Optoelectronic chip

Abstract

Optoelectronic chip for receiving a sample for optical investigation, comprising a substrate layer, a thin-film optical fiber with an active region in which the sample interacts with a guided mode of the thin-film optical fiber, wherein at least one scattering structure is arranged in the active region which scatters the light guided in the thin-film optical fiber, thereby generating a reference light field, wherein the chip is designed to be used to detect individual particles with a diameter smaller than the excitation wavelength in solution, on a surface of the thin-film optical fiber or in thin films with respect to a reference signal and to detect their individual scattering cross-section and / or particle mass in a parallelized imaging modality with spatial and temporal resolution.

Description

[0001] Interferometric scattering microscopy (iSCAT) is a technique that utilizes the interference of light fields to detect particles with subwavelength dimensions. For this purpose, light elastically scattered by particles is superimposed with a reference light field and projected onto a detector, such as a camera, where it interferes. Spatially resolved detection of the interference contrast allows information to be extracted about the positions of the particles as well as their scattering cross-section. The latter is related to the polarizability of the particles, which depends on both their mass and their chemical composition.The signal interference between the light scattered by the particle and the reference light enables optical detection of particles smaller than 5 nm with high temporal and spatial resolution, which is difficult or impossible with other non-interferometric optical imaging approaches.

[0002] iSCAT measurements can thus directly provide information about the relative distribution of different particle masses or their scattering cross-section in a sample solution, without requiring modification of the particles under investigation, such as with fluorescent labeling. Additionally, the absolute or relative concentration of particles, interactions between identical or different particles, or individual components from a sample with unknown composition and their diffusion behavior can be determined, providing valuable information for biology and environmental science.

[0003] In practice, iSCAT microscopy uses high numerical aperture (NA) objectives in combination with an immersion medium. In such a setup, the excitation light, scattered light, and reference light follow the same optical path. The reference light is generated by reflection of the excitation light at the interface between the sample holder and the sample. Since excitation and detection cannot be treated separately in this approach, the intensity of the reference light cannot be optimized to achieve the best interference contrast.

[0004] In addition, considerable effort must be made to create a homogeneous illumination surface, which is essential for quantitative measurements with additional optoelectronic elements such as acousto-optic deflectors.

[0005] Another disadvantage of approaches based on high numerical aperture (NA) and high magnification lenses combined with an immersion medium is their high temperature sensitivity. This means that temperature changes of just a few degrees Celsius significantly affect image quality, making temperature-dynamic investigations difficult. Integrating the illumination path onto a single chip, along with an integrated heating element, creates a monolithic device that is insensitive to temperature changes and allows for the investigation of an extended temperature range, typically from 0° to 100°C.

[0006] The scattering cross-section of particles in the subwavelength range scales with their radius to the power of 6 (Rayleigh scattering). Detecting small particles, such as individual proteins, using scattering microscopy is therefore a very challenging experimental task. Interferometric scattering microscopy (iSCAT) helps to overcome these limitations, as the interference contrast generated by this approach is proportional to the particle volume. However, conventional iSCAT systems rely on objectives with high numerical aperture (NA) and high magnification, which are expensive, user-unfriendly, and restrict the effective field of view. Furthermore, investigating the dynamic temperature behavior is difficult because focus shifts are frequently observed.A monolithic waveguide chip that ensures local excitation near its surface (evanescent field) as well as the generation of a reference field without the need for immersion oil opens up new avenues for robust, user-friendly and highly sensitive detection of individual biomolecules over an extended temperature range.

[0007] Against this background, it is an objective of the present invention to mitigate or even completely eliminate the problems of the prior art.

[0008] In particular, the present invention is based on the objective of creating a device for carrying out interferometric scattered light microscopy which eliminates the need for an objective with a very high numerical aperture (>1) in combination with immersion medium and preferably also enables the sample to be reliably and quickly set to a desired temperature as well as the observation of larger fields of view of up to several mm. 2 This enables a larger field of view. It allows for the parallel investigation of different sample areas, which can also be physically separated from each other.

[0009] This problem is solved by an opto-electronic chip having the features of claim 1 and an optical system having the features of claim 11.

[0010] Advantageous embodiments of the present invention are the subject of the dependent claims.

[0011] The present invention overcomes disadvantages of the prior art because the excitation and detection paths are fundamentally separate. The illumination profile is defined by the mode profile of the guided mode and can be adjusted to generate a very homogeneously illuminated active area. This approach allows the use of low-magnification lenses (20x, 40x, 60x) to illuminate large areas of up to several millimeters. 2This allows observation with a resolution of less than 100 nm, and thus well below half the wavelength of the excitation light, without the need for an immersion medium. The advantage of capturing the scattered light and the reference beam in the common path is retained. Since the evanescent field of the waveguide mode penetrates only a highly selective region of approximately 100 nm of the sample volume, background signals present in conventional iSCAT experiments are suppressed, and the total optical power required for illumination is reduced to a minimum. Undesirable effects such as sample heating, light-induced protein degradation, or cellular phototoxicity are thereby mitigated.

[0012] An optoelectronic chip according to the invention for receiving a sample for optical examination comprises a substrate layer and a thin-film optical fiber with an active region in which the sample interacts with a guided mode of the thin-film optical fiber, wherein at least one scattering structure is arranged in the active region which scatters the light guided in the thin-film optical fiber, thereby generating a reference light field (also referred to as a reference beam). The terms "optical fiber" and "waveguide" are used below and are preferably to be understood as synonyms within the scope of this application.

[0013] Preferably, the scattering structure is regular and / or irregular and extends partially or completely over the active area. The scattering structure can be regularly structured in some sections and irregularly in others. Examples of scattering structures include: a) A spatially periodic modulation of the effective refractive index of the optical fiber by a 1D grating structure. This grating structure can be achieved by modulating the thickness of the optical fiber layer or other layers near the optical fiber. b) a spatially periodic modulation of the effective refractive index of the optical fiber mode by a 2D periodic structure. c) A spatially random modulation of the effective refractive index of the optical fiber mode by a. Surface roughness of the waveguide layer caused by the coating process or surface roughness of the support structure (typ. < 10 nm rms) b. distributed, for example dispersed, scattering centers, such as nanoparticles, in the optical fiber layer or one of the layers near the optical fiber, which lead to a random or periodic modulation of the effective mode index.

[0014] In other words, the scattering structure is preferably formed by regularly and / or irregularly varying the effective mode index of the waveguide within a predetermined region. For example, the mode index of the waveguide can be locally varied in a regular, periodically recurring pattern.

[0015] According to one embodiment of the invention, the scattering structure is designed as surface roughness.

[0016] A periodic modulation of the effective refractive index of the optical fiber mode leads to selective diffraction or scattering of the light guided in the waveguide in the direction of a detector, e.g. a detector of an optical system or microscope.

[0017] Randomly distributed scattering centers, such as surface roughness, lead to a non-directional generation of the reference field.

[0018] An optoelectronic chip according to the invention can have several light guides, which can be arranged next to each other and / or one above the other.

[0019] For example, a first optical fiber, also known as a measuring optical fiber, can be provided and interacts with a sample. Additionally, a second optical fiber, also known as a reference optical fiber, can be provided, which extracts a specific amount of the light from the guided mode of the measuring optical fiber and directs it to a coupling area.

[0020] The scattering structure is preferably provided or arranged on or in the measuring light guide. The intensity of the light guided in the measuring light guide can be detected or monitored by means of the reference light guide and, for example, controlled or regulated by an optical system or microscope based on the detection results.

[0021] Furthermore, it has proven advantageous in practice if an optoelectronic chip according to the invention is equipped with a coupling area for extracting a guided mode from the thin-film optical fiber. The latter serves to monitor the intensity of the guided mode. The light extracted via this coupling area is, for example, supplied to a sensor for intensity measurement via a reference optical fiber. Subsequently, the intensity of the light from the guided mode of the thin-film optical fiber can be controlled or regulated by means of a control device.

[0022] The reference waveguide extracts, for example, a portion of the guided light from the measurement waveguide via evanescent coupling to the measurement waveguide. Any other type of coupling, such as via a splitter, is also conceivable.

[0023] Preferably, the light extraction for the reference waveguide, viewed along the propagation direction of the light in the measurement waveguide, takes place upstream of the active sample region of the measurement waveguide. This ensures that the amount of light transmitted into the sample volume remains constant regardless of the sample volume. It can be advantageous to perform the light extraction in a region of the measurement waveguide where only one mode is supported by the waveguide.

[0024] The detection of light scattered from the reference arm or reference optical fiber can be performed using a photodetector. Electronic signal amplification allows the power of a light source, such as a laser diode, which feeds light into the measurement waveguide, to be controlled. This compensates for intensity fluctuations caused by environmental influences, mechanical vibrations, and movements that the light, for example in an optical fiber, may experience between the light source and the measurement waveguide.

[0025] An optoelectronic chip according to the invention can be used to receive a sample for optical examination, wherein a sample, preferably an at least partially liquid, solid or gel-like sample, is applied to the optoelectronic chip in such a way that the sample partially or completely surrounds the active area of ​​the thin-film optical fiber.

[0026] The sample preferably contains at least one or a plurality of particles which are capable and / or designed to interact with a guided mode of the thin-film optical fiber.

[0027] Another aspect of the present invention relates to an optical system designed to be used with an optoelectronic chip according to the invention, and designed to generate interference between the scattered light of at least one particle located in the sample space and the reference light generated by the scattering structure, i.e., the light deflected by the scattering structure.

[0028] Furthermore, an optical system according to the invention, which is designed to be used with an opto-electronic chip according to the invention, is designed to image the generated interference on a detector.

[0029] An optical system according to the invention comprises at least one light source for feeding light into the at least one thin-film optical fiber, preferably the measuring waveguide. If an optoelectronic chip according to the invention with several measuring waveguides is used, the optical system preferably comprises several light sources, each of which is assigned to a measuring waveguide. The light sources can emit light of the same or different wavelengths.

[0030] In other words, light from one or more light sources of the same or different wavelengths is directed onto the coupling areas of the waveguides, either via free-space optics or using optical fibers and micro-optics. This allows different or the same wavelengths to be coupled into different measurement waveguides, either simultaneously or with a time delay. Each measurement waveguide preferably has at least one reference waveguide, which enables the intensity of the guided light in the respective measurement waveguides to be measured and individually controlled via separate photodetectors.

[0031] The reference light and the scattered light, which is scattered orthogonally to the propagation direction in the waveguide in the active region, are preferably imaged onto a 2D array detector via optics, and the optical system is preferably a microscope.

[0032] An optoelectronic chip and / or an optical system according to the invention can be used, for example, to determine the antigen-antibody binding affinity, to investigate antibody-antibody cross-linking and / or multi-sided binding processes, to analyze protein-protein interactions, to estimate protein sizes, in the context of investigations into protein degradation and denaturation properties, and to optimize and characterize formulations.

[0033] In other words, the present application describes the technical details of an optical chip designed for use within an optical microscope to detect individual particles (e.g., antibodies, viruses, etc.) with a diameter smaller than the excitation wavelength in solution or thin films with respect to a reference signal and to detect their individual scattering cross-section and / or particle mass in a parallelized imaging modality with spatial and temporal resolution.

[0034] A key component is an optical or optoelectronic chip that incorporates a thin-film optical fiber (also called a waveguide). Within an active region of the waveguide, the supported waveguide mode can interact with nanoparticles near the waveguide's surface (evanescent field). The light scattered by the nanoparticles is collected by a lens system and directed onto a detector (e.g., a camera).

[0035] The scattering signal of the particles is amplified by utilizing an optical reference field that is generated on the chip near the position of the nanoparticle by means of a scattering structure.

[0036] Both the reference light (also called reference field or reference beam) generated by the scattering structure and the scattered light of the particles (also called scattering field) are collected with the same optics and detected on the same detector where they interfere.

[0037] The excitation light, required to generate the reference field and interact with the nanoparticles, preferably follows the same optical path to the detector (e.g., a camera). This ensures that the phase relationship between the scattering and reference fields remains independent of external influences, making the system robust. Spatially and temporally resolved detection of the interference pattern allows for the determination of time-dependent particle positions and their scattering cross-section.

[0038] The reference light field for interference is generated on the chip by defined (e.g. periodic or regular) or undefined (random structures, surface roughness) structures within and / or along the active area of ​​the waveguide structure.

[0039] The strength of the reference light field is selected to optimize the interference contrast on the detector, the signal-to-noise ratio and / or the maintenance of a propagation mode within the waveguide chip.

[0040] The light intensity of the reference light field can be adjusted, in conjunction with the intensity of the mode guided in the waveguide, by the type of scattering structure, so that the resulting interference signal enables optimal localization of the particle under analysis in all three spatial directions at any given time. Parameters that determine the ideal strength of the reference field include, for example, the wavelength of the light, the scattering cross-section of the particle, the integration time of the detector, the signal strength at the detector, shot noise, and the diffusion velocity or residence time of the particle.

[0041] The average signal detected by the detector is several orders of magnitude higher than the scattering signal of the nanoparticle alone due to the combined detection of the scattered light from the particles and the reference light from the scattering structures, which increases the contrast, reduces the detection time and therefore also enables the detection of fast-moving, small particles (< 5 nm).

[0042] The measurement can be performed with multiple wavelengths to increase precision and to avoid absorption in the medium / particle and / or can be combined with a fluorescence detection channel.

[0043] The scattering cross-section is a function of the wavelength. Shorter wavelengths have the advantage that the scattering cross-section is increased for a constant particle size, thus leading to a stronger signal. At the same time, different wavelengths exhibit different penetration depths into the sample volume, so that the axial position of the particle can also be determined by a wavelength-dependent measurement.

[0044] An optoelectronic chip according to the invention serves, for example, to hold a sample when visualizing temperature-dependent processes and can generally be regarded as a microscope slide.

[0045] An optoelectronic chip according to the invention, in an embodiment optimized for the visualization of temperature-sensitive processes, preferably comprises a carrier layer, a light guide (hereinafter also referred to as a waveguide), preferably a thin-film light guide, and a heating element, preferably a thin-film heating element, wherein the light guide and the heating element are preferably arranged on opposite sides of the carrier layer.

[0046] When the term thin-film optical fiber is used, it should be understood that this reflects only a preferred embodiment and that other optical fibers are also encompassed by the invention. When the term thin-film heating element is used, it should be understood that this reflects only a preferred embodiment and that other heating elements are also encompassed by the invention.

[0047] The heating element and / or the light guide is / are preferably optically transparent. In a chip according to the present invention, such a heating element is optional.

[0048] Optically transparent material is preferably more transparent to light in the visible spectrum, with the transmission of light through the optically transparent material preferably being at least 0.5, and in particular at least 0.8. Optically opaque material is preferably more opaque to light in the visible spectrum, with the transmission of light through the optically opaque material preferably being a maximum of 0.49, and in particular a maximum of 0.3.

[0049] The light guide and / or the heating element can be located directly on a surface of the substrate layer or spaced away from it by one or more intermediate layers.

[0050] Furthermore, the light guide and / or the heating element and / or the carrier layer can each be designed as a single layer or as a composite of two or more sub-layers.

[0051] Preferably, the support layer consists entirely or at least partially of an opaque or transparent material, preferably Si or a SiO2-based glass or crystal.

[0052] The support layer therefore consists, for example, of glass, in particular borosilicate glass, and is preferably designed to provide mechanical stability to the opto-electronic chip.

[0053] Furthermore, another transparent layer can be located between the support layer and the thin-film waveguide, which has a lower refractive index than the support layer, preferably a refractive index between 1.0 and 1.5.

[0054] According to one embodiment of the invention, the support layer is made entirely or at least partially of a semiconductor material, preferably SiO2, and preferably a transparent layer, in particular a separating layer, is further provided between the support layer and the light guide, preferably the thin-film light guide.

[0055] Furthermore, the thin-film heating element is preferably connected and / or equipped with a temperature sensor, preferably in the form of a thin-film temperature sensor, which is designed to come into direct or indirect contact with a sample.

[0056] For example, the temperature sensor can include a sensor layer for detecting the temperature of the sample, which preferably comprises metal and / or consists of metal and preferably at least partially covers an outer surface of the optoelectronic chip and is further preferably designed to come into contact with a sample. The temperature sensor can be in direct or indirect contact with the sample.

[0057] Preferably, the temperature is measured using the temperature sensor at at least one location in the sample, preferably at a plurality of locations, in order to obtain a more reliable measurement value.

[0058] Preferably, a four-wire measurement is used as part of the temperature sensor.

[0059] Another option is to determine the temperature from a distance using an infrared sensor.

[0060] Furthermore, the opto-electronic chip preferably includes a control unit to control and / or regulate the thin-film heating element based on the measurement data regarding the sample temperature acquired by means of the temperature sensor.

[0061] Preferably, a thin-film heating element used within the scope of the invention is a resistance heating element or comprises one. For example, carbon nanotubes can also be used within the heating element.

[0062] In order to ensure that particles and / or objects and / or molecules under investigation can be localized close to a surface of the optoelectronic chip and thus in the range of evanescent waves, it has proven advantageous in practice if an outer surface of the optoelectronic chip, which is designed to come into contact with the sample, has at least partially or completely a surface modification, a surface functionalization or the possibility of surface functionalization to bind molecules (or other particles and / or objects) contained in the sample, in particular biological molecules.

[0063] Surface functionalization can, for example, involve adding specific functional chemical groups, such as hydroxyl groups, to the surface in order to selectively bind a desired class of molecules to the surface.

[0064] Furthermore, the present invention relates to the use of an optoelectronic chip according to the invention for receiving a sample during the visualization of temperature-dependent processes, wherein a sample, preferably an at least partially liquid, solid, or gel-like sample, is applied to the optoelectronic chip such that the sample partially or completely covers the thin-film optical fiber and preferably also the sensor layer of the temperature sensor. A chip according to the invention can also be used with a microfluidic system.

[0065] For example, an optoelectronic chip according to the invention can be used to observe a temperature-sensitive process at a precisely controlled sample temperature. Furthermore, an optoelectronic chip according to the invention can be used to investigate the temperature dependence of a process by observing the process at different precisely controlled sample temperatures.

[0066] The sample used in the present invention preferably contains at least one or more particles and / or objects and / or molecules which are capable and / or designed to interact with a guided mode (also referred to as mode) of the thin-film optical fiber. For example, the molecules are excited to fluoresce by the light guided or directed by the optical fiber, deflect this light, and / or absorb the light.

[0067] Another aspect of the invention relates to an optical system, preferably a microscope, particularly preferably a TIR microscope, which is designed to be used with an optoelectronic chip according to the invention.

[0068] An optical system according to the invention preferably has at least one emitter which sends light into the thin-film waveguide for optical excitation and at least one detector which detects light deflected by the sample and / or emitted normal to the plane of the thin-film waveguide.

[0069] This setup physically separates the light paths for sample excitation and light detection, thereby reducing general stray light generated when coupling the light into or guiding it within the waveguide, as well as stray light resulting from local scattering by the sample and background light. This leads to an improved ratio between the desired detected signals from the sample and unwanted signals caused by the measurement setup.

[0070] To guide light in a mode typical for the optical waveguide, coupling modules such as grating couplers, prism couplers, and / or direct coupling mechanisms between two optical waveguides are preferably used. These coupling modules serve to introduce external light into the waveguide. More efficient coupling modules can reduce the overall scattering background.

[0071] One or more optical fibers (measuring waveguides) direct the light onto or through an optoelectronic chip according to a preferred embodiment and thus also through the volume of the sample.

[0072] The light guided by the measuring waveguide can be reflected back and / or coupled out. For this purpose, coupling modules such as grating couplers, prism couplers, and / or direct coupling mechanisms between two optical fibers are preferably used.

[0073] It is also conceivable that additional coupling modules could be used to guide an optical mode of a different wavelength, different optical modes of the same wavelength, or a combination thereof, simultaneously propagating through the waveguide. The interaction of these modes within the waveguide and their detection could be used for highly sensitive measurements of the refractive index on the chip surface.

[0074] The temporal analysis of the transmission or reflection signal of one or more measuring waveguides can be used in addition to correlation measurements and fluctuation measurements (similar to dynamic light scattering - DLS).

[0075] Preferably, in an optical system according to the invention, the detector is an array detector and / or the optical system is a microscope.

[0076] Furthermore, the invention relates to the use of an optoelectronic chip and / or an optical system according to the invention for determining a phase transition of a particle (organic or inorganic) or a spatially extended material contained in the sample. This phase transition can, for example, involve the change of a biological molecule, such as an enzyme, a protein, or deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0077] Another aspect of the invention relates to the use of an opto-electronic chip and / or an optical system according to the invention in the context of high-throughput sequencing, preferably based on the analysis of individual molecules.

[0078] Another aspect of the invention relates to the use of an optoelectronic chip and / or an optical system according to the invention for investigating the binding affinities between at least one protein and at least one antibody as a function of temperature and / or other external stimuli, such as salt or buffer concentrations. Furthermore, the invention relates to the use of an optoelectronic chip and / or an optical system according to the invention for investigating living cells under temperature-controlled conditions and their interactions with individual particles. Interactions between proteins and / or interactions between proteins and small molecules can also be investigated using an optoelectronic chip and / or an optical system according to the invention.

[0079] In summary, the present invention offers at least the following advantages:

[0080] An optical microscope can be created that incorporates an optical chip according to the invention, which amplifies the scattering signal of small particles such as viruses, proteins, and other nanoparticles located in the evanescent field of the waveguide. This is achieved by generating a reference light path that interferes with the scattered light originating from the analytes in the sample at the detector. When the interference signal is detected, for example, by a camera, small biomolecules with a low mass (<500 kDa) or a small radius of less than 5 nm can be detected.

[0081] A chip according to the invention is particularly user-friendly and compact due to its monolithic chip design, in particular its compact and robust design, for the evanescent excitation of scattering particles (nanoparticles) and for the generation of reference light.

[0082] Furthermore, a chip according to the invention is suitable for generating a homogeneous sample illumination area (active area) and a reference beam.

[0083] Furthermore, a chip according to the invention can be used in combination with an optical system that projects the scattered and the reference beam onto a detector, where the interference signal is analyzed with temporal and spatial resolution.

[0084] Furthermore, a chip according to the invention enables highly selective excitation of a small sample volume above the waveguide in the axial direction (evanescent field, typically 100 nm above the waveguide) and over up to several mm 2 at the sample level. The optical chip can contain multiple or one measuring waveguide with different active areas and can be used in combination with fluorescence measurements.

[0085] Decoupling the excitation beam path from the detection beam path enables particularly clean optical detection using a chip according to the invention.

[0086] The use of a chip according to the invention also enables the use of lenses with low magnification (e.g. 20x, 40x, 60x) for imaging large areas up to several mm. 2 , without the need for the use of immersion oil, but it remains possible.

[0087] Furthermore, the reference beam was optimized for optimal interference contrast generation within a chip according to the invention. Additionally, a chip according to the invention enables dynamic temperature studies over an extended temperature range (preferably from 0°C to 100°C).

[0088] Furthermore, within the scope of the present invention, the spatial resolution is maintained down to the diffraction limit and / or even below (super resolution).

[0089] Furthermore, the present invention is preferably characterized by at least one or more of the following technical features: Preferably, a chip according to the invention comprises a thin-film waveguide with a thickness of preferably less than 5 µm (typ. <300 nm, in particular between 30-300 nm) on a transparent (e.g. SiO2) or non-transparent material (e.g. Si), the latter must be combined with a transparent separating layer (e.g. as an SOI system).

[0090] Preferably, a chip according to the invention further comprises at least one scattering structure within the active region of the waveguide, which generates a reference light field that enables interference with the light scattered by the particle on a detector. Furthermore, a chip according to the invention comprises an active region of the optical waveguide in which the sample volume can interact with the evanescent field of the guided mode.

[0091] It is also possible to integrate multiple measuring waveguides with separate active areas on one chip.

[0092] Furthermore, a collecting optic or detection optic is preferably provided to direct the scattered and reference light fields onto a detector (e.g. camera).

[0093] Preferably, the emitted and scattered light from the active area is detected using a 2D array detector (e.g., camera) that detects the light via the collecting optics perpendicular to the waveguide plane.

[0094] In a chip according to the invention, coupling modules for coupling free-space light modes into the supported waveguide mode and / or output coupling modules are preferably provided, which enable active intensity feedback of the light guided in the waveguide.

[0095] A chip according to the invention can be designed to accommodate a sample volume in the range of 0.1 µL up to several hundred µL (e.g. microfluidic channels).

[0096] Optionally, simultaneous detection of fluorescence is carried out via a separate detection path or at different wavelengths in the same detection path alongside interference microscopy, in particular interferometric scattering microscopy (iSCAT).

[0097] If a chip according to the invention is equipped with a heating element, this enables local and direct heating and thus the utilization of rapid temperature dynamics. This results in high temperature stability and high heating and cooling rates of up to 100 K / s.

[0098] Furthermore, within the scope of the present invention, optical excitation of parts of the sample volume can be provided via a free-jet optic.

[0099] Optionally, optical or electrical manipulation of the sample (e.g. by a laser trap, electrostatic trap, etc.) can also be provided within the scope of the present invention.

[0100] Preferably, the present invention utilizes at least one or more of the following physical effects: Interference: A small scattering signal from nanoparticles is amplified by interference between scattered light from the particle and reference light generated on the chip. The interference signal is detected by a photodetector (e.g., a camera). Spatial and temporal information about the positions of the particles and their scattering cross-section is extracted from this signal.

[0101] Fixed phase relationship: The on-chip generation of reference fields and their imaging via a common collecting optic ensures a fixed phase relationship between the light scattered by the particles and the reference field.

[0102] Evanescent excitation field: Preferably, scatterers / absorbers / emitters are excited in the optical near field of a thin-film waveguide. A highly confined and well-defined excitation volume is preferably created by the evanescent wave generated at the waveguide surface (penetration depth into the sample volume approximately 100 nm). The decay of the evanescent wave in the direction normal to the propagation of the waveguide mode can be smaller than the free-space wavelength of the excitation light.

[0103] Further advantages, features, and effects of the present invention will become apparent from the following description of preferred embodiments with reference to the figures, in which identical or similar components are designated by the same reference numerals. Here, the figures show: Fig. 1 a schematic representation of the layer structure of a chip according to the invention; Fig. 2 an opto-electronic chip according to the invention with a representation of the light coupling interfaces and the interfaces required for the measurement of light intensity in the waveguide; Fig. 2a an optoelectronic chip according to the invention with a mode purification structure; Fig. Figure 3 shows the principle of interferometric on-chip detection as carried out within the scope of the present invention. Fig. Figure 3a also shows a scattering structure for generating a reference light field; and Fig. Figure 4 shows the construction of an optical system according to the invention, in particular a microscope.

[0104] Fig. Figure 1 shows an optoelectronic chip 1 according to the invention, which has up to seven layers (L1-L7). In this embodiment, preferably all layers have a surface roughness of less than 5 nm RMS. All layers can be structured independently of one another in the substrate plane, e.g., with grating couplers for diffracting the incident free beam into one of the guided modes. The waveguide layer L5 can, for example, be chemically functionalized for the specific binding of biomolecules.

[0105] Layer L1 has a support material, in particular a transparent glass substrate (e.g. borosilicate, quartz glass, etc.) with a thickness between 50 and 1000 µm and a refractive index of n. sup or a semiconductor material (e.g. Si) in combination with a transparent separating layer on or consists of these.

[0106] Optionally, a transparent layer L2 can be provided as a separating layer, wherein the layer L2 has a refractive index of nsp1, preferably n sp1 <n wg The L2 layer can be composed of several sublayers.

[0107] Alternatively or additionally, an optional transparent layer L3 can be provided as a separating layer, wherein the layer L3 has a refractive index of n sp2 exhibits, where n sp2 <n wg The L3 layer can be composed of several sublayers.

[0108] To integrate a thin-film resistance temperature sensor, an additional layer L4, preferably a metal layer, can be applied either to the separating layer L2, the separating layer L3, or to the substrate of the support layer L1. The layer L4 can consist of metallic sublayers. The layer L4 preferably extends only over a portion of the adjacent layers, in this example, layers L1 and L4.

[0109] Layer L5 has or consists of a waveguide. Layer L5 serves as a high-refractive-index, non-absorbing layer with a refractive index of preferably n. wg >n sup The layer thickness is preferably between 30 and 600 nm. Layer L5 preferably comprises or consists of materials such as TiO2, Ta2O5, Al2O3, Nb2O5, Si3N4, GaP, ZrO2, SiO2, etc. Layer L5 can consist of several sublayers of different materials.

[0110] Optionally, layer L6 can be provided as a heating element. Layer L6 is preferably a transparent conductive layer with a thickness of 1 nm to 100 nm and is designed as a resistance heater, thus preferably comprising materials such as ITO, carbon nanotubes, etc. for resistance heating.

[0111] Layer L7 represents the sample volume. This volume contains particles that interact with the guided mode of the waveguide layer. The sample can be liquid, solid, or gel-like and preferably partially or completely surrounds the waveguide.

[0112] The functionality of an opto-electronic chip according to the invention is described below with reference to the illustration in Fig. 2 explained.

[0113] In Fig. Figure 2 shows a top view of an opto-electronic chip 1 according to the invention, in which a waveguide structure 2 and an active area 3 of the chip 1 are visible.

[0114] At the in Fig. The chip shown in Figure 2 is a substrate material coated on one side with a structured waveguide layer that supports single or multiple waveguide modes with a significant power ratio outside the waveguide layer itself (>1%). Within the active region 3 of the waveguide 2, the guided light can be scattered, absorbed, and / or re-emitted by particles within the sample volume. A reference light field is generated within the active region 3 of the waveguide 2 by selectively or non-selectively coupling portions of the guided mode toward the collecting optics (also referred to as detection optics). The waveguide (light guide) 2 corresponds to a measurement waveguide (measurement light guide).

[0115] This can be achieved, for example, by introducing a specific surface roughness or a periodic structure (e.g., a grid) within the active area 3, preferably within layers L1, L2 and / or L5, which serve as a scattering structure and generate a reference light field.

[0116] The scattering structure preferably overlaps spatially with the active area, especially when viewed along the light used as a reference beam.

[0117] The amount of light coupled into the reference beam path is preferably chosen such that the interference signal of the nanoparticles on the detector is optimized with respect to contrast, signal-to-noise ratio and shot noise for a given integration time of the detector.

[0118] The width of a waveguide of an optoelectronic chip according to the invention (for example, the dimension from the upper edge of the waveguide 2 or 9 to the lower edge of the waveguide 2 or 9 in Fig. 2) 2 is preferably between 100 nm and 1000 µm.

[0119] The dimensions of a chip 1 according to the invention are preferably 30 x 20 mm. It has proven advantageous if a chip 1 according to the invention is smaller than 50 mm x 50 mm and larger than 5 mm x 5 mm.

[0120] The on / off coupling of waveguide modes in the chip 1 according to the invention. Fig. 2 preferably as follows: Coupling regions 4 enable the coupling of free-space modes into and out of the waveguide mode. The chip 1 can contain one or more coupling regions 4 as well as one or more waveguides 2. One coupling region 4 is preferably provided to couple in a waveguide mode.

[0121] An additional coupling region can be used as a reference coupling region 5 to re-couple a specific fraction of the guided light into free-space modes, thus monitoring the light intensity propagating within the guided mode. In other words, the reference coupling region 5 selectively couples light out of the waveguide 2 to monitor the light intensity. A reference waveguide (reference optical fiber) 9 is provided for this purpose. The coupled-out light can be used to stabilize the intensity within the guided mode either before or after the active area.

[0122] Fig. Figure 2a shows an optoelectronic chip according to the invention with a mode purification structure. Mode purification can be achieved via a single-mode taper. Here, the guided mode in the measuring waveguide can be purified by an adiabatic transition 11 into the single-mode regime, so that multi-modal interferences are avoided and homogeneous sample illumination can be ensured. Reference numeral 10 designates in Fig. 2a a single-mode region in which a single mode is present. After conversion to the single-mode regime, the measurement waveguide 2 can be adiabatically expanded again by an adiabatic transition 11, so that a sample area from a few hundred µm² to several mm² can be excited. The single-mode region 10 can simultaneously be used, for example via evanescent coupling, to extract a specific amount of light from the waveguide for intensity monitoring using a reference waveguide 9.

[0123] The detection of particles in the sample volume is schematically shown in Fig. 3 shown and preferably carried out as follows: Particles 3 in the sample volume L7 and in the immediate vicinity of the waveguide 2 (layer L5) of the chip 1, i.e. within the evanescent field 4 of the waveguide 2, can interact with the propagation mode.

[0124] The light 5 scattered by the particles 3, as well as any fluorescence signals of the particles in combination with the reference beam or the reference light 6 generated by the scattering structure, is collected with optical elements (e.g. a lens 7) in an orthogonal direction to the propagation direction of the waveguide 2 on one or both sides of the waveguide (for example above and below the chip).

[0125] The signals are then projected onto a detector, e.g., camera 8, where the coherent signals, in particular the light scattered by the particles 5 and the reference light 6, interfere. The optionally still present fluorescence signal can be separated with optical filters and simultaneously projected onto another detector (not shown here).

[0126] In Fig. Figure 3a shows a scattering structure 12, which is arranged on the surface of the waveguide 2 (measuring waveguide) and generates the reference light field 6. The scattering structure is, for example, embedded in the waveguide 2 and can be created by surface etching of the waveguide 2. In principle, the scattering structure can be formed by applying and / or introducing surface modifications onto or into a surface of the waveguide 2, e.g., in the form of tiny protrusions or recesses.

[0127] Optionally, an optically transparent heating element can be used to control the chip's temperature via a resistance heater. Furthermore, an additional temperature sensor could be integrated into the waveguide structure to provide direct temperature feedback. This embodiment is particularly advantageous when monitoring temperature-sensitive processes.

[0128] The probability of finding particles within the excitation volume can be based on Brownian motion, convection, gravity, or determined via a specific or non-specific interaction potential caused by special surface properties (e.g., coatings, functionalizations, etc.) or external optical or electrical forces.

[0129] On a chip according to the invention, preferably one or more of the following functional elements are arranged: Waveguide input and output coupling structures: The waveguide mode is excited via coupling structures or coupling modules such as grating couplers, prism couplers, or direct fiber coupling mechanisms. The waveguide mode is preferably transmitted across the chip, including the sample volume. The transmitted mode can be back-reflected or output using similar arrangements to the input coupling module. A waveguide mode propagating simultaneously or separately in another direction can be coupled in using additional coupling modules.

[0130] Preferably, a special output coupling structure for measuring the intensity of the guided light is implemented in the waveguide, allowing the light intensity guided in the waveguide to be monitored in interaction with the sample volume. This intensity reference can be detected via a photosensitive element and used for active intensity stabilization.

[0131] The intensity of the measuring waveguide can also be detected in transmission and used for autocorrelation measurements similar to dynamic light scattering (DLS).

[0132] In the active region of the chip, the sample volume is positioned close to the waveguide. Particles within the sample volume that interact with the guided light (evanescent field) generate fluorescence and / or scattered light. A specific structure within this region creates a reference light field that can also be detected by the detection system and that allows interference with the light scattered by the nanoparticles in the sample volume.

[0133] Furthermore, a heating element can be provided in a chip according to the invention. The heating element preferably consists of, or comprises, an optically transparent, conductive thin film (e.g., ITO). Heat is generated, for example, by applying a direct current, and this heat is transported through the substrate material into the sample volume. The heating element is preferably localized to the sample volume. Metallic electrodes, for example, allow connection to an external electronic heating circuit.

[0134] A temperature sensor may also be present. Temperature measurement for the heating circuit can be implemented, in particular, with a thin-film resistance temperature sensor (e.g., a Pt sensor) integrated on the chip. The temperature sensor is preferably read via a four-point measurement. The sensor is preferably positioned between the heating element and the sample volume or on the top surface or on a side of the waveguide layer facing away from the substrate (separators are required).

[0135] Fig. Figure 4 schematically shows the structure of an optical system according to the invention, preferably a microscope. The microscope has at least one light source 13 which feeds light into a measuring waveguide 2 of the optoelectronic chip 1. The intensity of the light guided into the measuring waveguide 2 is detected by means of two photodetectors 14, for example by coupling the light out of the measuring waveguide 2 via a reference waveguide 9 and feeding it to a photodetector. The reference numeral 16 in Fig. The optical signals designated as 4 can be transmitted as a free beam and / or, for example, in fibers.

[0136] Both the light source 13 and the photodetectors 14 are connected to a control unit 15, which can control or regulate the light source 13, for example, based on the light intensities detected by the photodetectors 14. For this purpose, the control unit 15 is connected to the light source 13 and the photodetectors 14 via bidirectional data lines 17. The camera 8 or imaging optics are also connected to the control unit via bidirectional data lines 17.

[0137] The present invention is particularly useful for detecting individual particles and analyzing particle dynamics. Exemplary applications include: determining antigen-antibody binding affinity, antibody-antibody cross-linking and multi-sided binding processes, analyzing protein-protein interactions, estimating protein sizes (hydrodynamic radius), investigating protein degradation and denaturation properties, and optimizing and characterizing formulations (e.g., adeno-associated virus (AAV) vectors, nanoparticles, etc.).

Claims

[1] Opto-electronic chip for receiving a sample for optical examination, comprising a support layer, a thin-film optical fiber with an active region in which the sample interacts with a guided mode of the thin-film optical fiber, wherein at least one scattering structure is arranged in the active region which scatters the light guided in the thin-film optical fiber, thereby generating a reference light field. [2] Opto-electronic chip according to claim 1, wherein the scattering structure is regular or irregular and extends partially or completely over the active area. [3] Opto-electronic chip according to claim 1 or 2, wherein the scattering structure comprises local variations in the effective refractive index of the thin-film optical fiber or of a layer interacting with the guided mode. [4] Opto-electronic chip according to one of the preceding claims, wherein the reference light field is generated by light-scattering particles that interact with the mode guided in the light guide layer. [5] Optoelectronic chip according to any one of the preceding claims, characterized by that the thin-film waveguide further comprises a mode-cleaning structure which preferably limits the light guided in the thin-film waveguide to a single mode at least section by means of at least one adiabatic junction. [6] Opto-electronic chip according to one of the preceding claims, comprising at least two coupling areas, one for coupling the mode guided in the thin-film optical fiber and one for coupling a part of the mode guided in the thin-film optical fiber out of the thin-film optical fiber for monitoring the intensity of the guided mode. [7] Opto-electronic chip according to claim 6, characterized by , that the coupling area for coupling out a part of the mode guided in the thin-film optical fiber from the thin-film optical fiber for monitoring the intensity of the guided mode runs at least sectionally adjacent to the structure for mode purification, so that light from the thin-film optical fiber can preferably be transferred to a reference optical fiber for monitoring the intensity of the guided mode in the thin-film optical fiber by means of a preferably evanescent coupling. [8] Opto-electronic chip according to one of the preceding claims, which in addition to the thin-film waveguide includes an optically transparent thin-film heating element. [9] Use of an optoelectronic chip according to one of the preceding claims for receiving a sample for optical examination, wherein a sample, preferably a sample that is at least partially liquid, solid or gel-like, is applied to the optoelectronic chip in such a way that the sample partially or completely surrounds the active area of ​​the thin-film optical fiber. [10] Use according to claim 9, characterized in that the sample contains at least one or a plurality of particles which are capable and / or designed to interact with a guided mode of the thin-film optical fiber. [11] Optical system designed to be used with an optoelectronic chip according to any one of claims 1 to 8 above, and designed to generate interference between scattered light from a particle located in the sample space and reference light generated by the scattering structure. [12] Optical system according to claim 11, which is further designed to detect the generated interference by means of a detector. [13] Optical system according to claim 12, characterized by that the detector is an array detector and / or the optical system is a microscope. [14] Optical system according to one of claims 11 to 13, further comprising a light source designed to introduce light into a thin-film optical fiber of an optoelectronic chip, so that a sample taken up in an active region of the thin-film optical fiber can interact with the light. [15] Use of an opto-electronic chip according to any one of claims 1 to 8 and / or an optical system according to any one of claims 11 to 14 for determining antigen-antibody binding affinity, investigating antibody-antibody cross-linking and / or multi-sided binding processes, analyzing protein-protein interactions, estimating protein sizes, investigating protein degradation and denaturation properties, and optimizing and characterizing formulations.

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