Enzyme electrode probes

A survey of the field of enzyme electrode probes is presented. Probes for the analysis of over 40 different substrates and 25 different enzymes are described. Some of the basic properties of enzyme electrodes, their response characteristics, sensitivity, lifetime and specificity are likewise discussed, as is the future of such electrodes in the fields of medicine, manufacturing, biology and chemistry.     

Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry

The measurement of fluorescence lifetimes is known to be hindered by the wavelenght-dependent and photocathode area-dependent time response of photomultiplier tubes. A simple and direct method is described to minimize the effects in photomultiplier tubes for phase-modulation fluorometry. Reference fluorophores of known lifetime were used in place of the usual scattering reference. The emission wavelenghts of the reference and sample were matched by either filters or a monochromator, and the use of a fluorophore rather than a scatter decreases the differences in spatial distribution of light emanating from the reference and sample. Thus photomultiplier tube artifacts are minimized. Five reference fluorophores were selected on the basis of availability, ease of solution preparation, and constancy of lifetime with temperature and emission wavelenght. These compounds are p-terphenyl, PPO, PPD, POPOP and dimethyl POPOP. These compounds are dissolved in ethanol to give standard solutions that can be used over the temperature range from ?55 to +55°C. Purging with inert gas is not necessary. The measured phase and modulation of the reference solution is used, in conjunction with the known reference, lifetime, to calculate the actual phase and modulation of the exictation beam. The use of standard fluorophores does not require separate experiments to quantify photomultiplier effects, and does not increase the time required for the measurement of fluorescence lifetimes. Examples are presented which demonstrate the elimination of artifactual photomultiplier effects in measurements of the lifetimes of DADH (0.4 ns) and indole solutions quenched by iodide. In addition, the use of these reference solutions increases the accuracy of fluorescence lifetime measurements ranging ranging to 30 ns. We judge this method to provide more reliable lifetime measurements by the phase and modulation method. The test solutions and procedures we describe may be used by other laboratories to evaluate the performance of their phase fluorometers.     

Fast and easy preparation of an amperometric glucose biosensor

Summary Electrochemical polymerization of pyrrole in aqueous KCl solution containing glucose oxidase produces adherent films at platinum electrode surface. Such coated electrodes are prepared in 20 min and can determine glucose in the range 0 to 100mm. The useful lifetime of the electrode is 85 assays. Its stability is at least 75 days under storage in PBS at 4°C.     

The use of isochronal reference standards in phase and modulation fluorescence lifetime measurements

Errors in phase and modulation lifetime measurements observed with the only commercially available instrument are readily apparent when the Debye-Sears modulation tank is not perfectly tuned. Unfortunately, we have found that exact tuning was extremely difficult to achieve and maintain. We report that these errors could be reduced by using single-lifetime (homogeneous) reference standards whose fluorescence lifetime approximated that of the unknown sample (isochronal standards). A number of useful standards are suggested. In the proposed method, the phase shift and relative modulation of the sample emission are measured using the isochronal standard as a reference to determine the effective characteristics of the sinusoidal excitation. The importance of the improvement in accuracy accomplished by the proposed methods is illustrated by the accurate resolution of fluorescence lifetime heterogeneity for two known heterogeneous samples.     

Composite electrochemical biosensors: a comparison of three different electrode matrices for the construction of amperometric tyrosinase biosensors

A comparison of the behaviour of three different rigid composite matrices for the construction of amperometric tyrosinase biosensors, which are widely used for the detection of phenolic compounds, is reported. The composite electrode matrices were, graphite-Teflon; reticulated vitreous carbon (RVC)-epoxy resin; and graphite-ethylene/propylene/diene (EPD) terpolymer. After optimization of the experimental conditions, different aspects regarding the stability of the three composite tyrosinase electrode designs were considered and compared. A better reproducibility of the amperometric responses was found with the graphite-EPD electrodes, whereas a longer useful lifetime was observed for the graphite-Teflon electrodes. The kinetic parameters of the tyrosinase reaction were calculated for eight different phenolic compounds, as well as their corresponding calibration plots. The general trend in sensitivity was graphite-EPD>graphite-Teflon>RVC-epoxy resin. A correlation between sensitivity and the catalytic efficiency of the enzyme reaction for each phenolic substrate was found. Furthermore, differences in the sensitivity order for the phenolic compounds were observed among the three biocomposite electrodes, which suggests that the nature of the electrode matrix influences the interactions in the tyrosinase catalytic cycle.     

On the conversion of solid cancer excess relative risk into lifetime attributable risk

Risk coefficients representing the lifetime radiation-induced cancer mortality (or incidence) attributable to an exposure to ionizing radiation, have been published by major international scientific committees. The calculations involve observations in an exposed population and choices of a standard population (for risk transportation), of suitable numerical models, and of computational techniques. The present lack of a firm convention for these choices makes it difficult to inter-compare risk estimates presented by different scientific bodies. Some issues that relate to a necessary harmonization and standardization of risk estimates are explored here. Computational methods are discussed and, in line with the approach utilized by ICRP, conversion factors from excess relative risk (ERR) to lifetime attributable risk (LAR) are exemplified for exposures at all ages and for occupational exposures. A standard population is specified to illustrate the possibility of a simplified standard for risk transportation computations. It is suggested that a more realistic perception of lifetime risk could be gained by the use of coefficients scaled to the lifetime spontaneous cancer rates in the standard population. The resulting quantity lifetime fractional risk (LFR) is advantageous also because it depends much less on the choice of the reference population than the lifetime attributable risk (LAR). Received: 5 April 2001 / Accepted: 1 July 2001     

Protein modified- and membrane electrodes: strategies for the development of biomolecular sensors

Different procedures used for constructing protein/enzyme-modified electrodes are examined, in particular adsorption, covalent attachment and film deposition. The performances of such modified electrodes with electroactive proteins or enzymes attached to their active surface are examined, especially in the case of c-type cytochromes, hydrogenases and glucose oxidase. Another strategy presented in this review consists of the use of membrane electrodes with an electroactive protein imprisoned between a dialysis membrane and the electrode surface. The versatility and other advantages of such a procedure are underlined. Applications of membrane electrodes to the bioremediation of soils and effluents and as models for investigating interactions between proteins and soils are described.     

A potentiometric rapid method of determining the level of phenylacetic acid during the production of beta-lactam antibiotics

A potentiometric rapid method for control of phenylacetic acid (PAA) concentration in production of ++beta-lactam antibiotics is described. The method is based on ion selective electrodes with a film membrane. The results of the theoretical and experimental studies on estimation of the electrode selectivity specific of PAA in the presence of various interfering ions are presented. It was shown possible to use the electrodes for PAA control in the media containing nitrates, bicarbonates and chlorides. Recommendations how to use the ion selective electrodes at various stages in production of ++beta-lactam antibiotics are given. Prospects for improving the method and designing an instrument for rapid assay of phenyl acetate ion activity are discussed.     

Strategies to extend the lifetime of bioelectrochemical enzyme electrodes for biosensing and biofuel cell applications

Enzymes are powerful catalysts for biosensor and biofuel cell electrodes due to their unique substrate specificity. This specificity is defined by the amino acid chain's complex three-dimensional structure based on non-covalent forces, being also responsible for the very limited enzyme lifetime of days to weeks. Many electrochemical applications, however, would benefit from lifetimes over months to years. This mini-review provides a critical overview of strategies and ideas dealing with the problem of short enzyme lifetime, which limits the overall lifetime of bioelectrochemical electrodes. The most common approaches aim to stabilize the enzyme itself. Various immobilization techniques have been used to reduce flexibility of the amino acid chain by introducing covalent or non-covalent binding forces to external molecules. The enzyme can also be stabilized using genetic engineering methods to increase the binding forces within the protein or by optimizing the environment in order to reduce destabilizing interactions. In contrast, renewing the inactivated catalyst decouples overall system lifetime from the limited enzyme lifetime and thereby promises theoretically unlimited electrode lifetimes. Active catalyst can be supplied by exchanging the electrolyte repeatedly. Alternatively, integrated microorganisms can display the enzymes on their surface or secrete them to the electrolyte, allowing unattended power supply for long-term applications.     

Electrodes in external electrohysterography: a systematic literature review

BackgroundIn low-income countries, pregnant women do not have easy access to health care, especially in rural and peri-urban areas. In this context, they can be surprised by the uterine contractions that precede childbirth and sometimes find themselves giving birth at home or on the way to the nearest health facility (located miles away from their home). In view of the development of an external uterine electrohysterogram acquisition system for labour prediction, a review of the literature on electrodes and their characteristics is necessary.MethodsA comprehensive literature review was conducted to collate information on the use of electrodes in external EHG recording and their characteristics.ResultsWet electrodes based on Ag/AgCl redox chemistry are the most common type of electrodes for EHG, employed in different configurations on the pregnant woman’s abdomen. All positioning configurations are around the vertical median axis if they are not placed directly on it. Positioning below the navel seems to be the most efficient. The number of source, reference, and ground electrodes used varies from one author to another, as does the distance between the electrodes.ConclusionTwo well-positioned source electrodes on the vertical median axis, with ground electrode on the right side of the hip and reference one on the left side, are able to generate a good external EHG recording signal. The minimum allowed inter-electrode distance is approximately 17.5 to 25mm.     

Effect of Resonant Radiation Transport on the Parameters of RF and DC Discharges in a He-Ar-Xe Mixture

A study is made of the effect of the transport of Xe 147-nm resonant radiation on the parameters of a low-temperature plasma of DC and RF discharges in gas mixtures used as the working medium in lasers based on infrared transitions in xenon. RF discharges are treated in the planar geometry typical of slab lasers. DC discharges in tubes are treated in cylindrical geometry. The trapping of resonant radiation is described using different approximate models: the decay time approximation for a plasma slab (the Holstein approximation) and the effective lifetime approximation (the Biberman approximation). The transport equation for resonant radiation is solved numerically. The effect of the radiation transport on both the current-voltage characteristics of a discharge and the spatial distribution of the excited Xe atoms is investigated. The current-voltage characteristics calculated for a DC discharge with allowance for the resonant radiation transport agree well with the experimental characteristics. It is found that, for an RF discharge, the effective lifetime approximation overestimates the density of the excited Xe atoms near the electrodes by several times and underestimates this density at the midplane of the discharge gap.     

Lifetime Risk of Fatal Occupational Injuries within Industries,by Occupation,Gender, and Race

Estimates of risk accumulated over a working lifetime are used to assess the significance of many workplace health hazards. Most studies which have estimated this risk have focused on a worker's lifetime risk of dying of a stated illness based on exposure to a hazard in a specific job. The concept, however, has not been widely applied to occupational injury deaths. This study examines the use of lifetime risk based on national fatal injury data from the Bureau of Labor Statistics (BLS) Census of Fatal Occupational Injuries (CFOI). Lifetime risks are defined by specific causal events for those groups identified as having the highest general lifetime risks. The lifetime risk model for injury used in this work can be compared with risk assessments for occupational illnesses. Fatal injury lifetime risk estimates will be useful in defining traumatic injury exposures that are appropriate for targeting research and prevention efforts needed to reduce the burden of work-related death within the United States. These estimates also provide a means of prioritizing traumatic injury research with fatal illness research, while providing the additional benefit of providing a means of informing workers of their fatal injury risks.     

Single-cell bioelectrical impedance platform for monitoring cellular response to drug treatment

The response of cells to a chemical or biological agent in terms of their impedance changes in real-time is a useful mechanism that can be utilized for a wide variety of biomedical and environmental applications. The use of a single-cell-based analytical platform could be an effective approach to acquiring more sensitive cell impedance measurements, particularly in applications where only diminutive changes in impedance are expected. Here, we report the development of an on-chip cell impedance biosensor with two types of electrodes that host individual cells and cell populations, respectively, to study its efficacy in detecting cellular response. Human glioblastoma (U87MG) cells were patterned on single- and multi-cell electrodes through ligand-mediated natural cell adhesion. We comparatively investigated how these cancer cells on both types of electrodes respond to an ion channel inhibitor, chlorotoxin (CTX), in terms of their shape alternations and impedance changes to exploit the fine detectability of the single-cell-based system. The detecting electrodes hosting single cells exhibited a significant reduction in the real impedance signal, while electrodes hosting confluent monolayer of cells showed little to no impedance change. When single-cell electrodes were treated with CTX of different doses, a dose-dependent impedance change was observed. This enables us to identify the effective dose needed for this particular treatment. Our study demonstrated that this single-cell impedance system may potentially serve as a useful analytical tool for biomedical applications such as environmental toxin detection and drug evaluation.     

Electrical pulses appear in the inferior vena cava and abdominal aorta at contraction of leg muscles.

A vascular-interstitial neuromuscular closed circuit is described anatomically and electrically. The neuromuscular unit has a high resistance due to its axons. The "outer" vascular-interstitial branch has a low resistance due to a large cross section area. When a rat spontaneously contracts leg muscles after pinching a toe, electric potential pulses appear inside the inferior vena cava, between vena cava and peritoneum, between aorta and peritoneum, between vena cava and peritoneum and between aorta and subcutis. Minor pulses between electrodes in the peritoneal fluid are interpreted as induced by the intravascular potential pulses. The use of Ag-AgCl electrodes or platinum electrodes does not appreciably change the results.     

Fluorescence lifetime imaging: multi-point calibration, minimum resolvable differences, and artifact suppression

BACKGROUND: Frequency-domain fluorescence lifetime imaging microscopy (FLIM) is finding increasing use in the analysis of biological systems. However, the calibration, determination of resolvable lifetime differences, and evaluation of artifacts have not been extensively treated. We describe a multi-point method for calibrating a frequency-domain FLIM system, characterize the minimum detectable heterogeneity and intra- and inter-image lifetime differences, discuss the statistical treatment of FLIM data, and suggest methods for minimizing artifacts. METHODS: A set of solutions exhibiting single-component lifetimes suffice for accurately calibrating a reference material with a single-component lifetime, even in the absence of accurate data on the lifetimes of the individual solutions or the reference material. We used a set of rhodamine 6G solutions quenched with varying concentrations of iodide, leading to lifetimes of 0.5--4.0 ns, to calibrate a 1 microM reference solution of rhodamine 6G in water. RESULTS: We measured a value of 4.11 ns with an estimated absolute error of +/-0.05 ns for the rhodamine 6G reference solution. With 57.7 MHz modulation, the minimum detectable inter-image lifetime difference was 0.1--0.15 ns and the minimum detectable intra-image lifetime difference was 4--5 ps, allowing solutions differing in lifetime by 40 and 70 ps to be easily distinguished. The minimum detectable lifetime heterogeneity was 50--80 ps. Evaluation of replicate measurements of single solutions demonstrated that inter-image instrument errors exceeded those predicted from intra-image statistics by more than an order of magnitude. We also measured lifetimes and heterogeneity in 4 GFP variants (WTGFP, EGFP, S65T, and EYFP) with the technique. CONCLUSION: The multi-point calibration method is applicable to any system consisting of single-component lifetimes. Applying the method in our FLIM microscope allowed us to demonstrate a previously unreported degree of lifetime resolution in a FLIM microscope. Cytometry 43:248-260;2001.     

The use of nonmetal electrodes in electroejaculation of restrained but unanesthetized macaques

A successful technique for electroejaculation with nonmetallic electrodes cut from defibrillation pads is described. Twenty-six adult male cynomolgus and eleven adult male rhesus macaques were electroejaculated while immobilized with chair restraint. From 123 attempted electroejaculations in both species of macaques, 119 semen specimens were obtained. The volume, concentration, % motility, and % normal forms of cynomolgus and rhesus macaque semen are presented. The use of nonmetallic electrodes provides a high quality ejaculate while minimizing the risks of adverse affects on valuable populations of macaques.     

Whole Body Scanning in Medicine: I. Physical Aspects

Fundamental physical aspects of scanning the human body to determine in vivo radioisotope distribution are discussed, with special reference to the importance of collimator design and data display system. The pertinent details of a sensitive whole body scanner which has proved useful in clinical practice are described. The main features contributing to its versatility are: (1) fully automatic operation, (2) use of a 5″ diameter detector crystal with a focusing collimator, (3) provision to scale the scintiscan presentation to a convenient size, and (4) photographic recording of the scan.     

An adaptable spectroelectrochemical titrator: the midpoint reduction potential of the iron-sulfur center in lysine 2,3-aminomutase

Elaborations to an earlier design of an electron paramagnetic resonance (EPR) spectroelectrochemical titrator are described. While maintaining the anaerobic capabilities of the original design, a number of modifications and revisions have been introduced. The most significant modification is the use of a detachable spectral cell, making the apparatus modular and adaptable for multiple forms of spectroscopy. Additional modifications include removable reference, auxiliary, and working electrodes; modifications to facilitate sample transfer; and adaptations for operation within an anaerobic chamber. This apparatus has been used successfully in the coulometric titration of a [4Fe-4S] enzyme, as measured by EPR spectroscopy. The midpoint reduction potential for the 2+/1+ couple in the [4Fe-4S] cluster of lysine 2,3-aminomutase is -479+/-5mV, a value that falls within the range typical of ferredoxin-like iron-sulfur clusters.