DE2551004A1 - Determn. of glucose by oxidn. on supported glucose oxidase - and polarographic determn. of hydrogen peroxide formed, esp. for analysis of blood serum - Google Patents

Abstract

Glucose is determined in an aq. sample by (a) diluting the sample in an aq. diluent buffered to pH 4-8 (pref. 5-7), (b) passing the dil. sample through a bed of glucose oxidase held on a solid carrier, with a residence time sufficient to oxidise the glucose, with formation of H2O2, (c) removing the mixt. from the bed and passing it into direct contact with a system of polarographic electrodes, (d) determining the H2O2 polarographically by determn. of the electric current, which is directly proportional to the H2O2 concn., and (e) calculating the glucose concn. from this current. Claimed apparatus consists of a reaction chamber, with sample inlet and prod. outlet, and contg. the bed of supported enzyme, and a polarographic cell attached to the prod. outlet. The cell consists of a tubular chamber, with inlet and outlet, contg. a system of polarographic electrodes which are in direct contact with the sample; the electrodes are connected to an ampere meter for determn. of the current developed in the cell.

Description

Glucose analysis

The invention relates to a method and an apparatus for the determination and analysis of glucose. In particular, this relates to Invention to the analysis of glucose in physiological media, such as Elut and urine, and other aqueous specimens from medical and industrial Are interested.

In the medical field, there is a great need for a fast one and accurate analytical methods for determining glucose in blood serum nowadays before. As a result of recent advances in enzyme chemistry, it has been developing analytical methods for glucose based on the enzymatic oxidation of glucose Much attention has been paid to them based in the presence of glucose oxidase. The reaction runs according to the following equation: Glucose + oxygen Glucose oxidase gluconic acid + hydrogen peroxide.

This equation is a simplification of the complex equilibrium, that between the alpha and beta anomers of glucose, hydrogen peroxide, glucolactone, and gluconic acid is present in the presence of glucose oxidase, and zeiot that the glucose as a function of oxygen consumption or the increase in hydrogen peroxide or Gluconic acid can be measured, Many methods based on this equation have been described so far. As can be seen from the following explanation, these procedures are subject to one or more serious limitations, the affect their use in the clinical laboratory. Many of these styles involve a mass transport of either oxygen or glucose through a semipermeable membrane and through a membrane-liquid interface and are thereby only applicable with restrictions.

Such a method is described in the article "An Enzyme Electrode for the Amperometric Determination of Glucose "by G. G, Guilbault and G.J. Lubrano in Analytica Chimica Acta 64, (1973) pp. 439-455. This article describes a Enzyme-electrode method for the determination of glucose by direct amperometric Measurement of hydrogen peroxide. The enzyme electrode is constructed in such a way that glucose oxidase in or under a glucose-permeable membrane on a polarographic electrode is immobilized. When this electrode is placed in a glucose solution, it diffuses Glucose crosses the membrane, where it passes through the Glucose oxidase oxidizes to give hydrogen peroxide.

While this device is suitable for many purposes, it is of is limited by the permeation of glucose through the membrane. Also makes the immobilization of glucose oxidase directly on the electrode means a replacement or repair of the complete electrode arrangement is necessary in the event that the glucose oxidase is denatured.

This can be done in a modern analytical laboratory where a few hundred Blood samples processed daily can be time consuming and expensive.

U.S. Patent 3,539,455 discloses another polarographic membrane electrode method for glucose analysis. In this embodiment, the glucose permeates the sample a membrane to react with a quantity of a solution of soluble glucose oxidase, to produce hydrogen peroxide, which is measured polarographically. This way of working is dependent on my band and accordingly limited and does not set any immobilized Enzymes.

Many other polarographic membrane methods are suggested which involve the enzymatic oxidation of glucose to gluconic acid and hydrogen peroxide is measured as a function of the amount of oxygen consumed. Use such procedures usually an oxygen electrode with a membrane of the type described in U.S. Patent 2,913,836. Such procedures are in US patents 3,512,517; 3,542,662; as well as in the articles "A New Principle of Enzymatic Analysis" by H.U.

Bergmeyer and A. Hagen, which in the Z. Anal. Chem.

261, 333-336, (1972); as well as "The Enzyme Electrode" by S.J. Updike and G.P. Hicks, published in Nature, Vol. 214, 986-988, (1967); Studies on Conditions for the Polarographic Determination of Glucose with Glucose Oxidase "by M. Jemmali and R. Rodriquez-Kabana, published in Clinica Chimica Acta 42 (1972) 153-159; and "Immobilized Enzymes A Prototype Apparatus for Oxidase Enzymes in Chemical Analysis Utilizing Covalently Bound Glucose Oxidase "by M.K.

Weibel, W. Dritschilo, H.J. Bright and A.E. Humphrey, published in Analytical Biochemistry 52, 402-414 (1973).

These measuring methods are inherent in the diffusion of oxygen limited by a permiselective membrane.

In U.S. Patent 3,707,455, potentiometric rather than polarographic is used Method shown in which the glucose penetrates a membrane to the reaction chamber, which contains soluble glucose oxidase, and the resulting increase in gluconic acid is determined potentiometrically.

Other types of more general interest involving glucose measure as a function of various secondary chemical reactions are in the U.S. Patents 3,591,480; 3,623,960; 3,770,607 and in the articles "Electrochemical-Enzymatic Analysis of Blood Glucose and Lactate "by D.L. Williams, A.R. Doig, Jr., and A. Korosi, published in Analytical Chemistry, Vol. 42, No. 1, (1970); "Ion-Electrode Based Automatic Glucose Analysis System? 1 by R.A. Lienado and G.A. Rechnitz, published in Analytical Chemistry, Vol. 45, No. 13, (1973); and enzymes Electrode for Glucose Based on an Iodide Membrane Sensor "by G. Nagy, L.H. Von Storp and G.G. Guilbault, published in Analytical Chimica Acta, 66, (1973), 443-455.

In the known polarographic systems described above for Measurement of glucose, at which hydrogen peroxide is determined directly, is always a membrane because of the possible interference and poisoning of the electrode with foreign matter from the sample. The present invention provides an analytical one Method for glucose by direct polarographic determination of hydrogen peroxide that does not require a membrane.

In the present invention, a small amount of the glucose sample passes through compared to the volume of a steady flowing aqueous, buffered dilution stream, through a reaction chamber containing a layer of immobilized glucose oxidase, where it is oxidized to hydrogen peroxide as it penetrates the layer or leaked. The reaction chamber has a sample inlet and a reaction product outlet. The flow of the reaction product leads from the chamber to a polarographic one Cell. This causes a short contact time between the electrodes and the glucose sample, and electrode interference and poisoning are not essential, so that a Membrane is not needed. the Electrode speaks much faster on, and the sensitivity and reproducibility are with the present Invention increased, compared with the method by a membrane diffusion step are limited.

Accordingly, the present invention overcomes these disadvantages of the prior art in that it provides an apparatus and a method for Oxidation of glucose creates in which the glucose is immobilized in a layer Glucose oxidase is oxidized to form mass peroxide and gluconic acid, then the resulting hydrogen peroxide in a polarographic cell directly polarographic determined and then the polarographic measured value for the glucose equivalent the original sample is converted.

One of the essential features of the present invention is that that the layer of immobilized glucose oxidase is physically different from the polarographic Cell is disconnected. This represents a significant improvement over the above discussed types of enzyme electrodes, in which hydrogen peroxide forms a membrane must penetrate, which shields the sensing part of the polarographic electrode.

The invention is illustrated below with reference to exemplary embodiments in Connection with the drawings described in more detail.

1 shows a schematic flow diagram of a method according to the present invention: Figs. 2 and 3 schematic flow diagrams, which describes the details of polarographic cells used in connection with Figs can be used to show.

According to Fiq. 1, an aqueous sample containing glucose flows into a Reaction chamber containing a layer of immobilized glucose oxidase where the Sample is kept long enough at a temperature sufficient to remove the glucose to oxidize to gluconic acid and hydrogen peroxide. The reaction chamber is with a sample inlet and a reaction product outlet.

Typically, this oxidation takes place within a few seconds up to 30 minutes or longer at temperatures in the range from 0 ° C to about 500C completely away. A period of time of less than 1 minute at paum temperature is usually sufficient to oxidize glucose.

Because glucose oxidase works its best to oxidize glucose in the; If the pH range is from 4 to 8, the glucose sample is pre-treated with the glucose oxidase diluted with an aqueous thinner that is buffered to prevent its pH migrates from the pH range in which the glucose oxidase its Has the best effect on the oxidation of glucose, preferably for the purpose of uten efficiency the oxidation of glucose by glucose oxidase the pH of the buffered diluent in the range from about 5 to 7. Aqueous, buffered diluents are particularly suitable Solutions of sodium acetate and acetic acid, Other suitable buffers include sodium citrate and water-soluble phosphates and phthalate salts in 0.001 to 0.5 molar concentration.

The oxygen required for this oxidation reaction is through the Oxygen concentration that is normally in the glucose sample and the buffered Thinner is dissolved at room temperature, supplied. At this concentration lies Usually enough oxygen in front of enough glucose for a reliable one and oxidize reproducible polarographic detection. It's not necessary, that all of the glucose is oxidized to form hydrogen peroxide. It is only necessary that the percentage conversion is reliable and of a standard reproducible on unknown samples within a given number of laboratory conditions is.

In this way a reproducible value creates a 60 ° conversion of glucose an accurate polarographic response for the purpose of calibration and Analysis. In the preferred embodiment, substantially all of the glucose is oxidizes to form hydrogen @ eroxide so that every possible error by alteration the sample-to-sample conversion rate is eliminated. To such a complete To achieve conversion of glucose, additional oxygen can if desired an external source (e.g. an oxygen sparger or an oxygen containing gas) may be introduced into the sample, although this is seldom required is, slight fluctuations can occur in the concentration of the dissolved Oxygen in the glucose sample and in the buffered diluent as a function of the changes occur in pressure or temperature. Such fluctuations are insignificant, and the presence of sufficient oxygen is ensured if the detection method 1 the sample amount and concentrations specified here are used will. This is a significant advantage over procedures that control glucose levels measure as a function of the decrease in oxygen.

The ratio of the dilution of the glucose sample in the buffered diluent changes with the concentration of glucose in the sample. With physiological Media, such as blood and urine, that have an unknown concentration within of the expected concentration range is a dilution of 2.5 microliters of the sample in a stream of the buffered pre-diluent running at a rate flows from 0.1 to 5 and preferably 0.1 to 2 ml per minute, for a significant amount polarographic sensitivity suitable. Usually because of effectiveness and the economy of a small glucose sample (e.g. approximately 2 to 10 microliters) injected into the stream of the buffered diluent, which at a rate from 0.1 to 10 ml per minute when entering the layer of immobilized glucose oxidase flows.

The buffered thinner becomes more common with a small metering pump Design made to flow.

It is desirable to add salts in addition to buffering, such as e.g. potassium chloride or sodium chloride, which are used to a reference potential when silver-silver chloride reference electrodes are used that have the Use the sample as a filling solution. A bacteria inhibitor can be buffered with the Thinners have been combined to reduce bacterial impact.

Any of the known methods of immobilizing glocose oxidase on an insoluble support to form a layer of immobilized glucose oxidase can be used in the practice of the present invention. For example can glucose oxidase covalently to a porous glass slide with an amino-functional Silane coupling reagent may be bound as described in the article "Immobilized Enzymes: A Prototype Apparatus for Oxidase Enzyme in Chemical Analysis Utilizing Covalently Bound Glucose Oxidase "by M.K. Weibel et al, published in Analytical Biochemistry, 52, 402-414 (1973); Glucose oxidase can be on a full-body column immobilized as described in the article "A New Principle of Enzymatic Analysis" by H.V. Bergmeyer and A. Hagh, published in Z. Anal. Chem. 261, 333-336 (1972) will; Glucose oxidase can be immobilized in polyacrylic polymers, as in that Article "Enzyme Electrodes for Glucose Based on an Iodide Membrane Sensor by G. Nagy et al, published in Analytica Chemica Acta, 66, (1973), 443-455 will; Glucose oxidase can be immobilized in a polyacrylamide gel, such as in the article "An Enzyme Electrode for the Amperometric Determination of Glucose" by G. Guilbault et al, published in Analytica Chemica Acta 64, (1973), 439-455 or in U.S. Patent 3,542,662 is described; Glucose oxidase immobilized on nickel silica alumina, as described in the article "Immobilization of Glucose Oxidase on Nickel-Silica-Alumina "by W.M. Herring et al, published in Biotechnology and Bioengineering, Vol.

XIV, pp. 975-984, (1972); glucose oxidase can also be used immobilized with cyanogen bromide according to the method of US Pat. No. 3,645 852 "Method of Binding Water-Soluble Proteins and Water-Soluble Peptides to Water Insoluble Polymers Using Cyanogen Halide "by R. Axen, J.

Porath and E. Ernbach. The revelations of these prior publications are now incorporated by reference / this application so it is in the art known, the layer of immobilized glucose oxidase with selection of the carrier materials to form, such as, for example, porous glass; occurring in particles and preferably porous refractory oxide, such as aluminum oxide, titanium oxide, zirconium oxide, silicon oxide, magnesium oxide, Magnesium silicate and thorium oxide; Glass mass; porcelain occurring in particles; compacted and sintered refractory oxides; Volume; water-insoluble polymers, and that the glucose oxidase is immobilized thereon by chemical or physical means will.

It is only necessary that the layer is permeable to the sample must be, while a large area in relation to the volume is created in order to ensure adequate contact between the glucose oxidase and the glucose sample. A dense layer of glucose oxidase, which occurs on an inert particle porous, fibrous or gel-like carrier is immobilized preferred for this purpose. When fire-resistant oxides appearing in particles are used, the particle size and percent permeability are not particularly good critical as long as there is sufficient contact between glucose and glucose oxidase is created. It has been found that a volume permeability in the Range from about 10 to about 60% with an average diameter of the Pore size in the range of about 0.01 to 10 microns effectively and without waher available is . Refractory oxides occurring in particles that have a diameter of Particle size in the range of about 1 to about 1000 microns without another with a diameter particle size in the range of about 100 Microns to 400 microns available and very useful for the purposes at hand.

After the oxidation of glucose in the layer of immobilized Glucose oxidase, the resulting reaction mixture flows to the polarographic cell.

According to the present invention, the aqueous buffered Thinner is continuously pumped through the reaction chamber, which is the layer of the immobilized glucose oxidase and the polarographic cell. The glucose sample is placed with an injection needle in the layer of immobilized glucose oxidase injected through an injection port that is in the shape of a mixing valve with a rubber diaphragm can be covered.

While the glucose is the bed of immobilized glucose oxidase flows through and permeates, it is oxidized to hydrogen peroxide and gluconic acid, which are considered Reaction products remain in the sample. This reaction mixture then flows into the polarographic cell where hydrogen peroxide is polarographic by direct contact is detected with the polarographic electrodes. The polarographic approach from the measurement of hydrogen peroxide is measured by a current measuring device, such as a current recorder. This value is then applied to the glucose equivalent of the original sample converted.

The glucose equivalent of the original sample will normally be expressed in mg glucose / 100 ml (i.e. mg percent) of the sample.

These units are common in clinical applications.

Two embodiments of the polarographic shown in FIG Cells are shown schematically in FIGS. These cells become "flow" cells called because the sample being analyzed continuously passes through the cell flows.

In Fig. 2, the cell body 10 is made of a solid, inert electrical insulating material, such as glass or plastic. Polymethyl methacrylate plastics have proven very satisfactory for this purpose. The cell body 10 is provided with a narrow passage 11 through which the sample of the reaction mixture, containing hydrogen peroxide, flows into the cell cavity 15. The cavity 15 is sized so that there is effective contact between the sample and the polarographic Electrodes is ensured, while the dead volume is kept as low as possible will. The one used in the examples Cell is made of plastic and has a cavity approximately 0.25 cm wide, 1.25 cm long and approximately 0.05 cm depth.

The electrodes are inserted from the top of the cell and are attached along the longitudinal direction.

In Fig. 2 is a set of two polarographic electrodes 12 and 12 13 mounted in the cell body 10 so that the sensing tips of the electrodes in direct Are in contact with the sample in cavity 15. The electrode 12 functions as both Counter electrode as well as reference electrode. The electrode 12 is conventional Design and can be a silver wire that is drawn over a silver chloride, or Be a calomel electrode. A silver-silver chloride reference electrode is preferred, since the sample to be measured can act as a reference electrode filling solution by Chloride ions are entered in it. The other types of reference electrodes, who use their own bottled filling solution can, if so desired, be used.

The electrode 12 is connected to a source of constant DC potential connected (usually about 0.6 volts), as in normal polarography. The electrode 13 is a working electrode in the form of platinum, palladium, gold, Graphite or carbon or other inert conductive material.

The hydrogen peroxide in the sample depolarizes slightly polarographic Electrodes, and the current flow through the current measuring device at a given applied voltage is measured is proportional to the hydrogen peroxide concentration. This current flow is converted to the glucose equivalent to the original sample. In the polarographic cell shown in FIG. 2, the Reference electrode also act as a counter electrode and takes part in the redox reaction, which takes place in the cell. The IR voltage drop is not compensated, and the potential difference between the reference electrode and the working electrode can change.

The cell of FIG. 3 is preferred over the cell of FIG. 2. In the cell of FIG. 2, the reference electrode is the only other electrode in the Circuit so that current is forced to flow through it. This can be one Change in the potential difference between the electrodes due to the IR voltage drop effect through the sample. In addition, the silver chloride coating, if a silver-silver chloride reference electrode used, eventually attacked by the redex reaction. Accordingly is the use of such a cell, even if the electrode is close to the working electrode is not as accurate as the cell of FIG. 3.

If the cell of Figure 3 is used in a routine laboratory, then a peak detector and sample and hold circuit can be used measure the highest current over a baseline of the current, and this difference is then proportional to the hydrogen peroxide concentration in the sample and is therefore proportional to the glucose concentration.

In a preferred polarographic cell shown in FIG the polarographic cell is potentiostatic.

This is the so-called "three-electrode polarography", they in which Article "The Renaissance in Polarographic and Voltammetric Analysis" by Jud B. Flato, published in Analytical Chemistry, Vol. 44, September 1972, the teachings of which are incorporated herein by reference.

Fig. 3 shows a cell body 10 and a cavity 15, like the cell of Fig. 2. The cell of Fig. 3 is provided with a reference electrode 23 in the form of a Equipped with silver wire, which is coated with silver chloride and as dense as possible on the working electrode 21, which consists of a platinum wire.

The applied voltage (+ 0.6 volts DC) is at the input of the control amplifier 30 is applied, with which the reference electrode 23 also through the voltage follower 31 is connected. The output of the control amplifier 30 is connected to the counter electrode 20 made of platinum wire. Through this Construction ensures that there is essentially no current through the reference electrode 23 flows and sufficient compensation voltage is applied to the counter electrode 20, by the voltage difference between the reference electrode 23 and the working electrode 21 to maintain. The working electrode 21 is provided with a small conventional current measuring device connected, which provides a current measurement based on the glucose equivalent of the original Sample is converted. Other common electrodes, as described in connection with Fig. 2 can be used in the embodiment of FIG. if so desired.

In the following examples all parts are parts by weight, all percentages Weight percentages, and all temperatures are in ° C, unless otherwise indicated will.

Example 1 Part A Immobilized Glucose Oxidase on Alumina Powder 1 g of divided alumina is washed thoroughly with distilled water. The divided aluminum oxide has a particle size in the range from minus 40 to + 70 mesh (US sieves) and an average pore size diameter of 0.1 to 0.2 microns.

50 mg glucose oxidase (with a stated activity of 140 IU / mg) is added to the wet divided aluminum oxide in 40 ml of an aqueous solution, which has been buffered to a pH of 5> 5 with a standard buffer, the one Contains a mixture of potassium dihydrogen phosphate and disodium hydrogen phosphate. the The resulting mixture is gently stirred for half an hour at 6 to 80C to sorb the enzyme.

A crosslinking reagent is added to this mixture, which by Mixing 20 ml of methanol, 10 ml of distilled water, 0.15 ml of concentrated Hydrochloric acid, 0.08 ml of diaminopropane and 0.02 ml of dibromoethane is formed. The united Mixing of aluminum oxide, glucose oxidase and the wetting reagent becomes easy stirred with a magnetic stirrer at 6 to 80C overnight to oxidize the glucose on the alumina too immobilize. The resulting immobilized Glucose oxidase / alumina composition is distilled with approximately 2 liters Washed water and stored in distilled water.

Part B Calibration of the immobilized glucose oxidase / alumina composition for The catalytic activity of the immobilized glucose oxidase / alumina composition of part A is derived from the measured rate of oxidation of β-D-glucose to gluconic acid by parabenzoquinone in the presence of the glucose oxidase / alumina composition calculated. The reaction is represented by the following equation: ß-D-glucose + parabenzoquinone + H2O Glucose oxidase gluconic acid + hydroquinone.

The reaction is based on the potentiometric measurement of the change in concentration of hydroquinone followed over time. A platinum detector electrode is connected to a Dual compound calomel silver / silver chloride reference electrode used. Standard solutions To calibrate the electrode system, hydroquinone with approximately 100 molar Excess of parabenzoquinone, which is also present in the aqueous solutions, manufactured. A calibration curve is drawn by taking the hydroquinone concentration plotted against the millivolt reading of the potentiometer.

The reaction medium in which the glucose oxidation takes place is an aqueous solution that is 0.1 molar in dextrose and 0.01 molar in phosphate buffer at a pH of 5> 5 is. It is stirred overnight to ensure that the equilibrium between the alpha and beta-D-glucose forms has been reached. Sufficient parabenzoquinone and hydroquinone are added to make the final Solution 1.0 x 10-² molar and 1.0 x 10-4 molar with respect to the last components close. A known narrow of the glucose oxidase / alumina composition is added to a certain volume of the reaction medium, and the change the potential of the electrode immersed in the solution will decrease over time Followed the use of the electrode system described above.

The corresponding hydroquinone concentration is calculated from the nillivolt readings determined from the calibration curve and a diagram of the concentration of the test solution made against time. The slope of this curve represents the rate of oxidation the p-D-glucose which has been catalyzed by the immobilized enzyme. the Activity is then calculated from the following relationship: activity = oxidation rate Alumina Volume Using this calibration procedure, it turns out that the clucose oxidase / alumina composition of Part A has an activity of 995 IU glucoso-oxidase per ml of glucose = oxidase / aluminum oxide composition owns. 10 days later after adding the composition in distilled water 4 ° C, the activity is 825 i.U.

Glucose oxidase per ml of the glucose oxidase / alumina composition.

An international unit of biological activity is called that Amount of an active enzyme has been established that is a substrate to a product converts at a rate of one micromole per minute.

Part C Preparation of a layer of immobilized glucose oxidase for use in Fig. 1 A glass column is made from a 50 mm borosilicate glass tube with an inner diameter of 2.8 mm and an outer diameter of 6 minutes. A 400 mesh nylon washer is attached to one end of the column. The immobilized Glucose oxidase / aluminum oxide composition of Part A is entered in it, to fill the pipe. The glucose oxidase / aluminum oxide is made by gravity entered tightly in the column, and the other end of the column is also with a 400 mesh nylon disc after the column has been filled. That Volume of the glucose oxidase / alumina composition is approximately 0.3 cc.

The ends of the columns are then fitted with plastic connecting pipes, one of which is T-shaped for sample injection.

The injection T comes with a rubber membrane for sample injection provided with a hypodermic needle.

Part D Analysis of Glucose A buffered diluent for the glucose sample is made from: 50 ml of a saturated aqueous solution of 2-chloro-4- Phenylphenol; 10 ml of 1.0 normal potassium chloride; 8.2 g (0.1 mole) sodium acetate; sufficient 1.0 molar acetic acid to adjust the pH to 5.6; and in addition water to to make one liter of solution. The buffer is approximately 0.1 molar this way of acetate, 0.01 molar and potassium chloride and contains a trace of the bactericide 2-chloro-4-phenylphenol.

The polarographic cell is that shown in Figure 3 and is already has been described above. The electrodes come with a polarographic analyzer tied together.

The standard solution used to calibrate the polarographic cell is a 0.01 molar aqueous glucose solution containing 0.1 molar of sodium chloride as Reference electrode filling solution is.

Both chemicals are suitable as reagents. The standard soil solution is made well in advance of use to ensure chemical equilibrium. In other words, in general clinical usage, this standard indicates a glucose concentration of 180 mg of glucose per 100 ml of the sample (i.e. 180 mg% glucose).

The buffered diluent is produced by the apparatus described in FIG pumped at a rate of 1.0 m1 per minute. This results in. a baseline stream of approximately one nanoampere, a 3.0 microliter sample of the 0.1 molar glucose standard solution comes with a hypodermic needle through the injection T into the Column of immobilized enzyme as shown in Fig. 1 is injected. The current rises rapidly to a maximum current of 250 nanoamps over a 10 second period increases rapidly over a 30-second period to a value that is equal to approaches the baseline value. The calibration factor from current to concentration is therefore 1.39 nanoamps per mg% glucose for a 3 microliter sample.

Ten separate 3.0 microliter samples of a standard blood serum sample (which is given @@@ @ 3.5 mg% glucose) are successively in the stream of the buffered Injected thinner that flows into the layer of immobilized glucose oxidase 1 and the resulting peroxide is polarographically analyzed as above, and the corresponding Follow-up readings are recorded. Using the calibration factor the current is converted to the concentration; it turned out that the Samples an average value of 80 mg glucose / 100 ml sample (80 mg-%) with a Contained deviation coefficients of 0.7%.

A second, unusual standard blood crum sample (with 197 mg glucose content approached) is analyzed in the procedure described above. Of the entire 20 analyzes results in an average value of 200.5 mg glucose / 100 ml with a Standard deviation coefficients of 1.3%.

Example II Part A Preparation of Immobilized Glucose Oxidase 5 grams of particulate alumina that has a particle size in the range from -70 to + 80 mesh (US sieve) and an average pore diameter of 0.1-0.2 microns is dried by heating at 10,600C for 2 hours. After cooling, the alumina is in 1N hydrochloric acid for 2 to 4 hours soaked and washed with distilled water to remove absorbed air. The washed alumina is mixed with approximately 10 ml of a 0.01 molar phosphate buffer (pH 6.0) placed in a beaker.

As in Example I (250 mg), glucose oxidase becomes that in particles occurring aluminum oxide with enough distilled water added to the Bring the volume of the mixture to approximately 40 ml. The glucose oxidase / aluminum oxide mixture becomes easy with a magnetic stirrer at room temperature for about half an hour touched.

A wetting reagent is made from 20 ml of methanol, 10 ml of water, 0.15 ml concentrated hydrochloric acid, 0.05 ml of diaminopropane and 3 ml of a 10% by weight solution made from glutaraldehyde.

This reagent is added drop by drop to the glucose oxidase / aluminum oxide mixture added as described above. The addition is made at room temperature, and the finished reaction mixture is left to stand at OOC overnight. Then it will be them in one 0.001 molar phosphate buffer (pH 5.5) and washed stored in distilled water until the sample.

The sample sequence is the same as described in Example I, Part B. has been. The activity was found to be approximately 150 IU per ml of glucose oxidase / alumina composition.

Part B Glucose Analysis A layer of immobilized glucose oxidase is, as has been described in Example I, using the glucose oxidase / aluminum oxide composition, which has been described in Part A above.

The procedure for the analytical system is essentially the same as that in Part D of Example I.

The flow rate of the buffered diluent is 1.0 ml per minute; it has the same composition as in Example I. A 3.0 microliter Standard glucose solution (containing 180 mg% glucose) gives a reading of 590 nano amps. The calibration factor is therefore 3.28 nanoamps per mg% glucose for the 3 microliter sample.

Ten separate 3.0 microliter samples of a standard blood serum sample (indicated with 83.5 mg glucose / 100 ml) are sequentially in the layer of the immobilized Glucose oxidase as described above, injected, and the corresponding Current readings are recorded. Using the calibration factor for conversion the current to the glucose concentration, the samples are analyzed and ethäll an average of 82 mg glucose per 100 ml with a coefficient of deviation of 1.5%.

A second, unusual, standard blood serum sample (the 197 mg or similar) Should contain glucose) is analyzed as described above.

A total of ten attempts becomes an average of 197.2 mg glucose / 100 ml with a standard coefficient of deviation of 1.0% was obtained.

Example III Part A Preparation of Immobilized Glucose O @idase 5 g in particles of alumina having a particle size in the range of -60 to +70 mesh (US sieve) and an average pore diameter of 0.1 up to 0.2 microns are heated to 1150 ° C for two hours. After cooling down the aluminum oxide lying in the particles becomes overnight in 1N hydrochloric acid softened. Then it is washed with distilled water and placed in a beaker brought with 50 ml of a 0.001 molar phosphate buffer (pH 6.0) for half an hour, before adding 25 ml of the glucose oxidase solution if. Glucose oxidase (Asperilligus niger) is available in the art in a buffered solution (pH 4.0) and has a stated activity of 1000 IU / ml.

The resulting glucose oxidase / aluminum oxide mixture is used for a Stirred gently for half an hour at 3 to 80C.

A crosslinking reagent is made by mixing 20 ml of methanol, 0.01 ml of diaminopropane, 0.15 ml of concentrated hydrochloric acid, 0.03 ml of dibromoethane and 10 ml Water produced. This reagent is dispensed at a rate of 0.15-0.20 ml per minute added to the glucose oxidase / alumina mixture while using a magnetic stirrer is gently stirred and the temperature at 3 to 80C for 16 to Is held for 20 hours. The resulting immobilized glucose oxidase / alumina composition is washed with 2 l of distilled water and in distilled water up to Sample kept.

The sampling procedure is the same as that in Example I. has been described. The activity of the sample is 2,300 IU / ml immobilized Glucose oxidase / alumina composition.

Part B Glucose Analysis A layer of immobilized glucose oxidase is as described in Example I using the glucose oxidase / alumina composition which has been described in Part A above. The volume of the column is however in this case only 0.2 com.

The standardization procedure for the analytical system is in the essentially the same as that in Part D of Example I.

The flow rate of the buffered diluent of the composition of Example I is 0. ml per minute. A 2.5 microliter sample of the glucose standard, containing 180 mg% of glucose gives a current of 150 nanoamps. The calibration factor is therefore 0.83 nanoamps per mg% glucose for the 2.5 microliter sample.

Nine separate 2.5 microliter samples of a standard blood sample are made successively into the layer of immobilized glucose oxidase as described above is injected and the corresponding current reading is recorded. Under use of the calibration factor for converting the current to the concentration results for the samples averaged 79.7 mg glucose / 100 ml with a standard deviation coefficient of 1.5%. The stated value is 83.5 mg glucose / 100 ml.

A second, different standard blood serum sample containing 197 mg of glucose is analyzed as described above. From a total of 11 attempts becomes an average value of 205.6 mg glucose / 100 ml with a standard deviation coefficient of 1.3% obtained.

Similar results are obtained from the procedure described above for glucose analysis when the layer of immobilized glucose oxidase thereby is produced that the glucose oxidase is produced on a cross-linked polydextran will.

A 1.25 g sample of polydextran is mixed with 2 liters of distilled water washed and a uniform gel is made by mixing with water. 4 g of Brom @ yan are crushed in a mortar and quickly transferred to the polydextran gel.

The pH of the dispersed gel quickly increases to approximately 10.5 with a 3-normal hydroxide solution and adjusted at this pH by the gradual Maintain addition of more 3 normal sodium hydroxide.

At intervals, crushed ice is made from deionized water has been added to the temperature of the reaction mixture at about 200 to keep. The introduction of base will be discontinued if there is no further change occurs in pH and the cyanogen bromide crystals have been consumed. The mix will stirred continuously during the reaction. A total of about 13 ml Sodium hydroxide solution needed.

A large amount of ice is added and the mixture becomes one sintered glass filter funnel, the product is washed under vacuum, First, 300 ml of a 0.01 molar tri-hydroxymethyl aminomethane buffer (with HCl adjusted to pH 7) and then 300 ml of a 0.01 molar phosphate buffer (pH 5.6) can be used. Both washing solutions were previously cooled in an ice bath. The bottom of the filter funnel is then closed with a piece of wax film.

To the resulting polydextran activated by bromocyan, which still on the filter funnel is a cold solution of 50 mg glucose oxidase in 20 ml of a 0.01 molar phosphate buffer (pH 5.6) were added. The resulting product is stirred with a glass rod on the filter funnel and then to a beaker convicted. The washing of the polydextran activated by bromocyan and the subsequent Mixing with the glucose oxidase solution on the funnel is done very quickly, i.e. in a total time of about 90 seconds.

The glucose oxidase / polydextran mixture is then stirred with a magnetic stirrer Stirred gently for 16 to 20 hours at 0 ° C.

It is then washed with 1 to 2 liters of distilled water and stored in a 0.1 molar phosphate buffer, pH 5.6.

After 65 days the activity is 450 IU / ml glucose oxidase polydextran gel composition. The glucose oxidase / polydextran gel composition is packed tightly into a column for the glucose samples are analyzed as in Example I.

Similar results are also obtained with the procedure described above for glucose analysis obtained when the immobilized enzyme layer glucose oxidase comprises those on a porous particulate anion exchange resin has been immobilized. The resin used is the chloride form of a styrene-divinylbenzene copolymer with exchange groups of a quaternary amine ion.

A 250 mg sample of an anion resin is 0.1 molar with 30 ml Sodium borate buffer (pH 8.5) stirred vigorously.

It is then centrifuged for 20 minutes at 12,000 times the earth's concentration. Any excess is decanted and the washing process is repeated twice. the Final wash is carried out in the same way using a 0.1 molar Accet @ t buffer, pH 5.0.

The washed resin is dispersed in approximately 50 ml of the acetate buffer and gently stirred for 15 to 20 minutes at 5 to 100C before adding 100 mg of glucose oxidase is added in small steps. 0.5 ml of dibromopropane is then added to this mixture, dissolved in 2 ml of acetone was added. The final mix is then for a Stirred gently at 5 to 100C for an hour. The reaction product is at 120 times the gravitational pull centrifuged and discarded excess. The glucose-oxidaso-ion exchange resin composition is further treated with 40 ml of an acetate buffer solution (pH 5.0) which is 0.05 molar in 2-aminoethanol. It is stirred for one hour at 0 °, filtered and with 1 to 2 1 of distilled deionized water is washed in which it is then subsequently is kept at 4 ° C.

After one week the activity is 15 IU / ml immobilized Glucose Oxidase / Resin Composition. The gel composition is in a column entered, and the glucose samples are analyzed as in Example I.

Claims (10)

n 5 p r ii c h e Method for determining glucose in an aqueous Sample that contains glucose, characterized in that the sample with an aqueous buffered diluent adjusted to a pH in the range of about 4 to about 8 is buffered, is diluted that the diluted sample is in a layer of glucose oxidase, which is immobilized on a solid support, is passed over that the diluted Sample with the glucose oxidase for a time sufficient to oxidize the glucose is kept in contact to form hydrogen peroxide that the resulting reaction mixture is removed from the layer that the resulting reaction mixture to a direct Contact with a set of polarographic electrodes is passed that during direct contact with the polarographic electrodes is hydrogen peroxide an electric current is determined polarographically in the reaction mixture which is proportional to the concentration of hydrogen peroxide in the sample, that the electrical current is determined, and the current determination on the glucose concentration the sample is transferred. 2. The method according to claim 1, characterized in that essentially all of the glucose is oxidized to form hydrogen peroxide. 3. The method according to claim 1, characterized in that the aqueous buffered diluent is buffered to a pH in the range of approximately 5 to 7. 4. The method according to claim 1, characterized in that the aqueous Sample that contains glucose is blood serum. 5. The method according to claim 1, characterized in that the solid Carrier is a refractory oxide occurring in particles. 6. Device for determining glucose in an aqueous sample that Contains glucose, characterized in that it comprises a reaction chamber which contains a layer of glucose oxidase, which is on a solid support of the reaction chamber, which has a sample inlet and a reaction product outlet is that it is a polarographic cell connected to the reaction product outlet comprises that the cell comprises a tubular sample chamber having a sample inlet and a sample outlet that the chamber has a set of polarographic electrodes possesses that for direct electrical connection with the sample in the chamber therein are attached, with the electrodes for amperometric measurement of the in the cell generated electric current are electrically connected. 7. Apparatus according to claim 6, characterized in that the electrode set includes three electrodes: a working electrode, a counter electrode and a reference electrode. 8. Apparatus according to claim 6, characterized in that the electrode set a pair of electrodes comprises: a reference electrode and a working electrode. 9. Apparatus according to claim 7, characterized in that the working electrode Platinum is that the counter electrode is platinum, and that the reference electrode is with Silver chloride is coated silver. 10. Apparatus according to claim 6, characterized in that the fixed Carrier comprises a particulate refractory oxide.