ABSTRACT
An urea biosensor based on urease-BSA (bovine serum albumin) membrane immobilised on the surface of an ion-sensitive field effect transistor (ISFET) has been studied in a mix buffer solution composed of potassium phosphate, Tris, citric acid and sodium tetraborate. In this mix buffer, the biosensor showed a dynamic larger than the one observed in a phosphate or Tris buffer. Investigation of the individual effect of each component of the buffer solution on the biosensor response has shown that tetraborate anion acts as a strong competitive inhibitor for the hydrolysis reaction of urea catalysed by urease. The biosensor response was investigated in a phosphate buffer with different concentrations of tetraborate anion. The results showed that the apparent constant of Michaelis-Menten, K(m(app)), increases from 4.3 to 79.3 mM, for experiments realised without and with 0.5 mM sodium tetraborate, respectively. The mean value, determined graphically, for the inhibition constant, K(i), was 29 microM. The graphical representation of biosensor calibration curves in semilogarithmic co-ordinates showed that the linear range of the biosensor can be extended up to three orders of magnitude, allowing an urea detection in a concentration range 0-100 mM.
Subject(s)
Biosensing Techniques/instrumentation , Borates/chemistry , Electrochemistry/instrumentation , Membranes, Artificial , Serum Albumin, Bovine/chemistry , Urea/analysis , Urease/chemistry , Biosensing Techniques/methods , Coated Materials, Biocompatible/chemical synthesis , Electrochemistry/methods , Enzyme Activation , Enzymes, Immobilized , Equipment Design , Hydrogen-Ion Concentration , Ions , Reproducibility of Results , Sensitivity and Specificity , Transistors, Electronic , Urea/antagonists & inhibitors , Urea/chemistryABSTRACT
The bli-1 gene of Caenorhabditis elegans has been previously described as a mutation which disrupts the structure of the adult-stage cuticle causing the formation of fluid-filled blisters. We investigated the blistering phenotype exhibiting n361 allele and observed a gradual blister formation in adult nematodes. In the course several fine changes occurred including a high electron density granulous material filling the intermediate layer, alterations on struts structure, and finally the total disappearance of the fibrous and basal layers. With the ethanolic phosphotungstic acid technique (E-PTA), which localizes basic proteins, reaction product was observed in the cortical layer of the wild strain, whereas in the mutant strain an irregular labelling pattern was observed in this region. The granulous material inside the intermediate layer of the mutant strain showed also a strong reaction. An imidazole-buffered osmium tetroxide solution was used to visualize lipids at ultrastructural level, however no dense product was detected in the cuticle of both strains of C. elegans. Based on these results we postulated that the blistering phenotype is due to an altered function of bli-1 gene, which is probably enzymatic.
Subject(s)
Caenorhabditis elegans/ultrastructure , Epidermis/ultrastructure , Animals , Blister/genetics , Blister/metabolism , Blister/pathology , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/metabolism , Epidermis/metabolism , Histocytochemistry , Life Cycle Stages , Microscopy, Electron , Mutation , Phenotype , Phosphotungstic Acid , Staining and LabelingABSTRACT
Enzyme-based field effect transistors (ENFETs) for urea determination were developed based on the immobilization of urease within two different clay matrixes, one cationic (Laponite) and the other anionic (layered double hydroxide (LDH)), cross-linked with glutaraldehyde. The biosensor based on the enzyme immobilized in Laponite shows a greater sensitivity and smaller dynamic linear range, because the enzymatic reaction is protected from the effect of the buffer capacity of the outer medium. The apparent Michaelis-Menten constant, Km(app), is quite similar for both biosensors. Inhibition of the enzyme by sodium tetraborate was investigated. Tetraborate acts as a competitive inhibitor for urease in the two different types of clay, the inhibitor effect being stronger for the LDH/urease biosensor. In particular, the maximum limit of the dynamic linear range extends from 1.4 mM in the absence of the inhibitor to 12 mM in the presence of 0.5 mM tetraborate. The Km(app) values in the presence of 0.5 mM tetraborate for Laponite and LDH biomembranes were 10 and 62 mM, respectively. Comparison of the inhibition constant values, Ki 0.16 and 0.05 mM for Laponite and LDH biosensors, respectively, clearly indicates a stronger enzyme-inhibitor interaction in the LDH/urease biomembrane.
Subject(s)
Biosensing Techniques/methods , Urea/analysis , Aluminum Silicates/chemistry , Biosensing Techniques/standards , Calibration , Clay , Enzymes, Immobilized , Sensitivity and Specificity , UreaseABSTRACT
The sqt-1 gene is 1 of the several loci in Caenorhabditis elegans that primarily affects organismal morphology. Certain mutations in the sqt-1 gene can produce left roller animals, i.e., they rotate around their long axis and move in circular paths. We describe the morphological alterations seen in the cuticle of the left roller sqt-1(sc13). Deep-etched replica analyses showed that the fibrous layer is composed of a unique strand of parallel fibers, instead of the 2 meeting at an angle of 60 degrees as observed in the wild-type strain. In addition, honeycomb elements, fibers organized in a pentagonal fashion above the fibrous layer, completely fill the intermediate layer that is empty spaces in the wild type. These morphological alterations are likely to be involved in generating the helical twist of the sqt-1(sc13) left roller mutant.
Subject(s)
Caenorhabditis elegans/ultrastructure , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/physiology , Collagen/genetics , Freeze Etching , Freeze Fracturing , Microscopy, Electron , Microscopy, Electron, Scanning , Movement , Mutation , PhenotypeABSTRACT
Roller mutants of Caenorhabditis elegans rotate around their long axis and move in circular paths. Isolation and sequence of the rol-6 gene of C. elegans have shown that it encodes a cuticle collagen. In this paper, we describe the morphological alterations seen in the cuticle of the right roller mutant rol-6 (su1006) at the ultrastructural level. Deep-etched replica analyses showed that the honeycomb elements, fibers organized in a pentagonal fashion above the fishbone fibrous layer, completely fill the intermediate layer, which is observed to be largely empty spaces in the wild-type strain. The honeycomb fibers appear to connect the cortical and basal regions of the mutant cuticle. These fibers are likely to be involved in generating the helical twist of the mutant animals. Deep-etched replicas also revealed a delicate network of filaments on the nematode surface.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans/ultrastructure , Collagen/genetics , Helminth Proteins/genetics , Animals , Caenorhabditis elegans/genetics , Freeze Etching , Microscopy, Electron , Microscopy, Electron, Scanning , Mutation , PhenotypeABSTRACT
The nonlinear-optical properties of a semi-interpenetrating polymer network of poly(vinyl alcohol) glutaralde-hyde-polyaniline were studied. Large (>/=10(-12) cm(2)/W) and fast (<50 ps) refractive optical nonlinearites were observed. The potential of this novel material for photonic applications is evaluated.
