Method for determining actual concentration of a substrate using an array of self-calibrating biosensors and apparatus for carrying out said method

Abstract

The present invention relates to a method for determining, in a time-stable manner, the region in a medium where the actual concentration of a substrate (S1), consisting of any molecule susceptible to catalytic oxidation-reduction by a catalyst, is present, characterized in that it comprises the steps of: (a) acquiring at least one group of at least two biosensors, each having a calibration curve of a signal induced by an oxidation-reduction reaction, the biosensors in one group having an identical initial portion of their calibration curve up to a concentration value of the substrate (S1), called the separation concentration (SC), from which the signal measurements differ between the biosensors in the group, and in the case of several groups, the biosensors in different groups have different calibration curves without an identical initial portion; (b) placing the biosensors in contact with the medium; (c) measuring the signal induced by the oxidation or reduction reaction for each biosensor in the one or more groups; and (d) comparing all the signal values ​​generated by all the biosensors, according to the method described in the present specification.

Description

[Technical Field]

[0001] The present invention relates to a method for determining, in a time-stable manner, the area in a medium where the actual concentration of a substrate susceptible to an enzymatic redox reaction is present, using an array of biosensors, and to an apparatus for carrying out the method. [Background technology]

[0002] Diabetes is a global epidemic that affected 422 million people in 2014, compared with 108 million in 1980. If current incidence trends continue, it is expected to become the seventh leading cause of death worldwide (Non-Patent Documents 1, 2, 3). In 2016, approximately 12% of the French population was diagnosed with diabetes (Non-Patent Document 4). In addition to increased mortality, diabetes and its complications result in considerable economic losses and high social security costs (work stoppage, treatment, hospitalization, etc.) (Non-Patent Document 4). Indeed, in France, the cost of diabetes treatment is approximately 10 billion euros, 80% of which is related to the treatment of complications. These complications can be predicted and / or avoided with the use of reliable real-time blood glucose monitoring devices (Non-Patent Document 5).

[0003] The advent of glucose sensors has enabled patients to manage their insulin levels, helping to reduce diabetes-related mortality. Conventional glucose testing devices include glucose sensors (glucometers) based on electrochemical methods. Glucose biosensors consist of a working electrode based on a conductive material associated with an enzyme capable of catalyzing glucose oxidation, such as glucose oxidase (GOx) or glucose dehydrogenase (GDH), and a counter electrode, which can have platinum, gold, or carbon. The enzyme is immobilized on or near the surface of the electrode. Often, the enzyme is associated with a redox mediator that enables electron transfer between the enzyme and the electrode. The most commonly used mediators for GOx are ferrocene, ferrocyanide, and osmium complexes. The working and counter electrodes are placed in contact with the test sample. When a voltage is applied between the working and counter electrodes, a current flows through the circuit created by the electrodes and the test sample. This current is induced by the enzyme-catalyzed oxidation of glucose at the working electrode, and the value of this current depends on the concentration of glucose in the test sample.

[0004] The most common blood glucose testing involves performing multiple tests daily or weekly by analyzing small blood samples (5, 6), which is inconvenient and uncomfortable, thus reducing patient acceptance. Furthermore, such tests do not take into account rest periods and lead to approximations of measurements. Furthermore, this type of monitoring does not provide real-time information and therefore cannot prevent hypoglycemic (<3.0 mM) and hyperglycemic (>11.1 mM) events in advance.

[0005] Continuous and autonomous blood glucose monitoring is essential to understand the trend, direction, and frequency of changes in blood glucose levels. Interstitial blood glucose meters are available that allow diabetic patients to monitor their blood glucose levels in real time by wearing a patch on their skin. However, these devices need to be replaced every two weeks due to biosensor drift. Indeed, the time drift of enzyme biosensors in general, and sugar biosensors in particular, is a major key to developing enzyme biosensors that can maintain sensitivity over long periods of time without the need for reconditioning or replacement.

[0006] Electrochemical enzyme biosensors degrade over time due to a decrease in enzyme stability, a decrease in catalytic activity, and electrode degradation.

[0007] [Figure 1] illustrates this phenomenon and its direct impact on sugar quantification.

[0008] Figure 1 shows three graphs showing the reaction rate occurring at the working electrode of an electrochemical biosensor as a function of sugar concentration. In the left graph, the sugar concentration at a given rate is determined (rates β and δ correspond to the blood glucose rates (BGR) of α and γ). Over time, enzymatic degradation changes the reaction rate. This can be seen in the middle graph of Figure 1: BGRα is no longer defined by rate β, but by rate δ. Therefore, the values given by the biosensor are no longer reliable. Therefore, a calibration is required, as shown in the right graph. Calibration is performed as follows: (1) The user measures his / her BGR via a drop of blood and transmits his / her current BGR, which is equal to α, to the biosensor. (2) The velocity δ measured by the sensor at the same instant is recorded. (3) The program contained in the sensor that correlates speed with BGR is reset after two measurements.

[0009] Catalytic activity does not recover, and after a certain period of time, the accuracy of the biosensor decreases significantly, requiring replacement.

[0010] Therefore, enzyme biosensor measurements inevitably drift and require external calibration. This makes the development of implantable sugar biosensors that can operate for long periods of time currently out of reach. Furthermore, it is generally believed that, for all enzyme biosensors, this measurement drift is the main reason why enzyme biosensors have not yet reached the market.

[0011] Current glucose biosensors designed to measure blood glucose levels are limited by biosensor drift over time, which is a major obstacle to the development of implantable or non-implantable glucose biosensors that can operate over long periods of time to continuously measure blood glucose levels in diabetic patients.

[0012] Therefore, there is a need for an enzymatic glucose biosensor that "self-calibrates" away from the measurement drift of existing biosensors and provides stable, long-term continuous monitoring of blood glucose without the need for replacement or external calibration. This is achieved by comparing measurements from a series of biosensors of known properties, where the properties are not based on the values at which they are measured. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Global report on diabetes (2016). World Health Organization [Non-patent document 2] International Diabetes federation (2016) [Non-patent document 3] Mechanisms of diabetic complications. Forbes JM1, Cooper ME. Physiol Rev. 2013 Jan;93(1):137-88. doi: 10.1152 / physrev.00045.2011 [Non-patent document 4] Institut de veille sanitaire (France) [Non-Patent Document 5] Home Blood Glucose Biosensors: A Commercial Perspective. Jeffrey D. Newman & Anthony PF Turner. Biosensors and Bioelectronics, Volume 20, Issue 12, 20th Anniversary of Biosensors and Bioelectronics, 15 June 2005, Pages 2435-2453 [Non-patent document 6] Electrochemical Glucose Biosensors. J. Wang. Chem. Rev., 2008, 108 (2), pp 814-825 Summary of the Invention [Means for solving the problem]

[0014] The present invention therefore provides a method for determining, in a time-stable manner, the region in a medium where the actual concentration of a substrate (S1), composed of any molecule susceptible to catalytic oxidation-reduction by a catalyst, is present, comprising: a) acquiring at least one group of at least two biosensors, each biosensor having a calibration curve of a signal induced by a redox reaction; - the biosensors in one group have the same initial part of their calibration curve up to the concentration value of the substrate (S1), called the separation concentration (SC), from which the signal measurements differ between the biosensors in the group; When there are multiple groups, the biosensors in different groups have different calibration curves without having the same initial portion; Steps and b) placing the biosensor in contact with the medium; c) measuring a signal induced by an oxidation or reduction reaction for each biosensor in one or more groups; d) comparing all signal values generated by all biosensors, in the case of a single group of biosensors: - if all signal values are equal, the concentration of substrate (S1) is less than or equal to the minimum SC; - If all signal values are different, the concentration of substrate (S1) is higher than the highest SC. - if a part of the biosensors has the same signal value, the concentration of the substrate (S1) is less than or equal to the lowest SC of the biosensor in that part and greater than the SC of the biosensor with the next lowest SC, For multiple groups of biosensors, - if all signal values in each group are equal, the concentration of substrate (S1) is less than or equal to the minimum SC; - If all signal values are different in all groups, the concentration of substrate (S1) is higher than the highest SC. - if some of the biosensors in a group have the same signal value, the concentration of the substrate (S1) is less than or equal to the lowest SC of the biosensors in that group and greater than the SC of the biosensor with the next lowest SC in the relevant group, - if in some of the groups of biosensors all biosensors in one group have the same signal value and in the remaining part all biosensors in one group have different signal values, the concentration is less than or equal to the lowest SC of one or more groups with the same signal value in each group and greater than the highest SC of one or more groups with different signal values in each group; Steps and The present invention relates to a method comprising the steps of:

[0015] Separate concentrations are determined by successive comparison between two calibration curves, so for a group with n biosensors and n corresponding calibration curves, there are n-1 separate concentrations in the group.

[0016] For each biosensor, the working electrode can be a carbon, gold, or platinum electrode; OppositeThe electrodes can be platinum, gold, or diamond electrodes, and the reference electrode can be a silver chloride electrode.

[0017] For each biosensor, the working electrode can be a carbon, gold, or platinum electrode, the reference electrode can be a platinum, gold, or diamond electrode, and the reference electrode can be a silver chloride electrode.

[0018] The catalyst may be an enzyme catalyst or a chemical catalyst, and the chemical catalyst is an abiotic catalyst, particularly selected from platinum, platinum nanostructures, platinum alloy nanostructures, gold nanostructures, and gold alloy nanostructures, or a molecular catalyst, particularly selected from porphyrin-gold complexes and porphyrin-rhodium complexes.

[0019] When the catalyst is an enzyme catalyst, a mediator may be associated with the catalyst, the mediator being selected in particular from among ferrocene, ferrocyanide, osmium complexes, quinone derivatives such as naphthoquinone, and phenothiazine derivatives.

[0020] The substrate (S1) transporter can be disposed on one or more biosensors.

[0021] The biosensors in each group are p1: amount of catalyst; p2: the oxidation-reduction Km of the catalyst if the catalyst is an enzyme catalyst, or the saturation limit of the catalyst if the catalyst is a chemical catalyst; p3: if the catalyst is an enzyme catalyst, the amount of mediator of the catalyst, if any; or p4: If present, transport Km of substrate (S1) transporter At least one parameter selected from may be different.

[0022] The Km of an enzyme-catalyzed oxidation-reduction reaction is the Michaelis constant of the catalyst, which represents the substrate concentration at which the reaction rate is half of its maximum rate.

[0023] The Michaelis constant of an enzyme catalyst is specific to the catalyst and depends on the organism from which the catalyst is derived and the extraction process.

[0024] For example, for commercially available glucose oxidase enzymes, the Michaelis constant is 1.34 mM for glucose oxidase from P. ostreatus, 5.7 mM for glucose oxidase from P. amagasakiense, 6.2 mM for glucose oxidase from P. pinophilum, 10.2 mM for glucose oxidase from T. flavus, 30 mM for glucose oxidase from A. niger, and greater than 38 mM for glucose oxidase from P. chrysosporium.

[0025] The saturation limit of a chemical catalyst is the concentration of substrate (S1) at which the measured signal induced by the oxidation-reduction reaction of the substrate (S1) reaches a maximum limiting value. Km is the substrate concentration required for the signal induced by the oxidation-reduction reaction of the substrate to reach half of the saturation limit. The saturation limit depends on the nature of the chemical catalyst used and can therefore vary.

[0026] The transport Km of a substrate transporter is the Michaelis constant of the transporter, which represents the substrate concentration at which the transport rate of the substrate is half of its maximum rate.

[0027] As an example, the transport Km of the glucose transporter Glu1 is 3 mM, Glu2 is 17 mM, Glu3 is 1.8 mM, Glu4 is 5 mM, Glu8 is 2.4 mM, and Glu9 is 0.5 mM.

[0028] Biosensors (BCs) are B.C. 11 .........BC 1i .........BC 1n ... B.C. 21 .........BC 2i .........BC2n ... B.C. j1 .........BC ji .........BC jn ... B.C. m1 .........BC mi .........BC mn ... can be written as m and n are integers, m is the number of groups, n is the number of biosensors in each group, and biosensor BC 11 ~BC 1n belongs to group 1, and biosensor BC m1 ~BC mn belongs to group m, A parameter selected from p1 to p4 is changed between each biosensor in one group, and another parameter selected from these parameters p1 to p4 is changed between biosensors in two different groups.

[0029] As an example, the amount of catalytic mediator is varied between each biosensor in the first group so that each biosensor in that group has a different calibration curve.

[0030] To obtain a second, different group of biosensors, the same variation in the amount of catalytic mediator can be maintained among each biosensor in the second group, and a second parameter, such as the catalytic redox Km, can be varied between the two groups to differentiate between the first and second groups. For example, the Km can be selected to be a first value for each biosensor in the first group and a second value different from the first value for each biosensor in the second group.

[0031] especially, the substrate (S1) can be glucose or lactate, the catalyst is an enzymatic catalyst selected from glucose oxidase, glucose dehydrogenase, and cellobiose dehydrogenase, or lactate oxidase or lactate dehydrogenase, the enzyme catalytic mediator, if present, is selected from ferrocene, ferrocyanide, osmium complexes, quinone derivatives such as naphthoquinone, and phenothiazine derivatives; The substrate (S1) transporter, if present, may optionally be a glucose transporter selected from, in particular, GLUT1, GLUT2, GLUT3, GLUT4, GLUT6, GLUT8, GLUT10 and GLUT12, or a lactate transporter selected from, in particular, MCT1, MCT2, MCT3, MCT4.

[0032] The invention also provides an apparatus for carrying out the aforementioned method, comprising at least one group of at least two biosensors, each biosensor having a calibration curve for a signal induced by a redox reaction, - the biosensors in one group have the same initial part of their calibration curve up to a concentration value of the substrate (S1), called the separation concentration (SC), at which the signal measurements differ between the biosensors in the group; - if there are multiple groups, the biosensors in different groups have different calibration curves without the same initial portion between the groups, and each biosensor is capable of measuring a signal induced by a catalytic redox reaction of the substrate (S1); and Each biosensor is a catalyst; in the case of enzymatic catalysis, its mediator, if applicable; - Substrate (S1) transporters, if applicable; Equipped with The present invention also relates to an apparatus characterized in that

[0033] For each biosensor, the working electrode can be a carbon, gold, or platinum electrode; OppositeThe electrodes can be platinum, gold, or diamond electrodes, and the reference electrode can be a silver chloride electrode.

[0034] For each biosensor, the working electrode can be a carbon, gold, or platinum electrode, the reference electrode can be a platinum, gold, or diamond electrode, and the reference electrode can be a silver chloride electrode.

[0035] The catalyst may be an enzyme catalyst or a chemical catalyst, which may be an abiotic catalyst, particularly selected from platinum, platinum nanostructures, platinum alloy nanostructures, gold nanostructures, and gold alloy nanostructures, or a molecular catalyst, particularly selected from porphyrin-gold complexes and porphyrin-rhodium complexes.

[0036] When the catalyst is an enzyme catalyst, a mediator may be associated with the catalyst, the mediator being selected from, inter alia, ferrocene, ferrocyanide, osmium complexes, quinone derivatives such as naphthoquinone, and phenothiazine derivatives.

[0037] The biosensors in each group are: p1: amount of catalyst; p2: the oxidation-reduction Km of the catalyst if the catalyst is an enzyme catalyst, or the saturation limit of the catalyst if the catalyst is a chemical catalyst; p3: if the catalyst is an enzyme catalyst, the amount of mediator of the catalyst, if any; or p4: Transport Km of substrate (S1) transporter, if present; At least one parameter selected from may be different.

[0038] Biosensors (BCs) are B.C. 11 .........BC 1i .........BC 1n ... B.C. 21 .........BC 2i .........BC 2n ... B.C.j1 .........BC ji .........BC jn ... B.C. m1 .........BC mi .........BC mn ... It can be written as m and n are integers, m is the number of groups, n is the number of biosensors in each group, and biosensor BC 11 ~BC 1n belongs to group 1, and biosensor BC m1 ~BC mn belongs to group m, A parameter selected from p1 to p4 is changed between each biosensor in one group, and another parameter selected from these parameters p1 to p4 is changed between biosensors in two different groups.

[0039] especially, the substrate (S1) can be glucose or lactate, the catalyst may be an enzymatic catalyst selected from glucose oxidase, glucose dehydrogenase and cellobiose dehydrogenase, or lactate oxidase or lactate dehydrogenase; the enzyme catalytic mediator, if present, is selected from ferrocene, ferrocyanide, osmium complexes, quinone derivatives such as naphthoquinone, and phenothiazine derivatives; The substrate (S1) transporter, if present, may optionally be a glucose transporter selected from, in particular, GLUT1, GLUT2, GLUT3, GLUT4, GLUT6, GLUT8, GLUT10 and GLUT12, or a lactate transporter selected from, in particular, MCT1, MCT2, MCT3, MCT4.

[0040] In certain embodiments, the biosensor may be disposed on a substrate, with the working electrode, reference electrode, and counter electrode screen printed onto the substrate.

[0041] The support on which the biosensor is placed can be selected from a glass plate, a plastic plate such as a polyethylene terephthalate plate, a ceramic plate such as alumina, or a composite between alumina and another ceramic, a nylon plate, a silicon plate, a polystyrene-based film, or a polyester sheet.

[0042] The catalyst can be deposited on the working electrode of each biosensor by encapsulation, grafting, absorption, or entrapment.

[0043] If the catalyst is an enzyme catalyst, it may be present on the surface of the working electrode of each biosensor by being coated on the surface of the working electrode inside a protective layer, the protective layer comprising, inter alia, chitosan, Nafion, polypyrrole, or polyacrylic acid, or a conductive polymer such as polyaniline, polylactic acid, polydopamine, or polyethylene glycol. An enzyme catalyst mediator may be encapsulated with the enzyme catalyst within the protective layer.

[0044] The substrate (S1) transporter can be present by being applied as a layer to the working electrode or optionally onto a protective layer containing the enzyme catalyst, and the mediator is optionally present by depositing a layer of proteoliposomes surrounding the substrate (S1) transporter.

[0045] The support on which the biosensor is disposed and the biosensor may be coated with a layer of chitosan, poly(2-hydroxyethyl methacrylate), poly(4-vinylpyridine-co-styrene), or alumina.

[0046] The device of the present invention can be placed on the skin of a user or implanted in the user to determine the area where the actual concentration of a substrate (S1) in a medium is present, the substrate being sugar and the medium being blood.

[0047] In particular, for an implantable device for determining glucose concentration, a biosensor may be selected that can determine the actual glucose concentration with an accuracy of 0.5-1 mM within a concentration range of 2-10 mM, i.e., a biosensor with a separation concentration of 2-10 mM with a step size of 0.5 or 1 mM should be selected. [Brief explanation of the drawings]

[0048] [Figure 1] FIG. 1 shows the reaction rate occurring at the working electrode of an electrochemical biosensor as a function of sugar concentration. [Figure 2] FIG. 1 shows the arrangement of the working electrode, counter electrode, and reference electrode in each biosensor. [Figure 3] FIG. 1 shows calibration curves determined for each of the six groups of biosensors. [Figure 4] FIG. 1 shows calibration curves determined for each biosensor in the 10 groups. [Figure 5] FIG. 1 shows the working electrode 1 of the biosensor and the approach of sugar G to the surface of the working electrode 1. [Figure 6] FIG. 10 shows calibration curves for the four biosensors in Example 3. [Figure 7] FIG. 10 is a plot of the ratio of the current intensities measured for biosensors A1 and A2 as a function of lactate concentration over time. DETAILED DESCRIPTION OF THE INVENTION

[0049] The following examples illustrate the invention without limiting its scope.

[0050] Example 1: Self-calibrating biosensor based on the variation of the amount of mediator [M] and the amount of enzyme [E] An array of six pairs of biosensors is fabricated on the surface of a glass plate. The electrodes are printed onto the glass plate by screen printing. This deposition method involves printing carbon electrodes onto a solid support from carbon ink using a printing device. Each biosensor consists of a carbon working electrode, a platinum counter electrode, and an Ag / AgCl reference electrode.

[0051] Figure 2 shows the arrangement of the working electrode, counter electrode, and reference electrode in each biosensor. The working electrode of each biosensor is fabricated by screen printing from a carbon ink containing 40% by weight of carbon powder dispersed in an organic solution composed of terpineol and ethyl cellulose. The reference and counter electrodes of each biosensor are fabricated by screen printing from a platinum ink containing 40% by weight of platinum powder dispersed in an organic solution composed of terpineol and ethyl cellulose, and a silver ink containing 40% by weight of silver powder dispersed in an organic solution composed of terpineol and ethyl cellulose, respectively.

[0052] On each working electrode, 10 μL of a 5% by volume solution of the enzyme glucose oxidase (GOx) and its mediator naphthoquinone in Nafion-tetrabutylammonium bromide (TBAB) was applied, in the amounts shown in Table 1. This was allowed to air dry at room temperature for 6 hours.

[0053] This results in an array of 12 biosensors in six groups of two biosensors. The biosensors in the first group are labeled BC 11 and B.C. 12 and the biosensors in the second group are denoted as BC 21 and B.C. 22 and for the third group, BC 31 and B.C. 32 For the fourth group, BC 41 and B.C. 42 and for the fifth group, BC 51 and B.C. 52 For the sixth group, BC 61 and B.C.62 It is written as follows.

[0054] Within each group of two biosensors, the biosensors have the same amount of GOx enzyme and exhibit different amounts of mediator, with the amounts of GOx enzyme and mediator varying from group to group.

[0055] The biosensor is then calibrated. This initial calibration consists in measuring the intensity of the current induced by the redox reaction in sugar standard solutions with known sugar concentrations. Sugar is oxidized at the working electrode, while oxygen is reduced at the counter electrode.

[0056] Figure 3 shows the calibration curves determined for each of the six groups of biosensors. These calibration curves allow us to obtain separation concentrations where the current intensity of the sugar redox reaction differs between two biosensors in the same group.

[0057] Table 1 also shows the separation concentration (SC) values found. [Table 1]

[0058] To determine the sugar concentration range of a sample to be analyzed, a group of biosensors is contacted with the sample, the current value measured by each biosensor is measured, and the sugar concentration value is determined according to the following method.

[0059] Two biosensors BC 11 and B.C. 12 If the values measured by the biosensor are identical to those of the other biosensors, it means that the sugar concentration in the sample is higher than that measured by the biosensor BC. 11 and B.C. 12 This means that the isolation concentration of this group is lower than 1 mM, which is the lowest isolation concentration in the group.

[0060] Two biosensors BC 11 and B.C. 12If the values measured by biosensor BC are different, it means that the sugar concentration in the sample is higher than that measured by biosensor BC. 11 and B.C. 12 This means that the concentration is higher than 1 mM, which is the isolation concentration of this group.

[0061] Then, the biosensor, i.e., BC 21 and B.C. 22 The values measured by the next group of biosensors are compared: two biosensors BC 21 and B.C. 22 If the values measured by biosensor group B and C are identical, this indicates that the sugar concentration in the sample is the same as that in biosensor group B and C. 21 and B.C. 22 This means that the sugar concentration in the sample is between 1mM and 2mM.

[0062] The same procedure is followed for the following biosensors: BC 21 and B.C. 22 The values measured by the biosensor are different, and the BC 31 and B.C. 32 When the values measured by the biosensors are the same, the sugar concentration is 2 mM to 3 mM, and the same is true for the following biosensor groups.

[0063] If all the values of all the biosensors are different, it means that the sugar concentration in the sample is higher than the biosensor BC 61 and B.C. 62 This means that the highest separation concentration is one of the last group, i.e., higher than 6 mM.

[0064] This method therefore allows for the estimation of sugar concentrations by comparing the sugar redox reaction intensity values measured in each biosensor for each group.

[0065] This process avoids biosensor drift, as it is not a directly measured value that allows the glucose concentration to be established, but rather a comparison between the values measured by each biosensor.

[0066] Example 2: Self-calibrating biosensor based on the variation of Km of enzyme and the amount of mediator An array of 10 groups of two biosensors is deposited on the surface of a glass plate. Working, counter and reference electrodes are printed as in Example 1.

[0067] On each working electrode, 10 μL of Nafion-TBAB solution containing 3 mg / mL glucose oxidase enzyme and its mediator naphthoquinone was deposited in the amounts shown in Table 2. The Km of the glucose oxidase enzyme varies depending on the biosensor and is shown in Table 2.

[0068] Thus, we create an array of 20 biosensors belonging to 10 sets of biosensors. This network can be written as follows: B.C. 11 ......... 12 B.C. 21 ......... 22 B.C. 31 ......... 32 B.C. 41 ......... 42 B.C. 51 ......... 52 B.C. 61 ......... 62 B.C. 71 ......... 72 B.C. 81 ......... 82 B.C. 91 ......... 92 B.C. 101 ......... 102

[0069] Within each set, the Km of glucose oxidase is the same, but the amount of mediator is different. Only the Km of glucose oxidase differs between sets.

[0070] Figure 4 shows the calibration curves determined for each of the 10 groups of biosensors. These calibration curves allow us to obtain separation concentrations where the current intensity of the sugar redox reaction differs between two biosensors in the same group. [Table 2]

[0071] The same procedure as in Example 1 is used to determine the sugar concentration range.

[0072] For example, biosensor vs. BC 11 ,BC 12 ;BC 21 ,BC 22 ;BC 31 ,BC 32 ;BC 41 ,BC 42 ;BC 51 ,BC 52 If all the values of the signals of are the same between each pair of biosensors, it means that the concentration of sugar is lower than the lowest separation concentration of these pairs. 51 ,BC 52 The concentration of is 3.1 mM.

[0073] Therefore, the signal values of the other biosensors are different, which means that the sugar concentration is higher than the highest separated concentration of these sets. 61 ,BC 62 The concentration of is 2.7 mM.

[0074] Therefore, the sugar concentration ranges from 2.7 mM to 3.1 mM.

[0075] Example 3: Biomimetic "self-calibrating" biosensors A group of four biosensors is deposited on the surface of a glass plate. The working, counter, and reference electrodes are printed as in Example 1.

[0076] On each carbon working electrode 1, a layer 2 of polyaniline (PANI) is deposited by electropolymerization from potassium phosphate buffer (100 mM, pH 7) containing 2 mM aniline and 2 mg / mL glucose oxidase (GOx) enzyme 3.

[0077] Electropolymerization is carried out in a three-electrode cell by cyclic voltammetry from −0.5 V to 1 V (5 cycles, scan rate 10 mV / s), with a platinum wire as counter electrode and a silver wire as reference electrode.

[0078] On each working electrode on which the polyaniline layer was deposited, 100 µL of potassium phosphate buffer (100 mM, pH 7) containing proteoliposomes containing glucose transporter protein 5 was applied with a Km of 0.3 mM on the first carbon electrode, 0.6 mM on the second carbon electrode, 2 mM on the third carbon electrode, and 5 mM on the fourth carbon electrode. The concentration of the glucose transporter in the phosphate buffer was 0.11 mg / mL. The diameter of the proteoliposomes was 100 nm to 200 nm. Wait 20 min to allow the fusion of the proteoliposomes 4 to the surface of each working electrode.

[0079] Fusion of the proteoliposomes 4 allows the formation of a planar lipid bilayer 6 on the surface of the working electrode where the glucose transporter 5 is located.

[0080] FIG. 5 shows the working electrode 1 of a biosensor and the approach of sugar G to the surface of the working electrode 1. In the first step I, sugar G approaches glucose transporter protein 5. In step II, sugar G is taken up by glucose transporter protein 5. In step III, sugar G is released from glucose transporter protein 5 and migrates to polyaniline layer 2, where GOx enzyme 3 is located in the direction of arrow F, where sugar G is oxidized. In step IV, glucose transporter protein 5 is again ready to receive sugar.

[0081] The glucose transporter protein regulates the access of sugars to glucose oxidase (Gox) deposited on the surface of the working electrode.

[0082] Calibration allows the determination of the separation concentration between biosensors. In this case, a calibration curve is presented in the form of a histogram for each biosensor. The separation concentration corresponds to the concentration at which the biosensor current plateaus because the glucose transporter can no longer transport any more glucose to the working electrode.

[0083] [Figure 6] shows the calibration curves of the four biosensors in Example 3.

[0084] Table 3 shows the Km of glucose transporters and the separation concentrations between biosensors. [Table 3]

[0085] To determine the sugar concentration range of the sample to be analyzed, four biosensors are brought into contact with the sample, the current values measured by each biosensor are measured, and the sugar concentration values are determined by the following method.

[0086] If all values measured by each biosensor are equal, it means that the sugar concentration is below the minimum separation concentration of 0.2 mM.

[0087] If the value measured for the first biosensor is different from the values measured for the other biosensors, but the values for each of the other three biosensors are the same, then the sugar concentration is greater than the separation concentration for the first biosensor and less than or equal to the separation concentration for the second biosensor, i.e., greater than 0.2 mM and less than or equal to 0.5 mM.

[0088] If all the values measured by the biosensors are different, this means that the sugar concentration is higher than the separation concentration of the third biosensor, ie 1.5 mM.

[0089] Example 4: Implantable biosensors A set of ten groups of two biosensors is deposited on the surface of a silicon wafer. The working, counter, and reference electrodes are printed as in Example 1. The enzyme glucose oxidase and its mediator are then applied to each working electrode in the amounts shown in Example 1.

[0090] The silicon plate is then covered with a biocompatible chitosan film by immersing it in a 2% chitosan solution at room temperature for 10 seconds, and then dried at 4°C for 8 hours.

[0091] This results in a biosensor that can be implanted under the skin of a user to enable measurement of the sugar concentration in the user's blood.

[0092] Example 5: Self-calibrating biosensor for measuring lactate concentration Two biosensors are deposited on the surface of a glass plate. The working, counter, and reference electrodes are printed as in Example 1.

[0093] On each working electrode, 10 μL of a 1% volume chitosan solution containing the enzyme lactate oxidase (LOx) and its mediator naphthoquinone was applied in the amounts shown in Table 4. The solution was air-dried at room temperature for 6 hours. [Table 4]

[0094] [FIG. 7] is a plot of the ratio of the current intensities measured for biosensors A1 and A2 as a function of lactate concentration over time.

[0095] As can be seen from FIG. 7, the ratio of the current intensities measured for biosensors A1 and A2 is similar on the first day of measurement and 10 days after the first measurement.

[0096] This means that the drift over time of the A1 and A2 biosensors is identical, and therefore it is possible to use the set of biosensors to determine the lactate concentration range of the sample to be analyzed.

Claims

1. A substrate (S) consisting of any molecule susceptible to catalytic oxidation-reduction by a catalyst １ ) a method for determining in a medium the area in which the actual concentration is present in a time-stable manner, comprising: a) acquiring a plurality of groups of at least two biosensors, each biosensor having a calibration curve of a signal induced by a redox reaction; - the biosensors in one group are separated by a concentration of the substrate (S １ ) and from that point on, the signal measurements differ between biosensors within the group; - the biosensors in different groups do not have the same initial portion and have different calibration curves; Steps and b) placing the biosensor in contact with the medium; c) measuring the signal induced by the oxidation or reduction reaction for each of the biosensors in a plurality of the groups; d) comparing all signal values generated by all said biosensors, If all signal values are equal, the substrate (S １ ) is equal to or less than the minimum SC, If all signal values in each group are equal, the substrate (S １ ) is equal to or less than the minimum SC, If all signal values are different in all groups, the substrate (S １ ) concentration is higher than the highest SC, - if some of the biosensors in a group have the same signal value, the substrate (S １ ) is less than or equal to the lowest SC of the biosensor in that group and greater than the SC of the biosensor with the next lowest SC in the same group; - if in some groups of said plurality of groups of biosensors, all biosensors in one group have the same signal value and in the remaining groups, all biosensors in one group have different signal values, the concentration of said substrate (S 1 ) is less than or equal to the lowest SC of said one or more groups with the same signal value in each group and greater than the highest SC of said one or more groups with different signal values in each group; Steps and A method comprising:

2. The signal is an electrochemical signal, where each biosensor comprises a working electrode, a reference electrode and a counter electrode between which a current induced by an oxidation or reduction reaction passes, and the electrochemical signal is determined by the intensity of this current or the substrate (S １ 2. The method according to claim 1, wherein the potential difference between the electrodes during the oxidation-reduction reaction is either

3. 3. The method of claim 2, wherein for each biosensor, the working electrode is a carbon, gold, or platinum electrode, the counter electrode is a platinum, gold, or diamond electrode, and the reference electrode is a silver chloride electrode.

4. 4. The method of claim 2 or 3, wherein the catalyst is an enzyme catalyst or a chemical catalyst.

5. The method of claim 4, characterized in that the chemical catalyst is an abiotic catalyst or a molecular catalyst, the abiotic catalyst is selected from platinum, platinum nanostructures, platinum alloy nanostructures, gold nanostructures, and gold alloy nanostructures, and the molecular catalyst is selected from porphyrin-gold complexes and porphyrin-rhodium complexes.

6. 5. The method according to claim 4, characterized in that the catalyst is an enzyme catalyst and a mediator is associated with the catalyst, the mediator being selected in particular from ferrocene, ferrocyanide, osmium complexes, quinone derivatives such as naphthoquinone, and phenothiazine derivatives.

7. Substrate (S １ 7. The method according to claim 1, wherein a transporter is disposed on one or more of the biosensors.

8. The biosensors in each group are: p1: amount of catalyst; p2: Michaelis constant of the catalyst if the catalyst is an enzyme catalyst, or saturation limit of the catalyst if the catalyst is a chemical catalyst; p3: if the catalyst is an enzyme catalyst, the amount of mediator of the catalyst, if any; or p4: If present, the substrate (S １ ) Michaelis constant of the transporter, 8. The method according to claim 1, wherein at least one parameter selected from:

9. The biosensor (BC) BC １１ .........BC １ｉ .........BC １ｎ ... BC ２１ .........BC ２ｉ .........BC ２ｎ ... BC ｊ１ .........BC ｊｉ .........BC ｊｎ ... BC ｍ１ .........BC ｍｉ .........BC ｍｎ ... It is written, m and n are each an integer, m is the number of groups, n is the number of biosensors in each group, and biosensor BC １１ ~BC １ｎ belongs to group 1, and biosensor BC ｍ１ ~BC ｍｎ belongs to group m, 9. The method according to claim 8, wherein a parameter selected from p1 to p4 is varied between each biosensor in one group, and another parameter from these parameters p1 to p4 is varied between two different groups of biosensors.

10. - the substrate (S １ ) is glucose, the catalyst is an enzyme catalyst selected from glucose oxidase, glucose dehydrogenase, and cellobiose dehydrogenase; the mediator of the enzyme catalyst, if present, is selected from ferrocene, ferrocyanide, osmium complexes, quinone derivatives such as naphthoquinone, and phenothiazine derivatives; - the substrate (S １ ) If present, the transporter is in particular a glucose transporter selected from GLUT1, GLUT2, GLUT3, GLUT4, GLUT6, GLUT8, GLUT10 and GLUT12; 10. The method according to any one of claims 6 to 9, characterized in that

11. - the substrate (S １ ) is lactate, - the catalyst is an enzymatic catalyst selected from lactate oxidase or lactate dehydrogenase, the enzyme catalytic mediator, if present, is selected from ferrocene, ferrocyanide, osmium complexes, quinone derivatives such as naphthoquinone, and phenothiazine derivatives; - the substrate (S １ ) If present, the transporter is a lactate transporter selected in particular from MCT1, MCT2, MCT3, MCT4; 10. The method according to any one of claims 6 to 9, characterized in that

12. 12. An apparatus for carrying out the method according to any one of claims 1 to 11, comprising at least one group of at least two biosensors, each biosensor having a calibration curve for a signal induced by a redox reaction, The biosensors within a group are at a concentration of the substrate (S), called the separation concentration (SC), at which the signal measurements differ between the biosensors within the group. １ ) have the same initial portion of their calibration curves leading up to the concentration value - If there are multiple groups, the biosensors in different groups have different calibration curves without having the same initial part between groups, and each biosensor has a different calibration curve for the substrate (S １ ) can be measured the signal induced by the catalytic redox reaction of Each biosensor is Equipped with a catalyst, An apparatus characterized in that

13. Each biosensor is capable of measuring an electrochemical signal and detecting the substrate (S １ The electrochemical signal is measured by the intensity of the current or the amount of the substrate (S). １ 13. The device according to claim 12, wherein the potential difference between the electrodes during the oxidation-reduction reaction is any one of the following:

14. 14. The device of claim 13, wherein for each biosensor, the working electrode is a carbon, gold, or platinum electrode, the counter electrode is a platinum, gold, or diamond electrode, and the reference electrode is a silver chloride electrode.

15. 15. The device according to any one of claims 12 to 14, characterized in that the catalyst is an enzyme catalyst or a chemical catalyst, which can be an abiotic catalyst, in particular selected from platinum, platinum nanostructures, platinum alloy nanostructures, gold nanostructures and gold alloy nanostructures, or a molecular catalyst, in particular selected from porphyrin-gold complexes and porphyrin-rhodium complexes.

16. 16. The device according to claim 12, wherein the catalyst is an enzyme catalyst and a mediator is associated with the catalyst, the mediator being selected in particular from ferrocene, ferrocyanide, osmium complexes, quinone derivatives such as naphthoquinone, and phenothiazine derivatives.

17. The biosensors in each group are: p1: amount of catalyst; p2: Michaelis constant of the catalyst if the catalyst is an enzyme catalyst, or saturation limit of the catalyst if the catalyst is a chemical catalyst; p3: if the catalyst is an enzyme catalyst, the amount of mediator of the catalyst, if any; or p4: If present, the substrate (S １ ) Michaelis constant of the transporter, 17. The device according to claim 12, wherein at least one parameter selected from the group consisting of:

18. The biosensor (BC) BC １１ .........BC １ｉ .........BC １ｎ ... BC ２１ .........BC ２ｉ .........BC ２ｎ ... BC ｊ１ .........BC ｊｉ .........BC ｊｎ ... BC ｍ１ .........BC ｍｉ .........BC ｍｎ ... It is written, m and n are each an integer, m is the number of groups, n is the number of biosensors in each group, and biosensor BC １１ ~BC １ｎ belongs to group 1, and biosensor BC ｍ１ ~BC ｍｎ belongs to group m, 18. The device according to claim 17, wherein a parameter selected from p1 to p4 is varied between each biosensor in one group, and another parameter from these parameters p1 to p4 is varied between biosensors in two different groups.

19. - the substrate (S １ ) is glucose, the catalyst is an enzyme catalyst selected from glucose oxidase, glucose dehydrogenase, and cellobiose dehydrogenase; the mediator of the enzyme catalyst, if present, is selected from ferrocene, ferrocyanide, osmium complexes, quinone derivatives such as naphthoquinone, and phenothiazine derivatives; - the substrate (S １ ) If present, the transporter is in particular a glucose transporter selected from GLUT1, GLUT2, GLUT3, GLUT4, GLUT6, GLUT8, GLUT10 and GLUT12; 19. Device according to any one of claims 16 to 18, characterized in that

20. - the substrate (S １ ) is lactate, - the catalyst is an enzymatic catalyst selected from lactate oxidase or lactate dehydrogenase, the mediator of the enzyme catalyst, if present, is selected from ferrocene, ferrocyanide, osmium complexes, quinone derivatives such as naphthoquinone, and phenothiazine derivatives; - the substrate (S １ ) If present, the transporter is a lactate transporter selected in particular from MCT1, MCT2, MCT3, MCT4; 19. Device according to any one of claims 16 to 18, characterized in that

21. 21. The device according to any one of claims 12 to 20, characterized in that the biosensor is disposed on a support, and the working electrode, the reference electrode and the counter electrode are screen-printed onto the support.

22. 22. The device according to claim 21, wherein the support on which the biosensor is placed is selected from a glass plate, a plastic plate such as a polyethylene terephthalate plate, a ceramic plate such as alumina, or a composite between alumina and another ceramic, a nylon plate, a silicon plate, a polystyrene-based film, or a polyester sheet.

23. 23. The device according to claim 21 or 22, characterized in that the catalyst is deposited on the working electrode of each biosensor by encapsulation, grafting, absorption or entrapment.

24. 24. The device according to claim 23, characterized in that the catalyst is an enzyme catalyst and is present on the surface of the working electrode of each biosensor by being applied to the surface of the working electrode inside a protective layer, the protective layer comprising in particular chitosan, Nafion, polypyrrole or polyacrylic acid, or comprising a conductive polymer such as polyaniline, polylactic acid, polydopamine or polyethylene glycol.

25. 25. The device of claim 24, wherein the mediator of the enzyme catalyst is encapsulated with the enzyme catalyst within the protective layer.

26. Substrate (S １ ) transporter is present by being applied as a layer to the working electrode or on the protective layer optionally containing an enzyme catalyst, and the mediator is optionally １ 26. A device according to any one of claims 24 to 25, characterized in that it exists by depositing a layer of proteoliposomes surrounding the transporter.

27. 27. The device according to any one of claims 24 to 26, characterized in that the support and the biosensor carried by the support are coated with a layer of chitosan, poly(2-hydroxyethyl methacrylate), poly(4-vinylpyridine-co-styrene), or alumina.

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