Electrochemical biosensor and method for detecting or quantifying an analyte

Abstract

The present invention relates to an electrochemical biosensor for detecting and / or quantifying at least one analyte of a liquid sample, the biosensor comprising a liquid sample inlet (10), an area with electrical contacts (19), a support substrate (11) comprising at least one set of electrodes (12), and an insulating cover (13). Between the substrate (11) and the insulating cover (13) there is a segment (18) connected in fluid communication with the inlet (10), said segment (18) comprising a detection chamber (16) wherein the set of electrodes (12) is arranged, the biosensor (100) comprising a liquid sample outlet (17) that is in fluid communication with the segment (18), such that it allows the liquid sample (10) to circulate by capillarity between the inlet (10) and the outlet (17) via the segment (18) and the detection chamber (16). The invention also relates to an associated method.

Description

[0001] DESCRIPTION

[0002] Electrochemical biosensor and method for the detection or quantification of an analyte

[0003] TECHNICAL SECTOR

[0004] The present invention relates to electrochemical biosensors.

[0005] PRIOR STATE OF THE ART

[0006] Electrochemical sensors are well-known for the determination and / or quantification of analytes present in liquid samples. An electrochemical sensor is a device that records changes in electrical current or potential resulting from its interaction with the analyte of interest. These changes are translated into an electrical signal that can be correlated with the quantity, concentration, or level of the analyte of interest in a sample.

[0007] Portability, ease of use, the ability to perform rapid analyses with little sample pretreatment and small sample volumes, a simple procedure, and low cost are some of the prerequisites for successful application in any analytical method. Enzymatic electrochemical biosensors based on the use of printed electrodes are often a good solution to address these needs. However, depending on the analyte being measured, for example, in the case of nitrites, the oxygen present in the sample and / or reaction zone can interfere with the measurement, giving an erroneous analytical result.

[0008] Purging argon or degassing the working solution prior to analysis is usually the most commonly employed technique, but other than this, various oxygen removal mechanisms have been attempted, such as the use of chemical and enzymatic scavengers.

[0009] W02005119237A1 describes various solutions to the problem of oxygen interference. As an example, it describes the use of sodium sulfite as an oxygen scavenger. DISCLOSURE OF THE INVENTION

[0010] The object of the invention is to provide an electrochemical biosensor and a method for the detection and / or quantification of at least one analyte, as defined in the claims.

[0011] A first aspect of the invention relates to an electrochemical biosensor for the detection and / or quantification of at least one analyte present in a liquid sample, the biosensor comprising an inlet for the liquid sample, an electrical contact area, a support substrate comprising at least one set of electrodes, said set comprising at least one working electrode and a reference electrode, wherein the working electrode comprises at least one enzyme and optionally a mediator whose redox processes (electrochemical oxidation and / or reduction) occur at a potential with respect to the reference electrode, and an insulating cover.The biosensor of the invention comprises between the substrate and the insulating cover a section connected in fluid communication with the inlet, said section comprising a detection chamber where the set of electrodes is arranged, and a liquid sample outlet that are in fluid communication with the section, in such a way that it allows the circulation of the liquid sample by capillarity between the inlet and outlet through the section and the detection chamber.

[0012] A second aspect of the invention relates to a method for the detection or quantification of at least one analyte dissolved in a liquid sample by means of the electrochemical biosensor of the invention, the method comprising the following steps: a) Introducing a quantity of liquid sample to be analyzed through the inlet until the section is completely filled; b) Applying a potential to the set of electrodes for a certain period of time with respect to the reference electrode; c) Measuring the current intensity produced by the redox reaction; and d) Translating the current intensity data obtained in the previous step by means of a calibration curve obtained from standard samples comprising different concentrations of the analyte to be determined. Thanks to the invention, possible interferences in the enzyme-mediator-analyte reaction process due to the effect of contaminants or environmental elements on the sample are reduced or eliminated, as the case may be.As shown in the examples provided, the invention minimizes interference due to oxygen or other environmental elements present during the measurement. The invention also offers other advantages, such as greater structural simplicity of the biosensor and the method.

[0013] These and other advantages and features of the invention will become apparent from the figures and the detailed description of the invention.

[0014] DESCRIPTION OF THE DRAWINGS

[0015] Figures 1A and 1B show an exploded (Fig. 1A) and non-exploded (Fig. 1B) view of a biosensor according to an embodiment of the invention.

[0016] Figures 2A and 2B show an exploded (Fig. 2A) and non-exploded (Fig. 2B) view of a biosensor according to another embodiment of the invention.

[0017] Figures 3A and 3B show exploded (Fig. 3A) and non-exploded (Fig. 3B) views of a biosensor according to another embodiment of the invention.

[0018] Figure 4 shows graphs depicting the operation of a sensor according to embodiments of the invention.

[0019] Figure 5 shows graphs depicting the comparative performance of a nitrite biosensor according to an embodiment of the invention versus a reference biosensor.

[0020] Figure 6 shows graphs of the calibration line obtained with the biosensors in Figure 5.

[0021] Figure 7 shows graphs depicting the comparative operation of a nitrite biosensor according to the embodiment of Figure 5 in the presence (Fig. 7B) and absence (Fig. 7A) of an oxygen scavenger. DETAILED EXPOSURE OF THE INVENTION

[0022] As shown in Figures 1A to 3B, the electrochemical biosensor of the invention for the detection and / or quantification of at least one analyte present in the liquid sample, comprises an inlet 10 for the liquid sample comprising the analyte, an area 19 of electrical contacts, and a support substrate 11, in the form of a sheet, comprising at least one set of screen-printed electrodes 12 and an insulating cover 13 in the form of a sheet. The set of electrodes comprises at least one working electrode, a reference electrode and optionally a counter electrode, wherein the working electrode comprises at least one enzyme and optionally an electrochemical mediator deposited, preferably immobilized, thereon, and whose redox processes occur at a determined potential with respect to the reference electrode.Between the substrate 11 and the insulating cover 13 there is a section 18 connected in fluid communication with the inlet 10 for the liquid sample, with a detection chamber 16 in section 18 where the set of electrodes 12 is arranged. The electrochemical reaction occurs in the detection chamber. The biosensor also comprises an outlet 17 for the liquid sample that is in fluid communication with the section 18, such that it allows the circulation of the liquid sample by capillarity between the inlet 10 and outlet 17 through the section 18 and the detection chamber 16. The inlet 10 and the outlet 17 consist of openings in the biosensor that fluidly communicate with the section 18.

[0023] "Immobilized" refers to entrapped on, or chemically bound to, the electrode surface. The enzyme and mediator are "immobilized" in a sensor, for example, when they are covalently, ionically, or coordinately bound to the sensor components and / or trapped in a polymeric matrix, membrane, or sol-gel that prevents their mobility.

[0024] In a preferred embodiment, as shown in Figures 1A to 3B, in a longitudinal direction X of the biosensor, the biosensor has a proximal region X1, a distal region X2 and a central region X3. The electrical contact region 19 is in the distal region X2 of the biosensor, the liquid sample inlet 10 is in the proximal region X1 and the liquid sample outlet 17 is arranged in the central region X3, in a position upstream of the electrical contact region 19. In one embodiment, shown in Figure 1A and 1B, the outlet 17 consists of an opening in the insulating cover 13, in a position upstream of the electrical contact region 19 and downstream of the electrode array.

[0025] As shown in Figures 2A, 2B and 3A, 3B, for better control of the liquid sample outlet, the section 18 defining the detection chamber 16 is in fluidic communication with the liquid sample outlet 17 through a lateral fluidic channel 16a, the lateral fluidic channel 16a therefore comprising a first end in fluidic communication with the section 18 and a second end in fluidic communication with the liquid sample outlet 17, and the outlet 17 being on a side 17a of the biosensor. The first end of the lateral fluidic channel 16a is narrower than the section 18. Preferably, the lateral fluidic channel 16a is narrower than the section 18. In a preferred embodiment, in the longitudinal direction X, the first end of the lateral fluidic channel 16a is arranged in a position downstream of the electrode set 12.In a preferred embodiment, the outlet 17 is in a longitudinal position equal to or prior to the longitudinal position of the first end of the lateral fluidic channel 16a. In a preferred embodiment, said outlet 17 is arranged in a longitudinal position close to the proximal zone X1, preferably at a longitudinal distance of between 10 and 25% of a distal end of the proximal zone X1 with respect to the total length of the section 18. In this way, in a preferred embodiment, the lateral fluidic channel 16a is arranged at an angle with respect to the longitudinal direction X of between 90° and 175° on one side or the other of the section 18.

[0026] The arrangement of these sample outlets ensures that the liquid sample covers the detection chamber and that any liquid sample leaks during use of the biosensor are away from the electrical contact area 19. The design of the lateral fluidic channel 16a, with the angles described above, further prevents the effect of oxygen on the reaction.

[0027] In a preferred embodiment, the detection chamber 16 is in fluid communication with the inlet 10 via a fluidic channel 16b. This type of configuration allows for the generation of an internal inlet channel for liquid samples into the detection chamber 16.

[0028] Figures 2A, 2B and 3A, 3B show biosensors with at least one separating sheet 14 divided into two parts 14a and 14b, which define the geometry that the section 18 will have with its detection chamber and the fluidic channels 16a and 16b, once the sheet is joined to the support and the cover.

[0029] In a preferred embodiment, the biosensor comprises at least one separator sheet 14 between the substrate 11 and the cover 13. The separator sheet 14, together with the substrate 11 and the cover 13, define the section 18 and its geometry. The separator sheet 14 has two faces, being joined or adhered on one side to the substrate 11 and on the other side to the cover 13. In the embodiment that does not include the separator sheet 14, the substrate 11 and the cover 13 are joined or adhered to each other, such that they leave an unjoined space which defines the section 18 and its geometry.

[0030] With respect to materials, in a preferred embodiment:

[0031] The support substrate 11 comprises an insulating, non-porous material. By way of non-limiting example, the substrate comprises or consists of polyethylene terephthalate (PET), polycarbonate (PC), polyimide, alumina or other ceramic materials, paper for printed electronics, polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), preferably PET, PC or alumina.

[0032] The insulating cover 13 comprises a hydrophilic area that is in contact with the section 18. By way of example, but not limitation, the cover comprises or consists of polyethylene terephthalate (PET), polycarbonate (PC), polyvinyl chloride (PVC) or polyester (PL). The insulating cover 13 can be translucent or transparent such that it allows viewing the set of electrodes 12 and the detection chamber 16. The separator sheet (14) comprises an electrically insulating material selected from pressure sensitive adhesives (PSA) and / or thermal adhesives and / or polydimethylsiloxane (PDMS), and / or polyethylene (PE), polyethylene terephthalate (PET), polycarbonate (PC) and / or polymethylmethacrylate (PMMA) coated with an adhesive on both sides, such that they adhere to the substrate and to the insulating cover.

[0033] In a preferred embodiment, the substrate 11 comprises PET, the insulating cover comprises PL and the separator sheet 14 comprises PE.

[0034] With respect to the detection chamber 16, in a preferred embodiment, the detection chamber holds between 5 and 50 pL, preferably 40 pL, with the additional advantage that large volume samples of liquid analysis sample are not required.

[0035] Regarding the origin of the liquid sample, it can be of biological, food, or environmental origin, preferably food. The source sample can be pretreated to allow the dissolution of the analyte to be quantified or detected. By way of example, but not limited to, the sample can be pretreated, for example, by filtration, dilution, or pretreatment with chemical compounds.

[0036] Regarding the dimensions of the biosensor, it preferably comprises the following dimensions:

[0037] The biosensor has a size between 15 and 20 mm wide and 25 and 40 mm long and 150 pm and 2.4 mm thick, preferably 300 pm.

[0038] The entrance has a width between 2 and 5 mm, preferably 2 mm.

[0039] The outlet has a width between 2 and 5 mm, preferably 1.5 mm.

[0040] The detection chamber is between 12 and 18 mm long and 6 and 9 mm wide.

[0041] The width of the fluidic channel is smaller than the length, the width being between 2 and 5 mm, preferably 1.5 mm.

[0042] The thickness of the substrate, cover and separator sheet is between 50 and 800 pm each, preferably 100 pm thick each.

[0043] The biosensor of the invention, therefore, comprises dimensions that allow easy transport and handling by the user.

[0044] Regarding the mediators, these can be selected from organic, inorganic, organometallic or redox polymer mediators.

[0045] In the case of organic mediators, in a preferred embodiment, this is selected from viologens, methylene blue, methylene green, bromophenol blue, phenothiazines, phenoxazines, Wurster salts, cytochrome C, quinoids and their derivatives, fulvalenes and derivatives and / or anilines, among others.

[0046] In the case of inorganic mediators, these are selected from ruthenium, cobalt, and hexacyanoferrate (II and / or III) complexes, among others. In the case of organometallic mediators, these are selected from ferrocenes and derivatives, osmium, iron, and / or ruthenium complexes, pentacyanoferrate with pyridine, pyrazole, imidazole, or heterocycles, among others.

[0047] In the context of the invention, a mediator is understood to mean a compound with redox activity that facilitates the transfer of electrons from the enzyme to the electrode surface once the enzymatic reaction has occurred. The use of mediators to facilitate enzymatic catalysis is necessary when the enzyme does not have direct electron transfer with the electrode or when this is ineffective. In the first case, the mediator allows the transport of electrons from the active site of the enzyme to the electrode surface. In the second case, the redox mediator improves the electron transfer between the enzyme and the electrode surface, increasing the catalytic efficiency and therefore the sensitivity of the device.

[0048] With respect to the enzyme, in a preferred embodiment, the enzyme is selected from reductases, oxidases, dehydrogenases, and / or oxidoreductases. In one embodiment, the enzyme is a reductase and is selected from nitrite reductase, nitrate reductase, cytochrome C reductase, aldehyde reductase, glutathione reductase, ketone reductase, or acetoacetyl-CoA reductase, among others. In another embodiment, the enzyme is an oxidase and is selected from glucose oxidase, NAD(P)H oxidase, alcohol oxidase, or cytochrome oxidase, among others. In another embodiment, the enzyme is a dehydrogenase and is selected from glucose dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase, or glutamate dehydrogenase, among others. In another embodiment, the enzyme is an oxidoreductase and is selected from peroxidases, hydroxylases, oxidases and reductases, among others.

[0049] With respect to the electrode set, in a preferred embodiment the electrode set 12 are screen-printed on the substrate, where the reference electrode is screen-printed with Ag / AgCl or Ag inks, preferably Ag / AgCl, the working electrode is made of graphite, graphene, carbon, carbon nanotubes, gold, platinum, preferably graphite, and in the embodiments comprising the counter electrode, of graphite, graphene, carbon, carbon nanotubes, gold, platinum, preferably graphite. The electrode set 12 may comprise more than one working electrode.In a preferred embodiment, the working electrode comprises at least one enzyme and optionally a mediator whose redox processes (electrochemical oxidation and / or reduction) occur at a negative potential with respect to the reference electrode, and more specifically, the reduction of the mediator and / or the enzyme occurs at a negative potential with respect to the reference electrode, preferably at potentials less than -0.4 V when the reference electrode is Ag / AgCl. In a preferred embodiment, the enzyme is a nitrite reductase or a nitrate reductase.

[0050] Ammeter enzymatic biosensors operating at working reduction potentials, and more specifically, at negative potentials with respect to the reference electrode, such as enzymatic nitrite or nitrate reductase biosensors, are susceptible to being affected by the presence of oxygen in the medium, which leads to a decrease in the accuracy and precision of the analytical result. This interference is caused by the appearance of background currents due to the direct reduction of oxygen on the electrode surface or the interaction of oxygen with the redox process of the mediator and / or enzyme. In the first case, the current recorded in the presence of oxygen is greater than the current in its absence. In the second case, oxygen interacts with the reduced form of the mediator, consuming it chemically and preventing it from participating in the redox process.This means that even the smallest amount of oxygen, whether from the sample or present in the biosensor, can produce inaccurate quantitative values. Therefore, it is common to include an oxygen scavenger in the system to completely eliminate oxygen from the medium and ensure the precision and accuracy of the analytical result, since oxygen present in the sample itself or in the biosensor can cause this interference effect to appear. Surprisingly, as shown in the results provided in Example 3, the biosensor and method of the invention achieve results comparable to the use of scavengers, facilitating the user's operation.

[0051] A second aspect of the invention relates to a method for the detection or quantification of at least one analyte dissolved in a liquid sample by means of the electrochemical biosensor of the invention, which comprises the following steps: a) introducing a quantity of liquid sample to be analyzed through inlet 10 until completely filling section 18, b) applying a potential, preferably a constant potential, to the set of electrodes 12 for a certain period of time, preferably between 20 and 60 seconds, c) measuring the current intensity produced by the electrochemical reaction, and d) translating the current intensity data obtained in step c) by means of a calibration curve obtained from standard samples comprising different concentrations of the analyte to be determined.

[0052] The potential applied relative to the reference electrode will be determined by the enzyme and mediator used. In a preferred embodiment, the enzyme is a reductase, preferably a nitrite reductase or a nitrate reductase, the analyte being nitrite and nitrate, respectively, and the applied potential is a negative potential relative to the reference electrode, preferably between -0.4 V and -0.8 V.

[0053] Translating means correlating the current intensity data with the concentration value.

[0054] In a preferred embodiment, in step a) a quantity of liquid sample to be analyzed is introduced through inlet 10 until the detection chamber 16 is completely filled, forcing the exit of part of the liquid sample to be analyzed through the liquid sample outlet 17, the user ensuring that the detection chamber is completely covered or filled with the liquid sample and that any presence of bubbles inside the detection chamber is eliminated.

[0055] Thanks to the biosensor and the method of the invention, the presence of oxygen in the detection chamber is minimized, thus minimizing oxygen interference in the reaction. Some illustrative examples are described below, which demonstrate the features and advantages of the invention; however, they should not be construed as limiting the scope of the invention as defined in the claims.

[0056] Example 1: Functioning of different mediators in a sensor of the invention without enzyme

[0057] Sensors of the invention were manufactured, each sensor comprising a mediator deposited on the working electrode: Substrate: PET

[0058] Insulating cover: PL

[0059] Separator sheet: PE

[0060] Screen-printed electrodes:

[0061] - Working electrode: graphite

[0062] - Reference electrode: Ag / AgCl

[0063] - Graphite counter electrode

[0064] Mediator (one per biosensor): methyl viologen (A), methylene blue (B), safranin (C), bromophenol blue (D).

[0065] Electrolyte: 1 M KCI solution.

[0066] Sample volume: 40 pL

[0067] The result was compared with reference biosensors, which comprise the same elements except that they do not have a cover.

[0068] Measuring equipment: commercial potentiostat.

[0069] Figure 4 shows the voltammograms obtained with sheathed sensors (solid line) with respect to the reference (dashed line) for each mediator. The x-axis shows the applied potential data, and the y-axis shows the recorded current. As can be seen in the graphs in Figure 4, sheathed sensors produce curves with better-defined reduction peaks and less negative potentials than unsheathed sensors. The redox processes recorded with the sheathed sensor show a better definition of the electrochemical reduction processes. This is mainly due to the lower background current recorded, due to the elimination of the contribution of direct reduction of oxygen on the electrode surface and / or the interaction of oxygen with the redox process of the mediator / enzyme.

[0070] Example 2: Nitrite biosensor

[0071] Sheathed and unsheathed biosensors were fabricated for nitrite quantification.

[0072] Materials:

[0073] Substrate: PET

[0074] Insulation cover: PL Separator sheet; PE

[0075] Screen-printed electrodes:

[0076] - Working electrode: graphite

[0077] - Reference electrode: Ag / AgCl

[0078] - Graphite counter electrode

[0079] Mediator and enzyme: methyl viologen and nitrite reductase

[0080] Sample volume: 40 L Applied potential: -0.725 V Potential application time: 60 s

[0081] Measuring equipment: BIO 7000

[0082] The cyclic voltammograms obtained for the coated biosensors (B) with respect to those without a coating (A) are shown in Figure 5. The signal obtained with the coated biosensors (B) is better defined than with the uncoated biosensors (A). This better performance is described by a lower background current for the blanks (buffer represented as 0 ppm nitrite) in the presence of the coating and a greater signal difference at the measurement potential in the presence of the coating than in its absence, all derived from the elimination of the background currents generated by the reduction of oxygen on the electrode surface and / or the interaction of oxygen with the redox process of the mediator / enzyme.

[0083] If we also look at the calibration curves in Figure 6, we see that reproducibility is better in the biosensor with cover (B) than without cover (A).

[0084] Example 3: Nitrite biosensor. Measurement with and without the use of an oxygen scavenger.

[0085] The results obtained with a biosensor manufactured according to example 2 in the presence of an oxygen scavenger and without the presence of an oxygen scavenger are compared.

[0086] Figure 7 shows the results of the calibration line, where curve B) represents the results of a sample pretreated with sodium sulfite as an oxygen scavenger and curve A) represents the results of the same sample without sodium sulfite pretreatment. The observed effect is the decrease of the ordinate at the origin in the presence of oxygen scavenger while its slope remains stable with values ​​of R 2 around 0.99 for both cases. This demonstrates that the cover is capable of providing data similar to those obtained using an oxygen scavenger.

Claims

CLAIMS 1. Electrochemical biosensor for the detection and / or quantification of at least one analyte in a liquid sample, comprising a liquid sample inlet (10), an electrical contact area (19), a support substrate (11) comprising at least one set of electrodes (12) comprising at least one working electrode and a reference electrode, wherein the working electrode comprises at least one enzyme and optionally a mediator, and an insulating cover (13), characterized in that between the substrate (11) and the insulating cover (13) there is a section (18) connected in fluid communication with the inlet (10), said section (18) comprising a detection chamber (16) in which the set of electrodes (12) is arranged, the biosensor (100) comprising a liquid sample outlet (17) that is in fluid communication with the section (18),in such a way that it allows the circulation of the liquid sample (10) by capillarity between the inlet (10) and the outlet (17) through the section (18) and the detection chamber (16).

2. Electrochemical biosensor according to claim 1, wherein in a longitudinal direction (X) the biosensor has a proximal zone (X1), a distal zone (X2) and a central zone (X3), wherein the zone (19) of electrical contacts is located in the distal zone (X2), the inlet (10) of liquid sample is located in the proximal zone (X1), and the outlet (17) of liquid sample is located in the central zone (X3) in a position before the zone (19) of electrical contacts.

3. Electrochemical biosensor according to claim 2, wherein the detection chamber (16) is in fluid communication with the liquid sample outlet (17) through a lateral fluidic channel (16a), the outlet (17) being on one side of the biosensor.

4. Electrochemical biosensor according to claim 3, wherein in the longitudinal direction (x), the lateral fluidic channel (16a) is arranged in a position downstream of the set of electrodes (12) at an angle with respect to the longitudinal direction (x) of between 90° and 175°.

5. Electrochemical biosensor according to any of the preceding claims, wherein the biosensor comprises at least one separating sheet (14) between the substrate (11) and the cover (13) which together with the substrate (11) and the cover (13) defines the section 6. Electrochemical biosensor according to claim 5, wherein the separating sheet (14) comprises an electrically insulating material selected from pressure-sensitive adhesives and / or thermal adhesives and / or polydimethylsiloxane, and / or polyethylene, polycarbonate and / or polymethylmethacrylate coated with an adhesive.

7. Electrochemical biosensor according to any of the preceding claims, wherein the support substrate (11) comprises an insulating and non-porous material.

8. Electrochemical biosensor according to any of the preceding claims, wherein the insulating cover (13) comprises a hydrophilic area that is in contact with the section (18).

9. Electrochemical biosensor according to any of the preceding claims, wherein the detection chamber (16) comprises at least one fluidic channel that is in fluidic communication with the liquid sample inlet (10).

10. Electrochemical biosensor according to any of the preceding claims, wherein the mediator is selected from organic, inorganic, organometallic and / or redox polymer mediators.

11. Biosensor according to claim 10, wherein the mediator is organic and is selected from viologens, methylene blue, methylene green, bromophenol blue, phenothiazines, phenoxazines, Wurster salts, cytochrome C, quinoids and their derivatives, fulvalenes and derivatives and / or anilines.

12. Biosensor according to claim 10, wherein the mediator is inorganic and is selected from ruthenium, cobalt and / or hexacyanoferrate (II and / or III) complexes.

13. Biosensor according to claim 10, wherein the mediator is organometallic and is selected from ferrocenes and derivatives, osmium, iron and / or ruthenium and / or pentacyanoferrate complexes with pyridine, pyrazole, imidazole or heterocycles.

14. Electrochemical biosensor according to any of the preceding claims, wherein the enzyme is selected from reductases, oxidases, dehydrogenases, and / or oxidoreductases.

15. Electrochemical biosensor according to any of the preceding claims, wherein the enzyme is a nitrite reductase or a nitrate reductase.

16. Electrochemical biosensor according to any of the preceding claims, wherein the detection chamber holds between 5 and 50 pL.

17. Electrochemical biosensor according to any of the preceding claims, wherein the set of electrodes (12) are screen-printed on the substrate, and comprise the Ag / AgCl or Ag reference electrode, the working electrode of graphite, graphene, carbon, carbon nanotubes, gold, and / or platinum and optionally a counter electrode of graphite, graphene, carbon, carbon nanotubes, gold and / or platinum.

18. Method for the detection or quantification of at least one analyte dissolved in a liquid sample by means of an electrochemical biosensor according to any of the preceding claims, comprising the following steps: a) introducing a quantity of liquid sample to be analyzed through the inlet (10) until the section (18) is completely filled, b) applying a potential to the set of electrodes (12) for a certain period of time, c) measuring the current intensity produced by the redox reaction, and d) translating the current intensity data obtained in step c) by means of a calibration curve obtained from standard samples comprising different concentrations of the analyte to be determined.

19. Method according to claim 18, wherein in step a) a quantity of liquid sample to be analyzed is introduced through the inlet (10) until the detection chamber (16) is completely filled, forcing the exit of part of the liquid sample to be analyzed through the liquid sample outlet (17).

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