Biosensor system for continuous flow determination of enzyme activities. I. Determination of glucose oxidase and lactic dehydrogenase activities

A biosensor system for continuous flow determination of enzyme activity was developed and applied to the determination of glucose oxidase and lactic dehydrogenase activities. The glucose oxidase activity sensor was prepared from the combination of an oxygen electrode and a flow cell. Similarly, the lactic dehydrogenase activity sensor was prepared from the combination of a pyruvate oxidase membrane, an oxygen electrode, and a flow cell. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane, and glutaraldehyde. Glucose oxidase activity was determined from the oxygen consumed upon oxidation of glucose catalyzed by glucose oxidase. Lactic dehydrogenase activity was determined from the pyruvic acid formed upon dehydrogenation of lactic acid catalyzed by lactic dehydrogenase. The amount of pyruvic acid was determined from the oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. Calibration curves for activity of glucose oxidase and lactic dehydrogenase were linear up to 81 and 300 units, respectively. One assay could be completed within 15 min for both sensors and these were stable for more than 25 days at 5°C. The relative errors were ±4 and ±6% for glucose oxidase and lactic dehydrogenase sensors, respectively. These results suggest that the sensor system proposed is a simple, rapid, and economical method for the determination of enzyme activities.     

Modeling of spherical fluorescent glucose microsensor systems: design of enzymatic smart tattoos

A two-substrate mathematical model of microspherical optical enzymatic glucose sensors is presented. The sensors are based on the well-known oxidation of glucose by glucose oxidase, and are constructed by the encapsulation of glucose oxidase within hydrogel microspheres coated with ultrathin polyelectrolyte multilayer films. In order to measure glucose via changes in oxygen concentration, a fluorescent oxygen indicator is co-encapsulated with the enzyme. The model was used to predict the temporal and spatial distributions of glucose and oxygen within the sphere for step increases in bulk glucose concentration. In addition, the model was used to observe the effect of varying sensor parameters, namely sphere size, film thickness, enzyme concentration, and mass transport of substrate and co-substrate within the sphere and film coatings, on the response of the sensors. A major finding was that the application of {PSS/PAH} films as thin as 12 nm can drastically improve the sensor performance over uncoated sensors based on calcium alginate microspheres. The model is proposed as an important tool for a priori design of these complex sensor structures.     

Biosensor for continuos glucose monitoring

A potentially implantable glucose biosensor for continuous monitoring of glucose levels in diabetic patients has been developed. The glucose biosensor is based on an amperometric oxygen electrode and glucose oxidase immobilized on carbon powder held in a form of a liquid suspension. The enzyme material can be replaced (the sensor recharged) without sensor disassembly. Recharging of the biosensor is achieved by injecting fresh immobilized enzyme into the sensor using a septum. Diffusion membranes made of silastic latex-rubber coatings over a microporous polycarbonate membrane are used. Calibration curves of the amperometric signal show linearity over a wide range of glucose concentrations-up to 500 mg/dL (28 mM), covering hypoglycemic, normoglycemic, and hyperglycemic conditions. Preliminary in vitro studies of the biosensor show stable performance during several recharge cycles (of 14 days each) over a period of 4 months. (c) 1994 John Wiley & Sons, Inc.     

Glucose sensor for flow injection analysis of serum glucose based on immobilization of glucose oxidase in titania sol-gel membrane

A novel amperometric glucose sensor was constructed by immobilizing glucose oxidase (GOD) in a titania sol-gel film, which was prepared with a vapor deposition method. The sol-gel film was uniform, porous and showed a very low mass transport barrier and a regular dense distribution of GOD. Titania sol-gel matrix retained the native structure and activity of entrapped enzyme and prevented the cracking of conventional sol-gel glasses and the leaking of enzyme out of the film. With ferrocenium as a mediator the glucose sensor exhibited a fast response, a wide linear range from 0.07 to 15 mM. It showed a good accuracy and high sensitivity as 7.2 microA cm(-2) mM(-1). The general interferences coexisted in blood except ascorbic acid did not affect glucose determination, and coating Nafion film on the sol-gel film could eliminate the interference from ascorbic acid. The serum glucose determination results obtained with a flow injection analysis (FIA) system showed an acceptable accuracy, a good reproducibility and stability and indicated the sensor could be used in FIA determination of glucose. The vapor deposition method could fabricate glucose sensor in batches with a very small amount of enzyme.     

A novel wireless glucose sensor employing direct electron transfer principle based enzyme fuel cell

In this paper we present a novel wireless glucose biosensing system employing direct electron transfer principle based enzyme fuel cell. Using the glucose dehydrogenase complex, which is composed of a catalytic subunit containing FAD, the cytochrome c subunit that harbors heme c as the electron transfer subunit, and chaperone-like subunit, a direct electron transfer-type glucose enzyme fuel cell was constructed. The enzyme glucose fuel cell generated electric power, and the open-circuit voltage showed glucose concentration dependence, which suggests potential applications for this glucose-sensing system. We constructed a miniaturized "all-in-one" glucose enzyme fuel cell, which represents a compartmentless fuel that is based on the direct electron transfer principle. This involved the combination of a wireless transmitter system and a simple and miniaturized continuous glucose monitoring system, which operated continuously for about 3 days with stable response. This is the first demonstration of an enzyme-based direct electron transfer-type enzyme fuel cell and fuel cell-type glucose sensor which can be utilized as a subcutaneously implantable system for continuous glucose monitoring.     

Tonometric biosensor with a differential pressure sensor for chemo-mechanical measurement of glucose

A tonometric biosensor for glucose was constructed using a chemo-mechanical reaction unit and a differential pressure sensor. The reaction unit was fabricated by using both liquid and gas cells separated by an enzyme diaphragm membrane, in which glucose oxidase was immobilized onto the single (gas cell) side of the dialysis membrane. By applying glucose solution (0, 25.0, 50.0, 100, 150 and 200 mmol/l) into the liquid cell of the chemo-mechanical reaction unit, the pressure in the gas cell decreased continuously with a steady de-pressure slope because the oxygen consumption in the gas cell was induced by the glucose oxidase (GOD) enzyme reaction at the enzyme side of the porous diaphragm membrane. The steady de-pressure slope in the gas cell showed the linear relationship with the glucose concentration in the liquid cell between 25.0 and 200.0 mmol/l (correlation coefficient of 0.998). A substrate regeneration cycle coupling GOD with l-ascorbic acid (AsA: 0, 1.0, 3.0, 10.0 and 50.0 mmol/l; as reducing reagent system) was applied to the chemo-mechanical reaction unit in order to amplify the output signal of the tonometric biosensor. 3.0 mmol/l concentration of AsA could optimally amplify the sensor signal more than 2.5 times in comparison with that of non-AsA reagent.     

Wireless enzyme sensor system for real-time monitoring of blood glucose levels in fish

Periodic checks of fish health and the rapid detection of abnormalities are thus necessary at fish farms. Several studies indicate that blood glucose levels closely correlate to stress levels in fish and represent the state of respiratory or nutritional disturbance. We prepared a wireless enzyme sensor system to determine blood glucose levels in fish. It can be rapidly and conveniently monitored using the newly developed needle-type enzyme sensor, consisting of a Pt-Ir wire, Ag/AgCl paste, and glucose oxidase. To prevent the effects of interfering anionic species, such as uric acid and ascorbic acid, on the sensor response, the Pt-Ir electrode was coated with Nafion, and then glucose oxidase was immobilized on the coated electrode. The calibration curve of the glucose concentration was linear, from 0.18 to 144mg/dl, and the detection limit was 0.18mg/dl. The sensor was used to wirelessly monitor fish glucose levels. The sensor-calibrated glucose levels and actual blood glucose levels were in excellent agreement. The fluid of the inner sclera of the fish eyeball (EISF) was a suitable site for sensor implantation to obtain glucose sample. There was a close correlation between glucose concentrations in the EISF and those in the blood. Glucose concentrations in fish blood could be monitored in free-swimming fish in an aquarium for 3 days.     

Expression of glucose transporter isoform GLUT-2 and glucokinase genes in human brain

The glucose transporter isoform-2 (GLUT-2) and glucokinase are considered to be components of a glucose sensor system controlling several key processes, and hence may modulate feeding behaviour. We have found GLUT-2 and glucokinase mRNAs in several brain regions, including the ventromedial and arcuate nuclei of the hypothalamus. GLUT-2, glucokinase and glucokinase regulatory protein mRNAs and proteins were present in these areas as determined by biochemical approaches. In addition, glucose-phosphorylating activity with a high apparent Km for glucose that displayed no product inhibition by glucose-6-phosphate was observed. Increased glycaemia after meals may be recognized by specific hypothalamic neurones due to the high Km of GLUT-2 and glucokinase. This enzyme is considered to be the true glucose sensor because it catalyses the rate-limiting step of glucose catabolism its activity being regulated by interaction with glucokinase regulatory protein, that functions as a metabolic sensor.     

Immobilization of glucose oxidase on polymer membranes treated by low-temperature plasma

Low-temperature plasma was employed for activation of polymer membranes as a carrier for enzyme immobilization. Glucose oxidase was immobilized on polypropylene (PP), polyvinylidene fluoride (PVDF), or polytetrafluoroethylene (PTFE) membrane surfaces treated by nitrogen or ammonia gas plasma using glutaraldehyde as a linking agent. Enzyme activity was evaluated by the response of glucose sensor composed of the immobilized enzyme membrane and a dissolved oxygen electrode. The sensor response was found to depend on the kind of carrier membrane and to become maximum at suitable conditions of plasma treatment.     

Optical-transparent and flexible glucose sensor with ITO electrode

The glucose sensor was constructed by immobilizing glucose oxidase (GOD) with glutaraldehyde solution onto the sensitive area of the transparent oxygen electrode. The oxygen electrode was fabricated by sealing KCl electrolyte solution including the Indium-Tin Oxide (ITO)-electrode with both metal-weldable film and gas-permeable membrane coated with Ag/AgCl electrode. The sensor behavior was evaluated using standard glucose solutions in a batch measurement system with a computer-controlled potentiostat at a reduction potential of -900 mV. The sensor device has flexible structure and good optical transparency (less than 0.6 abs) at the visible wavelength from 400 to 700 nm. The sensor was possible to be used for measuring glucose from 0.06 to 1.24 mmol/l (correlation coefficient: 0.999), including the reported concentration of tear glucose in normal (0.14 mmol/l), with good reproducibility.     

Biocompatible glucose sensor prepared by modifying protein and vinylferrocene monomer composite membrane

This paper proposes a very simple procedure for preparing a biocompatible sensor based on a protein (bovine serum albumin, BSA), enzyme and vinylferrocene (VF) composite membrane modified electrode. The membrane was prepared simply by first casting vinylferrocene and then coating it with BSA and glucose oxidase immobilised with glutaraldehyde. The sensor response was independent of dissolved oxygen concentration from 3 to 10 ppm and showed good stability for serum sample measurement, unlike the commonly used BSA/enzyme modified electrode. The sensor response was almost unchanged over the measurement time (>10 h) whereas the responses of a BSA and glucose oxidase modified platinum electrode and an osmium-polyvinylpyridine wired horseradish peroxidase modified electrode (Ohara et al., 1993) fell to 68% of their initial value in a serum sample containing 10mM glucose.     

Amperometric determination of total assimilable sugars in fermentation broths with use of immobilized whole cells

A microbial sensor consisting of immobilized living whole cells of Brevibacterium lactofermentum and an oxygen electrode was prepared for continuous determination of total assimilable sugars (glucose, fructose and sucrose) in a fermentation broth for glutamic acid production. Total assimilable sugars were evaluated from oxygen consumption by the immobilized microorganisms. When a sample solution containing glucose was applied to the sensor system, increased consumption of oxygen by the microorganisms caused a decrease in the dissolved oxygen around the Teflon membrane of the oxygen electrode and the current of the electrode decreased markedly with time until steady state was reached. The response time was ≈ 10 min by the steady state method and 1 min by the pulse method. A linear relationship was found between the decrease in current and the concentration of glucose (<1 mM), fructose (<1 mM) and sucrose (<0.8 mM). The ratio of the sensitivity of the microbial sensor to glucose, fructose and sucrose was 1.00:0.80:0.92. The decrease in current was reproducible to within 2% of the relative standard deviation when a sample solution containing glucose (0.8 mM) was employed for experiments. The selectivity of the microbial sensor for assimilable sugars was satisfactory for use in the fermentation process. The additivity of the response of the microbial sensor for glucose, fructose and sucrose was examined. The difference between the observed and calculated values was within 8%. The microbial sensor was applied to a fermentation broth for glutamic acid production. Total assimilable sugars can be determined by the microbial sensor which can be used for more than 10 days and 960 assays.     

Development of biosensors for the simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose

Sensors for the simultaneous determinations of sucrose and glucose, lactose and glucose, and starch and glucose were prepared by a combination of the enzyme system shown below and an oxygen electrode: The mechanism for separating the substrates with the proposed sensors is based on the time lag arising from reaction and diffusion. Invertase, beta-galactosidase, amyloglucosidase, mutarotase, and glucose oxidase were covalently immobilized on triacetyl cellulose membranes containing 1,8-diamino-4-aminomethyloctane. A glucose oxidase membrane, mutarotase membrane, three sheets of triacetyl cellulose membranes, and invertase, or beta-galactosidase or amyloglucosidase membrane were placed in that order on the tip of the oxygen electrode. Calibration curves for sucrose, lactose, and starch were linear up to 40 mM, 60-180 mM, and 10%, respectively. The simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose was possible when the amount of glucose coexised was in the range of 2-16% sucrose, 2.8-8.3% lactose, or 0.1-1% starch. The relative errors were +/-4% for sucrose and +/-3% for lactose in 100 assays. The starch sensor was reused only five times. Each enzyme membrane was fairly stable for more than 10 days.     

The Application of Chemiluminescence of a Cypridina Luciferin Analog to Immobilized Enzyme Sensors

Chemiluminescence of a Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydro-imidazo[l,2-a]pyrazin-3-one, was applied to immobilized enzyme sensors. Xanthine oxidase, peroxidase, glucose oxidase, uricase and cholesterol oxidase were immobilized by using photo-crosslinkable resin prepolymer or ion-exchangeable cellulose beads. The immobilized enzyme sensor system was composed of a photoncounter and a test tube in which the immobilized enzyme membrane or particles were placed. A linear relation between the concentration of substrates and luminescence rate was obtained on a logarithmic scale. This immobilized enzyme sensor system could be used repeatedly. Hydrogen peroxide, xanthine and hypoxanthine were measured sensitively and rapidly within 100 sec. Glucose, cholesterol and uric acid were measured sensitively within 10 min but could be measured within 100 sec, although less sensitive. The detection limits for xanthine, hypoxanthine, hydrogen peroxide, glucose, cholesterol and uric acid were 0.02, 0.02, 0.2, 0.4, 2 and 2 μM, respectively. Concentrations of hypoxanthine in tuna muscle, and glucose and cholesterol in serum measured using this sensor system were comparable with those measured by the standard methods.     

酶电极流动注射分析法测定葡萄糖

 一种酶电极流动注射分析系统(EFIA)用于血糖和发酵葡萄糖的快速测定。研究了酶电极及其工作系统的性能和各种影响参数,,奠定了实用化基础。     

Monitoring of low concentrations of glucose in fermentation broth

A highly sensitive glucose sensor, operating in flow-injection analysis (FIA) mode, was developed for the detection of glucose in fermentation broth. The assay system is based upon the post-column reaction of the peroxide formed in the glucose-oxidase-catalysed reaction and subsequent spectrophotometric detection of the coloured product formed. The sensor system was characterised and calibrated using standard solutions, and later used for quantification of glucose in fermentation media. Two types of enzyme column were used: one operated in packed-bed mode and the other in expanded-bed mode. Both columns were integrated into a FIA system and were found to give good analytical results. Glucose concentrations as low as 0.1 mg/l and 5 mg/l could be detected in packed- and expanded-bed modes respectively. Glucose concentrations were measured during typical fed-batch fermentation conditions in this system, and the results are presented.     

Containment sensors for the determination of L-lactate and glucose

This paper reports some new results on enzyme based silicon containment sensors. For the first time an L-lactate sensor in containment technology is presented. Through optimization of the buffer system the stability of the lactate sensor was enhanced and the linear response of over 10 mM was achieved. The glucose sensor has also been optimized for a large linear measurement range exceeding 30 mM. A two-enzyme chip with glucose and lactate sensor elements which were integrated on one silicon chip is presented. The response behaviour of the two-enzyme chip was very similar to the single chip behaviour. No cross-talking effects could be observed. A fabrication process for mass-production is described.     

Glycosylated proline-rich peptide of human parotid saliva. Circular dichroism study.

The direct monitoring of sugars such as lactose, maltose, saccharose is not only useful at the applied point of view but also at the fundamental point of view for studying enzymology, especially in microbiology and fermentation. Benzyme systems were extensively used in solution for analytical applications in industry and medicine. The progress in the field of immobilization of bienzyme systems [1-3], especially within membranes [4-5], makes possible the production of new analytical devices. From the studies dealing with concentration profiles in artificial enzyme membranes [14], evidence was obtained for a well defined relationship between the local concentration of a metabolite and concentration of the first substrate in the bulk solution. In the described systems a substrate is transformed into glucose within a membrane, the glucose is then transformed in gluconic acid with a local oxygen consumption. The local pO2 level is linked to the glucose oxidase velocity, which is only linked to the glucose production, that is to say to the concentration of the first substrate. The enzyme electrode is based on the transformation of kinetic phenomena (reaction rates) into absolute values (local concentrations) through the diffusion-reaction coupling process. The manufacture of magnetic enzyme electrodes [6] allows convenient use of the active sensors. The pO2 electrode has some adventages, namely the specificity based on the selectivity of the gas permeable membrane and the linear relationship between the oxygen and the output of the electrode. pCO2, pH, ion electrodes give a logarithmic response as a function of the concentration. The grafting of a multienzyme system on a sensor allows a study of sequential systems in a defined context with a measurement of the local concentration of the metabolites. The tool is useful for both kinetics [4] and regulation studies [5].     

Determination of phosphate Ions with an enzyme sensor system

An enzyme sensor system for the determination of phosphate ions was constructed using immobilized enzymes and an oxygen electrode. The principle of this method is based upon the nucleoside phosphorylase catalyzed reaction for which the presence of inorganic phosphorus is indispensable. One assay could be completed within 3 min. This enzyme sensor was able to withstand at least 70 assays. This system was applicable to simple, rapid and continuous determination of phosphate ions in food.     

An autoclavable glucose biosensor for microbial fermentation monitoring and control

The design, construction, and characterization of a prototype-regenerable glucose biosensor based on the reversible immobilization of glucose oxidase (GOx) using cellulose binding domain (CBD) technology is described. GOx, chemically linked to CBD, is immobilized by binding to a cellulose matrix on the sensor-indicating electode. Enzyme immobilization can be reversed by perfusing the cellulose matrix with a suitable eluting solution. An autocavable sensor membrane system is employed which is shown to be practical for use in real microbial fermentations. The prototype glucose biosensor was used without failure or deterioration during fed-batch fermentations of Escherichia coli reaching a maximum cell density of 85 g (dry weight)/L. Medium glucose concentration based on sensor output correlated closely with off-line glucose analysis and was controlled manually at 0.44 +/- 0.2 g/L for 2 h based on glucose sensor output. The sensor enzyme component could be eluted and replaced without interrupting the fermentation. To our knowledge, no other in situ biosensor has been used for such an extended period of time in such a high-cell-density fermentation. (c) 1995 John Wiley & Sons, Inc.