HK1213994B - Device for use in the detection of binding affinities - Google Patents

Description

Device for detecting binding affinity

Technical Field

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

Such devices are used, for example, as biosensors in a wide variety of applications. One particular application is the detection or monitoring of binding affinity or progress. For example, with the aid of such biosensors, various assays for detecting the binding of a target sample to a binding site can be performed. Typically, a large number of such assays are performed on a biosensor at spots arranged in a two-dimensional microarray on the biosensor surface. The use of microarrays provides tools for the simultaneous detection of binding affinities or the processing of different target samples in high throughput screening, where large target samples like molecules, proteins or DNA can be analyzed rapidly. To detect the affinity of the target sample for binding to a specific binding site (e.g. the affinity of the target molecule for binding to a different capture molecule), a large number of binding sites may be immobilized on the surface of the biosensor at the applied spots, e.g. by inkjet printing or photolithography. Each spot forms a separate 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 on the binding affinity of the target sample.

Background

Known techniques for detecting binding affinity of a target sample use labels capable of emitting fluorescence upon excitation. For example, a fluorescent label may be used as a label for labeling the target sample. Upon excitation, the fluorescent label causes emission of fluorescence having a characteristic emission spectrum. Detection of this characteristic emission spectrum at a particular spot indicates that the labeled target molecule has been bound to the particular type of binding moiety present at the corresponding spot.

Sensors for detecting labeled target samples are described in "Zeptosens protein microarrays" in the following articles: a novel high performance microarray platform for low abundance protein analysis ", proteomics 2002, 2, s.383-393, willey-VCH press gmbh, 69451 weiinheim, germany. The described sensor includes a planar waveguide disposed on a substrate and a grating for coupling coherent light of a predetermined wavelength into the planar waveguide. The other grating is arranged at the end of the planar waveguide remote from the grating for coupling light into the waveguide. Coherent light that has propagated through the planar waveguide is coupled out of the waveguide by the further grating. The coupled-out light is used for the adjustment of the coupling of coherent light of a predetermined wavelength into the planar waveguide. Coherent light propagates through the planar waveguide under total reflection using an evanescent field of the coherent light propagating along an outer surface of the planar waveguide. The evanescent field penetrates the low index medium at the outer surface of the planar waveguide to a depth on the order of a fraction of the wavelength of coherent light propagating through the planar waveguide. The evanescent field excites the fluorescent labels of the labeled target samples that are bound to binding sites provided on the surface of the planar waveguide. Due to the very small penetration of the optically thinner medium by the evanescent field at the outer surface of the planar waveguide, only the labelled sample bound to the binding sites immobilized on the outer surface of the planar waveguide is excited. The fluorescence emitted by these markers is then detected with the aid of a CCD camera.

Although in principle it is possible to detect binding affinity using fluorescent labels, the disadvantage of this technique is that: the detected signal is generated by the label and not by the binding partner itself. Furthermore, marking the target specimen requires additional work steps. Furthermore, the marked target samples are relatively expensive. Another disadvantage is that: at the target sample, the steric hindrance of the fluorescent label results in a distortion that may interfere with the binding of the target sample to the capture molecules. A further disadvantage is the falsification of results caused by the photo-bleaching or quenching effect of the marking.

Disclosure of Invention

It is an object of the present invention to provide a device for use in detecting binding affinities of target samples and a system capable of detecting such binding affinities which overcomes or at least greatly reduces the disadvantages of the prior art sensors described above.

According to the present invention, the object is achieved by a device for detecting binding affinity, the device comprising a planar waveguide disposed on a substrate, and further comprising an optical coupler having a predetermined length for coupling coherent light of a predetermined wavelength into the planar waveguide such that a parallel beam of coherent light propagates through the planar waveguide, wherein an evanescent field of coherent light propagates along an outer surface of the planar waveguide, the outer surface of the planar waveguide comprising a binding site thereon, the binding site being capable of binding a target sample to the binding site such that light of the evanescent field is diffracted by the target sample bound to the binding site. The binding sites are arranged along a plurality of predetermined straight lines extending parallel to each other at a constant distance between adjacent straight lines. A predetermined straight line of the plurality of predetermined straight lines is arranged at an angle β with respect to the propagation direction of the evanescent field such that coherent light diffracted by the target sample bound to the binding site impinges at a diffraction angle α with respect to the predetermined straight line on another optical coupler arranged in a portion of the planar waveguide which is outside the beam of coherent light propagating through the planar waveguide. The further optical coupler couples the diffracted coherent light out of the planar waveguide to interfere with an optical path length difference at a predetermined detection location that is an integer multiple of a predetermined wavelength. Technically, the term "diffraction" describes the interference of coherent light of the evanescent field that has interacted with the target sample bound to the binding site. Diffraction causes the coherent light to propagate through the planar waveguide with an evanescent field at the outer surface for constructive interference in a predetermined direction within the planar waveguide.

The detection of binding affinity according to the present invention is not 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 of the planar waveguide. Detection of the binding site may be achieved in a label-free manner. Alternatively, diffraction enhancers (e.g., diffraction labels) that strongly scatter light may be used to enhance detection sensitivity. Such diffraction enhancing bodies may be nanoparticles (alone or with a binder) or, in another example, colloidal particles. Advantageously, the binding characteristics to be analyzed may be of a static type (e.g. it may be analyzed whether the target sample is bound to the binding site) or of a dynamic type (e.g. the dynamic of the binding process over time may be analyzed). According to the invention, the device comprises a planar waveguide on a substrate, the planar waveguide having a high refractive index with respect to a medium formed on an outer surface of an upper side of the planar waveguide. For example, the index of refraction of the planar waveguide may be in the range of 1.6 to 2.8, where the plane is planarThe refractive index of the medium at the waveguide surface is typically in the range of 1 to 1.6, in particular 1.33 to 1.4 for water or water buffers and 1 for air. The effective index N of the guided mode, the index of the medium at the surface of the planar waveguide and the predetermined wavelength of light determine the penetration depth of the evanescent field into the medium on the outer surface of the planar waveguide (the outer surface of the planar waveguide and the decrease in intensity of the evanescent field by 1/e)²Inter-distance). The penetration depth is such that an evanescent field penetrating out of the outer surface of the planar waveguide is diffracted at the target sample bound to the binding sites arranged at the outer surface. In use, coherent light of a predetermined wavelength (preferably monochromatic) is coupled into the planar waveguide via the optical coupler such that a parallel beam of coherent light propagates through the planar waveguide and the evanescent field propagates along the outer surface. The parallel light beams have a width corresponding to a predetermined length of the optical coupler, which in the case of a grating coupling coherent light into a planar waveguide is the length of the lines defining the grating. The predetermined wavelength is not limited to a specific value, but is preferably in the range of visible light. The outer surface of the planar waveguide includes binding sites thereon. The binding site is a location on the outer surface of the planar waveguide to which the target sample can bind. For example, the binding sites may comprise capture molecules that may be immobilized to the outer surface of the planar waveguide, or the binding sites may simply comprise activation sites on the outer surface of the planar waveguide to which the planar waveguide is capable of binding the target sample, or the binding sites may be embodied in any other suitable manner to bind the target sample at a desired location on the outer surface of the planar waveguide. In principle, the binding sites are capable of binding the target sample such that light of the evanescent field is diffracted by the target sample bound to the binding sites. According to the present invention, the binding sites are arranged along a plurality of predetermined straight lines. The arrangement of binding sites "along a predetermined straight line" represents the best case, where all binding sites are exactly arranged on the predetermined straight line. Such an optimal arrangement of binding sites results in a maximum signal at the detection site. As will be apparent to those skilled in the art: indeed, the placement of the binding sites may deviate to some extent from such an optimal placement without loss of detectable signal in the detection site. For example, byThe distance between adjacent predetermined lines is preferably in the range of about 100 nanometers to about 1000 nanometers, particularly between 300 nanometers to 600 nanometers the mentioned range allows visible, near infrared and soft ultraviolet light of wavelength range from 350 nanometers to 1500 nanometers to be used so that diffracted light can be detected by standard optical means the predetermined line is evanescent at an β angle in the range of 10 DEG to 70 DEG with respect to the direction of propagation of the evanescent field, the direction of propagation is defined as starting from the optical coupler and extending in the direction of coherent optical coupling into the planar waveguide, the planar waveguide is generally perpendicular to the lines forming the optical grating of the optical coupler, the diffracted light from a target sample bound to the target site is incident on another planar waveguide at an angle α relative to the line, the diffracted light from the target sample bound to the target site is guided by the diffraction medium along the predetermined line, the diffraction medium is relatively sensitive to the diffraction field of the diffraction of the predetermined line, the diffraction medium is relatively low, the diffraction medium is relatively sensitive to the diffraction of the diffracted light from the target sample bound to the target siteAnother key point of the invention is that another optical coupler is disposed in a portion of the planar waveguide that is outside of the beam of coherent light propagating through the planar waveguide. This allows for detection of signals from diffracted light that does not come from the background of coherent light propagating through the planar waveguide. Thus, the signal detected in the detection position has a background signal, a better detection sensitivity may be achieved, which allows to detect signals resulting from fewer diffraction centers. Since the other optical coupler is formed as a grating so that optical path lengths of light diffracted by different lines of the grating differ by integral multiples of the wavelength of the light at the predetermined detection position, the maximum value of the diffracted light is positioned at the predetermined detection position. For the maximum signal at the detection position, light from the optical coupler to the predetermined straight line, from the optical coupler to another optical coupler, and from the optical coupler to the predetermined detection position is also an integral multiple of the predetermined wavelength. Therefore, light diffracted by the target sample bound to the binding site constructively interferes at the predetermined detection position. The need for constructive interference is met by diffracted light added to the detectable signal in the detection position.

According to an advantageous aspect of the invention, the constant distance d between adjacent straight lines is chosen such as to satisfy the bragg condition 2Ndsin (α) k λ, where N is the effective index of the guided mode of the planar waveguide, d is the distance between adjacent predetermined straight lines, α is the diffraction angle, k is the number of intensity maxima and λ is the vacuum wavelength of the propagating light. It is important to note that the distance d between adjacent predetermined straight lines where constructive interference occurs at the predetermined detection location depends on the effective index of refraction N, which in turn depends on the index of refraction of the medium at the outer surface of the waveguide. Advantageously, the distance d between adjacent predetermined lines is selected to be a factor in the variation in the index of refraction of different samples applied to the outer surface. The constant distance d between adjacent lines explicitly comprises small changes in the distance between adjacent lines. Such a gradient in the inter-line distance between adjacent lines on the plurality of predetermined lines allows the bragg condition to be satisfied in only a small portion of the plurality of predetermined lines.

According to another advantageous aspect of the invention, the predetermined straight line is arranged at an angle β in the range of 10 ° to 70 ° with respect to the propagation direction of the evanescent field. Coherent light diffracted by the target sample bound to the binding site impinges on the other optical coupler at a diffraction angle α (which is equivalent to β) with respect to the straight line. It is advantageous for the predetermined straight line on the outer surface of the preparation device to be arranged at a fixed angle to the further optical coupler, with a fixed orientation thereon.

According to another advantageous aspect of the invention, the further optical coupler comprises a plurality of grating lines. Each of the plurality of grating lines has a respective curvature and a distance between adjacent grating lines such that another optical coupler is capable of coupling diffracted coherent light out of the planar waveguide so as to interfere at a predetermined detection location with an optical path length difference that is an integer multiple of a predetermined wavelength. The plurality of grating lines may have an axis of symmetry extending at a diffraction angle α with respect to the predetermined straight line. The symmetry applies to a plurality of grating lines in another optical coupler, with a symmetrically curved grating-like structure between adjacent grating lines having a reduced distance such that light of a single predetermined wavelength coupled out of the planar waveguide satisfies the condition that the optical path length difference is an integer multiple of the single predetermined wavelength in the detection position. Setting the axis of symmetry at the diffraction angle allows the detection position to include the central axis of the circumference forming the grating.

According to yet another advantageous aspect of the invention, the plurality of predetermined straight lines are arranged in an active area on the planar waveguide. The active area has a width equal to the length of the optical coupler such that the entire active area is illuminated by an evanescent field of coherent light coupled into the planar waveguide by the optical coupler. The light beam propagating in the waveguide has a small divergence angle so that the increase in beam width is negligible compared to other dimensions of the device. Thus, the width of the active area may generally be selected to coincide with the length of the optical coupler used to illuminate the entire active area. In practice, however, the width of the active area is small compared to the length of the optical coupler. As an example, the width of the active area is 310 microns and the length of the optocoupler is 400 microns.

According to another advantageous aspect of the invention, at least two predetermined straight lines are arranged successively on the planar waveguide in the propagation direction of the evanescent field. A respective further optical coupler is arranged relative to each set of predetermined straight lines such that coherent light diffracted by the target sample diffractively binding to binding sites arranged along the straight lines of the respective set of straight lines impinges on the respective further optical coupler at a diffraction angle α. By arranging the sets of predetermined lines one after the other in the propagation direction of the evanescent field, the evanescent field of the light beam impinges onto all the plurality of predetermined lines (or diffracts at all the sets of predetermined lines) thereby allowing for the simultaneous detection of binding affinities in the plurality of samples.

According to a preferred alternative aspect of the invention, each of the at least two plurality of predetermined straight lines has the same constant inter-adjacent straight line distance d. The same constant distance d between adjacent straight lines of each plurality of predetermined straight lines allows for redundant detection of binding affinities in multiple samples.

In another preferred alternative aspect of the invention, each of the at least two plurality of predetermined straight lines has a different constant distance d between adjacent straight lines_1…n. Different constant distances d_1…nA constant distance range may be covered which corresponds to the range of detectable refractive indices in the medium at the outer surface of the waveguide. The range of detectable refractive indices allows detection of binding affinity to samples in media having different or unknown refractive indices. Due to the different compositions, the index of refraction in the sample in contact with the sensor surface may vary within a range of a few percent. In preferred other aspects of the present invention, the constant distance d between adjacent straight lines of adjacent ones of the predetermined straight lines_1…nVarying in steps of 0.5 to 3 nm. Varying in equal steps with different constant distances d_1…nAllows for convenient quantification of binding affinities of samples of different or unknown refractive indices in a range of known detectable refractive indices. Constructive interference occurs at the predetermined detection location when the distance d of the plurality of predetermined straight lines matches the bragg condition for the refractive index of the applied sample.

In yet another preferred alternative aspect of the invention, the at least two plurality of predetermined straight lines comprises a plurality of groups of the plurality of predetermined straight lines, each group having an equal constant distance d between adjacent straight lines. Different groups of a plurality of predetermined straight lines have different constant distances d between adjacent straight lines_1…n. Multiple sets of equal constant adjacent interline distances dpin for other alternatives the discussed advantages allow for redundant detection of binding affinities as well as detection of binding affinities to samples in media having different or unknown refractive indices in a range of known detectable refractive indices.

In yet another preferred aspect of the invention, the optical coupler comprises at least two separate sections for coupling coherent light of a predetermined wavelength into the planar waveguide. Each individual section has a predetermined length and is separated from the other individual sections by a predetermined interval such that at least two parallel-beam coherent lights propagate through the planar waveguide separated by the predetermined interval. The separate portions of the optical coupler allow one or more sets of predetermined straight lines to be arranged in the propagation direction of each light beam coupled into the planar waveguide via the respective separate portion. By separating the parallel light beams coupled into the planar waveguide by a predetermined interval, a portion of the planar waveguide is caused to lie outside the parallel light beams of coherent light. Another optical coupler disposed in the section improves the detected signal by reducing background light in the detection location. In an example, the other optical coupler has a size of 400 microns, with the predetermined spacing being selected to be 600 microns.

According to an advantageous aspect of the invention, the binding sites comprise capture molecules attached to the outer surface of the planar waveguide only along a predetermined straight line. The capture molecules are capable of binding to the target sample. Two embodiments of how the binding sites are arranged along a predetermined set of straight lines are specifically envisaged. According to a first embodiment, the binding site comprises capture molecules attached to the surface of the planar waveguide only along a predetermined straight line. These capture molecules are capable of binding to the target sample and are immobilized on the outer surface of the planar waveguide (although, as mentioned above, the binding sites may be formed by the active surface of the planar waveguide itself). The immobilization of the capture molecules along the predetermined lines on the outer surface of the planar waveguide may generally be performed by any suitable method, for example using a photolithographic method using a photolithographic mask with straight lines. It goes without saying that the arrangement of the binding sites along a predetermined straight line is understood in any embodiment of the invention as: the majority of the binding sites-in this embodiment the capture molecules-are located along a predetermined straight line and include exactly some binding sites located at different positions than these.

According to a second embodiment, the binding sites comprise capture molecules capable of binding the target sample, the capture molecules being capable of binding the target sample arranged along the predetermined straight line by immobilizing the capture molecules capable of binding the target sample to the outer surface of the planar waveguide and by deactivating these capture molecules which are not arranged along the predetermined straight line. The term "deactivation" in this respect refers to any suitable method for altering the binding capacity of the capture molecule before or after it is immobilized onto the outer surface of the planar waveguide. In order to achieve that they are no longer able to bind the target sample, deactivation can be achieved, for example, by exposing the capture molecules to UV light. The deactivation of the capture molecules immobilized between the predetermined straight lines can be achieved, for example, by replacement of the binding regions of the capture molecules. According to this embodiment of the invention, the capture molecules may be applied uniformly or statistically uniformly to the outer surface of the planar waveguide. Only after the deactivation of the capture molecules arranged between the predetermined straight lines, the capture molecules arranged along the predetermined straight lines (which have not been deactivated) 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 further advantage of increasing the contribution of the signal generated by light diffracted by the target sample bound to the capture molecules to the total signal at the detection location. Typically, the difference between the signal of light diffracted by small target molecules bound to the capture molecules and light diffracted by capture molecules to which no target molecules are bound is small compared to light diffracted by the capture molecules alone. Assuming that the diffraction properties of the capture molecules arranged along the predetermined straight lines (which have not been deactivated) and the deactivated capture molecules arranged between the predetermined straight lines are almost the same, and further assuming that the capture molecules are uniformly distributed on the outer surface of the planar waveguide, ideally no signal is generated at the detection site after the capture molecules have been immobilized on the outer surface of the planar waveguide and after the capture molecules arranged between the predetermined straight lines have been deactivated. However, in practice, the deactivation of the capture molecules slightly changes the diffraction properties of the capture molecules, so that it does not ideally deactivate all the capture molecules arranged between the predetermined straight lines. Instead, only the vast majority of the capture molecules disposed between the predetermined straight lines may be deactivated. The deactivation of the capture molecules is performed to such an extent that the overall signal generated at the detection position by these capture molecules arranged along the predetermined straight lines and by these deactivated and non-deactivated capture molecules arranged between the predetermined straight lines is minimal and preferably zero. It is assumed that the signal obtained at the detection site can be reduced to zero, which means that after addition of the target sample, the signal generated at the detection site is only caused by the target sample bound to the capture molecules. If no target sample binds to the capture molecules, the signal at the detection site remains zero. This increases the sensitivity of the detector for signals generated by light diffracted by target molecules that bind to the capture molecules at the detection location.

Another aspect of the invention relates to a system for detecting binding affinity, comprising a device according to any one of the preceding claims, and further comprising a light source for emitting coherent light of a predetermined wavelength. The light source and the device are arranged relative to each other such that coherent light emitted by the light source is coupled into the planar waveguide via the optical coupler.

According to other aspects of the invention, the light source and the device are arranged to be adjustable relative to each other to change a coupling angle at which coherent light emitted by the light source is coupled into the planar waveguide via the optical coupler. The light source emits coherent light of a predetermined wavelength, preferably visible light, near infrared light or soft UV with a spectral range of (tunable) wavelengths in the range from 350 nm to 1500 nm.

According to another aspect of the invention, the light source is tunable to emit coherent light having a predetermined wavelength with a tuning range of about 1 nanometer to 5 nanometers. The tuning range of the light source allows the light source and the device to be set at a fixed in-coupling angle. When the wavelength of the emitted light in the tunable range matches the wavelength at which coupling occurs at a fixed incoupling angle, the light emitted by the tunable light source is coupled into the planar waveguide via an optical coupler (e.g. a grating).

The tunable light source may be used in a second advantageous mode of operation of the device in a system for detecting binding affinity. The bragg condition describing the maximum intensity of constructive interference involves: distance between adjacent predetermined lines; an angle of diffraction at the target sample bound to an evanescent field of binding sites disposed along a predetermined straight line; a wavelength of light propagating through the planar waveguide; and the effective index N of the guided mode. Considering the example where the index of refraction of the sample is not exactly known, the tunable light source allows to vary the wavelength at which the coupling occurs so that the bragg condition for the maximum intensity of constructive interference is satisfied (even for a fixed distance between adjacent straight lines and a fixed diffraction angle associated with a predetermined line). The wavelength of the tunable light source and the change in the incoupling angle (under which light is coupled into the waveguide via the optical coupler) allow the wavelength at which coupling into the waveguide to occur to be tuned to a wavelength that satisfies the bragg condition for a fixed distance between adjacent predetermined straight lines.

Drawings

Further advantageous aspects of the invention will become apparent from the following description of embodiments of the device with reference to the accompanying drawings, in which:

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

Fig. 2 shows a plan view of a planar waveguide of the device of fig. 1 showing different angles according to the present invention.

Fig. 3 shows a plan view of a planar waveguide of the device of fig. 1 showing the placement of the binding sites.

Fig. 4 shows a plan view of a planar waveguide of the device of fig. 1 showing the active area.

Fig. 5 shows a plan view of a planar waveguide of the apparatus of fig. 1 showing different optical paths.

Fig. 6 shows a plan view of a planar waveguide of the device of fig. 1 having two plurality of predetermined straight lines.

Fig. 7 shows three plural predetermined straight lines having a difference in constant distance d between adjacent predetermined straight lines.

Fig. 8 shows a plan view of a mask for preparing a device according to a second embodiment of the present invention, having a pattern of 24 predetermined lines thereon.

Fig. 9 shows a plan view of a non-fabricated device according to a second embodiment of the present invention fabricated using the mask of fig. 8.

FIG. 10 shows a plan view of a prepared device according to a second embodiment of the invention, as prepared for use in a binding affinity assay in the device of FIG. 9.

Fig. 11 shows a schematic diagram visualizing the optical path length difference for diffraction of light of an evanescent field on a target sample bound to binding sites arranged along a plurality of predetermined straight lines.

FIG. 12 shows the schematic of FIG. 11, wherein the binding sites comprise deactivating molecules along and between a plurality of predetermined lines for achieving minimal background signal.

Fig. 13 shows the schematic of fig. 12, wherein a target sample applied to the capture molecules capable of binding.

Detailed Description

FIG. 1 shows a perspective view of an embodiment of a device for use in detecting binding affinity. Structurally, the device comprises a substrate 3, a plurality of predetermined straight lines 7 (each line shown representing a plurality of lines) disposed on an outer surface 21 of a planar waveguide 2, an optical coupler 41, a sensing location and another optical coupler 8. It is further shown that: according to the working principle of the device, coherent light 1 is coupled into planar waveguide 2 such that as evanescent field 11 (represented by the parallel arrows) propagates, evanescent field 11 is diffracted such that diffracted coherent light 12 (represented by the parallel arrows) propagates at an angle with respect to predetermined line 7 coupled out of parallel waveguide 2 such that coupled light 13 coupled out of planar waveguide 2 interferes at detection position 9.

In the example shown, the planar waveguide 2 is arranged on a substrate 3, the substrate 3 allowing visible coherent light to propagate through it. Since the planar waveguide 2 has a thickness in the range of about 10 nm to about several hundred nm, it is drawn together with a line from the upper surface of the substrate 3. Coherent light 1 provided by a light source (not shown) has a predetermined wavelength. In practice, the predetermined wavelength is not limited to a specific value for this wavelength, but is specifically selected according to the effective index of refraction of the guided mode and the size, position and geometry of the optical coupler 41, the predetermined line 7 and further the optical coupler 8. In order to couple coherent light 1 of a predetermined wavelength into the planar waveguide 2, the optical coupler 41 employs a grating having a straight line of a predetermined length in the illustrated example, thereby allowing coherent coupling of the coherent light 1 into the planar waveguide 2 at a predetermined coupling angle. Due to the predetermined length of the coupler 41, a parallel beam of coherent light having a width according to the length of the optical coupler 41 propagates through the planar waveguide 2. The parallel beam of coherent light has an evanescent field 11 with a characteristic penetration depth. Evanescent field 11 enters planar waveguide 2 (distance between outer surface 21 of planar waveguide 2 and intensity drop 1/e of evanescent field 11²) Depends on the effective index N of the guided mode, on the index of the medium at the surface of the planar waveguide, and on the wavelength λ of the light. The light of evanescent field 11 is diffracted by the target sample (not shown in fig. 1) bound to the binding sites (not shown in fig. 1). In principle, the binding sites are arranged along a plurality of predetermined straight lines 7 extending parallel to each other at a constant distance between adjacent straight linesAnd (4) placing. The predetermined straight line 7 is arranged on the outer surface 21 of the planar waveguide 2 at an angle with respect to the propagation direction of the evanescent field 11. The light of the evanescent field 11 is diffracted to impinge on another optical coupler 8 formed in the planar waveguide 2 at diffraction angles with respect to the straight line. The diffracted lights interfere in the other optical coupler 8 with an optical path length difference of an integral multiple of the predetermined wavelength. Advantageously, the internal diffraction of the light propagation through the planar waveguide 2 is of higher efficiency than the diffraction of the guided light by the planar waveguide 2. This provides sufficient detection sensitivity, which allows detection of a relatively small number of diffraction centers. In theory, there may be other diffraction angles relative to the line having the maximum intensity of the diffracted light, so that the further optical coupler 8 may also be arranged at other diffraction angles. A further advantage of the present invention with respect to the arrangement of the further optical coupler 8 can be seen in fig. 1. Another optical coupler 8 and thus detection location 9 is arranged on the planar waveguide 2 and oriented with respect to each other such that no optical beam propagating through the planar waveguide 2 is detected. Thus, the further optical coupler 8 is arranged in a portion 10 of the planar waveguide 2, the portion 10 being located outside the beam of coherent light propagating through the planar waveguide 2 starting from the optical coupler 41. This allows the signal from the diffracted light to be detected without the background from the beam of coherent light propagating through the planar waveguide. A further advantage relates to the fact that the signal detected in the detection location 9 has less background signal because of the location of the further optocoupler 8 in the section 10. Thus, a better detection sensitivity is achieved, which allows the detection of signals resulting from fewer diffraction centers. The other optical coupler 8 is shown as a phase grating lens oriented about the axis of symmetry in the direction of the diffraction angle. The phase grating lens magnifies any optical device to couple the diffracted coherent light 12 out of the planar waveguide 2 while focusing it in the detection position 9 with sufficient intensity to bind affinity detection.

Fig. 2-6 are plan views of the outer surface 21 of the planar waveguide 2 of fig. 1, respectively, fig. 1 having described the planar waveguide 2, the optical coupler 41, the further optical coupler 8 and the plurality of predetermined straight lines 7 disposed on the outer surface 21 of the planar waveguide 2.

In fig. 2, an angle α with respect to the predetermined straight line 7 and an angle β with respect to the propagation direction of the evanescent field 11 are shown. In the present embodiment, the angle β is 22.5 ° and the angle α is 22.5 °. The fixed angle is significantly advantageous for the preparation of the device. The evanescent field 11 (represented by the arrow starting from the optical coupler 41 and ending the light beam in the center of the predetermined straight line 7) propagating along the outer surface 21 of the planar waveguide 2 is diffracted on the target sample (not shown) bound to the binding site (not shown). The diffracted coherent light 12 (represented by an arrow starting from the center of the predetermined straight line 7 and propagating along the symmetry axis of the other optical coupler 8) constructively interferes so as to impinge on the other optical coupler 8 at an angle α of 22.5 °. The angle α is according to the bragg condition 2Ndsin (α) ═ k λ, depends on the distance between adjacent predetermined straight lines 7 and depends on the predetermined wavelength λ and may vary to satisfy the bragg condition. N is the effective index of the guided mode in the planar waveguide and λ is the vacuum wavelength of the light propagating through the planar waveguide 2.

In fig. 3 a plan view of the planar waveguide 2 of the device of fig. 1 is provided, fig. 1 is an enlarged schematic view of the binding sites 5 arranged along a predetermined straight line 7. In the enlarged schematic view, the light of evanescent field 11 is represented by parallel arrows approaching a straight line 7 predetermined to be arranged at a known angle β. The predetermined straight lines 7 are arranged parallel to each other at a constant distance d. The diffracted coherent light 12 diffracted on the target specimen 6 bound to the binding sites 5 arranged along the predetermined straight line 7 has an optical path length difference of an integral multiple of the wavelength for a predetermined angle. The diffraction angle described is the first angle at which the intensity maximum occurs. In practice this is a well-known picture that depicts the bragg diffraction principle, where light is diffracted at the "crystal structure" to constructively interfere in a specific direction. The description is not corrected to the extent of the binding sites 5, and in a manner that the target samples 6 bound to the binding sites 5 are not arranged along the predetermined straight line 7 in the regular order shown. These settings deviate to some extent in the direction along and perpendicular to these lines without losing the maximum intensity of the diffracted coherent light 12.

In fig. 4, the arrangement of the predetermined straight line 7 in the effective region 71 on the planar waveguide 2 is exemplarily described. The configuration of the active area 71 is shown with respect to the coherent light propagating through the planar waveguide 2. Assuming a uniform density of diffraction centers of the effective region 71, in principle, the larger the area of the effective region 71, the more diffraction centers will contribute to diffracting the coherent light 12. The area of the active area 71 is selected primarily based on the intensity of the detected signal suitable for detecting binding affinity. Since the length of the optical coupler 41 is fixed, the width of the effective area 71 is fixed to be equal thereto. This allows the entire active area 71 to be shown by evanescent field 11, evanescent field 11 being shown by the parallel arrows laterally bounding the width of active area 71. The width of the active area 71 is such that on the one hand the diffracted coherent light 12 impinges completely on the other optical coupler 8 while on the other hand the other optical coupler 8 is illuminated only by the diffracted coherent light 12 from the diffraction center of the active area 11. The lateral spacing of the diffracted coherent light 12 from evanescent field 11 limits the light impinging on the other optical coupler 8 to diffracted coherent light 12 from the extended center in active area 71 and avoids other background light in area 10, except that no other light propagates through area 10 from diffracted light 12.

In fig. 5, two examples of light of different optical paths are shown by evanescent field 11 arrows, i.e. arrows for diffracted coherent light 12 and light interfering in the detection position 9. In principle, a plurality of parallel light beams start at the optical coupler 41 to be diffracted over the entire area of the effective region 71 provided with the predetermined straight line 7. The diffracted coherent light 12 propagates toward the other optical coupler 8 with an optical path difference of an integral multiple of a predetermined wavelength. The diffracted coherent light 12 impinges on the other optical coupler 8 such that it is coupled out of the planar waveguide 2. The other optical coupler 8 is depicted as a grating with a plurality of grating lines 81. The grating lines 81 are formed such that the diffracted coherent light 12 impinging thereon is coupled out of the planar waveguide 2 and focused into the detection position 9, each of the plurality of grating lines 81 having a respective curvature and being arranged in such a way that the distance between adjacent grating lines 81 in the propagation direction of the diffracted coherent light 12 decreases. This allows diffraction of light of a predetermined wavelength to "ideally" enter a single focal spot by way of optical path length differences that are integer multiples of the predetermined wavelength. A blank section 82 is formed in the further optical coupler 8 to avoid 2nd order bragg diffraction or similar optical effects that might reduce the overall intensity of the detected signal.

One advantageous aspect of the invention is illustrated in fig. 6, where the planar waveguide 2 of the device of fig. 1 comprises two plurality of predetermined straight lines 7. The two plurality of predetermined straight lines 7 have different distances between adjacent predetermined straight lines 7. In general, the distance between different adjacent predetermined straight lines 7 allows to detect the binding affinity for a sample having different refractive index indices at the same "fixed" diffraction angle. Each different diffraction index of the sample results in a different effective index of refraction for light propagating through the planar waveguide 2. In general, the effective index of the guided mode in the planar waveguide 2 depends on the thickness and index of refraction of the planar waveguide 2, the index of refraction of the substrate, the index of refraction of the medium on the outer surface 21 of the planar waveguide 2, and the polarization of the guided mode. Hence evanescent field 11 of light propagating through planar waveguide 2 has different specific optical path lengths between adjacent lines for different samples on the waveguide. In practice, the index of refraction of the medium on the outer surface 21 of the planar waveguide 2 is not completely known. Advantageously, the plurality of predetermined straight lines 7 with different distances allows to detect the signal for an unknown refractive index within a range of known detectable refractive indices, which for different samples may vary in the second or third decimal point of the refractive index. For the detection of binding affinity, a single plurality of predetermined straight lines 7 shows a detectable signal. As shown, at least two plurality of predetermined straight lines 7 are arranged on the planar waveguide 2 in the propagation direction of the evanescent field 11. The coherent light 12 coupled into the planar waveguide 2 is diffracted by the target sample 6 bound to the binding sites 5 of each set of predetermined straight lines 7. Another optical coupler 8 is arranged at each of the plurality of predetermined straight lines 7 so that light impinges at a diffraction angle relative to the straight lines in an outer region 10 of the beam of coherent light.

Fig. 7 again relates to the provision of at least two pluralities of predetermined straight lines at the planar waveguide 2The design of the wire 7. This is illustrated by the arrangement of three predetermined lines 7, the lines 7 on the left being marked with a distance d between the first constant adjacent straight lines 7 out of 24 constant distances_1…24. This is a design involving 24 predetermined straight lines 7 arranged each with a different constant distance between adjacent straight lines. As an example, the distance d between adjacent predetermined lines₁Is 446 nm and adjacent predetermined inter-line distance d₂Is 447 nanometers. The 24 plurality of predetermined lines is an arbitrarily selected number provided in this example in 24 different distance ranges between 446 nm to 469 nm in steps of 1 nm. The mentioned steps provide a sufficient range to cover the expected change in effective index of refraction in the second or third decimal place (corresponding to effective index of refraction changes in the percentage to thousandth range).

A second embodiment of the invention is provided in the apparatus shown in figures 9 and 10, which depicts the apparatus before preparation and when ready for use. The device was prepared using the mask 14 shown in fig. 8.

Fig. 8 shows a mask 14 used in photolithography for arranging the binding sites 5 along the predetermined straight lines 7 to the outer surface 21 of the planar waveguide 2. Such a mask 14 comprises a pattern thereon adapted to convey a predetermined straight line 7 on the outer surface 21. The pattern is used in a lithographic step to attach binding sites in a predetermined straight line 7 on the outer surface 21 of the planar waveguide 2. Shown in fig. 9 is an apparatus that has not yet been prepared. The photolithographic method exemplifies any suitable method for disposing the predetermined straight line 7 at the outer surface 21 of the planar waveguide 2. Generally, each method known in the art suitable for structuring binding sites on the nanometer to micrometer scale may be used for disposing binding sites thereon. In fig. 10, the prepared device is shown to have 24 predetermined straight lines 7. The 24 pluralities of predetermined straight lines 7 are arranged in columns with respect to the three separate portions 411, 412, 413 such that coherent light coupled via each of said separate portions is diffracted on the successively arranged 8 pluralities of predetermined straight lines 7. The plurality of predetermined lines 7 of 24 are arranged in three parallel rows with a distance between them forming a portion 10 of the planar waveguide 2 outside the parallel beams of coherent light propagating through the planar waveguide. The optical coupler 41 comprises three separate parts 411, 412, 413 for coupling three parallel beams of coherent light into the planar waveguide 2. The three separate portions 411, 412, 413 forming the optical coupler are arranged in a row and laterally spaced from adjacent separate portions by a predetermined distance. Thus, the parallel beams of coherent light propagate through the planar waveguides 2 separated by the predetermined distance. Each of the individual portions 411, 412, 413 has a predetermined length equal to the width of a plurality of predetermined straight lines among a plurality arranged in a single row. Each individual section 411, 412, 413 couples a coherent optical beam into the planar waveguide. Between which are three portions 10 on the outer surface 2 of the planar waveguide 2, which are located outside the coherent light beam. The portion 10 is used to set another optical coupler 8 to each of the plurality of predetermined straight lines 7, respectively. Coherent light that is not diffracted by the target sample bound to the binding sites arranged along the predetermined straight line 7 propagates through the planar waveguide to a further optical coupler 42 for coupling out light propagating through the planar waveguide 2 that is not diffracted at the target sample bound to the binding sites arranged along the predetermined straight line 7.

Fig. 11, 12 and 13 show examples for diffracting the light of the evanescent field 11. Light 11 is diffracted at the target specimen 6 bound to the binding sites 5 arranged along the predetermined straight line 7 with distance d, thereby contributing a maximum in the predetermined detection position. The schematic shown is well known from the description of bragg diffraction in "crystal structures". In principle, the bragg condition 2Ndsin (α) ═ k λ describes an angle at which the maximum intensity of diffracted light can be detected. Due to the parallel arrangement of the predetermined straight lines 7 with a constant distance d between adjacent lines, light of evanescent fields 11 diffracted on subsequent lines interferes at a predetermined diffraction angle, having an optical path length difference of an integer multiple of the predetermined wavelength of light propagating through the planar waveguide 2. Thus, the illustrated parallel beams 12 of diffracted light interfere at those diffraction angles to have an optical path length difference that is an integer multiple of the predetermined wavelength of the propagating light. The illustrated example illustrates the target sample 6 bound to the binding site without any predetermined conditions for the type of binding site and the type of target sample 6. For constructive interference, it is crucial that the binding sites to which the target sample may or may not bind are arranged along the predetermined straight line 7 so that the light constructively interferes under the prescribed conditions.

In fig. 11, the binding site includes a single type of capture molecule. The detection of binding affinity tests whether the capture molecules bind to the target sample 6 by actually observing the binding of the target sample 6 to the capture molecules. The capture molecules are in this first example attached to the outer surface of the planar waveguide to be arranged only along the predetermined straight line 7.

According to another example shown in fig. 12 and 13, the capture molecules 5 can bind to the target sample 6 disposed along the predetermined straight line 7 by disposing the capture molecules 5 capable of binding to the target sample 6 onto the entire outer surface of the planar waveguide and by deactivating these capture molecules 51 which are not disposed along the predetermined straight line 7.

This may be achieved by immobilizing the capture molecules on the (entire) outer surface of the planar waveguide such that there is no arrangement of capture molecules only along the plurality of predetermined lines 7. Thus, the light of evanescent field 11 diffracted by capture molecules 5 and capture molecules 51 does not interfere at the other optical coupler in the manner described above for diffracted coherent light 12.

Subsequently, the capture molecules 51 arranged between the predetermined lines 7 are deactivated so that no sample 6 can re-bind to these deactivated capture molecules 51. As shown in fig. 12, the deactivation is performed such that after the deactivation at the other optical coupler (the target sample 6 has no longer been added) all signals generated by the deactivated capture molecules 51 and the capture molecules 5 capable of binding the target sample are set or adjusted to the tuned minimum signal (and thus destructive interference), ideally zero, at the detection position. The light 121 diffracted at the deactivated capture molecules 51 and the capture molecules 51 has a difference in optical path length, increasing to a minimum in the predetermined detection position. The lines of deactivated capture molecules 5 and capture molecules 51 shown are "ideal" lines but provide a sufficient approximation, since light diffracted from capture molecules 5 and deactivated capture molecules 51 arranged differently from (or in the vicinity of) the plurality of predetermined "ideal" straight lines 7 is in principle self-eliminating.

Alternatively, a minimum signal before application of the target sample may be achieved by subsequently applying the capture molecules 5 and the deactivated capture molecules 51 such that in a first step the capture molecules 5 are applied to the outer surface of the planar waveguide along a plurality of predetermined straight lines 7 (similar to fig. 11). In a subsequent step, the deactivating capture molecules 51 are applied between lines of a plurality of predetermined straight lines 7.

In a final step, a target sample is added to the outer surface of the planar waveguide. Since only capture molecules arranged along the predetermined straight line 7 are able to bind to the target sample 6, the target sample 6 binds to these capture molecules along the predetermined line 7, as shown in fig. 13. Since the signal at the detection location caused by the deactivated capture molecules 51 and the capture molecules has been set or adjusted to a minimum value before, the target sample 6 binds to the capture molecules arranged along the predetermined line 7, after the signal at the detection location is then mainly caused by (or entirely caused by, if the signal generated by the deactivated capture molecules 51 and the capture molecules has been reduced to zero before) the light 12 that has been diffracted by the target sample 6.

Whereas embodiments of this invention have been described with the aid of the accompanying drawings, various modifications and changes to the described embodiments are possible without departing from the underlying general teachings of the invention. Therefore, the invention is not to be understood as being limited to the described embodiments, but the scope of protection is defined by the claims.

Claims (15)

1. An apparatus for detecting binding affinity, the apparatus comprising a planar waveguide (2) arranged on a substrate (3), and further comprising an optical coupler (41), the optical coupler (41) having a predetermined length for coupling coherent light (1) of a predetermined wavelength into the planar waveguide (2) such that a parallel beam of coherent light propagates through the planar waveguide (2), wherein an evanescent field (11) of coherent light propagates along an outer surface (21) of the planar waveguide (2), the outer surface (21) of the planar waveguide (2) comprising binding sites (5) thereon, binding sites (5) being capable of binding target samples (6) to the binding sites (5) such that light of the evanescent field (11) is diffracted by target samples (6) bound to the binding sites (5), wherein the binding sites (5) are arranged along a plurality of predetermined straight lines (7), the plurality of predetermined straight lines (7) extend parallel to each other with a constant inter-adjacent straight line distance, a predetermined straight line of the plurality of predetermined straight lines (7) being arranged at an angle (β) with respect to a propagation direction of the evanescent field (11) such that coherent light (12) diffracted by the target sample bound to the binding site (5) impinges under a diffraction angle (α) with respect to the predetermined straight line on a further optical coupler (8) arranged in a portion (10) of the planar waveguide (2) located outside of a beam of coherent light propagating through the planar waveguide (2), the further optical coupler (8) coupling the diffracted coherent light (12) outside the planar waveguide (2) for interfering with an optical path length difference of an integer multiple of a predetermined wavelength at a predetermined detection position (9).

2. The device according to claim 1, wherein the constant distance (d) between the adjacent predetermined straight lines (7) is selected such that a bragg condition 2Ndsin (α) ═ k λ is achieved

Wherein:

n is the effective index of the guided mode in the planar waveguide;

d is the distance between adjacent predetermined straight lines;

α is the diffraction angle;

k is the number of intensity maxima; and

λ is the vacuum wavelength of the propagating light.

3. The apparatus of claim 2, wherein the predetermined straight line (7) is arranged at an angle (β) of 22.5 ° with respect to the propagation direction of the evanescent field (11), and wherein the coherent light (12) diffracted by the target sample (6) bound to the binding site (5) impinges on the further optical coupler (8) under a diffraction angle (α) of 22.5 ° with respect to the predetermined straight line (7).

4. The apparatus of any one of claims 1 to 3, wherein the further optical coupler (8) comprises a plurality of grating lines (81), each of the plurality of grating lines (81) having a respective curvature and a distance between adjacent grating lines (81), such that the further optical coupler (8) is capable of coupling diffracted coherent light (12) out of the planar waveguide (2) for interference at a predetermined detection position (9) with an optical path length difference being an integer multiple of a predetermined wavelength, and wherein the plurality of grating lines (81) have an axis of symmetry extending below a diffraction angle (a) with respect to the predetermined straight line (7).

5. The device according to any one of claims 1 to 3, wherein said plurality of predetermined straight lines (7) are arranged in an active area (71) on said planar waveguide (2), said active area (71) having a width equal to the length of said optical coupler (41), such that the whole of said active area (71) is illuminated by an evanescent field (11) of coherent light coupled into said planar waveguide (2) by said optical coupler (41).

6. The device according to any one of claims 1 to 3, wherein at least two plurality of predetermined straight lines (7) are arranged successively on the planar waveguide (2) in the propagation direction of the evanescent field (11), the respective other optical coupler (8) being arranged with respect to each plurality of predetermined straight lines (7) such that coherent light (12) diffracted by a target sample (6) bound to a binding site (5) arranged along a straight line of the respective plurality of predetermined straight lines (7) impinges on the respective other optical coupler (8) under a diffraction angle (α).

7. The device according to claim 6, wherein adjacent straight lines of each plurality (7) of said at least two plurality (7) of predetermined straight lines have the same constant distance d between them.

8. The device according to claim 6, wherein each of said at least two plurality of predetermined straight lines (7) is more than oneAdjacent ones of the predetermined straight lines (7) have different constant distances d therebetween_1...n。

9. The device according to claim 8, wherein a constant distance d between adjacent straight lines of adjacent predetermined straight lines (7)_1...nWith an equal step difference in the range of 0.5 nm to 10 nm.

10. The device according to claim 6, wherein the at least two plurality of predetermined straight lines (7) comprises a plurality of groups of predetermined straight lines (7), adjacent straight lines of each group having an equal constant distance d between them, and wherein adjacent straight lines of different groups of predetermined straight lines (7) have different constant distances d between them_1...n。

11. The device according to any one of claims 1 to 3, wherein the optical coupler (41) comprises at least two separate portions (411, 412, 413) for coupling parallel beams of coherent light (1) of a predetermined wavelength into the planar waveguide (2), each separate portion (411, 412) having a predetermined length and being laterally spaced from an adjacent separate portion (411, 412, 413) of the optical coupler (41) by a predetermined distance, such that parallel beams of coherent light propagate through the planar waveguide (2) spaced by the predetermined distance.

12. The device according to any one of claims 1 to 3, wherein the binding sites (5) comprise capture molecules attached to the outer surface (21) of the planar waveguide (2) only along the predetermined straight lines (7), the capture molecules being capable of binding to the target sample (6).

13. The device according to any one of claims 1 to 3, wherein the binding sites (5) comprise capture molecules capable of binding the target sample (6), said capture molecules being capable of binding target samples (6) arranged along the predetermined straight line (7) by immobilizing capture molecules capable of binding target samples to the outer surface (21) of the planar waveguide (2) and by deactivating those capture molecules not arranged along the predetermined straight line (7).

14. A system for detecting binding affinity, comprising a device according to any one 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) emitted by the light source is coupled into the planar waveguide (2) via the optical coupler (41).

15. The system according to claim 14, wherein the light source and the device are arranged adjustable with respect to each other to change an incoupling angle under which the coherent light (1) emitted by the light source is coupled into the planar waveguide (2) via the optical coupler (41), and wherein the light source is tunable to emit light of a predetermined wavelength within a predetermined range.