Method and device for optical and label-free recognizing and identifying cells from liquid

Abstract

The present invention relates to a rapid and accurate optical method and a device for detecting cells from liquid samples in a label-free way. The method is based on the interference of parts of the divergent light beam, preferably a conical laser beam, wherein a first part comes from a light source, preferably from a single-mode optical fiber, directly, and a second part reflects from a flat glass surface, wherein the glass is functionalized by antibodies against the cells to be detected from the liquid sample. Cells bound to that surface modify the reflection, and by that also change the resulted interference pattern. By registering and interpreting the variation in the image, the presence of cells from the sample can be detected.

Description

[0001] Method and device for optical and label-free recognizing and identifying cells from liquid samples

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a rapid and accurate optical method and a device for detecting cells from liquid samples in a label-free way. The method is based on the interference of parts of the divergent light beam, preferably a conical laser beam, wherein a first part comes from a light source, preferably from a single-mode optical fiber, directly, and a second part reflects from a flat glass surface, wherein the glass is functionalized by antibodies against the cells to be detected from the liquid sample. Cells bound to that surface modify the reflection, and by that also change the resulted interference pattern. Registering and interpreting the variation of the image, the presence of cells from the sample can be detected.

[0004] BACKGROUND ART

[0005] Detection of cells from body fluids is traditionally accomplished in a microbiological laboratory, requiring incubators, skilled assistance and sterile conditions for cell-culturing. Subsequently, cells are identified by some labelling techniques like ELISA [Engvall, E. and Perlmann, P., 1972], Both steps are rather costly and time-consuming, while usually there is a significant time pressure for early diagnosis. On the contrary, label-free techniques, regardless of utilizing electrical or optical effects, generally do not need high-tech equipment, and normally can be applied on-site, albeit they are less sensitive [Maldonado, J. et al., 2020; Mathesz A. et al., 2015; Idil, N. et al., 2023; Pefrovszki, D. et al., 2021; Tran, H. V. et al., 2013; Ravalli, A. et al., 2015; Sepulveda, B.et al., 2009; Estevez, M. C. et al., 2012; Tertis, M. et al., 2019],

[0006] Maldonado et al. [Maldonado, J. et al., 2020] disclose two highly sensitive methodologies using an ultrasensitive photonic biosensor based on a bimodal waveguide interferometer (BiMW) for the fast detection of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA). The authors have developed a biofunctionalization strategy based on the use of a PEGylated silane (silane-PEG- COOH) which provides a highly resistant and bacteria-repelling surface, which is crucial to specifically detect each bacterium.

[0007] Mathesz et al. [Mathesz A. et al., 2015] have shown that an integrated optical Mach-Zehnder interferometer, a highly sensitive all-optical device made of a cheap photopolymer, can be used as a powerful lab-on-a-chip tool for specific, label-free detection of proteins. By modifications of said technique, the author has combined their interferometric biosensor with a microfluidic system allowing the rapid and specific detection of bacteria from solutions, having the surface of the sensor functionalized by bacteriumspecific antibodies. In said publication it is disclosed that the operation of an integrated optical interferometric biosensors is based on the specific binding of the target molecule or bacteria to the measuring arm of the interferometer. This binding is realized by appropriate recognition elements - for example receptors, antibodies, enzymes - immobilized to the waveguide surface. The binding of target objects causes a local refractive index change at the sensor surface which effects the propagation of the incoupled light in the waveguide via the evanescent field. The resulting phase difference between the propagating mode in the measuring and reference arms can be detected as an output intensity change due to the interference.

[0008] N. Idil et al [Idil, N. et al., 2023] provide a review about the recent advances in optical sensing for the detection of microbial contaminants. Said publication mentions the interferometric optical sensors, which are based on an optical method for measuring refractive index changes. Biomolecular interactions such as antigen-antibody, enzyme-substrate, or DNA hybridization and chemical reactions cause a change in refractive index. The basic principle of an interferometer is that it uses two equivalent light paths. One light gives the refractive index change caused by bioconjugate interaction, and the other light acts as a reference that equalizes nonspecific interactions.

[0009] Petrovszki et al [Petrovszki, D. et al., 2021] disclose an integrated electro-optical biosensor system for rapid and low-cost detection of bacteria, where the disclosed integrated microsystem consisting of dielectrophoretic surface-electrodes, a rib waveguide and a microfluidic channel. The quantitative measurement carried out by said biosensor system is based on the recorded scattered light intensity images, while an inhomogeneous alternating electric field is switched on and then off, inducing bacteria movement and change in the scattered image patterns.

[0010] BRIEF DESCRIPTION OF THE INVENTION

[0011] 1. The invention relates to an all-optical device for detecting an analyte in a sample, said device comprising a support (2) a test surface (9) on the support (2), a sample holding space (4) for accommodating the sample, the sample holding space (4) being in direct contact with the test surface (9), a window plate (8) having a transparent light-exit window, a light source (1) providing coherent light in the form of a divergent light beam (5), preferably extending along a longitudinal direction, said light source (1) being arranged to allow propagation of the light beam (5) from the light source ( 1) to the light-exit window through the sample holding space (4), wherein a first part of the light beam (5) propagates in the sample holding space (4) to the light-exit window without illumination of the test surface (9), forming a direct light (5a), and a second part of the light beam (5) propagates in the sample holding space (4) whereas it illuminates the test surface (9) with the light to provide a reflected light (5b) reflected from the test surface (9), wherein the reflected light (5b) and the direct light (5a) interfere to provide a detectable interference pattern (10).

[0012] In the device of the invention, the analyte, if present, is presented on test surface (9) to the second part of the light beam (5). Alternatively, the second part of the light beam (5) illuminates the test surface wherein the analyte intended to be present on the test surface (9).

[0013] Thus, in the all-optical device of the invention, the test surface on the support is capable of providing said analyte, once sedimented thereon, to light.

[0014] In the device, the interference pattern (10) is dependent on the presence of the analyte on the test surface (9). The interference pattern is different when the analyte is present on the test surface (test interference) and when the analyte is not present on the test surface (reference interference). Thereby, detecting a difference in the test interference and the reference interference, this is indicative of the presence of the analyte. This presence can be quantified according to the invention.

[0015] Preferably, in the all-optical device of the invention, e.g. any of the paragraphs described herein, the sample holding space (4) arranged in the pathway of the light beam (5) is transparent to the light beam (5) emitted by the light source.

[0016] 2. Preferably, in the all-optical device of paragraph 1 the light source (1) is a single-mode optical fiber and the light beam (5) is a light cone. In this embodiment the half-angle of the maximum cone (0) measured from a symmetry axis of the cone is defined by the numerical aperture of the optical fiber and the index of refraction of the medium.

[0017] 3. Preferably, in the all -optical device of any one of paragraphs 1 to 2, the test surface (9) comprises binding molecule for specifically binding the analyte, wherein the size of the analyte is commensurate with the wavelength of light or larger, and wherein the analyte is selected from the group consisting of: cells, extracellular vesicles, exosomes, viruses of at least 200 nm in at least one direction, preferably cells.

[0018] Preferably the size or diameter of the analyte is comparable to the wavelength (it is in the same order of magnitude) or larger that the wavelength.

[0019] 4. Preferably, in the all-optical device of paragraph 3, the binding molecule is a binding molecule for binding of a cell, preferably an antibody or antibody fragment.

[0020] 5. Preferably, the all-optical device according to any of paragraphs 1 to 4, also comprises a screen (6) wherein the interference pattern (10) is detectably formed on the screen (6). 6. Preferably, in the all-optical device according to any of paragraphs 1 to 5, the sample holding space (4) has an opening for filling the sample holding space (4) and an opening for removing the content of the sample holding space (4); optionally the two openings are the same.

[0021] 7. Preferably, in the all-optical device according to any of paragraphs 1 to 6, wherein the sample holding space (4) is a flow-through sample holding space (4) having an inlet for entering a fluid into the sample holding space (4) and an outlet for leaving / removing the fluid from the sample holding space (4), the flow- through configuration allowing, after being present in the sample holding space (4) for a sufficient time binding of the analyte to the test surface (9).

[0022] In a variant of the method the screen can be about 10 to 50 cm distance from the window plate.

[0023] The screen can be e.g. a plain surface, preferably a white surface.

[0024] The interference pattern projected on the screen can be recorded by a camera e.g. a ccd camera.

[0025] In an alternative embodiment, in a compact device the screen may be closer to the window plate the requirement is that the interference pattern is formed and visible with a sufficient resolution.

[0026] In an embodiment, in a compact device, the screen may comprise an image sensor on its surface, which directly detects the light passing through the sample volume and the window plate. In this embodiment the screen may be closer to the window plate, but at a distance that ensures that the interference pattern is formed and detectable with a sufficient resolution.

[0027] In an embodiment the interference pattern is detected by a camera e.g. a ccd camera.

[0028] Preferably the analyte is selected from the group consisting of: cells, extracellular vesicles, exosomes, viruses of at least 200 nm in at least one direction, preferably cells.

[0029] 8. Preferably, in the all-optical device according to any of paragraphs 1 to 6, wherein said sample holding space (4) has a cover, preferably a reflecting cover, wherein the light beam (5) is reflected both from the test surface (9) and the reflecting cover.

[0030] In a variant of the all-optical device all the walls reflect the light beam, and when the analyte sediments to the one of the walls the interference pattern changes. Typically, if a square cross section capillary is applied, the interference pattern in this embodiment is similar to a square-like checkered matrix, which may change into a series of lines once the analyte is sedimented to one of the wall serving as a test surface (9).

[0031] In a particular embodiment the test surface may be coated with an analyte-binding film in patterns. For example the test surface may be divided into part and reflection from different parts may result in different interference patterns.

[0032] 9. Preferably, in the all-optical device according to any of paragraphs 1 to 8, said sample holding space (4) is delimited by walls joined to each other by curved edges.

[0033] 10. Preferably, in the all-optical device according to any of paragraphs 1 to 7, the device also comprises

[0034] - a reference surface (29) provided on the support (2), and - a reference sample holding space (24) for accommodating a reference sample, the reference sample holding space (24) being in direct contact with the reference surface (29),

[0035] - a further window plate (8) having a transparent light-exit window,

[0036] - a further light source (21) providing coherent light in the form of a further divergent light beam (25), preferably extending along a longitudinal direction, said further light source (21) being arranged to allow propagation of the further light beam (25) from the further light source (21) to the light-exit window through the reference sample holding space (24) wherein a first part of the further light beam (25) propagates in the reference sample holding space (24) to the light-exit window without illumination of the reference test surface (29), forming a direct light (25 a), and a second part of the light beam (25) propagates in the reference sample holding space (24) whereas it illuminates the reference test surface (29) with the light to provide a reflected light (25b) reflected from the reference test surface (29), wherein the reflected light (25b) and the direct light (25 a) interfere to provide a detectable interference pattern (20)

[0037] In the device of the invention, the analyte, if present, is presented on reference test surface (29) to the second part of the light beam (25). Alternatively, the second part of the light beam (25) illuminates the test surface wherein the analyte intended to be present on the test surface (29).

[0038] The reference interference pattern (20) is dependent on the presence of the analyte on the reference test surface (29).

[0039] 11. Preferably, the all -optical device according to paragraph 10, also comprises means to illuminate said test surface (9) and said reference surface with a light parallel to surfaces, to provide reflected light from each surfaces.

[0040] Preferably the test interference pattern (10) and the reference interference pattern (20) are formed and recorded separately.

[0041] 12. The invention also relates to a method of all-optical detection of an analyte in a sample, in an all- optical device comprising a support (2) a test surface (9) on the support (2), a sample holding space (4) for accommodating the sample, the sample holding space (4) being in direct contact with the test surface (9), a window plate (8) having a transparent light-exit window, a light source (1) providing light in the form of a divergent light beam (5), said light source

[0042] (1) being arranged to allow propagation of the light beam (5) from the light source (1) to the light-exit window through the sample holding space (4), said method comprising accommodating said sample in a sample holding space (4) being in direct contact with the test surface (9), contacting said sample accommodated in the sample holding space (4) with the test surface (9), allowing said analyte in the sample to accumulate at the test surface (9), providing a coherent light in the form of a divergent light beam (5) propagating through the sample holding space (4), the surfaces delimiting the sample holding space (4) being transparent to the light emitted by said light beam (5), wherein a first part of the light beam (5) propagates in the sample holding space (4) to the light-exit window without illumination of the test surface (9), forming a direct light (5a), and a second part of the light beam (5) propagates in the sample holding space (4) whereas it illuminates the test surface (10) with the light to provide a reflected light (5b), and detecting interference between the direct light (5a) and the reflected light (5b) from the test surface (9), to provide a detectable interference pattern (10), analyzing said interference pattern (10) to assess whether the analyte is present on the test surface (9).

[0043] Preferably the analyte is selected from the group consisting of: cells, extracellular vesicles, exosomes, viruses of at least 200 nm in at least one direction, preferably cells.

[0044] 13. Preferably in the method according to paragraph 12, the light source (1) is a single-mode optical fiber and the light beam (5) is a light cone.

[0045] 14. Preferably in the method according to paragraphs 12 to 13, the test surface (9) comprises binding molecule for specifically binding the analyte, wherein the size of the analyte is commensurate with the wavelength of light or larger, and the analyte is selected from the group consisting of: cells, extracellular vesicles, exosomes, viruses of at least 200 nm in at least one direction, preferably cells.

[0046] 15. Preferably, in the method according to paragraph 14, the binding molecule is a binding molecule for binding of a cell, preferably an antibody or antibody fragment.

[0047] 16. Preferably, in the method according to paragraphs 12 to 15, the all-optical device also comprising a screen (6) wherein the interference pattern (10) is detectably formed on the screen (6). 17. Preferably, in the method according to paragraphs 12 to 16, the sample holding space (4) has an opening for filling the sample holding space (4) and an opening for removing the content of the sample holding space (4); optionally the two openings are the same.

[0048] 18. Preferably, in the method according to paragraphs 12 to 17, the sample holding space (4) is a flowthrough sample holding space (4) having an inlet for entering a fluid into the sample holding space (4) and an outlet for leaving / removing the fluid from the sample holding space (4), the flow-through configuration allowing, after being present in the sample holding space (4) for a sufficient time binding of the analyte to the test surface (9).

[0049] 19. Preferably, in the method according to paragraphs 12 to 17, said sample holding space (4) has a cover, preferably a reflecting cover, wherein the light beam (5) is reflected both from the test surface (9) and the reflecting cover.

[0050] 20. Preferably, in the method according to paragraph 12, the all-optical device also comprising

[0051] - a support (2) having a reference surface (29), and

[0052] - a reference sample holding space (24) for accommodating a reference sample, the reference sample holding space (24) being in direct contact with the reference surface (29),

[0053] - a further window plate (8) having a transparent light-exit window,

[0054] - a further light source (21) providing further light in the form of a further divergent light beam (25), preferably extending along a longitudinal direction, said further light source (21) being arranged to allow propagation of the further light beam (25) from the further light source (21) to the further light-exit window through the reference sample holding space (24), said method further comprising accommodating a reference sample in a reference sample holding space being in direct contact with the reference surface, contacting the reference sample accommodated in the reference sample holding space (24) with the reference surface (29), providing a coherent light in the form of a further divergent light beam (25) propagating through the reference sample holding space (24), wherein a first part of the further light beam (25) propagates in the reference sample holding space (24) to the light-exit window without illumination of the reference test surface (29), forming a further direct light (25 a), and a second part of the further light beam propagates in the reference sample holding space (24) whereas it illuminates the reference test surface (29) with the light to provide a reflected light (25b) reflected from the reference test surface (29), and detecting interference between the further direct light (25 a) and the further reflected light (25b) from the reference test surface (29), to provide a reference interference pattern (20), using the reference interference pattern (20) in analyzing said interference pattern (10) to assess whether the analyte is present on the test surface (9), preferably comparing the interference pattern (10) with the reference interference pattern (20), and assessing the binding of the analyte from the difference between the interference pattern (10) with the reference interference pattern (20).

[0055] DEFINITIONS

[0056] An “all -optical device”, as used herein, is a device for detection of an analyte wherein up to the formation of the detectable signal optical methods are used only in a specific material arrangement, i.e. no electronic or computer assisted steps are included. Such steps electronic or computer assisted steps may be useful in processing of the detectable signal e.g. the interference pattern formed upon the use of the device.

[0057] The “test surface” refers to a layer / an area on the bottom of the sample holding space, which can reflect the light illuminating it and on which the analyte is located during the analysis. In an embodiment the analyte is bound to a binding agent located on the test surface. In a further embodiment test surface may have a preliminary defined pattern.

[0058] A “chamber” is an enclosed space or compartment, preferably with an opening to allow communication with the environment.

[0059] A “walled chamber” as used herein is a chamber with walls to separate the enclosed space of the chamber from the environment or space outside the chamber. A wall is a barrier between the enclosed space of the chamber and the environment allowing separation of the former from the latter; preferably the barrier is a layer of any material suitable for this separation, i.e. providing the enclosure of the space of the chamber.

[0060] A “sample holding chamber” is a walled chamber comprising a “sample holding space”.

[0061] A “sample holding space” is the inner space of a compartment used to accommodate samples (e.g. biological sample) during an analysis. In an embodiment the sample holding space may be formed as a straight channel or a labyrinth (a zigzag channel). In a further embodiment the sample holding space may be formed in a flow-through configuration, in which the sample holding space having an inlet for entering the fluid sample and an outlet for evacuating the fluid sample. The sample holding space may be opened or closed from the top.

[0062] A “window” is an opening in a wall of a chamber to allow communication between the enclosed space of the chamber with the environment, preferably transfer of material, particles and / or waves or information. The window may be an opening which is an open space of the wall, or a part of the wall made of a material allowing said communication. In an embodiment the window may be made of a transparent material. In another embodiment the window can be opened and closed by a barrier or cover. “Transparent” refers to the property of a material that allows light to pass through it essentially without significant scattering or absorption. The degree of transparency depends on the wavelength of light and the structure of the material. The transparent materials include, for example, the clear glass.

[0063] The “light” or “visible light” is electromagnetic radiation that can be detected by the human eye. By definition, visible light refers to the wavelength range of 400-700 nanometers (run).

[0064] The “light ray” is a straight line that represents the path along which light travels. Light ray is an imaginary construct, that is, it has not physical existence, but provides a useful tool for understanding how light behaves, for example, it is used to describe the direction of light propagation.

[0065] The “light beam” is a directional projection of light energy radiating from a light source and comprising a plurality of light rays; wherein preferably the light rays are propagating within a given angle from the light source, e.g. in essentially the same direction in a light cone. A light beam is often considered together as a single entity. Light beams have a certain width or diameter and can be narrow or wide depending on the source of light and the optical system through which they pass.

[0066] The “coherent light” refers to a light in which the electromagnetic waves have the same frequency and their wavelengths must be in phase with each other. The coherent light has ability to exhibit “interference” effects. If at least two coherent light beams are combined, then there will be points in space where the waves amplify each other or weaken each other, which result an interference pattern.

[0067] The light traveling in any one direction in a straight line is called a ray of light.

[0068] A group of light rays given out from a source is called a beam of light.

[0069] The “analyte” refers to a substance as a component of a liquid sample which is to be detected by the device of the invention. The analyte includes, for example, the biological cells, extracellular vesicles or exosomes.

[0070] The singular forms “a”, “an” and “the”, or at least “a”, “an”, include plural reference unless the context clearly dictates otherwise.

[0071] The term “comprises” or “comprising” or “including” are to be construed here as having a non- exhaustive meaning and allow the addition or involvement of further features or method steps or components to anything which comprises the listed features or method steps or components. “Comprising” can be substituted by “including” if the practice of a given language variant so requires or can be limited to “consisting essentially of’ if other members or components are not essential to reduce the invention to practice.

[0072] BRIEF DESCRIPTION OF THE FIGURES

[0073] Figure 1 - Schematic 3D figure of the device. The side blocks (black) and the glass plates on top and the bottom (light gray) form the sample holding chamber 3. Thanks to the leveled end faces and edges of the blocks and glass plates the sample liquid (medium grey) has a flat and vertical surface, which is at the same time the output optical window. The laser light (not shown) enters the sample holding chamber 3 from a single-mode optical fiber (black with dark grey ending). The figure is not to scale.

[0074] Figure 2 - Schematic representation of the working principle, a) Side view of the light path. The light gray region shows the laser beam originated from the single-mode optical fiber, forming a coaxial light cone, and a part of it (in this example the most of it; see the darker gray) is reflected from the bottom surface of the sample holding space 4, i.e., the interface functionalized by the analyte cells (gray dots). In the overlapping area of the two parts, there occurs an interference which can be visualized / recorded by a screen

[0075] 6 as an interference pattern 10 (or a sensor of an imaging device). In order to make easier to see the concept, the drawing in the figure is not up-to-scale (actually in this example the diameter of the optical fiber is 125 pm, while its distance from the end of the glass is about 4600 pm), b) A 3D representation of the conical light beam. The colour code is the same as used in Figure 2a). The lower part of the direct beam hits the test surface 9 of the sample holding space 4 in a parabolic region (shown in lighter gray) and gets reflected from it. Eventually, the direct and the reflected parts of the beam are stopped by the screen 6, where the interference is detected. The local change of the reflectance in the elliptic region can be monitored as a variation in the interference stripes on the surface of the screen / detector. (The thin black line in the figure represents the edge of the substrate.)

[0076] Figure 3 - The optical path difference for a reflected and a direct ray.

[0077] Figure 4 - Illustration of the working principle of the invention: The exit end of the core of the optical fiber acts as a 1 light source in this case, and emits a light cone. The lower part of the cone of the incident light beam touches the test surface 9 of the support 2 wherefrom most of the light is reflected and it eventually meets the directly arriving light, resulting in interference which may be detected e.g. on a screen 6.

[0078] Figure 5 - Schematic representation of the evaluation procedure. As shown in the figure, 104cells / mL concentration was used in the experiment, selected as an example to demonstrate the process (for details for all of the concentrations see Figures 6, 7 and 9). Interference pattern of the reference (functionalized surface in pure PBS, without cells) labeled as “Reference” in the Figure 5a. Interference pattern recorded after the completed measuring cycle (after the cells bound to the test surface 9 and the sample holding chamber 3 was flushed 3X with PBS) is in the Figure 5c. After a simple image processing procedure (see Figures 9 / A and 9 / B), represented by the middle panel, Fourier spectra were calculated for quantification of the effect.

[0079] Figure 6 - Grayscale images of the interference patterns recorded at various cell concentrations from 103to 106cells / mL, set in quasi-exponential-scale steps, and for the cell-free reference. (The resolution of the original images is 2100 x 750 pixels).

[0080] Figure 7 - Cell-concentration dependence of the size of the effect, and a logarithmic fit to the measured

[0081] 7 points (appearing as a straight line in the semi-logarithmic representation). The horizontal and vertical error bars represent a pessimistic error estimate based on the accuracy of the cell concentrations (for details, see Materials and Methods), and 3 successive measurements per concentration, respectively.

[0082] Figure 8 - Ray representation of the light paths in the case when the light beam (red line) arrives from an optical fibre (b) into the material (a), and hits the surface of the other material (c) under the angle a, i.e., under angle of incidence 0i . The refracted part penetrates to the material (c) and, according to the corresponding refractive indices and ni, under the transmitted angle 0t.

[0083] Figure 9 / A and Figure 9 / B - On the chart all the concentrations that were studied is presented. As it has been described, colour images were taken by a photo camera and smaller parts were cropped for later analysis. In the end of the process for each concentration a 2100 by 750 pixels size, 8-bit images were produced. A small Matlab script was run and two graphs (and data series) were generated for each concentration. On the chart at left the vector is presented, it is an array of integers. Each number comes from adding up the pixel values of a column. It has been done for each column of the pixels of the input picture, and the result can be seen on the graph. The other graph next to that shows the amplitudes of the Fourier frequencies of that data array. Since it is a relatively big number of data points, a semi-logarithmic representation was chosen. This way, all the data points (i.e., the whole spectrum) are presented yet the important parts (peaks) at low frequencies can be well visualized. The default colours of the Matlab was used, the blue line represents the Fourier spectrum of the reference (with no cells) and the red one represents the sample with cells. Note, how the high amplitude waves in the left graphs (signs of clearly visible interference stripes in the image) disappear gradually as the cell concentration increases. This degradation is quantified by determining the ratio of the main peaks of the Fourier spectra.

[0084] Figure 10 - Rectangular capillaries [CM Scientific: VifroCom Glass Capillaries and Tubing, Square Capillaries]

[0085] Figure 11 - Interference fringes recorded in the case of rectangular channels, without cells (a), filled with cells during sedimentation (b), and when sedimented cells were attached to the bottom of the measuring cuvette (c). One can see that the horizontal part of the pattern has disappeared. Since the cells have sedimented at the bottom, only the interference fringes in the horizontal direction were affected (cancelled) and this did not happen to the vertical walls and so did not influence the interference in the vertical direction.

[0086] Figure 12 - Phase contrast images (40X objective, Nikon Eclipse Ti) show the cells attached on the surface. From left to right: the cells sedimented to the bottom, cells attached after 30 minutes, cells remained after washing.

[0087] Figure 13 - A perspective view of an embodiment of the all-optical device according to the invention.

[0088] Figure 14 - A perspective view of another embodiment of all-optical device according to the invention being provided with a flow-through configuration.

[0089] Figure 15 - A perspective view of another embodiment of all-optical device according to the invention provided with an opening. Figure 16 - A perspective view of another embodiment of all-optical device having a sample holding space 4 and a reference sample holding sample each having a flow-through configuration.

[0090] Figure 17 - A front view of the all-optical device as shown in figure 16.

[0091] DETAILED DESCRIPTION OF THE INVENTION

[0092] The present description discloses a novel all-optical, label-free technique for recognizing and identifying cells from liquid samples. The method of detection is based on optical interference of parts of a laser beam. As a laser light mediator, a single-mode optical fiber was inserted into a fluidic channel of straight walls created from flat glass plates. Since the outcoupled beam is divergent, part of it is reflected from the flat glass substrate at the bottom of the channel, eventually meeting with the directly propagating, unreflected light, giving rise to interference, accordingly. Since any object close to the glass surface disturbs the beam reflected from it, a change in the interference can be observed upon adsorption of the analytes. E.g., cells attached to the reflecting bottom of the fluidic channel can bring forth a significant change in the interference pattern.

[0093] The invention relates to a rapid and accurate optical method for detecting cells from liquid samples in a label-free manner. The working principle of the method is based on the interference of parts of a conical laser beam, coming from a single-mode optical fiber directly, and reflected from a flat glass surface. The glass is functionalized by antibodies against the cells to be detected from the liquid sample. Cells bound to that surface modify the reflected beam, and hence, change the resulting interference pattern, too. By registering and interpreting the variation in the image, the presence of cells from the sample can be detected. As for a demonstration, cell suspensions from a U937 cell line were used in glass chambers functionalized by antibodies (TMG6-5 (mlgGl)) to which the cells specifically bind [Sandor N. et al., 2013], The limit of detection (LOD) of the method was also estimated. This proof-of-concept setup offers a cost-effective and easy-to-use way of rapid and specific detection of any type of cells (including pathogens) from suspensions (e.g., body fluids). The possible portability of the device predicts its applicability as a rapid test in clinical diagnostics.

[0094] The specific identification of the cells is based on an antibody-antigen reaction. If, namely, the surface is functionalized by antibodies against a certain cell type, only that type of cell can be anchored to it [Tertis, M. et al., 2018; Sandor, N. et al. 2013], To demonstrate the principle of detection and identification by our method, here we used the human U937 lymphocyte cell line and respective antibodies, while the results are discussed in terms of further potential general utilization [Tertis, M. et al., 2018], In a particular advantageous embodiment of the method, for its operation there is no need of an upper plate of the measuring chamber, therefore the sample volume is theoretically not limited, hence the limit of detection can be arbitrarily extended. The detected interference is determined by the reflectivity of the functionalized surface that depends on the number of cells attached to the bottom plate, irrespective of the sample volume they were sediment from. Hence, by increasing the sample volume, when possible, the limit of detection (LOD) can be improved, practically at will. Since there is no need for a top wall for the sample holding chamber 3, it can be extended upward to hold higher sample volumes. For example, the sample volume in our experiments was ca. 100 pL, but it can be easily expanded several-fold, if there is enough sample available.

[0095] In such cases, however, where a lower sensitivity is sufficient (e.g., only the presence or absence of a microorganism is the question), conventionally available, horizontally aligned glass capillaries of a rectangular cross-section can also be used as sample holding chambers 3 (Figure 10). In this case, reflections of the divergent beam take place from four adjacent walls, and a more complex, “chessboard-like” interference pattern 10 is generated on the screen 6, in case of a clean, reference solution. However, having the cells (sediment) cover the bottom wall, the interference pattern 10 is reduced to stripes (Fig. 11). In this case, an evaluation software could apply either two ID or one 2D Fourier-transforms, to quantify the effect (that is the change in the interference pattern 10). An advantage of the interference-based detection principle of the method is that no fluorescent or absorbent labeling is required, hence, serious time- and cost-intensive steps are avoided.

[0096] Since the device is a relatively small, handheld one, no special laboratory equipment and expertise are needed for performing test [Prieto, F. et al., 2003; Kaur, B. et al., 2023], Considering the easy transportability of the device, the method can be ideally suited for outdoor applications and point-of-care diagnostics [Nath, P. et al., 2020; Der A. et al., 2010],

[0097] On the whole, the inventors have established a simple and robust label-free method for the detection of cells from suspension. The operation principle of the method allows the rapid and cheap detection of biological objects, including pathogenic microorganisms from body fluids, while its simple and user- friendly implementation makes it a promising tool for point-of-care applications.

[0098] The working principle of the device of the present invention is that the light coming from the optical fiber is slightly divergent, having a cone shape and Gaussian intensity distribution as a function of distance from the axis of the optical fiber (Figure 2). The numerical aperture (NA=0.12) of the optical fiber determines the half-angle of that light cone. The exit end of the core of the optical fiber acts as a light source in this case, and it is centered at the half-diameter of the optical fiber. There is an area where the incident light beam reflected from it. Since the angle of the light cone is really small (less than 5 degrees), obeying the optical laws, most of the light incident on the surface is reflected from it, and eventually meets the directly propagating light. Due to geometrical reasons, the reflected light travels a longer way to a certain point on the screen than the directly propagating one, in the same medium. Since in this case the light is actually a (coherent, monochromatic) laser beam, the differences in the optical path length result in an interference pattern (Figure 2). For this particular arrangement, it is a series of parallel bright strips separated by dark ones (Figure 2b).

[0099] According to the notations of Figure 3, A stands for the optical path difference, up to the distance 2Z, between the reflected light leaving the optical fiber under angle a, and the one that comes directly in the axis. It can be calculated as:

[0100] In the interference pattern, bright stripes occur when A is the multiple of ' / . = 673 run, the wavelength of the light, and there are dark stripes in between, corresponding to a A of an odd multiple of the halfwavelength. If there is any disturbance in the reflected beam due to absorption or light scattering by particles on the surface, the interference lines become changed or distorted. In this way, any object of size comparable to or larger than the wavelength on the reflecting surface can be detected by observing the variation in the interference fringes. In our case, these objects are the cells attached to the functionalized surface of the glass plate.

[0101] To avoid the aberration of the reflected light from the upper plate with no cells attached, its surface can be made ground (matte). However, this may not be necessary, if the arrangement provides that no part of the light beam is reflected from the upper plate of the sample holding chamber 3.

[0102] In another embodiment, the distance of the optical fiber should be adjusted carefully, so as to avoid its hitting the top surface of the sample holding chamber 3.

[0103] As to the measurement method, an interference image is detected with sample comprising an analyte (or assumable comprising said analyte). This may be called a test sample. Thereby an interference image (test image) is obtained. As a reference an interference image can be detected with a reference sample or even without sample (reference image). In an embodiment, as a result of the adherence of cells to the lower surface of the fluidic channel, however, the interference pattern is distorted.

[0104] The detection of the presence of the cells is carried out based on the difference between the test or sample image and the reference image.

[0105] In an embodiment, to quantify the difference, a one-dimensional array was created by column-wise summarizing the values of the image pixels, yielding sinusoidal curves along the dimension perpendicular to the stripes as “reduced interferograms” (Fig. 5). Subsequently a Fourier-transform was carried out, yielding the intensity distribution as a function of space frequency (Figure 5d). The magnitude of the effects was then defined by the ratio of the amplitudes of the main peaks of the Fourier-components determined from the interference fringes recorded at the beginning and end of the measuring cycle.

[0106] Note that the frequency values assigned to the maxima of the main peaks might occasionally be shifted during the measuring cycle, by effects due to the possibly different meniscus curvatures at the beginning and the end of the measurement. However, such a virtual change in the magnification of the fringe pattern does not alter the intensity distribution among the stripes of the main component and the rest, so it does not influence the weights of the Fourier components, either.

[0107] To determine the cell-concentration dependence of the method, a concentration series of cell suspension can be prepared, and the above procedure can be carried out for each concentration (Figure 6). In an embodiment the ratio of the sample and reference values depicted in a semi-logarhythmic plot can be used as a calibration curve. It has been found that this curve (Figure 7) shows a strongly linear character in the examined concentration range, suggesting a logarhythmic relationship between their signal size and cell concentration, apparently strictly following the Weber-Fechner law [Hecht, S., 1824],

[0108] We found this method superior to other methods attempting to determine visibility. However, other methods of evaluation and image analysis are available for a person skilled in the art.

[0109] For example, similarity of any test image to a reference image can be used by computer based image analysis. Optionally such similarity can be scored and thereby quantified [Chalom, E. et al., 2013; Katukam R. and Sindhoora P., 2015],

[0110] As an example, the structural similarity index measure (SSIM) method can be used. This is a method developed for predicting the perceived quality of digital television and cinematic pictures, as well as other kinds of digital images, but also used for measuring the similarity between two images. In this method the measurement or prediction of image quality is based on an initial uncompressed or distortion-free image as reference [Zhang, L. et al., 2012; Dossehnann, R. et al., 2011],

[0111] Fresnel’s Equations

[0112] When a light wave arrives at a flat interface of two materials with different index of refraction, a part of it is reflected and the other part is continuing its way to the other side, but in a different direction (in other words, it is refracted) as it is shown schematically in Figure 8.

[0113] The angle of incidence is the same as the angle of reflection (0!= 0r) while the angle of refraction 0tis governed by the Snell’s law.

[0114] The intensities (and amplitudes) of these two partial beams are described by the Fresnel’s equations. For both the p and .s' polarization (p stands for the E vector oscillating in the plane of incidence, while .s' for perpendicular to that). where r refers to the amplitude and R to the intensity of reflected light waves. Our laser source has a linearly polarized beam, and it was set to be s-polarized (’’horizontal”) where the reflection is the highest. Although the single-mode optical fibre we used was not a polarization-maintaining one, yet, the beam coming out from that was mostly s-polarized. Hence, the actual reflectivity was between the Rpand Rs (but closer to the latter). In order to determine the pessimistic estimate of the reflectivity, substituting the refractive indices (ni=1.333, n2=l,51) and the highest angle corresponding to the nominal NA=0. 12 of the optical fibre in water, considered at the beam waist (ca. 5°), the angle of incidence is 85°, from which we get Rs= 0.526, Rp=0.434. However, the laser beam from the optical fibre is a Gaussian beam, so, most of the light energy propagates in the middle part, under even higher angles of incidence. For example, at 01=88°, we get Rs= 0.772, Rp=0.716, etc. So, it can be concluded that most of the intensity which was reflected from the first surface took part in forming the interference.

[0115] Opportunity for increasing the Limit of Detection (LOD) of the method

[0116] As it can be seen on the images of the interference fringes (lines), even a few cells make a detectable change. From practical point of view, the device, we presented sort of 'counts' the cells present on the surface where the light is reflected from.

[0117] Let's suppose that there is a given output signal that belongs to 10 cells, and we have two different devices. Both of them have the same area of reflection but one of them has 5 times higher sample volume (i.e., the sample chamber is 5 times higher).

[0118] According to the working principle, in this case we will have the same output signal for the 10 cells but in the case of the device with bigger volume it belongs to a 5 times less concentration. This is true for all the concentrations including the one belonging to the LOD. In this example, the LOD of the bigger device is also less by the factor 5.

[0119] With proper modifications of the sample chamber, the volume, and hence the sensitivity, can be increased.

[0120] EXAMPLES

[0121] EXAMPLE 1 - Device with rectangular capillaries

[0122] 1. 1 Materials and Methods

[0123] U937 cells from a human lymphatic model cell line were grown in a humidified, 37 °C incubator with 5% CO2, under normal cultivating conditions [Sundsfrom, C. and Nilsson, K., 1976], The cell counting of the stock suspension (3 x 106cells / mL) was performed via the traditional Burker-chamber method, and its error was less than 5% [Rustichelli, D. et al., 2013], Prior to starting the measurement procedure, a concentration series was prepared by successive dilutions of the stock suspension, with final concentrations of 106, 3 x io5, io5, 3 x io4, io4, 3 x io3, and 103cells / mL. As for the possible change in the cell concentrations during the measurement procedure, the total measuring time for the whole concentration series was about two hours. During the measurement, regarding the doubling time of these cells (48-72 h, under optimal conditions (e.g., constant, 37 °C temperature) [Chen, X. and Zuckerman, S., 2004], the increment - calculating with a pessimistic approach, assuming rouble = 48 h doubling time and optimal culturing conditions - is less than 6%. Rectangular capillaries have been purchased [CM Scientific: VitroCom Glass Capillaries and Tubing, Square Capillaries] and used for initial experiments. (Figure 10) The inner width and height of the capillaries were 0.50 mm, whereas the wall thickness about 0. 10 mm.

[0124] The capillaries served as sample holding chamber 3 as the samples were introduced into the capillaries by capillary attraction (capillarity) at the measuring end of the capillary.

[0125] Optical fiber was inserted into the capillaries from the other, distal end up to a few mm, e.g. 1-10 mm, preferably 2-5 mm (e.g. 5 mm) to the measuring end leaving space for the sample.

[0126] One of the inner side of the capillary was / can be functionalized by antibodies as described in Example 2.

[0127] For binding the analyte particles (i.e., cells in this case), in order to sense their presence, the test surface 9 of the sample holding chamber 3 was functionalized with specific antibodies.

[0128] As a next step, the sample holding chamber 3 was emptied, and flushed three times with phosphate saline buffer (PBS).

[0129] Right after it was filled with the antibody (TMG6-5) solution, it was incubated for an hour at room temperature.

[0130] Another device was prepared for control measurements in the same way,

[0131] Just before the measurement, the antibody solution was removed and the sample holding chamber 3 was washed three times by PBS.

[0132] First, the reference interference image was recorded, while the device was filled with PBS. Next, the PBS was exchanged by the sample liquid, i.e., a suspension of U937 cells, carefully, in order to avoid any air bubble(s) remained in the sample holding chamber 3.

[0133] Therefore, the solution was deaerated (degassed) before application.

[0134] 1.2. Sample detection, recording interference

[0135] The output 10 interference pattern was recorded at regular intervals, to follow the process of sedimentation of the cells. After approximately 15 minutes, the output interference pattern 10 was not changing any more, indicating the sedimentation of the cells on the bottom surface.

[0136] Then the sample was removed from the sample holding chamber 3, and it was washed with PBS, before filling it up again with PBS. In this state, with the cells attached to the surface ( / via the antibodies?), was the final interference pattern 10 recorded.

[0137] Interference fringes were recorded in the case of rectangular channels, (a) without cells, (b) filled with cells during sedimentation, and (c) when sedimented cells were attached to the bottom of the measuring cuvette. One can see that the horizontal part of the pattern has disappeared. Since the cells have sedimented at the bottom, only the interference fringes in the horizontal direction were affected (cancelled) and this did not happen to the vertical walls and so they did not influence the interference in the vertical direction. (Figure 11)

[0138] EXAMPLE 2 - Device with a sample chamber (cuvette) with a detecting window (top plate)

[0139] 2.1. Materials and Methods

[0140] 2. 1. 1. Building of the device

[0141] A fluidic chamber comprising a sample holding space 4 was built from microscope slides, forming a support 2 and a top slide with two spacers in between forming sample holding chamber 3. The top slide (upper glass plate) may be shorter, allowing an easy way to insert the optical fiber as a light source 1 and the inner surface is ground, in order to avoid too much light reflected back to the direct-light region. Alternatively, the top slide may cover the sample holding chamber 3, however, the hight of the sample holding chamber 3 may provide that reflection from the top slide is avoided. As spacers, slices of microscope slides that were a bit longer than the upper glass plate were used. The schematic representation of the device is shown in Figure 1. Their thickness was around 1 mm, while the diameter of the stripped optical fiber was 125 pm. The window plate 8 plays a dual role. It holds the liquid sample in the channel, and its edge maintains the liquid surface flat and perpendicular to the surface of the bottom plate by means of surface tension. Note that although both the back and the front ends of the channel are open, being the walls of the sample chamber hydrophilic, for such a height of the liquid layer (1 mm), the capillary forces keep the liquid in. This technique has already been utilized successfully for building a flat-ended optical waveguide out of a photopolymer liquid at the end of a single-mode optical fiber [Valkai, S. et al., 2009], At the final assembly, a UV-curable optical adhesive (N0A81, ThorLabs, Newton, New Jersey, USA), and a transparent liquid were added between the parts, and the ending edges of the upper and lower glass plates were precisely aligned together. As for the final step, a light flash from a mercury arc lamp (of 100 W power for 5 s) was applied to cure the N0A81. The leveled ends of the glass plates - with the help of the surface tension - defined the end surface of the liquid with which the chamber is filled. The (stripped) optical fiber serving as a 1 light source was pushed and fixed onto the bottom surface, ensuring its optical axis was parallel with it. (See Figure 4.)

[0142] The test surface 9 on support 2 is functionalized, thereby, when a part of the conical coherent light emitted from the optical fiber serving as a light source 1 is reflected from the test surface 9 then the reflected light 5b encounters with the direct light 5 a in a given space part, and after passing through the top plate (8 window plate having a light-exit window) is detected as an interference pattern 10 by detecting means 7.

[0143] A red laser light beam (RLT650-100MGS laser, Roithner LaserTechnic, Vienna, Austria, 658 nm, 100 mW output beam) was coupled into a single-mode optical fiber (ThorLabs Inc., Newton, New Jersey, USA, SM600, NA = 0.12). The chosen wavelength is one of the most common ones for cheap lasers, but the same principles used in the present description apply to other wavelengths in the visible, as well. Label-free biosensing is accomplished by the interference of two parts of a divergent laser light beam exiting a singlemode optical fiber. The lower part of this conical Gaussian measuring light beam hits the channel-substrate interface, and the vast majority of the intensity is reflected from it (for more details, see Figure 8, Equations 2 and 3). The two coherent beams interfere, and the resulting interference pattern 10 can be seen at the surface of a screen 6 (white rectangle). When biological cells (circles) are attached to the test surface 9, they modify the reflected light 5b, which results in a variation in the interference pattern 10 relative to the situation without cells.

[0144] In order to bind the analyte particles (i.e., cells in this case) onto the test surface 9 of the sample holding space 4, it was functionalized with specific antibodies [Petrovszki, D. et al., 2022], The assembled device was cleaned by ultrasonic treatment in isopropanol (IP A), and then dried. The sample holding space was filled up with AnteoBindTM Biosensor (AnteoTech, Eight Mile Plains, Queensland, Australia) and incubated for 15 minutes at room temperature to facilitate antibody binding to the glass surface as a chelator. After that, the sample holding space 4 was emptied, and flushed three times with PBS. Subsequently, it was filled with the antibody (TMG6-5, mlgGl) solution and incubated for an hour at room temperature. Devices for control measurements were prepared in the same way, except for the antibody in the last step, which was IgG antigoat-antimouse. The latter antibody covered the pretreated surface, but did not specifically bind the U937 cells.

[0145] 2.1.2. Sample detection, recording interference

[0146] As the first step of the measurements, the measuring channel of the device, functionalized with antibodies specific to the human lymphatic cell line, was filled up with a liquid suspension of U937 cells, and the output interference pattern 10 was recorded at regular intervals, to follow the process of sedimentation of the cells. After 15 minutes, the output interference pattern 10 was not changing anymore, indicating that the sedimentation of the cells on the test surface 9 was complete. The following 15 minutes were left for the formation of the antibody -cell binding. Then, the sample holding space 4 was gently flushed with PBS three times, in order to remove the cells not anchored specifically to antibodies. Eventually, the final interference pattern 10 was recorded in PBS. For reference, the same procedure was repeated with a device functionalized with non-specific antibodies (IgG goat / anti-mouse).

[0147] Special care had to be taken during the measurements, to avoid any air bubble(s) remaining in the sample chamber. On the one hand, these might prevent cell adhesion, while on the other, they distort the measuring laser beam, introducing artifacts in the interference pattern 10. 2.2. Results and evaluation

[0148] 2.2. 1. Working principles

[0149] The light coming from the optical fiber is slightly divergent, having a cone shape and Gaussian intensity distribution as a function of distance from the axis of the optical fiber (Figure 2). The numerical aperture (NA = 0. 12) of the single-mode optical fiber determines the half-angle of that light cone. The exit end of the core of the optical fiber acts as a light source 1 in this case, and it is centered at the half-diameter of the optical fiber (62.5 pm) from the surface of the glass plate. There is an area from where the incident light beam is reflected. Since the angle of the light cone is rather small (less than 5 degrees), obeying the optical laws, most of the light incident on the surface is reflected from it, and eventually meets the directly propagating light. Note that some parts of the secondary reflected light from the lower surface of the glass substrate might also contribute to the interference to a lesser extent, but this does not represent a practical limitation of our method, since the evaluation procedure does not make use of the which-way information of the signal beam.

[0150] Due to geometrical reasons, the reflected light travels a longer way to a certain point on the screen 6 than the directly propagating one, in the same medium. Since in this case the light is a (coherent, monochromatic) laser beam, the differences in the optical path-length result in an interference pattern (Figure 2). For this particular arrangement, it is a series of parallel bright stipes separated by dark ones (Figure 2b).

[0151] 2.2.2. Evaluation procedure

[0152] At first, the reference experiment was carried out when the inner wall of the device was coated with nonspecific antibodies. Having completed the measuring cycle (i.e., after final washing with pure PBS buffer), no cells were found to be attached to the surface of the channel, as checked by a microscope; however, the parallel stripes were recorded as a reference image when the channel was filled up with the buffer, shown as Figure 5a. During the next experiments, another device functionalized by the specific antibodies was used with the same procedure. In this case, as a result of the adherence of cells to the lower surface of the fluidic channel (i.e. the test surface 9), the interference pattern 10 is distorted (Figure 5c,). To quantify the difference, utilizing a MATLAB script (for details see Figures 9 / A and 9 / B and the script below), we created a one-dimensional array by column-wise summarizing the values of the digitized image pixels, yielding sinusoidal curves along the dimension perpendicular to the stripes as “reduced interferograms” (Figure 5). Subsequently, a Fourier-transform was carried out, yielding the intensity distribution as a function of space frequency (Figure 5d). The magnitude of the effects was then defined by the ratio of the amplitudes of the main peaks of the Fourier-components determined from the interference fringes recorded at the end of the measuring cycle, and in the reference image. We found this method preferred to other methods attempting to determine visibility.

[0153] The Matlab script, used for the graphs is copied here as a text: start=imread('ref_.JPG'); finish=imread('le6_.JPG'); start_vector=sum(start(:, 1:2100)); % reference vector finish_vector=sum(fmish(:, 1:2100)); % 'cells in' vector figure plot([start_vector' finish_vector']) % graphs of the two vectors legend('Reference', 'Cells in') f_start=fft(start_vector); % FFT on control vector f_finish=fft(finish_vector); % FFT on 'cells in' vector len_start=length(start_vector); % length of the reference vector len_finish=length(finish_vector); % length of the 'cells in' vector ff_start=abs(f_start(l:len_start / 2+l)); % Freq spectrum, reference vector ff_finish=abs(f_finish(l:len_start / 2+l)); % Freq spectrum, 'cells in' vector max( [ff_start( 10 : end) ' ff_fmi sh( 10 : end) '] ) ;

[0154] Peak_ref=max([ff_start(10:end)]) %MAx value over the 10 position in Reference

[0155] Peak_cells=max([ff_finish(10:end)])% Max value of sample over the 10 position

[0156] Peak cells in per Peak_ref=Peak_cells / Peak_ref % Ratio of peak amplitudes figure % graph of the freq spectras (recalculated)

[0157] % The axis X is logaritmic semilogx([abs(f_start(l:len_start / 2+l)') abs(f_finish(l:len_finish / 2+l)')]) legend('Reference', 'Cells in')

[0158] 2.2.3. Calibration of the device

[0159] To determine the cell -concentration dependence of the method, a concentration series of cell suspension, from 103to 106cells / mL, was prepared, and the above procedure was carried out for each concentration (Figure 6).

[0160] Note that the frequency values assigned to the maxima of the main peaks might occasionally be shifted during the measuring cycle (Figures 9 / A and 9 / B), by effects due to the, possibly different meniscus curvatures at the beginning and the end of the measurement. However, such a virtual change in the magnification of the fringe pattern does not alter the intensity distribution among the stripes of the main component and the rest, so it does not influence the weights of the Fourier components, either.

[0161] Ratio of the main peak values of the reference and sample and reference spectra, respectively(Table 1), depicted in a semi-logarithmic plot (Figure 7) shows a linear character in the examined concentration range, implying a logarithmic relationship between their signal size and cell concentration, apparently strictly following the Weber-Fechner law [Hecht, S., 1824], The values plotted in Figure 7 are listed in Table 1. Table 1: The values ploted in Figure 7. The ratios are presented in four-digit precision showing the degradation and distortion of the interference pattern as the concentration of cells increases.

[0162] ,Tx Amplitude Ratio (Cells

[0163] Concentration (Cells / niL) . _ „ - . in / Reference)

[0164] 1030.9105

[0165] 3 x 1030.7479

[0166] 1040.5857

[0167] 3 x 1040.4570

[0168] 1050.3749

[0169] 3 x 1060.1172

[0170] 1060.0888

[0171] EXAMPLE 3 - Embodiments of the device according to the invention

[0172] For demonstration of the principles of the method, an all-optical device was built as illustrated on figure 4. The all-optical device comprises a support 2 constituted in the present example by a lower microscope slide. The support 2 has a test surface 9 capable of binding the analyte. The test surface 9 of support 2 may be functionalized. The all-optical device has a sample holding space 4 with preferably a main direction formed as a fluidic sample holding chamber 3, which may be built from the aforementioned lower microscope slide and upper microscope slide spaced apart from each other by two spacers in between. Thus the sample holding space 4 is in direct contact with the test surface 9.

[0173] The all-optical device has a light source 1 built in the present example as an optical fiber housed in a sheath. The light source 1 provides coherent light in the form of a divergent light beam 5 preferably extending along a longitudinal direction, preferably along a symmetry axis, preferably a light cone. Said light source 1 is arranged to allow propagation of the light beam 5 from the light source 1 to the light-exit window through the sample holding space 4. .

[0174] The top slide may be shorter, allowing an easy way to insert the light source 1. As spacers, slices of microscope slides were used, which may be a bit longer than the upper glass plate. Here their thickness is around 1 mm, while the diameter of the stripped optical fiber is 125 pm.

[0175] The sample holding chamber 3 is also delimited by a window plate 8 having a light-exit window, which may be arranged perpendicularly to the longitudinal direction of the light beam 5 and to the test surface 9. The window plate 8 plays a dual role. It holds the liquid sample in the channel, and also its edge maintains the liquid surface flat and perpendicular to the test surface 9 by means of surface tension. The light-exit window of the window plate 8 located in the pathway of the light beam 5 is transparent to the light beam 5 emited by the light source 1. The light source 1 is arranged so that a first part of the light beam 5 passes across the sample holding space 4 to the light-exit window of the window plate 8 without illumination of the test surface 9, i.e. forming a direct light 5 a, and a second part of the light beam 5 passes across the sample holding space 4 whereas it illuminates the test surface 9 with the light to provide reflected light 5b from the test surface 9. The second part of the light beam 5 illuminates the analyte intended to be on the test surface 9. The test surface 9 may be ground, in order to avoid too much light reflected back to the direct-light region. The reflected light 5b and the direct light 5a interfere to provide a detectable interference pattern 10. The interference pattern 10 is dependent on the presence of the analyte on the test surface 9. In the present example the light emitted by the light source 1 passes through the light-exit window of the window plate 8. A detecting means 7 may be placed at a suitable distance from the light source 1 to detect the interference pattern 10. The detecting means 7 may be provided with an adjacent screen 6 on which the interference pattern 10 may detectably form.

[0176] The all-optical device comprises a support 2 having a test surface 9. The test surface 9 is in direct contact with a sample holding space 4 for accommodating a sample to be analysed. The test surface 9 may be provided with binding molecule for specifically binding the analyte. The size of the analyte is preferably commensurate with the wavelength of light (in the same order of magnitude) or larger. The analyte may be selected from the group consisting of: cells, extracellular vesicles, exosomes, viruses of at least 200 nm in at least one direction, preferably cells. The binding molecule may be a binding molecule for binding of a cell, preferably an antibody or antibody fragment.

[0177] As shown in the embodiment depicted in figure 13 the sample holding space 4 may be formed as a channel extending along a main direction. The sample holding space 4 may be delimited vertically by a cover plate, for example a microscope slide spaced apart from the test surface 9 of the support 2 by two spacers / spacing blocks. The two spacers / spacing blocks may delimit the sample holding space 4 laterally.

[0178] The all-optical device comprises a light source 1 to provide coherent light in the form of a divergent light beam 5 preferably extending along a longitudinal direction, preferably a light cone. The 1 light source is arranged to allow penetration of the light beam 5 into the sample. The light source 1 is preferably a singlemode optical fiber located at one end of the sample holding space 4 such that the longitudinal direction is preferably arranged parallelly to the test suraface 9. The optical fiber may be arranged in a sheath, which may be tightly inserted between the two spacers / spacing blocks.

[0179] The all-optical device has a window plate 8 having a transparent light-exit window, which may be provided opposite to the first end face and perpendicular to the test surface 9. The light exit end of the optical fiber sheath and the window plate 8 may constitute respectively a first end face and a second end face delimiting axially the sample holding space 4 so as to retain the sample accommodated in the sample holding space 4. The light source 1 is arranged such that in an operating state a first part of the light beam 5 passes across the sample holding space 4 accommodating the sample to the to the light-exit window of the window plate 8 without illumination of the test surface 9, i.e. by forming a direct light 5a. The second part of the light beam 5 passes across the sample holding space 4 whereas it illuminates the test surface 9 with the coherent light to provide reflected light 5b from the test surface 9. The second part of the light beam 5 illuminates the analyte intended to be on the test surface 9.

[0180] The reflected light 5b and the direct light 5 a interfere to provide a detectable interference pattern 10, which is dependent on the binding of the analyte on the test surface 9 (if the analyte is present in the sample).

[0181] As shown on figure 14 another embodiment of the all -optical device according to the invention may be provided with a flow-through sample holding space 4. In this embodiment three spacers / spacing blocks may be provided to delimit laterally the sample holding space 4. The sample holding space 4 may be formed as a labyrinth having an inlet for entering a fluid into the sample holding space 4 and an outlet for evacuating the fluid from the sample holding space 4. The labyrinth may have for example three sections perpendicular to each other, with one section extending along the longitudinal direction of the light beam 5. The flow- through configuration enables to maintain the sample in the sample holding space 4 for a certain time sufficient to enable the binding of the analyte to the test surface 9.

[0182] The sample holding space 4 may have an opening for filling the sample holding space 4 and for removing the content of the sample holding space 4. As depicted on figure 15 the opening may be provided on the cover plate. In this case the two openings are the same.

[0183] Moreover, the wall delimiting the sample holding space 4 may be joined to each other by curved edges in order to facilitate the cleaning of the sample holding space 4 .

[0184] The surface of the cover plate delimiting the sample holding space 4 may have light reflecting properties with respect to the light emitted by the light source 1. In this case a third part of the emitted light beam 5 may pass across the sample holding space 4 whereas it illuminates the test surface 9 with the (coherent) light to provide reflected light 5d from the test surface 9.

[0185] The sample holding space 4 may be delimited by walls joined to each other by curved edges. This configuration facilitates the flushing and cleaning of the sample holding space 4. In another embodiment, the sample holding space 4 may be part a cuvette, which may be accommodated in the all-optical device. The base of the cuvette may then form a removable test surface 9.

[0186] Referring to figure 16 and figure 17 the all-optical device may further comprise a support 2 with a reference surface 29 and a reference sample holding space 24 for accommodating a reference sample formed as a fluidic reference sample holding chamber 23. The reference sample holding space 24 may be in direct contact with the reference surface 29. The sample holding space 4 as well as the reference sample holding space 24 may be delimited by respectively four vertical walls arranged perpendicularly to each other. The all-optical device may be thus partitioned into two spaces. The partitioned device may comprise a further window plate 28 having a transparent light-exit window. A further light source 21 may be disposed at the reference sample holding space 24 for providing coherent light in the form of a divergent light beam 25 preferably extending along a longitudinal direction. The further light source 21 may be arranged to allow propagation of the light beam 25 from the further light source 21 to the light-exit window through the reference sample holding space 24.

[0187] The first part of the light beam 25 may propagate in the reference sample holding space 24 to the lightexit window without illumination of the reference test surface 29, forming a direct light (25 a). A second part of the light beam 25 may propagate in the reference sample holding space 24 whereas it illuminates the reference test surface 2) with the light to provide a reflected light 25b reflected from the reference test surface 29. Thus the second part of the light beam 25 may illuminate the analyte intended to be on the reference test surface 29. The reflected light 25b and the direct light 25a may interfere to provide a detectable reference interference pattern 20. The interference pattern 20 may similarly to the reference pattern 10 depend on the presence of the analyte on the reference test surface 29.

[0188] In the embodiment depicted in figures 16 and 17 both the sample holding space 4 and the reference sample holding space 24 may be provided with a fluid ingress opening for filling respectively the sample holding space 4 and the reference sample holding space 24 and a fluid egress opening for removing the content of respectively the sample holding space 4 and the reference sample holding space 24. The respective fluid ingress opening and fluid egress opening may be provided on a lateral wall of the sample holding chamber 3 and the reference sample holding chamber 23, with the fluid ingress opening being preferably below the fluid egress opening.

[0189] LIST OF DESIGNATIONS

[0190] 1 light source

[0191] 2 support

[0192] 3 sample holding chamber

[0193] 3 a cover

[0194] 3b wall

[0195] 4 sample holding space

[0196] 5, 25 light beam

[0197] 5 a, 25 a direct light

[0198] 5b, 25b reflected light

[0199] 6 screen

[0200] 7 detecting means 8, 28 window plate

[0201] 9 test surface

[0202] 10 interference pattern

[0203] 21 light source

[0204] 23 reference sample holding chamber

[0205] 24 reference sample holding space

[0206] 29 reference test surface

[0207] 20 reference interference pattern

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Claims

CLAIMS1 An all-optical device for detecting an analyte in a sample, said device comprising a support (2) a test surface (9) on the support (2), a sample holding space (4) for accommodating the sample, the sample holding space (4) being in direct contact with the test surface (9), a window plate (8) having a transparent light-exit window, a light source (1) providing coherent light in the form of a divergent light beam (5), said light source (1) being arranged to allow propagation of the light beam (5) from the light source (1) to the lightexit window through the sample holding space (4), wherein a first part of the light beam (5) propagates in the sample holding space (4) to the light-exit window without illumination of the test surface (9), forming a direct light (5a), and a second part of the light beam (5) propagates in the sample holding space (4) whereas it illuminates the test surface (9) with the light to provide a reflected light (5b) reflected from the test surface (9), the analyte, if present, is presented on test surface (9) to the second part of the light beam (5) or the second part of the light beam (5) illuminates the analyte intended to be on the test surface (9) and wherein the reflected light (5b) and the direct light (5a) interfere to provide a detectable interference pattern (10), and wherein the interference pattern (10) is dependent on the presence of the analyte on the test surface (9).2 The all-optical device of claim 1 wherein the light source (1) is a single-mode optical fiber and the light beam (5) is a light cone extending along a longitudinal direction, preferably along a symmetry axis.3 The all-optical device of any one of claims 1 to 2, wherein the test surface (9) comprises binding molecule for specifically binding the analyte, wherein the size of the analyte is commensurate with the wavelength of light or larger, and wherein the analyte is selected from the group consisting of: cells, extracellular vesicles, exosomes, viruses of at least 200 nm in at least one direction, preferably cells.4 The all-optical device of claim 3, wherein the binding molecule is a binding molecule for binding of a cell, preferably an antibody or antibody fragment.5 The all-optical device according to any of claims 1 to 4, also comprising a screen (6) wherein the interference pattern (10) is detectably formed on the screen (6).6 The all-optical device according to any of claims 1 to 5, wherein the sample holding space (4) has an opening for filling the sample holding space (4) and an opening for removing the content of the sample holding space (4); optionally the two openings are the same.7 The all-optical device according to any of claims 1 to 6, wherein the sample holding space (4) is a flow-through sample holding space (4) having an inlet for entering a fluid into the sample holding space (4) and an outlet for leaving / removing the fluid from the sample holding space (4), the flow -through configuration allowing, after being present in the sample holding space (4) for a sufficient time binding of the analyte to the test surface (9).8 The all-optical device according to any of claims 1 to 6, wherein said sample holding space (4) has a cover, preferably a reflecting cover, wherein the light beam (5) is reflected both from the test surface (9) and the reflecting cover.9 The all-optical device according to any of claims 1 to 8, wherein said sample holding space (4) is delimited by walls joined to each other by curved edges.10 The all-optical device according to any of claims 1 to 7, wherein the device also comprises- a reference surface (29) provided on the support (2), and- a reference sample holding space (24) for accommodating a reference sample, the reference sample holding space (24) being in direct contact with the reference surface (29),- a further window plate (8) having a transparent light-exit window,- a further light source (21) providing coherent light in the form of a divergent light beam (25) preferably extending along a longitudinal direction, said further light source (21) being arranged to allow propagation of the light beam (25) from the further light source (21) to the light-exit window through the reference sample holding space (24) wherein a first part of the light beam (25) propagates in the reference sample holding space (24) to the light-exit window without illumination of the reference test surface (29), forming a direct light (25a), and a second part of the light beam (25) propagates in the reference sample holding space (24) whereas it illuminates the reference test surface (29) with the light to provide a reflected light (25b) reflected from the reference test surface (29),the analyte, if present, is presented on test surface (29) to the second part of the light beam (25) or the second part of the light beam (25) illuminates the analyte intended to be on the test surface (29) and wherein the reflected light (25b) and the direct light (25a) interfere to provide a detectable reference interference pattern (20), and wherein the reference interference pattern (20) is dependent on the presence of the analyte on the reference test surface (29).11 The all-optical device according to claim 10, also comprising means to illuminate said test surface (9) and said reference surface with a light parallel to surfaces, to provide reflected light from each surfaces.12 A method of all-optical detection of an analyte in a sample, in an all-optical device comprising a support (2) a test surface (9) on the support (2), a sample holding space (4) for accommodating the sample, the sample holding space (4) being in direct contact with the test surface (9), a window plate (8) having a transparent light-exit window(def: including opening), a light source (1) providing light in the form of a divergent light beam (5), said light source (1) being arranged to allow propagation of the light beam (5) from the light source (1) to the ligt-exit window through the sample holding space (4), said method comprising accommodating said sample in a sample holding space (4) being in direct contact with the test surface (9), contacting said sample accommodated in the sample holding space (4) with the test surface (9), allowing said analyte in the sample to accumulate at the test surface (9), providing a coherent light in the form of a divergent light beam (5) propagating through the sample holding space (4), the surfaces delimiting the sample holding space (4) being transparent to the light emitted by said light beam (5), wherein a first part of the light beam (5) propagates in the sample holding space (4) to the light-exit window without illumination of the test surface (9), forming a direct light (5a), and a second part of the light beam (5) propagates in the sample holding space (4) whereas it illuminates the test surface (10) with the light to provide a reflected light (5b), anddetecting interference between the direct light (5 a) and the reflected light (5b) from the test surface (9), to provide a detectable interference pattern (10), analyzing said interference pattern (10) to assess whether the analyte is present on the test surface (9).13 The method according to claim 12, wherein the light source (1) is a single-mode optical fiber and the light beam (5) is a light cone.14 The method according to claims 12 to 13, wherein the test surface (9) comprises binding molecule for specifically binding the analyte, wherein the size of the analyte is commensurate with the wavelength of light or larger, and wherein the analyte is selected from the group consisting of: cells, extracellular vesicles, exosomes, viruses of at least 200 nm in at least one direction, preferably cells.15 The method according to claim 14, wherein the binding molecule is a binding molecule for binding of a cell, preferably an antibody or antibody fragment.16 The method according to claims 12 to 15, wherein the all-optical device also comprising a screen (6) wherein the interference pattern (10) is detectably formed on the screen (6).17 The method according to claims 12 to 16, wherein the sample holding space (4) has an opening for filling the sample holding space (4) and an opening for removing the content of the sample holding space (4); optionally the two openings are the same.18 The method according to claims 12 to 17, wherein the sample holding space (4) is a flow-through sample holding space (4) having an inlet for entering a fluid into the sample holding space (4) and an outlet for leaving / removing the fluid from the sample holding space (4), the flow-through configuration allowing, after being present in the sample holding space (4) for a sufficient time binding of the analyte to the test surface (9).19 The method according to claims 12 to 17, wherein said sample holding space (4) has a cover, preferably a reflecting cover, wherein the light beam (5) is reflected both from the test surface (9) and the reflecting cover.20 The method according to claim 12,wherein the all-optical device also comprising- a support (2) having a reference surface, and- a reference sample holding space for accommodating a reference sample, the reference sample holding space being in direct contact with the reference surface,- a further transparent light-exit window on the window plate (8),- a further light source providing further light in the form of a further divergent light beam, said further light source being arranged to allow propagation of the further light beam from the further light source to the further light-exit window through the reference sample holding space, said method further comprising accommodating a reference sample in a reference sample holding space being in direct contact with the reference surface, contacting the reference sample accommodated in the reference sample holding space with the reference surface, providing a coherent light in the form of a further divergent light beam propagating through the reference sample holding space, wherein a first part of the further light beam (25) propagates in the reference sample holding space (24) to the light-exit window without illumination of the reference test surface (29), forming a further direct light (25 a), and a second part of the further light beam propagates in the reference sample holding space (24) whereas it illuminates the reference test surface (29) with the light to provide a reflected light (25b) reflected from the reference test surface (29), and detecting interference between the further direct light (25 a) and the further reflected light (25b) from the reference test surface (29), to provide a reference interference pattern (20), using the reference interference pattern (20) in analyzing said interference pattern (10) to assess whether the analyte is present on the test surface (9), preferably comparing the interference pattern (10) with the reference interference pattern (20), and assessing the binding of the analyte from the difference between the interference pattern (10) with the reference interference pattern (20).

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