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ABSTRACT: An automated sample preparation system was developed and tested for the rapid detection of Bacillus anthracis endospores by gas chromatography-mass spectrometry (GC-MS) for eventual use in the field. This reactor is capable of automatically processing suspected bio-threat agents to release and derivatize unique chemical biomarkers by thermochemolysis (TCM). The system automatically controls the movement of sample vials from one position to another, crimping of septum caps onto the vials, precise delivery of reagents, and TCM reaction times and temperatures. The specific operations of introduction of sample vials, solid phase microextraction (SPME) sampling, injection into the GC-MS system, and ejection of used vials from the system were performed manually in this study, although they can be integrated into the automated system. Manual SPME sampling is performed by following visual and audible signal prompts for inserting the fiber into and retracting it from the sampling port. A rotating carousel design allows for simultaneous sample collection, reaction, biomarker extraction and analysis of sequential samples. Dipicolinic acid methyl ester (DPAME), 3-methyl-2-butenoic acid methyl ester (a fragment of anthrose) and two methylated sugars were used to compare the performance of the autoreactor with manual TCM. Statistical algorithms were used to construct reliable bacterial endospore signatures, and 24 out of 25 (96%) endospore-forming Bacillus species were correctly identified in a statistically designed test.
Analytica chimica acta 05/2013; 775:67-74. · 4.31 Impact Factor
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ABSTRACT: Aims and background. As a powerful technique allowing analysis of large numbers of cells, fluorescence-activated cell sorting (FACS) is used more and more widely. For FACS analysis, adherent cells are usually detached by trypsinization, followed by centrifugation and resuspension. However, trypsinization can cut off some receptors from the cell surface like fine scissors, which will affect the accuracy of FACS results. Though non-enzymatic methods such as citric saline buffer have been used to determine cell surface receptors, how much of the receptors is cut off by trypsinization has been rarely studied. This work aimed to investigate whether different methods of detaching adherent cells could affect the detection of cell surface receptors. Methods. Human hepatocellular carcinoma cell lines (HepG2, Huh7 and Hep3B) were detached enzymatically with trypsin-EDTA solution or non-enzymatically with citric saline buffer, and then the receptors of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were detected by FACS analysis. Cell viability, cell cycle and apoptosis (sub-G1 fraction detected by FACS) of the trypsin-EDTA group and citric saline buffer group were also studied. Results. Different methods of detaching adherent cells could significantly affect the detection of TRAIL receptors. Compared to the conventional trypsin-EDTA group, the non-enzymatic group showed a 3.42-fold increase in the mean fluorescence intensity index of DcR HepG2 and a 1.25-fold increase in DR Huh 7 (P <0.05). However, the viability, cell cycle and apoptosis of these cells were not affected. Conclusions. Citric saline buffer might be recommended as the first choice to detach adherent cells for FACS analysis of cell surface receptors.
Tumori. 11/2012; 98(6):800-3.
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ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been demonstrated to induce cell apoptosis in many types of tumors, while many hepatocellular carcinoma (HCC) cells display high resistance to TRAIL. Another outstanding limitation of TRAIL is the short half-life in vivo. Stem cell-based therapies provide a promising approach for the treatment of many types of tumors because of the ability of tropism. Therefore, as a new therapeutic strategy, the combination of chemotherapeutic agents and TRAIL gene modified MSCs (TRAIL-MSCs) would improve the therapeutic efficacy of HCC in vivo. This is the first time to show the potential of combination of chemotherapeutic agents and MSCs as a gene vector in the therapy of HCC.
Cancer biology & therapy 08/2012; 13(12):1175-84. · 2.64 Impact Factor
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ABSTRACT: The anti-norovirus (anti-NoV) effect of grape seed extract (GSE) was examined by plaque assay for murine norovirus 1 (MNV-1), cell-binding reverse transcription-PCR for human NoV GII.4, and saliva-binding enzyme-linked immunosorbent assay for human NoV GII.4 P particles, with or without the presence of interfering substances (dried milk and lettuce extract). GSE at 0.2 and 2 mg/ml was shown to reduce the infectivity of MNV-1 (>3-log PFU/ml) and the specific binding ability of NoV GII.4 to Caco-2 cells (>1-log genomic copies/ml), as well as of its P particles to salivary human histo-blood group antigen receptors (optical density at 450 nm of >0.8). These effects were decreased as increasing concentrations of dried milk (0.02 and 0.2%) or lettuce extract were added. Under an electron microscope, human NoV GII.4 virus-like particles showed inflation and deformation after treatment with GSE. Under conditions that simulated applications in the food industry, the anti-NoV effect of GSE using MNV-1 as a target organism was shown to be limited in surface disinfection (<1-log PFU/ml, analyzed in accordance with EN 13697:2001). However, a 1.5- to 2-log PFU/ml reduction in MNV-1 infectivity was noted when 2 mg of GSE/ml was used to sanitize water in the washing bath of fresh-cut lettuce, and this occurred regardless of the chemical oxygen demand (0 to 1,500 mg/ml) of the processing water.
Applied and environmental microbiology 08/2012; 78(21):7572-8. · 3.69 Impact Factor
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ABSTRACT: The effects of 13 food extracts and juices, including shellfish, fruits, and vegetables, on the binding ability of human norovirus (NoV) were examined, using P particles of human NoV GII.4 as a research surrogate. The enhancements (positive values) or reductions (negative values) of NoV P particle detection (changes in optical density at 450 nm) in the presence of different food extracts and juices as compared with P particles diluted in phosphate-buffered saline were tested by saliva-binding, enzyme-linked immunosorbent assay in triplicate. In the presence of different food extracts and juices at different concentrations, an increase or decrease of the receptor-binding ability of the NoV P particles was observed. Due to a higher specific binding and thus a higher accumulation of the viral particles, oysters may be contaminated with human NoV more often than other shellfish species (mussel, hard clams, and razor clams). Cranberry and pomegranate juices were shown to reduce the specific binding ability of human NoV P particles. No such binding inhibition effects were observed for the other tested extracts of fresh produce (strawberry, blackberry, blueberry, cherry tomato, spinach, romaine lettuce) or, notably, for raspberry, which has been associated with human NoV outbreaks.
Journal of food protection 07/2012; 75(7):1350-4. · 1.94 Impact Factor
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ABSTRACT: The presence, locations and composition of simple sequence repeats (SSRs) in Herpes simplex virus type 1 (HSV-1) genome were extracted and analyzed by using the software Imperfect Microsatellite Extractor (IMEx). There were 663 mon-, 502 di-, 184 tri-, 20 tetra-, 4 penta- and 4 hexanucleotide SSRs that were observed in different distribution between coding and noncoding regions in the HSV-1 genome. G/C, GC/CG, and (GGC)(n) were predominant in mononucleotide, dinucletide, trinucleotide repeats respectively. Indeed, the results showed that GC content in simple sequence repeats was notably higher than that in entire HSV-1 genome. Our data might be helpful for studying the pathogenesis, genome structure and evolution of HSV-1.
Gene 03/2012; 499(1):37-40. · 2.34 Impact Factor
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ABSTRACT: Growth of silicalite with graphene oxide (GO) nanosheets occurred via attachment of GO onto the silicalite surface, and entrapment of GO nanosheets inside single crystals. Electrically conductive composites were produced by calcination in nitrogen whereas silicalite crystals with slit-like mesopores of sizes 20-25 Å were obtained after GO burn-off.
Chemical Communications 02/2012; 48(16):2249-51. · 6.17 Impact Factor
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ABSTRACT: Optimization of the unsaturated fatty acid composition of ruminant milk and meat is desirable. Alteration of the milk and fatty acid profile was previously attempted by the management of ruminal microbial biohydrogenation. The aim of this study was to identify the group of ruminal trans-vaccenic acid (trans-11 C18:1, t-VA) hydrogenating bacteria by combining enrichment studies in vitro.
The enrichment culture growing on t-VA was obtained by successive transfers in medium containing t-VA. Fatty acids were detected by gas chromatograph and changes in the microbial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE). Prominent DGGE bands of the enrichment cultures were identified by 16S rRNA gene sequencing.
The growth of ruminal t-VA hydrogenating bacteria was monitored through the process of culture transfer according to the accumulation of stearic acid (C18:0, SA) and ratio of the substrate (t-VA) transformed to the product (SA). A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Bacteria corresponding to predominant DGGE bands in t-VA enrichment cultures clustered with t-VA biohydrogenated bacteria within Group B.
This study provides more insight into the pathway of biohydrogenation. It also may be important to control the production of t-VA, which has metabolic and physiological benefits, through management of ruminal biohydrogenation bacterium.
BMC Research Notes 02/2012; 5:97.
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ABSTRACT: A simple method was developed for detection of Bacillus anthracis (BA) endospores and for differentiation of them from other species in the Bacillus cereus group. Chemical profiles that include lipids (i.e., fatty acids), carbohydrates (i.e., sugars), and the spore-specific biomarker, dipicolinic acid, were generated by one-step thermochemolysis (TCM) at 140 °C in 5 min to provide specific biomarker signatures. Anthrose, which is a biomarker characteristic of the B. cereus group of bacteria, was determined from a fragment produced by TCM. Surprisingly, several virulent BA strains contained very low levels of anthrose, which confounded their detection. A statistical discrimination algorithm was constructed using a combination of biomarkers, which was robust against different growth conditions (medium and temperature). Fifteen endospore-forming Bacillus species were confirmed in a statistically designed test (~90%) using the algorithm, including six BA strains (four virulent isolates), five B. thuringiensis (BT) isolates, and one isolate each for B. cereus (BC), B. mycoides (BM), B. atrophaeus (BG), and B. subtilis (BS). The detection limit for B. anthracis was found to be 50,000 endospores, on the basis of the GC/MS detection limits for 3-methyl-2-butenoic acid methyl ester, which is the biomarker derived from TCM of anthrose.
Analytical Chemistry 02/2012; 84(3):1637-44. · 5.86 Impact Factor
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Mingming Jia,
Kunxue Hong,
Jianping Chen,
Yuhua Ruan,
Zhe Wang,
Bing Su,
Guoliang Ren,
Xiaoqing Zhang,
Zhen Liu,
Quanbi Zhao, Dan Li,
Hong Peng,
Marcus Altfeld,
Bruce D Walker,
Xu G Yu,
Yiming Shao
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ABSTRACT: It is generally believed that CD8(+) cytotoxic T lymphocytes (CTLs) play a critical role in limiting the replication of human immunodeficiency virus type 1 (HIV-1) and in determining the outcome of the infection, and this effect may partly depend on which HIV product is preferentially targeted. To address the correlation between HIV-1-specific CTL responses and virus replication in a cohort of former plasma donors (FPDs), 143 antiretroviral therapy naive FPDs infected with HIV-1 clade B' strains were assessed for HIV-1-specific CTL responses with an IFN-γ Elispot assay at single peptide level by using overlapping peptides (OLPs) covering the whole consensus clade B proteome. By using a Spearman's rank correlation analysis, we found that the proportion of Gag-specific CTL responses among the total virus-specific CTL activity was inversely correlated with viral loads while being positively correlated to CD4 counts, as opposed to Pol- and Env-specific responses that were associated with increased viral loads and decreased CD4 counts. In addition, Vpr-specifc CTL responses showed a similar protective effect with Gag responses, but with a much lower frequency of recognition. Significantly, we also observed an association between HLA-A*30/B*13/Cw*06 haplotype and lower viral loads that was probably due to restricted Gag-specific CTL responses. Thus, our data demonstrate the prominent role of Gag-specific CTL responses in disease control. The advantage of HLA-A*30/B*13/Cw*06 haplotype in viral control may be associated with the contribution of Gag-specific CTL responses in the studied individuals.
Cell Research 01/2012; 22(5):903-14. · 8.19 Impact Factor
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ABSTRACT: Attempts were made to evaluate methods measuring the capsid integrity and/or functions of noroviruses (NoVs) following heat treatment. Intact viruses (Murine Norovirus-1 [MNV-1] and human NoV GII.4), virus like particles (VLPs) and P particles (expressed in vitro from the protruding domain of the viral capsid) of NoVs were used in this study. Following heat treatment, no significant difference of viral titer of MNV-1 versus NoV GII.4 was observed by RNase One RT-PCR or cell-binding RT-PCR, although cell-binding RT-PCR (to measure the capsid functions) revealed higher reductions than RNase One RT-PCR (to measure the capsid integrity). These results indicate that the function assay for receptor binding is more sensitive than the capsid integrity assay to measure the protected viral RNA. MNV-1 could be used as a surrogate for human NoVs by heat inactivation from the perspective of capsid integrity and/or functions. The heat resistance varied among different GI and GII NoV strains when their P particles were studied.
Journal of virological methods 01/2012; 181(1):1-5. · 2.13 Impact Factor
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ABSTRACT: To construct dual fusion reporter gene expression vector containing enhanced green fluorescence protein (EGFP) and human transferrin receptor (TfR), and validate the reconstructed plasmid, which will provide experimental foundation for in vivo dual-modality optical/Magnetic Resonance (MR) imaging.
Clone TfR into the pEGFP-C1 vector to construct pEGFP-C1-TfR plasmid.pEGFP-C1-TfR plasmid was transfected into 293T cells for 48 h, then investigate EGFP expression under a fluorescence microscope; detect TfR expression through PT-PCR; inspect the subcellular location of EGFP-TfR fusion protein through Confocal Scanning Laser Microscopy; evaluate the function of EGFP-TfR fusion protein through Tf probe uptake and competition assays.
DNA sequencing analysis confirmed that EGFP-TfR gene sequence was correct, and there was no mutation and deletion. After transfecting the reconstructed plasmid into 293T cells, fluorescence microscope observation and RT-PCR results demonstrated that EGFP and TfR were expressed efficiently. EGFP-TfR fusion protein was located predominantly in the cellular membrane, and could specifically mediate internalization of Tf.
EGFP-TfR dual fusion reporter gene expression vector has been successfully constructed, and could be expressed efficiently with functional features. Thus, the expression vector could be applied for in vivo dual-modality optical/Magnetic Resonance (MR) imaging.
Zhonghua yi xue za zhi 12/2011; 91(47):3363-6.
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ABSTRACT: To create far-red fluorescence protein reporter gene mKate2 lentivirus, label human liver cancer cell line HepG2 with lentivirus and explore the feasibility of in vitro fluorescence imaging of labeled tumor cells so as to provide experimental rationales for in vivo fluorescence tumor imaging.
mKate2 gene was amplified from pmKate2-N plasmid. Then the fragment was inserted into the lentivirus expression vector pLenti6.3/V5-DEST. The expression plasmids pLenti6.3-mKate2 and the packaging plasmids were cotransfected into 293T cells. The biological titer of lentivirus was determined. HepG2 cells were infected with mKate2 lentivirus at a MOI (virus multiplicity of infection) of 6 for 96 hours. The infection efficiency was assayed through fluorescence microscope and fluorescent-activated cell scanning (FACS). And 2 × 10(6) mKate2-HepG2 cells were collected for fluorescence imaging through an optical imaging system. And the optimal imaging parameters were determined.
DNA sequencing analysis confirmed that mKate2 gene sequence was correct and there was no mutation or deletion. The biological titer of produced mKate2 lentivirus was 1.6 × 10(6) TU/ml. At 96 hours after mKate2 lentivirus infection, fluorescence microscope showed that mKate2 was expressed in a large percentage of cells. FACS assay showed that the mKate2 positive rate was 93.8% ± 0.4%. Excitation light 530 ± 15 nm and emission light 710 ± 28 nm were the optimal imaging parameters for mKate2-HepG2 cells.
Lentivirus can mediate efficiently the mKate2 reporter gene labeling of human liver cancer cell line HepG2. The mKate2-labeled HepG2 cells can be detected through in vitro fluorescence imaging. Further tracing studies of in vivo tumor fluorescence imaging are technically feasible.
Zhonghua yi xue za zhi 05/2011; 91(19):1344-7.
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ABSTRACT: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines and has been shown to induce cell apoptosis in many types of tumors, but not in normal cells. This tumor-selective property has made TRAIL a promising approach for the development of cancer therapy. However, hepatocellular carcinoma (HCC) cells display a striking resistance to TRAIL. Although some chemotherapeutic agents can overcome this resistance, safety issues remain a concern because the combination of these agents and TRAIL has been reported to induce toxicity in normal hepatocytes. In this study, we examined whether cisplatin could reverse TRAIL resistance in HCC cells with different p53 status and evaluated the toxicity of combination TRAIL and cisplatin to normal hepatocytes and mesenchymal stem cells (MSCs). We observed that cisplatin could efficiently sensitize HCC cells, but not hepatocytes and MSCs to TRAIL-induced apoptosis within a wide therapeutic window. The apoptosis of HCC cells only partially depended on the upregulation of DR5 and the status of p53. In addition, we provide favorable evidence supporting the feasibility of the combination of chemotherapy and MSCs transduced with TRAIL.
Oncology Reports 02/2011; 25(2):461-8. · 1.84 Impact Factor
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ABSTRACT: To explore the effect of enhanced green fluorescence protein (EGFP) labeling mediated by lentivirus on the biophysical properties of mesenchymal stem cells (MSC), and whether the EGFP gene expression is permanent and stable.
MSC were infected with EGFP lentivirus at different virus multiplicity of infection (MOI). EGFP positive rate was measured with fluorescent-activated cell scanning (FACS) analysis, and EGFP expression in MSC was investigated under a fluorescence microscope. Cell viability, proliferation, apoptosis and cell cycle were detected with trypan blue stain, MTT colorimetric assay, Hoechst stain and FACS analysis respectively. To evaluate the stability of EGFP expression, EGFP lentivirus infected MSC were harvested after cultured continuously in vitro for 2, 4, 8 or 16 weeks, and EGFP positive rate and fluorescence strength were detected with FACS analysis.
After infected with EGFP lentivirus (MOI = 20) for 96 h, EGFP positive rate of MSC was 97.39% +/- 0.68%. Cell viability, proliferation, apoptosis and cell cycle of MSC infected with EGFP lentivirus were unaffected, as compared with control MSC (P > 0.05). When cultured in vitro continuously for 2, 4, 8 or 16 weeks, EGFP positive rates of EGFP-MSC were 97.50% +/- 0.54%, 97.32% +/- 0.51%, 97.39% +/- 0.11%, and 97.48% +/- 0.13% respectively, while EGFP fluorescence strength were 440 +/- 13, 445 +/- 12, 458 +/- 13 and 456 +/- 16 respectively. Both EGFP positive rate and fluorescence strength kept in a stable level.
EGFP lentivirus can efficiently label MSC and has no significant effect on the biophysical properties of MSC. EGFP gene expression in MSC is permanent and stable. EGFP-MSC can be used for further cell tracing research.
Zhonghua yi xue za zhi 05/2010; 90(19):1357-61.
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ABSTRACT: In vivo magnetic resonance (MR) tracking of magnetically labeled bone marrow mesenchymal stem cells (BMSCs) administered via the mesenteric vein to rats with liver fibrosis.
Rat BMSCs were labeled with superparamagnetic iron oxide (SPIO) and the characteristics of the BMSCs after labeling were investigated. Eighteen rats with CCL4-induced liver fibrosis were randomized to three groups to receive SPIO-labeled BMSCs (BMSC-labeled group), cell-free SPIO (SPIO group), or unlabeled BMSCs (control group). MR imaging of the liver was performed at different time points, and signal-to-noise ratio (SNR) of the liver was measured. In vivo distribution of delivered BMSCs was assessed by histological analysis.
Labeling of BMSCs with SPIO did not significantly alter cell viability and proliferation activity. In BMSC-labeled group, the liver SNR immediately decreased from 8.56+/-0.26 to 3.53+/-0.41 at 1 h post injection and remained at a significantly lower level till 12 days (P<.05 versus the level before). By contrast, the liver SNR of the SPIO group almost recovered to the preinjection level (P=.125) at 3 days after a transient decrease. In control group, the liver SNR demonstrated no significant difference at the tested time points. Additionally, Prussian blue-positive cells were mainly distributed in the liver parenchyma, especially in injured areas.
The magnetically labeled BMSCs infused through the mesenteric vein can be detected in the fibrotic liver of rats using in vivo MR imaging up to 12 days after injection.
Magnetic Resonance Imaging 04/2010; 28(3):394-9. · 1.99 Impact Factor
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ABSTRACT: To evaluate the dual-labeling efficiency of magnetic and luminescent quantum dots bifunctional nanoparticles to rat bone mesenchymal stem cells (BMSC).
Rat BMSC were isolated, purified, and expanded. Magnetic/luminescent bifunctional nanoparticles (Fe(3)O(4)@CdTe@SiO(2)), were prepared by using silicon dioxide (SiO(2)) to encapsulate simultaneously Fe(3)O(4) and CdTe quantum dots. BMSC were incubated with the Fe(3)O(4)@CdTe@SiO(2) nanoparticles which iron concentration was 25 microg/ml. Fluorescence microscope was used to detect the fluorescence of the intracellular nanoparticles. The dual-labeled BMSC with various concentration underwent ex vivo MR imaging with T(1)WI, T(2)WI and T(2)(*)WI sequences. To show the intracellular iron of labeled cells, prussian blue stain was performed. Spectrophotometer was used to detect the iron concentration in the cells.
Intracellular red fluorescence of Fe(3)O(4)@CdTe@SiO(2) can be observed via fluorescent microscopy. Rat BMSC could be effectively dual-labeled with approximately 90% efficiency. The MR images with T(2)WI and T(2)(*)WI sequences, especially with T(2)(*)WI sequence, showed that the signals of the dual-labeled BMSC were lower than those of the unlabeled cells. Cellular total iron is 14.05 + or - 4.15 pg per cell. Iron containing intracytoplasmic vesicles could be observed with Prussian blue staining.
Rat BMSC can be dual-labeled successfully with Fe(3)O(4)@CdTe@SiO(2) magnetic/luminescent bifunctional nanoparticles successfully, and might serve as a tool for magnetic resonance imaging and in vivo optical imaging.
Zhonghua yi xue za zhi 01/2010; 90(1):56-60.
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ABSTRACT: This communication describes a facile but effective method to prepare graphene film electrodes with tunable dimensions with Vaseline as the insulating binder. Cyclic voltammetry (CV) studies reveal that the as-prepared graphene film electrodes have tunable dimensions ranging from a conventional electrode to a nanoelectrode ensemble, depending on the amount of graphene dispersed into the insulting Vaseline matrix. A large amount of graphene (typically, 10.0μg/mL) leads to the formation of the film electrodes with a conventional dimension, while a small amount of graphene (typically, 1.0μg/mL) essentially yields the graphene film electrodes like a nanoelectrode ensemble. As one new kind of carbon-based film electrodes with tailor-made dimensions and a good electrochemical activity as well as a high stability, the graphene film electrodes are believed to be potentially useful for fundamental electrochemical studies and for practical applications.
Electrochemistry Communications - ELECTROCHEM COMMUN. 01/2009; 11(10):1912-1915.
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CrystEngComm 01/2008; 10(8). · 3.84 Impact Factor
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CrystEngComm 01/2008; 10(5). · 3.84 Impact Factor
