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An amperometric biosensor based on the coimmobilization of horseradish peroxidase and methylene blue on a beta-type zeolite modified electrode.

A new biosensor for the amperometric detection of hydrogen peroxide was developed based on the coimmobilization of horseradish peroxidase (HRP) and methylene blue on a beta-type zeolite modified glassy carbon electrode without the commonly used bovine serum albumin-glutaraldehyde. The intermolecular interaction between enzyme and zeolite matrix was investigated using FT-IR. The cyclic voltammetry and amperometric measurement demonstrated that methylene blue co-immobilized with HRP in this way displayed good stability and could efficiently transfer electrons between immobilized HRP and the electrode. The sensor responded rapidly to H2O2 in the linear range from 2.5 x 10(-6) to 4.0 x 10(-3) M with a detection limit of 0.3 microM. The sensor was stable in continuous operation.

Biosensing Techniques↗

Cell-based biosensors.

Different classes of cell-based biosensors are introduced. These include devices to measure cell-cell contact and set-ups to determine metabolic products. Main emphasis is put on sensors based on nerve-cell networks which are able to detect neuro-active compounds. The different experimental set-ups are explained and examples for typical applications are given. A main point concerns new achievements and prospects for future developments.

Animals↗

Biosensors as new analytical tool for detection of Genetically Modified Organisms (GMOs).

Three different biosensors for detection of Genetically Modified Organisms (GMOs) are presented. The sensing principle is based on the affinity interaction between nucleic acids: the probe is immobilised on the sensor surface and the target analyte is free in solution. The immobilised probes are specific for most inserted sequences in GMOs: the promoter P35S and the terminator TNOS. Electrochemical methods with screen-printed electrodes, piezoelectric and optical (SPR) transduction principles were applied.

Biosensing Techniques↗

Chlorophyll fluorescence biosensor for the detection of herbicides.

A biosensor is described for the detection of triazine and phenylurea herbicides in drinking water by kinetic measurements of endogenous chlorophyll fluorescence in isolated chloroplasts from higher plants. The pocket-size device uses a diode laser for simultaneous excitation of sample and reference channels, and photodiodes for detection of the emitted light. The biological material can be supplied as a freeze-dried powder, stable for 21 days at room temperature, for 6 weeks at 4 ( degrees )C or for at least 9 months at -20 ( degrees )C. The detection of 0.1 microg/l of a single herbicide, as required by European Community legislation on drinking water quality, can be achieved without prior extraction of the sample.

Journal Article↗

Biosensors of inorganic lead exposure and effect in an adult amphibian.

Lead (Pb) is a ubiquitous environmental pollutant, widely distributed, representing a high toxicological and ecotoxicological risk. Several morphological, functional, and biochemical parameters have been proposed as biomarkers of effect and exposure to Pb. The information related to adverse effects of Pb is not abundant for adult amphibians. These animals are of interest, because during their development they move from aquatic to terrestrial habitats, which may be polluted by the metal since they are receptors of products generated by anthropogenic activities. Previous studies carried out on the adult South American toad Bufo arenarum (Amphibia, Anura) showed that it has a high tolerance to lead and studied the effect of sublethal doses of the metal on the erythrocyte osmotic fragility and delta-ALAD activity. It was also shown that after a single injection of Pb, a significant increase in the number of reticulocytes was produced, suggesting the suitability of those cell counts as a biomarker of exposure to the metal; its impact on the immune system of the toads was also studied. In this work we extend our early studies on the same species evaluating the chronic effect of sublethal Pb (equivalent to 5.6% of the 120-h LD-50) on free erythrocyte protoporphyrin (FEP) and blood Pb and delta-ALAD activity; blood lead was positively associated with a significant decrease in the enzyme activity and to an increase in the FEP level. Pb concentration in target organs (liver, spleen, femur, and kidney) and the total cumulated amount as well as its impact over the mass of those organs were also determined. In addition, the magnitude of the possible depuration through urine and intestine was evaluated. Our results showed that FEP, delta-ALAD, and blood Pb are reliable biosensors of chronic metal intoxication, the former being the marker with the highest sensitivity.

Animals↗

A portable array biosensor for detecting multiple analytes in complex samples.

The Multi-Analyte Array Biosensor (MAAB) has been developed at the Naval Research Laboratory (NRL) with the goal of simultaneously detecting and identifying multiple target agents in complex samples with minimal user manipulation. This paper will focus on recent improvements in the biochemical and engineering aspects of this instrument. These improvements have enabled the expansion of the repertoire of analytes detected to include Salmonella typhimurium and Listeria monocytogenes, and also expanded the different sample matrices tested. Furthermore, all components of the biochemical assays could be prepared well in advance of sample testing, resulting in a "plug-and-play" methodology. Simultaneous detection of three toxins (ricin, staphylococcal enterotoxin B, and cholera toxin) was demonstrated using a novel fluidics cube module that limits the number of manipulations to only the initial sample loading. This work demonstrates the utility of the MAAB for rapid analysis of complex samples with multianalyte capability, with a minimum of operator manipulations required for either sample preparation or final analysis.

Antibodies↗

Assessment of heavy metal bioavailability using Escherichia coli zntAp::lux and copAp::lux-based biosensors.

To determine the amount of metals detectable by bacteria, two plasmids were constructed in which the metal-inducible zntA and copA promoters from Escherichia coli were fused to a promoterless Vibrio fischeri luxCDABE operon. The luminescence response of E. coli bearing these constructs was studied as a function of the concentration of several heavy metals and was shown to be influenced by cell growth phase. The zntAp::lux fusion is induced mainly by salts of cadmium, lead, mercury and zinc, with significant induction by other metal ions, whereas the specificity of copA induction is restricted to copper and silver. In optimized assay conditions, metals could be detected at threshold concentrations ranging from nanomolar to micromolar, with maximal induction observed after only 60-100 min incubation. The ability of these biosensor strains to distinguish bioavailable quantities of metals in a sample makes them good candidates as useful tools to monitor metal contamination in environmental samples.

Adenosine Triphosphatases↗

A Pseudomonas aeruginosa biosensor responds to exposure to ultraviolet radiation.

We fused the Pseudomonas aeruginosa recA promoter to a promoterless Vibrio fisheri lux operon. This recA-lux fusion (pMOE15) was introduced into wild-type P. aeruginosa strain FRD1 and recA expression was monitored by measuring 490-nm light production. The RM4440 strain responded to increasing doses of ultraviolet radiation by an increase in its bioluminescence. RM4440 has the potential to be useful as a biosensor for the presence of DNA-damaging agents in the environment.

Biosensing Techniques↗

Mediated microbial biosensor using a novel yeast strain for wastewater BOD measurement.

Two new yeast strains (SPT1 and SPT2) were isolated and immobilized on glassy carbon electrodes to form microbial biosensors for estimation of biochemical oxygen demand (BOD). Ferricyanide was proven to be the most efficient mediator to shuttle electrons from the redox center of reduced microbial enzymes to the electrode in the presence of excess glucose/glutamic acid (GGA). With a 3-fold greater metabolic assimilation capability and greater responses to various effluent samples, SPT1 was selected for sensor-BOD measurements. BOD estimations for the GGA standard resulted in an extended linear range: 2-100 mg/l. Response reproducibility was +/-10% for a GGA standard containing 10 mg BOD/l. For analysis of pulp mill effluents, the BOD detection limit was 2 mg/l with a response time of 5 min.

Biosensing Techniques↗

Development of a rapid pH-based biosensor to monitor and control the hygienic quality of reclaimed domestic wastewater.

The re-use of treated domestic wastewater necessitates a rigorous control and rapid monitoring of the hygienic quality of the reclaimed water. For this purpose, a new pH-based biosensor was developed. The essence of the methodology of the sensor is the monitoring of the acidification due to bacterial metabolism of added glucose. To improve the sensitivity, the alkalinity of the water sample is reduced prior to monitoring the acidification. This is done by stripping CO2 at a neutral or acidic pH value. The hygienic aspect of the sensor lies in the applied temperature (37 degrees C) and the use of N2 as decarbonizing gas, thus creating conditions favorable for enteric bacteria. The developed sensor could be used onsite at an advanced stage of treatment, as an endpoint or intake quality control device. For both applications, a useful correlation was obtained between log total plate count and lag time or acidification rate, respectively. Absolute detection limits lay in the ranges of either 10(3) colony-forming units (CFU)/ml in 6 h (endpoint quality control), or less than 10(5) CFU/ml in 1 h (intake quality control).

Biosensing Techniques↗

Cell surface measurements of ATP release from single pancreatic beta cells using a novel biosensor technique.

To examine the possibility that ATP modulates insulin secretion by an autocrine mechanism, we measured the local concentration of released ATP at the surface of a single pancreatic beta cell by a new biosensor technique, using PC12 cells expressing ligand-gated cation channels, P2X2 receptors. Upon application of glucose or glibenclamide, a series of current spikes, whose amplitude equates to an ATP concentration of over 25 microM, were recorded from a PC12 cell using the whole-cell patch-clamp technique, when placed near a rat pancreatic beta cell at 37 degrees C. The current response was inhibited by cooling (below 30 degrees C) or by applying an ATP-hydrolysing enzyme (apyrase) or a P2 receptor blocker (suramin). Thus, it is concluded that pancreatic beta cells secrete ATP in response to glucose stimulation, thereby increasing the ATP concentration close to the cell surface sufficiently high enough to enhance insulin secretion from the pancreatic beta cells.

Adenosine Triphosphate↗

Addressing a whole bioprocess in real-time using an optical biosensor-formation, recovery and purification of antibody fragments from a recombinant E. coli host.

The use of biosensor technology is described to address in real-time the production and subsequent purification of a bioactive recombinant protein product. The product, D1.3 Fv antibody fragment, was expressed in Escherichia coli and purified via two process routes, one for extracellular and one for intracellular product material. The cells were harvested by centrifugation in a solid bowl CARR Powerfuge and stored at -70 degrees C. Clarification of the supernatant was performed by depth filtration, followed by affinity chromatography for final purification of the extracellular product. To purify the intracellular product the harvested cells were resuspended and homogenised. Removal of debris in the CARR Powerfuge was followed by depth filtration and affinity chromatography. In this work we have shown the rapid determination of bioactive product levels, and the impact this has on improved accountability and confidence is demonstrated in process mass balances on the product using the data acquired during process operation.

Bioreactors↗

PCR-based ribosomal DNA detection technique for microalga (Heterosigma carterae) causing red tide and its application to a biosensor using labeled probe.

A technique for detecting Raphidophycean, a bloom-forming genus of algae, was developed using a specific DNA probe. The design of the probe was based on a sequence polymorphism within the small subunit (SSU) ribosomal RNA gene (rDNA) of this strain by using fluorescence polarization (FP) analysis and the BIAcore 2000 biosensor, which utilized surface plasmon resonance (SPR). The specific sequence in SSU rDNA for Heterosigma carterae was determined by sequence data analysis. One pair of polymerase chain reaction (PCR) probes was designed for use in making the identification. H. carterae SSU rDNA was amplified by PCR. Using a fluoroscein isothiocyanate-labeled or biotin-labeled oligonucleotide probe, the PCR-amplified rDNA was selectively detected as an FP-intensity change via FP analysis or as a resonance-unit change via SPR. Although total time for final detection after sampling was within 3 hours, specific rDNA could be detected within 10 minutes after PCR through these detection methods.

Base Sequence↗

Use of chitosan membrane from the carapace of the soldier crab Mictyris brevidactylus for biosensor construction.

Glucose oxidase (EC 1.3.4.3) was immobilized on chitosan membrane (<0.1 mm in thickness) prepared from the carapace of the soldier crab Mictyris brevidactylus. A glucose electrode was constructed by covering a platinum electrode (2.0 mm in diameter) with the enzyme membrane. The enzyme electrode sensed glucose amperometrically (1.0 micro A/mM, with linear range up to 0.5 mM, r = 0.999) when positively imposed with 0.6 V against an Ag/AgCl reference electrode. The glucose biosensor was sensitive (<0.1 micro M, S/N > 3), reproducible (CV for 55 micro M glucose <3%, n = 5), reagentless, and durable for months.

Animals↗

Titration biosensors for the estimation of the biochemical nitrate demand of municipal and industrial wastes.

An anoxic titrimetric test was investigated for measuring denitrification potential of different wastewaters, both municipal and industrial, and to quantify the denitrifying activity in an activated sludge system. The method measures the amount of acid that is required to maintain the pH set-point value in a batch denitrification experiment, and it was performed using a DENICON (denitrification controller) biosensor. The amount of acid is proportional to the nitrate used to oxidise the biodegradable chemical oxygen demand present in the wastewater, while the acid consumption rate is used to derive the denitrifying activity. The wastewaters tested were a municipal wastewater (MW), an industrial-municipal wastewater (MIW; 70% and 30%, respectively), and four industrial wastewaters drawn from an ice-cream factory (IW1), a beet-sugar factory (IW2), a brewery (IW3), and a tuna cannery industry (IW4). Good correlation between titration data and analyses was found.

Biosensing Techniques↗

Development of an oxygen-rich biosensor using enzymatic reaction.

This work reports a novel strategy for the development of an O2-rich biosensor. The principle is based on an enzymatic reaction between catalase and H2O2 to release O2, thus to increase the O2 amount in the enzyme matrix. This method improves the determination reliability by alleviating the O2 dependence.

Animals↗

Nanoparticles, molecular biosensors, and multispectral confocal microscopy.

Complex, multilayered nanoparticles hold great promise for more sophisticated drug/gene delivery systems to single cells. Outermost layers can include cell targeting and cell-entry facilitating molecules. The next layer can include intracellular targeting molecules for precise delivery of the nanoparticle complex inside the cell of interest. Molecular biosensors can be used to confirm the presence of expected molecules (for example, reactive oxygen species (ROS) as a surrogate molecule for signs of infection, or for activation in radiation damage, etc.) prior to delivery of counter-measure molecules such as drugs or gene therapy. They can also be used as a feedback control mechanism to control the proper amount of drug/gene delivery for each cell. Importantly, the full nanoparticle system can be used to prevent any cells from encountering the drug unless that cell is specifically targeted. Thus, if a cell is initially non-specifically targeted, a secondary check for other molecular targets which must also be present inside the target cell of interest can be used to catch initial targeting mistakes and prevent subsequent delivery of treatment molecules to the wrong cells. The precise intracellular location of nanoparticles within specific regions of a cell can be confirmed by 3D multispectral confocal microscopy. These single cell molecular morphology measurements can be extended from individual cells, to other cells in a tissue in tissue monolayers or tissue sections.

Biocompatible Materials↗