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At least 433 records · Page 24Linked to original sources

PubMed2007

Serum cytochrome c detection using a cytochrome c oxidase biosensor.

Within the last ten years it has emerged that the release of cytochrome c plays a critical role in the important process of programmed cell death. It has also been shown that this protein is released into the circulating blood following MIs (myocardial infarctions). Methods for the detection of this protein have therefore become important. The enzyme cytochrome c oxidase is specific for cytochrome c. Bovine cytochrome c oxidase was successfully immobilized in a didodecyldimethylammonium bromide vesicular system on to gold electrodes and its interaction with cytochrome c was studied. Square-wave-voltammetric analysis of the biosensor showed two redox couples with midpoint potential, E(0)', values of +182 and +414 mV compared with Ag/AgCl. The redox couple with E(0)' of +414 mV showed a cathodic sensitivity to the presence of cytochrome c in both buffer solution and human serum. Responses of the cytochrome c oxidase biosensor to oxidized cytochrome c followed hyperbolic electrochemical Michaelis-Menten kinetics with a K(m) of 1.57 microM and maximum current (I(max)) of 1.38 x 10(-6) muA. The detection limit of the biosensor in human serum was 0.2 microM, which is well below the lowest physiological concentration of 0.8 muM previously reported for MIs [Alleyne, Joseph and Sampson (2001) Appl. Biochem. Biotechnol. 90, 97-105]. These results indicate that the cytochrome c oxidase biosensors could be used to determine variations in cytochrome c concentration and thus have potential to be used as a diagnostic tool in the detection of MIs and possibly also in the study of programmed cell death.

Biosensing Techniques↗

PubMed2002

Characterization of autoantibodies from patients with Goodpasture's disease using a resonant mirror biosensor.

Goodpasture's disease is characterized by the binding of IgG autoantibodies to the glomerular basement membrane, leading to glomerular inflammation. The autoantigen has been identified as the noncollagenous domain of the alpha3 chain of type IV collagen (alpha3(IV)NC1). We have used the IAsys resonant mirror biosensor to analyse the extent and affinity of binding of anti-GBM antibodies from sera of patients to purified alpha3(IV) NC1. alpha3(IV) NC1 monomers were immobilized to a carboxylate cuvette, with the simultaneous use of a control well. The binding of serum from patients with Goodpasture's disease (n = 12), normal controls (n = 14) and disease controls with vasculitis (n = 14) was analysed. Antibody binding was detected in sera from all patients with Goodpasture's disease but not from controls. IAsys measurements of binding correlated with antibody levels assessed by the standardized ELISA used for clinical assays. Both ELISA and biosensor measurements showed declining antibody levels in serial serum samples from treated patients; however, the biosensor detected antibody recrudescence when ELISA remained negative. Autoantibodies from patients' serum had average affinity constants (Kd) of 6.5 x 10-11M to 52.07 x 10-10M, as determined by an inhibition assay, indicating high affinity. Sips analysis showed that the antibody response was relatively homogeneous (values of 0.46-1). Biosensor techniques can therefore be used to detect and characterize anti-GBM antibodies in serum from patients, with high sensitivity and without need for antibody purification. This technique may be useful in diagnosis and monitoring of patients with Goodpasture's disease, and may be applicable to other autoantibody mediated diseases.

Adolescent↗

PubMed2006

Development of glucose biosensor by using gelatin and gelatin-polyacrylamide supporting systems.

In this work an amperometric glucose biosensor based on surface immobilization method was developed. Glutaraldehyde was used as cross-linker to establish the immobilization of glucose oxidase onto gelatin (carrier/coating reagent). In order to increase the porosity of coating material, immobilization media was further treated by polyacrylamide. Although this treatment increased the performance of biosensor to a large extent with respect to current densities obtained, it negatively affected the long-term stability. Our biosensor showed linear response in the physiological range of blood glucose (0.05 to 6 mM), had an acceptable response time (60 seconds) and was stable for 17 repeated usages in 51 days. We obtained best results with pH values very close to physiological pH and our biosensor could work efficiently in the tested temperature range 15 to 65 degrees C.

Acrylic Resins↗

PubMed2006

Detection of glucose and related analytes by biosensors: a fractal analysis.

A fractal analysis is used to model the binding and dissociation kinetics of connective tissue interstitial glucose, adipose tissue interstitial glucose, insulin, and other related analytes on biosensor surfaces. The analysis provides insights into diffusion-limited analyte-receptor reactions occurring on heterogeneous biosensor surfaces. Numerical values obtained for the binding and the dissociation rate coefficients are linked to the degree of heterogeneity or roughness [fractal dimension (D(f))] present on the biosensor chip surface. The binding and dissociation rate coefficients are sensitive to the degree of heterogeneity on the surface. For example, for the binding of plasma insulin, as the fractal dimension value increases by a factor of 2.47 from D(f1)=0.6827 to D(f2)=1.6852, the binding rate coefficient increases by a factor of 4.92 from k(1)=1.0232 to k(2)=5.0388. An increase in the degree of heterogeneity on the probe surface leads to an increase in the binding rate coefficient. A dual-fractal analysis is required to fit the binding kinetics in most of the cases presented. A single fractal analysis is adequate to describe the dissociation kinetics. Affinity (ratio of the binding to the dissociation rate coefficient) values are also presented. Interferents for glucose, such as uric acid and ascorbic acid, were also detected by using glucose biosensors based on carbon nanotube (CNT) nanoelectrode ensembles (NEEs) (Lin Y, Lu F, Tu Y, Ren Z).

Adipose Tissue↗

PubMed2006

Fractal binding and dissociation kinetics of heart-related compounds on biosensor surfaces.

A fractal analysis is presented for the binding and dissociation of different heart-related compounds in solution to receptors immobilized on biosensor surfaces. The data analyzed include LCAT (lecithin cholesterol acyl transferase) concentrations in solution to egg white apoA-I rHDL immobilized on a biosensor chip surface (1), native, mildly oxidized, and strongly oxidized LDL in solution to a heparin-modified Au-surface of a surface plasmon resonance (SPR) biosensor (2), and TRITC-labeled HDL in solution to a bare optical fiber surface (3). Single-and dual-fractal models were used to fit the data. Values of the binding and the dissociation rate coefficient(s), affinity values, and the fractal dimensions were obtained from the regression analysis provided by Corel Quattro Pro 8.0 (4). The binding rate coefficients are quite sensitive to the degree of heterogeneity on the sensor chip surface. Predictive equations are developed for the binding rate coefficient as a function of the degree of heterogeneity present on the sensor chip surface and on the LCAT concentration in solution and for the affinity as a function of the ratio of fractal dimensions present in the binding and the dissociation phases. The analysis presented provided physical insights into these analyte-receptor reactions occurring on different biosensor surfaces.

Animals↗

PubMed1998

Biosensors for the enantioselective analysis of S-enalapril and S-ramipril.

S-Enalapril, and S-ramipril are angiotensin-converting enzyme (ACE) inhibitors which are used for treatment of hypertension. Due to the fact that only the S enantiomer possesses the ACE inhibiting activity, it is necessary to develop an enantioselective analytical method for its discrimination from the less active R-enantiomer. An amperometric biosensor, based on L-amino acid oxidase, was developed and proved reliable for the analysis of the S-enantiomer of these ACE inhibitors. The working range of the biosensor for S-enalapril assay (A) is 0.4-120 mumol/L, and for S-ramipril assay (B) is 0.2-100 mumol/L, with a limit of detection of 163 nmol/L (A) and 107 nmol/L (B), respectively. It is of interest to mention that the biosensors demonstrated enantioselectivity versus D-proline (1.4 x 10(-3) mol/L(A), 5.3 x 10(-3) mol/L(B) and also the selectivity versus the polyvinylpyrolidone (3.0 x 10(-3) mol/L(A), 3.2 x 10(-3) mol/L(B), respectively. The working pH ranges are: 6.8-7.4 (A), and 6.2-7.0 (B), respectively. The RSD < 1% assured by using the amperometric biosensors for S enantiomers assay in raw materials, in tablet formulations, and their suitability for the analysis of these drug enantiomers.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2003

Use of biosensors to screen urine samples for potentially toxic chemicals.

Forensic toxicology laboratories are often required to implicate or exclude poisoning as a factor in a death or unexplained illness. An analytical tool which enables toxicologists to screen a wide variety of common poisons would be extremely useful. In this paper, we describe the use of a bacterial biosensor for detecting the presence of commonly encountered potentially toxic chemicals in urine. The biosensor responds to any chemical that causes metabolic stress to the bacterial cell and the response is in direct proportion to the concentration of the stressor. This allows a measure of the concentration of a toxicant in urine, without knowing exactly what the toxic compound(s) may be. This affords a distinct advantage over conventional analytical techniques, which require an extensive screening program before it is even known that a toxic compound is present. This preliminary investigation has shown that this biosensor can indicate the presence, in urine, of herbicides such as glyphosate, 2,4-dichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxyacetic acid; the biocide pentachlorophenol; or inorganic poisons such as arsenic, mercury, and cyanide. The biosensor was also shown to be sensitive to a concentration range of these toxicants likely to be found in samples submitted for toxicological analysis.

Biosensing Techniques↗

PubMed1995

Monitoring of mixed venous oxygen saturation and pressure from biosensors in the right ventricle. A 24 hour study in patients with heart failure.

Right ventricle venous oxygen saturation was studied in 10 heart failure patients over 24 h using a lead equipped with an oxygen biosensor inserted temporarily. At the same time a pressure sensor, incorporated in the same lead to measure right ventricular pulse pressure and maximum positive and negative rate of pressure development (RV dP/dt), was tested to see whether it would interfere with the oxygen sensor. Data from the biosensor lead were continuously compared with mixed venous oxygen saturation obtained from a fibreoptic Swan-Ganz catheter with the tip in the pulmonary artery. For reference, blood samples were drawn at regular intervals from this catheter. A provocative protocol was used to cause haemodynamic changes. There was good correlation between oxygen saturation in the right ventricle (biosensor-derived) and mixed venous oxygen saturation in the pulmonary artery (fibreoptic-derived) (r = 0.86) and between sensor-obtained right ventricular oxygen saturation and oxygen saturation in the blood samples from the pulmonary artery (r = 0.90). Changes in central haemodynamics were also well reflected by changes in pulse pressure and dP/dt derived from the pressure sensor. As it was not possible to obtain absolute pressures no attempt was made to compare the pressure changes with conventional haemodynamics. These 24 h haemodynamic measurements suggest that a biosensor-equipped lead placed in the right ventricle could be a valuable tool for long-term monitoring of mixed venous oxygen saturation and pressure in patients with congestive heart failure.

Aged↗

PubMedDate not supplied

Assay development for a portable fiberoptic biosensor.

The fiberoptic biosensor with tapered optical probes has been developed to perform rapid and sensitive fluoroimmunoassays. A number of assays for biologic analytes were developed using a laboratory breadboard device that employed a large, 514 nm argon ion laser. These assays, with limits of detection of 5-50 ng/ml for protein antigens, showed promise for clinical use because of their demonstrated lack of matrix effects from plasma, seru, or blood. However, such a large device was impractical for on-site diagnostics, so a new, portable, multichannel biosensor was developed. To test this new biosensor, which uses 635 nm laser diodes, the assays were converted to use the cyanine dye, Cy5. The detection antibodies were labeled with Cy5 and assays performed to detect the F1 antigen of Yersinia pestis and the protective antigen of Bacillus anthracis. The limit of detection was found to improve by a factor of 10 for each assay. The portable biosensor was then evaluated in a blind test containing F1 antigen spiked into 30 of 173 serum samples. One hundred percent detection was achieved for samples with 100 ng/ml or more F1 antigen, with a specificity of 88%.

Animals↗

PubMed2006

Computational modeling of a new fluorescent biosensor for caspase proteolytic activity improves dynamic range.

The class of fluorescence resonance energy transfer (FRET) protein biosensors that are useful for measuring protease activity is composed of a tandem fusion of yellow fluorescent protein (YFP), a cleavage recognition sequence, and cyan fluorescent protein (CFP). The dynamic range of these FRET-based protein biosensors is often weak, but applications such as high throughput drug screening require stronger dynamic ranges. Using the biosensor for the caspase-3 protease as an example, here we showed a computational approach to improve the FRET dynamic range based on the atomic structure of caspase-3 bound to its inhibitor. This result was verified from our experiments where the FRET dynamic range improved by at least 60% on average in both in vitro and in vivo contexts. In concept, the same strategy can be applied to improve dynamic range of other FRET-based protein biosensors for protease activity where there exist solved atomic structures for protein complexes.

Caspase 3↗

PubMed2004

The role of host organism, transcriptional switches and reporter mechanisms in the performance of Hg-induced biosensors.

AIMS: The purpose of this study was to comprehensively compare the response of nine biosensors capable of being induced by Hg. Induction by Hg was based upon the insertion of merR, merB, zntA and zntR promoter genes. LuxCDABE or lucFF reporter genes expressed luminescence, and host organisms were Escherichia coli, Vibrio anguillarum and Pseudomonas fluorescens. The role of transcriptional switches, reporter mechanism and host organism was to be investigated. METHODS AND RESULTS: All biosensors were subjected to the same assay conditions. Sensors had their own individual growth characteristics and response to the doses of Hg tested. Maximum bioluminescence response was induced by concentrations of Hg between 2.5 nm and 5 microM. E. coli pRB28 was found to detect levels of Hg as low as 1.6 nm and yet was capable of operating in a concentration range of up to 12.5 microM. CONCLUSIONS: The response of the sensors demonstrated their suitability for analysis under environmentally relevant concentrations. The sensitivity of the sensors, the optimum range and the expediency of the assay could not be related to a single sensor trait. It may be concluded that biosensor performance is dependent on more than one of the single factors studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show that comparative testing of sensors is an important step in evaluating the relevance and performance of biosensors prior to routine environmental application.

Biological Availability↗

PubMed2006

Quantitative in situ assay of salicylic acid in tobacco leaves using a genetically modified biosensor strain of Acinetobacter sp. ADP1.

Salicylic acid (SA) plays important roles in plants, most notably in the induction of systemic acquired resistance (SAR) against pathogens. A non-destructive in situ assay for SA would provide new insights into the functions of SA in SAR and other SA-regulated phenomena. We assessed a genetically engineered strain of Acinetobacter sp. ADP1, which proportionally produces bioluminescence in response to salicylates including SA and methylsalicylate, as a reporter for salicylate accumulation in the apoplast of plant leaves. SA was measured quantitatively in situ in NN genotype tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaves inoculated with tobacco mosaic virus (TMV). The biosensor revealed accumulation of apoplastic SA before the visible appearance of hypersensitive response (HR) lesions. When the biosensor was infiltrated into TMV-inoculated leaves displaying HR lesions at 90 and 168 h post-inoculation, salicylate accumulation was detected predominantly in tissues surrounding the lesions and in veins adjacent to HR lesions. These images are consistent with previous data demonstrating that SA accumulation occurs prior to and following the onset of visible HR lesions. We also used the biosensor to observe apoplastic SA accumulation in tobacco leaves inoculated with virulent and HR-eliciting strains of the bacterial plant pathogen Pseudomonas syringae. The work demonstrates that the Acinetobacter sp. ADP1 biosensor is a useful new tool to non-destructively assay salicylates in situ and to map their spatial distribution in plant tissues.

Acinetobacter↗

PubMed2006

Application of an Escherichia coli green fluorescent protein-based lysine biosensor under nonsterile conditions and autofluorescence background.

AIMS: To examine the utility of an Escherichia coli green fluorescent protein (GFP) containing biosensor for quantification of bioavailable lysine in selected feed samples under nonsterile conditions and to estimate the background fluorescence of analyzed feed samples and evaluate the risk of confounding GFP emission from the lysine assay organism. METHODS AND RESULTS: Escherichia coli lysine auxotroph GFP based biosensor was used to determine the percentage of bioavailable lysine in two samples of soybean-, cottonseed-, and meat and bone meal under nonsterile conditions. The fluorescence emitted by GFP was successfully measured using a spectrofluorimeter to monitor bacterial growth response to protein-derived lysine and lysine containing small peptides. The autofluorescence of analyzed feed samples at different concentrations could also be estimated. CONCLUSIONS: When feed protein concentrations are decreased, autofluorescence interference can be avoided. SIGNIFICANCE: The E. coli lysine auxotroph GFP-based biosensor can successfully be used for the determination of bioavailable lysine in these selected animal feed proteins under nonsterile conditions. IMPACT OF THE STUDY: E. coli GFP biosensor for lysine has potential for routine application in animal feeds.

Animal Feed↗

PubMed1998

Biosensors for environmental monitoring.

Environmental monitoring is of great importance for its protection. Conventional monitoring methods are often slow and complex and require expensive equipment, making them unsuitable for in situ, real-time monitoring of pollutants. Biosensors based on a combination of a biological sensing element and an electronic signal-transducing element are alternative methods to conventional ones. Biosensors have a number of advantages, such as high selectivity, high stability, and short response time. Various kinds of biosensors have been developed and employed for detection of pollutants such as phosphate, cyanide, and herbicides. Some of these have already been exploited as real-time monitoring in situ. In this article, some of the applications of biosensors for environmental control are described.

Biosensing Techniques↗

PubMed2001

Detection of oxytetracycline production by Streptomyces rimosus in soil microcosms by combining whole-cell biosensors and flow cytometry.

Combining the high specificity of bacterial biosensors and the resolution power of fluorescence-activated cell sorting (FACS) provided qualitative detection of oxytetracycline production by Streptomyces rimosus in soil microcosms. A plasmid containing a transcriptional fusion between the tetR-regulated P(tet) promoter from Tn10 and a FACS-optimized gfp gene was constructed. When harbored by Escherichia coli, this plasmid produces large amounts of green fluorescent protein (GFP) in the presence of tetracycline. This tetracycline biosensor was used to detect the production of oxytetracycline by S. rimosus introduced into sterile soil. The tetracycline-induced GFP-producing biosensors were detected by FACS analysis, enabling the detection of oxytetracycline encounters by single biosensor cells. This approach can be used to study interactions between antibiotic producers and their target organisms in soil.

Biosensing Techniques↗

PubMed2002

A novel fluorescent protein-based biosensor for gram-negative bacteria.

Site-directed mutagenesis of enhanced green fluorescent protein (EGFP) based on rational computational design was performed to create a fluorescence-based biosensor for endotoxin and gram-negative bacteria. EGFP mutants (EGFP(i)) bearing one (G10) or two (G12) strands of endotoxin binding motifs were constructed and expressed in an Escherichia coli host. The EGFP(i) proteins were purified and tested for their efficacy as a novel fluorescent biosensor. After efficient removal of lipopolysaccharide from the E. coli lysates, the binding affinities of the EGFP(i) G10 and G12 to lipid A were established. The K(D) values of 7.16 x 10(-7) M for G10 and 8.15 x 10(-8) M for G12 were achieved. With high affinity being maintained over a wide range of pH and ionic strength, the binding of lipid A/lipopolysaccharide to the EGFP(i) biosensors could be measured as a concentration-dependent fluorescence quenching of the EGFP mutants. The EGFP(i) specifically tagged gram-negative bacteria like E. coli and Pseudomonas aeruginosa, as well as other gram-negative bacteria in contaminated water sampled from the environment. This dual function of the EGFP(i) in detecting both free endotoxin and live gram-negative bacteria forms the basis of the development of a novel fluorescent biosensor.

Biosensing Techniques↗

PubMed2005

Beta-adrenergic- and muscarinic receptor-induced changes in cAMP activity in adult cardiac myocytes detected with FRET-based biosensor.

beta-Adrenergic receptor activation regulates cardiac myocyte function through the stimulation of cAMP production and subsequent activation of protein kinase A (PKA). Furthermore, muscarinic receptor activation inhibits as well as facilitates these cAMP-dependent effects. However, it has not always been possible to correlate the muscarinic responses with the direct measurement of changes in cellular cAMP activity. Genetically encoded biosensors have recently been developed, making it possible to monitor real-time changes in cAMP and PKA activity at the single cell level. One such biosensor consists of the regulatory and catalytic subunits of PKA labeled with cyan and yellow fluorescent proteins, respectively. Changes in cAMP activity affecting the association of these labeled PKA subunits can be detected as changes in fluorescence resonance energy transfer. In the present study, an adenovirus-based approach was developed to express this recombinant protein complex in adult cardiac myocytes and use it to monitor changes in cAMP activity produced by beta-adrenergic and muscarinic receptor activation. The biosensor expressed with the use of this system is able to detect changes in cAMP activity produced by physiologically relevant levels of beta-adrenergic receptor activation without disrupting normal functional responses. It was also possible to directly demonstrate the complex temporal pattern of inhibitory and stimulatory changes in cAMP activity produced by muscarinic receptor activation in these cells. The adenovirus-based approach we have developed should facilitate the use of this biosensor in studying cAMP and PKA-dependent signaling mechanisms in a wide variety of cell types.

Adenoviridae↗

PubMed2005

Immobilization of glucose oxidase onto gelatin for biosensor construction.

The properties of a glucose biosensor made by immobilization of glucose oxidase onto gelatin in a layer of electrochemically deposited polyaniline have been investigated. Glucose oxidase was immobilized within gelatin cross-links with chromium(III) acetate. The glucose oxidase biosensor was developed by forming a polyaniline-deposited electrode surface as support for the immobilized enzyme gel, in order to increase its durability. The polyaniline/gelatin/glucose oxidase biosensor has been characterized using chemical and electrochemical methods. Temperature, pH, cross-linking agent concentration, enzyme concentration, kinetic properties, reusability and the effect of electro-active compounds were among the parameters studied. The response time of the glucose oxidase biosensor is 90 s, the detection limit is below 1 mmol/dm3 and the sensor can be used 20 times within a 2-month period without losing its stability.

Acetates↗