Methods and apparatus for label-free detection of analytes - Patents.com

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]

[0001] A method and device for label-free detection of analytes in fluids is presented. [Background technology]

[0002] At least one dielectric microsensor is used, comprising a microresonator and an adsorbent layer applied to the microresonator for binding the analyte. The microresonator is composed of a dielectric material and particles having a fluorescent marker. Furthermore, the microresonator has an optical refractive index greater than the optical refractive index of the fluid to be analyzed. The microresonator is adapted to allow a plurality of resonant modes to be characterized when the fluorescent marker is excited therein. An optical thickness of the adsorbent layer of the microsensor is determined from the spectral positions of at least two detected optical resonant modes of the microsensor, and the extent to which the analyte has bound to the at least one microsensor is determined therefrom.

[0003] Whispering Gallery Mode (WGM) based sensors are suitable for the determination of physical, chemical and biochemical parameters of fluids. Fluorescent microparticles with a diameter of a few micrometers are used as microscopic optical sensors. The microparticles and their surfaces can be tailored accordingly to fulfill the specific task in question, for example by specifically functionalizing their surface using applied biochemical layers (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 longer wavelength compared to the excitation light. This light is emitted in any spatial direction and may even be totally reflected inside the microparticle (i.e. inside the microparticle) at an oblique incidence on the microparticle's walls if the refractive index of the microparticle is greater than that of the surrounding medium. Depending on the diameter of the microparticle, the microparticle becomes an optical cavity of a specific size, which is filled by the individual wavelengths of the spectrally broad fluorescence spectrum in the form of resonant modes. The spectrum of the external emission from the microparticle is characterized by these modes.

[0005] If the optical diameter of a microparticle changes, for example due to binding (or adhesion) to the outer surface of a substance to be analyzed (analyte), the characteristic spectrum of the microparticle also changes. By analyzing the characteristic spectrum of the microparticle using spectral analysis, conclusions can be drawn about the effective optical diameter of the microparticle and thus indirectly about the surface occupancy of the analyte. Microparticles suitable for this procedure are also referred to below as "microresonators".

[0006] The microresonator can be wrapped with an adsorbent layer that is physically and / or chemically applied or bonded to the surface of the microresonator and has a function suitable for the respective application of the microresonator. For example, if the microresonator is used as a biosensor, this can be an adsorbent layer (e.g. an organic layer) suitable for specifically binding a particular analyte. The adsorbent layer is usually chemically different from the material of the microresonator and therefore can have a different optical index of refraction than the substrate of the microresonator. The microresonator and adsorbent layer are also referred to as a "microsensor".

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

[0008] For this reason, in previously recognized methods for quantifying the surface occupancy of each microresonator, the spectrum determined after adsorption had to always be referenced to a spectrum from the same microresonator before adsorption, i.e., the microresonator had to be measured at least once before and after binding of the analyte (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 expensive, and significantly limits the applicability of WGM-based microsensors. For example, the need to measure one and the same microresonator multiple times means, among other things, that it must be detectable several times during the measurement process. Therefore, previous methods have used immobilized microsensors, for example adsorbed on rigid surfaces, biological cells or attached to microstructures.

[0010] Therefore, a measurement method is desired where the microsensor can move freely in the medium to be analyzed, but this is not possible with the methods of quantifying the surface occupancy known in the prior art due to the immobilization of the microsensor. Furthermore, the immobilization of the microsensor in the prior art methods does not allow for continuous (i.e. continuous) measurements. As a result, existing implementations of WGM-based microsensor technology are so far only suitable for specific laboratory systems and not for applications in the field of continuous process monitoring. An example of a laboratory system that distinguishes itself from other state-of-the-art systems due to the use of a microchannel integrated with a holding structure was presented by Bischler et al. (R. Bischler et al.Phys. J. Special Topics, 2014, Vol. 223, pages 2041-2055 and DE 10 2014 104 595 A1). Here, the microsensor is only temporarily fixed in position by the holding structure and can be repeatedly moved to the measurement position by an automatic mechanical displacement unit. Once the measurement is completed, the microfluidic channel can be washed and refilled. The significance of the measurement results can be increased by continuously moving multiple microsensors during the measurement. Nevertheless, this system is not suitable for use in continuous process monitoring, since both the volume flow rate through the microfluidic channel and the number of microsensors available for one measurement are limited and far below the requirements for continuous process monitoring. Moreover, the relative shift of the resonance modes is also used to draw conclusions about the surface occupancy, as is the case in the state of the art, so multiple measurements of one and the same microsensor are 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 not only applies to previous implementations of WGM-based microsensor technology, but also affects other common methods for label-free detection of biological species, such as surface plasmon resonance (SPR), quartz crystal microbalance (QMC), and / or ellipsometry. In all of these methods, the sensor surface, which reacts specifically with the analyte, is usually formed as a wall in a microchannel. The sensor surface can be supplied with the analyte and the medium required for surface conditioning through the microchannel. However, since laminar flows are usually formed in the microchannel, the flow on the sensor surface is practically stationary and the transport of the analyte to the sensor surface is diffusion-limited. This results not only in long measurement times, but also in distorted (i.e. inaccurate) binding kinetics when the diffusion zone is exhausted, leading to incorrect determination of the affinity or affinity of the analyte to its binding partner.

[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 there, the microparticles flow freely with the medium to be analyzed and are randomly read out when detected by the optical system. Since each microparticle is measured only once, it is considered that the necessary referencing cannot be done on the same microparticle but must be done by other means. To this end, US 8,779,389 B2 proposes comparing one characteristic spectrum of the microparticle detected in each case with a set of appropriately pre-determined characteristic spectra and using this comparison to determine the best-fitting pre-defined spectrum. Analytes adsorbed to the microparticles are detected via the deviation of the measured spectrum from an optimal pre-defined spectrum. It is not explained in more detail how the optimal pre-defined spectrum can be determined without additional measurement criteria if the measured spectrum deviates from the spectrum measured in the presence of the analyte, such as before the interaction of the microparticle with the analyte. Furthermore, the refractive index of the medium can be pre-determined and the optimal spectrum can be compared with the measured refractive index with reference. To determine the refractive index of the medium, a quantity of microparticles sufficient for statistical evaluation in the selected medium is measured beforehand in the absence of analyte, obtaining a statistical reference value from which the refractive index of the medium can be determined. Deviations from the reference value are evaluated as indicative of the presence of the analyte in the medium. Thus, in each embodiment, the method only provides qualitative information on the presence of the analyte in the analyzed medium, in the sense of a "yes / no sensor", and therefore does not meet the requirements of the quantitative biosensor technology described above, which also allows, for example, the measurement of binding kinetics and, hence, the determination of affinity and avidity. In contrast, the method of the invention determines the optical thickness of an adsorbent layer located on the microresonator, which also contains a potential analyte. The optical thickness of this adsorbent layer is determined independently of the refractive index of the medium, providing a quantitative measure of the surface occupancy of the microresonator and allowing a quantitative description of the concentration of the analyte in the medium and of the measurement of binding kinetics. Summary of the Invention [Problem to be solved by the invention]

[0013] Based on this, it was the object of the present invention to provide a method and an apparatus which do not have the drawbacks known from the prior art, in particular by which it is possible to quantitatively detect analytes in unlabelled samples in a rapid, economical and continuous manner, without the risk of falsifying the results, and which also makes it possible to detect the pure (i.e. correct) binding kinetics of the analyte and the target molecule. [Means for solving the problem]

[0014] This problem is solved by a method having the features of claim 1 and by a device having the features of claim 8. The dependent claims indicate advantageous further developments.

[0015] According to the invention there is provided a method for label-free detection of an analyte in a fluid comprising (or consisting of) the following steps: a) providing at least one dielectric microsensor in the container, the at least one microsensor consisting or consisting of a microresonator and an adsorbent layer applied to the microresonator for binding the analyte, the microresonator consisting or consisting of particles of a dielectric material and a fluorescent marker, the microresonator having an optical refractive index greater than the optical refractive index of the fluid to be analyzed, the microresonator being suitable for enabling the emergence of a plurality of resonance modes within the microresonator upon excitation of fluorescence of the fluorescent marker; b) contacting at least one microsensor with a fluid to be analyzed, the fluid possibly containing an analyte; c) illuminating at least one microsensor in the fluid with light, the light having a wavelength suitable for exciting a fluorescent marker of the at least one microsensor into fluorescence; d) detecting at least two optical resonant modes of the at least one microsensor from the detected fluorescent light of the at least one microsensor; e) determining, via a numerical algorithm, an optical thickness of an adsorbent layer of the at least one microsensor in the fluid from the spectral positions of the at least two detected resonant modes; and f) determining the extent to which the analyte in the fluid is bound to the at least one microsensor based on the previously determined optical thickness of the adsorbent layer of the at least one microsensor.

[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. The absolute thickness of the adsorbent layer can then be determined based on the position of the detected modes relative to one another. By comparing the thickness of the adsorbent layer thus obtained with the known thickness of the adsorbent layer applied to the microresonator during the manufacturing process, a qualitative conclusion can be drawn, without being influenced by tampering, about the level of the analyte bound (or accumulated) on the surface of the microsensor, i.e. the level of the analyte in the fluid bound to the microsensor. By determining the absolute thickness of the adsorbent layer from individual measurements on the microsensor, the method according to the invention relates to a generic measurement that can be determined under a wide variety of process conditions. As a result, the method according to the invention is suitable for use under constantly changing process conditions and also for continuous (i.e. continuous) process monitoring. Since the requirement of maintaining identical process conditions as in the prior art methods is unnecessary, the method according to the invention can also be carried out more simply, faster, more economically and with less equipment.

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

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

[0019] Furthermore, the adsorbent layer can have 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, in particular 2 nm to 8 nm, whereby the thickness is understood to be the spatial extension of the adsorbent layer in the radial direction from the central point of the microresonator. In the case of very thin adsorbent layers in the range of a few nanometers (for example thickness ≦ 2 nm), the separation of the optical refractive index and the geometric layer thickness can no longer be reliably implemented. The main result for determining the optical thickness of the adsorbent layer of at least one microsensor is the so-called "optical layer thickness", i.e. a product of the type nAds*dAds, where nAds represents the optical refractive index of the adsorbent layer and dAds represents the radial thickness. This definition is comparable to the optical path length known in optics, which represents the product of the optical refractive index and the geometric optical path length. For the purposes of the present invention, knowledge of the optical layer thickness is sufficient, since the optical layer thickness changes with the adsorption of the analyte to the adsorbent layer, even if the geometric layer thickness only changes slightly due to the diffusion of the analyte or its exchange with previously non-specifically bound molecules, etc. Moreover, the optical refractive index of most substances (such as biomolecules) associated with the structure of the adsorbent layer is very similar to the optical refractive index of the analyte to be bound. The optical refractive index is usually in the range of 1.43 to 1.48.

[0020] In this method, preferably, prior to step b), there is no step of determining the optical thickness of the adsorbent layer of the at least one microsensor, which embodiment allows the method to be carried out more quickly and economically.

[0021] The optical thickness of the adsorbent layer can be determined in a process according to rigorous classical field theory.

[0022] In this method, the optical thickness of the adsorbent layer can be determined by using a numerical algorithm from the spectral positions of the at least two detected resonant modes and at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes. This embodiment has the advantage that the optical thickness of the adsorbent layer can be determined more accurately. By using a microresonator suitable for multiple resonant modes to appear inside the microresonator when the fluorescence of the fluorescent marker is excited, the linewidth of the individual modes can be made smaller than the spectral interval, making it possible to spectrally separate the modes.

[0023] Further, in the method, further parameters of the at least one microsensor can be determined from the spectral positions of the at least two detected resonant modes, preferably using a numerical algorithm, from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes. The further parameter is 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 adsorbent layer of the at least one microsensor, and combinations thereof.

[0024] Alternatively, a parameter of the fluid can be determined in the method using a numerical algorithm from the spectral positions of the at least two detected resonant modes, preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes, preferably an optical property of the fluid can be determined.

[0025] At least one microsensor used in the method can be designed as an essentially spherical particle. This is advantageous because the property of the microsensor's microresonator to form resonant modes depends on the shape of the microresonator. Due to their high symmetry, spherical particles form resonant modes that are independent of each propagation plane within the particle, facilitating the detection of particles moving freely in fluids in particular. Particles with very strong asphericity (e.g. irregular shapes, i.e. shapes without an axis of symmetry) do not show observable resonant modes because light is scattered from the particle too quickly. Therefore, the lifetime of the resonant modes of such particles is very short and they are no longer observable due to linewidth overlap.

[0026] The fluorescent markers of the micro-resonator can be located inside the micro-resonator or on the outer surface of the micro-resonator.

[0027] The detection of the at least two optical resonant modes of the at least one microsensor may be performed by detecting light scattered by the at least one microsensor and / or light emitted by the at least one microsensor.

[0028] The at least one microsensor used in the method can be freely movable, which is preferred as this allows the method to be carried out more quickly and economically, or the at least one microsensor can be fixed, in particular fixed to an inner surface of a fluid flow path in which contact between the at least one microsensor and the fluid to be analysed (which may contain the analyte) takes place.

[0029] In this method, the detection of at least two optical resonance modes of the at least one microsensor can be repeated at least once, and optionally several times, in order to determine, using a numerical algorithm, the time course of the optical thickness of the adsorbent layer of the at least one microsensor 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 amplitudes of the at least two detected resonance modes and the line widths of the at least two detected resonance modes. This embodiment has the advantage that the binding kinetics of the analyte to the microsensor can be detected. In contrast to the prior art methods using immobilized microsensors, the determination of the binding kinetics using the method according to the invention is not subject to errors. In addition to the binding kinetics itself, it is also possible to determine whether it has already reached a stationary state. From the determination of the amount of analyte adsorbed at 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 the at least one microsensor can also be determined from the spectral positions of the at least two detected resonant modes, preferably using a numerical algorithm, from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes. The at least one further parameter can be 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 adsorbent layer of the at least one microsensor, the refractive index of the fluid, and combinations thereof. An advantage of this embodiment is that the time course of other parameters of the at least one microsensor or the fluid of the analyte can also be recorded.

[0031] According to the invention there is further provided an apparatus for label-free detection of an analyte in a fluid, comprising (or consisting of): a) a container comprising at least one dielectric microsensor, the at least one microsensor comprising or consisting of a microresonator and an adsorbent layer applied to the microresonator for binding an analyte, the microresonator consisting of particles comprising or consisting of a dielectric material and a fluorescent marker, the microresonator having an optical refractive index greater than the optical refractive index of the fluid to be analyzed, the microresonator being suitable for allowing the appearance of a plurality of resonance modes inside the microresonator upon excitation of the fluorescence of the fluorescent marker; b) a light source for illuminating the at least one microsensor with light, the light source having a wavelength suitable for exciting a fluorescent marker of the at least one microsensor into fluorescence; c) a spectral analysis unit configured to detect at least two optical resonance modes of the at least one microsensor from the detected fluorescent light; d) an algorithm unit configured to determine, via a numerical algorithm, an optical thickness of the adsorbent layer of the at least one microsensor from the spectral positions of the at least two detected resonant modes; and e) an analysis unit configured to determine the extent to which the analyte is bound to the at least one microsensor based on the determined optical thickness of the adsorbent layer of the at least one microsensor.

[0032] The device according to the invention can be used to draw qualitative and even quantitative conclusions about the extent to which the analyte has been bound (or adsorbed) to the surface of the microsensor, i.e. the extent to which the analyte in the fluid has been bound to the microsensor, without being subject to falsification. The device allows the determination of the analyte under a wide range of process conditions, since the absolute thickness of the adsorbate layer can be determined from the individual measurements on the microsensor. As a result, the device according to the invention is also suitable for use under constantly changing process conditions and for continuous (i.e. continuous) process monitoring. Since it is not necessary to maintain the same process conditions as in the prior art devices, the detection of the analyte with the device according to the invention can also be performed more simply, faster, more economically and with less equipment.

[0033] The device according to the invention can also detect the binding of analytes to non-immobilized (i.e. freely movable) microsensors. As a result, the device according to the invention does not require, for example, a movable stage, which was necessary in prior art devices to fix and specifically position the microsensor above the detection unit. The use of costly mechanical positioning axes and, for example, air gaps between the detection optics and the microstructure holding the microsensor can thus be omitted. By eliminating the air gap, immersion objectives with numerical apertures of 1.0 or more (theoretical limit of air gap objectives) can be used, allowing a higher proportion of the fluorescence emitted by the microsensor to be detected and fed to downstream spectroscopic optics. This increased signal strength allows a significant reduction in the time required to detect the emission spectrum of the microsensor under investigation, so that even fast flowing microsensors can be detected with sufficient signal strength 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, particularly preferably 4 μm to 10 μm. The adsorbent layer can have 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, particularly preferably 2 nm to 8 nm, whereby the thickness is understood to be the spatial extension of the adsorbent layer in the radial direction from the central point of the microresonator.

[0035] The spectral analysis unit may be configured to perform detection of the at least two optical resonant modes of the at least one microsensor only after the at least one microsensor has come into contact with a fluid possibly containing the analyte. This embodiment has the advantage that detection of the analyte can be performed more quickly and economically with the device.

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

[0037] Further, the algorithm unit may be configured to determine, via a numerical algorithm, an optical thickness of the adsorbent layer of the at least one microsensor from the spectral positions of the at least two detected resonant modes and at least one further parameter selected from the group consisting of the relative amplitudes of a minimum of the two detected resonant modes and the linewidths of the at least two detected resonant modes.

[0038] Alternatively, the algorithm unit may be configured to determine, using a numerical algorithm, a further parameter of the at least one microsensor from the spectral positions of the at least two detected resonant modes, preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes. The further parameter is 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 adsorbent layer of the at least one microsensor, and combinations thereof.

[0039] Further, the algorithm unit may be configured to determine a parameter of the fluid, preferably an optical property of the fluid, from the spectral positions of the at least two detected resonant modes using a numerical algorithm, preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes.

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

[0041] The fluorescent marker of the microresonator can be located inside the microresonator or on the outer surface of the microresonator.

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

[0043] The container of the device may further contain a fluid, which may include an analyte. The container is optionally a fluid flow path.

[0044] The at least one microsensor is freely movable within the container and, optionally, within the fluid flow path of the device. This embodiment is advantageous in that it allows for a faster and more economical detection of the analyte. Alternatively, the at least one microsensor can be present in a fixed state in the fluid flow path of the device, in particular fixed to an inner surface of the fluid flow path of the device, the fluid flow path being particularly suitable for supplying the at least one microsensor to the fluid to be analyzed, which may contain the analyte.

[0045] The spectral analysis unit may be configured to repeat the detection of the at least two optical resonance modes of the at least one microsensor at least once and optionally several times, and the algorithm unit is configured to determine the time course of the optical thickness of the adsorbent layer of the at least one microsensor from the spectral positions of the at least two detected resonance modes, preferably using a numerical algorithm, and 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. This embodiment has the advantage that the binding kinetics of the analyte to the microsensor can be detected.

[0046] Furthermore, the spectral analysis unit may be configured to repeat the detection of the at least two optical resonant modes of the at least one microsensor at least once, optionally several times, and the algorithm unit is configured to determine, via a numerical algorithm, the time course of at least one further parameter of the at least one microsensor from the spectral positions of the detected resonant modes, preferably from the group consisting of the relative amplitudes of the at least two detected resonant modes and the line widths of the at least two detected resonant modes, where the at least one further parameter is selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the line widths of the at least two detected resonant modes, which 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 adsorbate layer of the at least one microsensor, the refractive index of the fluid and combinations thereof. An advantage of this embodiment is that the time course of other parameters of the at least one microsensor or the fluid of the analyte can also be recorded.

[0047] The device may include a fluid flow path. The fluid channel preferably includes a supply line suitable for supplying at least one microsensor to the fluid channel. Additionally, the fluid flow path preferably includes an outlet suitable for expelling the at least one microsensor from the fluid flow path, the outlet preferably including a separator for the at least one microsensor.

[0048] The fluid flow path may have at least one transparent wall that is transparent to light having a wavelength in the range of the emission wavelength of the fluorescent marker, at least in certain areas, the detection optical system is preferably arranged between the transparent wall and the spectral analysis unit, and the concave reflector is particularly preferably arranged on the side of the container facing the transparent wall.

[0049] Additionally, the fluid channel may have at least one transparent wall that is transparent to light of wavelengths in the range of the excitation wavelengths of the fluorescent markers, at least in certain regions.

[0050] Alternatively, the fluid flow path may have at least one transparent wall which is transparent, at least in certain regions, to light having wavelengths within the range of excitation and emission wavelengths of the fluorescent markers, and detection optics having a coupling element for the light of the light source is arranged between the transparent wall and the spectral analysis unit, the coupling element being reflective to light of wavelengths in the range of excitation wavelengths of the fluorescent markers and transparent to light of wavelengths in the range of emission wavelengths of the fluorescent markers.

[0051] Furthermore, the fluid flow path may have at least one transparent wall that is transparent, at least in certain areas, to light of wavelengths in the range of the emission wavelengths of the fluorescent markers, allowing the implementation of additional sensor technology, preferably a photodetector.

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

[0053] The microresonators used in the method and / or device may consist of or consist of a material selected from the group consisting of polymers, preferably polystyrene, melamine resin, polydivinylbenzene, polymethylmethacrylate, poly(styrene-co-divinylbenzene), poly(styrene-co-methylmethacrylate) or polydimethylsilane. The microresonators used in the method and / or device may also consist of or consist of a material selected from the group consisting of inorganic materials, preferably inorganic dielectric materials such as silica (SiO2) or titanium dioxide.

[0054] The adsorbent layer of the microresonator used in the method and / or device may comprise or consist of a dielectric material, in particular an organic material, such as an oligomer, polymer, peptide, protein, oligonucleotide, DNA or cellular components, as well as mixtures or compounds of these materials, but may also be made of metals, semiconductors, metamaterials or combinations or compounds thereof, provided that they are not so strongly damped that the resonant modes become undetectable. Additionally, the adsorbent layer of a microresonator used in the present methods and / or devices can include or consist of a material having a refractive index in the range of 1.43 to 1.47.

[0055] Furthermore, the adsorbent layer of the microresonator used in the method and / or device may be bonded to the microresonator via non-covalent interactions (e.g., via ionic interactions, dipole-dipole interactions, van der Waals interactions and / or hydrogen bonds). Furthermore, the adsorbent layer may alternatively or additionally 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 thereon.

[0056] Alternatively, the adsorbent layer of the microresonator used in the method and / or device may be divided into several sublayers. Depending on the physical model and the contrast of the different optical refractive indices of the individual sublayers of the adsorbent layer, the algorithm unit may be configured to characterize the adsorbent layer as a homogeneous layer with 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 description of the drawings]

[0057] The following figures and examples are intended to more fully illustrate the objects of the present invention and are not intended to limit the invention to the specific embodiments shown.

[0058] [Figure 1]FIG. 1 shows diagrammatically a device according to the invention with different transparent walls 50, 55 in a container (here a microchannel) for excitation of the microsensor 40 by an excitation light 60 and readout of the fluorescence light 100 of the microsensor 40. A fluid 20 moves in a flow path 10 where it is checked for the presence and concentration of a desired analyte by adding a WGM-based microsensor 40 via a dosing system 30. The microsensor 40 flows a certain distance with the fluid and can be excited to fluoresce through the transparent wall 55 with the help of an excitation light 60. The fluorescence emission 100 is further guided via the transparent wall 50 and downstream detection optics 90 to a spectral analysis unit 110, where the light is read out spectroscopically. As the flow continues, the microsensor 40 can be optionally removed again from the fluid by a suitable capture device 70 in combination with an outlet 80. For non-critical fluids, such as wastewater for example, the microsensor can remain at the outlet of the fluid, so that the components 70 and 80 are not necessary. [Diagram 2] FIG. 2 shows diagrammatically a further device according to the invention, designed similarly to FIG. 1, but having only a single transparent wall 50 for exciting the microsensor 40 with excitation light 60 and for reading out the fluorescent light 100 from the microsensor 40. [Diagram 3] FIG. 3 shows diagrammatically a further device according to the invention, which is designed similarly to FIG. 1, but with a different orientation of the excitation light 60 and a different beam shaping (collimated in FIG. 3a, focused in FIG. 3b). [Figure 4A] FIG. 4A shows an example of the fluorescence emission from a microsensor as a function of wavelength, showing various resonant modes with different polarization states TM, TE. [Figure 4B] FIG. 4B shows a schematic structure of a microsensor 40 consisting of a microresonator 43 and an adsorption layer 45. [Figure 4C] FIG. 4C shows an example of the increase in the spectral resonant modes TM, TE with increasing microsensor diameter. [Figure 4D]FIG. 4D shows an example of an increase in the resonant modes TM, TE in the spectrum of a microsensor with increasing thickness of the adsorbent layer of the microsensor (due to the binding of analyte to the adsorbent layer). [Figure 5A] 5A shows an example simulation of the WGM spectrum 170 of a microresonator (i.e., a dielectric microsensor) with radius R=3400 nm, internal refractive index 1.59 (an approximate value for polystyrene), and no adsorbent layer (i.e., a dielectric microsensor). The WGM spectra 170 in FIG. 5A were background corrected and normalized to their respective maximum values ​​before correction. [Figure 5B] FIG. 5B shows an exemplary numerical simulation of the WGM spectrum 180 of a microresonator (i.e., a dielectric microsensor) with radius R=3400 nm, internal refractive index 1.59 (approximate value for polystyrene), and with no adsorbent layer (adsorbent layer thickness 185 was set to 0 nm) or with an adsorbent layer of a certain thickness 185 (i.e., adsorbent layer thickness 185 was set to 5 nm, 20 nm, 60 nm, 100 nm, 140 nm, 180 nm or 200 nm) in the medium surrounding the microresonator with refractive index 1.33, in which the adsorbent layer had a refractive index m = 1.48. The WGM spectrum 180 in FIG. 5B was background corrected and normalized to its respective maximum value before correction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0060] In order to detect analytes in a fluid (analyte sample) and to detect binding events on the microsensor surface, it is essential to be able to determine the change in thickness of the adsorbent layer on the microresonator caused by the binding of the analyte of interest.

[0061] In contrast to particle size, the lot-to-lot scattering of the thickness of the specifically functionalized adsorbent layer is relatively small due to its low total thickness, typically a few to tens of nanometers, and therefore can be easily distinguished from adsorption events on the microsensor surface within the evanescent field of a resonant mode with an extension of about 50-100 nm from the surface of the microresonator, even without statistical or direct references. If the thickness of the adsorbent layer is known, it is possible to draw direct qualitative and quantitative conclusions about the binding events, even without a reference.

[0062] The separation of individual parameters such as the size of the microresonator and the thickness of the adsorbed layer can be achieved by their different influence on the mode positions of the various resonant modes of the microsensor. In dielectric (i.e. non-conductive and non-adhesive or only slightly adhesive) microresonators, two different types of resonant modes arise with different orientations of the electric field (see Figure 4B). In one case, the electric field of the mode is radially polarized and the simultaneously occurring magnetic field is tangential to the particle surface ("transverse magnetic mode", abbreviated "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 encapsulated microparticles (AL Aden & M. Kerker, Scattering of electromagnetic waves from two concentric spheres, Journal of Applied Physics, 1951, Vol.22, pages 1242-1246).

[0063] The key to separating individual parameters, such as the thickness of the adsorbent layer and the diameter of the microresonator, is that while the TM and TE modes show different shifts with increasing adsorbent layer thickness, they behave in a good approximation and in the same way with respect to the change in sensor size (see Figures 4C and 4D). For example, in polystyrene-based microsensors adsorbing biomolecules in aqueous solutions, the TM modes show a stronger shift than the TE modes (see Figure 4D). Thus, by modern computing techniques, e.g. numerical modeling and fitting of Mie spectra to measured data, it is possible to clearly separate the size of the microresonator and the thickness of the adsorbent layer.

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

[0065] Thus, with the aid of computer techniques, it is possible to draw conclusions from the measured spectrum very quickly (typically within a few seconds) about the system microresonator 43 together with the adsorbate layer 45 in the fluid 20 and to determine the parameters essential for the respective problem (sensor size, layer thickness, optical refractive index of the adsorbate and optical refractive index of the environment, etc.). The individual parameters can be separated by their different influence on the mode positions of the various resonant modes of the microsensor 40.

[0066] Besides the Mie theory, there are other optical models for the excitation of resonant modes in spheres, such as the Debye theory and the Airy model, which are only approximations but 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 the Mie theory.

[0067] Example 2 – Process or Equipment Variations

[0068] 1 and 2 show two different embodiments of the device according to the invention or the method according to the invention for the fluorescence excitation of a microsensor 40. In FIG.

[0069] In FIG. 1, the excitation light 60 is irradiated into the fluid through a separate access window 55 and irradiates at least one passing microsensor 40. The access window 55 can be positioned either opposite the access window 50 used for detection or arranged such 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 in terms of transmission, reflection and absorption can be precisely adapted to the respective radiation (fluorescence excitation or emission). At least one microsensor 40 that happens to pass through the light cone is fluorescently excited and can be spectroscopically analyzed through the access window 50. For this purpose, the detection optics 90 captures a part of the fluorescence light 100 emitted by the at least one microsensor 40 and transfers it to a 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 with regard to characteristic variables such as the spectral positions (optionally also the relative amplitudes and / or linewidths) of the resonance modes of different polarizations, and these results are then evaluated in the analysis unit 130 with regard to the presence and / or concentration of the analyte sought. The information on the analyte thus determined is then provided by the device to an operator of the device via a suitable interface.

[0070] However, in Fig. 2, the excitation of fluorescence 100 of 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 the excitation light 60 through the access window 50 via the coupling element 140. Again, at least one microsensor 40 flowing in the fluid 20 and which happens to pass through the light cone of the excitation light 60 is excited to fluorescence 100, which is read out via the detection optics 90 and analysed with respect to the desired parameters with the help of the units 110, 120, 130, as described above. It is important here 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 Fig. 1). Although the demands on the access window 50 are higher since it must be transparent not only to the fluorescence emission 100 but also to the excitation light 60, 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 fluorescence emission 100 of the microsensor 40 (see FIG. 3a), allowing the detection optics 90 to collect the fluorescence light over a larger solid angle, resulting in higher signal strength, shorter measurement times and finally a wider range of possible flow velocities of the fluid.

[0071] Figures 1 and 2 show two basic embodiments of the invention, which can be modified in various ways, for example Figure 3 shows two different modifications.

[0072] In the embodiment shown in FIG. 3, the flow direction of the fluid also corresponds to the x-axis of this figure. Now, the excitation light 60 is no longer guided along the optical axis of the detection optics 90, but is irradiated into the fluid 20 at a different angle. The 90 degree angle shown here with respect to the optical axis of the detection optics 90 is only an example. FIGS. 3a and 3b also differ in the shape of the excitation light 60 used for the fluorescence excitation 100. In FIG. 3a, the excitation light 60 is parallel, whereas in FIG. 3b, it is focused at the focal point of the detection optics 90. The latter embodiment has the advantage that only microsensors in the immediate vicinity of the focal point of the detection optics 90 are significantly excited by the fluorescence 100, thus reducing potential interfering signals from at least one microsensor 40 at a position in the fluid 20 that is not suitable for detection. As already mentioned above with respect to FIG. 2, in the embodiment shown in FIG. 3, the channel wall opposite the access window 50 can also serve as a reflector 160 to increase the signal of the fluorescence light of at least one microsensor 40 collected by the detection optics 90.

[0073] Furthermore, Fig. 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 about the solids content of the fluid and thus assisting the analysis unit 130 in interpreting the measurement results. In the absence of the further access window 58 in the beam path of the excitation light 60, as shown in Fig. 3b, the excitation light 60 can be modified, for example absorbed or reflected, via the rear wall 15 of the flow channel 10 in order to suppress undesired 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 Figures 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 again a flow path corresponding to Figures 1-3 after appropriate separation or preparation of the portion of the fluid 20 selected for analysis. Thus, the embodiments shown here have a good general validity also for more complex applications of the microsensor technology shown here.

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

[0076] The method according to the invention utilizes the measurement of the optical thickness of the adsorbent layer of at least one microsensor in a fluid that may contain the analyte, and its change caused by the binding of the sought analyte, in order to determine the presence of the analyte in the fluid. The advantages of using the optical thickness of the adsorbent layer compared to other possible parameters such as the refractive index m of the medium in which the microsensor is placed are explained below.

[0077] FIG. 5A shows the WGM spectrum 170 of a microcavity (i.e., a dielectric microsensor) with radius R = 3.4 μm, internal refractive index 1.59 (approximate value for polystyrene), and no adsorbent layer (i.e., the thickness of the adsorbent layer was set to 0 nm) numerically simulated in a medium surrounding the microcavity with different refractive indexes 175. The 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 Par-ticles, 1998, Wiley-VCH-Verlag, ISBN 9780471 293408, implemented in Matlab® 2021b) as per the usual state of the art. For better comparison, the WGM spectrum 170 was background corrected. After background correction, the spectra were normalized to their original maximum, i.e., the maximum before background correction, in order to keep the respective WGM amplitudes comparable. Spectrum 180 shown in Figure 5B was treated similarly. The choice of a microresonator size of 6.8 μm diameter represents a proven compromise between the size of the resonator surface and the quality of the excitable WGMs. Larger microresonators reduce the relative area occupied by the adsorbed analyte on the microsensor surface and correspondingly reduce the mode shift. Smaller microresonators result in increased losses of WGM excitation due to surface scattering and radiation losses, leading to poorer properties of the individual WGMs and more difficult fabrication.

[0078] Figure 5A clearly shows that the linewidth of this microcavity sized WGM increases rapidly in m0 with increasing refractive index 175 of the medium, and that this leads to the TM and TE modes starting to overlap even at values ​​of refractive index 175 that are still lower than those expected for biological materials (as shown by M0 = 1.40), making it practically impossible to experimentally determine M0 in a range suitable for the detection of (biological) analytes or adsorbates (such as bacteria as in US 8,779,389 B2) as these modes start to overlap. In practice, there is a limit to using the refractive index 175 of the medium as an indicator for the detection of (biological) analytes or adsorbates. This means that the refractive index of the adsorbates is usually high, 1.43-1.48, and therefore only acceptable when the surface coverage is low, i.e. the adsorbate concentration is low and the analyte concentration in the medium (i.e. the liquid) is also low.

[0079] In contrast, when using the optical thickness of the adsorbent layer of at least one microsensor, as in the method and device according to the invention, any refractive index of the analyte and the adsorbent layer is possible. This is illustrated in FIG. 5B. FIG. 5B shows a numerical simulation of the WGM spectrum 180 of a microresonator (i.e., a dielectric microsensor) with a radius of 3.4 μm, an internal refractive index of 1.59 (approximate value for polystyrene), and either no adsorbent layer (adsorbent layer thickness 185 set to 0 nm) or an adsorbent layer with a thickness 185 (i.e., adsorbent layer thickness 185 set to 0 nm). The refractive index of the adsorbent layer was mAds=1.48. Simulations were performed in Matlab® 2021b as before, following the theory of coated spheres by Bohren & Hofmann (Craig F. Bohren & Donald R. Huffman, Absorption and Scattering of Light by Small Particles, 1998, Wiley-VCH-Verlag, ISBN 9780471293408). Despite the high refractive index of the ADS adsorbent layer, m = 1.48, the WGM is surprisingly easily distinguishable up to an adsorbent layer thickness of 185. Due to this differentiability, all relevant parameters, especially the optical thickness of the adsorbent layer, can be determined based on a rigorous theory in which all parameters have a physical meaning, as described above for Figure 4D, and in principle can be determined for any refractive index of the analyte and the adsorbent layer. At the same time, it can be seen that ignoring the adsorbent layer on the microresonator in the simulation of the scattering spectrum based on the Mie theory will lead to different, potentially misleading results, since a change in the refractive index m of the environment will always change the entire environment of the microresonator, and it will not be possible to describe only the partial filling of the near field around the microresonator. Therefore, prior art methods and devices that do not take into account the adsorbent layer on the microresonator may lead to erroneous results if the refractive index of the environment (e.g., the surrounding medium) changes.

[0080] The use of the optical thickness of the adsorption layer is therefore a more general and versatile parameter for the detection of analytes in fluids, and thus the method and device according to the invention are more versatile and reliable than those of the prior art, and avoid the risk of erroneous results due to changes in the refractive index of the microresonator's environment.

[0081] (Additional Note) (Appendix 1) 1. A method for label-free detection of an analyte in a fluid, comprising: (a) providing at least one dielectric microsensor in a container; the at least one microsensor includes or consists of a microresonator and an adsorbent layer applied to the microresonator for binding an analyte; the microresonator is made of particles comprising or consisting of a dielectric material and a fluorescent marker, has an optical refractive index higher than the optical refractive index of the fluid to be analyzed, and is suitable for developing a plurality of resonant modes inside the microresonator when the fluorescence of the fluorescent marker is excited; (b) contacting the at least one microsensor with a fluid to be analyzed that may contain an analyte; (c) shining light onto the at least one microsensor in the fluid, the light having a wavelength suitable to excite the fluorescent marker of the at least one microsensor to produce fluorescence; (d) detecting at least two optical resonance modes of the at least one microsensor from the detected fluorescence of the at least one microsensor; and (e) determining via a numerical algorithm an optical thickness of the adsorbed layer of the at least one microsensor in the fluid from the spectral positions of the at least two detected resonant modes; (f) determining the extent to which an analyte in the fluid is bound to the at least one microsensor based on the predetermined optical thickness of the adsorbent layer of the at least one microsensor.

[0082] (Appendix 2) The particles of the microresonator have a diameter in the range of 1 μm to 20 μm, preferably 2 μm to 15 μm, 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 particularly preferably 2 nm to 8 nm, the thickness being the spatial extent of the adsorption layer in the radial direction from the center point of the microresonator. The method according to claim 1,

[0083] (Appendix 3) The step of determining the optical thickness of the adsorption layer of the at least one microsensor is not performed prior to step (b). 3. The method according to claim 1 or 2,

[0084] (Appendix 4) The optical thickness of the adsorbed layer is determined using a numerical algorithm from the spectral positions of the at least two detected resonant 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 resonant modes and the linewidths of the at least two detected resonant modes. The method according to any one of claims 1 to 3,

[0085] (Appendix 5) moreover, (i) determining, using a numerical algorithm, further parameters of the at least one microsensor from the spectral positions of the at least two detected resonant modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes; The further parameter is 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 adsorbent layer of the at least one microsensor and combinations thereof; and / or (ii) from the spectral positions of the at least two detected resonant modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes, a parameter of the fluid is determined using a numerical algorithm, preferably an optical property of the fluid. The method according to any one of claims 1 to 4,

[0086] (Appendix 6) The at least one microsensor includes: (i) Have free movement; or (ii) in particular fixed to an inner surface of a fluid flow path in which said contact between said at least one microsensor and a fluid to be analyzed, which may contain an analyte, occurs; 6. The method according to any one of claims 1 to 5,

[0087] (Appendix 7) said detection of at least two optical resonance modes of said at least one microsensor is repeated at least once, and optionally several times, using a numerical algorithm from the spectral positions of said at least two detected resonance modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of said at least two detected resonance modes and the linewidths of said at least two detected resonance modes, in order to obtain a time course of the optical thickness of the adsorbed layer of said at least one microsensor; Optionally, a time course 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 resonant modes, and preferably from at least one further parameter selected from the group consisting of: relative amplitudes of the at least two detected resonant modes and linewidths of the at least two detected resonant 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 adsorbent layer of the at least one microsensor, the refractive index of the fluid, and combinations thereof. The method according to any one of claims 1 to 6,

[0088] (Appendix 8) 1. An apparatus for label-free detection of an analyte in a fluid, comprising: (a) a container containing at least one dielectric microsensor, the at least one microsensor includes or consists of a microresonator and an adsorbent layer applied to the microresonator for binding an analyte; The microresonator comprises particles that contain or consist of a dielectric material and a fluorescent marker, has an optical refractive index higher than the optical refractive index of the fluid to be analyzed, and is suitable for generating a plurality of resonant modes inside the microresonator when the fluorescence of the fluorescent marker is excited; (b) a light source for shining light onto the at least one microsensor, the light having a wavelength suitable for exciting the fluorescent marker of the at least one microsensor to produce fluorescence; and (c) a spectral analysis unit configured to detect at least two optical resonance modes of the at least one microsensor from the detected fluorescence; and (d) an algorithm unit configured to determine, from the spectral positions of the at least two detected resonant modes, an optical thickness of the adsorbed layer of the at least one microsensor via a numerical algorithm; (e) an analysis unit configured to determine an extent to which an analyte is bound to the at least one microsensor based on the determined optical thickness of the adsorbent layer of the at least one microsensor.

[0089] (Appendix 9) The particles of the microresonator have a diameter in the range of 1 μm to 20 μm, preferably 2 μm to 15 μm, 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 particularly preferably 2 nm to 8 nm, the thickness being taken to mean the spatial extent of the adsorption layer in the radial direction from the center point of the microresonator. 9. The device of claim 8.

[0090] (Appendix 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 possibly containing an analyte. 10. The device according to claim 8 or 9,

[0091] (Appendix 11) The algorithm unit is configured to determine an optical thickness of the adsorbed layer of the at least one microsensor from the spectral positions of the at least two detected resonant modes and from at least one further parameter via a numerical algorithm, the parameter being selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes. 11. The device according to any one of claims 8 to 10.

[0092] (Appendix 12) The algorithm unit comprises: (i) determining, using a numerical algorithm, a further parameter of the at least one microsensor from the spectral positions of the at least two detected resonant modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes; The further parameter is 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 adsorbent layer of the at least one microsensor and combinations thereof; and / or (ii) determining a parameter of the fluid, preferably an optical property of the fluid, from the spectral positions of the at least two detected resonant modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes by a numerical algorithm; It is configured as follows: 12. The device according to any one of claims 8 to 11,

[0093] (Appendix 13) The vessel further comprises a fluid, which may contain an analyte, and the vessel is optionally a fluid flow path. 13. The device according to any one of claims 8 to 12.

[0094] (Appendix 14) The at least one microsensor includes: (i) freely movable within the container and, optionally, within a fluid flow path of the device; or (ii) fixed within a fluid flow path of the device, in particular on an inner surface of a fluid flow path of the device, the fluid flow path being particularly suitable for supplying the at least one microsensor to a fluid to be analyzed, the fluid possibly containing an analyte; 14. The device according to any one of claims 8 to 13,

[0095] (Appendix 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 optionally several times; and said algorithm unit is configured to calculate via a numerical algorithm the time course of the optical thickness of the adsorbed layer of said at least one microsensor from the spectral positions of said at least two detected resonance modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of said at least two detected resonance modes and the linewidths of said at least two detected resonance modes, the spectral analysis unit is optionally configured to repeat the detection of the at least two optical resonance modes of the at least one microsensor at least once, and optionally several times; and said algorithm unit is configured to determine via a numerical algorithm a time course of at least one further parameter of said at least one microsensor from the spectral positions of said at least two detected resonant modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of said at least two detected resonant modes and the linewidths of said at least two detected resonant 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 adsorbent layer of the at least one microsensor, the refractive index of the fluid and combinations thereof. 15. The device according to any one of claims 8 to 14.

[0096] (Appendix 16) Further comprising a fluid flow path; The fluid flow path preferably comprises: (i) a supply line suitable for supplying the at least one microsensor to the fluid flow path; and / or (ii) an outlet adapted to eject the at least one microsensor from the fluid flow path, the outlet preferably comprising a separator for the at least one microsensor; 16. The device according to any one of claims 8 to 15,

[0097] (Appendix 17) The fluid flow path has at least one transparent wall that is transparent to light having a wavelength within the following range at least in a portion of the area: That is, the range is: (i) the range of emission wavelengths of the fluorescent marker; In this case, preferably a detection optical system is arranged between the transparent wall and the spectral analysis unit, and particularly preferably a concave reflector is arranged on the side of the container facing the transparent wall, and / or (ii) the range of excitation wavelengths of said fluorescent markers; and / or (iii) the excitation and emission wavelength ranges of the fluorescent markers; In this case, a detection optical system having a coupling element for the light of the light source is arranged between the transparent wall and the spectral analysis unit, the coupling element being reflective to light having a wavelength in the range of the excitation wavelength of the fluorescent marker and transparent to light having a wavelength in the range of the emission wavelength of the fluorescent marker; and / or (iv) the range of emission wavelengths of said fluorescent markers, and thereby allowing the implementation of additional sensor techniques, preferably photodetectors; 17. The device of claim 16.

[0098] (Appendix 18) The algorithmic unit and the analysis unit, preferably the spectral analysis unit, the algorithmic unit and the analysis unit are preferably designed together as a single unit. 18. The device according to any one of claims 8 to 17. [Explanation of symbols]

[0099] 10: Fluid channel; 15: Walls of the fluid channel that have special properties to absorb or reflect the excitation light; 20: Fluid in flow (e.g. aqueous solution in flow); 30: supply line for at least one microsensor; 40: Microsensor 43: Microresonators (particles containing or consisting of dielectric materials and fluorescent markers); 45: Adsorption layer; 50: transparent-walled fluidic channel for fluorescence detection; 55: transparent wall of the fluid flow channel for fluorescence excitation; 58: Transparent walls of the fluid flow path for implementing additional sensor technology, e.g. photodetectors; 60: Excitation light; 70: A separator for at least one microsensor; 80: outlet for at least one microsensor; 90: Detection optics; 100: fluorescence from at least one microsensor; 110: Spectral analysis unit; 120: Algorithmic Unit; 130: Unit of Analysis; 140: Coupling element for light from a light source (excitation light); 150: photodetector; 160: concave reflector; 170: WGM spectrum of a microcavity without an adsorbed layer in a medium with different refractive index; 175: refractive index of the medium surrounding the microresonator; 180: WGM spectra of a microresonator without and with an adsorbed layer in a medium with a refractive index of 1.33; 185: Thickness of the adsorption layer

Claims

1. 1. A method for label-free detection of an analyte in a fluid, comprising: (a) providing at least one dielectric microsensor within a container; the at least one microsensor includes or consists of a microresonator and an adsorbent layer applied to the microresonator for binding an analyte; the microresonator is made of particles comprising or consisting of a dielectric material and a fluorescent marker, has an optical refractive index higher than the optical refractive index of the fluid to be analyzed, and is suitable for developing a plurality of resonant modes inside the microresonator when the fluorescence of the fluorescent marker is excited; (b) contacting the at least one microsensor with a fluid to be analyzed, the fluid possibly containing an analyte; (c) shining light onto the at least one microsensor in the fluid, the light having a wavelength suitable to excite the fluorescent marker of the at least one microsensor to produce fluorescence; (d) detecting at least two optical resonance modes of the at least one microsensor from the detected fluorescence of the at least one microsensor; (e) determining, via a numerical algorithm, an optical thickness of the adsorbed layer of the at least one microsensor in the fluid from the spectral positions of the at least two detected resonant modes; (f) determining the extent to which an analyte in the fluid is bound to the at least one microsensor based on the predetermined optical thickness of the adsorbent 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, preferably 2 μm to 15 μm, 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, very particularly preferably 2 nm to 8 nm, the thickness being taken to mean the spatial extent of the adsorption layer in the radial direction from the center point of the microresonator.

2. The method of claim 1 .

3. The step of determining the optical thickness of the adsorption layer of the at least one microsensor is not performed prior to step (b).

3. The method according to claim 1 or 2.

4. The optical thickness of the adsorbed layer is determined using a numerical algorithm from the spectral positions of the at least two detected resonant 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 resonant modes and the linewidths of the at least two detected resonant modes. The method according to any one of claims 1 to 3.

5. moreover, (i) determining, using a numerical algorithm, further parameters of the at least one microsensor from the spectral positions of the at least two detected resonant modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes; The further parameter is 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 adsorbent layer of the at least one microsensor and combinations thereof; and / or (ii) from the spectral positions of the at least two detected resonant modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes, a parameter of the fluid is determined using a numerical algorithm, preferably an optical property of the fluid. The method according to any one of claims 1 to 4.

6. The at least one microsensor (i) Have freedom of movement; or (ii) fixed to an inner surface of a fluid flow path, in particular where said contact between said at least one microsensor and a fluid to be analyzed, which may contain an analyte, occurs; The method according to any one of claims 1 to 5.

7. said detection of at least two optical resonance modes of said at least one microsensor is repeated at least once, and optionally several times, using a numerical algorithm from the spectral positions of said at least two detected resonance modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of said at least two detected resonance modes and the linewidths of said at least two detected resonance modes, in order to obtain a time course of the optical thickness of the adsorbed layer of said at least one microsensor; Optionally, a time course 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 resonant modes, and preferably from at least one further parameter selected from the group consisting of: relative amplitudes of the at least two detected resonant modes and linewidths of the at least two detected resonant 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 adsorbent layer of the at least one microsensor, the refractive index of the fluid, and combinations thereof. The method according to any one of claims 1 to 6.

8. 1. An apparatus for label-free detection of an analyte in a fluid, comprising: (a) a container containing at least one dielectric microsensor, the at least one microsensor includes or consists of a microresonator and an adsorbent layer applied to the microresonator for binding an analyte; The microresonator comprises particles that contain or consist of a dielectric material and a fluorescent marker, has an optical refractive index higher than the optical refractive index of the fluid to be analyzed, and is suitable for generating a plurality of resonant modes inside the microresonator when the fluorescence of the fluorescent marker is excited; (b) a light source for shining light onto the at least one microsensor, the light having a wavelength suitable for exciting the fluorescent marker of the at least one microsensor to produce fluorescence; and (c) a spectral analysis unit configured to detect at least two optical resonance modes of the at least one microsensor from the detected fluorescence; and (d) an algorithm unit configured to determine, via a numerical algorithm, an optical thickness of the adsorbed layer of the at least one microsensor from the spectral positions of the at least two detected resonant modes; (e) an analysis unit configured to determine an extent to which an analyte is bound to the at least one microsensor based on the determined optical thickness of the adsorbent 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, preferably 2 μm to 15 μm, 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, very particularly preferably 2 nm to 8 nm, the thickness being taken to mean the spatial extent of the adsorption layer in the radial direction from the center point of the microresonator.

9. The apparatus of claim 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 possibly containing an analyte.

10. Apparatus according to claim 8 or 9.

11. The algorithm unit is configured to determine an optical thickness of the adsorbed layer of the at least one microsensor from the spectral positions of the at least two detected resonant modes and from at least one further parameter via a numerical algorithm, the parameter being selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes. Apparatus according to any one of claims 8 to 10.

12. The algorithm unit comprises: (i) determining, using a numerical algorithm, a further parameter of the at least one microsensor from the spectral positions of the at least two detected resonant modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes; The further parameter is 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 adsorbent layer of the at least one microsensor and combinations thereof; and / or (ii) determining a parameter of the fluid, preferably an optical property of the fluid, by a numerical algorithm from the spectral positions of the at least two detected resonant modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of the at least two detected resonant modes and the linewidths of the at least two detected resonant modes. It is configured as follows: Apparatus according to any one of claims 8 to 11.

13. The vessel further comprises a fluid, which may contain an analyte, and the vessel is optionally a fluid flow path. Apparatus according to any one of claims 8 to 12.

14. The at least one microsensor (i) freely movable within the container and, optionally, within a fluid flow path of the device; or (ii) fixed within a fluid flow path of the device, in particular on an inner surface of a fluid flow path of the device, the fluid flow path being particularly suitable for supplying the at least one microsensor to a fluid to be analyzed, the fluid possibly containing an analyte; Apparatus according to any one of claims 8 to 13.

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 optionally several times; and the algorithm unit is configured to calculate via a numerical algorithm the time course of the optical thickness of the adsorbed 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 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 repeat the detection of the at least two optical resonance modes of the at least one microsensor at least once, and optionally several times; and said algorithm unit is configured to determine via a numerical algorithm a time course of at least one further parameter of said at least one microsensor from the spectral positions of said at least two detected resonant modes and preferably from at least one further parameter selected from the group consisting of the relative amplitudes of said at least two detected resonant modes and the linewidths of said at least two detected resonant 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 adsorbent layer of the at least one microsensor, the refractive index of the fluid and combinations thereof. Apparatus according to any one of claims 8 to 14.

16. Further comprising a fluid flow path; The fluid flow path preferably comprises: (i) a supply line suitable for supplying the at least one microsensor to the fluid flow path; and / or (ii) an outlet adapted to eject the at least one microsensor from the fluid flow path, the outlet preferably comprising a separator for the at least one microsensor. Apparatus according to any one of claims 8 to 15.

17. The fluid flow path has at least one transparent wall that is transparent to light having a wavelength within the following range at least in a portion of the area: That is, the range is: (i) the range of emission wavelengths of the fluorescent marker; In this case, preferably a detection optical system is arranged between the transparent wall and the spectral analysis unit, and particularly preferably a concave reflector is arranged on the side of the container facing the transparent wall, and / or (ii) the range of excitation wavelengths of the fluorescent markers; and / or (iii) the excitation and emission wavelength ranges of the fluorescent markers; In this case, a detection optical system having a coupling element for the light of the light source is arranged between the transparent wall and the spectral analysis unit, the coupling element being reflective to light having a wavelength in the range of the excitation wavelength of the fluorescent marker and transparent to light having a wavelength in the range of the emission wavelength of the fluorescent marker; and / or (iv) the range of emission wavelengths of said fluorescent markers, thereby allowing the implementation of additional sensor techniques, preferably photodetectors; 17. The apparatus of claim 16.

18. The algorithmic unit and the analysis unit, preferably the spectral analysis unit, the algorithmic unit and the analysis unit are preferably designed together as a single unit. Apparatus according to any one of claims 8 to 17.

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