X-ray photoelectron spectroscopy (XPS) measurements confirms n-doping of the films by BV molecules. Since thin films of MoS2 and MoSe2 are typically more resistive than their exfoliated and co-evaporation based CVD counterparts, the decrease in resistance by BV doping represents a significant step in the utilization of these samples in electronic devices. Using selective BV doping, we simultaneously fabricated many lateral heterojunctions in 1 cm2 MoS2 and 1 cm2 MoSe2 films. The electrical transport measurements performed across the heterojunctions exhibit current rectification behavior due to a band offset created between the doped and undoped regions of the material. Almost 84% of the fabricated devices showed rectification behavior demonstrating the scalability of this technique.Scalable heterojunctions based on two-dimensional transitional metal dichalcogenides are of great importance for their applications in the next generation of electronic and optoelectronic devices. However, reliable techniques for the fabrication of such heterojunctions are still at its infancy. Here we demonstrate a simple technique for the scalable fabrication of lateral heterojunctions via selective chemical doping of TMD thin films. We demonstrate that the resistance of large area MoS In particular, heterojunctions of 2D van der Waals materials involving graphene and transition metal dichalcogendies (TMDs) semiconductors have created enormous attention due to their potential opportunities of designing novel device structures for the discovery of novel physics as well as for their potential applications in the next generation of electronic and optoelectronic devices17. However, the majority of the techniques that has been demonstrated for heterojunction fabrications are complex and suffer from low yield making them not suitable for scalable device fabrication. Van der Waals heterojunction was first demonstrated in a graphene—tungsten disulfide (WS2) system and was fabricated by stacking graphene and mechanically exfoliated flakes of WS2 via deterministic transfer method18. Since then, many different techniques have been demonstrated for heterojunction fabrication including mechanical exfoliation and transfer24, vertical and lateral heterojunction via direct CVD growth32, and vertical heterojunction in TMD thin films35. The stacking of 2D layered materials by mechanical exfoliation is a widely used method to create van der Waals heterostructures for laboratory research. In this process the mechanically exfoliated thin layer of one TMD is transferred to another type of TMD by using intermediate polymer to form a heterojunction37. This process requires days in preparing one heterojunction device due to the complexity in placement of one material on top of the other, thus cannot be scalable, a critical requirement for semiconductor device manufacturing technologies. Selective doping via oxygen and fluorine based plasma, and radiation have also been used in mechanically exfoliated samples to fabricate lateral heterojunctions40 demonstrating proof of concepts and they are not scalable due to difficulties in achieving good yields of samples from mechanical exfoliation.Two dimensional (2D) layered materials are considered to be promising candidates for applications in electronics, optoelectronics, catalysis, sensor, and energy storage devices41. A few successful examples of vertically stacked TMD heterostructures have been demonstrated, however, these heterojunctions are limited to very small areas lacking a control of their location, size, and coverage hindering their scalability. The direct growth of 2D heterostructure has also been made via chemical vapor deposition (CVD) method32. The co-evaporation of metal/metal oxides and chalcogen produces many TMDs heterostructures at random locations on the growth substrate43. However, electrical connections to these heterojunction requires complex fabrication steps. Therefore, the scalability of the co-evaporation based CVD technique for heterojunction device fabrication is uncertain.Individual layer of co-evaporation based chemical vapor deposition (CVD) grown TMDs along with post growth transfer process have been used for the fabrication of heterojunctions45, could be useful in the fabrication of heterojunctions, development of reliable techniques for scalable fabrication is still at its infancy. One useful technique could be the selective chemical doping of 2D TMD thin films for the fabrication of lateral heterojunctions. However, it is not known if the chemical doping can be successfully implemented in TMD thin films. In particular, benzyl viologen (BV) molecules are suggested to be promising candidates for the chemical doping of carbon nanotubes, graphene and other 2D materials47. BV is a good choice for charge transfer doping, as it has one of the lowest reduction potential among the electron-donating organic molecules48 and can transfer electrons to a suitable host acceptor material through a direct redox reaction, resulting in a change of carrier concentration in the host material. Therefore, doping of 2D TMD thin films by using BV molecules could change their charge carriers and transport properties and selective doping could open up the possibility of creating a band offset with undoped TMD thin films to fabricate many TMD based heterojunctions in a massively parallel fashion.While large area TMD thin films, prepared via chalcogenization of metal  or metal oxide films2 thin films were grown via low pressure sulfurization of Mo films. The electrical characterization performed before and after BV doping show that the resistance of the samples decreased by up to two orders of magnitude upon doping. X-ray photoelectron spectroscopy (XPS) measurements showed a relative shift of peak positions towards higher binding energy values for the BV doped samples suggesting n-doping of the films. The amount of doping can be controlled by sample immersion time to the BV solution. We then implemented a selective area doping method to create doped and undoped-MoS2 regions to fabricate many lateral heterojunctions simultaneously on a 1 cm2 chip. The fabricated junctions showed excellent current rectification behavior demonstrating the effectiveness and scalability of this doping method. Similar results for BV doping of MoSe2 thin film along with parallel fabrication of heterojunctions between doped and undoped MoSe2 via selective doping were also demonstrated.In this paper, we demonstrate an effective chemical doping of 2D TMD thin films via BV molecules and use a selective doping technique to fabricate many lateral heterojunctions simultaneously on a single chip. The large area MoS2 thin films were prepared via low pressure sulfurization of molybdenum film deposited on a Si/SiO2 substrate, as described in the method section. Raman characterization confirmed the fabricated samples to be a few layer MoS2  characteristics of an undoped MoS2 sample (red curve). The resistance of this sample is calculated to be 215 MΩ. We have used highly doped Si as a back gate, however, no gate dependence was observed consistent with a few other reports52. We have measured 47 MoS2 devices fabricated on two chips. The resistance of these devices varied from 185 to 490 MΩ with an average of 347 MΩ as shown in the box plot of Fig. 2 devices are consistent with what has been reported in the literature54. After characterizing the as-prepared samples, we doped the MoS2 film by immersing it in BV solution for 36 h (see “Method” section for detail) and measured their electrical transport properties. Figure 2 thin film into BV solution indicates that the MoS2 thin film is strongly doped by the BV molecules due to the transfer of electrons from BV to MoS255 via surface charge transfer process resulting in an increase of charge carrier concentration. The charge transfer occurs through the redox reaction shown in supplementary Figure 0 , BV+ and BV2+ with a reduction potential of − 0.790 V (BV+  →  BV0) and -0.332 for (BV2+  →  BV+) vs the standard hydrogen electrode (SHE). On the other hand, MoS2 is known to have a conduction band edge at around 0 V vs SHE55. The lower reduction potential of BV compared to the MoS2 conduction band edge gives rise to electron transfer from BV to the MoS2 surface which increases the electronic carrier concentration (decrease of resistance) of MoS2 channel.Large area MoS2 devices after immersing them in BV solution and found that the resistance for all the samples decreased. This is more clearly shown in the box plot of Fig. 2 devices is 347 MΩ, which drops to 22 MΩ after doping. The average resistance of BV doped MoS2 devices is 94% lower than as prepared MoS2 devices. Since thin film of MoS2 is typically more resistive than their exfoliated and co-evaporation based CVD counterparts, the decrease in resistance by BV doping represents a significant step in the utilization of these samples in electronic devices.We have measured a total of 47 MoS2 thin film, X-ray photoelectron spectroscopy (XPS) measurements were performed for the as prepared and BV doped MoS2 samples 57. The presence of BV2+ peak suggest charge transfer from BV to MoS2 occurred  of S 2s, Mo 3d5/2 and Mo 3d3/2 electrons of MoS2, respectively  increases (decreases) with increasing immersion time and then saturates. We observed the current at Vds = 1 V increased from ~ 2.9 × 10–9 A to 7.0 × 10–9 A after 12 h immersion of the MoS2 device in BV solution resulting in a decrease of resistance from 360 MΩ to 142 MΩ. With further 12 h  soaking of the MoS2 device in BV solution, the current increased to ~ 8.2 × 10–8 A (resistance decreased to 12.2 MΩ). The current further increased to ~ 2.5 × 10–7 A with an additional 12 h  of immersion resulting in a decrease of resistance to ~ 4.1 MΩ. With further immersion of the MoS2 device for another 12 h  in BV solution, resistance decreased to 3.9 MΩ which is a small fraction of change compared to the MoS2 device with 36 h immersion. The variation of current and resistance with immersion time is more clearly shown in Fig. 2 device is immersed in BV solution, BV molecules adsorb on the MoS2 surface. The BV donates an electron to MoS2 and changes to another oxidation state (BV+). The BV+ further donates an electron to MoS2 surface and becomes BV2+. More BV molecules adsorb on the MoS2 surface with increase of immersion time which causes more charges transfer from BV to MoS2. When the MoS2 surface is saturated by BV2+ molecules, further charge transfer stops. Notably, the BV doped MoS2 thin film devices are relatively stable with only minimal change in the current over a period of a week . The resistance of this sample is calculated to be 388 MΩ. We have measured 27 MoSe2 devices that were fabricated on the same chip and the resistance of the devices varies from 150 MΩ to 410 MΩ with an average of 320 MΩ as shown in the box plot of Fig. 2 samples were then immersed in BV solution for 36 h for charge transfer doping process following which the electrical characterizations were carried out. Figure 2 devices. This suggests that, similar to MoS2 thin film, BV molecules can effectively dope MoSe2 thin film by transferring electrons from BV molecules to MoSe2. XPS measurement of MoSe2 films further confirms the charge transfer based doping. For as prepared MoSe2 thin film, the XPS peaks correspond to Mo 3d5/2 and 3d3/2 were observed at binding energies 229.1 and 232.2 eV respectively  of this device is calculated to be ~ 78. As the individual MoS2 devices did not show any gate dependence, the heterojunction devices did not show any gate dependence characteristics either when highly doped Si was used as back gate. In order to ensure that the measured electrical properties are originated from the fabricated heterojunctions, we monitored the effect of the fabrication process by measuring I–V curves after every fabrication step  between the doped and undoped regions of MoS2 resulting in the formation of lateral heterojunctions. This band offset prevents the flow of charge carriers at the negative bias voltages and leads to a rectification behavior. Similar selective doping method has been implemented to simultaneously fabricate many MoSe2 heterojunction devices on a 1 cm2 chip which also showed excellent current rectification behavior, demonstrating the scalability of the technique  and an alumina boat containing sulfur (S) powder was placed in the furnace upstream side. The system was pumped down to a base pressure of ∼30 mTorr and purged argon (Ar) gas to remove oxygen and water vapor. The Ar gas flow of ∼130 standard cubic centimeters per minute (sccm) was maintained during entire growth process. The furnace was heated to the growth temperature of 800 °C with ramping rate of 15°/min and was maintained at the growth temperature for 55 min. The sulfur vapor was carried by Ar gas to the central zone where it reacts with the Mo to form MoS2 films. The chamber was allowed to cool down to room temperature naturally after growth. MoSe2 film was prepared by selenization of the 2 nm Mo metal film using same method as MoS2 film preparation.Molybdenum (Mo) film of 6 nm thickness was deposited by an electron beam evaporator at a low evaporation rate of 0.05 Å/s on a Si/SiORaman characterization was performed using a Witec alpha 300 RA confocal Raman microscope with laser source of excitation wavelength of 532 nm and power of < 1 mW in ambient conditions at room temperature. A 100 × objective was used to focus the laser beam at a spot. Raman emission was collected and dispersed by a grating of 1,800 lines-per-mm with the data accumulation duration of 3 s.2 and carbon (C) 1 s reference line at the binding energy of 284.8 eV was used to calibrate the charging effect.XPS measurements of the TMD films were performed using a Thermo Scientific  XPS system with a monochromatic Al Kα radiation source. Pass energy of 20 eV with 0.1 eV scanning step was used for photoelectron detection. XPS spectra were taken onto the sample surface with a scan area 300 × 300 µm2 and MoSe2 films, 5 nm/40 nm Cr/Au electrodes were patterned on top of MoS2 and MoSe2 films using a shadow mask. The electrodes were deposited with a deposition rate of 0.05 Å/s at a base pressure of 5 × 10–7 mBar. The electrical transport measurements of the devices were performed in a probe station using a current preamplifier (DL instruments 1211) interfaced with Lab View program. All electrical measurements were carried out at room temperature in ambient conditions.For the electrical transport characterization of as prepared and BV doped MoS2 and MoSe2 devices was immersed into the extracted BV solution for doping of the thin films. The doping was performed by immersion of the MoS2 and MoSe2 devices into the BV solution for 12 to 48 h. The samples were then taken out of the solution and allowed to dry naturally.25 mg benzyl viologen dichloride (Sigma-Aldrich) was dissolved into 5 ml of nanopure DI water. 5 ml of toluene was added to benzyl viologen dichloride and water to make a bilayer solution. ~ 3.7 gm of Sodium borohydride (Sigma-Aldrich) was added to the water/toluene bilayer solution. The solution was then kept for one day to form benzyl viologen (BV). The BV accumulated in the toluene solution. The toluene solution with BV molecules was carefully extracted using pipette. The MoSSupplementary Information.


Recent studies have suggested that estrogen (E2) plays an important role in epithelial ovarian cancer (EOC). However, the mechanism of E2 in ovarian cancers is unclear. The purpose of this study was to investigate the effect of E2 on ovarian cancers and illuminate the mechanism of E2 in promote ovarian cancers proliferation.+. Moreover, siRNA-mediated silencing of TRPC3 expression inhibited the ability of E2 to stimulate the influx of Ca2+.We demonstrated that E2 stimulated the proliferation and invasion of ovarian cancer cells. In this study, ovarian cancer specimens were also analyzed for transient receptor potential channel C3 (TRPC3) expression; TRPC3 expression levels were higher in ovarian cancer samples than in normal ovarian tissue samples. Previous studies have shown that TRPC3 contributes to the progression of human ovarian cancer. In this study, we further investigated the interaction between E2 and TRPC3. We found that E2 stimulation enhanced the expression of TRPC3 at both the mRNA and protein levels. E2 stimulation enhanced the influx of Ca2In conclusion, TRPC3 plays a significant role in the stimulatory activity of E2 and could be a therapeutic target for the treatment of EOC. Furthermore, this study elucidates the molecular mechanism by which E2 promotes the proliferation and migration of EOC cells. Epithelial ovarian cancer (EOC) is the most common type of gynecological cancer. Annually, 230,000 women worldwide will be diagnosed with EOC, and 150,000 will die –8. Thus,The ovaries are the main source of female reproductive steroid hormones. Several lines of evidence support the view that the growth of many ovarian cancers is regulated by estrogen E2, , 10). Mo. Mo10]).2+ is a second messenger that plays a major role in the regulation of cellular functions such as secretion, cell growth and death, and contraction [2+, can be involved in calcium influx and downstream pathways, regulating cell survival, proliferation and carcinogenesis via intracellular translocation induced by hormones and growth factors [Catraction , 16. The factors –19. TRPC factors .Here, we aimed to identify the expression of TRPC3 in EOC tissue samples and ES2, SKOV3, OVCAR, and HEY cells treated with E2 to determine the roles of TRPC3 in the E2-mediated regulation of EOC-related cell proliferation using TRPC3-specific siRNA interference. In addition, we compared the expression of TRPC3 genes in human ovarian cancer tissue samples from patients with that in normal ovarian tissue samples.Normal ovarian tissue comes from 6 patients with benign uterine lesions and normal ovaries who has had a hysterectomy and has had the ovarian tissue removed. Seven ovarian cancer patient tissue samples were obtained from the gynecology and obstetrics department of Xijing Hospital . Prior to the experiment, all patients were informed of the purpose of the study as well as the procedures and voluntarily agreed to provide tissue. Written consent was obtained from all participants, and all protocols were approved by the Ethics Committee of Xijing Hospital, which is affiliated with the Air Force Medical University.2. All cells were purchased from GENE . E2 was purchased from Sigma-Aldrich . E2 was dissolved into DMSO. FBS was treated with activated carbon when added during E2 stimulation, and the medium used was phenol-free red medium. Cells were treated with 10 nM, 100 nM or 1 μM E2 for 24 h, 48 h or 72 h. DMSO was used as a negative control.ES2 and SKOV3 cells were cultivated in McCoy’s 5A medium ,and OVCAR and HEY cells were cultured in 1640 medium supplemented with 10% fetal calf serum  and 2 mM L-glutamine at 37 °C and 5% CO3 cells were grown in a 96-well plate with or without different concentrations of E2 . DMSO was used as a negative control. After incubation for 24 h, 48 h or 72 h, 20 μL of 5 mg/ml 3--dimethylthiahiazo-3, 5-di-phenytetrazoliumromide (MTT) was added to each well. The cells were incubated for 4 h at 37 °C, and then the culture medium was replaced with 150 μL of DMSO (Sigma). The absorbance of the formazan product at 490 nm was measured with a plate reader. All experiments were performed with three replicates.In total, 5 × 104 cells) in the upper compartment of a Transwell plate  with 1 μM E2. McCoy’s 5A medium supplemented with 10% FBS and 1 μM E2 was added to the bottom of the well to serve as a chemoattractant for the SKOV3 cells. For the control group, an equal volume of DMSO instead of 1 μM E2 was added to the medium. Next, the cells were allowed to migrate for 24 h, after which the cells were fixed, stained with crystal violet and quantified by ImageJ software. For each biological replicate, at least three random fields were imaged. The influence of E2 on SKOV3 cells transfected with TRPC3 SiRNA was also investigated by chemotaxis assays. For this experiment, cells were allowed to migrate for 48 h.SKOV3 cell migration was examined with chemotaxis assays. SKOV3 cells were starved for 12 h in McCoy’s 5A medium without FBS. Then, the cells were seeded . The wells without siRNA (no siRNA) or with a scrambled siRNA or a nonspecific pool of siRNAs were set as negative controls in parallel.5 cells/well) in McCoy’s 5A medium and 1640 medium. After reaching confluency, cells were starved with serum-free medium for 12 h. Then the cells monolayer was scratched horizontally with a yellow pipette tip to obtain a monolayer culture with a space without cells. Cellular debris was removed by gently washing twice with PBS and then cultured with or without 100 nM E2. Three randomly selected fields along the scraped line were photographed using a phase contrast inverted microscope. After incubation for 12, 24, 36, 48, 72 h, images were acquired and cell migration distances were evaluated by ImageJ.SKOV3 and HEY cells transfected with or without TRPC3 SiRNA were cultured on 6-well plates . One thousand SKOV3 and HEY cells transfected with or without TRPC3 SiRNA were cultured on 96-well plates and 100 nM E2 was added to the culture solution and cultured for 24 h, then 10 μL CCK-8 reagent was added to medium and incubated for 1 h. OD  at 450 nm was read with a microplate reader. Each experiment was performed three times independently.For immunocytofluorescence analysis, cells were fixed with 4% paraformaldehyde. Then, the cells were permeabilized with Triton X-100 and blocked with BSA. The cells were incubated with an anti-TRPC3 antibody  at 4 °C overnight and with a CY3–555-conjugated IgG antibody at 37 °C for 2 h in the dark on the following day. DAPI was used for nuclear staining. Images were captured with an FSX100 microscope . Tissue samples obtained from patients were fixed in 4% paraformaldehyde and serially cut into sections of 16-μm thickness using a cryostat. For immunocytofluorescence, the frozen sections were subjected to antigen retrieval. An anti-TRPC3 antibody was employed as the primary antibody at a dilution of 1:200; and a cyanidin-3-labeled goat anti-rabbit secondary antibody was used to visualize TRPC3 expression. All stained slides were scanned using a confocal laser scanning microscope, and images were analyzed using ImageJ software .OVCAR, ES2, HEY and SKOV3 cells were seeded in 6-well dishes. After 48 h, the cells were treated with E2. Total RNA was extracted from the different cells using RNAiso Plus  and was subsequently reverse transcribed into cDNA using PrimeScript™ RT.− 1), 10 μL of SYBR green PCR Master Mix, and 0.2 μL of ROX Reference Dye II, and distilled water was added to reach a final reaction volume of 20 μL. Taq DNA polymerase was activated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s. Quantitative RT-PCR data were normalized to the expression of the housekeeping gene GAPDH using the 2-ΔCt method. The primers used in this study are shown in Table Master Mix with random primers  was used according to the manufacturer’s protocol. The expression of different genes was analyzed using SYBR® PremixEx Taq™ II and the Bio-Rad CFX System. For real-time PCR, the reaction mixtures contained 2 μL of cDNA, 0.8 μL of each primer 10 mmol0 mmol− 1Cell lysates were extracted with a lysis buffer. Western blotting was performed with 30 μg of total protein after total protein concentrations were estimated by a BCA assay, and then the protein samples were mixed with 5X SDS-PAGE protein loading buffer  and boiled. The samples were transferred to a PVDF membrane  by using the wet transfer method after being separated on 10% SDS-PAGE gels . After blocking with 5% milk in TBST for 2 h, the membranes were incubated with an anti-TRPC3  or anti-β-actin  antibody overnight at 4 °C. To verify the specificity, the membranes were incubated with control peptide antigen and the primary antibody. β-actin was used as an internal control. The membranes were incubated with a horseradish peroxidase-conjugated anti-rabbit antibody at room temperature for 1 h after washing with TBST. The blots were visualized by ECL Western blotting substrate  according to the manufacturer’s instructions.2, 1.8 mM CaCl2, 10 mM HEPES, 10 mM glucose, and NaOH added to achieve a pH of 7.4) three times, continuously perfused throughout the experiment at room temperature with a nominal-calcium indicator loading buffer and challenged with ATP and E2. To investigate the effect of E2 on the expression of TRPC3 in ovarian cancer cells, SiTRPC3 and Sicon were transfected into SKOV3 and HEY cells, which were then incubated on a glass coverslip for 48 h. Then, 1 μM E2 was added to the medium at the start of detecting intracellular Ca2+. Excitation light was supplied via a Polychrome II polychromator , and emission was detected by a Sensicam CCD camera . Ca2+-sensitive Fura-2 AM fluorescence was measured ratiometrically at 340/380-nm wavelengths with an emission wavelength of 510 nm. Recordings were analyzed by using Axon Imaging Workbench .SKOV3 and HEY cells were grown on coverslips and loaded with the calcium indicator Fura-2 AM  for 30 min in McCoy’s 5A medium at room temperature and away from light, washed with normal calcium desklop fluid  and SPSS20 . A To investigate the function of ER-α in ovarian cancer, we detected the expression of ER-α in different ovarian cancer cell lines. The cell line ES2 had the highest expression level among the four ovarian cancer cell lines, while the cell line OVCAR had the lowest expression level Fig.a. Next, To detect the expression of TRPC3 in ovarian cancer tissue samples, ovarian tissue samples from ovarian cancer and uterine fibroid patients were collected to analyze the expression of TRPC3. The ovarian tissue samples from the patients with ovarian cancer showed higher protein expression of TRPC3 Fig. .Fig. 3TTo investigate whether E2 promotes the proliferation of ovarian cancer cells through TRPC3, we detected the expression of TRPC3 in different ovarian cancer cell lines through Western blot and immunocytofluorescence analyses. The expression of TRPC3 in SKOV3 and OVCAR cells was higher than that in HEY and ES2 cells  induced the upregulation of TRPC3 expression at the mRNA level Fig. a. E2, esPrevious studies have shown that TRPC3 acts as a Ca2 + −permeable channel in E2-responsive cells. To test whether E2 regulates TRPC3-mediated Ca2+ uptake, cells induced with 1 μM E2, ATP, and SiTRPC3 were analyzed using Ca2+ imaging. Treatment with 1 μM E2 facilitated intracellular calcium influx, which was consistent with the result of for ATP treatment in different cells Fig. a and c. EOC is the most lethal gynecological cancer and represents the seventh most commonly diagnosed cancer among women worldwide . ApproxiTo improve the treatment of EOC, disease pathogenesis is still being explored. Numerous studies have demonstrated the associations of E2 with the development and/or progression of various types of cancer, including cancers of the ovaries, lungs, and colon , 23. Pre+ signaling is believed to play a central role in the signaling cascades involved in tumorigenesis and neoplastic progression [+-dependent signaling suppress the proliferation of cancer cells in vitro and solid tumors in vivo [+ channels (ROCs) or internal Ca2+ store-operated channels (SOCs), which mediate the Ca2+ signaling pathway activated by many hormones and growth factors [Ca2gression . Inhibit in vivo , 28. The factors , 29. TRP factors –32. Prev factors . ConsistBecause of the possibility of TRPC heteromultimerization, biological activities may involve more than one TRPC, which makes it challenging to identify the function of a single subtype . InteresIn summary, the results presented in this study demonstrated that the molecular mechanism of E2 involved promoting the proliferation of ovarian cancer cells. We demonstrated that E2 stimulated proliferation and migration by upregulating the expression of TRPC3, which enhanced intracellular calcium influx.Additional future experiments might include overexpressing TRPC3 to observe the effects and experiments exploring the molecular mechanism by which estrogen regulates TRPC3 expression.Additional file 1: Figure S1. Specific verification of TRPC3 used control peptide antigen. Control peptide antigen was used to demonstrate specificity of the band around 97 kDa. It showed that band around 97 kDa specifically represent TRPC3.


Oryza sativa L.). Furthermore, it is known that Si supply reduces the concentration of Fe in rice shoots. We hypothesized that the Si-enhanced CB formation in the exodermis reduces in the flux of Fe in the apoplast and the uptake of Fe loaded deoxymugineic acid. Thus, the effect of silicic acid supply at varied Fe concentrations and Fe forms was investigated in nutrient solution. The Fe concentrations in the shoot and apoplastic Fe concentrations in the root were determined and an Affymetrix GeneChip experiment was carried out together with qRT-PCR measurements for observation of transcriptomic reactions. Additionally, the Fe uptake of an overexpression mutant of OsABCG25 with an enhanced exodermal CB formation was investigated. The application of silicic acid reduced the Fe concentrations in shoot DM independently of the supplied Fe concentration and Fe form. As a reaction to the Fe shortage, the full cascade of Fe-homeostasis-related genes in the roots was upregulated. Silicic acid supply also decreased the apoplastic Fe concentrations in roots. In addition, an overexpression mutant of OsABCG25 with an enhanced CB formation showed a reduced uptake of Fe in excess Fe conditions. The results suggest that the Si-induced CB formation in the exodermis hampers the flux of Fe into the apoplast of the cortex and, thus, Fe uptake of rice grown in nutrient solution which is reflected in the upregulation of Fe homeostasis-related genes.Gramineous plants take up silicon (Si) that enhances the formation of exodermal Casparian bands (CBs) in the roots of rice ( Another effect of Si is the earlier formation of the exodermal Casparian bands (CBs) which appear in submerged grown rice6. The mechanism is not yet clear7 but it is suggested that Si(OH)4 forms crosslinks with phenols within cell walls leading to an enhanced CB development. The CBs are depositions of suberin and lignin in anticlinal cell walls of the endodermis of all higher plants and of the exodermis of many plants9. Endodermal CBs control the unselective bypass flow of water and ions into the vascular bundle10. Exodermal CBs are described in rice as controlling radial oxygen loss and water flux12. It is also indicated in the literature that the exodermal CBs could affect in nutrient acquisition. Due to the late occurrence of CBs during root development and the disruptions of the exodermis from lateral roots, it is mentioned that the effect of exodermal CBs in ion exchange should not be overestimated14. Accordingly, the flux of apoplastic tracer, such as trisodium 3-hydroxy-5,8,10-pyrene trisulfonat or cis-absicid acid was not hampered by the exodermal CBs15. But, the exodermal CBs are also described as a diffusion barrier reducing the flux of ions into the cortex19 and to have a function in controlled substance exchange22, while positive effects of the exodermal CBs for water management are also described8. Furthermore, it has been shown that Si supply reduces the bypass flow of sodium and, thus, the sodium uptake declines24.Silicon (Si) is the second most abundant element in the soil crust and many plants can take up and accumulate Si26. Exodermal CBs block the radial oxygen loss in adventitious roots6. Previously it was shown that Si enhances the formation of exodermal CBs in adventitious roots of rice and clearly reduces the radial oxygen loss27. The exodermal CB formation starts with/ without Si application 4–5/8–10 cm from the root tip in the anticlinal cell walls and is completed at 7–8/12–13 cm distance from the root tip, respectively28.Rice has exodermal CBs which have a specific function regarding the aerenchyma, which is a root tissue developing from collapsing cortical cells to ensure the oxygen supply of the root29 reported that Si supply reduced the Fe uptake of plants grown in nutrient solution and under Fe excess the toxicity symptom leaf bronzing clearly decreased30 but causal relationships are still not clear. Iron acquisition in plant kingdom follows two strategies31. In dicotyledonous and monocotyledonous non-graminaceous species (strategy I) Fe(III) precipitates are dissolved in the rhizosphere by release of protons and excreted phenols such as coumarins chelate Fe(III)32. This complex diffuses to the root surface where the membrane bound ferric reductase reduces Fe(III) to Fe(II) which is taken up by iron related transporter (IRT). The activity of the ferric reductase is enhanced by low pH. Graminaceous plants such as rice follow strategy II in Fe uptake33. In strategy II, plants secrete via TOM1 phytosiderophores, such as deoxymugineic acid (DMA), into the rhizosphere that bind to hardly available FeIII+. This complex is taken up by yellow stripe-like (YSL) transporters at the root epidermis and, most probably, in the root cortex36. Under Fe deficiency iron homeostasis genes are regulated and the expression of genes involved in the synthesis of DMA from methionine such as nicotianamine synthase (NAS), nicotianamine aminotransferase (NAAT), and deoxymugineic acid synthase (DMAS) is enhanced. Also genes encoding for mugineic efflux transporters (TOM) and influx transporters of Fe chelates (YSL) are upregulated41. The rhizosphere is different in plants grown in nutrient solution to those grown in soil since the whole pot volume can be defined as rhizosphere, because of the high diffusion coefficient in water. Thus, DMA is extremely diluted and less effective for Fe acquisition. In nutrient solution, the binding to DMA and the subsequent uptake is discussed to take place in the apoplastic space of the cortex42.Silicon also affects nutrient uptake in rice. Previous studiesThe aim of this work is to identify the reasons for the lower Fe content in the leaves of plants grown with Si. We hypothesize that the earlier CB formation in the exodermis reduces the amount of Fe in the apoplast and, thus, reduces the uptake of DMA-loaded Fe.−1) or low Si  in nutrient solution for 28 d. The zone 2–6 cm from the tip of adventitious roots was harvested, since here CB development in the exodermis is clearly enhanced by Si supply28, and an Affymetrix GeneChip was used to identify differentially regulated genes. We observed 172 upregulated and 207 downregulated genes due to Si treatment with a minimal fold change of 2 at a p-value cut-off of 0.01  and OsHRZ1 (LOC_Os01g49470.2).Rice plants were grown with high , nicotianamine aminotransferase 1  and deoxymugineic acid synthase  were upregulated while OsNAS3 was not regulated differentially. The mugineic acid (MA) efflux transporter OsTOM1 (LOC_Os11g04020.1) was similarly upregulated, while the expression level of OsTOM3 was decreased. The nicotianamine efflux transporter OsENA1 responsible for Fe-MA mobilization in the vacuole was also upregulated. An increased expression level was furthermore observed for genes related to the influx of Fe-MA, such as OsYSL2 (LOC_Os02g43370.2), OsYSL9 (LOC_Os04g45860.1), OsYSL15 (LOC_Os02g43410.2) and OsYSL16 (LOC_Os04g45900.1). The Fe2+ transporter 1 (OsIRT1) was also upregulated. A differential regulation of genes involved in Fe-MA storage and sequestration was not observed .Additionally, genes for the desoxymugineic acid (DMA) biosynthesis, such as nicotianamine synthase 1/2 (II)SO4 than with FeEDDHA and FeEDTA. The Fe supply in the form of Fe(II)SO4 enhanced the Fe concentration in shoot DM by a factor of two compared to the Fe chelates.Silicic acid supply also reduced the Fe concentrations in shoot DM independently of the Fe form, as can be seen in Fig. EDTA or Fe(II)SO4 showed chlorotic leaves at the beginning of the experiment. This effect was not measurable with the SPAD meter due to the small leaf area of the plantlets.There was no difference in the chlorophyll concentrations (SPAD) at harvest between treatments  having an enhanced CB development in the exodermis was used and grown under excessive Fe supply to reinforce the hypothesis27. The overexpression mutant grown without Si contained clearly less Fe in shoot DM compared to WT , and finally the Fe-MA uptake transporters YSL2 and YSL15. The expression of OsENA1 coding for a Fe-DMA/nicotianamine (NA) transporter from the vacuole was also enhanced.Silicic acid supply hampered the Fe nutrition of rice and this was reflected in the expression of Fe homeostasis genes. About half of the genes investigated were upregulated at the lowest Fe level without Si supply Fig. , but notA similar pattern of Si-induced gene expression was observed in the experiment with different Fe forms Fig. .27. Si supply also reduced the Fe concentration in shoot DM of rice grown in nutrient solution under Fe excess nutrition44. We further observed a reduction of the Fe concentration in the root apoplast  at optimal Fe conditions, as observed in other studies51. Furthermore, a mutant overexpressing the ABC transporter OsABCG25 resulting in enhanced CB development27 also had a decreased concentration of Fe in the shoot DM 4 in terms of interactions with enzymes or other intracellular constituents but that such reactions are based on indirect effects caused by Si deposition and reactions in the apoplast. In a certain way this agrees with a lack of specifity of Si-transporters which originate according to the horizontal gene transfer model from bacterial As-transporters2.A scheme displaying the suggested functions of Si in Fe acquisition of plants grown in nutrient solution is presented in Fig. IDEF1 or 2, which both are regulating elements of OsTOM1, OsIRO2, OsNAS1 +2, OsNAAT1 and OsDMAS. But not OsNAS3 which is only active under excess Fe conditions53. The transcript level of OsIDEF1 +2 is not influenced by Fe availability, but only the protein binding to the DNA is enhanced during Fe deficiency54. This agrees well with our results that Si did not affect their expression levels  was highly upregulated, which agrees with the literature35. An upregulation of the TOM3 homologon was found, but not of OsTOM3 in the epidermis of rice roots in Fe-deficient conditions.TOM3 was not affected by Si in rice roots, while the TOM3 homologon 2 × 4 × H2O, 1.6 mM MgSO4 × 7 × H2O, 1.82 µM MnSO4 × H2O, 0.03 µM (NH4)6Mo7O24 × 4 × H2O, 9 µM H3BO3, 0.6 µM ZnSO4 × 7 × H2O and 0.15 µM CuSO4 × 5 × H2O. Iron was added as Sequestren™  for Fe gradient experiment in three different levels, low Fe (3.58 µM FeEDDHA), optimal Fe (35.81 µM FeEDDHA) and high Fe (179.05 µM FeEDDHA) corresponding to 0.2, 2 and10 mg Fe, respectively, in nutrient solution. An amount of 2 mg Fe L−1 was added as FeEDDHA, Fetrilon™  and iron sulfate  for the Fe form experiments. Plants were cultivated for three weeks as in the Fe gradient experiment, and the last week with 4.475 mM Fe supplied as FeIISO4 × 7 × H2O for the growth of the mutants of OsABCG25. Please see Hinrichs et al.27 for characterization of OsABCG25. The Si was applied as silica gel . A stock solution was prepared by addition of 5 kg silica gel in 1000 L H2Odest. at RT in the dark. The silica gel dissolved slowly until a Si-concentration of 50 mg*l−1 was reached. This stock solution was diluted with H2Odest. to a working-solution of 30 mg*l−1 according to Fleck et al.6. The H2Odest. had a basal Si-concentration of 3 mg*l−1 due to technical reasons. The resulting Si concentrations in the plant are in the range of 0.1 and 60 mg g−1 shoot DM according to previous findings57. The pH value of the nutrient solution was adjusted to 6.0 by the addition of 10% H2SO4 or 5 M KOH. The nutrient solution was renewed weekly for the first two weeks, after two weeks, and subsequently the nutrient solution was changed twice a week until harvest. Plants were cultivated in a climate chamber  for 28 d in nutrient solution.Seeds of After 28 d in nutrient solution, the root zones 4–6 cm from the the root tip were harvested, transferred immediately to liquid nitrogen and stored at −80 °C for transcript analysis. Only adventitious roots were harvested for Fe determination in the apoplast. Shoot and root were separated, dried at 60 °C for 4 d and ground for element analysis. The content of chlorophyll was measured spectrometrically using an N-Tester .2S2O4, 10 mM MES, 0.5 mM Ca(NO3)2, pH = 5.5) for 5 s. Subsequently, the roots were washed three times in H2O and transferred into 21 ml anaerobe reaction solution 2, pH = 5.5)51. The reaction solution with the samples was deoxygenized by N2 bubbling in an Erlenmeyer flask covered with a cotton plug for 2 min. After 2 min, 1 ml of a cooled 250 mM Na2S2O4-solution was added using a syringe and incubated by gently shaking for 5 min at room temperature. After incubation, the roots were removed, and the reaction was stopped. The washing solution and reaction solution were stored on ice with constant N2-bubbling until usage. The Fe was determined by measuring the A520 of the filtrated reaction solution.Adventitious roots were separated and Fe on the root surface was removed through rinsing in anaerobe washing solution , following the manufacturer’s instructions. The RNA quality was determined electrophoretically by 1% Agarosegel and fluoretically using a Nanophotometer . The RNA (1 µg), oligo(dT)18-Primer (0.25 µg) and random hexamer primer (0.25 µg) were used to synthesize first-strand cDNA using the Revert Aid™ H Minus First Strand cDNA Synthesis Kit , following the manufacturer’s instructions for GC-rich templates.The RNA was additionally measured by capillary electrophometric by a bioanalyzer  using a RNA 6000 nano assay in the GeneChip experiment. Only samples with a RNA integrity number over 9.5 were used for the GeneChip experiment. A set of sixteen Genechips were used for the experiment, consisting of two treatments (+Si/−Si), four biological repeats and two technical repeats of the Affymetrix GeneChip experiment. One biological repeat consisted of a pool of the RNA of five plants grown in the same 5-L pot.An amount of 50 ng cDNA was used as a template in 10 µl reaction mix of the SYBR Premix Ex Taq (Tli RNase H Plus)  and 0.2 µM forward and reverse primer in the quantitative real time PCR (qRT-PCR) experiments. The qRT-PCR runs were performed in the CFX96 cycler , using an initial 95 °C step for 30 s, followed by 40 cycles of 95 °C for 5 s, and 60 °C for 30 s, and a final melting curve procedure with a stepwise increment of 0.5 °C ranging from 65 to 95 °C.59. The geometric mean of the Ct values of the two endogenous control genes was calculated according to the literature60, and this was used to calculate the foldover reference of the different samples (2-delta Ct). A list of primer sequences used can be found in Supplementary Table 61, based on the 2−ΔΔCT method.The eukaryotic elongation factor 1-alpha  and Ubiquitin-40S ribosomal protein S27a-1  were used as an endogenous control due to its stable transcript abundance in rice3, 2 ml H2O and 2 ml 30% H2O2 in a microwave for 12 min at 190 °C, then diluted with 20 ml 10% NaOH, neutralized with HNO362 and filled up to a final volume of 100 ml.For the determination of the Si concentrations in the shoot and root, 200 mg dried plant matter was digested in 3 ml 65% HNO3, 2 ml H2O and 0.5 ml 30% H2O2 in a microwave for 25 min at 190 °C and then diluted with distilled water to 25 ml.In order to determine the Fe, 50 mg of dried and ground shoot matter was digested in 2 ml 65% HNOThe Si and Fe concentrations in the plant extracts and nutrient solutions- were measured by ICP-MS (7500c Agilent Technologies).63. Freshly prepared nutrient solution without Fe and with Si and nutrient solution with FeEDDHA with and without Si were measured immediately or incubated for three and seven days in the same conditions as used for growth of rice in the dark. An aliquot of 1.6 ml was transferred in a cuvette and measurement was performed with ten runs each partitioned in 20 cycles, whereby the mean is represented in the figure.The hydrodynamic particle size of possible precipitates of the elements Si and Fe in the nutrient solution was determined by the Zeta potential analyzer with light scattering technique et al.61. A students t-test was used with unpaired correlated p-Value for the raw Affymetrix GeneChip data using “GeneSpring” Software . A ranking of candidates was carried out using the “Omics Explorer” .All treatments were replicated four times unless stated otherwise and the mean of the treatments were compared using t-test, Tukey or Bonferroni test utilizing Sigma Plot . The statistical qRT-PCR analysis was performed using the R-Macro of Steibel The Affymetrix experiment, including the RNA quality control, GeneChip hybridization, normalization with GeneSpring Software and ranking with the Omics Explorer, was carried out by Dr. Dittrich-Breiholz at the Research Core Unit “Transcriptomics,” Institute of Cell Biochemistry, Hannover Medical School.Supplementary information


The thioester moiety is introduced as a lithium binding unit for the hierarchical formation of titanium(IV) catecholate‐based lithium‐bridged helicates. In solution, the coordination compounds show a monomer–dimer equilibrium which —in comparison to the oxo esters— is significantly shifted towards the monomers. In addition, the influence of the thioester side chain on the dimerization behavior is investigated and an expansible/compressible molecular switch is synthesized. In the latter case expansion and compression are performed reversibly in methanol, whereas in DMSO spontaneous expansion occurs. Titanium springs: Titanium (IV) triscatechol thioester complexes form in the presence of lithium cations dinuclear helicates, showing a monomer–dimer equilibrium with dimerization tendencies lying between those of the corresponding ketones and oxoesters. Based on this principle, it is possible to obtain a switchable spring‐type helicate which shows some spontaneous expansion in [D6]DMSO. In comparison to the oxygen homologous, they are more reactive regarding the cleavage of the C(=O)−S bond. The C=O double bond is less polarized. The high reactivity of the thioester functionality is important in synthesis as well as in nature for In 2005, we described the first examples of hierarchically formed helicatesIn more recent studies, the lithium‐dependent dimerization of carbonyl substituted titanium(IV) triscatecholate complexes has been used as a molecular switch to control the selectivity of a Diels–Alder reactionIn the present study a series of thioester‐substituted catechols has been synthesized and has been used to prepare the corresponding titanium(IV) complexes.6]DMSO.In addition, an example for an expansible/compressible helicate based on an alkyl‐bridged bis(catechol thioester) is described and a special spontaneous expansion behavior is observed in .Preparation of the catechol thioesters follows a modified protocol as described for the oxoesters Scheme .10 Dimet3(1 p)6Ti2] were obtained, the molecular structure of the anion [Li3(1 p)6Ti2]− is shown in Figure X‐Ray quality crystals of the benzyl derivative Li[Li3 p6Ti2] warom=2.8–3.2 Å) contributing significantly to the stability of the dimer. This has been also observed for the oxygen homologues.The X‐ray structure analysis shows the dimeric central part of the hierarchically formed helicate with two titanium and three lithium cations  very similar to the one observed for the corresponding aldehyde, ketone, or oxoester derivatives.4]MeOH solution both, the dimer as well as the monomer can be observed by 1H NMR spectroscopy  complex are found due to the high lithium cation‐coordinating ability of the solvent. Removal of lithium cations from the dimer results in its full dissociation. Characteristic for the monomer is the observation of the CH2 group at sulfur as one single peak due to fast inversion of the stereochemistry at the complex unit. In the dimer, the stereochemistry at the complex is locked and diastereotopic protons are detected for SCH2.3(1 c)6Ti2]/[Li2(1 c)3Ti] in [D4]MeOH. The minor species is the monomer which does not show this diastereotopic behavior. At the room‐temperature equilibrium state of the monomer/dimer equilibrium in [D4]MeOH, the dimerization constant can be easily obtained from the NMR integration (see Table m is used as reference state in the determination of Kdim).In [Dy Figure . In [D6]Following remarkable aspects can be realized from the collection of dimerization constants of the thioester‐derived complexes:4]MeOH of the corresponding dimeric coordination complexes raises dramatically from methyl (25 600) to ethyl (only dimer observed) due to stronger electron‐donating properties of the ethyl compared to the methyl substituent.3(1 a)6Ti2], Kdim=6650), ethyl (Li[Li3(1 b)6Ti2], Kdim=7380) and n‐propyl thioester dimers (Li[Li3(1 c)6Ti2], Kdim=8170) are significantly lower and are only slightly increasing with the chain length. This shows a strong electronic isolation of the ester carbonyl and the alkyl substituent by the sulfur atom.1) In case of the methyl versus the ethyl oxoesters, the dimerization constant in MeOH, whereas the hexyl thioester results in a dramatic decrease. This drop has been also observed for the corresponding hexyl oxoester in [D6]DMSO,n‐hexyl chain.2) For the nNon and nDodec thioesters, low dimer stability is observed. A similar effect has been observed for the oxoesters and there has been attributed to a loss of entropy due to side‐chain aggregation.3) For iPr and iBu, as well as the cyclic cyPent and cyHex, thioester derivatives show relatively high dimerization constants which may be due to some dispersive interactions4) The branched 4]MeOH and thus Kdim is low.5) Aromatic units  show high solphophilicity in [DThe observed lithium‐dependent dimerization of the thioester‐derived triscatechol titanium(IV) complexes show some behavior as observed for the oxoesters3(2)3Ti2] has been prepared by tethering two thioesters by a decyl‐bridge. Coordination of three internal lithium cations results in a compressed structure. Corresponding complexes with oxoesters have been earlier prepared and show the ability to be switched from the compressed to the expanded state or vice versa by simply removing or adding lithium cations. With the oxoesters the switching proceeds smoothly in [D6]DMSO solution.In addition to the hierarchical helicates, a classical helicate Li[Li3(2)3Ti2] displays a highly dominating signal in the negative ESI MS at m/z=1539.294 for [Li3(2)3Ti2]− . The 1H NMR in [D4]MeOH reveals characteristic signals for aromatic protons at δ=7.25  and 6.56  and of the diastereotopic SCH2 protons at δ=2.59 , and 1.99 ppm . Addition of [2.1.1]cryptand changes the spectrum significantly which can be best observed following the aromatic protons 3Ti2]. This indicates the expansion of the spring type complex to form [(2)3Ti2]4−. Upon addition of an excess of lithium chloride, the spectrum of the compressed compound [Li3(2)3Ti2]− is restored again. This behavior is the same as has been observed for the oxoesters in DMSO.3(2)3Ti2] in [D6]DMSO, a typical spectrum of the compressed helicate Li[Li3(2)3Ti2] is observed with aromatic catechol signals found at δ=7.1, 6.5, and 6.4 ppm. With time, the signal intensity decreases at room temperature and a new set of signals grows in at δ=6.8, 6.2, and 6.1 ppm until full conversion is obtained. The new signals are similar to those observed for the expanded potassium complex K4[(2)3Ti2]. Following the kinetics of the expansion for a solution of 2×10−3 mol L−1, a rate constant of k=2.2±0.4×10−4 s−1 is obtained for the first‐order expansion reaction at room temperature  based helicates. Dimerization constants of the thioester derivatives are much lower than observed for corresponding oxoesters but higher than for corresponding ketones. Dimerization constants strongly depend on the solvent as well as on the kind of thioester substituent. For the thioesters, the substituents are electronically well isolated from the carbonyl unit and Regarding the oxoesters, a molecular switch was created by introducing an alkyl tether at the complex units which easily can be switched in methanol between an expanded and compressed state. However, DMSO as solvent is a strong competitor for lithium‐cation binding so that in this solvent the complex spontaneously releases lithium cations and expands. The kinetics of this process can be easily followed by NMR spectroscopy.Herein, a new class of compounds is added to the family of hierarchically formed dinuclear titanium(IV) catecholates. Those show a distinct different dimerization behavior compared to the earlier investigated oxoesters, ketones, and aldehydes, which is important for the development of functional switches and switchable catalysts with tunable switching properties.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Single‐crystal X‐ray diffraction data on the neutral bis‐triarylborane precursor 2 N confirm its rod‐like dumbbell structure, which is shown to be important for DNA/RNA targeting and also for BSA protein binding. Fluorimetric titrations with DNA/RNA/BSA revealed the very strong affinity of 4+2 and indicated the importance of the properties of the linker connecting the two triarylboranes. Using the butadiyne rather than a bithiophene linker resulted in an opposite emission effect (quenching vs. enhancement), and 4+2 bound to BSA 100 times stronger than 4+3. Moreover, 4+2 interacted strongly with ss‐RNA, and circular dichroism (CD) results suggest ss‐RNA chain‐wrapping around the rod‐like bis‐triarylborane dumbbell structure like a thread around a spindle, a very unusual mode of binding of ss‐RNA with small molecules. Furthermore, 4+2 yielded strong Raman/SERS signals, allowing DNA or protein detection at ca. 10 nm concentrations. The above observations, combined with low cytotoxicity, efficient human cell uptake and organelle‐selective accumulation make such compounds intriguing novel lead structures for bio‐oriented, dual fluorescence/Raman‐based applications.A bis‐triarylborane tetracation . The use of fluorescent labels to image specific small molecules in live cells is an essential tool in biology, medicinal chemistry, and many other related fields of research. However, common fluorescent dyes also have many drawbacks. Due to the considerable size and aromatic nature, and also often being H‐bond donors/acceptors, they may alter the biological activity, cellular localization, and dynamics of the targeted bio‐molecules.Therefore, further development in the increasingly complex bioimaging field essentially requires new fluorophore structures for better bio‐target diversification. Even more useful would be new optical probes based on intrinsically different methods at least approaching similar sensitivities of common fluorophores. Such an aim recently led to the development of vibrational spectroscopy methods, particularly Raman bioimaging microscopy, due to its compatibility with aqueous biological samples.Phenyl end‐capped polyynes have been employed as alkyne tags for Raman visualization of mobile, small molecules in cellsAlthough fluorescence, under certain experimental conditions, interferes with Raman measurements, by causing significant background signal,z‐orbital, the three‐coordinate boron in a triarylborane is a strong π‐acceptor. However, it is also Lewis‐acidic and sensitive to hydrolysis. Bulky substituents can stabilize three‐coordinate boron against decomposition by air and moisture, while maintaining its π‐acceptor strength.4+3 , and it is completely inert, in terms of covalent and non‐covalent interactions, to DNA/RNA/protein. Even more importantly, it exhibits an intense Raman band with a narrow linewidth in the Raman‐silent spectral region (2250–2000 cm−1) convenient for Raman‐based probing in aqueous solutions. Additionally, the intensity of response can be strongly increased by surface‐enhanced Raman scattering (SERS) spectroscopy and related techniques.The question arose to which extent the chromophoric linker (bithiophene) is responsible for the observed spectroscopic responses and affinity. In a search for linkers which could also be non‐conventional response probes, 1,3‐butadiyne attracted our attention for several reasons. It is a rigid, rod‐like symmetrical linker, excellent for precise orientation of terminal triarylborane cations  of the molecules when physisorbed on a surface, and from the transfer of electrons from the metal to the molecule and vice versa for chemisorbed molecules.A, which was previously only accessible via Sandmeyer type reactions in moderate yields,A, using CuBr2 as a brominating agent. Compound A was then cross‐coupled with trimethylsilylacetylene, at the sterically less hindered bromine, giving B. For the synthesis of triarylborane C, compound B was lithiated and bisfluoroboraneC was deprotected using KOH in MeOH/THF to give the neutral terminal alkyne compound 1 N. The neutral 1,3‐butadiyne compound 2 N was formed by oxidative homo‐coupling of 1 N in a Glaser type reaction, using I2 as an oxidant. The two neutral compounds 1 N and 2 N were methylated at the amine groups using methyltriflate in CH2Cl2. The two cationic species 2+1 and 4+2 were precipitated by addition of Et2O; 2+1 was washed with CH2Cl2/Et2O, and 4+2 with acetone/Et2O.The key precursor 1 N  and 2 N. The solid state molecular structure of 2 N, along with selected bond lengths and angles, are depicted in Figure Single crystals suitable for X‐ray diffraction analysis were obtained for compounds 2+1, 4+2) are moderately soluble in water (c=1×10−3 m), their solutions being stable for longer periods when stored in the dark. The neutral analogue of 1 N was dissolved in DMSO to prepare a stock solution (c=5×10−3 m) which was further diluted with aqueous buffer prior to every experiment, yielding stable solutions up to the 0.1 m range. The poor solubility of neutral analogue 2 N under biorelevant conditions hampered further experiments.Both positively charged compounds , and changes in their UV/Vis absorption spectra upon temperature increase up to 90 °C were minor . Reproducibility of the UV/Vis spectra upon cooling to 25 °C was excellent. These findings indicate that the compounds do not aggregate by intermolecular stacking under the experimental conditions used. Absorption maxima and corresponding molar extinction coefficients (ϵ) are given in Table The absorbances of the studied compounds Figure  were pro1 N and its charged analogue 2+1. The neutral molecule 1 N shows an absorption band at 408 nm while the charged analogue 2+1 shows a hypsochromically shifted absorption with a maximum at 315 nm and a shoulder at 335 nm. This absorption behavior is well documented in previous publications from our group for a series of analogous neutral and charged compounds−1) in absorption, when comparing the monomer 2+1 and the dimer 4+2, is also consistent with our previous studies4+2. All photophysical data is summarized in Table Comparison of their UV/Vis absorption spectra reveals distinct differences between the neutral compound 1 N showed pronounced emission quenching upon heating to 90 °C, whereas the temperature‐induced emission changes of 2+1 and 4+2 were negligible .Both the neutral and charged compounds are strongly fluorescent, with significant apparent Stoke's shifts in all tested solvents . Naturally occurring calf thymus (ct)‐DNA represents a typical B‐helix structure with a balanced ratio of GC‐(48 %) and AT‐(52 %) base pairs. Synthetic alternating polynucleotides poly (dGdC)2 and poly (dAdT)2 represent two extreme situations , differing significantly in their secondary structures as well as in the availability of the minor groove for small molecule binding . For comparison between double stranded (ds) DNA and ds‐RNA, poly rA ‐ poly rU RNA was chosen as an A‐helix structure characterized by a major groove available for the binding of bulky small molecules.To study the interactions of Furthermore, to explore the DNA/RNA binding of the novel chromophore to a greater extent, we also studied the single stranded synthetic ss‐RNA polynucleotides poly G, poly A, poly U and poly C, each of them characterized by different properties. Adenine ss‐RNA mimics 50 to 250 adenine nucleotides at the 3′ end of mRNA, poly G is related to guanine‐rich sequences in both DNA and RNA, whereas poly C and poly U are significantly more flexible than purine‐RNAs, and with less organized secondary structures.Due to the possibility that the compounds studied could interact with proteins, we examined the most naturally abundant protein, bovine serum albumin (BSA), taking into account its versatility of binding sites.Tm value). Non‐covalent binding of small molecules to ds‐polynucleotides usually increases the thermal stability of the ds‐helices thus resulting in increased Tm values, and the increase (ΔTm) can (corroborated by other methods) be related to the various binding modes.It is well known that double stranded (ds)‐helices of polynucleotides dissociate into two single stranded polynucleotides upon heating at well‐defined temperatures , whereas monomer 2+1 induced only minor stabilization  and the neutral analogue 1 N did not affect the thermal stability of ds‐DNA . The results show a direct proportionality of ds‐DNA stabilization to the number of positive charges. However, tetra‐cation 4+3 stabilized ds‐DNA much more strongly , pointing to the significant impact of the bithiophene linker.Tetra‐charged 2+1  or 4+2 . Intriguingly, addition of ss‐RNA or BSA caused negligible changes in the emission from monomer 2+1 , in contrast to the behavior of dimer 4+2, which revealed a strong fluorimetric response .The strong intrinsic fluorescence of the compounds studied allowed fluorimetric titrations with various ds‐DNA/RNA, single‐stranded (ss)‐RNA and BSA. Addition of ds‐DNA or ds‐RNA caused strong, non‐selective quenching of the emission of 4+3 showed an emission increase upon DNA/RNA/BSA complexation, characterized by distinct differences in fluorescence maxima between DNA/RNA and protein (BSA), whereas 4+2 showed only non‐selective emission quenching. Such a difference in fluorimetric response between 4+2 and 4+3 might be attributed to the fact that bithiophene is a weak donor, whereas the diyne unit does not have an electron‐donating effect.Furthermore, 4+2:BSA) stoichiometry model, pointing to only one dominant binding site of 4+2 at BSA.The non‐linear fitting of the DNA or RNA titration data by means of the Scatchard equation 4+2 to ds‐DNA and ds‐RNA were within the same order of magnitude, while its affinity to ss‐RNA was approximately an order of magnitude lower. The affinity of 4+2 to BSA was an order of magnitude higher than its affinity towards ds‐DNA/RNA  within their absorption spectra upon binding to polynucleotides, which could provide useful information about modes of interaction.Thus far, we had studied the non‐covalent interactions at 25 °C by monitoring the spectroscopic properties of the compounds upon addition of the polynucleotides. In order to obtain insight into the changes of polynucleotide properties induced by small molecule binding, we chose CD spectroscopy as a highly sensitive method for the examination of conformational changes in the secondary structure of polynucleotides.2+1 did not significantly change the CD spectra of ds‐DNA or ds‐RNA , and no induced (I)CD bands >300 nm were observed. In contrast, tetracation 4+2 induced a significant decrease in intensity in the CD spectra of all ds‐DNA/RNA 2]; whereby for r<0.2 ICD bands were negative and for r ≥0.3 ICD bands were positive. Such a change of the ICD sign is commonly attributed to single molecule binding at an excess of DNA (r<0.2) and molecular aggregation within the DNA grooves at an excess of the small molecule (r>0.3).Only poly (dAdT)2 Figure  revealedλmax=360–380 nm), giving negligible ICD band, were attributed to the electronic transition along the long axis of 4+2, parallel to the diyne‐linker (see Table S5).Further, dominant ICD bands at 300–330 nm could be partially attributed to electronic transition vectors along the boron‐nitrogen axes (see Table S5), which seem to be well oriented with respect to the DNA chiral axis. The maxima at 4+2/GC‐DNA complex could be attributed to the sterically crowded minor groove with the amino groups of guanine, not allowing deep insertion of 4+2 and, thus, diminishing the induced CD effect.4+2. However, the large depth of the major groove allows heterogeneous orientation of 4+2 molecules with respect to the ds‐RNA chiral axis, thus resulting in negligible ICD bands.The almost negligible intensity of ICD bands for the analogue 4+2/ss‐RNA complexes  and Na‐cacodylate buffer,  . Hence, the band around 2220 cm−1 was assigned to the stretching of the C≡C triple bonds in bisaryl‐substituted diynes,−1, overlapped by the water band, was attributed to a phenyl ring stretching mode (Table S4).4+2 , the band around 1355 cm−1 was associated with mixed stretching vibrations of the triple bonds and phenyl rings, whereas stretching of the bonds between the boron atom and three aromatic substituents contributed to the band around 1070 cm−1. The strong Raman scattering of 4+2 was attributed to the diyne moiety conjugated to the aromatic rings,Raman spectra of 4+2 were measured in the 5×10−8 ‐ 5×10−6 m concentration range . The bands at 1286 cm−1 and 1155 cm−1 which appeared at low 4+2 concentrations (1×10−7 m and 5×10−7 m) were attributed to the symmetric stretchings of ‐CF3 and ‐SO3 groups, respectively, of trifluoromethanesulfonate anions.4+2, the counterions came close to the enhancing silver surface and, consequently, their vibrational modes were enhanced.The SERS spectra of −1) and phenyl (1596 cm−1) stretching bands implied that, at a concentration of 1×10−6 m, the molecules adopted an optimal position with the triple bonds and phenyl rings oriented perpendicular to the silver surface  was assigned to Ag−N stretching, indicating interaction between the nitrogen atom and the silver surface  in Na‐cacodylate buffer (pH 7.0) was acquired  c(4+2)=1×10−6 m, which produced the strongest SERS response, and b) at the lowest concentration at which 4+2 was detected (5×10−8 m)  Figure  a,b. In 4+2/ct‐DNA complex than for 4+2 alone. For example, at c(4+2)=5×10−8 m, the intensity of the triple C≡C stretching band at 2214 cm−1 for the complexes of r[4+2]/[ct‐DNA]=1 and 0.2 was enhanced 1.4 and 1.3 times, respectively, relative to that of the free 4+2 molecules. The increase in intensity obtained indicated interactions of 4+2 with the nucleic acid, owing to which the small molecules adopted a more optimal orientation with respect to the enhancing silver surface and/or were placed closer to the silver nanoparticles  upon binding to BSA was studied for the 4+2/BSA complexes prepared in r[4+2]/[BSA] molar ratios of 1, 0.2 and 0.1  in the simply prepared and widely used silver colloid, even at a nanomolar concentration range. Thereby the distinctive band of the C≡C triple bond at 2215 cm−1, which does not interfere either with the bands of its own, or with the bands of the other species, in the measured system, allowed easy detection of the small molecules. The intense SERS response obtained for 4+2 interacting with DNA, and its loss upon binding with BSA, is characteristic of binding with the nucleic acid and the protein, respectively. Owing to the sensitivity obtained and minimal spectral interference, the molecules studied could potentially be used as alkyne‐coded SERS tags for live cell imaging.It is interesting to note that the characteristic SERS response of The biological experiments aimed to verify the actual capability of the DNA/RNA/protein binder studied to penetrate the cells, to visualize its intracellular location and subcellular targets. Evaluation of the anti‐proliferative effect was conducted in order to identify further potential applications, either as a cytotoxic lead compound toward theranostic4+2 on the proliferation of human cell lines, we used the MTT test. The compound was tested on two human cell lines (HeLa and HEK 293). As an indicator of anti‐proliferation activity we used the IC50 value, which corresponds to the concentration of the compound that inhibits proliferation to 50 % compared to the control cells (proliferating without the compound in the medium). The results we obtained showed negligible anti‐proliferative activity of 4+2 even at the highest concentration used in the test .To examine toxicity, i.e., the effect of 4+2 in fluorescent confocal microscopy experiments. Confocal microscopy also allows us to propose the possible intracellular localization of the compound. The compound 4+2 entered cells very efficiently within 2 hours of cell immersion in c(4+2)=1×10−6 m. The observed fluorescence of the compound coincided with the area of the cell where the endoplasmic reticulum is located, and the grainy dispersion of the fluorescence signal indicates possible endosomal accumulation ‐C≡C−C≡C‐ with its corresponding monomeric ‐C≡CH (4+1) analogue.As a follow‐up to our work on novel applications of bis‐triarylboranes as DNA/RNA/protein binders and bright intra‐cellular probes,4+2 retains structural features very similar to those of its neutral analogue 2 N  is essential for efficient binding to the hydrophobic pocket of BSA. Moreover, the linker structure (4+2 diyne vs. 4+3 bithiophene) strongly influenced the BSA affinity (4+2 logKs=8.2 vs. 4+3 logKs=5.9), suggesting that a linear, aliphatic low‐volume linker (4+2 diyne) is highly preferred for insertion into BSA binding site.One of the main aims of the 4+2, 2+1) showed remarkably high affinity toward various types of DNA/RNA (logKs=6–7.5 range) and, intriguingly, dicationic monomer 2+1 showed only an order of magnitude lower affinity than tetracationic 4+2. This implies only a minor contribution of electrostatic interactions with the negatively charged DNA/RNA backbone and suggested binding within ds‐DNA/RNA grooves, within which the highly hydrophobic linker and extensive interactions of aromatic units surrounding the boron atoms strongly contribute to overall DNA/RNA affinity.Both cationic compounds  and consequently the intracellular accumulation of a probe. The Raman‐response of 4+2‐analogues in SRS microscopy could, thus, efficiently complement the fluorescence response when the studied system is turbid/non‐suitable for fluorescence imaging (e.g. cell‐organoid agglomerates), some other fluorescence probe is used simultaneously, or one of the cellular targets quenches the fluorescence emission. Moreover, the unique binding mode of ss‐RNAs with very high affinity makes bis‐triarylborane tetracations very promising ss‐RNA delivery systems, particularly as some of them have already shown very efficient cellular uptake and negligible toxicity.Our results strongly support further development of bis‐triarylborane Raman/fluorescence chromophores as dual probes for simultaneous confocal fluorescence microscopy and SRS microscopy of cell lines, whereby careful choice of a linker can finely tune DNA‐protein selectivity  should be addressed to the authors.SupplementaryClick here for additional data file.


Drosophila melanogaster Dα1 subunit on the agonist activity of imidacloprid and thiacloprid for Dα1/chicken β2 nAChRs expressed in Xenopus laevis oocytes. The S221A mutation hardly affected either the affinity or efficacy for ACh and imidacloprid, whereas it only slightly reduced the efficacy for thiacloprid on the nAChRs with a higher composition ratio of β2 to Dα1 subunits. The S221Q mutation markedly reduced the efficacy of the neonicotinoids for the nAChRs with a higher composition of the β2 subunit lacking basic residues critical for binding neonicotinoids. Hence, we predict the possibility of enhanced neonicotinoid resistance in pest insect species by a mutation of the serine when it occurs in the R81T resistant populations lacking the basic residue in loop D of the β1 subunit.Neonicotinoids selectively modulate insect nicotinic acetylcholine receptors (insect nAChRs). Studies have shown that serine with ability to form a hydrogen bond in loop C of some insect nAChR α subunits and glutamate with a negative charge at the corresponding position in vertebrate nAChRs may contribute to enhancing and reducing the neonicotinoid actions, respectively. However, there is no clear evidence what loop C properties underpin the target site actions of neonicotinoids. Thus, we have investigated the effects of S221A and S221Q mutations in loop C of the  They represent high selective toxicity to insect over vertebrate nAChRs and show high selective toxicity to insects with diverse actions6. They show high systemicity in crop plants, enabling seed treatments and now make up >25% of global pesticide sales7. Their potential risks to non-target pests, including pollinators, has been demonstrated8. The use of some neonicotinoids in the field is now restricted in the EU10. It remains of interest to understand the mechanism of target-site actions of neonicotinoids in order to assist in the design of new, more eco-friendly pesticides.Neonicotinoids are insecticides that modulate competitively insect nAChRs13. Indeed, the R81T mutation in loop D of aphids was first predicted13 then shown14 to reduce the affinity of neonicotinoids, thus resulting in resistance. We found earlier that a mutation of serine at position 221 to glutamate in the YXCC motif in loop C of the fruitfly (D. melanogaster) Dα1 subunit also markedly reduced the agonist action of imidacloprid and thiacloprid on Dα1/chicken β2 hybrid nAChR expressed in Xenopus laevis oocytes, pointing to a contribution of the serine221 to the selective action of the neonicotinoids tested15. A plausible explanation of this result is that repulsion of the electrostatically negative nitro group of imidacloprid and cyano group of thiacloprid by electronegative glutamate residue led to the reduced agonist actions. However, this mutation also changes the size of the residue.Studies of target site actions show that basic residues in loop D of the nAChRs binding site play a key role in electronic interactions with the nitro or cyano group of neonicotinoidsIn this study, the Ser221 of the Dα1 subunit was mutated to alanine or glutamine and then imidacloprid and thiacloprid as representatives of neonicotinoids carrying a nitro and cyano group, respectively  and chicken β2 subunit (Accession number NM_204813) were cloned into the pcDNA3.1 (+) vector . The S221A mutation was introduced with forward primer 5′- TTCTACGCATGCTGCGAGGAGCCG-3′, reverse primer 5′- GCAGCATGCGTAGAACTTCTCGTT-3′, whereas the S221Q mutations was introduced with forward primer 5′- TGCTGCGAGGAGCCGTATCTGGACA-3′, reverse primer 5′- CTGGTAGAACTTCTCGTTCCGCACC-3′. The entire nucleotide sequence of the S221A mutant cDNA was confirmed by automated sequencing using a 3130xl genetic analyzer (Thermo Fisher Scientific).XhoI. cRNAs encoding wild type and mutant Dα1 subunits as well as chicken β2 subunit was prepared from the cDNA construct using a mMESSAGE mMACHINE T7 ULTRA Kit (Thermo Fischer Scientific). cRNA was dissolved in RNase free water at a concentration of 1 μg/μL. To express (Dα1)3(β2)2 and (Dα1)2(β2)3 nAChRs in Xenopus oocytes, cRNAs of Dα1 and β2 subunits were mixed at ratios of 5:1 and 1:5, respectively16.Each cDNA construct was linearized by digesting with X. laevis) were anesthetised and oocytes were obtained according to the U.K. Animals (Scientific Procedures) Act, 1986. After treatment with collagenase and the follicle cell layers were removed from oocytes18. Each oocyte was injected with 50 nL of the cRNA solution and incubated for 4 or 5 days at 16 °C in the standard oocyte saline  supplemented with penicillin (100 units/mL), streptomycin (100 μg/mL), gentamycin (20 μg/mL) and sodium pyruvate (2.5 mM) at 16 °C16. Each data was obtained using oocytes from at least two frogs.Female frogs  at a membrane potential of −100 mV. The membrane current data were digitized by a Digidata 1550B A/D converter (Molecular Devices) and analyzed by Clampfit 10 (Molecular Devices).Agonist activity of ACh and neonicotinoids were evaluated by voltage-clamp electrophysiology as previously described. Oocytes were secured in a chamber and perfused with a SOS containing 0.5 μM atropine at a flow rate of 5–10 mL/min16. When recording the peak amplitude of the responses to imidacloprid and thiacloprid at concentrations higher than 1 μM, one oocyte was used for recording one response and abandoned to prevent underestimation of agonist responses resulting from irreversible nAChR desensitization.The compound was bath-applied in SOS for 3–5 s at an interval of 3–5 min with increasing agonist concentrationsmax is the maximum normalized response, EC50 is the half maximal response, X is the log [agonist concentration (M)] and nH is the Hill coefficient. Experiments were repeated (n = 5). Imax and pEC50 (= −logEC50) values were obtained as mean ± standard error of the mean. Concentration-response curve illustration and statistical analyses were performed using Prism 6.Peak current amplitude of each response to all the agonists were normalized to that of the response to 100 μM ACh and fitted by non-linear regression using a Prism 6  according to the following equation:3(β2)2 nAChR complexed with thiacloprid were built using Modeller19 with the structure coordinate of acetylcholine binding protein from Lymnaea stagnalis complexed with thiacloprid (PDB id 3WTK)20. Amino acid sequences of Dα1 and β2 subunits were aligned with the sequence of AChBP, and the models in complex with thiacloprid were built using automodel algorism of Modeller by taking into considerations water molecules observed frequently in the AChBP-neonicotinoid complexes. Models are illustrated using PyMOL 2.3 .The homology models of wild type and S221A mutant of the orthosteric agonist binding domain of (Dα1)3(β2)2 and (Dα1)2(β2)3 nAChRs concentration-dependently with pEC50 values of 6.81 ± 0.05 and 6.79 ± 0.04 3(β2)2 and (Dα1)2(β2)3 nAChRs 3(β2)2 and (Dα1)2(β2)3 nAChRs hRs Fig. . Thus, ttio Fig. , Table 2max value of thiacloprid for the nAChRs with increased β2 subunit composition ratio , but P < 0.05 by one-tailed t-test). The result suggests that thiacloprid does not rely mainly on the hydrogen bond with Ser221 when it binds to the Dα1/Dα1 subunit interface containing basic residues in loop E and loop G16, but does when it binds to the Dα1/β2 subunit interface lacking basic residues necessary for the interactions with neonicotinoids20.Thiacloprid evoked lower amplitude response than imidacloprid in oocytes expressing the nAChRs tested. The S221A mutation only slightly reduced the I15, as explained for the reduced agonist actions of imidacloprid on the Dα2/β2 nAChR by the P242E mutation in loop C21. To test this hypothesis, we investigated a mutation of Ser221 to glutamine having a similar size to glutamate but lacking a negative charge on the agonist activity of imidacloprid and thiacloprid on the hybrid nAChRs. The mutation was ineffective in changing the affinity but reduced the efficacy of imidacloprid in the (Dα1)3(β2)2 nAChR ). Such effect on the agonist actions not only of imidacloprid, but also of thiacloprid, became more evident by increasing the β2 subunit composition ratio ). Hence, the reduced neonicotinoid affinity by the S221E mutation is mainly due to electrostatic repulsion by the glutamate residue, and that the reduced neonicotinoid efficacy in the (Dα1)2(β2)3 nAChR by Gln221 is due to its interference with neonicotinoid access to the Dα1/β2 interface by either steric interactions, or omission of the hydrogen bond or both.We previously hypothesized that the reduced affinity of the neonicotinoids by the S221E mutation in the Dα1/β2 nAChRs stems from electrostatic repulsion of the neonicotinoids containing a negative charge by the glutamate residue containing a negative charge3(β2)2 nAChRs in complex with thiacloprid rid Fig. , since trid Fig. . Thiazolrid Fig. . The hydrid Fig. . Thiaclorid Fig. , explainDrosophila Dα1 subunit on the agonist actions of ACh, imidacloprid and thiacloprid on the (Dα1)3(β2)2 and (Dα1)2(β2)3 nAChRs. Both mutations hardly lowered the affinity of the neonicotinoids. Hence, hydrogen bond capability of the serine residue has a minor contribution to the interactions with neonicotinoids in the insect/vertebrate hybrid nAChRs. However, the effects of these mutations on the efficacy of the neonicotinoids were evident in the (Dα1)2(β2)3 nAChR, pointing to a role for the serine in determining the neonicotinoid actions at the Dα1/β2 interface lacking the basic residues involved in the interactions with neonicotinoids. Although studies are needed to confirm this using nAChRs that are composed completely of insect nAChR subunits, the present results provided a new insight in the mechanism of selective actions of neonicotinoids and predicted resistance which may arise from the mutations tested in pest populations, notably with the R81T mutation.In conclusion, we have investigated the effects of S221A and S221Q mutations in loop C of the


Furthermore, two D–A aggregates exhibit high photostability as evidenced by 4% and 8% of the fluorescence decreasing after 6 h of continuous irradiation in air, which is in sharp contrast to ≈95% of fluorescence decreasing in a reference compound. Importantly, with these molecules, ultrasensitive detection of sulfur mustard (SM) with a record limit of 10 ppb and selective detection of SM in complex matrices are achieved.Real‐time, high signal intensity, and prolonged detection is challenging because of the rarity of fluorophores with both high photostability and luminescence efficiency. In this work, new donor–acceptor (D–A) molecules for overcoming these limitations are reported. A hybridized local and an intramolecular charge‐transfer excited state is demonstrated to afford high photoluminescence efficiency of these D–A molecules in solution (≈100%). The twisted molecular structure and bulky alkyl chains effectively suppress  1 porous films to ultrasensitively and selectively detect 10 ppb sulfur mustard vapor.Two new donor–acceptor molecules that exhibit high photostability and photoluminescence efficiency (close to unity) both in solution and in the solid state are developed. Their application in fluorescence sensor is well illustrated by utilizing  In particular, under high (de)excitation light intensity  and long‐term light exposure , fluorophores with high photostability and luminescence efficiency enable the improvement of imaging resolution and a prolonged observation window. In contrast, fluorophores with low photostability and weak luminescence cause the loss of imaging resolution and signal intensity as well as a reduced working life. While substantial progress has been made in several organic systems to improve fluorophore brightness and photostability, it remains a great challenge to develop fluorophores that exhibit high photostability and emission efficiency in an aggregated state.For the application of organic fluorophores, high photostability and strong luminescence are significantly important parameters.3, 4 First, introduction of the appropriate A moiety can effectively lower the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the resulting D–A molecules, which can enhance their photostability. Second, the twisting geometry of D–A molecules allows for a certain extent of orbital mixing, producing a hybridized local excited‐state (LE) and intramolecular charge‐transfer (CT) state  excited state). The resulting HLCT state can provide a high‐efficiency luminescent pathway for D–A molecules. Furthermore, optimal D and/or A moieties offer molecular recognition of a specific analyte, which, along with high photostability and luminescence efficiency, makes D–A molecules suitable for constructing fluorescence sensors. Despite the promising characteristics described above, few D–A molecules haven been reported to show high photostability and luminescence efficiency of >70% both in solution and in the solid state. This is mainly because π‐π and dipole‐dipole interactions of D–A molecules can greatly reduce their luminescence in the solid state, even though they are highly luminescent in solution. Therefore, rational design of D–A molecules with high photostability and luminescence efficiency both in solution and in the solid state is highly desirable.As a widely explored class of fluorophores, organic donor–acceptor (D–A) molecules have the potential for the construction of fluorophores with high photostability and luminescence efficiency.22 Fir1 and 2  of solid films of molecules 1 and 2. On the other hand, the benzothiadiazole groups (A moieties) effectively lower the HOMO and LUMO of molecules 1 and 2, thus offering greatly enhanced photostability. The high luminescence efficiency and photostability make these molecules suitable for fluorescence sensors. In particular, porous 1 films that have optimized sulfur‐π and dipole‐dipole interactions with sulfur mustard (SM) enable the ultrasensitive detection of 10 ppb SM. Furthermore, porous 1 films exhibit no responses to most potential interferents, which thereby allow for the selective detection of SM in complex environments. These results provide important insights into the synthesis and application of organic fluorophores with high photostability, as well as, high luminescent efficiency.In the present work, we design new D–A molecules 2 Figure 1 that e1–2  and A (benzothiadiazole) moieties but are different in their linking sequence, while molecule 3 has only the same D moiety. Here, the designed molecules 1 and 2 that have bulky side chains and different electronic moieties were expected to exhibit a twisted molecular structure. As shown in Figure S1, Supporting Information, density‐functional theory (DFT) calculations confirm that the twisted angles between the D or A moieties in molecules 1 and 2 fall within the range of 30°–35°. Similar to other D–A molecules, the twisted molecular structure of 1 and 2 may allow for the formation of an intercrossed LE and CT state that can provide a pathway for high emission efficiency. To support this hypothesis, we performed optical measurements of 1 and 2 in solvents with different polarities. As shown in Figure 1 centered at 411–420 nm, compared to that of 3 centered at ≈366 nm, can be ascribed to the CT state of 1. The CT characteristic is further confirmed by the obvious redshift of the fluorescence spectra with the increasing solvent polarity. For example, the fluorescence peak of 1 redshift from 522 nm in cyclohexane to 567 nm in dimethylformamide. Similarly, molecule 2 exhibits a CT character of the excited state, as evidenced by the emergent absorption peak centered at 436–441 nm and the obvious redshift of the fluorescence spectra with increasing solvent polarity  of ≈100% in the different solvents , which is an uncommon phenomenon for the pure CT excited state. We measured the extinction coefficient (ε) of 1 and 2 in chloroform solution to be 82 640 and 77 000 L mol−1 cm−1 , which thereby gave rise to the brightness (ε × Φ) of 82 640 and 77 000 for 1 and 2 in chloroform, respectively.The detailed synthesis and characterization of 2 Figure  are provd method.24 As y Figure . The obv1) properties, we used the Lippert‐Mataga equation to fit the Stokes’ shift and the orientation polarizability . As shown in Figure S3, Supporting Information, the linear relation for 1 and 2 is different in low‐polarity and high‐polarity solvents. The corresponding excited‐state dipole moment (µe) for 1 and 2 is calculated to be 17.6 D and 17.2 D in low‐polarity solvents and 24.4 D and 23.7 D in high‐polarity solvents, respectively. The small µe of 17.6 D and 17.2 D can be attributed to the usual LE‐like state of 1 and 2, while the large µe of 24.4 D and 23.7 D can be attributed to the CT‐like state of 1 and 2. The nonlinear relationship between Stokes’ shift and solvent polarity  indicates that 1 and 2 possess an intercrossed excited state of LE and CT, that is, HLCT, in moderate polarity solvents. Other experimental evidence for the formation of the HLCT state was observed from fluorescence lifetime measurements of 1 and 2 in a moderately polar solvent. The fluorescence of 1 and 2 in chloroform follows single‐exponential decay , giving lifetimes of 6.41 and 4.54 ns, respectively; these results indicate that 1 and 2 have an HLCT state rather than two separate states. The results are also consistent with the frontier orbital data calculated by Gaussian 09 package. As shown in Figure 1 and 2 is distributed on the whole molecular backbone, whereas the LUMO is mainly located on the benzothiadiazole unit with some extent of distribution on the fluorene unit. The insufficient separation of the HOMO and LUMO levels likely leads to the intercrossed characteristics of the LE and CT states. Importantly, the formed HLCT state could provide access to a radiative process and thus gives rise to the high emission efficiency of 1 and 2 .To further explore the low‐lying excited state (S equation34 to 1 and 2. Aggregates from 1 or 2 were fabricated by simply injecting 0.3 mL of 1 or 2 in a chloroform solution (6 mg mL−1) into a vial containing 3 mL of methanol followed by 1 day of aging. Scanning electron microscopy (SEM) revealed that molecules 1 and 2 formed irregular colloidal nanoparticles , further indicating that the twisted structure and bulky alkyl chains resulted in the amorphous structure of colloidal NPs from 1 and 2.We next explored the solid‐state emission of s Figure 2a,b. Whes Figure ,d. The f The photostability of colloidal NPs from 1 and 2 was thus evaluated by monitoring the fluorescence intensity as a function of the continuous irradiation time. As shown in Figure 1 decreases by only 4% after 6 h of continuous irradiation in air , and the fluorescence intensity of colloidal NPs from 2 decreases by ≈8% under identical irradiation conditions. In sharp contrast, the fluorescence intensity of aggregates from 3 decreases by ≈95% under identical irradiation conditions . Notably, even under irradiation in air by a 365 nm LED lamp (100 mW cm−2), the fluorescence intensity of colloidal NPs from 1 and 2 decreases by only ≈8% and ≈10%, respectively, after 6 h of continuous irradiation . These results indicate the high photostability of colloidal NPs from 1 and 2. Such high photostability is also reflected by the time‐dependent fluorescence spectra of these colloidal NPs, where the fluorescence spectra only slightly decreases under continuous UV irradiation , the introduction of benzothiadiazole moieties in molecules 1 and 2 largely lowers the LUMO energy level by approximately 1 eV. The lowered LUMO energy level can greatly reduce the possibility of losing an electron from the LUMO to oxygen; this thereby can greatly enhance the stability of 1 and 2 in the excited state. In addition, inefficient intersystem crossing from the singlet state to the triplet state in 1 and 2, consistent with their high FQYs described above, can also play an important role in enhancing photostability.For practical applications, photostability is another important parameter.35 The2 Figure ,e. Compa1 and 2, which can also form porous colloidal NPs once deposited on substrates, enable them to be suitable fluorescence sensors for vapor analytes. Here, we chose a powerful blistering agent, SM, as the target analyte to illustrate the sensing application of these D–A molecules. SM belongs to a typical class of chemical warfare agents. Furthermore, there is no treatment or antidote for injuries caused by SM gas. To date, examples of fluorescent detection of trace SM vapor remain very limited. Therefore, ultrasensitive detection of SM vapor by a reliable fluorescence sensor is critical. Quantitative evaluation of the detection sensitivity was performed at the Chemical Defense National Laboratory, where SM vapors at different concentrations were available. The sensing experiments of colloidal NPs from 1 and 2 were performed using a home‐built optical chamber coupled with an Ocean Optics USB4000 fluorometer.The high photostability and luminescence efficiency of e agents.38 FurFigure 1 and 2 to trace SM vapors leads to obvious irreversible fluorescence quenching responses. Such fluorescence quenching was also reflected clearly in the fluorescence spectral changes . The noncovalent interactions mainly originate from sulfur−π interactions and dipole‐dipole interactions between the D–A molecule and SM. Therefore, the stronger interactions of 1 with SM than of 2 with SM enhanced the detection sensitivity of colloidal NPs from 1.As shown in s Figure ,d. Notab1 exhibited no responses to a nerve agent simulant, diethyl cyanophosphonate, and most common organic solvents . However, the quenching is reversible, which is distinct from the irreversible responses quenching by SM vapor and thereby does not interfere with SM detection . Upon exposure to the mixture of trace SM and hair spray, irreversible fluorescence quenching responses of colloidal NPs from 1 were clearly observed  into a vial containing 3 mL methanol and aged for 1 day at room temperature. Aggregates from 3 were self‐assembled by injecting 0.3 mL of the dichloromethane solution of 3 (5 mg mL−1) into a vial containing 3 mL methanol and aged for 1 day at room temperature. The resulting assemblies that were suspended in solution could be cast onto the substrate and into a quartz tube.Colloidal NPs from −2). The fluorescence quantum yields of the colloidal NPs from 1 and 2 drop‐cast on a PTFE film and the solution (2.5 µM) of 1 and 2 were determined by using a Hamamatsu Absolute PL Quantum Yield spectrometer C11347 coupled with an integrating sphere. Time‐dependent fluorescence profiles of colloidal NPs from 1 and 2 that were drop‐cast one a glass slide as a function of irradiation time were obtained using an Ocean Optics USB4000 fluorometer with a 385 nm LED lamp (0.053 mW cm−2) and 365 nm LED lamp (100 mW cm−2) as the light source.SEM images were obtained on a Hitachi SU8010 field‐emission microscope. SEM samples were prepared by drop‐casting the suspending aggregates in solution onto a silica substrate followed by sputtering Pt on the surface (The sputtered Pt layer was ≈5.4 nm). The fluorescence‐mode optical microscopic images were obtained on an inverted fluorescence microscope (Olympus × 71). UV–vis absorption spectra were obtained on a PerkinElmer Lambda 35. Fluorescence spectra and time‐dependent fluorescence profiles were obtained using an Ocean Optics USB4000 fluorometer with a 385 nm LED lamp as the light source (0.053 mW cm A jar (40 mL) containing 1 mL of an analyte was sealed for overnight to obtain the saturated vapors. The diluted vapor concentrations of different sulfides and other VOCs were obtained by injecting a small volume of the saturated vapor into a sealed vial (40 mL). Colloidal NPs from 1, 2, and aggregates from 3 inside the quartz tube were prepared by casting 10 µL methanol suspension of aggregates into a quartz tube, which were kept to be about 3.3 cm far from the tube air‐inlet. Solvent in the quartz tube was removed with a capillary and then the resulting quartz tube with colloidal NPs inside was dried for 5 min by a blower. Real‐time fluorescence responses were monitored by blowing 5 mL of the analyte vapor at certain concentration into the quartz that would be pumped into the chamber containing the predeposited colloidal NPs by an air pump (150 mL min−1). The fluorescence sensing of real SM vapors were performed at the Chemical Defense National Laboratory, where SM vapors at different concentrations were available. The time‐dependent fluorescence intensity and spectra were recorded with an Ocean Optics USB4000 fluorometer using a 385 nm LED lamp as the light source.The fluorescence sensing of different sulfides and other VOCs was performed using a previously reported home‐built detector.45 A jTheoretical calculations were performed using the B3LYP/6‐31g  level of theory in Gaussian 09 package.49 that reveals interactions between solvent and solute.f is the orientational polarizability of a solvent; µe is the excited‐state dipole moment; µg is the ground‐state dipole moment; ɑ is the solvent cavity (Onsager) radius. The ɑ value of both 1 and 2 was calculated to be 0.85 nm by using Equation 35]35[3µg values of 1 and 2 were calculated to be 3.88 D and 4.82 D, respectively, by using Gaussian 09 package. ∆f could be calculated by using Equation ( as shown below:The The authors declare no conflict of interest.Supporting InformationClick here for additional data file.