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1.
Lab Chip ; 9(11): 1601-8, 2009 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-19458869

RESUMO

The interactions between charged molecules and channel surfaces are expected to significantly influence the electrokinetic transport of molecules and their separations in nanochannels. This study reports the effect of wall-molecule interactions on flow control of negatively charged Alexa 488 and positively charged Rhodamine B dye molecules in an array of nanochannels (100 nm wx 500 nm dx 14 mm l) embedded in fluidic field effect transistors (FETs). For FET flow control, a third electrical potential, known as a gate bias, is applied to the channel walls to manipulate their zeta-potential. Electroosmotic flow of charged dye molecules is accelerated or reversed according to the polarity and magnitude of the gate bias. During FET flow control, we monitor how the electrostatic interaction between charged dye molecules and channel walls affects the apparent velocity of molecules, using laser-scanning confocal fluorescence microscopy. We observe that the changes in flow speed and direction of negatively charged Alexa 488 is much more pronounced than that of positively charged Rhodamine B in response to the gate bias that causes either repulsive or attractive electrostatic interactions. This observation is supported by calculations of concentration-weighted velocity profiles of the two dye molecules during FET flow control. The velocity profile of negatively charged Alexa 488 is much more pronounced at the center of each nanochannel than near its walls since Alexa 488 molecules are repelled from negatively charged channel walls. This pronounced center velocity further responds to the gate bias, increasing the average velocity by as much as 23% when -30 V is applied to the gate (zeta-potential = -80.6 mV). In contrast, the velocity profile of positively charged Rhodamine B is dispersed over the entire channel width due to dye-wall attraction and adsorption. Our experimental observations and calculations support the hypothesis that valence-charge-dependent electrostatic interaction and its manipulation by the gate bias would enhance molecular separations of differentially charged molecules in nanofluidic FETs.


Assuntos
Eletricidade , Eletro-Osmose/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Adsorção , Corantes Fluorescentes/química , Microscopia Confocal , Propriedades de Superfície , Transistores Eletrônicos
2.
Lab Chip ; 9(11): 1609-17, 2009 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-19458870

RESUMO

We have fabricated multiple-internal-reflection Si infrared waveguides integrated with an array of nanochannels sealed with an optically transparent top cover. The channel walls consist of a thin layer of SiO2 for electrical insulation, and gate electrodes surround the channel sidewalls and bottom to manipulate their surface charge and zeta-potential in a fluidic field effect transistor (FET) configuration. This nanofluidic device is used to probe the transport of charged molecules (Alexa 488) and to measure the pH shift in nanochannels in response to an electrical potential applied to the gate. During gate biasing for FET operation, laser-scanning confocal fluorescence microscopy (LS-CFM) is used to visualize the flow of fluorescent dye molecules (Alexa 488), and multiple internal reflection-Fourier transform infrared spectroscopy (MIR-FTIRS) is used to probe the characteristic vibrational modes of fluorescein pH indicator and measure the pH shift. The electroosmotic flow of Alexa 488 is accelerated in response to a negative gate bias, whereas its flow direction is reversed in response to a positive gate bias. We also measure that the pH of buffered electrolyte solutions shifts by as much as a pH unit upon applying the gate bias. With prolonged application of gate bias, however, we observe that the initial response in flow speed and direction as well as pH shift becomes reversed. We attribute these anomalous flow and pH shift characteristics to a leakage current that flows from the Si gate through the thermally grown SiO2 to the electrolyte solution.


Assuntos
Eletricidade , Técnicas Analíticas Microfluídicas/instrumentação , Nanotecnologia/instrumentação , Eletro-Osmose/instrumentação , Corantes Fluorescentes/análise , Corantes Fluorescentes/isolamento & purificação , Concentração de Íons de Hidrogênio , Microscopia Confocal , Proteínas/análise , Proteínas/isolamento & purificação , Dióxido de Silício/química , Transistores Eletrônicos
3.
Lab Chip ; 9(2): 219-31, 2009 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-19107277

RESUMO

Lab-on-a-chip (LOC) technology provides a powerful platform for simultaneous separation, purification, and identification of low concentration multicomponent mixtures. As the characteristic dimension of LOC devices decreases down to the nanoscale, the possibility of containing an entire lab on a single chip is becoming a reality. This research examines one of the unique physical characteristics of nanochannels, in which native pH shifts occur. As a result of the electrical double layer taking up a significant portion of a 100 nm wide nanochannel, electroneutrality no longer exists in the channel causing a radial pH gradient. This work describes experimentally observed pH shifts as a function of ionic strength using the fluorescent pH indicator 5-(and-6)-carboxy SNARF-1 and compares it to a model developed using Comsol Multiphysics. At low ionic strengths (approximately 3 mM) the mean pH shift is approximately 1 pH unit whereas at high ionic strengths (approximately 150 mM) the mean pH shift is reduced to 0.1 pH units. An independent analysis using fluorescein pH indicator is also presented supporting these findings. Two independent non-linear simulations coupling the Nernst-Planck equation describing transport in ionic solutions subjected to an electric field and Poisson's equation to describe the electric field as it relates to the charge distribution are solved using a finite element solver. In addition, the effects of chemical activities are considered in the simulations. The first numerical simulation is based on a surface zeta-potential which significantly underestimates the experimental results for most ionic strengths. A modified model assuming that SNARF and fluorescein molecules are able to diffuse into the hydrolyzed SiO2 phase, and in the case of the SNARF molecule, able to bind to neutral regions of the SiO2 phase agrees quantitatively with experimental results.


Assuntos
Benzopiranos/química , Análise em Microsséries/métodos , Naftóis/química , Rodaminas/química , Algoritmos , Fluoresceína/química , Concentração de Íons de Hidrogênio , Concentração Osmolar , Dióxido de Silício/química
4.
Mol Cell Biochem ; 319(1-2): 189-201, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18682896

RESUMO

The existence of several model systems with which to investigate a particular disease is advantageous for researchers. This is especially true for ovarian cancer, which, due to its complex and heterogeneous nature, inherently requires a large number of model systems. Here, we report a new ovarian serous adenocarcinoma cell line, designated YDOV-157, and characterized via post genomics and post proteomics. In this study, primary culture of tumor cells from ascites was performed and the cells were immortalized up to at least 60 passages in vitro. We studied the morphologies, cell proliferation, BRCA1/2 mutations, tumorigenesis capacity, and chemosensitivity of YDOV-157. Using a cDNA microarray, differentially expressed genes were identified and some of them were validated. Using proteomic analysis, we identified proteins that were differentially expressed in YDOV-157. The newly derived cell line, designated YDOV-157, grew as a monolayer and the doubling time was 102 h. When transplanted into nude mice, it initiated the formation of tumor masses with microscopic findings identical to those of the primary tumor. Chemosensitivity test showed that paclitaxel induced the highest chemosensitivity index. In microarray analysis, 2,520 probes were differently expressed, compared to human ovarian surface epithelial cells (HOSEs). In SYBR Green real-time PCR, the expression of E2F2 (P = 0.040) and CRABP2 genes (P = 0.030) was significantly higher in the ovarian cancer cell lines than in HOSEs. Furthermore, proteomic analysis showed that expression of 28 spots was significantly altered between YDOV-157 and HOSE. In conclusion, the newly derived YDOV-157 cell line may be an important research resource for studying cancer cell biology and should also be very useful for developing new strategies that inhibit cancer cell growth and progression.


Assuntos
Adenocarcinoma/metabolismo , Linhagem Celular Tumoral/metabolismo , Regulação Neoplásica da Expressão Gênica , Modelos Biológicos , Proteínas de Neoplasias/biossíntese , Neoplasias Ovarianas/metabolismo , Adenocarcinoma/patologia , Linhagem Celular Tumoral/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/patologia , Proteômica/métodos
5.
Lab Chip ; 8(2): 251-8, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18231663

RESUMO

Using Si as the substrate, we have fabricated multiple internal reflection infrared waveguides embedded with a parallel array of nanofluidic channels. The channel width is maintained substantially below the mid-infrared wavelength to minimize infrared scattering from the channel structure and to ensure total internal reflection at the channel bottom. A Pyrex slide is anodically bonded to the top of the waveguide to seal the nanochannels, while simultaneously enabling optical access in the visible range from the top. The Si channel bottom and sidewalls are thermally oxidized to provide an electrically insulating barrier, and the Si substrate surrounding the insulating SiO(2) layer is selectively doped to function as a gate. For fluidic field effect transistor (FET) control, a DC potential is applied to the gate to manipulate the surface charge on SiO(2) channel bottom and sidewalls and therefore their zeta-potential. Depending on the polarity and magnitude, the gate potential can accelerate, decelerate, or reverse the flow. Here, we demonstrate that this nanofluidic infrared waveguide can be used to monitor the FET flow control of charged, fluorescent dye molecules during electroosmosis by multiple internal reflection Fourier transform infrared spectroscopy. Laser scanning confocal fluorescence microscopy is simultaneously used to provide a comparison and verification of the IR analysis. Using the infrared technique, we probe the vibrational modes of dye molecules, as well as those of the solvent. The observed infrared absorbance accounts for the amount of dye molecules advancing or retracting in the nanochannels, as well as adsorbing to and desorbing from the channel bottom and sidewalls.


Assuntos
Corantes Fluorescentes/química , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Adsorção , Microscopia Confocal/instrumentação , Microscopia Confocal/métodos , Sensibilidade e Especificidade , Silício/química , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Propriedades de Superfície , Transistores Eletrônicos
6.
J Cancer Res Clin Oncol ; 134(8): 909-17, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18210151

RESUMO

PURPOSE: New diagnostic markers, other than squamous cell carcinoma (SCC) antigen, are needed for the detection of cervical cancer. Osteopontin (OPN) is a candidate frequently associated with several human malignancies. The purpose of this study was to evaluate the clinical significance of OPN expression as a diagnostic and prognostic biomarker for cervical cancer. METHODS: Immunohistochemical staining of tissue from 97 cervical cancer cases and 22 healthy subjects was performed in order to determine the source of elevated plasma OPN levels. In addition, plasma OPN levels of 81 patients with cervical cancer, 34 patients with carcinoma in situ (CIS) of the uterine cervix, and those of 283 healthy women were measured with a commercially available solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). The correlation between OPN levels and clinical features were examined and compared to SCC antigen levels in the cervical cancer cases. RESULTS: Immunohistochemical staining revealed OPN immunoreactivity in 67.0% (65/97) of cervical cancer tissues, and the immunostaining score in the cervical cancer tissue sections was 2.06 (95% CI, 1.70-2.42). There was no significant difference in immunostaining scores based on age, tumor size, and tumor stage, but higher scores (3.0< score 215.5 ng/ml) were also correlated with disease-free survival (P = 0.038). CONCLUSIONS: These results suggest that plasma OPN levels are potentially useful as a diagnostic and prognostic biomarker for cervical cancer.


Assuntos
Biomarcadores Tumorais/sangue , Osteopontina/biossíntese , Osteopontina/sangue , Neoplasias do Colo do Útero/sangue , Neoplasias do Colo do Útero/metabolismo , Intervalo Livre de Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Prognóstico , Sensibilidade e Especificidade , Neoplasias do Colo do Útero/mortalidade
7.
J Korean Med Sci ; 22(2): 283-9, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17449938

RESUMO

Minoxidil induces hair growth in male pattern baldness and prolongs the anagen phase. All-trans retinoic acid (ATRA) has been reported to act synergistically with minoxidil in vivo: they can enhance more dense hair regrowth than either compound alone. We evaluated the effect of minoxidil combined with ATRA on hair growth in vitro. The effect of co-treatment of minoxidil and ATRA on hair growth was studied in hair follicle organ culture. In cultured human dermal papilla cells (DPCs) and normal human epidermal keratinocytes, the expressions of Erk, Akt, Bcl-2, Bax, P53 and P21 were evaluated by immunoblot analysis. Minoxidil plus ATRA additively promoted hair growth in vitro, compared with minoxidil alone. In addition, minoxidil plus ATRA elevated phosphorylated Erk, phosphorylated Akt and the ratio of Bcl-2/Bax, but decreased the expressions of P53 and P21 more effectively than by minoxidil alone. Our results suggest that minoxidil plus ATRA would additively enhance hair growth by mediating dual functions: 1) the prolongation of cell survival by activating the Erk and Akt signaling pathways, and 2) the prevention of apoptosis of DPCs and epithelial cells by increasing the ratio of Bcl-2/Bax and downregulating the expressions of P53 and P21.


Assuntos
Cabelo/efeitos dos fármacos , Cabelo/crescimento & desenvolvimento , Minoxidil/administração & dosagem , Tretinoína/administração & dosagem , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Cabelo/citologia , Humanos , Ceratolíticos/administração & dosagem
8.
Photochem Photobiol ; 82(3): 645-50, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16478297

RESUMO

Thrombospondin 1 (TSP1) is an extracellular glycoprotein and a recognized inhibitor of angiogenesis. Recent studies have demonstrated that UV radiation induces an angiogenic switch, by which it alters the balance between pro- and anti-angiogenic factors in the skin. Here we describe the effects of acute UV exposure on TSP1 expression in human skin epidermis, primary keratinocytes and the epidermal cell line HaCaT. We found that protein and mRNA expressions of TSP1 are significantly reduced in human skin in vivo and in keratinocytes in vitro by a single UV exposure. In human skin and keratinocytes, UV exposure induced the phosphorylation of Akt, a downstream target of the PI3K pathways. Specific inhibitors of PI3K, wortmannin and LY294002, completely blocked Akt activation and UV-induced TSP1 downregulation in keratinocytes. We showed that a specific Akt phosphorylation inhibitor and small interfering RNA-mediated Akt depletion were also blocked by UV-induced TSP1 downregulation in keratinocytes. In conclusion, our findings demonstrate that acute UV exposure downregulates TSP1 expression via PI3K-Akt activation in human keratinocytes. These novel findings may help us understand the regulatory mechanisms of UV-induced skin angiogenesis.


Assuntos
Queratinócitos/metabolismo , Trombospondina 1/genética , Raios Ultravioleta , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/efeitos da radiação , Regulação para Baixo/genética , Regulação para Baixo/efeitos da radiação , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Trombospondina 1/efeitos da radiação
9.
Artigo em Coreano | WPRIM (Pacífico Ocidental) | ID: wpr-90750

RESUMO

OBJECTIVE: Ovarian cancer is the most lethal disease among gynecologic malignancies. Although many efforts have been made to explore the mechanisms involved in its development, the genetic events in the pathogenesis of ovarian cancer are still unclear. We characterized a cell line (designated OHK) established from a malignant Brenner tumor cell. METHODS: The cells were obtained during the operation of a 43-year-old Korean woman with ovarian cancer. The OHK cells continuously propagated in vitro over a period of about 36 months and, to date, have undergone over 200 passages, without being infected by either Mycoplasma or any bacteria. We measured the doubling time of OHK cells. To investigate the tumorigenecity of OHK, cells were inoculated subcutaneously into the back of nude mice. Several tumor markers were analyzed using culture media and lysates of cytosol. Morphology and ultrastructure were analyzed by phase-contrast microscopy and electron microscopy. OHK was also analyzed for gene mutation, the typing of human leukocyte antigen and Flow cytometric cell cycle analysis and DNA index. RESULTS: They proliferated in a monolayered sheet showing a pavement-like arrangement without suppression by intercellular contacts. They also formed epithelial cell lining in shapes of polymorphism and polygons. Doubling time was 38.4 hour which was relatively slow compared to other cancer cells. Microscopic view revealed intranuclear infoldings which are typical in malignant Brenner tumors. The OHK cells secreted significantly high level of CA 125 into the culture medium. A 215th codon at exon 4 of p53 was mutated to C/C in OHK. BRCA 1 was a wild type and polymorphisms were detected in exons 2, 10, 11, 14 and 17 of BRCA 2. The cells showed aneuploidy with DNA index of 1.589 measured by flow cytometry. When transplanted into nude mice, OHK cells successfully induced tumor which was histopathologically resembled malignant Brenner tumor. CONCLUSION: These results strongly suggest that OHK is a typical cell line of malignant Brenner tumor. This may provide a useful cellular resource for studying the pathogenesis of malignant Brenner tumor.


Assuntos
Adulto , Animais , Feminino , Humanos , Camundongos , Aneuploidia , Bactérias , Tumor de Brenner , Ciclo Celular , Linhagem Celular , Códon , Meios de Cultura , Citosol , DNA , Células Epiteliais , Éxons , Citometria de Fluxo , Leucócitos , Camundongos Nus , Microscopia Eletrônica , Microscopia de Contraste de Fase , Mycoplasma , Neoplasias Ovarianas , Biomarcadores Tumorais
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