Method for analysing interactions between biomolecules

Abstract

Method for analysing interactions between biomolecules A method for analysing interactions between biomolecules is provided. The method is suitable to measure interactions of biomolecules within the membrane of a cell and can be used advantageously in binding assays, affinity measurement methods and kinetics measurement methods.

Description

[0001] Method for analysing interactions between biomolecules

[0002] TECHNICAL FIELD

[0003] The present invention relates to the field of measuring molecular interactions between biomolecules. In particular, it relates to the measurement of interactions between membrane proteins in a cell and further biomolecules. Further, the present invention relates to the use of such measurement methods in binding assays, affinity measurements, and kinetics measurements.

[0004] BACKGROUND

[0005] In the development of new drugs, it is required to determine a binding state, i.e. , if a potential drug molecule binds to a target, or to determine binding affinity and binding kinetics between the drug molecule and its intended target. Certain drug molecules act on membrane proteins, resulting in binding between the drug molecule and the membrane protein. In such case, determining a binding state means to evaluate if a specific drug molecule, such as an antibody, binds to a specific membrane protein. Similarly, determining binding affinity and binding kinetics means to evaluate how quickly and strongly a specific drug molecule, such as an antibody, binds to a specific membrane protein and how quickly this binding is released again.

[0006] In the prior art, several techniques for measuring biomolecular interactions are known. For instance, interactions between antigens and antibodies can be measured with Bio-Layer Interferometry (BLI), Surface Plasmon Resonance (SPR), or various kinds of ELISA assays. In BLI or SPR, a capture molecule (e.g., antigen) is isolated and coated onto the surface of a biosensor. The biosensor is then brought into contact with the sample (e.g., antibody), either by moving the biosensor into the sample or by flowing the sample over the biosensor. In the case of BLI, the measurement is performed by means of interferometry, whereas SPR measures changes in the properties of a surface plasmon. In an ELISA assay, a capture molecule (e.g., antigen) is isolated and coated onto the bottom of a well within a microtiter plate. The sample (e.g., antibody) is then added into the well, and an enzyme is bound to the sample, often by using a detection antibody. The measurement is performed by analysing a colour change caused by a reaction between the enzyme and a substrate. BLI and SPR are surface sensitive, i.e. , only interactions within a distance of around 200 nm from the surface can be measured. ELISA does not suffer from the same distance limitations, but does not provide time resolved binding information.

[0007] Alternatively, ELISA methods have been developed, wherein the enzymesubstrate reaction is not measured by colorimetric or fluorescent detection but by electrochemical detection. Further, cell-based ELISA assays are known, where the capture molecule is not isolated, but entire cells are fixed in the wells of a mictrotiter plate.

[0008] For measuring binding affinities and kinetics, most commonly a SPR system is used today. With a standard SPR method the behaviour of the surface plasmon is read out with a coherent light source. However, SPR systems have the disadvantage that they are incompatible with entire cells due to limitations of the technology. In particular, SPR cannot be used for measurements using a whole cell because cells are too large for this surface sensitive technology, because cells move, and because cells absorb and release salts. This results in the occurrence of interfering signals and additionally, leads to the problem that liquid flow channels of the apparatus, which are very thin, are blocked by the cells. Therefore, SPR systems usually work with isolated molecules. However, many membrane proteins cannot be isolated as they lose their shape, and thus their function, outside of the membrane. For SPR, isolated membrane proteins must be stabilized, which is very laborious and time-consuming.

[0009] It would therefore be advantageous to measure the binding to the membrane protein directly on the cell. This is quicker and also more accurate, as the isolated protein may not behave in the same way as in the cell membrane.

[0010] Therefore, it would be desirable to measure molecular interactions between biomolecules without the need to isolate molecules from a cell. Further, for determining binding affinity and binding kinetics, it would be advantageous if this measurement could be performed continuously without the need to replace any solutions during measurement.

[0011] SUMMARY

[0012] To solve the above problems, the present invention provides a method for analysing interactions between biomolecules, wherein the measurement can be performed without isolating molecules from a cell. The method of the invention is described in the claims. Preferred embodiments are described in the dependent claims. The inventive method is a method for analysing an interaction between at least one type of first biomolecule(s) and at least one type of second biomolecule(s), comprising the following steps:

[0013] Step (S1 ): Attaching cells comprising at least one type of first biomolecule(s) onto the surface of a sensor.

[0014] Step (S2): Providing a solution or dispersion containing at least one type of second biomolecule(s), wherein the at least one type of second biomolecule(s) is capable of forming a bonding with the at least one type of first biomolecule(s).

[0015] Optionally, before or after step (S2), the following step (S21 ) can be performed. Step (S21 ): Binding an enzyme to the at least one type of second biomolecule(s).

[0016] Preferably, step (S21) is performed before step (S2), i.e., in step (S2), a solution or dispersion containing at least one type of second biomolecule(s) linked to an enzyme is provided. Connecting the second biomolecule and the enzyme can be performed by any suitable technique known to the skilled person, e.g., by conjugation chemistry. Step (S21 ) can also be done in advance, e.g., several hours or days before providing the solution in step (S2).

[0017] Alternatively, instead of in step (S21), binding of an enzyme can be performed later during step (S31 ).

[0018] Step (S3): Dipping the sensor from step (S1 ) into the solution from step (S2) or step (S21 ).

[0019] Optionally, the following step (S31 ) can be performed after step (S3). In the inventive method, either step (S21 ) or step (S31 ) is performed.

[0020] Step (S31 ): Binding an enzyme directly or via a linker to the at least one type of second biomolecule(s).

[0021] After steps (S1 ) to (S3) or (S1 ) to (S31 ), on the sensor surface, first and second biomolecule(s) are bound together, wherein the first biomolecules are contained on the surface of a cell, i.e., within the membrane of a cell, and an enzyme is bound to the second biomolecules. Next, there are three alternatives how to proceed:

[0022] Option 1 : The binding between first and second biomolecules is investigated, i.e., how strong, how fast (kon), or how long the first biomolecule binds to the second kind of biomolecule. For this purpose, step (S4) is performed after step (S3) / (S31 ). Option 2: The release of binding between first and second biomolecules is investigated, i.e., how quickly the binding is released (koff). For this purpose, step (S5) is performed after step (S3) / (S31 ).

[0023] Option 3: First, the binding between first and second biomolecules is investigated, and subsequently, the release of binding between first and second biomolecules is investigated. For this purpose, step (S4) is performed after step (S3) / (S31 ) and subsequently, step (S5) is performed.

[0024] Step (S4) comprises the following steps (S41 ) and (S42).

[0025] Step (S41 ): Dipping the sensor from step (S3) or (S31 ) into a solution containing a substrate capable of undergoing an electrochemical reaction with the enzyme.

[0026] Step (S42): Performing electrochemical measurement in the solution of step (S41 ).

[0027] Step (S5) comprises the following steps (S51 ), (S52) and (S53).

[0028] Step (S51 ): Dipping the sensor from step (S3) or (S31 ) of from step (S42) into a buffer solution.

[0029] Step (S52): Dipping the senor into a solution containing a substrate capable of undergoing an electrochemical reaction with the enzyme.

[0030] Step (S53): Performing electrochemical measurement in the solution of step (S52).

[0031] After step (S4) (Option 1 ), or after step (S5) (Option 2), or after steps (S4) and (S5) (Option 3), the following step (S6) is performed. Of course, step (S6) can also be performed as immediate step whenever electrochemical measurements have been performed, e.g., between steps (S4) and (S5) in Option 3. Step (S6): Calculating at least one interaction between the at least one type of second biomolecule(s) and the at least one type of first biomolecule(s) based on the measured values of electrochemical measurement performed in step (S42) and / or step (S53). The measured values of electrochemical measurement are also referred to as “electrochemical changes”.

[0032] Another aspect of the present invention is the use of the inventive method in a binding assay, an affinity measurement method, or a kinetics measurement method.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG .1 is a diagram showing the individual steps of a method according to the invention.

[0035] FIG. 2 is a sketch illustrating the performance of kinetics measurement with the inventive method as described in Example 1 below. In this example, method steps (S3) and (S4) are repeated each three times, but this is only exemplary, and any suitable number of repetitions can be employed.

[0036] FIG. 3 illustrates a time-dependent signal resulting from the measurement shown in FIG. 2. The obtained signal can be fitted to a binding model.

[0037] DETAILED DESCRIPTION

[0038] The method of the present invention allows to measure binding events between biomolecules, e.g., if a specific antibody binds to membrane proteins in a cell. Further, with the inventive method, binding affinity and binding kinetics of biomolecules can be measured, e.g., how quickly and strongly a specific antibody binds to membrane proteins in a cell.

[0039] Therefore, the present invention further relates to the use of the inventive method in a binding assay, an affinity measurement method, or a kinetics measurement method. Biomolecular binding events are commonly described mathematically by a binding or affinity constant Ka[M-1] and a dissociation constant Kd [M], The binding reaction is characterized by the on-rate constant konor association rate (ka) [M-1s-1], and the unbinding reaction is characterized by the off-rate constant koff or dissociation rate (kd) [s-1], respectively.

[0040] The binding constant Ka and the dissociation constant Kd are defined as follows: where [R], [L] and [RL] represent the concentration of unbound free receptors R, the concentration of unbound free ligand L and the concentration of receptorligand complexes RL for the reaction R + L «-► RL.

[0041] When investigating a biomolecule which is a potential drug molecule, it is important to know how fast / strong / long it binds to the target, usually a protein. Just as important is the determination of how quickly the binding is released. To perform corresponding kinetics and affinity measurements related to the formation of bonds, e.g., determination of kon, preferably step (S3), step (S41 ), step (S42), and optionally step (S31 ) are repeated until a defined end condition is reached. An example for determining kon is shown in Example 1 and FIG.2 and 3. To perform corresponding kinetics and affinity measurements related to the release of bonds, e.g., determination of koff, preferably step (S51 ), step (S52), and step (S53) are repeated until a defined end condition is reached.

[0042] Any suitable end condition can be defined, but it is preferred that the defined end condition is reaching a steady-state condition or expiry of a predefined period of time. At the beginning of measurement, a certain amount of second biomolecules is contained in solution. When the sensor with the attached cells is dipped into the solution with second biomolecules, the second biomolecules start binding to the first biomolecules comprised in the cells on the sensor. For example, after 1 minute about 10% of first biomolecules have formed a binding to the second biomolecules, after 5 minutes about 20% of first biomolecules have formed a binding to the second biomolecules, etc. After a certain amount of time, an equilibrium condition is reached, such that no further increase in bound second biomolecules can be detected (steady-state as end condition). When measuring binding kinetics, the measurement can be repeated until a steady-state is reached, or it can be aborted beforehand if it takes too long (expiry of a defined period of time as end condition).

[0043] The time span between repetition of measurements in step (S42) or step (S53) is not particularly limited. However, typically the formation and release of a binding between biomolecules is a rather slow interaction, such that the measurement of about one data point per 20 seconds is sufficient. Preferably, when repeating step (S3), step (S41), step (S42), and optionally step (S31 ) until a defined end condition is reached, or when repeating step (S51 ), step (S52), and step (S53) until a defined end condition is reached, a measurement is performed every 120, every 90, every 60, every 40, every 30, every 20, or every 10 seconds, preferably every 60, or every 20 seconds.

[0044] When measuring the release of bonds (e.g., determination of koff) a pure buffer solution is used in step (S51 ), and during the measurement the signal becomes smaller. It possible to measure the release of bonds (e.g., determination of koff) after measuring the formation of bonds, (e.g., determination of kon) (Option 3), or to measure the release of bonds directly, i.e. , without measuring the formation of bonds beforehand, (Option 2). To do this, the sensor is immersed in the solution in step (S3) until binding has occurred (without measurement), after which the release of bonds is measured.

[0045] Usually, the measured values of electrochemical measurement performed in step (S42) or step (S53), i.e., the electrochemical changes, are proportional to the number of binding events between the at least one type of first biomolecule(s) and the at least one type of second biomolecule(s). Preferably, the interaction between the at least one type of second biomolecule(s) and the at least one type of first biomolecule(s) calculated in step (S6) is at least one selected from the group consisting of the number of binding events, the association rate (ka), the dissociation rate (kd), and the binding affinity.

[0046] Preferably, performing electrochemical measurement in step (S42) and / or step (S53) is performed by common electrochemical measurement techniques, particularly preferably by EIS (electrochemical impedance spectroscopy), chronoamperometry, or voltammetry.

[0047] The inventive method is not limited to a specific type of electrochemical measurement, but various types of electrochemical measurements can be facilitated including, but not limited to reduction-oxidization electrochemical measurements. For performing a reduction-oxidization electrochemical measurement, an electrode and a counter-electrode are submerged into the solution. Then, a potential difference or a current between the electrode and the counter-electrode are measured. Preferably, the sensor includes at least one of the electrodes, preferably both electrodes.

[0048] In step (S1 ), the cells can be attached to the sensor by any suitable technique known to the skilled person. Preferably, step (S1 ) is performed by dipping the sensor into a solution comprising the cells, for a time t5 sufficient for the cells to form a bonding to the surface of the sensor, or by growing the cells onto the surface of the sensor.

[0049] Preferably, at least part of the sensor surface, e.g., a sensing region of an electrode that is arranged on the sensor, is bio-functionalized to facilitate the attachment of cells on the sensor surface. There are various methods available for bio-functionalizing the electrodes on the sensor, such as forming selfassembled monolayers. Here, sulphur in an alkanethiol adheres to the electrode surface while the other end of the molecule can be functionalized with various chemical groups. In another option, biomolecules can be directly adsorbed onto the surface, using the affinity between metal and certain functional chemical groups. In yet a further option, a layer-by-layer-assembly is possible where oppositely charged polyelectrolytes, proteins, or nanoparticles form a multi-layer structure. In yet a further scenario, biomolecules can be covalently bonded to the surface of electrodes via bio-conjugation. A reactive group of the biomolecule can be used or a linker molecule can be relied upon that links the electrode surface and the biomolecules. Another option is electrochemical grafting where certain organic molecules can be grafted onto the surface of the electrodes using electrochemical process. Once a linker layer is established, additional biomolecules can be added to the sensor surface to facilitate adhesion of cells.

[0050] In the method of the present invention, preferably, the sensor is biofunctionalized before performing step (S1 ). A preferred method of biofunctionalizing the sensor is as follows: The surface of the sensor is made of gold. The first step is to add a linker layer onto the gold surface, e.g., a suitable linker is a short polymer with a sulphur group on one end and a carboxylic acid on the other end. Attaching the linker layer onto the gold surface can be achieved with common techniques known to the skilled person. Once this linker layer is established it is possible to link additional biomolecules, often proteins, to the carboxylic acid group. There are a large number of proteins that adhere to cells, and which are commercially available. Such proteins will be linked to the carboxylic acid group. In other words, the sensor surface is functionalized with a linker and proteins capable of being linked to the carboxylic acid groups of the linker and capable of adhering to the cell surface are brought into contact with the functionalized sensor surface to form a linkage between the proteins and the carboxylic acid groups of the linker. Next, in step (S1 ), the cells are attached to the sensor surface by bringing the cells into contact with the functionalized sensor surface under conditions allowing the proteins to adhere to the cells, such that the proteins bound to the linker adhere to the cells.

[0051] Alternatively, the sensor surface can be made hydrophobic by attaching a functional polymer onto the surface. In this case, the cells can be attached to the sensor surface without using adhering proteins. Preferably, in step (S1 ), the cells comprising at least one type of first biomolecule(s) are contained in a well of a mictrotiter plate in suspension and the sensor (which has preferably been bio-functionalized in advance) is dipped into this well to attach the cells to the sensor surface.

[0052] Alternatively, attaching the cells to the sensor surface can be achieved by growing the cells onto the surface of the sensor. That means, the cells are grown directly on the sensor ahead of the experiment.

[0053] Preferably, after step (S1 ), an additional step (S11 ) is performed, wherein the number of cells attached to the sensor surface is determined. This determination of surface coverage is important for standardization, as the signal strength depends on the number of cells. Step (S11 ) is preferably performed by electrochemical impedance spectroscopy (EIS).

[0054] A further advantage of the present invention is the possibility to use the method in an easy set-up. A common measurement system for performing electrochemical measurements of assays in wells of a multi-well mictrotiter plate can be employed. A suitable measurement system is disclosed in DE 10 2023 123 620. Preferably, the solutions in step (S2), and / or step (S21 ), and / or step (S3), and / or step (S31 ), and / or step (S41 ), and / or step (S51 ), and / or step (S52) are contained in wells of a mictrotiter plate. More preferably, the solutions of all steps, i.e. (S2), step (S3), step (S41 ) and / or step (S52), step (S21) or step (S31 ) and optionally step (S51) are contained in wells of a mictrotiter plate. Thus, the sensor can be dipped, for instance by a robotic actuator, into the individual wells of a mictrotiter plate. Usually, steps (S2) to (S4) or steps (S2) to (S5) can be repeated 1 to 100 times without replacing the solution(s) in step (S3), and / or step (S31 ) and / or step (S41 ) and / or step (S51 ) and / or step (S52).

[0055] When the measurement is finished, the sensor can be regenerated and used for the next measurement.

[0056] As described above, after steps (S1 ) to (S3) or (S1 ) to (S31), on the sensor surface, first and second biomolecules are bound together, wherein the first biomolecules are contained within the membrane of a cell and an enzyme is bound to the second biomolecules. Binding an enzyme to the second biomolecules in step (S21 ) or step (S31 ) can be achieved by any suitable method known to the skilled person. When the binding of the enzyme is achieved in step (S31 ), the enzyme can be bound directly or via a linker to the at least one type of second biomolecule(s). Preferably, in step (S31 ) the enzyme is bound to the at least one type of second biomolecule(s) via a linker, wherein the linker is capable of forming a bond to the at least one type of second biomolecule(s). A preferred example for the linker is a secondary antibody (detection antibody). Binding the enzyme to the at least one type of second biomolecule(s) is preferably performed by dipping the sensor from step (S3) into a solution containing the linker tagged with the enzyme. Preferably, dipping is performed for a time t4 sufficient for the linker to bind to the at least one type of second biomolecule(s).

[0057] Any suitable combination of enzyme and substrate can be employed, and such combinations are known in the art. For example, the enzyme can be selected from the group consisting of Horseradish Peroxidase (HRP) and Alkaline Phosphatase (AP), and the substrate can be selected from the group consisting of Tetramethylbenzidine (TMB) and Nitroblue Tetrazolium (NT).

[0058] The duration of the individual method steps is not particularly limited. However, to obtain reliable results , preferably, step (S3) is performed for a time t1 sufficient for at least part of the second biomolecules to be captured by at least part of the first biomolecules, steps (S41 ) and (S52) are performed for a time t2 sufficient for the enzyme to react with the substrate, and step (S51 ) is performed for a time t3 sufficient for at least part of the second biomolecules to be released from at least part of the first biomolecules.

[0059] Preferred ranges for the duration of individual method steps are a range of 0.1 sec to 24 h for time t1 , preferably 1 sec to 1 h, more preferably 10 sec to 30 min, more preferably 10 sec to 5 min; a range of 0.1 to 20 sec time for t2, preferably 0.5 to 15 sec, more preferably 1 to 10 sec; and a range of 0.1 sec to 24 h for t4, preferably 10 sec to 12 h, more preferably 10 sec to 2h, more preferably 10 sec to 15 min. For instance, t1 can be 10 sec, t2 can be 5 sec, and t4 can be 30 sec, see also the Examples below.

[0060] The duration of step (S51 ), time t3, is not particularly limited and depends on the specific types of first and second biomolecules. A skilled person knows how to determine a suitable duration t3 for dipping the sensor into a buffer solution to allow the release of second biomolecules. An exemplarily range is from 0.1 sec to 2 h, preferably 10 sec to 30 min, more preferably 10 sec to 15 min.

[0061] Optionally, between any steps of dipping the sensor in the individual wells, as described above, it can be dipped into a well containing a buffer solution for washing, as required.

[0062] In the present invention, the buffer solution is not particularly limited, and the skilled person knows how to choose an appropriate buffer solution for particular biomolecules.

[0063] In general, the method of the present invention is suitable to probe any molecular interaction between biomolecules. Preferably, the first and second biomolecule is each a protein. Preferably, the first biomolecule is a surface membrane protein. Exemplary surface membrane proteins are protein A or ionchannel proteins, such as GPCR (G protein-coupled receptor). An example for an interaction according to the present invention is an interaction between an antibody and a membrane protein. In this case, the at least one type of second biomolecule(s) is an antibody, antigen binding region or fragment thereof, and the at least one type of first biomolecule(s) is an antibody binding membrane protein. Other interactions can also be measured, for example interactions between cells. Preferably, the at least one type of second biomolecule(s) contained in the solution or dispersion of step (S2) are contained on the surface of a cell. Preferably, the second biomolecule is a surface membrane protein. Performing a measurement for interactions between first and second biomolecules which are each contained on the surface of a cell, i.e. , within the membrane of a cell, is for instance valuable in the field of T-cell therapy, where the binding of T-cells to cancer cells needs to be evaluated. Other examples of interactions between first and second biomolecules that can be measured with the inventive method are interactions between: first biomolecule second biomolecule sugar group on a cell surface protein surface receptor on a cell surface RNA fragment membrane protein on a cell surface small molecule small molecule on a cell surface protein

[0064] An example for cells comprising at least one type of first biomolecules are breast cells comprising a surface membrane protein called Her2.

[0065] Overexpression of Her2 is linked to certain types of breast cancer, thus making it a target for breast cancer therapeutics. In the early stages of finding suitable antibodies as a drug to inhibit overexpression of Her2, researchers would have a large panel, e.g., 100+, of similar antibodies all targeting Her2. It is then important to compare the binding affinities and kinetics between all the antibodies. This can be done with the inventive method. An example for the at least one type of second biomolecules capable of forming a bond with the at least one type of first biomolecules is Trastuzumab, a commercially available antibody drug, that targets breast cells comprising Her2. Thus, during the development of Trastuzumab the method according to the invention would have been very useful to evaluate the suitability of Trastuzumab to inhibit overexpression of Her2.

[0066] The kind of sensor is not particularly limited as long as the sensor is able to measure electrical signals in solution. However, particularly suitable is a dip-in- well sensor for electrochemical measurements by being dipped into an assay at a distal end. This dip-in-well sensor preferably comprises (i) a base ; (ii) at least one metal electrode arranged on a first surface or a second surface of the base and extending between the distal end and a proximal end of the dip-in-well sensor ; (iii) a substrate slab attached, at its second surface, to the first surface of the base and extending along the at least one metal electrode between the distal end and the proximal end ; and (iv) a further metal electrode arranged on a first surface of the substrate slab and extending between the distal end and the proximal end .Such sensor is disclosed in DE 10 2023 123 620 and is particularly advantageous because it can be bio-functionalized, is easy-to- manufacture, and can be conveniently attached to mounting brackets of a measurement system.

[0067] Preferably, the cells comprising at least one type of first biomolecules are attached to a sensing region of the further metal electrode that is arranged at the distal end of the sensor. The further metal electrode also forms a connection region that extends away from the sensing region towards the proximal end of the sensor. The width of the sensing region and the connection region of the further metal electrode is defined in the plane of the respective metal layer perpendicular to the longitudinal direction of sensor extending between the distal end and the proximal end. In other words, the connection region can form a constriction of the further metal electrode. The further metal electrode includes a narrow center part. This has the effect that the surface of the further metal electrode facing the surrounding - and, accordingly, potentially in contact with the assay - is reduced in the connection region if compared to a scenario in which the connection region has the same width as the sensing region. Thereby, the electrochemical measurement is less sensitive to varying cover ratios of the assay on the connection region. Thus, the signal of the electrochemical measurement predominantly stems from the sensing region of the further metal electrode.

[0068] Thus, a more reproducible signal is provided, and the electrochemical measurement is more accurate. EXAMPLE 1

[0069] In the following, the determination of kon for binding of an antibody to a surface membrane protein of cells (kinetics measurement) is described as an example of the inventive method with reference to FIG. 2 and 3.

[0070] A common measurement system for performing electrochemical measurements of assays in wells of a multi-well mictrotiter plate is used. Cells comprising a surface membrane protein as first biomolecule, such as breast cells comprising Her2, are attached to the surface of a biosensor (step (S1 ) in FIG. 2). The number of cells attached to the sensor surface is determined by electrochemical impedance spectroscopy (EIS) (step (S11 ), not shown in FIG. 2).

[0071] Next, a solution containing an antibody, such as Trastuzumab, as second biomolecule is provided in a well of the mictrotiter plate. The antibody is conjugated with an enzyme, such as HRP (Horseradish Peroxidase) (steps (S2) and (S21 ), not shown in FIG. 2), and the sensor is dipped into the solution for 10 seconds. During this time, some of the antibody molecules bind to the surface membrane proteins (step (S3) in FIG. 2)

[0072] Then, the sensor is dipped for 5 seconds into another well of the mictrotiter plate containing a solution of a substrate, such as Tetramethylbenzidine (TMB). Electrochemical measurement is performed by chronoamperometry to measure the electrochemical reaction between HRP and TMB in the solution (steps (S41) and (S42) in FIG. 2). A signal intensity p1 dependent on the number of antibodies bound to the surface membrane protein is obtained. By using the results from step (S11 ), the signal strength can be standardized in dependency on the number of cells attached to the sensor.

[0073] The sensor is dipped again for 10 seconds into the well with the solution containing the antibody (repetition of step (S3)); and for 5 seconds into the well with the substrate solution and electrochemical measurement is performed (repetition of steps (S41 ) and (S42)). A signal intensity p2 is obtained. Then, the procedure is repeated again, and a signal intensity p3 is obtained.

[0074] Optionally, between any steps of dipping the sensor in the individual wells, as described above, it can be dipped into a well containing a buffer solution for washing, as required.

[0075] The values p1 , p2, and p3 are plotted against the total duration of dipping the sensor into the solution containing the antibody. A curve fit is performed and the parameter kon for the binding reaction between surface membrane protein and antibody can be obtained from the following equation (step (S6), see FIG. 3):

[0076] S(t) oc exp (-fcont) ,

[0077] S(t): signal intensity at time t t: total duration of dipping the sensor into the solution containing the antibody.

[0078] The time t is calculated by adding up the times of step (S3), i.e. , after the first dipping of the sensor into the solution with the antibody for 10 seconds, t = 10 sec, after the second dipping of the sensor into the solution with the antibody for 10 seconds, t = 20 sec, etc.

[0079] EXAMPLE 2

[0080] Example 2 is performed in the same way as Example 1 . However, step (S31 ) is performed instead of providing a solution containing an antibody which is conjugated with an enzyme (step (S21 )).

[0081] Cells comprising a surface membrane protein as first biomolecule, such as breast cells comprising Her2, are attached to the surface of a biosensor (step (S1 )). The number of cells attached to the sensor surface is determined by electrochemical impedance spectroscopy (EIS) (step (S11 )). A solution containing an antibody (sample antibody), such as Trastuzumab, not conjugated with an enzyme, as second biomolecule is provided in a well of the mictrotiter plate (step (S2)). The sensor is dipped into this solution for 10 seconds. During this time, some of the antibody molecules bind to the surface membrane proteins (step (S3)).

[0082] Next, the sensor is dipped into a well containing a detection antibody (i.e. , a linker) tagged with an enzyme, such as HRP, for 30 seconds. The tagged detection antibody binds to the sample antibody (step (S31 )).

[0083] Then, the sensor is dipped for 5 seconds into another well of the mictrotiter plate containing a solution of a substrate, such as Tetramethylbenzidine (TMB). Electrochemical measurement is performed by chronoamperometry to measure the electrochemical reaction between HRP and TMB in the solution (steps (S41) and S42)). A signal intensity p1 dependent on the number of sample antibodies bound to the surface membrane protein is obtained. By using the results from step (S11 ), the signal strength can be standardized in dependency on the number of cells attached to the sensor.

[0084] The sensor is dipped again for 10 seconds into the well with the solution containing the sample antibody (repetition of step (S3)); for 30 seconds into the well containing the detection antibody (repetition of step (S31 )); and for 5 seconds into the well with the substrate solution, and electrochemical measurement is performed (repetition of steps (S41 ) and (S42)). A signal intensity p2 is obtained. Then, the procedure is repeated again, and a signal intensity p3 is obtained.

[0085] Optionally, between any steps of dipping the sensor in the individual wells, as described above, it can be dipped into a well containing a buffer solution for washing, as required.

[0086] The values p1 , p2, and p3 are plotted against the total duration of dipping the sensor into the solution containing the sample antibody. A curve fit is performed and the parameter kon for the binding reaction between surface membrane protein and sample antibody can be obtained (step (S6)).

Claims

1 . A method for analysing an interaction between at least one type of first biomolecule(s) and at least one type of second biomolecule(s), comprising:(51 ) attaching cells comprising at least one type of first biomolecule(s) onto the surface of a sensor,(52) providing a solution or dispersion containing at least one type of second biomolecule(s), wherein the at least one type of second biomolecule(s) is capable of forming a bond with the at least one type of first biomolecule(s),(S21 ) binding an enzyme to the at least one type of second biomolecule(s), wherein step (S21 ) is performed before or after step (S2), wherein either step (S21 ) or step (S31 ) is performed,(53) dipping the sensor from step (S1 ) into the solution from step (S2) or step (S21 ),(S31 ) binding an enzyme directly or via a linker to the at least one type of second biomolecule(s), wherein either step (S21 ) or step (S31 ) is performed,(54) (S41 ) dipping the sensor from step (S3) or (S31 ) into a solution containing a substrate capable of undergoing an electrochemical reaction with the enzyme, and(S42) performing electrochemical measurement in the solution of step (S41 ), and / or(55) (S51 ) dipping the sensor from step (S3) or (S31 ) or from step (S42) into a buffer solution,(552) dipping the senor into a solution containing a substrate capable of undergoing an electrochemical reaction with the enzyme, and(553) performing electrochemical measurement in the solution of step (S52), wherein step (S5) is performed subsequently to step (S4) or instead of step (S4), and(56) calculating at least one interaction between the at least one type of second biomolecule(s) and the at least one type of first biomolecule(s) based on the measured values of electrochemical measurement performed in step (S42) and / or step (S53).

2. The method of claim 1 , wherein step (S3) is performed for a time t1 sufficient for at least part of the second biomolecules to be captured by at least part of the first biomolecules, and / or step (S41 ) and / or step (S52) is performed for a time t2 sufficient for the enzyme to react with the substrate, and / or step (S51 ) is performed for a time t3 sufficient for at least part of the second biomolecules to be released from at least part of the first biomolecules.

3. The method of claim 1 or 2, whereinafter step (S1 ), an additional step (S11 ) is performed, wherein the number of cells attached to the sensor surface is determined, preferably by electrochemical impedance spectroscopy (EIS).

4. The method of any one of claims 1 -3, wherein step (S3), step (S41 ), step (S42), and optionally step (S31 ), are repeated until a defined end condition is reached, and / or step (S51 ), step (S52) and step (S53) are repeated until a defined end condition is reached, preferably wherein the defined end condition is reaching a steady-state condition or expiry of a predefined period of time.

5. The method of any one of claims 1 -4, wherein in step (S31 ) the enzyme is bound to the at least one type of second biomolecule(s) via a linker, wherein the linker is capable of forming a bonding to the at least one type of second biomolecule(s), and step (S31 ) is performed by dipping the sensor from step (S3) into a solution containing the linker tagged with the enzyme, for a time t4 sufficient for the linker to bind to the at least one type of second biomolecule(s), preferably wherein the linker is a secondary antibody.

6. The method of any one of claims 1 -5, wherein step (S1 ) is performed by dipping the sensor into a solution comprising the cells, for a time t5 sufficient for the cells to form a bonding to the surface of the sensor, orby growing the cells onto the surface of the sensor.

7. The method of any one of claims 1 -6, wherein steps (S2) to (S4) or steps (S2) to (S5) are repeated 1 to 100 times without replacing the solution(s) in step (S3), and / or step (S31 ) and / or step (S41 ) and / or step (S51 ) and / or step (S52).

8. The method of any one of claims 1 -7, wherein the measured values of electrochemical measurement performed in step (S42) or step (S53) is proportional to the number of binding events between the at least one type of first biomolecule(s) and the at least one type of second biomolecule(s) and the interaction between the at least one type of second biomolecule(s) and the at least one type of first biomolecule(s) calculated in step (S6) is at least one selected from the group consisting of the number of binding events, the association rate (ka), the dissociation rate (kd), and the binding affinity.

9. The method of any one of claims 1 -8, wherein time t1 is in the range of 0.1 sec to 24 h, and / or time t2 is in the range of 0.1 to 20 sec, and / or time t4 is in the range of 0.1 sec to 24 h.

10. The method of any one of claims 1 -9, wherein the solutions in step (S2), and / or step (S21 ), and / or step (S3), and / or step (S31 ), and / or step (S41 ) and / or step (S51 ) and / or step (S52) are contained in wells of a mictrotiter plate, preferably wherein the solutions of step (S2), step (S3), step (S41 ) and / or step (S52), step (S21 ) or step (S31 ) and optionally step (S51 ) are all contained in wells of a mictrotiter plate.11 . The method of any one of claims 1 -10, wherein performing electrochemical measurement in step (S42) and / or step (S53) is performed by electrochemical measurement techniques, preferably EIS, chronoamperometry, or voltammetry.

12. The method of any one of claims 1 -11 , wherein the at least one type of first biomolecule(s) is a surface membrane protein.

13. The method of any one of claims 1 -12, wherein the at least one type of second biomolecule(s) contained in the solution or dispersion of step (S2) are contained on the surface of a cell.

14. The method of any one of claims 1 -13, wherein the sensor is a dip-in-well sensor for electrochemical measurements by being dipped into an assay at a distal end, the dip-in- well sensor comprising: a base, at least one metal electrode arranged on a first surface or a second surface of the base and extending between the distal end and a proximal end of the dip-in-well sensor, a substrate slab attached, at its second surface, to the first surface of the base and extending along the at least one metal electrode between the distal end and the proximal end, and a further metal electrode arranged on a first surface of the substrate slab and extending between the distal end and the proximal end.

15. Use of the method according to any one of claims 1 -14 in a binding assay, an affinity measurement method or a kinetics measurement method.

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