Method for removing dissolved oxygen from an electrochemical biosensor using an oxidase as the sensor enzyme

Description

State of the art

[0001] The present invention is based on a method for determining the concentration of an analyte in a sample. Such methods are generally known from the prior art and are performed millions of times a day. Examples include controls in food production and blood glucose measurements. In Germany alone, 24 million of the latter are performed per day on patients with diabetes mellitus.

[0002] To measure the concentration of an analyte, such as glucose or lactose, electrochemical biosensors are typically used. These biosensors are suitable for determining the concentrations to be measured with the aid of enzymatic reactions. A major challenge with these methods is that dissolved oxygen in the sample influences the measurement results. In electrochemical biosensors, the presence of dissolved oxygen short-circuits the electron transfer process from the analyte to the electrode. This leads to significant inaccuracies at low analyte concentrations.

[0003] While the use of tests to determine the glucose concentration in the blood of diabetic patients is urgently needed and can save lives, the methods used are repeatedly controversial due to their lack of accuracy. The most widely available biosensors for glucose on the market are based on the enzymes glucose dehydrogenase or glucose oxidase. Both enzymes have different disadvantages that can affect the accuracy of the measurement. For example, some glucose dehydrogenase-based systems produce false readings in the presence of certain medications and react nonspecifically with other sugars such as maltose, galactose, and xylose. This results in falsely elevated readings, which can lead to the failure to detect hypoglycemia and fatal outcomes.

[0004] Glucose oxidase-based test systems are less susceptible to medications, varying pH levels, and the temperature of the patient's blood, and due to their specificity for glucose, they are not susceptible to other sugars. Furthermore, glucose oxidase is inexpensive to produce. However, glucose oxidase test systems are sensitive to elevated or decreased oxygen concentrations. For example, elevated oxygen concentrations lead to lower readings, whereas decreased oxygen concentrations result in elevated readings. Furthermore, in the presence of oxygen, hydrogen peroxide is formed, which can deactivate the glucose oxidase used.

[0005] State-of-the-art methods for oxygen removal are known in which glucose oxidase, galactose oxidase, or pyranose oxidase are combined with catalase (US9187779B2). These two enzymes enable the removal of oxygen. The oxidase catalyzes the oxidation of a sugar (glucose, galactose) to a hemiacetal (glucose oxidase), ketone (pyranose oxidase), or aldehyde (galactose oxidase), and hydrogen peroxide. The resulting hydrogen peroxide is degraded by the added catalase to oxygen and water. This combination allows the effective removal of oxygen in the presence of the corresponding sugars. However, this system cannot be used to develop a biosensor for detecting glucose, as the oxidoreductases used (except galactose oxidase) are specific for glucose. Degradation of glucose would, however, distort the measurement results of a glucose biosensor.A further disadvantage of the described method is that the oxidoreductases used (glucose oxidase, galactose oxidase and pyranose oxidase) can also transfer the electrons to a mediator of the measurement process, which would lead to a falsification of the measurement results.

[0006] The state of the art also includes a method in which an oxidase was used to remove oxygen in an oxidase-based electrochemical sensor system. This involves spatial separation of both processes (O2 removal and analyte detection) ("A Polymer Multilayer Based Amperometric Biosensor for the Detection of Lactose in the Presence of High Concentrations of Glucose" by F. Lopez et al., "Electroanalysis" 2017). The spatial separation is achieved by using multiple layers. A lower layer is electrically connected to an electrode and can transfer electrons. This layer contains an enzyme specific for the analyte. An upper layer is applied to the lower layer, in which enzymes for the removal of oxygen or other molecules such as glucose and hydrogen peroxide are embedded.However, this design has the disadvantage that the sensor enzyme is located in the lower layer. This design can limit the analyte concentration in the lower layer, and the application of the upper layer can impair the activity of the enzymes in the lower layer.

[0007] Furthermore, methods for oxygen removal using alcohol oxidase are already known from the state of the art. Alcohol oxidase is produced in bacteria or fungi. These cells are then used as whole cells, as cell lysates, or as cell extracts to remove oxygen. In this application, the cells or extracts are packaged in an oxygen-permeable membrane. However, the use of cells or cell extracts is not practical for developing a biosensor, as they are difficult to integrate into a miniaturized sensor and are more suitable for oxygen removal in packaging.

[0008] US 2012 / 0211372 A1 discloses systems and methods for the enzymatic removal of oxygen from aqueous solutions. However, these systems and methods are not suitable for use in biosensors in which the content of an analyte is determined using oxidases. Disclosure of the invention

[0009] It is therefore an object of the present invention to provide a process which removes dissolved oxygen from an aqueous solution and does not have the disadvantages of the prior art described above.

[0010] This object is achieved by a method according to claim 1.

[0011] The process according to the invention uses an oxidase for oxygen removal.

[0012] The dissolved oxygen is removed via a coupled enzyme reaction with an oxidase and a catalase. InIn a first step, an oxidase catalyzes a reaction. In the first reaction, oxygen reacts with a substrate of the oxidase, e.g., a short-chain alcohol or formaldehyde, to form an aldehyde or an acid and hydrogen peroxide. In a second step, in a second reaction, catalyzed, for example, by a catalase, the hydrogen peroxide is disproportionated to water and ½ oxygen. The resulting oxygen is finally converted to water by cycling through the first and second steps. The oxidase used is highly specific for oxygen as an electron acceptor, and no electrons are transferred to mediators or other non-specific molecules (Azevedo, Biosensors&Bioelectronics, 2005).This special property of oxidase ensures that the removal of oxygen has no influence on the measurement results of enzyme-based biosensors, or more precisely, oxidase-based biosensors. The process described here removes dissolved oxygen quickly and completely. The coupled enzyme reaction makes it possible to effectively remove dissolved oxygen even from small sample volumes without causing a more than negligible direct reaction at the electrode of an electrochemical biosensor due to oxygen diffusion processes.

[0013] The method according to the invention is advantageously suitable for removing dissolved oxygen from aqueous solutions, in particular for removing dissolved oxygen from aqueous solutions to improve the quality of results of electrochemical analyses that use oxidases specific for the desired analyte as sensor enzymes, for example in the determination of glucose concentrations.

[0014] All components required for oxygen removal and analyte determination can be deposited as a solid on the electrode of an electrochemical measuring cell. The enzymes used can be applied by drop casting and then dried. The substrate for the oxidase for oxygen removal—for example, methanol, ethanol, or propanol—can be processed into a water-soluble solid, for example, by mixing it with maltodextrin. Substrate within the meaning of the present invention includes, in particular, the reducing agent. Methanol can also be processed into a water-soluble gel by adding calcium acetate. Paraformaldehyde (CAS No. 30525-89-4), another substrate for the oxidase for oxygen removal, is a solid that can also be used in an electrochemical measuring cell.The method according to the invention has the advantage that by adding an aqueous sample containing the analyte, all necessary components are dissolved, thus eliminating the need for labor-intensive sample preparation. Furthermore, the use of solids advantageously improves the efficiency of analyte concentration determination using an analyte-specific oxidase, since the solids have no influence on the oxidation reaction of the sensor enzyme or the electrochemical processes of the measuring cell.

[0015] It is also conceivable to use the method according to the invention in other chemical processes in which dissolved oxygen is an interfering factor, for example, in the concentration analysis of phosphates or lactose using oxidase-based biosensors. The quality of the results of electrochemical analyses with oxygen-sensitive sensor enzymes, especially when the enzymes used are oxidases, is improved by the method according to the invention.

[0016] Advantageous embodiments and further developments of the invention can be found in the dependent claims and the description with reference to the drawings.

[0017] According to a preferred embodiment of the present invention, a short-chain alcohol, preferably methanol, ethanol, propanol, butanol, pentanol, formaldehyde, or paraformaldehyde, is used as the reducing agent. It is conceivable to use the alcohol in a concentration of 5 to 100 mmol / l, preferably 45 mmol / l to 55 mmol / l. Paraformaldehyde can be used as another possible substrate for the oxidase to remove oxygen. Paraformaldehyde is a polymer of formaldehyde and exists as a solid.

[0018] According to a further preferred embodiment of the present invention, an alcohol oxidase isolated from Pichia pastoris, Candida boidinii, Hansenula sp., or other organisms is used as the oxidase for oxygen removal. These oxidases advantageously improve the effect of the method according to the invention. These oxidases have a high specific activity for short-chain alcohols and formaldehyde and are also inexpensive and commercially available. The intended oxidase is selected such that the enzymatic activity for the oxidation of an analyte is significantly lower than for the oxidation of the reducing agent. The enzymatic activity for the oxidation of the analyte is less than 5% of the enzymatic activity for the oxidation of the reducing agent.

[0019] According to a further preferred embodiment of the present invention, the disproportionation is carried out using a catalyst, preferably using catalase, particularly preferably using catalase isolated from bovine liver. Catalase isolated from bovine liver advantageously improves the efficiency of the process according to the invention. Furthermore, this catalase is commercially available. However, it is also conceivable that Aspergillus niger, Corynebacterium glutamicum, or Micrococcus lysodeikticus are used as the catalase.

[0020] The present invention further relates to a measuring system for the electrochemical determination of an analyte in an aqueous solution in an electrochemical measuring cell, wherein the measuring cell comprises electrodes and the chemical reactants for carrying out a method according to the invention. The measuring system enables electrochemical measurements to be carried out, wherein, to improve the accuracy of the measurement, dissolved oxygen is removed using the method according to one of claims 1 to 4. Dissolved oxygen is removed without the oxygen removal process influencing the electrochemical determination using an oxidase.

[0021] According to a preferred embodiment of the present invention, the chemical reactants are integrated into the measuring cell.

[0022] According to a preferred embodiment of the present invention, the electrodes are screen-printed electrodes. These are inexpensive and flexible in manufacture and use. It is also conceivable to use glassy carbon electrodes as electrodes. The electrodes can be a few millimeters in size, preferably up to 3 mm.

[0023] According to a preferred embodiment of the present invention, the volume of the electrochemical measuring cell is ≤ 150 µl, preferably ≤ 100 µl. In a likewise preferred embodiment of the present invention, the volume can be in the range of ≥ 0.5 µl to ≤ 100 µl, preferably ≥ 0.5 µl to ≤ 50 µl, particularly preferably ≥ 0.5 µl to ≤ 10 µl.

[0024] According to a further preferred embodiment of the present invention, the measuring system comprises 1,1-ferrocenedimethanol as an electron mediator. It is also conceivable that the sensor enzyme comprises an enzyme of the oxidase class (EC number: 1.1.3) and an electron mediator compatible with the sensor enzyme. It is further conceivable that the electron mediator has a similar redox potential to that of the sensor enzyme. It is also conceivable that other ferrocene derivatives are used as electron mediators. However, it is also conceivable to use other classes of electron mediators such as ferricyanides, conductive organic salts, redox hydrogels with a complexed metal (e.g. iron, osmium, or ruthenium), phenothiazines, phenoxazines, or viologens. By using an oxidase from P.Pastoris can use the above-mentioned electron mediators because this enzyme does not react with them, but is specific for oxygen as an electron acceptor.

[0025] According to a further preferred embodiment of the present invention, the measuring system comprises a redox polymer with an osmium complex as electron mediator.

[0026] According to a further preferred embodiment of the present invention, the analyte is glucose, lactose, and / or phosphate, wherein the sensor enzyme for oxidizing the analyte comprises glucose oxidase, lactose oxidase, or a combination of xanthine oxidase and purine nucleoside phosphorylase. This makes it possible to measure the analyte concentration, e.g., the glucose concentration, lactose concentration, or phosphate concentration, of an aqueous solution. The respective oxidase, e.g., glucose oxidase, is selected such that the enzymatic activity for oxidizing the reducing agent is significantly lower than for oxidizing the analyte. The enzymatic activity for oxidizing the reducing agent is less than 5% of the enzymatic activity for oxidizing the analyte.

[0027] According to a preferred embodiment of the invention, the transfer of electrons to the electron mediator by the oxygen scavenging oxidase results in a measured current that is ≤5% of the current measured during analyte oxidation. To ensure this, a specific concentration ratio between the oxygen scavenging oxidase and the sensor enzyme must be determined.

[0028] It would also be conceivable, however, for the measurement system to comprise lactose oxidase for the determination of lactose or a combination of xanthine oxidase and purine nucleoside phosphorylase for the determination of phosphate. Furthermore, it is also conceivable for an embodiment of the present invention to comprise an oxidase that is specific for an analyte not yet mentioned in the previous embodiments.

[0029] A further subject of the present invention is a method for the electrochemical determination of a concentration of an analyte in an aqueous solution using a measuring system according to one of claims 5 to 11, characterized in that dissolved oxygen in the aqueous solution is removed using a method according to one of claims 1 to 4. The method makes it possible to determine the concentration of an analyte in an aqueous solution without the presence of dissolved oxygen leading to undesirable deviations in the measurement results.

[0030] According to a preferred embodiment of the present invention, the analyte is oxidized in an oxidation reaction catalyzed by an oxidase, whereby electrons from the oxidation reaction are transported by an electron mediator to an electrode, and the current generated by the transport of the electrons by the electron mediator is measured. This makes it possible to determine the concentration of the analyte based on a current between the electrodes of the measuring system used.

[0031] According to a further preferred embodiment of the present invention, the analyte is glucose, with glucose oxidase being used as the oxidase. This provides a method for determining a glucose concentration in an aqueous solution without bound oxygen or the production of hydrogen peroxide leading to falsified results. It would also be conceivable, however, for the method to use lactose oxidase for the determination of lactose or a combination of xanthine oxidase and purine nucleoside phosphorylase for the determination of phosphate. Furthermore, it is also conceivable to use an oxidase that is specific for an analyte not yet mentioned in the previous embodiments.

[0032] According to a further preferred embodiment of the present invention, the analyte is lactose or phosphate, wherein lactate oxidase or a combination of xanthine oxidase and purine nucleoside phosphorylase is used as the oxidase.

[0033] According to a further preferred embodiment of the present invention, 1,1-ferrocenedimethanol is used as the electron mediator. According to a further preferred embodiment of the present invention, a redox polymer with an osmium complex is used as the electron mediator. It has been shown that 1,1-ferrocenedimethanol or a redox hydrogel with complexed osmium as the electron mediator increases the quality of the measurement results of the method. It is also conceivable that other classes of electron mediators, such as ferricyanides, conducting organic salts, metal complexes (e.g., iron, osmium, or ruthenium complexes), phenothiazines, phenoxazines, or viologens, are used.

[0034] Further details, features, and advantages of the invention will become apparent from the drawings and the following description of preferred embodiments with reference to the drawings. The drawings merely illustrate exemplary embodiments of the invention, which do not limit the essential inventive concept. Short description of the drawing

[0035] Figure 1 shows the basic principle of the method for removing dissolved oxygen according to an exemplary embodiment of the present invention. Figure 2 shows the operating principle of the electrochemical biosensor for determining the concentration of the analyte of the aqueous sample at which dissolved oxygen is removed according to an exemplary embodiment of the present invention. Figure 3 shows the electrochemical characterization of various short-chain alcohols as reducing agents for the oxidase for oxygen removal. Figure 4 shows the electrochemical characterization of maltodextrin-methanol solid as a reducing agent for the oxidase for oxygen removal. Figure 5 shows the electrochemical characterization of calcium acetate-methanol solid as a reducing agent for the oxidase for oxygen removal. Figure 6 shows the electrochemical characterization of paraformaldehyde as a reducing agent for the oxidase for oxygen removal.Figure 7 shows the electrochemical characterization of the electron mediator 1,1-ferrocenedimethanol together with the oxygen scavenging oxidase. Figure 8 shows the electrochemical characterization of the oxygen scavenging oxidase with the electron mediator 1,1-ferrocenedimethanol in solution. The oxidase cannot transfer electrons to this mediator. Figure 9 shows the electrochemical characterization of the oxygen scavenging oxidase with glucose in solution. The oxygen scavenging oxidase cannot oxidize glucose. Figure 10 shows the determination of a glucose concentration series under different conditions in a sample volume of 1000 µl. Figure 11 shows the influence of different concentration ratios of the oxygen scavenging oxidase and the sensor enzyme. Figure 12 shows the determination of a glucose concentration series under different conditions in a sample volume of 100 µl. Exemplary embodiment of the invention

[0036] In Figure 1 The basic principle of the method for removing dissolved oxygen 12 according to an exemplary embodiment of the present invention is shown schematically as a flow diagram. In the first step 1, a reducing agent 15, here methanol, reacts with oxygen 12 to form an aldehyde 16 and hydrogen peroxide 13. An oxidase for oxygen removal 14, here alcohol oxidase, serves as the catalyst for this reaction. In the second step 2, the hydrogen peroxide 13 is reduced to water 18 and the catalase 17 is oxidized. Furthermore, the oxidized catalase 17 and further hydrogen peroxide 13 are reduced to water 18 and oxygen 12. The overall equation for the second step 2 is 2 H 2 O 2 → O 2 + 2 H 2 O. After completing the first step 1 and the second step 2, 1 O 2 has reduced to ½ O 2. By repeatedly passing through the first step 1 and the second step 2, the dissolved oxygen 12 is removed.

[0037] In Figure 2 the basic principle of the method for removing dissolved oxygen 12 according to an exemplary embodiment of the present invention is schematically illustrated as a flow chart.

[0038] In the first step 1 of the process, electrode 4 absorbs an electron 5 and converts the reduced mediator 7 into its oxidized form. The oxidized sensor enzyme 8 (e.g., glucose oxidase) oxidizes the analyte 11 (e.g., glucose) and is thereby reduced to the reduced sensor enzyme 9. If the analyte 11 is glucose, gluconolactone is formed as the oxidized analyte 10.

[0039] As a side reaction, the reduced sensor enzyme 9 can be oxidized by dissolved oxygen 12, releasing an electron 5. This releases hydrogen peroxide 13, which causes interference at the electrode 4.

[0040] The method according to the invention allows the dissolved oxygen 12 to be completely removed. The reducing agent 15, here an alcohol, reacts with the oxygen 12 to form the aldehyde 16 and hydrogen peroxide 13. In various alternative embodiments, the reducing agent 15 can be ethanol, propanol, butanol, pentanol, and preferably methanol. An oxygen removal oxidase 14, here alcohol oxidase, serves as the catalyst for this reaction. The oxygen removal oxidase 14 used is highly selective for oxygen 12 and does not use artificial electron mediators to transfer electrons 5. This property makes it possible to remove oxygen 12 in oxidase-based biosensors without interfering with it. In the second step 2, the hydrogen peroxide 13 is reduced to water 18, and the catalase 17 is oxidized. Furthermore, the oxidized catalase 17 and the hydrogen peroxide 13 are reduced to water 18 and ½ oxygen 12.After completing the first step 1 and the second step 2, 1 oxygen 12 has been reduced to ½ oxygen 12. By repeatedly cycling through the first step 1 and the second step 2, the dissolved oxygen 12 is completely removed.

[0041] The oxygen removal oxidase 14 oxidizes a substrate and removes dissolved oxygen 12. In Figure 3 The removal of oxygen 12 using various substrates is shown. According to an exemplary embodiment of the present invention, methanol, ethanol, propanol, butanol, or pentanol can be used as the substrate at a concentration of 50 mmol / l. According to a preferred embodiment, the alcohol is provided in solid form. In Figure 4The removal of oxygen 12 using the reducing agent 15, methanol bound in maltodextrin, is shown. For this purpose, the reducing agent 15 is added to maltodextrin until the powder can no longer bind liquid. The excess methanol is removed by evaporation. The reducing agent maltodextrin bound for the process preferably contains approximately 5% by weight of methanol. The solid is added to the reaction mixture before the start of the reaction (1 mg per 100 µl). The oxygen 12 is removed just as effectively as by the addition of a liquid substrate (cf. Figure 3 ).

[0042] In Figure 5The removal of oxygen 12 bound in calcium acetate using the reducing agent 15 methanol is shown. For this purpose, a saturated solution of calcium acetate is prepared (3 g Ca(CH 3 COO) 2 to 10 g water). 40 ml of methanol is then added while stirring. 2 mg of this solid is then added to 100 µl of the reaction mixture. The oxygen 12 is removed just as effectively as by the addition of liquid substrate.

[0043] In another exemplary embodiment of the present invention, paraformaldehyde, a polymer of formaldehyde, is used as a solid reducing agent 15 for oxygen removal. For this purpose, 1 mg of paraformaldehyde is added to 100 µl of the reaction mixture. Figure 6 shows the electrochemical characterization of oxygen removal. Oxygen 12 is removed just as effectively as by adding liquid substrate.

[0044] The oxygen removal oxidase 14 is unable to transfer electrons 5 to an electron mediator. Figure 7 The diagram shown contains experimental data from an experiment investigating the influence of oxidase on oxygen removal. This experiment showed that the oxidase does not transfer electrons 5 to the electron mediator 1,1 ferrocenedimethanol for oxygen removal 14, but can still remove oxygen 12 from the solution.

[0045] The oxygen removal oxidase 14 is unable to oxidize the analyte 11. Figure 8 shows a diagram with data from an experiment in which glucose is added as a substrate to a biosensor according to the invention. The oxygen removal oxidase 14 is capable of completely removing the oxygen 12 from the solution. The added glucose shows no effect on the background current of a control experiment (cf. Figure 3).

[0046] To demonstrate the suitability of oxidase for oxygen removal 14 in a biosensor system, a glucose biosensor is tested under various conditions. The data from this test series are presented in Figure 9 The current for the oxidation of the analyte 11 glucose by the sensor enzyme was investigated in an air atmosphere, an argon atmosphere, and a room air atmosphere in combination with the oxygen removal oxidase 14 at a glucose concentration of 50 mmol / l. Comparing these measurements, it can be seen that the measured current under an argon atmosphere is identical to the current measured when the oxygen removal oxidase was used in a room air atmosphere. In comparison, the measured current is lower when the oxygen removal oxidase 14 is not present in the system. Figure 10A concentration series of a glucose biosensor is shown. The presence of oxygen 12 in the solution leads to up to 30% lower measured currents because the sensor enzyme transfers electrons 5 to the oxygen 12 present in the solution.

[0047] A preferred ratio between the sensor enzyme and the oxidase for oxygen removal 14 is 1:10. In Figure 11 The experimental evidence of this optimization is shown. This can be demonstrated by a constant current over a measurement time of 200 minutes. The constant current indicates that oxygen 12, which diffuses from the air into the solution, can be removed immediately and completely. Figure 12shows the detection of a glucose concentration series in a reaction volume of 100 µl using an oxygen-insensitive biosensor. The interference of oxygen 12 in the solution results in a measured current that is on average 25% lower and a maximum of 90% lower (~10 mmol / L glucose concentration). List of reference symbols

[0048] Fig. 1 1First step 2Second step 3Third step Fig. 2 4 Electrode 5 Electron 6 Oxidized mediator 7 Reduced mediator 8 Oxidized sensor enzyme 9 Reduced sensor enzyme 10 Oxidized analyte 11 Analyte 12 Oxygen 13 Hydrogen peroxide 14 Oxidase for oxygen removal 15 Reducing agent 16 Aldehyde 17 Catalase 18 Water Fig. 319Y axis: current in µA 20X axis: potential in V (E vs Ag / AgCl 3M KCl) 21Control in phosphate buffer (100 mM, pH 7.0-7.5) 221 ml phosphate buffer + 50 mmol / l methanol 231 ml phosphate buffer + 50 mmol / l ethanol 241 ml phosphate buffer + 50 mmol / l propanol 251 ml phosphate buffer + 50 mmol / l butanol 261 ml phosphate buffer + 50 mmol / l pentanol Fig. 4 27Y-axis: Current in µA 28X-axis: Potential in V (E vs Ag / AgCl 3M KCI) 29Control in 1 ml phosphate buffer (100 mM, pH 7.0-7.5) 301 ml phosphate buffer + maltodextrin-methanol solid Fig. 5 31Y-axis: Current in µA 32X-axis: Potential in V (E vs Ag / AgCl 3M KCI) 33Control in 1 ml phosphate buffer (100 mM, pH 7.0-7.5) 341 ml phosphate buffer + calcium acetate-methanol solid Fig. 6 35Y-axis: Current in µA 36X-axis: Potential in V (E vs Ag / AgCl 3M KCl) 37Control in 1 ml phosphate buffer (100 mM, pH 7.0-7.5) 381 ml phosphate buffer + paraformaldehyde (0.1 mg / ml) Fig. 739Y-Achse: Strom in µA 40X-Achse: Potential in V (E vs Ag / AgCl 3M KCI) 411 ml Phosphatpuffer (100 mM, pH 7,0-7,5), 10U AOX, 2000U CAT, 100 µmol / l 1,1-Ferrocendimethanol 421 ml Phosphatpuffer (100 mM, pH 7,0-7,5), 10U AOX, 2000U CAT, 100 µmol / l 1,1-Ferrocendimethanol, 50 mmol / I Ethanol Fig. 8 43Y-Achse: Strom in µA 44X-Achse: Potential in V (E vs Ag / AgCl 3M KCI) 451 ml Phosphatpuffer (100 mM, pH 7,0-7,5), 10U AOX, 2000U CAT, 50 mmol / I Glukose 461 ml Phosphatpuffer (100 mM, pH 7,0-7,5), 10U AOX, 2000U CAT, 50 mmol / I Glukose, 50 mmol / I Ethanol Fig. 947Y-axis: Current in µA 48X-axis: Potential in V (E vs Ag / AgCl 3M KCl) 49Control without glucose: 1 ml phosphate buffer (100 mM, pH 7.0-7.5), 10 U AOX, 2000 U CAT, 15 U GOX, 100 µmol / l 1,1-ferrocenedimethanol, 50 mmol / l ethanol 50Glucose determination in air atmosphere: 1 ml phosphate buffer (100 mM, pH 7.0-7.5), 15 U GOX, 100 µmol / l 1,1-ferrocenedimethanol, 50 mmol / l glucose 51Glucose determination with oxygen removal in air atmosphere: 1 ml phosphate buffer (100 mM, pH 7.0-7.5), 10U AOX, 2000U CAT, 15U GOX, 100 µmol / l 1,1-ferrocenedimethanol, 50 mmol / l ethanol, 50 mmol / l glucose 52Glucose determination Argon atmosphere: 1 ml phosphate buffer (100 mM, pH 7.0-7.5), 15U GOX, 100 µmol / l 1,1-ferrocenedimethanol, 50 mmol / l glucose Fig. 1053Y-axis: Current in µA 54X-axis: Glucose concentration in mmol / l 55Control air atmosphere: 1 ml phosphate buffer (100 mM, pH 7.0-7.5), 15 U GOX, 100 µmol / l 1,1-ferrocenedimethanol 56Glucose determination with oxygen removal air atmosphere: 1 ml phosphate buffer (100 mM, pH 7.0-7.5), 10 U AOX, 2000 U CAT, 15 U GOX, 100 µmol / l 1,1-ferrocenedimethanol, 50 mmol / l ethanol 57Glucose determination argon atmosphere: 1 ml phosphate buffer (100 mM, pH 7.0-7.5), 15 U GOX, 100 µmol / l 1,1-ferrocenedimethanol Fig. 1158Y-axis: Current in µA 59X-axis: Time in minutes 60Glucose determination with oxygen removal Air atmosphere: 1 ml phosphate buffer (100 mM, pH 7.0-7.5), 10U AOX, 2000U CAT, 15U GOX, 100 µmol / l 1,1-ferrocenedimethanol, 100 mmol / l ethanol 61Glucose determination with oxygen removal Air atmosphere: 1 ml phosphate buffer (100 mM, pH 7.0-7.5), 20U AOX, 2000U CAT, 10U GOX, 100 µmol / l 1,1-ferrocenedimethanol, 100 mmol / l ethanol 62Glucose determination with oxygen removal Air atmosphere: 1 ml phosphate buffer (100 mM, pH 7.0-7.5), 50U AOX, 2000U CAT, 5U GOX, 100 µmol / l 1,1-ferrocene dimethanol, 100 mmol / l ethanol Fig. 1263Y-axis: Current in µA 64X-axis: Glucose concentration in mmol / L 65Glucose determination with oxygen removal Air atmosphere: 1 ml phosphate buffer (100 mM, pH 7.0-7.5), 10 U AOX, 2000 U CAT, 15 U GOX, 100 µmol / L 1,1-ferrocenedimethanol, 100 mmol / L ethanol 66Glucose determination with oxygen removal Argon atmosphere: 1 ml phosphate buffer (100 mM, pH 7.0-7.5), 2000 U CAT, 10 U GOX, 100 µmol / L 1,1-ferrocenedimethanol

Claims

1. Method for removing dissolved oxygen (12) in an electrochemical biosensor which uses a first oxidase for oxygen removal (14) and a second oxidase as a sensor enzyme (8 / 9), wherein i) in a first step (1), a first reaction is catalyzed by the first oxidase for oxygen removal (14), wherein in the first reaction, oxygen reacts with a reducing agent (15) to form hydrogen peroxide (13) without electrons (5) being transferred to an acceptor other than oxygen (12), wherein no other components of the electrochemical biosensor are oxidized, ii) in a second step (2), in a second reaction, the hydrogen peroxide (13) is disproportionated to water (18), iii) in a third step (3), in a third reaction, the second oxidase, which serves as a sensor enzyme (8 / 9), catalyzes the oxidation of the analyte (11) without the third step (3) oxidizing other components of an aqueous solution of the electrochemical biosensor, wherein the oxidase that serves as a sensor enzyme (8 / 9) is specific for the analyte (11), wherein the electrons (5) released by the oxidation of the analyte (11) are transferred exclusively to an electron mediator.

2. Method according to claim 1, wherein a short-chain alcohol, preferably methanol or ethanol or propanol or butanol or pentanol, or formaldehyde or paraformaldehyde is used as the reducing agent (15).

3. Method according to one of the preceding claims, wherein an oxidase isolated from Pichia pastoris or Candida boidinii or Hansenula sp. is used as the first oxidase for oxygen removal (14).

4. Method according to one of the preceding claims, wherein the disproportionation of hydrogen peroxide (13) to water (18) is carried out by means of a catalyst, preferably by means of catalase (17); and a catalase isolated from bovine liver is preferably used as catalase (17).

5. Measuring system for the electrochemical determination of an analyte (11) in an aqueous solution in an electrochemical measuring cell, wherein the measuring cell has electrodes (4), wherein the measuring cell has the chemical reaction partners for carrying out a method according to one of the preceding claims.

6. Measuring system according to claim 5, wherein the chemical reaction partners are integrated into the measuring cell.

7. Measuring system according to one of claims 5 or 6, wherein the electrodes (4) are screen-printed electrodes.

8. Measuring system according to one of claims 5 to 7, wherein the volume of the measuring cell is less than ≤ 150 µl, preferably ≤ 100 µl.

9. Measuring system according to one of claims 5 to 8, wherein the measuring system comprises 1,1-ferrocendimethanol as an electron mediator.

10. Measuring system according to one of claims 5 to 9, wherein the measuring system comprises a redox polymer with an osmium complex as an electron mediator.

11. Measuring system according to one of claims 5 to 10, wherein the analyte (11) is glucose, lactose and / or phosphate, wherein the sensor enzyme for oxidizing the analyte comprises glucose oxidase, lactose oxidase or a combination of xanthine oxidase and purine nucleoside phosphorylase.

12. Method for the electrochemical determination of a concentration of an analyte (11) in an aqueous solution using a measuring system according to one of claims 5 to 11, characterized in that dissolved oxygen (12) in the aqueous solution is removed using a method according to one of claims 1 to 4.

13. Method according to claim 12, wherein the analyte (11) is oxidized in an oxidation reaction catalyzed by the oxidase, wherein electrons (5) from the oxidation reaction are transported from the electron mediator to an electrode (4), wherein the current generated by the transport of the electrons (5) through the electron mediator is measured.

14. Method according to claim 12 or 13, wherein the analyte (11) - is glucose, wherein glucose oxidase is used as the oxidase; or - lactose or phosphate, wherein lactate oxidase or a combination of xanthine oxidase and purine nucleoside phosphorylase is used as the oxidase.

15. Method according to any of claims 12 to 14, wherein the electron mediator is - 1,1-ferrocenedimethanol is used as the electron mediator; or - a redox polymer with an osmium complex is used.