calculated using 22.4The total protein of HNSCC cells and PDLC cells was extracted with SDS lysis buffer (Beyotime) and determined by BCA kit (Thermo). The amount of protein in each swimming lane was 30 µg in the premade gel  and was electrophoresed at a constant pressure of 140 V for 1 hours, followed by a transmembrane with a constant 400‐mA flow. The PVDF membrane (Merck Millipore) was incubated with primary antibody at 4°C overnight. The antibodies are listed as follows: a rabbit anti‐human LASP1(8636), polyclonal primary antibody; rabbit anti‐human P‐AKT (4060), polyclonal primary antibody; rabbit anti‐human AKT (2920), polyclonal primary antibody; rabbit anti‐human β‐tublin (2148), polyclonal primary antibody ; and rabbit anti‐human HSPA1A, polyclonal primary antibody . HRP‐labelled rabbit antibody  was used for incubation for 1h, and an ECL  developing solution was added and developed on a chemiluminometer (Amersham Imager 600).2.5LASP1 interference control lentivirus and three interfering lentiviruses, overexpression control and overexpression lentivirus were constructed by Shanghai Jiman Biotechnology Co, Ltd. The target strand sequences of human shRNAs for LASP1 and HSPA1A were as follows: LASP1, shRNA1: GAA CTA CAA GGG CTA CGA GAA; shRNA2: GCA TAA AGC ATG CTT CCA TTG; shRNA3: GCG CTA CAA GGA GGA GTT TGA; NC shRNA: TTC TCC GAA CGT GTC ACG T. HSPA1A: shRNA1: GGA TCC AGT GTT CCG TTT CCA; shRNA2: GTC AGT TCT CAA TTT CCT GTG; shRNA3: GCC ATC TTA CGA CTA TTT CTT; NC shRNA: TTC TCC GAA CGT GTC ACG T. The cDNA sequence of LASP1 is in accordance with the Genebank (PUBMED ID: NM 001271608.1) and the transduct into PGMLV‐6751. The empty lentiviruses were selected as the negative control for OE‐DNA.5 cells CAL27 and HN6 cells per well were seeded in the six‐well plate, cultured until the cell density was about 40%. After 24 hours of infection, the complete medium was replaced. The infection efficiency was observed after 48 hours, and the cells were screened with puromycin‐containing medium after 72 hours. Two weeks later, CAL27, HN6 interference and overexpression stable cell lines were constructed. The HSPA1A interfering cell line was constructed in the same manner in the CAL27 (sh‐LASP1 and OE‐LASP1) and HN6 (sh‐LASP1 and OE‐LASP1) stable cell lines using the blasticidin.The average 1 × 102.6The cells in logarithmic growth phase were inoculated into the 96‐well plates at 1000 cells/well. The CCK‐8 reagent  was added at 10 µL per well and placed in the incubator for 2 hours, and then, it was detected by a microplate reader  at a wavelength of 450 nm. Absorbance (OD) values were recorded to indicate the cell viability.2.7The cells in logarithmic growth phase were inoculated into six‐well plates at 500 cells/well, and the culture medium was changed every two days. When the cells showed obvious clonal proliferation, the culture was ceased. The culture plates were washed three times with PBS, fixed with paraformaldehyde for 30 minutes and washed with PBS for another three times. The cells were stained with 0.1% crystal violet staining for 1 hours, followed by wash with PBS three times. The images were recorded with a scanner (CanoScan 5600F), which quantified the number of cell clones with over 50 cells.2.85/well. The cell invasion assay was implemented with Matrigel (BD Biosciences) in chamber. After culturing for 24 hours, the cells were fixed and stained with 0.1% crystal violet for 1 hour, followed by washing with PBS 3 times. Images of three fields were captured, and the cells were counted.The cells in the logarithmic growth phase were cultured in a double‐free (serum‐free and antibiotic‐free) DMEM medium and then inoculated into the transwell chamber  at the density of 5 × 102.95 cells in logarithmic growth phase were seeded into each well of the six‐well plates for 24 hours. The cells were collected and fixed in 70% ethanol for 24 hours at 4°C overnight, followed by washing with PBS 3 times. Next, cell cycle detection reagent (556547) was used to stain for 0.5 hours on ice, and the cells were analysed by flow cytometry .The 2 × 102.106) were injected into nude mice through the tail vein to evaluate the tumorigenic ability. Five mice were used in each group. After 42 days, the mice were sacrificed by cervical dislocation. The number of metastatic lung nodules in each mouse was counted under the microscope . H‐E staining and immunohistochemistry  were used to analyse tumours in the lung, followed by a statistical analysis of the differences between the CAL27 scramble control and interference groups.All animal experiments were approved by the Animal Research Committee of the Experimental Animal Research Center of the Ninth People's Hospital of Shanghai and in accordance with the guidelines for international animal research. Tumour cells CAL27 (Scramble or sh‐LASP1)  and HSPA1A antibody  at 4°C overnight. The following day, the slips were incubated with the corresponding secondary antibody. The cells were contained with 4′,6‐diamidino‐2‐phenylindole  to detect nuclei. The cells were observed and imaged using a TCS SP2 laser‐scanning confocal microscope (Leica Microsystems).2.12g for 30 seconds, and the proteins were then separated from the beads using immunoblotting loading buffer for 10 minutes at 95°C. The supernatants were collected for subsequent immunoblotting analysis with the indicated antibodies.The cells were lysed with IP buffer . After the protein concentration was determined to be 1 mg/mL, the cell lysates were incubated with rabbit anti‐human LASP1 or HSPA1A  antibody, as well as the protein A/protein G‐coated agarose beads (Merck) overnight at 4°C; the cell lysates were then washed with PBS 3 times and centrifuged each time at 1000 × In the LC/MS/MS assay, CAL27(OE‐LASP1) cells were lysed with IP buffer, and anti‐FLAG beads  were added into the cell lysates at 4°C overnight; as mentioned above, we used Protein Stains Kit  and noticed an obvious band, and this band was analysed by .2.132 tests of independence. The Kaplan‐Meier test was used for univariate survival analysis. The Cox regression test was used for multivariate analyses. Student's t test and one‐way ANOVA were used to compare the means of 2 groups or more. All of the experiments were independently performed 3 times. P < .05 was considered as significant.The data were analysed using SPSS19.0. The interrelationship between LASP1 expression and histology or clinical factors was analysed using Fisher's exact test and χ33.1P = .0482) and TNM stage (P = .0174) but not age, sex or smoking history. The mRNA expression of HSPA1A was not associated with any clinical parameters , as detected by flow cytometry, whereas there was no significant change in the G1 and S phase, indicating that LASP1 promotes the cell cycle G2/M phase transition , as measured with flow cytometry, whereas there was no significant change in the G1 and S phase. These findings indicate that LASP1 positively promotes the cell cycle G2/M phase transition   and control cells were injected into nude mice through the tail vein. After 42 days, we found that the number of surface nodules in the lung tissue in the interference group was significantly lower than that in the control group  cells significantly promoted cellular proliferation compared with lower cells (sh‐LASP1), whereas knocking down HSPA1A attenuated the influence of overexpressing LASP1 (Figure 4In this study, we examined LASP1 levels through a publicly available database and our native data. We observed that a high expression of LASP1 is closely related to shorter survival and a poor prognosis in HNSCC tissue. Furthermore, the overexpression of LASP1 promoted proliferation, the formation of cell clones, metastasis and invasion, the acceleration of the transition of G2/M phase in the HNSCC cell lines and the formation of lung metastasis in nude mice in vivo. To identify the binding partner of LASP1 that mediated the pro‐oncogenic effect, we performed a Co‐IP assay followed by LC/MS/MS examination. Interestingly, we confirmed that the heat shock protein family A member, HSPA1A, is the most significant binding partner of LASP1. Moreover, HSPA1A is critical for the promotion of the biological behaviour of HNSCC cell lines by enhancing the interaction between LASP1 and P‐AKT.There is increasing evidence that LASP1 is not only a specific focal adhesion protein with related cytoskeletal proteins but also a signalling protein which performs its biological function with multiple chaperones, dynamically participating in a wide range of cellular behaviours.4 exposure can activate the hippocampal PI3K/AKT signalling pathway in the rat hippocampus, leading to apoptosis of the central nervous cells, accompanied by enhanced HSPA1A transcription and translation.Therefore, after we confirmed the direct binding of LASP1 with HSPA1A, we speculated the interaction between LASP1 and the P‐AKT/AKT pathway and that inhibiting HSPA1A would greatly reduce the biological behaviour of HNSCC cell lines. HSPA1A is widely present in the cytoplasm, exosomes and cell membrane, and this protein induces tumour cell growth, cell migration as well as therapeutic resistance.In summary, LASP1 and HSPA1A are both up‐regulated in HNSCC, and HSPA1A directly binds to LASP1, which synergistically affects the development of HNSCC. We therefore expect that the double inhibition of LASP1 and HSPA1A expression may be an effective method for the treatment of HNSCC.The authors confirm that there are no conflicts of interests.All animal experiments were approved by the Animal Research Committee of the Experimental Animal Research Center of the Ninth People's Hospital of Shanghai and in accordance with the guidelines for international animal research.This manuscript is consent for publication.


This is the first hydroxide solvate that is not in contact with a cation and furthermore one of rare known water‐stabilized hydroxide anions. Thermolysis in vacuum results in the decomposition of the hydroxide salt and quantitative liberation of the free phosphazene base. This approach was used to synthesize the Schwesinger base from its hydrochloride salt after anion exchange in excellent yields of over 97 %. This deprotonation method can also be used for the phosphazene‐base‐catalyzed preparation of the Ruppert–Prakash reagent Me3SiCF3 using fluoroform (HCF3) as the trifluoromethyl building block and sodium hydroxide as the formal deprotonation agent.The reaction of a strongly basic phosphazene (Schwesinger base) with water afforded the corresponding metastable hydroxide trihydrate [OH(OH No contact with the cation: The metastable [OH(OH2)3]− anion is generated by treatment of a strongly basic phosphazene (Schwesinger base) with four equivalents of water. Thermolysis under vacuum results in the decomposition of the hydroxide salt and quantitative liberation of the free phosphazene base. This process was used to synthesize the Schwesinger base on a larger scale. Thus, currently a considerable number of reports focus on oxonium ions of the general formula [OH3)nOHm)]n+ sucnOHm)]n+ 2)]−.− anion. Thus, it is obvious that the presence of weakly Lewis acidic cations is a prerequisite for the observation of well‐separated hydroxide ions solvated by a shell of water molecules.Considering the many variations of hydrated oxonium salts in the liquid or solid state, information on isolated hydrates of the hydroxide anion is rare.MeCNpKBH+values, such as 26.91 up to 42.72  was prepared from phosphorus pentachloride and tert‐butylamine in a Kirsanov‐type reaction.2N)3PNH (4) is accessible in analogy to the synthesis of (Me2N)3PNH as previously devised by Schwesinger et al.4 and 5 resulted in a nucleophilic substitution with the formation of phosphazenium chloride [{Et2N)3P=N}3P=NH(tBu)]Cl ([3H]Cl) in high yield. In the 31P NMR spectrum of salt [3H]Cl the central phosphorus atom was observed as a quartet of doublets at δ=−33.9 ppm with coupling constants of 2JPP=70 Hz and 2JPH=8 Hz. The phosphorus atoms of the three peripheral phosphazenyl substituents gave rise to a signal at δ=7.4 ppm, which is split into a doublet of tridecets with a 2JPP coupling constant of 70 Hz and a 3JPH coupling constant of 10 Hz to the 12 methylene protons of the ethyl units.Compound Cl[3H]Cl was elucidated by X‐ray crystallography. Suitable single crystals were grown from a saturated ethereal solution at −28 °C  pm, representing a double bond. The bonds P1−N2, P1−N3, and P1−N4 range from 163.2(2) to 163.8(2) pm and also point to some degree of multiple bonding.The byproduct a Scheme .17 The mation of HCl to th3 in n‐hexane. The resulting phosphazenium hydroxides are soluble in chlorobenzene. At room temperature, however, such solutions slowly decompose. This product separates from polar solvents as an oil, whereas amorphous or crystalline samples are obtained from apolar solvents like n‐hexane. This finding may be rationalized by the various amounts of water incorporated in the precipitates, which hampered the isolation of a well‐defined bulk material. However, colorless crystals were obtained after slow evaporation of a methanol/water solution of 3 at room temperature and atmospheric pressure. The result of the elemental analysis revealed a possible hydroxide hexahydrate salt of the protonated phosphazene, [OH(OH2)6]− . Since the crystals did not show any diffraction pattern, they could not be analyzed by X‐ray diffraction. Nevertheless, a single crystal of 2)3][3H][OH with 339.4(3) pm. This distance is clearly greater than the van der Waals radii and shows that this is the first example of an isolated hydroxide hydrate anion that is not in direct contact to a cation. The O−O distances are in the range of 251.6(1) pm to 260.2(3) pm. The calculated value of the C3‐symmetrical hydroxide shows a slightly longer distance of 261.2 pm 3][3H][OH3][3H][OH3][3H][OH(OH. Following a procedure by Taylor and Hupfield2)3][3H][OH(OH by exposing solutions of [3H]Cl to a strongly basic OH− ion‐exchange resin (Scheme 3 was isolated after thermolysis of 2)3][3H]Cl.A viable and elegant conversion of HCl into 3SiCl to generate the Ruppert–Prakash reagent, as shown in Scheme 3SiCF3 is the most commonly used trifluoromethylation agent in laboratory and industry,We used this method for the phosphazene‐base‐catalyzed trifluoromethylation reaction of Me3) is an excellent and cheap source of the trifluoromethyl building block, since it is a waste product of Teflon production.3 can be performed by using n‐butyllithium or potassium tert‐butanolate; however, the high fluorophilicity of alkali cations leads to a carbenoidic bond situation and rapid difluorocarbene elimination at low temperatures.3SiCF3. Prakash et al. managed the synthesis of Me3SiCF3 in high yields from Me3SiCl, HCF3, and potassium bis(trimethylsilyl)amide (KHMDS) in toluene at low temperatures.2 and fluoroform.Fluoroform  with yields of up to 75 % amine and use of 2 in catalytic amounts of about 20 mol %.[3H]Cl in the trifluoromethylation reaction 3]−Cl in excellent yields of over 98 % and reused for the trifluoromethylation reaction without a loss of reactivity. Since the exchange resin can be regenerated with aqueous sodium hydroxide solution, in the overall reaction the trifluoromethylation is carried out using sodium hydroxide as base.In conclusion, we have reported the first structurally characterized metastable hydroxide trihydrate The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


We present the temperature dependent density, fluorescence emission and absorption spectroscopic data, that are needed for an evaluation of the excited state dipole moment of anisole in ethyl acetate via the methods of thermochromic shifts. Furthermore, the rotationally resolved electronic Stark spectrum of anisole in the molecular beam is presented. Finally, the Cartesian coordinates of the CC2/cc-pVTZ optimized structures of anisole are given in bohr units. For details about the evaluation of the dipole moments from the given data, see the connected research article: Lindic et al. (2018)  Specifications tableValue of the data•Temperature dependent absorption and emission data can be used for alternative evaluation schemes for dipole moments etc.•The temperature dependent density data can be used for own determinations of cavity sizes.•ab initio structures give the optimized structures at CC2/cc-pVTZ level of theory and can be used as good starting geometries for other levels of theory.The 1••-1. The field free spectrum had been obtained before by Pasquini et al. •3.•Table 2.dat•Table 3.dat••S0 in Bohr units from the CC2/cc-pVTZ calculations using the Turbomole program package [3].•S1 in Bohr units from the CC2/cc-pVTZ calculations using the Turbomole program package [3].2w in a temperature range of 263 K and 343 K with an increment of 2 K using a density meter (Anton Paar DMA4500M). The cavity volume is determined from the slope of the graph of ρ-1 vs. the mole fraction w at each temperature The density ρ of the solution of anisole in ethyl acetate was measured at different mass fractions Absorption spectra of anisole, dissolved in ethyl acetate have been recorded in a temperature range of − 10 °C and + 70 °C with 5 °C increment using a Varian Cary 50 Scan UV spectrometer


NxN matrix A, whose elements estimate the dependence within each possible pair of signals. Such matrix can be used as a feature vector for (un)supervised subject classification. Yet if N is large, A is highly dimensional. Little is known on the effect that different strategies to reduce its dimensionality may have on its classification ability. Here, we apply different machine learning algorithms to classify 33 children (age [6-14 years]) into two groups  using EEG FC patterns obtained from two phase synchronisation indices. We found that the classification is highly successful (around 95%) if the whole matrix A is taken into account, and the relevant features are selected using machine learning methods. However, if FC algorithms are applied instead to transform A into a lower dimensionality matrix, the classification rate drops to less than 80%. We conclude that, for the purpose of pattern classification, the relevant features should be selected among the elements of A by using appropriate machine learning algorithms.Functional connectivity (FC) characterizes brain activity from a multivariate set of N brain signals by means of an  The redundant connections found by this strategy are: O1 − O2)α and the later one with When applying FCBF, 12 out of the 280 features have non-zeron SU correlation values. Then, the strategy selected 10 of them as predominant features. The selected connections are shown in T3 − Fp2)β and O1 − C4)γ, As we can see in E1 and E2 in a given band is represented in the figure as Now we will analyse the BN classifier models generated with the connections selected by FCBF and SS. O1 − C4)γ depend on values of (O1 − O2)α. We can also see that connection T3 − Fp2)β and (C3 − C4)β and C3 − C4)β. It is worth noting that (C3 − C4)β influences two different connections. O1 − C4)γ and (O1 − O2)α is inverted. Another difference is that (C3 − C4)β receives influences from (T3 − Fp2)β and C3 − C4)β and (T4 − Fp1)β are influenced by other four connections each. The influence of (C3 − C4)β comes from T3 − Fp2)β, (Fp2 − C4)δ and (T4 − Fp1)β. Finally, (T4 − Fp1)β is influenced by Fp2 − C4)δ, (T3 − Fp2)β and In C3 − C4)β and O1 − C4)γ and (O1 − O2)α is also present in all models although the direction of the arc is different in the first model.Note that in all models generated with connections selected by FCBF, we found that O1 − O2)α and (O1 − C4)γ. Finally, in The models obtained with SS are much simpler than those obtained with FCBF, since SS only selected five connections. As we can see in O1 − C4)γ and (O1 − O2)α is also presented in two of the three models generated with the connections selected by SS. This dependence is also found in the models generated previously with FCBF. Thus, we think that this is a robust result.Finally, it is noteworthy that the dependence between , ape, e.g., . In othee, e.g., , 67) to e, e.g., . In bothAnother interesting issue that we have investigated is the effect of the segment selection procedure and the estimation of the statistical significance of the FC indices on classification accuracy. The main conclusion in this regard was that the optimal combination consists in selecting the most stationary segments among those available ones and at the same time use the values of the indices as such, without any thresholding from the multivariate surrogate data. The result concerning the need for a careful selection of the segments according to their stationarity is hardly surprising. In fact, stationarity is known to be one of the prerequisites to estimate many interdependence indices such as correlation, coherence, mutual information or those based in the concept of generalized synchronisation , and theAdmittedly, we cannot guarantee that these results will held for other sets of subject / neuroimaging modalities. It may be, for instance, that, contrary to what we have found here, there are instances where the original FC features, even after conveniently selected, do not outperform FC-based methods of dimensionality reduction such as SCA. Or that the use of surrogate data may be useful when the number of electrodes is high. Besides, it may be that BNC could no be always the best classifier. Different algorithms such as SVM , linear A very recent metanalysis of neuroimaging biomarkers in ADHD  has warnThat said, our results suggest that the combination of a careful selection of the data segments, a suitable feature selection method and a machine learning algorithm such as BNC, able to cope with high dimensional data, can turn the curse of dimensionality into a blessing, as the availability of many features allows selecting an optimal subset of meaningful, information-rich variables that can accurately classify subjects from their brain connectivity patterns even from scalp EEG data.In conclusion, the present outcomes indicate that the use of machine learning algorithms on EEG patterns of FC represents a powerful approach to investigate the underlying mechanisms contributing to changes, as regard to controls, in FC among different scalp electrodes, while allowing at the same time the use of this information for subject classification. They also suggest that this approach may not only be relevant for clinical applications (as it is the case for the theta/beta ratio in ADHD ), but al