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PubMed1997

Detection and epitope mapping of immunoreactive human endothelin-1 using ELISA and a surface plasmon resonance-based biosensor.

A surface plasmon resonance-based biosensor (BIA technology) and enzyme-linked immunosorbent assays (ELISA) have been used for detecting and characterizing human endothelin (ET), a potent vasoactive 21 amino acid polypeptide. Antibodies produced against the isoform ET-1 and its C-terminal eptapeptide ET-1(15-21) have been characterized with respect to their binding capacity to the two isoforms ET-1 and ET-3, the non-secreted portion of the precursor molecule Big.ET-1(22-38), the C-terminal of ET-1, six analogues of ET-1(16-21) each containing a substitution with Ala of a single amino acid in positions 16-21, respectively, and three synthetic cyclic peptides mimicking the N-terminal portion of ET-1. Antibodies reacting with ET-1 also bound to ET-1(16-21) and, with less affinity, to ET-3 but did not cross-react with Big.ET-1(22-38). Ala substitution in positions 16, 17 and 19 of ET-1(16-21) hardly affected the antibody binding capacity of ET-1(16-21), whereas Ala substitution of Asp18, Ile20 and, in particular, Trp21, inhibited its immunoreactivity. The C-terminus thus represents an immunodominant epitope in ET-1 and is important for antibody binding. Epitope mapping using as antibody pairs polyclonal anti-ET-1 and monoclonal anti-ET-1(15-21) antibodies indicated the presence of another immunogenic domain in the N-terminal portion of the molecule. There was excellent agreement between the epitopes determined using ELISA and BIA analyses.

Amino Acid Sequence↗

PubMed1998

Electrosynthesized non-conducting polymers as permselective membranes in amperometric enzyme electrodes: a glucose biosensor based on a co-crosslinked glucose oxidase/overoxidized polypyrrole bilayer.

A glucose amperometric biosensor based on glucose oxidase immobilized on an overoxidized polypyrrole (PPyox) platinum modified electrode, by glutaraldehyde co-crosslinking with bovine serum albumine, is described. The advantages of covalent immobilization techniques (e.g. high loading and long-term stability of the enzyme) are coupled with the excellent interferent rejection of electrosynthesized non-conducting polymers. The sensor showed an apparent Michaelis-Menten constant of 16 +/- 0.8 mM, a maximum current density of 490 microA/cm2 and a shelf lifetime of at least 3 months. Ascorbate, urate, cysteine and acetaminophen at their maximum physiological concentrations produced a glucose bias in the low micromolar range. Flow-injection response was linear up to 20 mM glucose with typical sensitivity of 84.0 +/- 1.5 nA/mM. The sensor was tested for glucose determination of untreated serum samples from both normal and diabetic subjects; results of amperometric assay compared well with those obtained by a standard enzymatic-colorimetric method.

Animals↗

PubMed1998

Amperometric biosensor with a composite membrane of sol-gel derived enzyme film and electrochemically generated poly(1,2-diaminobenzene) film.

A sol-gel silicate-based biosensor for glucose was made by utilizing a composite membrane of sol-gel enzyme film and electrochemically generated poly(1,2-diaminobenzene) film to improve the selectivity of the sol-gel enzyme sensors. The stability of the sensor was improved by exposing the enzyme layer to glutaraldehyde vapor. The glucose sensor responded rapidly (ca. 15 s) to glucose at 0.6 V (versus Ag/AgCl), without any interferences from electroactive species such as L-ascorbate and urate below 0.2 mM. Reliable results were obtained in the assays of glucose in controlled human sera, with both the steady-state and flow-injection measurements. Subsequently, the same sol-gel procedure was applied to the preparation of the sensors for galactose and cholesterol. The galactose and cholesterol sensors responded rapidly to galactose and cholesterol, respectively, although the sensitivity of these sensors was inferior to that of glucose sensor.

Biocompatible Materials↗

PubMed1998

Application of a linear alkylbenzene sulfonate biosensor to river water monitoring.

A novel whole cell biosensor was constructed for the detection of anionic surfactants in aquatic environments. The analysis was rapid, convenient and did not require organic reagents. In this report, the application of this sensor to river water samples was investigated when applied to environmental samples; other organic substances present in river water may affect the measurement of linear alkylbenzene sulfonates. In order to deal with this problem, a correction system was developed using whole cells of Trichosporon cutaneum. This system was applied to in situ 24 h continuous monitoring in the Saka river.

Benzenesulfonates↗

PubMed1998

Influence of calcium on glucose biosensor response and on hydrogen peroxide detection.

Of small species capable of reaching a platinum working electrode from biological samples, calcium cations have been found to inhibit significantly glucose biosensor responses. The sensitivities to glucose of sensors immersed in carbonate buffer saline solutions decreased when 0.5 mM calcium chloride was added. The degree of inhibition was proportional to the glucose response in the absence of calcium (0-17% of the normalized current). Likewise, sensor sensitivities to hydrogen peroxide decreased, in the 5-90% range, in the presence of 0.5 mM calcium. Bare Pt-lr wires show a reversible inhibition of hydrogen peroxide sensitivity. This reversible inhibition is directly related to the decrease of hydrogen peroxide oxidation rate at the platinum anode: this has been evidenced, using rotating disk electrodes, by plotting Koutecky-Levich plots. Such inhibition has been found both for free and chelated calcium cations at levels below 1 mM. Several hypotheses for possible reactions between platinum, hydrogen peroxide and calcium are discussed.

Animals↗

PubMed1998

Genetic engineering of a single-chain antibody fragment for surface immobilization in an optical biosensor.

The development of a biosensor based on a genetically engineered biomolecule offers many potential advantages to sensors that rely on natural proteins only. Here we present how protein engineering techniques can be used to introduce a functional unit for surface immobilization into a single-chain antibody fragment (scFv). A peptide known to mimic the binding properties of biotin was fused to the carboxyterminus of the phosphorylcholine-binding scFv fragment of IgA McPC603. This fusion protein could be immobilized on a streptavidin monolayer. The resulting scFv monolayer was capable of binding a fluorescently labeled phosphorylcholine analog, as detected by total internal reflection fluorescence. In contrast, an scFv monolayer formed by introducing biotin through chemical modification was not capable of binding phosphorylcholine. These results demonstrate the utility of site-specific, oriented attachment strategies in the formation of protein monolayers in optical sensors, made possible by the use of protein engineering techniques.

Adhesiveness↗

PubMed1998

H+ISFET-based biosensor for determination of penicillin G.

A biosensor based on an H+ ion sensitive field effect transistor (H+(-)ISFET) and penicillin G acylase has been developed. The response time of the sensor to different concentrations of penicillin G was 30 s. In a 20 mM phosphate buffer at pH 7.0, the linear range of the calibration curve was from 0.5 to 8 mM. The coefficients of variation for three samples with 20 repeated measurements were below 5%. Stability of the sensor could reach about 6 months and more than 1000 runs were performed without a significant decrease of the output value. The sensor was tested for measurement of the penicillin G content in penicillin fermentation both. Forty samples with low and high concentrations of penicillin G were chosen for the correlation test. The values assayed by the sensor method were compared with the values assayed by HPLC method, the correlation coefficient (r) was 0.9944 and the regression equation was y = 1.034X - 2083.7, respectively. The different measuring methods are discussed in the text.

Biosensing Techniques↗

PubMed1998

Surface plasmon resonance biosensors as a tool in antibody engineering.

Modern gene technology combined with efficient microbial expression systems provides tools to produce antibodies with reduced functional size and improved binding properties as well as antibody fusions or novel antibodies. Surface plasmon resonance based biosensors, which measure antigen-antibody interactions in real-time, can be used for a diverse characterization of the modified antibodies. To date, the majority of published work originates from real-time biospecific interaction analysis based on the BIAcore instruments. This article describes the range of applications in antibody engineering in which BIAcore has been applied.

Animals↗

PubMed1998

A biosensor based on magnetoresistance technology.

We are developing a biosensor that will measure, at the level of single molecules, the forces that bind DNA-DNA, antibody-antigen, or ligand-receptor pairs together. The Bead Array Counter (BARC) will use these interaction forces to hold magnetic microbeads to a solid substrate. Microfabricated magnetoresistive transducers on the substrate will indicate whether or not the beads are removed when pulled by magnetic forces. By adapting magnetoresistive computer memory technology, it may be possible to fabricate millions of transducers on a chip and detect or screen thousands of analytes. The multi-analyte capability of this portable sensor would be ideal for on-site testing, while the potential to directly gauge intermolecular interaction strengths suggests drug discovery applications.

Biosensing Techniques↗

PubMed1998

DNA biosensors based on peptide nucleic acid (PNA) recognition layers. A review.

Peptide nucleic acid (PNA), originally developed as a gene-targeting drug, has demonstrated remarkable hybridization properties towards complementary oligonucleotides. Biosensors based on replacement of the DNA recognition layer with a PNA one, offer greatly improved distinction between closely related sequences, as well as several other attractive advantages. The present review discusses the unique structural, hybridization, and recognition features of PNA probes, along with the opportunities accrued from the use of PNA recognition layers in DNA diagnostics.

Animals↗

PubMed1998

Amperometric phenol biosensor based on a thermostable phenol hydroxylase.

Phenol hydroxylase (EC 1.14.13.7) was produced using Bacillus stearothermophilus in a 5-1 batch fermentation leading to approximately 17 units after 6 h. The partially purified phenol hydroxylase was entrapped in a sol-gel matrix. The enzyme-loaded silica gel was attached to the sensitive top of a Clark-type oxygen electrode and its application as a phenol biosensor was tested. There was linearity between the maximal rate of oxygen consumption and phenol concentration in the range between 2.5 and 400 microM at 40 degrees C and pH 7.6. The signal could be read off after 10 s at a concentration of 400 microM phenol. The sensor lost 20% of its activity within 7 days. Para-substituted phenols were not detectable.

Biosensing Techniques↗

PubMed1998

A new interference-free lysine biosensor using a non-conducting polymer film.

An electrochemical biosensor for the determination of lysine to be used for rapid evaluation of food quality has been developed. Platinum electrodes have been coated by electropolymerisation with 1,2-diaminobenzene (1.2-DAB) using cyclic voltammetry. The reduction in the oxidation of interferents compared with the bare platinum electrode was 100% for ascorbic acid, 99% for acetaminophen and 99% for cysteine. The enzyme L-lysine-alpha-oxidase was then immobilised onto the polymer layer by passive adsorption and a calibration curve for lysine constructed. This gave a linear range of 1 x 10(-5) mol/l to 1 x 10(-3) mol/l and a limit of detection of 2 x 10(-7) mol/l.

Biosensing Techniques↗

PubMed1999

Detecting staphylococcal enterotoxin B using an automated fiber optic biosensor.

The Man-portable Analyte Identification System (MANTIS), the first fully automated, self-contained, portable fiber optic biosensor, was utilized for the detection of Staphylococcal Enterotoxin B (SEB), a bacterial toxin produced by Staphylococcus aureus that commonly causes food poisoning. Because of its remarkable toxicity and stability, SEB is considered a prime threat as a biological weapon of mass destruction. The assay for SEB was used to evaluate the MANTIS' ability to function in the presence of various environmental interferents. The sensor could reliably detect SEB spiked into liquid samples containing a variety of smoke particles. However, substantial interference occurred when SEB was mixed into matrices capable of adsorbing SEB, such as 1% solutions of clay, topsoil, or pollen. Of equal importance, none of the interferents produced false positives in the MANTIS. The MANTIS demonstrated the capability to perform simultaneous immunoassays rapidly in the field with little or no user intervention.

Biosensing Techniques↗

PubMed1999

Needle-type lactate biosensor.

A needle-type lactate biosensor has been developed for continuous intravascular lactate monitoring. The sensor employs poly(1,3-phenylenediamine) as the inner layer on the platinum electrode in order to eliminate the interference from oxidizable physiological substances. Cross-linking with glutaraldehyde was used for enzyme immobilization. Dithiothreitol was used as the stabilizer of lactate oxidase. PVC (polyvinyl chloride) was chosen as the external diffusion control membrane. Sensor performance was evaluated in vitro and the sensor shows a sensitivity of 10-15 nA/mM, and a linear range from 1 mM to at least 15 mM lactate. Evaluation of the sensor response in blood plasma showed similar sensitivity and linear range as indicated by the calibration curves obtained in buffer solution. The sensor has a short response time of approximately 1 minute. The sensors were operated continuously for 7 days in phosphate buffer containing solution with a concentration at the physiological lactate level. No significant change in sensor sensitivity and its linear range has been observed. Sensors show a minimum change in its performance when stored in buffer at 4 degrees C for at least 9 months.

Biosensing Techniques↗

PubMed1999

Characterisation of a thermophilic L-glutamate dehydrogenase biosensor for amperometric determination of L-glutamate by flow injection analysis.

Carbon paste wax electrodes incorporating thermophilic L-glutamate dehydrogenase, NADP and a polymeric toluidine blue O (poly-TBO) mediator have been characterised for the amperometric determination of L-glutamate at 313-318 K in a flow injection analysis (FIA) system. The biosensors exhibit good sensitivity, mechanical stability and reproducibilty, unlike carbon paste- or carbon wax-based electrodes under the same conditions. The carbon paste wax electrode responds linearly to L-glutamate up to 40 mM, the detection limit is 0.3 mM and the RSD (n = 10) for 5 mM L-glutamate was 7.6%. The response to some potential interferents has been quantified. Addition of finely ground hexaammineruthenium (III) trichloride ([Ru(NH3)6]Cl3) to the carbon paste wax electrodes decreases the FIA peak width and increases the peak current. The metal complex appears to accelerate the rate of oxidation of NAD(P)H by poly-TBO.

Biosensing Techniques↗

PubMed1999

A contactless surface acoustic wave biosensor.

Surface acoustic wave sensors operating in liquid generally cause problems resulting from wire bonding. The authors present an approach for a biosensor where the need for bonding wires is eliminated by utilizing inductive coupling of the sensor device to the RF circuitry. Protection of the electrodes from the liquid is achieved by coating the device surface with a SiO2 layer, resulting in a simplified handling of the devices. The first measurements with a sensor operating at 420 MHz are presented, demonstrating the potential of this operating principle for biosensing.

Acoustics↗

PubMed1999

A microbial biosensor based on Yarrowia lipolytica for the off-line determination of middle-chain alkanes.

A microbial biosensor based on immobilised psychrotrophic yeast Yarrowia lipolytica integrated to FIA for the determination of middle chain alkanes was developed. The system responded very well to middle chain alkanes even at low operational temperatures down to +5 degrees C. The maximum sensitivity was obtained at 15 degrees C. A linear relationship was observed between the sensor response and dodecane concentration up to 100 microM.

Alkanes↗

PubMed2000

Multi-analyte interrogation using the fiber optic biosensor.

The capabilities of the portable, automated fiber optic biosensor, RAPTOR, have recently been evaluated. Developed to perform rapid fluoroimmunoassays in the field, the RAPTOR was designed to test samples for up to four different target analytes simultaneously. Assay time could be varied from a 3-min rapid screen to a standard 10-min test. A trial of 203 blind samples tested for Staphylococcal enterotoxin B, ricin, Francisella tularensis, and Bacillus globigii has been conducted. Sensitivities obtained were 10, 50 ng/ml, 5 x 10(5), and 5 x 10(4) cfu/ml, respectively.

Bacillus↗

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