HK1199304B

HK1199304B - Device for use in the detection of binding affinities

Info

Publication number: HK1199304B
Application number: HK14112863.3A
Authority: HK
Inventors: 克里斯托夫．法蒂蒙尔
Original assignee: 弗.哈夫曼-拉罗切有限公司
Priority date: 2012-01-17
Filing date: 2013-01-17
Publication date: 2017-10-27

Description

Device for detecting binding affinity

Technical Field

The present invention relates to a device for detecting binding affinity and a method of a system for detecting binding affinity according to the respective independent claims.

Background

For example, such devices are used as biosensors in a variety of applications. One particular application is the detection and monitoring of binding affinity (binding affinity) or processes. For example, with the aid of such biosensors, various tests for detecting binding of a target sample to a binding site can be performed. Typically, a large number of such assays are performed on biosensors at sites arranged at a two-dimensional microarray on the biosensor surface. The use of microarrays provides a tool for simultaneously detecting the binding affinity or process of different target samples in high-throughput drug screening, where large numbers of target samples, such as molecules, proteins or DNA, can be analyzed quickly. In order to detect the affinity of a target sample bound to a specific binding site, such as the affinity of target molecules bound to different capture molecules, a large number of binding sites are immobilized on the surface of the biosensor at a site where they can be applied, e.g. by inkjet assay sites (ink-jet spotting). Each site forms an independent measurement zone for a predetermined type of capture molecule. The affinity of the target sample for a particular type of capture molecule is detected and used to provide information about the binding affinity of the target sample.

Known techniques for detecting binding affinity of a target sample use labels that fluoresce upon excitation. For example, fluorescent labels may be used as labels for labeling target samples. When excited, the fluorescent label is caused to emit fluorescent light having a characteristic emission spectrum. Detection of this characteristic emission spectrum at a particular site indicates that the labeled target molecule has bound to a specific type of binding site present at the respective site.

Sensors for the detection of labelled target samples are described in the following article "Zeptosens' protein microorganisms" A novel high performance microorganism platform for low absorption of protein analysis ", Proteomics2002,2, S.383-393, Wiley-VCH Verlag GmBH 69451Weinheim, Germany. The described sensor comprises a planar waveguide arranged on a substrate, and a grating for coupling coherent light of a predetermined wavelength into the planar waveguide. Further a grating is arranged at that end of the planar waveguide remote from the grating to couple light into the waveguide. Coherent light propagating through the planar waveguide is coupled out of the planar waveguide through the further grating. The coupled-out light is used to condition coherent light of a predetermined wavelength to be coupled into the planar waveguide. Coherent light propagates through the planar waveguide under total reflection conditions, wherein an evanescent field (evanescent field) of the coherent light propagates along an outer surface of the planar waveguide. The depth of penetration of the evanescent field into the low refractive index medium at the outer surface of the planar waveguide is of the order of a fraction of the wavelength of coherent light propagating through the planar waveguide. The evanescent field excites fluorescent labels of the labeled target sample bound to binding sites arranged on the outer surface of the planar waveguide. Due to the very small penetration of the evanescent field into the optically thinner medium at the outer surface of the planar waveguide, only the labelled sample bound to the binding sites arranged on the outer surface of the planar waveguide is excited. The fluorescence emitted by these labels is then detected by means of a CCD camera.

Although binding affinity can be detected in principle by using fluorescent labels, this technique is disadvantageous in that: the detected signal is generated by the label and not by the binding partner itself. Furthermore, marking the target sample requires additional work steps. Furthermore, the labeled target samples are relatively expensive. Another disadvantage is the distortion of the results caused by the photo-bleaching or quenching effect.

Disclosure of Invention

It is an object of the present invention to provide a device for detecting binding affinities, as well as a system and a method capable of detecting such binding affinities, which overcome or at least substantially reduce the above-mentioned disadvantages of the prior art sensors.

According to the invention, this object is achieved by a device for detecting binding affinity. The apparatus includes a planar waveguide disposed on a substrate, and further includes an optical coupler for coupling coherent light of a predetermined wavelength into the planar waveguide such that the coherent light propagates through the planar waveguide, wherein a evanescent field of the coherent light propagates along an outer surface of the planar waveguide. The outer surface of the planar waveguide includes binding sites thereon capable of binding the target sample to the binding sites such that light of the evanescent field is scattered by the target sample bound to the binding sites. The binding sites are arranged along a plurality of predetermined lines arranged such that light scattered by a target sample bound to the binding sites interferes with a difference in optical path length at a predetermined detection position, the difference being an integer multiple of a predetermined wavelength of the light.

According to the present invention, the detection of binding affinity is neither limited to a specific type of target sample nor to any type of binding site, but rather the binding properties of molecules, proteins, DNA, etc. can be analyzed with respect to any type of binding site on the planar waveguide. The detection of binding affinity can be achieved in a label-free manner. Alternatively, a scattering enhancing agent that strongly scatters light (such as a scattering label) may be used to improve detection sensitivity. Such scattering enhancers may be nanoparticles (alone or with a binder) or in another example colloidal particles. The binding characteristics to be analyzed may be of a static type (e.g. it may be analyzed whether the target sample has bound or not to a binding site) or of a dynamic type (e.g. the kinetics of the binding process over time may be analyzed). The binding sites are locations on the outer surface of the planar waveguide to which the target sample can bind. For example, the binding sites may comprise capture molecules immobilized on the outer surface of the planar waveguide, or may simply comprise activation sites on the outer surface of the planar waveguide to enable binding of the target sample to the activation sites, or may be embodied in any other form suitable for binding of the target sample at the desired location on the outer surface of the planar waveguide. The plurality of predetermined lines may include individual lines or may include a line pattern in which the lines are connected to form a single line, for example, a bent single line pattern. The distance between adjacent predetermined lines along which the bonding sites are arranged is selected in relation to the predetermined wavelength of the light. A preferred distance between adjacent predetermined lines is on the order of greater than 100 nanometers (nm). A range of about 100 nanometers to about 1000 nanometers for the distance between adjacent predetermined lines is preferred for using visible light in a planar waveguide so that scattered light can be detected by standard optical means. Furthermore, it is preferred that the planar light waveguide has a high refractive index relative to the medium on the outer surface of the planar waveguide, so that the depth of penetration of the evanescent field is only small and the fraction of coherent light propagating in the evanescent field is high. For example, the refractive index of the planar waveguide may be in the range of 1.6 to 2.5, whereas the refractive index of the medium at the surface of the planar waveguide is typically in the range of 1 to 1.5. By way of example, the binding sites may comprise capture molecules immobilized on the outer surface of the planar waveguide. The immobilized capture molecules, together with the target sample bound thereto, form a plurality of scattering centers that scatter the coherent light of the evanescent field. Coherent light propagating along the planar waveguide has a predetermined wavelength and is preferably monochromatic (theoretically at a single wavelength). Since the evanescent field light propagating along the surface of the planar waveguide is coherent with the light propagating within the planar waveguide, the evanescent field coherent light is coherently scattered by scattering centers formed by target molecules bound to capture molecules arranged on different predetermined lines (or more generally by target samples bound to binding sites). Scattered light at any location can be determined by adding contributions from each of the individual scattering centers. The maximum value of the scattered light is located at the predetermined detection position because the predetermined line is arranged such that the optical path lengths of the light scattered by the different scattering centers differ by an integral multiple of the wavelength of the light at the predetermined detection position. The optical path length of light from the optical coupler to the predetermined line and from the predetermined line to the predetermined detection position is also an integral multiple of the predetermined wavelength for the maximum signal at the detection position. Thus, light scattered by the target sample bound to the binding sites interferes at the predetermined detection position. The need for constructive interference is met by adding any scattered light of a detectable signal in the detection site. The predetermined detection position is not limited to a specific shape, and may have a dot or strip shape, for example. The arrangement of the bonding sites "along a predetermined route" represents the following optimization scenario: in which all the bonding sites are arranged exactly on a predetermined route. This optimized design of the binding sites results in a maximum signal at the detection location. It will be apparent to those skilled in the art that in practice the arrangement of the bond sites may deviate somewhat from this optimized arrangement. Such a deviation may for example be caused by a method for arranging the bonding sites on the outer surface of the planar waveguide, as will be explained in more detail below.

According to an aspect of the device according to the invention, the distance between adjacent predetermined lines decreases in the propagation direction of the evanescent field of light. In general, the angle at which the scattered light of the evanescent field interferes at a predetermined detection position is different for different scattering centers (target samples bound to the binding site) arranged along a predetermined line. Since scattered light is to interfere to the maximum value at a predetermined detection position, the difference in optical path length of light scattered by a plurality of kinds of scattering centers must be an integral multiple of the wavelength of light. The reduction in the distance between adjacent predetermined lines explains that fact and causes the light to interfere to a maximum at the predetermined detection position, which need not have a spot shape or a small spot shape but may have a stripe shape or any other desired shape.

According to a further aspect of the device according to the invention, the plurality of predetermined lines on which the bonding sites are arranged comprise curves. The curvature of the lines is such that evanescent field light scattered by target samples bound to binding sites arranged along these predetermined lines interferes to a maximum at predetermined detection positions. The detection position preferably has a spot shape. Each of the respective predetermined lines may have a curvature different from the curvatures of the other predetermined lines. In practice, the detection location is not a point but may be a small spot (spot) or a strip having a smaller length than the predetermined line length along which the binding sites are arranged. The curvature of each respective curved predetermined line is selected such that the optical path length of light propagating from the optical coupler to the respective predetermined line and from the respective predetermined line to the predetermined detection position is an integral multiple of the predetermined wavelength of the propagating light for the entire curve. This advantage is also: light scattered by scattering centers located on the outside of the predetermined line contributes to the signal in the spatially reduced area of the spot-like detection location (or spot-like or strip-like).

According to still a further aspect of the apparatus of the present invention, the plurality of predetermined lines are arranged on the outer surface of the planar waveguide in the following manner: in such a way that they are at X_j,Y_jThe position in the coordinate system is geometrically defined by the following equations,

wherein

λ is the vacuum wavelength of the propagating light,

n is the effective refractive index of the guided mode in the planar waveguide; n depends on the thickness and refractive index of the planar waveguide, the refractive index of the substrate, the refractive index of the medium on the outer surface of the planar waveguide and the polarization of the guided mode,

n_Sis the refractive index of the substrate and,

f is the thickness of the substrate,

A₀is an integer selected to approximate the refractive index n of the substrate_SThe product of the thickness f of the substrate divided by the wavelength λ, an

j is a running integer representing the index of the corresponding line.

Selected integer A₀A negative j value is assigned to the negative x value at the center of the line and a positive j value is assigned to the positive x value at the center of the line. Or in other words the integer A₀Defining an origin of an x, y coordinate frame for locating the line on the outer surface of the planar waveguide; selected A₀The values place the detection position at x-0, y-0, and z-f.

As outlined above, for improving the signal at the predetermined detection positions, it is preferred that the plurality of predetermined lines are arranged in such a way that the scattering centers arranged along these predetermined lines are located on a curved grid-like structure with a decreasing distance between adjacent predetermined lines. This arrangement achieves the following conditions: for light propagating from the optical coupler to each predetermined line and scattered by the scattering center at a predetermined detection position, the difference in optical path length is an integral multiple of a predetermined wavelength of the light propagating in the waveguide. Further, the optical path length of the light propagating from the optical coupler to each predetermined line and from each predetermined line to the predetermined detection position is an integral multiple of the predetermined wavelength of the propagating light with respect to the entire curve. Thus, since the bonding sites are arranged on the outer surface of the planar waveguide, a compact device can be formed, while the detection position can be formed at the bottom surface of the substrate carrying the planar waveguide.

Both embodiments are specifically seen as how the bonding sites may be arranged along a plurality of predetermined lines. According to a first embodiment, the binding sites comprise capture molecules attached to the surface of the planar waveguide only along a predetermined line. These capture molecules are capable of binding to the target sample and being immobilised on the outer surface of the planar waveguide (although as mentioned above, the binding sites may be formed by an activated surface of the planar waveguide itself). The immobilization of the capture molecules on the outer surface of the planar waveguide along the predetermined line may generally be performed by any suitable method, such as by using a lithographic method using a lithographic mask having a curved profile. Of course, the placement of the bonding sites along the predetermined route is to be understood by the following physician in any embodiment of the invention: most binding sites (i.e. capture molecules in the present embodiment) are placed along a predetermined line and specifically comprise: some of the bonding sites are arranged at positions different from the predetermined route.

According to a second embodiment, the binding sites further comprise capture molecules capable of binding to the target sample, which is not limited to a specific type of binding site or to a specific type of target sample. The capture molecules are again able to bind to the target sample. However, the arrangement of capture molecules capable of binding target molecules along the predetermined line is performed by distributing and immobilizing capture molecules capable of binding target samples on the (entire) surface of the planar waveguide and by subsequently deactivating those capture molecules that are not arranged along the predetermined line. The term "deactivating" in this respect refers to any suitable method for altering the binding capacity of the capture molecules (e.g. by exposing the capture molecules to light for a predetermined period of time) to achieve that they are no longer able to bind to the target sample. According to this embodiment of the invention, the capture molecules may be uniformly or statistically applied on the outer surface of the planar waveguide. After deactivating the capture molecules arranged between the predetermined lines, only the capture molecules (which are not activated) arranged along the predetermined lines are able to bind to the target sample. However, the deactivated capture molecules remain immobilized on the outer surface of the planar waveguide.

This embodiment has the following additional benefits: the contribution of the signal generated by light scattered by the target molecules bound to the capture molecules to the overall signal at the detection location increases. In general, the difference between the signal of light scattered by the target molecules bound to the capture molecules and the signal of light scattered by the capture molecules without any target molecules bound to the capture molecules is small compared to the light scattered by the capture molecules alone. Assuming that the scattering properties of the capture molecules arranged along the predetermined lines (the capture molecules have not been deactivated) and the scattering properties of the deactivated capture molecules arranged between the predetermined lines are equivalent and further assuming that the capture molecules are uniformly distributed on the outer surface of the planar waveguide, after the capture molecules are immobilized on the outer surface of the planar waveguide and after the capture molecules arranged between the predetermined lines are deactivated, ideally no signal is generated at the detection site. In practice, however, deactivation of the capture molecules slightly changes the scattering properties of the capture molecules, so that deactivation of all capture molecules arranged between the predetermined lines may not be ideal. Instead, only a majority of the capture molecules disposed between the predetermined lines may be deactivated. The deactivation of the capture molecules was performed to the following extent: such that the overall signal generated at the detection position by those capture molecules arranged along the predetermined lines, and by those deactivated and minimally non-deactivated capture molecules arranged between the predetermined lines, is at a minimum, and preferably 0. It is assumed that the signal obtained at the detection site can be reduced to 0, which means that after addition of the target sample, the signal generated at the detection site is generated only by the target sample bound to the capture molecule. If no target sample binds to the capture molecules, the signal at the detection position remains 0. This increases the sensitivity of the detector to signals generated at the detection location by light scattered by target molecules bound to the capture molecules.

According to a further aspect of the device according to the invention, the planar waveguide has a refractive index n_wSubstantially higher than the refractive index n of the substrate_sAnd which is substantially higher than the refractive index n of the medium on the outer surface of the planar waveguide_medSo that for a predetermined wavelength of light, it gradually becomesThe evanescent field has a penetration depth of 40nm to 200 nm. The term "substantially higher" should be understood to indicate a difference in refractive index that allows coupling of light into the planar waveguide where the light propagates under total reflection. Light propagating along the planar waveguide has an evanescent field propagating along an outer surface of the planar waveguide. The evanescent field has a penetration depth which depends on a factor n_medThe effective refractive index n of the guided mode, and the wavelength of the propagating light, the penetration depth may thus be adapted such that the evanescent field of light is coherently scattered by a target sample bound to a binding site on (or close to) the predetermined line at the outer surface. The above approximation of the penetration depth is understood to explicitly include the exact boundary values thereof.

According to a further aspect of the device according to the invention, the device comprises a further optical coupler for coupling out light that has propagated through the planar waveguide. Both the optical coupler for coupling light into the planar waveguide and the further optical coupler for coupling out light that has propagated through the planar waveguide may comprise a grating for coherently coupling light into and out of the planar waveguide. The optical coupler and the further optical coupler comprise gratings for coherently coupling light into and out of the planar waveguide under a respective predetermined in-coupling (in-coupling) angle or out-coupling (out-coupling) angle. The in-coupling angle or the out-coupling angle is determined by the wavelength of the light and the characteristics of the optical coupler. However, it is within the scope of the invention that light may also be coupled into and out of the planar waveguide by any other means suitable for coupling light into and out of the planar waveguide, wherein the thickness of the planar waveguide is in the range of a few nanometers to a few hundred nanometers. By way of example only, an optional optical coupler may be an optical prism.

According to a further aspect of the device according to the invention, the planar waveguide has a first end portion and a second end portion arranged at opposite ends of the planar waveguide with respect to a propagation direction of light through the planar waveguide. The first end portion and the second end portion include a material that is absorptive at a wavelength of light propagating through the planar waveguide. The absorptive material minimizes reflection of light propagating along the planar waveguide toward the respective end portion and back into the planar waveguide. This improves the detection signal when the light that can be reflected from the end of the planar waveguide is removed or at least largely minimized.

According to a further aspect of the device according to the invention, the plurality of measurement zones are arranged on an outer surface of the planar waveguide. In each measurement zone, the bonding sites are arranged along a plurality of predetermined lines. For high throughput screening, transient detection of binding affinity can be achieved for different types of binding sites and samples of target samples by arranging the respective target samples bound to the binding sites in separate measurement areas. Each measurement zone has a corresponding respective detection position to allow for independent detection of scattered light of the evanescent field.

According to a further aspect of the device according to the invention, the plurality of measurement zones comprises measurement zones of different sizes. All dimensions of the measurement zone are known. At the respective detection positions, the light scattered in the respective measurement zones of different sizes may be compared, wherein target samples of the same type are bound to binding sites of the same type. The intensity of the scattered light at the detection location correlates twice with the number of scattering centers in the respective measurement zone on the planar surface of the waveguide. Thus, for a uniform distribution and areal density of scattering centers in different size measurement zones, the intensity of scattered light at the respective detection positions of the respective measurement zones of different sizes is secondarily correlated with the size of the respective measurement zone. Thus, the intensity of scattered light at the detection locations of different size measurement zones can be used to verify: the measured intensity is really representative of the light scattered by the scattering centers arranged on the predetermined line.

According to a further aspect of the device according to the invention, each measurement zone has a size greater than 25 μm²Wherein the plurality of predetermined lines have a distance of less than 1.5 μm, in particular less than 1.0 μm, between adjacent predetermined lines. This allows to obtain a height with a large number of measurement zonesHighly integrated devices, e.g. having 1000, 10000, 100000 … … up to 4 × 10 per square centimetre⁶A measurement area.

Advantageously, the combination sites are arranged along at least two pluralities of predetermined lines in a single measurement zone. Each of the two plurality of predetermined lines is arranged such that light scattered by a target sample bound to a binding site arranged along the respective plurality of lines interferes with a difference in optical path length that is an integer multiple of a predetermined wavelength of light at the respective detection position for each of the plurality of predetermined lines. The individual detection positions are spatially separated from each other. More than one predetermined line in the measurement zone arranged to provide spatially separated detection locations allows for performing additional methods for detecting binding events, such as detecting cooperative binding or detecting a cascade of reactions.

According to a further aspect of the device according to the invention, the device comprises a membrane having an aperture arranged such that light at the detection location is allowed to pass through the aperture, while light at a location different from the detection location is blocked by the membrane. The mechanical membrane as well as the electronic membrane may be adapted to blind out (blind out) all light except light scattered to the detection location. Advantageously, the diaphragm may be formed on the outer surface of the substrate on the side remote from the planar waveguide. For example a non-transparent material such as a chrome layer, may be applied to the surface of the substrate remote from the waveguide. The non-transparent chromium layer has a transparent hole in the detection direction, through which light scattered at the detection location can pass, while remaining light not scattered into the hole is blocked.

According to a further aspect of the device according to the invention, the membrane further comprises at least one further hole, which is arranged adjacent to the above-mentioned hole when the membrane is seen in the propagation direction of the light through the planar waveguide. The further aperture is located adjacent to the aperture such that incoherent light scattered into the further aperture may pass through the further aperture. Advantageously, the detected incoherent background light can be corrected by using a diaphragm with an additional aperture. Further the aperture itself does not detect incoherent background light at the detection position but allows to determine the amount of incoherent light at the detection position by measuring incoherent light at a position different from the detection position. Such determination of the amount of incoherent light at the detection position cannot be separated from the light at the detection position, but can be subtracted from the overall signal at the detection position once it is measured by the detector. For an improved correction, the first further aperture is located in front of the detection location and the second further aperture is located behind the detection location with respect to the direction of the propagating light. This configuration allows the average value of the incoherent light to be detected at the detection position to correct the signal at the detection position.

Another aspect of the invention relates to a system for detecting binding affinity, the system comprising a device for detecting binding affinity according to the invention. The system further comprises a light source for emitting coherent light of a predetermined wavelength, wherein the light source and the device are arranged relative to each other such that the coherent light is coupled into the planar waveguide by the optical coupler. Optionally, since the exact coupling angle of the optical coupler may vary from device to device, the system further comprises optical means for scanning and/or adjusting the angle of the light affecting the optical coupler. Optionally, the wavelength of the light emitted by the light sources in the system may be tuned, which may be advantageous in case the angle of the light affecting the optical coupler is fixed for structural reasons.

According to yet another aspect of the system of the present invention, the system further comprises an optical imaging unit, wherein the optical imaging unit is focused to produce an image of the device detection location. The optical imaging unit is capable of providing an image of the predetermined detection position in which light scattered by the target sample bound to the binding site interferes with a difference in optical path length that is an integer multiple of the predetermined wavelength of light. The optical imaging unit may be used to image light present at the detection position to the observation position. The optical imaging unit may be adapted to image light from the detection position and light from the further aperture or apertures, since such light may be used to subtract incoherent background light from the total light present at the detection position. Alternatively or additionally, the optical imaging unit may be used to select only light at the detection position by focusing the optical imaging unit to the detection position. Subsequently, the diaphragm is no longer required.

Another aspect of the invention relates to a method of detecting binding affinity. The method comprises the following steps:

there is provided an apparatus comprising a planar waveguide and an optical coupler disposed on a substrate,

coupling coherent light of a predetermined wavelength into the planar waveguide such that the coherent light propagates along the planar waveguide, wherein an evanescent field of the coherent light propagates along an outer surface of the planar waveguide,

attaching a target sample to bonding sites arranged along a plurality of predetermined lines on an outer surface of the planar waveguide,

light of evanescent fields scattered by target samples bound to binding sites arranged along a predetermined line is detected at predetermined detection positions, the light scattered by the target samples bound to the binding sites having a difference in optical path length at the predetermined detection positions that is an integer multiple of a predetermined wavelength of the light.

Drawings

Further advantageous aspects of the invention will become apparent from the embodiments of the invention described below with reference to the description of the schematic drawings, in which:

fig. 1 shows a perspective view of an embodiment of the device according to the invention;

FIG. 2 shows a cross-sectional view of the device of FIG. 1;

FIG. 3 shows a graphical representation of different optical paths for light propagating along an outer surface and scattered into an evanescent field at a detection location;

FIG. 4 shows a measurement zone comprising a plurality of predetermined course arrangements of apparatus according to the invention, wherein the bond sites are fixed along the predetermined courses;

FIG. 5 shows the measurement of FIG. 4, wherein some of the target sample is bound to the binding sites;

fig. 6 shows a measurement zone comprising a plurality of predetermined course arrangements of devices according to the invention, wherein the bond sites are fixed along and between the predetermined courses;

FIG. 7 shows the measurement zones of FIG. 6 with those bond sites disposed between predetermined lines deactivated;

FIG. 8 shows the measurement zone of FIG. 7 with a target sample added;

FIG. 9 shows the measurement zone of FIG. 8 with a target sample bound to a binding site fixed along a predetermined line;

FIG. 10 shows a diagrammatic representation of a blank section configuration in which predetermined lines of the measurement zone are to be removed;

FIG. 11 shows the measurement zone of FIG. 10 with the predetermined lines removed in the blank section;

FIG. 12 shows a top view of a further embodiment of a device comprising a plurality of measurement zones according to the present invention;

FIG. 13 shows a bottom view of the embodiment of the device of FIG. 10, wherein for each measurement zone, a hole is arranged at a detection location and two further holes are arranged at locations before and after the detection location;

figures 14 to 17 show a portion of a measurement zone of a device in different stages of a process of binding a target sample according to the invention;

FIG. 18 shows a cross-sectional view of a further embodiment of the device, wherein the device comprises an additional carrier substrate;

FIG. 19 shows a graphical representation of different optical paths for evanescent fields of light scattered at two different predetermined lines arranged in a single measurement zone;

FIG. 20 shows a top view of the apparatus of FIG. 18 with 12 measurement zones disposed thereon, wherein three predetermined lines are disposed in each measurement zone; and

fig. 21 shows a top view of the device of fig. 18 with holes in a non-transparent layer formed on top of an additional carrier substrate.

Fig. 1 shows a perspective view of an embodiment of a device for detecting binding affinity of a sample according to the present invention. The device comprises a substrate 3 of transparent material, which in the embodiment shown has the shape of a rectangular cube, but is not limited to this shape. A planar waveguide 2 (see also fig. 2) is arranged on the upper side of the substrate 3, into which planar waveguide 2 the coherent light 1 is coupled such that the coherent light propagates through the planar waveguide 2 under total reflection conditions. Since the planar waveguide 2 has a thickness only in the range of a few nanometers to a few hundred nanometers, it is not shown in fig. 1 as a separate layer, but is exaggerated in fig. 2. As indicated by the parallel arrows in fig. 1, coherent light 1 of a predetermined wavelength is coupled into a planar waveguide 2 through a substrate 3 by means of a grating 4 acting as an optical coupler. Coherent light coupled into the planar waveguide 2 propagates along the planar waveguide 2, with evanescent fields 6 (represented by arrows) penetrating into the medium of the upper surface of the planar waveguide 2 (see again fig. 2). The evanescent field 6 propagates along the outer surface 5 of the planar waveguide 2. The measurement zone 10 arranged on the outer surface of the planar waveguide 2 comprises a plurality of predetermined lines 9 (each of the illustrated lines represents a plurality of lines, in particular 50 lines in the present example of such an arrangement; and wherein for the sake of clarity only one such measurement zone is shown in fig. 1). Binding sites (not shown in fig. 1) to which target samples can be bound are arranged along these predetermined lines 9. The coherent light of the evanescent field 6 is scattered by the target sample bound to the binding sites within the measurement zone 10. Some of the light scattered by the target sample bound to the binding sites is directed to the detection position, in which the membrane 11 comprising the hole 21 is arranged. The membrane 11 is made of a non-transparent material and may for example be a layer of chrome applied to the lower surface of the substrate 3.

Fig. 2 shows a cross-sectional view of the device of fig. 1, wherein the thickness of the planar waveguide is shown enlarged for explaining the basic operating principle. As can be seen, the light coupled into the planar waveguide 2 propagates through the planar waveguide 2 by means of the grating 4 under total reflection conditions until reaching a further grating 13 arranged at the opposite end of the planar waveguide 2. This further grating acts as a further optical coupler for coupling light out of the planar waveguide. In order to avoid reflections and to minimize incoherent background light, the first end portion 14 and the second end portion 15 of the planar waveguide 2 comprise an absorbing material. The evanescent field 6 propagates along the outer surface 5 of the planar waveguide 2 corresponding to light propagating in the planar waveguide.

Refractive index n of planar waveguide 2_wSubstantially higher than the refractive index n of the substrate 3_sAnd also substantially higher than the refractive index n of the medium on the outer surface 5 of the planar waveguide 2_med. Refractive index n of the medium on the outer surface 5_medMay vary depending on the type of sample applied to the medium. For example, in case a target sample is present in an aqueous solution applied to the outer surface 5 of the planar waveguide 2, the refractive index n of the medium on the outer surface 5_medThe refractive index of water may be of the order of magnitude, or in case of a dry target sample, the refractive index of air may be reached, or in case the bonding site to which the target sample 8 may be bonded is accommodated in the hydrogel layer 16 on the outer surface 5, the refractive index of the hydrogel layer 16 may be of the order of magnitude. Depth of penetration of evanescent field 6 into the medium on the outer surface 5 of the planar waveguide 2 (1/e of the outer surface 5 of the planar waveguide 2 and evanescent field 6)²The distance between intensity drops) depends on the refractive index n of the medium on the outer surface 5 of the planar waveguide 2_medThe effective refractive index N of the guided mode, and the wavelength λ of the light.

The light in the evanescent field 6 propagating along the outer surface 5 of the planar waveguide 2 is scattered by the target sample 8 bound to the binding sites and these binding sites may comprise capture molecules 7 capable of binding the target sample 8 and arranged in a measurement zone 10 (fig. 1) along a predetermined line 9. In fig. 2, the arrows of decreasing length indicate: the distance between adjacent predetermined lines along which the capture molecules 7 are arranged decreases when viewed in the propagation direction of the light. As can be further seen, in the embodiment shown in fig. 2, the target sample 8 is applied to the measurement zone by dispensing droplets containing the target sample 8. Some of the light scattered by the target sample 8 bound to the capture molecules 7 is directed to the detection location, in which the hole 21 of the membrane 11 is arranged. Alternatively, the light at the detection position may be imaged onto the photodetector 20 by the optical imaging unit 19. Since the optical imaging unit 19 and the photodetector 20 may be provided selectively or in combination, and may be provided in combination in one unit in particular, the optical imaging unit 19 and the photodetector 20 are shown as being surrounded by a box indicated by a dotted line.

Although it has been shown in fig. 2 that the optical path lengths of the light scattered by the target sample 8 bound to different capture molecules 7 to the evanescent field 6 at the detection position are different, this becomes clearer when viewing fig. 3, in which fig. 3 a number of such optical paths are clearly shown. Some of the coherent light of the evanescent field 6 is scattered by the target sample 8 bound to different capture molecules 7, for example in a manner interfering at the detection location, which is the location of the aperture 21 of the membrane 11. The arrangement and geometry of the predetermined lines 9 and the thickness of the substrate 3 are selected for the predetermined detection positions such that at the detection positions the difference in optical path length is an integer multiple of the predetermined wavelength of the coherent light. Thus, the interference of light at the detection position is a coherent addition overlap of light scattered to the detection position by the target sample 8 bound to different capture molecules 7.

For the embodiment shown in fig. 3, a plurality of predetermined curves 9 are arranged on the outer surface 5 of the planar waveguide in the following manner: so that their position in the plane of the outer surface 5 of the planar waveguide passes through the equation

To start with x_j,y_jA coordinate system is geometrically represented, wherein

λ is the vacuum wavelength of the propagating light,

n is the effective index of refraction of the guided mode in the planar waveguide; n depends on the thickness and refractive index of the planar waveguide, the refractive index of the substrate, the refractive index of the medium on the outer surface of the planar waveguide and the polarization of the guided mode,

n_Sis the refractive index of the substrate and,

f is the thickness of the substrate,

j is a running integer representing the index of the corresponding line.

Fig. 4 shows an enlarged view of a measurement zone 10 comprising the predetermined line 9 and the binding sites represented by the capture molecules 7, wherein the capture molecules 7 are immobilized on the outer surface of the planar waveguide 5 along the predetermined line 9 (see fig. 1). The immobilization of the capture molecules 7 only along the predetermined lines may be performed by means of lithographic techniques, which have been discussed above. In fig. 5, the target sample 8 is bound to some capture molecules 7. Since the capture molecules 7 are arranged along the plurality of predetermined lines 9, the target samples 8 bound to the capture molecules 7 are also arranged along the plurality of predetermined lines 9. At the detection position this results in coherent addition of the light scattered by the scattering centers formed by the target sample 8 bound to the capture molecules 7, as explained above.

Fig. 6, 7, 8 and 9 again show the measurement zone 10 in an enlarged view. However, the way how the capture molecules 7 capable of binding to the target sample 8 are immobilized along the predetermined line is different.

As can be seen in fig. 6, in a first step the capture molecules 7 are immobilized on the (entire) outer surface of the planar waveguide in the measurement zone 10, such that there is no arrangement of capture molecules along the plurality of predetermined lines 9. Therefore, the light of the evanescent field scattered by the capture molecules 7 does not interfere at the detection position in the above-described manner.

As can be seen from fig. 7, the capture molecules arranged between the predetermined lines 9 are deactivated such that no more target sample can bind to these deactivated capture molecules 12. Accordingly, only capture molecules 7 capable of binding to the target sample are arranged along the plurality of predetermined lines 9. The accuracy of the immobilization of the capture molecules 7 along the predetermined line 9 depends on the method of attaching, immobilizing and deactivating the capture molecules 7. Thus, the position of the immobilized capture molecules 7 capable of binding the target sample 8 may not be exactly "on" the predetermined line 9, but may deviate to some extent from the exact position on the predetermined line 9. In practice, the deviation from the exact position on the predetermined line 9 may be within a range which is less than a quarter of the distance of the adjacent predetermined line 9. This still causes structural interference of the light scattered to the detection location.

As explained in the introductory part, the deactivation of the capture molecules 12 arranged between the predetermined lines 9 is performed such that after deactivation the overall signal generated by the deactivated capture molecules 12 and the capture molecules 7 capable of binding to the target sample 8 at the detection position (not yet target sample 8 has been added) is set or adjusted to the tuned minimum signal at the detection position, ideally to 0.

The next step is to add the target sample 8 to the measurement zone 10 on the outer surface of the planar waveguide, as shown in fig. 8. Since only the capture molecules 7 arranged along the predetermined line 9 are able to bind to the target sample 8, the target sample 8 is bound to those capture molecules 7 along the predetermined line 9, as shown in fig. 9. Since the tuning signal previously caused by the deactivated capture molecules 12 and capture molecules 7 at the detection position has been set or adjusted to a minimum (see above), the signal at the detection position is subsequently caused mainly (or entirely if the signal generated by the deactivated capture molecules and capture molecules has been reduced to 0 before) by light scattered by the target sample 8 bound to the capture molecules 7 arranged along the predetermined line.

Fig. 10 shows a part of the measurement zone 10 used for elucidating the construction of the blank section described above, in which the predetermined line 9 has been removed to avoid second order Bragg reflection in the planar waveguide. Bragg reflections should be avoided since they cause a decrease in the intensity of the light propagating along the planar waveguide. This is particularly disadvantageous in case a plurality of measurement zones 10 are arranged one after the other on the outer surface of the planar waveguide in the direction of the propagating light. The reduction in the intensity of the scattered propagating light in the subsequent measurement zones is therefore not only due to the scattering process described in the respective measurement zone, but also due to the bragg reflection in the planar waveguide. Since in each measurement zone the predetermined line 9 in the circular part of the measurement zone has the following distance between adjacent lines: which fulfils the conditions for the second order bragg reflection and thus the second order bragg reflection in the planar waveguide defines further locations 22 for structural interference of the light reflected by the bragg reflection. In the example shown, the intersections of the arcs of the circle 21 with the predetermined course 9 indicate those points of the predetermined course 9 which are used to achieve the bragg condition accurately, so that light is reflected back and the structures interfere at the further locations 22. This reflected light is not available for scattering in the subsequently arranged measurement zones 10.

Fig. 11 shows a measurement zone 10 comprising a region 23, which region 23 is close to the arc of the circle 21, in which region 23 the predetermined line 9 has been removed to avoid such second order bragg reflections.

Fig. 12 and 13 show a top view and a bottom view of a further embodiment of the device according to the invention. In this embodiment, the device comprises a plurality of measurement zones 10 of a first size and measurement zones 17 of different sizes. Each measurement zone 10 comprises a region 23 in which the plurality of predetermined lines 9 are removed to avoid bragg reflections (see above). In general, the measurement zone may also not include the area 23. A grating 4 for coupling light into the planar waveguide and a further grating 13 for coupling light out of the planar waveguide are provided. Between the grating 4 and the further grating 13, a plurality of measurement zones 10 of a first size and a plurality of measurement zones 17 of different sizes are arranged, wherein the bonding sites are arranged along the predetermined line 9, which has been described in detail above. The plurality of measurement zones 10 of the first size and the plurality of measurement zones 17 of different sizes allow for simultaneous detection of different combinations of target samples and binding sites so that the binding affinity of the plurality of combinations of target samples and binding sites for a particular target sample to a particular binding site can be analyzed simultaneously. Alternatively, redundant measurements may be performed for the same combination of target sample and binding site.

As can be seen from the bottom view of fig. 13, a hole 21 is provided for each measurement zone at the detection location, wherein the scattered light has a difference in optical path length which is an integer multiple of the wavelength of the light propagating in the waveguide to and from the scattering center on the predetermined line to the predetermined detection location, as has been described in detail above. It goes without saying that an optical imaging unit can be arranged, which has already been discussed in detail in connection with fig. 2.

Measurement zones 17 of different sizes are arranged between the measurement zones 10. The measurement zone 17 has known dimensions that are different from the dimensions of the measurement zone 10, wherein all dimensions are known. The light scattered in the detection area 10 and in the corresponding detection area 17 at the respective detection positions may be compared (for the same type of target sample bound to the same type of binding site). The intensity of the scattered light at the detection location correlates secondarily with the number of scattering centers in the measurement zone on the planar surface of the waveguide. Assuming an areal density and a uniform distribution of scattering centers in different sized measurement zones, the intensity of scattered light at the respective detection positions of the different sized corresponding measurement zones correlates twice with the size of the respective measurement zone. Thus, at the detection locations of different size measurement zones, the intensity of scattered light can be used to verify: the measured intensity is truly representative of the light scattered by scattering centers arranged on the predetermined line.

For improved detection of binding affinity, two further holes 18 are formed on the substrate 3, wherein the two further holes 18 are located in front of and behind each hole 21 and are dedicated to a respective measurement zone 10. While coherent light propagating through the planar waveguide 2 may also be scattered incoherently by the planar waveguide 2 in its direction, the contribution of this incoherently scattered light is also detected at the detection location by the aperture 21. The aperture 18 in front of the aperture 21 and at a predetermined distance behind the aperture 21 is used to determine an average signal representative of this incoherent scattered light, which can be used to correct the detected signal at the detection position by subtracting the average signal of the incoherent light from the overall signal detected at the detection position. This correction of the signal at the detection location is particularly advantageous in combination with the above-mentioned reduction of the background signal caused by scattering at the binding site without any target molecules attached thereto.

Fig. 14 to 17 show a part of a measurement zone formed on the outer surface 5 of the planar waveguide according to the invention. Different stages of the process of binding the target sample 8 to the capture molecules 7 are shown. In this process, the binding of the target sample 8 to the capture molecules 7 is enhanced. The capture molecules 7 are immobilized at the outer surface 5. Subsequently, the target sample 8 and the connector 24 are applied. The applied target sample 8 is allowed to bind to the capture molecules 7 until an equilibrium condition is reached, in which the target sample 8 is bound to the capture molecules 7 and the target sample 8 is released from the capture molecules 7 in equilibrium. The connector is then activated (e.g. by light) to enhance the binding between the target sample 8 and the capture molecules 7. Subsequently, the unbound target sample 8 and the unused connector 24 are washed away. Due to the enhanced binding between the target sample 8 and the capture molecules 7 caused by the connector 24, inadvertent washing away of the target sample 8 bound to the capture molecules 7 is prevented or at least greatly reduced. Therefore, the signal at the detection position can be further enhanced. Examples of such processes for using optically activated connectors are described in detail in the following documents: "Capture Compound Mass Spectrometry: A Technology for the investigation of Small molecular Protein Interactions", ASSAY and Drug Development Technologies (Assay and Drug Development Technologies), Vol.5, No. 3, 2007.

Fig. 18 shows a cross-sectional view of a device, which is mainly shown in fig. 1, but according to a further example has a layered structure, which is used, for example, in highly integrated systems (that is to say up to about 4 × 10 per square centimeter)⁶Individual measurement zones). In the example shown, the measurement zone 10 has approximately 25 μm²Size area. This dimension allows a large number of measurement zones 10 to be arranged on the outer surface 5 of the planar waveguide 2 to perform a large number of measurements by using a single device. For example, by essentially "cutting out" 25 μm from a larger measurement zone²To achieve a reduced size measurement zone 10. However, making the distance between predetermined lines in such a reduced-size measurement zone 10 constant will cause: the cone formed by light scattered at the target sample bound to the binding sites in the reduced size detection sites 10 will have an aperture angle which is substantially smaller than the aperture angle of the larger size measurement zone. A smaller aperture angle of the cone of light will cause: the same optical detection unit (compare with fig. 2) that is used to measure a larger measurement zone and with a given aperture angle, measures not only the light at the detection location but also some incoherent background light. This may degrade the signal-to-noise ratio (S/N-ratio). In order to prevent deterioration of the signal-to-noise ratio, the distance between the measurement zone 10 and the detection position must ideally be reduced so that the aperture angle of the cone formed by the light scattered by the target sample bound to the binding site of the measurement zone 10 of reduced size and interfering at the detection position is identical to the aperture angle of the optical detection unit. In order to reduce the distance between the reduced-size measurement zone 10 and the detection location, the arrangement of the plurality of predetermined lines in the reduced-size measurement zone 10 must be determined according to the formula described above with respect to fig. 3 so that light scattered by the target sample 8 bound to the binding site interferes at the new detection location. Since the distance between the reduced-size measurement zone 10 and the new detection position is only in the range of 10 micrometers to several hundred micrometers, the thickness of the substrate 3 may become impractically thin.Especially under laboratory conditions, it may be disadvantageous to process a device comprising a substrate 3 having a thickness in the range of 10 to several hundred micrometers. In order to improve the operation of such a device, the device according to the present embodiment has the following layered structure (from the lower side to the upper side): an additional carrier substrate 24, a layer of non-transparent material 111, a substrate 3 and a planar waveguide 2. The additional carrier substrate 24 is made of a transparent material (e.g. glass, plastic) and has a thickness (e.g. up to 3 mm) that makes the device suitable for handling. A layer of non-transparent material 111 is formed on top of the additional carrier substrate 24. The layer 111 of non-transparent material is for example a black chrome layer in which the holes 21, 18 are lithographically formed. The substrate 3 is of a transparent material and has a thickness corresponding to the distance between the reduced-size measurement zone 10 and the detection location. As mentioned above, the planar waveguide 2 and the measurement zone 10 are similar in principle. Each measurement zone 10 may include more than one of a plurality of predetermined lines, as will be discussed in more detail below in conjunction with fig. 19.

The diagram of the optical path in fig. 19 is similar to that in fig. 3. However, two different pluralities of predetermined lines 9, 91 are arranged in a single measurement zone, and in each such zone light is scattered by target samples bound to the different pluralities of predetermined lines 9, 91 to different, spatially separated detection positions (focal distances). Light of the evanescent field 6 propagating along the outer surface 5 is scattered at the target sample joined to the junction points along the first plurality of predetermined lines 9 so as to interfere at the right-hand focal point (bold line) and scattered at the target sample joined to the junction points along the second plurality of predetermined lines 91 so as to interfere at the left-hand focal point (dashed line). This principle is applied to each of the plurality of predetermined lines 9, 91 associated with the respective detection locations, so that an additional plurality of predetermined lines may be arranged in such a measurement zone (e.g. three as shown in fig. 20). Target samples that can be bound to binding sites arranged at two predetermined lines 9, 91 (fig. 19) may form a cooperative binding via a plurality of binding interactions at the intersection of the lines 9, 19. Such multiple binding interactions have high intensity. The two combinations may be formed simultaneously or sequentially in a short period of time (transient). Such multiple binding interactions are optically detected at two separate detection locations to provide correlated signals at the two detection locations.

Fig. 20 shows a top view of the arrangement of fig. 18, wherein 12 measurement zones 10 are arranged on the outer surface of the planar waveguide. In each measurement zone 10, three pluralities of predetermined lines are provided, and a target sample bonded to a bonding site along the three pluralities of predetermined lines scatters light coupled into the planar waveguide via the optical coupler 4 to three spatially individually separated individual detection positions. Three multiple line arrangements are beneficial when process cascades are detectable. This cascade of processes is present, for example, when the target sample is cleaved as individual products at the arranged first type of capture molecules to provide a signal at the first detection position. The first product of this reaction then binds to a second type of capture molecule to provide a signal at the second detection site. The second product of this reaction binds to a third type of capture molecule to provide a signal at a third detection site.

Fig. 21 shows a bottom view of the device of fig. 20, from below, through a transparent additional carrier substrate 24, a layer 111 of non-transparent material arranged on top of the additional carrier plate 24 can be seen. A set of 9 holes is formed in the layer of non-transparent material 111. Structurally, the layer of non-transparent material 111 includes a plurality of voids having a shape that blocks any light except for scattered light required for measurement at the respective detection locations. In order to optimally suppress the diffuse incoherent background light at the detection position, the diameter of the circular aperture is selected to be larger than the diameter d of the focal spot generated by the scattered light interfering at the detection position₀. In principle, the dimensions are given by the Abbe's formula (Abbe's formula) to calculate the resolution of a theoretically possible microscope:

d₀＝λ/2n_Ssinα＝λf/n_SD，

wherein

Lambda is the vacuum wavelength of coherent light propagating in the planar waveguide,

alpha is half the opening angle of the measurement zone,

n_Sis the refractive index of the substrate 3,

f is the focal length of the measurement zone, and

d is the diameter of the measuring zone.

Further holes are formed in the non-transparent layer 111 in front of and behind the holes 21 (see fig. 18) to determine the average background signal. The shape of the hole may be chosen so as to correspond to the shape of the focal spot formed by the light interfering at the detection position. It may be advantageous to provide an elongated aperture 21 (extending in the direction of evanescent field propagation) to avoid cutting off the light to be detected at the detection position with the edges of the aperture, e.g. to prevent variations in focal spot position caused by variations in the refractive index of the sample applied to the outer surface of the planar waveguide or by small variations in the thickness of the planar waveguide.

Having thus described the embodiments of the present invention by way of the accompanying drawings in the specification, many modifications and variations to the described embodiments are possible without departing from the broad teachings of the present invention. Therefore, the invention should not be construed as being limited to the described embodiments, but rather construed according to the below claims.

Claims

1. Apparatus for detecting binding affinity, the apparatus comprising a planar waveguide (2) arranged on a substrate (3), and further comprising an optical coupler (4), the optical coupler (4) for coupling coherent light (1) of a predetermined wavelength into the planar waveguide (2) such that the coherent light propagates through the planar waveguide (2), wherein an evanescent field (6) of the coherent light propagates along an outer surface (5) of the planar waveguide (2), the outer surface (5) of the planar waveguide (2) comprising binding sites on the outer surface (5) to which binding sites a target sample (8) can be bound such that light of the evanescent field (6) is scattered by the target sample (8) bound to the binding sites, wherein the binding sites are arranged along a plurality of predetermined lines (9), the predetermined line (9) is arranged such that light scattered by a target sample (8) bound to the binding sites interferes with a difference in optical path length at a predetermined detection position, the difference being an integer multiple of a predetermined wavelength of the light.

2. The device according to claim 1, wherein the distance between adjacent predetermined lines (9) decreases in the direction of propagation of the evanescent field of light.

3. The apparatus according to claim 2, wherein the plurality of predetermined lines (9) on which the binding sites are arranged comprise curves, the curvature of the lines being arranged such that light of evanescent fields (6) scattered by target samples (8) bound to the binding sites interferes at predetermined detection points as detection positions.

4. The device according to claim 3, wherein the plurality of predetermined lines (9) are arranged on the outer surface (5) of the planar waveguide (2) in such a way that their positions are governed by the equation

Is geometrically defined in which

λ is the vacuum wavelength of the propagating light,

n is the effective refractive index of the guided mode in the planar waveguide; n depends on the thickness and refractive index of the planar waveguide, the refractive index of the substrate, the refractive index of the medium on the outer surface of the planar waveguide, and the polarization of the guided mode,

n_Sis the refractive index of the substrate and,

f is the thickness of the substrate,

j is a running integer representing the index of the corresponding line.

5. The device according to any of claims 1 to 4, wherein the binding sites comprise capture molecules (7), the capture molecules (7) being attached to the surface of the planar waveguide (2) only along the predetermined line (9), the capture molecules being capable of binding to the target sample (8).

6. The device according to any of claims 1 to 4, wherein the binding sites comprise capture molecules (7) capable of binding to the target sample (8), wherein capture molecules (7) capable of binding to a target sample (8) are arranged along the predetermined line (9) by dispensing the capture molecules (7) capable of binding to a target sample (8) onto the outer surface (5) of the planar waveguide (2) and by deactivating those capture molecules (12) which are not arranged along the predetermined line (9).

7. The device according to any of claims 1 to 4, wherein the planar waveguide (2) has a refractive index (n)_w) Substantially higher than the refractive index (n) of the substrate (3)_s) And which is also substantially higher than the refractive index (n) of the medium on the outer surface (5) of the planar waveguide (2)_med) Such that the evanescent field (6) has a penetration depth in the range of 50 to 200nm for the predetermined wavelength of the light.

8. The device according to any one of claims 1 to 4, comprising a further optical coupler (13) for coupling out light propagating through the planar waveguide (2), wherein the optical coupler (4) for coupling light into the planar waveguide (2) and the further optical coupler (13) for coupling out light that has propagated through the planar waveguide (2) each comprise a grating (4, 13) for coherently coupling light into the planar waveguide (2) and coherently coupling light out of the planar waveguide (2).

9. The device according to any one of claims 1 to 4, wherein the planar waveguide (2) has, in relation to the propagation direction of light through the planar waveguide, a first end portion (14) and a second end portion (15) arranged at opposite ends of the planar waveguide (2), each of the first end portion (14) and the second end portion (15) comprising a material that is absorptive at the wavelength of the light propagating through the planar waveguide (2).

10. The device according to any one of claims 1 to 4, wherein a plurality of measurement zones (10, 17) are arranged on the outer surface (5) of the planar waveguide (2), wherein in each measurement zone (10) the bonding sites are arranged along the plurality of predetermined lines (9).

11. The device according to claim 10, wherein the plurality of measurement zones comprises measurement zones (10, 17) of different sizes.

12. The device according to claim 10, wherein each measuring zone (10) has more than 25 μ ι η²And wherein the plurality of predetermined lines (9) have a distance of less than 1.5 μm, in particular a distance of less than 1 μm, between adjacent predetermined lines (9).

13. The apparatus according to claim 10, wherein the binding sites are arranged along at least two pluralities of predetermined lines (9) in a single measurement zone (10), wherein each of the two pluralities of predetermined lines (9) is arranged such that a difference in optical interference path length of light scattered by target samples (8) bound to binding sites arranged along the respective plurality of predetermined lines (9), the difference being an integer multiple of a predetermined wavelength of the light at the respective detection position for each plurality of predetermined lines (9), and wherein the respective detection positions are spatially separated from each other.

14. The device according to any one of claims 1 to 4, further comprising a membrane (11) having an aperture (21) arranged such that light at a detection location is allowed to pass through the aperture (21) while light at a location different from the detection location is blocked by the membrane (11).

15. The device according to claim 14, wherein the membrane (11) further comprises at least one further hole (18), the at least one further hole (18) being arranged adjacent to the hole (21) when viewed in the propagation direction of the light through the planar waveguide (2).

16. System for detecting binding affinity, comprising a device according to any of the preceding claims, and further comprising a light source for emitting coherent light (1) of a predetermined wavelength, the light source and the device being arranged relative to each other such that the coherent light (1) is coupled into the planar waveguide (2) via the optical coupler (4).

17. The system according to claim 16, further comprising an optical imaging unit (19), the optical imaging unit (19) being focused to produce an image of the detected position of the device.

18. The system according to claim 16 or 17, wherein the system further comprises a photodetector (20) for measuring the intensity of the light at the detection location.

19. A method for detecting binding affinity, the method comprising the steps of:

-providing a device comprising a planar waveguide (2) and an optical coupler (4) arranged on a substrate (3),

-coupling coherent light (1) of a predetermined wavelength into the planar waveguide (2) such that the coherent light propagates along the planar waveguide (2), wherein an evanescent field (6) of the coherent light propagates along an outer surface (5) of the planar waveguide (2),

-attaching a target sample (8) to binding sites arranged along a predetermined line (9) on the outer surface (5) of the planar waveguide (2),

-detecting evanescent field light scattered by said target sample (8) bound to binding sites arranged along said predetermined line (9) at predetermined detection positions, wherein the light scattered by the target sample (8) bound to said binding sites has a difference in optical path length at the predetermined detection positions being an integer multiple of a predetermined wavelength of the light.