WO2012101539A1

WO2012101539A1 - Localization of detection spots

Info

Publication number: WO2012101539A1
Application number: PCT/IB2012/050156
Authority: WO
Inventors: Ron Martinus Laurentius Van Lieshout; Joannes Baptist Adrianus Dionisius Van Zon; Toon Hendrik Evers; Derk Jan Wilfred Klunder
Original assignee: Koninklijke Philips Electronics N.V.
Priority date: 2011-01-27
Filing date: 2012-01-12
Publication date: 2012-08-02

Abstract

The invention relates to a method and a sensor device(100) that allow for the determination of geometrical properties like the position and/or area of at least one detection spot(153) on a contact surface(152), wherein said detection spot(153) has a height that is different from the height of the residual surface. The detection spot(153) may for example be a region coated with binding sites for target molecules. The localization of the detection spot(153) is achieved by illuminating the contact surface(152) with evanescent waves and by observing an output light beam(L2, L2') which is affected by an interaction between test particles(1), which cover the detection spot(153) and the contact surface(152), and said evanescent waves. The test particles may for example be magnetic particles(1) that are attracted to the contact surface(152) by magnetic forces, or they may be transported to the contact surface(152) by sedimentation. The location of the detection spot(153) may be determined at the end or at the beginning of an assay and be used during the evaluation of measurements.

Description

LOCALIZATION OF DETECTION SPOTS

FIELD OF THE INVENTION

The invention relates to a method and a sensor device with which geometrical properties of at least one detection spot on a contact surface can be

determined.

BACKGROUND OF THE INVENTION

The WO 2009/112905 A2 discloses a biosensor device that applies frustrated total internal reflection (FTIR) of a light beam to detect magnetic particles bound to binding sites on a contact surface. Markers are used to infer the position of the binding sites at the beginning of an assay, i.e. while magnetic particles are not yet bound.

Moreover, it is mentioned that the binding sites can be tracked in images of the contact surface after they have become visible.

SUMMARY OF THE INVENTION

Based on this background, it was an object of the present invention to provide means that allow for more accurate measurements of processes occurring at particular detection spots on a contact surface.

This object is achieved by a method according to claim 1, a sensor device according to claim 2, and a use according to claim 15. Preferred embodiments are disclosed in the dependent claims.

According to a first aspect, the invention relates to a method for determining (one or more) geometrical properties like the position, shape, area etc. of at least one particular region or body which lies on a contact surface adjacent to a sample chamber. Said particular region/body will be called "detection spot" in the following (without any prejudice with respect to the shape or size of this region/body). The "sample chamber" is typically an open cavity, a closed cavity, or a cavity connected to other cavities by fluid connection channels. The term "contact surface" shall denote some part of the (inner) surface of the sample chamber. Based on these prerequisites, the method for determining geometrical properties of at least one detection spot comprises the following steps, which may be executed in the listed or any other appropriate order:

a) Covering the contact surface (where it is freely accessible) and the detection spot with test particles (e.g. nano-particles). The coverage will typically not be complete (gapless), but it preferably has a known (e.g. uniform) density. Hence each point on the contact surface and on the detection spot has some probability to be contacted by a test particle.

b) Illuminating the contact surface with an input light beam, wherein this illumination typically comprises also some (small) volume adjacent to the contact surface.

c) Detecting an output light beam that comes from the contact surface and that is affected by an interaction between the mentioned test particles and the input light beam. Said interaction may particularly comprise the scattering or absorption of light energy from the input light beam by the test particles.

d) Determining geometrical properties of the detection spot from the detected output light beam.

The input light beam may preferably have some spatially non-uniform characteristic, for example a non-uniform intensity, yielding a position-dependent interaction between test particles and the input light beam.

According to a second aspect, the invention relates to a sensor device for investigating a sample in a sample chamber, said sample chamber having a contact surface with at least one detection spot on it. The sensor device comprises the following components:

a) A light source for generating an input light beam that illuminates the contact surface.

b) A light detector for detecting an output light beam that is generated by the input light beam at the contact surface.

c) An evaluation unit that is adapted to determine geometrical properties of the detection spot from signals of the light detector that were generated while the contact surface and the detection spot were covered with test particles interacting with said input light beam. With the sensor device, a method of the kind described above can be executed. The explanations and definitions provided above with respect to the method are therefore analogously valid for the sensor device, too.

The method and the sensor device allow to localize detection spots on a contact surface by evaluating an interaction between an input light beam and test particles covering the contact surface and the spots. It is important to notice in this context that the test particles need not (or even must not) specifically interact with the detection spots or the contact surface. In particular, it is not required that the test particles are (specifically or non- specifically) bound to the detection spots. All that is needed is that the test particles are distributed all over the contact surface and the detection spots. At a detection spot, the (set of) test particles will then usually differ in some property from the (set of) test particles next to the spot. For example, the set of test particles may cover the detection spot with a lower density, or the test particles at the spot may have another distance from the contact surface than next to the spot. All these differences lead to an altered interaction with the input light beam. As a result, the location of the detection spots is revealed in the output light beam that is generated and affected by the interaction between test particles and input light beam.

In the following, various preferred embodiments of the invention will be described that relate to both the method and the sensor device defined above.

In a first preferred embodiment of the invention, the input light beam or a part thereof consists of evanescent waves that illuminate the contact surface (i.e. the surface itself and a small layer adjacent thereto). Preferably, the evanescent waves are generated at the contact surface. The intensity of evanescent waves depends strongly on their distance from the emission plane (e.g. the contact surface). On the other hand, test particles at a detection spot will typically have another distance from the contact surface than next to a detection spot. The location of the detection spots is therefore revealed in the output light beam that is generated and affected by the interaction between test particles and evanescent waves.

According to another embodiment of the invention, the output light beam is used to generate an image of the contact surface. In this case the light detector is typically realized by an imaging device, for example a CCD or CMOS camera. In an image of the contact surface, brightness can be determined and observed simultaneously for the whole surface, and known image processing techniques can be applied to localize the detection spot (e.g. via an intensity contrast).

In general, the detection spot will distinguish in some property from the remainder of the contact surface that helps or that is even needed to execute the desired processing or investigation of a sample in the sample chamber adjacent to the contact surface. In a particularly preferred embodiment of the invention, the detection spot comprises binding sites for target particles of a sample, i.e. components by which said target particles are (specifically) immobilized at the detection spot. After such an immobilization, the target particles can qualitatively or quantitatively be detected at the detection spots. The target particles may optionally be the same as the test particles. The target particles may inter alia comprise atoms, molecules, complexes, nanoparticles, or microparticles, particularly biological substances like biomolecules, cell fractions or cells.

The contact surface may particularly be a surface of a transparent wall (or body). This allows to illuminate and/or observe processes at the contact surface through said transparent wall.

In a further development of the aforementioned embodiment, the evanescent waves are generated at the contact surface by total internal reflection of an input light beam propagating through said transparent wall. Total internal reflection provides a readily feasible way to generate evanescent waves at the contact surface, wherein the intensity of said waves drops strongly with increasing distance from the place of their generation (i.e. the contact surface).

The light that constitutes the output light beam may have various origins. In one embodiment, the output light beam may comprise totally internally reflected light of an input light beam. In a typical realization of this embodiment, said input light beam is directed through the above mentioned transparent wall to the contact surface, where it is totally internally reflected into the output light beam. When the evanescent waves generated at the reflecting surface interact with test particles (or other entities) close to the contact surface, this leads to frustrated total internal reflection (FTIR) due to the scattering of light. The scattered light energy misses in the output light beam, and the corresponding intensity drop reveals the presence (and amount) of scattering particles at the contact surface.

In another embodiment, the output light beam may comprise light that was scattered by test particles (or other entities) close to the contact surface. This approach is complementarily to the aforementioned one in the sense that now scattered light is measured directly (instead of indirectly via its absence in the reflected light beam). This procedure is particularly advantageous for low concentrations of scattering particles because it can determine the small quantity of scattered light more accurately.

Knowledge of geometrical properties like the position or area of the detection spot is usually important for a precise execution and evaluation of processes and measurements at the contact surface. For this reason, it is often desirable to know the exact position of the detection spot in advance, i.e. before an assay is performed. According to a preferred embodiment of the invention, the geometrical properties of the detection spot are therefore determined at the being of an assay, and measurements during the assay are evaluated based on these determined properties. The brightness of an output light beam is for example only evaluated at image positions (pixels) that have been determined as belonging to the detection spot.

In another embodiment, geometrical properties of the detection spot are determined at the end of an assay, wherein measurements during the assay are recorded (stored) and evaluated afterwards based on said determined properties. This approach requires to intermediately store all measurements, but it has the advantage that the localization of the detection spot can be deferred to the end of the assay.

There are different possibilities how the contact surface can be covered with test particles. In one preferred embodiment, the test particles are transported to the contact surface by sedimentation. This approach has the advantage that no specific features of the test particles are required (besides having a higher density than the surrounding medium). Moreover, the sedimentation typically yields a very homogeneous distribution of test particles, thus allowing a good localization of the detection spot.

In another embodiment of the invention, the test particles comprise magnetic particles, i.e. particles which are magnetic or magnetizable. Magnetic test particles can be actuated and particularly be attracted to the contact surface by the action of an appropriate magnetic field, which allows to control and accelerate the coverage of the contact surface with test particles. Most preferably, a combination of actuation and sedimentation is applied because actuation can speed up the sedimentation process, while subsequent sedimentation provides a uniform distribution of test particles.

In a related embodiment of the invention, a magnet is provided for actuating magnetic particles in the sample chamber. The actuation may particularly comprise an attraction to the contact surface, allowing the aforementioned forced coverage of the contact surface with magnetic test particles. In general, the actuation may comprise the forced movement of magnetic particles in any convenient direction, particularly a movement away from the contact surface in order to separate surface-bound from unbound particles.

The invention further relates to the use of the sensor device described above for molecular diagnostics, biological sample analysis, chemical sample analysis, food analysis, and/or forensic analysis. Molecular diagnostics may for example be accomplished with the help of magnetic beads or fluorescent particles that are directly or indirectly attached to target molecules.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. These embodiments will be described by way of example with the help of the accompanying drawings in which:

Figure 1 schematically shows a side view of a sensor device according to the present invention that is adapted to perform FTIR and Evanescent Dark Field

Microscopy (EDFM) measurements;

Figure 2 shows an EDFM image of the contact surface with a detection spot when test particles have sedimented;

Figure 3 shows an EDFM image of the contact surface with a detection spot when test particles have been magnetically attracted;

Figure 4 shows an EDFM image of the contact surface with a detection spot to which magnetic particles have specifically bound, while non-bound particles have been removed.

Like reference numbers in the Figures refer to identical or similar components. DESCRIPTION OF PREFERRED EMBODIMENTS

The measurements in many biosensors are based on nanoparticle labels, particularly magnetic (nano-) particles or beads that can be actuated with electromagnetic fields. Typically, the magnetic beads are functionalized with antibodies that can bind a specific analyte molecule of a sample. The beads are attracted to the sensor surface, where the number of bound beads is directly or inversely related to the amount of analyte molecules present in the sample. The beads can then be detected using any technique that is more sensitive to beads that are close to the surface. For example, the detection technique may be based on evanescent optical fields, e.g. Frustrated Total Internal Reflection (FTIR).

Figure 1 shows a biosensor device 100 of the aforementioned kind in a schematic side view. The sensor device 100 comprises a reader 110 and a disposable cartridge 150 in which a sample with target components of interest can be provided. The cartridge 150 may for example be made from glass or transparent plastic like poly-styrene. It comprises a sample chamber 151 in which a sample fluid with target components to be detected (e.g. drugs, antibodies, DNA, parathyroid hormone PTH etc.) can be provided. The sample further comprises magnetic particles 1, for example superparamagnetic beads, wherein these particles 1 are usually bound as labels to the aforementioned target components (for simplicity only the magnetic particles 1 are shown in the Figure). It should be noted that instead of magnetic particles other label particles, for example electrically charged or fluorescent particles, could be used as well.

The cartridge 150 has a transparent wall 154 with a "contact surface" 152 that (partially) borders the sample chamber 151. A plurality of "detection spots" 153 is disposed on the contact surface 152. They comprise binding sites, e.g. antibodies, which can specifically bind the target components.

The reader 110 comprises a light source 120 for emitting an "input light beam" LI, a light detector 130 for detecting and measuring an "output light beam" L2, and an evaluation unit 135 for evaluating the signals of the light detector. The input light beam LI generated by the light source 120 propagates through the wall 154 and arrives at the contact surface 152 at an angle larger than the critical angle of total internal reflection (TIR) and is therefore totally internally reflected as the output light beam L2. The output light beam L2 leaves the cartridge 150 and is detected by the light detector, e. g. by the light-sensitive pixels of a camera 130. The light detector 130 thus generates an image of the contact surface, which is further processed in the evaluation unit 135.

The reader 110 further comprises a magnetic field generator, for example electromagnets 140 with a coil and a core disposed at the bottom and/or at the top (not shown) of the cartridge, for controllably generating a magnetic field at the contact surface 152 and in the adjacent space of the sample chamber 151. With the help of this magnetic field, the magnetic particles 1 can be manipulated, i.e. be magnetized and particularly be moved (if magnetic fields with gradients are used). Thus it is for example possible to attract magnetic particles 1 to the contact surface 152 in order to accelerate the binding of the associated target component to said surface.

The described sensor device 100 applies optical means for the detection of magnetic particles 1 and the target components one is actually interested in. For

eliminating or at least minimizing the influence of background (e.g. of the sample fluid, such as saliva, blood, etc.), the detection technique should be surface-specific. As indicated above, this is achieved by using the principle of frustrated total internal reflection. This principle is based on the fact that an evanescent wave propagates (exponentially dropping) into the sample chamber 152 when the incident light beam LI is totally internally reflected. If this evanescent wave then interacts with another medium having a different refractive index from water like the magnetic particles 1 , part of the input light will be coupled into the sample fluid (this is called "frustrated total internal reflection"), and the reflected intensity will be reduced (while the reflected intensity will be 100% for a clean interface and no interaction). Further details of this procedure may be found in the

WO 2008/155723 Al, which is incorporated into the present text by reference. It should be noted that, in the context of the present invention, the "input light beam" LI shall comprise the light from its emission by the light source 120 to its interaction with the contact surface or with particles at said surface; the input light beam hence comprises also the evanescent waves. The "output light beam" L2, on the contrary, shall comprise the rest of the light, i.e. light after total internal reflection at the contact surface and/or after interaction of evanescent waves with particles at the contact surface.

By adding a high Numerical Aperture (NA) imaging means - e.g. an objective lens plus camera 130' - to the described FTIR setup, microscope images from the contact surface 152 can be made in real time and the position and height of individual beads 1 can be determined as a function of time by analyzing the images with suitable software. Said imaging is based on Dark Field Microscopy (DFM) with evanescent field illumination, i.e. the recorded output light beam L2' originates from light scattering in the evanescent field. As "Dark Field Microscopy" basically means a condition where a specular reflected light beam is outside the numerical aperture of a lens (irrespective of the applied illumination principle), the term "Evanescent Dark Field Microscopy" or "EDFM" will be used in the following to denote the particular approach of the present invention which combines DFM with evanescent field illumination. Due to the decreasing evanescent field intensity, beads at a certain distance from the surface give less light in EDFM than beads close to the surface. This effect can be used to determine geometrical properties like position, size, and shape of a detection spot.

In the sensor device 100 of Figure 1, light detectors for both FTIR measurements (camera 130) and EDFM measurements (objective and camera 130') are provided. It should however be noted that in a commercial apparatus typically only one of these alternatives would be present for reasons of cost. Moreover, the objective lens 130' is in practice typically positioned below the contact surface to allow focusing on the beads there. To this end, the objective lens 130' may be integrated with the electromagnet 140.

It should further be noted that FTIR and EDFM differ in the way they determine the presence (and density) of beads at the contact surface:

- FTIR measures intensities, wherein the measured intensity is related to the absorption of evanescent waves by beads. Since the detection camera 130 is under a small angle with respect to the contact surface 152, the numerical aperture is very small and hence the resolution low. It is therefore not possible to resolve individual beads of typically used sizes (e.g. about 500 nm), so bead counting is usually not possible.

- In contrast to this, the detection camera 130' is perpendicular to the contact surface 152 in EDFM. Hence the numerical aperture is large, yielding a high resolution that allows to resolve individual beads and to generate a signal based on bead counting.

When the described sensor device 100 is applied, the detection of the biomarker particles 1 occurs through a specific reaction between the analyte and the binding sites (capture probes) deposited on the contact surface 152. This reaction is confined to the detection spots 153 where the binding sites are deposited. Hence the image analysis of the images generated with the light detectors 130, 130' is preferably carried out on this area or Region-Of-Interest (ROI). Especially at low analyte concentrations or even when no analyte is present at all, there is no (or little) specific binding and thus no means of determining whether the area where the binding sites are deposited is in the field of view or within the ROI. Without knowing the presence of the binding sites it is not possible to state that a measurement is negative (no analyte present) or that the correct measurement has been performed. Hence there is a need to accurately know the size and position of the area of the binding sites and/or to know whether the binding sites are present.

Accordingly, the main problems addressed by the present invention can be summarized as follows:

1. For an accurate readout of the signal change obtained during or after an assay, it is important that a predefined region of interest (ROI) is positioned over the detection spots 153 occupied with the binding sites (e.g. inkjet printed antibodies). Using set values for the positions of the detection spots is however problematic because there are many causes for a deviation of the detection spots from their required position. For example, during inkjet-printing, the position of the spots can deviate from their required positions. Other additional causes of such a misalignment can be a deviation in the cartridge geometry or a distorted optical path of the device.

2. Usually the position of a detection spot becomes clear when a positive signal build-up in time is achieved. However at the start or in the beginning of the assay no clear binding is yet seen.

3. When measuring low concentrations or negative samples (i.e. no analyte present), the reaction between the analyte and binding sites is not always clearly distinguishable. To ensure that the ROI is in the same area where the binding sites are printed, a positive control is needed. A positive control confirms the presence of the binding sites.

4. The size of the detection spot printed with antibodies determines the absolute number of beads which can be bound within the spot. In practice there will be variations in spot size and/or spot position. In order to be tolerant against these variations, a ROI has to be chosen which is (much) smaller than the average printed spot size to prevent the counting of non-specifically bound beads outside the printed spot. However a much smaller ROI gives a statistical disadvantage since not all beads are counted.

Therefore preferably the ROI matches the exact size and position of the deposited spot. Since the concentration of a target substance is directly related to the surface density of beads, the counted number of beads has to be divided by the size of the ROI to obtain the correct sensor signal in case of EDFM. To solve the mentioned problems, the present invention makes use of the fact that the illumination of the magnetic beads 1 near the contact surface 152 is accomplished by an evanescent field, generated under total internal reflection conditions of the incoming light beam LI . The intensity of the evanescent standing wave drops exponentially with the distance to the surface 152. Therefore beads which are close to the surface are illuminated stronger and scatter more light (i.e. they appear brighter in the output light beam L2' used for EDFM and darker in the output light beam L2 used for FTIR) than beads which are further away from the surface. Based on this feature, geometrical properties like the position, area, shape etc. of a detection spot 153 can be determined by bringing magnetic "test" particles 1 in close contact with the surface 152 and said spot, respectively. The actual height of (non-bound) magnetic test particles 1 above a surface is influenced by the height of the (bio)molecule layer between the test particle and the surface and also the charge of the surface and the test particle. As an antibody layer differs from the surrounding layer (containing e.g. surface blocking material such as bovine serum albumin or no other material at all) both in height and in charge, the average distance of test particles above a detection spot 153 is different from that above the surrounding area. This is schematically illustrated in Figure 1.

To give an example, the printed antibodies in a detection spot 153 typically have a size of about 15 nm, where the blocking agent (e.g. BSA and polysorbate 20) generally has a smaller size (about 8 nm). Test beads in a detection spot 153 which are on top of these antibodies are therefore further away from the contact surface 152 (i.e. from an ideal plane defining this surface) than test beads which are on top of the non functionalized surface. Using this difference in height between test beads directly above the printed binding sites (antibodies) and test beads above the blocked non printed area, one can determine the positions where the binding sites are printed based on a difference in measured light intensity.

There are different ways of how magnetic test particles can be brought in close contact with the surface. For example, test particles can be brought to the contact surface by means of sedimentation. As can be seen from the EDFM image of Figure 2, this results in a very homogeneous dispersion of the test particles. Sedimentation is preferably performed after an assay. If the position of the ROIs needs to be adjusted after the assay, all required information to calculate the results of the newly defined ROIs needs to be stored during the entire assay as well. This may pose a problem with respect to memory capacity, particularly if highly detailed information during the entire time of the assay (e.g. 25 frames per second for 5 minutes) is needed. Sedimentation is therefore preferably used at low (or zero) analyte concentrations to ensure that there are binding sites available on the contact surface (positive control for presence of the binding sites).

As an alternative, the magnetic test particles 1 may be attracted towards the contact surface 152 at the start of the assay using magnetic forces, i.e. by activation of the magnet 140. Figure 3 shows the resulting EDFM image of the contact surface, from which the position of the detection spot can be determined. Although Figure 3 shows only a single image, a better determination could be obtained by averaging over several images. These images can be simply successive images. However, it can also be chosen to only average images that were obtained during the same phase of a pulse (sequence) of the magnet (e.g. only when the bottom magnet is on, or only just after the bottom magnet has been switched off). If the real positions of the detection spots are determined early in the assay, only a limited amount of images needs to be stored. Since the approximate position of the spots is known (only relatively small deviations are expected), the amount of stored data can be further reduced by storing only the information of a relatively small area surrounding the expected position of the spots.

As an example, a cartridge was injected with 1 mg/ml 1000 nm antibody (mAb2) conjugated superparamagnetic beads 1 , where after a continuous magnetic attraction was applied for 10 seconds. This attraction period was also used to focus a high NA lens on the contact surface to obtain a sharp image. After this continuous attraction the actuation protocol used for the assay was activated. Again the beads 1 were attracted to the contact surface (e.g. 3 seconds), thereafter a diffusion step was allowed (no magnetic actuation) for e.g. 3 seconds. The last step of this actuation cycle consisted of a washing step where a top magnetic field was active during 4 seconds to remove the non-bound beads from the evanescent field. During the attraction and/or diffusion step the contrast can be clearly seen between the area where the detection spot is printed and the blocked non functionalized area surrounding it (Figure 3). As a comparison the EDFM image of Figure 4 shows the detection spot when beads are specifically bound to the binding sites on the contact surface.

The approach of the present invention can particularly be used within an immuno-assay platform based on magnetic bead actuation and Frustrated Total Internal Reflection (FTIR), optionally in combination with (evanescent) Dark-Field optical detection. This platform can be operated in a decentralized setting while it still has the performance of a central lab analysis system which can detect analyte concentrations in the range from fM (10^~15 M) up to nM (10^~9 M). The assay performed on this platform consists of a specific chemical reaction between antibody conjugated superparamagnetic beads that can bind to the analyte to be detected, and binding sites deposited on a certain area of the surface (detection spot). The analyte is detected by measuring the presence of specifically bound beads on the surface. By counting the beads within the detection spot, the analyte concentration can be determined. For a high analyte concentration, many beads are present on the surface and the measurement spot is clearly visible. However for low analyte concentrations, it is not clear where the binding sites are deposited in the field of view. By using the height difference between the functionalized area and the non-functionalized blocked area, an intensity difference can be created when accumulating test beads on the surface by means of magnetic actuation and/or sedimentation. This creates a clear contrast which can be used to determine the position of the detection spot, allowing further signal processing such as bead counting within the spot. Simultaneously the detection of the spot can be used as a positive control that the deposited antibodies are present.

IN SUMMARY, THE PRESENT INVENTION THEREFORE PROVIDES:

- A method to determine the size and the position of the area (detection spot) where the binding sites are present without a (specific or non-specific) reaction.

- A method of automatically detecting and positioning a ROI before or closely after the assay has started.

- A method of automatically detecting a ROI after the end of the assay and analyzing the previously recorded images with the knowledge of the accurate position of the ROI.

- A method to determine the presence of an area where binding sites are printed inside the field of view (positive control).

- A method to correct for variations in spot size and detect irregular printed spots, thus reducing the statistical spread in the determined analyte concentration.

- A difference in physical properties (e.g. height) is used between the printed detection spot and the non functionalized area.

Finally it is pointed out that in the present application the term "comprising" does not exclude other elements or steps, that "a" or "an" does not exclude a plurality, and that a single processor or other unit may fulfill the functions of several means. The invention resides in each and every novel characteristic feature and each and every combination of characteristic features. Moreover, reference signs in the claims shall not be construed as limiting their scope.

Claims

CLAIMS:

1. A method for determining geometrical properties of at least one detection spot (153) that lies on a contact surface (152), comprising the following steps:

a) covering the contact surface (152) and the detection spot (153) with test particles (1);

b) illuminating the contact surface (152) with an input light beam (LI); c) detecting an output light beam (L2, L2') that comes from the contact surface (152) and that is affected by an interaction between the test particles (1) and the input light beam (LI):

d) determining geometrical properties of the detection spot (153) from the detected output light beam (L2, L2').

2. A sensor device (100) for investigating a sample in a sample chamber (151), said sample chamber having a contact surface (152) with at least one detection spot (153) on it, comprising:

a) a light source (120) for generating an input light beam (LI) that illuminates the contact surface (152);

b) a light detector (130, 130') for detecting an output light beam (L2, L2') that is generated by the input light beam at the contact surface (152);

c) an evaluation unit (135) that is adapted to determine geometrical properties of the detection spot (153) from signals of the light detector (130, 130') that were generated while the contact surface (152) and the detection spot (153) were covered with test particles (1).

3. The method according to claim 1 or the sensor device (100) according to claim 2,

characterized in that the input light beam or a part thereof consists of evanescent waves that illuminate the contact surface.

4. The method according to claim 1 or the sensor device (100) according to claim 2,

characterized in that the output light beam (L2, L2') is used to generate an image of the contact surface (152).

5. The method according to claim 1 or the sensor device (100) according to claim 2,

characterized in that the detection spot (153) comprises binding sites for target particles (1).

6. The method according to claim 1 or the sensor device (100) according to claim 2,

characterized in that the contact surface (152) is the surface of a transparent wall (154).

7. The method or the sensor device (100) according to claim 6,

characterized in that evanescent waves are generated by total internal reflection of an input light beam (LI) at the contact surface (152).

8. The method according to claim 1 or the sensor device (100) according to claim 2,

characterized in that the output light beam (L2) comprises totally internally reflected light of an input light beam (LI).

9. The method according to claim 1 or the sensor device (100) according to claim 2,

characterized in that the output light beam (L2') comprises light that was scattered by the test particles (1).

10. The method according to claim 1 or the sensor device (100) according to claim 2,

characterized in that geometrical properties of the detection spot (153) are determined at the beginning of an assay, and that measurements during the assay are evaluated based on these determined properties.

11. The method according to claim 1 or the sensor device (100) according to claim 2,

characterized in that geometrical properties of the detection spot (153) are determined at the end of an assay, and that measurements recorded during the assay are evaluated based on these determined properties.

12. The method according to claim 1 or the sensor device (100) according to claim 2,

characterized in that the test particles (1) are transported to the contact surface (152) by sedimentation.

13. The method according to claim 1 or the sensor device (100) according to claim 2,

characterized in that the test particles (1) comprise magnetic particles.

14. The method according to claim 1 or the sensor device (100) according to claim 2,

characterized in that a magnet (140) is provided for actuating magnetic particles (1) in the sample chamber (151).

15. Use of the sensor device according to any of the claims 2 to 14 for molecular diagnostics, biological sample analysis, chemical sample analysis, food analysis, and/or forensic analysis.