Electrochemical regeneration of immunosensors

Abstract

The present invention relates to the field of reusable immunosensors. In particular, the present invention is a method for regenerating an immunosensor comprising at least one polypeptide attached to a conductive surface of the immunosensor, wherein the at least one polypeptide can specifically bind to an analyte detected by the immunosensor, and the method comprises applying a positive potential of about 0.3 V on the conductive surface of the immunosensor at a temperature selected from the temperature range of about 35 °C to about 42 °C for a time sufficient to enable regeneration. Further, the present invention also relates to a regenerated immunosensor obtainable by the method of the present invention, as well as systems and devices comprising the immunosensors described herein, the device being capable of applying a positive potential of about 0.3 V to the conductive surface of the immunosensor for a time sufficient to enable regeneration. The present invention also generally contemplates the use of a temperature selected from the temperature range of about 35 °C to about 42 °C and a positive potential of about 0.3 V on the conductive surface of an immunosensor according to any one of claims 1 to 13 for the regeneration of an immunosensor.

Description

Technical Field

[0001] The present invention relates to the field of reusable immunosensors. In particular, the present invention is a method for regenerating an immunosensor comprising at least one polypeptide attached to the conductive surface of the immunosensor, wherein the at least one polypeptide can specifically bind to an analyte detected by the immunosensor, and the method comprises applying a positive potential of about 0.3 V on the conductive surface of the immunosensor at a temperature selected from the temperature range of about 35°C to about 42°C for a time sufficient to enable regeneration. Further, the present invention relates to a regenerated immunosensor obtainable by the method of the present invention, as well as systems and devices comprising the immunosensors described herein, which devices can apply a positive potential of about 0.3 V to the conductive surface of the immunosensor for a time sufficient to enable regeneration. The present invention also generally contemplates the use of a temperature selected from the temperature range of about 35°C to about 42°C and a positive potential of about 0.3 V on the conductive surface of the immunosensor for the regeneration of the immunosensor.

Background Art

[0002] Regeneration of the sensor surface is an important but difficult problem in immunosensing, especially when the sensor surface has to be used for multiple subsequent measurements. Non-invasive surface regeneration of immunosensor platforms is important in order to reduce costs and to be able to perform multiple measurements without significantly impairing the sensitivity and / or specificity of the immunosensor. An immunosensor suitable for multiple measurements further provides the possibility to be adapted for continuous measurements, is embeddable and can even be adapted for use in in vivo measurements.

[0003] Many of the current playback approaches apply harsh conditions to disrupt the interaction between the analyte in the immune complex and its binding partner. These include the application of extreme pH, temperature, and chaotropic agents to dissociate the immune complex that forms the basis of immunosensing. In most of these approaches, an additional regeneration and / or wash solution is required to disrupt the interaction between the analyte and its binding partner and / or to remove the released analyte from the sensor surface. As a result, these approaches are inconsistent with the possibility of in vivo application. In many cases, these approaches significantly impair the specificity of the sensor and / or its affinity for the analyte, thus affecting the overall sensor function. Other currently known approaches utilize strong electric fields to dissociate immune complexes, which are associated with the drawbacks of undesirable oxygen reduction and electrochemical desorption, electrolysis of water on the electrodes, and non-specific adsorption of chemical components. Therefore, these approaches are also inconsistent with the possibility of in vivo application.

[0004] U.S. Patent No. 7,118,922 discloses a method for regenerating a flexural plate wave (FPW) immunosensor. The method includes the step of applying at least one electrical pulse to the bound antibody-antigen complex of the immunosensor. The electrical pulse can be applied by an electrophoresis manipulator equipped with an electrical pulse applicator.

[0005] Many studies have reported dissociating antibodies bound to surface-immobilized antigens for regenerating the immunosensor surface. However, the dissociation of antigens from surface-bound antibodies by applying a defined voltage has not been reported.

[0006] Asanov et al. reported using an electric field to enhance the dissociation of antibodies bound to surface-immobilized antigens to regenerate the surface of an immunosensor that is part of a total internal reflection fluorescence (TIRF) flow cell (Asanov et al., 1998, Anal. Chem. 70, 1156 - 1163). A polarization change swept from -0.7 V to +1.1 V was applied to achieve dissociation of the antibodies for sensor regeneration. Different sweep modes, namely "square wave" polarization and "sawtooth" polarization, were applied.

[0007] The electrical control of antibody / antigen binding has also been reported by Brod et al., 2008 (Sensors and Actuators B, 128, 560 - 565). The authors immobilize the antigen on the working electrode while the antibody is in solution. Antibody - antigen binding is monitored by electrochemical surface plasmon resonance measurements. After binding, application of a negative potential (-0.5 V versus an Ag / AgCl reference electrode) causes rapid dissociation of the antibody molecules from the antigen. When the potential is removed, the antibody rebinds to the antigen.

[0008] To enable repeated use of the immunosensor, Khoo and co - authors applied a negative potential to the working electrode to remove the antibodies bound to the epitope / antigen (Khoo, et al., 2016, Sensors and Actuators B, 224, 683 - 691). To regenerate the sensor surface of the immunosensor, the electrode polarization was set to -0.8 V (versus Ag / AgCl, 3M KCl) over a 10 - minute time interval. In this way, the authors were able to dissociate the antibodies from the epitope / antigen immobilized on the surface and achieve regeneration of the immunosensor.

[0009] So far, for the dissociation of immune complexes with antigens immobilized on the electrode surface, negative potentials or potential sweeps covering a wide voltage range have been used successfully. However, the dissociation of antigens from surface - bound antibodies would offer a much wider range of applications since many commercially available immunosensor systems are based on surface - immobilized antibodies as specific binding partners for the analyte (i.e., antigen).

[0010] Furthermore, like currently known methods, applying a negative potential of -0.6 V or less, in particular, may have the drawback of promoting undesirable oxygen reduction and electrochemical desorption of biological components related to the electrode or sensor surface. On the other hand, applying a high positive potential of +1 V or more is also disadvantageous because it promotes electrolysis of water and non-specific adsorption of chemical components on the electrode.

[0011] Therefore, there is a strong desire to provide a method for regenerating an immunosensor that overcomes these drawbacks.

Summary of the Invention

[0012] The technical problem underlying the present invention can be regarded as providing means and methods for meeting the above-mentioned needs. This is solved by the embodiments characterized below in the claims and in this specification. This problem is solved in particular by a method for regenerating an immunosensor having the features of the independent claims, a regenerated immunosensor obtained by the method, and a device comprising the same. Advantageous embodiments that can be realized alone or in any combination are listed in the dependent claims and throughout this specification.

[0013] The method according to the present invention is advantageous for at least the following reasons. According to the approach of the present inventors, the need to apply a high-intensity potential to the immunosensor is reduced. The present invention provides an improved electrochemical regeneration method by regulating the temperature at which a moderate positive potential is applied. Further, the regeneration method of the present invention has been demonstrated in a configuration in which a polypeptide capable of specifically binding to an analyte detected by an immunosensor is attached to a conductive surface, like many commercially available sensor configurations. This provides a wide range of commercial applications. The method according to the present invention can also be applied to immunosensors that are continuously measured in vivo in assays where it is important to monitor not only an increase in analyte but also a decrease in analyte concentration.

[0014] When used hereinafter, the terms "have", "comprise", or "include", or any grammatical variations thereof, are used in a non-exclusive manner. Thus, these terms can refer to both situations where there are no additional features in the entity being described in this context in addition to the features introduced by these terms, and situations where there are one or more additional features. As an example, the expressions "A has B", "A includes B", and "A contains B" can refer to both situations where there are no other elements in A besides B (i.e., the situation where A consists only of B), and situations where there are one or more additional elements such as element C, elements C and D, or additional elements in entity A besides B.

[0015] Furthermore, note that the terms "at least one" or "one or more" or similar expressions indicating that a feature or element can be present one or more times are typically used only once when introducing each respective feature or element. Hereinafter, in most cases, when referring to each respective feature or element, the expressions "at least one" or "one or more" are not repeated, despite the fact that each respective feature or element can be present one or more times.

[0016] As used herein, the term "standard conditions", unless otherwise specified, refers to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e., preferably, with respect to a temperature of 25 °C and an absolute pressure of 100 kPa. Also preferably, standard conditions include a pH of 7. Furthermore, unless otherwise indicated, the term "about" relates to the indicated value with the technical precision generally accepted in the relevant art, preferably relating to ±20% of the indicated value, more preferably ±10%, and most preferably ±5%. With respect to temperature values expressed in °C, the term "about" typically refers to a deviation of + / -0.5 °C from a given value.

[0017] Furthermore, the term "essentially" means that there are no deviations that affect the indicated result or use, i.e., even if there are deviations, the indicated result does not deviate by more than ±20%, more preferably ±10%, and most preferably ±5%. Thus, "consisting essentially of" means including the specified components, but excluding other components such as materials present as impurities, inevitable materials resulting from the processes used to provide the components, and components added for purposes other than achieving the technical effects of the present invention. For example, a composition defined using the phrase "consisting essentially of" includes any known acceptable additives, additives, diluents, carriers, etc.

[0018] Furthermore, when used hereinafter, the terms "preferably", "more preferably", "most preferably", "in particular", "more specifically", "specifically", "more specifically", "typically", "more typically" or similar terms are used in combination with any feature without further limiting possibilities. Thus, the features introduced by these terms are optional features and are not intended to limit the technical scope of the claims in any way. The present invention may be practiced, as would be understood by those skilled in the art, by using alternative features. Similarly, features introduced by the expression "in one embodiment" or similar expressions are any features, without any limitation regarding further embodiments of the present invention, without any limitation regarding the scope of the present invention, and without any limitation regarding the possibility of combining such introduced features with any other optional or non-optional features.

[0019] The above definitions apply hereinafter with the necessary modifications. The additional definitions and explanations made hereinafter apply mutatis mutandis to all embodiments described herein (unless otherwise specified).

[0020] The present invention relates to a method for regenerating an immunosensor comprising at least one polypeptide attached to a conductive surface of the immunosensor, wherein the at least one polypeptide can specifically bind to an analyte detected by the immunosensor, and at a temperature selected from the temperature range of about 35°C to about 42°C, applying a positive potential of about 0.3 V to the conductive surface of the immunosensor for a time sufficient to enable regeneration.

[0021] As used herein, the term "sensor" is a broad term and should be given its ordinary customary meaning to those skilled in the art and should not be limited to a special or customized meaning. This term specifically, but not limited to, any element or device configured to detect at least one condition or measure at least one measured variable, typically referring to an analyte as defined elsewhere in this specification. A sensor may specifically be, or include, an analyte sensor for at least partial implantation into a user's body tissue, more specifically an analyte sensor for continuous monitoring of an analyte. In particular, a sensor according to the present invention is understood to refer to an immunosensor as defined elsewhere in this specification.

[0022] As used herein, the term "immunosensor" relates to a sensor capable of detecting an analyte by specific binding to a polypeptide attached to a conductive surface. Typically, specific binding results in the formation of an immune complex on the conductive surface comprising an analyte detected by the immunosensor and at least one polypeptide capable of specifically binding to the analyte. Thus, an immunosensor can be regarded as a sensor that detects an analyte based on the formation of an immune complex. Further details regarding such sensors are described elsewhere in this specification.

[0023] The term "specific binding" can typically relate to the binding of an antibody to an antigen. However, alternatively, it also includes the binding of an enzyme to its substrate, the binding of a matrix protein to its binding partner, or the binding between a receptor protein and its ligand. More typically, "specific binding" is characterized by a specific binding affinity of the at least one polypeptide for the analyte. The binding affinity can typically be in the sub-molar range, more typically in the nanomolar range. Methods for determining the binding affinity of the at least one polypeptide for the analyte are known in the art and are typically based on ligand binding assays such as fluorescence quenching, isothermal titration calorimetry or surface plasmon resonance measurements (The Immunoassay Handbook, Fourth Edition, Elsevier 2013, ISBN 978-0-08-097037-0, chapter 2.12).

[0024] As referred to herein, an "immune complex" relates to the at least one polypeptide bound to the analyte. Thus, an immune complex according to the invention typically comprises at least the following two components: (i) the at least one polypeptide capable of specifically binding to the analyte detected by the immunosensor, and (ii) the analyte. More typically, the immune complex is immobilized on a conductive surface. The immobilization can typically be due to the at least one polypeptide being bound to the conductive surface of the immunosensor. More typically, at least one polypeptide that is attached to the conductive surface of the immunosensor and capable of specifically binding to the analyte detected by the immunosensor is selected from the group consisting of antibodies, receptor proteins, ligand proteins, matrix proteins, enzymes, and nucleic acid binding proteins, and / or fragments thereof, and even more typically, the polypeptide is an antibody or a fragment thereof. Thus, the formation of an immune complex according to the invention typically occurs on the conductive surface of the immunosensor.

[0025] The conductive surface may be particularly related to the surface of the working electrode of the sensor. More specifically, the sensor surface may be exposed to an analyte, or a sample or body fluid suspected of containing the analyte.

[0026] As used herein, the term "conductive surface" refers to a surface that comprises or consists of a material capable of conducting or transmitting an electric current. Suitable materials for use as a conductive surface may be, or may include, one or more metals of gold, copper, silver, palladium or platinum, particularly gold, or at least one conductive compound, for example, at least one conductive organic or inorganic compound. Examples include compounds such as Ag / AgCl, indium tin oxide (ITO). Additionally or alternatively, at least one conductive surface may be, or may include, at least one non-metallic conductive material such as graphite, carbon paste, graphene or quartz. The conductive surface may be, or may include, a layer of a suitable conductive material. Typically, the conductive surface is one that comprises gold, or a gold layer, a gold coating, or a coating that includes gold and other compounds such as nanoparticles or ITO. The terms "gold layer" and "gold coating" are used interchangeably herein. Typically, this term refers to at least one layer of gold atoms deposited on a solid surface such as an electrode surface. Methods of atomic deposition of gold are well known in the art and include chemical vapor deposition techniques such as atomic layer deposition. The gold layer or coating is understood to at least partially cover the conductive surface. A typical conductive surface according to the present invention is the surface of a gold-coated crystal or a gold electrode as commercially available from Biolin Scientific AB (Stockholm, Sweden). A more typical conductive surface according to the present invention is a graphene surface, or the surface of a carbon electrode or an indium tin oxide (ITO) electrode.

[0027] Typically, an immunosensor regenerated in accordance with the present invention includes at least one working electrode and at least one polypeptide capable of binding to an analyte. More typically, the immunosensor may further include a counter electrode, or may further include a counter electrode and a reference electrode. Even more typically, an immunosensor regenerated in accordance with the present invention includes at least one working electrode, at least one polypeptide capable of binding to an analyte, and a counter electrode. An immunosensor including two electrodes, a working electrode and a counter electrode, may sometimes be referred to elsewhere in this specification as a two-electrode system.

[0028] As used herein, the term "regenerating an immunosensor" refers to dissociating an analyte bound to the surface of the immunosensor from that surface. In particular, the term refers to dissociating an analyte from at least one polypeptide attached to the conductive surface of the sensor. Thus, this term can be understood to restore the binding capacity of the conductive surface of the immunosensor. More specifically, this term refers to disrupting the specific binding of an analyte to at least one polypeptide. In other words, the term "regenerating an immunosensor" in accordance with the present invention can be understood to refer to the non-binding or detachment of an analyte from the at least one polypeptide attached to the conductive surface of the sensor. More specifically, the term "regenerating" refers to dissociating an immunocomplex comprising a polypeptide capable of specifically binding to an analyte detected by the immunosensor and an analyte bound to the polypeptide. Dissociation restores the binding capacity of the immunosensor, particularly the binding capacity of at least one polypeptide attached to the conductive surface of the sensor and capable of specifically binding to an analyte detected by the immunosensor. Typically, regeneration by the method of the present invention restores the binding capacity of the immunosensor to at least 60%, at least 70%, or at least 75% compared to a control immunosensor. The "recovered binding capacity" is also referred to herein as the "regenerated binding capacity". The relative recovery [%] of the sensor may be calculated by multiplying the ratio of the amount of analyte (e.g., TSH) bound to the regenerated immunosensor after regeneration to the amount of analyte bound to a fresh immunosensor by 100%.

[0029] The term "control immunosensor" typically refers to an immunosensor that has not previously been in contact with a sample and / or analyte. In this specification, "control immunosensor" is also referred to as "fresh immunosensor". The term "control immunosensor" may also refer to an immunosensor that has previously been in contact with a sample and / or analyte, but has then been treated such that substantially all bound analyte molecules have been removed from at least one polypeptide bound to the conductive surface of the immunosensor by chemical and / or physical means.

[0030] Binding capacity can be measured by various methods known to those skilled in the art. Suitable methods include microbalance detection, particularly quartz crystal microbalance (QCM) detection, surface plasmon resonance measurement, electrochemical impedance spectroscopy (EIS) measurement, total internal reflection fluorescence measurement. Typically, a microbalance detection method, particularly a quartz crystal microbalance (QCM) detection method, can be implemented to measure the binding capacity of an immunosensor. Briefly, an immunosensor can be contacted with a sample containing a known amount; that is, typically an amount of analyte sufficient to achieve sensor saturation. In particular, sensor saturation can be achieved with a sample containing an analyte concentration more than 10 times the K D of the polypeptide that can specifically bind to the analyte detected by the immunosensor. Thereby, it is considered that 100% of the binding sites on the sensor surface are bound to the analyte. This means that essentially all of the available polypeptides that can specifically bind to the analyte detected by the immunosensor and are attached to the conductive surface are bound to the analyte. The weight of the bound analyte is typically measured by microbalance detection and reflects the binding capacity of the immunosensor. More typically, the total amount of analyte is the mass binding per area (ng / cm 2) is specified as; this value specifically reflects the mass of the analyte bound to the conductive surface of the immunosensor. The value measured using a fresh immunosensor under the sensor saturation conditions shall be set to 100% when compared with the binding capacity of the regenerated immunosensor. Equipment suitable for microbalance measurements is, for example, the commercially available Q-sense E4 from Biolin Scientific AB (Stockholm, Sweden) combined with an electrochemical module. The mass deposited on the surface can be extracted using a suitable software tool such as Dfind software (Stockholm, Sweden).

[0031] The terms "protein" or "polypeptide" are used interchangeably and refer to a molecule consisting of a chain of amino acids without referring to a specific mode of action, size, three-dimensional structure or origin.

[0032] The phrase "capable of specifically binding to the analyte detected by the immunosensor" generally refers to the binding affinity of at least one polypeptide for the analyte detected by the immunosensor.

[0033] As is generally known, the affinity of a polypeptide, as defined elsewhere in this specification, for its binding partner, such as an antigen or analyte, is inversely proportional to the dissociation constant (K D ). Therefore, a high affinity of at least one polypeptide for the analyte detected by the immunosensor is characterized by a low K D . Typically, a K D of less than 10 μM represents an appropriate affinity for the binding of the polypeptide to the analyte detected by the immunosensor according to the present invention. More typically, a K -15 in the range of 10 -5 M to 10 D M may represent an appropriate affinity in accordance with the present invention.

[0034] "At least one polypeptide capable of specifically binding to an analyte detected by an immunosensor" is assumed to be attached to a conductive surface. Typically, when a polypeptide is attached to a conductive surface, the polypeptide is immobilized on the surface.

[0035] Thus, according to the present invention, it can be understood that "at least one polypeptide capable of specifically binding to an analyte detected by an immunosensor" is immobilized on or configured to be immobilized on the conductive surface of the sensor. More typically, the polypeptide is bound to the conductive surface by a covalent chemical bond. Even more typically, the covalent bond can be achieved by a covalent bond to the conductive surface via a chemical linker such as the widely used EDC / NHS chemistry including N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Further methods for attaching a polypeptide to a conductive surface by a covalent chemical bond include thiol coupling or conjugation. Those skilled in the art are well aware of the procedures necessary to establish the bond by the chemical linker molecule. The type of chemical linker molecule used can be adapted based on the conductive surface and the polypeptide that binds to the conductive material of the surface.

[0036] Typically, the at least one polypeptide is bound to the analyte before performing the method according to the present invention. In other words, typically, before performing the method according to the present invention, the analyte detected by the immunosensor is bound to a polypeptide capable of specifically binding to the analyte. More typically, the immunosensor has been in contact with a sample or body fluid suspected of containing the analyte before performing the method according to the present invention. Even more typically, at least one polypeptide of the immunosensor is specifically bound to the analyte contained in the sample or body fluid. Even more typically, the binding results in the detection of the analyte by the immunosensor.

[0037] The term "sample" is understood by those skilled in the art and typically relates to any portion of a body fluid. A sample can be obtained by well-known techniques including, for example, venipuncture or arterial puncture, epidermal puncture, etc. The sample may be processed by any technical means including, but not limited to, heating, dilution, centrifugation, filtration, pH adjustment, etc. before being brought into contact with the immunosensor. Alternatively, especially in the case of in vivo measurements, the immunosensor may be in direct contact with the body fluid suspected of containing the analyte. More specifically, in the case of an implantable or implanted immunosensor, the immunosensor may be in direct contact with the body fluid suspected of containing the analyte.

[0038] As used herein, the term "body fluid" relates to all body fluids of a subject known or suspected to contain the analyte of the present invention, including interstitial fluid, blood, plasma, tears, urine, lymph fluid, cerebrospinal fluid, bile, feces, sweat, and saliva. Generally, any type of body fluid may be used. Preferably, the body fluid is a body fluid present in the body tissues of the subject such as interstitial tissue. Thus, by way of example, the body fluid can be selected from the group consisting of blood and interstitial fluid, typically interstitial fluid. However, additionally or alternatively, one or more other types of body fluids may be used. The body fluid can generally be contained in body tissues.

[0039] Typically, the polypeptide capable of specifically binding to an analyte detected by an immunosensor is selected from the group consisting of antibodies, receptor proteins, ligand proteins, matrix proteins, enzymes, and nucleic acid-binding proteins, and / or fragments thereof. More typically, the polypeptide comprises an antibody or a fragment thereof, and even more specifically an IgG or Fab fragment. Even more typically, the polypeptide is a Fab fragment that specifically recognizes a polypeptide analyte. In particular, the analyte may be selected from TSH and TnT, and suitable examples of polypeptides capable of specifically binding to an analyte detected by an immunosensor include, for example, anti-TSH F(ab’)2 antibody fragments and anti-TnT fab antibody fragments available from Roche Diagnostics GmbH, Penzberg, Germany.

[0040] According to the method of the present invention, a positive potential is applied to an immunosensor, particularly to at least one polypeptide attached to a conductive surface. The potential is in the range of about +0.1 to about +0.5 V, typically in the range of about +0.2 to +0.4 V. More typically, the potential is about +0.3 V. In particular, a two-electrode system can be used as defined elsewhere in this specification. The potential or a potential within the range is advantageous because it can contribute to the dissociation of the immune complex without harming or damaging the polypeptide and / or without changing its binding ability and / or binding specificity. It is believed that the potential, the secondary structure and / or the net charge of the polypeptide can be modified. This may interfere with the electrostatic interactions involved in the binding of the at least one polypeptide to the analyte and is expected to promote the non-binding and dissociation of the immune complex. Furthermore, the inventors have found that applying a negative potential results in irreversible changes on the sensor surface.

[0041] The appropriate positive potential applied by the method of the present invention avoids or reduces undesirable side effects including undesirable oxygen reduction, electrochemical desorption, electrolysis of water on the electrode, and non-specific adsorption of chemical components. Therefore, it is advantageous to apply an appropriate positive potential within the range described herein.

[0042] The potential shall be applied by using a conductive surface as one electrode and at least one additional electrode, particularly a counter electrode. Alternatively, at least one polypeptide attached to the conductive surface may be placed in the electric field between two electrodes that apply the potential. Those skilled in the art well know how to apply a potential to at least one polypeptide bound to the conductive surface of the immunosensor according to the present invention. Typically, the potential is applied and can be controlled by using an analog output such as a voltage source, a current source, a voltmeter, an ammeter, an impedance meter, an impedance spectrometer, a frequency analyzer, a potentiostat, a frequency generator or a battery.

[0043] The potential is applied for a time sufficient to enable regeneration of the immunosensor. Typically, the potential is applied for a time selected from the range of about 5 minutes to about 30 minutes, more typically for a time selected from the range of about 7 minutes to about 20 minutes, even more typically for a time selected from the range of about 8 minutes to about 12 minutes. Depending on the degree of regeneration envisaged, the time for which the potential is applied can be shortened or extended. More typically, the time is about 10 minutes.

[0044] Furthermore, the potential shall be applied at a temperature selected from the temperature range of about 35°C to about 42°C. The temperature can be applied and / or controlled actively, i.e., by applying heat to the immunosensor until the desired temperature within the temperature range is reached and maintained. Alternatively, the immunosensor may be placed in an environment having the desired temperature such that the temperature of the immunosensor is passively adjusted to the ambient temperature. Typically, this is the case when the immunosensor is used in in vivo applications within a subject having a body temperature within the aforementioned temperature range. More typically, the temperature assumed is within the range of 36°C to 40°C, and even more typically, the temperature assumed is about 37°C. Applying the temperature is advantageous as it contributes to dissociating the analyte from at least one polypeptide that can specifically bind to the analyte detected by the immunosensor. The inventors hypothesize that the temperature can cause or promote structural and / or conformational changes in the polypeptide and / or the analyte that may loosen the interaction between the polypeptide and the analyte in the immune complex. The effect of temperature on the dissociation of the immune complex is particularly pronounced in combination with the applied moderate potential. In other words, applying the temperature usually enables efficient dissociation of the immune complex that is already at a moderate potential. This is advantageous as it can avoid the use of harsh conditions that may be potentially harmful to the components of the immune complex, i.e., the at least one polypeptide that can specifically bind to the analyte detected by the immunosensor and the analyte. Thereby, the immunosensor can potentially be regenerated multiple times using relatively mild conditions while maintaining sensitivity and specificity.

[0045] When the immunosensor is used ex vivo, the immunosensor may typically also be treated with a washing solution to remove the analyte separated from the immunosensor. Thus, typically, the immunosensor is treated with a pH-neutral buffer solution, and more typically, at least the conductive surface of the immunosensor is treated with a pH-neutral buffer solution. Even more typically, the buffer solution is applied to remove the separated analyte. Most typically, the buffer solution is a 50 mM 4-morpholineethanesulfonic acid (MES) solution adjusted to pH 7.0. Specifically, the buffer solution is applied for a period sufficient to remove the separated analyte, for example, in the range of about 1 minute to about 30 minutes. More specifically, the buffer solution is applied to the conductive surface in a microfluidic system such as a flow cell. When using a microfluidic system, the surface of the immunosensor may be continuously rinsed with the buffer solution. Typically, the buffer solution may be contained in a reservoir that is in fluid connection with the conductive surface of the sensor. More typically, the sensor or at least the conductive surface of the sensor is disposed within a flow cell that is in fluid connection with the reservoir. Such a microfluidic system is advantageous because it can assist in the particularly efficient removal of the separated analyte.

[0046] It will be understood that regeneration in an immunosensor used in vivo does not require such washing with a pH-neutral buffer solution.

[0047] In particular, when used in vivo, the immunosensor according to the present invention may be fully or partially implantable and can thus be adapted to detect analytes in body fluids, especially interstitial fluid, in subcutaneous tissue. Other parts or components may remain outside the body tissue. For example, as used herein, the terms "implantable" or "subcutaneous" refer to being placed fully or at least partially within the body tissue of a subject. For this purpose, the immunosensor may include an insertable part, where the term "insertable part" generally may refer to a part or component of an element configured to be insertable into any body tissue. Typically, the insertable part may fully or partially include a biocompatible surface layer or coating, which causes minimal harm to the user or body tissue, at least during a typical period of use. For this purpose, the insertable part may be fully or partially covered with at least one biocompatible membrane layer, such as at least one polymer membrane, for example a gel membrane, which may be permeable to body fluids on the one hand, or at least to the analyte to be detected by the immunosensor, and impermeable to the immunosensor, especially the compounds contained in the working electrode, on the other hand, thus preventing its movement into the body tissue. Biocompatible materials suitable for use as biocompatible membrane layers are known in the art and may include one or more of the following materials: methacrylate-based polymers and copolymers, acrylamide-methacrylate-based copolymers, hyaluronic acid (HA), agarose, biodegradable polysaccharides such as dextran and chitosan, and polyvinylpyrrolidone.

[0048] In the method according to the present invention, the at least one polypeptide capable of specifically binding to the analyte detected by the immunosensor specifically recognizes the analyte. Specifically, the polypeptide is an antibody or a fragment thereof. The terms "specific recognition" and "specific binding" are used interchangeably and are explained in more detail elsewhere in this specification.

[0049] As used herein, the term "analyte" is a broad term and should be given its ordinary and customary meaning to one of ordinary skill in the art and should not be limited to a special or customized meaning. Specifically, this term may refer to any element, component or compound that may be present in a body fluid, the concentration of which may be of interest, for example, to a subject or a healthcare professional. Specifically, an analyte may be or may include any chemical substance or chemical compound that may be involved in the metabolism of a subject or patient. As an example, at least one analyte may be selected from the group consisting of polypeptides, small molecules, carbohydrates, and lipids. More specifically, the analyte is a protein or polypeptide. However, additionally or alternatively, other types of specimens may be used and / or any combination of specimens may be determined.

[0050] Typical polypeptide analytes include cardiac troponin molecules or derivatives thereof, particularly troponin T or troponin I and derivatives thereof, brain natriuretic peptide (BNP) molecules or derivatives thereof, particularly BNP or nt-proBNP and derivatives thereof; D-dimer molecules; peptide hormones such as thyreotropin (TSH), human chorionic gonadotropin (hCG), erythropoietin (EPO), insulin, somatotropin (human growth hormone, hGH), adrenocorticotropic hormone (ACTH).

[0051] More specifically, in the method according to the present invention, the at least one polypeptide specifically recognizes cardiac troponin as an analyte, and the analyte may be cardiac troponin. Typically, the cardiac troponin is troponin T. In this case, the at least one polypeptide that can specifically bind to the analyte detected by the immunosensor is an anti-troponin antibody or a fragment thereof, such as a fragment of an anti-TnT fab antibody.

[0052] Alternatively, in the method according to the present invention, the at least one polypeptide specifically recognizes TSH as an analyte, and the analyte may be TSH. In this case, the at least one polypeptide capable of specifically binding to the analyte detected by the immunosensor is an anti-TSH antibody or a fragment thereof, for example, a fragment of an anti-TSH F(ab’)2 antibody.

[0053] The method according to the present invention may include a further step of measuring using the regenerated immunosensor at least once, and a further step of applying a positive potential of about 0.3 V to the conductive surface of the immunosensor at a temperature selected from the temperature range of about 35°C to about 42°C for a time sufficient to enable regeneration after measurement. In other words, the method may include a further step of detecting the analyte using the regenerated immunosensor and a further step of regenerating the immunosensor by applying the described temperature and potential. These further steps may be regarded as a second regeneration cycle. Therefore, the present invention provides means for regeneration of one or more cycles. The number of regeneration cycles is not typically limited in principle. Typically, the method according to the present invention may provide means for regenerating the immunosensor multiple times, for example, 2 times, 3 times, 4 times, 5 times, up to 10 times, or more.

[0054] The present invention also relates to the regenerated immunosensor obtained by the method of the present invention. The regenerated immunosensor typically has a binding capacity of at least 60%, at least 70%, or at least 75% of that of a control immunosensor. Those skilled in the art know how the binding capacity of an immunosensor can be measured. An exemplary procedure for measuring the binding capacity is described in more detail elsewhere in this specification.

[0055] The present invention also relates to a method for detecting an analyte in a sample or body fluid using the above-described regenerated immunosensor.

[0056] Furthermore, the present invention relates to an apparatus comprising an immunosensor as described herein, the apparatus being capable of applying a positive potential of about 0.3 V to the conductive surface of the immunosensor for a time sufficient to enable regeneration. Further, the apparatus can typically apply the potential at a temperature selected from a temperature range of about 35°C to about 42°C. More typically, the apparatus may comprise a heat source capable of actively heating the immunosensor to control the temperature. Alternatively, the apparatus may comprise a heat source capable of heating the environment around the immunosensor to passively adjust the temperature of the immunosensor to the ambient temperature. If the immunosensor is embeddable and / or is embedded, the heat source may not be necessary.

[0057] The apparatus may further comprise at least one analog output, such as a voltage source, a current source, a voltmeter, an ammeter, an impedance meter, an impedance spectrometer, a frequency analyzer, a potentiostat, a frequency generator, or a battery. The analog output can be used, in particular, to apply and control the specific positive potential described above.

[0058] Typically, the apparatus comprises at least one evaluation unit comprising at least one data processing unit, such as a microcontroller.

[0059] The apparatus typically comprises a housing and may further comprise a user interface or means for data storage and / or data transfer. The user interface may comprise a display. More typically, the apparatus comprises one or several control elements. The apparatus is typically part of a system adapted to perform a method for regenerating the immunosensor of the present invention.

[0060] Furthermore, the apparatus may comprise an analysis channel or area in which the conductive surface of the immunosensor is arranged to contact a sample, typically a body fluid.

[0061] The sensor can be combined with further sensing techniques including quartz crystal microbalance (QCM), surface plasmon resonance (SPR), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), etc. Specifically, the sensor may further be combined with a microbalance unit. In particular, when used in in vitro embodiments, the sensor may be part of a fluid system, particularly a microfluidic system, that includes at least a pump and one or more valve selectors.

[0062] In particular, when used in in vitro embodiments and when part of a fluid system, the device may typically include a reservoir for a pH neutral buffer solution as specified elsewhere in this specification. The device is typically associated with a peristaltic pump and may further comprise at least one flow cell, typically at least one electrochemical flow cell. The reservoir may be microfluidically connected to the flow cell and the peristaltic pump.

[0063] Furthermore, the device may be a device that can be fully or partially implanted. Thus, the device may further include a biocompatible membrane layer as described elsewhere in this specification. Typically, the device is adapted for in vivo measurement of an analyte that is specifically recognized by at least one polypeptide of the immunosensor. More typically, the in vivo measurement is a continuous or semi - continuous measurement. The semi - continuous measurement is preferably an important one for subsequent measurements at defined time intervals (e.g., every 0.1 - 1000 seconds). More typically, the in vivo measurement is a real - time in vivo measurement. Even more typically, the analyte may be present and detectable in the interstitial fluid of the subject and in the device. Further, the device may be implanted in a tissue suitable for contact with the interstitial fluid.

[0064] The present invention further relates to a system adapted to perform a method for regenerating an immunosensor. The system comprises an apparatus as outlined elsewhere in this specification and at least one electronic control element. The electronic control element can control at least the parameters applied to the method of the present invention. The parameters include a positive potential, the time for applying the potential, and typically also the temperature. The electronic control element is typically a data processing unit, such as a microcontroller.

[0065] The present invention also generally intends to use a temperature selected from a temperature range of about 35°C to about 42°C and a positive potential of about 0.3 V on the conductive surface of the immunosensor for the regeneration of the immunosensor, as described elsewhere in this specification. Such use is advantageous as it enables efficient regeneration of the immunosensor.

[0066] The following are the preferred embodiments contemplated by the present invention.

[0067] Embodiment 1: A method for regenerating an immunosensor comprising at least one polypeptide attached to the conductive surface of the immunosensor, wherein the at least one polypeptide can specifically bind to an analyte detected by the immunosensor, the method comprising applying a positive potential of about 0.3 V to the conductive surface of the immunosensor for a time sufficient to enable regeneration at a temperature selected from a temperature range of about 35°C to about 42°C.

[0068] Embodiment 2: The method according to Embodiment 1, wherein the at least one polypeptide is bound to the analyte before the method is carried out.

[0069] Embodiment 3: The method according to Embodiment 1 or 2, wherein the polypeptide capable of specifically binding to the analyte detected by the immunosensor is selected from the group consisting of an antibody, a receptor protein, a matrix protein, an enzyme, and a nucleic acid-binding protein.

[0070] Embodiment 4: The method according to any one of Embodiments 1 to 3, wherein the time is selected from the range of about 8 to about 12 minutes.

[0071] Embodiment 5: The method according to Embodiment 4, wherein the time is about 10 minutes.

[0072] Embodiment 6: The method according to any one of Embodiments 1 to 5, wherein the immunosensor is treated with a pH neutral buffer solution.

[0073] Embodiment 7: The method according to Embodiment 6, wherein the buffer solution is applied to remove the separated antigen.

[0074] Embodiment 8: The method according to Embodiment 6 or 7, wherein the buffer solution is a 50 mM 4-morpholineethanesulfonic acid (MES) solution adjusted to pH 7.0.

[0075] Embodiment 9: The method according to any one of Embodiments 1 to 8, wherein the binding capacity of the immunosensor can be restored to at least 60%, at least 70%, or at least 75% compared to a control immunosensor.

[0076] Embodiment 10: The method according to any one of Embodiments 1 to 9, wherein the at least one polypeptide specifically recognizes cardiac troponin and the analyte is cardiac troponin.

[0077] Embodiment 11: The method according to Embodiment 10, wherein the cardiac troponin is troponin T.

[0078] Embodiment 12: The method according to any one of Embodiments 1 to 9, wherein the at least one polypeptide specifically recognizes TSH and the analyte is TSH.

[0079] Embodiment 13: A method according to any one of Embodiments 1 to 11, further comprising a further step of measuring using the regenerated immunosensor, and a further step of applying a positive potential of about 0.3 V onto the conductive surface of the immunosensor for a time sufficient to enable regeneration after measurement, at a temperature selected from the temperature range of about 35°C to about 42°C, at least once.

[0080] Embodiment 14: A regenerated immunosensor obtained by the method according to any one of Embodiments 1 to 13.

[0081] Embodiment 15: An apparatus comprising the immunosensor according to Embodiment 14, wherein the apparatus can apply a positive potential of about 0.3 V onto the conductive surface of the immunosensor for a time sufficient to enable regeneration.

[0082] Embodiment 16: The apparatus according to Embodiment 15, wherein the positive potential is applied at a temperature selected from the temperature range of about 35°C to about 42°C.

[0083] Embodiment 17: The apparatus according to Embodiment 15 or 16, comprising a reservoir for a pH-neutral buffer solution according to any one of Embodiments 6 to 8.

[0084] Embodiment 18: The apparatus according to Embodiment 15 or 17, wherein the sample is an implantable device.

[0085] Embodiment 19: The apparatus according to any one of Embodiments 15 to 18, wherein the apparatus is adapted for in vivo continuous measurement of an analyte specifically recognized by at least one polypeptide of the immunosensor.

[0086] Embodiment 20: The apparatus according to Embodiment 19, wherein the in vivo measurement is a real-time in vivo measurement.

[0087] Embodiment 21: A system suitable for carrying out the method according to any one of Embodiments 1 to 13, i) At least one immunosensor comprising at least one polypeptide attached to the conductive surface of the immunosensor, wherein the at least one polypeptide is capable of specifically binding to an analyte detected by the immunosensor; and at least one control element capable of applying a positive potential of about 0.3 V on the conductive surface of the immunosensor for a time sufficient to enable regeneration; and ii) A system comprising at least one electronic control element capable of controlling at least said parameters applied in the method according to any one of Embodiments 1 to 13.

[0088] Embodiment 22: The system according to Embodiment 21, wherein at least one electronic control element is further capable of controlling temperature.

[0089] Embodiment 23: The system according to Embodiment 21 or 22, wherein the polypeptide capable of specifically binding to the analyte detected by the immunosensor is selected from the group consisting of an antibody, a receptor protein, a matrix protein, an enzyme, and a nucleic acid-binding protein.

[0090] Embodiment 24: The system according to any one of Embodiments 21 to 23, wherein the immunosensor is a regenerated immunosensor obtained by the method according to any one of Embodiments 1 to 13.

[0091] Embodiment 25: The system according to any one of Embodiments 21 to 24, wherein the at least one electronic control element is a microcontroller capable of controlling at least a positive potential and a time for applying the potential.

[0092] Embodiment 26: Use of a temperature selected from the temperature range of about 35°C to about 42°C and a positive potential of about 0.3 V on the conductive surface of the immunosensor according to any one of Embodiments 1 to 13 for regeneration of the immunosensor.

[0093] All references cited in this specification are hereby incorporated by reference in their entirety and as specifically referred to herein for their entire disclosure content.

Brief Description of the Drawings

[0094]

Figure 1-1

Figure 1-2

Figure 2

Figure 3-1

Figure 3-2

Figure 4

Figure 5

Example

[0095] The present invention will be described by way of examples. However, the examples are merely illustrative of the present invention and should in no way be construed as limiting the scope of the present invention.

[0096] Example 1: Regeneration of the sensor Chemicals: Hydrogen peroxide (30%), ammonia solution (25%), sodium hydroxide pellets (NaOH), 4-morpholineethanesulfonic acid (MES), N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and bovine serum albumin (BSA) were obtained from Sigma-Aldrich (Germany). SH-PEG-COOH (0.5 kDa) was obtained from IrisBiotech, Germany. Anti-TSH F(ab’)2 antibody fragment, anti-TnT fab antibody fragment, recombinant TSH antigen, and recombinant TnT antigen were obtained from Roche Diagnostics GmbH, Penzberg, Germany. Gold-coated quartz crystals were obtained from Biolin Scientific AB (Stockholm, Sweden).

[0097] Equipment: Measurements were performed using a Q-sense E4 from Biolin Scientific AB (Stockholm, Sweden) combined with an electrochemical module. Lumps deposited on the surface were extracted using Dfind software (Stockholm, Sweden). An Ivium-n-Stat was used for applying electrochemical voltage pulses.

[0098] Modification of QCM Chip: The quartz crystal microbalance (QCM) chip was washed in a base piranha solution (1 part of 30% H2O2 + 1 part of 25% NH3 + 8 parts of deionized (DI) water) at 70 °C for 10 minutes. Subsequently, it was thoroughly rinsed with DI water and dried with N2. The washed chip was treated with a UV lamp before modification. For the modification of the chip, the reagent was introduced into the microfluidic channel connected to the QCM (flow rate: 80 μL / min). Unless otherwise specified, all reagents were prepared in 50 mM MES buffer pH 7. Furthermore, the same buffer was used throughout the measurement for sensor stabilization and for the intermediate washing steps. The buffer-stabilized gold chip was treated with a solution of 0.5 kDa SH-PEG-COOH (40 μM) for 40 minutes (flow rate: 80 μL / min). The thiol-modified sensor chip was rinsed with buffer for 10 minutes. To activate the -COOH groups on the sensor, a mixture of EDC (0.4 mg / mL) - NHS (0.6 mg / mL) prepared in 50 mM MES buffer pH 4.7 was introduced into the flow channel for 20 minutes. The activated sensor was modified with either anti-TSH F(ab’)2 (170 μg / mL) or anti-TnT Fab antibody fragment (21 μg / mL) in 50 mM MES buffer (pH 4.7) for 30 minutes. To remove the unbound antibody fragments, buffer was flowed through the fluid channel for 10 minutes. Bovine serum albumin (BSA; 10 mg / mL in MES pH 7) was flowed through for 10 minutes to block the remaining active sites on the gold chip.

[0099] Regeneration Procedure: For glycine regeneration, 50 mM glycine pH 2 buffer was flowed over the analyte-saturated sensor for 10 minutes (flow rate of 80 μL / min). This was followed by flowing 50 mM MES buffer pH 7. When the regenerated sensor was stabilized, i.e., the sensor drift was less than about 2 Hz / 10 minutes, it was used for the detection of the analyte.

[0100] For electrochemical regeneration, a two - electrode setup was used, and a voltage of +0.3 V (for 10 minutes) was applied between the gold sensor chip as the working electrode and the built - in CE electrode of the QCM electrochemical cell. During this process, 50 mM MES buffer pH 7 was continuously flowed for 10 minutes to wash away the analyte separated from the surface of the sensor. During the whole process, the temperature of the QCM chamber was adjusted to the desired temperature (25 °C or 37 °C) with the built - in temperature controller.

[0101] A potential of 300 mV (the working electrode consists of a conductive surface to which a Fab antibody fragment, anti - TSH F(ab’)2 or anti - TnT Fab antibody fragment is conjugated via EDC - NHS chemistry) was applied, and at a temperature of 25 °C or higher, the immunosenor consisting of antibodies covalently bound to the conductive surface could be regenerated in a short incubation time of 10 minutes (the recovery rate at the tested temperature was 60% or more. See Example 2 and Figures 2 and 4).

[0102] Example 2: Regeneration of TSH - immunosensor 45 nM of TSH was used for binding to new and regenerated sensors. Using a new, i.e., fresh sensor, 57 ng of TSH was bound to the sensor as determined by electrochemical QCM (e - QCM, see Figure 1A). The sensor was regenerated using either the electrochemical regeneration protocol or the conventional glycine regeneration. The electrochemical regeneration of the TSH sensor was carried out by applying 0.3 V at 40 °C for 10 minutes according to the regeneration protocol described in detail above in this specification. After regeneration, 46 ng of TSH could be bound to the regenerated sensor, showing a regeneration binding capacity of 80.7% compared to the new sensor (Figure 1B). As a comparative example, the TSH sensor was regenerated using the conventional glycine regeneration protocol as described in more detail above in this specification. Briefly, regeneration was carried out using a glycine treatment at pH 2 for 10 minutes. Then, the regenerated sensor could bind 51 ng of TSH, representing a binding capacity of 89.5% compared to the new sensor (Figure 1C).

[0103] The regeneration binding capacity was evaluated using various regeneration approaches (Figure 2). A potential of +0.3 V was applied in combination with a temperature of 25 °C, 37 °C, or 40 °C. As an additional experiment, a temperature of 40 °C was applied without applying a potential. The sensor recovery rate was calculated by multiplying by 100% the ratio of the amount of analyte (TSH) bound to the regenerated immunosensor to the amount of analyte bound to the fresh immunosensor. The results of the present inventors indicate that higher temperatures result in an increase in TSH sensor regeneration.

[0104] Example 3: Regeneration of a cardiac troponin T immunosensor Regeneration of the sensor for cardiac troponin T (TnT). 44 nM of TnT was used for binding to new and regenerated sensors. Using a new, i.e., fresh sensor, 394 ng of TnT was bound to the sensor as measured by e-QCM (see Figure 3A). The sensor was regenerated using either an electrochemical regeneration protocol or a conventional glycine regeneration. As a comparative example, the TnT sensor was regenerated using a conventional glycine regeneration protocol as described in more detail above herein. Briefly, regeneration was performed using a glycine treatment at pH 2 for 10 minutes. Thereafter, the regenerated sensor was able to bind 304.4 ng of TnT and showed a regenerated binding capacity of 77.25% compared to the new sensor (Figure 3B). Electrochemical regeneration of the TnT sensor was carried out by applying 0.3 V at 25 °C for 10 minutes according to the regeneration protocol described in more detail above herein. After regeneration, 63.26 ng of TnT was able to bind to the regenerated sensor and showed a regenerated binding capacity of 16.05% compared to the binding capacity of the new (fresh) sensor (Figure 3C). When the temperature of the electrochemical regeneration was set to 37 °C (Figure 3D), 285.94 ng of TnT was able to bind to the regenerated sensor. The regenerated binding capacity increased significantly to 76.34%.

[0105] Figure 4 shows a comparison of various regeneration methods of the TnT sensor. The sensor recovery rate is calculated by multiplying by 100% the ratio of the amount of analyte (TSH) bound to the regenerated immunosensor to the amount of analyte bound to the fresh immunosensor. According to the results of the present inventors, regarding the regeneration of the TSH sensor (Example 2), it is shown that the higher the temperature, the more significantly the regeneration of the TnT sensor increases.

[0106] Example 4: Multiple regeneration cycles The TnT sensor was electrochemically regenerated as in Example 3, but multiple times, that is, up to 3 times. For the second and third regenerations, the regeneration procedures outlined above were repeated 2 times and 3 times, respectively. The results are shown in Figure 5. The results show that even multiple regeneration cycles of the same sensor are possible using the regeneration approach of the present inventors.

[0107] Cited references: U.S. Patent No. 7,118,922 Asanov et al., 1998, Anal. Chem. 70, 1156 - 1163) Brod et al., 2008 Sensors and Actuators B, 128, 560 - 565 Khoo et al., 2016, Sensors and Actuators B, 224, 683 - 691 The Immunoassay Handbook, Fourth Edition, Elsevier 2013, ISBN 978 - 0 - 08 - 097037 - 0, chapter 2.12

Claims

1. A method for regenerating an immunosensor comprising at least one polypeptide attached to a conductive surface of the immunosensor, wherein the at least one polypeptide can specifically bind to an analyte detected by the immunosensor, the method comprising applying a positive potential of about 0.3 V to the conductive surface of the immunosensor for a time sufficient to enable regeneration at a temperature selected from the temperature range of about 35°C to about 42°C.

2. The method according to claim 1, wherein the at least one polypeptide is bound to the analyte before the method is carried out.

3. The method according to claim 1 or 2, wherein the polypeptide that can specifically bind to the analyte detected by the immunosensor is selected from the group consisting of an antibody, a receptor protein, a matrix protein, an enzyme, and a nucleic acid binding protein.

4. The method according to any one of claims 1 to 3, wherein the time is selected from the range of about 8 to about 12 minutes.

5. The method according to claim 4, wherein the time is about 10 minutes.

6. The method according to any one of claims 1 to 5, wherein the binding capacity of the immunosensor can be restored to at least 60%, at least 70%, or at least 75% compared to a control immunosensor.

7. The method according to any one of claims 1 to 6, wherein the at least one polypeptide specifically recognizes cardiac troponin and the analyte is the cardiac troponin.

8. The method according to claim 7, wherein the cardiac troponin is troponin T.

9. A further step of measuring using the regenerated immunosensor, and a further step of applying a positive potential of about 0.3 V on the conductive surface of the immunosensor for a time sufficient to enable regeneration after measurement at a temperature selected from a temperature range of about 35°C to about 42°C, the method according to any one of claims 1 to 8, comprising at least once.

10. A system suitable for implementing the method according to any one of claims 1 to 9, i) at least one immunosensor comprising at least one polypeptide attached to the conductive surface of the immunosensor, the at least one polypeptide being capable of specifically binding to an analyte detected by the immunosensor; An apparatus comprising at least one control element capable of applying a positive potential of about 0.3 V on the conductive surface of the immunosensor for a time sufficient to enable regeneration; and ii) at least one electronic control element capable of controlling at least the parameters applied in the method according to any one of claims 1 to 9 A system comprising.

11. The system according to claim 10, wherein the at least one electronic control element is further capable of controlling the temperature.

12. The polypeptide capable of specifically binding to the analyte detected by the immunosensor is selected from the group consisting of an antibody, a receptor protein, a matrix protein, an enzyme, and a nucleic acid binding protein, the system according to claim 10 or 11.

13. The immunosensor is a regenerated immunosensor obtained by the method according to any one of claims 1 to 9, the system according to any one of claims 10 to 12.

14. The system according to any one of claims 10 to 13, wherein the at least one electronic control element is a microcontroller capable of controlling at least the positive potential and the time for applying the potential. Claim 15 Use of a temperature selected from a temperature range of about 35°C to about 42°C and a positive potential of about 0.3 V on the conductive surface of the immunosensor for regeneration of the immunosensor.

Images