Surface Plasmon Resonance Measurement System and Method for Injecting a Sample Using a Cuvette Injection Flow System for Surface Plasmon Resonance Measurement

Abstract

The surface plasmon resonance measurement method using a so-called cuvette injection flow system includes: i. at least one sensor having a surface; ii. at least one flow cell in liquid contact with the surface of the sensor; iii. at least one cuvette (20) connected to the inlet of the flow cell via a central channel as a micro bioreactor; iv. an optical unit for measuring the reflection of surface plasmon resonance of incident light on the surface of the sensor; v. liquid transport means via suction or dispensing; and vi. sampling means (22, 23) for supplying at least one sample from an open cuvette at the upper end of the flow path.

Description

Cross - reference to related applications

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 351,478, filed on June 13, 2022, and is hereby incorporated by reference in its entirety.

Technical Field

[0002] The present invention relates to surface plasmon resonance measurement and injection systems, and a surface plasmon resonance measurement method using a so - called cuvette injection flow system.

Background Art

[0003] Surface plasmon resonance is a photoelectronic technique for detecting interactions with a thin metal film. When the Kretschmann configuration is applied, polarized light is irradiated through a prism onto a thin metal film. The incident angle can be changed, and the intensity of the reflected light is observed using an optical unit. During the scan of the incident angle, the intensity of the reflected light passes through a minimum due to the excitation of surface polaritons. The angle at which the maximum loss of the reflected light intensity occurs is called the SPR angle or SPR - dip. The SPR angle depends on the refractive index of the medium present on the metal surface and thus on the accumulation or desorption of molecules, such as proteins, on the thin metal layer. In Chapter 3 of the Surface Plasmon Resonance Handbook 2017, edited by R.B.M. Schasfoort of the RSC London, some configurations of SPR devices can be known.

[0004] SPR is mainly used to measure changes in refractive index in the evanescent field, and the phenomenon occurs at a very short distance from the sensor surface. When (bio)molecular interactions occur at the sensor surface, changes in refractive index can be measured in real - time and label - free.

[0005] SPR sensors are generally not selective with respect to the molecular interactions of target compounds. This is because changes in the angle of surface plasmon resonance of incident light on the sensor surface can be due to differences in the medium, such as the composition and concentration of the buffer, absorption of non-target substances on the surface, or, for example, temperature.

[0006] Selectivity may be achieved by modifying the sensor surface with a binding ligand, thereby capturing the target compound. Common-mode effects such as temperature differences and volume changes at the SPR angle of incident light on the sensor surface can be compensated for by using channels or spots where specific biomolecular interactions do not occur. SPR measurements are preferably performed while a buffer or sample flows continuously along the sensor surface to reduce mass diffusion to the sensor surface. Various fluid configurations are applied to bring the analyte into contact with the ligand bound to the sensor surface. The main systems are lateral flow cells with inlets and outlets connected to, for example, sample loops or hydrodynamic separation principles, address flow principles, open cuvette configurations, reciprocating flow systems, air park systems, etc.

[0007] Non-specific binding of non-target compounds or components may still occur. Therefore, SPR measurements preferably include an initial binding step in which the sample solution flows continuously along the sensor surface, followed by a dissociation step in which a buffer solution or another solution flows continuously along the sensor surface. Thereby, non-target compounds are dissociated while the target compound continues to bind to the specific ligand attached to the sensor surface. If partial dissociation of the target compound also occurs, correction is possible by measuring at appropriate reference positions on the same sensor surface.

[0008] In an SPR imaging system, a polarized light source irradiates the sensor surface through a prism, and the reflected light is observed using a camera. When the sensor surface is imaged by the camera, different portions of the sensor surface can be observed in real time at positions where the same or different ligands are attached to the sensor surface. This enables the measurement of different target compounds within the same sample simultaneously in real time.

[0009] The sensor surface is generally provided with ligands that are biological elements, such as single cells, microorganisms, cell organelles, cell receptors, enzymes, antibodies, antigens, proteins, DNA, RNA, peptides, or other biologically active compounds.

[0010] The SPR fluid system should be provided with means to prevent air or bubbles from coming into contact with the sensor surface. The sample should be injected immediately for stepwise conversion between buffer and sample. For rapid biomolecular interactions, the conversion from buffer to sample should be made as fast as possible, preferably within 1 second, from pure buffer to 100% sample contact. If the injection tube is long, the sample is injected slowly rather than immediately. Laminar flow within the tube causes mixing of the buffer and sample during transport of the sample within the tube. In commercial injection systems, air bubbles are used to prevent mixing of the buffer and sample during transport. Just prior to injection, the air bubbles remain in the T-connected tube. Then, the sample and buffer are injected immediately upon connection. In other devices, an injection loop very close to the sensor surface is applied to enable immediate bubble-free injection of the sample. In the 1990s, a cuvette-based system with injection and drainage tubes was available on the market. An open cuvette was pressed directly onto the sensor surface, and mixing was applied using a free-wall jet system or a piston mixer. In the first edition of the Surface Plasmon Resonance Handbook, that cuvette system is described in Chapter 3.3.2.

[0011] The core of the present invention is to apply a so-called cuvette injection flow (CIF) system to an SPR imaging apparatus. The cuvette portion of the CIF system of the present invention can be considered as a controlled micro bioreactor. The injection and discharge tubing lines connected to the cuvette have beneficial functions. This enables mixing of samples, diluting samples in a controlled manner, culturing, pre-reacting, preventing precipitation using cells, temperature-adjusting samples before injection, and generating a gradient of ligand density on the sensor surface of the flow cell portion of the system. In this patent application, other advantages and functions for pre-treating samples before contact with the sensor surface are described. For example, two cuvettes are connected to one flow cell to generate two consecutive ligand density gradients. The core of the present invention is characterized in that the distance between the cuvette and the flow cell is relatively short, for example, a few millimeters with a volume of less than 10 microliters. In this way, immediate injection of the sample into the flow cell is possible.

[0012] The flow cell portion of the CIF system of the present invention is generally composed of a closed space formed by a support, which is applied to the sensor surface, thereby forming a flow cell with inlet and outlet ports. The flow cell is connected to a system for sucking buffer solution, sample, or other related liquids, such as a regeneration solution, etc. Liquid transport means are also present to maintain the flow of liquid across the sensor surface during measurement. Thus, this substantially avoids the possibility that changes in composition, concentration, pH, etc. will bring about changes in the reflectivity of surface plasmon resonance. The measurement includes not only the initial binding step and the subsequent dissociation step as described above. Clearly, a pre-adjustment step and / or a final regeneration step may also be included. Generally, under flowing conditions, the measurement may be carried out in a period from 1 second to 1 day, or preferably from 30 seconds to 1 hour, for example, from 30 seconds to 5 minutes. The measurement time depends, inter alia, on the concentration of the target compound, and / or the reactivity of the ligand, and the applied flow conditions.

[0013] The flow cell may have a flow cell volume in the range of 1 nanoliter to 1 milliliter, such as 10 nanoliters to 1 milliliter, 100 nanoliters to 500 microliters, like 1 to 100 microliters, depending on the selectivity and sensitivity of the measurement. Surface plasmon resonance (SPR) is a representative method for detecting in real time the label-free biomolecular interaction between a ligand immobilized on a sensor in the form of a microchip and a specific analyte.

[0014] The microarray of spotted ligands can be used for analysis at various optimized concentrations. However, not only the concentration but also the affinity / avidity can be implemented on the chip. The sensor can be used for comparison and prediction of (pre)clinical, initial, and diseased states.

[0015] The CIF system can be used to generate a gradient of ligand density on the sensor surface. After the ligand is injected into the cuvette, the ligand solution is slowly poured into the flow cell manually by the user or automatically using an autosampler, by suction and diffusion. The ligand binds to the sensor surface (e.g., by pre-activation using EDC / NHS), but due to this slow injection / diffusion, the contact time of the ligand varies across the entire sensor surface area. The closer to the cuvette injection line, the longer the contact time. When the ligand solution reaches near the outlet, the ligand can be returned, thereby forming a gradient of ligand density from the beginning (high ligand density) to the end (low or zero ligand density) of the flow cell. An important application of the label-free sensing device is the kinetic measurement of the on-rate and off-rate of the analyte binding to the ligand. This is described in the section "CIF Devices for Determining Dynamic Parameters Using Ligand Density Gradients". In a special version of the CIF system, two cuvettes are connected to the flow cell. The two cuvettes can be applied to generate two different ligand density gradients using less volume. A large amount of analyte can be injected into the cuvette, and the two small cuvettes are treated with the same analyte. The double biomolecular interaction with two ligand density gradients can be applied to this fluid CIF configuration.

Summary of the Invention

[0016] In one aspect, the present invention aims to further improve SPR measurement and maintain a (substantially continuous) reciprocating flow on the sensor surface.

[0017] In another aspect, the present invention aims to further improve SPR measurement and maintain substantially continuous flow conditions on the sensor surface.

[0018] This object of the present invention is i. at least one sensor having at least one sensor surface, ii. A controlled microreactor, or a dual-tube combined with one cuvette, and at least one flow cell in contact with the liquid on the sensor surface, iii. An optical unit for measuring the angle (change) of surface plasmon resonance of incident light on the sensor surface, iv. Sampling means for supplying at least a sample and a buffer, v. Liquid transport means for liquid transport, vi. Means for generating a reciprocating flow of the sample or buffer on the sensor surface, vii. Means for generating a gradient of ligand density on the sensor surface, viii. Means for operating a cuvette equipped with an injection line or a discharge line or both, is achieved by providing a surface plasmon resonance measurement system including.

Brief Description of the Drawings

[0019] The present invention will be described in more detail below merely by way of example with reference to the accompanying drawings listed below.

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DETAILED DESCRIPTION OF THE INVENTION

[0020] According to the present invention, a surface plasmon resonance measurement system includes means for generating a reciprocating flow during measurement on the sensor surface, thereby maintaining the flow conditions during measurement. However, due to the reciprocating flow, the amount of liquid required, particularly the amount of sample and further the amount of buffer and any regeneration liquid, remains relatively small. In particular, it is important to note that since the sample travels back and forth across the entire sensor surface, the amount of sample does not substantially depend on the time required to perform the measurement. Due to the reciprocating motion, the transport of the target compound from the sample solution to the sensor surface, where the target compound binds to the ligand, does not substantially depend on the diffusion rate through the stationary liquid film layer on the sensor surface. Further, a transport or injection loop is not required, nor is liquid transport means provided with a valve for limiting the amount of sample required for SPR measurement.

[0021] Regarding the reliable reciprocating flow of the sample and / or buffer on the sensor surface during measurement, it is important to note that it is essential that the sample and buffer are separated by a separation liquid (for example, the bubbles shown in WO 2012 / 045325). However, if the volume of the channel between the flow cell and the cuvette is less than the volume of the sample injected into the cuvette, a separation liquid is not required. More specifically, if the sum of the volume of the flow cell and the volume of the channel between the cuvette and the flow cell is substantially less than the volume of the injected sample, stable injection of the sample can be achieved. The movement of the sample or buffer due to internal diffusion also occurs outside the flow cell in the tube connected to the pump via the flow cell. The injected sample maintains its concentration in the flow cell and is not diluted by the buffer through internal diffusion of the buffer into the sample during the measurement time of the sample. If a very long contact time is to be applied, a larger sample volume should also be applied to avoid internal diffusion of the buffer. Internal diffusion of the buffer dilutes the sample in contact with the sensor surface.

[0022] For reliable and simple generation of reciprocating flow conditions, the sampling means preferably includes a flow cell and a tube or microchannel connected to the reciprocating flow means. Thus, the same tube may be used to generate the reciprocating flow of the buffer solution and the sample solution simultaneously. In this regard, more preferably, the reciprocating flow means includes the reciprocation of a movable actuator, such as a piston or a pressure unit, etc. In this way, the reciprocating flow may be generated using a piston or a pressure unit. Such a pressure unit may apply pressure to the tube, thereby generating a reciprocating flow of the sample and the buffer solution in the tube.

[0023] As described above, SPR measurement requires monitoring of changes in the SPR angle, or changes in reflectivity corresponding to an increase or decrease in the mass of the material on the sensor surface, and / or due to the presence of the sample, buffer solution, and regeneration solution on the sensor surface. It can be used to calculate the change in the angle of surface plasmon resonance of the incident light on the sensor surface. The monitoring may be performed by individual optical methods, such as a photodiode or a camera, etc. However, a normal camera may also be used to photograph the state of surface plasmon resonance on the sensor surface or multiple measurement ranges of the sensor surface.

[0024] The calibration routine can be applied to calculate the reflectance (%R) in refractive index units (RIU) or 10 -6~ times resonance units (RU) (alternatively microrefractive index units (μRIU)). The calibration routine includes connecting and injecting a solution of a refractive index buffer, such as X% to 10% glycerol, into the flowing buffer solution. In a controlled microbioreactor connected to two lines, a gradient of glycerol can also be generated for the calibration procedure. The non-linearity of the reflectance curve regarding the measurement range of the sensor surface can be fitted to the response of the X% glycerol injection. In this way, the change in reflectance can be recalculated as a change in resonance units (RU) or microrefractive index units (μRIU).

[0025] As indicated above, SPR measurements may be sensitive to temperature changes. To avoid the influence of temperature on SPR measurements, it is preferable that a thermostatic unit is present for the sample, buffer, wash, mixing and / or calibration solution, and that it is in contact with the sensor for the measurement during the reciprocating movement. Such a thermostatic unit is suitable for maintaining the temperature of the sample and / or buffer at a constant temperature of + or - 0.1 °C, preferably + / - 0.01 °C, more preferably less than + / - 0.01 °C.

[0026] In an example of such a thermostatic unit, the liquid from the cuvette can be aspirated into a temperature-controlled compartment. This compartment includes a metal block with a channel structure having a certain length of channel or tube, i.e., capable of holding the liquid at a constant volume and maintained at a precisely constant temperature. The volume of the liquid contained in the tube within the thermohead is selected such that the liquid entering the cuvette has the same temperature as the liquid in the flow cell before being injected into the flow cell. This prevents volume changes due to temperature differences in the liquid in contact with the sensor surface.

[0027] Another aspect of the present invention relates to means and methods for measuring (bio)molecular interactions by SPR measurement, for example, in an SPR measurement system according to the present invention, which is the subject matter of the present invention described above. According to the present invention, the means and methods for this SPR measurement include the following features. i. Sampling means for the sample in a cuvette or micro bioreactor closely connected via a channel of small volume to the flow cell ii. The volume of the channel between the cuvette and the flow cell, which is usually less than the sample volume, for example, between 1 and 20 microliters iii. An open cuvette equipped with a tube connected to the bottom of the cuvette for discharging (or emptying) the cuvette iv. An open cuvette or container equipped with an injection line for storing the sample v. An injection line equipped with a temperature-controlled storage line capable of injecting the temperature-controlled sample from the cuvette into the flow cell vi. An injection line for injecting a part of the sample volume to dilute the sample in the cuvette vii. For a series of injections, injecting the sample into the cuvette in a controlled manner via an autosampler viii. Mixing the sample in the cuvette by reciprocating flow via a storage line or a discharge line ix. Mixing the particles in the sample in the cuvette or the cells in the culture medium and avoiding precipitation of the particles or cells before being injected into the flow cell x. Slowly injecting a ligand into the flow cell to generate a gradient of ligand density on the sensor surface xi. Optionally, slowly injecting two ligands simultaneously from two small cuvettes connected to the flow cell (the two small cuvettes can be applied to be filled with one analyte) xii. Contacting the sensor surface with a buffer solution xiii. Measuring the reflectivity of surface plasmon resonance on the sensor surface while in contact with the buffer solution during the reciprocating operation xiv. Injecting the sample quickly directly from the cuvette into the flow cell without liquid separation, i.e., without bubbles separating the buffer solution from the sample xv. Contacting the sensor surface with the sample in the flow cell xvi. Measuring the change in the angle of surface plasmon resonance of the incident light on the sensor surface while in contact with the sample during the reciprocating operation, as an option hereinafter xvii. Returning the sample and then returning the buffer solution along the sensor surface including a diffusion range for separating the sample and the flowing buffer solution xviii. Contacting the sensor surface with the buffer solution while the sample returns to the cuvette and can be removed via 1. the discharge line or 2. the injection line xix. Measuring the change in the angle of surface plasmon resonance of the incident light on the sensor surface while in contact with the buffer solution (so-called dissociation phase) during the reciprocating operation xx. Optionally washing the sensor surface with a regeneration solution to regenerate the sensor surface Measuring a refractive index-controlled buffer solution for calibrating the sensor by injecting a calibration solution from the cuvette

[0028] The cuvette injection flow device determines dynamic parameters using a gradient of ligand density.

[0029] The cuvette injection flow device is the core of the present invention and can also generate a steep gradient of ligand density on the sensor surface. This is very advantageous for measuring affinity parameters. Because the values of the affinity constants (k d , k a , and K D ) determined by the label-free interaction analysis method are affected by ligand density. By generating a gradient of ligand density, SPR image generation using the cuvette injection flow device of the present invention can measure the analyte ligand by combining the gradient of ligand density in a spatially analyzed manner. Dynamic titration measurements that can be automatically performed in a cuvette flow cell without a regeneration step can be applied to various binding antibodies with a gradient ligand density that binds to one antigen. For various ligand densities and analyte concentrations, the overall suitable ratios (k d and k a ) and dissociation equilibrium (K D ) constants are measured, and the parameters can be determined at a fixed ligand density (a value of fixed R max would be fine). For example, R max = 100 μRIU response level (K D R100 ), or extrapolation can be performed at R max = 0 μRIU.

[0030] These molecular binding constants obtained from the current immobilized ligands based on the analysis are affected by the immobilized state of the ligands. Thus, apparent constants deviated from the true "solution" constants are determined and generated due to the interference effects caused by the immobilization of the ligands. These interference effects include rebinding effects, mass transport limitations, non-specific binding, and biases from the 1:1 model binding. As the ligand density increases, these interference effects become more prominent. Therefore, it is generally accepted that the ligand density should be applied just above the detection limit of the biosensor device. The same applies to the analyte concentration. When multiple analyte molecules compete for the interaction with one immobilized ligand molecule, interference effects occur.

[0031] The calculation of the "true" affinity equilibrium constant is more reliable at lower densities. It is preferable to have only the "density" of a single immobilized ligand molecule acting as a free ligand [1]. At this time, the contribution of the interference effect is zero and does not affect the ratio and the affinity equilibrium constant. Substantially, it cannot be measured under this condition. By reducing the ligand density, the sensorgram becomes noisier and less reliable. Furthermore, the quality of the fitting to the noisy curve cannot be properly judged. It should be noted that the heterogeneity of the immobilization artifacts and surface binding sites should be prevented, for example, by applying a high-affinity anti-ligand antibody or by using a tag-antitag interaction to capture the orientation of the ligand.

[0032] The so-called method for determining the affinity constant K D R0 was published in 2011. This minimizes the contribution of the interference effect or makes it theoretically zero, and the constant can well estimate the true constant of the interaction of biomolecules in solution. This method is based on extrapolating the number of immobilized ligand and analyte molecules to zero, resulting in an interaction that mimics the true 1:1 binding model with no theoretical interference effects for one ligand and one analyte molecule.

[0033] The recognized actual effects are the additional ligand immobilization artifacts and the heterogeneity of the surface binding sites. This method cannot compensate for this, and the alternative route is to capture the ligand and subsequently perform the target interaction. If bad regeneration steps are included, the R max value decreases after subsequent injections of the analyte concentration and can affect the dynamic affinity constant again. Preferably, any regeneration steps of the surface should be avoided, which is achieved using kinetic titration.

[0034] The calculation of the kinetic constants was performed from spots with discrete ligand densities. Recently, many users of the SPR platform have adjusted the ligand density so that the interaction with the analyte is very small but still measurable. The sensitivity of the device is determined by how low the ligand density can be. The users decide what is a low value, and the values generated by the users deviate from each other. This is because there are no rules for interpreting the quality of the fitting of the binding curves.

[0035] The cuvette injection flow device can generate a steep gradient of ligand density, and the device measures the analyte binding of the ligand gradient. All densities from very high to zero low density are available. If the gradient in the flow cell is divided, for example, in a measurement range of 1000, or a more adjusted or dynamic measurement range, the device automatically, for example, R max= 100 μRIU or any value of a similar set of biomolecular interactions, the binding results can be known. The proven methods published in Reference 1 and Reference 2 can be performed with a gradient ligand density instead of a discrete low ligand density, but the number of spots is limited. Interpretation of the quality of fitting by the user, for example, by applying a 1:1 Langmuir binding algorithm, is no longer necessary. The software generates biomolecular affinity parameters measured in the same way using the same ligand density always at some position with a gradient. Interpretation of the curve by the user, laboratory technician, or device operator is no longer necessary. Always, the parameters are generated in the same way using a dynamic gradient method that greatly improves the analysis of the data.

[0036] When a controlled gradient of ligand density can be generated on the sensor surface, there are more applications. For example, particles such as cells, viruses, organelles, vesicles, etc. contain a certain number of cell surface receptors (CDs) (which bind to anti-CD antibodies). These antibodies bind to these particles, and tests such as inhibition tests can be performed with a gradient. The greater the binding affinity (the better the binding force for multivalent interactions), the more these particles are present at a low ligand density. At zero ligand density, there is no binding.

[0037] The T / S measurement method published on page 447, Chapter 12.8.1 of the second edition of the Surface Plasmon Resonance Handbook can be applied to a gradient. As described on page 463, Chapter 12.8.4, this can be an important method for the affinity ranking of interactions using an increased flow protocol. These detection methods can be better applied to a sensor surface with a gradient of ligand density.

[0038] After injecting cells into the flow cell, the cells bind to the sensor surface. Companies developing antibodies for various cell applications need to characterize the affinity of monoclonal antibodies for live cell receptors. Direct detection of antibodies binding to the precipitated cell line was not possible due to the high unstable baseline caused by cell activity. However, the inventors noticed that the release of cells from the sensor surface depends on several factors. For example, flow rate, the number of cell receptors, the affinity between the immobilized ligand and the cell receptor, ligand density, etc. are important parameters. When applied in combination with the flow rate (shear rate) at which ligand density increases, affinity ranking can be measured with multiple receptor and antibody combinations (Ab combinations). Shearing in cells depends on the local velocity profile of the buffer flow in the immobilized cells. In a specific region of ligand density, the cells still bind, but by increasing the buffer velocity, the cells are pulled out from the surface and the cells no longer bind. The higher the velocity, the greater the ligand density required to maintain the cells on the surface. This process using SPR imaging can be tracked in real time. By applying a uniform force to the cells, the ligand density of a series of anti-membrane antigens adjusts the position where cells at a specific velocity dissociate from the gradient. In this way, when different antibodies are immobilized on the ligand gradient simultaneously, the affinity of cell receptors is compared and ranked against each other. This SPR imaging application has a great impact.

[0039] A reliable multi-functional SPR imaging measurement method is obtained when the sensor surface includes a plurality of active sites (e.g., spots or gradients or gradient spots) where changes in the angle of surface plasmon resonance of incident light at the sensor surface are individually observed (preferably equipped with a camera).

[0040] In a special configuration of the CIF, two small containers within the cuvette are connected to one flow cell to generate two different ligand density gradients according to FIG. 8.

[0041] As shown in FIG. 1, SPR measurement may be performed with only one flow cell or, for example, with a plurality of two to six or more flow cells. When a plurality of flow cells are used, each flow cell may be supplied by its own pumping means for generating ligand density either with or without a gradient on the sensor surface. However, preferably, the flow cell into which the analyte is injected is supplied by normal pumping means such that all spots are under the same conditions (flow rate and sample transport and passage), and thus the buffer enables reliable automated measurements at spots, gradients, or gradient spots. This is the so-called "one over all" method.

[0042] The features mentioned and other features of the SPR measurement system and SPR measurement method according to the present invention are further illustrated by various embodiments. These are for informational purposes only and are not intended to limit the present invention in any way. Referring to the accompanying drawings, FIG. 2 represents a schematic view of a cuvette injection flow system according to the present invention. The cuvette 20 is connected to an injection line 22 and a discharge line 23. The cuvette is connected to the inlet of the flow cell by a low-capacity channel. The sample is injected into the cuvette manually or by an autosampler. FIG. 3 represents details of an enlarged area of FIG. 2. This figure is a view of the instant of injection including a ligand density gradient. FIG. 4 shows the sample in the cuvette. The cuvette is operated with one or two injection or discharge lines via a dispensing valve of a syringe pump. The cuvette is connected to the flow cell by a low-capacity channel. The outlet of the channel (at the back of the figure) is connected to the syringe pump.

[0043] To avoid the influence of temperature, it is preferable that all liquids (samples, cleaning solutions, calibration solutions, etc.) flow back and forth across the entire sensor surface. This can be achieved by using a temperature-controlled injection line as shown in Figure 5. Specifically, the cuvette is connected to the flow cell via a low-volume channel, and the outlet of the flow cell is connected to a pump. The injection line of the cuvette is wound around the periphery of the device in contact with a temperature-controlled tube for injecting the temperature-controlled sample into the cuvette.

[0044] When injected into the cuvette, the cells may settle. Figure 6 shows the procedure for resuspending again with a back-and-forth flow. To obtain a cell suspension, a back-and-forth flow can be applied to the injection line again. The cell suspension can be injected directly into the flow cell without delay. This mixing method can be applied to dilute the sample in the cuvette.

[0045] The SPR image of the injection of the flow cell from the cuvette is shown in Figure 7.

[0046] Figure 8 represents a cuvette designed with two injection lines to the flow cell / chamber at the bottom and equipped with two additional containers. (Analysis cycle)

[0047] As an example, the process of operating a cuvette injection flow system for SPR image generation is described. First, the baseline measurement is performed using a flowing buffer that fills the flow cell, irradiating polarized light and observing the reflected light using a camera to measure the angle of surface plasmon resonance of the incident light on the sensor surface over time (see Figure 7, panel A). The measurement is performed according to the present invention during the back-and-forth flow. Subsequently, the flow cell is initially partially filled with the sample by suction through the cuvette (see Figure 7, panel B and panel C). In this way, a gradient of ligand density can also be constructed.

[0048] When the sample completely fills the flow cell (see Panel D of Figure 7), the SPR measurement is again performed under reciprocating flow. The volume of the sample is large enough to prevent internal diffusion of the buffer flowing into the sample during the measurement time of the sample. Thereafter, the sample is removed from the flow cell, the flow cell is refilled with flowing buffer, and the dissociation step of the SPR measurement is performed to first measure the change in the angle of the incident light due to dissociation of non-specific compounds and subsequent dissociation from the ligand-bound target compound. Then, under reciprocating flow, or reciprocating flow with continuous inflow of buffer into the flow cell, the situation of Panel A of Figure 7 is reached again.

[0049] Finally, the sample may be removed from the system and the procedure for SPR measurement according to the present invention may be resumed. Obviously, the sensor surface may be in contact with a calibration solution for calibration. The calibration solution is known for the change in the angle of surface plasmon resonance of the incident light on the sensor surface (resulting in refractive index), and the solution may be a mixture of water / glycerol.

[0050] Although not yet described, note that after sample measurement and desorption measurement using buffer, it is necessary to regenerate the active sites present in the flow cell. The regeneration fluid may be aspirated, for example, after discharge of the sample from the SPR measurement system, and the active sites are applied to the regeneration medium, thereby supplying the flow cell and its active sites in a regeneration format for measurement of possible target compounds. The injection of the regeneration solution can also be operated manually or by autosampler means via a cuvette, or via the tubes of the flow cell port, injection line port, or discharge line port. One of the tubes of the syringe pump can be connected, for example, to a regeneration solution of 100 mM phosphoric acid pH 3.0.

[0051] The cuvette supplied with two additional containers can generate gradients of two different ligand densities on the sensor surface (see Figure 8). The volume of each container with ligand is similar to half of the flow cell chamber. Both of the two ligand density gradients can be generated simultaneously by timely contact of the ligand covering the length of the flow cell of the activated sensor surface.

[0052] Although the present invention has been described with reference to specific embodiments, those skilled in the art should recognize that many variations can be made therefrom (e.g., under the specific experimental conditions described herein). It should be understood and appreciated that the present disclosure related to the present invention only shows some preferred embodiments, objects, and advantages of the present invention without departing from the broader scope and spirit of the present invention. These discoveries related to the present invention only help in understanding many additional potential applications that can be envisioned by a person of ordinary skill in the art, and thus it should be understood and appreciated that there is no intention to limit the present invention in any way. Therefore, other objects and advantages of the present invention will be apparent to those skilled in the art from the claims and the specification. (References)

[0053] 1. R.B.M.Schasfoort, W.de Lau, A.van der Kooi, H.Clevers and Gerard H.M.Engbers. Method for estimating the single molecular affinity, Anal.Biochem.421(2012),794 - 796

[0054] 2. Schuck P. and Zhao H. The Role of Mass Transport Limitation and Surface Heterogeneity in the Biophysical Characterization of Macromolecular Binding Processes by SPR Biosensing. In Methods in Molecular Biology, Springer protocols.627(2010),15 - 54

[0055] 3. Myszka D.G.et al.Extending the range of rate constants available from BIACORE:interpreting mass transport-influenced binding data.Biophys J.75(2)(1998):583-94

[0056] 4. R.Karlsson,P.S.Katsamba,H.Nordin,E.Pol and D.G.Myszka,Analyzing a kinetic titration series using affinity biosensors.Anal.Biochem.349(2006)136-147

Claims

1. A surface plasmon resonance measurement system, i. At least one sensor having a surface, ii. At least one flow cell that is in liquid contact with the surface of the sensor, iii. A microbioreactor comprising at least one cuvette connected to the inlet of the flow cell via a central channel, iv. An optical unit for measuring the reflection of surface plasmon resonance of incident light at the surface of the sensor, v. A means of transporting liquids via suction or dispensing, vi. A sampling means for supplying at least one sample from an open cuvette at the upper end of the flow path, A system that includes this.

2. The system according to claim 1, wherein the channel between the cuvette and the flow cell has a small sample volume applied to the cuvette.

3. The system according to claim 1 or 2, wherein the cuvette is injected and / or discharged independently without the transport of liquid to the flow cell.

4. The system according to claim 1 or 2, wherein the round-trip volume applied to the flow cell is less than the sample volume.

5. The system according to claim 1 or 2, wherein the cuvette can be emptied using at least one discharge connection without emptying the flow cell.

6. The system according to claim 1 or 2, wherein the cuvette is manually injected with a sample using a pipette.

7. The system according to claim 1 or 2, wherein the cuvette can be automatically injected with a sample using an autosampler.

8. The system according to claim 1 or 2, wherein the sample injected into the cuvette can be transported by the liquid transport means to a discharge or injection connection for storage of the sample.

9. The system according to claim 8, wherein the stored samples are temperature-controlled.

10. The system according to claim 1 or 2, wherein the cuvette includes the flow cell to the cuvette injection line, the discharge line, or the sample storage line.

11. The system according to claim 1 or 2, wherein the cuvette comprises two additional containers having two injection lines to the flow cell / chamber at its bottom.

12. The system according to claim 5, further comprising one or more flow cells and cuvettes.

13. The system according to claim 8, wherein the stored sample may be mixed with a buffer injected through the flow cell to dilute the stored sample.

14. The system according to claim 13, wherein the stored sample is used in a dynamic titration experiment in which the stored sample is injected after being diluted at least twice.

15. The system according to claim 8, wherein the sample is automatically injected from the discharge line.

16. The system according to claim 8, wherein the sample is a regeneration solution for automatic injection into the flow cell via the cuvette for regeneration.

17. The system according to claim 8, wherein the sample is a calibration solution for automatic injection into the flow cell via the cuvette for calibration.

18. The system according to claim 5, for controlling the injection of ligand to generate a gradient in ligand density by at least one reciprocating flow of the sample in the channel that contacts the surface of the sensor.

19. The system according to claim 11, for controlled injection of two ligands in the channel in contact with the surface of the sensor, by at least one reciprocating flow of the sample to generate two gradients in ligand density.

20. The system according to claim 5, for injecting a suspension of particles or cells pre-mixed in the cuvette using a syringe pump in a reciprocating flow through an injection line or discharge line for injecting the suspension of particles or cells pre-mixed in the cuvette.

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