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At least 919 records · Page 51Linked to original sources

PubMed2006

A hydrogen peroxide biosensor based on the direct electrochemistry of hemoglobin modified with quantum dots.

Direct electron transfer of hemoglobin modified with quantum dots (QDs) (CdS) has been performed at a normal graphite electrode. The response current is linearly dependent on the scan rate, indicating the direct electrochemistry of hemoglobin in that case is a surface-controlled electrode process. UV-vis spectra suggest that the conformation of hemoglobin modified with CdS is little different from that of hemoglobin alone, and the conformation changes reversibly in the pH range 3.0-10.0. The hemoglobin in a QD film can retain its bioactivity and the modified electrode can work as a hydrogen peroxide biosensor because of its peroxidase-like activity. This biosensor shows an excellent response to the reduction of H2O2 without the aid of an electron mediator. The catalytic current shows a linear dependence on the concentration of H2O2 in the range 5x10(-7)-3x10(-4) M with a detection limit of 6x10(-8) M. The response shows Michaelis-Menten behavior at higher H2O2 concentrations and the apparent Michaelis-Menten constant is estimated to be 112 microM.

Biosensing Techniques↗

PubMed2005

Using co-cultures expressing fluorescence resonance energy transfer based protein biosensors to simultaneously image caspase-3 and Ca2+ signaling.

Fluorescence resonance energy transfer (FRET)-based protein biosensors allow the spatial and temporal imaging of signaling events in living cells. However, the simultaneous correlation of multiple events of a signaling pathway is hindered by the spectral cross-talk between fluorescent proteins. Here, we show, for signaling pathways that progress synchronously, multiple events can be correlated by using co-cultures expressing different FRET-based protein biosensors. As a demonstration, we investigated the simultaneous caspase-3 and Ca2+ signaling events involved in cell death of COS-7 cells induced by 10 mM H2O2. Interestingly, this H2O2 stimulus induced synchronous caspase-3 activation and Ca2+ signaling. In parallel to caspase-3 activation, cytosolic Ca2+ concentration, [Ca2+]c, gradually rises to its peak and then slowly drops. As cell shrinkage and rounding ensues, [Ca2+]c again gradually rises to its peak and then reaches a plateau. These observations reveal the relative timing and location of these signaling events in cell death induced by this stimulus of H2O2. Finally, our approach offers an exciting opportunity for spatial and temporal imaging of multiple events in a signaling pathway in living cells.

Animals↗

PubMed2005

Conformational biosensor for diagnosis of prion diseases.

A fluorescence technology to monitor the proliferation of amyloidogenic neurological disorders is proposed. A crude brain homogenate (0.01%) from animals infected with a transmissible spongiform encephalopathy is employed as a catalytic medium initiating conformational changes in 520 nM polypeptide biosensors (Tris/trifluoroethanol 50% mixture at pH 7). The fluorescence methods utilize pyrene residues covalently attached to the peptide ends. The coil-to-beta-strand transitions in biosensor molecules cause elevation of a distinct fluorescence band of the pyrene aggregates (i.e. excimers). This approach enables the detection of infectious prion proteins at fmol, does not require antibody binding or protease treatment. Technology might be adopted for diagnosing a large variety of conformational disorders as well as for generic high-throughput screening of the amyloidogenic potential in plasma.

Amyloid↗

PubMed2006

Development of a fiber optic enzymatic biosensor for 1,2-dichloroethane.

There is a significant need for devices capable of measuring water contaminant concentrations in situ--continuously, rapidly, and without reagents, extraction, or other pretreatment. Toward this goal, we constructed and tested fiber optic biosensors for measurement of 1,2-dichloroethane (DCA) in aqueous solutions. The biocomponent was the haloalkane dehalogenase, DhlA, in whole cells of Xanthobacter autotrophicus GJ10. These cells were immobilized in calcium alginate on the tip of a fiber optic fluoresceinamine-based pH optode. The resulting biosensor could quantify DCA at 11 mg/l and had a linear response up to at least 65 mg/l. Total signal change was reached in 8-10 min, and measurements were reproducible (SE <9%). The sensor's small size, potential for remote operation, and low cost make it of interest for further development.

Biosensing Techniques↗

PubMed2005

Evaluation of microfluidic biosensor development using microscopic analysis of molecular beacon hybridization kinetics.

Molecular beacons, oligonucleotide probes that fluoresce upon hybridization to a target nucleic acid, can be used in microfluidic devices to detect and quantify nucleic acids in solution as well as inside bacterial cells. Three essential steps towards the development of such devices as integrated microfluidic biosensors using molecular beacons were investigated in the present study. First, experiments using real-time confocal microscopy indicated that diffusion of DNA molecular beacons across a 100-mum diameter microfluidic channel took less than one minute after the flow of reagents was stopped. Second, experiments to evaluate hybridization kinetics of DNA molecular beacons with target nucleic acids in solution showed that DNA molecular beacons can be used to characterize hybridization kinetics in real time in microfluidic channels and that hybridization signals approached their maximum in approximately three minutes. Finally, it was demonstrated that peptide nucleic acid molecular beacons can be used to detect bacterial cells in microfluidic devices. These results suggest that the use of microfluidic devices to detect nucleic acids in solution and in bacterial cells is promising and that further development of an integrated microfluidic biosensor for bacterial detection based on this concept is warranted.

Biosensing Techniques↗

PubMed2004

Nano-biosensor development for bacterial detection during human kidney infection: use of glycoconjugate-specific antibody-bound gold NanoWire arrays (GNWA).

Infectious disease, commonly caused by bacterial pathogens, is now the world's leading cause of premature death and third overall cause behind cardiovascular disease and cancer. Urinary Tract Infection (UTI), caused by E. coli bacteria, is a very common bacterial infection, a majority in women (85%) and may result in severe kidney failure if not detected quickly. Among hundreds of strains the bacteria, E. coli 0157:H7, is emerging as the most aggressive one because of its capability to produce a toxin causing hemolytic uremic syndrome (HUS) resulting in death, especially in children. In the present study, a project has been undertaken for developing a rapid method for UTI detection in very low bacteria concentration, applying current knowledge of nano-technology. Experiments have been designed for the development of biosensors using nano-fabricated structures coated with elements such as gold that have affinity for biomolecules. A biosensor is a device in which a biological sensing element is either intimately connected to or integrated within a transducer. The basic principle for the detection procedure of the infection is partly based on the enzyme-linked immunosorbent assay system. Anti-E. coli antibody-bound Gold Nanowire Arrays (GNWA) prepared on anodized porous alumina template is used for the primary step followed by binding of the bacteria containing specimen. An alkaline phosphatase-conjugated second antibody is then added to the system and the resultant binding determined by both electrochemical and optical measurements. Various kinds of GNWA templates were used in order to determine the one with the best affinity for antibody binding. In addition, an efficient method for enhanced antibody binding has been developed with the covalent immobilization of an organic linker Dithiobissuccinimidylundecanoate (DSU) on the GNWA surface. Studies have also been conducted to optimize the antibody-binding conditions to the linker-attached GNWA surfaces for their ability to detect bacteria in clinical concentrations.

Antibodies, Bacterial↗

PubMed1992

Mapping of viral epitopes with conformationally specific monoclonal antibodies using biosensor technology.

An automated biosensor system (BIAcore) designed for measuring molecular interactions in real time and without labelling any of the reactants was used for mapping the epitopes of tobacco mosaic virus protein using conformationally specific monoclonal antibodies (MAbs). Some of the MAbs used as capturing antibody on the sensor chip allowed a conformational change to occur in the viral protein. As a result, MAbs specific for the quaternary structure of polymerized viral protein were able to bind to monomeric viral subunits. Compared with classical solid-phase enzyme immunoassay, the biosensor technology possesses several advantages for epitope mapping of viral proteins.

Antibodies, Monoclonal↗

PubMed1994

Enzyme-based biosensor as a selective detection unit in column liquid chromatography.

A reagentless enzyme electrode based on co-immobilized alcohol oxidase and horseradish peroxidase was used as the working electrode in an amperometric flow-through cell connected to a column liquid chromatographic (CLC) system for the selective detection of methanol and ethanol. The enzymes were covalently immobilized in carbon paste (graphite-phenylmethylsilicone oil) in the presence of polyethylenimine. Electrodes prepared from the enzyme-modified carbon paste were optimized with respect to their sensitivity and selectivity. Different membranes were cast or electropolymerized directly on the surface of the electrode to increase the long-term stability of the biosensor. The compatibility with the reversed-phase chromatographic system was established. A PLRP-S polymer-based separation column was used with phosphate buffer as the mobile phase. The selectivity of the enzyme electrode was also determined by injecting some easily oxidizable and possibly interfering species normally present in biological samples. The enzyme electrode was also used in an on-line system, consisting of a microdialysis probe as the sampling unit, the CLC system and the biosensor detection device, for the selective following of the ethanol produced when a paper pulp industrial waste water was fermented with Saccharomyces cerevisiae.

Biosensing Techniques↗

PubMed1994

Cross-reactivity of monoclonal antibodies to a chimeric V3 peptide of HIV-1 with peptide analogues studied by biosensor technology and ELISA.

The reactivity of monoclonal antibodies (Mabs) raised against a cyclic peptide representing a chimeric V3 loop of HIV-1 gp120 with different peptide analogues was studied with a biosensor system (BIAcore) and by ELISA. In both assays, the Mabs cross-reacted extensively with the V3 regions of different HIV-1 strains and recognized the cyclic form of the peptide immunogen better than its linear form. The highest degree of cross-reactivity was observed with peptides that shared a Lys312 with the chimeric sequence. Dissociation rate constants of ten Mabs measured with the BIAcore with respect to different peptides increased with increasing numbers of substitutions in the flanking regions of the V3 tip sequence Gly Pro Gly Arg. Immobilization of the cyclic peptide on the sensor chip via a thiol group added near the end of the loop structure preserved the conformation of the peptide. In view of the good correlation between the BIAcore and ELISA results, biosensor data should be useful for selecting peptides to be used in diagnostic solid phase assays.

Amino Acid Sequence↗

PubMed1996

Selection of binders from phage displayed antibody libraries using the BIAcore biosensor.

In this report we show that phage displayed antibodies can be selected based on dissociation rate constants, using a BIAcore biosensor. To demonstrate the principle, two Fab phage stocks displaying antibodies specific for hen egg lysozyme or phenyloxazolone were mixed in a ratio of 1:10 and injected over the biosensor chip containing immobilized lysozyme. Antigen-specific bound phages were eluted and analysed for specificity and phage titer. This procedure enriched for phages carrying specific antibodies. Selection of high affinity binders from phage libraries was then demonstrated with the BIAcore when phages were eluted and collected at different time points. Soluble antibody fragments were subsequently expressed and their kinetic parameters were determined. The time of elution was directly proportional to the affinity, due to decreased dissociation rate constants. This procedure offers a rapid and simple approach for selecting binders from phage libraries differing in antibody dissociation rate constants.

Bacteriophages↗

PubMed1995

Immobilised yeast cells biosensor for total toxicity testing.

An immobilised yeast cell biosensor has been developed for the total toxicity testing of a sample that may contain a number of different pollutant species; the biosensor uses an amperometric gas diffusion oxygen as indicator electrode. The method is based on the perturbation of the respiratory activity of a yeast, Saccharomyces cerevisiae, immobilised on an agar gel containing the culture medium (i.e., 'agarised medium'), by the toxic test substance. Glucose is used as substrate while the toxic substances tested consist of several metallic ions, phenol and cationic or anionic surfactants.

Biosensing Techniques↗

PubMed1995

Photo-crosslinked copolymers of 2-hydroxyethyl methacrylate, poly(ethylene glycol) tetra-acrylate and ethylene dimethacrylate for improving biocompatibility of biosensors.

A copolymer containing 88% 2-hydroxyethyl methacrylate (HEMA), 9% poly(ethylene glycol) (MW 18.5 kDa) tetra-acrylate and 3% ethylene dimethacrylate was prepared and evaluated for use as a biocompatible interface between glucose biosensors and tissue in the rat. The glucose sensor utilizes glucose oxidase that is electrically 'wired' to a gold current collector by a reduction-oxidation polymer. Coatings of the copolymer were crosslinked in situ on the sensors using long wavelength ultraviolet light and 2,2-dimethoxy-2-phenyl-acetophenone as the initiator. The effect these films had on the current response to glucose was measured. Over a glucose concentration range of 0-30 mM, the average percentage decrease in response was 45 +/- 28% (mean +/- 95% confidence interval) at 37 degrees C for films that were about 0.1 mm thick, an acceptable value. Copolymer-treated and control electrodes were implanted in the intrascapular subcutaneous tissue of male Sprague-Dawley rats for three days. The explanted samples were evaluated using scanning electron microscopy. The control electrodes were highly encapsulated with fibrous material, while the copolymer-treated electrodes induced much less encapsulation. The results show this copolymer to be a candidate as a biocompatible coating for electrically wired oxidoreductase-based subcutaneous biosensors.

Acrylates↗

PubMed1992

Flow injection analysis and in-line biosensors for bioprocess control: a comparison.

Miniaturization will unify the different approaches chosen for the application of biosensors in bioprocess control. The most versatile system, which in our opinion is flow injection analysis will be the method of choice for the introduction of biosensors in bioprocess control. A lot of experience will be gained for the future development of miniaturized total chemical analysis systems.

Biosensing Techniques↗

PubMed1993

Amperometric biosensor for the determination of phenolic compounds using a tyrosinase graphite electrode in a flow injection system.

Selective and sensitive devices for the monitoring of phenol and phenolic compounds are required in clinical and environmental analysis. This paper describes a biosensor for the analysis of phenolic compounds in a flow injection system. The enzyme electrode is based on the use of immobilized tyrosinase and the amperometric detection of the enzymatic product at -50 mV vs. SCE. The enzyme is covalently immobilized on the surface of a carbodiimide-activated graphite electrode. The biosensor responds to a variety of phenolic substrates with different conversion efficiencies. The detection limit for phenol is 0.003 microM (S/N = 3), a quantification limit of 0.01 microM (rsd 3.7%), and an extended dynamic range up to 5 microM is achieved with a sample frequency of 110 samples per hour.

Biosensing Techniques↗

PubMed1994

Fluorimetric determination of alkaline phosphatase activity in human serum by use of a flow-through biosensor.

A fluorimetric method for the determination of alkaline phosphatase activity based on the use of a flow-through biosensor is reported. The biochemical basis of the method is the hydrolysis of 4-methyl-umbelliferone-phosphate catalyzed by the analyte with fluorimetric monitoring of the 4-methyl-umbelliferone formed (lambda ex = 365 nm, lambda em = 445 nm). The enhancement of sensitivity achieved when the reaction product is retained on the support packed in the flow-cell makes the method suitable for determination of the analytes in serum samples after 1:50 dilution. The linear range is found to be between 0.1 and 20 U l-1, with relative standard deviation less than 2.2%. The use of this biosensor was tested by the determination of the analyte in human serum from healthy and sick individuals with excellent recoveries (94-103%).

Alkaline Phosphatase↗

PubMed1985

Diabetes mellitus: biosensors for research and management.

The condition of diabetes mellitus is described with particular reference to the parameters that it would be desirable to monitor in order to improve management and understanding of the disease. Previous attention has largely focused on analysis of glucose, but many other intermediates of carbohydrate, fat and protein metabolism are deranged in diabetes and may be alternative measures of control. The need for laboratory analysers, self-monitoring, closed-loop devices and alarms are detailed and the problems associated with implantable sensors discussed. Progress in the development of biosensors is reviewed using glucose sensors as the main example. Electrochemical, optoelectronic and calorimetric approaches to sensing are considered and it is concluded that configurations based either on hydrogen peroxide detection or on mediated electron transfer are most likely to provide a raid route to in vivo monitoring. The extension of biosensor technology to tackle other important substrates is discussed, the principal hurdle to success being seen as the lack of long-term stability of the biological component.

Blood Glucose↗

PubMed1986

The development and application of FET-based biosensors.

After having considered the general definition of biosensors, the specifications of one type are discussed here in more detail, namely the pH-sensitive ISFET, which is at present being clinically investigated for intravascular blood pH recording. Results, advantages and possible improvements will be discussed, as well as a prediction with respect to future developments of FET-based biosensors.

Biological Products↗

PubMed1992

Biosensors and enzyme immobilized electrodes.

A biosensor is a device which consists of a biological sensing element connected to a transducer. The transducer can be electronic, optical, electrical, etc. This emerging technology offers us a powerful tool which is radically altering our approach to analytical methods. It was realised that enzymes are natural sensors on account of their highly selective nature. Much of the impetus to the work has come from medical requirements. Instant analysis of clinical samples has an obvious appeal to physicians and patients alike. Of particular interest is the possibility of continuous 'in-vivo' monitoring of metabolites, drugs and proteins using miniature, portable systems. In recent years, there has been a growing demand for biosensors in the fields of veterinary science, animal husbandry, the food industry and environmental monitoring. However, the possibility of successful application rests upon future developments. Increasing attention will have to be paid to the engineering of both the basic components and the device on the whole. New biochemical reactions will either have to be discovered or engineered through genetic manipulation or chemical techniques. Optimization of response time, selectivity, stability and low costs should receive priority considerations.

Biosensing Techniques↗