Clinical uses of enzyme electrode probes

Clinical applications of enzyme electrochemical sensors are reported; they are based on the coupling of enzymes with potentiometric membrane electrodes (pH, iodide) or potentiometric probes (ammonia, carbon dioxide) or amperometric devices (oxygen, hydrogen peroxide). The most popular and successful immobilization procedures for enzymes are reviewed, namely physical entrapments and chemical methods for binding enzymes to solid support like collagen and nylon net; procedures specifically developed for clinical uses of enzyme probes.The simplicity of the apparatus is evidenced, and it is explained how a single instrument can be useful for all kind of measurements. Practical suggestions for constructing a typical probe are given. Single paragraphs are devoted to the determination of urea, cholesterol, creatinine, amino acids, glucose, lactate, protein, choline and acetylcholine to clarify the sequence of enzymatic and electrochemical reactions in order to elucidate the application range the sensitivity and the selectivity as well as the relevant interferences for each metabolite either in the enzymatic or in the electrochemical step. The applications performed in vitro, in vivo and ex vivo and the commercial availability of some instruments are reported.     

Determination of glucose,urea and penicillin using enzyme-pH-electrodes

Conventional hydrogen ion glas electrodes have been used for the preparation of enzyme-pH-electrodes by either entrapping the enzymes within polyacrylamide gels around the electrode or as liquid layer trapped within a cellophane membrane. The enzymes were glucose oxidase, urase and penicillinase.The pH response to glucose concentration was about linear within 10^?1–10^?3 M glucose and for urea linear within 5·10^t—–5·10^?5M. The pH response to penicillin was about linear in the range from 10^?3–10^?2 M resulting in a pH shift of 1.4 units; reproduceable pH response was obtained down to concentrations of 3·10^?5 M.Studies as to the effect of buffer using an urease–pH-electrode showed a buffer concentration of 10^?2 M a substantial shift of about one pH-unit in the range of 10^?4 to 10^?2 M urea. Both urease- and penicillinase–pH-electrodes were tested as to stability showing no decrease in pH response except at high substrate concentration (1·10^?2 M) over a period of 2–3 weeks kept at room temperature.     

Transporters,channels, or simple diffusion? Dogmas,atypical roles and complexity in transport systems

The recent breakthrough discoveries of transport systems assigned with atypical functions provide evidence for complexity in membrane transport biochemistry. Some channels are far from being simple pores creating hydrophilic passages for solutes and can, unexpectedly, act as enzymes, or mediate high-affinity uptake, and some transporters are surprisingly able to function as sensors, channels or even enzymes. Furthermore, numerous transport studies have demonstrated complex multiphasic uptake kinetics for organic and mineral nutrients. The biphasic kinetics of glucose uptake in Saccharomyces cerevisiae, a result of several genetically distinct uptake systems operating simultaneously, is a classical example that is a subject of continuous debate. In contrast, some transporters display biphasic kinetics, being bona fidae dual-affinity transporters, their kinetic properties often modulated by post-translational regulation. Also, aquaporins have recently been reported to exhibit diverse transport properties and can behave as highly adapted, multifunctional channels, transporting solutes such as CO₂, hydrogen peroxide, urea, ammonia, glycerol, polyols, carbamides, purines and pyrimidines, metalloids, glycine, and lactic acid, rather than being simple water pores. The present review provides an overview on some atypical functions displayed by transporter proteins and discusses how this novel knowledge on cellular uptake systems may be related to complex multiphasic uptake kinetics often seen in a wide variety of living organisms and the intriguing diffusive uptake of sugars and other solutes.     

An immersible manometric sensor for measurement of humidity and enzyme mediated changes in dissolved gas

An immersible manometric sensor was made by covering the gaseous cavity of a pressure transducer with a 1 microm controlled pore membrane. Transfer of gas across the membrane allowed the pressure transducer to record changes in humidity or dissolved gas when immersed in solution. By immersing the sensor in distilled water, atmospheric humidity could be estimated by the deficit of atmospheric vapor pressure from saturation. In another application of the sensor, CO(2) was monitored continuously. This was not possible in previous closed-reactor type manometric sensors, and may allow the new technology to be used in applications requiring continuous monitoring of a process or stream. By coupling the sensor with enzymes liberating or consuming dissolved gas, different chemicals could be estimated. Urea was estimated by first hydrolyzing it with urease and then measuring the resulting CO(2) gas in solution. Glucose was measured through its enzymatic oxidation by glucose oxidase. The sensitivity to urea over the range 0-2.5 mM was about 1.02 kPa/mM, and the standard error was 0.086 mM. Due to the lower solubility of oxygen, the sensitivity to glucose in a range from 0 to 10 microM was over 100 kPa/mM, with a standard error of only 0.76 microM. This sensitivity was not possible in closed-reactor type manometric sensors due to constraints of dimensioning the head space gas volume for reproducibility and effective mass transfer. The 90% rise times for the sensor ranged from about 1-60 min for the different applications. The dynamic characteristics of the device may be improved by using a membrane with greater porosity, higher rigidity and lower thickness, and by reducing the dimensions of the cavity volume in the sensor through integrated microfabrication of the membrane onto the transducer.     

Glucose controls multiple processes in Saccharomyces cerevisiae through diverse combinations of signaling pathways

We have studied how the lack of glucose sensors in the plasma membrane, or of the enzymes Hxk1, Hxk2, Glk1, which catalyze the first intracellular step in glucose metabolism, affect the different responses of Saccharomyces cerevisiae to glucose. Lack of the G-protein-coupled receptor Gpr1 or of Snf3/Rgt2 did not affect glucose repression of different genes or activation by glucose of plasma membrane ATPase, whereas lack of Gpr1 decreased, in an additive manner with lack of Mth1, the degradation of fructose 1,6-bisphosphatase that takes place in the presence of glucose. In an hxk1 hxk2 glk1 strain, unable to phosphorylate glucose, all of these responses to the sugar were suppressed or strongly reduced. In the absence of Hxk2 (or Hxk1 and Hxk2), glucose repression of SUC2, GAL1 and GDH2 was relieved, but that of FBP1 and ICL1 was maintained. Hxk1 or Hxk2 were needed for activation of plasma membrane ATPase but not for degradation of FbPase.     

Fabrication of a sensing module using micromachined biosensors

Micromachining is a powerful tool in constructing micro biosensors and micro systems which incorporate them. A sensing module for blood components was fabricated using the technology. The analytes include glucose, urea, uric acid, creatine, and creatinine. Transducers used to construct the corresponding sensors were a Severinghaus-type carbon dioxide electrode for the urea sensor and a Clark-type oxygen electrode for the other analytes. In these electrodes, detecting electrode patterns were formed on a glass substrate by photolithography and the micro container for the internal electrolyte solution was formed on a silicon substrate by anisotropic etching. A through-hole was formed in the sensitive area, where a silicone gas-permeable membrane was formed and an enzyme was immobilized. The sensors were characterized in terms of pH and temperature dependence and calibration curves along with detection limits. Furthermore, the sensors were incorporated in an acrylate flow cell. Simultaneous operation of these sensors was successfully conducted and distinct and stable responses were observed for respective sensors.     

Penicillin production by glucose-derepressed mutants ofPenicillium chrysogenum

Summary Wild-type strains ofPenicillium chrysogenum produce lower penicillin V titers in media containing excess glucose. Two mutant strains were isolated and shown to produce normal penicillin V titers in the presence of excess glucose. These strains, designated as glucose-repression insensitive (GRI) mutants, produced higher penicillin V titers than the wild-type strain in media containing lactose as the main carbohydrate source. In lactose-based media, the production of penicillin V was depressed to a much lesser extent by in-cycle additions of glucose with the GRI mutants when compared to the wild-type strain. In short-term biosynthesis experiments using washed cells in a medium containing glucose as the sole carbon source, the GRI mutants produced penicillin V at a faster rate than the wild-type strain. In fed-batch fermentations in 14-liter fermentors, where glucose was fed continuously and pH controlled, both GRI mutants produced more than 10% higher penicillin V titers than the wild-type strain. These results suggest that isolation of GRI mutants is an effective way to select for higher producing strains and that the synthesis of penicillin synthesizing enzymes in GRI mutants may be less repressed by glucose than in wild-type strains.     

The immobilization of enzymes on nylon structures and their use in automated analysis

1. Glucose oxidase (EC 1.1.3.4) and urease (EC 3.5.1.5) were covalently attached through glutaraldehyde to low-molecular-weight nylon powder. 2. Immobilized derivatives of glucose oxidase and urease were prepared by cross-linking the respective enzymes within the matrix of a nylon membrane. 3. An improved process is described for the immobilization of glucose oxidase and urease on the inside surface of partially hydrolysed nylon tube. 4. Automated analytical procedures are described for the determination of glucose with each of the three immobilized glucose oxidase derivatives and for the determination of urea with each of the three immobilized urease derivatives. 5. The efficiencies of the three immobilized enzyme structures as reagents for the automated determination of their substrates were compared.     

Immobilization of biocatalysts with bombyx mori silk fibroin by several kinds of physical treatment and its application to glucose sensors

Biocatalyst-immobilized Bombyx mori silk fibroin membrane was prepared. The insolubilization of the water-soluble membranes was performed by physical treatments only, i.e. stretching, compressing and standing under high humidity and methanol-immersion treatment, without any use ofcovalently binding reagent. All physical treatments performed were effective for the purpose of the immobilization of the enzymes in the membranes. The structural characterization of the glucose oxidase (GOD) immobilized membrane was performed in detail. The permeability of the substrate depends on the crystalline structure, i.e. the fraction of Silk I and Silk II of the membrane. The activity yield of the immobilized GOD was more than 80% of the value of free enzyme when 0–002% of the enzyme was entrapped in the membrane, but it decreased with increasing the concentration of the GOD in the membrane. This seems to result from diffusion limitation of the substrate. The pH and thermal stabilities of the immobilized enzyme were much improved, and were essentially independent of the methods of the immobilization. Development of the GOD or microorganism, Pseudomonas fluorescens immobilized silk fibroin membranes as glucose sensors are described.     

Amperometric enzyme electrodes

Three different types of amperometric enzyme electrode are described. The first type uses a conducting organic-salt electrode to oxidize NADH. Results for sensors for ethanol and for bile acids are presented. In the second type of sensor, flavoenzymes are directly oxidized on the surface of the conducting organic-salt electrode. Results for five different enzymes are described. The mechanism of the enzyme oxidation is discussed and the reaction is shown to take place by heterogeneous redox catalysis and not by homogeneous mediation. The enzymes are strongly adsorbed on the electrode; microelectrodes for in vivo studies can be constructed without a membrane. Results for in vivo studies of changing glucose levels in the brain of a freely moving rat are presented. The third type of sensor is designed to measure low levels of toxic gases such as H2S and HCN. This is done by monitoring the inhibition by the toxic gas of the activity of the respiratory enzyme cytochrome oxidase.     

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 new amperometric glucose microsensor: in vitro and short-term in vivo evaluation

For biosensor fabrication, it is important to optimize materials and methods in order to create predictable function in vitro and in vivo. For this reason, we designed a new glucose sensor ('revised protocol') that utilized an outer permselective membrane made of amphiphobic polyurethane which allows glucose passage through hydrophilic segments. An inner polyethersulfone membrane, stabilized with a trimethoxysilane, provided specificity. Before application of the inner membrane, it was necessary to etch the platinum electrode with a radio frequency oxygen plasma. The revised protocol sensors (n=185) were compared with sensors fabricated with an earlier ('original') protocol (n=204) which used an outer polyurethane without hydrophilic segments and a complex inner membrane of cellulose acetate and Nafion. The function of revised protocol sensors was more predictable in vitro as evidenced by a much lower variation of glucose sensitivity than the original protocol sensors. Revised and original protocol sensors were nearly linear up to a glucose concentration of 20 mM. In vitro interference from 0.1 mM acetaminophen was minimal in both groups of sensors and would be expected to represent about 2% of the total sensor response at normal glucose levels for revised protocol sensors. Prolonged testing of the revised protocol sensors for 11 days during immersion in buffer revealed stable sensitivities (day 1: 6.12+/-1.34 nA/mM; day 3: 6.33+/-1.40; day 8: 7.13+/-1.39; and day 11: 7.56+/-1.47; sensitivity for day 1 vs. each other day: not significant) and no critical loss of glucose oxidase activity. The response of the revised protocol sensors (n=7) to intraperitoneal glucose was tested in rats approximately one day after subcutaneous implantation and the sensors tracked glucose closely with a slight lag of 3-6 min.     

Intravascular glucose/lactate sensors prepared with nitric oxide releasing poly(lactide-co-glycolide)-based coatings for enhanced biocompatibility

Intravenous amperometric needle-type enzymatic glucose/lactate sensors intended for continuous monitoring are prepared with a novel nitric oxide (NO) releasing layer to improve device hemocompatibility. To create an underlying NO release coating, the sensors with immobilized enzymes (either glucose oxidase or lactate oxidase) are prepared with a thin layer of poly(lactide-co-glycolide) (PLGA) loaded with lipophilic diazeniumdiolate species that slowly release NO via a proton driven reaction. An outer thin layer (ca. 30 μm) of PurSil (polyurethane/dimethylsiloxane copolymer) limits the flux of glucose and lactate to the inner layer of enzyme, to provide the desired linear amperometric response. A 30 μm coating of PLGA containing 33 wt% of the appropriate NO donor (N-diazeniumdiolated dibutylhexanediamine, DBHD/N?O?) can release NO at a physiologically relevant rate > 1 × 10?1?mol min?1 cm?2 for at least 7 days without influencing the analytical performance of the glucose/lactate sensors. In vitro, the sensors exhibit relatively stable amperometric response over a one-week period with high selectivity over interferences (e.g., ascorbic acid) required for blood monitoring applications. Glucose sensors implanted in the veins of rabbits for 8h exhibit significantly enhanced hemocompatibility for the NO release sensors vs. corresponding controls (without NO release in same animals), with greatly reduced thrombus formation on their surfaces. Further, the analytical performance of the NO release glucose sensors are superior to controls placed in the veins of the same animals, with a greater accuracy in measuring blood glucose levels as evaluated using a Clarke error grid type analysis.     

Endocytosis and Vacuolar Degradation of the Yeast Cell Surface Glucose Sensors Rgt2 and Snf3

Sensing and signaling the presence of extracellular glucose is crucial for the yeast Saccharomyces cerevisiae because of its fermentative metabolism, characterized by high glucose flux through glycolysis. The yeast senses glucose through the cell surface glucose sensors Rgt2 and Snf3, which serve as glucose receptors that generate the signal for induction of genes involved in glucose uptake and metabolism. Rgt2 and Snf3 detect high and low glucose concentrations, respectively, perhaps because of their different affinities for glucose. Here, we provide evidence that cell surface levels of glucose sensors are regulated by ubiquitination and degradation. The glucose sensors are removed from the plasma membrane through endocytosis and targeted to the vacuole for degradation upon glucose depletion. The turnover of the glucose sensors is inhibited in endocytosis defective mutants, and the sensor proteins with a mutation at their putative ubiquitin-acceptor lysine residues are resistant to degradation. Of note, the low affinity glucose sensor Rgt2 remains stable only in high glucose grown cells, and the high affinity glucose sensor Snf3 is stable only in cells grown in low glucose. In addition, constitutively active, signaling forms of glucose sensors do not undergo endocytosis, whereas signaling defective sensors are constitutively targeted for degradation, suggesting that the stability of the glucose sensors may be associated with their ability to sense glucose. Therefore, our findings demonstrate that the amount of glucose available dictates the cell surface levels of the glucose sensors and that the regulation of glucose sensors by glucose concentration may enable yeast cells to maintain glucose sensing activity at the cell surface over a wide range of glucose concentrations.     

The interaction of rat liver carbamoyl phosphate synthetase and ornithine transcarbamoylase with inner mitochondrial membranes

The intramitochondrial localization of the urea cycle enzymes, carbamoyl phosphate synthetase and ornithine transcarbamoylase, has been examined by both in vitro and in situ studies. The following three lines of evidence are presented to establish that significant fractions of the rat liver enzymes are loosely associated with the inner mitochondrial membrane: 1) when the mitochondrion is fractionated, the enzymes partition between the matrix and membrane fractions in the absence of detergent and partition solely to the matrix in the presence of detergent; 2) the purified enzymes associate with purified inner membrane preparations; and, 3) protein A-gold electron microscopic immunocytochemical analysis of rat liver sections reveals a nonrandom arrangement of the enzyme, with the maximal enzyme density adjacent to the inner mitochondrial membrane. These findings serve as the basis for novel potential mechanisms for regulation of the activity of the enzymes and provide additional evidence for the extensive organization of the mitochondrial matrix. The membrane interaction might also serve as the organizing factor for a carbamoyl phosphate synthetase-ornithine transcarbamoylase or other multienzyme complex.     

Calorimetric biosensors with integrated microfluidic channels

A microfluidic device capable of measuring real-time enthalpy changes of biochemical reactions and thermal properties of biological fluids is presented in this paper. The device consists of a freestanding microthermopile integrated with a glass microfluidic reaction chamber. The p-type polysilicon/gold microthermopiles fabricated on a 2 μm thick thermally isolated membrane showed a sensitivity of 0.94 V/W and a thermal time constant of less than 100 ms. Although the device is not restricted to enzymatic reactions, in this paper measurements of the heat of reaction from the catalytic action of glucose oxidase, catalase, and urease on glucose, hydrogen peroxide, and urea, respectively, are reported. Reactions were performed in open air using liquid batch testing and in enclosed fluidic reaction chamber by continuous flow experiments. A sensitivity of 53.5 μV/M for glucose, 26.5 μV/M for hydrogen peroxide and 17 μV/M for urea was obtained. Detection limit for glucose in the continuous flow mode is 2 mM (30 pmol). The aim of this work is to demonstrate the potential of the integrated calorimetric microfluidic device for fundamental thermodynamic studies in biochemical reactions. Using arrays of such devices with immobilized enzymes multi-analyte detection can be accomplished and the effects of interferents from competing substrates can be compensated. This paper presents the design, fabrication and initial testing results from such a microthermopile-based thermal biosensor.     

Effect of retinol deficiency on the activity and solubility of various enzymes of the endoplasmic reticulum membranes in the rat liver

Solubilization by sodium deoxycholate and trypsin of some metabolic enzymes of unrelated compounds associated with endoplasmic reticulum membranes was carried out. The effects of urea, butanol and detergents on the retinol content in the membranes were studied. It was shown that retinol deficiency causes changes in the interactions of NADH-arylesterase with microsomal membrane components that are manifested in the decrease of the activating effect of butanol and low detergent concentrations on the NADH-reductase activity as well as in the increase in the damaging effect of urea and high detergent concentrations on the enzyme activity. Under conditions of retinol deficiency, the degree of solubilization of NADH-reductase, hydroxylase and arylesterase in the presence of sodium deoxycholate is enhanced. After treatment of liver microsomes of retinol-deficient animals with trypsin or with a trypsin-sodium cholate mixture, the content of these enzymes in the supernatant becomes much greater than that in liver microsomes of vitamin A-deficient rats. It is assumed that retinol deficiency causes of weakening of hydrophobic interactions within the membrane as well as partial translocation of the enzymes from the hydrophobic to the hydrophilic layer.     

Structured bienzymatical models formed by sequential enzymes bound into artificial supports: Active glucose transport effect

Summary Two different artificial membrane systems bearing two built-in sequential enzymes are studied and compared in this communication.The first is a nonstructured membrane bearing two mixed enzymes: -galactosidase and glucose-oxidase. Its use enables a mathematical model to be formulated describing the heterogeneous phase kinetics of a bienzymatic system. The second is a multi-layer membrane system in which the structural dissymmetry involves a spatial orientation of the reacting metabolites, resulting in active glucose transport.The latter system consists of two active leaflets, the first phosphorylating glucose (hexokinase+ATP), the second dephosphorylating glucose-6 phosphate (phosphatase). On either side of this system, a perm-selective proteic layer allows the passage of glucose but not of glucose-6 phosphate. When positioned between two compartments containing glucose, such a membrane accumulates glucose on its phosphatase side, while degrading ATP.The accumulation of glucose as a function of the initial concentration shows the classical saturation of the transport system. Fructose competes with glucose transport.The chemical balance of these two reactions has the appearance of hydrolysis of ATP. Vectorial catalysis is a result of the dissymmetry in distribution of active sites and can be explained by an oscillatory concentration profile of glucose inside the membrane.The bienzymatic mechanism, a model of which is given here, is valid for any thickness of active layers and applicable to a system where both active sides are part of the same molecule as soon as it forms a uniformly oriented monolayer throughout the membrane structure.