Microparticle for immobilizing analytes from a fluid; method and apparatus for fabricating the microparticle

Abstract

A microparticle for immobilizing analytes from a fluid is provided. The microparticle comprises a body. The body comprises a hydrophobic surface. The microparticle further comprises detection elements. The detection elements comprise a first detection element and a second detection element. The first detection element comprises a first hydrophilic surface. The second detection element comprises a second hydrophilic surface. The detection elements are mechanically interconnected by means of the body. The first detection element comprises, at the first hydrophilic surface, a first molecule composition for immobilizing analytes from the fluid at the first hydrophilic surface. The second detection element comprises, at the second hydrophilic surface, a second molecule composition for immobilizing analytes from the fluid at the second hydrophilic surface. The first molecule composition is different from the second molecule composition. The microparticle is non-mirror symmetric about any plane which does not intersect all of the detection elements.

Description

Technische Universitat Miinchen, in Vertretung des Freistaates BayernU31245WOMicroparticle for immobilizing analytes from a fluid; method and apparatus for fabricating the microparticleFIELD OF THE DISCLOSURE

[0001] The present disclosure relates to a microparticle for immobilizing analytes from a fluid selectively at a predefined region along the microparticle. Specifically, the present disclosure pertains to a microparticle with a hydrophobic body and hydrophilic detection elements, the hydrophilic detection elements comprising molecule compositions for immobilizing the analytes. The disclosure further relates to a method and an apparatus for fabrication of the microparticle using parallel fluid flows in a flow channel.BACKGROUND

[0002] For the compartmentalization of aqueous specimen, micro well plates are commonly applied. Microwell plates typically provide up to 1536 compartmentalized spaces in the form of microwells, each typically providing a volume in a range of tens of nanoliters up to several milliliters.[ooo3]For high-throughput or high-resolution (i.e., in terms of analyzing smaller volumes or sub volumes of the aqueous specimen) investigations on aqueous specimen, a compartmentalization into compartments of smaller volumes and / or larger number is desirable.SUMMARY OF THE DISCLOSURE

[0004] It is an object of this invention to provide microparticles that overcome one or more of the disadvantages of known systems, as well as a method and an apparatus for providing such microparticles.[ooo5]According to a first aspect of the disclosure, a microparticle for immobilizing analytes from a fluid is provided. The microparticle comprises a body. The body comprises a hydrophobic surface. The microparticle further comprises detection elements. The detection elements comprise a first detection element and a second detection element. The first detection element comprises a first hydrophilic surface. The second detection element comprises a second hydrophilic surface. The detection elements are mechanically interconnected by means of the body. The first detection element comprises, at the first hydrophilic surface, a first molecule compo-sition for immobilizing analytes from the fluid at the first hydrophilic surface. The second detection element comprises, at the second hydrophilic surface, a second molecule composition for immobilizing analytes from the fluid at the second hydrophilic surface. The first molecule composition is different from the second molecule composition. The microparticle is non-mir-ror symmetric about any plane which does not intersect all of the detection elements.

[0006] When introduced into the fluid with the analytes, a respective microparticle provides for selective mobilization of the analytes from the fluid at the (first and second) hydrophilic surfaces of the detection elements. The selectivity is achieved by the hydrophobic / hydrophilic character of the body and the detection elements, i.e., analytes which are soluble in aqueous solutions wet the hydrophilic surfaces of the detection elements rather than the hydrophobic surface of the body. At the (first and second) hydrophilic surfaces of the detection elements, the analytes encounter the (first and second) molecule compositions, which immobilize the analytes at said surfaces.

[0007] As the molecule compositions of the detection elements, or at their hydrophilic surfaces, respectively, differ, analytes from the fluid are immobilized selectively at either of the detection elements, or at the hydrophilic surfaces thereof, respectively. Thus, the microparticle according to the invention allows for selectively immobilizing the analytes at predefined positions along the microparticle, or along its surface, respectively, so that the analytes become accessible for investigations, for example assays such as a bioassay or an immunoassay, at well-defined positions along the microparticle, for example using microscopic techniques.[ooo8]The difference in molecule composition between the first and second detection elements thus allows for the simultaneous detection of multiple analytes, making the microparticle highly versatile for multiplex assays.

[0009] However, degrees of freedom which give rise to an uncertainty regarding the position of the immobilized analyte (or of the detection element that the analyte has been immobilized at, respectively) along the microparticle result from the orientational degrees of freedom of the microparticle, including the rotational degrees of freedom. In many cases, the orienta-tional / rotational degrees of freedom of the microparticle are constrained as a microparticle typically has an anisotropic, flattened shape. When the fluid with the analytes and the microparticle are placed on a substrate defining a lower boundary of the fluid (e.g., the bottom of a well plate or a petri dish, or the surface of a microscope slide), the microparticle tends to align with its flattened side along and / or on that lower boundary. This is an immediate consequenceof the flattened shape of the microparticle, because of which the potential energy of the microparticle is minimized when the microparticle is aligned with its flattened side along and / or on the lower boundary. Consequently, the remaining orientational degrees of freedom of the microparticle are the orientation around the surface normal of the lower boundary, and the binary degree of freedom as to which side of the microparticle faces up or down. Typically, a microparticle exhibits two flattened surfaces on opposite sides, and when the microparticle settles on the lower boundary of the substrate due to its high density (as compared to the density of the aqueous fluid), either of the flattened surfaces may point upwards or downwards, similar to the situation after flipping a coin.[ooio] As a consequence of the aforementioned degrees of freedom, and, in particular, for a microparticle having an overall highly symmetric shape (e.g., similar to a cylinder or a square), observing a microparticle with several detection elements from above or below (e.g., along the surface normal of said lower boundary of the substrate) typically does not allow for an unambiguous assignment of the observed detection elements to specific molecular compositions, or to immobilized analytes, respectively.[oon] The microparticle according to the invention solves this problem by providing the microparticle with a non-mirror symmetric shape. As a consequence, the orientation of the microparticle can be determined unambiguously when the microparticle is observed from above or below (e.g., along the surface normal of said lower boundary of the substrate). This is beneficial for accurate analysis in diagnostic applications.

[0012] In the context of this disclosure, the term microparticle may refer to an object having a size of less than 1 mm along at least one spatial direction, in particular having a thickness of less than 1 mm.

[0013] Alternatively, the term microparticle may refer to an object having a size of less than 1 mm along any spatial direction.

[0014] The analytes may be biomolecules such as ligands or proteins or antigens.

[0015] The fluid maybe an aqueous fluid.

[0016] Notably, the microparticle may exhibit a mirror symmetry about a mirror plane intersecting all of the detection elements and / or about a mirror plane perpendicular to a thickness direction of the microparticle.

[0017] The thickness direction may correspond to a direction, along which the spatial extension of the microparticle is the smallest.

[0018] The thickness direction maybe a direction from the top surface to the bottom surface.

[0019] According to an embodiment, the body comprises a marker structure. The marker structure is non-mirror symmetric about any plane which does not intersect all of the detection elements.

[0020] The inclusion of a marker structure that is non-mirror symmetric enhances the microparticle's ability for its orientation to be identified during analysis. This asymmetry in the marker structure aids in distinguishing the orientation of the microparticle and ensures a correct assignment of the individual detection elements.

[0021] In particular, since the marker structure is part of the body, it can be observed in bright-held microscopy, for example in reflected light microscopy or in transmitted light microscopy. In other words, a respective marker structure facilitates a quick detection of the orientation of the microparticle using well-established and simple techniques.

[0022] According to some embodiments, the marker structure is associated with an outer surface of the body. In particular, the marker structure may be formed by the outer surface of the body or may be arranged on the outer surface of the body.

[0023] Optionally, the body has a top surface and a bottom surface opposite to the top surface, and at least part of the marker structure is associated with the top surface and / or with the bottom surface. In particular, the at least part of the marker structure may be associated with a circumferential shape of the top surface and / or with a circumferential shape of the bottom surface. In particular, the marker structure may comprise recesses in the circumferential shape of the top surface and / or in the circumferential shape of the bottom surface. In particular, at least two of said recesses may have different shapes and / or at least two of said recesses may be comprised in the same one of the circumferential shape of the top surface and the circumferential shape of the bottom surface. In particular, said at least two of the recesses may have different shapes and may both be comprised in both the circumferential shape of the top surface and the circumferential shape of the bottom surface.

[0024] The marker structure being associated with the outer surface of the body allows for easy identification and differentiation of the microparticle, in particular under a microscope andusing brightfield microscopy. This can be particularly useful in applications where multiple microparticles are used, as it enables quick visual or automated recognition.

[0025] According to an embodiment, the microparticle comprises at least two different marker materials having different optical properties, wherein the at least two different marker materials are arranged in the microparticle non-mirror symmetrically about any plane which does not intersect all of the detection elements.

[0026] Similar to the marker structure, the inclusion of at least two different marker materials with different optical properties allows for detecting the orientation of the microparticle. The non-mirror symmetrical arrangement ensures that the detection elements can be uniquely identified based on their optical signatures, enhancing the accuracy and reliability of analyte detection.

[0027] The different marker materials may be applied as an alternative to the marker structure, or they may be applied additionally, to make the orientation accessible to a larger variety of detection techniques, including photoluminescence microscopy.

[0028] In respective embodiments, the first detection element may comprise a first marker material of the at least two different marker materials at a higher concentration, in particular in terms of weight percent or in terms of molar concentration, than the second detection element, and the second detection element may comprise a second marker material of the at least two different marker materials at a higher concentration, in particular in terms of weight percent or in terms of molar concentration, than the first detection element. In particular, said higher concentrations may be higher at least by a factor of two or at least by a factor of five or at least by a factor of ten.

[0029] In respective embodiments, the first detection element may comprise a first marker material of the at least two different marker materials at a higher concentration, in particular in terms of weight percent or in terms of molar concentration, than the body. In particular, this higher concentration maybe higher at least by a factor of two or at least by a factor of five or at least by a factor of ten.

[0030] Alternatively, or in addition, the second detection element may comprise a second marker material of the at least two different marker materials at a higher concentration, in particular in terms of weight percent or in terms of molar concentration, than the body. Inparticular, this higher concentration may be higher at least by a factor of two or at least by a factor of five or at least by a factor of ten.

[0031] The different optical properties may comprise at least one of the following: different photoluminescence emission wavelengths, and maxima at different wavelengths in corresponding optical absorption spectra.

[0032] Alternatively, or in addition, the at least two different marker materials may comprise or maybe at least two different photoluminescent dyes. In particular, the at least two different marker materials may comprise or may be at least two different fluorescent dyes.

[0033] Using different photoluminescent or fluorescent dyes as marker materials enhances the optical detection capabilities of the microparticle. These dyes provide strong and distinct optical signals, which improve the sensitivity in determining the orientation of the microparticle.

[0034] According to an embodiment, the analytes comprise analytes of a first type, and the first molecule composition comprises immobilization molecules of a first type at a higher concentration than the second molecule composition. In respective embodiments, the immobilization molecules of the first type may be adapted to immobilize analytes of the first type at the respective surface comprising the respective molecule composition comprising the immobilization molecules of the first type.

[0035] Respective embodiments beneficially ensure that analytes of the first type get immobilized at the first hydrophilic surface, providing them at a predefined position along the microparticle for further analysis.

[0036] In respective embodiments, the immobilization molecules of the first type may be adapted to selectively immobilize the analytes of the first type at the respective surface comprising the respective molecule composition comprising the immobilization molecules of the first type.

[0037] The higher concentration may refer to a higher concentration in terms of weight percent or in terms of molar concentration.

[0038] The higher concentration may be higher at least by a factor of two, or at least by a factor of five, or at least by a factor of ten.

[0039] The immobilization molecules of the first type may be essentially absent in the second molecule composition.

[0040] According to an embodiment, the analytes comprise analytes of a first type and analytes of a second type. In respective embodiments, the first molecule composition may comprise immobilization molecules of a first type for selectively immobilizing the analytes of the first type from the fluid at the first hydrophilic surface, and the second molecule composition may comprise immobilization molecules of a second type for selectively immobilizing the analytes of the second type from the fluid at the second hydrophilic surface.

[0041] A respective microparticle can effectively differentiate and capture multiple types of analytes from a fluid. This selective immobilization enhances the specificity and efficiency of the microparticle in capturing target analytes, providing a significant technical improvement in applications such as diagnostics and assays such as biochemical assays.

[0042] The microparticle for immobilizing the analytes from the fluid may refer to a microparticle for immobilizing analytes of the first type and of the second type different from the first type from the fluid, in particular, selectively at different surface regions along the microparticle.

[0043] The first molecule composition may comprise the immobilization molecules of the first type at a higher concentration, in particular in terms of weight percent or in terms of molar concentration, than the second molecule composition and optionally than the body, in particular, at least by a factor of two or at least by a factor of five or at least by a factor of ten.

[0044] The second molecule composition may comprise the immobilization molecules of the second type at a higher concentration, in particular in terms of weight percent or in terms of molar concentration, than the first molecule composition and optionally than the body, in particular, at least by a factor of two or at least by a factor of five or at least by a factor of ten.

[0045] According to an embodiment, the first detection element comprises a first cavity, and at least part of the first hydrophilic surface is a surface of the first cavity, and the second detection element comprises a second cavity, and at least part of the second hydrophilic surface is a surface of the second cavity.

[0046] The first detection element and / or the second detection element may have a width (such as a diameter) in a range from 50 pm to 120 pm.

[0047] The first detection element and / or the second detection element may have a thickness (e.g., from a top surface of the microparticle to a bottom surface of the microparticle) in a range from 100 pm to 150 pm.

[0048] The first cavity and / or the second cavity may have a volume of no more than 1 nanoliter, preferably, a volume in a range from 200 pL to 1 nL.

[0049] The presence of cavities in the detection elements allows for an increased surface area for analyte immobilization, enhancing the immobilization capability of the microparticle. In particular, as an analyte having diffused into one of the cavities is surrounded by the corresponding hydrophilic surface on multiple sides, the probability that the analyte gets immobilized is improved (i.e., enhanced) significantly.

[0050] Further, the first and second hydrophilic surfaces interact with an aqueous fluid that the microparticle is placed in. The aqueous fluid wets the respective surfaces. The aqueous fluid having wetted the first / second hydrophilic surfaces is sealed by the body and / or by the first / second detection element. Preferably, the microparticle is applied in a fluid comprising an oil, more specifically, and / or is transferred to a fluid comprising an oil after exposure to the fluid comprising the analyte. The oil seals the cavities, and thus the fluid having wetted the first / second hydrophilic surfaces. This results in sealed nano-liter compartments within microparticles.

[0051] The first cavity and the second cavity may be separated from one another by means of the body.

[0052] Separating the cavities by the body ensures that there is no cross-contamination between the different molecule compositions on the first and second hydrophilic surfaces. This separation maintains the integrity of the detection process for each type of analyte, leading to more accurate and reliable results.

[0053] The compartments within the microparticle and separated by the body also allow for signal accumulation for different types of analytes within corresponding cavities of the microparticle, while minimizing cross-contamination at the same time.

[0054] Said surface of the first cavity may refer to an inner surface of the first cavity and said surface of the second cavity may refer to an inner surface of the second cavity.

[0055] A respective geometry enhances the probability for successful immobilization further.

[0056] The first cavity and / or the second cavity may be formed as a through hole extending from a bottom surface of the microparticle to a top surface of the microparticle, the top surface and the bottom surface being opposite surfaces of the microparticle.

[0057] A respective geometry enhances the probability for successful immobilization further.

[0058] In addition, the through-hole geometry allows for subsequent analysis of the analyte immobilized on the respective hydrophilic surface using transmission microscopy, as light can be shone through the through hole.

[0059] According to some embodiments, the detection elements maybe embedded in the body, in particular, the first cavity and the second cavity may be embedded in the body.[oo6o]According to an embodiment, the microparticle has a thickness of less than 1 mm, in particular from a top surface of the microparticle to a bottom surface of the microparticle, the bottom surface being located opposite to the top surface, in particular, wherein the top surface and the bottom surface are parallel to each other and / or wherein an aspect ratio between the thickness of the microparticle and a width of the top surface and / or of the bottom surface is no more than 1:1 or no more than 1:2 or no more than 1:2.5 or no more than 1:3.

[0061] A respective geometry beneficially ensures that the microparticle aligns on a substrate (e.g., at the bottom of the well plate or on a microscope slide) with one of the top surface and the bottom surface pointing towards the substrate, and the other one of the top surface and the bottom surface pointing in the opposite direction, which is typically the opposite direction with respect to an external reference frame defined by the local direction of the gravitational field of the earth. Thus, the rotational degrees of freedom of the microparticle get constrained from three rotational degrees of freedom to a single rotational degree of freedom plus the binary degree of freedom of either the top surface or the bottom surface pointing upwards along the local direction of the gravitational field of the earth.

[0062] The microparticle may have a width of less than 3 mm, in particular of less than 1 mm and / or along any spatial direction.

[0063] A microparticle with a respective width can beneficially be fabricated using the stop flow lithography method described below.

[0064] Moreover, a microparticle with the respective width allows for compartmentalization into detection volumes, as defined by the detection elements, on the nanoliter scale.

[0065] The microparticle may be shaped to permit, when the microparticle is placed in the fluid, the fluid to reach the first hydrophilic surface and to reach the second hydrophilic surface.

[0066] The body may comprise or may consist of a body material, the body material being hydrophobic.

[0067] The body material may be a polymer.[oo68]The body material maybe a first hydrogel material.

[0069] The hydrophobic surface may be a surface of the body material and / or of the first hydrogel material.

[0070] The hydrophobic body material ensures a hydrophobic surface of the body.

[0071] A hydrophobic surface of the body prevents non-specific binding of analytes to the body, directing them instead to the hydrophilic detection elements. This selective interaction improves the accuracy of analyte detection by reducing background noise and enhancing signal clarity.

[0072] The body having the hydrophobic surface ensures a separation between the detection elements, thus ensuring that the detection elements can be resolved individually, e.g., in a microscopic measurement.

[0073] The body having the hydrophobic surface also ensures a separation between the detection elements and the outer circumference of the microparticle, thus ensuring that detection elements of different microparticles can be resolved individually, even if the microparticles are in contact with each other, e.g., in a microscopic measurement.

[0074] The body having the hydrophobic surface also assists, when the aqueous fluid comprising the analyte has wetted the hydrophilic surfaces of the detection elements, and when the microparticle is exposed to the fluid comprising oil, the oil of the fluid in sealing the aqueous fluid. This sealing beneficially supports signal accumulation. The inventors have found that,after sealing, the signal accumulation increases with time, providing an even further amplified signal, e.g., for a diagnostic immunoassay.

[0075] The body having the hydrophobic surface thus aids in signal accumulation, allowing to increase the signal over time, which can be then clearly distinguished from a reference background. The body having the hydrophobic surface therefore gives rise to an improved contrast between the signal and the background.

[0076] Microparticle, and in particular, the combination of the detection elements with the body having the hydrophobic surface, thus improve the accuracy and sensitivity of an assay performed with the microparticle.

[0077] The improved accuracy can be attributed to better statistical confidence, in particular, when multiple, e.g., hundreds of microparticles are applied simultaneously.

[0078] The increased signal improves the detection limit of the assay by providing a quantifiable signal, even from a small amount of analytes, or in case of a fluid comprising the analyte at very low concentration, respectively, for example, at a picomolar or femtomolar concentration. The microparticle thus provides an improved detection of analytes.

[0079] The first detection element may comprise or may consist of a first material, the first material being hydrophilic.[oo8o]The first material may be a polymer.

[0081] The first material maybe a second hydrogel material.

[0082] The first hydrophilic surface maybe a surface of the first material and / or of the second hydrogel material.

[0083] The second detection element may comprise or may consist of a second material, the second material being hydrophilic, in particular, wherein the second material is the same as the first material.[oo84]The second material maybe a polymer.

[0085] The second material maybe a third hydrogel material.[oo86]The second hydrophilic surface maybe a surface of the second material and / or of the third hydrogel material.

[0087] The term “hydrogel material” (i.e., in the context of the first hydrogel material, of the second hydrogel material, and of the third hydrogel material) may refer to a covalently crosslinked hydrogel.[oo88]The term “covalently cross-linked hydrogel” (i.e., in the context of the first covalently cross-linked hydrogel material, of the second covalently cross-linked hydrogel material, and of the third covalently cross-linked hydrogel material) may refer to a hydrogel network where the polymer chains are interconnected through covalent bonds.[oo89]These bonds are strong chemical bonds that provide stability and robustness to the hydrogel structure, making it less likely to dissolve or degrade in the presence of fluids. This stability ensures that the hydrogel can effectively immobilize analytes and withstand the mechanical stresses of fluid flow within the capillary tube.

[0090] The first hydrogel material may comprise at least one of the following or may be composed of one of the following: Allyl Methacrylate, Phenyl acrylate, Divinyl benzene, Benzyl Methylacrylate, 1,3-Butanediol Dimethacrylate, 1,4-Butanediol Dimethacrylate, Butyl Acrylate, n-Butyl Methacrylate, Ethyl Acrylate, Ethyl Methacrylate, 2-Ethyl Hexyl Acrylate, 1,6-Hexanediol Dimethacrylate, Isobutyl Methacrylate, Lauryl Methacrylate, Methyl Acrylate, Methyl Methacrylate, Stearyl Methacrylate, 2,2,3,3,4,4,5,5-Octafluoropentyl Acrylate, 2,2,2-Tri-fluoroethyl 2-methylacrylate, Polypropylene glycol) dimethacrylate (PPGDMA).

[0091] The second hydrogel material may be a covalently cross-linked hydrogel, formed from at least one of the following monomers or from a combination of the following monomers: Acrylamide, N,N-methylene-bisacryl-amide, Methacrylic Acid, Monoethylene Glycol, 4-Hy-droxybutyl Acrylate, Hydroxyethyl Acrylate (HEA), 2-Hydroxyethyl Methacrylate (HEMA), 2-Hydroxypropyl Acrylate, Diethyleneglycol Diacrylate (DEGDA), Diethyleneglycol Dimethacrylate (DEGDMA), Triethylene Glycol, Triethylene Glycol Dimethacrylate, Polyethylene Glycol (200 / 400 / 600) Diacrylate (PEGDA), Polyethylene Glycol (200 / 400 / 600) Dimethacrylate (PEGDMA).

[0092] The third hydrogel material may be a covalently cross-linked hydrogel, formed from at least one of the following monomers or from a combination of the following monomers: Acryla-mide, N,N-methylene-bisacryl-amide, Methacrylic Acid, Monoethylene Glycol, 4-Hydroxy-butyl Acrylate, Hydroxyethyl Acrylate (HEA), 2-Hydroxyethyl Methacrylate (HEMA), 2-Hy-droxypropyl Acrylate, Diethyleneglycol Diacrylate (DEGDA), Diethyleneglycol Dimethacrylate (DEGDMA), Triethylene Glycol, Triethylene Glycol Dimethacrylate, Polyethylene Glycol (200 / 400 / 600) Diacrylate (PEGDA), Polyethylene Glycol (200 / 400 / 600) Dimethacrylate (PEGDMA).

[0093] According to some embodiments, the first hydrogel material is a covalently cross-linked hydrogel comprising polypropylene glycol) diacrylate (PPGDA), the second hydrogel material is a covalently cross-linked hydrogel formed at least in part from poly( ethylene glycol) diacrylate (PEGDA), and the third hydrogel material is a covalently cross-linked hydrogel formed at least in part from poly( ethylene glycol) diacrylate (PEGDA).

[0094] The hydrophilic first material of the first / second detection element ensures a (first / second) hydrophilic surface of the first / second detection element.

[0095] The hydrophilic surface of the first / second detection element promotes strong interactions with analytes in the fluid, facilitating their immobilization on the surface. This characteristic enhances the efficiency and effectiveness of the immobilization process and of a further analysis and detection process.

[0096] Forming at least part of the first and second detection element from the same first / second material ensures efficient fabrication of the detection elements.

[0097] The first detection element and the second detection element maybe encircled by a top surface of the microparticle, in particular, wherein the first detection element and the second detection element are encircled by the top surface of the microparticle and by a bottom surface of the microparticle, the top surface and the bottom surface being opposite surfaces of the microparticle.[oo98]The first detection element and the second detection element may be encircled by a circumferential shape of the top surface of the microparticle and / or the first detection element and the second detection element maybe encircled by a circumferential shape of the top surface of the microparticle and by a circumferential shape of the bottom surface of the microparticle. In particular, the circumferential shape of the bottom surface of the microparticle and / or the circumferential shape of the top surface of the microparticle may be defined by the body ofthe microparticle.The encircling may refer to or may be an encircling in a plane defined by the bottom surface or defined by the bottom surface.

[0099] Encircling the detection elements with the top and bottom surfaces provides structural integrity and protection, ensuring that the detection elements remain intact and functional during use.

[0100] The detection elements may further comprise a third detection element comprising a third hydrophilic surface, wherein the third detection element at the third hydrophilic surface comprises a third molecule composition for immobilizing analytes from the fluid at the third hydrophilic surface, wherein the third molecule composition is different from the first molecule composition and from the second molecule composition.

[0101] Including a third detection element with a distinct molecule composition allows for the simultaneous detection of multiple analytes within the same microparticle. This feature increases the versatility and application range of the microparticle, enabling it to be used in complex analytical scenarios.

[0102] The detection elements may further comprise a fourth detection element comprising a fourth hydrophilic surface, wherein the fourth detection element at the fourth hydrophilic surface comprises a fourth molecule composition for immobilizing analytes from the fluid at the fourth hydrophilic surface, wherein the fourth molecule composition is different from the first molecule composition, from the second molecule composition and from the third molecule composition.

[0103] The addition of a fourth detection element further expands the microparticle's capability to detect a wider range of analytes simultaneously. This feature enhances the microparticle's utility in comprehensive analytical applications, providing a robust tool for multi-analyte detection.

[0104] The detection elements may be separated from one another by means of the body.

[0105] Separation of detection elements by the body prevents cross-contamination and interference between different analyte interactions. This spatial separation ensures that each detection element functions independently, improving the specificity and accuracy of the detection process.[oio6] Moreover, a separation between the detection elements facilitates reliable, separate detection of optical signals selectively from one of the detection elements.

[0107] According to a second aspect of the disclosure, a plurality of microparticles is provided. Each microparticle of the plurality comprises the marker structure according to any of the embodiments described above, and / or each microparticle comprises the at least two different marker materials according to any of the embodiments described above.

[0108] The arrangement of the marker structure and / or marker materials is consistent across the microparticles in the plurality. This uniformity ensures that the microparticles behave predictably and produce reliable results in assays. It also simplifies the manufacturing process, as each microparticle is produced to the same specifications, reducing variability and increasing the reproducibility of results.

[0109] In preferred embodiments, the arrangement of the marker structure and / or of the marker materials may be the same in the microparticles of the plurality of microparticles.

[0110] In some embodiments, a kit is provided, comprising a first plurality of microparticles and a second plurality of microparticles. Each microparticle of the first plurality comprises a first marker structure, the first marker structure being a marker structure according to any of the embodiments described above, and / or each microparticle of the first plurality comprises a first set of different marker materials, the first set of different marker materials comprising the at least two different marker materials according to any of the embodiments described above. Each microparticle of the second plurality comprises a second marker structure, the second marker structure being a marker structure according to any of the embodiments described above, and / or each microparticle of the second plurality comprises a second set of different marker materials, the second set of different marker materials comprising the at least two different marker materials according to any of the embodiments described above. In respective embodiments, the first set of different marker materials may be different from the second set of different marker materials. Alternatively, or in addition, the first marker structure may be different from the second marker structure.

[0111] Respective embodiments are particularly beneficial if, for example, a first vial Vi has the first plurality of microparticles with a first marker structure, lets call it Pi. A second vial V2 has the second plurality of microparticles with similar detection elements to the detection elements of the microparticles of the first plurality, but with different marker structure, P2. A biosample coming from patient 1 having a specific analyte concentration is mixed with Pi invial Vl, and a biosample from patient 2 is mixed with P2 in vial V2. After performing the first steps of an assay in separate vials Vi and V2, Pi will bind analyte concentration corresponding to patient i and P2 will bind analyte concentration specific to patient 2. Then Pi and P2 can be mixed and the remaining steps of the assay can be performed together. This will save effort, materials, and time. Upon imaging within one detection element containing two populations of particles Pi and P2 that can be distinct based on marker arrangement, the signal from one detection element demonstrates the concentration of analytes for two patients.

[0112] The plurality of microparticles may comprise at least five or at least 10 or at least 100 microparticles.

[0113] The microparticle according to the first aspect and / or the plurality of microparticles according to the second aspect maybe adapted for a uniform encapsulation of discrete aqueous droplets in the detection elements (such as for a uniform encapsulation of a first aqueous droplet in the first detection element and of a second aqueous droplet in the second detection element).

[0114] In other words, the microparticle according to the first aspect and / or the plurality of microparticles according to the second aspect may be adapted for the formation of consistent aqueous droplets in the detection elements (such as for the formation of a first aqueous droplet in the first detection element and for the formation of a second aqueous droplet in the second detection element).

[0115] According to a third aspect of the disclosure, a method is provided for fabricating the microparticle according to any of the embodiments above. The method comprises providing parallel flows of fluids in a flow channel. The parallel flows of fluids comprise a flow of a fluid comprising a precursor for a hydrophobic material. The parallel flows of fluids further comprise a flow of a first additional fluid and a flow of a second additional fluid. The first additional fluid comprises a precursor for a hydrophilic material, and the second additional fluid comprises a precursor for a hydrophilic material. The method further comprises curing the precursor for the hydrophobic material into a hydrophobic material forming at least part of the body, and curing the precursor for the hydrophilic material comprised in the first additional fluid into a hydrophilic material forming at least part of the first detection element. Additionally, the method comprises curing the precursor for the hydrophilic material comprised in the second additional fluid into a hydrophilic material forming at least part of the second detection element.

[0116] Optionally, the provided parallel flows of the fluids comprise a common solvent.

[0117] Any of the curing processes described above may comprise a photochemical reaction, and / or may refer to a curing using illumination, in particular illumination with UV radiation.

[0118] Any of the said hydrophilic materials may be a hydrophilic polymer, and / or the hydro-phobic material may be a hydrophobic polymer.

[0119] The term flow channel may refer to an object adapted to permit a fluid flowthrough the flow channel along a flow direction defined by the flow channel, the flow channel comprising sidewalls that enclose the fluid flow in a fluid-tight manner in the plane perpendicular to the flow direction.

[0120] The first additional fluid and the second additional fluid may comprise immobilization molecules, such that the first molecule composition comprises immobilization molecules from the first additional fluid and the second molecule composition comprises immobilization molecules from the second additional fluid. In particular, the immobilization molecules comprised in the first additional fluid may comprise immobilization molecules of a first type and the immobilization molecules comprised in the second additional fluid may comprise immobilization molecules of a second type.

[0121] Providing parallel flows of fluids in a flow channel ensures that the different precursor materials can be accurately controlled and directed. This allows for precise fabrication of the microparticle's structure, ensuring that each material is correctly positioned.

[0122] The flow of a first / second additional fluid comprising a precursor for a hydrophilic material allows for the formation of a (first / second) detection element with a (first / second) hydrophilic surface. This hydrophilic layer can be functionalized to interact with specific biomolecules, making it useful for applications such as biomarker detection. Having multiple detection elements, or multiple hydrophilic surfaces, respectively allows the microparticle to simultaneously detect multiple biomarkers, enhancing its diagnostic capabilities.

[0123] Curing the precursors into hydrophobic / hydrophilic material ensures that the microparticle is solidified and stable, providing the structural integrity of the microparticle, and well-defined positions of the detection elements along the fabricated microparticle.

[0124] The common solvent improves the parallelicity of the flows and thus the reliability and resolution of the fabrication method.

[0125] The common solvent allows hydrophobic and hydrophilic material to coflow in parallel streamlines without phase separation. The common solvent makes them partially miscible. The hydrophobic and hydrophilic material flowing parallel to each other can diffuse to some extent at the interface. This improves the quality and the resolution of the fabricated microparticles.

[0126] In the context of this disclosure, the term precursor may refer to a precursor for polymerization, or to a precursor for forming a polymer therefrom, respectively. In particular, the term precursor may refer to a photochemical precursor suitable for polymerization under illumination, such as illumination with UV light.

[0127] The hydrophobic material of the method may be the first hydrogel material described above.

[0128] The hydrophilic material for which the first additional fluid comprises the precursor may be the second hydrogel material described above.

[0129] The hydrophilic material for which the second additional fluid comprises the precursor may be the third hydrogel material described above.

[0130] According to some embodiments, the hydrophobic material is the first hydrogel material, the hydrophilic material for which the first additional fluid comprises the precursor is the second hydrogel material, and the hydrophilic material for which the second additional fluid comprises the precursor is the third hydrogel material.

[0131] The method for fabricating the microparticle according to this disclose may comprise forming the first covalently cross-linked hydrogel in the process step of curing of the precursor for the hydrophobic material into the hydrophobic material forming the at least part of the body, forming the second covalently cross-linked hydrogel in the process step of curing the precursor for the hydrophilic material comprised in the first additional fluid into the hydrophilic material forming the at least part of the first detection element, and forming the third covalently cross-linked hydrogel in the process step of curing the precursor for the hydrophilic material comprised in the second additional fluid into the hydrophilic material forming the at least part of the second detection element.

[0132] A precursor for a hydrogel material (as in the context of the precursor for the first, second, and / or third hydrogel material described above) may comprise at least one of the following or may be one of the following: Allyl Methacrylate; Benzyl Methylacrylate; 1,3-Butanediol Dimethacrylate; 1,4-Butanediol Dimethacrylate 745 Butyl Acrylate n-Butyl Methacrylate; Diethyleneglycol Diacrylate; Diethyleneglycol Dimethacrylate; Ethyl Acrylate 750 Ethyleneglycol Dimethacrylate; Ethyl Methacrylate; 2-Ethyl Hexyl Acrylate; 1,6-Hexanediol Dimethacrylate; 4- Hydroxybutyl Acrylate 755 Hydroxyethyl Acrylate; 2-Hydroxyethyl Methacrylate; 2-Hydrox-ypropyl Acrylate; Isobutyl Methacrylate; Lauryl Methacrylate 760 Methacrylic Acid; Methyl Acrylate; Methyl Methacrylate; Monoethylene Glycol; 2,2,3,3,4,4,5,5-Octafluoropentyl Acrylate 765 Pentaerythritol Triacrylate; Polyethylene Glycol (200) Diacrylate; Polyethylene Glycol (400) Diacrylate; Polyethylene Glycol (600) Diacrylate; Polyethylene Glycol (200) Dimethacrylate 770 Polyethylene Glycol (400) Dimethacrylate; Polyethylene Glycol (600) Dimethacrylate; Stearyl Methacrylate; Triethylene Glycol; Triethylene Glycol Dimethacrylate 775 2,2,2-Trifluoroethyl 2-methylacrylate; Trimethylolpropane Triacrylate; Acrylamide; N,N-meth-ylene-bisacryl-amide; Phenyl acrylate 780 Divinyl benzene.

[0133] A precursor for a hydrophilic material (such as the precursor comprised in the first additional fluid and / or the precursor comprised in the second additional fluid) may comprise at least one of the following or maybe one of the following: Acrylamide, N,N-methylene-bisacryl-amide, Methacrylic Acid, Monoethylene Glycol, 4-Hydroxybutyl Acrylate, Hydroxyethyl Acrylate (HEA), 2-Hydroxyethyl Methacrylate (HEMA), 2-Hydroxypropyl Acrylate, Diethyleneglycol Diacrylate (DEGDA), Diethyleneglycol Dimethacrylate (DEGDMA), Triethylene Glycol, Triethylene Glycol Dimethacrylate, Polyethylene Glycol (200 / 400 / 600) Diacrylate (PEGDA), Polyethylene Glycol (200 / 400 / 600) Dimethacrylate (PEGDMA).

[0134] According to a further aspect of the disclosure, an apparatus is provided for fabricating the microparticle according to any of the embodiments described above. The apparatus comprises a flow channel. The flow channel comprises at least three inlet openings for providing parallel flows of fluids in the flow channel. Each of the at least three inlet openings is adapted to provide a respective one of the parallel flows of the fluids in the flow channel. The flow channel further comprises a reducing section located downstream from the at least three inlet openings. A cross-sectional area of the flow channel at a downstream end of the reducing section is smaller than a cross-sectional area of the flow channel at an upstream end of the reducing section. The flow channel further comprises tapered elements arranged in the flow channel and at least partially in the reducing section of the flow channel. Each of the tapered elements has a smaller cross-sectional area at a downstream end of the respective tapered element thanat an upstream end of the respective tapered element. The at least three inlet openings comprise a first inlet opening located in the flow channel at a first lateral position and a second inlet opening located in the flow channel at a second lateral position laterally offset from the first lateral position. The tapered elements comprise a first tapered element arranged downstream from the first inlet opening at a lateral position corresponding to the first lateral position, to reduce a cross-sectional area of the one of the parallel flows of the fluids provided by the first inlet opening. The tapered elements further comprise a second tapered element arranged downstream from the second inlet opening at a lateral position corresponding to the second lateral position, to reduce a cross-sectional area of the one of the parallel flows of the fluids provided by the second inlet opening.

[0135] According to an embodiment, the flow channel comprises at least four inlet openings for providing the parallel flows of fluids in the flow channel.

[0136] According to an embodiment, the flow channel comprises at least five inlet openings for providing the parallel flows of fluids in the flow channel.

[0137] According to an embodiment, the flow channel comprises at least six inlet openings for providing the parallel flows of fluids in the flow channel.

[0138] According to an embodiment, the flow channel comprises at least seven inlet openings for providing the parallel flows of fluids in the flow channel.

[0139] The one of the parallel flows of the fluids provided by the first inlet opening may refer to the flow of the first additional fluid described above in the context of the method.

[0140] The one of the parallel flows of the fluids provided by the second inlet opening may refer to the flow of the second additional fluid described above in the context of the method.

[0141] The apparatus comprises a flow channel with at least three inlet openings, enabling parallel flows of fluids. This configuration allows for the simultaneous introduction of multiple fluid streams, which is beneficial for creating complex multi-layered particles, with the advantages described above in the context of the microparticle.

[0142] Tapered elements within the flow channel, particularly in the reducing section, further refine the flow by reducing the cross-sectional area of each stream. This design enhances the precision of particle formation, since the flows can be sculpted with a larger cross-section, andthen their cross sections can be “compressed” into the one of the desired microparticle. This approach allows for the creation of particles with complex geometries at high resolution, and thus for the production of the microparticle described above in the context of the first aspect.

[0143] The arrangement of the first and second inlet openings at laterally offset positions, along with corresponding tapered elements, allows for the independent manipulation of fluid streams. This spatial configuration is beneficial for creating particles with distinct regions or compartments, each potentially carrying different functional materials, thereby increasing the versatility and functionality of the particles.

[0144] Notably, in the context of this disclosure, the terms “downstream” and “upstream” do not require an actual flow of a fluid through the channel to be well-defined. To the contrary, the terms “downstream” and “upstream” are to be understood relative to each other, i.e., they indicate opposing directions along the flow channel, and are defined this way also in the absence of the flow of a fluid through the channel. In other words, the terms “downstream” and “upstream” are used for illustrative purposes and could alternatively be replaced by the terms “first direction” and “second direction opposite to the first direction”. If the terms were to be replaced accordingly, the reducing section may serve as a reference, i.e., the cross-sectional area of the flow channel at the end of the reducing section in the first direction would be smaller than a cross-sectional area of the flow channel at the end of the reducing section in the second direction opposite to the first direction, and the reducing section would be located along the first direction after / behind the at least three inlet openings.

[0145] Each of the tapered elements may extend along a direction parallel to a flow direction defined by the flow channel or may enclose a line parallel to a flow direction defined by the flow channel.

[0146] According to an embodiment, the tapered elements further comprise an outer tapered element around the first tapered element and the second tapered element.

[0147] The outer tapered element works in conjunction with the first and second tapered elements to further refine the flow of fluids within the flow channel. This additional tapered element provides an extra level of control over the fluid dynamics, ensuring that the parallel flows are more precisely directed and reduced in cross-sectional area, leading to an improved resolution and reliability of the method and of the microparticles being fabricated.

[0148] The outer tapered element may surround the first tapered element and the second tapered element in a plane perpendicular to a flow direction defined by the flow channel.

[0149] The outer tapered element may have an asymmetric shape around a centerline of the flow channel, in particular, wherein the asymmetric shape refers to a shape without a rotational symmetry around the center line, in particular, to a shape without a discrete or continuous rotational symmetiy around the centerline.

[0150] The outer tapered element may comprise at least one notch, in particular, at least two notches at different lateral positions along the outer tapered element.

[0151] The at least two notches at the different lateral positions along the outer tapered element may have different sizes and / or the at least two notches may further comprise a third notch (or maybe at least three notches, respectively).

[0152] The symmetrical shape of the outer tapered element or the notch(es) beneficially break any mirror symmetry of the fabricated microparticle about any plane which does not intersect all of the detection elements, with the advantages described above in the context of the microparticle according to the first aspect of this disclosure.

[0153] According to an embodiment, the at least three inlet openings comprise at least one outer inlet opening. In respective embodiments, according to a projection onto a plane perpendicular to a flow direction defined by the flow channel, each of the at least one outer inlet opening may be arranged around the first inlet opening and around the second inlet opening.

[0154] The outer inlet opening(s) beneficially provide further streams of fluids, in particular of the fluid, which is cured to form the body, and / or a stream of a carrier fluid surrounding the other parallel streams of fluids and having a concentration of precursors lower than any of the other streams of the fluids.

[0155] In respective embodiments, the at least one outer inlet opening may comprise a first outer inlet opening. In respective embodiments, the outer tapered element may be arranged downstream from the first outer inlet opening and, according to the projection onto the plane perpendicular to the flow direction defined by the flow channel: the upstream end of the outer tapered element may be arranged around the first outer inlet opening and / or the outer tapered element may overlap the first outer inlet opening.

[0156] A respective first outer inlet opening beneficially provides a stream of a fluid suitable for forming the body therefrom. Positioning the outer tapered element downstream from the first outer inlet opening and arranging it around or overlapping the first outer inlet opening ensures that the fluid flow from the outer inlet is effectively guided and constricted by the tapered element, resulting in a more focused and controlled flow, improving the precision at which the shape of the body is provided.

[0157] The first outer inlet opening may be adapted to provide the flow of the fluid comprising the precursor for the hydrophobic material as described above in the context of the method.

[0158] The flow of the fluid comprising the precursor for the hydrophobic material, as described above in the context of the method, may be provided by the first outer inlet opening.

[0159] The at least one outer inlet opening may comprise a second outer inlet opening.

[0160] The outer tapered element may be arranged downstream from the second outer inlet opening and, according to the projection onto the plane perpendicular to the flow direction defined by the flow channel: the second outer inlet opening maybe arranged around the downstream end of the outer tapered element and / or the outer tapered element may overlap the second outer inlet opening.

[0161] A respective second outlet opening beneficially provides an additional stream of a fluid, namely of a carrier fluid which avoids that the material of the body and / or of the detection elements sticks to the walls of the channel and clogs the channel when cured.

[0162] According to an embodiment, the apparatus further comprises a lithographic mask arranged in a lithographic masking section of the apparatus, wherein a section of the flow channel passes through said lithographic masking section, and wherein the lithographic mask is arranged in the lithographic masking section such that, under illumination of the mask with a UV radiation having a direction towards said section of the flow channel, said UV radiation is masked with the lithographic mask before reaching said section of the flow channel.

[0163] The masking enables precise control over the exposure of the fluids within the flow channel to UV radiation, thereby allowing for accurate patterning and structuring of the microparticles being fabricated.

[0164] The lithographic mask may comprise slits having segments with an extension perpendicular to a flow direction defined by the flow channel in the lithographic masking section, said slits being adapted to allow said UV radiation to reach said section of the channel, wherein the lithographic mask is adapted to block at least a part of the UV radiation or the UV radiation not passing through the slits.

[0165] The apparatus may further comprise a UV source adapted to provide said UV radiation having the direction towards said section of the flow channel.

[0166] According to a further aspect of the disclosure, the method described above in the context of the third aspect of this disclosure applies the apparatus according to any of the embodiments described above; wherein the parallel flows of the fluids in the flow channel are provided via the at least three inlet openings; wherein the flow of the first additional fluid is provided from the first inlet opening; and wherein the flow of the second additional fluid is provided from the second inlet opening.

[0167] In respective embodiments, the flow of the fluid comprising the precursor for the hydrophobic material maybe provided from one of the at least one outer inlet opening described above, in particular, may be provided from the first outer inlet opening.

[0168] The parallel flows of the fluids provided in the flow channel may further comprise a carrier flow, in particular, wherein the carrier flow may be provided from one of the at least one outer inlet opening described above, in particular, maybe provided from the second outer inlet opening described above.

[0169] In the context of this disclosure, a carrier flow may refer to one of the parallel flows of the fluids, the carrier flow being a flow of a fluid comprising a lower concentration of the precursor for the hydrophobic material than the flow of the fluid comprising the precursor for the hydrophobic material; and comprising a lower concentration of the precursor for the hydrophilic material than the flow of the first additional fluid; and comprising a lower concentration of the precursor for the hydrophilic material than the flow of the second additional fluid. In particular, said higher concentrations may refer to higher concentrations in terms of weight percent or in terms of molar concentration and / or said higher concentrations maybe higher at least by a factor of two or at least by a factor of five or at least by a factor of ten.LIST OF FIGURES

[0170] In the following, a detailed description of the present disclosure and examples thereof is given with reference to the figures, wherein

[0171] Fig. la and Fig. ib give a schematic illustration of a microparticle according to an example of the present disclosure;

[0172] Fig. 2a and Fig. 2b, Fig. 3a and Fig. 3b, Fig. 4a and Fig. 4b, Fig. 5a and Fig. 5b, Fig. 6a and Fig. 6b, Fig. 7a and Fig. 7b, Fig. 8a and Fig. 8b each give schematic illustrations of a microparticle according to a further example of the present disclosure;

[0173] Fig. 9 schematically illustrates a method according to an example of the present disclosure;

[0174] Fig. 10a to Fig. ion schematically illustrate an apparatus according to an example of the present disclosure;

[0175] Fig. 11 schematically illustrates an apparatus according to a further example of the present disclosure;

[0176] Fig. 12 and Fig. 13 schematically illustrate lithographic masking sections of apparatus according to different examples of the present disclosure;

[0177] Fig. 14 schematically illustrates a lithographic mask of an apparatus according to an example of the present disclosure;

[0178] Fig. 15 schematically illustrates a lithographic masking section and a reducing section of a flow channel, of an apparatus according to an example of the present disclosure;

[0179] Fig. 16 and Fig. 17 schematically illustrate the parallel flows of fluids inside the flow channel in different planes perpendicular to the flow direction;

[0180] Fig. 18 and Fig. 19 schematically illustrate outer tapered elements of apparatus according to different examples of the present disclosure;

[0181] Fig. 20 schematically illustrates the parallel flows of fluids inside the flow channel in a plane perpendicular to the flow direction, in apparatus having outer tapered elements with different shapes;

[0182] Fig. 21 gives a photograph of microparticles according to an example of the present disclosure;

[0183] Fig. 22 schematically illustrates an apparatus according to a further example of the present disclosure;

[0184] Fig. 23 schematically illustrates an apparatus according to a further example of the present disclosure;

[0185] Fig. 24a and Fig. 24b schematically illustrate a method of using a microparticle according to an example of the present disclosure;

[0186] Fig. 25 to Fig. 34 illustrate exemplary ELISA results;

[0187] Fig. 35 shows a microparticle according to a further example;

[0188] Fig. 36 to Fig. 38 illustrate acrylic acid functionalization;

[0189] Fig. 39 schematically illustrates another method of using a microparticle according to an example of the present disclosure; and

[0190] Fig. 40a and Fig. 40b schematically illustrate results of the method of Fig. 24a.DESCRIPTION OF EXAMPLES

[0191] Fig. ia and Fig. ib illustrate a microparticle 100, comprising a body 110 and detection elements 120.

[0192] The microparticle 100 can beneficially be applied in an assay, for example in a bioassay or in an immunoassay, as will be described in detail below in the context of Fig. 24a to Fig. 40b. Generally speaking, for this purpose, the microparticle 100 is placed in a fluid arranged over / above a substrate, such as a well of a microplate, a petri dish or a suitable microscope slide. Analytes from the fluid are immobilized selectively at the detection elements 120, or at- ‘2^ -hydrophilic surfaces thereof, respectively. Consequently, the concentration of the analytes is drastically increased at the detection elements 120, or at their hydrophilic surfaces, respectively, as compared to what the concentration of the analytes would be in the fluid in the absence of the microparticle 100. The increased concentration of the analytes at the detection elements 120 facilitates measurements and analysis thereon with increased signal strength and thus with an increased accuracy, in particular, when a spatially resolved measurement and analysis technique is used, for example making use of a microscope. In other words, the microparticle too facilitates a high reliability measurement / analysis even on analytes comprised in the fluid in small amounts or at small concentration. This concerns, on the one hand, fluids with a large overall volume but a small concentration of the analyte, as the microparticle too can immobilize, capture and concentrate the analyte spatially distributed in the fluid. On the other hand, signals from small amounts of analytes, resulting from a small overall amount of the liquid they are comprised in, can be amplified and the microparticle too thus permits measurements on fluid volumes on the nanoliter scale.

[0193] In the depicted embodiment, the body 110 is formed from a hydrophobic polymer without any further coating, thus providing a hydrophobic surface of the body 110.

[0194] In the depicted embodiment, the detection elements 120 each comprise a hydrophilic polymer. The detection elements 120 are formed without any further coating, such that the hydrophilic polymers provide hydrophilic surfaces of the detection elements 120.

[0195] The body's 110 hydrophobic nature minimizes non-specific binding at / to the body 110, allowing effective immobilization of analytes from an aqueous solution at the detection elements 120.

[0196] The material of at least one, and preferably of both of the detection elements 120 further comprises a molecule composition for immobilizing analytes at the hydrophilic surface of the respective detection element 120.

[0197] More specifically, said molecule compositions are configured such that, when the microparticle too is placed in a fluid, such as an aqueous fluid, analytes comprised in the fluid are immobilized at the molecule compositions at the hydrophilic surfaces.

[0198] For this purpose, the molecule composition of at least one, and preferably of each of the detection elements comprises immobilization molecules of at least one type, for example proteins, antibiotics, antigens, lectins, biotin, streptavidin, aptamers, or enzymes.

[0199] In the depicted embodiment, the detection elements 120 are each formed from a mixture of the hydrophilic polymer and said molecule composition. Consequently, the detection elements 120 comprise the molecule compositions not only at the hydrophilic surfaces, but also in their bulk materials.

[0200] The molecule compositions of the detection elements 120 differ.

[0201] According to an embodiment, the detection elements 120 comprise the same type of immobilization molecules, but at different concentrations.

[0202] A respective embodiment allows to realize an assay with a wide dynamic range. After having introduced the microparticle too into the fluid with the analytes, the detection element 120 with the most suitable amount of analyte’s binding sites can be used for the subsequent measurements and analysis, improving the accuracy of the assay.

[0203] In a similar embodiment, one of the detection elements 120 comprises the immobilization molecule at a significant concentration, whereas the immobilization molecule is essentially absent in another one of the detection elements 120.

[0204] In a respective embodiment, the other detection element 120 without any significant concentration of the immobilization molecules serves as a reference, for example when an assay is performed using the microparticle too.

[0205] According to another, preferred embodiment, one of the detection elements 120 (or the molecule composition thereof, respectively) comprises immobilization molecules of a first type for immobilizing analytes of a first type, and another one of the detection elements 120 comprises immobilization molecules of a second type different from the first type for immobilizing analytes of a second type different from the first type.

[0206] Respective embodiments enable providing a multiplexed immunoassay platform, i.e., when an assay is performed using a respective microparticle too, several analytes are selectively localized at the different detection elements 120 and thus become separately available at significant concentration for further analysis and measurements.

[0207] The microparticle too has a flattened shape, i.e., the thickness of the microparticle too along the z-axis is smaller than the width of the microparticle too along any direction in a plane x-y perpendicular to the z-axis along which the thickness of the microparticle too is defined.

[0208] Since the thickness t of the microparticle too is defined along the z-axis (i.e., along the direction along which the spatial extension of the microparticle 100 is the smallest), this direction is referred to as the thickness direction of the microparticle too.

[0209] In the context of this disclosure, the wider sides of the microparticle 100 are also referred to as the top surface and the bottom surface of the microparticle 100. Notably, the terms bottom surface and top surface are used interchangeably throughout this disclosure. In other words, the terms “top” and “bottom” in the words top surface and bottom surface refer to the depiction of the microparticle 100 in the figures, and can be interchanged, e.g., by flipping the orientation of the figure, or by turning the depicted x, y, z coordinate system. In yet other words, the terms top surface and bottom surface do not imply an orientation of the respective surfaces with respect to a real-world reference frame, except for where the orientation of the particle with respect to the real-world reference frame is made explicit in the claims or in the following description.

[0210] When the microparticle too is used for analysis, for example in an assay, it is desirable to know the orientation of the microparticle too and thus the positions of the detection elements 120 relative to the position of the microparticle too, for example relative to its center position or center-of-mass position.

[0211] Due to its flattened shape, when the microparticle too is used in the fluid, for example for an assay, the microparticle too will align with its wider sides facing up and down, and its thickness direction facing up, i.e., being orientated along the direction of the gravitational field. This orientation brings the center of mass of the microparticle too to the lowest possible position in the gravitational field of the earth, thus posing an energetically favorable arrangement. The remaining degrees of freedom of the microparticle too are the rotation around the direction of the gravitational field of the earth, and the binary degree of freedom as to whether the top surface of the bottom surface of the microparticle too faces up with respect to the gravitational field of the earth.

[0212] Conventional microparticles are formed with a highly symmetric shape, since a microparticle with a highly symmetric shape is technically easier to produce than a microparticle having a lower symmetry. For example, when the conventional microparticle is produced using stop-flow lithography, its highly symmetric shape allows for the usage of a fluid channel or several interconnected fluid channels having a highly symmetric cross-section, such as a circular cross-section, a rectangular cross-section, or a square shaped cross-section.

[0213] As a consequence of the highly symmetric shape of a conventional microparticle, when the conventional microparticle aligns with its wider sides facing up and down, and its thickness direction facing up as described above for the microparticle 100, the two remaining degrees of freedom (rotation around the direction of the gravitational field of the earth, binary degree of freedom) give rise to an uncertainty about the orientation of the conventional microparticle. For example, if a conventional microparticle had a flattened cylindrical shape, observation of this conventional microparticle aligned as explained above under a microscope, which typically views along (or opposite to) the earth’s gravitational field cannot determine the azimuthal angle related to the rotation of the conventional microparticle around the direction of the earth’s gravitational field. A respective observation cannot determine either, whether the top surface or the bottom surface of the conventional microparticle faces upwards in the earth’s gravitational field.

[0214] In contrast, the microparticle too, and more specifically, the body 110 of the microparticle too according to the embodiment of Fig. la, Fig. lb has a low-symmetry shape. In other words, the microparticle too, or its body 110, respectively, does not have any mirror symmetry about any mirror plane other than the x-y plane intersecting all the detection elements 120 at a position along the z axis corresponding to half the thickness t of the microparticle too.

[0215] Hence, when the microparticle too according to the embodiment of Fig. la, Fig. lb is allowed to align in the earth’s gravitational field and is then observed along the direction of the earth’s gravitational field, for example using a microscope, this observation immediately and fully determines the orientation of the microparticle too. A respective observation thus determines uniquely and unambiguously the positions of the detection elements 120 with respect to the microparticle too, which may, for example be expressed in terms of relative positions with respect to the circumference of the microparticle too or its body 110, or in terms of relative positions with respect to the center of mass of the microparticle too or of its body 110, respectively.

[0216] In particular, since the microparticle too, or its body 110, respectively, does not have any mirror symmetiy about any mirror plane other than the x-y plane intersecting all the detection elements 120, the shape of the microparticle too is chiral, and it is thus immediately obvious from the observation explained above whether the top surface or the bottom surface of the microparticle too faces upwards with respect to the earth’s gravitational field.

[0217] In the example of Fig. la, Fig. ib this becomes obvious when following the circumference of the microparticle too, or its body 110, respectively, in a clockwise direction. Startingfrom the topmost position along the circumference, this topmost position is part of the longest edge of the circumference. Along the clockwise direction, an acute angle and a shorter edge follow, followed by an optuse angle and an even shorter edge. If the microparticle too was flipped upside down, this order of elements would be reversed, or would be observed along the counterclockwise direction, respectively.

[0218] In other words, a user performing an assay or another optical measurement on the microparticle IOO immediately knows that the detection element 120 closer to the aforementioned acute angle is the first detection element, and the detection element 120 closer to the aforementioned obtuse angle is the second detection element, independent of the orientation (i.e., rotation about the z axis and up / down-flipping) of the microparticle 100.

[0219] This is particularly beneficial as the detection elements 120 comprise different molecule compositions for the immobilization of the analytes. Knowing which molecule composition is associated with which of the detection elements 120, the user can immediately identify which of the detection elements 120 carries which molecule composition.

[0220] Fig. 2a, Fig. 2b show an embodiment of a microparticle similar to the microparticle too of Fig. la, Fig. ib. Similar elements are described with identical reference numerals. To avoid repetition, they will not be described again. Instead, the following description focuses on the modifications over the foregoing embodiment.

[0221] In Fig. 2a, Fig. 2b, the thickness t and the width w of the microparticle too are indicated.

[0222] Preferably, the width w is at least 1.5 times as large as the thickness t, and more preferably at least twice as large as the thickness t, and even more preferably at least 2.5 or at least three times as large as the thickness t.

[0223] The inventors have realized that a respective aspect ratio between width w and thickness t beneficially ensures a reliable orientation of the microparticle too with its top surface or bottom surface pointing up when the microparticle too is allowed to align in the earth’s gravitational field.

[0224] Each of the detection elements 120 of the microparticle too of Fig. 2a, Fig. 2b comprises a cavity 122 in the form of a through hole extending from the top surface of the microparticle too to its bottom surface.

[0225] On the one hand, the cavity 122 ensures that an analyte which enters therein is surrounded from several sides by the hydrophilic surface of the detection element 120, improving the probability of successful immobilization of the analyte.

[0226] In addition, the through hole in the detection element 120 beneficially allows for measurements and analysis of analytes immobilized at the detection element 120 in the cavity 122 in transmittance geometry.

[0227] The cavity 122 is particularly beneficial for cell encapsulation in the microparticle too to perform a cell-section assay. Similar to analyzing biofluid from patients, proteins are also secreted by cells. By adding cell-attachment motifs to the detection element 120, cells can entrap in the cavity 122 of the detection element 120, which will then release proteins, that are detected by similar same ELISA (Enzyme-linked immunosorbent assay) steps.

[0228] According to some embodiments, the first / second molecule composition is adapted to provide analyte binding sites and are mixed in the fluid streams that the detection elements 120 are formed from before the fabrication of the microparticle too as described below. In respective embodiments, the first / second molecule composition is cross-linked in the fabrication process all over the detection element 120 at some specific concentration. Therefore, a microparticle too without any cavity 122 can immobilize analyte on a top and bottom surface of the respective detection element 120, where the detection element 120 is in direct contact with the fluid.

[0229] Provision of the cavity 122 enlarges the contact area between the microparticle too and the fluid, so that more potential binding sites are provided.

[0230] For example, for a round detection element 120 without a cavity 122, the provided contact area is pi*r2at both the top surface and the bottom surface of the detection element 120 embedded in the body, where r is the radius of the round detection element 120. Providing the detection element 120 with the cavity 122 in the form of a through hole enlarges the contact area by the area of the side wall of the through hole, which is 2*pi*r*t. In an exemplary embodiment, the thickness t is 150 pm, and the radius r = 60 pm.

[0231] Fig. 3a, Fig. 3b show an embodiment of a microparticle similar to the microparticle too according to the foregoing embodiments. Similar elements are indicated with identical reference numerals. To avoid repetition, they will not be described again. Instead, the following description focuses on the modifications over the foregoing embodiments.

[0232] In contrast to the microparticles 100 according to the foregoing embodiments, the microparticle 100 according to the embodiment of Fig. 3a, Fig. 3b has a highly symmetric outer shape, or circumferential shape, respectively.

[0233] To break the mirror symmetry of the microparticle 100, the detection elements 120 comprise different marker materials having different optical properties. For this purpose, in the depicted embodiment, the detection elements 120 each comprise a different fluorescent dye.

[0234] Thus, when the microparticle 100 is allowed to align in the earth’s gravitational field and is then observed with a microscope along the direction of the earth’s gravitational field, the orientation of the microparticle 100 can beneficially be determined by performing a fluorescence microscopy measurement with the microscope.

[0235] The microparticle too according to the embodiment of Fig. 3a, Fig. 3b has further been modified over the microparticles too according to the foregoing embodiments to comprise three detection elements 120. This improves the multiplexing capabilities of the microparticle too, for example when the microparticle too is used in an assay as described above.

[0236] Fig. 4a, Fig.4b show an embodiment of a microparticle similar to the microparticle too according to the foregoing embodiments. Similar elements are indicated with identical reference numerals. To avoid repetition, they will not be described again. Instead, the following description focuses on the modifications over the foregoing embodiments.

[0237] The microparticle too according to the depicted embodiment comprises a marker structure 112 associated with the outer surface of the body 110, breaking the mirror symmetry of the microparticle too. More specifically, the marker structure 112 breaks the mirror symmetry of the outer shape, or of the circumferential shape, respectively, of the microparticle too.

[0238] In the depicted embodiment, the marker structure 112 comprises a plurality of at least three recesses, and in the depicted embodiment exactly three recesses, arranged at unequal distances on the outer surface of the body 110, i.e., the distance between a first pair of recesses from the plurality of at least three recesses is different from the distance between a second pair of recesses from the plurality of at least three recesses.

[0239] When the microparticle 100 is allowed to align in the earth’s gravitational field and is then observed with a microscope along the direction of the earth’s gravitational field, the orientation of the microparticle 100 can beneficially be determined by performing a bright-light observation of the outer shape of the microparticle 100 with the microscope.

[0240] Fig. 5a, Fig.5b show an embodiment of a microparticle similar to the microparticle 100 according to the foregoing embodiments. Similar elements are indicated with identical reference numerals. To avoid repetition, they will not be described again. Instead, the following description focuses on the modifications over the foregoing embodiments.

[0241] The microparticle too according to the depicted embodiment comprises the marker structure 112 associated with the outer surface of the body 110, breaking the mirror symmetry of the microparticle too. More specifically, the marker structure 112 breaks the mirror symmetry of the outer shape, or of the circumferential shape, respectively, of the microparticle too.

[0242] In the depicted embodiment, the marker structure 112 comprises two recesses with different sizes on the outer surface of the body 110.

[0243] In the depicted embodiment, the marker structure 112 comprises two recesses with different shapes on the outer surface of the body 110.

[0244] When the microparticle too is allowed to align in the earth’s gravitational field and is then observed with a microscope along the direction of the earth’s gravitational field, the orientation of the microparticle too can beneficially be determined by performing a bright-light observation of the outer shape of the microparticle too with the microscope.

[0245] Fig. 6a, Fig. 6b show an embodiment of a microparticle similar to the microparticle too according to the foregoing embodiments. Similar elements are indicated with identical reference numerals. To avoid repetition, they will not be described again. Instead, the following description focuses on the modifications over the foregoing embodiments.

[0246] While the microparticles too according to the foregoing embodiments have parallel top and bottom surfaces, the top surface and the bottom surface of the microparticle too according to the embodiment of Fig. 6a, Fig. 6b are not parallel. Both configurations are possible, although the parallel top and bottom surfaces are preferred, as they provide an improved reliability of the alignment of the microparticle too in the earth’s gravitational field (i.e., with the top surface or the bottom surface pointing up).

[0247] While the microparticles 100 according to the foregoing embodiments have flat top and bottom surfaces, the top surface and the bottom surface of the microparticle 100 according to the embodiment of Fig. 6a, Fig. 6b are not flat. Both configurations are possible, although the flat top and bottom surfaces are preferred, as they provide an improved reliability of the alignment of the microparticle 100 in the earth’s gravitational field (i.e., with the top surface or the bottom surface pointing up).

[0248] Fig. 7a, Fig. 7b show an embodiment of a microparticle 100 similar to the microparticle 100 according to the foregoing embodiments. Similar elements are described with identical reference numerals. To avoid repetition, they will not be described again. Instead, the following description focuses on the modifications over the foregoing embodiments.

[0249] While in the microparticles too according to the foregoing embodiments, the detection elements 120 are embedded in the body 110, this is not the case for the microparticle too according to the embodiment of Fig. 7a, Fig. 7b.

[0250] Both configurations are possible, although the detection elements 120 embedded in the body 110 are preferred, as they provide an improved structural integrity of the microparticle too.

[0251] Fig. 8a, Fig. 8b show an embodiment of a microparticle too similar to the microparticle too according to the foregoing embodiments. Similar elements are described with identical reference numerals.

[0252] The microparticle too according to the embodiment of Fig. 8a, Fig. 8b comprises four detection elements. This improves the multiplexing capabilities of the microparticle too, for example when the microparticle too is used in an assay as described above.

[0253] Fig. 9 illustrates a method 200 for fabricating the microparticle too described above.

[0254] The method 200 comprises providing 210 parallel flows of fluids in a flow channel, said parallel flows of fluids comprising a flow of a fluid comprising a precursor for a hydrophobic material, a flow of a first additional fluid, the first additional fluid comprising a precursor for a hydrophilic material, and a flow of a second additional fluid, the second additional fluid comprising a precursor for a hydrophilic material.

[0255] The method 200 comprises curing 220 the precursor for the hydrophobic material into a hydrophobic material forming at least part of the body 110.

[0256] The method 200 comprises curing 230 the precursor for the hydrophilic material comprised in the first additional fluid into a hydrophilic material forming at least part of the first detection element.

[0257] The method 200 comprises curing 240 the precursor for the hydrophilic material comprised in the second additional fluid into a hydrophilic material forming at least part of the second detection element.

[0258] Fig. 10a to Fig. ion illustrate an apparatus 300 for fabricating the microparticle 100 described above, or for carrying out the method 200 described above, respectively.

[0259] Therein, Fig. 10a gives a perspective view of a three-dimensional model of the apparatus 300. Fig. 10b gives a photograph of a prototype corresponding to the three-dimensional model, wherein the photograph has been recorded in bright-field microscopy. Fig. 10c to Fig. ion give cross-sectional views through the apparatus 300, as indicated by planes po to pio indicated in Fig. 10c and in Fig. 10m.

[0260] The apparatus 300 comprises a flow channel 302. The flow channel 302 defines a flow direction for the parallel flows of the fluids therein. In the depicted embodiment, the coordinate system is orientated such that the z-axis coincides with the flow direction. The flow channel 302 comprises walls enclosing the parallel flows of the fluids in the plane perpendicular to the flow direction, which is the x-y plane in the depicted embodiment.

[0261] In the following, the apparatus 300, or the flow channel 302, respectively is described following along the z direction, or along the downstream direction of the flow channel 302, respectively.

[0262] In the upstream part of the flow channel 302, several inlet openings 310 are arranged inside of the flow channel 302, as is best visible in Fig. 10a to Fig. 10c and in Fig. 10m.

[0263] Amongst the inlet openings 310, most upstream, a second outer inlet opening 318 with a large width is provided, as is best visible in Fig. tod showing a cross section in plane pi.

[0264] The second outer inlet opening 318 serves to provide a carrier flow, or a flow of a carrier fluid, respectively, into the flow channel 302. This carrier flow is essentially free of precursors, i.e., it comprises significantly less (i.e., in terms of a molar concentration) of the hydrophobic precursor than the flow of the fluid comprising the precursor for a hydrophobic material described above in the context of the method 200, and it comprises significantly less (i.e., in terms of a molar concentration) of the precursor for the hydrophilic material than the flow of the first additional fluid and than the flow of the second additional fluid. Thus, when the aforementioned precursors are cured to form solid materials, essentially no solid material is formed in the carrier flow. Due to its large width, the carrier flow thus forms a liquid carrier flow around the formed solids, even if the carrier flow is or was exposed to UV illumination. The carrier flow thus serves to transport the formed solids and to prevent clogging of the flow channel 302.

[0265] Amongst the inlet openings 310, next (i.e., along the downstream direction, or along the positive z direction, respectively), a first outer inlet opening 316 with a width smaller than the one of the second outer inlet opening 318 is provided in the flow channel 302, as is best visible in Fig. toe showing a cross section in plane p2.

[0266] This first outer inlet opening 316 serves to provide the flow of the fluid comprising the precursor for the hydrophobic material described above in the context of the method 200.

[0267] In other words, the first outer inlet opening 316 serves to provide the precursor that the material of the body 110 of the microparticle too is to be formed from.

[0268] In the upstream portion of the flow channel 302, the first outer inlet opening 316 is separated from the flow of the fluid from the first outer inlet opening 316, or from the carrier flow, respectively, by an outer tapered element 336.

[0269] In the upstream portion of the flow channel 302, the outer tapered element 336 surrounds the first outer inlet opening 316 in a plane (x-y plane) perpendicular to the flow direction (z direction) defined by the flow channel 302, and the flow of the fluid from the second outer inlet opening 318 (carrier flow) surrounds the outer tapered element 336 in said plane.

[0270] Despite being referred to as the outer “tapered” element 336, the outer tapered element 336 is not necessarily tapered in this upper section of the flow channel 302. In the depicted embodiment, the outer tapered element 336 instead maintains its width, or diameter, respec-tively, in the section of the flow channel 302 with the inlet openings 310. In the depicted embodiment, the outer tapered element 336 only tapers in a section of the flow channel 302 downstream from the inlet openings 310.

[0271] Amongst the inlet openings 310, next (i.e., along the downstream direction), a first inlet opening 312 is provided in the flow channel 302, as is best visible in Fig. lof showing a cross section in plane p3.

[0272] This first inlet opening 312 serves to provide the flow of the first additional fluid described above in the context of the method 200.

[0273] In other words, this first inlet opening 312 serves to provide the material that a first one of the detection elements 120 of the microparticle 100 is to be formed from.

[0274] The first inlet opening 312 is offset from the center of the flow channel 302 in the plane (x-y plane) perpendicular to the flow direction (z direction).

[0275] The first inlet opening 312 is surrounded by the outer tapered element 336, by the flow of the fluid from the second outer inlet opening 318 (carrier flow), and by the flow of the fluid from the first outer inlet opening 316, namely in the plane (x-y plane) perpendicular to the flow direction (z direction).

[0276] Amongst the inlet openings 310, next (i.e., along the downstream direction), at least one second inlet opening 314 is provided in the flow channel 302.

[0277] In the depicted embodiment, the at least one second inlet opening 314 comprises a second inlet opening 314 (shown, e.g., in Fig. 10g showing a cross section in plane p4), a third inlet opening 314 (shown, e.g., in Fig. toh showing a cross section in plane ps), and a fourth inlet opening 314 (shown, e.g., in Fig. toi showing a cross section in plane p6). Thus, three respective inlet openings 314 are provided in addition to the first inlet opening 312.

[0278] In alternative embodiments (not shown), the at least one second inlet opening 314 comprises only the second inlet opening 314 (shown, e.g., in Fig. 10g) and the third inlet opening 314-

[0279] In alternative embodiments (not shown), the at least one second inlet opening 314 comprises only the second inlet opening 314 (shown, e.g., in Fig. 10g).[o28o]The second inlet opening 314 serves to provide the flow of the second additional fluid described above in the context of the method 200. In other words, the second inlet opening 314 serves to provide the material that a second one of the detection elements 120 of the microparticle 100 is to be formed from.

[0281] In embodiments with a third inlet opening 314, the third inlet opening 314 serves to provide a flow of a third additional fluid (similar to the flow of the first additional fluid and / or to the flow of the second additional fluid described above in the context of the method 200). In other words, the third inlet opening 314 serves to provide the material that a third one of the detection elements 120 of the microparticle 100 is to be formed from.

[0282] In embodiments with a fourth inlet opening 314, the fourth inlet opening 314 serves to provide a flow of a fourth additional fluid (similar to the flow of the first additional fluid and / or to the flow of the second additional fluid described above in the context of the method 200). In other words, the fourth inlet opening 314 serves to provide the material that a fourth one of the detection elements 120 of the microparticle too is to be formed from.

[0283] In embodiments, wherein the at least one second inlet opening 314 are actually several second inlet openings 314, such as in the depicted embodiment having three second inlet openings 314, the respective second inlet openings 314 are each laterally offset from each other and from the first inlet opening 312 in the plane (x-y plane) perpendicular to the flow direction (z direction).

[0284] Amongst the inlet openings 310, next (i.e., along the downstream direction), an inner inlet opening 304 is provided in the flow channel 302, as is best visible in Fig. toj showing a cross section in plane p .

[0285] The inner inlet opening 304 serves to provide an inner flow of a fluid essentially free of precursors, similar to the carrier fluid (or to the carrier flow, respectively) described above. Thus, when the precursors are cured to form solid materials, essentially no solid material is formed in the inner flow.

[0286] The inner flow thus serves to provide the cavities 122 of the microparticle too.

[0287] Fig. 10k shows a cross section through the flow channel 302 in the plane p8 after the last one of the at least three inlet openings 310, or, more specifically, just after the inner inlet opening 304.

[0288] Up to this plane p8 (i.e., along the downstream direction), both the flow channel 302 and the outer tapered element 336 have a constant width, or a constant cross-sectional area, respectively.

[0289] Fig. 10I shows a cross section through the flow channel 302 in the plane p9 in a reducing section 320 of the flow channel 302 downstream from the inlet openings 310.

[0290] As is visible from Fig. 10I, and also in Fig. 10a - Fig. 10c and Fig. ion, in the reducing section 320, the cross-sectional area (or the width, respectively) of both the flow channel 302 and of the outer tapered element 336 decreases in the downstream direction.

[0291] Thus, the pattern formed by the parallel flows of the fluids in the flow channel 302 is “compressed” into a pattern with a smaller cross-section area (or the width, respectively).

[0292] The flow channel 302 according to the depicted embodiment thus allows for sculpting the pattern formed by the parallel flows of the fluids in the flow channel 302 upstream of the reducing section 320 using inlet openings 310 with sizes on a scale of several millimeters, and only thereafter compressing the pattern into a much smaller structure for the microparticle too.

[0293] This approach permits to shape the microparticle too with a high (submillimeter) accuracy, without a need to manufacture the inlet openings 310 with the same level of accuracy.

[0294] Also shown in Fig. 10I are notches 360 formed in the outer tapered element 336. These notches 360 shape the flow of the fluid from the first outer inlet opening 316 (i.e., the fluid comprising the precursor for the hydrophobic material for forming the body 110 of the microparticle too) to provide the marker structures 112 in the form of recesses 112, as described above in the context of the microparticle too according to Fig. 4a - Fig. 6b, Fig. 8a, and Fig.8b.

[0295] Fig. 10m repeats the cross-sectional view already depicted in Fig. 10k, with an indication of the plane po (shown in Fig. 10c) and of the plane pio (shown in Fig. ion).

[0296] Fig. 11 illustrates an embodiment of an apparatus 300 similar to the apparatus 300 according to the foregoing embodiment. Similar elements are indicated with identical reference numerals. To avoid repetition, they will not be described again. Instead, the following description focuses on the modifications over the foregoing embodiment.

[0297] In addition to the flow channel 302 described in detail above, the apparatus 300 of Fig.11 comprises a pump and reservoir system 344 for providing the aforementioned fluids to the inlet openings 310.

[0298] In the depicted embodiment, the pump and reservoir system 344 comprises a plurality of syringe pumps, including at least one syringe pump for providing each of the aforementioned fluids.

[0299] The apparatus 300 according to the embodiment of Fig. 11 further comprises a lithographic masking section 350.

[0300] A section 302m of the flow channel 302 passes through the lithographic masking section 350.

[0301] A lithographic mask 352 is arranged in the lithographic masking section 350 above said section 302m of the flow channel 302.

[0302] A UV source 356 is arranged in the lithographic masking section 350 above the lithographic mask 352.

[0303] The apparatus 300 of Fig. 11 further comprises a valve 346 for selectively shutting off the flow channel 302, or for selectively shutting off the parallel flows of the fluids in the flow channel 302, respectively. In other words, the valve 346 is adapted to selectively stop the parallel flows of the fluids in the flow channel 302.

[0304] The apparatus 300 of Fig. 11 further comprises a particle collection system 348. In the depicted embodiment, the particle collection system 348 comprises a filter for filtering microparticles too out of a stream of fluids through the flow channel 302.

[0305] In the depicted example of the particle collection system 348, the microparticles too are collected in a commercially available 15 ml falcon tube. Then a washing buffer (ethanol / wa-ter / PBS) is added, which results in diluting the uncured material and making it less dense compared to the cured microparticles too which settle down. The uncured material is removed and the process is repeated with fresh wash solution 3 times. PBS refers to phosphate-buffered saline.[O3o6]The apparatus 300 of Fig. 11 further comprises a computer system 340 comprising controllers 342 for controlling the pump and reservoir system 344, the UV source 356, and the valve 346.

[0307] To fabricate particles, the computer system 340 controls the other components of the apparatus 300 in the following manner:

[0308] In a first step, the computer system 340 controls the valve 346 to be in an open state, and the computer system 340 controls the pump and reservoir system 344 to provide the aforementioned fluids (i.e., the fluid comprising the precursor for the hydrophobic material, the first additional fluid, and the second additional fluid as explained above in the context of the method 200; and in embodiments with the carrier flow also the carrier fluid; and in embodiments with the inner flow, also the fluid of the inner flow) to the at least three inlet openings 310 of the flow channel 302.

[0309] In response to this first step, the parallel flows of the fluids (as described above in the context of the method 200) are provided in the flow channel 302 via the at least three inlet openings 310.

[0310] In a second step after the first step, the computer system 340 controls the valve 346 to be in a closed state. Optionally, in the second step, the computer system 340 controls the pump and reservoir system 344 to stop providing the aforementioned fluids. In an alternative embodiment, the pump and reservoir system 344 remains switched on, and only the valve 346 is controlled to be in a closed state.

[0311] In response to this second step, the parallel flows of the fluids (as described above in the context of the method 200) are stopped in the flow channel 302, 302m.

[0312] In a third step after the second step, the computer system 340 controls the UV source 356 to produce UV radiation 354.

[0313] In response to this third step, the UV source 356 shines the UV radiation 354 through the lithographic mask 352 and into the section 302m of the flow channel 302.

[0314] Under the resulting irradiation with UV radiation 354, the precursor for the hydrophobic material, the precursor for the hydrophilic material comprised in the first additional fluid, and the precursor for the hydrophilic material comprised in the second additional fluid (andin embodiments with a third and / or a fourth additional fluid, also the precursors for the hydrophilic material comprised in the third and / or fourth additional fluid) get cured, i.e., photo-chemically cured. In other words, those precursors solidify under the exposure to the UV radiation 354, such that the precursor for the hydrophobic material solidifies into material of the body no, the precursor for the hydrophilic material comprised in the first additional fluid solidifies into material of a first detection element 120, and the precursor for the hydrophilic material comprised in the second additional fluid solidifies into material of a second detection element 120 (and, in embodiments with a third and / or a fourth additional fluid, the precursor for the hydrophilic material comprised in the third and / or the fourth additional fluid solidifies into material of a third and / or a fourth detection element 120).

[0315] Notably, the first additional fluid is provided with the molecule composition of the first detection element 120 as described above in the context of the microparticle too according to the embodiments of Fig. la to Fig. 8b. Thus, the molecule composition comprised in the first additional fluid is comprised in the material of a first detection element 120 formed therefrom.

[0316] Correspondingly, the second additional fluid is provided with the second molecule composition of the second detection element 120 as described above. Thus, the molecule composition prized in the second additional fluid is comprised in the material of a second detection element 120 formed therefrom.

[0317] In embodiments with a third and / or a fourth additional fluid, the third / fourth additional fluid is provided with the third / fourth molecule composition of the third / fourth detection element 120 as described above. Thus, the molecule composition comprised in the third / fourth additional fluid is comprised in the material of a third / fourth detection element 120 formed therefrom.

[0318] In a fourth step after the third step, the computer system 340 controls the UV source 356 to stop producing the UV radiation 354.

[0319] The computer system 340 then controls the apparatus 300 to iterate the process, starting again from the first step.

[0320] Fig. 12 and Fig. 13 illustrate lithographic masking sections 350 of the apparatus 300 according to two different embodiments.

[0321] In the embodiment of Fig. 12, the lithographic mask 352 is arranged on a substrate 16, and the section 302m of the flow channel 302 is arranged over / above the lithographic mask 352.

[0322] The substrate 16 is at least partially transparent to the UV radiation 354 from the UV source 356. Thus, the UV radiation 354 passes through the substrate 16, through slits (not shown) in the mask 352, and into the section 302m of the flow channel 302.

[0323] During operation, the aforementioned fluids with the precursors flow are located in the section 302m of the flow channel 302. Upon irradiation with the UV radiation 354, the precursors are cured / solidify to form solid material of the body 110 and of the detection elements 120.

[0324] The embodiment of Fig. 13 is similar to the embodiment of Fig. 12. However, in the embodiment of Fig. 13, the wall of the flow channel 302 is thinner or even absent in a region adjacent to the lithographic mask 352. Hence, the fluid is constrained by the lithographic mask 352 and / or by the substrate 16 in the respective region.

[0325] In the embodiments depicted in Fig. 12, Fig. 13, the section 302m of the flow channel 302 in the lithographic masking section 350 has a rectangular or quadratic cross-section. In alternative embodiments, the section 302m of the flow channel 302 in the lithographic masking section 350 has an ellipsoidal or, preferably, round cross-section (not shown).

[0326] Fig. 14 illustrates a lithographic mask 352 of the apparatus 300 according to an embodiment.

[0327] The lithographic mask 352 according to the embodiment of Fig. 14 comprises slits 20 having segments 20a with an extension (along the x-direction) perpendicular to a flow direction (corresponding to the z direction) defined by the flow channel 302 in the lithographic masking section 350.

[0328] Fig. 15 schematically illustrates a lithographic masking section 350 and a reducing section 320 of a flow channel 302, of an apparatus 300 according to an example of the present disclosure.

[0329] In Fig. 15, elements similar to elements described above are indicated with the same reference numerals. For a detailed description of the respective elements, reference is made to their detailed description above.

[0330] Further, planes p20 (between the reducing section 320 and the lithographic masking section 350, or at the transition between the reducing section 320 and the lithographic masking section 350, respectively) and p22 (at the upstream end of the reducing section 320) are indicated in Fig. 15.

[0331] According to the embodiment of Fig. 15, the section 302m of the flow channel 302 in the lithographic masking section 350 has a circular cross-section.

[0332] Notably, the square- and ring-shaped elements to the left of the reducing section 320 in Fig. 15 depict the parallel flows of the fluids in the flow channel 302 upstream of the reducing section 320 (rather than physical structures of the flow channel 302 itself), similar to the parallel flows of the fluids shown schematically in Fig. 10k.

[0333] Fig. 16 shows a cross section through the parallel flows of the fluids in the flow channel 302 in a plane p20 between the reducing section 320 and the lithographic masking section 350, or at the transition between the reducing section 320 and the lithographic masking section 350, respectively. The cross section through the parallel flows of the fluids in the flow channel 302 is similar in the section 302m of the flow channel 302 in the lithographic masking section 350.

[0334] Reference numerals in Fig. 16 indicate the inlet that the respective parallel flow of the fluid in the flow channel 302 is provided from. For a detailed description of the indicated inlets and the provided parallel flows of the fluids, reference is made to the respective detailed description in the context of Fig. 10a to Fig. ion.

[0335] Fig. 17 shows a cross section through the parallel flows of the fluids in the flow channel 302 in a plane p22 at the upstream end of the reducing section 320.

[0336] Reference numerals in Fig. 17 indicate the inlet that the respective parallel flow of the fluid in the flow channel 302 is provided from. For a detailed description of the indicated inlets and the provided parallel flows of the fluids, reference is made to the respective detailed description in the context of Fig. 10a to Fig. ion.

[0337] Fig- 18 and Fig. 19 depict two different examples of outer tapered elements 336.

[0338] The detailed description of the outer tapered elements 336 given above applies also to the tapered elements 336 of Fig. 18 and Fig. 19. The following description focuses on modifications implemented in the tapered elements 336 of Fig. 18 and Fig. 19.

[0339] The tapered elements 336 of Fig. 18 and Fig. 19 are provided with notches 360 each having a length D, referring to the length D of the notches along the flow direction defined by the flow channel 302. In the depicted embodiments, the length D refers to the length as measured from the downstream end of the tapered element 336.

[0340] In the depicted embodiments, the tapered elements 336 is provided with two notches 360 having different lengths D, resulting in the fabrication of a microparticle 100 similar to the one of Fig.5a, Fig.5b. In the specific example, one of the notches 360 has a length D of 1.5 mm, and the other notch has a length D of 0.5 mm.

[0341] In alternative embodiments (not shown), the tapered element 336 is provided with at least three notches 360. In respective embodiments, distances between neighboring ones of the at least three notches 360 differ, resulting in the fabrication of a microparticle 100 similar to the one of Fig. 4a, Fig. 4b. In respective embodiments, the lengths of the at least three notches 360 may be the same or they may be different.

[0342] Fig. 20 shows cross sections through the parallel flows of the fluids in the flow channel 302 in a plane p20 between the reducing section 320 and the lithographic masking section 350, or at the transition between the reducing section 320 and the lithographic masking section 350, respectively; similar to the presentation in Fig. 16. As in Fig. 16, reference numerals in Fig. 20 indicate the inlet that the respective parallel flow of the fluid in the flow channel 302 is provided from. For a detailed description of the indicated inlets and the provided parallel flows of the fluids, reference is made to the respective detailed description in the context of Fig.10a to Fig. ion.

[0343] In Fig. 20, the cross sections through the parallel flows of the fluids are given for different apparatus, each having a tapered element 336 each with two notches 360 (as depicted in Fig. 18, Fig. 19) of equal, lengths D. Those lengths D are different for the different apparatus 300. The lengths D are indicated for the different apparatus 300, with the lengths being 2.75 mm, 1.75 mm, 1.25 mm, and 0.75 mm.

[0344] Fig- 21 gives a microscopy image of microparticles 100.

[0345] The microparticles 100 are each formed with a first detection element 120 comprising a first fluorescent dye, with a second detection element 120 comprising a second fluorescent dye, with a third detection element 120 comprising a third fluorescent dye, and, with a fourth detection element 120 comprising a fourth fluorescent dye. Each of the first, second, third and fourth fluorescent dye is a different fluorescent dye, or has a different photoluminescence (or fluorescence, respectively) emission wavelength, respectively.

[0346] As can be seen from the different apparent brightnesses of the detection elements 120 within each of the microparticles 100, the individual detection elements 120 within each of the microparticles 100 can be distinguished and identified in the microscopy image, with the advantages described above in the context of the microparticles according to the embodiments of Fig. la to Fig. 8b.

[0347] Actually, the photoluminescence (or fluorescence, respectively) emission wavelengths from the first, second, third and fourth fluorescent dye differ, although this may not be apparent from the grayscale photograph, and hence the individual detection elements 120 within each of the microparticles 100 can be distinguished and identified based on their photoluminescence (or fluorescence, respectively) emission wavelengths, or colors, respectively.

[0348] Fig. 22 shows an apparatus 300 similar to the apparatus of Fig. 10a to Fig. ion. Similar elements are indicated with identical reference numerals as in Fig. 10a to Fig. ion. For a detailed description of those elements, reference is made to the corresponding description in the context of Fig. 10a to Fig. ion.

[0349] In contrast to the apparatus 300 according to the embodiment of Fig. 10a to Fig. ion, the flow channel 302 of the apparatus 300 according to the embodiment of Fig. 22 is formed with four inlet openings (rather than with seven inlet openings) 310, namely with the first outer inlet opening 316, with the second outer inlet opening 318, with the first inlet opening 312, and with the second inlet opening 314.

[0350] In other words, the apparatus 300 according to the embodiment of Fig. 22 does not comprise the third inlet opening, the fourth inlet opening, and the inner inlet opening.

[0351] Fig. 23 shows an apparatus 300 similar to the apparatus of Fig. 22. Similar elements are indicated with identical reference numerals. For a detailed description of those elements, reference is made to the corresponding description in the context of Fig. 10a to Fig. ion.

[0352] The apparatus 300 of Fig. 23 is similar to the apparatus 300 of Fig. 22. As compared to the apparatus 300 of Fig. 22, the flow channel 302 of the apparatus 300 of Fig. 23 further comprises the inner inlet opening 304. For a detailed description of the inner inlet opening 304, reference is made to the corresponding description in the context of Fig. 10a to Fig. ion.

[0353] Fig. 24a illustrates a process involving microparticle 100, detailing a sequence of steps for immobilizing analytes. The figure shows a series of stages, including biotinylation, streptavidin binding, and antibody immobilization, followed by biomarker detection and amplification. Each step is depicted with corresponding reagents and conditions, highlighting the complex interactions required to achieve successful analyte immobilization.

[0354] The microparticle 100 serves as the substrate for these interactions, providing hydrophilic surfaces for the binding and detection of analytes.

[0355] The detection elements 120 of the microparticle 100 of Fig. 24a are formed of hydrophilic material comprising biotin, i.e., from first and second additional fluids each comprising a precursor for a hydrophilic material and biotin (more specifically, acrylate-PEG-biotin) in addition. According to some embodiments, the concentration of biotin in the different detection elements 120 of the microparticle too differ. The inventors have successfully performed experiments to demonstrate that the measured signal intensity indeed increases with biotin concentration, in an approximately linear manner.

[0356] The steps shown in Fig. 24a follow a known protocol of sandwich ELISA, as previously reported in: Ghulam Destgeer, Mengxing Ouyang, Chueh-Yu Wu, and Dino Di Carlo: “Fabrication of 3D concentric amphiphilic microparticles to form uniform nanoliter reaction volumes for amplified affinity assays,” Lab on a Chip, 2020, 20, 3503-3514. This document is incorporated herein in its entirety by reference.

[0357] The sequence of steps shown in Fig. 24a demonstrates the functionality of the microparticle 100 in a practical application. Each stage of the process builds upon the previous one, ultimately leading to the amplification with oil encapsulation and detection of the target analytes. This process highlights the importance of the microparticle's 100 design and composition in achieving reliable and reproducible results.

[0358] Fig. 24b illustrates a process similar to the one of Fig. 24a. The detailed description of Fig. 24a applies accordingly.

[0359] According to alternative embodiments (not shown), the detection elements 120 could be designed to bind different types of molecules, such as nucleic acids or small molecules, depending on the application.

[0360] A first example of immobilization molecules to be applied is given in the following:

[0361] Biomarker to be detected: Recombinant human IL-6 protein (R&D Systems 206-IL). Capture Anti-bodies: Human IL-6 MAb (Clone 973132) (R&D Systems MAB 9540). Detection Anti-bodies: Human IL-6 antibody (R&D Systems MAB2063).

[0362] A second example of immobilization molecules to be applied is given in the following:

[0363] Biomarker to be detected: Recombinant human TNF-alpha protein (R&D Systems 210-TA). Capture Anti-bodies: Human TNF-alpha antibody (R&D Systems MAB610). Detection Anti-bodies: Human TNF- alpha antibody (R&D Systems BAF210).

[0364] A third example of immobilization molecules to be applied is given in the following:

[0365] Biomarker to be detected: Recombinant human IL-8 / CXCL8 protein (R&D Systems 208-IL). Capture Anti-bodies: Human IL-8 / CXCL8 antibody (R&D Systems MAB2081). Detection Anti-bodies: Human IL-8 / CXCL8 antibody (R&D Systems MAB2082).

[0366] A fourth example of immobilization molecules to be applied is given in the following:

[0367] Biomarker to be detected: Recombinant human C-reactive protein / CRP protein (R&D Systems 1707-CR). Capture Anti-bodies: Human C-reactive protein / CRP antibody (R&D Systems MAB17073). Detection Anti-bodies: Human C-reactive protein / CRP antibody (R&D Systems MAB17072).

[0368] A fifth example of immobilization molecules to be applied is given in the following:

[0369] Biomarker to be detected: Recombinant anti-cardiac troponin I (cTnl) (Hytest Finland Cat.# RC4T21). Capture Anti-bodies: Troponin I cardiac (cTnl), antibody (Hytest Finland 4T21 / 4T21CC, Mi8cc). Detection Anti-bodies: Troponin I cardiac (cTnl), antibody (Hytest Finland 4T21 / 4T21CC, 4C2CC).

[0370] A sixth example of immobilization molecules to be applied is given in the following:

[0371] Biomarker to be detected: NT-proBNP (HyTest, Finland 8NT2). Capture Anti-bodies: Anti-NT-proBNP (HyTest, Finland 4NT1CC 15C4.cc). Detection Anti-bodies: Anti-NT-proBNP (HyTest, Finland 4NT1CC 13G12CC). NT-proBNP refers to N-terminal pro b-type natriuretic peptide.

[0372] Fig. 25 and Fig. 26 show the results of IL- 2 detection at the first hydrophilic surface 150 of the first detection element 160 using amphiphilic microparticles 100 functionalized with a first molecule composition 170.

[0373] In Fig. 25, the horizontal axis is IL2 Concentration [ng / mL], and for each IL-2 concentration 240, specifically o, 0.1, 1, and 10 ng / mL, multiple bars are depicted, each corresponding to a different incubation time in minutes (o, 15, 30, 45, and 60 min). The vertical axis represents fluorescence signal intensity 290 in arbitrary units. The graph demonstrates that, for each IL- 2 concentration 240, the signal intensity 290 increases with incubation time, and higher IL-2 concentrations 240 yield higher signal intensities 290 at each time point.

[0374] Fig. 26 presents a bar graph with the horizontal axis depicting IL2 Concentration [ng / mL] and the vertical axis showing fluorescence signal intensity 290 at t = 45 min. The bars correspond to IL-2 concentrations 240 of o, 0.1, 1, and 10 ng / mL, with measured values of 3,006.97, 5,137.81, 4,890.59, and 13,253.69 arbitrary units, respectively, indicating a clear positive correlation between IL-2 concentration 240 and signal intensity 290 at the first hydrophilic surface 150.

[0375] In the experiments performed to obtain the results shown in Fig. 25 and Fig. 26, biotinylated amphiphilic microparticles 100 are fabricated by adding acrylate-PEG-biotin, which was 1.12 mg / mL, into PEGDA (Poly(ethylene glycol) diacrylate). The acrylate-PEG-biotin 5K was purchased from Biopharma PEG. For IL-2 detection, the protocol is as follows: microparticles 100 are seeded in 4 separate wells 200 inside a well-plate, washed three times with PBS + 0.5% Pluronic F127 (PBSP), incubated with streptavidin 210 (10 pg / mL) for 30 min for all wells 200, and washed three times with PBSP. The microparticles too are then incubated with biotinylated IL-2 capture antibody 220 (5 pg / mL) for 1 h for all wells 200, followed by incubation with protein-free blocking buffer 230 for 1 h. The microparticles too are incubated for 1 h with IL- 2 protein 240 at different concentrations (o, 0.1, 1, and 10 ng / mL) in the separate wells 200, and washed three times with PBSP. All wells 200 are then incubated for 1 h with 0.25 pg / mL IL- 2 detection antibody 250 linked with HRP 260 through a linking kit, followed by washing three times with PBSP. QuantaRed mixture is seeded and removed, oil 280 is seeded-5i-on top to create droplet compartments 270, and measurement of fluorescence signal 290 is performed for 1 h under a microscope. Herein, PBSP refers to PBS with 0.5% Pluronic F127.

[0376] The results show that the IL-2 protein 240 positive signal (0.1, 1, and 10 ng / mL) is higher than the negative control (o ng / mL).

[0377] Fig. 27 and Fig. 28 show the results of IL-6 detection at the first hydrophilic surface 150 of the first detection element 160 using biotinylated amphiphilic microparticles 100, consistent with the first example described above.

[0378] In Fig. 27, the horizontal axis is IL6 Concentration [ng / mL], and for each IL-6 concentration 240, specifically o, 0.01, 0.1, 1, 10, and 100 ng / mL, multiple bars are depicted, each corresponding to a different incubation time in minutes. The vertical axis represents fluorescence signal intensity 290 in arbitrary units. The graph demonstrates that, for each IL-6 concentration 240 up to 10 ng / mL, the signal intensity 290 increases with incubation time, and higher IL-6 concentrations 240 yield higher signal intensities 290 at each time point.

[0379] Fig. 28 presents a bar graph with the horizontal axis depicting IL6 Concentration [ng / mL] and the vertical axis showing fluorescence signal intensity 290 at t = 30 min. The bars correspond to IL-6 concentrations 240 of o, 0.01, 0.1, 1, 10, and too ng / mL, with measured values of 577.87, 3,195.07, 3,667.59, 3,468.71, 18,089.99, and 22,733.31 arbitrary units, respectively, indicating a clear positive correlation between IL-6 concentration 240 and signal intensity 290 at the first hydrophilic surface 150.

[0380] In the experiments performed to obtain the results shown in Fig. 27 and Fig. 28, biotinylated amphiphilic microparticles 100 are used, and the protocol follows the process described for the first example described above.

[0381] In more detail, microparticles 100 are seeded in separate wells 200 inside a well-plate, washed three times with PBS + 0.5% Pluronic F127 (PBSP), and incubated with streptavidin 210. The microparticles 100 are then incubated with 5 pg / mL biotinylated IL-6 capture antibody 220, followed by incubation with protein-free blocking buffer 230. The microparticles too are incubated for 1 h with IL-6 protein 240 at different concentrations (o, 0.01, 0.1, 1, 10, and too ng / mL) in the separate wells 200. After washing, all wells 200 are incubated for 1 h with 0.25 pg / mL IL-6 detection antibody 250 linked with HRP 260 through a linking kit, followed by washing three times with PBSP. QuantaRed mixture is seeded and removed, oil 280is seeded on top to create droplet compartments 270, and measurement of fluorescence signal 290 is performed for 1 h under a microscope.

[0382] The results show that the IL-6 protein 240 positive signal (0.01, 0.1, 1, 10, and 100 ng / mL) is higher than the negative control (o ng / mL), and concentrations of 1, 10, and 100 ng / mL are clearly detectable at the first hydrophilic surface 150 of the first detection element 160.

[0383] Fig. 29 and Fig. 30 show the results of IL-8 detection at the first hydrophilic surface 150 of the first detection element 160 using biotinylated amphiphilic microparticles 100, consistent with the third example described above.

[0384] In Fig. 29, the horizontal axis is IL8 Concentration [ng / mL], and for each IL-8 concentration 240, specifically o, 0.01, 0.1, 1, 10, and 100 ng / mL, multiple bars are depicted, each corresponding to a different incubation time in minutes. The vertical axis represents fluorescence signal intensity 290 in arbitrary units. The graph demonstrates that, for each IL-8 concentration 240, the signal intensity 290 increases with incubation time, and higher IL-8 concentrations 240 yield higher signal intensities 290 at each time point.

[0385] Fig. 30 presents a bar graph with the horizontal axis depicting IL8 Concentration [ng / mL] and the vertical axis showing fluorescence signal intensity 290 at t = 30 min. The bars correspond to IL-8 concentrations 240 of o, 0.01, 0.1, 1, 10, and too ng / mL, with measured values of 1,275.42, 1,275.32, 1,773.61, 1,923.29, 2,236.17, and 20,737.21 arbitrary units, respectively, indicating a clear positive correlation between IL-8 concentration 240 and signal intensity 290 at the first hydrophilic surface 150.

[0386] In the experiments performed to obtain the results shown in Fig. 29 and Fig. 30, biotinylated amphiphilic microparticles 100 are used, and the protocol follows the process described for the third example described above.

[0387] More specifically, the microparticles 100 are seeded in separate wells 200 inside a wellplate, washed three times with PBS + 0.5% Pluronic F127 (PBSP), and incubated with streptavidin 210. The microparticles 100 are then incubated with 5 pg / mL IL-8 capture antibody 220, followed by incubation with protein-free blocking buffer 230. The microparticles too are incubated for 1 h with IL-8 protein 240 at different concentrations (o, 0.01, 0.1, 1, 10, and too ng / mL) in the separate wells 200. After washing, all wells 200 are incubated for 1 h with 0.25 pg / mL IL-8 detection antibody 250 linked with HRP 260 through a linking kit, followed bywashing three times with PBSP. QuantaRed mixture is seeded and removed, oil 280 is seeded on top to create droplet compartments 270, and measurement of fluorescence signal 290 is performed for 1 h under a microscope.

[0388] The results show that the IL-8 protein 240 positive signal (10 and 100 ng / mL) is higher than the negative control (o ng / mL), 0.01 ng / mL shows similar signal to negative control, and 0.1-10 ng / mL show slightly increasing signal.

[0389] Fig. 31 and Fig. 32 show the results of NT-proBNP detection at the first hydrophilic surface 150 of the first detection element 160 using biotinylated amphiphilic microparticles 100, consistent with the sixth example described above.

[0390] In Fig. 31, the horizontal axis is NT-proBNP Concentration [ng / mL], and for each NT-proBNP concentration 240, specifically o, 0.01, 0.1, 1, 10, and 100 ng / mL, multiple bars are depicted, each corresponding to a different incubation time: 20min, 35mm, and 6omin. The vertical axis represents fluorescence signal intensity 290 in arbitrary units. The graph demonstrates that, for each NT-proBNP concentration 240, the signal intensity 290 increases with incubation time, and higher NT-proBNP concentrations 240 yield higher signal intensities 290 at each time point.

[0391] Fig. 32 presents a bar graph with the horizontal axis depicting NT-proBNP Concentration [ng / mL] and the vertical axis showing fluorescence signal intensity 290 at t = 35 min. The bars correspond to NT-proBNP concentrations 240 of o, 0.01, 0.1, 1, 10, and 100 ng / mL, with measured values of 314.75, 345.78, 376.62, 1,466.33, 18,125.60, and 18,888.17 arbitrary units, respectively, indicating a clear positive correlation between NT-proBNP concentration 240 and signal intensity 290 at the first hydrophilic surface 150.

[0392] In the experiments performed to obtain the results shown in Fig. 31 and Fig. 32, biotinylated amphiphilic microparticles 100 are used, and the protocol follows the process described above for the sixth example.

[0393] In more detail, the microparticles 100 are seeded in separate wells 200 inside a wellplate, washed three times with PBS + 0.5% Pluronic F127 (PBSP), and incubated with streptavidin 210. The microparticles 100 are then incubated with 5 pg / mL NT-proBNP capture antibody 220, followed by incubation with protein-free blocking buffer 230. The microparticles 100 are incubated for 1 h with NT-proBNP protein 240 at different concentrations (o, 0.01, 0.1, 1, 10, and 100 ng / mL) in the separate wells 200. After washing, all wells 200 are incubated for1 h with 0.25 pg / mL NT-proBNP detection antibody 250 linked with HRP 260 through a linking kit, followed by washing three times with PBSP. QuantaRed mixture is seeded and removed, oil 280 is seeded on top to create droplet compartments 270, and measurement of fluorescence signal 290 is performed for 1 h under a microscope.

[0394] The results show that the NT-proBNP protein 240 positive signal (10 to 100 ng / mL) is much higher than the negative control (o ng / mL), and concentrations of 0.01, 0.1, and 1 ng / mL show increasing signal intensities 290, demonstrating the sensitivity and dynamic range of the assay at the first hydrophilic surface 150 of the first detection element 160.

[0395] Fig. 33 and Fig.34 show the results of CRP detection at the first hydrophilic surface 150 of the first detection element 160 using biotinylated amphiphilic microparticles 100, consistent with the fourth example described above.

[0396] In Fig. 33, the horizontal axis is CRP Concentration [ng / mL], and for each CRP concentration 240, specifically o, 0.1, 1, 10, too, and 1000 ng / mL, multiple bars are depicted, each corresponding to a different incubation time: tomin, t30min, and t6omin. The vertical axis represents fluorescence signal intensity 290 in arbitrary units. The graph demonstrates that, for each CRP concentration 240, the signal intensity 290 increases with incubation time, and higher CRP concentrations 240 yield higher signal intensities 290 at each time point.

[0397] Fig. 34 presents a bar graph with the horizontal axis depicting CRP Concentration [ng / mL] and the vertical axis showing fluorescence signal intensity 290 at t = 30 min. The bars correspond to CRP concentrations 240 of o, 0.1, 1, 10, too, and 1000 ng / mL, with measured values of 4,775.04, 5,273.26, 6,017.76, 22,918.99, 27,989.34, and 26,621.68 arbitrary units, respectively, indicating a clear positive correlation between CRP concentration 240 and signal intensity 290 at the first hydrophilic surface 150.

[0398] In the experiments performed to obtain the results shown in Fig. 33 and Fig. 34, biotinylated amphiphilic microparticles 100 are used, and the protocol follows the process described for the fourth example described above.

[0399] In more detail, the microparticles 100 are seeded in separate wells 200 inside a wellplate, washed three times with PBS + 0.5% Pluronic F127 (PBSP), and incubated with streptavidin 210. The microparticles 100 are then incubated with 5 pg / mL CRP capture antibody 220, followed by incubation with protein-free blocking buffer 230. The microparticles 100 are incubated for 1 h with CRP protein 240 at different concentrations (o, 0.1, 1, 10, 100, and 1000ng / mL) in the separate wells 200. After washing, all wells 200 are incubated for 1 h with 0.25 pg / mL CRP detection antibody 250 linked with HRP 260 through a linking kit, followed by washing three times with PBSP. QuantaRed mixture is seeded and removed, oil 280 is seeded on top to create droplet compartments 270, and measurement of fluorescence signal 290 is performed for 1 h under a microscope.

[0400] The results show that the CRP protein 240 positive signal (0.1, 1, 10, 100, and 1000 ng / mL) is higher than the negative control (o ng / mL), and concentrations of 10, 100, and 1000 ng / mL are clearly detectable at the first hydrophilic surface 150 of the first detection element 160.

[0401] In the following, exemplary application cases (not depicted in the figures) of the microparticle too are described.

[0402] First exemplary application case: detection of COPD-relevant biomarkers.

[0403] The application of amphiphilic microparticles too for the detection of COPD biomarkers is demonstrated using immunoassays targeting C-reactive protein (CRP), as described in the fourth example and illustrated in Fig.33 and Fig. 34. Amphiphilic microparticles too are functionalized with biotinylated CRP capture antibody 220, enabling the immobilization of CRP at the first hydrophilic surface 150 of the detection element 160. C-reactive protein is a relevant indicator of systemic inflammation, closely associated with COPD severity and the risk of exacerbations. The clinically relevant range for CRP detection in COPD patients is between 1 and too pg / mL, with normal levels defined as less than 1 pg / mL and moderate to high inflammation indicated by concentrations between 10 and 50 pg / mL.

[0404] Second exemplary application case: detection of interleukins-relevant biomarkers.

[0405] The application of amphiphilic microparticles too for the detection of interleukins uses immunoassays targeting IL-2, IL-6, and IL-8, as described in the first and third examples and illustrated in Fig. 25, Fig. 26, Fig. 27, Fig. 28, Fig. 29, and Fig. 30. Amphiphilic microparticles too are functionalized with biotinylated capture antibodies 220 specific for each interleukin, enabling immobilization of the target analyte at the first hydrophilic surface 150 of the detection element 160.

[0406] For Interleukin-6 (IL-6), the clinically relevant range in healthy individuals is typically between 1 and 10 pg / mL, with levels increasing to the nanogram per milliliter range duringinflammation, infection, or cancer. The ELISA protocol on amphiphilic microparticles 100, as shown in Fig. 27 and Fig. 28, demonstrates detection of IL-6 at concentrations as low as 10 pg / mL (0.01 ng / mL). To further improve sensitivity and enable reliable detection within the clinically relevant range, the density of binding sites on the microparticles 100 and the incubation times may further be increased.

[0407] For Interleukin-2 (IL-2), the protocol and results are shown in Fig. 25 and Fig. 26, following a similar approach as described for IL-6 and IL-8. This approach enables sensitive, specific, and rapid detection of interleukins for clinical and research applications, with ongoing optimization to achieve detection limits suitable for healthy and disease states.

[0408] Third exemplary application case: cardiac patients.

[0409] The application of amphiphilic microparticles 100 for cardiac patients focuses on the detection of C-reactive protein (CRP), a biomarker of systemic inflammation that is linked to cardiovascular disease severity and risk of exacerbations. The clinically relevant range for CRP in cardiac patients is generally considered to be between 1 and 100 pg / mL, with normal levels defined as less than 1 pg / mL and moderate to high inflammation indicated by concentrations between 10 and 50 pg / mL. The ELISA protocol on amphiphilic microparticles 100, as described in the fourth example and illustrated in Fig.33 and Fig. 34, enables the immobilization and quantification of CRP at the first hydrophilic surface 150 of the detection element 160 using biotinylated CRP capture antibody 220.

[0410] Fourth exemplary application case: heart patients.

[0411] The application of amphiphilic microparticles 100 for heart patients uses the detection of NT-proBNP, a clinically important biomarker for cardiac function, as described in the sixth example and illustrated in Fig. 31 and Fig. 32. NT-proBNP levels below 125 pg / mL in individuals under 75 years old indicate a low probability of heart failure, while elevated levels above 900 pg / mL are associated with heart failure or other cardiac conditions. The ELISA protocol on amphiphilic microparticles 100 enables sensitive detection of NT-proBNP at the first hydrophilic surface 150 of the detection element 160, with results showing reliable quantification from 10 pg / mL up to 100 ng / mL, encompassing the clinically relevant range for heart patients.

[0412] Fifth exemplary application case: alternative application for cardiac patients.

[0413] A further application of amphiphilic microparticles 100 for cardiac patients is the detection of cardiac troponin I (cTnl), a highly specific biomarker for myocardial injury and acute cardiac events. The clinically relevant range for cTnl is between o and 0.04 ng / mL in healthy individuals, while concentrations can increase up to 10 ng / mL in cardiac patients experiencing myocardial infarction or other cardiac conditions. The application case uses cTnl detection using amphiphilic microparticles 100 functionalized with biotinylated cTnl capture antibody 220. The protocol involves seeding microparticles 100 in wells 200, washing, sequential incubation with streptavidin 210, biotinylated cTnl capture antibody 220, protein-free blocking buffer 230, and cTnl protein 240 at defined concentrations, followed by incubation with cTnl detection antibody 250 linked with HRP 260, addition of QuantaRed mixture, and oil 280 to create droplet compartments 270. Measurement of fluorescence signal 290 at the first hydrophilic surface 150 of the first detection element 160 enables sensitive and specific quantification of cTnl, supporting rapid and reliable point-of-care diagnostics for cardiac patients.

[0414] Fig. 35 illustrates an alternative microparticle 100. The microparticle 100 is generally similar to the microparticle 100 described above in the context of the foregoing embodiments.

[0415] As compared to the biotin-containing molecule composition of the foregoing embodiments, the microparticle 100 of Fig. 35 comprises detection elements comprising molecule compositions based on alternative species.

[0416] In the depicted embodiment, different detection elements of the microparticle 100 comprise molecule compositions based on different species. A respective microparticle 100 is also referred to as a multi-functionalized microparticle 100, or as a microparticle 100 comprising a multi-functionalization, respectively. This is in contrast to a microparticle 100 comprising a single-functionalization, where the molecule compositions of the detection elements use a common species (e.g., biotin such as acrylate-PEG-biotin) for immobilization of analytes at the hydrophilic surfaces 150 of the detection elements 160.

[0417] The microparticle 100 comprising the multi-functionalization is fabricated using copolymerizing acrylate-PEG-biotin and acrylate-PEG-azide into PEGDA during microparticle synthesis, each at 1.12 mg / mL. This results in a microparticle 100 presenting both biotin and azide functional groups at the first hydrophilic surface 150.

[0418] The microparticle 100 of Fig. 35 further comprises a detection element functionalized with acrylate-PEG-N3, enabling azide-based conjugation at at least one of the hydrophilic surfaces (in other words: at a hydrophilic surface of at least one of the detection elements). Forthis purpose, aciylate-PEG-N3 is incorporated into the PEGDA matrix at a concentration of 1.12 mg / mL during microparticle synthesis, resulting in the presentation of azide functional groups at the first hydrophilic surface 150 of the detection element. This azide functionalization allows for the covalent immobilization of DBCO-modified capture antibody 220 via strain-promoted azide-alkyne cycloaddition (SPAAC), a copper-free click chemistry reaction that is highly specific and efficient. The protocol involves seeding the microparticle 100 in wells 200, washing, incubation with DBCO-modified capture antibody 220, blocking buffer 230, addition of target analyte 240, incubation with detection antibody 250 linked with HRP 260, Quan-taRed mixture, and oil 280 to create droplet compartments 270. Measurement of the fluorescence signal 290 at the first hydrophilic surface 150 confirms successful azide-based functionalization and analyte detection, supporting multiplexed and orthogonal assay design on the microparticle too.

[0419] The microparticle too of Fig. 35 further comprises a detection element functionalized with acrylic acid, providing carboxyl groups at the first hydrophilic surface 150.

[0420] According to an example, acrylic acid is incorporated into the PEGDA matrix during microparticle synthesis to introduce carboxyl functionalities at the first hydrophilic surface 150 of the detection element. These carboxyl groups enable covalent immobilization of amine-con-taining capture antibody 220 through carbodiimide chemistry, specifically using EDC / NHS activation. The protocol involves seeding the microparticle too in wells 200, washing, activation of carboxyl groups with EDC / NHS, incubation with capture antibody 220, blocking buffer 230, addition of target analyte 240, incubation with detection antibody 250 linked with HRP 260, QuantaRed mixture, and oil 280 to create droplet compartments 270. Measurement of the fluorescence signal 290 at the first hydrophilic surface 150 confirms successful acrylic acidbased functionalization and analyte detection, enabling robust and flexible covalent attachment of biomolecules for immunoassay applications on the microparticle too.

[0421] The microparticle too of Fig. 35 further comprises a hydrophilic element serving as a control group, where no capture antibody 220 is attached at the first hydrophilic surface 150 of the detection element 160. These control regions are used (e.g., in ELISA) in parallel with functionalized regions but remain unmodified with respect to antibody attachment, allowing for the measurement of background or non-specific signal. The control group provides a reference for evaluating the specificity and accuracy of the immunoassay, ensuring that any detected fluorescence signal 290 at the first hydrophilic surface 150 is attributable to specific analyte binding rather than non-specific interactions or assay artifacts.

[0422] The microparticle 100 comprising the multi-functionalization facilitates orthogonal conjugation strategies: For example, streptavidin 210 can be immobilized via biotin, and DBCO-modified capture antibody 220 can be conjugated via azide-DBCO click chemistry (DBCO referring to Dibenzocyclooctyne). This allows for the simultaneous and spatially controlled immobilization of different capture molecules at the first hydrophilic surface 150, further enhancing the possibilities of multiplexed detection. This multi-functionalization strategy extends the capabilities of the microparticle 100, enabling multiplexed and flexible assay design by utilizing both biotin and azide chemistries or any combination of biotin, Acrylate-PEG-N3, and / or acrylic acid chemistry at the first hydrophilic surface 150 of the first detection element 160.

[0423] Fig. 36, Fig. 37, and Fig. 38 show the functionalization of the microparticle 100 with aciylic acid, providing carboxyl groups at the first hydrophilic surface 150 of the first detection element 160. These figures collectively illustrate the process and the results of covalent immobilization of capture antibody 220 via carbodiimide chemistry, enabling robust and flexible attachment of biomolecules for analyte detection at the first hydrophilic surface 150.

[0424] Fig. 36 depicts the chemical strategy for introducing carboxyl (-COOH) groups into the microparticle 100 by incorporating acrylic acid (25%) into the PEGDA 575 (40%) matrix, with ethanol (35%) as a solvent. Acrylic acid crosslinks with PEGDA and introduces -COOH groups, which can be activated by EDC / NHS chemistry for covalent attachment to amine (-NH2) groups present in proteins and antibodies. The process is based on the procedure described in detail in Chen, L., An, H.Z., Haghgooie, R., Shank, A. T., Martel, J.M., Toner, M. and Doyle, P.S. (2016), “Flexible Octopus-Shaped Hydrogel Particles for Specific Cell Capture.” Small, 12: 2001-2008. https: / / d0i.0rg / 10.1002 / smll.201600163.

[0425] Fig. 37 and Fig. 38 show experimental results (fluorescence data) on the functionalization of a single-cavity O-shape microparticle 100 with acrylic acid at the first hydrophilic surface 150 of the detection element 160.

[0426] To obtain the results shown in Fig. 37 and Fig. 38, the microparticles 100 are seeded in three wells 200, ethanol is removed, and the microparticles 100 are washed three times with PBSP. EDC / NHS solutions are prepared at o mg / mL, 3 mg / mL, and 30 mg / mL. The washed microparticles 100 are incubated with these solutions for 30 minutes, rendering the first hydrophilic surface 150 reactive to -NH2 groups. Streptavidin (containing -NH2) is then incubated on the microparticles 100, and immobilization is confirmed using 0.01 mg / mL biotin-4-- 6o -fluorescence. The fluorescence signal 290 shows that a 3 mg / mL concentration of EDC / NHS provides higher immobilization of streptavidin.

[0427] Fig. 38 analyzes the cross-reactivity of EDC / NHS treatment on three sets of microparticles 100: Set 1, fabricated without acrylic acid and without acrylate PEG biotin; Set 2, fabricated without acrylic acid and with acrylate PEG biotin; and Set 3, fabricated with 25% acrylic acid). The results show that EDC / NHS treatment has no effect on particles lacking acrylate groups (signal ~773 vs ~799 a.u.), and does not affect biotin functionality (signal ^1317 vs ~1353 a-u. for biotin particles). In contrast, acrylic acid (-COOH) particles show a very high affinity to streptavidin after EDC / NHS activation (signal increases from ~3O32 to ~26385 a.u.), confirming effective covalent functionalization.

[0428] Alternatively or in addition to the above-described biotine-based functionalization and acrylic acid-based functionalization, azide-based functionalization (not shown in the figures) of the microparticle too maybe applied to provide the molecule composition of at least one of the detection elements (or at at least one of the first hydrophilic surfaces, respectively).

[0429] Azide-based functionalization of the microparticle too provides an alternative and complementary strategy to the biotin-based and acrylic acid-based approaches. In this approach, the first molecule composition 170 comprises acrylate-PEG-azide, which is incorporated into the PEGDA matrix during microparticle synthesis, typically at a concentration of 1.12 mg / mL. This results in the presentation of azide functional groups at the first hydrophilic surface 150 of the first detection element 160.

[0430] The azide groups at the first hydrophilic surface 150 enable orthogonal conjugation of DBCO-modified capture antibody 220 via strain-promoted azide-alkyne cycloaddition (SPAAC), commonly referred to as "click chemistry." This covalent immobilization is highly specific and efficient, allowing for robust attachment of biomolecules without the need for copper catalysis or additional activation steps. The protocol involves seeding the microparticle too in wells 200, washing, incubation with DBCO-modified capture antibody 220, blocking buffer 230, addition of target analyte 240, incubation with detection antibody 250 linked with HRP 260, QuantaRed mixture, and oil 280 to create droplet compartments 270.

[0431] Azide-based functionalization is fully compatible with multi-functionalization strategies, where acrylate-PEG-azide is co-polymerized with acrylate-PEG-biotin to create a microparticle too presenting both azide and biotin functionalities at the different detection elements(or at the corresponding hydrophilic surfaces, respectively) of the microparticle 100. This enables simultaneous immobilization of DBCO-modified capture antibody 220 and streptavidin 210, supporting multiplexed detection of multiple analytes within a single droplet compartment 270. The use of azide chemistry thus expands the overall teaching and flexibility of the platform, providing an efficient, orthogonal, and highly specific method for functionalizing the first hydrophilic surface 150 of the first detection element 160.

[0432] A suitable route for Azide-based functionalization is, for example, described in Ji-Hyeon Kim, Ji Hong Kim, Hye-Seon Jeong, Sei- Jung Lee, Jong Pil Park, Chang-Hyung Choi, Color-encoded multicompartmental hydrogel microspheres for multiplexed bioassays, Ta-lanta, Volume 279, 2024, 126571, ISSN 0039-9140, https: / / doi.org / io.ioi6 / j.ta-lanta.2024.126571.

[0433] Fig. 39 shows an example of the microparticle 100, illustrating the process of immobilizing analytes from a fluid. The figure includes various stages of the process, each indicated by different symbols and annotations. The components shown in this figure include the microparticle 100, streptavidin, biotin, biotinylated antibodies (Abi and Ab2), biomarkers (Biomarker 1 and Biomarker 2), horseradish peroxidase HRP-conjugated antibodies (HRP-Abi and HRP-Ab2), and ADHP (io-Acetyl-3,7-dihydroxyphenoxazine) for the readout. Alternatively, 3,3',5,5'-Tetramethylbenzidine TMB can be applied. As a further alternative, a luminol substrate gives a fluorescence signal in the microscope that can be used in ELISA for readout, such as ADHP, TMB, P-galactosidase. Some of these are sensitive (like ADHP), while some are more stable (like P-galactosidase). HRP refers to horseradish peroxidase.

[0434] A microparticle too having detection elements 120 with molecule compositions comprising immobilization molecules of different types can be achieved by including acrylate-PEG-biotin in the first additional fluid (as described above in the context of the method 200), including acrylate-PEG- streptavidin in the second additional fluid (as described above in the context of the method 200). The preparation process of acrylate-PEG-streptavidin is demonstrated in Langmuir 2015, 31, 48, 13165-13171. This document is herewith incorporated herein in its entirety by reference. Acrylate-PEG-streptavidin is prepared by mixing streptavidin and Acrylate-PEG-Succinimidyl Valerate (ACRL-PEG-SVA) at a mole ratio of 1:1. Streptavidin is chemically inert to ethanol and UV radiation, like biotin is, making it suitable for the method 200 described above.

[0435] The microparticle 100 comprises a body 110 with hydrophobic surfaces and detection elements 120 with hydrophilic surfaces. The detection elements 120 are shown to interact withdifferent molecules during the incubation and binding processes. Streptavidin binds to biotin, which in turn binds to biotinylated antibodies (Abi and Ab2). These antibodies capture specific biomarkers (Biomarker 1 and Biomarker 2) from the fluid. HRP-conjugated antibodies (HRP-Abi and HRP-Ab2) then bind to these biomarkers, and the presence of HRP is detected using TMB, which produces an optical signal.

[0436] According to an embodiment, the process steps of Fig. 39 are similar to the process steps of Fig. 24a, however, with an additional step of adding capture antibody-i after particle fabrication. This results in a microparticle 100 having detection elements 120 with different molecule compositions at the respective surfaces, wherein the different molecule compositions comprise different immobilization molecules for capturing analytes of different types.

[0437] Mixing PEG linker (ACRL-PEG-SVA, Lysan Bio) with antibody results in ACR-PEG-Ab, EDC / NHS is a well-reported linking mechanism allowing acrylate linker to attach to antibody. This ACR-PEG-Ab is then cross-linked with hydrophilic PEGDA during the photo-polymerization of the stop-flow lithography process, making the hydrophilic layer functionalized with antibody. For further details, reference is made to Anal. Chem. 2011, 83, 1, 193-199 and to Nature Protocols volume 6, pagesi76i-i774 (2011). Both documents are herewith incorporated herein in their entirety by reference.

[0438] In a modification of the method of Fig. 39, nanoparticles coated with different antibodies are comprised in the first additional fluid and the second additional fluid (as described above in the context of the method 200), such that the resulting first and second detection element 120 of the same microparticle 100 fabricated comprise the nanoparticles coated with the different antibodies. The application of coated nanoparticles during photopolymerization has, for example been reported in Lab Chip, 2010,10, 3335-3340. This document is herewith incorporated herein in its entirety by reference.

[0439] Moreover, hydrogel droplet microarrays with trapped antibody- functionalized beads have been demonstrated in Lab Chip, 2011,11, 528- 534. This document is herewith incorporated herein in its entirety by reference.

[0440] Fig. 40a shows an example of the microparticle 100 during the incubation process, illustrating the time-dependent binding of analytes. The figure includes images taken at different time points (20 min, 60 min, and 100 min) to show the progression of the binding process.

[0441] The microparticle 100 is shown to have a body with multiple detection elements 120, half of them serving as detector, the other ones (not equipped with the molecule composition with immobilization molecule) serving as a reference. The detection elements 120 comprise cavities 122 which get filled with the surrounding fluid containing the analytes, and the binding process is monitored over time. The images show the increasing intensity of the signal as more analytes are captured by the detection elements.

[0442] Fig. 40b shows the quantitative analysis of the binding process illustrated in Fig. 40a, by giving the time-dependent increase in signal intensity. The figure includes a graph with time t on the x-axis and signal intensity on the y-axis, showing the increase in signal over time for (1) the cavities with the molecule composition with the immobilization molecules (upper data points 370) and (2) the reference cavities without the molecule composition with the immobilization molecules (lower data points 372). The data points represent the mean signal intensity, and the error bars represent the standard deviation, indicating the variability of the measurements.

[0443] The graph shows that the signal intensity increases over time as more analytes are captured by the detection elements 120.

[0444] The advantage of quantitative analysis is that it provides a precise measurement of the amount of analyte captured by the detection elements 120, allowing for accurate quantification of the analytes in the fluid sample. This is particularly important for applications where the concentration of the analyte is critical, such as in clinical diagnostics or environmental monitoring.

[0445] By monitoring the intensity of signal under the microscope, the concentration of the analyte can be estimated.

[0446] The examples of the present disclosure disclosed herein only constitute specific examples for illustration purposes. The present invention can be implemented in various ways and with many modifications without altering the underlying basic properties. Therefore, the present invention is only defined by the claims as stated below.

Claims

Technische Universitat Munchen, in Vertretung des Freistaates BayernU31245WOClaims1. A microparticle (100) for immobilizing analytes from a fluid, the microparticle (100) comprising:a body (no) comprising a hydrophobic surface; anddetection elements (120), the detection elements (120) comprising a first detection element comprising a first hydrophilic surface and a second detection element comprising a second hydrophilic surface;wherein the detection elements (120) are mechanically interconnected by means of the body (no);wherein the first detection element comprises, at the first hydrophilic surface, a first molecule composition for immobilizing analytes from the fluid at the first hydrophilic surface, and the second detection element comprises, at the second hydrophilic surface, a second molecule composition for immobilizing analytes from the fluid at the second hydrophilic surface;wherein the first molecule composition is different from the second molecule composition; andwherein the microparticle (100) is non-mirror symmetric about any plane which does not intersect all of the detection elements (120).

2. The microparticle (100) according to claim 1, wherein the body (110) comprises a marker structure (112), wherein the marker structure (112) is non-mirror symmetric about any plane which does not intersect all of the detection elements (120).

3. The microparticle (100) according to claim 2, wherein the marker structure (112) is associated with an outer surface of the body (110).

4. The microparticle (100) according to claim 3, wherein the marker structure (112) is formed by the outer surface of the body (110).

5. The microparticle (100) according to claim 3, wherein the marker structure (112) is arranged on the outer surface of the body (110).

6. The microparticle (100) according to any of claims 3 to 5, wherein the body (110) has a top surface (114, 116) and a bottom surface (114, 116) opposite to the top surface (114, 116), wherein at least part of the marker structure (112) is associated with the top surface (114, 116) and / or with the bottom surface (114, 116).

7. The microparticle (100) according to claim 6, wherein the at least part of the marker structure (112) is associated with a circumferential shape of the top surface (114, 116) and / or with a circumferential shape of the bottom surface (114, 116).

8. The microparticle (100) according to claim 7, wherein the marker structure (112) comprises recesses in the circumferential shape of the top surface (114, 116) and / or in the circumferential shape of the bottom surface (114, 116).

9. The microparticle (100) according to claim 8, wherein at least two of said recesses have different shapes.

10. The microparticle (100) according to claim 9, wherein the at least two of said recesses are comprised in the same one of the circumferential shape of the top surface (114, 116) and the circumferential shape of the bottom surface (114, 116).

11. The microparticle (100) according to claim 8, wherein at least two of said recesses have different shapes and are comprised in the same one of the circumferential shape of the top surface (114, 116) and the circumferential shape of the bottom surface (114, 116).

12. The microparticle (100) according to claim 8, wherein at least two of the recesses have different shapes and are both comprised in both the circumferential shape of the top surface (114, 116) and the circumferential shape of the bottom surface (114, 116).

13. The microparticle (100) according to any of the preceding claims, comprising at least two different marker materials having different optical properties, wherein the at least two different marker materials are arranged in the microparticle (100) nonmirror symmetrically about any plane which does not intersect all of the detection elements (120).

14. The microparticle (100) according to claim 13, wherein the first detection element comprises a first marker material of the at least two different marker materials at a higher concentration than the second detection element, and the second detectionelement comprises a second marker material of the at least two different marker materials at a higher concentration than the first detection element.

15. The microparticle (too) according to claim 14, wherein the first detection element comprises the first marker material of the at least two different marker materials at the higher concentration, in terms of weight percent, than the second detection element.

16. The microparticle (too) according to claim 14, wherein the first detection element comprises the first marker material of the at least two different marker materials at the higher concentration, in terms of molar concentration, than the second detection element.

17. The microparticle (too) according to any of claims 14 to 16, wherein the second detection element comprises the second marker material of the at least two different marker materials at the higher concentration, in terms of weight percent, than the first detection element18. The microparticle (too) according to any of claims 14 to 16, wherein the second detection element comprises the second marker material of the at least two different marker materials at the higher concentration, in terms of molar concentration, than the first detection element.

19. The microparticle (too) according to any of claims 14 to 18, wherein said higher concentrations are higher at least by a factor of two or at least by a factor of five or at least by a factor of ten.

20. The microparticle (too) according to any of claims 14 to 19, wherein the first detection element comprises the first marker material of the at least two different marker materials at a higher concentration than the body (110).

21. The microparticle (too) according to claim 20, wherein the first detection element comprises the first marker material of the at least two different marker materials at the higher concentration, in terms of weight percent, than the body (110).

22. The microparticle (too) according to claim 20, wherein the first detection element comprises the first marker material of the at least two different marker materials at the higher concentration, in terms of molar concentration, than the body (110).

23. The microparticle (100) according to any of claims 20 to 22, wherein the first detection element comprises the first marker material of the at least two different marker materials at the higher concentration than the body (110) at least by a factor of two or at least by a factor of five or at least by a factor of ten.

24. The microparticle (100) according to any of claims 14 to 23, wherein the second detection element comprises a second marker material of the at least two different marker materials at a higher concentration than the body (110).

25. The microparticle (100) according to claim 24, wherein the second detection element comprises the second marker material of the at least two different marker materials at the higher concentration, in terms of weight percent, than the body (no).

26. The microparticle (100) according to claim 24, wherein the second detection element comprises the second marker material of the at least two different marker materials at the higher concentration, in terms of molar concentration, than the body (110).

27. The microparticle (100) according to any of claims 24 to 26, wherein the second detection element comprises the second marker material of the at least two different marker materials at the higher concentration than the body (110), at least by a factor of two or at least by a factor of five or at least by a factor of ten.

28. The microparticle (100) according to any of claims 13 to 27, wherein the different optical properties comprise at least one of the following: different photoluminescence emission wavelengths, and maxima at different wavelengths in corresponding optical absorption spectra.

29. The microparticle (100) according to any of claims 13 to 28, wherein the at least two different marker materials comprise or are at least two different photoluminescent dyes.

30. The microparticle (100) according to claim 29, wherein the at least two different marker materials comprise or are at least two different fluorescent dyes.

31. The microparticle (100) according to any of the preceding claims, wherein the analytes comprise analytes of a first type;wherein the first molecule composition comprises immobilization molecules of a first type at a higher concentration than the second molecule composition, wherein the immobilization molecules of the first type are adapted to immobilize analytes of the first type at the respective surface comprising the respective molecule composition comprising the immobilization molecules of the first type.

32. The microparticle (too) according to claim 31, wherein the immobilization molecules of the first type are adapted to selectively immobilize the analytes of the first type at the respective surface comprising the respective molecule composition comprising the immobilization molecules of the first type.

33. The microparticle (too) according to claim 31 or 32, wherein the higher concentration refers to a higher concentration in terms of weight percent or in terms of molar concentration.

34. The microparticle (too) according to any of claims 31 to 33, wherein the higher concentration is higher at least by a factor of two, or at least by a factor of five, or at least by a factor of ten.

35. The microparticle (too) according to any of claims 31 to 34, wherein the immobilization molecules of the first type are essentially absent in the second molecule composition.

36. The microparticle (too) according to any of the preceding claims, wherein the analytes comprise analytes of a first type and analytes of a second type;wherein the first molecule composition comprises immobilization molecules of a first type for selectively immobilizing the analytes of the first type from the fluid at the first hydrophilic surface; andwherein the second molecule composition comprises immobilization molecules of a second type for selectively immobilizing the analytes of the second type from the fluid at the second hydrophilic surface.

37. The microparticle (too) according to claim 36, wherein the microparticle (too) for immobilizing the analytes from the fluid refers to a microparticle (too) for immobilizing analytes of the first type and of the second type different from the first type from the fluid.

38. The microparticle (100) according to claim 36, wherein the microparticle (100) for immobilizing the analytes from the fluid refers to a microparticle (100) for immobilizing analytes of the first type and of the second type different from the first type from the fluid selectively at different surface regions along the microparticle (100).

39. The microparticle (100) according to any of claims 36 to 38, wherein the first molecule composition comprises the immobilization molecules of the first type at a higher concentration than the second molecule composition.

40. The microparticle (100) according to claim 39, wherein the first molecule composition comprises the immobilization molecules of the first type at the higher concentration, in terms of weight percent, than the second molecule composition.

41. The microparticle (100) according to claim 39, wherein the first molecule composition comprises the immobilization molecules of the first type at the higher concentration, in terms of molar concentration, than the second molecule composition.

42. The microparticle (100) according to any of claims 39 to 41, wherein the first molecule composition comprises the immobilization molecules of the first type at the higher concentration than the second molecule composition, at least by a factor of two or at least by a factor of five or at least by a factor of ten.

43. The microparticle (100) according to any of claims 39 to 42, wherein the first molecule composition comprises the immobilization molecules of the first type at a higher concentration than the body (110).

44. The microparticle (100) according to claim 43, wherein the first molecule composition comprises the immobilization molecules of the first type at the higher concentration, in terms of weight percent, than the body (110).

45. The microparticle (100) according to claim 43, wherein the first molecule composition comprises the immobilization molecules of the first type at the higher concentration, in terms of molar concentration, than the body (110).

46. The microparticle (100) according to any of claims 43 to 45, wherein the first molecule composition comprises the immobilization molecules of the first type atthe higher concentration than the body (no), at least by a factor of two or at least by a factor of five or at least by a factor of ten.

47. The microparticle (too) according to any of claims 36 to 46, wherein the second molecule composition comprises the immobilization molecules of the second type at a higher concentration than the first molecule composition.

48. The microparticle (too) according to claim 47, wherein the second molecule composition comprises the immobilization molecules of the second type at the higher concentration, in terms of weight percent, than the first molecule composition.

49. The microparticle (too) according to claim 47, wherein the second molecule composition comprises the immobilization molecules of the second type at the higher concentration, in terms of molar concentration, than the first molecule composition.

50. The microparticle (too) according to any of claims 47 to 49, wherein the second molecule composition comprises the immobilization molecules of the second type at the higher concentration than the first molecule composition, at least by a factor of two or at least by a factor of five or at least by a factor of ten.

51. The microparticle (too) according to any of claims 47 to 50, wherein the second molecule composition comprises the immobilization molecules of the second type at a higher concentration than the body (110).

52. The microparticle (too) according to claim 51, wherein the second molecule composition comprises the immobilization molecules of the second type at the higher concentration, in terms of weight percent, than the body (110).

53. The microparticle (too) according to claim 51, wherein the second molecule composition comprises the immobilization molecules of the second type at the higher concentration, in terms of molar concentration, than the body (110).

54. The microparticle (too) according to any of claims 51 to 53, wherein the second molecule composition comprises the immobilization molecules of the second type at the higher concentration than the body (110), at least by a factor of two or at least by a factor of five or at least by a factor of ten.55- The microparticle (100) according to any of the preceding claims,wherein the first detection element comprises a first cavity (122), and at least part of the first hydrophilic surface is a surface of the first cavity (122), and the second detection element comprises a second cavity (122), and at least part of the second hydrophilic surface is a surface of the second cavity (122).

56. The microparticle (100) according to claim 55, wherein the first cavity (122) and the second cavity (122) are separated from one another by means of the body (110).

57. The microparticle (100) according to claim 55 or 56, wherein said surface of the first cavity (122) refers to an inner surface of the first cavity (122) and said surface of the second cavity (122) refers to an inner surface of the second cavity (122).

58. The microparticle (100) according to any of claims 55 to 57, wherein the first cavity (122) and / or the second cavity (122) is formed as a through hole extending from a bottom surface (114, 116) of the microparticle (100) to a top surface (114, 116) of the microparticle (100), the top surface (114, 116) and the bottom surface (114, 116) being opposite surfaces of the microparticle (100).

59. The microparticle (100) according to any of the preceding claims, wherein the detection elements (120) are embedded in the body (110).

60. The microparticle (100) according to claim 59, wherein the first cavity (122) and the second cavity (122) are embedded in the body (110).

61. The microparticle (100) according to any of the preceding claims, wherein the microparticle (100) has a thickness (t) of less than 1 mm.

62. The microparticle (100) according to any of claims 1 to 60, wherein the microparticle (100) has a thickness (t) of less than 1 mm from a top surface (114, 116) of the microparticle (100) to a bottom surface (114, 116) of the microparticle (100), the bottom surface (114, 116) being located opposite to the top surface (114, 116).

63. The microparticle (100) according to claim 62, wherein the top surface (114, 116) and the bottom surface (114, 116) are parallel to each other.

64. The microparticle (100) according to claim 62 or 63, wherein an aspect ratio between the thickness (t) of the microparticle (100) and a width (w) of the bottomsurface (114, 116) is no more than 1:1.5 or no more than 1:2 or no more than 1:2.5 or no more than 1:3.

65. The microparticle (100) according to claim 62 or 64, wherein an aspect ratio between the thickness (t) of the microparticle (100) and a width (w) of the top surface (114, 116) is no more than 1:1.5 or no more than 1:2 or no more than 1:2.5 or no more than 1:3.

66. The microparticle (100) according to any of the preceding claims, wherein the microparticle (100) has a width (w) of less than 3 mm.

67. The microparticle (100) according to any of claims 1 to 65, wherein the microparticle (100) has a width (w) of less than 1 mm.

68. The microparticle (100) according to claim 66 or 67, wherein the microparticle (100) has said width along any spatial direction.

69. The microparticle (100) according to any of the preceding claims wherein the microparticle (100) is shaped to permit, when the microparticle (100) is placed in the fluid, the fluid to reach the first hydrophilic surface and to reach the second hydrophilic surface.

70. The microparticle (100) according to any of the preceding claims, wherein the body (110) comprises or consists of a body material, the body material being hydrophobic, optionally, the body material being a first hydrogel material.

71. The microparticle (too) according to any of the preceding claims, wherein the first detection element comprises or consists of a first material, the first material being hydrophilic, optionally, the first material being a second hydrogel material.

72. The microparticle (too) according to any of the preceding claims, wherein the second detection element comprises or consists of a second material, the second material being hydrophilic, optionally, the second material being a third hydrogel material.

73. The microparticle (too) according to claim 72, wherein the second material is the same as the first material, optionally, the first material and the second material being a same hydrogel material.74- The microparticle (100) according to any of the preceding claims, wherein the first detection element and the second detection element are encircled by a top surface (114, 116) of the microparticle (100), in particular, wherein the first detection element and the second detection element are encircled by the top surface (114, 116) of the microparticle (100) and by a bottom surface (114, 116) of the microparticle (100), the top surface (114, 116) and the bottom surface (114, 116) being opposite surfaces (114, 116) of the microparticle (100).

75. The microparticle (100) according to any of the preceding claims, wherein the detection elements (120) further comprise a third detection element comprising a third hydrophilic surface, wherein the third detection element at the third hydrophilic surface comprises a third molecule composition for immobilizing analytes from the fluid at the third hydrophilic surface, wherein the third molecule composition is different from the first molecule composition and from the second molecule composition.

76. The microparticle (100) according to claim 75, wherein the detection elements (120) further comprise a fourth detection element comprising a fourth hydrophilic surface, wherein the fourth detection element at the fourth hydrophilic surface comprises a fourth molecule composition for immobilizing analytes from the fluid at the fourth hydrophilic surface, wherein the fourth molecule composition is different from the first molecule composition, from the second molecule composition and from the third molecule composition.

77. The microparticle (100) according to any of the preceding claims, wherein the detection elements (120) are separated from one another by means of the body (110).

78. A plurality of microparticles (100) according to any of the preceding claims,wherein each microparticle (100) of the plurality of microparticles (100) comprises the marker structure (112) according to any of claims 2 to 12.

79. The plurality of microparticles (100) according to claim 78, wherein each microparticle (100) of the plurality of microparticles (100) comprises the at least two different marker materials according to any of claims 13 to 30.

80. A plurality of microparticles (100) according to any of claims 1 to 77, wherein each microparticle (100) of the plurality of microparticles (100) comprises the at least two different marker materials according to any of claims 13 to 30.

81. The plurality of microparticles (100) according to claim 80, wherein each microparticle (100) of the plurality of microparticles (100) comprises the at least two different marker materials according to any of claims 13 to 30 with a same arrangement thereof in the microparticles (100) of the plurality of microparticles (100).

82. A method (200) for fabricating the microparticle (100) according to any of the preceding claims, the method (200) comprising:providing (210) parallel flows of fluids in a flow channel (302), said parallel flows of fluids comprising:a flow of a fluid comprising a precursor for a hydrophobic material;a flow of a first additional fluid, the first additional fluid comprising a precursor for a hydrophilic material; anda flow of a second additional fluid, the second additional fluid comprising a precursor for a hydrophilic material;curing (220) the precursor for the hydrophobic material into a hydrophobic material forming at least part of the body (110);curing (230) the precursor for the hydrophilic material comprised in the first additional fluid into a hydrophilic material forming at least part of the first detection element; andcuring (240) the precursor for the hydrophilic material comprised in the second additional fluid into a hydrophilic material forming at least part of the second detection element;wherein, optionally, the provided parallel flows of the fluids comprise a common solvent and / or the first additional fluid and the second additional fluid comprise immobilization molecules, such that the first molecule composition comprises immobilization molecules from the first additional fluid and the second molecule composition comprises immobilization molecules from the second additional fluid, in particular, wherein the immobilization molecules comprised in the first additional fluid comprise immobilization molecules of a first type and theimmobilization molecules comprised in the second additional fluid comprise immobilization molecules of a second type, and / orwherein at least one of the following three applies: the hydrophobic material is a first hydrogel material, the hydrophilic material for which the first additional fluid comprises the precursor is a second hydrogel material, and the hydrophilic material for which the second additional fluid comprises the precursor is a third hydrogel material;preferably, the hydrophobic material being the first hydrogel material, the hydrophilic material for which the first additional fluid comprises the precursor being the second hydrogel material, and the hydrophilic material for which the second additional fluid comprises the precursor being the third hydrogel material.

83. An apparatus (300) for fabricating the microparticle (too) according to any of claims 1 to 77, the apparatus (300) comprising a flow channel (302), the flow channel (302) comprising:at least three inlet openings (310) for providing (210) parallel flows of fluids in the flow channel (302), wherein each of the at least three inlet openings (310) is adapted to provide a respective one of the parallel flows of the fluids in the flow channel (302);a reducing section (320) downstream from the at least three inlet openings (310), wherein a cross-sectional area of the flow channel (302) at a downstream end of the reducing section (320) is smaller than a cross- sectional area of the flow channel (302) at an upstream end of the reducing section (320);tapered elements (330) arranged in the flow channel (302) and at least partially in the reducing section (320) of the flow channel (302), each of the tapered elements (330) having a smaller cross-sectional area at a downstream end of the respective tapered element than at an upstream end of the respective tapered element;wherein the at least three inlet openings (310) comprise a first inlet opening (312) located in the flow channel (302) at a first lateral position and a second inlet opening(314) located in the flow channel (302) at a second lateral position laterally offset from the first lateral position; andwherein the tapered elements (330) comprise:a first tapered element (332) arranged downstream from the first inlet opening (312) at a lateral position corresponding to the first lateral position, to reduce a cross-sectional area of the one of the parallel flows of the fluids provided by the first inlet opening (312), anda second tapered element (334) arranged downstream from the second inlet opening (314) at a lateral position corresponding to the second lateral position, to reduce a cross-sectional area of the one of the parallel flows of the fluids provided by the second inlet opening (314).

84. The apparatus (300) according to claim 83, wherein the tapered elements (330) further comprise an outer tapered element (336) around the first tapered element (332) and the second tapered element (334).

85. The apparatus (300) according to claim 84, wherein the outer tapered element (336) surrounds the first tapered element (332) and the second tapered element (334) in a plane perpendicular to a flow direction defined by the flow channel (302).

86. The apparatus (300) according to claim 84 or 85, wherein the outer tapered element (336) has an asymmetric shape around a centerline of the flow channel (302).

87. The apparatus (300) according to claim 86, wherein the asymmetric shape refers to a shape without a rotational symmetry around the center line.

88. The apparatus (300) according to claim 86, wherein the asymmetric shape refers to a shape without a discrete or continuous rotational symmetry around the centerline.

89. The apparatus (300) according to any of claims 84 to 88, wherein the outer tapered element (336) comprises at least one notch.

90. The apparatus (300) according to any of claims 84 to 88, wherein the outer tapered element (336) comprises at least two notches at different lateral positions along the outer tapered element (336).

91. The apparatus (300) according to claim 90, wherein the at least two notches at the different lateral positions along the outer tapered element (336) have different sizes.

92. The apparatus (300) according to claim 90 or 91, wherein the at least two notches further comprise a third notch.

93. The apparatus (300) according to any of claims 83 to 92,wherein the at least three inlet openings (310) comprise at least one outer inlet opening (316, 318); andwherein, according to a projection onto a plane perpendicular to a flow direction defined by the flow channel (302), each of the at least one outer inlet opening (316, 318) is arranged around the first inlet opening (312) and around the second inlet opening (314).

94. The apparatus (300) according to claim 93, wherein the at least one outer inlet opening (316, 318) comprises a first outer inlet opening (316).

95. The apparatus (300) according to claim 94, further with the features of claim 84, wherein the outer tapered element (336) is arranged downstream from the first outer inlet opening (316) and, according to the projection onto the plane perpendicular to the flow direction defined by the flow channel (302): the upstream end of the outer tapered element (336) is arranged around the first outer inlet opening (316).

96. The apparatus (300) according to claim 94, further with the features of claim 84, wherein the outer tapered element (336) is arranged downstream from the first outer inlet opening (316) and, according to the projection onto the plane perpendicular to the flow direction defined by the flow channel (302): the outer tapered element (336) overlaps the first outer inlet opening (316).

97. The apparatus (300) according to claim 95 or 96, wherein the at least one outer inlet opening (316, 318) comprises a second outer inlet opening (318).

98. The apparatus (300) according to claim 97, wherein the outer tapered element (336) is arranged downstream from the second outer inlet opening (318) and, according to the projection onto the plane perpendicular to the flow direction defined by the flow channel (302): the second outer inlet opening (318) is arranged around thedownstream end of the outer tapered element (336) and / or the outer tapered element (336) overlaps the second outer inlet opening (318).

99. The apparatus (300) according to any of claim 83 to 98,wherein the apparatus (300) further comprises a lithographic mask (352) arranged in a lithographic masking section (350) of the apparatus (300), wherein a section (302m) of the flow channel (302) passes through said lithographic masking section (350), and wherein the lithographic mask (352) is arranged in the lithographic masking section (350) such that, under illumination of the mask with a UV radiation (354) having a direction towards said section (302m) of the flow channel (302), said UV radiation (354) is masked with the lithographic mask (352) before reaching said section (302m) of the flow channel (302).too.The apparatus (300) according to claim 99, wherein the lithographic mask (352) comprises slits (20) having segments (20a) with an extension perpendicular to a flow direction defined by the flow channel (302) in the lithographic masking section (350), said slits (20) being adapted to allow said UV radiation (354) to reach said section (302m) of the channel, wherein the lithographic mask (352) is adapted to block at least a part of the UV radiation (354) or the UV radiation (354) not passing through the slits (20).

101. The apparatus (300) according to claim 99 or too, wherein the apparatus (300) further comprises a UV source (356) adapted to provide said UV radiation (354) having the direction towards said section (302m) of the flow channel (302).

102. The method (200) according to claim 82,wherein the method (200) applies the apparatus (300) of any of claims 83 to 101;wherein the parallel flows of the fluids in the flow channel (302) are provided via the at least three inlet openings (310);wherein the flow of the first additional fluid is provided from the first inlet opening (312); andwherein the flow of the second additional fluid is provided from the second inlet opening (314).103- The method (200) according to claim 102, further with the features of claim 93, wherein the flow of the fluid comprising the precursor for the hydrophobic material is provided from one of the at least one outer inlet opening (316, 318).

104. The method (200) according to claim 102, further with the features of claim 93, wherein the flow of the fluid comprising the precursor for the hydrophobic material is provided from the first outer inlet opening (316).

105. The method (200) according to any of claims 102 to 104, wherein the parallel flows of the fluids provided in the flow channel (302) further comprise a carrier flow.

106. The method (200) according to claim 105, further with the features of claim 93, wherein the carrier flow is provided from one of the at least one outer inlet opening (316, 318).

107. The method (200) according to claim 106, wherein the carrier flow is provided from the second outer inlet opening (316).

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