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Analytical and Bioanalytical Chemistry (v.377, #3)
The potential of mechanism-based bioanalytical tools in ecotoxicological exposure and effect assessment by Rik I. L. Eggen; Helmut Segner (pp. 386-396).
The current challenge to ecotoxicology is to develop tools that allow rapid and cost-efficient detection of those environmental chemicals or their combinations that are responsible for sublethal, chronic toxic effects in exposed organisms. Bioanalytical tools may meet these challenges, particularly if they are mechanism-based. Technically, bioanalytical tools allow rapid and cost-efficient analysis of environmental matrices. Mechanism-based, bioanalytical tools, however, do not only indicate that certain chemicals are there, but—and this is the major advantage of mechanism-based bioanalytical tools (MBBTs)—they indicate that chemicals with a specific mode of toxic action or a specific toxic potential are there. In this way MBBTs bridge exposure and effect assessment and help in a faster identification of the causative agent(s). Several principles of MBBTs, including immunoassays, enzyme inhibition assays, receptor assays and gene induction assays are briefly discussed and their application in processes such as bioassay-directed fractionation is illustrated. The focus of this manuscript is the analytical power of MBBTs in exposure and effect assessment. MBBTs have, however, a much broader potential and can support research on other challenges in ecotoxicology such as mixture effects or multiple effects caused by single pollutants or by various stresses simultaneously.
Keywords: Bioassay; Biomarker; Immunoassay; Nuclear receptor; Reporter gene; Biosensor
Effect-directed analysis: a promising tool for the identification of organic toxicants in complex mixtures? by Werner Brack (pp. 397-407).
Wastewater effluents, groundwater, surface water, sediments, soils and air particulate matter are often contaminated by a multitude of chemicals. Since often no a priori knowledge of relevant toxicants exists, chemical analysis alone is not an appropriate tool for hazard assessment. Instead, a linkage of effect data and hazardous compounds is required. For that purpose, effect-directed analysis (EDA) was developed, which is based on a combination of biotesting, fractionation procedures and chemical analytical methods. Since a controversial discussion about the prospects of success in relation to the expense exists, the current methodological state of EDA for organic toxicants in complex mixtures and important results are reviewed in this paper with the aim of establishing criteria for the successful use of this promising tool. While EDA is a powerful tool to identify specifically acting individual toxicants close to the source of emission, it is inappropriate for screening purposes and often may fail in remote areas where the concentrations of specific toxicants are too low relative to the nonspecific toxicity of the whole mixture of natural and anthropogenic compounds. The biological tools have to be carefully selected with respect to their ability to detect specific effects and their significance in hazard assessment. Sophisticated chemical tools are required to identify individual toxicants in mixtures of thousands of compounds, which are typical for contaminated environments.
Approaches to bioresponse-linked instrumental analysis in water analysis by Gerald Brenner-Weiß; Ursula Obst (pp. 408-416).
A new concept based on hyphenation of biotests, for biological selection, and chemical analysis is introduced for water analysis. Biomolecular recognition components such as receptors, enzymes, and nucleic acids integrated in biological reaction chains are used for binding and selective enrichment of known and unknown biologically active substances in water samples; this is followed by identification and quantitation. The coupling of biomolecular recognition and binding to chemical analysis can be achieved either in discrete analytical steps, e.g. binding and elution of bioactive ligands from affinity columns followed by chemical analysis, or by methods capable of monitoring the binding of the ligand and simultaneous verification of its identity. This analytical strategy, denoted bioresponse-linked instrumental analysis (BLIA), enables detection of potential biological effects and identification of the analyte causing these effects. Several examples are presented.
Keywords: Bioresponse-linked instrumental analysis; Water analysis; Biomolecular recognition; Chemical analysis
Recombinant antibodies for environmental analysis by K. Kramer; B. Hock (pp. 417-426).
Initial steps of antibody engineering in the late eighties revolutionized the technology of antibody production, particularly in the area of immunotherapy and diagnostics. Hallmarks that seemed to be out of reach for a long time are now the state of the art, e.g. tailoring of antibodies to match particular needs or by-passing immunization by use of antibody libraries. Despite the apparent benefits of recombinant antibody technologies, this field has been opened up hesitantly for other applications. This review addresses the development of recombinant antibody synthesis in environmental analysis. Examples are given of the molecular evolution of pesticide antibodies and their application for the analysis of real samples.
Keywords: Recombinant antibodies; Environmental analysis; Pesticides; Molecular evolution
Olfactory receptors: molecular basis for recognition and discrimination of odors by Heinz Breer (pp. 427-433).
The daunting task of our nose to detect and discriminate among thousands of low-molecular-weight organic compounds with diverse chemical structures and properties requires an enormous molecular recognition capacity. This is based on distinct proteins, capable of recognizing and binding odorous compounds, including odorant-binding proteins, which are supposed to shuttle odorous compounds through the nasal mucus, and most notably the odorant receptors, which are heptahelical membrane proteins coupling via G-proteins onto intracellular transduction cascades. From more than a thousand genes each olfactory neuron is supposed to express only one receptor subtype. Receptors appear to be selective but rather non-specific—i.e. a distinct odorant activates multiple receptors and individual receptors respond to multiple odorants. It is the molecular receptive range of its receptor type which determines the reaction spectrum of a sensory neuron. Populations of cells equipped with the same receptor type project their axons to common glomeruli, thereby transmitting the molecular receptive range of a receptor type into the receptive field of glomerulus. Recent insight into the molecular basis of odor recognition and the combinatorial coding principles of the olfactory system may provide some clues for the design and development of technical sensors, electronic noses. In this review more emphasis has been placed on physiological rather than analytical aspects.
Keywords: Odorant receptors; Binding-proteins; Gene family; Sensory neurons; Projection; Plfactory bulb
Biosensors for environmental pollutants and food contaminants by Antje J. Baeumner (pp. 434-445).
This review article provides an overview of the most recent literature on biosensors for environmental pollutants and food contaminants. Due to the large number of publications, only papers published between 2000 and January 2003 were considered. Also, while not all of the published literature could be reviewed here, over 200 references are cited to provide a good overview of research undertaken in the last two years. Older publications are covered by a number of earlier review articles. This article provides an introduction into the field including specific consideration of the application areas, describes the typical biosensor assay format used, and is subsequently structured according to the biorecognition elements used (i.e., nucleic acids, enzymes, whole cells, tissue and whole organisms, antibodies and receptors, and biomimetic materials). In addition, a section on microbiosensing systems is provided. Since only very few microbiosensors with applications in environmental and food systems have been published, enabling technology is also covered in this article.
Keywords: Biosensor; Environmental analysis; Food contaminants; Microfabrication
Current research activity in biosensors by Hideaki Nakamura; Isao Karube (pp. 446-468).
Biosensors consist of a molecular recognition element and a transducer. Since the first biosensor was developed by Updike and Hicks [1] many biosensors and their associated techniques have been studied and developed. In this review current research activity on the fundamentals and applications of biosensors is summarized. In discussion of the former, molecular recognition elements, techniques and tools for biosensor construction, and basic biosensor devices are introduced. Coverage of the latter includes description of biosensors for environmental, food, and clinical fields. Chemical sensors developed in our laboratory for environmental monitoring are also described. This review mainly summarizes work performed by our group and by our colleagues, and refers to the main review articles summarizing each field of biosensor research in recent years.
Keywords: Biosensor; Molecular recognition element; Array; Environmental biosensor; Food biosensor; Clinical biosensor
Array biosensor for detection of toxins by Frances S. Ligler; Chris Rowe Taitt; Lisa C. Shriver-Lake; Kim E. Sapsford; Yura Shubin; Joel P. Golden (pp. 469-477).
The array biosensor is capable of detecting multiple targets rapidly and simultaneously on the surface of a single waveguide. Sandwich and competitive fluoroimmunoassays have been developed to detect high and low molecular weight toxins, respectively, in complex samples. Recognition molecules (usually antibodies) were first immobilized in specific locations on the waveguide and the resultant patterned array was used to interrogate up to 12 different samples for the presence of multiple different analytes. Upon binding of a fluorescent analyte or fluorescent immunocomplex, the pattern of fluorescent spots was detected using a CCD camera. Automated image analysis was used to determine a mean fluorescence value for each assay spot and to subtract the local background signal. The location of the spot and its mean fluorescence value were used to determine the toxin identity and concentration. Toxins were measured in clinical fluids, environmental samples and foods, with minimal sample preparation. Results are shown for rapid analyses of staphylococcal enterotoxin B, ricin, cholera toxin, botulinum toxoids, trinitrotoluene, and the mycotoxin fumonisin. Toxins were detected at levels as low as 0.5 ng mL−1.
Keywords: Biosensor; Immunosensor; Array; Multi-analyte; Toxin; Detection
Are biosensor arrays in one membrane possible? A combination of multifrequency impedance measurements and chemometrics by Britta Lindholm-Sethson; Josefina Nyström; Paul Geladi; Roger Koeppe; Andrew Nelson; Conor Whitehouse (pp. 478-485).
A novel concept of a membrane-based micro-array biosensor is presented. The methodology is based on a single lipid membrane interrogated with electrochemical impedance techniques followed by multivariate data analysis. A single membrane is designed so that relaxation processes with a range of time constants can be probed at different potentials. A range of other approaches cited in the literature is reviewed.
Keywords: Membrane-based biosensor; Micro array; Electrochemical impedance spectroscopy; Chemometrics
Biological application of microelectrode arrays in drug discovery and basic research by Alfred Stett; Ulrich Egert; Elke Guenther; Frank Hofmann; Thomas Meyer; Wilfried Nisch; Hugo Haemmerle (pp. 486-495).
Electrical activity of electrogenic cells in neuronal and cardiac tissue can be recorded by means of microelectrode arrays (MEAs) that offer the unique possibility for non-invasive extracellular recording from as many as 60 sites simultaneously. Since its introduction 30 years ago, the technology and the related culture methods for electrophysiological cell and tissue assays have been continually improved and have found their way into many academic and industrial laboratories. Currently, this technology is attracting increased interest owing to the industrial need to screen selected compounds against ion channel targets in their native environment at organic, cellular, and sub-cellular level.As the MEA technology can be applied to any electrogenic tissue (i.e., central and peripheral neurons, heart cells, and muscle cells), the MEA biosensor is an ideal in vitro system to monitor both acute and chronic effects of drugs and toxins and to perform functional studies under physiological or induced pathophysiological conditions that mimic in vivo damages. By recording the electrical response of various locations on a tissue, a spatial map of drug effects at different sites can be generated, providing important clues about a drug's specificity.In this survey, examples of MEA biosensor applications are described that have been developed for drug screening and discovery and safety pharmacology in the field of cardiac and neural research. Additionally, biophysical basics of recording and concepts for analysis of extracellular electrical signals are presented.
Keywords: Drug discovery; Safety pharmacology; Organotypic tissue culture; Cell culture; Ion channel; Microelectrode array; Field potential; Electrophysiology
Biosensors based on enzyme field-effect transistors for determination of some substrates and inhibitors by Sergei V. Dzyadevych; Alexey P. Soldatkin; Yaroslav I. Korpan; Valentyna N. Arkhypova; Anna V. El'skaya; Jean-Marc Chovelon; Claude Martelet; Nicole Jaffrezic-Renault (pp. 496-506).
This paper is a review of the authors' publications concerning the development of biosensors based on enzyme field-effect transistors (ENFETs) for direct substrates or inhibitors analysis. Such biosensors were designed by using immobilised enzymes and ion-selective field-effect transistors (ISFETs). Highly specific, sensitive, simple, fast and cheap determination of different substances renders them as promising tools in medicine, biotechnology, environmental control, agriculture and the food industry.The biosensors based on ENFETs and direct enzyme analysis for determination of concentrations of different substrates (glucose, urea, penicillin, formaldehyde, creatinine, etc.) have been developed and their laboratory prototypes were fabricated. Improvement of the analytical characteristics of such biosensors may be achieved by using a differential mode of measurement, working solutions with different buffer concentrations and specific agents, negatively or positively charged additional membranes, or genetically modified enzymes. These approaches allow one to decrease the effect of the buffer capacity influence on the sensor response in an aim to increase the sensitivity of the biosensors and to extend their dynamic ranges.Biosensors for the determination of concentrations of different toxic substances (organophosphorous pesticides, heavy metal ions, hypochlorite, glycoalkaloids, etc.) were designed on the basis of reversible and/or irreversible enzyme inhibition effect(s). The conception of an enzymatic multibiosensor for the determination of different toxic substances based on the enzyme inhibition effect is also described.We will discuss the respective advantages and disadvantages of biosensors based on the ENFETs developed and also demonstrate their practical application.
Keywords: Biosensors; ENFETs; Enzyme; Substrates; Inhibitors; Multibiosensor
Biosensors based on electropolymerized films: new trends by Serge Cosnier (pp. 507-520).
Electropolymerized films have received considerable attention in the development of biosensors and biochips, and are advancing rapidly. This paper reviews recent advances and scientific progress in electrochemical immobilization procedures for biological macromolecules on electrodes via electrogenerated polymer films. Biomolecule immobilization is classified as covalent linkage, attachment by affinity interactions, and physical entrapment. The last approach entails the use of conducting and non-conducting films, composite polymer films, and templates for the electropolymerization process. Some advances in the electrochemical transduction of biological events (enzymatic reaction, immunoreaction, or oligonucleotide hybridization) involving the redox properties or the conductivity of electropolymerized films are also presented.
Keywords: Biosensors; Biochips; Polypyrrole; Electropolymerized films; Enzyme electrode; Protein immobilization
Electrical biochip technology—a tool for microarrays and continuous monitoring by Joerg Albers; Thomas Grunwald; Eric Nebling; Gundula Piechotta; Rainer Hintsche (pp. 521-527).
Based on electrical biochips made in Si-technology cost effective portable devices have been constructed for field applications and point of care diagnosis. These miniaturized amperometric biosensor devices enable the evaluation of biomolecular interactions by measuring the redox recycling of ELISA products, as well as the electrical monitoring of metabolites. The highly sensitive redox recycling is facilitated by interdigitated ultramicroelectrodes of high spatial resolution. The application of these electrical biochips as DNA microarrays for the molecular diagnosis of viral infections demonstrates the measurement procedure. Self-assembling of capture oligonucleotides via thiol-gold coupling has been used to construct the DNA interface on-chip. Another application for this electrical detection principle is continuous measuring with bead-based biosensors. Here, paramagnetic nanoparticles are used as carriers of the bioanalytical interface in ELISA format. A Si-micromachined glucose sensor for continuous monitoring in interstitial fluid ex vivo shows the flexibility of the electrical platform. Here the novel approach is a pore membrane in micrometer-dimensions acting as a diffusion barrier. The electrochemical detection takes place in a cavity containing glucose oxidase and a Pt-electrode surface. The common hydrogen peroxide detection, together with Si technology, enable precise differential measurements using a second cavity.
Keywords: Electrical biochips; Microsystems; Magnetic beads; DNA array; Glucose biosensor
Present and future of surface plasmon resonance biosensors by Jiří Homola (pp. 528-539).
Surface plasmon resonance (SPR) biosensors are optical sensors exploiting special electromagnetic waves—surface plasmon-polaritons—to probe interactions between an analyte in solution and a biomolecular recognition element immobilized on the SPR sensor surface. Major application areas include detection of biological analytes and analysis of biomolecular interactions where SPR biosensors provide benefits of label-free real-time analytical technology. This paper reviews fundamentals of SPR affinity biosensors and discusses recent advances in development and applications of SPR biosensors.
Keywords: Optical sensor; Biosensor; Affinity biosensor; Immunosensor; Surface plasmon resonance
Sensor strategies for microorganism detection—from physical principles to imprinting procedures by Franz L. Dickert; Peter Lieberzeit; Oliver Hayden (pp. 540-549).
Detecting cells and microorganisms in different matrices is becoming an increasingly important task in a variety of fields including bioprocess control, food technology, health care, and environmental analysis. In this review, fast on-line detection methods for this purpose are presented including different recognition and transducer strategies.
Keywords: Microorganism detection; Sensor; QCM; SAW; SPR; imprinting,
Non-aerosol detection of explosives with a continuous flow immunosensor by Lisa C. Shriver-Lake; Paul T. Charles; Anne W. Kusterbeck (pp. 550-555).
Contamination of groundwater, soil, and the marine environment by explosives is a global issue. Identification, characterization and remediation are all required for a site recognized as contaminated with 2,4,6-trinitrotoluene (TNT) or hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). For each step, a method to accurately measure the contaminant level is needed. This paper reviews some of the current methods with emphasis on a single biosensor developed in our laboratory. Current regulatory methods require samples to be sent off-site to a certified laboratory resulting in time delays up to a month. A continuous flow biosensor for detection of explosives has been developed and tested for the rapid field screening of environmental samples. The detection system is based on a displacement immunoassay in which monoclonal antibodies to (TNT) and RDX are immobilized on solid substrates, allowed to bind fluorescently labeled antigens, and then exposed to explosives in aqueous samples. Explosive compounds present in the sample displace proportional amounts of the fluorescent label, which can then be measured to determine the original TNT or RDX concentration. The system can accurately detect ppb to ppt levels of explosives in groundwater or seawater samples and in extracts of contaminated soil. The biosensor has applications in environmental monitoring at remediation sites or in the location of underwater unexploded ordnance.
Keywords: Sensor; Explosives; TNT; RDX; Immunoassay
Biochemical analysis with microfluidic systems by Ursula Bilitewski; Meike Genrich; Sabine Kadow; Gaber Mersal (pp. 556-569).
Microfluidic systems are capillary networks of varying complexity fabricated originally in silicon, but nowadays in glass and polymeric substrates. Flow of liquid is mainly controlled by use of electroosmotic effects, i.e. application of electric fields, in addition to pressurized flow, i.e. application of pressure or vacuum. Because electroosmotic flow rates depend on the charge densities on the walls of capillaries, they are influenced by substrate material, fabrication processes, surface pretreatment procedures, and buffer additives. Microfluidic systems combine the properties of capillary electrophoretic systems and flow-through analytical systems, and thus biochemical analytical assays have been developed utilizing and integrating both aspects. Proteins, peptides, and nucleic acids can be separated because of their different electrophoretic mobility; detection is achieved with fluorescence detectors. For protein analysis, in particular, interfaces between microfluidic chips and mass spectrometers were developed. Further levels of integration of required sample-treatment steps were achieved by integration of protein digestion by immobilized trypsin and amplification of nucleic acids by the polymerase chain reaction. Kinetic constants of enzyme reactions were determined by adjusting different degrees of dilution of enzyme substrates or inhibitors within a single chip utilizing mainly the properties of controlled dosing and mixing liquids within a chip. For analysis of kinase reactions, however, a combination of a reaction step (enzyme with substrate and inhibitor) and a separation step (enzyme substrate and reaction product) was required. Microfluidic chips also enable separation of analytes from sample matrix constituents, which can interfere with quantitative determination, if they have different electrophoretic mobilities. In addition to analysis of nucleic acids and enzymes, immunoassays are the third group of analytical assays performed in microfluidic chips. They utilize either affinity capillary electrophoresis as a homogeneous assay format, or immobilized antigens or antibodies in heterogeneous assays with serial supply of reagents and washing solutions.
Keywords: Enzymes; Immunoassays; Nucleic acid analysis; Capillary electrophoresis; Flow-through analysis
Simultaneous measurement of the maximum oscillation amplitude and the transient decay time constant of the QCM reveals stiffness changes of the adlayer by C. Galli Marxer; M. Collaud Coen; H. Bissig; U. F. Greber; L. Schlapbach (pp. 570-577).
Interpretation of adsorption kinetics measured with a quartz crystal microbalance (QCM) can be difficult for adlayers undergoing modification of their mechanical properties. We have studied the behavior of the oscillation amplitude, A 0, and the decay time constant, τ, of quartz during adsorption of proteins and cells, by use of a home-made QCM. We are able to measure simultaneously the frequency, f, the dissipation factor, D, the maximum amplitude, A 0, and the transient decay time constant, τ, every 300 ms in liquid, gaseous, or vacuum environments. This analysis enables adsorption and modification of liquid/mass properties to be distinguished. Moreover the surface coverage and the stiffness of the adlayer can be estimated. These improvements promise to increase the appeal of QCM methodology for any applications measuring intimate contact of a dynamic material with a solid surface.
Keywords: QCM; Amplitude; Viscoelasticity; Decay time constant; Protein; Cell
Cell spreading on quartz crystal microbalance elicits positive frequency shifts indicative of viscosity changes by Carine Galli Marxer; Martine Collaud Coen; Thomas Greber; Urs F. Greber; Louis Schlapbach (pp. 578-586).
Cell attachment and spreading on solid surfaces was investigated with a home-made quartz crystal microbalance (QCM), which measures the frequency, the transient decay time constant and the maximal oscillation amplitude. Initial interactions of the adsorbing cells with the QCM mainly induced a decrease of the frequency, coincident with mass adsorption. After about 80 min, the frequency increased continuously and after several hours exceeded the initial frequency measured before cell adsorption. Phase contrast and fluorescence microscopy indicated that the cells were firmly attached to the quartz surface during the frequency increase. The measurements of the maximal oscillation amplitude and the transient decay time constant revealed changes of viscoelastic properties at the QCM surface. An important fraction of these changes was likely due to alterations of cytosolic viscosity, as suggested by treatments of the attached cells with agents affecting the actin and microtubule cytoskeleton. Our results show that viscosity variations of cells can affect the resonance frequency of QCM in the absence of apparent cell desorption. The simultaneous measurements of the maximal oscillation amplitude, the transient decay time constant and the resonance frequency allow an analysis of cell adsorption to solid substratum in real time and complement cell biological methods.
Keywords: Cell; QCM; Adsorption; Cytoskeleton; Amplitude; Decay time constant