This functional contrast agent is intended for the specific and non-invasive detection of prostate cancer cells that are PSMA positive, a marker implicated in prostate tumor progression and metastasis. The two-step carbodiimide reaction used to conjugate the antibody to the nanoparticle was efficient and we obtained an elemental iron content of 1958±611 per antibody. Immunofluorescence microscopy and flow cytometry showed that the conjugated muJ591:MIRB complex specifically binds to PSMA-positive (LNCaP) cells. The muJ591:MIRB complex reduced cell adhesion and cell proliferation on LNCaP cells and caused apoptosis as tested by Annexin V assay, suggesting anti-tumorigenic characteristics. Measurements of the T2 relaxation time of the muJ591:MIRB complex using a 400 MHz Innova NMR and a multi-echo spin-echo sequence on a 3T MRI  showed a significant T2 relaxation time reduction for the muJ591:MIRB complex, with a reduced T2 relaxation time as a function of the iron concentration. PSMA-positive cells treated with muJ591:MIRB showed a significantly shorter T2 relaxation time as obtained using a 3T MRI scanner. The reduction in T2 relaxation time for muJ591:MIRB, combined with its specificity against PSMA+LNCaP cells, suggest its potential as a biologically-specific MR contrast agent.In this study we developed, characterized and validated  In the last 10 years, the incidence of prostate cancer has steadily risen, remaining the second most common cancer diagnosed in men worldwide. In Canada alone, it accounts for roughly 27% of newly diagnosed cancers in 2012 in vivo prostate cancer specific targeting strategies Studies have suggested that, by specifically targeting prostate specific membrane antigen (PSMA) expressing cells, both localized and aggressive conditions can be treated PSMA receptors are localized primarily to the apical plasma membrane of prostate epithelium and play an integral role in the progression of prostate malignancies, probably by promoting anti-apoptotic signaling, ensuring cell resilience and promoting cell proliferation Endorectal magnetic resonance imaging (MRI) is becoming common part of the local work-up for prostate cancer. Dynamic studies using gadolinium-DTPA contrast agents have demonstrated useful enhancement of tumor images In this study, we developed a functional super-paramagnetic iron oxide (SPIO)-based MR contrast agent that specifically attaches to PSMA receptors for MR imaging of prostate cancer cells. We present a method to develop this functional MR contrast agent using a commercially available SPIO, Molday ION Rhodamine-B Carboxyl (MIRB), conjugated to the anti-PSMA receptor antibody J591. We characterized and validated this antibody:SPIO-nanoparticle complex for specific detection of PSMA positive prostate cancer tumor cells using immunofluorescence, flow cytometry and 3T clinical MRI. Our new contrast agent can be used for the specific and non-invasive detection of prostate cancer cells with PSMA, a marker implicated in prostate tumor progression and metastasis Murine J591 mAb, at a concentration of 5 mg/mL and with known specificity for PSMA, was procured from Dr. Neil H Bander's laboratory  through an institutional material transfer agreement. The isotype control antibody for synthesis and cell culture experiments was a murine IgG1  at a concentration of 5 mg/mL in 1× phosphate buffered saline without calcium or magnesium ions , as per manufacturer's recommendation.N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-(4-Morpholino)ethanesulphonic acid hydrate (MES), sodium bicarbonate, HPLC-grade water (ChromoSolv Water), chloroform, acetonitrile, and deuterium oxide, were purchased from Sigma Aldrich, Oakville, ON, Canada, unless otherwise stated.Molday ION Rhodamine Carboxyl  was used to prepare the antibody:SPIO complexes. All the reagents required for the conjugation reactions and analysis, including Antibodies were conjugated to MIRB based on a modification of the manufacturer's method . Briefly, MIRB (0.2 mg of iron) was added to 100 µL of muJ591 or muIgG at 1 mg/mL in 1× PBS buffer (pH 7.65) in sterile 1.5 mL microfuge tubes. The antibody with MIRB solution was lightly vortexed prior to the addition of 100 µL of 0.1 M sodium bicarbonate buffer (pH 8.0), ice incubated for 15 min with occasional vortexing, then kept 24 h at 4°C in a rotator mixer. The overnight incubated antibody:MIRB were mixed with 50 µL of 2 mg/mL EDC solution in deionized water plus 100 µL of MES buffer (pH 5.6) and kept at 37°C for 5 min with occasional vortexing, prior to addition of 50 µL of 2 mg/mL NHS and incubation at 37°C for further 15 min, with occasional mixing. The solution was then cooled on ice for 15 min and kept at 4°C for 48 h in a rotator mixer. After 48 h the mixture was transferred to a sterile 15 mL conical tube and rinsed twice with 500 µL 1× PBS buffer to extract out the antibody:MIRB conjugates attached to the tube wall. In order to remove any unbound antibodies, the reaction mixture was transferred to a fresh sterile 15 mL conical tube containing 50 µL of 2 mg/mL NHS plus 150 µL of 0.25 M MES buffer and vortexed. This tube was kept overnight at 4°C in a rotator mixer, washed with an equal volume of 0.1 M Sodium bicarbonate buffer (pH 8.0) and centrifuged using 10 K Amicon Ultra Centrifugal Filters , based on the manufacturer's protocol for ultrafiltration. The solution retained in the filtration column, containing the remaining antibody:MIRB conjugates, was washed with 0.01 M sodium bicarbonate (pH 7.65) using the buffer exchange steps described in the manufacturer's protocol. Each of the eluants was analyzed for both antibody and iron contents using the Bradford protein assay kit and inductive coupled plasma atomic emission spectroscopy (ICP-AES) based assay, respectively. The total protein concentration of the conjugates was determined by Bradford protein assay using gradient concentration of BSA and human γ-globulin as standards for indirect estimation of antibody content.ICP-AES was used to determine the elemental iron contents of the antibody:MIRB conjugates. Antibody:MIRB conjugates were acid digested using an equal volume of 70% nitric acid in deionized LC-MS grade water, in a final sample volume of 6 mL; a buffer only (negative control) was similarly digested. All the samples were run on the Varian Vista Pro CCD simultaneous ICP-OES instrument .The complex muJ591:MIRB was immobilized on a Nucleopore membrane  and heated at 57°C in a hot air oven for 25 min to remove moisture. The immoblized complex was then mounted and sputter-coated with carbon. SEM images were acquired at 5000 and 10,000× magnification using a Hitachi Su-70 Schotty Field Emission SEM  with a resolution of 1.5 nm at 1 kV. The same system was used to perform Energy Dispersive X-ray (EDX) spectroscopy at 5000× magnification to confirm the presence of organic matter and SPIO nanoparticles.Particle size of muJ591:MIRB conjugate was determined by dynamic light scattering. The muJ591:MIRB conjugate was dispersed in 0.9% saline at a protein concentration of 0.01% w/v. Measurements were carried out using a Zetasizer Nano ZS instrument . The particle size results were reported as the harmonic intensity averaged particle diameter (or Z-average) and they are expressed in nanometers.To determine whether the muJ591:MIRB conjugate generated from the labelling reactions retained the super-paramagnetic characteristics required of an MR contrast agent, we measured the T2 relaxation time in NMR. Samples with equal total protein concentration (0.04 µg/µL) of muIgG:MIRB and muJ591:MIRB complexes were separately prepared in 600 µL of deuterium oxide and transferred into Wilmad NMR tubes . T2 relaxation times were obtained after optimizing the magnetic field of the Innova Unity 500 NMR machine and samples were run as per the manufacturer's recommendation.max) using a Synergy4 fluorimetric plate reader . The peak λmax and λmax shift were determined by plotting the fluorescence intensity values at different excitation and emission wavelengths.To test whether the conjugation reaction caused fluorescence enhancement or quenching of the Rhodamine-B fluorophore on the MIRB conjugates we performed a fluorimetric analysis. MIRB nanoparticles and muJ591:MIRB complexes samples with an equal iron concentration were analyzed for fluorescence intensity and wavelength at peak intensity  and 1% antibiotics/antimycotics. The cells were grown to 80% confluence in 75 cm2 vented cap, canted neck falcon flasks  with media replenished every three days.PSMA-positive LNCaP cells (CRL-1740) and PSMA-negative DU145 cells (HTB-81) were procured from American Type Cell Culture, Manassas, VA, USA. All cells were grown in a 37°C, 5% COCells were seeded at an initial concentration of 1000 (for DU145) and 2000 cells (for LNCaP) per well in 96 well plates and grown for 3 days before performing the experiments. Immunofluorescence studies were then conducted on both LNCaP and DU145 after treatment with 0.1 µg/µL muJ591:MIRB for 1 h with or without addition of secondary antibody conjugated to AlexaFluor-488 fluorophore. Then, cells were incubated with warm 0.5% BSA in RPMI media to encourage cells to internalize the antibody. The cells were then visualized at different time points  under a standard inverted fluorescence microscope  attached to a closed circuit camera .A flow cytometric assay was performed based on a modification of the method described by Peldschus K et al (2010) 2 for 40 minutes. Cells were finally washed with serum-free RPMI-1640 until cells in the uncoated wells stopped detaching. All cells were then fixed using ice-cold methanol and stained with 0.1% crystal violet. Filtered 2% SDS was added and the plates were incubated with agitation at room temperature for 30 minutes, after which time, absorbance readings at 550 nm were taken using the PowerWave XS plate reader . We compared the absorbance readings from the muJ591- and muJ591:MIRB-coated plates with respect to the untreated control wells to analyze adhesion.Cell adhesion after treatment of plates with muJ591 and muJ591:MIRB was evaluated. The muJ591:MIRB complex and muJ591 mAb was prepared in 1× phosphate buffered saline (PBS) at a concentration of 0.01 µg/mL. We then precoated 96-well plates with 100 µL of muJ591:MIRB complex and muJ591 mAb in quadruplicates, and incubated overnight at 4 °C for immobilization of the complex on the plate surface. After overnight incubation, we aspirated out the solution and blocked the wells with 1% BSA in 1× PBS for at least 10 minutes and aspirated out again. Non-treated wells were used as control. About 100,000 LNCaP or DU145 cells were then seeded in the precoated and control wells and incubated at 37°C, 5% CO2 for 2 hours followed by 20 min incubation with agitation in the dark at room temperature. The fluorescence intensities were then measured at 2, 6, 12 and 24 h post muJ591:MIRB treatment using the FLx800 plate reader at excitation of 540 nm and emission of 590 nm.For cell proliferation assessment, a resazurin-based fluorimetric assay  was performed. After the 96 well plates were precoated with muJ591:MIRB or 1× phosphate buffered saline (control) and blocked with 1% BSA as described above, 1000 DU145 or 2000 LNCaP cells were seeded. The cells were grown until control wells reached 80% confluence. The LNCaP and DU145 cells were further treated with muJ591:MIRB complex (0.01 µg/mL) or serum free media (control) for 1 h, after which media was replaced with serum free media. The Resazurin solution was added, as recommended by the manufacturer's protocol, and plates were further incubated at 37°C, 5% COFor determining cell apoptosis and death caused by the muJ591:MIRB complexes on LNCaP cells, an Annexin V-FITC assay was performed . Cells were dislodged from the flasks using 2 mM EDTA in 1× phosphate buffer saline, pelleted and resuspended in sterile, supplemented media, and then seeded in 6 well culture plates at a concentration of 10,000 cells per well. Once cells reached 80–90% confluence, muJ591:MIRB or control  treatment was added to the wells for 1 h followed by 3 washes in phosphate-buffered saline (PBS). Flow cytometric analysis was then performed based on method described by Richard et al (2010) To validate we could detect changes on the T2 relaxation time with a clinical MR scanner we performed MR imaging of the conjugates first and then conjugate-treated cells. The muJ591:MIRB and muIgG:MIRB complexes, as well as muJ591, muIgG and MIRB alone were suspended on PBS at different concentrations . Then, 100 µL of serum free media were added to 96-well plates (n = 3) and placed on the MR scanner.in vitro cell MR imaging, both LNCaP and DU145 cells were plated, in triplicate, at concentrations of 105 cells/well in 96 well plates and grown for 36–48 h. The cells were then treated with 100 µL of antibody:MIRB conjugates or non-conjugated antibodies for 1 h, rinsed 2× times with serum free media, 1× time with 1× Tris-EDTA buffered saline, and 1× times with 0.01 M Sodium bicarbonate buffer to remove unbound conjugates, and then resuspended in 100 µL serum free media and placed on the MR scanner. Different concentrations of muJ591:MIRB, muJ591, muIgG:MIRB and muIgG  were used.For 5, 7.55, 55, 2.55 and 1.255 cells/well and grown for 48 h. The cells were then treated with 100 µL of muJ591:MIRB for 1 h, rinsed 2× times with serum free media, 1× time with 1× Tris-EDTA buffered saline, and 1× times with 0.01 M Sodium bicarbonate buffer to remove unbound conjugates, resuspended in 100 µL serum free media and placed on the MR scanner.To determine the limit of detection LNCaP cells were plated in 96 well plates, in triplicate, at concentrations of 10A clinical 3T MRI scanner  with a small surface Flex coil was used. Images were acquired with a multi-echo spin echo sequence with 64 echo times spaced by 5.8 ms . The intensity as a function of echo time was fitted to:Z is the intensity at echo time TE; A, B and T2 are the fitting parameters Where MAll statistical analyses were performed using Graphpad Prism software . All data were tested for normality and homogeneity of variances using a Shapiro-Wilks test and a Bartlett's test, respectively, before choosing a suitable parametric or non-parametric statistical test. A resulting p-value of less than 0.05 was considered to be significant. Parametric data sets were analysed using Student's t-tests or one-way ANOVAs, followed by an appropriate post-hoc such as a Tukey HSD. Non-parametric data sets were analysed using one-way Kruskal Wallis ANOVA followed by a Nemenyi's post hoc, and Friedman's tests followed by a pair-wise comparison using a Wilcoxon signed rank test with a Bonferroni adjustment.ICP-AES was used to estimate the amount of elemental iron present in each of the conjugated muIgG:MIRB and muJ591:MIRB complexes. We observed that muJ591:MIRB complexes had 1958±611 (n = 8) elemental iron per antibody while muIgG:MIRB complexes had 2906±631 (n = 8) elemental iron per antibody. The iron nanoparticles presence on the muJ591:MIRB complex was further confirmed by Energy Dispersive X-ray spectroscopy . The preThe presence of the muJ591:MIRB complex immobilized on salt crystals from the buffer can be observed in the superposed EDX-SEM images . EDX mapThe average particle sizes for the muJ591:MIRB conjugates were 34.75±14.41 nm with a polydispersity index (PdI) value of 0.23±0.01. For comparison, we performed particle size measurements for the MIRB nanoparticles alone and obtained 34.64±10.83 nm with a PdI of 0.25±0.02 which are within the values reported by the manufacturer (35 nm).Immunofluorescence microscopy was used to determine whether PSMA positive LNCaP cells were specifically detected by the muJ591:MIRB complex. max of MIRB alone and muJ591:MIRB complex  treated LNCaP cells. The complex antibody:MIRB demonstrated faster T2 relaxation time with increasing iron concentration . For nonLive PSMA-positive (LNCaP) prostate cancer cells showed a reduced T2 relaxation time when treated with the muJ591:MIRB complex , and T2 5 or lower seeded cell counts, showing there is a limit on the detection provided by the MRI that depends on the cell population.For live PSMA-positive (LNCaP) prostate cancer cells treated with muJ591:MIRB the T2 relaxation time became faster for higher seeded cell counts . No chanLNCaP and DU145 prostate cancer cells also showed a reduced T2 relaxation time when treated with the MIRB nanoparticle alone, with a faster T2 for higher iron concentrations. LNCaP cells treated with MIRB alone at an iron concentration of 0.125 µg/µL showed a T2 of 385±33 ms, whereas for muJ591:MIRB-treatment at 0.1 µg/µL, the T2 was 272±18 ms, showing that at a similar iron concentration, we can still differentiate between MIRB uptake and muJ591:MIRB binding by the T2 relaxation time values. DU145 cells treated with MIRB alone at an iron concentration of 0.125 µg/µL had a T2 of 161±18 ms, and this value was consistently faster than DU145 cells treated with muJ591:MIRB. This showed that both cell lines were taking up MIRB when it was not conjugated, and once MIRB was conjugated to antibody, only the PSMA-positive LNCaP cells were detected as changing their T2 relaxation time values. This confirmed that muJ591:MIRB complex detection of PSMA-positive LNCaP cells using a clinical 3T MRI was feasible and sensitive for iron concentrations as low as 0.02 µg/µL (0.035 µg/µL protein concentration).in vitro, flow cytometry, and immunofluorescence based methods.We were able to stably conjugate the prostate cancer-detecting muJ591 antibody and MIRB to generate antibody:SPIO complexes with an iron to antibody concentration that is sufficient to serve as a functional MR contrast agent detectable by clinical MRI Several studies have shown how antibodies conjugated to super-paramagnetic iron oxide particles were used for specific MR imaging of human lymphocytes, the Her-2/neu receptor in breast cancer cells, and apoptotic cells When 1 µg/µL of muIgG antibody or muJ591 antibody were conjugated to MIRB using our two-step carboiimide reaction, we recovered in average 0.45±0.03 µg/µL of muIgG:MIRB and 0.56±0.05 µg/µL of muJ591 antibody:MIRB complex. This protein recovery after the conjugation was estimated by Bradford protein assays performed before and after the reaction.The MIRB cluster-conjugated muJ591 antibody had similar values of elemental iron per antibody to the muIgG antibody. It is therefore likely that we have similar nanoparticle per antibody ratio and the muIgG is therefore an acceptable control. However, when the T2 relaxation time was obtained, the muIgG:MIRB had a slower T2 which was very closer to the value obtained for the MIRB nanoparticle alone. The conjugation with the muJ591 antibody seems to have an effect on the relaxation time in a favorable direction to its detection since the T2 relaxation time becomes faster.Note that, due to limitations in sample preparation and the resolution of the SEM instrument which we used, we were not able to accurately estimate the particle size of the conjugates on the SEM images. The drying step performed during the sample preparation step to immobilize the muJ591:MIRB conjugates on to the Nucleopore filters resulted in formation of crystalline buffer salts which are prominently seen in the SEM images as large micrometer-size structures. However, by performing the EDX elemental mapping we were able to confirm the presence of the muJ591:MIRB conjugates immobilized within these structures. We therefore used dynamic light scattering to obtain particle sizes of the conjugates.When equal protein concentrations of antibody:MIRB conjugates were subjected to NMR analysis, we found that the T2 relaxation time of the muJ591:MIRB was similar to the one for the muIgG:MIRB complex. This could be attributed to the similar amount of elemental iron contents and molecular size of the conjugates. The T2 relaxation time measurements with the MRI showed differences between the muIgG:MIRB and the muJ591:MIRB complexes. The difference between NMR and MRI results can be explained by the use of a standard multi echo spin echo sequence on the MRI and calculations based on processed MR images intensity changes. Nevertheless, our findings showed that the antibody:MIRB complex presents a significantly faster T2 relaxation time that makes it a good candidate as MR contrast agent.max values as well as the fluorescence intensity of Rhodamine-B fluorophore in the MIRB nanoparticles. The observed Rhodamine-B quenching was exploited to indirectly determine the success of antibody conjugation to MIRB nanoparticles. Fluorescence intensity measurements were performed routinely as quality control assessment measure following each synthesis experiments and to determine the overall loading concentration. Our results agree with previous reports that indirectly suggest that the solvent polarity and proximity of quenching species to the metal-fluorophore conjugation system may play a role in the fluorescence quenching The success of our two-step carboiimide conjugation could be confirmed by determining the changes in peak λin vitro experiments, the muIgG:MIRB conjugates were easily cleared following the washes during the immunostaining steps while the muJ591:MIRB conjugates were found to remain adherent to the PSMA expressing LNCaP cells. Thus by using muIgG antibody as a control, we ensured the overall efficacy and functionality of the complexes.Our findings suggest that the binding of the conjugate muJ591:MIRB to the PSMA-positive cells is highly specific. The control muIgG:MIRB conjugate could not be detected in the MRI images or T2 relaxation time maps of the cells. We have used a muIgG antibody as an experimental control antibody for both synthesis and validation studies. Several studies used IgG antibody to demonstrate the specificity of their functional MR contrast agents in vivo studies. We first determined the best buffer in which the antibody:MIRB conjugates would maintain stable surface charge and remain in solution without flocculation or aggregation during storage. We performed a zeta potential analysis (data not shown) and observed that the 0.01 M sodium bicarbonate buffer could maintain uniform surface charge of antibody:MIRB complex. Preliminary in vivo tests were performed  and confirmed 0.01 M sodium bicarbonate as the best buffer for in vivo use.We additionally determined potential stability issues while formulating the conjugates in different buffers in order to maximize the functionality and specificity of the antibody:MIRB conjugates for future in vitro. Flow cytometry-based detection showed as well that the muJ591:MIRB was binding to LNCaP cells.Androgen-dependent LNCaP, which specifically express PSMA receptors when compared to other prostate cancer cell-lines Flow cytometric and immunofluorescence approaches were both successful in discerning the binding capacity and also the relative uptake capacity of muJ591:MIRB conjugates in both DU145 and LNCaP cells. Flow cytometric-based detection was more accurate in determining the extent of binding, which is especially useful when picogram level of contrast agents are used. A future line of experiments will be to determine the antibody:MIRB uptake kinetics using confocal microscopy.We could detect the presence of muJ591:MIRB on live PSMA positive cells using a clinical 3T MR scanner. The change in T2 relaxation time was significant and standard multi-echo, spin-echo sequences allowed us to quantify it. It should therefore be possible to obtain images using standard T2-weighted imaging at echo times selected by the T2 relaxation time value of the complex, as well as T2-mapping techniques. There is however a limit on the detection capabilities of the technique related to the amount of cells and therefore SPIO signal.Because prostate cancer cell lines take up non-conjugated MIRB and this also modifies the T2 relaxation time, special caution should be taken when comparing non-conjugated SPIO-based contrast agents to the antibody:SPIO agents. However, the fact that non-conjugated MIRB is taken up by multiple cells and the antibody:MIRB only by specific cell types should give the necessary specificity to detect PSMA-positive cells only.in vitro detection using the MR scanner we suspended cells on serum-free media during the imaging. Other more tissue-like experimental settings such as agar or agarose were preliminary investigated but they were found to strongly modify the T2 relaxation time and reduce the sensitivity of the detection.For live cell Our findings showed that the muJ591:MIRB treated LNCaP cells showed significantly reduced cell adhesion and increased Annexin V expression, along with significant reduction in cell proliferation. Tumor cells that are less adherent may be more likely to cause metastasis as there is enhanced migratory and invasive potential. At the same time, some studies also show that certain tumor cells that become less adherent may undergo anoikis, a form of apoptosis induced by anchorage-dependent cells detaching from extracellular matrix (ECM) in vivo images should be done at different time points. A longer time course study would be necessary for long term in vivo effects targeting PSMA-expressing cells.Knowing that muJ591:MIRB likely has pro-apoptotic effects on prostate cancer cells, care should be taken with the imaging timing to strike a balance between detection of treated cells and their death due to the muJ591. The Recent years have given rise to the field of theranostics, whereby non-invasive detection and treatment are possible due to advances in nanoparticle synthesis and advanced imaging methods. Theranostic nanomedicines, mostly nanoparticles carrying therapeutic or diagnostic markers in form of targeted small interference RNA or antibodies, have been designed to improve current cancer therapies thus addressing specific limitations of current treatment and diagnostics approaches in vivo prostate cancer MRI studies.In conclusion, we demonstrated the generation and characterization of muJ591:MIRB nanoparticle conjugates, which can specifically detect PSMA expressing LNCaP cells using 3T Clinical MRI. Our findings suggest that muJ591:MIRB could be a promising diagnostic agent that shows therapeutic effects —a possible prostate cancer theranostic agent. This study also paves the way for our next goal, to appropriately validate the MR contrast agent for


In metastatic breast cancers, the acquisition of metastatic ability, which leads to clinically incurable disease and poor survival, has been associated with acquisition of epithelial-mesenchymal transition (EMT) program and self-renewing trait (CSCs) via activation of PI3K/AKT and IL6/JAK2/STAT3 signaling pathways. We found that TrkB is a key regulator of PI3K/AKT and JAK/STAT signal pathway-mediated tumor metastasis and EMT program. Here, we demonstrated that TrkB activates AKT by directly binding to c-Src, leading to increased proliferation. Also, TrkB increases Twist-1 and Twist-2 expression through activation of JAK2/STAT3 by inducing c-Src-JAK2 complex formation. Furthermore, TrkB in the absence of c-Src binds directly to JAK2 and inhibits SOCS3-mediated JAK2 degradation, resulting in increased total JAK2 and STAT3 levels, which subsequently leads to JAK2/STAT3 activation and Twist-1 upregulation. Additionally, activation of the JAK2/STAT3 pathway via induction of IL-6 secretion by TrkB enables induction of activation of the EMT program via induction of STAT3 nuclear translocation. These observations suggest that TrkB is a promising target for future intervention strategies to prevent tumor metastasis, EMT program and self-renewing trait in breast cancer. There are several discrete steps in the biological cascade of metastasis, loss of cellular adhesion, increased motility and invasiveness, entry and survival into circulation, exit into new tissue, and eventual colonization of a distant site , 2. Durihigh/CD24low antigenic phenotype, which results in their having stem cell-like characteristics (CSCs). There is accumulating evidences of a role of PI3K/AKT and JAK/STAT3 signaling pathways in tumor metastasis and EMT. Activation of the PI3K/AKT pathway, IL-6/JAK2/STAT3 pathway, and stem cell-like characteristics contribute to the poor outcomes of metastatic breast cancers [Metastatic breast cancers are more chemoresistant forms of breast cancer with expression of high levels of EMT markers and exhibit a CD44 cancers , 9. More cancers , while i cancers , 12.Independent of these findings, TrkB, a member of the tropomyosin-related kinase (Trk) family of neurotrophin receptors, is critical to biological processes in the developing and mature nervous systems, including neuronal growth, differentiation, and survival . Also, TIt is reported that ectopic TrkB expression in RIE-1 cells suppresses anoikis through AKT activation . HoweverWe initially examined whether the elevated levels of TrkB associated with the increase in phosphorylation of TrkB. As shown in Figure These observations led us to speculate that TrkB might modulate expression or activation of c-Src. To determine whether TrkB contributes to regulation of c-Src in MAD-MB-231 cells, we selected highly metastatic MDA-MB-231 and Hs578T cells stably expressing the TrkB-shRNAs. TrkB-shRNA partially suppressed the expression of endogenous TrkB  in MDA-MB-231 and Hs578T cells . We nextTo further investigate the induction of c-Src phosphorylation by TrkB, we examined whether TrkB directly interacts with c-Src in MDA-MB-231 cells. Interestingly, endogenous TrkB was able to interact with endogenous c-Src under physiological conditions Figure . To confTherefore, we examined whether c-Src activation is involved in the TrkB-induced Mek1 and AKT activation as well as cyclin D1 expression. To test this notion, we selected MDA-MB-231 cells stably expressing the c-Src-shRNAs. As shown in Activation of STAT3 is required for c-Src-mediated proliferation, survival and transformation –31, and Given-mentioned role of TrkB as c-Src activator via direct interaction, it is possible that the TrkB/c-Src complex could activate JAK2 via interaction. Indeed, we found that both TrkB and c-Src interacted with JAK2 after transient transfection . MoreoveInterestingly, MDA-MB-231 TrkB-shRNA cells demonstrated much lower Twist-1 and Twist-2 mRNA levels than MDA-MB-231 cells treated with SU6656 Figure . AdditioWe next attempted to investigate whether TrkB knockdown, which was identified above as being activated by the JAK2/STAT3/Twist-1 axis without c-Src, affects activation of the JAK2/STAT3/Twist-1 axis in Hs578T and MDA-MB-231 cells. Immunoblotting analysis revealed that JAK2 and STAT3 phosphorylation levels were markedly reduced in Hs578T and MDA-MB-231 TrkB-shRNA cells relative to its control-shRNA cells. Interestingly, the JAK2, STAT3, and Twist expression levels were also significantly reduced in Hs578T and MDA-MB-231 TrkB-shRNA cells Figure . AdditioBecause TrkB knockdown reduced the JAK2 protein levels, we initially focused on JAK2 expression. TrkB knockdown significantly reduced the JAK2 protein levels, but had no effect on JAK2 mRNA levels Figure , suggest+CD24+ and CD44+CD24− breast cancer cells have high phospho-STAT3 via their expression of genes such as IL6, PTGIS, and HAS1, which activate an autocrine loop [STAT3 phosphorylation by IL-6 promotes its nuclear translocation and DNA binding followed by the transactivation of genes involved in a number of cellular functions, including proliferation, differentiation, and survival . Moreover, the IL-6/STAT3/JAK2 pathway is involved in the maintenance of stem cell–like cancer cells  because ine loop . Furtherine loop . We specTo further examine the TrkB-mediated regulation of the JAK2/STAT3 pathway, we assessed IL-6-induced JAK2 and STAT3 activation in MBA-MB-231 and Hs578T control-shRNA or TrkB-shRNA cells. Relative to the MDA-MB-231 and Hs578T control-shRNA cells, TrkB-shRNA cells showed significantly reduced activation and expression of JAK2/STAT3, as well as Twist-1 expression after stimulation with or without IL-6 Figure . FurtherThe results of a recent study suggests that Twist-1 and Twist-2 induce activation of the EMT program to maintain the resulting mesenchymal state –40. MoreDuring the process of tumor metastasis, which is often enabled by EMT program, pleiotropically acting transcription factors, such as E12, Goosecoid, SIP1, Snail, Twist-1, and Twist-2, are induced that orchestrate EMT programs , 41. TheAlso, relative to the parental MDA-MB-231 control-shRNA cells, TrkB-shRNA cells significantly downregulated the expression of mesenchymal markers such as N-cadherin and fibronectin, whereas they upregulated the expression of epithelial markers (such as E-cadherin and α-catenin). Moreover, the levels of Twist-1 was significantly downregulated in MDA-MB-231 TrkB-shRNA cells relative to MDA-MB-231 control-shRNA cells . FurtherTo determine the contribution of TrkB to primary tumor formation, we injected MDA-MB-231 cells expressing either TrkB-shRNA or control-shRNA into the mouse mammary fat pads of BALB/c Nu/Nu mice and examined the resulting primary tumors 30 days later. Control MDA-MB-231 cells formed primary mammary tumors at identical rates, whereas primary tumor formation by MDA-MB-231 TrkB-shRNA cells was markedly decreased Figure . These rTo determine if the loss of TrkB expression affected the ability of MDA-MB-231 cells to metastasize, MDA-MB-231 cells expressing either TrkB-shRNA or the control shRNA were injected into the tail veins of BALB/c Nu/Nu mice, and their lungs were examined for metastases 35 days after injection. Suppression of TrkB expression strongly reduced the number of metastatic nodules relative to MDA-MB-231 control-shRNA cells Figure . Additiohigh/CD24low cells, which show a stem cell-like phenotype, are enriched in highly metastatic breast cancer cells  have been proposed to be resistant to cancer therapies through activation of the PI3K/AKT pathway, IL-6/JAK2/STAT3 pathway, and EMT [TrkB is overexpressed in several human cancers, ranging from neuroblastomas to pancreatic ductal adenocarcinomas, and its overexpression suppresses anoikis as an EMT inducer by regulation of Zeb1 , 42. Ind and EMT , 43–47. In the present study, we found that activation of the MEK and PI3K/AKT pathway through TrkB-mediated c-Src activation after TrkB-c-Src complex formation induced tumorigenicity and metastatic potential of breast cancer. These our results are supported by those of a previous study in which the phosphatidylinositol-3-OH kinase/protein kinase B signaling pathways required for EMT, anokis suppression, c-Src activated PI3K/AKT and Ras/MAPK cascades , 48, 49.in vitro and was required for cell transformation of a number of oncogenes and activation of STAT3 by interleukin-6 or expression of activated c-Src induced Twist expression at the protein and mRNA levels [We further dissected the role of TrkB in tumorigenicity and metastasis. Activated STAT3 was able to transform cells A levels , 50–52. A levels , 53–57. Recent studies showed that the IL-6 inflammatory feedback loop leads to CSC self-renewal and induction of EMT, both of which are implicated in tumor metastasis and poor outcomes by therapeutic resistance , 37, 58.Recent evidence indicates transcription factors Twist-1 and Twist-2, which are master regulators of embryonic morphogenesis, play an essential role in metastasis, CSCs and EMT of breast cancer , 60–66. Human breast cancer , SYF, 293T, and MDCK cell lines were maintained as previously described , 69, 70.Each of the two shRNA-encoding oligonucleotides against mouse and human TrkB was designed and verified to be specific to TrkB through BLAST searches against the mouse and human genomes, respectively. The primers corresponding to TrkB were cloned into the pLKO lentiviral vector to generate the TrkB-shRNA expression plasmid . shRNA tAssays were performed as previously described, with modification , 69. TheAll assays were performed as previously described , 71. TheThe primer sequences are listed in the supplemental experimental procedures . Total RE-cadherin, Twist-1, Twist-2, or STAT3 reporter gene constructs and 0.5 μg of pCMV-β-gal were cotransfected per well. Cell extracts were prepared 48 hrs after transfection, and the luciferase activity was quantified using an Enhanced Luciferase Assay Kit (Promega). All experiments were performed in triplicate.Cells that were 50% confluent in 12-well dishes were transfected using Lipofectamine 2000 (Invitrogen). A total of 0.5 μg 5 cells suspended in 50 μl PBS/Matrigel (BD Biosciences) were injected subcutaneously into the left and right hind flank regions under anesthesia. Mice were euthanized at 5 weeks and primary tumors were excised for analysis. For tail-vein injection, 1 × 105 cells suspended in 50 μl PBS were injected into the tail vein of 7-week-old BALB/c Nu/Nu mice.BALB/c Nu/Nu mice were purchased from the Korea Research Institute of Bioscience and Biotechnology  and handled in compliance with the IACUC. For tumorigenicity studies, 1 × 10Mice were given intraperitoneal injections of 150 ng/g D-Luciferin  and anesthetized with 2.5% isoflurane. At 7–8 min after injection, animals were imaged using the Xenogen Spectrum (IVIS-200) imaging system.The equal numbers of MDA-MB-231 control-shRNA or TrkB-shRNA cells were plated and cultured for 3 days. Subsequently, conditioned media from these cell cultures were collected and analyzed by the Human IL-6 Quantikine ELIZA kit (R&D systems) according to the manufacturer's instructions.t test (two-tailed).Data are expressed as the means ± SEM. Statistical analyses of these data were conducted via a Student's


We investigate optical field enhancement for a wide mid-infrared range, originating from the excitation of graphene plasmons, by introducing a graded dielectric grating of varying period underneath a graphene monolayer. Excitation of the plasmonic mode can be achieved by illuminating a normal-incidence plane wave on the gratings due to guided-mode resonance. The gratings of varying period enable the excitation of the plasmonic mode with a very high field enhancement factor (to the order of magnitude of 1000) within a wide spectral band, which leads to the frequency-dependent spatially separated localization of the infrared spectrum modes. We also demonstrate that the excitation position of the plasmonic mode can be freely tuned by varying the thickness of the interlayer as well as the chemical potential of the graphene monolayer. This structure enables the design of two-dimensional plasmonic photonic circuits and metamaterials targeted towards numerous potential applications including optoelectronic detectors, light-harvest devices, on-chip optical interconnects, biosensors, and light-matter interactions. The efficient concentration of optical energy into nanoscale objects and structures and the consequent enhancement of the optical field by optical antennas224It would be hugely advantageous to extend the concept of energy concentration to the infrared and THz regime, which would enable significantly increased applications including probingRecent concerns regarding plasmonics have been partly shifted to graphene plasmons (GPs)1415142210d1, is assumed to be 500 nm. In this case, the grating layer can be treated as infinitely thick since the silicon substrate below the grating does not affect the field distribution of the GPs in the grating/silica/graphene/air system. As the grating period is much smaller than the wavelength of the incident wave, the grating can be approximately modeled as an effective medium with the equivalent permittivity24εsilica = 3.9 and εsilicon = 11.9 are the respective permittivities of silica and silicon at the mid-infrared rangef = w2/Λ is the filling ratio of the silica.We first consider the dispersive relationship of the plasmonic mode supported by a graphene monolayer on a uniform diffractive grating substrate with a silica layer as the interlayer 1/2  are the permittivities and wavevectors of the grating layer (i = 1), silica (i = 2), graphene (i = g) and air (i = 0), respectively, while β is the propagation constant of the GPs along the x axis. The dispersion relation of the GPs by equation μc = 0.65 eV is represented by solid lines in j is the diffraction order and θ is the incident angle. To excite the plasmonic waves with wavelength λ and the fundamental order j = 1 under the condition of normal incidence θ = 0, the following expression should be satisfiedFor the four-layer grating/silica/graphene/air system, the dispersion relation of the GPs can be derived by combining the Maxwell equations and continuous boundary conditions26d2, with μc = 0.65 eV and w1 = 62 nm. It can be clearly seen from d2 = 10 nm, which can be attributed to the excitation of the fundamental (j = 1) and third-order (j = 3) plasmonic modes on the graphene surface. The missing second-order (j = 2) mode is here caused by the vanishing dipole moment. The scaling of the resonant wavelength, associated with the grating period, is consistent with equation d2 increases due to the fact that the overlap between the GPs and the grating reduces as d2 increases and hence reduces the excitation efficiency. The scaling of the fundamental-mode resonant wavelength with respect to the thickness of the silica layer, d2, by finite element method (FEM) simulation agrees with the theoretical results of equation d2. This is because the effective index of the plasmonic mode in the grating/silica/graphene/air system reduces with the increased thickness of the silica layer, d2. From 2) and light source (|E0|2). The enhancement level presented here is quantitatively comparable to that reported in plasmonic nanocavity gratings made of nanogrooves in a gold filmw1, along the x direction, while keeping the width of the silica layer, w2, constant. Wide-band plasmonic field enhancement can be anticipated under the illumination of a normal-incidence plane wave. The simulation result shown in 102/|E0|2) is independent of the power enhancement on the graphene surface, which is proportional to the power of incident light. The average electric field enhancement for the first 3 um in the left region is approximately 1000, but this does not mean the power enhancement is larger than 1 as the power of the incident light source is unchanged. The GPs excited by the silicon grating propagate along the graphene surface and hence suffer an absorption loss from the graphene monolayer. This is why the electric field intensity reaches its maximum value at the center position for each excitation wavelength . The calculated results show that the propagation loss by a UDGS is almost equal for the three wavelengths. For example, the values of Im(β) with λ = 7, 8 and 9 μm at w1 = 20 nm are 1.81 × 105, 1.62 × 105 and 1.46 × 105 m−1, respectively. Additionally, the left-going plasmon for a longer wavelength undergoes a much larger propagation distance between the excitation position and the 0 ~ 2 μm region. As a result, the field enhancement for λ = 7 μm is higher than that for a longer wavelength in the first 0 ~ 2 μm region.The UDGS can only excite the GPs on a graphene monolayer within a very narrow bandwidth around the resonant frequency. Bearing in mind that the resonant frequency is dependent on the grating period , we furtd2. According to equation λ = 8 μm in our case), a much wider w1 is required to excite the GPs as d2 increases due to the gradual reduction of the value of n ) is the Fermi-Dirac distribution, ε is the energy, μc is the chemical potential, T is the temperature, e is the electron charge, kB is the Boltzmann constant, ω is the radian frequency, and τ is the momentum relaxation time representing the loss mechanism due to the carrier intraband scattering.The optical properties of graphene are typically described by its surface conductivity. The Kubo formula has been widely employed to retrieve its surface conductivity in a series of theoretical and experimental studies on graphene313233μc| ≫ kBT, the surface conductivity of graphene could be approximated as32In the mid-infrared range with |T = 300 K and τ = 1 ps. Considering that in high-quality suspended graphene a DC mobility of μ > 100000 cm2V−1s−1 has been experimentally achievedτ > 1.5 ps, our setting of τ = 1 ps can be said to conservatively reflect the practical transport loss of graphene. However, as a graphene monolayer with a carrier mobility of more than 100000 cm2V−1s−1 is rarely seen in graphene samples in practice, we have also studied the electric field enhancement utilizing a much more realistic scattering time of graphene [see In our study, hene see .η0 (≈377 Ω) is the impedance of air, k0 is the light wavevector in free space, and dg is the thickness of the graphene monolayer (assumed to be 0.5 nm in our work).By treating the graphene monolayer as an ultra-thin metal film, graphene’s equivalent permittivity can be written as14To calculate the dispersion relation of the GPs, two-dimensional FEM simulation based on the mode solver of COMSOL is employed. In the simulation, 5-layer meshes are employed to denote the graphene layer while non-uniform meshes with a maximum element size of 500 nm are adopted to represent the other regions besides graphene.How to cite this article: Zhang, T. et al. Tunable broadband plasmonic field enhancement on a graphene surface using a normal-incidence plane wave at mid-infrared frequencies. Sci. Rep.5, 11195; doi: 10.1038/srep11195 (2015).


AoB PLANTS was conceived. I hope that the range of papers included in the special issue will reveal some of the complexities in soil community-mediated plant invasion.Soil microbial communities are one of the multiple factors that facilitate or resist plant invasion. Regional and biogeographic studies help to determine how soil communities and the processes mediated by soil microbes are linked to other mechanisms of invasion. Both the success of plant invasions and their impacts are profoundly influenced by a wide range of soil communities and the soil processes mediated by them. With an aim to better understand the mechanisms responsible for the soil community-driven routes, a special issue of  Soil microbial communities play an important role in explaining why some exotic species become aggressive invaders and form mono-dominant communities in non-native ranges but coexist with their neighbours in species-diverse native communities. Research on invasion ecology during last two decades has made a significant contribution in understanding how soil communities can either resist or facilitate invasions a. van deIn the boom phase, the positive benefits of introduction phase increase to facilitate invasive species by evolving increased competitive ability , invasivAlliaria petiolata (M. Bieb.) Cavara & Grande) suppress mycorrhizae and disrupt mutualistic associations between mycorrhizae and native tree seedlings  by disrupting mutualistic root-fungal symbioses in native plant species. Similar results were obtained in a long-term field experiment: where garlic mustard was present, vital rates of native perennials were suppressed relative to weeded plots, suggesting a general link between the chemically driven disruption of the mutualism and plant physiological declines, carbon stress and demographic declines.One of the hypotheses that links novel chemicals and suppression of root-fungal symbioses is disruption of mutualistic hypothesis  and the University of Delhi supported this work.The None declared.


The effect of hygrothermal conditions (air temperature and relative humidity) on the dehydration of theophylline monohydrate was investigated. Firstly, the equilibrium states of theophylline were investigated. The data from gravimetric analysis at constant temperature and humidity were reported as desorption isotherms. The PXRD analysis was used to identify the different polymorphic forms of theophylline: the monohydrate, the metastable anhydrate, and the stable anhydrate. Solid-solid phase diagrams for two processing times were proposed. Secondly, the dehydration kinetics were studied. The water content evolutions with time were recorded at several temperatures from 20°C to 80°C and several relative humidities from 4% to 50%. Different mathematical models were used to fit the experimental data. The spatially averaged solution of 2D Fickian transient diffusion equation best represented the water mass loss versus time experimental relationship. The dehydration rate constant was found to increase exponentially with air temperature and to decrease exponentially with air relative humidity. Its hydration behaviour and solid-state transitions have been extensively studied and presently the compound is known to exist as a monohydrate (M) and four anhydrous polymorphs  [Theophylline (CThe M form has been shown to lose water in low relative humidity (or water activity for solvents) to produce form II which is the most prevalent form and has been long considered as the only stable form at room temperature , 5. FormThe dehydration kinetics of theophylline monohydrate has been the subject of several investigations. Airaksinen et al.  investign of 0.33, respectively. Lower temperatures resulted in higher concentration of form III in the dehydrated product.Nunes et al.  investign = 0.25 and that at 40°C the dehydration of theophylline was a two-step process. Each step could be linearized using the Avrami-Erofeev's relationship with n = 0.25. The same conclusion was given by Agbada and York [Suzuki et al.  reportedand York .Matsuo and Matsuoka  have fouIt follows from the above short literature review, that the dehydration rates of theophylline were studied mainly with temperature as the controlling parameter and the influence of relative humidity has not been much investigated. The aim of this work was to experimentally study the influence of both parameters, varying in a broad range, on the solid phase transition pathways and kinetics during dehydration of theophylline. From equilibrium data, a phase diagram was determined and from kinetic data a simple predictive model for dehydration progress with time was derived. The model proposed here was compared to the common ones from the literature concerning the dehydration of theophylline and was proven to interpret better the experimental curves. Unlike the others, this model included explicitly the relative humidity and was based on water diffusion as rate controlling transition mechanism.2. 6H2O, Mg(NO3)2, K2CO3, NaBr, NaCl and KCl salts used for hygrothermal conditioning of hydrated theophylline samples were also supplied by Sigma-Aldrich.Anhydrous theophylline was purchased from Sigma-Aldrich . Hydrated theophylline was recrystallised from a saturated solution of anhydrous theophylline. Anhydrous theophylline was first dissolved in hot (60°C) distilled water. The solution was then filtered and allowed to cool overnight. Hydrated theophylline crystals were harvested and dried at room temperature. CsF, LiCl, MgClA differential scanning calorimeter  was used. The instrument was calibrated with pure indium. About 4-5 mg theophylline sample was packed in aluminum pans, crimped with lids having several pinholes. Tests were run from room temperature to 300°C, at a heating rate of 5°C/min under dry nitrogen purge.The thermogravimetric analyser (TGA Netzsch TG 209 F1 Iris ASC) was used. The sample was heated in an open aluminum pan from room temperature to 200°C, under nitrogen purge, at a rate of 1°C/min.α radiation (λ = 1.54 Å) using Göbel mirror bent gradient multilayer optics. The scattered intensities were measured with a scintillation counter. The angular range was 5–50° with a scan speed of 5° 2θ min−1.X-Ray powder (XRPD) theta-theta diffractometer  was used. The XRPD experiments were performed in symmetrical reflection mode with CuK2·6H2O, Mg(NO3)2, K2CO3, NaBr, NaCl, and KCl were prepared and stored at 10, 20, 30, 40, 50, and 80°C providing the different required relative humidity conditions. The relative humidity values were verified with an Activity-meter  and are detailed in Saturated solutions of CsF, LiCl, MgClHitachi S-800 Field Emission Gun (FEG) Scanning Electron Microscope (SEM) was used. All samples were coated with gold for increased signal/noise ratio.In order to interpret and fit the experimental dehydration curves, several equations were proposed in the literature. Among them, the models A and B are the most widely used for solid-state transitions see . ConcernDSC and TGA curves for the recrystallized and commercial theophylline are shown in Figures The DSC curve of commercial theophylline showed only one endothermic peak at 272°C due to melting and no weight loss was observed on the TGA curve. Therefore, the commercial product appeared effectively as the anhydrous form of theophylline. Similar results were reported by Suzuki et al. .The powder X-ray diffraction patterns of the recrystallized and commercial theophylline are shown in X versus air relative humidity RH) in The final equilibrium contents obtained from gravimetric experiments at constant temperature and constant relative humidity were used to plot the desorption isotherms of theophylline. The plot of desorption equilibriums  and low temperatures. The three forms of theophylline coexisted for moderate temperatures and humidities.θ).The evolution of PXRD patterns of theophylline during processing at 3.8% and 20°C (conditions which favor the occurrence of the anhydrous metastable form) is presented in The transition between the monohydrate and the metastable form corresponds to the reversible loss of water through channels in the crystal lattice according to Phadnis and Suryanarayanan . The cryA second solid-solid phase diagram corresponding to equilibrium states (after one month) of theophylline monohydrate processing at different relative humidities and different temperatures is presented on Our results did not agree closely with the above cited literature data. Ticehurst et al.  used aceX  versus time plots for the dehydration of theophylline monohydrate at 11.61% relative humidity and at different temperatures  is shown in The water content X  versus time plots for the dehydration of theophylline monohydrate at 40°C, and at different relative humidities  is presented in The water content These experiments showed that both temperature and humidity affected the dehydration rate of theophylline monohydrate. The same kinds of curves were also obtained for all temperatures and relative humidities considered, but these results are not shown here.X/X0 ≤ 0.8) with a slow quasilinear trend and a second stage (0.8 ≤ X/X0 ≤ 0) with a fast quasiexponential trend. The first stage was practically neither observed for the highest applied temperatures when probably form II was exclusively produced nor for the lowest relative humidity when probably the form III was exclusively produced. According to the literature [Most of the experimental curves exhibited two dehydration stages: the first stage  and a linear expression for lnk0 could be a possible mathematical representation of the theophylline's dehydration curves during the second stage. This representation would include the influence of both temperature and relative humidity in a quite simple way. The predicted dehydration curves of the theophylline monohydrate, according to different models are shown in Thus, the Fickian diffusion-based model (C) coupled with the Arrhenius expression for Except for the lowest relative humidity (2.4%) and the highest temperature (80°C), the points are rather narrowly distributed along the bisecting line and thus the correlation between the two sets of data can be considered satisfactorily.The capability of the model C to interpret correctly the experimental data may indicate that the dehydration rate of theophylline in the second  stage of the process is controlled by water transfer within the crystal rather than the nucleation and growth of the new crystal phase or the progression of the interface between the new and old phases. This corroborates the hypothesis of a “WET4” dehydration mechanism with a process rate controlled by diffusion suggested at the end of Section 3.3.Theophylline is classified as a lattice channel (or tunnel) hydrate, which class is known for releasing water rather easily. However, unlike in many others channel hydrates, theophylline's molecules are very tightly packed and its channels are particularly narrow (roughly of the size of the water molecule) according to Ahlqvist and Taylor . Water mThe effect of hygrothermal conditions (air temperature and relative humidity) on the dehydration of theophylline monohydrate was investigated. The results of gravimetric experiments provided the conditions where the monohydrate transforms to the anhydrate. The analysis of PXRD patterns allowed the determination of the conditions where only one of the anhydrate forms was obtained or where the two forms could coexist. The metastable form appeared preferably for low temperatures and low humidities, while the stable form was preferred for high temperatures. The coexistence occurred only for short processing times (few days). The proposed solid-solid phase diagram can be very useful for predicting the state of theophylline during drying or during storage.The dehydration rate was found to increase strongly with air temperature and to decrease with air relative humidity. Three mathematical models: Avrami-Erofeev's solid-state transition, first order chemical kinetic, and Fick's 2-D diffusion were used to fit the experimental data. The measured evolutions of water mass with time during dehydration of theophylline for all considered conditions were found to be best represented by the spatially averaged solution of 2-D Fickian transient diffusion equation. The parameters of this equation were correlated to temperature and relative humidity by exponential expressions, and finally a simple but comprehensive model for prediction of the dehydration progress with time was obtained. This kind of procedure of fitting semi-empirical equations to dehydration curves can provide a fast way for drying process modeling and optimal control in pharmaceutical industry.


This constitutes a demonstration of an optoelectronically active device integrated directly atop graphene and paves the way towards a generation of flexible highly performing hybrid two-dimensional (2D)/0D optoelectronics.The realization of low-cost photodetectors with high sensitivity, high quantum efficiency, high gain and fast photoresponse in the visible and short-wave infrared remains one of the challenges in optoelectronics. Two classes of photodetectors that have been developed are photodiodes and phototransistors, each of them with specific drawbacks. Here we merge both types into a hybrid photodetector device by integrating a colloidal quantum dot photodiode atop a graphene phototransistor. Our hybrid detector overcomes the limitations of a phototransistor in terms of speed, quantum efficiency and linear dynamic range. We report quantum efficiencies in excess of 70%, gain of 10 The combination of fast photo-response and high gain plays a pivotal role in photodetector devices. Here the authors combine a colloidal quantum dot photodiode with a graphene phototransistor to overcome the speed, quantum efficiency and linear dynamic range limitations of available phototransistors. These parameters eventually determine the sensitivity of a photodetector quantified via the specific detectivity D*. D* is defined as NEP is the noise equivalent power, R is the responsivity, A is the optically active area of the detector, fB is the noise bandwidth and Sn is the noise spectral density of the detector.A vast number of applications calls for highly sensitive detectors that can sense light from the ultraviolet to the short-wave infrared (SWIR) range, covering a broad spectrum of 300–3,000 nm24678EQE) of these systems is a trade-off between light absorption, which increases for thicker sensitizing layers, and charge transfer, which decreases for thicker layers as it is limited by carrier diffusion towards the graphene channel. This has limited the quantum efficiency to about 25% for an optimized layer of CQDs of thickness 80 nmGraphene's unique properties such as high charge carrier mobility, atomically thin profile and broadband absorption has made this material a very attractive platform for photodetectors11121418EQE as high as 70–80% (limited by reflection), a sub-millisecond temporal response, a gain-bandwidth product on the order of 108 and a linear dynamic range in excess of 110 dB. This highly performing detector also exhibits very high sensitivity with experimentally measured D* of 1 × 1013 Jones.In this letter, we address these three main challenges by presenting a photodetector architecture that is essentially a graphene–CQD photodiode and a high-gain phototransistor. This is realized by transforming the electrically passive sensitizing layer to an active one, where the electric field in the photodiode significantly enhances charge collection due to carrier drift instead of solely relying on diffusion. Our devices operate in the visible, near-infrared and SWIR range and show an Our photodetector platform integrates a CQD photodiode atop a graphene transistor. The CQD photodiode consists of an indium tin oxide (ITO) top-contact acting as the cathode of the CQD photodiode, whereas graphene acts as the hole acceptor contact and the charge transport channel for the phototransistor. VTD). Considering first operation without VTD: on carrier photogeneration in the CQD layer, holes are transferred to the graphene via drift from the narrow depletion region formed in the PbS QD solid near the graphene–QD interface and via diffusion further away from the quasi-neutral region. Prior studies have shown that efficient charge collection in this configuration occurs at an optimum thickness of 80–90 nm of PbS QDs, limited by the short carrier diffusion length22The essence of the device operation is captured in the band structure, shown in τlifetime is the carrier lifetime of the trapped electrons in the sensitizing centres of PbS QDs and τtransit is the transit time of holes in the graphene channel.where VTD bias extends the depletion region in the 300-nm-thick CQD layer, amplifying thus the zone of efficient charge collection and concomitantly quantum efficiency. This enhancement is evident in VBD) and the top-contact bias (VTD), and VTD=1.2 V, as compared with photoresponse at VTD=0 V. Further increase of VTD results in opposite effects and fully suppresses the photo-induced voltage change at VTD=4 V. This phenomenon is observed for any VBD value for both n-doped and p-doped graphene channel, and this is due to increased leakage current from the CQD photodiode (as discussed below). It is important to note that changing the potential of the top electrode induces a corresponding change in graphene doping; however, this change is weak and cannot account for the observed change in photocurrent. This is clear in VTD as a result of suppressed gating via the CQD layer. g/∂n) extracted from the corresponding resistance curves that is not affected by the electric bias of the top-contact, from which we conclude that the photoresponse enhancement is not due to increasing transconductance . To corroborate our findings we also extracted the EQE of the phototransistor using the formula−1) of photoconductive detectors:To quantify the impact of the e on VTD . For a pλ the wavelength, e the elementary charge, h is the Planck constant, c the speed of light, RΩ is the detector's resistance in dark and G is the photoconductive gain given by the ratio of carrier lifetime over the transit time. Here the carrier lifetime is governed by the time constant of the graphene–CQD–ITO photodiode response, in contrast with the case of the passive QD layer, in which carrier lifetime is determined by the sensitizing traps of the PbS QDs. To estimate the carrier lifetime τlifetime in our devices, we measured the photoresponse bandwidth of the phototransistor by sweeping the light modulation frequency fc as demonstrated in VTD, reaching an electrical 3 dB bandwidth of 1.5 kHz for the optimum VTD, which corresponds to an effective τlifetime of 106 μs. The time response of the graphene–CQD–ITO photodiode when measured independently yields a temporal response composed of two time constant components . This yields a photoconductive gain G on the order of 105 and gain-bandwidth product of >1.5 × 108 Hz. The extracted EQE of the phototransistor  and RΩ and compensate for the slight gating effect, induced by VTD bias. Considering first the passively sensitized photodetector  wafer using a direct laser writer lithography. Ti/Pd electrodes (2 nm/40 nm thickness) were e-beam evaporated at a base pressure 10−3 mbar and lifted-off in warm acetone bath.Metal contacts were designed on a precleaned Si/SiOCVD graphene grown on copper foil was transferred by a poly(methyl methacrylate) (PMMA)-assisted wet process onto the substrate with metal contacts. Ammonium persulfate was used to etch the copper substrate. After the transfer, the substrate with metal contacts and PMMA/graphene film was dried overnight at room temperature. PMMA was then removed by warm acetone, followed by annealing in forming gas at 300 °C to remove residues.2 plasma etching were used to pattern the graphene layer. Graphene stripes had dimensions of 70 × 10 nm with 10 × 10 μm area between V+ and V− electrodes.Laser writer lithography and Ar/OThe synthesis of PbS nanocrystals was carried out under inert conditions using a Schlenk line as previously described in literature31Electron beam lithography (EBL) was used to pattern 30 × 30 μm windows in PMMA resist at the centre of the graphene Hall bars. Desired PbS CQDs were then spin-coated right on the PMMA. Lift-off in warm acetone performed directly after spin-coating removed PMMA together with the excess of CQDs. As a result, the CQDs remained only in the areas defined by the lithography. The areas were designed to ensure isolation of the graphene channel from the ITO electrode introduced next and to fully cover the central region of the graphene Hall-bar.−1 in octane) was spin-cast onto the substrate at 2,500 r.p.m. for 15 s. Deposition of each layer was followed by spin-casting of EDT solution (2 vol.% in isopropanol) and two rinse-spin steps with octane and isopropanol. All the spin-coating steps were performed under ambient condition and at room temperature. The resulting thicknesses of nine PbS–EDT layers were 300±20 nm, as determined by a profilometer (Alpha-Step IQ).PbS QD layers were fabricated by layer-by-layer spin-coating. For each layer, PbS solution  to form a transparent electrode on top of the PbS layer. EBL lithography allowed to form the overlapping area of ITO, CQD and graphene precisely between the V+ and V− electrodes and avoid shortening of ITO and graphene channels. Each fabricated chip contained up to seven devices with top-electrodes.−3 mbar) to improve the device stability. Device illumination was done through a viewport by a 635 nm wavelength diode laser, modulated with a mechanical chopper or turned on and off. The laser was coupled to a digital variable optical attenuator to provide output power tunability. In addition, a supercontinuum Vis/NIR laser (SuperK Select) was used to measure photoresponse spectra. Ithaco 1211 amplifier was used to measure ITD. Ithaco 1201 and Stanford SR810 amplifier were used for voltage read-out of photoresponse in combination with a mechanical light chopper with 250–3,000 Hz range.All transport measurements were carried out at room temperature, in a shielded enclosure and vacuum , where n is the charge carrier concentration in graphene, by taking a derivative of the RΩ (VBD) curve. Capacitive coupling of the 285-nm-thick SiO2 gate is 7 × 1010 cm−2 V−1.We calculate the transconductance of our phototransistor  and negative (hole) transconductance maximums using: ∂σ/∂n=e × μ, where e is elementary charge. Maximum transconductance of 2.4 S cm2 corresponds to 1,500 cm2 V−1 s−1 of electron mobility in graphene.Charge carrier mobility τtransit of charge carriers in the graphene channel is calculated as:Transit time The data that support the findings of this study are available from the corresponding authors upon request.How to cite this article: Nikitskiy, I. et al. Integrating an electrically active colloidal quantum dot photodiode with a graphene phototransistor. Nat. Commun. 7:11954 doi: 10.1038/ncomms11954 (2016).Supplementary Figures 1-5 and Supplementary Note 1


The resistance of WO3−x crystal sensors in response to relative humidity could be tuned from a negative to positive one by increasing the compositional x. And it was revealed that the positive humidity impedance was driven by the defects of oxygen vacancy. This result will extend the application field of humidity sensors, because the positive humidity impedance sensors would be more energy-efficient, easier to be miniaturized and electrically safer than their negative counterparts for their lower operation voltages. And we believe that constructing vacancies in semiconducting materials is a universal way to fabricate positive impedance humidity sensors.Resistivity-type humidity sensors have been investigated with great interest due to the increasing demands in industry, agriculture and daily life. To date, most of the available humidity sensors have been fabricated based on negative humidity impedance, in which the electrical resistance decreases as the humidity increases, and only several carbon composites have been reported to present positive humidity impedance. However, here we fabricate positive impedance humidity sensors only via single-component WO Resistivity-type humidity sensors, which can perceive and record the change in electrical resistance in response to that in environmental humidity, have been investigated with great interest due to the increasing demands in industry, agriculture and daily life1234672S, O2, NOx, COx, NH3 and so on12132O (humidity) based on WO3 (the only reported tungsten oxide based sensors in literature) are WO3 nanowire humidity sensor on chip manufactured using CMOS-MEMS technique3 thin-film sensor fabricated using deposition technology3 composites2O3 and WO33 composites3 to relative humidity (RH) is generally attributed to the water dissociative chemisorptions process that would result in the formation of hydroxyl groups on the surface of WO3 crystals; and then, electrons are accumulated on the WO3 surface. As a result, the resistance of WO3 crystals decreases with increasing RH17In sensing materials, semiconductor metal oxides are one of the most promising candidates for solid-state chemical sensors due to their high sensitivity, and quick response and recovery93−x crystals with a variety of oxygen-deficient stoichiometries, such as WO2.72, WO2.8, WO2.83 and WO2.9, can be easily prepared, since they are stable, ordered phases with precise stoichiometries. And the early studies revealed that oxygen vacancy can consistently account for the defect level and trap assisted conduction in semiconducting oxides2122233 would be affected by water vapor when the experiments were performed in air. These facts inspire us to design and fabricate various WO3−x humidity resistors in which the different densities of oxygen vacancy might induce and modulate the humidity sensitivity.Furthermore, unlike most of the oxygen-deficient metal oxides, which are not stable , WO3−x) nano-/micron-structures (NMS) only by heating WO3 powder in S atomsphere in a vacuum tube furnace, and with the structured WO3−x crystals, humidity sensors were fabricated simply by screen-printing them onto ceramic substrates with Ag-Pd interdigital electrodes. Surprisingly, a positive humidity-sensitive property was found in the sensors prepared by single-component WO3−x crystals with high density of oxygen vacancies. And the resistance of WO3−x crystal sensors in response to relative humidity could be tuned from a negative to positive one by increasing the compositional x. We believe that our method not only provides a new avenue for fabricating highly effective positive humidity sensors by various metal oxides, but also creates a powerful platform to understand and design desirable semiconducting oxides humidity sensors. In addition, the findings on the positive resistance characteristics of single-component material humidity sensors can not only extend the application of humidity-sensitive resistor in different types of miniaturized devices, but also enrich and compensate for the humidity-sensing principles.Therefore, here we developed an approach to prepare oxygen-deficient tungsten oxides . The XRD pattern of the sample prepared at 1050 °C matches well with that of the monoclinic W19O55 . And the sample synthesized at 1150 °C consists of monoclinic W18O49 . However, the diffraction peaks of their corresponding annealed samples are all well assigned to those of the identical phase, monoclinic WO3 (JCPDS card no. 43-1035).After a systematical investigation7/2 and W4f5/2 lines, respectively, which can be assigned to those of the W6+ oxidation state7/2 and W4f5/2 lines, respectively, which can be attributed to those of the W5+ oxidation state7/2 line at 32.7 eV and W4f5/2 at 35.2 eV, indicating the existence of W4+ oxidation state on the sample surface6+ over W5+ and W4+ in the spectra, it was calculated that the formula of the three samples are WO2.9 (synthesized at 950 °C), WO2.89 (at 1050 °C) and WO2.72 (at 1150 °C), respectively, in which the mean valence of their W ions decreased from 5.58 to 5.27  analysis was carried out. The results are shown in 5.27 see . In addi3−x crystals exhibit a sharp signal at g =  2.28, while that of WO3 presents no obvious signals. The signal of the present oxygen-deficient WO3−x compounds could be attributed to oxygen vacancies. In literature, it was reported that the peak assigned to oxygen vacancies in metal oxides always has a g factor of 2.012829303−x compounds is more complex. Di Valentin and Pacchioni3−x, indicating that sometimes the oxidation state of the under-coordinated W ions is still formally of a chemical valence of + 6, while two extra electrons are trapped in the vacancy voids, which can be explained by the following synthetic expression: W6+/VO(2e−)/W6+ (VO is the oxygen vacancy). Thus, the shift of the g value for the present oxygen-deficient WO3−x compounds to a higher one might be owing to the presence of two charge centers (two unpaired electrons) trapped in the oxygen vacancy. Moreover, the sharp g signal of metal oxides becomes stronger with increasing concentration of oxygen vacancies2829303−x compounds, as the value of x increased, the sharp g signal gets stronger and stronger, indicating that the density of oxygen vacancies in them increases. But there is no signal in the ESR spectrum of WO3, implying that the annealed samples were almost completely oxidized, containing very little or even no oxygen vacancies. This result is also in good agreement with the XPS spectra.The electron spinning resonance (ESR) spectra of the as-prepared tungsten oxides NMS recorded at room temperature are displayed in 3−x crystals , the sensors fabricated with WO2.72 present the lowest impedance, and the impedance of the sensors fabricated with WO2.89 is lower than that with WO2.9, while the resistance of the sensors with WO3 is the highest. That is to say, under low RH environment, the higher the density of oxygen vacancies in tungsten oxide crystals, the lower the impedance of the senores fabricated with them, which is similar with the conduction behavior of many semiconducting oxidesThe dependence of impedance on RH was measured for the sensors fabricated with the obtained WOtals see , in whic2.72 NMS increases remarkably at low humidity (11~75% RH), and still increases somewhat at higher humidity (75~95% RH), presenting a completely positive resistance sensitivity to RH. With decreasing density of oxygen vacancies in tungsten oxide crystals, however, the fabricated sensors will present different sensing behaviors. The sensors fabricated with the as-prepared WO2.89 nanorods exhibits a positive resistance response to humidity in the range of 11–85% RH with slightly increased resistance. But at the humidity higher than approximately 85% RH, a negative response could be observed in such sensors, presenting an extreme point there (see the inset of 2.9 nanorods), the response curve recorded from the sensors still exhibits a similar sensing profile with that of WO2.89 sensors, but the extreme point was found at a lower humidity of about 54% RH. When it comes to WO3 NMS, with increasing RH, the impedance of the sensors fabricated with the annealed NMS rapidly decreased monotonously, presenting a completely negative resistance characteristic to RH, which is in accordance with the already reported humidity sensitivity of the sensors fabricated with other forms of WO3 materials153−x samples with x >  0.11 in this study), they will present a completely positive humidity-sensitivity in the entire RH range (here from 11–99%). With a medium x (0.1 ≤  x ≤  0.11 in this work), they will exhibit a positive humidity-sensitive property at low RH, but still a negative one at high RH. In such case, the extreme point may gradually move to a lower RH with decreasing x. When the density of oxygen vacancies is low (in samples with x <  0.1), for example with x =  0 as in the present WO3 NMS, the fabricated sensors may show an almost completely negative humidity sensitive characteristics in the entire RH range.Moreover, from 2.72 and WO2.89 increased, showing a positive resistance characteristic, but the humidity-sensitive resistors fabricated with WO2.9 and WO3 presented a negative humidity impedance characteristics, i.e., the impedance of the sensors decreased as the humidity increased. For the sensors with WO2.72, the impedance increased from 6.9–26 kΩ, presenting a gain of 276.8% as the humidity increased from 11–95% RH. As the density of oxygen vacancies decreased, the impedance gain of the fabricated sensors would decrease. For example, the sensors with WO2.89 still had a gain of about 33% with the impedance from 837–1114 kΩ. But when it comes to WO2.9, the impedance of the as-fabricated sensors decreased from 936–627 kΩ under similar RH environment. And the resistance of the sensors with WO3 even presented a sharp decrease from 20716–424 kΩ  and their recovery time  can be calculated. 3−x crystals, both of the response and recovery times changed dramatically as the environment humidity varied. The response time of the sensors with WO2.72 NMS was about 6 s, indicating that such sensors have a very good response to humidity, which is comparable with those of the well-known WO3 based sensors17183 NMS also displayed a smiliar, quick response time of 4 s in the humidification process. But for the sensors with WO2.89 and WO2.9 NMS, they should take a longer response time  in the humidification process. The sensors with WO2.72 or WO3 NMS would display a quick response time, because both of them simply present a completely positive or negative humidity sensitive characteristics in the entire RH range (11–99% RH). However, because the sensors with WO2.89 and WO2.9 NMS exhibit a positive humidity-sensitive property at low RH but still a negative one at high RH, their response times become longer. This phenomenon might be correlated with the strong competition between the oxygen vacancies induced positive humidity sensibility and water-related negative humidity sensitive property. As for the recovery time of the sensors fabricated with the obtained WO3−x crystals, as shown from the 3 is generally smaller than the one of hydroxyl groups from the oxygen vacancies33From the response and recovery characteristic curves as shown in 2.9 , the oxygen vacancies induced electrical conduction of the sensors is the primary mechanism at low humidity (lower than 85% RH), but the water-related electrolytic conduction would become dominating at high humidity (85–95% RH in this case), thus resulting in the turning point of impedance at 85% RH. As the density of oxygen vacancies is further reduced, the turning point of the sensor impedance will move toward a lower humidity (here at 54% RH for the WO2.9 sensors), and even disappear, presenting a totally negative humidity impedance characteristic (as shown in the present WO3 sensors).On the other hand, for the sensors with the present structured oxygen-deficient WO3−x crystalline NMS of different densities of oxygen vacancies. The resistance of WO3−x crystal sensors in response to relative humidity could be tuned by changing the compositional x. When the density of oxygen vacancies is high (in WO3−x samples with x >  0.11 in this study), they will present a completely positive impedance humidity-sensitivity in the entire RH range (here from 11–99%). With 0.1 ≤  x ≤  0.11, they will exhibit a positive impedance humidity-sensitive property at low RH, but still a negative one at high RH. In such case, the extreme point may gradually move to a lower RH with decreasing x. When the density of oxygen vacancies is low (in samples with x <  0.1), for example with x =  0 as in the present structured WO3 crystals, the fabricated sensors may show an almost completely negative impedance humidity sensitive characteristics in the entire RH range. Moreover, the sensors fabricated with WO2.72 crystals possess high sensitivity with a short response time of about 6 s, a recovery time of approximately 100 s and the largest humidity hysteresis of about 45.8% at 33% RH. The humidity sensitivity of the present tungsten oxides sensors may be controlled by the combination of oxygen vacancy induced electrical conduction and water-related electrolytic conduction.In summary, humidity sensors were successfully fabricated with WO3 powder  was loaded in a quartz boat located at the center of the furnace, while another boat with 1 g Aladdin-reagent S powder was located at the upstream from the WO3 powder. Before heating, the quartz tube was evacuated and flushed with Ar gas repeatedly for several times to deplete the residual gases. Then the furnace was heated up to the selected temperatures of 950, 1050 and 1150 °C with a dwelling time of 1 h. After that, the furnace was cooled down naturally to room temperature. Finally, powder-like products could be collected. For the purpose of comparison, a portion of the collected powders was annealed in air at 500 °C for 2 h in a muffle furnace.To fabricate the proposed tungsten oxides NMS, a high-temperature thermal evaporation process via a horizontal quartz tube furnace was usedg value calibration.The morphology of the obtained samples was examined by a field emission scanning electron microscope . The chemical composition was measured by an energy-dispersive X-ray (EDX) spectroscope attached to the SEM. The phase structure and composition were identified by XRD  in continuous scanning mode with a rate of 6°/min. The chemical state of the elements in the samples was investigated by XPS , and the results were calibrated by C1s line . The unpaired electron and defect in the samples were investigated by ESR  at room temperature with a microwave frequency of 9.44 GHz. Diphenylpicrylhydrazyl was used for the 3−x materials.Extended Data 2, Mg(NO3)2, NaCl, KCl and KNO3, which could present a relative humidity at 25 °C of approximately 11%, 33%, 54%, 75%, 85% and 95%, respectively. In typical measurement, each sensor was soaked at 25 °C in an atmosphere of different RH levels in the six chambers with different salt solutions till it reached the adsorption-desorption equilibrium for water, and then the impedances of the sensors with the RH and time were measured, respectively.The characteristic humidity sensitivity of the as-fabricated sensors, including impedance vs. relative humidity, response property and humidity hysteresis, was examined by a Keithley 2410 analyzer (USA). During the measurement, the applied operation voltage was AC 1 V and operation frequency was 1000 Hz. And the controlled humidity environment was achieved by a series of super-saturation aqueous solutions with different salts of LiCl, MgClHow to cite this article: Qian, J. et al. Positive impedance humidity sensors via single-component materials. Sci. Rep.6, 25574; doi: 10.1038/srep25574 (2016).


There is a huge interest in doped graphene and how doping can tune the material properties for the specific application. It was recently demonstrated that the effect of doping can have different influence on the electrochemical detection of electroactive probes, depending on the analysed probe, on the structural characteristics of the graphene materials and on the type and amount of heteroatom used for the doping. In this work we wanted to investigate the effect of doping on graphene materials used as platform for the detection of catechin, a standard probe which is commonly used for the measurement of polyphenols in food and beverages. To this aim we compared undoped graphene with boron-doped graphene and nitrogen doped graphene platforms for the electrochemical detection of standard catechin oxidation. Finally, the material providing the best electrochemical performance was employed for the analysis of real samples. We found that the undoped graphene, possessing lower amount of oxygen functionalities, higher density of defects and larger electroactive surface area provided the best electroanalytical performance for the determination of catechin in commercial beer samples. Our findings are important for the development of novel graphene platforms for the electrochemical assessment of food quality. Heteroatom doped graphene has been lately considered as an ultimate candidate for numerous applications due to the possibility to tailor the material characteristics and to improve the physicochemical, optical, structural and electronic properties123456791012131412In this work we investigate the effect of heteroatom doping on the detection of catechin, a polyphenol generally used as an index of food and beverage quality. A part from traditional techniques based on tedious and expensive chromatographic analysis161719We found that, for the examined case, the best electroanalytical response was provided by the undoped graphene which was the material possessing the highest C/O ratio and the largest D/G ratio and electroactive surface area as compared to both heteroatom doped graphene materials. This opens new possibilities in the choice of the best suited graphene platform for electrochemical applications.Glassy carbon (GC) electrodes, (diameter = 3 mm), Ag/AgCl reference electrode and platinum counter electrode were obtained from CH Instruments . Boron – doped diamond electrode with a doping level of 1000 ppm of B and an H terminated surface was purchased from Windsor Scientific.N,N-dimethylformamide (DMF), hydrochloric acid (37%), ethanol, sodium hydroxide, sodium chloride, sodium phosphate monobasic, sodium nitrate, potassium phosphate dibasic, potassium permanganate, potassium chloride, and (+)-catechin hydrate were purchased from Sigma Aldrich (Singapore). Milli-Q water (resistivity: 18.2 MΩcm) was used throughout the experiments. Beer samples were obtained from a local supermarket. Sample n.1 and sample n.2 are lager beers from different brands whilst sample n.3 is a stout beer.Graphite was provided by Asbury Carbons. Fuming nitric acid (>90%) was purchased from by J.T. Baker. Sulfuric acid (95–98%), potassium chlorate (98%), hydrogen peroxide (3%), A μAutolab type III electrochemical analyzer  connected to a personal computer and controlled by General Purpose Electrochemical Systems, GPES Version 4.9 software (Eco Chemie) was used to perform differential pulse voltammetry measurements.A Thermoscientific Finnpipette  was employed for the electrode modification with graphene materials.Thermally reduced graphene was prepared first by synthesising graphite oxide through the Staudenmaier method−1. The sample was flushed with nitrogen repeatedly before it was inserted by the magnetic manipulator into the preheated furnace and held for 3 minutes. The nitrogen flow rate was maintained at 1000 °C min−1 to remove any exfoliation by-products from the procedure.Thermally reduced graphene was prepared by introducing 0.2 g of graphite oxide into a quartz capsule connected to a magnetic manipulator, which was inside a vacuum tight tube furnace with controlled atmosphere. The magnetic manipulator created a temperature gradient of over 1000 °C min3Et2O). Exfoliation was performed in a bubbler filled with the liquid boron precursor at 20 °C and 1000 mbar. Nitrogen carrier gas with a flow rate of 100 mL/min was used and dilution was performed with 1 L/min nitrogen and hydrogen/nitrogen mixture (0.5 L/min N2 and 0.5 L/min H2). The reactor was continuously flushed with nitrogen and the flow of boron precursor was stabilised for 5 minutes before it was introduced into the hot region of the reactor. Exfoliation was then performed for 12 minutes at 1000 °C.Boron-doped graphene (BDG) was prepared from graphite oxide synthesized through Staudenmaier method. Graphite oxide was thermally exfoliated in the presence of a boron precursor, namely boron trifluoride diethyl etherate  into a flask under continuous stirring. The mixture was cooled in an ice bath before the addition of 15 g of potassium permanganate. Vigorous stirring was maintained for 2 hours to obtain a homogenous solution. After that, the mixture was cooled down and then reheated to 35 °C for 30 minutes. Subsequently, the mixture was diluted with 250 mL of deionised water and it was further heated to 70 °C. The temperature of the mixture was maintained for 15 minutes before it was further diluted with 1000 mL of deionised water. The removal of unreacted manganese dioxide and potassium permanganate was carried out by adding hydrogen peroxide (3%) into the mixture and decanting. Repeated centrifugation and redispersion into distilled water was performed with barium nitrate until a negative reaction to sulphate ions was observed. Graphite oxide slurry was then dried at 60 °C for 48 hours in a vacuum oven.Nitrogen-doped graphene was prepared by exfoliation of produced graphite oxide in ammonia atmosphere. A quartz glass capsule was filled with 100 mg of graphite oxide before it was connected to a magnetic manipulator and placed in a horizontal quartz glass reactor. The reactor was flushed continuously with nitrogen before it was introduced into the hot region. Then the nitrogen flow was changed to ammonia. The temperature of the mixture was maintained for 12 minutes at 600 °C and the ammonia flow rate of 300 mL/min was used to remove any exfoliation by-products. The synthesised thermally reduced graphene (TRG), boron-doped graphene (BDG) and nitrogen-doped graphene (NDG) were ultrasonicated for few minutes before each use. After ultrasonication, 1 μL of the material was deposited onto the surface of a glassy carbon (GC) working electrode. The solvent was left to evaporate at room temperature in order to obtain a randomly distributed film of the desired material on the electrode surface. After each measurement the surface of GC was cleaned by polishing with 0.05 μm alumina powder on a polishing cloth.Electrochemical experiments were performed in a 4 mL voltammetric cell at room temperature (25 °C) using a three electrode configuration.Differential pulsed voltammetry parameters used for the experiment were applied as follows: 3 s equilibration time, 50 ms modulation time, 0.5 s interval time, 25 mV modulation amplitude, and 4 mV step. The raw data obtained were treated by a baseline correction with a peak width of 0.01, using GPES software. Measurements were performed in a 4 mL solution containing various concentrations of standard analyte in 100 mM phosphate buffer solution (PBS) at pH 7.3. Catechin hydrate in increasing concentrations from 1.2 μM to 12.0 μM was used for the measurements, similarly to previous findingsThe analysis of commercial lager beer sample was performed by using a dilution factor of 1:10. Standard addition method was used for the analysis of real samples.In this study we wanted to compare the electrochemical performance of undoped and doped graphene platforms namely thermally reduced graphene (TRG), boron doped graphene (BDG), and nitrogen doped graphene (NDG) for the detection of catechin, an important polyphenol which is correlated to food quality. Unmodified glassy carbon (GC) electrode and boron doped diamond (BDD) electrode were also used as reference materials. We wanted to investigate if the presence of heteroatoms with different electronegativity could have an influence on the electrochemical response provided by the graphene platform, as it could be expected from previous works1213141524−1 is correlated to the presence of defects due to sp3 hybridized carbon whilst the G band at around cm−1 1560 indicates the sp2 hybridized carbon. The ratio between the intensities of D and G band provide information on the degree of disorders in the carbon structure of the material. As depicted in In order to gain more insight into the material properties, characterization was performed by XPS, Raman spectroscopy and prompt gamma-activation analysis−2 cm2 for GC, 2.58 × 10−2 cm2 for BDD, 4.72 × 10−2 cm2 for BDG, 8.62 × 10−2 cm2 for NDG and 1.78 × 10−1 cm2 for TRGk0 = 2.30 × 10−4 cm s−1, k0 = 2.03 × 10−4 cm s−1, k0 = 1.73 × 10−2 cm s−1, k0 = 6.49 × 10−2 cm s−1, k0 = 4.63 × 10−2 cm s−1 for GC, BDD, BDG, NDG, and TRG respectively. To summarize the characterization results, TRG is the material with lowest content of oxygen functionalities, highest amount of structural disorders and largest electroactive surface area.Moreover, from a previous electrochemical study to evaluate the electroactive surface area of all materials, the following results were obtained: 2.28 × 10The oxidation of catechin occurs sequentially at the catechol and resorcinol group respectivelyThe oxidation process is pH dependant, with a shift towards lower oxidation potentials when the pH of the solution is increased from 3.5 to 8.03 hybridized carbon and therefore lacks of the sp2 network that would be necessary to form π-π stacking interactions with the aromatic polyphenol used as probe. Such interactions, which are very likely to occur on both doped and undoped graphene materials, are able to promote an accelerated heterogeneous electron transferThe observed trend could be attributed to the structure of BDD which contains sp2) and the peak width at half height (W1/2) are consolidated in −1 on TRG, followed by BDG at 88.283 nA μM−1, NDG at 81.282 nA μM−1, GC at 68.267 nA μM−1 and finally BDD at 1.6946 nA μM−1.The response from the oxidation of catechin on bare GC, BDD, TRG, BDG and NDG was studied between 1.2–12.0 μM and the voltammograms were displayed in 2 ≥ 0.9797 for all graphene platforms. The influence of different materials on peak width at half height (W1/2) for 12.0 μM catechin was also investigated to correlate the parameter to the selectivity of the materials in the presence of interferences. With the data collated, BDD has the highest W1/2 while the other materials have similar W1/2 with slight improvement for TRG and NDG as compared to GC.Overall, both undoped and doped graphene materials showed enhanced sensitivity on the detection of catechin as compared to bare GC, whilst among the doped graphenes, BDG showed a better sensitivity than NDG. All materials presented good linearity of response with RAs observed form the material characterization, TRG showed the lowest content of oxygen functionalities, the highest amount of structural disorders and the largest electroactive surface area. All these factors contributed to improve the material electroanalytical performance, thus resulting in enhanced sensitivity of the electrochemical signal. As for a comparison between the doped graphenes, BDG showed better calibration sensitivity as compared to NDG despite the lowest amount of defects and electroactive surface area presented by the former. Clearly, among the doped graphenes, the electrochemical response is mostly influenced by the kind of heteroatom rather than the properties of the materials. In fact, as recently demonstrated, the favourable thermodynamic interactions between the electron withdrawing boron and the electron donating oxygen groups of the analysed probe strongly influences the oxidation processGiven the best electrochemical performance of TRG in terms of sensitivity, selectivity and linearity of response, the material was chosen for the application to real sample analysis.2 ≥ 0.9589) and repeatability of results (RSD ≤ 10.44%) were achieved.The results obtained for three commercial beer samples and represented as catechin equivalents are depicted in Finally, in order to confirm the selectivity of the response towards catechin in real samples, a study on TRG was performed by measuring the concomitant current response of luteolin, another polyphenol present in beer. As it can be seen in 2 network which favours the interactions with the analyte by π-π stacking. As a result of that, an increased intensity of the peak current and a lower oxidation potential was observed on both undoped and doped graphene platforms.We investigated the influence of structural properties of doped and undoped graphene materials on their electrochemical performance for the assessment of catechin, a standard polyphenol commonly used as index of food quality. We observed that in general, graphene materials show an enhanced electroanalytical response when compared to bare glassy carbon and boron doped diamond electrodes. This is because of the larger electroactive surface area they possess, together with the spOverall, the undoped graphene namely thermally reduced graphene (TRG) provided the best analytical performance in terms of sensitivity, selectivity and linearity of response due to the intrinsic properties of the material such as lower content of oxygen functionalities, higher amount of structural disorders and larger electroactive surface area as compared to doped graphenes. We demonstrated that in the reported case, the outstanding material properties play a major role towards the oxidation of catechin rather than the nature of heteroatom used for the doping. In addition we found out that within the heteroatom doped materials, the best performance was provided by the boron doped graphene because of the favourable effect of boron in promoting the thermodynamic interactions between the analytical probe and the graphene platform. Finally, we demonstrated the suitability of TRG platform for the real sample analysis by determining the amount of polyphenols, expressed as catechin equivalents, in three commercial beer samples. These findings provide an insight into doped and undoped graphene suitability for food science application.How to cite this article: Chng, C.E. et al. Doped and undoped graphene platforms: the influence of structural properties on the detection of polyphenols. Sci. Rep.6, 20673; doi: 10.1038/srep20673 (2016).