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Simple route of caspase-3 FRET sensor synthesis using “click chemistry”
Lišková, Marcela ; Křenková, Jana ; Klepárník, Karel ; Pazdera, P. ; Foret, František
Programmed cell death or apoptosis is regulated process of cell suicide. The central role in apoptosis play cysteine proteases called caspases. Caspases recognize tetra-peptide sequences Asp-Glu-Val-Asp (DEVD) on their substrates and hydrolyze peptide bonds after aspartic acid residues. Various techniques for the determination of caspase-3 are commercially available e.g. Enzyme Linked Immuno-Sorbent Assay (ELISA), Western blotting or flow cytometric analysis. The products of the cleavage can be detected by spectrophotometry, fluorimetry, chemiluminescence (CL) or ELISA. In this work, we suggested fluorescent sensor based on easily prepared Förster Resonance Energy Transfer (FRET). We use very simple chemistry called “click”. This type of chemistry takes advantages of quickness, simplicity and cheapness. “Click chemistry” is based on usage of various functional group and cross-linkers to combine individual molecules together.
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Multi-step synthesis of caspase-3 sensor based on Förster resonance energy transfer
Lišková, Marcela ; Klepárník, Karel ; Pazdera, P. ; Foret, František
Programmed cell death or apoptosis is regulated process of cell suicide. The central role in apoptosis play cysteine proteases called caspases. Caspases recognize tetra-peptide sequences Asp-Glu-Val-Asp (DEVD) on their substrates and hydrolyze peptide bonds after aspartic acid residues. Various techniques for the determination of caspase-3 are commercially available e.g. Enzyme Linked Immuno-Sorbent Assay (ELISA), Western blotting or flow cytometric analysis. The products of the cleavage can be detected by spectrophotometry, fluorimetry, chemiluminescence (CL) or ELISA. In this work, we suggested fluorescent sensor based on Förster Resonance Energy Transfer (FRET) to determine caspase-3 in cell nucleus or cytoplasm by apoptosis for very fast analysis by fluorescence microscopy without sample destroying.
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Sensor based on Förster resonance energy transfer with quantum dot
Lišková, Marcela ; Datinská, Vladimíra ; Klepárník, Karel ; Foret, František
Förster resonance energy transfers (FRET) are based on the nonradiative transfer of energy between a donor and acceptor dye in the distance 1-10 nm. With respect to this, quantum dots (QDs) with a high absorbance can serve as suitable donors of energy in FRET. Here, we present architectures of two sensors based on QD conjugates useful in imaging and diagnostics of cancer. One sensor, based on an oligonucleotide conjugate, can be used for the detection of complementary oligonucletide chains. The other was designed for the detection of enzymes inside cells.
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