Mikroraménka, používaná jako flexibilní nosič hrotu u AFM skenovacích technik, jsou představena jako nanomechanický převodník vhodný pro konstrukci biosensorů. Pro afinitní reakce se využívá ohyb raménka v důsledku změny povrchového napětí po vazbě komplementárních biomolekul na jedné straně nesoucí ligand; postup je demonstrován na vazbě protilátky na protein A. Jinou možností je přímé kontaktní snímání pohybu biologických systémů. Tento přístup byl velmi efektivní ve spojení s tepajícímí kardiomyocyty pro vývoj buněčného biosensoru vhodného pro testování látek ovlivňujících srdeční činnost.

Microcantilevers, used as flexible support for the scanning tip in AFM techniques, are introduced as nanomechanical transducer suitable for construction of biosensors. For affinity interactions, the deflection of the cantilever due to the change of surface stress following the binding of complementary biomolecules on one side with immobilised ligand; this approach is demonstrated on the interaction of antibody with protein A. An alternative application is the direct contact monitoring of mechanically active biological systems. This method was quite effectively linked with beating cardiomyocytes in order to develop cellular biosensor suitable for testing of drugs modifying heart activity.

• Klíčová slova
• mikromechanický biosenzor, afinitní reakce,
• MeSH
• biomechanika MeSH
• biosenzitivní techniky * MeSH
• kardiomyocyty MeSH
• mikroskopie atomárních sil * využití   MeSH
• nanotechnologie * MeSH

Cell volume and its regulation are key factors for cellular integrity and also serve as indicators of various cell pathologies. SPR sensors represent an efficient tool for real-time and label-free observations of changes in cell volume and shape. Here, we extend this concept by employing the use of long-range surface plasmons (LRSP). Due to the enhanced penetration depth of LRSP (~1μm, compared to ~0.4μm of a conventional surface plasmon), the observation of refractive index changes occurring deeper inside the cells is possible. In this work, the responses of a confluent normal rat kidney (NRK) epithelial cell layer to osmotic stress are studied by both conventional and long-range surface plasmons. Experiments are conducted in parallel using cell layers grown and stimulated under the same conditions to enable direct comparison of the results and discrimination of the osmotic stress-induced effects in different parts of the cell.

• MeSH
• analýza selhání vybavení MeSH
• barvení a značení MeSH
• biosenzitivní techniky přístrojové vybavení   MeSH
• buněčné linie MeSH
• design vybavení MeSH
• krysa rodu rattus MeSH
• ledviny cytologie   fyziologie   MeSH
• osmotický tlak MeSH
• počítačové systémy MeSH
• povrchová plasmonová rezonance přístrojové vybavení   MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• velikost buňky MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

DNA methylation, i.e., addition of methyl group to 5'-carbon of cytosine residues in CpG dinucleotides, is an important epigenetic modification regulating gene expression, and thus implied in many cellular processes. Deregulation of DNA methylation is strongly associated with onset of various diseases, including cancer. Here, we review how DNA methylation affects carcinogenesis process and give examples of solid tumors where aberrant DNA methylation is often present. We explain principles of methods developed for DNA methylation analysis at both single gene and whole genome level, based on (i) sodium bisulfite conversion, (ii) methylation-sensitive restriction enzymes, and (iii) interactions of 5-methylcytosine (5mC) with methyl-binding proteins or antibodies against 5mC. In addition to standard methods, we describe recent advances in next generation sequencing technologies applied to DNA methylation analysis, as well as in development of biosensors that represent their cheaper and faster alternatives. Most importantly, we highlight not only advantages, but also disadvantages and challenges of each method.

• MeSH
• 5-methylcytosin metabolismus   MeSH
• biosenzitivní techniky metody   MeSH
• epigeneze genetická genetika   MeSH
• lidé MeSH
• metylace DNA genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

The piezoelectric sensor (quartz crystal microbalance, QCM) was used to monitor cell adhesion in real time. Two cell lines, rat epithelial cells (WB F344) and lung melanoma cells (B16F10) were used. The cells were adhered and grown on the gold surface of the sensor pre-coated with adsorbed layer of extracellular matrix proteins as vitronectin and laminin. The process of cell attachment and spreading on the gold surface was continuously monitored and displayed by changes of the resonant frequency Deltaf and resistance DeltaR values of the piezoelectric resonators. The initial phase of cell attachment and spreading induced a decrease of frequency and increase of resistance relating viscoelastic properties of the cell monolayer on the sensing surface. The steady-state of both shifts was achieved after a few hours. The presence and state of cells on the surface was confirmed by fluorescent microscopy. The obtained results demonstrate that the piezoelectric sensor is suitable for studies of the cell adhesion processes. Thus obtained cell-based biosensor has potential for identification and screening of biologically active drugs and other biomolecules affecting cellular shape and attachment.

• MeSH
• biosenzitivní techniky přístrojové vybavení   MeSH
• buněčná adheze fyziologie   MeSH
• buněčné linie MeSH
• financování organizované MeSH
• krysa rodu rattus MeSH
• nádorové buněčné linie MeSH
• vitronektin MeSH
• zlato MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH

Src kinase plays an important role in a multitude of fundamental cellular processes and is often found deregulated in tumors. Active Src adopts an open conformation, whereas inactive Src is characterized by a very compact structure stabilized by inhibitory intramolecular interactions. Taking advantage of this spatial regulation, we constructed a fluorescence resonance energy transfer (FRET)-based Src biosensor and analyzed conformational changes of Src following Src activation and the spatiotemporal dynamics of Src activity in cells. We found that activatory mutations either in regulatory or kinase domains induce opening of the Src structure. Surprisingly, we discovered that Src inhibitors differ in their effect on the Src structure, some counterintuitively inducing an open conformation. Finally, we analyzed the dynamics of Src activity in focal adhesions by FRET imaging and found that Src is rapidly activated during focal adhesion assembly, and its activity remains steady and high throughout the life cycle of focal adhesion and decreases during focal adhesion disassembly.

• MeSH
• biosenzitivní techniky metody   MeSH
• fokální adheze metabolismus   MeSH
• FRAP MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mutageneze MeSH
• rezonanční přenos fluorescenční energie MeSH
• skupina kinas odvozených od src-genu antagonisté a inhibitory   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Although phosphatidic acid (PA) is structurally the simplest membrane phospholipid, it has been implicated in the regulation of many cellular events, including cytoskeletal dynamics, membrane trafficking and stress responses. Plant PA shows rapid turnover but the information about its spatio-temporal distribution in plant cells is missing. Here we demonstrate the use of a lipid biosensor that enables us to monitor PA dynamics in plant cells. The biosensor consists of a PA-binding domain of yeast SNARE Spo20p fused to fluorescent proteins. Live-cell imaging of PA dynamics in transiently transformed tobacco (Nicotiana tabacum) pollen tubes was performed using confocal laser scanning microscopy. In growing pollen tubes, PA shows distinct annulus-like fluorescence pattern in the plasma membrane behind the extreme tip. Coexpression studies with markers for other plasmalemma signaling lipids phosphatidylinositol 4,5-bisphosphate and diacylglycerol revealed limited colocalization at the shoulders of the apex. PA distribution and concentrations show distinct responses to various lipid signaling inhibitors. Fluorescence recovery after photobleaching (FRAP) analysis suggests high PA turnover in the plasma membrane. Our data show that a biosensor based on the Spo20p-PA binding domain is suitable for live-cell imaging of PA also in plant cells. In tobacco pollen tubes, distinct subapical PA maximum corroborates its involvement in the regulation of endocytosis and actin dynamics.

• MeSH
• biosenzitivní techniky metody   MeSH
• buněčná membrána chemie   metabolismus   MeSH
• diglyceridy metabolismus   MeSH
• fluorescence MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• fosfolipasa D metabolismus   MeSH
• fotovybělování MeSH
• kyseliny fosfatidové analýza   metabolismus   MeSH
• počítačové zpracování obrazu MeSH
• proteiny Qb-SNARE genetika   metabolismus   MeSH
• proteiny Qc-SNARE genetika   metabolismus   MeSH
• pylová láčka genetika   růst a vývoj   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• tabák cytologie   metabolismus   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Neopterin is a valuable biomarker of cellular immunity associated with various pathological situations such as viral and bacterial infections, autoimmune, cardiovascular, neurodegenerative and malignant disorders. To produce specific antibodies against neopterin for a rapid multi-biomarker-based diagnosis, a novel hapten derivative was synthesized and attached to carrier proteins. The thoroughly characterized conjugates were used for immunization of BALB/c mice and rabbits. The produced monoclonal antibody reached in both direct and indirect enzyme-linked immunosorbent assay (ELISA) format LoD of 0.18 and 0.45 μg L(-1), respectively, and was a superior immunoreagent for further biosensor developments with regard to assay sensitivity and material availability. The best polyclonal antibody was somewhat more sensitive in direct ELISA with LoD of 0.05 μg L(-1). The optimized ELISA method was evaluated with blood samples collected from patients with renal insufficiency, patients with sepsis, patients without confirmed clinical diagnosis, and healthy volunteers. In plasma samples, neopterin concentrations ranging from 3.2 to 103 μg L(-1) could be determined with the monoclonal ELISA whereas twofold lower results were obtained with the polyclonal ELISA. A satisfactory correlation of results was found between the polyclonal ELISA and IBL Neopterin ELISA kit within the concentration range 0.5-16 μg L(-1) (R = 0.874; n = 40), and slightly lower correlation was found for monoclonal-based ELISA (R = 0.819; n = 40). These data show that the generated antibodies may be used as functional analytical reagents for the integration into multianalyte biochip detection systems.

• MeSH
• biosenzitivní techniky metody   MeSH
• ELISA metody   MeSH
• hapteny chemie   imunologie   MeSH
• králíci MeSH
• lidé MeSH
• monoklonální protilátky imunologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• neopterin krev   imunologie   MeSH
• protilátky imunologie   MeSH
• renální insuficience krev   MeSH
• senzitivita a specificita MeSH
• sepse krev   MeSH
• specificita protilátek MeSH
• tvorba protilátek MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH

Cardiomyocyte contraction and relaxation are important parameters of cardiac function altered in many heart pathologies. Biosensing of these parameters represents an important tool in drug development and disease modeling. Human embryonic stem cells and especially patient specific induced pluripotent stem cell-derived cardiomyocytes are well established as cardiac disease model.. Here, a live stem cell derived embryoid body (EB) based cardiac cell syncytium served as a biorecognition element coupled to the microcantilever probe from atomic force microscope thus providing reliable micromechanical cellular biosensor suitable for whole-day testing. The biosensor was optimized regarding the type of cantilever, temperature and exchange of media; in combination with standardized protocol, it allowed testing of compounds and conditions affecting the biomechanical properties of EB. The studied effectors included calcium , drugs modulating the catecholaminergic fight-or-flight stress response such as the beta-adrenergic blocker metoprolol and the beta-adrenergic agonist isoproterenol. Arrhythmogenic effects were studied using caffeine. Furthermore, with EBs originating from patient's stem cells, this biosensor can help to characterize heart diseases such as dystrophies.

• MeSH
• agonisté adrenergních beta-receptorů farmakologie   MeSH
• antagonisté beta-1-adrenergních receptorů farmakologie   MeSH
• biomechanika účinky léků   MeSH
• biosenzitivní techniky přístrojové vybavení   metody   MeSH
• buněčné kultury přístrojové vybavení   metody   MeSH
• buněčné linie MeSH
• design vybavení MeSH
• isoprenalin farmakologie   MeSH
• kardiomyocyty cytologie   účinky léků   metabolismus   MeSH
• kontrakce myokardu účinky léků   MeSH
• lidé MeSH
• metoprolol farmakologie   MeSH
• mikroskopie atomárních sil přístrojové vybavení   metody   MeSH
• pluripotentní kmenové buňky cytologie   MeSH
• preklinické hodnocení léčiv přístrojové vybavení   metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

A novel and facile in vitro cell sensing system has been developed with one-step electropolymerization of the conducting polypyrrole(PPy) polymer using RGD peptide as the sole dopant on an indium tin oxide (ITO) surface. The resulted RGD peptide-doped polypyrrole (PPy/RGD) composite film had a robust surface, in which PPy provided a biocompatible matrix for cell growth and a conducting interface for electrical detection, while the RGD peptide entrapped in the PPy matrix conferred the desired biomimetic properties. Using the human lung cancer cell A549 as a model, this system can be used to monitor cell behaviors of proliferation and cytotoxicity.

• Klíčová slova
• RGD peptidy, elektropolymerizace, polypyrrole,
• MeSH
• biomimetické materiály MeSH
• biosenzitivní techniky * MeSH
• oligopeptidy MeSH
• polymery MeSH
• proliferace buněk MeSH
• protinádorové látky toxicita   MeSH
• pyrroly * MeSH
• skenovací elektrochemická mikroskopie MeSH
• Publikační typ
• práce podpořená grantem MeSH

Guanine-rich sequences of DNA are able to create tetrastranded structures known as G-quadruplexes; they are formed by the stacking of planar G-quartets composed of four guanines paired by Hoogsteen hydrogen bonding. G-quadruplexes act as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplexes form a complex with anionic porphyrin hemin and exhibit peroxidase-like activity. This review focuses on overview of sensing techniques based on G-quadruplex complexes with anionic porphyrins for detection of various analytes, including metal ions such as K+, Ca2+, Ag+, Hg2+, Cu2+, Pb2+, Sr2+, organic molecules, nucleic acids, and proteins. Principles of G-quadruplex-based detection methods involve DNA conformational change caused by the presence of analyte which leads to a decrease or an increase in peroxidase activity, fluorescence, or electrochemical signal of the used probe. The advantages of various detection techniques are also discussed.

• MeSH
• biosenzitivní techniky * MeSH
• delece genu MeSH
• DNA katalytická chemie   MeSH
• DNA chemie   MeSH
• G-kvadruplexy * MeSH
• ionty analýza   chemie   MeSH
• kovy analýza   chemie   MeSH
• lidé MeSH
• nádorový supresorový protein p53 chemie   genetika   MeSH
• nanočástice chemie   MeSH
• nukleové kyseliny analýza   chemie   MeSH
• organické látky analýza   chemie   MeSH
• proteiny analýza   chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH