Search

The present study describes the development of materials suitable for environmental control of algae. Molecularly imprinted polymers (MIPs) were used as simazine carriers able to provide the controlled release of simazine into water. Three polymers were designed using computational modelling. The selection of methaerylic acid (MA) and hydroxyethyl methacrylate (HEM) as functional monomers was based on results obtained using the Leapfrog algorithm. A cross-linked polymer made without functional monomers was also prepared and tested as a control. The release of simazine from all three polymers was studied. It was shown that the presence of functional monomers is important for polymer affinity and for controlled release of herbicide. The speed of release of herbicide correlated with the calculated binding characteristics. The high-affinity MA-based polymer released similar to 2% and the low-affinity HEM-based polymer released similar to 27% of the template over 25 days. The kinetics of simazine release from HEM-based polymer show that total saturation of an aqueous environment could be achieved over a period of 3 weeks and this corresponds to the maximal simazine solubility in water. The possible use of these types of polymers in the field of controlled release is discussed. (c) 2005 Elsevier B.V. All rights reserved.

A new simple method for the post-polymerisation treatment of molecularly imprinted polymers was proposed. A layer of mineral oil was deposited onto the surface of the polymer in order to create a hydrophobic environment in the binding sites and to improve the recognition properties of the polymer in polar solvents. The testing of polymers performed in acetonitrile showed that the modified polymers possessed significantly increased selectivity as compared with non-treated ones. The three-fold improvement of recognition of the template (cocaine) was achieved; the same time, for non-specific molecule (morphine) the improvement was only 1.3 times. The investigation of the stability of mineral oil coating on the polymer surface suggested that the effect produced is stable over a long period of time. This approach could be used to broaden the range of experimental conditions where molecularly imprinted polymers can perform successfully. (c) 2005 Elsevier B.V. All rights reserved.

Twenty years on from a review in the first issue of this journal, this contribution revisits glucose sensing for diabetes with an emphasis on commercial developments in the home blood glucose testing market. Following a brief introduction to the needs of people with diabetes, the review considers defining technologies that have enabled the introduction of commercial products and then reviews the products themselves. Drawing heavily on the performance of actual instruments and publicly available information from the companies themselves, this work is designed to complement more conventional reviews based on papers published in scholarly journals. It focuses on the commercial reality today and the products that we are likely to see in the near future. (c) 2004 Elsevier B. V. All rights reserved.

A DNA-based surface plasmon resonance (SPR) biosensor has been developed for the detection of TP53 mutation using the inexpensive and commercially available instrument, SPREETA (TM) SPR-EVM-BT, from Texas Instruments. A direct immobilisation procedure, based on the coupling of thiol-derivatised oligonucleotide probes (Probe-C6-SH) to bare gold sensor surfaces, was optimized using synthetic oligonucleotides. Hybridisation reactions between the immobilised probe and a short sequence (26 mer) complementary, non-complementary and one-point mutation DNA were then investigated. The main analytical parameters of the sensor system were studied in detail including selectivity, sensitivity, reproducibility and analysis time. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples, DNA extracted from both normal, wild-type, (Jurkat) and mutated (Molt 4), carrying the mutation at codon 248 of the TP53 cell lines. The results obtained demonstrate that the DNA-based SPR biosensor was able to distinguish sequences present in the various samples that differ only by one base; and hence, it appears to be a strong candidate technique for the detection of gene mutation. (c) 2004 Elsevier B.V. All rights reserved.

A molecularly imprinted polymer (MIP) film for domoic acid (DA) was synthesised by direct photo-grafting onto a gold chip suitable for a surface plasmon resonance (SPR) based bioanalytical instrument system, the BlAcore 3000(TM). The gold surface was first functionalised with a self-assembled monolayer of 2-mercaptoethylamine and subsequent carbodiimide chemistry was performed for covalent attachment of the photoinitiator, 4,4-azobis(cyanovaleric acid). This ensured that the formation of the MIP thin film, comprising 2-(diethyl amino) ethyl methacrylate as functional monomer and ethylene glycol dimethacrylate as cross-linker, occurred only at the surface level. Optimisation and control over the grafting procedure were achieved using contact angle measurements and atomic force microscope (AFM) imaging. The surface grafting resulted in the formation of thin and homogeneous MIP film with thickness of 40 nm. A competitive binding assay was performed with free DA and its conjugate with horseradish peroxidase, which was used as a refractive label. The sensor was evaluated for its sensitivity, cross-reactivity, and robustness by using a BlAcore 3000(TM). Likewise, monoclonal antibodies acting as natural receptors for the toxin were studied with the same BlAcore system. Results of a comparison between the artificial and natural receptors are reported. In contrast to monoclonal antibodies, the regeneration of MIP chip did not affect its recognition properties and continuous measurement was possible over a period of at least 2 months. (C) 2004 Elsevier B.V. All rights reserved.

Genosensor technology relying on the use of carbon and gold electrodes is reviewed. The key steps of each analytical procedure, namely DNA-probe immobilisation, hybridisation, labelling and electrochemical investigation of the surface, are discussed in detail with separate sections devoted to label-free and newly emerging magnetic assays. Special emphasis has been given to protocols that have been used with real DNA samples. (C) 2003 Elsevier B.V. All fights reserved.

Microsystin-LR is one of the most widespread and dangerous toxins produced by the freshwater Cyanobacteria. The contamination of water supplies with microcystin-LR has been reported in several areas around the world and the development of an easy-to-use, rapid, robust and inexpensive sensor for this toxin is urgently required. In this work an artificial receptor for microcystin-LR was synthesised using the technique of molecular imprinting. The composition of the molecularly imprinted polymer (MIP) was optimised using computer modelling. The synthesised polymer was used both as a material for solid-phase extraction (SPE) and as a sensing element in a piezoelectric sensor. Using the combination of SPE followed by detection with a piezoelectric sensor the minimum detectable amount of toxin was 0.35 nM. The use of MIP-SPE provided up to 1000 fold preconcentration, which was more than sufficient for achieving the required detection limit for microcystin-LR in drinking water (I nM). This work is the first example where the same MIP receptor has been used successfully for both SPE and the corresponding sensor. (C) 2002 Elsevier Science B.V. All rights reserved.

An immunosensor for the determination of okadaic acid (OA) using a quartz crystal microbalance (QCM) was developed and optimised in standard solutions. Several coupling techniques, protein A, protein G and polyethylenimine (PEI) with glutaraldehyde (GA) cross-linking, were investigated for the determination of okadaic acid and a very good result was obtained with PEI coupling. With the PEI coupling method, the optimisation of incubation time for the activation of PEI on the crystal surface using GA, the effect of the dilution factor of OA-bovine serum albumin (BSA) conjugate and the amount of antibody on crystal frequency were studied. Different molar ratios (4:1, 14:1, 30:1) of OA to bovine serum albumin for the conjugation were examined and the results using ELISA and a QCM showed that a ratio of 14:1 was slightly better than the other two. The strong attachment of the cross-linked complex to the gold surface resulted in an excellent storage lifetime of 38 days. However, the detection limit (1.9 mug/ml) and the sensitivity of the sensor were not satisfactory. Significant improvement of the performance of the device was obtained by incorporating an antibody-BSA hydrogel. Initial results showed that the minimum amount of analyte detectable and the sensitivity of the device were improved by 524- and 80-fold, respectively. (C) 2002 Elsevier Science B.V. All rights reserved.

Measurement of ecological, climatic and anthropogenic changes underpins the formulation of effective management strategies for sustainable use and protection of the marine environment. Sensors are traditionally used in marine studies to determine physical parameters, but there is increasing demand for real-time information about chemical and biological parameters. These parameters are currently measured in samples collected at sea and subsequently analysed in the laboratory. Biosensors fuse the exquisite sensitivity and specificity of living systems with the processing power of microelectronics to deliver simple, inexpensive measurement systems for use in the field or deployment in situ. While their potential for use in the marine environment is enormous, much published work to date has focussed on applications in freshwater and wastewater. Marine applications pose a substantial challenge in the robustness required for remote application, but recent developments in portable medical devices and receptor design suggest that these demands can now be realistically tackled. (C) 2002 Elsevier Science Ltd. All rights reserved.

The high sensitivity and specificity of DNA hybridisation techniques makes them powerful tools for environmental or clinical analysis. This work describes the development of a DNA piezoelectric biosensor for the detection of the hybridisation reaction. Attention was focused on the choice of the coating chemistry that could be used for the immobilisation of oligonucleotides onto the gold surface of the quartz crystal. Four immobilisation procedures were tested and compared considering the amount of immobilised probe, the extent of the hybridisation reaction, the possibility of regeneration and the absence of non-specific adsorption. All the experiments were performed with oligonucleotides of 25 bases (probe, target and non-complementary oligonucleotide). The four coating methods were all based on the use of self-assembled monolayers (SAM). Three of them employed the interaction between streptavidin and biotin for the immobilisation of a biotinylated probe. Results indicated that immobilisation of a biotinylated probe on streptavidin linked to a layer of carboxylated dextran provides higher sensitivity for the detection of the hybridisation reaction, absence of non-specific adsorption and a higher stability with respect to the regeneration step. (C) 2002 Elsevier Science B.V. All rights reserved.

The use of volatile production patterns produced by bacterial contaminants in urine samples were examined using electronic nose technology. In two experiments 25 and 45 samples from patients were analysed for specific bacterial contaminants using agar culture techniques and the major UTI bacterial species identified. These samples were also analysed by incubation in a volatile generation test tube system for 4-5 h. The volatile production patterns were then analysed using an electronic nose system with 14 conducting polymer sensors. In the first experiment analysis of the data using a neural network (NN) enabled identification of all but one of the samples correctly when compared to the culture information. Four groups could be distinguished, i.e. normal urine, Escherichia coli infected, Proteus spp. and Staphylococcus spp. In the second experiment it was again possible to use NN systems to examine the volatile production patterns and identify 18 of 19 unknown UTI cases. Only one normal patient sample was mis-identified as an E coli infected sample. Discriminant function analysis also differentiated between normal urine samples, that infected with E coli and with Staphylococcus spp. This study has shown the potential for early detection of microbial contaminants in urine samples using electronic nose technology for the first time. These findings will have implications for the development of rapid systems for use in clinical practice. (C) 2002 Elsevier Science B.V. All rights reserved.

A new technique for coating microtitre plates with molecularly imprinted polymers (MIP), specific for low-molecular weight analytes (epinephrine, atrazine) and proteins is presented. Oxidative polymerization was performed in the presence of template; monomers: 3-aminophenylboronic acid (APBA), 3-thiopheneboronic acid (TBA) and aniline were polymerized in water and the polymers were grafted onto the polystyrene surface of the microplates. It was found that this process results in the creation of synthetic materials with antibody-like binding properties. It was shown that the MIP-coated microplates are particularly useful for assay development. The high stability of the polymers and good reproducibility of the measurements make MIP coating an attractive alternative to conventional antibodies or receptors used in enzyme linked immunosorbent assay (ELISA). (C) 2001 Elsevier Science B.V. All rights reserved.

A method for the selective detection of creatinine is reported, which is based on the reaction between polymerised hemithioacetal formed by allyl mercaptan, o-phthalic aldehyde, and primary amine leading to the formation Of fluorescent isoindole complex. This method has been demonstrated previously for the detection of creatine using creatine-imprinted molecularly imprinted polymers (MlPs) Since MlPs created using traditional methods were unable to differentiate between creatine and creatinine, a new approach to the rational design of a molecularly imprinted polymer (MIP) selective for creatinine was developed using computer simulation. A virtual library of functional monomers was assigned and screened against the target molecule, creatinine, using molecular modelling software. The monomers giving the highest binding score were further tested using simulated annealing in order to mimic the complexation of the functional monomers with template in the monomer mixture. The result of this simulation gave an optimised MIP composition. The computationally designed polymer demonstrated superior selectivity in comparison to the polymer prepared using traditional approach, a detection limit of 25 muM and good stability. The Bite-and-Switch approach combined with molecular imprinting can be used for the design of assays and sensors, selective for amino containing substances. (C) 2001 Elsevier Science B.V. All rights reserved.

A new polymer capable of reacting with primary amines was synthesised from allyl mercaptan, o-phthalic dialdehyde and ethylene glycol dimethacrylate by radical polymerisation. Reactive hemithioacetal formed by allyl mercaptan and dialdehyde can bind primary amino groups without additional pre-activation forming the fluorescent isoindole complex. It gives a great opportunity to monitor binding and perform loading of the amino compounds onto the reactive surface. The reactive polymer is found to be an effective matrix for immobilisation of the proteins and other amino-containing compounds in affinity chromatography and could be used for their detection in solution. (C) 2001 Elsevier Science Ltd. All rights reserved.

Molecularly imprinted polymers (MIPs) represent a new class of materials that have artificially created receptor structures(1-3). Since their discovery in 1972, MIPs have attracted considerable interest from scientists and engineers involved with the development of chromatographic adsorbents, membranes, sensors and enzyme and receptor mimics.

In this paper, we report the realisation of a DNA piezoelectric biosensor coupled with the polymerase chain reaction (PCR) for the detection of a specific bacterial toxicity factor. Biotinylated 23-mer probes were immobilised on the streptavidin coated gold surface of a quartz crystal; streptavidin was covalently bound to the thiol/dextran modified gold surface. The hybridisation of the immobilised probe with a synthetic oligonucleotide was investigated; the absence of non-specific adsorption was verified using a non-complementary oligonucleotide. Many cycles of measurements can be performed on the same crystal surface by regenerating the single strand with 1 mM HCl. The same hybridisation reaction was then performed using real samples of DNA extracted from bacteria and amplified by PCR. The PCR product was a fragment of a specific gene of Aeromonas hydrophila. The piezoelectric biosensor was able to distinguish samples containing the gene or not; in this way it was possible to determine the pathogenicity of different Aeromonas strains isolated from water, vegetables or human specimens. Experiments with non-specific samples confirmed the absence of adsorption or non-specific effects on the quartz crystal treated with the reported procedure. (C) 2000 Elsevier Science B.V. All rights reserved.

In this paper we report the coupling of the Polymerase Chain Reaction (PCR) with a piezoelectric biosensor to detect a point mutation in a human gene. Biotinylated 23-mer probes were immobilised on the streptavidin coated gold surface of a quartz crystal; streptavidin was covalently bound to the thiol/dextran modified gold surface. The hybridisation of the immobilised probes with a short sequence (23 mer) complementary, non-complementary and mismatched DNA was investigated: the device was able to distinguish the different synthetic oligonucleotides. Many cycles of measurements can be performed on the same crystal surface regenerating the single strand of DNA with 1 mM of HCl. The same hybridisation reaction was then performed using real samples of human DNA extracted from blood and amplified by PCR, following a standard procedure for genetic detection of the polymorphism of the apolipoprotein E (apoE) gene. The procedure was able to distinguish the sequences present in the different samples, which differ only in one base: in this way it was possible distinguish between different groups of genotypes with apoE typing. Experiments with blank samples confirmed the absence of adsorption or non-specific effects on the quartz crystal treated with the reported procedure. (C) 2000 Elsevier Science S.A. All rights reserved.

Two series of experiments are reported which result in the discrimination between Helicobacter pylori and other bacterial gastroesophageal isolates using a newly developed odour generating system, an electronic nose and a hybrid intelligent odour recognition system. In the first series of experiments, after 5 h of growth (37 degreesC), 53 volatile sniffs were collected over the headspace of complex broth cultures of the following clinical isolates: Staphylococcus aureus, Klebsiella sp., H. pylori, Enterococcus faecalis (10(7) ml(-1)), Mixed infection (Proteus mirabilis, Escherichia coli, and E. faecalis 3 x 10(6) mi each) and sterile cultures. Fifty-six normalised variables were extracted from 14 conductive polymer sensor responses and analysed by a 3-layer back propagation neural network (NN). The NN prediction rate achieved was 98% and the test data (37.7% of all data) was recognised correctly. Successful clustering of bacterial classes was also achieved by discriminant analysis (DA) of a normalised subset of sensor data. Cross-validation identified correctly seven unknown samples. In the second series of experiments after 150 min of microaerobic growth at 37 degreesC, 24 volatile samples were collected over the headspace of H. pylori cultures in enriched (HPP) and normal (HP) media and 11 samples over sterile (N) cultures. Forty-eight sensor parameters were extracted from 12 sensor responses and analysed by a 3-layer NN previously optimised by a genetic algorithm (GA). GA-NN analysis achieved a 94% prediction rate or unknown data. Additionally the genetically selected 16 input neurones were used to perform DA-cross validation that showed a clear clustering of three groups and reclassified correctly nine sniffs. It is concluded that the most important factors that govern the performance of an intelligent bacterial odour detection system are: (a) an odour generation mechanism, (b) a rapid odour delivery system similar to the mammalian olfactory system, (c) a gas se

The present review highlights some modern aspects of biosensor revelation, a detection method which has already found a large number of applications in healthcare, food industry and environmental analysis. First, the concept of bio-recognition, which is at the heart of biosensor technology, is discussed, with emphasis on host-guest-like recognition mechanisms. This detection device has been successfully coupled, in its first applications, to chromatographic columns, which allow a high resolution of complex mixtures of analytes prior to interaction with the biosensing unit. The properties of the transducing elements, which should generate a signal (e.g., electrochemical, thermal, acoustic, optical) of proper intensity and of relative fast rise, are additionally evaluated and discussed. The review then focuses on potential applications of biosensing units in capillary electrophoresis (CE) devices. CE appears to be an excellent separation methodology to be coupled to biosensor detection, since it is based on miniaturized electrophoretic chambers, fast analysis times, complete automation in sample handling and data treatment and requires extremely small sample volumes. Although only a few applications of CE-based biosensors have been described up to the present, it is anticipated that this hyphenated technique could have a considerable expansion in the coming years, (C) 2000 Elsevier Science B.V. All rights reserved.

The possibility of developing a simple, inexpensive and specific personal passive "real-time" air sampler incorporating a biosensor for formic acid was investigated. The sensor is based on the enzymatic reaction between formic acid and formate dehydrogenase (FDH) with nicotinamide adenine dinucleotide (NAD(+)) as a co-factor and Meldolas blue as mediator. An effective way to immobilise the enzyme, co-factor and Meldolas blue on screen-printed, disposable, electrodes was found to be in a mixture of glycerol and phosphate buffer covered with a gas-permeable membrane. Steady-state current was reached after 4-15 min and the limit of detection was calculated to be below 1 mg/m(3). However, the response decreased by 50% after storage at -15 degreesC for 1 day. (C) 2000 Elsevier Science B.V. All rights reserved.

Solvent resistant screen-printed three-electrode devices were employed to assess the behaviour of free and immobilised glucose oxidase in water-miscible organic solvent/aqueous buffer mixtures. Three alcoholic solvents were examined, methanol, ethanol and isopropanol. A rhodinised-carbon electrocatalyst was employed to facilitate hydrogen peroxide oxidation at a decreased operating potential. The devices had a linear response range of 1-10 mU GOX and allowed detection of down to 2 x 10(-14) mol GOX. The measurement of 0-1 (v/v) glucose in 30% (v/v) ethanol was achieved in 2 min using these highly storage-stable enzyme electrodes. Such devices are likely to have widespread applications in the diagnostic and on-line monitoring fields where rapid analysis in alcoholic matrices is desirable. (C) 1998 Elsevier Science B.V. All rights reserved.

This paper reports the synthesis and screening of a combinatorial peptide library for new affinity ligands for glycosylated haemoglobin (HbA(1c)), which is an important indicator of diabetes control. The new ligands are suitable for large-scale synthesis and overcome the disadvantages of antibodies (unstable and expensive to produce etc.), while remaining as efficient as antibodies in binding to the analyte. The library consisted of 262 144 hexapeptides synthesised using the one-bead-one-compound technique. The hexapeptides attached onto beads were screened with glycosylated haemoglobin HbA(1c). The structures of the peptides exhibiting high affinity were characterised by Edman microsequencing. Computer modelling simulation of one of the lead sequences has shown that this class of ligand has a high affinity and specificity for glycosylated haemoglobin. (C) 1998 Elsevier Science S.A. All rights reserved.

A three electrode design using solvent resistant materials is reported, enabling the production of screen printed electrodes for use in biosensor applications involving water-miscible organic solvents. Heat stabilised polyester sheets were used as base materials in combination with carbon basal tracks and an epoxy-based polymer protective coating. The new electrodes avoid problems with solvent induced baseline shifts displayed by conventional screen-printed electrodes. The material for the underlying basal track has been changed from silver to carbon ink to reduce the number of materials involved in the production and avoid possible interferences. In addition, the design is readily applied in biosensor applications. Different methods for the incorporation of a rhodium catalyst are discussed. The application of the electrode system modified with the rhodium catalyst for the amperometric detection of hydrogen peroxide in different concentrations of methanol, isopropanol and ethanol is demonstrated.

Analysis of silage effluent identified glucose and lactic acid as suitable markers for diagnosis of silage effluent pollution in river water. The use of biosensors, utilising the reactions of glucose oxidase and lactate oxidase to detect glucose and lactic acid respectively, in silage effluent, was investigated. The lactate sensor was able to detect effluent from mature silage at 1/1000 dilution, whilst the glucose sensor proved more suitable for detecting effluent from freshly ensiled grass, which contains higher levels of sugar than mature silage. In both cases the sensor response was within 60 s of exposure to the effluent. The potential of biosensors for rapid monitoring in the water industry was demonstrated in this work. Copyright (C) 1996 Elsevier Science Ltd

A flow-injection analysis system was combined with a mass producible, disposable biosensor and was used to monitor glucose concentrations during several microbial fermentations. The biosensor was manufactured using thick film, screen printing technology. Unlike previous devices of this type, the biosensor was designed to make multiple measurements over extended periods rather than to operate as a one-shot sensor. One yeast, one lactic acid, and three E. coli bioreactor cultures were tested using either defined or complex media. Results from the sensor were compared with a standard spectrophotometric test kit. In samples containing glucose concentrations within the range of the biosensor and the test kit, good correlations were obtained between the two methods. In addition to glucose, microbial growth and pH were recorded. (C) 1997 by Elsevier Science Inc.

We report a novel procedure for the electrochemical modification of graphite disc electrodes with potassium hexacyanoferrate(III) using cyclic voltammetry. Cyclic voltammograms of the modified electrodes in 0.1 M potassium chloride showed two redox couples, at 0.22 and 0.148V and at 0.923 and 0.798 V. These redox potentials correspond to those cited for Prussian blue type films. These modified electrodes exhibited excellent stability during repeated potential cycling in 0.1 M potassium chloride with no difference in peak heights between the second and thirtieth scans. We used glucose oxidase as the model enzyme to investigate application of these novel modified graphite disc electrodes in the development of biosensors. The resulting glucose sensors exhibited a response to glucose at potentials as low as 450 mV vs. saturated calomel electrode with a linear range up to 1.5 mM glucose and sensitivity of 50 +/- 10 nA mM(-1) cm(-2) (geometric area). The modified graphite disc electrodes displayed an electrocatalytic effect on hydrogen peroxide with sensitivity of 0.70 mu A mM(-1) cm(-2) (geometric area). The effect of ascorbic acid, 4-acetamidophenol and uric acid, common interferents found in blood, is also reported. (C) 1996 Published by Elsevier Science Limited

A choline amperometric biosensor based on screen-printed electrodes was assembled and used to assess the inhibitory effect of organophosphorus and carbamic pesticides on acetylcholinesterase activity both in standard solutions and real samples, Acetylcholinesterase catalyses the cleavage of acetylcholine to choline and acetate, therefore the amount of choline measured using the biosensor is directly related to the enzyme activity. The extent of enzyme inhibition can be used as an index of the amount of anticholinesterase pesticides present. The hydrophobicity of organophosphorus and carbamic pesticides led to the evaluation of organic, water miscible solvents for use in the proposed method. Berate buffer containing 1% v/v acetonitrile was selected since it exhibited the least influence on enzyme activity from the tested solvents (acetonitrile, acetone, tetrahydrofuran and ethylacetate). Other solvents (dimethylsulfoxide and methanol) were avoided as they exhibited electrochemical interferences. An inhibition calibration curve was obtained using carbofuran, a strong inhibitor of acetylcholinesterase. The lowest detectable standard solution (mean +/-3 standard deviation of the blank) was 2 mu gl(-1) following an incubation time of 10 min. The method was then applied to real samples (fruits and vegetables) showing its suitability as a rapid screening assay (12 min per test) for the assessment of anticholinesterase pesticides, The biosensor results were compared with a standard analytical technique (gas chromatography with nitrogen phosphorus detector, GC-NPD).

Screen printed amperometric glucose biosensors were constructed and optimised for use in a flow injection analysis (FIA) system. The sensors were fabricated using a catalytic metallised carbon based ink, allowing a working potential of +350 mV (Ag/AgCl). Overall the sensors displayed a high degree of reliability and robustness. A linear response was obtained over the range 0.1 to 25 mM glucose with a slope of 1.35 +/- 0.20 mu A/mM. The within electrode coefficient of variation at each concentration tested (n = 10) was less than 2% (or 0.1 mM for concentrations below 5 mM). In addition, it was demonstrated that a single sensor can be used in the FIA system over a seven day period indicating a high level of operational stability. These sensors fulfil the criteria necessary for a mass produced device, which would be suitable for the on-line monitoring of glucose consumption during microbial fermentation.

Prototype electrochemical lactate electrodes based on lactate oxidase were produced by screen-printing and application of membranes by dip-coating or ink-jet printing. The link between enzyme and electrode was made by electro-deposition of platinum onto graphite pads. The linear range of the sensors was small; up to 2 mM lactate. Electrodes retained their activity when stored dry; activity remained high for 244 days. The properties of the electrodes indicate that screen- and ink-jet printing are feasible techniques for the machine production of lactate sensors.

The presence of toxic substances in water and water treatment plants is a major environmental problem. Numerous test methods have been developed to assess the effect of chemical pollutants on the environment, including bioassays. Bioassays in which test organisms are exposed to various doses of the pollutant are used for toxicity evaluations. This is achieved by monitoring the biological integrity of these organisms and comparing them with those which have not been exposed to the pollutant. The need for simple, rapid and relatively inexpensive aquatic toxicity tests has influenced the expansion in research in this area and accelerated the development and application of biological toxicity tests.

There is a widespread need for commercial instrumentation for the rapid and inexpensive detection of microbial contamination of food, industrial waste water and clinical samples. A large number of detection methods have been developed utilizing the optical, electrochemical, biochemical and physical properties of microorganisms. The need for a device which can produce a rapid, accurate, sensitive, real-time analysis for clinical, industrial and environmental applications has led to considerable progress being achieved in recent years in the development of biosensors for microbial detection. This intense research has resulted in the commercialization of several instruments. Techniques used for the quantification of microorganisms are reviewed under the general categories of non-bioelectrochemical and bioelectrochemical methods.

The principle of sandwich-type biosensors based on liquid-crystalline dispersions formed from [DNA-polycation] complexes is outlined. These biosensors will find application in the determination of a range of compounds and physical factors that affect the ability of a given polycationic molecule to maintain intermolecular crosslinks between neighbouring DNA molecules. In the case of liquid-crystalline dispersions formed from [DNA-protamine] complexes the lowest concentration of hydrolytic enzyme (trypsin) detectable was approximate to-10(-14) M. (C) 1996 Elsevier Science Limited.

This work investigates the feasibility of constructing a bioelectrochemical sensor that can operate directly in gases. A series of experiments are described, resulting in a sensor that is responsive to phenol vapours. The sensor was based on ionically conducting films that incorporate a biological redox catalyst at the surface of an array of interdigitated microband electrodes. Exposure to phenol vapour drives the bioelectrochemical reaction, providing a basis for a current signal under constant potential conditions. Ionic materials included Nafion and films based on tetrabutylammonium toluene-4-sulphonate (TEATS). The quasi-reversible electrode reaction of potassium hexacyanoferrate (II) within TEATS was investigated as a function of drying time. E(0) and k(0) were determined at a TEATS modified microdisc electrode under steady-state conditions. Drying time (water loss) from the TEATS film had the effect of increasing the film ionic strength. It was shown that as the film ionic strength increased, E(0) for potassium hexacyanoferrate (II) shifts toward positive potentials (because of ion pairing) and there was a corresponding increase in the heterogeneous rate constant, k(0). The latter effect was attributed to increasing ion-ion (cation-ferrocyanide ion) interactions as the film dried and the enhancing effect this had on the prevention of surface poisoning reactions at the electrode. These factors are discussed in relation to sensor design.

Amperometric biosensors (based on rhodinised carbon electrodes) for glucose, glutamine and glutamate were constructed. The sensors were incorporated into a three cell parallel FIA system and used to monitor the three analytes on-line during two mammalian cell perfusion cultures. All measurements were made simultaneously from undiluted media sample. Use of the FIA system enabled easy and rapid exchange of the sensors, during cultivation. The inclusion of a calibration step, regularly for all sensors, helped to maintain the accuracy of all measurements. Comparison with off-line measurements indicated that all three biosensors operated successfully, providing accurate information.

Interest in operating bioelectrochemical sensors in unconventional environments such as non-polar water-immiscible organic solvents has led to new concepts in biosensor design. This brief review considers the evolution of an approach to electrochemical measurement using thin ionically conducting films at the surface of closely spaced micro-electrodes. The ability to support ail the elements normally associated with the bioelectrochemistry of catalytic proteins at a semi-rigid ionic matrix leads to a robust sensor configuration capable of operating in non-polar solvents such as hexane or directly in gases. Such multi-phase sensors are expected to be of particular utility in environmental monitoring where measurements need to be made in water and air samples.

Methylamine dehydrogenase, a tryptophan tryptophyl quinone (TTQ) containing quinoprotein, catalyzes the oxidation of a variety of primary aliphatic monoamines and diamines to their respective aldehydes and ammonia. This paper reports the construction and characterization of an enzyme electrode capable of detecting histamine and methylamine at +200 mV versus a saturated calomel reference electrode. The methylamine dehydrogenase isolated from Paracoccus denitrificans was used in conjunction with the insoluble mediator tetracyanoquinodimethane (TCNQ) to construct enzyme electrodes which will potentially provide simple rapid analysis of histamine without the need for the extensive sample pretreatments currently required in HPLC and GLC analysis. The linear response of this amperometric sensor, between 0 and 200 mu M, correlates well with elevated histamine levels predominant in patients with chronic myelogenous leukaemia, whilst the observed limit of detection, 4.8 mu M, compares favourably with the lower limits of detection reported for a potentiometric histamine sensitive enzyme electrode.

A disposable choline biosensor based on ruthenized carbon screen-printed electrodes has been prepared and its use for monitoring organophosphorus pesticides and carbamates is described. The presence of 0.5% ruthenium on activated carbon mixed to form a simple graphite-based ink for the working electrode surface increased the sensitivity towards hydrogen peroxide. The choline biosensor is based on such an electrode coupled with choline oxidase immobilized by adsorption. The inhibition effect of carbamates and organophosphorus pesticides on acetylcholinesterase was detected using this biosensor. With the optimized procedure described (pH, buffer composition, incubation time, substrate concentration) we are able to detect concentrations of pesticides (carbofuran) as low as 1 nM.

Amperometric enzyme sensors for lactate, glutamate and glutamine were constructed using rhodinised carbon electrodes. All three sensors operated at a potential of +400 mV (Ag/AgCl). Lactate sensors were constructed using lactate oxidase immobilized in hydroxyethylcellulose, overlaid with a cellulose acetate membrane. Using a flow-injection system, the sensors had a linear range of 0.1 to 1.5 mM. Measurements of lactate concentrations from a mammalian cell culture were compared with results obtained from a commercial instrument. A correlation coefficient of r=0.982 (n = 15) was obtained. Glutamate and glutamine sensors were fabricated based on glutaraldehyde immobilization; the former by incorporating glutamate oxidase and the latter based on glutamate oxidase and glutaminase (both had a linear range of 0.1 to 1.5 mM). The feasibility of using these sensors, in conjunction with a previously described glucose sensor, for mammalian cell culture monitoring is discussed

n/a

Micropollutants in waste water streams can be a serious problem for the anaerobic digestion process. A short-term acute bioassay system is described for testing the effects of new and potentially toxic compounds on anaerobic digestion processes. Change in methanogenic activity was used as the monitored process parameter and the performance of intoxicated inocula was compared to activity prior to adding test compounds and to the activity of a parallel control assay. The performance of the bioassay was tested with chlorinated solvents and heavy metals. Trichloroethane caused a 50% reduction in methanogenic activity at 7 mg/l assay. The performance of suspended and fixed biomass assays were compared; the suspended growth was found to be five times more sensitive to trichloroethane. There was no clear inhibition with the heavy metals even at the highest concentration used (up to 750 mg Cu/l). The duration of assay was found to be an important parameter in the evaluation of anaerobic toxicity.

This paper describes the first successful application of polymeric ferrocenes as mediators in amperometric biosensors. The results have implications for the design of stable biosensors and bioelectronic devices involving electron transfer from oxidoreductases to electrode surfaces. The behaviour of two ferrocene polymers in enzyme electrodes was explored and a distinctive pH profile was noted. It is suggested that changes in either the enzyme conformation or the polymer in response to hydrogen ion concentration may explain the difference in behaviour between monomeric and polymeric ferrocenes.

Ink-jet printing has been demonstrated as a manufacturing technique that facilitates the rapid, reproducible and economical production of amperometric glucose biosensors. Glucose was chosen as the analyte for demonstrating the process. For eight electrodes produced, the relative standard deviation of the response was less than 5%. The technique is extremely versatile, and will enable a wide variety of reagents to be placed on virtually any sensor design. This technique will be of particular benefit for the mass manufacture of intricate devices, where existing production techniques, such as screen-printing, may not be suitable.

An amperometric horseradish peroxidase electrode is described for the determination of hydrogen peroxide in organic solvents. The enzyme was co-adsorbed with an electron mediator, hexacyanoferrate(II), to the surface of a graphite foil electrode making reagentless measurement possible. The electrochemical reduction of the enzymatically oxidized mediator was utilized as the analytical signal. The electrode can be operated in dioxane, chloroform and chlorobenzene, the presence of a small quantity of aqueous buffer being essential for activity. On this basis a small, probe-type sensor has been assembled the response of which is linearly related to hydrogen peroxide concentration between 0.05 and 1 mM. A tyrosinase sensor has been constructed by combining a Clark-type oxygen electrode with a membrane bearing adsorbed enzyme. The sensor is capable of measuring between 0.1 and 5 mM phenol in chloroform saturated with aqueous buffer.

The properties of glucose oxidase (GOD) are described in relation to the widespread use of this enzyme in biosensors, The shortcomings of other enzymes that oxidize glucose are indicated. Isolation of and early work on the enzyme are briefly reviewed. The sources and the physical characteristics of the enzyme are listed. The structure of GOD and how it relates to the immobilization and stability of the enzyme is described along with other factors that influence stability. The role of flavin adenine dinucleotide and attempts to use it in immobilization of the enzyme are described. Inhibitors of the enzyme are listed, and substrates of the enzyme and the reaction mechanism are considered in detail. The role of electron acceptors in amperometric biosensors is described, and alternatives to their use are highlighted. Other uses of the enzyme are described and the properties of the enzyme are summarized with reference to a commercially successful biosensor.

Biosensors have been extensively studied during the last 20 years, and a myriad of laboratory biosensors have been developed. Improvements are required in biosensor design and performance before they become widely accepted in industrial process monitoring. However, as the biotechnology industry expands, biosensors may become more acceptable because, despite their limitations, they are the only devices capable of delivering the information required.

Organic phase enzyme electrodes (OPEEs) offer new possibilities both for the design of biosensors for use in organic solvents and in the construction of devices incorporating an organic phase for use with aqueous samples. The advantages that accrue from performing biocatalytic and electrochemical reactions in non-aqueous systems are discussed in relation to possible applications in medicine. pharmaceuticals, petrochemicals, the food industry, environmental monitoring and defence. This critical but speculative review considers the detailed implications of operating enzyme electrodes in organic solvents and briefly examines the broader issues of optical, calorimetric and piezoelectric transduction coupled with both catalytic and affinity systems, such as nucleic acid probes and antibodies.

The latest workshop of the European Community (EC) Concerted Action on Chemical sensors for in vivo monitoring was held in Nauplion, Greece, in April this year. This fifth workshop focused on The design and development of new sensors for in vivo monitoring, and was organized into five sessions: design and development of new sensors; operational considerations; performance of analytical systems; novel sensors/tissue heterogeneity; and infra-red spectroscopy.

An amperometric enzyme electrode incorporating horseradish peroxidase is described for the determination of hydrogen peroxide in organic solvents. The enzyme was co-adsorbed with an electron mediator, potassium hexacyanoferrate(II), on the surface of a graphite foil electrode, making reagentless measurement possible. The electrochemical reduction of the enzymatically oxidized mediator was utilized as the analytical signal. Studies in different solvent systems revealed that the electrode could be operated in dioxane, chloroform and chlorobenzene, the last two providing approximately double the sensitivity of the former. The presence of a small amount of aqueous buffer was essential for sensor activity. During 2 weeks of intermittent use, the sensitivity of the electrode decreased to 40% of its initial value. At least 50 assays could be performed with a single sensor.

n/a

n/a