Electrochemical regeneration of immunosensors

Abstract

The invention relates to the field of reusable immunosensors. In particular, the present invention relates to a method for regenerating an immunosensor comprising at least one polypeptide attached to an electrically conductive surface of the immunosensor wherein the at least one polypeptide is capable of specifically binding to an analyte to be detected by the immunosensor, the method includes the step of applying a positive potential of about 0.3 V to the immunosensor on the conductive surface of the immunosensor at a temperature selected from a temperature range of about 35 DEG C to about 42 DEG C for a time sufficient to allow regeneration. Furthermore, the invention relates to a regenerated immunosensor obtainable by the method of the invention, as well as to systems and devices comprising the immunosensor described herein wherein the device is capable of applying a positive potential of about 0.3 V on the conductive surface of the immunosensor for a time sufficient to allow regeneration. In general, the present invention also contemplates the use of a temperature selected from the temperature range of about 35 DEG C to about 42 DEG C as set forth in any of claims 1 to 13 and a positive potential of about 0.3 V on a conductive surface of an immunosensor for regeneration of the immunosensor.

Description

[0001] The present invention relates to the field of reusable immunosensors. In particular, 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 is capable of specifically binding to an analyte to be detected by the immunosensor, the method comprising the step of applying a positive potential of about 0.3 V to the immunosensor on the conductive surface of the immunosensor for a time sufficient to allow regeneration at a temperature selected from the temperature range of about 35°C to about 42°C. In addition, the present invention also relates to a regenerated immunosensor obtainable by the method of the present invention, and a system and device comprising the immunosensor described herein, wherein the device is capable of applying a positive potential of about 0.3 V on the conductive surface of the immunosensor for a time sufficient to allow regeneration. In general, the present invention also 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.

[0002] In immunosensing, regeneration of the sensor surface is an important but difficult task, especially when the sensor surface has to be used for multiple subsequent measurements. Non-invasive surface regeneration of immunosensor platforms is important to reduce costs and to enable multiple measurements without significant loss of immunosensor sensitivity and / or specificity. Immunosensors suitable for multiple measurements would further offer the possibility of being adapted for continuous measurements and could even be adapted to be implantable and used for in vivo measurements.

[0003] Many regeneration methods currently followed involve the application of harsh conditions to destroy the interaction between the analyte and its binding partner in the immune complex. These methods include the application of extreme pH, temperature and chaotropic agents to dissociate the immune complex in immunosensing. In most of these methods, additional regeneration and / or washing solutions are required to destroy the interaction between the analyte and its binding partner and / or remove the released analyte from the sensor surface. This makes these methods inconsistent with potential in vivo applications. Typically, these methods suffer from significant loss of sensor specificity and / or affinity for the analyte, and therefore affect overall sensor function. Other currently known methods utilize strong electric fields to dissociate immune complexes, which are associated with the disadvantages of undesirable oxygen reduction and electrochemical desorption, water electrolysis on the electrode, and nonspecific adsorption of chemical components. Therefore, these methods are also inconsistent with potential in vivo applications.

[0004] US 7,118,922 B1 discloses a method for regenerating a bending plate wave sensor (FPW) immunosensor. The method comprises the step of applying at least one electric pulse to a bound antibody-antigen complex of the immunosensor. The electric pulse may be applied by an electrophoresis manipulator comprising an electric pulse applicator.

[0005] Many studies have reported the dissociation of antibodies bound to surface-immobilized antigens to regenerate immunosensor surfaces. However, the dissociation of antigens from surface-bound antibodies by applying a defined voltage has never been reported.

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

[0007] Brod et al. also reported electrical control of antibody / antigen binding in 2008 (Sensors and Actuators B, 128, 560–565). The authors immobilized the antigen on the working electrode while the antibody was in solution. Antibody-antigen binding was monitored by electrochemical surface plasmon resonance measurements. After binding, application of a negative potential (-0.5 V relative to an Ag / AgCl reference electrode) resulted in rapid dissociation of the antibody molecule from the antigen. When the potential was removed, the antibody re-binded to the antigen.

[0008] To ensure that the immunosensor can be reused, Khoo and co-authors applied a negative potential to the working electrode to remove the epitope / antigen-bound antibodies (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 (vs. Ag / AgCl, 3M KCl) for a time interval of 10 min. In this way, the authors were able to dissociate the antibodies from the surface-immobilized epitopes / antigens and achieve regeneration of the immunosensor.

[0009] So far, negative potentials or potential scans covering a wide voltage range have been successfully used to dissociate immune complexes in which the antigen is immobilized on the electrode surface. However, dissociation of antigens from surface-bound antibodies would offer a 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, applying a negative potential, especially a negative potential of -0.6 V or less, in currently known methods may have the disadvantage that biological components associated with the electrode or sensor surface may promote undesired oxygen reduction and electrochemical desorption. On the other hand, applying a high positive potential of more than +1 V or more is also disadvantageous because this favors water electrolysis and nonspecific adsorption of chemical components on the electrode.

[0011] Therefore, it would be highly desirable to provide a method for regenerating immunosensors that overcomes these drawbacks.

[0012] The technical problem underlying the present invention can be seen as providing means and methods that meet the aforementioned needs. This technical problem is solved by the embodiments characterized in the claims and below. In particular, this problem is solved by a method for regenerating an immunosensor, a regenerated immunosensor obtainable by said method, and a device comprising the regenerated immunosensor, having the features of the independent claims. In the dependent claims and throughout the description, advantageous embodiments are listed that can be realized individually or in any combination.

[0013] The method of the present invention is advantageous for at least the following reasons: The method employed by the inventors reduces the need to apply high intensity potentials to the immunosensor. The present invention provides an improved electrochemical regeneration method by adjusting the temperature at which a moderately positive potential is applied. In addition, the regeneration method of the present invention has been demonstrated in a configuration in which a polypeptide capable of specifically binding to an analyte to be detected by the immunosensor is attached to a conductive surface, as in many commercially available sensor configurations. This provides a wide range of commercial applications. The method according to the present invention can even be used with immunosensors that perform in vivo measurements in a continuous manner in an assay for which it is important to monitor not only the increase in analyte but also the decrease in analyte concentration.

[0014] As used below, the terms "having", "comprising" or "including" or any grammatical variations thereof are used in a non-exclusive manner. Thus, these terms may refer to the absence of other features in the entity described in this context in addition to the features introduced by these terms, or the presence of one or more other features. As an example, the expressions "A has B", "A includes B" and "A contains B" may refer to the absence of other elements in A in addition to B (i.e., the presence of A solely and exclusively consisting of B); or to the presence of one or more other elements in entity A in addition to B (such as element C, element C and element D, or even other elements).

[0015] Further, it should be noted that the terms "at least one", "one or more" or similar expressions indicating that a feature or element may be present once or more than once are usually used only once when introducing the corresponding feature or element. In the following, in most cases, when referring to the corresponding feature or element, the expression "at least one" or "one or more" is not repeated even though the corresponding feature or element may be present only once or more than once.

[0016] As used herein, if not otherwise noted, the term "standard conditions" relates to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e. preferably, a temperature of 25°C and an absolute pressure of 100 kPa; also preferably, standard conditions include a pH of 7. In addition, if not otherwise stated, the term "about" relates to the indicated value with a technical accuracy recognized in the relevant field, preferably to the indicated value ±20%, more preferably ±10%, most preferably ±5%. With respect to temperature values ​​given in °C, the term "about" generally refers to a deviation of + / -0.5°C of the given value.

[0017] In addition, the term "substantially" means that there is no deviation that has an impact on the indicated result or use, that is, the potential deviation will not cause the indicated result to deviate by more than ±20%, more preferably ±10%, and most preferably ±5%. Therefore, "substantially consisting of..." means including the specified components, but excluding other components, except for materials present as impurities, unavoidable materials present as a result of the process for providing 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 "substantially consisting of..." encompasses any known acceptable additives, excipients, diluents, carriers, etc.

[0018] In addition, as used hereinafter, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically", "typically", "more typically" or similar terms are used in conjunction with optional features without limiting other possibilities. Therefore, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As the skilled person will recognize, the present invention can be implemented by using alternative features. Similarly, the features introduced by "in one embodiment" or similar expressions are intended to be optional features without any limitation on other embodiments of the invention, without any limitation on the scope of the invention, and without any limitation on the possibility of combining the features introduced in such a manner with other optional or non-optional features of the invention.

[0019] The definitions made above apply mutatis mutandis to the following. The additional definitions and explanations made further below also apply mutatis mutandis to all embodiments described in this specification (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 is capable of specifically binding to an analyte to be detected by the immunosensor, the method comprising the step of applying a positive potential of about 0.3 V to the immunosensor 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 allow regeneration.

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

[0022] The term "immunosensor" as used herein relates to a sensor capable of detecting an analyte by specifically binding to a polypeptide attached to a conductive surface. Typically, the specific binding results in the formation of an immune complex on the conductive surface, the immune complex comprising at least one polypeptide capable of specifically binding to the analyte to be detected by the immunosensor and the analyte. Thus, an immunosensor can be considered as a sensor in which analyte detection is based on the formation of an immune complex. More details about such sensors will be described in detail elsewhere herein.

[0023] The term "specific binding" may generally refer 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 certain binding affinity of the at least one polypeptide to the analyte. The binding affinity may generally be in the submolar range; more typically in the nanomolar range. Methods for determining the binding affinity of the at least one polypeptide to the analyte are known in the art and are generally 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] The "immune complex" mentioned herein relates to the at least one polypeptide that has bound to the analyte. Therefore, the immune complex according to the present invention generally comprises at least the following two components: (i) the at least one polypeptide that is capable of specifically binding to the analyte to be detected by the immunosensor, and (ii) the analyte. More typically, the immune complex is immobilized on a conductive surface. The immobilization can generally be produced by the at least one polypeptide attached to the conductive surface of the immunosensor. More typically, the at least one polypeptide attached to the conductive surface of the immunosensor and capable of specifically binding to the analyte to be detected by the immunosensor is selected from the group consisting of the following items: 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. Therefore, the formation of the immune complex according to the present invention generally occurs at the conductive surface of the immunosensor.

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

[0026] The term "conductive surface" as used herein refers to a surface containing or consisting of a material capable of conducting or transmitting electric current. Suitable materials for use as conductive surfaces may be or may include at least one metal, such as one or more of gold, copper, silver, palladium or platinum, in particular gold. Additionally or alternatively, the conductive surface may be or may include at least one conductive compound, such as at least one conductive organic or inorganic compound. For example, compounds such as Ag / AgCl and indium tin oxide (ITO) may be mentioned. Additionally or alternatively, the 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 suitable conductive material. Typically, the conductive surface includes gold or a gold layer, a gold coating or a coating comprising gold and other compounds such as nanoparticles or ITO. The terms "gold layer" and "gold coating" are used interchangeably herein. Typically, the term refers to at least one layer of gold atoms deposited on a solid surface such as an electrode surface. Atomic deposition methods of gold are well known in the art and include chemical vapor deposition techniques, such as atomic layer deposition. A gold layer or coating is understood to be at least partially covering a conductive surface. Typical conductive surfaces consistent with the present invention are gold-coated quartz crystals, such as commercially available from Biolin Scientific AB (Stockholm, Sweden), or the surface of a gold electrode. Other typical conductive surfaces consistent with the present invention are graphene surfaces, or the surface of a carbon electrode or an indium tin oxide (ITO) electrode.

[0027] Typically, the immunosensor to be regenerated according to the present invention comprises at least one working electrode and at least one polypeptide capable of binding to an analyte. More typically, the immunosensor further comprises a counter electrode, alternatively it may further comprise a counter electrode and a reference electrode. More typically, the immunosensor to be regenerated according to the present invention comprises at least one working electrode and at least one polypeptide capable of binding to an analyte and a counter electrode. An immunosensor comprising two electrodes (a working electrode and a counter electrode) may also be referred to elsewhere herein 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 said surface. In particular, the term refers to dissociating the analyte from the at least one polypeptide attached to the conductive surface of the sensor. Thus, the term can be understood as restoring the binding capacity of the conductive surface of the immunosensor. More specifically, the term refers to destroying the specific binding of the analyte to the at least one polypeptide. In other words, the term "regenerating an immunosensor" in accordance with the present invention can be understood as meaning to disentangle or dissociate the analyte from the at least one polypeptide attached to the conductive surface of the sensor. More specifically, the term "regeneration" refers to dissociating an immune complex comprising a polypeptide capable of specifically binding to an analyte to be detected by the immunosensor and an analyte bound to the polypeptide. Dissociation results in restoration of the binding capacity of the immunosensor, in particular the binding capacity of the at least one polypeptide attached to the conductive surface of the sensor and capable of specifically binding to the analyte to be detected by the immunosensor. Typically, the regeneration according to 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 “restored binding capacity” is also referred to herein as “regenerated binding capacity.” The relative amount [%] of sensor recovery can be calculated as the ratio of the amount of analyte (e.g., TSH) bound to the immunosensor after regeneration to the amount of analyte bound to the new immunosensor multiplied by 100%.

[0029] The term "control immunosensor" generally refers to an immunosensor that has not been previously contacted with a sample and / or analyte. A "control immunosensor" is also referred to herein as a "fresh immunosensor". The term "control immunosensor" may also refer to an immunosensor that has been previously contacted with a sample and / or analyte, but has since been treated by chemical and / or physical methods in such a way that substantially all bound analyte molecules have been removed from the at least one polypeptide attached to the conductive surface of the immunosensor.

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

[0031] The terms "protein" or "polypeptide" are used interchangeably and refer to a molecule composed of a chain of amino acids, without regard to a particular mode of action, size, 3-dimensional structure or origin.

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

[0033] It is well known that the affinity of a polypeptide, as defined elsewhere herein, for its binding partner, such as an antigen or analyte, is related to the dissociation constant (K D ). Thus, the high affinity of the at least one polypeptide for the analyte to be detected by the immunosensor is characterized by a low K D Typically, K below 10 μM Drepresents the suitable affinity of the polypeptide for binding to the analyte to be detected by the immunosensor according to the present invention. More typically, -15 M to 10 -5 K in the M range D Suitable affinities may be indicated in accordance with the present invention.

[0034] The "at least one polypeptide capable of specifically binding to an analyte to be detected by the immunosensor" should be attached to a conductive surface. Typically, attaching a polypeptide to a conductive surface results in the immobilization of the polypeptide on the surface.

[0035] Therefore, according to the present invention, the "at least one polypeptide capable of specifically binding to an analyte to be detected by the immunosensor" can be understood to be fixed or configured to be fixed on the conductive surface of the sensor. More typically, the polypeptide is attached to the conductive surface by covalent chemical bonds. Even more typically, the covalent bonding can be achieved by chemical linkers such as the widely used EDC / NHS chemistry involving N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) covalently linked to the conductive surface. Further ways to attach polypeptides to conductive surfaces by covalent chemical bonds include thiol coupling or conjugation. The skilled person is well aware of the procedures necessary to establish the attachment by the chemical linker molecule. The type of chemical linker molecule used can be adjusted based on the polypeptide to be attached to the conductive surface and the conductive material of the surface.

[0036] Typically, prior to the implementation of the method according to the invention, the at least one polypeptide has bound the analyte. In other words, typically, prior to the implementation of the method according to the invention, the analyte to be detected by the immunosensor is bound to a polypeptide capable of specifically binding to the analyte. More typically, prior to the implementation of the method according to the invention, the immunosensor has been contacted with a sample or body fluid suspected of containing the analyte. Even more typically, the at least one polypeptide of the immunosensor specifically binds 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 refers to any sub-portion of a body fluid. The sample can be obtained by well-known techniques, including, for example, venous or arterial puncture, epidermal puncture, etc. Prior to contact with the immunosensor, the sample can be processed by any technical means, including but not limited to: heating, dilution, centrifugation, filtration, pH adjustment, etc. Alternatively, particularly in the case of in vivo measurements, the immunosensor can 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 can be in direct contact with the body fluid suspected of containing the analyte.

[0038] As used herein, the term "body fluid" refers to all body fluids of a subject known to contain or suspected of containing an analyte of the present invention, including interstitial fluid, blood, plasma, tears, urine, lymph, cerebrospinal fluid, bile, feces, sweat and saliva. Generally, any type of body fluid can be used. Preferably, the body fluid is a body fluid present in a body tissue of the subject, such as an interstitial tissue. Thus, as an 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 can be used. Body fluids can generally be contained in body tissues.

[0039] Also typically, the polypeptide capable of specifically binding to the analyte to be 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. More typically, the polypeptide comprises an antibody or a fragment thereof, even more particularly an IgG or Fab fragment. Even more typically, the polypeptide is a Fab fragment that specifically recognizes the polypeptide analyte. In particular, the analyte can be selected from TSH and TnT, and suitable examples of polypeptides capable of specifically binding to the analyte to be detected by the immunosensor include, for example, anti-TSH F(ab′)2 antibody fragments and anti-TnT fab antibody fragments available from Roche Diagnostics GmbH in Penzberg, Germany.

[0040] According to the method of the present invention, a positive potential should be applied to the immunosensor and in particular to the at least one polypeptide attached to the conductive surface. The potential should be in the range of about +0.1V to about +0.5V, typically in the range of about +0.2V to +0.4V. More typically, the potential should be about +0.3V. In particular, a two-electrode system as defined elsewhere herein can be used. The potential or a potential within the range is advantageous because it can contribute to the dissociation of the immune complex without harming or destroying the polypeptide and / or changing its binding capacity and / or binding specificity. It is believed that the application of the potential can modify the secondary structure and / or net charge of the polypeptide. This may interfere with the electrostatic interaction involving the binding of the at least one polypeptide to the analyte, and is expected to promote the unwinding and dissociation of the immune complex. In addition, the inventors have found that the application of a negative potential can lead to irreversible changes in the sensor surface.

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

[0042] The potential should be applied by using the conductive surface as one electrode and at least one additional electrode, in particular as a counter-electrode. Alternatively, the at least one polypeptide attached to the conductive surface can be placed in the electric field between the two electrodes to which the potential is applied. The skilled person is well aware of how to apply the potential to the at least one polypeptide attached to the conductive surface of the immunosensor according to the invention. Typically, the potential is applied and controlled by using an analog output such as, for example, 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 allow the immunosensor to regenerate. Typically, the potential is applied for a time selected from the range of about 5 min to about 30 min; more typically for a time selected from the range of about 7 min to about 20 min, and even more typically for a time selected from the range of about 8 min to about 12 min. Depending on the degree of regeneration envisioned, the time for which the potential is applied can be reduced or increased. More typically, the time is about 10 min.

[0044] Furthermore, the potential should be applied at a temperature selected from the temperature range of about 35°C to about 42°C. The temperature can be actively applied and / or controlled, i.e., by applying heat to the immunosensor until the desired temperature within the temperature range is reached and maintained. Alternatively, the immunosensor can be placed in an environment having a desired temperature, so that the temperature of the immunosensor passively adapts to the ambient temperature. Typically, this is the case if the immunosensor is used for in vivo application in a subject whose body temperature is within the above temperature range. More typically, the envisaged temperature is in the range of 36°C to 40°C, and even more typically, the envisaged temperature is about 37°C. Applying the temperature is advantageous because it helps to dissociate the analyte from the at least one polypeptide capable of specifically binding to the analyte to be detected by the immunosensor. The inventors assume that temperature can cause or promote structural and / or conformational changes in the polypeptide and / or the analyte, which can weaken 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 evident in combination with the moderate potential applied; in other words, applying the temperature typically allows the immune complex to effectively dissociate at a moderate potential. This is advantageous because the use of harsh conditions that are potentially harmful to the components of the immune complex, i.e. the at least one polypeptide capable of specifically binding to the analyte to be detected by the immunosensor and the analyte, can be avoided. This provides the possibility of regenerating the immunosensor multiple times using relatively mild conditions while maintaining its sensitivity and specificity.

[0045] If the immunosensor is used in vitro, the immunosensor can also be treated with a washing solution to remove the dissociated analyte from the immunosensor. Therefore, 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 used to remove the dissociated analyte. Most typically, the buffer solution is a 50mM 4-morpholinoethanesulfonic acid (MES) solution adjusted to pH 7.0. Specifically, the buffer solution is applied for a time period sufficient to remove the dissociated analyte, for example, for a time period in the range of about 1min to about 30min. More specifically, the buffer solution is applied to a conductive surface in a microfluidic system such as a flow cell. In the case of using a microfluidic system, the surface of the immunosensor can be rinsed in a continuous manner with a buffer solution. Typically, the buffer solution can be contained in a reservoir connected to the conductive surface fluid of the sensor. More typically, the sensor or at least the conductive surface of the sensor is located in a flow cell connected to the reservoir fluid. Such microfluidic systems are advantageous since they can support a particularly efficient removal of the dissociated analytes.

[0046] It will be appreciated that regeneration of the immunosensor for in vivo use does not require such washing using a pH neutral buffer solution.

[0047] In particular, in the case of in vivo use, the immunosensor according to the present invention can be fully or partially implantable and can therefore be suitable for detecting analytes in body fluids, especially interstitial fluids, in subcutaneous tissue. Other parts or components may remain outside the body tissue. For example, as used herein, the term "implantable" or "subcutaneous" refers to being arranged completely or at least partially in the body tissue of the subject. For this purpose, the immunosensor may include an insertable portion, wherein the term "insertable portion" generally refers to a part or component of an element configured to be inserted into any body tissue. Typically, the insertable portion may include a biocompatible surface layer or coating that has as little harmful effect as possible on the user or body tissue, at least during the typical duration of use. To this end, the insertable portion may be fully or partially covered with at least one biocompatible film layer, such as at least one polymer film, such as a gel film, which can be permeable to body fluids or at least to the analyte to be detected by the immunosensor on the one hand, and can not be permeable to the immunosensor, especially the compounds contained in the working electrode, thereby preventing it from migrating into the body tissue. Suitable biocompatible materials for use as the biocompatible film layer are known in the art and may include one or more of the following materials: methacrylate-based polymers and copolymers, acrylamide-methacrylate-based copolymers, biodegradable polysaccharides such as hyaluronic acid (HA), agarose, dextran and chitosan, and polyvinyl pyrrolidone.

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

[0049] The term "analyte" as used herein is a broad term and is given a common and customary meaning to those of ordinary skill in the art, and is not limited to a special or custom meaning. The term specifically refers to any element, component or compound that may be present in a body fluid and whose concentration may be of interest to, for example, a subject or a health care professional. Specifically, the analyte may be or may include any chemical substance or chemical compound that may participate in the metabolism of a subject or patient. For example, the at least one analyte may be selected from the group consisting of: polypeptides, small molecules, carbohydrates, lipids. More specifically, the analyte is a protein or a polypeptide. However, additionally or alternatively, other types of analytes may be used and / or any combination of analytes may be determined.

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

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

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

[0053] The method according to the present invention comprises at least once: a further step of measuring using the regenerated immunosensor, and a further step of applying a positive potential of about 0.3V to the immunosensor on the conductive surface of the immunosensor for a time sufficient to allow regeneration at a temperature selected from the temperature range of about 35°C to about 42°C after the measurement. In other words, the method may include a further step of detecting an analyte using the regenerated immunosensor and a further step of regenerating the immunosensor by applying the temperature and potential. These further steps can be regarded as a second regeneration cycle. Therefore, the present invention provides means for more than one regeneration cycle. The number of regeneration cycles is in principle not limited. Typically, the method according to the present invention can provide means for regenerating the immunosensor multiple times, such as twice, three times, four times, five times, up to ten times or more.

[0054] The present invention also relates to a regenerated immunosensor obtainable by the method of the present invention. The regenerated immunosensor typically has at least 60%, at least 70% or at least 75% of the binding capacity of the control immunosensor. Those skilled in the art know how to determine the binding capacity of an immunosensor. Exemplary procedures for determining binding capacity are described in more detail elsewhere herein.

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

[0056] However, the present invention relates to a device comprising an immunosensor as described herein, wherein the device is capable of applying a positive potential of about 0.3V on the conductive surface of the immunosensor for a time sufficient to allow regeneration. In addition, the device typically may be capable of applying the potential at a temperature selected from a temperature range of about 35°C to about 42°C. More typically, the device may include a heat source capable of actively heating and controlling the immunosensor to the temperature. Alternatively, the device may include a heat source capable of heating the environment surrounding the immunosensor to passively adjust the temperature of the immunosensor to the ambient temperature. In the case where the immunosensor is implantable and / or implanted, the heat source may not be necessary.

[0057] The device 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 pack. The analog output may be particularly useful for applying and controlling the positive potential specified above.

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

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

[0060] Furthermore, the device may comprise an analytical channel or region wherein the conductive surface of the immunosensor is placed in contact with a sample, typically a bodily fluid.

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

[0062] In particular when used in an in vitro embodiment, and when as part of a fluid system, the device can typically include a reservoir for a pH neutral buffer solution as described elsewhere herein. The device can further include at least one flow cell, typically at least one electrochemical flow cell, typically connected to a peristaltic pump. The reservoir can be connected to the flow cell and the peristaltic pump microfluidically.

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

[0064] The present invention further relates to a system suitable for carrying out the method for regenerating an immunosensor. The system comprises an apparatus as outlined elsewhere herein and at least one electronic control element. The electronic control element is capable of controlling at least the parameters applied in the method of the present invention. The parameters include the positive potential, the time of application of the potential, and typically also the temperature. The electronic control element is typically a data processing unit, such as a microcontroller.

[0065] In general, the present invention also 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 regeneration of the immunosensor as described elsewhere herein. Said use is advantageous because it allows for efficient regeneration of the immunosensor.

[0066] The following are preferred embodiments of the present invention:

[0067] Example 1: A method for regenerating an immunosensor, the immunosensor comprising at least one polypeptide attached to a conductive surface of the immunosensor, wherein the at least one polypeptide is capable of specifically binding to an analyte to be detected by the immunosensor, the method comprising the step of applying a positive potential of about 0.3 V to the immunosensor on the conductive surface of the immunosensor for a time sufficient to allow 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 has bound to the analyte before the method is performed.

[0069] Embodiment 3: The method according to embodiment 1 or 2, wherein the polypeptide capable of specifically binding to the analyte to be detected by the immunosensor is selected from the group consisting of antibodies, receptor proteins, matrix proteins, enzymes and nucleic acid binding proteins.

[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 min to about 12 min.

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

[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 dissociated antigens.

[0074] Embodiment 8: The method according to embodiment 6 or 7, wherein the buffer solution is a 50 mM 4-morpholinoethanesulfonic 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 up 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 the 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, wherein the method comprises at least once: a further step of performing a measurement using the regenerated immunosensor, and a further step of applying a positive potential of about 0.3 V to the immunosensor 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 allow regeneration after the measurement.

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

[0081] Embodiment 15: A device comprising the immunosensor of embodiment 14, wherein the device is capable of applying a positive potential of about 0.3 V to the conductive surface of the immunosensor for a time sufficient to allow regeneration.

[0082] Embodiment 16: The device of 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 device of embodiment 15 or 16, wherein the device comprises a reservoir for a pH neutral buffer solution as described in any one of embodiments 6 to 8.

[0084] Embodiment 18: The device of any one of Embodiments 15 or 17, wherein the device is an implantable device.

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

[0086] Embodiment 20: The device of embodiment 19, wherein the in vivo measurement is a real-time in vivo measurement.

[0087] Embodiment 21: A system suitable for performing the method according to any one of embodiments 1 to 13, the system comprising:

[0088] i) an apparatus comprising:

[0089] at least one immunosensor comprising at least one polypeptide attached to a conductive surface of said immunosensor, wherein the at least one polypeptide is capable of specifically binding to an analyte to be detected by the immunosensor, and

[0090] at least one control element capable of applying a positive potential of about 0.3 V to said conductive surface of the immunosensor for a time sufficient to allow regeneration;

[0091] ii) at least one electronic control element capable of controlling at least a parameter applied in the method according to any one of embodiments 1 to 13.

[0092] Embodiment 22: The system of Embodiment 21, wherein the at least one electronic control element is further capable of controlling temperature.

[0093] Embodiment 23: The system according to any one of embodiments 21 or 22, wherein the polypeptide capable of specifically binding to the analyte to be 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

[0094] 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.

[0095] Embodiment 25: The system of any one of Embodiments 21 to 24, wherein the at least one electronic control element is a microcontroller capable of controlling at least the positive potential and the time when the potential is applied.

[0096] 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 as described in any one of embodiments 1 to 13 for regeneration of the immunosensor.

[0097] The entire disclosures of all references cited in this specification and the disclosures specifically mentioned in this specification are incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 : Electrochemical regeneration of TSH sensor (e-QCM data). Shown are mass per unit area [ng / cm 2 ] relative to time [min].

[0099] (A): TSH detection with a new (fresh) non-regenerated sensor, binding 57ng TSH (this value generated with a new immunosensor was used as a reference value for calculating the regeneration efficiency)

[0100] (B): TSH detection using electrochemically regenerated TSH sensor, regenerated at 40°C and 0.3V applied for 10 min, 46ng TSH bound, representing a regenerated binding capacity of 80.7% compared to the fresh sensor measurement of (A)

[0101] (C): TSH was detected using a conventional TSH sensor, which was regenerated using glycine treatment at pH 2 for 10 minutes, binding 51 ng TSH, indicating a binding capacity of 89.5% after regeneration compared to the new sensor measurement of (A).

[0102] Figure 2 : TSH sensor recovery for different regeneration conditions. The recovery rate is expressed in %. The recovery percentage refers to the regenerated binding capacity of the sensor compared to a new, i.e., fresh sensor. The sensor recovery percentage is calculated as the ratio of the amount of analyte (TSH) bound to the immunosensor after regeneration to the amount of analyte bound by the new immunosensor multiplied by 100%. The amount of analyte bound by the new immunosensor is shown in Figure 1 middle.

[0103] Figure 3 : Evaluation of different regeneration methods for cardiac troponin T (TnT) sensors by e-QCM measurements. Shown are the mass per unit area [ng / cm 2 ] relative to time [min].

[0104] (A): Detection of TnT using the new non-regenerated sensor, which bound 394ng TnT;

[0105] (B): Detection of TnT using conventionally regenerated TnT sensor, regenerated for 2 min at pH 2 using glycine treatment, binding of 51 ng TSH, indicating 89.5% binding capacity after regeneration compared to the new sensor.

[0106] (C): Detection of TnT using an electrochemically regenerated TnT sensor; regeneration was performed at 25 °C and 0.3 V was applied for 10 min, binding 62.26 ng of TnT, indicating a regenerated binding capacity of 16.05% compared to the new sensor;

[0107] (D): Detection of TnT using electrochemically regenerated TnT sensor; regeneration was performed at 37°C and 0.3 V was applied for 10 min, 285.94 ng TnT was bound, representing a regenerated binding capacity of 76.34% compared to the new sensor.

[0108] Figure 4 : Recovery of TnT sensor for different regeneration conditions. The recovery rate is expressed in %. The percentage of recovery is as shown in Figure 2 Calculation is performed in .

[0109] Figure 5 : Multiple repetitions of sensor TnT regeneration. The bar graph shows the binding capacity after regeneration as % of sensor recovery. For new (fresh) sensors, the efficiency was set to 100% and the sensor recovered after 1st, 2nd and 3rd rounds of regeneration.

[0110] Examples

[0111] The present invention will be described by way of examples. However, these examples are only intended to illustrate the present invention and should not be construed as limiting the scope of the present invention in any way.

[0112] Example 1: Sensor Regeneration

[0113] Chemicals: H2O2 (30%), NH3 solution (25%), sodium hydroxide pellets (NaOH), 4-morpholinoethanesulfonic acid (MES), N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), and bovine serum albumin (BSA) were from Sigma-Aldrich, Germany. SH-PEG-COOH (0.5 kDa) was purchased from IrisBiotech, Germany. Anti-TSH F(ab')2 antibody fragments, anti-TnT fab antibody fragments, recombinant TSH antigen, and recombinant TnT antigen were from Roche Diagnostics GmbH, Penzberg, Germany. Gold-coated quartz crystals were purchased from Biolin Scientific AB (Stockholm, Sweden).

[0114] Instrumentation: Measurements were performed on a Q-sense E4 coupled to an electrochemical module from Biolin Scientific AB (Stockholm, Sweden). Species deposited on the surface were extracted using Dfind software (Biolin Scientific AB (Stockholm, Sweden)). Ivium-n-Stat was used for electrochemical voltage pulse applications.

[0115] Modification of QCM chip: The gold quartz crystal microbalance (QCM) chip was cleaned in a piranha base solution (1 part 30% H2O2 + 1 part 25% NH3 + 8 parts deionized (DI) water) at 70°C for 10 min. Subsequently, the chip was thoroughly rinsed with DI water and dried with N2. The cleaned chip was treated with UV light before modification. To modify the chip, the reagents were introduced into the microfluidic flow channel coupled to the QCM (flow rate: 80 μL / min). Unless otherwise stated, all reagents were prepared in 50 mM MES buffer pH 7. In addition, the same buffer was used throughout the measurement process to stabilize the sensor and perform intermediate washing steps. The buffer-stabilized gold chip was treated with a 0.5 kDa SH-PEG-COOH (40 μM) solution for 40 min (flow rate: 80 μL / min). The thiol-modified sensor chip was rinsed with buffer for 10 min. In order 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 min. The activated sensor was modified for 30 min with anti-TSH F (ab') 2 (170 μg / mL) or anti-TnT Fab antibody fragments (21 μg / mL) in 50 mM MES buffer (pH 4.7). In order to remove unbound antibody fragments, the buffer was allowed to flow in the fluid channel for 10 min. Bovine serum albumin (BSA; 10 mg / mL in MES pH 7) flowed for 10 min to block the remaining active sites on the gold chip.

[0116] Regeneration procedure: For glycine regeneration, 50 mM glycine pH 2 buffer was allowed to flow over the analyte-saturated sensor for 10 min (flow rate of 80 μL / min). This was followed by a flow of 50 mM MES buffer pH 7. Once the regenerated sensor was stable, i.e., once the sensor drift was about 2 Hz / 10 min or less, it was used to detect analytes.

[0117] For electrochemical regeneration, a two-electrode setup was used and a voltage of +0.3 V (10 min) 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 allowed to flow continuously for 10 min to wash away the dissociated analytes from the surface of the sensor. During the entire process, the temperature of the QCM chamber was adjusted to the desired temperature (25°C or 37°C) with the built-in temperature controller.

[0118] Immunosensors comprising antibodies covalently attached to a conductive surface (as working electrodes comprising a conductive surface to which Fab antibody fragments, anti-TSH F(ab')2 or anti-TnT Fab antibody fragments were coupled via EDC-NHS chemistry, as indicated above) under an applied potential of +300 mV l resulted in regeneration of the mentioned immunosensors (recovery of more than 60% for the tested temperatures, see Examples 2 and Figure 2 and Figure 4 ).

[0119] Example 2: TSH immunosensor regeneration

[0120] 45 nM TSH was used for binding to new and regenerated sensors. Using new, i.e., fresh sensors, electrochemical QCM (e-QCM, see Figure 1 A) It was determined that the sensor bound 57ng TSH. The sensor was regenerated using either an electrochemical regeneration protocol or conventional glycine regeneration. Electrochemical regeneration of the TSH sensor was performed at 40°C and 0.3V applied for 10 minutes, following the regeneration protocol described in more detail above. After regeneration, 46ng TSH could bind to the regenerated sensor, representing a regenerated binding capacity of 80.7% ( Figure 1 B). As a comparative example, the TSH sensor was regenerated using the conventional glycine regeneration protocol described in more detail above. Briefly, regeneration was performed at pH 2 for 10 minutes using glycine treatment. Thereafter, the regenerated sensor could bind 51 ng TSH, representing a regenerated binding capacity of 89.5% ( Figure 1 C).

[0121] The regenerated binding capacity using different regeneration methods has been evaluated ( Figure 2 ). A potential of +0.3 V was applied in conjunction with a temperature of 25°C, 37°C, or 40°C. As an additional experiment, a temperature of 40°C was applied without an applied potential. The percentage of sensor recovery was calculated as the ratio of the amount of analyte (TSH) bound to the immunosensor after regeneration to the amount of analyte bound to the new immunosensor multiplied by 100%. Our results indicate that higher temperatures lead to increased TSH sensor regeneration.

[0122] Example 3: Regeneration of cardiac troponin T immunosensor

[0123] Regeneration of cardiac troponin T (TnT) sensors. 44 nM TnT was used to bind to new and regenerated sensors. Using new, i.e., fresh sensors, the e-QCM (see Figure 3A) It was determined that the sensor bound 394 ng TnT. 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 the conventional glycine regeneration protocol described in more detail above. Briefly, regeneration was performed at pH 2 for 10 minutes using glycine treatment. Thereafter, the regenerated sensor could bind 304.4 ng TnT, representing a regenerated binding capacity of 77.25% ( 0.004 ng TnT) compared to a fresh sensor. Figure 3 B). Electrochemical regeneration of the TnT sensor was performed at 25°C and 0.3 V applied for 10 minutes, following the regeneration protocol described in more detail above. After regeneration, 63.26 ng of TnT could bind to the regenerated sensor, representing a regenerated binding capacity of 16.05% ( 0.05 %) compared to that of a new (fresh) sensor. Figure 3 C). The temperature of electrochemical regeneration was set at 37°C ( Figure 3 D), then 285.94ng TnT can bind to the regenerated sensor. The regenerated binding capacity increased significantly to 76.34%.

[0124] A comparison of different regeneration methods for TnT sensors is shown in Figure 4 The percentage of sensor recovery was calculated as the ratio of the amount of analyte (TSH) bound to the immunosensor after regeneration to the amount of analyte bound to the new immunosensor multiplied by 100%. Our results showed that for TSH sensor regeneration (Example 2), higher temperatures resulted in a significant increase in sensor regeneration for the TnT sensor.

[0125] Example 4: Multiple regeneration cycles

[0126] The TnT sensor was electrochemically regenerated as in Example 3, but multiple times, i.e., up to three times. For the second and third regenerations, the regeneration procedure outlined above was repeated two and three times, respectively. Figure 5 The results show that even multiple regeneration cycles of the same sensor are possible using our regeneration method.

[0127] References:

[0128] US 7,118,922 B1

[0129] Asanov et al., 1998, Anal. Chem. 70, 1156-1163)

[0130] Brod et al., 2008 Sensors and Actuators B, 128, 560–565

[0131] Khoo, et al., 2016, Sensors and Actuators B, 224, 683–691

[0132] The Immunoassay Handbook, 4th edition, Elsevier 2013, ISBN 978-0-08-097037-0, chapter 2.12

Claims

1. A method for regenerating an immunosensor, the immunosensor comprising at least one polypeptide attached to a conductive surface of the immunosensor, wherein the at least one polypeptide is capable of specifically binding to an analyte to be detected by the immunosensor, the method comprising the step of applying a positive potential of about 0.3 V to the immunosensor 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 allow regeneration.

2. The method according to claim 1, wherein the at least one polypeptide is already bound to the analyte prior to carrying out the method.

3. The method according to claim 1 or 2, wherein the polypeptide capable of specifically binding to the analyte to be detected by the immunosensor is selected from the group consisting of: antibodies, receptor proteins, matrix proteins, enzymes and nucleic acid binding proteins.

4. The method according to any one of claims 1 to 3, wherein the time is selected from the range of about 8 min to about 12 min. The method according to claim 4 , wherein the time is about 10 min.

6. The method of any one of claims 1 to 5, wherein the binding capacity of the immunosensor can be restored up 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 of claim 7, wherein the cardiac troponin is troponin T.

9. The method according to any one of claims 1 to 8, wherein the method comprises at least once: a further step of performing a measurement using the regenerated immunosensor, and a further step of applying a positive potential of about 0.3 V to the immunosensor 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 allow regeneration after the measurement.

10. A system suitable for carrying out the method according to any one of claims 1 to 9, the system comprising: i) an apparatus comprising: at least one immunosensor comprising at least one polypeptide attached to a conductive surface of the immunosensor, wherein the at least one polypeptide is capable of specifically binding to an analyte to be detected by the immunosensor, and at least one control element capable of applying a positive potential of about 0.3 V to said conductive surface of said immunosensor for a time sufficient to allow regeneration; ii) At least one electronic control element which is capable of controlling at least a parameter used in the method according to any one of claims 1 to 9.

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

12. The system according to any one of claims 10 or 11, wherein the polypeptide capable of specifically binding to the analyte to be 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.

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

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 of applying the potential.

15. 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 for regeneration of said immunosensor.

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