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PubMed2006

A novel system for environmental monitoring through a cooperative/synergistic scheme between bioindicators and biosensors.

This paper addresses environmental monitoring through a robust dynamic integration between biomonitor and biosensor systems, a strategy that has not been attempted before. The two systems are conceptually interrelated and methodologically correlated to a cooperative/synergistic scheme (CSS) with a view to minimise uncertainty and monitoring costs and increase reliability of pollution control and abatement. The structures and operations of the biosensor component (in terms of sensitivity, device and method versatility, nature-mimicking physicochemical mechanisms, prospects and technological input) are such that they reinforce or promote the structures and operations of the natural component (in terms of bio-surveillance, impact assessment, environmental quality indexing, stress responses, metabolic pathways, etc.) and vice versa. The bioindicator ontology presented herein, including concepts, relations and controlled vocabulary aiming at establishing an integrated methodology for mapping/assessing negative environmental externalities, provides a useful tool for the design/development/implementation of an environmental network for the monitoring of a variety of pollutants over time and space and the assessment of environmental quality; the collection of the available information and its classification into taxonomic and partonomic relations allows the construction of a database that links pollutants with organisms' response, at a phenomenological and in-depth level, considering ecological parameters, relations and geomorphologic characteristics. As a result, a computer program has been designed/developed as a decision support system and has been successfully tested on a representative population of species indigenous to southern Greece. Significantly, a novel system in the form of a rational framework at the conceptual design level has been developed, that actually contributes towards achieving a cost-effective long-term monitoring program, with the flexibility to counter on-course any (anticipated or not) variations/modifications of the surveillance environment. This novel and pioneering approach will further offer a dynamic system utilised in (a) environmental impact studies and risk assessment (positive/analytic approach), (b) decision-making in the short-run (normative/tactic approach), and (c) policy-making in the long-run (normative/strategic approach). The proposed CSS, based on the integration of multiple data sources, can establish a local area network, incorporated into/expanding to a wide area network, thus offering the potential of better predictive ability and greater lead-time warning at alarm conditions than that provided by separate, stand-alone surveillance modalities.

Biosensing Techniques↗

PubMed2003

Characterization and application of a Listeria monocytogenes reactive monoclonal antibody C11E9 in a resonant mirror biosensor.

Typical detection of Listeria monocytogenes involves selective enrichment, isolation and biochemical testing. Development of antibodies to Listeria species has improved detection; however, most antibodies detect all species of Listeria. A previously developed monoclonal antibody (MAb)-C11E9 was examined for its reaction to 13 L. innocua and 40 L. monocytogenes strains representing all 13 serotypes by ELISA. Absorbance values for L. monocytogenes strains were 0.44-3.58 and for L. innocua 0.22-1.44. ELISA reactions were divided into three arbitrary groups of high (Abs 1.0 or higher), intermediate (0.6-0.99) and low (0.18-0.59). Most L. monocytogenes strains (32/41, 78%) were in the high group while only 23% (3/13) of L. innocua were in the same group. In the Western blot assay, antibody reacted with phosphate-buffered saline (PBS) extracted protein preparations of 52, 66 and 97 kDa. Ribopattern of all strains was analyzed and no clear relationship was observed for antibody reaction and ribotype of a given strain. MAb C11E9 was used in a resonant mirror biosensor (IAsys sensor), but failed to detect any viable intact L. monocytogenes cells at levels as high as 10(8) cells/ml; however, it showed binding (85-150 arc/s) with the surface protein preparations containing the 97-, 66- and 52-kDa proteins at 208 mug/ml. Binding kinetics of L. monocytogenes and L. innocua surface protein extracts showed significantly (p<0.05) higher responses than the three other Listeria species (L. ivanovii, L. welshimeri and L. grayi), which could be detected in 10-20 min. These data corroborate with ELISA results. In summary, this study suggest that MAb-C11E9 is suitable for detection of all serotypes of L. monocytogenes despite cross-reaction with L. innocua and could be used for detection of soluble protein extracts in the resonant mirror (IAsys) biosensor.

Antibodies, Monoclonal↗

PubMed2004

Monitoring an antibody affinity chromatography with a label-free optical biosensor technique.

In the production of polyclonal antibody, a purification step is necessary which is often done by affinity chromatography. We present a biosensor system based on reflectometric interference spectroscopy (RIfS) to monitor the quantity and quality in terms of affinity and kinetic constants of the antibody during this procedure. Biosensors are rapid compared with ELISA, which is done in practice and can work fully automated. They provide additional information about the active antibody to protein concentration ratio and the affinity of the antibody. We show how to determine these values very accurate. In addition, we describe a new rapid method to monitor the affinity chromatography in process. This gives the possibility to select antibody fractions with best properties in respect to the application.

Antibody Affinity↗

PubMed2005

Electrochemical reduction of cytochrome P450 as an approach to the construction of biosensors and bioreactors.

In the present review an attempt was made to present an up-to-date amount of the data on electrochemical reduction of the hemoprotein cytochrome P450. The concept and potentialities of enzyme electrodes--transducers--as the main element for construction of electrochemical biosensors were discussed. Different types of electrodes for bioelectrochemistry were analysed. New nanotechnological approaches to cytochrome P450 immobilisation were reported. It was shown that nanobiotechnology in electrochemistry has potential application in manufacturing biosensors and bioreactors for clinical medicine and pharmacology.

Bioreactors↗

PubMed2004

Development of a bacterial biosensor for nitrotoluenes: the crystal structure of the transcriptional regulator DntR.

The transcriptional regulator DntR, a member of the LysR family, is a central element in a prototype bacterial cell-based biosensor for the detection of hazardous contamination of soil and groundwater by dinitrotoluenes. To optimise the sensitivity of the biosensor for such compounds we have chosen a rational design of the inducer-binding cavity based on knowledge of the three-dimensional structure of DntR. We report two crystal structures of DntR with acetate (resolution 2.6 angstroms) and thiocyanate (resolution 2.3 angstroms), respectively, occupying the inducer-binding cavity. These structures allow for the construction of models of DntR in complex with salicylate (Kd approximately or = 4 microM) and 2,4-dinitrotoluene that provide a basis for the design of mutant DntR with enhanced specificity for dinitrotoluenes. In both crystal structures DntR crystallises as a homodimer with a "head-to-tail" arrangement of monomers in the asymmetric unit. Analysis of the crystal structure has allowed the building of a full-length model of DntR in its biologically active homotetrameric form consisting of two "head-to-head" dimers. The implications of this model for the mechanism of transcription regulation by LysR proteins are discussed.

Base Sequence↗

PubMed2005

A bacterial biosensor of endocrine modulators.

The nuclear hormone receptors comprise one of the largest classes of protein targets for drug discovery, as their function has been linked to a variety of serious diseases, including several forms of cancer. Identifying novel compounds with the ability to modulate the function of these targets could lead to the development of effective therapeutics. In vivo sensors of ligand binding have emerged as tools that can greatly accelerate the lead identification process, allowing new drugs to be discovered more rapidly and cheaply. In this work, a novel sensor of nuclear hormone binding has been developed in Escherichia coli by constructing a fusion of the ligand-binding domain of the human estrogen receptor with a thymidylate synthase enzyme (TS). Expression of this fusion protein in TS-deficient bacterial cells resulted in growth phenotypes that were dependent on the presence of estrogen. Subsequent replacement of the estrogen receptor with the ligand-binding domain of the human thyroid hormone receptor led to specific thyroid hormone-enhanced growth that was insensitive to estrogen. This biosensor was then challenged with a small library of estrogen and thyroid hormone analogues, and it was observed that levels of cell growth correlate well with ligand-binding affinity. Remarkably, this simple biosensor was able to discriminate between agonistic and antagonistic activities, as combinations of estrogen agonists had an additive impact on cell growth, whereas known estrogen antagonists were found to neutralize agonist effects. This system constitutes a technique for facile selection of lead compounds with potential medical applications.

Biosensing Techniques↗

PubMed2004

On-line assay of the S-enantiomers of enalapril, ramipril and pentopril using a sequential injection analysis/amperometric biosensor system.

A sequential injection analysis/amperometric biosensor system is proposed for the enantioselective analysis of the S-enantiomer of enalapril, ramipril and pentopril. The amperometric biosensor used as detector in the sequential injection analysis was designed by immobilization of l-amino acid oxidase in carbon paste. The proposed SIA system can be utilized reliably for the enantioanalysis of the S-enantiomer from the raw materials as well as from their pharmaceutical formulations, with a rate of 75 samples per hour and R.S.D. values better than 0.1% (n = 10).

Biosensing Techniques↗

PubMed2004

Amperometric biosensor based on horseradish peroxidase for biogenic amine determinations in biological samples.

An amperometric biosensor for total biogenic amine determinations, using a carbon paste electrode modified with horseradish peroxidase (HRP) enzyme is described. The HRP immobilization on graphite was made using bovine serum albumin, carbodiimide and glutaraldehyde. The biosensor response was optimized using serotonin and it presented the best performance in 0.1 mol l(-1) phosphate buffer (pH=7.0) containing 10 micromol l(-1) of hydrogen peroxide. Under optimized operational conditions at -50 mV versus SCE, a linear response range from 40 to 470 ng ml(-1) was obtained. The detection limit was 17 ng ml(-1) and the response time was 0.5s. The proposed sensor presented a stable response during 4h under continuous monitoring. The difference of the response between six sensor preparations was <2%. The sensor was applied in the determination of total biogenic amines (neurotransmitters) in rat blood samples with success, obtaining a recovery average of 102%.

Animals↗

PubMed2006

Magnetic biosensor for the detection of Yersinia pestis.

A novel type of magnetic-beads based magnetic biosensor is described for the detection of Yersinia pestis. Experiments were performed with the antigen fraction F1 of these bacteria. The magnetic sensor platform offers easy and reliable detection of Y. pestis by the use of magnetic beads for labelling and quantification in a single step due to their paramagnetic features. The system uses antiYPF1 antibodies as capture element on ABICAP columns as core element of the magnetic sensor. Several immobilization methods for antibodies on polyethylene were exploited. The established biosensor has a linear detection range of 25-300 ng/ml Y. pestis antigen F1 and a detection limit of 2.5 ng/ml in buffer and human blood serum. The presented sensor system is small, simple, portable and therefore usable as off-lab detection unit for medical and warfare analytes.

Animals↗

PubMed2005

Shining light on signaling and metabolic networks by genetically encoded biosensors.

Fluorescent labels have revolutionized cell biology. Signaling intermediates and metabolites can be measured in real time with subcellular spatial resolution. Most of these sensors are based on fluorescent proteins, and many report fluorescence resonance energy transfer. Because the biosensors are genetically encoded, a toolbox for addressing cell biological questions at the systems level is now available. Fluorescent biosensors are able to determine the localization of proteins and their dynamics, to reveal the cellular and subcellular localization of the respective interactions and activities, and to provide complementary data on the steady state levels of ions, metabolites, and signaling intermediates with high temporal and spatial resolution. They represent the basis for cell-based high-throughput assays that are necessary for a systems perspective on plant cell function.

Biosensing Techniques↗

PubMed2005

Developing implantable optical biosensors.

Nanobiotechnologists are developing devices that can measure specific enzymes and proteins. These devices are expected to detect single enzyme or protein molecules accurately, providing highly sensitive biosensing applications. A recent study by Strano and co-workers shows that single-walled carbon nanotubes (SWNTs) hold great promise as implantable biosensors. Although most researchers have focused on substrate-oriented biosensors, Strano and colleagues have shown that the inherent fluorescent properties of suspended individual SWNTs can be used for solution-phase beta-D-glucose sensing.

Animals↗

PubMed2002

Biosensor analysis of dynamics of interleukin 5 receptor subunit beta(c) interaction with IL5:IL5R(alpha) complexes.

To gain insight into IL5 receptor subunit recruitment mechanism, and in particular the experimentally elusive pathway for assembly of signaling subunit beta(c), we constructed a soluble beta(c) ectodomain (s(beta)(c)) and developed an optical biosensor assay to measure its binding kinetics. Functionally active s(beta)(c) was anchored via a C-terminal His tag to immobilized anti-His monoclonal antibodies on the sensor surface. Using this surface, we quantitated for the first time direct binding of s(beta)(c) to IL5R(alpha) complexed to either wild-type or single-chain IL5. Binding was much weaker if at all with either R(alpha) or IL5 alone. Kinetic evaluation revealed a moderate affinity (0.2-1 microM) and relatively fast off rate for the s(beta)(c) interaction with IL5:R(alpha) complexes. The data support a model in which beta(c) recruitment occurs with preformed IL5:R(alpha) complex. Dissociation kinetics analysis suggests that the IL5-alpha-beta(c) complex is relatively short-lived. Overall, this study solidifies a model of sequential recruitment of receptor subunits by IL5, provides a novel biosensor binding assay of beta(c) recruitment dynamics, and sets the stage for more advanced characterization of the roles of structural elements within R(alpha), beta(c), and cytokines of the IL5/IL3/GM-CSF family in receptor recruitment and activation.

Animals↗

PubMed2002

Biosensor detection of triplex formation by modified oligonucleotides.

Due to the instability of DNA oligonucleotides in biological solutions, antisense or antigene therapies aimed at modulation of specific gene expression will most likely require the use of oligonucleotides with modified backbones. Here, we examine the use of a surface plasmon resonance biosensor (BIAcore) to compare triplex-directed binding of modified oligonucleotides targeted to a region of the murine c-myc promoter. We describe optimization of experimental conditions to minimize nonspecific interactions between the oligonucleotides and the sensor chip surface, and the limitations imposed by certain backbones and sequence types. The abilities of pyrimidine oligonucleotides with various modified backbones to form specific triple helices with an immobilized hairpin duplex were readily determined using the biosensor. Modification of the third-strand oligonucleotide with RNA or 2(')-O-methyl RNA was found to enhance triplex formation, whereas phosphorothioate or phosphotriester substitutions abrogated it. A comparison of these results to DNase I footprinting experiments using the same oligonucleotides showed complete agreement between the two sets of data.

DNA↗

PubMed2002

Development of a biosensor-based method for detection and isotyping of antibody responses to adenoviral-based gene therapy vectors.

A biosensor-based assay, using a surface plasmon resonance detection system, was developed to detect and isotype anti-adenoviral antibodies in patients dosed with an adenoviral-based gene therapy vector. In the assay, whole, intact virus was immobilized onto the sensor chip surface. Electron microscopy and monoclonal antibody studies provide evidence that the virus remains intact after immobilization. The patients tested had preexisting serum levels of anti-adenoviral antibodies. A classic anamnestic response was observed in patients dosed with the gene-therapy agent. Isotyping experiments indicated that IgG antibodies predominated in serum even at the predose time point. Analysis of ascites fluid samples from some patients indicated detectable levels of IgA in addition to IgG. Results obtained using the biosensor-based assay corresponded to an existing enzyme-linked immunosorbent assay. The assay was easy to perform and the automated instrument reduced the required "hands on" time. In addition to studying the development of anti-adenoviral antibodies, the techniques described may be applied to virus:receptor interaction studies or antiviral drug:virus interaction studies.

Adenoviridae↗

PubMed2003

Hybridization biosensor using di(2,2'-bipyridine)osmium (III) as electrochemical indicator for detection of polymerase chain reaction product of hepatitis B virus DNA.

A novel hepatitis B virus (HBV) DNA biosensor was developed by immobilizing covalently single-stranded HBV DNA fragments to a gold electrode surface via carboxylate ester to link the 3(')-hydroxy end of the DNA with the carboxyl of the thioglycolic acid (TGA) monolayer. A short-stranded HBV DNA fragment (181bp) of known sequence was obtained and amplified by PCR. The surface hybridization of the immobilized single-stranded HBV DNA fragment with its complementary DNA fragment was evidenced by electrochemical methods using [Os(bpy)(2)Cl(2)](+) as a novel electroactive indicator. The formation of double-stranded HBV DNA on the gold electrode resulted in a great increase in the peak currents of [Os(bpy)(2)Cl(2)](+) in comparison with those obtained at a bare or single-stranded HBV DNA-modified electrode. The mismatching experiment indicated that the surface hybridization was specific. The difference between the responses of [Os(bpy)(2)Cl(2)](+) at single-stranded and double-stranded DNA/TGA gold electrodes suggested that the label-free hybridization biosensor could be conveniently used to monitor DNA hybridization with a high sensitivity. X-ray photoelectron spectrometry technique has been employed to characterize the immobilization of single-stranded HBV DNA on a gold surface.

2,2'-Dipyridyl↗

PubMed2003

Aptamer-based biosensor arrays for detection and quantification of biological macromolecules.

We have developed a chip-based biosensor for multiplex analysis of protein analytes. The biosensor utilizes immobilized DNA and RNA aptamers, selected against several different protein targets, to simultaneously detect and quantify levels of individual proteins in complex biological mixtures. Aptamers were each fluorescently labeled and immobilized on a glass substrate. Fluorescence polarization anisotropy was used for solid- and solution-phase measurements of target protein binding. We show that solid-phase aptamer-protein interactions recapitulate binding interactions seen in solution. Furthermore, we demonstrate specific detection and quantitation of cancer-associated proteins (inosine monophosphate dehydrogenase II, vascular endothelial factor, basic fibroblast growth factor) in the context of human serum and in cellular extracts. It is expected that this technology could speed diagnosis of cancer by enabling direct detection of the expression and modification of proteins closely correlated with disease.

Anisotropy↗

PubMed2001

Synthesis of the chelator lipid nitrilotriacetic acid ditetradecylamine (NTA-DTDA) and its use with the IAsys biosensor to study receptor-ligand interactions on model membranes.

This work describes the synthesis and use of the chelator lipid, nitrilotriacetic acid ditetradecylamine (NTA-DTDA). This lipid is readily dispersed in aqueous media, both alone and when mixed with carrier lipids like dimyristoylphosphatidylcholine (DMPC). Fluorescence microscopic examination of membranes deposited from NTA-DTDA-containing liposomes shows that NTA-DTDA mixes uniformly with the carrier lipid, and does not phase separate. NTA-DTDA-membranes deposited onto the sensing surface of IAsys biosensor cuvettes show good stability, permitting use of the biosensor to study protein interactions. Hexahistidine-tagged proteins including recombinant forms of the extracellular regions of murine B7.1 (B7.1-6H) and of the human erythropoietin receptor (EPOR-6H) bind to NTA-DTDA-membranes; the stability of binding is dependent on both protein concentration, and density of NTA-DTDA. Kinetic measurements show that high stability of anchored proteins (t(1/2) approximately 10-20 h, apparent K(d) approximately 1 nM) can be achieved using membranes containing 25 mol% NTA-DTDA, but low levels of bound protein (<200 arc seconds). The system is used to study the interaction of human EPO with the EPOR anchored onto NTA-DTDA-membranes. In addition to the biological applications reported recently, the results show that NTA-DTDA can be a useful reagent in the study of receptor-ligand interactions.

Amines↗

PubMed2003

Application of a real-time biosensor to detect bacteria in platelet concentrates.

A spore-based biosensor for detecting low levels of bacteria in real-time has been recently developed. The system (termed LEXSAS, label-free exponential signal-amplification system) exploits spore's ability to produce fluorescence when sensing neighboring bacterial cells. We studied the LEXSAS as a possible approach for identifying bacterially contaminated platelet concentrates prior to transfusion because the system offers rapid analysis, high sensitivity, and low cost. If successful, this approach could reduce the risk of morbidity and mortality from transfusion-related bacteremia and sepsis. In this study, we used the LEXSAS for detecting bacteria in platelet concentrates spiked with Bacillus cereus, Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, or Streptococcus pyogenes. Bacteria were separated from platelets using a 2-min procedure based on bacterial resistance to detergents and osmotic shock. The results indicate that the LEXSAS could be used to design a practical biosensor for identifying bacterially contaminated platelets in real-time.

Aminopeptidases↗