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Biomedical subjects

M E Meyerhoff

Publications and source records attributed to M E Meyerhoff.

At least 55 records · Page 3Linked to original sources
PubMed1994

Adapting homogeneous enzyme-linked competitive binding assays to microtiter plates.

Recently devised homogeneous enzyme-linked binding assays useful for the rapid detection of carbohydrate structure/content of intact glycoproteins (via use of lectins as binders) and for quantitating given vitamins (e.g., biotin; using soluble binding proteins) are adapted successfully to a microtiter plate reader format. The problem of nonspecific adsorption of the binders and enzyme-saccharide/vitamin conjugates is solved via the addition of Tween 20 to the assay buffer. More convenient and reliable photometric detection of the preferred labeling enzyme, glucose-6-phosphate dehydrogenase (G6PDH), is accomplished by monitoring the rate of generation of reduced thio-NAD (from thio-NAD) at 405 nm instead of NADH (from NAD) at 340 nm. By employing these modifications it is shown that homogeneous enzyme-linked binding assays can be readily adapted to microtiter plates without loss in analytical assay performance. Results further suggest that other homogeneous assays based on G6PDH, including commercial EMIT assays used routinely in clinical chemistry laboratories for detecting drugs of abuse, could, in principle, be run on microtiter plates to significantly enhance sample throughput.

Binding, Competitive↗
PubMed1993

Preparation and characterization of covalently bound tetraphenylporphyrin-silica gel stationary phases for reversed-phase and anion-exchange chromatography.

A tetraphenylporphyrin-based stationary phase for high-performance liquid chromatography is prepared by attaching a p-carboxyphenyl derivative of the porphyrin to aminopropyl silica gel through an amide bond. This stationary-phase support is used for the reversed-phase separation of several polycyclic aromatic hydrocarbons and is shown to exhibit novel shape selectivity toward planar aromatic solutes. Subsequent metalation of the immobilized tetraphenylporphyrin with either tin(IV) or indium(III) results in a stationary phase with anion-exchange characteristics. Both the tin(IV) and indium(III) metalloporphyrin-based stationary phases exhibit useful selectivity for the separation of aromatic sulfonates and aromatic carboxylates.

Chromatography, Ion Exchange↗
PubMed1993

Electrochemical sensor for heparin: further characterization and bioanalytical applications.

Further studies regarding the potentiometric response to heparin of polymeric membranes doped with lipophilic quaternary ammonium salts are reported. Among a wide range of membrane formulations examined, optimum response toward macromolecular heparin is achieved using tridodecylmethylammonium chloride as the active membrane component within poly(vinyl chloride) or poly(vinyl chloride)/(vinyl acetate) films plasticized with dioctyl sebacate. Although such membranes are shown to exhibit a typical Hofmeister potentiometric selectivity pattern toward small inorganic and organic anions, a very large and reproducible response to heparin at submicromolar levels is observed in the presence of physiological saline (0.12-0.15 M NaCl), as well as in citrated whole blood. Equal response on a mass basis occurs with fragments of heparin's as small as 2500 Da. Complexation of heparin's anionic sites by macromolecules that bind heparin with high affinity (e.g., protamine and poly(L-lysine)) reduces the membrane electrode's heparin response, indicating that the electrode detects biologically available (unbound) heparin levels in solution. Quantitative potentiometric titrations of protamine with porcine mucosa heparin followed by the sensor yield stoichiometric values in good agreement with the literature. The biomedical utility of the sensor is demonstrated by measuring its response in whole blood from patients undergoing open heart surgery before and after heparin therapy and correlating such response to conventional blood clotting time measurements.

Electrochemistry↗
PubMed1993

Shape-selective separation of polycyclic aromatic hydrocarbons by reversed-phase liquid chromatography on tetraphenylporphyrin-based stationary phases.

The reversed-phase chromatographic behavior of planar and non-planar polycyclic aromatic hydrocarbons (PAHs) is investigated on tetraphenylporphyrin and two metallotetraphenylporphyrin [Sn(IV), In(III)] bonded stationary phases using methanol-water and acetonitrile-water as mixed solvent mobile phases. Large differences in the capacity factors of aromatic solute pairs having the same number of carbon atoms, but differing in three-dimensional shape (e.g., triphenylene/o-terphenyl and perylene/alpha, alpha'-binaphthyl), suggest that the three tetraphenylporphyrin-based supports possess shape selectivity toward small planar aromatic solutes. Capacity factors for planar PAH solutes on these supports are significantly greater than for non-planar polyaryls having the same number of carbon atoms.

Chromatography, High Pressure Liquid↗
PubMed1993

Polymer membrane-based ion-, gas- and bio-selective potentiometric sensors.

Recent progress in the design of new polymer membrane-based potentiometric ion-, gas- and bio-selective electrodes in chemistry laboratories at the University of Michigan (Ann Arbor) is reviewed. Emphasis is placed on describing the performance of devices for measuring anions (e.g., salicylate, thiocyanate, chloride and heparin) and gases (e.g., ammonia, carbon dioxide and oxygen) in biological samples, both in vitro and in vivo. Beyond direct measurement of key ions and gases in complex matrices, some of the new membrane electrode systems reported can serve as base transducers for the development of biosensors containing integrated biological reagents, including enzymes and antibodies. New approaches for mass fabricating solid-state ion and biosensor devices as well as future directions for research in the entire field of polymer membrane sensors are also described.

Anions↗
PubMed1993

Simultaneous determination of ammonia nitrogen and L-glutamine in bioreactor media using flow injection.

A novel split stream flow injection (FI) system suitable for the simultaneous determination of L-glutamine and ammonia nitrogen (ammonia-N) in cell culture media is described. Potentiometric detection of ammonia-N in one portion of the manifold is achieved using a commercial ammonia gas-sensing electrode fitted with a wall-jet cap. L-Glutamine is quantified in the other part of the split sample by potentiometric detection of ammonium ions (by an ammonium-selective polymer membrane electrode), liberated from the hydrolysis of glutamine after the sample flows through a glass bead reactor containing immobilized glutaminase. Endogenous ammonia-N and potassium ions that would normally interfere with the glutamine measurement are removed upstream using a unique tubular cation-exchange unit. Using 50 microliters sample volumes and mixed solutions of ammonium chloride and L-glutamine in Iscove's Modified Dulbecco's Medium to calibrate the FI measuring system, values for ammonia-N and L-glutamine determined for 22 media samples obtained from a bioreactor growing retroviral producer cells correlate well with those measured with commercial, manual enzymic-spectrophotometric assay kits.

Ammonia↗
PubMed1992

Reversible potentiometric oxygen sensors based on polymeric and metallic film electrodes.

Various materials and sensor configurations that exhibit reversible potentiometric responses to the partial pressure of oxygen at room temperature in neutral pH solution are examined. In one arrangement, platinum electrodes are coated with plasticized poly(vinyl chloride) films doped with a cobalt(II) tetraethylene pentamine complex. For such sensors, potentiometric oxygen response is attributed to a mixed potential originating from the underlying platinum electrode surface as well as a change in redox potential of the Co(II)-tetren-doped film as the complex binds oxygen reversibly. The response due to the platinum surface is prolonged by the presence of the Co(II)-tetren/PVC film. Alternately, thin films of metallic copper, electrochemically deposited on platinum and/or sputtered or vapor deposited on a single crystal silicon substrate, may be used for reversible oxygen sensing. The long-term reversibility and potentiometric stability of such copper film-based sensors is enhanced (up to 1 month) by preventing the formation of cuprous oxide on the surfaces via the application of an external nonpolarizing cathodic current through the working electrode or by specifically using sputtered copper films that have [100] preferred crystal structures as determined by X-ray diffraction. The implications of these findings in relation to fabricating analytically useful potentiometric oxygen sensors are discussed.

Cobalt↗
PubMed1992

Lectin-based homogeneous enzyme-linked binding assay for estimating the type and relative amount of carbohydrate within intact glycoproteins.

The feasibility of using a new lectin-based homogeneous enzyme-linked binding assay for estimating the type and relative amount of specific carbohydrate structures within intact glycoproteins is examined. Malate dehydrogenase-galactose, -mannose, and -N-acetylglucosamine conjugates are utilized in conjunction with Jacalin, concanavalin A, and wheat germ agglutinin, respectively. The catalytic activity of the glyco-enzyme conjugates is inhibited significantly (greater than 60%) in solution in the presence of the respective lectins. The observed inhibition for each reagent set is reversed in proportion to the type and relative amount of specific carbohydrates present within test glycoproteins added to the assay mixture. Competitive binding ED50 values for a number of synthetic and native model glycoproteins correlate well with the known carbohydrate content of these species. The proposed method is much faster than previous solid-phase lectin-based enzyme-linked methods used to probe carbohydrate content/structure (less than 15 min) and has the potential to be fully automated.

Acetylglucosamine↗
PubMed1991

Operation of ion-selective electrode detectors in the sub-Nernstian/linear response range: application to flow-injection/enzymatic determination of L-glutamine in bioreactor media.

A novel approach for eliminating positive errors from endogenous ionic interferences when using ion-selective electrodes as detectors in flow-injection enzyme-based blosensing configurations is described. The method involves using a high background level of interfering ions in the sample diluent/carrier stream to convert the normally logarithmic potentiometric sensor into a linear detector over a given concentration range of primary ions. A split-stream single-detector arrangement provides a convenient means to compensate for varying levels of background interferent ions in the injected samples. One portion of the split stream passes directly to the ion-electrode detector, yielding a signal linearly related to the concentration of endogenous primary ions in the sample. The second portion of the split sample is delayed while passing through an immobilized enzyme that generates electrode detectable primary ions in proportion to the concentration of the substrate analyte in the sample. Two linear equations with two unknowns describe the twin potentiometric responses observed. The concept is demonstrated by the accurate determination of L-glutamine in hybridoma bioreactor media via the use of an ammonium-ion-selective membrane electrode detector and immobilized glutaminase enzyme.

Biosensing Techniques↗
PubMed1991

Electrochemical performance, biocompatibility, and adhesion of new polymer matrices for solid-state ion sensors.

Ammonium and potassium ion-selective membranes formulated with PVC/hydroxylated PVC, polyurethane/hydroxylated PVC, and moisture-curable silicone rubber matrices are studied in an effort to extend the lifetime of solid-state ion sensors through improved membrane adhesion. The PVC/membranes exhibit electrochemical performance equivalent to that of conventional PVC membranes in terms of slope, detection limit, and selectivity. The polyurethane- and silicone-rubber-based membranes have better adhesion to silicon nitride than do PVC or hydroxylated PVC matrices. Incorporating a silanizing reagent (silicon tetrachloride) significantly improves the adhesion of the polyurethane matrix. The use of silicon tetrachloride in membrane matrices also enhances the electrochemical stability of the interfacial potential between ion-selective polymer-matrix membranes and silver epoxy inner reference electrodes of solid-state sensors. The biocompatibility of the polymer matrices is examined via radiotracer protein adsorption studies and whole blood clotting time measurements. The polyurethane- and silicone-rubber-based membranes exhibit less overall nonspecific protein adsorption than the PVC or hydroxylated PVC matrices.

Adhesiveness↗
PubMed1991

Potentiometric enzyme channeling immunosensor for proteins.

A potentiometric immunosensor for the detection of human IgG has been developed using an asymmetric, ion-selective membrane with immobilized adenosine deaminase and IgG. A protein A-alkaline phosphatase conjugate binds to the immobilized IgG, creating a bienzymatic catalytic layer. In the presence of sample IgG, the conjugate does not bind to the membrane. Instead, the intermediate in the two-step reaction (adenosine) must diffuse to the membrane surface, reducing the rate of product (ammonium) formation within the diffusion layer detected by the membrane. The immunosensor demonstrated is for the determination of IgG. A simplified model is described to predict the maximum rate enhancement for the 'channeled' versus 'unchanneled' reaction mechanisms.

Biosensing Techniques↗
PubMed1990

Homogeneous enzyme-linked binding assay for studying the interaction of lectins with carbohydrates and glycoproteins.

A simple and rapid homogeneous enzyme-linked binding assay method for studying lectin-carbohydrate interactions is described. The method is based on the homogeneous inhibition of appropriate enzyme-saccharide conjugates by specific carbohydrate-binding lectins. In the presence of carbohydrate structures recognized by the lectins, enzyme activity is regained in an amount of proportional to the concentration of carbohydrate. The new method can be used to rapidly assess the relative carbohydrate specificity of the various lectins and for the selective analytical detection of simple saccharides and complex glycoproteins. Indeed, when Jacalin lectin is used in conjunction with a malate dehydrogenase-galactose conjugate, selective measurement of human IgA (immunoglobulin A) at microgram per milliliter levels in less than 10 min is possible. The potential for using this analytical methodology for determining changes in the carbohydrate structure of intact recombinant glycoproteins is also discussed.

Carbohydrates↗
PubMed1990

Use of ionomer membranes to enhance the selectivity of electrode-based biosensors in flow-injection analysis.

The use of ionomer membranes to enhance the selectivity of potentiometric enzyme electrodes in flow-injection measurement arrangements is examined. The ionomer membranes employed are permeable to analyte substrates but relatively impermeable to detectable ions that would normally interfere with the measurement of the substrates if the enzyme electrodes were in direct contact with the sample. As a model system, the selectivity of enzyme electrodes prepared with nonactin-based ammonium-sensitive polymeric membranes is evaluated. In the preferred configuration, a thin hydrophilic anion-exchange membrane is incorporated within a flow-through dialysis unit upstream from the enzyme-electrode detector. As the sample passes through the dialysis unit, neutral or anionic analyte molecules (urea or glutamine) move through the membrane while the permeation of endogenous ammonium ions and other cations in the sample is retarded. A flowing recipient buffer on the other side of the membrane carries the analyte substrate to the enzyme-electrode detector. Enhancements in selectivity for analyte substrates over endogenous ammonium and potassium ions are greater than or equal to 9-fold when compared to enzyme-electrode flow-injection analysis (FIA) systems assembled without the ionomer membrane unit. The analytical utility of the proposed system is demonstrated by the accurate measurements of urea in blood serum and L-glutamine in hybridoma bioreactor media.

Biosensing Techniques↗
PubMed1990

Current and future directions in the technology relating to bedside testing of critically ill patients.

Significant progress has been made recently in the measurement methods and instrumental approaches applicable to bedside testing of critically ill patients. While the "ideal" technology would involve the ability to obtain accurate stat profile values on a continuous basis via noninvasive methods, given the present state of noninvasive sensing technologies, this capability is unlikely to be achieved in the foreseeable future. In principle, invasive and on-line techniques offer more hope for future success in continuous bedside monitoring of all the key critical care analyses. However, success in these directions will come only when issues regarding sensor stability and sampling device/sensor biocompatibility are completely solved. Until then, it appears that the user-friendly point of care type stat analyzers that can provide accurate values for all the key analytes, used in conjunction with existing noninvasive trend monitors (eg, pulse oximetry), will offer the most attractive approach for the effective treatment of critically ill patients.

Clinical Laboratory Techniques↗