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)

Many-sided investigations of urease immobilization methods were carried out to create the biosensor devices on the base of semiconductor structures. Special attention was concentrated on the biomembrane formation by means of urease and bovine serum albumin (BSA) cross-linking by gaseous glutaraldehyde. Optimal conditions for the formation process were selected which preserve about 20% of total urease activity after the cross-linking. The properties of enzyme immobilized by the above-mentioned method have been comprehensively studied. They included the urease activity dependence on pH, ionic strength, incubation buffer capacity as well as the enzyme stability during its functioning, storing and thermoinactivation. As was shown, for immobilized ureas Km value for urea at pH 7.0 and 20 degrees C is 1.65 time less than for free enzyme. In the presence of EDTA (1 mM) the enzyme activity in the biomembrane is practically unchanged under a month storing. Biomembrane possesses good adhesion to silicon surface and its swelling level under different conditions does not exceed 35%. The conclusion is made about the prospects of the used method of biomembrane formation for biosensor technology based on semiconductor structures.
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PMID:[Study of immobilization and properties of urease for creation of a biosensor based on semiconductor structures]. 151 49

The nicotinic acetylcholine receptor, purified from Torpedo electric organ, was coupled to a light addressable potentiometric sensor (LAPS) to form a LAPS-receptor biosensor. Receptor-ligand complexes containing biotin and urease were captured on a biotinylated nitrocellulose membrane via a streptavidin bridge and detected with a silicon-based sensor. Competition between biotinylated alpha-bungarotoxin and nonbiotinylated ligands formed the basis of this assay. This biosensor detected both agonists (acetylcholine, carbamylcholine, succinylcholine, suberyldicholine, and nicotine) and competitive antagonists (d-tubocurarine, alpha-bungarotoxin, and alpha-Naja toxin) of the receptor with affinities comparable to those obtained using radioactive ligand binding assays. Consistent with agonist-induced desensitization of the receptor, the LAPS-receptor biosensor reported a time-dependent increase in affinity for the agonist carbamylcholine as expected, but not for the antagonists.
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PMID:Detection of nicotinic receptor ligands with a light addressable potentiometric sensor. 162 72

Sensitive immunoassays are essential for establishing the efficacy of recombinant vaccines to hepatitis B virus (HBV). These experimental vaccines include the PreS2 and S domains of the HBV envelope protein. To facilitate measurement of antibody against HBV PreS2, we employed the immuno-ligand assay with silicon sensor-based detection. Labeling of immune reagents with the haptens biotin and fluorescein allows adaptation to the immunofiltration light addressable potentiometric sensor (LAPS) system. A biotinylated monoclonal anti-PreS2 antibody and anti-PreS2 in clinical serum samples competitively bind in liquid phase to a fluorescein labeled PreS2 + S antigen. Streptavidin mediates the immobilization on biotinylated nitrocellulose membranes. Fluorescein mediates binding of an anti-fluorescein urease conjugate to the immune complex. Urease serves as the signal-generating component which subsequently is measured in the LAPS reader. In comparison to a competitive RIA, the immuno-ligand assay demonstrated a four-fold improved sensitivity using a smaller sample volume. The higher sensitivity resulted in earlier detection of seroconversion during a clinical vaccine study.
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PMID:Detection of antibody to the PreS2 sequence of the hepatitis B virus envelope protein using an immuno-ligand assay with a silicon sensor detection system. 176 51

Rapid, quantitative hybridization assays with good sensitivity are needed in many applications, for example, determining the amount of specific product from PCR. We have developed an assay which relies on the hybridization of a biotinylated oligomer and a fluoresceinated oligomer to a single-stranded target in solution. The hybridized complex is captured by streptavidin to a biotinylated membrane. After capture, the hybridization complex is detected by an antifluorescein-urease conjugate which binds to the fluoresceinated probe. The membrane-bound urease conjugate is exposed to urea and assayed with a pH-sensitive silicon sensor. The total assay time is less than 2 h and the sensitivity limit is 20 x 10(6) molecules with a coefficient of variation, CV, of less than 10%. The assay was applied to the analysis of a model target using PCR. We were able to measure the amount of specific product and the amplification factor during the exponential phase of PCR. Using extrapolation from the measured amounts of amplified product, the initial amounts of target molecules were calculated to be 1.2 x 10(6) and 4.0 x 10(2) when the added quantities were 3 x 10(6) and 3 x 10(3), as determined by serial dilution.
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PMID:Quantitation of DNA hybridization in a silicon sensor-based system: application to PCR. 179 56

A sensitive sandwich immunoassay for human chorionic gonadotropin (hCG) was developed with biotin-mediated filtration capture and silicon sensor detection. A high density of biotin on the membrane assured efficient capture of complexes containing streptavidin and analyte. Capture efficiency was not affected over a wide range of filtration flow rates or biotin concentrations. The assay utilized the pH sensing ability of the light addressable potentiometric sensor (LAPS) for the detection of urease-antibody conjugates. A LAPS reader was constructed which allowed the enzyme conjugate to be detected in approximately 1 microliter volumes. Effects from variations in detection volume were studied. 10 pg of hCG could be detected in an assay time of 20 min with four standard deviations separation from background. Comparison to a commercial RIA was made.
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PMID:A silicon sensor-based filtration immunoassay using biotin-mediated capture. 223 Jan 51

Grafting of SH-groups to the silica surface through the hydrolytically stable Si-C-bond is conducted by gamma-mercaptopropyltrimethoxysilane. After 2,2'-dithiobis-p-nitrobenzoic acid (Ellman's reagent) activation of sulphydryl groups urease of microbial origin was immobilized by these carriers. Certain properties of the preparations obtained were studied. The Km of the enzyme during nonporous silicon aerosil immobilization is shown to remain without considerable changes. The found variations in properties of silochrome-immobilized urease are caused by the diffusion inhibition for the substrate and product of the reaction observed even when the substrate concentration is two orders higher than Km.
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PMID:[Properties of urease immobilized on silochrome by means of disulfide bonds]. 632 34

Simple solid-phase optoelectronic sensors for penicillin, urea, and glucose are described. Triphenylmethane dyes such as bromcresol green and bromthymol blue were derivatized with glutathione and co-immobilized with appropriate enzymes to a transparent membrane sandwiched between a red-light-emitting diode and a silicon photodiode with integral amplifier. In the presence of the corresponding substrates, catalytic action in the enzyme-dye membrane perturbs the local pH and causes characteristic color changes in the membrane which are monitored as a rise or fall in the output voltage of the detector system. With enzymes such as penicillinase, urease, and glucose oxidase, the response of the optoelectronic sensors is extremely reproducible over the concentration range 0-10 mM penicillin G, urea, or D-glucose, respectively. This report describes the construction and operation of these simple, inexpensive, and reagentless optoelectronic sensors.
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PMID:Solid-phase optoelectronic sensors for biochemical analysis. 674 21

A new kind of calorimetric biosensor for the measurement of the heat (molar enthalpy change) of enzymatic reactions is presented. The device operates according to the Seebeck effect, the same principle on which thermocouples are based. The thermopile used in this work consists of an array of p-type silicon/aluminium strips integrated on a thin silicon membrane (5 microns). Its sensitivity is about 1 V output voltage per watt of heating power, corresponding to a temperature resolution in the order of 10(-5) K and a heating power resolution of some tenths of a mu W in the flow system used. Furthermore, this performance is obtained without any control of external temperature because of the high common-mode thermal noise rejection ratio of the thermopile. The universal technique of calorimetry combined with the specificity of biochemical reactions makes this biosensor very versatile, with a broad range of possible applications. Glucose oxidase together with catalase for the determination of glucose, urease and penicillinase for the monitoring of urea and penicillin G, respectively, were immobilized directly onto the back side of the thermopile. The sensor was operated in conjunction with flow injection analysis which, in addition to its traditional advantages, allows preconditioning of the samples. Thus, artefacts due to mixing effects were suppressed and interference caused by differences in ionic strength between sample and carrier was strongly decreased. Detection limits between 1 and 2 mM were reported in the flow injection conditions described.
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PMID:An integrated silicon thermophile as biosensor for the thermal monitoring of glucose, urea and penicillin. 831 96

A liquid-phase immuno-ligand assay has been developed for quantitative determination of recombinant tick anticoagulant protein (rTAP) secreted in yeast fermentations. A polyclonal anti-TAP antibody was labeled with biotin or fluorescein. Labelled antibodies were used in a non-competitive sandwich format to capture rTAP from solution, then reacted with urease-conjugated anti-fluorescein antibody. Detection of the immune complex was by a commercially available silicon-based potentiometric sensor which measures urease activity. Sample throughput was 90 samples per 7 h with a 2 h incubation time. The range of the standard curve was 0.1-10 ng/ml with an assay sensitivity of 0.025 ng/ml. For a mid-range concentration of 1 ng/ml, intraday and interday method precision was determined to be 1.031 +/- 0.061 and 1.077 +/- 0.026 ng/ml, respectively. Typically, spiked samples of 1 microgram rTAP/ml fermentation medium required dilutions of 1/1000 to generate a response in the mid-range of the standard curve. This assay provides a convenient method to quantitate product expression in multiple fermentation samples within 3 h after sampling. In addition, a modified version of the assay was developed which provided accurate results within 1 h of sample acquisition.
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PMID:Nanogram quantitation of secreted protein in a recombinant yeast fermentation using an immuno-ligand assay. 844 55

Highly specific detection of human alpha 1-acid glycoprotein (AGP) and asialo-alpha 1-acid glycoprotein (asialo-AGP) was made possible by use of a sandwich immunoassay. The glycoproteins were sandwiched between biotinylated and fluoresceinated polyclonal rabbit anti-human AGP antibodies. Additionally, asialo-AGP could be distinctly detected, apart from AGP, via the formation of a heterosandwich immunoassay using biotinylated polyclonal rabbit anti-human AGP and the lectin, fluoresceinated ricin toxin. Streptavidin was added to the formed immunocomplexes and the immunocomplexes captured on a biotinylated nitrocellulose membrane. The signal generator, urease conjugate of an anti-fluorescein antibody, was then bound to the complex on the membrane. The rate of pH change under microvolume conditions (0.6 microliters) was monitored using a silicon chip-based, light addressable potentiometer sensor. Results indicated that AGP and asialo-AGP can be detected to the 2 pg level when two antibodies are used to form the immunocomplex. Asialo-AGP can be detected down to 250 pg when the heterosandwich immunoassay is used; this assay exhibited no response up to 10 ng for native AGP or asialofetuin. Both immunoassays can be used to quantify the level of AGP and asialo-AGP in solution. Although the assay presented is very specific for AGP, asialo-AGP and terminal galactose, it is readily adaptable for the detection of any glycoprotein and terminal carbohydrate (or branched structure) by use of a protein-specific antibody and various lectins.
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PMID:Detection of human asialo-alpha(1)-acid glycoprotein using a heterosandwich immunoassay in conjunction with the light addressable potentiometric sensor. 887 21


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