Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.3.4.6 (urease)
7,490 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a nonisotopic RNase protection assay using RNA probes that are dual-labeled with biotin and fluorescein for detection. This system utilizes capture of the protected RNA probe hybrids to streptavidin-coated membranes attached to plastic dipsticks, complexing of anti-fluorescein-urease conjugate with the labeled RNA probe, and quantitative detection of the membrane-bound complex by a potentiometric silicon sensor. The dual-label RNase protection (RP) assay was capable of measuring beta-actin mRNA in cellular RNA samples at the 27- to 45-amol level (10-17 pg) with high precision (%CV < 7). We have used this method to quantitate the levels of erbB-2 mRNA in the human tumor cell lines SKBR-3, SKOV-3, and MCF-7. The levels of erbB-2 mRNA in these cells were 105, 190, and 0.9 amol per microgram of cellular RNA, respectively. The dual-label RP method should be useful for measuring the mRNA expression for other erbB-2 homologs such as erbB-3 and erbB-4 in tumor cells and tissues and can be a generally useful mRNA quantitative method for laboratories wishing to minimize radioisotope use.
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PMID:Nonisotopic quantitation of mRNA using a novel RNase protection assay: measurement of erbB-2 mRNA in tumor cell lines. 893 64

Intact plasmids, plasmid fragments, and cDNA were detected using two DNA or RNA probes of varying lengths, each containing only biotin or fluorescein molecules. The probes were hybridized with the target plasmid/cDNA, bound with streptavidin, captured on nitrocellulose membranes, and detected using the urease-conjugate of an anti-fluorescein antibody via the light-addressable potentiometric sensor. The output of the silicon-chip sensor is in the form of rate, microV/s, and is directly proportional to the quantity of hybridized DNA captured on the membrane. Use of the larger probes (for beta-actin cDNA target) results in detection of intact plasmid at the level of approximately 106 target molecules; complementary DNA could be detected at similar levels. When the smaller probes are utilized (pGEM or pBSActB targets), plasmid fragmented targets could be detected only at levels 200 times greater than those observed when using the larger RNA probes.
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PMID:Detection of plasmids using DNA and RNA probes and the light-addressable potentiometric sensor. 947 98

Salmonella typhimurium was detected to levels as low as 119 CFUs using the Threshold Immunoassay System. This immunoassay system utilizes solution-based binding of the biotin and fluorescein labeled antibodies to salmonella, followed by filtration-capture of the immunocomplex on a biotin-coated nitrocellulose membrane. Lastly, an anti-fluorescein urease conjugate is bound to the immunocomplex. Detection of the bound immunocomplex is made possible via the silicon chip-based light-addressable potentiometric sensor. In the presence of the urea, urease converts the substrate to ammonia and CO2 and this results in a pH change at the silicon surface. The resultant pH change is monitored with time and the signal output is reported in microV s(-1). An experiment whereby chicken carcass washings were fortified with salmonella showed a recovery of 90%, indicating that the technique can be used to test for salmonella under these conditions. Precautions must be used with this instrument as sample debris will affect sample flow through the membrane and hence the signal output.
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PMID:Detection of salmonella in poultry using a silicon chip-based biosensor. 1051 39

An ion-sensitive field effect transistor (ISFET)-based immunosensor was developed to detect/quantitate beta-Bungarotoxin (beta-BuTx), a potent presynaptic neurotoxin from the venom of Bungarus multicinctus. A murine monoclonal antibody (mAb 15) specific to beta-BuTx was immobilized onto silicon nitride wafers after silanization and activation with glutaraldehyde. A chip based enzyme linked-immunosorbantassay (ELISA) was performed to ascertain antigen binding to the immobilized antibody. To develop an electrochemical immunosensing system for the detection/quantitation of beta-BuTx, an ISFET was used as a solid phase detector. MAb 15 was immobilized on the gate region of the ISFET. The antigen antibody reaction was monitored by the addition of urease conjugated rabbit anti-beta-BuTx antibodies. The sensor can detect toxin level as low as 15.6 ng/ml. The efficacy of the sensor for the determination of beta-BuTx from B. multicinctus venom was demonstrated in mouse model. Toxin concentration was highest at the site of injection (748.0+/-26 ng/ml) and moderate amount was found in the plasma (158.5+/-13 ng/ml).
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PMID:An ISFET-based immunosensor for the detection of beta-Bungarotoxin. 1219 31

Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon. The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of >90% were observed.
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PMID:Immobilized enzyme studies in a microscale bioreactor. 1505 11

Urease has been immobilized and layered onto the walls of manufactured silicon microchannels. Enzyme immobilization was performed using layer-by-layer nano self-assembly. Alternating layers of oppositely charged polyelectrolytes, with enzyme layers "encased" between them, were deposited onto the walls of the silicon microchannels. The polycations used were polyethylenimine (PEI), polydiallyldimethylammonium (PDDA), and polyallylamine (PAH). The polyanions used were polystyrenesulfonate (PSS) and polyvinylsulfate (PVS). The activity of the immobilized enzyme was tested by pumping a 1 g/L urea solution through the microchannels at various flow rates. Effluent concentration was measured using an ultraviolet/visible spectrometer by monitoring the absorbance of a pH sensitive dye. The architecture of PEI/PSS/PEI/urease/PEI with single and multiple layers of enzyme demonstrated superior performance over the PDDA and PAH architectures. The precursor layer of PEI/PSS demonstrably improved the performance of the reactor. Conversion rates of 70% were achieved at a residence time of 26 s, on d 1 of operation, and >50% at 51 s, on d 15 with a six-layer PEI/urease architecture.
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PMID:Activity and lifetime of urease immobilized using layer-by-layer nano self-assembly on silicon microchannels. 1591 90

The principles of attenuation of the light intensity due to multiple reflections are realised in a planar silicon oxide (SiO(2))silicon nitride (Si(3)N(4)) waveguiding structure for the purpose of developing optical biosensors with improved sensitivity. The analysis of the experimental data shows that the large difference in refractive indices of core and cladding layers gives rise to an increase in sensitivity by a factor of 3 over previously reported structures. Composite polyelectrolyte self-assembled thin films containing cyclo-tetra-chromotropylene as an indicator and enzymes glucose oxidase or urease were employed in the superstrate as a sensing membrane. Individual enzyme reactions as well as their inhibition by pesticides were studied by monitoring the intensity of light output from the planar waveguide. The results were compatible with those obtained by conventional ultraviolet-visible absorption spectroscopy. The instrument detection limit for Imidacloprid pesticide was found to be as low as 10 ppb in concentration.
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PMID:Planar silicon nitride waveguides for biosensing. 1646 26

A three layer waveguiding silicon dioxide (SiO(2))/silicon nitride (Si(3)N(4))/SiO(2) structure on silicon substrate was proposed as an optically efficient biosensor for calibration of heavy metal ions in drinking water. The catalytic activities of urease and acetylcholine esterase (AchE) were inhibited by the presence of cadmium (Cd(2+)) and lead (Pb(2+)) ions. The detection limit as low as 1 ppb was achieved by employing the technique of total reflection at the interface between the Si(3)N(4) core and composite polyelectrolyte self-assembled (PESA) membranes containing cyclotetrachromotropylene (CTCT) as an indicator.
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PMID:Optical biodetection of cadmium and lead ions in water. 1701 58

The microenvironments of the sol-gel-derived urease biosensors in terms of elemental ratio, surface morphology, specific surface area and pore size were investigated to characterize the physicochemical properties of poly(vinyl alcohol) (PVA)-modified sol-gel materials. X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and surface area analyzer were used to identify the surface species, topography and pore distribution of the organically doped sol-gel network. XPS results showed that stoichiometric ratios of oxygen-to-silicon in sol-gel materials were in the range 2.08-2.11. The sol-gel materials were partially dried and negatively charged, which retained 6-8% water content to maintain urease activity. The surface morphology of the sol-gel altered obviously when macromolecules were encapsulated, resulting in the increase in surface mean roughness from 0.207 to 2.636 nm. The specific surface area decreased dramatically after the immobilization of biomolecules and organic additives, which clearly depicts that PVA and urease were co-encapsulated into the sol-gel network. However, there still exist enough pore volumes for analytes to mass transport. The apparent Michaelis-Menten constant value (Km) of the encapsulated urease was similar to that in solution and the overall catalytic efficiency in PVA-doped sol-gel-derived glasses only decreased by a factor of 3.2 relative to the value in solution. In addition, the analytical performance of the entrapped urease in PVA-doped sol-gel materials was examined by determining the Cu(II) concentration in aqueous solution. The analytical range of Cu(II) was in the range 2x10(-6) to 2x10(-2) M with a detection limit of 1.5 microg L(-1). Results obtained in this study demonstrate a strategy for maintaining urease activity for biomedical and environmental applications.
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PMID:Preparation and characterization of urease-encapsulated biosensors in poly(vinyl alcohol)-modified silica sol-gel materials. 1747 71

We describe a simple method for the covalent immobilisation of proteins to hydrogen-terminated silicon surfaces and demonstrate various protein detection strategies. Using hydrosilation chemistry, 1-undecenylaldehyde is attached to the surface through stable Si-C bonds; the reaction occurs primarily via the vinyl group and mainly aldehyde groups are presented at the top surface of the monolayer. Proteins are then captured by reaction with their surface lysines. The proteins are bound via a Schiff base, whose formation is reversible, but can be fixed by reduction with cyanoborohydride in a one-pot reaction. Using standard methods of patterning, we were able to specifically localise proteins (urease, amyloid beta (Abeta1-42), GFP and TolAIII-GFP) with little non-specific adsorption at non-reactive sites. We characterised the immobilised proteins by X-ray photoemission spectroscopy, atomic force microscopy and FTIR, and showed that they retain their functionality using potentiometry, fluorescence and coupled antibody systems with chromogenic substrates. We also exploited the conductivity of the silicon substrate to demonstrate electrochemical detection of surface-bound proteins. These protocols will aid the development of protein biochips based on silicon, which gives rise to the possibility of detecting protein-protein and protein-small molecule interactions electronically. Such chips would be expected to be of utility for comparative proteomics and in molecular medicine, drug discovery and diagnostics.
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PMID:Immobilisation of proteins at silicon surfaces using undecenylaldehyde: demonstration of the retention of protein functionality and detection strategies. 1923 99


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