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Introduction to the principles and applications of biosensors.

A biosensor is an analytical device that responds to an analyte in an appropriate sample and interprets its concentration as an electrical signal via a suitable combination of a biological recognition system and an electrochemical transducer. As a result of recent scientific and technological progress, such devices are likely to play an increasingly important role in generating analytical information in all sectors of human endeavour, from medicine to the military. In particular, biosensors will form the basis of cheap, simple devices for acquiring chemical information, bringing sophisticated analytical capabilities to the non-specialist and general public alike. The market opportunities for the rapid exploitation of novel developments in this sector are substantial. Biosensor research is also likely to have a significant impact on the development of modern electronics.

Biotechnology

Sensitivity and dynamics of bioreceptor-based biosensors.

Bioreceptors can provide the basis for specific and sensitive biosensors. There are many sources of bioreceptors in nature for biochemicals of interest in biotechnology and biomedicine. These bioreceptors (antibodies, enzymes, membrane proteins, binding proteins) can be modified and produced in large quantities using modern biotechnological techniques. The characteristics of the biosensor can also be fine-tuned by modifying the structure of the analog-analyte, which will also provide several orders of magnitude of range sensitivity with a given bioreceptor. The ultimate sensitivity of the biosensor may be limited by the dissociation kinetics of the reaction between analyte and bioreceptor because there is a trade-off between sensitivity and sensor response rate to changes in analyte concentration.

Antibodies

Microdialysis and glucose biosensor for in vivo monitoring.

Microdialysis coupled to a glucose biosensor led to a continuous monitor system in vivo for glucose. Several microdialysis probes were used to stabilize the biosensor response. In vivo experiments, especially when the probe was placed subcutaneously, showed that the sensitivity of the biosensor decreased continuously; various kinds of fibers with a molecular weight cut-off (MWCO) ranging from 6,000 to 20,000 were compared. A wall-jet flow cell as detector for glucose showed less interference when compared to a thin layer cell. Glucoday, a new commercial instrument based on this principle, is presented.

Biosensing Techniques

[Biosensors for the determination of phenol and benzoate on the basis of Rhodococcus cells and enzyme extracts].

An amperometric biosensor for determination of phenol, cresol, benzoate and 2-methyl-4-chlorophenol using Rhodococcus-cells and enzyme extracts of Rhodococcus has been developed. The influence of cultivation of Rhodococcus-cells and preincubation of the biosensor with desired substrate on sensibility and specificity was been investigated. In relation to cultivation and preincubation the Rhodococcus sensor was high specific to benzoate or phenol and cresol. A linear range was obtained for phenol and benzoate up to 80 mumol and for 2-methyl-4-chlorophenol up to 400 mumol. The biosensor using enzyme extracts show a higher specificity, it is but necessary NADPH. A further disadvantage is the little measuring range of this sensor.

Benzoates

[Nucleic acids as a basis for creating biosensors].

Here we present a brief conception of biosensors. Structural peculiarities and properties of single- and double-stranded nucleic acids that are to be taken into account when creating biosensors on the basis of these biomolecules are considered. On the example of two biologically active compounds a possibility is shown for constructing biosensors on the basis of liquid-crystalline dispersions of low molecular mass DNA and on the basis of liquid-crystalline DNA dispersions immobilized due to their inclusion into the synthetic polymeric matrix.

Biosensing Techniques

Determination of the physical structure of biological materials at biosensor interfaces by techniques of increasing magnification from microscopic to molecular scale.

Chemical selectivity of biosensors is derived from biological materials interfaced to the surface of transducing devices. Molecular recognition events lead to macroscopic function suitable for analytical measurements. The structure-function relationships of biochemical species at interfaces must be established to characterize and optimize biosensor operation. The techniques of ellipsometry, fluorescence microscopy, electron microscopy, and scanning tunneling microscopy are used to investigate the structure of monolayers and multilayers of proteins and lipids at interfaces that are prepared by Langmuir-Blodgett techniques and by self-assembly from bulk solution. The relative merits and limitations of the measurement techniques in the determination of aspects of interfacial structure are considered.

1,2-Dipalmitoylphosphatidylcholine

Biosensors for process monitoring.

A short review about the biosensor research activities for bioprocess monitoring in the F.R.G. after its reunification is given. The principles of biosensor applications are presented. In situ sensors and sensors based on the principles of flow injection analysis are studied. Some applications of a four-channel enzyme thermistor, bio-field effect transistors, and immunoanalysis systems for real process monitoring are presented.

Biosensing Techniques

Modified electrode surface in amperometric biosensors.

The electron transfer reactions of biological molecules are frequently very slow at ordinary electrodes. To overcome this problem, and thus to facilitate the direct coupling of biological redox reactions to electrodes for biosensor or bioelectronic applications, various types of modified electrode have been used. These include electrodes modified by the covalent attachment of species to the surface, by the reversible adsorption of promotors, or by the deposition of polymeric species, and the use of conducting polymers or conducting organic salts as electrode materials. Some of these different approaches are reviewed and their applications to biosensors and bioelectrochemistry are discussed.

Biosensing Techniques

A biosensor for L-proline determination by use of immobilized microbial cells.

A biosensor to quantify L-proline within 10(-5)-10(-3) mole/L concentration is described. Immobilized Pseudomonas sp. cells grown in a medium containing L-proline as the only source of carbon and nitrogen were used to create the biosensor. The cells oxidized L-proline specifically consuming O2 and did not react with other amino acids and sugars. The change in oxygen concentration was detected with a Clark oxygen membrane electrode. The cells were immobilized by entrapment in polyvinyl alcohol (PVA) cryogel. The resultant biocatalyst had a high mechanical strength and retained its L-proline-oxidizing ability for at least two months.

Adenosine Triphosphate

Mapping of viral epitopes with conformationally specific monoclonal antibodies using biosensor technology.

An automated biosensor system (BIAcore) designed for measuring molecular interactions in real time and without labelling any of the reactants was used for mapping the epitopes of tobacco mosaic virus protein using conformationally specific monoclonal antibodies (MAbs). Some of the MAbs used as capturing antibody on the sensor chip allowed a conformational change to occur in the viral protein. As a result, MAbs specific for the quaternary structure of polymerized viral protein were able to bind to monomeric viral subunits. Compared with classical solid-phase enzyme immunoassay, the biosensor technology possesses several advantages for epitope mapping of viral proteins.

Antibodies, Monoclonal

Flow injection analysis and in-line biosensors for bioprocess control: a comparison.

Miniaturization will unify the different approaches chosen for the application of biosensors in bioprocess control. The most versatile system, which in our opinion is flow injection analysis will be the method of choice for the introduction of biosensors in bioprocess control. A lot of experience will be gained for the future development of miniaturized total chemical analysis systems.

Biosensing Techniques

Biosensors and enzyme immobilized electrodes.

A biosensor is a device which consists of a biological sensing element connected to a transducer. The transducer can be electronic, optical, electrical, etc. This emerging technology offers us a powerful tool which is radically altering our approach to analytical methods. It was realised that enzymes are natural sensors on account of their highly selective nature. Much of the impetus to the work has come from medical requirements. Instant analysis of clinical samples has an obvious appeal to physicians and patients alike. Of particular interest is the possibility of continuous 'in-vivo' monitoring of metabolites, drugs and proteins using miniature, portable systems. In recent years, there has been a growing demand for biosensors in the fields of veterinary science, animal husbandry, the food industry and environmental monitoring. However, the possibility of successful application rests upon future developments. Increasing attention will have to be paid to the engineering of both the basic components and the device on the whole. New biochemical reactions will either have to be discovered or engineered through genetic manipulation or chemical techniques. Optimization of response time, selectivity, stability and low costs should receive priority considerations.

Biosensing Techniques

A fast responding fibre optic glucose biosensor based on an oxygen optrode.

A fast responding glucose biosensor for the continuous determination of glucose is presented. The biosensor is based on an oxygen optrode, which measures the consumption of oxygen via dynamic quenching of the fluorescence of an indicator by molecular oxygen. Glucose oxidase (GOD) is immobilised onto the surface of this oxygen optrode by adsorption to carbon black and by crosslinking with glutardialdehyde. Carbon black is used as an optical isolation to protect the optrode from the interference of ambient light and sample fluorescence. The measurements were performed in a flow through cell with air saturated glucose standard solutions (phosphate buffered saline pH 6.9). The effect of four different qualities of GOD in relation to response times (the time required to reach 90% of the steady-state signal, tau 90, was 8-60 s, the linear analytical range (0.01 to 2 mM glucose) and the long-term stability (tau 1/2 was 1-20 weeks) were investigated. A simple device is presented capable of enlarging the analytical range up to 200 mM glucose concentration.

Biosensing Techniques

Piezoelectric crystal biosensors.

The recent development of piezoelectric devices as biosensors is reviewed. Biological materials, like enzymes, lipids, antibodies and antigens, have been used as specific coatings and were utilized for the determination of different substrates. Methods of protein coating and several applications are reported including microgravimetric immunoassays, microbial assays, DNA hybridization, enzyme detections and gas phase biosensors. Although the piezoelectric immunochemical sensor is convenient to use and very promising, a thorough understanding of the different phenomena associated with crystals frequency measurement in biological reactions is still lacking and deserves further investigation.

Antibodies

The potential role of biosensors in the food and drink industries.

Despite their apparent potential as analytical tools in the food and drink industries, only a few biosensors are used routinely. This article describes the development of biosensors for these sectors and discusses the technical and economic problems of applying this technology to the monitoring of food and drink products.

Beverages

An FIA biosensor system for the determination of phosphate.

A flow injection analysis (FIA) biosensor system for the determination of phosphate was constructed using immobilized nucleoside phosphorylase and xanthine oxidase and an amperometric electrode (platinum vs silver/silver chloride, polarized at 0.7 V). When a phosphate-containing sample was injected into the detection cell, phosphate reacted with inosine in the carrier buffer to produce hypoxanthine and ribose-1-phosphate in the presence of nucleoside phosphorylase. Hypoxanthine was then oxidized by xanthine oxidase to uric acid and hydrogen peroxide, which were both detected by the amperometric electrode. The response of the FIA biosensor system was linear up to 100 microM phosphate, with a minimum detectable concentration of 1.25 microM phosphate. Each assay could be performed in 5-6 min and the system could be used for about 160 repeated analyses. This system was applicable for the determination of phosphate in various food products and plasma, and the results obtained agreed well with those of the enzymatic assay.

Biosensing Techniques

Internal supply of coenzyme to an amperometric glucose biosensor based on a chemically modified electrode.

A biosensor for glucose using glucose dehydrogenase immobilized on a chemically modified graphite electrode was supplied with coenzyme, nicotinamide adenine dinucleotide (NAD+), through pores in the material. A graphite rod was hollowed out, leaving 0.3 mm at the end contacting the solution, filled with 10 mM NAD+ and pressurized. The response factor was 40% of that obtained when 2 mM NAD+ was mixed with the sample solution in a flow system. The coenzyme consumption was 11 microliters h-1 representing a 500-fold saving compared to supply through the bulk solution. The biosensor had a linear calibration curve from the detection limit, 1 microM, to 2 mM glucose and a repeatability of 0.3%. The graphite electrode was modified by adsorption of a bis-(benzophenoxazinyl)-terephthaloyl derivative in order to be able to oxidize NADH at 0 mV versus Ag/AgCl, 0.1 M KCl.

Biosensing Techniques

A chemiluminescence fiber-optic biosensor system for the determination of glutamine in mammalian cell cultures.

A chemiluminescence fiber-optic biosensor system has been developed for determining glutamine in hybridoma cell cultures producing monoclonal antibodies against viral surface antigens. Glutaminase and glutamate oxidase (GLO) were immobilized onto aminopropyl glass beads via glutaraldehyde activation separately and packed in a column. Two separate columns containing immobilized GLO and catalase were placed upstream to eliminate endogenous glutamate. In the presence of ferricyanide, luminol reacted with hydrogen peroxide released from the enzymatic reactions to produce a chemiluminescence (CL) light signal which was detected and quantitated with a fiber-optic system. In combination with flow injection analysis it was possible to process samples virtually identically, thus avoiding difficulties in reproducing the CL signal. There was an excellent linear relationship between the CL response and standard glutamine concentration in the range 10(-6) to 10(-3) M. A complete analysis could be performed in 2 min including sampling and washing. Each immobilized enzyme column was stable for at least 300 repeated analyses without any loss of activity. When the biosensor system was used for the determination of glutamine in spent mammalian cell cultures, the values obtained compared well with those of high-performance liquid chromatography, thus validating the applicability of the CL fiber-optic system.

Animals