WO2012045325A1

WO2012045325A1 - Surface plasmon resonance measuring system and a method for surface plasmon resonance measurement

Info

Publication number: WO2012045325A1
Application number: PCT/EP2010/006159
Authority: WO
Inventors: Gerardus Henricus Maria Engbers; Richard Bernardus Maria Schasfoort
Original assignee: Ibis Technologies Bv
Priority date: 2010-10-04
Filing date: 2010-10-04
Publication date: 2012-04-12

Abstract

The invention relates to surface plasmon resonance (SPR) measuring system comprising: at least one sensor having at least one sensor surface; at least one flow cell which is in liquid contact with the sensor surface; an optical unit for measuring the surface plasmon resonance angle of light incidence at the sensor surface; sampling means for supplying at least a sample and a buffer separated by separation fluϊdum into the flow cell; liquid transport means for liquid transport; and means for generating a back and forth flow of sample or buffer at the sensor surface, and to a SPR method.

Description

SURFACE PLASMON RESONANCE MEASURING SYSTEM AND A METHOD FOR SURFACE PLASMON RESONANCE MEASUREMENT

The present invention relates to a surface plasmon resonance measuring system and to a method for surface plasmon resonance measurement.

Surface Plasmon Resonance is an optoelectronic technique for detecting interactions at a thin metal film. Polarized light is shown through a prism onto a thin metal film applied on a sensor. The angle of incidence is changed and the intensity of the reflected light is monitored using an optical unit. The intensity of the reflected light passes through a minimum due to excitation of so called surface plasmons. The angle at which maximum loss of the reflected light intensity occurs is called the SPR angle. The SPR angle is dependent on the refractive index of the medium present on the metal surface and, thus dependent on the accumulation or desorption of molecules such as proteins on the thin metal layer.

SPR is predominantly used for measuring the shift in the SPR angle, which is due to the occurrence of (bio) molecular interaction at the sensor surface. Thus, (bio)molecular interactions may be studied in real time.

SPR sensors are generally not selective in relation to the molecular interaction of the target compound. Because the changes in the surface plasmon resonance angle of light incidence at the sensor surface may be due to differences in the medium, such as the composition and concentration of the buffer, due to absorption of non-target material on the surface, and also to for instance the temperature.

Selectivity may be achieved by modifying the sensor surface by binding ligands which selectively capture the target compound. Common mode effects like temperature differences and bulk changes in the surface plasmon resonance angle of light incidence at the sensor surface can be compensated by using a channel or spot where specific bio-molecular interactions do not occur. It is preferred that the SPR measurement is carried out while the buffer or sample continuously flows along the sensor surface for reducing mass diffusion relative to the sensor surface.

Non-specific binding of non-target compound or components may still take place, thereby it is preferred that the SPR measurement comprises a first association step by continuously flushing the sample solution along the sensor surface, followed by a dissociation step in which continuously buffer solution or another solution is flushed along the sensor surface, thereby dissociating non-target compound while the target compound remains bound to the specific ligands adhered to the sensor surface. If partial dissociation of the target compound also may occur then correction would be possible by measurement of appropriate references.

The SPR measuring system comprises generally a source for polarized light that shines via a prism onto the sensor surface. The reflected light is monitored using an optical unit, such as a photo diode or camera. When using a camera it is possible to monitor real time and separate and individual parts of the sensor surface at which the same or different ligands are adhered to the sensor surface. Hereby it becomes possible to real time and at the same time measure for different target compound in one and the same sample.

The sensor surface is provided with a ligand which is generally a biological element, such as a tissue, a microorganism, an organelle, a cell receptor, an enzyme, an antibody, an antigen, protein, DNA, RNA, peptide or other biologically active compound.

The flow cell consists generally of a confined space formed in a support, which is applied onto the sensor surface thereby forming the flow cell. The flow cell is connected to a system for aspirating buffer, sample or other relevant liquid such as a regeneration solution. Liquid transport means are also present in order to maintain a flow of liquid over the sensor surface during the measurement. Accordingly, this substantially avoids that changes in composition, concentration, pH and the like will result in a change in the surface plasmon resonance angle. The measurement not only comprises, as stated above, a first association step followed by a dissociation step. Obviously a pre-accommodation step and/or a last regeneration step may also be included. Generally, under flushing conditions, the measurement may take place during 1 second to 1 day, or preferably 30 seconds to 1 hour, such as 30 seconds to 5 minutes. The measuring time is inter alia dependent on the concentration of the target compound and/or the reactivity of the ligand and the applied flow conditions.

The flow cell may have a flow cell volume ranging from 1 nanolitre to 1 millilitre, such as 10 nanolitre to 1 millilitre, like 100 nanolitre to 500 microlitre, like 10-100 microlitre dependent on selectivity and sensitivity of the measurement. Surface plasmon resonance (SPR) is the golden standard for detecting in real-time and label free biomolecular interactions of a specific analyte to a ligand that is immobilized on a sensor in the form of a microchip. For instance the imaging feature of the IBIS-iSPR instrument enables to detect e.g. 120 interactions

simultaneously. A microarray of spotted ligands can be utilized in different and optimized concentrations for analysis. However, not only the concentration but also affinity/avidity can be implemented on the chip. Such sensor may be used for the comparison and prediction of the status of (pre)clinical, early and established disease.

The present invention has for its object to further improve the SPR measurement while maintaining a back and forth flow (which is quasi continuous) at the sensor surface.

The present invention has for his object to further improve the surface plasmon resonance measurement while maintaining a substantially continuous flow condition at the sensor surface.

This object of the invention is met by providing a surface plasmon resonance measuring system comprising:

i. at least one sensor having at least one sensor surface;

ii. at least one flow cell which is in liquid contact with the sensor surface; iii. an optical unit for measuring (the shift in) the surface plasmon resonance angle of light incidence at the sensor surface;

iv. sampling means for supplying at least a sample and a buffer separated by separation fluidum into the flow cell;

v. liquid transport means for liquid transport; and

vi. means for generating a back and forth flow of sample or buffer at the sensor surface.

The surface plasmon resonance measuring system according to the invention comprises means for generating a back and forth flow during measurement at the sensor surface, thereby maintaining the flow conditions during measurement. However, due to the back and forth flow the required amount of liquid, in particular the amount of sample and further the amount of buffer and optional regeneration liquid are kept relatively small. It is important to note that the amount of in particular the sample is substantially independent of the time required for carrying out the measurement, because in particular the sample is moved back and forth over the sensor surface. Due to the back and forth movement the transport of target compound from the sample solution towards the sensor surface where the target compound is to bind to the ligand, is substantially independent on the diffusion rate through the stationary liquid film layer on the sensor surface. Furthermore, no transport or injection loops are required and no liquid transportation means comprising valves for otherwise limiting the amount of sample required for doing the SPR measurement.

It is noted that the sensor or multiple sensors may form with the flow cell (or multitude of flow cells) an exchangeable cartridge.

It is important to note that for a reliably back and forth flow sample and/or buffer at the sensor surface during the measurement, it is essential that the sample and the buffer are separated by a separation fluidum. Accordingly, it is avoided that during measurement of the sample or the buffer the sensor surface may come into contact with respectively the buffer or the sample. Even a migration of sample or buffer along the wall of the tubing towards and into the flow cell is substantially avoided.

Preferably, the separation fluidum has a different refractive index in comparison to the sample and the buffer. Accordingly, passage of the separation fluidum over the sensor surface will result in an abrupt change in the surface plasmon resonance angle of light incidence at the sensor surface which can be used as an electronic trigger for starting or terminating the measurement of the sample or the buffer as well as a reference in data processing.

A very reliable SPR measuring system is obtained when the back and forth flow volume is less than the volume of the separation fluidum. Thereby it is relatively impossible that during the back and forth flow during the measurement of the sample, buffer solution could get into contact with the sensor surface. In turn, it is also substantially impossible that during the measurement of the buffer solution (when measuring the base line or during the dissociation step) that sample liquid may get into contact with the sensor surface.

For a reliable a rapid SPR system for measuring a sample relative to a buffer, it is very convenient when the sampling means comprises a microchannel, such as a tubing, connected to the flow cell for taking up buffer and sample separated by separation fluidum. Accordingly buffer and sample separated by the separation fluidum are one after the other brought into contact with the sensor surface for measurement during back and forth flow conditions, while it is not necessary to use several tubings connected by a multi valve with the inlet of the flow cell. For a reliable and simple generation of the back and forth flow conditions, it is preferred that the sampling means comprises a tubing or microchannel connected to the flow cell and to the back and forth flow means. Accordingly, the same tubing may be used for generating the back and forth flow of simultaneously the buffer solution and the sample solution. In this respect it is further preferred that the back and forth flow means comprise a back and forth moving actuator, such as a piston or pressure unit. In this way the back and forth flow may be generated using a piston or a pressure unit. Such pressure unit may exercise a pressure on the tubing, thereby generating in the tubing the back and forth flow of sample and buffer, and also of the separation fluidum.

The separation fluidum may be any suitable fluidum that is capable of operating sample and buffer during the measurement of the sample and of the buffer, and substantially avoid any contact between the sample and the buffer during their measurement and during the transport. In addition it is preferred that separation fluidum has a refractive index that is substantially different from the refractive index of the sample and of the buffer and thus also a substantial difference in the surface plasmon resonance angle of the light incidence at the sensor surface. Accordingly it is preferred that the separation fluidum is a gas, such as air, or a liquid substantially immiscible with sample and buffer.

A relatively simple SPR measuring system is obtained when the sensor surface comprises two or more different active sites. Accordingly, the back and forth flow means are integrated in the liquid transportation means, so that the back and forth flow is directly generated in the liquid transportation means.

As stated above, the SPR measurement requires the monitoring of a shift of the SPR angle, that is the shift in the angle of light incidence which is dependent on an increase or decrease of material mass at the sensor surface and/or due to the presence at the sensor surface of a sample, buffer, regeneration liquid or separation fluidum, and can be used for calculating a change or shift in the surface plasmon resonance angle of light incidence at the sensor surface. The monitoring may take place with individual optical means, such as photodiode or camera. However, a common camera may be used for monitoring the surface plasmon resonance angle of light incidence of one the sensor surface or a plurality of sensor surfaces. Still, it is preferred that the plurality of flow cells uses separate sampling means but common transportation means and/or means for generating the back and forth flow. This results in a reliable SPR measuring system and also in a possibility of real time and at the same time monitoring of a change in the surface plasmon resonance angles of light incidence at the sensor surface in various flow cells. However, if in relation to a sample various target compounds are to be measured, then it is preferred that the sensor surface comprises two or more different active sites. These individual active sites may then be monitored by separate optical means or by a common camera monitoring different individual active sites present on the sensor surface of one or more flow cells.

It may be required that the sample to be measured is a reaction product. Accordingly, it is then preferred that the sampling means comprise a unit for mixing sample with a reagent.

As indicated above the SPR measurement may be sensitive to temperature changes. In order to avoid an influence of temperature on the SPR measurement it is preferred that a thermostatic unit is present for the sample,the buffer, washing, mixing and/or calibration solutions, which will be in contact with the sensor for measurement during the back and forth movement. Such thermostatic unit is suitable for

maintaining the temperature of the sample and/or buffer at a constant temperature + or - 0.1 °C, preferably +/- 0.01°C, more preferably less than +/- 0.01°C. In an example of such a thermostatic unit, the so called thermo-head, the liquid that enters the flow cell is first passed through the thermohead that comprises a metal block with a channel structure that can have a specific length of channels or tubing and therefore can hold a specific volume of liquid and that is precisely maintained at a specific temperature. The comprised volume of liquid in the tubing in the thermohead is chosen such that the liquid that enters the flow cell has the same temperature as the liquid in the flow cell. This prevents a bulk shift due to temperature differences of liquids that come into contact with the sensor surface.

Another aspect of the invention relates to a method for measuring a (bio)molecular interaction by SPR measurement such as in the SPR measuring system according to the invention, which has been discussed above and is subject of claim 1. This method for SPR measurement comprises according to the invention the steps of: i. filling sample means with buffer and sample which sample is separated by separation flui^'dum from the buffer;

ii. contacting at least one sensor surface with the buffer; iii. measuring the surface plasmon resonance angle of light incidence at the sensor surface while in contact with the buffer being in back and forth movement;

iv. passing the separation fluidum along the sensor surface;

v. contacting the sensor surface with the sample;

vi. measuring the change in the surface plasmon resonance angle of light incidence at the sensor surface at the sensor surface while in contact with the sample being in back and forth movement; and optionally the step of:

vii. passing back the separation fluidum along the sensor surface

viii. contacting the sensor surface with the buffer

ix. measuring the change in the surface plasmon resonance angle of light incidence at the sensor surface while in contact with the buffer being in back and forth movement; and

vii. optionally washing the sensor surface with a regeneration liquid to

regenerate the sensor surface and/or calibrating the sensor using a calibration liquid.

As argued above the start of the contact of sensor surface with the sample, with the buffer and/or with the separation fluidum may be triggered as a result of a change in the surface plasmon resonance angle of light incidence at the sensor surface when separation fluidum, sample or buffer becomes into contact with the sensor surface and results in a change in the surface plasmon resonance angle of light incidence at the sensor surface. When injection cycles are applied this trigger moment is essential for aligning all individual injections.

A reliable and multi-functional SPR measuring method is obtained when preferably the sensor surface comprises a plurality of active sites monitored individually for change in the surface plasmon resonance angle of light incidence at the sensor surface, preferably with a camera.

The SPR measurement may be carried out in one single flow cell or in a plurality of flow cells. When a plurality of flow cells is used, then each flow cell may be served by its own pump means. However, it is preferred that the plurality of flow cell is served by common pump means such that all flow cells are subjected to the same conditions (flow rate and transport and passage of sample, buffer and separation fluidum) therefore making it possible to do a reliable automatic measurement in the plurality of flow cells.

Mentioned and other features of the SPR measuring system and of the method for SPR measurement according to the invention will be further illustrated by various embodiments which are given for information purposes only and are not intended to limit the invention to any extent, while making reference to the annexed drawings, wherein:

Figure 1 : a schematic presentation of a first SPR measuring system according to the invention;

Figure 2: at a larger scale detail II of figure 1 ;

Figures 3 and 4: show the measurement with the SPR system of figure 1 in the association step and dissociation step;

Figure 5: a schematic overview of the SPR measuring system;

Figures 6-8: other embodiments of the SPR measuring system according to the invention;

Figure 9: a sensor surface provided with an array of different active sites; and

Figure 1 shows a SPR measuring system 1 according to the invention. The SPR system 1 comprises a sensor 2 which is connected on the one hand to a hemispheric or triangular prism 3 and to the other to a flow cell 4. The flow cell comprises a support 5 (made of Delrin™, a polyoxymethylene). The support is provided with several measuring locations 6-9 which are connected in series to a flow cell inlet 10 and a flow cell outlet 1 1. The flow cell inlet 10 is connected via a tubing 14 to sampling 12 comprising a sampling needle 13.

The outlet 1 1 of the flow cell is connected via a tubing 15 and a multivalve 16 to means 18 for generating a back and forth flow 17 at the sensor surface 19 applied on a glass support 20. The sensor surface 19 is provided on the measuring locations 6-9 with active sites 21 -24, comprising different ligands specific for various target compounds that are potentially present in a sample 25. As shown in figure 1, the sensor surface 19 is in contact with buffer 26. The sensor 2, the prism 3 and at least part of the sampling means 12 and tubings 14 and 15 are present in a thermostatic unit 27 controlled at a desired temperature, such as 37° + or - 0.01 °C. In order to avoid temperature effects it is preferred that all liquids (sample, washing solution, calibration solution and the like) subject to back and forth flow over the sensor surface.

Figure 2 shows more in detail the sensor 2, the flow cell 4 and the optical unit 28. A source for polarized light 29 shines via the hemispherical or triangular prism 3 through the glass support 20 onto the gold metal layer 19 (50 nanometer) and to the active site 21-24, present in the channels 6-9, respectively. The reflected light 30 is monitored by a camera 31 which at the same time monitors the reflected light coming from all four active sites 21-24. As shown in figure 2 the SPR measurement of the sample 25 takes place while it is under the back and forth flow 17. In comparison to figure 1 , is in figure 3 the buffer 26 removed out of the flow cell 4 and substituted for the sample 25 using the transportation means 33, which are incorporated in the means 18 for generating the back and forth flow. Accordingly, the retraction of the piston 34 results in a liquid flow through the flow cell 4. First the buffer 26 is removed from the flow cell 4 and before the sample 25 could enter the flow cell 4 a separation fluidum 32 is passed through the flow cell, as it is present in between the buffer 26 and the sample 25. It is noted that the volume of the separation fluidum 32 is larger than the back and forth flow volume. Accordingly, during the back and forth movement buffer 26 and sample 25 cannot make contact via the inner surface of the tubings 14 and 15, which have been in contact and therefore wetted with either sample or buffer. The separation fluidum is in this case air aspirated after the aspiration of the buffer but before the aspiration of the sample.

Figure 4 shows a further step in the SPR measurement, in which sample 25 and separation fluidum 32 are removed from the flow cell 4. The flow cell is again filled with buffer 26 by a flow 36 carried out with the transportation means 33 comprising the piston 34. When having filled the flow cell 5 with buffer 26, then the dissociation step of the SPR measurement can take place measuring the release of non-specifically bound compound in combination with the (gradual) dissociation of the target compound from the ligand under real time conditions. By subtraction of a signal from a reference solution a discrimination can be made between the non- specific and specific release.

Figure 5 shows an instrument 37 comprising the SPR measuring system 1 according to the invention. The instrument 37 comprises a thermostatic sample rack 38 from which the sample means 12 aspirate with the needle 13 a sample (about 80 micro litre) and transports the sample using the transport means 33 to the flow cell 4. After measurement the sample may be returned into the sample well or in to a waste well 39 connected with a waste bottle 40. As described above the tubing 14 and 15 has been prefilled with buffer via the valve 16 and aspirated from the buffer bottle 41. The separation fluidum could be taken from the fluidum well 42. However, when the separation fluidum is air, then air could be aspirated (for example 25 micro litre) from the surrounding air via the needle 13 before aspirating the sample.

Figure 6 shows an alternative SPR measuring system 43 according to the invention. In this case the means 18 for generating the back and forth movement are not integrated with the transportation means 33. The means 18 for the back and forth flow 17 are connected via the valve 16 with the inlet 1 1 of the flow cell 4, whereas the transport means 33 for the transport flow 36 are connected via the valve 44 to the inlet 1 1 of the flow cell 4. Accordingly, as shown, the dissociation step of measuring the surface plasmon resonance angle of light incidence at the sensor surface after the measurement of the surface plasmon resonance angle of light incidence of the sample 25 takes place under the back and forth movement while transporting the buffer 26 through the flow cell 4 and remove continuously with the flow 36 dissociated target and a-specific compounds.

Figure 7 shows another SPR measuring system 45 according to the invention. In this case the means 18 for the back and forth flow 17 comprises a back and forth moving actuator 46. The actuator 46 comprises a jacket 47 which forces 48 onto a flexible part 49 of the tubing 15 thereby generating a back and forth flow 17 of the buffer 26, the separation fluidum 32 and the sample 25 during the measurement of the sample 25 and of the buffer 26.

Figure 8 shows an instrument 50 according to the invention comprising four parallel SPR systems 51-54 according to the invention, for which the details have been described in relation to the figures 1-4. A common optical unit in the form of a non-shown camera monitors the changes in the surface plasmon resonance angle of light incidence at the sensor surface in the active sites 21-24 of each of the flow cells 4. Accordingly, it is possible to measure in parallel for samples 25 or to measure for one sample 25 4x times different target compounds. The measurement of the sample of the buffer in the dissociation step and the for the determination of the base line is triggered by a change in the surface plasmon resonance angle of light incidence at the sensor surface the separation fluidum passing through the flow cells when changing in between the buffer, the sample and the buffer again. This provides a very accurate manner in triggering the measurements of each of the active sites independently by a non-shown computer assistant program.

Figure 9 shows a flow cell 55 of the invention. The flow cell comprises a glass support 20 provided with a gold metal layer (about 50 nanometer), which is covered by support 56 provided with the inlet 10 and the outlet 1 1. The inlet 10 and the outlet 1 1 communicate with a channel 58 covering the active sites provided on the gold metal layer 19. The glass support 20 is covered by the hemispheric or triangular prism 59 via which the polarised light is directed towards the gold metal layer and via which passes the reflected light towards the camera (not shown) for measuring a shift in the SPR angle of light incidence which is dependent on an increase or decrease of material mass at the sensor surface and/or due to the presence at the sensor surface of a sample, buffer, regeneration liquid or separation fluidum, and can be used for calculating a change of the surface plasmon resonance angle of light incidence at the sensor surface..

With reference to figures 1 , 3 and 4 the SPR measuring method according to the invention is described. For example, buffer 26 is aspirated from a bottle containing buffer and the needle, tubing 14 and 15, and flow cell 4, as well as back and forth flow means 18 are filled with buffer. Subsequently, separation fluidum 32 is aspirated (25 micro litre). When the separation fluidum is air, it can be aspirated from the surrounding air. Otherwise, separation fluidum is aspirated from a reservoir comprising the separation fluidum. The amount and volume of the separation fluidum aspirated is such, that its volume is larger than the back and forth volume generated with the back and forth flow means 18. Subsequently, the sample is aspirated from the sample rack (for instance 70-80 micro litre).

First, a base line measurement is carried out with the buffer 26 filling the flow cell and measuring the surface plasmon resonance angle of light incidence at the sensor surface with lapse of time by shining polarised light and monitoring the reflective light with the camera. The measurement takes place according to the invention with the back and forth flow at a back and forth flow volume smaller than the volume of the separation fluidum. Subsequently, the separation fluidum is passed through the flow cell and thereafter the flow cell is filled with sample. Then, SPR measurement takes place again under back and forth flow generated with back and forth flow means. Thereafter, the sample is removed out of the flow cell and the flow cell is refilled with buffer for carrying out the dissociation part of the SPR measurement for first measuring shift in the angle of light incidence due to a dissociation of non-specific compounds and subsequently the dissociation from the ligand bound target compounds. Again under back and forth flow or even with back and forth flow with a continuous flushing of the flow cell with buffer (see in this respect the embodiment 43 of figure 6).

Finally, the sample is removed from the system and the procedure for SPR measurement according to the invention may be restarted. Obviously, for calibration the sensor surface may be contacted with a calibration solution of which the shift of the surface plasmon resonance angle of light incidence at the sensor surface (and thus the refractory index) known, such solution may be water/glycerol mixture.

It is noted, although not yet described, that it is required to regenerate the active sites present in the flow cell after a sample measurement and the desorption measurement with buffer, then a regeneration fluidum may be aspirated after for instance the release of the sample from the SPR measuring system, and subjecting the active sites to the regeneration medium, thereby providing the flow cell and its active sites in a regeneration form for measurement of target compounds considered.

A glass substrate of 10x20mm (thickness 1mm) is provided by electron beam deposition with a gold layer (thickness 500 A). An immobilization layer of polyethyleneoxide (PEO) is applied using thiol-Cl 1-carcoxylate) (technology disclosed the article of E. A. Smith et al., JACS 125, 6140-6148, (2003). The PEO has a chemical functionality of a COOH group for binding to an NH₂ group of an antibody for a specific antigen. Using a spotter, preferably a continuous flow micro arrayer a sensor comprising several different ligand spots and reference and blank spots can be applied.

When applied in an IBIS-iSPR® imaging instrument this sensor provided excellent results using specific ligands for the specific diseases to be tested on patient samples.

Claims

1. Surface plasmon resonance measuring system comprising: i. at least one sensor having at least one sensor surface;

ii. at least one flow cell which is in liquid contact with the sensor

surface;

iii. an optical unit for measuring the surface plasmon resonance angle of light incidence at the sensor surface;

v. liquid transport means for liquid transport; and

2. System according to claim 1 , wherein the back and forth flow volume is less than the volume of the separation fluidum.

3. System according to claim 1 or 2, wherein the sampling means comprises a channel, such as a tubing, connected to the flow cell for taking up buffer and sample separated by separation fluidum.

4. System according to claim 1-3, wherein the sampling means comprises a channel, such as a tubing, connected to the flow cell and to the back and forth flow means, preferably the back and forth flow means comprise a back and forth moving actuator, such as a piston or pressure unit.

5. System according to claim 1-4, wherein the separation fluidum is a gas, such as air, or a liquid substantially immiscible with sample and buffer.

6. System according to claim 1-5, wherein the liquid transport means and the back and forth flow means are integrated.

7. System according to claims 1-6, comprising a plurality of flow cells preferably having separate sampling means and/or transport means.

8. System according to claims 1-7, wherein the sensor surface comprises two or more different active sites.

9. System according to claims 1 -8, wherein the sampling means comprise a unit for mixing sample with a reagent.

10. System according to claims 1-9, comprising a thermostatic unit for the sample and/or the buffer.

1 1. Method for surface plasmon resonance measurement comprising the steps of:

i. filling sample means with buffer and sample which sample is separated by separation flu^'fdum from the buffer;

ii. contacting at least one sensor surface with the buffer;

iii. measuring measuring the surface plasmon resonance angle of light

incidence at the sensor surface while in contact with the buffer being in back and forth movement;

iv. passing the separation flui^'dum along the sensor surface;

v. contacting the sensor surface with the sample;

vi. measuring the measuring the surface plasmon resonance angle of light incidence at the sensor surface while in contact with the sample being in back and forth movement; and optionally the step of:

vii. passing the separation fluidum along the sensor surface

viii. contacting the sensor surface with the buffer

ix. measuring the surface plasmon resonance angle of light incidence at the sensor surface while in contact with the buffer being in back and forth movement; and

vii. optionally washing the sensor surface with a regeneration liquid to

regenerate the sensor surface.

12. Method according to claim 1 1, wherein the start of step v) and/or step viii) is triggered by a change in surface plasmon resonance angle in step iv) and/or vii).

13. Method according to claim 1 1 or 12, wherein the sensor surface comprises a plurality of active sites monitored individually for change in surface Plasmon resonance angle, preferably with a camera.

14. Method according to claims 1 1-13, wherein the method is carried out in at least one flow cell, or in a plurality of flow cells served by common pomp means.