extfieldsofstudy:
Materials Science
Medicine

text:
Integration of CdSe/CdSexTe1−x Type-II Heterojunction Nanorods into Hierarchically Porous TiO2 Electrode for Efficient Solar Energy Conversion

Semiconductor sensitized solar cells, a promising candidate for next-generation photovoltaics, have seen notable progress using 0-D quantum dots as light harvesting materials. Integration of higher-dimensional nanostructures and their multi-composition variants into sensitized solar cells is, however, still not fully investigated despite their unique features potentially beneficial for improving performance. Herein, CdSe/CdSexTe1−x type-II heterojunction nanorods are utilized as novel light harvesters for sensitized solar cells for the first time. The CdSe/CdSexTe1−x heterojunction-nanorod sensitized solar cell exhibits ~33% improvement in the power conversion efficiency compared to its single-component counterpart, resulting from superior optoelectronic properties of the type-II heterostructure and 1-octanethiol ligands aiding facile electron extraction at the heterojunction nanorod-TiO2 interface. Additional ~32% enhancement in power conversion efficiency is achieved by introducing percolation channels of large pores in the mesoporous TiO2 electrode, which allow 1-D sensitizers to infiltrate the entire depth of electrode. These strategies combined together lead to 3.02% power conversion efficiency, which is one of the highest values among sensitized solar cells utilizing 1-D nanostructures as sensitizer materials.

Optical Properties of NRs and HNRs. Absorption and photoluminescence (PL) spectra of NRs and
HNRs used in this study are shown in Fig. 1. Each sample is synthesized from CdSe NR seeds, with the second components grown at the tips. TEM images of NRs and HNRs are presented in Fig. 2, showing that HNRs synthesized from ~15 nm long CdSe NR seeds are ~25 nm long in average. The absorption spectrum for CdSe-only NRs shows a peak near 600 nm with a sharp PL peak at 615 nm. When CdTe is introduced as the second component, an absorption shoulder appears at 650 nm due to the smaller bandgap of CdTe. The absorption tail extends beyond 700 nm as a result of the charge transfer transition from the CdTe valence band to the CdSe conduction band. Recombination across the CdSe/CdTe interface occurs at energies lower than the bandgap of either component, shifting the PL peak to ~800 nm and broadening it considerably. Alloyed CdSe 0.4 Te 0.6 also forms a type-II junction with CdSe, therefore many of the absorption and PL features for these HNRs are similar. The difference between the PL peak position of the HNRs and that of the CdSe seeds, measured to be 0.37 eV, was used to determine the concentration of Te in the alloy following the calibration introduced in our previous work 34, 35 . The CdSe/CdSe 0.4 Te 0. 6 HNRs recapped with 1-octanethiol retain the absorption features seen with the native ligands, but have higher PL intensity and better stability in air, similar to the 1-octanethiol capped CdSe/ CdTe HNRs we reported recently 36 . For every heterostructured sample, the absorption spectrum extends beyond 700 nm, and should allow a greater portion of the incident solar spectrum to be collected in a photovoltaic device compared to single-component CdSe.

Photovoltaic Properties of NR-and HNR-Sensitized PV Devices. Photocurrent-voltage (J-V)
characteristics, absorbance spectra of sensitized films, incident photon-to-current efficiency (IPCE), and absorbed photon-to-current efficiency (APCE) spectra for various NR-and HNR-sensitized PV devices are presented in Fig. 3. Photovoltaic parameters of the devices are given in Table 1. Every device exhibits similar open-circuit voltages (V oc ) and fill factors (FF), but short-circuit current density (J sc ) values show remarkable differences. As the time constant of the electron transfer from CdTe (or CdSe 0.4 Te 0.6 ) tips to CdSe in HNRs (<400 fs) is much shorter than that of the electron transfer from CdTe to TiO 2 (~1 ns) 34,36,37 , we believe that most of the photocurrent would come from the electron transfer at the CdSe-TiO 2 interface (for CdSe NRs and HNRs). If a major portion of HNRs are anchored to TiO 2 only by their tips, electron extraction at the CdTe/TiO 2 interface should compete with more efficient electron Scientific RepoRts | 5:17472 | DOI: 10.1038/srep17472 transfer to CdSe. Moreover, electron transfer from CdSe to CdTe is not favored considering the band diagram in Fig. 1b, leading to poor fill factor as we previously reported in the case of organic-inorganic hybrid solar-cell structure utilizing curved CdSe/CdTe HNRs as inorganic light harvesters 38 . Such a loss of fill factor was not observed in HNR-sensitized PV devices, and therefore we believe that most of HNRs incorporated in the mp-TiO 2 frame have some portion of CdSe directly anchored to the TiO 2 nanoparticles. The TEM images of HNRs anchored on mp-TiO 2 in Fig. 2f partially support this argument.
The TOPO-CdSe/CdTe HNR device delivers slightly higher J sc compared to the TOPO-CdSe device. Considering that the photocurrent generated from single-component CdTe sensitizers is generally far lower than the single-component CdSe sensitizers presumably due to the recombination loss from the charge-carrier trapping [38][39][40][41] , higher J sc of the TOPO-CdSe/CdTe HNRs than that of the TOPO-CdSe NRs (with the same dimension of ~25 nm) cannot be explained simply by summing up the photocurrent generated from individual CdSe and CdTe components in HNRs. Due to the unique feature of type-II band offset in HNRs, where photoexcited electron-hole pairs are innately separated, the electron extraction process at the TiO 2 -sensitizer interface becomes more efficient with HNRs than with the single-component NRs 39 . This explains the increase of IPCE over the entire wavelength region for the TOPO-CdSe/CdTe HNR-SSCs compared to the case of TOPO-CdSe single-component NRs working as light harvesters. Furthermore, the type-II interface of TOPO-CdSe/CdTe HNR leads to a charge-separated state (CSS) absorption 39 , enabling the utilization of less energetic photons close to the near-infrared region. The effect of CSS absorption inherent for the HNR sensitizers is reflected in the extended absorption tail of the sensitized films ( Fig. 3b) compared to that of the TOPO-CdSe NR device.
In terms of the performance of TOPO-CdSe/CdTe HNR device, the stability of CdTe component in polysulfide electrolyte is a major concern, and therefore should be discussed. It has been well known that in the case of CdTe-sensitized SSCs using polysulfide electrolyte, anodic corrosion due to the ineffective hole scavenging from CdTe leads to the degradation of CdTe and low photovoltaic performance 42,43 . This degradation can be lessened by forming a semiconductor shell preventing CdTe cores from directly facing the polysulfide electrolyte 25 . Even though the CdTe component of the CdSe/CdTe HNRs does not have such a core-shell structure, our devices are finally treated to have ZnS passivation layer and therefore CdSe/CdTe HNR devices are not much affected by the anodic corrosion and exhibit reasonable performance.
In the case of TOPO-CdSe/CdSe 0.4 Te 0.6 HNR device, ~11% extra enhancement of J sc compared to that of the TOPO-CdSe/CdTe NR device is observed. CdSe/CdSe 0.4 Te 0.6 HNRs allow faster charge separation compared to the CdSe/CdTe HNRs and have lower valence band position in the alloyed tip 34 , which might lead to the J sc improvement by aiding facile charge extraction to TiO 2 . Furthermore, in the case of HNRs with alloyed tips, enhanced chemical stability of alloyed tips is also thought to contribute to achieve the improved photocurrent 39 . A noticeable further enhancement in the photocurrent density is observed by exchanging the surface ligands on the CdSe/CdSe 0.4 Te 0.6 HNRs, from native TOPO, ODPA, and TOP (simply referred to here as TOPO) ligands to 1-octanethiol (OT). As the absorption spectra obtained from mp-TiO 2 films sensitized with TOPO-CdSe/CdSe 0.4 Te 0.6 and OT-CdSe/CdSe 0.4 Te 0.6 are quite similar (Fig. 3b), the J sc upsurge may be attributed to the enhancement of electron injection/collection efficiency rather than the difference in the light harvesting from both sensitizers. Such an interpretation is further supported by the trend shown in the APCE spectra ( Fig. 3d): APCE is represented as the product of charge collection efficiency (η col ) and charge injection efficiency (η inj ), which can be calculated from the following relation (equation (1)) 44 : where the term LHE represents light harvesting efficiency. By comparing Fig. 3c,d, it is found that the APCE onset of the OT-CdSe/CdSe 0.4 Te 0.6 device is extended to ~760 nm compared to that of the TOPO-capped HNR devices. This result is in line with the previous report of the photocurrent spectra extended over ~700 nm for the 1-octanethiol capped CdSe/CdTe HNRs 36 . Moreover, the difference of IPCE among the TOPO-capped sensitizer devices almost vanishes from the ~580-nm wavelength in the APCE spectra, showing that the charge collection and injection efficiency of the TOPO-capped samples are quite similar in this wavelength region. However, APCE near ~480 nm corresponding to the X 2 excitonic band of CdSe still shows composition dependence, which we might attribute to the aforementioned effect from the faster charge-separation kinetics of type-II band offset and higher photocurrent obtainable from alloyed tips than CdTe tips. However, clear interpretation for this phenomenon is not established yet and further study is needed.
It is readily deduced that η col can be improved from the 1-octanethiol capping, as it has been well known that the thiol recapping of CdTe reduces the surface defect sites responsible for any unwanted recombination of photo-generated charge carriers 36,40,41 . This feature is also found in Fig. 1a showing higher PL intensity of OT-CdSe/CdSe 0.4 Te 0.6 than TOPO-CdSe/CdSe 0.4 Te 0.6 , which is presumably resulted from the suppressed recombination by 1-octanethiol capping. Improvement of carrier collection from enhanced electric-field throughout the HNR absorber layer is another possible factor leading to the improved J sc , but it may hardly be the case in our devices considering photocurrent saturation under the reverse-biased condition in the J-V curve (Fig. 3a) and our sensitized solar cell structure utilizing monolayer HNRs as light absorber layer 45 .
However, the difference of η inj from 1-octanethiol recapping is not self-evident and further investigation on the TiO 2 -OT-HNR interface is needed. To elucidate the interface properties, electrochemical impedance spectroscopy (EIS) was carried out 46,47 . The mp-TiO 2 electrodes sensitized with TOPO-capped CdSe/CdSe 0.4 Te 0.6 HNR and OT-capped CdSe/CdSe 0.4 Te 0.6 HNR sensitizers were chosen for the EIS measurement, to examine the capping ligand effect by excluding any side effects from the HNR composition and different loading amount of HNRs on mp-TiO 2 electrodes. Figure 4 presents the   52 , faster electron injection is predicted for this case as the CBM offset serves as the driving force for the electron transfer at the TiO 2 -CdSe interface [53][54][55] . Less energetic electrons generated in the HNRs may become extractable by this larger injection driving force, which also explains the origin of red-shifted APCE onset for the OT-CdSe/CdSe 0.4 Te 0.6 device. The higher recombination resistance is believed to result from the reduced recombination by thiol-capping, as explained in the previous section. Therefore, it is concluded that both η col and η inj (equation (1)) are improved through the 1-octanethiol recapping, and the J sc increase from the OT-CdSe/CdSe 0.4 Te 0.6 device with the red-shifted IPCE onset compared to the TOPO-CdSe/CdSe 0.4 Te 0.6 can happen without remarkable increase of the light absorption. The OT-CdSe/CdSe 0.4 Te 0.6 HNR-SSC shows J sc of 6.238 ± 0.212 mA cm −2 with 2.202 ± 0.131 overall PCE, approximately 33% improvement over the single-component CdSe NR devices with native ligands.

The Effect of Polystyrene Bead-Induced Percolating Pores on the PV Performance.
Even though the power conversion efficiency of the HNR device is much enhanced by utilizing both the type-II HNR sensitizers and ligand exchange, utilization of such 1-D long nanostructures as light harvesters for SSCs is still limited by their spatial incompatibility with the TiO 2 nanoparticle-based porous photoelectrodes. This type of electrode commonly provides ~20-nm sized pores (BET scale) 56 , which is smaller than the length of these 1-D nanostructures (~25 nm long). However, strategies to circumvent such limitations have been investigated only by a few researchers 28,57 .
To render the internal pore structures of mp-TiO 2 electrode more suitable for the infiltration of 1-D sensitizers, we utilized polystyrene (PS) microbeads as sacrificial additives for the conventional TiO 2 paste. Figures 5 and 6b show cross-sectional SEM images and EDS profiles of mp-TiO 2 electrodes derived from the pastes of different TiO 2 :PS weight ratios. For the electrode without PS beads, Cd/Ti ratio is not homogeneous throughout the electrode cross-section, and abruptly decreases at the ~2-μ m depth, suggesting that ~20-nm sized pores in mp-TiO 2 electrodes are easily clogged by the NR or HNR sensitizers. However, when the PS beads are incorporated into the TiO 2 pastes and calcined to leave large pores (Fig. 7a), the Cd/Ti ratio at every given depth of the electrode cross-section is highly increased, and the distribution of Cd/Ti values becomes much more homogeneous throughout the entire electrode depth. Figure 6a shows the J-V characteristics of HNR devices with different TiO 2 to PS ratios, the photovoltaic parameters of which are presented in Table 2. Incorporation of PS beads leads to a significant increase in both J sc and PCE. From these results, we presume that the incorporation of PS beads leads to It is predicted that with ~32.5% volumetric occupation of 200-nm sized PS beads in the TiO 2 nanoparticle electrode, percolation of beads extends throughout the entire electrode depth (8 μ m). Assuming the porosity of mp-TiO 2 films to be in the range of 0.5-0.6 56 , the PS beads account for the 27.5-32.1% of the total volume in the electrodes with TiO 2 :PS = 4:1 after calcination, which is close to 32.5%. Therefore, the homogeneous Cd/Ti ratio throughout the electrode depth and the improvement of PV performances from the PS-derived hierarchically porous TiO 2 electrodes can be explained by the percolation of large pores and subsequently facilitated infiltration of HNRs. Furthermore, dS/dz values increase even further after the formation of electrode-penetrating percolation channel, as seen in Fig. 7b, meaning that the infiltration of HNRs would be more promoted by the addition of more PS beads. Therefore, the Cd/  Ti ratio exhibits a further increase for the TiO 2 :PS = 2:1 electrode, and the optimal PV performance is obtained at this ratio. We also believe that this strategy can benefit from the Mie-type light-scattering effects produced by spherical pores filled with electrolytes, as previously explored theoretically (in the case of dye-sensitized solar cells) and experimentally 58,59 . However, with a larger amount of PS beads the electrode structure collapses from the aggregation of PS beads forming large voids (Fig. 5d), leading to inferior J sc and FF due to the loss of active mp-TiO 2 volume to accommodate HNRs and deteriorated recombination at the direct electrolyte-FTO contact.
Our simple approach utilizing sacrificial spherical additives results in ~32% additional enhancement in J sc compared to the OT-CdSe/CdSe 0.4 Te 0.6 HNR-SSC from the mp-TiO 2 electrode without polystyrene   microbead-induced percolating pores, yielding a 3.02% efficient PV device. This is one of the highest values among the SSCs with 1-D sensitizers, and a promising result proving that multi-composition variants of 1-D nanostructures can be fully utilized for this type of photovoltaic device with sensible engineering.

Discussion
In this work, CdSe/CdSe x Te 1−x HNRs were effectively utilized as a light absorber material for semiconductor-sensitized solar cells. Tailored nano-heterostructures and surface passivation with suitable ligand proved to be crucial factors to achieve the enhanced photocurrent density, by utilizing the superior optoelectronic properties of type-II heterostructures and modifying the conduction band level with surface ligands. Furthermore, to circumvent the spatial incompatibility of 1-D sensitizers with the conventional mp-TiO 2 electrodes, spherical PS bead additives were incorporated to render a hierarchical pore structure in the mp-TiO 2 electrode. Electrodes with PS-modified pore structures showed highly enhanced PV performance by the formation of large percolating pores inside the electrode, which was also supported by a Monte-Carlo simulation. Consequently, 3.02% efficient HNR-SSC was achieved by integrating the aforementioned approaches, exhibiting that 1-or higher-dimensional nanostructures and their multi-composition variants can potentially be fascinating alternatives as light harvesters for sensitized PVs. during the growth of seeds, limiting the average overall length of the HNRs to ~25 nm. After 5 additional minutes of growth, the reaction mixture was cooled by removing the heating mantle. The NR/ HNR suspensions were cleaned once by precipitation with chloroform and methanol, and redissolved in hexanes. Any insoluble precipitates were discarded, and the NR/HNRs were precipitated again by the addition of chloroform and methanol, dissolved in anhydrous toluene, and stored under N 2 . Recapping with 1-octanethiol followed our previous work on the CdSe/CdTe HNRs 36 . Briefly, 12 mL of 1-octanethiol was added to the flask containing approximately 4 mL of reaction mixture under Ar atmosphere. The reaction vessel was heated to 110 °C, and allowed to cool slowly to 60 °C. After stirring overnight, the HNRs were purified and stored as above. Each immersion took 1 min, and the films were rinsed with DIW after each immersion to remove any remaining ions. This cycle was repeated twice to complete the ZnS passivation. To fabricate cuprous sulfide counter electrodes, thoroughly polished brass foil was first etched by hydrochloric acid for 20 min in an 80 °C oven. Etched foil was then sulfurized by adding a polysulfide solution (2 M Na 2 S and 2 M S in DIW) droplet, immediately after which the foil turned black 62 . The NR-or HNR-sensitized mp-TiO 2 electrode and cuprous sulfide counter electrode were finally assembled into a sandwich-type cell using a binder clip and 60 μ m thick scotch tape as a spacer. A solution of 1 M Na 2 S, 1 M S, and 0.2 M KCl in MeOH:DIW = 7:3 solvent was used as the working electrolyte.

Preparation of Polystyrene
Characterization. Transmission electron microscopy (TEM) was carried out on a JEOL 2100 TEM operating at 200 kV with samples that were prepared by drop-drying a dilute solution of NRs or HNRs in chloroform onto a Cu grid with a thin carbon film (Electron Microscopy Sciences). The UV-vis absorption spectra were collected with an Agilent 8453 photodiode array spectrometer, and photoluminescence spectra were collected with a Horiba Jobin Yvon FluoroMax-3 fluorometer. The morphologies of mp-TiO 2 electrodes were analysed using a scanning electron microscope (JSM-6360: Hitachi). The photocurrent-voltage (J-V) curves of SSCs were obtained with a potentiostat (CHI 608C: CH Instrumental Inc., Austin, USA) under AM 1.5 illumination (K3000: McScience, Korea, intensity at 100 mW cm −2 ). An incident photon-to-current conversion efficiency (IPCE) measurement system (K3100: McScience, Korea) was used to obtain IPCE spectra. Electrochemical impedance spectra (EIS) were obtained by a potentiostat with 20 mV sinusoidal perturbation and frequencies ranging from 10 −1 to 10 5 Hz.


extfieldsofstudy: Chemistry Medicine

text:
Crystal Structure of Human Importin-α1 (Rch1), Revealing a Potential Autoinhibition Mode Involving Homodimerization

In this study, we determined the crystal structure of N-terminal importin-β-binding domain (IBB)-truncated human importin-α1 (ΔIBB-h-importin-α1) at 2.63 Å resolution. The crystal structure of ΔIBB-h-importin-α1 reveals a novel closed homodimer. The homodimer exists in an autoinhibited state in which both the major and minor nuclear localization signal (NLS) binding sites are completely buried in the homodimerization interface, an arrangement that restricts NLS binding. Analytical ultracentrifugation studies revealed that ΔIBB-h-importin-α1 is in equilibrium between monomers and dimers and that NLS peptides shifted the equilibrium toward the monomer side. This finding suggests that the NLS binding sites are also involved in the dimer interface in solution. These results show that when the IBB domain dissociates from the internal NLS binding sites, e.g., by binding to importin-β, homodimerization possibly occurs as an autoinhibition state.

Importin-α works as an adaptor molecule to connect importin-β and the NLS cargo, which makes it possible for the limited variety of importin-βs to bind to a large variety of NLS cargoes. The hetero-trimer is then transported into the nucleus through the NPC, with the importin-β working as an active engine. Such a transportation system ensures that larger cargoes (Mw > 40 kDa) can travel through the NPC into the nucleus [9][10][11][12]. There seems to be some mechanism in the isoforms of human importin-αs that is responsible for the diversity of binding to a variety of NLS cargoes, even though the whole structures of the subfamilies closely resemble each other. On the other hand, some importin-αs are known to form homodimers. The crystal structure of yeast importin-α was first reported as a homodimer [13]. In the homodimer, major and minor NLS binding sites are still exposed to solution; thus, NLS peptides can bind to the sites. In addition, Xenopus importin-α seems to form homodimers or multimers during purification [14]. However, the functional aspects of these multimerization properties of importin-αs have not been resolved.
Here, we report homodimeric human importin-α1 (h-importin-α1) by the use of X-ray crystallography and shed light on the solution state of the homodimerization in relation to NLS binding by using analytical ultracentrifugation (AUC) and isothermal titration calorimetry (ITC).

Protein preparation
h-importin-α1 (1-526) and ΔIBB-h-importin-α1 were overexpressed by E. coli strain BL21 (DE3) Codonplus-RIL (Stratagene) and purified as follows: Frozen cells (10 g) obtained from a 1 L culture were suspended in 100 mL of sonication buffer (50 mM tris(hydroxymethyl)aminomethane (Tris)-HCl, 500 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0) and lysed by sonication using an Ultrasonic Processor VCX 500 (Sonics & Materials, Inc.). The lysate was collected and centrifuged at 15,000 rpm for 1 h. The supernatant was filtered through a 0.45 μm HT Tuffryn membrane syringe filter (Pall Corporation). A GSTrap FF column (GE Healthcare) packed with Glutathione Sepharose 4 Fast Flow (GE Healthcare) was pre-equilibrated with phosphate-buffered saline (PBS) buffer. This lysate was then loaded onto a column at a flow rate of 1 mL/min. After washing with 50 mL wash buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 10% glycerol, 1 mM EDTA, pH 8.0), bound GST-tagged h-importin-α1 was eluted with 30 mL elution buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 10% glycerol, 1 mM EDTA, 5 mM reduced glutathione, pH 8.2). Elution fractions containing GST-h-importin-α1 and GST-ΔIBB-h-importin-α1 were pooled. The PreScission protease (GE Healthcare) was incubated with the pooled fractions according to the manufacturer's instructions, and the solution was dialyzed against 2 L of digestion buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.01% Triton X-100, pH 7.5) for 12 h at 277 K. The digested protein was filtered through a 0.45 μm HT Tuffryn membrane syringe filter and loaded onto a GSTrap FF column (GE Healthcare). The cleaved h-importin-α1 and ΔIBB-h-importin-α1 were collected in the flow-through and washed fractions. The protein samples were diluted to obtain an NaCl concentration of 75 mM and a pH of 8.0 with 20 mM Tris-HCl, 1 mM DTT, 1 mM EDTA, and 10% glycerol. The samples were loaded onto a Resource-Q column (GE Healthcare) packed with SOURCE 15Q (GE Healthcare) (S1 Fig.). For elution, the column was developed with a linear gradient from 0 to 500 mM NaCl in 20 mM Tris-HCl, 1 mM DTT, 1 mM EDTA, 10% glycerol, pH 8.0, at a flow rate of 0.2 mL/min. The eluted fractions of interest were pooled and concentrated with a Centriprep YM-10 centrifugal device (Amicon/Millipore) to a concentration of 10 mg/mL and used for the ITC and AUC-SV experiments. For the crystallization experiments, these fractions were filtered and loaded in a pre-equilibrated HiLoad 16/60 Superdex 75 size exclusion column (GE Healthcare) with 20 mM Tris-HCl, pH 8.0, 200 mM NaCl, and 5 mM DTT. The eluted fractions were collected and concentrated with the Centriprep YM-10 to 10 mg/mL.

Analytical ultracentrifugation (AUC)
AUC sedimentation velocity (AUC-SV) experiments were carried out in AUC buffer (0.1 M Tris-HCl, pH 8.0, 0.2 M NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP)), using an Optima XL-I analytical ultracentrifuge equipped with two optical systems, the Rayleigh interference and absorbance systems (Beckman Coulter). For AUC-SV measurements, 10 mg/mL h-importin-α1 and ΔIBB-h-importin-α1 samples were diluted with the AUC buffer. Then, 4 mM SV40 and nucleoplasmin NLS solutions, dissolved in the AUC buffer, were added to the sample solution. The AUC-SV measurements were conducted at 50,000 rpm at a temperature of 293 K, using an An-50 Ti rotor featuring cells with a standard 12-mm charcoal-epon double sector centerpiece and sapphire windows. During the runs, changes in the concentration gradient were monitored with a Rayleigh interference optical system or absorbance at 280 nm. All of the AUC-SV raw data were analyzed by the program SEDFIT14.1, with the continuous C(s) distribution model [16]. The SEPHAT 10.58d program was used for analysis of the isotherm of weight-average s-values. A monomer-dimer self-association model was applied [17].

Isothermal titration calorimetry (ITC)
We investigated the binding of SV40 NLS and nucleoplasmin NLS to h-importin-α1 and ΔIBB-h-importin-α1. ITC measurements were carried out using a MicroCal iTC 200 (GE Healthcare) in 0.1 M Tris-HCl, pH 8.0, 0.2 M NaCl, and 5 μM DTT at 293 K. The cell chamber was loaded with 200 μL of 15 μM h-importin-α1 and titrated 20 times with 2 mM of SV40 NLS solution or 2 mM nucleoplasmin NLS solution, while stirring at 1,000 rpm. The data analysis was performed with Origin software equipped with the ITC (Fig. 3A). ΔIBB-h-importin-α1 (17 μM) was used for the ITC experiment with 40 titrations for SV40 NLS binding and 30 titrations for nucleoplasmin NLS (Fig. 3B, C).

Diffraction data collection, structure determination and validation
All diffraction data were collected at the BL26B2 station of SPring-8 using the mail-in data collection system [18], [19]. The crystals were soaked in the solution containing 30% (w/v) PEG 8000 as a cryoprotectant and flash-frozen at 100 K. The diffraction data were processed using the HKL2000 package [20]. The molecular replacement technique was executed using the MrBump package [21] using the truncated molecular model (84-476) of importin-α5 (PDB ID: 2JDQ) as a template, which gave two solutions in the asymmetric unit. The initial model was improved using RESOLVE [22] and further refined using LAFIRE [23] and ARP/wARP packages [24]. The model was manually rebuilt with the help of XtalView [25] and Coot [26], and refined with CNS [27] and PHENIX [28]. The current model of the ΔIBB-importin-α1 involves the residues of 75-496 (chain A) and 75-497 (chain B) with an R/R free factor of 0.193/ 0.221. The structural validation was performed with the Procheck [29] and MolProbity [30] programs. The coordinate and structure factors of ΔIBB-h-importin-α1 have been deposited at the Protein Data Bank with PDB ID: 3WPT.

AUC-SV experiments
shows that it is in a concentration-dependent monomer-dimer equilibrium (Fig. 1A, B). The C(s) distributions are characterized by two peaks, one at 3.1-3.3 S and one at 3.9 S; the relative sizes of the peaks vary with protein concentration, and they are interpreted as corresponding to monomer and dimer, respectively. The C(s) distributions were used to create a weight average S value (sw) isotherm. We calculated the sw by integration of the C(s) curves between 2.5 S and 5.0 S for each protein concentration, and then plotted the sw vs protein concentration using SEDPHAT (Fig. 1C). The monomer-dimer self-association model was applied for the curve fitting. The monomerdimer KD is estimated to be 8 ± 3 μM. The NLS ligands, SV40 and NP, shift the equilibrium to the monomer side (Fig. 1D, E). These results show that the NLS binding sites are involved in the dimer interface in solution.
h-importin-α1. However, h-importin-α1 remains monomeric in a variety of protein concentration (Fig. 1F). Neither of the SV40 nor NP NLS peptides changed the monomeric state (Fig. 1G, H). These results show that the self-bound IBB domain autoinhibits not only NLS binding but also homodimerization.

X-ray crystallography of ΔIBB-h-importin-α1
Only the diffractive crystals of ΔIBB-h-importin-α1 were obtained, not those of h-importin-α1. The crystals diffracted X-rays to 2.6 Å resolution in SPring-8. The molecular replacement technique was used to solve the structure ( Table 1). The crystal structure of ΔIBB-importin-α1 is shown in Fig. 2A. The pseudo 2-fold axis lies in close proximity to the N239 of chains A and Table 1. Crystallographic data collection and refinement statistics of ΔIBB-h-importin-α1.

Space group
Tetragonal P4  B, which are in the disallowed region in the Ramachandran plot. Structural stress is probably put on them owing to the dimerization. The K108 residue is extensively involved in the dimer formation and is represented in the close-up views. The side chain of K108 enters the P1 0 site of the minor NLS binding site [31], making hydrogen bonds with D325, T328, and Q369 of another protomer and one water molecule (Fig. 2B). The P1 0 site comprises V321, T322, D325, T328, N361, I362, G365, and Q369, which are conserved in the minor NLS binding site of importin-α proteins (Fig. 3). The major (W142, N146, W184, N188, W231, N235, W273,  Y277) and minor (W357, N361, W399, N403) NLS binding sites are buried in the dimer interface (Fig. 2C), thus NLS peptides cannot access the NLS binding sites. Because the major NLS binding site involves typically P1-P7 binding pockets [32], it has higher NLS binding affinity than the minor NLS binding site with P1 0 -P2 0 . The P1 0 -binding pocket in the minor NLS binding site plays a common role in a variety of importin-αs; the P1 0 accepts the K/R residues of the typical NLS signal motif KRXX [31][32][33], (Fig. 4). In the present crystal structure of ΔIBB-h-importin-α1, each of the K108 residues enters into the P1 0 -binding pocket of another protomer so that the minor NLS binding sites are in a state of autoinhibition (Fig. 2B), and, together with the major NLS binding sites, are buried in the dimer interface (Fig. 2C). As a result, the closed homodimer is stabilized owing to the limited access of NLS peptides to the major and minor NLS binding sites.

Isothermal titration calorimetry (ITC) assay for binding of NLS peptides to h-importin-α1 and ΔIBB-h-importin-α1
h-importin-α1. We carried out a binding assay of h-importin-α1 and ΔIBB-h-importin-α1 to study autoinhibition resulting from homodimerization. SV40 NLS bound to h-importin-α1 with a KD value of~100 μM (Table 2). While the fitting to the ITC data, one-site binding model was used because two-site model did not converge to adequate values. The AUC-SV experiments reveal that h-importin-α1 is monomeric during SV40 NLS binding (Fig. 1G). This result suggests that both the IBB domain and the NLS peptides bind to the internal NLS binding sites and prevent homodimerization.
On the other hand, the bipartite nucleoplasmin NLS peptide did not bind to h-importin-α1 (Fig. 3D). This result suggests that NLS binding is restricted by the IBB domain. ΔIBB-h-importin-α1. ΔIBB-h-importin-α1 bound to SV40 NLS with KD =~6 μM and to nucleoplasmin NLS peptides with KD =~0.14 μM (Table 2). During the NLS binding, the monomer-dimer equilibrium shifted to the monomer side (Fig. 1D, E), which suggests that the NLS binding sites are involved in the dimer interface even in solution, as in the crystal structure.

Homodimerization found in importin-αs
It has been shown or suggested that the importin-αs from yeast and Xenopus eggs form dimers. Yeast importin-α (PDB IB: 1BK6) provided the first crystal structure of an importin-α that was shown to dimerize not only in the crystal but also in solution, as revealed by dynamic light scattering (DLS) [13]. The SV40 NLS peptides are, however, able to bind to the NLS binding sites because the homodimer is an "open" structure where the NLS binding sites are exposed to the solution (Fig. 4A). The open homodimerization probably attributes to H119 at the top of the loop; this loop corresponds to the K108 loop of ΔIBB-importin-α1. H119 cannot enter the P1 0binding pocket in the minor NLS binding site, and this inability causes the protomers to slide along the long axis of the molecules. The NLS binding parameters of yeast importin-α were analyzed by fluorescence depolarization assay and it was found that yeast importin-α (1-542) binds to neither SV40 NLS-GFP nor Myc NLS-GFP [34]. On the other hand, yeast ΔIBBimportin-α (89-530) binds to both SV40 and Myc NLS, with KDs of 9 ± 4 nM and 6 ± 3 nM, respectively [35]. In addition, an enzyme-linked immunosorbent assay (ELISA)-based assay revealed that yeast importin-α (1-542) does not bind to SV40 NLS, whereas yeast ΔIBB-importin-α (88-530) binds very tightly [35]. These results suggest that the IBB domain is mainly responsible for the autoinhibition activity of yeast importin-α, while the "open homodimerization" does not restrict the NLS binding. Thus, the NLS binding affinity of yeast importin-α changes dramatically from 0% (off), when the IBB autoinhibits itself, to 100% (on), when it does not, that is keenly contrast to the closed-homodimer of h-importin-α1.
Moreover, it has been reported that wild importin-α from Xenopus eggs is homodimerized during purification [14], and a variety of studies have revealed that purified Xenopus importinα is in dimer-monomer equilibration in the concentration range of 0.6~1.2 mM, with a dissociation constant KD of~20 μM [36]. We found that the Xenopus importin-α has an arginine residue (R105) that is aligned to the K108 of importin-α1 (S2 Fig.). Thus, the R105 possibly interacts with the P1 0 -binding pocket of another protomer, promoting the closedhomodimerization.
The first structural knowledge about the autoinhibition mechanism was obtained from the crystal structure of mouse-importin-α2 (m-importin-α2) [37], [38]. In that crystal structure, the IBB domain binds to the major, but not the minor, NLS binding site; thus the NLS binding affinity is partly autoinhibited. It has been unclear whether m-importin-α2 forms a homodimer or not [37], although the m-importin-α2 has a K102 and the P1 0 -binding pocket consists of the same residues as in h-importin-α1 (S2 Fig.). Further studies should be addressed about the multimerization property of the m-importin-α2.

A possible autoinhibition state of h-importin-α1
Together with the results obtained from the AUC-SV, ITC, and crystallography, we can add a potential autoinhibition mode of the h-importin-α1 to the conventional scenario, as shown in Fig. 5. In the canonical scheme, the self-bound IBB domain autoinhibits NLS binding to the NLS binding sites in h-importin-α1. Our current study reveals that the self-bound IBB domain also autoinhibits homodimerization. The IBB domain is dissociated from the NLS binding sites, e.g., by the competitive binding of NLS peptides to the NLS binding sites and/or the binding of the IBB domain to importin-β (Fig. 5A). Upon the IBB domain dissociation, the himportin-α1 can homodimerize as an autoinhibition state (Fig. 5B), which possibly lowers the KD (~6 μM) for NLS comparing to that of yeast (~10 nM) [34]. Through the association/dissociation of the IBB domain and molecular h-importin-α1, the NLS binding to the h-importin-α1 is controlled by both of the self-binding of IBB domain and homodimerization.

Conclusions
In h-importin-α1, NLS binding affinity is autoinhibited by the self-binding of IBB domain. In addition, the closed homodimerization possibly works as an autoinhibition state when the IBB domain dissociates from the NLS binding sites. There still remains unresolved subjects whether the current closed-homodimer is physiologically relevant in vivo. Further studies are required to address the functional aspects of the closed-homodimer, by mutagenesis and cellular biological experiments.


text:
Visible light promoted thiol-ene reactions using titanium dioxide †

The radical addition of thiols to alkenes is reported under photoredox conditions, using visible light and TiO 2 as a cheap and readily available photocatalyst. tolerated at the 2- and 3-positions of the alkene (entries 3 and 4), but 1,2-disubstitution was restricted to cyclic alkenes. Vinyl acetate and acrylonitrile were both productive, interestingly undergoing no reaction at all in the absence of PRC (entries 6 and 7). More reactive alkenes such as styrenes gave very high yields of the thiol ene products, but background reaction was high for 2i and 2j .

Visible light promoted thiol-ene reactions using titanium dioxide † Venugopal T. Bhat, Petar A. Duspara, Sangwon Seo, Nor Syazwani Binti Abu Bakar and Michael F. Greaney* The radical addition of thiols to alkenes is reported under photoredox conditions, using visible light and TiO 2 as a cheap and readily available photocatalyst.
Recent developments in visible light photoredox catalysis (PRC) have substantially expanded the scope and application of organic photochemistry. 1 Using a domestic lightbulb as the light source, rather than specialist UV equipment, the process uses a photoactive catalyst to mediate electron transfer between reactants, resulting in novel transformations that frequently proceed under mild conditions. The vast majority of PRC in organic synthesis to date is based on two classes of catalyst; transition metal complexes, such as the archetypal Ru(bpy) 3 Cl 2 and Ir(ppy) 3 systems, or organic dyes such as methylene blue or eosin-Y. The Noble metals of the first class are expensive and a limited resource, but are highly optimisable in terms of ligand-metal tuning. Organic dyes lack this particular quality, but may be tuned via conventional synthesis and are usually less expensive. We were interested in exploiting a third class of photoredox catalyst; semi-conducting metal oxides such as titania (TiO 2 ). With 4 million tons of titania being produced per annum, the material is cheap, readily accessible, and non-toxic (commonly ingested as food additive E 171), as well as offering the process benefits of heterogeneous catalysis such as simple removal by filtration. 2 The UV photoredox properties of TiO 2 have been extensively investigated and applied on an industrial scale to areas such as water treatment and photovoltaics. Applications to organic synthesis, however, remain under-developed, particularly in the area of visible light photochemistry. 3,4 We were interested in developing a titania-mediated PRC reaction using visible light, and chose to investigate the thiol-ene reaction for this purpose.
The addition of thiols to alkenes is a radical reaction that is typically initiated with UV light. 5 Although an old reaction, 6 its reliability, substrate scope, and compatibility has led to renewed interest in the context of click chemistry, where the facile C-S bond formation can be used as a powerful ligation strategy in biological and materials science. 7 Indeed, the largest field of application for thiol-ene chemistry is currently in polymer science, where thiols are cross-linked with alkenes under UV curing conditions, producing polymers with very uniform step-growth properties. 8 Recent literature reports have established PRC for thiol-ene addition using both Ru(II) and eosin-Y photocatalysis, providing an important frame of reference for our inchoate titania process. [9][10][11] We were aware at the outset that titania visible light PRC would likely require a nanoparticulate form of the catalyst. Unmodified titania absorbs very weakly in the visible region, but there is evidence that substrate binding to the surface can shift the absorption and enable electron transfer. 4a,c-e Rueping and co-workers, for example, have recently demonstrated visible light photoredox activity in dehydrogenative coupling reactions using commercially available titania P25 nanoparticles, 4d,e and we chose to begin our studies using this material (aeroxide s from Sigma-Aldrich). Using the addition of benzyl mercaptan (1a) to cyclohexene (2a) as a test reaction, we were pleased to observe very high conversions into adduct 3a using one equivalent of TiO 2 in MeCN under irradiation with a domestic light bulb ( Table 1). The reaction was also viable in DCM (entry 2), under neat conditions (entry 3), and using blue LEDs as an alternative light source (entry 4). Control experiments established that titania in the dark gave no reaction (entry 5), but there was a small background reaction with ambient light in the absence of titania (20% conversion). We were pleased to see that the titania loading could be reduced to catalytic quantities with little drop in overall yield (entries 8-10), and we established that oxygen was beneficial for the reaction. Use of an inert atmosphere and de-gassed solvent halved the efficiency of the process (entry 11). This was an expected requirement, as an oxidative quencher is needed to balance charge and prevent hole-electron recombination in the titania photocatalyst (vide infra).
With a titania PRC system in place, we moved on to investigate substrate scope, beginning with the alkene partner. We were pleased to find that a variety of primary and 1,1-disubstituted substrates underwent successful thiol-ene reaction with benzyl mercaptan ( Table 2). The reaction was tolerant of a variety of functional groups (ester, alcohol, silane, nitrile, halogen) giving the thiol ene adducts 3 in generally high yields. Substitution was   tolerated at the 2-and 3-positions of the alkene (entries 3 and 4), but 1,2-disubstitution was restricted to cyclic alkenes. Vinyl acetate and acrylonitrile were both productive, interestingly undergoing no reaction at all in the absence of PRC (entries 6 and 7). More reactive alkenes such as styrenes gave very high yields of the thiol ene products, but background reaction was high for 2i and 2j.
Thiol scope was initially trialled (Table 3) using catalytic amounts of titania (0.1 equiv.) and vinyl acetate (2h), an alkene with zero background reaction for the addition of benzyl mercaptan (Table 1, entry 7). We were pleased to find that the catalytic protocol was successful, with primary alkyl thiols (entries 1-3) and thiophenols (entry 4) undergoing thiol-ene reaction under the longer reaction time of 40 h. Increasing steric hindrance around the thiol was deleterious, with cyclohexyl thiol reacting in moderate yield (entry 5) and tert-butyl thiol undergoing no reaction, even with stoichiometric titania (entry 6). We could extend the reaction to a double thiol-ene reaction, with ethane and propane dithiol undergoing addition to styrenes in good yield (entries 7-9). p-Methoxystyrene proved less reactive, and required stoichiometric quantities of titania to yield 3t in an acceptable timeframe.
A mechanism for the reaction is outlined in Scheme 1, based upon the photo-excitation of electrons to the conduction band of the titania catalyst. The resultant holes are reductively quenched by the thiol, generating a thiyl radical cation, which can lose a proton to give a thiyl radical. Oxygen acts as a sacrificial electron acceptor, enhancing reaction efficiency by reducing hole-electron recombination in the titania. The thiyl radical can then initiate the thiol-ene cycle through addition to an alkene and generation of an alkyl radical, which propagates the reaction by abstracting a hydrogen atom from the thiol starting material.
In conclusion, we have developed a titania photoredox system for the thiol-ene reaction that uses visible light. The photocatalyst is cheap, robust, readily available, and easily processed out of the reaction for re-use. Applications of this method to the broad remit of thiol-ene chemistry are ongoing in our laboratory.
We thank the University of Manchester and the EPSRC for funding.


Ru-N-C Hybrid Nanocomposite for Ammonia Dehydrogenation: Influence of N-doping on Catalytic Activity

For application to ammonia dehydrogenation, novel Ru-based heterogeneous catalysts, Ru-N-C and Ru-C, were synthesized via simple pyrolysis of a mixture of RuCl3·6H2O and carbon black with or without dicyandiamide as a nitrogen-containing precursor at 550 °C. Characterization of the prepared Ru-N-C and Ru-C catalysts via scanning transmission electron microscopy, in conjunction with energy dispersive X-ray spectroscopy, indicated the formation of hollow nanocomposites in which the average sizes of the Ru nanoparticles were 1.3 nm and 5.1 nm, respectively. Compared to Ru-C, the Ru-N-C nanocomposites not only proved to be highly active for ammonia dehydrogenation, giving rise to a NH3 conversion of >99% at 550 °C, but also exhibited high durability. X-ray photoelectron spectroscopy revealed that the Ru active sites in Ru-N-C were electronically perturbed by the incorporated nitrogen atoms, which increased the Ru electron density and ultimately enhanced the catalyst activity.

Introduction
To address the growing demand for clean and sustainable technologies for portable, automobile, and stationary fuel cell applications, the development of potentially feasible hydrogen storage materials has become a bourgeoning field of research over the last decade. Relevant materials include metal hydrides [1,2], metal-organic frameworks [3][4][5], and chemical hydrides [6,7]. Among these materials, chemical hydrogen storage materials, such as sodium borohydride [8][9][10] and ammonia borane [11,12], are of particular interest because they can store a large quantity of hydrogen in a safe manner [8][9][10][11][12]. In addition to the solid materials, liquid or gaseous fuels, such as formic acid [13] and ammonia [14], are promising candidates for chemical hydrogen storage applicable to fuel cells since they can provide economically viable hydrogen storage and delivery methods for on-site power generation using existing infrastructure [15,16].
Ammonia, a gas that can be condensed under mild conditions (20 °C and 0.8 MPa), has attracted increasing attention as a hydrogen carrier due to its significantly high hydrogen storage density of 17.8 wt%; furthermore, hydrogen can be produced on demand from ammonia in the presence of an appropriate catalyst [14,17]. In addition, H2-release from ammonia is essentially CO-free, which allows direct feeding of the product stream into polymer electrolyte membrane fuel cells (PEMFCs), which is a potential advantage over conventional hydrocarbon reforming that requires an expensive, external hydrogen purification system. Moreover, nitrogen gas, the spent-fuel produced upon ammonia dehydrogenation, can readily be regenerated into ammonia by the Haber-Bosch process [18].
For ammonia to be useful in fuel cell applications, complete conversion (>99%) is required to circumvent poisoning of the PEMFC catalysts by unreacted ammonia, although an efficient adsorbent can decrease the quantity of residual ammonia to <200 ppb [19,20]. To achieve such high conversion, high reaction temperatures (>400 °C) are generally required to overcome the high activation barriers. In this context, numerous Ru-based heterogeneous catalysts, including Ru/carbon nanotubes (CNT) [21][22][23][24][25], Ru/Al2O3 [21,[26][27][28][29], and Ru/SiO2 [27] have been developed to promote ammonia dehydrogenation. Notably, Ru catalysts on carbon-based supports, such as CNTs and carbon nanofibers (CNFs), are highly active for the desired reaction. The activity of Ru/CNT [22,30] and Ru/CNF [31] has been further enhanced by increasing the charge density of the carbon supports via nitrogen doping. In addition to the catalysts for NH3 decomposition, we recently developed a novel Fe-N-C electrocatalyst for the oxygen reduction reaction (ORR) that possesses a number of Fe-N-C interfaces [32]. In such nanostructures, the incorporated nitrogen atoms were proposed to donate electron density to the Fe metal centers, ultimately enhancing the ORR activity. The previous results obtained with the use of the N-doping strategy, along with our recent discovery, inspired us to develop a Ru-based catalyst supported on N-doped carbon materials given that the Ru-N-C interface is expected to provide improved catalyst activity and durability via the electronic interactions between Ru and the N-C sites.
In this contribution, we report the synthesis, characterization, and H2-release properties of a Ru-N-C heterogeneous catalyst for ammonia dehydrogenation. The catalyst was prepared by pyrolysis of the precursors containing Ru, N, and C atoms. Numerous analytical techniques indicated the formation of hollow, hybrid nanostructures of Ru-N-C. In addition, the prepared Ru-N-C nanocomposite exhibited enhanced activity and durability compared to Ru-C at the temperatures ranging from 475 to 550 °C.
The influence of nitrogen doping on enhancing the catalytic performance of Ru-N-C was further proposed, based on the results obtained by a number of analyses.

Results and Discussion
Preparation of the Ru-N-C hybrid nanostructure was achieved by a modified procedure based on a previous report [32]. Ru metal ions were chemically chelated on carbon black spheres by thermal treatment of a mixture of dicyandiamide (HN-(CN)2), RuCl3, and carbon in aqueous medium at 100 °C in air. The resulting Ru 3+ -chelated composites were further calcined at 550 °C for 4 h in a N2 atmosphere, which facilitated the formation of hybrid and graphitized Ru-N-C structures (Scheme 1). A possible mechanism for the formation of Ru-N-C is that the added Ru 3+ (aq) ions could initially be interacted with nitrogen atom (HN-(CN)2) at dicyandiamide via an attractive force between Ru 3+ and nitrogen lone pairs. The weakly interacted species, Ru 3+ -NH-(CN)2 may then be transformed into the graphitized Ru-N-C structure in the presence of excess carbon species upon calcination at 550 °C by forming new chemical bonds between Ru-N and C via doping. Ru-C and C-N materials were likewise synthesized in the absence of the nitrogen-containing precursor, dicyandiamide, or RuCl3 as control catalysts. The Ru content of the Ru-N-C and Ru-C materials were determined to be 0.97 wt% and 0.86 wt%, respectively, by using inductively coupled plasma optical emission spectroscopy (ICP-OES). In addition, the N content of Ru-N-C was found to be 14.2 wt%, as measured by SEM-EDX. The Ru, C and N contents of Ru-N-C and Ru-C are presented in Table S1. The transmission electron microscope (TEM) image of Ru-N-C demonstrates the formation of hollow graphitized structure even in the absence of a sacrificial template (Figure 1a,b); this approach to achieving such structures may potentially contribute to the reduction of chemical wastes [32] given that the formation of hollow carbon structures usually requires multistep processes with different templates [33,34]. The Ru-C catalyst also had a hollow structure, but aggregated Ru particles were observed with sizes of ~20 nm (Figure 1c,d). These results imply that the added nitrogen-containing precursor, dicyandiamide, facilitated uniform dispersion of the Ru species during pyrolysis. Given that Fe species promote the formation of hollow carbon nanostructures [32], the Ru species employed in the current synthetic procedure likewise played important roles in catalyzing the generation of hollow, N-doped graphitized nanostructures upon pyrolysis of the precursors containing carbon and/or nitrogen [35,36].
Elemental mapping of Ru-N-C using high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and energy-dispersive X-ray (EDX) indicated that Ru, C, and N were well dispersed over the catalyst (Figure 2, Figures S1 and S2). X-ray diffraction (XRD) analysis of Ru-N-C and Ru-C showed broadened peaks centered at 25.2° (Figure 3), which is attributed to the graphitic stacking of C3N4 [37]. Ruthenium carbide, previously synthesized under high pressure (50,000 bar) and temperature (2000 K), appeared at 18.67°, 20.22°, and 21.23° [38]. Such peaks were not found in the XRD spectra, indicating that no ruthenium carbide species were formed under the reaction conditions employed in this study. A broad peak centered at 42.2° corresponding to metallic Ru was detected in the EDX profile of the Ru-N-C hybrid nanocomposite, indicating the formation of Ru nanoparticles with a size of 1.3 nm, calculated using the Scherrer equation. In contrast, additional sharp peaks centered at 38.5°, 44.2°, 58.5°, 69.7°, 78.7°, and 86.3° were observed in the spectrum of Ru-C, which are clearly attributed to the metallic Ru species (PDXL ver. 2.3.1). The average size of the Ru particles in Ru-C was likewise calculated to be 5.1 nm using the Scherrer equation, which is larger than that of the Ru particles in Ru-N-C. These results are consistent with the TEM observations (Figure 1b vs. 1d).  The textural properties of both catalysts were further determined from the N2 adsorption-desorption isotherms. The BET surface area of Ru-N-C (898 m 2 ·g −1 ) was lower than that of Ru-C (1110 m 2 ·g −1 ) and there was also a slight reduction of the pore size and pore volume of Ru-N-C ( Table 1). The stabilities of the as-prepared catalysts were also evaluated by thermogravimetric analysis (TGA). The TGA profile of Ru-N-C indicated reasonable stability up to 550 °C, after which there was a sharp loss of mass (Figure 4, red). The slight loss of mass at <100 °C is attributed to the desorption of adsorbed water species from the catalyst surface, while the sharp decrease beyond 550 °C may be associated with decomposition of the C-N support structure [39][40][41]. In contrast, decomposition of Ru-C was initiated at 380 °C, followed by a continual mass loss with increasing temperature (Figure 4, black) that may be attributable to collapse of the hollow carbon structure. The results suggest that the doped N atoms played an additional role in increasing the thermal stability of the catalyst, which can likely affect catalytic activity (vide infra).   graphitic peak centered at 284.6 eV (Figure 5a). Additional peaks for C-O species were found in the range of 284-290 eV, which presumably originated from adsorbed oxygen species from the precursor employed [42][43][44]. In the case of the Ru 3p state, a dominant peak centered at 463.3 eV was observed in the deconvoluted spectrum of Ru-C (Figure 5b), which is likely attributed to the RuO2 phase [31]. An additional peak was observed at 466.1 eV for the Ru-C catalyst, presumably corresponding to RuO2·xH2O [31]. The Ru 3d spectrum of Ru-C exhibits a single peak centered at 281.2 eV ( Figure S3a). Similar to the case of Ru-C, a dominant, narrow sp 2 graphitic carbon peak centered at 284.6 was found in the XPS spectrum of Ru-N-C (Figure 6a). Other peaks were also detected at 285-290 eV. In contrast to the peaks observed for Ru-C, these peaks may be derived from the formation of carbon species possessing C(sp 2 )-N and C(sp 3 )-N networks [45], indicating that nitrogen atoms were introduced into the graphitic structure upon pyrolysis. Moreover, Ru-N-C exhibited dominant N 1s peaks at binding energies ranging from 396 to 408 eV (Figure 6b), suggesting the formation of pyridinic-, nitrile-, pyrrolic-, and/or graphitic-like nitrogen species [32,45,46]. The Ru 3p XPS spectrum of Ru-N-C showed a major peak centered at 462.7 eV (Figure 6c). Notably, the peak at 462.7 eV for Ru-N-C appeared at lower binding energy compared to that of Ru-C, suggesting that the incorporated nitrogen atoms donated electron density into the Ru active sites. Ru-N-C also presented a broad peak centered at 466.6 eV, again attributable to RuO2·xH2O. As in the case of the Ru 3p spectrum, the Ru 3d peak of Ru-N-C occurred at a slightly lower binding energy (281.0 eV) than that of Ru-C ( Figure S3b), again supporting the proposed electron transfer from N to Ru in the Ru-N-C nanocomposite.
The increased Ru electron density in Ru-N-C induced by nitrogen doping could potentially improve the activity and durability of the catalyst for NH3 dehydrogenation, although its surface area is lower than that of Ru-C. To prove this hypothesis, the H2-release properties of the as-developed catalysts were evaluated by varying the temperature and/or GHSV. Nearly exclusive ammonia dehydrogenation (≥98%) is thermodynamically achievable even at temperatures as low as 425 °C [47]. In addition, the equilibrium ammonia conversion at 500 °C and 1 bar is close to 99.7% [15]. Due to the high kinetic barrier, however, only a limited number of catalysts show high activity at ≤500 °C. We initially assessed the catalytic activity of the as-prepared Ru catalysts for ammonia dehydrogenation as a function of temperature. As depicted in Figure 7, the activity of Ru-N-C proved to be higher than that of Ru-C at temperatures ranging from 475 to 550 °C. For example, at 550 °C, 95% NH3 conversion was achieved with Ru-N-C versus 68% conversion obtained with Ru-C. The conversion obtained at 500 °C with Ru-N-C (56%) was likewise found to be higher than that with Ru-C (20%). In the presence of C-N, negligible dehydrogenation activity was found at temperatures ≤600 °C (Figure 7, green triangles). For all the catalysts, the ammonia conversion increased with increasing temperature (Table S2), which is congruent with the endothermic nature of the dehydrogenation process.  Temperature ( o C)

Ru-N-C
Since the Ru-N-C hybrid nanocomposite showed enhanced activity compared to Ru-C, the influence of the GHSV on NH3 dehydrogenation in the presence of Ru-N-C was evaluated at different temperatures. As illustrated in Figure 8, the NH3 conversion obtained with Ru-N-C declined as the GHSV increased at all temperatures employed (Table 2). Compared to the NH3 conversion achieved with Ru-N-C at 550 °C using a GHSV of 2234 mL·g −1 ·h −1 (>99%), the NH3 conversion decreased slightly to 94.5% when the GHSV was increased to 7448 mL·g −1 ·h −1 . With a further increase of the GHSV to 11,172 mL·g −1 ·h −1 at 550 °C, the Ru-N-C catalyst still exhibited a high conversion of 81.5%. However, the decrease in NH3 conversion observed for Ru-N-C with increasing GHSV was found to be pronounced at temperatures of 450 °C and 500 °C. The conversion (45%) achieved with a GHSV of 2234 mL·g −1 ·h −1 decreased to 0% at 450 °C upon utilization of a GHSV of 7448 mL·g −1 ·h −1 . Likewise, the conversion (>97%) acquired using a GHSV of 2234 mL·g −1 ·h -1 decreased to 31% at 500 °C when a GHSV of 11,172 mL·g −1 ·h −1 was employed. Based on the results described above, it is reasonable that the improved activity for H2-release from NH3 over Ru-N-C may originate from the incorporation of nitrogen species that contributed to: (i) the formation of the small-sized Ru nanoparticles by initially anchoring the Ru 3+ precursor to prevent them from sintering ( Figure 1); (ii) enhancing the thermal stability of the catalyst ( Figure 4); and (iii) increasing the Ru electron density induced by the interaction between Ru and N ( Figure 6).  Long-term stability is a desirable property for a number of catalytic applications. In this context, long-term experiments with Ru-N-C were carried out under dehydrogenation conditions employing a GHSV of 7448 mLg −1 h −1 and temperatures of 500 °C and 550 °C. Initially at 550 °C, the initial NH3 conversion of 95% obtained with Ru-N-C decreased slightly to 88% after 80 h ( Figure S4). Upon dehydrogenation at 500 °C, the initial NH3 conversion of ca. 60% was maintained for 80 h without apparent deactivation (Figure 9, red). Surprisingly however, the initial NH3 conversion of 19% obtained with Ru-C increased gradually to 36% after 80 h under the identical conditions (Figure 9, black). Considering that NH3 has previously been employed as the N-doping agent for carbon nanotubes [22,30], the unexpected, increased activity of Ru-C strongly suggests the transformation of Ru-C into a N-incorporated Ru-C structure analogous to that of Ru-N-C via N-doping upon NH3 dehydrogenation. Consistently, elemental mapping with the spent Ru-C catalyst, obtained at randomly selected four different areas, revealed the presence of N element over Ru-C with an average quantity of 0.82 wt% ( Figure 10 and Figure S5), again confirming the incorporation of N atoms into Ru-C during NH3 dehydrogenation. The results suggest that the as-developed Ru-N-C catalyst could be employed for a long-term fuel cell application.

Synthesis of Ru-N-C, Ru-C, and C-N Materials
RuCl3·xH2O (0.25 g, Sigma-Aldrich, St. Louis, MO, USA), carbon black (1.0 g, Ketjen, Akzo Nobel, Chicago, IL, USA), and dicyandiamide (1.0 g, Sigma-Aldrich, St. Louis, MO, USA) were combined with distilled water (50 mL), followed by heating at 100 °C for 4 h with vigorous stirring to vaporize water. The resulting black solids obtained subsequent to complete drying were ground and pyrolyzed at 550 °C for 4 h under a N2 flow, yielding the final product, Ru-N-C as a black powder. The Ru-C and C-N materials were also synthesized using an otherwise identical procedure except for the absence of either dicyandiamide or RuCl3·xH2O. The schematic synthetic procedure is depicted in Scheme 1.

Catalytic NH3 Dehydrogenation
The performance of the catalyst was evaluated under a flow of 10% NH3/N2 at a rate of 37.2 mL·min −1 at atmospheric pressure using a fixed-bed quartz reactor. In a typical experiment, the catalyst (0.1 g, 0.05 g, 0.03 g, 0.01 g; 100-150 μm) was positioned in the reactor, followed by reduction using 50% H2/N2 at a flow rate of 30 mL·min −1 using a temperature of 550 °C and a duration of 1 h. The in-situ reduced catalyst was then flushed with N2 for 1 h, and the NH3 dehydrogenation reactions were then executed at temperatures ranging from 550 to 450 °C while decreasing the temperature in 25 °C increments. The compositions of the effluent gases were determined by using an online gas chromatograph (Agilent 7890A, Santa Clara, CA, USA) equipped with Porapak Q and Molecular sieve capillary columns as well as a thermal conductivity detector (TCD, Agilent, Santa Clara, CA, USA). The content of unreacted NH3 was analyzed using a tunable diode laser ammonia gas analyzer (Model Airwell+7, KINSCO Technology, Seoul, Korea). The dehydrogenation activity of the catalysts was monitored as a function of the GHSV and the temperature.

Catalyst Characterization
The crystalline patterns of the as-prepared materials were initially determined by XRD studies using a Rigaku X-ray diffractometer with CuKα radiation at 40 kV and 20 mA. Continuous scanning at a rate of 0.5°·min −1 was performed in the 2θ range of 20°-80°. The specific surface area, pore size, and pore volume of the materials were determined using a nitrogen adsorption instrument (Micrometrics ASAP 2000, Dr. Norcross, GA, USA) at 77 K. The thermal stability of the catalysts was evaluated by TGA at a heating rate of 10 °C·min −1 in the temperature range of 25-1000 °C.
The morphology of the synthesized catalysts was characterized using a HRTEM (FEI Tecnai F20, FEI Corporate, Hillsboro, OR, USA) and a HAADF-STEM (FEI Titan, FEI Corporate, Hillsboro, OR, USA) at 200 kV. Electron energy loss spectra (EELS) of C (K edge, 284 eV), N (K edge, 401 eV), and Fe (L3 edge 708 eV) were collected by using a Quantum 996 instrument (Quantum instrument, Bartlett, IL, USA) to determine the atomic distribution of the elements.
The electronic structure and local bonding environment of Ru-N-C were further identified by using an XPS instrument equipped with a PHI 50000 Versa Probe (Ulvac-PHI, Kanagawa, Japan) using a monochromated Al Kα X-ray (1486.6 eV) beam calibrated by the C 1s peak (284.6 eV) with a background pressure of 6.7 × 10 −8 Pa. The Ru contents of the catalytic materials were determined by means of inductively coupled plasma-optical emission spectrometry (ICP-OES) (Varian 720ES Agilent, Santa Clara, CA, USA) and scanning electron microscopy-energy dispersive X-ray (SEM-EDX, FEI Corporate, Hillsboro, OR, USA) analysis. CO chemisorption experiments were conducted to evaluate the Ru dispersion.

Conclusions
The simple synthetic strategy presented herein provides an economical route for large-scale production of the highly active Ru-N-C catalyst. The Ru-N-C catalyst displayed excellent performance for NH3 dehydrogenation with high stability. The incorporated nitrogen atoms were proposed to play pivotal roles in: (i) generating uniformly distributed, small-sized Ru nanoparticles; (ii) improving the thermal stability of the catalyst; and (iii) donating electron density to Ru via electronic interactions between Ru and N. For the undoped Ru-C catalyst, the reactant NH3 further revealed to act as a N-doping agent to convert Ru-C into N-doped Ru-C structure during NH3 dehydrogenation, which ultimately enhancing the desired activity. The as-developed Ru-N-C hybrid nanocomposite is thus applicable for on-site hydrogen production from ammonia with relevant catalyst optimization, and further provides insight for the development of various M-N-C catalysts (M = transition metals) for a number of chemical transformations.


extfieldsofstudy:
Medicine
Materials Science

text:
Light-induced pyroelectric effect as an effective approach for ultrafast ultraviolet nanosensing

Zinc oxide is potentially a useful material for ultraviolet detectors; however, a relatively long response time hinders practical implementation. Here by designing and fabricating a self-powered ZnO/perovskite-heterostructured ultraviolet photodetector, the pyroelectric effect, induced in wurtzite ZnO nanowires on ultraviolet illumination, has been utilized as an effective approach for high-performance photon sensing. The response time is improved from 5.4 s to 53 μs at the rising edge, and 8.9 s to 63 μs at the falling edge, with an enhancement of five orders in magnitudes. The specific detectivity and the responsivity are both enhanced by 322%. This work provides a novel design to achieve ultrafast ultraviolet sensing at room temperature via light-self-induced pyroelectric effect. The newly designed ultrafast self-powered ultraviolet nanosensors may find promising applications in ultrafast optics, nonlinear optics, optothermal detections, computational memories and biocompatible optoelectronic probes.

A s one of the most widely used wurtzite semiconductor materials, ZnO has been extensively investigated as building blocks for nano/microdevices in various applications, including energy harvester 1 , optoelectronics 2,3 , field-effect transistors 4 and logic computations 5,6 . Among these applications, room temperature ultraviolet detection is drawing tremendous attentions for defence technology, flame warning and chemical/biological analysis 7,8 because of the wide bandgap (3.3 eV) and high exciton-binding energy (60 meV) of ZnO (refs 9-12). Although numerous ZnO-based ultraviolet photodetectors (PDs) have been demonstrated with high photoresponsivity and/or specific detectivity, the relatively long response time and fluctuations in performance have always been an inevitable obstacle to improving the general performances of these PDs for practical applications. For example, the typical rising and falling times of previously reported ZnO-based ultraviolet PDs are ranging from a few seconds to few hours in most cases [13][14][15][16][17] . Therefore, it is necessary and essential to develop ultrafast response ultraviolet sensing devices based on ZnO micro/nanostructures.
Owing to the non-central symmetric crystal structures of wurtzite ZnO, pyroelectric potentials are induced by temperature changes across the material [18][19][20] . On light illuminations, a rapid rise in temperature is naturally induced within ZnO nanowires (NWs), leading to a distribution of pyroelectric potentials along the crystal with polarization pyrocharges presenting at both ends of the NW. A new approach is thus introduced to modify the charge-carrier transport process during the optoelectronic processes within ZnO-based devices by utilizing the pyroelectric polarizations.
In this work, we utilize this light-self-induced pyroelectric effect in ZnO to modulate the optoelectronic processes and thus enhance the performances of ultraviolet sensing. To maximize the enhancements of pyroelectric effect, low background temperature is maintained by minimizing the dark current (o0.1 nA) via designing and fabricating a self-powered PD that operates under zero bias 8,21,22 . Considering the simple synthesizing process and intrinsic optoelectronic properties, such as direct bandgap, large absorption coefficient and high carrier mobility [23][24][25][26][27][28][29] , CH 3 NH 3 PbI 3 (MAPbI 3 ) perovskite is applied to form a heterostructure with ZnO as self-powered PDs. The response time of the self-powered ZnO/perovskite-heterostructured (ZPH) PD is improved from 5.4 s to 53 ms in the rising time, and 8.9 s to 63 ms in the falling time by five orders in magnitudes. The specific detectivity D* and the responsivity R of the PD are both enhanced by 322%. The light-self-induced pyroelectric effect is attributed to these enhancements, and the working mechanism is carefully investigated. This work provides a novel design approach to achieve ultrafast response ultraviolet-sensing devices at room temperature enhanced by pyroelectric effect. The demonstrated self-powered PDs may become prospective candidates for ultrafast and energy-efficient ultraviolet sensing in different research and commercial fields.

Results
Design mechanism of self-powered ZPH PDs. The structure of self-powered ZPH PD is schematically illustrated in Fig. 1a. Side-view and top-view scanning electron microscopy images presented in Fig. 1b confirm the PD configuration, showing fluorine-doped tin oxide (FTO) glass uniformly and compactly covered by ZnO-NW array with diameters of 50-70 nm and lengths of 500 nm (top right panel of Fig. 1b, Supplementary  Fig. 1a and Supplementary Note 1), and then partially covered by MAPbI 3 perovskite with an average thickness of 500 nm (bottom right panel of Fig. 1b, Supplementary Fig. 1b and Supplementary Note 1), followed by a complete cover of hole transport material (HTM) spiro-OMeTAD with thickness of 500-1,000 nm (Supplementary Fig. 1c and Supplementary Note 1) and Cu top electrode with a thickness of 250 nm. The phase of MAPbI 3 perovskite is verified by taking XRD spectra (Fig. 1c) of the perovskite film-covered ZnO-NW array 30 . Detailed fabrication processes and measurement set-up are found in the Methods.
Relevant energy levels of the materials used to fabricate the self-powered ZPH PDs are depicted in Fig. 1d. The valence band and conduction band of the MAPbI 3 perovskite are À 5.4 and À 3.9 eV, versus vacuum, respectively 25,31 . On ultraviolet illumination, excitons are generated in the ZnO layer according to the absorption spectra of ZnO-NW array and ZnO/MAPbI 3 perovskite film as shown in Supplementary Fig. 1d and in Supplementary Note 1. The energy band alignment of the device ensures electrons are injected from the photoexcited layer of the ZnO and collected by FTO electrode while the valence band holes reach the Cu electrode through the hole transport layer.
Ultraviolet illumination produces a fast temperature increase within pyroelectric ZnO NWs for the light absorption, due to the non-central symmetric crystal structures; pyroelectric potentials are induced across the NWs by polarizations, with positive potentials at the HTM/Cu electrode side and negative potentials at the FTO electrode side based on the c axis direction of ZnO-NW array, as shown in Fig. 1e. This light-self-induced pyroelectric potentials are aligning with the photovoltaic direction over the external circuit and thus enhance the transient short-circuit current and open-circuit voltage. More importantly, the pyroelectric potential presenting in ZnO NWs may speed up charge-carrier separation at the interface, preventing photoexcited electrons in ZnO from recombining with holes at the heterojunctions, further improving the performance of ultraviolet PD in response time. I-V characteristics of the self-powered ZPH PDs under 325-nm illumination (red curve in Fig. 1f) were measured and plotted in Fig. 1f in comparison with dark current (black curve in Fig. 1f), showing a good ultraviolet response of the device in output current, increasing from 1.5 mA (dark) to 143 mA (3.7 Â 10 À 3 W cm À 2 ) when the HTM/Cu electrode was biased at 0.5 V. The self-powered operations are observed from the enlarged I-V curves (right panel of Fig. 1f) with a measurable (B1 mA) photocurrent under ultraviolet illumination at zero bias. This can be attributed to the well-aligned band structure of the device (see Fig. 1d).
General performances of the self-powered ZPH PDs. The shortcircuit current response of ZPH PDs to ultraviolet illumination was systematically investigated and summarized in Fig. 2a by varying the power densities from 3.7 Â 10 À 3 to 9 Â 10 À 6 W cm À 2 . Under each power density, obvious two-stage shortcircuit currents were derived. In the first stage, a sharp peak was induced by the combination of photovoltaic effect and pyroelectric effect on illumination which lead to an instantaneous temperature rise within ZnO. The corresponding short-circuit current is labelled as I pyro þ photo ; subscripts 'pyro' and 'photo' indicate the output current induced by pyroelectric effect and photovoltaic effect, respectively. In the second stage, the pyroelectric potentials disappeared as the temperature stayed constant by retaining the ultraviolet illumination, and the short-circuit current reached a steady plateau until shutting the laser beam, labelled as I photo . The corresponding currents of both stages were extracted and plotted in Fig. 2b as black and red dots, respectively, to demonstrate the pyroelectric enhancements on output signals of ZPH PDs. It is clear that the short-circuit currents I pyro þ photo and I photo monotonically increase with the power density of illumination in both ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms9401 stages, with the current of the first stage larger than that of the second stage for all illumination conditions.
To better understand the enhancements by pyroelectric effect on the performances of ZPH PDs, specific detectivity D* is demonstrated in Fig. 2c for currents obtained from both stages, defined as D* ¼ R/(2e Á I dark /S) 0.5 to describe the smallest detectable signal by considering the dark current I dark , which is in the order of 10 À 11 A ( Supplementary Fig. 3), as the major noise 32 . S is the effective area of the PDs, R is the corresponding photoresponsivity ( Supplementary Fig. 2) calculated as R ¼ S is the illumination power on PDs; I light represent the short-circuit current with illumination, respectively; g G is the internal gain; Z ext is the external quantum efficiency; q is electronic charge; h is Planck's constant; v is the frequency of the light; and I ill is the excitation power density. Both D* and R derived from the first stage with a combination of pyroelectric effect and photovoltaic effect (labelled as D* pyro þ photo ) display larger values than those obtained from the second stage, where only photo-excitation process contributes (labelled as D* photo ). The maximum value of D* is observed as 4.0 Â 10 10 and 1.3 Â 10 10 Jones for the first and second stages at the power density of 1.9 Â 10 À 5 W cm À 2 , respectively. Under the same condition, the maximum value of R is 26.7 and 8.3 mA W À 1 for two stages (Supplementary Note 2).
These results indicate 332% enhancements on both detectivity and photoresponsivity by pyroelectric effect.
The stability and repeatability of the self-powered ZPH PDs are studied and presented in Supplementary Figs 4 and 5 and in Supplementary Note 3. Short-circuit I-t curves of a typical device were measured under a power density of 3.7 Â 10 À 3 W cm À 2 after keeping the device in air for 1 and 3 weeks ( Supplementary  Fig. 4), showing impressive stability of these PDs. Besides, the repeatability of the PD was also investigated by monitoring the I-t performances of four different devices fabricated through the same procedures as shown in Supplementary Fig. 5. Considering the self-powered operation mechanism of ZPH PDs, open-circuit voltage, similar to short-circuit current indicated above, can also be utilized to detect the illumination intensities as shown in Supplementary Fig. 6. The open-circuit V-t curves present a sharp peak followed by a stable plateau as well, with the open-circuit voltage in both stages increased monotonously with the power density (Supplementary Note 4).
Working mechanism of the self-powered ZPH PDs.  On the basis of the absorbance spectra presented in Supplementary Fig. 1d, ZnO NWs are transparent to 442-nm illumination, and therefore the pyroelectric effect was only observed under 325 nm as clearly shown in Fig. 3a,b. The signal of 325-nm illumination displays a sharp rising edge, a stable plateau and a falling edge (black solid curve in Fig. 3b), while the signal of 442-nm illumination (red dash curve in Fig. 3b) is the same as those derived from traditional ZnO-based PDs reported previously [13][14][15][16] . Additional measurements were conducted to investigate the frequency dependence of self-powered ZPH PDs under both 325-and 442-nm illuminations. The corresponding I-t curves are presented in Supplementary Fig. 7, derived by varying the optical chopper frequency from 100 to 1,000 Hz. No obvious dependence on frequency was observed under 325-nm illumination, suggesting a potential ultrafast response of PDs enhanced by pyroelectric effect. As a comparison, the output signals were clearly affected by the frequency of light source under 442-nm illumination, indicating a slow response of these PDs without pyroelectric enhancements (Supplementary Note 5). One cycle of the short-circuit I-t curve under 325 nm was divided into four stages, labelled as I, II, III and IV in Fig. 3b,c, to elaborate the physical mechanism of the pyroelectric effectcombined photon-sensing performances of self-powered ZPH PDs. In stage 'I', the 325-nm ultraviolet illumination generates free carriers as photocurrent to transport through the external circuits from Cu to FTO electrode; meanwhile, an instantaneous temperature increase inside ZnO is produced on ultraviolet illumination, leading to a distribution of polarization pyroelectric charges across the NW with positive pyro-potential at the HTM/Cu electrode side, and negative pyro-potential at the FTO electrode side. The light-self-induced pyro-potential follows the photocurrent direction in external circuits and thus enhances the output signals with a sharp rising edge (I, Fig. 3b,c). In stage 'II', the illumination is retained and the temperature stays at constant. Therefore, the pyroelectric potential disappears rapidly (in less than 1 ms) due to the leakage, and the output current reaches a stable plateau (II, Fig. 3b,c). In stage 'III', the illumination is eliminated and the photocurrent thus disappears, while an instantaneous temperature decrease induced during this process results in a pyro-potential distribution in the opposite direction to that in 'I', leading to current flows from FTO to Cu electrode as a falling peak with negative current magnitudes (III, Fig. 3b,c). In stage 'IV', the temperature falls back to room temperature and stays steady; thus, the pyro-potential vanishes again because of leakage and the output returns to dark current (IV, Fig. 3b,c).
The heating process of ultraviolet illumination was simulated by using finite element method based on the transient heat conduction equation as demonstrated in Supplementary Note 6. The temperature profiles, temperature variation rate dT/dt of the ZnO NW and the corresponding output currents induced by the pyroelectric effect were calculated and presented in Supplementary Fig. 8a,b. Furthermore, a nanosecond-pulsed laser with wavelength of 355 nm was applied to experimentally verify the pyroelectric response of ZPH PD as shown in Supplementary Fig. 8c,d. These theoretical simulations and experimental results provide convinced proofs to establish the direct correlation between observed optoelectronic performances and the ultraviolet illumination-induced pyroelectric effect in ZnO NWs (Supplementary Note 6). More control experiments were conducted to confirm the role played by the pyroelectric effect on the photon-sensing performances of the self-powered ZPH PDs. Figure 4a presents the short-circuit current response of the PDs under 325-nm illumination (power density of 3.7 Â 10 À 3 W cm À 2 ) in different background environmental temperatures ranging from 27 to 70°C. The sharp peak of the short-circuit currents related to pyroelectric effect I pyro þ photo decreased with increasing the environmental temperature, while the stable plateau of the current controlled by photo-excitation process I photo remained unchanged. These results further verify the mechanism of light-self-induced pyroelectric effect in ZnO NWs. Moreover, by removing the perovskite layer, a new configuration of Si/ZnO PD was fabricated and tested as shown in Fig. 4b. Similar I-t characteristics (Fig. 4b) were obtained under 325-nm illumination, confirming the pyroelectric effect was induced within ZnO instead of other materials.  Fig. 9d) and 8.9 s ( Supplementary Fig. 9e), respectively. Therefore, the photon response time of ZPH PDs was reduced from several seconds to few microseconds (by 10 5 time), by light-self-induced pyroelectric effect in ZnO NWs. Furthermore, these results also indicate essentiality to configure such PDs in a self-powered manner without applying any bias voltages to maximize the enhancements of pyroelectric effect.

Discussion
These self-powered ZPH PDs possess novel, unique and complement features in comparison with conventional photosensing devices on the basis of top-down or bottom-up approaches. First, the long-overlooked pyroelectric effect induced by ultraviolet illuminations was employed to enhance the performances of photosensing. This is a fundamentally new mechanism that relies on the naturally existing temperature changes on ultraviolet illuminations. Compared with traditional methods 16,17 to improve the response time and enhance the general performances of PDs, the pyro-phototronic approach demonstrated in this work is easier, faster and less costly. Moreover, this approach can be extended to other pyroelectric semiconductor-material combinations, including visible and/or infrared-sensitive materials, and thus broaden the spectra ranges for more practical applications. Second, selfpowered PDs operated under zero bias were achieved by forming heterostructures between ZnO and perovskite. This configuration not only provides energy-efficient ultravioletsensing performances but also maintains low background temperatures by minimizing the dark current (o0.1 nA) to maximize the enhancements of pyroelectric effect. Third, the response time of these self-powered ZPH PDs was improved through pyroelectric effect from 5.4 s to 53 ms at the rising edge, and 8.9 s to 63 ms at the falling edge by five orders of magnitudes. The specific detectivity D* and the responsivity R of the PDs were both enhanced by 322%. The newly designed ultrafast ultraviolet PDs operating under zero bias may lead to further explorations in ultrafast optics, nonlinear optics, optothermal detections, computational memories and biocompatible optoelectronic probes.

Methods
Fabrication process of ZPH PDs. CH 3 NH 3 I was synthesized and purified following the previously reported method 34 . The synthesized CH 3 NH 3 I powder was mixed with PbI 2 (Aldrich) at 1:1 mol ratio in N,N-Dimethylmethanamide (DMF) at 60°C for 12 h, followed by filtering twice using 13-mm diameter and 0.45-mm pore polyvinylidene difluoride syringe filter (Whatman). The derived product was used as a coating solution for the formation of MAPbI 3 . Then, MAPbI 3 perovskite DMF solution with 40 wt% was prepared for further usages. Meanwhile, the FTO glasses (Sigma-Aldrich, 13 O À 2 ) were ultrasonically cleaned for 5 min in acetone, distilled water and ethanol, consecutively. Next, a layer of 50-nm ZnO seed layer was deposited on FTO glasses by radio frequency magnetron sputtering at room temperature (PVD75 system, Kurt. J. Lesker Company). The coated FTO glasses were then placed into the mixed nutrient solutions (0.02 M Zn(NO 3 ) 2 and 0.02 M HMTA) for ZnO NWs growth via a hydrothermal method in a mechanical convection oven (model Yamato DKN400, Santa Clara, CA, USA) at 85°C for 30 min. To get separated ZnO NWs, 0-5 ml ammonium hydroxide (Sigma-Aldrich) was added per 100 ml mixing solution.
After cooling down the whole system, the product was washed with ethanol and distilled water, collected and vacuum-dried at 100°C for 1 h. A 40-wt% perovskite DMF solution was spin-coated on the dried ZnO array at 4,000 r.p.m., followed by spin-coating the spiro-OMeTAD as a hole transport thin layer with a spin coater (SCS 6800). Another thin layer of Cu (250 nm) was deposited as the bottomelectrode afterwards. Testing wires were connected to top and bottom electrodes by silver paste. Finally, a thin layer of kapton tape was employed to fix the testing wires and to improve its resistance to environmental contaminations and corrosions.
Material characterizations. Detailed microscopic structures of ZnO NWs, perovskite, spiro-OMeTAD and copper film were characterized using scanning electron microscope (Hitachi SU8010), PANalytical X'Pert PRO diffractometer (Almelo, the Netherlands) with Cu KR radiation (l ¼ 0.15418 nm) for X-ray diffraction patterns of the MAPbI 3 perovskite.
Optical and electrical measurements. Transmission spectra of ZnO and ZnO/perovskite were measured by a ultraviolet-visible spectrophotometer (JLSCO V-630). I-V characteristics of the device were measured and recorded with a self-developed, computer-controlled measurement system with Stanford SRS Low-noise current preamplifier (SR570)/SRS low-noise voltage preamplifier (SR560) in conjunction with a GPIB controller (GPIB-USB-HS, NI 488.2). The optical input stimuli were provide by a He-Cd dual-colour laser (wavelength ¼ 325 and 442 nm, Model No. KI5751I-G, Kimmon Koha Co, Ltd). A continuously variable filter was used to control the light power density, which was measured with a thermopile powermeter (Newport 818 P-001-12). An objective was used to expand 325 and 442 nm laser to illuminate the whole device. The effective size of the device is 3 Â 3 mm. A hot plate was used to control and change the background environmental temperatures.


FLAVONOIDS FROM THE ROOTS OF DIOCLEA REFLEXA ( HOOK F . )

Reflevone (1), a new flavone, together with, mearnsetin (2), 7,4'-dihydroxyflavone (3) and two known phytosterols, β-sitosterol (4) and stigmasterol (5) were isolated from ethanol soluble part of the roots of Dioclea reflexa (Hook F.). Their structures were elucidated by analysis of spectroscopic data involving 1D and 2D NMR, MS, IR and UV experiments. The crude roots extract showed significant activity with IC50 = 5.18 μg/mL against the prostate cancer cell line.

INTRODUCTION
Dioclea (Leguminosae-Fabacea) is a small genus containing 40 species, widely distributed in Africa and South America [1]. The pharmacological activities of this genius include anticonvulsant, anxiolytic [2], antioxidant [3], antiarrhythmogenic [4], immunodulatory [5], analgesic and central antinociceptive effects [6], which have been identified with Dioclea plants. Dioclea reflexa (Hook F.) is a woody climber, which produces flowers and pods on long ropelike stems that hang from the forest canopy. It is known as 'Agbaarin' in Western Nigeria. The stem extract of this species is used by the local populace as an anti-tuberculosis (TB), and antiinflammatory agents; and also in the treatment of rheumatism and itching [7,8]. Previous studies of the seed have led to the isolation of dioclimidazole and two steroids: taraxasterol and stigmasterol [7,9]. In our earlier work on the root of D. reflexa, we characterized 15 volatile compounds comprising monoterpenes and diterpenes [8]. Although, several secondary metabolites have been isolated from the seeds [7,8] of D. reflexa, none have been reported from the roots of this species. In our search for the active constituents of D. reflexa, we hereby report an investigation of the cytotoxic activity of the crude extracts of leaves, stems and roots of D. reflexa against prostate cancer cell (PC-3). This led to the isolation and characterization of a new flavone, reflevone (1), together with mearnsetin (2), 7,4'-dihydroxyflavone (3), β-sitosterol (4) and stigmasterol (5) from roots extract. This is the first reported isolation of compounds 1-3 from this species.

General experimental procedures
The 1 H-NMR spectra (δ ppm, J in Hz) were recorded in deuteriated acetone or methanol on a Bruker AM-400 spectrometer (400 MHz), while 13 C-NMR spectra were recorded on the same instrument using the same solvent at 100 MHz. IR and UV were recorded using a JASCO 302-A and SHIMADZU UV-240 spectrometers respectively. HR-EIMS were carried out using MAT 95XP spectrometer. For the detection of samples on TLC plates, ceric sulfate spray reagent and UV light were used. TLC was carried out to check the purity of samples on pre-coated Merck silica gel F 254 aluminium sheets (0.25 mm thickness). Column chromatography was performed on silica gel (70-230 mesh) and Sephadex LH-20 (Fluka 25-100 mm). Melting points were determined on a Buchi 510-K melting point apparatus and were uncorrected. Enzyme-linked immonosorbent assays were recorded on Spectra Max plus 384, Molecular Devices, CA, USA, reader.

Plant material
The whole plant of Dioclea reflexa was collected from a farm land at Eruwa in Ibarapa East Local Government area of Oyo state, Nigeria, in May, 2011, and authenticated by Mr Bolu Ajayi of Herbarium Section, Plant Biology Department, University of Ilorin, Nigeria and a voucher specimen (UIL-0027) was deposited at the herbarium of the University.

Extraction and isolation
The air-dried and powdered roots of D. reflexa (1 kg) were extracted with 5 L of ethanol at room temperature for two weeks. Evaporation of the solvent under vaccuo yielded crude root ethanol extract (85 g). The same extraction procedure was adopted for the leaves and stem ethanol extracts. These extracts were first subjected to preliminary anticancer assay. The crude root ethanol extract was then subjected to silica gel column chromatography, eluted with gradient of n-hexane, dichloromethane, ethyl acetate and methanol in increasing polarity. The 332 fractions (100 mL/vial) collected were combined according to TLC profile.

In vitro cytotoxicity assay
The cytotoxic activity against Prostrate Cancer (PC-3) cells using test extract was performed in 96-well flat-bottomed micro plates by employing 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [10]. The PC-3 cells were grown in Dulbecco's Modified Eagle Medium, supplemented with 5% fetal bovine serum (FBS) and antibiotics (100 µg/mL of penicillin and 100 µg/mL of streptomycin) under a 5% CO 2 humidified atmosphere at 37 o C incubation. Cells culture with a density of 1 x 10 5 cells/mL were exposed to plant extract dissolved in DMSO at various concentrations of 1-30 µM. After 48 h, cells were washed 3 times with FBS, and then 200 µL of 0.5 mg/mL MTT solution in FBS was added and incubated for 4 h. Finally, 100 µL of DMSO was added to each well. In the experiment, Doxorubicin was used as positive control. The optical density was measured spectrophotometrically at 570 nm in an enzyme-linked immonosorbent assay reader (Spectra Max plus 384, Molecular Devices, CA, USA). The 50% inhibition concentration (IC 50 ) was calculated as the compound concentration required to reduce MTT signals by 50% compared with untreated control cultures.

RESULTS AND DISCUSSION
The IC 50 values of various parts extract of D. reflexa when tested against PC-3 cell lines are presented in Table 1. The roots extract showed cytotoxic activity against the cell lines (IC 50 5.18 µg/mL) when compared to the positive control, doxorubicin (2.8 µg/mL). This strong cytotoxic activity prompted further investigation of the roots ethanol extract of D. reflexa. The roots extract was then subjected to repeated column chromatography which yielded compound 1. Compound 1 was isolated as a yellow powder. The molecular formula was established as C 16 H 12 O 6 (300.0621) corresponding to 11 units of unsaturation based on the HREIMS, 13 C and DEPT NMR data ( Table 2). The UV spectrum of 1 showed maxima at λ max (logε) nm 212 (1.45) and 311(0.9) similar to those of flavones skeleton with two absorption bands. The IR spectrum suggested the presence of hydroxyl (3377 cm -1 ), conjugated carbonyl (1776 cm -1 ) and aromatic system (1570 cm -1 ).  (Table 2), DEPT and HMBC spectra showed resonances for all 16 carbons comprising one methoxy, six methine and nine quartenary carbons. The most downfield shifted peak at δ C 177.15 was assigned to carbonyl group (C-4). Two peaks at δ C 106.58 and δ C 151.83 showed double intensities and were assigned to C-2′/6′ and C-3′/5′, respectively [11,12]. The HMBC spectrum of 1 showed cross-peaks from H-2' to C-3', C-4', and C-2; from H-3 to C-4, C-2 and C-1' confirming the assignments for C-2, H2'/6' and the B-ring. In addition, the methoxy at δ H 3.88 showed correlation with carbon resonance at δ C 139.0 on the HMBC spectrum and was assigned C-4′ ( Figure 1). Furthermore, the 1 H-1 H COSY spectrum demonstrated correlations of H-5 to H-6 ( Figure 1). The spectral data were in accordance with reported data on flavonoids of 7-hydroxy A-ring [11,12].  The 2H singlet at δ 7.07, 2H broad singlet at δ 8.48 and 3H singlet of methoxy at δ 3.88 in ring B were assigned to H-2′/6′, OH-3′/5′ and H-4′, respectively; this suggested a 3',4',5' trioxygenated symmetrical substituted B-ring, typical of mearnsetin B-ring [13]. In HMBC, C-4 showed long-range connectivity to the 1 H peak at 7.95 ppm which was directly attached to the 13 C peak at 127.66 ppm (HSQC spectrum) and was assigned as C-5. In addition, the 1 H at 7.95 ppm showed long-range coupling to 13 C signals at 163.3 ppm and 158.74 ppm, these were assigned as C-7 and C-9, respectively. The pronounced upfield shift of 13 C peak at 163.3 ppm further supported the presence of a hydroxyl at C-7. The COSY spectrum as well as the HMQC spectrum confirmed the proposed structure (Figure 1). The complete assignments of the 1 H and 13 C-NMR data are listed in Table 2. These observations suggested that compound 1 is a 7,3′,5′trihydroxy-4′-methoxyflavone. Extensive literature search further revealed that compound 1 is new and has not been reported in the chemical literature, hence it was given the trivial name reflevone. Scheme 1. Fragmentation pattern for compound 1.
The retro-Diels-Alder fragments of ring C led to diagnostic peaks at m/z 137 and 164, supporting the presence of one hydroxyl in ring A, a methoxy and two hydroxyls in ring B (Scheme 1). Another diagnostic fragment at m/z 149 (C 8 H 5 O 3 ) indicated methoxy moiety at C-4′ which was demethylated (Scheme 1). Other significant peaks in addition to molecular ion at m/z 300.1 are 285 [M + -CH 3 ], 257, 229 and 121.  Compound 2, obtained as a yellow powder was assigned the molecular formula C 16 H 12 O 8 with molecular ion peak at m/z 332 from HR-EIMS and supported by its NMR spectra data ( Table 3). The UV spectrum in methanol showed maximum absorptions at 365, 260 and 212 nm. The 1 H-NMR spectrum ( Figure 2) showed two meta coupled 1H doublets at 6.26 (J = 2 Hz) and 6.52 (J = 2 Hz) assigned to H-6 and H-8 respectively corresponding to δc 99.18 ppm and δc 94.44 ppm respectively in the HSQC spectrum, characterised the H-6 and H-8 of a flavonoid 5,7-dihydroxyl A-ring [14]. The 13 C-NMR spectrum revealed all the sixteen carbons which were deduced as one methoxyl, four methines and eleven quartenary carbons. The broad singlet 1 H peaks at δ H 8.36 ppm which was not directly attached to any carbon in HSQC, was assigned to 3′/5′-OH. In the 1 H-NMR spectrum, 3H singlet at δ H 3.88 revealing a methoxyl group correlated with the carbon resonance at 138.02 (C-4′) on the HMBC spectrum. The 2H singlets at δ H 7.38 was attributed to the one proton each at 2′ and 6′ positions of 3′,4′,5′ tri-O-substituted B ring. Long-range COSY spectrum in conjuction with the HMBC spectrum allowed the assignments of H-6 and H-8 ( Figure 2). A singlet at δ H 12.1 corresponding to 13 C peak at δ C 162.3 ppm in HSQC, implied the presence of an OH at C-5, since H-bonding with the C=O (C-4) causes this type of substituted flavonoid to give signal between 159.8 and 162.3. Also, the signals for C-5 unsubstituted flavonoids are usually found between 106.9 and 127.75 [12]. Thus compound 2 was identified as 3,5,7,3′,5′-pentahydroxy-4-methoxyflavone (mearnsetin) by comparison of it spectra data with the literature [13,14].  (Table 3) with those reported in literature [11,[15][16][17] led to its identification as 7,4′-dihydroxyflavone. Compounds 4 and 5 were identified as β-sitosterol and stigmasterol respectively [18]. This is the first reported isolation of compounds 1-3 from this species.
In conclusion, the present study has shown that roots extracts of D. reflexa could be a lead in discovery of anti-cancer drug as demonstrated by its cytotoxic effect. Purification of the roots extracts led to the isolation of a new flavone compound and four others. Further studies are suggested on screening the isolated compounds against PC-3 cell lines, particularly the new compound, reflevone.