Method and apparatus for label-free detection of analytes

Abstract

The present invention relates to a method and device for label-free detection of an analyte in a fluid. At least one dielectric microsensor is used, which comprises a microresonator and an adsorption layer for binding the analyte, which is applied to the microresonator. The microresonator consists of particles comprising a dielectric material and a fluorescent marker. Furthermore, the microresonator has an optical refractive index higher than the optical refractive index of the fluid to be analyzed. The microresonator is suitable for forming a plurality of resonant modes when the fluorescent marker is excited therein. An optical thickness of the adsorption layer of the microsensor is determined from the spectral positions of at least two detected optical resonant modes of the microsensor, which thickness is used to determine the extent to which the analyte is bound to the at least one microsensor.

Description

Technical Field

[0004] , , , fluid

[0001] In a fluid Analytes A method and apparatus for label-free detection are introduced.

Background Art

[0002] At least one dielectric microsensor is used and is applied to a microresonator to couple with , analyte and has adsorption layer The microresonator is composed of particles having a dielectric material and a fluorescent marker. Further, the microresonator has an optical refractive index greater than that of the fluid to be analyzed. The microresonator is suitable for enabling a plurality of resonance modes to be characterized when the fluorescent marker is excited inside it. The optical thickness of the adsorption layer of the microsensor is determined from the spectral positions of at least two detected optical resonance modes of the microsensor , analyte The degree to which is coupled to at least one microsensor will be determined hereafter.

[0003] Whispering gallery mode (WGM)-based sensors are suitable for determining physical, chemical, and biochemical parameters of fluids. Fluorescent microparticles with a diameter of several micrometers are used as microscopic optical sensors. The microparticles and their surfaces can be appropriately adjusted to meet a specific task of interest, for example, by specifically functionalizing their surfaces using a coated biochemical layer (M. Himmel-haus, Microsensors on the Fly, Optik & Photonik, 2016, Vol.11, pages 43-47).

[0004] When the fluorescence in the microparticle is excited, the dye emits light with a wavelength longer than that of the excitation light. Since this light is emitted in an arbitrary spatial direction, the refractive index of the microparticle is the surrounding fluidIf the refractive index is greater than that of the microparticle, light may be incident obliquely on the wall of the microparticle and undergo total internal reflection within the microparticle (i.e., inside the microparticle). Depending on the diameter of the microparticle, the microparticle becomes a light cavity of a certain size, which is filled with individual wavelengths of a spectrally broadband fluorescence spectrum in the form of resonant modes. The spectrum emitted from the microparticle to the outside is characterized by these modes.

[0005] The optical diameter of the fine particles is, for example, that of the analyte. (Analyte When the bonding (or adhesion) to the outer surface of the microparticles changes, the characteristic spectrum of the microparticles also changes. By analyzing the characteristic spectrum of the microparticles using spectral analysis, the optically effective diameter of the microparticles, and indirectly, the Analytes We can draw conclusions about the surface occupancy rate. Fine particles suitable for this procedure will also be referred to as "microresonators" below.

[0006] Microresonators are physically and / or chemically coated or bonded to the surface of the microresonator, and have functions suitable for each application of the microresonator. adsorption layer It can be wrapped in a material. For example, if a microresonator is used as a biosensor, this can be an adsorption layer (e.g., an organic layer) suitable for specifically binding to a particular analyte. . adsorption layer Because it is typically chemically different from the material of the microresonator, it can have a different optical refractive index than the substrate of the microresonator. and adsorption layer It is also called a "microsensor".

[0007] When biological substances (such as proteins, antibodies, peptides, oligonucleotides, DNA, RNA, viruses, bacteria, and / or their components) adhere to the surface of a microsensor, the optical diameter of the microsensor typically changes by only a few nanometers. The diameter tolerance associated with the manufacture of the microresonator alone is at least on the order of 50 nm (standard deviation for commercially available microparticles: 1-5%), and is therefore already considerably larger than the change expected due to the adsorption of biomolecules.

[0008] Therefore, conventional methods for quantifying the surface occupancy of each microresonator required that the spectrum determined after adsorption always refer to the spectrum from the same microresonator before adsorption; that is, the microresonator , analyte Measurements had to be taken at least once before and after coupling (see, e.g., WO 02 / 13337 A1, WO 2005 / 116615 A1, Foreman et al., Advances in Optics and Photonics, 2015, Vol. 7, pp. 168-240).

[0009] This requirement in known prior art methods is time-consuming and uneconomical, significantly limiting the applicability of WGM-based microscopic sensors. For example, one Micro resonator The need to measure multiple times means, in particular, that detection must be possible several times during the measurement process. Therefore, previous methods have used immobilized microsensors, such as those adsorbed onto rigid surfaces, biological cells, or held in microstructures.

[0010] Therefore, microsensors are the target of analysis. fluidWhile a measurement method that allows for free movement within the microfluidic channel is desired, this has been impossible with existing methods for quantifying surface occupancy due to the immobilization of the microsensor. Furthermore, the immobilization of the microsensor in these prior art methods makes continuous measurement impossible. As a result, existing implementations of WGM-based microsensor technology have so far been suitable only for specific laboratory systems and not for applications in the field of continuous process monitoring. Bischler et al. (R. Bischler et al. Phys. J. Special Topics, 2014, Vol. 223, pages 2041-2055 and DE 10 2014 104 595 A1) presented an example of a laboratory system that sets itself apart from other state-of-the-art systems by using a microfluidic channel integrated with a holding structure. Here, the microsensor is only temporarily fixed in position by the holding structure and can be repeatedly moved to the measurement position by an automated mechanical displacement unit. Once the measurement is complete, the microfluidic channel can be cleaned and refilled. Moving multiple microsensors continuously during measurement can increase the importance of the measurement results. However, the volumetric flow rate through the microfluidic channel and the number of microsensors that can be used in a single measurement are limited and far below the requirements for continuous process monitoring, making this system unsuitable for continuous process monitoring. Furthermore, as is common with state-of-the-art technology, the relative shift of the resonance mode is also used to derive conclusions about surface occupancy, making multiple measurements of the same microsensor absolutely essential.

[0011] However, the lack of suitability of label-free biosensors for use in the field of continuous process monitoring is a drawback that applies not only to previous implementations of WGM-based microsensor technology but also to other common methods for label-free detection of biological species, such as surface plasmon resonance (SPR), quartz microbalance (QMC), and / or ellipsometry. , analyteThe sensor surface that reacts specifically with the analyte is typically formed as a wall within the microchannel. The sensor surface receives the analyte and the surface conditioning necessary through the microchannel. fluid This can supply the analyte. However, since laminar flow is usually formed within the microchannel, the flow on the sensor surface becomes virtually stationary, and the transport of the analyte to the sensor surface is diffusion-limited. As a result, not only is the measurement time longer, but when the diffusion zone is depleted, the coupling dynamics become distorted (i.e., inaccurate). , analyte The affinity and affinity for its binding partners will be incorrectly determined.

[0012] An attempt to overcome this drawback of existing implementations of WGM-based microsensor systems is described in US 8,779,389 B2. In the method disclosed therein, the particles are analyzed fluid They flow freely and are randomly read out when detected by the optical system. Since each particle is measured only once, the necessary reference cannot be performed with the same particle and must be done by other means. For this purpose, US 8,779,389 B2 proposes comparing one characteristic spectrum of the particle detected in each case with a set of appropriately predetermined characteristic spectra, and using this comparison to determine the most suitable predetermined spectrum. Adsorbed onto the particle Analytes This is detected through the deviation between the measured spectrum and the optimal predetermined spectrum. (Particles) and analytes The measured spectrum, such as before the interaction Analytes It is not explained in more detail how the optimal given spectrum can be determined without additional measurement criteria if it deviates from the spectrum measured in the presence of the analyte. Furthermore, the refractive index of the medium can be determined in advance and compared with the refractive index obtained by measurement by referring to the optimal spectrum. To determine the refractive index of the medium, a sufficient amount of fine particles for statistical evaluation in the selected medium is measured in advance in the absence of the analyte to obtain a statistical reference value that can determine the refractive index of the medium. The deviation from the reference value is ,fluid Medium AnalytesIt is evaluated as evidence of the presence of [something]. Thus, in each embodiment, this method is analyzed in the sense of "yes / no sense". fluid Medium Analytes This only provides qualitative information regarding the presence of and therefore does not meet the requirements of the quantitative biosensor technology described above, which would enable, for example, the measurement of binding dynamics and the determination of affinity and avidity derived therefrom. In contrast, the method of the present invention provides information on the presence of a microresonator located on the microresonator. adsorption layer Determine the optical thickness, adsorption layer This also includes potential analytes. adsorption layer The optical thickness is ,fluid Determined independently of the refractive index, it provides a quantitative measure of the surface occupancy of the microresonator. ,fluid Medium Analytes This also enables quantitative descriptions of the concentration and binding dynamics of the substance. [Overview of the Initiative] [Problems that the invention aims to solve]

[0013] Based on this, the object of the present invention was to provide a method and apparatus that does not have the drawbacks known in the prior art. In particular, this method and apparatus allows for the processing of unlabeled samples in Analytes It is possible to quantitatively detect it in a rapid, economical, and continuous manner, without the risk of results being tampered with. , analyte This also makes it possible to detect the pure (i.e., correct) binding dynamics with the target molecule. [Means for solving the problem]

[0014] This problem is solved by the method having the features of claim 1 and the apparatus having the features of claim 8. Dependent claims represent advantageous and further developments.

[0015] According to the present invention, in a fluid Analytes A method for detecting a label-free substance is provided, which includes (or consists of) the following steps. a) providing at least one dielectric microsensor within a container, said at least one microsensor being , micro resonator and , analyte for bonding to The micro resonator applied to adsorption layer consisting of or comprising these, The micro resonator being particles consisting of or comprising a dielectric material and a fluorescent marker, the microresonator having an optical refractive index greater than that of the fluid to be analyzed, and the microresonator being suitable for enabling the manifestation of a plurality of resonance modes inside the microresonator upon excitation of the fluorescence of the fluorescent marker; b) contacting the at least one microsensor with an analyte-containing fluid to be analyzed; c) irradiating the at least one microsensor in the fluid with light having a wavelength suitable for exciting the fluorescent marker of the at least one microsensor to fluorescence; d) detecting at least two optical resonance modes of the at least one microsensor from the detected fluorescence light of the at least one microsensor; e) determining, via a numerical algorithm, the optical thickness of the at least one microsensor in the fluid from the spectral positions of the at least two detected resonance modes; and adsorption layer f) determining the degree to which an analyte in the fluid has bound to the at least one microsensor based on the previously determined optical thickness of the at least one microsensor. adsorption layer

[0016] The method according to the invention provides for the detection of at least two emission modes (i.e., one or more emission modes) of the microsensor. And , adsorption layer the absolute thickness of can be determined based on the positions of the detected modes relative to each other. The thickness thus obtained adsorption layer is compared with the known thickness applied to the microresonator during the manufacturing process adsorption layer and if , analyte ​​The level at which it binds (or accumulates) on the surface of the microsensor, i.e., in the fluid Analytes Qualitative conclusions can be drawn about the level coupled to the microsensor without being affected by tampering. By determining the absolute thickness of the adsorbent layer from individual measurements on the microsensor, the method according to the present invention relates to general measurements that can be determined under a wide variety of process conditions. As a result, the method according to the present invention is suitable for use under constantly changing process conditions and is also suitable for continuous process monitoring. Since the requirement to maintain identical process conditions, as in the prior art method, is not required, the method according to the present invention can be implemented more easily, more quickly, more economically, and with fewer devices.

[0017] According to the present invention, the term "fluorescent marker" specifically refers to quantum dots.

[0018] In this process, the microresonator particles can have a diameter in the range of 1 μm to 20 μm, preferably 2 μm to 15 μm, and particularly preferably 4 μm to 10 μm.

[0019] moreover , adsorption layer The thickness can be in the range of 0.5 nm to 30 nm, preferably 1 nm to 20 nm, particularly preferably 1.5 nm to 10 nm, and especially 2 nm to 8 nm, thereby the thickness is , micro resonator In the radial direction from the center point adsorption layer It is understood to be a spatial extension of a very thin material in the range of a few nanometers. adsorption layer For example, when the thickness is ≤ 2 nm, the separation of the optical refractive index and the geometric layer thickness can no longer be reliably achieved. at least one microsensor adsorption layer The main result for determining the optical thickness is the so-called "optical layer thickness," which is a product of the type n Ads* dAds, where nAds is the adsorption layerdAds represents the optical refractive index, and dAds represents the radial thickness. This definition is analogous to the optical path length known in optics, which is the product of the optical refractive index and the geometric optical path length. For the purposes of this invention, knowledge of the thickness of the optical layer is sufficient, because the thickness of the optical layer changes only slightly due to the diffusion of the analyte or exchange with molecules that were previously nonspecifically bound, even if the geometric layer thickness changes only slightly. adsorption layer This is because it changes due to adsorption to it. , adsorption layer The optical refractive index of most substances related to the structure (such as biomolecules) is very similar to that of the analyte being bonded. The optical refractive index is typically in the range of 1.43 to 1.48.

[0020] In this method, preferably, before step b), at least one microsensor adsorption layer There is no step to determine the optical thickness. This embodiment allows the method to be implemented more quickly and economically.

[0021] adsorption layer The optical thickness can be determined by a process following rigorous classical field theory.

[0022] In this method , adsorption layer The optical thickness can be determined using a numerical algorithm from the spectral positions of at least two detected resonance modes and at least one further parameter selected from the group consisting of the relative amplitudes of at least two detected resonance modes and the linewidths of at least two detected resonance modes. In this embodiment, , adsorption layer This has the advantage of allowing for a more precise determination of the optical thickness. When the fluorescence of the fluorescent marker is excited... , micro resonator Suitable for multiple resonance modes to emerge within it Micro resonator By using this method, the linewidth of each mode can be made smaller than the spectral interval, making it possible to spectrally separate the modes.

[0023] Furthermore, in this method, additional parameters of at least one microsensor can be determined from the spectral positions of at least two detected resonance modes, preferably using a numerical algorithm, from at least one additional parameter selected from the group consisting of the relative amplitudes of at least two detected resonance modes and the linewidths of at least two detected resonance modes. The additional parameters are preferably the diameter of at least one microsensor, the refractive index of at least one microsensor, and at least one microsensor adsorption layer The refractive indices are selected from the group consisting of refractive indices and combinations thereof.

[0024] Separately, the parameters of the fluid can be determined in a method using a numerical algorithm, preferably from the spectral positions of at least two detected resonance modes, and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of at least two detected resonance modes and the linewidths of at least two detected resonance modes, thereby determining the optical properties of the fluid.

[0025] At least one microsensor used in this method can be designed as an essentially spherical particle. Micro resonator Characteristics that form a resonant mode is a micro resonator This is advantageous because it depends on the particle's shape. Spherical particles have high symmetry, allowing them to form resonance modes independent of each propagation surface within the particle, which facilitates the detection of particles moving freely in a fluid. Particles with very strong asphericity (e.g., irregular shapes, i.e., shapes without an axis of symmetry) do not exhibit observable resonance modes because light is scattered from the particle too quickly. Therefore, the lifetime of the resonance modes of such particles is very short, and the linewidths overlap, making them unobservable.

[0026] The fluorescent marker for the microresonator can be placed inside the microresonator or on its outer surface.

[0027] Detection of at least two optical resonance modes of at least one microsensor can be performed by detecting light scattered by at least one microsensor and / or light emitted by at least one microsensor.

[0028] At least one microsensor used in this method can be moved freely. This is preferable because it allows the method to be implemented more quickly and economically. Alternatively, at least one microsensor can be fixed, in particular, when at least one microsensor is connected to the fluid to be analyzed. (Analyte It can be fixed to the inner surface of a fluid channel that comes into contact with (which may include)

[0029] In this method, the detection of at least two optical resonance modes of at least one microsensor is performed from the spectral positions of the at least two detected resonance modes, preferably from at least one further parameter selected from the group consisting of the relative amplitude of the at least two detected resonance modes and the linewidth of the at least two detected resonance modes, of the at least one microsensor adsorption layer The time course of the optical thickness can be determined using a numerical algorithm, and this can be repeated at least once, and arbitrarily several times. In this embodiment, the optical thickness of the microsensor is Analytes This method has the advantage of being able to detect the binding dynamics. In contrast to prior art methods that use immobilized microsensors, the determination of binding dynamics using the method according to the present invention is not affected by errors. In addition to the binding dynamics themselves, it is also possible to determine whether they have already reached a stationary state. From the quantitative determination of the amount of analyte adsorbed in the stationary state, conclusions can be drawn about the content of the analyte in the analyzed fluid in a more accurate and less error-prone manner.

[0030] In this method, the time characteristics of at least one further parameter of at least one microsensor can also be determined from the spectral positions of at least two detected resonance modes, preferably using a numerical algorithm, from at least one further parameter selected from the group consisting of the relative amplitudes of at least two detected resonance modes and the linewidths of at least two detected resonance modes. The at least one further parameter is the diameter of at least one microsensor, the refractive index of at least one microsensor, and at least one microsensor adsorption layer The refractive index can be selected from the group consisting of the refractive index of the fluid, the refractive index of the fluid, and combinations thereof. The advantages of this embodiment are , analyte It is also possible to record the time course of at least one microsensor or other parameters of the fluid.

[0031] According to the present invention, further, in a fluid Analytes A device for detecting a label-free substance is provided, comprising (or comprising) the following: a) A container comprising at least one dielectric microsensor, wherein the at least one microsensor is , micro resonator and , analyte To combine The micro resonator It includes, or consists of, an adsorption layer coated on, The micro resonator A container, a microresonator comprising a dielectric material and a fluorescent marker, or particles made thereof, having an optical refractive index greater than that of the fluid being analyzed, and suitable for enabling the emergence of multiple resonance modes within the microresonator when the fluorescence of the fluorescent marker is excited; b) A light source for irradiating at least one microsensor with light, the light source having a wavelength suitable for exciting a fluorescent marker of at least one microsensor to fluorescence, c) A spectral analysis unit configured to detect at least two optical resonance modes of at least one microsensor from the detected fluorescence light; d) From the spectral positions of at least two detected resonant modes via a numerical algorithm, at least one microsensor adsorption layer An algorithm unit configured to determine the optical thickness of; and e) At least one microsensor adsorption layer Based on the determined optical thickness , analyte An analysis unit configured to determine the extent to which at least one microsensor is coupled.

[0032] The apparatus according to the present invention , analyte The degree to which it is bonded (or adsorbed) to the surface of the microsensor, i.e., in the fluid Analytes The degree to which the adsorbent is coupled to the microsensor can be used to derive qualitative and even quantitative conclusions without being affected by tampering. This device can determine the absolute thickness of the adsorbent layer from individual measurements on the microsensor, thus enabling the determination of the analyte under a wide range of process conditions. As a result, the device according to the present invention is suitable for use under constantly changing process conditions and for continuous process monitoring. Since there is no need to maintain identical process conditions as with prior art devices, the device according to the present invention... Analytes Detection can also be performed more easily, more quickly, more economically, and with fewer devices.

[0033] Furthermore, the device according to the present invention can be used with non-fixed (i.e., freely movable) microsensors. AnalytesThe coupling of these elements can be detected. As a result, the apparatus according to the present invention does not require, for example, a movable stage, which was necessary in the prior art apparatus to fix and specifically position the microsensor above the detection unit. Therefore, the use of costly mechanical positioning axes and, for example, an air gap between the detection optical system and the microstructure holding the microsensor can be omitted. By eliminating the air gap, an immersion objective lens with an numerical aperture of 1.0 or more (the theoretical limit of air-gap objective lenses) can be used, allowing for the detection of a higher proportion of fluorescence emitted from the microsensor and supplying it to the downstream spectroscopic optical system. By increasing the signal intensity in this way, the time required to detect the emission spectrum of the microsensor under investigation can be significantly reduced, and even high-speed microsensors can be detected with sufficient signal intensity and accurately analyzed.

[0034] The particles of the microresonator can have a diameter in the range of 1 μm to 20 μm, preferably 2 μm to 15 μm, and particularly preferably 4 μm to 10 μm. . adsorption layer The thickness can be in the range of 0.5 nm to 30 nm, preferably 1 nm to 20 nm, particularly preferably 1.5 nm to 10 nm, and particularly preferably 2 nm to 8 nm, so that the thickness is in the radial direction from the center point of the microresonator. adsorption layer It is understood to be a spatial expansion of [the original space].

[0035] The spectral analysis unit has at least one microsensor Analytes It can be configured to perform detection of at least two optical resonance modes of at least one microsensor only after contact with a fluid that may contain such a fluid. , analyte This has the advantage that detection can be carried out more quickly and economically using the device.

[0036] The algorithm unit follows rigorous classical field theory. adsorption layer It may be configured to perform the determination of the optical thickness.

[0037] Furthermore, the algorithm unit, via a numerical algorithm, uses at least one microsensor to determine the spectral positions of at least two detected resonant modes and at least one additional parameter selected from the group consisting of at least two relative amplitudes of the detected resonant modes and at least two linewidths of the detected resonant modes. adsorption layer It can be configured to determine the optical thickness.

[0038] Separately, the algorithm unit may be configured to use a numerical algorithm to determine further parameters of at least one microsensor from the spectral positions of at least two detected resonance modes, preferably from at least one further parameter selected from the group consisting of the relative amplitudes of at least two detected resonance modes and the linewidths of at least two detected resonance modes. The further parameters are preferably the diameter of at least one microsensor, the refractive index of at least one microsensor, and at least one microsensor adsorption layer The refractive indices are selected from the group consisting of refractive indices and combinations thereof.

[0039] Furthermore, the algorithm unit may be configured to use a numerical algorithm to determine fluid parameters, preferably the optical properties of the fluid, from the spectral positions of at least two detected resonance modes, and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of at least two detected resonance modes and the linewidths of at least two detected resonance modes.

[0040] At least one microsensor is preferably designed as an essentially spherical particle.

[0041] The fluorescent marker for the microresonator can be placed either inside the microresonator or on its outer surface.

[0042] The spectral analysis unit may be configured to perform detection of at least two optical resonance modes of at least one microsensor by detecting light scattered by at least one microsensor and / or light emitted by at least one microsensor.

[0043] The apparatus container may further contain a fluid that may contain the analyte. The container is optionally a fluid channel.

[0044] At least one microsensor is freely movable within the container and optionally within the fluid flow path of the device. This embodiment is , analyte This is advantageous in that it can detect more quickly and economically. Alternatively, at least one microsensor can be fixed in the fluid flow path of the device, in particular fixed on the inner surface of the fluid flow path of the device, and the fluid flow path is , analyte It is particularly suitable for supplying at least one microsensor to the fluid to be analyzed, which may contain [specific substances / components].

[0045] The spectral analysis unit can be configured to detect at least two optical resonance modes of at least one microsensor at least once, and optionally several times, and the algorithm unit uses the spectral positions of at least two detected resonance modes to determine the position of at least one microsensor adsorption layer A numerical algorithm is preferably used to determine the time course of the optical thickness of the microsensor, which is configured to determine at least one further parameter selected from the group consisting of the relative amplitudes of at least two detected resonance modes and the linewidths of at least two detected resonance modes. Analytes It has the advantage of being able to detect the binding dynamics.

[0046] Furthermore, the spectral analysis unit may be configured to repeat the detection of at least two optical resonance modes of at least one microsensor at least once, and optionally several times, and the algorithm unit is configured to determine the time course of at least one further parameter of at least one microsensor from the spectral position of the detected resonance modes, preferably from at least one further parameter selected from the group consisting of the relative amplitudes of at least two detected resonance modes and the line widths of at least two detected resonance modes, via a numerical algorithm, where the at least one further parameter is selected from the group consisting of the relative amplitudes of at least two detected resonance modes and the line widths of at least two detected resonance modes, and the at least one further parameter is particularly preferably selected from the group consisting of the diameter of at least one microsensor, the refractive index of at least one microsensor, the refractive index of the adsorbent layer of at least one microsensor, the refractive index of the fluid, and combinations thereof. The advantages of this embodiment are , analyte It is also possible to record the time course of at least one microsensor or other parameters of the fluid.

[0047] The apparatus may include a fluid channel. The fluid channel preferably includes a supply line suitable for supplying at least one microsensor to the fluid channel. Furthermore, the fluid channel preferably includes an outlet suitable for discharging at least one microsensor from the fluid channel, and the outlet preferably includes a separator for at least one microsensor.

[0048] The fluid channel may have at least one transparent wall that is transparent to light having wavelengths in the range of emission wavelengths of the fluorescent marker in at least a specific region, the detection optical system is preferably positioned between the transparent wall and the spectral analysis unit, and the concave mirror is particularly preferably positioned on the side of the container facing the transparent wall.

[0049] Furthermore, the fluid channel may have at least one transparent wall in at least a specific region that is transparent to light of wavelengths in the excitation wavelength range of the fluorescent marker.

[0050] Separately, the fluid channel may have at least one transparent wall that is transparent to light having wavelengths within the range of the excitation and emission wavelengths of the fluorescent marker in at least a specific region, and a detection optical system having a coupling element for light from a light source is placed between the transparent wall and the spectral analysis unit, wherein the coupling element is reflective to light with wavelengths within the range of the excitation wavelengths of the fluorescent marker and transmittance to light with wavelengths within the range of the emission wavelengths of the fluorescent marker.

[0051] Furthermore, the fluid channel may have at least one transparent wall that is transparent to light of wavelengths in the range of emission wavelengths of the fluorescent marker in at least a specific region, and that allows for the implementation of additional sensor technology, preferably a photodetector.

[0052] The algorithm unit and the analysis unit, preferably a spectral analysis unit, can be designed as a single unit, preferably monolithically.

[0053] The microresonators used in the method and / or apparatus are made of, or may be made of, materials selected from the group consisting of polymers, preferably polystyrene, melamine resin, polydivinylbenzene, polymethyl methacrylate, poly(styrene-co-divinylbenzene), poly(styrene-co-methyl methacrylate), or polydimethylsilane. Micro resonator It may also be composed of a material selected from the group consisting of inorganic materials, preferably a material selected from the group consisting of inorganic dielectric materials such as silica (SiO2) or titanium dioxide, or may consist of these materials.

[0054] Microresonators used in this method and / or apparatus adsorption layer This includes, or can be composed of, materials selected from dielectric materials, particularly organic materials, such as oligomers, polymers, peptides, proteins, oligonucleotides, DNA or cellular components, and mixtures or compounds of these materials. Furthermore, metals, semiconductors, metamaterials, or combinations or compounds thereof can also be used, provided that they do not attenuate so strongly that the resonance mode becomes undetectable. Furthermore, the microresonator used in this method and / or device adsorption layer This may include or consist of materials having a refractive index in the range of 1.43 to 1.47.

[0055] Furthermore, the microresonators used in the method and / or device adsorption layer It can be coupled to the microresonator via non-covalent interactions (e.g., via ionic interactions, dipole-dipole interactions, van der Waals interactions and / or hydrogen bonds). , adsorption layer Alternatively or additionally, these can be bonded to the microresonator via covalent bonds (i.e., chemical bonds). The type of chemical bond, and therefore the choice of molecules used for bonding, depends on the surface of the microresonator and the functional groups available therein.

[0056] Separately, microresonators used in the method and / or apparatus adsorption layer It may be divided into several sublayers. The algorithm unit is a physical model and , adsorption layer The contrast of the different optical refractive indices of the individual sublayers and , adsorption layer It can be configured to characterize a uniform layer having an average optical refractive index (AL Aden & M. Kerker, Scattering of electromagnetic waves from two concentric spheres, Journal of Applied Physics, 1951, Vol.22, pp. 1242-1246), or as a composite layer with different optical refractive indices (R. Bhandari, Scattering coefficients for a multilayered sphere: Analytic expressions and algorithms, Applied Optics, 1985, Vol.24, pp. 1960-1967). [Brief explanation of the drawing]

[0057] The following figures and examples are intended to illustrate the objectives of the present invention in more detail and are not intended to limit the invention to the specific embodiments shown herein.

[0058] [Figure 1]Figure 1 schematically shows an apparatus according to the present invention having different transparent walls 50, 55 within a container (here a microchannel) for excitation of a microsensor 40 by excitation light 60 and readout of fluorescence light 100 from the microsensor 40. Fluid 20 moves through a channel 10 in which the presence and concentration of a desired analyte are inspected by adding a WGM-based microsensor 40 via a dosing system 30. The microsensor 40 flows a distance with the fluid and can be fluorescence-excited through the transparent wall 55 with the help of excitation light 60. The fluorescence emission 100 is then led to a spectral analysis unit 110 via the further transparent wall 50 and a downstream detection optical system 90, where the light is readout spectroscopically. As the flow continues, the microsensor 40 can optionally be removed from the fluid again by a suitable capture device 70 combined with an outlet 80. For example, in the case of a non-critical fluid such as wastewater, the microsensor can remain at the fluid outlet, so components 70 and 80 are not necessary. [Figure 2] Figure 2 schematically shows a further apparatus according to the present invention, which is designed similarly to Figure 1 but has only a single transparent wall 50 for exciting the microsensor 40 with excitation light 60 and reading out the fluorescent light 100 from the microsensor 40. [Figure 3] Figure 3 schematically shows a further apparatus according to the present invention, which is designed similarly to Figure 1, but with a different orientation of the excitation light 60 and different beam shaping (collimated in Figure 3a and focused in Figure 3b). [Figure 4A] Figure 4A shows an example of fluorescence emission obtained from a microsensor as a function of wavelength, illustrating various resonance modes with different polarization states TM and TE. [Figure 4B] Figure 4B schematically shows the structure of a microsensor 40, which consists of a microresonator 43 and an adsorption layer 45. [Figure 4C] Figure 4C shows an example of the increase in the spectral resonance modes TM and TE due to an increase in the diameter of the microsensor. [Figure 4D]Figure 4D shows an example of the increase in resonance modes TM and TE in the microsensor spectrum with increasing thickness of the adsorption layer of the microsensor (due to binding of the analyte to the adsorption layer). [Figure 5A] Figure 5A shows an exemplary simulation of the WGM spectrum 170 of a microresonator (i.e., dielectric microsensor) with radius R=3400nm, internal refractive index of 1.59 (approximate value for polystyrene), and no adsorption layer (i.e., dielectric microsensor). The WGM spectrum 170 in Figure 5A has been background-corrected and normalized to the respective maximum values ​​before correction. [Figure 5B] Figure 5B shows an exemplary numerical simulation of the WGM spectrum 180 of a microresonator (i.e., dielectric microsensor) having a radius R = 3400 nm, an internal refractive index of 1.59 (approximate value for polystyrene), and either having no adsorption layer in the medium surrounding the microresonator (adsorption layer thickness 185 set to 0 nm) or having an adsorption layer with some thickness 185 (i.e., the adsorption layer thickness 185 set to 5 nm, 20 nm, 60 nm, 100 nm, 140 nm, 180 nm, or 200 nm). In the medium surrounding the microresonator with a refractive index of 1.33, the adsorption layer had a refractive index m = 1.48. The WGM spectrum 180 in Figure 5B was background-corrected and normalized to the respective maximum values ​​before correction. [Modes for carrying out the invention]

[0059] Example 1 - Principle of the method according to the present invention

[0060] In the fluid (analytical sample) Analytes To detect this and the coupling phenomenon on the microsensor surface, the problem Analytes On the microresonator generated by the coupling adsorption layer It is essential to be able to determine the change in thickness.

[0061] In contrast to particle size, it is specifically functionalized. adsorption layer Lot-to-lot scattering of thickness is relatively small due to the low overall thickness, typically a few to tens of nanometers, and therefore, without statistical or direct reference. , micro resonator Within the evanescent field of a resonant mode extending approximately 50-100 nm from the surface, it can be easily distinguished from adsorption events on the microsensor surface. . Adsorption layer If the thickness is known, it is possible to draw direct qualitative and quantitative conclusions about the binding event without reference.

[0062] Micro resonator The isolation of individual parameters such as size and thickness of the adsorption layer can be achieved by their different effects on the mode position of the various resonant modes of the microsensor. Dielectrics (i.e., non-conductive and non-adsorbent, or only slightly adsorbent) Micro resonator This results in two distinct types of resonant modes with different electric field directions (see Figure 4B). In one case, the electric field of the mode is radially polarized, and the simultaneously generated magnetic field is tangential to the particle surface ("transverse magnetic mode," abbreviated as "TM"). These two fundamental types of resonant modes perfectly capture the interface between the microsensor and its environment and are described by rigorous optical theories such as the Mie theory for enclosed microparticles (AL Aden & M. Kerker, Scattering of electromagnetic waves from two concentric spheres, Journal of Applied Physics, 1951, Vol.22, pages 1242-1246).

[0063] Adsorption layer In separating individual parameters such as thickness and micro-resonator diameter, the important thing is that TM mode and TE mode are , adsorption layerWhile different shifts are observed with increasing thickness, they behave similarly and closely with respect to changes in sensor size (see Figures 4C and 4D). For example, in polystyrene-based microsensors that adsorb biomolecules in aqueous solutions, the TM mode shows a stronger shift than the TE mode (see Figure 4D). Therefore, modern computing techniques, such as numerical modeling and fitting of Mie spectra to measurement data, , micro resonator It is possible to clearly separate the size of the particles from the thickness of the adsorption layer.

[0064] An example of a fluorescence spectrum obtained from the microsensor 40 is shown in Figure 4A. The resonance mode of the microsensor 43 is superimposed on the fluorescence background of the dye, and the spectral position, amplitude, and linewidth can be determined by operations corresponding to the art (e.g., subtracting the background or numerically fitting a theoretical resonance curve to the measured spectrum). From these quantities, which are characteristic of each microsensor (40) and its environment at the time of measurement, conclusions can be drawn about both the size of the sensor and the thickness of the adsorption layer by numerically adapting a suitable model, such as the Mie theory of a dielectric sphere surrounded by at least one envelope.

[0065] Therefore, with the help of computer technology, it is possible to draw conclusions about the system microresonator 43 together with the adsorbent layer 45 in the fluid 20 from the measured spectrum in a very short time (typically within a few seconds), and to determine the parameters essential to each problem (such as sensor size, layer thickness, optical refractive index of the adsorbent, and optical refractive index of the environment). The individual parameters can be separated by the differences in their influence on the mode position of the various resonance modes of the microsensor 40.

[0066] Besides the Mie theory, other optical models for the excitation of resonance modes in a sphere include the Debye theory and the Airy model. While these are only approximations, they have the advantage of being able to be expressed analytically. Since all of these existing models describe the same physical system, the aforementioned parameters can be determined in the same way as in the Mie theory.

[0067] Example 2 - Process or apparatus variations

[0068] Figures 1 and 2 show two different embodiments of the apparatus or method according to the present invention relating to the fluorescence excitation of the microsensor 40.

[0069] In Figure 1, excitation light 60 is irradiated into the fluid from a separate access window 55 and irradiates at least one passing microsensor 40. Access window 55 can be positioned on the opposite side of access window 50 used for detection, or it can be positioned so that at least one microsensor 40 located in the detection area is irradiated. The advantage of this arrangement is that the dielectric properties of the two access windows 50, 55 with respect to transmission, reflection, and absorption can be precisely matched to their respective radiation (fluorescence excitation or emission). At least one microsensor 40 that happens to pass through the light cone is fluorescence excited and can be spectroscopically analyzed through access window 50. For this purpose, the detection optics 90 captures a portion of the fluorescence light 100 emitted by at least one microsensor 40 and transfers it to the spectral analysis unit 110. The spectral profile of the fluorescence emission 100 of the microsensor 40 obtained by the spectral analysis unit 110 is then analyzed by the algorithm unit 120 for characteristic variables such as the spectral positions (and optionally relative amplitude and / or linewidth) of resonance modes of different polarizations. These results are then evaluated in the analysis unit 130 for the presence and / or concentration of the analyte to be determined. Analytes Information regarding this is then provided by the device to the device operator via an appropriate interface.

[0070] However, in Figure 2, the excitation of fluorescence 100 from at least one microsensor 40 is performed from the same side as the subsequent fluorescence detection through the access window 50. The detection optics 90 is additionally used to irradiate the fluid 20 with excitation light 60 through the access window 50 via the coupling element 140. In this case as well, at least one microsensor 40 flowing through the fluid 20 and passing through the light cone of excitation light 60 is excited to fluorescence 100, read out via the detection optics 90 as described above, and analyzed with respect to desired parameters with the help of units 110, 120, and 130. The important point here is that the coupling element 140 is transparent so that the fluorescence emission 100 of the microsensor 40 captured by the detection optics 90 can reach the spectral analysis unit 110 (as in Figure 1). The access window 50 must be transparent not only to the fluorescent emission 100 but also to the excitation light 60, thus increasing the requirements for the access window 50. However, the advantage of this embodiment is that the wall of the flow channel 10 facing the access window 50 can be designed as a reflector 160 for the fluorescent emission 100 of the microsensor 40 (see Figure 3a). As a result, the detection optical system 90 can collect the fluorescent light with a larger solid angle, which increases the signal intensity, shortens the measurement time, and ultimately widens the range of possible fluid velocities.

[0071] Figures 1 and 2 illustrate two basic embodiments of the present invention, and these embodiments can be modified in various ways. For example, Figure 3 shows two different examples of modifications.

[0072] In the embodiment shown in Figure 3, the direction of fluid flow also corresponds to the x-axis in this figure. Here, the excitation light 60 is no longer guided along the optical axis of the detection optical system 90, but is irradiated into the fluid 20 at a different angle. The 90-degree angle with respect to the optical axis of the detection optical system 90 shown here is just one example. Figures 3a and 3b also show different shapes of the excitation light 60 used for fluorescence excitation 100. In Figure 3a, the excitation light 60 is parallel light, while in Figure 3b, it is focused to the focal point of the detection optical system 90. The latter embodiment has the advantage that only the microsensors located very close to the focal point of the detection optical system 90 are significantly excited to fluorescence 100, thus reducing potential interference signals from at least one microsensor 40 located in the fluid 20 that are unsuitable for detection. As already described for Figure 2, in the embodiment shown in Figure 3, the flow channel wall facing the access window 50 can also function as a reflector 160 to increase the fluorescence signal of at least one microsensor 40 collected by the detection optical system 90.

[0073] Furthermore, Figure 3a shows a potentially useful additional sensor technology in the form of a further access window 58 and a photodetector 150 located behind it, which can be used for further characterization of the fluid 20, for example, turbidity measurement, providing information on the solids content of the fluid and thus assisting the analysis unit 130 in interpreting the measurement results. As shown in Figure 3b, if there is no additional access window 58 in the beam path of the excitation light 60, the excitation light 60 can be modified, for example, by being absorbed or reflected through the rear wall 15 of the flow path 10, to suppress undesirable effects such as excitation of at least one poorly positioned microsensor 40, or to achieve desired effects such as increasing the intensity of the excitation light 60 in the detection volume of the detection optics 90.

[0074] Depending on the fluid 20, its composition, its environment, its application, or other influencing factors related to the analysis of the fluid 20, it may not be possible to directly introduce the microsensor into the fluid, as shown in Figure 1-3, and a portion of the fluid 20 must be separated from the flow path 10 for analysis. However, even in such cases, it is always possible to obtain the flow path corresponding to Figure 1-3 again after appropriately separating or preparing the portion of the fluid 20 selected for analysis. Therefore, the embodiments shown herein have sufficient general applicability even in more complex applications of the microsensor technology shown herein.

[0075] Example 3 - Advantages of the method according to the present invention

[0076] The method according to the present invention involves measuring the presence of an analyte in a fluid by using at least one microsensor in the fluid which may contain the analyte. adsorption layer This method utilizes the measurement of the optical thickness and the changes that occur due to the bonding of the analyte to be sought. 0 adsorption layer The optical thickness is determined by the placement of a microsensor. fluid The advantages of using this parameter compared to other possible parameters such as the refractive index m are explained below.

[0077] Figure 5A shows a radius R = 3.4 μm and an internal refractive index of 1.59 (approximate value for polystyrene). ,adsorption layer None (that is, ,adsorption layer (The layer thickness was set to 0 nm) Micro resonator The WGM spectrum 170 of (i.e., dielectric microsensor) is obtained using different refractive indices of 175. Micro resonatorThis was a numerical simulation performed in the surrounding medium. Spectrum 170 was calculated according to the Mie theory of a dielectric sphere embedded in a medium (Craig F. Bohren & Donald R. Huffman, Absorption and Scattering of Light by Small Spheres, 1998, Wiley-VCH-Verlag, ISBN 9780471 293408, implemented in MATLAB® 2021b), as is typical in the realm of technology. For better comparison, WGM spectrum 170 was background-corrected. After background correction, the spectra were normalized to their respective original maximum values, i.e., the maximum values ​​before background correction, in order to maintain equivalent WGM amplitudes. Spectrum 180, shown in Figure 5B, was processed similarly. Diameter: 6.8 μm Micro resonator I chose the size because , resonator This value has been proven to be a compromise between surface size and the quality of excitable WGMs. Micro resonator If it is large, it will adsorb Analytes The relative area occupied by the micro-resonator on the microsensor surface decreases, and the mode shift also decreases accordingly. When the micro-resonator is small, the loss of WGM excitation increases due to surface scattering and radiative loss, so the characteristics of individual WGMs deteriorate and processing becomes difficult.

[0078] Figure 5A shows this Micro resonator The linewidth of the WGM size clearly shows that m0 increases rapidly with increasing refractive index of the medium (175), and that this leads to the TM and TE modes beginning to overlap even at refractive index values ​​still lower than the expected 175 for biological materials (as shown from M0 = 1.40). Because these modes begin to overlap, it becomes virtually impossible to experimentally determine M0 within a range suitable for detecting (biological) analytes or adsorbents (such as bacteria like US 8,779,389 B2). In practice, there are limitations to using a refractive index of 175 for the medium as an indicator for detecting (biological) analytes or adsorbents. This is because the refractive index of adsorbents is usually high (1.43-1.48), resulting in low surface coverage, i.e., low adsorbent concentration. ,fluidThis means that it is only acceptable if the concentration of the analyte in the liquid is also low.

[0079] In contrast, as in the method and apparatus according to the present invention, at least one microsensor adsorption layer When using the optical thickness, the analyte and adsorption layer Any refractive index is possible. This is shown in Figure 5B. Figure 5B shows a radius of 3.4 μm and an internal refractive index of 1.59 (approximate value for polystyrene). , adsorption layer none (Adsorption layer (Setting the layer thickness of 185 to 0nm) or a layer with a thickness of 185 adsorption layer (that is) , adsorption layer (Having either a layer thickness of 185 set to 0 nm) Micro resonator This shows a numerical simulation of the WGM spectrum 180 of a dielectric microsensor. . adsorption layer The refractive index was mAds = 1.48. The simulation was performed using Matlab® 2021b, as before, following the covered sphere theory by Bohren & Hofmann (Craig F. Bohren & Donald R. Huffman, Absorption and Scattering of Light by Small Particles, 1998, Wiley-VCH-Verlag, ISBN 9780471293408). s adsorption layer Despite its high refractive index of m = 1.48, WGM is surprisingly , adsorption layer It is easily distinguishable up to a thickness of 185. This differentiability allows all relevant parameters, especially the optical thickness of the adsorbent layer, to be determined based on a rigorous theory in which all parameters have physical meaning, as previously mentioned for Figure 4D, and in principle, it is possible to determine whatever the refractive indices of the analyte and the adsorbent layer may be. Simultaneously, in the simulation of the scattering spectrum based on Mie theory, on the microresonator adsorption layerIgnoring this leads to different, potentially misleading results, as changes in the refractive index m of the environment always alter the entire environment of the microresonator, making it impossible to describe only the partial filling of the near-field around the microresonator. Therefore, prior art methods and apparatus that do not consider the adsorption layer on the microresonator may lead to erroneous results if the refractive index of the environment (e.g., the surrounding medium) changes.

[0080] Therefore, the use of the optical thickness of the adsorption layer is a more general and versatile parameter for detecting analytes in a fluid. Consequently, the method and apparatus according to the present invention are more versatile and reliable than the methods and apparatus of the prior art, and can avoid the risk of erroneous results due to changes in the refractive index of the microresonator environment.

[0081] (Note) (Note 1) A method for detecting analytes in a fluid in a label-free manner, (a) A step of supplying at least one dielectric microsensor into a container, The at least one microsensor comprises or consists of a microresonator and an adsorption layer coated on the microresonator for binding the analyte. The microresonator comprises a dielectric material and a fluorescent marker, or consists of particles made thereof, has an optical refractive index higher than the optical refractive index of the fluid being analyzed, and is suitable for generating multiple resonance modes inside the microresonator when the fluorescence of the fluorescent marker is excited, and the process is as follows: (b) The step of bringing at least one microsensor into contact with the fluid to be analyzed, which may contain the analyte, (c) A step of irradiating light onto the at least one microsensor in the fluid, wherein the light has a wavelength suitable for exciting the fluorescent marker of the at least one microsensor to produce fluorescence, (d) A step of detecting at least two optical resonance modes of the at least one microsensor from the fluorescence detected by the at least one microsensor, (e) A step of determining the optical thickness of the adsorption layer of the at least one microsensor in the fluid via a numerical algorithm from the spectral positions of the at least two detected resonance modes, (f) A method comprising the step of determining the extent to which an analyte in the fluid is bound to the at least one microsensor based on a predetermined optical thickness of the adsorption layer of the at least one microsensor.

[0082] (Note 2) The particles of the microresonator have a diameter in the range of 1 μm to 20 μm, preferably 2 μm to 15 μm, and particularly preferably 4 μm to 10 μm, and / or the adsorption layer has a thickness in the range of 0.5 nm to 30 nm, preferably 1 nm to 20 nm, particularly preferably 1.5 nm to 10 nm, and even more preferably 2 nm to 8 nm, where the thickness refers to the spatial extent of the adsorption layer in the radial direction from the center point of the microresonator. The method described in Appendix 1, characterized by the features described herein.

[0083] (Note 3) The step of determining the optical thickness of the adsorption layer of the at least one microsensor is not performed before step (b). The method according to Appendix 1 or 2, characterized by the features described herein.

[0084] (Note 4) The optical thickness of the adsorption layer is determined using a numerical algorithm from the spectral positions of the at least two detected resonance modes and at least one further parameter, the parameter being selected from the group consisting of the relative amplitude of the at least two detected resonance modes and the linewidth of the at least two detected resonance modes. The method described in any one of the appendices 1 to 3, characterized by the features described herein.

[0085] (Note 5) moreover, (i) From the spectral positions of the at least two detected resonance modes, and preferably from at least one further parameter selected from the group consisting of the relative amplitude of the at least two detected resonance modes and the linewidth of the at least two detected resonance modes, further parameters of the at least one microsensor are determined using a numerical algorithm. The further parameters are preferably selected from the group consisting of the diameter of the at least one microsensor, the refractive index of the at least one microsensor, the refractive index of the adsorption layer of the at least one microsensor, and / or combinations thereof. (ii) The parameters of the fluid, preferably the optical properties of the fluid, are determined using a numerical algorithm from the spectral positions of the at least two detected resonance modes and, preferably, from at least one further parameter selected from the group consisting of the relative amplitude of the at least two detected resonance modes and the linewidth of the at least two detected resonance modes. The method described in any one of the appendices 1 to 4, characterized by the following:

[0086] (Note 6) The at least one microsensor is (i) It is freely movable, or (ii) In particular, the at least one microsensor is fixed to the inner surface of the fluid channel through which the contact between the fluid to be analyzed, which may contain the analyte, takes place. The method described in any one of the appendices 1 to 5, characterized by the following:

[0087] (Note 7) The detection of at least two optical resonance modes of the at least one microsensor is performed at least once, and optionally several times, using a numerical algorithm to obtain the temporal evolution of the optical thickness of the adsorption layer of the at least one microsensor, from the spectral positions of the at least two detected resonance modes and preferably, from at least one further parameter selected from the group consisting of the relative amplitude of the at least two detected resonance modes and the linewidth of the at least two detected resonance modes. Optionally, the temporal evolution of at least one further parameter of the at least one microsensor is determined using a numerical algorithm from the spectral positions of the at least two detected resonance modes, and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonance modes and the linewidths of the at least two detected resonance modes. The at least one further parameter is particularly preferably selected from the group consisting of the diameter of the at least one microsensor, the refractive index of the at least one microsensor, the refractive index of the adsorption layer of the at least one microsensor, the refractive index of the fluid, and combinations thereof. The method described in any one of the appendices 1 to 6, characterized by the features described herein.

[0088] (Note 8) A device for detecting analytes in a fluid in a label-free manner, (a) A container comprising at least one dielectric microsensor, The at least one microsensor comprises or consists of a microresonator and an adsorption layer coated on the microresonator for binding the analyte. The microresonator comprises a container and particles containing or comprising a dielectric material and a fluorescent marker, having an optical refractive index higher than the optical refractive index of the fluid being analyzed, and being suitable for generating multiple resonance modes inside the microresonator when the fluorescence of the fluorescent marker is excited. (b) A light source for irradiating light onto the at least one microsensor, wherein the light has a wavelength suitable for exciting the fluorescent marker of the at least one microsensor to produce fluorescence, (c) A spectral analysis unit configured to detect at least two optical resonance modes of at least one microsensor from the detected fluorescence, (d) An algorithm unit configured to determine the optical thickness of the adsorption layer of the at least one microsensor via a numerical algorithm from the spectral positions of the at least two detected resonance modes, (e) an analysis unit configured to determine the extent to which an analyte has bonded to the at least one microsensor based on the determined optical thickness of the adsorption layer of the at least one microsensor.

[0089] (Note 9) The particles of the microresonator have a diameter in the range of 1 μm to 20 μm, preferably 2 μm to 15 μm, and particularly preferably 4 μm to 10 μm, and / or the adsorption layer has a thickness in the range of 0.5 nm to 30 nm, preferably 1 nm to 20 nm, particularly preferably 1.5 nm to 10 nm, and even more preferably 2 nm to 8 nm, where the thickness refers to the spatial extent of the adsorption layer in the radial direction from the center point of the microresonator. The apparatus as described in Appendix 8, characterized by the features described herein.

[0090] (Note 10) The spectral analysis unit is configured to perform the detection of the at least two optical resonance modes of the at least one microsensor only after the at least one microsensor has come into contact with a fluid that may contain the analyte. The apparatus according to appendix 8 or 9, characterized in that it is a device.

[0091] (Note 11) The algorithm unit is configured to determine the optical thickness of the adsorption layer of the at least one microsensor via a numerical algorithm from the spectral positions of the at least two detected resonance modes and at least one further parameter, the parameter being selected from the group consisting of the relative amplitude of the at least two detected resonance modes and the linewidth of the at least two detected resonance modes. The apparatus according to any one of the appendices 8 to 10, characterized in that it is a device.

[0092] (Note 12) The aforementioned algorithm unit is (i) The system is configured to determine further parameters of the at least one microsensor using a numerical algorithm from the spectral positions of the at least two detected resonance modes and, preferably, from at least one further parameter selected from the group consisting of the relative amplitude of the at least two detected resonance modes and the linewidth of the at least two detected resonance modes. The further parameters are preferably selected from the group consisting of the diameter of the at least one microsensor, the refractive index of the at least one microsensor, the refractive index of the adsorption layer of the at least one microsensor, and / or combinations thereof. (ii) From the spectral positions of the at least two detected resonance modes, preferably with respect to at least one further parameter selected from the group consisting of the relative amplitude of the at least two detected resonance modes and the linewidth of the at least two detected resonance modes, a numerical algorithm is used to determine the parameters of the fluid, preferably the optical properties of the fluid. It is configured to The apparatus according to any one of the appendices 8 to 11, characterized by the features described herein.

[0093] (Note 13) The container further contains a fluid which may contain the analyte, and the container is optionally a fluid channel. The apparatus according to any one of the appendices 8 to 12, characterized by the features described herein.

[0094] (Note 14) The at least one microsensor is (i) It is freely movable within the container and optionally freely movable within the fluid flow path of the apparatus, (ii) Fixed within the fluid channel of the apparatus, and in particular fixed on the inner surface of the fluid channel of the apparatus, the fluid channel is particularly suitable for supplying the at least one microsensor to a fluid of analysis which may contain an analyte. The apparatus according to any one of the appendices 8 to 13, characterized by the features described herein.

[0095] (Note 15) The spectral analysis unit is configured to optionally repeat the detection of the at least two optical resonance modes of the at least one microsensor at least once several times, and The algorithm unit is configured to calculate the time course of the optical thickness of the adsorption layer of the at least one microsensor via a numerical algorithm, from the spectral positions of the at least two detected resonance modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonance modes and the linewidths of the at least two detected resonance modes. The spectral analysis unit is optionally configured to optionally repeat the detection of the at least two optical resonance modes of the at least one microsensor at least once, several times, and The algorithm unit is configured to determine, via a numerical algorithm, the temporal progression of at least one additional parameter of the at least one microsensor from the spectral positions of the at least two detected resonance modes, and preferably, at least one additional parameter selected from the group consisting of the relative amplitudes of the at least two detected resonance modes and the linewidths of the at least two detected resonance modes. The at least one further parameter is particularly preferably selected from the group consisting of the diameter of the at least one microsensor, the refractive index of the at least one microsensor, the refractive index of the adsorption layer of the at least one microsensor, the refractive index of the fluid, and combinations thereof. The apparatus described in any one of the appendices 8 to 14, characterized by the features described herein.

[0096] (Note 16) Further equipped with fluid channels, The fluid passage is preferably, (i) including a supply line suitable for supplying at least one microsensor to the fluid flow path, and / or (ii) The device includes an outlet suitable for discharging the at least one microsensor from the fluid flow path, the outlet preferably having a separator for the at least one microsensor. The apparatus according to any one of the appendices 8 to 15, characterized in that it is a device.

[0097] (Note 17) The fluid channel has at least one transparent wall that is transparent to light of wavelengths within the following range in at least a portion of the region, In other words, the said range is, (i) The range of emission wavelengths of the fluorescent marker, In this case, preferably the detection optical system is positioned between the transparent wall and the spectral analysis unit, and particularly preferably the concave reflector is positioned on the side of the container facing the transparent wall, and / or (ii) The range of excitation wavelengths of the fluorescent marker, and / or (iii) The range of the excitation wavelength and emission wavelength of the fluorescent marker, In this case, a detection optical system having a coupling element for the light of the light source is placed between the transparent wall and the spectral analysis unit. The coupling element is reflective to light having wavelengths in the excitation wavelength range of the fluorescent marker, and transmittance to light having wavelengths in the emission wavelength range of the fluorescent marker, and / or (iv) A range of emission wavelengths of the fluorescent marker, which thereby enables the implementation of additional sensor technology, preferably a photodetector. The apparatus as described in Appendix 16, characterized by the features described herein.

[0098] (Note 18) The algorithm unit and the analysis unit, preferably the spectral analysis unit, the algorithm unit and the analysis unit are designed as a single unit, preferably as an integrated unit. The apparatus according to any one of the appendices 8 to 17, characterized by the features described herein. [Explanation of Symbols]

[0099] 10: Fluid channel (flow path); 15: Walls of fluid channels with special properties for absorbing or reflecting excitation light; 20: Fluid in flow (such as aqueous solution in flow); 30: A supply line for at least one microsensor; 40: Microsensor 43: Microresonators (particles containing or composed of dielectric material and a fluorescent marker); 45: Adsorption layer; 50: Transparent wall for a fluid channel used for fluorescence detection; 55: Transparent wall of a fluid channel for fluorescence excitation; 58: Additional sensor technologies, such as transparent walls for fluid channels to implement photodetectors; 60: Excitation light; 70: Separator for at least one microsensor; 80: An outlet for at least one microsensor; 90: Detection optics; 100: Fluorescence from at least one microsensor; 110: Spectral analysis unit; 120: Algorithm Unit; 130: Analysis Unit; 140: Coupling element for light (excitation light) from a light source; 150: Photodetector; 160: concave reflector; 170: Adsorption layer in media with different refractive indices Micro resonator WGM spectrum; 175: Refractive index of the medium surrounding the microresonator; 180: WGM spectra of microresonators with or without an adsorption layer in a medium with a refractive index of 1.33; 185: Thickness of the adsorption layer

Claims

1. A method for detecting analytes in a fluid in a label-free manner, (a) A step of supplying at least one dielectric microsensor into a container, The at least one microsensor comprises or consists of a microresonator and an adsorption layer coated on the microresonator for binding the analyte. The microresonator comprises a dielectric material and a fluorescent marker, or consists of particles made thereof, has an optical refractive index higher than the optical refractive index of the fluid being analyzed, and is suitable for generating multiple resonance modes inside the microresonator when the fluorescence of the fluorescent marker is excited, and the process is as follows: (b) The step of bringing at least one microsensor into contact with the fluid to be analyzed, which may contain the analyte, (c) A step of irradiating light onto the at least one microsensor in the fluid, wherein the light has a wavelength suitable for exciting the fluorescent marker of the at least one microsensor to produce fluorescence, (d) A step of detecting at least two optical resonance modes of the at least one microsensor from the fluorescence detected by the at least one microsensor, (e) A step of determining the optical thickness of the adsorption layer of the at least one microsensor in the fluid via a numerical algorithm from the spectral positions of the at least two detected resonance modes, (f) A method comprising the step of determining the extent to which an analyte in the fluid is bound to the at least one microsensor based on a predetermined optical thickness of the adsorption layer of the at least one microsensor.

2. The particles of the microresonator have a diameter in the range of 1 μm to 20 μm, and / or the adsorption layer has a thickness in the range of 0.5 nm to 30 nm, where the thickness refers to the spatial extent of the adsorption layer in the radial direction from the center point of the microresonator. The method according to feature 1.

3. The step of determining the optical thickness of the adsorption layer of the at least one microsensor is not performed before step (b). The method according to 1 or 2, characterized by the features described above.

4. The optical thickness of the adsorption layer is determined using a numerical algorithm from the spectral positions of the at least two detected resonance modes and at least one further parameter, the parameter being selected from the group consisting of the relative amplitude of the at least two detected resonance modes and the linewidth of the at least two detected resonance modes. The method according to feature 1.

5. moreover, (i) From the spectral positions of the at least two detected resonance modes, further parameters of the at least one microsensor are determined using a numerical algorithm, The further parameters are selected from the group consisting of the diameter of the at least one microsensor, the refractive index of the at least one microsensor, the refractive index of the adsorption layer of the at least one microsensor, and / or combinations thereof. (ii) The parameters of the fluid are determined using a numerical algorithm from the spectral positions of the at least two detected resonance modes. The method according to feature 1.

6. The at least one microsensor is (i) It is freely movable, or (ii) Fixed on the inner surface of the fluid channel through which the contact between the at least one microsensor and the analyte fluid which may contain the analyte is made The method according to feature 1.

7. The detection of at least two optical resonance modes of the at least one microsensor is repeated at least once to obtain the temporal evolution of the optical thickness of the adsorption layer of the at least one microsensor using a numerical algorithm based on the spectral positions of the at least two detected resonance modes. The method according to feature 1.

8. A device for detecting analytes in a fluid in a label-free manner, (a) A container comprising at least one dielectric microsensor, The at least one microsensor comprises or consists of a microresonator and an adsorption layer coated on the microresonator for binding the analyte. The microresonator comprises a container and particles containing or comprising a dielectric material and a fluorescent marker, having an optical refractive index higher than the optical refractive index of the fluid being analyzed, and being suitable for generating multiple resonance modes inside the microresonator when the fluorescence of the fluorescent marker is excited. (b) A light source for irradiating light onto the at least one microsensor, wherein the light has a wavelength suitable for exciting the fluorescent marker of the at least one microsensor to produce fluorescence, (c) A spectral analysis unit configured to detect at least two optical resonance modes of at least one microsensor from the detected fluorescence, (d) An algorithm unit configured to determine the optical thickness of the adsorption layer of the at least one microsensor via a numerical algorithm from the spectral positions of the at least two detected resonance modes, (e) an analysis unit configured to determine the extent to which an analyte has bonded to the at least one microsensor based on the determined optical thickness of the adsorption layer of the at least one microsensor.

9. The particles of the microresonator have a diameter in the range of 1 μm to 20 μm, and / or the adsorption layer has a thickness in the range of 0.5 nm to 30 nm, where the thickness refers to the spatial extent of the adsorption layer in the radial direction from the center point of the microresonator. The apparatus according to feature 8.

10. The spectral analysis unit is configured to perform the detection of the at least two optical resonance modes of the at least one microsensor only after the at least one microsensor has come into contact with a fluid that may contain the analyte. The apparatus according to feature 8 or 9.

11. The algorithm unit is configured to determine the optical thickness of the adsorption layer of the at least one microsensor via a numerical algorithm from the spectral positions of the at least two detected resonance modes and at least one further parameter, the parameter being selected from the group consisting of the relative amplitudes of the at least two detected resonance modes and the linewidths of the at least two detected resonance modes. The apparatus according to feature 8.

12. The aforementioned algorithm unit is (i) The system is configured to determine further parameters of the at least one microsensor from the spectral positions of the at least two detected resonance modes using a numerical algorithm, The further parameters are selected from the group consisting of the diameter of the at least one microsensor, the refractive index of the at least one microsensor, the refractive index of the adsorption layer of the at least one microsensor, and / or combinations thereof. (ii) From the spectral positions of the at least two detected resonance modes, the parameters of the fluid are determined by a numerical algorithm. It is configured to The apparatus according to feature 8.

13. The container further contains a fluid which may contain the analyte. The apparatus according to feature 8.

14. The at least one microsensor is (i) It is freely movable within the container, (ii) Fixed within the fluid flow path of the apparatus The apparatus according to feature 8.

15. The spectral analysis unit is configured to repeat the detection of the at least two optical resonance modes of the at least one microsensor at least once, and The algorithm unit is configured to calculate the time course of the optical thickness of the adsorption layer of the at least one microsensor via a numerical algorithm, based on the spectral positions of the at least two detected resonance modes. The apparatus according to feature 8.

16. Further equipped with fluid channels, The fluid channel is, (i) Includes a supply line suitable for supplying the at least one microsensor to the fluid flow path, and / or (ii) Including an outlet suitable for discharging the at least one microsensor from the fluid flow path The apparatus according to feature 8.

17. The fluid channel has at least one transparent wall that is transparent to light of wavelengths within the following range in at least a portion of the region, In other words, the said range is, (i) The range of emission wavelengths of the fluorescent marker, and / or, (ii) The range of excitation wavelengths of the fluorescent marker, and / or (iii) The range of the excitation wavelength and emission wavelength of the fluorescent marker, In this case, a detection optical system having a coupling element for the light of the light source is placed between the transparent wall and the spectral analysis unit. The coupling element is reflective to light having wavelengths in the excitation wavelength range of the fluorescent marker, and transmittance to light having wavelengths in the emission wavelength range of the fluorescent marker, and / or (iv) The range of emission wavelengths of the fluorescent marker, and thereby enabling the implementation of additional sensor technology, The apparatus according to feature 16.

18. The algorithm unit and the analysis unit, or the spectral analysis unit, the algorithm unit and the analysis unit are designed as a single unit. The apparatus according to feature 8.

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