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Parallel single-cell analysis of active caspase-3/7 in apoptotic and non-apoptotic cells
Ledvina, Vojtěch ; Klepárník, Karel
Caspases are proteases that play key role in the process of apoptosis, the programmed\ncell death. Among them, caspase-3 and -7 are main executioner caspases that cleave\nmany vital proteins during apoptosis and after their widespread activation, the process\ncannot be reversed. To analyze caspase-3/7 activation within single cells, a miniaturized\ndevice for parallel analysis of eight samples was developed. The assay is based on the\nmodified luciferin-firefly luciferase bioluminescence (BL) system. Individual\nsuspended cells were collected and transferred into detection microvials using a\nmicromanipulator. The bioluminescence was detected using a photon counting head\nwith cooled photcathode. The LOD suitable for detection of active caspase-3/7 in both\napoptotic and non-apoptotic cells was reached.
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Development of microfluidic device for droplet generation and microparticle encapsulation
Křivánková, Jana ; Foret, František
Microfluidic devices combined with various bioanalytical and optical sensors proved\ntheir potential in chemical, biological, and biomedicine applications. At a droplet\nmicrofluidic device a fluid is divided into numerous droplets surrounded by a\ncontinuous immiscible fluid phase. Generated droplets serve then as\nvessels/microcapsules for delivering drugs, nutrients as well as for encapsulating of\nbiologically active particles and cells.\nDescribed experimental study deals with the development of a practical dropletbased\nmicrofluidic chip useful for production of monodisperse water/oil emulsion and\nfor encapsulation of thin-metal magnetic microsheets.
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CE-nESI/MS in electrode-free design: narrowing the separation channel to 5 μm
Týčová, Anna ; Foret, František
Capillary electrophoresis-nanospray/mass spectrometry (CE-nESI/MS) in electrodefree\narrangement represents a very simple instrumentation for analysis of biomolecules.\nSo far, CE-nESI/MS analyses in this design were limited to 10-75 μm ID of the\nseparation channel. Although the work with narrower dimensions brings several\nchallenges, there is a great potential for better sensitivity, improved separation power\nand lower sample consumption. This work is devoted to some practical aspects of\nexperiments in narrow bore channels and systematic evaluation of CE-nESI/MS\nanalyses conducted in capillaries of 25, 15 and 5 μm ID.
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Characterization of FRET sensor
Datinská, Vladimíra ; Klepárník, Karel ; Belšánová, B. ; Minárik, M. ; Foret, František
In this study, we present characterization of sensor based on Fӧrster resonance energy\ntransfer (FRET). The sensor is composed of ssDNA chain attached to a laboratory\nsynthesized quantum dot (QD). A complementary chain of a sample is labeled by a\nluminescent dye. When the dsDNA hybrid is formed, the energy from the QD (donor)\nis transferred to the dye (acceptor) and FRET is observed as a decrease of QD\nluminescence emission intensity and an increase of dye luminescence emission\nintensity.
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A novel nanospray liquid junction interface for versatile CE-MS
Křenková, Jana ; Haselberg, R. ; Somsen, G. W. ; Foret, František
In this work we present a new polyimide-based liquid junction nanospray interface, combining the advantages of sheath flow and sheathless designs. The liquid junction interface was prepared as a hybrid microfabricated plastic liquid junction sprayer allowing to perform analysis in standard (30-100 micro m ID) separation capillaries. The interface incorporates a self-aligning port for coupling of the separation capillary as well as ports for automated flushing of both the liquid junction and the separation capillary. The interface permits performing CE both with and without electroosmotic flow. The use of large bore capillaries allows injection and preconcentration of larger sample volumes, favoring detection sensitivity. Different compositions of the spray liquid and BGE can be used when needed.
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