BIOS sensor, method for detection using this BIOS sensor, method for manufacturing and using the BIOS sensor

Description

[0001] The present invention relates to a biosensor. In particular, the present invention relates to a biosensor with improved resistance to the influence of oxygen. The following invention further relates to a method for manufacturing such a sensor. Furthermore, the present invention relates to a method for detecting an analyte.

[0002] Electrochemical biosensors are generally well-known. Currently, they are mainly used to determine, for example, the glucose or lactose concentration of an aqueous sample using an enzymatic reaction.

[0003] The most widely used electrochemical biosensors are employed to determine the glucose content of various samples. They are of great importance in fields such as food analysis and medical diagnostics. In Germany, several million blood glucose measurements (approximately 24 million measurements per day) are performed daily on patients with diabetes mellitus. This practice has led to improved diabetes management and is now an indispensable part of daily treatment plans. However, the accuracy of commercially available devices is a subject of debate, as it can lead to undesirable misdiagnosis / misinterpretations, which in some cases have already resulted in patient deaths (Olansky, Diabetes Care, 2010).According to DIN EN ISO 15197:2015, the following rules for the accuracy of blood glucose meters have recently come into effect: Measurements may deviate from the laboratory value by a maximum of ± 15% for readings ≥ 100 mg / dl and ± 15 mg / dl for readings below 100 mg / dl. Reducing these tolerances can improve individual treatment success. The most widely used biosensors for glucose on the market are based on the enzymes glucose dehydrogenase (GDH) or glucose oxidase (GO). Both enzymes have various drawbacks that can affect measurement accuracy. For example, some GDH-based systems (test strips with GDH-PQQ) produce false readings in the presence of certain medications and react non-specifically with other sugars such as maltose, galactose, and xylose. Otherwise, falsely elevated readings can occur, which can lead to undetected hypoglycemic episodes and potentially fatal outcomes.Therefore, in 2009 the FDA issued a warning advising against the use of GDH-PQQ systems in treatment with drugs containing these sugars (FDA Public Health Notification: Potentially Fatal Errors with GDH-PQQ* Glucose Monitoring Technology). Meanwhile, mutated variants of the enzyme, which have a reduced affinity for sugars other than glucose, are also being used.

[0004] However, there are also reports that mutated forms of the enzyme have led to elevated readings in the presence of galactose (Ceriotti, J. Clin. Sci. Tech., 2015). Other GDH-based test systems are also used in which the enzyme's specificity for other sugars does not distort the results (GDH-FAD, GDH-NAD). GO-based test systems are less susceptible to medications, pH, and temperature in the patient's blood and, due to their specificity for glucose, are not susceptible to other sugars. Furthermore, GO is inexpensive to produce and highly sensitive (Aggidis, Biosensors and Bioelectronics, 2015).

[0005] However, glucose tolerance (GO) test systems are sensitive to elevated or decreased oxygen concentrations, a phenomenon known as the oxygen effect. Elevated oxygen concentrations lead to lower readings, while decreased oxygen concentrations result in elevated readings (Schmid, Diabetes Technol Ther., 2014). Furthermore, the presence of oxygen leads to the formation of hydrogen peroxide, which can deactivate the GO test system (Prévoteau, Electrochimica Acta, 2012).

[0006] According to the current state of technology, biosensors are known in which the influence of oxygen, the so-called oxygen effect, is to be eliminated by correction methods.

[0007] A disadvantage of this correction method is the need for an additional electrode, which makes the sensor design more complex and therefore more expensive. Furthermore, the oxygen effect can be accounted for by simultaneously using GO and GDH. However, the disadvantage of this method is also the need for an additional electrode, which makes the sensor design more complex and expensive. Moreover, the described methods do not remove oxygen; they only measure its influence and then correct for it. These systems only work for glucose determination because they utilize the properties of glucose-specific sensor enzymes.

[0008] Key publications in this field include approximately Aggidis AG, Newman JD, Aggidis GA. Investigating pipeline and state of the art blood glucose biosensors to formulate next steps. Biosens Bioelectron. 2015 Dec 15; 74:243-62; Cass AE, Davis G, Francis GD, Hill HA, Aston WJ, Higgins IJ, Plotkin EV, Scott LD, Turner AP., Ferrocene-mediated enzyme electrode for amperometric determination of glucose, Anal Chem. 1984 Apr;56(4):667-71; Dicks JM, Aston WJ, Davis G, Turner APF, Mediated amperometric biosensors for d-galactose, glycolate and 1-amino acids based on a ferrocene-modified carbon paste electrode, Analytica Chimica Acta, Volume 182, 1986, Pages 103-112; Ceriotti F, Kaczmarek E, Guerra E, Mastrantonio F, Lucarelli F, Valgimigli F, Mosca A. Comparative performance assessment of point-of-care testing devices for measuring glucose and ketones at the patient bedside. J Diabetes Sci Technol. 2015 Mar; 9(2):268-77. Olansky L, Kennedy L. Finger-stick glucose monitoring: issues of accuracy and specificity. Diabetes Care.2010 Apr; 33(4):948-9; Van Hecke W, Salaheddin C, Ludwig R, Dewulf J, Haltrich D, Van Langenhove H. Biocatalytic cascade oxidation using laccase for pyranose 2-oxidase regeneration. Bioresour Technol. 2009 Dec; 100(23):5566-73; Plumeré N, Henig J, Campbell WH. Enzyme-catalyzed O2 removal system for electrochemical analysis under ambient air: application in an amperometric nitrate biosensor. Anal Chem. 2012 Mar 6; 84(5):2141-6; Prévoteau A., Mano N., Oxygen reduction on redox mediators may affect glucose biosensors based on "wired" enzymes, Electrochimica Acta 68 (2012) 128-133; Schmid C, Baumstark A, Pleus S, Haug C, Tesar M, Freckmann G. Impact of partial pressure of oxygen in blood samples on the performance of systems for self-monitoring of blood glucose. Diabetes Technol Ther. 2014 Mar; 16(3):156-65; sowie .

[0009] US 7,955,484; US 9,187,779; US 761162; US 9,546,390; US 5,431,800A; US 5,628,890A; US 5,997,817A; US 6,299,757 B1; US 8,475,638 B2; US 7,727,467 B2; and US; 7,906,009 B2.

[0010] Furthermore, according to the current state of technology, methods are known that remove oxygen or other molecules from a biosensor.

[0011] For example, biosensors are known from Maidan R, Heller A. Elimination of electrooxidizable interferants in glucose electrodes, JACS, 1991, 113 (23), 9003-9004, or Lopez F, Ma S, Ludwig R, Schuhmann W, Ruff A. A Polymer Multilayer Based Amperometric Biosensor for the Detection of Lactose in the Presence of High Concentrations of Glucose, Electroanalysis, 2017, 29 (1), 154-161. The biosensors described here consist of several layers. The bottom layer is electrically connected to the electrode and can transfer electrons. This layer contains an enzyme specific to the analyte. A further layer is then applied to this layer, in which enzymes for the removal of oxygen (Maidan, JACS, 1991) or other molecules such as glucose (Lopez, Electroanalysis, 2017) and hydrogen peroxide are embedded. On a laboratory scale, this approach is suitable for producing interference-resistant biosensors.However, the production of a multilayer for mass production (approx. 750 million test strips per day (worldwide), 24 million test strips per day (Germany)) is less suitable because it is more expensive.

[0012] Lopez F, Ma S, Ludwig R, Schuhmann W, Ruff A. A Polymer Multilayer Based Amperometric Biosensor for the Detection of Lactose in the Presence of High Concentrations of Glucose, Electroanalysis, 2017, 29 (1), 154-161 further describes a method for oxygen removal using a specific enzyme system described in Plumeré et al., Anal. Chem., 2012 (see above). This enzyme system is used here as part of a multilayer. This means that a sensor enzyme is covered by a polymer layer in which the enzymes for oxygen removal are embedded. However, this method has the disadvantage that, on the one hand, the production of such a test strip or biosensor is comparatively complex and therefore costly, and on the other hand, the performance of the biosensor may not be optimal.

[0013] Maidan R, Heller A. Elimination of electrooxidizable interferants in glucose electrodes, JACS, 1991, 113 (23), 9003-9004 further describes how, in order to generate hydrogen peroxide, oxygen is removed as a side reaction by an enzyme system (lactate oxidase). Comparatively large sample volumes are used in this process.

[0014] In Monteiro T, Rodrigues PR, Gonçalves AL, Moura JJ, Jubete E, Añorga L, Piknova B, Schechter AN, Silveira CM, Almeida MG. Construction of effective disposable biosensors for point of care testing of nitrite, Talanta. 2015 Sep 1;142:246-51, the enzyme system for oxygen removal, which was already described in the publication Plumeré et al., Anal. Chem. 2012, is also used. The sample volume used here, as demonstrated by the experiment, is ≥100 µl.

[0015] In the method described in Quan D, Shim JH, Kim JD, Park HS, Cha GS, Nam H, Electrochemical determination of nitrate with nitrate reductase-immobilized electrodes under ambient air, Anal. Chem., 2005, 77(14):4467-4473, oxygen is removed using sulfite. The described screen-printed electrode is integrated into a sample vessel. The volume of this vessel is designed for a volume ≥100 µl.

[0016] Document US 2013 / 0153416 A1 deals specifically with an arrangement of numerous microfluidic biosensors. These biosensors utilize enzymes that are catalytically active for specific targets and can analyze sample volumes ranging from 0.1 to 20 µl. They also feature channels smaller than 500 µl, enabling rapid analysis. Furthermore, the document describes the inclusion of sorption materials for absorbing and desorbing liquid to allow for the adjustment of a defined liquid concentration.

[0017] However, such solutions known from the prior art may still have potential for improvement, especially with regard to effective and safe measurement with biosensors in very small sample volumes.

[0018] It is therefore an object of the present invention to provide a measure by which at least one disadvantage of the prior art can be at least partially overcome. In particular, it is an object of the present invention to provide a solution that enables effective and reliable measurement with biosensors even with very small sample volumes.

[0019] According to the invention, the problem is solved by a biosensor having the features of claim 1. The problem is further solved according to the invention by a method having the features of claim 6, by a method having the features of claim 8, and by use having the features of claim 10. Preferred embodiments of the invention are disclosed in the dependent claims, in the description, and in the figures, wherein further features described or shown in the dependent claims, in the description, in the figures, or in the example may, individually or in any combination, constitute subject matter of the invention unless the context clearly indicates otherwise.

[0020] The present invention relates to an electrochemical biosensor for the detection of at least one analyte dissolved in an analyte solvent, comprising a capillary detection chamber and a plurality of electrodes, wherein the plurality of electrodes comprises at least one working electrode having a measuring area positioned in the capillary detection chamber and provided with an immobile detection agent for interacting with the analyte, and wherein the plurality of electrodes further comprises a counter electrode extending into the capillary detection chamber, and wherein the plurality of electrodes are electrically contactable outside the capillary detection chamber, wherein the capillary detection chamber has a volume in the range of ≤ 10 µl, and wherein an oxygen-binding or oxygen-reactive inerting agent is further provided in the capillary detection chamber.which is positioned at least partially between the working electrode and an inlet opening of the capillary detection chamber, and wherein the length of the capillary detection chamber between the working electrode and the inlet opening and the type and quantity of the inerting agent are selected and adapted to one another such that oxygen diffusing from the inlet opening towards the working electrode in the analyte solvent arranged in the detection chamber can be completely removed from the analyte solvent by the inerting agent before reaching the working electrode, and the inerting agent is at least partially soluble in the analyte solvent, so that when the analyte solvent flows in, the inerting agent is immediately at least partially, and in particular completely, soluble.

[0021] Such a biosensor can easily enable reliable and, in particular, oxygen-independent detection of an analyte, where the analyte is dissolved in an analyte solvent. Detection can be either qualitative, quantitative, or both.

[0022] To enable this, the biosensor is designed to have a capillary detection chamber. A capillary detection chamber is defined as a space or volume in which the analyte is detected and which is designed as a capillary. A capillary is defined as a space with a comparatively small volume that allows for capillary action. In the biosensor described here, this volume is particularly small, in the range of ≤ 10 µl, for example, ≤ 5 µl.

[0023] Furthermore, the biosensor has a plurality of electrodes, each of which includes at least one working electrode. This working electrode has a measuring area positioned in the capillary detection chamber and is equipped with an immobile detection agent for interaction with the analyte. In other words, the working electrode, known per se for electrochemical biosensors, is equipped with a detection agent that is immobile and thus remains in contact with the analyte or the analyte solvent at the desired position. The detection agent should be, in a manner known per se, a reagent that interacts with the analyte, for example, reacts with it, such that the reaction with the analyte, and thus the analyte itself, can be detected using the working electrode.

[0024] Accordingly, most electrodes also have a counter electrode that extends into the capillary detection space and thus also comes into contact with the analyte solvent.

[0025] Furthermore, a so-called reference electrode can be provided, creating a three-electrode arrangement. Such an arrangement can be chosen, in particular, to minimize the influence of current-dependent processes at the counter electrode during an exemplary amperometric measurement. This three-electrode arrangement thus comprises a working electrode, a counter electrode, and a reference electrode. The reaction or interaction of analytical interest takes place at the working electrode, with the potential being monitored by a high-resistance reference electrode. The low-resistance counter electrode serves only as a current contact. Any deviations from the preselected setpoint of the working voltage can thus be automatically corrected by a potentiostat.

[0026] Accordingly, if the working electrode, the counter electrode, and any other electrodes located outside the capillary detection space are electrically contactable, electrochemical detection of the analyte can be performed using amperometric measurements. For this purpose, the current flow can be measured using an ammeter that is ideally very sensitive, with the current being proportional to the analyte concentration. This functionality is fundamentally known for electrochemical biosensors. However, various electrochemical methods, such as coulometry, amperometry, voltammetry, or potentiometry, can be used to determine the analyte.

[0027] The biosensor described here further provides for an oxygen-binding or oxygen-reactive inerting agent in the capillary detection chamber, which is positioned at least partially between the working electrode and an inlet opening of the capillary detection chamber. Such an inerting agent removes oxygen present in the analyte solution. For this purpose, the oxygen can, for example, bind to the inerting agent or react with it. An inlet opening is also defined as an opening in the detection chamber through which the analyte solution to be analyzed enters the detection chamber.

[0028] Furthermore, it is provided that the length of the capillary detection space between the working electrode and the inlet opening and the inerting agent are selected and adapted to each other in such a way that oxygen diffusing from the inlet opening towards the working electrode in the analyte solvent can be completely removed from the analyte solvent by the inerting agent before reaching the detection agent.

[0029] In other words, the system does not simply involve placing a freely selectable inerting agent in a freely selectable quantity into a detection chamber of a freely selectable size. Instead, a multitude of parameters are specifically adapted to one another to ensure that no oxygen can pass from the inlet opening to the working electrode and the inerting agent via diffusion. The adaptable parameters include, in particular, the length of the capillary detection chamber between the working electrode and the inlet opening, and the inerting agent itself. Adapting the inerting agent refers to both its type and quantity.

[0030] Such adjustments to the respective parameters, in order to optimize oxygen removal and the distance of the electrode to the inlet or capillary opening, can be carried out experimentally, for example through trials. Furthermore, this can be achieved through computer simulation, which provides information about the precise dimensions and the concentrations of the inerting agent, such as an enzyme, to be used. This enables optimized and therefore cost-effective design of a biosensor and thus improved manufacturability.

[0031] Regarding the oxygen concentration in the analyte solvent, this can be taken into consideration, although its influence is rather small. Under normal ambient conditions, the oxygen concentration should fluctuate. One can assume a value of approximately 8 mg / L, which corresponds roughly to a concentration at 25°C. The operating range of a sensor is typically between 10°C and 37°C. Within this range, the concentration of dissolved oxygen is approximately 11.3 to 6.7 mg / L at standard pressure (1013 hPa). These values ​​have little impact on the performance of the inerting agent.

[0032] In particular, the design described above can offer significant advantages over solutions from the prior art.

[0033] The biosensor described above can address the problem that electrochemical biosensors are affected by dissolved oxygen and additional dissolution processes from the ambient air. Specifically, in electrochemical biosensors, the presence of dissolved oxygen leads to a short circuit in the electron transfer process from the analyte to the electrode. This results in significant inaccuracies at low analyte concentrations. Therefore, particularly in small volumes of the detection chamber or analyte solvent, the detection medium can be protected from oxygen exposure.

[0034] For large volumes, oxygen diffusion to the electrode is limited by the large distances between the solution surface and the electrode; therefore, oxygen can be removed relatively easily before interference can occur. However, for small volumes, it has been found that the distance from the inlet opening to the working electrode or the detection medium is so small that diffusion processes become significant. Therefore, the biosensor described here is particularly relevant and advantageous for small sample volumes. For effective measurement and to achieve the aforementioned advantages, it can be particularly advantageous if the volume of the capillary detection chamber is in a range of ≤ 10 µl, approximately ≤ 5 µl, for example in a range of ≥ 0.5 µl to ≤ 10 µl, approximately in a range of ≥ 1 µl to ≤ 5 µl, approximately in a range of ≥ 1.5 µl to ≤ 3 µl, and particularly preferably in a range of ≥ 0.3 µl to ≤ 0.5 µl.

[0035] Therefore, the present invention is of particular relevance for biosensors, since biosensors are characterized on the one hand by a very specific detection of the analyte and on the other hand by a particularly high sensitivity and a very low detection limit for the analyte, and are therefore particularly suitable for small volumes.

[0036] A biosensor is proposed that can remove dissolved oxygen from a system for determining an analyte with a small analyte volume in a particularly safe and effective manner. It has been found that the design and construction of the biosensor, for example as a test strip, allows for a corresponding methodology, whereby the influence of oxygen can be significantly reduced or even completely eliminated.

[0037] In summary, the biosensor described here is designed so that the dimensions of the test strip and the biosensor itself are chosen such that the oxygen can be completely removed by the interfering agent before it reaches the working electrode or the detection agent through diffusion. This prevents oxygen from negatively affecting the analysis or the biosensor's methodology.

[0038] Following the above, a particularly safe and reliable measurement or detection can thus be permitted, whereby in particular an oxygen-containing environment may have no or only a substantially negligible influence on the measurement result.

[0039] In this way, corresponding biosensors can be produced simply and particularly cost-effectively, since the manufacturing principle can be easily adapted from known biosensors by selecting the appropriate parameters, as described above.

[0040] Regarding applicability, improved biosensors can be produced in an unlimited manner for applications such as glucose determination in blood and other aqueous solutions. Furthermore, biosensors can be developed for new fields of application, such as nitrate determination, without being limited to the examples above.

[0041] Preferably, the distance between the working electrode and the inlet opening, and in particular the nearest inlet opening when multiple inlet openings are provided, can be in the range of ≥ 2 mm to ≤ 15 mm, preferably in the range of ≥ 3 mm to ≤ 8 mm. It has been found that, particularly in this configuration, the inerting agent, and especially the quantity and type or activity of oxygen binding or the oxygen reaction, can be advantageously configured. In other words, it can be ensured that oxygen cannot reach the working electrode through diffusion processes. This applies particularly to the previously described volumes of the capillary detection chamber in the range of ≥ 0.5 µl to ≤ 10 µl.

[0042] It is further evident from the above that this configuration, in particular, can achieve a particularly high level of oxygen removal efficiency. Even if oxygen begins to diffuse through the analyte solvent, interaction with the solvent can occur at any point. Therefore, the oxygen can be removed very effectively, or a comparatively small amount of inerting agent is sufficient to remove the oxygen. This allows, for example, a conventionally sized sensor with little inerting agent or a sensor with a particularly compact design, both of which can be advantageous in specific applications.

[0043] Furthermore, it can be advantageous for the inerting agent to be enzyme-based. Such inerting agents, in particular, can effectively remove oxygen from the analyte solvent, thereby reducing the influence of an oxygen environment on the biosensor. Moreover, in relevant applications, these systems can leave the actual measurement unaffected, thus combining effective oxygen removal with reliable measurement results.

[0044] An example of such enzyme systems that can be used as inerting agents includes pyranose 2-oxidase (P2Ox) with a catalase (Pox CAT), but is not limited to this.

[0045] Furthermore, it may also be preferred that the detection agent be an enzyme-based detection agent. The selection of the detection agent is fundamentally, and also in this application, dependent on the desired application in a manner apparent to those skilled in the art. For the exemplary case of a glucose sensor, an enzyme-based detection agent may, for example, comprise glucose dehydrogenase (GDH) or glucose oxidase (Gox), but is not limited to these.

[0046] The sensor enzymes can be, for example, both reductases and oxidases, regardless of whether the sensor enzyme is used as an inerting agent or as a detection agent.

[0047] The present invention relates, in summary, to an electrochemical biosensor, designed, for example, as a test strip, for various analytes, wherein the detection agents used, such as sensor enzymes, can also be oxygen-sensitive or oxygen itself interferes in the potential range of the analyte. The oxygen dissolved in the sample is effectively removed, so that an analyte can be determined in an aqueous solution such as whole blood, plant juice, beverages, or other solutions without the need to protect the sample from atmospheric oxygen. This allows for significant advantages in applicability and in the potential field of application.

[0048] Regarding further advantages and technical features of the biosensor, reference is made hereto to the description of the system, the method for manufacturing a biosensor, the method for detecting an analyte and its use, as well as to the figures and the description of the figures, and vice versa.

[0049] Furthermore, a system not belonging to the invention is described for the qualitative or quantitative detection of at least one analyte dissolved in an analyte solvent, comprising a biosensor as described above in detail, and further comprising an analyte solvent arranged in the capillary detection chamber, wherein at least one analyte to be detected is provided in the analyte solvent.

[0050] The system described here, which is not part of the invention, therefore initially comprises a biosensor, with reference to the preceding descriptions regarding the biosensor. It is further provided that an analyte solvent is provided in the capillary detection chamber, wherein at least one analyte to be detected is provided in the analyte solvent.

[0051] Such a system thus enables the reliable detection of the analyte without oxygen diffusing through the analyte solvent negatively affecting or falsifying the detection, for example, in an amperometric measurement. The analyte solvent and / or the analyte itself can be selected in a manner understandable to those skilled in the art with regard to the specific application. The same applies to the detection agent, as described in detail above.

[0052] According to the invention, the inerting agent is at least partially soluble or dissolved in the analyte solvent. It is particularly preferred that the inerting agent is completely soluble or dissolved in the analyte solvent. In this configuration, a particularly safe and effective removal of oxygen present in the analyte solvent can be achieved.

[0053] Firstly, this ensures that oxygen diffusing through the analyte solvent comes into contact with the inerting agent, thus guaranteeing an interaction between oxygen and inerting agent.

[0054] Furthermore, this design allows for particularly simple manufacturing of the biosensor. This is because the inerting agent can be easily and locally positioned at any desired location within the detection space, especially between the working electrode and the output, without requiring highly precise positioning over a defined area. This allows for positioning of the inerting agent with a comparatively high tolerance. Since the inerting agent is then dissolved in the analyte solvent, it distributes itself naturally through dissolution and subsequent mixing, ensuring that the inerting agent is present throughout the entire detection space.

[0055] Thus, particularly in this configuration, effective oxygen removal can be achieved in a simple way.

[0056] It may further be preferred that the system also includes an evaluation unit which electrically contacts the majority of electrodes and through which, based on information supplied by the electrodes, at least one aspect of the quantity and type of analyte can be determined. Such an evaluation unit can thus electrically contact the electrodes. For this purpose, it may be provided, for example, that the sensor can be inserted into a receiving area of ​​the evaluation unit, so that, at a defined position of the biosensor in the evaluation unit, the electrodes are contacted and the quantity and / or type of analyte can be determined, or in other words, a measurement result can be generated. The information supplied by the electrodes can, for example, be understood to be a current or a voltage, which can be detected by appropriate measurement methods.

[0057] If the evaluation unit is designed, for example, for amperometric measurement or for carrying out other measurement procedures as described above, an evaluation can be carried out simply by inserting the biosensor, designed as a test strip, into the evaluation unit, so that the presence of the analyte can be indicated quantitatively and / or qualitatively.

[0058] Regarding further advantages and technical features of the system, reference is made hereto to the description of the biosensor, the method for manufacturing a biosensor, the method for detecting an analyte and its use, as well as to the figures and the description of the figures, and vice versa.

[0059] Furthermore, a method for detecting at least one analyte dissolved in a solvent is described, comprising the following process steps: a) Providing a biosensor as described in detail above; b) Filling the capillary detection chamber with analytes dissolved in the analyte solvent, wherein c) during filling of the capillary detection chamber the analyte solvent is guided together with the analyte from the inlet opening to the detection medium; d) Externally contacting the at least two electrodes; and e) Determining at least one piece of information supplied by the electrodes regarding the quantity and type of analyte.

[0060] The method described here is therefore used to detect at least one analyte dissolved in a solvent. This detection can include qualitative detection, quantitative detection, or a combination of both.

[0061] To make this possible, the procedure includes the following procedural steps.

[0062] First, according to procedure step a), a biosensor is provided, as described in detail above. Therefore, reference is made to the preceding explanations regarding the respective characteristics of the biosensor.

[0063] In step b), the capillary detection chamber is then filled with analytes dissolved in the analyte solvent. In other words, the analyte solvent, along with the dissolved analyte, is introduced or poured into the detection chamber. This can be achieved, for example, solely through capillary action within the capillary detection chamber. The biosensor, with its inlet opening, is immersed in the analyte solvent, thus drawing the solvent into the solution to be analyzed, and filling the capillary detection chamber almost completely.

[0064] In particular, step b) of the procedure should be carried out in such a way that the analyte dissolved in the analyte solvent is present at the detection medium and the counter electrode in order to perform the corresponding detection. This is generally unproblematic as long as the detection chamber is completely filled with analyte solvent, as described above.

[0065] According to process step c), it is further provided that, when filling the capillary detection chamber, the analyte solvent is guided together with the analyte from the inlet opening to the detection medium. In particular, it may be provided that the analyte solvent is guided along or flows alongside the inerting medium and thus comes into contact with the inerting medium when the detection chamber is filled.

[0066] This can help to remove oxygen from the analyte solvent, for example through a reaction, or to immobilize it. Furthermore, the inerting agent is soluble in the analyte solvent, so that when the analyte solvent flows in, the inerting agent can be dissolved immediately, at least partially, and in particular completely. The latter allows for a particularly effective reduction of oxygen exposure and also improves the manufacturability of the biosensor.

[0067] Finally, according to process steps d) and e), the at least two electrodes are externally contacted; and furthermore, at least one of the quantity and type of analyte is determined from the information supplied by the electrodes, thus enabling a qualitative and / or quantitative determination of the analyte. In these steps, for example, contacting the electrodes, and in particular at least the working electrode and the counter electrode, makes evaluation possible. This can be achieved using fundamentally different electrochemical methods such as coulometry, amperometry, voltammetry, or potentiometry, so that the information from the electrodes is, in particular, electrical or electrochemical information, such as current, voltage, etc.

[0068] Furthermore, this process step can be achieved, for example, by inserting the biosensor into an evaluation unit, as described in more detail above.

[0069] The method described here thus allows for effective and reliable quantitative and / or qualitative detection of the analyte dissolved in the analyte solvent. In particular, it prevents interference from oxygen diffusing into the detection medium.

[0070] In accordance with the foregoing, it may be particularly preferred that oxygen diffusing from the inlet opening towards the detection agent in the analyte solvent is completely removed from the analyte solvent by the inerting agent.

[0071] In this configuration in particular, effective detection can be enabled, which is independent of the oxygen surrounding the biosensor.

[0072] Regarding further advantages and technical features of the method for detecting an analyte, reference is hereby made to the description of the system, the biosensor, the method for manufacturing a biosensor, and its use, as well as to the figures and the description of the figures, and vice versa.

[0073] Furthermore, a method for producing a biosensor for detecting at least one analyte dissolved in an analyte solvent, in particular a biosensor as described above in detail, is described, wherein the method comprises the following process steps: i) Providing a base body with a capillary detection chamber, wherein the detection chamber has a volume in the range of ≤ 10 µl; ii) Arranging a plurality of electrodes such that they are externally electrically contactable and extend into the detection chamber; iii) Arranging a detection agent on an electrode serving as a working electrode;and iv) arranging an inerting agent in the detection chamber, in particular between an inlet opening of the detection chamber and the electrode serving as the working electrode, wherein v) the length of the capillary detection chamber between the electrode serving as the working electrode and the inlet opening and the inerting agent are selected and adapted to each other in such a way that oxygen diffusing from the inlet opening towards the electrode serving as the working electrode in the analyte solvent can be completely removed from the analyte solvent by the inerting agent before reaching the detection agent.

[0074] The process steps described above may be carried out in the sequence described above or in a different sequence. Furthermore, the individual process steps may each be single-step or comprise a plurality of sub-steps, whereby the sub-steps of different process steps may overlap in time, or where one or more sub-steps of a process step may at least partially occur between sub-steps of another process step without departing from the scope of the invention.

[0075] Furthermore, with regard to the individual features, reference is made to the description and the corresponding properties and designs as they are described elsewhere, for example in the description of the biosensor.

[0076] The process for manufacturing the biosensor comprises the following steps. First, according to step i), a base body with a capillary detection chamber is provided, the detection chamber having a volume of ≤ 10 µl, for example, ≤ 5 µl. In this step, the basic structure of the biosensor is thus formed, which can support the functional components described below.

[0077] In principle, the basic body can be formed from a single part, or it can be constructed from multiple individual parts. For example, the following process steps may be used to form the basic body: vi) Providing a sensor cover; vii) Applying two lateral boundaries to the sensor cover such that the lateral boundaries define a space between the lateral boundaries forming the detection space, and that the lateral boundaries form an inlet opening on at least one side; ix) Providing a sensor base; and xii) Fixing the sensor base to the side parts.

[0078] Care should be taken to ensure that any previously applied electrodes or detection medium are present within the detection space. This can be easily achieved by appropriately dimensioning the individual components or by correctly positioning the electrodes and detection medium.

[0079] Furthermore, the basic body, including the sensor cover, the sensor base, and the side boundaries, can be made of paper, cardboard, plastic, or other materials. The sensor cover, the sensor base, and the side boundaries can also be plate-like. With the design described here, the capillary detection space can be easily created by joining the sensor base, side panels, and sensor cover together, for example, by gluing.

[0080] Furthermore, in the method described here, according to process step ii), a plurality of electrodes are arranged such that they are electrically contactable externally and extend into the detection space. Thus, the electrodes have a region that is arranged such that it is positioned in the detection space when a sensor is generated, and furthermore, they have a region that is externally electrically contactable.

[0081] Only one working electrode and one counter electrode can be applied, or a reference electrode can be added, as described above. Furthermore, the electrodes can be applied, for example, by means of a printing process, such as screen printing, or by creating a structured, electrically conductive layer, such as a layer of carbon or a metal like gold.

[0082] For example, the electrodes can be applied to the sensor cover according to process step x) before it is fixed to the side panels or the lateral boundaries.

[0083] In addition, in the method described here, a detection means is arranged on an electrode serving as a working electrode according to process steps iii) and xi). This step can, for example, take place after the electrode has been applied, as described above, and / or before the sensor base is attached to the lateral boundaries in the described embodiment.

[0084] In addition to the application of the detection agent described above, the method further comprises, according to process step iv), the arrangement of an inerting agent in the detection chamber, in particular between an inlet opening of the detection chamber and the detection agent. The inerting agent can be selected, as described in detail above, and can, in principle, be applied by a selectable method. For example, it can be applied by a conventional coating process.

[0085] For example, in the embodiment of the manufacture of the base body described above in process step viii), the inerting agent may be applied after the lateral boundaries have been applied to the sensor cover and before the sensor base is fixed to the lateral boundaries and thus placed in the space between. The inerting agent can then also be applied to the sensor cover.

[0086] Based on the above, it may therefore be the case that the process for manufacturing the biosensor comprises the following process steps: vi) Providing a sensor cover; vii) Applying two lateral boundaries to the sensor cover such that the lateral boundaries define a space between the lateral boundaries forming the detection space, and that the lateral boundaries form an inlet opening on at least one side; viii) Applying an inerting agent to the space; ix) Providing a sensor base; x) Applying at least two electrodes to the sensor base; xi) Applying a detection agent to an electrode to be used as a working electrode; and xii) Fixing the sensor base to the side parts such that the electrodes extend into the detection space formed by the space between the lateral boundaries and the detection agent is present in the detection space.

[0087] In principle, according to process step v), it is further stipulated that the length of the capillary detection space between the electrode serving as the working electrode and the inlet opening and the inerting agent are selected and adapted to each other in such a way that oxygen diffusing from the inlet opening towards the electrode serving as the working electrode in the analyte solvent can be completely removed from the analyte solvent by the inerting agent before reaching the electrode serving as the working electrode.

[0088] This feature, as described in greater detail above with reference to the sensor, prevents oxygen diffusing through the analyte solvent present in the detection chamber from reaching the working electrode. This allows for a reliable and effective measurement that is also highly independent of the presence of oxygen outside the sensor.

[0089] Regarding further advantages and technical features of the method for producing a biosensor, reference is hereby made to the description of the system, the biosensor, the method for detecting an analyte, and its use, as well as to the figures and the description of the figures, and vice versa.

[0090] The use of a sensor for detecting at least one analyte dissolved in a solvent is also described. Detection can be qualitative, quantitative, or both qualitative and quantitative.

[0091] This use enables safe and effective detection of the analyte, which is also very independent of the presence of oxygen outside the sensor.

[0092] For example, at least one of a sensor, system or method, as described in detail above, is used for glucose determination or nitrate determination.

[0093] Regarding further advantages and technical features of the use, reference is hereby made to the description of the system, the biosensor, the method for detecting an analyte, and the method for manufacturing a biosensor, as well as to the figures and the description of the figures, and vice versa.

[0094] The invention is explained below by way of example with reference to the accompanying drawings and examples, wherein the features shown below can represent an aspect of the invention both individually and in combination, and wherein the invention is not limited to the following drawing, the following description and the following embodiment.

[0095] They show: Fig. 1 a schematic view of a biosensor and its manufacture; Fig. 2 a schematic, partially transparent view in various forms to visualize the dimensions of the biosensor; Fig. 3 Various cross-sections through the detection space of the biosensor to describe oxygen removal by an enzyme system; Fig. 4 Diagrams depicting a simulation of oxygen removal by an enzyme system; Fig. 5 another diagram depicting a simulation of oxygen removal by an enzyme system Fig. 6 a diagram illustrating the behavior of a biosensor without an inerting agent; Fig. 7 Diagrams showing the measurement behavior of a biosensor at different analyte concentrations; Fig. 8 another diagram showing the measurement behavior of a biosensor at different analyte concentrations; and Fig. 9Another diagram showing the measurement behavior of a biosensor.

[0096] In the Figure 1 A biosensor 10 and a method for manufacturing one are described.

[0097] In particular, an electrochemical biosensor 10 is generated for the detection of at least one analyte dissolved in an analyte solvent. Such a biosensor 10 comprises a capillary detection chamber 12 having a volume in the range of ≤ 10 µl, for example ≤ 5 µl, preferably in the range of ≥ 1.5 µl to ≤ 3 µl. Furthermore, the biosensor 10 comprises a plurality of electrodes, namely a working electrode 14, a counter electrode 16, and a reference electrode 18. The working electrode 14 has a measuring area 20 positioned in the capillary detection chamber 12, which is provided with an immobile detection agent 22 for interaction with the analyte. Furthermore, all electrodes 14, 16, and 18 extend into the detection chamber 12 and are electrically contactable from the outside. For this purpose, the biosensor has a contact area 24 in which the electrodes 14, 16, 18 are exposed.For example, the contact area 24 can be inserted into an evaluation unit to enable evaluation of the detection or measurement.

[0098] It is further provided that an oxygen-binding or oxygen-reactive inerting agent 26 is provided in the capillary detection chamber 12, which is positioned at least partially between the immobile detection agent 22 or the working electrode 14 and an inlet opening 28 of the capillary detection chamber 12.

[0099] For effective and, in particular, oxygen-independent detection, the length of the capillary detection chamber 12 between the working electrode 14 and the inlet opening 28 and the inerting agent 26 are selected and adapted to each other such that any oxygen diffusing from the inlet opening 28 towards the working electrode 14 in the analyte solvent present in the detection chamber 12 is completely removed from the analyte solvent by the inerting agent 26 before reaching the working electrode 14. For example, as shown below, the distance between the working electrode 14 and the inlet opening 28 may be in the range of ≥ 2 mm to ≤ 15 mm.

[0100] Such a sensor is particularly useful in the Figure 1The final product of the process shown there is presented as stage VIII. Such a design can be described as a test strip, which is particularly flat in comparison to its width and has a multi-layered structure.

[0101] The process for manufacturing such a biosensor 10 can proceed approximately as follows, as described in the Figure 1 as shown. Basically, according to Figure 1 A first part is created as stage IV and a second part as stage VII, which are then combined to form the biosensor 10 as stage VIII.

[0102] To produce stage IV, a sensor cover 30 and two lateral boundaries 32, 34 are first provided as stage I. Then, to create stage II, the lateral boundaries 32, 34 are applied to the sensor cover 30 and fixed there, for example by gluing, such that the lateral boundaries 32, 34 define an intermediate space 36 between the lateral boundaries 32, 34 forming the detection space 12, wherein the lateral boundaries 32, 34 form the entrance opening 28 on at least one side.

[0103] The inerting agent 26 is then applied to the space 36 to create stage IV. As an intermediate stage III, depending on the application method (e.g., coating), the inerting agent 26 may initially be moist and, after drying, converted into the dried inerting agent 26. As described above, the inerting agent 26 serves primarily to remove oxygen. For example, as shown in arrow 38, pyranose 2-oxidase (P2Ox) can be applied with a catalase (Pox CAT), for instance, by dropcasting. In this example, the enzymes are embedded in a polyvinyl alcohol matrix (5 mg / ml PVA, 25 mM phosphate, 20 µM EDTA, pH 7.3).

[0104] According to arrow 40, an additional reagent 42, in this case glucose, can be applied, which can improve or enable the removal of oxygen as described below. This configuration is advantageous, for example, for nitrate sensors as biosensors 10. Glucose can be applied as reagent 42 at a concentration of 9 mg / ml in ultrapure water. The glucose should be applied separately to prevent the oxidase from immediately converting the substrate. After these process steps, stage IV can be completed.

[0105] In principle, it can be intended that the reagent 42 is considered part of the inerting agent 26, so that in principle, when an inerting agent 26 is mentioned, this can be a substance or, for example, in addition to an enzyme, it can have a substrate or reagent 42.

[0106] To form stage VII, a sensor base 44 can first be provided. A plurality of electrodes 14, 16, 18 can be applied to this base, configured as working electrode 14, counter electrode 16, and reference electrode 18, respectively. Application can be achieved, for example, by printing or other coating processes. Furthermore, the electrodes 14, 16, 18 can be made of a metal or an electrically conductive carbon material. This can generate stage V.

[0107] The detection agent 22 is then applied to the working electrode 14 according to arrow 45 to generate stage VII. Depending on the application method, such as coating, the detection agent 22 may initially be moist as an intermediate stage VI and, after drying, be transferred to the dried inerting agent 22 according to stage VII. Specifically, a nitrate reductase (NaR), embedded in a viologen-polyvinyl alcohol polymer, can be applied as the detection agent 22 to a working electrode 14 designed as a screen-printed electrode by dropcasting.

[0108] Subsequently, stages IV and VII can be connected such that the side of the sensor base 44 equipped with electrodes 14, 16, 18 faces the lateral boundaries 32, 34 and the space 36, respectively. Specifically, the sensor base 44 is fixed to the lateral boundaries 32, 34, for example by gluing, such that the electrodes 14, 16, 18 extend into the detection space 12 formed by the space 36, and the detection medium 22 is located within the detection space 12. Furthermore, a contact area 24 is provided, at which the electrodes 14, 16, 18 can be externally connected, for example by an evaluation unit. This can be achieved by making the sensor cover 30 shorter than the sensor base 44.

[0109] Furthermore, it is provided that the length of the capillary detection chamber 12 between the working electrode 14 and the inlet opening 28 and the inerting agent 26 are selected and adapted to each other in such a way that oxygen diffusing from the inlet opening 28 towards the working electrode 14 in an analyte solvent present in the detection chamber 12 can be completely removed from the analyte solvent by the inerting agent 26 before reaching the working electrode 14.

[0110] Exemplary, but in no way limiting, dimensions of such a biosensor 10 are shown in the Figure 2 shown for clarification, whereby Figure 2a ) the top view of stage V shows, where Figure 2b ) a transparent top view of part of stage VIII, namely without electrodes 14, 16, 18, without inerting agent 26, without reagent 42 and detection agent 22, i.e. only of the basic body of the biosensor 10, and wherein Figure 2C A partially transparent view of level VIII is shown for clarification.

[0111] In detail, in Figure 2a It is shown that the length of the sensor base 44, shown as length 46, is 20 mm, and that the width of the sensor base 44, shown as width 48, is 5 mm. Furthermore, the width, shown as width 50, is 1 mm for the reference electrode 18 as an example. The working electrode 14 can also have a width of 3.5 mm.

[0112] In Figure 2b It has been shown that the capillary detection chamber 12 has a diameter or width 52 of 1 mm or 2 mm. Such configurations can be particularly suitable for creating a detection chamber 12 with a volume in the range of ≤ 10 µl, for example, ≤ 5 µl.

[0113] The dimensions of the detection chamber 12 are crucial for the effective removal of dissolved oxygen from the vicinity of the working electrode 14. Based on the activity of the inerting agent 26, such as its enzyme activity, and the diffusion coefficient for oxygen in the analyte solvent, such as aqueous solutions, a model can be created to estimate the time required and determine the distance between the working electrode 14 and the inlet opening 28. To achieve effective oxygen removal, the working electrode 14 can be located approximately 5 mm from the inlet opening 28. This results, for example, in a minimum length of 15 mm for the detection chamber 12. The detection chamber 12 can have a diameter of 1–2 mm and a height of 100 µm. With a length of 15 mm, this results in a total volume of 1.5 or 3.0 µl.

[0114] In the Figure 3A cross-section through the detection chamber 12 is shown, in particular the inerting agent 26 and glucose as an additional reagent 42. The oxygen removal of the inerting agent 26 when the biosensor 10 is used as a nitrate sensor is shown.

[0115] The analyte (nitrate) is drawn into the detection chamber 12 along with the analyte solvent by capillary action, as shown in Figure 1a). However, the sample is in constant exchange with the ambient air, so atmospheric oxygen continuously enters the sample and must be removed. The dissolved oxygen is removed via a coupled enzymatic reaction, as described below.

[0116] Reagent 42 and inerting agent 26 are soluble in the analyte solvent and are dissolved by filling the detection chamber 12. In this process, reagent 42 and inerting agent 26 are combined, and the enzyme system (P2OxCAT), which in this configuration serves as inerting agent 26, can begin removing the dissolved oxygen by reacting glucose (reagent 42) with oxygen, as described in the Figure 3b ) is shown. Oxygen that dissolves from the ambient air is completely removed by the inerting agent 26 in conjunction with its reagent 42. The detection agent 22 is immobilized on the working electrode 14. Due to the anaerobic conditions, an interference-free measurement of the analyte can be carried out, as shown in the Figure 3c ) is shown. 3D figureFigure 54 further shows the oxygen content in the analyte solvent in µM on the Y-axis relative to the distance to the inlet opening 28, which is shown in mm on the X-axis. It is shown that from a distance 54 of approximately 1.5 mm from the inlet opening 28, all oxygen is removed from the analyte solvent, thus creating anaerobic conditions for measurement.

[0117] The following examples are shown to demonstrate the effective action of a biosensor 10 designed as described above.

[0118] Before the first experiments were performed, simulations were used to determine the minimum concentrations of the enzyme system as inerting agent 26 for oxygen removal, in order to achieve effective oxygen removal after ≤ 10 seconds with given electrode dimensions 12, 14, 16 as described above. The simulation was performed based on enzyme kinetic data of the enzyme pyranose oxidase (Rungsrisuriyachai, ABB, 2008; https: / / doi.org / 10.1016 / j.abb.2008.12.018). Furthermore, the following values ​​were assumed for the diffusion of oxygen in water as the analyte solvent (2.4 × 10⁻⁵ cm² / s, Fourmond, JACS, 2015; DOI 10.1021 / jacs.5b01194). To ensure full saturation of the inerting agent 26 with its substrate or reagent 42, the concentration of glucose was set to 10 mmol / l as the concentration of reagent 42.

[0119] The concentration of pyranose oxidase required for complete oxygen removal in ≤ 10 seconds was determined to be 10 µmol / l. The simulation is approximately in the Figure 4 shown, where the x-axes represent the distance of the respective position in the detection chamber 12 to the inlet opening 28 and the y-axis indicates a dimensionless concentration, and where diagram a) shows the starting point of the measurement, diagram b) a time after 10 seconds, and diagram c) a time after 300 seconds. Furthermore, the dashed lines describe the position of electrodes, with the left line showing the position of the reference electrode 18 and the right line the working electrode 14. The counter electrode can be located directly at the inlet opening, as is the case, for example, with the Figures 1 to 3This can be seen from the diagrams. Line A further describes the concentration of oxygen and line B describes the concentration of glucose as reagent 42. The diagrams according to Figure 4 This shows that after oxygen removal, the solution remains oxygen-free for at least 300 seconds.

[0120] Assuming constant oxygen diffusion and constant enzyme activity as inerting agent 26, the substrate or reagent 42 (glucose) at the concentration of 10 mmol / l is sufficient to theoretically keep the solution oxygen-free for ≤1200 minutes. This is in the Figure 5This shows a diagram where the x-axis represents the distance to the inlet opening 28 and the y-axis represents a dimensionless concentration, and where the diagram further shows a time after 1440 minutes. The dashed lines also describe the position of electrodes, with the left line showing the position of the reference electrode 18 and the right line the working electrode 14. The counter electrode can be located directly at the inlet opening 28, as is the case, for example, with the Figures 1 to 3 This can be seen from the graph. Line A describes the concentration of oxygen and line B describes the concentration of glucose as reagent 42.

[0121] The increase in oxygen concentration on the right side of the diagrams of the Figures 4 and 5This can be explained by the fact that the detection chamber 12 in this configuration was open on both sides, the axis thus strictly speaking shows the distance to an entrance opening 28, but another is located at 15mm, so the detection chamber has a length of 15mm, as also described above.

[0122] In an initial experiment, the liquid to be tested was drawn up by capillary action, and the detection chamber 12 was thus completely filled with liquid, i.e., analyte solvent with the analyte dissolved therein. For the example experiments shown in the following figures, a biosensor 10 was used for nitrate detection. Nitrate reductase was used as the sensor enzyme. An enzyme system comprising pyranose oxidase and catalase was used to remove oxygen (as described in US 9,187,779 B2).

[0123] A gold electrode was used for the working electrode 14, although other materials, such as glassy carbon, are also conceivable for the working electrode 14 or the other electrodes 16 and 18. A silver / silver chloride system was used as the reference electrode 18.

[0124] In the first experiment, the screen-printed electrodes 14, 16, 18 were tested without the inerting agent 26 for oxygen removal. This is in the Figure 6 shown, whereby this attempt is shown by the curves A of the in Figure 6 as shown in the diagram. In the diagram in Figure 6The current in µA is shown on the Y-axis, and the voltage in volts across the electrode against Ag / AgCl paste as a reference electrode 18 is shown on the X-axis. This experiment clearly demonstrates the influence of oxygen on the measurement signal in the range of 0.8 V to 0.3 V. Curves B and C, on the other hand, show the removal of oxygen by glucose oxidase (curve B) and by pyranose oxidase (curve C), respectively, as inerting agents 26. A closed test strip or biosensor 10 was used for oxygen removal by pyranose oxidase. The other two experiments were performed with open biosensors 10, i.e., without sensor caps. A reaction buffer of 25 mM phosphate and 20 µM EDTA at pH 7.3 was used. The enzymes for oxygen removal were each used at 1 mg / ml glucose oxidase, pyranose oxidase, and catalase. Glucose 9 mg / ml was used as substrate or reagent 42.It has been shown that the influence of oxygen can be essentially eliminated by the inerting agent 26, possibly again with its reagent 42.

[0125] To demonstrate the effective operation of a biosensor 10, for example designed as a test strip, a concentration series with different concentrations of potassium nitrate (0.5 to 16 mM) in the solution was measured on an open system, i.e. without sensor cover 30, as described in the Figures 7 and 8As shown in diagrams a) and b), curve A shows a concentration of 0 mM, curve B shows a concentration of 0.05 mM, curve C shows a concentration of 0.1 mM, curve D shows a concentration of 0.25 mM, curve E shows a concentration of 0.5 mM, curve F shows a concentration of 0.75 mM, curve G shows a concentration of 1 mM, curve H shows a concentration of 2 mM, curve I shows a concentration of 4 mM, curve J shows a concentration of 8 mM, and curve K shows a concentration of 16 mM.

[0126] The enzyme system for oxygen removal, used as an inerting agent 26, was also added directly to the solution. The current in µA is shown on the Y-axis, and the voltage in volts across the electrode against the Ag / AgCl paste as a reference electrode 18 is shown on the X-axis.

[0127] In the Figure 8Furthermore, the current in µA at a voltage of -0.8 V is plotted on the Y-axis against the known nitrate concentrations in mM on the X-axis. This plot allows the linear range of the biosensor to be determined. A linear range of 0-500 µM was identified. The measurement signal then quickly reaches saturation at a concentration of 4,000 µM. This enables particularly sensitive detection of the analyte.

[0128] Both Figures 7 and 8A reaction buffer of 25 mM phosphate and 20 µM EDTA at pH 7.3 was used. The enzymes for oxygen removal were pyranose oxidase and catalase, respectively, at a concentration of 1 mg / ml each. Glucose was used as substrate and reagent 42, respectively, at a concentration of 1 mg / ml. Nitrate reductase was used at a concentration of 1 mg / ml. Each cycle was performed at a feed rate of 2 mV / s, thus completing one cycle in 3.3 minutes. Various concentrations of potassium nitrate, ranging from 0.05 to 16 mM, were used for the concentration series. Diagram b) shows the measurement of the concentration series repeated from Diagram a) using a new biosensor 10 to demonstrate reproducibility.

[0129] In Figure 8 The current at a voltage of -0.8V is plotted against the known concentrations. The linear region can be determined from this plot.

[0130] To demonstrate the functionality of a biosensor 10 as a nitrate test strip, a closed strip containing the sensor enzyme nitrate reductase as the detection agent 22 was prepared, and this test strip was used to detect potassium nitrate in a solution. This is in Figure 9 The graph shows the voltage in volts on the x-axis and the current in µA on the y-axis. Curve A represents a potassium nitrate concentration of 0 mM, and curve B represents a potassium nitrate concentration of 10 mM.

[0131] In this experiment, a reaction buffer of 25 mM phosphate and 20 µM EDTA at pH 7.3 was used. The enzymes for oxygen removal were pyranose oxidase and catalase, each at a concentration of 1 mg / ml. Glucose was used as the substrate at a concentration of 9 mg / ml. Nitrate reductase was used at a concentration of 1 mg / ml. Effective detection of nitrate was demonstrated.

[0132] From the above, it is evident that the biosensor 10 described here makes it possible to produce sensors for bioanalytical applications (e.g., blood glucose, nitrate), such as test strips, in which dissolved oxygen is completely removed from the sample. This has the advantage that the removed oxygen cannot affect the sensor enzymes and thus the measurement. This makes it possible to produce biosensors with higher accuracy, since the oxygen effect is completely eliminated. Furthermore, this method of oxygen removal and the specific design of the biosensor 10 can also be used to produce novel biosensors 10 that utilize oxygen-sensitive sensor enzymes. Reference sign

[0133] 10 Biosensor 12 Detection chamber 14 Working electrode 16 Counter electrode 18 Reference electrode 20 Measuring range 22 Detection agent 24 Contact area 26 Inerting agent 28 Entrance opening 30 Sensor cover 32 Lateral limit 34 Lateral limit 36 ​​Intermediate space 38 Arrow 40 Arrow 42 Reagent 44 Sensor base 45 Arrow 46 Length 48 Width 50 Width 52 Width 54 Distance

Claims

1. Electrochemical biosensor for detecting at least one analyte dissolved in an analyte solvent, comprising a capillary detection chamber (12) and a plurality of electrodes, wherein the plurality of electrodes (14, 16, 18) comprise at least one working electrode (14) with a measurement region (20) which is positioned in the capillary detection chamber (12) and provided with an immobile detection means (22) for interacting with the analyte, and wherein the plurality of electrodes further comprise a counter electrode (16) which extends into the capillary detection chamber (12), and wherein the plurality of electrodes (14, 16, 18) are electrically contactable outside the capillary detection chamber (12), wherein the capillary detection chamber (12) has a volume of ≤10 µl, and wherein, furthermore, an oxygen-binding or oxygen-reactive inerting agent (26) is provided in the capillary detection chamber (12) and positioned at least in part between the working electrode (14) and an inlet (28) of the capillary detection chamber (12), and wherein the length of the capillary detection chamber (12) between the working electrode (14) and the inlet (28) and also the type and amount of the inerting agent (26) are chosen and matched to one another such that oxygen diffusing from the inlet (28) in the direction of the working electrode in the analyte solvent disposed in the detection chamber (12) can be removed completely from the analyte solvent by the inerting agent (26) before reaching the working electrode (14), and wherein the inerting agent (26) is at least partially soluble in the analyte solvent such that, in the event of an influx of the analyte solvent, the inerting agent (26) is immediately soluble, at least in part and in particular in full.

2. Biosensor according to Claim 1, characterized in that the volume of the capillary detection chamber (12) is ≤5 µl.

3. Biosensor according to Claim 1 or 2, characterized in that the distance between the working electrode (14) and the inlet (28) is in a range from ≥2 mm to ≤15 mm.

4. Biosensor according to any of Claims 1 to 3, characterized in that the inerting agent (26) is an enzyme-based inerting agent.

5. Biosensor according to any of Claims 1 to 4, characterized in that the detection means (22) is an enzyme-based detection means.

6. Method for detecting at least one analyte dissolved in a solvent, comprising the method steps of: a) providing a biosensor (10) according to any of Claims 1 to 5, b) filling the capillary detection chamber (12) with analyte dissolved in the analyte solvent, wherein c) when filling the capillary detection chamber (12), the analyte dissolved in the analyte solvent is guided from the inlet (28) to the detection means (22); d) externally contacting the electrodes (14, 16, 18); and e) determining at least one of the amount and type of analyte using information supplied by the electrodes (14, 16, 18), wherein at or after method step b), the inerting agent (26) dissolves in the analyte solvent at least in part.

7. Method according to Claim 6, characterized in that oxygen diffusing from the inlet (28) in the direction of the detection means (22) in the analyte solvent is removed completely from the analyte solvent by the inerting agent (26).

8. Method for producing a biosensor (10) according to any of Claims 1 to 5 for detecting at least one analyte dissolved in an analyte solvent, wherein the method comprises the method steps of: i) providing a main body with a capillary detection chamber (12), wherein the detection chamber (12) has a volume of ≤10 µl; ii) arranging a plurality of electrodes (14, 16, 18) such that they can be electrically contacted externally and run into the detection chamber (12); iii) arranging a detection means (22) on an electrode serving as a working electrode (14); and iv) arranging an inerting agent (26) in the detection chamber (12) between an inlet (28) of the detection chamber (12) and the electrode serving as the working electrode (14), wherein v) the length of the capillary detection chamber (12) between the electrode serving as the working electrode (14) and the inlet (28) and also the type and amount of the inerting agent (26) are chosen and matched to one another such that oxygen diffusing from the inlet (28) in the direction of the electrode serving as the working electrode (14) in the analyte solvent disposed in the detection chamber (12) can be removed completely from the analyte solvent by the inerting agent (26) before reaching the electrode serving as the working electrode (14) and wherein the inerting agent (26) is at least partially soluble in the analyte solvent such that, in the event of an influx of the analyte solvent, the inerting agent (26) is immediately soluble, at least in part and in particular in full.

9. Method according to Claim 8, characterized in that the method comprises the following method steps: vi) providing a sensor cover (30); vii) applying two lateral boundaries (32, 34) to the sensor cover (30) in such a way that the lateral boundaries (32, 34) define an intermediate space (36), which forms the detection chamber (12), between the lateral boundaries (32, 34) and that the lateral boundaries (32, 34) form an inlet (28) on at least one side; viii) introducing the inerting agent (26) into the intermediate space (36); ix) providing a sensor base (44); x) applying at least two of the electrodes (14, 16) to the sensor base (44); xi) applying the detection means (22) to an electrode to be used as a working electrode (14); and xii) securing the sensor base (44) to the lateral parts (32, 34) in such a way that the electrodes (14, 16) run into the detection chamber (12) formed by the intermediate space (36), and the detection means (22) is present in the detection chamber (12).

10. Use of the biosensor (10) according to any of Claims 1 to 5 for the detection of at least one analyte dissolved in a solvent.

11. Use according to Claim 10, characterized in that the biosensor (10) according to any of Claims 1 to 5 is used for the determination of glucose or for the determination of nitrate.