Space filtering in optical biomolecule interaction analysis

Abstract

The invention is directed to a device for the detection of target molecules, comprising - a transparent substrate provided with binding sites on one surface of the substrate, wherein the binding sites are capable of binding at least one target molecule, - a light source, - means for coupling light provided by the light source into the substrate, wherein at least a part of the light generates an evanescent field of light propagating along the surface provided with the binding sites generating scattered signal light - at least one detector, - means to measure signal light under at least two different phase conditions or positions wherein the evanescent field of light is diffracted by target molecules bound to the binding sites, thereby creating a detection signal which is detected by the at least one detector. The signal (i.e. the diffracted light) is measured at the at least two different positions which are used to calculate its relative phase distribution.

Description

SPACE FILTERING IN OPTICAL BIOMOLECULE INTERACTION ANALYSIS

[0001] The invention is associated to the field of sensor instruments for optical and label-free detection of biomolecule interactions.BACKGROUND

[0002] Label -free detection of biomolecule interactions can be realized by a variety of methods, which can be classified according to their respective detection principle, e.g., calorimetric, electrochemical, acoustic and optical methods. Optical methods have in common that the association of biomolecules or analytes to their ligands or binders induces a change of light guided along or through the active sensor surface. For example, the intensity of light or its phase is altered due to biomolecule association and this change can be detected in real-time to determine binding kinetics and analyte concentration in solution (1) the data are called sensorgramms.

[0003] The most widely used method in the field of label-free biomolecule interaction analysis (BIA) with optical transducer is based on a technology referred to as “Surface Plasmon resonance” (SPR). In this method, the change in the refractive index measured by light reflected from a metal surface is detected (refractometric sensor. Binding of biomolecules to the other side of this surface leads to a change in the refractive index, which is proportional to the mass added to the sensor surface. This method offers high sensitivity (appr. 10 pg / mL), but it is prone to changes in media conditions (media composition, temperature) and non-specific binding and requires a reference channel (2). SPR-based Biacore instruments, distributed by Cytiva Life Sciences, are leading the market in the field of optical BIA (https: / / www.cytivalifesciences.com / en / us / about-us / our-brands / biacore). These instruments can have a limit of detection (LOD) down to 0.01 ng / cm2, depending on the molecular weight of analytes and binders.

[0004] Another method that is widely employed in the field is called Biolayer Interferometry (BLI). Here, the interference pattern of light reflected from two surfaces, a layer of immobilized protein and an internal reference layer, is analyzed. Specific interferometric wave patterns can be attributed to changes in protein association on an optical fiber surface, which is dipped into the analyte solution (3). Main vendor of BLI instruments is Sartorius with its Octet system series (https: / / www.sartorius.com / en / products / protein-analysis / octet- 1 ab el -free-detecti on- sy stem s) . Although these instruments have some advantages, as nofluidics are necessary and shaking of the microtiter plate keeps analyte solutions homogeneous, but compared to other technologies in the field, the LOD of BLI with 0.1 ng / cm2is rather high and only analytes with a molecular weight higher than 150 Da can be detected. Another variant of interferometric BIA, the waveguide-based “Grating-Coupled Interferometry” (GCI) offers kinetic measurements with faster off-rates than classical BLI (kd=10 s'1vs. 0.1 s'1) and is commercialized by Creoptix (https: / / www.creoptix.com / technologies / gci). GCI was proven to detect analytes with a molecular mass of 59 Da at signals lower than 1 pg / mm2(4). A new interferometric method commercialized by Refeyn (https: / / www.refeyn.com / ) is called “Mass Photometry”. The technology is based on “Interferometric scattering microscopy” (iSCAT,) (5) and detects light scattered from single analytes coming in close proximity to their respective binder. Compared to other systems, it can also detect smaller analytes down to 30 kDa.

[0005] Another group of optical BIA instruments utilize planar waveguides guiding coherent light to diffractive gratings. The associated technology named “Focal Molography” was originally published by ETH Zurich University in cooperation with Roche Diagnostics GmbH. Here, Molograms, nanopattems of molecular recognition sites, create light diffraction. Specific binding of target molecules to the mologram increases light diffraction, forming a coherent signal. The technology offers many advantages, as measurements are not affected in crude samples, independent on refractive index of the analyte solution and temperature changes. The LOD of 0.01 ng / cm2is comparable to Biacore devices. As the sensitivity of this technology can be seen as a function of the molecular mass of binders and analytes, small analytes with a low molecular weight need to be introduced into the system in high concentration, limiting its usability e.g. in cytokine diagnostics from blood samples. Another hurdle is that not only binders have to be attached to specific regions of the Mologram, but the adjacent regions need to be backfilled with “non-binders” of equal molecular weight. This complicates the production of the required sensor chips. Furthermore, the invention disclosures filed in this subfield claim that the use of coherent light is unavoidable for the respective optical setups, increasing light source costs (WO2013107811, WO2014086789, WO20 15004264, WO2015007674, WO2019166562, EP3428622B1, EP3428622B1, EP3835764A1).

[0006] The prior art related to “Label-free detection of biomolecules” is further for example disclosed in the following publications:(1) Nirschl M, Reuter F, Vbrbs J. Review of transducer principles for label-free biomolecular interaction analysis. Biosensors (Basel). 2011;l(3):70-92. Published 2011 Jul 1. doi: 10.3390 / biosl030070(2) Nguyen HH, Park J, Kang S, Kim M. Surface plasmon resonance: a versatile technique for biosensor applications. Sensors (Basel). 2015; 15(5): 10481-10510. Published 2015 May 5. doi: 10.3390 / sl50510481(3) Concepcion J, Witte K, Wartchow C, et al. Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization. Comb Chem High Throughput Screen. 2009; 12(8):791-800. doi: 10.2174 / 138620709789104915(4) Jankovics H, Kovacs B, Saftics A, et al. Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers. Sci Rep 10, 22253 (2020). doi: 10.1038 / s41598-020-79226-w(5) Young G, Kukura P, Interferometric Scattering Microscopy. Ann Rev Phys Chem 70, 301-322 (2019). doi: 10.1146 / annurev-physchem-050317-021247(6) Gatterdam, V, Frutiger, A, Stengele, KP, et al. Focal molography is a new method for the in situ analysis of molecular interactions in biological samples. Nature Nanotech 12, 1089-1095 (2017). Doi: 10.1038 / nnano.2017.168OBJECT OF THE INVENTION

[0007] The aim of the invention disclosed in this application is related to the detection of scattered light from an ensemble of binding molecules where the measured signal changes if analytes bind to a sub-ensemble of the ensemble of molecules.

[0008] In the prior art, coherent light is needed to confine the signal and to increase the signal to noise ratio. Signal enhancement is achieved by scattered light from the individual molecules arranged in a well-defined pattern contributing coherently to one point at a detector which is typically a camera sensor. This also requires a spectral coherence of the excitation light in units of spectral bandwidth divided by the center wavelength of approximately 1 / 1000. Spectral coherence results in a prominent speckle background at the detector. The origin of the speckles is either scattered light from molecules non-specifically bound to the surface and from excitation light scattered and reflected within the sensor chip or apparatus. Another contribution of the speckle background is related to the finite roughness of the surfaces which are passed by the exciting light. This also includes the surface where the analytes are bound. The latter results in signal which is indistinguishable from the scattered signal generated by the molecules immobilized on the surface and the analytes (targetmolecules) to be detected since the roughness is equivalent to a structured adherence of molecules on the surface. In order to distinguish signal from speckle background, the speckle pattern has to be well characterized. Here, the most prominent problem is that only the background speckle intensity pattern can be measured and not its phase. There are suggestions to measure the local phase shifts of the light by means of a switchable coherent reference beam with a known phase pattern on the detector surface (DE102019219473A1). For this purpose a planar wave with a tilted angle of incidence with respect to the signal on the detector can be used to generate a set of reference images from which a local relative phase could be derived. A well calibrated reference beam is required which has to be also tuned in its intensity to generate an interference pattern with the signal which can be analyzed properly. This result is in a substantial optical effort and adds further requirements to the light source.OBJECT OF THE INVENTION

[0009] One aspect of the invention was to improve the known method for detection of the mass of target molecules bound to a substrate, for example by reducing the need of spectrally coherent light and to remove speckles at the detectors as a source of errors.

[0010] In the technology of detection of biological targets by light scatting, it is known from US20210270736A1 to obtain phase information using a reference wave.

[0011] It was found that phase retrieval can be used to obtain corrected scattered light intensities which provide an improved basis for further calculations by removing the background in relation to the phase distribution of relevant signal.

[0012] Accordingly, object of the invention is a method for detection the total mass of target molecules bound to a substrate, comprising providing a transparent substrate having binding sites on one surface, wherein the binding sites are capable of binding at least a part of the target molecules and coupling the light provided by a light source into the substrate, wherein at least a part of the light generates an evanescent field of light propagating along the surface provided with the binding sites thereby generating scattered light intensities characterized in providing the transparent substrate with a fluid comprising target molecules wherein at least a part of the target molecules bind to the binding sites and detecting the scattered light intensities of at least two different occupancies of the binding sites with target molecules, each under at least two different phase conditions and calculating the amplitude and phase information of the scattered light intensities by phase retrieval and correcting the scattered light intensities with the amplitude and phase information and calculating the totalmass of the target molecules bound to the substrate from the corrected scattered light intensities.

[0013] By calculating the corrected scattered light intensities, the interactions kinetics between molecules and sensorgramms may be obtained.

[0014] The method of the invention can be conducted for example by providing a (one) fluid comprising target molecules to the transparent surface and determining first scattered light intensities at the beginning at the end of the measurement i.e. at a time where no or only a minor amount of binding sites are occupied by target molecules and at another time where binding sites are nearly saturated.

[0015] In practice, the least two scattered light intensities are preferable obtained at occupancies of the binding sites with the target molecules of 0 to 10 % and 80 to 100 %, respectively.

[0016] The at least two scattered light intensities i.e. the least two different occupancies of the binding sites with target molecules can further be obtained by providing the transparent substrate at least two fluids provided with different concentrations of target molecules.

[0017] In a variant thereof, the at least two scattered light intensities can further be obtained by providing a first fluid without target molecules and a second fluid comprising target molecules to the transparent surface. The first fluid may be a washing fluid.

[0018] Accordingly, in the method of the invention, the transparent substrate can be provided with a first fluid having a first concentration of target molecules and detecting first scattered light intensities under at least two different phase conditions and providing the transparent substrate with a second fluid having a second concentration of target molecules and detecting second scattered light intensities under at least two different phase conditions wherein the first concentration is between 0 and 50% of the second concentration and detecting the mass of the target molecules bound to the substrate by calculating amplitude and phase information by phase retrieval of the first and second scattered light intensities and calculating corrected scattered light intensities from the amplitude and phase information, thereby obtaining the mass of the target molecules bound to the substrate.

[0019] For phase retrieval, interference with a reference beam is explicitly not performed.

[0020] The invention can also be applied if different subareas are addressed subsequently. The subsequent detection can be achieved by coupling the light subsequently to the subareas of the substrate such that light of the evanescent field is diffracted by targetmolecules bound to binding sites located in the subareas and the detection signals of the subareas are detected subsequently.

[0021] Alternatively, the light is coupled simultaneously to the subareas of the substrate such that light of the evanescent field is diffracted by target molecules bound to binding sites located in the subareas and the detection signals of the subareas are detected subsequently.

[0022] In one aspect of the invention the wavelength is repeatedly swapped in its center wavelength and a set of data is acquired for various wavelengths of the source. This further reduces noise of the signal by combining the data.

[0023] In another aspect of the invention spatio-temporal modulation of the light is used to eliminate the speckles generated at the detector.

[0024] A further aspect of the invention is an increase of sensitivity by a more distinct immobilization pattern and the readout of more than one diffraction order of the diffracted light.

[0025] The following description of the drawings disclose the main aspects of the invention. The aspects shown can be combined by persons skilled in the art in various ways. Thus the invention covers also all combination of the disclosed aspects.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 : A lens setup for a focal imaging.

[0027] Figure 2: Specific means for increasing the optical path length.

[0028] Figure 3 : Device for label-free biomolecule interaction analysis.

[0029] Figure 4: By specific means, the optical path length can be increased (28) and the focus can be translated relative to the detector (29).

[0030] Figure 5: A diffractive sensor as described in Figure 4, bundling the diffracted light (01) from different binding zones (04).

[0031] Figure 6: Diffractive sensor with non-coherent light source.

[0032] Figure 7: A simplified version of a diffractive sensor as described in Figure 6 with direct light (01) guiding from grating (13) to binding zone (04).

[0033] Figure 8: Diffractive sensor with non-coherent light source and 3D volume holographic grating for filter integration.

[0034] Figure 9: Diffractive sensor with coherent or non-coherent light source and grating outside of the sensor chip (02).

[0035] Figure 10: Diffractive sensor with grating and binding zones positioned in row on the sensor chip with planar waveguide.

[0036] Figure 11 : Diffractive sensor with grating downstream of binding zones on a sensor chip.

[0037] Figure 12: Diffractive sensor with immersion layer.

[0038] Figure 13: Diffractive sensor with dual function.

[0039] Figure 14: Sketch of the measurement.

[0040] Figure 15: Sketch of the phase retrieval process.

[0041] Figure 16: Sketch of the reconstruction process of the relative scaling (Sc) between signal and background.DESCRITION OF THE INVENTION

[0042] The method of the invention is directed to detect the mass of target molecules bound to a substrate. Target molecules are especially RNA, DNA, c-DNA, single stranded DNA, peptides, proteins and oligonucleotides.

[0043] The involves providing a transparent substrate having binding sites on one surface, wherein the binding sites are capable of binding at least a part of the target molecules.

[0044] Obviously, the binding sites need to be adapted to the target molecules. For example bindings sites may be antibodies, or part of antibodies or oligonucleotides designed or selected to bind the desired target molecules. Accordingly, the binding sites should be designed to bind such target molecules as specifically as possible. In general, the device is not restricted to any binding chemistry and a person skilled in the art is well aware of the binding chemistry which can be used for such purposes. For example, the binding sites provided on the surface of the substrate may be selected from the group of antibodies or fragments thereof of nucleic acids.

[0045] Further, the method involves coupling the light provided by a light source into the substrate, wherein at least a part of the light generates an evanescent field of light propagating along the surface provided with the binding sites thereby generating scattered light intensities.

[0046] The substrate used in the device of the invention is not of particular importance as long as the light coupled into the substrate generates an evanescent field of light propagating along the surface provided with the binding sites. Suitable substrates are glass or transparent polymers.

[0047] In the following, the terms “binding zones” and “subareas” are used interchangeable.

[0048] In one embodiment of the invention, the at least one light source provides low coherent or non-coherent light and the dispersion of the detection signal generated by the diffraction of low coherent or non-coherent light is reduced by at least 50%, preferable by at least 80%, more preferable by at least 95% and most preferred by at least 99% by one or more dispersive elements.

[0049] The light provided by the light source can be low coherent or non-coherent light. Such light has preferable a spectral bandwidth wider than 1 / 100, more preferable wider than 1 / 1000 and most preferred wider than 1 / 10000 of the center wavelength of the light. Independent from the spectral bandwidth, the light may have a wavelength in the visible range (so called white light), i.e. between 250 and 800 nm.

[0050] The device of the invention may comprise more than one light source (e.g., 2) which may provide light beams with the same or a different wavelength or range of wavelengths.

[0051] The reduction of dispersion is calculated by the ratio of dispersion obtained with and without using dispersive elements. In absolute values, the dispersion should be less than 10 um / nm wavelength of the light.

[0052] The dispersive elements can be located in the path of light before and / or after the light is diffracted by the target molecules bound to the binding sites.

[0053] In all embodiments described hereinafter, the dispersive elements may be gratings and / or coupling gratings and / or prisms and / or volumetric holograms and / or tilted interfaces. Further, holographic gratings, volume gratings spatial light modulators or DMDs can be used as dispersive elements.

[0054] In one embodiment of the invention, a plurality of binding sites is provided at different locations on the substrate, thereby generating a plurality of detection signals and wherein the detection signals are individually focused on the detector using at least one array of optical elements.

[0055] The arrays of optical elements may comprise lenses and / or microlenses and / or facetted elements and / or rotating apertures or sliding apertures. It is possible to use several arrays of optical elements, which can have the same or different optical properties. In a variant thereof, different arrays of optical elements with different optical properties are provided wherein the different arrays of optical elements are exchangeable. In another variant,the arrays of optical elements are provided with at least one aperture plate and / or an aperture plate array.

[0056] In another embodiment, the detection signal is space-filtered in a focus plane by means of a diaphragm and / or a pinhole. The space filtering should select at least one diffraction order of the detection signal.

[0057] The substrate may comprise a planar waveguide and / or a prism and / or independent thereof, at least one coupling surface for means for coupling a beam of light.

[0058] Optionally, the binding sites are arranged on one surface of the substrate in a plurality of lines having the same or different pitch. The substrate itself may be produced from glass or transparent polymers.

[0059] In a further embodiment, light provided by the light source is spatio-temporal modulated with a variable mask, a spatial light modulator or a scanner.

[0060] In a variant of the invention, the optical path length can be increased (28) and the focus can be translated relative to the detector (29). In other words, the at least two different phase conditions can be obtained by providing means to change the optical path of light between the substrate and the at least one detector.

[0061] The means to change the optical path may be selected from the group consisting of movable glass sheet (element 28 in figure 2A), prism, mirror (element 31 in figure 2B or element 32 in figure 2C), lens (element 7 and 28 in figure 3C), lens-array (element 9 and 28 in figure 3C), grating (element 13 in multiple figures).

[0062] Further, the optical path may be changed by moving at least one detector relative to the substrate surface and / or by splitting the first and second scattered light intensities into at least two beams and detect them separately. This embodiment is shown in figure 2C.

[0063] The binding sites may be provided in a non-stochastic distribution to the transparent substrate. More preferred is that the non-stochastic distribution of the binding sites on the transparent substrate results in coherent scattered light intensities.Embodiments of the invention

[0064] The invention itself and various embodiments thereof are explained in the following by referring to the figures.

[0065] Figure 1 shows a broadband light source (11) is coupled from a pupil on a dispersive element (13) onto the coupling grating of the waveguide (13). The dispersion is selected to ensure coupling conditions for all wavelengths. The diffracted signal light from thebinding zones (04) falls on another grating (13) in the detection beam path, the grating constant is chosen so that the light from all wavelengths is concentrated in one point (08) and consequently focused (10) by means of a microlens array (09), or one lens, on one point or multiple points of the detector (11). By specific means, the optical path length can be increased (28) and the focus can be translated relative to the detector (29). Data acquired at different optical path lengths are then used for phase retrieval.

[0066] This variant of the invention uses a broadband light source (11) which is coupled from a pupil on a dispersive element (13) onto the coupling grating of the waveguide (13). The dispersion is selected to ensure coupling conditions for all wavelengths. The diffracted signal light from the binding zones (04) falls on another grating (13) in the detection beam path, the grating constant is chosen so that the light from all wavelengths is concentrated in one point (08) and consequently focused (10) by means of a microlens array (09), or one lens, on one point or multiple points of the detector (11). By specific means, the optical path length can be increased (28) and the focus can be translated relative to the detector (29). Data acquired at different optical path lengths are then used for phase retrieval.

[0067] Another embodiment is depicted in Figure 2, which shows specific means for increasing the optical path length. A: retarder plate (30), B: movable wedge prism (31), C: image splitting prism (32) resulting in multiple signal images on a single detector (11). Optionally, the detector can also be translated (29).

[0068] In another one variant of the invention, different binding zones (04) are defined at specific positions of a continuous pattern of straight lines with binding sites (03) on a sensor chip equipped with a planar waveguide ((02), Figure 1).

[0069] In this embodiment, the optical path length can be increased (28) and the focus can be translated relative to the detector (29). A: Diffractive sensor with different binding zones (04) within a continuous pattern of straight lines (03) on a sensor chip with planar waveguide (02) with microlens array (09) for photodetection (11). Upper panel: Top view of the waveguide (02). Lower panel: Side view of the waveguide (02) with optical detection pathway. B: Alternative setup according to A: Lines (03) are only generated inside the binding zones (04). C: Variants for microlens array (09): Upper panel with 3 microlenses for formation of 3 foci, middle panel with 5 microlenses for formation of 5 foci, lower panel with one lens for formation of a single focus.

[0070] Scattered coherent light (01) is optionally passed through an aperture plate (06) before it is focused by a lens (07). After passing a pinhole (08), light is collected by a lens (07) and directed to a microlens array (09), generating single foci (10) on a detector (11). Themicrolenses focus the light scattered from the binding zones to the detector. Optionally, the lines are only generated inside the binding zones (04) to reduce background signal. The microlens array (09) might also consist of other pluralities of microlenses than depicted in the figure Figure 1 A. Alternatively, in this and all following embodiments, the microlens array (09) could be replaced by facetted elements or rotating or sliding apertures selecting the light from individual binding zones sequentially (not depicted). In a more elaborated embodiment, the array of microlenses (09) can be changed to a microlens array with a higher or lower number of individual microlenses or just removed to adapt the device to different sensor chips with lower or higher numbers of binding zones Figure IB and C). The binding zones might be generated by spotting different binders to the binding zones or by optically activating a crosslink between the binders and the surface. The pattern of the binding zones has to be designed according to the microlens array (09) used in the detection beam path. The advantage of the embodiment is, that the structure of the sensor chip is simple. The generation of one planar wave enables simple remote detection, where the distance between the sensor and the detector unit can be large. This reduces the contribution of scattered light on the sensor. Different types of apertures (Figure 1 A, 06 and or 08) may be inserted to further reduce scattered light. One further advantage is that the simple structure of the sensor chip also enables an easy production of the chip since the pattern may be generated just by an interference of two planar beams of activating light (not depicted).

[0071] In another version of the previous embodiment shown in Figure 4, the diffractive sensor does not contain a continuous pattern of straight lines with binding structures (03), but an alternative grating structure with individual straight gratings generating individual diffracted planar wavefronts (03) generated by the binding zones (04).

[0072] Diffractive sensors are generating individual diffracted planar wavefronts (03) restricted to binding zones (04). The waveguide (02) directs the light (01) to a hologram with lens-phase pattern containing binding structures (03) positioned with increasing proximity to each other, causing a different diffraction pattern for each binding zone (04), guiding the light (01) diffracted from different binding zones (04) to a common center, but not bundling it. A telecentric lens (12) generates single foci (10) on the photodetector (11)

[0073] The angle and grating period is designed in a way that the signal light (01) scattered from the binding zones (04) enters a common aperture (06). After passing the aperture (06), light is directed to a lens ideally with a telecentric design (12), generating single foci (10) of the light from the binding zones (04) on the photodetector (11). In thisembodiment the straight beamlets emitted from the individual binding zones can be directed to a far common aperture. This enables the reduction of scattered light.

[0074] In another embodiment shown in Figure 5, the diffractive sensor is coupled to another detection pathway by an alternative hologram with individual gratings (03).

[0075] By specific means, the optical path length can be increased (28) and the focus can be translated relative to the detector (29).

[0076] The waveguide (02) directs the light (01) to a collimating pattern containing binding structures (03) generating a common spherical wave of diffracted light from all different binding zones (04) focused to a common first focus (10) within a pinhole (08) with acts as a global space filter. After passing the pinhole (08), the signal light is directed to a lens (07) ideally with telecentric design (12) and to a subsequent microlens array, generating single foci (10) that are focused onto a detector (11). The signal light (01) is optionally passed through an aperture plate (06) before or after it is focused by the lens (07) to remove background signal. The embodiment has the advantage that a real space filter eliminates most of the stray light. Therefore in this setup, the lines do not need to be restricted to the binding zones (04) to reduce background light. As described in one of the other embodiments the microlens array (09) could be exchangeable to adapt the device for different geometries of binding zones. The additional lens (07) is used to collimate the bundle of light and to hit the microlens array along its axis. The lens might also be eliminated at the cost of telecentricity on the detector (11). In this embodiment the production of the sensor chip can be done by an interference of one planar activating beam with one spherical activating beam resulting in the required segment of a Fresnel-zone type pattern (not depicted).

[0077] In further embodiments, specific optical setups allow to simplify the sensor chip. They also allow for the use of non-coherent light and include one or more gratings that can be for example, relief structures, holographic amplitude of phase gratings, volumeholographic gratings, i.e. a spatial light modulator that is used as holographic grating. The additional gratings are inserted in the excitation beam path directly or as part of the sensor chip or in the detection beam path directly. The light sources used might be lasers, super luminescent diodes, broad band Vertical Emitting Surface Emitting Laser (VESCL), LEDs, spectral lamps or white light lasers e.g. generated in a photonic crystal fiber. In all embodiments where grating structures are used to extend the spectral range of the light which can be transmitted and focused on the detector it is also possible to use a tunable or spectrally switchable light source. Then a series of data for different wavelength can be acquired and accumulated to further increase the signal to background ratio. The advantage of a serialreadout for different wavelengths is that remaining dispersion leading to different positions of the signals for different wavelengths can be corrected by numerical methods.

[0078] In another embodiment, the grating (04) and binding zone are positioned in a row, so-called “single-side replication” as depicted in Figure 6. Here, light (01) from a white light source (14) is filtered (15) to become monochromatic red light. Monochromatic light (01) is reflected by a binding zone-shaped grating (13), creating a dispersed first or higher order diffracted beam. A minor portion of light (01) is not reflected and serves as internal reference. The major portion of light (01) is reflected again by the rear surface (16) of the sensor chip to guide it to the binding zones (04) inside the flow cell (18) and create a signal in a light-covered detection space (19) subsequently. Excess light (dotted lines) is led out of the system into beam dumps (17). By specific means, the optical path length can be increased (28) and the focus can be translated relative to the detector (29).

[0079] Accordingly , light (01) from a light source (14) is filtered (15) to become sufficiently monochromatic light or light with a well-defined bandwidth. The light (01) is diffracted by a grating (13), creating a dispersed first or higher order diffracted beam. A portion of the light (01) preferentially the minus first order or the transmitted or reflected zero order might serve as internal reference. The major portion of light (01) is reflected by a the rear surface (16) of the sensor chip and is guided to the binding zones (04) inside the flow cell (18) and creates the signal light in the detection space (19) subsequently. Excess light (dotted lines) is led out of the system into beam dumps (17). Alternatively, to the setup depicted in Figure 4, binding zones (04) can be designed in different shapes, as shown in Figures 1-3. The beam path can be modulated accordingly.

[0080] A simpler embodiment of the device illustrated in Figure 6 is depicted in Figure 5. Here, the light (01) is diffracted by a grating (13) and directed directly to the binding zone (04) inside the flow cell (18). In this setup, grating (13) and binding zones (04) are positioned on the sensor chip (02) on opposing sides. Alternatively, to the setup depicted in Figure 7, binding zones (04) can be designed in different shapes, as shown in Figures 3-5. The beam path can be modulated accordingly.

[0081] A more advanced version of the previous embodiments (described in Figure 6 and Figure 7) contains a 3D volume holographic grating (20) for shaping the excitation light (Figure 8).

[0082] Figure 8 shows how monochromatic light (01) is guided into a volume hologram (20), creating dispersion and redirecting it to the binding zones (04) inside the flow cell (18). Focal spots (10) are created in a light-covered detection space (19) subsequently. Asecond volume hologram (20) assures scattering and can be used as reference. By specific means, the optical path length can be increased (28) and the focus can be translated relative to the detector (29).

[0083] The light (01) is guided into a volume hologram (20), creating dispersion and redirecting it to the binding zones (04) inside the flow cell (18). Focal spots (10) are created in a detection space shielded from external light (19) subsequently. A second volume grating (20) assures scattering and can be used as reference. This setup may result in high efficiency, since a better support of only selected diffraction orders is possible with volume-holographic gratings. The volume-holographic grating may include additional functions, such as collimating and filtering the excitation light. Then, part of the excitation optics (08, 07, 15) could be eventually omitted.

[0084] In another embodiment, the chip containing the binding zones does not include gratings (Figure 9).

[0085] Figure 9 shows how light (01) is guided by a mirror (16) onto a grating (13), creating dispersion and reflecting it to the binding zones (04) inside the flow cell (18). By specific means, the optical path length can be increased (28) and the focus can be translated relative to the detector (29).

[0086] Here, the diffractive sensor is adapted for the use of both, coherent and noncoherent light, by installation of a grating outside the sensor chip. The light (01, coherent or non-coherent) is guided onto a grating outside of the chip (02), creating dispersion and reflecting it to the binding zones (04) at the interface between the chip and the flow cell (18). The dispersion of the light (01) at the external grating (13) is adjusted to compensate the dispersion added at the interface to the chip (02) and the dispersion added by the binding zones (04) itself resulting in a signal beam, where all wavelengths from the light source propagate along one axis. The lens (08) has to be sufficiently achromatic to not introduce further chromatic errors. Same is true for the microlens arrays (09). The microlens array may be also replaced by or be replaceable within the device by other types of microlens arrays one or two dimensionally arranged or even by just a single lens (not depicted). By this means the system can be adopted to different arrangements of binding zones.

[0087] In a further embodiment shown in Figure 10, light (01) is directed from a first grating by a planar waveguide (black arrow) to the binding zones (04). A: diffracted light from the binding zones (04) is focused by two telecentric lenses (12) between an aperture (06) before it reaches a grating (13), guiding it to the photodetector (11). B: Light (01) formbinding zones (04) is directly guided to the grating (13). By specific means, the optical path length can be increased (28) and the focus can be translated relative to the detector (29).

[0088] The dispersion of the first grating is done in a manner that the angle of coupling is optimized for all wavelengths of the light. The first grating may be imaged to the coupling grating of the waveguide (not shown in the Figure).

[0089] In another embodiment not specifically shown in a figure but like Figure 10A movement of the pinhole (08) along the direction of dispersion would allow for selection of the detection wavelength (not illustrated in the figure). A similar configuration could be also realized in the excitation beam path if the first grating (13) is imaged to the coupling grating forming an intermediate dispersed focus of the exciting light (01). A dynamic motion of even oscillation of one or both pinholes (08) - one in excitation and one in detection - supports a wavelength modulated readout of the sensor chip (02). The motion might be synchronous or asynchronous. In case an asynchronous motion of the pinholes (08) would be applied, e.g., at different frequencies generates a modulation of the signal with mixed frequencies, since the setup acts as two independent monochromators, where the efficiency of the second monochromator is given by the binding of molecules at the binding zones (04). This modulation might help to further suppress straylight especially speckles on the camera or detector. The camera or detector readout must be synchronized or at least sample the modulation generated by the motion of one or both pinholes. In a similar approach modulated wavelength or even only some discrete or only two wavelengths might be applied. The resulting modulation of the signal at the detector can be used to suppress background. In a very simple version of the system, the detector might be replaced by a position sensitive detector or a linear array with two or more elements. The signal may be than extracted from the raw signal by extracting the position modulation.

[0090] To simplify the optical setup of the device, in one embodiment a grating is positioned downstream of binding zones on the sensor chip (Figure 11).

[0091] Here, light (01) is guided to the binding zones (04) on the upper side of the sensor chip (02). Diffracted light (01) is then directed to a grating (13) on the lower side of the chip (02, parallel to the binding zone), diffracting it towards a lens (07) that focusses the signal on a photodetector (11). By specific means, the optical path length can be increased (28) and the focus can be translated relative to the detector (29).

[0092] The detection beam path might include other variants shown in the alternative embodiments including multiplexing with microlenses. In a variant of the embodiment not shown in the figures the second grating might be a transmission grating positioned in theinstrument in proximity to the chip generating a transmitted beam identical to the one shown in Figure 9. In a further embodiment of the invention the transmission grating either on the chip or in the instrument may include the focusing function and dispersion function. In this setup, further optics can be omitted.

[0093] Furthermore, in one embodiment of the of the invention, signal-to-noise ratio of the device can be enhanced on various levels, enabling the signal extraction from the noisy environment: In one case, a spectral-temporal modulation is performed by shifting the spectrum of light send into the beam path of the device in a temporal course (not depicted). For this, several methods would be feasible, as for example switching between different coherent light sources, using a monochromator with modulated transmission wavelength, utilizing a rotating grid or a rotating multilayer filter in the excitation beam path. In another case, a better signal-to-noise ratio can be achieved by modulating the intensity of the excitation light over time (locked-in amplification detection). In a similar manner also the angle of incidence of the excitation light might be modulated. This could be done by galvanometric scanner tilting a mirror, glass plate or grating, acousto-optical deflector, movable slit or diaphragm or other means to change the angle of incidence. The resulting modulation of the signal at the detector is then analyzed. The advantage might be that modulation of the stray -light varies in a different manner than the modulation of the signal contributed by the binding molecules. This allows for a further increase of the sensitivity.

[0094] Here, the light (01) coming from the reference gratings (13') is shifted in phase and therefor can be used as reference. As the diffracted beams coming out of detection zones and reference gratings are excited with the same incidence wave, a fixed phase relationship is achieved. The different patterns of the focal point (10) projected onto the photodetector (11) provides a specific intrinsic signal, which reveals the relative phase of the signal light (01). The knowledge of the phase of the signal light allows the analysis whether the contribution of the signal is predominantly from the ridges versus the groves of the grating. This feature can be used also for control of quality during an experiment (e.g., analyte binding to binding structures) or also during production. Quality control during production is achieved in case of an inline analysis while the photochemical functionalization or specific binding of the analytes to the ridges or backfilling of molecules to the groves of the grating is done.

[0095] In another embodiment shown in Figure 12, a grating (13) is optically coupled to the chip (02) with an immersion fluid (23). In case that the refractive index of the fluid is higher than the refractive index in the liquid within the flow cell (18) the grating (13) could be arranged in a simple geometry according to the embodiments shown in Figure 4 and 5where the grating (13) is in a plane parallel to the plane of the binding zone (04). The immersion liquid can also be an adhesive which is supplied with the chip. It can also be supplied with microfluidic channels within the element coupled to the chip. The grating (13) can be holographic, volume holographic or relief grating. The position of the grating can be at the rear or frontside or in the volume of the element coupled to the chip. This element might also contain part of the detection optics (not shown in the figure). The shown embodiment may include other methods of detection as described within the description of the alternative embodiments. Other methods to couple the chip to the instruments might also include prisms with or without generating dispersion at the coupling point of the light.

[0096] A different embodiment not shown in the figures defines a method to generate a binder modulation on a sensor chip on binding zone. The state-of-the-art technique to generate a modulated grating like structure is photochemistry. Typically, UV light at a wavelength in the range between 300 and 450 nm is used. The readout of the sensor chips is then performed at red or infrared light (>600 nm). This is done to reduce autofluorescence in the optical path and from the sample excited by the evanescent field. It might be advantageous to generate the modulated grating with a similar or identical wavelength than the detection wavelength. The advantage would be that the identical beam path could be utilized for the production of the detection zones as well as for the analysis. The invention includes a setup where a laser beam is split to two beams where one is sent along the excitation beam path and the other in backward direction along the detection beam path. The two beams then interfere at the binding zone generating an interference which can be used to generate the binding zones if the laser has a similar or identical wavelength as the detection light source used for the read out of the sensor. The advantage of the approach is that optical errors in the excitation and detection beam path and also in the sensor are intrinsically eliminated by this method. Photochemical reactions can be excited if two or more low energy photons combine in a multiphoton excitation process. This is possible if the electric field is high enough. Femto- and picosecond lasers at low repetition rate are able to provide sufficient electric fields even at larger areas. Short laser pulses require a finite spectral bandwidth of the laser. Some of the described embodiments describe versions which eliminate the need to use coherent light for the readout of the sensor (e.g., Figure 1). The setup utilizing gratings generate pulse stretching of short pulse lasers. A proper setup of the parameters of the laser including pulse length, repetition rate and power is necessary. It is also of advantage to subsequently illuminate individual binding zones and thus limit the addressed area. Here, ascanner might be utilized. Methods of temporal focusing can help to confine the multiphoton process to the surface of the chip.

[0097] To compensate for the spectral bandwidth a grating might be inserted upstream the binding zone and is used to create dispersion of the laser pulse. The wavelength of the pulsed light can be e.g. between 650 to 1000 nm. The photochemical effect is at least proportional to the square of the intensity generating a grating with higher order components. In this example, un-pulsed light at a similar wavelength of 650-1000 nm from another e.g., continuous light source can be used for the detection of analytes binding in a diffractive sensor, as described e.g. in Figure 6. This approach has the advantage that errors occurring during the chip preparation or due to an uneven chip surface are automatically corrected in the detection step. Thus, it even allows to print diffractive structures as an arbitrary speckle pattern with binding sites onto the chip surface instead of a line pattern with binding sites (03) as depicted e.g., in Figure 3.

[0098] In another embodiment shown in Figure 13, the production of the chip and the analysis is done synchronously. With the same device, binding sites and non-binding sites (04) can be printed on chips (02) by stepwise spatial illumination and subsequently detection of analyte binding. Two separate optical paths are used for excitation light and for printing (01') and for detection (01). A beam splitter (21) separates the detection light (01) into two beams and guides them into the sensor chip (02), passing a dispersive element, i.e. a grating (13). By specific means, the optical path length can be increased (28) and the focus can be translated relative to the detector (29).

[0099] This can be done if the light activating the photochemical reactions and the detection are performed in a pulsed alternating manner, e.g., by switching between the modes at e.g., 0.1 to 100 Hz. It is also possible to use bandwidth filters to suppress the activation light and autofluorescence from the chip and sample. The attachment of the binders or adapter molecules could then be directly monitored. Implementing a phase-shifter (e.g. piezo on a mirror (16), tilting plate (21 or 16)), SLM = Spatial Light Modulator ) in one of the two excitation beam paths allows a switching of the activations from the bright areas (constructive interference lines) to the dark areas (destructive lines). Thus, by exchanging the binders in the liquid from specific ones to unspecific ones or between different adapter molecules in combination with shifting the phase and a readout of the chip allows to generate a well characterized chip with maximum sensitivity. This is true not only for the case of multiphoton excitation but can also be utilized in single photon UV photochemistry. Then, there are different beam path needed for the beam activating the photochemistry and the light for thedetection. A device which is able to perform the immobilization of binders and the readout can be used by customers designing their own experimental setup. The coherent photoactivation light source (OT) can be split into two beams of identical intensity. Both can be directed towards the chip (02) and interfere at the surface. This directly generates the binding zone (04) necessary to bind the analytes. By shifting the beam splitter (21) or tilting one of the directing mirrors (16) the phase of the interference at the sample can be changed. By this means different molecules can be bound with different modulation phases. It also allows first binding of the analyte with a first modulation phase and a second ‘backfilling’ molecule at a modulation phases shifted with pi / 2. The reaction time or intensity can be chosen so that the overall coverage with scattering molecules (analytes and backfilling molecules) is finally homogenous. An in situ control of the diffracted light during the subsequent binding may help to tune the homogeneity of the activated chip and thus to minimize the initial diffraction.[000100] It is of advantage to combine the functionalization of the chip with a direct readout of the immobilized molecules. To achieve this it might be useful to bind a small amount of fluorescent dye molecules with the same grating structure. After washing the additional application of UV or pulsed IR light generates a fluorescence from the dye. The intensity of the fluorescence can then be detected with respect to the phase between the interference structure and the grating. This allows to shift phase so that the intensity is minimized. This process allows a repositioning of the substrate for the sensor chip and still do a proper backfilling of the grit. Thus large areas can be produced at the same time.[000101] In another embodiment of the invention not shown in a figure the angle of incidence of the two light beams which interfere at the surface can be changed and selected. To achieve this a scanner or SLM may be used in a pupil of the illumination optics. This pupil is than imaged with appropriate lenses over the split beam path to the surface of the chip generating gratings with variable grating modulation. This allows for generation of overlaying multiple grating structures subsequently or in the case of the use of an SLM also simultaneously. The use of an SLM or DLP (digital light processor, a MEMS device) also allows a generation of spatially separated sets of gratings arranged e.g. in an array. This makes the production of different kinds of chips versatile and easy. When an SLM or DLP is used different diffraction orders may be used to directly generate the two beams necessary to generate the interference at the sample surface. The use of overlapping gratings in multiplexed assays helps to reduce mass effects, since a larger surface is used for detection. This reduces the need of diffusion or flow in the chip.[000102] Background of the invention is the fact that the overall signal detected at the detector contains light from the molecules of interest arranged in the grid like structure (the ‘real signal’) and light from nonspecific bound molecules arranged statistically on the surface and straylight within the instrument (background signal). Both parts of the signal can be regarded as a sum over the light from all contributing molecules, whereas the ‘real signal’ adds with a well-defined phase and all other contributions add with a statistical distributed phase which generates speckles. These speckles change if the illumination amplitude distribution or polarization distribution with respect to position on the surface changes. [000103] In an embodiment of the invention the ‘real signal’ interferes at the detector (11) coherently at one focus point. This is achieved by focusing the light scattered from the surface with the focusing lens (07). The pupil is arranged at a position with respect to the surface that a diffraction order (typically the first order of diffraction) of the light scattered from the grid like structure is collected by the pupil. The spatio-temporal modulation now introduces in a simple embodiment a spatial varying intensity modulation which effects the distribution of light at the area of the grid like structure on the chip surface (02). In the easiest embodiment a transmission structure is inserted in the illumination beam path and varied. This can be done just by rotating a structured mask in the excitation beam path. The result is, that the signal light which is accumulated at a given time contains contributions of a subpopulation of the molecules of interest and also a subpopulation of the molecules contributing to the background signal and also contributions of the light reflected and scattered within the apparatus. All these contributions change over time in case a spatiotemporal modulation is applied but still maintain the focus of the focus on the detector. In the case the average transmission of the mask would be e.g. 30% the overall signal at the detector would be reduced to an average of 30%. The ‘real signal’ is still a sum over contributions with well-defined phases and would thus be in a first order consideration independent of the position of the mask. The background signal is also reduced to 30% but adds up to a speckle distribution which is changing with the position of the mask. This is also the case for the contribution of the light reflected and scattered within the apparatus. If the detector is temporarily integrating over all or a large enough entity of the time (i.e. positions of the mask) the speckle distribution will average to a spatially smooth signal at the detector. The integration can be either done directly on the detector by accumulating light for a long enough time or by adding several readouts of the detector. In case several readouts are taken correction on the individual data may be undertaken, do further increase the sensitivity. Among those might be shifting the data to correct for aberrations, means to deconvolve theintroduced artefacts generated by the modulation, normalization to the actual overall excitation and transmission. The final integration of the speckle-reduced signal can be performed over a moving interval to increase the temporal resolution. The interval might be synchronized to an overall modulation period of the mean used to spatio-temporal modulate the light. The background signal at the position where the ‘real signal’ occurs can be than estimated by an average of the signal over areas where only speckles are expected. The uncertainty of this estimation is much lower for the integrated signal. This holds especially in case that a substantial contribution of the signal originates from reflected and scattered light within the apparatus. This makes the ‘real signal’ distinguishable in space with respect to the background also in cases where the absolute signal of the background exceeds the ‘real signal’. Without the temporal integration the uncertainty of the determination of the background equals the modulation depth of the speckles, which is in the order of the speckle intensity itself. The sensitivity of the system would be limited by shot noise in case a complete constant background is achieved by introducing the spatio-temporal modulation and temporal integration.[000104] In a more precise description the spatial and temporal modulation M(x,y,t) might be a complex vectorial (for the two polarization directions) function multiplied to the field vector components of the wave S(x,y) in the pupil of the detector (or at the surface of the chip). The detector than detects the intensity derived from the Fourier transform of the modulated wave in the pupil which can be calculated as a convolution of the Fourier transform of the M with the Fourier transform of S for each vector component. The time dependent signal at the detector is thus smoothed out and spatially and temporarily modulated. It is thus important, that the Fourier transform of M contains enough low frequency components to keep the final signal still confined on the detector. The overall structure of the mask has to be large with respect to the pitch of the lines in the grating it is ideally in the order of the overall size of the grid structure itself and should not contain Fourier-components close to the Fourier-component of the grit structure. Thus simple macroscopic multiple holes, macroscopic gratings ideally approximately perpendicular to the grit and macroscopic random structures are suitable for the de-speckling of the background. The mask could also contain phase structures if a fraction of the molecules would be still illuminated an undisturbed phase. The mask may be in the excitation beam path close to or also apart from a position conjugated to the grid structure. It can be also in the detection beam path between the chip and the focusing lens or also after the focusing lens.[000105] The generation of the mask may be also done by means of an SLM of DLP device. A SLM might be also used to focus different subareas of the chip related to fractions of the aperture of the SLM to different areas of the detector and modulating the phase of the signal in the fractions of the SLM individually. This can be used to configure the system for different multiplexed applications and maintain the ability to de-speckle the background signal. A DLP may be sued in a similar way. The spatio-temporal modulation might be also just an introduction of a spatial distributed and modulated rotation of the polarization by either 0 or 90°. At a given fraction of the area of approximately 50% of the area the signal interference at the detector is split into two independent subsets generating two independently speckled background signals and ‘real signals’. This already reduces the overall modulation depth of the combined speckle background. The full amount of light is uses and further speckle reduction is achieved by modulating the subarea where the polarization is rotated. A polarization rotation might be introduced by an SLM like using liquid crystals of other masks introducing polarization rotation.[000106] The method to reduce the speckles on the detector might be combines with all other aspects describes in the different embodiments of the invention and added in various means as a person skilled in the art can easily recognize. It is also possible to introduce an angular modulation of the incident light in the case of a chip based on total internal reflection. This Modulation of the Angle results in a modulation of the position of the ‘real signal’ on the detector. In a simple embodiment not shown in a figure this modulation is readout with just a dual pad photodiode or position sensitive detector. A similar result would be obtained by inserting an oscillating mirror in the detection path. This could also be applied for chips with planar waveguide. The modulation of the difference or ratio of the two signals would be dominated by the ‘real signal’ especially in cased where the de-speckling of the background in the vicinity of the real signal is done at a higher frequency than the modulation of the angle of incidence. This is a method to get a very simple and sensitive device for point of care diagnostic or environmental or food analysis.[000107] There are two contributions to the speckles at the detector. A: scattering of light from unspecific bound statistically distributed molecules and B: scattering from surfaces of the apparatus and from the sensor chip. In a first step the contribution A is considered in a simplified presentation. The number of unspecific bound molecules shell be ‘n’. Each individual molecule contributes with a probability p = 0.5 constructively to any given spatial frequency transmitted through the optical system and with q = 1- p = 0.5 destructively. This is also true for the spatial frequency of the grid The expectation value of the electrical field atthis spatial frequency is thus 0.5 n and thus the expectation value sum of the electrical field over the constructive and destructive subset of molecules is zero. The electrical field is gaussian distributed for sufficiently large number of molecules n ( with a variance sigmaA2 = npq = 0.25n. The Intensity is proportional to the square of the electric field. Therefore the speckle amplitude scales with sqrt(npq). In the given case that ‘m’ would be the number of specifically bound molecules the contribution would be proportional to m since all electric field vectors add coherently. The overall detection limit thus limited at m >~ sqrt(npq) = 0.5 sqrt(n) This reflects the statistical contribution of molecules arbitrarily bound to the spatial frequency selected for the specific binding of the target molecules. In one embodiment of the invention not shown in a figure the detection limit is increased by changing the factor p towards larger or smaller values. As an example if p = 0.01 the detection limit would be m >~ sqrt(n*0.01*0.99) =~ 0.099 sqrt(n) which is already an increase of a factor of 5 in sensitivity. Reducing of increasing the probability p is achieved by narrowing of widening of the lines of the grid. This could be done by several means. Dedicated masks, multiphoton supported immobilization, saturation could be used. In case activation is saturated with an illumination pattern generated by an interference of two planar waves for a long time only small lines would not be covered in the vicinity of the lines where the electrical field of the interference is zero. This consideration holds only if the stronger confinement of the structure is taken into account in the readout system. This requires to read all added spatial frequencies i.e. additionally not only the first but a series of higher diffraction orders. These could be collected as a series of foci on the detector. Each focus represents one order of diffraction and has its own independent speckle background. The signal of all detected diffraction orders has to be accumulated to achieve the signal with increased sensitivity.[000108] In another embodiment not shown in a figure a different mean of speckle reduction is introduced. This can be combined with most of the embodiments described in this disclosure and addresses the light scattered within the apparatus In case that the incoming and outcoming beam light used to excite and detect the signal is approximately collimated a displacement of the chip does only effect absolute phases between incoming and outcoming beams but not relative phases within the scattered beam. Thus a displacement of the chip introduces phase differences between scattered with origin from the excitation beam path and scattered light with origin from the apparatus after emitted from the chip since the overall beam pathlength of the scattered light changes due to the displacement of the chip with respect to the apparatus. A random or vibrational displacement introduced for example by an piezo-transducer thus eliminates speckles from scattered light with origin from the apparatus.A pure displacement without any tilt of the chip preserves the position of the focus of the diffraction orders on the detector and thus increases the signal-to-noise ratio significantly in cases of a dominant contribution of light scattered from the apparatus. In another embodiment of the invention, enhancement of the signal to noise ratio of the device can be achieved by a combination of inline holographic phase retrieval of the speckle background (in the following denoted as background) and a model of the signal generated by the light illuminating the sensor chip with the binding sites (in the following denoted as signal) in combination with a nonlinear optimization procedure to extract the relative phase shift and intensity weight of background and signal.[000109] Inline holography makes use of the interference patterns generated by light transmitting the sample and light scattered by the sample [D. Gabor, A New Microscopic Principle, Nature, 161(4098):777-778, 1948], Hence, no reference beam is required. Several of these interference patterns assuring sufficient phase diversity are necessary for successful phase retrieval.[000110] In another embodiment of the invention, phase diversity is generated by placing the detector at different positions (11 and 33) along the optical axis relative to the focus (Fl) of the mologram (Figure 14, left) or defocussing the latter by an additional optical device (29) and using a fixed detector (33), see Figure 14, right.[000111] The complex signal (solid line) from the sensor chip (02) with binding sites (04) is superimposed by a complex speckle background (wavy dotted lines). An aperture (08) is placed within the beam path (optionally at different positions (33) inside and outside the focal plane (Fl)) filtering light scattered under high angles or truncating the field of view. A detector (11) is placed at different positions (33) along the optical axis. Intensities containing contributions from signal and background are measured. Alternatively, the movable detector can be replaced by a fixed one and a means to defocus the molograms (29). The movable (33) aperture (08) can be placed in the conjugated planes of the sensor chip (focal length Fl) and / or the conjugated planes of the device to defocus the molograms (focal length F2).[000112] Further, to allow reconstruction of the complex valued field, the (de-)focused molograms must sufficiently decay within the field of view. This is necessary for accurate numerical propagation and can be achieved by a pinhole aperture (08) placed inside or outside the focal plane or in the conjugated planes with respect to the focal planes of the sensor chip (02) with the binding sites (04) and / or the device to defocus the mologram (Fl / F2). The aperture filters high spatial frequency components of the light or truncates the field of view (see Figure 14).[000113] Phase retrieval can be carried out by a variant of the Gerchberg-Saxton algorithm designed in 1972 to determine phase information from measured intensities and the knowledge of light propagation [R. W. Gerchberg and W.O, Saxton. A Practical Algorithm for the Determination of Phase from Image and Diffraction Plane Pictures, OPTIK,35(2):237- 246, 1972],[000114] While the original Gerchberg-Saxton algorithm uses two intensity measurements, this is not sufficient for the problem at hand. In one embodiment reconstructed intensities are therefore iteratively adapted at several defocus positions until a common complex valued wave field is found that reproduces the measured intensities at each (de-)focus position. Figure 15 illustrates the phase retrieval process.[000115] A guess of the complex field to be reconstructed is propagated back and forth by optical near-field propagation (P) between the focal plane (F) and the defocus planes (Dl- DN) where intensities have been measured. At each position, the intensities of the guessed field are replaced by the measured intensities while phases are kept unconstrained. Finally, the complex valued speckle background (B) and the complex valued signal (S) are reconstructed and sketched within a dashed frame each.[000116] A numerical model of the complex wavefield of either signal (S) or background (B) is propagated back and forth (P) between the focal plane (F) and the defocus positions (Dl-DN) of the detector where intensities have been recorded. Reconstructed intensities are replaced by measured ones; phases are kept unconstrained.[000117] The described phase retrieval can performed at least for the speckle background , preferably for both background and the most dominant signal resulting from fully occupied binding sites. If due to performance issues only the background can be reconstructed, the signal must be modeled from the geometry of the scattering patterns on the sensor chip.[000118] In a further embodiment, a nonlinear optimization procedure illustrated in Figure 16 can be used to determine the position of the signal (S) within the background (B), the phase shift between signal and background and most importantly the relative scaling between background and signal intensities.[000119] The latter is a measure of the number of molecules binding to the diffracting structures. With this procedure the appearance of the signal from the background speckle pattern can be determined at very early stages even when background and signal are not yet distinguishable by pointwise comparison of the measured intensities.[000120] Here, a complex model of the background (B, dashed frame) and the signal (S, dashed frame), as well as a measured intensity distribution (M, solid frame) consisting of a superposition of background and signal with unknown relative scaling (Sc), positioning (O) and phase shift (P) are fed into an iterative (round arrow) nonlinear optimization framework, during which different combinations of signal and background are tested against the measurement until a best match is found. The corresponding parameters O, Sc and P are returned. The scaling parameter Sc describes the relative strength of signal and background and is related to the number of binding molecules.GLOSSARY TO THE FIGURES[000121] Structures & symbols01 Light for binding detection0T Light for chip surface activation02 Sensor chip (with waveguide)03 Line with binding structures04 Binding zone05 Beam path06 Aperture plate07 Lens or objective08 Pinhole aperture09 Microlens array10 Focus11 Detector12 Telecentric lens13 Grating13' Reference grating13" Spatial light modulator grating14 Light source for binding detection14' Light source for chip surface modulation15 Filter16 Mirror or reflective surface17 Beam dump18 Flow cell19 Light-protected room20 Volume-holographic grating21 Beam splitter22 Digital light processing (DLP) projector23 Glass slide with immersion liquid24 Binder24' Deactivated binder25 Analyte26 Variable mask or spatial light modulator27 Scanner28 Means to increase the optical path length29 Means to translate the focus relative to the detector30 Retarder plate31 Wedge prisms32 Image splitting prism33 Means to translate an element in the beam path

Claims

CLAIMS1. Method for detection the total mass of target molecules bound to a substrate, comprising providing a transparent substrate having binding sites on one surface, wherein the binding sites are capable of binding at least a part of the target molecules and coupling the light provided by a light source into the substrate, wherein at least a part of the light generates an evanescent field of light propagating along the surface provided with the binding sites thereby generating scattered light intensities characterized in providing the transparent substrate with a fluid comprising target molecules wherein at least a part of the target molecules bind to the binding sites and detecting the scattered light intensities of at least two different occupancies of the binding sites with target molecules, each under at least two different phase conditions and calculating the amplitude and phase information of the scattered light intensities by phase retrieval and correcting the scattered light intensities with the amplitude and phase information and calculating the total mass of the target molecules bound to the substrate from the corrected scattered light intensities.

2. Method according to claim 1 characterized in that at least two scattered light intensities are obtained at occupancies of the binding sites with the target molecules of 0 to 10 % and 80 to 100 %, respectively.

3. Method according to claim 1 or 2 characterized in that the least two different occupancies of the binding sites with target molecules are obtained by providing the transparent substrate at least two fluids provided with different concentrations of target molecules.

4. Method according to any of claims 1 to 3 characterized in providing the transparent substrate with a first fluid having a first concentration of target molecules and detecting first scattered light intensities under at least two different phase conditions and providing the transparent substrate with a second fluid having a second concentration of target molecules and detecting second scattered light intensities under at least two different phase conditions wherein the first concentration is between 0 and 50% of the second concentration and detecting the mass of the target molecules bound to the substrate by calculating amplitude andphase information by phase retrieval of the first and second scattered light intensities and calculating corrected scattered light intensities from the amplitude and phase information, thereby obtaining the mass of the target molecules bound to the substrate.

5. Method according to any of claims 1 to 4, characterized in that by calculating the the corrected scattered light intensities, the interactions kinetics between molecules and sensorgramms is obtained.

6. Method according to any of claims 1 to 5, characterized in that the at least two different phase conditions are obtained by providing means to change the optical path of light between the substrate and the at least one detector.

7. Method according to claim 6, characterized in that the means to change the optical path is selected from the group consisting of movable glass sheet, prism, , lens , lens-array , grating .

8. Method according to any of claims 1 to 7, characterized in that the optical path is changed by moving at least one detector ( 29 ) relative to the substrate surface.

9. Method according to any of claims 1 to 8, characterized in that the optical path is changed by splitting the scattered light intensities into at least two beams and detect them as first and second scattered light intensities separately.

10. Method according to any of claims 1 to 9, characterized in that the light provided by the light source into the substrate is coherent light.

11. Method according to any of claims 1 to 10, characterized in that the binding sites are provided in a non-stochastic distribution to the transparent substrate12. Method according to any of claims 1 to 11, characterized in that the non-stochastic distribution of the binding sites on the transparent substrate results in coherent scattered light intensities.

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