Bioreporter pseudomonas fluorescens HK44 immobilized in a silica matrix

The bioluminescent bioreporter Pseudomonas fluorescens HK44, the whole cell bacterial biosensor that responds to naphthalene and its metabolites via the production of visible light, was immobilized into a silica matrix by the sol-gel technique. The bioluminescence intensities were measured in the maximum of the bioluminescence band at X = 500 nm. The immobilized cells (>105 cells per g silica matrix) produced light after induction by salicylate (cone. > 10 g/l), naphthalene and aminobenzoic acid. The bioluminescence intensities induced by 2,3-dihydroxynaphthalene 3-hydroxybenzoic acid and 4-hydroxybenzoic acid were comparable to a negative control. The cells in the silica layers on glass slides produced light in response to the presence of an inductor at least 8 months after immobilization, and >50 induction cycles. The results showed that these test slides could be used as assays for the multiple determination of water pollution

Hybrid coatings as transducers in optical biosensors

Sensitive coatings are described for a novel enzyme-based optical sensor for in-situ continuous monitoring of reactants, such as glucose, in biotechnological production processes. Glucose oxidase, incorporated into suitable coating materials that are applied on lenses or optical fibers, is used to catalyze oxidization of glucose to gluconic acid in the presence of oxygen. The presence and consumption of oxygen is determined by measuring the fluorescence signal of incorporated metal organic ruthenium complexes, which is quenched by oxygen. Inorganic-organic hybrid polymers, synthesized via sol-gel processing, were used as coating material. Due to the hybrid character of the coating, good adhesion is achieved on both glass and polymer surfaces. Good compatibility is also given with enzymes and ruthenium complexes. The sensitive optical coating was built up as double-layer and single-layer structures. The double layer comprised a primary coating containing the oxygen-sensitive ruthenium complex, and a secondary coating containing the enzyme. The single layer comprised a single coating containing both the ruthenium complex and the enzyme

Optical fibre biosensors for oxygen and glucose monitoring

An optical fibre biosensor that uses an oxygen sensitive coating; Ruthenium complex [Dichlorotris(1,10-phenantroline)-ruthenium(II) hydrate], incorporated into an adhesive inorganic-organic hybrid polymer coating (ORMOCER®) is described. The Ruthenium/ ORMOCER® layer is used with optical fibres to form an extrinsic or intrinsic sensor. It can be applied to a microscope slide or lens to be interrogated by optical fibres, or form a cladding layer for an evanescent field optical fibre sensor. The Ruthenium complex is caused to fluoresce by a high brightness blue LED at 470 nm and the excitation light at 600nm is detected by a photomultiplier tube used as a photon counter, to measure fluorescence lifetime. The fluorescence is quenched by oxygen depletion within the layer, which can be linked to glucose by incorporation of suitable enzymes. The detection threshold is 0.7mg(O2)/litre, and the sensitivity is 70 ns/mg per litre

The metastasis promoting protein S100A4 is increased in idiopathic inflammatory myopathies

Objectives. The S100A4 protein is known as a metastasis promoting factor; however, its involvement in non-malignant diseases such as RA and psoriasis has been recently described. The aim of this study was to investigate the expression and possible role of S100A4 in idiopathic inflammatory myopathies. Methods. S100A4 protein expression was detected by immunohistochemistry in muscle tissue from control individuals (n = 11) and patients with PM and DM (n = 8/6). IF staining was used to co-localize S100A4 with selected cells. Cytokine expression and protein synthesis in S100A4-treated cells were analysed by RT-PCR and ELISA. Results. S100A4 protein was significantly up-regulated in muscle tissue of patients with inflammatory myopathies compared with control individuals and was associated particularly with the presence of mononuclear infiltrates. Only few regenerating muscle fibres in PM/DM expressed S100A4. Then we analysed the effect of S100A4 on human myocytes and peripheral blood mononuclear cells (PBMCs). Although S100A4 did not affect myocytes, stimulation of PBMCs with S100A4 significantly induced the expression and synthesis of TNF-α, IL-1β and IL-6, but not of IFN-α. We showed that S100A4 is not directly involved in perforin/granzyme B-induced apoptosis and that it does not modulate the expression of Bax and Bcl2 mRNA in myocytes and PBMCs. Conclusion. Increased expression of S100A4 in inflamed muscle tissue highlights its potential role in the pathogenesis of inflammatory myopathies. S100A4 may act as a cytokine-like factor indirectly promoting muscle fibre damage by stimulating mononuclear cells to increase the synthesis of pro-inflammatory cytokines

Membrane-based electrochemical nanobiosensor for escherichia coli detection and analysis of cells viability

A sensitive and selective membrane-based electrochemical nanobiosensor is developed for specific quantitative label-free detection of Escherichia coli (E. coli) cells and analysis of viable but nonculturable (VBNC) E. coli cells which remain mostly undetected using current methods. The sensing mechanism relies on the blocking of nanochannels of a nanoporous alumina-membrane modified electrode, upon the formation of immune complexes at the nanoporous membrane. The resulting obstacle to diffusive mass transfer of a redox probe in the analysis solution to the underlying platinum electrode reduces the Faradaic signal response of the biosensor, measured using cyclic voltammetry. Antibody loading under conditions of varying antibody concentrations and pHs are optimized. The biosensor gives a low detection limit of 22 cfu mL ̄¹ (R² = 0.999) over a wide linear working range of 10 to 10⁶cfu mL ̄¹. It is specific toward E. coli with minimal cross-reactivity to two other pathogenic bacteria (commonly found in waters). Relative standard deviation (RSD) for triplicate measurements of 2.5% indicates reasonably useful level of reproducibility. Differentiation of live, VBNC, and dead cells are carried out after the cell capture and quantitation step, by simple monitoring of the cells’ enzyme activity using the same redox probe in the analysis solution, in the presence of glucose.Accepted versio